NOTE TO USERS

This reproduction is the best copy available.

® UMI

STUDIES ON HIGH PRESSURE PROCESSING AND PRESERVATION OF MANGO JUICE: PRESSURE DESTRUCTION KINETICS, PROCESS VERIFICATION AND QUALITY CHANGES DURING STORAGE

by

Nikhil Davangere Hiremath

Department of Food Science and Agricultural Chemistry Macdonald Campus ofMcGill University Montreal, Canada

February, 2005

A thesis submitted to McGill University in partial fulfillment of the requirements for the degree ofMasters of Science

©Nikhil Davangere Hiremath, 2005 Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition

395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada

Your file Votre référence ISBN: 0-494-12462-8 Our file Notre référence ISBN: 0-494-12462-8

NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats.

The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission.

ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse.

While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ABSTRACT

Among a number emerging technologies considered by FDA, high pressure (HP) processing is very promising and gaining considerable importance. HP processing is a novel technology and has proven to be effective in eliminating microorganisms and enzymes while retaining quality characteristics of food. Mango, one of the commercially important tropical fruits in the world, is widely accepted by consumers for its aroma and flavor. Currently, thermal processing is the primary mode of preservation of mango products, which can be considerably damaging to the delicate quality of mango. HP processing could thus be a potential alternative for extending the shelf-life of mango products. Establishing HP processing technology need data on microbial inactivation kinetics and shelf-life study of the product. The objective ofthis research was therefore to evaluate the application of HP treatment for inactivation of microorganisms (pathogenic and spoilage type) and to evaluate the shelf-life of HP treated mango juice.

HP destruction kinetics of three common spoilage microorganisms, Leuconostoc mesenteroides, Zygosaccharomyces bailii and Pichia membranaefaciens and two pathogenic microorganisms, Escherichia coli 0157:H7 and Listeria monocytogenes Scott

A, were evaluated at 250-550 MPa with 0- 60 min holding time at room temperature with species specifie initial counts between 106 to 108 CFU/mL. Microbial destruction by pressure was evaluated based on (i) an initial kill due to a pressure pulse (pulse effect) (ii) a subsequent semi-Iogarithmic destruction during the pressure hold time characterized by

Dp value (time required to kill 90% of population). Pressure sensitivity of Dp values was evaluated by pressure death time approach characterized by Zp value (pressure range required to affect a lO-fold change in Dp). Species specifie Dp values decreased with an increase in pressure. Pathogenic E. coli 0157:H7 was the most resistant bacteria among aIl the strains tested. L. mesenteroides was found to be most resistant spoilage microorganism and yeast ceIls were less resistant to pressure than bacteria. HP processes for mango juice were therefore developed based on destruction of E. coli OI57:H7.

Mango juice was treated at different process lethality (0, 6, 8 and 10D at 400 MPa), and stored 4-20°C to monitor the growth and recovery of pressure killed/injured ceUs. The results showed that no growth in test samples subjected to 6-10D pressure processes.

Quality (color - L, a, b; pH, acidity, °Brix ) and stability (microbial growth) of high pressure processed (O-IOD treatment at 400 MPa) fresh mango juice was likewise evaluated at 4, 12 and 20°C for up to 60 days. The results showed a decrease in pH and b values, increase in acidity and sorne microbial growth. The changes were dependent of storage temperature and pressure severity. While microbial stability could be achieved in

10D processed products stored at 4°C, quality was not stable. Additional treatments carried out at 550 MPa for 1 min and similar storage tests were carried out. The samples pressure treated at 550 MPa maintained their superior quality throughout the 60-day test period and showed no microbial growth (at 4°C). HP pasteurization therefore can be used as a sound and alternative method for the preservation of mango juice.

11 RÉSUMÉ

Parmi les nombreuses technologies émergentes considérées par la FDA, Le procédé de la haute est une technologie attractive et très prometteuse. La haute pression est une nouvelle technologie très efficace pour l'élimination des microorganismes et des enzymes en conservant les caractéristiques de qualité des aliments. La mangue, un des fruits tropicaux commerciaux les plus importants dans le monde, est largement apprécié par les consommateurs pour son arôme et sa saveur. Actuellement, le traitement thermal est le mode primaire de conservation des produits de mangue, qui peut être considérablement destructeurs pour la qualité délicate de la mangue. Le traitement par la haute pression pourrait ainsi être une alternative potentielle pour prolonger la durée de vie d'un produit de produits de mangue. L'utilisation de ce traitement nécessite des des données sur les cinétiques d'inactivation microbienne et sur l'étude de la durée de vie du produit. L'objectif de ce travail était donc d'évaluer l'application de traitement de haute pression pour inactiver des microorganismes (des pathogènes et des microorganismes responsables de la détérioration des aliments) et d'évaluer la durée de vie du jus de mangue traité par haute pression.

La cinétique de destruction par faute pression de trois microorganismes responsables de la détérioration des aliments, Leuconostoc mesenteroides,

Zygosaccharomyces bailii et Pichia membranaefaciens et deux microorganismes pathogènes, Escherichia coli 0157:H7 et Listeria monocytogenes Scott A, a été évaluée à

250-550 MPa à la température ambiante avec des temps de traitement variant de 0-60 minute et en utilisant des comptes initiaux spécifiques aux souches variant entre 106 à 108

CFU/mL. La destruction microbienne par la haute pression a été évaluée en se basant sur

111 (i) un initial tuent en raison d'une impulsion de pression (l'effet d'impulsion) (ii) une destruction semi-Iogarithmique consécutive pendant le temps d'application de la pression caractérisé par la valeur de Dp (la durée de temps requise pour détruire 90 % de population). La sensibilité de pression des valeurs de Dp a été évaluée par l'approche de temps de mort de pression caractérisée par la valeur de Zp (la gamme de pression requise pour affecter un changement à dun facteur 10 de Dp). Les valeurs de Dp spécifiques aux espèces microbiennes ont diminué lorsque la pression a augmenté. Le pathogène E. coli

0157:H7 était le plus résistant parmi toutes les souches testées. L. mesenteroides était le plus résistant parmi les microorganismes responsables de la détérioration des aliments et les cellules de levure étaient moins résistantes à la pression que les bactéries. Les procédés de la haute pression pour le jus de mangue ont été donc développés en se basant sur la destruction du pathogène E. coli 0157:H7. Le jus de mangue a été traité à différents traitements de mortalité (0, 6, 8 et 10D à 400 MPa) et stocké à 4-20°C pour contrôler la croissance et le rétablissement des cellules détruites ou endommagées. Les résultats ont montré qu'aucune croissance dans des échantillons d'essai soumis aux traitements de pression 6-1 OD.

La qualité (couleur - L, a, b; pH, l'acidité, °Brix) et la stabilité (la crOIssance microbienne) de jus de mangue frais traité à la haute pression (traitements O-IOD à 400

MPa) a été évalué à 4, 12 et 20°C pour une période allant jusqu'à 60 jours. Les résultats ont montré une diminution du pH et des valeurs de b, une augmentation de l'acidité et une certaine croissance microbienne. Les changements étaient dépendants de la température de stockage et la sévérité de la pression. Tandis que la stabilité microbienne pourrait être atteinte dans les produits traités au 10D et stockés à 4°C, la qualité n'était pas stable. Des

lV traitements supplémentaires à 550 MPa pendant 1 minute et des essais de stockages semblables ont été effectués. Les échantillons traités à 550 MPa ont maintenu leur qualité supérieure pendant la période d'essai de 60 jours et n'ont montré aucune croissance microbienne (à 4°C). La pasteurisation par haute pression peut donc être utilisée comme une méthode alternative pour la conservation du jus de mangue.

v ACKNOWLEDGEMENTS

1 feel honored in expressing my sincere thanks to my thesis supervisor Dr. H. S

Ramaswamy for his continuous guidance, timely constructive suggestions and constant encouragement at every stage of my master's programme. 1 am grateful to him for accepting me into the food processing group and also providing me financial support to carry out my research studies. His thoughtfulness is appreciated and will be remembered forever. His tireless efforts to do the final editing of the thesis in time for the final submission is greatly appreciated.

1 express profound sense of gratitude to Dr lP Smith for providing me the facilities and laboratory space to carry out the microbiology work, critical comment s, review and timely help during the course of investigation and preparation of thesis.

1 avail this opportunity to thank Mr. Bernard Cayouette for his assistance and teaching me the microbiological procedures, Mrs. Lise Stiebel and Mrs. Barbara Laplaine and the Food Science Department for their extensive help and support.

1 would also like to thank to all my lab mates of the food processing group, Dr.

Songming Zhu Dr. Jasim Ahmed, Mr. Kevin Shao, Mr. Yang Meng, Mrs. Neda

Maftoonazad, Mr. Heping Li, Mr. Alli Taherian, Mr. Manguang Lin, Mr. Shafi Zaman,

Mr. Minli Chi and Mrs. Anuradha Gundurao for their cooperation and friendship.

1 convey my special thanks to all my friends Dr. Venkatesh, Dr. Vikram, Mr.

Santosh, Ms. Jasmine, Mr. Arun, Mr. Baljinder and Ms.Vidya for their moral support and to make an enjoyable student life during my stay at Macdonald campus.

Last but not the least 1 pay my deepest regards to my father, mother, brother and sister in law for their love, inspiration and encouragement.

vi TABLE OF CONTENTS

ABSTRACT

RESUME 111

ACKNOWLEDGEMENTS VI

TABLE OF CONTENTS Vll

LIST OF TABLES Xl

LIST OF FIGURES Xli

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW 5

Mango and Mango Products 5 History, description and importance 5 Production statistics 5 Important mango cultivars 7 Post harvest technology 7 Processing of mango 11 Raw mango products 12 Ripe mango products 13 Waste utilization 15 High pressure processing 16 Introduction 16 Dimension and nomenclature used for pressure 17 High pressure processing: Basic principles 18

Vu Advantages of HP processing 19 Historical time line for HP processing 19 Commercial HP processed products 20 High pressure processing equipment 20 Batch processing system 21 Semi continuous HP system 22 High pressure pro cess equipment manufacturers 22 High pressure effects on microorganisms 23 Inactivation mechanism 23 Critical factors for HP destruction of microorganisms 25 Inactivation kinetics 26 High pressure effects on microorganisms in fruit and fruit products 30 Spoilage microorganisms 31 Pathogens and contaminant microorganisms 32 Quality improvement and shelf life extension 33 High pressure processing ofmango juice 36 Outlook ofhigh pressure technology 37

CHAPTER 3: BIGB PRESSURE DESTRUCTION KINETICS OF SPOILAGE AND PATBOGENIC MICROORGANISMS IN MANGO JUICE 39

Abstract 39 Introduction 40 Materials and methods 44 Cultures, media and preparation of inoculums 44 Preparation of mango juice 45 Preparation of cell suspension and test samples 46 High pressure equipment and treatment 46 Enumeration 47

Vlll Kinetic Data Analysis 49 Process establishment and verification 51 Results and Discussion 53 Dual effect pressure destruction 53 Pulse pressure effect 55 Spoilage microorganisms 56 Pathogens 57 Pressure hold time kinetics 58 Pressure Sensitivity ofDp values 60 Process establishment / verification / challenge 62 Conclusions 65

CHAPTER 4: EVALUATION OF QUALITY CHANGES IN HIGH PRESSURE PROCESSED MANGO JUICE DURING STORAGE 67

Abstract 67 Introduction 68 Materials and Methods 71 Product 71 Preparation and processing of mango juice 71 Storage 72 Microbiological analysis 73 Total soluble solids 73 pH 73 Total titrable acidity 74 Color 74 Data analysis 74 Results and Discussion 75 Microbial growth 75 Aerobic plate counts 79

IX Yeast and mold counts 80 Total soluble solids (OBrix) 82 pH 85 Total titrable acidity 88 Color changes during storage 91 L values 91 b values 94 Temperature sensitivity ofD values 97 Conclusions 100

CHAPTER 5: GENERAL CONCLUSIONS 101 General conclusion 101 Suggestion for future work 103

CHAPTER6:REFERENCES 104

x LIST OF TABLES

2 .. 1 Distribution ofworld mango production in year 2000 by continent 6 2 .. 2 Main mango producing countries in year 2001 6 2 .. 3 Important mango cultivars in major producing countries 8 2.4 Utilization ofmango for processing (products range) 15 2.5 Units of atmospheric pressure 17 2.6 Historical time line ofhigh pressure processing offoods 20 2.7 Pressure induced decimal reduction times (Dp values) ofvarious microorganisms 28 3.1 Media for cultivation and incubation temperature and time of spoilage and 45 pathogenic microorganisms 3.2 Processing conditions for spoilage and pathogenic microorganisms 48 3.3 Enumeration media and incubation temperature and time for spoilage and 49 pathogenic microorganisms 2 3.4 PE, ND, D and DPE values together with the associated R values for spoilage and 56 pathogenic microorganisms in mango juice 3.5 Zp values together with the associated R2 values for spoilage and pathogenic 61 . .. . . mlcroorgamsms ln mango JUlce 4.1 Lag period and decimal multiplication time for total aerobic bacteria and yeast 81 /mold in pressure treated mango juice 4.2 DO.5 values for the changes in eBrix) values of HP treated mango juice during 84 storage at different temperatures 4.3 D5 values for the changes in pH values of HP treated mango juice during storage at 87 different temperatures 4.4 Dl.2 values for the changes in total titrable acidity of HP treated mango juice 90 during storage at different temperatures 4.5 D20 values for the color changes (L values) of HP treated mango juice during 93 storage at different temperatures 4.6 D20 values for the color changes (b values) of HP treated mango juice during 96 storage at different temperatures 4.7 Temperature sensitivity data for different qu al ity parameters of HP treated mango 99 JUlce

Xl LIST OF FIGURES

3.1 Effeet of pressure level and pressurization time on the destruction of spoilage 54 . .. . . mleroorgamsms ln mango JUlee 3.2 Effeet of pressure level and pressurization time on the destruction of pathogenie 55 . .. . . mleroorgamsms ln mango JUlee

3.3 Pressure sensitivity ofDp values for spoilage pathogenie mieroorganisms in 62 mangojuiee 3.4 Growth of E. coli 0157: H7 in untreated mango juiee during storage at 20, 12 and 63 4°C 3.5 Effeet of applied pressure lethalities on growth of E. coli 0157 :H7 in mango 64 during storage at 20, 12 and 4°C 4.1 Effeet of applied pressure lethality on the growth of aerobie baeteria during 77

storage ofmango juiee at 20 oC (.), 12°C (.) and 4°C (.~) 4.2 Effeet of applied pressure lethality on the growth ofyeast and mold during 78 storage ofmango juiee at 20 oC (.), 12°C (.) and 4°C (.) 4.3 Effeet of pressure treatment (550MPa) on the growth ofaerobie baeteria and 79 yeast and mold during storage ofmango juiee at 20 oC (.), 12°C (.) and 4°C (.) 4.4 Effeet of applied pressure lethality on the °Brix values during storage of mango 83 juiee at 20 oC (.), 12°C (.) and 4°C (.) 4.5 Effeet of pressure treatment (550MPa) on °Brix values during storage ofmango 85 juiee at 20 oC (.), 12°C (.) and 4°C (.) 4.6 Effeet of applied pressure lethality on the pH during storage of mango juiee at 20 86 oC (.), 12°C (.) and 4°C (.) 4.7 Effeet of pressure treatment (550MPa) on pH during storage ofmango juiee at 20 88 oC (.), 12°C (.) and 4°C (.) 4.8 Effeet of applied pressure lethality on the total titrable aeidity during storage of 89 mango juiee at 20 oC (.), 12°C (.) and 4°C (.) 4.9 Effeet of pressure treatment (550MPa) on total titrable aeidity during st orage of 91 mango juiee at 20 oC (.), 12°C (.) and 4°C (Â)

Xll 4.10 Effect of applied pressure lethality on the L values during storage of mango 92 juice at 20 oC (.), 12°C (.) and 4°C ( .. ) 4.11 Effect of pressure treatment (550MPa) on L values during storage ofmango 94 juice at 20 oC (.), 12°C (.) and 4°C ( .. ) 4.12 Effect of applied pressure lethality on the b values during storage of mango 95 juice at 20 oC (.), 12°C (.) and 4°C ( .. ) 4.13 Effect of pressure treatment (550MPa) on b values during st orage ofmango 96 juice at 20 oC (.), 12°C (.) and 4°C ( .. ) 4.14a Temperature sensitivity ofthe Dm values for the °Brix, pH and total 97 titrable acidity, color (L and b values) 4.14b Temperature sensitivity of the Dm values for the changes in total 98 titrable acidity and color (L and b values) of pressure treated mango juice

xiii CHAPTERI

INTRODUCTION

Non-thermal food processmg technologies, such as high pressure (HP) processing, high electric field pulses and high intensity ultrasound applications or the utilization of antimicrobial agents, have been the focus of research in recent years due to their potential as novel methods of food preservation with minimal loss of organoleptic and nutritional quality to food. Thermal processing is the main method to achieve microbial stability and safety in food products. Although this technology is effective, economical and readily available, it has, in many cases, undesirable effects on food quality that must be minimized. This can be achieved by HP processing at refrigerated, ambient or moderately high temperature conditions which inactivates pathogenic and spoilage microorganisms with little or no change to product "freshness" as compared with conventional thermal processing. Therefore, HP processing is considered the main non-thermal alternative technology since the instant and homogeneous transmittance of pressure throughout a food system represents a major advantage over conventional thermal processing in which large temperature gradients exist and the process is based on minimal sterility achieved at the coldest point in the container.

Righ pressure processing was first discovered in the 19th century by Rite who found that an exposure of approximately 600 MPa for one hour at room temperature couid extend the sheif-life of raw milk by four days. Aimost a century Iater, first commercial food products that incorporated HP processing into its treatment were introduced in Japan Gams and jellies). Currently, HP products such as apple juice, orange

1 JUlce, avocado, oysters, ham etc are commercialized in many countries such as USA,

Japan, Canada and France (Cheftel, 1995; Knorr, 1993).

Mango is one of the most important fruits in Asia, and currently ranks fifth in

total production among major fruits world wide, after bananas and plantains, citrus (aU

types), grapes and apples. It is one of the most popular and widespread tropical fruits in

the world, and according to F AO statistics it has increased in worldwide production by

nearly 50% between the years of 1971 and 1993. Mangos are of tropical origin and are

sensitive to chilling injury if stored below 10 oc. Because of the short shelf-life of fresh

mango which limits its marketability, the surplus must be converted to other stable forms

by processing and incorporation to various mango products in order to minimize the post

harvest losses. Most mangos are consumed fresh and as a fruit slice, but sorne non­

fibrous pulpy varieties of mango are used for processing. The fibrous ones make a good

choice for juice since they cannot be effectively consumed in any other way. Both ripe

and unripe mangos are processed into a variety of products, and are also frozen in sucrose

syrup or as a puree, and are popular in beverages (Nanjundaswamy, 1997). The market

for processed mango products especiaUy juice, nectar, puree and other mango juice

beverages has considerably increased in recent years although it is generally insignificant

when compared with the fresh fruit trade. This is because the combination of aIl

processed mango products currently represents less than 0.2% of the total mango

production (Victor et al., 2004).

Currently, mango is processed and preserved by thermal pasteurization. The

conventional thermal processing of mangoes leads to quality degradation in terms

physico-chemical characteristics and thus presents a challenge to the food industry. High-

2 pressure processing is an alternative to heat and/or chemicals and may have potential as a food processing method. There have been sorne studies on HP processing of several fruit juices such as apple (Riahi et al., 2003), orange (Basak et al., 2001; Boffet al., 2003 Bull et al., 2004 Nienaber et al., 2001; Garcia et al., 2001; Goodner, et al., 1998), pineapple

(Aleman et al., 1994, 1996) guava, strawberry and grape fruit juice (Butz et al., 1997;

Cano et al., 1997).

The mechanism of HP and its effects on food components, microorganisms are different from the conventional processing methods. A first order reaction kinetics is commonly employed for microbial destruction. In HP, a two-step destruction behavior is possible (Basak et al., 2002; Mussa et al., 1998, 1999; Pandey et al., 2003; Ramaswamy et al., 2003). Establishment and understanding of high pressure action on microorganisms and food components are necessary. Inactivation studies on the HP destruction of pathogenic and spoilage microorganisms should be carried out, not only after processing but also during storage, to demonstrate safety and stabil ity of high pressure treated foods.

When considering microbial safety and stability, the required pressure treatment is dependent on the target pathogenic microorganisms to be eliminated as weIl as its pressure resistance characteristics.

The exploration of high-pressure processing on mango juice, especially, has not received much attention and only a couple of studies have focused on HP processing of mango juice (Boynton et al., 2002; Otero et al., 2000). None of the studies focus on the detailed evaluation of HP destruction kinetics of spoilage and pathogenic microorganisms, or their changes during storage.

3 Therefore the objectives of this research were

1) To evaluate the effects of HP processing conditions (pressure level and holding

time) on the inactivation kinetics of selected spoilage and pathogenic . .. mlcroorgamsms ln mango Jutce.

2) To establish and verify high pressure treatment conditions of mango juice based

on the kinetic data of the most pressure resistant pathogen; to carry out challenge

studies by subjecting the product inoculated with the pathogen to the established

pressure process and evaluate their growth and recovery at various storage

conditions.

3) To evaluate growth of indigenous microorganisms and changes in sensory quality

attributes of high pressure treated mango juice during storage.

4 CHAPTER2

REVIEW OF LITERA TURE

Mango and Mango Products

History, Description and Importance

Mango (Mangifera indica L.) is a member offamily Anacardiaceae has been cultivated in lndia for more than 4000 years. Beginning in the 16th Century, mangos were gradually distributed around the world, reaching the Americas in the 18th Century. The first recorded introduction into Florida was Cape Sable in 1833. Mangos become very large trees. In height a mango tree may reach 35-40 meters (130 feet) with a span of 10 meters (33 feet) at the top.

Botanically, mango fruit, is a large, fleshly drupe, and as such the pericarp is divided into three layers a thin outer skin: the epicap, an edible fleshy middle layer: the mesocarp and an inner hard shell: the endocarp which surrounds the single seed . They are variable in size, from 10-25 cm long and 7-12 cm diameter, and may weigh up to 2.5 kg . Ripe mango are said to be excellent source ofvitamins C, Bl, B2 and provitamin A (Mukherjee, 1997).

Production Statistics

Major world producers of mango inc1ude India, Pakistan, Indonesia, Mexico, Brazil, and the Philippines. Other important producers are Australia, South Africa, Israel, Egypt, and the

D.S. According to FAO (2000), mango is cultivated in at least 87 countries worldwide with Asia being the main producer which comprised about 75% of the world' s production of fresh mangoes. lndia continues to be the main producing country followed by China and other

5 countries. Nine countries account for almost 86% of the total world production (Tables 2.1 and

2.2).

