Application of ISO22000 and comparison to HACCP for processing of

Volume 43 , Issue 10 , pp.1729-1911(October 2008) Original articles Application of ISO22000 and comparison to HACCP for processing of ready to eat vegetables: Part I (p 1729-1741) Theodoros H. Varzakas, Ioannis S. Arvanitoyannis Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2007.01675.x

Safety evaluation of individual non-fried and fried sunflower oil, paraffin oil, jojoba oil and their binary mixtures on rat health (p 1742-1753) Radwan S. Farag, Mostafa M. Farag, Amany M. Basuny, Rehab F. Mohammed Published Online: Aug 12 2008 8:56AM DOI: 10.1111/j.1365-2621.2007.01679.x

Nutritional potential and functional properties of tempe produced from mixture of different legumes. 1: Chemical composition and nitrogenous constituent (p 1754-1758) Ahmad G. Nassar, Adel E. Mubarak, Alaa E. El-Beltagy Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2007.01683.x

Effect of cold pre-treatment duration before freezing on frozen bread dough quality (p 1759-1762) Yuthana Phimolsiripol, Ubonrat Siripatrawan, Vanna Tulyathan, Donald J. Cleland Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2007.01685.x

Optimisation of supercritical carbon dioxide extraction of lutein esters from marigold (Tagetes erect L.) with soybean oil as a co-solvent (p 1763-1769) Qingxiang Ma, Xiang Xu, Yanxiang Gao, Qi Wang, Jian Zhao Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2007.01694.x

Effect of domestic cooking on the red cabbage hydrophilic antioxidants (p 1770-1777) Anna Podsędek, Dorota Sosnowska, Małgorzata Redzynia, Maria Koziołkiewicz Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2007.01697.x

The effect of par-baking and frozen storage time on the quality of cup cake (p 1778-1785) Mehmet Murat Karaoğlu, Halis Gürbüz Kotancilar, Kamil Emre Gerçekaslan Published Online: Jul 25 2008 5:50AM DOI: 10.1111/j.1365-2621.2007.01698.x

Effect of preliminary and culinary processing on amino acid content and protein quality in frozen French beans (p 1786-1791) Waldemar Kmiecik, Zofia Lisiewska, Jacek Słupski, Piotr Gębczyński Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2007.01702.x

Effect of regular and hydrolysed dairy proteins on texture, microstructure and colour of lean poultry meat batters (p 1792-1797) Shai Barbut Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2007.01705.x

Quality characteristics and storage stability of patties from buffalo head and heart meats (p 1798-1806) Arun K. Verma, Veerappa Lakshmanan, Arun K. Das, Sanjod K. Mendiratta, Anne Sita Ram Anjaneyulu Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2007.01707.x

Antioxidant activity of pomegranate rind powder extract in cooked chicken patties (p 1807-1812) Basappa M. Naveena, Arup R. Sen, Rose P. Kingsly, Desh B. Singh, Napa Kondaiah Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2007.01708.x

Antioxidant activities of red pepper (Capsicum annuum) pericarp and seed extracts (p 1813-1823) Ki Hyeon Sim, Han Young Sil Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2008.01715.x

Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic (p 1824-1831) Mariana A. Moraes, Gabriela S. Rosa, Luiz A. A. Pinto Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01716.x

Determination of oil palm fruit phenolic compounds and their antioxidant activities using spectrophotometric methods (p 1832-1837) Yun Ping Neo, Azis Ariffin, Chin Ping Tan, Yew Ai Tan Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01717.x

Comparison of polyamine, phenol and flavonoid contents in plants grown under conventional and organic methods (p 1838-1843) Giuseppina Pace Pereira Lima, Suraya Abdallah da Rocha, Massanori Takaki, Paulo Roberto Rodrigues Ramos, Elizabeth Orika Ono Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01725.x

Equilibrium moisture of kale seed (Brassica oleracea var. acephala): an alternative method for choice of the best model (p 1844-1849) Marcos A. S. Barrozo, Natália C. Bego, Daniel T. Oliveira Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01728.x

Effect of flavonoids on the oxidative stability of corn oil during deep frying (p 1850-1854) Shahina Naz, Rahmanullah Siddiqi, Syed Asad Sayeed Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01731.x

(+)-Catechin and (−)-epicatechin levels of concentrated and ready-to-drink grape juices through storage (p 1855-1859) Andréa Pittelli Boiago Gollücke, Jane Cristina de Souza, Débora de Queiroz Tavares Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2008.01733.x

Synthesis and in vitro digestion of resistant starch type III from enzymatically hydrolysed cassava starch (p 1860-1865) Calvin Onyango, Christopher Mutungi Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01764.x

A comparison of antioxidant properties between artisan-made and factory-produced chocolate (p 18661870) Rinaldo Cervellati, Emanuela Greco, Stefano Costa, Maria Clelia Guerra, Ester Speroni Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01765.x

Banana: cultivars, biotechnological approaches and genetic transformation (p 1871-1879) Ioannis S. Arvanitoyannis, Athanassios G. Mavromatis, Garyfalia Grammatikaki-Avgeli, Michaela Sakellariou Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2008.01766.x

Effect of the side chain size of 1-alkyl-pyrroles on antioxidant activity and 'Laba' garlic greening (p 18801886) Dan Wang, Xiaosong Hu, Guanghua Zhao Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2008.01778.x

Comparative study on composition and antioxidant properties of mint and black tea extract (p 1887-1895) Ekambaram Padmini, Krishnan Prema, Bose Vijaya Geetha, Munuswamy Usha Rani Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01782.x

The effect of shape, blanching methods and flour on characteristics of restructured sweetpotato stick (p 1896-1900) Joko S. Utomo, Yaakob B. Che Man, Russly A. Rahman, Mohd. Said Saad Published Online: Sep 10 2008 5:05AM DOI: 10.1111/j.1365-2621.2008.01792.x

Short communication Fluid dynamic gauging studies of swelling behaviour of whey protein gels in NaOH/NaCl solutions (p 19011907) Pradeepta K. Sahoo, Y.M. John Chew, Ruben Mercadé-Prieto, D. Ian Wilson, Xiao W. Dai Published Online: Sep 10 2008 5:06AM DOI: 10.1111/j.1365-2621.2007.01689.x

Book reviews Modifying lipids for use in food (p 1908-1909) Paul Wassell Published Online: Dec 21 2007 12:00AM DOI: 10.1111/j.1365-2621.2007.01584.x

Bakery Products Science and Technology (p 1910-1911) Narpinder Singh Published Online: Jun 28 2008 6:13AM DOI: 10.1111/j.1365-2621.2007.01653.x

International Journal of Food Science and Technology 2008, 43, 1729–1741

Original article Application of ISO22000 and comparison to HACCP for processing of ready to eat vegetables: Part I Theodoros H. Varzakas1 & Ioannis S. Arvanitoyannis2* 1 Department of Technology of Agricultural Products, School of Agricultural Sciences, Technological Educational Institute of Kalamata, Hellas, Greece 2 Department of Agriculture, Animal Production and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, 38446 Volos, Hellas, Greece (Received 18 April 2007; Accepted in revised form 1 August 2007)

Summary

Preliminary Hazard Analysis was used to analyse and predict the occurring failure modes in a food chain system (ready to eat vegetables processing plant), in conjunction with ISO22000, the new Food Safety Management System, based on the functions, characteristics and ⁄ or interactions of the ingredients or the processes, upon which the system depends. Critical control points (CCPs) have been identified and implemented in the Hazard Analysis Critical Point Control plan. The decision table for CCP determination during processing of ready to eat vegetables is shown and compared with the ISO22000 Analysis Worksheet for determination of the prerequisite programmes. The prerequisite programmes are the main difference between the two systems. The incorporation of PrPs in the ISO22000 made the system more flexible as a smaller number of CCPs was introduced.

Keywords

CCPs, GHPs, GMPs, HACCP, ISO22000, Preliminary Hazard Analysis, PrPs, ready to eat vegetables manufacturing.

Introduction

As food safety continues to be a worldwide public health issue, epidemiological studies have shown a significant increase in the number of produce-related foodborne illnesses over the past three decades. The Centers for Disease Control and Prevention (CDC) have reported that the mean number of outbreaks associated with fruits and vegetables more than doubled from 1973 to 1987 (4.3 per year) and again from 1988 to 1991 (9.75 per year). Salmonella spp. were the most common aetiological agents linked to these outbreaks. During 1995 alone, major outbreak investigation linked infections with Salmonella serotype Stanley to alfalfa sprouts, Salmonella Hartford to unpasteurised orange juice, Shigella spp. to lettuce and green onions, Escherichia coli O157:H7 to lettuce and hepatitis A virus to tomatoes (Tauxe, 1997). More recently in the USA, The Center for Science in the Public Interest ranked fresh produce the fourth highest cause of all food illness since 1990, behind seafood, eggs and beef. This data excluded bagged salads, fruit salad, or other processed produce. Sprouts and lettuce were the most frequent culprits. In a related study, labelled ‘multi-ingredient foods’, 14 outbreaks over the 10-year period (1990–2000) were *Correspondent: Fax: +302421093137; e-mail: [email protected]

attributed to bagged salads as well as salad bars and processed items not devoted exclusively to produce items. The importance of fresh produce as a vehicle for pathogen transmission and those specific pathogens epidemiologically implicated in fresh produce-related diseases have been extensively reviewed and documented (Doyle, 1990; Nguyen-The & Carlin, 1994; Beuchat & Ryu, 1997; Tauxe et al., 1997; DeRoever, 1999; Gillian et al., 1999; Garg et al., 1990; Stevenson & Combas, 1999; Stevenson, 1993; Fain, 1996; Zagory et al., 1996; Hurst, 2002; Notermans et al., 1995b; Brackett, 1987). Processors of fresh-cut produce have long understood their responsibility for providing a microbiologically safe, high-quality product to the consumer. They have always taken a proactive attitude toward safety. In 1987, they established a sanitation task force to develop model sanitary guidelines. The culmination of this work was a publication entitled ‘Recommended Sanitary Guidelines for the Produce Processing Industry’ that was released in 1992. This document set forth basic sanitary Good Manufacturing Practices (GMPs) and standardised processing procedures (SOPs) to ensure consistent quality and improved a processor’s credibility with local, state and federal food inspectors for ensuring a safe product to the consumer (Hurst, 1992). Despite the many technological and educational advances within the fresh-cut industry in its short history, the challenge remains how to best ensure

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product safety. The purpose of this study is to review some control measures for safety based on HACCP and ISO22000 and provide a preventative, advantageous strategy for minimising food safety hazards. The Hazard Analysis Critical Point Control approach

As indicated above, monitoring a finished food product is no guarantee of safety because unsafe samples may escape detection. What is needed is a more focussed approach toward controlling food safety. Such a programme is the Hazard Analysis Critical Point Control (HACCP) concept. HACCP is a structured approach to the identification, assessment of risk (likelihood of occurrence and severity), and control of hazards associated with a food production process or practice. HACCP addresses the root causes of food safety problems in production, storage, transportation, etc. and is preventative (FDA, 1994). It aims to identify possible problems before they occur and establish control measures at stages in production that are critical to product safety. One of the purposes of HACCP is to desing safety into the process, thereby reducing the need for extensive microbiological testing of inline samples and finished product (Silliker, 1995). Design and implementation of a HACCP system involves the well-known seven basic principles or steps (Stevenson & Bernard, 1999). HACCP for the fresh-cut industry must be built around a series of preservative factors (hurdles) to control pathogen growth, because there is no definitive kill step in the processing operation. Hurdle technology uses a combination of suboptimal growth conditions in which each factor alone is insufficient to prevent the growth of pathogens, but when combined in additive fashion give effective control (Gorris & Tauscher, 1999). Fresh-cut hurdles include purchasing produce from certified grower ⁄ packers, implementing comprehensive plant sanitation programmes, using numerous antimicrobial agents (Beuchat, 1998) in the wash water, using modified atmosphere packaging (MAP) techniques (Gorris & Tauscher, 1999), and following consistent low temperature management. The message is clear – all food processors will have HACCP in their future (Stier & Blumenthal, 1995). HACCP undoubtedly will be the food safety system of the future for regulatory use (Corlett, 1998). HACCP is a proven, cost-effective method of maximising food safety, because it focuses on hazard control at its source. It offers systematic control by covering all aspects of production and handling from raw materials to consumer preparation. HACCP builds customer confidence that food safety is being effectively managed at a fresh-cut operation. Because of its stringent controls, HACCP will bring about improvements in product quality. It demonstrates where to target

International Journal of Food Science and Technology 2008

resources to reduce risks. HACCP implementation will reduce losses from recalled or reworked product. HACCP complements total quality management because it offers continuous problem prevention. Although HACCP implementation may lead to product quality improvement, it should be a distinctly separate programme, not incorporated with quality control in a fresh-cut operation. HACCP is a food safety system and should focus solely on safety issues. For example, HACCP would be designed to prevent E. coli O157:H7 contamination in fresh-cut lettuce but would not guarantee the absence of brown leaves in a 2-lb bag (Hurst, 1995). In the early days of HACCP development, too many areas were designated as ‘critical’, causing the overlap of safety and quality points. The result was frustration and overwork among personnel who were responsible for monitoring and documenting these areas. Mixing safety and quality aspects in the same plan caused a dilution of the really critical areas and failures of many HACCP plans. HACCP is not a substitute for the FDA’s Sanitary GMPs. In fact, effective sanitation must be a prerequisite for successful implementation of a plant HACCP programme. Sanitary procedures may, however, become incorporated as a control tool in HACCP plans to prevent a hazard from becoming a reality. For example, scarred cutting boards on a lettuce trim line may have shown to be a niche for microbiological pathogens based on equipment audits, if careful sanitation does not exert control over this area. Moreover, HACCP plans, unlike GMPs, are not designed to cover all areas of sanitary control in a food operation. Instead, they narrowly focus on specific areas where hazards might be introduced. All GMP requirements are equal from a regulatory perspective. So, dust is filth under GMPs, and its presence on equipment is a violation. However, under HACCP, control of dust is a sanitary step but is not critical, because its presence is unlikely a safety hazard. As HACCP is integrated into food industry management systems, it becomes evident that HACCP cannot exist as a stand-alone programme. Sperber (1998) point out that HACCP cannot be successfully applied in a vacuum, but rather, it must be supported by a strong foundation of prerequisite programmes. While not a formal part of HACCP, prerequisite programmes must be developed and implemented in a food processing operation before attempting to put a HACCP plan in place. Prerequisite programmes are written, implemented procedures that address operational conditions and provide the documentation to help an operation run more smoothly to maintain a comprehensive food-safety assurance programme. Fresh-cut processors should develop written prerequisite programmes for the following operations: raw material receipt and storage; wash

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ISO22000 & HACCP in ready to eat vegetables T. H. Varzakas and I. S. Arvanitoyannis

water quality; equipment maintenance; production controls for grading, washing, cutting, drying and packaging; temperature and microbiological controls; chemical control; sanitary control for the facility, equipment and employees; product coding and traceability; recall procedure control; and finished product storage and distribution control. The heart of any prerequisite programme is the SOPs. SOPs are written references used to describe a specific sequence of events necessary to perform a task (Harris & Blackwell, 1999). In other words, they are step-by-step instructions that outline how an operation is to be carried out. SOPs must be written for both safety and non-safety operational tasks. For example, a detailed procedure should be written for monitoring and maintaining correct disinfectant levels of fresh-cut wash water to insure product safety. Likewise, a specific SOP for checking product temperature should be developed that gives instruction on where and how often to perform the task to maintain product quality and shelf life. SOPs are used to assure that critical processing steps are accomplished and can also be used to train employees. Sanitation SOPs (SSOPs) focus more narrowly on specific procedures that allow a fresh-cut processing plant to achieve sanitary process control in its daily operation. SSOPs can be categorised as two types. SSOPs refer to the sanitary procedures used prior to the start of production (preoperational sanitation). Operational SOPs (OSOPs) refer to sanitary actions taken during production to prevent product contamination or adulteration (Stevenson & Bernard,1999). Preoperational sanitary procedures are written references that describe the cleaning of equipment, utensils, the processing line and the facility area. Specific instructions must include a description of equipment disassembly, use of approved chemicals according to label directions, cleaning techniques, reassembly of equipment and proper use of approved sanitizers. Operational SOPs are sanitary practices that must be performed and documented daily to validate that freshcut product safety was maintained during production. There are five key elements to writing an OSOP. They include a written action plan identifying the task, the frequency of the task, the person responsible for the task, the person responsible for verifying the activity and corrective actions taken if the expected outcome is not met. By establishing effective prerequisite programmes prior to designing and implementing HACCP, the number of critical control points (CCPs) intended in a plan for fresh-cut plants may be reduced. HACCP becomes more ‘user friendly’ and manageable because resources can be concentrated on the hazards associated with the product, not the processing environment. If SSOPs and ⁄ or OSOPs are included as part of the HACCP plan, they must lend themselves to all aspects

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of a CCP, including established critical limits and monitoring, corrective action, record keeping and verification procedures. Good agricultural practices (GAPs) should also be employed to minimise these hazards (Center for Food Safety and Applied Nutrition (CFSAN), 1998). Specifically, they address potential contamination from water sources, fertiliser use (manure or compost), worker health and hygiene, and field and packing shed sanitation, and calls for the development of trace-back procedures for fresh produce. Moreover GMPs should be used i.e. the minimum sanitary and processing requirements necessary to insure the production of wholesome food (Harris & Blackwell, 1999). FDA requirements for GMPs are listed in Title 21, Part 110 of the Code of Federal Regulations. GMPs are written for the following plant areas: building and facilities, equipment and utensils, employee practices, pest control, production and process controls and warehousing practices. GMPs are broadly written, general in nature, and not intended to be plant specific. GMPs can be used to explain tasks that are part of many jobs (e.g. GMPs are written for personal hygiene and dress regardless of job title, management, production, quality assurance, maintenance, etc.). GMPs differ from HACCP in a number of ways. First, they are not designed to control specific hazards; second, they do not provide methods for monitoring hazards; and third, they do not require specific recordkeeping procedures. GMPs are not used to establish deviation limits and do not describe corrective action requirements. Advantages of ISO22000

1 Optimum distribution of resources inside the food chain organisation. 2 Effective communication of suppliers, clients, authorities and other involved authorities. 3 Focus on the prerequisite programmes, conditions and hygiene measures, planning of preventive actions with the aim to eliminate any possible failures. 4 Better documentation. 5 Creation of trust with the prerequisite the credibility of the management system based on the provision of the conditions for the accomplishment of solid results i.e. the management processes and provision of resources and visual operations.

The new standard ISO22000 ‘Food safety management systems – Requirements for food chain organisations’ aims at the proper implementation worldwide of the internationally well-known principles of HACCP from the food chain organisations to provide safe food to the consumers.

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Prerequisite programmes are written, implemented procedures that address operational conditions and provide the documentation to help an operation run more smoothly to maintain a comprehensive food-safety assurance programme. Processors should develop written prerequisite programmes for the following operations: raw material receipt and storage; wash water quality; equipment maintenance; production controls for grading, washing, cutting, drying and packaging; temperature and microbiological controls; chemical control; sanitary control for the facility, equipment and employees; product coding and traceability; recall procedure control and finished product storage and distribution control (Fig. 1). ISO22000 specifies the requirements of a Food Safety Management System, encompassing all the range of food organisations involved in the food chain from farmers to catering businesses. ISO22000 creates a uniform and homogeneous platform of requirements, acceptable to all authorities worldwide. The adoption of ISO22000 was carried out in the year 2005. These food organisations involve the following categories: 1 The directly involved organisations with the food chain, i.e. primary production, food additives manufacturers, raw and auxiliary raw materials for the food industries, food manufacturers, food services, food distributors, pest control companies as well as distribution and warehousing companies. 2 The indirectly involved such as suppliers of raw materials, equipment, cleaning and disinfectant solutions, packaging materials and other materials that come directly or indirectly into contact with food (Arvanitoyannis & Tzouros, 2006). Product preparation

Ready-To-Use (RTU) fruits and vegetables is a rapidly developing category of foods. This product category concerns the food industries a great deal because it serves big restaurants, catering units and modern households where lack of time is the result of the modern way of living. The processing of minimally processed foods or RTU fruits and vegetables consists of cleaning, dehusking and cutting so that they become ready to use. The most important characteristics of these products regarding quality and safety are listed below: 1 Plant tissue could be damaged during processing 2 The product is raw, unprocessed and plant tissue remains live during its shelf life 3 Packaging protects the product from microbial contamination and allows the extension of its shelf life 4 Fresh-cut vegetables are being processed under quality management systems to assure quality.

International Journal of Food Science and Technology 2008

Minimally processed vegetables are in danger of increasing the percentage of metabolic processes which cause the aggravation of fresh products. The natural damage of freshly cut products caused by processing increases respiration and ethylene production in a few minutes, accelerates the ripening of climacteric fruits and vegetables (e.g. tomatoes, melons), and the accumulation of phenolic substances, with the immediate result of an increase in biochemical reactions responsible for colour change, taste, nutritional composition such as loss of vitamins. The greater the processing, the greater the distress of the vegetable tissues. All these reactions could be minimised by cooling the products before processing. The strict temperature control following processing is critical for the reduction of the negative effects in metabolic activities. Other preventative measures leading to the reduction of the damage in RTU products are the correct use of sharp knives and their cutting quality, the strict hygienic rules, the effective washing and drying (humidity removal on the vegetable surface). RTU vegetables maintain a big part of the microflora after processing. Pathogenic microorganisms could be part of this microflora and consist of Listeria monocytogenes detected in a fresh-cut salad product (Nguyen-The & Carlin, 1994), E. coli O157:H7 found in whole cabbage heads from the supplier and Enterohaemorrhagic E. coli in raw vegetable salads in Mexico (Nguyen-The & Carlin, 1994), Clostridium botulinum found in cut cabbage in the USA and Vibrio cholerae detected in cabbage in Peru (Nguyen-The & Carlin, 1994). Changes in the environmental conditions of a product (temperature, humidity, etc.) could lead to important changes in the microflora. The danger of occurrence of pathogenic microorganisms could increase with the packaging film [high relative humidity and low oxygen (O2) percentage], packaged products with low salt concentration and high pH and with storage of packaged products at high temperatures (>5 C). Modified atmosphere packaging

Ready-To-Use vegetables are usually packaged in semipermeable packaging where they modify atmosphere due to respiration. The level of O2 gets reduced from 21% to 2–5% and CO2 level increases from 0.03% to 3–10%. These levels of gases in conjunction with cooling, reduce the respiration rate and microbial growth, delay the physiological ageing and extend the shelf life. Polyvinylchloride (PVC) can be used in the perimetric coverage whereas polypropylene (PP) and polyethylene (PE), used in bags, comprise films used widely in packaging of minimally processed products. Multilayer films, developed from plastic sheets of selective permeability in respiratory gases, can be used with different

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ISO22000 & HACCP in ready to eat vegetables T. H. Varzakas and I. S. Arvanitoyannis

Are the technical infrastructure and the preventative maintenance program adequate?

Corrective actions

No

Yes

Is it feasible to evaluate it?

Yes

No

Do they contribute in the control of recognisable food safety hazards?

No

Included in the prerequisite programs

Yes

Does the effectiveness of the remaining control measures depend on them?

No

Yes

To be included in HACCP plan as a control measure Figure 1 Recognition of prerequisite programmes.

injection gas percentages in the product. Products are usually packaged under vacuum or following injection of different mixtures of gases [O2, carbon dioxide (CO2), carbon monoxide and ⁄ or nitrogen (N2)]. Modified atmospheric packaging creates an atmosphere inside

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packaging poor in O2 and rich in CO2 hence, there is control of enzymatic and chemical reactions as well as bacterial growth. Packaging under vacuum and with gas addition accomplishes the modified atmosphere rapidly increasing the shelf life and quality of processed

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product. In products with high respiration rate, such as broccoli, non-permeable films are used in combination with a permeable membrane to modify the atmosphere through respiration. The ideal films as well as the ideal atmospheric conditions for minimally processed products have not yet been agreed. Moreover, different products require different atmospheric conditions and each processing line has its own peculiarity especially with time delays and temperature deviations. Modified atmosphere packaging for RTU salads has an O2 percentage of 2–8% and CO2 level of 5–15%. Carbon dioxide concentration of 5–15% under low O2 percentages (0.8 mm) and if found they are immediately removed. Casing and

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M: yes Sanitation programme Regular maintenance FIFO system M: yes Personnel hygiene rules M, P: yes Cleaning programme Right equipment Personnel hygiene rules M, P: yes Cleaning programme Right equipment Personnel hygiene rules M, C, P: yes Cleaning programme for equipment Addition of disinfectant solution Control of the mechanism for removal of foreign matter M, C, P: yes Cleaning programme for equipment Control of the mechanism for removal of foreign matter

2. Storage

International Journal of Food Science and Technology 2008

M: yes Regular maintenance of equipment Macroscopic control

M, C, P: yes Cleaning programme for equipment Quality certificates of packaging materials Personnel hygiene rules

8. Drying by centrifugation

9. Packaging

7. Second washing

6. First washing, disinfection

5. Slicing

3. Macroscopic control 4. Prewashing and preparation

M, C, P: yes

1. Receiving of raw materials and vegetables

Q1 Do preventative control measures exist? (Yes ⁄ no)

M, C, P: no

M: no

P: yes Foreign matter removal by washing M, C: no

M: yes Disinfection to reduce microbial load P, C: no

M, P: no

M, P: no

M: no

M: no Storage under controlled conditions

M, C, P: no

Q2 Is the step specifically designed to eliminate or reduce the likely occurrence of hazard to an acceptable level? (Yes ⁄ no)

M: no Personnel training M: no Personnel training P: yes Metal pieces from cutting machine M: no Personnel training P: yes Metal pieces from cutting machine M: ) C: no P: yes Presence of foreign matter in the product M, C: no Operation control of washing machine Water specifications Regular maintenance of equipment P: ) M: yes Presence of water film on the surface of the food due to inadequate drying M, C, P: no Equipment maintenance Macroscopic control

M: yes Pathogenic microorganisms P: yes Foreign matter M: no GMPs Right equipment

Q3 Could contamination with identified hazards(s) or could this increase to unacceptable levels? (Yes ⁄ no)

Decision table for critical control point determination during processing of ready to eat vegetables

Processing stage

Table 1

M, C, P: no

CCP4M

) M, C, F: yes

M: no

CCP3M

)

M, C: no P: yes Second washing follows

M: yes P: yes Metal detector

)

)

M: ) M: yes F: yes Metal detector

CCP2M

CCP1M

Is this step a critical control point? (Yes ⁄ no)

M: no

M, P: no

Q4 Will a subsequent step eliminate identified hazard(s) or reduce likely occurrence to acceptable levels? (Yes ⁄ no)

1736 ISO22000 & HACCP in ready to eat vegetables T. H. Varzakas and I. S. Arvanitoyannis

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CCP5M

) CCP5M

)

) P: )

M: )

P: )

M: no Personnel training ) M: yes Growth of microorganisms in case of increase in temperature M, P: no Personnel training Temperature control of transportation vehicle

M, P: no M, P: no M, P: yes Right transportation vehicles Cleaning programme

The CCP Decision Tree is a tool used to determine the right CCPs for each processing stage. However, according to Wedding (1999a), it is not the perfect tool and cannot replace common sense and processing knowledge and can sometimes lead to false conclusions. This can be seen in Table 1. In Table 2 CCPs, critical limits, process control, corrective actions and verification in cut vegetables have been described. As described earlier the requirements for ISO22000 assume the determination of the prerequisite programmes (Table 3, Fig. 1). The questions frequently asked for each processing step involve questions regarding the adequacy of the technical infrastructure and preventative maintenance, the feasibility for their evaluation, their contribution in the control of recognisable food safety hazards, whether the effectiveness of the remaining control measures depends on them. These questions lead to the answer of a programme being prerequisite or not. Control of CCPs in RTU vegetables

To develop a HACCP plan it is required to get a product description for the product designed to be produced. Information to be included in the product description involves (intended use, packaging materials, ingredients, shelf life), will aid the risk analysis and determination of CCPs. Following product description, flow diagrams, determination of dangers and their risk analysis follows as well as CCPs determination, critical limits determination and their monitoring (accompanied by recording of the measurements), corrective actions and verification procedures. Conclusions

14. Distribution

) M: no ) M: no 12. Paletising 13. Storage of final product

11. Labelling

P: yes Control of the right operation of equipment M: yes Macroscopic control no M: yes Cleaning programme

P: yes Removal of metallic objects M: no

Determination of CCPs in RTU vegetables – Decision tree

10. Metal detection

Processing stage

Q1 Do preventative control measures exist? (Yes ⁄ no)

Storage and product distribution – CCP5

Following paletising product is stored in refrigerators (4–6 C), until loading in tracks operating under the same temperature.

Q4 Will a subsequent step eliminate identified hazard(s) or reduce likely occurrence to acceptable levels? (Yes ⁄ no) Q2 Is the step specifically designed to eliminate or reduce the likely occurrence of hazard to an acceptable level? (Yes ⁄ no)

Continued Table 1

palletising then follows and cartons are ready to be loaded.

Q3 Could contamination with identified hazards(s) or could this increase to unacceptable levels? (Yes ⁄ no)

Is this step a critical control point? (Yes ⁄ no)

ISO22000 & HACCP in ready to eat vegetables T. H. Varzakas and I. S. Arvanitoyannis

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

In this work comparison of ISO22000 analysis with HACCP is carried out over ready to eat salads processing and packaging. Critical control points, critical limits, process control, corrective actions and verification in cut vegetables have been identified and implemented in the HACCP plan.

International Journal of Food Science and Technology 2008

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M

International Journal of Food Science and Technology 2008

Concentration of disinfectant in washing water at 100 ppm (90–110) Absence of foreign matter in washing water (clean water) Absence of foreign matter from cut vegetables Water temperature 1–5 C

Absence of humidity on the surface of the cut vegetables (removed humidity during drying: 25–30% of the initial weight of the washed product)

CPM

P

Concentration of residual chlorine in water: 0.2–0.8 ppm (Limit for taking measures: >1 ppm & S + P (9:1, v/v) > P > S + J (8:2, v/v) > J > S + J (9:1, v/v) > S + P (8:2, v/v) > commercial chip sample. This sequence indicates that potato chips resulted from heated sunflower oil alone had the highest overall acceptability score. On the contrary, the commercial Chrispy had the lowest score of overall acceptability. Table 1 indicates the score values of the sensory evaluation tests of potato chips produced from different heated single and binary oils after 4-h heating period. The potato chips taste score range from 7.76 [S + P (8:2, v ⁄ v)] to 8.46 for neat P + S. The texture and appearance scores were highest for potato fried in S alone, whereas the lowest score was recorded for potato fried in a mixture of S + P (8:2, v ⁄ v). The colour score was highest for potato samples produced from heated S + J (9:1, v ⁄ v), whereas the lowest colour score was for chips obtained from heated S + P (8:2, v ⁄ v). The judges felt that the odour of potato chips obtained from heated

Results and discussion

Sensory evaluation of deep-fried potato chips

The objective of this study was to evaluate the sensory of deep-fried chips produced from frying in sunflower, paraffin and jojoba oils separately and binary mixtures of these oils. Commercial potato chips (Chrispy) was obtained from local market and used to characterise the basis attributes of potato chips and their values were compared with that obtained from the potato chips fried in single and binary mixtures of the oils under study at 2 and 4 h from the beginning of deep-fat process. Table 1 shows the sensory evaluation of potato chips in single and binary mixed oils after 2-h frying period as well as the commercially prepared potato chips (Chrispy). The taste scores of the samples suggested that potato chips obtained by frying in single oil, in general, were higher than that obtained from mixed binary oils. It is worth noting that potato chips resulted from frying in sunflower oil had the highest taste score. In addition, potato chips produced from single and binary mixed oils under study had higher taste scoring values than that of the commercial chip sample. The texture scores for the various potato chips indicate that the highest score was for potato chips produced from heated sunflower oil and the lowest score was for potato chips obtained from heated S + P oil mixture (8:2, v ⁄ v). Generally, the texture score of fried potato chip samples were little bit

Table 1 Sensory evaluation of fried potato chips in different oils and oil mixtures after two and four hours frying periods and the commercially prepared potato chips (Chrispy)

Attribute 2-h heating period Taste Texture Appearance Colour Odor Overall Quality 4-h heating period Taste Texture Appearance Colour Odor Overall Quality

Sunflower oil mixed with jojoba oil

Sunflower oil mixed with paraffin oil

Sunflower oil (S)

Jojoba oil (J)

Paraffin oil (P)

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

Commercial Sample (Chrispy)

9.20 9.33 9.20 9.66 9.86 9.45 Excellent

8.20 7.96 8.33 9.03 9.16 8.53 Good

8.60 8.46 8.50 8.63 8.93 8.62 Good

7.86 8.16 8.46 8.70 9.10 8.45 Good

8.13 8.16 8.33 8.86 9.40 8.57 Good

8.60 8.36 8.86 9.23 8.52 8.71 Good

7.76 7.90 8.63 8.03 9.40 8.34 Good

7.66 8.10 8.26 8.66 9.00 8.33 Good

8.46 8.93 9.03 8.90 8.93 8.85 Good

8.03 8.20 8.66 8.90 9.06 8.57 Good

8.46 8.56 8.73 8.96 9.00 8.74 Good

8.33 8.53 8.70 9.00 9.10 8.73 Good

7.86 8.13 8.20 8.80 9.03 8.40 Good

8.26 8.60 8.73 8.96 9.10 8.73 Good

7.76 7.90 8.63 8.03 9.40 8.34 Good

7.80 7.93 8.10 8.70 8.83 8.27 Good

Each value represents the parameter mean value from 20 panelists. Overall quality of fried potato chips was rated on a 10-point scale. The overall quality scale was 1,2 bad; 3,4 poor, 5,6 fair; 7,8 good and 9,10 excellent.

International Journal of Food Science and Technology 2008

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Safety evaluation of non-fried and fried sunflower, paraffin, jojoba oils and their mixtures on rats R. S. Farag et al.

S + P (8:2, v ⁄ v) was the highest record, whereas the lowest one came from potatoes produced from heated sunflower oil alone. The total acceptability of the fried potato chips was ranked in the decreasing order according to the type of frying medium as follows: S > P > S þ Pð9:1; v=vÞ > S þ Jð9:1; v=vÞ > J > S þ Jð8:2; v=vÞ > S þ Pð8:2v=vÞ: This sequence demonstrates that the potato chips obtained by frying using sunflower oil alone had the highest acceptability, whereas potato chips obtained from binary heated oils especially S + P (8:2, v ⁄ v) had the lowest record. It is worth noting that the results of organoleptic tests indicate that all types of chips were categorised good and had little bit better records in total acceptability than the commercially produced potato chips (Chrispy). Biochemical evaluation of non-fried and fried sunflower oil, jojoba oil, paraffin oil and their mixtures on rat liver and kidney functions Activity of rat sera enzymes

Table 2 shows sera AST activity of rats fed on non-fried and fried S, J, P and mixtures of them. There were slight

and non-significant increases in the activity of AST during the whole experiment (8 weeks) for rats fed on non-fried S and J. The administration of P alone caused gradual and significant increase in rat sera starting from the second week and towards the end of the experimental period. The data for the administration of S + J mixtures at ratios 9:1 and 8:2 (v ⁄ v) indicate that the activity of AST paralleled with the non-fried S. This means that the addition of J to S did not cause any changes in AST activity. On the contrary, the binary oil mixtures of S + P (9:1 and 8:2, v ⁄ v) induced gradual and significant increase in AST activity. However, this increase was obviously lower than that rats fed on fried P alone. These results highlight the potential effect of P on rising AST activity. The data in Tables 3 and 4 for rat sera activities of ALT and ALP of non-fried and fried S, J, P, S + J (9:1 and 8:2, v ⁄ v) and S + P (9:1 and 8:2, v ⁄ v) indicate similar results for AST enzyme activity. Total lipids

Table 5 indicates the changes of total lipid contents of rats administered non-fried and fried S, J, P and mixtures of them. The results demonstrate that the administration of non-fried and fried S, J and their mixtures (9:1 and 8:2, v ⁄ v) caused gradual and

Table 2 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the activity of sera aspartate aminotransferase (IU L)1) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

Paraffin oil (P)

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

40.13a ± 40.33a ± 41.17a ± 40.87a ± 41.77a ± 40.53a ± 40.80a ± 40.67a ± 40.33a ± 2.42

0.15 0.25 0.76 0.23 0.68 0.21 0.10 0.00 0.58

41.00a ± 40.33a ± 40.67a ± 41.00a ± 40.10a ± 40.33a ± 41.00a ± 41.33a ± 41.41a ± 1.47

1.00 0.25 0.35 1.00 0.10 0.58 1.00 0.58 0.15

41.33a ± 43.67b ± 50.33c ± 53.33d ± 57.33e ± 60.33f ± 63.67g ± 70.67h ± 72.67h ± 2.07

153 0.58 0.58 0.58 0.58 0.58 2.08 0.58 0.58

41.10a ± 41.00a ± 41.00a ± 41.40a ± 40.67a ± 40.93a ± 41.50a ± 41.67a ± 40.67a ± 1.40

0.85 1.00 1.00 0.58 0.58 0.93 0.58 0.58 0.58

41.67a ± 40.67a ± 40.53a ± 40.77a ± 43.00a ± 42.00a ± 40.63a ± 42.10a ± 41.67a ± 1.52

1.53 0.58 0.21 0.21 1.00 1.00 0.35 1.15 0.58

40.67a ± 41.00a ± 43.00b ± 44.67c ± 45.60c ± 44.67c ± 45.67c ± 45.76c ± 46.67c ± 1.38

0.58 1.73 1.00 0.58 0.00 0.58 0.58 0.58 0.58

41.67a ± 42.00a ± 45.67b ± 47.33c ± 48.33c ± 52.67d ± 52.67d ± 53.33d ± 54.67e ± 1.45

1.15 1.00 0.58 0.58 0.58 .58 0.58 0.58 0.58

41.00a ± 43.00a ± 44.67b ± 45.33bc ± 46.67bc ± 47.17cd ± 47.70cd ± 47.67cd ± 50.67cd ± 2.42

1.00 1.00 0.58 0.58 0.58 0.15 0.36 4.04 0.58

40.67a ± 41.00a ± 42.43b ± 43.37bc ± 44.67c ± 47.43d ± 50.133e ± 51.77f ± 52.00g ± 1.47

0.58 1.00 0.49 0.64 0.58 0.51 0.15 0.40 2.00

40.67a ± 44.33b ± 52.67c ± 55.33d ± 60.00e ± 63.33f ± 66.67g ± 75.67h ± 77.33i ± 2.067

1.15 1.53 0.58 1.15 1.00 0.58 1.53 2.08 0.58

40.33a ± 40.33a ± 43.00b ± 44.33b ± 44.67b ± 46.67c ± 47.00c ± 47.20c ± 47.67c ± 1.40

0.58 0.58 1.00 0.58 0.58 0.58 1.00 0.58 0.58

41.00a ± 41.67a ± 43.67b ± 44.60b ± 45.67b ± 46.00b ± 47.67c ± 47.33c ± 45.00b ± 1.52

1.00 1.53 0.58 0.58 0.58 0.00 0.58 0.58 0.00

41.33a ± 42.33a ± 45.33b ± 45.67b ± 47.67c ± 48.83c ± 49.67d ± 54.00e ± 56.67f ± 1.38

0.58 0.58 0.58 0.58 0.58 0.29 0.58 1.00 0.58

40.33a ± 43.00b ± 45.67c ± 47.33d ± 50.00e ± 52.00f ± 54.00g ± 57.33h ± 60.67i ± 1.45

0.58 1.00 0.58 0.58 1.00 1.00 1.00 0.58 0.58

IU refers to aspartate aminotransferase activity as international units. The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

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Table 3 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the activity of serum alanine aminotransferase (IU L)1) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

44.00a ± 43.77a ± 44.00a ± 43.77a ± 44.53a ± 44.33a ± 45.33a ± 45.33a ± 45.67a ± 3.18

1.00 0.68 1.00 1.53 1.50 2.08 0.58 2.08 0.58

46.00a ± 45.00a ± 45.5a ± 45.87a ± 46.00a ± 45.33a ± 45.67a ± 44.67a ± 45.33a ± 2.39

1.00 1.00 0.17 0.15 1.00 1.53 0.58 1.53 1.53

46.00a ± 46.33a ± 48.67a ± 52.33b ± 57.33c ± 61.00d ± 63.67d ± 68.00e ± 73.00f ± 3.01

44.33a ± 46.00a ± 47.00a ± 48.50b ± 51.33b ± 52.50c ± 55.00d ± 56.67d ± 58.33e ± 3.2

3.79 1.00 1.00 0.50 1.58 0.50 1.00 1.15 0.58

45.00a ± 45.20a ± 47.77b ± 49.10b ± 53.00c ± 54.33d ± 56.24d ± 57.33d ± 58.35e ± 2.39

1.00 0.35 0.25 0.17 1.00 0.58 0.58 1.53 0.58

45.67a ± 46.33a ± 50.00a ± 54.77b ± 59.23c ± 59.67c ± 66.50d ± 71.67e ± 74.97e ± 5.01

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

1.00 1.53 0.58 2.08 0.58 1.73 0.58 1.00 3.00

45.63a ± 45.00a ± 46.00a ± 44.67a ± 45.00a ± 44.67a ± 44.76a ± 45.00a ± 45.00a ± 2.51

035 1.00 1.00 1.53 1.00 2.08 1.66 1.00 1.73

45.00a ± 44.93a ± 45.53a ± 44.77a ± 46.00a ± 44.33a ± 45.33a ± 44.00a ± 45.17a ± 2.68

1.00 1.83 1.50 1.69 1.00 1.53 2.08 1.00 0.76

45.00a ± 45.67a ± 46.00a ± 47.00a ± 48.33b ± 49.00b ± 50.00c ± 51.67c ± 54.00d ± 2.70

1.00 0.58 1.00 0.00 0.58 1.00 1.00 3.79 1.00

44.00a ± 45.00a ± 45.67a ± 46.50b ± 47.73b ± 48.33b ± 48.80c ± 51.00c ± 52.33c ± 2.33

2.65 1.00 0.58 0.50 0.15 0.58 0.62 1.00 0.58

1.53 0.58 1.00 1.66 0.68 1.15 1.37 1.53 1.00

45.00a ± 44.33a ± 47.00a ± 48.33b ± 50.67c ± 55.17d ± 55.67d ± 56.83d ± 59.67e ± 2.68

1.00 0.58 1.00 0.58 0.58 1.26 0.58 0.76 0.58

45.17a ± 44.33a ± 48.00b ± 48.50b ± 52.67c ± 53.33c ± 55.67d ± 59.33e ± 59.33e ± 2.51

0.76 0.58 0.00 0.50 0.58 0.58 0.58 0.58 0.58

45.00a ± 44.67a ± 47.00a ± 49.33b ± 51.33c ± 51.46c ± 56.68d ± 59.68e ± 59.69e ± 2.33

7.00 0.58 1.00 0.58 0.58 0.58 0.58 0.58 0.58

44.33a ± 46.00a ± 47.00a ± 48.50b ± 51.33b ± 52.50c ± 55.00d ± 56.67d ± 58.33e ± 3.2

3.79 1.00 1.00 0.50 1.58 0.50 1.00 1.15 0.58

Paraffin oil (P)

IU refers to alanine aminotransferase activity as international units. The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

significant increases in the levels of total lipids. On the contrary, the administration of non-fried, fried P and the binary mixtures of S + P (9:1 and 8:2, v ⁄ v) caused significant decreases in total lipid contents of rat sera starting from the third week of the commencement and towards the end of the experiment. It seems that the application of all types of oil samples caused slight decrease or increase in the values of total lipids irrespective of the significance of statistical analysis.

Meanwhile, the administration of non-fried and fried P caused gradual and slight decreases in the levels of LDL-C during the experiment. High-density lipoprotein-cholesterol content

Table 8 shows the changes in high-density lipoproteincholesterol (HDL-C) levels of rats fed on diets containing non-fried and fried S, J, P and mixtures of them. The results demonstrate that the above mentioned oils possessed very little change on the levels of HDL-C.

Total cholesterol

Table 6 shows the sera total cholesterol contents of rats fed on non-fried, fried S, J and P besides their binary mixtures (S + J (9:1 and 8:2, v ⁄ v) and S + P (9:1 and 8:2, v ⁄ v). The results indicate very little increases (S, P and their binary mixtures) and decreases (P) because of the administration of these oils throughout the entire nutritional experiment. Low-density lipoprotein-cholesterol content

Changes in the levels of low-density lipoprotein-cholesterol (LDL-C) of rats administered non-fried and fried S, J, P and mixtures of them are shown in Table 7. The results indicate that there were very little changes in the LDL-C levels of rats fed on diets containing non-fried and fried S, J and their mixtures (9:1 and 8:2, v ⁄ v).

International Journal of Food Science and Technology 2008

Urea and uric acid contents

Tables 9 and 10 show the changes of urea and uric acid contents (mg dL)1) of rats fed on non-fried and fried S, J, P and mixtures of them. The results show that the administration of non-fried and fried S, J and their mixtures induced very little change on the sera levels of urea and uric acid during the whole experiment (8 weeks). On the contrary, the administration of nonfried, fried P and its mixtures with S at ratios of 2:8 and 1:9 caused gradual and significant increase in urea and uric acid levels starting from the second week and towards the end of the experiment. It is worth noting that paraffin oil alone exhibited higher increases on sera uric acid contents than that of the binary mixed oils. In general, non-fried and fried P alone or mixed with

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Safety evaluation of non-fried and fried sunflower, paraffin, jojoba oils and their mixtures on rats R. S. Farag et al.

Table 4 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the activity of serum alkaline phosphatase (IU L)1) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

81.00a ± 81.83a ± 82.00a ± 81.43a ± 81.33a ± 80.23a ± 81.67a ± 81.33a ± 82.00a ± 3.30

1.00 0.57 1.00 0.98 1.53 0.21 1.53 1.53 1.73

81.33a ± 82.33a ± 83.67a ± 82.67a ± 83.33b ± 84.67b ± 84.33b ± 84.00a ± 84.67a ± 2.97

1.53 0.58 0.58 0.58 0.58 0.58 1.15 0.00 1.53

81.00a ± 83.00a ± 83.67a ± 84.67a ± 86.33b ± 88.00b ± 88.33b ± 91.67c ± 101.67d ± 4.75

78.00a ± 80.67a ± 79.67a ± 85.67b ± 85.67b ± 84.67b ± 85.00b ± 89.67c ± 91.67c ± 3.30

2.65 0.58 0.58 3.06 0.58 0.58 0.00 0.58 2.89

80.33a ± 81.00a ± 79.00a ± 85.67b ± 85.67b ± 85.67b ± 85.00b ± 88.33c ± 91.33d ± 2.97

0.58 1.00 3.61 0.58 0.58 0.58 1.00 1.53 3.21

79.67a ± 80.00a ± 85.33b ± 89.33b ± 93.33c ± 106.67d ± 125.00e ± 126.67e ± 136.67f ± 4.75

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

1.00 1.00 1.15 1.15 1.15 0.00 3.79 0.58 2.89

83.67a ± 83.00a ± 83.67a ± 83.67a ± 83.00a ± 84.67a ± 84.67a ± 84.00a ± 84.00a ± 2.27

0.58 1.00 0.58 0.58 1.73 0.58 1.53 1.73 1.00

82.33a ± 84.00a ± 83.00a ± 82.67a ± 83.00a ± 84.00a ± 84.67a ± 83.33a ± 84.33a ± 2.57

1.15 1.00 1.00 0.58 0.00 1.00 1.15 1.53 2.00

81.01a ± 81.67a ± 81.00a ± 82.33a ± 82.67a ± 82.67a ± 83.33b ± 83.67b ± 84.67c ± 1.86

0.58 0.58 0.00 0.58 0.58 0.58 0.58 0.58 0.58

81.01a ± 81.33a ± 81.33a ± 84.33b ± 85.67b ± 84.33b ± 86.33b ± 87.33c ± 88.67c ± 2.64

2.89 0.58 1.15 1.15 0.58 0.58 0.58 0.58 0.58

0.58 1.00 0.58 0.58 2.89 5.77 5.00 2.89 2.89

80.00a ± 83.33b ± 80.00a ± 84.67b ± 84.00b ± 83.67b ± 85.33c ± 88.67d ± 88.67d ± 2.28

1.00 0.58 0.00 0.58 1.00 0.58 0.58 0.58 0.58

79.33a ± 81.00a ± 81.13a ± 84.00b ± 85.33b ± 85.67b ± 85.33b ± 88.33c ± 90.33c ± 2.57

0.58 0.00 2.89 1.00 .58 0.58 0.58 0.58 0.58

80.33a ± 80.67a ± 80.33a ± 83.67b ± 85.67c ± 87.33d ± 88.33d ± 92.00e ± 95.00f ± 1.86

0.58 0.58 0.58 0.58 0.58 0.58 0.58 1.00 0.00

80.00a ± 81.00a ± 81.67a ± 84.33b ± 86.67b ± 90.00c ± 92.67d ± 94.33d ± 101.67e ± 2.64

1.00 1.00 0.58 0.58 1.00 0.58 0.58 0.58 2.89

Paraffin oil (P)

IU refers to alkaline phosphatase activity as international units. The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

non-fried and fried S (1:9 and 2:8, v ⁄ v) caused deleterious effects on rat liver and kidney functions and vice versa with J alone and its mixtures with S. Histopathological examination results

The influence of non-fried and fried S, J, P and their mixtures on rat liver and kidney tissues of male albino rats was microscopically examined. Histopathological examination of liver tissues

Microscopical examination of liver tissues of rats given basal diet consisting of non-fried S showed no histopathological alterations except slight hydropic degeneration of some hepatocytes (Fig. 1). The examination of liver tissues of rats given basal diet containing unheated P revealed an activation of Kupffer cells and vacuolar degeneration of hepatocytes (Fig. 2) as the vacuoles pushing the nucleus to the side of cells with the appearance of cell signet ring. This means that the histological examination showed signs of toxicity. However, the liver tissues of rats administered basal diet comprises of unheated J indicate apparent normal hepatocytes. In other words, the histological examination did not reveal any microscopic sign of toxicity. Sections of liver tissues from rats given basal diet includes non-fried S and J mixtures (9:1 and 8:2, v ⁄ v)

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

showed no histopathological changes except slight Kupffer cell activation. On the contrary, examination of liver tissues of rats administered basal diet comprises non-fried S and P mixtures (8:2, v ⁄ v) revealed sinusoidal dilatation as well as hydropic degeneration of hepatocytes (Fig. 3). Minute intracytoplasmic vacuoles were observed in hepatocytes in sections of rats administered basal diet includes non-fried S and P mixture (9:1, v ⁄ v). This case indicates signs of toxicity. Swelling of hepatocytes as well as hydropic degeneration of the hepatocytes were noticed in the liver tissues of rats given basal diet comprises fried S (Fig. 4). Histology of liver tissues of rats administered basal diet containing fried J showed vacuolar degeneration of few hepatocytes in the hepatic lobule as well as Kupffer cell activation. Massive vacuolation of hepatocytes with the appearance of signet ring hepatocytes associated with karyomegaly of some hepatocytic nuclei (Fig. 5) were found in examined liver tissues of rats administered basal diet includes fried P. Here again, the microscopical examination indicates the occurrence of toxicity signs. Liver tissues of rats given basal diet containing of fried S and P mixtures (9:1 and 8:2, v ⁄ v) showed no histopathological changes expect slight portal infiltration with leucocytic inflammatory cells. Figure 6 illustrates the presence of minute vacuoles in the cytoplasm of some hepatocytes in liver

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)1

Table 5 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on sera total lipid contents (mg dL ) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

0.43a ± 0.43a ± 0.43a ± 0.44a ± 0.45a ± 0.46a ± 0.50b ± 0.51b ± 0.57c ± 0.07

0.03 0.03 0.01 0.01 0.00 0.00 0.01 0.01 0.02

0.42a ± 0.43a ± 0.44a ± 0.44a ± 0.42a ± 0.44a ± 0.45b ± 0.47b ± 0.47b ± 0.03

0.41a ± 0.41a ± 0.42a ± 0.43a ± 0.47a ± 0.50b ± 0.53b ± 0.52b ± 0.55c ± 0.07

0.02 0.02 0.02 0.01 0.01 0.09 0.02 0.02 0.01

0.39a ± 0.43b ± 0.42b ± 0.43b ± 0.44b ± 0.45c ± 0.46c ± 0.49d ± 0.55e ± 0.03

Paraffin oil (P)

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.03

0.43a ± 0.42a ± 0.41a ± 0.39b ± 0.39b ± 0.36c ± 0.35c ± 0.36c ± 0.35c ± 0.03

0.02 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01

0.45a ± 0.42a ± 0.44a ± 0.42a ± 0.44a ± 0.47b ± 0.52c ± 0.47b ± 0.52c ± 0.04

10.01 0.01 0.01 0.01 0.02 0.00 0.06 0.02 0.01

0.45a ± 0.44a ± 0.44a ± 0.45a ± 0.44a ± 0.46a ± 0.49b ± 0.53c ± 0.58d ± 0.03

0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.03 0.01

0.41a ± 0.41a ± 0.41a ± 0.41a ± 0.46b ± 0.45b ± 0.46b ± 0.49c ± 0.50c ± 0.03

0.01 0.01 0.01 0.01 0.03 0.00 0.03 0.01 0.01

0.45a ± 0.45a ± 0.44a ± 0.45a ± 0.44a ± 0.45a ± 0.48b ± 0.49b ± 0.54c ± 0.02

0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01

0.01 0.02 0.02 0.01 0.1 0.00 0.01 0.01 0.01

0.41a ± 0.41a ± 0.41a ± 0.39a ± 0.37b ± 0.35b ± 0.34c ± 0.36b ± 0.36b ± 0.03

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01

0.41a ± 0.41a ± 0.42a ± 0.42a ± 0.45b ± 0.47b ± 0.52c ± 0.51c ± 0.52c ± 0.04

0.01 0.01 0.01 0.01 0.01 0.01 0.06 0.00 0.01

0.40a ± 0.39a ± 0.41a ± 0.42a ± 0.45b ± 0.46c ± 0.51d ± 0.51d ± 0.54d ± 0.03

0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.42a ± 0.41a ± 0.41a ± 0.42a ± 0.42a ± 0.43a ± 0.46b ± 0.45b ± 0.50c ± 0.03

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.40a ± 0.39a ± 0.43b ± 0.42b ± 0.46c ± 0.47c ± 0.50d ± 0.51d ± 0.53e ± 0.02

0.01 0.02 0.02 0.01 0.01 0.00 0.01 0.01 0.01

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

Table 6 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the sera total cholesterol contents (mm)

of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

Paraffin oil (P)

S+J (8:2, v ⁄ v)

S+J (9:1, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

161.53a ± 161.35a ± 161.62a ± 161.23a ± 161.10a ± 161.58a ± 161.43a ± 161.49a ± 161.78a ± 1.78

0.32 0.38 0.33 1.07 0.92 0.93 2.22 0.12 0.34

161.25a ± 161.00a ± 160.63a ± 160.80a ± 161.10a ± 161.50a ± 160.00a ± 157.87b ± 157.89b ± 1.65

0.49 0.78 0.78 1.14 0.85 0.50 1.08 0.35 0.55

160.70a ± 161.50a ± 161.60a ± 161.37a ± 160.70a ± 159.50a ± 157.93b ± 154.77c ± 152.50c ± 2.92

0.84 0.44 0.10 0.57 0.61 0.50 2.27 0.59 0.50

160.70a ± 161.53a ± 160.65a ± 161.12a ± 161.13a ± 161.40a ± 160.43a ± 160.62a ± 160.47a ± 1.49

0.85 0.50 0.41 0.77 0.76 0.72 0.83 0.28 0.50

161.10a ± 161.00a ± 161.47a ± 161.01a ± 160.88a ± 161.27a ± 160.31a ± 161.35a ± 161.45a ± 1.41

0.14 0.62 0.21 0.88 0.78 0.68 0.53 0.16 0.05

160.50a ± 160.43a ± 160.90a ± 161.30a ± 161.23a ± 161.63a ± 160.71a ± 160.41a ± 159.80a ± 1.51

0.71 0.38 0.82 0.61 1.07 0.40 0.60 0.10 0.46

160.60a ± 161.30a ± 161.47a ± 161.57a ± 161.83a ± 160.97a ± 160.90a ± 160.73a ± 160.67a ± 1.05

0.71 0.26 0.47 0.31 0.15 0.21 0.18 0.15 0.32

160.55a ± 161.13a ± 161.10a ± 161.30a ± 161.47a ± 162.07a ± 162.00a ± 163.40b ± 163.763b ± 1.78

0.78 0.06 0.10 0.10 0.21 0.38 1.34 0.20 0.31

160.27a ± 160.53a ± 160.37a ± 160.77a ± 161.27a ± 161.47a ± 161.67a ± 162.07b ± 162.13b ± 1.65

0.25 0.49 0.64 0.67 0.06 0.15 0.12 0.06 0.49

160.93a ± 160.80a ± 160.6a ± 159.33a ± 159.97a ± 158.47a ± 157.77b ± 157.0b ± 158.80a ± 2.92

0.07 0.17 0.10 1.33 0.40 0.42 0.15 0.21 5.29

161.23a ± 161.30a ± 161.60a ± 161.57a ± 162.17a ± 162.30a ± 162.43a ± 163.07b ± 163.03b ± 1.49

0.15 0.10 0.26 0.15 0.15 0.20 0.45 0.06 0.06

161.10a ± 161.30a ± 161.60a ± 161.57a ± 162.17a ± 162.30a ± 162.43a ± 163.07b ± 163.03b ± 1.49

0.17 0.10 0.26 0.15 0.15 0.20 0.45 0.06 0.06

160.40a ± 160.70a ± 161.30a ± 161.40a ± 161.77a ± 161.83a ± 162.03b ± 162.13b ± 162.33b ± 1.51

0.69 0.61 0.10 0.40 0.32 0.15 0.06 0.15 0.06

160.33a ± 161.07a ± 161.10a ± 161.30a ± 161.38a ± 161.47b ± 161.87b ± 162.27b ± 162.5c ± 1.05

1.26 0.06 0.10 0.00 0.06 0.06 0.15 0.06 0.06

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

International Journal of Food Science and Technology 2008

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Safety evaluation of non-fried and fried sunflower, paraffin, jojoba oils and their mixtures on rats R. S. Farag et al.

Table 7 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the sera levels of low-density lipoproteins (LDL-C) (mg dL)1) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

Paraffin oil (P)

S+J (8:2, v ⁄ v)

S+J (9:1, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8 : 2, v ⁄ v)

100.60a ± 100.71a ± 100.7a ± 101.35a ± 101.529a ± 101.63a ± 101.76a ± 101.73a ± 101.71a ± 1.17

0.30 0.20 0.10 0.35 1.00 0.20 1.00 0.10 1.00

100.51a ± 100.62a ± 100.30a ± 100.28a ± 100.26a ± 100.18a ± 100.15a ± 100.13a ± 100.11a ± 1.14

0.00 0.02 0.30 0.10 0.20 0.10 0.24 0.13 0.11

101.10a ± 100.67a ± 101.00a ± 100.81a ± 98.56a ± 96.55b ± 96.40b ± 95.46b ± 95.40b ± 4.39

1.00 1.53 0.00 5.00 0.00 1.00 1.00 0.00 1.00

100.50a ± 100.46a ± 100.70a ± 101.30a ± 101.20a ± 101.30a ± 101.50b ± 101.66b ± 101.60b ± 0.83

0.20 0.00 0.10 1.00 0.20 0.00 0.30 0.10 0.00

100.01a ± 100.01a ± 100.14a ± 100.20a ± 100.25a ± 100.28a ± 100.30a ± 100.41a ± 100.42a ± 0.72

0.00 0.00 0.14 0.20 0.25 0.28 0.24 0.00 0.02

101.51a ± 101.53a ± 101.41a ± 101.20a ± 100.59a ± 100.50a ± 100.40a ± 100.20a ± 100.10a ± 1.05

0.00 1.00 1.00 1.00 0.00 0.00 0.74 0.00 0.10

100.91a ± 100.90a ± 100.71a ± 100.66a ± 100.50a ± 100.55a ± 100.43a ± 100.41a ± 100.41a ± 1.1

0.00 0.10 0.10 0.30 0.10 0.00 0.31 0.01 0.00

100.30a ± 100.40a ± 100.50a ± 100.10a ± 101.20a ± 101.30a ± 101.129a ± 101.45a ± 101.56a ± 1.17

0.00 0.40 0.10 0.10 1.00 0.10 0.55 0.05 0.06

101.67a ± 101.65a ± 101.70a ± 101.85a ± 101.85a ± 101.90a ± 101.95a ± 101.96a ± 101.11a ± 1.19

1.00 0.20 0.70 1.00 0.00 1.00 0.05 1.00 0.00

100.30a ± 100.35a ± 100.35a ± 100.90a ± 100.60a ± 98.91a ± 98.54a ± 98.21a ± 94.10b ± 3.03

1.00 1.00 0.00 2.00 0.00 2.00 2.00 0.00 1.00

100.40a ± 100.40a ± 100.35a ± 100.31a ± 100.30a ± 100.20a ± 100.20a ± 100.10a ± 100.10a ± 0.83

0.00 0.40 0.35 0.01 0.00 0.20 0.16 0.10 0.00

100.40a ± 100.60a ± 100.70a ± 100.70a ± 100.50a ± 100.60a ± 100.60a ± 100.80a ± 100.80a ± 0.72

0.40 0.20 0.10 0.00 0.20 0.20 0.00 0.00 0.10

100.60a ± 100.20a ± 100.10a ± 100.40a ± 100.60a ± 100.50a ± 100.50a ± 100.40a ± 100.60a ± 1.05

0.20 0.00 0.10 0.10 0.10 0.20 0.00 0.00 0.00

101.60a ± 101.60a ± 101.40a ± 101.30a ± 101.20a ± 101.50a ± 101.60a ± 101.50a ± 100.83a ± 1.11

1.00 0.00 1.00 0.00 1.00 1.00 0.00 0.00 0.58

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time. Table 8 Influence of non- fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the sera levels of high -density lipoproteins (HDL-C) (mg dL)1) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

40.41a ± 40.42a ± 40.50a ± 41.30a ± 41.40a ± 41.45a ± 41.61a ± 41.65a ± 41.65a ± 0.74

1.00 0.02 0.20 0.30 0.40 0.00 0.00 0.00 0.15

41.56a ± 40.561a ± 40.619a ± 40.60a ± 40.709a ± 40.509a ± 41.159a ± 41.40a ± 41.45a ± 0.86

0.07 0.00 0.30 0.20 1.00 0.20 0.50 0.30 0.10

40.60a ± 40.60a ± 40.65a ± 40.70a ± 40.75a ± 40.70a ± 40.60a ± 40.70a ± 40.75a ± 0.69

40.60a ± 40.30a ± 40.50a ± 40.40a ± 40.40a ± 40.30a ± 40.41a ± 40.30a ± 40.30a ± 0.74

0.00 0.30 0.50 0.40 0.00 0.30 0.13 0.30 0.00

40.41a ± 40.45a ± 40.40a ± 40.40a ± 40.35a ± 40.30a ± 40.36a ± 40.20a ± 40.20a ± 0.86

0.20 1.00 0.40 0.00 0.00 0.10 0.00 0.10 0.00

40.70a ± 40.70a ± 40.60a ± 40.60a ± 40.40a ± 40.60a ± 40.50a ± 40.60a ± 40.40a ± 0.69

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

0.00 0.30 1.00 0.30 0.05 0.20 0.00 0.10 0.05

40.31a ± 40.51a ± 40.52a ± 40.66a ± 40.60a ± 41.31a ± 41.13a ± 41.65a ± 41.65a ± 0.95

0.00 1.00 0.20 0.10 0.00 1.00 0.56 0.00 0.00

40.20a ± 40.25a ± 40.30a ± 40.35a ± 40.45a ± 41.00a ± 40.87a ± 41.00a ± 41.20a ± 1.28

0.00 0.25 0.30 0.35 0.45 0.00 0.46 0.00 0.20

40.16a ± 40.15a ± 40.20a ± 40.20a ± 40.25a ± 40.30a ± 40.30a ± 40.50a ± 40.50a ± 0.82

0.07 0.15 1.00 0.10 0.25 0.30 0.38 0.00 0.00

40.30a ± 40.35a ± 40.35a ± 40.30a ± 40.36a ± 40.40a ± 40.46a ± 40.45a ± 40.40a ± 0.92

0.00 0.35 0.00 0.30 0.10 0.30 0.14 0.45 0.00

0.00 0.10 0.10 0.00 0.00 0.30 0.10 0.10 0.00

40.30a ± 40.30a ± 40.40a ± 40.60a ± 40.10a ± 40.10a ± 40.00a ± 40.00a ± 40.10a ± 1.28

0.30 1.00 1.00 0.20 0.10 0.00 1.00 2.00 0.00

30.40a ± 30.08a ± 30.04a ± 30.24a ± 29.78a ± 29.07a ± 29.88a ± 30.10a ± 29.33a ± 0.55

0.05 0.08 0.06 0.03 0.26 0.59 0.10 0.09 0.23

41.40a ± 41.30a ± 40.85a ± 40.85a ± 40.70a ± 40.75a ± 40.70a ± 40.70a ± 40.60a ± 0.92

1.00 0.00 0.05 0.00 0.00 1.00 1.00 0.00 0.00

30.37a ± 30.45a ± 31.25a ± 32.23a ± 35.22cb ± 37.22c ± 39.47d ± 39.47d ± 40.18d ± 1.47

0.10 0.09 0.23 1.05 0.15 0.86 1.12 0.56 0.08

Paraffin oil (P)

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

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)1

Table 9 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the sera urea levels (mg dL ) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

29.89a ± 30.27a ± 29.41a ± 29.47a ± 28.92a ± 28.65a ± 29.51a ± 29.34a ± 29.88a ± 0.91

0.68 0.64 0.10 0.02 1.08 0.31 0.42 0.29 0.38

29.12a ± 29.41a ± 29.31a ± 29.75a ± 29.78a ± 29.75a ± 29.75a ± 29.133a ± 28.68a ± 2.79

0.11 0.24 0.76 0.56 0.26 0.53 0.66 0.23 0.30

30.26a ± 30.49a ± 35.41b ± 43.67c ± 45.31d ± 46.78e ± 48.17f ± 50.119g ± 51.34h ± 1.08

29.88a ± 29.47a ± 28.71a ± 30.15a ± 29.19a ± 28.48a ± 29.68a ± 29.45a ± 29.42a ± n.s.

0.69 0.60 0.44 0.05 0.06 0.59 0.61 1.20 0.23

30.32a ± 30.08a ± 29.17a ± 29.26a ± 30.01a ± 29.81a ± 28.04a ± 29.28a ± 30.18a ± n.s.

0.06 0.08 1.20 0.98 0.19 0.70 2.50 0.72 0.31

29.42a ± 29.30a ± 29.38a ± 33.13a ± 39.57b ± 43.14b ± 47.75c ± 51.65d ± 52.53d ± 4.00

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

0.05 0.03 1.14 0.26 0.11 0.59 0.08 0.10 0.42

30.40a ± 30.08a ± 30.04a ± 30.24a ± 29.78a ± 29.07a ± 29.88a ± 30.10a ± 29.33a ± 0.55

0.05 0.08 0.06 0.03 0.26 0.59 0.10 0.09 0.23

29.08a ± 24.42a ± 29.82a ± 0.30a ± 29.78a ± 28.75a ± 29.79a ± 29.12a ± 29.75a ± n.s.

0.93 1.07 0.63 0.05 0.59 1.09 9.58 0.86 0.52

29.75a ± 29.90a ± 29.57a ± 30.19a ± 29.91a ± 31.51b ± 37.07d ± 34.21c ± 36.47d ± 1.50

0.53 0.68 0.65 0.80 0.55 0.43 1.01 0.27 0.56

30.37a ± 30.45a ± 31.25a ± 32.23a ± 35.22cb ± 37.22c ± 39.47d ± 39.47d ± 40.18d ± 1.47

0.10 0.09 0.23 1.05 0.15 0.86 1.12 0.56 0.08

0.63 1.14 1.20 2.66 1.47 3.61 0.52 0.48 0.45

30.42a ± 29.70a ± 29.04a ± 30.20a ± 29.95a ± 29.76a ± 29.42a ± 29.48a ± 30.12a ± n.s.

1.23 0.37 1.025 1.31 1.57 0.66 0.50 0.33 0.03

29.18a ± 29.67a ± 30.08a ± 29.36a ± 29.48a ± 29.47a ± 31.10a ± 30.41a ± 30.04a ± n.s.

1.20 0.55 0.15 0.40 1.30 0.58 1.08 2.08 1.94

29.82a ± 28.57a ± 29.72a ± 34.24b ± 34.43b ± 36.13c ± 38.86d ± 40.08d ± 40.03d ± 1.98

0.45 0.55 1.44 1.42 0.67 0.13 0.65 0.08 0.95

29.20a ± 29.35a ± 30.17a ± 30.75a ± 34.43b ± 37.09c ± 38.08c ± 39.75d ± 40.72d ± 2.00

1.08 0.41 0.73 1.08 1.58 0.95 0.93 0.56 0.52

Paraffin oil (P)

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time. n.s., not significant. )1

Table 10 Influence of non-fried and fried sunflower oil, jojoba oil, paraffin oil and mixtures of them on the levels of sera uric acid (mg dL ) of rats Blood withdrawal period (week) Non-fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01 Fried oils 0 1 2 3 4 5 6 7 8 LSD = 0.01

Sunflower oil (S)

Jojoba oil (J)

4.53a ± 4.54a ± 4.48a ± 4.53a ± 4.49a ± 4.50a ± 4.51a ± 4.52a ± 4.53a ± 0.26

0.01 0.03 0.03 0.03 0.03 0.01 0.03 0.03 0.01

4.48a ± 4.54a ± 4.58b ± 4.61b ± 4.52a ± 4.50a ± 4.50a ± 4.59b ± 4.59b ± 0.07

4.55a ± 4.49a ± 4.55a ± 4.61a ± 4.81b ± 5.01b ± 5.31c ± 5.39c ± 5.41d ± 0.26

0.01 0.02 0.04 0.01 0.01 0.18 0.18 0.05 0.43

4.49a ± 4.46a ± 4.53a ± 4.61b ± 4.50a ± 4.53a ± 4.49a ± 4.85c ± 5.10d ± 0.07

Paraffin oil (P)

S+J (9:1, v ⁄ v)

S+J (8:2, v ⁄ v)

S+P (9:1, v ⁄ v)

S+P (8:2, v ⁄ v)

0.03 0.01 0.02 0.01 0.03 0.02 0.04 0.01 0.03

4.48a ± 4.51a ± 4.93b ± 5.21c ± 5.71d ± 6.55e ± 7.53f ± 8.07g ± 8.57h ± 0.30

0.03 0.01 0.04 0.02 0.01 0.05 0.03 0.06 0.05

4.48a ± 4.55a ± 4.53a ± 4.50a ± 4.51a ± 4.53a ± 4.24a ± 4.53a ± 4.53a ± 0.14

0.02 0.01 0.04 0.03 0.01 0.02 0.04 0.03 0.03

4.48a ± 4.51a ± 4.52a ± 4.60b ± 4.50a ± 4.47a ± 4.50a ± 4.49a ± 4.55ab ± 0.07

0.03 0.01 0.01 0.00 0.03 0.06 0.03 0.04 0.05

4.48a ± 4.52a ± 4.51a ± 4.58b ± 4.65c ± 4.73d ± 4.85e ± 4.88e ± 4.94f ± 0.07

0.03 0.02 0.02 0.21 0.02 0.02 0.02 0.03 0.05

4.48a ± 4.54a ± 4.65b ± 4.66b ± 4.81c ± 4.95d ± 5.12e ± 5.26f ± 5.33g ± 0.07

0.03 0.03 0.02 0.06 0.02 0.05 0.03 0.03 0.03

0.03 0.04 0.02 0.03 0.02 0.03 0.01 0.05 0.10

4.53a ± 4.52a ± 4.53a ± 4.86b ± 5.20c ± 6.85d ± 7.70e ± 8.15f ± 9.67g ± 0.30

0.06 0.01 0.02 0.15 0.00 0.57 0.26 0.00 0.29

4.45a ± 4.57a ± 4.51a ± 4.54a ± 4.54a ± 4.57a ± 4.81b ± 4.70b ± 5.00c ± 0.14

0.02 0.01 0.03 0.06 0.04 0.03 0.07 0.00 0.28

4.50a ± 4.49a ± 4.49a ± 4.58b ± 4.56b ± 4.65c ± 4.73d ± 4.89e ± 5.10f ± 0.07

0.02 0.03 0.01 0.03 0.04 0.05 0.03 0.03 0.10

5.13a ± 5.18a ± 4.50b ± 4.50b ± 4.60c ± 4.80d ± 5.00e ± 5.22f ± 5.61g ± 0.07

0.03 0.03 0.01 0.01 0.01 0.01 0.00 0.03 0.01

4.72a ± 4.82b ± 4.51a ± 4.50a ± 4.71a ± 4.93b ± 5.53c ± 5.62d ± 6.03e ± 0.07

0.03 0.03 0.02 0.01 0.01 0.06 0.12 0.03 0.06

The data are expressed as mean values ± standard error. Values in the column followed by the same letter are not significantly at P = 0.01 compared with the value of zero time.

International Journal of Food Science and Technology 2008

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Safety evaluation of non-fried and fried sunflower, paraffin, jojoba oils and their mixtures on rats R. S. Farag et al.

Figure 1 Liver of rats from group 1 administered non-fried sunflower

Figure 4 Liver of rats from group 8 administered fried sunflower oil

oil showing slight hydropic degeneration of some hepatocytes (H & E, ·200).

showing swelling of hepatocytes and clear cytoplasm (hydropic degeneration) (H & E, ·200).

Figure 2 Liver of rats from group 2 administered non-fried paraffin

Figure 5 Liver of rats from group 9 administered fried paraffin oil

oil showing hepatocellular vacuolations (H & E, ·200).

showing karyomegaly of some hepatocytes (H & E, ·400).

Figure 3 Liver of rats from group 7 administered non-fried sunflower

Figure 6 Liver of rats from group 12 administered fried sunflower

oil and paraffin oil at ratio (8:2, v ⁄ v) showing sinusoidal dilatation as well as hydropic degeneration of hepatocytes (H & E, ·200).

oil and jojoba oil at ratio (8:2, v ⁄ v) showing Kupffer cell activation (H & E, ·200).

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Table 11 Relative liver and kidney tissues deterioration using different non-fried, fried oils and binary oil admixtures Relative deterioration value Liver tissues

Kidney tissues

Oil

Fried oils

Non-fried oils

Fried oils

Non-fried oils

Sunflower oil (S) Paraffin oil (P) Jojoba oil (J) S + P(9:l, v ⁄ v) S + P (8:2, v ⁄ v) S + J (9:1, v ⁄ v) S + J (8:2, v ⁄ v)

15–20 15–20 15–20 15–20 15–20 10 10

0 20–30 0 15–20 15–20 0 0

10 70 50 20–30 50–60 10 10

0 50 0 10 20 0 0 Figure 7 Kidney of rats from group 2 administered non-fried paraffin

tissues of rats administered fried S and J mixture (9:1, v ⁄ v). Histological changes observed in liver tissues of rats given fried S and J mixture (8:2, v ⁄ v) a slight activation of Kupffer cells. In order to envisage the influence of non-fried, fried and oils mixed with J and P at ratios of 9:1 (v ⁄ v) and 8:2 (v ⁄ v) on the human health, the histological examination of non-fried S was taken as a guide for comparison and given a value of 100%. The relative deterioration values were calculated from the following equation: Relative deterioration ¼ Dr  Ds/Dr  100 where Dr and Ds refer to deterioration of reference and sample, respectively. Table 11 shows the relative deterioration of liver tissues using different oil samples compared with non-fried sunflower oil. The administration of various oils and oil mixtures induced variable changes in liver tissues. The deterioration order for liver tissue using the non-fried oils can be arranged as follows: P > S þ Pð8:2; v=vÞ > S þ Pð9:1; v=vÞ

oil showing vacuolar degeneration of endothelial lining the glomerular tufts (H & E, ·200).

However, kidney tissues of rats administered basal diet comprises non-fried P showed vacuolar degeneration of endothelial lining the glomerular tufts (Fig. 7). Histological examination of kidney tissues of rats given diet containing non-fried J and S and J mixtures (9:1 and 8:2, v ⁄ v) showed no histopathological changes, whereas kidney tissues of rats administered basal diet includes non-fried S and P mixtures (9:1 and 8:2, v ⁄ v) showed mild changes described as slight vacuolation of endothelial lining the glomerular tufts and hydropic degeneration of epithelial lining the proximal convoluted renal tubules (Fig. 8). Examination of kidney tissues of rat groups given basal diet comprises S + P oil mixtures (9:1 and 8:2, v ⁄ v) showed similar histopathological changes described as vacuolations of some renal tubular epithelium associated with the appearance of intratubular flocculent easimphilic proteinaceous casts.

> S þ Jð8:2; v=vÞ ¼ S þ Jð9:1; v=vÞ ¼ J ¼ S: This arrangement indicates that P caused the most harmful effect on liver tissues followed by S + P mixtures, whereas S, J and their mixtures exhibited slight alteration on liver tissues. In case of fried oils and fried oil mixtures, the deterioration order was as follows: P > J > S þ Pð8:2; v=vÞ > S þ Pð9:1; v=vÞ > S þ Jð8:2; v=vÞ ¼ S þ Jð9:1; v=vÞ > S: Here, the fried paraffin and its mixtures induced the highest deterioration on liver tissues. Hence, paraffin oil alone and its mixtures with sunflower oil have to be abandoned from use in frying foods. Histopathological examination of kidney tissues

No histopathological changes were observed in kidney tissues of rats given basal diet containing non-fried S.

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Figure 8 Kidney of rats from groups 6 and 7 administered non-fried

sunflower and paraffin oil at ratio (9:1, and 8:2, v ⁄ v) showing hydropic degeneration of epithelial lining the proximal convoluted renal tubules (H & E, ·400).

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Safety evaluation of non-fried and fried sunflower, paraffin, jojoba oils and their mixtures on rats R. S. Farag et al.

While, kidney tissues of rats given basal diet includes fried S and J oil mixtures (9:1 and 8:2, v ⁄ v) showed mild changes described as cloudy swelling of epithelial lining proximal convoluted tubules with the appearance of star-shaped lumen. The administration of various oils and oil mixtures induced variable changes in kidney tissues. The deterioration order for kidney tissues using the non-fried oils can be arranged as follows: P > S þ Pð9:1; v=vÞ ¼ S þ Pð8:2; v=vÞ > S þ Jð9:1; v=vÞ ¼ S þ Jð8:2; v=vÞ ¼ J ¼ S: This arrangement indicates that P caused the most harmful effects on kidney tissues followed by the mixtures of S + P (8:2) and S + P (9:1, v ⁄ v), whereas S, J and mixtures of them did not show any obvious alteration on kidney tissues. In case of fried oils and fried oil mixtures, the deterioration order was as follows: P ¼ S þ Pð9:1; v=vÞ ¼ S þ Pð8:2; v=vÞ ¼ J ¼ S > S þ Jð9:1; v=vÞS þ Jð8:1; v=vÞ: Here, the fried P and its mixtures with sunflower oil induced the largest deterioration on kidney tissues. It is worth mentioning that histopathological examination of rat liver and kidney tissues indicates that the observed changes paralled the biochemical data. These results suggest a ban on paraffin oil at all in deep-fat processes. There is some evidence that mineral oil exposure may be associated with human disease. Subcutaneous injection of mineral oil induces scalloping lipogranulomas, a chronic local inflammatory reaction (Di Benedetto et al., 2002), and aspiration causes a severe chronic pneumonitis termed ‘lipoid pneumonia’ (Spickard & Hirschmann, 1994). The oil is absorbed through the intestine and distributes throughout the body, causing lipogranulomas in the lymph nodes, liver and spleen of healthy individuals (Dincsoy et al., 1982). Oral or intraperitoneal administration of mineral oil induces similar lesions in laboratory animals (Firriolo et al., 1995; Shaheen et al., 1999). Our previous study (Farag et al., 2008) suggests to mix paraffin oil with sunflower oil to increase its shelf life during deep-fat frying process. In addition, the scores of overall acceptability of potato chips produced from paraffin oil alone and sunflower oil + paraffin oil mixtures were categorised good and had little higher score in the total acceptability than the already product commercially chips (Chrispy). On the contrary, the results of biochemical tests and histopathological examinations have led to proposals to ban the use of paraffin oil in various frying processes. References AOAC (2000). Official Methods of Analysis of the Association of Official Analytical Chemists, 17th edn (edited by W. Horwitz).Washington: AOAC.

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Assmann, G. (1979). Cholesterol determination in high density lipoproteins separated by three different methods. The Internist, 20, 559–604. Barham, D. & Trinder, P. (1972). Enzymatic, colorimetric method for determination uric acid in serum plasma and urine. The Analyst, 97, 142–146. Belfield, A. & Goldberg, D.M. (1971). A colorimetric method for determination of alkaline phosphatase in serum. Enzyme, 12, 561– 565. Bergmeyer, H.U. & Harder, M. (1986). A colorimetric method of determination of serum glutamic oxaloacetic and pyruvic tranasminase. Clinical Biochemistry, 24, 28–34. Canoira, L., Alcantara, R., Garcia-Martinez, M.J. & Carrasco, J. (2006). Biodiesel from jojoba oil-wax: transesterification with methanol and properties as a fuel. Biomass and Bioenergy, 30, 76–81. Carpenter, R.P., Lyon, D.H. & Hasdell, T.A. (2000). Guidelines for Sensory Analysis in Food Product Development and Quality Control. Pp. 71–91. Gaithersburg: Aspen Publishers, Inc. Cochran, W.G. & Cox, G.M. (1992). Experimental Designs, 2nd edn. New York: Wiley. Culling, C.F.A. (1965). Handbook of Histopathological Techniques, 2nd edn. London: Butterworth. Di Benedetto, G., Pierangeli, M., Sealise, A. & Bertani, A. (2002). Paraffin oil injection in the body: an obsolete and destructive procedure. Annals Plastic Surgery, 49, 391–396. Dincsoy, H.P., Weesner, R.E. & MacGee, J. (1982). Lipogranulomas in non-fatty human livers: a mineral oil induced environmental disease. American Journal of Pathology, 78, 35–41. Farag, R.S., Farag, M.M. & Rehab, F.M.A. (2008). Use of sunflower oil mixed with jojoba and paraffin oils in deep-fat frying process. International Journal of Food Science and Technology, 43, 1306– 1315. Fawcett, J.K. & Scott, J.E. (1960). Enzymatic, colorimetric method for determination urea in serum, plasma and urine. Journal of Clinical Pathology, 13, 156–162. Firriolo, J.M., Morris, C.F., Trimmer, G.W., Twitty, L.D., Smith, J.H. & Freeman, J.J. (1995). Comparative 90-day feeding study with low viscosity white mineral oil in Fisher-344 and Sprague–Dawleyderived CRL: CD rats. Toxicological Pathology, 23, 26–33. Frings, C.S. & Dunn, R.T. (1970). Colorimetric method for determination of total serum lipids based on the sulphophosphovanillin reaction. American Journal of Clinical Pathology, 53, 89–91. Gayol, M.F., Labuckas, D.O., Oberti, J.C. & Guzma´n, C.A. (2004). Chemical characterization of jojoba seeds (Simmondsia Chinensis, Link Schneider) proceeding from ‘‘Ban˜ado De Los Pantanos’’, La Rioja, Argentina. Journal of Argentina Chemical Society, 92, 4–6. Heimbach, J.T., Bodor, A.R., Douglass, J.S. et al. (2002). Dietary exposures to mineral hydrocarbons from food-use applications in the United States. Food and Chemical Toxicology, 40, 555–571. Milthorpe, P.L. & Dunstone, R.L. (1989). The potential of jojoba (Simmondsia chinensis) in New South Wales. 1 and 2. Australian Journal of Experimental Agriculture, 29, 389–395. Nash, J.F., Gettings, S.D., Diembeck, W., Chudowski, M. & Kraus, A.L. (1996). A toxicological review of topical exposure to white mineral oils. Food and Chemical Toxicology, 34, 213–225. Roechlau, P., Bernt, E. & Gruber, W.L. (1974). Kinetics of the cholesterol sulfuric acid reaction: a fast kinetic method for serum cholesterol. Clinical Chemists and Clinical Biochemistry, 12, 403–408. Shaheen, V.M., Satoh, M., Richards, H.B. et al. (1999). Todo Sobre la Jojoba. Chemical Technician, 25, 49–54. Spickard, A. & Hirschmann, J.V. (1994). Exogenous lipoid pneumonia. Archives of International Medicine, 154, 686–692. Tobares, L., Frati, M., Guzma´n, C. & Maestri, D. (2004). Agronomical and chemical traits as descriptors for discrimination and selection of jojoba (Simmondsia chinensis) clones. Industrial Crops and Products, 19, 107–111.

