international journal of food and fermentation technology

PRINT ISSN NO. : 2249-1570 ONLINE ISSN NO.: 2277-9396

INTERNATIONAL JOURNAL OF FOOD AND FERMENTATION TECHNOLOGY VOL. 1, NO. 2, DECEMBER 2011

Editor-in-Chief Prof. VK Joshi Professor and Head, Fermentation Technology Lab, Department of Food Science and Technology, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India [email protected]

NEW DELHI PUBLISHERS 90, Sainik Vihar, Near Lakshmi Narayan Mandir, Mohan Garden, New Delhi – 110059 (India) Email: [email protected] Website: www.ndpublisher.in Phone: 91-11-25372232 Mobile: 9971676330, 9582248909

INTERNATIONAL JOURNAL OF FOOD AND FERMENTATION TECHNOLOGY

ABOUT THE JOURNAL The Journal would publish research papers on all the subjects related to the Food and Fermentation Technology. All aspects of food and fermentation technology would be considered. Papers on bio-technology, bio-chemical, toxicological aspects having direct bearing or are related with food would be welcomed. R & D work related to the fermentation covering microbiology, biochemical, genetics, indigenous fermented foods, toxicology or nutritive value would also be included. Articles highlighting the food standards and safety issues will be given special emphasis. Preparation and evaluation of alcoholic beverages would be a important aspect of the articles published. The review on any aspect of food processing, composition, nutrition, and fermentation would be considered. Management of food processing industrial waste would be an integral component of the papers. The other aspects of food processing like low temperature preservation, by dehydration, thermal processing, irradiation, emerging technologies viz., ohmic preservative, pulse electric field, high pressure preservation, enzymology, microbiological quality, food safety and standards and food engineering, will also be considered. International Journal of Food and Fermentation Technology, half yearly journal, publishing original research papers, short communications and review papers on topics which include: • Food microbiology • Bio-chemical aspects of food • Genetic and genetically modified foods • Enology • Indigenous fermented foods • Toxicology, safety and quality • Food processing • Fermentation technology • Food engineering • Quality assurance • Food preservation • Food additives

SUBSCRIPTION RATES Institutional

Individual

INR

USD

INR

USD

Print

1500

300

1500

245

Online

1500

200

1000

200

Print +Online

2800

400

2500

400

Kindly Send: Demand Draft/Cheques in favour of NEW DELHI PUBLISHERS payable at New Delhi

Authors can submit their scientific articles/research papers and review articles in Electronic Media in MS Office Compatible software by Email: [email protected]/[email protected] For details please see instructions to authors Research manuscript can also be submitted online : www.ndpublisher.in

NEW DELHI PUBLISHERS 90, Sainik Vihar, Near Lakshmi Narayan Mandir, Mohan Garden, New Delhi – 110059 (India) Email: [email protected] Website: www.ndpublisher.in Phone: 91-11-25372232 Mobile: 9971676330, 9582248909

EDITORIAL BOARD Editor-in-Chief Prof. V K Joshi Professor and Head, Fermentation Technology Lab, Department of Food Science and Technology, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India [email protected] / [email protected]

Honorary Editor Dr (Mrs) Sunita Garg, IJNPR, NPARR and Wealth of India Division National Institute of Science Communication and Information Resources, CSIR, New Delhi, India [email protected] Dr J K Gupta Department of Entomology and Apiculture Dr YS Parmar University of Horticulture and Forestry Nauni-Solan, Himachal Pradesh, India [email protected]

Dr N S Thakur Department of Food Science and Technology Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India [email protected]

Executive Editorial Board Dr (Mrs) Devina Vaidya Department of Food Science and Technology Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India [email protected] Dr B L Attri Central Institute of Temperate Horticulture, Regional Station, Mukteshwar, Kumaun, Nainital, Uttarakhand, India. [email protected],

Dr Shashi Bhushan Division of Biotechnology, Institute of Himalayan Bio-resource Technology (CSIR), Palampur, Distt Kangra, Himachal Pradesh, India [email protected] Dr Satish Kumar Sharma G B Pant University of Agriculture & Technology, Hill campus, Ranichauri, Distt Tehri-Garhwal, Uttarakhand, India [email protected]

Dr Wamik Azmi Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, Himachal Pradesh, India [email protected]

Dr Om Prakash Chauhan Fruits and Vegetables Technology Division Defence Food Research Laboratory Siddarthanagar, Mysore, India [email protected]

Dr Neerja S. Rana Department of Basic Sciences Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni-Solan, Himachal Pradesh, India [email protected]

Dr P S Panesar Department of Food Engineering and Technology SL Institute of Engineering and Technology, Longowal, Punjab, India [email protected]

EDITORIAL ADVISORY BOARD Dr Sumit Arora ( Dairy Science) Dairy Chemistry Division, National Dairy Research Institute Karnal, Haryana, India [email protected] Prof Tek Chand Bhalla (Food Fermentation & Enzyme Tech.) Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India [email protected] Dr M C Pandey, (Meat and Meat products) Department of Freeze Drying and Animal Product Technology, Defense Food Research Laboratory, Mysore, India [email protected] Dr V M Pratape, (Grain Science and Technology), Department of Grain Science and Technology, Central Food Technological Research Institute, Mysore, India [email protected] Dr Pura Naik J (Plantation Crops) Division of Plantation, Spices and Flavour Tech., Central Food Technological Research Institute, Mysore, India [email protected] Professor Pradeep Khanna (Mushroom Production) College of Basic Science, PAU Ludhiana, Punjab, India [email protected] Dr Y S Dhaliwal (Food and Nutrition) Department of Food Science and Nutrition, College of Home Science, CSK HPKV, Palampur, Himachal Pradesh, India [email protected] Dr R S Singh ( Food Fermentation and Enzyme Technology) Department of Biotechnology, Punjabi University, Patiala, Punjab, India. [email protected] Dr S S Kanwar Department of Microbiology, CSK HPKV, Palampur, Distt Kangra Himachal Pradesh, India [email protected] Dr RC Ray (Food Fermentation) Principal Scientist (Microbiology) Regional Centre of Central Tuber Crops Research Institute Dumuduma Housing Board, Bhubaneswar, Orissa, India

Dr. Rintu Banerjee (Microbial Technology) Microbial Biotechnology and Downstream Processing Laboratory, Agricultural & Food Engineering Department Indian Institute of Technology, Kharagpur,West Bengal, India rin_ [email protected] Dr Eveline Bartowsky (Wine Microbiology) The Australian Wine Research Institute P.O. Box 197, Glen Osmond, Australia. [email protected] Dr L. Rebordinos ( Food Microbiology) Laboratorio de Microbiología y Genética. Facultad de Ciencias del Mar y Ambientales. Universidad de Cádiz. Polígono del río San Spain. [email protected] Dr Aline Lonvaud (Wine and Brandy) Faculty of Enology, University Victor Segalen Bordeaux 2, France. [email protected] Dr Creina S. Stockley ( Wine and Health) The Australian Wine Research Institute Australia. [email protected] Dr M. Remedios Marín (Wine Modelling) Universidad Publica de Navarra Nafarroako Unibertsitste Publikoa Area de Tecnologia de Alimentos Universidad Publica de Navarra Campus Arrosadia Pamplona, (Navarra), Spain. [email protected] Dr Philippe Jeandet (Sparkling Wine) Laboratory of Enology and Applied Chemistry, Unité de Recherche sur la Vigne et le Vin de Champagne, Research Unit N°2069 University of Reims, Faculty of Science, France. [email protected] Dr Vasudeo P. Zambare Sequence Biotech. Pvt. Ltd. Nashik, Maharashtra [email protected]

Dr Luca Cocolin (Wine Microbiology) Dipartimento di Scienze degli Alimenti, Università degli studi di Udine, Facoltà di Agraria, via Marangoni, Udine, Italy. [email protected] Dr Ginés Navarro (Wine Fermentation) Departamento de Química Agrícola, Geología y Edafología, Facultad de Química. Universidad de Murcia. Campus Universitario de Espinardo, Murcia. Spain. [email protected]

Dr R K Gupta (Statistics) Department of Basic Sciences Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, India. [email protected] Dr S K Patyal Department of Entomology and Apiculture, DR. Y S Parmar University of Horticulture and Forestry, Nauni, Solan [email protected]

Contents International Journal of Food and Fermentation Technology Vol. 1, No. 2, December 2011 Message Inaugural Editorial

i iii

Review Paper Anti-oxidant properties and other functional attributes of tomato: An overview

Ramesh C. Ray, Aly F. El Sheikha, Smita H. Panda and Didier Montet

139

Prebiotics: Current status and perspectives

Parmjit S. Panesar, Shweta Kumari and Reeba Panesar

149

Antimicrobial activity of essential oils : A review

VK Joshi, Rakesh Sharma and Vikas Kumar

161

Use of fermentation technology on vegetable residues for value added product development : A concept of zero waste utilization

Anshu Singh, Arindam Kuila, Sunita Adak, Moumita Bishai and Rintu Banerjee

173

White button mushroom (Agaricus bisporus): Composition, nutritive value, shelf-life extension and value addition

Surabhi Sharma and Devina Vaidya

185

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables

VK Joshi, Somesh Sharma, Arjun Chauhan and N.S. Thakur

201

Comparative evaluation of different cell disruption methods for the release of L-asparaginase from Erwinia carotovora MTCC 1428

Sarita and Wamik Azmi

211

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead

Ghan Shyam Abrol and VK Joshi

221

Olive press cake - proximate composition and quality of extracted oil

Rakesh Sharma, PC Sharma and VK Joshi

231

Development of wild pomegranate drink and its evaluation during storage

NS Thakur, Girish S Dhaygude and VK Joshi

237

Research Paper

Beneficial effects of pigment from Monascus purpureus as an alternative treatment on preventing atherosclerosis, hypercholesterolemia and lipid modificaction

Marzieh Rezaei1, Rasoul Roghanian, Iraj Nahvi, and Jamal Moshtaghian

247

Preparation and evaluation of custard apple wine: Effect of dilution of juice on physico-chemical and sensory quality characteristics

Vikas Kumar, P. Veeranna Goud, J. Dilip Babu and R. Subhash Reddy

255

L.V.A. Reddy and L.P.A. Reddy

261

Ranjeeta Bhari

267

Short Communication Preliminary study on preparation and evaluation of wine from guava (Psidium guajava l.) fruit Book Review Handbook of Enology: Principles, Practices and Recent Innovations

Message

Fermentation, the anaerobic way of life, has attained a wider meaning in the biotransformations resulting in a wide variety of fermented foods and beverages. Fermented products made with uncontrolled natural fermentations or with defined starter cultures, achieve their characteristic flavour, taste, consistency and nutritional properties through the combined effects of microbial assimilation and metabolite production, as well as from enzyme activities derived from food ingredients. Fermented foods and beverages span a wide diversity range of starchy root crops, cereals, pulses, vegetables, nuts and fruits, as well as animal products such as meats, fish, seafood, and dairy. The science of chemical, microbiological and technological factors and changes associated with manufacture, quality and safety is progressing and is aimed at achieving higher levels of control of quality, safety and profitability of food manufacture. Both producer and consumer benefit from scientific, technological and consumer-oriented research. Small-scale production needs to be better controlled and safeguarded. Traditional products need to be characterized and described to establish, maintain and protect their authenticity. Medium- and large-scale food fermentation required selected, tailor-made or improved processes that provide sustainable solutions for the future conservation of energy and water, and responsible utilization of resources and disposal of byproducts in the environment. The newly launched International Journal of Food and Fermentation Technology will have an important role to play in the dissemination and exchange of views and scientific and technological developments in India and abroad. I wish the newborn a long and prosperous career of strong science, profitable spin-off’s to entrepreneurs, and flavourful exchanges of opinions. M.J. Robert Nout Laboratory of Food Microbiology Wageningen University Wageningen, the Netherlands (i)

Inaugural Editorial It gives me a lot of pleasure to place before you the inaugural issue of ‘International Journal of food and Fermentation Technology.’ The launching of a new journal is a signal of the maturity of a scientific field. In the field of food and fermentation, this maturity is derived principally from the consolidation of general and scientific interest in the field, and from its practical relevance, rather than from the genesis of new methodologies. The food and fermentation technology is popular as it relates to the common man, has pivotal role to play in the economy of any country rather globally. It influences the intake of various nutrients to the enhancement of health or to the onset of disease. The upsurge of interest in the processes and dynamics by which food is processed largely related to ensuring that these foods are safer, packed and delivered in appropriate packages. All over the world especially in the developing countries the postharvest losses are staggering high and are nothing short of colossal wastage of national resources. The preservation of food is essential for saving the hard earned crops from wastage. These are considered as the complementary means of production of the food commodities may be milk, fruits and vegetables. or meat. To preserve these crops, a multitude of methods are employed viz; thermal processing, low temperature storage, use of radiations, aseptic packaging, chemical preservation, preservation by fermentation or the use of emerging technologies such as ohmic heating, pulsed electric field, microwave heating, hurdle technologies etc. The food preservation saves the hard earned produce that can be used to feed millions of hungry mouths. Besides this role, food processing plays an important role in providing jobs to a large number of people, gives diversified food products even in non-accessible areas including to the armed forces. Indigenous fermented foods are prepared and consumed world-over. These are prepared by the technologies developed over the time using the collective wisdom of the people which passed on to the next generation. Rather, it can be called the oldest biotechnology or food preservation technology. Recent developments have boosted scientific research in the process of the indigenous fermented foods due to diversity of taste and presence of antioxidant and antimicrobial compounds in the fermented foods. Food fermentation as a method of preservation is being employed from time immemorial. Now-a-days, due to development of new techniques of preservation, the food fermentation is employed mainly to enhance the taste and flavour. Globally, production of wines, brandies, beers and other alcoholic beverages constitute a big chunk of the production. Besides, a large number of fermented products be olive pickles, sauerkraut, curd, cheese, yoghurt, sausages, fermented vegetables like kimchi, is the contribution of fermentation. Now-a-days, the focus is to use fermentation technology for waste utilization, production of flavour, colour, enzymes and other bio-preservatives. The biggest factor in the enhanced interest in processed food products is due to the role these play in combating several diseases. The nutraceutical foods are increasingly becoming a part of human diet. These relate to the effects of small quantities of substances present in such foods that can have beneficial effects on health over and above their values as nutrients. The advent of regulations requiring claims for nutraceutical effects should be based on sound scientific evidence . But a consistent nutraceutical effect can only be assured if the ‘dose’ of the beneficial (iii)

the nutraceutical agent contained in a given food is too low that the dose and form required to elicit the reported effect is difficult to determine. Such type of issues needs more attention of the active researchers and needs appropriate solution. Man has been striving hard to develop such techniques/methods that give maximum safety, minimum loss of nutrients and the products can be stored without spoilage for larger time. The new methods being developed need to be highlighted and brought to the notice of industrialists. The endeavour of the scientific community is to employ as natural products as possible as usage of synthetic products, may be colour, flavour or other additives has resulted in several diseases/disorders to the consumers. The production of biocolour, bioflavour, bioactive compounds, is getting a justified attention both from the consumers as well as researchers. While on one hand, the scientific community is striving hard to develop the safe and nutritious food, the unscrupulous people are trying hard to earn profits by preparing and selling the adulterated foods, which is nothing short of crime against the mankind. The excessive pesticide residues in the food need to be highlighted and force such people to stop such activities. At scientific level, different developing of different methods developed to check adulteration would be highlighted. The journal intends to provide a forum to the active researchers, academicians, the policy makers and the industrialists. We recognize that the success of a journal depends on the quality and scientific merit of papers that are published therein. We aim to publish papers of high scientific quality and to maintain a high level of professional integrity in assessment and publication. We have appointed an editorial board of prestigious and reputable members who are prepared to commit their time and intellect to achieve this end. The New Delhi publisher is also not lagging behind in our efforts to achieve these goals. I cordially invite the academic, industrial and consumers to use this journal as a powerful vehicle for communication of their scientific endeavours. The scientific journals always serve as strong link between the academia and the industry and we would strive hard to achieve these goals.

V.K. Joshi Professor and Head, Department of Food Science and Technology, Dr Y.S.Parmar University of Horticulture & Forestry, Nauni-Solan (H.P.), India [email protected]; [email protected] Ph. : 01792-252342(R) 01792-252410(O) Fax : 01792-252844

(iv)

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 139-148

Review Paper

Anti-oxidant properties and other functional attributes of tomato: An overview Ramesh C. Ray1*, Aly F. El Sheikha2,3, Smita H. Panda4 and Didier Montet3 1Regional

Centre, Central Tuber Crops Research Institute, Dumduma PO Bhubaneswar, India. University, Faculty of Agriculture, Department of Food Science and Technology, 32511 Shibin El Kom, Minufiya Government, Egypt. 3Centre de Coopération Internationale en Recherche Agronomique pour le Développement, CIRAD, UMR Qualisud, TA 95B/16, 34398 Montpellier Cedex 5, France. 4Agri-Bioresource Research Foundation, 81, District Centre, Chandrasekharpur, Bhubaneswar, Orissa, India. 2Minufiya

Abstract. Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetables, both nutritionally and economically. The edible part, fruit, known as the power house of nutrition, is a rich source of carotenoids (lycopene, ß-carotene and lutein, etc) and phenolics, vitamins and minerals. Dietary intake of tomatoes and tomato products containing lycopene has been shown to be associated with decreased risk of chronic diseases such as cancers and cardiovascular diseases. Serum and tissue lycopene levels have been inversely related with the chronic disease risk. The antioxidant properties of lycopene are thoughts to be primarily responsible for its beneficial properties. Keywords: Tomato, Anti-oxidant, Lycopene, Nutrient Composition, Health benefits

Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetables, both nutritionally and economically (Karavina et al., 2009). The edible part, fruit, is known as the power house of nutrition (Weisburger, 2002; Tyssandier et al., 2004). It contains a multitude of vitamins and minerals that act to support human health (Kris-Etherton et al., 2002). It is widely acknowledged that the intake of fruits and vegetables such as tomato diminishes the prevalence of cancers (Ito et al., 2003; Campbell et al., 2004) and cardiovascular diseases (Khan and Yeole, 2005). Further, the antioxidant microconstituents present in tomato such as vitamin C and E, carotenoids, polyphenols, and trace metals, i.e. selenium, copper, manganese and zinc, which are cofactors of antioxidant *E-mail of corresponding author : 1c_rayctcricrediffmail.com MS Received on : 30th May, 2011 Accepted on : 2nd Sept,. 2011

enzymes (Martinez-Valverde et al., 2002), play a key role in the health protection mechanisms by scavenging free radicals(Subhash et al., 2007). In the United States, consumption of tomatoes and tomato products ranks number two to potatoes among vegetables (Martinez-Valverde et al., 2002). As far as their consumption is concerned tomatoes and related products rank as the number three contributor of vitamin C and the number four contributor of provitamin A and are the ninth highest contributor of potassium to the U.S. diet. In Italy, tomatoes have been estimated as the second most important source of vitamin C after oranges (Acquaah, 2002). Tomato products are also an essential part of the Mediterranean diet (Khan and Yeole, 2005). In contrast, tomato

Ray et al.

consumption in some populations appears to be too low in Belgium (Tuyns et al. 1992). This review addresses the antioxidant properties and other health attributes of tomato and tomato products.

Nutrient and Phytochemical of Tomatoes Tomato products are excellent sources of potassium, folate and vitamins A, C and E (Table 1) (Campbell et al., 2004). Fiber is another dietary component that has been associated with decreased cancer risk and appreciable amounts are found in tomato products. The higher amount of fibers (11.8 g fiber per cup) has been found in tomato paste (Campbell et al., 2004). Tomatoes also contain a variety of phytochemicals, including carotenoids and polyphenols. In tomatoes and tomato products, lycopene is the carotenoid with the highest concentration, but tomatoes also contain other carotenoids, including phytoene, phytofluene, and the provitamin A carotenoid, β-carotene (Table 2) (Tonucci et al., 1995). Other sources of lycopene

include fresh watermelon (45.3µg/g) and pink grapefruit (14.2 µg/g), but 85% of lycopene exposure comes from tomato sources, such as canned tomato sauces (287.6 µg/g) (Levy and Sharoni, 2004). Tomatoes are also a concentrated source of flavonols, and contain 98% of the total flavonols in tomato skin as conjugated forms of quercetin and kaempferol (Stewart et al., 2000). The flavanone naringenin is present in small quantities in tomatoes in its conjugated form (Campbell et al., 2004). Many of these nutrients and phytochemicals present in tomatoes have antioxidant properties and in combination with lycopene may contribute to the numerous health benefits. Antioxidants in Tomatoes In the recent years, increasing attention has been paid to the role of antioxidants in human health. Antioxidants are protective agents that inactivate ‘Reactive Oxygen Species’ (ROS) and thereby, significantly delay or prevent oxidative damage (Martinez-Valverde et al., 2002). Fruits and vegetables contain numerous different compounds, many of which have antioxidant properties (Heinonen et al., 1998). These include ascorbic

Table 1. Nutrient composition of fresh tomatoes and tomato products* Nutrient

Tomato products (100 g) Raw tomatoes

Potassium (mg)

Catsup

Tomato juice

Tomato sauce

Tomato soup

237.00

382.00

229.00

331.00

181.00

0.54

1.46

0.32

2.08

0.50

Vitamin A (IU)

833.00

933.00

450.00

348.00

193.00

Vitamin C (mg)

12.70

15.10

18.30

7.00

27.30

Total folate (µg)

15.00

15.00

20.00

9.00

70.00

α-tocopherol (mg)

*Source: Campbell et al.( 2004). Table 2. Carotenoid content of tomatoes and related tomato products* Carotenoids (µg/100g)

Tomato products (100g) Raw tomatoes

Catsup

Tomato juice

Tomato sauce

Tomato soup

Lycopene

2573

17007

9037

15152

5084

ß-carotene

449

560

270

290

75

α-carotene

101

0

0

0

0

Lutein/Zeaxanthin

123

0

60

0

1

1860

3390

1900

2950

1720

820

1540

830

1270

720

Phytoene Phytofluene

*Source: Tonucci et al. (1995).

IJFFT 1(2) 2011 : 140

Anti-oxidant properties and other functional attributes of tomato: An overview

acid, α-tocopherol, carotenes and a wide variety of phenolic compounds (Grolier et al., 2001; Martinez-Valverde et al., 2002). Tomatoes are widely consumed either raw or after processing and can provide a significant proportion of the total antioxidants in the diet. This is largely in the form of carotenes (Scholz et al., 2000; Khachik et al., 2002) and phenolic compounds (RiceEvans et al., 1997). Amongst the carotenoids, lycopene predominates (Table 2) and is mainly responsible for the red colour of tomato fruits and their derived products. The lycopene content varies significantly with ripening and the variety of tomato, and is associated with the content of insoluble solids (Sahlin et al., 2004; Jamal and Chieri, 2006). In addition, lycopene appears to be relatively stable during food processing and cooking. Several mechanisms underlying the protective effects of lycopene and other carotenoids have been postulated. These include antioxidant activities, such as the quenching of singlet oxygen or the scavenging of peroxyl radicals, induction of cell-cell communication and growth control (Wills and Ku, 2002; Soto-Zamora et al., 2005). Much of the research on antioxidant action has focused on phenolic compounds such as the flavonoids and hydroxycinnamic acids (Rice-Evans et al., 1997). Quercetin is one of over 4000 naturally available plant phenolics with pharmacological effects that have been reported as antioxidant compounds (Anese et al., 1999; Martinez-Valverde et al., 2002). In addition, hydroxycinnamic acids, such as caffeic, chlorogenic, ferulic, sinapic and p-coumaric acids, contribute to variable extents to the antioxidant capacity of many plantderived products (Ahn et al., 2005). Tomatoes contain quercetin, naringenin, and chlorogenic acid as the main phenolic compounds (Abushita et al., 2000). Lipid peroxidation occurs by oxidation of fatty acids in the presence of enzymes and by exposure to reactive oxygen species and to transition metal ions in a free radical chain reaction. Phenolic compounds, because of their structure, are very efficient scavengers of peroxyl radicals. Moreover, the action of phenolic compounds can be related to their capacity to reduce and chelate ferric iron which catalyzes lipid peroxidation (Subhash et al., 2007), whereas lycopene is the most efficient singlet oxygen quencher amongst the biological carotenoids (Khachik et al., 2002). Although natural antioxidants appear important in disease prevention, only limited data are available on their occurrence and distribution in tomatoes.

Lycopene as an Effective Antioxidant in Tomato Tomatoes and tomato-based food products are the major source of lycopene and a number of other carotenoids, such as phytoene, phytofluene, α-carotene, γ-carotene, β-carotene, and neurosporene (Ajlouni et al., 2001; Martinez-Valverde et al., 2002). Lycopene is a carotenoid, a potent antioxidant, found in high concentrations in vegetables such as tomato. Lycopene constitutes 60-74% of the carotenoids present in tomatoes and tomato products. This compound can exist as different conformational isomers, but the predominant form found in tomatoes and tomato products (around 95%) is all-translycopene (Thompson et al., 2000; Ajlouni et al., 2001). Many factors affect the lycopene content, such as maturity, cultivar and heat treatment. As tomatoes develop from immature green to ripe, the increase in carotenoid content is related to the increase in lycopene content within the plastids (Jamal and Chieri, 2006). In our diet, 95% of lycopene intake comes from tomatoes and tomato products (Yokota et al., 2003). The powerful antioxidant effects of lycopene may be due to the fact that lycopene is not converted to vitamin A (α-carotene or β-carotene). This suggests that red tomatoes do not have enzymes to convert lycopene to α-carotene or β-carotene (Giovannucci et al., 2002). Therefore, lycopene is available to act as a potent antioxidant in the body, which will promote health (Sesso et al., 2004).

Mechanism of Antioxidant Activity of Lycopene Several possible mechanisms of action have been proposed for the health protective effects of lycopene or tomato phytochemicals (Campbell et al., 2004). These include antioxidant potential, altering xenobiotic mechanism, modulation of the IGF-I axis, inhibiting cell-cycle progression and increasing the function of gap junctions. A brief account of these mechanisms is discussed below. 

First by low-density lipoprotein (LDL) mediated antioxidant activity, i.e. prevent the oxidative modification of LDL, because of its singlet oxygen quenching activity. It donates an electron to the peroxyl radical and thus inhibits the chain propagation and the formation of lipidhydroperoxide (Sahlin et al., 2004; Ahn et al., 2005).



Second by tissue or cell mediated antioxidant activity where there is increased uptake of lycopene by the cells and decreased release of reactive oxygen species (Khan and Yeole, 2005). IJFFT 1(2) 2011 : 141

Ray et al.







Lycopene has been shown to alter xenobiotic metabolism. It significantly induces phase I enzymes such as cytochrome P450-dependent enzymes in a dosedependent manner and increase hepatic quinine reductase (QR), a phase II enzyme, by 2-fold (Brienholt et al., 2000). The lycopene induced phase II detoxification enzymes have been demonstrated in a variety of animal model (Bhuvaneswari et al., 2002; Velmurugan et al., 2002). This class of enzymes is important for the removal of foreign substances and carcinogens from the body. Tomato flavonoids, such as kaempferol, quercetin, and naringenin, have demonstrated high potencies and selectivities for the inhibition of cytochrome P450-IA isoforms (Birt et al., 2001). Lycopene has also been hypothesized to modulate hormone and growth factor signaling in prostate cells. Alternations in IGF-I (tumour) activity, which stimulates proliferation and apoptotic resistance in cells, were examined in a case-control study of 112 men (Mucci et al., 2001). Cooked tomato consumption was associated with a 31.5% decrease in serum IGF-1 levels. Lycopene supplementation was found to decrease tumor (IGF-1) expression in rats (Siler et al., 2004). A significant trend toward lower serum IGF-1 and higher insulin growth factor binding protein-3 (IGFBP-1) was found with higher weekly consumption of catsup and tomato juice in 344 disease free men (Gunnell et al., 2003), and a similar decrease in the ratio of IGF-1 to IGFBP3 was found in ferrets fed lycopene (Liu et al., 2003). Both lycopene and tomato polyphenols, including quercetin, kaempferol, and rutin were shown to interfere with IGF1 signaling in vitro, thus preventing the growth factor from stimulating cell proliferation (Karas et al., 2000). Lycopene and lycopene metabolites were also proposed to increase gap junction communication between cells by increasing the levels of connexin 43 (Stahl et al., 2000; Aust et al., 2003). Formation of gap junctions allows for cell-to-cell communication, which is important in the regulation of uncontrolled, rapid cell growth. In PC-3 MM2 (metastatic prostate cancer cells) and normal oral mucosal cells, lycopene failed to inhibit growth or increase connexin 43 levels, whereas in PC 3 prostate MCF-7 breast, and KB-1 oral cancer cells, the inhibition of growth by lycopene was demonstrated by increased connexin 43 levels (Forbes et al., 2003;

IJFFT 1(2) 2011 : 142

Livny et al., 2002, 2003), thus, suggesting that upregulation of connexin 43 may be important to the anticancer action of lycopene.

Health Benefits of Tomatoes Tomatoes contain a wide variety of nutrients and non-nutrient substances (Tables 1 and 2) that may benefit a person’s health. It is believed that these components work together to create the health benefits. Tomato components and their associated health benefits are discussed below.

Lycopene Lycopene is a type of carotenoid having a strong antioxidant with antiproliferative actions. Among the health benefits attributed to lycopene, the risk of prostate and breast cancer decreases substantially (Anese et al., 1999; Tyssandier et al., 2004). Lycopene appears to have a favorable effect in treating many other cancers such as lung, stomach, colorectal, oral, esophageal, pancreatic, bladder and cervical (Giovannucci, 1999; Giovannucci et al., 1995, 2002). Also, research has shown that lycopene lowers the oxidation of LDL cholesterol, reduces heart disease and increases the resistance to lung cancer and exercise induced asthma (Subhash et al., 2007). There are also evidences that lycopene in tomatoes may help to prevent cataracts (Pollack et al., 1997; Stahl et al., 2001), age-related macular degeneration (Khachik et al., 2002) and sunburns (Stahl et al., 2001). More and more research appears to show that lycopene assists the immune system in protecting the body from illness (Chew and Park, 2004; Anese et al., 1999; Jane et al., 2000). The lycopene content in different processed tomato products is given in Table 3. Table 3. Lycopene content in different tomato products* Tomato Products

Lycopene content (µg/g wet product)

Fresh tomato

8.8- 42.0

Cooked tomato

37

Tomato sauce

62

Tomato paste

54-1500

Tomato powder Tomato soup (condensed) Tomato juice *Source: Gartner et al. (1997).

1126- 1265 80 50-116

Anti-oxidant properties and other functional attributes of tomato: An overview

C40H56 Octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene

Fig. 1. Structure of Lycopene

C40H56O2 ß- -carotene-3, 3′-diol (3R,3′R,6′R) [127-40-2]. Fig. 2. Structure of Lutein Lycopene has a structure (Fig. 1) similar to that of the wellknown antioxidant ß-carotene, but its antioxidant activity is much stronger (Herber and Lu, 2002; Jamal and Chieri, 2006). Tsen et al. (2006) were able to explain why lycopene is more potent than ß-carotene in neutralizing singlet oxygen, a strong free radical. The antioxidant capacity of lycopene offers protection against gamma-radiation induced damage to cells (Subhash et al., 2007) during radiotherapy of cancer patients (Ito et al., 2003; Kris-Etherton et al., 2002). This in vitro study was carried out on rat hepatocytes, which were pretreated with lycopene and exposed to different levels of gamma-radiation. The pre-treatment resulted in significant reduction of DNA damage and lipid peroxidation (as measured by thiobarbituric acid reactive substances) (Porrini and Riso, 2000). Other components may also play a role in the protective effects of tomatoes. A study by Gitenay et al. (2007) showed that tomatoes, even yellow varieties without lycopene, showed a stronger antioxidant effect in rats. Rats with mild oxidative stress, caused by low vitamin E level, were fed with placebo, yellow tomato extract, red tomato extract or lycopene. All

diets had no effect on plasma cholesterol but only the red tomato diet reduced triglycerides (Tsen et al., 2006). Rats fed with yellow or red tomato extract showed lower levels of thiobarbituric reactive species in the heart than those fed with placebo or lycopene. This led the researchers to conclude that tomatoes, containing lycopene or not, have a higher potential than lycopene alone to attenuate oxidative stress parameters in a mild oxidative stress context (Herber and Lu, 2002; Khan and Yeole, 2005). Lycopene reduces myocardial damage by suppressing the oxidative stress. High iron levels in blood can cause oxidation and may be carcinogenic (Tsen et al., 2006; Bose and Agrawal, 2007). A Brazilian study led by Matos (2006) demonstrated that rats treated with a high level of iron (ferric nitrilotriacetate) significantly increased DNA damage and malondialdehyde (indicator of lipid oxidation) level in the prostate. The pretreatment of lycopene to the rats reversed these effects; lipid and DNA damage was almost completely prevented (Gitenay et al., 2007; Stahl et al., 2001). A study on adult asthma patients showed that the intake of tomato juice or a tomato extract (both corresponding to a daily intake of 45 mg lycopene)

IJFFT 1(2) 2011 : 143

Ray et al.

inverted the unfavorable effect of a low anti-oxidant diet on asthma and airway inflammation (Wood et al., 2008). Intake of tomato juice or lycopene extract resulted in a reduced influx of white blood cells in the airway. Treatment with the lycopene extract also reduced the activity of neutrophil elastase, which plays a role in degenerative and inflammatory diseases (Tyssandier et al., 2004).

cholesterol from the body, by helping the body to remove it more efficiently (Beecher, 1998). Fiber in tomato slows gastric emptying and, therefore, absorption of sugar into the bloodstream, positively impacting diabetes. In addition to its indirect effects on blood sugar, fiber also assists in removing carcinogenic compounds in the colon (Tuyns et al., 1992).

Lutein Vitamin C Tomatoes are an excellent source of vitamin C, a nutrient known for its antioxidant action (Acquaah, 2002; Karavina et al., 2009). It may help reduce blood pressure and cholesterol levels. Vitamin C helps to form connective tissue, keeps capillaries healthy to help prevent easy bruising, and keeps the gums healthy. High intakes of vitamin C and ß-carotene may prevent atherosclerosis, diabetes, colon cancer and asthma (Wood et al., 2008).

Vitamin A The tomato also contains an abundance of other carotenoids, besides ß-carotene, making it a rich source of vitamin A (Tyssandier et al., 2004). Some researchers believe that it is the diverse carotenoid compounds in the tomato which enhance the action of lycopene (Campbell et al., 2004). It aids in the development of healthy skin, hair and mucous membranes. It also aids proper vision, development of bones and teeth, and reproduction (Ito et al., 2003).

Vitamin K Potassium, vitamin B6, folate and niacin are all present in tomato and work together to help fight atherosclerosis (He and MacGregor, 2003). The potassium in the tomato works against heart disease by lowering blood pressure. Vitamin K helps to build bone and is important to maintain blood clotting, thus, helping to prevent hemorrhaging (excessive, uncontrollable bleeding). Vitamin K is also important to maintain bone health through the act of mineralization (Bugel, 2003; He and MacGregor, 2003).

Dietary fiber In addition, tomatoes are rich in fiber, the undigestible part of a plant. While vitamins C and A deter free radical damage to cholesterol, the fiber in the tomato lowers the amount of

IJFFT 1(2) 2011 : 144

Lutein is a plant pigment that belongs to the well-known group of carotenoids. Lutein along with ß-carotene is one of the most widely distributed carotenoids in fruits and vegetables frequently consumed by different populations (Granado et al., 2003; O’Neill et al., 2001). Tomatoes are a rich source of lutein. Chemically, lutein (Fig. 2) and its structural isomer zeaxanthin are the dihydroxy derivatives of α-carotene and β-carotene, respectively, presenting two hydroxyl groups at the terminal rings of the molecule. In foods, lutein can be found either in its free form, bound to proteins, or esterified as a monoester or di-ester. After being released from the food matrix, it is incorporated into micelles to be absorbed by passive transport by enterocytes and, along with other carotenoids and fat-soluble dietary components, is incorporated into nascent chylomicrons for transport to the liver.

Epidemiologic and Lycopene The antioxidant properties of lycopene, have raised interest in tomato as a food with potential anticancer properties (Bose and Agrawal, 2007). Higher consumption of tomatoes is in fact compatible with current general recommendations aimed at increasing intake of fruits and vegetables (Gardner et al., 2000). These items include tomato and spaghetti sauce, tomato soup, salsa, ketchup, and tomato paste. Moreover, many of these processed foods are better sources of bioavailable lycopene than are fresh tomatoes (Giovannucci, 1999). Epidemiologic evidences regarding consumption of tomato and related products with the risk of cancer at various body sites and on coronary heart diseases are discussed briefly.

Lung and Pleural Cancers One of the cancer sites for which benefit of fruits and vegetables has been most apparent is for cancers of the lung, the leading cause of cancer death worldwide. A case study in Hawaii by Le Marchand et al. (1989) found that tomato intake substantially reduced the risk of lung cancer.

Anti-oxidant properties and other functional attributes of tomato: An overview

Stomach Cancer

Esophageal Cancer

Stomach or gastric cancer remains one of the major causes of cancer death in the world. Several case studies from a variety of populations, including the United States, Japan, Israel, Italy, Spain, Poland, Belgium, and Sweden have reported data on tomato on lycopene intake and stomach cancer risk (Giovannucci, 1999). Inverse associations between tomato consumption and risk of gastric cancer were observed in all these diverse population except for Spain and Japan. Another study in Japan that examined plasma levels of various nutrients in samples of populations in various regions found that regions high in plasma lycopene had the lowest gastric cancer rates and regions low in lycopene had the highest rates (Herber and Lu, 2002; Jamal and Chieri, 2006). Chronic infection by Helicobacter pylori is a major established risk factor for gastric cancer. Chronic infections may increase cancer risk by increasing the oxidative load. Elevated DNA oxidation occurs early during H. pylori infection (Baik et al., 1996). Dietary antioxidants, including lycopene, may potentially reduce the impact of oxidative load from H. pylori infections in the stomach (Giovannucci, 1999).

Esophageal cancers have received little study regarding tomatoes and lycopene. One study in Iran, which has extremely high rates of esophageal cancer particularly in men, found significant reduction (39%) in risk for the men who consumed tomatoes frequently, but no relationship was apparent for women (Nomura et al., 1997; Giovannucci, 1999).

Colorectal Cancer Cancers of the colorectum are common in economically developed countries. Five studies have reported on tomato intake in relation to colorectal cancer risk (Centonze et al., 1994). One study in the United States reported statistically significant inverse associations between tomato consumption and colon cancer risk for men and women. A study in Belgium (Hu et al., 1991) found no overall association but did find a suggestion of an inverse association between consumption of tomato puree and colon cancer risk. The consumption of tomato products was low in this population. In a rodent model of N-methyl nitrosourea induced colonic aberrant crypt foci, lycopene and lutein, but not ß-carotene, in relatively small doses demonstrated efficacy against this premalignant lesion (Boileau et al., 2003; Gitney et al., 2007).

Oral/Laryngeal/Pharyngeal Cancer Very few case studies have been reported on tomato intake in relation to oral cancers. Studies in China reported that high consumption of tomatoes was related to approximately half the risk of oral cancer (Zheng et al., 1992; 1993).

Prostate Cancer Prostate cancer is the second leading cause of cancer death in American men (Campbell et al., 2004). Epidemiological evidences within the past decade strongly suggest that the consumption of fresh and processed tomato products is associated with a reduced risk of prostrate cancer (Giovannucci et al., 1995; Gann and Khachik, 2003, Campbell et al., 2004). In a cohort of 14 000 Seventh-day Adventist men, only tomato intake and intake of beans, lentils, and peas were significantly related to lower prostate cancer risk in a multivariate analysis. ß-Carotene-rich foods were unrelated to risk (Khachik et al., 2002). In a larger, more comprehensive dietary study, intake of the carotenoids, β-carotene, α-carotene, lutein, and βcryptoxanthin was not associated with risk of prostate cancer, but high lycopene intake was related to a significant reduction (21%) in risk (Liu et al., 2006; Sesso et al., 2004). High intake of tomatoes and tomato products, which accounted for 82% of lycopene, reduced risk of total prostate cancer by 35% and aggressive prostate cancer by 53%.

Coronary Heart Diseases Coronary heart disease (CHD) is one of the principal causes of deaths in world. Although genetic factors and age are important in determining the risk due to CHD, other factors such as hypertension, hypercholesterolemia, insulin resistance and life style factors such as diet and smoking caro tenoids are protective effect against CHD by preventing the oxidation of LDL, because of its singlet oxygen quenching activity (Ahn et al., 2005) and can suppress cholesterol synthesis. For example, in J-774A1 macrophage like cell line, lycopene at a concentration of 10 μM induced 73% inhibition in cholesterol synthesis from (3H) acetate (Khan and Yeole, 2005). In another study, six healthy male subjects consumed 60mg/day lycopene for three months. At the end of the treatment period, a significant 14% reduction in plasma LDL cholesterol levels were observed with concomitant decrease in the activity of

IJFFT 1(2) 2011 : 145

Ray et al.

macrophage 3-hydroxyl-3- methyl glutaryl coenzyme A (HMGC-A) reductase, a rate limiting enzyme in cholesterol synthesis (Fuhrman et al., 1997).

Bhuvaneswari V, Velmurugan B and Nagini S. 2002. Induction of glutathione-dependent hepatic biotransformation enzymes by lycopene in the hamster cheek pouch carcinogenesis model. J. Biochem. Mol. Biol. Biophys., 6: 257-260.

Conclusion

Birt DE, Hendrich S and Wang W. 2001. Dietary agents in cancer prevention: flavonoid and isoflavonoids. Pharmacol. Ther., 90: 157-177.

Tomato and tomato-based products are important sources of many established nutrients and are predominant sources of some phytochemicals, which render many health benefits. Anticancer properties for several of these nutrients have been hypothesized. Among the most prominent phytochemicals in tomatoes are the carotenoids, i.e. lycopene, zeaxanthin, phytoene and phytofluene. Tomato product consumption can affect not only the lycopene status, but also that of other antioxidant microconstituents (β-carotene and lutein). Lycopene as well as β-carotene are apparently the main tomato microconstituents responsible for the effect of tomato products on antioxidant status. There are many epidemiological studies and evidences to suggest that lycopene and other phytochemicals in tomato are beneficial in preventing cancers and other diseases; however, there are few human clinical trials. Animal experiments and cell culture studies show promising results but the value of lycopene in preventing diseases remains suggestive rather than conclusive. References Abushita AA, Daood HG and Biacs PA. 2000. Change in carotenoids and antioxidant vitamins in tomato as a function of varietal and technological factors. J. Agric. Food Chem., 48: 2075-2081. Acquaah G. 2002. Horticulture: Principles and Practices. New Jersey, Prentice Hall. Ahn T, Oke M, Schofield A and Paliyath G. 2005. Effects of phosphorus fertilizer supplementation on antioxidant enzyme activities in tomato fruits. J. Agric. Food Chem., 53: 1539- 1545.

Boileau TW, Liao Z, Kim S, Lemeshow S, Erdman JWJr and Clinton SK. 2003. Prostate carcinogenesis in N-methyl-N-nitrosourea (NMU)-testosterone-treated rats fed tomato powder, lycopene, or energy-restricted diets. J. Natl. Cancer Inst., 95: 1578-1586. Bose KS and Agrawal BK. 2007. Effect of lycopene from tomatoes (cooked) on plasma antioxidant enzymes, lipid peroxidation rate and lipid profile in grade-I hypertension. Ann. Nutr. Metab., 51: 477-481. Breinholt V, Lauridsen ST, Daneshvar B and Jakobsen J. 2000. Doseresponse effects of lycopene in selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett., 154: 201-210. Bugel S. 2003. Vitamin K and bone health. Proc. Nutr. Soc., 62: 839843. Campbell JK, Canene-Adams K, Lindshield BL, Boileau TWM, Clinton SK and Erdman JW. 2004. Tomato phytochemicals and prostate cancer risk. J. Nutr., 134: 3486S–3492S. Centonze S, Boeing H, Leoci C, Guerra V and Misciagna G. 1994. Dietary habits and colorectal cancer in a low-risk area. Results from a population-based case–control study in southern Italy. Nutr Cancer., 21: 233-246. Chew BP and Park JS. 2004. Carotenoid action on the immune response. J. Nutr., 134: 257S-261S. Forbes K, Gillette K and Sehgal I. 2003. Lycopene increases urokinase receptor and fails to inhibit growth or connexin expression in a metastatically passaged prostate cancer cell line: a brief communication. Exp. Biol. Med., 228: 967-971.

Ajlouni S, Kerner S and Masih L. 2001. Lycopene content of hydroponic and non-hydroponic tomatoes during post harvest storage. Food Australia, 5: 195-196.

Fuhrman B, Elis A and Aviram M. 1997. Hypocholesterolemic effect of lycopene and beta carotene is related to suppression of cholesterol synthesis and augmentation of LDL receptor activity in macrophase. Biochem. Biophys. Res. Commun., 233: 658662.

Anese M, Manzocco L, Nicoli MC and Lerici CR. 1999. Antioxidant properties of tomato juice as affected by heating. J. Sci. Food Agric., 79: 750-754.

Gann PH and Khachik F. 2003. Tomatoes or lycopene versus prostate cancer: Is evolution anti-reductionist. J. Natl. Cancer Inst., 95: 1563-1565.

Aust O, Ale-Agha N, Zhang L, Wollersen H, Sies H and Stahl W. 2003. Lycopene oxidation product enhances gap-junction communication. Food Chem. Toxicol., 41: 1399-1407.

Gardner PT, Tamsin AC, White D, McPhail B and Duthie GG. 2000. The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices. Food Chem., 68: 471-474.

Baik SC, Youn HS, Chung MH, Lee WK, Cho MJ and Ko GH. 1996. Increased oxidative DNA damage in Helicobacter pyloriinfected human gastric mucosa. Cancer Res., 56: 1279-1282. Beecher GR. 1998. Nutrient content of tomatoes and tomato products. Proc. Soc. Exp. Biol. Med., 218: 98-100.

IJFFT 1(2) 2011 : 146

Gartner C, Stahl W and Sies H. 1997. Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am. J. Clin. Nutr., 66: 116-122.

Anti-oxidant properties and other functional attributes of tomato: An overview

Giovannucci E. 1999. Tomatoes, tomato-based products, lycopene, and cancer: Review of the epidemiologic literature. J. Natl. Cancer Inst., 91: 317-331. Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. 1995. Intake of carotenoids and retinol in relation to risk of prostate cancer. J. Natl. Cancer Inst., 87: 1767-1776. Giovannucci E, Rimm EB, Liu Y, Stamper MJ and Illett WC. 2002. A prospective study of tomato products, lycopene and prostrate cancer risk. J. Natl. Cancer Inst., 94: 391-398. Granado F, Olmedilla B and Blanco I. 2003. Nutritional and clinical relevance of lutein in human health. Br. J. Nutr., 90: 487-502. Gitenay D, Lyan B, Rambeau M, Mazur A and Rock E. 2007. Comparison of lycopene and tomato effects on biomarkers of oxidative stress in vitamin E deficient rats. Eur. J. Nutr., 46: 468475. Grolier P, Bartholin G, Broers L, Caris-Veyrat C, Dadomo M, DiLucca G, Dumas Y, Meddens F, Sandei L and Schuch W. 2001. Compositions of tomato and tomato products in antioxidants. In: The White Book on Antioxidants in Tomatoes and Tomato Products and Their Health Benefits, (Eds. R Bilton, M Gerber, P Grolier and C Leoni), FAIR RTD Programme (FAIR CT 97-3233), pp 1-100. Gunnell D, Oliver SE, Peters TJ, Donovan JL, Persad R, Maynard M, Gillatt D, Perarce A, Hamdy FC, Neal DE and Holly JMP. 2003. Are diet-prostate cancer associations mediated by the IGF axis? A cross-sectional analysis of diet, IGF-1 and IGFBP-3 in healthy middle-aged men. Br. J. Cancer., 88: 1682-2686. He FJ and MacGregor GA. 2003. Potassium: more beneficial effects. Climacteric. Suppl., 3: 36-48. Herber D and Lu QY. 2002. Overview of mechanisms of action of lycopene. Exp. Biol. Med. 227: 920-923. Heinonen IM, Pekka JL and Hopia AI. 1998. Antioxidant activity of berry and fruit wines and liquors. J. Agric. Food Chem., 46: 2531. Hu JF, Liu YY, Yu YK, Zhao TZ, Liu SD and Wang QQ. 1991. Diet and cancer of the colon and rectum: a case–control study in China. Int. J. Epidemiol., 20: 362-367. Ito Y, Wakai K, Suzuki K, Tamakoshi A, Seki N, Ando M, Nishino Y, Kondo T, Watanabe Y, Ozasa K and Ohno Y. 2003. JACC Study Group. Serum carotenoids and mortality from lung cancer: a case-control study nested in the Japan Collaborative Cohort (JACC) study. Cancer Sci., 94: 57-63. Jamal J and Chieri K. 2006. Variation of lycopene, antioxidant activity, total soluble solids and weight loss of tomato during postharvest storage. Post Harvest Biol. Technol., 36: 151-155. Jane E, Upritchard WHF and Sutherland JIM. 2000. Effect of supplementation with tomato juice, vitamin E and C on LDL oxidation and products of inflammatory activity in type- II diabetes. Diabetes Care, 23: 733-738.