Table 2.1: Distribution ofworld mango production in year 2000 by continent (Source www.FAO.org)

Continent Production 103 tones % Asia 19,262 76.9 America North and Central America 2,322 9.27 South America 1,124 4.49 Africa 2,253 9.00 Oceania 44 0.18 Europe ( includes Israel) 35 0.14 World 25,040

Table 2.2: Main mango producing countries in year 2001 (Source www.FAO.org)

Country Production 10;J tones 1996 1998 2001 India Il,000 Il,000 11,500 China 2,008 2,142 3,215 Thailand 1,400 1,350 1,350 Mexico 1,189 1,461 1,1459 Pakistan 908 914 938 Indonesia 1,000 605 950 Philippines 480 700 884 Nigeria 500 500 729 Brazil 610 456 540

6 Mexico is the main mango producing country in the American continent and is the largest mango exporting country supplying 41 % of the world market. The quantity of processed mango fruit that reaches the world market is insignificant compared with the fresh fruit trade. Export figures for mango pulp (13-18° Brix) according to the FAO statistics reached 6,885 tons compared with 4,711 tons in 1997. Thailand is the main exporter, with 6,671 tons. Sorne concentrated pulp, at 28° Brix, also reached the European market mainly from India and

Colombia. Mango juice exports, increased from 16.9 tons in 1997 to 18.6 in 2000, with India and Egypt supplying in bulk. Within Latin America, Mexico exports mango juice to Brazil

(Victor, 2004).

Important Mango Cultivars

Several hundreds of mango cultivars exist (Table 2.3). In mango orchards, several cultivars are always grown intermixed to improve cross-pollination. In India, the commonest cultivar is 'Alphonso', known as the King of Mangoes. The best 'Alphonso' mangos are reputed to come from the town of Ratnagiri in Maharashtra. About 80% of mangos in UK supermarkets are of the single cultivar 'Tommy Atkins', which dominates the world export trade. It travels well and has a good shelf-life, but does not have the same flavour as sorne less common varieties obtained from Asian shops. (adapted from http://www.horticultureworld.net/cultivars.htm).

Post Barvest Technology

Mangoes are generally harvested at physiologically mature stage and ripened for optimum quality. Fruits are hand-picked or plucked with a harvester. After harvest the fruits are usually heaped under a tree on the ground. Bruised and injured fruits develop brown to black

7 Table 2.3: Important mango cultivars in major producing countries

Country Cultivars Australia 'Kensington Pride', 'Banana', 'EarIT)gold', 'Glenn', 'Haden', 'Irwin', 'Keitt', 'Kent', 'Zill' Bangladesh 'Aswina', 'Fazli', 'Gopal Bhog', 'Himsagar','Khirsapati', 'Langra', 'Kishan Bhog', 'Kohinoor', 'Kua Pahari', 'Mohan Bhog' Brazil 'Bourbon', 'Carlota', 'Coracao', 'Espada', 'Itamaraca', 'Maco', 'Magoada', 'Rosa', 'TommT) Atkins'

China 'Bahu', 'Guixiang', 'Huangpi', 'Huang-jU" 'Macheco', 'Sannian', 'Yuexi No. l' Costa Rica 'Haden', 'Irwin', 'Keitt', 'Mora', 'TommT) Atkins' Ecuador 'Haden', 'Keitt', 'Kent', 'TommT) Atkins' Eg-jpt 'Alphonso', 'Bullock's Heart', 'Hindi Be Sennara', 'Langra', 'Mabrouka', 'Pairie', 'Taimour', 'Zebda'

Guatemala 'Haden', 'Kent', 'TommT) Atkins' Haiti 'Francine', 'Madame Francis' India 'Alphonso', 'Banganapalli', 'BombaT)" 'BombaT) Green', 'Chausa', 'Dashehari', 'Fazli', 'Fernandian', 'Himsagar', 'Kesar', 'Kishen Bhog', 'Langra', 'Mallika', 'Mankurad, 'Mulgoa', 'Neelum', 'Pairi', 'Samar Behisht Chausa, 'Suvarnarekha', 'Totapuri', 'Yanraj', 'Zardalu', 'Amrapali', 'Bangalora', 'Gulabkhas' Indonesia 'Arumanis', 'Dodol', 'Gedong', 'Golek', 'Madu', 'Manalagi', 'Cengkir', 'Wangi' Israel 'Haden','TommT) Atkins','Keitt', 'MaTja', 'Nimrod', 'Kent', 'Palmer' KenTja 'Boubo','Ngowe','Batawi' MalaT)sia 'Arumanis', 'Kuala Selangor 2', 'Golek', 'Apple Rumani', 'Malgoa', 'Apple Mango', 'Maha-65', 'Tok Boon' Mali 'Amelie', 'Kent' Mexico 'Haden', 'Irwin', 'Kent', 'Manila" 'Palmer', 'Sensation', 'TommT) Atkins', 'Yan DTjke' MTjallmar 'Aug Din', 'Ma Chit Su', 'Sein Ta Lone', 'Shwe Hin Tha' Pakistan 'Anwar Ratol', 'Baganapalli', 'Chausa', 'Dashehari', 'Gulab Khas', 'Langra', 'Siroli', 'Sindhri', 'Suvarnarekha', 'Zafran' Peru 'Haden', 'Keitt', 'Kent', 'TommT) Atkins' Philippines 'Carabao', 'Manila Super', 'Pico', 'BinoboT)', 'Carabao', 'Dudul', 'Pahutan', 'Senora' Singapore 'Apple Mango', 'Arumanis', 'Golek', 'Kaem Yao', 'Mangga Dadol' South Africa 'Fascell', 'Haden', 'Keitt', 'Kent', 'Sensation', 'TommT) Atkins', 'Zill, Sri Lanka 'Karutha Colomban, Willard, Yellai Colomban, Petti amba, Malwana amba, Parrot Mango and Peterpasand, Dapara, Hingurakgoda Thailand 'Nam Doc Mai', 'Ngar Cham', 'Okrong', 'Rad', 'Choke Anand', 'Kao Keaw', 'Keow SavoeT)', 'Pimsenmum' USA 'Keitt', 'Kent', 'TommT) Atkins' Yenezuela 'Haden' 'Keitt' 'Kent' 'TommT) Atkins Yietnam Combodiana

8 spots during st orage making the fruits unattractive. Moreover, injuries to the peel or to the stalk end serve as avenues for invasion of microorganism and lead to rotting of the fruits. The post harvest losses in mangoes have been estimated in the range of 25-40 per cent from harvesting to consumption stage. If proper methods of harvesting, handling, transportation and storage are adopted, such losses could be minimized. (Johnson et al., 1997)

a) Maturity: The mango fruits should be harvested at green mature stage. The harvest maturity in Dashehari and Langra cultivars reaches 12 weeks after fruit set, while in Chausa and

Mallika it takes about 15 weeks. The best way to observe maturity in mango is the color of the pulp which turns cream to light yellow on maturity and hardening of the stone.

b) Harvesting: The harvesting in mango should be done in the morning hours and fruits should be collected in plastic trays and kept in shades. The fruits should not be allowed to fall on the ground as the injured fruits cause spoilage to other healthy fruits during packaging and storage. Fruits harvested with 8-10 mm long stalks appear better on ripening as undesired spots on skin caused by sap bum are prevented. Such fruits are less prone to stem-end rot and other storage diseases.

Fruits harvested by stick are injured / bruised due to impact resulting in decay, poor quality and attract low priee. Traditional methods consume a lot of energy. To overcome these problems, a simple, low cost and portable mango harvesting device has been designed and developed at CISH, Lucknow, India. Mango fruits are taken into the pouch and held between the divider and knife and as the device is pulled the blade cuts the pedicel. The fruits are then conveyed through a nylon chute to collecting boxes without bringing down the device every time. This saves time and protects fruits from mechanical damage due to impact. It also protects operator's hand from the sap which oozes out from the point of detachment.

9 c) Grading: If the fruits are graded according to their size, weight, color and maturity, both the producer and consumer are benefited. It has been observed that bigger size fruits take 2-

4 days more time in ripening than smaller ones. Rence, packaging of smaller fruits with larger ones should be avoided to achieve uniform ripening. Immature, overripe, damaged and diseased fruits should be discarded.

d) Packaging: Wooden boxes are commonly used for packaging and transportation of mango fruits. Under dynamic transport conditions nails come out due to vibration and puncture the fruits, which result in bruising, decay and low price of fruits. Further, tao much ventilation affects the quality of fruits due to shrinkage, loss in weight, color, etc. To overcome these problems, CFB (carton fireboard) Boxes of 5 kg and 10 kg capacity for packing and shipping of mango fruits successfully as an alternative to traditional nailed wooden boxes. The use of CFB boxes for packaging for the domestic market is also the need of the hour due to scarcity of the wood and environmental concerns. For export purposes, CFB boxes are already in extensive use.

Paper scraps, newspapers, etc., are commonly used as cushioning material for the packaging of fruits which prevent them from getting bruised and spoiled during storage and transportation.

Low-density polyethylene (LDPE) lining has also been found beneficial as it maintains humidity, which results in lesser shrinkage during storage. Wrapping of fruits individually (Unipack) with newspaper or tissue paper and packing in honeycomb structure helps in getting optimum ripening with reduced spoilage. Normally the lid of the wooden boxes is nailed with an area of 5 to 7 cm high in the middle. This puts· pressure on the fruits during transport and results into reduced quality.

e) Storage: Storage is essential for extending the consumption period of fruits, regulating their supply to the market and also for transportation to long distances. The mature green fruits

10 can be kept at room temperature for about 4-10 days depending upon the variety. Shelf life of fruits could be extended by pre-cooling, chemical treatments, low temperature, etc.

The harvested fruits are precooled to 10-12°C and then stored at an appropriate temperature. The fruits of Dashehari, Mallika and Arnrapali should be stored at 12°C, Langra at

14°C and Chausa at 8°C with 85-90 per cent Relative Humidity. The fruits could be stored for 3-

4 weeks in good condition at low temperature. The problem of chilling injury at low temperature can be overcome by keeping the fruits in 0.5 per cent ventilated polythene bags.

Calcium infiltration is an improved technique of extending the st orage life of Dashehari fruits. The fruits are kept in calcium chloride solution (4%) at sub- atmospheric pressure of 500 mm Hg for 5 minutes. The treated fruits can be stored at low temperature (12°C) for 27 days.

It is a general practice to harvest fruits early in the season (premature stage) to capture early market. These fruits do not ripe uniformly without any ripening aid. Su ch fruits could be ripened uniformly by dipping in 750 ppm ethrel (1.8 ml/litre) in hot water at 52+2°C for 5 minutes within 4-8 days under ambient conditions. Mature fruits can similarly be ripened with lower doses of ethrel for uniform color development.

f) Transportation: The truck has been adopted as the most convenient mode of transport due to its easy approach from the orchards to the market. Refrigerated vans are commonly for long distance transport and export purposes as they would help in reducing the post harvest losses.

Processing of Mango

In most mango growing countries the fruit is generally consumed fresh. Non-fibrous pulpy mango varieties are normally used for processing. Mangoes are processed at both the ripe

11 and unripe stages of maturity for conversion into various products Table (2.4). Green mangoes are processed into pickles, brine stock and chutney and drum dried green mango powder. Most recent developments are instant mango pickles, drum-dried green mango powder and raw mango beverage base. Ripe mangoes are processed into canned slices, pulp, beverages like ready- to­ serve (RTS), nectar andjam etc. (Nanjundaswamy, 1991).

Raw Mango Products

i. Mango slices in brine - Raw mango slices dipped in 1.5 per cent potassium

metabisulphite solution for 5 minutes, drained and then mixed with equal amounts of

powdered salt are stored in polythene pouches. These slices could be used later for

product preparation.

iÏ. Pickle: Different kinds of pickle are made from raw mango fruits. Small slices from

mature green fruits are mixed with sufficient quantity of powdered salt and kept in sun

till green color disappears from the slices. Spices are then mixed in it and the slices

dipped in boiled but cool oil. The prepared pickle is filled in c1ean glass jars and cured for

couple of days in sun. Oilless pickle is another popular form of pickle from mango. The

fruits are peeled, sliced into small pieces and mixed with 20 per cent salt, 7.5 per cent

chilli powder and 1 per cent asafoetida according to the weight of pieces. The whole

mixture is then kept in sun for few days with frequent stirring and then filled in c1ean

glass jars. The jars are stored in c1ean dry place.

iii. Dried green mango powder: Mango slices from peeled mangoes are dipped in 1.5 per

cent solution of potassium metabisulphite for 5 minutes and dried in sun or solar

12 dehydrator. It is commonly known as "Amchoor" and is obtained by grinding the dried

slices and thereafter, stored in air tight containers.

iVe Green mango beverage: Whole raw mangoes roasted or boiled with equal amount of

water and the pulp is extracted. To this extract, sugar, water, salt, mint, cumin, black

pepper and citric acid are mixed. Mixture is heated and filled in clean glass bottles. It

may be used as raw mango squash.

Ripe Mango Products

i. Frozen products: Pasteurized Tothapuri mango pulp frozen in the form of slab in

polythene bag with added ascorbic acid was found to keep well for an year when stored at

-18 oC (Ramana et al., 1984).

ii. Mango slices in syrup: Firm ripe mango fruits are peeled and cut into slices. The slices

are then transferred to boiling syrup containing 40 per cent sugar, 0.3 per cent citric acid

and 350 ppm sulphur dioxide for 5 minutes. The slices are filled in clean jars and boiling

syrup is poured until aIl the slices are covered. FinaIly, the jars are covered with lids and

sealed with wax. iii. Canned man go pulp: The development of mango beverage industry has increased the

demand of canned mango pulp. Addition of ascorbic acid to the pulp is reported to be

helpful in better retention of quality. Bulk preserved mango pulp in barrels using 1000

ppm sulfur dioxide could be later used in preparation of jam, fruit bar and squash. iVe Mango Beverages (Juice and Nectar): Mango juice may be prepared by mixing 1/3rd

of fresh or stored pulp with 2/5th of water. Sugar and citric acid are so added that total

13 soluble si ides (T.S.S.) and acidity of the product reaches to 15 per cent and 0.3 per cent,

respectively. The mixture is heated to 95°C, filled hot in clean, sterilized bottles and

crown corked. The bottles are sterilized in boiling water for 10-15 minutes, cooled to

room temperature and stored. The procedure for nectar preparation is similar to that of

juice except that the pulp percentage is reduced to 15 per cent. The choice of most of the

suitable beverages is the subject of investigation of in many countries (Krishnamurthy et

al., 1984).

v. Mango Leather or Aam Papad: Homogenized mango pulp is taken and potassium

metabisulphite is added to it @ 2 g per kg of pulp. The pulp is then spread on trays and

kept for drying in solar dehydrator or sun. After drying of one layer, another layer is

spread over it and kept for drying. The process is repeated as per desired thickness.

Finally they are cut into pieces and wrapped in butter paper or polythene cellophane

sheet. Many other dehydrated products like intermediate moi sture mango, osmotic

dehydrated mango, dehydrated mango slices, mango cereal flakes, and strained baby food

and mango powder has been developed. vi. Mango jam and Mango TofTee: Mango jam is conventionally produced in lndia for

export. Mango toffee can be prepared by mixing mango pulp with sucrose, glucose, skim

milk powder and hydrogenated fat and cooking in a steam jacketed vessel. The cooked

mass is transferred to a smoothly greased tray and allowed to cool.

14 Table: 2.4: Utilization of mango for processing (products range)

Green mangoes Ripe mangoes Mango waste

Peel Stone Mango pickle Mango slices in syrup Pectin Starch

Mango chutney Mangojuice Mango Syrup Fat

Mango slices in brine Mango nectar Aroma Concentrate Colorant Mango pulp Biogas

Mango Squash Dehydrated slices or powder Mango RTS beverage Mango syrup Raw mango beverage Mangojam Mango fruit bar Mango powder Strained baby foods Mango cereal flakes Mango concentrate Mango aroma concentrate Sturctured mango products

Waste Utilization

During the processing of mango, peel and stone are generated as waste (40-50% of total fruit weight). They are rich in various nutrients and many value added products could be obtained from them. Good quality jelly grade pectin (6.1%) and edible fibre (5.4%) could be extracted from ripe mango peel. Acceptable quality vinegar (5.2% acetic acid) and citric acid (20 g / kg peel) could be obtained from mango peel through microbial fermentation. Mango peel having low protein value (3.9 %) is a poor quality animal feed. The peel could be protein enriched more than five times (20 %) by solid state fermentation using Aspergillus niger. Mango

15 peel has lignocellulosic composition and hence its complete break down is difficult. It' s co­ composting with cowdung in 3: 1 ratio results in its successful biodegradation.

Mango kernel contains high amount of fat and starch. The oil extracted from kernel is of good quality and could be used in cosmetic and soap industries. The kernel flour (starch) after mixing with wheat or maize flour is used in chapaties. About ten per cent alcohol could be obtained from mango kernel by co-culture fermentation. In food processing industries, various enzymes are invariably used for pulp liquefaction, juice clarification, etc. Enzymes such as cellulase and pectinase from mango peel and amylase from mango kernel could be produced by microbial fermentation (Nanjundaswamy, 1991).

High Pressure Processing

Introduction

High pressure (HP) processing, also described as high hydrostatic pressure (HHP), or ultra high pressure (UHP) processing, subjects liquid or solid food s, with or without packaging, to pressures in the range of 100-1000 MPa. The processing temperature during pressure treatments can be adjusted from below O°C to ab ove 100°C. High pressure processing of foods is one of the non-thermal processing methods, which is rapidly gaining in popularity worldwide as an alternative to conventional food processing. The ability of HP to inactivate pathogenic microorganisms at low temperatures without the use of chemical preservatives, resulting in almost complete retenti on of nutritional and sensory characteristics of fresh foods without sacrificing shelf-life is one of its major advantages. The technology of high pressure originated from applications in material science such as ceramics, super alloys and artificial diamonds. The

16 first application of high pressure processing of foods was done in the 19th century when scientists discovered that high pressures could kill bacteria.

A brief review on high pressure (HP) applications is presented in this part of the thesis.

Special emphasis is placed on the application ofthis technology to fruit and fruit products. Sorne basic principles ofhigh pressure technology, advantages and applications are dealt with briefly.

Dimension and Nomenclature used for Pressure

Pressure is a measure of the force exerted per unit area and in the International System of units

(SI), the units are N/m2 or commonly designated as Pa. Other units (Metric and British) are also frequently used of pressure. Table 2.5 shows sorne commonly used units for designating a unit atmospheric pressure. The pressure levels used in HP application for food processing are too high to be measure in Pascals (Pa). Rence they are mostly measured in mega-pascals or MPa. 1

3 6 MPa = 10 kPa = 10 Pa.

Table 2.5: Units for atmospheric pressure

One atmosphere 1.0 atm SI Unit l.0l3 x 10'Pa 101.3 kPa or 0.10l3 MPa Barometric l.0l3 bar Metric 1.034 kg/cm:.! British 14.7 lb/in:.! or 14.7 psi

17 High Pressure Processing: Basic Principles

Two fundamental principles, which determine the behavior of materials under pressure, are of particular interest for food application.

The isostatic principle which states that pressure acts directly and uniformly on the entire sample. The product thus undergoes a complete homogeneous treatment process in which, in contrast to the thermal or mechanical processes, no part of the product is subjected to over treatment. This is the most unique property of HP processing. Rence pressure is transferred instantly and uniformly throughout the food system. Thus the application of HP processing is independent of sample size and geometry. This property of HP processing allows not only the flexibility ofusing different product packages, but also the compliance ofprocess scale-up. If the temperature gradient within the pressure vessel is not significant, the optimum process condition acquired from laboratory scale experiments can be directly translated to the pilot or plant scale processes, because pressure itself does not make any energy gradient within the pressure vessel.

The work of compression during the HP treatment is accompanied by a uniform temperature increase. This adiabatic heating rate is specific for each chemical compound (water -3°C/100

MPa, fats and oils -6-8 °C/100 MPa) and is reversible upon pressure release.

Le Ch ateliers princip le, which states that any phenomenon that is linked to a reduction in volume, is enhanced by pressure and vice versa. HP processing affects non-covalent bonds

(hydrogen, ionic, and hydrophobic bonds) as they are sensitive to pressure. This means that low molecular weight components responsible for nutritional and sensory quality are not affected, whereas the high molecular weight components are sensitive because their secondary and tertiary structures are important in their functionality determination. Pressure leads to increased ionization, since water molecules arrange more compactly around electric charges. This results

18 in more or less pronounced negative and reversible pH shifts dependant on the chemical nature of the buffer.

Advantages of HP Processing

According to Knorr (1993), the following advantages have been credited to High pressure processing of foods:

1) High pressure is transferred instantly and uniformly throughout food system.

Thus the application of HP processing is independent of sample size and

geometry.

2) HP processing allows not only the flexibility of product package but also the

compliance of process scale-up.

3) HP processing has no energy gradients and hence can be easily scaled up from

laboratory to industrial applications.

4) HP processing inactivates most vegetative microorganisms.

5) HP processing affects biopolymers, but allows quality retention, such as color

and flavor.

7) HP processing can be used to improve product functionality.

8) HP processing pro duces no effiuents, and hence is environmentally friendly.

Historical Timeline for HP Processing

The first scientific report on the application of HP processing is generally traced back to

1895/1899 depending on the source citations. The origin is generally credited to Bert Hite of

West Virginia, although sorne literature suggests it could have come earlier. The following Table

2.6 gives a historical time line for the HP processing offoods (grad.fst.ohio-state.edu/hpp).

19 Table 2.6: Historical timeline of high pressure processing of foods

1895 H. Robert uses high pressure to kill bacteria

1899 Bert H. Rite at the West Virginia Agricultural Experimental Station examined pressure effects on milk, meat, fruits and vegetables.

1914 P. W. Bridgman coagulated egg albumen under high pressure

1990 First commercial products like fruit juices, jams, fruit toppings and tenderized meats introduced in Japan

1995 Orange juice commercialized in France

1997 Market introduction of guacamole in the US and sliced cooked ham in Spain.

1999 Oysters introduced in the US

2000 Range of salsas launched in the US market

Commercial HP Processed Products

The reported commercial products inc1ude: apple cider, apple juice, apple sauce (single serving packs), avocado (halves, pulp) beef, chic ken, cod (both dried and salted), fruit purees, fruit smoothies, guacamole, jams/jellies, limeade/lemonade, mussels, onions (chopped), orange juice, oysters (shucked, in-shell, and shooters), ham, ready-to-eat meal kits (beef and chicken), salsa, seafood salads, strawberry juice (Source: Virginia tech web site www.hpp.vt.edu).

High Pressure Processing Equipment

HP applications in food processing make use of pressures in the range of 100-1000 MPa, which ranges from the level at the bottom of the sea to ten times higher. Special equipment is

20 needed to generate and endure such high pressures. In deep sea, the hydrostatic pressure increases by one atmospheric pressure for every ten-meter depth of water.

An iso-static HP unit consists mainly of HP enclosures (chamber, lids and closure pins), pressurization and depressurization devices, appropriate valves, control s, temperature control devices, and a handling system. The HP vessel and its closures can be of different designs, and obviously each will having its own advantages and limitations. The pressurization system can be of the direct type (with ram piston, assembled in the vessel), indirect type (with an external high pressure pump) or in a limited way, one with both the se types in combination, and is usually combined with appropriate devices for heating of the pressure medium. The pressure build up time is determined by the pump capacity, the compressibility of the pressurization medium and of the product to be compared, and more importantly the size of the pressure chamber. The components of a high pressure processing system can be configured to treat foods packaged in a flexible container in a batch process or arranged to process unpacked liquid foods in a semi­ continuous manner (USDA, 2000).