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Original article Nutritional potential and functional properties of tempe produced from mixture of different legumes. 1: Chemical composition and nitrogenous constituent Ahmad G. Nassar,1 Adel E. Mubarak2 & Alaa E. El-Beltagy3* 1 Food Science and Technology Department, Faculty of Agriculture, Al-Azhar University, Assiut, Egypt 2 Home Economic Department, Faculty of Specific Education, Menofiya University, Ashmoon, Egypt 3 Food Science and Technology Department, Faculty of Agriculture, Menofiya University, 32516-Shibin El-Kom, Egypt (Received 26 May 2005; Accepted in revised form 6 August 2007)

Summary

Fermented foods such as Tempe represent a technological alternative for a great variety of legumes and combination of them to improve their nutritional quality and to obtain edible products with palatable sensorial characteristics. The chemical composition, carbohydrate fraction and nitrogenous constituents were investigated for individual different legumes, i.e. faba bean; lupine, chickpea; peas and their mixture before and after fermentation by Rhizopus oligosporus. Tempe had a higher (P < 0.05) protein and fibre content compared with legume mixtures before fermentation, while it had a lower fat, ash and carbohydrate contents. Also, reducing and non-reducing sugars, stachyose as well as raffinose were reduced after fermentation of legume mixtures. A significant reduction was observed in non-protein nitrogen and protein nitrogen, while total nitrogen and true protein were increased.

Keywords

Chemical composition, chick pea, faba bean, lupine, peas, raffinose, stachyose, tempe.

Introduction

Legumes are an important source of protein in the Egyptian diet and in many developing countries. Higher meat price in recent years and the need for protein rich foods have led people in most developing countries to shift their consumption to certain legumes (Askar, 1986). Fermented foods may be defined as those foods which have been subjected to the action of microorganisms or enzymes so that desirable biochemical changes cause a significant modification to the food. Using fermentation the food may be made more nutritious, more digestible, and safer or have better flavour. Fermented foods constitute a major part of the diet in all parts of the world and can be divided into many classes: beverages; cereal and diary products; fish products; fruit and vegetable products, legumes and meat products (Campbell-Platt, 1987). Tempe is a traditional Indonesian solid-substrate fermentation in which soybean are hydrated and acidified, dehulled, cooked and then fermented with Rhizopus spp. The cotyledon become covered and penetrated by denes white non-sporulated mycelium that binds them into a compact, sliceable mass. Tempe is obtained *Correspondent: E-mail: [email protected]

through a two stage fermentation, which consists of a soaking process and solid-substrate fermentation with different strains of Rhizopus spp. (R. oligosporus, R. arrhizus and R. stolonifer) as reported by Nout & Rombouts (1990) and Steinkraus (1996). Other substrates have been used to elaborate tempe: common bean, chickpeas for animal consumption, rapeseed, lupine, horsebean, groundnut, wheat and corn ⁄ soybean. The process to prepare tempe requires a relatively simple infrastructure and can produce profound chemical changes and improve the nutritional quality. The tempe manufacture could be an appropriate method for small and medium scale processing of locally available legumes and or cereals into wholesome products of high nutritional value in developing countries (Hachmeister & Fung, 1993). Tempe is widely consumed in Indonesia, the Netherlands and North America. The high protein content and pleasant, relatively bland taste has led to it occupying a small, but expanding part of the vegetarian market in Japan, USA and Europe (Mital & Garg, 1990; Liu, 1997). The present work was aimed to introduce tempe technology in Egypt to produce tempe in small scale using mixtures of different Egyptian traditional legumes and evaluate the chemical and nutritional attributes of the produced tempe.

doi:10.1111/j.1365-2621.2007.01683.x  2008 Institute of Food Science and Technology

Tempe production and evaluation of chemical and nutritional attributes A. G. Nassar et al.

Materials and methods

Methods Inoculum preparation

Materials

Four different kinds of most popular traditional Egyptian legume: Green pea (Pisum sativum) variety Lencolen, broad bean (Vicia faba L.) variety Giza 3, chickpea (Cicer arietinum) and termis (Lupinus termis) were obtained from the Seed Department, Agricultural Research Center, Giza, Egypt. Strains

Rhizopus oligosporus (NRRL 2710) were supplied by Northern Regional Research Laboratory, Peoria, IL, USA. The strain were maintained on slants of potato-dextrose-agar at 5 ± 1 C and used after 7 days.

Table 1 Chemical composition (% on dry weight basis) of legumes used in tempe preparation

Inoculum was prepared by soaking each slant with 4 mL of sterile distilled water for 2 min and 1 mL (1 · 106 spores mL)1) of such suspension was used to inoculate the legume mixtures grits (about 50 g dry weight). Tempe preparation

Broad bean and Green peas as well as whole Sweet Termis and Chick pea seeds were soaked in tap water (1:20 w ⁄ v) at room temperature (about 25 C) for 16 h, then dehulled manually and ground into grits using household blender and mixed using 75% faba bean and 25% of each other legumes as well as 50% faba bean and 50% of the other legumes. Faba bean (the most popular Egyptian legume) were used as a control, also a mixture (25% of each four legumes) were used. All the previous mixtures were cooked in tap water (1:3 w ⁄ v)

Legumes

Crude protein

Crude fat

Crude fibre*

Ash

Total carbohydrates

Faba bean Lupine Chickpea Peas l.s.d. 5%

25.35 ± 0.85 37.72 ± 0.88 21.54 ± 0.50 32.26 ± 0.80 0.88

1.05 ± 0.15 13.62 ± 0.20 5.75 ± 0.21 2.45 ± 0.10 0.23

3.48 ± 0.40 6.39 ± 0.42 4.23 ± 0.36 4.15 ± 0.50 0.50

3.59 ± 0.21 2.73 ± 0.25 2.81 ± 0.12 3.42 ± 0.20 0.25

66.53 ± 1.40 39.90 ± 0.82 65.67 ± 0.93 57.72 ± 1.10 1.21

*Calculated by difference

Table 2 Chemical composition (% on dry weight basis) of legume mixtures and their produced tempe

Legumes

Crude protein

Crude fat

Crude fibre*

Ash

Total carbohydrates

100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture Fermented 100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture l.s.d. 5%

25.35 ± 0.85

1.05 ± 0.15

3.48 ± 0.40

3.59 ± 0.21

66.53 ± 1.40

28.44 ± 1.20 24.40 ± 0.95 27.07 ± 0.76

4.20 ± 0.10 2.22 ± 0.08 1.40 ± 0.07

4.35 ± 0.50 3.74 ± 0.45 3.66 ± 0.40

3.30 ± 0.22 3.46 ± 0.30 3.58 ± 0.21

59.87 ± 0.95 66.31 ± 1.30 64.33 ± 1.12

31.53 23.45 28.80 29.21

7.30 3.40 1.75 5.71

4.97 3.78 3.83 4.57

4.50 3.26 3.51 3.16

53.22 66.10 62.14 57.45

± ± ± ±

1.47 1.10 0.65 0.83

± ± ± ±

0.27 0.12 0.07 0.15

± ± ± ±

0.50 0.38 0.40 0.43

± ± ± ±

0.44 0.22 0.31 0.21

± ± ± ±

0.96 1.15 1.00 0.86

31.41 ± 1.25

0.62 ± 0.04

4.09 ± 0.42

2.25 ± 0.16

61.63 ± 0.90

31.60 ± 0.96 30.85 ± 0.86 31.46 ± 0.80

2.17 ± 0.09 1.05 ± 0.05 0.53 ± 0.03

7.36 ± 0.55 5.72 ± 0.47 7.73 ± 0.60

2.05 ± 0.18 1.74 ± 0.17 1.92 ± 0.12

56.82 ± 1.10 60.64 ± 0.75 58.36 ± 0.66

34.89 ± 1.30 28.38 ± 0.65 34.30 ± 0.72 32.25 ± 0.80 1.47

4.89 ± 0.17 2.13 ± 0.13 1.06 ± 0.07 4.21 ± 0.16 0.27

6.53 ± 0.36 6.46 ± 0.42 5.13 ± 0.36 7.27 ± 0.58 0.60

2.17 ± 0.20 1.39 ± 0.17 2.38 ± 0.26 2.00 ± 0.12 0.44

51.52 ± 0.97 61.64 ± 0.72 57.13 ± 0.82 54.27 ± 0.58 1.46

*Calculated by difference

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analysis of variance using a completely randomised design. Differences between any two means were determined using l.s.d with a P < 0.05 significance level (Steel & Torrie, 1980).

Table 3 Carbohydrate fractions (% on dry weight basis) of legumes used in tempe preparation

Legumes

Reducing sugars

Non-reducing sugars

Stachyose

Raffinose

Faba bean Lupine Chickpea Peas l.s.d. 5%

4.98 ± 0.41 1.13 ± 0.12 2.10 ± 0.21 3.75 ± 0.18 0.35

61.55 ± 1.23 38.77 ± 0.78 63.57 ± 0.85 53.97 ± 0.66 0.93

1.85 ± 0.18 2.93 ± 0.26 1.63 ± 0.18 2.19 ± 0.16 0.26

1.06 ± 0.15 1.46 ± 0.16 0.99 ± 0.14 0.81 ± 0.10 0.16

Results and discussion

Proximate composition

Proximate composition of raw, mixtures and fermented legume mixtures by R. oligosporus are presented in Tables 1 and 2. Lupine had the highest amount of protein, fat and fibre compared with other legumes used in this study (P £ 0.05). After mixing legumes, treatment of 50% faba bean and 50% lupine had the highest amount of protein (31.53%), fat (7.30), fibre (4.97%) and ash (4.50%) content. On the other hand, fermentation increased significantly (P £ 0.05) the crude protein and crude fibre content in all legume mixtures. The increasing rate of protein and fibre ranged between 2.50–26.34% and 17.52–111.20% respectively, while fat, ash and carbohydrates were significantly decreased (P £ 0.05). These changes may be due to leaching out some compounds during boiling (before fermentation) and to fungi growth which have consumed carbohydrate and fat as an energy sources and the development of a fibre-rich fungous mycelium (Shurtleff & Aoyagi, 1979). The obtained results agreed with those obtained by Njoku et al. (1991), De-Reu et al. (1995) and El-Bagoury et al. (2001).

acidified by adding 1% of 85% lactic acid for 25 min then cooled to 37 C and inoculated with spores suspension, mixed, packed in petri dishes, incubated for 48 h at 37 ± 1 C (El-Bagoury et al., 2001). Proximate analysis

Moisture content, crude fat, ash, total proteins and nonprotein nitrogen were determined according to AOAC (1990). Reducing sugar contents were determined in 70% ethanol extracts by phenol-sulfuric acid method according to Dubois et al. (1956). Starch was determined as glucose after hydrolyzation by HCl (Sp.gr 1.18). Statistical analysis

Results are expressed as the mean values of three separate determinations (±SD). Data were subjected to

Legumes 100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture Fermented 100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture l.s.d. 5%

Reducing sugars

Non-reducing sugars

Stachyose

Raffinose

4.98 ± 0.41

61.55 ± 1.23

1.85 ± 0.18

1.06 ± 0.15

4.06 ± 0.38 4.26 ± 0.41 4.67 ± 0.36

55.85 ± 1.10 62.05 ± 1.46 59.65 ± 1.12

2.12 ± 0.21 1.79 ± 0.20 1.94 ± 0.18

1.16 ± 0.13 1.00 ± 0.12 0.96 ± 0.11

3.06 3.54 4.36 3.00

50.16 62.56 57.76 54.46

2.39 1.74 2.02 2.15

1.30 1.03 0.92 1.08

± ± ± ±

0.25 0.37 0.44 0.28

± ± ± ±

0.96 1.23 1.05 0.86

± ± ± ±

0.30 0.25 0.26 0.23

± ± ± ±

0.16 0.13 0.15 0.14

2.21 ± 0.23

59.42 ± 0.95

0.91 ± 0.21

0.63 ± 0.08

1.43 ± 0.21 1.51 ± 0.24 1.26 ± 0.20

53.39 ± 0.82 59.13 ± 0.87 57.10 ± 0.88

0.97 ± 0.17 0.82 ± 0.16 0.73 ± 0.12

0.59 ± 0.07 0.44 ± 0.09 0.31 ± 0.08

1.20 ± 0.17 1.17 ± 0.15 1.39 ± 0.21 1.41 ± 0.19 0.44

49.32 ± 0.75 60.47 ± 1.15 55.71 ± 0.66 52.86 ± 0.76 1.47

1.03 ± 0.15 0.64 ± 0.12 0.72 ± 0.20 0.85 ± 0.17 0.30

0.60 ± 0.10 0.49 ± 0.07 0.36 ± 0.09 0.21 ± 0.06 0.16

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Table 4 Carbohydrate fractions (% on dry weight basis) of legume mixtures and produced tempe

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Table 5 Nitrogenous constituents (% on dry weight basis) of legumes used in tempe preparation

Legumes

Total nitrogen

Non-protein nitrogen

Protein nitrogen

True protein

Faba bean Lupine Chickpea Peas l.s.d. 5%

4.05 ± 0.25 6.03 ± 0.30 3.44 ± 0.25 5.16 ± 0.22 0.30

0.98 ± 0.10 1.37 ± 0.13 1.36 ± 0.12 1.30 ± 0.10 0.13

3.07 ± 0.25 4.67 ± 0.41 2.07 ± 0.27 3.86 ± 0.26 0.42

19.18 ± 1.10 29.15 ± 1.00 12.93 ± 0.60 24.13 ± 0.85 1.02

Carbohydrate fractions

Reducing and non-reducing sugars as well as oligosaccharides (stachyose and raffinose) of raw beans are illustrated in Table 3. Chickpea and faba bean contained the highest amount of non-reducing sugars (63.57 and 61.55% respectively) followed by pea (53.97%) and lupine (38.77%), While, faba bean and peas had the highest content of reducing sugars. On the other side, lupine and peas had the highest amount of stachyose (2.39 and 2.19% respectively), while lupine and faba bean had the highest amount of raffinose. As shown in Table 4 a significant (P £ 0.05) decrease was noticed in the reducing, non-reducing, stachyose and raffinose in the different legume mixtures fermented with R. oligosporus. The reduction rates of reducing, non-reducing, stachyose and reffinose ranged 41–63%, 3–7%, 47–57% and 34–65% respectively. Similar results were obtained by Egounlety & Aworh (2003) who reported that about 50% of raffinose and >55% of stachyose were lost Table 6 Nitrogenous constituents (% on dry weight basis) of legume mixtures and their produced tempe

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during pretreatment and fermentation of some legumes. The reduction in carbohydrate fraction might be due to their utilisation as a carbon and energy source by the fungi during fermentation (Omafuvbe et al., 2000). Nitrogenous constituents

Results in Tables 5 and 6 illustrate the changes in nitrogenous compounds of raw, mixtures and fermented legume mixture. Table 5 reveals that lupine had the higher nitrogenous constituents followed by peas and faba bean, while chickpea had the lowest. Fermentation increased significantly (P £ 0.05) total nitrogen and non-protein nitrogen content (Table 6), the maximum increasing in total nitrogen and nonprotein nitrogen were found in the mixture of 50% faba bean + 50% peas 4.604 lupine 5.07–5.58%). Conversely, a significant (P < 0.05) decrease in protein nitrogen content and hence true protein were noticed in all fermented legume mixtures. The highest decrease in true protein was noticed in the mixture of 25% of each

Legumes

Total nitrogen

Non-protein nitrogen

Protein nitrogen

True protein

100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture Fermented 100% faba bean 75% faba bean + 25% lupine 25% chickpea 25% peas 50% faba bean + 50% lupine 50% chickpea 50% peas Mixture l.s.d. 5%

4.05 ± 0.25

0.98 ± 0.10

3.07 ± 0.25

19.18 ± 1.10

4.55 ± 0.23 3.90 ± 0.36 4.33 ± 0.31

1.08 ± 0.11 1.06 ± 0.12 1.05 ± 0.15

3.46 ± 0.10 2.83 ± 0.18 3.27 ± 0.21

21.63 ± 1.05 17.68 ± 0.95 20.42 ± 1.03

5.07 3.74 4.60 4.66

1.77 1.16 1.02 1.23

3.30 2.58 3.58 3.43

20.65 16.13 22.37 21.44

± ± ± ±

0.42 0.35 0.46 0.31

± ± ± ±

0.16 0.15 0.10 0.15

± ± ± ±

0.46 0.31 0.36 0.40

± ± ± ±

1.17 0.86 0.90 0.96

5.02 ± 0.50

2.18 ± 0.17

2.84 ± 0.30

17.75 ± 1.06

5.05 ± 0.38 4.93 ± 0.22 5.03 ± 0.39

3.04 ± 0.20 2.43 ± 0.18 2.30 ± 0.15

2.01 ± 0.21 2.50 ± 0.26 2.73 ± 0.20

12.56 ± 0.75 15.62 ± 0.70 17.06 ± 1.15

5.58 ± 0.50 4.54 ± 0.40 5.48 ± 0.25 5.16 ± 0.30 0.50

2.40 ± 0.16 2.52 ± 0.10 3.19 ± 0.20 3.73 ± 0.25 0.20

3.18 ± 0.38 2.02 ± 0.17 2.29 ± 0.20 1.43 ± 0.15 0.45

19.87 ± 1.13 12.63 ± 0.65 14.31 ± 0.80 8.94 ± 0.65 1.18

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legumes (decreased from 21.35 to 8.94%). The increase in total nitrogen is attributed to producing the fungal protein. The increase in non-protein nitrogen and the decrease in protein nitrogen may be due to hydrolysis of legume proteins by proteolysis of fungi enzymes during fermentation. These results agree with the findings of Nowak & Szebiotko (1992), Komari (1993) and El-Sayed & El-Bagoury (2003). Conclusion

In conclusion tempe can be produced from mixtures of different Egyptian traditional legumes. Fermentation process improved the nutritional quality of legume mixtures by increasing the protein and fibre content and by reducing the flatulence sugars (stachyose and raffinose). Legume mixtures (25% of each legumes) had the highest nutritional quality. References AOAC (1990). Official Methods of Analysis, of the Association of Official Analytical Chemists, 16th edn. Arlington, Virginia: AOAC. Askar, A. (1986). Faba beans (Vicia faba L.) and their role in the human diet. Food and Nutrition Bulletin, 8, 15–24. Campbell-Platt, G. (1987). Fermented Foods of the World. A Dictionary and Guide. London: Butterworths. De-Reu, J.C., Wold, R.M.T., De-Groot, J., Nout, M.J., Ronibouts, F.M. & Gryppen, H. (1995). Protein hydrolysis during soybean tempe fermentation with Rhizopus oligosporus. Journal of Agricultural and Food Chemistry, 43, 2235–2241. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. Egounlety, M. & Aworh, O.C. (2003). Effect of soaking, dehulling, cooking and fermentation with Rhizopus olyosporus on the oligo-

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saccharides, trypsin inhibitor, phytic acid and tannins of soybean (Glycine max Merr), cowpea (Vigna unguiculata L. Walp) and ground beana engineering. Journal of Food Engineering, 56, 249–254. El-Bagoury, A.A., El-Sayed, H. & Mohamed, G.H. (2001). Chemical and nutritional evaluation of mung bean tempe. Journal of Agricultural Science Mansoura University, 26, 2969–2981. El-Sayed, H.E. & El-Bagoury, A.A. (2003). Evaluation of tempe produced by fermentation of soybean using two species of Rhizopus. Journal of Agricultural Science Mansoura University, 28, 1911–1926. Hachmeister, K.A. & Fung, D.Y.C. (1993). Tempeh: a mold modified indigenous fermented food made from soybean and ⁄ or cereal grains. Critical Reviews in Microbiology, 19, 137–188. Komari, T. (1993). Composition of lencaena tempe. ASEAN Food Journal, 8, 157–158 [C.F., F. S. T. A., 26 (2): J78]. Liu, K. (1997). Fermented oriental soy foods. In: Soybeans: Chemistry, Technology and Utilization (edited by K. Liu). Pp. 218–296. New York: Chapman and Hall. Mital, B.K. & Garg, S.K. (1990). Tempeh-technology and food value. Food Review International, 6, 213–224. Njoku, H.O., Ofuya, C.O. & Ogbulie, J.N. (1991). Production of tempeh from the African yam bean (Sphenostylis stenocarpa Hams). Food Microbiology, 8, 209–214. Nout, M.J.R. & Rombouts, F.M. (1990). Recent developments in tempe research. Journal of Applied Bacteriology, 69, 609–633. Nowak, J. & Szebiotko, K. (1992). Some biochemical changes in tempe manufacture-isolation of vitamin B12 producing bacteria Jarja. Food Microbiology, 22, 310–316. Omafuvbe, B.O., Shonukan, O.O. & Abiose, S.H. (2000). Microbiological and biochemical changes in the traditional fermentation of soybean for daddawa-Nigerian food condiment. Food Microbiology, 17, 469–474. Shurtleff, W. & Aoyagi, A. (1979). The Book of Tempeh, a Super Soy from Indonesia. New York: Harper and Row. Steel, R.G. & Torrie, J.H. (1980). Principles and Procedures of Statistics, 2nd edn. New York: McGraw-Hill Book Co. Steinkraus, K.H. (1996). Indonesian tempe and related fermentation. In: Handbook of Indigenous Fermented Foods, 2nd edn (edited by K.H. Steinkraus). Pp. 7–110. New York: Marcel Dekker.

 2008 Institute of Food Science and Technology

International Journal of Food Science and Technology 2008, 43, 1759–1762

Original article Effect of cold pre-treatment duration before freezing on frozen bread dough quality Yuthana Phimolsiripol,1 Ubonrat Siripatrawan,1* Vanna Tulyathan1 & Donald J. Cleland2 1 Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 2 Institute of Technology and Engineering, Massey University, Private Bag 11-222, Palmerston North, New Zealand (Received 24 January 2007; Accepted in revised form 14 August 2007)

The effect of cold pre-treatment (CT) duration prior to freezing on the quality of a standard bread dough was investigated. Doughs held at 0 C or 10 C for 1 h or 3 h before air-blast freezing were compared with standard dough frozen after 0.5 h at 0 C (0 C ⁄ 0.5 h) and fresh (unfrozen) dough. Cumulative gas production measured in a risograph was used to quantify the dough quality after storage at )18 ± 0.1 C for 1, 7 or 17 days. Relative to fresh dough, gas production significantly reduced after freezing for all treatments. The doughs with CT at 0 C for 1 or 3 h or 10 C for 1 h had significantly higher gas production after freezing and less rapid decline in gas production during frozen storage than the doughs with the 0 C ⁄ 0.5 h CT. The 10 C ⁄ 3 h CT gave no gas production benefit after freezing and had the most rapid decline in gas production during frozen storage.

Summary

Keywords

Cold pre-treatment, freezing, frozen bread dough, frozen storage.

Introduction

The bakery industry is increasingly using frozen bread dough because it offers added value in terms of both convenience (ready for use) and storage life. The dough can be frozen immediately after preparation or after a short pre-fermentation. In either case, final proofing (CO2 production by yeast fermentation) is required before baking. Maintenance of yeast viability and dough gas production properties during freezing and frozen storage are important if proofing is to be fast and effective leading to high quality bread (Hino et al., 1987). A key parameter is the gas production rate during proofing. The gas production depends on yeast strain, number of yeast cells, cell activity and amount of fermentable sugars (Autio & Mattila-Sandholm, 1992; El-Hady et al., 1996; Teunissen et al., 2002). Yeast cells can be injured during freezing by physical factors such as ice crystal formation and dehydration (Mazur, 1970; Wolfe & Bryant, 1999). During frozen storage, ice recrystallisation deteriorates the cell membrane and impairs activity in many cellular systems (Tanghe et al., 2003). Cells can also suffer biochemical damage, e.g. oxidative stress by reactive oxygen species formed during the thawing process (Hermes-Lima & Storey, 1993). Yeast cells show differ*Correspondent: Fax: +662 254 4314; e-mail: [email protected]

ent degrees of tolerance to such stresses depending on their growth state (Mager & Ferreira, 1993). The net effect is that the gas production of yeast cells is reduced by freezing and frozen storage. This leads to increasing proofing time and lowering bread volume (Aibara et al., 2001). There are two main approaches to improve yeast viability and dough quality after freezing and frozen storage. The first is yeast selection based on studies of gene expression. Oda et al. (1986) selected eleven yeast strains suitable for frozen dough from over 300 Saccharomyces strains. Teunissen et al. (2002) used repetitive freezing and thawing for up to 200 cycles to isolate freeze-resistant yeast strains from frozen dough. The second is to use a physical treatment to induce freeze tolerance in normal baker’s yeast. Berry & Foegeding (1997) indicated that most microorganisms must accommodate a variety of changes in their environment in order to survive and multiply. Adaptations to fluctuations in temperature are possibly the most common because of the impact of temperature on all cellular reactions. Beales (2004) stated that the application of physical stress to microorganisms is the most widely used method to induce cell inactivation and promote food stability. Nakagawa & Ouchi (1994) showed the improvement of freeze-tolerance of commercial baker’s yeast in dough by heat treatment of the dough before freezing. Diniz-Mendes et al. (1999) found that a mild cold shock (10 C for 3 h) before freezing

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increased yeast survival in a cell suspension stored at )20 C for 28 days. However, there are no reports of cold pre-treatment being applied to dough before freezing. Phimolsiripol et al. (2006) reported good control of dough rheological properties by holding the dough at 0 C prior to proofing. Therefore, the objective of this work is to examine the effect of cold pretreatment at 0 and 10 C before freezing and frozen storage on dough quality as measured by gas production for a standard bread dough. Materials and methods

120 min. The freezing rate was estimated to be about )0.34 C min)1 between )5 and )20 C and was unaffected by the CT regimes. After freezing, the dough pieces were stored at )18 ± 0.1 C for 1, 7 or 17 days. Longer storage durations were not used due to time constraints and because previous experience for the dough formula suggested that if there were significant differences between treatments then they were likely to be apparent within this time frame. For each CT and frozen storage regime combination, three replications were used. After frozen storage, dough samples were thawed to 0 C, prior to quality assessment, by transferring them to a water bath at 0 C for 90 min.

Dough preparation

Dough samples were prepared using a straight dough formula. The dough formula comprised 60% w ⁄ w commercial wheat flour (12% moisture content db, 13% protein db, 0.67% ash db), 2% w ⁄ w compressed yeast, 1% w ⁄ w salt, 2% w ⁄ w sugar, 2% w ⁄ w canola oil and 33% w ⁄ w water (40% of this water as ground ice). This corresponded to 3.3 g yeast, 1.7 g salt, 3.3 g sugar, 3.3 g oil and 55 g water for each 100 g of flour. Standard baker’s compressed yeast (Pinnacle brand, Auckland, New Zealand) was used. This yeast is known not to be particularly freeze tolerant. All ingredients were mixed in a dough mixer (Model 7MX; Delta Food Equipment, Auckland, New Zealand) for 4 min at low speed and for 10 min at high speed. The dough temperature was 18 ± 1 C at the end of mixing as recommended by Basaran & Gocmen (2003) as a compromise between excessive pre-fermentation and adequate development of the gluten network during mixing. After mixing, the dough was divided into 100 ± 2 g pieces, manually moulded into round shapes (about 5 cm diameter), and placed into 170 mm · 180 mm snaplock polyethylene bags and then cooled for 30 min in a water bath at 0 C. Cold treatment

Further cold treatment (CT) of the dough was achieved by further holding the dough pieces in a water bath before freezing. Two temperature levels (0 C or 10 C) and two holding times (1 or 3 h) were used. Fresh dough (non-frozen) and standard dough frozen after 0.5 h at 0 C (0 C ⁄ 0.5 h) were used as controls. The small size of the dough pieces and the use of water immersion with flexible packaging meant that heat transfer was rapid and the pieces quickly equilibrated to the water bath temperature. Freezing, frozen storage and thawing

The dough pieces were frozen in an air blast freezer operated at about )25 C with air speed of 2.5 m s)1 for

International Journal of Food Science and Technology 2008

Gas production measurement

Gas production was measured using a risograph (R-Design, Pullman, WA, USA) according to the method of El-Hady et al. (1996). Fifty gram samples of dough were placed into fermentation jars, and then in a water bath at 30 C. The gas volume was measured every minute for 180 min after a 10-min delay (time zero was taken at the end of the 10 min delay). Total gas production was expressed as mL over the 180 min. Measurements were made in triplicate for each treatment. Statistical analysis

Data collected were subjected to analysis of variance using proc glm (SAS Institute, Cary, NC, USA). Duncan’s multiple range test (P < 0.05) was used to detect differences among treatment means. Results and discussion

Figure 1 gives the total gas production for all CTs and storage period. Gas production of frozen dough decreased significantly (P < 0.05) with increasing storage time for all the treatments. The doughs with cold treatment at 0 C for 1 or 3 h or 10 C for 1 h had higher gas production than the standard doughs with 0.5 h cold treatment before freezing. Fresh dough produced 338.6 ± 3.8 mL total gas production. The higher gas production for the cold treatments was probably because of fermentation starting during the holding period prior to the risograph measurement. The decline in gas production during frozen storage was less rapid for the additional cold treatments than the standard dough (0 C ⁄ 0.5 h) except for the 10 C ⁄ 3 h treatment which had the most rapid decline (33% after 17 days). The rapid decline for the 10 C ⁄ 3 h treatment was probably because of significant prefermentation during the treatment. Van Dijck et al. (2000) indicated that a major disadvantage of a prefermentation period is that it gives a rapid loss of the

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Cold pre-treatment effect on frozen dough quality Y. Phimolsiripol et al.

yeast strains give similar effects and to consider prolonged storage durations.

Total gas production (mL)

380 Standard 0 °C/0.5 h 0 °C/1.5 h 0 °C/3.5 h 0 °C/0.5 h+10 °C/1 h 0 °C/0.5 h+10 °C/3 h

360 340

Acknowledgments

We gratefully acknowledge the financial support of the Royal Thai Government. Technical assistance by the Institute of Food, Nutrition, Human and Heath, Massey University, New Zealand is gratefully appreciated.

320 300 280 260

References 240 220

0

2

4

6

8

10

12

14

16

18

Storage period (day) Figure 1 Total gas production of doughs during frozen storage for different durations of cold pre-treatment (CT).

freeze resistance of the yeast cells. In contrast, Rasanen et al. (1995) reported that a short pre-fermentation at 34 C for 25 or 40 min improved the freeze–thaw stability of frozen dough. Holding the dough at 0 C for 1 h or 3 h or 10 C for 1 h before freezing seems to be an effective approach to improve frozen dough quality as a result of lower gas production loss during subsequent freezing and frozen storage for up to 17 days. One explanation of this result is that yeast cells adapt to the slow freezing process during cold treatment. Evidence for cold-induced expression changes associated with improved cryoresistance has also been found by Sahara et al. (2002). According to Kaul et al. (1992), such protection is probably to be due to retardation of membrane fluidity. Matsutani et al. (1990) suggested that an increase in freeze-tolerance was caused by the altered permeability of cell membranes at cold temperature, resulting in an increase in the rigidity of the cell surface. An alternative explanation is that the cold treatments allow better water adsorption into the dough (the water becomes more tightly bound to other dough constituents). Therefore, fewer ice crystals will form around yeast cells, leading to less damage to the yeast during freezing and frozen storage. Conclusions

Cold treatments of dough at 0 C for 1 or 3 h or 10 C for 1 h significantly improved the dough quality after freezing and frozen storage for up to 17 days. The advantages of cold treatment before freezing of dough are reduced freezing time by pre-cooling the dough and improved dough quality by either inducing yeast freeze tolerance or changes in water availability in the dough. Future work is required to optimise the temperature and duration of the cold treatment, to determine if other

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Aibara, S., Nishimura, K. & Esaki, K. (2001). Effects of shortening on the loaf volume of frozen dough bread. Food Science and Biotechnology, 10, 521–528. Autio, K. & Mattila-Sandholm, T. (1992). Detection of active yeast cells (Saccharomyces cerevisiae) in frozen dough sections. Applied and Environmental Microbiology, 58, 2153–2157. Basaran, A. & Gocmen, D. (2003). The effects of low mixing temperature on doughrheology and bread properties. European Food Research and Technology, 217, 138–142. Beales, N. (2004). Adaptation of microorganisms to cold temperature, weak acid preservatives, low pH, and osmotic stress: a review. Comprehensive Reviews in Food Science and Food Safety, 3, 1–20. Berry, E.D. & Foegeding, P.M. (1997). Cold temperature adaptation and growth of microorganisms. Journal of Food Protection, 60, 1583–1594. Diniz-Mendes, L., Bernardes, E., de Araujo, P.S., Panek, A.D. & Pascoalin, V.M.F. (1999). Preservation of frozen yeast cells by trehalose. Biotechnology and Bioengineering, 65, 572–578. El-Hady, E.A., El-Samahy, S.K., Seubel, W. & Brummer, J.-M. (1996). Change in gas production and retention in non-prefermented frozen wheat doughs. Cereal Chemistry, 73, 472–477. Hermes-Lima, M. & Storey, K.B. (1993). Antioxidant defenses in the tolerance of freezing and anoxia by garter snakes. American Journal of Physiology, 265, R646–R652. Hino, A., Takano, H. & Tanaka, Y. (1987). New freeze-tolerant yeast for frozen dough preparations. Cereal Chemistry, 64, 269–275. Kaul, S.C., Obuchi, K. & Komatsu, Y. (1992). Cold shock response of yeast cells: induction of 33 kDa protein and protection against freezing injury. Cellular and Molecular Biology, 38, 553–559. Mager, W.H. & Ferreira, P.M. (1993). Stress response of yeast. Biochemistry Journal, 290, 1–13. Matsutani, K., Fukuda, Y., Murata, K., Kimura, A., Nakamura, I. & Yajima, N. (1990). Physical and biochemical properties of freezetolerant mutants of a yeast Saccharomyces cerevisiae. Journal of Fermentation and Bioengineering, 70, 275–276. Mazur, P. (1970). Cryobiology: the freezing of biological systems. Science, 168, 939–949. Nakagawa, S. & Ouchi, K. (1994). Improvement of freeze tolerance of commercial baker’s yeasts in dough by heat treatment before freezing. Bioscience Biotechnology and Biochemistry, 58, 2077– 2079. Oda, Y., Uno, K. & Ohta, S. (1986). Selection of yeast for breadmaking by the frozen-dough method. Applied and Environmental Microbiology, 52, 941–943. Phimolsiripol, Y., Siripatrawan, U., Tulyathan, V. & Cleland, D. J. (2006). Effect of holding time on CO2 production and rheological properties in yeasted frozen dough. In: Proceeding of the 34th International Symposium on Agricultural Engineering (edited by S. Kosutic). Pp. 587–592. Opatija, Croatia: HINUS, Zagreb. Rasanen, J., Harkonen, H. & Autio, K. (1995). Freeze-thaw stability of prefermented frozen lean wheat doughs: effect of flour quality and fermentation time. Cereal Chemistry, 72, 637–642.

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Sahara, T., Goda, T. & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277, 50015– 50021. Tanghe, A., Van Dijck, P. & Thevelein, J.M. (2003). Determinants of freeze tolerance in microorganisms, physiological importance, and biotechnological applications. Advanced in Applied Microbiology, 53, 129–176. Teunissen, A., Dumortier, F., Gorwa, M.-F. et al. (2002). Isolation and characterization of a freeze-tolerant diploid derivative of an

International Journal of Food Science and Technology 2008

industrial baker’s yeast strain and its use in frozen doughs. Applied and Environmental Microbiology, 68, 4780–4787. Van Dijck, P., Gorwa, M.-F., Lemaire, K. et al. (2000). Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications. International Journal of Food Microbiology, 55, 187–192. Wolfe, J. & Bryant, G. (1999). Freezing, drying, and ⁄ or vitrification of membrane-solute-water systems. Cryobiology, 39, 103–129.

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International Journal of Food Science and Technology 2008, 43, 1763–1769

Original article Optimisation of supercritical carbon dioxide extraction of lutein esters from marigold (Tagetes erect L.) with soybean oil as a co-solvent Qingxiang Ma,1 Xiang Xu,1 Yanxiang Gao,1* Qi Wang1 & Jian Zhao2 1 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China 2 School of Wine and Food Sciences, EH Graham Centre for Agricultural Innovation, Charles Sturt University, NSW, Australia (Received 18 March 2007 Accepted in revised form 21 September 2007)

Summary

Response surface methodology was used to optimise the conditions of supercritical carbon dioxide extraction of lutein esters from marigold (Tagetes erect L.) with soybean oil as a co-solvent. The effect of pressure, temperature, CO2 flow rate and soybean oil concentration, and their interactions on the yield of lutein was investigated. Results showed that the data could be well fitted to a second-order polynomial model with a R2-value of 0.9398. The independent parameters of flow rate, pressure, temperature, all quadratics as well as the interactions between flow rate and pressure, and between flow rate and temperature affected the yield significantly (P £ 0.05). The model predicted that the optimal conditions were 35.5 MPa, 58.7 C, CO2 flow rate of 19.9 L ⁄ h with 6.9% of soybean oil as a co-solvent, and under such conditions, the maximum yield of 1039.7 mg lutein ⁄ 100 g marigold could be achieved.

Keywords

Co-solvent, lutein, marigold, response surface methodology, supercritical carbon dioxide extraction.

Introduction

Marigold (Tagetes erecta L.), belonging to the compositae family, is a common ornamental plant available in many parts of the world (Sowbhagya et al., 2004). The flower of marigold is rich in carotenoids which are attracting growing attention for their health benefiting properties (Bartlett & Eperjesi, 2003; Alves-Rodrigues & Shao, 2004). The major carotenoid present in marigold is lutein, which is a dihydroxylated compound, accounting for over 90% of the total carotenoids in the flower (Quackenbush, 1973). Olmedilla et al. (2001) suggested that lutein supplementation may improve visual function in age-related macular degeneration patients. Epidemiological studies have shown that people with a high intake of a-carotene, b-carotene and lutein had a lower risk of lung cancer (Chew et al., 1996; Landrum & Bone, 2001). Therefore, there is a growing interest in extracting lutein from various plant materials. Conventional methods for extracting carotenoids from natural matrices are mainly based on solvent extraction. These methods are time-consuming since they require multiple extraction steps, and use large quantities of *Correspondent: Fax: +861062737986; e-mail: [email protected]

organic solvents (Delgado-Vargas & Paredes-Lo´pez, 1997a,b; Barzana et al., 2002; Navarrete-Bolan˜os et al., 2005), which are often expensive and potentially harmful. Therefore, it is highly desirable to develop alternative extraction techniques that can overcome those shortcomings. Previous studies have demonstrated the feasibility of extracting carotenoids with supercritical carbon dioxide (SC-CO2). For example, Jaren-Galan et al. (1999) and Daood et al. (2002) reported a SC-CO2 process for extracting oleoresin from red pepper while Spanos et al. (1993), Barth et al. (1995) and Vega et al. (1996) modelled SC-CO2 extraction of b-carotene from carrots. Some studies have investigated the use of co-solvents such as organic solvents, to increase the extraction efficiency of SC-CO2. Added properly, co-solvents have been found to improve the extraction efficiency (Sovova´ et al., 2004). However, ethanol as a co-solvent has no effect on the SC-CO2 extraction of lutein esters from marigold (Naranjo-Modad et al., 2000), because the majority of xanthophylls in marigold are lutein esters (Tsao et al., 2004). The effect of vegetable oils as cosolvents was studied on SC-CO2 extraction of lycopene from tomato and the results demonstrated that the yield of lycopene can increase up to 60% (Vasapollo et al., 2004). Canola oil as a continuous co-solvent for SC-CO2 extraction of b-carotene, a-carotene and lutein from

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carrot was also reported (Sun & Temelli, 2006), the yields of a- and b-carotene and lutein were more than twice and four times higher, respectively, than those obtained with SC-CO2 extraction alone. A few studies have been reported on the extraction of lutein esters from marigold with SC-CO2 (Ambrogi & Eggers, 1997; Rao & Reddy, 2004). However, the effect of SC-CO2 extraction parameters on the yield of lutein esters and the optimum extraction conditions has not been subjected to a thorough study. Furthermore, there has been no report on using soybean oil as a co-solvent for SC-CO2 extraction of lutein esters from marigold. The objective of the present work was to investigate the effect of pressure, temperature, CO2 flow rate and, particularly, soybean oil as a co-solvent on the yield of lutein, and to find the optimal extraction conditions using the surface response methodology (RSM). Materials and methods

Materials

Marigold petals were kindly supplied by Yantai Natural Pigments Co., Shandong, China, with an initial moisture content of 22%. The material was dried using a vacuum drying chamber (50 C) to a final moisture content of 7.35%, and then ground to small particles ranging from 0.15 to 0.30 mm in size. They were stored in dark at room temperature until used. Chemicals

Lutein standard (>90%) was purchased from Sigma (St. Louis, MO, USA). Carbon dioxide with a purity of 99.5% was supplied by Beijing Lingyun Jiancai Co., Beijing, China. HPLC grade ethyl acetate, acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Soybean oil was purchased from a local supermarket and no carotenoids were detected in it by HPLC analysis. Supercritical CO2 extraction

The supercritical fluid extraction system (1 L sample capacity) used in this study was purchased from Nantong Hua’an Co. Ltd. (Model HA220-50-06, Jiangsu, China). Fig. 1 shows a schematic diagram of the system. Samples (200 g) of ground marigold with the corresponding soybean oil concentration (w ⁄ w) according to the experimental design (see Table 2 below for detail) were loaded into the extractor, and then the extractor and the two separators were heated to designed temperatures. After an initial air purge, liquefied carbon dioxide was pumped into the extraction vessel by a high pressure pump to a given pressure, and the temperature inside the vessel was raised to, and maintained at the desired level by a heating

International Journal of Food Science and Technology 2008

Figure 1 A Schematic diagram of the SC-CO2 extraction apparatus used in the study. 1, CO2 feed tank; 2, Filter; 3, Cold bath; 4, Pump; 5, Extractor; 6, 7, Separators I and II; 8, Pressure manometer; 9, Flow meter.

jacket encasing the vessel. The pressure and temperature were controlled to an accuracy of ±0.5 MPa and ±0.5 C, respectively. The flow rate of CO2 was regulated by adjusting the length of the pump stroke. To investigate the optimum conditions for the extraction of lutein esters, a series of trials were carried out with varying conditions of pressure, temperature, flow rate and soybean oil concentration (see below for detail). Each extraction run lasted for 3 h; later, the extract was collected in the first separator (set at 40 C and 6–8 MPa) while water and volatile components were recovered in the second separator (set at 20 C and 4–6 MPa). The extract was then weighted to obtain the yield and analysed for lutein content. Analytical methods

Moisture content was determined gravimetrically using a moisture analyzer (MF-50, A&D Co. Ltd, Japan). Lutein content of the extract was determined by first saponifying the lutein esters in the extract according to the method of AOAC (1990). Briefly, the entire extract was diluted with n-hexane and made up to 50 mL. Aliquots (0.1 mL) of the diluted extract was added into 100 mL flask and mixed thoroughly with 30mL hexane– acetone–absolute alcohol –toluene (v:v:v:v, 10:7:6:7) and 2 mL of 40% methanolic KOH was pipetted into the flask. It was placed at 56 C water bath for 20 min in the dark. Then the flask was cooled in dark for 1 h. n-Hexane (30 mL) was added and it was diluted to 100 mL with 10% Na2SO4 solution. The upper phase (50 mL) was used to determine the total free lutein content according to the procedure described by Piccaglia et al. (1998). HPLC was performed with an Agilent 1100 Series (Agilent Corporation, MA, USA) HPLC system equipped with a diode array detector (DAD), and separation of lutein was carried out in a DIKMA ODS C18 (Dikma Technology, Beijing, China) column (250 mm · 4.6 mm i.d.; particle size, 5 lm) set at a

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SC-CO2 Extraction of Lutein from Marigold Q. Ma et al.

column temperature of 30 C. The mobile phase was a gradient elution system consisting of ethyl acetate (solvent A) and acetonitrile–methanol (9:1, v ⁄ v, solvent B) with the gradient program running from 0% A to 100% A in 30 min at a constant flow rate of 1.0 mL ⁄ min. All solvents were filtered through a 0.45 lm syringe filter prior to analysis. The injection volume was 10 lL and the detector was set at 450 nm. Lutein was identified by comparing the retention time of the peak in the extract with that of standard compound. Lutein content was determined by referring to a calibration curve established by running lutein standard at varying concentrations through the HPLC system under the same conditions. The standard curve was linear from 12.06 to 60.48 lg ⁄ mL (R2 = 0.9994). Experimental design

Response surface methodology was used to optimise temperature, pressure, flow rate and soybean oil concentration for the extraction of lutein esters. The coded and uncoded independent variables used in the RSM design are listed in Table 1. The levels of the independent variables selected were based on the results of Table 2 Four factor, five level central composite design with the observed responses and predicted values of lutein yield

Experiment numbera 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 a

Pressure (MPa) 40 20 20 30 14.5 30 20 20 30 40 20 45.5 30 20 30 30 20 30 30 20 40 40 40 40 40 30 40

Table 1 Uncoded and coded independent variables and their levels used in the RSM design Coded levels Independent

Variable

)1.55

)1

0

1

1.55

X1 X2 X3 X4

Pressure (MPa) Temperature (C) Flow rate (L ⁄ h) Co-solvent (%)

14.5 33.2 7.3 0

20 40 10 2.2

30 52.5 15 6.1

40 65 20 10

45.5 71.8 22.7 12.1

preliminary experiments. The experimental design was based on the central composite design using a 24 factorial and star design with three central points as shown in Table 2. A second-order polynomial equation was used to express the yield of lutein as a function of the independent variables: Y ¼ a0 þ a1 X1 þ a2 X2 þ a3 X3 þ a4 X4 þ a11 X21 þ a22 X22 þ a33 X23 þ a44 X24 þ a12 X1 X2 þ a13 X1 X3 þ a14 X1 X4 þ a23 X2 X3 þ a24 X2 X4 þ a34 X3 X4 ð1Þ

Temperature (°C) 65 65 40 52.5 52.5 52.5 65 65 33.2 65 40 52.5 52.5 65 52.5 71.8 40 52.5 52.5 40 65 40 65 40 40 52.5 40

Flow rate (L ⁄ h) 10 20 10 15 15 15 20 10 15 10 20 15 15 10 7.27 15 20 22.7 15 10 20 10 20 10 20 15 20

Co-solvent (%) 10 2.2 2.2 0 6.1 12.1 10 2.2 6.1 2.2 10 6.1 6.1 10 6.1 6.1 2.2 6.1 6.1 10 2.2 2.2 10 10 2.2 6.1 10

Yield Observed (mg ⁄ 100 g)

Predicted (mg ⁄ 100 g)

375.43 472.39 273.24 490.24 395.19 756.24 493.45 165.94 490.33 263.33 301.24 593.20 962.04 212.38 632.02 650.12 274.26 689.44 951.29 233.45 872.93 170.79 921.96 253.16 543.21 962.32 643.29

385.59 415.37 265.66 587.86 375.89 708.36 464.05 202.30 495.95 263.69 284.59 662.24 931.90 251.98 487.37 694.24 250.65 883.83 931.90 300.59 792.34 186.74 913.24 293.89 487.32 931.89 593.48

Experiments were performed in random order.

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where Y represents the yield of lutein expressed as mg ⁄ 100 g marigold (on a dry matter basis) and aij are coefficients of the equation. The coefficients of the equation were estimated by using the software Statgraphics plus 5.1 (Statpoint Inc., Northern Virginia, USA). Statistical analysis

Experimental data were analysed by multiple regressions to fit the second-order polynomial equation to all independent variables. Analysis of variance (anova) was performed to evaluate significant differences between independent variables (P £ 0.05). To visualise the relationships between the responses and the independent variables, surface response plots of the fitted polynomial regression equation were generated using Statgraphics plus 5.1.

Table 3 Estimated coefficients of the second-order regression model for the yield of lutein Term

Coefficient

Value

P-value

Intercept X1 X2 X3 X4 X1*X1 X1*X2 X1*X3 X1*X4 X2*X2 X2*X3 X2*X4 X3*X3 X3*X4 X4*X4

a0 a1 a2 a3 a4 a11 a12 a13 a14 a22 a23 a24 a33 a34 a44

)3335.600 71.571 77.172 54.021 87.469 )1.726 0.281 1.578 0.463 )0.901 0.912 0.076 )4.118 )0.013 )7.799

– 0.0008 0.0091 0.0000 0.0838 0.0000 0.1621 0.0058 0.4580 0.0003 0.0322 0.8782 0.0030 0.9918 0.0011

R2 = 0.9398.

Results and discussion

Fitting the model

The yields of lutein obtained from all the experiments are listed in Table 2. The experimental data were used to calculate the coefficients of the response surface equation [Eq. (1)] and the resultant second-degree polynomial model was used to predict the yield of lutein. Fig. 2 is a plot of the predicted versus experimental values of lutein yield, and it showed that the two sets of data agreed with each other quite well (R2 = 0.9398). Analysis of variance (anona) was used to evaluate the significance of the coefficients of Eq. (1). Table 3 gives the regression coefficients of the equation, which showed that all the independent parameters, except the concentration of co-solvent, had a significant (P < 0.01) 1000

positive linear effect on lutein yield. Furthermore, the quadratics of all the independent parameters (P < 0.005), as well as the interactions between CO2 flow rate and pressure (P < 0.01) and between CO2 flow rate and temperature (P < 0.05) had a significant effect on the lutein yield. The effect of pressure

The best way to visualise the effect of each parameter on the yield of lutein within the experimental range was to generate response surface plots of the equation. Response surface plots showing the effect of pressure on the yield are given in Figs 3–5, which showed that the yield of lutein increased initially with the rise of extraction pressure. However, when the pressure was over 30 MPa (the value varied dependent on the value of other parameters), the yield began to decline with further

800 600 990

Y (mg 100 g–1)

Observed values

1766

400 200

890 790 690 590 490 20

0 0

200

400 600 Predicted values

800

1000

Figure 2 Predicted versus experimental values for the yields of lutein

(R2 = 0.9398).

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28 32 36 Pressure (MPa)

65 60 55 50 45 40 Temperature (°C) 40

Figure 3 Response surface of the effects of pressure and temperature on the extraction yield (Y) at the flow rate 15 L ⁄ h and co-solvent concentration 6.1%.

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20

24

28 32 36 Pressure (MPa)

20 18 16 14 12 10 Flow rate (L h–1) 40

Figure 4 Response surface of the effects of pressure and flow rate on the extraction yield (Y) at 52.5 C and the co-solvent concentration 6.1%.

Y (mg 100 g–1)

980 880 780 680 580 480 380 40

45

50 55 60 Temperature (°C)

65

0

2

4

6

8

10

Co-solvent (%)

Figure 6 Response surface of the effects of temperature and co-solvent on the extraction yield (Y) at 30 MPa and the flow rate 15 L ⁄ h.

1050

1140 940 740 540 340 20

Y (mg 100 g–1)

1110 1010 910 810 710 610 510

24

28 32 36 Pressure (MPa)

40

0

2

4

6

8

10

Co-solvent (%)

Figure 5 Response surface of the effects of pressure and co-solvent on the extraction yield (Y) at 52.5 C and the flow rate 15 L ⁄ h.

increases in pressure, a finding similar to that reported by Sun & Temelli (2006). This could be explained by the change in lutein solubility in SC-SO2 as a function of pressure. According to Clifford (1999), the solubility of a solute generally increases with an initial rise of pressure since the density of the supercritical solvent increases with pressure. As the pressure continues to rise, however, the solvent becomes highly compressed and, for some solutes, the repulsive solute–solvent interactions also become important. At a certain pressure level, the repulsive solute–solvent interactions may become greater than the increase in the solubility gained from the increased solvent density, with a net lowering of solubility. The effect of temperature

Temperature had a significant effect on the yield of lutein (Figs 3, 6 and 7). The yield of lutein increased initially with the rise of temperature, and reached maximum values at approximately 55 C, then decreased with further increases in temperature. A similar finding was reported by Sovova´ et al. (2004) for the

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Y (mg 100 g–1)

Y (mg 100 g–1)

SC-CO2 Extraction of Lutein from Marigold Q. Ma et al.

950 850 750 650 550 40

45 50 55 60 Temperature (°C)

65

20 18 16 14 12 10 Flow rate (L h-1)

Figure 7 Response surface of the effects of temperature and flow rate on the extraction yield (Y) at 30 MPa and the co-solvent concentration 6.1%.

extraction of oleoresin from stinging nettle leaves by SCCO2. Increasing temperature increases the solubility of the solute, which results in higher yields. While at high temperature throughout the extraction, the decline in the yield could be explained by the degradation of lutein esters. In previous study, the loss of lutein esters during the SC-CO2 extraction increased in proportion to the temperature and approximately half of the esters was lost at 36.4 MPa and 55 C (Naranjo-Modad et al., 2000). The effect of CO2 flow rate

The yield of lutein was significantly affected by CO2 flow rate (Figs 4, 7 and 8). Similar to the effect of pressure and temperature, the yield of lutein increased initially with the rise of CO2 flow rate. However, under certain conditions, further increases in flow rate resulted in a decline in the yield. A similar observation was also reported by Sun & Temelli (2006) for the extraction of carotenoids from carrots using canola oil as a cosolvent. This could be explained from the balance

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SC-CO2 Extraction of Lutein from Marigold Q. Ma et al.

Used as a co-solvent at low to medium concentrations, soybean oil was able to substantially increase the yield of lutein. The model predicted that the maximum yield of 1039.7 mg lutein ⁄ 100 g marigold could be achieved under the optimal conditions: 35.5 MPa, 58.7 C, CO2 flow rate of 19.9 L ⁄ h with 6.9% of soybean oil as a cosolvent.

1140 Y (mg 100 g–1)

1768

940 740 540 340 10

12

14 16 18 Flow rate (L h-1)

20

0

2

4

6

8

10

Co-solvent (%)

Figure 8 Response surface of the effects of flow rate and co-solvent on the extraction yield (Y) at 30MPa and 52.5 C.

between the opposing effects of the amount of fresh solvent and solvent–solute contact time. Initially, as the flow rate increased, the lutein yield increased as greater amounts of fresh solvent came into contact with the material. However, as the flow rate continued to rise, there was less and less contact time between the solvent and the material, and the solvent may exit the system without dissolving all the solute it could. Eventually, it may reach a point, at which the contact time was so short that the yield began to decline. The effect of soybean oil concentration

The concentration of soybean oil had a significant (P < 0.01) quadratic effect on the lutein yield. At low to medium concentration, using soybean oil as a co-solvent improved the yield of lutein by SC-CO2 extraction (Figs 5, 6 and 8). Under the extraction condition of 30 MPa, 52.5 C and a CO2 flow rate of 10 L ⁄ h with 6.1% soybean oil as co-solvent, the yield of lutein was more than twice higher than that without the co-solvent. This was consistent with the findings of Sun & Temelli (2006), who reported that yield of lutein could be increased by up to three times when canola oil was used as a continuous co-solvent, at low to medium concentrations (1–3%), for the SC-CO2 extraction of lutein from carrots. Conclusion

The current study showed that the second-order polynomial model was sufficient to describe and predict the responses of lutein yield to changes in the extraction conditions, within the experimental ranges, of the SCCO2 extraction of lutein esters from marigold. The independent parameters of CO2 flow rate, pressure and temperature (P < 0.01), all quadratics (P < 0.005) as well as the interactions between flow rate and pressure (P < 0.01) and between CO2 flow rate and temperature (P < 0.05) significantly influenced the yield of lutein.

International Journal of Food Science and Technology 2008

Acknowledgments

We gratefully acknowledge Dr. Xiaojun Liao, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China, for his helpful advice. References Alves-Rodrigues, A. & Shao, A. (2004). The science behind lutein. Toxicology Letters, 150, 57–83. Ambrogi, A. & Eggers, R. (1997). Extraction and concentration of xanthophyll pigments from marigold (Tagetes erecta). In: The 4th Italian Conference on Supercritical Fluids and Their Applications (edited by E. Reverchon). Pp. 129–135. Italy: Univ degli Studi di Salerno. AOAC (1990). Official Methods of Analysis, 16th edn. Pp. 1048–1049. Washington, DC: Association of Official Analytical Chemists. Barth, M.M., Zhou, C., Kute, K.M. & Rosenthal, G.A. (1995). Determination of optimum conditions for supercritical fluid extraction of carotenoids from carrot (Daucus carota L.) tissue. Journal of Agricultural and Food Chemistry, 43, 2876–2878. Bartlett, H. & Eperjesi, F. (2003). Age-related macular degeneration and nutritional supplementation: a review of randomised controlled trials. Ophthalmic and Physiological Optics, 23, 383–399. Barzana, E., Rubio, D., Santamaria, R.I. et al. (2002). Enzymemediated solvent extraction of carotenoids from marigold flower. Journal of Agricultural and Food Chemistry, 50, 4491–4496. Chew, B.P., Wong, M.W. & Wong, T.S. (1996). Effects of lutein from marigold extract on immunity and growth of mammary tumors in mice. Anticancer Research, 16, 3689–3694. Clifford, T. (1999). Fundamentals of Supercritical Fluids. Pp. 51. New York, NY: Oxford University Press. Daood, H.G., Illesb, V., Gnayfeed, M.H., Meszaros, B., Horvath, G. & Biacs, P.A. (2002). Extraction of pungent spice paprika by supercritical carbon dioxide and subcritical propane. Journal of Supercritical Fluids, 23, 143–152. Delgado-Vargas, F. & Paredes-Lo´pez, O. (1997a). Effect of enzymatic treatments of marigold flowers on lutein isomeric profiles. Journal of Agricultural and Food Chemistry, 45, 1097–1102. Delgado-Vargas, F. & Paredes-Lo´pez, O. (1997b). Effect of enzymatic treatments on carotenoid extraction from marigold flowers. Journal of Agricultural and Food Chemistry, 58, 255–258. Jaren-Galan, M., Nienaber, U. & Schwartz, S.J. (1999). Paprika (Capsicum annuum) oleoresin extraction with supercritical carbon dioxide. Journal of Agricultural and Food Chemistry, 47, 3558–3564. Landrum, J.T. & Bone, R.A. (2001). Lutein, zeaxanthin, and the macular pigment. Archives of Biochemistry and Biophysics, 385, 28– 40. Naranjo-Modad, S., Lopez-Munguia, A., Vilarem, G., Gaset, A. & Barzana, E. (2000). Solubility of purified lutein diesters obtained from Tagetes erecta in supercritical CO2 and the effect of solvent modifiers. Journal of Agricultural and Food Chemistry, 48, 5640– 5642. Navarrete-Bolan˜os, J.L., Rangel-Cruz, C.L., Jime´nez-Islas, H., Botello-Alvarez, E. & Rico-Martı´ nez, R. (2005). Pre-treatment

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SC-CO2 Extraction of Lutein from Marigold Q. Ma et al.

effects on the extraction efficiency of xanthophylls from marigold flower using hexane. Food Research International, 38, 159– 165. Olmedilla, B., Granado, F., Blanco, I., Vaquero, M. & Cajigal, C. (2001). Lutein in patients with cataracts and age related macular degenerations: a long term supplementation study. Journal of the Science of Food and Agriculture, 81, 904–909. Piccaglia, R., Marotti, M. & Grandi, S. (1998). Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Industrial Crops and Products, 8, 45–51. Quackenbush, F.W. (1973). Use of heat to saponify xantophyll esters and speed analysis for carotenoids in feed material: collaborative study. Journal of the Association of Official Analytical Chemists, 56, 748–753. Rao, J.R. & Reddy, G.B.S. (2004). Extracton of Lutein From Marigold Meal. US patent: PCT ⁄ IB01 ⁄ 02057. Washington, DC: US Patent and Trademark Office. Sovova´, H., Sajfrtova´, M., Ba´rtlova´, M. & Opletal, L. (2004). Nearcritical extraction of pigments and oleoresin from stinging nettle leaves. Journal of Supercritical Fluids, 30, 213–224.

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Sowbhagya, H.B., Sampathu, S.R. & Krishnamurthy, N. (2004). Natural colorant from marigold-chemistry and technology. Food Reviews International, 20, 33–50. Spanos, G.A., Chen, H. & Schwartz, S.J. (1993). Supercritical CO2 extraction of b-carotene from sweet potatoes. Journal of Food Science, 58, 817–820. Sun, M. & Temelli, F. (2006). Supercritical carbon dioxide extraction of carotenoids from carrot using canola oil as a continuous cosolvent. Journal of Supercritical Fluids, 37, 397–408. Tsao, R., Yang, R., Young, J.C., Zhu, H. & Manolis, T. (2004). Separation of geometric isomers of native lutein diesters in marigold (Tagetes erecta L.) by high-performance liquid chromatography– mass spectrometry. Journal of Chromatography A, 1045, 65–70. Vasapollo, G., Longo, L., Rescio, L. & Ciurlia, L. (2004). Innovative supercritical CO2 extraction of lycopene from tomato in the presence of vegetable oil as co-solvent. Journal of Supercritical Fluids, 29, 87– 96. Vega, P.J., Balaban, M.O., Sims, C.A., O’Keefe, S.F. & Cornell, J.A. (1996). Supercritical carbon dioxide extraction efficiency for carotenes from carrots by RSM. Journal of Food Science, 61, 757–759, 765

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Original article Effect of domestic cooking on the red cabbage hydrophilic antioxidants Anna Podse˛dek,* Dorota Sosnowska, Małgorzata Redzynia & Maria Koziołkiewicz Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz´, 90-924 Lodz´, Stefanowskiego 4 ⁄ 10, Poland (Received 18 July 2006; Accepted in revised form 21 September 2007)

Summary

The contents of vitamin C, total phenolics, anthocyanins, hydroxybenzoic and hydroxycinnamic acids as well as 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) monocation radical scavenging activity were evaluated in two varieties of red cabbage before and after conventional and steam-cooking. During the conventional cooking 32.7–64.5% of vitamin C and 45.7–66.9% of total phenolics were retained in cooked tissue. Decreasing cooking water volume by half led to better retention of both phenolics (by 2.7–14.5%) and vitamin C (by 14.2–18.4%). However, shortening the cooking time by half affected the retention of phenolics and vitamin C only by 3.8–6.7% and 0–2.2%, respectively. Steam-cooking is recommended to prevent the major loss of scavenging activity, because under these conditions, the corresponding TEAC (Trolox Equivalent Antioxidant Capacity) values were reduced only by 5–20%. Moreover, the content of vitamin C was decreased by 2.1–22.7% while losses of total phenolics were up to 10%. The phenolics are the major source of free radical scavenging activity of red cabbage, since the contribution of vitamin C to TEAC for fresh and cooked red cabbage was from 18.2 to 28.5%.

Keywords

antioxidant activity, Brassica oleracea var capitata rubra, food processing, phenolic compounds, vitamin C.

Introduction

Many reports link Brassica vegetable intakes with reduced risk of chronic diseases like cardiovascular disease and cancer (Cohen et al., 2000; Chu et al., 2002; Uhl et al., 2004). The beneficial biological properties of these vegetables have been partially attributed to their dietary antioxidants. These antioxidants include vitamins C and E, carotenoids and phenolics. In addition to antioxidant compounds, Brassica vegetables provide a large group of glucosinolates, which according to Plumb et al. (1996) possess rather low antioxidant activity, but products of their hydrolysis can protect against cancer (Steinkellner et al., 2001; Keum et al., 2004). Brassica vegetables include different heading cabbages, such as white, red and Savoy cabbage. Red cabbage (Brassica oleracea var capitata rubra) is widely cultivated in North and Central Europe, North America, China and Japan. In Poland, cabbage is a very popular vegetable. In 2003, annual production of different types of cabbage was 1237 thousand tons and that was 1.5-fold or 5.3-fold more than production of *Correspondent: Fax: +4842 6366618; e-mail: [email protected]

carrot or tomato, respectively (Statistical Year Book of the Republic of Poland, 2003). Red cabbage is a less popular vegetable than white cabbage, because its contribution to total cabbage production was about 10% in 2003, while for white cabbage, it was 85%. Red cabbage can be cooked in many ways and it may be eaten raw also as an ingredient of various salads. According to literature data, red cabbage is considered as a vegetable of high antioxidant activity. For example, the antioxidant capacity of aqueous extract of red cabbage, tested by the Briggs-Rauscher method, was similar to that for Brussels sprouts, and about 7-fold and 15-fold higher than broccoli and cauliflower activity, respectively (Honer & Cervellati, 2002). Among 11 vegetables tested, Hassimotto et al. (2005) noticed that in the liposome oxidation method, methanolic extract of red cabbage showed the highest antioxidant activity. The very high antioxidant activity of red cabbage was confirmed by Proteggente et al. (2002) in three different assays: ORAC (Oxygen Radical Absorbance Capacity), FRAP (Ferric Reducing Ability of Plasma) and TEAC (Trolox Equivalent Antioxidant Capacity). The ORAC value for red cabbage was also the highest among the Brassica vegetables tested by Wu et al. (2004). In this study, the total antioxidant capacity for red cabbage was

doi:10.1111/j.1365-2621.2007.01697.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Red cabbage antioxidants A. Podse˛dek et al.

22.5 lmol of Trolox ⁄ g, for broccoli )15.9 lmol ⁄ g, for common cabbage )13.6 lmol ⁄ g and for cauliflower only 6.5 lmol ⁄ g. The total antioxidant capacity for red cabbage was similar to that of hydrophilic antioxidants (phenolic compounds + vitamin C) because the contribution of lipid-soluble extracts was only 0.9% (Wu et al., 2004). Such a low scavenging capacity of a lipidsoluble extract could be attributed to a low level of carotenoids (0.20 mg ⁄ 100 g) and vitamin E (0.05 mg ⁄ 100 g) in red cabbage (Piironen et al., 1986; Muller, 1997). Therefore, vitamin C and phenolic compounds seem to be the major antioxidants in red cabbage. Red cabbage as well as red onion, red radish, eggplant and red potato are sources of anthocyanins, which belong to flavonoids – subgroup of phenolic compounds. Their content in red cabbage ranges from 25 to 495 mg ⁄ 100 g of fresh weight (Mazza & Miniati, 1993; Wang et al., 1997; Piccaglia et al., 2002). In Japan, red cabbage is a source of red food colourants and the preparation of these pigments is described in several patents (Bridle & Timberlake, 1997). Relatively, little has been reported about other phenolics of red cabbage. Most reports published so far concern the content and properties of antioxidants of fresh vegetables. On the other hand, some studies have indicated that vegetable processing such as cooking, influences dietary antioxidant activities. Some reports have focused mainly on the preservation of phenolic compounds in broccoli (Vallejo et al., 2003; Zhang & Hamauzu, 2004) and vitamin C in broccoli and Brussels sprouts (Howard et al., 1999; Czarniecka-Skubina, 2002; Zhang & Hamauzu, 2004). The above-mentioned studies have shown that the loss of dietary antioxidants in the vegetables tested is caused by the cooking conditions, such as type of cooking (conventional, steaming, microwaving, etc.), cooking time and amount of water. The present study was carried out to estimate the vitamin C and phenolic compounds contents in red cabbage as well as the changes in their scavenging capacities towards 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical monocation (ABTS•+) caused by different cooking conditions. To the best of our knowledge, this is the first such a study carried out for red cabbage.

Cabbage (500 g) was placed on a tray in a pot, and then covered with a lid and steamed over boiling water for 5 or 20 min. The samples were homogenised in a blender (Tefal). One part of the homogenised samples was used for the determination of dry matter and ascorbic acid contents. The second part was lyophilised and ground. The resulting powder was stored at )20 C until extraction of phenolic compounds.

Materials and methods

Dry matter determination

Plant materials

Dry matter content was determined by drying a sample (2 g) at 105 C to constant weight.

Red cabbage (Brassica oleracea var capitata) varieties Koda and Kissendrup were cultivated and harvested under normal commercial conditions at farms of the PlantiCo Horticulture Breeding and Seed Production, Gole˛biew, Ltd. (central region of Poland). Exterior damaged leaves and heart were removed, while edible

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parts of cabbage were chopped, and then cooked in one of the five different ways. Each of the cooking procedures was repeated in triplicate. The raw cabbage was used as a reference sample. Chemicals

Chlorogenic acid, gallic acid, 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), 2,2¢-azinobis (3-ethylbenzo-thiazoline-6 sulphonic acid) (ABTS), DL-dithiothreitol, potassium persulphate and metaphosphoric acid were purchased from Sigma–Aldrich (Steinheim, Germany) and rutin, L-ascorbic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cyanidin 3-glucoside was purchased from Extrasynthese (Genay, France). HPLC grade methanol, acetonitrile and formic acid were purchased from J.T. Baker (Deventer, Holland). All other chemicals were reagent grade products purchased from POCh (Gliwice, Poland). Ultra pure water was produced in the laboratory using a SimplicityTM Water Purification System (Millipore, Marlborough, MA, USA). Conventional cooking

Red cabbage (500 g) was added to 500 or 1000 mL of boiling water and cooked for 10 or 20 min. Then excess water was dripped off and the cooked cabbage was homogenised in a blender (Braun GmbH, Kronberg, Germany). One part of the homogenised samples was used for the determination of dry matter and ascorbic acid contents. The second part was lyophilised and ground. The resulting powder was stored at )20 C until the phenolic compounds were extracted. Steaming

Vitamin C extraction and analysis

The extraction method used in this study was a modification of the method described by Howard et al. (1999). Raw and cooked cabbage samples (10 g) were

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extracted with a 1% solution of meta-phosphoric acid (25 mL) for 15 min at room temperature and centrifuged at 2500 · g for 10 min. The residue was reextracted with 10 mL of the extracting solution and centrifuged. The combined supernatants were diluted to 50 mL with 1% meta-phosphoric acid. One milliliter of this solution and 1 mL of dithiothreitol (5%) were mixed and kept in the dark for 18 h at room temperature. Then, after dilution with meta-phosphoric acid, the samples were analysed using a Knauer high performance liquid chromatograph equipped with a Eurospher-100 C-18 column (25 cm · 4.6 mm; 5 lm; Knauer, Berlin, Germany) fitted with the same guard column. The HPLC method was adapted from Gliszczynska-Swiglo & Tyrakowska (2003). For the analysis of vitamin C, the following gradient of methanol (solvent A) and 5 mmol ⁄ L KH2PO4 pH 2.6 (solvent B) was used: linear increase of solvent A from 5% to 22% in 6 min and then return to the initial conditions within the next 9 min with a flow rate of 1 mL ⁄ min with a UVVis detector set at 245 nm. Ascorbic acid was identified by comparison with its genuine sample.

tives were quantified at 280 nm and expressed as GAEs’, hydroxycinnamic acid derivatives – at 320 nm as chlorogenic acid equivalents, flavonols – at 360 nm as rutin equivalents, and anthocyanins – at 520 nm as cyanidin 3-glucoside equivalents. ABTS radical cation scavenging activity

The scavenging activity of ABTS•+ was determined according to the procedure described by Re et al. (1999). 2,2¢-Azinobis(3-ethyl-benzothiazoline-6-sulphonic acid) radical cation (ABTS•+) was produced by the reacting of 7 mmol ⁄ L ABTS water solution with 2.45 mmol ⁄ L potassium persulphate (final concentration) followed by an incubation of the mixture in the dark for 12–16 h at room temperature. Later, water phenolic extract (20 lL) was mixed with 1 mL of diluted ABTS•+ solution and after 6 min at 30 C, this mixture was measured at 734 nm. Trolox (6-hydroxy-2,5,7,8-tetramethychroman2-carboxylic acid) was used as a standard and the capacity of free radical scavenging was expressed as lmoles of Trolox in 1 g fresh vegetable weight (TEAC Trolox Equivalent Antioxidant Capacity).

Phenolics extraction and determination

Lyophilised raw or cooked vegetables (2 g) were extracted twice with 50 mL of a 70% solution of MeOH for 15 min at room temperature (Vallejo et al., 2003). The mixture was then centrifuged at 2500 · g for 15 min, and the resulting supernatant was evaporated under reduced pressure (T < 40 C). The aqueous extracts were diluted to 20 mL with water, and analysed to quantify total phenolics content and to determine phenolic profiles. Total phenolics were analysed spectrophotometrically by the Folin-Ciocalteu procedure (Peri & Pompei, 1971). In brief, 10 mL of water, 0.1– 0.6 mL of the sample, 0.5 mL of Folin-Ciocalteu reagent and 5 mL of 20% Na2CO3 were mixed and diluted to 50 mL with water. After 20 min of incubation in the dark, the absorbance was measured at 700 nm. The total phenolics amount in the extract was expressed as gallic acid equivalent (GAE) in miligrams per 100 g of fresh weight. Phenolic profiles were determined using a HPLC Knauer system equipped with a UV–Vis detector and a Eurospher-100 C-18 column (25 cm · 4.6 mm; 5 lm). The binary mobile phase consisted of water ⁄ formic acid (90:10, v ⁄ v) (solvent A) and water ⁄ acetonitrile ⁄ formic acid (40:50:10, v ⁄ v ⁄ v) (solvent B), according to Dyrby et al. (2001). Samples were eluted at a flow rate of 1 mL ⁄ min with the following gradient program: 0 min: 88% A + 12% B, 26 min: 70% A + 30% B, 40– 43 min: 0% A + 100% B, 48–50 min: 88% A + 12% B. Phenolics were qualified into one of the four subclasses and quantified on the base of maximum of UV–Vis absorption. The hydroxybenzoic acid deriva-

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Statistical analysis

The analysis of variance was performed on data for differences between the cooking methods using the anova (Statistica Ver. 6.0, StatSoft Inc., Tulsa, OK), followed by the Tukey’s post hoc test with significance level P £ 0.05. Results and discussion

The effect of the cooking method on vitamin C content

The vitamin C content in the two varieties of red cabbage (Koda and Kissendrup) cooked under different conditions is presented in Table 1. The fresh vegetables contain 62.00–72.56 mg of vitamin C ⁄ 100 g of fresh weight and these values are higher than the total vitamin C content (37 mg ⁄ 100 g) determined by Proteggente et al. (2002). The vitamin C content was reduced after all the cooking procedures tested. The highest vitamin C content was found for steamed red cabbage. Steaming for 5 min is used to prepare red cabbage salad and this process leads to the inactivation of the endogenous enzymes and to the softening of the cabbage. In this case, the losses of vitamin C were 2.1% for Kissendrup and 15.6% for Koda (Fig. 1). Further decrease in vitamin C content was observed after the steaming for 20 min, but although the steaming time increased fourfold, the content of this vitamin decreased only by 6.4– 7.1%. Puupponen-Pimia et al. (2003) reported that up to one-third of vitamin C content was lost during the blanching of cabbage in water and steam for 3 min at 96 C. Other authors (Lisiewska & Kmiecik, 1996;

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Red cabbage antioxidants A. Podse˛dek et al.

Table 1 Effect of different cooking methods on the antioxidants content in two varieties of red cabbage Koda Cooking method Raw cabbage In boiling water

In steam

Kissendrup

Cooking time (min)

Vegetable: water (g ⁄ mL)

Dry matter (%)

Vitamin C (mg ⁄ 100 g)

Phenolics (mg ⁄ 100 g)

Dry matter (%)

Vitamin Ca (mg ⁄ 100 g)

Phenolicsb (mg ⁄ 100 g)

20 20 10 20 5

1:2 1:1 1:1 – –

10.42 9.36 9.13 8.23 10.75 10.79

72.56 23.74 33.61 31.74 56.29 61.50

134.73 61.61 81.18 90.18 123.09 137.07

11.48 6.91 8.64 8.28 10.39 10.38

62.00 26.77 38.36 38.72 56.14 60.06

171.36 68.71 90.45 97.02 140.39 154.15

± ± ± ± ± ±

a

0.21 0.36 0.13 0.10 0.48 0.10

± ± ± ± ± ±

7.99a 2.81e 4.57d 0.53d 4.94c 0.75b

b

± ± ± ± ± ±

3.35a 1.77e 3.89d 5.48c 2.36b 3.02a

± ± ± ± ± ±

0.58 0.16 0.06 0.11 0.08 0.14

± ± ± ± ± ±

2.74a 2.53d 1.00c 1.72c 1.79b 0.45a

± ± ± ± ± ±

13.77a 6.87d 5.66c 4.66c 6.02b 12.32b

Mean ± SD, n ‡ 3. Different letters indicate significant differences within columns by the Tukey post hoc test at P £ 0.05. a Vitamin C values are expressed as ascorbic acid equivalents. b Phenolics values are expressed as gallic acid equivalents.

Koda

Contribution (%)

100 80

20

44.7

77.3

84.4

3

4

5

34.2

36.2

18.9

19.1

32.7

46.9

1

2

40.3

60 40

22.7

15.6

27.0

0

Kissendrup 2.1

Contribution (%)

100 80 60

8.5

18.6

16.3

17.8

19.2

45.2

63.6

64.5

91.5

1

2

3

4

25.8 29.0

40 20 97.9

0 Edible part

Cooking water

5

When the water volume was reduced by half (1:1 v ⁄ w), retention of vitamin C increased to 46,9% (Koda) to 63.6% (Kissendrup). On the other hand, shortening the cooking time from 20 to 10 min only slightly affected retention of vitamin C in edible parts of the cabbage. Similar retention of vitamin C has been reported for broccoli florets and stems cooked conventionally for 5 min (Zhang & Hamauzu, 2004) and for Brussels sprouts cooked for 26 min in a pot of boiling water (Czarniecka-Skubina, 2002). The loss of vitamin C during the cooking was caused by its thermal degradation and leaching into the cooking water. Conventional cooking resulted in significant loss of vitamin C in cooking water for both the varieties tested (Fig. 1). As expected, the lower volume of cooking water led to better retention of vitamin C in the cooked tissue. On the other hand, prolongation of the cooking time from 10 to 20 min did not influence the distribution of vitamin C in the cooked cabbage and in the cooking water.

Lost

Figure 1 Distribution of vitamin C in edible part, cooking water and lost observed for two varieties of red cabbage cooked in one of the five different methods: 1, conventional cooking for 20 min, ratio vegetable:water = 1:2 (w ⁄ v); 2, conventional cooking for 20 min, ratio vegetable:water = 1:1 (w ⁄ v); 3-conventional cooking for 10 min, ratio vegetable:water = 1:1 (w ⁄ v); 4, steaming for 20 min; 5, steaming for 5 min.

Howard et al., 1999) obtained similar results for other Brassica vegetables, the losses in vitamin C content during the blanching reaching 30% in cauliflower and 30–40% in broccoli. In the case of conventional cooking, the amount of water strongly influenced the content of vitamin C. When the water to vegetable ratio was 2:1 (v ⁄ w) and the cabbage was cooked for 20 min, 32.7% (Koda) to 45.2% (Kissendrup) of vitamin C was retained (Fig.1).

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

The effect of the cooking method on phenolics content

The total phenolics content in red cabbage cooked under different conditions was determined by FolinCiocalteu assay (Table 1). The level of total phenolics in fresh vegetable was 134.73 mg ⁄ 100 g for Koda and 171.36 mg ⁄ 100 g for Kissendrup. Our results are in accordance with Hassimotto et al. (2005) and Proteggente et al. (2002). However, some authors reported higher phenolics content (217–679 mg ⁄ 100 g) in red cabbage (Chun et al., 2004; Wu et al., 2004; Amin & Lee, 2005; Karadeniz et al., 2005). As it is in the case of vitamin C, a decrease in the content of the red cabbage phenolics was detected for all the cooking procedures. The phenolics level in both the cultivars tested decreased in the following order: fresh > 5–min steamed > 20-min steamed > 10-min cooked in boiling water > 20-min cooked in boiling water. The Koda

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red cabbage cooked in steam for 5 and 20 min retained 101.7% and 91.4% of the total phenolics present in fresh vegetable, respectively (Fig. 2). In the case of Kissendrup red cabbage, higher losses (ca. 10%) of phenolic compounds were observed. A higher decrease in total phenolics content after blanching of red cabbage was reported by Amin & Lee (2005). The red cabbage blanched for 5, 10 or 15 min had 95.4%, 64.1% and 63.2% of total phenolics as compared with the fresh vegetable. Our results were similar to those previously reported by Vallejo et al. (2003) for broccoli. Steamcooking of broccoli for 3.5 min caused slight losses (8.8%) of total phenolics. For the conventional cooking (20 min and 2:1 ratio of water : cabbage), retention of phenolics ranged from 45.7% (Koda) to 50.1% (Kissendrup). Fifty percent decrease in water volume led to better retention of phenolics in the cooked tissue (Table 1 and Fig. 2). On the other hand, shortening the conventional cooking time from 20 to 10 min increased the retention of phenolics in the cooked cabbage only by 3.8–6.7%. Wu et al. (2004) noticed a significant decrease (26.4%) in total phenolics content during the cooking of red cabbage for 3–4 min in boiling water. In the study carried out by Turkmen et al. (2005), broccoli cooked for 5 min in boiling water retained 94% of total

Koda Contribution (%)

100 80 60

21.9

27.3

17.9

19.1 14.0

8.6

27.0

40 20

45.7

60.2

66.9

91.4

100.0

1

2

3

4

5

0

100 Contribution (%)

1774

21.1

19.1

Kissendrup 10.0

80

30.9

29.4

60

19.0

17.8

50.1

52.8

56.6

81.9

90.0

1

2

3

4

5

22.3

40 20 0 Edible part

Cooking water

Lost

Figure 2 Distribution of total phenolics in edible part, cooking water and lost observed for two varieties of red cabbage cooked in one of the five different methods: 1, conventional cooking for 20 min, ratio vegetable:water = 1:2 (w ⁄ v); 2, conventional cooking for 20 min, ratio vegetable:water = 1:1 (w ⁄ v); 3, conventional cooking for 10 min, ratio vegetable:water = 1:1 (w ⁄ v); 4, steaming for 20 min; 5, steaming for 5 min.