Karas M, Amir H, Fishman D, Danilenko M, Segal S, Nahum A, Koifmann A, Giat Y, Levey J and Sharoni Y. 2000. Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr. Cancer., 36: 101-111. Karavina C, Chihiya J, Tigere TA and Musango R. 2009. Assessing the effects of fermentation time on tomato. J. Sustain. Develop. Africa., 10: 106-111. Khachik F, Carvalho L, Bernstein PS and Muir GJ. 2002. Chemistry, distribution and metabolism of tomato carotenoids and their impact on human health. Exp. Biol. Med., 227: 845-851. Khan S and Yeole PG. 2005. Protective effects of tomato in coronary heart diseases: a nutraceutical perspective. Health Administrator, 20: 104-108. Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM, Binkoski AE, Hilpert KF, Griel AE and Etherton TD. 2002. Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am. J. Med., 113: 71S-88S. Le Marchand L, Yoshizawa CN, Kolonel LN, Hankin JH and Goodman MT. 1989. Vegetable consumption and lung cancer risk: a population-based case– control study in Hawaii. J. Natl. Cancer Inst., 81: 1158-1164. Levy J and Sharoni Y. 2004. The functions of tomato lycopene and its role in human health. Herbal Gram, 62: 49-56. Liu C, Lian F, Smith DE, Russell RM and Wang XD. 2003. Lycopene supplementation inhibits lung squamous metaplasia and induces apoptosis via up-regulating insulin-like growth factorbinding protein 3 in cigarette smoke-exposed ferrets. Cancer Res., 63: 3138-3144. Liu A, Pajkovic N, Pang Y, Zhu D, Calamini G, Mesecar AL and Van Breemen RB. 2006. Absorption and sub-cellular localization of lycopene in human prostate cancer cells. Mol. Cancer Ther., 5: 2879-2885. Livny O, Kaplan I, Reifen R, Polak-Charcon S, Madar Z and Schwartz B. 2002. Lycopene inhibits proliferation and enhances gapjunction communication of KB-1 human oral tumor cells. J. Nutr., 132: 3754-3759. Livny O, Kaplan I, Reifen R, Polak-Charcon S, Madar Z and Schwartz B. 2003. Oral cancer cells differ from normal oral epithelial cells in tissue like organization and in response to lycopene treatment: an organotypic cell culture study. Nutr. Cancer., 47: 195-209. Martýnez-Valverde I, Periago MJ., Provan G and Chesson A. 2002. Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato. J. Sci. Food Agric., 82: 323-333. Matos HR, Marques SA, Gomes OF, Silva AA, Heimann JC, Di Mascio P and Medeiros MH. 2006. Lycopene and beta-carotene protect in vivo iron-induced oxidative stress damage in rat prostate. Braz. J Med. Biol. Res., 39: 203-210.

IJFFT 1(2) 2011 : 147

Ray et al.

Mucci LA, Tamimi R, Lagiou P, Trichopoulous A, Benetou V, Spanos E and Trichopoulos D. 2001. Are dietary influences on the risk of prostate cancer mediated through the insulin-like growth factor system? BJU Int., 87: 814-820.

Subhash K, Bose C and Agrawal BK. 2007. Effect of short term supplementation of tomatoes on antioxidant enzymes and lipid peroxidation in type –II Diabetes. Ind. J. Clin. Biochem., 22: 9598.

Nomura AM, Ziegler RG, Stemmermann GN, Chyou PH and Craft NE. 1997. Serum micronutrients and upper aerodigestive tract cancer. Cancer Epidemiol. Biomarkers Prev., 6: 407-412.

Thompson KA, Marshall MR, Sims CA, Wei CI, Sargent, SA and Scott JW. 2000. Cultivar, maturity and heat treatment on lycopene content in tomatoes. J. Food Sci., 65: 791-795.

O’Neill ME, Carroll Y and Corridan B. 2001. A European carotenoid database to assess carotenoid intakes and its use in a fivecountry comparative study. Br. J. Nutr., 85: 499-507.

Tonucci LH, Holden JM, Beecher GR, Khachik F, Davis CS and Mulokozi G. 1995. Carotenoid content of thermally processed tomato-based food products. J. Agric. Food Chem., 43: 579586.

53.

Pollack A, Madar Z, Eisner Z, Nyska A and Oren P. 1997. Inhibitory effect of lycopene on cataract development in galactosemic rats. Metab. Pediatr. Syst. Ophthalmol., 19: 3136.

Porrini M and Riso P. 2000. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J. Nutr., 130: 189-192. Rice-Evans CA, Miller NJ and Paganga G. 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci., 2: 152-159. Sahlin E, Savage GP, Lister CE. 2004. Investigation of the antioxidant properties of tomatoes after processing. J. Food Comp. Anal., 17: 635-647. Scholz TA, Hata TR, Pershing LK, Gellermann W, McClane R, Alexeeva M, Irmakov I and Khachik F. 2000. Non-invasive Raman spectroscopic detection of carotenoids in human skin. J. Invest. Dermatol., 115: 441-448. Sesso HD, Buring JE, Norkus EP, Gaziano JM. 2004. Plasma lycopene, other carotenoids, and retinol and the risk of cardiovascular disease in women. Am. J. Clin. Nutr., 79: 47-53. Siler U, Barella L, Spitzer V, Schnorr J, Lein M, Goralczyk R. and Wertz K. 2004. Lycopene and vitamin E interfere with autocrine/ paracrine loops in the Dunning prostrate cancer model. FASEB J., 18: 1019-1021. Soto-Zamora G, Yahia EM, Brecht JK and Gardea A. 2005. Effects of post harvest hot air treatments on the quality and antioxidants levels in tomato fruit. Lebensm Wiss. Technol., 38: 657-663. Stahl W, von Laar J, Martin HD, Emmerich T and Sies H. 2000. Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene. Arch. Biochem. Biophys., 373: 271-274. Stahl W, Heinrich U, Wiseman S, Eichler O, Sies H, Tronnier H. 2001. Dietary tomato paste protects against ultraviolet light-induced erythema in humans. J Nutr., 131: 1449-1451. Stewart AJ, Bozonnet S, Mullen W, Jenkins GI, Lean MEJ and Crozler A. 2000. Occurrence of flavonols in tomatoes and tomato-based products. J. Agric. Food Chem., 48: 2663-2669.

IJFFT 1(2) 2011 : 148

Tsen KT, Tsen SW and Kiang JG. 2006. Lycopene is more potent than beta carotene in the neutralization of singlet oxygen: role of energy transfer probed by ultrafast Raman spectroscopy. J. Biomed. Opt., 11: 064025. Tuyns AJ, Kaaks R, Haelterman M and Riboli E. 1992. Diet and gastric cancer. A case–control study in Belgium. Int. J. Cancer., 51: 1-6. Tyssandier V, Feillet-Coudray C, Caris-Veyrat C, Guilland J, Coudray C, Bureau S, Reich M, Amiot-Carlin M, Bouteloup-Demange C, Boirie Y and Borel P. 2004. Effect of tomato product consumption on the plasma status of antioxidant microconstituents and on the plasma total antioxidant capacity in healthy subjects. J. Am. Coll. Nutr., 23: 148-156. Velmurugan B, Bhuvaneswari V, Burra UK and Nagini S. 2002. Prevention of N-methyl-N’-nitro-N-nitrosoguanidine and saturated sodium chloride-induced gastric carcinogenesis in Wistar rats by lycopene. Eur. J. Cancer Prev., 11: 19-26. Weisburger JH. 2002. Lycopene and tomato products in health promotion. Exp. Biol. Med. 227: 924-927. Wills RBH and Ku VVV. 2002. Use of 1-MCP to extend the time to ripen of green tomatoes and post harvest life of ripe tomatoes. Biores. Technol., 26: 85-90. Wood LG, Garg ML, Powell H and Gibson PG. 2008. Lycopene-rich treatments modify noneosinophilic airway inflammation in asthma: Proof of the concept. Free Radic. Res., 2: 94-102. Yokota T, Etoh H, Oshima S, Hayakawa K and Ishiguro Y. 2003. Oxygenated lycopene and dehydrated lutein in tomato puree. Biosci. Biotechnol. Biochem., 67: 2644-2647. Zheng T, Boyle P, Willett WC, Hu H, Dan J and Evstifeeva TV. 1993. A case–control study of oral cancer in Beijing, People’s Republic of China. Associations with nutrient intakes, foods and food groups. Eur. J. Cancer B. Oral Oncol., 10: 45-55. Zheng W, Blot WJ, Shu XO, Gao YT, Ji BT and Ziegler RG. 1992. Diet and other risk factors for laryngeal cancer in Shanghai, China. Am. J. Epidemiol., 136: 178-191.

IJFFTJ.Vol. No. 1,Technol. June 2011,1(2) pp. 181-192 New Delhi Publishers Int. Fd.1,Ferm. 2011 : © 149-160

Review Paper

Prebiotics: Current status and perspectives Parmjit S. Panesar*, Shweta Kumari and Reeba Panesar Biotechnology Research Laboratory, Department of Food Engineering & Technology, Sant Longowal Institute of Engineering & Technology, Longowal, Punjab, India.

Abstract : Prebiotics are food ingredients that are good for the health. The prebiotics stimulate the growth of healthy bacteria such as Bifidobacterium and Lactobacillus in the gut and increase resistance to invade pathogens. This effect is induced by consuming functional foods that contain prebiotics. These compound cannot be digested in the small intestines but stimulate the growth and activity of strains of bacteria in the large intestines. Prebiotics are widely used to prepare fermented dairy products like yoghurt or freeze-dried cultures. Futhermore, prebiotics are heat resistant, which keep them intact during the baking process and allow them to be incorporated into every day food choices. By consuming a nondigestible ingredient, it allows the growth of bio-cultures by reaching the intestine unaffected by the digestion processes. These foods induce metabolic activity, leading to health improvement. Healthy bacteria, in the intestine can combat unwanted bacteria, providing a number of health benefits. Keywords : Prebiotics, Probiotics, Yoghurt, Bifidobacterium, Lactobacillus

A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and activity of one or a limited number of bacteria in the colon, which can improve the host health. These prebiotics are digested by the good microorganisms in the gastrointestinal tract to increase the number or activity of these health promoting microorganisms, such as Bifidobacterium bifidum and Lactobacillus acidophilus (Zimmer and Gibson, 1998; Manning and Gibson, 2004; Panesar et al., 2009). Inulin, galacto-oligosaccharides, fructo-oligosaccharides, lactulose and its hydrolysates, malto-oligosaccharides and resistant starch are common prebiotics used in human nutrition. The main end products of carbohydrate metabolism are short-chain fatty acids, namely acetate, butyrate and propionate, which are further used by the host organism as an energy source (Grajek et al., 2005). *E-mail of corresponding author : [email protected] MS Received on : 2nd April, 2011 Accepted on : 5th Sept., 2011

Prebiotics are functional foods that exert a beneficial effect on host health and reduce the risk of chronic diseases beyond their nutritive value (Ziemer and Gibson, 1998; Saarela et al., 2002). A food can be made functional by addition of a potential health promoting entity, reducing or removing concentrations of harmful components and modifying the nature or the bioavailability of one or more components (Ziemer and Gibson, 1998; Saarela et al., 2002). The first generation of functional foods was based on fortification with vitamins and minerals (mainly calcium). However, the concept has recently moved towards food ingredients exerting a positive effect on the gut microbiota, introducing probiotics and prebiotics (Ziemer and Gibson, 1998). Various aspects connected with prebiotiocs have been reviewed here.

Panesar et. al.

Sources of Prebiotics Traditional dietary sources of prebiotics include soybeans, bananas, garlic, barley, onion, jerusalem artichoke tuber, wheat, asparagus, rye, and chicory root.

Inulin Inulins are a group of naturally occurring polysaccharides produced by many plants (Roberfroid, 2005). They belong to a group of fibers well-known as fructans. Inulin is used by some plants as a source of storing energy and is usually found in roots or rhizomes (Table 1). Inulin is gradually more used in processed foods because of its low calorie content. In terms of nutritive value, it is considered as soluble fiber and a prebiotic that promotes the growth of health promoting intestinal bacteria. Table 1. Inulin content on fresh weight basis of some important sources. Source

Inulin (%)

Asparagus root

10-15

Banana

0.3-0.7

Camas

12-22

Chicory root

15-20

Dahlia tubers

15-20

Dandelion

12-15

Salisfy

15-20

Jerusalem artichoke

15-20

Leek

3-10

Onion

2-6

Source : Gupta and Kaur, (1997); Van Loo et al., (1995).

Soybean Oligosaccharide Soybean seed is a rich source of non-digestible galactooligosaccharides (GOS). Galactooligosaccharides (GOS) are naturally occurring non-digestible carbohydrates present in different foods and legumes, such as soybeans. This oligosaccharide is not hydrolyzed by humans due to lack of αgalactosidase enzyme, so they cannot be digested when consumed. Intact oligosaccharides reach the colon, where they are preferentially hydrolyzed by beneficial bifidogenic microorganisms that contain the enzyme (Liu, 1999).

IJFFT 1(2) 2011 : 150

Fermentation of nondigestible oligosaccharides results in the production of gases such as carbon dioxide, hydrogen, methane, etc and short chain fatty acids, which are beneficial because of their prebiotic activity and have associated health benefits (Roberfroid and Slavin, 2000; Gibson and Roberfroid, 1995; Tomomatsu, 1994).

Human Milk Oligosaccharides Human milk oligosaccharides (HMOs) are the growth factors that also contain a significant level (5 to 8 g/L) of oligosaccharides (similar to the fructo-oligosaccharides). These sugar-like molecules provide various health-promoting properties for the infant. These carbohydrates found in breast milk, which act as prebiotics, protect the baby’s intestinal tract from unwanted pathogenic bacteria, lower the risk of diarrhea, and modulate important immune responses in the baby (www.vegetarian-nutrition.info ; Kunz et al., 2000; Ward et al., 2006).

Major Synthetic Prebiotics Prebiotic can be produced by the transgalactosylation activity of respective carbohydrate by both chemical and enzymatic methods.

Galacto-oligosaccharides Galacto-oligosaccharides (GOS), the most promising nondigestible oligosaccharides have drawn attention in the field of functional foods owing to their known health benefits and potential to improve the quality of many foods. At present, GOS are also used as low-calorie sweetener in various food products such as yoghurt, confectioneries, breads and beverages (Park and Oh, 2010). GOS generally comprises of di(galactosylglucose), tri(galactosylgalactosylglucose), tetra(galactosylgalactosyl- galactosylglucose), penta (galactosylgalactosylgalactosylgalactosylgalactosysglucose) and hexasaccharides (galactosylgalactosylgalactosyl galactosyl glucose). Galacto-oligosaccharides (GOS) are produced by transgalactosylation activity during the hydrolysis reaction of lactose with β-galactosidase. GOS production from lactose by the transgalactosylation reaction of β-galactosidase has also been previously reported (Prenosil et al., 1987; Ekhart and Timmermans, 1996). Commercially GOS is produced by using β-galactosidases isolated from several sources such as

Prebiotics: Current status and perspectives

bacteria, yeast and fungi (Crittenden and Playne, 1996; Ekhart and Timmermans, 1996). β-galactosidase from Aspergillus oryzae (Iwasaki et al., 1996), Sirobasidium magnum (Onishi & Tanaka 1997), Penicillium simplicissimum (Cruz et al., 1999), Bacillus circulans (Boon et al., 1999), Bifidobacterium infantis (Hung and Lee, 2002), Bullera singularis (Cho et al., 2003), Escherichia coli (Chen et al., 2003), Kluyveromyces lactis (Kim et al., 2004) and Kluyveromyces marxianus (Panesar et al., 2006) used for transgalactosylation reaction. Galacto-oligosaccharides production from lactose using microbial β-galactosidases is shown in Table 2.

Fructooligosaccharides Fructooligosaccharides (FOS) also sometimes called oligofructose or oligofructan is a class of oligosaccharides used as an artificial alternative sweetener. FOS can be considered as a small dietary fiber with low caloric value. The fermentation of FOS results in the production of various gases and acids, and also provides some energy to the body. FOS is extracted from fruits and vegetables such as bananas, onions, chicory root, garlic, asparagus, barley, wheat, tomatoes, and leeks. The Jerusalem artichoke and yacon have been found to have the highest concentrations of FOS among cultured plants.

Table 2 : Production of Galacto-oligosaccharides using β-galactosidases. Biocatalyst Permeabilized Cells

Crude enzymes

Purified enzymes

Immobilized enzymes

Recombinant enzymes

Enzyme sources Rhodotorula minuta Sirobasidium magnum Sirobasidium magnum Bacillus circulans Aspergillus oryzae Penicillium sp. Lactobacillus reuteri Talaromyces thermophilus Bifidobacterium longum Geobacillus stearothermophilus Saccharopolyspora rectivirgula Sterigmatomyces elviae Penicillium simplicissimum Enterobacter agglomerans Lactobacillus acidophilus Bacillus circulans Bullera singularis Aspergillus oryzae Kluyveromyces lactis Talaromyces thermophilus Penicillium expansum F3 Thermus sp. Bifidobacterium infantis Pyrococcus furiosus Thermotoga maritime Sulfolobus solfataricus Geobacillus stearothermophilus R109W

Temp. (ºC) 60 50 50 40 40 55 30 40 45 37 60 60 50 50 30 40 45 40 40 40 50 70 60 80 80 80 37

pH Lactose (g/L) GOS (% w/v) Reference 6.0 – – 6.0 4.5 4.0 6.5 6.5 6.8 6.5 6.5 5.0 6.5 7.5 6.5 6.0 3.7 4.5 7.0 6.5 5.4 7.0 7.5 5.0 6.0 6.0 6.5

200 360 360 46 380 400 205 200 400 180 600 200 600 125 205 6.0 3.7 4.5 400 200 380 300 300 270 500 600 180

38 38 67 24 31 40 38 50 33 2 44 39 31 38 39 46 300 400 25 50 28.7 30 63 22 19 53 23

Onishi and Yokozeki (1996) Onishi et al., (1996) Onishi et al.,(1996) Mozaffar et al., (1985) Iwasaki et al., (1996) In and Chae (1998) Splechtna et al., (2006) Nakkharat et al., (2006) Hsu et al., (2007) Placier et al., (2009) Nakao et al., (1994) Onishi and Tanaka (1995) Cruz et al., (1999) Lu et al., (2007) Nguyen et al., (2007) Mozaffar et al., (1986) Shin and Yang (1998) Albayrak and Yang (2002) Chockchaisawasdee et al., (2004) Nakagawa et al., (2006) Li et al. (2008) Akiyama et al.,(2001) Hung and Lee (2002) Bruins et al., (2003) Ji et al., (2005) Park et al., (2008) Placier et al., (2009)

Source: Li et al., (2008); Park and Oh, (2010).

IJFFT 1(2) 2011 : 151

Panesar et. al.

FOS can be produced by degradation of inulin or polyfructose, which is a polymer of D-fructose residues linked by β (2-1) bonds with a terminal α (1-2) linked D-glucose. Commercially FOS is prepared by the transfructosylation action of a βfructosidase on saccharose (Hartemink, 1999).

Isomaltose Oligosaccharide Isomaltose oligosaccharide (IMO) is also known as bifidusfactor because of its powerful growth promoting effects on intestinal beneficial bacteria. IMO are composed of glucose monomers linked by α-1,6 (and rarely α-1,4) glucosidic linkages (Chen et al., 2001). It occurs naturally in various fermented foods and sugars such as sake, soybean sauce and honey (Yun et al., 1994). IMO is produced from starch by the action of three separate enzymes. Firstly, starch is hydrolysed to maltooligosaccharides by α-amylase (EC 3.2.1.1) and pullulanase (EC 3.2.1.41). After that, α-glucosidase (EC 3.2.1.20) is added to catalyze a transfer reaction that convert the α-1,4 linked maltooligosaccharide into α-1,6 linked IMO. Further, glucose is removed to produce a higher concentration of product (Kohmoto et al., 1998). The α-transglucosidase (EC 2.4.1.24) from A. niger catalyzes the synthesis of pantose and higher IMO from maltose rich starch hydrolysates. In this case, maltose plays the role of both glucosyl donor and acceptor. IMO have an attractive physicochemical characteristic such as relatively low sweetness, low viscosity and bulking properties (Lee et al., 2002; Mountzouris et al., 1999; Robyt, 1992).

XOS are synthesized by enzymatic hydrolysis of xylan from corn cobs (Crittenden and Playne, 1996). Oat spelt xylan and wheat arabinoxylan can be used as raw material for XOS production. The raw materials for XOS production are hardwoods, corn cobs, straws, bagasses, hulls, malt cakes and bran (Vazquez et al., 2000). Three different methods have been used for production of XOS: (1) enzymic treatment of native xylan-containing lignicellulosic material; (2) chemical fractionation of a suitable lignicellulosic material to isolate xylan, followed by enzymatic hydrolysis to produce XOS; and (3) hydrolytic degradation of xylan by steam, water or mineral acids (Vazquez et al., 2000). In the enzymatic production of XOS, enzyme complexes with low exo-xylanase or βxylosidase activity are required, to avoid the production of xylose. Many microorganisms are known for the production of different types of xylanases. But, the nature of the enzymes varies from organism to organism. Among different Trichoderma spp., Trichoderma reesei, T. harzianum, T. viride and T. koningii are known to be good producers of both cellulolytic and xylanolytic enzymes (Chen et al., 1997). Enzymatic synthesis of some important food-grade oligosaccharides and details of their synthesis are shown in Table 3. Table 3. Enzymatic synthesis of some important food-grade oligosaccharides Class of oligosaccharides

Production process

Galacto-oligosaccharides

transgalactosylation of lactose by β-galactosidase transfructosylation of sucrose by β-fructofuranosidase or hydrolysis of inulin by endoinulinase debranching of starch by isoamylase/ pullulanase, followed by hydrolysis by specific oligosaccharide forming α-amylase hydrolysis of xylan by the action of β-xylanase isomerization of lactose by β-galactosidase and β-glycosidase

Fructooligosaccharides

Xylo-oligosaccharides Xylo-oligosaccharides (XOS) are a chains of xylose molecules linked by β-1,4 bonds and mainly consists of xylobiose, xylotriose and xylo-tetraose (Hopkins et al., 1998) and are found naturally in bamboo shoots, fruits, vegetables, milk and honey (Vazquez et al., 2000). XOS promotes the selective growth of Bifidobacterium sp., which have important biological effects since as they suppress the growth of entero-putrefactive and pathogenic intestinal bacteria due to the production of short chain fatty acids and enhance nutrient absorption. Xylooligosaccharides can also be used as ingredients of functional foods, cosmetics, pharmaceuticals or agricultural products (Alonso et al., 2003).

IJFFT 1(2) 2011 : 152

Isomaltooligosaccharides

Xylooligosaccharides Lactulose

Source : Singh and Singh, (2010).

Lactulose Lactulose (4-0-β-D-galactopyranosyl-D-fructofuranose), a synthetic disaccharide composed of two smaller sugar molecules fructose and galactose bonded together with β-

Prebiotics: Current status and perspectives

1,4-glycosidic bond (Panesar et al., 2008). The β-glycosidic linkage of the disaccharide lactulose is not hydrolyzed by mammalian digestive enzymes (Ruttloff et al., 1967), and ingested lactulose passes the stomach and small intestine without degradation. Lactulose is 1.5 times sweeter than lactose and can be crystallized from alcoholic solution. It is characteristically utilized by all the species of Bifidobacterium, which resides in the human intestine tract. Lactulose can be produced by the isomerization of lactose by regrouping the glucose residue to the fructose molecule (Kochetkov and Bochkov, 1967). A large number of complex reagents such as aluminate and borate (Carobbi and Innocenti, 1990; Krumbblolz and Dashed, 1991; Zokaee et al., 2002), alkalies i.e. sodium hydroxide (De Haar and Plump, 1991; Deya and Takahashi, 1991; Dendene et al., 1994 and Zokaee et al., 2002), magnesium oxide (Carobbi et al., 1985), tertiary amines (Parrish et al., 1970) can be used. Lactulose can also be produced by enzymatic transgalactosylation of lactose to fructose by using β-galactosidase from Aspergillus oryzae and the hyperthermostable β-glycosidase from Pyrococcus furiosus (Mayer et al., 2004). But, the cost of β-galactosidase

production is the main hurdle for the commercialization of the enzymatic method, several attempts have been made to develop cost-effective system. Whole cells biocatalyst can be used for the production of lactulose because it has several advantages over purified enzymes. However, the permeability barrier of the cell envelope for substrates and products often causes very low reaction rates in whole cells, especially yeast cells. In order to reduce the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilized yeast cells can be used (Panesar et al., 2006; Panesar et al., 2007; Panesar et al., 2008). The ethanol (50%, v/v) permeabilized Kluyveromyces marxianus cells were found to be effective for lactulose production (Lee et al., 2004). Further, gene encoding a thermostable β-galactosidase from Sulfolobus solfataricus has been cloned and expressed in Escherichia coli for lactulose production. The transgalactosylation reaction caused by the β-galactosidase was applied to produce lactulose using lactose as a galactose donor and fructose as an acceptor (Kim et al., 2006).

Fig. 1. Potential benefits of prebiotics (Source: Olmstead et al., 2008)

IJFFT 1(2) 2011 : 153

Panesar et. al.

Potential Benefits of Prebiotics Prebiotics are non-digestible fiber in the diet. Because the body does not possess the necessary enzymes to break down the prebiotic as it travels down the digestive tract. When it reaches the large intestine and the colon, the bacteria that are naturally present there start to break down the prebiotics as the bacteria have the enzymes necessary to do so. Bifidobacteria use prebiotics and foods that promote the growth of the bifidobacteria are good for health. Prebiotics increase the amount of beneficial bacteria in the lower intestine, and improve the digestion and absorption of nutrients, detoxification and elimination processes, and stimulates the immune system. Prebiotics may also help to relieve constipation and other gastrointestinal disorders including problems that are related to irritable bowel syndrome, inflammatory bowel disease, and lactose intolerance (Fig. 1).

Short Chain Fatty acid Production Prebiotics act as an energy sources for health benefit intestinal bacteria that ferment them into short-chain fatty acids (Swennen and Courtin, 2006; De Preter et al., 2007). Shortchain fatty acids play essential roles in the growth and physiology of intestinal tissue as well as in systemic metabolism (Topping and Clifton, 2001; Säemann et al., 2002).

Improvement of Constipation All prebiotic that reach the large intestine have a laxative effect on bowel habit. The mechanism of action by means of stimulation of microbial growth, increase in bacterial cell mass and thus stimulation of peristalsis by the increased bowel content (Cummings, 1994). Intake of isomalto-oligosaccharides has been shown to increase stool frequency and wet stool output in chronic constipated elderly men (Chen et al., 2001).

of secretory IgA and immune-stimulating cytokines and enhanced production of short chain fatty acids and other antimicrobial substances that create an unfriendly environment for pathogen growth (Gibson et al., 2005; Hosono et al., 2003). Reduce Carcinogenicity The potential of prebiotics to enhance detoxification processes in colon cells, reduce toxic metabolite production in the gut, and protect against colonic tumor development has been recognized. The prebiotic like inulin-type fructans, galactooligosaccharides, and xylooligosaccharides have been shown to suppress chemically induced colon cancer and precancerous colon lesions (Wijnands et al., 2001; Pool-Zober, 2005). Apart from this, inulin and oligofructose are also helpful in reducing the risk of colon cancer, dietary supplementation which reduces the incidence of chemically-induced mammary cancer, slows the growth of implanted tumors, decreases metastases of implanted cancers, and enhances the efficacy of cancer chemotherapy (Taper, 2005).

Regulation of lipid metabolism The food industries take interest in developing functional foods to modulate blood lipids level, such as cholesterol and triglycerides. The principal hypotriglyceridemic mechanism of action appears to be a decrease in liver lipogenesis through increased production of short-chain fatty acids by intestinal microorganism in the large bowel. Short-chain fatty acid production leads to increased portal concentrations of propionate relative to acetate which inhibits lipogenesis in hepatocytes (Letexier et al., 2003; Wong et al., 2006; Swenmen et al., 2006; Delzenne and Kok, 2001). Galactooligosaccharides and xylooligosaccharides were found to be helpful to decrease serum cholesterol and triglycerides, respectively, in animal (Chonon et al., 1995; Beylot et al., 2005).

Improve Immune Response The gastrointestinal tract (GIT) is an important component of the body’s defensive system. The intestinal tract not only provides non-specific protection in the form of a physical barrier against toxins and pathogenic organisms, but it also provides specific protection in the form of gut-associated lymphoid tissue (Watzl et al., 2005). Prebiotic support the growth of health beneficial intestinal microorganisms, which increases the competition with pathogens for colonization sites, upregulated gut-associated lymphoid tissue (GALT) expression

IJFFT 1(2) 2011 : 154

Enhancement of Mineral Absorption Prebiotic substantially enhance mineral absorption, especially calcium and magnesium. (Beynen et al., 2002; Chonan et al., 2002; Coudray et al, 2003; Mineo et al., 2002; Younes et al, 2001; Raschka and Daniel, 2005). The combination of inulin and oligofructose has been shown to increase calcium and magnesium absorption more effectively than either oligosaccharide alone. Increased colonic mineral absorption results from fermentation of inulin-type fructans which leads

Prebiotics: Current status and perspectives

to higher concentrations of short-chain fatty acids, a lower colon pH, and enhanced mineral solubility and bioavailability. In addition to increasing mineral absorption, research suggests inulin-type fructans enhance calcium secretion and improve bone mineralization and density in young adults (Abrams et al., 2005).

Side Effects of Prebiotics Generally, oligosaccharides are well tolerated. Some individuals report increased flatulence initially. Tolerance depends on the dose and individual sensitivity factors. In sensitive individuals, such as the elderly or people with reduced kidney function, excess dosage can result in dehydration and high sodium levels. Fructans of small degree of polymerisation are osmotically active and rapidly fermented. Common side effects are abdominal cramping, gas, and flatulence. Less common side effects are nausea and vomiting. Excessively high dosage can cause explosive and uncontrollable diarrhea. In normal individuals, overdose is considered uncomfortable, but not life threatening (www. Food-Info.net).

Global Status of Prebiotics Prebiotics in food and beverage products are useful in a wide variety of applications, as they have properties for enhancing texture, general fiber content, which corresponds with increasingly health-driven market in Europe and the growing importance of digestive health to consumers. The majority of the market is governed by the fructans manufacturers (inulin and fructo-oligosaccharide) but the important part of prebiotics market are the lactose-derived prebiotics (galactooligosaccharide and galacto-fructose), and there is enormous potential application for the emerging segment of resistant starch prebiotics in the next few years. Recent study from Frost and Sullivan (http://www.food.frost.com) on the European human food and beverage prebiotics market, finds that the market earned revenues of $295.5 million in 2008, representing a volume of 91,905 tonnes. The prebiotics market is expected to reach $766.9 million in 2015, with overall volumes of 204,895 tonnes and a compound annual growth rate of 14.0 percent. In this research, Frost and Sullivan’s expert analysts thoroughly examined the following segments: fructans (Inulin, Fructo-oligosaccharides) and lactose-derived prebiotics (galacto-oligosaccharides and galacto-fructans), with separate forecasts for each type of ingredient prebiotics are

also considered. Market share of prebiotics as industrial food ingredient main countries has been shown in Fig. 2.

Rest of Western Europe 17%

Fig. 2. Percentage share of market value for prebiotics as industrial food ingredient by main country, 2005 (Source: www.ingredientsdirectory.com/ repotrs, RTS resources Ltd).

According to a new report from Global Industry Analysts (GIA), the European and the U.S. market for prebiotics is expected to reach nearly $1.2 billion and $225 million, respectively, by the year 2015. Europe and U.S. are the largest markets for inulin and fructo-oligiosaccharide in term of production as well as demand and contributes nearly 92 % of global market. The daily per capita intake of inulin and fructooligosaccharides in the USA varies between 1 and 4 g and in Europe between 2 and 12 g (Van Loo et al., 1995). The market has since taken-off and witnessed very high growth internationally. The functional food market in India is poised for growth. Inulin, fructo-oligosaccharides, polydextrose, galactooligosaccharides, xylooligosaccharides, lactulose, raffinose etc. are the well known prebiotics fibres available in the market. Commercially, they are derived from chicory roots. In India, chicory is grown in Gujarat, Andhra Pradesh, Assam and South India. In India, the market for inulin and fructo-oligosaccharides was valued at $ 0.2 million including both food and neutraceutical applications in 2007-08.

IJFFT 1(2) 2011 : 155

Panesar et. al.

Conclusions and Future Prospects Prebiotics, a type of dietary fiber has been associated with a numerous health benefits including lowering blood lipid and sugar levels and reducing the risk of heart disease and colon cancer. Fiber supplements have also been used to improve a variety of gastrointestinal disorders including hemorrhoids and constipation. They have wide range of applications in food and pharmaceutical sector, including prebiotic properties, which can stimulate the growth and development of beneficial gastrointestinal microflora. These organisms provide specific health benefits for the host that include normalization of colonic transit time, increased production of short-chain fatty acids, improved pathogen resistance, enhanced mineral absorption, favorable modulation of blood lipids, improved gut mucosal barrier and immune function, and possible regulation of blood glucose and insulin levels.

References Abrams SA, Griffin IJ, Hawthorne KM, Liang L, Gunn SK, Darlington G and Ellis KJ. 2005 A combination of prebiotic short- and longchain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am J Clin Nut.r, 82:471-6. Akiyama K, Takase M, Horikoshi K and Okonogi S. 2001 Production of galactooligosaccharides from lactose using a β-glucosidase from Thermus sp. Z-1. Biosci Biotechnol Biochem., 65: 438– 441. Albayrak N and Yang ST. 2002. Production of galactooligosaccharides from lactose by Aspergillus oryzae βgalactosidase immobilized on cotton cloth. Biotechnol Bioeng., 77: 8-19. Alonso JL, Domínguez H, Garrote G, Parajó JC, José Vázquez M. 2003. Xylooligosaccharides: Properties and Production Technologies. J Elec Environ Agric Food Chem., 2: 230-232. Beylot M. 2005. Effects of inulin-type fructans on lipid metabolism in man and in animal models. Br J Nutr., 93: 63-68. Beynen AC, Baas JC, Hoekemeijer PE, Kappert HJ, Bakker MH, Koopman JP and Lemmens AG. 2002. Faecal bacterial profile, nitrogen excretion and mineral absorption in healthy dogs fed supplemental oligofructose. J Anim Physiol Anim Nutr., 86: 298-305. Boon MA, Janssen AEM and Van der Palt A. 1999. Modelling and parameter estimation of the enzymatic synthesis of oligosaccharides by β-galactosidase from Bacillus circulans. Biotechnol Bioeng., 64: 558-567. Bruins ME, Strubel M, Van Lieshout, JFT, Janssen AEM and Boom RM. 2003. Oligosaccharide synthesis by the hyperthermostable

IJFFT 1(2) 2011 : 156

β-glucosidase from Pyrococcus furiosus: kinetics and modelling. Enzyme Microbiol Technol., 33: 3-11. Carobbi R and Innocenti F. 1990. Process for preparing lactulose from lactose by epimerisation with sodium aluminate. European patent 0320670. Carobbi R, Miletti S and Franci V. 1985. Process for preparing lactulose from lactose, in the form of syrup or a crystalline product. US Patent 4536221. Chen C, Chen JL and Lin TY. 1997. Purification and characterization of a xylanase from Trichoderma longibrachiatum for xylooligosaccharide production. Enzyme Microb Technol., 21: 91–96. Chen CW, Ou-Yang CC and Yeh CW. 2003. Synthesis of galactooligosaccharides and transgalactosylation modeling in reverse micelles. Enzyme Microb Technol., 33: 497-507. Chen H-L, Lu, Yu-Ho, Lin J-J, and Ko L-Y. 2001. Effects of isomaltooligosaccharides on bowel functions and indicators of nutritional status in constipated elderly men. J Am College Nutri., 20: 44-49. Cho YJ, Shin HJ and Bucke C. 2003. Purification and biochemical properties of a galactooligosaccharide producing βgalactosidase from Bullera singularis. Biotechnol. Letts., 25: 2107-2111. Chockchaisawasdee S, Athanasopoulos VI, Niranjan K and Rastall RA. 2004. Synthesis of galacto-oligosaccharide from lactose using β-galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane-fitted bioreactors. Biotechnol Bioeng., 89: 434–443. Chonan O, Matsumoto K, Watanuki M. 1995. Effect of galactooligosaccharides on calcium absorption and preventing bone loss in ovariectomized rats. Biosci Biotechnol Biochem., 59: 236-139. Coudray C, Tressol JC, Guex E and Rayssiguier Y. 2003. Effects of inulin-type fructans of different chain length and type of branching on intestinal absorption and balance of calcium and magnesium in rats. European J Nutri., 42: 91-98. Crittenden RG and Playne MJ. 1996. Production, properties and applications of food grade oligosaccharides. Trends Food Sci Technol., 7: 353-361. Cruz RD, Arcadia Cruz V, Belote JG, de Oliveira Khenayfes M, Dorta C, dos Santos Oliveira LH, Ardiles E, and Galli A. 1999. Production of transgalactosylated oligosaccharides (TOS) by galactosyltransferase activity from Penicillium simplicissimum. Biores Technol., 70: 165-171. Cummings JH. 1994. Non-starch polysaccharides (dietary fibre) including bulk laxatives in constipation. In: Constipation. (Eds. MA Kamm and JE Lennard-Jones), Wrightson Biomedical Publishing Ltd, Petersfield, UK, pp. 307-314. De Harr WT and Pluim H. 1991. Method of preparing lactulose. European patent 0339749.

Prebiotics: Current status and perspectives

Delzenne NM and Kok N. 2001. Effects of fructans-type prebiotics on lipid metabolism. Am J Clin Nutr., 73: 456-458. Dendene K, Guihard L, Nicolas S and Bariou B. 1994. Kinetics of lactose isomerisation to lactulose in alkaline-medium. J Chem Technol Biotechnol., 61: 37- 42. De Preter V, Vanhoutte T, Huys G, Swings J, De Vuyst L, Rutgeerts P and Verbeke K. 2007. Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-Enriched inulin on colonic nitrogen-protein metabolism in healthy humans. Am J Physiol Gastrointest Liver Physiol., 292: 358-368. Deya, E. and Takahashi K. 1991. Production process of high purity lactulose syrup. US Patent 5034064. Ekhart PF and Timmermans E. 1996. Techniques for the production of transgalactosylated oligosaccharides (TOS). Bull. IDF, 313: 59-64. Gibson GR, McCartney AL and Rastall RA. 2005. Prebiotics and resistance to gastrointestinal infections. Br J Nutr., 93: 31-34. Gibson GR and Roberfroid MB. 1995. Dietary modulation of the human colonic microflora: Introducing the concept of prebiotics. J Nutr., 125: 1401-1412. Grajek W, Olejnik A and Sip A. 2005. Probiotics, prebiotics and antioxidants as functional foods. Acta Biochemica polonica., 52: 665-671. Gupta A K and Kaur N. 1997. Fructan storing plants-A potential source of high fructose syrups. J Sci Ind Res., 56: 447–452 Hartemink R. 1999. Prebiotic effects of Non-digestible oligo- and polysaccharides. PhD thesis, Wageningen University, the Netherlands, pp. 218. Hopkins MJ, Cummings JH and Macfarlane GT. 1998. Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources. J Appl Microbiol., 85: 381–6. Hosono A, Ozawa A, Kato R, Ohnishi Y, Nakanishi Y, Kimura T and Nakamura R. 2003. Dietary fructooligosaccharides induce immunoregulation of intestinal IgA secretion by murine peyer’s patch cells. Biosci Biotechnol Biochem., 67: 758-64. Hsu CA, Lee SL and Chou CC. 2007. Enzymatic production of galactooligosaccharides by β-galactosidase from Bifidobacterium longum BCRC 15708. J Agric Food Chem., 55: 2225-2230. Hung MN and Lee BH. 2002. Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96. Appl Microbiol Biotechnol., 58: 439-445. In M.J. sand Chae, H.J. 1998. Characterization of β-galactosidase with high transgalactosylation activity produced by Penicillium sp. KFCC 10888. Korean J Appl Microbiol Biothechnol., 26: 40-44. Iwasaki K, Nakajima M and Nakao S. 1996. Galacto-oligosaccharide production from lactose by an enzymic batch reaction using βgalactosidase. Process Biochem., 31: 69-76.

Ji ES, Park NH and Oh DK. 2005. Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from Thermotoga maritima. World J Microbiol Biotechnol., 21: 759764. Kim CS, Ji ES and Oh DK. 2004. A new kinetic model of recombinant β–galactosidase from Kluyveromyces lactis for both hydrolysis and transgalactosylation reactions. Biochem Biophy Res Commun., 316: 738–746. Kim YS, Parkand CS and Oh DK. 2006. Lactulose production from lactose and fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme Microb Technol 39: 903-908. Kochetkov NK and Bochkov AF. 1967. Chemistry of carbohydrates. Moscow, pp 672. Kohmoto T, Fukui F, Takaku H, Machida Y, Arai M and Mitsuoka T. 1998. Effect of isomaltooligosaccharides on human fecal flora. Bifidobact Microflora, 7: 61-69. Krumbbloz RE and Dorscheid MG. 1991. Method of manufacturing lactulose. European patent 0375040. Kunz C, Rudloff S, Baier W, Klein N and Strobel S. 2000. Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annual Review Nutr., 20: 699-722. Letexier D, Diraison F and Beylot M. 2003. Addition of inulin to moderately high carbohydrate diet reduces hepatic lipogenesis and plasma triacylglycerol concentrations in humans. Am. Clinical Nutri., 77: 559-564. Lee YJ, Kim CS and Oh DK. 2004. Lactulose production by βgalactosidase in permeabilized cells of Kluyveromyces lactis. Appl Microbiol Biotechnol., 64: 787-793. Lee HS, Auh JH, Yoon HG, Kim MJ, Park JH, Hong SS, Kang MH, Kim TJ, Moon TW, Kim JW, Park KH. 2002. Cooperative action of α-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production. J Agric Food Chem., 50: 2812-2817. Liu KS. 1999. Chemistry and nutritional value of soybean components. In Soybeans: Chemistry, technology and utilization. Aspen Publ. Inc: Gaithersburg, Maryland, USA, pp. 25-113. Lu L, Xiao M, Xu X, Li Z and Li Y. 2007. A novel β-galactosidase capable of glycosyl transfer from Enterobacter agglomerans B1. Biochem Biophys Res Commun., 356: 78-84. Li Z, Xiao M, Lu L and Li Y. 2008. Production of non-monosaccharide and high-purity galactooligosaccharides by immobilized enzyme catalysis and fermentation with immobilized yeast cells. Process Biochemistry, 43: 896-899. Manning TS and Gibson GR. 2004. Prebiotics. Best Pract Res Clin Gastroenterol, 18: 287-298. Mayer L, Kranz B and Fischer L. 2010. Continuous production of lactulose by immobilized thermostable β-glycosidase from Pyrococcus furiosus. J Biotechnol., 145: 387-393.

IJFFT 1(2) 2011 : 157

Panesar et. al.

Mineo, H., Hara, H. and Tomita, F. 2002. Sugar alcohols enhance calcium transport from rat small and large intestine epithelium in vitro. Dig Dis Sci., 47: 1326-33. Mountzouris KC, Gilmour SG, Grandison AS and Rastall RA. 1999 Modelling of oligodextran production in an ultrafiltration stirredcell membrane reactor. Enzyme Microb Technol., 24: 75–85. Mozaffar Z, Nakanishi K and Matsuno R. 1986. Mechanism for reversible inactivation of immobilized β-galactosidase from Bacillus circulans during continuous production of galactooligosaccharides. Appl Microbiol Biotechnol., 25: 229231. Nakagawa T, Ikehata R, Uchino M, Miyaji T, Takano K and Tomizuka N. 2006. Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans. Microbiol Res., 161: 75-79. Nakao M., Hatada M and Kodama Y, Nakayama T, Shibano Y and Amachi T. 1994. Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula. Appl Microbiol Biotechnol., 40: 657-663 Nakkharat P, Kulbe, K.D., Yamabhai, M. and Haltrich, D. 2006. Formation of galacto-oligosaccharides during lactose hydrolysis by a novel β-galactosidase from the moderately thermophilic fungus Talaromyces thermophilus. Biotechnol J., 1: 63363. Nguyen TH, Splechtna B, Krasteva S, Kneifel W, Kulbe KD, Divne C, Haltrich D. 2007. Characterization and molecular cloning of a heterodimeric β-galactosidase from the probiotic strain Lactobacillus acidophilus R22. FEMS Microbiol Lett., 269: 136-144 Olmstead, S., David Wolfson, M.D., Dennis Meiss, N.D. and Janet Ralston, BS, 2008. www.KLAIRE.com. Onishi N and Tanaka T. 1997. Purification and characterization of galacto-oligosaccharide-producing β–galactosidase from Sirobasidium magnum. Letts Appl Microbiol., 24: 82-86. Onishi N and Tanaka T. 1995. Purification and properties of a novel thermostable galacto-oligosaccharide-producing βgalactosidase from Sterigmatomyces elviae CBS8119. Appl Environ Microbiol., 61: 4026-4030. Onishi N and Yokozeki K. 1996. Gluco-oligosaccharide and galactooligosaccharide production by Rhodotorula minuta IFO879. J Ferment Bioeng., 82: 124-127. Onishi N, Kira I and Yokozeki K. 1996. Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803. Lett Appl Microbiol., 23: 253-256. Panesar PS, Kumari S, Bera MB and Chopra H. 2008. Lactulose: Production strategies and its potential applications. J Punjab Acad Sci., 5-6: 61-66. Panesar PS, Kaur G, Panesar R and Bera MB. 2009. Synbiotics: potential dietary supplements in functional foods. FST Bulletin, Food Science Central, IFIS Publishing UK. (http:// www.foodsciencecentral.com/fsc/ixid15649)

IJFFT 1(2) 2011 : 158

Panesar PS, Panesar R, Singh RS, Kennedy JF and Kumar H. 2006. Microbial production, immobilization and applications of β-Dgalactosidase. J Chem Technol Biotechnol., 81: 530-543. Panesar R, Panesar PS, Singh RS, Kennedy JF and Bera MB. 2007 Production of lactose hydrolyzed milk using ethanol permeabilized yeast cells. Food Chem., 101: 786-790. Panesar PS. 2008. Application of response surface methodology in the permeabilization of yeast cells for lactose hydrolysis. Biochem Engg J., 39: 91-96. Park A-R and Oh D-K. 2010. Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives. Appl Microbiol Biotechnol., 85: 1279-1286. Park HY, Kim HJ, Lee JK, Kim D and Oh DK. 2008. Galactooligosaccharide production by a thermostable βgalactosidase from Sulfolobus solfataricus. World J Microbiol Biotechnol., 24: 1553–1558. Parrish FW, Hicks K and Doner L. 1980. Analysis of lactulose preparations by spectrophotometric and high performance liquid chromatographic methods. J Dairy Sci., 63: 1809-1814. Placier G, Watzlawick H, Rabiller C and Mattes R. 2009. Evolved βgalactosidases from Geobacillus stearothermophilus with improved transgalactosylation yield for galactooligosaccharides production. Appl Environ Microbiol., 19: 6312-6321. Pool-Zobel BL. 2005. Inulin-type fructans and reduction in colon cancer risk: review of experimental and human data. Br J Nutr., 93: 73-90. Prenosil JE, Stuker E and Bourne JR. 1987. Formation of oligosaccharides during enzymatic lactose hydrolysis. Part, II, Modeling. Biotechnol Bioeng, 30: 1026-1031. Raschka L and Daniel H. 2005 Mechanisms underlying the effects of inulin-type fructans on calcium absorption in the large intestine of rats. Bone, 37: 728-735. Roberfroid M. 2005. Introducing inulin-type fructans. Br J Nutr., 93: 13-25. Roberfroid MB and Slavin J. 2000 Non digestible oligosaccharides. Crit Rev Food Sci Nutr., 40: 461-480. Robyt JF. 1992. Structure, biosynthesis, and uses of non-starch polysaccharides: Dextran, alternan, pullula and algin. In: Developments in Carbohydrate Chemistry (Eds. RJ Alexander, HF Zobel and MN St Paul): American Association of Cereal Chemists, pp. 261-292. Ruttloff H, Taufel A, Krause W, Haenel H and Taufel K. 1967. Enzymatic hydrolysis of galacto-oligosaccharides in the human and animal intestine with particular regard to L. bifidus. Behavior of lactulose in the intestine. Nahrung., 11: 39-46. Saarela M, Lähteenmäki L, Crittenden R, Salminen S and MattilaSandholm T. 2002. Gut bacteria and health foods-the European perspective. Inter J Food Microbiol., 78: 99-117. Säemann MD, Böhmig GA and Zlabinger GJ. 2002. Short-chain fatty acids: bacterial mediators of a balanced host-microbial

Prebiotics: Current status and perspectives

relationship in the human gut. Wien Klin Wochenschr., 114: 289-300. Shin HJ and Yang JW. 1998. Enzymatic production of galactooligosaccharide by Bullera singularis β-galactosidase. J Microbiol Biotechnol., 8: 484–489. Singh R.S. and Singh R. P. 2010. Production of fructo-oliosaccharides from inulin by endoinulinase and their prebiotic potential. Food Technol Biotechnol 48 : 435-450. Splechtna B, Nguyen TH, Steinbock M, Kulbe KD, Lorenz W, Haltrich D. 2006. Production of prebiotic galactooligosaccharides from lactose using β-galactosidases from Lactobacillus reuteri. J. Agric. Food Chem., 54: 4999–5006. Swennen K, Courtin CM and Delcour JA. 2006. Non-digestible oligosaccharides with prebiotic properties. Crit Rev Food Sci Nutr., 46: 459-71. Taper HS and Roberfroid MB. 2005. Possible adjuvant cancer therapy by two prebiotics—inulin or oligofructose. In Vivo, 19: 201204. Tomomatsu, H. 1994. Health effects of oligosaccharides. Food Technol., 18: 61-65. Topping, D.L. and Clifton, P.M. 2001. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev., 81: 1031-1064. Van Loo J, Coussement P, De Leenheer L, Hoebregs H and Smits G. 1995. On the presence of inulin and oligofructose as natural ingredients in the Western diet. Critical Reviews Food Sci. Nutr., 35: 525-552. Vazquez MJ, Alonso JL, Dominguez H and Parajo JC. 2000. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol., 11: 387–93.