Batch processing system

For batch operations, the packaged food is loaded into the pressure vessel, vessel is sealed and the process water is pumped into the vessel to displace the air. The pumping of the fluid in to the chamber builds up the pressure after closing the vessel. After the desired holding time at an appropriate pressure level, the pressure is release using a pressure release valve, the water in the chamber can be drained or reused after the product is be removed from the pressure vessel. The displacement of air prior to the pressure treatment is done to reduce the pumping costs by eliminating air compressing (USDA, 2000).

21 Semi-continuous HP system

Semi-continuous systems are currently used for treating liquid or viscous food products without packaging them prior to treatment. In the semi-continuous system, three or more batch cylinders are working together in a synchronized way to maintain a continuous flow of the product. The liquid product after being filled in to the treatment chamber is treated the same way.

Rather than pumping the HP fluid into the chamber, the fluid pushes a movable piston in the chamber to result in compression and pressurization of the liquid. Upon depressurization, the piston retracts to its original position. The treated liquid will be discharged by moving the piston again using with the discharge valve open which moves it from the pressure vessel to a sterile hold tank and then filled into a pre-sterilized containers. To complete the cycle, the piston then is pu shed back to its original position by the product to be treated. Pulsed HP processing systems can be adapted from semi-continuous and batch processing systems by programming a series of treatment cycles of short duration (USDA, 2000).

High Pressure Proeess Equipmellt Mallufacturers

A detailed listing of the different manufactures of HP processing equipment is available at a web site maintained by the Ohio-State University: www.grad.fst.ohiostate.edu/hpp.

The following are sorne listed:

ACB Pressure Systems has developed a range of high-pressure equipment, the HYPERBAR pasteurizers, specially designed to treat food products.

Elmhurst Researeh, Ille. designs and manufactures ofultra high pressure vessels in general and for food processing.

Ellgilleered Pressure Systems Ille. supplies laboratory scale and industrial high­ pressure processing equipment.

22 Flow/Avure Pressure Systems manufactures both batch presses and continuous systems.

Kobelco (Kobe Steel) offers a whole range of laboratory and industrial high­ pressure processors.

Mitsubishi Heavy Industries manufactures a high-pressure food processing test system.

National Forge supplied high-pressure research equipment in the past.

Resato specializes in the design and manufacture of high-pressure components, high-pressure test equipment, and complete systems.

Stansted Fluid Power manufactures high-pressure engineering equipment and systems for a wide range of industrial and research applications.

Stork Food and Dairy System and Uhde Hochdrucktechnik offer high­ pressure processmg equipment for the treatment of food, pharmaceuticals or cosmetics.

High Pressure EfTects on Microorganisms

Inactivation Mechanism

High pressure affects the morphology of cell membrane or biochemical reactions of microorganisms, which leads to the inactivation of microorganisms. Primarily, the cell membrane is considered to be the major target for the pressure-induced inactivation of microorganism, and it is generally accepted that the leakage of intracellular constituents through the permeabilized cell membrane is the most direct reason of cell death by high-pressure treatment. However, if the applied pressure is not severe enough to induce a total permeabilization of cell, permeabilization takes place only on the outer membrane and, as in the case of Gram-negative bacteria, the permeabilized membrane can be rapidly restored after pressure release (Hauben et al., 1996). This result was also verified by Ritz et al. (2000) with the

23 membrane proteins of Salmonella spp. They observed that most of the outer membrane proteins disappeared after HP treatment, whereas the cytoplasmic membrane bounded proteins were still retained after HP treatment. The fluidity of cell membrane plays an important role in the susceptibility of microorganisms to pressure treatments. Conversely, increased membrane fluidity can protect the ceUs against pressure inactivation (Steeg et al., 1999).

The denaturation of key enzymes in microorganism by pressure has been regarded another important reason of celI death. Because intracellular enzymes seemed not to be a determining factor of pressure resistance (Simpson and Gilmour, 1997), membrane bounded

ATPase was considered to be a key enzyme (Wouters et al., 1998). Indeed, the observations on acid effiux and ATP pool suggest that ATPase is affected by HP treatment faster than the intracelIular enzyme system related to glycolysis.

Bacterial spores are known to be pressure resistant, and the inactivation mechanism is different from that of other vegetative microorganisms. Under moderate and pulsating pressures at moderate temperatures, it was assumed that pressure caused inactivation of spores by first initiating germination and then inactivating germinated forms (Sale et al., 1970). Biochemically, binding of a germinant to it' s receptor site is be1ieved to promote the germination process

2 followed by the efflux of Ca + and other ions and the influx of water into the spore, resulting in the activation of spore specific cortex lytic enzyme (Wuytack, et al., 2000). This germination process can be enhanced by pressure treatments because the volume of system decreases during germination as a result of increased solvation of spore' s components (Clouston and Wills, 1969;

Heinz and Knorr, 1998). According to Le Chatelier's princip le, any reaction which results in a decrease in activation volume is accelerated by the HP process. It was also suggested that pressure-induced germination involves activation of physiological pathways and is therefore not

24 merely a physico-chemical process in which water is forced into the spore protoplast, because inhibitors of nutrient-induced germination also inhibit pressure-induced germination (Wuytack, et al., 2000).

Critical Factors for the HP Destruction ofMicroorganisms

There are many factors, which affect the inactivation of microorganisms by HP treatment.

Pressure level, pressure mode, treatment time, pH, water activity (aw) and treatment temperature are aIl considered critical parameters and various combinations of these parameters have been investigated and the general deductions are found to be as follows:

1) The pressure level and pressurization time is directly proportional to the rate of

inactivation.

2) High pressure can be applied in either static mode or in a dynamic mode. The effect of

mode is directly related to the level of pressurization and the pressurization time.

Dynamic pressurization can be either pulse pressurization or cyclic pressurization, which

is a combination of pulse and/or static pressurization. Pulse pressurization has significant

effect on the destruction of microorganisms (Hayakawa et al., 1994; Basak and

Ramaswamy, 1996; Aleman et al., 1996; Palou et al., 1998; Mussa et al., 1999; Basak et

al., 2003; Ramaswamy et al., 2003; Riahi et al., 2003).

3) As the treatment time increases, the destruction of microorganisms increases. Longer

ho Id times with higher pressures result in higher inactivation of microorganisms.

4) Microorganisms are more susceptible to pressure at lower pH. Further sub lethally

injured microorganisms induced by HP can be reactivated in nutrition rich environments,

but fail to repair at acidic conditions (Linton, 1999b; Basak et al., 2003).

25 5) Reduction in water activity exerts a protective effect on microorganisms against pressure

treatments (Oxen and Knorr, 1993; Palou et al., 1997).

6) The treatment temperature above or below room temperature tends to increase the

inactivation rate of microorganisms (Knorr and Heinz, 1999).

Inactivation Kinetics

There are several ways in which the rate of microbial inactivation can be reported. It has been generally recognized that when a suspension of microorganisms is heated at constant temperature, the decrease in the number of viable organisms is described by a first order reaction:

- dN/dt = k.N (2.1)

where N is number of viable organisms and k is the first order rate constant for microhial inactivation. Integrating equation (1) using the initial condition, N=No at t=O

ln (NINo) = -k t (2.2)

Equation (2.2) suggests a linear plot of N against t in a semi-Iogarithmic scale. Equation (2) can he expressed in the common decimallogarithm:

ln (NINo) = 2.303 log (NINo) (2.3)

And the decimal reduction time (D value), the time required to reduce the viable organisms by a factor of 10, is defined as:

D = - t / log (NINo) (2.4)

26 The first order inactivation rate (k) and the decimal reduction time (D value) are the most frequently used concept to explain the thermal inactivation of microorganisms. By analogy with

D value for thermal inactivation, decimal reduction times for pressure treatment (Dp value) can be defined. Indeed, several studies have been conducted to calculate the Dp values of various microorganisms, in the same manner as the thermal D values. In this case time represents treatment time at a constant pressure (since temperature can play a role in inactivation that must be fixed as well). Table 2.7 summarized the thermal kinetic parameter computed for different microorganisms in various studies. However, the inactivation curves can also be non-linear when represented on a semi-Iogarithmic scale. Mathematical models based on Gompertz equation

(Heinz and Knorr, 1996; Patterson and Kilpatrick, 1998) or quadratic polynomial expressions

(Reyns et al., 2000) can be used to predict the inactivation of microorganisms with non-linear inactivation characteristics. Such models can also be used predict the inactivation of microorganisms at various pressure-temperature combinations and provide flexibility in selecting optimum processing conditions without compromising microbiological safety.

If a set of kinetic parameters was collected, the pressure sensitivities of the kinetic parameter can be evaluated. The inactivation kinetics of microorganisms or enzymes by HP can be analyzed either by Arrhenius type mode1 or pressure death time model (Basak et al., 1996;

Mussa et al., 1999). The pressure dependence of the kinetic parameters by pressure destruction time model (analogous to the TDT model used in thermal processing) is determined by plotting the decimal logarithm of Dp values versus pressure. From the regression slope of log (Dp) vs. pressure data, the pressure Z-value (Zp) can be determined as negative reciprocal of the slope:

Zp= - (l/slope) (2.5)

27 Table 2.7: Pressure induced decimal reduction times (Dpvalues) ofvarious microorganisms

Microorganism Medium Temp Pressure Dp Value Reference (OC) (MPa) (min) Lactobacillus sake 25 345 10.1 Kalchyanand Leuconostoc mesenteroides 0.1 % peptone 25 345 5.6 et al., 1998. Listeria monocytogenes solution 25 345 4.0 Escherichia coli 0157:H7 25 345 3.0 Staphylococcus aureus 25 345 1.7 Pseudomonas flourescens 25 345 0.6 Salmonella typhimurium 25 345 0.6 Serritia liquefaciens 25 345 0.6

Salmonella seftenberg Chicken medium 23 340 7.1 Metrick et al., Phosphate buffer 23 340 4.2 1989. (pH 7.0) 23 340 7.6 Salmonella typhimurium 23 340 7.4 Pseudomonas flourescens Minced beef 20 150 24.0 Calrez et al., Listeria innocua muscle 20 330 6.5 1993. Citrobacter freundii 20 230 15.0 Broth (pH 3.5;aw 21 310 2.0 Paulo et al., Zygosaccharomyces bailii 0.98) 21 431 2.1 1997. Saccharomyces cerevisiae Orange juice Ns 350 0.6 Parish, 1998a Ns 500 0.1 Listeria monocytogenes Fresh pork loin 25 414 2.2 Ananth et al., Salmonella typhimurium 20 414 1.5 1998.

Saccharomyces cerevisiae Orangejuice (pH 3.9) 37 350 2.8 Zook et al., (in ascopore) Apple juice (pH 3.9) 37 350 2.5 1999. Pseudomonas flourescens Ewe's milk (pH 2 250 3.9 Gervilla et al., 6.7; fat 7.6%) 25 250 4.6 1999. 50 250 2.8 Escherichia. coli 405 CECT Ewe's milk (pH 2 300 5.4 6.7; fat 7.6%) 25 300 5.2 50 300 2.5 Tris-HCl buffer -1 180 79.1 Reyns et al., Zygosaccharomyces bailii (pH 6.5) 20 180 237 2000. 40 180 55.5 -1 260 3.7 20 260 12.8 40 260 1.8

28 Escherichia coli KUEN Broth Ns 300 6.10 Dogan and 1584 400 3.94 Erkmen, 600 1.35 2004 b.

Milk 300 7.63 400 3.85 600 1.66 300 5.38 Peaehjuiee 400 3.38 600 1.22 300 2.42 400 1.57 Orange juiee 600 0.68

Listeria monocytogenes Broth Ns 300 8.22 Dogan and 400 3.79 Erkmen, 600 1.63 2004 a. Milk 300 10.99 400 6.00 600 2.43 300 6.17 Peaehjuice 400 3.39 600 1.52 Orange juiee 300 2.87 400 1.80 600 0.87 Zygosaccharomyces bailii Applejuiee 25 200 3.19 Riahi et al., 300 Nd 2003. Pichia membranaefaciens 25 200 18.5 300 4.1 Leuconostoc mesenteroides 25 200 4.74 300 Nd 10 350 18.7 Escherichia coli 0157:H7 400 14.3 30 300 9.43 350 3.22 Leuconostoc mesenteroides Single strength 20 350 2.0 Basak et al., Saccharomyces cerevisiae Orange juiee 20 250 5.2 2002.

Saccharomyces cerevisiae Pineapple juiee 270 5.1 Aleman et al., 1996. General microflora Fresh eut pineapple 4 340 3.0 Aleman et al., 21 3.1 1994. 38 >2.5

29 The Zp value, thus, represents the pressure range that results in a 10 fold change in Dp value.

In the Arrhenius model, the pressure dependence of k values is computed as the volume change of activation (~y*) from the regression slope oflog e (k) vs. pressure as shown in Eq. (2.6):

~y# = - RT (slope) or -RT [~(1ogek) / ~P] (2.6)

l where, P is the pressure (MPa), k is the rate constant (min- ), T is the absolute temperature (K), R

6 3 1 1 is the gas constant (8.314 x 10- m mole- MPaK ) and ~ y# is the volume change of activation

3 l (m mole- ) (Eyring and Magee, 1942).

Volume change of activation (~y#) and pressure Zp of L. monocytogenes were calculated to be 4.17'10-5 m3morl and 134MPa respectively (Mussa et al., 1999).

High Pressure Effects on Microorganisms in Fruits and Fruit Products

The application of HP to fruit products has been considered to be the most realistic, because the inherent low pH of fruits can inhibit the growth of most spoilage and pathogenic bacteria. Further, the yeasts and molds which survive at such low pHs are highly susceptible to

HP treatment. Many studies have been done on the evaluation of the effect of HP treatment on fruit juices (Aleman et al., 1996; Basak and Ramaswamy, 1996; Palou et al., 1998; Raso et al.,

1998; Garcia- Linton et al., 1998, 1999a; Prestamo et al., 1999; Zook et al., 1999; Graells et al.,

2001; Basak et al., 2003; Ramaswamy et al., 2003; Riahi et al., 2003).

Reyns et al. (2000) reported Zp values of various microorganisms: 66.5 MPa for Z. bailii; 68 MPa for S. cerevisiae; 130-200 MPa for Listeria spp; 155 MPa for S. typhimurium; 134

MPa for S. seftenberg; 75 MPa for E. coli; 100 MPa for L. casei.

30 Spoilage Microorganisms

Ogawa et al. (1990) investigated the HP effect on 9 different species of yeast in orange juice. Results showed that a 5-log reduction was achieved at 300 MPa for a hold time of 30 min or 400 MPa for 5 min. They also reported that the destruction of microorganisms in mandarin juice was influenced by the sugar concentration and not by its pH (2.5-4.5). Aleman et al. (1994,

1996) reported the destruction of S. cerevisiae in fresh cut pineapple and pineapple juice. They reported cyc1ic pressurization was more effective than static pressurization to destroy the target microorganism. Similar results were reported by Palou et al. (1998) for Z. bailii in laboratory model systems.

Parish (1998a) studied the effect of pressure on native microflora and S. cerevisiae in non-pasteurized Hamlin orange juice at pressures ranging between 500- 300 MPa. The Dp values ranges between 4 to 76 s for ascospores and 1 to 38 s for vegetative cells of S. cerevisiae. High pressure destruction kinetics of L. mesenteroides and S. cerevisiae in single and concentrated orangejuice by HP was evaluated at se1ected pressures (100-400 MPa) and holding times (0-120 min) (Basak et al., 2003). They reported that L. mesenteroides was more pressure resistant than

S. cerevisiae especially in the concentrated orange juice. Riahi et al. (2003) studied the effect of

HP on destruction of spoilage microorganisms L. mesenteroides, S. cerevisiae and P. membranaefaciens in apple juice. They achieved a >5 log reduction in counts of L. mesenteroides at 400MPa and Z. bailii and P. membranaefaciens at 300MPa by pulse pressurization. Inactivation of Z. bailii ascospores and vegetative cells in different fruit juices

(apple, orange, pineapple, cranberry and grape juices) by HP was also investigated by Raso et al.

31 (1998). They reported a 5 log cycle reduction of vegetative cells at 300 MPa after 5 mm pressurization, while the population of ascospores decreased between 0.5-1 log cycles.

The effect ofcontinuous (689 MPa with holding times of5, 15 or 25 min) and oscillatory

(one, three or five cycles at 689 MPa with holding time of 1 s) HP treatment on the viability of

Byssochlamys nivea ascospores suspended in apple juice and cranberry juice concentrates was evaluated by Palou et al. (1998). According to these authors, inactivation of the initial spore inocula was achieved after three or five cycles. Inactivation was not observed within 25 min with continuous pressurization a 60°C. No effect of spore viability was observed with continuous or oscillatory treatments at 21 oC.

Pathogenic and Contaminant Microorganisms

In general, the growth of food borne pathogens in fruit products are considered minimal because of its acidic pH. However, their presence in unpasteurized fruit juices may result from pre- or post-contamination of the fruit products. Recent outbreaks of food poisoning by

Salmonella spp. in orange juice and E. coli 0157:H7 in apple juice indicate that pathogens could survive in acidic products (Morgan et al., 1983; Besser et al., 1993; Weagent et al., 1994).

Furthermore, the resistance of Salmonella spp, E. coli 0157:H7, L. monocytogenes and S. aureus to HP has been reported (USDA, 2000). Their resistance to HP is of key concern in effective development of high pressure processing treatment for such juice products.

Pressure destruction kinetics of L. monocytogenes and E. coli in broth, milk, a peach and orange juice was reported by Dogan and Erkem (2004 a&b). They observed that HP was more effective against bacteria in fruit juices than in milk and broth due to a synergist effect of low pH and HP in juices. HP inactivation kinetics of E. coli was evaluated in the pressure range of300 to

700 MPa and reported D values were 3.94 and l.35 min at 400 and 600 MPa, respectively. On

32 the other hand, the D values were 0.83 and 0.68 min for aerobic bacteria and E. coli, respectively in orange juice while D values of L. monocytogenes ranged between 12.1 to 1.14 min under similar conditions. Garcia-Graells et al. (2001) observed the destruction of a pressure resistant mutant of E. coli in orange, apple and mango and reported that the destruction was not significant immediately after pressurization (up to 500 MPa), but a significant decrease in the number of survivors was noticed upon subsequent refrigerated storage. Riahi et al. (2003) studied the effects of HP on the destruction of E. coli 0157: H7 in apple juice. They reported that pulsed pressure was more effective at higher-pressure levels and resulted in almost a four-log cycle reduction in E. coli 0157: H7 cells at 400 MPa. During the pressure hold as the associated

D values decreased with an increase in pressure and followed a first order model of inactivation.

Quality Improvement and Shelf-Life Extension

HP processing does not alter low molecular weight components of food such as pigments, flavor components and vitamins. Pressure modifies the secondary and tertiary bonds, and hence, macromolecules such as proteins and carbohydrates are denatured upon pressurization.

The effect of HP on shelf-life of different fruit juices (orange, grapefruit and apple) and apple cut salad has been studied extensively. A treatment of 450 MPa during 5 min at room temperature was shown to result in complete destruction of aerobic microtlora of citrus juices while a pressure of 500 MPa made apple juice shelf-stable (Tonello et al., 1997). The shelf life of the three juices when treated with HP was 5 weeks when stored at refrigerated temperatures.

Total microflora and lactic acid bacteria reductions of apple cuts salad were around 1 log cycle just after a HP treatment of 200 MPa during 1h.

33 Kimura et al. (1994) compared the quality of pressure treated and heat-treated jams during storage at 5 and 25 °e for 1-3 months. The quality parameters evaluated were volatile components, anthocyanins, browning index, furfural, sucrose and vitamin e content. They observed that the freshly pressure processed jam maintained an initial bright red color due to the non-degradative effect of anthocyanins. Pressure- treated jam had a better fresh quality than the heat treated jam immediately after processing and the quality was maintained at low temperature storage but not at room temperature. Furthermore, the pressure treated jam could be stored at refrigerated conditions with minimal loss in sens ory and nutritional characteristics for up to 3 months. The deterioration in the quality ofjam at room temperature was believed to be due to the presence of dissolved oxygen and enzymes.

The quality and shelf-life of pressure treated and thermally pasteurized (88-900 e for 24 s) guava puree was studied by Gow and Hisin (1996). At 600 MPa, substantial microbial inactivation was noticed and the pressure treated samples did not show any change in color, cloud formation, degradation of pectin and ascorbic acid content compared to the fresh samples.

The pressure treated guava puree maintained good quality (similar to freshly extracted guava puree) at 4°e for 40 days. HP inhibited enzymatic browning reactions to sorne extent.

Donsi et al. (1996) studied the high pressure stabilization of orange juice, by evaluating microbial activity and the chemical composition of orange juice treated at different pressure levels for various processing times. They obtained a 2-month shelf-life for pressure treated juice at 350 MPa for 1 min at 300 e stored under refrigerated storage. Several other studies have demonstrated the effect of HP on orange juice. (Basak et al., 2003 ; Donsi et al., 1996; Goodner et al., 1999; Parish 1998 a, b; Takahashi et al., 1993). These reports suggest a minimal effect on the sensory and nutritional quality of orange juice. The beta-carotene, total carotenoides and L, a,

34 b values remained unaltered during pressurization and no significant changes were observed during 60 days of storage at refrigerated conditions. High pressure treated orange juice retained its characteristic flavor when treated at 500 MPa at ambient temperature (Takahashi et al., 1993).

The shelf-life of high pressure processed Valencia and Nave1 orange juices were compared to fresh and thermally pasteurized juices. The juices were pressurized at 600 MPa at

20°C for 60seconds and stored at 4 and 12°C for up to 12 weeks. The microbial, physical and chemical properties were evaluated during storage. Juices stored at 4°C were microbially stable and the pectin methyl esterase (PME) was not completely inactivated in Valencia orange juice

(pH 4.3) by HP or thermal treatment whereas the PME was reduced significantly with thermal treatment and HP in Navel orange juice (pH 3.7). Other quality parameters °Brix, viscosity, titratable acid content, alcohol insoluble acids, browning index , color, ascorbic acid and ~­ carotene concentrations of the juices were not significantly affected by HP (Bull et al., 2004).

Butz et al. (1997) reported the effects oh HP on antimutagenic activity of strawberry and grapefruit juice and was compared with the raw and heated samples. They reported that the antimutagenic activity strawberry and grapefruit juice was not affected by heat and pressure.

The antioxidative capacity, nutrient content and sensory quality of orange juice and orange-Iemon - carrot juice (OLC) product after high pressure treatment and storage in different packaging materials (glass, polypropylene, Barex®, teflon flasks and PE foil) were studied. High pressure treatment and st orage up to 21 days at 4°C caused no significant differences in oxidative capacity, vitamin C, sugar or carotene content. However the odor and flavor of orange juice were slightly altered, depending upon treatment and cultivar. No difference between non treated and pressure treated (500MPa) OLC mixture. Overall quality of the pressure treated juice was less altered (Garcia et al., 2001).

35 Stabilization of fresh orange juice at pressures between 500 MPa and 800 MPa and temperatures between 25 and 50 oC were evaluated. The juice was processed at 800 MPa and

25°C for 1 min and use of thermally pasteurized pulp yielded the lowest level of residual pectinmethylesterase activity (3.9%) and good cloud stability at 4 and 37 oC over a period of more than 2 months. Color values were stable during storage at 4, 15, and 26 oC. Ascorbic acid loss was less than 20% after storage for3 months at 4°C or 2 months at 15°C (Nienaber et al.,

2001).