International Journal of Food Science and Technology 2008

phenolics. Conversely, Vallejo et al. (2003) and Zhang & Hamauzu (2004) reported higher losses (about 70%) of total phenolics content after conventional cooking of broccoli for 5 min. The contents of different groups of phenolics in the two varieties of red cabbage before and after cooking are presented in Table 2. Anthocyanins dominated in raw as well as in cooked cabbage. In raw cabbage, the average content of hydroxycinnamic and hydroxybenzoic acids was 2.6- and 4.1-fold lower than the content of anthocyanins, respectively. Literature data reported a wide range of anthocyanins content (25–495 mg ⁄ 100 g fresh weight) for red cabbage (Mazza & Miniati, 1993; Piccaglia et al., 2002). Cooking procedures affected the levels of all the phenolics groups tested. The highest reduction in their content was observed in the case of conventional cooking (20 min in boiling water in a 2:1 ratio to weight of cabbage). In this case, the cooked cabbage contained, depending on the variety 39.8– 43.2% anthocyanins, 28.6–29.8% hydroxybenzoic acids, and 72.9–79.6% hydroxycinnamic acids as compared to the raw vegetable. For the other cooking procedures, we observed similar stabilities of phenolic acids. In addition, we observed an increase in the content of hydroxycinnamic acids, which were probably released from their conjugates. According to Wu & Prior (2005), red cabbage contains 23 different anthocyanins which are cyanidin derivatives highly conjugated with sugars (glucose and xylose) and acyl groups (caffeoyl, p-coumarolyl, feruloyl, p-hydroxybenzoyl, sinapoyl and oxaloyl). The major acylated anthocyanins in red cabbage are cyanidin 3-diglucoside-5-glucoside derivatives (Dyrby et al., 2001; Giusti & Wrolstad, 2003; Wu & Prior, 2005). In our study, we did not determine the changes in flavonols, since their content in fresh cabbage was below 0.3 mg ⁄ 100 g (data not presented). The effect of the cooking method on free radicals scavenging activity

The antioxidant activity, which reflects the ability of water phenolic extracts and vitamin C to scavenge the 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical monocation (ABTS•+), is shown in Fig. 3. The vitamin C activity expressed as TEAC was calculated on the basis of its concentration in the vegetables studied and the TEAC value which is 0.95 mm for ascorbic acid. We could not use ascorbic acid extract because it also contains phenolic compounds, which are extracted with meta-phosphoric acid. On the other hand, we did not find vitamin C in the phenolic extracts tested. The results clearly show a decrease in the ABTS•+ scavenging activity for all the samples cooked conventionally in boiling water (Fig. 3). After 10 and 20 min of the cooking with volume of water equal to the weight of cabbage, the radical scavenging activity of red cabbage decreased by

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Red cabbage antioxidants A. Podse˛dek et al.

a

Table 2 Hydroxybenzoic acids, hydroxycinnamic acids and anthocyanins contents (mg ⁄ 100 g) in raw and cooked red cabbage Koda

Kissendrup

Cooking method

Cooking time (min)

Cabbage: water (g ⁄ mL)

Hydroxybenzoic acidsb

Hydroxycinnamic acidsc

Raw cabbage In boiling water

– 20 20 10 20 5

– 1:2 1:1 1:1 – –

15.48 4.62 9.41 9.76 9.77 10.06

25.59 20.38 26.05 27.64 28.05 28.15

In steam

Anthocyaninsd

Hydroxybenzoic acidsb

Hydroxycinnamic acidsc

Anthocyaninsd

59.76 23.81 31.47 40.55 44.95 47.30

19.91 5.71 12.17 12.78 12.03 12.98

28.87 21.07 31.47 32.37 32.63 33.20

85.08 36.76 47.30 63.31 62.82 71.71

a

Data expressed as means of two samples. Content based upon gallic acid as a standard. c Content based upon chlorogenic acid as a standard. d Content based upon cyanidin 3-glucoside as a standard. b

Koda

TEAC (micromole g–1)

12 10 8 6 4 2 0 Fresh

1

2

3

5

Kissendrup

14 TEAC (micromole g–1)

4

12 10 8 6 4 2 0 Fresh

1 Phenolics

2

3

4

5

Vitamin C

Figure 3 Effect of different cooking methods on TEAC values for red cabbage phenolic compounds and vitamin C: 1, conventional cooking for 20 min, ratio vegetable:water = 1:2 (w ⁄ v); 2, conventional cooking for 20 min, ratio vegetable:water = 1:1 (w ⁄ v); 3, conventional cooking for 10 min, ratio vegetable:water = 1:1 (w ⁄ v); 4, steaming for 20 min; 5, steaming for 5 min.

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

38% and 42%, respectively. The 20 min cooking with two volumes of water caused a 50% decrease in the total TEAC value. However, steaming for 5 and 20 min affected the total TEAC value only by 5% and 20% for Koda, or 8% and 12% for Kissendrup, respectively. Amin & Lee (2005) observed that radical scavenging activity of red cabbage dropped from 72% to 61%, 29% and 32% after 5, 10 and 15 min of blanching, respectively. Previous studies have indicated the decrease in antioxidant activity during cooking of other Brassica vegetables, such as broccoli, Swamp cabbage and kale (Ismail et al., 2004; Lin & Chang, 2005). Zhang & Hamauzu (2004) reported that scavenging activity of broccoli towards 1,1-diphenyl-2-picryl-hydrazl (DPPH) radicals declined during conventional and microwave cooking and showed similar trends for these two cooking methods. However, other studies indicated that boiling, steaming and microwaving increased free radical scavenging activity by 40% for red cabbage and by 15.8–16.7% for broccoli (Wu et al., 2004; Turkmen et al., 2005). In the present study, the changes of antioxidant activity were caused by the losses of both phenolics and vitamin C. A comparison of TEAC values of phenolic extracts with TEAC values for vitamin C suggests that the phenolics are the major source of free radical scavenging activity of fresh and cooked red cabbage. The contribution of vitamin C to total TEAC of fresh cabbage was 28.5% for Koda and 20.9% for Kissendrup, respectively, and remained at a similar level in the cooked vegetable. Moreover, TEAC value for phenolic extracts was highly correlated both with total phenolics content determined by the Folin-Ciocalteu method (R2 = 0.98) and anthocyanins content determined by the HPLC method (R2 = 0.81). On the other hand, the correlation between hydroxybenzoic or hydroxycinnamic acids contents and TEAC values was rather poor (R2 = 0.17–0.59), therefore, total phenolics content

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and ⁄ or anthocyanins content can be used as indicators of free radical scavenging activity for fresh and cooked red cabbage. Conclusions

Our studies have shown that losses of vitamin C and phenolic compounds in red cabbage during cooking vary depending on the cooking treatment and, to a lesser extent, on the cabbage variety. Steaming is recommended to preserve the hydrophilic antioxidants content and to prevent a loss of their free radical scavenging activity in red cabbage. In order to minimise the losses of vitamin C and phenolic compounds during conventional cooking, red cabbage should be cooked with as small amount of water as possible. Moreover, in the case of conventional cooking, the amount of water and not the cooking time influenced retention of antioxidants. In addition, total phenolics content and ⁄ or anthocyanins content can be used as indicators of free radical scavenging activity for fresh and cooked red cabbage due to their high content and good correlation with TEAC value. The effects observed in our study should be helpful for calculating the dietary intake of hydrophilic antioxidants from cooked red cabbage. Acknowledgments

The authors acknowledge financial support from the State Committee for Scientific Research (PBZ-KBN094 ⁄ P06 ⁄ 2003 ⁄ 03). References Amin, I. & Lee, W.Y. (2005). Effect of different blanching times on antioxidant properties in selected cruciferous vegetables. Journal of the Science of Food and Agriculture, 85, 2314–2320. Bridle, P. & Timberlake, C.F. (1997). Anthocyanins as natural food colours – selected aspects. Food Chemistry, 58, 103–109. Chu, Y.-F., Sun, J., Wu, X. & Liu, R.H. (2002). Antioxidant and antiproliferative activities of common vegetables. Journal of Agriculture and Food Chemistry, 50, 6910–6916. Chun, O.K., Smith, N., Sakagawa, A. & Lee, C.Y. (2004). Antioxidant properties of raw and processed cabbages. International Journal of Food Sciences and Nutrition, 3, 191–199. Cohen, J., Kristal, R. & Stanford, J. (2000). Fruit and vegetable intakes and prostate cancer. Journal of the National Cancer Institute, 9, 61–68. Czarniecka-Skubina, E. (2002). Effect of the material form, storage and cooking methods on the quality of Brussels sprouts. Polish Journal of Food and Nutrition Sciences, 11, 52, 75–82. Dyrby, M., Westergaard, N. & Stapelfeldt, H. (2001). Light and heat sensitivity of red cabbage extract in soft drink model system. Food Chemistry, 72, 431–437. Giusti, M. & Wrolstad, R.E. (2003). Acylated anthocyanins from edible sources and their applications in food systems. Biochemical Engineering Journal, 14, 217–225. Gliszczynska-Swiglo, A. & Tyrakowska, B. (2003). Quality of commercial apple juices evaluated on the basis of the polyphenol

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content and the TEAC antioxidant activity. Journal of Food Science, 68, 1844–1849. Hassimotto, N.M.A., Genovese, I. & Lajolo, F.M. (2005). Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps. Journal of Agricultural and Food Chemistry, 53, 2928– 2935. Honer, K. & Cervellati, R. (2002). Measurements of the antioxidant capacity of fruits and vegetables using the BR reaction method. European Food Research and Technology, 215, 437–442. Howard, L.A., Wong, A.D., Perry, A.K. & Klein, B.P. (1999). b-carotene and ascorbic acid retention in fresh and processed vegetables. Journal of Food Science, 64, 929–936. Ismail, A., Marjan, Z.M. & Foong, C.W. (2004). Total antioxidant activity and phenolic content in selected vegetables. Food Chemistry, 87, 581–586. Karadeniz, F., Burdurlu, H.S., Koca, N. & Soyer, Y. (2005). Antioxidant activity of selected fruits and vegetables grown in Turkey. Turkish Journal of Agriculture and Forestry, 29, 297–303. Keum, Y.-S., Jeong, W.-S. & Kong, A.N.T. (2004). Chemoprevention by isothiocyanates and their underlying molecular signaling mechanisms. Mutation Research, 555, 191–202. Lin, Ch.-H. & Chang, Ch.-Y. (2005). Textural change and antioxidant properties of broccoli under different cooking treatments. Food Chemistry, 90, 9–15. Lisiewska, Z. & Kmiecik, W. (1996). Effects of level of nitrogen fertilizer, processing conditions and period of storage of frozen broccoli and cauliflower on vitamin C retention. Food Chemistry, 57, 267–270. Mazza, G. & Miniati, E. (1993). Anthocyanins in Fruits, Vegetables, and Grains. Pp, 283–288, Boca Raton, FL: CRC Press. Muller, H. (1997). Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Zeitschrift fur Lebensmittel – Untersuchung und – Forschung A, 204, 88–94. Peri, C. & Pompei, G. (1971). An assay of different phenolic fractions in wines. American Journal of Enology and Viticulture, 22, 2. Piccaglia, R., Marotti, M. & Baldoni, G. (2002). Factors influencing anthocyanin content in red cabbage (Brassica oleracea var capitata L f rubra(L) Thell). Journal of the Science of Food and Agriculture, 82, 1504–1509. Piironen, V., Syvaoja, E.-L., Varo, P., Salminen, K. & Koivistoinen, P. (1986). Tocopherols and tocotrienols in Finnish foods: vegetables, fruits, and berries. Journal of the Agricultural and Food Chemistry, 34, 742–746. Plumb, G.W., Lambert, N., Chambers, S.J. et al. (1996). Are whole extracts and purified glucosinolates from cruciferous vegetables antioxidants?. Free Radical Research, 25, 75–86. Proteggente, A.R., Pannala, A.S., Paganga, G. et al. (2002). The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin C composition. Free Radicals Research, 36, 217–233. Puupponen-Pimia, R., Hakkinen, S.T., Aarni, M. et al. (2003). Blanching and long-term freezing affect various bioactive compounds of vegetables in different ways. Journal of the Science of Food and Agriculture, 83, 1389–1402. Re, R., Pellergini, N., Protegente, A., Pannal, A., Yang, M. & RiceEvans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Steinkellner, H., Rabot, S., Freywald, C. et al. (2001). Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens. Mutation Research, 480–481, 285–297. Turkmen, N., Sari, F. & Velioglu, Y.S. (2005). The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chemistry, 93, 713–718. Uhl, M., Kassie, F., Rabot, S. et al. (2004). Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development

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of preneoplastic lesions induced by 2-amino-3-methylimidazol [4,5-f] quinoline (IQ) in liver and colon of Fisher 344 rats. Journal of Chromatography B, 802, 225–230. Vallejo, F., Tomas-Barberan, F.A. & Garcia-Viguera, C. (2003). Phenolic compound contents in edible parts of broccoli inflorescences after domestic cooking. Journal of the Science of Food and Agriculture, 83, 1511–1516. Wang, H., Cao, G. & Prior, R.L. (1997). Oxygen radical absorbing capacity of anthocyanins. Journal of Agricultural and Food Chemistry, 45, 304–309. Wu, X. & Prior, R.L. (2005). Identification and characterization of anthocyanins by high-performance liquid chromatography – elec-

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trospray ionization – tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains. Journal of Agricultural and Food Chemistry, 53, 3101–3113. Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D., Gebhardt, S.E. & Prior, R.L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agricultural and Food Chemistry, 52, 4026–4037. Zhang, D. & Hamauzu, Y. (2004). Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. Food Chemistry, 88, 503–509.

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International Journal of Food Science and Technology 2008, 43, 1778–1785

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Original article The effect of par-baking and frozen storage time on the quality of cup cake Mehmet Murat Karaog˘lu,* Halis Gu¨rbu¨z Kotancilar & Kamil Emre Gerc¸ekaslan Food Engineering Department, Faculty of Agriculture, Atatu¨rk University, 25240, Erzurum, Turkey (Received 4 July 2007; Accepted in revised form 19 October 2007)

Summary

The effects of frozen storage and initial baking time of par-baked cake on baking loss, volume, moisture, colour and textural properties of cake obtained after thawing and rebaking were investigated. Cakes, parbaked at 175 C for 15, 20 and 25 min, were stored at )18 C for 3, 6 and 9 months. After storage, parbaked cakes were thawed and rebaked at 175 C for 10, 15 and 20 min. Baking loss, moisture content, L and +b colour values, firmness, gumminess and chewiness of the resulting full-baked cakes were significantly affected by both par-baking and frozen storage time, while specific volume, cohesiveness, springiness and resilience values were significantly affected by frozen storage time. The increase in the time of frozen storage of the par-baked cake leads to a decrease in the quality of the rebaked cake, namely an increase of baking loss and cake crumb firmness, and a loss in the moisture content and specific volume. Moisture of cake crumb, L and +b colour values, firmness, gumminess and chewiness significantly increased as the par-baking time increased. However, regarding baking loss, specific volume, moisture content and textural properties, 3-month intermediate storage at )18 C and 20-min initial baking time gave the best result among the cakes produced by using the two-step baking procedure.

Keywords

Cake attributes, frozen storage, par-baking, physical properties, texture.

Introduction

Cakes are chemically and mechanically leavened bakery products that are consumed in high quantities by all ages. The quality of industrial cakes may dramatically change depending on shelf-life and tolerance to staling. The shelf-life of cakes is generally from 1 to 4 weeks or more, depending on formulation, packaging, water activity and storage temperature. High-quality cakes have various properties, including high volume and shelf-life, low cake crumb firmness, and uniform crumb structure. These properties depend on the balanced formulas, aeration of cake batters, stability of fluid batters in the early stage of baking and thermal-setting stage. In general, cakes with higher specific volumes have lower firmness values (Gelinas et al., 1999; Sheetharaman et al., 2002; Dog˘an et al., 2007; Gomez et al., 2007). The starch properties such as gelatinization temperature, paste viscosity and retrogradation of starch affect cake baking. Consequently, the quality of finished cake can markedly be influenced by both time and temperature of baking and storage, because baking *Correspondent: Fax: +90 442 2360958; e-mail: [email protected];[email protected]

and storage conditions modify the pasting and crystallization properties of starch (Mizukoshi et al., 1979, 1980; Shittu et al., 2007). Bakery products during storage are subject to changes, resulting in adverse effects on quality, ranging from staling to total spoilage. The shelf-life of these products, defined as the period of time during which quality losses do not exceed a tolerated level, can be decisively influenced by its storage conditions (Kotsianis et al., 2002). The staling of bakery products is a process of chemical and physical changes such as moisture redistribution, drying, starch retrogradation, increased firmness as well as loss of aroma and flavour (He & Hoseney, 1990). Cake staling has been connected with the intrinsic firming process of the cake crumb and the migration of moisture from the crumb to the drier regions of the cake (Guy, 1983). In order to delay staling in cakes, numerous studies have been conducted by several researchers (Lahtinen et al., 1998; Rodriguez et al., 2002; Seyhun et al., 2003; Gomez et al., 2007; Ji et al., 2007). But, staling is still a significant limiting factor in the keeping quality of cakes. A different approach for increasing the shelf-life of bakery products is to modify the baking method (Karaog˘lu & Kotancılar, 2006). In this sense, partial

doi:10.1111/j.1365-2621.2007.01698.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Par-baking and frozen storage time on cup cake quality M. M. Karaog˘lu et al.

baking and further frozen storage and rebaking processes help keep the cakes fresh all time. The par-baking process includes three stages: a par-baking phase, a storage period, and a reheating or rebaking step performed by the consumer prior to consumption. During the prebaking or par-baking step, the products are baked until a structure sets that makes the products suitable for storage conditions. Par-baked products are packaged and frozen. Then, reheating or rebaking reverses certain product properties typically developed during storage, generating the characteristics of a freshly baked product (Grau et al., 1999; Vulicevic et al., 2004). Product formulation, processing, packaging and storage conditions affect storage life of bakery products (Vulicevic et al., 2004). Cake is not a product of daily consumption such as bread, so long-term storage of partially baked cake is necessary and frozen storage could be preferred to positive temperature storage with respect to quality of cake. The chemical composition of the cake influences the quality preservation and crumb properties. Moreover, these are also affected by the cake composition during the freezing process and storage. Par-bakery products are thus often frozen to delay staling and prolong the shelf-life (Barcenas et al., 2004; Ribotta & Le Bail, 2007). The lower storage temperature remarkably reduces the rate of crumb hardening resulting from staling (Cauvain, 1998). Much research has been undertaken regarding the qualitative and textural properties of fresh cakes. However, no information is available on quality and storage life of par-baked and frozen-stored cakes. The objective of the present study was to evaluate textural properties such as firmness, cohesiveness, springiness, gumminess, chewiness, resilience, and other quality characteristics such as specific volume, baking loss, crumb moisture and crust colour for experimental par-baked cakes, rebaked and frozen-stored up to 9 months at frozen temperature. Materials and methods

Materials

Cake flour containing 28.5% wet gluten, 13.5% moisture, 10.6% protein and 0.55% ash was supplied by Birlik Un (Erzurum, Turkey). All the other ingredients (fresh eggs, milk, semisolid margarine, baking-powder, salt and sugar) were obtained from local commercial markets. Methods Cake baking

The cakes were prepared according to the procedure reported by Karaog˘lu & Kotancılar (2007). A cake batter recipe containing 100% cake flour, 90% sugar, 40% egg white, 60% milk, 40% margarine, 1.5% salt

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

and 1.7% baking powder (all percentages are given on flour weight basis). A three-stage mixing method was used to prepare the batter using a mixer (Arc¸elik, ARK 99 RS, Istanbul, Turkey). At first, the egg white and sugar were mixed at speed 8 for 3 min. Then, milk and margarine were added and mixed at the same speed for 2 min. Finally, flour and baking powder were added and mixed at speed 6 for 3 min. Then for production of parbaked cake (baking phase I), 60 g of batter was placed in greased pans (diameter 75 mm and depth 35mm) and baked at 175 C for 15, 20, 25 min. Par-baked cakes were cooled for 1 h at room temperature (20 C), packed in double-folded polyethylene pouches and stored for 3, 6, 9 months at )18 C (intermediate storage). After storage, frozen cake samples were thawed overnight at refrigerator temperature (4 C), and acclimated for 2 h at room temperature (20 C). Then, par-baked cakes were re-baked in the same oven (175 C) for 20, 15, 10 min. Baking times of the parbaked cakes were completed to baking time of the control cakes by rebaking process (baking phase II). Control cakes were baked at 175 C for 35 min. Flour analysis

Protein, ash and moisture contents were determined according to American Association of Cereal Chemists (AACC)-Approved Methods 46-12, 08-01 and 44-15A, respectively (Anonymous, 1983). Protein and ash were expressed on a dry weight basis. Wet gluten content were determined according to the International Association for Cereal Science and Technology (ICC) Standard Method 137-1 (Anonymous, 2000) using the Glutomatic 2200 system (Perten, Huddinge, Sweden). Zeleny sedimentation volumes were determined according to the AACC method 56-81. Cake analysis

The cakes were weighed after removal from the pan and cake volumes were determined by the rape seed displacement method. The specific volume was calculated by the ratio of volume to weight (Lee et al., 1982). Colour was measured using a Minolta Colorimeter CR200 (Minolta Camera Co., Osaka, Japan) (Elgu¨n et al., 1999). Crust colour was checked at five different points on each cake and every point was measured three times. Results were expressed in the CIE L*a*b* colour space. Symmetry index was determined by the modified AACC-10 91 procedure (Anonymous, 1967). Texture analyses

In cake texture determinations, the crust was removed, and each cake crumb was sliced into four (40 · 40 · 20) mm samples to be measured. Textural parameters were measured using a texture analyzer (TA-XTplus; Stable Micro Systems, Godalming, Surrey, UK) equipped with a 25-mm probe (P ⁄ 25) and according to texture profile

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analysis (TPA). The TPA method was carried out under these conditions: pre-test speed: 1 mm s)1, test speed: 2 mm s)1, post-test speed: 1 mm s)1, distance: 8 mm, trigger type: outo-20 g, time: 3 s. Texture parameters such as firmness, cohesiveness, springiness, chewiness, gumminess and resilience were calculated from a force– time graph according to the methods described by Carr & Tadini (2003). Firmness was defined as the peak force during the first compression cycle and cohesiveness as the ratio of the positive force area during the second compression portion to that during the first compression. Springiness was defined as the distance (mm) the sample recovers after the first compression. Chewiness is work, that is the product of hardness · cohesiveness · springiness, mJ. Gumminess is hardness · cohesiveness.

general, baking loss of rebaked cakes were greater than that of the once-baked (control group) standard product. Baking loss rate of control group and rebaked cakes which had 3-month storage time was the lowest (approximately 16%) but that of rebaked cakes parbaked for 15 min and stored for 9 months at )18 C was the highest (23.90%) in that. During storage, baking loss of all rebaked cakes was not significantly different (P < 0.01) from that of control group in the first 3 month. Rebaked samples with 15-min initial baking time lost less weight during storage than with 20 and 25 min initial baking time (Table 2). That is, long parbaking times during first baking phase led to lower values for baking loss than short par-baking time. High baking loss in cakes rebaked after par-baking and storing can be explained by high moisture loss in cakes baked in two stages (par-baking and storing) when compared with control samples (baked in one stage), as more water is evaporated during the processes such as par-baking, storing and rebaking (Karaog˘lu & Kotancılar, 2007). Anyway, correlation analysis revealed a negative correlation between baking loss and specific volume, moisture content of crumb (Table 3). That is, baking loss increased as moisture content and specific volume decreased, indicating an inverse relationship. Specific volume of rebaked cakes, which were parbaked for 10, 15 and 20 min and stored for 3 months at )18 C, were statistically the same as that of control cake (Tables 1 and 2). All rebaked cakes, which were stored for 6 and 9 months at )18 C, had lower specific volume than control group and rebaked cakes stored for 3 months. The specific volume of cakes stored for 6 and 9 months did not differ significantly (P < 0.05). The change in par-baking time did not significantly affect specific volume of rebaked cakes. The destabilization of

Statistical analysis

The experiments were carried out in duplicate and the analysis was performed in triplicate. SPSS 10.0 software for Windows was used to perform statistical analyses (SPSS., 1999). Differences in rebaked cakes after parbaked and stored because of par-baking time and intermediate storage time were tested for significance using analysis of variance techniques (anova). Duncan’s multiple-range test was used when anova indicated significant difference in means. A level of significance of P < 0.05 was used throughout the analysis. All data were presented as the mean ± SE (standard error). Result and discussion

Par-baking and intermediate storage time had a significant effect on baking loss of cakes (Tables 1 and 2). In

Table 1 Effects of par-baking and intermediate storage time on baking loss, specific volume, moisture of cake crumb and crust colour of rebaked cakes (mean ± SE)a Colour of cake crust PBT (min)

15

20

25

IST (month) C 3 6 9 3 6 9 3 6 9 P

n

Baking loss (%)

Specific volume (cm3 g)1)

2 2 2 2 2 2 2 2 2 2

16.41 16.99 22.32 23.90 16.18 17.23 19.47 16.28 17.80 18.96 **

2.52 2.53 2.35 2.32 2.51 2.33 2.33 2.57 2.24 2.21 **

± ± ± ± ± ± ± ± ± ±

0.16e 0.01e 0.02b 0.82a 0.00e 0.92e 0.37c 0.02e 0.17de 0.76cd

± ± ± ± ± ± ± ± ± ±

0.04a 0.12a 0.03b 0.02b 0.03a 0.01b 0.02b 0.01a 0.01b 0.03b

Moisture of cake crumb (%)

L

21.5 20.5 18.5 17.50 20.5 20.0 18.0 21.0 20.5 19.0 **

47.07 45.84 44.95 41.98 47.83 46.63 45.01 48.22 46.73 43.72 **

± ± ± ± ± ± ± ± ± ±

0.5a 0.4ab 0.5cd 0.5d 0.5ab 0.0ab 0.2d 0.0a 0.5ab 0.0bcd

+a ± ± ± ± ± ± ± ± ± ±

0.15ab 0.40abc 0.25bc 1.25d 0.10a 0.95ab 1.14bc 0.56a 1.21ab 0.53cd

17.38 15.84 13.95 13.12 15.61 15.09 15.24 15.08 14.66 14.76 **

+b ± ± ± ± ± ± ± ± ± ±

0.45a 0.57ab 0.66cd 0.88d 0.26bc 0.23bc 0.25bc 0.16bc 0.54bcd 0.48bcd

21.65 17.99 17.66 16.92 20.52 20.10 17.00 21.09 19.84 17.05 **

± ± ± ± ± ± ± ± ± ±

1.11a 0.02cd 0.02cd 0.31d 0.43ab 0.56ab 0.02d 0.84ab 0.33bc 0.47d

a

Mean values with different letters in the same column are statistically different at (P < 0.05). C, control group; PBT, par-baking time; IST, intermediate storage time. **P < 0.01.

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Par-baking and frozen storage time on cup cake quality M. M. Karaog˘lu et al.

Table 2 The general effects of par-baking and intermediate storage time on baking loss, specific volume, moisture of cake crumb and crust colour of rebaked cakes (mean ± SE)a Colour of cake crust n

Baking loss (%)

Par-baking time 15 min 8 19.91 20 min 8 17.33 25 min 8 17.36 P ** Intermediate storage time 0 (C) 6 16.41 3 months 6 16.49 6 months 6 19.12 9 months 6 20.78 P **

Specific volume (cm3 g)1)

Moisture of cake crumb (%)

L

+a

+b

± 1.24a ± 0.52b ± 0.44b

2.43 ± 0.04a 2.42 ± 0.03a 2.39 ± 0.05a

19.5 ± 0.6b 20.0 ± 0.5ab 20.5 ± 0.3a *

44.96 ± 0.75b 46.64 ± 0.48a 46.44 ± 0.68a **

15.07 ± 0.67a 15.83 ± 0.36a 15.47 ± 0.45a

18.56 ± 0.52b 19.82 ± 0.60a 19.82 ± 0.63a *

± ± ± ±

2.52 2.54 2.31 2.29 **

21.5 20.6 19.6 18.6 **

47.07 47.30 46.10 43.57 **

17.38 15.51 14.56 14.37 **

21.65 19.87 19.08 16.99 **

0.07c 0.16c 1.04b 1.03a

± ± ± ±

0.02a 0.03a 0.02b 0.02b

± ± ± ±

0.2a 0.2a 0.4b 0.4c

± ± ± ±

0.08a 0.50a 0.54a 0.71b

± ± ± ±

0.20a 0.21b 0.31c 0.48c

± ± ± ±

0.53a 0.64b 0.49b 0.14c

a Mean values with different letters in the same column are statistically different at (P < 0.05). C, control group. *P < 0.05; **P < 0.01.

Table 3 Correlation coefficients of baking loss, specific volume, moisture content and textural properties of rebaked cakes after par-baking and

storing

Baking loss (%) Specific volume (cm3 g)1) Moisture of cake crumb (%) Firmness (N) Cohesiveness Springiness Gumminess Chewiness

Specific volume (cm3 g)1)

Moisture of cake crumb (%)

Firmness (N)

Cohesiveness

Springiness

Gumminess

Chewiness

Resilience

)0.530** – – – – –

)0.817** )0.574** – – – – –

0.475* )0.707** )0.660** – – – – –

)0.703** 0.592** 0.844** )0.737** – – – –

)0.468* 0.276 0.388 )0.233 0.481* – – –

0.298 )0.601** )0.502* 0.892** )0.606** )0.186 – –

0.258 )0.589** )0.468* 0.883** )0.562** )0.096 0.995** –

)0.773** 0.721** 0.878** )0.812** 0.944** 0.512* )0.692** )0.651**

Correlation is significant at the *0.05 and **0.01 level.

air cells during par-baking, storing and rebaking processes would result in lowered specific volume. However, 15-min par-baking and 3-month intermediate storage time resulted in satisfactory cake volume. The decrease in specific volume can be correlated to weight loss, moisture loss, and gas escape from cake during longer storage period, par-baking and rebaking processes. However, in this study, baking loss and moisture loss were more pronounced than volume shrinkage. The specific volume of rebaked cakes showed a significant positive correlation with moisture of cake crumb, cohesiveness and resilience, while a significant negative correlation was obtained between specific volume and textural properties such as firmness, gumminess and chewiness (Table 3). As shown Tables 1 and 2, an increase in frozen storage period of par-baked cakes steadily decreased crumb moisture content of rebaked cakes. The 3-month intermediate storage time caused a slight decrease in

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moisture content. The crumb moisture content of rebaked cakes was significantly lower than that of control cake, except that of rebaked cake par-baked for 25 min and stored for 9 months. As par-baking time of rebaked cakes increased from 15 to 25 min, crumb moisture content of rebaked cakes significantly increased. Moisture losses from cake samples were the result of the potential difference in water vapour pressure between the storage atmosphere and the cake. Moisture loss in rebaked cakes can also be attributed a reduction of water retention capacity of cake constituents during par-baking and frozen storage (Karaog˘lu & Kotancılar, 2007). The high moisture loss in twice-baked cakes has also been reported by other researches (Grau et al., 1999; Karaog˘lu & Kotancılar, 2007). Therefore, we suggest here that it is necessary to do further researches to make modifications which will increase the moisture retention capacity of finished product in parbaking process. Because, moisture content is the most

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(a)

Firmness (N)

1782

(b)

15

(c)

15

15

10

10

10

5

5

5

0

0 C

3

6

9

0 C

3

6

9

C

Intermediate storage time (month)

important factor affecting the crumb softness of cakes (Lahtinen et al., 1998). In our study, a significant negative correlation was found between moisture content of cake crumb and cake crumb firmness (P < 0.01). Thus, crumb moisture content seems to be an important parameter explaining the textural properties of rebaked cakes after par-baking and storing. Par-baking time and intermediate storage time significantly (P < 0.01) affected the colour values of rebaked cakes (Tables 1 and 2). In general, the surface brightness (L value), yellowness (+b value) and redness (+a value) of rebaked cakes were decreased with increasing intermediate storage time. Similar results were obtained by Karaog˘lu & Kotancılar (2007), who studied quality and textural behaviour of par-baked and rebaked cake during refrigerator storage. This is expected result, because the rate of brown pigment formation decreases with intermediate frozen storage time. Rebaked cakes with 20 and 25 min par-baking and 6-month storage time had statistically the same L colour value as control cake. L and +b colour values of rebaked cake crust increased up to a par-baking time of 20 min and then (25-min par-baking time) remained statistically unchanged. Colour is one of the most important appearance attributes of bakery foods, as it influences consumer acceptability. Colour is also used for process controlling, because brown pigments increase as the browning and caramelization reactions progress (Moss & Otten, 1989; Kahyaog˘lu & Kaya, 2006). It has been reported that instrumental measurement of baked products’ colour is a predictable quality check that could be used in determining the effects of ingredient or product formulation, process variable as well as storage conditions of baked products (Gallagher et al., 2003; Erkan et al., 2006). Cake crumb firmness was evaluated on rebaked cakes after 24, 48 and 72 h of ageing at room temperature (20 C) (Fig. 1). The crumb firmness of cakes, rebaked

International Journal of Food Science and Technology 2008

3

6

9

Figure 1 Effect of par-baking times [15 min (o), 20 min (h), 25 min (D)] and intermediate storage time on changes in hardness of cake crumb stored at room temperature for (a) 24, (b) 48 and (c) 72 h after rebaking (C: control group).

after par-baking and frozen storage, was significantly (P < 0.01) higher than that of the control group (baked with one step), which was expected because of the significantly lower moisture content of the control cake. The inverse relationship between the firmness and the crumb moisture has been previously reported (Barcenas & Rosell, 2006; Karaog˘lu & Kotancılar, 2007). Water acts as a plasticizer in the bakery product; the decrease in the moisture content supports the formation of hydrogen bonds among the starch polymers or between the starch and the proteins yielding greater firmness (Karaog˘lu & Kotancılar, 2006). The crumb firmness of rebaked cakes showed a significant (P < 0.01) increase with the duration of intermediate frozen storage. During storage, a similar increase in the firmness was observed in breads, produced with par-baking and rebaking method (Vulicevic et al., 2004; Karaog˘lu, 2006a,b; Karaog˘lu & Kotancılar, 2006). The increase in the firmness as a consequence of frozen storage indicates that the damage of the cake constituents produced during frozen storage might produce some effects during the rebaking and later cooling that favour the firmnessl; 15 and 20 min par-baking time showed statistically the same effect on firmness of rebaked cake, while 25 min of par-baking time increased firmness (Table 4). For this reason, it can be advised that par-baking (initial baking) time should not be very long regarding the cake crumb firmness. On the other hand, the first baking step should not be very short. If not, the cake crumb is not stable enough to prevent volume loss through shrinkage after par-baking stage. Firmness or softness is the texture property, which has attracted most attention in bakery product assessment because of its close association with human perception of freshness (Car & Tadini, 2003; Giannou & Tzia, 2007). The increase in cake crumb firmness was an expression of the staling process during which modifications in the starch molecule took place. Cake crumb firmness showed a positive correlation with

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Par-baking and frozen storage time on cup cake quality M. M. Karaog˘lu et al.

a

Table 4 The general effects of par-baking and intermediate storage time on textural properties of rebaked cakes (mean ± SE) n Par-baking time 15 min 8 20 min 8 25 min 8 P Intermediate storage time 0 (C) 6 3 months 6 6 months 6 9 months 6 P

Firmness (N)

Cohesiveness

Springiness

Gumminess

Chewiness

Resilience

6.48 ± 0.49b 6.47 ± 1.05b 7.86 ± 0.71a **

0.590 ± 0.017a 0.604 ± 0.017a 0.601 ± 0.016a

0.831 ± 0.007a 0.832 ± 0.007a 0.835 ± 0.004a

3.77 ± 0.17c 4.22 ± 0.49b 4.68 ± 0.35a **

3.13 ± 0.14c 3.51 ± 0.38b 3.92 ± 0.30a **

0.269 ± 0.012a 0.275 ± 0.012a 0.274 ± 0.010a

4.82 6.49 7.27 9.17 **

0.640 0.617 0.610 0.526 **

0.823 0.856 0.840 0.813 **

3.09 4.05 4.43 5.32 **

2.54 3.47 3.74 4.33 **

0.306 0.294 0.267 0.225 **

± ± ± ±

0.06d 0.50c 0.43b 0.68a

± ± ± ±

0.008a 0.004b 0.008b 0.005c

± ± ± ±

0.000c 0.003a 0.002b 0.004d

± ± ± ±

0.07d 0.36c 0.24b 0.71a

± ± ± ±

0.06c 0.29b 0.20b 0.30a

± ± ± ±

0.002a 0.002b 0.004c 0.004d

a Mean values with different letters in the same column are statistically different at (P < 0.05). C, control group. **P < 0.01.

a

Table 5 Effects of par-baking and intermediate storage time on textural properties of rebaked cakes (mean ± SE) PBT (min)

15

20

25

IST (months) C 3 6 9 3 6 9 3 6 9 P

n 2 2 2 2 2 2 2 2 2 2

Cohesiveness

Springiness

Gumminess

Chewiness

Resilience

0.640 0.609 0.599 0.514 0.611 0.634 0.529 0.632 0.598 0.535 **

0.823 0.858 0.841 0.803 0.860 0.837 0.809 0.850 0.842 0.827 **

3.09 3.68 3.97 4.34 3.31 4.19 6.28 5.16 5.14 5.35 **

2.54 3.16 3.34 3.49 2.86 3.57 5.08 4.39 4.33 4.43 **

0.306 0.290 0.265 0.216 0.292 0.279 0.222 0.298 0.257 0.238 **

± ± ± ± ± ± ± ± ± ±

0.018a 0.001bc 0.001c 0.009d 0.003bc 0.001ab 0.008d 0.003ab 0.004c 0.007d

± ± ± ± ± ± ± ± ± ±

0.001de 0.006a 0.005bc 0.001f 0.004a 0.004bc 0.003ef a0.008b 0.002bc 0.006cd

± ± ± ± ± ± ± ± ± ±

0.17e 0.14de 0.03cd 0.02c 0.04de 0.38c 0.42a 0.17b 0.02b 0.05b

± ± ± ± ± ± ± ± ± ±

0.14e 0.14cd 0.04cd 0.02c 0.03de 0.27c 0.35a 0.10b 0.03b 0.07b

± ± ± ± ± ± ± ± ± ±

0.003a 0.005bc 0.000d 0.001f 0.003b 0.006c 0.001f 0.001ab 0.004d 0.001e

a Mean values with different letters in the same column are statistically different at (P < 0.05). C, Control group; PBT, par-baking time; IST, intermediate storage time. **P < 0.01.

baking loss and a negative correlation with specific volume and crumb moisture (Table 3). The internal resistance of cake crumb is evaluated by cohesiveness which is a characteristic of mastication. Cohesiveness is defined as ‘how well the product withstands a second deformation relative to how it behaved under the first deformation’ (Wang et al., 2006). Compared with control cake, cohesiveness values were found to be significantly lower for all rebaked cakes. Although par-baking times did not significantly influence the rebaked cake cohesiveness, cohesiveness value of rebaked cake decreased significantly as the intermediate frozen storage time increased (Tables 4 and 5). The observed decrease in cohesiveness may be explained by a transformation in the cake crumb during frozen storage resulting in weak internal bonds which stabilizes the cake structure. Because, once these bonds are broken, they can not reform.

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Springiness is a measurement of how much the cake springs back after being compressed one time and it can be defined as the elasticity of the cake. All treatments (par-baking, frozen storage and rebaking) significantly improved springiness of cakes in comparison with control cake, except the rebaked cake after 15- and 20-min par-baking and 9-month frozen storage (Table 5). A similar result was also obtained in rebaked cakes, par-baked for 15, 20, 25 min and stored for 30, 60, 90 days at refrigerator temperature (Karaog˘lu & Kotancılar, 2007). The highest springiness was obtained when cake was par-baked for 15 and 20 min and stored for 3 months at )18 C. During storage, cake springiness increased significantly up to 9 months at frozen conditions and then significantly decreased (Table 4). Gumminess and chewiness increased significantly (P < 0.01) with increasing initial (par-baking) time and intermediate storage time (Tables 4 and 5).

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Gumminess is a parameter dependent on hardness and cohesiveness; chewiness depends on gumminess and springiness. That is, both, gumminess and chewiness are parameters dependent on firmness. For this reason, these values in rebaked cakes followed a similar trend with the firmness. For consumer, gumminess presents the density that persists throughout chewing; chewiness describes how long it takes to chew a sample of cake to the consistency suitable for swallowing. High values are associated with dense, rubbery characteristics, certainly not desirable in rebaked cakes (Svec & Hruskova, 2004; Karaog˘lu & Kotancılar, 2007). Intermediate storage time affected the resilience values of rebaked cakes significantly, while the effect of initial baking time was not statistically significant. All rebaked cakes had lower resilience values than that of control cake and resilience of rebaked cakes decreased in parallel to the increase in intermediate storage time. Gumminess and chewiness were negatively correlated with specific volume and crumb moisture content, while resilience was positively correlated with specific volume and crumb moisture content (Table 3). Conclusions

The two-step baking procedure was successfully used to bakery products such as cake. The par-baking time of cake prior to their freezing and intermediate frozen storage time have a significant effect on their quality after thawing and rebaking. The increase in the time of frozen storage of the par-baked cake leads to a decrease in the quality of the resulting full baked cake, namely an increase of baking loss and the cake crumb firmness, and a loss in the moisture content and specific volume. Moisture content is the most important factor affecting the crumb softness of cakes. Therefore, we suggest here that it is necessary to do further researches to make modifications which will increase the moisture retention capacity of cake produced by the two-step baking procedure. Although specific volume and textural properties such as cohesiveness, springiness and resilience did not significantly change, moisture of cake crumb, L and +b colour values, firmness, gumminess and chewiness increased significantly as the par-baking time increased. It can be advised that par-baking (initial baking) time should not be very long regarding the cake crumb firmness. On the other hand, the first baking step should not be very short. If not, the cake crumb is not stable enough to prevent volume loss through shrinkage after par-baking stage. The results obtained in this study will be helpful for future studies of bakery products. References Anonymous, (1967). Standard Methods of The International Association for Cereal Chemists. Detmold: IACC.

International Journal of Food Science and Technology 2008

Anonymous (1983). Approved Methods of the AACC. Methods 10-09, 44-15, 44-18. St Paul, MN: American Association of Cereal Chemists (AACC). Anonymous. (2000). The Standard Methods of the ICC, 2000 ed. ICC standard No. 137 ⁄ 1, approved 1982 and revised 1994.Vienna: International Association for Cereal Science and Technology. Barcenas, M.E. & Rosell, C.M. (2006). Effect of frozen storage time on the bread crumb and aging of par-baked bread. Food Chemistry, 95, 438–445. Barcenas, M.E., Benedito, C. & Rosell, C.M. (2004). Use of hydrocolloids as bread improvers in interrupted baking process with frozen storage. Food Hydrocolloids, 18, 769–774. Carr, L.G. & Tadini, C.C. (2003). Influence of yeast and vegetable shortening on physical and textural parameters of frozen part baked French bread. Lebensmittel-Wissenschhaft und-Technologie, 36, 609– 614. Cauvain, S.P. (1998). Improving the control of staling in frozen bakery products. Trends in Food Science and Technology, 9, 56–61. _ Dog˘an, I.S., Javidipour, I. & Akan, T. (2007). Effects of interesterified palm and cottonseed oil blends on cake quality. International Journal of Food Science and Technology, 42, 157–164. Elgu¨n, A., Ertugay, Z., Certel, M. & Kotancılar, H.G. (1999). Guide Book for Analytical Quality Control and Laboratory for Cereal and Cereal Products. Erzurum: Atatu¨rk Univ. Publication No: 335. Pp. 245 Erkan, H., C¸elik, S., Bilgi, B.D. & Ko¨ksel, H. (2006). A new approach fort he utilization of barley in food products: barley tarhana. Food Chemistry, 97, 12–18. Gallagher, E., Kunkel, A., Gormley, T.R. & Erendt, E.K. (2003). The effect of dairy powder addition of loaf and crumb characteristics, and on shelf-life (intermediate and long term) of gluten-free breads stored in modified atmosphere. European Journal of Food Research, 218, 44–48. Gelinas, P., Roy, G. & Guillet, M. (1999). Relative effects of ingredients on cake staling based on an accelerated shelf-life test. Journal of Food Science, 64, 937–940. Giannou, V. & Tzia, C. (2007). Frozen dough bread: Quality and textural behavior during prolonged storage – Prediction of final product characteristics. Journal of Food Engineering, 79, 929–934. Gomez, M., Ronda, F., Caballero, P.A., Blanco, C.A. & Rosell, C.M. (2007). Functionality of different hydrocolloids on the quality and shelf-life of yellow layer cakes. Food Hydrocolloids, 21, 167–173. Grau, H., Wehrle, K. & Arendt, E.K. (1999). Evaluation of a two-step baking procedure for convenience sponge cakes. Cereal Chemistry, 76, 303–307. Guy, R.C.E. (1983). Factors affecting the staling of Maderia slab cake. Journal of Scieince of Food and Agriculture, 34, 477–491. He, H. & Hoseney, R.C. (1990). Changes in bread firmness and moisture during long-term storage. Cereal Chemistry, 67, 603–605. Ji, Y., Zhu, K., Qian, H. & Zhou, H. (2007). Staling of cake prepared from rice flour and sticky rice four. Food Chemistry, 104, 53–58. Kahyaog˘lu, T. & Kaya, S. (2006). Modelling of moisture, colour and texture changes in sesame seeds during the conventional roasting. Journal of Food Engineering, 75, 167–177. Karaog˘lu, M.M. (2006a). Effect of Initial Baking And Storage Time On Pasting Properties And Aging Of Par-Baked And Rebaked Rye Bread. International Journal of Food Properties, 9, 583–596. Karaog˘lu, M.M. (2006b). Effect of baking procedure and storage on the pasting properties and staling of part-baked and rebaked wheat bran bread. International Journal of Food Science and Technology, 41(supplement 2), 77–82. Karaog˘lu, M.M. & Kotancılar, H.G. (2006). Effect of par-baking, storage and rebaking process on the quality of white pan bread. International Journal of Food Science and Technology, 41(supplement 2), 108–114. Karaog˘lu, M.M. & Kotancılar, H.G. (2007). Quality and textural behaviour of par-baked and rebaked cake during prolonged storage. International Journal of Food Science and Technology, (in press).

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Par-baking and frozen storage time on cup cake quality M. M. Karaog˘lu et al.

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Seyhun, N., Sumnu, G. & Sahin, S. (2003). Effects of different emulsifier types, fat contents, and gum types on retarding of staling of microwave baked cakes. Die Nahrung, 47, 248–251. Sheetharaman, K., Chinnapa, N., Waniska, D. & White, P. (2002). Changes in textural, pasting and thermal properties of wheat buns and tortillas during storage. Journal of Cereal Science, 35, 215– 223. Shittu, T.A., Raji, A.O. & Sanni, L.O. (2007). Bread from composite cassava-wheat flour: I. Effect of baking time and temperature on some physical properties of bread loaf. Food Research International, 40, 280–290. SPSS. (1999). SPSS for Windows Release 10.01. Chicago, IL: SPSS Inc. Svec, I. & Hruskova, M. (2004). Image data of crumb structure of bread from flour of Czech spring wheat cultivars. Czech Journal of Food Science, 22, 133–142. Vulicevic, I.R., Abdel-Aal, E.S.M., Mittal, G.S. & Lu, X. (2004). Quality and storage life of par-baked frozen breads. LebensmittelWissenschaft und -Technologie, 37, 205–213. Wang, R., Zhou, W., Yu, H.H. & Chow, W.F. (2006). Effects of green tea extract on the quality of bread made from unfrozen and frozen dough process. Journal of the Science of Food and Agriculture, 86, 857–864.

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Original article Effect of preliminary and culinary processing on amino acid content and protein quality in frozen French beans Waldemar Kmiecik, Zofia Lisiewska,* Jacek Słupski & Piotr Ge˛bczyn´ski Department of Raw Materials and Processing of Fruit and Vegetables, Agricultural University of Krakow, 122 Balicka, 30-149 Krakow, Poland (Received 10 May 2006; Accepted in revised form 30 October 2007)

The aim of the study was to evaluate the content of amino acids and protein quality of French bean pods. The investigated material consisted of the raw vegetable, fresh pods cooked to consumption consistency, and two kinds of frozen products stored for 12 months at )20 C and then prepared for consumption: frozen beans obtained using the traditional method (blanching before freezing) and frozen products of the ready-toeat type (cooking before freezing). A comparison of the amino acid content in the product prepared for consumption showed that the lowest quantities were found in French beans obtained using the traditional method; products obtained using the modified technology and beans cooked directly after harvest had similar levels of most amino acids. The content of amino acids in 16 g N was less varied than in 100 g of the product. The protein in all the three products prepared for consumption as well as that in fresh bean pods hardly differs, as confirmed by the values in the essential amino acid index (EAA). The first limiting amino acid was methionine with cystine and the second was lysine.

Summary

Keywords

Amino acids, culinary treatment, freezing, French bean

Introduction

As a result of their chemical composition, which determines nutritive and functional quality as well as sensory traits, leguminous vegetables are recommended as indispensable constituents of the human diet (Iqbal et al., 2006). The edible parts of most leguminous vegetables are seeds. French bean is an exception because its edible part is the pod. The seeds, whose size approximates that of wheat seeds, constitute about 10% of the pod weight. However, French bean is regarded as a vegetable of high nutritive value, containing protein, carbohydrates, minerals and B group vitamins (Dinelli et al., 2006). The season for harvesting raw bean pods is short and in order to prolong their consumption, the food industry supplies them in the form of frozen and canned products. Frozen beans retain the constituents of the raw material to a higher degree than canned products (Lisiewska et al., 1999; Korus et al., 2003). However, frozen beans produced using the traditional method have to undergo culinary processing before consumption, resulting in further losses of chemical constituents and additional time being spent in the preparation of meals at home. *Correspondent: Fax: +48 12 6624757; e-mail: [email protected]

To meet the requirements of consumers preferring to eat a home, the food industry supplies products of the do-it-for-me or ready-to-eat type (Sloan, 2005), which, in this case of frozen products, require only defrosting and heating in a microwave oven (Datta et al., 2005). As microwave heating does not involve water and is faster than other methods of heating, it would be expected to cause less damage than hot water and steam. The aim of the present study was to evaluate amino acid content and protein quality in French bean pods. The investigation included: (i) raw material; (ii) fresh pods cooked to consumption consistency; (iii) frozen beans produced using the traditional method (pods blanched before freezing, and then cooked to consumption consistency); and (iv) the do-it-for-me type product (pods cooked to consumption consistency before freezing and then defrosted and heated to consumption temperature in a microwave oven). Before being prepared for consumption, frozen products were stored for 12 months at )20 C. Material and methods

Material

The investigated material was composed of pods of French beans Phaseolus vulgaris cv. Delfina. French bean was grown in the experimental field of the

doi:10.1111/j.1365-2621.2007.01702.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Amino acids in French beans W. Kmiecik et al.

Department. The harvest of French bean was carried out during the beginning of August. The material from harvesting to beginning of the freezing was stored about 4 h in temperature 10 C.

microwave type NN-F621MB (Matsushita Electric, Cardiff, UK). The time of defrosting and heating to 75 C (Codex Alimentarius, 1993) was 7 min 45 s. Analytical procedures

Production of frozen products

Two variants of processing the raw material before freezing were used. In variant I, the traditional technology was to blanch the raw material; after freezing and frozen storage the frozen product was cooked in salted water to consumption consistency (sensory evaluated). In variant II, the raw material was cooked to consumption consistency to obtain a do-it-for-me product, which after freezing and frozen storage only had to be defrosted and heated in a microwave oven to consumption temperature. In variant I, pods were blanched in a stainless steel vessel in water, the proportion of the blanched material to water being 1:5. The blanching temperature was 95– 98 C and the time was 3 min (Canet & Alvarez, 2005). These conditions permitted a decrease in the activity of catalase and peroxidase to a level below 5% of the initial value. After blanching, the material was immediately cooled in cold water, slightly shaken and left for 30 min on sieves to drain the water remaining on the surface. In variant II, pods were cooked to consumption consistency in water with 2% added salt (NaCl). The cooking was carried out in a stainless steel vessel, with the proportion of the weight of the raw material to water being 1:1. The material was placed in boiling water. The cooking time measured from the moment when the water came to the boil again was 9 min. After cooking, the pods were drained, placed in sieves and cooled in a stream of cold air. Blanched and cooked vegetable was packed in 500-g portions in polyethylene bags and frozen at )40 C in a Feutron 3626-51 (Greiz, Germany) blast chamber. The time required for the inside of the product to reach the storage temperature of )20 C was 90 min (freezing rate 0.52 C min)1). The products thus obtained were stored for 12 months. Preparation of frozen product for evaluation

Frozen blanched product (variant I) was cooked in water with 2% added salt (NaCl), the proportion in weight of brine to plant material being 1:1. As in the case of cooking, the frozen material was put in boiling water. The time of cooking, measured from the moment when the brine was boiling again, was 6 min. After cooking, the water was immediately drained and the product was cooled to 20 C for analyses. As regards frozen product cooked before freezing (variant II), a portion of 500 g in a heat-resisting vessel covered with a lid, was defrosted and, was heated in a Panasonic

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The content of dry matter and total N were determined according to procedures described by the AOAC (1990). The content of amino acids (except for triptophane) was determined using an AAA-400 amino acid analyser (INGOS, Prague, Czech Republic). The analytical procedure applied was in accordance with the recommendations of the producer. The freeze-dried material was hydrolysed in 6 m HCl for 24 h at 110 C. After cooling, filtering and washing, the hydrolyte was evaporated in a vacuum evaporator, the dry residue being dissolved in a buffer of pH 2.2. The prepared sample was analysed using the ninhydrin method. Buffers of pH 2.6, 3.0, 4.25 and 7.9 were applied. The ninhydrine solution was buffered at pH 5.5. A column 370 mm in length was filled with Ostion ANB INGOS ionex. The temperature of the column was maintained at 55–74 C and that of the reactor at 120 C. The determination of the sulphur-containing amino acids, methionine and cystine, was carried out by means of oxygenating hydrolysis, using a mixture of formic acid and hydrogen peroxide (9:1) at 110 C for 24 h. After cooling, the sample was processed as with acid hydrolysis. Buffers of pH 2.6 and 3.0 were used; the temperature of the column was 60 C and that of the reactor 120 C. The calculations were carried out according to the external standard. All determinations were carried out in three experimental replications each in two parallel samples. The level of amino acids was given in 100 g of edible parts of the products in order to compare the amino acid content in French bean pods according to the culinary and technological processing applied. The composition of amino acids was also expressed as grams per 16 g of N to estimate the quality of the protein French bean pods by comparing it with the FAO ⁄ WHO pattern (FAO ⁄ WHO, 1991; Institute of Medicine, 2002). On the basis of the amino acid composition, the chemical score (CS) index was calculated using the Mitchell and Block method (Osborne & Voogt, 1978), and the integrated essential amino acid (EAA) index using the Oser method (Oser, 1951). Statistical analysis

Statistical analysis allowing a comparison of the content of amino acids in the fresh raw material, boiled raw material and frozen goods after preparation for consumption was carried out using single-factor analysis of variance (anova) on the basis of the Snedecor F-test and Student’s t-test; the least significant difference (LSD) was calculated at the probability level P < 0.01 and P < 0.05 (Snedecor & Cochman, 1980). The Statistica

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6.1 program was used (StatSoft Polska Sp. z o.o., Krakow, Poland). Results and discussion

In the total content of amino acids in the raw material, aspartic acid and glutamic acid dominated: 20% and 12% respectively (Tables 1 and 2). The content of the remaining amino acids was distinctly lower: 2% cystine and methionine; 3–4% isoleucine, tyrosine, phenylalanine, treonine, histidine, proline and glycine; the content of the remaining amino acids was 5–6%. Cruz-Garcia et al. (1999) found a similar proportion of aspartic and glutamic acids in the total content of amino acids: 25% and 14% respectively. Different authors have reported a similar proportion of glutamic acid; the content of aspartic acid, however, varies to a considerable degree: according to Eppendorfer & Bille (1996) it was only 11% and according to Gonzalez-Castro et al. (1997) 18%. All authors agree that the level of methionine and cystine was the lowest in the total content of amino acids, making them the first limiting amino acids. According to the sources quoted above, the ratio of essential amino acids (TEAA) to non-essential amino acids (TNEAA) varied from 1:1.1 to 1:1.8. The ratio

found in raw French bean pods was in the middle of this range, being 1 to 1.4. After cooking, fresh French beans contained significantly more (P < 0.01) amino acids in 100 g of the product, with the exception of cystine and phenylalanine, whose level did not change (Table 1). It is certain that the increase in the amounts of amino acids was only an apparent one, as the dry matter content in the bean pods increased to a similar degree as a result of tissue shrinkage and water loss. A higher level of dry matter after cooking has also been observed in spinach, carrot and cauliflower (Kmiecik & Budnik, 1997; Jaworska & Kmiecik, 2000; Kala & Prakash, 2004). Thermal processing in water also brings about losses in soluble constituents, resulting in a relative increase in the proportion of non-soluble compounds in the total content of constituents (Kmiecik et al., 2004; Lisiewska et al., 2004). In a study on some methods used in preparing French beans for consumption, Cruz-Garcia et al. (1999) found that a large amount of cooking water in relation to the weight of beans (2.5:1) and a long cooking time (30 min) brought about the loss of amino acids by diffusion and thermal degradation. In the present study, the ratio of brine to pods was 1:1 and the time required to obtain consumption consistency was

Table 1 Amino acid composition of raw and processed French bean, in mg per 100 g of product

Pods

French bean prepared for consumption from frozen pods

LSD

P < 0.01

P < 0.05

8.3 8.3 12.8 3.6 5.8 7.3 6.4 ns 13.3 9.7 12.0 5.9 59.0 9.4 33.7 25.4 14.4 9.1 9.1 10.1 107.6 169.1

5.9 5.9 9.2 2.6 4.2 5.2 4.6 ns 9.5 6.9 8.6 4.2 42.1 6.7 24.1 18.1 10.3 6.5 6.5 7.2 76.8 120.6

Amino acid

Raw

Cooked

Blanched before freezing

Cooked before freezing

Isoleucine Leucine Lysine Cystine Methionine Total sulphur amino acids Tyrosine Phenylalanine Total aromatic amino acids Threonine Valine Histidine Total essential amino acids (TEAA) Arginine Aspartic acid Glutamic acid Serine Proline Glycine Alanine Total non-essential amono acids (TNEAA) Total amino acids Dry matter g per 100 g fresh matter

70 ± 4 122 ± 6 108 ± 3 25 ± 2 21 ± 2 46 46 ± 3 78 ± 7 124 79 ± 4 94 ± 5 49 ± 3 692 97 ± 4 343 ± 11 198 ± 7 119 ± 6 67 ± 4 69 ± 5 92 ± 6 985 1677 9.25

82 ± 4 143 ± 4 126 ± 3 28 ± 2 26 ± 4 54 62 ± 4 83 ± 3 145 92 ± 5 109 ± 5 60 ± 3 811 111 ± 3 422 ± 21 268 ± 8 143 ± 8 78 ± 6 83 ± 3 111 ± 3 1216 2027 11.02

79 ± 4 130 ± 3 104 ± 10 20 ± 1 21 ± 2 41 43 ± 2 76 ± 4 119 73 ± 4 98 ± 4 55 ± 2 699 97 ± 4 287 ± 11 230 ± 15 96 ± 6 70 ± 3 77 ± 5 90 ± 4 947 1646 9.51

90 ± 4 157 ± 1 127 ± 4 27 ± 2 28 ± 2 55 53 ± 2 85 ± 4 138 91 ± 5 118 ± 7 57 ± 3 833 111 ± 6 390 ± 17 273 ± 15 126 ± 7 80 ± 4 90 ± 4 114 ± 5 1184 2017 11.45

LSD, least significant difference; ns, not significant.

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Table 2 Amino acid composition of raw and processed French bean, in g per 16 g N

Pods

French bean prepared for consumption from frozen pods

LSD

P < 0.01

P < 0.05

0.402 0.413 ns 0.171 ns ns 0.302 ns ns ns ns 0.277 ns ns 1.564 1.220 0.684 ns ns ns ns ns

0.287 0.295 ns 0.122 ns ns 0.216 ns ns ns ns 0.198 ns ns 1.116 0.870 0.488 ns 0.321 ns ns ns

Amino acid

Raw

Cooked

Blanched before freezing

Cooked before freezing

Isoleucine Leucine Lysine Cystine Methionine Total sulphur amino acids Tyrosine Phenylalanine Total aromatic amino acids Threonine Valine Histidine Total essential amino acids (TEAA) Arginine Aspartic acid Glutamic acid Serine Proline Glycine Alanine Total non-essential amono acids (TNEAA) Total amino acids Total N g per 100 g fresh matter

3.63 ± 0.21 6.29 ± 0.30 5.57 ± 0.18 1.27 ± 0.09 1.08 ± 0.10 2.35 2.39 ± 0.14 4.04 ± 0.36 6.43 4.10 ± 0.19 4.85 ± 0.28 2.52 ± 0.14 35.74 5.00 ± 0.21 17.67 ± 0.56 10.20 ± 0.34 6.13 ± 0.32 3.46 ± 0.18 3.58 ± 0.24 4.76 ± 0.29 50.8 86.54 0.31

3.64 ± 0.18 6.37 ± 0.16 5.60 ± 0.15 1.23 ± 0.08 1.15 ± 0.19 2.38 2.76 ± 0.19 3.72 ± 0.13 6.48 4.13 ± 0.23 4.86 ± 0.20 2.67 ± 0.11 36.13 4.97 ± 0.12 18.83 ± 0.94 11.94 ± 0.36 6.37 ± 0.35 3.48 ± 0.26 3.72 ± 0.15 4.95 ± 0.15 54.26 90.39 0.36

4.15 ± 0.20 6.79 ± 0.16 5.45 ± 0.52 1.03 ± 0.06 1.08 ± 0.11 2.11 2.26 ± 0.12 4.00 ± 0.21 6.26 3.84 ± 0.19 5.15 ± 0.23 2.88 ± 0.11 36.63 5.09 ± 0.23 15.03 ± 0.59 12.06 ± 0.77 5.01 ± 0.31 3.65 ± 0.13 4.01 ± 0.26 4.71 ± 0.20 49.56 86.19 0.31

3.93 ± 0.16 6.88 ± 0.05 5.57 ± 0.20 1.18 ± 0.09 1.23 ± 0.08 2.41 2.30 ± 0.09 3.72 ± 0.18 6.02 3.97 ± 0.23 5.16 ± 0.32 2.51 ± 0.15 36.45 4.85 ± 0.24 17.08 ± 0.75 11.97 ± 0.66 5.54 ± 0.29 3.52 ± 0.19 3.94 ± 0.16 5.01 ± 0.24 51.91 88.36 0.37

LSD, least significant difference; ns, not significant.

only 9 min, resulting in a better retention of amino acids compared with that in the above-quoted studies. However, Chau et al. (1997) showed that doubling the cooking time did not always result in further decreases in amino acid content, while sometimes, on the contrary, the amounts of amino acids increased, the response not being identical in all species. Similarly, Candela et al. (1997) stress that thermal processing in water brought about various changes, showing significant losses or increases in the amounts of individual amino acids depending on the vegetable species. Parihar et al. (1996) also found that changes in the level of amino acids were affected by the method of thermal processing and by the investigated species. According to Ziena et al. (1991), an increase in the temperature of thermal processing from 100 to 125 C also contributed towards higher losses. In comparison with fresh bean pods prepared for consumption, cooked French beans from the frozen product obtained using the traditional method contained significantly less (P < 0.01 or P < 0.05) amino acids except for isoleucine, phenylalanine and glycine, whose level did not change. In frozen French beans obtained using the modified method and prepared for consumption in a microwave oven, the amino acid content generally remained unchanged. However, at

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P < 0.01 the results showed more leucine and less serine and at P < 0.05 more isoleucine, valine and glycine and less aspartic acid. Phenylalanine was the only amino acid which did not change during any of the culinary or technological processes applied. The amino acid content obtained expressed in 16 g N showed that the cooking of raw bean pods did not affect the content of most amino acids (Table 2). The exceptions were tyrosine and glutamic acid, whose content increased at P < 0.01, and aspartic acid, with an increase at P < 0.05. In comparison with the protein in cooked fresh French bean pods, the protein in cooked pods obtained from the traditional frozen product contained less (P < 0.01) cystine, tyrosine, aspartic acid and serine, more (P < 0.01) isoleucine, and also more leucine and histidine but only at the significance level of P < 0.05. The protein in frozen bean pods obtained using the modified method and prepared for consumption, again compared with cooked fresh pods, differed only in having a higher content of leucine (P < 0.01) and isoleucine (P < 0.05) and a lower (P < 0.01) content of tyrosine, aspartic acid and serine. The culinary and technological processing applied did not affect the level of lysine, methionine, phenylalanine, treonine, valine,

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arginine, proline or alanine if their content was expressed in 16 g N. The total sulphuric amino acids, aromatic, essential, non-essential and total amino acids were also stable during the cooking, freezing and preparation of frozen products for consumption. Neither did the preparation for consumption of fresh and frozen broad beans and grass pea result in significant changes in protein quality (Kmiecik et al., 1999; Korus et al., 2003). Changes in amino acid content may be affected by protein solubility in water and by damage to plant tissue. The changes in the level of amino acids may be relative or merely apparent rather than absolute changes (Ziena et al., 1991). During the preparation of food the side chains of some protein-bound amino acids can react chemically with each other or with other molecules present in the food and these reactions can result in a change in the composition of amino acids (Sherr et al., 1989). Baxter (1995) argues that the initial reaction in the Maillard process involves the formation of new components based on sugar and amino acid, although the reaction is reversible. However, subsequent reactions are not reversible and amino acids are destroyed. The author observed that amino acids suffered some degree of loss when material is submitted to sterilization temperature. Heat denaturation breaks covalent disulphide bonds, liberating hydrogen sulphide. Heat may also alter amino acid residues chemically and lead to the formation of new bonds (Espe & Lied, 1999). Methionine and cystine can suffer thermal breakdown, producing sulphur compounds (Marshall et al., 1982). Decomposition of some protein fractions rich in TEAA can result in higher values for TNEAA when they are calculated on the basis of 100% protein (Ziena et al., 1991). The protein in all the three

products prepared for consumption as well as that in fresh beans pods hardly differs, as confirmed by the values in the EAA index (Table 3). It is difficult to compare the values in the EAA and CS indexes with those given in other papers because the pattern for protein has changed over time (Pysz & Pisulewski, 2004). However, irrespective of the pattern for leguminous vegetables, the first limiting amino acid is cystine with methionine (Eppendorfer & Bille, 1996; Lisiewska et al., 2001; Prakash et al., 2001). The lowest (84) CS index for methionine with cystine was found for protein in the frozen bean product obtained using the traditional method and prepared for consumption; it was similar for the remaining samples, varying between 94 and 96. Lysine was the second limiting amino acid; however, the values of the CS index for this amino acid were high, varying in the range of 94–97. As P. Ge˛bczyn´ski (unpublished data) reports, compared with the traditional frozen product, frozen French beans obtained using the modified method contained higher amounts of antioxidative constituents such as vitamin C, total carotenoids, beta-carotene and polyphenols, and showed higher total antioxidative activity. Conclusions

The new type of product (do-it-for-me) provides the consumer with higher levels of amino acids in 100 g of edible portion than that obtained using the traditional method, with comparable protein quality. The method of producing the do-it-for-me frozen French bean is described in detail in the Material and methods section so that it could be easily introduced by manufacturers.

Pods

French bean prepared for consumption from frozen pods Cooked before freezing 140 104 96 96 96 117 147 132 114

Index

Amino acid

Raw

Cooked

Blanched before freezing

CS

Izoleucine Leucine Lysine Cystine + methionine Tyrosine + phenylalanine Threonine Valine Histidine

130 95 96 94 102 121 139 133 112

130 97 97 95 103 121 139 141 114

148 103 94 84 99 113 147 152 115

EAA

Table 3 Amino acids indexes of raw and processed French bean according to FAO ⁄ WHO (1991)

CS, chemical score index; EAA, essential amino acid index.

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References AOAC. (1990). Official Methods of Analysis of the Association of Official Analytical Chemists, 15th edn. Washington, DC: Association of Official Analytical Chemists. Baxter, J.M. (1995). Free amino acid stability in reducing sugar systems. Journal of Food Science, 60, 405–408. Candela, M., Astiasaran, I. & Bello, J. (1997). Cooking and warmholding: Effect on general composition and amino acids of kidney beans (Phaseolus vulgaris), chickpeas (Cicer arietinum), and lentils (Lens culinaris). Journal of Agricultural and Food Chemistry, 45, 4763–4767. Canet, W. & Alvarez, M.D. (2005). Quality and safety of frozen vegetables. In: Handbook of Frozen Food Processing and Packaging (edited by S. Da-Wen). Pp. 377–415. Salisbury: Techset Composition Limited. Chau, Ch.F., Cheung, P.C.K. & Wong, Y.S. (1997). Effects of cooking on content of amino acids and antinutrients in three Chinese indigenous legume seeds. Journal of the Science of Food and Agriculture, 75, 447–452. Codex Alimentarius. (1993). Code of Hygienic Practice for Precooked and Cooked Foods in Mass Catering. CAC ⁄ RCP 39. Available at: http://www.codexalimentarius.net/web/standard_list.jsp. Cruz-Garcia, C.D., Lopez-Hernandez, J., Gonzalez-Castro, M.J., Rodriguez-Bernaldo de Queiros, A.I. & Simal-Lozano, J. (1999). Effects of various culinary treatments on the amino acid content of green beans. Deutsche Lebensmittel Rundschau, 95, 12. Datta, A.K., Geedipalli, S.S.R. & Almeida, M.F. (2005). Combining microwaves with other modes of heating-infrared and jet-impingement – can provide selective heating to improve food quality and cooking speed. Food Technology, 1, 36–40. Dinelli, G., Bonetti, A., Minelli, M, Marotti, I., Catizone, P. & Mazzanti, A. (2006). Content of flavonols in Italian bean (Phaseolus vulgaris L.) ecotypes. Food Chemistry, 99, 105–114. Eppendorfer, W.H. & Bille, S.W. (1996). Free and total amino acid composition of edible parts of beans, kale, spinach, cauliflower and potatoes as influenced by nitrogen fertilization and phosphorus and potassium deficiency. Journal of the Science of Food and Agriculture, 71, 449–458. Espe, M. & Lied, E. (1999). Fish silage prepared from different cooked and uncooked raw materials: chemical changes during storage at different temperatures. Journal of the Science of Food and Agriculture, 79, 327–332. FAO ⁄ WHO. (1991). Protein quality evaluation. Report of the joint FAO ⁄ WHO expert consultation, FAO Food and Nutrition Paper, 51. Rome: FAO. Gonzalez-Castro, M.J., Lopez-Hernandez, J., Simal-Lozano, J. & Oruna-Concha, M.J. (1997). Determination of amino acids in green beans by derivatization with phenylisothiocianate and high-performance liquid chromatography with ultraviolet detection. Journal of Chromatographic Science, 35, 181–185. Institute of Medicine (2002). Protein and amino acids. In: Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty acids, Cholesterol, Protein, and Amino Acids. Pp. 589–768. Washington, DC: Food and Nutrition Board, National Academies Press. Iqbal, A., Khalil, I.A., Ateeq, N. & Khan, M.S. (2006). Nutritional quality of important food legumes. Food Chemistry, 97, 331–335. Jaworska, G. & Kmiecik, W. (2000). Comparison of the nutritive value of frozen spinach and New Zealand spinach. Polish Journal of Food and Nutritional Sciences, 9, 79–84.

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Kala, A & Prakash, J. (2004). Nutrient composition and sensory profile of differently cooked green leafy vegetables. International Journal of Food Properties, 7, 659–669. Kmiecik, W. & Budnik, A. (1997). Wpływ dwo´ch sposobo´w gotowania brokuła nła poziom wybranych wskaz´niko´w fizykochemicznych. Bromatologia i Chemia Toksykologiczna, 30, 303–309. Kmiecik, W., Lisiewska, Z. & Ge˛bczyn´ski, P. (1999). Content of amino acids in fresh and frozen and cooked broad bean seeds (Vicia faba var. Major) depending on cultivar and degree of maturity. Journal of the Science of Food and Agriculture, 79, 555–560. Kmiecik, W., Korus, A. & Lisiewska, Z. (2004). Evaluation of physicochemical and sensory quality of frozen green grass pea (Lathyrus sativus L.). International Journal of Food Science and Technology, 39, 149–155. Korus, A., Lisiewska, Z. & Kmiecik, W. (2003). Content of amino acids in fresh and preserved physiologically immature grass pea (Lathyrus sativus L.) seeds. European Food Research and Technology, 217, 148–153. Lisiewska, Z., Kmiecik, W. & Ge˛bczyn´ski, P. (1999). Effect of maturity stages on the content of ash components in raw, frozen and canned broad beans. Food Chemistry, 67, 155–162. Lisiewska, Z., Kmiecik, W. & Korus, A. (2001). Content of nitrogen compounds in raw and preserved seeds of grass pea (Lathyrus sativus L.). European Food Research and Technology, 213, 343– 348. Lisiewska, Z., Słupski, J., Kmiecik, W. & Ge˛bczyn´ski, P. (2004). Amino acid profiles and protein quality of fresh and frozen dill depending on usable part of raw material, pre-treatment before freezing, and storage temperature of frozen products. Electronic Journal of Polish Agricultural Universities, Food Science and Technology, 7, 1, http://www.ejpau.media.pl. Marshall, H.F., Chang, K.C., Miller, K.S. & Satterlee, L.D. (1982). Sulfur amino acid stability. Effects of processing on legume proteins. Journal of Food Science, 47, 1170. Osborne, D.R. & Voogt, P. (1978). The Analysis of Nutrients in Food. London: Academic Press. Oser, B.L. (1951). Method for integrating essential amino acid content in the nutritional evaluation of protein. Journal of the American Dietetic Association, 27, 396–399. Parihar, P., Mishra, A., Gupta, O.P. & Singh, A. (1996). Effect of cooking on limiting essential amino acid content of common pulses. Advances in Plant Sciences, 9, 165–169. Prakash, D., Niranjan, A., Tewari, S.K. & Pushpangadan, P. (2001). Underutilised legumes: potential sources for low-cost protein. International Journal of Food Sciences and Nutrition, 53, 337–341. Pysz, M. & Pisulewski, P.M. (2004). Wspo´łczesne pogla˛dy na zapotrzebowanie człowieka na białko, wartos´ c´ od_zywcza˛ białek _ z_ ywnos´ ci i metody jej oceny. Zywienie Człowieka i Metabolizm, 3, 254–264. Sherr, B., Lee, C.M. & Jelesciewicz, C. (1989). Absorption and metabolism of lysine Maillard products in relation to utilization of l-lysine. Journal of Agricultural and Food Chemistry, 37, 119– 122. Sloan, A.E. (2005). The new face of frozen. Food Technology, 12, 21. Snedecor, G.W. & Cochman, W.G. (1980). Statistical Methods. 7th edn. Ames, IA: Iowa State University Press. Ziena, H.M., Youssef, M.M. & El-Mahdy, A.R. (1991). Amino acids composition and some antinutritional factors of cooked faba beans (Medammis): effects of cooking temperature and time. Journal of Food Science, 56, 1347–1349.

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Original article Effect of regular and hydrolysed dairy proteins on texture, microstructure and colour of lean poultry meat batters Shai Barbut* Food Science Department, University of Guelph, Guelph, Ontario, NIG 2W1, Canada (Received 20 March 2007; Accepted in revised form 19 October 2007)

Summary

The use of 2% milk protein isolate (MPI), and some of its fractions which included caseinate, whey protein isolate (WPI), two whey protein hydrolysates (5.2% and 8.5%; WPH-I and WPH-II respectively) and blactoglobulin (b-lac) was evaluated in lean chicken breast meat batters. Adding caseinate and MPI resulted in the highest fracture force values, and caseinate also provided higher yield compared with the control. Both proteins were observed to form distinct protein islands embedded within the meat protein matrix, which appeared to enhance the gel structure. The two hydrolysates provided the highest yield compared with all other treatments. However, adding WPH-II also resulted in the lowest fracture force and hardness values, while WPH-I provided similar values to the control. The low hardness value could be explained by the light micrograph which showed WPH-II interfering with the binding of the meat proteins. The WPI and b-lac provided similar yield, fracture and hardness values as the control. The colour of the products was most affected by the WHP-I and WHP-II; both resulted in lower lightness, yellowness and overall spectra reflectance curves. A cost analysis revealed that caseinate addition was the most economical in this lean meat system.

Keywords

Chicken, colour, dairy, meat, microstructure, poultry, protein, texture, whey.

Introduction

Various non-meat additives, such as milk proteins and hydrocolloid gums are currently utilised by the meat industry to improve texture, moisture retention and control cost. The meat industry, like other sectors of the food industry, continues to evaluate new and modified (e.g. hydrolysate) non-meat ingredients to enhance yield, texture and address the changing market needs (Ensor et al., 1987; Kerry et al., 1999; Pszczola, 2006). Current changes include consumer demand for low fat ⁄ low calorie meat products that taste and have a mouth-feel of full fat products. In most cases, extra moisture is part of the formulation used to replace fat, and therefore, it is important to find ingredients that would contribute to moisture retention. Overall, water is a major constituent of lean meat (c. 70%) and the ability of a meat product to retain its own and additional moisture is very important. This is especially true when the product is heated and the moisture retention of meat proteins is significantly reduced. This reduction affects yield and other quality attributes such as flavour and texture (Offer et al., 1984; Tsai et al., 1998). Currently, many *Correspondent: E-mail: [email protected]

commercial ground ⁄ chopped ⁄ whole-muscle meat products rely on non-meat additives to enhance the texture and water binding properties. These additives commonly include proteins (e.g. dairy, soy) and polysaccharides (e.g. starch, carrageenan) that are used as binders, fillers or extenders to improve meat products’ characteristics and control cost (Comer, 1979; Endres & Monagle, 1987; Smith & Rose, 1995; Barbut, 2002a). The additives must be compatible with the meat proteins otherwise they disrupt the structure and can also lower yield. Beuschel et al. (1992) indicated that the contribution of whey protein concentrate to a meat system depends on the meat pH, solubility of the whey protein and heating temperature. They reported that gel hardness increased as whey protein solubility decreased at pH 6.0, 7.0 and 8.0, when heated to 65 C; however, the opposite trend was observed when heated to 90 C. Hongsprabhas & Barbut (1999) reported on the beneficial effect of using cold-set whey proteins (pre-heated under low ionic strength conditions prior to actual gelling) in improving the texture and water binding of minced poultry meat; however, salt concentration was a key factor in the degree of improvement. Beuschel et al. (1992) and others have indicated that in order to optimise the contribution of non-meat ingredients and

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Regular and modified dairy proteins in lean chicken meat S. Barbut

balance benefit vs. cost, it is essential to understand the interactions within the meat system. The objective of this study was to evaluate the effects of two traditional and four new dairy protein preparations (four native and two hydrolysates), used at a commonly prescribed level of 2% (w ⁄ w), on yield, texture, microstructure and colour of lean chicken breast meat batters.

USA) was used to monitor the core temperature. Cook loss was determined as the amount released (decanted into 15-mL test tubes) divided by the initial meat batter weight. Solid loss was determined as the volume of sediments accumulating at the bottom of the 15-mL test tubes, after an overnight refrigerated storage. Texture analysis

Materials and methods

Meat and meat batter preparation

Fresh skinless chicken breast meat was obtained from a local processing plant and brought to the laboratory within 10-h postmortem. All connective tissue and visible fat was removed and the meat chopped for 30 s in a bowl chopper (Schneidmeister SMK 40, Berlin, West Germany), packed under vacuum and frozen for up to 4 weeks at )18 C. The chemical composition of the raw meat determined in duplicates (AOAC 1990) was: 73.9% water, 22.4% protein, 2.3% fat and 1.0% ash. Meat for each of the trials was thawed overnight at 4 C. Each treatment consisted of 100 g minced meat mixed with one of six dry dairy proteins (2% w ⁄ w) which included: sodium caseinate (87% protein) (Herman Laue Spice Co., Uxbridge, ON, Canada), milk protein isolate (MPI, 90% protein) (New Zealand Milk Products, Auckland, New Zealand), whey protein hydrolysate I (WPH-I, 97.2% protein; degree of hydrolysis 5.2%) (Bio Zate 1; Davisco Inc., Eden Prairie, MN, USA), whey protein hydrolysate II (WPH-II, 97.2% protein; degree of hydrolysis 8.5%) (Bio Zate 3; Davisco Inc.), b-lactoglobulin (b-lac, 95% protein) (BioPure; Davisco Inc.) and whey protein isolate (WPI, 98.3% protein; Davisco Inc.). Sodium tri-poly-phosphate (TPP) was added (0.3%) to all the dairy protein treatments and one control. A second control, without TPP was also evaluated. Water (51%) was added to bring the meat protein level to 14%, which is a common level in further processed poultry products on the market. Salt (2.5%) and TPP (0.3%) were also added to duplicate the common level used by the industry. The meat batters were mixed by hand for 3 min (dry ingredients initially dissolved in water), equilibrated in a refrigerator for 1 h and then mixed for another 1 min.

Following an overnight refrigeration period, texture profile analysis (TPA) parameters were determined using six centre cores (16 mm diameter, 10 mm high) per treatment, which were compressed twice to 75% of their original height by a texture analyser (Stable Micro Systems TA.XT2; Texture Technologies Corp., Scarsdale, NY, USA) employing a moving flat plate descending at 1.5 mm s)1. The TPA parameters of fracturability, hardness, springiness, cohesiveness and chewiness were determined (Park et al., 1990). Microstructure

Samples (10 · 10 · 4 mm) were cut from the centres of cooked meat batters. Samples were fixed in 10% formalin for 10 h, dehydrated with a series of increasing alcohol solutions (50–100%) followed by xylene (100%), using an automated system (Tissue-Tek VIP # 5; Sakura Finetechnical, Tokyo, Japan). Samples were later embedded in paraffin, cut into 4 to 6-lm-thick sections, allowed to float on water and transferred onto glass slides. Slides were air dried and stained with haematoxylin ⁄ eosin. Specimens were observed using a light microscope (Olympus Optical BX60, Tokyo, Japan) at ·100 magnification. Images were captured by a computerised image analysis system (image-pro plus version 4.5–1.29; Media Cybernetics Inc., Silver Spring, MD, USA). Colour

A colour meter (Minolta Spectrophotometer CM-1000, Osaka, Japan) with a window diameter of 10 mm was used to evaluate three freshly cut surfaces from each cooked sample to obtain reflectance curves as well as the CIE L* (lightness), a* (redness) and b* (yellowness) values.

Cooking and cook loss

Two 35 g portions were stuffed into 50-mL test tubes and centrifuged (Model 225, Fisher Scientific, Pittsburgh, PA, USA) for 30 s at the low speed setting to pack the meat and remove small air bubbles. The tubes were heated (30–75 C) in a water bath (model W26, Haake, Dieselstr, Germany) within 1.25 h, followed by cooling at room temperature for 15 min. A thermocouple (Model # 52 K ⁄ J; Fluke Co, Inc., Everett, WA,

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Statistical analysis

The experiment was designed as a complete randomised block with three separate replications. Statistical analysis was performed using a software package (sas version 8.02; SAS Institute, Cary, NC, USA). The SAS General Linear Model procedure was used for analysis of variance. Tukey multiple comparison analysis was performed to separate the means (P < 0.05).