Ward RE, Niñonuevo M, Mills DA, Lebrilla CB and German JB. 2006. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl Environ Microbiol., 72: 4497-4499. Watzl B, Girrbach S and Roller M. 2005. Inulin, oligofructose and immunomodulation. Br J Nutr., 93: 49-55. Wijnands MVW, Schoterman HC, Bruijntjes JP, Hollanders VMH and Woutersen RA. 2001. Effect of dietary galactooligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in fischer 344 rats. Carcinogenesis, 22: 127-132. Wong JM, De Souza R, Kendall CW, Eman A and Jenkins DJ. 2006. Colonic health: fermentation and short chain fatty acids. J Clinical Gastroenterol, 40: 235-243. Yun JW, Lee MG and Song SK. 1994. Continuous production of isomalto-oligosaccharides from maltose syrup by immobilized cells of permeabilized Aureobasidium pullulans. Biotechnol Lett., 16: 1145-50. Younes H, Coudray C, Bellanger J. and Demigne C. 2001. Rayssiguier Y and Remesy C. Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnesium balance in rats. Br J Nutri, 86: 479-485. Zheng P, Yu H, Sun Z, Ni Y, Zhang W, and Fan Y, Xu Y. 2006. Production of galacto-oligosaccharides by immobilized recombinant βgalactosidase from Aspergillus candidus. Biotechnol J., 1: 14641470. Zimmer CJ and Gibson GR. 1998. An overview of probiotics, prebiotics and synbiotics in the functional food concept: perspectives and future strategies. Inter Dairy J., 8: 473-479. Zokaee F, Kaghazchi T, Zare A. and Soleimani M. 2002. Isomerization of lactose to lactulose-study and comparison of three catalytic systems. Process Biochem, 37: 629-635.

IJFFT 1(2) 2011 : 159

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 161-172

Review Paper

Antimicrobial activity of essential oils : A Review V K Joshi*, Rakesh Sharma and Vikas Kumar Department of Food Science and Technology, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, HP, India

Abstract : Of late, there is a rapidly growing demand of environmental friendly, safe preservatives for food preservation because some of the traditional food preservation techniques have undesirable effects on the quality of food products and the artificial preservatives are increasingly being banned. This has necessitated the need to exploit the natural biological substances from plants which are safe and are capable of preserving the food products. Antimicrobial is a chemical compound present in or added to food, food packaging, food contact surfaces or food processing environments that inhibits the growth of or inactivate pathogenic or spoilage causing microorganisms. Many plants contain compounds that have some antimicrobial activity, collectively referred to as “green chemicals”. Volatile oils of many plants are known to have antimicrobial activity, which could probably act as chemical defense against plant pathogen. Among the essential oil components, the volatiles monoterpenes and aldehydes have attracted the recent interest of researchers and food industries for their use as food preservatives. Various herbs and spices have been found to have broad spectrum activity against a number of bacteria and fungi, hence showing their potential for their use in foods as antimicrobial additives. In this article, efforts have been made to present the information pertaining to various plant species especially essential oils having some antimicrobial compound, their antimicrobial activity, mode of action, application in food preservation and other related aspects. Keywords : Essential oil, Volatile oils, Antimicrobial compounds, Antimicrobial activity, Biopreservatives

The spoilage and poisoning of foods by microorganisms is a problem that is not yet under adequate control despite the availability of a range of robust preservation techniques (e.g. sterilization, freezing, drying, and use of preservatives). In many countries worldwide, there is a rapidly growing demand for environmentally friendly but safe preservatives to be used for mild food preservation. Traditional food preservation techniques have undesirable effects on the appeal of fresh food products and artificial preservatives are increasingly being banned. To meet the growing consumer demands, food manufactures are searching for new more natural alternative substances or chemicals that sufficiently assure the safety of *E-mail of corresponding author : [email protected] MS Received on : 23rd March, 2011 Accepted on : 28th Oct., 2011

their products in the retail chain by acting as antimicrobial compound. Antimicrobial is a chemical compound present in or added to food, food packaging, food contact surfaces or food processing environments that inhibits the growth of or inactivate pathogenic or spoilage causing microorganisms. Antimicrobials are also some times called “preservatives” however, the latter term is more precisely defined as chemical agents that act as antimicrobial, antioxidant or anti-browning (e.g., prevention of enzymatic browning) agents (Davidson, 2006). Many plants contain compounds that have some antimicrobial activity, collectively referred to as “green chemicals”. Species and herbs, for instances, are well known to inhibit bacteria, yeasts and molds and have traditionally

161

Joshi et. al.

found wide applications in food preservation. (Roller, 2003; Draughon, 2004) Volatile oils are very complex mixtures of compounds. The constituents of the oils are mainly monoterpenes and sesquiterpines which are hydrocarbons with the general formula (C5H8)n. The oxygenated compounds derived from these hydrocarbons include alcohols, aldehydes, esters, ethers, ketones, phenols and oxides. It is estimated that there are more than 1000 monoterpene and 3000 sesquiterpene structures. Other compounds include phenylpropenes and specific compounds containing sulphur or nitrogen. Volatile oils of many plants are known to have antimicrobial activity (Corner and Beuchet, 1984; Deans et al., 1992; Piccaglia et al., 1993), which could probably act as chemical defense against plant diseases. Pathogens can readily penetrate at wound sites caused, for example, by herbivores. Wounding of leaves which are covered with volatile oil glands results in the rupture of glands causing the oil to flow over the wound. The existence, therefore, of antimicrobial activity in the oil, would be of considerable benefit to the plant. Indeed, a good majority of aromatic and medicinal plants do not succumb to many of the commonest diseases. It is also suggested that complex oil presents a greater barrier to pathogen adaptation than would a more simple mixture of monoterpenes. This theory is well documented in the detailed study of Myrica gale volatile oil and its inhibitory properties against a broad spectrum of fungal species (Carlton et al., 1992; Svoboda et al., 1998). The complicated mixtures of monoterpenes and sesquiterpenes in the whole oil represented the strongest barrier to fungal infection. Volatile oils exhibited various reductions in growth of microorganisms, depending on the oil concentration and chemical composition. Food microorganisms (e.g. Salmonella enteritidis and Listeria monocytogenes) are of particular interest (Fyfe et al., 1998; Tassou et al., 1995). The review focuses on the antimicrobial spectrum of essential oils, their mode of action, sources and other relevant issues.

Nakatani,1994). Plants have enormous potential as a source of antimicrobial compounds, with over 1389 plants that have been recognized as potential green chemical sources (Wilkins and Board, 1989) and more specifically by the identification of over 250 new anti-fungal metabolites in plants between 1982 and 1993 (Grayer and Harborni, 1994).

Essential Oils and their Components Essential oils, also called ‘volatile oils’, are volatile, odoriferous substances, widely distributed throughout the plant kingdom. These are mixture of volatile compounds isolated from spices and herbs but can also be extracted from fruits, roots and stem of plants. They occur in some 60 families and are rather frequent or abundant in several unrelated plant families such as Labiatea, Rutaceae, Geraniaceae, Umbelliferae, Compositae, Lauraceae, Gramineae and Leguminosae. Some oils and isolated plant compounds are used in food as flavouring agents. Derived from their functionality in plants, these compounds show a wide range of interesting biological activities (Nakalani, 1994). Essential oil components may be generally classified into four main groups: 1. Terpenes/Terpenoids 2. Straight chain compounds 3. Benzene derivatives 4. Miscellaneous Composition of some of the essential oils is presented in Table 1. The amount of oils ranges from a minute trace to as one or 2 per cent or even more (FRI, 1972). Compounds that are approved for use in food and combine antimicrobial activity with low mammalian toxicity have great potential for application as natural food preservatives. Structure of some of these compounds are depicted in Fig.1

Natural Antimicrobials of Plant Origin

Plant Sources

Plants have for centuries been appreciated for their antimicrobial or medicinal activity. Some of these plants would be suitable to cultivate instead of lower value crops, thus, improving revenues of the growers. Since ancient times, herbs and spices have been used not only as tastemakers, but also as preservatives or antioxidants (Culter, 1995; Beuchat, 1994;

Most of the herbs and spices are the plant substances that contribute to flavour and are generally used as seasoning in various foods rather than as source of nutrition. Since ancient times, spices and herbs have been at least suggested to have healing or disinfectiong properties. The most potent of the spices and herbs are cinnamon (Cinnamomum zeylanicum),

IJFFT 1(2) 2011 : 162

Antimicrobial activity of essential oils

Fig. 1. Structure of some important components of essential oils. IJFFT 1(2) 2011 : 163

Joshi et. al.

Table 1. Composition of some essential oils Common name

Botanical name

Major components of the oils

Anise Seed Oil

Pimpinella anisum

Trans-anethole (95%) Methyl Chavicol (2%)

Bergamot oil

Citrus aurantium

Linalyl acetate (35%) Limonene (30%) Linalol (5%)

Bitter Almond oil

Prunus amygdalus

Benzaldehyde (97.5%) Hydrogen cyanide (2%)

Cardamom oil

Elettaria cardamomum

Linalol (0.2%), decanol (0.2%) Limonene (93%)

Peppermint oil

Mentha pipereta

Levo-menthol (50%) Levo-menthone (20%)

Rose oil

Rosa damescena

Citronellol (50%)

Rosemary oil

Rosmarinus officinalis

Sweet basil oil

Ocimum basilicum

Sweet orange oil

Citrus sinensis

Limonene (94%)

Thyme oil

Thymus vulgaris

Thymol (50%)

Ginger oil

Zingiber officinale

Lemon grass oil

Cymbopogen flexuosus

Lemon oil

Citrus limon

Geraniol (18%) Alpa-pinene (20%) 1,8-cineole (20%) Linalol (45%) Methyl chavicol (25%)

Para-cymene (15%) Zingiberene (35%) A-R-Curcumene (10%) Geranial (40%) Neral (30%) Limonene (63%) Beta-pinene (12%) Geranium oil

Pelargonium gravestens

Citronellol (32%) Geranio (12%)

Garlic

Allium sativm

daillyl disulfide (30%) daillyl trisulfide (30%) daillyl sulfide (15%)

Eucalyptus oil

Eucalyptus globus

1,8-Cineols (75%) Alpha-pinene (10%)

Davana oil

Artemisia pallens

Davanone (40%)

Coriander oil

Coriandrum sativum

Linalol (74%)

Cassia oil

Cinnamonum cassia

Cinnamaldehyde (85%)

Cinnamon oil

Cinnamonum

Eugenol (80%)

(a) Cinnamon leaf oil

zeylanicum

Cinnamaldehyde (76%)

(b) Cinnamon bark oil

IJFFT 1(2) 2011 : 164

Antimicrobial activity of essential oils

clove (Syzygium aromaticum), thyme (Thymus vulgaris), oregano (Origanum vulgare) and vanilla (Vanilla planifolia, Vanilla pompon, Vanilla tahitensis) and their respective principle antimicrobial components, cinnamic aldehyde, eugenol, thymol, carvacrol and vanillin (Bullerman et al, 1977, Davisson, 2001). These spices and spices components have broad spectrum activity against a number of bacteria and fungi and have potential for their use in compatible foods as antimicrobial additives. Some of the spices and herbs are recognized for their specific properties which are responsible for microbial inhibition in various foods. The preservative effect of these plant substances is mainly due to the presence of variety of antimicrobial substances present in them (Table 2).

certain levels of dosage, the volatile oils saturate the membranes and show effects similar to those of local anaesthetics. They can interact with the cell membranes by means of their physiochemical properties and molecular shapes, and can influence their enzymes, carriers, ion channels and receptors.

Mode of Action of Essential Oils

1. Distillation: Essential oils can be extracted by using different methods of distillation. The oldest and simplest method of distillation is boiling in water or water distillation. In this method, the plant material is allowed to stand in water in a copper still and is then heated to a boiling point. The essential oil vaporizes and together with the steam passes in to condenser. Upon cooling, the oil or essence collects on the

An excellent survey on the uses of fragrances and essential oils as medicaments was published by Buchbauer and Jirovetz (1994). It has been suggested that volatile oils, either inhaled or applied to the skin, act by means of their lipophilic fraction reacting with the lipid parts of the cell membranes, and as a result, modify the activity of the calcium ion channels. At

Extraction of Essential Oils Essential oils are extracted from plant tissues by a variety of methods depending upon the quantity and stability of compound. Some need delicate techniques as they are unstable and become altered under drastic treatments. The different methods which are used for extraction of essential oils are discussed here.

Table 2. Antimicrobial compound, action and inhibitory effect of some plant sources. Plant Source

Antimicrobial compound

Action

Concentration used

Inhibitory effect

Garlic (aqueous extract)

Allicin

Bactericidal, Antibacterial

5% in NA

Inhibited the growth of Bacillus cereus by 58.2%

Garlic (aqueous extract)

Allicin

Bactericidal, Antibacterial

10% in NA

Inhibited the growth of Bacillus cereus by 100%

Garlic / onion oil

Allicin

Bactericidal, Antibacterial/ Growth inhibitor Bacricidal

1500mg/g

Inhibited toxin production in meat slurry by Clostridium botulinum

Thyme

Thymol

Inhibits growth of pathogenic fungi

0.4mg/g

Completely inhibited the growth of Asperigillus flavus and A. versicolor

Oregano

Thymol

Inhibits growth production

2% PDA

Completely inhibited the growth of mycotoxigenic moulds, pathogenic and non-pathogenic fungi and moulds

Clove oil

Eugenol

Antimycotic/Antifungal

250 ppm in culture medium

Inhibited the growth and production of several moulds

Cinnamon

Cinnamic aldehyde

Inhibits mould growth mycotoxin production

2% in medium

Toxin production by Aldehyde

and

toxin

and

culture

toxin

Aspergillus parasiticus reduced by 99%

NA : Nutrient Agar, PDA : Potato Dentrose Agar Source : Singh and Thompkinson (2001), Sharma et al. (2003)

IJFFT 1(2) 2011 : 165

Joshi et. al.

surface of the water and is removed and filtered. However, distillation using steam is the most widely used method. The steam is normally generated in a separate boiler and then, blown through the botanical material in a still. Essential oils components, with boiling points normally ranging upto 300oC, evaporate at a temperature close to the boiling point of water. The steam and essential oil are condensed and separated. Further, for a small range of essential oils e.g. oil from exudates such as balsams, dry distillation method is often used (EIRI, 2008). 2. Expression of Oils: In case where heat would destroy the odours, expression technique is resorted to, as in the production of citrus oil from the juices and waste rinds from citrus-canning factories. Expression usually involves squeezing the material at great pressure in order to press out the oil. Citrus oils are often isolated from the peel by expression (“cold pressing”). This process involves the abrasion of peel and the removal of the oil in the form of an aqueous emulsion that is subsequently, separated in a centrifuge (EIRI, 2008). 3. Solvent extraction: Most flowers contain too little volatile oil to undergo expression and their chemical components are too delicate and easily denatured by the high heat used in steam distillation. Instead, a solvent such as hexane or supercritical carbon dioxide is used to extract the oils. Extracts from hexane and other hydrophobic solvent are called concretes, which are a mixture of essential oil, waxes, resins, and other lipophilic (oil soluble) plant material. Although highly fragrant, concretes contain large quantities of non-fragrant waxes and resins. Often, another solvent, such as ethyl alcohol, which is more polar in nature, is used to extract the fragrant oil from the concrete. Supercritical carbon dioxide is used as a solvent in supercritical fluid extraction. This method has many benefits, including avoiding petrochemical residues in the product and the loss of some “top notes” when steam distillation is used (EIRI, 2008). The supercritical carbon dioxide extracts both the waxes and the essential oils that make up the concrete. The lower temperature process prevents the decomposition and denaturing of compounds. When the extraction is complete, the pressure is reduced to ambient and the carbon dioxide reverts to a gas, leaving no residue.

IJFFT 1(2) 2011 : 166

4. Maceration: Maceration actually creates more of an “infused oil” rather than an “essential oil” and is most often used for creating extracts and resins. The plant matter is soaked in vegetable oil, water, or another solvent. Antimicrobial Activities of Essential Oils Microbial pathogens are responsible for an estimated 3,23,000 hospitalizations each year at a cost of $ 7 to 10 billion annually (Mead et al, 1999). In the year 2006, about seven serotypes accounted for 66% of the laboratory confirmed Salmonella infections in humans as follow; Typhimurium 19%, Enteritides 19%, Newport 9%, Heidelberg 6%, Javiana 5%, Montevideo 4%, and Heidelberg 4% (CDC, 2007). The effects of essential oils and their components on bacterial growth and survival have been studied for many years. Aromatic essential oils have been known since antiquity to possess biological activity including antibacterial, antifungal, antiviral and anti-inflammatory effects (Thompson, 1989; Smith et al, 1998). These oils can also be active against higher organisms such as nematodes, helminthes, insects etc. Generally, they are composed of terpenoid structures and their effect is the result of the combination of all their constituents, which in some oils may number over 100 compounds. One of the well-established properties of plant essential oils is their antimicrobial activity. They are active against a a wide range of microorganisms including food spoilage microorganism, potentially pathogenic microbes of human, environment or animal origin and some microorganism in the gastrointestinal tract of animals (Svoboda et al., 2006). Essential oils from citrus offer the potential for all natural antimicrobials for use in improving the safety of organic or all natural foods. Subba et al, (1967) determined that orange and lemon oil had in vitro antibacterial effects on Salmonella and other foodborne microorganisms. Deans and Richie (1987) reported that Gram-positive and Gram-negative bacteria were equally sensitive to citrus essential oils and components. However, Fisher and Phillips (2006), on the other hand, found that Gram-positive bacteria were more sensitive than Gramnegative in vitro. Seven citrus essential oils were screened by disc diffusion assay for their antibacterial activity against 11 serotypes/strains of Salmonella (Bryan et al, 2008). The 3 most active oils were selected to determine the minimal

Antimicrobial activity of essential oils

inhibitory concentration (MIC) against the some Salmonella. Orange terpenes (C4), singles-folded d-limonene (C5), and orange essence terpenes (C6) all exhibited inhibitory activity against the Salmonella spp. On the disc diffusion assay orange terpens and d-limonene both had MICs of 1%. The most active compound, terpenes from orange essence, produced MIC that ranged from 0.125% to 0.5% against the 11 salmonella tested (Table 3). Table 3: MIC (in per cent, v/v) of orange oils against 11 Salmonella spp. Salmonella spp

C4

C5

C6

S. enteritidis 1773-92

1

1

0.25

S. senftenberg 43845

1

1

0.5

S. senftenberg 1402-94

1

1

05.

S.tennessee 825-94

1

1

0.5

S. kentucky 1271-94

1

1

0.25

S. eidelberg 8326

1

1

0.25

S. enteritidis 13076

1

1

0.13

S. montevideo G4639

1

1

0.25

S. michigan

1

1

0.25

S. typhimurium (Copenhagen)

1

1

0.5

S. stanfey H1256

1

1

0.5

Moss 2002). In a study screening of 26 essential oils against Botrytis cinera, causal organism of grey mould of grapes was carried out (Tripathi et al, 2008). Out of 16 essential oils, Ocimum sanctum, Prunus persica & Zingiber offianale were selected for further investigation as the MIC of these oils were lower as compared to other fungitoxic oils (Fig 2). The MIC of O. sanctum, P. persica and Z. officinale oils was found to be 200, 100 and 100 ppm, respectively. It was found that all these essential oils were fungistatic at their respective MIC (Table 4). The fungicidal effects of the three oils appeared at higher concentrations (2000, 1500 and 2500 ppm, respectively). When the essential oils were compared with some prevalent synthetic fungicides they were found to be more effective than the synthetic fungicides (Fig 3). Antibacterial activity of Carvacrol, Citral and Geraniol against Salmonella typhimurium and its rifampicin-resistant (Rifr) strain in cultures medium and on fish cubes were studied by Kim et al., (1995). It was found that Carvacrol was the most potent bactericidal activity with minimum inhibitory and bactericidal concentrations (MIC and MBC) of 250 ug/mL for both the tester strains.

Fig. 2. Screening of some essential oils for fungitoxicity against B. cinerea Source: Bryan et al., (2008)

Post-harvest diseases render heavy losses to perishable during transit and storage. Fruits due to their low pH are spoiled primarily by fungi which in addition to causing rot, may also contaminate the fruits by producing mycotoxins (Phillips, 1984;

Fig. 3. Comparative efficacy of the oils with some prevalent synthetic fungicides

IJFFT 1(2) 2011 : 167

Joshi et. al.

Table 4. Effect of increased inoculums density on fungitoxicity of the oils Number of fungal diseases

Approximate number of spores

1

2358 - 103

-

+

-

+

-

+

2

47174 - 103

-

+

-

+

-

+

4

94348 - 103

-

+

-

+

-

+

8

1888696 - 103

-

+

-

+

-

+

16

377392 - 103

-

+

-

+

-

+

32

754784 - 103

-

+

-

+

-

+

64

1509568 - 103

-

+

-

+

-

+

O. sanctum Treatment Control

Growth of the test fungus P. persica Treatment Control

Z. officinale Treatment Control

- Indicates no growth of test fungus, + indicates growth of test fungus Source: Tripathi et al. (2008)

Ocimum is widely cultivated and extensively used for food, perfumery, cosmetics, pesticides, medicine and traditional rituals because of their natural aroma and flavour, and other properties (Alburguergus, 1996). Literature reports that O. basilicum leaf essential oils or leaf powder have effective insecticidal and pesticidal activities against Vigna ungiuculata pests Callosobruchus macuktus (Keita et al, 2001; Keita et al, 2002). The aerial parts essential oils of Ocimum basilicum (Lamiaceas) from Togo were steam-distilled and investigated for their composition (GC and GC/MS) and in vitro antimicrobial activities. Five oil Cheno-types were identified and classified as follows in line with their principal components; estragols type, linalool (estragole type; methyl eugenol type; methyl eugenol/t-anethole type; t-anethole-type. The in vitro microbiological experiments revealed that only the “methyl eugenol” and “methyl eugenol / t-anethole” chemotypes were active against tested fungi and bacteria (Koba et al, 2009). The minimum inhibitory concentration (MIC) ranged from 8015 µlL-1 and from 200-500 µlL-1 respectively. These findings are supportive of the potential of both the basil oil chemotypes for use as active ingradients in natural antibiotic drugs. Dacryodis edulis named “African pear” is a topical tree producing a consumable fruit, which softens when heated and then, is eaten with cassava or as a dessert. In a study to find out antimicrobial properties of the essential oil of Dacryodis edulis (G.Don), it was revealed that the essential oil of D. edulis possesses potential antimicrobial activity (Obame et al, 2008). Twenty-four components were identified that constituted 98.5% of the total oil. Sabinene (21.8%), terpinene-

IJFFT 1(2) 2011 : 168

4-01(19.8%), pinene (17.5%) and p-cymene 11.3% were the major components comprising the 70.3% of the essential oil. The antimicrobial activity of the essential oil of D. edulis against 16 species of microorganisms by the disc diffusion method and the broth micro dilution is reported (Obame et al 2008). The results showed that the essential oil of D.edulis had an antimicrobial activity against all the tested microorganisms and it can be used as natural preservatives in food against the well known causal agents of food borne diseases and food spoilage.

Fig. 4. Minimal Inhibitory Concentratuion (MIC) values of different essential oils (ml/100, v/v)

Antimicrobial activity of essential oils

A study was conducted to find out to the effectiveness of natural essential oils in extending the shelf-life of leafy vegetables (Ponce et al, 2003). The essential oils utilized were eucalyptus (Eucalyptus globules), tea tree (Melalcuca atternifolia), melisa (Melissa officinalis), rosemary (Rosmarinus officinalis), clove (Syzygium aromaticum) and lemon (Citrus limonus). The MIC values of these essential oils is presented in Fig 4. The impact of essential oils on bacteria, especially on pathogens has been extensively studied in the laboratory and significant variations have been noted. For example, Escherichia coli was found to be more vulnerable than Pseudomones fluorescens or Serratia marcescens to the essential oils of sage, rosemary, cumin, caraway, clove and thyme (Forag et al, 1989), whereas Salmonella typhimurium was more sensitive to oregano and thyme oils than Pseudomonas aerogenose (Paster et al, 1988). Mold growth on black table olives was found to be suppressed by methyl eugenol and the essential oil from Echinophora sibthorpiana (Kivanc and Akgul, 1990). Plant oils can be used to cure mycotic infections and plant oils may have role as pharmaceutical and preservatives (Bansod and Rai, 2008). It was found that maximum antimycotic activity was demonstrated by oils of Cymbopogon martini, Eucalyptus globulus and Cinnamomum zylenicum as compared to control, followed by Cymbopogon citratus which showed activity similar to control (miconazole nitrate). The oils of Mentha spicata, Azadirachta indica, Eugenia caryophyllata, Withania somnifera and Zingiber officinale exhibited moderate activity. The oils of Cuminum cyminum, Allium sativum, Ocimum sanctum, Trachyspermum copticum, Foeniculum vulgare and Elettaria cardamomum demonstrated comparatively low activity against A. niger and A. fumigatus as compared to control. Mixed oils showed maximum activity as compared to standard.

Application of Antimicrobials from Plants Among the essential oil components, the volatiles monoterpenes and aldehyde have attracted the recent interest of researchers and food industries because they can be ued as food preservatives that leave a negligible amount of residues. For instance, with carvons, the prims monoterpone in essential oil of caraway (Carum carvi L.) seeds, a powerful antifungal effect has been found, which is already exploited for the protection of potato tubers under storage conditions.

Cinnamaldehyde, the major compound in cassia oil shows potent antifungal activity against several food associated fungi like Penicillium sp.; Fusarium sp., and Aspergillus sp. (Pauli and knobloch, 1987). Cinnamaldehdye has also been shown to posses antiaflatoxigenic properties (Mahmoud, 1994). An example of the use of cinnamoldhyde in food preservation is its potential use as a surface disinfectant for tomatoes (Smid, et al., 1996). Tomatoes are particularly vulnerable to microbial spoilage at calyx and on the fruit surface. The major pathogens affecting the postharvest life of tomato fruit are; Alternaria alternate, Botrytis cinerea and Rhizopus stolonifer. Smid et al, (1996) has investigated the reduction of spoilageassociated fungi and bacteria on whole tomatoes packed under modified atmospheres conditions. Tomatoes were treated for 30 minutes with a solution containing 13mM cinnamaldehdye and stored at 18oC in sealed plastic bags. The calyx of cinnamadehdye-treated tomatoes remained free from visible fungal growth for at least 9 days. However, on day 4, visible fungal growth was observed on calyex of untreated fruits. Penicillum sp. was found to be the dominant fungal species on the calyx. Further, after 2 days of storage, pronounced growth of the bacterial population was observed on control tissues treated with 0.85% NaCl, whereas after 4 days of storage, a significant increase in the size of the bacterial population was detected on untreated tomatoes. In contrast hardly any development of the bacterial population was detectable on cinnamadehyde-treated tomatoes.

Conclusion Consumers are demanding safer foods and they also prefer foods that do not have synthetic additives, which pave the way for the use of natural preservatives such as essential oils. Spices and essential oils are being researched more extensively for their contributions as natural agents to use for food preservation. A wide range of natural products from plants and microorganisms have been found to be useful for extending the shelf-life of foods and reducing or eliminating pathogens at the same time. Using essential oil components for the inhibition of food pathogens may find a wider application in food processing. Since the use of synthetic fungicides is becoming increasingly limited and regulated, alternative methods of controlling postharvest pathogens are constantly being sought. Plant essential oils have been extensively investigated in vitro, and demonstrated to have antibacterial and antifungal activities. However, much work remains to be done to develop a formulation that maintains the fungicidal IJFFT 1(2) 2011 : 169

Joshi et. al.

activity of the material, and yet does not induce undesirable effects. Although, the essential oils look promising as alternative to antibiotics or chemical preservative, there is scarce information available on the effective dose that can be used without the risk of toxic effects in consumers or imparting unwanted taint to meat and milk products. Essential oils are lipophillic in nature, so have poor water solubility. They also tend to associate with food components such as fat or proteins. Their use as food preservative is therefore problematic. Nevertheless, the benefits of using essential oils reside in their breadth of antimicrobial activity and lack of any apparent build up of any microbial resistance. The challenge therefore, is to more clearly establish the target range for each essential oil, the minimum inhibitory concentration, any potential residue from their use (either directly or as transformed products), any potential allergenicity or toxicity, their efficacy in vivo and ultimately their cost effectiveness. The result will be a range of natural products that are safe to use, effective and free of the side effects encountered with the use of antibiotics and other chemical preservatives in the food supply.

References Albuquerque UP. 1996. De Taxonomia e ethanobatanica do genero Ocimum L. (Lamiaceae) no Nordeste do Brasil – renferencia espeinl para Pernambuco. Recife. Dissertacao (Mestradoern Biologia Vegital) – Centrods Ciencias Biologias, Universidede Federal de Pernamber Co. p.125. Bansod Sunita and Rai Mahendra. 2008. Antifungal activity of essential oils from Indian medicinal plants against human pathogenic Aspergillus fumigatus and A. niger. World Journal of Medical Sciences, 3: 81-88. Beuchat LR. 1944. Antimicrobial properties of species and their essential oils. In: Natural antimicrobial systems and food preservation. (Eds. VM Dillon and RG Board), CAB International: Walligford. p.167. Bryan CA, Crandall PG, Chalova VI and Ricke SC. 2008. Orange essential oils antimicrobial activities against Salmonella spp. J Food Sci., 73: 264–267. Buchbauer G and Jirovetz L. 1994. Aromatherapy – use of fragrances and essential oils as medicaments. Flav. Fragr. J., 9: 217 – 222. Bullerman LB, Lieu FY, and Seier SA. 1977. Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldhyde and eugenol. J. Food Sci., 42:1107-1109, 1116. Carlton RR, Waterman PG, Gray AI, Deans SG. 1992. The antifungal activity of the leaf gland volatile oil of sweet gale (Myrica gale) (Myricaceae). Chemoecology, 3: 55-59.

IJFFT 1(2) 2011 : 170

CDC. 2007. Prelimianry Food Net data on the incidence of infection with pathogens transmitted commonly through food-10 States. MMWR, 56: 336-339. Conner DE and Beuchat LR. 1984. Effects of essential oils from plants on growth of food spoilage yeasts. J. Food Sci., 49: 429434. Culter HG. 1995. Natural product flavor compounds as potential antimicrobials, insecticides and medicinal. Agro-Food-IndustryHi-Tech, 6: 19. Davidson PM. 2001. Chemical preservatives and natural antimicrobial compounds. In: Food microbiology: fundamental and frontiers. 2 nd ed. (Eds. MP Doyle, LR Beuchat and TJ Montville), Washington DC: American Society for Microbiology. p. 593627 Davidson PM. 2006. Food Antimicrobial: Back to nature. Acta Hort., 709: 29-33. Deans SG and Richie G. 1987. Antimicrobial properties of plant essential oils. Int. J. Food Microbiol., 5: 165-180. Deans SG, Svoboda KP, Gundidza M, Brechany EY. 1992. Essential oil profiles of several temperate and tropical aromatic plants: their antimicrobial and antioxidant activities. Acta Hortic., 306: 229-232. Draughon A. 2004. Use of botanicals as biopreservaties in foods. Food Technol., 58: 820. EIRI. 2008. Modern Technology of essential oils. EIRI Board of consultants and Engineers. Engineers India Research Institute. New Delhi, India, pp. 465. Farag RS, Daw ZY, Hewedi FM and El-Baroty GSA. 1989. Antimicrobial activity of some Efyptian spice essential oils. J. Food Prot., 52: 665. 1Fisher K and Phillips CA. 2006. The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter jejuni, Escherichia coli 0157:H7, Listeria monocytogenes, Bacillus cerecis and Staphylococcus aureus in vitro and in food systems. J. Appl. Microbiol., 101: 12321240. FRI. 1972. Essential oils, In: Forest product utilization. The Manager of Publications, New Delhi. pp. 660-685 Fyfe L, Armstrong F, Stewart J. 1998. Inhibition of Listeria monocytogenes and Salmonella enteriditis by combination of plant oils and derivatives of benzoic acid: the development of synergistic antimicrobial combinations. Inter J. Antimicrobial Agents, 9: 195-199. Grayer RJ and Harborne JB. 1994. A survey of antifugal compounds from higher plant. 1982-1993. Phyto-chemistry, 37: 19 Keita KG, Glitoh AI and Huignard J. 2002. Susceptibility of the bruchus Callosobruchus maculates (Colcoptera: Bruchidai) and its parasitoid Dinarmus basalis (Hymenopteral: pteromatidae) to three essential oils. J. Econ. Enotomol., 95: 174-182. Keita SM, Vincent C, Schmit J, Arnason JT and Belanger A. 2001. Efficacy of oil of Ocimum basilicum L. and O. gratissimum L. asan insecticidal fumigant and power to control Callosobruchus maculates (Fab). J. Stored Prod Res. 37: 339-349.

Antimicrobial activity of essential oils

Kim JM, Marshall MR, Cornell JA, Preston III JF and Wei CI. 1995. Antibacterial activity of carvacrol, citral and geraniol against Salmonella typhimurium in culture medium and on fish cubes. J. Food Sci., 60: 1364-1368, 1374. Kiranc M and Akgul A. 1990. Mould growth on black table olvies and prevention by sorbic acid, methyl eugenol and spice essential oil. Nahrung., 34:369. Koba Koffi, Poutouli PW, Raynaud Christens, Chaumont Jean-Pierre and Sanda Komla. 2009. Chemcial composition and antimicrobial properties of different basil essential oils chemotypes from Togo. Bangladesh J. Pharmocol., 4: 1-8. Mahmaud ALE. 1994. Antifungal action and antiaflatoxigenic properties of some essential oil constituents. Lett. Appl. Microbiol., 19: 110. Mead PS, Stutsker L, Dietz V, McCaig F, Bresee JS, Shapiro C, Griffio PM, and Tauxe RV. 1999. Food related illness and death in the United States. Emerg. Inf. Dis., 5: 607-625. Moss MO. 2002. Mycotoxins review: Aspergillus and Penicillium. Mycologist, 16: 116-119. Nakatani N. 1994. Antioxidative and antimicrobial constitutents of herbs and speices. In: Spices, herbs and edible fungi. (Ed. G. Charalambous) Elsevier Science: Amsterdam. pp. 251. Obame LC, Edou P, Bassole IHN, Koudou J, Agnaniet H, Eba F and Traore AS. 2008. Chemical composition, antioxidant and antimicrobial properties of the essential oil of Dacryodes edilus (G. Don) H.J.Lam from Gabon. African J. Microbial. Research., 2: 148-152. Paster N, Juven BJ and Harshemesh H. 1988. Antimicrobial activity and inhibitin of aflatoxin BI formation by olive plant tissue constituents. J. Appl. Bacteriol., 64-293. Pauli A and Knobloch K. 1987. Inhibitory effects of essential oil components on growth of food contaminating fungi. Z. Lebensm. Unters. Fossch., 184: 10. Phillips DJ. 1984. Mycotoxins as a postharvest problem. In: Postharvest pathology of fruit and vegetables: postharvest losses in perishable crops (Ed. HE Molins), University of Californa, Berkeley Publcations N.E., pp. 50-54. Piccaglia R, Marotti M, Giovanelli E, Deans SG, Eaglesham E. 1993. Antibacterial and antioxidant properties of Mediterranean aromatic plants. Ind. Crops and Prod., 2: 47-50.

Ponce AG, Fritz R, Delvalle CE, and Roura SI. 2003. Antimicrobial activity action of essential oils on native microbial population of organic swiss chard. Lebensm wiss Technol., 36:679-684. Roller S. (Ed). 2003. Natural antimicrobials for the minimal processing of food. Woodhead Publishing: Cambridge, U.K. Sharma Rakesh, Sharma SK, Kumar Rajesh and Kamboj Prashant. 2003. Food preservation of biological origin-an overview. Beverage and Food World. July: 36-39. Singh AK and Thompkinson DK. 2001. Food bio-preservatives. In: Lecture compendium on Refresher Course on Advances in Food Science and Technology, Department of Food Technology, Guru Lambeshwar University, Hisar, Haryana, Nov 19-Dec 09, 2001, pp. 269-279. Smid EJ, Hendriks L, Boerrigter HAM and Gorris LGM. 1996. Surface disinfection of tomatoes using the natural plant compound transcinnamaldehyde, Postharvest Biol. Technol., 9: 343. Smith-Palmer A, Steward J, and Fyfe L. 1998. Antimcirobial properties of plant essential oils and essences against five important food borne pathogens. Lett. Appl. Microbiol., 26: 122-188. Subba MS, Soumithri TC and Rao RS. 1967. Antimicrobial action of citrus oils. J. Food Sci., 32: 225-227. Svoboda K, Brooker JD and Zrustova J. 2006. Antimicrobial and antioxidant properties of essential oils: their potential application in food industry. Acta Hort., 709: 35-41. Svoboda KP, Inglis A, Hampson J, Galambosi B, Asakawa Y. 1998b. Biomass production, essential oil yield and composition of Myrica gale L. harvested from wild populations in Scotland and Finland. Flav. Fragr. J., 13: 367 – 372. Tassou CC, Drosinos EH, Nychas GJE. 1995. Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4°and 10° C. J. Appl. Bact., 78: 593-600. Thompson DP. 1989. Fungitoxic activity of essential oil components on food storage fungi. Mycologia., 81: 151-153. Tripathi-Pramila Dubey NK and Shukla AK. 2008. Use of some essential oils as postharvest botanical fungicides in the management of greymould of grapes caused by Botrytis cinerea. World J. Microbiol. Biotechnol., 24: 39-46. Wilkins KM and Board RG. 1989. Natural antimicrobial systems. In: Mechanisms of action of food preservation procedure. (Ed. GW Gould), Elsevier Applied Science: London. pp.285.

IJFFT 1(2) 2011 : 171

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 173-184

Review Paper

Use of fermentation technology on vegetable residues for value added product development : A concept of zero waste utilization Anshu Singh, Arindam Kuila, Sunita Adak, Moumita Bishai and Rintu Banerjee* Microbial Biotechnology and Downstream Processing Laboratory, Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, West Bengal, India.

Abstract: The intensification of consumer demand for healthy diet has opened multiple resources for food processing industries. Vegetable industries are coming up with innovative products as vegetables are common choice of major portion of world population. During vegetable processing, unused residues and effluents come out as waste. Due to high moisture content of these biological wastes, there is rapid decomposition resulting in foul odour and dispersal of pathogens. Many strategies were approached towards the zero waste concept and aimed for waste minimization to its reusability. Process designing on zero waste concept has given a new turn to industries to meet the need of the consumers with no longer waste generation. Zero waste approach has integrated the waste of one operation to be the resource/raw material for another. Application of fermentation techniques to waste reusability have been an ecofriendly and economically feasible process for bioconversion of the waste to valuable products. Keywords: Vegetable, Waste, Fermentation, Value added products, Bioconversion, Effluent

Human health ultimately depends upon the basic diet, which is necessary for good health and productive livelihood. Among the various components of diet, vegetables have a prominent place as these play a crucial role in the human nutrition by providing phenolics, proteins, minerals, dietary fibers, carbohydrates etc. to the body system. All these constituents found in vegetables are known for their health promoting effects. Vegetables being perishable in nature get easily spoiled at farm during harvesting, postharvest handling, transportation, storage and marketing (Basediya et al., 2011). A large amount of vegetable wastes generated at earlier mentioned stages and even during processing are mostly discarded. Though a portion of waste is utilized as animal feed and manure but this application is still at a very small scale. Disposal of the vegetable matter creates economic and environmental problems due to

*E-mail of corresponding author : [email protected] MS Received on : 24th May, 2011 Accepted on : 1st Oct., 2011

lack of proper utilization. A new concept has emerged as a solution for industries to minimise and recycle the waste, i.e. zero waste concept. It forms the closed loop adding profitability with the integration and designing of processes to convert the raw material and waste into value added products (Ngoc and Schnitzer, 2008). From zero waste concept view point, there are two strategies to eliminate the waste. One is minimisation of waste by redesigning of manufacturing system and the other is reusability of generated waste. The combination and integration of all these approaches in food processing system seems to be the best strategy to overcome the waste. The fermentation process

Singh et al.

has evolved as an efficient approach as it doesn’t require the redesigning of whole system processes. Using this technology, large amount of wastes can be recycled with no requirement of pre-processing steps. Application of this technique has been employed for several years to accelerate the decomposition of residues for favourable outcome. Microbial system possesses several hydrolytic enzymes and has become a potential candidate for the treatment of vegetable wastes. Bioconversion of vegetable residues to valuable products is highly dependent upon the biochemical composition of the left-over material. Therefore, the characterization of the waste generated through the systems is an essential step. The next step concentrates on identifying, analyzing and designing for potential recovery and reuse of it. Even the remaining wastes need to be treated properly before discharging into the environment. Together all these steps, form a systematic fermentation methodology that develops a model for a zero waste. Fig. 1 shows the schematic diagram for value added product development from processing of vegetable wastes. Researchers and industries are now engaged in a number of projects involving the technology of “waste to best” method, which also reduces the impact of waste on the environment. In this article, the waste from vegetable, their characteristics and utilization has been reviewed.

Domestic/household wastes These wastes are released during the scrapping/peeling off and cooking process. The household released residues are produced in smaller quantities and often mixed with non-organic materials, which requires a tedious process of separation for efficient bioconversion.

Agricultural wastes Growing populations across the globe demand both fresh produce and processed horticultural products. The lack of processing and improper post-harvest operation is responsible for the release of fruits and vegetable waste. Major causes of vegetable waste generation are poor handling, improper packing, high moisture and nutrients content.

Agro-Industrial wastes Consumer’s awareness of diet related health problems (Gilbert, 1997) had made a large group of the population dependent on intake of fruits and vegetables. Agro-food industries are utilizing the vegetable raw materials to provide the better product with good nutritional profile. During manufacturing processes, release of residues comes as a waste in large amount. Growth of processing industries produces a variety of products and significantly reduces the post-harvest wastage but the major problems being faced by all industries across the world is the waste generation. Agro-industrial waste consists of both solid and liquid residues; rotten, peels, shells and scraped portions of vegetables form the solid portion, slurries and washed water are the liquid part.

Characterization of vegetable waste: assessment for reusability

Fig. 1. Value added products derived from processing of vegetable wastes.

Sources of vegetable wastes Vegetable wastes are plant tissue residues generated right from agricultural field to processing factories. The main forms of these organic wastes are domestic/household, industrial and agricultural waste.

IJFFT 1(2) 2011 : 174

Evaluation of various characteristics of waste is essentially required for the management of waste and residues. Fig. 2 shows the systematic approach for waste characterization (Fiehn, 2007; Huys and Gielen, 2007). It has been reported that the chemical composition of material is the determining factor for decomposition or Nitrogen mineralization (Rahn et al., 2009). Waste characterization is an essential step to review the potentials of the waste whether it should be disposed or recycled for developing various value-added components. Tables 1 and 2 show the physical and chemical characterization of several types of vegetable waste, respectively. Further, the waste from vegetable industries is organic in nature with high

Fermentation technology on vegetable residues

Table 1. Physical characteristics of the various types of vegetable waste Waste

Moisture (%)

Ash (%)

Total solid (%)

Volatile solid (%)

Potato

87.6

11.7

19

95

Tomato

4.3

3.1

11.9

93.2

Onion

50-60

4.7±0.1

-

-

Benítez, 2011

Peas

88.8

11.11

91.22

CPCB, 2007

Sugar beet

-

3.36

11

84

Asparagus

-

1.91

9.5

-

References Afifi, 2011; Paravira, 2005; Yamada, 2009 Hills and Nakano, 1984

Hampannavar and Shivayogimath, 2010 Lane, 1984

Table 2. Chemical characteristics of the various types of vegetable waste Waste

Starch (%)

Cellulose (%)

Hemicellulose (%)

Pectin (%)

Protein/amino acids (%)

References

Potato

37

17

14

17

4

Mayer, 1998

Tomato

-

31.5

17.8

-

17

Sarada and Joseph, 1993

Apple

-

43.6

24.4

11.7

-

Nawirska 2005

and

Kwaniewska,

Carrot

-

51.6

12.3

3.88

-

Nawirska 2005

and

Kwaniewska,

Pear

-

34.5

18.6

13.4

-

Nawirska 2005

and

Kwaniewska,

BOD (Biological Oxygen Demand) ranging from 817-1,927 in carrot, 43-1,400 in green beans and 454-1,575 in tomatoes. Due to high BOD, the waste is highly decomposable and is a rich source of several nutrients like vitamins, minerals, fibres, etc. (Joshi et al., 1999).

Fermentation technology: method of processing vegetable residue

Fig. 2. Systematic approach for waste characterization.