The impact of ultra high pressure (UHP) processing on anti-mutagenic and anti-oxidative factors, sugars, ascorbic acid and carotenoids, in products from oranges, apples, peaches, mixed citrus juices, carrots, tomatoes, strawberries and raspberries were studied. High pressure did not induce loss ofbeneficial substances in the fruit and vegetable matrices in most cases. Incomplete pressure inactivation of invertase resulted in total disappearance of sucrose during storage of HP treated raspberries (Butz et al., 2003).

High Pressure Processing of Mango

Sliced mangoes were vacuum-sealed, and processed at 300 MPa or 600 MPa for 1 min.

Fresh mango flavor declined and off-flavor increased during storage at 3°C, but color, texture, and other sensory attributes changed slightly. The microbial levels in the control were 2 and 3 log cfu/mL greater than the 300 and 600 MPa treatments, respectively after 9 weeks of storage.

Pressure treatments also slightly reduced fresh mango flavor and increased off flavor and sweetness. Carambolas were sliced, vacuum-sealed, and processed at 600 MPa for 2,4, or 6 min; at 800MPa for 1, 3, or 5 min; or untreated. Samples were stored at 3°C for 2 and 4 wk and color

36 was evaluated after air exposure. AU the 800 MPa treatments reduced browning compared to the control, but the 600 MPa was less effective (Boynton et al., 2002).

A histological technique was used to evaluate modifications on the microstructure of peach and mango due to c1assical methods of freezing and those produced by high-pressure-shift freezing (HPSF). With the high-pressure-shift method, samples are cooled under pressure (200

MPa) to -20°C without ice formation, then pressure is released to atmospheric pressure (0.1

MPa). The high level of super cooling (approximately 20°C) leads to uniform and rapid ice nuc1eation throughout the volume of the specimen. This method maintained the original tissue structure to a great extent. Since problems associated with thermal gradients are minimized, high-pressure-shift freezing prevented quality losses due to freeze-cracking or large ice crystal presence. (Otero, L et al., 2000).

Demand for processed mango products such as mango pulp, juice and concentrates have been on arise because these are used in the fruit juice beverage industry as a base material, in the dairy industry as a flavoring ingredient and in baby food formulations. Currently, mango pulp and mango juice are preserved by heat pasteurization (Nanjundaswamy, 1997). However, heat processing can cause changes in the color, and also contribute to the quality loss during storage.

High pressure processing is considered to be emerging technology ofthe new millennium, which has shown the potential to preserve fruit and fruit products.

Outlook of High Pressure Technology

High pressure processing offers the food industry a number of possibilities inc1uding food preservation and processing (pasteurization and sterilization). At present, high pressure processing has been successfuUy applied for pasteurization of food products but sterilization is

37 not yet achieved. Many researchers have proposed high pressure processing for fruit juices, such as orange juice. Less information is available on the processing of mango juice by high pressure.

Although basic knowledge high pressure effects are understood, critical information is still required with respect to kinetic aspects of microorganisms and quality of fruit juices after processing and during storage. Rence, continuing research in academia and industry together is necessary to understand the pressure effects on fruit products which will promote the possibilities of commercialization of high pressure processing for different food products.

38 CHAPTER3

HIGH PRESSURE DESTRUCTION KINETICS OF SPOILAGE AND

PATHOGENIC MICROORGANISMS IN MANGO JUICE

Abstract

High-pressure destruction kinetics of three common spoilage microorganisms,

Leuconostoc mesenteroides, Zygosaccharomyces bailii, Pichia membranaefaciens and two pathogenic microorganisms Escherichia coli 0157:H7 and Listeria monocytogenes

Scott A were evaluated in processed mango juice at pressures ranging between 250-550

MPa and a hold time 0- 60 min at 20-25°C with species specific initial counts in the 106 to 108 CFU/mL range. Microbial destruction was evaluated based on (i) an initial kill due to a pressure pulse (pulse effect) and (ii) a subsequent semi-Iogarithmic destruction during the pressure hold time characterized by Dp value (time causing a 90% reduction in survivors). Dp values were species specific and decreased with an increase in pressure.

Pressure sensitivity of Dp was evaluated by a Zp value (pressure range that results in one decimal change in Dp). Among the spoilage bacteria and yeast, L. mesenteroides was found to be most resistant, yet its complete destruction was achieved by a 5 min treatment at 400 MPa. Pathogenic E. coli was the most resistant of aU the bacterial strains tested. A six-log reduction in E. coli 0157: H7 was observed at 400 MPa when treated for 10 min while at 500 and 550 MPa, there were no survivors after the 1 min hold time. HP processes were established for 6, 8 and lOD treatments based on E. coli 0157:H7 and counts monitored after treatment and during subsequent storage at 20, 12 and 4°C for 28 days. Results showed that these pro cesses were satisfactory from pathogen point of view since no growth of E. coli 0157:H7 was observed during the entire storage time.

39 Introduction

Mango pulp is a popular beverage and is used as a base to prepare a wide variety of products. Currently, thermal pasteurization is considered to be the most common method of preservation of mango pulp. Although this method extends the shelf-life with assured safety of mango pulp, it may have a detrimental effect on the nutritional quality of the product.

As consumer demands for products with inherently superior sensory qualities has always been rising, high pressure (HP) processing is being intensely studied as an alternative to thermal processing as it can inactivate microorganisms without altering the flavor and nutritional contents of foods (Drake et al., 1997; Furukawa et al., 2001).

Research efforts in both academia and industry have recently shifted the focus on high pressure processing offoods especially on evaluating its effect on food safety and quality.

High pressure processmg was discovered in the 19th century. Initially, high­ pressure research was a qualitative trail and error process, but today it is characterized by a more systematic fundamental approach in which attention is given to kinetic aspects and to specifie products (Hendrickx et al., 1998). A number of studies have reported the effect ofpressure on destruction of vegetative cells ofbacteria, yeast and molds as well as the more resistant spores in foods. Early studies have mostly focused on fruit juices, as preservation of acid foods was reported to be the most likely application of high pressure processing (Lechowich, 1993). The ability of HP processing to inactivate microorganisms and enzymes while leaving other quality attributes intact has encouraged food industries

40 around the world (Japan, America and Europe) to introduce high pressure preserved foods in the commercial market (Mermelstein, 1997).

Fruit juices are usually acidic with a pH of lower than 4.5. High pressure processing of different fruit juices has been investigated as method to preservation by several researchers (Aleman et al., 1994, 1996; Basak et al., 1996, 2002; Butz et al.,

1997; Dogan and Erkmen, 2004 a&b; Donsi et al., 1997; Riahi et al., 2003; Gow and

Hisin, 1996; Ogawa et al., 1990; Parish, 1998; Takahashi et al., 1994; Zook et al., 1999).

Most of these studies have focused on citrus, peach, guava, strawberry and apple juices.

Very little work, however, has been done on the HP preservation of mango juice.

The spoilage microorganisms encountered in mango JUlce are Leuconostoc mesenteroides, Zygosaccharomyces bailii, and Pichia membranaefaciens. Since fruit juices are lower in pH < 4.5, it supports the growth ofyeast and molds while preventing the growth of most bacteria with the exception of aciduric bacteria. Z. bailii and P. membranaefaciens can grow in acidic conditions and form ascospores. Z. bailii has been reported to be the most pressure resistant strain among eight food borne yeasts screened under a limited number of pressure- temperature combinations (Kristein et al., 2000)

Raso et al. (1998) reported a 5 log cycle reduction in Z. bailii in different fruit juices after a 5 min treatment at 300 MPa. Riahi et al. (2003) reported that a pulse effect resulted in complete destruction of L. mesenteroides and Z. bailii at 400 MPa and P. membranaefaciens at 300 MPa. Basak et al. (2002) reported about 3-4 log cycles reduction in L. mesenteroides and S. cerevisiae by a pressure pulse at 350-400 MPa.

41 Many diseases can be transmitted through the consumption of foods contaminated with these microorganisms or from a wide range ofinfected animaIs (Doyle et al., 2001).

E. coli 0157:H7 and L. monocytogenes Scott A are important food borne pathogens. E. coli 0157:H7, with an optimum growth at pH around 7.0, can grow in the pH range between 4.5 and 9.0 (Glass et al., 1992). It has been reported that this pathogen can survive in sorne acidic foods, such as apple cider pH 3.7 (Miller and Kaspar, 1994) mustard pH 3.1, sweet pickle pH 2.8 and ketchup pH 3.6 (Tsai and Ingham, 1997) and mayonnaise pH 3.65 (Weagant et al., 1994) for various periods of storage. The risk of E. coli 0157:H7 infection is a particular problem in the food industry. E. coli 01S7:H7 is a major food-borne pathogen, causing hemorrhagic colitis and hemolytic ureamic syndrome (Doyle, 1991). It has been a public health concern for food processors and consumers since its first recognition in 1982, with incidents of food-borne disease associated with this pathogen occurring in the US, Canada and UK (Doyle, 1991;

Anonymous, 1995). In recent years, many outbreaks of food borne illnesses and diseases have been related to the consumption of E. coli contaminated acidic foods. The pressure resistances of these pathogens are of key concern in development of effective HP processing treatments. USDA requires five log reductions of the most resistant organisms ofpublic health significance in fruit juices (USDA, 2000).

The destruction kinetics has of E. coli 0157: H7 and L. monocytogenes Scott A have been reported in apple, peach, and orange juices (Riahi et al., 2003; Dogan and

Erkmen, 2004 a&b). Alpas et al. (2000) reported an 8-log reduction of E. coli and L. monocytogenes at 345 MPa and SO°C for 5 min. A 3-log reduction of L. monocytogenes

42 was obtained after a treatment of 15 min at 379 MPa in phosphate buffer (Patterson et al.,

1995a).

Although HP can successfully control microorganisms, there is lack of quantitative inactivation data on the kinetics of microbial inactivation especially regarding pressure, microbial type and suspending medium effects (Earnshaw et al.,

1995; Mussa et al., 1996; Palou et al., 1997; Zook et al., 1999). The patterns of HP inactivation kinetics observed with different microorganisms are quite variable. Sorne investigators have indicated first order kinetics in case of several bacteria and yeast

(Cariez et al., 1993; Palou et al., 1997; Zook et al., 1999). Non-log-Iinear regression has been recommended by several others (Heinz and Knorr, 1996; Patterson and Kilpatrick,

1998; Reyns et al., 2000). Sorne have analyzed the pressure destruction kinetics as a dual effect (Basak et al., 2002; Riahi et al., 2003 Mussa et al., 1999). The pattern of destruction kinetics is influenced by pressure, temperature and composition of the medium (Palou et al., 1997; Zook et al., 1999).

High-pressure technology is eventually expected to result in a commercially sterile product just as the product from their conventional counter parts. Traditionally, commercial sterility is established using thermal death time approach coupled with the results of the heat penetration tests which are mathematically combined to form a scheduled thermal process (NCA, 1968). A standard operating protocol must be developed to verify the HP processes. Su ch protocols should involve microbiological testing to demonstrate the effect of HP treatment on pathogens as weIl as spoilage microorganisms in the HP treated foods. The dispersing medium for microorganisms may also have an influence on pressure destruction ofmicroorganisms (Hoover et al., 1989).

43 To ensure microbial safety and stability of processed juices, the HP treatment must ensure a satisfactory reduction in initial microbial counts; thus, pressure dependence of microbial inactivation rates are needed. Such process establishment requires data on pressure destruction kinetics of spoilage and pathogenic bacteria in the product being tested. There is no detailed published study on the pressure inactivation kinetics of spoilage and pathogenic bacteria in mango juice. Rence, the objective of this study was to investigate and establish the destruction kinetic parameters of common spoilage microorganisms and pathogenic microorganisms in mango juice. A further objective was to evaluate the practical applicability of generated kinetic data for establishing the HP process and their verification through storage studies.

Materials and Methods

Cultures, Media and Preparation of Inoculum

Zygosaccharomyces bailii (ATCC 2333), Pichia membranaefaciens (ATCC

2085) Leuconostoc mesenteroides (ATCC 8293) were obtained from the American Type

Culture Collection (ATCC; Manassas, V A, USA). The culture of Escherichia coli

0157:R7 (ATCC 43894) and Listeria monocytogenes Scott A was obtained from microbiology laboratory Department of Food Science and Agricultural Chemistry,

Macdonald Campus, McGill University, Ste-Anne-De-Bellevue, QC, Canada were revived in their respective broths (Table 3.1)

44 Table 3.1: Media for cultivation and incubation temperature and time of spoilage and pathogenic microorganisms

Microorganism Media used Incubation temperature and time Zygosaccharo1.nyces Yeast malt broth 27°C for 48 hours bailiii (Difco Laboratories, with agitation (ATCC 2333) Detroit, MI, USA) 150rev/min Pichia Yeast malt broth 27°C for 48 hours 1.ne1.nbranaefaciens (Difco Laboratories, with agitation (ATCC 2085) Detroit, MI, USA) 150rev/min Leuconostoc MRS broth (Difco 27°C for 48 hours 1.nesenteroides (ATCC Laboratories, Detroit, 8293) MI, USA) Escherichia coli 0157: Tryptose soya agar 37°C for 24 hours H7 (ATCC 43894) broth + 0.6% yeast extract (Difco Laboratories, Detroit, MI, USA) Listeria 1.nonocytogenes Brain heart infusion 37°C for 48 hours (Scott A) broth (Difco Laboratories, Detroit, MI, USA)

Preparation of Mango Juice

Commercially sterile Alphonso mango pulp (Brix 28.0) was purchased from local supermarket (Cedar Brand, Marche Victoria, Montreal). A sufficient amount of sterilized distilled water was added to the pulp to adjust the total soluble solids content (TSS) to

45 15°Brix (the natural TSS found in mango juice). The samples were stored at 4°C before use. Prior to inoculation the samples were thawed to 20-23°C.

Preparation of Cell Suspension and Test Samples

Each microbial strain was grown on its respective agar plates (Table 3.1) and then a loop full was transferred to a fresh broth and incubated. This procedure was repeated two more times to maintain cells predominantly in their stationary phase of growth. This constituted the stock culture which was refreshed every 48 h. The resulting enriched culture was centrifuged at 4000Xg, at 10°C, for 15 min and sedimented cells were aseptically suspended in 100 mL freshly prepared juice. In preparation of test samples the media for cell culture and incubation times were so adjusted that the initial counts were

~106 CFU/mL for Z. bailii and P. membranaefaciens and ~108 CFU/mL for L. mesenteroides, E. coli 0157: H7 and L. monocytogenes. The inoculated test samples

(25mL) were then transferred aseptically into sterile polyethylene pouches and heat sealed with hand sealer, twice, with minimal headspace. AlI test samples were stored at

4°C overnight for the cells to adjust in the new environment. All samples were equilibrated to the desired temperature (15-23°C) before pressurization.

High- Pressure Equipment and Treatment

The hydrostatic pressure vessel used for this study was a batch type ABB Isostatic

Press Model # CIP42260 (ABB Autoclave System, Autoclave Engineers, and Erie, PA) with a lOcm diameter and 55 cm height stainless steel cylindrical pressure chamber. The maximum pressure tolerance level of the equipment is 414 MPa. The pressure come up

46 times varied from 1 to 3 min depending on the selected pressure. The higher the pressure the higher were the come up times. The depressurization time was < 20 s. Samples were submerged in water containing 2% mineraI oil that acted as a hydrostatic fluid medium.

AU treatments were carried out at room temperature 20-23°C. Isothermal conditions were maintained by circulation ofwater around the pressure chamber. The medium and sample temperature increased during the treatment as a result of adiabatic heating. Renee, the temperature of medium and sample were adjusted to a lower (than the desired target) temperature before treatment. The temperature of medium was monitored by a thermocouple attached to a temperature logger during treatment. AlI the treatments were carried out in duplicates and results were averaged for each sample. The various combinations for pressure treatments of the prepared pouches are shown in Table 3.2.

Additional pressure treatments at 450, 500 and 550 MPa (for E. coli 0157: R7) were carried out using another HP unit capable of operation at pressures up to 650 MPa

(model ACIP 6500-5-12VB, ACB, Nantes, France).

Enumeration

Samples were aseptically opened and each sample was seriaUy diluted using

0.1 % sterile peptone water (Difco Laboratories, Detroit, MI, USA). Non-inoculated control (for contamination check), untreated (control) and pressure treated samples were enumerated by spread plating the appropriate dilution on to the respective agar. Two plates were used for each dilution and were incubated at the times and temperatures shown in Table (3.3). The results were averaged from four measurements (two sachets/ treatment x two replicates).

47 Table 3.2: Processing conditions for spoilage and pathogenic microorganisms

Microflora Pressure Pressure holding times (MPa) (min) Zygosaccharon1yces 200 0 10 20 30 40 bailiii 250 0 5 10 15 20 (ATCC 2333) 300 0 5 10 15 20 350 0 1 2 3 4 Pichia 200 0 5 10 15 20 n1en1branaefaciens 250 0 1 2 3 4 5 (ATCC 2085) 300 0 1 2 3 4 Leuconostoc 300 0 15 30 45 60 n1esenteroides 350 0 5 10 15 20 (ATCC 8293) 400 0 2 4 6 8 Escherichia coli 250 0 5 15 30 45 0157: H7 ATCC 300 0 5 10 15 (43894) 350 0 5 10 15 400 0 5 10 15 450 0 1 2 3 4 500 0 1 2 3 550 0 1 2 3 Listeria 250 0 5 10 15 20 n1onocytogenes 300 0 5 10 15 (Scott A) 350 0 2 4 6 8 400 0 1 2 3 4 5

48 Table 3.3: Enumeration media and incubation temperature and time for spoilage and pathogenic microorganisms

Microorganism Enumeration Media Incubation temperature and time Zygosaccharor.nyces Yeast malt agar 27°C for 3 days bailiii (Difco Laboratories, (ATCC 2333) Detroit, MI, USA) Pichia Yeast malt agar 27°C for 3 days r.ner.nbranaefaciens (Difco Laboratories, (ATCC 2085) Detroit, MI, USA) Leuconostoc De Man Rogosa and 27°C for 3 days r.nesenteroides ATCC Sharpe agar (Difco (8293) Laboratories, Detroit, MI, USA) Escherichia coli 0157: Violet red bile agar 37°C for 24 hours H7 (ATCC 43894) (Difco Laboratories, Detroit, MI, USA) Listeria r.nonocytogenes Lister selective agar 37°C for 48 hours (Scott A) + Listeria supplement (Difco Laboratories, Detroit, MI, USA)

Kinetic Data Analysis

The pressure destruction of microorganisms was analyzed as a dual effect as described by (Mussa et al., 1999; Basak et al., 2001; Riahi et al., 2003) which involved a pulse pressure effect (PE) i.e. change in viability due to rapid pressurization and depressurization which was obtained by subtracting the survivors (logarithmic scale) after

49 one pressure pulse from the initial count (also on logarithmic scale) followed by a first order rate of destruction during the pressure hold time. Pressure destruction of microorganisms during the hold time was assumed to follow the first-order rate kinetics expressed as;

Log NlNo = -k t (3.1) where N = number of surviving microorganisms after pressure treatment for time t (min), l No = Initial count (t = 0 min) and k = inactivation rate constant (min- )

When Dp values at different pressures were plotted on a semi logarithmic scale, the pressure range for one log cycle change in Dp value represents the Zp value. Thus pressure Z value or Zp value can be defined as the pressure required for a tenfold change in the D value, and can be expressed by the following equation where DpI and Dp2 are decimal reduction times at pressures Pl and P2

Zp = (P2- Pl) / log (DpdDP2) (3.2)

Sorne small variations in the initial counts are inevitable, although variations are rarely large enough to move them to the next decimal logarithm. Within the limits of a decimal logarithm, it is possible to normalize the counts so that the survivor plots are better represented for comparative purposes. This is normally done by dividing all survivor counts by their initial counts and then multiplying them by their nominal logarithmic starting counts. This will result in appropriately shifting of the survivor curves along the vertical ordinate scale so that they all deem to start with the same initial count and this shift will not affect the slopes ftom which the kinetic parameters are obtained. When adjusted for initial count this way, the computed Dp value from survivor

50 curves plotted as log (N) vs. t will yield the same result as the as plotting original

10g(N/No) vs. t.

The relative magnitude of the pressure pulse and pressure hold time effects was

investigated using ND and Dp values (Mussa et al., 1999). ND represents the number of

pressure pulses required to achieve one decimal reduction in microbial population (thus

equivalent to D value) and can be expressed by using the following equation.

ND = I/LogPE (3.3)

DpE represents decimal reduction time equivalent of PE which can be defined as

the holding time in minutes which results in an equivalent destruction achieved by one

pressure cycle at a given pressure level. These values were obtained as follows:

DpE = Dp value x [Log (PE)] (3.4)

Process Establishment and Verification

The reference pressure for the application of pressure processing was set at 400

MPa, the maximum that could be accommodate with the equipment used so that the treatment times are kept lower. The kinetic data (Dp values and Zp value), obtained at various pressure levels, was analyzed and the more resistant bacteria identified based on the pressure destruction time at 400 MPa. The process lethality was defined as the

treatment time in minutes at a reference pressure that would result in selected multiples of

logarithmic cycle reductions (multiples of Dp values) for resistant pathogenic

microorganism. The process lethality was defined mathematically as

Fop=nxDp (3.5)

51 Where Fop= pro cess lethality, min; n= the target number oflogarithmic cycle reductions,

D= decimal reduction time, min, for the target pathogen.

The processing conditions were selected to represent OD for control samples, 6D,

8D and 10D for pressure processes. For example, if Dp value of a microorganism was 5 min, the 6D pro cess wou Id be estimated by multiplying 5 x 6 =30min. Similarly, the 10D process would be 10 x 5 = 50 min etc.

Sachets of inoculated mango juice samples with an initial population 108 CFU/mL of the resistant pathogen were subjected to pressure treatment (400 MPa) equivalent to destroy the pathogen by OD, 6D, 8D and 10D. The treated samples were then stored at

20°C, 12 oC and 4°C for a period of 30 days. Two pouches were randomly withdrawn and samples were plated in duplicate immediately after processing and during storage at an interval of 3, 6 and 9 days at 20, 12 and 4°C and for maximum of 30 days. The minimum level of detection was 25 CFU/mL.

Selective media are traditionally used to suppress the growth of the non-selective background bacteria. Since they are selective, sorne of the species specifie injured cells might also fail to grow. In the present study, the strains were also maintained on the selective media prior to making the stock culture and hence, it is possible that they may have acquired sorne prior resistance to the media. The plate eount technique as commonly used is only useful for enumerating counts greater than 20. Renee, microbial concentrations below 10-100 CFU/mL in the original sample normally fail to show on the plate, although they may be present. These are generally considered gray zones and are not easily countable. The is one of the reasons for the challenge study in which the inoculated samples, trom the terminal treatments (6-10 D treated samples), were stored

52 for different periods of time to allow for the recovery and growth of both the residual population (low probability after the 6, 8 and 10 D treatments) as weB as prevailing injured cells.

ResuUs and Discussion

Dual Errect Pressure Destruction

Survivor curves of spoilage microorganisms Z. bailii, P. membranaefaciens, L. mesenteroides and pathogenic microorganisms E. coli and L. monocytogenes as a function of ho Id time are shown ln Figures 3.1 and 3.2, respectively.