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Results and discussion

The addition of caseinate, WPH-I and WPH-II resulted in significantly lowest (P < 0.05) cook loss ⁄ highest yield compared with the control and the other treatments (Table 1), meaning that these proteins were gelling and helping to bind moisture. Adding MPI and b-lac did not contribute to moisture binding (Table 1), and WPI slightly increased cook loss compared with the control. The control without phosphate (Treatment 8) showed the highest cook loss, which was the result of lower salt-soluble protein extraction from the meat (Offer et al., 1984; Smith & Rose, 1995). Previous work has also indicated the beneficial effect of adding certain dairy proteins to a meat system. Tsai et al. (1998) showed a 6% reduction in cook loss when 3.5% caseinate was added to a restructured beef product cooked to 65 C. Atughonu et al. (1998) reported that adding 3.5% whey protein concentrate (34% protein content) improved cook yield by 1.6%, while 2% caseinate (93% protein) improved yield by 7% in beef and pork frankfurters. Examining the contribution of the dairy proteins to the texture of the meat batters revealed that caseinate and MPI provided significantly higher fracturability compared with the control. The micrographs of these treatments show that distinct dairy protein islands were formed within the meat protein gel (Fig. 1b and c). These unique structures were not observed in the control treatment (Fig. 1a). The latter shows a typical microstructure of a lean meat batter composed of small sections of muscle fibres embedded within a coagulated salt-soluble protein matrix (Barbut, 1997; Su et al., 2000). The dairy protein islands appear to help in moisture binding as well as act as reinforcement components to strengthen the gel structure. The formation of distinct dairy protein islands in an emulsified meat product containing caseinate was also reported by Barbut (2006). Aguilera & Kessler (1989) discussed the formation of a gel matrix using two different gelling systems, and illustrated the creation of

simple, mixed, filled and filled ⁄ mixed gels. One of the filled gels they described, termed ‘reinforcing-matrix’, seems to fit the structure produced by the addition of caseinate and MPI added to the lean chicken meat batters studied here. In such a system, the ‘filler’ occupies regions within the main structure that enhance the overall firmness of the structure. Other researchers have also reported that the addition of 2% caseinate significantly increased the hardness of beef frankfurters compared with the control (Hung & Zayas, 1992). Su et al. (2000) reported that 2% caseinate significantly increased shear force values of reduced-fat frankfurters. In the present study, the size of the caseinate protein islands was 40–50 lm whereas for the MPI it was in the rage of 10–30 lm. This might be associated with the higher yield value obtained by the caseinate treatment; however, more research is needed to prove ⁄ disprove this point. It should also be pointed out that the dairy protein islands might have been the result of lower initial water solubility (i.e. all dry powders were mixed with water prior to adding to the meat). Most processors do the same or just add the dry ingredients directly to the ground ⁄ chopped meat batter, meaning that our procedure was representative. In any case, it would be interesting to further examine different hydration methods. When examining the contribution of the two WPHs to texture, it is interesting to note that the one with the higher degree of hydrolysis (8.5%) resulted in the lowest fracturability value (Table 1), while the one with 5.2% hydrolysis (WPH-I) provided a high fracturability value. However, both showed the highest yield values (i.e. indicating that when gelling they held the same amount of moisture). The explanation for the poor texture of the WPH-II treatment can be seen in the micrograph showing that it interfered with the meat protein forming a continuous matrix ⁄ good binding among the cooked gel components. Figure 1e shows the disconnection ⁄ separation between certain muscle fibres and the protein matrix, which was not seen in any of the other

Table 1 Effect of regular and modified dairy protein (2%) on cook loss and colour of lean chicken breast meat batters

Treatment

Cook loss (g)

Liquid loss (mL)

Solid loss (mL)

Lightness (L*)

Redness (a*)

Yellowness (b*)

1. 2. 3. 4. 5. 6. 7. 8.

3.05cd 1.89e 2.88d 1.30f 1.05f 3.23bc 3.42b 4.80a

2.93cd 1.70e 2.78d 1.20f 0.96f 3.25bc 3.30b 4.70a

0.23d 0.18e 0.21ed 0.03f 0.03f 0.31c 0.35b 0.42a

80.4a 79.2bc 78.8c 76.2d 75.9d 80.3a 80.2ab 80.3a

1.1b 1.0b 1.0b 1.7d 1.8d 0.9b 1.4c 0.4a

12.7b 12.4b 12.2b 11.2c 10.8c 12.4b 12.1b 13.5a

Control Caseinate Milk protein isolate Whey protein hydrolysate I Whey protein hydrolysate II b-lactoglobulin Whey protein isolate Control no-phosphate

a–d

Means in the same column followed by different letters are significantly different (P < 0.05).

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Regular and modified dairy proteins in lean chicken meat S. Barbut

(a)

(d)

(b)

(e)

(c)

(f)

Figure 1 Light micrographs of lean poultry meat batters with and without 2% (w ⁄ w) dairy proteins: (a) control, (b) with sodium caseinate, (c) isolated milk proteins, (d) whey protein hydrolysate I, (e) whey protein hydrolysate II and (f ) b-lactoglobulin. MF, muscle fibres; DPI, dairy protein island; S, separation. Bar = 100 lm.

treatments. This separation resulted in only 30% of the fracturability and 40% of the hardness values observed for the control. The WPH-II also resulted in significantly lower springiness and cohesiveness values compared with the control and the WPH-I. This indicates that once the cooked gel structure of the WPH-II treatment was deformed (75% compression) it experienced major structural damage and could not rebound like the control and the WPH-I treatments. The chewiness and gumminess values, which include the hardness component, were also significantly lower by WPH-II addition compared with control and WPH-I treatments. The results suggest that a whey protein with a high degree of hydrolysis should not be used in such meat products, because the smaller whey protein peptides interfere with the natural binding of the meat proteins. Overall, the use of the TPA evaluation was also reported useful in other studies dealing with meat batter reformulation. Park et al. (1990) used TPA to develop frankfurters with elevated levels of oleic acid which also included additional moisture, and identified a few formulations acceptable to a consumer panel. Later,

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Ordonez et al. (2001) used the test to develop low-fat (10%) frankfurters with soy and carrageenan that had similar texture to regular products (30% fat) on the market. The b-lac, which is an expensive dairy protein isolate (see price comparison below), did not improve yield or texture compared with the control (Tables 1 and 2); all values similar to the control, expect solid loss. In terms of its effect on the microstructure, it appears that its contribution was in some filling of the spaces around muscle fibres (Fig. 1f; see the smooth protein gel around the muscle fibres at the bottom left). However, it did not form the distinct dairy protein islands seen in the caseinate and MPI treatments discussed above. WPI resulted in a similar microstructure seen in the b-lac treatment (note: the WPI not presented here). Like the b-lac, WPI did not affect any of the textural parameters compared with the control (Table 2). Similar results were reported by Hung & Zayas (1992), who indicated that adding 3.5% regular whey protein concentrate (i.e. pretty close to the actual protein level in the 2% WPI used here) did not affect hardness of beef frankfurters.

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Table 2 Effect of regular and modified dairy protein (2%) on texture profile analysis parameters of cooked lean chicken meat batters Fracturability (N)

Fracture dist. (mm)

Hardness (N)

Springiness (mm)

Cohesiveness (ratio)

Chewiness (N mm)

Gumminess

Treatment 1. 2. 3. 4. 5. 6. 7. 8.

19.9bc 23.9a 23.2a 22.7ab 5.93d 18.1c 19.0c 7.36d

5.53ab 5.72ab 5.53ab 5.96a 3.95c 5.46b 5.40b 4.21c

35.9ab 36.8ab 39.6a 31.2bc 14.1b 35.1ab 37.6a 25.6c

0.72c 0.76bc 0.77b 0.81a 0.50e 0.74bc 0.75bc 0.55d

0.37a 0.35ab 0.36ab 0.37a 0.25c 0.37a 0.38a 0.34b

9.7a 10.1a 11.0a 9.5a 1.8c 9.6a 10.7a 4.9b

13.7ab 13.2ab 14.2a 11.5b 3.6d 12.8ab 14.3a 9.0c

Control Caseinate Milk protein isolate Whey protein hydrolysate I Whey protein hydrolysate II b-lactoglobulin Whey protein isolate Control no-phosphate

a–d

Means in the same column followed by different letters are significantly different (P < 0.05).

However, using WPI in the present study reduced cook loss (Table 1), which is one of the reasons it is used by the industry, as an additive in meat products (Comer, 1979; Pszczola, 2006). The control with no-phosphate showed the highest cook loss. It also resulted in low TPA parameters compared with all the other treatments except the WPHII (Table 2). The effect of the no-phosphate was clearly due to the low level of salt-soluble meat protein extraction, and this was not similar to disrupting the binding (among the meat proteins) observed in the WPH-II treatment (Fig. 1). The microstructure of the no-phosphate product was similar to the control but slightly denser because of the higher moisture loss. In terms of colour, the addition of caseinate and MPI caused lower lightness values (Table 1). The two hydrolysates further reduced lightness as well as increased redness and decreased yellowness values. The b-lac did not affect colour and WPI only increased redness. However, the control with no phosphate showed a marked reduction in redness and increased yellowness. This was because of higher cook loss and more specifically a higher loss of the water-soluble myoglobin (red) pigment. The spectra curves (Fig. 2) show a typical profile of a cooked chicken breast meat product Reflectance spectra

1796

65 60 55 50 45 40 35 30 400

450

500

550

600

650

700

Wavelength (nm) Figure 2 Effect of dairy proteins on cooked meat batter reflectance spectra: ¤, control; , caseinate; , milk protein isolate; · and *, whey protein hydrolysate I and II respectively; d, b-lactoglobulin; +, whey protein isolate, V control no-phosphate.

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(Swatland, 1989; Barbut, 2002b). The two whey hydrolysate treatments with the lowest lightness values (Table 1) also showed the lowest reflectance curves (Fig. 2); the WPH-II with a lower L* value also showed the lowest spectra curve compared with all other treatments. The spectra curves of the other treatments aggregated within the same region. Evaluating the cost benefit of various additives is of interest to the food industry when examining the additives’ contribution to water holding, texture and price of the final product. Comparing the relative costs of the six milk ingredients tested here (based on current Canadian market values) and setting the regular WPI as 100%, MPI is 115%, sodium caseinate is 125%, WPH-I is 160%, WPH-II is 300% and b-lac is 400%. Therefore, the most cost-effective ingredient tested here was the caseinate, which provided low cook loss, and significantly higher fracturability value compared with the control (Tables 1 and 2). The WPH-I, which provided the lowest cook loss and a fracturability value similar to the control, will be less cost effective because of its higher price. The b-lac, which was tested here, is expensive (i.e. not commonly used in meat products) but evaluated to see if this fraction of the whey proteins can make any unique contribution to the lean chicken meat formulation. Overall, different dairy proteins are used by the meat industry, and their selection should be based on contribution to products’ performance and cost. Employing texture, microstructure and colour evaluations can help understand the unique contribution of each non-meat additive. In a water added lean meat system, such as studied here, caseinate was the most cost-effective dairy ingredient in enhancing both water retention and texture. Acknowledgments

The authors would like to thank the Dairy Farmers of Ontario, and Ontario Ministry of Agriculture and Food for financial support and J. Thompson for technical assistance.

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References Aguilera, J.M. & Kessler, H.J. (1989). Properties of mixed and filled type dairy gels. Journal of Food Science, 54, 1213–1217. AOAC (1990). Official Methods of Analysis of the Official Analytical Chemists, 15th edn. Arlington, VA: Association of Analytical Chemists. Atughonu, A.G., Zayas, J.F., Herals, T.J. & Harbers, L.H. (1998). Thermo-rheology, quality characteristics, and microstructure of frankfurters prepared with selected plant and milk additives. Journal of Food Quality, 21, 223–238. Barbut, S. (1997). Microstructure of white and dark turkey meat batters as affected by pH. British Poultry Science, 38, 175–182. Barbut, S. (Ed.) (2002a). Protein Gelation. Poultry Products Processing: An Industry Guide. Pp. 277–282. New York, NY: CRC Press. Barbut, S. (2002b). Cold meat cuts: effect of retail light on preference. Journal of Food Science, 67, 2781–2784. Barbut, S. (2006). Effects of caseinate, whey and milk powders on the texture and microstructure of emulsified chicken meat batters. Journal of Food Science and Technology, 39, 660–664. Beuschel, B.C., Partridge, J.A. & Smith, D.M. (1992). Insolubilized whey protein concentrate and ⁄ or chicken salt-soluble protein gel properties. Journal of Food Science, 57, 852–855. Comer, F.W. (1979). Functionality of fillers in comminuted meat products. Canadian Institute of Food Science and Technology Journal, 12, 157–165. Endres, J.G. & Monagle, C.W. (1987). Nonmeat protein additives. In: Advances in Meat Research: Restructured Meat and Poultry Products (edited by A.M. Pearson & T.M. Dutson). Pp. 331–350, Vol 3. New York, NY: Van Nostrand Reinhold. Ensor, S.A., Mandigo, R.W., Calkins, C.R. & Quint, L.N. (1987). Comparative evaluation of whey protein concentrate, soy proteins isolate and calcium-reduced nonfat dry milk as binders in an emulsion-type sausage. Journal of Food Science, 52, 1155–1158. Hongsprabhas, P. & Barbut, S. (1999). Effect of pre-heated whey protein level and salt on texture development of poultry meat batters. Food Research International, 32, 145–149.

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Hung, S.C. & Zayas, J.F. (1992). Functionality of milk proteins and corn germ protein flour in comminuted meat products. Journal of Food Quality, 15, 139–152. Kerry, J.F., Morrisey, P.A. & Buckley, D.J. (1999). The rheological properties of exudates from cured porcine muscle: effects of added polysaccharides and whey protein ⁄ polysaccharide blends. Journal of Science and Food Agriculture, 79, 1260–1266. Offer, G., Restall, D. & Trinick, J. (1984). Water holding in meat. In: Recent Advances in the Chemistry of Meat (edited by A.J. Bailey). Pp. 71–86. London: The Royal Society of Chemistry, Burlington House. Ordonez, M., Rovira, J. & Jaime, I. (2001). The relationship between the composition and texture of conventional and low-fat frankfurters. International Journal of Food Science and Technology, 36, 749– 758. Park, J., Rhee, K.S. & Ziprin, Y.A. (1990). Low-fat frankfurters with elevated levels of water and oleic acid. Journal of Food Science, 55, 871–872. Pszczola, D. (2006). Which starch is on first. Food Technology, 60, 51– 64. Smith, D.M. & Rose, A.J. (1995). Properties of chicken salt-soluble protein and whey protein concentrate gels as influenced by sodium tripolyphosphate. Poultry Science, 74, 169–175. Su, Y.K., Bowers, J.A. & Zayas, J.F. (2000). Physical characteristics and microstructure of reduced-fat frankfurters as affected by salt and emulsified fats stabilized with nonmeat proteins. Journal of Food Science, 65, 132–128. Swatland, H.J. (1989). A review of meat spectrophotometry (300 to 800 nm). Canadian Institute of Food Science and Technology Journal, 13, 481–484. Tsai, S.-J, Unklesbay, N., Unklesbay, K. & Clarke, A. (1998). Water and absorptive properties of restructured beef products with five binders at four isothermal temperatures. Journal of Food Science and Technology, 31, 78–83.

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Quality characteristics and storage stability of patties from buffalo head and heart meats Arun K. Verma,1 Veerappa Lakshmanan,1 Arun K. Das,2* Sanjod K. Mendiratta1 & Anne Sita Ram Anjaneyulu1 1 Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP 243 122, India 2 Goat Products Technology Laboratory, Central Institute for Research on Goats, Makdoom, Farah, Mathura 281 122, India (Received 9 July 2007; Accepted in revised form 17 October 2007)

Summary

The study was conducted to evaluate the suitability of using buffalo head and heart meat in emulsion based products preparation and to assess their quality during refrigerated storage. The whole study was carried out in three phases. In phase I, head meat patties (HMP) (treatment I, II and III) were prepared in which head meat was substituted with 15%, 20% and 25% skeletal meat and compared with patties from skeletal meat (control). Treated patties had higher emulsion stability, cooking yield, pH and lower shrink percentage and chewiness than control. Sensory attributes of control and treated patties did not differ significantly. In phase II, HMP were prepared in which head meat was substituted with 20%, 30% and 40% heart meat and similarly compared with control as in phase I. Addition of heart meat in HMP had significantly (P < 0.05) increased pH, cooking yield, moisture, lower protein and fat content than control patties. Hardness, gumminess and chewiness values of control patties were higher than treated patties. HMP with heart meat had higher tenderness than control. Control patties rated better than treated patties during sensory evaluation. In phase III, quality of patties was assessed at refrigerated storage (4 ± 1 C) for 15 days. The patties remained stable with minor changes in physico-chemical, microbiological and sensory quality during refrigerated storage for 15 days. Buffalo head and heart meat effectively be utilised in developing patties.

Keywords

Head meat, heart meat, patties, quality, shelf-life, texture analysis.

Introduction

India has the world’s largest buffalo population containing 98.81 million buffaloes. Buffalo meat production is 1.78 million tonnes from slaughter of 10.94 million animals and this account for 25.85% of total meat production in the country (FAO, 2006). India is the fifth largest exporter of buffalo meat in the global market with exports of 4.60 lakh tonnes valued at Rs. 2629 crores in 2005–06 (APEDA, 2006). The slaughter of large numbers of buffaloes for export results in the production of buffalo offal meats at a much higher quantity than the demand for local consumption. This leads to price-cut of buffalo offal meats and even considerable wastage. Hence, efficient utilisation of these meats adopting cost effective way with modern technologies is essentially needed to support an eco*Correspondent: Fax: +91 565 2763246; e-mail: [email protected]

nomical and viable meat production system. A great deal of efforts and research are being focused on appropriate technologies for better utilisation of these offal meats. Both these meats are available at a comparatively cheaper price than skeletal meat. Krishnan & Sharma (1991) prepared highly acceptable buffalo meat sausages by incorporating buffalo offal (rumen and heart meat) meats. Anjaneyulu & Kondaiah (1990) reported that up to 20% combination of rumen meat and heart meat in the ratio of 3:1 can be incorporated in comminuted buffalo meat products as a replacement of lean without impairing the quality of the final product. Pearson & Gillett (1997) have indicated that heart meat and head meat of cattle can be used as ingredients for comminuted meat products. In this study, an attempt was made for efficient utilisation of buffalo head and heart meats in comminuted meat product for preparation of quality meat patties and its shelf-life evaluation during refrigerated storage.

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Quality of patties from buffalo head and heart meat A. K. Verma et al.

Materials and methods

Table 2 Formulation for control and treated patties (combination of buffalo head and heart meat)

Meat and additives

Ingredients

Buffalo head meat and heart meats obtained freshly from local Slaughterhouse were brought to the laboratory. The connective tissue layers of head meat and top fatty portion of heart meat were carefully separated out and discarded. They were cut into chunks of about 2 · 3 · 3-cm size. Meats were chilled overnight, ground through 8-mm plate and used for meat patties preparation. Similarly, buffalo skeletal meat was chilled overnight and then ground through 8-mm plate. Other additives used were sodium chloride, sodium tripolyphosphate, sodium nitrite, spice mix, refined wheat flour, condiments (onion and garlic paste) and refined vegetable oil. The whole study was conducted in three phases.

Skeletal meat (%) 67.3 Head meat (%) – Heart meat (%) – Ice flakes (%) 10.0 Refined vegetable 10.0 oil (%) Condiments (%) 5.0 Refined wheat 3.5 flour (%) Dry spice mix (%) 2.0 Salt (%) 1.7 Tripolyphosphate (%) 0.5 Sodium nitrite (ppm) 100

– 61.7 15.6 – 10.0

– 54.1 23.2 – 10.0

– 46.4 30.9 – 10.0

5.0 3.5

5.0 3.5

5.0 3.5

2.0 1.7 0.5 100

2.0 1.7 0.5 100

2.0 1.7 0.5 100

TI (80% head meat and 20% heart meat); TII (70% head meat and 30% heart meat); TIII (60% head meat and 40%heart meat); Condiments – onion and garlic (4:1).

Phase I

In this phase, standardisation of the process to prepare acceptable quality of meat patties using head meat and skeletal meat was studied. Three different combinations of head meat and skeletal meat were assessed and compared with control patties prepared from skeletal meat. Three different treatments of this experiment were: treatment I (85% head meat + 15% skeletal meat), treatment II (80% head meat + 20% skeletal meat) and treatment III (75% head meat + 25% skeletal meat). The formulation and processing of control and treated patties were standardised by preliminary trials (Table 1). Phase II

In this study, three different proportions of head meat and heart meat were assessed and compared with Table 1 Formulation for control and treated patties using combination of buffalo head and skeletal meat Ingredients

Control Treatment I Treatment II Treatment III

Control Treatment I Treatment II Treatment III

Head meat (%) – Skeletal meat (%) 67.3 Ice flakes (%) 10.0 Refined vegetable 10.0 oil (%) Condiments (%) 5.0 Refined wheat 3.5 flour (%) Dry spice mix (%) 2.0 Salt (%) 1.7 Tripolyphospate (%) 0.5 Sodium nitrite (ppm) 100

65.6 11.7 – 10.0

61.7 15.6 – 10.0

57.8 19.5 – 10.0

5.0 3.5

5.0 3.5

5.0 3.5

2.0 1.7 0.5 100

2.0 1.7 0.5 100

2.0 1.7 0.5 100

T I (85% head meat and 15% skeletal meat); T II (80% head meat and 20% skeletal meat). T III (75% head meat and 25% skeletal meat); condiments – onion and garlic (4:1).

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control patties from skeletal meat of buffaloes (Table 2). Three different treatments of this experiment were: treatment I (80% head meat + 20% heart meat), treatment II (70% head meat + 30% heart meat) and treatment III (60% head meat + 40% heart meat). On the basis of various quality parameters the best combinations from par I and Phase II were selected for storage study. Phase III

The best meat patties developed in phase I (treatment I) and phase II (treatment II) were packed in low density polyethylene (LDPE) pouches and stored at (4 ± 1 C). They were evaluated for pH, thiobarbituric acid reacting substances (TBARS) number, microbial quality and sensory attributes at 0, 3, 6, 9, 12 and 15 days of storage to determine the keeping quality. Preparation of patties

The problem of dark-colored product using head meat was successfully overcome by washing head meat chunks in chilled water for 7 min to reduce the content of total pigments. As head meat absorbed chilled water to an extent of 10% of its weight, no added water (in the form of ice flakes) was used in all the formulations containing head meat (Table 1). Buffalo skeletal, head and heart meat were minced in Eletrolux mincer (Electrolux, Italy) and patties were prepared by blending different minced meat in a Hobart paddle type mixer (Hobart Corporation, Troy, OH, USA) with salt, phosphate and other ingredients as per the requirement of each treatment. The mixing was continued until a viscous emulsion was formed. About 66 g of emulsion was moulded in a Petri dish (72-mm

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diameter and 15-mm height) to form patties. Moulded patties were cooked in a oven at 180 ± 5C for about 15 min. Then, they were turned upside down and cooked further for about 10 min till the internal temperature reached 75–80 C. The temperature was recorded by a digital probe thermometer (Oakton, Shanghai, China). The patties were cooled, weighed and dimensions were measured.

load cell and 75-mm compression platen probe (P75). Shear force of samples was estimated with a WarnerBratzler blade attached to the same texture analyzer. Five cores (1.5-cm height and 1.5-cm diameter) were taken from patties of each treatment. The crosshead speed was 2 mm s–1. Maximum force required to cut the sample (shear force) was recorded. Thiobarbituric acid reacting substances number

Analysis of raw and cooked patties

pH determination

pH of emulsion and cooked products was determined by blending 10 g of sample with 50 mL of distilled water for 1 min using an Ultra Turrax T 25 tissue homogeniser (Janke and Kunkel, IKA Labortechnik, Staufen, Germany) at 8000 rpm for 1 min. The pH of the suspension was recorded by dipping combined glass electrode of Elico digital pH meter, Model LI 127 (Elico Limited, Hyderabad, India). Proximate composition

The moisture, protein and fat content of meat emulsion and buffalo meat patties were determined by the methods of AOAC (1995). Product yield

Product yield was determined by measuring weight of patties for each treatment and calculating the ratio of cooked weight to raw weight and expressed as a percentage. The diameter and height of the cooked patties were recorded by use of a Vernier caliper at three different points on each patty. The gain in height and decrease in diameter were expressed as percentage. The shrinkage was determined according the equation reported by El-Magoli et al. (1996). Emulsion stability (ES) was determined by heating 25-g emulsion samples at 80 C in a thermostatically controlled water bath for 20 min. After draining out the exudate, the cooked mass was cooled, weighed and the yield was expressed as ES percent (Kondaiah et al. 1985). Texture profile analysis

The textural properties of patties were evaluated using the texturometer (Stable Micro System Model TA.XT 2i ⁄ 25, UK) at Post-Harvest Technology, Central Avian Research Institute, Izatnagar. Texture profile analysis (Bourne, 1978) was performed using central cores of five pieces of each sample (1.5 · 1.5 · 1.5 cm) which were compressed twice to 80% of the original height. A crosshead speed of 2 mm s–1 was used applying 25-kg

International Journal of Food Science and Technology 2008

The TBARS number was determined using the distillation method described by Tarladgis et al. (1960). The OD was multiplied by the factor of 7.8 and TBARS value was expressed as mg malonaldehyde per kilogram of sample as suggested by Koniecko (1979). Microbiological analysis

A 10-g sample of patties was ground in a sterile pestle and mortar with 90-mL sterile 0.1% peptone water. Appropriate dilutions of samples were prepared in sterile 0.1% peptone water and plated, in duplicate, on the growth media by using the pour plate method. Plate Count Agar was used for total plate count and for Psychrotrophs count. The plates were incubated at 35 ± 2 C for 24 h for total plate count and at 4 ± 1 C for 10–14 days for Psychrotrophs count. Following incubation, plates showing 30-300 colonies were counted and expressed as log10 CFU g–1 sample (APHA, 1984). Sensory evaluation

Sensory evaluation method using an 8-point descriptive scale (Keeton, 1983) was followed, where 8 = excellent; 1 = extremely poor. The sensory panel consisted of ten experienced scientists of the division. The panelists were explained about the nature of experiments with out disclosing the identity of samples and were asked to rate their preference on 8-point descriptive scale on the sensory evaluation proforma for different traits. Samples were warmed using microwave oven for 1 min, cut across their centre to make eight equal size and shape (triangular) pieces per patty and served to the panelists. Water was provided to rinse mouth between the samples. The panelists judged the samples for general appearance, flavour, juiciness, texture, binding and overall acceptability. Statistical analysis

Each experiment was replicated three times and the data obtained were analyzed using standard statistical procedures (Snedecor & Cochran, 1994). anova was used to determine significant differences (P < 0.05) among

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Quality of patties from buffalo head and heart meat A. K. Verma et al.

means for the different treatments. Two-way analysis of variance was conducted to analyze the results of the storage studies to determine the effect of treatment and storage period. Results and discussion

Phase I – buffalo meat patties with head meat Physico-chemical properties Emulsion quality. Physico-chemical characteristics of the different emulsions and cooked patties are presented in Table 3. The ES of the control group was significantly (P < 0.05) lower than the treated groups. However, among treatment groups, there were no significant differences in the ES. ES of skeletal meat emulsion recorded in the present study was in agreement with the value reported by Krishnan & Sharma (1990) and Devatkal et al. (2004) as 95.89% in buffalo skeletal meat loaf. Lower ES of skeletal meat emulsion (control) than treated groups might be due to low salt soluble proteins (8.25%) in skeletal meat as compared to head meat (12.02%), as reported by Verma et al. (2008). pH of control emulsion was significantly (P < 0.05) lower than the pH of treatment groups. Low pH of control than treatment

Table 3 Effect of different levels of skeletal meat incorporation on the quality of raw and cooked buffalo head meat patties (HMP)

Parameters

emulsions might be due to lower pH value (5.85) of buffalo skeletal meat than head meat (6.41), as reported by Verma et al. (2008). There was no significant difference in moisture, protein and fat content of control and treated emulsions. Cooking yield and pH. Cooking yield of buffalo skeletal meat patties was significantly (P < 0.05) lower than the cooking yield of patties from treatment groups. However, no significant differences were observed for the cooking yield among treatments. Higher ES of treatment emulsions might be the contributing factor for higher cooking yield of head meat patties (HMP). Patties incorporated with different level of skeletal meat had significantly (P < 0.05) higher pH than control. However, pH of patties among the treatments did not differ significantly. Low pH of skeletal meat patties might be due to lower pH value of buffalo skeletal meat than head meat (Verma et al., 2008).

The moisture content of control patties, and patties from treatment groups were found to be non-significant. Moisture content in all meat patties recorded in the present study was in agreement with the value reported by Pati et al. (1992) in buffalo meat

Proximate composition.

Control

Treatment I

Raw patties (n = 6) ES* (%) 95.16 ± 0.28b pH 6.11 ± 0.04b Moisture (%) 63.11 ± 0.61 Protein (%) 14.83 ± 0.09 Fat (%) 11.03 ± 0.61 Cooked HMP (n = 6) CY (%)** 91.19 ± 0.46b pH 6.18 ± 0.03b Moisture (%) 61.67 ± 0.62 Protein (%) 17.49 ± 0.33 Fat (%) 11.91 ± 0.03 Diameter Decrease (%) 13.70 ± 1.27 Height gain (%) 19.24 ± 1.87b Shrink (%) 7.98 ± 1.29a Texture profile analysis (n = 12) 76.95 ± 5.99 Hardness (N cm–2) Adhesiveness (Ns) )3.78 ± 1.82 Springiness (cm) 0.82 ± 0.01 Cohesiveness 0.28 ± 0.01 Gumminess (N cm–2) 21.61 ± 1.71 17.73 ± 1.44a Chewiness (N cm–1) Shear force (N) 12.01 ± 0.58

Treatment II

Treatment III

96.73 6.46 61.56 14.26 10.73

± ± ± ± ±

0.24a 0.03a 1.05 0.56 0.25

96.44 6.48 61.55 14.27 10.79

± ± ± ± ±

0.25a 0.03a 0.53 0.09 0.09

96.82 6.47 60.87 14.31 9.93

± ± ± ± ±

0.23a 0.04a 4.02 0.41 0.18

93.37 6.46 60.75 17.00 12.81

± ± ± ± ±

0.23a 0.11a 0.33 0.27 0.78

92.71 6.48 61.26 17.08 12.25

± ± ± ± ±

0.28a 0.16a 0.50 0.12 0.48

92.52 6.45 60.88 17.41 11.21

± ± ± ± ±

0.35a 0.13a 0.23 0.13 0.24

13.49 ± 0.79 38.32 ± 2.47a 4.33 ± 0.68b

13.20 ± 0.70 40.46 ± 3.58a 3.54 ± 0.71b

15.02 ± 0.57 46.43 ± 1.10a 6.39 ± 1.52ab

67.96 )0.83 0.77 0.27 18.05 13.89 11.64

66.95 )4.23 0.75 0.27 17.02 12.80 10.99

58.00 ± 3.41 )3.16 ± 2.56 0.76 ± 0.03 0.28 ± 0.01 16.23 ± 2.10 12.46 ± 1.09b 10.40 ± 0.83

± ± ± ± ± ± ±

4.85 2.14 0.02 0.01 1.35 1.14b 0.77

± ± ± ± ± ± ±

5.33 3.04 0.02 0.01 1.54 1.20b 0.52

Mean values bearing same superscripts row-wise do not differ significantly (P < 0.05). ES, emulsion stability; CY, cooking yield; *n = 12; **n = 16.

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patties. There were no significant differences in the protein content between control and treated groups. Devatkal et al. (2004) reported similar results in buffalo meat loaf. Similarly fat content was not significantly between control and treated patties. The degree of shrinkage is important in maintaining quality of meat patties prepared for food service establishments. Therefore, change in diameter and thickness must be considered when benefits of meat additives were evaluated (Das et al., 2008). Control patties did not differ significantly from treated patties. The results in the present study were in agreement with the findings of Suman & Sharma (2003) in low fat ground buffalo meat patties. Buffalo head meat significantly (P < 0.05) increased the gain in height of patties compared with that of control. The higher height gain recorded in treatment groups might be due to higher thermal expansion of head meat. Shrink percentage in treated patties was significantly (P < 0.05) increased because of head meat in the patties formulation. However, no significant difference was observed in the shrink percent of control and patties of treatment III. Shrink percentage in the control patties recorded in present study was in agreement with the results of Suman & Sharma (2003) in low fat ground buffalo meat patties. Shrinkage.

There were no significant differences in the textural attributes except chewiness measured in the present study between control and treated patties (Table 3). Control patties showed significantly (P < 0.05) higher chewiness than that of treated patties. Textural attributes of meat patties recorded in the present study were in agreement with the value reported by Devatkal et al. (2004) in buffalo skeletal meat loaf. In this study, texture profile analysis showed a general declining trend for any of the parameters for treatment patties as compared to control with increasing level of skeletal meat incorporation, except cohesiveness which was almost similar between control and treatments.

Texture profiles.

Sensory properties. Results of sensory evaluation showed that there were no significant differences in all the sensory attributes between skeletal meat patties (control) and patties from treatment groups (Table 4). However, all the sensory attributes among treatment groups did not differ significantly. Krishnan & Sharma (1990) reported similar observations. Overall sensory scores were slightly lower in HMP than skeletal meat patties, but the difference did not turn out to be statistically significant. However, results of sensory evaluation indicated that addition of skeletal meat affected the sensory attributes of HMP.

International Journal of Food Science and Technology 2008

Table 4 Sensory attributes of buffalo head meat patties with different levels of skeletal meat Parameters

Control

Treatment I

Treatment II

Treatment III

Appearance Flavour Juiciness Texture Binding Overall acceptability

6.88 6.84 7.00 6.78 6.97 6.78

6.91 6.72 6.69 6.72 6.88 6.66

6.94 6.84 6.91 6.91 6.94 6.91

6.94 6.78 6.75 6.78 6.94 6.72

± ± ± ± ± ±

0.09 0.11 0.00 0.12 0.12 0.12

± ± ± ± ± ±

0.05 0.10 0.12 0.12 0.13 0.12

± ± ± ± ± ±

0.04 0.09 0.08 0.07 0.12 0.05

± ± ± ± ± ±

0.04 0.09 0.11 0.09 0.12 0.08

Mean values bearing same superscripts row-wise do not differ significantly (P < 0.05). n = 30.

Phase II – head meat patties with heart meat Physico-chemical properties

The physico-chemical characteristics of head meat (raw and cooked) patties with different level of heart meat incorporation are presented in Table 5. ES of treatment groups was not significantly different from control emulsion. Thomas et al. (2006) reported similar results. Heart meat incorporated emulsions had significantly (P < 0.05) higher pH compared to control and this is due to higher pH value of buffalo heart meat (Verma et al., 2008). Similarly higher moisture content of heart meat (78.42%) than skeletal meat (75.85) as reported by Verma et al. (2008) might be the reason for higher moisture content in treated emulsions then control one. However, protein and fat content of control group were significantly higher than treated emulsion. There were no difference in protein and fat content among treatment groups. Emulsion quality.

Cooking yield of HMP with heart meat incorporation was significantly (P < 0.05) higher than patties prepared from skeletal meat. Higher pH and consequently higher water holding capacity of head and heart meat might be the contributing factors for higher cooking yield. However, cooking yield of patties from treatment I and II did not differ significantly. Krishnan & Sharma (1991) reported similar results in buffalo meat products. pH of skeletal meat patties was significantly (P < 0.05) lower than patties from treatment groups. pH of cooked patties followed the similar trend as it was in raw emulsions.

Cooking yield and pH.

Treated patties showed significantly (P < 0.05) higher moisture content than control patties. Moisture content in treated patties was in accordance with value reported by Anjaneyulu et al. (1989) in buffalo meat patties. Higher moisture could be due to

Proximate composition.

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Quality of patties from buffalo head and heart meat A. K. Verma et al.

Table 5 Quality of raw and cooked buffalo head meat patties (HMP) with different levels of heart meat

Parameters

Control

Raw patties (n = 6) ES (%)* 94.89 ± 0.42 pH 6.14 ± 0.03b Moisture (%) 62.85 ± 0.36b Protein (%) 14.43 ± 0.23a Fat (%) 10.83 ± 0.22a Cooked HMP (n = 6) CY (%)** 90.69 ± 0.25b pH 6.18 ± 0.02b Moisture (%) 60.83 ± 0.73b Protein (%) 17.08 ± .29a Fat (%) 13.01 ± 0.23a Diameter** Decrease (%) 11.89 ± 0.98c Height gain (%) 20.46 ± 1.17b Shrink (%) 6.34 ± 0.47 Texture profile analysis (n = 12) Hardness (N) 74.58 ± 2.13a Adhesiveness (Ns) )3.36 ± 1.82 Springiness (cm) 0.79 ± 0.02a Cohesiveness 0.27 ± 0.01a Gumminess (N cm–2) 20.06 ± 1.61a Chewiness (N cm–1) 16.61 ± 1.14a Shear force (N) 11.67 ± 0.96

Treatment I

Treatment II

Treatment III

95.56 6.51 64.61 13.34 9.35

± ± ± ± ±

0.27 0.06a 0.50a 0.11b 0.11b

94.78 6.57 64.25 12.98 9.10

± ± ± ± ±

0.27 0.04a 0.40a 0.10b 0.16b

94.85 6.52 64.42 12.89 9.20

± ± ± ± ±

0.26 0.04a 0.08a 0.32b 0.07b

92.04 6.53 63.78 14.42 10.65

± ± ± ± ±

0.35a 0.03a 0.65a 0.13b 0.47b

91.79 6.59 63.89 15.08 10.61

± ± ± ± ±

0.81a 0.05a 0.21a 0.32b 0.36b

90.78 6.57 62.73 14.56 10.27

± ± ± ± ±

1.13b 0.03a 0.44a 0.28b 0.26b

13.23 ± 0.61cb 36.86 ± 2.21a 4.69 ± 0.36

15.69 ± 0.83ab 41.94 ± 2.37a 6.54 ± 0.61

16.73 ± 0.99a 42.92 ± 2.62a 6.33 ± 0.59

53.97 ± 6.34b )3.17 ± 1.14 0.75 ± 0.01ab 0.26 ± 0.01ab 13.32 ± 1.80b 8.19 ± 0.93b 11.73 ± 1.20

49.69 ± 3.94b )2.03 ± 1.12 0.72 ± 0.01b 0.26 ± 0.01a 11.02 ± 0.95b 7.31 ± 0.60b 10.86 ± 0.93

45.48 ± 3.21b )0.29 ± 0.48 0.64 ± 0.02ab 0.24 ± 0.01b 11.38 ± 0.81b 6.50 ± 0.79b 9.80 ± 1.30

Mean values bearing same superscripts row-wise do not differ significantly (P < 0.05). ES, emulsion stability; CY, cooking yield, * = 12, **n = 16.

incorporation of heart meat (Verma et al., 2008). However, treated patties had significantly lower protein and fat content than control one. Krishnan (1988) reported similar results. Lower fat content in treated patties with heart meat might be due to increased fat loss during broiling as heart meat has lower emulsifying capacity than skeletal meat (Verma et al., 2008). Shrinkage. Heart meat addition in HMP significantly increased percent of diameter decrease and height gain. These results were in agreement with the findings of Kumar (2004). Higher rate of height gain in treated meat patties might be due to poor binding capacity of heart meat in emulsion based meat products and biophysical properties of heart and ⁄ or head meat. There were no significant differences in the shrink percentage of skeletal meat patties and patties from treatment groups. Similar shrink percentage was reported by Suman & Sharma (2003) in low fat ground buffalo meat patties.

Addition of heart meat in the formulations significantly (P < 0.05) decreased all textural parameters of the patties except shear force (Table 5). Hardness significantly decreased with increasing level of heart meat because of soft texture of heart meat

Texture profiles.

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and higher moisture content in compared to skeletal meat. Various workers have reported that product hardness was decreased with increase in moisture (Colmenero et al., 1995; Chin et al., 2004). Springiness value decreased significantly (P < 0.01) with heart meat because of compact binding resulting in loss of elasticity in the product. Similar findings of decrease in hardness, springiness of patties with potato starch were reported (Kumar et al., 2004). Gumminess and chewiness followed the same declining trend as other texture profile parameters on addition of heart meat in HMP. Though there was no significant difference in shear force of control and treated patties but control patties required higher shear force than all other treatments. These results indicated that increased incorporation of heart meat enhanced tenderness of the patties and this was also reflected in texture scores by the panelists (Table 6). Sensory properties. All the sensory attributes except juiciness recorded in the present study indicated significantly lower scores for HMP with different levels of heart meat than control patties (Table 6). Texture of treated patties showed a progressive decrease at higher levels of heart meat inclusion, which was also reflected in the lower Warner-Bratzlar shear force value of these patties.

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Among the treatment groups, scores for various sensory attributes were higher in treatment I i.e. 80% HMP with 20% heart meat.

patties might be due to addition of heart meat and higher microbial load (Table 7). Microbiological quality

Phase III – shelf-life evaluation pH

Effect of refrigerated storage on the quality of different buffalo meat patties is presented in Table 7. There were significant (P < 0.05) differences in the pH of patties between treatments and storage period. Overall days means revealed that pH value significantly (P < 0.05) increased up to 12 days as compared to zero day. pH increase during storage period was reported by Thomas et al. (2006) in buffalo meat patties and Rajkumar et al. (2004) in goat meat patties. Similar finding was also reported by Biswas et al. (2004) in pork patties. Overall treatment means revealed that pH value was significantly (P < 0.05) lower in control than treatment I and treatment II. TBARS number

TBARS number gradually increased from 0.53 to 0.77 mg kg–1 of meat sample as storage period advanced (Fig. 1). Increase in TBARS number with advancement of storage period was also reported by Das et al. (2008). According to Ockerman (1985), rancid flavour can be detected by sensory evaluation at a TBARS number above 1 mg MDA kilogram per sample. These values were well below the acceptable limits of 1–2 mg malonaldehyde per kilogram meat (Witte et al., 1970). Among treatment means, TBARS number of control and treatment II patties did not differ significantly. However, treatment I patties had significantly (P < 0.05) lower TBARS number as compared to control and treatment II patties. Low TBARS number for treatment I patties could be due to washing of head meat. Reduction in TBARS value of washed meat had also been reported by Kulkarni (1989). Similar findings were also reported by Colmenero & Matamoros (1981) in mechanically deboned pork. Higher TBARS number in head-heart meat Table 6 Sensory attributes of buffalo head mead patties with different levels of heart meat Parameters

Control

Appearance Flavour Juiciness Texture Binding Overall acceptability

7.17 7.03 7.00 7.03 7.07 7.17

± ± ± ± ± ±

0.12a 0.03a 00 0.03a 0.07a 0.06a

Treatment I 6.63 6.57 6.70 6.67 6.70 6.60

± ± ± ± ± ±

0.11b 0.12b 0.12 0.11b 0.13b 0.01b

Treatment II 6.57 6.40 6.67 6.57 6.70 6.47

± ± ± ± ± ±

0.12b 0.10b 0.12 0.11b 0.14b 0.08bc

Treatment III 6.33 6.30 6.57 6.47 6.63 6.33

± ± ± ± ± ±

0.15b 0.14b 0.15 0.11b 0.11b 0.09c

Mean values bearing same superscripts row-wise do not differ significantly (P < 0.05), n = 30.

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Microbiological evaluation of meat patties during storage indicated that there was no significant increase in total plate count up to 6th day (Table 7). Total plate count significantly (P < 0.05) increased from 9th day of storage. Overall treatment means of total plate count revealed significant (P < 0.05) lower counts in control and treatment I patties than treatment II patties. Higher ultimate pH of heart meat compared to head and skeletal meat might be the contributing factor for higher microbial counts in treatment II patties (Verma et al., 2008). There was a gradual increase in the psychotropic count of meat patties with advancement of refrigerated storage. However, the increase in counts observed up to 6th day were found to be statistically non-significant. Counts on 9th day were significantly (P < 0.05) higher than zero day. Overall mean of psychotropic counts for control patties was significantly (P < 0.05) lower than treatment I and treatment II patties. Higher psychotropic counts in treated patties could be due to poor handling of raw head and heart meat and their higher ultimate pH than skeletal meat. The microbial counts were well within the limits of log CFUg)1 prescribed for cooked meat products (Shapton & Shapton, 1991). Sensory properties

Sensory analysis during storage showed that HMP with skeletal and heart meat incorporation received lower sensory scores in all the sensory attributes than control patties (data not presented). All the sensory attributes followed a decreasing trend with increasing storage period. There was a significant difference between HMP with skeletal meat (treatment I) and patties with heart meat (treatment II) in case of overall acceptability. The overall acceptability score of patties remained almost unchanged up to day 9 of storage, whereas it decreased significantly with progressive increase in storage. All meat patties were acceptable up to 15 days of storage. Conclusions

An acceptable product with good quality characteristics can be prepared from buffalo head meat with combination of skeletal meat (20%) and heart meat (20%). Almost all physico-chemical and sensory characteristics of HMP incorporated with different proportions of skeletal meat were almost similar to the skeletal meat patties. Among HMP, those containing 20% skeletal meat was found organoleptically most desirable. HMP containing different proportions of heart meat had comparatively lower physico-chemical and sensory properties than skeletal meat patties but, all were organoleptically acceptable. Among these, HMP having

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Quality of patties from buffalo head and heart meat A. K. Verma et al.

Table 7 Changes in pH and microbial quality of head meat patties during refrigerated storage (4 ± 1C) Storage days Parameters

0

3

pH Control 6.16 Treatment I 6.46 Treatment II 6.57 Days mean ± SE 6.39 TPC (log10 CFU g–1) Control 2.79 Treatment I 2.67 Treatment II 2.92 Days mean ± SE 2.79 Psychrotrophic count (log10 Control 0.67 Treatment I 2.04 Treatment II 2.69 Days mean ± SE 1.80

6

9

12

Treatment mean ± SE

15

0.04 0.01 0.01 0.04c

6.19 6.49 6.58 6.40

± ± ± ±

0.03 0.02 0.02 0.04c

6.18 6.50 6.52 6.42

± ± ± ±

0.03 0.03 0.03 0.04bc

6.20 6.50 6.59 6.43

± ± ± ±

0.03 0.03 0.01 0.04abc

6.21 6.52 6.62 6.45

± ± ± ±

0.05 0.03 0.01 0.05ab

6.28 6.53 6.60 6.47

± ± ± ±

0.05 0.03 0.01 0.04a

6.20 ± 0.02c 6.50 ± 0.01b 6.58 ± 0.01a

± 0.06 ± 0.07 ± 0.06 ± 0.04c CFU g–1) ± 0.43 ± 0.42 ± 0.25 ± 0.29c

3.09 2.79 3.30 3.06

± ± ± ±

0.17 0.06 0.24 0.11c

2.99 2.87 3.39 3.09

± ± ± ±

0.08 0.09 0.31 0.12c

3.26 3.62 4.38 3.75

± ± ± ±

0.10 0.09 0.23 0.14b

3.28 3.88 4.65 3.94

± ± ± ±

0.22 0.09 0.22 0.17b

4.60 4.98 4.99 4.86

± ± ± ±

0.19 0.28 0.33 0.15a

3.33 ± 0.11b 3.47 ± 0.15b 3.94 ± 0.16a

0.73 2.38 2.98 2.03

± ± ± ±

0.46 0.17 0.16 0.28bc

0.88 2.49 3.18 2.19

± ± ± ±

0.56 0.14 0.13 0.30bc

0.89 2.94 3.40 2.41

± ± ± ±

0.57 0.14 0.11 0.32ab

0.91 3.20 3.38 2.50

± ± ± ±

0.58 0.13 0.02 0.33ab

1.75 3.32 3.35 2.81

± ± ± ±

0.56 0.03 0.03 0.25a

0.97 ± 0.21c 2.73 ± 0.11b 3.16 ± 0.07a

± ± ± ±

Mean values within a row with different superscripts are significantly (P < 0.05) different Treatment I: 80% head meat + 20% skeletal meat Treatment II: 80% head meat + 20% heart meat.

References

TBARS number (mg malonaldehyde kg–1)

0.9 Control Treatment-I Treatment-II

0.8 0.7 0.6 0.5 0.4

0

2

4 6 8 10 12 Storage period (days)

14

16

Figure 1 Changes in thiobarbituric acid reacting substances number of control and treated patties during storage (––, contorl; ÆÆÆsÆÆÆ, treatment I; –.–, treatment II).

20% heart meat were most desirable. Storage at refrigerated temperature (4 ± 1C) indicated that all meat patties were acceptable up to 15-day storage. Acknowledgments

We wish to thank Head, Division of Post-Harvest Technology, CARI, Izatnagar for providing facilities to evaluate texture analysis of patties. The authors also gratefully acknowledge the Indian Council of Agricultural Research (ICAR), New Delhi for providing research grant in the form of Junior Research Fellowship to the first author.

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Anjaneyulu, A.S.R. & Kondaiah, N. (1990). Quality of buffalo meat nuggets and rolls containing edible byproducts. Journal of Food Science and Technology, 3, 95–99. Anjaneyulu, A.S.R., Sharma, N. & Kondaiah, N. (1989). Evaluation of salt, polyphosphates and their blends at different levels on physicochemical properties of buffalo meat patties. Meat Science, 25, 293–306. AOAC (1995). Official Methods of Analysis. Pp. 1–23. Washington, DC: Association of Official Analytical Chemists. APEDA (2006). Agriculture and Processed Food Products Export Development Authority. New Delhi. Available at: http://www. apeda.com (last accessed 7 January 2008). APHA (1984). Compendium of Methods for the Microbiological Examination of Foods. 2nd edn, (edited by M.L. Speck). Pp. 5–99. Washington, D.C.: American Public Health Association. Biswas, A.K., Keshri, R.C. & Bisht, G.S. (2004). Effect of enrobing and antioxidants on quality characteristics of precooked pork patties under chilled and frozen conditions. Meat Science, 66, 733–741. Bourne, M.C. (1978). Texture profile analysis. Food Technology, 32, 62–72. Chin, K.B., Lee, H.L. & Chun, S.S. (2004). Product characteristics of comminuted sausages as affected by various fat and moisture combinations. Asian-Australasian Journal of Animal Science, 17, 538–542. Colmenero, J.F. & Matamoros, G.E. (1981). Effect of washing on the properties of mechanically deboned meat. Processing of the European Meeting of Meat Research workers, No. 27, I. C. 43, 351– 354. Colmenero, J.F., Carballo, J. & Solas, M.J. (1995). Effect of use of freeze-thawed pork on the properties of Bologna sousages with two fat levels. International Journal of Food Science and Technology, 30, 335–345. Das, A.K., Anjaneyulu, A.S.R., Verma, A.K. & Kondaiah, N. (2008). Physico-chemical, textural, sensory characteristics and storage stability of goat meat patties extended with full-fat soy paste and soy granules. International Journal of Food Science and Technology, 43, 383–392.

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Devatkal, S., Mendiratta, S.K. & Kondaiah, N. (2004). Quality characteristics of loaves from buffalo meat, liver and vegetables. Meat Science, 67, 377–383. El-Magoli, S.B., Laroia, S. & Hansen, P.M.T. (1996). Flavor and texture characteristics of low-fat ground beef patties formulated with whey protein concentrate. Meat Science, 42, 179–193. FAO (2006). FAO Statistics on Livestock Population of the Countries in Asia-Pacific Region. Rome: FAO. Available at: http://www.fao.org (last accessed 7 January 2008). Keeton, J.I. (1983). Effect of fat and NaCl ⁄ phosphate levels on the chemical and sensory properties of pork patties. Journal of Food Science, 48, 878–881. Kondaiah, N., Anjaneyulu, A.S.R., Kesava Rao, V., Sharma, N. & Joshi, H.B. (1985). Effect of salt and phosphate on the quality of buffalo and goat meats. Meat Science, 15, 183–192. Koniecko, E.K. (1979) Other meat properties. In: Handbook for Meat Chemists. Pp 68–69. Wayne, NJ: Avery Publishing Group Inc. Krishnan, K.R. (1988). Development of buffalo meat sausages incorporating offal meat. PhD Thesis, IVRI, Izatnagar, UP, India: Submitted to Deemed University. Krishnan, K.R. & Sharma, N. (1990). Studies on emulsion type buffalo meat sausages incorporating skeletal and offal meat with different levels of pork fat. Meat Science, 28, 51–60. Krishnan, K.R. & Sharma, N. (1991). Studies on the quality characteristics of buffalo skeletal, offal meats and their combinations. Journal of Food Science and Technology, 28, 304–307. Kulkarni, V.V. (1989). Effect of washing on quality of ground buffalo meat. PhD Thesis, Izatnagar, UP, India: Submitted to Indian Veterinary Research Institute, Deemed University. Kumar, R.R. (2004). Effect of some unconventional non-meat extenders on the quality of patties from meat of spent hens. MVSc Thesis, Izatnagar, UP, India: submitted to Indian Veterinary Research Institute, Deemed University. Kumar, M., Sharma, B.D. & Nanda, P.K. (2004). Studies on pork patties: 2. Effect of potato starch on textural, processing and microstructural quality. Fleischwirtschaft International, 84, 66–70.

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Ockerman, H.W. (1985). Quality Control of Post Mortem Muscle Tissues (I): Meat and Additive Analysis. Pp. 90–93. OH, USA: The Ohio State University, Dephasement of Animal Science. Pati, P.K., Anjaneyulu, A.S.R. & Kondaiah, N. (1992). Effect of buffalo fat premix on the quality of quality of patties. Journal of Food Science and Technology, 29, 167–169. Pearson, A.M. & Gillett, T.A. (1997). Raw Materials. In: Processed Meats, 3rd edn. Pp. 126–143. New Delhi: CBS Publisher & Distributors. Rajkumar, V., Agnihotri, M.K. & Sharma, N. (2004). Quality and shelf-life of vacuum and aerobic packed chevon patties under refrigeration. Asian-Australian Journal of Animal Science, 17, 548– 553. Shapton, D.A. & Shapton, N.F. (1991). Principles and Practices for the Safe Processing of Foods. Pp. 377–444.Oxford: Butterworth-Heineman Ltd. Snedecor, G.W. & Cochran, W.G.. (1994). Analysis of Variance. In: Statistical Methods, 9th edn. Pp. 72–148. Ames, Iowa: Iowa State University Press. Suman, S.P. & Sharma, B.D. (2003). Effect of grind size and fat levels on the physico-chemical and sensory characteristics of low-fat ground buffalo meat patties. Meat Science, 65, 973–976. Tarladgis, B.G., Watts, B.M., Younathan, M.T. & Dugan, L.R. Jr (1960). A distillation method for quantitative determination of malonaldehyde in rancid foods. Journal of the American Oil Chemists Society, 37, 44–48. Thomas, R., Anjaneyulu, A.S.R. & Kondaiah, N. (2006). Quality and shelf-life evaluation of emulsion and re-structured buffalo meat nuggets at cold temperature. Meat Science, 72, 373–379. Verma, A.K., Lakshmanan, V., Das, A.K., Mendiratta, S.K. & Anjaneyulu, A.S.R. (2008). Physico-chemical and functional quality of buffalo head meat and heart meat. American Journal of Food Technology, 3, 134–140. Witte, V.C., Krouze, G.F. & Bailey, M.E. (1970). A new extraction method for determining 2-thiobarbituric acid values of pork and beef during storage. Journal of Food Science, 35, 482–585.

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International Journal of Food Science and Technology 2008, 43, 1807–1812

Original article Antioxidant activity of pomegranate rind powder extract in cooked chicken patties Basappa M. Naveena,1* Arup R. Sen,1 Rose P. Kingsly,2 Desh B. Singh2 & Napa Kondaiah1 1 National Research Centre on Meat, Chengicherla, PB No. 19, Uppal (Po), Hyderabad 500039, Andhra Pradesh, India 2 Central Institute of Post Harvest Engineering & Technology, Abohar 152116, Punjab, India (Received 19 June 2007; Accepted in revised form 6 November 2007)

Summary

The radical scavenging activity, reducing power and phenolic composition of pomegranate rind powder extract (RP) were determined and antioxidant properties of RP was evaluated in cooked chicken patties compared with vitamin C (VC) during refrigerated storage. Freshly minced chicken meat were assigned to one of the following six treatments: control (meat without any antioxidant); RP 5, RP 10, RP 15 and RP 20 (5, 10, 15 and 20 mg equivalent RP phenolics 100 g)1 meat, respectively) and VC 50 (50 mg VC 100 g)1 meat). The RP exhibited significantly (P < 0.05) higher reducing power and 1,1-diphenyl-2picrylhydrazyl radical scavenging activity. Incorporation of RP into chicken patties significantly (P < 0.05) reduced the HunterLab L* values compared with control and VC patties. Total phenolic content (as tannic acid equivalent) significantly (P < 0.05) increased from 308 in control to 441 lg g)1 in RP 20 patties. Addition of RP to chicken patties did not affect any of the sensory attributes. The values of thiobarbituric acid reactive substances were significantly (P < 0.05) reduced from 1.530 in control patties to 0.135 mg malonaldehyde kg)1 samples in RP patties. Pomegranate rind powder extract treatment (RP 10, RP 15 and RP 20) substantially inhibited (P < 0.05) lipid oxidation in cooked chicken patties to a much greater extent than VC treatment. Therefore, pomegranate rind powder can be utilized as an excellent natural antioxidant source.

Keywords

Chicken patties, natural antioxidants, phenolics, pomegranate, rind powder.

Introduction

Lipid oxidation may produce changes in meat quality parameters such as colour, flavour, odour, texture and even the nutritional value and therefore is a major cause of quality deterioration in meat products (Fernandez et al., 1997). Minced meats undergo oxidative changes and develop rancidity more quickly than intact muscle as grinding exposes more of the muscle surface to air and microbial contamination. The synthetic phenolic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used as effective additives to retard the development of rancidity in meat products (Barlow, 1990). Many attempts have been made to reduce pigment and lipid oxidation in meats through endogenous and exogenous treatments with antioxidants, in particular with vitamins E and C. Vitamin C has been used as an antioxidant in a great variety of products and is extensively used in the food *Correspondent: E-mail: naveenlpt@rediffmail.com

doi:10.1111/j.1365-2621.2007.01708.x  2008 Institute of Food Science and Technology

industry (Larsen, 1997) The synthetic antioxidants currently used have been found to exhibit various health effects (Shahidi et al., 1992). In addition, there has been growing interest in natural antioxidant because they have greater application in food industry for increasing the stability and shelf-life of food products (Shahidi et al., 1992). Consequently, search for natural additives, especially of plant origin, has notably intensified in recent years. Constant research endeavours have been going on to screen the edible plant materials (Patel & Rajorhia, 1979; Guleria et al., 1983) that could be explored for extracting the antioxidant principles residing in them naturally. Compounds obtained from natural sources such as grains, oilseeds, spices, fruits and vegetables have been investigated (Chen et al., 1996). Extracts from spices, rosemary, thyme and sage are reported to possess antioxidant properties comparable with or greater than BHA & BHT (Kramer, 1985). Pomegranate is an important source of bioactive compounds and has been used for folk medicine for centuries. Most pomegranate fruit parts are known to

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possess enormous antioxidant activity. In India, pomegranate arils are used as such or are made into juice. Fresh juice contains small amount of pectin, ascorbic acid and polyphenolic flavonoids (Aviram et al., 2000). Ozkal & Dinc (1994) reported the presence of tannins, anthocyanins and flavonoids in pomegranate rind. Pomegranate peel is a rich source of tannins and other phenolic compounds (Ozkal & Dinc, 1994). Pomegranate peel ⁄ rind extract has markedly higher antioxidant capacity than the pulp extract in scavenging or preventive capacity against superoxide anion, hydroxyl and peroxyl radicals as well as inhibiting low-density lipoprotein oxidation (Li et al., 2006). Pomegranate peel extract showed an increase in total phenolics, antioxidant capacity and antimutagencity with increase in concentration. (Ghasemian et al., 2006). Pomegranate peel extract has both antioxidant and antimutagenic properties and may be exploited as biopreservative in food applications and neutraceuticals. However, so far, there has been no attempt to investigate the antioxidant properties of pomegranate in meat products. The purpose of this work was to compare the effects of addition of different levels of pomegranate rind powder extract and vitamin C on colour and lipid stability of cooked chicken patties during chilled storage. Materials and methods

Materials

Fresh chicken leg and breast muscles were obtained five times at intervals of 3 to 4 weeks from local poultry processing plants of Hyderabad. Meat samples were stored at 4 C for approximately 4 h before use. Ascorbic acid (vitamin C: VC) was obtained from Merck (Mumbai, India). Preparation of pomegranate rind powder and extract (RP)

Mature and healthy pomegranate fruits (wild type) were washed and cut manually to separate the seeds and rind. Rind was cut into 1 cm2 pieces using a sharp plastic knife and dried in an air circulatory tray drier (Narang Scientific Works, New Delhi, India) at 60 C for 48 h. Dried pieces were cooled and powdered in a heavy duty grinder and sieved using a 60 mesh sieve and packed and stored at room temperature in high-density polyethylene bags till extraction. About 20 g of dried rind powder was mixed with 500 mL boiled distilled water and left for 5 min. The extract was obtained by filtration and analysed for pH, total phenolic content (Escarpa & Gonzalez, 2001), reducing power (Jayaprakasha et al., 2001) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (Singh et al., 2002). Freshly prepared extract was used for each replication.

International Journal of Food Science and Technology 2008, 43, 1807–1812

Preparation of chicken patties

Chilled chicken leg and breast muscles were minced using 8-mm plate followed by 4-mm plates in a meat mincer (Model TC 22; RIO INOX, Sirman, Italy). Six different chicken patty treatments were prepared using rind powder extract (RP) and vitamin C (VC); (i) Control, no additive; (ii) RP 5, 5 mg equivalent RP phenolics 100 g)1 meat; (iii) RP 10, 10 mg equivalent RP phenolics 100 g)1 meat; (iv) RP 15, 15 mg equivalent RP phenolics 100 g)1 meat; (v) RP 20, 20 mg equivalent RP phenolics 100 g)1 meat; (vi) VC 50, 50 mg VC 100 g)1 meat. Sodium chloride (1% w ⁄ w) dissolved in distilled water was added to all samples. The RP was substituted with distilled water in control and VC samples. Immediately after adding distilled water, sodium chloride and RP, samples were thoroughly shaken. After mixing, chicken samples (100 g portions) were formed into patties and cooked in convection type microwave oven (Model MC-767 w ⁄ w; LG Electronics India Pvt. Ltd., New Delhi, India) under grilling with 900 W power for around 20 min until internal temperature reaches 80 C. After cooling to room temperature, the patties were aerobically packaged in a low-density polyethylene pouches and stored at 4 C for 15 days and analysed for total phenolic content (Escarpa & Gonzalez, 2001), pH, instrumental colour, water activity (aw), sensory attributes and thiobarbituric acid reactive substances. pH, water activity and cooking yield

The pH of cooked patty was determined by blending 10 g sample with 50 mL distilled water for 60 s in a homogenizer (Model: MICCRA D8-Si, ART Moderne Labortechnik, D-79379 Mullheim, Germany). The pH values were measured using a standardised electrode attached to a digital pH meter (Model 420 A+; Thermo Orion, Beverly, MA, USA). The water activity (aw) was determined with a Rotronic AG analyzer (Model Hygrolab 3; Grinddelstr. 6, CH-8303 Bassersdorf, UK) at 25 C. Cooking yield was determined by dividing cooked product weight by the raw uncooked weight and multiplying by 100. Instrumental colour

Colorimetric analysis was performed using a HunterLab Miniscan XE Plus colorimeter (Hunter Associates Laboratory Inc., Reston, VA, USA) at day 0 with 25 mm aperture set for illumination D65, 10  standard observer angle. CIE L* (lightness), a* (redness) and b* (yellowness) were measured on the outer surface of cooked chicken patties from five randomly chosen spots.

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Pomegranate and antioxidant activity B. M. Naveena et al.

Thiobarbituric acid reactive substances

The thiobarbituric acid reactive substances (TBARS) value (mg malonaldehyde kg)1) of chicken patties was determined by using the extraction method described by Witte et al. (1970) with slight modifications as the slurry was centrifuged at 3000 · g for 10 min (Centurion Scientific, Model K2 series, East Preston, UK) instead of filtration through Whatman No. 42. Sensory evaluation

A semitrained sensory panel of eight to ten members evaluated the cooked chicken patties on day 0 of storage. The panelists rated each sample for three characteristics (appearance, juiciness and overall palatability) on an eight-point descriptive scale (Keeton, 1983) and three characteristics (off-odour, sweet flavour and chicken flavour) on a five-point rating scale (Leheska et al., 2006; Vasavada et al., 2006). Water was served for cleansing the mouth between samples. Statistical analysis

All data were analysed using SPSS (SPSS version 13.0 for windows; SPSS, Chicago, IL, USA). The cooking yield, pH, aw, instrumental colour and sensory attributes were analysed using one-way anova. A 6 · 4 factorial design with five replicates was employed for storage data (TBARS), with treatments and storage time as main effects using two-way anova. The least significant difference (LSD) was calculated at P < 0.05. Results and discussion

2 1.8

84 82

OD at 700 nm

% Radical scavenging activity

The results of radical scavenging activity of pomegranate rind powder extract (RP) along with vitamin C (VC) are presented in Fig. 1. Increasing the concentration of RP did not significantly increase the radical scavenging

activity up to 300 lg phenolics; however, 400 lg RP phenolics and vitamin C (VC) significantly increased (P < 0.05) the scavenging activity compared with 50 lg RP. Negi & Jayaprakasha (2003) have reported a sharp increase in radical scavenging activity with an increase in the concentration of pomegranate peel extracts up to 25 ppm after which a little increase in radical scavenging activity was observed. The antioxidant activity has been reported to be concomitant with the reducing power (Amarowicz et al., 2000). Figure 2 shows the reducing powers of different concentration of phenolic compounds in RP using potassium ferricyanide method. There was a significant (P < 0.05) increase in reducing power of RP with increase in concentration. At 200–400 lg phenolics, the reducing power of RP is greater than (P < 0.05) those of VC. Negi & Jayaprakasha (2003) have also reported a significant increase in reducing power of pomegranate peel extracts with increase in concentration from 50 to 400 ppm. Reducing properties are generally associated with the presence of reductones (Pin-Der, 1998). Gordon (1990) reported that the antioxidative action of reductones is based on the breaking of free radical chain by the donation of hydrogen atom. Reductones also react with certain precursors of peroxides thus preventing peroxide formation. The pH and phenolic content of fresh RP were found to be 3.9 and 10 mg equivalent mL)1 extract, respectively. The pH, total phenolic content and water activity (aw) of cooked patty is given in Table 1. Raw patty pH and cooking yield did not differ (P > 0.05) between control, RP and VC patties. However, cooked pH significantly (P < 0.05) reduced in RP 15 and RP 20 patties compared with control and RP 5 patties. The total phenolic content of cooked chicken patties was significantly higher in RP 15 and RP 20 patties compared with others. The higher level of phenolics

80 78 76 74 72

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

70 RP 50

RP 100 RP 200 RP 300 RP 400

VC 50

VC 100

Concentrations Figure 1 Radical scavenging activity of pomegranate rind powder extract (RP) at different concentrations of phenolic compounds and vitamin C (VC) by 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. RP 50, 50 lg RP phenolics; RP 100, 100 lg RP phenolics; RP 200, 200 lg RP phenolics; RP 300, 300 lg RP phenolics; RP 400, 400 lg RP phenolics; VC 50, 50 lg VC; VC 100, 100 lg VC.

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0 RP 50

RP 100 RP 200 RP 300 RP 400 Concentration

VC 50

VC 100

Figure 2 Reducing power of pomegranate rind powder extract (RP) at different concentrations of phenolic compounds and vitamin C (VC). RP 50, 50 lg RP phenolics; RP 100, 100 lg RP phenolics; RP 200, 200 lg RP phenolics; RP 300, 300 lg RP phenolics; RP 400, 400 lg RP phenolics; VC 50, 50 lg VC; VC 100, 100 lg VC.

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Table 1 The pH, total phenolics content and water activity (aw) of cooked chicken meat patties with the addition of pomegranate rind powder extract (RP) and vitamin C (VC)

Cooked pH Control RP 5 RP 10 RP 15 RP 20 VC 50

6.39 6.38 6.33 6.28 6.10 6.33

± ± ± ± ± ±

0.00c 0.01c 0.01bc 0.04b 0.04a 0.01bc

Total phenolics (as tannic acid equivalent) (lg g)1) 308.00 309.50 316.00 435.00 441.00 311.50

± ± ± ± ± ±

25.62a 23.16a 33.21a 36.81b 41.78b 32.36a

Table 2 Instrumental colour values of cooked chicken meat patties with the addition of pomegranate rind powder extract (RP) and vitamin C (VC) L* (lightness)

aw 0.929 0.908 0.908 0.906 0.905 0.925

± ± ± ± ± ±

0.01 0.01 0.01 0.00 0.01 0.00

Mean values in the same column bearing the same superscripts do not differ significantly (P < 0.05); each value is a mean ± SE of five replicates. RP 5, 5 mg equivalent (eq.) RP phenolics 100 g)1 meat; RP 10, 10 mg eq. RP phenolics 100 g)1 meat; RP 15, 15 mg eq. RP phenolics 100 g)1 meat; RP 20, 20 mg eq. RP phenolics 100 )1 meat; VC 50, 50 mg VC 100 g)1 meat.

may indicate that this product is nutritionally fortified with the addition of pomegranate rind powder (Leheska et al., 2006). The phenolic compounds are of great interest as they are involved in biochemical and pharmacological effects, including anticarcinogenic and antioxidant effects (Doshi et al., 2006). Several studies have also reported on the relationship between phenolic content and antioxidant activity (Velioglu et al., 1998). Phenolic compounds have attracted an increased attention in the field of nutrition, health and medicine largely because of their anticarcinogenic ⁄ antimutagenic, antiulceric, antiallergic, antiatherogenic, antinflammatory, antimicrobial properties and very importantly, antioxidant activity (Teissedre et al., 1996; Bagchi et al., 1998; Siato et al., 1998; Catterall et al., 2000). Addition of RP did not (P > 0.05) affect the aw of cooked chicken patties even though the values have slightly reduced in RP added patties compared with control. Treatment with RP (P < 0.05) reduced the HunterLab L* (lightness) values compared with control and VC containing patties (Table 2). The VC patties also had lower (P < 0.05) L* values compared with control. No significant difference was observed in HunterLab a* (redness) values. The RP 10 and VC 50 patties showed significantly (P < 0.05) lower and higher HunterLab b* (yellowness) values, respectively compared with control. With the addition of RP, the chicken patties became slightly darker, which might have resulted in lower L* values. Mitsumoto et al. (2005) have reported the discolouration of chicken meat patties with addition of natural antioxidants like tea catechins. In contrast to instrumental colour values, sensory evaluation scores did not reveal any significant difference in appearance scores between control and treated samples. The juiciness scores were reduced (P < 0.05) from 7.26 in

International Journal of Food Science and Technology 2008, 43, 1807–1812

Control RP 5 RP 10 RP 15 RP 20 VC 50

63.80 57.21 57.65 55.03 56.71 60.84

± ± ± ± ± ±

d

0.73 0.51b 0.42b 0.95a 0.74ab 0.90c

a* (redness)

b* (yellowness)

5.01 5.31 5.47 6.01 5.57 5.79

23.34 22.40 21.73 22.50 22.69 24.95

± ± ± ± ± ±

0.17 0.24 0.19 0.20 0.11 0.51

± ± ± ± ± ±

0.50b 0.37ab 0.39a 0.58ab 0.43ab 0.46c

Mean values in the same column bearing the same superscripts do not differ significantly (P < 0.05); each value is a mean ± SE of five replicates. See details of RP and VC at different treatments in Table 1.

control to 7.20 in RP 20 patties compared with others. This might be because of slight reduction in aw values at higher level which may cause dryness. No significant difference was observed in off-odour, sweet flavour, chicken flavour and overall palatability scores at any level. However, there was a slight reduction in chicken flavour and overall palatability scores in RP 20 patties compared with others. Thus, sensory evaluation scores revealed that RP can be incorporated up to 20 mg phenolics 100 g)1 chicken meat patties without affecting any of the sensory attributes. Effect of RP and VC treatments on thiobarbituric acid reactive substances (TBARS) values in cooked chicken patties are shown in Table 3. The RP and VC treatments significantly (P < 0.05) reduced the TBARS values compared with control throughout the storage. The RP treatment (RP10, RP15 and RP20) greatly (P < 0.05) suppressed lipid oxidation in cooked chicken patties compared with VC treatment. However, no difference was observed between RP 10, RP 15 and RP 20 treated patties, indicating that addition of 10 mg equivalent RP

Table 3 Thiobarbituric acid reactive substances (TBARS) values of cooked chicken meat patties with the addition of pomegranate rind powder extract (RP) and vitamin C (VC) during refrigerated storage (mg of malonaldehyde kg)1 meat) Day 0 Control RP 5 RP 10 RP 15 RP 20 VC 50

0.286 0.100 0.075 0.078 0.08 0.110

Day 5 ± ± ± ± ± ±

bA

0.05 0.02aA 0.00aA 0.01aA 0.00aA 0.01aA

0.866 0.410 0.181 0.11 0.132 0.344

Day 10 ± ± ± ± ± ±

cB

0.03 0.07bB 0.02aAB 0.02aAB 0.01aB 0.07bB

1.15 0.371 0.11 0.110 0.123 0.418

± ± ± ± ± ±

Day 15 cC

0.01 0.06bB 0.02aA 0.02aAB 0.00aB 0.02bB

1.53 0.598 0.272 0.135 0.145 0.513

± ± ± ± ± ±

0.08dD 0.15cB 0.07abB 0.01aB 0.01aB 0.08cbB

a–d

Treatments within the same storage conditions (column-wise) with different superscripts are significantly different (P < 0.05). A–DStorage conditions within the same treatment (row-wise) with different superscript are significantly different (P < 0.05); each value is a mean ± SE of five replicates. See details of RP and VC at different treatments in Table 1.

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Pomegranate and antioxidant activity B. M. Naveena et al.

phenolics 100 g)1 meat would be sufficient to inhibit lipid oxidation in chicken meat patties. The TBARS values significantly (P < 0.05) increased in control patties throughout the storage; however, in treated patties, the values increased (P < 0.05) up to 5th of storage only. The increase in TBARS values in all treated samples was very slow and remained low (100 mg kg)1). Based on this, the red pepper pericarp and seed extracts were classified as having high flavonoid content. Conclusions

The antioxidant properties of both red pepper pericarp and red pepper seed are deemed a part of the reason why they are pharmacologically useful, but their mechanisms for antioxidant activity remain unclear. In addition, our study suggests major differences in the mechanisms of action between red pepper pericarp and red pepper seed. In summary, the antioxidant activities of red pepper pericarp and seed extracts were evaluated with various antioxidant assays. All the extracts showed strong antioxidant activity with the testing methods. Overall, the red pepper pericarp extract showed higher scavenging activity than the red pepper seed extract. The red pepper pericarp extract was highly effective in scavenging free radicals, such as DPPH and hydroxyl radicals, and in chelating ferrous ion. It also has high phenolic compounds and flavonoids. The red pepper seed extract was highly effective for superoxide anion scavenging and SOD activity. Generally, the red pepper pericarp and seed extracts were highly effective for the antioxidant properties assayed, with the exceptions of ferrous chelating activity, hydroxyl radical scavenging and SOD activity. These results suggest that red pepper pericarp and seed are healthy foods, having radical scavenging and antioxidant activities. Finally, further studies are warranted for the isolation and identification of their individual phenolic compounds, and in vivo studies are needed to better understand their mechanisms of action as antioxidants. In addition, it would be extremely useful to utilise red pepper seed waste in meat and fish products, as an alternative natural antioxidant to synthetic antioxidants used in the food industry. However, more research is necessary to examine the functionality of red pepper in meat and fish products. References Ahn, J., Grcu¨n, I.U. & Fernando, L.N. (2002). Antioxidant properties of natural plant extracts containing polyphenolic compounds in cooked ground beef. Journal of Food Science, 67, 1364–1369. Amarowicz, R., Naczk, M. & Shahidi, F. (2000). Antioxidant activity of crude tannins of canola and rapeseed hulls. Journal of the American Oil Chemists’ Society, 77, 957–961.

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Aruma, O.I. (1994). Deoxyribose assay for detecting hydroxyl radicals. Methods in Enzymology, 233, 57–66. Barz, W. & Hoesel, W. (1977). Metabolism and degradation of phenolic compounds in plants. Phytochemistry, 12, 339–369. Block, G. & Langseth, L. (1994). Antioxidant vitamins and disease prevention. Food Technology, 48, 80–84. Botsoglou, N.A., Christaki, E., Fletouris, D.J., Florou-Paneri, P. & Spais, A.B. (2002). The effect of dietary oregano essential oil on lipid oxidation in raw and cooked chicken during refrigerated storage. Meat Science, 62, 259–265. Brand-Williams, W., Cuvelier, M.E. & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LebensmittelWissenschaft und Technologie, 28, 25–30. Branen, A.L. (1975). Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. Journal of the American Oil Chemists’ Society, 52, 59–63. Carbonaro, M., Virgili, F. & Carnovale, E. (1996). Evidence for protein-tannin interaction in legumes: implications in the antioxidant properties of faba bean tannins. Lebensmittel-Wissenschaft Technologie, 29, 743–750. Chung, S.K., Osawa, T. & Kawakishi, S. (1997). Hydroxyl radical scavenging effects of spices and scavengers from brown mustard (Brassica niger). Bioscience, Biotechnology, and Biochemistry, 61, 118–123. Cotelle, N., Bernier, J.L., Henichart, J.P., Catteau, J.P., Gaydou, E. & Wallet, J.C. (1992). Scavenger and antioxidant properties of tensynthetic flavoned. Free radical Biology and Medicine, 13, 211– 219. Deepa, N., Kaura, C., Singh, B. & Kapoor, H.C. (2006). Antioxidant activity in some red sweet pepper cultivars. Journal of Food Composition and Analysis, 19, 572–578. Dinis, T.C.P., Madeira, V.M.C. & Almeida, L.M. (1994). Action of phenolic derivatives (acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics, 315, 161–169. Diplock, A.T. (1997). Will the good fairies please prove us that vitamin E lessens human degenerative disease?. Free Radical Research, 27, 511–532. Duh, P.D. (1998). Antioxidant activity of burdock (Arctium lappa Linne): its scavenging effect on free radical and active oxygen. Journal of the American Oil Chemists’ society, 75, 455–461. Gnayfeed, M.H., Daood, H.G., Biacs, P.A. & Alcaraz, C.F. (2001). Content of bioactive compounds in pungent spice red pepper (paprika) as affected by ripening and genotype. Journal of the Science of Food and Agriculture, 81, 1580–1585. Hagerman, A.E., Riedl, K.M., Jones, G.A., Sovik, K.N., Ritchard, N.T. & Hartzfeld, P.W. (1998). High molecular weight plant polyphenolics (tannins) as biological antioxidants. Journal of Agricultural and Food Chemistry, 46, 1887–1892. Hasler, C.M. (1998). Functional foods: their role in disease prevention and health. Food Technology, 52, 63–69. Hornero-Me´ndez, D., Guevara, R.G.L. & Mı´ nguez-Mosquera, M.I. (2000). Carotenoid biosynthesis changes in five red pepper (Capsicum annuum, L.) cultivars during ripening. Cultivar selection for breeding. Journal of Agricultural and Food Chemistry, 48, 3857– 3864. Howard, L.R., Talcott, S.T., Brenes, C.H. & Villalon, B. (2000). Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as influenced by maturity. Journal of Agricultural and Food Chemistry, 48, 1713–1720. Iqbal, S., Bhanger, M.I. & Anwar, F. (2007). Antioxidant properties and components of bran extracts from selected wheat varieties commercially available in Pakistan. Lebensmittel-Wissenschaft Technologie, 40, 361–367. Ito, N., Fukushima, S., Hasegawa, A., Shibata, M. & Ogiso, T. (1983). Carcinogenicity of butylated hydroxyanisole in F344 rats. Journal of the National Cancer Institute, 70, 343–347.

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International Journal of Food Science and Technology 2008, 43, 1824–1831

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Original article Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic Mariana A. Moraes, Gabriela S. Rosa & Luiz A. A. Pinto* Unit Operations Laboratory, Department of Chemistry, Fundac¸a˜o Universidade Federal do Rio Grande – FURG, P.O. Box 474, 96201-900, Rio Grande, Rio Grande do Sul, Brazil (Received 9 July 2007; Accepted in revised form 6 December 2007)

Summary

The moisture equilibrium isotherms of garlic and apple were determined at 50, 60 and 70 C using the gravimetric static method. The experimental data were analysed using GAB, BET, Henderson–Thompson and Oswin equations. The isosteric heat and the differential entropy of desorption were determined by applying Clausius–Clapeyron and Gibbs–Helmholtz equations, respectively. The GAB equation showed the best fitting to the experimental data (R2 > 99% and E% < 10%). The monolayer moisture content values for apple were higher than those for garlic at the studied temperatures; the values varied from 0.050 to 0.056 and from 0.107 to 0.168 for garlic and apple, respectively. The isosteric heat and the differential entropy of desorption were estimated in function of the moisture content. The values of these thermodynamic properties were higher for apple (in range 48–100 kJ mol)1 and 14–150 J mol)1 K)1) than for garlic (in range 43– 68 kJ mol)1 and 0–66 J mol)1 K)1). The water surface area values decreased with increasing temperature. The Kelvin and the Halsey equations were used to calculate the pore size distribution.

Keywords

Apple, drying, fruits ⁄ vegetables, garlic, physicochemical properties.