Reusability of perishable vegetable waste is one of the most important approaches to convert the whole process into zero emission models. Biodegradable nature of vegetable wastes makes the component easily accessible to microorganisms and provides a viable solution to detrimental environmental effects. Microbes are potential candidate for reprocessing and eventual utilization of vegetable processing residues. The use of microorganisms on these potential polluting materials IJFFT 1(2) 2011 : 175

Singh et al.

requires a proper skill and environment, which are provided by the efficient methods of fermentation process applied for biological degradation. Application of the fermentation principles provides the vital information about the optimal parameters controlling the processes (Petre et al., 1999), which helps to maximise the bioconversion to valuable product in cost effective and eco- friendly manner.

Aerobic digestion of wastes Aerobic digestion is a biological process, in which, organisms use available organic matter to support biological activity (Jin et al., 2009). The process uses organic matter, nutrients and dissolved oxygen, and produces stable solids and carbon dioxide. The aerobic digestion process mainly consists of two steps; the direct oxidation of biodegradable matter and endogenous respiration where cellular material is oxidized. For the purpose of reducing the volume of waste, anaerobic digestion has been widely used in the large scale industries, since it generates biogas and has high efficiency to treat waste. However, in case of small and medium scale industry, aerobic digestion is mostly used due to low investment cost, reduction in volume and mass of waste, reduction of pathogens, lower capital investment and simple operation. There are several aerobic treatment processes but the most popular among all are activated sludge and trickling filter method. For any biological process, microorganisms are mainly acting on organic matter only when the environmental conditions are favourable. Some of the factors are pH, acidity, alkalinity, solid-to-liquid ratio (vegetable waste to liquid ratio), food to micro organism ratio, temperature, toxic elements, biological oxygen demand, chemical oxygen demand etc. The overall aerobic digestion process can be illustrated in the following equations: Organic matter + O2 + NH4 Organic matter + O2

CO2 + H2O + Cellular material Bacteria

CO2 + H2O + NO3 + Digested waste

For efficient bioconversion, the process should operate at optimum conditions for maximum yield. Liu et al. (2010) used autothermal thermophilic aerobic digestion system for digestion of organic waste. Maximum digestion of volatile suspended solid (50.4%) at 55 oC after 264 h of incubation has been reported. Chang et al. (2006) reported composting of vegetable

IJFFT 1(2) 2011 : 176

wastes resulted in maximum carbon conversion of 14.54% after 4 days of incubation. Sarkar et al. (2010) used two amylolytic and three cellulolytic thermophilic bacteria for composting of vegetable wastes. They reported a significant reduction in C/N ratio after 10 days of incubation. Vermicomposting involve the bio-oxidation and stabilization of organic matter through the joint action of earthworms and microorganisms under aerobic and mesophilic conditions. There are several reports on vermicomposting of vegetable wastes. Suther (2009) reported vermicomposting of vegetable market solid waste causing a decrease in organic Carbon (12.7–28%) and C:N ratio (42.4–57.8%), while increase in total N (50.6– 75.8%), available P (42.5–110.4%) and exchangeable K (36.0–78.4%) contents. Aerobic treatment can successfully integrated into the vegetable waste management with the enforcement of legislative measures, and technical and managerial support.

Anaerobic digestion of wastes Anaerobic digestion is a biological process that happens naturally when bacteria break down organic matter in environment with little or no oxygen. It is a well studied technique for organic waste biodegradation (Mata-Alvarez et al., 2000). It was concluded from several research activities that anaerobic digestion can provide the biogas, a renewable form of energy (Angelidaki et al., 2005; Bolzonella et al., 2006). Vegetable market waste was explored by Gavrilescu (2009) as feedstock for biphasic biomethanation process. Simultaneously, the solid slurry left after gas production can be reutilized as soil conditioner with minor treatment (Converti et al., 1999). Fig. 3 shows the diagram for conversion of waste to energy by anaerobic treatment. Important factors effecting the efficiency and stability of anaerobic digestion are feed, type (Converti et al., 1999), temperature (Kettunen and Rintala, 1997), incubation time, pH (Landine et al., 1983), organic overloading and material flow. The three stages of anaerobic digestion are controlled by the bacteria; therefore, the product varies with the type of bacterial population. Total solid content is also an important factor for anaerobic digestion. Anaerobic batch digestion of vegetable waste with 5% total solid (Rajeshwari et al., 1998) resulted in better methane production than 8% total solid in batch digester due to more volatile fatty acid which were inhibitory component effecting pH (Marouani et al., 2002). Any variation at any step will hinder the overall production pattern.

Fermentation technology on vegetable residues

Fig. 3. Scheme for conversion of waste to energy by anaerobic treatment (a zero waste concept).

Value added product derived from vegetable waste Vegetable wastes have proved to be an efficient raw material for production of several value added products due to their easy availability and abundance in supply. Different scientific approaches have been employed for this purpose.

Organic acid production Among the organic acids, lactic acid, acetic acid, oxalic acids are of greater importance because of their varied applications from food industry to pharmaceuticals. They can be produced by fermentation process by the activity of different microorganisms including fungi and bacteria. Various organic acids are produced by the microbial fermentation e.g. citric acid, lactic acid and acetic acid by Aspergillus niger, Lactobacillus delbrueckii and Acetobacter aceti, respectively (Sethi and Maini, 1999). Although several substrates have been used, but in respect of large scale production, an agricultural waste has high demand due to its abundant availability (Soccol et al., 2006). Among them, citric acid is an important chemical having world wide demand due to its high usage and low toxicity. Beet molasses have been

used for citric acid production. Different attempts have been made to enhance the productivity. El-Abyad et al. (1992) investigated various treatments of beet molasses necessary for improving the production of citric acid using Aspergillus niger var Tieghem strain 599. Addition of 0.05% N as (NH4)2SO4 was found to improve the yield (22.81%) and conversion coefficient (24.52%). In a similar study, by Nehad et al. (2008) addition of natural oils with high unsaturated fatty acids to beet molasses increased the citric acid yield by Aspergillus niger A20. At 4% olive oil concentration, 12 day old surface culture gave maximum yield of 72.9 g/L (Adham, 2002). Lactic acid, a unique substrate in medical field for production of biodegradable polymer, can also be produced from residual waste with the help of fungus and bacteria. Afifi, (2011), used liquid potato waste (LPW) having high starch content upto 50% which produces 16.09 g/L lactic acid by selecting factors like temperature, pH, incubation time, etc. Zhang and Jin (2009), used Rhizopus arrhizus under nutrient limited condition in presence of potato starch waste which increased the lactic acid production upto 103.8 g/L in 48 h fermentation. Among other organic acids, oxalic acid also has high demand in many sectors. Sugar beet molasses have been used as low cost substrate for oxalic acid production using IJFFT 1(2) 2011 : 177

Singh et al.

different reactors. High production was obtained in the reactor having nitrogen oxide (Guru et al., 2001). Acetic acid, another organic acid can be produced by carrots and white radish leafage. Carrots have been used as the substrate in the hydrothermal two stage production of acetic acid which resulted in high yield (Jin et al., 2005). Thus, organic acid production by utilizing vegetable waste serves two purpose; reduction in the cost of raw material and recycling the waste, thus decreasing the pollution problem.

Bioenergy Biofuel production from vegetable wastes is one of the interesting approaches from the economic and environment protection point of view. Global energy demand (Schenk et al., 2008) has stimulated recent interest to explore alternative sources for petroleum-based fuels. Various alternative fuel options are mainly biogas, bioethanol, biodiesel etc. Biogas, a renewable energy source is produced by the biomethanation process. This flammable gas is produced by fermentation of organic material in oxygen depleted conditions. Biomethanation technology is gaining wider acceptance in waste management as it not only provides the renewable energy, but also leaves a

residue in form of organic manure. Several workers reported the use of fruit and vegetable wastes as substrate for the biogas production as mentioned in Table 3. Kale and Mehetre, (2006) developed kitchen waste biogas plant using thermophilic bacteria. Socio-economic aspect studies of anaerobic digestion of vegetable waste by Cojolon et al. (2008) prove that daily domestic wastes produce sufficient biogas for daily cooking. Mixed vegetable waste anaerobic batch digestion after 47 days resulted in 0.16m3 biogas/kg TS (Rajeshwari et al., 1998). There are certain limitations of biomethanation process, such as start-up of the system is too long unavailability of raw materials prolongs the standardization. Biodiesel is alcohol ester of fatty acids produced when oil reacts with alcohol in presence of a catalyst. The process is known as transesterification. Different approaches followed for this purpose, differ in the type of catalyst used for transesterification (Gomez-Castro et al., 2010; Kiss et al., 2010). Several reports are available on utilization of various type of substrates for biodiesel production (Bajaj et al., 2010; Balat and Balat, 2010). One of the potent and efficient substrate for production of biodiesel is waste vegetables. Presence of fatty acid is the main criteria for the production

Table 3. Biogas production from vegetable wastes Wastes

Asparagus, carrots, green peas, French beans, spinach and strawberries Carrot peel

Loading rate

Incubation time (days)

Biogas quantity

References

0.80 - 1.60 kg VS m? 3 day? 1

32 days

0.30 to 0.58 m3 kg-1 VS day? 1

Knol et al., 1978

12g VS L-1 day- 1

2 day

0.42 L g-1 (carrot)

Verrier et al., 1987

-1

French bean Tomato

Tomato-processing waste

0.32 L g (French bean) VS 40 kg TS m? 3 day? 1

20 day

0·6 m3/kg VS

Viswanath et al.,1992

4.3 kg VS

24 day

0.42 m-3 kg-1

Sarada and Joseph, 1994

-3

m d Cassava

Cabbage, carrot, lettuce

IJFFT 1(2) 2011 : 178

-3

VS

0.588 g L-1d-1

100

0.42 L L-1d-1

Ishizuka et al., 1995

3.07 kg VS m? 3 day? 1

19 day

0.63 m3 kg-1 VS

Lin et al., 2011

Fermentation technology on vegetable residues

of biodiesel. Alcohol esters of vegetable oils possess characteristics that are very close to that of diesel fuel (Ghassan et al., 2003). Su et al. (2010) reported high temperature boiling pretreatment of kitchen garbage for biodiesel production. Zhang et al. (2003) carried out an economic study on the use of waste cooking oil and vegetable oils for four continuous alkali-acid catalyzed processes. Bioethanol is another one alternative fuel source. It has been found to serve considerably as transportation fuel. Ethanol production from vegetable waste mainly includes pretreatment, hydrolysis and fermentation (Chandel et al., 2007). Several reports are there on bioethanol production from various food wastes by using Saccharomyces cerevisiae (Izmirlioglu and Ali, 2010; Yan et al., 2011). Arapoglou et al. (2010) reported 7.6 g/L bioethanol production from potato peel after 2 days of incubation at 32 oC.

Polyhydroxybutrate production (PHB) Polyhydroxybutyrate (PHB) is a biopolymer that can be used as a biodegradable thermoplastic material for waste management strategies and biocompatibility in medical devices (Gouda et al., 2001). It has wide applications in different areas such as packaging, pharmaceuticals, chemical and cosmetic industries. In almost all established industrial processes for PHB production, the substrates used are sugar-based compounds, which have a high market price (Serafim et al.,

2008). Therefore, more cost-effective technologies are required. In recent years, vegetable and food wastes have been employed for production of PHB in a cost effective way (Carucci et al., 2001; Koller et al., 2008). Rusendi and Sheppard, (1995) reported the use of potato processing waste from the potato-chip plant for the production of PHB. Hafuka et al. (2011) reported 87% PHB yield from food wastes after 259 h of incubation.

Single cell protein production The increasing world deficiency of protein is becoming a main problem of humankind. The world availability of protein can be increased in the following three basic ways: (1) by increasing the efficiency of the existing methods of protein production, (2) by making increased use of the new available technologies and (3) by utilizing new probable sources of protein and developing new technologies for them. In this respect, several vegetable wastes can be employed for single cell protein (SCP) production. Compared to conventional methods, microbial production of SCP has several advantages such as high protein content and short growth times leading to rapid biomass production, which can be continuous and is independent of the environmental conditions (Bekatorou et al., 2006). The use of fungi, especially yeasts, for SCP production is more convenient, as they can be easily propagated using cheap raw materials and easily harvested due to their

Table 4. Different value added product development from vegetable waste Vegetable waste

Value added products

References

Asparagus waste

Mushroom

Wang et al., 2010

Cabbage waste

Single cell protein

Choi et al., 2002; Choi et al. 1999

Carrot waste

Biohydrogen, Mass production entomopathogenic fungi

Onion waste

Biomethane, Vinegar

Horiuchi et al., 1999; Romano and Zhang 2008,

Peas waste

Biomethane, Biohydrogen

Kalia and Joshi, 1995

Tomato waste

Biomethane, Biogas, Single cell protein

Fernandez-Gomez et al., 2010; Trujillo et al., 1993; Viswanath, 1992

Sugar beet waste

Single cell protein, arabinofuranosidase

of

α-L-

Sahayaraj and Namasivayam, 2008; Vrije et al., 2010;

Barrocal, 2010; Roche et al., 1995

IJFFT 1(2) 2011 : 179

Singh et al.

bigger cell sizes and flocculation abilities. Many strains of Saccharomyces, viz., S. cerevisiae, S. lipolytica, S. fibuligera and some other yeasts like Kluyveromyces fragilis and some strains of Candida and Torulopsis are also used in biomass production (Bhalla et al., 1999). Zhao et al. (2010) reported single cell protein production (6.8 g/L) by Candida utilis from waste of capsicum powder. Gao et al. (2007) achieved a maximum 53 g crude protein/100 g of cell dry weight from Jerusalem artichoke extract. They reported maximum protein production in C. utilis. Stabnikova et al. (2005) reported production of selenium enriched Saccharomyces cerevisae biomass by growing the organism in extracts of cabbage, watermelon, a mixture of residual biomass of green salads and tropical fruits. Filamentous fungi (Aspergillus, Fusarium, Rhizopus, etc.), Alga (Spirullina, Chlorella etc.) and many bacterial species (Bacillus, Lactobacillus, Pseudomonas etc.) are extensively used in SCP production (Bhalla et al., 1999). The alarming prospects of world scarcity lead to great expectations about the social and economic relevance of microbial production of single cell protein by utilizing vegetable wastes. Table 4 shows value added products from different types of vegetable waste.

Health, environmental and social aspect of vegetable waste management Tapping the potential of vegetable wastes for production of value added products-organic acids, PHBs, SCP etc and for generation of biofuels, is an efficient mode of waste treatment. For accomplishment of better waste utilization, strategies for efficient waste management need to be adopted. The best approach for waste management is reduction of the waste at its source before its entry into the waste stream i.e. integrated waste management. Socio-economic aspect of waste generation and handling is yet another area that needs to be considered for adopting an efficient strategy of integrated waste management. Vegetable wastes are generated as a part of human society either at small domestic level or at large industrial level. The composition and type of waste generated and the waste management strategies depend on income level of the country and extent of industrialization. In developed countries, better, waste management practices are followed like sanitary landfills, composting, incineration etc. Wastes are collected and mostly dumped in open or burnt in open (Sandra, 2006). This leads to serious hazardous impact on both environment and human

IJFFT 1(2) 2011 : 180

health. When dumped in open or in landfills, vegetable wastes being rich in organic matter get easily decomposed by the action of various microbes. This produces different gases like methane and carbon dioxide both of which contribute to the green house effect leading to global warming (Brown and David, 1994). Open air incineration also generates noxious gases causing air pollution. Apart from this, the open decomposed organic matter acts as breeding ground of various disease causing organisms and their vectors thereby leading to a spread of air borne diseases at an alarming rate. Leachate from dumped waste contain high amount of total dissolved solid, ammonia, nitrate, phosphate, calcium, sulfate, and iron, as well as numerous heavy metals which have the potential to cause water pollution of surface and ground water (Salem et al., 2008), hence, necessitating the application of alternative strategies. Alternative methods like recovery and use of landfill gas for combustion purposes help to reduce the emission of green house gases and simultaneously, act as a source of renewable fuel. Composting and anaerobic digestion of organic wastes are yet another available promising alternative which helps to mitigate the health and emission related problems (Pipatyi and Savolainen, 1996). The remaining biomass after anaerobic digestion could be used as a fertilizer. Having highlighted the various techniques and plausible options available for waste management it may be re-emphasized that a prudent selection of waste management scheme is essential to ensure human and environment safety. This can be done by educating people about waste handling, proper municipal organization and similar activities. Implementation of cheap and ecofriendly techniques add advantage for better utilization of significant amount of the generated vegetable waste. Recycling and reuse of these organic wastes for production of useful products could be the most sought after approach for economic sustenance of a society with an ecofriendly touch.

Conclusion The present article addresses the problems associated with the vegetable wastes generated in a huge amount and it’s recycling in an environmental friendly manner. The review also highlights on some of the research activities being carried out globally where vegetable waste has been explored for valuable product generation. Waste characterization makes them a suitable candidate for energy generation in an economically viable way to meet the global demand of energy

Fermentation technology on vegetable residues

instead of dumping them in landfills. Combination of value added product development with the aerobic composting is considered to be the best possible alternatives for vegetable waste management. Emphasis on the suitable implementation of fermentation technologies for value added product generation adopting the zero waste concept needs to be laid.

Carucci A, Dionisi D, Majone M, Rolle E and Smurra P. 2001. Aerobic storage by activated sludge on real wastewater. Water Res., 35: 3833–3844. Chang JI, Tsai JJ and Wu KH. 2006. Composting of vegetable waste. Waste Manage. Res., 24: 354–362.

References

Chandel AK, Chan ES, Rudravaram R, Narasu ML, Rao LV and Ravindra P. 2007. Economics and environmental impact of bioethanol production technologies: an appraisal. Biotechnol. Mol. Biol. Rev., 2: 14-32.

Adham NZ. 2002. Attempts at improving citric acid fermentation by Aspergillus niger in beet molasses medium. Bioresource Technol., 84: 97–100.

Choi MH, Ji GE, Koh KH, Ryu YW, Jo DH and Park YH. 2002. Use of waste Chinese cabbage as a substrate for yeast biomass production. Bioresource Technol., 83: 251–253.

Afifi MM. 2011. Enhancement of lactic acid production by utilizing liquid potato wastes. Int. J. Biol. Chem., 5: 91–102.

Choi MH and Park YH. 1999. Growth of Pichia guilliermondii A9, an osmotolerant yeast, in waste brine generated from kimchi production. Bioresource Technol., 70: 231–236.

Angelidaki I, Boe K and Ellegaard L. 2005. Effect of operating conditions and reactor configuration on efficiency of full-scale biogas plants. Water Sci. Technol., 52: 189–194. Arapoglou D, Varzakas T, Viyssides A and Israilides C. 2010. Ethanol production from potato peel waste (PPW). Waste Mange., 30: 1898–1902. Bajaj A, Lohan P, Jha PN and Mehrotra R. 2010. Biodiesel production through lipase catalyzed transesterification: an overview. J. Mol. Catal. B: Enzymatic, 62: 9–14. Balat M and Balat H. 2010. Progress in biodiesel processing. Appl. Energ., 87: 1815–1835. Barrocal VM, Garcia-Cubero MT, Gonzalez-Benito G and Coca M. 2010. Production of biomass by Spirulina maxima using sugar beet vinasse in growth media. New Biotechnol., 27: 851–856. Basediya AL, Samuel DVK and Beera V. 2011. Evaporative cooling system for storage of fruits and vegetables: a review. J. Food Sci. Technol., doi: 10.1007/s13197-011-0311-6. Bekatorou A, Psarianos C and Koutinas AK. 2006. Production of food grade yeasts. Food Technol. Biotechnol., 44: 407–415. Benítez V, Mollá E, Martín-Cabrejas MA, Aguilera Y, López-Andréu FJ, Cools K, Terry LA and Esteban RM. 2011. Characterization of industrial onion wastes (Allium cepa L.): dietary fibre and bioactive compounds. Plant Foods Hum. Nutr., 66: 48–57. Bhalla TC, Gajju Harish and Agrawal Hari Om. 1999. Production of single cell proteins. In: Biotechnology: Food Fermentaton, Microbiology, Biochemistry and Technology, Vol II. (Eds. VK Joshi and Ashok Pandey), Educational Publishers and Distributors, New Delhi, pp. 1003-1022.

Cojolon CL, Estrada TE and Ciferri A. 2008. Technical, socio/ economical and environmental aspects of biogas generation using rural household waste. La Chimica & l’Industria, Milan, 90: 130–134. Converti A, Del-Borghi A, Zilli M, Arni S and Del-Borghi M. 1999. Anaerobic digestion of the vegetable fraction of municipal refuses: mesophilic versus thermophilic conditions. Bioprocess Eng., 21: 371–376. CPCB. 2007. Sponge Iron Industry. Comprehensive industry document series. Central pollution control board (Ministry of Environment and Forests, Govt. of India), pp. 87–98. EI-Abyad MS, Hamissa FA and Gad AS. 1992. Treatment of beet molasses for citric acid production by a potent strain of Aspergillus niger van Tieghem. Bioresource Technol., 39: 191– 197. Fernandez-Gomez MJ, Nogales R, Insam H, Romero E and Goberna M. 2010. Continuous-feeding vermicomposting as a recycling management method to revalue tomato-fruit wastes from greenhouse crops. Waste Manage., 30: 2461–2468. Fiehn H. 2007. A case study on alternative approaches to waste characterization analysis amid salvaging and recycling issues in South Africa. Eleventh international waste management and landfill symposium, Environmental Sanitary Engineering Centre, Italy, Leal W (eds.), October1–5, pp 1-11. Gao LM, Chi ZM, Sheng J and Ni XM. 2007. Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast Cryptococcus aureus G7a and its nutritive analysis. Appl. Microbiol. Biotechnol., 77: 825–832.

Bolzonella D, Pavan P, Mace S and Cecchi F. 2006. Dry anaerobic digestion of differently sorted organic municipal solid waste: a full-scale experience. Water Sci. Technol., 53: 23–32.

Gavrilescu M. 2009. Environmental biotechnology: achievements, opportunities and challenges. Dyn. Biochem. Process Biotechnol. Mol. Biol., 4: 1–36.

Brown KA and David MH. 1994. Using landfill gas: a UK perspective. Renew. Energ., 5: 774–781.

Ghassan T, Al-Widyan MI and Al-Shyoukh AO. 2003. Combustion performance and emissions of ethyl ester of a waste vegetable oil in a water-cooled furnace. Appl. Therm. Eng., 23: 285–293.

IJFFT 1(2) 2011 : 181

Singh et al.

Gilbert L. 1997. The consumer market for functional foods. J. Nutraceut. Function. Med. Foods, 1: 5–21. Gomez-Castro FI, Rico-Ramirez V, Segovia-Hernandez JG and Hernandez S. 2010. Feasibility study of a thermally coupled reactive distillation process for biodiesel production. Chem. Eng. Process., 49: 262–269. Gouda MK, Swellam AE and Omar SH. 2001. Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen source. Microbiol. Res. J., 156: 201–204. Guru M, Bilgesu AY and Pamuk V. 2001. Production of oxalic acid from sugar beet molasses by formed nitrogen oxides. Bioresource Technol., 77: 81–86. Hafuka A, Sakaida K, Satoh H, Takahashi M, Watanabe Y and Okabe S 2011. Effect of feeding regimens on polyhydroxybutyrate production from food wastes by Cupriavidus necator. Bioresource Technol., 102: 3551–3553. Hampannavar US and Shivayogimath CB. 2010. Anaerobic treatment of sugar industry wastewater by upflow anaerobic sludge blanket reactor at ambient temperature. Int. J. Environ. Sci., 1: 631–639. Hills DJ and Nakano L. 1984. Effects of particle size on anaerobic digestion of tomato solid waste. Agric. Wastes, 10: 285–295. Horiuchi JI, Kanno T and Kobayashi M. 1999. New vinegar production from onions. J. Biosci. Bioeng., 88: 107–109. Huys T and Gielen P. 2007. Systematic approach to radioactive waste characterization at belgoprocess. The 11th international conference on environmental remediation and radioactive waste management. Bruges, Belgium, pp. 655–658. Ishizuka K, Hisajima S and Macer DRJ. 1995. Sago palm, a promising renewable carbohydrate resource, a material for environmental conservation and sustainable development. In: Proceeding of the international seminar on traditional technology for environmental conservation, development in the Asia-pacific region, UNESCO, Tsukuba Science City, Japan, pp. 75–76. Izmirlioglu G. and Ali C. 2010. Ethanol Production from waste potato mash by using Saccharomyces cerevisiae. ASABE. Jin Y, Li H, Mahar RB, Wang Z and Nte Y. 2009. Combined alkaline and ultrasonic pretreatment of sludge before aerobic digestion. J. Environ. Sci., 21: 279–284. Jin F, Zhou Z, Moriya T, Kishida H, Higashijima H and Enomoto H. 2005. Controlling hydrothermal reaction pathways to improve acetic acid production from carbohydrate biomass. Environ. Sci. Technol., 39: 1893–1902. Joshi VK, Pandey A. and Sandhu DK.1999. Fermentation technology for food industry waste utilization. In: Biotechnology: Food Fermentaton, Microbiology, Biochemistry and Technology. Vol II. (Eds. VK Joshi and Ashok Pandey). Educational Publishers and Distributors, New Delhi, pp. 1291-1348.

IJFFT 1(2) 2011 : 182

Kale SP and Mehetre ST. 2006. Kitchen waste based biogas plant. Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, India. Kalia VC and Joshi AP. 1995. Conversion of waste biomass (peashells) into hydrogen and methane through anaerobic digestion. Bioresource Technol., 53: 165-168. Kettunen R and Rintala J. 1997. The effect of low temperature (5 – 29 °C) and adaptation on methanogenic activity of biomass. Appl. Microbiol. Biotechnol., 48: 570–576. Kiss FE, Jovanoviæ M and Boškoviæ GC. 2010. Economic and ecological aspects of biodiesel production over homogeneous and heterogeneous catalysts. Fuel Process. Technol., 91: 1316– 1320. Knol MM, Most V and Wart JD. 1978. Biogas production by anaerobic digestion of fruit and vegetable waste: A preliminary study. J. Sci. Food Agric., 29: 822–830. Koller R, Chiellini BE, Fernandes EG, Horvat P, Kutschera, C, Hesse P and Braunegg G. 2008. Polyhydroxy alkanoate production from whey by Pseudomonas hydrogenovora. Bioresource Technol., 99: 4854–4863. Landine RC De-Gairie CJ, Cocci AA, Steeves AL, Brown GJ and Viraraghavan, T. 1983. Anaerobic pretreatment facility also provides sludge disposal capability and source of renewable energy for food processor. In: Proceedings of the 38th industrial waste conference, Lafayette, Purdue University, pp. 805–815. Lane AG. 1984. Laboratory scale anaerobic digestion of fruit and vegetable solid waste. Biomass, 5: 245–259. Lin J, Zuo J N, Gan L L, Li P, Liu F L, Wang K J et al., 2011. Effects of mixture ratio on anaerobic co-digestion with fruit and vegetable waste (FVW) and food waste (FW) of China. J. Environ. Sci., 23. Liu S, Song F, Zhu N, Yuan H and Cheng J. 2010. Chemical and microbial changes during autothermal thermophilic aerobic digestion (ATAD) of sewage sludge. Bioresource Technol., 101: 9438–9444. Marouani L, Bouallagui H, Ben CR and Hamdi M. 2002. Biomethanation of green wastes of wholesale market of Tunis. In: Proceedings of the international symposium on environmental pollution control and waste management, Tunis, Bennaceur R (eds.), pp. 318–323. Mata-Alvarez J, Mace S and Liabres P. 2000. Anaerobic digestion of organic solid wastes. an overview of research achievements and perspectives. Bioresource Technol., 74: 3–16. Mayer F. 1998. Potato pulp: properties, physical modification and applications. Polym. Degrad. Stabil., 59: 231–235. Nawirska A and Kwaniewska M. 2005. Dietary fibre fractions from fruit and vegetable processing waste. Food Chem., 91: 221– 225.

Fermentation technology on vegetable residues

Nehad ZA, Essam MA and Haba AR. 2008. Metabolic inhibitors as stimulating factors for citric acid production. Pak. J. Sci. Ind. Res., 51: 245-249 Ngoc NU and Schnitzer H. 2008. Waste management towards zero emissions approach in the fruit juice processing industry. 1st WSEAS international conference on environmental and geological science and engineering, Malta, Sokolov V (eds.), September, 11–13. Nigam P and Vogel M. 1991. Bioconversion of sugar Industry byproducts-molasses and sugar beet pulp for single cell protein production by yeasts. Biomass Bioenerg., 1: 339–345. Paravira W, Murto M, Zvanya R and Mattiasson B. 2005. Comparative performance of a UASB reactor and an anaerobic packed-bed reactor when treating potato waste leachate. Renew. Energ., 12: 1–11. Patle S and Lal B. 2007. Ethanol production from hydrolyzed agricultural wastes using mixed culture of Zymomonas mobilis and Candida tropicalis. Biotechnol. Lett., 29: 1839–1843. Petre M, Zarnea G, Adrian P and Gheorghiu E. 1999. Biodegradation and bioconversion of cellulose wastes using bacterial and fungal cells immobilized in radiopolymerized hydrogels. Resour. Conserv. Recy., 27: 309–332. Pipatyi R and Savolainen I. 1996. Role of energy production in the control of greenhouse gas emissions from waste management. Energy Convers. Mgmt., 37: 1105–1110. Rajeshwari KV, Panth DC, Lata K and Kishore VVN. 1998. Studies on biomethanation of vegetable market waste. Biogas Forum, 3: 4–11. Rahn CR, Bending GD, Lillywhite RD and Turner MK. 2009. Coincorporation of biodegradable wastes with crop residues to reduce nitrate pollution of groundwater and decrease waste disposal to landfill. Soil Use Manag., 25: 113–123. Roche N, Berna P, Desgranges C and Durand A. 1995. Substrate use and production of á-L-arabinofuranosidase during solid-state culture of Trichoderma reesei on sugar beet pulp. Enzyme Microb. Technol., 17: 935–941.

solid waste management. The World Bank Group, Urban papers. Washington, DC. Sarada R and Joseph R. 1994. Studies on factors influencing methane production from tomato-processing waste. Bioresource Technol., 47: 55–57. Sarada R and Joseph R. 1993. Biochemical changes during anaerobic digestion of tomato processing waste. Proc. Biochem., 28: 461– 466. Sarkar S, Banerjee R, Chanda S, Das P, Ganguly S and Pal S. 2010. Effectiveness of inoculation with isolated Geobacillus strains in the thermophilic stage of vegetable waste composting. Bioresource Technol., 101: 2892–2895. Schenk PM, Thomas-Hall SR, Stephens E, Marx UC, Mussgnug JH, Posten C, Kruse O and Hankamer B. 2008. Second generation biofuels: high efficiency microalgae for biodiesel. Bioenergy Res., 1: 20–43. Serafim LS, Lemos PC, Albuquerque MGE and Reis MAM. 2008. Strategies for PHA production by mixed cultures and renewable waste materials. Appl. Microbiol. Biotechnol. 81: 615–628. Sethi Vijay and Maini SB. 1999. Production of organic acids. In: Biotechnology: Food Fermentaton, Microbiology, Biochemistry and Technology, Vol II. (Eds. VK Joshi and Ashok Pandey). Educational Publishers and Distributors, New Delhi, pp. 12591290. Sharma Nivedita.1999. Industrial ethanol production. In: Biotechnology: Food Fermentaton, Microbiology, Biochemistry and Technology, Vol II. (Eds. VK Joshi and Ashok Pandey). Educational Publishers and Distributors, New Delhi, pp. 12011230. Soccol CR, Vandenberghe LPS, Rodrigues C and Pandey A. 2006. New perspectives for citric acid production and application. Food Technol. Biotechnol., 44: 141–149. Stabnikova O, Wang JY, Ding HB and Tay JW. 2005. Biotransformation of vegetable and fruit processing wastes into yeast biomass enriched with selenium. Bioresource Technol., 96: 747–751.

Romano R and Zhang R. 2008. Co-digestion of onion juice and wastewater sludge using an anaerobic mixed biofilm reactor. Bioresource Technol., 99: 631-637.

Su W, Ma H, Gao M, Zhang W and Wang Q. 2010. Research on biodiesel and ethanol production from food waste. The project was supported by the 11th five years key programs for science and technology support of China (2006BAJ04A06).

Rusendi D and Sheppard JD. 1995. Hydrolysis of potato processing waste for the production of poly-ß-hydroxybutyrate. Bioresource Technol., 54: 191–196.

Suther S. 2009. Vermicomposting of vegetable-market solid waste using Eisenia fetida: impact of bulking material on earthworm growth and decomposition rate. Ecol. Eng., 35: 914–920.

Sahayaraj, K. and Namasivayam, S.K.R. 2008. Mass production of entomopathogenic fungi using agricultural products and by products. Afr. J. Biotechnol., 7: 1907–1910.

Trujillo D, Perez JF and Cebre FJ. 1993. Energy recovery from wastes: anaerobic digestion of tomato plant mixed with rabbit wastes. Bioresource Technol., 45: 81–83.

Salem Z, Hamouri K, Djemaa R and Allia K. 2008. Evaluation of landfill leachate pollution and treatment. Desalination, 220: 108–114.

Verrier, D.; Roy, F. and Albagnac, G. 1987. Two-phase methanization of solid vegetable wastes. Biol. Waste., 22: 163–177.

Sandra, C. 2006. Occupational and environmental health issues of

Viswanath P. 1992. Anaerobic digestion of fruit and vegetable processing wastes for biogas production. Bioresource Technol., 40: 43–48.

IJFFT 1(2) 2011 : 183

Singh et al.

Vrije T, Budde MAW, Lips SJ, Bakker RR, Mars AE and Claassen PAM. 2010. Hydrogen production from carrot pulp by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. Int. J. Hydrogen Energ., 35: 13026– 13213. Wang Q, Li BB, Li H and Han JR. 2010. Yield, dry matter and polysaccharides content of the mushroom Agaricus blazei produced on asparagus straw substrate. Sci. Hort., 125: 16–18. Yamada S, Shinomiya N, Ohba K, Sekikawa M and Oda Y. 2009. Enzymatic hydrolysis and ethanol fermentation of by-products from potato processing plants. Food. Sci. Technol. Res., 15: 653–658.

IJFFT 1(2) 2011 : 184

Yan S, Wang P, Zhai Z and Yao J. 2011. Fuel ethanol production from concentrated food waste hydrolysates in immobilized cell reactors by Saccharomyces cerevisiae H058. J. Chem. Technol. Biotechnol., 86: 731–738. Zhang Z and Jin B. 2009. Proceedings of the conference of the Australian society of sugar cane technologists, Ballina, New South Wales, Australia, pp. 372–380. Zhang Y, Dube MA, McLean DD and Kates M. 2003. Biodiesel production from waste cooking oil: 2. Economic assessment and sensitivity analysis. Bioresource Technol., 90: 229–240. Zhao G, Zhang W and Zhang G. 2010. Production of single cell protein using waste capsicum powder produced during capsanthin extraction. Lett. Appl. Microbiol., 50: 187–191.

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 185-199

Review Paper

White button mushroom (Agaricus bisporus): Composition, nutritive value, shelf-life extension and value addition Surabhi Sharma* and Devina Vaidya Department of Food Science and Technology, Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan, H.P., India.

Abstract : Agaricus bisporus is the most widely cultivated species of edible mushrooms comprising about 32% of the world production. It is a good source of high quality proteins, amino acids, carbohydrates, vitamins (mainly vitamin C and vitamin B-complex) and minerals, such as iron, calcium, phosphorous, potassium, selenium and dietary fibre. The dietary fibre content of Agaricus bisporus is attributed to the presence of polysaccharide chitin in its cell walls. Mushrooms are rich sources for antioxidants like lectins, terpenoids, beta-glucans, ascorbic acid, tocopherols and carboxylic acids. Agaricus bisporus is highly perishable and has a short shelf life of 3–5 days at 20C and around 1–2 days under ambient (Temp. 25 ± 20C, RH 70%) conditions. The short shelf-life of white button mushroom is an impediment to the distribution and marketing of the fresh produce. Accelerated physiological, morphological and microbial changes lead to browning and sliminess in addition to early breaking of the veil, expansion and darkening of the cap and gills and elongation of the stem. To increase the postharvest life of button mushroom different treatments like precooling, packing, irradiaition and processing techniques have been successfully utilized. The present review highlights the nutritional quality, health promoting phytonutrients, functional properties and shelf-life extension of white button mushroom along with their potential application. Keywords: Agaricus bisporus, White button mushroom, Processing, Shelf-life extension

Mushrooms are popularly known as functional foods (Liu and Wang 2009). World production of cultivated edible mushroom increased from 170 MT in 1960 to 10,995.5×103 MT in 2007 (Chang, 2007). China has become a giant producer and consumer of mushrooms and a large share (70%) of the total world production comes from China (Chang,2007). India’s share is estimated to be around 70,000 MT per annum in which major share is contributed by button mushroom (Agaricus spp.), while the rest being speciality one (Singh et al. 2003). *E-mail of corresponding author : [email protected] MS Received on : 27th May, 2011 Accepted on : 1st Oct., 2011

Diversified agro-climatic conditions in India offer vast potential for growing different types of mushrooms. There are about 20 varieties of mushrooms being cultivated throughout the world for food. In India, only white button mushroom (Agaricus bisporus), oyster mushroom (Pleurotus spp.) and paddy straw mushroom (Volvariella volvacea) are grown commercially. Out of these, white button mushroom contributes ~90% of the total production. Mushrooms have been recognized as the alternate source of good quality protein and produce the highest

Sharma and Vaidya

quantity of protein per unit area from agro-wastes. Besides, they provide potentiality for generating employment, improving economic status of growers, help in checking pollution and earn foreign exchange (Rai and Arumuganathan,2005).

Nutritional quality White button mushrooms (Agaricus bisporus) are a valuable source of several micronutrients in a low energy, nutrientdense food. Bano (1976) suggested that food value of mushrooms lies between meat and vegetables. Mushrooms are also low in sodium and kilojoules. They are excellent source of proteins, vitamins and minerals (Prakash and Tejaswin, 1991; Ghosh and Singh, 1995). Crisan and Sands (1978) observed that mushrooms in general contain 90% water and 10% dry matter (Table 1). More so, the protein content varies between 27 and 48%. Carbohydrates are less than 60% and lipids are between 2 to 8%. Orgundana and Fagade (1981) indicated that an average mushroom is about 16.5% dry matter out of which 7.4% is crude fibre, 14.6% is crude protein and 4.48% is fat and oil. Gruen and Wong (1982) indicated that edible mushrooms were highly nutritional and compared favourably with meat, egg and milk food sources. It provides 29-34% of the recommended daily intake (RDI) for Vitamin B2 (riboflavin) and 23-26% of the RDI for niacin per 100g. Mushrooms are also one of the very few foods that provide a natural source of vitamin D which can be significantly enhanced by sunlight or irradiation. In addition, mushrooms provide 22-26% of the RDI for selenium and 2029% of the adequate intake (AI) for copper. Carbohydrates: Dominating carbohydrates present in mushroom are raffinose, sucrose, glucose, fructose and xylose (Singh and Singh, 2002). Water soluble polysaccharides of mushrooms have antitumor effect (Yoshioka et al., 1975).The carbohydrate content of mushrooms represents the bulk of fruiting bodies accounting for 50 to 65% on dry weight basis. Free sugars amounts to about 11%. Florezak et al. (2004) reported that Coprinus atramentarius (Bull.: Fr.) Fr. contain 24% of carbohydrate on dry weight basis. The mannitol, also called as mushroom sugar constitutes about 80% of the total free sugars, hence it is dominant (Tseng and Mau, 1999; Wannet et al., 2000). Mc-Connell and Esselen (1947) reported that fresh mushroom contains 0.9% mannitol, 0.28% reducing sugar, 0.59% glycogen and 0.91% hemicellose. Carbohydrates of Agaricus bisporus were also reported by Crisan and Sands (1978). IJFFT 1(2) 2011 : 186

Proteins: In mushrooms dry matter is mainly represented by proteins (Aletor, 1995; Alofe et al., 1995; Florczak and Lasota, 1995; Zrodlowski, 1995; Chang and Buswell, 1996). Lintzel (1941) reported the digestibility of mushroom protein to be as high as 72 to 83%. Protein content of mushrooms depends on the composition of the substratum, size of pileus, harvest time and species of mushrooms (Bano and Rajarathnam,1982). Haddad and Hayes (1978) indicated that protein in A. bisporus mycelium ranged from 32 to 42% on the dry weight basis. Abou et al. (1987) found 46.5% protein on dry weight basis in A. bisporus. Purkayastha and Chandra (1976) found 14 to 27% crude protein on dry weight basis in A. bisporus, Lentinus subnudus, Calocybe indica and Volvariella volvacea. On dry matter basis, the protein content of mushrooms varies between 19/100 and 39/100 g (Weaver et al., 1977; Breene, 1990). In terms of the amount of crude protein, mushrooms rank below animal meats but well above most other foods including milk (Chang, 1980). Verma et al. (1987) reported that mushrooms are very useful for vegetarian because they contain some essential amino acids which are found in animal proteins. Rai and Saxena (1989) observed decrease in the protein content of mushroom on storage. Friedman (1996) reported that the total nitrogen content of dry mushrooms is contributed by protein, amino acids and also revealed that crude protein is 79% compared with 100% for an ideal protein. Fats: The fat content in mushroom is very low in comparison to carbohydrates and proteins. The fats present in mushroom fruiting bodies are dominated by unsaturated fatty acids. Mushrooms are considered good source of fats and minerals (Jiskani, 2001). Yilmaz et al. (2006) and Pedneault et al. (2006) reported that fat fraction in mushrooms is mainly composed of unsaturated fatty acids. Hugaes (1962) observed that mushrooms are rich in linolenic acid which is an essential fatty acid. Total fat content in A bisporus was reported to be 1.66 to 2.2/100 g on dry weight basis (Maggioni et al., 1968). Ogundana and Fagade (1981) indicated that mushrooms have 4.481% fats on dry weight basis. In 100 g fresh matter of A. bisporus (Lange) Sing and Pleurotus ostreatus (Jacq: Fr.) Kumm, the content of fatty compounds were found to be 0.3 and 0.4 g, respectively (Manzi et al., 2001), but on dry weight basis, it is 2 and 1.8 g, respectively (Shah et al., 1997). Aletor (1995), Manzi et al. (2001), Sanme et al. (2003) and Manzi et al. (2004) worked on the fibre content of different mushrooms.

Composition, nutritive value, shelf-life extension and value addition of mushroom

Table 1: Chemical composition of Fresh Mushroom (Agaricus bisporus) Moisture (%)

Protein

Fat

(%)

(%)

A

B

A

Carbohydrates (%)

B

A

B

Ash

Crude fibre

Ascorbic Acid

(%)

(%)

(%)

A

B

A

B

A

14.5

16.2

5.0

92.8

42.5

89.5

26.3

1.8

59.9

12.0

10.4

89.5

26.3

1.8

59.9

12.0

10.4

88.60-92.50

3.10-3.90

0.88-0.92

3.92-4.07

90.0-92.5

3.5-3.9

0.28-0.36

3.87-4.50

90.7-92.5

3.5-3.9

0.28-0.36

3.78-4.36

91.2

3.85

0.18

3.63

A = Fresh Weight basis

0.80-1.40

0.96-1.9

Reference B Pruthi et al. (1984) 82

Bano and Rajarathana m (1986) Bano et al. (1981)

0.73-1.0

5.69-6.20

0.83-1.12

5.69-8.3

Sethi et al. (1991)

0.34-1.13

5.80-8.69

Choudhary (2000)

1.14

Tomar (1998)

Tyagi

B = Dry Weight basis

Vitamins: Mushrooms are one of the best sources of vitamins especially Vitamin B (Breene, 1990; Mattila et al., 1994; Zrodlowski, 1995; Chang and Buswell, 1996; Mattila et al., 2000). Vitamin content of edible mushrooms has been reported by Esselen and Fellers (1946), Block et al. (1953) and Litchfield (1964). Manning (1985) gave a comprehensive data of vitamin content of mushrooms and some vegetables. According to Mattila et al. (1994), wild mushrooms contain much higher amounts of vitamin D2 than dark cultivated A. bisporus. Mushrooms also contain vitamin C in small amounts (Sapers et al., 1999; Mattila et al., 2001). Mineral constituents: The fruiting bodies of mushrooms are characterized by a high level of well assimilated mineral elements. Major mineral constituents in mushrooms are K, P, Na, Ca, Mg and elements like Cu, Zn, Fe, Mo, Cd form minor constituents (Bano and Rajarathanum, 1982; Bano et al., 1981; Chang, 1982). K, P, Na and Mg constitute about 56 to 70% of the total ash content of the mushrooms (Li and Chang, 1982) while potassium alone forms 45% of the total ash. Abou-Heilah et al. (1987) found that content of potassium and sodium in A. bisporous was 300 and 28.2 ppm, respectively. A. bisporus

ash analysis showed high amount of K, P, Cu and Fe (Anderson and Fellers, 1942). Varo et al. (1980) reported that A. bisporus contains Ca (0.04 g), Mg (0.16 g), P (0.75 g), Fe (7.8 g), Cu (9.4 mg), Mn (0.833 mg) and Zn (8.6 mg) per kilogram fresh weight. Mushrooms have been found to accumulate heavy metals like cadmium, lead, arsenic, copper, nickel, silver, chromium and mercury (Schmitt and Sticher, 1991; Mejstrick and Lepsova, 1993; Wondratschek and Roder, 1993; Kalac and Svoboda, 2000; Svoboda et al., 2001; Issilogglu et al., 2001; Malinowska, 2004). The mineral proportions vary according to the species, age and the diameter of the fruiting body. It also depends upon the type of the substratum (Demirbas, 2001). The mineral content of wild edible mushrooms has been found higher than cultivated ones (Aletor, 1995; Mattilla et al., 2001; Rudawska and Leski, 2005).