High pressure had a pronounced effect on the destruction of spoilage microorganisms compared to that of pathogenic microorganisms in mango juice. Experimental data suggested that the survival curves of the microorganisms tested did not originate from their nominal initial counts but rather from the reduced count levels which is a result of pressure pulse treatment which existed at all pressure levels. The second part is the conventional tirst order rate destruction during the pressure hold time. Similar observations were reported by Riahi et al. (2003) for HP destruction of spoilage and pathogenic microorganisms in apple juice, Basak et al. (1996) for the inactivation of

PME in orange juice and Mussa et al. (1999) for destruction of L. monocytogenes and natural microflora in milk and pork.

53 Figure 3.1: EfTect of pressure level and pressurization time on the destruction of

spoilage microorganisms in mango juice

6 Z. bailii

5~~~t:::~~==~~ __~;- ______-t __ ~R:2~=~0~.9~1~

z~- 4- ... 200MPa CI:; 3 RZ = 0.94 0i'­ 88 250MPa ...Jo _2 .300MPa 1111111350MPa

O+-----.-----~----~----~----~----,_----,_----~--~ o 5 10 15 20 25 30 35 40 45 Time(min)

P. membranaefaciens 6

5 -4 z...J R2 =0.97 .2ooMPa E R2 = 0.96 ~:; 3 !III 250MPa ...Ji'- ~2 ",300 MPa

A 0 0 5 10 15 20 25 Time (min)

L. mesenteroides 9 8 7 R2 = 0.90 3 6 Z E 5 .300MPa a- o ::J 4 R2 =0.95 li! 350 MPa ...Ji'- 2.3 .400 MPa R2 =0.93 2 1 • 0 0 5 10 15 20 25 30 35 40 45 50 lime (min)

54 Figure 3.2: EtTect of pressure level and pressurization time on the destruction of

pathogenic microorganisms in mango juice

E.coli 0157:H7

.250MPa _6 .. 300MPa ..J Z E 5 Il 350MPa CI- 0::)4 .\ 400MPa ..JIL. 2. 3 ~ 450MPa 2 2 R = 0.98 .500MPa 2 o 550MPa R = 0.90

o 5 10 15 20 25 30 35 40 45 50 Time (min)

L. monocytogenes 7

R2 =0.87

.250MPa .300MPa

l1li' 350MPa "" 400MPa 2

o+------~------~------~------~------~ o 5 10 15 20 25 Time (min)

Pulse Pressure EtTect

PE, ND, Dp and DpE values together with the associated R2 for Z. bailii, P. membranaefaciens, L. mesenteroides, and E. coli 0157: H7 and L. monocytogenes are presented in Table 3.4.

55 2 Table 3.4: PE, ND, Dp and DpE values together with the associated R values for

spoilage and pathogenic microorganisms in mango juice

Microflora Pressure PE ND Dpvalue DpE R2 (MPa) (Log) (min) Zygosaccharor.nyces 200 0.77 1.29 31.6 24.4 0.91 bailii 250 0.75 1.33 9.78 7.33 0.94 (ATCC 2333) 300 0.99 1.01 1.68 1.66 0.93 350 1.85 0.54 0.62 1.14 0.96 Pichia 200 0.56 1.78 15.9 8.90 0.97 r.ner.nbranaefaciens 250 0.67 1.49 2.12 1.42 0.96 (ATCC 2085) 300 0.92 1.08 1.02 0.94 0.97 Leuconostoc 300 0.09 11.1 16.3 1.46 0.90 r.nesenteroides 350 0.15 6.67 3.15 0.47 0.95 (ATCC 8293) 400 0.16 6.25 0.69 0.11 0.93 Escherichia coli 250 0.2 5.0 8.73 1.74 0.95 0157: H7 300 0.5 2.0 5.36 2.68 0.94 (ATCC 43894) 350 1.5 0.67 3.43 5.15 0.98 400 1.6 0.62 2.88 4.60 0.89 450 2.1 0.47 0.72 1.52 0.99 500 2.8 0.35 ND ND --- 550 3.5 0.28 ND ND --- Listeria 250 0.05 20.0 13.6 0.68 0.87 r.nonocytogenes 300 0.07 14.2 5.23 0.36 0.91 (Scott A) 350 0.09 11.1 2.01 0.18 0.96 400 0.12 8.33 0.80 0.09 0.98

Spoilage microorganisms: A numher of studies have shown that the effect of HP

can he grouped into cell envelope related effects; pressure induced cellular changes, hiochemical aspects and effects on genetic mechanisms. The effect of pulse pressure is

56 due to the rapid pressurization and depressurization. It is believed that rapid decompression invokes cavitations in the cell that result in physical disruption and death.

Hayakawa et al. (1994) has shown the pulse pressure treatment to be effective in case of spores, bacteria and vegetative cells. PE values were dependent on the pressure level.

Among the spoilage microorganisms studied yeasts were more sensitive than bacteria. Z. bailii was the most sensitive to pulse pressure when compared to P. membranaefaciens and L. mesenteroides (Figure 3.1)

The PE values ranged from 0.77 log cycle reduction at 200 MPa to 1.85 log cycle reduction at 350 MPa. With pressure pulse inactivation, Z. bailii was the least resistant spoilage microorganism. L. mesenteroides was found to be the most pressure resistant among the spoilage microorganisms studied in mango juice. Riahi et al. (2003) reported complete destruction of Z. bailii, P. membranaefaciens and L. mesenteroides with a single pulse at 400 MPa and 300 MPa, respectively, in apple juice. Basak et al.

(2002) reported about a 3 to 4 log cycle reduction in the population of L. mesenteroides and S. cerevisiae, respectively. Hayakawa et al. (1994) reported that a four-cycle pressure treatment with a 5 min holding time at 600 MPa decreased the spore count of Baci/lus stearothermophillus by 5 log cycles. Aleman et al. (1994; 1996) studied static versus pulsed pressure applications in the inactivation of S. cerevisiae in pineapple juice and found that pulse treatments were more effective than static applications over comparable lengths of time.

Pathogens: E. coli 0157:H7 was found to be more sensitive to pulse pressure when compared to that of L. monocytogenes. At 400 MPa a reduction of 1.6 log cycle and

0.12 log cycle was noticed for E. coli 0157:H7 and L. monocytogenes, respectively

57 (Figure 3.2). Riahi et al. (2003) reported a 4-log reduction of E. coli 0157:H7 at 400 MPa in apple juice. Vachon et al. (2002) studied the effect of pressure levels from 100-300

MPa with a combination of one or more pressure pulse(s) at pH 7.2 in O.OlM phosphate buffered saline for the inactivation of three major food borne pathogens, L. monocytogenes, E. coli 0157:H7 and Salmonella enterica. Complete destruction of L. monocytogenes was achieved after three successive pulses at 300 MPa, while treatments with three and five successive pulses at 200 MPa eliminated E. coli 0157: H7 and

Salmonella, respectively.

Pressure Hold Time Kinetics

Figures 3.1 and 3.2 show the pressure destruction of Z. bailii, P. membranaefaciens, L. mesenteroides, E. coli 0157 :H7 and L. monocytogenes at different pressure levels and they aU followed the first order kinetic model with a good R2 value

(>0.9). Logarithm of microorganism survival (N) in mango juice decreased linearly with time. The cells were destroyed more rapidly with increase in pressure. The calculation of

D p values . from the survivor curves were used to compare the resistance of microorganisms in the comparison of the effectiveness of pressure treatments. Calculated

Dp values (Table 3.4), which was obtained from the regression of logarithmic survivors versus time shows that an increase in pressure level resulted in lowering of D p values.

Several authors have reported pressure destruction of microorganisms as a first order rate.

Inactivation parameters may be influenced by several factors: culture homogeneity, microbial strain, level of pressure, culture medium, and temperature (Earnshaw et al.,

1995; Palou et al., 1997).

58 The Dp values of vegetative yeast cells of Z. bailii and P. membranaefaciens at

300 MPa was 1.68 and 1.02 min, respectively and 3.15 min for L. mesenteroides at 350

MPa Thus L. mesenteroides was more pressure resistant than the yeasts in mango juice.

Basak (2001) and Riahi (2003) reported Dpvalues of2 min and 0.80min at 350 MPa for destruction of L. mesenteroides in orange juice and apple juice respectively. Raso et al

(1998) investigated the effect of pressure on Z. bailii in different fruit juices and reported that after 5 min of pressurization the population of vegetative cells decreased almost 5 log cycles.

The Dp values of E. coli 0157:H7 and L. monocytogenes at 400 MPa were 2.88 mm and 0.80 min, respectively. It was noticed that E. coli 0157:H7 was the most resistant of aIl the microorganisms evaluated in mango juice in this study. Dogan and

Erkmen (2004 a&b) reported Dp values of 2.43, 1.52,0.87 and 1.63 min at 600 MPa for

L. monocytogenes in raw milk, peach juice, orange juice and BHI broth, respectively and

1.66, 1.22, 0.68 and 1.35 min at 600 MPa for E. coli in raw milk, peach juice, orange juice and BHI broth, respectively, indicating L. monocytogenes to be more pressure resistant in orange juice. However, Riahi (2003) found the opposite in apple juice processed at 250-400 MPa.

The net effect of pressure cycle on microbial destruction is due to the combination of pressure pulse and pressure hold time. ND and DpE provide sorne relative comparisons between these two parameters. ND value is an informative parameter indicating the number of pressure cycles required to achieve log cycle destruction in microbial population (equivalent of a decimal reduction). DpE is another beneficial indicator, which shows the relative effect effectiveness of pressure pulses versus pressure hold time

59 approaches. It was observed that as pressure level increased the ND value decreased

(Table 3.4). Similar observations have been reported by Basak et al. (2001), Riahi et al.

(2003) and Mussa et al. (1999). For example, the associated PE is about 1.5-log cycle reduction at 400 MPa in case of E. coli 0157:H7 (ND about 0.6) with a Dp value of around 3 min. If one wanted to achieve an 8-log reduction in microbial count, a 5-cycle pressure pulse would be sufficient. Alternatively, a hold time of 24 min will result in 8 log reductions. DpE value is calculated by multiplying Dp value with log PE. For the case of E. coli 0157:H7, DpE value at 400 MPa is 4.60 min. In other words, a holding time of approximately 5 min will have the same effect as a single pressure pulse. Riahi et al.,

(2003) reported much smaller ND value «0.2) and 0.88 at 400 MPa for spoilage microorganisms and E. coli 0157:H7 in apple juice respectively.

Pressure Sensitivity of Dp Values

The pressure death time plot, which is analogous to the TDT concept in thermal processing, was used to evaluate the pressure sensitivity of the reaction rate constant (Dp value) for spoilage and pathogenic microorganisms.

The Zp values of the spoilage and pathogenic microorganisms are shown in Table

3.5. The Dp value curves are shown in Figure 3.3. The high R2 values for an the microorganisms indicate that the PDT approach is applicable to de scribe the pressure sensitivity parameters. The associated Zp value for P. membranaefaciens and Z. bailii in mango juice was almost similar, while L. mesenteroides was more resistant in mango juice compared to other spoilage microorganisms. Among the pathogenic microorganisms E. coli was found to be highly resistant to pressure having a Zp value of

60 263 MPa. In general pathogenic microorganisms were found to be more resistant in mango. Factors affecting the pressure sensitivity of microorganisms include type/strain of microorganism, composition of the dispersion medium as well as rate of pressurization and depressurization, cell growth and handling, method of inoculum preparation, handling procedures. (Basak et al., 2002; Hoover et al., 1989; Hayakawa et al., 1994; and

Palou et al., 1998).

Table 3.5: Zp values together with the associated R2 values for spoilage and

pathogenic microorganisms in mango juice

Zp values R2 Microorganism (MPa) Zygosaccharomyces bailii 84 0.98 (ATCC 2333) Pichia membranaefaciens 84 0.93 (ATCC 2085) Leuconostoc mesenteroides 72 0.99 (ATCC 8293) Escherichia coli 0157: H7 204 0.92 (ATCC 43894) Listeria monocytogenes 121 0.99 (Scott A)

61 Figure 3.3: Pressure sensitivity of Dp values for spoilage pathogenic microorganisms

in mango juice

Spoilage microorganisms

R2 = 0.98 1.2 R2 = 0.99 i8I P.membranaefaciens 0.8 c • Z.bailii CI 0 ... L. mesenferoides ....1 0.4 0

-0.4 150 250 350 450 Pressure (MPa)

Pathogenic microorganisms 1.2 0.9

c 0.6 • E.coli o 157:H7 CI 0 iII! L. monocytogenes ....1 0.3 0.0 -0.3 200 250 300 350 400 450 500 Pressure (MPa)

Process EstablishmentNerification/Challenge

From the preceding sections, it is evident that amongst the microorganisms studied, the pathogenic E. coli 0157:H7 is the most pressure resistant and therefore

62 should be used as the target for establishing the pressure processing. The Dp value of E. coli 0157:H7 was found to be 2.88 min at 400 MPa (the highest pressure level studied under this set of experiments). This was rounded to 3 min for the purpose of process establishment. Hence the required pressure treatment times were 18, 24 and 30 min for the 6, 8 and IOD processes. The growth curves of E. coli 0157:H7 in untreated mango

(OD) juice were also included for comparison (Figure 3.4). Figure 3.5 shows the growth of E. coli 0157:H7 in mango juice subjected to HP treatments equivalent ofresulting 6, 8 and 10 logarithmic cycle reductions in E. coli 0157:H7 and then stored at different temperatures.

Figure 3.4: Growth of E. coli 0157: H7 in untreated mango juice during storage at

20, 12 and 4°C

OD

7 6 5 z~ 4 tII- o~ ..JLL 3 0 2 1 1 0 0 3 6 8 9 12 15 Da ys

As shown in Figure 3.4, the viable population of E. coli 0157:H7 in untreated mango juice declined when stored at 20, 12 and 4°C. Similar findings were reported by

Cheng et al. (2001). Immediately after HP treatment at 6D the viable count decreased

63 from 108 CFU/mL to a maximum of 102 CFU/mL. However, samples treated by 8D and

10D processes showed no survivors. Since the Dp value used was slightly higher than the actual and since the pro cess will also benefit from the PE value (1.6 log) at 400 MPa, it was expected that the E. coli 0157:H7 counts in test samples (with 108 CFU/mL initial count) immediately after processing would be below 10 CFU/mL even for the 6D process. However, sorne discrepancies were observed and maximum counts in the 102

CFU/mL regions were observed. However, no colonies were observed in the other treated samples.

Figure 3.5: Effect of applied pressure lethalities on growth of E. coli 0157:H7 in

mango during storage at 20, 12 and 4°C

20, 12 and 4°C

3

z-g- 2 CI-o=> 0100 ..JU- 0 1 1180 - 1160 DIII6I lfIIIII lJ9JI6I lJ9JI6I .. 0 0 6 12 18 24 30 Da ys

However, during the entire remaining storage period of 30 days, none of the pressure treated samples showed viability at 20, 12 or 4°C (Figure 3.5). Hence the counts observed immediately after the 6D processes could be an experimental error. Sorne

64 challenge studies have previously demonstrated that during storage, the pressure injured ceUs (which fail to show when counted immediately on a harsh medium like VRBA) could recover and show growth and activity along with any residual survivors (Zaman et al., 2005). However, in this study, such a behavior was not observed, possibly because of the acidic nature of the fruit pulp. Hence pressure processes 6-10D were considered safe.

Conclusions

Pressure destruction of spoilage and pathogenic microorganisms were studied as influenced by pressure levels and pressure hold time. This study showed dual destruction behaviour with a step change in the number of survivors due to the application of pressure pulse, and a first order rate of destruction during the pressure hold. The pulse effect was pronounced in the case of E. coli 0157:H7 and Z. bailii. The most resistant microorganism in mango juice from those studied was E. coli 0157:H7 while L. mesenteroides was the most resistant spoilage microorganism. From a design point of view, kinetic data on pressure resistant pathogenic microorganisms are indispensable.

Storage studies were found to be a safety assessment tool for HP processing that may cause sub-lethal injuries to microbial ceUs. This study showed that the kinetic data for E. coli 0157:H7 would be useful in estimating the HP process time aimed at assuring safety in mango juice. Since no growth of E. coli 0157:H7 was observed during storage regardless of pressure lethality and temperature, pressure processes equivalent of 6-1 OD with respect to E. coli 0157:H7 would ensure complete destruction. Since 6D process would be a border line case especially with initial counts as high as 108 CFU/mL, 8 and

65 10D processes were considered safe. This is equivalent of a treatment time of 24-30 min at 400 MPa. Since the Zp value for E. coli 0157:H7 is ~200 MPa, this would also me an, at around 600 MPa, these times could be reduced to one tenth of their value giving a short

2-3 min process to assure pathogen safety.

66 CHAPTER4

EVALUATION OF QUALITY CHANGES IN HIGH PRESSURE PROCESSED

MANGO JUICE DURING STORAGE

Abstract

Fresh mango juice pressure treated at 400 MPa to achieve different process lethalities (0-100 based on Escherichia coli 0157:H7 destruction rate) were stored at 4,12 and 20°C for se1ected times (up to 60 days) and the associated quality changes evaluated.

AH samples were analyzed for their color, pH, acidity, °Brix and microbial growth. A decrease in the pH and b values, increase in acidity and microbial growth were observed for sorne pressure treated samples at 400 MPa. QuaHty changes were evaluated based on zero and first order models and the rate constants were obtained. Temperature sensitivity of rate constants was evaluated using the Z value approach. The result showed that the rate constants associated with microbial growth and changes in quality factors increased as the storage temperature and applied process lethality increased. The results indicated that while these pressures processed samples were microbiologically safe as determined previously, they failed to provide quality stability. Hence, additional treatments carried out at 550 MPa for 1 min and similar storage tests were carried out. Test samples pressure treated at 550

MPa maintained their superior quality throughout the 60-day test period and showed no microbial growth (at 4°C). High pressure (HP) pasteurization therefore has the potential as an alternative method for the preservation of mango fruit juice.

67 Introduction

Mango (Mangifera indica L.) of the family Anacardiaceae is a big flesh drupe with edible pulp and a stony layer around the seed. It is known to be among one of the most nutritious fiuits. Over 1000 varieties of mango exist, among which are Alphonso, Pairi,

Banganpalli, Bombai, Palmer, Saigon, Edward and Julie (Hill et al., 1999). Each variety of mango is distinct trom the other in color and flavor and therefore, varies in its suitability for processing into different products such as juices and chutney (Adriana et al., 1998;

Murthy et al., 1984).

Mango with its many versatile properties, has naturally found application for processing into various products. Green mangoes are processed into many traditional products such as pickle, brine stock and chutney, drum dried mango powder, and productions of raw mango based beverages are new developments. Ripe mangoes are processed into canned slices, pulp, beverages like RTS (ready-to-serve) beverage, nectar and juice, dehydrated products like mango fruit bar, mango cereal flakes, mango powder, strained baby foods etc.

Mango is one of the most important tropical fruits and is becoming extremely significant in countries of temperate regions of the world. It is called "King of fruits".

Mango pulp is used as a base for many products. The demand for processed mango products especially mango juice, mango pulp, mango pulp concentrate and mango jam is increasing. Export of these items from India, Brazil, Mexico, Philippines, Taiwan etc., is targeted to reach more than 1,500,000 metric tonnes in the next five years. There is also considerable demand for low viscosity low pulp containing mango juice in the world market. Mango, with its intense flavor, has the potential also as a natural concentrate.

68 Mango juice blended with other juices could be a novel item in the world market. Extruded products using mango pulp and cereal flours can be developed to produce snack items.

Mango nectar can be prepared and could be marketed in either a frozen form or botding or bulk aseptic packing. There is considerable potential for export of mango products but it is imperative that the physico- chemical properties and quality of the pulp are maintained

(Nanjundaswamy, 1991).

Today's consumer demand are for high quality foods that are convenient, nutritious, with freshly prepared flavor, texture and color, with minimal or no chemical preservatives, and above an safe. Conventional thermal processing although ensures safety and extends shelf life of the product it is often leads to detrimental changes in food quality. HPP has shown to be an attractive process because the pressure treatments required to inactivate bacterial cells, yeasts and molds have minimal effect 0 the sensory qualities such as texture, color and flavor (Arroyo et al., 1999).

Spoilage of fruit juices due to microbial growth may lead to off flavors, odors, turbidity and gas production. Acid foods such as fruit juices have been considered safe, however recent food borne diseases outbreaks attributed to consumption JUlce contaminated with pathogens such as E.coli 0157:R7 and Salmonella spp. have demonstrated that unpasteurized juice can be a vehicle for food borne illnesses. Rence appropriate control measures should be taken during juice manufacture. HPP could be used to avoid the detrimental effects of traditional high temperature thermal pasteurization of many foods.

The pressure treatment required for a microbiologically safe and stable product is dependent on the target microorganism to be inactivated. Bacterial vegetative cells, yeasts

69 and moulds are sensitive to pressures between 200 and 700 MPa but bacterial spores may

survive pressurization above 1000 MPa (Arroyo et al., 1999; Sale et al., 1970). Spoilage of foods, due to the outgrowth ofbacterial spores, can be controlled via complementary means

such as refrigeration and or acidification. Various factors influence the pressure-resistance

of microorganisms, including the target microorganism and its physiological state, the intrinsic properties of the menstruum, and the processing temperature, time and magnitude

of pressure treatment (Alpas et al., 2000; Benito et al., 1999; Kalchayanand et al., 1998).

The feasibility of high pressure processing as a preservative method for citrus juices have been extensively investigated (Basak 2001; Boff et al., 2003; Bull et al., 2004;

Fernandez Garcia et al., 2001; Goodner et al., 1998; Nienaber et al., 2001; Ogawa et al.,

1990; Parish et al., 1998 (a); Sanchez-Moreno et al., 2003; Takahashi et al., 1993). Several

studies have been reported on the quality of HP processed juices like tomato, carrot and guava juice (Gow and Hsin, 1998; Park et al., 2002; Porretta et al., 1995). The effect of high pressure on the quality of pre-cut mangos has been studied by Boynton et al. (2002).

At present, high pressure processed orange juices under refrigerated conditions and a range of other food products have been introduced into in the Japanese, European and North

American markets.

However, there is not much information available on high-pressure preservation of mango juice. In the previous chapter, a detailed report on the evaluation of the HP

destruction kinetics of spoilage and pathogenic microorganisms in mango juice. It was

shown that from pro cess establishment point of view, one should consider the most resistant pathogen (a non-pathogen of higher resistance may overrule this in order to provide better shelf-life). Based on E. coli 0157:H7 destruction kinetics, 6-lOD pressure

70 pro cesses (18-30 min treatment at 400 MPa) were designed and tested to be effective for pathogen control. Since the samples were inoculated with pathogen, no quality studies were carried out in the experimental plan. Moreover, processed juice was used as the base for the kinetic studies to avoid variations in the test sample during the course of investigation.

Hence, the objective of this study was to pressure process freshly prepared mango juice according to the pre-established processing schedule and evaluate microbial (indigenous microorganisms, no pathogens) and quality changes in the treated product during storage selected temperatures (4, 12 and 20°C) for up to 60 days.

Materials and Methods

Product

Good quality mangoes (Kent variety) were obtained from the local supermarket

(Costco, Quebec Canada). Mangoes were selected in anticipation of the processing date and were allowed to ripen for 5 days at room temperature (20°C). Cold st orage was avoided to prevent any chilling injury. Mangoes were chosen based on uniformity and consistency in ripeness (coler and softness).