Introduction

Garlic (Allium sativum) has been cultivated for centuries all over the world on account of its culinary and medicinal properties. Garlic is dehydrated into different products such as powders, flakes and slices. Dehydrated garlic has great commercial value and is used as a spice or standard ingredient in prepared foods and formulations (Pezzutti & Crapiste, 1997). Apple cultivation is a relevant economic activity in south region of Brazil, being that cultivates Fuji and Gala constitutes about 95% of the Brazilian production (Wosiacki et al., 2004). The apples are consumed either fresh or in the form of various processed products such as juice, jam, marmalade and dried (Sacilik & Elicin, 2006). The knowledge of the relationship between moisture content and water activity is essential for drying and storage. At the end of the drying process, the product moisture content reaches a value that corresponds to the equilibrium with the surrounding atmosphere and its mass becomes stationary. This thermodynamic equilibrium is characterised for the equilibrium isotherms, and its determination is essential for the better understanding of modelling problems on drying operations *Correspondent: Fax: +55 53 3233 8745; e-mail: [email protected]

(Kouhila et al., 2001). Knowledge of the sorption equilibrium is also important for predicting stability and quality changes during packaging and storage of dehydrated foods and formulations (Pezzutti & Crapiste, 1997). The widely accepted criteria used to select the most appropriate sorption model were the degree of fit to the experimental data and the simplicity of the model (Simal et al., 2007). Thermodynamic parameters such as isosteric heat and differential entropy of sorption determine the end-point to which food must be dehydrated in order to achieve a stable product with optimal moisture content, and yield the theoretical minimum amount of energy required to remove a given amount of water from the food. These parameters also provide an insight into the microstructure associated with the food as well as the theoretical interpretation of physical phenomena occurring at the food–water interface (Togrul & Arslan, 2007). The isosteric heat of sorption is the required energy to remove water from the mass unit of a solid matrix. It can be considered an indicative of the intermolecular attraction forces between sorption sites and water. The change in the isosteric heat of sorption with the moisture content of the sample indicates the availability of polar sites to water vapour as desorption ⁄ adsorption proceeds (Kumar et al., 2005). The heat of sorption of water is important for modelling various food processes and

doi:10.1111/j.1365-2621.2008.01716.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic M. A. Moraes et al.

food storage, as well as for designing the equipment of some processes (Delgado & Sun, 2002). The differential entropy of a material is proportional to the number of available sorption sites at a specific energy level (McMinn & Magee, 2003). The Kelvin equation is the most commonly employed model for evaluation of the pore size distribution in porous materials with mesopores size, but it does not take into account the thickness of the layers formed on the porous surface prior to condensation. The Halsey equation has been studied by many researchers to predict the thickness of these layers (Lastoskie et al., 1993). The aims of the present study were to determine the equilibrium isotherms of garlic and apple at 50, 60 and 70 C, to fit a suitable model for describing the sorption characteristics and to determine thermodynamic functions as the isosteric heat and the differential entropy of desorption, the surface area and the pores sizes distribution from experimental data. Material and methods

Material

The utilised raw materials were garlic (Allium sativum L.) cultivar amarante and apple Fuji (Mallus percicae). The moisture analyses of samples were realised according to AOAC (1995) by the vacuum oven method.

in the literature. Some of these models are based on theories of the mechanism of sorption, others have been purely empirical, or semi-empirical (Kaymak-Ertekin & Gedik, 2004). Al-Muhtaseb et al. (2002) reviewed moisture sorption characteristics of food products, and the applicability of various mathematical models was discussed. However, because of complex composition and structure of foods, mathematical prediction of sorption behaviour is difficult. Some of the mostly applied equations are GAB, BET, Henderson–Thompson and Oswin (Kaya & Kahyaoglu, 2007). The GAB and the BET models have a theoretical basis, whereas the other models are empirical or semiempirical (Adebowale et al., 2007). The parameters of the GAB (eqn 1) and BET (eqn 2) models have a physical meaning. In the GAB and BET models, Me is the equilibrium moisture content, aw is the water activity, Mm is the water content corresponding to saturation of all primary sites by one water molecule (namely monolayer moisture content), and CG, K and CB are energy constants. The Henderson–Thompson (eqn 3) and the Oswin (eqn 4) equations are empirical models and a1, a2, b1 and b2 are constants. All equations are shown below. Me ¼

Mm  CG  K  aw ð1  K  aw Þ  ð1  K  aw þ CG  K  aw Þ Me ¼

M m  C B  aw ð1  aw Þ  ð1  aw þ CB  aw Þ

ð1Þ

ð2Þ

Experimental procedure

Equilibrium moisture contents of samples were determined at 50, 60 and 70 C. The static gravimetric method with sulphuric acid solutions at different concentrations was used. Initially, the samples of garlic and apple were peeled and cut uniformly in small pieces of c. (0.5 · 0.5 · 0.3) cm of size. The samples were put in glass jars. It was used as an initial mass of c. 3 g in each jar. Eleven jars were filled until a quarter depth with different concentrations of sulphuric acid solutions (20– 70% p ⁄ p) to keep the water activity of 0.06–0.89 inside the jars, according to Perry (1984). The samples, placed on support in each jar, were not in contact with the acid solution. The jars were placed in an incubator with controlled temperature (±1 C) for c. 20 days. Samples were weighed at regular intervals and the equilibrium was judged to have been attained when the difference between three consecutive weighing did not exceed 0.001 g. When the equilibrium conditions were reached, the moisture content analysis was carried out. Each experiment was accomplished in triplicate. Calculation procedure

Numerous mathematical models for the description of the moisture sorption behaviour of foods are available

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

 Me ¼

 lnð1  aw Þ 1=b1 a1 

M e ¼ a2

aw 1  aw

ð3Þ

b2 ð4Þ

Non-linear regression analysis was done with the experimental data of equilibrium isotherms of garlic and apple, using the statistica for Windows 5.1 (StatSoft, Inc., Tulsa, OK, USA), which estimates the parameters of eqns 1, 2, 3 and 4. The fit of different models to observations was evaluated with the determination coefficient (R2) and mean relative error (E%), which is defined in eqn 5.  1 0   n  Mi  Mi  X e p 100 @ A ð5Þ E% ¼ N Mie i¼1 where Mei is the experimental value, Mpi is the predicted value and N is the number of experimental points. The net isosteric heat of sorption can be determined from moisture sorption data using the equation derived from the Clausius–Clapeyron equation (eqn 6), and the

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Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic M. A. Moraes et al.

isosteric heat of sorption was calculated by eqn 7 (Arslan & Togrul, 2005). dðln aw Þ qst ¼ dð1=TÞ R

ð6Þ

Qst ¼ qst þ DHvap

ð7Þ

where aw is the water activity, T is the temperature (K), qst the net isosteric heat of sorption (kJ mol)1), R is the universal gas constant (kJ mol)1 K)1), Qst the isosteric heat of sorption (kJ mol)1) and DHvap the heat of vaporisation of water (kJ mol)1 water). The differential entropy of desorption (Sd) can be calculated from Gibbs–Helmholtz equation, as presented in eqn 8 (Simal et al., 2007). qst  G Sd ¼ T

ð8Þ

where the free Gibbs energy is calculated as: G ¼ RT ln aw

ð9Þ

Substituting eqn 9 in eqn 8, and after rearranging, the final form is eqn 10. qst Sd  ð10Þ  ln aw ¼ RT R where Sd is the differential entropy (kJ mol)1 K)1), qst is the net isosteric heat of sorption (kJ mol)1 K)1), G is the free Gibbs energy (kJ mol)1), R the universal gas constant (kJ mol)1 K)1), T is absolute temperature (K) and aw is the water activity. The net isosteric heat (qst) and differential entropy Sd of desorption can be calculated by plotting ln(aw) vs. 1 ⁄ T, for a specific moisture content of the material, and determining the slope ()qst ⁄ R) and intercept (Sd ⁄ R). This procedure is repeated for many values of moisture content to determine qst and Sd dependency with the moisture content (Delgado & Sun, 2002; Kaya & Kahyaoglu, 2007). Specific surface area is an important role in determining the water-binding properties of particulate materials. The values of water surface area, given in m2 g)1 of solid, can be determined from eqn 11, using the monolayer moisture values (Cassini et al., 2006). S0 ¼ Mm

1 N0 AH2 O ¼ 3:5  103 Mm PMH2 O

ð11Þ

where S0 is the surface area (m2 g)1), Mm is the monolayer moisture content, PMH2 O is the molecular weight of water (18 g mol)1), N0 is the number of Avogadro (6 · 1023 molecules per mole) and AH2O is the area of water molecule (10.6 · 10)20 m2). The average pore size at any given moisture content was determined by the Kelvin and the Halsey equations.

International Journal of Food Science and Technology 2008

The Kelvin equation, eqn 12, is used for the calculation of critical radius (Singh et al., 2001). rc ¼

2rVM RT lnð1=aw Þ

ð12Þ

The thickness of the adsorbed layer of water was calculated from the Halsey equation (eqn 13).   5 1=3 t ¼ 0:354  ð13Þ ln aw Pore radius Rp is obtained by the sum of the critical radius rc, and the multilayer thickness, t, showed in eqn 14. R p ¼ rc þ t

ð14Þ

where rc is the critical radius (m), r is the surface tension (N m)1), VM is the molar volume of sorbate (m3 mol)1), R is the universal gas constant (kJ mol)1 K)1), T is the temperature (K), aw is the water activity, t is the multilayer thickness (m) and Rp is the pore radius (m). The volume values of the liquid (with relation to 1 kg of dry material) contained in the pores with a radius Rp, was calculated according to eqn 15. V¼M

1 q

ð15Þ

where V is the volume of liquid (m3 kg)1 dried material), M is the moisture content and q is the density (kg m)3). Results and discussion

The initial moisture content values for the samples of garlic and apple were 1.94 ± 0.09 and 5.66 ± 0.41 g water per gram dry matter (dry basis), respectively. Through the values of water activity (aw) obtained from Perry (1984) and the experimental data of equilibrium moisture content (Me) of the materials, at the three studied temperatures, was plotted the curves Me vs. aw, showed in Fig. 1 for garlic and in Fig. 2 for apple. The sigmoid shapes of the isotherm curves at different temperatures, which are typical of food isotherms, can be observed in Figs 1 and 2. In the first segment (with low aw) of the S-shaped sorption isotherm curves, garlic and apple adsorbed relatively lower amounts of moisture per unit increase in water activity. However, larger amount of moisture was adsorbed at higher aw. Similar behaviour has been reported by other authors for different foods (Sanni et al., 1997; McLaughlin & Magee, 1998; Togrul & Arslan, 2007). The data for garlic (Fig. 1) also indicate that the equilibrium moisture content decreased with increasing temperature, at constant aw, thus indicating that the materials became

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic M. A. Moraes et al.

Figure 1 Sorption isotherms of garlic at different temperatures, utilising the GAB model.

the water activity. The same behaviour was observed by Simal et al. (2007) for pineapple, Kaymak-Ertekin & Gedik (2004) for grapes and apricots and Roman et al. (1982) for apples. Non-linear regression was done by the least square method, utilising GAB, BET, Henderson–Thompson and Oswin models, with the isotherm data obtained experimentally. The adjustment results at different temperatures are shown in Tables 1 and 2 for garlic and apple, respectively. Analysing Tables 1 and 2, it can be verified that the GAB model presented the best fitting, showed in Figs 1 and 2, with high determination coefficients (R2 > 99%) and low mean relative error. The fit of a model is good enough practical purposes when E% is less than 10% (Kaymak-Ertekin & Gedik, 2004). The GAB model has been considered the best fit model for majority of food materials up to aw > 0.9, whereas the BET model gives a good fit to data for aw < 0.5 (Ariahu et al., 2006). According to Lomauro et al. (1985), the GAB equation gave the best fit for more than 50% of the fruits, meats and vegetables analysed. In Tables 1 and 2, it can be verified that the monolayer moisture content decrease with the increase of the temperature. This behaviour is not so clear for garlic (Table 1) although a strong trend is shown. The monolayer moisture content indicates

Table 1 Adjustment parameters of isotherms models of garlic at selected temperatures Garlic Constants Figure 2 Sorption isotherms of apple at different temperatures, utilising the GAB model.

less hygroscopic. This trend may be due to a reduction in the total number of active sites for water-binding as a result of physical and ⁄ or chemical changes in the product induced by temperature. In Fig. 2, the isotherms for apples present the same behaviour that the isotherms for garlic in low water activities range and then the isotherms crosses at a water activity range 0.6–0.8, this phenomenon occurs in food rich in sugars (Simal et al., 2007). For water activities at 0.8 or above, data of equilibrium moisture content at 50 C are the same or below those at 70 C. The latter behaviour may be attributed to the effect of temperature on the physicochemical state and the solubility of the sugars (Pezzutti & Crapiste, 1997). The solubility of sugars increases with the temperature converting the crystalline sugar into sugar solution, and thus lowering

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

50 °C

GAB Mm (d.b.) 0.056 ± 0.004 7.17 ± 1.77 CG K 0.98 ± 0.01 E (%) 9.76 99.36 R2 (%) BET 0.051 ± 0.003 Mm (d.b.) 10.08 ± 2.95 CB E (%) 13.25 94.93 R2 (%) Henderson–Thompson 4.305 ± 0.224 a1 0.76 ± 0.04 b1 E (%) 28.53 97.43 R2 (%) Oswin a2 0.095 ± 0.003 0.72 ± 0.02 b2 E (%) 12.45 R2 (%) 99.12

60 °C

70 °C

0.052 ± 0.003 3.55 ± 0.86 0.98 ± 0.02 9.31 99.29

0.050 ± 0.002 1.75 ± 0.40 0.99 ± 0.01 9.61 99.44

0.048 ± 0.004 4.26 ± 1.05 16.01 96.84

0.030 ± 0.006 6.06 ± 3.96 23.77 83.18

4.169 ± 0.171 0.703 ± 0.03 27.68 98.15

3.835 ± 0.111 0.61 ± 0.02 26.76 98.83

0.084 ± 0.002 0.77 ± 0.02 9.24 99.24

0.068 ± 0.002 0.87 ± 0.02 17.05 99.45

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Table 2 Adjustment parameters of isotherms models of apple at selected temperatures Apple Constants

50 °C

GAB Mm (d.b.) 0.168 ± 0.006 26.33 ± 7.23 CG K 0.98 ± 0.01 E (%) 9.02 99.07 R2 (%) BET 0.146 ± 0.002 Mm (d.b.) 39.51 ± 5.54 CB E (%) 3.92 99.2 R2 (%) Henderson–Thompson 1.793 ± 0.04 a1 0.84 ± 0.06 b1 E (%) 89.47 R2 (%) 94.92 Oswin 0.332 ± 0.014 a2 0.67 ± 0.03 b2 E (%) 24.39 97.86 R2 (%)

60 °C

70 °C

0.130 ± 0.002 25.39 ± 8.78 0.99 ± 0.02 5.77 99.70

0.107 ± 0.003 15.39 ± 5.48 0.99 ± 0.01 8.56 99.53

0.119 ± 0.005 46.11 ± 19.78 9.79 92.62

0.106 ± 0.004 21.65 ± 6.35 9.23 95.54

1.866 ± 0.030 0.63 ± 0.04 282.05 95.80

1.703 ± 0.029 0.53 ± 0.04 472.94 95.54

0.237 ± 0.012 0.83 ± 0.03 53.64 98.31

0.211 ± 0.015 1.04 ± 0.05 98.82 97.51

the quantity of water strongly adsorbed in material sites, and is considered an important measure to know the stable conditions of conservation of food materials. Iglesias & Chirife (1982) studied this phenomenon and verified that it occurs because of the enzymatic reactions and the protein alteration contained in the material. The values of the two GAB parameters, CG and K (Tables 1 and 2) have been found to decrease with increasing temperature. Such decreasing trends to reveal that the binding energies associated with the mono and multilayer sorption of water to the garlic and apple decrease with increase in temperature. These trends have been to be quite common for many foods (Chen & Jayas, 1998; Das & Das, 2002). CG is related to the difference of the chemical potential in the upper layers and in the monolayer, while K is related to this difference in the pure liquid adsorbed and in the upper layers. Thus, the K parameter is, practically without exception, near but less than unity. This fact constitutes a definitive characteristic of the GAB isotherm (Simal et al., 2007). Figures 3 and 4 present the results of )ln(aw) vs. (1 ⁄ T) at the three analysed temperatures, varying the equilibrium moisture content (Me) from 5% to 40%, in dry basis (d.b.), for garlic and apple, respectively. The values of net isosteric heat of desorption, qst, were calculated from the data of angular coefficients in Figs 3 and 4, according to eqn 10. The values of heat of

International Journal of Food Science and Technology 2008

Figure 3 )ln(aw) vs. (1 ⁄ T) curves of garlic, at the analysed temperatures.

Figure 4 )ln(aw) vs. (1 ⁄ T) curves of apple, at the analysed temperatures.

desorption (Qst), in function of the equilibrium moisture content, calculated by eqn 7, are shown in Fig. 5 for garlic and apple, at 60 C (drying temperature). It can be verified through Fig. 5 that the values of heat of desorption for apple were higher than those for garlic. In this figure, we can also verify for the samples that with the increase of equilibrium moisture content, the heat of desorption approaches the heat of vaporisation of pure water at the same temperature. This occurs because as more water is presented in the material the binding energy between water molecules becomes weaker, approaching that of the water molecules in the liquid state. When the material presents low moisture contents, the energy of interaction between water molecules and primary sorption sites in the food solids is greater than the energy that binds the water molecules together in succeeding layers of water.

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic M. A. Moraes et al.

Figure 5 Isosteric heat of desorption of garlic and apple as a function of equilibrium moisture content and heat of vaporisation of pure water, at 60 C.

Isosteric heat of desorption of apple increased to a maximum and then decreased with the increase in moisture content according to Fig. 5. The same phenomenon was observed in various studies of food materials, as the case of mulberry (Maskan & Gogus, 1998) and cookies and corn snacks (Palou et al., 1997). Decrease of the heat of desorption below this value of moisture content was qualitatively explained by Maskan & Gogus (1998) in function of the binding energy of water contained in the material sites. The maximum isosteric heats of apple were obtained in the moisture content of 10% d.b. and were 98.9 kJ mol)1. The maximum enthalpy value indicates the covering of the strongest binding sites and the greatest water–solid interaction. The covering of less favourable locations and the formation of multilayers then follow, as shown by the decrease in enthalpy with increasing moisture content. Differential entropy values (Sd) were calculated from the y-intercept of these straight lines, determined through linear regression for the six values of equilibrium moisture content, according to eqn 10. Figure 6 shows the differential entropy for garlic and apple as a function of moisture content, at 60 C. In Fig. 6, the values of differential entropy of apple were lower than those of garlic. These values increase with the increase in moisture content, but remain negative values. This is an indication of thermodynamic compensation between the heat and entropy of moisture sorption by the materials (garlic and apple). At lower moisture content values, the water molecules are tightly bound on the sorbent surfaces and therefore have low degree of freedom resulting in low entropy of sorption. At higher moisture contents, the water molecules are sorbed on multilayer on the top of the tightly bound first layer. The multilayer

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Figure 6 Differential entropy of desorption of garlic and apple as a function of equilibrium moisture content, at 60 C.

water molecules have greater degree of freedom and hence higher entropy of sorption. Moisture adsorption is essentially exothermic while desorption is mainly endothermic (Ariahu et al., 2006). Analysing Fig. 6 can be verified that the differential entropy of apple was lower at lower moisture content, but it then increased rapidly with increase in moisture content in moisture of 10% [dry basis (d.b.)]. Below a moisture content of 10%, the differential entropy of apple increased sharply with decrease in moisture content. Table 3 presents the water surface area of garlic and apple between 50 and 70 C. These values were estimated using eqn 11 and the monolayer moisture contents were obtained by GAB model. The results presented in Table 3 indicate that the total surface area available for desorption decreased with increasing temperature. The values of surface area for garlic were lower than those for apple as the monolayer moisture values for garlic were lower in comparison with apple. The large surface area of many foodstuffs is due to the existence of an intrinsic microspore structure in these materials (Calzetta Resio et al., 2000). Tolaba et al. (2004) for quinoa grains, reported the surface area values of 303.45, 297.85 and 206.5 m2 g)1 for adsorption and 349.65, 303.1 and 200.55m2 g)1 for Table 3 Water surface area (S0) of garlic and apple, at different temperatures

T (°C)

S0 garlic (m2 g)1 solid)

S0 Apple (m2 g)1 solid)

50 60 70

315 301 196

588 455 374.5

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References

Figure 7 Integral curve of liquid volume in the pores vs. its radius of garlic and apple, at 60 C.

desorption, for the temperatures of 20, 30 and 40 C, respectively. Figure 7 gives the relation of the pore volume vs. its radius. It can be verified in Fig. 7 that the pores sizes varied from 0.5 to 30 nm during desorption of garlic and apple depending on moisture content. At higher moisture levels, the pores sizes were larger. According to these values, garlic and apple pores can be classified as microspores and mesopores according to IUPAC (International Union of Pure & Applied Chemistry) classification (Lastoskie et al., 1993). Conclusion

The GAB model presented the best fitting the equilibrium isotherms data of garlic and apple, with high determination coefficient values (R2 > 99%) and low mean relative error values (E% < 10%). The monolayer moisture content values (Mm) for garlic and apple, at the analysed temperatures, were estimated in the range 5.0–5.6% and 10.7–16.8%, in d.b., respectively. The heat of desorption values for apple were higher than those for garlic calculated through the Clausius– Clapeyron equation. The Gibbs equation was used to calculate the differential entropy. The values of differential entropy for garlic were found to be greater than those for apple, and the differential entropy for apple decrease with decreasing moisture content until reach a minimum value ()150 J mol)1 K)1), and then increase sharply. The water surface area values decreased with increasing of temperature. The pores of garlic and apple were determined with the Kelvin and the Halsey equations, and they were found to be microspores and mesopores according to IUPAC and the pores sizes varied from 0.5 to 30 nm.

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Adebowale, A.R., Sanni, L., Awonorin, S., Daniel, I. & Kuye, A. (2007). Effect of cassava varieties on the sorption isotherm of tapioca grits. International Journal of Food Science and Technology, 42, 448–452. Al-Muhtaseb, A.H., McMinn, W.A.M. & Magee, T.R.A. (2002). Moisture sorption isotherm characteristics of food products: a review. Trans IChemE, 80, 118–128. AOAC (1995). Official Methods of Analysis, 16th edn. Chapter 37, p. 4. Washington: Association of Official Analytical Chemists Inc. Ariahu, C.C., Kaze, S.A. & Achem, C.D. (2006). Moisture sorption characteristics of tropical fresh water crayfish (Procambarus clarkii). Journal of Food Engineering, 75, 355–363. Arslan, N. & Togrul, H. (2005). Modelling of water sorption isotherms of macaroni stored in a chamber under controlled humidity and thermodynamic approach. Journal of Food Engineering, 69, 133–145. Calzetta Resio, A.N., Tolaba, M.P. & Suarez, C. (2000). Some physical and thermal characteristics of amaranth starch. Food Science and Technology International, 6, 371–378. Cassini, A.S., Marczak, L.D.F. & Norena, C.P.Z. (2006). Water adsorption isotherms of texturized soy protein. Journal of Food Engineering, 77, 194–199. Chen, C. & Jayas, D.S. (1998). Evaluation of the GAB equation for the isotherms of agricultural products. Transaction of the ASAE, 41, 1755–1760. Das, M. & Das, S.K. (2002). Analysis of moisture sorption characteristics of fish protein myosin. International Journal of Food Science and Technology, 37, 223–227. Delgado, A.E. & Sun, D. (2002). Desorption isotherms for cooked and cured beef and pork. Journal of Food Engineering, 51, 163–170. Iglesias, H.A. & Chirife, J. (1982). Handbook of food isotherms. Pp. 3, 283, 292. New York, NY: Academic Press. Kaya, S. & Kahyaoglu, T. (2007). Moisture sorption and thermodynamic properties of safflower petals and tarragon. Journal of Food Engineering, 78, 413–421. Kaymak-Ertekin, F. & Gedik, A. (2004). Sorption isotherms and isosteric heat of sorption for grapes, apricots, apples and potatoes. Lebensmittel-Wissenschaft und Technologie, 37, 429–438. Kouhila, M., Belghit, A., Daguenet, M. & Boutaleb, B.C. (2001). Experimental determination of the sorption isotherms of mint (Mentha viridis), sage (Salvia officinalis) and verbena (Lippia citriodora). Journal of Food Engineering, 47, 281–287. Kumar, A.J., Singh, R.R.B., Patil, G.R. & Patel, A.A. (2005). Effect of temperature on moisture desorption isotherms of kheer. Food Science and Technology, 38, 303–310. Lastoskie, C., Gubbins, K.E. & Quirke, N. (1993). Pore size distribution analysis of microporous carbons: a density functional theory approach. The Journal of Physical Chemistry, 97, 4786– 4796. Lomauro, C.J., Bakshi, A.S. & Labuza, T.P. (1985). Evaluation of food moisture sorption isotherm equations. Part I. Fruit vegetable and meat products. Lebensmittel-Wissenschaft und Technologie, 18, 111–117. Maskan, M. & Gogus, F. (1998). Sorption isotherms and drying characteristics of Mulberry (Morus alba). Journal of Food Engineering, 37, 437–449. McLaughlin, C.P. & Magee, T.R.A. (1998). The determination of sorption isotherm and the isosteric heats of sorption for potatoes. Journal of Food Engineering, 35, 267–280. McMinn, W.A.M. & Magee, T.R.A. (2003). Thermodynamic properties of moisture sorption of potato. Journal of Food Engineering, 60, 157–165. Palou, E., Lopes, M.A. & Argaiz, A. (1997). Effect of temperature on the moisture sorption isotherms of some cookies and corn snacks. Journal of Food Engineering, 31, 85–93. Perry, R.H. (ed.) (1984). Chemical Engineers’ Handbook, 6th edn. Pp. 3/65. New York, NY: McGraw-Hill.

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Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic M. A. Moraes et al.

Pezzutti, A. & Crapiste, G.H. (1997). Sorptional equilibrium and drying characteristics of garlic. Journal of Food Engineering, 31, 113–123. Roman, G.N., Urbician, M.J. & Rotstein, E. (1982). Moisture equilibrium in apples at several temperatures: experimental data and theoretical considerations. Journal of Food Science, 47, 1484–1488. Sacilik, K. & Elicin, A.K. (2006). The thin layer drying characteristics of organic apple slices. Journal of Food Engineering, 73, 281–289. Sanni, L.O., Atere, A. & Kuye, A. (1997). Moisture sorption isotherms of fufu and tapioca at different temperatures. Journal of Food Engineering, 34, 203–212. Simal, S., Femenia, A., Castell-Palou, A. & Rossello´, C. (2007). Water desorption thermodynamic properties of pineapple. Journal of Food Engineering, 80, 1293–1301.

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Singh, R.R.B., Rao, K.H., Anjaneyulu, A.S.R. & Patil, G.R. (2001). Moisture sorption properties of smoked chicken sausages from spent hen meat. Food Research International, 34, 143–148. Togrul, H. & Arslan, N. (2007). Moisture sorption isotherms and thermodynamic properties of walnut kernels. Journal of Stored Products Research, 43, 252–264. Tolaba, M.P., Peltzer, M., Enriquez, N. & Pollio, M.L. (2004). Grain sorption equilibria of quinoa grains. Journal of Food Engineering, 61, 365–371. Wosiacki, G., Pholman, B.C. & Nogueira, A. (2004). Quality profile of 15 apple cultivars. Cieˆncia e Tecnologia em Alimentos, 24, 347– 352.

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International Journal of Food Science and Technology 2008, 43, 1832–1837

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Original article Determination of oil palm fruit phenolic compounds and their antioxidant activities using spectrophotometric methods Yun Ping Neo,1,2 Azis Ariffin,1 Chin Ping Tan,1 & Yew Ai Tan2* 1 Food Technology Department, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2 Malaysian Palm Oil Board, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia (Received 18 September 2007; Accepted in revised form 19 November 2007)

Summary

There is scarce information on the phenolics of oil palm fruits (Elaeis guineensis). In this study, phenolics were extracted from oil palm fruits and analysed using spectrophotometry for information on the different types of palm phenolics and their antioxidative activities. Analyses of the total phenolic content (TPC), total flavonoid content (TFC), o-diphenols index, hydroxycinnamic acid index, flavonols index and phenol index showed ranges between 5.64 and 83.97 g L)1 gallic acid equivalent (GAE), 0.31–7.53 g L)1 catechin equivalent, 4.90–93.20 g L)1 GAE, 23.74–77.46 g L)1 ferulic acid equivalent, 3.62–95.33 g L)1 rutin equivalent and 15.90–247.22 g L)1 GAE, respectively. The antioxidant assay, 2,2-diphenyl-2-picrylhydrazyl radical scavenging assay, showed antioxidative activities in all the extracts with results ranging from 4.41 to 61.98 g L)1 trolox equivalent. The high antioxidant activities of the oil palm fruit phenolics were also found to increase with increasing TPC and TFC.

Keywords

Antioxidant activities, Elaeis guineensis, palm fruit, phenolic compounds, spectrophotometric indices.

Introduction

In recent years, oil extracted from the mesocarp of the oil palm (Elaeis guineensis) has gained wide recognition in the world because of its health implications and wide applications. Currently, Southeast Asia, particularly Malaysia and Indonesia are the world’s largest producers of palm oil. As a fruit, the oil palm contains the common constituents of fruits, such as sugar, water and various bioactives. However, the oil palm fruit is unique in that its major component is the oil. The oil soluble compounds in the palm fruit are well documented (Sundram et al., 2003; Tan et al., 2007) but there is scarce information on the water-soluble components of the fruit. The palm oil extracted from the mesocarp of the oil palm fruit is a good source of lipophilic antioxidants such as tocols (tocopherols and tocotrienols), carotenoids and the cholesterol-lowering phytosterols. Tocopherols are known to be strong antioxidants that trap the peroxyl radicals in vivo, and the carotenoids are the precursors of vitamin A. Most of the past studies were focused on tocols and carotenoids of palm oil (Choo, 1994; Choo et al., 1996). *Correspondent: Fax: +60 3 89221742; e-mail: [email protected]

The extraction of palm oil is a wet process. Life steam is injected during the sterilisation and digestion steps of palm oil milling resulting in several classes of watersoluble compounds in the oil palm fruit leaching into the steriliser condensate and the aqueous by-products of the palm oil milling process (Tan et al., 2007). This aqueous stream from the palm oil mill is a source of novel natural antioxidants and value-added food ingredients as it contains phenolic compounds, several of which are found to possess health-benefiting properties (Sundram et al., 2003). Balasundram et al. (2005) reported that the total phenolic content (TPC) of the extract isolated from the aqueous by-products contained 40.3 ± 0.5 mg gallic acid equivalent (GAE) per gram of the extract and this extract showed an antioxidant activity comparable to that of ascorbic acid. It is to be noted that the phenolics analysed by Balasundram et al. consisted mainly of the soluble free (SF) phenolics, which were co-extracted into the aqueous stream of the palm oil milling process together with suspended solids and residual oil. Phenolic compounds are often difficult to quantify accurately as they can either be in the free or insolublebound (ISB) forms (Bravo, 1998). Spectrophotometric methodologies have been widely applied for the determination of phenolic compounds. The most common method is the determination of TPC using

doi:10.1111/j.1365-2621.2008.01717.x  2008 Institute of Food Science and Technology

Determination of oil palm fruit phenolic Y. P. Neo et al.

Folin-Ciocalteau colorimetry, which has been widely used for food products such as tea, wines, fruits, vegetables and herbs (Alonso et al., 2002; Kaur & Kapoor, 2002; Will et al., 2002; Del Rio et al., 2004; Juntachote et al., 2006). Although spectrophotometric methodologies are not able to give specific information on individual phenolics like the high performance liquid chromatography (HPLC) methods, they do provide rapid information on the total amount of a phenolic group. The present study outlines the extraction and spectrophotometric investigation of SF, ISB and esterified (E-F) phenolic compounds of the oil palm fruits. As most phenolic substances absorb UV light, the extracted phenolics were analysed at different wavelengths in order to determine the different phenolic groups. The comparative antioxidative activities of these palm phenolics were also investigated. Materials and methods

Chemicals and apparatus

Acetone, ethanol, hydrochloric acid (HCl), diethyl ether, ethyl acetate and sodium hydroxide (NaOH) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Methanol, n-hexane, anhydrous sodium sulphate, sodium carbonate, Folin-Ciocalteau reagent, sodium nitrite (NaNO2) and aluminium chloride (AlCl3 6H2O) were obtained from Merck (Darmstadt, Germany). Gallic acid, catechin, ferulic acid, rutin, 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), were purchased from Sigma Chemical Co. (St Louis, MO, USA). All reagents used were of analytical grade unless otherwise specified. Spectrophotometric analyses were performed using a PerkinElmer Lambda 12 UV-visible spectrophotometer (PerkinElmer, Uberlingen, Germany). Sample preparation

Ripe palm fruits (E. guineensis) from the same plot and row at the Malaysian Palm Oil Board, Kajang were collected, composited and divided into four lots. The palm mesocarp was separated manually from the nut by using a small knife, then cut into small pieces and subjected to Soxhlet extraction using n-hexane until the mesocarp was free from oil. Isolation of soluble free, esterified and insoluble-bound phenolic compounds

Soluble free, esterified and insoluble-bound phenolic compounds in the deoiled mesocarp were extracted according to the method of Krygier et al. (1982), with a slight modification in the hydrolysis time, from 4 to 20 h. In brief, the dried, deoiled mesocarp (1 g) was

 2008 Institute of Food Science and Technology

extracted six times with methanol ⁄ acetone ⁄ water (7:7:6, v ⁄ v ⁄ v) at room temperature. The pooled extracts, concentrated by evaporation, was adjusted to pH 2 with HCl before washing with hexane to remove lipid contaminants. The SF phenolics in this washed mixture were then extracted six times using diethyl ether ⁄ ethyl acetate (DE-EA, 1:1, v ⁄ v). The E-F phenolics remaining in the aqueous acidic fraction after removal of the SF phenolics were hydrolysed with 4 m NaOH for 20 h at room temperature under a stream of nitrogen. The resulting hydrolysate was then acidified to pH 2 with HCl, washed three times with hexane and extracted another six times with DE-EA to obtain the E-F phenolics. The deoiled mesocarp, after the first extraction step with methanol ⁄ acetone ⁄ water, was similarly hydrolysed and the ISB phenolics in this hydrolysate were isolated by extraction with DE-EA (six times) after washing with hexane. The extracted SF, ISB and E-F phenolics were all evaporated to dryness and redissolved separately in methanol for subsequent analyses. Isolation of phenolic compounds

Phenolic compounds were extracted from the deoiled mesocarp according to the method described by Wang & Helliwell (2001). One gram of deoiled mesocarp was mixed with 40 mL of 60% ethanol. Five millilitres of 6 m HCl was added to the mixture and refluxed for 2 h. The extract (PE) was cooled, filtered and made up to 50 mL with 60% ethanol. UV measurement of phenolic compounds

Spectrophotometric determinations of the extracted phenolic compounds were conducted as reported by Bonoli et al. (2004). The extract was diluted with methanol. The absorbance readings were taken at 280, 320 and 370 nm. Determination of total phenolic content

Total phenolic content of the extracts were estimated colometrically using the Folin-Ciocalteau method (Singleton & Rossi, 1965). To 0.1 mL of extract, 0.5 mL of Folin-Ciocalteau reagent was added followed by 7 mL of distilled water. The mixture was left standing for 5 min at room temperature after which 1.5 mL of sodium carbonate solution was added. This solution was left at room temperature for 2 h before absorbance measurement at 765 nm with gallic acid as reference standard and methanol as blank. Determination of total flavonoid content

The determination of flavonoids was performed according to the colorimetric assay of Liu et al. (2002). The extract (0.5 mL) taken in methanol was diluted with 2.5 mL of distilled water. After the addition of 150 lL, 5% NaNO2 solution, the mixture was left to stand for 6 min at room temperature and then another 5 min after

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adding 300 lL of 10% AlCl3 6H2O solution. The mixture was made up to 5 mL with distilled water after adding 1 mL of 1 m NaOH. The solution was thoroughly vortexed and the absorbance was measured at 510 nm with catechin as reference standard and methanol as blank.

1.00

4321

0.8 0.6 0.4

2,2-diphenyl-2-picrylhydrazyl radical scavenging assay

where AC(0) is the absorbance of the control (DPPH without antioxidant) at t = 0 min and AA(t) is the absorbance of the antioxidant at plateau. The scavenging activity was measured as g L)1 TE. Statistical analysis

All experiments and measurements were replicated four times and the results were expressed as mean values ± SD. The data were compared using analysis of variance (anova) and Pearson’s linear correlations with 5% significance level (P < 0.05) using minitab 13.0 (Minitab Inc., State College, PA, USA). The average values were compared by Tukey’s test. Results and discussions

The UV spectra of all the extracts were recorded using a spectrophotometer and Fig. 1 shows that all four oil palm fruit extracts have a maxima around the region of 280 nm. The phenol indices (PIs) of the oil palm fruit extracts determined by spectrometric analysis, ranged

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0.2 0.00 200.0 250

300

350

400 nm

450

500

550 600.0

Figure 1 UV ⁄ Vis spectra of soluble free (SF, 4), insoluble-bound (ISB, 3), esterified (E-F, 2) and phenolic extract (PE, 1) of ripe palm fruit.

Phenol indices (PI) g L–1 GAE g–1 extract

2,2-diphenyl-2-picrylhydrazyl assay (DDPH) was performed according to Thaipong et al. (2006) and Yen & Duh (1994), with modifications on sample volume and standing hour. The antioxidant activities of the extracts by DPPH assay were quantified as trolox equivalent antioxidant capacity (TEAC) by referring to the percentage of inhibition of trolox standard. The incubation time of the DPPH methanolic solution with the extracts or standards generally ranged from 10 min to 24 h (Thaipong et al., 2006; Kanatt et al., 2007; Oszmianski et al., 2007). In this study, 24 h of incubation time was applied as it provided a sufficient time period for the extracts or standards to reach plateau. The DPPH stock solutions were prepared by dissolving 24 mg DPPH in 100 mL methanol and stored at )3 C. The working solution contained 10 mL of the stock solution and 45 mL methanol, with an absorbance of 1.10 ± 0.03 units at 515 nm. The extract (150 lL) was added to 2.85 mL of the working solution and this was left to react for 24 h in dark. The absorbance was measured at 515 nm and the percentage of inhibition of the DPPHscavenging activity was calculated as following:   ðACð0Þ AAðtÞ Þ  100 %Inhibition ¼ ACð0Þ

350 300 250 200 150 100 50 0 SF

ISB

E-F

PE

Figure 2 Phenol indices of soluble free (SF), insoluble-bound (ISB), esterified (E-F) and phenolic extract (PE) of ripe palm fruit.

Flavonols indices (FI) 140 g L–1 RE g–1 extract

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120 100 80 60 40 20 0

SF

ISB

E-F

PE

Figure 3 Flavonols indices of soluble free (SF), insoluble-bound (ISB), esterified (E-F) and phenolic extract (PE) of ripe palm fruit.

from 15.90 to 247.22 g L)1 GAE per gram extract of dried deoiled oil palm fruit mesocarp (Fig. 2). Figure 3 shows the flavonols indices (FIs) of the extracts, which ranged from 3.62 to 95.33 g L)1 rutin equivalent (RE) per gram of extract, while the hydroxycinnamic acid indices (HCAI) of the extracts ranged from 23.74 to 77.46 g L)1 ferulic acid equivalent (FAE) per gram of extract (Fig. 4). O-diphenol indices (ODPI) were measurements of the

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Determination of oil palm fruit phenolic Y. P. Neo et al.

100

gL

–1

FAE g

–1

extract

Hydroxycinnamic acid indices (HCAI) 120

Folin-Ciocalteau† g L)1 Total flavonoidà g L)1 DPPH§ g L)1 GAE per gram extract CE per gram extract TE per gram extract

80 60 40 20 0 SF

ISB

E-F

PE

Figure 4 Hydroxycinnamic acid indices of soluble free (SF), insoluble-

bound (ISB), esterified (E-F) and phenolic extract (PE) of ripe palm fruit.

g L–1 GAE g–1 extract

o -diphenol indices (ODPI) 140 120 100 80 60 40 20 0

Table 1 Total phenolic content, total flavonoid content and antioxidant activities of the soluble free (SF), insoluble-bound (ISB), esterified (E-F) and phenolic extract (PE) of ripe palm fruit

SF

ISB

E-F

PE

Figure 5 o-diphenol indices of soluble free (SF), insoluble-bound (ISB), esterified (E-F) and phenolic extract (PE) of ripe palm fruit.

phenolic compounds with o-diphenol structure which reacted with molybdate to form yellow solutions (Maillard et al., 1996). Figure 5 shows that the ODPIs of the extracts ranging from 4.90 to 93.20 g L)1 GAE per gram of extract. According to Bonoli et al. (2004), different groups of phenolic compounds can be quantified by measuring absorbance at various wavelengths. Total phenol, flavonols, hydroxycinnamic acids and o-diphenols of barley flour were quantified by reading the absorbance at 280, 320 and 370 nm, respectively. Their findings suggested that phenolic groups have absorbance at particular wavelength, indicating the usefulness of the indices, which can be defined as the total amount of each phenolic group that may present in the extracts. The oil palm extracts showed high values for each of the index reported by Bonoli et al. It is an analytical challenge to measure the various groups of phenolic compounds in oil palm fruit extracts and report meaningful values, as it is believed that different individual phenolics will not respond equally in direct spectral analysis. In addition, the occurrence of some interfering compounds in the extract’s matrix can absorb significantly at particular wavelengths, and this must be taken into account. Not withstanding this, the ODPIs may provide more accurate values since it is based on the reaction of a specific phenolic structure.

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SF 5.64 ISB 6.88 E-F 7.31 PE 83.97

± ± ± ±

0.44a 0.64b 0.76b 20.08

0.31 0.31 0.42 7.53

± ± ± ±

0.02a 0.03a 0.04b 0.44

4.41 5.58 6.05 61.98

± ± ± ±

0.23a 0.74b 0.67b 2.45

†,à,§ The data are displayed with means ± SD of four measurements Numbers with different letters in the same column are significantly different (P < 0.05)

The TPC of the extracts were found to range from 5.64 to 83.97 g L)1 GAE per gram of extract (Table 1). The Folin-Ciocalteau method provides a more accurate measurement of TPC compared with PIs as the Folin-Ciocalteau reagent reacts equally with various groups of phenolic compounds (Waterhouse, 2002). The Folin-Ciocalteau method is based on the reduction of the reagent where the product of reduction exhibits a blue colour with maximum absorption at 765 nm (Singleton & Rossi, 1965). The TPC of SF fraction was significantly lower compared with ISB and E-F portions. The total flavonoid contents (TFC) of the extracts are shown in Table 1. TFC of E-F fraction was significantly (P < 0.05) higher than the SF and ISB fractions. Two extraction methods were applied in this study in order to better understand the phenolic compounds present in the oil palm fruit. The PE can be considered as a sum of free and bound phenolic compounds in palm mesocarp as it is extracted as a whole by refluxing with acid. The sums of TPC and TFC in SF, ISB and E-F were 19.83 g L)1 GAE and 1.04 g L)1 catechin equivalent (CE), respectively. From Table 1, TPC and TFC of the PE were higher than the sum in the three fractions (SF, ISB and E-F). The TPC and TFC of PE were 83.97 ± 20.08 g L)1 GAE and 6.86 ± 1.40 g L)1 CE, respectively per gram of extract. The sum of the three fractions (SF, ISB and E-F) gave a lower amount of TPC and TFC compared with PE because of losses incurred during the complicated extraction process. However, Krygier et al. (1982) protocol provided a more complete picture of the phenolic content in the mesocarp of oil palm fruits by determining the SF, ISB and E-F phenolic compounds. Thus, the PE could be used as an indicator of the amount of total phenolic compounds present in the oil palm fruit. Shahidi & Naczk (2004) reported that E-F phenolic acids were the dominant form of phenolics in rapeseed and canola. The high amount of both TPC and TFC in PE suggested that the phenolic compounds in palm fruits are mostly present as bound glycosides. This would account for the

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E-F fractions having significantly higher (P < 0.05) amounts of TPC and TFC compared with the SF fraction. All four extracts of the palm phenolics showed antioxidative potential. The antioxidant activities of the extracts determined by DPPH assay were in the order of E-F > ISB > SF and were found to range from 4.41 to 6.05 g L)1 TE. The SF fraction, the fraction with the lowest TPC showed significantly lower antioxidant activities in comparison to the other fractions at P < 0.05 as shown in Table 1. The high antioxidant activity of the PE, which was 62 g L)1 TE per gram of extract, indicated the total antioxidant power of the oil palm fruit mesocarp extract. Positive Pearson’s linear correlations were found between DPPH and TPC (R2 = 0.96, P < 0.05) and DPPH and TFC (R2 = 0.98, P < 0.05). Such relationships have also been observed in other studies (Kaur & Kapoor, 2002; Tsao et al., 2005; Azizah et al., 2007; Yemis et al., 2007). Conclusion

This study on the phenolic compounds in the mesocarp of palm fruit is important to the palm oil industry as well as the nutrition sector, because it provides new information, which can lead to new edible applications of palm products. The spectrophotometric methods investigated were found to be useful and rapid methods for screening phenolics in oil palm fruits. The preliminary set of results can be used later to support more detailed characterisation of phenolics. The confirmation of the antioxidative potential of phenolics in oil palm fruits implies that the palm fruit can be used as a source of antioxidants for both food and non-food applications. Acknowledgments

The first author is funded under the GSAS program of Malaysian Palm Oil Board. The sampling and analysis were carried out in the laboratory at Malaysian Palm Oil Board, Kajang, Malaysia. References Alonso, A.M., Guillen, D.A., Barroso, C.G., Puertas, B. & Garcia, A. (2002). Determination of antioxidant activity of wine byproducts and its correlation with polyphenolic content. Journal of Agricultural and Food Chemistry, 50, 5832–5836. Azizah, O., Amin, I., Nawalyah, A.G. & Ilham, A. (2007). Antioxidant capacity and phenolic content of cocoa beans. Food Chemistry, 100, 1523–1530. Balasundram, N., Tan, Y.A., Sambanthamurthi, R., Sundram, K. & Samman, S. (2005). Antioxidant properties of palm fruit extracts. Asia Pacific Journal of Clinical Nutrition, 14, 319–324. Bonoli, M., Verardo, V., Marconi, E. & Caboni, M.F. (2004). Antioxidant phenols in barley (Hordeum vulgare L.) flour:

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comparative spectrophetometric study among extraction methods of free and bound phenolic compounds. Journal of Agricultural and Food Chemistry, 52, 5195–5200. Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56, 317–333. Choo, Y.M. (1994). Palm oil carotenoids. Nutrition Bulletin, 15, 130– 137. Choo, Y.M., Yap, S.C., Ooi, C.K., Ma, A.N., Goh, S.H. & Ong, A.S.H. (1996). Recovered oil from palm-pressed fibre: a good source of natural carotenoids, vitamin E and sterols. Journal of American Oil Chemist Society, 73, 599–602. Del Rio, D., Stewart, A.J., Mullen, W. et al. (2004). HPLC-MS analysis of phenolic compounds and purine alkaloids in green and black tea. Journal of Agricultural and Food Chemistry, 52, 2807– 2815. Juntachote, T., Berghofer, E., Bauer, F. & Siebenhandl, S. (2006). The application of response surface methodology to the production of phenolic extracts of lemon grass, galangal, holy basil and rosemary. International Journal of Food Science and Technology, 41, 121–133. Kanatt, S.R., Chander, R. & Sharma, A. (2007). Antioxidant potential of mint (Mentha spicata L.) in radiation-processed lamb meat. Food Chemistry, 100, 451–458. Kaur, C. & Kapoor, H.C. (2002). Anti-oxidant activity and total phenolic content of some Asian vegetables. International Journal of Food Science & Technology, 37, 153–161. Krygier, K., Sosulski, F. & Hogge, L. (1982). Free, esterified and insoluble bound phenolic acids 1. Extraction and purification procedure. Journal of Agricultural and Food Chemistry, 30, 330– 334. Liu, M., Li, X.Q., Weber, C., Lee, C.Y., Brown, J. & Liu, R.H. (2002). Antioxidant and antiproliferative activities of raspberries. Journal of Agricultural and Food Chemistry, 50, 2926–2930. Maillard, M.N., Soum, M.H., Boivin, P. & Berset, C. (1996). Antioxidant activity of barley and malt: relationship with phenolic content. Lebensmittel Wissenschaft und Technologie, 29, 238–244. Oszmianski, J., Wojdylo, A., Lamer-Zarawska, E. & Swiader, K. (2007). Antioxidant tannins from Rosaceae plant roots. Food Chemistry, 100, 579–583. Shahidi, F. & Naczk, M. (2004). Phenolic compounds of major oilseeds and plant oils: Phenolics in Food and Nutraceuticals. Pp. 83–128. Boca Raton: CRC Press. Singleton, V.L. & Rossi, J.A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Sundram, K., Sambanthamurthi, R. & Tan, Y.A. (2003). Palm fruit chemistry and nutrition. Asia Pacific Journal of Clinical Nutrition, 12, 355–362. Tan, Y.A., Sambanthamurthi, R., Sundram, K. & Mohd Basri, W. (2007). Valorisation of palm by-products as functional components. European Journal of Lipid Science and Technology, 109, 380–393. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L. & Byrne, D.H. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extract. Journal of Food Composition and Analysis, 19, 669– 675. Tsao, R., Yang, R., Xie, S., Sockovie, E. & Khanizadeh, S. (2005). Which polyphenolic compounds contribute to the total antioxidant activities of apple? Journal of Agricultural and Food Chemistry, 53, 4989–4995. Wang, H. & Helliwell, K. (2001). Determination of flavonols in green and black tea leaves and green tea infusion by highperformance liquid chromatography. Food Research International, 34, 223–227. Waterhouse, A.L. (2002). Polyphenolics determination of total phenolics. In: Current Protocols in Food Analytical Chemistry. Pp. 1–4. New York: John Wiley & Sons, Inc.

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Will, F., Schulz, K., Ludwig, M., Otto, K. & Dietrich, H. (2002). The influence of enzymatic treatment of mash on the analytical composition of apple juice. International Journal of Food Science & Technology, 37, 653–660. Yemis, O., Bakkalbasi, E. & Artik, N. (2007). Antioxidative activities of grape (Vitis vinifera) seed extracts obtained from different

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varieties grown in Turkey. International Journal of Food Science & Technology (OnlineEarly Articles). doi:10.1111/j.1365-2621.2007. 01415.x. Yen, G.C. & Duh, P.D. (1994). Scavenging effect of methanolic extracts of peanut hulls on the free-radical and active-oxygen species. Journal of Agricultural and Food Chemistry, 42, 629–632.

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Original article Comparison of polyamine, phenol and flavonoid contents in plants grown under conventional and organic methods Giuseppina Pace Pereira Lima,1* Suraya Abdallah da Rocha,1 Massanori Takaki,2 Paulo Roberto Rodrigues Ramos1 & Elizabeth Orika Ono1 1 Universidade Estadual Paulista, Instituto de Biocieˆncias, Departamento Quı´ mica e Bioquı´ mica, CP 510, CEP 18618-000, Botucatu, Sa˜o Paulo, Brazil 2 Universidade Estadual Paulista, Instituto de Biocieˆncias, CP 199, 13506-900, Rio Claro, Sa˜o Paulo, Brazil (Received 13 September 2007; Accepted in revised form 17 December 2007)

Summary

The objective of this work was to compare the contents of polyamines (putrescine, spermidine and spermine), and total soluble phenols and flavonoids in parts of plants grown under either organic or conventional cropping, commonly discarded during food preparation. The contents of free polyamines, total phenols and total soluble flavonoids in peels (zucchini squash, banana, potato, eggplant, orange, lime, mango, passion fruit and radish), leaves (zucchini squash, broccoli, carrot, collard, cassava, radish and grape), stalks (broccoli, collard and spinach) and zucchini seeds were analysed. Most analysed vegetables presented higher contents of polyamines and total phenols under organic cropping, contrary to the results obtained for total flavonoids, possibly because of the cultural practices adopted.

Keywords

Leaves, peels, putrescine, spermidine, spermine, stalk.

Introduction

Usually, non-conventional edible parts of vegetable as peel, seeds, stalks and leaves are discarded during meal preparation. Those parts could contain large amounts of substances important for human metabolism and could be used as alternative nutritional sources and decrease of global hunger. The use of non-conventional parts can also decrease the urban solid waste. An increasingly larger part of the population is giving preference to organic cultivation products, mainly because of the absence of contaminants in the production process (Pussemier et al., 2006). The number of rural areas dedicated to organic agriculture has increased worldwide. According to the Stiftung Oekologie and Landbau (SOEL, 2003), c. 23 million hectares are cultivated under the organic system. Latin America has c. 4.7 million hectares under organic or non-conventional cultivation (including biodynamic cultivation), of which 3.2 million hectares are in Argentina alone (Yussefi & Willer, 2003). Consequently, the market is turning to the consumption of organic products, and several studies have been conducted on this subject, but still little attention is paid *Correspondent: Fax: +55 14 38116255; e-mail: [email protected]

to the quality of foods. Many researches have revealed a higher nutritional value in organic foods, with smaller nitrate contents (Siderer et al., 2005) and better organoleptic quality. However, these products have been insufficiently evaluated with regard to their contents of other substances that are important for the metabolism, such as polyamines and phenolic compounds. Polyamines are molecules that have two or more amine groups, mainly including putrescine, spermidine and spermine. They can be synthesised in situ or obtained from the diet and from intestinal flora microorganisms (Bardo´cz et al., 1993, 1996). The absorption of polyamines by intestinal cells has been suggested as a mechanism that regulates their endogenous concentrations (Bardo´cz et al., 1993; Seiler et al., 1998; Eliassen et al., 2002). It is believed that polyamines influence growth, possibly acting on cell proliferation and differentiation (Bardo´cz et al., 1993, 1995, 1996), and are intimately associated with the growth of tumours (Bardo´cz et al., 1993; Seiler et al., 1998; Eliassen et al., 2002). Significant polyamine levels come from the diet (Bardo´cz et al., 1995). In view of the importance of these substances in the development of cancer and in growth, it is in the general interest to perform analyses of their contents in foods (Lima et al., 2006). Few papers have been developed in order to verify the impact of cultivation systems, either organic or

doi:10.1111/j.1365-2621.2008.01725.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Polyamine, phenol and flavonoid contents in plants G. P. P. Lima et al.

conventional, on secondary metabolism (Asami et al., 2003). Several factors interfere with the quality of foods; among them are phenolic compounds, which have been widely studied because of their influence on food quality. The presence of these compounds in plants has been extensively studied as they present pharmacological and antinutritional properties and inhibit the oxidation of lipids and the proliferation of fungi (Fernandez et al., 1998). Among phenolic compounds, an increasing interest has been demonstrated towards the study of the activity of flavonoids, which play an important role in human health. The properties of flavonoids are known in antioxidative, antimicrobial, antimutagenic and anticarcinogenic processes. Considering these factors, studies on the natural sources of flavonoids are thus relevant. Biochemical analyses in vegetables are commonly performed using pulp or leaves; in this work, plant parts generally discarded by the consumer at meal preparation time were analysed, such as peels, stalks and leaves, in view of their intended utilisation as nutritional sources. The utilisation of these parts, which are normally discarded, could mean the ingestion of substances that are essential for cell metabolism. Therefore, in this work, we propose an analysis of polyamines (putrescine, spermidine and spermine), phenols, and flavonoids in peels, stalks and leaves of plants cultivated under organic and conventional systems. Material and methods

We analysed edible parts of the food plants most consumed by the Brazilian population (State of Sa˜o Paulo), which are generally discarded every day. Peel samples of zucchini squash (Cucurbita pepo L.), banana (Musa sp sub-group Cavendish), potato (Solanum tuberosum L.), eggplant (Solanum melongena L.), peˆra orange (Citrus sinensis L. Osbeck), Tahiti lime (Citrus limon L.), mango (Mangifera indica L.), sour passion fruit (Passiflora edulis Sims.), radish (Raphanus sativus L.), leaf samples of zucchini squash (C. pepo L.), broccoli (Brassica oleracea var. italica L.), carrot (Daucus carota L.), collard (B. oleracea var. acephala L), yellow cassava (Manihot esculenta Crantz), radish (R. sativus L.) and grape (Vitis vinifera L.); stalk samples of broccoli (B. oleracea var. italica L.), collard (B. oleracea var. acephala L.), and spinach (Spinacia oleracea L.); and seed samples of zucchini squash (C. pepo L.) were divided into lots containing four replicates, consisting of three specimens each; the produce was purchased directly from the producers grown under either conventional or organic cultivation (Associac¸a˜o de Certificac¸a˜o ‘‘Instituto Biodinaˆmico’’, Botucatu, Sa˜o Paulo, Brazil), washed in running water with a brush and immersed in a chlorine solution (20 mL sodium

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hypochlorite to 1 L distilled water, 2% active chlorine) for 15 min and then prepared for the proposed analyses. For the polyamine and flavonoid determinations, the samples were frozen in liquid nitrogen and stored in freezer for later analysis. For the determination of total phenols, the samples were dried in a forced air circulation oven at 60 C, until constant weight. All analyses were performed with three replications. Polyamine contents

Samples of each food were analysed according to the method proposed by Flores & Galston (1982), with modifications (Lima et al., 2006) as follows. The fresh material was homogenised for 1 min in 5% (v ⁄ v) cold perchloric acid (Merck, NJ, USA), using a food homogenizer. After centrifugation for 20 min at 4 C, dansyl chloride [Sigma (Sigma-Aldrich, Sa´o Paulo, Brazil), 95%] and saturated sodium carbonate were added to the supernatant. Proline (Sigma, min. 99%) was added after 1 h at 60 C and the mixture was maintained in the dark for 30 min, at room temperature. Toluene was used to extract the dansylated polyamines and aliquots were applied onto thin-layer chromatography plates [glass plates coated with 60G silica Gel – Merck (20 · 20 cm)] and were separated in laboratory bowls containing chloroform:triethylamine (Merck) (10:1). Putrescine (Sigma, min. 98%), spermidine (Sigma, min. 98%) and spermine (Sigma, min. 95%) standards were submitted to the same process. The entire procedure was monitored under u.v. light (254 nm). The polyamines were quantified by comparison against the standards, which were also applied onto the plates, by fluorescence emission spectroscopy (excitation at 350 nm and emission measurement at 495 nm), in a Video Documentation System, using the Image Master version 2.0 software program by Amersham Pharmacia Biotech 1995, 1996 (Amersham Pharmacia Biotech, Uppsala, Sweden). The free polyamine contents were expressed as lg g)1 fresh matter. Total phenols content

The analysis was performed following the spectrophotometric method using the Folin–Ciocalteau reagent (Singleton & Rossi, 1965). Samples of dry and ground material were weighed and placed into centrifuge tubes. Acetone (70%) (Merck) in distilled water was added to each tube, and the tubes were then taken to an ultrasound bath for 20 min and later centrifuged for 10 min. The supernatant was placed in vials and maintained in ice. Aliquots were transferred to test tubes and the volume was completed with distilled water. The vials were allowed to sit for 45 min and a reading was then obtained at A725 nm. The results were expressed as microgram of phenols per gram dry matter equivalent in tannic acid (Sigma).

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Total flavonoids content

The analysis was performed according to the spectrophotometric method adapted from Santos & Blatt (1998) and Awad et al. (2000). Fresh material samples were weighed and then macerated in a solution consisting of 70% methanol and 10% acetic acid. The samples were then taken to an ultrasound bath for 30 min. After filtering, they were centrifuged for 20 min at 12 500 · g. Upon transferring the supernatant to test tubes, aluminium chloride was added and the volume was completed with 10% acetic acid; the tubes were then agitated and allowed to sit for 30 min. The absorbance reading was performed at 425 nm and the results were expressed as microgram of flavonoids per gram fresh mass [rutin equivalent (Sigma)]. The results were submitted to anova (Tukey, P < 0.05). Results and discussion

Apparently, one of the primary reasons for the consumption of organic products is the perception that they are more nutritious when compared with those grown under conventional cropping (Magkos et al., 2003). However, studies have focused on analyses of vitamins and minerals, and do not present references with regard to some substances that are important for nutrition, such as polyamines and phenolic compounds.

Polyamine levels may show alterations in the plant, depending on the management practices adopted (Bouchereau et al., 1999). The polyamine contents found in this work showed variations in relation to the cultivation methods (organic or conventional), with the exception of mango peel, where the levels of polyamines (putrescine, spermidine and spermine) were very low (trace). It can be seen that in produce grown under conventional cultivation, putrescine (Table 1) was only higher in banana and eggplant peels, radish skin, pumpkin seeds and cassava leaves. No significant difference occurred in pumpkin leaves. In all other samples, organic cultivation increased the putrescine levels. The spermidine analysis showed higher levels only in banana and eggplant peels (Table 1) derived from conventional cultivation. No significant difference occurred for this triamine between radish skin, pumpkin seeds and cassava and grape leaves, although a tendency for higher concentrations can be noticed under organic cultivation. For spermine (Table 1), higher concentrations were observed in pumpkin and banana peels, pumpkin seeds and cassava leaves; only pumpkin leaves did not show significant differences between the values found. In this analysis, as well as for the other polyamines studied, a tendency for higher contents can also be observed in plants cultivated under the organic method. This tendency can possibly be attributed to the fact that

Putrescine (lg g)1 fresh matter)

Spermidine (lg g)1 fresh matter)

Spermine (lg g)1 fresh matter)

Vegetables

Conventional

Organic

Conventional

Organic

Conventional

Organic

Pumpkin peel Banana peel Potato peel Eggplant peel Orange peel Lime peel Mango peel Passion fruit rind Radish skin Pumpkin seeds Pumpkin leaves Broccoli leaves Carrot leaves Collard leaves Cassava leaves Radish leaves Grape leaves Broccoli stalks Collard stalks Spinach stalks

96 b 666 a 327 b 416 a 493 b 89 b tr 411 b 349 a 169 a 302 a 486 b 123 b 187 b 350 a 277 b 540 b 294 b 124 b 82 b

213 a 571 b 408 a 276 b 8795 a 590 a 268 1509 a 239 b 64 b 382 a 1917 a 241 a 598 a 195 b 617 a 685 a 1026 a 421 a 354 a

375 a 962 a 556 b 600 a 254 b 117 b tr 875 b 512 a 365 a 835 b 359 b 257 b 202 b 486 a 397 b 899 a 613 b 129 b 98 b

436 a 333 b 829 a 447 a 1290 a 750 a 467 2442 a 516 a 314 a 1070 a 1658 a 529 a 967 a 466 a 940 a 968 a 793 a 669 a 617 a

1000 a 1967 a 593 b 545 a 130 b 131 b tr 676 595 b 269 a 580 b 289 b 236 b 86 b 617 a 508 b 1205 b 677 b 144 b 71 b

384b 1275 b 1150 a 557 a 5804 a 947 a 614 3596 a 752 a 166 b 749 a 4542 a 496 a 1209 a 289 b 1288 a 1999 a 2157 a 814 a 799 a

Table 1 Putrescine, spermidine and spermine (lg g)1 fresh matter) in produce grown under conventional and organic production systems

Different lower case letters in the rows indicate a significant difference (P < 0.05) between means; tr – trace.

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Polyamine, phenol and flavonoid contents in plants G. P. P. Lima et al.

organically grown plants show higher stress levels, as they do not usually receive any phytosanitary treatment and are more susceptible to the attack of pathogens and other damages (Legaz et al., 1998). Several papers have reported an increase in polyamine levels as a response to different types of stress (Flores & Galston, 1984). The studied plants possibly suffered some sort of damage, either by the attack of pathogens (Legaz et al., 1998) or by mechanical damages, thus showing higher levels of these substances. The tendency observed for higher levels of polyamines in organically grown foods can also be attributed to their greater longevity, a statement that is very common and popular among consumers. In general, polyamines have been considered as possible senescence inhibitors and seem to decrease during the maturation of some fruits; its exogenous application may delay chlorophyll loss in R. sativus L. leaves (Altman, 1982). On the contrary, increases in these substances have been verified in other fruits like tomato and cherimoya, suggesting that changes in polyamine levels after harvesting depend on the species (Escribano & Merodio, 1994). High polyamine levels can be detrimental to human health in some cases, such as certain types of cancer, as it is a known fact that they are substances related to the growth of tumours. Polyamines can be absorbed and distributed through the body and then used during cell growth in organs and tissues (Thomas & Thomas, 2003). One of the most abundant polyamines in the human body is putrescine, which can be metabolised into spermidine and spermine (Kalac & Krausova´, 2005). The literature shows several papers on polyamine contents in fruits and vegetables, and putrescine seems to be the most common among them, found at higher quantities among the most studied (Eliassen et al., 2002; Lima et al., 2006). Bardo´cz et al. (1995) stated that all foods in the diet contribute in a similar way towards spermidine contents, with the highest levels being found in leafy and green vegetables, whereas putrescine is found at higher contents and is the most common in fruits and vegetables that are not green. Different results were found at the polyamine analysis in this work. Bardo´cz et al. (1995) stated that more than 80% of ingested putrescine can be converted into other polyamines and other metabolites, such as amino acids, whereas for spermidine and spermine, around 70–80% of the ingested doses remained in their original forms. The broad polyamine content variations observed in this work lead us to declare that precautions must be taken in the preparation of diets for persons that suffer from certain diseases, as these substances are associated with a potential growth of tumours (Quemener et al., 1994), and more in-depth studies dealing with cultivated plants, grown either organically or under the conventional method, must be carried out in Brazil.

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A higher content of total phenols measured in milligram equivalent of tannic acid per gram fresh matter was found in organically grown pumpkin, banana, and mango peels, pumpkin seeds, and cassava and grape leaves, whereas higher values were obtained for eggplant peel, broccoli and radish leaves, and stalks grown under conventional cultivation. No significant differences were verified in the other foods analysed, but a tendency was observed for foods produced organically to show higher total phenol contents (Table 2). With regard to total flavonoid contents (Table 2), some species did not show significant differences between the cultivation systems studied, such as pumpkin, eggplant and orange peels, passion fruit rind, radish leaves and broccoli and collard stalks. Only banana peel and carrot and grape leaves grown organically showed higher flavonoid contents. Flavonoids, such as quercetin and kaempferol, are phenolic compounds that can be synthesised by plants as a response to the attack of pathogens, and the level of phenolic compounds in plants depends on their maturity stage, variety, storage and genetic factors, among others (Nicolas et al., 1994), and may be present at different levels in the same plant. In this work, we found variations among the species studied, but it can be

Table 2 Flavonoids [microgram of flavonoids (rutin) per gram green mass]) and phenols [microgram of phenols (tannic acid) per gram dry matter] in produce grown under conventional and organic production systems Total flavonoids

Total phenols

Plant parts

Conventional

Organic

Conventional

Organic

Pumpkin peel Banana peel Potato peel Eggplant peel Orange peel Lime peel Mango peel Passion fruit rind Radish skin Pumpkin seeds Pumpkin leaves Broccoli leaves Carrot leaves Collard leaves Cassava leaves Radish leaves Grape leaves Broccoli stalks Collard stalks Spinach stalks

1.3 a 0.1 b 0.5 a 2.5 a 11.4 a 13.9 a 6.1 a 0.1 a 4.4 a 6.5 a 4.7 a 6.5 a 3.0 b 7.4 a 10.3 b 6.4 a 5.2 b 2.71 a 0.3 a 1.6 a

1.4 a 0.6 a 0.2 b 2.7 a 11.4 a 6.6 b 3.3 b 0.3 a 1.9 b 2.4 b 3.2 b 4.00 b 6.2 a 1.6 b 11.8 a 5.4 a 11.0 a 1.6 a 0.17 a 0.6 b

13 b 33 b 43 a 63 a 52 a 56 a 58 b 49 a 55 a 9b 47 a 55 a 50 a 53 a 37 b 49 a 40 b 62 a 53 a 57 a

19 a 61 a 48 a 51 b 48 a 62 a 63 a 46 a 63 a 36 a 52 a 37 b 64 a 60 a 63 a 30 b 53 a 51 b 42 a 57 a

Different lower case letters in the rows indicate a significant difference (P < 0.05) between means.

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noted that only a few showed higher flavonoid contents under organic cultivation. Mitchell & Chassy (2006) reported higher quercetin content in two varieties of organically grown tomato. Similar results were described by Ren et al. (2001) in spinach, onion and pumpkin, a tendency for higher flavonoid contents was found in organically grown species. Some papers have also reported differences in phenolic compounds between the agricultural practices used with some species (Asami et al., 2003). The results found in this work show that there are variations between the foods analysed, with regard to both total phenol and flavonoid contents, which may have been influenced not only by the cropping conditions (organic or conventional), but also by the fact that the foods were purchased by simulating the consumer’s mind, i.e., at the ideal physiological maturity for consumption. Studies have demonstrated that the phenolic compounds makeup in peach, pear and apple show differences with regard to their commercial maturity (Lee et al., 1990; Nicolas et al., 1994). Our data demonstrate a tendency for a higher total phenols content under organic cultivation, corroborating the hypothesis (Carbonaro & Mattera, 2001; Asami et al., 2003) that organic foods show higher endogenous phenol contents because of the cultivation method, with low amounts or even absence of pesticides, a common practice in conventional agriculture, as in many plants the use of pesticides and fertilizers has been responsible for a significant decrease of phenols, like in cider and apple (Lea & Beech, 1978; Nicolas et al., 1994). On the contrary, Hakkinen & Torronen (2000) reported that organic cultivation did not show a positive effect on the levels of phenolic compounds in strawberry. The data we found, showing higher phenol contents in almost all plants analysed and their possible relation with plant stress, could confirm the data found for polyamines in these plants, possible indicators of stress. Fruits and vegetables are sources of antioxidant phenolic compounds for man. Epidemiological studies indicate that an inverse correlation exists between the consumption of certain vegetables and diseases, such as cancer, cardiovascular problems, diabetes and early aging (Hollman et al., 1996). Antioxidant phenolic compounds, especially flavonoids, neutralise reactive oxygen species (ROS) before they can cause damage to cells and the cultivation method may interfere with the amounts of these compounds (Mitchell & Chassy, 2006), although few studies have been conducted to investigate these differences. Knekt et al. (2002) found an inverse relation between the consumption of flavonoids in the diet and the development of tumours. Therefore, the consumption of pesticide-free foods containing higher contents of antioxidant compounds would be advisable.

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Conclusions

Our results have demonstrated that most vegetables analysed showed a tendency to present higher polyamine and total phenol contents in foods obtained under organic cultivation, in contrary to what was observed for total flavonoids, possibly because of the cultural practices adopted during cultivation. Acknowledgments

The authors would like to thank CNPq (Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico) for granting a Master’s scholarship to the second author. References Altman, A. (1982). Retardation of radish leaf senescence by polyamines. Physiologia Plantarum, 54, 189–193. Asami, D.K., Hong, Y-J., Barrett, D.M. & Mitchell, A.E. (2003). Comparison of the total phenolics and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional, organic, and sustainable agricultural practices. Journal of Agriculture and Food Chemistry, 51, 1237–1241. Awad, A.M., de Jager, A. & van Westing, L.M. (2000). Flavonoid and chlorogenic acid levels in apple fruit: characterization of variation. Scientia Horticulturae, 83, 249–263. Bardo´cz, S., Grant, G., Brown, D.S., Ralph, A. & Pusztai, A. (1993). Polyamines in food – implications for growth and health. Journal of Nutritional Biochemistry, 4, 66–71. Bardo´cz, S., Duguid, T.J., Brown, D.S., Grant, G., Pusztai, A. & Ralph, A. (1995). The importance of dietary polyamines in cell regeneration and growth. British Journal of Nutrition, 73, 819–828. Bardo´cz, S., White, A., Grant, G., Brown, D.S., Duguid, T.J. & Pusztai, A. (1996). Effects of dietary polyamines and clofibrate on metabolism of polyamines in rats. Journal of Nutritional Biochemistry, 10, 700–708. Bouchereau, A., Aziz, A., Larher, F. & Martin-Tanguy, J. (1999). Polyamines and environmental challenges: recent development. Plant Science, 140, 103–125. Carbonaro, M. & Mattera, M. (2001). Polyphenoloxidase activity and polyphenol levels inorganically and conventionally grown peach (Prunus persica L., cv. Regina Bianca) and pear (Pyrus communis L., cv. Williams). Food Chemistry, 72, 419–424. Eliassen, K.A., Reistad, R., Risoen, U. & Ronning, H.F. (2002). Dietary polyamines. Food Chemistry, 78, 273–280. Escribano, M.I. & Merodio, C. (1994). The relevance of polyamine levels in cherimoya (Annona cherimola Mill.) fruit ripening. Plant Physiology, 143, 207–212. Fernandez, M.A., Saenz, M.T. & Garcia, M.D. (1998). Antiinflamatory activity in rats and mice of phenolic acids isolated from Scrophularia frutescens. Journal of Pharmacy and Pharmacology, 50, 1183–1186. Flores, H.E. & Galston, A.W. (1982). Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiology, 69, 701–706. Flores, H.E. & Galston, A.W. (1984). Osmotic stress-induced polyamine accumulation in cereal leaves. II. Relation to amino acid pools. Plant Physiology, 75, 110–113. Hakkinen, S.H. & Torronen, A.R. (2000). Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique. Food Research International, 33, 517–524.

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Polyamine, phenol and flavonoid contents in plants G. P. P. Lima et al.

Hollman, P.C., Hertog, M.G. & Katan, M.B. (1996). Role of dietary flavonoids in protection against cancer and coronary heart disease. Biochemical Society Transactions, 24, 785–789. Kalac, P. & Krausova´, P. (2005). A review of dietary polyamines: formation, implications for growth and health and occurrence in foods. Food Chemistry, 90, 219–230. Knekt, P., Kumpulainen, J. & Jarvinen, R. (2002). Flavonoid intake and risk of chronic disease. The American Journal of Clinical Nutrition, 76, 560–569. Lea, A.G.H. & Beech, F.W. (1978). The phenolic of ciders: effect of cultured conditions. Journal of the Science of Food and Agriculture, 29, 493–496. Lee, C.Y., Kagan, V., Jaworski, A.W. & Brown, S.K. (1990). Enzymatic browning in relation to phenolic compounds and polyphenoloxidase activity among various peach cultivars. Journal of Agricultural and Food Chemistry, 38, 99–101. Legaz, M.E., De Armas, R., Pinon, D. & Vicente, C. (1998). Relationships between phenolics-conjugated polyamines and sensitivity of sugarcane to smut (Ustilago scitaminea). Journal of Experimental Botany, 49, 1723–1728. Lima, G.P.P., Rocha, S.A., Takaki, M. & Ramos, P.R.R. (2006). Polyamines contents in some foods from Brazilian population basic diet. Cieˆncia Rural, 34, 1294–1298. Magkos, F., Arvaniti, F. & Zampelas, A. (2003). Organic food: nutritious food or food for though. A review of evidence. Journal of Food Sciences and Nutrition, 54, 357–371. Mitchell, A.E. & Chassy, A.W. (2006). Antioxidants and the Nutritional Quality of Organic Agriculture. Available at http://mitchell.ucdavis. edu/Is%20Organic%20Better.pdf (last accessed 13 February 2008). Nicolas, J.J., Richard-Forget, F.C., Goupy, P.M., Amiot, M.J. & Aubert, S. (1994). Enzymatic browning reactions in apple and apple products. Critical Reviews in Food Science and Nutrition, 34, 109–157.

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Pussemier, L., Larondelle, Y., Peteghem, C.V. & Huyghebaert, A. (2006). Chemical safety of conventionally and organically produced foodstuffs: a tentative comparison under Belgian conditions. Food Control, 17, 14–21. Quemener, V., Blanchard, Y., Chamaillard, L., Havouis, R., Cipolla, B. & Moulinoux, J.P. (1994). Polyamine deprivation: a new tool in cancer treatment. Anticancer Research, 14, 443–448. Ren, H., Bao, H., Endo, H. & Hayashi, T. (2001). Antioxidative and antimicrobial activities and flavonoids contents of organically cultivated vegetables. Nippon Shokuhin Kagaku Kagaku Kaushi, 48, 246–252. Santos, M.D. & Blatt, C.T.T. (1998). Teor de flavono´ides e feno´is totais em folhas de Pyrostegia venusta Miers. de mata e cerrado. Revista Brasileira de Botaˆnica, 21, 135–140. Seiler, N., Atanossov, C.L. & Raul, F. (1998). Polyamine metabolism as target fir cancer chemoprevention (review). International Journal of Oncology, 13, 993–1006. Siderer, Y., Maquet, A. & Anklam, E. (2005). Need for research to support consumer confidence in the growing organic food market. Trends in Food Science e Technology, 16, 332–343. Singleton, V.L. & Rossi, J.A. Jr (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. SOEL (2003). Stiftung Oekologie and Landbau, available at http:// www.soel.de/inhalte/publikationen/s/s_74.pdf (last accessed 13 February 2008) Thomas, T. & Thomas, T.J. (2003). Polyamine metabolism and cancer. Journal of Cellular and Molecular Medicine, 7, 113–126. Yussefi, M. & Willer, H. (2003). The World of Organic Agriculture 2003: Statistics and Future Prospect. SOL, 5th, August 26. Available at http://www.soel.de/inhalte/publikationen/s/s_74.pdf (last accessed 13 February 2008).

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Original article Equilibrium moisture of kale seed (Brassica oleracea var. acephala): an alternative method for choice of the best model Marcos A. S. Barrozo,* Nata´lia C. Bego & Daniel T. Oliveira Chemical Engineering School, Federal University of Uberlaˆndia, P.O. Box 593, Bloco K, Santa Moˆnica, 38400-902 Uberlaˆndia, Minas Gerais, Brazil (Received 30 May 2007; Accepted in revised form 9 January 2008)

Summary

As majority of the sorption equilibrium equations are nonlinear, care should be taken in estimating their parameters, because in some situations, the confidence interval of the least-squares (LS) estimators may not be appropriate. In these cases, some procedures are available in the literature to validate the statistical properties of the LS estimators of nonlinear models. In this work, the nonlinear measures are used to discriminate, between six equations that represent the sorption equilibrium isotherms of kale seeds (Brassica oleracea L. var. acephala D.C.). Results show that the Halsey modified equation is the best one to describe the experimental data.

Keywords

Drying, nonlinearity measures, seeds, sorption.

Introduction

Kale plants are native to the Mediterranean or to Asia Minor. They have been in cultivation for so long and have been so shifted about by prehistoric traders and migrating tribes, that it is not certain in which of those two regions the species originated. Wild forms have become widely distributed from their place of origin and are found on the coasts of northern Europe and Britain. It was introduced to America from Europe as early as the 17th century. Apparently, all the principal forms of kales that we know today have been known for at least 2000 years (Sawazaki et al., 1997). Kale (Brassica oleracea var. acephala) is derived from the cabbage of the mustard family. The name ‘acephala’ means ‘headless’ separating it from cabbage which is B. oleracea capitata. It is grown for its leaves and fleshy midribs. The genus includes broccoli, brussels sprouts, cabbage, cauliflower, collards, kohlrabi, mustard, petsai, rape, turnip, rutabaga and other crucifers (Kresovich et al., 1992). Seeds are living organisms that require specific storage conditions in order to remain capable of producing healthy, vigorous plants. Temperature and moisture are the primary factors that cause seeds to lose their ability to germinate. Excessive seed moisture increases its respiration rate, can contribute to the growth of *Correspondent: Fax: +55 34 32394188; e-mail: [email protected], [email protected]

micro-organisms, attract insect attack, and reduced viability. Kale seeds intended for long-term storage should kept at less than 10% moisture. Thus, drying kinetic parameters (Barrozo et al., 2006) as well as the moisture sorption isotherm should be well known for these seeds. The equilibrium relationship between the relative humidity (RH) and the moisture content at constant temperatures and pressures is expressed by the sorption isotherms. The traditional method for measuring sorption properties is the static method. The advantage of the static method is its ability to maintain constant conditions easily (Arnosti et al., 1999). To obtain experimentally the isotherms of equilibrium using the static method, it is possible to use acid solutions of different concentrations or saturated salt solutions. The salt solutions are used very often because they offer security and facility to maintain the constant RH. When there is evaporation of water, some salt precipitate, but the RH does not modify. As majority of the sorption equilibrium equations in the literature are nonlinear, care should be taken when estimating their parameters from experimental data. Note that, in some situations, the estimators (especially, confidence intervals) may not be appropriate. Therefore, some procedures are available in the literature to validate the statistical properties of the least-squares (LS) estimators of nonlinear models. In this work, the nonlinear measures (Bates & Watts, 1980) are used to select, from six model equations, the best one to represent the sorption equilibrium isotherms of kale seeds.

doi:10.1111/j.1365-2621.2008.01728.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Equilibrium moisture of kale seed: method for choice of the best model M. A. S. Barrozo et al.