Functional Properties Experimental evidence indicates that mushrooms contain many biologically active components that offer health benefits and protection against degenerative diseases (Barros et al. 2008). Mushrooms are rich sources for compounds like lectins,

IJFFT 1(2) 2011 : 187

Sharma and Vaidya

terpenoids, beta-glucans, ascorbic acid, tocopherols, carboxylic acids and various dietary fibers (Parslew et al., 1999; Mau et al., 2001; Wasser and Weis, 1999; Wasser, 2002). Mushrooms have been found effective against cancer, cholesterol reduction, stress, insomnia, asthma, allergies and diabetes (Bahl, 1983). Due to high amount of proteins, they can be used to bridge the protein malnutrition gap. Mushrooms as functional foods are used as nutrient supplements to enhance immunity in the form of tablets. Due to low starch content and low cholesterol, they suit diabetic and heart patients. One third of the iron in the mushrooms is in available form. Their polysaccharide content is used as anticancer drug. Even, they have been used to combat HIV effectively (Nanba, 1993; King, 1993). Biologically active compounds from the mushrooms possess antifungal, antibacterial, antioxidant and antiviral properties, and have been used as insecticides and nematicides as well. DPPH radical scavenging activity: Babu and Rao (2011) studied the methanolic extracts of the three commercially cultivated mushrooms and found increasing scavenging effect with increased concentration. The activity was moderate (43.5–59.0%) even at low concentration of 1.5 mg/ml. However, the scavenging effect of TROLOX at 6 ìg/ml was 65.1 per cent. The methanolic extracts showed excellent scavenging activity at 1.5 mg/ml compared to several commercial mushrooms reported by Yang et al. (2002). Of the three species studied, Hypsizygus ulmarius cap exhibited maximum scavenging activity (59.0%) followed by Agaricus bisporus cap (58.2%). Reducing power: Agaricus bisporus cap (2.18%) exhibited excellent reducing power (Babu and Rao, 2011). The reducing power of Agaricus bisporus is close to the value reported by Barros et al. (2008). The high reducing power exhibited by the extracts might be indicative of the hydrogen donating ability of the active species present in the extracts. H2O2 scavenging activity: Agaricus bisporus cap exhibited excellent scavenging activity (76.1%),(Babu and Rao, 2011). Antioxidant components: Phenols such as tocopherols, BHT and gallate are found in mushrooms and are known to be effective antioxidants (Yang et al. 2002). Babu and Rao, (2011) reported total flavonoids in Agaricus bisporus cap were 2.173±0.007c and 1.533±0.005 which might account for better results found in ferrous ion chelation and superoxide scavenging activities. The total phenols in the extracts ranged from 14.73–

IJFFT 1(2) 2011 : 188

21.17±0.01mg/g . Total flavonoid content of Agaricus bisporus is similar to the value reported by Barros et al. (2008). Many species of mushrooms have been found to be highly potent immune enhancers, potentiating animal and human immunity against cancer (Wasser and Weis,1999; Borchers et al., 1999; Kidd, 2000; Feng et al.,2001). Tyrosinase from A. bisporus is antioxidant (Shi et al., 2002). Antimicrobial activity: Antimicrobial activity of A. bisporus must has been due to the presence of essential bioactive components. Catechin which is one of phenolic components, has been found to exhibit antimicrobial, antioxidant, anticancer and antiallergy properties (Baise et al.,2002; Shimamura et al., 2007). Caffeic acid and rutin has been shown to exhibit antimicrobial activity (Baise et al.,2002). Gallic acid is a bioactive compound which is widely present in plants (Lee et al.,2000). It is a strong natural antioxidant; it has also been shown to have anti-inflamatory, antitumor, antibacterial and antifungal activity (Li et al.,2000; Kroes et al.,1992; Miki et al.,2001; Panizzi et al., 2002). The antioxidant activity of A. bisporus methanolic extract was also due to these bioactive compounds as most of them exhibited both antimicrobial and antioxidant activity. Flavonoid and phenolic compounds are potent water soluble and free radical scavenger which prevent oxidative cell damage (Del-Rio et al., 1997; Okwu, 2004; Harja and Anu, 1999). Presence of ascorbic acid and phenolic compound in A. bisporus comfirms its antioxidant activity (Niki et al., 1994). A number of studies have been focused on biological activities of phenolic compound as a potential antioxidant and free radical scavengers (Harja and Anu, 1999). Presence of phenols in A.bisporus made this macrofungus a good candidate for formulating antioxidant products. The methanolic extracts of A. bisporus scavenge more DPPH radical than hydroxyl radical. A. bisporus acts as a natural source of antimicrobial against the tested organisms and a validation of its antioxidant activity. Some of the bioactive components as revealed in the research, like catechin had been labeled as anticancer, thus the possibility of A. bisporus as anticancer agent.

Shelf-life Extension Mushrooms are generally harvested after 3 weeks of casing. Button mushrooms are to be harvested when the CAP size is 30 – 45 mm in diameter. Shelf-life of mushrooms are mainly dependent upon harvesting time and storage. Pre-cooling: Pre-cooling of mushroom is a major traditional

Composition, nutritive value, shelf-life extension and value addition of mushroom

application of vacuum cooling. The porous structure and higher moisture content of mushroom have made this possible (Frost et al., 1989). Burton et al. (1987) indicated that the advantage of vacuum cooling was equivalent to a prolonged shelf life of 24 h after 102 h storage. The influence of vacuum cooling on mushroom quality was also investigated. For high quality mushroom, no significant difference of product quality was found between vacuum and conventional cooling if the mushrooms were stored at 50C after cooling (Frost et al., 1989). However, if the mushrooms were slightly deteriorated prior to cooling, vacuum cooling appeared to have an adverse effect by accelerating the enzymatic browning of mushroom caps in comparison with conventional cooling methods if the products were stored at 1°C for 8–10 days. Enzymatic browning of mushroom caps caused by the enzyme polyphenol oxidase is a major criteria of mushroom quality, however, pre and post packaging of similar type mushrooms reduces activity of the polyphenol oxidase and lowers the incidence of browning (Gormley and MacCanna, 1967). Vacuum cooling of mushroom was also found to result in around 3.6% of weight loss, which was higher than 2% for air blast chilling (Wang and Sun, 2001). However, during storage, vacuum cooled mushroom experienced less weight loss than air blast cooled ones (Sun, 1999a), which, therefore, helped to compensate cooling loss. Pre-wetting mushroom prior to vacuum cooling was demonstrated as an effective method to increase product yield (Sun, 1999b). Vacuum cooling has been adopted commercially in the United States, the United Kingdom, Ireland and other parts of Europe, and has been found to cool mushrooms uniformly within a stack (McDonald and Sun, 2000). Packing: Mushrooms are sensitive to desiccation and drought, consequently a suitable package is very important during storage. Mushrooms are usually packed in polypropylene bags of 250-500 g capacities. Quantities more than this have a tendency to lose acceptability. For transportation, these small packs are stacked in large containers (Sethi and Anand, 1976; 1984-85). Saxena and Rai (1988) stored button mushrooms in polypropylene bags of less than 100 gauge thickness with perforations having vent area of about 5 per cent and they observed exacerbated veil-opening, browning and reduction in weight during the storage in the perforated bags kept at 15°C; mushrooms were best preserved in non-perforated bags kept at 5°C. They also suggested that button mushroom should be stored in polystyrene or pulp-board punnets for transporting

to the long distances, instead of using polythene bags. Plastic punnets with size of 130 x 130 x 72 mm, cardboard chip of 4 lb capacity with size of 305 x 125 x 118 mm, plastic trays of 5 lb capacity with size of 400 x 300 x 100 mm, expanded polystyrene containers of 5 lb capacity with size of 330 x 280 x 145 mm and expanded polystyrene container of 10 lb capacity with size of 400 x 333 x 167 mm packs are used for bulk packaging in the developed countries. Dhar (1992) also found that fruit bodies of summer white button mushroom (Agaricus bitorquis) could be stored without significant loss of quality for 6 days at 15°C in nonperforated packs without any chemical treatment or washing in water. De la Plaza et al. (1995) found that use of oriented polypropylene (OPP) film can double the storage period compared to PE film, and maintains mushroom quality for at least 2 days at 18°C. Transportation: The positive effects of pre-cooling and packing will be partially neutralized if the product thereafter is stored and transported in a hot environment. Mushrooms, therefore, need complete cool-chain for storage and transport. To keep the precooled mushrooms cool during the transport to the short distances under the ambient conditions, the polypacks of mushrooms are stacked in small wooden cases or boxes with sufficient crushed ice in polypacks (overwrapped in paper) by the small growers. For transport of the large quantities to the long distances, refrigerated trucks, though costlier, are indispensable (Rai & Arumuganathan, 2008) as shown in Fig 1. Modified Atmosphere Packaging: During postharvest storage, the main processes related to mushroom deterioration have been related to the development of sporophore, such as breaking of the veil, elongation of the stipe, opening of the pileus, expansion of gill-tissue and spore formation (LopezBriones, 1992; Braaksma et al., 1994, Eastwood and Burton 2002). These phenomena, together with browning of the cap and gills and a general loss of appearance, have been considered negative quality characteristic and limit the shelf life of this type of mushroom. The MAP technology for extending the shelf life of button mushroom was tried at the Institute of Horticultural Research, Littlehampton (UK). The incorporation of a small area of a microporous film into the overwrapping film created modified atmospheres in mushroom punnets with moderately low but acceptable oxygen levels. The technique delayed mushroom development, reduced browning and incidence of visible symptoms of diseases on mushrooms at 18°C storage (Burton, 1988).

IJFFT 1(2) 2011 : 189

Sharma and Vaidya

phenol oxidase activity was reduced and there was less brown discolouration of the cap. An atmosphere containing 5 per cent CO2 with or without 1 per cent O2 level prevented cap opening of mushroom up to five weeks at 0°C (Murr and Morris, 1974). Nichols and Hammond (1975) reported that 14 pinholes (one mm diameter) in overwrapping PVC film could control the degree of modification of the atmosphere. Bush and Cook (1976) reported that the optimum conditions for retaining the most acceptable colour and appearance of mushrooms were to store them in perforated plastic packs at 4-7°C and 40-50 per cent relative humidity. According to Nichols (1985) the overwrap films should be perforated to keep the oxygen levels above 4 per cent to prevent anaerobiosis. Burton and Twyning (1989) compared the modified atmosphere storage of button mushroom at the ambient (18°C) and lower storage temperature (10°C and 2°C) and reported that the combination of low temperature storage with modified atmosphere delayed the post harvest development of mushroom. Maini et al. (1987) reported that washing of mushrooms prior to packing is very important for enhancing the shelf-life and extending its marketing period. Washing of fresh mushroom in water containing sodium sulfite solutions resulted in lower bacterial counts and improved initial appearance, but more rapid bacterial growth and browning occurred during subsequent storage compared to unwashed controls (Guthrie and Bellman, 1989).

Fig. 1. Post Harvest Practices of Mushroom

Nichols and Hammond (1975) packed the mushrooms in six different types of films, stored them at two different temperatures (2°C and 18°C) and evaluated the effect of modified atmospheres on the quality. They found that at 2°C, equilibrium was established roughly after 24 h at 4 to 10 per cent CO2 and 11 to 17 per cent O2; the mean concentration was dependent on the type of film. At 18°C, equilibrium was established at 8 to 15 per cent CO2 and 1 to 2 per cent O2.They suggested that film should be chosen according to the storage temperature of the product. The poor keeping quality of mushroom was mainly attributed to enzyme and microbial activity. The high levels of polyphenol oxidases present in the mushroom reduced the keeping quality in the presence of oxygen and resulted in discoloration. In absence of oxygen,

IJFFT 1(2) 2011 : 190

Fresh Sliced Mushroom: Production of sliced mushroom is growing because of increased consumer demand for fresh ready to use products and convenience food. Some of the workers have reported the process for sliced mushrooms as whole mushroom soaked for 10 minutes in the solution of citric acid or hydrogen peroxide then sliced, packed and stored at 4 o C for a period of 19 days. Both treatments reduced the number of Pseudomonas bacteria and improved the keeping quality of the sliced mushroom when compared to water soaked slices. Steeping Preservation: Fresh mushrooms were washed and blanched in 0.05 per cent of KMS for 5 min. After draining off, mushrooms were washed with cold water for 4-5 times and then filled in the bottles or cans. Hot brine of 18 to 20% NaCl and 0.1 % citric acid was filled in the bottles. After proper lidding, the bottles were kept for storage at room temperature. This method of steeping preservation is known as unexhausted steeping preservation and was good for storing

Composition, nutritive value, shelf-life extension and value addition of mushroom

mushrooms upto 3 months (NRCM, Annual Report-2000-01). Only water-blanched mushrooms impart yellow colour to lesser degree and whiteness is maintained excellently in the treated mushrooms (Pruthi et al., 1984). Freezing: Individual quick freezing is another popular processing method followed in large industrial units. In this process, raw materials are washed at processing units after receipt from farm, and then the mushrooms are inspected, sliced and graded according to quality. After that, blanched and water cooled mushroom are subjected to tunnel freezing stage. At this stage are cooled in a system having temperature around -40°C and core areas of mushroom pieces acquire a temperature of around -180C. Subsequently packed in multilayer poly-bags and stored in a cold storage having temperature – 20°C to -35°C. Vacuum freeze drying (V.F.D) is a further development in mushroom processing. In this process the original shape, quality, colour size, texture, freshness properties of thermal produce are retained. This process technique involves the cooling of mushroom mush below the freezing point i.e. -40°C where moisture present in mushroom is connected to tiny ice molecules which further directly sublime into vapour when subjected to vacuum with a slight rise in temperature resulting a dried end product.

Canning: Canning is technique by which the mushrooms can be stored for longer periods up to a year and most of the international trade in mushrooms is done in this form. The canning process can be divided into various unit operations namely cleaning, blanching, filling, sterilization, cooling, labeling and packaging (Fig. 2). In order to produce good quality canned mushrooms, these should be processed as soon as possible after the harvest. In case a delay is inevitable , mushrooms should be stored at 4 to 5 °C till processed. The mushrooms with a stem length of one cm are preferred and are canned whole, sliced and stems-and-pieces as per demand (Beelman and Edwards ,1989) . Longitudinal (mushroom shape) slicing is common (Mudhahar and Bains, 1982; Pruthi et al., 1984). Azad et al. (1987) recommended a brine solution with 2 % common salt, 1 % sugar and 0.05 % citric acid for filling the cans for better results. Adsule et al. (1983) suggested a novel double purpose preservation of tomato juice in place of brine solution for canning of mushrooms; unlike brine solutions there is no need to add citric acid to tomato juice for lowering the pH of the filling medium. Further, the nutrients of the mushrooms could be retained in the tomato juice for human consumption. Arumuganathan et al. (2004) obtained improved quality of the canned button mushroom when the mushrooms were pre-treated with EDTA. Agar-agar, methyl cellulose, carboxy methyl cellulose, pectin and pectin-calcium chloride have been used by various workers in increasing the drained weigh (reducing the shrinkage) of canned products (Singh et al.,1982). The shrinkage losses can also be decreased by the vacuum treatment of the fresh mushroom, cutting the blanching time and also prehydration treatments. The level of the vacuum achieved and the quality of the mushroom will affect the shrinkage losses of 5 to 10 per cent (Steinbuch, 1978). Kapoor (1989) documented a standardized canning process and recommended steam blanching for low loss in weight. Konanayakam et al.(1987) developed a method to determine the shrinkage of mushrooms during processing based on liquid displacement method. The method consists of immersing the sample in a glass container with an over flow spout. Water treated with a surfactant was used as the displacement liquid in the glass container.

Fig. 2. Flow chart for canning of mushroom

Pickling: Pickling of mushroom is also a popular method of preservation (Fig 3). It is more economically viable way during the surplus periods. Joshi et al. (1991 and 1996) reported the preparation of sweet chutney and mushroom sauce from edible

IJFFT 1(2) 2011 : 191

Sharma and Vaidya

mushroom having a shelf life of over a year with better sensory qualities. Pickle prepared from paddy straw mushroom was also reported with better quality. The pickle can be stored upto one year in the lug bottles (Saxena and Rai, 1990).

Fig. 3. Flow chart for mushroom pickle (Saxena and Rai, 1990) Drying: Mushrooms contain about 90 per cent moisture at the time of harvesting and are dried to a moisture level down below 10-12 per cent. At a drying temperature of 55-600C, the insects and microbes on the mushrooms will be killed in a few hours, which gives us the dehydrated final product of lower moisture content with longer shelf-life. The temperature, moisture of the mushroom and humidity of the air affect the colour of the dried product (Yapar et al., 1990). Dehydrated mushrooms are used as an important ingredient in several food formulations including instant soups, pasta, snack seasonings, casseroles, meat and rice dishes (Tuley, 1996; Gothandapani et al., 1997). Shreshtha (2005) recommended the water blanching of mushroom (Agaricus bisporus) for 5 minutes along with 0.5 per cent citric acid, 0.1 per cent KMS and 125 ppm Ethylene IJFFT 1(2) 2011 : 192

diamine tetra acetic acid to improve colour and texture of mushroom slices. Pruthi et al. (1984) reported that longitudinally sliced and blanched but ton mushrooms when dried at 60 0C for 5 h, had a drying ratio of 10.8:1 and rehydration ratio of 2.78 as against cross slit mushrooms with drying time of 8 h, drying ratio of 10.9:1 and rehydration ratio of 2.80. According to Arora et al. (2003), blanching of both button and oyster mushroom in boiling water for one min and treating in solution containing 0.1 % citric acid and 0.25% KMS for 15 min at room temperature resulted in lowest browning index and the activation energy values of button and oyster mushroom were determined to be 19.79 and 23.59 kJ/mol in the cabinet drying method. Drying in mechanical dehydrator was reported to be fastest by Katiyar (1985) because of high air temperature and forced air circulation. Mean dehydration time was 8.4 h where as 16.8 sun hours were needed in sundrying. However, Kumar (1992) dehydrated Agaricus bisporus for 9 h at 60±20C to a constant weight. Lidhoo and Agrawal (2006) dried white button mushroom in a hot air oven and observed that minimum browning index was recorded at 650C and rehydration ratio obtained at this temperature was 2.9. Yang and Le Maguer (1992) conducted studies on osmotic dehydration of the button mushroom in a continuously circulated contacting reactor and recommended 1 per cent NaCl as the optimum. Pretreatments of the mushrooms in high concentrations of sucrose, followed by high salt concentration was most effective method to remove water and loading salt to further lower the water activity in the mushroom. Kar and Gupta (2001) reported that osmosis using 15 per cent brine solution could remove about 35 per cent of initial moisture in one hour. Mushrooms are freeze dried at -200C and the moisture is removed by sublimation at a very low vacuum (0.012 mbar) for 12-16 h. The freeze dried mushrooms have superior flavour and appearance but are brittle (Kapoor, 1989). The appearance of freeze dried mushrooms is very similar to fresh mushrooms but as the product is brittle, it is packed in sturdy packings and cushion-packs flushed with nitrogen for better keeping quality (Saxena and Rai, 1990). The product can be stored upto 6 months without any change in its quality and appearance. However, this is a very costly and energy-intensive process and the venture depends upon the demand and price for such products. Freeze-drying has been tried by the following method also. The sliced mushrooms are immersed in a solution of 0.05 per cent KMS and 2 per cent salt for about 30 min. The pretreated mushrooms are then blanched in boiling water for

Composition, nutritive value, shelf-life extension and value addition of mushroom

two min followed by cooling. The product is frozen at -220C for one min. The frozen mushrooms are dried to moisture content of 3 per cent in a freeze drier and packed under vacuum (Kannaiyan and Ramsamy, 1980). Singh et al. (2001) studied the drying characteristics of the fluidized bed drying of the button mushroom and found that quality of the dehydrated mushrooms was significantly influenced by the pretreatments as well as temperature; the samples treated with 1 per cent KMS, 0.2 per cent citric acid and 3 per cent salt solution and dried at 500C gave satisfactory results. Dried mushrooms can

be further utilized for pickling, soup development, curry preparation and further grinding to make flour for the development of other value added products (Fig 4). Irradiation: Radiation preservation offers a method of “cold sterilization” where the mushrooms may be preserved without marked change in their natural characters. Low dosages of γradiation could be used to reduce the microbial contamination and extend the shelf-life of mushrooms. However, irradiation should be given immediately after harvest for optimum benefits. Various types of beneficial effects of radiation have been

Fig. 4. Utilization of mushroom flour for the preparation of value-added products (Shrestha, 2005)

IJFFT 1(2) 2011 : 193

Sharma and Vaidya

observed in preserving the button mushroom (Staden, 1967; Campbell et al., 1968; Wahid and Kovacs, 1980; Roy and Bahl, 1984 a; Lescane, 1994). Irradiation has been found to delay the maturation i.e. development of cap, stalk, gill and spore and also reduce the loss of water, colour, flavour, texture and finally the quality losses. Cobalt 60 (Co60) has been used as a common source of γ-rays. A dose of 400 krad gave whiter buttons than the controls when the atmospheric temperature during growth and subsequent handling was slightly lower than 200C (Roy and Bahl, 1984 b). A dose of 10 kGy (Kilo Gray) is reported to completely destroy microorganisms. Enhancement in shelf-life of Agaricus bisporus upto a period of 10 days was achieved by application of gamma rays close to 2 kGy and storage at 100C (Lescane, 1984). Irradiation reduces the incidence of fungal and bacterial infection and also retards the breakdown of mannitol and trehalose. However, the loss of flavor components has been noticed in irradiated mushrooms. But amino acids in fresh mushrooms were better preserved by γ irradiation and this showed that irradiation at low levels proved better than irradiation levels of 1 & 2 kGy (Roy and Bahl, 1984 a). Koorapati et al. (2004) evaluated the effect of electron-beam irradiation on quality of white button mushroom and observed that irradiation levels above 0.5 kGy prevented microbes induced browning. A study was conducted by Escriche et al. (2001) to determine the effect of ozone on postharvest quality of mushroom. Ozone treatment (100 mg / h) of mushrooms prior to packaging increased the external browning and reduced the internal browning rates. The ozone treatment exhibited no significant differences in terms of texture, maturity index and weight loss of mushrooms.

Research Gaps As aforementioned, mushrooms with its huge health benefits can solve many problems of undernutrition and malnutrition. Despite this fact mushroom cultivation and its utilization is not catching up fast because of one or more of the following reasons: 



Mushrooms are not popular in India. Both produces and consumers are not aware of its intrinsic worth. This can be overcome by organizing campaigns, training programmes, workshops, propagating its virtues through media etc. Being highly perishable, lack of immediate access to markets is a major bottleneck in mushroom farming. The seasonality and wide fluctuation in collection results

IJFFT 1(2) 2011 : 194

into erratic procurement and supply. The collection therefore, may be organized by farming co-operatives or by NGOs/traders. 

As mushrooms have to be immediately processed to increase its shelf life period, lack of storage facilities like processing units, cold storage, refrigerated transport etc are also some of the deterrents.

Conclusion It can be inferred that production of highly perishable commodities such as mushroom need a lot more than interim infrastructural facilities. It needs a synergy between various segments of cultivators, productions such as production centres, pre-cooling units, cold storages and export processing units or export processing zones and more so the ultimate consumers. Further, value added products from mushroom are a promising enterprise. Mushroom being highly perishable forces the producer to preserve and process it. Preservation is essential to make it available throughout the year to retain maximum nutrients, texture and flavour and to increase its per capita consumption in developing countries. The value added products will not only cater to the protein and micronutrient requirement but at the same time will enable the population to live a healthy life. But food processing in India is not only far behind the developed countries of the world but is much less than developing countries like Philippines and China where value addition is 45 and 23 per cent, respectively, as compared with 7 per cent in India. Presently the mushroom products available are bakery products (biscuits, bread, cakes), pickles, chutneys, nuggets, papads & fast food items like burgers, cutlets and pizza etc.

References Abou-Heilah A N, Kasionalsim M Y and Khaliel A S. 1987. Chemical composition of the fruiting bodies of Agaricus bisporus. Int J Expt Bot, 47: 64-68. Adsule PG, Onkaraya H, Tewari RP and Girija V.1983. Tomato juice as a new canning medium for button mushroom (Agaricus bisporus (Lange) Sing.). Mushroom J, 124: 143-145. Aletor VA.1995. Compositional studies on edible tropical species of mushrooms. Food Chem, 54: 265-268. Alofe FV, Odeyemi O and Oke OL.1995. Three edible mushrooms from Nigeria: their proximate and mineral composition. Plant Foods for Hum Nutr, 49: 63-73. Anderson EE and Fellers CR. 1942. The food value of mushrooms (A.Campestris). Proc Am Soc Hort Sci, 41: 301.

Composition, nutritive value, shelf-life extension and value addition of mushroom

Arora S, Shivhare US, Ahmed J and Raghavan GSV. 2003. Drying kinetics of Agaricus bisporus and Pleurotus florida mushrooms. Transactions of ASAE, 46 : 721-724.

Braaksma A, Schaap DJ, de Vrije T, Jongen WMF and Woltering EJ.1994. Ageing of mushroom (Agaricus bisporus) under postharvest conditions. Postharvest Biol and Techn, 4: 99-110

Arumuganathan T, Rai RD, Chandrasekar V and Hemakar Anil Kumar. 2004. Studies on canning of button mushroom, Agaricus bisporus for improved quality. Mushroom Res., 12(2): 117-120.

Breene WM. 1990. Nutritional and medicinal value of speciality mushrooms. J Food Protect, 53: 883-894.

Azad KC, Srivastava MP, Singh RC and Sharma PC.1987. Commercial preservation of mushrooms - 1. A technical profile of canning and its economics. Indian J of Mushrooms, XII-XIII: 21-29. Babu DR and Rao GN. 2011. Antioxidant properties and electrochemical behavior of cultivated commercial Indian edible mushrooms. J Food Sci Techn. Bahl N.1983. Medicinal value of edible fungi. In: Proceeding of the International Conference on Science and Cultivation Technology of edible Fungi. Indian Mushroom Science II, pp. 203-209. Baise HP, Walker TS, Stermitz FR, Hufbauer RS and Vivanco JM. 2002. Enantiometric dependent phytotoxic and antimicrobial activity of catechin; a rhizosecreted racemic mixture from Centaurea maculosa (spotted knapweed). Plant Physiol,128: 1127-1135.

Burton KS, Frost CE and Atkey PT.1987. Effect of vacuum cooling on mushroom browning. International Journal of Food Science and Technology, 22: 599–606. Burton KS. 1988. Effect of pre- and post- harvest development on mushroom tyrosinase. J of Horti. Sci, 63(2):255-260. Burton KS and Twyning RV. 1989. Extending mushroom storage life by combined modified atmosphere packaging and cooling. Acta Hortic, 258: 565-569. Bush P and Cook DJ. 1976. The purchase and domestic storage of pre-packed mushrooms. Mushroom J, 39: 76-80. Campbell JD, Stothers S, Vaisey M and Berck B.1968. Gamma radiation influence on the storage and nutritional quality of mushroom. J of Food Sci, 3: 540-542. Chang ST and Buswell JA. 1996. Mushroom Nutriceuticals. World J Microbiol Biotechnol, 12: 473-476.

Bano Z.1976.. Nutritive value of Indian mushrooms and medicinal practices. Eco Bot, 31: 367-371.

Chang ST. 1980. Mushroom as human food. Bio Science, 30: 339401.

Bano Z, Bhagya S and Srinivasan KS. 1981. Essential amino acid composition and proximate analysis of the mushroom Pleurotus edodus and Pleurotus florida. Mushroom Newsl Trop, 1(3):6-9.

Chang ST. 2007. Development of the World Mushroom Industry and its roles in human health. In: Rai RD, Singh SK, Yadav MC, Tewari RP (eds) Mushroom biology and biotechnology. Mushroom Society of India, National Research Centre for Mushroom, Solan

Bano Z and Rajarathnam S. 1986. Vitamin values of Pleurotus mushrooms. Qual Pl Fd Hum Nutri, 36:11. Bano Z and Rajarathanam S.1982. Pleurotus mushrooms as a nutritious food. In: Tropical mushrooms –Biological Nature and cultivation methods, (Chang ST, Quimio, TH, eds.) The Chinese University Press, Hongkong. Bano Z, Bhagya S and Srinivasan KS. 1981. Essential amino acid composition and proximate analysis of Mushroom, Pleurotus florida.Mushrooms News Lett Trop,1: 6-10. Barros L, Falcao S, Baptista P, Freire C, Vilas-Boas M and Ferreira ICFR.2008.Antioxidant activity of Agaricus sp. mushrooms by chemical, biochemical and electrochemical assays. Food Chem, 111:61–66 Beelman RB and Edwards CE. 1989. Variability in the protein content and canned product yield of four important processing strains of the cultivated mushrooms (Agaricus bisporus). Mushroom News, 37(7): 17-26. Block SS, Stearns TW, Stephens RH and McCandless RFJ. 1953.Mushroom mycelium experiments with submerged culture. J Agr Food Chem,1: 890-893. Borchers AT, Stern JS, Hackman RM.1999. Mushrooms, tumors, and immunity. Proc Soc Exp Biol Med, 221: 281-293.

Chang ST.1982. Prospects for mushroom protein in developing countries. In: Tropical Mushroom – Biological Nature and Cultivation Methods (Chang ST, Quimio TH. eds.), Chinese University Press, Hong Kong. Choudhary P. 2000. Development of process for manufacture of mushroom powder and its utilization in biscuits and cakes. M.Sc. (Food technology). Thesis. G. B. Pant Univ. Ag. Tech., Pantnagar. Crisan EW and Sands. 1978. A Nutritional value. In: Chang ST and Hayes WA (eds.). The biology and cultivation of edible mushrooms.Academic Press, New York. De la Plaza JL, Alique R, Zamorano JP, Calvo ML and Navarro MJ.1995. Effect of the high permeability to O2 on the quality changes and shelf-life of fresh mushrooms stored under modified atmosphere packaging. Mushroom Science, 14(2): 709716. Del-Rio ABG Obdululio, Castillo J, Marin FG and Ortuno A. 1997.Uses and properties of citrus flavonoids. J Agri Food Chem, 45: 4505-4515 Demirbas A. 2001. Concentrations of 21 metals in 18 species of mushrooms growing in the east Black Sea region. Food Chem, 75:453-457.

IJFFT 1(2) 2011 : 195

Sharma and Vaidya

Dhar BL.1992. Postharvest storage of white button mushroom Agaricus bitorquis. Mushroom Res, 1: 127-130. Eastwood D and Burton K. 2002. Mushrooms- a matter of choice and spoiling onself. Microbiology International, 2: 7-14 Escriche I, Serra JA, Gomez M and Galotto MJ. 2001. Effect of ozone treatment and storage temperature on physicochemical properties of mushrooms (Agaricus bisporus). Food Sci and Tech International, 7(3): 251-258. Esselen WB and Fellers CR. 1946. Mushrooms for food and flavor. Bull.Mass. Agric. Exp. Sta., p. 434. Feng W, Nagai J and Ikekawa T. 2001. A clinical pilot study of EEM for advanced cancer treatment with EEM for improvement of cachexia and immune function compared with MPA . Biotherapy, 15: 691-696. Florezak J and Lasota W. 1995. Cadmium uptake and binding by artificially cultivated cultivated (Pleurotus ostreatus). Bromatol Chem Toksykol, 28: 17-23. Florezak J, Karmnska A and Wedzisz A. 2004. Comparision of the chemical contents of the selected wild growing mushrooms. Bromatol Chem Toksykol, 37: 365-371.

Joshi VK, Seth PK, Sharma RC and Sharma R.1991. Standardization of a method for the preparation of sweet chutney from edible mushrooms Agaricus bisporus. Indian Food Packer, 45(2): 3943. Joshi VK, Kaur M. and Thakur NS. 1996. Lactic acid fermentation of mushroom (Agaricus bisporus) for preservation and preparation of sauce. Acta Alimentaria, 25(1): 1-13 Kalac P and Svoboda L. 2000. A review of trace element concentrations in edible mushrooms. Food Chem, 69: 273-281. Kannaiyan S and Ramaswamy K.1980. A handbook of edible mushrooms. Todays & tomorrow’s printers and publishers, pp. 44-50. Kar Abhijit and Gupta D K. 2001. Osmotic dehydration characteristics of button mushrooms. J. Food Sci and Techn, 38(4): 357-257. Katiyar RC.1985. Evaluation of drying characteristics and storage behaviour of cultivated mushroom (Agaricus bisporus (Lange) Sing.). M.Sc. thesis. Dept. of Pomology and Fruit Technology. HPKV College of Agriculture, Solan, India. Kidd PM. 2000. The use of mushroom glucans and proteoglycans in cancer therapy. Alternative Med Rev, 5: 4-27.

Friedman M.1996. Nutritional value of proteins from different food sources: review. J Agric Food Chem, 44 : 6-29.

King TA.1993. Mushrooms, the ultimate health food but little research in U. S to prove it. Mushroom News, 41: 29-46.

Frost CE, Burton KS and Atkey PT.1989. A fresh look at cooling mushroom. Mushroom Journal, 193 : 23–29.

Konanayakam M, Sastry S K and Anantheswaran RC.1987. A method to determine shrinkage of mushrooms during processing. J. Food Sci. and Techn, 24(5): 257-258.

Ghosh S and Singh S. 1995. Utilization of whey for the manufacture of ready-to-serve mushroom soup. Mushroom Res, 4:23-26. Gormley T R and MacCanna C.1967. Pre-packaging and shelf life of mushroom. Irish J. Agri Res, 6: 255–265.

Koorapati A, D Foley R Pilling and Prakash A.2004. Electron-beam irradiation preserves the quality of white button mushroom (Agaricus bisporus) slices. J. Food Science, 69(1): 25-29.

Gothandapani L, Parvathi K and Kennady Z J.1997. Evaluation of different methods of drying on the quality of oyster mushroom (Pleurotus sp.). Drying Technology, 15(6-8): 1995-2004.

Kroes BH, Van den Berg AJJ, Quarles van HC, Ufford HD Van and Labadie RP.1992. Anti-inflamatory of gallic acid. Planta Medica, 58:499-504

Gruen VEC and Wong HX.1982. Immunodulatory and Antitumour activities of a polysaccharide- peptide complex from a mycelial culture of Trichoderma sp. Sciences, 57: 269-281

Kumar Ashwani. 1992. Studies on storage and dehydration of white button mushroom, Agaricus bisporus. M.Sc. thesis, Dept. of Postharvest Technology, UHF, Solan, India.

Guthrie BD and Bellman RB. 1989. Control of bacterial deterioration in fresh washed mushrooms. Mushroom Sci, 12(2): 689-699.

Lee MW, Lee YA, Park HM, Toh SH, Lee E J, Jang H D and Bor YH.2000. Archives of Parmacal Res., 23: 455-458.

Hadded NA and Hayes WA. 1978. Nutritional factors and the composition of the Agaricus bisporus mycelium. Mushroom Sci, 10: 715-722.

Lescane C.1984. Extension of mushroom (Agaricus bisporus) shelf life by gamma radiation. Post Harvest Biology and Technology, 4: 255-260.

Hugaes DH. 1962. Preliminary Characterization of the lipid constituents of the cultivated mushroom Agaricus campestris. Mush Sci, 5: 540-546.

Li AS, Bandy B, Tsang SS and Davison AJ. 2000. DNA-breaking versus DNA-protecting activity of four phenolic compounds in vitro. Free Radical Res., 33: 551-566.

Issilogglu M, Yilmaz F and Merdivan M. 2001. Concentrations of trace elements in wild edible mushrooms. Food Chem, 73: 163175.

Li GSF and Chang ST. 1982. Nutritive value of Volvariella volvacea, In:Tropical mushrooms –Biological nature and cultivation methods (Chang ST, Quimio TH (eds)) Chinese university press Hong Kong, pp. 199-219.

Jiskani MM. 2001. Energy potential of mushrooms. Dawn Econ. Bus.Rev., p. 4.

IJFFT 1(2) 2011 : 196

Lidhoo CK and Agrawal YC. 2006. Hot-air over drying characteristics

Composition, nutritive value, shelf-life extension and value addition of mushroom

of button mushroom-safe drying temperature. Mushroom Res, 15(1): 59-62.

Mc Connel JE and Esselen WB. 1947. Carbohydrate in cultivated mushrooms. Food Res,12: 118-121.

Lietaniemi V, Kumpulainen J, Valtonen M and Piironen V.2001. Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. J Agric Food Chem, 49: 2343-2348.

McDonald K and Sun DW. 2000. Vacuum cooling technology for the food processing industry: A review. J Food Engineering, 45: 55–65.

Lintzel W. 1941. The nutritional value of edible mushroom proteins. Biochem. Acta, 308: 413-419.

Mejstric V and Lepsova A. 1993. Applicability of fungi to the monitoring of environmental pollution by heavy metals. In: B. Markert (ed) Plants as biomonitors. Germany: VCH weinheim.

Liu GQ and Wang XL.2009.Selection of a culture medium for reducing costs and intracellular polysaccharide production by Agaricus blazei AB2003. Food Technol Biotechnol, 47:210–214

Miki KR, Yasushi R, Yunmo L, Testuro Y, Hideyuki A, Toshihiro W, Motohiro Y, Norio A, Seigou T, Genzou M, Shinya G, Kohshi F Hisayoshi and Kazunori F. 2001. Antitumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice. Anti-Cancer Drugs, 12: 847-852

Lopez-Briones G, Varoquaux P, Yves C, Bouquant J, Bureau G and Pascat B. 1992. Storage of common mushroom under controlled atmospheres. Inter. J Food Science and Technology, 28: 57-68

Mudahar GS and Bains GS. 1982. Pre-treatment effect on quality of dehydrated Agaricus bisporus mushrooms. Indian Food Packer, 28(5): 19-27.

Maggioni A, Passera C, Renosto F and Benetti E. 1968. Composition of cultivated mushrooms (Agaricus bisporous) during the growing cycle as affected by the nitrogen source in compositing. J Agr Chem, 16: 517-519.

Murr DP and Morris LL. 1974. Influence of O2 and CO2 on O-diphenol oxidase activity in mushrooms. J Amer. Soc. Hortic Sci, 99: 272277

Litchfield JH.1964. Nutrient content of morel mushroom mycelium: B vitamin composition. J Food Sci, 29: 690-691.

Maini SB, Sethi V, Diwan B and Munjal RL. 1987. Pre-treating mushrooms to enhance their shelf life and marketability. Ind Mushroom Sci, 2:215 Malinowska E, Szefer P and Faradays J.2004. Metals bioaccumulation by bay Bolete, Xerocomos badius from selected sites. Poland Food Chem,84: 405-416. Manning K. 1985. Food value and chemical composition. Flegg PB Spencer DM, Wood DA (eds). The biology and technology of the cultivated Mushroom. John Willey and sons, New York, pp. 221-230. Manzi P A, Agguzzi A and Pizzoferrato L. 2001. Nutritional mushrooms widely consumed in Italy. Food Chem, 73: 321-325. Manzi PS, Marconi Aguzzi A and Pizzoferrato L. 2004. Commercial mushroom nutritional quality and effect of cooking. Food Chem, 84: 201-2006. Marja PK and Anu IH. 1999. Activity of plant extracts containing phenolic compounds. J Agric Food Chem, 47: 3954-3962 Mattila PH, Piironen VI, Uusi- R and Koivistoinen PE. 1994. Vit. D contents in edible mushrooms. J Agr Food Chem, 42: 24492453. Mattila PK, Konko M, Eurola J, Pihlava J, Astola L, Vahteristo V, Hietaniemi J, Kumpulainen N, Valtonen V and Piironen V. 2001. Contents of vitamins, mineral elements and some phenolic compounds in the cultivated mushrooms. J Agric Food Chem, 49:2343-2348. Mau JL, Chao GR and Wu KT. 2001. Antioxidant properties of methanolic extracts from several ear mushrooms. J Agric Food Chem, 49:5461–5467

Nanba H. 1993. Maitake mushroom the king mushroom. Mushroom News, 41: 22-25. Nichols R. 1985. Post harvest physiology and storage. In: the biology and cultivation of cultivated mushroom (Flegg, P.B.; Spencer, D.M. and Wood, D.A., eds.). John Wiley and Sons Ltd., New York. Nichols R and Hammond JBW. 1975. Storage of mushroom in prepacks. The effect of changes of carbon dioxide and oxygen on quality. J Food Sci Agric, 24:1371-1381. Nochols R and Hammond JBW.1973. Storage of mushrooms in prepacks: the effect of changes in carbon dioxide and oxygen on quality. J Food Sci and Agric, 24: 1371-1381 NRCM- Annual Report – 2000-01. Published by National Research Centre for Mushroom, Solan, H.P. Okwu DE.2004. Phytochemical and vitamin content of indigenous species of southern Nigeria. J Sustain Agric Environ, 6: 30-37 Orgundana SK and Fagade O. 1981. The nutritive value of some Nigerian edible mushrooms. In: Mushroom Science XI, Proceedings of the Eleventh International Scientific Congress on the Cultivation of Edible Fungi, Australia. Panizzi LC, Caponi S, Catalona PL, Cioni and Morelli I. 2002. In vitro antimicrobial activity of extracts and isolated constituents of Rubus ulmifolius. J Ethanopharmacol, 79: 165-168 Parslew R, Jones KT, Rhodes JM and Sharpe GR.1999. The antiproliferative effect of lectin from the edible mushroom (Agaricus bisporus) on human keratinocytes: preliminary studies on its use in psoriasis. Br J Dermatol, 140:56–60 Pedneault KP, Gosselia A and Tweddell RJ. 2006. Fatty acid composition of lipids from mushrooms belonging to the family

IJFFT 1(2) 2011 : 197

Sharma and Vaidya

Boletaceae. Mycolog Res, 110: 1179-1183. Prakash JD and Tejaswin MS. 1991. Mushroom for many uses. Food Digest, 22(1): 84-91. Pruthi J S, Manan JK, Raina BL and Teotia MS.1984. Improvement in whiteness and extension of shelf-life of fresh and processed mushrooms (Agaricus bisporus and Volvariella volvacea). Indian Food Packer, 38(2): 55-63. Purkayastha RP and Chandra A. 1976. Amino acid composition of protein of some edible mushroom growth in synthetic medium. J Food Sci Technol, 3: 13-17. Rai RD and Arumuganathan T. 2005. Nutritive value of mushrooms. In: Frontiers in mushroom biotechnology. National Research Centre for Mushroom, Solan, pp 27–36 Rai RD and Saxena S. 1989. Biochemical changes during the post harvest storage of button mushroom (Agaricus bisporus). Curr Sci, 58: 508-10. Rai RD and Arumuganathan T. 2008. Post Harvest Management of mushrooms. Technical Bulletin. National Research Centre for Mushroom.pp 1-92. Roy MN and Bahl Nita. 1984. Gamma radiation for preservation of Agaricus bisporus. Mushroom Journal, 136: 124-125. Roy MN and Bahl Nita. 1984. Studies on gamma radiation for preservation of Agaricus bisporus. Mushroom Journal, 144: 411-414. Rudawska M and Leski T. 2005. Macro and micro elemental contents in fruiting bodies of wild mushrooms from the Netecka forest in west –central Poland. Food Chem, 92: 499-502 Sanme RB, Dell Lumyoung P, Izumori K and Lumyoung S. 2003. Nutritive value of popular wild edible mushrooms from Northern Thailand. Food Chem, 82: 527-532. Sapers GM, Miller RL, Choi SW and Cooke PH. 1999. Structure and composition of mushrooms as affected by hydrogen peroxide wash. J Food Sci, 64: 889-892. Saxena S and Rai RD. 1988. Storage of button mushrooms (Agaricus bisporus). The effect of temperature, perforation of packs and pre-treatment with potassium metabisulphite. Mushroom J. Tropics, 8: 15-22.

Sethi Vijay and Anand JC.1984-85. Post harvest care of mushrooms. Indian J. Mushrooms, 10-11: 1-6. Shah H, Iqtidar A, Khalil and Jabeen S. 1997. Nutritonal composition and protein quality of Pleurotus mushroom. Sarhad J Agric, 13: 621-626. Shi YL, James AE, Benzie IFF, Buswell JA. 2002. Mushroom derivedpreparation in the prevention of H 2 O 2 –induced oxidative damage to cellular DNA. Teratoegensis Carcinogenesis Mutagenesis, 22: 103-111. Shimamura T, Zhao WH and Hu ZQ. 2007. Mechanism of action and potential for use of tea catechin in an antiinfective agent. Antiinfective Agents in Mechanical Chemistry, 6: 57-62 Singh NB and Singh P. 2002. Biochemical Composition of Agaricus bisporus. J Indian Bot Soc, 81: 235-237. Singh RP, Dang RL, Bhatia AK and Gupta AK.1982. Water binding additives and canned mushroom yield. Indian Food Packer, 36(1): 32-36. Singh Sudhir Kumar, Maharaj Narain and Kumbhar BK. 2001. Effect of drying air temperatures and standard pretreatments on the quality of fluidized bed dried button mushroom (Agaricus bisporus). Indian Food Packer, 55(5): 82-86. Staden OL.1967. Radiation preservation of fresh mushroom. Mushroom Science, 6: 457-461. Steinbuch E.1978. Factors affecting quality and shrinkage losses of processed mushrooms. Mushroom Sci., 10(2): 759-766. Sun DW.1999a. Comparison of rapid vacuum cooling of leafy and non-leafy vegetables. ASAE Paper. No. 996117, ASAE, 2950 Niles Road, St. Joseph, MI 49085 9659, USA. Sun DW.1999b. Effect of pre-wetting on weight loss and cooling times of vegetables during vacuum cooling. ASAE Paper. No.996119, ASAE, 2950 Niles Road, St. Joseph, MI 49085 9659, USA. Svoboda L, Zimmermannova K and Kallac P. 2001. Concentrations of Mercury, Cadmium, Lead, and Copper in the fruiting bodies of the edible mushrooms in an emission area of a copper smelter and a mercury smelter. Sci Total Environ, 246: 61-67.

Saxena S and Rai RD. 1990. Postharvest technology of mushrooms. Technical Bulletin No.2: NRCM, Solan.

Tomar P. 1998. optimization of process parameters for preparation of mushroom powder. M.Sc. (Food technology). Thesis. G. B. Pant Univ. Ag. Tech., Pantnagar.

Schmitt HW and Sticher H. 1991. Heavy metal compounds in soil .In Merian,E (ed.). Metals and their compounds in the environment.Weinheim: VCH Verlagsgessellschaft, pp. 311-326.

Tseng YH and Mau JL. 1999. Contents of sugars free amino acids and free 5- nucleotides in mushroom, Agaricus bisporus, during the post harvest storage. J Sci Food Agric, 79: 1519-1523.

Sethi Vijay and Anand JC. 1976. Processing of mushrooms. Indian Mushroom Sci., 1: 233-238.

Tuley L. 1996. Swell time for dehydrated vegetables. Intern Food Ingredients, 4(1): 23-27.

Sethi V, Bhagwan J, Dehal N and Lal S. 1991. Low cost technology for preserving mushroom (Agaricus bisporus). Indian Food Packer. 45(6): 22-26

Tyagi RK. 2004. Development of mushroom based ‘papad’ using response surface methodology, PhD (Food technology). Thesis. G. B. Pant Univ. Ag. Tech., Pantnagar.

IJFFT 1(2) 2011 : 198

Composition, nutritive value, shelf-life extension and value addition of mushroom

Varo P, Lahelman O, Nuurtamo M, Saari E, Koivistoinen P. 1980.Mineral element composition of Finish Food. VII Postal, Vegetables, fruits, berries, nuts and mushrooms. Acta Agric Scandinavica Supplement, 22: 107-113. Verma RN, Singh GB and Bilgrami KS. 1987. Fleshy fungal flora of N. E. H. India- I. Manipur and Meghalaya. Indian Mush Sci, 2: 414- 421. Wahid M and Kovacs E.1980. Shelf life extension of mushrooms (Agaricus bisporus) by gamma radiation. Acta Alimentaria, 9: 357-359. Wang LJ and Sun DW. 2001. Rapid cooling of porous and moisture foods by using vacuum cooling technology. Trends in Food Sci. and Techn., 12(5-6): 174-184. Wannet WJB, Hermans JHM, Vander Drift C and Op den Camp HJM.2000. HPCL detection of soluble carbohydrates involved in mannitol and trehalose metabolism in the edible mushroom, Agaricus bisporus. J Agric Food Chem, 48: 287-291. Wasser SP and Weis AL. 1999. Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol, 19:65-96. Wasser SP. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol, 60:258–274. Weaver KC, Kroger M and Kneebone LR.1977. Comparative protein studies on nine strains of Agaricus bisporus (Lange). J Food Sci, 42: 364-366.

Wondratschek I and Roder U.1993. Monitoring of heavy metals in soils by higher fungi.In B. Market (ed). Plants as biomonitors, pp. 365-378. Yang DC and Maguer M Le.1992. Mass transfer kinetics of osmotic dehydration of mushrooms. J Food Process Preservation, 16(3): 215-231. Yang QY, Hu YJ, Li XY, Yang SX, Liu JX, Liu TF, Xu GM and Liao LM. 1993. A New Biological Response Modifier - PSP. (In: Chang ST, Buswell JA , Chiu SW (eds.) Mushroom Biology and Mushroom Products. The Chinese University Press, Hong Kong, pp. 247259. Yapar S, Helvaci S S and Peker S. 1990. Drying behaviour of mushroom slices. Drying Technology, 8(1): 77-99. Yilmaz NM, Solamaz I and El Mastas M.2006. Fatty acid composition in some wild edible mushrooms growing in the Middle Black region of Turkey. Food Chem, 99: 168-174. Yoshioka Y, Ikekawa T, Nida M and Fukuoka F. 1975. Studies on antitumor activity of some fractions from basidiomycetes I. An antitumor acidic polysaccharide fraction of Pleurotus ostreatus (Fr.) Quel. Isolation and structure of a B- glu can,Carbohydrate Res., 140: 923-100. Zrodlowski Z. 1995. The influence of washing and peeling of mushrooms Agaricus bisporus on the level of heavy metal contaminations. Pol J Food Nutr Sci, 4: 23-33.

IJFFT 1(2) 2011 : 199

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 201-209

Research Paper

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables V.K. Joshi*, Somesh Sharma, Arjun Chauhan and N.S. Thakur Department of Food Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, India.