Preparation and Processing of Mango Juice

AlI utensils used in the preparation of mango juice were pre-sterilized in an autoclave. Fresh mangoes were washed thoroughly, peeled, stone removed manually by using a knife and the pulp portion was cut in to small slices. A juice extractor was used to obtain juice/pulp from the prepared fruit slices. The juice/pulp was strained using a strainer with 1 mm mesh screen to get mango juice ofuniform consistency and partic1e size.

71 The pressure treatments were conducted in two parts.

1) Treatment at 400 MPa - These treatments were given at a lethality of OD, 6D, 8D

and lOD based on E. coli destruction kinetics (Chapter 3). The treatment times

corresponded to 18,24 and 30 min for 6D, 8D and 10D processes, respectively.

2) 550 MPa - The treatment was done for 1 min.

The untreated juice and juice to be treated was immediately packed in polyethylene bags (Whirl-Pak®) for 400 MPa and retort pouches with vacuum sealing for 550 MPa, respectivelyand stored at 4°C prior to pressurization.

Fifteen pouches each containing 45 mL of juice, for each pressure lethality, were pressure treated in batch mode. This was done using an isostatic high-pressure machine

(Model # CIP 42260, ABB Autoclave System, Columbus, Ohio, USA) with chamber dimension of 0.56 m height and 0.1 m diameter. FortY five packets each containing 45 mL of juice was treated at 550 MPa for 1 min in batch mode using ACB, Nantes, France

(model ACIP 6500-5-12VB). The equipment and process have been detailed in Chapter 3.

Storage

After HP processmg, aH juices were stored at 4, 12 and 20°C and storage temperature was monitored and verified each day manuaHy. Two packets were randomly withdrawn and samples were analyzed, in duplicate for microbial and physico-chemical properties immediately after processing. Pressure treated samples at 400 MPa were analyzed for microbial growth and physico-chemical parameters and at an interval of 3, 6 and 9 days for 20, 12 and 4°C and for during storage up to 60 days. The samples treated at

72 550 MPa were analyzed for microbial growth and physicochemical parameters at an interval of 7 days for aU the temperatures during storage up to 60 days.

Microbiological Analysis

For microbiological analysis, two pouches of mango juice were cut asepticaUy

(before treatment, after treatment and at selected intervals during storage) and serially diluted with sterile peptone water and plated using the spread plate technique. Yeast and mold counts were done on Potato Dextrose Agar (Difco Laboratories, Detroit. MI). Total plate counts were on using Plate Count Agar (Difco Laboratories, Detroit. MI). The incubation temperature was 27°C for yeasts and molds, and 37°C for aerobic bacteria, respectively. The colonies were counted after 4 days of incubation and results were reported as CFU/mL. The minimum level of detection was 10 CFU/mL.

Total Soluble Solids

The total soluble sugars in juices before and after pressurization were measured using a hand held refractometer (Atago, Japan). °Brix is the unit used to designate the percent dissolved sugar. The sample was adjusted to 20°C and thoroughly mixed the before measurement.

pH

The pH of mango juice was measured using a pH meter (Accumet Basic Model no

AB 15) at room temperature after calibration to pH 7 with a buffer solution.

73 Total Titrable Acidity

The total titrable acidity was measured in terms of citric acid and expressed as g/100g of citric acid. Ten grams of juice was diluted with 100 ml distilled water and mixed with 3-4 drops of phenolphthalein indicator and titrated against O.lN NaOH until the desired end point (light pink color) was reached. The percent acidity was calculated using the following equation (AOAC, 1980).

Per cent acidity = Titre Value x 0.1 x 0.07 x 100 g/100g of citric acid (4.1) 10

Color

The visual color of the mango juices was measured using a Minolta reflectance chromameter (Minolta Corp, Japan Model CM-508d). L, a, b values were measured under standard illumination representing daylight. L values represent the lightness, a values the red to green shades and b values measure the yellow-blue shades. The colorimeter was calibrated using a white standard. Mango juice (20 mL) was poured into petri dish and the chromameter head was placed in juice to measure the L, a, b values. Five readings were taken for each sample and the values obtained were averaged.

Data Analysis

The changes in °Brix, pH, total titrable acidity and color (L and b values) during storage were modeled assuming a first order kinetic process expressed as:

ln [C/Co] = - kt (4.2)

74 where, C = values ofparameters (concentration) after a pressure treatment time t (min), Co l = initial value of the quality parameter and k = reaction rate constant (min- ). For microbial

growth during storage, a decimal multiplication time [Dm values] (i.e. the time required to

cause a change in the quality parameter ten times) was defined.

Dm = [1/slope] (4.3)

For most quality parameters the conventional approach was used since the parametric

values decreased with time. However, for acidity, an approach similar to microbial growth

was used since it increased with time during storage. For quality parameters, the magnitude

ofD value was too large and not meaningful. Henc~ fractional D values were considered in

these situation to get storage times that could be easily compared.

Similarly, the TDT concept was used to explain the relationship between the

decimal reduction (multiplication) times with temperature as

Z = [T2-Td / Log [Dl /D2] (4.4)

Where Dl and D2 are decimal reduction (multiplication) time (days) at Tl and T2

respectively and Z = temperature range required to change D by a factor 10. Values of Z

can be obtained as an inverse reciprocal of the slope of the log D values regressed against temperature (Ramaswamy et al., 1989).

Results and Discussion

Microbial growth

Changes in microbial growth and in physico-chemical characteristics were

evaluated during storage ofpressure processed juice and are discussed separately. Mango is

a highly perishable produce. While as a whole fruit, its shelf-life can be several weeks

75 depending on the packaging and storage conditions, once prepared as juice it becomes highly perishable. While the skin otIers barrier to microbial penetration and the intact cells prevent the enzyme-substrate contact and interaction, these go unchecked in the prepared juice. Hence, the untreated mango juice had a very limited shelf-life especiallY at 20°C. At this temperature, complete spoilage of the untreated juice occurred due to fermentation and no analysis was possible after 3 days. Packages showed hard swelling due to the accumulation of gas formed by anaerobic fermentation. However, sorne testing was possible on the untreated samples stored at 12 and 4°C, which were microbiologically acceptable for 7 days and 15 days, respectively.

The changes in microbial population of different test samples are shown in Figures

4.1- 4.3. The growth of bacteria as measured by the aerobic plate count in HP treated mango juice (0-10D) ditIerent storage temperatures is shown in Figure 4.1 as a function of time. Similar results for yeasts and molds are shown in Figure 4.2 and changes in both bacterial and mold counts in juice processed for 1 min at 550 are shown in Figure 4.3. The mean initial population of the total aerobic bacteria, and yeasts and molds for fresh juice

3 4 (no treatment, OD) were _10 - 10 CFU/mL (Figure 4.1-4.2). Pressure processing at 400

MPa (18-30 min) and 550 MPa (1 min) reduced the microbial population to below the detection limits immediately after the processing (Figures 4.1-4.3). However, it was clear that the process did not kill the entire microbial population (possibly spores survived) and depending on the severity of the treatment and temperature of storage, they revived their growth. Thus, microbial growth was evident in high-pressure processed juice over the time at 20, 12 and 4°C, but the process helped to suppress and delay the growth providing shelf­ life extension.

76 Figure 4.1: EtTect of applied pressure lethality on the growth of aerobic bacteria

during storage of mango juice at 20 oC (.), 12°C (.) and 4°C (.)

00 60

7 8 l' 7 :' 6 J /" ...J / .e 5 ",./" u..~ t :.... ~4 ! z i ~/ 8' 3 4- 1 ...J i f 1 2 i i ; i 1 .1 o+------,------~------_, ! o 10 20 30 o 10 20 30 40 50 60 70 Tlme (Days) Tlme (Days)

80 100

8 6 7 r 5 t~ i 6 ; ! :::ï :::ï i l l" 4 .e 5 1 .e -; ~ l' ~ .' u.. u.. 4 4 l*.# )' 3 lYS ~ / ~ r / ! z 4 Z i )1 CI 3 [ 1 CI 1 2 ... 1• 1 .9 i ; .9 ! i 2 i 1 ! 1 1 ! 1 ! 1 1 J 1 i 1 i ! i 1 i f 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (Days) Time (Days)

77 Figure 4.2: Effeet of applied pressure lethality on the growth of yeast and mold

during storage of mango juiee at 20 oC (.), 12°C (_) and 4°C ( ... )

OD 6D 7 4 6 ;*' BIt /;88 ; i ./ 1 5 ..,- ! 1 ::i 3 1 )If"" ::i i E /" 1 ::l E + 1 4 :::: 1 -LI.. ./ ::l 1 LI.. 1 2- 2 f 1 Z 3 2- j 1 z 1 8' f 1 ..J 8' 1• 2 ..J ! 1 1 1 1 i 1 1 ! i 1 1 ! 0 0 30 40 50 60 0 10 20 30 0 10 20 lime (Days) lime (Days)

8D 10D 5 4

3 ::i ~ .E ! li! ::l f ! LI.. f 2- 2 1 i z i CI 4 i 0 ! i ..J i f i f i i i i i 0 ~.J 0 10 20 30 40 50 60 70 o 10 20 30 40 50 60 70 Tlme (Days) Tlme (Days)

78 Figure 4.3: EfTect of pressure treatment (550MPa) on the growth of aerobic bacteria

and yeast and mold during storage of mango juice at 20 oC (.), 12°C (.) and

Aerobic bacterla Yeastand mold

3 4

_3 ,! .e....1 / ;:) u...... 1 -. 1 2,2 i z i 1 1:11 i o i ....1 i ! ! 1 o~~-~~~j~~~~~~~~~~ o 10 20 30 40 50 60 70 o 10 20 30 40 lime (Days) lime (Days)

Aerobic Plate Counts

The growth and activity of aerobic plate counts in pressure treated mango juice as shown in Figure 4.1 showed sorne characteristic patterns. As mentioned before, the untreated samples with initial counts ~ 104 CFU/mL reached their microbiologically unacceptable levels (106 CFU/mL) more quickly than treated samples. The st orage temperature had an effect on the growth rate as can be generally expected. Spoilage was evident at 105 CFU/mL and occurred after 3 days at 20°C, 12 days at 12°C and 21 days at

79 Compared to untreated juices (OD), all treatments had an effect on the growth of total aerobic bacteria at each storage temperature. In all the se the counts immediately after treatment were below detectable level. Test sampI es treated by a 6D process started showing the growth at the first test interval (3 days) when stored at 20°C which was delayed by 12 days at 12°C and 27 days at 4°C (Table 4.1). The decimal multiplication times ranged from 1 to 10 days between 4 and 20 oC, with the times increasing at higher temperatures. At a higher pressure lethality of 8D, these changes occurred more slowly taking 9, 24 and 36 days at 20, 12 and 4°C to register their first sign of growth (Figure 4.1).

However, once they started growing, their multiplication rates were similar to those subjected to 6D process (Table 4.1). At 10D further delays were observed especially at

12°C (27 days) and 4°C (45 days). Again, the temperature dependent multiplication rates were similar. However, when treated at 550 MPa for one min, the lag for microbial growth significantly increased to 28 days at 20°C and 49 days at 12°C while no growth was apparent at 4°C. Under this pressure, after the lag, the growth achieved was quicker, reaching spoilage levels within seven days.

Yeast and Mold Growth

Observations with respect to the growth of yeast and mold are somewhat similar to those observed with aerobic bacteria (Figure 4.2 and 4.3). With the control sample, the counts quickly increased to unacceptable levels within 3, 12 and 27 days at 20, 12 and 4°C, respectively. For the 6D process, their growth was delayed by 9, 18 and 36 days at the above temperature, respectively (Table 4.1). They multiplied quickly once started to grow, with the de ci mal multiplication time varying from 2 to 16 days between 4 and 20 oC , again

80 indicating a lower multiplication time at lower temperatures. Again, these growth were progressively delayed as the pressure lethality was increased, in each case the lag period for yeast and mold was higher than for aerobic bacteria discussed earlier (Table 4.1). When subjected to 1 min treatment at 550 MPa, again their growth was apparent after 28 and 63 days at 20 and 12C, respectively; however, the growth was completely suppressed up to 60 days at 4°C (Figure 4.3, Table 4.1).

Table 4.1: Lag period and decimal multiplication time for total aerobic bacteria and

yeast /mold in pressure treated mango juice at difTerent temperatures

HP treatment Storage Lag period Decimal Lag period Decimal Temperature Aerobic multiplication Yeast and multiplication (OC) bacteria time mold time (days) Aerobic (days) Yeast and bacteria mold (days) (days) 6D, 18 min at 20 3 1.6 9 2.9 400 MPa 12 12 5.2 18 6.8 4 27 10 36 17 8D, 24 min at 20 9 2.8 15 4.9 400 MPa 12 24 6.0 24 8.6 4 36 14 45 18 10D, 30 min 20 9 3.5 18 8.1 at400MPa 12 26 9.7 36 23 4 45 27 54 27 1 min at 550 20 28 - 28 - MPa 12 49 - 63 - 4 ND - ND - ND Not detected

Thus, high pressure processed mango juice demonstrated sorne microbiological activity during storage indicating the processed products were not completely free of

81 microorganism. The pressure severity and storage temperatures influenced their growth lag and rate with higher lethality and lower temperatures prolonging the lag time and growth rate. No growth of bacteria, yeasts and molds were observed in mango juice treated at 550

MPa, stored at 4°C for 60 days. Renee, only the pressure treatment at 550 MPa was effective for inhibition of microbial growth, perhaps a slightly higher treatment (time or pressure) may be warranted if the growth were to be prevented at storage between 12 and

20°C.

Total soluble solids (OBrix)

Figure 4.4 shows the changes ln the normalized soluble solids content [Log

{Brix(t)/Brix(o)XlOO} vs. time] of pressure processed juice during storage at different temperatures. In general, storage temperature had sorne consistent although small effect on change in soluble solids. This can be an important parameter for stability. Changes in soluble solids mostly imply sorne microbial fermentation or enzymatic breakdown of sugars. If one were to expect storage stability, the se changes need to be minimized. In comparison with the changes soluble solids in untreated juice where the microbial activity resulted in rapid spoilage and fermentation leading to the production of gasses (largely carbon dioxide), pressure treated samples had no significant change. Rowever, when the treated samples were compared amongst themselves systematic changes were observed sorne what indicating first order rate of change. Comparing all of them in a first order mode, the associated D value could be used as a parameter of comparison.

82 Figure 4.4: EfTect of applied pressure lethality on the °Brix values during storage of

mango juice at 20 oC (.), 12°C (.) and 4°C (A)

00 60 2.01

2.01

0' 2.00 ....o 0' >oC ~ 2.00 ~:=:::::::::_-'_-___ >oC ~ o ŒI 1.99 >oC >oC ŒI 1.99 ŒI >< ŒI ...... ~ 1.98 ~ 1.98

1.97 +------,--,------,---, 1.97 +-----,---..-----, o 5 10 15 20 o 10 20 30 lime (Days) lime (Days)

80 100

2.01 2.01 ..... 0 0' 0 2.00 .... '" Â Â 0 2.00 ...... i""'1I! Â >oC >oC ~ .-.. 0 0 >oC >oC ŒI 1.99 ŒI 1.99 ->oC >oC ŒI lB...... 1.98 1.98 ~ ~

1.97 1.97 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 lme (Oays) lime (Days)

Table 4.2 shows the kinetic data (Do.5 values) obtained by the semi- logarithmic regression analysis between the °Brix values against storage time. Since the normal D values indicate a 90% reduction in value of soluble solids which is rather meaningless. That

83 would mean a total spoilage by fermentation. Rence a fractional D value (DO.5) is used as an indicator of the associate changes. The DO.5 indicates a tenth of a D value and hence represents a 0.5% change in soluble solids concentration. The kinetic data show that the changes in mango °Brix values during storage were minimal as shown by large DO.5 values

(Table 4.2). The predicted time based on the log-linear model varied from 1-22 days at

200 e to 3-31 days at 12°e and 7-73 days at 4°e (Table 4.2). The DO.5 values increased with an increase in pro cess lethality and a decrease in temperature of storage. Mango juice pressure treated at 550 MPa for 1 min also showed no change in the °Brix values after storage at 4, 12 and 200 e (Figure 4.5).

Table 4.2: Do.5values for the changes in (OBrix) values of HP treated mango juice

during storage at ditTerent temperatures

Temperature Pressure lethality 0 0.5 values R" (oC) # Oays 20 0 1 0.89 20 6 2 0.75 20 8 5 0.82 20 10 22 0.57 12 0 3 0.89 12 6 4 0.99 12 8 7 0.75 12 10 31 0.57 4 0 7 0.75 4 6 11 0.75 4 8 24 0.57 4 10 73 0.31

84 Figure 4.5: EtTect of pressure treatment (550MPa) on °Brix values during storage of

mango juice at 20 oC (.), 12°C (.) and 4°C (.&.)

550

15

14.5

~ ·C !III == 14,···. o • 13.5

13 +---~----~--~----~--~----~--~ o 10 20 30 40 50 60 70 Time (Days)

pH

Figure 4.6 shows changes in the normalized pH [Log {pH (t)/pH (0) X lOO} vs. time] during storage at different temperatures. As with soluble solids the range of variation was comparatively minimal during storage; however the pH curves showed a c1ear first order trend with storage time for aB samples. Again, the changes depended largely on the storage temperature, increasing with an increasing temperature, and decreasing with pressure lethality.

85 Figure 4.6: EfTect of applied pressure lethality on the pH during storage of mango

juice at 20 oC (.), 12°C (.) and 4°C (Â)

00 60

2.01 2.01 2.00 ....0 0 ....0 2.00 .... 1.99 ....0 .-.>< >< 0 1.98 0 1.99 :J: :J: 0. 1.97 0. -:J: :J: 0. 1.96 - 0. 1.98 ...... - 1.95 -...... ~ 1.94 ~ 1.97 1.93 0 5 10 15 1.96 0 10 20 30 Tlme (Days) Tlme (Days)

80 100 2.01 2.01 .... 0.... 0 2.00 0 0...... >< ~ .-. 0 0 :J: 0. 1.99 =a. 1.99 -:J: 0. =a -...... 1.98 - ~ ~ 1.98

1.97 188 0 10 20 30 40 50 60 70 1.97 Time (Days) 0 10 20 30 40 50 60 70 Tlme (Days)

Since the changes were small and the magnitude of pH was much smaller than soluble solids (~4 vs. 15), a Ds value (storage time resulting in a 5% change in pH) was

86 used for comparison (Table 4.3). High R2 values were obtained for most treatment and

hence the decrease in the pH could be explained well by the logarithmic model. The

predicted times for resulting in a 5% change in pH varied from 1-22 days at 20°C to 4-44

days at 12°C and 10-75 days at 4°C (Table 4.3). Tables show 4.2 and 4.3 show large

similarities, only difference is that the time base is for a 5% change in pH while it is for a

0.5% in total soluble solids. Figure 4.7 the effect of pressure treatment at 550 MPa on the

pH changes during storage. Associated changes were small

Table 4.3: D5 values for the changes in pH values of HP treated mango juice during

storage at different temperatures

L Temperature Pressure lethality 0 5 values R (oC) # Oays 20 0 0.99 1 20 6 0.96 5 20 8 0.88 17 20 10 0.95 22 12 0 0.94 4 12 6 0.82 14 12 8 0.91 28 12 10 0.97 45 4 0 0.83 10 4 6 0.93 45 4 8 0.8 74 4 10 0.91 74

87 Figure 4.7: Effect of pressure treatment (550MPa) on pH during storage ofmango

juice at 20 oC (.), 12°C (_) and 4°C (.A.)

550

4.6

4.5 • • liI1 Â .. • Il • • • Â • Â 4.4 • ,. :J: Q. • • 4.3

4.2 4.1 a 20 40 60 80 Time (Days)

Total Titrable Acidity

The total titrable acidity of mango juice is often expressed as acidity in terms of citric acid. Figure 4.8 shows changes in the total titrable acidity during storage at different temperatures. The curves showed a general increase in total titrable acidity as the storage time increased. Rowever, the range of variation was comparatively minimal during storage.

These trends are consistent with a decrease in soluble solids and decrease in pH. In contrast with the previous two curves (Figures 4.4-4.7), the acidity curves would show an increase in value with time. Renee the conventional D has to be defined as a decimal multiplication time. Again a full D defined this way would mean doubling of acidity which is quite a drastic change. Renee, to get a meaningful comparison, considering the magnitude of acidity a Dl.2 value was defined as the time to increase the acidity by 20%.

88 Figure 4.8: EtTect of applied pressure lethality on the total titrable acidity du ring

storage of mango juice at 20 oC ( • ), 12°C (.) and 4°C (Â)

00 60

2.4 2.5

2.4 ::::: 2.3 .... 0 0 0 ....0 .... >< 2.3 >< 2.2 ..... 0 0 1\1 1\1 2.2 .!!. 2.1 .!!...... 2.1 ~ ~

1.9 1.9 0 5 10 15 0 10 20 30 Tlme (Days) Tlme (Days)

80 100

2.3 2.3

• ....0 2.2 ....0 2.2 0 0 ...... >< >< SIl: 0 0 1\1 1\1 2.1 2.1 1\1 -1\1 - :::::; ....-- ~ .9

1.9 1.9 0 20 40 60 80 0 20 40 60 80 Tlme (Days) Tlme (Days)

89 2 Table 4.4 shows the kinetic data D1.2 values obtained from the curves. High R values were obtained for most treatment and hence the increase in the total titrable acidity could be explained by the logarithmic model. The predicted times for resulting in a 20% change in acidity varied from 0.6-14 days at 20°C to 2-20 days at 12°C and 4-47 days at

4°C (Table 4.4) somewhat in line with other changes reported earlier. Thus acidity of the pressure processed product increased during storage increasing with temperature. Pressure lethality contributed to slowdown the increase in acidity during storage. Figure 4.9 shows the smaller change in acidity in mango juice pressure treated at 550 MPa. The product was considered to be chemically stable under after this treatment.

Table 4.4: D1.2 values for the changes in total titrable acidity of HP treated mango

juice during storage at different temperatures

Temperature Pressure lethality Du values R~ (oC) # Days 20 0 0.6 0.93 20 6 1.4 0.91 20 8 7 0.9 20 10 14 0.91 12 0 2 0.94 12 6 4 0.92 12 8 18 0.86 12 10 20 0.85 4 0 4 0.95 4 6 8 0.87 4 8 27 0.82 4 10 47 0.86

90 Figure 4.9: EfTect of pressure treatment (550MPa) on total titrable acidity during

storage of mango j uice at 20 oC ( • ), 12°C (.) and 4°C ( ... )

550 O~l 188 • • • lIIi !II! • !II! • • 0.30 ~::< ; r:~ t f::' !III ~:'. ",~ >- :t! :si! 0.20 ~ fi!.

0.10

0.00 +--~-~---,------,------.---,------, o 10 20 30 40 50 60 70 Time (Days)

Color Changes during Storage

L Value

Changes in L values were measured to investigate changes in the brightness of processed juice during storage. Overall, changes were noticed under aU conditions of storage. Changes in color of mango juice (L values) during storage at different temperatures are indicated in Figure 4.10 as a semi-Iogarithmic plot vs storage time. The changes were c1early dependent on storage tempe rature and pressure lethality. Looking at the time scale it becomes obvious the changes were slower at lower temperature and higher pressure lethality.