Table 1 Equations for predicting the equilibrium moisture content Name (reference) Henderson (Henderson, 1952) Henderson–Thompson (Thompson et al.,1968) Chung–Pfost (Chung & Pfost, 1967) Chen–Clayton (Chen & Clayton, 1971)

Equation h i RHÞ 1=b ð1Þ Meq ¼ lnð1 aTS h i RHÞ 1=b ð2Þ Meq ¼ lnð1 aðTS þcÞ Meq Meq

  ðTS þcÞ lnðRHÞ ð3Þ ¼ 1 a b ln   lnðRHÞ 1 ¼ cT d ln aT b ð4Þ S

S

b

Sabbah (Pfost et al., 1976)

Meq

Halsey Modified (Osborn et al., 1989)

Meq

¼ a RH TSc ð5Þ h i1=b  expðaTS þcÞ ¼ ð6Þ lnðRHÞ

Equilibrium moisture equations

Several equations have been proposed to correlate the equilibrium moisture content, Meq, of agricultural and food products as a function of the air RH and the solid material temperature, Ts. Among them, the theoretical equations are based on well-known sorption kinetic theories, such as the Kelvin, the Langmuir or the BET one. However, in practical applications, these theoretical equations cannot accurately predict the equilibrium moisture content of seeds over a wide range of temperatures and air RHs. This has led some researchers to develop empirical or semi-empirical models in order to improve the accuracy of the predicted Meq values. Table 1 presents some usual equations reported in the literature for estimating Meq = f(RH,Ts) of biological materials. The coefficients a, b, c and d are the model parameters to be fitted to experimental data. In the literature, other models are described and used. The models of Table 1 have been tested and their parameters are evaluated for many agricultural crops (Mazza & Jayas, 1991; Basunia & Abe, 2001; Hong et al., 2002; Chen, 2003; Aviara et al., 2004; Tolaba et al., 2004; Pagano & Mascheroni, 2005; Cordeiro et al., 2006). These equations, among others, have been adopted as standard equations by the American Society of Agricultural Engineers for describing sorption isotherms (ASAE, 2003). Mazza & Jayas (1991) described equilibrium moisture content data of pea seeds at four temperatures and proposed the Chung–Pfost equation (eqn 3) as the best model among the other four models studied. Chen (2003) showed that the Henderson–Thompson equation (eqn 2) was the best model to describe the relationship between RH, equilibrium moisture and temperature of the pea sorption data. Ajibola (1989) showed that the modified Halsey equation (eqn 5) was the best model to represent the equilibrium data of melon seed. The choice of the best equations in the works mentioned here was based on R2 analysis and in some situations, on residual analysis. In the literature, there are other methodologies about the use of statistical indices to evaluate the performance of predictive models

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(Giffel & Zwietering, 1999). In the present work, the statistical discrimination between these nonlinear models was based on nonlinearity measures. Materials and methods

Experimental methodology

The technique used to obtain the equilibrium data of kale seed was based on the static method with the use of saturated salt solutions. Various salts were chosen, to get a larger range of RH (Greenspan, 1977). The samples were sealed up in small glass cylindrical containers with base diameter of 40 mm and height of 60 mm. Each of these vessels contained a different saturated salt solution, corresponding to a range of RH of 11–84% (Table 2). The kale seeds were placed on a perforated tray, arranged 30 mm from the base of the vessel to avoid any contact between the saturated salt solutions and the samples of seeds. The initial seed mass used in each reservoir was of about 1 g. This value was chosen because it allowed us to obtain a monolayer of grains in the surface of the tray. The initial kale seed moisture content was always higher than the equilibrium value to guarantee the desorption process. The vessels were then placed in an oven at controlled temperatures of 30, 35, 40 and 50 C (maximum variation of 0.5 C), and kept under constant thermodynamic conditions. The equilibrium conditions Table 2 Relative humidity (decimal) for the salt solutions and experimental data for the equilibrium moisture content (gram of water per 100 g of dry solid) as a function of temperature Temperature (°C) 30

35

Salt solution

RH

LiCl

0.113

3.01 3.22

0.113

CH3CO2K

0.216

0.210

MgCl2Æ6H2O

0.324

K2CO3

0.432

NaNO2

0.635

4.46 3.42 5.21 5.52 5.48 5.95 6.22 5.93 7.88 7.53

NaCl

0.750

KCl

0.834

Meq

10.26 9.64 9.88 12.15 12.73

RH

40 Meq

RH

0.321

2.86 3.18 3.37 4.10 3.81 5.46

0.318

0.432

6.28

0.432

0.625

7.31 7.42 7.34 10.32 10.59 10.27 12.97 12.03 12.93

0.616

0.749

0.826

0.112

0.204

0.748

0.818

50 Meq 3.11 3.00 2.87 4.00 5.23 5.36 5.42 6.12 6.19 6.06 7.47 7.32 7.14 10.46 10.00 10.05 11.53 11.42

RH

Meq

0.111

2.63

0.192

3.10 3.91 5.16 5.20 5.26 6.63 5.40 5.70 7.00 7.32 7.30 10.87 10.17

0.312

0.433

0.597

0.746

0.802

11.46

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were considered to be attained when three subsequent measurements of the mass sample gave identical results. Measurements were taken at intervals of 48 h. Each sample was weighed on an analytical balance with precision of 1 mg. The equilibrium moisture content of each sample was determined by the oven-drying method at 105 C for 24 h. The assays were performed in three containers containing the same salt solution to verify the reliability of the experimental procedure. Statistical methodology Nonlinearity measures

In general, the technique used for estimating the unknown parameters in linear or nonlinear equations is the LS method. This method has some optimum properties when certain conditions are met. The LS estimators of the unknown parameters in linear models are unbiased, normally distributed and have the property of being minimum variance (^ r2 ) estimators (Seber & Wild, 1989). Given that the assumptions noted above are satisfied, the criterion of LS thus provides the best available estimates in practice. An important point in the LS estimators for the nonlinear models is that these estimators do not have the properties of the linear models estimators. Only ‘asymptotically’, i.e., as the sample size increases to infinity, do the properties of the estimators in a nonlinear model approach the properties of a linear model. For finite sample size, a LS estimator of a parameter in a nonlinear model has essentially unknown properties. So, in general, the nonlinear regression models differ from linear regression models in that the LS estimators of the parameters are biased, non-normally distributed, and have variances exceeding the minimum possible variance. The extent of the bias, the non-normality and the excess variance differs widely from model to model. Beale (1960) made the first serious attempt to measure nonlinearity. Box (1971) presented a formula for estimating the bias in the LS estimators, and Gillis & Ratkowsky (1978), using simulation studies, found that this formula not only predicted bias to the correct order of magnitude but also gave a good indication of the extent of nonlinear behaviour of the model. Bates & Watts (1980) developed measures of nonlinearity based on the geometric concept of curvature. They showed that the nonlinearity of a model can be separated into two components: (a) an ‘intrinsic’ nonlinearity (IN) associated with the curvature of the solution locus and (b) a ‘parameter-effects’ nonlinearity (PE) associated with the fact that the projections of the parameter lines on the tangent plane to the solution locus are, in general, neither straight, parallel nor equispaced. Bates & Watts (1980) demonstrated the relationship between their measures and those of Beale (1960), and explained why Beale’s measures generally tend to underestimate

International Journal of Food Science and Technology 2008

the true nonlinearity. In addition, they showed that the bias measure of Box (1971) is closely related to the measure of parameter-effects nonlinearity (PE). Curvature measures of nonlinearity proposed by Bates & Watts(1980)

A question that has occupied the attention of researchers is that of how well some specified model fits the data. Extensive methodology has been developed for investigating whether a proposed model provides a good description of the data. Comparisons of the R2 values and residuals analysis can be insufficient to discriminate between nonlinear regression models (Ratkowsky, 1983). Other properties are desirable for nonlinear models, such as, the LS estimators of its parameters are almost unbiased, normally distributed, and whose variances are close to the minimum variance. Such a model should have both a low IN and a low PE. A negligible IN will mean negligible bias in the predicted values of response; if, simultaneously, the PE is also negligible, the more valid will be statistical tests of the consistency of the fitted parameters. Details about the development, procedure and equations for determining IN and PE values are found in Bates & Watts (1980) work. The statistical significance of these measures can bep evaluated by comparing the IN ffiffiffiffi and PE values with 1=2 F , where F = F(a, n–p, p) is the inverse of the probability Fisher distribution pffiffiffiffi obtained at the significance level a. The value 1=2 F may be regarded as the radius of curvature of the 100 (1–a)% confidence region. So, the solution locus may be considered to be sufficiently linear over pan ffiffiffiffi approximate 95% confidence region pffiffiffiffi if IN < 1=2 F (a = 0.05). Similarly, if PE < 1=2 F , the projected parameter lines may be regarded as being sufficiently parallel and uniformly spaced, i.e., the LS estimates of the parameter do not depend on the user being able to supply a good initial prevision and the tests of parameters invariance will be adequate. For carrying out all calculations to determine the IN and PE values, a computer program in Fortran language, similar to one proposed by Ratkowsky (1983), has been developed for the present work. The bias measure proposed by Box (1971)

The formula derived by Box (1971) for calculating the bias in the LS estimates of the parameters in nonlinear regression models having a single response variate is: 2 !1 !1 3 n n n 2 X X X ^ r Fi FTi Fu tr4 Fi FTi Hu 5 Biasð^hÞ ¼  2 i¼1 u¼1 i¼1 ð7Þ where ^h represents the LS estimator, Fi (=Fu) is the px1 vector of first derivatives of f(Xi, h) and Hu is the pxp

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Equilibrium moisture of kale seed: method for choice of the best model M. A. S. Barrozo et al.

matrix of second derivatives with respect to each of the elements of h, evaluated at Xi (independent variable), ^2 are usually used where i = 1,2...,n. In practice, ^ h and r in place of the unknown quantities. The bias expressed as a percentage of the LS estimate is a useful quantity as an absolute value in excess of 1% appears to be a good thumb rule for indicating nonlinear behaviour (Ratkowsky, 1983). The percentage bias is given by: %Biasð^ hÞ ¼

100  Biasð^ hÞ ^ h

ð6Þ

Results and discussion

The experimental data for the equilibrium moisture content with the replicates for each operational condition are given in Table 2. This table also presents the saturated salt solutions used and the respective RH for the four temperature levels. The average SD of the equilibrium moisture data (replicates) was equal to 0.27. Table 3 presents the results obtained by the LS parameter estimation for the six model equations listed in Table 1. These results include the estimated parameter values (for Meq expressed in dry basis percentage, temperature in C and RH in the decimal fraction), as well as, the respective values of the quadratic regression coefficient (R2), the intrinsic curvature measure (IN), the parameter-effects measure (PE), the bias percentage, the

significance level of the parameters and the asymptotic 95% confidence intervals. It can be observed in Table 3 that three equations had the highest values of R2 and very close among them. As mentioned previously, the higher R2 value for the Halsey equation is not enough to guarantee the statistical validity of the parameters obtained in a nonlinear regression. Table 3 results show that all calculated values of the intrinsic p curvature measures (IN) are not significant ffiffiffiffi (IN < 1=2 F ), inferring a small deviation of the model solution locus from linearity (Ratkowsky, 1983). On the contrary, the calculated values of PE (the parameterseffects curvature measure) are significant for eqns 1–5 pffiffiffiffi (PE 1=2 F ), meaning that, at least, one parameter of these equations has a strong nonlinear behaviour. The best results of the nonlinearity measures are obtained for the Halsey modified model equation (eqn 6) as pffiffiffiffi PE < 1=2 F . Therefore, among the six equations, this Halsey modified equation behaves closest to the LS linear model approach, meaning valid inference results based on asymptotic approximations assumed in the LS nonlinear estimates. As eqns 1–5 present significant nonlinear parametereffects (PE), the bias measures should be used to identify parameters that are responsible for the nonlinear behaviour (% bias > 1%). The semi-empirical equation of Henderson (1952) is based on the Gibbs sorption model. Although this equation is commonly used to predict Meq of many biological products, it cannot describe well the grain

Table 3 Statistical results of the least-squares estimation

Equation

R2

Curvature

Parameter

Estimated value

% Bias box

Significance level (P)

95% CI

Henderson (eqn 1)*

R2 = 0.931

Henderson–Thompson (eqn 2)**

R2 = 0.941

IN = 0.024 PE = 1.592 IN = 0.059 PE = 6.123

Chung–Pfost (eqn 3)**

R2 = 0.971

IN = 0.027 PE = 3.648

Chen–Clayton (eqn 4)***

R2 = 0.972

IN = 0.130 PE = 62.203

Sabbah (eqn 5)**

R2 = 0.931

IN = 0.036 PE = 9.549

Halsey modified (eqn 6)**

R2 = 0.975

IN = 0.017 PE = 0.079

a b a b c a b c a b c d a b c a b c

3.52 · 10)4 2.043 4.47 · 10)4 1.728 19.262 1380.67 0.281 249.99 6.848 )0.098 0.262 )0.020 20.016 0.764 0.133 1.29 · 10)3 1.931 3.150

1.00 0.130 0.070 0.091 11.056 47.719 0.049 55.262 41.744 )3.495 11.366 5.459 3.670 0.058 0.016 0.988 0.043 0.066

0.000 0.000 0.238 0.167 0.001 0.157 0.000 0.221 0.300 0.710 0.043 0.882 0.001 0.000 0.082 0.575 0.000 0.000

2.3 · 10)4; 4.7 · 10)4 1.88; 2.21 )3.0 · 10)4;1.2 · 10)3 0.35; 3.11 18.1; 20.4 )549.6; 3311.0 0.269; 0.294 )154.7; 654.7 )6.17; 19.86 )0.62; 0.43 0.08; 0.515 )0.29; 0.25 9.2; 30.9 0.70; 0.83 )0.017; 0.283 )3.3 · 10)3; 5.9 · 10)3 1.84; 2.02 2.87; 3.43

*1=2

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Fð2;60;0:95Þ ¼ 0:282; **1=2 Fð3;59;0:95Þ ¼ 0:301; ***1=2 Fð4;58;0:95Þ ¼ 0:314.

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Equilibrium moisture of kale seed: method for choice of the best model M. A. S. Barrozo et al.

sorption equilibrium. To improve its application, Thompson et al. (1968) have modified this Henderson equation by introducing a new fitted parameter (eqn 2). Table 3 results show that the addition of one more parameter into the Henderson equation causes a greater model deviation from the linear behaviour. As this parameter is empirical, a parametric analysis leading to a new formulation (reparameterization) of eqns 2 and 3 can take their behaviour close to the linear one. As already predicted by the PE values, eqns 1–5 present, at least, one parameter with %bias higher than 1%. The Halsey modified equation, expressed by eqn 6, is the only one in which %bias values are not significant (%bias < 1% for all the three parameters). Therefore, the results of the curvature and bias measures showed that the modified Halsey model estimates are more robust. The parameter a of the Halsey equation is not significant (see significance level in Table 3), showing that the effect of temperature is very small and that this model is over parametrized for this case. The experimental values of the kale seed equilibrium moisture content (Meq) expressed as percent dry basis at the different temperatures and RHs, obtained in a range of 11 < RH (%) < 83 and 30 £ Ts (C) £ 50, are presented in Fig. 1. Conventionally, equilibrium moisture content was found to increase with decrease in temperature at constant RH and to increase with increase in RH of air when temperature was kept constant. The response surface given by the modified Halsey equation is also presented in Fig. 1. It can be

14

Meq (dry base × 100)

12 10 8 6 4 2

0.6 5 R 0. 4 H 0.

32 30

0.2

36 34

0.3

50 48 46 44 42 40 38

0.8 7 0.

)

Ts

(C

Figure 1 Experimental equilibrium moisture content of kale seeds as a function of RH and Ts and the response surface predicted by the Halsey modified equation (eqn 6 in Table 1).

International Journal of Food Science and Technology 2008

observed a good agreement between the experimental data and the predicted values. Conclusions

From results obtained in this present work, the following conclusion can be drawn: 1 From the discrimination approach based on nonlinear measures, it is possible to select the more robust model equation for describing the experimental sorption equilibrium data of kale seeds; 2 Among six models analyzed in this work, the Halsey modified equation is the only one that for which curvature measures and bias were not significant; the parameter a of the Halsey equation is not significant showing that the effect of temperature is very small 3 The statistical analysis, as well as, the computer program developed in this work can be easily extended to any other nonlinear regression model estimation. Acknowledgments

The authors would like to thank the Brazilian funding agencies, FAPEMIG and CNPq, for their financial support. References Ajibola, O.O. (1989). Thin layer drying of melon seed. Journal of Food Engineering, 9, 305–320. Arnosti, S. Jr, Freire, J.T., Sartori, D.J.M. & Barrozo, M.A.S. (1999). Equilibrium moisture content of Brachiaria brizantha. Seed Science and Technology, 27, 273–282. ASAE (2003). Moisture relationships of plant-based agricultural products. ASAE standard-2003, standard engineering practices data. ASAE yearbook. Pp. 538–550. St Joseph: ASAE D245.5. Aviara, N.A., Ajibola, O.O. & Oni, S.A. (2004). Sorption equilibrium and thermodynamic characteristics of soya bean. Biosystems Engineering, 87, 179–190. Barrozo, M.A.S., Henrique, H.M., Sartori, D.J.M. & Freire, J.T. (2006). The use of the orthogonal collocation method on the study of the drying kinetics of soybean seeds. Journal of Stored Products Research, 42, 348–356. Basunia, M.A. & Abe, T. (2001). Moisture desorption isotherms of medium-grain rough rice. Journal of Stored Products Research, 37, 205–219. Bates, D.M. & Watts, D.G. (1980). Relative curvature measures of nonlinearity. Journal of the Royal Statistical Society, 42, 1–25. Beale, E.M.L. (1960). Confidence regions in nonlinear estimation. Journal of the Royal Statistical Society, 22, 41–76. Box, M.J. (1971). Bias in nonlinear estimation. Journal of the Royal Statistical Society, 33, 171–201. Chen, C. (2003). Moisture sorption isotherms of pea seeds. Journal of Food Engineering, 58, 45–51. Chen, C.S. & Clayton, J.T. (1971). The effect of temperature on sorption isotherms of biological materials. Transactions of the ASAE, 14, 927–929. Chung, D.S. & Pfost, H.B. (1967). Adsorption and desorption of water vapour by cereal grains and their products Part II. Transactions of the ASAE, 10, 494–551.

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Cordeiro, D.S., Raghavan, G.S.V. & Oliveira, W.P. (2006). Equilibrium moisture content models for Maytenus ilicifolia leaves. Biosystems Engineering, 94, 221–228. Gillis, P.R. & Ratkowsky, D.A. (1978). The behaviour of estimators of the parameters of various yield-density relationships. Biometrics, 34, 191–198. Greenspan, L. (1977). Humidity fixed points of binary saturated aqueous solutions. Journal Res. Natl. Bureau of Standards, 81, 89– 93. Henderson, S.M. (1952). A basic concept of equilibrium moiture content. Agricultural Engineering, 33, 29–31. Hong, T.D., Ellis, R.H., Gunn, J. & Moore, D. (2002). Relative humidity, temperature, and the equilibrium moisture content of conidia of Beauveria bassiana (Balsamo) Vuillemin: a quantitative approach. Journal of Stored Products Research, 38, 33–41. Kresovich, S., Williams, J.G.K., Mcferson, J.R., Routman, E.J. & Chaal, B.A. (1992). Characterization of genetic identities and relationship of Brassica oleraceae L. via a random amplified polymorphic DNA array. Theoretical and Applied Genetics, 85, 190–196. Mazza, G. & Jayas, D.S. (1991). Evaluation of four three-parameter equations for the description of the moisture sorption data of Lathyrus pea seeds. Lebensmittel-Wissenschaft und-Technologie, 24, 562–565.

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Osborn, G.S., White, G.M., Sulaiman, A.H. & Welton, L.R. (1989). Predicting equilibrium moisture proportions of Soybeans. Transactions of the ASAE, 32, 2109–2113. Pagano, A.M. & Mascheroni, R.H. (2005). Sorption isotherms for amaranth grains. Journal of Food Engineering, 67, 441–450. Pfost, H.B., Maurer, S.G., Chung, D.S. & Milliken, G. (1976). Summarizing and reporting equilibrium moisture data for grains. Pp. 76–3520. St Joseph: ASAE. Ratkowsky, D.A. (1983). Nonlinear Regression Modeling. Pp. 13–47. New York, NY: Marcel Dekker Inc. Sawazaki, H.E., Nagai, H. & Sodek, L. (1997). Characterization of genetic variability of kale plants by enzymatic polymorphism and RAPD. Bragantia, 56, 9–19. Seber, G.A.F. & Wild, J. (1989). Nonlinear Regression. Pp. 120–128. New York, NY: Wiley. Te Giffel, M.C. & Zwietering, M.H. (1999). Validation of predictive models describing the growth of Listeria monocyogenes. International Journal of Food Microbiology, 46, 135–149. Thompson, T.L., Peart, R.M. & Foster, G.H. (1968). Mathematical simulation of corn drying – a new model. Transactions of the ASAE, 11, 582–586. Tolaba, M.P., Peltzer, M., Enriquez, N. & Pollio, M.L. (2004). Grain sorption equilibria of quinoa grains. Journal of Food Engineering, 61, 365–371.

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Original article Effect of flavonoids on the oxidative stability of corn oil during deep frying Shahina Naz,* Rahmanullah Siddiqi & Syed Asad Sayeed Department of Food Science & Technology, University of Karachi, Karachi –75270, Pakistan (Received 3 July 2007; Accepted in revised form 6 December 2007)

Summary

To study the effect of flavonoids on the stability of frying oil, refined corn oil was analysed periodically for its peroxide value (PV), p-anisidine value (p-AV) and iodine value (IV) after its use for deep-frying of French fries at 180 C for varying periods of time, namely 30, 60 and 90 min. PV and p-AV values increased with respect to time while a decrease in IV was observed with increase in time (P < 0.001). Deep-frying of French fries in corn oil was then carried out in the presence of flavonoids, viz. pelargonidin, cyanidin, quercetin, myrecetin and gallic acid as antioxidants. All antioxidants effectively reduced the oxidation rate in the oil, as detected by decrease in PVs and p-AVs and relatively low reduction rate in IVs (P < 0.001). The order of antioxidative activity was gallic acid > quercetin > myrecetin > cyanidin > pelargonidin.

Keywords

Corn oil, deep frying, flavonoids, iodine value, p-anisidine value, peroxide value.

Introduction

Almost all free and packaged foods and drinks undergo gradual changes during storage. Ignoring the degradation caused by microorganisms, the typical cause of spoiling is the presence of oxygen and the products of chemical oxidation (Gray et al., 1994; Du, 1997). The process of auto-oxidation and the development of rancidity involve a free radical chain mechanism with several steps (Rossignol-Castera, 1998). In addition, lipid quality deteriorates under photo-oxidative conditions (Xiaoying & Ahn, 1998) or oxidation under thermal conditions such as frying of food (Matalgyto & Al-Khalifa, 1998; O’Neill et al., 1998; Medina et al., 1999; Witting et al., 1999; Yuki & Sadaaki, 2001). For these reasons, preservatives with antioxidant activity have been added to packaged foods for many years. Major food antioxidants include tertiary butylhydroquinone, propyl gallate, butylated hydroxytoluene (BHT) and butylated hydroxyl anisole (BHA). The use of these synthetic antioxidants, however, is not without their problems. BHA and BHT have been suspected of being responsible for liver damage and carcinogenesis in laboratory animals (Grice, 1986; Wichi, 1988). The trend nowadays is towards improving shelf-life of products, without using synthetic preservatives. Thus interest is renewed in the potential of natural antioxidants. *Correspondent: E-mail: [email protected]

The inherent antioxidant properties of all plantbased polyphenols (Oelschlaeger et al., 2004; Gramza & Korczak, 2005; Osakabe, 2005; Scalbert et al., 2005; Loots et al., 2006) offer natural options to food manufacturers. Some of the most commonly occurring polyphenols are the flavonoids with a large number of phenolic hydroxyl groups attached to ring structures that confer antioxidant activity. They are widely distributed in the plant kingdom, being present in all vascular plants. They are major constituents of many fruits (Oelschlaeger et al., 2004; Loots et al., 2006), grains and beverages such as green and black tea (Gramza & Korczak, 2005), coffee (Caemmerer & Kroh, 2006) and chocolate (Osakabe, 2000; Keri, 2001). This exceeds their daily consumption and causes their presence in the human diet, at rather high levels. In view of the above facts, studies have been designed to evaluate the role of naturally occurring polyphenols in chemical stabilisation of edible oils and fats (Koketsu & Satoh, 1997; Basuny, 2004 and Farag et al., 2006). In one of our previous studies, the relative efficiency of caeffic, vanillic and ferulic acids in reducing oxidation rate in heated frying oil has already been evaluated (Naz et al., 2004). In this study the effects of flavonoids, viz. pelargonidin, cyanidin, quercetin, myrecetin and gallic acid on the stability of heated refined corn oil was evaluated. Although experiments have already been performed to evaluate the antioxidant activity of these compounds in different lipid-contain-

doi:10.1111/j.1365-2621.2008.01731.x  2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Flavonoids and oxidative stability of corn oil S. Naz et al.

ing models using different methods (Kaehkoenen & Heinonen, 2003), no study has yet evaluated the effect of these compounds on the oxidative stability of edible oils during heating. Refined corn oil was selected for the study for two major reasons. First, corn oil can play a very important role in human diet. It is a concentrated source of energy, very digestible and provides essential fatty acids (Hauman, 1985). Because of the health benefits, it is popular as a cooking medium. Secondly, its unsaturated fatty acids have relatively high reaction rates with oxygen (List & Erickson, 1985). Materials and methods

Analysis of the corn oil used for deep-frying

A volume of 2.5 L of refined corn oil (purchased from local market) was placed in a deep-fryer; 250 g of peeled potatoes were cut into cubical bars (10 · 10 · 90 mm) and then fried at 180 C for 30, 60 and 90 min in triplicate. After frying, oil samples were cooled to room temperature and then each of the replicates (triplicate) was analysed in triplicate for change in peroxide value (PV; IUPAC Standard Methods 2.501, 1987), p-anisidine value (p-AV; IUPAC Standard Methods 2.504, 1987) and iodine value (IV; IUPAC Standard Methods 2.505, 1987). The results were compared with the values obtained for the oil sample analysed immediately after opening the can (control). Analysis of corn oil used for deep-frying in presence of added antioxidants

An amount of 0.5 g of each antioxidant, namely pelargonidin (1-benzopyranium, 3,5,7-trihydroxy)-2-(4-hydroxyphenyl)-chloride (9Cl), Sigma (Sigma-Aldrich, St. Louis, MO, USA) [134-04-3], cyanidin (1-benzopyrylium, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-chloride (9Cl), Sigma [528-58-5]), myrecetin (4H-1-benzopyran-4-one, 3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-9(Cl), Sigma [529-44-2], quercetin(4H-1-benzopyran-4-one, 3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl), Sigma [117-39-5] and gallic acid (3,4,5-trihydroxy benzoic acid, Sigma [149-917]) was dissolved in 25 mL of absolute ethanol. The ethanolic solution of each antioxidant was then mixed with 2.5 L of the corn oil in triplicate to be used for deepfrying at 180 C for 90 min. Changes in PV, p-AV and IV of each replicate (triplicate) were analysed as described in the previous section. The results were compared with the values obtained for the oil samples heated without antioxidants (control). The amount of 0.5 g of the antioxidant and 90 min as the frying duration were selected after several trials with different concentrations and frying durations for significant changes in values of sample with antioxidants.

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Statistical analysis

The data were analysed for significant differences by one-way analysis of variance and multiple comparisons by the Bonferroni (1936) method using S-Plus 7.0 software. Results and discussion

Refined corn oil was selected for this study because of its composition and nutritional value. It is composed of 99% triacylglycerols with polyunsaturated fatty acid (PUFA) 59%, monounsaturated fatty acid 24% and saturated fatty acid (SFA) 13%. The PUFA is linoleic acid (C18:2n-6) primarily, with a small amount of linolenic acid (C18:3n-3) giving a n-6 ⁄ n-3 ratio of 83 (Dupont et al., 1990). No single source of salad or cooking oil provides an optimum fatty acid (FA) composition. Because of its low content of SFAs which raises cholesterol and its high content of PUFAs which lowers cholesterol, consumption of corn oil can replace SFAs with PUFAs, and the combination is more effective in lowering cholesterol than simple reduction of SFA. It has good sensory qualities for use as a cooking oil, is highly digestible and provides energy and essential fatty acids (EFA) (Dupont et al., 1990). To derive the oxidation rate in each oil sample, the samples were analysed periodically for PV, p-AV and IV, as a single reaction criterion is not enough to account for the oxidative changes at various stages under different conditions. PV and p-AV values increased with increase in frying time (Matalgyto & Al-Khalifa, 1998; Isabei & Mariano, 2001) (Fig. 1), whereas IVs decreased with respect to time (Fig. 2). The values were significantly different compared with control (P < 0.001), and differences among values of the samples fried at different temperatures were also highly significant (P < 0.001) except the difference between 60 and 90 min of frying (statistically insignificant). Oxidation, which consists of a complex series of 7 6

PV p-AV

5 4 3 2 1 0

Control

30 min

60 min

90 min

Figure 1 Effect of deep frying on the PV and p-AV of corn oil.

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134

7

132

6

130

5 PV(meqO2 Kg–1)

IV (I2 100g–1 oil)

128 126

4 3

124

2

122

1 0

120 Control

30 min 60 min Frying time

Gallic acid

90 min

Quercetin

Myrecetin

Cyanidin

Pelargonidin

Control

Figure 3 Effect of adding antioxidant on the PV of the corn oil.

Figure 2 Effect of deep frying on the IV of corn oil. 6

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5 4 p-VA

chemical reactions, is characterised by a decrease in the total unsaturated content of the oil because of abstraction of hydrogen adjacent to a double bond and the formation of free radicals. Hence the deep-frying, which accelerates the oxidation in the oil, and also causes a reduction of the IVs and increase in PVs and p-AVs. However, if the free radical exceeds a certain level, recombination of free radical (termination), may cause decline in PVs and p-AVs. This may be the reason for the insignificant difference in values between 60 and 90 min of frying. The effects of adding antioxidants, viz. pelargonidin, cyanidin, quercetin, myrecetin and gallic acid on PV, IV and p-AV of the corn oil were analysed after deep-frying for 90 min at 180 C. A decrease in the PVs and p-AVs, and relatively low reduction in IVs were observed with all antioxidants. As the results were significantly different compared with the control (P < 0.001) and as multiple comparisons among different antioxidants showed highly significant differences (P < 0.001 except insignificant IV difference between quercetin and myrecetin and PV difference between myrecetin and cyanidin), it is concluded that all antioxidants effectively reduced the oxidation rate in the oil (Figs 3–5). The order of antioxidative activity was found to be gallic acid > quercetin > myricetin > cyanidin > pelargonidin. This difference in activity may be accounted on the basis of their chemical structures (Fig. 6). In general, the antioxidant capacity of compounds possessing an o-diphenolic arrangement (catechol structure) is higher than in monophenols because of their ability to form o-quinones when reacting with free radicals. In addition, compounds having two -OH groups at adjacent positions act as chelators for most of the metal ions that act as pro-oxidants and that may catalyse the reaction even if present in trace amounts. Replacing the 3-hydroxyl group of protocatechuic acid by a methoxy group as in vanillic acid had a suppressive influence on the antioxidant capacity (Rosch et al.,

3 2 1 0

Gallic acid

Quercetin

Myrecetin

Cyanidin Pelargonidin

Control

Figure 4 Effect of adding antioxidant on the p-AV of corn oil.

128 127.5 127 IV (gl2 100g–1 oil)

1852

126.5 126 125.5 125 124.5 124 Gallic acid

Quercetin

Myrecetin

Cyanidin

Pelargonidin

Control

Figure 5 Effect of adding antioxidants on the IV of the corn oil.

2003; Naz et al., 2004, 2005). This explains the comparatively higher antioxidant capacity of cyanidin compared with pelargonidin. The highest antioxidant activity of gallic acid is because of the presence of an additional hydroxyl group. Because of its pyrogallol structure, it shows a greater oxidisability and the quinine formed can be stabilised by resonance structures. The importance of a pyrogallol structure for maximum antioxidant activity of hydroxyl benzoic acid derivatives was also described by Rice-Evans et al.

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Flavonoids and oxidative stability of corn oil S. Naz et al.

OH

OH HO

OH

+ O

HO

OH

+ O

OH

Cyanidin

Pelargonidin

OH OH

OH HO

HO

O

O

OH

OH OH

OH

OH

O

OH

Quercetin

O

Myrecetin

HO HO

COOH HO

Figure 6 Phenolic structures.

(1996) for the trolox equivalent antioxidant capacity (TEAC) assay and by Cao et al. (1997) for the oxygen radical absorbing capacity (ORAC) assay. A possible reason for the lower antioxidant activity of myricetin compared with quercetin could be the high oxidation sensitivity of myricetin which caused its rapid decomposition during measurement (Burda & Oleszek, 2001). References Basuny, A.M. (2004). Influence of grape seed phenolic compounds on thermal stability of frying oil. Egyptian Journal of Food Science, 32, 65–78. Bonferroni, C.E. (1936). Teoria statistica delle classi e calcolo delle probabilita. Pubblicazioni del R Instituto Superiore di Scienze Economiche Commerciali di Firenze, 8, 3–62. Burda, S. & Oleszek, W. (2001). Antioxidant and antibacterial activities of flavonoids. Journal of Agricultural Food Chemistry, 49, 2774–2779. Caemmerer, B. & Kroh, L.W. (2006). Antioxidant activity of coffee brews. European Food Research and Technology, 223, 469–474. Cao, G., Sofie, E. & Prior, R.L. (1997). Antioxidant and prooxidant behavior of flavonoids: structure–activity relationship. Free Radical Biology and Medicine, 22, 749–760. Du, W. (1997). The oxidation factors of high grade edible oil and its prevention. Zhongguo Youzhi, 22, 35–37. Dupont, J., White, P.J., Carpenter, M.P. et al. (1990). Food uses and health effects of corn oil. Journal of the American College of Nutrition, 9, 438–470.

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Koketsu, M. & Satoh, Y.-I. (1997). Antioxidant activity of green tea polyphenols in edible oils. Journal of Food Lipids, 4, 1–9. List, G.A. & Erickson, D.R., 1985. Relative reaction rates of unsaturated fatty acids with O2 and inherent stability of oil. In: Baily’s Industrial Oil and Fat Products, Vol. III, 4th edn (edited by T.H. Applewhite). pp. 275–277. New York, NY: Wiley. Loots, D.T., Vander-Westhuizen, F.H. & Jerling, J. (2006). Polyphenol composition and antioxidant activity of Kei –Apple (Dovyalis caffra) juice. Journal of Agricultural and Food Chemistry, 54, 1271–1276. Matalgyto, F.S & Al-Khalifa, A.S. (1998). Effect of microwave oven heating on stability of some oil and fats. Arab Gulf Journal of Scientific Research, 16, 21–40. Medina, I., Satue-Gracia, M.T., German, J.B. & Frankel, E.N. (1999). Comparison of natural polyphenol antioxidants from extra virgin olive oil with synthetic antioxidants in tuna lipids during thermal oxidation. Journal of Agricultural and Food Chemistry, 47, 4873– 4879. Naz, S., Sheikh, H., Siddiqi, R. & Sayeed, S.A. (2004). Oxidative stability of olive, corn and soybean oil under different conditions. Food Chemistry, 88, 253–259. Naz, S., Sheikh, H., Siddiqi, R. & Sayeed, S.A. (2005). Deterioration of olive, corn and soybean oils due to air, light, heat and deepfrying. Food Research International, 38, 127–134. O’Neill, L.M., Galvin, K., Morrissey, P.A. & Buckley, D.J. (1998). Comparison of effect of dietary olive oil, tallow and vitamin E on the quality of broiler meat and meat products. British Poultry Science, 39, 365–371. Oelschlaeger, C., Milde, J., Schempp, H. & Treutter, D. (2004). Polyphenols and antioxidant capacity of Sorbus domestica L. fruits. Journal of Applied Botany and Food Quality, 78, 112–116.

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Osakabe, M. (2000). Functions of polyphenols in chocolate and cocoa. Food Style 21, 4, 68–71. Osakabe, M. (2005). Cacao polyphenols: antioxidant activity and marketing development. Food Style 21, 9, 39–42. Rice-Evans, C.A., Miller, N.J. & Paganga, G. (1996). Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933–956. Rosch, D., Bergmann, M. & Knorr, D. (2003). Structure-antioxidant efficacy relationships of phenolic compounds and their composition to the antioxidant activity of sea buckthorn juice. Journal of Agriculture and Food Chemistry, 51, 4233–4239. Rossignol-Castera, A. (1998). Oxidation and antioxidation mechanisms. Rivista Italiana EPPOS, 9, 163–186. Scalbert, A., Johnson, I.T. & Saltmarch, M. (2005). Polyphenols: antioxidants and beyond. Amercian Journal of Clinical Nutrition, 81, 512S–217S. Wichi, H.P. (1988). Enhanced tumpr development by butylated hydroxyanisole (BHA) from the perspective effect on forestomach and oesophageal squamous epithelium. Food and Chemical Toxicology, 26, 717–723. Witting, P.K., Detlef, M. & Roland, S. (1999). Assesment of prooxidant activity of vitamin E in human low-density lipoprotein and plasma. Methods in Enzymology, 299, 362–375. Xiaoying, C. & Ahn, D.U. (1998). Antioxidant activities of six naturals phenolics against lipid oxidation induced by Fe+2 or ultraviolet light. Journal of the American Oil Chemists Society, 75, 1717–1721. Yuki, F. & Sadaaki, L. (2001). Temperature dependency of autoxidation rate constants of edible oils. Wayo Joshi Daigaku Kiyo, Kaseikei-Hen, 40, 25–35.

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International Journal of Food Science and Technology 2008, 43, 1855–1859

Original article (+)-Catechin and ())-epicatechin levels of concentrated and ready-to-drink grape juices through storage Andre´a Pittelli Boiago Gollu¨cke,* Jane Cristina de Souza & De´bora de Queiroz Tavares Food and Nutrition Department, Food Engineering Institute, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 80 CP 6121, CEP 13083-862, Campinas, SP, Brazil (Received 2 August 2007; Accepted in revised form 11 January 2008)

Summary

Commercial concentrated Concord (CCJ) and Isabel (CIJ) grapes juices were stored at 4–5 C while pasteurised ready-to-drink juices of the same grape cultivars (PCJ and PIJ) were kept at 20–25 C under indirect light for 10 months, simulating industrial storage conditions. (+)-catechin preservation during storage ranged between 63% (PCJ) and 52% (PIJ); ())-epicatechin retention was of 32% (CCJ) and 15% (CIJ). Total phenols retention ranged from 93% (CCJ) to 84% (PCJ) and radical scavenging activity (RSA) from 87% (PIJ) to 85% (CCJ and PCJ). Concentrated juices showed higher monomeric flavan-3-ols amounts and CCJ depicted superior phenolic contents. PIJ yielded the highest RSA during storage per phenolic unit. Process and storage impacted flavan-3-ols and not total phenolics and RSA during 10-month ageing.

Keywords

1,1-Diphenyl-2-picrylhydrazil, grape juice, phenolics, quality, storage.

Introduction

Epidemiologic studies have revealed that phenolic-rich diets reduce mortality by degenerative diseases caused by oxidative stress (Scalbert et al., 2005). Amongst the various classes of phenolic compounds, flavan-3-ols exert physiologic properties that may be the source of health benefits from wine consumption (Gu¨rbu¨z et al., 2007). Dietary intervention studies support flavan-3-olrich foods and beverages as being beneficial to cardiovascular health (Keen et al., 2005). In grape juices, flavan-3-ols are mostly found in the monomeric forms of catechins [(+)-catechin (CAT) and ())-epicatechin (EPI)] with large differences amongst cultivars (Jarowski & Lee, 1987; Lee & Jarowski, 1987; Spanos & Wrolstad, 1990; Auw et al., 1996). Flavan-3-ols are also present in other processed foods and beverages such as chocolate (2.00–14.89 mg g)1), chocolate milk (21.1 mg L)1); wines (27.3–95.5 mg L)1) and tea (100–800 mg L)1) (Arts et al., 2000; Tokusoglu & U¨nal, 2002; Manach et al., 2004). Monomeric flavan-3-ols belong to the group of three polyphenols most well-absorbed by humans, after gallic acid and isoflavones (Manach et al., 2005). In human intervention studies flavan-3-ols have been associated with increased plasma antioxidant activity, increased plasma ascorbate concentrations, increased resistance to LDL oxidation and decreased plasma lipid peroxide and *Correspondent: E-mail: [email protected]

malondialdehyde concentrations (Lotito & Fraga, 1997; Kampa et al., 2000; Kimura et al., 2002; Williamson & Manach, 2005). In an epidemiological study, Arts et al. (2001a,b) demonstrated a positive association between monomeric flavan-3-ol consumption and reduced mortality by chronic diseases. The principal property of polyphenols including flavan-3-ols is the antioxidant capacity, associated with product quality and health benefits. Vidal et al. (2004) investigated bitter and astringent properties of tannin-like polyphenols of wine. The authors found that monomeric flavan-3-ols contributed to astringency and bitterness when in sufficient concentration and that polymeric substances formed during wine ageing may lead to decrease in both sensory properties. Es-Safi et al. (2003) demonstrated that CAT incubated with glyoxylic acid (an oxidation product of tartaric acid present in grapes) formed brown products during ageing. Polyphenolic compounds are highly unstable and react with other substances and amongst themselves during food processing and storage. In wines, phenolics undergo changes during ageing resulting in known and unknown phenolic species (Cheynier, 2005). Such modifications are overlooked in most studies concerning food composition. Arts et al. (2000) noted that epidemiological research required further studies on flavan-3-ol changes during processing and ageing. The design of the present work contemplates two commercial products under particular storage conditions. Furthermore, knowledge of the radical scavenging activity (RSA)

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during storage may support prediction of the antioxidant capacity. Thus, the objectives of this work were to verify the impact of storage on total phenols, flavan-3-ol monomers and antioxidant activity of commercial grape juices. Materials and methods

absorbance at 517 nm was measured with a Beckman spectrometer before addition of the radical (blank sample) and after 30 min; the difference was plotted in a Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) (Sigma-Aldrich) standard curve. Analyses were carried out in duplicates and the results expressed in mm Trolox equivalent L)1 (mm TE).

Sample production and storage

Determination of (+) catechin and ()) epicatechin

Samples of concentrated and pasteurised ready-to-drink grape juices of Concord and Isabel cultivars (Vitis labrusca species) were received in February and March of 2006. Concentrated juices were supplied by a manufacturer after process which consists of pressing with simultaneous pasteurisation followed by concentration to 68Brix. Concentrated juices are produced during harvest and supplied along the year to other manufacturers for the production of reconstituted grape beverages. Storage conditions were between 4 and 5 C in the dark, simulating storage conditions at the industry. Pasteurised ready-to-drink grape juices of the same cultivars were obtained from another manufacturer after cold grape pressing, pasteurisation and bottling. Soluble solids ranged from 14 and 16Brix for Concord and from 17 to 19Brix for Isabel grape. Juices were stored in their own transparent green glass bottles under indirect lighting and room temperature (20–25 C), simulating storage conditions at manufacturer or retail. Every 30 days two samples from each combination were taken from the specific storage condition and placed at a freezer at )18 C in the dark until analysed, up to maximum ageing time of 10 months. Prior to analysis concentrated juices were reconstituted to 17Brix. Pasteurised juices were analysed at the natural Brix.

CAT and EPI were analysed by HPLC with fluorescence detection according to Arts & Hollman (1998) and Arts et al. (2000). The standards of CAT and EPI were obtained from Sigma-Aldrich. A Perkin Elmer HPLC (Perkin-Elmer, Newark, CT, USA) equipped with a 250 mm · 4.6 mm GL Science, Inertisil ODS – 3.5 lm column was used. Analyses were conducted in duplicates. A representative chromatogram is shown in Fig. 1. Detection limit was 0.05 mg L)1 and quantification limits were 1.00 mg L)1 for CAT and 2.00 mg L)1 for EPI.

Determination of total phenols

Total phenols (TP) were measured by the Folin– Ciocalteu assay (Singleton & Rossi, 1965) using gallic acid (Sigma-Aldrich, St Louis, MO, USA) for the standard curve and the results were expressed in mg gallic acid equivalents L)1 (GAE). Floating particles were removed and the juices were treated enzymatically with 200 ppm pectinase (Pectinex Ultra SP-L, Novozymes, Denmark), to avoid the formation of clouds particles. Samples were then diluted 1:100 with deionised water. Colorimetric results of duplicates were read at 760 nm with a Beckman spectrometer. Determination of radical scavenging activity

The 1,1-diphenyl-2-picrylhydrazil (DPPH) (Sigma-Aldrich, Steinheim, BW, Germany) assay was used to measure RSA based on the methods of Brand-Williams et al. (1995), as modified by Kim et al. (2002). The

International Journal of Food Science and Technology 2008

Statistical analysis

The data were treated with anova and the means were compared using Tukey’s test. Pearson correlation coefficients were used to investigate relationships between parameters. Data analyses were conducted with Excel 97 (Microsoft Corporation, Washington, D.C., USA). Results and discussion

In concentrated Concord grape juice (CCJ) CAT and EPI decreased from 33.51 to 19.93 mg L)1 and 19.12 to 6.15 mg L)1, respectively, in 10 months (Fig. 2). TP and RSA decreased from 2775.0 to 2587.6 mg GAE and from 8.77 to 7.45 mm TE, respectively. In terms of retention during storage, CAT and EPI demonstrated preservation of 59% and 32%, respectively, which was lower than preservation of TP or RSA (93% and 85%, respectively). The results for concentrated Isabel grape juice (CIJ) under the same storage conditions showed similar performance for CAT with reduction from 21.73 to 11.85 mg L)1 and lower preservation for EPI from 25.54 to 3.88 mg L)1 (55% and 15% retention, respectively) (Fig. 3). TP and RSA varied from 1615.0 to 1429.0 mg GAE and from 7.38 to 6.33 mm TE, respectively (88% and 86% retention, respectively). In pasteurised Concord juice (PCJ) (Fig. 4) CAT reduction was from 1.68 to 1.06 mg L)1 (63% retention) and EPI concentrations were below quantification limit (2.00 mg L)1). TP and RSA decreased from 1884.3 to 1582.3 mg GAE and from 8.02 to 6.83 mm TE (84% and 85% retention, respectively). Pasteurised Isabel juice (PIJ), under similar storage settings demonstrated similar modifications (Fig. 5). CAT showed reduction

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Storage of concentrated and ready-to-drink grape juices A. P. B. Gollu¨cke et al.

(+)-catechin

500

Intensity (mv)

400

300

200

(–)epicatechin

100

0

Figure 1 Chromatogram of concentrated 0

(+)-catechin

35

Total phenols

2

(–)-epicatechin RSA

25 20 15 10 5 0 2

3

4

5 6 7 Time (months)

6

8

10

12

14

16

18

20

22

24

26

28

Retention time (min)

30

1

4

8

9

10

Figure 2 Effect of concentrated Concord grape juice (CCJ) storage on

(+) catechin (CAT), ())-epicatechin (EPI), total phenols (TP) and radical scavenging activity (RSA).

from 3.89 to 2.01 mg L)1 (52% retention) and EPI concentrations were below quantification limit. TP and RSA decreased from 1306.9 to 1126.9 mg GAE and from 6.98 to 6.08 mm TE (86% and 87% retention, respectively). Considering the four investigated juices, CAT and EPI (detected only in concentrated juices), demonstrated the lowest preservation, regardless of cultivar and storage differences (Table 1). EPI depicted lower preservation than its isomer CAT, probably due to higher reactivity, earlier observed by Freitas et al. (1998) in a model experiment. Concentrated juices demonstrated higher retention of TP than pasteurised juices (93 and 88% for CCJ and CIJ vs. 84–86% for PCJ and PIJ, respectively). The low preservation of the monomeric flavan-3-ols contrast with the higher retention of TP and RSA, demonstrating that other phenolic compounds contributed to the antioxidant capacity. It was previously verified that theaflavins, originated from

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

CAT and EPI (mg L–1); TP (x100mg GAE) RSA (mM TE)

40

RSA (mM TE)

CAT and EPI (mg L–1); TP (x100 mg GAE)

Concord juice (CCJ) after 10 months of storage.

24 22

(+)vcatechin

(–)-epicatechin

Total phenols

RSA

20 18 16 14 12 10 8 6 4 2 0 1

2

3

4

5

6

7

8

9

10

Time (months) Figure 3 Effect of concentrated Isabel grape juice (CIJ) storage on (+) catechin (CAT), ())-epicatechin (EPI), total phenols (TP) and radical scavenging activity (RSA).

oxidised and dimerised flavan-3-ols in teas possessed the same RSA as the initial monomers (Leung et al., 2001). As for the decrease in phenolic content, this could be attributed to enzymatic and non-enzymatic reactions (Es-Safi et al., 2003). Talcott & Lee (2002) found that processing methods rather than storage conditions were important for retention of radical scavenging properties in Muscadine grape juices. This partially agrees with the present investigations, in which neither storage nor process impacted RSA, which was more related to cultivar, with Concord juices showing higher activity. Statistical differences (P < 0.05) amongst juices (10 months averages) were found: for CATs CCJ > CIJ > PCJ = PIJ; for EPIs CCJ = CIJ; for TPs CCJ > PCJ > CIJ > PIJ and for RSA CCJ > CIJ = PCJ > PIJ. Concentrated juices showed notably higher flavan-3-ol contents, possibly due to

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CAT (mg L–1); TP (x100mg GAE) RSA (mM TE)

Storage of concentrated and ready-to-drink grape juices A. P. B. Gollu¨cke et al.

25 (+) catechin

Total phenols

RSA

20 15 10 5 0 1

2

3

4

5

6

7

8

9

10

Time (months) Figure 4 Effect of pasteurised Concord grape juice (PCJ) storage on (+) catechin (CAT), total phenols (TP) and radical scavenging activity (RSA). Note: ()) epicatechin contents below quantification limit (2.00 mg L)1).

(+) catechin

14 CAT (mg L–1); TP (x100mg GAE) RSA (mM TE)

1858

RSA

Total phenols

12 10 8 6 4 2

Conclusions

0 1

2

3

4

5 6 7 Time (months)

8

9

10

Figure 5 Effect of pasteurised Isabel grape juice (PIJ) storage on (+) catechin (CAT), total phenols (TP) and radical scavenging activity (RSA). Note: ()) epicatechin contents below quantification limit (2.00 mg L)1).

Table 1 Retention (in %) of investigated parameters in concentrated Concord and Isabel juices (CCJ and CIJ, respectively), and pasteurised juices of the same cultivars (PCJ and PIJ) after 10 months of storage

CCJ CIJ PCJ PIJ

extraction of flavan-3-ols. Regarding TP contents, grape cultivar rather than process was the relevant factor in our study, with Concord grapes showing higher contents than Isabel grapes. Ageing was associated with decrease in CAT, EPI, TP and RSA in all four juices (P < 0.05). Despite the high reduction suffered by flavan-3-ols, retentions of TP and RSA were above 84%. Zafrilla et al. (2003) observed similar evolution in wines: antioxidant activity was maintained after 7 months storage, although decrease in the concentration of some phenols was observed. In CCJ, CIJ and PIJ, RSAs were strongly correlated with TP than CAT or EPI (P < 0.05). Both CAT and EPI were strongly correlated with each other (r > 0.96, P < 0.01). Great differences in the TP contents in grape juices found no correspondence in RSA. Hence, in order to verify radical scavenging potential of each product, the mean ratio TP:RSA was calculated. It decreased in the following order: PIJ > CIJ > PCJ > CCJ, indicating that specific phenolic compounds and not TP amounts were relevant in the radical scavenging power. Da´valos et al. (2005) called this assessment ‘antioxidant activity provided by a unit of polyphenol’ and in our study it indicated that Isabel juices contained or retained specific phenolic compounds with higher antioxidant activity per phenolic unit.

CAT

EPI

TP

RSA

59 55 63 52

32 15 n ⁄ da n ⁄ da

93 88 84 86

85 86 85 87

CAT, (+)-catechin; EPI, ())-epicatechin; TP, total phenols; RSA, radical scavenging activity. a Below detection limit of 2.00 mg L)1 throughout the 10 months of storage.

process (hot pressing). In contrast, pasteurised juices (cold pressing) showed only about 10% of that amount. The findings agree with Fuleki & Ricardo-da-Silva (2003) who demonstrated that hot pressing enhanced

International Journal of Food Science and Technology 2008

Storage impacted monomeric flavan-3-ols contents in grape juices, but not total phenolics and RSA. Both storage conditions revealed similar retention of RSA in vitro after 10 months. This study demonstrated that the antioxidant power per phenolic unit must be addressed when evaluating antioxidant activity of phenolic-rich foodstuffs. Acknowledgments

The authors thank the National Council for Scientific and Technological Development (CNPq), linked to the Brazilian Ministry of Science and Technology (MCT) for the postgraduate scholarship (process no. 131016 ⁄ 2006-7). References Arts, I.C.W. & Hollman, P.C.H. (1998). Optimization of a quantitative method for the determination of catechins in fruits and legumes. Journal of Agriculture and Food Chemistry, 46, 5156–5162. Arts, I.C.W., van de Putte, B. & Hollman, P.C.H. (2000). Catechin contents of foods commonly consumed in the Netherlands. 2. Tea, wine, fruit juices, and chocolate milk. Journal of Agriculture and Food Chemistry, 48, 1752–1757. Arts, I.C.W., Hollman, P.C.H., Feskens, E.J.M., Mesquita, H.B.B. & Kromhout, D. (2001a). Catechin intake might explain the inverse

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Storage of concentrated and ready-to-drink grape juices A. P. B. Gollu¨cke et al.

relation between tea consumption and ischemic heart disease: the Zupthen Elderly Study. American Journal of Clinical Nutrition, 74, 227–232. Arts, I.C., Jacobs, D.R.J., Harnack, L.J., Gross, M. & Folsom, A.R. (2001b). Dietary catechins in relation to coronary heart disease death among postmenopausal women. Epidemiology, 12, 668–675. Auw, J.M., Blanco, V., O’Keefe, S.F. & Sims, C.A. (1996). Effect of processing on the phenolics and colour of Cabernet Sauvignon, Chambourcin and Noble wines and juices. American Journal of Enology and Viticulture, 47, 279–286. Brand-Williams, W., Cuvelier, M.E. & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittlewissenschaft und Technologie, 28, 25–30. Cheynier, V. (2005). Polyphenols in foods are more complex than often thought. American Journal of Clinical Nutrition, 81, 223S–229S. Da´valos, A., Bartolome´, B. & Go´mez-Cordove´s, C. (2005). Antioxidant properties of commercial grape juices and vinegars. Food Chemistry, 93, 325–330. Es-Safi, N., Cheynier, V. & Moutounet, M. (2003). Implication of phenolic reactions in food organoleptic properties. Journal of Food Composition and Analysis, 16, 535–553. Freitas, V.A.P., Glories, Y. & Laguerre, M. (1998). Incidence of molecular structure in oxidation of grape seed procyanidins. Journal of Agriculture and Food Chemistry, 46, 376–382. Fuleki, T. & Ricardo-da-Silva, J.M. (2003). Effects of cultivar and processing method on the contents of catechins and procyanidins in grape juice. Journal of Agriculture and Food Chemistry, 51, 640–646. Gu¨rbu¨z, O., Go¨c¸men, D., Dagdelen, F. et al. (2007). Determination of flavan-3-ols and trans-resveratrol in grapes and wine using HPLC with fluorescence detection. Food Chemistry, 100, 518–525. Jarowski, A.W. & Lee, C.Y. (1987). Fractionation and HPLC determination of grape phenolics. Journal of Agriculture and Food Chemistry, 35, 257–259. Kampa, M., Hatzoglou, A., Notas, G. et al. (2000). Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines. Nutrition of Cancer, 37, 223–233. Keen, C., Holt, R.R., Oteiza, P.I., Fraga, C.G. & Schmitz, H.H. (2005). Cocoa antioxidants and cardiovascular health. American Journal of Clinical Nutrition, 81, 298S–303S. Kim, D-O., Lee, K.W., Lee, H.J. & Lee, C.Y. (2002). Vitamin C equivalent capacity (VCEAC) of phenolic phytochemicals. Journal of Agriculture and Food Chemistry, 50, 3713–3717. Kimura, M., Umegaki, K., Kasuya, Y., Sugisawa, A. & Higutchi, M. (2002). The relation between single ⁄ double or repeated tea catechin

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ingestions and plasma antioxidant activity in humans. European Journal of Clinical Nutrition, 56, 1186–1193. Lee, C.Y. & Jarowski, A.W. (1987). Phenolic compounds in white grapes grown in New York. American Journal of Enology and Viticulture, 38, 277–281. Leung, L.K., Su, Y., Chen, R., Zhang, Z., Huang, Y. & Chen, Z-Y. (2001). Theaflavins in black tea and catechins in green tea are equally effective antioxidants. Journal of Nutrition, 131, 2248–2251. Lotito, S.B. & Fraga, C.G. (1997). (+)-Catechin prevents human plasma oxidation. Free Radical Biology and Medicine, 24, 435–441. Manach, C., Scalbert, A., Morand, C., Re´me´sy, C. & Jime´nez, L. (2004). Polyphenols: food sources and bioavailability. American Journal of Clinical Nutrition, 79, 727–747. Manach, C., Williamson, G., Morand, C., Scalbert, A. & Re´me´sy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 81, 230S–242S. Scalbert, A., Johnson, I.T. & Saltmarsh, M. (2005). Polyphenols: antioxidants and beyond. American Journal of Clinical Nutrition, 81, 215S–217S. Singleton, V.L. & Rossi, J.A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Spanos, G.A. & Wrolstad, R.E. (1990). Influence of processing and storage on the phenolic composition of Thompson Seedless grape juice. Journal of Agriculture and Food Chemistry, 38, 1565–1571. Talcott, S.T. & Lee, J-H. (2002). Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. Journal of Agriculture and Food Chemistry, 50, 3186–3192. Tokusoglu, O¨. & U¨nal, M.K. (2002). Optimized method for simultaneous determination of catechin, gallic acid, and methylxantine compounds in chocolate using RP-HPLC. European Food Research Technology, 215, 340–346. Vidal, S., Francis, L., Noble, A., Kwiatkowski, M., Cheynier, V. & Waters, E. (2004). Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine. Analytica Chimica Acta, 513, 57–65. Williamson, G. & Manach, C. (2005). Bioavailability and bioefficacy of polyphenols in humans II. Review of 93 intervention studies. American Journal of Clinical Nutrition, 81, 243S–255S. Zafrilla, P., Morillas, J., Mulero, J. et al. (2003). Changes during storage in conventional and ecological wine: phenolic content and antioxidant activity. Journal of Agriculture and Food Chemistry, 51, 4694–4700.

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International Journal of Food Science and Technology 2008, 43, 1860–1865

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Original article Synthesis and in vitro digestion of resistant starch type III from enzymatically hydrolysed cassava starch Calvin Onyango* & Christopher Mutungi Kenya Industrial Research and Development Institute, PO Box 30650-00100, Nairobi, Kenya (Received 9 November 2007; Accepted in revised form 21 April 2008)

Summary

Resistant starch type III (RS III) was synthesised from cassava starch by autoclaving followed by debranching with pullulanase, at varied concentrations (0.4–12 U g)1) and times (2–8 h), and recrystallisation ()18 to 90 C for 1–16 h). The highest RS III yield (22 g ⁄ 100 g) was obtained at an enzyme concentration of 4 U g)1 after 8 h incubation, followed by recrystallisation at 25 C for 16 h. Varying the recrystallisation conditions indicated that higher RS III yields (30–35 g ⁄ 100 g) could be obtained at 90 C within 2 h. Thinning cassava starch using a-amylase prior to debranching using pullulanase did not further increase the RS III content. In vitro digestion data showed that whereas 44% RS III was digested after 6 h, the corresponding value for cassava starch was 89%.

Keywords

a-Amylase, cassava starch, in vitro digestion, pullulanase, resistant starch type III.

Introduction

Resistant starch type III (RS III) is typically quantified as a component of dietary fibre because it resists digestion in the small intestine and instead passes on to the colon where it is fermented by the microflora resulting in the production of short chain fatty acids, gaseous matter and biomass. These compounds confer positive physiological colonic and non-colonic effects when consumed (Xue et al., 1996; Jacobasch et al., 1999; Topping & Clifton, 2001; Topping et al., 2003; Lim et al., 2005; Nugent, 2005). Consequently the importance of RS III as a nutraceutical is rising and it is increasingly being industrially synthesised for use as a food additive (Sajilata et al., 2006). Production of RS III is a two-stage process that begins with hydration and gelatinisation of starch during which amylose is leached from the granules into solution as a random coil polymer. This process is enhanced by acids or enzymes, which partially hydrolyse the starch polymers (Kettlitz et al., 2000; Lehmann et al., 2002, 2003; Gonza´lez-Soto et al., 2004; Onyango et al., 2006). The second stage involves incubation of the material to allow the flexible linear amylose polymers to recrystallise as double helices and form tightly packed helical or spherical structures stabilised by hydrogen bonds. Thus, formation of RS III is a crystallisation process of amylose, into enzyme-

*Correspondent: E-mail: [email protected]

resistant double helices stabilised by hydrogen bonds, in a partially crystalline system. Enzyme-catalysed hydrolysis is preferable to acidcatalysed hydrolysis for synthesis of RS III because the former provide higher yields, significantly improves product quality and reduces energy consumption. Enzymatic methods for the synthesis of RS III involve hydrolysis with isoamylase or pullulanase to eliminate a-d-(1 fi 6) glycosidic linkages of amylopectin (Chiu et al., 1994; Kettlitz et al., 2000; Lehmann et al., 2002, 2003; Gonza´lez-Soto et al., 2004). Amylopectin does not favour formation of RS III because its a-d-(1 fi 6) glycosidic linkages are hindered in movement and they detract from ordering of amylose (Eerlingen et al., 1994; Thompson, 2000). Contrastingly, amylose favours formation of RS III because it has linear chain polymers whose degree of polymerisation can be optimised. When the degree of polymerisation of amylose is too low (35 glucose units) then synthesis of RS III is hindered either because the polymer lacks the minimum chain length required to form crystallites or it cannot reach the required alignment of polymer chains to form resistant crystallites (Schmiedl et al., 2000; Lehmann et al., 2002, 2003). Thus, the aim of this study was to synthesise RS III from cassava starch by eliminating a-d-(1 fi 6) glycosidic linkages of amylopectin and partially hydrolysing a-d-(1 fi 4) linkages of amylose polymers prior to controlled incubation. The in vitro rate of digestion of RS III was determined using pancreatic amylase.

doi:10.1111/j.1365-2621.2008.01764.x  2008 Institute of Food Science and Technology

Resistant starch from cassava starch C. Onyango and C. Mutungi

Materials and methods

Cassava starch extraction and composition

Cassava (Manihot esculenta Gaerth) tubers were purchased from Kenya Agriculture Research Institute. Starch was extracted from the tubers using pilot scale equipment as described earlier (Onyango et al., 2006). Protein (n · 6.25), moisture, ash, fibre and lipid contents were determined using standard AOAC (2000) methods. Starch and amylose contents were determined as described earlier (Onyango et al., 2006). Determination of pullulanase activity

Pullulanase activity was determined colorimetrically with pullulan as substrate. The enzyme stock solution (Sigma P-2986; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was diluted in sodium acetate buffer (20 mM sodium acetate trihydrate in deionised water adjusted to pH 5.0 using 1 M hydrochloric acid) to 10)1, 10)2 and 10)3 dilutions. A 2% w ⁄ v pullulan solution was prepared by adding 2 g pullulan (Sigma P-4516) in 100 mL sodium acetate buffer (pH 5). The solution (50 lL) was diluted further in 350 lL sodium acetate buffer before adding 100 lL pullulanase and the reaction allowed to proceed at 25 C. The reaction was stopped after exactly 10 min by adding 500 lL copper solution (4 g copper sulphate, 0.185 g sodium sulphate, 23.96 g sodium carbonate, 15.96 g sodium bicarbonate and 12.14 g sodium potassium tartrate dissolved in 1000 mL distilled water), heated in a boiling water bath for 15 min then reacted with 500 lL arsenomolybdate solution (49.43 g ammonium molybdate tetrahydrate, 5.93 g sodium arsenate dibasic heptahydrate and 756 mM sulphuric acid in 1000 mL distilled water). The content of reducing sugars was determined by reading the absorbance using a u.v.-spectrophotometer at 546 nm against glucose standards. Enzyme activity (U mL)1) was expressed as lmoles glucose released per minute of the reaction by 1 mL enzyme solution. Effect of debranching cassava starch using pullulanase on resistant starch type III content

Cassava starch (5 g) was suspended in 20 mL distilled water and gelatinised by autoclaving at 121 C for 15 min. The gel was diluted further by adding 20 mL sodium acetate buffer (pH 5) and cooled to 60 C. Enzyme solutions (2 mL) at different concentrations (1, 2.5, 5, 7.5, 10, 15, 20 and 30 U mL)1) were added to starch suspensions to give treatments equivalent to 0.4, 1, 2, 3, 4, 6, 8 and 12 U g)1. The samples were incubated at 60 C and 1 mL aliquots taken and analysed for reducing sugars, using 3,5-dinitrosalicyclic acid reagent after 2, 4, 6 and 8 h. The experiment was set as a 8 · 4

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factorial design with two replicates. Analysis of variance was done using GenStat (VSN International, Hertfordshire, UK) statistical software and the means separated using least significant difference (LSD) test. Starch hydrolysates treated with different pullulanase concentrations for 8 h and those from the enzyme concentration-treatment time combination giving optimal amount of reducing sugars were used to determine the RS III contents. The samples were heated in a boiling water bath for 10 min to denature the enzyme, cooled and incubated at 25 C for 16 h then dried in a hot air oven at 60 C. The samples were ground using a mortar and pestle and the RS III content determined by adding 200 mg sample to 80 mg porcine pancreatic a-amylase (Sigma A-3176) in a 50 mL centrifuge tube and mixing with 10 mL phosphate buffer (pH 6.0). The mixture was incubated at 37 C for 16 h after which the pH was adjusted to 4.5 using 2% v ⁄ v phosphoric acid before adding 200 lL amyloglucosidase (Sigma A7095). The mixture was incubated at 60 C for 30 min, centrifuged at 1500 g for 10 min and the supernatant decanted. The residue was washed with phosphate buffer (pH 7.5) and re-suspended in 10 mL of the same buffer before adding 200 lL protease (Sigma P-2143, 16 mg in 100 mL phosphate buffer at pH 7.5) and incubating at 42 C for 4 h. Samples were centrifuged at 1500 g for 10 min, washed twice with 10 mL distilled water and dried in an air oven at 60 C to constant weight. The content of RS III was determined from the difference in weight of centrifuge tube containing the dry residue and the corresponding tare weight and expressed as a percent of the untreated sample. Effect of incubation temperature and time on resistant starch type III content

Cassava starch (5 g) was suspended in 20 mL distilled water and gelatinised by autoclaving at 121 C for 15 min before adding 2 mL pullulanase (10 U mL)1) to give an enzyme concentration of 4 U g)1 of starch. The mixture was incubated in a water bath at 60 C for 8 h after which the samples were heated in a boiling water bath for 10 min to denature the enzyme. The hydrolysates were cooled under running tap water to 25 C then incubated at )18, 4, 25, 60 and 90 C for 1, 2, 4, 8 and 16 h. The samples were dried in an air oven at 60 C to constant weight, ground using a mortar and pestle and the RS III content determined as described earlier. The experiment was set as a 5 · 5 factorial design with two replicates. Analysis of variance was done using GenStat statistical software and means separated using LSD test. Effect of thinning cassava starch prior to debranching

Starch suspensions (10% w ⁄ v) were prepared in distilled water and gelatinised by autoclaving at

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121 C for 15 min then cooled under running tap water to 25 C before mixing with 1 mL enzyme solutions containing 100, 250, 500, 1000 or 2000 U mL)1 porcine pancreatic a-amylase to give enzyme concentrations of 20, 50, 100, 200, and 400 U g)1 of starch. The starch-enzyme mixture was incubated in a water bath at 37 C for 15, 30, 60 or 120 min after which it was heated in a boiling water bath to denature the enzyme. A 1 mL aliquot of the mixture was obtained and analysed for reducing sugars using 3,5-dinitrosalicylic acid. The degree of hydrolysis was expressed as mg maltose ⁄ 100 mg starch. The experiment was set up as a 5 · 4 factorial design with two replicates. Analysis of variance was done using GenStat statistical software and the means separated using LSD test. The enzyme concentration-treatment time combinations giving a reducing power of 10 mg maltose ⁄ 100 mg starch were selected and used to partially hydrolyse cassava starch slurries after which pullulanase (4 U g)1) was added and the mixtures incubated at 60 C for 1, 2, 4 or 8 h. Recrystallisation was done at 90 C for 4 h after which the hydrolysates were dried in a hot air oven at 60 C, ground and the RS III content determined. The experiment was set up as a 3 · 2 · 4 factorial design with two replicates. Analysis of variance was done using GenStat statistical software and the means separated using LSD test. In vitro digestibility of cassava starch and resistant starch type III

In vitro digestibility of RS III and cassava starch were determined according to the method of Marlett & Longacre (1996) with modification. The sample (50 mg) was added to 5 mL phosphate buffer (pH 6.9), heated in a boiling water bath for 10 min then cooled to 37 C. Pancreatin solution was prepared by mixing 1 g porcine pancreatin (Sigma P1745) with 5 mL phosphate buffer (pH 6.9) containing 0.04% (w ⁄ v) sodium chloride for 10 min and the mixture allowed to stand for 10 min before adding 50 lL of the supernatant to the RS III suspension. The mixture was incubated in a shaking water bath at 37 C for 6 h and samples collected periodically. For each collected sample, enzyme activity was terminated by heating the sample in a boiling water bath for 5 min. The samples were centrifuged at 1500 g for 10 min, the pellet washed twice with 80% ethanol (5 mL) and dried to constant weight in a hot air oven set at 60 C. Per cent hydrolysis was calculated by multiplying the difference in weight of undigested sample and dry pellet by 100 and dividing the product with the weight of the undigested sample. The tests were carried out in duplicate.

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Results and discussion

Composition of cassava starch

Moisture, protein, fibre, ash and lipid contents of extracted cassava starch were 9.30%, 1.07%, 1.04%, 0.38% and 0.30% respectively. The starch and amylose contents were 92% and 16.57%, respectively, on dry weight basis. These parameters were within values reported earlier for cassava starch extracted by a similar method (Onyango et al., 2006). Effect of pullulanase concentration on debranching cassava starch and resistant starch type III production

The amount of reducing sugars liberated in 1 min when pullulanase (1 mL) was reacted with pullulan was 0.65 lmol (measured as glucose), which translated to an enzyme activity of 325 U mL)1. This stock solution was appropriately diluted to obtain enzyme concentrations required for our study. The solids content of the starch suspension was adjusted to the highest feasible solids level (25 g ⁄ 100 g) in order to facilitate uniform blending of enzyme and starch suspension, subsequent to drying and maximise the RS III yields. Other authors reported optimum RS III yields from starch suspensions with at least 15 g ⁄ 100 g solids (Chiu et al., 1994; Lehmann et al., 2002, 2003; Gonza´lez-Soto et al., 2004). The interaction effects of enzyme concentration and hydrolysis time were insignificant (P > 0.05), whereas the rate of production of reducing sugars increased with time and enzyme concentration (Fig. 1). A similar pattern has been noted during enzymatic synthesis of RS III from banana starch and indicates that complete debranching of amylopectin with pullulanase is time and 50

40 Maltose (m/mL)

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30

20

10

0

0

1

3

2 0.4 U g–1

4 U g–1

4 Time (h) 1 U g–1 8 U g–1

5

6

2 U g–1

7

8

3 U g–1

12 U g–1

Figure 1 Effect of time and enzyme concentration on production of reducing sugars.

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Resistant starch from cassava starch C. Onyango and C. Mutungi

enzyme-concentration dependent (Gonza´lez-Soto et al., 2004). At enzyme concentrations exceeding 3 U g)1 the rate of production of reducing sugars tended to level off when the incubation time exceeded 4 h. Debranching starch increases the concentration of linear short chains that contribute to the formation of RS III. Enzyme catalysed hydrolysis is suitable for synthesis of RS III because it is high yielding, significantly improves product quality and reduces energy consumption. Pullulanase is specific to a-d-(1 fi 6) glycosidic linkages of amylopectin and partially depolymerises amylopectin chains resulting to increased polymer mobility for molecular rearrangement. The resulting RS III yields ranged from 8.98 to 35.25 g ⁄ 100 g. Contrastingly, acid hydrolysis of cassava starch gives lower RS III yields (0.59–9.97 g ⁄ 100 g) because its hydrolytic mode of action is non-specific as it randomly breaks both the a-d-(1 fi 4) and a-d-(1 fi 6) glycosidic bonds (Onyango et al., 2006). Similarly low RS III yields have been reported from banana starch debranched using acids rather than pullulanase (Lehmann et al., 2003); though these authors finally recommended an integrated acidenzyme hydrolysis for production of high RS III yields. The yields of reducing sugars were related to the RS III contents for cassava starch samples hydrolysed for 8 h at different pullulanase concentrations (Fig. 2). Synthesis of RS III correlated positively with increasing yields of reducing sugars up to an enzyme concentration of 4 U g)1 after which the RS III content declined while the amount of reducing sugars increased as enzyme concentration increased from 8 to 12 U g)1. Debranching of starch with pullulanase gives rise to linear low molecular weight polymers which promote retrogradation. However, there is also production of oligosaccharides whose chain lengths are not long enough to 30 25

40

20 30 15 20

RS III (g/100g)

Reducing sugars (mg mL–1)

50

10 10

5 0

0 0

2

4 6 8 10 Pullulanase concentration (U g–1)

Reducing sugars

12

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Effect of incubation temperature and time on resistant starch type III production

Synthesis of RS III is influenced by several factors such as nature of starch, amylose content, starch concentration in the gel, hydrolysis and incubation conditions and presence of lipids or low molecular weight substances such as sugars (Chiu et al., 1994; Skrabanja et al., 1999; Schmiedl et al., 2000; Lehmann et al., 2002, 2003; Gonza´lez-Soto et al., 2004). The most important variables during recrystallisation of amylose in a partially crystalline system are incubation temperature and time. We studied the influence of incubation, at various time-temperature combinations, on RS III production from cassava starch hydrolysed for 8 h at a pullulanase concentration of 4 U g)1 (Table 1). The interaction effect of incubation time and temperature significantly influenced the RS III yields (P < 0.05) with the highest yields being noted in samples incubated at 90 C for 2–8 h. Generally, higher RS III contents were obtained in shorter time periods when enzyme-mediated hydrolysis was compared to acidmediated hydrolysis (Onyango et al., 2006), though the yields from enzyme hydrolysed cassava starch could not be easily interpreted on the basis of nucleation, propagation and maturation. However, it appears that nucleation, propagation and maturation all occurred within the first 2 h for hydrolysates incubated at 90 C. Resistant starch type III yields from cassava starch incubated at 4 C did not differ (P > 0.05) from those at )18 C in the first 2 h but thereafter significantly increased (P < 0.05) with time whereas samples incubated at )18 C did not change with time (P > 0.05). The RS III contents from cassava starch incubated at intermediate temperature ranges (25 or 60 C) did not differ Table 1 Effect of incubation temperature and time on resistant starch type III content* Incubation time (h) Incubation temperature (°C)

1

2

4

8

16

)18 4 25 60 90

14.39b 14.52bc 22.83e 23.35e 30.90g

12.17ab 15.89cb 24.27e 23.48e 31.44gh

10.87a 16.04c 22.00e 24.27e 34.06h

11.52a 19.59d 23.66ef 27.78fg 35.25h

8.98a 21.45de 25.36f 27.14f 23.28e

Resistant starch yield

Figure 2 Effect of pullulanase concentration on reducing sugars and

resistant starch type III content.

recrystallise into RS III, and which possibly sterically impeded retrogradation at the high enzyme concentrations. In order to increase the RS III yield, the hydrolysate should be washed with water to remove these interfering substances (Lehmann et al., 2002, 2003).

Means sharing similar superscripts along the same row or column are not significantly different at P = 0.05. *g ⁄ 100 g starch.

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significantly (P > 0.05) irrespective of the incubation time. Synthesis of RS III is inhibited at low temperatures by the formation of ice crystals within the recrystallising linear chains that limit the mobility and realignment of the chains and result in a pulverous product that is more susceptible to amylolytic attack. Similarly to acid hydrolysed cassava starch incubated at freezing temperatures (Onyango et al., 2006), a large proportion of the RS III was probably synthesised during nucleation with no further increase in yields during maturation and propagation. Starch retrogradation temperature is important as it influences starch crystalline structure and subsequent nutritional and functional properties of RS III. X-ray diffraction analysis of RS III shows that retrogradation at low temperatures favours formation of B-type polymorph whereas mixtures of A-, with V-, or B-type polymorphs are formed at high temperatures (Shamai et al., 2003). These polymorph structures have implications on in vivo digestion since RS III with A-type patterns are attacked more rapidly than those with B-type patterns (Alonso et al., 1998). Effect of thinning cassava starch prior to debranching

The reducing sugars content of cassava starch, hydrolysed using a-amylase, was used to determine the optimum enzyme-time treatment combination required for thinning prior to debranching (Table 2). The interaction effect of a-amylase concentration and incubation time was significant (P < 0.05) and the reducing sugars contents increased with increasing levels of these factors. Hydrolysates with reducing sugars contents less than ten facilitate synthesis of RS III (Kettlitz et al., 2000). Consequently gelatinised cassava starch was thinned using a-amylase concentrations of 20, 50 and 100 U g)1 and incubated for 15 or 30 min prior to debranching with pullulanase and recrystallisation at 90 C for 4 h (Table 3). The interaction effect of thinning conditions (a-amylase concentration and treatment time) and debranching time was significant (P < 0.05) and RS III content was highest (33.13 g ⁄ 100 g) when gelatinised Table 2 Reducing sugars* contents of cassava starch thinned using a-mylase Treatment time (min) Concentration of a-amylase (U g)1)

15

30

60

120

20 50 100 200 400

1.49a 2.17b 7.79d 9.33d 30.44g

1.79a 3.21b 13.33e 12.21e 30.61g

2.55b 3.33b 12.69e 12.75e 31.64g

2.71b 4.56c 16.96f 14.77f 38.71h

Means sharing similar superscripts along the same row or column are not significantly different at P = 0.05. *mg maltose ⁄ 100 mg starch.

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Table 3 Effect of thinning and debranching cassava starch on resistant starch type III contents* Debranching time (h) Concentration of a-amylase (U g)1) 20 50 100

Treatment time (min) 15 30 15 30 15 30

1

2 a

14.18 19.31b 15.76a 19.47b 19.66b 18.63b

4 cd

24.17 25.60d 23.89c 21.89c 23.98c 19.31b

8 f

33.13 23.20c 27.64d 17.84b 18.58b 19.13b

29.24e 26.32d 28.21e 18.90b 17.43b 17.61b

Means sharing similar superscripts along the same row or column are not significantly different at P = 0.05. *g ⁄ 100 g starch.

cassava starch was thinned using an a-amylase concentration of 20 U g)1 for 15 min (Table 3). Generally, RS III yields from cassava starch thinned prior to debranching were not appreciably higher than those from cassava starch treated with pullulanase (Table 1). Thinning enhances the activity of pullulanase on amylopectin since unlike isoamylase it exhibits limited activity towards branched longer chain dextrins (Henrik & Allan, 2001). We had, therefore, anticipated that increased yields of low molecular weight fragments after amylolytic and pullulanase hydrolysis of cassava starch would facilitate recrystallisation and thus increase RS III yields. The degree of polymerisation of the glucose units determines the ease with which RS III can be synthesised. Excessive hydrolysis of amylose gives low RS III contents because the resultant polymers are not long enough to form enzyme-resistant crystallites, whereas insufficient hydrolysis is undesirable since the long chain polymers cannot effectively realign to form enzyme-resistant crystallites (Thompson, 2000). In vitro digestion of cassava starch and resistant starch type III

The course of in vitro digestion of cassava starch and its RS III derivative is given in Fig. 3. Resistant starch type III had a low digestion rate as only 31% was digested within 1 h and the amount digested increased to 44% after 6 h (P > 0.05). Contrastingly, 74% cassava starch was digested within 1 h and the amount increased to 89% after 6 h. The high digestion rate of cassava starch indicates easy accessibility of amylolytic enzymes to the starch polymers. Contrastingly, RS III is largely inaccessible to enzymatic hydrolysis because the linear a-d-(1 fi 4) linkages of amylose polymers have recrystallised as double helices and formed tightly packed helical or spherical structures stabilised by hydrogen bonds. The rate and extent of starch and RS III digestion affects several human physiological functions and consequently health and are therefore regarded as

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Resistant starch from cassava starch C. Onyango and C. Mutungi

100

Hydrolysis (%)

80

60

40

20

0 0

1 RS III

2

3 Time (h)

4

5

6

Gelatinised cassava starch

Figure 3 In vitro digestion of cassava starch and resistant starch type

III.

predictors of metabolic responses to complex carbohydrates in vitro. A slow rate of release of glucose in the blood stream results in low glycaemic and insulinaemic responses (Lund & Johnson, 1991; Xue et al., 1996) with associated benefits for weight management. Conclusion

Resistant starch type III was synthesised from cassava starch after hydrolysis using pullulanase and incubation at controlled conditions. Maximum RS III yields were obtained when the starch was hydrolysed with pullulanase (4 U g)1) for 8 h prior to incubation at 90 C for 2 h. Thinning of the starch hydrolysate with a-amylase prior to debranching with pullulanase did not further increase the RS III content. In vitro digestion of RS III revealed a slow rate of digestion and indicates that the product could be suitable for formulation of foods for diabetics. The economic viability of enzyme-mediated synthesis of RS III from cassava starch is highly promising because the specific nature of enzyme action facilitates synthesis of large quantities of RS III, and quality products that requires minimal downstream processing with minimal energy requirements. Acknowledgment

Calvin Onyango is grateful to International Foundation for Science (IFS) for the research grant. References Alonso, A.G., Calixto, F.S. & Delcour, J.A. (1998). Influence of botanical source and processing on formation of resistant starch type III. Cereal Chemistry, 75, 802–804.

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AOAC (2000). In: Official Methods of Analysis of the Association of Official Analytical Chemists, 17th edn (edited by W. Horwitz). Gaithersburg, MD: AOAC International. Chiu, C-W., Henley, M. & Altieri, P. (1994). Process for making amylase resistant starch from high amylose starch. U.S. Patent, 5,281,276. Eerlingen, R.C., Jacobs, H. & Delcour, J.A. (1994). Enzyme-resistant starch. V. Effect of retrogradation of waxy maize starch on enzyme susceptibility. Cereal Chemistry, 71, 351–355. Gonza´lez-Soto, R.A., Agama-Acevedo, E., Solorza-Feria, J., Rendo´nVillalobos, R. & Bello-Pe´rez, L.A. (2004). Resistant starch made from banana starch by autoclaving and debranching. Starch ⁄ Sta¨rke, 56, 495–499. Henrik, B. & Allan, S. (2001). Starch debranching enzymes. U.S. Patent, 6,265,197. Jacobasch, G., Schmiedl, D., Kruschewski, M. & Schmehl, K. (1999). Dietary resistance starch and inflammatory bowel diseases. International Journal of Colorectal Diseases, 14, 201–211. Kettlitz, B.W., Coppin, J.V.J.-M., Roper, H.W.W. & Bornet, F. (2000). Highly fermentable resistant starch. U.S. Patent, 6,043,229. Lehmann, U., Jacobasch, G. & Schmiedl, D. (2002). Characterisation of resistant starch type III from banana (Musa acuminata). Journal of Agricultural and Food Chemistry, 50, 5236–5240. Lehmann, U., Ro¨ssler, C., Schmiedl, D. & Jacobasch, G. (2003). Production and physicochemical characterisation of resistant starch type III derived from pea starch. Nahrung ⁄ Food, 47, 60–63. Lim, C.C., Ferguson, L.R. & Tannock, G.W. (2005). Dietary fibres as ‘‘prebiotics’’: implications for colorectal cancer. Molecular Nutrition and Food Research, 49, 609–619. Lund, E.K. & Johnson, I.J. (1991). Fermentable carbohydrate reaching the colon after ingestion of oats in humans. Journal of Nutrition, 121, 311–317. Marlett, J.A. & Longacre, M.J. (1996). Comparison of in vitro and in vivo measures of resistant starch in selected grain products. Cereal Chemistry, 73, 63–68. Nugent, A.P. (2005). Health properties of resistant starch. Nutrition Bulletin, 30, 27–54. Onyango, C., Bley, T., Jacob, A., Henle, T. & Rohm, H. (2006). Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid hydrolysed cassava starch. Carbohydrate Polymers, 66, 494–499. Sajilata, M.G., Singhal, R.S. & Kulkarni, P.R. (2006). Resistant starch: a review. Comprehensive Reviews in Food Science and Food Safety, 5, 1–17. Schmiedl, D., Ba¨uerlein, M., Bengs, H. & Jacobash, G. (2000). Production of heat-stable, butyrogenic resistant starch. Carbohydrate Polymers, 43, 183–193. Shamai, K., Bianco-Peled, H. & Shimoni, E. (2003). Polymorphism of resistant starch type III. Carbohydrate Polymers, 54, 363–369. Skrabanja, V., Liljeberg, H.G.M., Hedley, C.L., Kreft, I. & Bjork, I.M.E. (1999). Influence of genotype and processing on the in vitro rate of starch hydrolysis and resistant starch formation in peas (Pisum sativum L). Journal of Agricultural and Food Chemistry, 47, 2033–2039. Thompson, D.B. (2000). Strategies for the manufacture of resistant starch. Trends in Food Science and Technology, 11, 245–253. Topping, D.L. & Clifton, P. (2001). Short–chain fatty acids and human colonic function: roles of resistant starch and non-starch polysaccharides. Physiological Reviews, 81, 1031–1064. Topping, D.L., Fukushima, M. & Bird, A.R. (2003). Resistant starch as a prebiotic and symbiotic: state of the art. Proceedings of the Nutrition Society, 62, 171–176. Xue, Q., Newman, R.K. & Newman, C.W. (1996). Effects of heat treatment of barley starches on in vitro digestibility and glucose responses in rats. Cereal Chemistry, 73, 588–592.

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Original article A comparison of antioxidant properties between artisan-made and factory-produced chocolate Rinaldo Cervellati,1* Emanuela Greco,1 Stefano Costa,2 Maria Clelia Guerra2 & Ester Speroni2 1 Dipartimento di Chimica ‘‘G. Ciamician’’, Universita` di Bologna, Via Selmi 2, I-40126 Bologna, Italy 2 Dipartimento di Farmacologia, Universita` di Bologna, Via Irnerio 46, I-40126 Bologna, Italy (Received 14 December 2007; Accepted in revised form 2 April 2008)

Summary

The antioxidant capacities and the total phenolic content in cocoa liquor directly manufactured chocolate from an artisan manufacturer were measured using different in vitro methods (BR, TEAC, and Folin– Ciocalteu Reagent). These parameters were then compared with those of a chocolate made by a leading manufacturing company producing chocolate and cocoa-containing products. A statistical analysis of the collected data showed that the antioxidant properties of the artisan-made chocolate are significantly better than those of the factory-produced one. These results were ascribed to the fact that all the bioactive components in the cocoa beans are better preserved in the artisan-made chocolate.

Keywords

Antioxidant properties, Briggs–Rauscher reaction, chocolate, TEAC method, total phenolics.