Abstract : An attempt to develop and evaluate the instant chutney mix from fermented vegetables is reported. The vegetables were fermented using sequential culture of lactic acid bacteria viz., Lactobacillus plantarum (NCDC 020), Pediococcus cerevisiae (NCDC 038) and Streptococcus lactis Var. diacetylactis (NCDC 061) as per the conditions optimized earlier. Different combinations of fermented vegetables viz., carrot, radish and cucumber with anardana and amchoor powders were made separately and processed as per the routine practice. Different combinations under investigation were fermented vegetable powders + anardana or amchoor powders (1:1) alongwith only amchoor and anardana powder based chutneys. All the instant chutney powders were prepared using common recipe. The titrable acidity of carrot, radish and cucumber based instant chutneys ranged from 0.72 to 1.24%. The ratio of fermented vegetable powders to the fruit powders influenced the titrable acidity, brix-acid ratio, pH and colour of the products. The physico-chemical and sensory characteristics of all the products prepared met the specifications of Fruit Products Order (FPO), Govt. of India. Instant chutneys prepared by using fermented vegetables powders + anardana (1:1) were adjudged to be the most acceptable. However, among all the combinations used in the study, fermented carrot powder + anardana powder (1:1) based instant chutney mix had the highest acceptability and was rated the best. Production cost of fermented vegetable based instant chutney powders ranged between ` 16.95 – `. 27.09 per 200 g pouch. The lowest (` 16.95 per 200 g pouch) cost was recorded for carrot based Instant chutney mix containing fermented carrot + anardana powder (1:1), having the highest overall acceptibility followed by radish based Instant chutney powder. Keywords: Lactic acid, Fermentation, Lactic acid bacteria, Lactobacillus plantarum, Pediococcus cerevisiae, Strepto coccus lactis, Chutney, Anardana.

India is the second largest producer (129077 MT) of fruits and vegetables (NHB, 2009) in the world but unfortunately, due to the lack of postharvest infrastructure and processing capabilities, a lot of produce goes waste, resulting in a huge loss of these natural resources. Among different vegetables

*E-mail of corresponding author : [email protected] MS Received on : 4th July, 2011 Accepted on : 17th Oct., 2011

produced and consumed in India, carrot (Daucus carota), radish (Raphnus sativus) and cucumber (Cucumis sativus), occupy a significant place in Indian diet as salad and cooked food. These vegetables in their natural state can be preserved for a very short period only and thus, their availability to the consumers remains seasonal. Among different methods,

Joshi et al.

fermentation is one of the oldest methods of food preservation in the world. Fermentation using natural or starter culture has emerged to be a cheap method of preservation to increase the shelf-life of the products, besides preparation of new products with diversified taste and flavour. The tremendous increase in consumer’s demand for fresh-like products, containing natural ingredients, changing food patterns and convenience led to the development of minimally processed products using lactic acid bacteria (LAB) cultures. Recently, the presence of bacteriocin, an antimicrobial substance in these products has also attracted the attention of scientists (Joshi et al., 2006). Lactic acid fermentation using LAB cultures is normally employed to prepare fermented grape juice, fermented peanut milk, yoghurt, fermented corn meal and sweet potato lacto-pickle besides fermented beverages from wheat and maize (Bucker et al., 1979; Takagi et al., 1990; Cheng et al., 1990; Frazier and Westhoff, 1998; Sahlin, 1999; Joshi and Thakur, 2000; Panda et al., 2007). Fermented foods prepared by using lactic acid bacteria (LAB) have better acceptability (Hang and Jackson, 1967). Kimchi and sauerkraut are the well known lactic acid fermented products. However, there is scanty information on the preparation of products from lactic acid fermented vegetables though a fermented beverage traditionally known as kanji and ready-to-serve drink has been prepared from crimson coloured carrots (Sethi 1990; Sharma et al., 2008) while sauce was also developed from lactic acid fermented mushrooms (Joshi et al., 1996). In our earlier

attempt, a process of sequential LAB culture of vegetables was standardized (Sharma and Joshi, 2007; Joshi et al., 2008). To develop diversified products especially having healthful properties available to the consumers was considered desirable from fermented vegetable consumption point of view. Therefore, instant chutney mix was prepared from fermented vegetables and evaluated for nutritional quality, safety aspects and economic viability.

Materials and Methods Fermented vegetables Carrot, radish and cucumber were fermented with three different cultures viz. Lactobacillus plantarum (NCDC 020), Pediococcus cerevisiae (NCDC 038) and Streptococcus lactis var diacetylactis (NCDC 061) at the rate of 2 % sequentially at 26 oC containing 2.5 % salt (w/w), whereas cucumber was fermented at 32 oC in brine containing 3% salt (Joshi et al., 2008). Fermented vegetables after completion of fermentation were dried by spreading uniformly on aluminium trays @1 kg/tray of size 65 × 45 cm in a dehydrator at a temperature of 60 ± 20C. These were then grounded to powder in a grinder and packed in polyethylene pouches for storage till required. The combinations of vegetable powders with fruit powders used in different ratios for the preparation of instant chutney mix are described in Table 1.

Table 1. Details of ratios of fermented vegetables (carrot, radish and cucumber), anardana and amchoor used in the preparation of different instant chutney mix Treatments

Fermented vegetable powders (g)

Fruit powders (g) Anardana

Amchoor

T1C (Carrot control)

250

-

-

T2CA (Carrot + Anardana)

125

125

-

T3CAm (Carrot + Amchoor)

125

-

125

T1R (Radish control)

250

-

-

T2RA (Radish + Anardana)

125

125

-

T3Ram (Radish + Amchoor)

125

-

125

T1Cu (Cucumber control)

250

-

-

T2CuA (Cucumber + Anardana)

125

125

-

T3CuAm (Cucumber + Amchoor)

125

-

125

T3Am (Amchoor powder)

-

-

250

T5A (Anardana powder)

-

250

-

C – Carrot, R – Radish, Cu – Cucumber, A – Anardana, Am – Amchoor

IJFFT 1(2) 2011 : 202

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables

Fruit powders viz., anardana and amchoor powders were procured from the local market and were stored in polyethylene bags for later use.

tasting the next sample. Each sample was evaluated for various quality attributes viz., colour, consistency/texture, aroma, taste/ acid sugar blend, overall acceptability. Judges were asked to rate the samples on a prescribed sensory evaluation performa with earlier stated attributes (Joshi, 2006).

Preparation Method

Statistical analysis

The combination of fermented vegetable powders with anardana or amchoor powder was mixed with different proportions of ingredients like powdered onion, garlic, ginger, sugar, salt, chilli, blackpepper, clove, cardamom, cinnamon and mint etc. Details of ingredients used for the preparation of instant chutney mix as per standard recipe are : 250g, sugar250g, salt- 50g, onion powder- 40g, garlic powder- 10g, cardamom- 20g, black pepper- 10g, cumin- 20g, cinnamon10g, red chillies- 10g, cloves- 20g and mint- 20g. Dried chutney powder mix was reconstituted with water in the ratio of 1:3 and allowed to stand for 10 min prior to serving. The proportion of anardana in the final chutney (reconstituted) was each and fermented vegetable powder was 4.08 g. The chutney mixes were analysed for various chemical, nutritional and sensory characteristics.

The data of quantitative estimation of various physico-chemical characteristics of products were anlaysed by Completely Randomized Design (CRD) while the data of sensory evaluation were analysed by Randomized Block Designs (RBD) (O’Mahony, 1985).

Physico-Chemical analysis

Physico-chemical characteristics

Instant chutney mixes of different combinations were analyzed for various physico-chemical characteristics. The TSS was measured using Erma hand refractometer (0-32 oB) and titratable acidity was calculated as per standard method (AOAC, 1980) and expressed as % lactic acid. The pH was taken with HPG, G-2004 pH meter, after calibrating it with buffer solutions of pH 4 and 9.2 (Ranganna, 1986). Determination of reducing sugars was done as per NelsonSomogy method (Sadasivam and Manickam ,1996).Salt content was determined by titration method using silver nitrate solution and expressed as % NaCl (Ranganna, 1986). The rehydration ratio was calculated by the method as suggested by Ranganna (1986).

The red tintometer colour units (TCU) of the instant chutney mix ranged from 4.0 to 7.0 (TCU). While, yellow and blue tintometer colour units (TCU) were statistically non-significant (Table 2). The maximum red TCU (7.0) was recorded in T4 CA which were at par with T5 A. But the lowest (4.0) red TCU were recorded in T3 Am. This may be due to the colour of acidulants like amchoor and anardana powders used in instant chutney mix production. Total soluble solids ranged between 12.0 to 16.8oB having significant differences with maximum (16.8oB) in T4 CA having carrot and anardana in 1:1 ratio, which was statistically at par with T2 CAm, T3 Am and T5 A and minimum (12.0oB) in T1 C (control). It is discernible from the data (Table 2) that the titratable acidity of reconstituted instant chutney mixes ranged between 0.72 to 1.20 % as lactic acid which was within the range of FPO specifications. The highest value (1.20%) was recorded in T2 CAm containing carrot and amchoor in 1:1 ratio and lowest (0.72%) in T1 C (control). The higher titratable acidity observed in T2 CAm might be due to higher initial values in the fermented carrot powder and amchoor powder. The brixacid ratio of instant chutney mixes of different treatments

Fruit powders

Sensory Evaluation The sensory evaluation of different instant chutney mixes was conducted by a semi-trained panel of 10 judges. It was first reconstituted with water and then, served. Each judge was given a set of products separately in isolated booths and provided with a glass of fresh water to rinse their mouth before

Cost of production The economics of production of products at the laboratory scale was worked out based on the cost of all ingredients, 10 % overhead charges, including fermentation charges, 20 % processing charges (cost of making puree/pulp/slurry) and 20 % profit margin.

Results and Discussion

IJFFT 1(2) 2011 : 203

Joshi et al.

varied from 13.34 to 20.25 and highest (20.25) being in 50 per cent anardana (T2 A) instant chutney and lowest (13.34) in T2 CAm, which might be due to its higher acidity. The pH values ranged between 3.32 to 3.51 which are in consistence with the acidity of different treatments. The highest (1.50%) salt content was noted in T1 C (control) and lowest (1.02%) in T3 Am and T5 A which were statistically at par with T4 CA. The total sugars content of chutneys from different treatments were significantly different. Maximum (14.75%) was recorded in T5 A which was at par with T4 CA and minimum (9.27%) in T1 C (control). The higher total sugar content observed in T5 CA and T4 CA might be due to the higher initial values for total sugars in anardana. Non-significant difference for rehydration ratio among the treatments of fermented carrot

based instant chutneys was observed which ranged between 2.2 to 3.5 (Table 2). The data (Table 3) on physico-chemical characteristics of fermented radish instant chutney mix clearly revealed that there were significant differences among the treatments for titratable acidity, brix-acid ratio, pH, salt, total sugar and red tintometer colour units. However, no significant differences for yellow and blue tintometer colour units were observed. Whereas, T1 R was significantly different from others with lowest (4.0 ) red TCU. The differences in T1 R might be due to the preparation of radish chutney without addition of anardana or amchoor. It is clear that TSS of various treatments of radish instant chutney mix ranged between 14.0 to 16.0oB with non-significant differences. Further, titratable

Table 2. Physico-chemical and sensory characteristics of reconstituted fermented carrot based instant chutney mix Characteristics

Treatments T 1C

Physico-chemical Tintometer colour units Red Yellow Blue pH Salt (%) Total sugars (%) Rehydration ratio Total soluble solids (oBrix) Titratable acidity (% LA) Brix-acid ratio Sensory (Score out of 20) Colour Flavour Texture Taste Overall acceptability

Anardana

CD (d < 0.05)

T2CAm

T 3Am

T 4CA

T 5A

5.0 10.0 1.0 3.51 1.50 9.27 3.0 12.0 0.72 16.67

5.2 10.0 1.0 3.32 1.12 13.5 3.0 16.0 1.20 13.34

4.0 10.0 2.0 3.37 1.02 13.75 3.0 15.0 0.99 15.43

7.0 10.0 3.34 1.07 14.27 3.5 16.8 1.16 14.48

6.8 10.0 1.0 3.45 1.02 14.75 2.2 16.0 0.79 20.25

0.979 NS NS 0.052 0.053 0.047 NS 3.05 0.044 0.410

15.90 14.20 15.70 15.40

14.60 15.70 16.70 15.70

13.80 12.10 14.30 14.28

17.40 17.10 17.20 16.80

16.20 13.20 15.20 13.15

0.920 0.738 1.09 0.762

15.30

16.01

13.62

17.12

14.03

1.12

T1C: Control T2CAm: Carrot powder + Amchoor powder (1:1) T3Am: Amchoor powder T4CA: Carrot powder + Anardana powder (1:1) T5A: Anardana powder

IJFFT 1(2) 2011 : 204

Amchoor

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables

Table 3. Physico-chemical and sensory characteristics of reconstituted fermented radish based instant chutney mix Characteristics

Treatments T 1R

Physico-chemical Tintometer colour units Red Yellow Blue pH Salt (%) Total sugars (%) Rehydration ratio Total soluble solids (oBrix) Titratable acidity (% LA) Brix-acid ratio Sensory (score out of 20) Colour Flavour Texture Taste Overall acceptability

Amchoor

Anardana

CD (d < 0.05)

T2RAm

T 3Am

T 4RA

T 5A

4.0 10.0 1.0 3.42 1.12 11.48 3.0 14.0 0.82 17.07

7.0 10.0 1.0 3.30 1.10 13.15 2.6 16.0 1.22 13.11

7.0 10.0 2.0 3.37 1.02 13.75 3.0 15.0 0.99 15.16

7.0 10.0 3.32 1.15 14.75 2.6 16.0 1.20 13.34

7.0 10.0 1.0 3.46 1.05 14.75 3.2 16.0 0.79 20.25

2.58 NS NS 0.050 0.053 0.042 NS NS 0.038 0.440

13.50 12.10 13.80 12.70 13.02

13.70 12.90 14.10 14.30 13.75

13.70 12.10 14.30 13.28 13.34

14.30 14.40 14.40 14.51 14.40

14.60 13.20 15.20 13.15 14.03

NS 1.03 NS 1.06 0.948

T1R: Control T2RAm: Radish powder + Amchoor powder (1:1) T3Am: Amchoor powder T4RA: Radish powder + Anardana powder (1:1) T5A: Anardana powder

acidity of various treatments ranged from 0.79 to 1.22 per cent, being highest (1.22%) in T2 RAm, which was at par with T4 RA containing radish and anardana in 1:1 ratio. The higher titratable acidity of T2 RAm might apparently be due to higher initial values of titratable acid in fermented radish and amchoor powder. The lowest titrable acidity (0.79%) was recorded in T5 A. However, it was within the range of FPO specifications. Further it is clear that brix-acid ratio of fermented radish instant chutney mix varied from 13.11 to 20.25, with highest (20.25) in T5 A and lowest (13.11) in T2 RAm which was at par with T4 RA. Further, the pH values of various treatments ranged between 3.30 to 3.46, and was recorded highest (3.46) in T5 A and lowest (3.30) in T2 RAm which was at par with T4 RA. Salt content was recorded highest (1.15%) in T4 RA which was at par with T1 R (control) and T2 RAm, and lowest (1.02%) in T3 Am which was at par with T5 A. The total sugars content varied from 11.48 to 14.75 per cent being the highest (14.75%) in T4 RA and T5 A, and the lowest (11.48%) in T1 R (control). It is clear that rehydration ratio of various treatments of radish instant chutney mix ranged

between 2.6 to 3.2 and these parameters showed nonsignificant differences. The red tintometer colour units (TCU) of the cucumber instant chutney mix ranged from 6.0 to 9.0 (Table 4). The maximum red tintometer colour units (9.0 TCU) were recorded in T4 CuA, which was at par with T2 CuAm, T3 Am and T5 A. The lowest (6.0 TCU) red tintometer colour units were recorded in T1 Cu (control). The differences in colour units among different treatments may be due to the colour of fruit powders used. There were significant differences among the treatments for TSS which ranged between 12.0 to 16.0oB with maximum (16.0 o B) in T 2 CuAm, T 4 CuA and T 5 A which were statistically at par with T3 Am and minimum (12.0oB) in T1 Cu (control). The titratable acidity of various reconstituted instant chutney mixes ranged between 0.79 to 1.24 per cent. However, the highest (1.24%) titratable acid content was recorded in T2 CuAm which was at par with T4 CuA and the lowest (0.79%) was recorded in T5 A. The higher titratable acidity observed in T2 CuAm might apparently be due to higher initial values of

IJFFT 1(2) 2011 : 205

Joshi et al.

titratable acid in the fermented carrot and amchoor powder. Brix-acid ratio of fermented cucumber instant chutney mixes varied from 12.90 to 20.25, which was recorded the highest (20.25) in T5 A and lowest (12.90) in T2 CuAm which was at par with T1 Cu (control). The lower value observed in T2 CuAm might be due to its higher acidity. The pH values of various treatments ranged between 3.30 to 3.46 being highest in T5 A and lowest in T2 CuAm which was at par with T4 CuA. The highest (1.57%) salt content was recorded in T1 Cu (control) and lowest (1.02%) in T3 Am. The total sugar content of various chutneys was significantly different. The maximum total sugar content (15.16%) was found in T3 Am and lowest (9.84%) in T1 Cu (control). The higher total sugar content recorded in T3 Am might be due to higher initial values of amchoor powder. There were no significant differences among the treatments w.r.t. rehydration ratio of the fermented

cucumber based instant chutneys and the ratio ranged between 2.2 to 3.1 (Table 4).

Sensory characteristics Table 2 reveals that the colour score of fermented carrot based instant chutney of different treatments ranged from 13.80 to 17.40. Maximum score (17.40) was obtained in T4 CA and minimum (13.80) in T3 Am which was at par with T2 CAm. The highest flavour score (17.10) was obtained by T4 CA and the lowest (12.10) by T3 Am. The texture scores shown in the data varied from 14.30 to 17.20, highest being (17.20) recorded in T4 CA, which was at par with T2 CAm and lowest (14.30) in T3 Am which was at par with T5 A. Hence, treatment T3 Am and T5 A were least liked with respect to this attribute. It is also clear from the results that the taste scores varied from

Table 4. Physico-chemical and sensory characteristics of fermented cucumber based instant chutney Characteristics

Treatments T 1Cu

Physico-chemical Tintometer colour units Red Yellow Blue pH Salt (%) Total sugars (%) Rehydration ratio Total soluble solids (oBrix) Titratable acidity (% LA) Brix-acid ratio Sensory (score out of 20) Colour Flavour Texture Taste Overall acceptability

Anardana

CD (d < 0.05)

T2CuAm

T 3Am

T 4CuA

T 5A

6.0 10.0 1.0 3.40 1.57 9.84 2.5 12.0 0.86 13.95

7.0 10.0 1.0 3.30 1.42 12.48 2.2 16.0 1.24 12.90

8.0 10.0 2.0 3.37 1.02 15.16 3.1 15.0 0.99 15.16

9.0 10.0 1.0 3.32 1.35 13.16 2.2 16.0 1.21 13.23

7.0 10.0 1.0 3.46 1.05 14.82 3.1 16.0 0.79 20.25

2.16 NS NS 0.055 0.034 0.140 NS 2.93 0.041 0.289

14.00 12.40 12.90 12.05 12.83

13.80 13.80 13.30 14.10 13.75

13.70 12.10 14.30 13.28 13.34

14.00 14.10 14.70 14.60 14.35

14.60 13.20 15.20 13.15 14.03

NS 0.085 0.928 1.21 0.952

T1Cu: Control T2CuAm: Cucumber powder + Amchoor powder (1:1) T3Am: Amchoor powder T4CuA: Cucumber powder + Anardana powder (1:1) T5A: Anardana powder

IJFFT 1(2) 2011 : 206

Amchoor

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables

13.15 to 16.80; highest being (16.80) recorded in T4 CA, the least liked treatment with respect to this attribute was T5 A. The overall acceptability score of fermented carrot based instant chutneys ranged between 13.62 to 17.12. Maximum overall acceptability score (17.12) was recorded in T4 CA which was at par with T2 Cam. In consistence with the trend of various attributes discussed, the overall acceptability score of T4 CA confirmed the suitability of carrot + anardana (1:1) for fermented carrot based instant chutney. In case of fermented radish based chutney colour scores ranged between 13.50 to 14.60 (Table 3). The flavour scores

of various treatments ranged between 12.10 to 14.40, with highest (14.40) in T4 RA and lowest (12.10) in T1 R (control) and T2 Am. T4 RA was the most liked treatment with respect to this attribute. The texture scores ranged between 13.80 to 15.20 and were non significant. The highest taste score (14.51) was recorded in T4 RA which was at par with T2 RAm and lowest (12.70) was awarded to control (T1 R) which was at par with T3 Am and T5 A. It is clear from the data (Table 3) that overall acceptability scores ranged between 13.02 to 14.40 being highest score (14.40) in T4 RA, which was at par with T5 A. The lowest (13.02) overall acceptability score was

Table 5. Cost of production of fermented vegetable instant chutney mix Items

Rate (Rs.)

Carrot (Carrot + Anardana) (1:1) Quantity Amount (Rs)

Fermented carrot powder 108/ kg Fermented carrot powder 108/ kg Fermented radish powder 171.4/kg Fermented cucumber powder 277/kg Anardana powder 30/kg Sugar 17/kg Salt 7/kg Onion 8/kg Garlic 20/kg Ginger 32/kg Black pepper 120/kg Cumin 200/kg Cinnamon 200/kg Red chillies 80/kg Cloves 300/kg Cardamom 250/kg Electricity @ 1 unit/ 4 h 1.10 Packaging material (laminated pouches) 1/ pouch Total cost Overhead charges @ 10% Total cost Profit @20% Total cost Cost per 200 g pouch From 1 kg fermented carrot 110 g powder 1 kg fermented radish 70 g powder 1 kg fermented cucumber 45 g powder Total yield 2.34 kg (based on the actual lab. data)

500 g 500 g 500 g 1.00 kg 100 g 80 g 20 g 40 g 20 g 40 g 20 g 20 g 40 g 40 g 3 units 11 No.

54 54 15.0 17.0 0.70 0.64 0.40 1.28 2.40 8.00 4.00 1.60 12.0 10.0 3.30 11 141.32 14.13 155.45 31.09 186.54 16.95

Radish (Radish + (1:1) Anardana) Quantity 500 g 500 g 1.00 kg 100 g 80 g 20 g 40 g 20 g 40 g 20 g 20 g 40 g 40 g 3 units 11 No.

Cucumber (Cucumber + Anardana) (1:1)

Amount (Rs) Quantity Amount (Rs) 85 15.0 17.0 0.70 0.64 0.40 1.28 2.40 8.00 4.00 1.60 12.0 10.0 3.30 11 172.32 17.23 189.55 37.91 227.46 20.67

500 g 500 g 1 kg 100 g 80 g 20 g 40 g 20 g 20 g 20 g 20 g 40 g 40 g 3 units 11 No.

138.87 15.0 17.0 0.70 0.64 0.40 1.28 2.40 8.00 4.00 1.60 12.0 10.0 3.30 11 225.82 22.58 248.40 49.68 298.08 27.09

IJFFT 1(2) 2011 : 207

Joshi et al.

recorded in T1 R (control) which was at par with T2 RAm and T3 Am instant chutney. An overview of the results on physicochemical and sensory characteristics clearly revealed that T4 RA, instant chutney mix had higher TSS, titratable acidity, salt and total sugar which made a good blend in instant chutney. Further, sensory quality results reveal that this treatment had higher scores for texture, flavour taste and overall acceptability. Hence, based on these results T4 RA was ranked as the best blend for making fermented radish based instant chutney. It is evidently clear (Table 4) that there were significant differences among various treatments of cucumber instant chutney mix for flavour, texture, taste and overall acceptability scores. Although the colour scores of this product varied from 13.70 to 14.60 but statistically there were no significant differences among various treatments. The flavour scores of various treatments ranged between 12.10 to 14.10, with highest (14.10) in T4 CuA and lowest (12.10) in T3 Am, whereas, the highest texture score (15.20) was recorded in T5 A which was at par with T3 Am, T4 CuA and T2 CuAm. It is clear that taste scores of fermented cucumber based instant chutney mix ranged between 12.05 to 14.60. The maximum score (14.60) was recorded in T4 CuA which was at par with T2 CuAm. Whereas, the overall acceptability scores of various treatments ranged between 12.83 to 14.35, being highest (14.35) in T4 CuA, that was at par with T5 A. However, the lowest score (12.83) was awarded to T1 Cu (control) which was at par with T2 CuAm and T3 Am. An overview of the result of physico-chemical and sensory characteristics clearly revealed that cucumber anardana chutneys have more desirable characteristics than the control and amchoor based chutneys. Hence, based on physico-chemical and sensory characteristics T4 CuA was ranked as the best and was the most suitable blend for fermented cucumber based instant chutney making.

Cost of production The data in Table 5 showed that the cost of production for dried instant chutney mix was the lowest (Rs.16.95) in carrot based chutney having carrot and anardana in 1:1 ratio alongwith other ingredients. The costs of radish and cucumber based chutney mixes were higher due to their lower recovery of dried powder. As in radish and cucumber only 70 g and 45 g dried powder was obtained from 1.00 kg fermented radish and cucumber, whereas in carrot the recovery of dried powder was more (110 g).

IJFFT 1(2) 2011 : 208

Conclusion Based on physico-chemical and sensory characteristics of various chutneys, it is apparent that all the treatments were within the specifications of FPO. In consistence with the trend of various attributes discussed, the overall acceptability score of T4CA confirms the suitability of fermented carrot for preparation of fermented carrot based instant chutney. Further, among fermented radish based instant chutney T 4RA containing radish + anardana (1:1) was adjudged the best as this product along with optimum physico-chemical characteristics had reliable scores for colour, flavor, consistency, taste and overall acceptability. Whereas, the treatment T4CuA having fermented cucumber powder + anardana (1:1) got the best scores. Among all the chutneys made from fermented vegetable powders, carrot based chutney powder ranked the best. However, irrespective of fermented vegetable powders used, the anardana containing vegetable powder instant chutneys were better. Thus, it can be concluded that preparation of instant chutney powders is an exciting proposition for making healthful food from lactic acid fermented vegetables.

References AOAC. 1980, Official methods of analysis. Association of Official Analytical Chemists, (Ed. W Hortwitz), 13th edn., Washington DC. Bucker ER, Mitchell JH and Johnson MG. 1979, Lactic fermentation of Peanut milk. J. Food Sci., 44: 1534-1538. Cheng YJ, Thompson LD and Brittin HC. 1990. Sogurt–a Yogurt like soyabean product - Development and properties, J. Food Sci., 55: 1178. Frazier WC and Westhoff DC. 1998, Food Microbiology. Tata Mcgraw Hill Publi. Co., 7th edn., New Delhi. Hang YD and Jackson H. 1967, Preparation of soyabean cheese using lactic starter organisms- General characteristics of the finished cheese. Food Technol., 21: 1033. Joshi VK, 2006, Sensory Science: Principles and applications in food evaluation. Agrotech Publishing Academy, New Delhi. Joshi VK, Kaur M. and Thakur NS. 1996, Lactic acid fermentation of mushroom (Agaricus bisporus) for preservation and preparation of sauce. Acta Alimentaria, 25: 1-11. Joshi VK and Thakur S. 2000, Lactic acid fermented beverages. In: Postharvest Technology of Fruits and Vegetables. (Eds. LR Verma and VK Joshi). Vol. II. Indus Publ. Co., New Delhi, p. 1102.

Preparation and evaluation of instant chutney mix from lactic acid fermented vegetables

Joshi VK, Sharma S, Rana N. 2006, Production, purification, stability and efficacy of bacteriocin from the isolate of natural lactic acid fermentation of vegetables. Food Technol Biotechnol., 44: 435439. Joshi VK, Sharma S and Thakur NS, 2008, Effect of temperature, salt concentration and fermentation type (inoculated vs natural) on lactic acid fermentation behavior and quality of carrot. Acta Alimentaria, 37: 205-219. NHB. 2009, Horticulture Database. www.nhb.gov.in O’Mahony M. 1985, Sensory evaluation of foods – Statistical methods and procedures. Marcell Dekker Inc., New York, pp. 168-169. Panda SH, Parmanick M, and Ray RC. 2007, Lactic acid fermentation of sweet potato (Ipomoea batalas L.) into pickles. Food Process Preserv., 31: 83-101. Ranganna, S. 1986, Handbook of Analysis of Quality Control for Fruit and Vegetable Products. 2nd Edn. Tata McGraw Hill Publ. Co., New Delhi.

Sadasivam S and Manickam A. 1996, Biochemical methods. 2nd edn. New Age International, New Delhi. Sahlin P. 1999, Fermentation as a method of food processing: production of organic acids, pH-development and microbial growth in fermenting cereals. Licentiate thesis, Division of the Applied Nutrition and Food Chemistry, Lund University. Sethi V. 1990, Lactic fermentation of black carrot juice for spiced beverage. Indian Food Packer, 44: 7-12. Sharma S, and Joshi VK. 2007, Influence of temperature and salt concentration on lactic acid fermentation of radish. J. Food Sci. Technol., 44: 611-614. Sharma S, Joshi VK and Lal Kaushal BB. 2008, Preparation of readyto-serve drink from lactic acid fermented vegetables. Indian Food Packer, 62: 52-62. Takagi K, Toyoda M, Saito Y, Niwa M and Morimoto H. 1990, Composition of fermented grape juice continuously produced by immobilized Lactobacillus casei. J. Food Sci., 55: 455-457.

IJFFT 1(2) 2011 : 209

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 211-219

Research Paper

Comparative evaluation of different cell disruption methods for the release of L-asparaginase from Erwinia carotovora MTCC 1428 Sarita and Wamik Azmi* Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, Himachal Pradesh, India

Abstract : L-asparaginase (E.C. 3.5.1.1.) is a therapeutic enzyme extensively used in the treatment of acute lymphoblastic leukemia and its clinical preparations are of microbial origin. However, many Gram’s negative bacteria do not typically excrete proteins into the culture medium since the cell wall is covered by an outer membrane. L-asparaginase obtained from bacterial culture Erwinia carotovora MTCC1428 is an intracellular enzyme, hence cell disruption is mandatory to release the enzyme for further purification. Cells of E. carotovora were lysed by different physical (vortex, pestle and mortar, sonicator, Bead Beater), chemical (alkali lysis, acetone powder, guanidine-HCl and triton X-100) and enzymatic (lysozyme) methods. Among various methods evaluated, L-asparaginase release was found to be the best when cells were disrupted by sonication. Different parameters considered important include cell concentration, cell volume, number of cycles and amplitude of sonication that were optimized for the release of enzyme. The recovery of enzyme was found 70% and the specific activity of the released L-asparagianse was found to be 0.16 U/mg of protein with sonication. Moreover, the loss in enzyme activity was also found to be minimum (25%) when E. carotovora MTCC1428 cells were disrupted by sonication. Keywords : L-asparaginase, Erwinia carotovora MTCC1428, Intracellular enzyme, Cell disruption, Sonication

The importance of microorganisms as a source of commercially useful chemicals, antibiotics, and enzymes has been recognized for a very long time (Joshi and Pandey, 1999). Most of the chemicals of microbial origin produced industrially today are of the extracellular type. However, a much larger proportion of the potentially useful microbial products are retained within the cell. A vast majority of the enzymes are intracellular in nature. Even greater use of microbial products, many of which will be intracellular, can be expected from the predicted surge in biotechnology (Chisti and Moo-Young, 1986). L-Asparaginase (EC. 3.5.1.1) is an important component in the treatment of children with acute lymphoblastic leukemia. Its antineoplastic activity is associated with the property of *E-mail of corresponding author : [email protected] MS Received on : 24th March, 2011 Accepted on : 28th Sept., 2011

depleting the circulating pool of L-asparagine by the asparaginase catalytic activity. Malignant cells with low Lasparagine levels are killed due to lack of an exogenous supply of this amino acid combined with an impaired protein synthesis mechanism. However, normal cells are protected from Lasparagine-starvation due to their ability to produce this amino acid. Based on this, L-asparaginase has also been included in most contemporary, multi-agent regimens for adult acute lymphoblastic leukemia (Narta et al, 2007; Prakasham et al, 2007). L-asparaginase is an intracellular product in Erwinia carotovora MTCC1428. The isolation of intracellular products requires that the cell either be genetically engineered so that what would normally be an intracellular product is excreted

Sarita and Azmi

into the environment or it must be disintegrated by physical, chemical or enzymatic means to release its contents into the surrounding medium. The genetic manipulation of microbial cells to make them leaky is limited in scope. Making the cell fully permeable to any significant fraction of the intracellular products and enzymes would not only be difficult, but will also imply discontinued existence of the cell. It is in this context that the unit operation of microbial cell disruption or intracellular product isolation is of increasing importance (Chisti and MooYoung, 1986). There are several methods of partial or selective disruption of membranes to release intracellular proteins. These include the use of chelating agents (Marchesi et al, 1970), adjustment of ionic strength and pH (Schnebli and Abrams, 1970), organic solvents (Somerville et al, 1970) and detergents (Helenius and Simons, 1975). Cell disruption by mechanical methods, e.g. homoginization or bead milling, or non-mechanical methods such as chemical or enzymatic methods (Kula and Schutte, 1987; Asenjo and Patrick, 1990; Middleberg, 1994) is the first requirement for the purification of intracellular microbial enzymes but commonly initiates cellular and molecular degradation process analogous to those of natural cell death and lysis. In addition, the generation of cell debris may promote electrostatic and/or hydrophobic product-debris interactions. Such adverse effects will compromise the yield and molecular fidelity of protein products (Horst et al, 2001). Keeping in view the importance of the cell disruption, a comparative study was carried out to investigate the release of enzyme L-asparaginase from the resting cells of E. carotovora MTCC1428. Different cell disruption methods were evaluated in the present study and sonicating of the cells of E. carotovora MTCC 1428 led to the maximum release of L-asparaginase, and this technique was further modulated to maximize the release of enzyme. The results have been described in this communication.

Material and Methods Chemicals All the chemicals were procured from Himedia, Merck and SDfine, India and were of analytical grade.

Microorganism and maintenance of culture The culture of E. caratovora MTCC 1428 was procured from Department of Biotechnology, Himachal Pradesh University, Shimla-5. The culture of E. carotovora MTCC1428 IJFFT 1(2) 2011 : 212

was maintained on a medium containing (%, w/v) casein enzyme hydrolysate (tryptone) 1.5, peptone 0.5, NaCl 0.5 and agar 2.0 (pH 7.0) Slants containing 1.0% (w/v) L-asparagine or in 20% (v/v) glycerol stocks were used and sub-culturing was done periodically. Phenol Red (0.0012%) was used as an indicator for L-asparaginase activity. The production of Lasparaginase was generally accompanied by an increase in pH due to the formation of ammonia after the hydrolysis of Lasparagine. Phenol Red at acidic pH was yellow and turned pink at alkaline pH. Thus, a pink zone was formed around the colonies of E. carotovora MTCC1428.

Production of L-asparaginase The E. caratovora MTCC1428 strain was cultivated on soluble components of medium in 250 mL Erlenmeyer shake flasks under submerged conditions. The preculture was prepared in above said medium with L-asparagine (0.1%) by inoculating the medium with loopful of culture and incubated it in a temperature controlled orbital shaker (25 oC, 150 rpm). 12 h old, 4% (v/v). Preculture was used to inoculate the production medium (L-asparagine, 0.6%). The culture was allowed to grow for 14 h under controlled conditions. The cells were harvested by centrifugation (10,000g for 10 min. 4 oC) and were washed with potassium phosphate buffer (pH 8.5, 0.05 M). The production of biomass and L-asparaginase were monitored at 14 h interval.

L-asparaginase assay L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia and the amount of liberated ammonia was measured spectrophotometrically. The Lasparaginase from E. caratovora MTCC1428 was found to be intracellular in nature and hence, the resting cells suspended in potassium phosphate buffer (0.05 M, pH 8.5) were used for the enzyme assay. Potassium phosphate buffer 1.45 mL (0.05 M, pH 8.5), cell suspension (50 µL) of known dcw and 500 µL of 10 mM substrate (L-asparagine) prepared in potassium phosphate buffer (0.05 M, pH 7.5) were incubated at 37 °C for 15 min. The reaction was stopped by the addition of 500 µL chilled TCA (15%, w/v). A set of control was also run. From the reaction mixture, 1 mL was withdrawn and amount of released ammonia was measured by ammonia hypochloride method (Fawcett and Scott, 1960). Activity of the L-asparaginase from the whole cell of E. caratovora MTCC1428 was expressed in terms of units (U). The L-

Evaluation of different cell disruption methods

asparaginase unit has been defined as the µ moles of ammonia released by one mg of dry cell weight in one minute under standard assay condition. For cell free extract, the supernatant (50 µL) was taken in test tubes and 1.45 mL of buffer was added to make the volume to 1.5 mL. Rest of procedure was the same as for the whole cells. The L-asparagine unit (IU) has been defined as the µ moles of ammonia released /mL/ min under standard assay conditions, and specific activity is IU/mg of proteins. Protein estimation was done by Bradford assay (Bradford, 1976).

Extraction of L-asparaginase from E. carotovora MTCC1428 The cells of E. carotovora MTCC1428 was grown in 100 mL of medium for 14 h and the broth was centrifuged at 10,000g for 10 min at 4 0C to obtain the cell pellet. The pellet was washed thrice with potassium phosphate buffer (0.05 M, pH 8.5). In each method of extraction, 100 µL of PMSF (0.1 mM in ethanol) was added to quench the serine protease activity. Lysozyme treatment for cell disruption: Cell pellet of E. carotovora MTCC1428 (154 mg dcw) was suspended in 2 mL of solution A (50 mM glucose, 10 mM EDTA and 25 mM Tris, pH 8.0). To this, 0.5 mL of solution B (Lysozyme 20 mg/ mL, dissolved in solution A) was added and mixed the reaction mixture thoroughly. Tube was incubated in ice for 10 min and then 0.5mL of solution C (0.2 M NaOH and 1% SDS) was added. Tube was further incubated in ice for 10 min and centrifuged at 10,000g for 15 min at 4°C. L-asparaginase activity was measured in cell pellet as well as supernatant. Alkali lysis method: Resting cells (154 mg dcw) of E. carotovora MTCC1428 was suspended in 1 mL of solution A (50 mM glucose, 10 mM EDTA and 25 mM Tris, pH 8.0) and 2 mL of solution B (0.2 M NaOH and 1% SDS) was added to it. The tubes were closed tightly and the contents were mixed by inverting the tubes rapidly 5 times. The tubes were placed in ice bath and 1.5 mL of chilled solution C (5 M Potassium acetate 60 mL, Glacial acetic acid 11.5 mL and distilled water 28.5 mL) was added. The tubes were closed and gently vortexed for 10 seconds. Rest of the procedure was the same as for lysozyme treatment. Acetone powder method for cell disruption: 10 mL of chilled anhydrous acetone was added to the 154 mg (DCW) resting cells of E. carotovora MTCC1428. The slurry was incubated

for 30 min at 10°C and contents was mixed and centrifuged at 10,000g for 15 min. The cell pellet was resuspended in 10 mM sodium borate buffer (pH 6.5) and kept at 40 °C for 10 min. Rest of the procedure was the same as for lysozyme treatment. Triton X-100 and Guanidine-HCl treatment for cell disruption: To the 10 mL resting cell suspension of E. carotovora MTCC1428 in potassium phosphate buffer (0.05 M, pH 8.5) containing 15.4 mg/mL (dcw) cell mass, 4 mL of 2 M Guanidine-HCl and 0.24 mL of Triton X-100 (2%,v/v) was added. The reaction contents were mixed and incubated at room temperature for 15 min. Rest of the procedure was the same as for lysozyme treatment. Disruption of cells in pestle and mortar: The resting cells of E. carotovora MTCC1428 were suspended in potassium phosphate buffer (0.05 M, pH 8.5) and 5 mL of cell slurry containing 15.4 mg/mL (dcw) cell mass was subjected to disruption in pestle and mortar. 100 µL of PMSF (0.1 mM in ethanol) and 5 mL of glass beads (0.5 mm) was taken along with the resting cells in mortar. Cell slurry was crushed in mortar and pestle (in ice bath) for 15 min and rest of the procedure was the same as for lysozyme treatment. Disruption of cells by vortex with glass beads in culture tubes: The cells of E. carotovora MTCC1428 were suspended in potassium phosphate buffer (0.05 M, pH 8.5) and 5 mL chilled resting cells (15.4 mg/mL dcw) and 5 mL of glass beads (0.5 mm diameter) were placed in a culture tube and vortexed for 20 min. Rest of the procedure was the same as for lysozyme treatment. Disruption of cells by Bead Beater®: Resting cells (15 mL containing 15.4 mg/mL dcw) of E. carotovora MTCC1428 was placed in a Bead Beater® along with the 15 mL Zirconium beads (0.1 mm diameter). Cell disruption was carried out for 6 cycles of 1 min each. Samples were collected for each cycles, centrifuged at 10,000g for 15 min at 4 °C and used for L-asparaginase assay. Disruption of cells by sonication: Sonication was carried out with 15 mL resting cell suspension of E. carotovora MTCC1428 (15.4 mg/mL dcw) at an amplitude of 35% for 5 cycles of 1 min each in an ice bath. Sample was taken out after every cycle and centrifuged at 10,000g for 15 min at 4 °C. The pellet and supernatant were used for L-asparaginase assay.

IJFFT 1(2) 2011 : 213

Sarita and Azmi

Optimization of parameters for the release of Lasparaginase by sonication Resting cell suspension 15 mL of E. carotovora MTCC1428 (15.4 mg/mL dcw) was sonicated at a pulse of 1 min (1 min on, 1 min off) for 10 cycles in an ice bath at 35% amplitude. The different concentrations (7.7 mg/mL, 11.2 mg/mL, 15.4 mg/mL, 18.3 mg/mL, and 21.3 mg/mL dcw) of the resting cell suspension of E. carotovora MTCC1428 were disintegrated by sonication for 5 cycles and analyzed for L-asparaginase activity. The different volumes (10 to 25 mL) of resting cell suspension (15.4 mg/mL dcw) of E. carotovora MTCC1428 were subjected to sonication for 5 cycles at 35% amplitude in ice bath and enzyme activity was measured for each set of experiment. The 15 mL of resting cell suspension (15.4 mg/ mL dcw) of E. carotovora MTCC1428 was sonication at different amplitudes (30%, 35%, and 39%) for 5 cycles. The amount of released L-asparaginase was measured spectrophotometrically.

Results and Discussion Extraction of L-asparaginase by different methods Various methods (enzymatic, chemical and physical) were tried to release the intracellular L-asparaginase from E. carotovora MTCC1428 and a comparison of different cell disruption

methods have been made in Table 1. The enzymatic (lysozyme) digestion of the cell wall of the E. carotovora MTCC1428 released 0.74 U of L-asparaginase (Table 1). However, even after cell lysis, 1.76 U the enzyme activity was still within the unlysed cells. The overall recovery of Lasparaginase was found to be 8.1% and almost 73% loss in the enzyme activity was observed. De Jong (DeJong, 1972 ) reported the release of L-asparaginase from the cells of S. griseus ATCC 10137 when treated with lysozyme, however, the high concentrations of lysozyme could affect the enzyme activity adversely. Cell lysis of Gram negative bacteria requires passage through the outer membrane which is aided by the addition of EDTA to chelate the divalent cations (Schutte and Kula, 1993). This enzyme cleaves β (1-4) glycosidic linkage occurring in the polysaccharide protein of bacterial cell walls (Bucke, 1983). However, from process economics points of view, this method seems to be very costly at large scale. In an earlier work, the release of L-asparaginase from the cells of S. albidoflavus was found to increase from 5.93 IU to 7.1 and 7.51 IU per mg of cell dry weight when shifted from physical treatment (cell grinding) to chemical treatment with EDTA and lysozyme, respectively (Narayana et al, 2008). Among the different chemical methods of cell disintegration, significant amount of protein was released (3.1 mg/mL) with very little L-asparaginase activity (0.1 U) when the cells of E.

Table 1. The extraction of L-asparaginase from E. carotovora MTCC 1428 cells by different disruption methods Treatment

Methods

Total enzyme activity (U) Whole cells

After cell lysis In cells

In supernatant

Released protein (mg/mL)

Specific activity (U/mg)

Loss in enzyme activity (%)

Recovery (%)

1.

Lysozyme

9.10

1.76

0.74

2.6

0.08

73

8.1

2.

Alkali lysis

8.94

0.62

0.10

3.1

0.01

92

1.1

3.

Acetone powder

10.02

1.39

4.77

5.6

0.09

39

48

4.

Triton-X and Guanidine-HCl

10.24

2.17

5.10

5.5

0.08

29

50

5.

Mortar and Pestle

10.49

2.31

5.0

5.7

0.09

30

48

6.

Vortexing with glass beads

10.19

3.40

4.0

5.5

0.08

27

39

7.

Bead Beater®

10.64

2.16

5.1

4.9

0.11

32

48

8.