Table 4.5 shows the kinetic data (D2o values, time required to result in 20% change) obtained by the semi logarithmic regression analysis between the L values against storage

91 time. High R2 values were obtained for most treatment and hence the decrease in L values

could be explained weIl by the logarithmic model. These changes varied from 1-14 days at

20°C, 3-35 days at 12°C and 7-65 days at 4°C.

Figure 4.10: EfTect of applied pressure lethality on the L values during storage of

mango juice at 20 oC (.), l2°C (_) and 4°C (~)

00 60 2.05 2.05

2.00.·.·.1 ....0 1.95 0 ....0 .... 0 >oC 1.90 .... 1.95 >oC .9 1.85 ~ ::J 1.90 ...... ::J ..... 1.80 i 1.85 ~ 1.75 ...J 1.80 1.70

1.65 1.75 0 10 20 30 0 10 20 30 Tlme (Days) Tlme (Days)

80 100

2.05 2.05 .... S 2.00' 0 2.00 .:~ ....0 ....0 >oC >oC 1.95 .-. .9 .9 1.95 ::J 1.90 -...J ...... 1.90 ~ 1.85 ~

iII 1.80 • 1.85 • 0 20 40 60 0 10 20 30 40 50 60 70 Tlme (Days) Time (Days)

92 For samples without pressure treatment (OD), color changes were noticed soon after storage of the samples. The most severe changes in color were observed in samples stored at 20°C followed by the samples stored at 12 and 4°C. The higher pressure lethality and lower temperature resulted in better color stability. This decrease in L value during storage could be due to the higher activity of polyphenoloxidase in aH samples, which leads to the formation of dark pigments (conversion of phenolic compounds to quinone by polyphenol oxidase). Higher pressure may also reduce enzymatic browning, white lower pressures may activate sorne browning enzymes (Anese et al., 1995). Improved col or stability was also observed during storage at lower temperatures. However, this may be attributed to lower microbial growth in the se treatments.

Table 4.5: D20 values for the color changes (L values) of HP treated mango juice

during storage at ditTerent temperatures

L Temperature Pressure lethality 0 20 values R CC) # Oays 20 0 1 0.99 20 6 3 0.99 20 8 7 0.81 20 10 14 0.92 12 0 3 0.95 12 6 6 0.99 12 8 18 0.95 12 10 35 0.85 4 0 7 0.96 4 6 18 0.89 4 8 36 0.89 4 10 65 0.74

Figure 4.11 shows the effect of pressure treatment at 550 MPa on the color (L values). No changes in the values were observed during storage at aU the temperatures.

93 Figure 4.11: Effeet of pressure treatment (550MPa) on L values during storage of

mango juice at 20 oC (.), 12°C (_) and 4°C (.)

550 50

Time (Days)

b values

Changes b values were measured to investigate changes in the yellowness of processed juice during storage. OveraU, changes were noticed under aU conditions of storage. Figure 4.12 indicates the changes in b values of pressure treated mango juice during storage at different temperatures.

Table 4.6 shows the kinetic data (D20 values) obtained by the semi logarithmic regression analysis between the b values against storage time. Again, high R2 values were obtained for most treatment and hence, the decrease in the b values cou Id be eXplained weil by the logarithmic model. These changes varied from 0.7-24 days at 20°C, 2-44 days at

12°C and 5-61 days at 4°C, somewhat consistent with changes in L values. The extent of change was a function of pressure lethality level and storage temperature. Higher the pressure lethality and lower the temperature resulted in a better color retention. The effect

94 of pressure treatment at 550 MPa on the color b values is shown in Figure 4.13. No changes in the values were observed during storage at aH the temperatures.

Figure 4.12: EtTect of applied pressure lethality on the b values during storage of

mango juice at 20 oC (. ), 12°C (.) and 4°C (Â)

95 Table 4.6: D20 values for the color changes (b values) of HP treated mango juice

during storage at different temperatures

Temperature Pressure lethality D20 values Roi eC) # (Days) 20 0 1 0.99 20 6 2 0.99 20 8 11 0.96 20 10 24 0.87 12 0 2 0.92 12 6 4 0.91 12 8 17 0.82 12 10 44 0.82 4 0 5 0.97 4 6 11 0.98 4 8 37 0.87 4 10 61 0.87

Figure 4.13: Effect of pressure treatment (550MPa) on b values during storage of

mango juice at 20 oC (. ), 12°C (.) and 4°C (Â)

550

~OOi lIII lIII CI) 32.50 . • iIII -CIl • • • :::s ~ .,;;;. • ~ Ci 31.00 t • • • > • • e... 29.50 0 '0 28.00 0 26.50

25.00 0 10 20 30 40 50 60 70 Time (Days)

96 Temperature Sensitivity of D values

Figures 4.14a,b (i-v) show the temperature sensitivity plots for the changes in °Brix, pH, % acidity and color with different pressure lethalities. Table 4.7 shows the Zp values for each variable. A good log-linear relationship was found with D value for changes in each parameter vs storage temperature (shawn with high R2 values). The Zp values increased with an increase in applied pressure lethality. Higher Zp values indicate a better temperature stability (in fact a lower temperature dependence). Hence, the IOD pressure processing permits better temperature stability for all parameters and hence is a desirable condition ta use.

Figure 4.14a: Temperature sensitivity of the Dm values for the changes in °Brix and

pH of pressure treated mango juice

°Brix pH

2.0 2.0

1.5 1.5 .00 II> ~ d #60 CS 1.0 CI 1.0 CI 0 0 .:::.80 ..J ..J :.: 100 0.5 ~ 0.5 0.0 0.0 0 10 20 30 0 10 20 30 Tem perature" oc Temperature OC (i) (ii)

* OD (+), 6D (.), SD (Â), IOD (x)

97 Figure 4.14b: Temperature sensitivity of the Dm values for the changes in total

titrable acidity and color (L and b values) of pressure treated mango juice

%acidity Color ( L values)

2.0 2.0

1.5 :':~'.... 1.5 ;:;i: ..... N 6 1.0 ~ en en 1.0 0 0 ..J 0.5 ..J

0.0 ~ 0.5

-0.5 0.0 0 10 20 30 0 10 20 30 Temperature OC Temperature OC

(iii) (iv)

Color (b values)

2.0 ;~'.' 1.5 ..... " .~,,:::. ~ 1.0 en 0 ..J 0.5 ~ 0.0

-0.5 0 10 20 30 Temperature Oc

(v)

* OD (.), 6D (_), 8D (.), lOD (x)

98 Data for 550 MPa could not be inc1uded in the figure because the condition almost did not result in any appreciable change in any of the quality parameters. While from microbial destruction point of view, pressure processing at 550 MPa would be highly desirable since it would be more efficient against bacterial destruction especially bacterial spores which may be implicated in product spoilage.

Table 4.7: Temperature sensitivity data for different quality parameters of HP

treated mango juice during storage

Quality Pressure Zp Qarameter lethality # (oC) R2 °Brix 0 19.4 0.99 6 22.9 0.99 8 24.7 0.88 10 30.6 0.94 pH 0 15.9 0.99 6 16.1 0.99 8 25.1 0.96 10 30.6 0.99 %acidity 0 18.9 0.99 6 21.4 0.97 8 25.8 0.93 10 31.0 0.94 Color (L values) 0 19.8 0.93 6 21.0 0.75 8 21.9 0.98 10 24.3 0.98 Color (b values) 0 18.2 0.99 6 21.2 0.98 8 30.7 0.96 10 40.2 0.96

99 Conclusions The microbial and quality changes of high pressure processed mango juice processed at different pro cess lethalities at 400 MPa were small during st orage at 4, 12 and

20°C. Changes in quality parameters and microbial growth were influenced by the storage temperature and applied pressure lethality. A lOD process coupled with a storage temperature of 4°C, delayed these deteriorative changes in mango juice. Additional treatments at 550 MPa for 1min resulted in excellent juice quality and it was comparable to that offreshjuice for up to 60 days at 4°C.

One of the reasons for the relative1y more rapid changes in quality factors observed with pressure processed products at 400 MPa as compared with 550 MPa could be the choice of packaging material used. It was a polyethylene plastic bag not protected fully against light and gas exchange, while the one used after the 550 MPa treatment (carried out at a later time), the packaging material used was a total barrier retort pouch.

100 CHAPTER5

GENERAL CONCLUSIONS

General Conclusions

Non-thermal food processing technologies, such as HP processing, high electric field pulses and high intensity ultrasound applications and/or the utilization of

antimicrobial bacteriocins, have received great attention by food scientists due to their potential as new methods to preserve food with minimal 10ss of sensory and nutritional

qualities of foods. These non-thermal processing technologies may be applied to produce new food products, or as substitutes for conventional processes which could induce

significant losses in product quality. The objective of this thesis was to investigate the

applicability of HP processing to preserve mango juice, a popular beverage in the tropical

subcontinents and of high commercial value. For the application of HP processing to mango juice, the ability of HP processes to inactivate the microorganisms of spoilage and health concern while minimizing changes in physicochemical and other quality parameters in order to have a commercial viability. Therefore, our efforts were focused

on the gathering of the kinetic data on spoilage and pathogenic microorganisms and

subsequently on its effects on quality parameters both immediately after the process as well as during storage at room or refrigerated temperatures.

The research work was carried out in two stages. The first part focused on the effect of pressure level and pressure hold time on the destruction of spoilage and pathogenic microorganisms in mango juice and establishment/verification of the HP process. Based on the treatment conditions developed in the first part, the second part of

101 this research involved the changes in quality parameters during storage of mango juice at different temperatures. The findings ofthis research are summarized below.

The effect of pressure level and time on the destruction of spoilage and pathogenic microorganisms were characterized to be a dual effect. The first one was a

static effect consisted of a rapid reduction in the microbial population as a result of the application of a pulse pressure without any holding. The second was the first order rate of destruction during the pressure-hold time. The pressure inactivation kinetics of microorganisms was established with a wide range of process conditions (200- 550 MPa) and times (0-60min). In order to understand the pulse effect, two terms were used. ND represented the number of pulses required to achieve one log reduction and DpE represented the decimal reduction time equivalent of PE. For aB of the microorganisms studied, PE (pulse effect) increased with an increase in pressure. This effect was more pronounced in case of E. coli 0157:H7. The associated Dp values decreased with an increase in pressure level for aH the microorganisms. Pressure death time models were used to describe the pressure sensitivity of microbial destruction rate (Dp). Results showed that E. coli 0157:H7 was the most pressure resistant of aB the microorganisms tested in mango juice.

HP processes were developed based on the kinetic data for the most resistant microorganism. Mango juice was therefore treated to achieve different process lethalities with respect to E. coli 0157:H7 and the survival and growth E. coli 0157:H7 was monitored during storage at different temperatures for 30 days. No growth of E. coli

0157:H7 was observed during the entire storage period in mango juice subjected to 6-

10D processes with initial counts or 108 CFU/mL.

102 Storage studies of HP processed fresh mango juice by the established processes

showed that the shelf-life of mango juice was extended significantly by controlling

microbial growth. The results showed that the growth of microorganisms and changes in

quality parameters were influenced by interaction of applied pressure lethality and

storage temperature. Overall, changes in quality were small, and the product was not

completely stable wh en processed at 400 MPa even for a 10D process; they were

microbiologically safe however. Additional treatments at 550 MPa for 1 min showed that

the quality of mango juice was stabilized and was comparable to fresh untreated juice

even after 60 day storage at 4°C.

The ultimate objective of mango juice processing would be to ensure the safety of the consumer and provide sufficiently long shelf-life for the manufacturer while restoring

the quality properties. The results from the present study suggest that mango juice with

superior quality cou Id be obtained by the application of appropriate high pressure treatments and subsequent storage under refrigerated conditions.

Suggestions for Future Research

This work demonstrated several interesting results on the destruction of

microorganisms and the quality of mango juice. The following are recommended for

future research in HP processing of mango juice.

1. Destruction kinetics of microbial spores and enzymes responsible for spoilage of

mango pulp using different temperature-pressure combinations.

2. Evaluating the influence of packaging material on the shelf-life of HP treated

mango juice and other mango products such as puree, nectar etc.

103 CHAPTER6

REFERENCES

Adriana, Z.M., Delia, B.R. (1998). Effect ofripening cultivar differences and processing

on the carotenoid composition of mango. Journal of Agricultural and Food

Chemistry, 46:128-130.

Aleman, G., Farkas, D.F., Torres, A, Wilhelm sen, E. and McIntyre, S. (1994). Ultra high

pressure pasteurization of fresh cut pineapple. Journal of Food Protection,

57(10): 931- 934.

Aleman, G. D., Ting, E. Y, Mordre, S. C., Hawes, A C. O., Walker, M., Farkas, D. F.

and Torres, 1. A (1996). Pulsed ultra high pressure treatments for pasteurization

ofpineapple juice. Journal ofFood Science, 61(2): 388-390.

Alpas, H, Kalchayanand, N., Bozoglu, F. and Ray, B. (2000). Interactions of high

hydrostatic pressure, pressurization temperature and pH on death and injury of

pressure-resistant and pressure sensitive strains of foodborne pathogens.

International Journal ofFood Microbiology, 60: 33 -42.

Ananth, v., Dickson, 1. S., OIson, D. G. and Murano, E. A (1998). Shelf life extension,

safety, and quality of fresh pork loin treated with high hydrostatic pressure.

Journal ofFood Protection, 61(12): 1649-1656.

104 Anese, M., Nicoli, M., Aglio, G. and Lerici, C. (1995). Effect ofhigh-pressure treatments

on peroxidase and polyphenoloxidase activities. Journal of Food Biochemistry,

18:285- 293.

Anonymous, (1995). Escherichia coli 0157:H7 outlinked to commercially dry-cured

salami. Morbidity andMortality Weekly Report, 44:157-160.

AOAC, (1980). Official methods ofanalysis, 13th edn. Association of Official Analytical

Chemists, Washington, DC, p 271.

Arroyo, G., Sanz, P. D. and Prestamo, G. (1999). Response to high pressure, low­

temperature treatment in vegetables: determination of survival rates of microbial

populations using flow cytometry and detection of peroxidase activity using

confocal microscopy. Journal ofAppliedMicrobiology, 86(3): 544 -556.

Basak, S. and Ramaswamy, H. S. (1996). Ultra high pressure treatment of orange juice: a

kinetic study on inactivation of pectin methyl esterase. Food Research

International, 29(7): 601-607.

Basak S. (2001). Studies on high pressure processmg of orange juice: enzyme

inactivation, microbial destruction, and quality changes, process verification and

storage. High pressure processing of [Ph.D thesis]. Ste Anne de Bellevue,

Montreal: McGill University. Available from: McGill University.

Basak, S., Ramaswamy, H. S. and Piette, J. P. G. (2002). High pressure destruction

kinetics of Leuconostoc mesenteroides and Saccharomyces cerevisiae in single

105 strength and eoneentrated orange juiee. Innovative Food Science and Emerging

Technologies, 3: 223-231.

Benito, A., Ventoura, G., Casadei, M., Robinson, T. and Maekey, B. (1999). Variation in

resistanee of natural isolates of Escherichia coli 0157:H7 to high hydrostatie

pressure, mild heat, and other stresses. Applied and Environmental Microbiology,

65(4): 1564 -1569.

Besser, RE., Lett, S.M., Weber, J.T., Doyle, M.P., Barrett, T.J., Wells, J.G. and Griffin,

P.M. (1993). An outbreak of diarrhea and hemolytie uremie syndrome from

Escherichia coli 0157:H7 in fresh-pressed apple eider. The Journal of the

American Medical Association, 269: 2217-2220.

Boff, lM., Truong, T.T., Min, D.B. and Shellhammer, T.H. (2003). Effeet of thermal

proeessmg and earbon-dioxide-assisted high-pressure proeessing on

peetinmethylesterase and ehemieal changes in orange juiee. Journal of Food

Science, 68(4): 1179-1184.

Boynton, B.B., Sims, C.A, Sargent, S., Balaban, M.O., and Marshall, M.R (2002).

Quality and st ab ility of preeut Mangos and Carambolas subjeeted to high-pressure

processing. Journal ofFood Science, 67(1): 409-415.

Bull, M.K., Zerdin, K., Howe, E., Goieoeehea, D., Paramanandhan, P., Stoekman, R,

Sellahewa, l, Szabo, E. A., Johnson, R L. and Stewart, C. M. (2004). The effeet

of high pressure proeessing on the mierobial, physieal and ehemieal properties of

106 Valencia and Navel orange jUlce. Innovative Food Science and Emerging

Technologies, 5:135-149.

Butz, P., Koller, W. D., Tauscher, B. and Wolf, S. (1994). Ultrahigh pressure processing

of onions: chemical and sensory changes. Lebensmittel-Wissenschaft und

Technologie, 27: 463-467.

Butz, P., Edenharder, R., Fister, H. and Tauscher, B. (1997). The influence of high

pressure processing on antimutagenic activities offruit and vegetable juices. Food

Research International, 30 (3/4):287-291.

Butz, P., Femandez, A, Fister, H. and Tauscher, B. (1997). Influence ofhigh hydrostatic

pressure on aspartame: instability at neutral pH. Journal ofAgricultural and Food

Chemistry, 45(2): 302-303.

Butz, P., Femandez, A, Lindauer, R., Dieterich, S. and Tauscher, B., (2003). Influence of

ultra high pressure processing on fruit and vegetable products. Journal of Food

Engineering, 56:233- 236.

Cano, M. P., Hemandez, D.E. and Ancos, B. (1997). High pressure and temperature

effects on enzyme inactivation in strawberry and orange products. Journal of

Food Science, 62 (1): 85-88.

Cariez, A, Rosec, J. P., Richard, N. and Cheftel, J. C. (1993). High pressure

inactivationof Citrobacter freundii, Pseudomonas fluorescens and Listeria

107 innocua III inoculated minced beef muscle. Lebensmittel Wissenschaft und

Technologie, 26: 357-363.

Cariez, A., Veciana-Nogues, T. and Cheftel, 1. C. (1995). Changes in color and

myoglobin of minced beef meat due to high pressure processing. Lebensmittel

Wissenschaft und Technologie, 28(5): 528-538.

Cheftel, 1. C. and Culioli, 1. (1995). Review: High-pressure, microbial inactivation anf

food preservation. Food Science& Technology International, 1: 75-90.

Cheftel, 1. C. and Culioli, 1. (1997). Effect of high pressure on meat: A review. Meat

Science, 46: 211-236.

Cheng Hsin-Yi and Cheng-Chun Chou (2001). Acid adaptation and temperature effect on

the survival of E. coli0157:H7 in acidic fruit juice and lactic fermented milk

product. International Journal ofFood Microbiology, 70: 189-195.

Clouston, 1. G. and Will s, P.A. (1969). Initiation of germination and inactivation of

Bacil/us pumilus spores by hydrostatic pressure. Journal ofBacteriology, 97: 684-

690.

Dogan, C. and Erkmen, O. (2004a). High pressure inactivation kinetics of Listeria

monocytogenes inactivation in broth, milk, and peach and orange juices. Journal

ofFood Engineering, 62: 47-52.

Dogan, C. and Erkmen, O. (2004b). Kinetic analysis of Escherichia coli inactivation by

high hydrostatic pressure in broth and foods. Food Microbiology, 21: 181-185.

108 Donsi, G., Ferrari, G. and Matteo, M.D. (1996). High pressure stabilization of orange

juice: evaluation of the effects of process conditions. Italian Journal of Food

Science, 2:99-106.

Doyle, M.P., (1991). Escherichia coli 0157:H7 and its significance III foods.

International. Journal ofFood Microbiology, 12: 289-301.

Doyle, M. P., Beuchat, L. R, and Montville, T. 1. (2001). Food microbiology:

fundamentals and frontiers. Washington: ASM press, pp.383-410.

Drake, M.A, Harrison, S.L., Asplund, M., Barbosa Canovas, G. and Swan son, B.G.

(1997) High pressure treatment of milk and effects on microbiological and

sensory quality of cheddar cheese. Journal ofFood Science, 62(4): 843 845.

Earnshaw, RG., Appleyard, 1., and Hurst, RM. (1995). Understanding physical

inactivation processes: combined preservation opportunities using heat,

ultrasound and pressure. International Journal of Food Microbiology, 28: 197-

219.

Eyring, H. and Magee, 1.L. (1942). Application of the theory of ab solute reaction rates to

bacterialluminescence. Journal of Cellular and Comaprative Physiology, 20:

169-177.

Fernandez Garcia, A, Butz, P., Bognar, A and Tauscher, B. (2001). Antioxidative

capacity, nutrient content and sensory quality of orange juice and an orange-

109 lemon-carrot juice product after high-pressure treatment and storage in different

packaging. European Food Research and Technology, 213: 290-296.

Food and Agriculture Organization of the United Nations. (1993). FAO Quarterly

Bulletin ofStatistics. Rome: FAO, (6):1, p31. Available from: FAO, Rome

Furukawa, S., Noma, S., Yoshikawa, S., Furuya, H, Shimoda, M., and Hayakawa, I.

(2001). Effect of filtration of bacterial suspensions on the inactivation ratio in

hydrostatic pressure treatment. Journal ofFood Engineering, 50: 59-61.

Garcia, A, F., Butz, P., Bognar, A and Tauscher, B. (1998). Antioxidative capacity,

nutrient content and sensory quality of orange juice and orange - lemon - carrot

juice product after high pressure treatment and storage in different packaging.

European Food Research Technology, 213: 290-296.

Garcia-Graells, C., Hauben, K. l A and Michiels, C. W. (2001). High-pressure

inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in

fruit juices. Applied and Environmental Microbiology, 64: 1566-1568.

Gervilla, R, Mor-Mur, M., Ferragut, V. and Guamis, B. (1999). Kinetics of destruction

of Escherichia coli and Pseudomonas fluorescens inoculated in ewe's milk by

high hydrostatic pressure. FoodMicrobiology, 16: 173-184.

Glass, K.A, Loeffelholz, lM., Ford, lP., Doyle, M.P., (1992). Fate of Escherichia coli

0157:H7 as affected by pH or sodium chio ride and in fermented, dry sausage.

Applied and Environmental Microbiology, 58: 2513-2516.

110 Goodner, IK, Braddock, RI and Parish, M.E. (1998). Inactivation of peetinesterase in

orange and grapefruit juices by high pressure. Journal of Agricultural and Food

Chemistry, 46: 1997-2000.

Goodner, IK, Braddock RI, Parish, M.E. and Sims, C.A (1999). Cloud stabilization of

orange juice by high pressure processing. Journal of Food Science, 64(4): 699-

700.

Gow C.Y, Hisin T.L., (1996). Comparison of high pressure treatment and thermal

pasteurization effects on the quality and shelf life of guava puree. International

Journal ofFood Science and Technology, 31: 205-213.

Gow-Chin Yen and Hsin-Tang Lin (1998). Effects ofhigh pressure and heat treatment on

pectie substances and related characteristics in guava juice. Journal of Food

Science, 63(4): 684-687.

Hauben, K I A, Wuytack, E. Y, Soontjens, C. C. F. and Michiels, C. W. (1996). High

pressure transient sensitization of Escherichia coli to lysozyme and nisin by

disruption of outer-membrane permeability. Journal of Food Protection, 59(4):

350-355.