Introduction

In recent years cocoa and dark chocolate have attracted continuous interest not only for their palatable treat and nutritional properties, but especially for its potential health benefits. These beneficial effects on cardiovascular diseases (Kris-Etherton & Keen, 2002; Heiss et al., 2003), blood pressure (Taubert et al., 2007a,b), plasma LDL and HDL cholesterol (Baba et al., 2007), diabetes (Makoto Tomaru et al., 2007), platelet function (Rein et al., 2000; Pearson et al., 2002), breast cancer (Ramljak et al., 2005), antioxidant status and oxidative stress (Heiss et al., 2003), are mainly ascribed to the high flavanoid content of cocoa and chocolate. Many papers have been published about the antioxidant capacity and phenolic content of cocoa and cocoa related products (Waterhouse et al., 1996; Adamson et al., 1999; Arts et al., 1999; Vinson et al., 1999; Won Lee et al., 2003; Gu et al., 2006; Miller et al., 2006). A more recent study investigated the effect of some steps of manufacturing chocolate on single components and antioxidant activity of cocoa beans (Arlorio et al., 2008). The authors found that the roasting step caused a dramatic reduction in clovamide, parallel to an overall decrease of the antioxidant capacity. Before becoming cocoa powder or chocolate, cocoa beans undergo the following treatments: fermentation, *Correspondent: Fax: +39 051 209 9456; e-mail: [email protected]

drying, deshelling, sterilisation, roasting and grinding. The first three steps are usually done at the site of cultivation, while the others are factory-based. From the grinding a cocoa liquor (CoL) is obtained that will be transformed into cocoa powder or chocolate products (Cooper et al., 2007). The transformation process of CoL into dark chocolate commonly used by the multinational companies implies pressing, mixing, conching and tempering. Cocoa liquor is heated up to 95– 105 C and is then pressed. As a result, a great part of the fat (cocoa butter) is separated from cocoa paste. Cocoa paste can be alkalised to favour aggregation, but it is known that this step considerably reduces the polyphenol content (Gu et al., 2006). Cocoa paste, part of the cocoa butter, sugar and possible natural flavours are then mixed in specified proportions. After the mixing, the mass is ground. This chocolate mass is then subjected to conching which is intensive mixing at a high temperature. Conching is a very long process (up to 24 h) and, as a result, the superfluous moisture is evaporated from the chocolate mass. Finally this mass is tempered. In the tempering process, which is long and complex, the chocolate mass is gradually cooled to 27 C, heated again to 37 C and then slowly re-cooled into a solid state thus leading to the desired uniform crystallisation of the chocolate. In many countries, some artisan manufacturers make dark or baking chocolate directly conching cocoa liquor (maybe added with sugar and natural flavours). In this case the conching treatment requires much longer duration, more than 5 days.

doi:10.1111/j.1365-2621.2008.01765.x  2008 Institute of Food Science and Technology

Antioxidant properties between artisan-made and factory-produced chocolate R. Cervellati et al.

The aim of the present work is to compare the antioxidant capacity and the total phenolic content of a chocolate produced by a worldwide known company with those of an artisan manufacturer. The antioxidant capacity was evaluated using two tests: the Briggs-Rauscher (BR) oscillating reaction method that works at pH  2 and the trolox equivalent antioxidant capacity (TEAC) assay working at pH 7.4. Total phenolic content was determined using the Folin-Ciocalteu reagent. Materials and methods

Chemicals and apparatus

Malonic acid, manganese (II) sulphate monohydrate, NaIO3, Na2CO3 anhydrous, (all reagent grade ‡99%) were purchased from Merck (Darmstadt, Germany). Gallic acid (3,4,5-trihydroxy benzoic acid; Seelze, Riedel-de Hae¨n, Germany), 2,6-DHBA (2,6-dihydroxy benzoic acid; Aldrich, USA), K2S2O8, ABTS (2,2¢-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), Folin-Ciocalteu reagent (FC), HClO4 and H2O2 were purchased from Fluka (St. Louis, MO, USA) and Trolox (6-hydroxy-2,5,8-tetrametylchroman-2-carboxylic acid) from Aldrich (St. Louis, MO, USA). HClO4 was analysed by titration against a standard 0.1 M NaOH solution (from Merck). H2O2 was standardised daily by manganometric analysis. All stock solutions were prepared with double distilled (dd), deionised water. Potentiometric measurements were performed by recording the potential of an iodide ion selective electrode (model 9453; Orion, Beverly, MA, USA) using a multimeter (WTW model pH 540; GLP, Weilheim, Germany) controlled by an IBM-compatible PC. As a reference a double junction Ag ⁄ AgCl electrode (model 373-90-WTEISE-S7; Ingold, Urdorf, Switzerland) was used. Spectrophotometric measurements were made on a Shimadzu 1601 PC UV-vis spectrophotometer (Kyoto, Japan). Chocolate samples

Multinational manufactured (MM) 99% cocoa. Ingredients: cocoa paste + cocoa powder + cocoa butter 99%, sugar cane. Artisan manufactured (AM) classic 100% cocoa. Ingredients: cocoa liquor 99.98%, sweeteners (0.02%: sodium cyclamate, sodium saccharinate, acesulfame k). AM red pepper flavour 100% cocoa. Ingredients: cocoa liquor 99.96%, red pepper powder, sweeteners (0.02%: sodium cyclamate, sodium saccharinate, acesulfame k). AM rosemary flavour 100% cocoa. Ingredients: cocoa liquor 99.95%, rosemary powder, sweeteners (0.02%: sodium cyclamate, sodium saccharinate, acesulfame k). All samples were produced from cocoa beans Criollo variety, Ecuador origin.

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Sample preparation

Bioactive compounds from chocolate were extracted using the procedure suggested by Serafini et al. (2003). A sample of 2 g were ground in a mortar. Exactly one gram of this powder was defatted by adding 10 mL of n-hexane, the mixture was ultrasonicated for 10 min at 30 C and then centrifuged at 3000 rpm for 10 min. This operation was repeated twice. The residue was extracted with 5 mL of a mixture of acetone, water and acetic acid (70.0:29.8:0.2 by volume). The mixture was put in an ultrasound bath for 15 min at 30 C, then centrifuged for 4 min at 875 g. The supernatant liquid was separated and the operation was repeated with 2 mL of the acetone-water-acetic acid solution. The collected supernatant liquids were filtered with a PTFE 0.45 lm filter, then the organic solvent was removed in a water-bath at 45 C. After filtration doubly distilled water was added to a final volume of exactly 10.0 mL. Antioxidant activity assay based on the Briggs-Rauscher reaction

The chemical in vitro method reported by Cervellati et al. (2001), is based on the inhibitory effects by free radical scavengers on the oscillations of the BR reaction. In brief, when antioxidant scavengers of free radicals are added to an active oscillating BR mixture there is an immediate quenching of the oscillations, an inhibition time (tinhib) that linearly depends on the concentration of the antioxidant added, and a subsequent regeneration of the oscillations. Relative antioxidant activity (r.a.c.) with respect to a substance chosen as standard (2,6DHBA; Ho¨ner & Cervellati, 2005) is determined on the basis of concentrations of sample and 2,6-DHBA that give the same tinhib. r.a.c. is expressed as mg 2,6-DHBA equivalents ⁄ g chocolate. Antioxidant activity based on the trolox equivalent antioxidant capacity assay

The protocol suggested by Re et al. (1999) was used. Antioxidant activity is expressed as mg Trolox equivalents ⁄ g chocolate. Determination of total phenolics (antioxidant reducing capacity quantification)

This test is based on the oxidation of phenolic groups by phosphomolybdic and phosphotungstic acids (FC reagent). After oxidation the absorbance of a green-blue complex can be measured at 765 nm. The procedure for 20 mL total volume of the reacting mixture (Singleton & Rossi, 1965) was used. Total phenolic content is expressed as mg gallic acid equivalents (GAE) ⁄ g chocolate.

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ing chocolate and cocoa-containing products. The results are reported in Fig. 1. From the data in Fig. 1a,b it can be seen that the artisan-produced chocolate has significant higher antioxidant activity (with both methods) than the reference sample. As far as the total phenolic content is concerned only the two flavoured handicraft chocolates show significant higher GAE than the factory-based one (Fig. 1c). It can be excluded that these significant differences can be due to the 1% difference in the total cocoa content between MM and AM chocolates. There are no great differences among the three AM samples; this is not surprising because the main ingredient, cocoa liquor, is the same. Only the parameters of the AM rosemary flavoured chocolate seem a little higher than those of the other two chocolates. This can be due to some very high antioxidant capacity compounds contained in the rosemary: rosmarinic acid, carnosic acid and carnosol (Cervellati et al., 2002; Costa et al., 2007). As pointed out in the Introduction several authors have investigated antioxidant properties of cocoa and related products, but it is difficult to compare their results with those presented here. In fact the

Results and discussion

In Table 1, the first two columns show the antioxidant data for the examined chocolate samples. The third column shows the values of the total phenolic content (total reducing power). The statistical significance among the mean values of the different samples was evaluated using the Student’s t-test, taking the MM sample as reference because its Factory is considered one of the leaders in manufactur-

Table 1 Relative antioxidant capacity and total phenolic content of the chocolate extracts

Sample

BR (r.a.c.)m±r mg/g 2,6-DHBA

TEAC mg/g Trolox

TPC GAE mg/g GA

MM AM classic AM red pepper AM rosemary

2.9 5.8 6.2 6.4

20 33 35 40

13.2 15 15 16

± ± ± ±

0.3 0.4 0.4 0.5

± ± ± ±

2 3 7 5

± ± ± ±

0.8 1 1 1

Data in the table are average values of five measurements at different extract concentrations ± SD.

(a) 7 6

(b) 45 MM AM classic AM red pepper AM rosemary

***

***

***

MM AM classic AM red pepper AM rosemary

40 35

***

4 3 2

**

***

5

mg/g Trolox

mg/g 2,6-DHBA

30 25 20 15 10

1

5

0

0 MM

AM classic

AM red pepper

AM rosemary

MM

AM classic

(c)

18 16 14

AM red pepper

AM rosemary

Sample

Sample

mg/g GAE

1868

MM AM classic AM red pepper AM rosemary

*

*

12 10 8 6 4 2 0

MM

AM classic

AM red pepper

AM rosemary

Sample Figure 1 Statistical significances of the antioxidant parameters with respect to the MM sample. (a) BR r.a.c. values, ***P < 0.0001; (b) TEAC

values, **P < 0.002, ***P < 0.0001; (c) GAE values, *P < 0.01.

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Antioxidant properties between artisan-made and factory-produced chocolate R. Cervellati et al.

concentration of all polyphenols can vary tremendously among cocoa-containing foods, and this can vary depending on the source of the beans, the processing conditions, and how the chocolates are manufactured (Cooper et al., 2007). Moreover, different authors used different extraction procedures and different antioxidant testing methods. Some comparison can be made with the data recently reported by Miller et al. (2006) on the total polyphenols (FC method) of chocolate products in the US. For dark chocolate they found GAE values ranging from 11.7 to 14.9 and for baking chocolate from 27.2 to 29.7. These latter values are higher than those reported here. However, Waterhouse et al. (1996) reported a value of 8.4 mg GA g)1 baker’s chocolate. It must be taken into account that the FC reagent method suffers from a number of interfering substances (Huang et al., 2005) even if it is the recommended method to measure the total reducing capacity (Prior et al., 2005). Since different antioxidant testing methods give different ranking orders of antioxidant capacity due to different experimental conditions, we used two methods in order to obtain a realistic estimate of the antioxidant activity of the examined chocolates, as suggested by Prior et al. (2005). Overall, in vitro chemical antioxidant assays can only partially mimic physiological conditions. The BR method was adopted because it works at pH  2, similar to that of the fluids in the human stomach. Kanner & Lapidot (2001), observed that some plantderived antioxidants are able to prevent lipid peroxidation, amplified in the acidic pH of gastric juice. The conception of the stomach as a bioreactor, where reactive oxygen species and food nutrients interact, underlines the importance of determining antioxidant activity of dietary sources at acidic pH. The pH of TEAC method is 7.4, equal to that of the human plasma. Correlations between the different methods were obtained by Pearson’s correlation coefficient in bivariate correlation. Significant correlations were found between GAE and BR and TEAC tests: R = 0.9505 (P < 0.025), R = 0.9900 (P < 0.01), respectively. Those data are quite in agreement with the fact that the total reducing capacity is related to the antioxidant activity of the chocolate flavonoids. Good correlation was also observed between the BR and TEAC methods: R = 0.9753 (P < 0.025). Our results showed that the antioxidant properties of the artisan-made chocolate are significantly better than those of a factory-produced one. All the bioactive substances in the cocoa beans are better preserved because of the direct process used in artisan-made chocolate which leads to a higher quality product. Further studies are needed to investigate the in vivo plasma antioxidant status after the repeated oral administration of these chocolates in animal models in order to verify the in vitro chemical results.

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References Adamson, G.E., Lazarus, S.A., Mitchell, A.E. et al. (1999). HPLC Method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity. Journal of Agricultural and Food Chemistry, 47, 4184–4188. Arlorio, M., Locatelli, M., Travaglia, F. et al. (2008). Roasting impact on the contents of clovamide (N-caffeoyl-L-DOPA) and the antioxidant activity of cocoa beans (Theobroma cacao L.). Food Chemistry, 106, 967–975. Arts, I.C.W., Hollman, P.C.H. & Kromhout, D. (1999). Chocolate as a source of tea flavonoids. The Lancet, 354, 488. Baba, S., Natsume, M., Yasuda, A et al. (2007). Plasma LDL and HDL cholesterol and oxidized LDL concentrations are altered in normo- and hypercholesterolemic humans after intake of different levels of cocoa powder. Journal of Nutrition, 137, 1436–1441. Cervellati, R., Ho¨ner, K., Furrow, S.D., Neddens, C. & Costa, S. (2001). The Briggs-Rauscher reaction as a test to measure the activity of antioxidants. Helvetica Chimica Acta, 84, 3533–3547. Cervellati, R., Renzulli, C., Guerra, M.C. & Speroni, E. (2002). Evaluation of antioxidant activity of some natural polyphenolic compounds using the Briggs-Rauscher reaction method. Journal of Agricultural and Food Chemistry, 50, 7504–7509. Cooper, K.A., Campos-Gime´nez, E., Jimene´z Alvarez, D., Nagy, K., Donovan, J.L. & Williamson, G. (2007). Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentration in chocolate. Journal of Agricultural and Food Chemistry, 55, 2841– 2847. Costa, S., Utan, A., Speroni, E. et al. (2007). Carnosic acid from rosemary extracts: a potential chemoprotective agent against aflatoxin B1. An in vitro study. Journal of Applied Toxicology, 27, 152– 159. Gu, L., House, S.I., Wu, X., Ou, B. & Prior, R.L. (2006). Procyanidin and catechin contents and antioxidant capacity of cocoa and chocolate products. Journal of Agricultural and Food Chemistry, 54, 4057–4061. Heiss, C., Dejam, A., Kleinbongard, P., Schewe, T., Sies, H. & Kelm, M. (2003). Vascular effects of cocoa rich in flavan-3-ols. Journal of American Medical Association, 290, 1030–1031. Ho¨ner, K. & Cervellati, R. (2005). Evaluation of antioxidant activity of spirits using two different free radical scavenging testing methods. Italian Journal of Food Science, 17, 395–406. Huang, D., Ou, B. & Prior, R.L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856. Kanner, J. & Lapidot, T. (2001). The stomach as a bioreactor: Dietary lipidic peroxidation in the gastric fluid and the effects of plantderived antioxidants. Free Radical Biology and Medicine, 21, 1388– 1395. Kris-Etherton, P.M. & Keen, C.L. (2002). Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Current Opinion in Lipidology, 13, 41–49. Makoto Tomaru, D.D.S., Takano, H., Osakabe, N. et al. (2007). Dietary supplementation with cacao liquor proanthocyanidins prevents elevation of blood glucose levels in diabetic obese mice. Nutrition, 23, 351–355. Miller, K.B., Stuart, D.A., Smith, N.L. et al. (2006). Antioxidant activity and polyphenol and procyanidin contents of selected commercially available cocoa-containing and chocolate products in the United States. Journal of Agricultural and Food Chemistry, 54, 4062–4068. Pearson, D.A., Paglieroni, T.G., Rein, D. et al. (2002). The effects of flavanol-rich cocoa and aspirin on ex-vivo platelet function. Thrombosis Research, 106, 191–197. Prior, R.L., Wu, X. & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolic in foods and dietary. Journal of Agricultural and Food Chemistry, 53, 4290–4302.

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Ramljak, D., Romanczyk, L.J., Metheny-Barlow, L.J. et al. (2005). Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells. Molecular Cancer Therapy, 4, 537–546. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & RiceEvans, C.A. (1999). Antioxidant activity applying an improved ABTSd+ radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Rein, D., Paglieroni, T.G., Wun, T. et al. (2000). Cocoa inhibits platelet activation and function. American Journal of Clinical Nutrition, 72, 30–35. Serafini, M., Bugianesi, R., Maiani, G., Valtuena, S., De Santis, S. & Crozier, A. (2003). Plasma antioxidant from chocolate. Nature, 424, 1013. Singleton, W.L. & Rossi, J.A. (1965). Colorimetric of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158.

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Taubert, D., Roesen, R. & Scho¨mig, E. (2007a). Effect of cocoa and tea intake on blood pressure – a meta-analysis. Archives of Internal Medicine, 167, 626–634. Taubert, D., Roesen, R., Lehmann, C., Yung, N. & Scho¨mig, E. (2007b). Effects of low habitual cocoa intake on blood pressure and bioactive nitric oxide. Journal of American Medical Association, 298, 49–60. Vinson, J.A., Proch, J. & Zubik, L. (1999). Phenol antioxidant quantity and quality in foods: cocoa, dark chocolate, and milk chocolate. Journal of Agricultural and Food Chemistry, 47, 4821– 4824. Waterhouse, A.L., Shirley, J.R. & Donovan, J.L. (1996). Antioxidant in chocolate. The Lancet, 348, 834. Won Lee, K., Kim, Y.J., Lee, H.J. & Lee, C.Y. (2003). Cocoa has more phenolic phytochemicals and a higher antioxidant capacity than teas and red wine. Journal of Agricultural and Food Chemistry, 51, 7292– 7295.

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International Journal of Food Science and Technology 2008, 43, 1871–1879

Original article Banana: cultivars, biotechnological approaches and genetic transformation Ioannis S. Arvanitoyannis,1* Athanassios G. Mavromatis,2 Garyfalia Grammatikaki-Avgeli3 & Michaela Sakellariou2 1 Department of Agriculture, Ichithyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Fytokou Str, Nea Ionia Magnesias 38446 Volos, Hellas, Greece 2 Department of Agriculture, Crop Science and Rural Environment, School of Agricultural Sciences, University of Thessaly, Fytokou Str, Nea Ionia Magnesias 38446 Volos, Hellas, Greece 3 School of Agriculture Technology, TEI of Crete, Heraklion, Grete, Hellas, Greece (Received 8 June 2007; Accepted in revised form 28 March 2008)

Summary

Genetic modification of banana has been considered as a path towards increasing the value of this crop according to health and nutrition in developing countries. Banana as a crop is one of the most important and widely consumed fruits as a weaning food by children and as a starchy staple for all other consumers. As well as providing a low cost and easily produced source of energy, bananas are also rich in certain minerals and in vitamins A, C and B6. Growing urbanisation in many developing countries upgraded the crop importance as a source of revenue, occasionally providing the main source of income for rural communities. Genetically modified organism bananas have been advocated as carrier for vaccines and as a source of carotenoids that can counteract debilitating vitamin A deficiency. The rather high vulnerability of banana to pests and diseases triggered biotechnological applications in an attempt to produce new, more resistant banana cultivars. However, the potential biosafety of genetically modified banana and its applications should be taken into account prior to its extensive usage. The current survey summarises the most important biotechnological techniques (in vitro culture, DNA fingerprinting, somatic emrbyogenesis, DNA flow cytometry, etc.) and applications (micropropagation, in vitro selection, somaclonal variation, protoplast fusion, haploid production, etc.) in banana and emphasises on genetic transformation in conjunction with the expressed gene and modified trait aiming at a further improvement of this crop.

Keywords

Banana, biosafety, biotechnology, genetic transformation, genetically modified organism.

Introduction

Banana is the common name used for the herbaceous plants of the genus Musa which is cultivated in more than 100 countries throughout the tropics and subtropics, with an annual world production of around 98 million tonnes, of which around a third is produced in each of the African, Asia-Pacific, and Latin American and Caribbean regions (Frison & Sharrock, 1999). The oldest records of edible bananas come from India (600 bc), known only by hearsay in the Mediterranean region in the third century bc (Horry et al., 1997). It is believed that they were first introduced to Europe in the 10th century. Early in the 16th century, Portuguese mariners transported the plant from the West African coast to South America. The wild types found in cultivation in the Pacific have been traced to eastern *Correspondent: Fax: +30 24210 93137; e-mail: [email protected]

Indonesia from where they spread to the Marquesas and gradually to Hawaii. Nowadays, bananas and plantains are grown in every humid tropical region and constitute the fourth largest fruit crop of the world (Morton, 1987). Description

Banana is a monoecious plant having male flowers at the tip of inflorescence and female flowers behind. The fruit of banana or plantain is a product of parthenocarpy and characterised as berry with a leathery outer peel that contains much collenchyma (Daniells et al., 2001). The fruits are formed in layers called combs or hands, consisting of 10–20 bananas, and there are 6–15 combs per stalk. The latter equals 40–50 kg per stalk or ten or more tons per acre. If commercially grown, the large terminal bud and bracts are removed to redirect sugars to the developing fruits. An unripened banana and the plantain have high starch and low sugar levels plus

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copious amounts of bitter-tasting latex. Starch is converted to sugar as the fruit ripens, so that bananas can eventually contain about 25% of total sugars. As the banana ripens, the latex is also decomposed. Plantain has the stinging, bitter latex, so the peel is removed with a knife and the pulp is soaked in salt water for 5–10 min prior to cooking (http://www.crfg.org). Bananas are harvested unripe and green, because they can ripen and spoil very rapidly. The fruits are cleaned of old floral parts, combs and divided into smaller bunches. Poorly formed fruits are removed, and bunches are thrown into a water bath, where latex is washed away. Then fruits are dried and usually placed in a ripening room for several days before their transfer to market, or exported after storing and packing with cushion (usually paper). Presence of naturally formed ethylene gas, produced by ripe fruits, hastens considerably the ripening of surrounding, greener fruits (http://www.botgard.ucla.edu). Banana cultivars

The genus Musa is classified into four sections, the members of which include both seeded (wild) and nonseeded or parthenocarpic edible types (Ortiz, 1997). Two of the sections (Callimusa and Australimusa) contain species with chromosome number of 2n = 2x = 20 while the species in the other two sections (Eumusa and Rhodochlamys) have as basic chromosome number (n = 11) (http://www.inibap.org). The centre of diversity of the species is believed to be either Malaysia (Simmonds, 1962) or Indonesia (Horry et al., 1997). Bananas and plantains belong to the Eumusa section of the genus Musa, family Musaceae, and order Zingiberales (Gill, 1988). This section is the largest in the genus and the most geographically widespread, with

species being found throughout South East Asia from India to the Pacific Islands. The genomic groups proposed by Simmonds & Shepherd (1955), to classify the edible clones are (AA), (BB), (AB), (AAA), (AAB), (ABB), (AAAA) and (ABBB) (Fig. 1). Most cultivars are derived from two diploid wild species, Musa acuminata (AA genome) and Musa balbisiana (BB genome) (Osuji et al., 1997). Edible clones are classified as to the relative contribution of M. acuminata and M. balbisiana (Simmonds & Shepherd, 1955). Musa acuminata is the most widespread of the Eumusa species. Chromosome structural changes, either having occurred spontaneously or as a response to recombination events, resulted in the development of natural reproductive barriers within the species thereby causing subspecies divergence and genetic diversity in the species as a whole (Horry et al., 1997). In conjunction with chromosome restitution, this process gave rise to (i) autoploids and homogenomic hybrids which are essentially (AAA) dessert and beer bananas, and (ii) alloploids and heterogenomic hybrids comprising the plantains (AAB) and the cooking bananas (ABB) (Ortiz, 1997). The most important banana types, genomic groups and cultivars with their characteristic agronomical and quality traits, are presented in Table 1. Triploid (AAA) cultivars arose from diploids, perhaps following crosses between edible diploids and wild M. acuminata subspecies (Ortiz, 1995). In most parts of South East Asia, the triploid cultivars, which are more vigorous and have larger fruit, replaced the original diploids (AA). The diploid and triploid M. acuminata cultivars were taken by humans to areas where M. balbisiana was native (India, Myanmar, Thailand, Philippines) and natural hybridisation resulted in the formation of hybrid progeny with the genomes (AB),

Development of tetraploid (2n = 4x) banana cultivars AA w

X

AA cv

AA cv

X

AA cv

AA (w)cv

AA cv

X

BB w

AA cv AB (w) cv

AAAA cv Colchicine treatment

AABB cv

Development of triploid (2n = 3x) banana cultivars

AAA (w) cv

AA cv

X

AAAA cv

AA (w)

X

AAAA cv

AAA cv

BB w

X

AAAA cv

AAB (w) cv

AA cv

X

AABB cv

AAB cv

AA (w)

X

AABB cv

AAB (w) cv

International Journal of Food Science and Technology 2008

Figure 1 Types of banana according to genomic group and ploidy level. Possible crosses applied for the development of new triploid (2n = 3x) and tetraploid cultivars (2n = 4x). cv, Commercial cultivar; w, wild germplasm.

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Banana: cultivars & biotechnological approaches I. S. Arvanitoyannis et al.

Table 1 Common types and commercial cultivars of banana characterised by genomic group, agronomical and quality traits Type or species (genomic group) Dwarf Cavendish Robusta (AAA) Lacatan (AAA) Cavendish Nana Giant Cavendish Plantain (AAB) Musa balbisiana (ABB) Dwarf Prata (AAB) · SH 3142 (AA) (AAAB)

Cultivar

Agronomical and quality traits

Cavendish (AAA)

Accepted sensory & physicochemical traits Large bunches of high quality fruit High aromatic Good quality traits, high yielded

Mediate fruit size

Valery Pisang

Williams Grand Naine French Bluggoe Gold finger

Very good quality traits High yielded, very good quality traits Cooking bananas Drought tolerance Tolerance to low temperatures, drought tolerance, resistance to Fusarium oxysporum

(AAB) and (ABB) (Daniells & Smith, 1991). The Indian subcontinent is thought to have been the major centre for hybridisation of acuminata types with the indigenous M. balbisiana and the region is noted for the wide variation of (AAB) and (ABB) cultivars (Ortiz, 1997). Musa balbisiana is considered to be more resistant to diseases and tolerant to abiotic stress, than M. acuminata. Such characteristics often occurred in cultivars containing a ‘B’ genome. Hybridisation would have given rise to a wide range of edible types of banana, some of which would have survived and been multiplied under domestication (Simmonds, 1962). Consequently, a diverse selection of banana cultivars is thought to have arisen in South East Asia along with the earliest developments of agriculture many thousands of years ago (Price, 1995). The subsequent dispersal of edible bananas outside of Asia was most likely brought about solely by humans (Simmonds, 1962). This early dispersal of banana cultivars resulted in the development of distinct subgroups of cultivars. Secondary diversification within the major sub-groups of cultivated bananas is thought to have been the result of somatic mutations rather than

Sweet pulp Sensitivity to choke throat, sensitivity to Fusarium oxysporum Sweet taste, long size fruits Sensitivity to Mycosphaerella fijiensis Horn Long size fruits Fruit acidity, small fruit size

sexual reproduction. Mutations affecting traits of economic or horticultural interest were selected by farmers over the years and multiplied by vegetative propagation to produce morphotypes (http://www.inibap.org). Although the precise number of Musa cultivars is uncertain, Samson (1982) has estimated that they must be over 300 (Valmayor et al., 2000). A collection of most Musa accessions is maintained by the Plantain and Banana Improvement Programme of the International Institute of Tropical Agriculture in a field gene bank located at Onne near Port Harcourt, Nigeria (Swennen & Vuylsteke, 1991). Applications of biotechnology in banana

A high number of applied biotechnological techniques are increasingly being used worldwide towards improving the handling and properties of plantain and banana germplasm (Table 2). Tissue culture is used for germplasm exchange, conservation and rapid multiplication, while in vitro seed germination (based on embryo culture or rescue) plays a critical role in generating hybrid

Table 2 Biotechnological techniques and applications in banana Technique

Application

References

In vitro culture

Micropropagation, germplasm conservation, embryo culture, virus free meristem culture

Protoplast fusion Somatic embryogenesis In vitro mutagenesis Anther culture

Development of new hybrids Somaclonal variation Development of new cultivars Haploid production, development of new dilpoid cultivars Ploidy level estimation, study of somaclonal variation Development of new genetically modified organism cultivars Cultivar identification, genetic analysis Breeding (early selection) ⁄ MAB, genome mapping, detection of somaclonal variation

Arias (1992), Crouch et al. (1998), Vuylsteke (1989), Israeli et al. (1995), Creste et al. (2004) Sagi et al. (1995), Assani et al. (2005) Gimenez et al. (2001), Damasco et al. (1997) Damasco et al. (1997), Bhagwat & Duncan (1998) Assani et al. (2003)

DNA flow cytometry Genetic transformation DNA fingerprinting Molecular markers

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Roux et al. (2003) Coˆte et al. (1997), Ganapathi et al. (2002), Becker et al. (2000) Crouch et al. (2000) Ferreira et al. (2004), Crouch et al. (2000), Ramage et al. (2004)

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plants (Crouch et al., 1998). A reproducible anther culture method for producing haploid plants of banana would be useful in conventional breeding programs, as well as in somatic protoplast fusion procedures, to obtain directly triploid cells starting from diploid and haploid cells. Assani et al. (2003) reported successful regeneration of haploid plants (2n = x = 11) in four genotypes of the species M. balbisiana (BB), which is known to carry resistant genes against economically important banana diseases. A protoplast fusion technique to obtain somatic hybrids between triploid and diploid bananas, was put forward by Matsumoto et al. (2002). The technique is particularly useful in introducing disease resistance from wild relatives or other species into a cultivated variety. Assani et al. (2005) compared the most frequently used fusion method (electrofusion technique) with the chemical procedure using polyethylene glycol (PEG). According to their observations with regard to frequency of binary fusion, protoplast fusion with the fusogen polyethylene glycol was the best. Conversely, electric fusion was found to be better with respect to mitotic activities, somatic embryogenesis and plantlet regeneration rate. Mutation breeding could also contribute to genetic improvement of banana plants. However, there has been no practical implementation of a mutation breeding program for banana. Bhagwat & Duncan (1998) reported the use of gamma irradiation on explants of in vitro grown cultures of banana (AAA group) to evaluate the effectiveness of inducing mutations and also with the aim of producing variants tolerant to the fungus Fusarium oxysporum f. sp. Cubense well known as Panama disease. Somatic embryogenesis techniques in the genus Musa aimed at developing new, high performance micropropagation techniques and cell regeneration systems useful for genetic transformation and cultivar improvement (Kosky et al., 2002). Moreover, the cell culture could be associated with mutation induction, genetic transformation processes (biolistic or Agrobacterium systems) or selection methods based on somaclonal variation. The use of biotechnological approaches, such as in vitro mutagenesis and genetic transformation, are seriously impeded since the treatment of multicellular meristems results in a high degree of chimerism (Roux et al., 2003). The high incidence of ‘off-types’ produced by banana meristem culture is a major concern to commercial growers. At the same time, somaclonal variation is an important tool for the improvement of banana germplasm (Khayat et al., 2004). Vidal & Garcia (2000) managed to obtain a somaclonal variant (CIEN BTA03) resistant to Yellow Sigatoka from a susceptible banana clone (Williams clone), by increasing the production of adventious buds using 6-benzylaminopurine, at high concentrations. Molecular techniques have been applied to detect and partially characterise the somaclonal variants in banana. Ramage et al. (2004)

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used random amplified polymorphic molecular markers (RAPD) to detect dwarf off-types in micropropagated Cavendish bananas (AAA group). Aneuploidy, which involves an under- or over-representation of one or more chromosomes, is a frequent type of mutation in Musa. Flow cytometry is a convenient and rapid method for the detection of such aneuploidy (Roux et al., 2003). A substantial amount of research was carried out to distinguish and classify Musa accessions on the basis of morphological characteristics. However, the classification of certain accessions on this basis has been disputed (Crouch et al., 1998). Molecular markers were used to study diversity in Musa, as well as in Musa genotyping and mapping (Kaemmer et al., 1997). Ferreira et al. (2004) employed RAPD molecular markers for the characterisation of banana diploids (AA) with contrasting levels of Black Sigatoka (Mycosphaerella fijiensis) and Yellow Sigatoka (Mycosphaerella musicola) resistance. Creste et al. (2004) estimated genetic diversity of Musa diploid and triploid accessions by microsatellite markers. Genetic variability was investigated among forty genotypes of banana using B-genome derived SSRs molecular markers (Oreiro et al., 2006). In a comparative analysis of phenotypic and genotypic diversity among plantain landraces (Musa spp. AAB group), a rather poor correlation was found between RAPD-based estimates of genetic diversity and a phenotypic index based on agronomic characters. These results suggested that the traditional designations of plantain landraces based on morphotype do not provide a true reflection of overall genetic divergence (Crouch et al., 2000). Furthermore, this study was conducted to compare different PCR-based marker systems [RAPD, variable number tandem repeats (VNTR) and amplified fragment length polymorphism (AFLP)] for the analysis of Musa breeding populations. AFLP assays had by far the highest multiplex ratio while VNTR analysis detected the highest levels of polymorphism. In general, there was a poor correlation between estimates of genetic similarity based on different types of marker (Crouch et al., 2000). Genetically modified bananas

Genetic transformation is of great interest in banana because (i) the cultivated varieties are triploid and sterile, (ii) some resistance sources are not available among genetic resources (i.e. virus resistance) and (iii) the foreign gene within the genetically modified plant cannot be transferred to another plant because the triploid plants will not produce fertile pollen. Therefore, the risk of direct gene contamination is minimised both for other plants and for the environment. A sexual gene transfer methods such as transformation may be required for characteristics lacking in Musa genebanks, or for the genetic improvement of cultivars

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not amenable to conventional cross breeding such as Cavendish bananas or Horn plantains. Relative success in genetic engineering of bananas and plantains has been achieved recently to enable the transfer of foreign genes into plant cells (Table 3). Protocols for electroporation of protoplasts derived from embryogenic cell suspensions (Sagi et al., 1998), particle bombardment of embryogenic cells (Sagi et al., 1995; Coˆte et al., 1997; Becker et al., 2000), and co-cultivation of wounded meristems with Agrobacterium (May et al., 1995) are available for bananas and plantains. The Agrobacterium-mediated transformation method may be more widely applicable as it is based on the use of differentiated tissue that can be routinely regenerated into whole plants. In addition, it has been applied to a wide range of plantain and banana cultivars and synthetic hybrids (Bosque-Pe´rez et al., 1998). Moreover, Agrobacterium-mediated transformation offers several advantages over direct gene transfer methodologies like particle bombardment and electroporation (Gheysen et al., 1998; Hansen & Martha, 1999; Shibata & Liu, 2000). Various diseases caused by fungi and viruses have seriously endangered the production of banana and plantains. Through genetic transformation technologies, disease resistant varieties may be produced. Transgenic plants have been produced for the cultivars Williams, Gros Michel, Bluggoe and Three Hand planty, using gene constructs encoding for various antifungal peptides which have previously proved to be highly active in vitro against major pathogenic fungi of bananas (Remy et al., 2000). Fusarium wilt caused by Fusarium oxysporum f. sp. cubense and Black sigatoka, by the fungus Mycosphaerella fijiensis f. sp. cubense, which are the most devastating fungal diseases of Musa. The most attractive strategy for black sigatoka control in Musa is probably

the production of disease resistant plants through the transgenic approach. These approaches included the expression of genes encoding plant, fungal or bacterial hydrolytic enzymes (Lorito et al., 1998), genes encoding elicitors of defence response (Keller et al., 1999) and antimicrobial peptides (Cary et al., 2000). Efforts are presently focused on the recently described antimicrobial proteins (AMPs) which are stable, cysteine-rich small peptides isolated from seeds of diverse plant species (Sa´gi et al., 1998). AMPs have a broad-spectrum antimicrobial activity against fungi as well as bacteria and most are non-toxic to plant and mammalian cells. On the basis of their broad-spectrum activity against fungal pathogens, individual or combined expression of cecropin (identified from the cecropia moth and is very active against Gram-negative bacteria), magainin and their derivatives (acts on both Gram-positive and Gramnegative bacteria, fungi and protozoa) in Musa may result in enhanced resistance to several pathogens (Tripathi, 2003; Tushemereirwe et al., 2002). The AMPs of plant origin may be the potent candidates for fungal resistance in Musa as they have high in vitro activity to Mycospaerella fijiensis and Fusarium oxysporum f. sp. cubense and they are non-toxic to human or banana cells as well. Several hundreds of transgenic lines of Musa, especially plantains, expressing AMPs were developed using particle bombardment of embryogenic cell suspensions (Remy, 2000), but none of these transgenic plants was used in field trials due to the lack of biosafety guidelines in most tropical countries. Further research is anticipated to demonstrate whether these transgenic plants can express the functional antimicrobial peptides at levels high enough to control fungal leaf or root diseases in the field. It was shown that one AMP gene is also expressed in the fruit, thereby providing the

Table 3 Genetic modification in banana (methodology used, expressed gene and modified traits) Expressed gene

Method

Modified trait

References

MSI-99, a magainin analogue

Agrobacterium

Chakrabarti et al. (2003)

Antimicrobial peptides

Particle bombardment

Antimicrobial peptides

Particle bombardment

Protein engineered rice Cystatin (OcIdeltaD86) Cysteine proteinase inhibitor (oryzacystatin-I) Hepatitis B antigen, HBsAg (pHBS, pHER, pEFEHBS) Synthetic cercosporins Antiretroviral genes (adefovir, tenofovir) Human lysozyme gene

Agrobacterium

Resistance to Fusarium oxysporum f.sp. cubense and Mycosphaerella musicola Resistance to Fusarium oxysporum f. sp. cubense and Mycospaerella fijiensis Resistance against preharvest and postharvest diseases Verticillium theobromae or Trachysphaera fructigena Resistance to nematode Radopholus similis

Agrobacterium

Remy et al. (2000), Sa´gi et al. (1998), Tripathi (2003) Cary et al. (2000), Sa´gi et al. (1998)

Atkinson et al. (2004)

Agrobacterium

Resistance to banana weevil (Cosmopolites sordidus) Edible vaccine against hepatitis B

Agrobacterium Agrobacterium

Resistance to bacterial wilt (Xanthomonas spp.) Resistance to Banana Streak Virus

Ganapathi et al. (2002), Kumar et al. (2005) Rajasekaran et al. (2001) Helliot et al. (2003)

Agrobacterium

Resistance to Fusarium oxysporum

Pei et al. (2005)

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Kiggundu et al. (2002)

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opportunity to enhance the resistance against preharvest and postharvest diseases such as cigar-end rot and crown rot (Sa´gi et al., 1998). Magainin is one of the earliest reported antimicrobial peptides isolated from skin secretions of the African clawed frog Xenopus laevis. Chakrabarti et al. (2003) have recently reported successful expression of a synthetic substitution analogue of magainin, MSI-99 and enhanced disease resistance. MSI-99 was subcloned into plant expression vectors pMSI164 and pMSI168. Transgenic banana plants were obtained for both pMSI164 and pMSI168 transformations and displayed considerable resistance to F. oxysporum f. sp. cubense and Mycosphaerella musicola. The obtained results suggest that MSI-99 can be successfully applied in imparting enhanced disease resistance in transgenic banana plants (Chakrabarti et al., 2003). Xanthomonas campestris pv. musacearum is gradually spreading in East Africa and if uncontrolled could result in massive losses. To date, no banana germplasm exhibiting resistance to the disease was identified. Use of genetic transformation technology with bactericidal transgenes encoding for peptides such as cecropins and lysozyme, may offer an alternative and potential solution to these problems (Tripathi, 2003). Banana bunchy top, caused by Banana bunchy top virus (BBTV), genus Nanavirus is one of the most threatening diseases in the world, as infected plants do not produce fruit. Engineering resistance to BBTV is an obvious objective for banana transformation, since no natural resistance to this virus has so far been identified in the Musa genepool. One of the strategies currently being tested in several laboratories is the expression of various BBTV genes in transgenic banana plants in order to interfere with the normal replication, encapsidation or movement of the virus. Another approach is the expression of heterologous antiviral proteins that are known to act by the inhibition of viral replication or translation in bananas (Sa´gi et al., 1998). Similar strategies can also be considered against another recently emerged DNA virus, the Banana streak virus (BSV), whose molecular research in laboratories worldwide revealed a number of interesting features including its integration into the banana genome (Sa´gi et al., 1998). The BSV, genus Badnavirus, has a major impact on banana and plantain production in Africa (Swennen & Vuylsteke, 2001). Researchers at International Institute of Tropical Agriculture (Nigeria) in collaboration with John Innes Centre (UK) are experimenting with transgenic plants resistant to BSV (including expression of integrated sequences) based on the novel approach of gene silencing (http://www.inibap.org). Nematodes are recognised as severe production constraints to bananas and plantains (Gowen & Queneherve, 1990). There are several possible

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approaches for developing transgenic plants with improved nematode resistance. The use of proteinase inhibitors (PIs), as nematode antifeedants, is an important element of natural plant defence strategies (Ryan, 1990). Cysteine PIs (cystatins) are inhibitors of cysteine proteinases and were isolated from seeds of a wide range of crop plants consumed by humans including those of sunflower, cowpea, soybean, maize and rice (Atkinson et al., 1995). Atkinson et al. (2004) were the first to report transgenic resistance against the nematode Radopholus similis, a major pest of banana. Cavendish banana was effectively transformed by means of Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIdeltaD86) of value for control of plant parasitic nematodes. The banana weevil (Cosmopolites sordidus) is a pest of substantial importance in Africa and greatly affects banana and plantain production. Although remarkable progress was made in banana transformation, the identification and introduction of useful genes into banana to reduce losses caused by the banana weevil is still a major challenge. Among the various genes available for genetic engineering for pest resistance are (Carozi & Koziel, 1997; Sharma et al., 2000): PIs, Bacillus thuringiensis (Bt) toxins, plant lectins, vegetative insecticidal proteins and alpha-amylase inhibitors. The PIs if expressed in bananas, could greatly improve their resistance to the banana weevil. However, any strategy targeting to the use of a gene coding for an inhibitor should ideally include a design program for optimising the inhibitor action against the target enzyme. Cysteine proteinase activity was recently identified in the mid-gut of the banana weevil. In vitro studies revealed that these cysteine proteinases are strongly inhibited by both a purified recombinant rice (oryzacystatin-I) and papaya cystatin (Kiggundu et al., 2002). Nevertheless, the incorporation of genes on several occasions is random in plant genome thereby resulting in either non-anticipated expression or lower expression level. In view of the dynamic of plant genome, the insertion gene may interact with the other genes thus potentially having unexpected or disastrous effect upon the plant itself. Over the last years there has been a growing interest in ‘molecular farming’ for the production of added value compounds of pharmaceutical, cosmetic and industrial importance. Out of these, edible vaccines are of prime importance for human health care (Ganapathi et al., 2002). The production of antigens in genetically engineered plants is anticipated to provide an inexpensive source of edible vaccines and antibodies in the fight against infectious diseases such as hepatitis B (Prakash, 1996). In this regard, banana can serve as an ideal system for the production and delivery of edible vaccines. Ganapathi et al. (2002)

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sub-cloned hepatitis B surface antigen (HBsAg) and transformed embryogenic banana cells using Agrobacterium-mediated transformation in order to produce transgenic banana plants for edible vaccines against hepatitis B. Embryogenic cells of banana cv. Rasthali (AAB) were transformed with the ‘s’ gene of HBsAg using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilised to optimise the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen were confirmed by PCR, Southern hybridisation and reverse transcription (RT)-PCR. HBsAg obtained from transgenic banana plants was similar to human serum derived one in analogous density properties (Kumar et al., 2005). Biosafety

The potential biosafety risks of transgenic varieties have generated controversy in international community. According to the U.S. National Research Council (2002), these risks include: (i) the flow of transgenes, (ii) evolution of resistance in the targeted pest population, (iii) plant escape and establishment of self-reproducing populations, (iv) effects on non-target species, and (v) health hazards (Smale et al., 2006). Biosafety frameworks, laws, and regulations in developing countries have been developed in response to the implementation of the Cartagena Protocol on Biosafety. The Cartagena Protocol, a supplement to the Convention on Biological Diversity, addresses the safe transfer, handling, and use of living modified organisms (LMOs), especially those that may have an adverse effect on biodiversity, taking into account risks to human and animal health. Bananas are being considered as the vehicle of choice for phytoceuticals, particularly for applications in the developing countries, because of their worldwide popularity, abundance and baby-friendliness. On the contrary, bananas contain low protein levels and are unlikely to produce large amounts of recombinant proteins (i.e. vaccines). Banana trees also take a few years to mature and the fruit spoils fairly rapidly after ripening thereby making transportation and storage difficult (http://www.biosafety-info.net). It should be pointed out that clonal propagation and the system for disseminating plant material are responsible for engendering the risk of resistance evolution for transgenic bananas rather than gene flow. The risk of resistance evolution in the targeted pest population may be great with the soil and root borne problems of banana, since mats move slowly with new roots in a given location and farmer propagation reproduces the same trait. Largescale multiplication schemes, such as those envisaged for tissue culture systems, would contribute to genetic

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uniformity in the trait (Smale et al., 2006). Gene flow is possible among tetraploid bananas, such as recently developed hybrids, although normally these are reproduced through self – propagation. However, no flow of transgenes is possible among East African highland bananas because they are sterile triploids (http://photoscience.la.asu.edu). Extensive research over the past two decades has shown that a wide range of valuable proteins can be expressed efficiently in plants. Examples include human serum proteins and growth regulators, antibodies, vaccines, industrial enzymes, biopolymers and molecular biology reagents (Smale et al., 2006). One of the most important advantages of edible vaccines is the potential to drastically reduce or eliminate transport costs (http://www.biosafety-info.net). Furthermore, edible vaccines would not require the purification, strict refrigeration, and injections that make conventional vaccines expensive to use. The edible vaccines would encourage preventive medicine in the Third World. There are numerous regulatory and safety (dosage) issues associated with ‘being vaccinated via a banana’, and current research is directed more toward oral vaccination with more or less pure compounds that could be produced inexpensively (http://photoscience.la.asu.edu). However, the most important objections to edible vaccines arise from: (i) their inadequate absorbance from the gut and being eventually broken down by the gut enzymes, (ii) the fact that elimination of transport cost is not feasible and (iii) contradictory environmental repercussions (http:// www.biosafety-info.net). Conclusions

Plant biotechnology has the potential to play a key role in the sustainable production of Musa. However, there is enormous potential for genetic manipulation of Musa species in order to improve their disease and pest resistance. The use of appropriate constructs may allow the production of nematode, fungal, bacterial and virusresistant plants in a significantly shorter period of time than using conventional breeding, especially if several traits can be introduced simultaneously. It may also be possible to incorporate other characteristics such as drought tolerance, thereby extending the geographic spread of banana and plantain production, and thus contributing substantially to enhanced food security and poverty alleviation. Banana seems like a model plant for genetic modification due to its advantage of clonal propagation and lack of pollen fertility thereby ensuring optimal conditions for minimal cross-contamination. However, high caution is required for biosafety experiments and potential risk assessment bearing in mind that it is consumed by most humans and mainly by children.

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Ferreira, C.F., Oliveira Silva, S., Sobrinho, N.P.D., Damascena, S.C.S., Oliveira Alves, F.S. & Paz, O.P. (2004). Molecular characterization of Banana (AA) diploids with contrasting levels of Black and Yellow Sigatoka resistance. American Journal of Applied Sciences, 1, 276–278. Frison, E.A. & Sharrock, S.L. (1999). Introduction: the economic, social and nutritional importance of banana in the world. In: Bananas and Food Security (edited by C. Picq, E. Foure´ & E.A. Frison). Pp. 21–35. International Symposium, Douala, Cameroon, 10–14 November, 1998. France: INIBAP. Ganapathi, T.R., Chakrabarti, A., Sunil Kumar, G.B., Revathi, C.J., Prasad, K.S.N. & Bapat, V.A. (2002). Genetic transformation of banana for disease resistance and molecular farming. In: 3rd International Symposium on Molecular and Cellular Biology of Bananas Leuven. Gheysen, G., Angenon, G. & Montague, M.V. (1998). Agrobacterium mediated plant transformation: a scientifically intriguing story with significant application. In: Transgenic Plant Research (edited by K. Lindsey). Pp. 1–33. The Netherlands: Harwood Academic Press. Gill, L.S. (1988). Taxonomy of Flowering Plants. Pp. 106–109. Ibadan: Eds Africana Fep. Publishers Ltd. Gimenez, C., De Garcia, E., De Enrech, N.Z & Blanka, I. (2001). Somaclonal variation in banana: cytologenetic and molecular characterization of the somaclonal variants. CIEN BTA-03. In Vitro Cellular & Developmental Biology Plant, 37, 217–222. Gowen, S. & Queneherve, P. (1990). Nematode parasites of banana, plantains and abaca. In: Plant Parasitic Nematodes in Subtropical and Tropical Agriculture (edited by L.M. Sikora & R.A. Bridge). Pp. 431–460. Leuven CAB International. Hansen, G. & Martha, S.W. (1999). Recent advances in the transformation of plants. Trends in Plant Science, 4, 226–231. Helliot, B., Panis, B., Frison, E. et al. (2003). The acylic nucleoside phosphonate analoques, adefovir, tenofovir and PMEDAP, efficiently eliminate banana streak virus from banana (Musa sp.). Antiviral Research, 59, 121–126. Horry, J.P., Ortiz, R., Arnaud, E. et al. (1997). Banana and plantain. In: Biodiversity in Trust: Conservation and Use of Plant Genetic Resources in CGIAR Centres (edited by D. Fuccillo, L. Sears & P. Stapleton). Pp. 67–81. Cambridge: Cambridge University Press. Israeli, Y., Lahav, E. & Reuveni, O. (1995). In vitro culture of bananas. In: Bananas and Plantains (edited by S. Gowen). Pp. 147–178. London: Chapman & Hall. Kaemmer, D., Fischer, D., Jarret, R.L. et al. (1997). Molecular breeding in the genus Musa: a strong case for STMS marker technology. Euphytica, 96, 49–63. Keller, H., Pamboukdjian, N., Ponchet, N. et al. (1999). Pathogeninduced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance. Plant Cell, 11, 223–236. Khayat, E., Duvdevani, A., Lahav, E. & Ballesteros, B.A. (2004). Banana Improvement: cellular, molecular and induced mutations (edited by S.M. Jaine & R. Swennen). Enfield, NH: FAO Corporate Document Repository. Kiggundu, A., Kunert, K., Viljoen, A., Pillay, M. & Gold, C.S. (2002). In:3rd International Symposium on Molecular and Cellular Biology of Bananas. Designing Proteinase Inhibitors for Banana Weevil Control. Leuven. Kosky, R.G., Silva, M.deF., Pe´rez, L.P. et al. (2002). Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor. Plant Cell Tissue and Organ Culture, 68, 21–26. Kumar, G.B., Ganapathi, T.R., Revathi, C.J., Srivinas, L. & Bapat, V.A. (2005). Expression of hepatitis B surface antigen in transgenic banana plants. Planta, 222, 484–493. Lorito, M., Woo, S.L., Garcia, I. et al. (1998). Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proceedings of the National Academy of Science, 95, 7860–7865.

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Banana: cultivars & biotechnological approaches I. S. Arvanitoyannis et al.

Matsumoto, K., Vilarinhos, A.D. & Oka, S. (2002). Somatic hybridization by electrofusion of banana protoplasts. Euphytica, 125, 317– 324. May, G., Afza, R., Mason, H., Wiecko, A., Novak, F. & Arntzen, C. (1995). Generation of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation. Bio ⁄ Technology, 13, 486–492. Morton, J. (1987). Banana. In: Fruits of Warm Climates (edited by F. Julia). Pp. 29–46. Miami, FL: Morton. National Research Council (2002). Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation. Washington: National Academy Press. Oreiro, C.E., Odunola, O.A., Lokko, Y. & Ingelbrecht, I. (2006). Analysis of B-genome derived simple sequence repeat (SSR) markers in Musa spp. African Journal of Biotechnology, 5, 126–128. Ortiz, R. (1995). Musa genetics. In: Bananas and Plantains. (edited by S. Gowen). Pp 84–109. London: Chapman & Hall. Ortiz, R. (1997). Morphological variation in Musa germplasm, Genetic Resources and Crop Evolution, 44, 393–404. Osuji, J.O., Okoli, B.E., Vuylsteke, D. & Ortiz, R. (1997). Multivariate pattern of quantitative trait variation in triploid banana and plantain cultivars. Scientia Horticulturae, 71, 197–202. Pei, X.W., Chen, S.K., Wen, R.M., Ye, S., Huang, J.Q. & Zhang, Y.Q. (2005). Creation of transgenic bananas expressing human lysozyme gene for panama wilt resistance. Journal of Integrative Plant Biology, 47, 971–977. Prakash, C.S. (1996). Edible vaccines and antibody producing plants. Biotechnology and Development Monitor, 27, 10–13. Price, N.S. (1995). The origin and development of banana and plantain cultivars. In: Bananas and Plantains (edited by S. Gowen). Pp. 1–12. London: Chapman and Hall. Rajasekaran, K., Stromberg, K.D., Cary, J.W. & Cleveland, T.E. (2001). Broad-spectrum antimicrobial activity in vitro of the synthetic peptide D4E1. Journal of Agricultural and Food Chemistry, 49, 2799–2803. Ramage, C.M., Borda, A.M., Hamill, S.D. & Smith, M.K. (2004). A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA). Scientia Horticulturae, 103, 145–151. Remy, S. (2000). Genetic transformation of banana (Musa sp.) for disease resistance by expression of antimicrobial proteins. PhD Thesis, Belgium: KUL. Remy, S., Buyens, A., Cammue, B.P.A., Swennen, R. & Sa´gi, L. (2000). Production of transgenic banana plants expressing antifungal proteins. International Symposium on Banana in the Subtropics. Acta Horticulturae, 490, 219–277. Roux, N., Toloza, A., Radecki, Z., Zapata-Arias, F.J. & Dolezel, J. (2003). Rapid detection of aneuploidy in Musa using flow cytometry. Plant Cell, 21, 483–490. Ryan, C.A. (1990). Proteinase inhibitors in plants: genes for improving defences against insects and pathogens. Annual Review of Phytopathology, 28, 425–449. Sagi, L., Panis, B., Remy, S. et al. (1995). Genetic transformation of banana (Musa sp.) via particle bombardment. Bio ⁄ Technology, 13, 481–485.

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Sagi, L., Gregory, D.M., Remy, S. & Swennen, R. (1998). Recent developments in biotechnological research on bananas (Musa spp.). Biotechnology and Genetic Engineering Reviews, 15, 313–317. Sa´gi, L., Remy, S. & Swennen, R. (1998). Genetic transformation for the improvement of bananas – a critical assessment. In: INIBAP Annual Report. Pp. 33–36. Montpellier: FRA. Samson, J.A. (1982). Tropical Fruits. Edinburgh: Longman. Sharma, H.C., Sharma, K.K., Seetharama, N. & Ortiz, R. (2000). Prospects for using transgenic resistance to insects in crop improvement. EJB Electronic Journal of Biotechnology 3. Shibata, D. & Liu, Y.G. (2000). Agrobacterium-mediated plant transformation with large DNA fragments. Trends in Plant Science, 5, 354–357. Simmonds, N.W. (1962). Evolution of the Bananas. Pp. 170. London: Longman, Green & Co. Ltd. Simmonds, N.W. & Shepherd, K. (1955). Taxonomy and origins of cultivated bananas. Botanical Journal of Linnean Society, 55, 302– 312. Smale, M., Zambrano, P., Zepeda, J.F. & Gruere, G. (2006). Parables: applied economics literature about the impact of genetically engineered crop varieties in developing countries. In: Biosafety and Biodiversity Risks – Genetic Resource Policies. Discussion paper 158 of International Food Policy Research Institute. Swennen, R. & Vuylsteke, D. (1991). Bananas in Africa: Diversity, uses and prospects for improvement. In: Crop Genetic Resources of Africa, Vol II. Proceedings of an International Conference, Ibadan (Nigeria) (edited by P. Perrino, F. Attere & H. Zedah). Pp. 151–159. Swennen, R. & Vuylsteke, D. (2001). Banana. In: Crop Production in Tropical Africa (edited by R.H. Raemaekers). Pp. 530–552. Belgium: DGIC. Tripathi, L. (2003). Genetic engineering for improvement of Musa production in Africa. African Journal of Biotechnology, 2, 503–508. Tushemereirwe, W.K., Kangire, A., Smith, J. et al. (2002). An Outbreak of Banana Bacterial Wilt in Mukono and Kayunga Districts: A New and Devastating Disease. The First Updated Disease Report. NARO ⁄ KARI. Infomusa, 12, 4–8. Valmayor, R.V., Jamaluddin, S.H., Silayoi, B. et al. (2000). Banana cultivar names and synonyms in Southeast Asia. In: Proceedings of International Network for the Improvement of Banana and Plantain – Asia and the Pacific Office, pp. 24. Philippines: Los Ban˜os, Laguna. Vidal, M.C. & Garcia, E. (2000). Analysis of a Musa spp. Somaclonal variant resistant to Yellow Sigatoka. Plant Molecular Reports, 18, 23–31. Vuylsteke, D. (1989). Shoot-tip Culture for the Propagation, Conservation and Exchange of Musa Germplasm. Rome: IBPGR.

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Original article Effect of the side chain size of 1-alkyl-pyrroles on antioxidant activity and ‘Laba’ garlic greening Dan Wang, Xiaosong Hu & Guanghua Zhao* College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China (Received 22 February 2008; Accepted in revised form 30 April 2008)

Summary

Previous studies showed that 1-alkyl-pyrroles not only occur in fresh food products postulated as a natural antioxidant but also might be involved in garlic greening. In the present study, a series of 1-alkyl-pyrroles with different side chain size were synthesised to study the relationship of structure and antioxidative activity and their effects on ‘Laba’ garlic greening. The antioxidative activity of these compounds was evaluated by the method of scavenging ABTS· and DPPH·. Results showed that increasing the size of R groups on the side chain, the antioxidative activity decreased gradually against the two radicals. The 1-alkyl-pyrroles generally exhibited stronger scavenging activities against ABTS· than DPPH·. In contrast, their corresponding amino acids except for tyrosine showed almost no antioxidative activities while pyrrole exhibited much weaker activity as compared with the 1-alkyl-pyrroles, suggesting that the 1-alkyl-pyrroles donate H-atom from pyrrole moiety rather than side chain to quench the two radicals. On the other hand, all 1-alkyl-pyrroles can turn newly harvested garlic green but to a different extent. All these results suggested that these pyrrole derivatives occurring in foodstuff played an important role in either protecting foodstuff from oxidation or acting on pigment precursors during ‘Laba’ garlic greening.

Keywords

1-Alkyl-pyrroles, antioxidant activity, greening, ‘Laba’ garlic, side chain.

Introduction

Free radicals, especially those derived from oxygen, can react with various biological molecules because of their high reactivities. These radicals play a role in most major health problem of the industrialised world, such as cardiovascular diseases, cancer, neurological diseases and aging (Darley-Usmar & Halliwell, 1996; Halliwell, 1996). Although almost all organisms have evolved defense and repair systems, these systems alone are insufficient to entirely prevent the damage (Sies, 1997). Fortunately, antioxidants can help the human body prevent or reduce the oxidative damage caused by these toxic radicals (Halliwell, 1996; Sies, 1997). To date, a large number of antioxidants has been either synthesised or separated from naturally occurring resource such as fruit, vegetables, plant and marine animals, and many of which exhibit a good antioxidative activity against DPPH·, ABTS·, hydroxyl radical and so on (Huang et al., 2005). Particularly, many natural compounds containing a pyrrole-ring moiety are of great interests because of *Correspondent: Fax: +86 10 62737434 11; e-mail: [email protected]

their biological activities, which have been widely used in medicine and agriculture (Chin et al., 2003; Bellina & Rossi, 2006). Such compounds are also produced during peroxidation of lipids existing in foodstuff and biological systems. For example, lipid-derived aldehyde, 4,5(E)-epoxy-2(E)-heptenal, was isolated from both the Cu ⁄ a-tocopherol-induced auto-oxidation of either butterfat or cod liver oil and the thermal decomposition of methyl linolenate hydroperoxides, and subsequently this compound can react with either lysine or butylamine to produce a series of pyrrole derivatives including 1-alkyl-pyrroles such as e-N-pyrrolylnorlecucine, 1-(5¢-amino-1¢-carboxy-pentyl) pyrrole and 1-alkyl-2(1¢-hydroxyalkyl) pyrroles under different conditions such as microwave irradiation and thermal processing. Three of these pyrrole derivatives were found to protect against peroxidation and protein damage while amino acids lacked this activity for the systems assayed, suggesting that the pyrrole derivatives formed during lipid peroxidation might be an antioxidative defense mechanism by which oxidative stress is feed-backinhibited to fight with damage caused by the toxic radicals (Hidalgo & Zamora, 1995; Zamora & Hidalgo, 1995; Zamora et al., 1997; Hidalgo et al., 1998). Later, it was found that e-N-pyrrolylnorlecucine is not only a

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Antioxidant activity of 1-alkyl-pyrroles D. Wang et al.

final product in the reaction of 4,5(E)-epoxy-2(E)heptenal with lysine but also occurs in twenty-two different fresh food products (Zamora et al., 1999). Consistent with this observation, three pyrrole derivatives have been isolated from Lycium Chinese fruits (Chin et al., 2003). On the other hand, garlic greening is a major concern during garlic processing. Despite having been studied for about 50 years, and many methods have been found to prevent this phenomenon such as heat, the mechanism of garlic greening is still poorly understood (Rejano et al., 2004). Recently, two pyrrole derivatives, 1-(2¢-methyl-1¢-carboxyl-propyl)-3,4-dimethyl-pyrrole (PP-Val) and 1-(1¢-carboxyl-ethyl)-3,4-dimethyl-pyrrole (PP-Ala), were isolated and identified from a model reaction system consisting of trans-(+)-S-(1-propenyl)l-cysteine sulfoxide (1-PeCSO), alliinase, and either l-valine or l-alanine. This system represents blue–green discolouration that occurs when purees of onion (Allium cepa L.) and garlic (Allium sativum L.) are mixed (Imai et al., 2006). Based on this observation, it was proposed that 1-alkyl-pyrroles might be involved in garlic greening (Imai et al., 2006), however, more evidences were needed to verify this idea. All the above results suggested that pyrrole derivatives seem to be a common component which is distributed in foodstuff. It was proposed that these pyrrole derivatives might act as a natural antioxidant against oxidative stress (Zamora et al., 1997, 1999). However, so far, there are few reports on the relationship between structure and antioxidative activity of organic compounds, especially pyrrole derivatives. In the present study, a series of 1-alkyl-pyrroles were synthesised according to a published method to study effects of their side chain size on ‘Laba’ garlic greening and antioxidative activity (Gloede et al., 1968; Sircar et al., 1993). Materials and methods

Chemicals

Glycine, alanine, valine, leucine, isoleucine, tyrosine, methionine, phenylalanine and pyrrole were obtained from Xinjingke Biotechnological Co. (Beijing, China). 2,5-Dimethoxytretrahydrorufan was purchased from Fluka (Beijing, China). 1,1-Diphenyl-2-picrylhydrizyl (DPPH), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the crystallised diammonium salt form, and horseradish peroxidase were purchased from SigmaAldrich chemical Co. (Beijing, China). Methanol, hydrochloric acid, acetic acid, sodium acetate anhydrous, ethyl acetate, sodium sulfate anhydrous, charcoal, potassium hydroxide and hydrogen peroxide were purchased from Beijing Chemistry Co. (Beijing, China). All solvents ⁄ chemicals used were of analytical grade or purer.

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Instrumentals

Infrared spectra were recorded as KBr disks on a Perkin-Elmer spectrum 100 FT-IR spectrometer (PerkinElmer, Shelton, CT, USA). 1H NMR spectra were performed with a dpx-300 MHZ NMR spectrometer (Brucker BioSpin, Rheinstetten, Germany). DMSO was used as a solvent with tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained by using LC-MS ⁄ MS (Alliance2695 ⁄ Quattro Micro API; Waters Co., Milford, MA, USA), and detection was performed in the negative mode. The optimised MS instrument parameters obtained by the tuning were as follows: source temperature, 110 C; desolvation temperature, 200 C; desolvation gas flow, 450 L h)1 nitrogen; cone gas flow, 50 L h)1; argon collision gas pressure to 2 · 10)3 mbar for MS ⁄ MS. P-Gly and P-Tyr: capillary voltage, 3.08 kV cone voltage, 22.0 V; P-Ala: capillary voltage, 2.55 kV, cone voltage, 17.0 V; P-Val: capillary voltage, 3.22 kV cone voltage, 13 V; P-Leu, P-Ile and P-Met: capillary voltage, 2.90 kV, cone voltage, 22.0 V; P-Phe: capillary voltage, 2.55 kV, cone voltage, 23.0 V. Absorbance measurements were obtained on a UV spectrophotometer (Beijing Pgeneral Instrumental Co., Beijing, China). The determination of the pKa was carried out in ddH2O at 25 C by titration following standard methods (Albert & Serjeant, 1962). General procedure for synthesis of 1-alkyl-pyrroles

Eight compounds 1-(1¢-carboxy-methyl) pyrrole (P-Gly), 1-(1¢-carboxy-ethyl) pyrrole (P-Ala), 1-(2¢hydroxybenzene-1¢-carboxy-ethyl) pyrrole (P-Tyr), 1-(2¢-methyl-1¢-carboxy-propyl) pyrrole (P-Val), 1-(3¢-thiomethyl-1¢-carboxy-propyl) pyrrole (P-Met), 1-(3¢-methyl-1¢-carboxy-butyl) pyrrole (P-Leu), 1-(2¢methyl-1¢-carboxy-butyl) pyrrole (P-Ile), 1-(2¢-phenyl1¢-carboxy-ethyl) pyrrole (P-Phe) were synthesised as previously described with slight modification (Gloede et al., 1968; Sircar et al., 1993). Briefly, sodium acetate anhydrous (2.0 g) and alanine (27.2 mmol) were dissolved in 50 mL of glacial acetic acid. 2,5-Dimethoxytetrahydrofuran (27.2 mmol) was added to the above solution. The mixture was stirred under reflux for 40 min. After its temperature was reduced to room temperature, the mixture was poured into 300 mL of ice water. Resultant solution was then filtered through charcoal. The product was extracted into ethyl acetate, which was washed two times with distilled water to remove residual acetic acid and salt. Then the solvent was evaporated off under vacuum. Potassium hydroxide (10%) was added dropwise until pH 7.0, and then the solution was acidified by hydrochloric acid (2.0 m) to pH in the rage of 1.0–2.0. After keeping at 0–4 C overnight, the solution was filtered. Resulting filtrate was extracted with ethyl acetate again, which was dried

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with sodium sulfate anhydrous. After removing ethyl acetate under vacuum, finial product was kept in a desiccator for later use. P-Gly, P-Val, P-Tyr, P-Phe were prepared with a similar procedure as used for P-Ala except that reflux time was only for 5 min for P-Gly (Gloede et al., 1968). P-Met; P-Leu, P-Ile were prepared under similar conditions as described above except that after the solution was acidified to pH in the range of 1.0–2.0, oil residues appeared, which were separated as final products for future use. The structure of the synthesised compounds was determined by 1H NMR, IR and MS. Preparation of pickling solution of garlic and measurement of UV-vis spectra

The garlic bulbs used in the present study were harvested on May 2007. After cracking newly harvested garlic bulbs, the damaged and small cloves were discarded and the remaining cloves were peeled, rinsed with tap water, and then triple-rinsed with distilled water three times. The cloves were divided into eight equal parts (each containing 30 cloves) and were pickled in acetic acid solutions (50 mL, 5%, v ⁄ v) with pH = 2.0. Five 1-alkyl-pyrroles (P-Gly, P-Ala, P-Val, P-Ile and P-Phe) were added to the above acetic acid solution to make their final concentration as 2.5 mm respectively. Two acetic acid solutions containing either pyrrole (2.5 mm) or pyrrole (2.5 mm) plus corresponding amino acids (2.5 mm) were made as controls. A solution containing 5% acetic acid alone used as another control. All these samples were allowed to stand at room temperature for 10 days. The resulting solution was filtered and placed into a quartz cuvette for UV ⁄ vis spectral measurement. Scavenging DPPH· activity assay

Effects of eight 1-alkyl-pyrroles on scavenging DPPH· were evaluated as previously described (Sa´nchez-Moreno et al., 1998). Basically, 4.8 mL of 1-alkyl-pyrroles or corresponding free amino acids or pyrrole in methanol at different concentrations was mixed with 0.2 mL of DPPH· methanolic solution (1 mm). All of eight 1-alkyl-pyrroles had good solubility in methanol while free amino acids used their saturated concentration because of the limited solubility. After incubation for 30 min in the dark, the absorbance was measured at 517 nm as Asample. In parallel, a solution consisting of 4.8 mL of methanol and 0.2 mL of DPPH· methanolic solution (1 mm) was made and likewise allowed to stand for 30 min in the dark, and its absorbance at 517 nm was also determined as Acontrol. The scavenging activity of all samples was calculated by the equation of scavenging rate (%) = (1 ) Asample ⁄ Acontrol) · 100%.

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Scavenging ABTS· activity assay

The procedure for measuring antioxidant activity of 1alkyl-pyrroles was almost the same as previously reported for ABTS· method (Cano et al., 1998; Ferna´ndez-Pacho´n et al., 2004; Villan˜o et al., 2005). Free radicals were prepared by mixing 1.5 mm ABTS, 15 lm hydrogen peroxide and 0.25 lm peoxidase in 50 mm glycine-HCl buffer (pH 4.5). The mixture contains 30 lm of the ABTS cation radical. After the radical was formed, 4 mL of 1-alkyl-pyrroles or corresponding free amino acids was added to 0.5 mL of ABTS· solution in glycine-HCl buffer (0.5 mL) as samples while 4 mL of glycine-HCl buffer instead of sample solution was also mixed with ABTS· solution (0.5 mL) as control. After both sample and control solution were allowed to stand for 15 min, their absorbances (Asample and Acontrol) were measured on UV-spectrophotometer at 414 nm respectively. Scavenging rate was calculated by the formula of scavenging rate (%) = (1 ) Asample ⁄ Acontrol) · 100%. Because of the limited solubility of P-Phe, phenylalanine, pyrrole and tyrosine in buffer solution they were not measured by this method. In both methods above, all measurements were performed in triplicate, and the radical was prepared freshly and protected against light. In scavenging both DPPH· and ABTS· experiments, EC50 of all the 1-alkyl-pyrroles was also determined, which represents the effective concentration when their scavenging rate equals 50%. Statistics analysis

The data were analysed using the Statistic Analysis System (sas, 2002) package software for the analysis of variance, Duncan’s test, and F-test. All experiments were carried out in triplicate. The significance was established at P < 0.05. Results and discussion

Effects of 1-alkyl-pyrroles on garlic greening

1-Alkyl-pyrroles were prepared according to reported method (Gloede et al., 1968; Sircar et al., 1993), and their structures are outlined in Fig. 1. 1H NMR spectra and MS data of these 1-alkyl-pyrroles were identical to those by Wang et al. (2008), confirming their purity. As the structure of these pyrrole derivatives is similar to that of PP-Val, which was proposed as a pigment precursor for garlic greening (Imai et al., 2006), effect of these pyrrole derivatives on greening of intact garlic cloves (which were newly harvested) was first studied. Intact garlic clove was usually directly used for making ‘Laba’ garlic, a homemade Chinese food product (Bai et al., 2005). If they were pigment precursors, freshly harvested garlic clove would turn green upon addition

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that PP-Val might be involved in garlic and onion discolouration (Imai et al., 2006).

3

4

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Antioxidative activities of 1-alkyl-pyrroles

COOH

1' R = -H(P-Gly); -CH3 (P-Ala); -CH(CH3)2 (P-Val); -CH2CH(CH3)2 (P-Leu); -CH(CH3)CH2CH3 (P-Ile); -CH2CH2SCH3 (P-Met); H2C

(P-Phe)

OH (P-Tyr); H2C

Figure 1 Structures of 1-alkyl-pyrroles.

of these compounds. Indeed, the garlic cloves turned green but to a different extent upon addition of P-Gly, P-Ala, P-Val, P-Ile and P-Phe to their corresponding acetic acid-pickling solutions respectively, and their UV ⁄ vis spectra were displayed in Fig. 2. It was observed that there are two maximal absorptions at ca. 440 and 590 nm significantly appearing after garlic cloves were soaked in acetic acid-pickling solutions containing P-Gly, P-Ala and P-Val while other two 1-alkyl-pyrroles P-Ile and P-Phe turned the garlic cloves green to a much less degree. Consistent with present observation, recent studies showed that the UV-vis absorptions (440 and 590 nm) of pigment(s) responsible for garlic greening (Kubec et al., 2004; Bai et al., 2005; Kubec & Velı´ sˇ ek, 2007). Interestingly, with an increase of R group size located on their side chain, the ability of these derivatives to turn garlic cloves green decreased. In contrast, no colour change was observed when newly harvesting garlic cloves were immersed in acetic acid solution alone or acetic acid solutions containing pyrrole (2.5 mm) and glycine (2.5 mm), these results indicating that 1-alkylpyrroles can act as pigment precursors for ‘Laba’ garlic greening. The present study agrees with the proposal

0.5 P-Gly 0.4 P-Ala

Abs

0.3 P-Val P-Ile

0.2 0.1 P-Phe 0.0

Control 400

450

500

550

600

Wavelength (nm) Figure 2 UV-vis spectra of green color formation.

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650

700

Previous studies showed that 1-alkyl-pyrroles naturally occur in food stuff as a potential antioxidant (Zamora et al., 1999). To confirm this idea, antioxidant activities of eight 1-alkyl-pyrroles were evaluated, and the relationship of the structure–antioxidative activity was also established. Scavenging DPPH· (a stable neutral free radical) is one of the major methods commonly used to evaluate antioxidative activity. The method is based on reduction of alcoholic DPPH· in the presence of a hydrogen-donating antioxidant because of the formation of non-radical form DPPH-H by the reaction (Shon et al., 2003). The free radical scavenging activity against DPPH· of all eight 1-alkyl-pyrroles was found to be dose-dependent (Fig. 3a). To compare their free radical scavenging activities against DPPH·, the effective concentration for 50% scavenging rate (EC50) of all the 1-alkyl-pyrroles was determined from plots of scavenging rate vs. concentration (Fig. 3a), and results were given in Fig. 3b. It was found that these compounds exhibited pronouncedly distinct activity against DPPH·. The most interesting feature in their DPPH·-scavenging activity is that these compounds except for P-Tyr exhibits a good relationship of the structure–activity, namely, the activity correlates with molecular size. With an increase of size of R groups which are in the order of H(P-Gly) < CH3- (P-Ala) < (CH3)2CH- (P-Val)  (P-Met) < CH3CH2CH(CH3)CH3SCH2CH2(P-Ile)  (CH3)2CHCH2- (P-Leu) < phenyl (P-Phe), the compounds showed a gradually decreasing activities against DPPH·. Consequently, of the seven compounds, P-Gly showed the strongest scavenging DPPH· activity because of the smallest side chain size, whose EC50 is 0.791 ± 0.002 mm while P-Phe with the largest size side chain exhibited the weakest activity, and its EC50 is 77.15 ± 2.29 mm. The scavenging DPPH· activity of PGly is almost 100-fold stronger than that of its analogues P-Phe. It was well-known that antioxidants exerted their free radical scavenging activities against DPPH· by donating a hydrogen atom to DPPH·, resulting in quenching of DPPH· by forming DPPH-H (Shon et al., 2003). Our results showed that pyrrole only had a weak-scavenging activity against DPPH· with EC50 as 102 ± 3.12 mm (Fig. 3b) while eight free amino acids except for tyrosine lack antioxidative activities against DPPH· at their saturated concentration (data not shown). This result suggested that DPPH· can extract H-atom from pyrrole ring rather than from the side chain of amino acids. Therefore, it is reasonable to believe that the present 1-alkyl-pyrroles quenched DPPH· through donating

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(a)

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Scavenging rate of DPPH.(%)

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20 10

0 0 0

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4 Samples (mM)

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Samples

Figure 3 DPPH·-scavenging activity of eight synthesised 1-alkyl-pyrroles and pyrrole. (a) Plots of sample concentration vs. scavenging DPPH· rate. (b) EC50 of eight 1-alkyl-pyrroles and pyrrole against DPPH·. Each value represents the average of three independents measurements. Vertical bars represent the standard deviation.

H-atom from their pyrrole ring rather than the R groups located on the side chain to DPPH·. However, based on the fact that all 1-alkyl-pyrroles had much stronger ability to quench DPPH· as compared with pyrrole, it is concluded that the 1-alkyl groups greatly affect the H-donating property of the pyrrole moiety, namely, they enhance the ability of the pyrrole moiety to donate the hydrogen atom to quench DPPH·. As all the 1-alkylpyrroles exhibited quite different scavenging activity against DPPH·, this raises an interesting question of why the different R groups have different effects on the activity. It can be explained from a standpoint of chemical reaction that the larger the size of the R group, the harder DPPH· reaches the pyrrole moiety of 1-alkylpyrrole by collision to extract H-atom. In other words, the R group on the side chain might inhibit DPPH· from obtaining a hydrogen atom from the pyrrole moiety of these 1-alkyl-pyrroles, and the larger the R group, the stronger the inhibition. As a result, P-Gly has the strongest activity against DPPH· while the scavenging activity of P-Phe exhibits the weakest. Above results showed that the size of the side chain of the 1-alkyl-pyrroles have a negative effect on the antioxidative activity. However, from the standpoint of chemistry, if a compound has a stronger electrondonating power, it will exhibit a stronger ability to quench DPPH· in the absence of steric effect. On increasing the size of the side chains from H- (P-Gly), CH3- (P-Ala), (CH3)2CH- (P-Val), CH3SCH2CH2(P-Met), to (CH3)2CHCH2- (P-Leu), the electron-donating power of 1-alkyl-pyrroles increases. Consequently, it would be expected that the ordering of the antioxidative activity of the 1-alkyl-pyrroles follows the sequence P-Gly < P-Ala < P-Val < P-Met < P-Leu, but that

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was not the case. Thus, the steric effect plays a much larger role than electronic effect in determination of the reactivity of the 1-alkyl-pyrroles with DPPH·. P-Tyr possesses the stronger activity against DPPH· than P-Val, P-Met, P-Ile, P-Leu and P-Phe although its side chain has the largest size among its all analogous compounds. The most possible reason for the stronger activity of P-Tyr comes from its phenolic hydroxyl group of Tyr with a pKa value of 10.07 (Trudy & James, 2001). A high pKa value of the phenolic hydroxyl indicates that proton is hard to be dissociated from )OH under the present experimental condition, so the phenolic hydroxyl group of P-Tyr easily donates its H atom from )OH to quench DPPH·. Consistent with this idea, it had been reported that hydrogen atom of the phenolic hydroxyl group could donate to stabilise the DPPH free radicals (Villan˜o et al., 2005; Torres et al., 2007). It is reasonable to believe that the ability of the phenolic hydroxyl group to quench DPPH· is larger than that of pyrrole moiety; otherwise, P-Tyr would exhibit a lower antioxidative activity as compared with its analogues P-Val, P-Met, P-Ile, P-Leu and P-Phe because of its larger side chain size. On the other hand, the activity of P-Tyr against DPPH· is lower than those of both P-Gly and P-Ala, a result further indicating that its activity is a balance between the inhibition caused by the bulkyl structure of its side chain for interaction with DPPH· and the H-donating activity contributed by its phenolic hydroxyl group. To confirm above conclusion, the antioxidant activity of the seven 1-alkyl-pyrroles including P-Gly, P-Ala, P-Val, P-Met, P-Tyr, P-Ile and P-Leu and corresponding free amino acids was further evaluated by the ABTS· assay, and results were shown in Fig. 4. In the absence of antioxidants, ABTS· is rather stable, but it reacts

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Antioxidant activity of 1-alkyl-pyrroles D. Wang et al.