Sonication

10.64

1.79

5.6

4.6

0.12

31

53

IJFFT 1(2) 2011 : 214

Evaluation of different cell disruption methods

carotovora MTCC1428 were subjected to alkali lysis (Table 1). This might be due to the strong alkaline conditions and denaturation of enzyme by a protein denaturant (SDS) present in the lysis solution. The lower L-asparaginase recovery (1.1%) and higher loss (92%) in enzyme activity make alkali lysis method unsuitable for release of enzyme from E. carotovora MTCC1428 cells. The acetone powder of the resting cells of E. carotovora MTCC1428 was prepared to release the L-asparaginase and 5.6 mg/mL protein was released in the supernatant with 4.77 U enzyme activity. This method seems reasonably better for cell disintegration as the recovery of enzyme was high (48%) and the loss in activity after lysis was low (39%). Lee et al (1989) used acetone treatment of E. carotovora to increase the permeability of cell wall and enzyme recovery in cell free extract was reported to be 57%. Triton X-100 and Guanidine-HCl treatment has widely been applied for cell disruption (Helenius and Simons, 1975) and this method yielded 5.5 mg/mL protein and 5.1 U of enzyme activity in the supernatant (specific activity 0.08 U/ mg of protein) released from the cells of E. carotovora MTCC1428. The overall loss in the enzyme activity was low (29%) with 50% recovery of enzyme. This method was found to be the best among the different chemical methods used as far as recovery and loss in enzyme activity was concerned. The release of protein from microorganisms can also be accomplished by applying different mechanical disruption methods (Table 1). The cells of E. carotovora MTCC1428 were crushed in a mortar with pestle along with glass beads and the cell free extract contained 5.0 U of L-asparaginase activity and 5.7 mg/mL protein. The loss in enzyme activity was 30% with overall recovery of 48% of L-asparaginase. Disintegration of the cells of E. carotovora MTCC1428 was also carried out by vortexing the cell slurry with glass beads (Horst, 2001). The amount of protein released in supernatant was found to be 5.5 mg/mL with 4.0 U of L-asparaginase (specific activity 0.08 U/mg of protein). However, even after lysis, 1/3rd amount of L-asparaginase was released from the resting cells. Bead agitation or bead milling is frequently used in large to middle scale preparation of intracellular protein from microorganisms in which harvested cells are vigorously agitated with beads in a closed chamber (Kula and Schutte, 1987). The cells of E. carotovora MTCC1428 were disrupted

in a Bead Beater® along with zirconium beads and in the cell free extract 4.9 mg/mL protein and 5.1 U of L-asparaginase was obtained. The overall recovery was 48% with 32% loss in the enzyme activity. This method seems to be suitable for release of L-asparaginase as the loss in activity obtained after lysis was low and the specific activity was found to be reasonably high (0.11 U/mg of protein). The lower recovery among these physical methods of cells lysis might be due to incomplete disintegration of the resting cells of E. carotovora MTCC1428 as significant amount of enzyme was retained within the cells even after lysis (Table 1). Protein release in these devices depend upon the cell disruption caused by shear forces and collision between beads and can be described as first order process (Melenders et al, 1992). A simultaneous cultivation and disruption of E. coli cells using glass beads to release recombinant enzyme has also been reported (Hirose et al, 1999). The mechanical lysis leads to 90% release in the protein content. Protein release is governed by a number of factors in bead mill disruption process such as bead volume, bead size, number of cycles, time of pulse, cell concentration, cell volume and agitation speed. The kinetics of protein release from baker yeast by disruption in a high speed ball mill has been investigated (Dunnill and Lilly, 1975) and depend upon the construction of the mill. The rate of protein release depended on density and size of beads. The Zirconium beads have greater density in comparison to glass beads. Due to this, they generate more shearing force, resulting in harder crushing action on cell suspension and hence, greater cell disruption. For a fixed agitation rotor speed and bead load, the collision frequency of beads decline with increasing diameter of beads (Dunnill and Lilly, 1975). The disintegration of E. carotovora MTCC1428 cells was also carried out by sonication. After 5 cycles of sonication, 5.6 U of L-asparaginase was found in the supernatant. A total of 4.6 mg/mL protein was released after the sonication. Specific activity was found to be 0.12 U/mg of protein. The L-asparaginase recovery was 53% with a loss of only 31% of enzyme activity. Amongst all the methods used for the disruption of bacterial biomass, sonication was found to be the most effective for the release of intracellular Lasparaginase from E. carotovora MTCC1428. The recovery of L-asparaginase has found to be more than 80% by 10 min

IJFFT 1(2) 2011 : 215

Sarita and Azmi

sonication in case of a recombinant strain of E. coli (Krasotkina et al, 2004). Sonication being most efficient in the recovery of this membrane bound enzyme, recommended for its extraction from fresh bacterial biomass (Singh and Jhamb, 2004). Bead beater as well as sonicator was found to be the most efficient for cell lysis. Ultrasonics has been widely used in various biological and chemical applications. Zhang et al (2005) reported the use of ultrasonic treatment to enhance protein– starch separation for use in the wet-milling industry. Ultrasonics has also been employed to assist the extraction of resveratrol from grapes (Cho et al, 2005). Li et al (2004) utilized ultrasound treatment to enhance oil extraction from soybeans. Wood et al (1997), studied ultrasonics to enhance ethanol yield from simultaneous saccharification and fermentation of mixed office paper. They achieved a 20% increase in ethanol yield from their sonicated samples. Optimization of the release of L-asparaginase by sonication Cell slurry (5 mL) was sonicated for 10 cycles of a pulse of 1 min. The maximum enzyme activity (0.51 U) and specific activity (0.12 U/mg) was found at the 5th cycle of sonication (Fig.1). The activity of enzyme decreased after 5th cycle possibly due to the thermal denaturation. Therefore, it could be suggested that the 5 cycles of sonication were most suitable for the release of L-asparaginase from the cells of E. carotovora MTCC1428. Different concentrations of the cells

of E. carotovora MTCC1428 were sonicated for the release of L-asparaginase (Table 2). The amount of enzyme released became almost constant beyond the cell concentration of 15.4 mg/mL. The maximum protein was released at the cells of concentration of 15.4 mg/mL with maximum recovery of 54%. Therefore, it can be concluded that the cell concentration of 15.4 mg/mL was most suitable for the release of Lasparaginase by sonication. Decreased enzyme release with lower cell concentration was expected as decreased cell concentration resulted in lesser crushing action of sonicator against cells. Maximum protein was released at cell concentration (15.4 mg/mL and other higher concentrations). Varying volume (10-25 mL) of cells slurry of E. carotovora MTCC1428 (21.3 mg/mL) were lysed for 5 on/off cycles of sonication. The maximum enzyme (8.71 U) was released at 25 mL of cell slurry (Table 3). However, maximum recovery of the enzyme was obtained (52%) when 15 mL of the slurry was used for lysis. The decrease in cell volume resulted in lower protein release, which was expected because amount of protein to be released was proportional to amount of cells. The 15 mL cell slurry (containing 15.4 mg/mL cells) was sonicated at different amplitudes (30, 35 and 39%) for 5 on/ off cycles (Table 4). The most efficient amplitude was found to be 39%. Below this amplitude, the lysis was not very effective as the activity in pellet after lysis was found to be very high. Higher amplitude resulted in greater cell disruption. This is because at higher amplitude the shear force on cells is higher which causes an efficient cell lysis.

Table 2. Disruption of different concentrations of E. carotovora MTCC1428 cells by sonication Treatment

Cell concentration (mg/mL)

Total enzyme activity (U) Whole cells

After cell lysis In cells

In supernatant

Released protein (mg/ml)

Specific activity (U/mg)

Loss in enzyme activity (%)

Recovery (%)

1.

7.7

9.15

3.4

3.7

4.3

0.09

27

40

2.

11.4

9.15

2.9

4.0

4.5

0.09

25

44

3.

15.4

9.15

1.29

4.9

4.6

0.12

32

4.

18.3

9.15

1.36

4.8

4.6

0.11

33

53

5.

21.3

9.15

1.34

4.9

4.6

0.11

32

54

IJFFT 1(2) 2011 : 216

54

Evaluation of different cell disruption methods

7

0.14

0.6

5 0.5 4 0.4

0.10

0.08

0.06

Specific activity (U/mg)

0.12 6

Protein released (mg/mL)

L-asparaginase activity (IU)

0.7

0.04

3

0.02

0.3

2 0.00 0

2

4

6

8

10

12

Disruption cycles

Fig 1. Disruption of E. carotovora MTCC1428 cells by sonication at different cycles Table 3. Disruption of different volumes of E. carotovora MTCC1428 cells by sonication Treatment

Cell volume (mL)

Total enzyme activity (U) Whole cells

After cell lysis In cells

In supernatant

Released protein (mg/mL)

Specific activity (U/mg)

Loss in enzyme activity (%)

Recovery (%)

1.

10

6.7

2.0

3.08

4.4

0.09

24

46

2.

15

10.05

1.75

5.2

4.6

0.12

31

52

3.

20

13.4

2.45

6.80

5.5

0.12

31

51

4.

25

16.75

2.90

8.71

8.0

0.11

31

52

Table 4. Disruption of E. carotovora MTCC1428 cells at different amplitudes of sonication S. No.

Amplitude

Total Enzyme activity (U) Whole cells

After cell lysis In cells

In supernatant

Released protein (mg/mL)

Specific activity (U/mg)

Loss in enzyme activity (%)

Recovery (%)

1.

30

12

3.8

4.1

4.5

0.09

34

34

2.

35

12

2.2

6.3

4.6

0.13

30

53

3.

39

12

0.6

8.4

5.4

0.16

25

70

IJFFT 1(2) 2011 : 217

Sarita and Azmi

Conclusion Amongst all the methods used for disruption of cells, sonication was found to be the most effective for the release of intracellular L-asparaginase from resting cells of E. carotovora MTCC1428. The alkali lysis method was found to be the least efficient in terms of L-asparaginase recovery. The enzymatic method (lysozyme treatment) was also not found suitable as the enzyme recovery was low and was also very costly when applied at large scale.

Acknowledgements The research work was supported by University Grants Commission, Government of India, New Delhi, India. Authors also wish to acknowledge the facilities provided by SubDistributed Information Centre, Department of Biotechnology, Himachal Pradesh University, Shimla-5, India.

References Asenjo JA and Patrick I. 1990. Large scale protein purification. In: Protein Purification and Application (Eds. ELV Harris and S Angal), Oxford University Press, Oxford, pp 1-28. Bradford MM. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem., 72: 248-254. Bucke C. 1983. The biotechnology of enzyme isolation and purification In: Principles of Biotechnology, (Ed. A Wiseman). Surrey University Press Glasgow, Scotland, pp. 151-171. Chisti Y and Moo-Young M. 1986. Disruption of microbial cells for intracellular products. Enzyme Microb Technol., 8: 194-204. Cho Y, Hong J, Chun HS, Lee SK and Min H. 2005. Ultrasonicationassisted extraction of resveratrol from grapes. J Food Eng., 77: 725-730.

using glass beads to release recombinant á-amylase and other enzymes. Biotechnol Techniques, 13: 571-575. Horst, B. 2001. Process integration of cell disruption and fluidized bed adsorption of microbial enzymes: application to the retrodesign of the purification of L-asparaginase. Ph. D. Thesis, The University of Birmingham, Edgbaston, Birmingham, UK. Horst B, Roger JH and Andrew L. 2001. Direct process integration of cell disruption and fluidized bed adsorption in the recovery of labile microbial enzymes. Bioseperation, 10: 73-85. Joshi VK and Pandey, A. 1999. Biotechnology: Food fermentation. In: Biotechnology: Food fermentation Vol I (Eds. VK Joshi and A Pandey) Educational Publishers and Distributors, New Delhi, pp. 1-25. Krasotkina J, Anna AB, Yuri VG and Nikolay NS. 2004. One step purification and kinetic properties of the recombinant Lasparaginase from Erwinia carotovora. Biotechnol Appl Biochem., 39: 215-221. Kula MR and Schutte H. 1987. Purification of proteins and the disruption of microbial cells. Biotechnol Prog., 3: 31-42. Lee SM, Wroble MH and Ross JT. 1989. Bioconversion of amino acids into flavouring alcohols and esters by Erwinia carotovora subsp. Atroseptica. Appl Biochem Biotechnol., 22: 1-11. Li H, Pordesimo L and Weiss J. 2004. High intensity ultrasound – assisted extraction of oil from soybeans. Food Res Int., 37: 731738. Marchesi SL, Steers E, Marchesi VI and Tiliack TW. 1970. Physical and chemical properties of a proteins isolated from recombinant cell membranes. Biochem., 9: 50-57. Melenders AV, Unno H, Shiragami N and Honda H. 1992. A critical concept of critical velocity for cell disruption by bead mill. J Chem Eng Japan, 25: 354-356. Middleberg APJ. 1994. Process scale disruption of microorganisms. Biotechnol Adv., 3: 491-551.

DeJong P. 1972. L-Asparaginase production by Streptomyces griseus. Appl Microbiol., 23: 1163-1164.

Narayana KJP, Kumar KG and Vijayalakshmi M. 2008. L-asparaginase production by Streptomyces albidoflavus. Ind J Microbiol., 48: 331–336.

Dunnill P and Lilly MD. 1975. Protein extraction and recovery from microbial cells. In: Single Cell Protein II, (Eds, DR Tannenbaum and DIC Wang), The MIT Press Cambridge, pp. 179-207.

Narta UK, Kanwar SS and Azmi W. 2007. Pharmacological and clinical evaluation of L-asparaginase in the treatment of leukemia. Crit Rev Oncol Hematol., 61: 208–221.

Fawcett JK and Scott JE. 1960. A rapid and precise method for the determination of urea and ammonia. J Clin Path., 13: 156-159.

Prakasham RS, Rao CS, Rao RS, Lakshmi GS and Sarma PN. 2007. Lasparaginase production by isolated Staphylococcus sp.–6A: design of experiment considering interaction effect for process parameter optimization. J App Microbiol., 102: 1382-1391.

Helenius A and Simons K. 1975. Solublization of membranes by detergents. Biochem Biophy Acta, 415: 29-39. Hirose J, Komemoto K, Ishimura A, Yokoi H and Takasaki Y. 1999. Simultaneous cultivation and disruption of Escherichia coli

IJFFT 1(2) 2011 : 218

Schnebli HP and Abrams A. 1970. Membrane adenosine triphosphate from Streptococcus feacalis. J Biol Chem., 245: 1115-1121.

Evaluation of different cell disruption methods

Schutte H and Kula MR. 1993. Cell disruption and isolation of nonsecreted products. In: Biotechenology, (Eds. HJ Rehm and G Reed), Vol 3, pp 505-526 Singh RS and Jhamb K. 2004. Studies on extraction of intacellular Lasparaginase from E. coli. J Punjab Acad Sci., 1: 57-61. Somerville HJ, Delafield FR and Rittenberg SC. 1970. Uera_mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other spieces. J Bacteriol., 101: 551-560.

Wood BE, Aldrich HC and Ingram LO. 1997. Ultrasound stimulates ethanol production during the simultaneous saccharification and fermentation of mixed waste office paper. Biotechnol Prog., 13: 232-237. Zhang Z, Niu Y, Eckhoff S and Feng H. 2005. Sonication enhanced corn starch separation. Starch., 57: 240-245.

IJFFT 1(2) 2011 : 219

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 221-229

Research Paper

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead Ghan Shyam Abrol* and V K Joshi Department of Food Science and Technology, Dr Y S Parmar University of Horticulture and Forestry , Nauni, Solan, India.

Abstract : Influence of initial TSS (total soluble solids) level on physico-chemical and sensory evaluation of mead was investigated and is reported here. Wild apricot fruit is higher in titrable acidity (2.23 ± 0.16 %MA), lower in TSS (10.0 ± 0.24°B) but has appreciable amount of ascorbic acid (37.30 ± 0.13 mg/100gm). Honey was used to ameliorate the initial TSS to 22, 24 and 26°B of wild apricot must to prepare wine and compared with that made with sugar. Rate of fermentation (RF) of honey based must was found higher than sugar based must. Among all the musts, RF of 26ºB honey must was the highest but, the fermentation efficiency of 26ºB wild apricot sugar based wine was the highest. In general, the fermentation efficiency increased with increase in initial TSS of wild apricot must. Out of two sources of sugar used, wild apricot sugar based wine had higher TSS, total sugars, pH, volatile acidity, ethanol and total phenols while wild apricot honey wine (mead) recorded higher reducing sugars, titratable acidity and higher alcohols. With the increase in initial TSS level of the musts (22º to 26ºB), reducing sugars, total sugars, titratable acidity, volatile acidity, ethanol content, higher alcohols and total phenols increased, whereas pH was decreased, irrespective of sugar source used. Based on the various physico-chemical characteristics, initial TSS of 26ºB was found to be optimum for mead preparation. On the basis of sensory characteristics, wild apricot must of 26ºB was awarded with better score because of balanced astringency, alcohol, sugar and acid level. Keywords : Wild apricot, Mead, Wine, Fermentation, Saccharomyces cerevisiae, Total soluble solids, TSS

Wild apricot fruit is found growing naturally at higher altitude and is utilized for the production of hard liquor by the local people (Parmar and Kaushal, 1982). The alcoholic drink so prepared contains high quantity of alcohol but completely lacks in nutrients, consumption of which results in various disorders as well as malnutrition (Joshi et al., 1990). Wine on the other hand, is the oldest known beverage, and its representations appeared in the region of Udimu in Egypt, some 5000 years back (Petric, 1923). Wines have always been considered as safe and healthy drinks, besides an important adjunct to the diet. Consumption of red wine is considered as the miracle saviour since phenolic compounds in the wine help to combat *E-mail of corresponding author : [email protected] MS Received on : 20th April, 2011 Accepted on : 1st Oct., 2011

heart diseases and other ailments (Muller, 1995). Phytoalexins like resveratrol have been found in grapes which possess cancer chemoperventive activity (Michael et al., 1993; Meshing et al., 1997; Joshi and Devi, 2009). The fruit is also dried for making chalori, chaat etc. has no commercial utility. Honey is known for its excellent effect on digestion and metabolism (Ioyrish, 1974). Light honey has been reported to make mead, as the dark honey is necessary to add enough acid and tannins to the must made from honey which contains neither of it (Filipello and Marsh, 1934). There is no report on the physico-chemical and sensory quality characteristics of the wine made from wild apricot must ameliorated with honey.

Abrol and Joshi

So, attempt has been made to utilize the fruit to make mead by ameliorating the must with honey to raise the TSS to 22, 24 and 26°B of wild apricot must which was compared with the sugar ameliorated must wine.

Material and methods Raw materials

The fruits of wild apricot were procured from Kinnaur Distt. of Himachal Pradesh. Wild apricot was converted into pulp. To prepare pulp, 10 per cent water was added before cooking the fruit. The cooked fruits were passed through a pulper to remove skin and stones. The pulp was filled-in plastic barrels and preserved with addition of potassium metabisulphite (KMS) @ 2000 ppm and was used for fermentation after removal of KMS by heating. Honey was purchased from the Department of Entomology of the Dr Y S Parmar University of Horticulture and Forestry, for the preparation of wild apricot mead. Sucrose, the common sugar was procured from the local market for the preparation of wild apricot wine. The pectin esterase enzyme used in the studies was manufactured by M/S Triton Chemicals, Mysore, India under the brand name “Pectinol”.

Preparation of wine Yeast culture: The yeast culture viz. Saccharomyces cerevisiae var. ellipsoideus, (UCD 595) used in the study was originally obtained from Department of Enology and Viticulture, California, Davis, USA. It was maintained on yeast malt extract agar (YMEA) medium and re-cultured after every three months or whenever needed from the stock yeast culture. Activation of yeast culture: Culture of Saccharomyces cerevisiae var. ellipsoideus, (UCD 595) was made for the preparation of wild apricot mead and wine. The wild apricot pulp was heated to boiling point followed by cooling. The pulp was diluted in 1:2 ratios (Joshi et al., 1990) which was inoculated with yeast from the slant. Preparation of must: For conducting the experiment, the pulp stored with 2000 ppm KMS was heated to make it free from SO2 and then, diluted in the ratio of 1:2 with water. To the diluted pulp, 0.1% diamonium hydrogen phosphate (DAHP) as nitrogen source and 0.5% pectinase enzyme for clarification were added. The TSS of diluted wild apricot was raised with either sugar or honey to 22°B, 24°B and 26°B as per the treatments. The respective must were inoculated with 5% of IJFFT 1(2) 2011 : 222

activated culture of Saccharomyces cerevisiae var. ellipsoideus. The fermentation of each treatment was carried out in 10 l capacity narrow mouth glass carboys, filled up to 75% of their capacity. Fermentation: Fermentation for all the treatments (22°B, 24°B and 26°B) for mead and wine was carried over at room temperature (22-25°C). When a stable TSS was reached, the fermentation was considered completed. Air locks were fitted in the mouth of glass carboys near the end of fermentation. Siphoning/racking: When fermentation was complete, siphoning/racking was done after 15 days and then, after one month. Physico-chemical analysis The weight of 10 fruits was taken with physical balance while diameter and length were measured with vernier callipers and expressed in millimeters (mm). Colour of wild apricot pulp and mead was measured with Lovibond tintometer in terms of unit of red, yellow and blue as per the standard procedure described (Ranganna, 1986). Total soluble solids (TSS) were measured using an Erma hand refractometer (0 to 32oB) and the results were expressed as degree Brix (oB). The readings were corrected by incorporating the appropriate correction factor for temperature variation (AOAC, 1980). Titratable acidity was estimated by titrating a known aliquot of the sample against N/10 NaOH solution using phenolphthalein as an indicator. The titratable acidity was calculated and expressed as per cent malic acid (AOAC, 1980). The total phenols content in different wines were determined by Folin Ciocalteu calourimetric procedure given by Singleton and Rossi (1965). ELTOp-3030 pH meter was used to measure pH. The total and reducing sugars of fruit and mead were estimated by Lane and Eynon volumetric method (AOAC, 1980) by titrating the sample against Fehlings solutions. Ascorbic acid content was determined as per standard method (AOAC,1980) using 2, 6-dichlorophenol-indophenol dye. Volatile acidity of wild apricot mead was determined by the standard method (Amerine et al., 1980). Quantity of ethanol was estimated by spectrophotometric method (Caputi et al., 1968) whereas, fusel oil in wine was estimated by the method given by Guymon and Nakagiri (1952).

Sensory analysis For sensory analysis, chilled and coded samples were served to the judges who were asked for sensory evaluation,

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead

performed on a prescribed proforma as given by Amerine et al. (1980).

Statistical analysis Statistical analysis of the quantitative data of chemical parameters was done by Completely Randomized Design (CRD) Factorial (Cochran and Cox, 1963).

Results and discussion Physico-Chemical Characteristics of Wild Apricot Fruit/ pulp Physico-chemical characteristics of wild apricot fruit are shown in Table 1. The pulp content of fruit was 77%. The kernel of the fruits was sweet in taste. The reducing sugars and total sugars were found to be 4.83±0.22% and 6.16±0.03%, respectively. pH value of the pulp was estimated to be 3.57±0.01. Carotene content was recorded to be 2.50 ± 0.07 mg/100gm. Colour of the fresh fruit was ‘yellowish white with pink tinge’ whereas the tintometre reading of the pulp in terms tintometre colour units was 4, 9.3, 0 as Y (yellow), R (red), B (blue) units, respectively. The TSS of pulp was low whereas, the titratable acidity (as malic acid) was very high. Clearly, to make wine the total sugars content have to be raised to obtain ethyl alcohol content typical of table wine. Honey used for preparation of wild apricot mead had TSS of 78°B, whereas its titratable acidity was 0.26% as citric acid.

Fermentation behaviour of wild apricot musts The results (Fig. 1) depicted the fermentation behaviour of wild apricot must prepared at different initial TSS (22, 24 and 26°B) levels using two different sources of sugar (cane sugar and honey). The lowest TSS (8.4°B) of must in 22°B was attributed to the lower sugar content than that of 26°B wild apricot honey must (8.8°B) after 336 hrs of study. Irrespective of TSS levels wild apricot ameliorated must with honey had faster reduction in TSS than that of sugar used to ameliorate wild apricot must. It is attributed to easily availability of fermentable sugar to the yeast in wild apricot honey must. The higher decrease in TSS during initial fermentation is attributed to the higher fermentability of musts of different treatments (described earlier) because of more availability of sugar and less ethyl alcohol formation in the medium. With increase in time, however, the ethanol content increased exerting inhibitory effect on the fermentability (Mota et al., 1984; Nishino et al., 1985; Sharma and Joshi, 1996). So, the trend of ethanol increase or TSS fall during fermentation is as expected and discussed earlier for other wines also (Amerine et al., 1980; Joshi and Bhutani, 1990; Sharma and Joshi, 1996; Joshi et al., 1999).

Table 1 : Physico-chemical characteristics of fresh wild apricot fruit/ pulp Physico-chemical characteristics Fruit weight (g)

Mean ± SD (n=10) 12.42 ± 3.32

Fruit diameter (cm)

2.46 ± 0.38

Fruit volume (cm3)

10.55 ± 1.35

TSS (°B)

10.00 ± 0.24

Titratable acidity (%MA)

2.23 ± 0.16

Ascorbic acid(mg/100g)

37.30 ± 0.13

Reducing sugars (%)

4.83 ± 0.22

Total sugars (%)

6.16 ± 0.03

pH

3.57 ± 0.01

Carotenoids(mg/100g)

2.50 ± 0.07

Pulp content (%)

77.00 ± 0.2

SD: Standard deviation

Fig. 1. A comparison of fermentation behaviour of wild apricot musts of different treatments

A comparison of RF (rate of fermentation) revealed that the 26 °B wild apricot honey ameliorated must had the highest RF (1.21) while, 22°B wild apricot sugar must gave the least fermentation rate (0.90). In general, RF of honey based wine was higher than the sugar based wine. Fig. 2 shows the comparison between practical and theoretical yield of alcohol IJFFT 1(2) 2011 : 223

Abrol and Joshi

(%v/v) in wild apricot must of different treatments. Clearly, wild apricot honey ameliorated must had the lower practical yield of ethanol than that of wild apricot sugar must. Though, the practical yield of ethanol was lower both in sugar and honey ameliorated musts, than the theoretical yield. The difference was attributed to the utilization of sugar by the yeast for its metabolism and growth (Rebordinos et al., 2011)

Fig. 4. A comparison of titratable acidity(%MA) during fermentation of musts of different treatments

Fig. 2. A comparison between practical and theoretical yield of alcohol (%v/v) in wild apricot must of different treatments

Fig. 3. A comparison of efficiency of wild apricot musts of different treatments

In comparison to the efficiency of wild apricot musts of different treatments, (Fig. 3) 26ºB wild apricot sugar must showed the highest efficiency 22ºB wild apricot honey must the lowest. Irrespective of different initial TSS levels wild apricot sugar must had the higher efficiency than that of wild apricot honey musts. In general, the fermentation efficiency increased with increase in initial TSS.

IJFFT 1(2) 2011 : 224

The change in % titratable acidity (as malic acid) of different musts can be seen in Figure 4. It is clear that the acidity of wild honey must within the treatment was found more than the wild apricot sugar based wine. It is also clearly visualized that there was an increase in acidity at the peak of fermentation and then, a decrease in titratable acidity took place at the end of fermentation in all the treatments. The increase in acidity during peak of fermentation might have been caused by more CO2 evolution and excess retention of of CO2 resulting in the formation of carbonic ions in the must and ultimately increase in acidity, whereas at the end titratable acidity decreased in all the treatments because of less CO2 evolution that resulted in lower carbonic ions in the must and finally, low acidity. Besides production of some organic acid during fermentaion may have increased the acidity partly (Zoecklein et al., 1995). The highest titratable acidity (1.2%) was observed in 26ºB honey must after 144 hrs of fermentation. Physico-chemical characteristics of wild apricot mead Table 2 summarizes the effect on total soluble solids, total sugars and reducing sugars of wines of different treatments. The highest TSS was observed in 26°B wild apricot sugar based wine and mead (8.77, 8.60°B) and the lowest in 22°B wild apricot sugar and mead (8.33, 8.27°B). There were significant differences both between the treatments and subtreatments. The highest TSS (8.77°B) was observed in 26°B

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead

wild apricot sugar based wine followed by 26°B wild apricot mead (8.60°B) and lowest (8.27°B) in 22°B wild apricot mead. The wide variation in TSS of different treatments is apparently related to the difference in sources of sugars, initial TSS and as well as the fermentation behaviour of the different must. The must with honey as fermentable sugar reduced the highest TSS in all the treatments but produced less ethanol content. It could be due to the fact that honey had more reducing sugars content that are easily available to the yeast for fermentation thus, increasing the fermentability. The wide variation in TSS is also apparently related to the difference in fermentability behaviour of the musts. The results (Table 2) show that there was significant difference in treatments and sub-treatments for reducing sugars content. The reducing sugars of different treatments ranged between 0.30 to 0.48%. Amongst the treatments, 26°B wild apricot mead had the highest reducing sugar (0.48%) followed by 24°B wild apricot mead (0.44%) and (0.30%) lowest in 22°B wild apricot sugar based wine. With the increase in initial sugar level, reducing sugars content were increased. Out of two sources of sugar used, honey based wine was found to have more (0.44%) reducing sugars than sugar based wine (0.35%). There were also significant differences amongst treatments and sub-treatments for total sugars. In different wines, total sugars ranged between 1.33 to 1.11%. The highest total sugars (1.29%) was recorded in 26°B wild apricot sugar based wine and lowest (1.18%) in 22°B wild apricot mead. Sugar based wine had highest total sugars (1.29%) while honey based wine had the lowest (1.18%) total sugars. Clearly, both the reducing and total sugars left in the wild apricot mead and wine were very less i.e. residual sugar. The behaviour is obvious as after fermentation reduced quantity of sugars are left in the wine as observed earlier in plum and apricot wine also (Joshi and Sharma, 1993; Joshi et al., 1990). Perusal of the result (Table 3) revealed that the titratable acidity of different wines was significantly different from each other and ranged between 0.74 to 0.86%. The highest titratable acidity (0.86%) was recorded in 26°B wild apricot mead, while the lowest titratable acidity (0.74) was recorded in wild apricot sugar based wine made from 22ºB must. Overall, wild apricot sugar based wine had the lower acidity (0.80%) than the wild

apricot mead (0.83%) in all the treatments. Table 3 further revealed that the pH of different treatments and sub-treatments were significantly different from each other and ranged between 3.01 to 3.19. The titratable acidity and pH are known to be inversely related with each others as recorded here. Similar findings were obtained earlier also (Joshi and Sharma, 1994) in apricot wine. It is also discernible from the data (Table 3) that the volatile acidity ranged between 0.023 to 0.030 % (% acetic acid) among the different treatments. Similarly, significantly different volatile acidity was recorded in various sub-treatments in wild apricot wine. Mead recorded the lower volatile acidity than that of wild apricot sugar wine, which was less than that of earlier finding (Joshi et al., 1990). However, it was found within the range, which is desirable also. A sound wine, generally has volatile acidity less than 0.04% as acetic acid (Amerine et al., 1980). High volatile acidity indicates acidification of wine. The highest acidity was recorded in 26ºB honey wine. These findings are correlated with those obtained earlier (Joshi and Sharma, 1993) in apricot wine. Table 4 summarizes the results of alcohol, higher alcohols and total phenols in wines . There were significant differences among the treatments for ethanol levels. The highest ethanol content recorded in 26°B wild apricot sugar based wine (12.17%) and lowest in 22°B wild apricot sugar based wine (10.23%), whereas among the sub-treatments wild apricot sugar based wine had the highest (11.37%) ethanol content and wild apricot mead was the lowest (9.18%). Table wine contains alcohol ranging from 7 to 14 % (Amerine et al., 1980) and from this point of view based on ethanol content, all the wines fall with in the category of table wines. The amounts of higher alcohols in different wines (Table 4) were significantly different from each other. The highest content of higher alcohols was recorded in 26°B wild apricot mead (153.0 mg/l), whereas, the lowest was observed in 22°B wild apricot sugar based wine (113.0 mg/l). With the increase in initial TSS level from 22 to 26°B, increase in higher alcohols content was observed and it ranged between 117.0 to 155.5 mg/l. In wild apricot mead, the higher alcohols ranged between 121.0 to 158.0 mg/l, whereas, in wild apricot sugar based wine it ranged between 113.0 to 153.0 mg/l.

IJFFT 1(2) 2011 : 225

Abrol and Joshi

Table 2. Effect of initial TSS levels on TSS, reducing sugars and total sugars of wild apricot mead and wine TSS (°B)

Reducing Sugars

Total sugars (%)

Treatment

Wine

mead

Mean

Wine

mead

Mean

Wine

mead

Mean

Initial TSS 22°B (T1)

8.33

8.27

8.30

0.30

0.41

0.36

1.24

1.11

1.18

Initial TSS 24°B (T2)

8.57

8.37

8.47

0.35

0.44

0.40

1.28

1.18

1.23

Initial TSS 26°B (T3)

8.77

8.60

8.68

0.40

0.48

0.44

1.33

1.25

1.29

Mean

8.56

8.41

0.35

0.44

1.28

1.18

CD(p>0.05) TSS(B) Sub-treatment (S) BXS

0.08 0.07 0.12

CD(p>0.05) TSS(B) Sub-treatment (S) BXS

CD(p>0.05) TSS(B) Sub-treatment (S) BXS

0.01 0.01 0.02

0.01 0.01 0.02

Table 3. Effect of different initial TSS levels on titratable acidity, pH and volatile acidity of wild apricot sugar based wine and mead Treatment

Titratable acidity (as %malic acid)

pH

Volatile Acidity (as % acetic acid)

Sugar

Honey

Mean

Sugar

Honey

Mean

Sugar

Honey

Mean

Initial TSS 22°B (T1)

0.74

0.83

0.79

3.16

3.01

3.09

0.027

0.023

0.025

Initial TSS 24°B (T2)

0.80

0.81

0.81

3.17

3.09

3.13

0.028

0.024

0.026

Initial TSS 26°B (T3)

0.84

0.86

0.85

3.19

3.11

3.15

0.030

0.025

0.027

Mean

0.80

0.83

3.17

3.07

0.028

0.024

CD(p>0.05)

CD(p>0.05)

CD(p>0.05)

TSS(B)

0.01

TSS(B)

Sub-treatment (S)

0.01

Subtreatment (S)

0.01

Sub-treatment (S)

BXS

0.01

BXS

0.02

BXS

IJFFT 1(2) 2011 : 226

0.01

TSS(B)

NS 0.001 NS

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead

Table 4. Effect of different initial TSS levels on ethanol, higher alcohols and total phenols of wild apricot sugar based wine and mead Ethanol (%v/v)

Higher alcohols (mg/l)

Total phenols (mg/l)

Treatment

Sugar

Honey

Mean

Sugar

Honey

Mean

Sugar

Honey

Mean

Initial TSS 22°B (T1)

10.23

9.18

9.71

113.0

121.0

117.0

245.5

238.0

241.8

Initial TSS 24°B (T2)

11.70

9.86

10.78

131.5

136.5

134.0

264.0

255.0

259.5

Initial TSS 26°B (T3)

12.17

10.38

11.28

153.0

158.0

155.5

279.0

270.0

274.5

Mean

11.37

9.81

132.4

138.5

252.8

254.3

CD(p>0.05)

CD(p>0.05)

CD(p>0.05)

TSS(B)

0.06

TSS (B)

1.4

TSS (B)

4.3

Sub-treatment (S)

0.05

Sub- treatment (S)

1.1

Sub- treatment (S)

3.0

BXS

0.09

BXS

1.9

BXS

2.5

The total phenols (Table 4) of wines of different treatments were recorded significantly different and ranged between 241.8 mg/l to 274.5 mg/l. The highest (279.0 mg/l) total phenols were found in 26°B wild apricot sugar based wine and lowest in 22°B wild apricot mead (238.0 mg/l). Higher alcohols and total phenols in (honey wine) mead were found lesser than that of sugar based wine. At low concentration, the higher alcohols may play an important role in sensory quality (Amerine et al., 1980). Lesser quantity of higher alcohols denotes the non-oxidative conditions of all the wines (Guymon et al., 1961) which indirectly reflects the proper conditions of wine preparation adopted in this study. In earlier studies (Joshi et al.1990), the total phenols were recorded 240 mg/l which is lower than that of sugar wine but higher than of wild apricot mead. The differences can be attributed to the variation in the phenol content of the wild apricot fruit used for wine making.

Sensory quality

evaluation of wild apricot sugar and mead for various treatments (Table 5). In comparison of sensory scores of wild apricot wines of different treatments, wine of 26ºB scored the highest (1.71) for overall quality. The wine from initial TSS 24ºB was also liked by the judges and awarded with a good sensory score (1.67). Out of three initial TSS levels, 26ºB scored better because of balanced astringency, alcohol, sugar and acid. However, out of two sources of sugar used for wine preparation, honey based wine (mead) was better in overall acceptability, mead was found dry and higher in acidity which was preferred in wines having 26ºB initial TSS. Conclusion Rate of fermentation (RF) of honey based must was found higher than sugar based must. With the increase in initial TSS level of musts, TSS, reducing sugars, total sugars, titratable acidity, volatile acidity, ethanol content, higher alcohols and total phenols increased, whereas, pH decreased. It can be concluded that wild apricot wine of 26ºB was the best.

Composite scoring test was carried out for the sensory

IJFFT 1(2) 2011 : 227

IJFFT 1(2) 2011 : 228 1.58 1.75

24°B

26°B

TSS level Source of sugar

0.055 NS

1.71

Honey

CD(0.05)

1.67

Sugar

Source of sugar

1.74

Colour

22°B

TSS level

Treatments

0.061 0.049

1.72

1.66

1.69

1.63

1.75

Appearance

0.069 0.056

3.41

3.54

3.53

3.43

3.46

Aroma

NS NS

1.59

1.60

1.57

1.61

1.61

Volatile acidity

0.038 0.031

1.64

1.47

1.54

1.60

1.53

Total acidity

0.024 0.019

0.69

0.60

0.59

0.71

0.62

Sweetness

0.047 NS

0.82

0.79

0.85

0.79

0.77

Body

Table 5. A comparison of sensory scores of wild apricot wines of different treatments

0.047 0.038

1.75

1.70

1.77

1.68

1.72

Flavour

0.043 NS

0.81

0.79

0.85

0.77

0.78

Bitterness

0.045 0.037

0.75

0.64

0.73

0.70

0.66

Astringency

0.031 0.025

1.68

1.63

1.71

1.67

1.59

Over all impression

16.57

16.09

16.58

16.17

16.23

Total score

Abrol and Joshi

Effect of different initial TSS level on physico-chemical and sensory quality of wild apricot mead

References A.O.A.C. 1980. Assocation of Official Analytical Chemists. Official Methods of Analysis. Hortwitz, W. (ed.), 13th ed. Washington, D.C. p.1015. Amerine MA, Berg HW, Kunkee RE, Qugh CS, Singleton VL and Webb AD. 1980. The Technology of Wine Making, 4th edn. AVI Publishing Co., Inc. Westport, CT. Caputi A, Ueda M and Brown J. 1968. Spectrophtometric determintaion of ethanol in wine. Am. J. Enol. Vitic, 19: 160-165. Cockrane WG and Cox GM. 1963. Experimental Designs, 14th edn, 613 p., Asia Publishing House, Bombay. Filipello F and Marsh GL. 1934. Honey wine. J. Honey Wine, 14: 42. Guymon JF and Nakagiri J. 1952. Methods for the determination of fusel oil. Proc. Am. Soc. Enol. 3: 117-134. Ioyrish N. 1974. Bees and people. Mir Puplisher, Moscow. Joshi VK and Bhutani VP. 1990. Evaluation of plum cultivars for wine preparation. In: International Horticulture Cong. held in Italy in Aug. 1990. Abst. 3336. Joshi VK and Sharma SK. 1993. Effect of method of must preparation and initial sugar levels on the quality of Apricot Wine. J. Sci. Indust. Res, 39: 255-257. Joshi VK, Bhutani VP, Lal BB and Sharma R. 1990. A method for the preparation of wine from the wild apricot. Indian Food Packer, 44: 50-55. Joshi VK, Sandhu DK and Thakur NS. 1999. Fruit based alcoholic beverages. In: Biotechnology Food Fermentation, Joshi VK and Pandey Ashok eds. Asiatech Pub. House, New Delhi, pp.647744. Joshi VK and Devi MP. 2009. Resveratrol: importance, role, contents in wine and factors influencing its production. Proceedings of the National Academy of Sciences India, 79: 212-226. Meshiang J, Lininey G, George O, Thomas CF, Christopher WW, Harry HS, Richard CM and John MP. 1997. Cancer

chemopreventive activity of reserveratrol a natural product derived from grapes. Science, 275: 217-220. Michael GJ, Julie EB, Samuel ZG, Bernard R, Martin V, Walter W and Charles HH. 1993. Moderate alcohol intake, increased levels of high density lipoprotein and its subfractions and decreased risk of myocardial infraction. The New England Journal Medicine, 329: 1829-1834. Mota M, Strehaiano P and Goma G. 1984. Studies on conjugate effects of substrate (glucose) and product (ethanol) on cell growth kinetics during fermentation by different yeast strains. J. Inst. Brewing, 90: 359-362. Muller CJ. 1995. Wine and Health - It is more than alcohol. In: Wine analysis and production, (Zoecklein Bruce W ed.) Chapman & Hall, New York, pp.15-29. Nishino H, Miyazaki S and Tohji K. 1985. Effect of osmotic pressure on growth and rate and fermentation activity of wine yeasts. Am. J. Enol. Vitic., 36: 170-174. Parmar C and Kaushal MK. 1982. Prunus armenica. In : Wild Fruits. Kalyani Publishers, New Delhi, pp. 66–69. Petric WMF. 1923. Social life in Ancient Egypt. Methuen, London. Ranganna S. 1986. Handbook of analysis and quality control for fruit and vegetable products. Tata McGraw Hill Publishing Company Ltd., New Delhi, p.1112. Rebordinos L, Infante JJ, Rodriguez ME, Vallejo I and Cantoral JM. 2011. Wine yeast growth and factors affecting. In: Handbook of Enology: Principles, Practices and Recent Innovations, Joshi VK (eds.) Asiatech Publishers, Inc., New Delhi, pp 406-435. Sharma SK and Joshi VK. 1996. Optimization of some parameters of Secondary Fermentation for Production of Sparkling Wine. Indian J. Exper. Biol., 34: 235. Singleton VL and Rossi JA Jr. 1965. Colorimetry of total phenolics with phosphomolybadic phosphotungstic acid reagents. Am. J. Enol. Vitic., 16: 144-158. Zoecklein BW, Fugelsang KC, Gump BH and Nury FS. 1995. Wine analysis and production. Chapman Hall, New York.

IJFFT 1(2) 2011 : 229

IJFFT No. 1,Technol. June 2011, pp. 2011 251-256 © New Delhi Publishers, India Int. J. Vol. Fd. 1, Ferm. 1(2) : 231-236

Research Paper

Olive press cake - proximate composition and quality of extracted oil Rakesh Sharma*, PC Sharma and VK Joshi Department of Food Science and Technology, Dr Y S Parmar University of Horticulture and Forestry, Solan, India.

Abstract: Olive (Olea europaea L) is a drupe and made up of about 74% juice/ pulp and 26% solids. The pulp contains up to 75% oil, however, kernel contains 12-18% oil on dry weight basis. Olive oil is generally extracted from the fruits by grinding, crushing, centrifugation and pressing. In the present study, the oil was extracted by use of enzymatic treatments. The press cake left after oil extraction (about 32%), which still contains 9.70% of oil, was evaluated for proximate composition and further utilization. It was found in the preliminary experiments that extraction of oil by enzymatic treatments not only enhanced the oil yield but also reduced the amount of press cake as well as loss of oil with the press cake. Unlike, other oil cakes, olive cake had a low total protein (6.87-7.05%) and high crude fibre (40.86- 43.00 %) and fat (7.13-9.69 %) contents. Based on proximate composition of olive press cake, it could be utilized as a supplementary feed for animals. The oils extracted from olive cakes by solvent extraction method had their quality characteristics not in agreement with either the PFA standards or the standards of Codex Alimentarius Commission for edible purposes. However, these oils can be used for edible purposes after refining or as such can be utilized for various industrial purposes.

Keywords: Olive, Olive oil, Enzymatic extraction, Press cake, Oil quality

Olive oil is known for its anti-ulcer, rapid digestibility and plasma cholesterol lowering properties (Christakis et al., 1980). It is generally extracted from the fruits by different methods such as grinding, crushing, centrifugation and pressing (Kiritsakis and Markakis 1987). After the oil has been extracted from the olives, two main by-products viz. cake and the vegetable water are obtained. Olive cake consists of olive kernel crushed into fragments, skin, crushed pulp, about 25 per cent water and the remaining oil. On the other hand, vegetable water has the appearance of a brown watery residue, which has a bitter taste; relatively high organic matter content and considered as a source of pollution (Codounis, 1973; Cucurachi, 1975). Carola (1975) reported that up to 40 per cent cake is obtained after oil extraction, which still contains 4-5 per cent oil when

*E-mail of corresponding author : [email protected] MS Received on : 13th April, 2011 Accepted on : 10th Sept., 2011

well exhausted and 8-12 per cent when coming from traditional presses. Further, use of different enzymes has been reported by various researchers for enhancing olive oil recovery (Ranalli and De Mattia, 1997; Ranalli et al., 1998; Sharma et al., 2005). It has been reported that enzymatic treatments of olive paste during malaxation not only enhanced the oil recovery, but also reduced amount of press cake as well as oil in press cake, significantly (Sharma et al., 2005, Sharma et al., 2007). Fernandez Diez (1971) reported that oil could be extracted from the press cake using solvents for various industrial purposes. However, Raina (1995) suggested that olive oil extracted from press cake can be used for edible purposes after refining or as such can be used for industrial purposes like soap manufacture, in

Sharma et al.

textile, toilet, cosmetics and pharmaceuticals preparations. Olive cake as such is not very palatable and is not widely employed for feeding animals. However, some workers have reported its use up to 20 per cent for feeding dairy cattle (Carola, 1975; Belibasakis, 1982; Baskou, 2002). Hence, the present study was conducted to study the proximate composition of olive press cake and quality characteristics of the oil extracted from this cake obtained after enzymatic extraction of virgin olive oil.

Material and Methods Raw material: Olive fruits (Olea europaea L.) were collected from Olive Development Station, Zardari, Solan, (HP). The enzyme pectinase was procured from M/s Tritone Chemicals,

Mysore, India whereas cellulase and pectinase CCM enzymes were procured from M/s Biocon Ltd., Banglore, India. Extraction of oil from olive fruits Virgin oil was extracted by the application of enzymatic treatments standardized earlier (Sharma et al., 2005). Various enzyme and enzyme combinations viz. Pectinase (P) @ 0.10%, cellulase (C) @ 0.08%, P+C (1:1) @0.05% and pectinase CCM @ 0.08% were used for enhancing oil yield. After oil extraction, olive press cake was collected, packed in polyethylene bags and stored at refrigerated conditions for further studies. The schematic flow sheet for extraction of olive oil is given in Fig 1.

OLIVE FRUIT

SORTING & WASHING

CRUSHING/ MILLING

Enzyme Treatment (P+C @0.05%)

MALAXATION (To make paste)

HYDRAULIC PRESSING

Press cake

Further utilized

OIL - WATER MIXTURE

CENTRIFUGATION

Vegetable water

Discarded

OILY MUST

FILTERATION/ DECANTATION

PURE OLIVE OIL

Fig 1. Schematic flow sheet for extraction of olive oil and utilization of press cake (P = Pectinase, C = Cellulase) IJFFT 1(2) 2011 : 232

Olive press cake - proximate composition and quality of extracted oil

Extraction of oil from olive press cake Oil from olive press cake was extracted by using solvent extraction method (Ranganna 1997). The oils thus, obtained were analyzed for various quality characteristics and compared with the standards of Prevention of Food Adulteration Act and Codex Alimentarius Commission. Analyses The physical characteristics of fruit such as average fruit weight, size of fruit and stone (length and breadth), pulp recovery, pulp/stone ratio and fruit volume were estimated according to standard methods. The oil content in fruit and cake was estimated by using solvent extraction method (Ranganna 1997). Total solids, moisture and ash contents in fruit and cake were determined following the methods of AOAC (1995). Kjeldahl Nitrogen method was used to determine the total protein content of the samples (AOAC, 1984). Crude fibre which is the organic residue was estimated by method given by Sankaram (1966). Total phenols were determined by colorimetric method (Thimmaiah 1999). Quality characteristics like free fatty acids content was estimated by titrating known volume of sample against 0.1N KOH using phenolphthalein as an indicator (Thimmaiah, 1999), saponification value (SV) was estimated by titrating completely saponified sample against 0.5N HCl using phenolphthalein as indicator, whereas iodine value (IV) and peroxide value (PV) were estimated using standard methods (AOAC 1995). Fatty acid profile (consisting of oleic, palmitic, palmitoleic, linoleic and stearic acid) of different oils was estimated according to standard procedure of ISI (1984). The quality of oil was compared with PFA standards (Anon. 1996) and the standards of Codex Alimentarius Commission (CAC, 1993). Triplicate determinations were made for each physicochemical and sensory parameters. Complete Randomized Design (CRD) was used to analyse the data of quantitative estimation of various physico-chemical characteristics, whereas RBD was used for analysis of sensory data as described by Cochran and Cox (1967). Results and Discussion Physico-chemical composition of fruits The olive fruits used in the present study have about 74% juice (66 % water, 32% oil) and 26% solids. Pulp (74.10 % of

fruit) accounts for pulp:stone ratio of 2.86. It contains up to 75% oil, however, kernel contains 12-18% oil on dry weight basis. Various physico-chemical characteristics (Table-1) of fruit in this study are in corroboration with reported work (Singh et al., 1986, Katiyar, 1989; Raina, 1995). Fruits under the study gave 32.95 % (dry wt. basis) oil yield, whereas oil yield of olive fruits is reported to vary from 16.20 to 43.40 % on dry weight basis (Kumar et al., 1986, Singh et al., 1986, Sharma, 2006). Thus, the olive fruits used for these studies had good size, pulp: stone ratio and were well suitable for oil extraction due to their high oil contents. Table 1: Physico-chemical characteristics of olive fruits Parameters

Mean+ SD

Weight of fruit, g

2.60+ 0.08

Fruit length, mm

21.16+ 0.13

Fruit breadth, mm

15.36+0.29

Pulp, %

74.10+1.63

Stone, %

25.90+0.82

Pulp: stone ratio

2.86

Moisture, %

66.50+1.63

Ash, %

2.46+0.02

Total protein, %

2.73+0.08

Oil yield on whole fruit basis, %

11.95+1.46 (32.95+1.46)*

SD= Standard deviation in parenthesis are on dry weight basis

*Figures

Effect of application of enzymes for extraction of oil Data presented in Table 2 showed that the enzymatic treatment of olive paste prior to oil extraction brought a significant improvement in oil yield over the control (without enzymatic treatment). The enzyme combination of pectinase and cellulase (1:1) @ 0.05% resulted in maximum oil yield (9.9 %), thereby accounting for 11 % increase in oil yield over the control. Several workers have also obtained higher oil yields by using enzymes (Dominguez et al., 1994, Ranalli et al., 2001, Sharma et al., 2007). Further, the amount of cake and oil in cake obtained with various enzymatic treatments ranged from 29.7 to 32.0 and 7.1 to 9.7 %, respectively, with minimum values obtained in treatment having pectinase + cellulase @ 0.05 % and maximum was in the control (untreated). The rheological modifications of enzymatically treated olive paste might have favoured the release of liquids thereby reducing

IJFFT 1(2) 2011 : 233

Sharma et al.

the amount of residual oil in press cake. Similar findings have also been reported by Dominguez et al., (1994) and Ranalli et al., (2001). Table 2. Effect of enzymatic treatments on oil yield and yield of press cake after oil extraction from olive fruits Enzyme

Oil % Increase Amoyield in oil yield unt of (%) over the cake control left (%)

Oil in cake (%)

Pectinase (0.10%)

9.3

4.3

31.1

8.7

Cellulase (0.08%)

9.4

6.2

30.4

8.4

Pectinase + Cellulase (0.05%)

9.9

11.0

29.7

7.1

Pectinase CCM (0.08%)

9.4

5.8

30.9

8.3

Control (untreated)

8.9

-

32.0

9.7

Proximate analysis of press cake The total solids of olive cakes ranged from 76.65 to 79.14 per cent, with the maximum value obtained in treatment using pectinase + cellulase @ 0.05 per cent and minimum in control (Table 3). The higher values of total solids observed in cakes of enzymatic treatments compared to that of untreated control sample, might be due to the better rupture of cell wall to release the cellular constituents. All the enzymatic treatments were, however, found non-significant with each other. Further, the crude fats of press cakes obtained by different enzymatic treatments ranged from 7.13 (Pectinase + Cellulase) to 9.69 (Control) per cent. The better efficiency of enzymes for

extraction of oil resulted in comparatively lesser crude fats left in the cakes obtained from enzymatic treatments of olive paste. Similar results were also reported earlier (Ranalli and De Mattia 1997; Ranalli et al., 2001). The total protein and total ash contents of press cakes although varied from 6.87 to 7.05 per cent and 8.12 to 8.50 per cent, respectively, were at par with various enzymatic treatments (Table 2). Further, the crude fibres of olive cakes obtained by using pre-standardized enzymatic treatments ranged from 40.86 to 43.00 per cent with maximum in Pectinase + Cellulase (0.05%) and minimum in control. Among different enzymatic treatments, Pectinase (0.10%), Cellulase (0.08%) and Pectinase CCM (0.08%) were found non-significant with each other and differed significantly with rest of the treatments. The higher values of crude fibres obtained in Pectinase + Cellulase (0.05%) might be attributed to the respective lower moisture content and higher total solids as compared to other enzymatic treatments. Enzymatic treatments resulting in more effective separation of the liquid phases (oil and water) from the solid phase could be the reason for higher values of solids and crude fibres obtained in these treatments as has also been reported (Ranalli et al., 2001). Overall, it was found that the cake obtained after oil extraction from olive fruits was observed to be good source of crude fibres and ash, besides presence of some amounts of proteins and fats, thus can be used as supplementary animal feed. Bloemeyer (1977) had also obtained weight gains of 110 to 125g/ day with grazing sheep when fed with 500 g of hay and the concentrate (containing up to 40% olive cake with urea molasses) according to live weight (20-30 g/kg live weight).

Table 3: Proximate analysis of olive cake left after oil extraction Parameters

Control

P (0.10%)

C (0.08%)

P+C (0.05%)

PCCM (0.08%)

CD0.05

76.65

77.42

78.92

79.14

77.48

2.03

Crude fat (%)

9.69

8.74

8.39

7.13

8.34

1.15

Total protein (%)

6.87

6.89

6.92

7.05

6.90

NS

40.86

41.25

42.15

43.00

42.08

0.83

8.12

8.25

8.32

8.50

8.33

NS

Total solids (%)

Crude fiber (%) Total ash (%)

P= Pectinase, C= Cellulase, P + C= Pectinase + Cellulase, P

IJFFT 1(2) 2011 : 234

CCM=

Pectinase CCM

Olive press cake - proximate composition and quality of extracted oil

Quality characteristics of oil extracted from olive press cake Perusal of data (Table 4) showed that the free fatty acid contents of oil extracted from olive cakes obtained in different enzymatic treatments ranged from 4.90 (Pectinase + Cellulase @ 0.05%) to 5.35 (Pectinase CCM @ 0.08%) per cent, while the peroxide value varied between 21.75 (Pectinase + Cellulase @ 0.05%) and 26.76 (Control) meq/kg). The free fatty acids and peroxide values of all the oils were above the maximum permissible limits prescribed by PFA (Anon. 1996) and Codex Alimentarius Commission (CAC, 1993) for olive oil. Further, the iodine values and saponification values of various oils ranged from 70.90 to 71.20 and 199.50 to 205.10, respectively, although the differences among various enzymatic treatments were found to be non-significant. The unsaponifiable matter ranged from 1.56 to 1.93 per cent with maximum in control and minimum in the treatment using pectinase + cellulase (0.05 %). All the oils under the study exceeded the maximum

prescribed limits laid down by PFA and the Codex Alimentarius Commission for saponification value and unsaponifiable matter of olive oil, whereas the iodine value of these oils was below the standard limits (79-90, PFA and 75-94, Commission of Codex Alimentarius). Similar results were also reported by various workers (Raina, 1995, Ranalli and De Mattia, 1997, Ranalli et al., 2001). Therefore, it was found that all the oils extracted from olive cakes by solvent extraction method did not meet their quality characteristics as specified by PFA (Anon, 1996) and Codex Alimentarius Commission (CAC, 1993). Thus, conclusively it emerges out that based on proximate analysis of olive press cake obtained after enzymatic extraction of olive oil, it has the potential to be used as a supplementary feed and similar uses. Further, the oils extracted from these cakes could not be recommended for edible purposes, however, can be used for edible purposes only after refining or as such can be utilized for various industrial purposes like soaps, textiles, toilet, cosmetics preparations and other industrial purposes.

Table 4: Quality analysis of oil extracted from olive cake Parameters

Control (Untreated)

P (0.10%)

C (0.08%)

P+C (0.05%)

PCCM (0.08%)

5.12

4.92

4.99

4.90

5.35

0.38

Peroxide value (meq/Kg)

26.76

26.01

25.13

21.75

22.64

1.25

Iodine value

71.20

71.00

70.96

70.90

71.12

NS

200.80

200.35

199.50

203.00

205.10

NS

1.93

1.81

1.88

1.56

1.71

0.24

Free fatty acids (%)

Saponification value Unsaponifiable matter (%)

P= Pectinase, C= Cellulase, P + C= Pectinase + Cellulase, P

CCM

CD0.05

= Pectinase CCM

Parameters

PFA Standards

Codex Alimentarius Commission

Saponification value Iodine value Unsaponifiable matter Free fatty acids Peroxide value

185-196 79-90 Not more than 1.0% Not more than 3.3 % -

184-196 75-94 Not more than 1.5% Not more than 20 meq O2 per Kg

IJFFT 1(2) 2011 : 235

Sharma et al.

References Anonymous 1996. The Prevention of Food Adulteration Act, 1954 and PFA rules, 1955 (Act No. 37 of 1954, amended up to 31/03/ 1996). All India Food Preservation Association, New Delhi, India, p 260. AOAC 1984. Official Methods of Analysis of the Association of Official Analytical Chemists, 14th ed. Association of Official Analytical Chemists, Washington, DC, USA. AOAC 1995. Official methods of analysis. 16th edn. Association of Official Analytical Chemists, Washington, DC. Belibasakis N G 1982. The chemical composition of olive cake. Greek Veterinary 2: 106-114.

Katiyar S K, Kumar N and Bhatia A K 1989. Chemical evaluation of olive fruits of nine cultivars of Himachal Pradesh. J Food Sci Technol 26: 225-227. Kiritsakis A, Markakis P 1987. Olive oil- a review. Adv Food Res 31: 453-482. Kumar S, Goswami A K and Sharma T R 1986. Evaluation of oil from olives grown in Himachal Pradesh. J Food Sci Technol 23(4): 239-240 Raina B L 1995 Olive. In: Handbook of Fruit Science and Technology - Production, Composition, Storage and Processing. Salunkhe D K and Kadam S S (eds). Marcel Dekker, Inc, New York. pp 465473.

Bloemeyer A 1977. The feasibility of using by-products of olive for feeding lambs in Tunisia. Animal Res. and Dev 5: 84.

Ranalli A, De Mattia G 1997. Characterization of olive oil produced with a new enzyme processing aid. J Amer Oil Chem Soc 74: 1105-1113.

Boskou D 2002. Olive oil. In: Vegetable Oils in Food Technology, Composition, Properties and Uses. Gunston F D (ed). Blackwell Publishing Ltd, UK.

Ranalli A, De Mattia G, Ferrante M L 1998. The characteristics of percolation olive oils produced with a new processing enzyme aid. Int J Food Sci Technol 33: 247-258.

CAC 1993. Revised norm for olive oil, Cl 1993/15-FO. Codex Alimentarius Commission, WHO/FAO.

Ranalli A, Malfatti A, Cabras P 2001. Composition and quality of pressed virgin olive oil extracted with a new processing aid. J Food Sci 66: 592-603.

Carola C 1975. Products. In: Olive Oil Technology. Saurez J M M (ed). FAO, Rome. Christakis G, Fordyce MK, Kurtz CS 1980. The biological aspects of olive oil. Proc, 3rd Int Cong on the Biological Value of Olive Oil, Chanea, Greece, p. 85. Cochran W G, Cox C M 1967. Experimental designs. John Wiley and Sons, Inc, New York. Codounis M 1973. Report on the possibilities of utilizing residues of the Greek agricultural industries (Plant production) as feeding stuffs. Ministry of National Economy, Athens.

Ranganna S 1997. Handbook of analysis and quality control for fruits and vegetable products, 2nd Edn. Tata McGraw Hill Pub Co Ltd, New Delhi, India, p 1112. Sankaram A 1966. A Laboratory Manual for Agricultural Chemistry. Asia Publishing House, Madras, India. Sharma R 2006. Evaluation of olive (Olea europaea L.) cultivars grown in Himachal Pradesh for oil extraction purposes. Himachal J Agri Res, 32: 30-93.

Cucurachi A 1975. Final Operation. In: Olive Oil Technology. Suarez J M M (ed). FAO, Rome.

Sharma R, Kaushal B B and Sharma P C 2007. Development of cost effective commercial method for enhancing yield and quality of olive oil. J Food Sci Technol., 44: 133-137..

Dominguez H, Nunez M J, Lema J M 1994. Enzymatic pretreatment to enhance oil extraction from fruits and oilseeds- a review. Food Chem 49: 271-286.

Sharma R, Kaushal B B L and Sharma P C 2005. Refinement of oil extraction technology for maximizing yield and quality of olive oil. Indian Food Packer, 59: 179-188.

Fernandez Diez M J 1971. The olive. In: The Biochemistry of Fruits and their products. Vol. 2. Hulme AC (ed.) 255-280.

Singh R P, Rana H S and Chadha T R 1986 Studies on the physicochemical characteristics of some olive (Olea europaea L.) cultivars. In: Advances in Research on Temperate Fruits. Chadha T R, Bhutani V P and Kaul J L (eds). pp 55-59.

ISI 1984. Handbook of food analysis, SP: 18 (Part XIII). Indian Standards Institute, New Delhi, p 98-102.

Thimmaiah S K 1999. Standard methods of biochemical analysis. Kalyani Publishers, New Delhi, pp. 287-288.

IJFFT 1(2) 2011 : 236

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 237-246

Research Paper

Development of wild pomegranate drink and its evaluation during storage NS Thakur*, Girish S Dhaygude and VK Joshi Department of Food Science and Techhnology, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, HP, India.

Abstract : Studies were undertaken for the preparation of drink from wild pomegranate and its quality evaluation during storage. A product with 14% juice and 150B TSS was found to be the best on the basis of some physico-chemical characteristics like colour , titratable acidity , ascorbic acid and sensory quality of drink. Drink could safely be stored for a period of six months under both the ambient and refrigerated conditions without much changes in various quality characteristics. However the changes in the quality characteristics of the drink were slower in refrigerated storage conditions as compared to ambient conditions. Both the packaging materials viz., PET and glass bottles were found suitable, with comparatively less changes occurring in glass bottle under refrigerated conditions. Keywords: Wild pomegranate, Punica granatum, Drink, Polyethylene terephthalate

Wild pomegranate (Punica granatum L.) is a shrub of family punicaceae and is one of the most important wild fruit, which resembles with cultivated pomegranate for various morphological characters (Sharma and Sharma, 1990). Wild pomegranate is widely distributed in drier and sub-marginal land of mid-hill regions of outer Himalaya, where slightly hot climate characterized by dry summer and fairly pronounced winter prevails. In India, it is found in vast tract of the hill slopes of Himachal Pradesh, Jammu and Kashmir and Uttrakhand at an altitude of 900 to 1800 m above mean sea level. In Himachal Pradesh, it is distributed in some pockets of Solan, Sirmour, Mandi, Shimla, Kullu and Chamba districts (Bhrot, 1998). The arils of wild pomegranate are a rich source of organic acids apart from having appreciable amount of sugars, anthocyanins, phenols, ascorbic acid etc. It also contains good amount of minerals like phosphorus, calcium, potassium and *E-mail of corresponding author : [email protected] MS Received on : 30th May 2011 Accepted on : 2nd Sept. 2011

iron (Parmar and Kaushal, 1982). Citric acid is present in sufficient quantity in this fruit, besides other acids like malic, succinic and tartaric acid (Saxena et al., 1987). This fruit is also associated with various medicinal properties. It is laxative, diuretic and used for curing vomiting, sore throat, brain diseases, spleen complaints, chest troubles, scabies, bronchitis, earache, liver and kidney disorders (Kirtikar and Basu, 1935). Sherbet prepared from ripe fruit is useful in gastric and asthmatic fever and inflammation of urinary tract (Dastur, 1962). It is believed that pomegranate has cancer fighting properties and a glass full of pomegranate juice is said to contains more antioxidants than 10 cups of green tea (Anon, 2005). Wild pomegranate is too acidic by nature that is it cannot be used for table purpose but is being used as a good souring agent in curries, chutneys and other culinary preparations in dried form (Phadnis, 1974). It is a paradox that such a miracle fruit having enormous potential for therapeutic use has never

Thakur et al.

been utilized commercially for value addition except in the form of anardana. An important reason for this is being lack of awareness regarding its nutritive value, use and unavailability of technology for its processing. Keeping in view all these factors, the present studies were under taken to develop a drink from this fruit and study its storage life.

Material and Methods Wild pomegranate fruits harvested at optimum maturity were procured from Narag area of Sirmour district of Himachal Pradesh, India and brought to the department of Food Science and Technology Nauni, Solan, HP. Fruits after thorough washing in water were used for physico-chemical analysis and juice extraction. Packaging materials like PET bottles and glass bottles were procured from Chandigarh market.

Juice extraction and development of drink Fruits were cut into parts and arils were extracted manually. The extracted arils were used for extraction of juice and preparation of drink. Juice was extracted by food processor of Maharaja make. Drink was prepared by mixing different proportions viz., 8, 10, 12 and14% of wild pomegranate juice and adjusting the TSS 12oB (T1 to T4) and other set of TSS adjusted to 15oB with same juice proportions (T5 - T8) .

Packaging and storage

phenolphthalein as an indicator and expressed as per cent citric acid (AOAC, 1984). The pH of the drink was determined by using a digital pH meter (CRISON Instrument, Ltd, Spain).Total anthocyanins present in all the samples were determined by a‘ method given by Ranganna (1997). The phenols or tannin contents were determined by the FolinCiocalteu procedure given by Singleton and Rossi (1965).

Sensory evaluation Nine points hedonic rating test (Amerine et al. 1965) was followed for conducting the sensory evaluation of wild pomegranate drink. The panel of ten judges comprising of faculty members and postgraduate students were selected to evaluate the products for sensory parameters such as colour, body, taste, aroma and overall acceptability. Efforts were made to keep the same panel for sensory evaluation throughout the entire period of study.

Microbial evaluation Total plate count was estimated by serial dilution technique using total plate count/standard plate count agar medium prepared according to Ranganna (1997). The innoculated plates were then incubated at 280C for 72 h prior to counting of microbes. The results of the total plate count (TPC) were expressed as x 102 cfu/g of sample.

The drink prepared by following the best recipe was packed in presterilized glass and PET bottles of each of 200 ml capacity. After proper labelling of bottles they were kept for further analysis and storage studies. Packed bottles were stored in ambient (20-250C) and low temperature (4-70C) conditions for 6 months. The physico-chemical, microbiological and sensory quality characteristics of the product were determined at 0, 3 and 6 months of storage.

Statistical analysis

Analysis

Results and discussion

Wild pomegranate fruit drink was analyzed for different quality attributes. The colour of drink in terms of different units (Red & Yellow) was observed with Tintometer (Lovibond Tintometer Model-E). Titratable acidity was estimated by titrating a known volume of the sample against standard NaOH solution by using

IJFFT 1(2) 2011 : 238

Data on physico-chemical characteristics of drink was analysed by Completely Randomized Design (CRD) before and during storage, whereas, data pertaining to the sensory evaluation were analyzed by using Randomized Block Design (RBD) as described by Mahony (1985). The experiment for recipe standardization was replicated three times and for storage studies five times.

Physico-chemical and sensory characteristics Data (Table 1) pertains to physico-chemical characteristics of different recipes of wild pomegranate drink. It is apparent that with constant TSS of 12o B, red colour units titratable

Development of wild pomegranate drink and its evaluation during storage

acidity and ascorbic acid in the drink increased with the increase in the juice content in T1 to T4, whereas, yellow colour units decreased with the decrease in juice content in the drink. In the other set of treatments i.e. T5 to T8 similar trend was recorded. The pH in both the sets increased with the decrease in titratable acidity. Data on sensory characteristics of different recipes of wild pomegranate drink given in Table 2 indicate that the mean colour, body, taste, aroma and overall acceptability scores were obtained highest in T8 comparing to other treatments.

Wild pomegranate drink recipe with 14% juice and 150B TSS (T8) was found to be best on the basis of some physicochemical and sensory characteristics. The higher contents of colour, titratable acidity, and ascorbic acid were because of higher content of fruit juice used in this recipe. This recipe also obtained maximum scores for sensory parameters like colour, body, taste, aroma and overall acceptability which may also be due to higher juice content, best combination of juice and syrup, best sugar-acid blend in the product.

Table 1. Physico-chemical characteristics of different recipes of wild pomegranate drink Treatment

Physico-chemical characteristics TSS (0B)

Colour (TCU)

Titratable acidity (%)

Ascorbic acid (mg/100 ml)

pH

R

Y

T1

2.30

1.32

12.00

0.27

1.69

4.38

T2

2.50

1.23

12.00

0.34

2.35

4.33

T3

3.10

1.11

12.00

0.37

2.66

4.22

T4

3.50

1.00

12.00

0.41

3.03

4.11

T5

2.40

1.41

15.00

0.27

1.66

4.39

T6

2.60

1.33

15.00

0.34

2.34

4.33

T7

3.20

1.25

15.00

0.38

2.68

4.21

T8

3.63

1.13

15.00

0.41

3.03

4.11

0.74

0.16

-

0.01

0.22

0.01

CD

0.05

Table 2. Sensory characteristics (score) of different recipes of wild pomegranate drink Treatment

Colour

Body

Taste

Aroma

Overall acceptability

T1

6.00

5.70

6.00

5.70

5.25

T2

6.70

6.00

6.70

6.70

6.05

T3

7.10

6.30

7.30

6.85

6.45

T4

8.00

6.70

7.40

7.80

7.50

T5

6.50

5.50

6.20

6.00

5.55

T6

7.00

6.00

7.00

6.80

6.20

T7

7.30

6.20

7.30

6.90

6.85

T8

8.50

7.90

8.30

8.20

8.10

0.82

0.74

0.69

0.77

0.69

CD

0.05

IJFFT 1(2) 2011 : 239

Thakur et al.

Storage of wild pomegranate drink Physico-chemical characteristics

Titratable acidity

Colour There was a significant decrease in red and yellow TCU of wild pomegranate drink during storage (Table 3 and 4). More loss in red and yellow TCU of drink stored under ambient conditions was observed as compared to refrigerated storage conditions. The reason for decrease in colour units during storage might be due to degradation of anthocyanins pigment. However, non-significant difference among both the packaging materials shows their equal effect on colour of drink. Similar decreasing trend of red and yellow colour units of seabuckthorn drink during storage has been reported by Kaushal (2004).

The titratable acidity of drink showed a slight decrease during storage (Table 5) and it was observed more under ambient storage conditions as compared to refrigerated conditions. Decrease in titratable acidity during storage might be due to co-polymerization of organic acids with sugars and amino acids. Our results are in conformity with the findings of Pandey et al. (1995) in mango drink, Khurdiya and Roy (1983) in jamun drink and Kaushal (2004) in seabuckthorn drink. The more retention of titratable acidity in drink packed in glass bottle than PET bottle during storage might be due to slower rate of chemical reactions in glass bottle as compared to PET. The findings of the present study are in agreement with the results reported by Kansal (2003) in Kinnow drink and Krishnaveni et al. (2009) in jack fruit RTS beverage.

Table 3. Effect of packaging and storage on red TCU of wild pomegranate drink V S/T T1 T2 Mean TxS interaction Table Treatment T1 T2 Mean

Ambient storage (Months) 0 3 6

Mean 0

3.63 3.63 3.63

2.82 2.65 2.74

2.06 1.98 2.02

2.84 2.75 2.80

0

3

6

3.63 3.63 3.63

2.92 2.78 2.85

2.29 2.15 2.22

Refrigerated storage (Months) 3 6 2.51 2.32 2.42

Mean

3.63 3.63 3.63

3.02 2.90 2.96

3.05 2.95 3.00

Mean

T= NS

T×S= NS

T= packaging material

2.95 2.85

S= 0.24 V= 0.20

T×V= NS S×V= NS T×S×V= NS

S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle

CD0.05

Table 4. Effect of packaging and storage on yellow TCU of wild pomegranate drink V S/T T1 T2 Mean TxS interaction Table Treatment T1 T2 Mean

IJFFT 1(2) 2011 : 240

Ambient storage (Months) 0 3 6

Mean 0

1.13 1.13 1.13

0.99 0.90 0.95

0.75 0.56 0.66

0.96 0.86 0.91

0

3

6

1.13 1.13 1.13

1.00 0.91 0.96

0.79 0.66 0.73

Refrigerated storage (Months) 3 6 0.83 0.75 0.79

Mean

1.13 1.13 1.13

1.00 0.92 0.96

0.99 0.93 0.96

Mean

T= NS

T×S= NS

T= packaging material

0.97 0.90

S= 0.08 V= NS

T×V= NS S×V= NS T×S×V= NS

S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle T2= PET bottle

CD0.05

Development of wild pomegranate drink and its evaluation during storage

Table 5. Effect of packaging and storage on titratable acidity (%) of wild pomegranate drink V S/T

Ambient storage (Months) 0 3 6

Mean 0

Refrigerated storage (Months) 3 6

Mean

T1

0.413

0.383

0.371

0.389

0.413

0.398

0.392

0.401

T2

0.413

0.374

0.362

0.383

0.413

0.388

0.379

0.393

Mean

0.413

0.379

0.367

0.386

0.413

0.393

0.385

0.397

0

3

6

Mean

T= NS

T×S= NS

T= packaging material

T1

0.413

0.391

0.382

0.395

S= 0.10

T×V= 0.002

S= Storage period

T2

0.413

0.381

0.371

0.388

V= NS

S×V= 0.003

V= Storage conditions

Mean

0.413

0.386

0.377

T×S×V= NS

T1= Glass bottle

TxS interaction Table Treatment

CD0.05

T2= PET bottle

Anthocyanins A significant decrease in anthocyanins content of drink was recorded during the storage (Table 6) and more retention of anthocyanins was observed under refrigerated storage conditions than ambient conditions. Loss of anthocyanins in drink might be due to their high susceptibility to auto oxidative degradation during storage. More retention of this characteristic in the product might be due to slower rate of auto oxidation of anthocyanins in the product in refrigerated storage conditions as compared to ambient conditions. Anthocyanins in this product were retained at par in both the packaging materials during storage. Similar observations have

been reported by Kannan and Thirumaran (2004) in jamun drink.

Phenols A gradual decrease in phenols content of drink observed during storage (Table 7) which was slower under refrigerated storage conditions than ambient conditions. Significant decrease in phenols content during storage might be due to their involvement in the formation of polymeric compounds, complexing of phenols with protein and their subsequent precipitations as observed by Abers and Wrolstad (1979) in strawberry preserve and Premachandran (1982) in apple

Table 6. Effect of packaging and storage on anthocyanins (mg/100 ml) of wild pomegranate drink V S/T T1 T2 Mean TxS interaction Table Treatment T1 T2 Mean

Ambient storage (Months) 0 3 6

Mean 0

3.07 3.07 3.07

1.83 1.80 1.82

0.92 0.89 0.90

1.94 1.92 1.93

0

3

6

3.07 3.07 3.07

1.97 1.92 1.94

1.27 1.20 1.24

Refrigerated storage (Months) 3 6 1.62 1.52 1.57

Mean

3.07 3.07 3.07

2.10 2.04 2.07

2.26 2.21 2.24

Mean

T= NS

T×S= NS

T= packaging material

2.10 2.06

S= 0.10 V= 0.08

T×V= NS S×V= 0.14 T×S×V= NS

S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle

CD0.05

IJFFT 1(2) 2011 : 241

Thakur et al.

Table 7. Effect of packaging material and storage on phenols (as tannic acid mg/100 ml) of wild pomegranate drink V

Ambient storage (Months)

S/T

0

3

6

T1

16.72

12.97

9.74

T2

16.72

10.99

Mean

16.72

11.98

Mean

Mean

0

3

6

13.14

16.72

14.38

10.67

13.92

8.64

12.12

16.72

13.29

9.46

13.16

9.19

12.63

16.72

13.83

10.07

13.54

TxS interaction Table Treatment

Refrigerated storage (Months)

CD0.05 0

3

6

Mean

T= NS

T×S= NS

T= packaging material

T1

16.72

13.68

10.20

13.53

S= 0.37

T×V= NS

S= Storage period

T2

16.72

12.14

9.05

12.64

V= 0.30

S×V= 0.53

V= Storage conditions

Mean

16.72

12.91

9.63

T×S×V= NS

T1= Glass bottle T2= PET bottle

nectar. Slower rate of loss of phenols might be due to slower reaction rate in refrigerated storage conditions as compared to ambient. However, retention of more phenols of drink in glass bottle may also be the slower reaction rates in glass bottle, as glass material absorb heat at slower rate as compared to PET. Similar observations were recorded by Kansal (2003) in Kinnow drink, Kannan and Thirumaran (2004) in jamun drink.

Sensory characteristics Colour The colour scores of drink decreased significantly during storage (Table 8). However, they were retained better in refrigerated storage conditions than ambient conditions. Decrease in colour score during storage might be due to degradation of colour pigment (anthocyanins) and browning caused by copolymerization of organic acids of the product and this might have led the judges to award the lower scores during storage. The retention of higher colour scores of drink in refrigerated storage conditions might be due to lesser degradation of colour pigment which led the judges to award the higher scores as compared to ambient conditions. Decrease in colour scores have also been reported by Shrivastava (1998) in mango drink, Kotecha and Kadam (2003) in tamarind drink and Kaushal (2004) in seabuckthorn drink. Judges did not find any colour difference in the product among both the packaging materials during storage.

IJFFT 1(2) 2011 : 242

Body Body scores of drink decreased with advancement of storage period and retained higher in refrigerated storage conditions than ambient (Table 9). As far as packaging is concerned, there was no significant effect of packaging materials on the body scores of drink. The possible reason for decrease in body scores might be due to the formation of precipitates in the product as a result of interactions between phenols and protein as well as the formation of cation complexes with pectin and phenols during storage, which led the judges to award lower scores. Retention of higher body scores in refrigerated conditions might be due to better condition of the drink during storage as result of slower rate of above mentioned reactions. Our results of body scores are in conformity with the findings of Kaushal (2004) in seabuckthorn drink.

Taste There was a decrease in taste scores of drink with advancement of storage period (Table 10). However, this decrease was significantly lower under refrigerated storage conditions than ambient conditions. As far as the packaging material is concerned higher decrease in taste scores of drink was observed in the drink packed in PET bottle than glass bottle. The possible reason for decrease in taste scores might be due to the loss of sugar-acid blend responsible for taste during storage. Similar observations of decrease in taste scores of the product during storage were noticed by Kotecha and

Development of wild pomegranate drink and its evaluation during storage

Table 8. Effect of packaging and storage on colour score of wild pomegranate drink V S/T

Ambient storage (Months) 0 3 6

Mean 0

T1 T2 Mean

8.50 8.50 8.50

7.70 7.40 7.55

6.10 6.80 6.45

7.43 7.57 7.50

TxS interaction Table Treatment T1 T2 Mean

0 8.50 8.50 8.50

3 8.05 7.65 7.85

6 7.00 7.05 7.03

Mean 7.85 7.73

Refrigerated storage (Months) 3 6

8.50 8.50 8.50

8.40 7.90 8.15

7.90 7.30 7.60

Mean 8.27 7.90 8.09

CD0.05 T= NS S= 0.24 V= 0.20

T×S= NS T×V= 0.28 S×V= 0.35 T×S×V= 0.50

T= packaging material S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle

Table 9. Effect of packaging and storage on body score of wild pomegranate drink V S/T

Ambient storage (Months) 0 3 6

Mean 0

T1 T2 Mean

7.40 7.40 7.40

6.80 6.70 6.75

6. 20 6.00 6.10

6.80 6.70 6.75

TxS interaction Table Treatment T1 T2 Mean

0 7.40 7.40 7.40

3 6.95 6.80 6.88

6 6.60 6.30 6.45

Mean 6.98 6.83

Refrigerated storage (Months) 3 6

7.40 7.40 7.40

7.10 6.90 7.00

7.00 6.60 6.80

Mean 7.17 6.97 7.07

CD0.05 T= NS S= 0.26 V= 0.21

T×S= NS T×V= NS S×V= 0.37 T×S×V= NS

T= packaging material S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle

Table 10. Effect of packaging and storage on taste score of wild pomegranate drink V S/T

Ambient storage (Months) 0 3 6

Mean 0

T1 T2 Mean

8.30 8.30 8.30

7.80 7.50 7.65

6.90 6.50 6.70

7.67 7.49 7.55

TxS interaction Table Treatment T1 T2 Mean

0 8.30 8.30 8.30

3 7.90 7.65 7.78

6 7.30 6.90 7.10

Mean 7.83 7.62

Refrigerated storage (Months) 3 6

8.30 8.30 8.30

8.00 7.80 7.90

7.70 7.30 7.50

Mean 8.00 7.80 7.90

CD0.05 T= NS S= 0.25 V= 0.21

T×S= NS T×V= NS S×V= 0.36 T×S×V= NS

T= packaging material S= Storage period V= Storage conditions T1= Glass bottle T2= PET bottle

IJFFT 1(2) 2011 : 243

Thakur et al.

Kadam (2003) in tamarind drink, Shrivastava (1998) in mango drink and Kaushal (2004) in seabuckthorn drink. Retention of higher taste scores in refrigerated conditions might be due to the better retention of original sugar-acid blend. Higher score retained in glass bottle might be due to the better retention of sugar acid-blend as a result of slower reaction rate in glass bottle as compared to PET because glass absorbs heat at slower rate than PET.

storage might be due to degradation of aromatic compounds in the product. However, the retention of higher scores in refrigerated conditions might be due to the slower degradation of aromatic compounds which led the judges to award the higher scores to the product in refrigerated conditions as compared to ambient conditions. Decrease in aroma scores of mango drink during storage have been reported by Shrivastava (1998).

Aroma

Overall acceptability

Aroma scores of drink decreased during storage and this decrease was more in ambient storage conditions as compared to refrigerated (Table 11). The decrease in aroma scores during

The overall acceptability scores of drink decreased significantly during storage (Table 12). However, the drink stored at

Table 11. Effect of packaging and storage on aroma score of wild pomegranate drink V

Ambient storage (Months)

S/T

0

3

6

T1

8.20

7.90

6.60

T2

8.20

7.70

Mean

8.20

7.8

Mean

Mean

0

3

6

7.57

8.20

7.80

7.40

7.80

6.50

7.47

8.20

7.40

7.10

7.57

6.55

7.52

8.20

7.60

7.25

7.69

TxS interaction Table Treatment

Refrigerated storage (Months)

CD0.05 0

3

6

Mean

T= NS

T×S= NS

T= packaging material

T1

8.20

7.85

7.00

7.68

S= 0.22

T×V= NS

S= Storage period

T2

8.20

7.55

6.80

7.52

V= NS

S×V= 0.30

V= Storage conditions

Mean

8.20

7.70

6.90

T×S×V= NS

T1= Glass bottle T2= PET bottle

Table 12. Effect of packaging and storage on over all acceptability score of wild pomegranate drink V

Ambient storage (Months)

S/T

0

3

6

T1

8.10

7.60

6.80

T2

8.10

7.30

Mean

8.10

7.45

Mean

Mean

0

3

6

7.50

8.10

7.90

7.50

7.83

6.50

7.30

8.10

7.70

7.10

7.63

6.65

7.40

8.10

7.80

7.30

7.73

TxS interaction Table Treatment

Refrigerated storage (Months)

CD0.05 0

3

6

Mean

T= NS

T×S= NS

T= packaging material

T1

8.10

7.75

7.15

7.67

S= 0.25

T×V= NS

S= Storage period

T2

8.10

7.50

6.80

7.47

V= 0.21

S×V= 0.36

V= Storage conditions

Mean

8.10

7.63

6.98

T×S×V= NS

T1= Glass bottle T2= PET bottle

IJFFT 1(2) 2011 : 244

Development of wild pomegranate drink and its evaluation during storage

Table 13. Microbial population (X102 cfu/ml) of wild pomegranate drink I/T

Ambient storage ( months)

Refrigerated storage (months)

Initial

3

6

3

6

T1

0.0

0.2

0.4

0.09

0.15

T2

0.0

0.3

0.5

0.10

0.17

T= Treatment T1 = Glass bottle I= Storage interval

refrigerated conditions and also in both the packaging materials like PET and glass bottles, with comparatively less changes in glass bottle under refrigerated conditions.

refrigerated storage conditions was significantly better in overall acceptability scores than in ambient conditions. Decrease in overall acceptability scores might be due to the loss in appearance, flavour compounds and uniformity of the product. Higher overall acceptability scores retained in refrigerated conditions might be due to the minute loss of colour, uniformity of the product and minute loss of flavour compounds during storage. Shrera (2005) has also observed decrease in overall acceptability scores of drink prepared from hill lemon stored for six months.

Amerine MA, Pangborn RM and Roessler EB. 1965. Principles of sensory evaluation of food. Academic press, London.

Microbiological quality

Anonymous. 2005. Pomegranate: the poor man’s apple. Indian Food Indus, 24: 33

References Abers JE and Wrolstad RE. 1979. Causative factors of colour determination in strawberry preserves during processing and storage. J Food Sci Technol., 44:75.

The perusal of data in Table 13 indicates that microbial count increased in the drink during storage. The minimum microbial count of 15 cfu/ml was observed in glass bottles after six months in refrigerated storage conditions. The drink packed in PET bottles and stored under ambient conditions exhibited highest microbial load of 50 cfu/ml after six months of storage.

AOAC. 1984. Official methods of analysis of the association of official analytical chemist, (Ed. Hortwits W), Association of official analytical chemists, Washington DC, USA.

Increase in total microbial count (cfu/ml) was observed during storage (Table 13) which might be due to contamination during plating instead of contaminated product. The drink packed in glass bottle and stored under refrigerated storage conditions recorded minimum microbial load during storage. Kaushal (2004) in seabuckthorn drink and Sarvanan et al. (2004) in papaya drink have also reported a low microbial count at initial stage which increased slightly during storage. Except for this increase in microbial count no other spoilage symptoms like fermentation, discolouration, gas formation etc. were observed in drink which confirm that product was safe for consumption.

Dastur JF. 1962. Medicinal plants of India and Pakistan, Bombay Taraporevala Publishing. 139p.

Conclusion Best wild pomegranate drink can be prepared by using 14 per cent juice and fixing TSS 150B. Drink could safely be stored for a period of six months under both the ambient and

Bhrot NP. 1998. Genetical analysis of wild pomegranate (Punica granatum L.) for same growth ecological and quality characters. Ph D Thesis, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan (HP), India.

Kannan S and Thirumaran AS. 2004. Studies on the storage life of jamun (Syzygium cumini) fruit products. J Food Sci Technol., 42: 186-188. Kansal H. 2003. Studies on processing of kinnow juice extraction via modified method of bitter less kinnow juice extraction. MSc Thesis, DND University of Agriculture Technology, Faizabad (UP), India. Kaushal M. 2004. Utilization of seabuckthorn (Hippophae salicifolia D. Don.) for preparation and evaluation of some value added products. PhD Thesis, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan (HP), India. Khurdiya DS and Roy SK. 1984. Studies on stability of anthocyanin during storage. Indian Food Packer, 28: 5-8. Kirtikar KR and Basu BD. 1935. Indian medicinal plants, Dehradun. 1084p.

IJFFT 1(2) 2011 : 245

Thakur et al.

Kotecha PM and Kadam SS. 2003. Preparation of ready to serve beverage, syrup and concentrate from tamarind. J Food Sci Technol., 40: 76-79. Krishnaveni A, Manimegalai G and Sarvanakumar R. 2009. Storage quality of jackfruit RTS beverage. J Food Sci Technol., 38: 601602. Mahony MO. 1985. Sensory evaluation of food: statistical methods and procedures. Marcel Dekker, New York. Pandey DC, Tomar MC and Singh UB. 1995. Preparation processing and storage studies on raw mango pana. Indian Food Packer, 49: 5-9. Parmar C and Kaushal MK. 1982. Wild fruit of sub-himalayan region. Kalyani Publisher, New Delhi.

Ranganna S. 1997. Handbook of analysis and quality control for fruit and vegetable products. Tata McGraw Hill, New Delhi. Saravanan K, Godra RK, Goyal RK and Sharma RK. 2004. Processing of papaya fruit for the preparation of ready to serve beverage and its quality. Indian J Hill Farm, 17: 49-55. Saxena AK, Manan JK and Berry SK. 1987. Pomegranate: Post Harvest Technology, Chemistry and Processing. Indian Food Packer, 41: 43-60. Shrera SK. 2005. Utilization of hill lemon (Citrus pseudolimon Jan) and Tulsi (Oscimum sanctum L.) for the development of RTS and appetizer. MSc Thesis, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan (HP), India.

Phadnis NA. 1974. Pomegranate for dessert and juice. Indian Hort, 19: 9-13.

Shrivastava JS. 1998. Comparative study of RTS drinks prepared from Dasheri and Beganpalli mangoes. Indian Food Packer, 52: 38-40.

Premachandran U. 1982. Studies on the utilization of apple juice concentrate for nectar preparation. PhD Thesis, Division of Fruits and Horticulture Technology, IARI, New Delhi.

Singelton VL and Rossi JA. 1965. Colorimetry of total phenolics with phosphomolybedic phosphotungstic acid reagent. Amer J Enology and Vitic, 16: 144-158.

IJFFT 1(2) 2011 : 246

Int. J. Fd. Ferm. Technol. 1(2) 2011 : 247-253

Research Paper

Beneficial effects of pigment from Monascus purpureus as an alternative treatment in preventing atherosclerosis, hypercholesterolemia and lipid modificaction Marzieh Rezaei, Rasoul Roghanian, Iraj Nahvi, Jamal Moshtaghian Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran.

Abstract: Monascus purpureus is a filamentous fungus which produces secondary metabolites e.g. pigments used for colouring besides therapeutic and medicinal values. The current study was conducted to examine the efficacy of pigment in lowering cholesterol and triglyceride concentrations in the rats sera. Pigment of Monascus purpureus was produced through submerged fermentation under the conditions: pH 6-7, agitation 200-300 r.p.m , temperature 25 - 30°C for 5 - 7 days. The experimental groups were treated with Monascus purpureus pigment. Then, the level of cholesterol and triglyceride were measured after two weeks pigment regimen. The results showed that cholesterol level in rat’s sera were reduced down to 48.4% and 45.5% in comparison with control group. Also, the analysis of variance indicated that the effect of the M. purpureus pigment on the triglyceride in hypercholesterolemic rats was significantly different from the control group at the same week. Keywords : Monascus purpureus, Atherosclerosis, Pigment, hypercholesterolemia, lipid modification

People with hyperlipidemia have responded well to the lipidlowering agents including HMG-CoA reductase inhibitors (statins), fibrates, and nicotinic acids (Kreisberg, 2003). However, long-term safety and potential drug interaction between Statins and other hypolipidemic agents may become problematic (Moghadasian, 2002; Bellosta et.al., 2004 ) Nowadays, many people would like to use a natural product as an alternative to chemical drugs (Statin treatment). The objective of this study was to assess the beneficial effects of red pigment prepared from a filamentous fungus for prevention of hyperlipidemia and atherosclerosis. Filamentous fungi possess important properties which play a significant role in the human lifestyle and in the environment, *E-mail of corresponding author : [email protected] MS Received on : 10th Sept., 2011 Accepted on : 1st Oct., 2011

by participating in the production of food and health products. Their biochemical potential and their adaptation to extreme life conditions in liquid media have been exploited to produce molecules such as antibiotics (e.g., penicillin, cephalosporins), enzymes (á-amylase, cellulase), organic acids (e.g., citric acid) (Kubicek, 1986; Moreira et.al., 1996) and food colourants (e.g., Anka) (Lin, 1973). Monascus is a filamentous fungus which produces secondary metabolites e.g. pigments used for red colouring of rice wine, soybean cheese, fish and red meat. These pigments are principally used in the south of China, Taiwan, Japan and Indonesia. This fungus is traditionally cultivated on solid media, rice grains or bread, though such solid-state fermentation does not enable the environmental parameters to be controlled and

Rezaei et al.

submerged cultures in natural or synthetic media have been developed recently (Hajjaj et.al., 2000). The other types of secondary metabolites produced by Monascus spp. include a group of yellow, orange and red pigments (Blanc et.al., 1994; Wong, 1981), a group of antihypercholesterolemic agents, including monacolin K and the hypotensive agent C-aminobutyric acid (GABA) (Albert et.al., 1980; Juzlova et.al., 1996; Su et.al., 2003), and antibacterial compounds including pigments and citrinin (as monascidin) (Blanc et.al., 1995 a,b; Wong, 1981). Monacolin K (also known as Lovastatin, Mevinolin and Mevacor) is a secondary metabolite of Monascus with the molecular formula C24H36O5 and a molecular weight of 404.55 KD (Endo, 1979). Monacolin is a potent competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis (Albert, 1990). It not only inhibits cholesterol biosynthesis, but also lowers blood cholesterol level in both humans and animals. The results obtained in feeding the pigment of Monascus to rats on these parameters are reported here.

Materials and methods Microorganism and Pigment production The cultivation method applied in this study was submerged fermentation. Monascus purpurus DSM1603 was cultivated on slanted malt extract agar (MEA) at 25° C. Then, the seed culture was prepared by transferring a loop full of spore from MEA agar slanted into a 500-ml flask containing 100 ml basal medium containing 5 g/l peptone, 3g/l malt extract and 10 g/l glucose with the pH set at 5.0. Each 500 ml Erlenmeyer flask containing 80 ml of the produced medium (semi-synthetic) was incubated with 20 ml of the inoculums culture and incubated at 30º C, 300 rpm for 64 to 72 hours (Sanae et.al., 2005). The fermentation process was carried out under the conditions of pH, 6-7; agitation, 200-300 rpm; temperature, 25-30° C for a period of 5-7 days (Panagou et.al., 2005; Wang et.al., 2005). Pigments were released into the fermentation medium through mycelia (Su et.al., 2003). The fermentation broth medium containing red pigment was separated from the mycelia using whatman filter No 1 (Sanae et.al., 2005). The filtered broth was used for drinking for the treated rats or it was diluted using clean tap water to prepare lower dose of the pigment (i.e. 25%).

IJFFT 1(2) 2011 : 248

Rats and pigment treatment 25 male Wistar rats, 8 weeks of age with an average body weight of 250 g, were obtained from the laboratory animal unit of Chamran University, Ahvaz, Iran. The animals were housed in standard plastic cages and kept in a room under standard conditions of temperature, 22 2° C; hydrometry, 50 5%; 12-hour reverse light-dark cycle and free access to regular rat chow pellets and water. After 2 weeks of accommodation, the animals were randomly distributed into 3 groups of 5 each as indicated in following: Group 1 (Control group), Group 2 and Group 3 received red pigment with the concentrations of either 25% or 100%, respectively. Treated animals had only free access to pigment solutions for drinking while animals in the control group had only free access to regular drinking water.

Sample preparation At the end of the 14-day experimental period, the blood samples were collected from eye medial canthus of each rat while the animal was anaesthetized with ether. Blood samples were collected into the tubes containing no anticoagulant. Sera were separated via cold centrifugation at 3000×g for 10 minutes at 4°C. Cholesterol, TG (Total glycerides) , LDL (Low Density Lipids) and HDL (High Density Lipids) concentrations were determined using a commercial diagnostic kits provided by Pars Azmon Co., Iran. The Atherogenic Index (AI) was calculated using the following formula (Jeun et.al., 2008): AI= (Total cholesterol- HDL-cholesterol) / HDL-cholesterol

Statistical Analysis The data obtained in this study were analyzed using ANOVA and LSD’s test for multiple comparisons. Statistical significance was determined based on the p value being set at 0.05. SPSS, Version 15.0 was used to conduct statistical analysis.

Result and Discussion The effect of M.purpureus pigments on serum cholesterol level After 2-weeks period of treatment, blood factors were determined. Table 1 showed the alterations in sera Cholesterol levels. Comparing the treated animals with the control ones,

The effects of monascus purpureus pigment on lipid modififaction

the result indicated that Cholesterol level in rat’s sera reduced down to 48.4% and 45.5% influenced by use of 25% and 100% of M.purpureus pigment respectively. The analysis of variance (ANOVA) showed that the serum Cholesterol level was significantly lower in the groups treated with M.purpureus pigments, compared to hypercholesterelomia (p= 0.001) (Table 2). Also as revealed in the Table 3, the Athrogenic Index in animals after pigment treatment in comparision with Hypercholesterolemia shows that AI reduced to 79.27% and 64.58 % under the influence of treating the animals with 25% and 100% of M.purpureus pigment respectively. The other detailed results i.e. Athrogenic Index (AI) before and after hypercholesterolemia and Mean differences of Athrogenic

Index in pigment, treated rats in comparison with Control group are also shown. Table 1: LSD’s test for multiple comparisons: The effect of the Low and High dose of Monascus pigment on the Cholesterol concentration in comparison with control group PD3

p

PD2

PD1

Groups

- 2.4%

+64.3%

Control

*0.014

48.4%

- 50.9%

- 1.9%

Low dose

*0.000

45.5%

- 28.20%

+ 6%

High dose

PD1: percentage of differences of Cholesterol concentration compare to baseline PD2: percentage of differences of Cholesterol concentration compare to Hypercholesterolemia PD3: percentage of differences of Cholesterol concentration compare to Control group

Table 2 : The analysis of variance (ANOVA), the effect of the Monascus pigment on the Cholesterol concentration in Hypercholesterelomic rats

Changes compared to hypercholesterolemia Inner groups Total

SS

df

MS

F

Inter groups

5878.987

2

2939.493

12.745

2767.645

12

230.638 3.816

0.052

641/8646

14

1312.1648

2

6560.842

Inner groups

20632.563

12

1719.380

Total

33754.246

14

Changes compared to baselineInter groups

p *0.001

SS: some of square, df: degree of freedom, MS: Mean Square *Significantly different from control group at the same week (p