(b) 100

P-Gly

8

P-Ala

7

80

P-Tyr

60

P-Met

P-Val

40 P-Ile

20

P-Leu

. EC50 for ABTS (mM)

Scavenging rate of ABTS. (%)

(a)

6 5 4 3 2 1

0

0 0

2

4 Samples (mM)

6

8

P-Gly P-Ala P-Tyr P-Met P-Val Samples

P-Ile P-Leu

Figure 4 ABTS·-scavenging activity of seven synthesised 1-alkyl-pyrroles. (a) Plots of sample concentration vs. scavenging ABTS· rate. (b) EC50

of seven 1-alkyl-pyrroles against ABTS·. Each value represents the average of three independents measurements. Vertical bars represent the standard deviation.

energetically with an H-atom donor and gets converted into its non-coloured form (Roginsky & Lissi, 2005). The limited solubility in buffer solution excluded P-Phe, phenylalanine, pyrrole to be measured by this method. Agreeing with the above observation with DPPH· method, the seven 1-alkyl-pyrroles also exhibited free radical scavenging activity against ABTS·, and their activity was also dose-dependent (Fig. 4a) while corresponding free amino acids except for tyrosine didn’t have the ability to quench ABTS free radical even at concentrations of 100 mm (data not shown). Again, except for P-Tyr, the six 1-alkyl-pyrroles exhibited a very good relationship between their structure and activity, namely, with increasing the size of the R groups on the side chain the free radical scavenging activity against ABTS· of the 1-alkyl-pyrroles generally decreased (Fig. 4b). Consequently, P-Gly with the smallest side chain exhibited the strongest activity against ABTS· with EC50 as 0.831 ± 0.003 mm among them whereas P-Leu possessed the lowest activity with EC50 as 7.44 ± 0.24 mm because of the inhibition caused by its side chain for interaction with ABTS·. This result is in good agreement with the above observation with DPPH· (Fig. 4b). Likewise, of the seven 1-alkyl-pyrroles, the scavenging activity against ABTS· of P-Tyr ranked as third, confirming the above results obtained with DPPH· assay (Fig. 3b). It was worthy noting that the 1-alkyl-pyrroles generally have stronger ability to quench ABTS· than DPPH· as showed in Figs 3b and 4b. This can be explained from a standpoint of the structure of both ABTS· and 1-alkylpyrroles. All the 1-alkyl-pyrroles were weak monocarboxylic acids, which can be considered as analogues of acetic acid. Indeed, our results showed that their pKa are in the range 3.42–3.95 (Table 1), which is lower than

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Table 1 pKa of 1-alkyl-pyrroles Compounds

pKa at 25 °C

P-Gly P-Ala P-Tyr P-Met P-Val P-Ile P-Leu P-Phe

3.55 3.65 3.95 3.68 3.91 3.42 3.45 3.55

± ± ± ± ± ± ± ±

0.023 0.074 0.088 0.055 0.088 0.051 0.024 0.023

that of acetic acid (4.75), indicating that they are stronger acids than acetic acid. The calculation according to the Henderson–Hasselbach equation (eqn 1), indicated that most (>80%) of these 1-alkyl-pyrroles (monocarboxylic acids) exist in their dissociated forms in buffer solution at pH 4.5, which was used for ABTS· assay. In eqn 1 A- and HA are the dissociated and undissociated species respectively. It has been wellestablished that ABTS· is a stable cation radical while DPPH· is a neutral radical without taking any charge. pH ¼ pKa þlog½A =½HA

ð1Þ

Thus, their dissociated forms of the 1-alkyl-pyrroles facilitate their binding to ABTS· but have no influence on their binding to DPPH·. Conclusions

The present study indicate that 1-alkyl-pyrroles might be the precursors which were involved in ‘Laba’ garlic greening. These compounds exhibited a promising

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relationship of the structure–activity, namely, with increasing the size of side chain their free radical scavenging activities against DPPH· and ABTS· gradually decreased. The mechanism by which the 1-alkylpyrroles donate H-atom from their pyrrole moiety to quench both radicals was also demonstrated. The 1alkyl-pyrroles generally exhibited stronger scavenging activity against ABTS· than DPPH. Acknowledgments

This project was supported by the National Natural Science Foundation of China (30570181) and the Ministry of Education of the People’s Republic of China, Specialised Research Fund for the Doctoral Program of Higher Education (20070019004). References Albert, A. & Serjeant, E.P. (1962). Ionization Constants of Acids and Bases. London: Methuen. Bai, B., Chen, F., Wang, Z., Liao, X., Zhao, G. & Hu, X. (2005). Mechanism of the greening color formation of ‘‘Laba’’ garlic, a traditional homemade Chinese food product. Journal of Agricultural and Food Chemistry, 53, 7103–7107. Bellina, F. & Rossi, R. (2006). Synthesis and biological activity of pyrrole, pyrroline and pyrrolidine derivatives with two aryl groups on adjacent positions. Tetrahedron, 62, 7213–7256. Cano, A., Herna´ndez-Ruı´ z, J., Garcı´ a-Ca´novas, F., Acosta, M. & Arnao, M.B. (1998). An end-point method for estimation of the total antioxidant activity in plant material. Phytochemical Aanalysis, 9, 196–202. Chin, Y.-W., Lim, S.W., Kim, S.-H. et al. (2003). Hepatoprotective pyrrole derivatives of Lycium chinense fruits. Bioorganic Medicinal Chemistry Letters, 13, 79–81. Darley-Usmar, V. & Halliwell, B. (1996). Blood radicals: relative nitrogen species, relativeoxygen species, transition metal ions and the vascular system. Pharmaceutical Research, 13, 649–662. Ferna´ndez-Pacho´n, M.S., Villan˜o, D., Garcı´ a-Parrilla, M.C. & Troncoso, A.M. (2004). Antioxidant activity of wines and relation with their polyphenolic composition. Analytica Chimica Acta, 513, 113–118. Gloede, J., Podusˇ ka, K., Gross, H. & Rudinger, J. (1968). Amino acids and peptides. LXXIX. a-Pyrrolo analogues of a-amino acids. Collection Czechoslov. Chemical Communications, 33, 1307–1314. Halliwell, B. (1996). Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Radical Research, 25, 57–74. Hidalgo, F.J. & Zamora, R. (1995). Characterization of the products formed during microwave irradiation of the nonenzymatic browning lysine ⁄ (E)-4,5-Epoxy-(E)-2-heptenal model system. Journal of Agricultural and Food Chemistry, 43, 1023–1028. Hidalgo, F.J., Alaiz, M. & Zamora, R. (1998). A spectrophotometric method for the determination of proteins damaged by oxidized lipids. Analytical Biochemistry, 262, 129–136.

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Huang, D., Ou, B. & Prior, R.L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856. Imai, S., Akita, K., Tomotake, M. & Sawada, H. (2006). Identification of two novel pigment precursors and a reddish-purple pigment involved in the blue-green discoloration of onion and garlic. Journal of Agricultural and Food Chemistry, 54, 843–847. Kubec, R. & Velı´ sˇ ek, J. (2007). Allium discoloration: the color-forming potential of individual thiosulfinates and amino acids: structural requirements for the color-developing precursors. Journal of Agricultural and Food Chemistry, 55, 3491–3497. Kubec, R., Hrba´cˇova´, M., Musah, R.A. & Velı´ sˇ ek, J. (2004). Allium discoloration: precursors involved in onion pinking and garlic greening. Journal of Agricultural and Food Chemistry, 52, 5089–5094. Rejano, L., Castro, A.D., Sa´nchez, A.H., Casado, F.J. & Montan˜o, A. (2004). Thermal kinetics of pungency loss in relation to the quality of pickled garlic. International Journal of Food Science and Technology, 39, 311–317. Roginsky, V. & Lissi, E.A. (2005). Review of methods to determine chain-breaking antioxidant activity in food. Food Chemistry, 92, 235–254. Sa´nchez-Moreno, C., Larrauri, J.A. & Saura-Calixto, F. (1998). A procedure to measure the antiradical efficiency of polyphenols. Journal of the Science of Food and Agriculture, 76, 270–276. Shon, M.Y., Kim, T.H. & Sung, N.J. (2003). Antioxidants and free radical scavenging activity of Phellinus baumii (Phellinus of Hymenochaetaceae) extracts. Food Chemistry, 82, 593–597. Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Experimental Physiology, 82, 291–295. Sircar, I., Winters, R.T., Quin, J. III, Lu, G.H., Major, T.C. & Panek, R.L. (1993). Nonpeptide angiotensin II receptor antagonists. 1. Synthesis and in vitro structure-activity relationships of 4-[[[(1Hpyrrol-1-ylacetyl) amino] phenyl] methyl] imidazole derivatives as angiotensin II receptor antagonists. Journal of Medicinal and Chemistry, 36, 1735–1745. Torres, P., Pen˜alver, P. & Morales, J.C. (2007). Synthesis and evaluation of new phenolic-based antioxidants: structure-activity relationship. Food Chemistry, 103, 55–61. Trudy, M. & James, R.M. (2001). Biochemistry: An Introduction, (Second edition) [M]. McGraw-Hill Companies, China Science Press, Inc. Pp 86. Villan˜o, D., Ferna´ndez-Pacho´n, M.S., Troncoso, A.M. & Garcı´ aParrilla, M.C. (2005). Comparison of antioxidant activity of wine phenolic compounds and metabolites in vitro. Analytical Chimica Acta, 538, 391–398. Wang, D., Husile, N., Han, N., Chen, F. & Zhao, G. (2008). 2-(1HPyrrolyl)carboxylic acids as pigment precursors in garlic greening. Journal of Agricultural and Food Chemistry, 56, 1495–1500. Zamora, R. & Hidalgo, F.J. (1995). Linoleic acid oxidation in the presence of amino compounds produces pyrroles by carbonyl amine reactions. Biochimica et Biophysica Acta, 1258, 319–327. Zamora, R., Alaiz, M. & Hidalgo, F.J. (1997). Feed-back inhibition of oxidative stress by oxidized lipid ⁄ amino acid reaction products. Biochemistry, 36, 15765–15771. Zamora, R., Alaiz, M. & Hidalgo, F.J. (1999). Determination of e-Npyrrolylnorleucine in fresh food products. Journal of Agricultural and Food Chemistry, 47, 1942–1947.

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International Journal of Food Science and Technology 2008, 43, 1887–1895

Original article Comparative study on composition and antioxidant properties of mint and black tea extract Ekambaram Padmini,* Krishnan Prema, Bose Vijaya Geetha & Munuswamy Usha Rani P.G. Department of Biochemistry, Bharathi Women’s College, Affiliated to University of Madras, Chennai 600108, Tamilnadu, India (Received 26 September 2007; Accepted in revised form 9 May 2008)

Summary

The antioxidant properties of plants could be correlated with oxidative stress defence in different human diseases. The present study was undertaken to evaluate and compare the antioxidant potential and the phytochemical composition in the aqueous extracts of mint leaves, black tea and black tea enriched with mint extract. All the three preparations exhibited free radical-scavenging potential for nitric oxide (NO) radical, superoxide anion radical and hydroxyl radical, and the values were lesser than those of the antioxidants which acted as standards. In comparison, the mint extract exhibited higher free radical and NO scavenging effect. Hydroxyl radical and superoxide scavenging effects were more pronounced in tea with the mint extract, while the reducing power was exhibited more significantly by the black tea extract. The phytochemical compounds were identified and the total phenols and flavonoids were quantified and compared between these extracts.

Keywords

Antioxidant, black tea, flavonoids, free radicals, Mentha spicata, polyphenols.

Introduction

Oxidative stress is a central risk in gaining recognition as a key phenomenon in chronic diseases. It plays an important role in the pathogenesis of various diseases, like atherosclerosis, alcoholic liver cirrhosis, cancer and pre-eclampsia (Maxwell & Lip, 1997; Padmini & Sowmya, 2005). The condition of oxidative stress is initiated by reactive oxygen species (ROS), which can easily initiate the lipid peroxidation of the membrane lipids, like phospholipids, and lipoproteins by propagating a reaction chain thereby causing damage to the cell membrane (Staniek & Nobl, 1999). Thus, to counteract the oxidative damage from ROS, antioxidant defence systems have coevolved with aerobic metabolism. Diet plays a major role in environmental control of oxidative stress. The natural diet, like fruits, vegetables and red wine, can decrease oxidative stress (Leighton et al., 1999). Recent investigations have shown that the antioxidant properties of plants could be correlated with oxidative stress defence (Muchuweti et al., 2006; Zhu et al., 2006). In this respect, flavonoids and other polyphenolic compounds that are widely distributed in plants have received the greatest *Correspondent: Fax: +91 44 25280473; e-mail: [email protected] and [email protected]

attention as free radical scavengers, inhibitors of lipid peroxidation in addition to metal chelators (Staniek & Nobl, 1999). The leaves of the mint plant Mentha spicata have a pleasant, warm, fresh, aromatic, sweet flavour with a cool aftertaste. It is carminative, stimulative, stomachic, diaphoretic and antispasmodic. The extract of mint has long been used for digestive purposes and for relief from heavy colds. Mint extract also possesses polyphenolic and flavonoid contents, and hence, the antioxidant property (Kanatt et al., 2007). Similarily, the black tea constituents, like polyphenols, theaflavins and gallate esters, have been proposed to have antioxidant property and decrease the risk of heart disease, cancer and hypertension (Miller et al., 1996). Nagchavduri et al. (2003–2004) have also demonstrated that black tea pretreatment leads to the significant restoration of superoxide dismutase (SOD) and catalase, enzyme activity in gastric and liver tissue following ethanol-induced oxidative stress. Our previous study had reported that black tea extract enriched with mint is more efficient in protecting small dense lowdensity lipoprotein (LDL) against lipid peroxidation (Padmini & Geetha, 2007). The current study involves the evaluation and comparison of the antioxidant potential and the phytochemical composition of the aqueous extracts of mint, black tea and black tea enriched with mint extract. The

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present work will also investigate the relation between the biological properties of the active constituents with their antioxidant potential.

Measurements were accomplished using 10–50 lL volume of the sample and assayed in triplicates. Antioxidant potential assays

Materials and methods

Chemicals

Rutin was obtained from Acros Organics (New Jersey, USA). 1,1-diphenyl-2-picryl hydrazyl (DPPH) was purchased from Himedia, India. Nitro blue tetrazolium (NBT), nicotinamide adenine dinucleotide phosphate reduced (NADH), phenazine methosulphate (PMS), trichloro acetic acid (TCA), thiobarbituric acid (TBA), ethylene diamine tetra acetic acid (EDTA), hydrogen peroxide (H2O2), ferric chloride and butylated hydroxy toluene (BHT) were obtained from SRL Chemicals (Mumbai, India). Ascorbic acid and vitamin E were obtained from SD Fine Chem. Ltd. (Biosar, India). Naphthyl ethylene diamine dihydrochloride was obtained from Rochlight Ltd. (Suffolk, UK). Sodium nitro prusside was obtained from Ranbaxy Lab (Mohali, India). Potassium ferric cyanide was obtained from May and Backer (Dagenham, UK). 2-Deoxy-2-ribose was obtained from Fluka (Buchs, Switzerland). Preparation of mint extract

Mint was purchased from the local market. The leaves were separated and washed under tap water. About 100 g of mint leaves were refluxed using 1000 mL of distilled water. The filtrate was separated and further filtered using Whatman No. 4 filter paper. The filtered solution was diluted (1:100) with distilled water. Measurements were accomplished using 10–50 lL volume of the sample and assayed in triplicates. For phytochemical analysis, the mint leaves were air dried for 10 days, milled into powder and stored in airtight bottles till analysis. Preparation of black tea extract

About 10 g of commercially available South Indian black tea leaves were brewed and extracted in 100 mL of distilled water, and the temperature of water was kept below 80 C while brewing. The mixture was decanted and filtered using Whatman No. 2 filter paper. The resulting filtrate was diluted (1:100) with distilled water. Measurements were accomplished using 10–50 lL volume of the sample and assayed in triplicates. Preparation of black tea extract with mint

The prepared mint extract and black tea extract were taken in the ratio 1:1, mixed well for 5 min and filtered.

International Journal of Food Science and Technology 2008

DPPH radical-scavenging activity

The free radical-scavenging capacity of the extract was determined using DPPH. One millilitre of methanol DPPH solution (0.15 mm) was mixed with serial dilutions (10–50 lg) of mint extract, black tea extract and black tea extract with mint. After 10 min of incubation, the absorbance was read at 515 nm using a spectrophotometer (Perkin-Elmer Lambda 20 UV-visible spectrophotometer; Perkin-Elmer UV-visible spectrophotometer, Boston, MA, USA). Vitamin C was used as a standard. The inhibition curve was plotted and comparison was made for the black tea extract, the mint extract and black tea extract with mint (Viturro et al., 1999). Nitric oxide (NO) radical inhibition assay

The NO radical inhibition was estimated using Griess Illosvoy reaction (Garrat, 1964) with some modifications using naphthyl ethylene diamine dihydrochloride (0.1% w ⁄ v) and sulfanilic acid reagent (0.33% in 20% glacial acetic acid). The absorbance of the pink chromophore formed was measured at 540 nm against the corresponding blank solution. Rutin was used as a standard. Superoxide anion-scavenging activity

Measurement of superoxide anion-scavenging activity of three extracts was done based on the method of Nishimiki et al. (1972) using nitroblue tetrazolium (NBT) solution and PMS. The absorbance at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased superoxide anion-scavenging activity. Curcumin was used as a positive control. Hydroxyl radical-scavenging assay

The assay was performed by the method described by Halliwell et al. (1987) using 2-deoxy-2-ribose with minor changes. After an incubation period of 1 h at 37 C, the extent of deoxyribose degradation was measured by the TBA reaction (Nishimiki et al., 1972; Bouchet et al., 1998). The absorbance was measured at about 532 nm against the blank solution. Vitamin E was used as a positive control. Reducing power

The reducing power of the three extracts was determined according to the method of Oyaizu (1986) using potassium ferri cyanide. The absorbance was measured at 700 nm and the increased absorbance of the reaction mixture indicated increased reducing power. Butylated hydroxy toluene was used as a standard.

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Antioxidant properties of mint and black tea extract E. Padmini et al.

High-performance thin layer chromatography (HPTLC) analysis of tea and mint extracts for phytochemical composition

The mint extract and the black tea extracts were subjected to HPTLC analysis using CAMAG TLC scanner III (Camag, Muttenz, Switzerland) according to the procedure of Vilegas et al. (1998). An aliquot of about 10 lL of suitably diluted mint extract, the black tea extract and black tea extract with mint were applied as a 0.5-cm band on a 2-mm thick silica gel G coated aluminum plate (Merck, Silica Gel GF254, Germany). After that, the plate was developed using toluene ⁄ ethyl acetate ⁄ formic acid ⁄ methanol (30:30:8:2 by volume) as the mobile phase. The developed plate was dried at room temperature and the naturally colored spot was then scanned with a CAMAG TLC Scanner III in the ascending mode at 254 nm coupled with an SP 4100 integrator. Then the qualitative data from the HPTLC chromatogram were compared between the three different extracts. Determination of total phenols Preparation of fat-free sample for total phenol estimation

Two grams of the sample was defatted with 100 mL of diethyl ether using a Soxhlet apparatus for 2 h, and the resulting compound was used for the analysis of total phenols. After the phenolic components of the tea and mint extracts were extracted using ether, the colour was developed using ammonium hydroxide and amylalcohol in these extracts. The absorbance of the solution was read at 505 nm (Obadoni & Ochuko, 2001)

Statistical analysis

The results were expressed as mean value ± SD. The values were subjected to statistical analysis using normal tests of significance. The statistical significance was arrived at by comparing the results of the three different types of extract using Student’s t-test (Sokal & Rohlf, 1994). The spss software package version 7.0 was used to test the significance of the experiments performed. Differences were taken to be statistically significant for values of P < 0.001 and P < 0.01. Results

DPPH radical-scavenging activity

As shown in Fig. 1, the aqueous extract of M. spicata exhibits a significant dose-dependent inhibition of DPPH activity when compared with black tea extract and black tea extract with mint (P < 0.001), with a 50% inhibition (IC50) at a concentration of 50 lg mL)1. The result is mentioned in Fig. 2, and the IC50 value of extract is found to be lesser than the standard, vitamin C (IC50 70 lg mL)1 results not shown). NO radical inhibition assay

The NO scavenging by the aqueous extract of M. spicata is increased significantly (P < 0.001) when compared with other extracts in a dose-dependent manner as illustrated in Fig. 2. At a concentration of 45 lg mL)1 of the extract, 50% of NO generated was scavenged. This IC50 value of the mint extract is found to be lesser than the standard, rutin [IC50 70 lg mL)1 (results not shown). Superoxide anion-scavenging activity

Determination of flavonoids

About 10 g of the plant samples were extracted repeatedly with 100 mL of 80% aqueous methanol at room temperature. The whole solution was filtered through Whatman Filter paper no.42 (125 mm).The filtrate was later transferred into a crucible and evaporated to dryness over a water bath and weighed (Boham & Kocipai, 1994). Aluminium chloride colorimetric method was used for flavonoid determination (Chang et al., 2002). The dried filtrate was separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 m potassium acetate and 2.8 mL of distilled water and kept at room temperature for 30 min. The absorbance of the reaction mixture was then measured at 415 nm with a double beam Perkin Elmer UV ⁄ Visible spectrophotometer. The calibration curve was obtained by preparing quercetin solutions at concentrations 12.5 to 100 lg per mL in methanol.

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The superoxide anion derived from dissolved oxygen by phenazine methosulphate ⁄ NADH-coupling reaction reduces nitro blue tetrazolium. The decrease in the absorbance at 560 nm with the tea and mint extracts thus indicates the consumption of superoxide anion in the reaction mixture. As mentioned in Fig. 3, all the extracts and curcumin showed the scavenging activity, but the superoxide scavenging effect is significantly higher (P < 0.001) in the black tea extract when compared with the mint extract and the tea with mint extracts. IC50 values for the standard and the various extracts are 20 and 30 lg ml)1, respectively. Hydroxyl radical-scavenging assay

To attack the substrate, deoxyribose hydroxyl radicals were generated by the reaction of ferric and EDTA together with H2O2 and ascorbic acid. When the tea and mint extracts were incubated with the aforementioned

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Figure 1 Scavenging effect of aqueous extracts of mint extract, black tea extract, black tea with mint extract and standard vitamin C on 1,1¢-diphenyl-2-picryl hydrazyl radical. Results are mean ± SD of five parallel measurements.

Figure 2 Scavenging effect of aqueous extracts of mint extract, black tea extract, black tea with mint extract and standard rutin on nitric oxide radical. Results are mean ± SD of five parallel measurements.

Figure 3 Scavenging effect of aqueous extracts of mint extract, black tea extract, black tea with mint extract and curcumin on the scavenging of superoxide anion radical formation. Results are mean ± SD of five parallel measurements.

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Antioxidant properties of mint and black tea extract E. Padmini et al.

reaction mixture, it could prevent the damage against sugar. In Fig. 4, the concentrations of 50% inhibition are found to be 22, 34 and 48 lg mL)1 for the mint extract, black tea extract and mint-enriched black tea extract. The hydroxyl radical-scavenging effect for the tea enriched with mint extract is highly significant (P < 0.001) when compared with tea and mint extracts, and the inhibition value is found to be lesser than the standard. Reducing power

Figure 5 shows the reductive capabilities of the tea and the mint extracts when compared with butylated hydroxy toluene. The reducing power of these extracts is very potent and the power of the extract can reduce most Fe3+ ions, which have a lesser reductive activity than the standard of butylated hydroxy toluene. The black tea extract is shown to have significant reducing power (P < 0.001) when compared with tea and tea with mint extracts.

HPTLC analysis of the phytochemical constituents

As shown in Fig. 6a, the HPTLC fingerprint of black tea extract shows nine peaks at various Rf values 0.06, 0.15, 0.21, 0.33, 0.44, 0.53, 0.61, 0.76 and 0.86. The peak at the Rf 0.44 has major peak area. The major peak area compounds belong to the polyphenol family and flavonoid compounds. Figure 6b shows the HPTLC fingerprint of aqueous extract of mint, which exhibits five peaks in this chromatographic condition, the peaks corresponding to the Rf values 0.42 and 0.68 having major peak area. The major peak area compounds belong to the alkaloids, phenolic anthraquinones and quinine compounds. Figure 6c shows the HPTLC chromatogram of phytochemicals from the aqueous extract of tea with the mint extract which exhibits nine peaks in this chromatographic condition, the peaks corresponding to the Rf values 0.44 and 0.61 having major peak area which represents the polyphenolic compounds.

Figure 4 Scavenging effect of aqueous extracts of mint extract, black tea extract, black tea with mint extract and vitamin E on deoxyribose degradation assay. Results are mean ± SD of five parallel measurements.

Figure 5 The reductive ability of aqueous extracts of mint extract, black tea extract, black tea with mint extract and butylated hydroxytoluene. Results are mean ± SD of five parallel measurements.

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(a) 1000 900 800 700 [AU]

600

5 7

500 400 4

300 200

1

100 0 –0.07

2

6

3

0.13

9

8 0.33

0.72

0.53 Rf

0.93

1.13

(b) 200

[AU]

150

100 3

4

2

1

50

5

0 –0.07

0.13

0.33

0.53

0.72

0.93

Rf

(c) 1000 900 800 700 600 [AU]

1892

5 7

500 400 4

300 200 100 0 –0.07

1

2

0.13

8 9

6

3

0.33

0.53 Rf

0.72

Phytochemical composition analysis and quantification of total phenols and flavonoids

Table 1 shows the phytochemical analysis data of the black tea and the aqueous extract of mint. The compounds like phenolic compounds and anthraqui-

International Journal of Food Science and Technology 2008

0.93

1.13

Figure 6 High-performance thin layer chromatography (HPTLC) chromatogram of phytochemicals from the aqueous extract of black tea. (a) HPTLC chromatogram of phytochemicals from the aqueous extract of mint (Mentha spicata) leaves. (b) HPTLC chromatogram of phytochemicals from the aqueous extract of black tea with the mint extract.

nones are found to be present in higher levels in the black tea extract when compared with mint extract. The presence of flavonoids are found to be in higher concentration in the black tea extract with mint extract when compared with the mint extract and the black tea extracts. The black tea extract shows the presence of

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Antioxidant properties of mint and black tea extract E. Padmini et al.

Table 1 Phytochemical analysis of the black tea extract and the aqueous extract of mint Different types of extracts

S. no.

Phytochemicals

Black tea extract

Mint leaf extract

Black tea with mint extract

1 2 3 4 5 6 7 8 9 10

Steroids Phenolic compounds Reducing sugars Flavonoids Glycosides Saponins Alkaloids Anthraquinones Quinines Tannins

) +++ + + ) + ) ++ + ++

+ ++ + + + + + -

+ +++ ) ++ ) + + ++ + ++

) denotes absent. + denotes present (+ ⁄ ++ ⁄ +++ indicates the qualitative strength of the contents).

Table 2 Phytochemical composition of the aqueous extract of mint leaves, black tea extract and the black tea extract enriched with mint extracts expressed as mg per 100 g dry weight Different types of extracts

S. no.

Phytochemicals

Black tea extract

Mint leaf extract

Black tea with mint extract

1 2

Total phenols Flavonoids

1.51 ± 0.12* 1.38 ± 0.06

1.14 ± 0.13 1.59 ± 0.08

1.32 ± 0.09 1.73 ± 0.07*

Results are the mean of triplicate determinations on a dry weight basis ± standard deviation *P < 0.01 is statistically significant when compared between the three extracts.

tannins, but it is absent in the mint extract. The compounds like steroids and alkaloids that are found to be present in the mint extract are absent in the black tea extract. Glycosides are found to be absent in both the extracts. Table 2 summarises the quantitative determination of total phenols and flavonoids of the black tea extract, the aqueous extract of mint and the black tea extract enriched with mint extract. High quantities of total phenols were found in the black tea extract when compared with the mint extract. The flavonoids were present in high concentration in the black tea extract enriched with mint extract when compared with tea and mint extracts (P < 0.01). Discussion

Oxidative stress is the situation that ensues when the delicate balance between the production of ROS and antioxidant defence system is disturbed. As depleted

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endogenous antioxidant defence system is responsible for this situation, the use of antioxidant supplementation in reducing the level of oxidative stress and in slowing or preventing the development of complications associated with diseases is gaining importance (Rose et al., 1982). In the present study, the antioxidant potential present in the aqueous extract of mint, black tea and black tea extract with mint were examined using DPPH radical scavenging, NO radical scavenging, superoxide anion radical scavenging and hydroxyl radical-scavenging assays. Our results demonstrated that the extracts have phytochemical constituents, like flavonoids and polyphenols, which are proven to influence the antioxidant potential of these extracts. The aqueous extract of mint was observed for its maximum DPPH radical-quenching property, which is shown by the significant decrease in absorbance at 516 nm. Following mint, black tea extract with mint and black tea extract showed the DPPH radical-scavenging property in descending order, and hence, our results confirmed the overall protective effect of these extracts. Similarly, mint extract possessed the greater NO radical-scavenging property when compared with other two extracts. NO is one of the important radicals that regulates the function of mitochondria (Valdez et al., 2000), and the overproduction of such radical is involved in pathological cellular mechanism. Therefore, the supplementation of such extracts that are rich in NO radical-scavenging properties can be beneficial under stress situations. It has also been reported that the interaction of NO with polyphenolic antioxidants is highly relevant in physiological and pathological cellular mechanism (Moriel et al., 2000). In the PMS ⁄ NADH–NBT system, superoxide anion derived from dissolved oxygen by PMS ⁄ NADH coupling reaction reduces NBT. The decreased absorbance at 560 nm in the presence of antioxidants indicates the consumption of superoxide anion in the reaction mixture. Hence, we report an observed greater superoxide anion-scavenging property for black tea extract enriched with mint followed by mint extract and black tea extracts. In hydroxyl-scavenging assay, the use of Fe3+ in the presence of a reducing agent, such as ascorbate, produces hydroxyl ion which degrade 2-deoxy-2-ribose into fragments that on heating with TBA at low pH form a pink chromogen that shows absorbance at 532 nm (Aruoma et al., 1989; Aruoma, 1996). However, when a similar reaction was carried out in the presence of aqueous extract of mint, tea, black tea extract with mint or vitamin E, the production of hydroxyl radicals was inhibited with decrease in absorbance at 532 nm. Although all the three aqueous extracts exhibited strong scavenging effect of hydroxyl radicals, the black tea extract enriched with mint exhibited excellent hydroxyl

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radical-scavenging activity when compared with mint extract and black tea extract. For the measurements of the reductive ability, the Fe3+ to Fe2+ transformation was investigated in the presence of hydroalcoholic extract (Oyaizu, 1986). The reducing power was increased by increasing the amount of extract, and the reducing capacity of compound may serve as a significant indicator of its potential antioxidant activity (Meir et al., 1995). The presence of phenolic compounds as confirmed by phytochemical analysis may also contribute directly to the reducing capacity and hence the antioxidative action of extracts. In this investigation, we observed that black tea extract possessed higher reducing activity when compared with mint and tea enriched with mint extracts. Tea remains the most consumed drink in the world, and an accumulated number of population studies suggest that the consumption of green and black tea may bring positive health effects (Riemersma et al., 2001). One hypothesis explaining such effect is that the high levels of polyphenols and flavonoids in tea that can protect cells and tissues from oxidative damage by scavenging oxygen free radicals may therefore be active as antioxidants (Knekt et al., 1996). The radical-scavenging activity of these tea and mint extracts are mainly attributed to the presence of phenolic compounds in which the free hydroxyl group is mainly responsible for antioxidant activity (Weng & Wang, 2000; Cakir et al., 2003). The polyphenols are known to reduce the formation of free radicals by scavenging or chelating iron and copper and preventing them from participating in the Fenton reaction (Van Acker et al., 1998). Flavonoids are potent water soluble antioxidants and free radical scavengers which prevent oxidative cell damage and have strong anticancer activity and anti-inflammatory activity (Okwu, 2004). Mint leaves are rich in tocopherols, polyphenols and have a high content of provitamin A activity, which is not lost during boiling process (Bicudo et al., 2000). Hence, the greater antioxidant effect of black tea extract with mint may be attributed to the enrichment of flavonoids from the mint leaves, which is helpful for the protective role in treating diseases. Conclusion

Our study clearly indicates that it is important to measure both the antioxidant activity using various radicals and oxidation systems and the analysis of phytochemical composition into account while evaluating the anti-oxidant potential of the tea and plant extracts. The study also indicates that the tea and the mint extracts when combined may not necessarily exhibit cumulative effect. However, the increased consumption of mint, tea or tea enriched with mint may contribute to the improvement in quality of healthy life by increasing

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the antioxidant defence and delaying the onset of various degenerative diseases caused by oxidative stress. Acknowledgment

The project is funded by National Tea Research Foundation, NTRF: 115 ⁄ 07 Tea Board of India. References Aruoma, O.I. (1996). Characterization of drugs as antioxidant prophylactics. Free Radical Biology and Medicine, 49, 105–107. Aruoma, O.I., Laughton, M.J. & Halliwell, B. (1989). Carnosine, homocarnosine and anserine; could they act as antioxidants in-vivo? Biochemistry Journal, 264, 863–869. Bicudo, L., Muradian, A., Vanderlinde, D.W. & Sasaki, R. (2000). Provitamin activity of raw and cooked brazilian leaves. Cienciae Technologia de Alimentos, 20, 21–27. Boham, A.B. & Kocipai, A.C. (1994). Flavonoid and condensed tannins from leaves of Hawaiian vaccinium, vaticulum and vicalycinium. Pacific Science, 48, 458–463. Bouchet, N., Barrier, L. & Fauconneau, B. (1998). Radical scavenging activity and antioxidant properties of tannins from Guiera senegalensis (Combretaceae). Phytotherapy Research, 12, 159–162. Cakir, A., Mavi, A., Yildirim, A., Duru, M.E., Harmandar, M. & Kazaz, C. (2003). Isolation and characterization of antioxidant phenolic compounds from the aerial parts of Hypericum hyssopifoliump (L) by activity guided fractionation. Journal of Ethnopharmacology, 87, 73–83. Chang, C., Yang, M., Wen, H. & Chern, J. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food Drug Analysis, 10, 178–182. Garrat, D.C. (1964). The Quantitative Analysis of Drugs. 3rd edn. Pp. 456–458. Japan: Chapman and Hall Ltd. Halliwell, B., Gutteridge, J.M. & Aruoma, O.I. (1987). The deoxyribose method: a simple ‘test tube’ assay for determination of rate contents for reaction of hydroxyl radicals. Analytical Biochemistry, 165, 215–219. Kanatt, S.R., Chander, R. & Sharma, A. (2007). Antioxidant potential of mint (Mentha spicata L.) in radiation-processed lamb meat. Food Chemistry, 100, 451–458. Knekt, P., Jarvinen, R., Reunanen, A. & Maatela, J. (1996). Flavanoid intake and coronary mortality in Finland: a cohort study. British Medical Journal, 312, 478–481. Leighton, F., Cuevas, A., Gusasch, V. et al. (1999). Plasma polyphenols and antioxidants, oxidative DNA damage and endothelial function in a diet and wine intervention study in humans. Drugs Experimental and Clinical Research, 25, 133–141. Maxwell, S.R. & Lip, G.Y. (1997). Free radicals and antioxidants in cardiovascular disease. British Journal of Clinical pharmacology, 44, 307–317. Meir, S., Kanner, J., Akiri, B. & Hadas, S.P. (1995). Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. Journal of Agriculture and Food Chemistry, 43, 1813–1817. Miller, N.J., Castelluccio, C., Tijiburg, L. & Rice-Evans, C. (1996). The antioxidant properties of theoflavins and their gallate esterradical scavengers or metal chelators. FEBS Letters, 392, 40–44. Moriel, P., Plavnik, F.L., Zanella, M.T., Bertolami, M.C. & Abdalla, D.S.P. (2000). Lipid peroxidation and antioxidants in hyperlipidemia and hypertension. Biological Research, 33, 105–112. Muchuweti, M., Nyamukonda, L., Chagonda, L.S., Ndhlala, A.R., Mupure, C. & Benhura, M. (2006). Total phenolic content and antioxidant activity in selected medicinal plants of Zimbabwe. International Journal of Food Science and Technology, 41, 33–38.

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Nagchavduri, A.K., Sen, T. & Roy, D.K. (2003–2004). Pharmacological evaluation of the medicinal properties of Indian black tea, Annual Society Report, Kolkata, India: National Tea Research Foundation. Nishimiki, M., Appaji, N. & Yagi, K. (1972). The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochemistry and Biophysics Research Communication, 46, 849–854. Obadoni, B.O. & Ochuko, P.O. (2001). Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta States of Nigeria. Global Journal of Pure and Applied Science, 8, 203–208. Okwu, D.E. (2004). Phytochemicals and vitamin content of indegenious spices of south eastern Nigeria. Journal of Sustainability Agriculture Environment, 6, 30–37. Oyaizu, M. (1986). Studies on product of browning react prepared from glucose amine. Japanese Journal of Nutrition, 44, 307–315. Padmini, E. & Geetha, V.B. (2007). In vitro studies on the effect of black tea extracts on the small dense LDL oxidation in Preeclampsia. Biomedicine, 27, 168–172. Padmini, E. & Sowmya, S. (2005). Atherosis: a major event in the pathophysiology of preeclampsia. Biomedicine, 25, 6–12. Riemersma, R.A., Rice- Evans, C.A., Tyrrell, R.M., Clifford, M.B. & Lean, M.E.J. (2001). Tea flavonoids and cardiovascular health. Quaterly Journal of Medicine, 94, 277–282. Rose, W.M., Creigton, M.O., Stewart, D.H.P.J., Sanwal, M. & Trevithick, G.R. (1982). In vivo effects of vitamin E on cataractgenesis in diabetic rats. Canadian Journal of Ophthalmology, 17, 61–66.

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Sokal, R.R. & Rohlf, F.J. (1994). Biometry, 3rd edn. San Francisco: W.H. Freeman. Staniek, K. & Nobl, H. (1999). H2O2 detection from intact mitochondria as a measure for one electron reduction of dioxygen requires a non-invasive assay system. Biochimica Biophysica Acta, 1413, 70–80. Valdez, L.B., Arnaiz, S.L., Bustamante, J., Alvarez, S., Costa, L.E. & Boveris, A. (2000). Free radical chemistry in biological systems. Biological Research, 33, 65–70. Van Acker, S., Van Balen, G., Van den Berg, D., Bast, A. & Van der Vijh, W. (1998). Influence of iron chelation on the antioxidant activity of flavonoids. Biochemistry and Pharmacology, 56, 35–943. Vilegas, J.H.Y., Lancas, F.M., Wauters, J.N. & Angenot, L. (1998). Characterization of adulteration of ‘Espinheira santa’ (Maytenus ilicifolia and Maytenus aquifolium celastraceae) hydroalcoholic extracts with Sorocea bomplandii (Moraceae) by high performance thin layer chromatography. Phytochemical Analysis, 9, 263–266. Viturro, C., Molina, M. & Schmeda-Hischmann, G. (1999). Free radical scavengers from Mutisia friesiana (Asteraceae) and Sanicula graveolens (Apiacease). Phytotherapy Research, 13, 422–424. Weng, X.C. & Wang, W. (2000). Antioxidant activity of compounds isolated from Salvia plebia. Food Chemistry, 71, 489–493. Zhu, Y.X., Huang, H. & Tu, Y.Y. (2006). A review of recent studies in China on the possible beneficial health effects of tea. International Journal of Food Science and Technology, 41, 333–340.

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Original article The effect of shape, blanching methods and flour on characteristics of restructured sweetpotato stick Joko S. Utomo,1 Yaakob B. Che Man,1* Russly A. Rahman1 & Mohd. Said Saad2 1 Department of Food Technology, Faculty of Food science and Technology, University Putra Malaysia, 434400 UPM Serdang, Selangor DE, Malaysia 2 Department of Crops Science, Faculty of Agriculture, University Putra Malaysia, 434400 UPM Serdang, Selangor DE, Malaysia (Received 9 March 2005; Accepted in revised form 15 July 2005)

Summary

The blanching methods and controlling dry matter content used in the preparation of mashed sweetpotato (SP) for producing restructured sweetpotato stick (RSS) were studied. The two shapes of trimmed tuber were sliced and diced. Blanching was conducted by (i) boiling for 2 min in water at 100 C and (ii) blanching in water at 100 C containing 1% (w ⁄ v) sodium tripolyphosphate (STP) for 2 min. Controlling the dry matter content of dough was conducted by adding SP flour (0%, 5%, 10%, 15% and 20%). Chemical and physical characteristics were determined on the final product. The results from this study showed that blanching in 1% STP for 2 min improves colour and ash content of fried stick. Mixing of 5% SP flour with mashed SP produces suitable dough for further processing. RSS can be produced by a combination of blanching in 1% STP and mixing with 5% SP flour produce an intermediate hardness, high lightness and low redness colouration of RSS.

Keywords

Blanching, chemical evaluation, colour, hardness, sweetpotato flour, sweetpotato-stick.

Introduction

Sweetpotato (SP) is an important food crop in the world, especially in developing countries of the tropics and sub-tropics. It is well known that SP not only provide energy, but also an excellent source of provitamin A and vitamin C, minerals, dietary fibre and protein (Edmond & Ammerman, 1971; Lanier & Sistrunk, 1979; Picha, 1985). Despite these properties, SP is not a welldeveloped food item. Traditionally, SP is processed using basic conventional methods of cooking, such as baking, boiling, steaming and frying. The popular frying products of SP are French friedtype products or sticks and chips. SP French fries and chips were judged to possess both good quality and acceptability by a consumer panel (Hoover & Miller, 1973; Walter & Hoover, 1986 and Schwartz et al., 1987). Texture is one of the important attributes of the product. Walter et al. (1992, 1993) controlled the firmness of fries through managing the pH of SP tissue using acid and base media. Many published reports have described pureed SP products (Collins & Walter, 1992). However, there are few accounts of restructured products. Walter & Hoover *Correspondent: Fax: +603-89423552; e-mail: [email protected]

(1984) and Hoover et al. (1983) developed patties SP processed from SP puree. Collins & Washam-Hutsell (1987) introduced ‘vegetable leather’ prepared from SP puree. Che Man (1996) developed SP rounds made from SP patties. Development of texturized SP puree was reported by Truong & Walter (1994) and Truong et al. (1995). Restructured French fries were also developed by Walter et al. (2002) and Sylvia et al. (1997). The preparation of SP foods has several drawbacks. Further difficulties arise as a result of chemical and physical characteristics of the SP such as size, shape, sugar content, solid content, etc. All these variations affect the colour, texture and flavour of the finished products. Discolouration is a major problem to the quality of those products, which arises from two different sources. The first known is the formation of grey discolouration caused by the oxidase reaction of polyphenol group of enzymes; and the second is the non-enzymatic browning that results when reducing sugars condensed with amino groups. Several methods have been developed to eliminate discolouration. Hoover & Miller (1973) used sodium acid pyrophosphate blanch treatment to eliminate greying. Olorunda & Kitson (1977) eliminated discolouration in chips prepared from white flesh varieties by dipping in sodium sulphite (SO2). Langdon (1987) reported that phenolase enzymes could be eliminated by lowering the pH of the

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Restructured sweetpotato stick J. S. Utomo et al.

media and maintaining it below 3.0. Hannigan (1979) and Truong et al. (1998) eliminated non-enzymatic browning by water extraction and blanching treatment by decreasing of reducing sugars. To solve the difficulties controlling the inadequate qualities, an attempt can be made to prepare ‘fabricated’ French-fries or sticks from restructured SP roots. The essential ingredients of extrudable French-fries potato dough include dehydrated mashed potatoes and a sufficient amount of water to afford a malleable consistency. To prevent the browning of SP upon processing, dehydration cannot be carried out; SP flour may be added to increase dry matter content. Blanching and flour addition are the treatments which control the colour and solid content of the mashed roots, affecting the appearance and textural properties of the products. In this manner, the chemical and physical characteristics of final products can be carefully controlled. The objectives of the present study were to study the effect of shape of trimmed tubers, blanching methods and SP flour added on physical properties of dough; chemical and physical properties of restructured SP.

(iii) 10%; (iv) 15% or (v) 20% SP flour. Molding was conducted using Texture Analyzer (TA.TX2i, Godalming, UK) attached with a stainless steel tube (75 mm inner diameter) having three of 1 mm · 1 mm square holes and compression platen (SMS ⁄ P75), 50 kg load cell with 2 mm s-1 speed for 90% distance to produce sticks having dimension 1 cm · 1 cm and then cut into 5 cm length. The sticks then deep-fried at 163 C for 1 min, packaged in plastic bags and frozen at )20 C until final preparation and evaluation. The RSS were prepared by deep frying in 175 C for 2 min.

Materials and methods

Physical characteristics

Materials

White variety SP roots were purchased from a local market. Refined bleached and deodourized (RBD) palm olein was obtained from a local refinery. Carboxymethylcellulose (CMC) and sodium tripolyphosphate (STP) were of food grade. All chemicals and solvents used were of analytical grade unless otherwise specified. SP flour was prepared in our laboratory. SP tubers were peeled and shredded manually. Drying was conducted using a cabinet drier at 55 C for 24 h. Dried material was then milled and sifted through a 70 mesh sieve. The flour was packaged in plastic bag and stored at )20 C for further use. Preparation of restructured sweetpotato stick

Preparation of restructured sweetpotato stick (RSS) was arranged by factorial of Randomised Complete Block Design, 2 · 2 · 5 · 3 (shape of trimmed tubers · blanching methods · percentage of SP flour added · replication. The tubers were peeled and (i) sliced into 2.3 mm thickness and 2.5 cm width (Hoover & Miller, 1973) or (ii) diced into cubes of approximately 1 cm · 1 cm · 1 cm, washed and blanched. Blanching was done by dipping SP in (i) water for 2 min at 100 C or (ii) 1% (w ⁄ v) STP solution for 2 min at 100 C. The blanched materials were drained about 3 min to remove excess water, and then mashed and CMC was added (0.3%, w ⁄ w) as a binder. The mashed SP was mixed using universal mixer (Aikosha, AM-20) with (i) 0%; (ii) 5%;

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Proximate analyses

Dry matter content of root and dough before extrusion was determined by an oven drying method (AOAC, 1975). Proximate analysis was made on the final product. Moisture, protein, fat and ash were determined by the AOAC method (AOAC, 1975). Protein was calculated as nitrogen (Kjeldahl) · 6.25. Carbohydrate was expressed as the difference from moisture, protein, fat and ash.

The colour of fried stick was determined by the Hunter Color Instrument (Hunter Lab, Reston, VA, USA) and values (L, a, b) were collected. ‘L’ describes Lightness (0 = black, 100 = white), ‘a’ intensity in red (a > 0) and ‘b’ intensity in Yellow (b > 0). Textural properties of dough and fried stick were evaluated using a Texture Analyzer (TA.TX2i, Godalming, UK) fitted with the appropriate test accessories. Firmness of dough was recorded as a force needed during extruding the dough. Hardness of fried stick was expressed as force required to shearing and cutting the samples by the single downward action of the shear blade (HDP ⁄ BS). The test speed was 1 mm s-1 with reach 110% distance. Firmness of dough and hardness of fried stick were expressed in kilogram (kg). Data collection and analysis were accomplished by the EXTRAD Dimension Software of the Texture Analyzer. Statistical analysis

The data collected were analysed by the analysis of variance (anova) and significant differences among means were determined by Duncan’s multiple range test using MSTAT-C statistical software. Results and discussion

Physical characteristics of dough

Based on the anova, no correlation occurred among the shape of materials, blanching methods and amount of

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SP flour added to influence the firmness and dry matter content of dough. Table 1 shows that the blanching methods significantly influenced firmness and dry matter. Blanching in 1% (w ⁄ v) STP for 2 min produced a firmer dough (18.98 kg) than in water (16.38 kg) and a high dry matter content (32.80%) compared with 32.25%. This may be caused by cross-linkage between phosphates and hydroxyls-end of carbohydrates which decreases water binding capacity. The percentage of SP flour added into mashed SP significantly influenced firmness and dry matter; the higher the percentage of SP flour, the higher the firmness and dry matter. The range of dry matter content of dough was 25.98–38.83% which increased about 3% for every 5% of SP flour added. Without any addition, the dry matter content of dough was 25.98%, and too soft so that the shape of stick was not formed. According to Gutcho (1973), products containing more than 73% by weight of water tend to puff undesirably during frying. The dough itself is too soft to handle and it tends to disintegrate prior to completion of frying. This shows that 5% SP flour is the lowest amount needed to be added to mashed SP to produce a dough suitable for further processing. Proximate composition

The proximate composition of fried RSS is shown in Table 2. The shape of trimmed tuber before blanching did not affect the moisture, protein, fat and carbohydrate content of fried product. Blanching methods significantly influenced fat, ash and carbohydrate content. Fat absorption during frying was influenced by the blanching method. It was higher when trimmed tuber was blanched in water than in 1% STP. This Table 1 Effect of experimental factors on firmness and dry matter of

dough Treatment Shape Sliced Diced Blanching Water 1% STP % SP flour 0 5 10 15 20

Firmness (kg)

Dry-matter (%, w ⁄ w)

18.21a 17.14a

32.43a 32.62a

ns

16.38b 18.98c

32.25b 32.80c

9.77d 12.71e 15.67f 20.66g 29.58h

25.98d 29.46e 32.60f 35.75g 38.83h

ns

a–h

Mean within columns for each treatments (shape; blanching or % SP flour), followed by different superscript letters are significantly different at P < 0.05. ns Not significant.

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Table 2 Effect of experimental factors on proximate composition (%, w ⁄ w) of fried RSS Treatment Shape Sliced Diced Blanching Water 1% STP % SP flour 0 5 10 15 20

Moisture

Protein

Fat

34.83a 34.88a

ns

1.94a 2.09a

ns

15.29a 14.56a

33.88b 35.88b

ns

2.02b 2.01b

ns

1.97c 1.88c 1.90c 2.11c 2.23c

ns

34.15cd 38.43c 36.98c 34.09cd 30.62d

Ash

Carbohydrate

1.23a 1.44b

45.83a 47.03a

15.62b 14.22c

1.29c 1.38d

47.18b 45.68c

18.05d 15.43e 13.94e 13.44e 13.74e

1.31e 1.30e 1.29e 1.37e 1.40e

ns

ns

ns

42.03d 43.21d 45.90e 49.00f 52.01g

a–g Mean within columns for each treatments (shape; blanching or % SP flour), followed by different superscript letters are significantly different at P < 0.05. ns Not significant.

result agrees with Varela (1988) that in fried products, fat or oil absorption occurs as moisture is removed from the food during frying. The lower the moisture content, the higher the fat content. The ash content of product blanched in 1% STP was higher than that blanched in water. This may be ther result of the increase in phosphorous content absorbed from STP solution during blanching. Carbohydrate was expressed as the difference from moisture, protein, fat and ash. The moisture content of fried stick was affected by SP flour added, without any SP flour added; moisture content was low and was not significant from many other values. The lowest water content was in the sample containing 20% flour. The fat content of fried stick without SP flour was the highest and was significantly different from fried stick with SP flour added. It shows that the SP flour reduced the oil uptake during frying. The increase in SP flour added into mashed SP significantly increased carbohydrate content from 42.03 to 52.01%. The protein content of the fried product was not influenced by the method of preparation. The blanching method seems to be a more important factor affecting chemical characteristics of fried RSS than the amount of SP flour added. Blanching raw material in 1% STP for 2 min produces low fat and high ash content of fried product. Physical characteristics of RSS

Table 3 shows that the shape of raw materials (chips or cubes) did not affect the physical characteristics. However, blanching methods significantly affected Hunter L, a and b colour values of RSS. Blanching in 1% STP solution produced fried stick with higher L value (49.72)

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generates the darker RSS than 0% SP flour. Redness or a-value were not influenced by the percentage of SP flour mixed.

Table 3 Effect of experimental factors on hardness, L, a and b value of fried RSS Treatment Shape Sliced Diced Blanching Water 1% STP % SP flour 0 5 10 15 20

Hardness (kg)

L

1.97a 2.17a

ns

48.18 a 48.26a

2.19b 1.95b

ns

1.34c 1.72d 2.02d 2.37e 2.90f

a

ns

5.84 a 6.02a

b

ns

13.17 a 13.16a

46.72b 49.72c

5.45b 6.41c

12.17b 14.16c

52.03 d 47.09 e 47.14 e 47.43 e 47.44e

6.04de 5.36e 5.75e 5.99de 6.51d

16.29d 12.34e 12.30e 12.41e 12.49e

Correlation among parameters

ns

Table 4 shows that correlations between parameters determined in this study were evaluated. Dry matter has an important role in the characteristics of the products. The dry matter content of dough was highly correlated with carbohydrate, fat content and hardness of fried sticks. The dry matter of dough depended on the SP flour mixture; the higher the amount of SP flour added the higher the dry matter content. The dry matter of dough was dominated by carbohydrate. The increase in carbohydrate content decreased fat absorption during frying, but increased the hardness. There was no correlation between hardness and fat content, but shows a positive correlation with moisture content. Thus, hardness of product is influenced by carbohydrate and moisture content. L and a-values have a positive correlation with ash content. L and a values increased by the increase in ash content, whereas ash content was increased when tubers were blanched in 1% STP solution. From these findings, one can conclude that an increase in the percentage of SP flour added causes the increase in carbohydrate content and hardness, but a decrease in fat content. Moreover, blanching using 1% STP improves the lightness and red colouration of the products.

a–e Mean within columns for each treatments (shape; blanching or % SP flour), followed by different superscript letters are significantly different at P < 0.05. ns Not significant.

than blanching in water (46.72). Based on a and b values of fried sticks, red and yellow colouration decreased when chips were blanched in water. This shows that blanching in 1% STP improves the colour of fried RSS compared with blanching in water. The percentage of SP flour added significantly influenced all parameters measured. Hardness of RSS significantly increased with the increase in SP percentage added. Fried stick made without SP flour has an irregular shape and soft (low in hardness). Mixing 5 and 10% of SP flour produces fried product having uniform shape and intermediate hardness. Adding SP flour significantly affected L and b-values. RSS produced without SP flour has higher L and b-value than any other percentage of SP flour; however, there was no significant difference among the amount of SP flour added. That means that the addition of SP flour

Conclusion

The results from this study showed that blanching in 1% STP for 2 min improves in colour and ash content of fried sticks. The mixing of 5% SP flour to the mashed SP produces suitable conditions of the dough for further processing. RSS can be produced by the above combi-

Table 4 Correlation coefficients (r) between parameters measured of dough and RSS

Firmnessa Drymattera Moisture Protein Fat Ash Carbohydrate Hardness L a b

Firmnessa

Drymattera

Moisture

Protein

Fat

Ash

Carbohydrate

Hardness

L

a

b

1 0.89** )0.21 0.40** )0.52** 0.14 0.70** 0.30* 0.02 0.37* )0.07

1 )0.24 0.30* )0.53** 0.18 0.82** 0.53** )0.23 0.25 )0.35*

1 )0.03 )0.34* )0.13 )0.42** )0.38* 0.32* )0.27 0.21

1 )0.20 0.27* 0.17 )0.05 0.09 0.16 0.06

1 )0.06 )0.44** 0.19 )0.25 )0.05 )0.09

1 0.23 0.23 0.45** 0.45** 0.06

1 0.45** )0.35* 0.22 )0.42**

1 )0.49** 0.19 )0.50**

1 0.25 0.97**

1 0.35*

1

a Dough. Significance correlation *P < 0.05 and

**

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P < 0.01.

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Restructured sweetpotato stick J. S. Utomo et al.

nation with an intermediate hardness, high lightness and low redness colouration. Acknowledgments

The author wishes to thank Agency of Agriculture Research and Development Indonesia for providing the funds (PAAT Project ADB Loan No. 1526-INO) to support his study at Universiti Putra Malaysia (UPM). References AOAC (Association of Official Analytical Chemists). (1975). Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. Arlington, VA: AOAC, Inc. Che Man, Y.B. (1996). Development and evaluation of a snack food from sweet potato. ASEAN Food Journal, 11, 114–119. Collins, J.L. & Walter, W.M. Jr. (1992). Processing and processed products. In: Fifty Years of Cooperative Sweetpotato Research 19391989. (edited by A. Jones & J.C. Bowkamp). Pp. 71–87. No. 369. Baton Rouge, LA: Louisiana Agriculture Experimental Station Southern Cooperative Bulletin. Collins, J.L. & Washam-Hutsell, L. (1987). Physical, chemical, sensory and microbiological attributes of sweet potato leather. Journal of Food Science, 52, 646–648. Edmond, J.B. & Ammerman, G.R. (1971). Sweet Potato: Production, Processing, Marketing. Pp. 334. Westport, CT: AVI publishing. Gutcho, M.. (1973). Prepared Snack Foods. Pp. 288. New Jersey, NJ: Noyes Data Corporation. Hannigan, K.J. (1979). Sweetpotato chips. Food Engineering, 51, 26. Hoover, M.V. & Miller, N.C. (1973). Process for producing sweet potato chips. Food Technology, 27, 74, 76, 80. Hoover, M.W., Walter, W.M. & Giesbrecht, F.G. (1983). Method of preparation and sensory evaluation of sweet potato patties. Journal of Food Science, 48, 1568–1569. Langdon, T.T. (1987). Preventing of browning in fresh prepared potatoes without the use of sulfiting agents. Food Technology, 41, 64–67. Lanier, J.J. & Sistrunk, W.A. (1979). Influence of cooking method on quality attributes and vitamin content of sweet potatoes. Journal of Food Science, 44, 374–376, 380.

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Olorunda, A.O. & Kitson, J.A. (1977). Controlling storage and processing conditions help produce light colored chips from sweetpotatoes. Food Product Development, 11, 44–45. Picha, D.H. (1985). Crude protein, minerals, and total carotenoids in sweet potatoes. Journal of Food Science, 50, 1768–1769. Schwartz, S.J., Walter, W.M. Jr, Carrol, D.E. & Giesbrecht, F.G. (1987). Chemical, physical, and sensory properties of a sweet potato French-fry type products during frozen storage. Journal of Food Science, 52, 617–619. Sylvia, K.W., Walter, W.M. Jr & Giesbrecht, F.G. (1997). Alkaliprocessed sweetpotato French fries. Journal of Food Quality, 20, 17– 30. Truong, V.D. & Walter, W.M. Jr (1994). Physical and sensory properties of sweetpotato puree texturized with cellulose derivatives. Journal of Food Science, 59, 1175–1180. Truong, V.D., Walter, W.M. Jr & Giesbrecht, F.G. (1995). Texturization of sweetpotato puree with alginate: effect of tetrasodium pyrophosphate and calcium sulfate. Journal of Food Science, 60, 1054–1059. Truong, V.D., Walter, W.M. Jr & Bett, K.L. (1998). Textural properties and sensory quality of processed sweetpotatoes as affected by low-temperature blanching. Journal of Food Science, 63, 739–743. Varela, G. (1988). Current facts about the frying of food. In: Frying of Food, Principles, Changes, New Approaches (edited by G. Varela, A.E. Bender & I.D. Morton). Pp. 9–25. Chicester: Ellis Horwood. Walter, W.M. Jr & Hoover, M.W. (1984). ffect of pre-processing storage condition on the composition, microstructure, and acceptance of sweetpotato patties. Journal of Food Science, 49, 1258–1261. Walter, W.M. Jr & Hoover, M.W. (1986). reparation, evaluation, and analysis of a French-fry-type product from sweet potato. Journal of Food Science, 51, 967–969. Walter, W.M. Jr, Fleming, H.P. & McFeeters, R.F. (1992). Firmness control of sweetpotato French fry-type product by tissue acidification. Journal of Food Science, 57, 138–142. Walter, W.M. Jr, Fleming, H.P. & McFeeters, F.T. (1993). Basemediated firmness retention of sweetpotato products. Journal of Food Science, 58, 813–816. Walter, W.M. Jr, Truong, V.D. & Espinell, K.R. (2002). Textural measurements and product quality of restructured sweetpotato French fries. Lebensm-Wiss U.-Technology, 35, 209–215.

 2008 Institute of Food Science and Technology

International Journal of Food Science and Technology 2008, 43, 1901–1907

Short communication Fluid dynamic gauging studies of swelling behaviour of whey protein gels in NaOH ⁄ NaCl solutions Pradeepta K. Sahoo,1,2 Y.M. John Chew,1* Ruben Mercade´-Prieto,1 D. Ian Wilson1 & Xiao W. Dai1 1 Department of Chemical Engineering, Pembroke Street, New Museums Site, Cambridge, CB2 3RA, UK 2 Department of Food Engineering, BCKV, Mohanpur-741252, Nadia, West Bengal, India (Received 19 February 2007; Accepted in revised form 17 October 2007)

Keywords

b-Lactoglobulin, fluid dynamic gauging, heat-induced gel, swelling, whey protein gel.

Introduction

The thermal treatment of milk in dairy heat exchangers is often accompanied by chronic fouling caused by thermal denaturation of whey proteins and crystallisation of calcium phosphate (Visser & Jeurnink, 1997). Fouling deposits on exchangers operating in the temperature range of 70–95 C consist chiefly of heatinduced whey protein gels and particularly b-lactoglobulin (bLg) (Lalande et al., 1985). These deposits are often removed by contact with circulating alkaline solutions during cleaning-in-place cycles (Bird & Fryer, 1991). The alkali causes the proteinaceous material to swell as the interprotein repulsion increases, and it subsequently breaks down either via erosion or dissolution (Mercade´-Prieto & Chen, 2006). Bird & Fryer (1991) observed that the cleaning rate increased with NaOH concentration at low NaOH concentrations, but decreased above c. 0.5 wt% NaOH, indicating that there is an optimal concentration where cleaning is fastest. Recently, Mercade´-Prieto et al. (2007) have shown using gravimetric swelling studies that the reduced cleaning rate at high NaOH concentration is accompanied by a smaller equilibrium swelling ratio and swelling rate under these conditions. Both phenomena are attributed to the screening effect of the high concentration of base cation (Na+ in this case), which effectively diminishes the interprotein repulsion between the charged residues on the protein. The same behaviour is observed if the Na+ (aq.) concentration is increased by adding a salt, e.g. NaCl. In this study, we employ the technique of fluid dynamic gauging (FDG) to verify these observations for thin deposits of bLg and whey protein concentrate (WPC) gels, which serve to simulate the *Correspondent: Fax: +44 1223 334796; e-mail: [email protected]

proteinaceous milk fouling layer adhering to a heat transfer surface. Fluid dynamic gauging is a technique by which the thickness of a soft and fragile deposit layer immersed in liquid can be monitored in situ and in real time without touching the surface of layer and by measuring the flow through a nozzle located close to the deposit surface (Tuladhar et al., 2000, 2002). The position of the layer surface is calculated from the location of the nozzle and the flow rate, which is determined by the clearance between the nozzle and surface, h. This paper describes the development of an experimental methodology that combines FDG and confocal laser scanning microscopy (CLSM) to track structure and changes within WPC and bLg gels upon exposure to the cleaning agent NaOH. The gels are used as analogues for heat exchanger deposits (Hooper et al., 2006). FDG provides information on the thickness and the factors governing dynamic changes of such films, while CLSM yields information on the microstructure and chemical nature at different depths with the film. Successful implementation of such a methodology would allow long-term studies to characterise the structural changes associated with the milk fouling ⁄ cleaning cycle and to determine the effect of the gauging technique on the whey protein deposit microstructure. The practicalities of the work are as follows: an FDG flow cell has been built containing a viewing window within the reservoir. A protein film is attached to this viewing window, while the reservoir contains the cleaning solution. The dynamic gauge is placed just (100–200 lm) above the viewing window. This allows cleaning behaviour to be monitored from above the film, while microstructural data are obtained from below by using an inverted microscope to view the film through the viewing window. By scanning the protein film

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interface, in real time, it is possible to observe cleaning solution–film interactions. Materials and methods

Gel formation

Commercial WPC (WPC80, c. 80 wt% protein) was obtained from Volac International Ltd (Royston, Herts, UK), and pure (>90%) bLg was supplied by Davisco Foods International, Inc. (Lesueur, MN, USA). Deionised water was used in forming gels and preparation of solutions. Deposits were formed by heating 20 wt% WPC80 and 16 wt% bLg solutions on 50 mm diameter polystyrene Petri dishes, submerged in a water bath at 75 C for 30 or 90 min. Fresh gels of uniform thickness were used for each experiment. WPC80 gels were opaque and of a ‘particulate’ nature (pH 6.2), while bLg were transparent and defined as ‘fine stranded’ (pH 7.5) (Langton & Hermansson, 1992). Fluid dynamic gauging

A small dynamic gauging apparatus was constructed to hold the Petri dish samples and allow the deposit to be studied from beneath by microscopy or other techniques. Figure 1 shows a schematic of the FDG apparatus and the configuration of the device when combined with CLSM. The flow cell (i.d. 120 mm) holds c. 400 mL solution maintained at a constant hydrostatic head by a weir arrangement. The gauge is constructed from 316 stainless steel with a tube i.d. of 4 mm and a nozzle throat diameter, dt, of 1 mm. A perforated ring ⁄ mesh of diameter 60 mm is used to stabilise the flow near the nozzle. The nozzle may be moved vertically towards or across the sample using precision micrometers. The system is quasi-stagnant, in that the only flows present are the gauging flow and the weir discharge. The gauging flow rate-clearance profiles in Fig. 2 demonstrate that the edges of the Petri dish did not influence the FDG characteristics: the gauging flow is controlled by fluid behaviour in the immediate vicinity of the nozzle. The presence of the surface affects the flow significantly when the clearance, h, is such that h ⁄ dt < 0.30. This linear regime is employed in gauging experiments as the thickness of the layer is given by the difference between the clearance and the known location of the nozzle relative to the surface. The flow cell has been built containing a viewing window within a reservoir. A protein gel formed in a Petri dish is attached to this viewing window, while the reservoir contains the cleaning solution. The dynamic gauge is placed just above the viewing window (100– 200 lm, corresponding to 0.1 £ h ⁄ dt £ 0.2). This allows cleaning behaviour to be monitored from above the film, while microstructural data are obtained from below by

International Journal of Food Science and Technology 2008

the confocal microscope which captures the film through the viewing window. Experiments were performed at room temperature (20 ± 2 C). Gel thickness was monitored by measuring the flow rate, at 10 s intervals, with an electronic balance connected to a PC. The gauging flow also serves as a sampling stream, removing solution from the interface region. The concentration of eluted protein was measured at 3 min intervals using UV absorbance at 280 nm (UV1; Thermo Spectronic, Runcorn, UK), following appropriate calibrations. Experiments were performed in triplicate. The swelling ratio is calculated from the initial (d0) and final (df) gel thickness (eqn 1). The swelling rate is estimated as the thickness increase during the time that the deposits starts (t0) and finishes (tf) to swell, as shown in Fig. 3a; swelling ratio ¼

df  d0 d0

ð1Þ

swelling rate ¼

df  d0 tf  t0

ð2Þ

An attempt was made to image the WPC gels undergoing swelling by combining FDG with CLSM. The fluorophore dye Rhodamine 6G dye was added at 10 lm to the protein solution prior to gel formation and gels of initial thickness 0.20–0.25 mm were generated for this work. A IX70 CLSM (Olympus, Tokyo, Japan) equipped with a Fluoreview FV300 scanning unit (Olympus, Tokyo, Japan) was used, with emitted fluorescence detected in the wavelength range 565–630 nm using a 630 dichoric mirror with a BA565 long pass filter. Results and discussion

Swelling results of protein gels in alkali using fluid dynamic gauging

Figure 3 shows a typical set of thickness profiles obtained for the three gels tested here in 0.25 wt% NaOH (pH c. 12.8) in the absence of any added salt. The profiles exhibit the successive stages of induction, swelling and final plateau reported previously (Tuladhar et al., 2000). The swelling ratios and rates found for the bLg gels cured for 30 min (bLg-30 m) and for WPC80 heated for 30 min (WPC-30 m) are very similar, suggesting that the bLg in WPC defines the swelling behaviour. On the other hand, the WPC80 gels formed during 90 min (WPC-90 m) have a significantly lower swelling ratio, as should be expected for a more crosslinked system (Brannon-Peppas & Peppas, 1991). The swelling ratio in these gels depended on the initial thickness, as shown in Fig. 4, indicating that the plateau was not a true equilibrium state but rather an

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

Fluid dynamic gauging for cleaning studies P. K. Sahoo et al.

(a)

Micrometer

Siphon tube

Screw Perforated wall Solvent outlet Nozzle Deposit

Solvent inlet Petri dish

Perspex

±0.005g

Microscope (b)

CCD camera

FDG apparatus

Nozzle

Light source

Deposit

Objective lens

Figure 1 (a) Schematic of fluid dynamic gauging (FDG) apparatus; (b) experimental set up of FDG with confocal laser scanning microscopy.

intermediate stage in the reaction with hydroxide. Over the timescale of interest for cleaning, this is nevertheless an important process feature: the true equilibrium swelling ratio could be obtained by interpolating the data to vanishingly small initial thickness values such that swelling occurs rapidly, without any diffusion limitation, and before dissolution can occur. The effect of the addition of NaCl in reducing the swelling ratio and the rate is shown in Fig. 5 for WPC30 m. The effect of salt addition is summarised in Fig. 6

 2008 The Authors. Journal compilation  2008 Institute of Food Science and Technology

where the change inP concentration is expressed via the ionic strength (I ¼ 12 Ci z2i , where Ci is concentration of ion i and zi the charge on the ion). The swelling ratio decreases greatly with I (Fig. 6a), as does the swelling rate (Fig. 6b), in good agreement with our previous results (Mercade´-Prieto et al., 2007). While WPC-90 m certainly swelled less than the other two gels, there is little difference in the effect of ionic strength on the swelling rate. It is noteworthy that at high values of I (>1 m), the swelling ratio of all three gels is statistically

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0.6

1

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Fluid dynamic gauging for cleaning studies P. K. Sahoo et al.

0.4

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0

0.1

0

1

2 0

0

0

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h/dt

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(mm)

Figure 4 Effect of initial thickness on swelling ratio for whey protein concentrate-90 m gel in contact with 0.25 wt% NaOH, no added salt.

Figure 2 Calibration curves for fluid dynamic gauging apparatus using water with suction pressure difference of 390 Pa. Solid symbols, plane surface; open symbols, Petri dish. Vertical dashed line indicates end of pseudo-linear region used in gauging tests.

1.8

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f

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t0

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5

25

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35

Time (min) Figure 3 Fluid dynamic gauging swelling profiles of protein gels exposed to 0.25 wt% NaOH and no added salt, with construction lines shown for whey protein concentrate (WPC)-90 m. Symbols: solid triangles, WPC-90 m; open triangles, WPC-30 m; open circles, bLg-30 m.

the same, as expected when considering that the protein charges will be completely screened by the high concentration of NaCl (Skouri et al., 1995). In addition, it is also observed that the induction time (t0) before the gel starts to swell also increases with I (Fig. 6c).

International Journal of Food Science and Technology 2008

Figure 5 Fluid dynamic gauging swelling profiles of whey protein concentrate-30 m gels exposed to 0.25 wt% NaOH and different NaCl concentrations. Symbols: open triangles, no added salt; solid triangles, 0.1 wt% NaCl; solid circles, 0.5 wt% NaCl.

Fluid dynamic gauging also allows one to analyse the solution in the region in contact with the deposit. By measuring the protein concentration in the gauging flow, it is possible to determine when the deposit starts to dissolve. Figure 7 shows that this only happens after the gel has swollen significantly (as reported by Tuladhar et al., 2002), underlying the importance of swelling in the cleaning of dairy deposits. This behaviour was observed in all the gels tested, irrespective of the presence of added salt (results not shown).

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Fluid dynamic gauging for cleaning studies P. K. Sahoo et al.

Swelling ratio (mm min–1)

1.4

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0.6

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0 0

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(b) 0.05

Swelling rate (mm min–1)

[Protein] in gauge flow ( mg l–1)

0.04

1.5

(a) 0.8

0.04

Figure 7 Protein assay in gauge fluid during swelling of whey protein concentrate-90 m gel, 0.25 wt% NaOH and no added salt.

0.03

Confocal laser scanning microscopy

0.02

0.01

0 0

0.2

0.4

0.6

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1

1.2

I (M)

These experiments employed an initial gel thickness of about 210 lm as it proved difficult to scan through a thicker layer. The average fluorescence intensity of light and gel thickness was monitored using CLSM and FDG respectively throughout the swelling process. Figure 8 shows the profiles obtained for WPC-90 m; similar results, where the intensity matches the FDG thickness measurements, were obtained for the other gels. The change in intensity for the swollen deposit is attributed to dequenching with the Rhodamine 6G dye particles.

(c) 12

6 4 2 0 0

0.2

0.4

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I (M)

B C

0.30

60

0.25 0.20

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40

0.15 A 0.10

D

20

0.05 0.00 0

Figure 6 Effect of NaCl concentration on swelling behaviour of protein gels at 20 C: (a) thickness-swelling ratio; (b) swelling rate; (c) induction period. Symbols: solid triangles, whey protein concentrate (WPC)-90 m; open triangles, WPC-30 m; open circles, bLg-30 m. Error bars are the standard deviation of three repetitions.

80

0.35

Fluorescence intensity

0.40 8

Deposit thickness (mm)

Induction time (min)

100

0.45

10

10

20

30

40

0 50

Time (min) Figure 8 Fluorescence intensity and swelling profile during swelling of whey protein concentrate-90 m gel in 0.25 wt% NaOH and no added salt. Letters indicate times of the CSLM images in Fig. 9.

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(a)

(b) 7.5 min

0.0 min

10 m

(c)

15.0 min

10 m

(d) 37.5 min

10 m

As the film swelled, the extent of self-quenching (i.e. loss of fluorescence) between the fluorophore molecules decreased, therefore increasing the fluorescence intensity. As the gel is dissolved, the amount of fluorophore molecules decreased, therefore decreasing the intensity. CLSM images of the WPC gels at different times are shown in Fig. 9. It is clear that the structure within the WPC changes as the gel is swelled. The changes in intensity can also be correlated with the images. Conclusion

Fluid dynamic gauging has been used to verify that the polyelectrolyte screening effects observed with synthetic polymers at high ionic strengths are also observed in protein deposits, greatly affecting the dynamics of swelling. The strong contribution of bLg to WPC gel behaviour has also been confirmed. The combination of FDG with CLSM, to yield simultaneous local measurements of mechanical, chemical and microstructural information has been demonstrated and the areas for improvement of the imaging techniques identified. Acknowledgments

WPC80 and b-lactoglobulin powders were kindly donated by Volac and Davisco, respectively. A Seligman

International Journal of Food Science and Technology 2008

10 m

Figure 9 Confocal laser scanning microscopy images of whey protein concentrate-90 m gel in 0.25 wt% NaOH and no added salt at different times.

Fellowship for PKS from the SCI, a Royal Academy of Engineering Research Fellowship for YMJC and support for RM-P from the Cambridge European Trust are all gratefully acknowledged. References Bird, M.R. & Fryer, P.J. (1991). An experimental study of the cleaning of surfaces fouled by whey proteins. Food and Bioproducts Processing, 69, 13–21. Brannon-Peppas, L. & Peppas, N.A. (1991). Equilibrium swelling behaviour of pH-sensitive hydrogels. Chemical Engineering Science, 46, 715–722. Hooper, R.J., Paterson, W.R. & Wilson, D.I. (2006). Comparison of whey protein model foulants for studying cleaning of milk fouling deposits. Food and Bioproducts Processing, 84, 329–337. Lalande, M., Tissier, J.P. & Corrieu, G. (1985). Fouling of heat transfer surfaces related to b-lactoglobulin denaturation during heat processing of milk. Biotechnology Progress, 1, 131–139. Langton, M. & Hermansson, A.-M. (1992). Fine-stranded and particulate gels of b-lactoglobulin and whey protein at varying pH. Food Hydrocolloids, 5, 523–539. Mercade´-Prieto, R. & Chen, X.D. (2006). Dissolution of whey protein concentrate gels in alkali. AIChE Journal, 52, 792–803. Mercade´-Prieto, R., Sahoo, P.K., Falconer, R.J., Paterson, W.R. & Wilson, D.I. (2007). Polyelectrolyte screening effects on the dissolution of whey protein gels at high pH conditions. Food Hydrocolloids, 21, 1275–1284. Skouri, R., Schosseler, F., Munch, J.P. & Candau, S.J. (1995). Swelling and elastic properties of polyelectrolyte gels. Macromolecules, 28, 197–210.

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Fluid dynamic gauging for cleaning studies P. K. Sahoo et al.

Tuladhar, T.R., Paterson, W.R., Macleod, N. & Wilson, D.I. (2000). Development of a novel non-contact proximity gauge for thickness measurement of soft deposits and its application in fouling studies. Canadian Journal of Chemical Engineering, 78, 935–947. Tuladhar, T.R., Paterson, W.R. & Wilson, D.I. (2002). Investigation of alkaline cleaning-in-place of whey protein deposits using dynamic gauging. Food and Bioproducts Processing, 80, 199–214.

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Visser, J. & Jeurnink, T.J.M. (1997). Fouling of heat exchangers in the dairy industry. Experimental Thermal and Fluid Science, 14, 407– 424.

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Book review Modifying lipids for use in food Edited by Frank D. Gunstone. Cambridege, UK:

Woodhead Publishing Ltd. & Boca Raton, FL, USA: CRC Press. 2006. Pp. 609. ISBN 13: 978 1 85573 971 0. £150 ⁄ US$270 ⁄ €220.

Part I of the book essentially starts by introducing the reader to the understanding of food lipid structures, and from which sources these are found, ranging from animal, vegetable, marine and microbial sources. This sets a good background for both technologists and academics. Whether this book is used as a reference or not it will prove invaluable, because within most of many of the subject areas discussed – there is also a separate subheading dealing with future trends. In chapter 2, Gunstone presents possibilities of using oils, which are currently deemed ‘minor’ and shows how these lipid types – by nature of their unique fatty acid compositions – are likely to cause interest in the future. Chapters 4 and 5 highlight continuing opportunities surrounding raw material from marine and microbial sources. The subject of securing functional lipids from marine sources has been a discussion for several years. These chapters show that ultimately microbial lipids may result via use of GM bacterium, modified by the use of desaturase. Excellent information is given in chapter 8 dealing with Structure & Properties of Fat Crystal Networks. The reader is well informed and presented with a sound overview in determination of fractal dimensions of fat crystal networks using new counting techniques. Rheological methods are linked to solid fat content and crystal polymorph. Part II of the book deals with modifying lipids for use in food. Chapter 10 deals with Fractionation. Again the reader is informed as to present and new techniques the fractionation of numerous commodity oils to achieve lower saturates, and achieve special melting curves. A particularly excellent contribution is brought to the book by the Technical University of Denmark; chapter 11 reviews chemical and enzymatic interesterification of lipids for use in food, and offers current and new practices. The chapter also deals with modification of phospholipids, and conversions of MAG, DAG, TAG structures using enzyme technology. Subsection 11.4 – Remarks and Future trends brings the reader up-to-date with how new enzymatic

approaches are being commercially implemented, or are still at the ‘teething’ stage. In view of the need for healthier, nutritional requirements agro-biotechnology has and is making huge steps in the engineering of common edible oil sources. We are shown examples of the diversity and markets. Low trans containing oils are also discussed. Is it possible to obtain very long chain polyunsaturated fatty acids? – The most serious technical engineering challenges are discussed. Will Palm oil succumb to transgenic manipulation, and what of the future? – Current commodity oils will probably become more sophisticated, as plant breeders attempt to satisfy growing niche markets. Part III deals with application of modified lipids in food. Emulsifiers and surfactants for dairy and bakery use are the topic in chapter 16, which is largely centred on butter, cream variants where good information is provided highlighting physico-chemical defects of fresh, and UHT creams. It also touches on the subject of margarine and spreads. Flo¨ter and Duijn present structuring functionality of different fat compositions with special attention to the role of trans fatty acids and their elimination. This part of the book goes on to discuss application and use of zero calorie lipids, artificial dairy products, chocolate and confectionery fats and developments in frying oils. The book would not be complete without the section on Speciality oils and their applications in food. This section is most relevant given the food industry in general is continuously seeking to find novel application of lipid types, either for nutritional or other technical effect. Finally, the book revisits the subject of microbial derived oils, marine and fish oil, showing practical applications of these lipid types. This book will no doubt be found as an invaluable resource and reference in the near future. Reviewer comment

Chapter 9 has some very practical information to the reader, but the arguments to the pros and cons of palm oil vs. soybean oil are weak. It is not helped by comparing North American hydrogenated formulations and the gigantean consumer base with Denmark and its use of interesterified or fractionated components. Given a reference from the literature over a 7-year period (1995–2002) paragraph 1, p. 175 – it now might be considered potentially

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Book review

misleading. The paragraph ends with ‘Thus, it would appear that hydrogenation remains the technology of choice to formulate margarine ⁄ spread products throughout most of the world’. Given this was not published until 2006, the reader should use discernment.

 2007 Institute of Food Science and Technology

Paul Wassell Senior Application Specialist, Multiple Food Applications, Oils & Fats innovation group, Danisco A ⁄ S, Denmark

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1909

International Journal of Food Science and Technology 2008, 43, 1910–1911

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Book review Bakery Products Science and Technology

By Y.H., Hui, H., Corke, I.D, Leyn, W.K, Nip & N, Cross Blackwell Publishing Ltd. Pp. 592. ISBN: 0813801877. € 110 In this book 50 professionals from fifteen different countries working in industry, government and academic institutions have contributed their perspectives on the state of baking today. The book has been divided into five parts containing thirty chapters. Parts I and II deal with flours and baking ingredients, whereas Parts III, IV and V cover principles of baking, bread and special products. Chapter 1 deals with baking materials ⁄ ingredients and baking techniques. This chapter includes definitions of milling products, wheat milling and composition. The composition and functionality of various ingredients used in bakery products such as baking powders, fats, oils, sweetening agents, dairy products, eggs, etc. is presented. The dough making processes, soft wheat products and staling mechanism and factors affecting the staling of bakery products are also briefly discussed. Chapter 2 is devoted to wheat flour classification. An overview of world wheat production, consumption, export and import data is reported. The characteristics of Canadian and US wheat classes and their grading requirements are included in this chapter. Details about the characteristics of European and Asian wheat could also have been included. Chapter 3 is devoted to microbial and animal contaminants as well as physico-chemical and rheological characteristics of flours. Some details on quality evaluation of flour would have made this chapter more interesting. Chapter 4 briefs about the composition, isolation techniques, functional properties and uses of gluten proteins. Chapter 5 deals with production, consumption, composition of rye and its products. Flavour of native rye and formation of flavour during processing is also briefly discussed. Chapter 6 covers production and consumption of rice along with the composition, functional and baking properties of rice flour. Chapters 7–12 are devoted to sweeteners, eggs, yeast, fat replacers, water and functional additives. The properties of selected sugars and sweeteners are presented. Functionality of sweeteners as well as problems and possible solutions in reduced sugar or sugarless baking is very interesting as the population of diabetics

is increasing worldwide. The composition and functional properties of egg in baking is presented in Chapter 8. Major food fermentations involving yeasts, commercial yeast products, yeast strains for specific dough applications and behaviour of yeast in dough are focused in Chapter 9. Chapter 10 is devoted to classification and functional properties of fat replacers. Safety aspects of fat replacers and effects of fat replacements with fat replacers on bakery products are also focused in this chapter. Chapter 11 describes the role of water in baking whereas Chapter 12 covers oxidising and reducing agents, vital gluten, enzymes, emulsifiers and hydrocolloids. The role of these additives to compensate for variation in the processing characteristics of flour, improving the quality and extending the shelf life of baked products is discussed. Chapters 13–16 deal with dough mixing, fermentation, baking and sensory attributes of baker products. Chapter 14 briefly covers changes in dough during fermentation, methods of fermentation and factors influencing the dough fermentation. Chapter 15 focuses on the concept and knowledge of baking process. The frequent problems encountered during baking process due to deficiencies in oven operation are also discussed along with brief description of various types of ovens. The introduction to basic principles of sensory assessment, sensory attributes of bakery products and factors affecting the same have been discussed in Chapter 16. Chapters 17 relates to process of bread making, quality analysis and process monitoring techniques. The key factors involved in bread staling are focused in Chapter 18. In this chapter authors have tackled the issue of bread quality from European prospective and factors that are important in different types of breads popular in UK and Europe are covered. Methods have been outlined to explain how the process leading to deterioration in quality can be slowed down to enhance the perceived freshness in bread. Chapter 19 deals with a concise overview of the structure and properties of different constituents of flour and their functionality in bread making. The role of different enzymes in bread making is also covered in this chapter. The application of enzymes in improving the processes to minimise the effect of wheat flour variability and to replace the chemical additives is important. Chapter 20 reviews health benefits of sourdough, its process, microflora and functional aspects, while Chapter 21 deals with frozen dough.

doi:10.1111/j.1365-2621.2007.01653.x  2008 The Author. Journal compilation  2008 Institute of Food Science and Technology

Book review

Chapter 22 covers various aspects of cake production as a function of ingredients, mixing methods and baking requirements focusing on their effect on cake quality. Chapter 23 provides description of production, role of ingredients, changes during baking, quality as well as sanitary aspects of cracker. Also troubleshooting in cracker production is included. Chapter 24 is devoted to non-enzymatic browning of cookies, crackers and biscuits. Chapter 25 provides information limited to description of and basic baking procedures of the specialty baked products of 22 countries. Chapters 26 and 27 deal with dietetic bakery products and gluten-free cereal-based products, respectively. Chapter 26 discusses the food intolerances and bakery products for diabetics, special healthier life style, and special religious diet requirements as well as for various stages of human development. Chapter 28 deals with production, ingredients and evaluation of muffins

 2008 The Author. Journal compilation  2008 Institute of Food Science and Technology

and bagels. Chapter 29 covers structure, formulation and processing of pretzel. Chapter 30 provides details of Italian bakery products. The information of production, ingredients, types and quality evaluation of cookies has not been dealt, which could have been of greater interest. Every year environmental calamities such as sprouting, hailstorms, fungal infections, etc. adversely affect the baking quality of flours in one or other part of the world. Chapter related to the same could also have been included. The characteristics of wheat grown in Europe and Asia would have made this book more interesting. This book will serve as an excellent reference for professionals working in baking industry and academia. Narpinder Singh Guru Nanak Dev University, Amritsar, Punjab, India

International Journal of Food Science and Technology 2008

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