Hayakawa, 1., Kajihara, l, Morikawa, K, Oda, M. and Fujio, Y (1992). Denaturation of

bovine serum albumin (BSA) and ovalbumin by high pressure, heat and

chemicals. Journal ofFood Science, 57(2): 288-292.

111 Hayakawa, 1., Kanno, T., Yoshiyama, K., and Fujio, Y. (1994). Oscillatory compared

with continuous high pressure sterilization on Bacil/us stearothermophilus spores.

Journal ofFood Science, 59: 164-167.

Heinz, V. and Knorr, D. (1996). High pressure inactivation kinetics of Bacillus subtilis

cells by a three-state-model considering distributed resistance mechanism. Food

Biotechnology, 10(2): 149-161.

Hendrickx, M., Ludikhuyze, L., Van den Broeck, 1., and Weemaes, C. (1998). Effects of

high pressure on enzymes related to food quality. Trends in Food Science &

Technology, 9: 197-203.

Hill, D.S. and Waller, lM. (1999) Pests and diseases of tropical crops, vol. 2, 4th edn. pp

219-223.

Hoover, D.G., Metrick, c., Papineau, AM., Farkas, D.F. and Knorr, D. (1989).

Biological effects of high hydrostatic pressure on food microorganisms. Food

Technology, 43: 99-107.

Johnson, G. 1., Sharp, lL., Milne, D.L., Oosthuyse, S.A (1997). Postharvest technology

and quarantine treatments ln: The Mango Botany, Production and Uses, ed. R.

Litz, CAB international, New York, USA, 509-544.

Kaanane, A, Kane, D. and Labuza, T.P. (1988). Time and temperature effect on stability

of Moroccan processed orange juice during storage. Journal of Food Science,

53(5):1470-1473, 1489.

112 Kalchayanand, N., Sikes, T., Dunne, C.P. and Ray, B. (1998). Factors infuencing death

and injury of foodborne pathogens by hydrostatic pressure-pasteurization. Food

Microbiology, 15: 207- 214.

Kalchyanand, N., Sikes, A, Dunne, C. P. and Ray, B. (1998). Interaction of hydrostatic

pressure, time, and temperature of pressurization and pediocin AcH on

inactivation offoodborne bacteria. Journal ofFood Protection, 61(4): 425-431.

Kim, Y. S., Park, SJ., Cho,Y.H. and Park J. (2001). Effects of combined treatment of

high hydrostatic pressure and mi Id heat on the quality of carrot juice. Journal of

Food Science, 66(9): 1355-1360.

Ki mura, K., Ida, M., Yosida,Y., Ohki, K., Fukumoto, T. and Sakui, N. (1994).

Comparison of keeping quality between pressure processed jam and heat

processed jam: changes in flavor components, hue and nutrients during storage.

Bioscience, Biotechnology, Biochemistry, 58: 1386-1391.

Kirstien, M.F.A, Reyns., Carine. C.F., Kris, c., Carla, A, Hendrickx, M.E., and Chris,

W.E., (2000). Kinetic analysis and modeling of combined high pressure

temperature inactivation of the yeast Zygosaccharomyces bailii. International

Journal ofFood Microbiology, 56: 199-210.

Knorr, D. (1993). Effects of high-hydrostatic-pressure processes on food safety and

quality. Food Technology, 47(6): 156, 158-161.

113 Knorr, D. and Heinz, Y. (1999). Recent advances in high pressure processing of foods.

New Food, 2(3): 15-19.

Krishnamurthy, G.Y., Giridhar, N. and Raghuramiah, B. (1984). Suitability of sorne

mango varities for proeessing. Journal of Food Science and Technology, 21: 21-

25

Lechowich, R.Y. 1993. Food safety implications of high hydrostatic pressure as a food

proeessing method. Food Technology, 47(6): 170-172.

Linton, M., McClements, J. M. and Patterson, M. F. (1999a). Inactivation of Escherichia

coli 0157:H7 in orange juice using a combination ofhigh pressure and mild heat.

Journal ofFood Protection, 62(3): 277 -279.

Linton, M., MeClements, J. M. and Patterson, M. F. (1999b). Survival ofEscherichia coli

0157:H7 during storage in pressure-treated orange juice. Journal of Food

Protection, 62: 1038-1040.

Mermelstein, N. (1997). High pressure processmg reaches the U.S market. Food

Technology, 51: 95-96.

Metriek, C., Hoover, D. G. and Farkas, D. F. (1989). Effeets ofhigh hydrostatie pressure

on heat-resistant and heat-sensitive strains of Salmonella sp. Journal of Food

Science, 54(6): 1547-1549.

Miller, L.G., and Kaspar, C.W. (1994). Escherichia coli 0157:H7 aeid toleranee and

survival in apple eider. Journal ofFood Protection, 57: 460-464.

114 Morgan, D., Newman, c.P., Hutchinson, D.N., Walker, AM., Rowe, B. and Majid, F.

(1993). Verotoxin producing Escherichia coli 0157:H7 infections associated with

the consumption of yoghurt. Epidemiology and Infection, 111: 181-187.

Mukherjee, S. K., (1997) Introduction: Botany and Importance In: The Mango Botany,

Production and Uses, ed. R. Litz, CAB international, New York, USA, 509-544.

Murthy, G.Y.K., Giridhar, N. and Raghuramaiah, B. (1984). Suitabilty of sorne mango

varieties for processing, Journal of Food Science and Technology (India), 21(1):

21-25.

Mussa, D. M. and Ramaswamy, H. S. (1997). Ultra high pressure pasteurization ofmilk,

kinetics of microbial destruction and changes in physico-chemical characteristics.

Lebensmittel Wissenschaft und Technologie, 30: 551-557.

Mussa D. (1999). High pressure processing of milk and muscle foods: evaluation of

process kinetics, safety and quality changes [Ph.D thesis]. Ste Anne de Bellevue,

Montreal: McGill University. Available from: McGill University.

Mussa, D. M., Ramaswamy, H. S. and Smith, 1. P. (1999). High pressure destruction

kinetics of Listeria monocytogenes on pork. Journal of Food Protection, 62(1):

40-45.

Mussa, D.M., Ramaswamy, H.S., and Smith, 1.P. (1999). High pressure (HP) destruction

kinetics of Listeria monocytogenes Scott A in raw milk. Food Research

International, 31(5): 343-350.

115 Nanjundaswamy, A. M., (1991). Mango processing: Present status and future outlook.

Acta Horticulturae, 291: 525-545.

Nanjundaswamy, A. M., (1997) Processing In: The Mango Botany, Production and Uses,

ed. R. Litz, CAB international, New York, USA, 509-544.

National Canners association (NCA), (1968). Laboratory manual for food canners and

processors 3rd edition. A VI publishing Co., Wesport, CT.

Nienaber, U, and Shellhammer, T.H. (2001). High-pressure processing of orange juice:

combination treatments and a shelf life study. Journal of Food Science, 66(2):

332-336.

Ogawa, H., Fukuhisa, K., Kubo, Y, and Fukumoto, H. (1990). Pressure inactivation of

yeasts, molds, and pectinesterase in Satsuma mandarin juice: effects of juice

concentration, pH, and organic acids, and comparison with heat sanitation.

Agricultural and Biological Chemistry, 54(5): 1219-1225.

Otero, L., Martino, M., Zaritzky, N., Solas, M., Sanz, P.D. (2000). Preservation of

microstructure in peach and mango during high-pressure-shift freezing. Journal

ofFood Science, 65(3):466-470.

Oxen, P. and Knorr, D. (1993). Baroprotective effects of high solute concentrations

against inactivation of Rhodotorula rubra. Lebensmittel Wissenschaft und

Technologie, 26(3): 220-223.

116 Palou, E., Lopez Malo, A, Barbosa Canovas, G.Y, Welti Chanes, J, and Swanson, B.G.

(1997). Kinetic analysis of Zygosaccharomyces bailii inactivation by high

hydrostatic pressure. Lebensmittel-Wissenschaft und-Technologie, 30: 703-708.

Palou, E., Lopez-Malo, A, Barbosa-Canovas, G. Y, Welti-Chanes, J and Swan son, B.G.

(1997). Effect of water activity on high hydrostatic pressure inhibition of

Zygosaccharomyces bailii. Letters in Applied Microbiology, 24: 417- 420.

Palou, E., Lopez Malo, A, Barbosa Canovas, G.Y, Welti Chanes, J, Davidson, P.M and

Swan son, B.G. (1998). Effect of oscillatory high hydrostatic pressure treatments

on Byssochlamys nivea ascospores suspended in fruit juice concentrates. Letters

in AppliedMicrobiology, 27: 375-378.

Palou, E., Lopez Malo, A, Barbosa Canovas, G.Y, Welti Chanes, J, and Swan son, B.G.

(1998). Oscillatory high hydrostatic pressure inactivation of Zygosaccharomyces

bailii. Journal ofFood Protection, 61(9): 12l3-1215.

Pandey, P. K., Ramaswamy, H. S. and Idziak, E. (2003). High pressure destruction

kinetics of indigenous microflora and Escherichia coli in raw milk at two

temperatures. Journal ofFood Processing Engineering, 26: 265-283.

Parish, M.E. (1998a). High pressure inactivation of Saccharomyces cerevisiae,

endogenous microflora and pectinmethylesterase in orange juice. Journal of Food

Safety, 18(1): 57-65.

117 Parish, M.E. (1998b). Orange juice quality after treatment by thermal pasteurization or

isostatic high pressure. Lebensmittel-Wissenschaft und-Technologie, 31 :439-442.

Park, S.l, Lee, l 1. and Park (2002). Effects of a combined process of high-pressure

carbon dioxide and high hydrostatic pressure on the quality of carrot juice.

Journal ofFood Science, 67(5): 684-687.

Patterson, M. F. and Kilpatrick, D. J. (1998). The combined effect of high hydrostatic

pressure and mild heat on inactivation of pathogens in milk and poultry. Journal

ofFood Protection, 61(4): 432-436.

Patterson, M., Quinn, M., Simpson, R, and Gilmour, A (1995a). Sensitivity of

vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered

saline and foods. Journal ofFood Protection, 61 (4): 432-436.

Porretta, S., Birzi, A, Ghizzoni, C. and Vicini, E. (1995). Effects of ultra-high

hydrostatic pressure treatments on the quality of tomato juice. Food Chemistry,

52: 35-4l.

Prestamo, G., Sanz, P. D., Fonberg-Broczek, M. and Arroyo, G. (1999). High pressure

response of fruit jams contaminated with Listeria monocytogenes. Letters in

Applied andMicrobiology, 28(4): 313-316.

Ramana, K.Y.R, Ramaswamy, H. S., Prasad, B.A, Patwrdhan, M.Y. and Ranganna, S.

(1984). Freezing preservation of Totapuri mango pulp. Journal of Food Science

and Technology, 21: 282-285

118 Ramaswamy, H. S., van de Voort, F.R. and Ghazala, S. (1989). Analysis of TDT and

arrhenius methods for handling process and kinetic data. Journal ofFood Science,

54: 1322-1326.

Ramaswamy, H. S., Riahi, E. and Idziak, E. (2003). High pressure destruction kinetics of

Escherichia coli (29055) in apple juice. Journal of Food Science, 68(5): 1750-

1756.

Raso, 1., Calderon, M. L., Gongora, M., Barbosa-Canovas, G. V. and Swanson, B. G.

(1998). Inactivation of Zygosaccharomyces bailii in fruit juices by heat, high

hydrostatic pressure and pulsed electric fields. Journal of Food Science, 63(6):

1042-1044.

Reyns, K. M. F. A., Soontjens, C. C. F., Cornelis, K., Weemaes, C. A., Hendrickx, M. E.

and Michiels, C. W. (2000). Kinetic analysis and modelling of combined high­

pressure-temperature inactivation of the yeast Zygosaccharomyces bailii.

International Journal ofFoodMicrobiology, 56: 199-210.

Riahi E. (2003). Effect of high pressure treatment on the kinetics of enzyme inactivation

and microbial destruction in apple juice. [Ph.D thesis]. Ste Anne de Bellevue,

Montreal: McGill University. Available from: McGill University.

Riahi, E., Ramaswamy, H.S. and Idziak, E. (2003). High pressure destruction kinetics of

Leuconostoc mesenteroides, Pichia membranafaciens and Zygosaccharomyces

bailii in apple juice. Applied Biotechnology, Food Science and Policy, 1(1): 1-8.

119 Ritz, M., Freulet, M., Orange, N. and Federighi, M. (2000). Effects of high hydrostatic

pressure on membrane proteins of Salmonella typhimurium. International Journal

ofFood Microbiology, 55: 115-119.

Sale A. J. H, Gould, G. W. and Hamilton, W. A. (1970). Inactivation ofbacterial spores

by hydrostatic pressure. Journal o/General Microbiology, 60: 323-334.

Sanchez-Moreno, C., Plaza, L., de Ancos, B. and Cano, M.P. (2003). Vitamin C,

provitamin A carotenoids, and other carotenoids in high-pressurized orange juice

during refrigerated storage. Journal ofAgricultural and Food Chemistry, 51: 647-

653.

Sanchez-Moreno, C., Plaza, L., de Ancos, B. and Cano, M.P. (2003). Effect of high­

pressure processing on health-promoting attributes of freshly squeezed orange

juice (Citrus sinensis L.) during chilled storage. European Food Research and

Technology, 216: 18-22.

Schreck, C., Ludwig, H (1997). The inactivation of vegetative bacteria by pressure, in:

K. Heremans (Ed.), High pressure Research in Biosciences and Biotechnology,

Leuven University Press, Leuven, Belgium.

Simpson, R. K. and Gilmour, A. (1997). The effect of high hydrostatic pressure on the

activity of intracellular enzymes of Listeria monocytogenes. Letters in Applied

Microbiology, 25: 48-53.

120 Steeg, P. T., Hellemons, 1. C. and Kok, A. (1999). Synergistic actions of nisin, sublethal

ultrahigh pressure, and reduced temperature on bacteria and yeast. Applied and

Environmental Microbiology, 65(9): 4148-4154.

Styles, M.F., Hoover, D.G. and Farkas, D.F. (1991). Response ofListeria monocytogenes

and Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food

Science ,56: 1404-1407.

Takahashi, Y., Ohta, H, Yonei, H and Ifuku Y. (1993). Microbicidal effect of

hydrostatic pressure on Satsuma mandarin juice. International Journal of Food

Science and Technology, 28: 95-102.

Tewari, G., Jayas, D. S. and Horrey, R. A. (1999). High pressure processing offoods: an

overview. Science Des Aliments, 19: 619-621.

Tonello, C., Kesenne, S., Muterel, C. and Jolibert, F. (1997). The effect of high

hydrostatic pressure treatments on shelf life of different fruit products, in: K.

Heremans (Ed.), High pressure Research in Biosciences and Biotechnology,

Leuven University Press, Leuven, Belgium.

Tsai, Y.W., Ingham, S.C., (1997). Survival of Escherichia coli 0157:H7 and Salmonella

spp. in acidic condiments. Journal ofFood Protection, 60: 751-755.

USDA. (2000). Kinetics of microbial inactivation for alternative food processmg

technologies: High pressure processing. http://vm.cfsan.fda.gov/~comm/.

121 Vachon, IF., Kheadr, E.E., Giasson, l, Paquin, P., and Fliss, I. (2002). Inactivation of

foodborne pathogens in milk using dynamic high pressure. Journal of Food

Protection, 65(2): 345-352.

Victor, G. S. (2004). Mango production and world market: CUITent situation and future

propects. ACTA Horticulturae, 645: 107-112.

Weagant, S.D., Bryant, IL. and Bark, D.H., (1994). Survival of Escherichia coli

0157:H7 in mayonnaise-based sauces at room and refrigerated temperatures.

Journal ofFood Protection, 57: 629-631.

Wouters, P., Glaasker, E. and Smelt, I P. P. M. (1998). Effects of high pressure on

inactivation kinetics and events related to proton effiux in Lactobacillus

plantarum. Applied and Environmental Microbiology, 64(2): 509-514.

Wuytack, E. Y., Soons, l, Pochhet, F. and Micheiels, C. W. (2000). Comparative study

of pressure- and nutrient-induced germination of Bacillus subtilis spores. Applied

and Environmental Microbiology, 66(1): 257-261.

Zaman, S.u., Ramaswamy H.S. and. Smith, IP. (2005). High pressure destruction

kinetics of Escherichia coli (OI57:H7) and Listeria monocytogenes (Scott A) in

fish homogenate. Journal ofFood Science, (In print).

Zook, C. D., Parish, R. l, Braddock, R. land Balaban, M. O. (1999). High pressure

inactivation kinetics of Saccharomyces cerevisiae ascopores in orange and apple

juices. Journal ofFood Science, 64(3): 533-535.

122 McGill University

. University Biobazards Committee . . * APPLICATION TO USE BIOHAZARDOUS MATERIALS

No project should becommenced without prior approvalof an application to usebiohazardous materials. Submit this application to the Chair, Biohazards Committee, and one month before starting new projects or expiry of a previously approved application.

1. PRINCIPAL INVESTIGATOR: Dr. R.S. Ramaswamy

ADDRESS: Macdonald Campus (McGill) TELEPHONE: (514) 398-7919

FAX NUMBER: (514) 398-7977 DEPARTMENT: Food Science and Agricultural Chemistry E-MAll.,:[email protected] PROJECT TITLE: Advanced value-added applications of high pressure processing: High pressure sterilization, Thermodynamic, molecular andfunctional property evalutionlmodeling

2. FUNDING SOURCE: MRCO NSERC~ NIH 0 FCAR 0 . FRSQ 0

INTERNALO OTHER 0 (specify)

Grant No.: G205694/NSERC NSERC STPGP 269840 Beginning date: Nov 2003 End date Oct 2006 3. Indicate if tbis is ŒlRenewal use application: procedures have been previously approved and no alterations have been made to the protocol. Approval End Date: Original Approval End Date Oct 2003 REC;E'VEl.t o New funding source: project previously reviewed and approved under an application to another aCfiqy·1 9 2004

Agency Approval End Date RESEARCH GRANTS . OFFIC~ o New project: project not previously reviewed or procedures and/or microorganism altered from previously approved application.

CERTIFICATION STATEMENT: The Biohazards Committee approves the experimental procedures proposed and certifies with the applicant that the experiment will be in accordance with the principles outlined in the "Laboratory Biosafety Guidelines" prepared by Health Canada and the MRC, and in the "McGill Laboratory Biosafety Manual". ContainmentLevel(circle 1): 10 20 30 40 ~ (,1 _.,J{ 1 - Jb M.-"t If' ;)-00 If Principal Investigator or course director: ~ . . date: . ( }\. S.RJ\I'-\-A$\j" ;..:.,s) A (J . p. S..,...... I~) day month year

€bairpers6ft, Bi6hazards Committ@e: d.9!.l~f date: SIGNATURE day month year Approved period: beginning 1C) O~ t9 Cf ending 3 , 10 Ob day month year day month year

* as defined in the "McGiIl Laboratory Biosafety manual" 2'" REVISION, JANUARY 1996 q . l.~i:)~A.1'\....tl rEKlSUNNEL" (attach additional sheets ifpreferred) Name Department Check approprlate classification . Fellow Investigator Technician Student & Research Assistant Undergraduate Graduate ror. H. S. Ramaswamy Food Sei. &Agri. Chemistry ,(

. Dr. Jim Smith Food Sci. & Âgri. Chemistry ,(

.1Mr. Bernard Cayouette Food Sei. & Agri. Chemistry ,(

YanwenShao 1F00d Sei. & Agri. Chemistry ,(

;Nikhil Hiremath lFood Sci. & Agri. Chemistry ,(

Shafi Ullah Zaman 1F00d SeL & AgrL Chemistry· ,(

5. EMERGENCY: Person(s) designated to handle emergencies

Name: Dr. Jim Smith. . Phone No: work: (514) 398-7923/8623 home: (514) 457-2262

Name: Mr. Bernard Cayouette Phone No: work: (514) 398~8623 home: (514) 254-2434

6. Briefly describe:

i) the biohazard.ous material involve

Yeast: Zygosaccharomyces baiili (non pathogenie) Pichia membranafaciens (non pathogenie)

ii) the procedures involving biohazards AU stock cultures are prepared from frozeri cultures in glycerol and grown ovemight at 30 to 37°C by Mr. Bernard . . Cayouette. Working culture~ (- 10 mL) are prepared the foUowing day'and food (milk, cheese, juice, fish - 20g) arè

inoculated, in duplicate;in double lined plastic bags with various levels ofpathogens to giv~ final inoculum levels ofl06-: 108 CFU/g. The inoculated samples are transferred to the Pilot plant in Styrofoam© container \vith ice where·fuey are . subjected to various high pressure treatments. After each high pressure cycle, the bags are returned to the lab, again in a Styrofoam© container, for microbiological analysis. Plates are incubated under selected conditions and enumerated within

24-48 hours. AU contaminated containers, plat~, dilutjoo bottles etc., are sterilized by autoclavingprior to dis~ding and are clearly identified as "Autoclaved and Sterilized" before pick-up by janitorial staff. Preparation of aU cultures, dilutions and plates, as weIl as inoculation of aU food samples, are done uncie~ aseptic conditions in a biological safety cabinet. AU .stafflstudents wear face ~asks, labcoats and rubber gloves during aU microbiological procedures. No pipetting is done by mouth. Benches are routinely washed with a 1% hypochlorite solution at the end of each day while the safety cabinet is swabbed with 70% ethanol and the UV light left ovemight. . iii) The protocol for decontaminating spills Spills seldom occur in our laboratory since good house keeping is encouraged and monitored on a regular basisto prevent

. such events. However, in the event of a spill, the protocol for decontaminating spills of type 2 micro~rganisms is as outlined in bath the McGill and Department laboratory safety manuals. If the spill occurs on the bench, it is absorbed by the white paper bench cloth routinely placed on each bench. The c10th is removed and the "contaminated" area is soaked

with 1% hypochlorite solution for ~ 30 minutes and then wiped dry. Then the area wiped up with paper towels soaked in

70% ethanol. If spill occurs in the safety cabinet, the spill is wiped up and then the area is soaked with 70% .ethariol for ~

30 minutes, dried with clean papertowels and the U.V light left on for ~ 1 hour prior ta resumption ofwork. Studentsl staff do not enter into the lab for at least 1 hour after a spill. All.towels,.contuninated paper clothes etc, lab coats,masks are placed in autoclavable bags. and decontaminated by sterilization. An bags are c1early labeled "Autoclaved and . Sterilized".

. If a spill occurs on the body, the clothing is removed and sterilized. Splashes to the face are washed with germicidal soap . and hot water. AU spills are reported to the !aboratory supervisor and a follow up session is done with the students to go over the cause(s) of the spill and to re-enforce preventive measures.

Copies of the McGiU. and. departmental safety protocols are in the lab all the times for pemsal by the students.

Furthermore most of the work done in our l~boratory is under thé constant supervision ofMr. Bernard Cayouette.

7.. Does the protocol present conditions (e.g. handling oflarge volumes or high concentrations of pathogens) which could increase the hazards of the infectious agent(s)? No, Cultures are prepared in 10 ml amounts, in triplicate, for use in the inoculat.ion studies.

8. Do the specific procedures to be employed invol:ving genetically engin~red organisms hàve a history of sare use? NIA

9. What precautions are being taken to reduce production of infectious droplets and aerosols? AIl inoculationlenumeration procedure are done under the strictest of aseptic conditions in a biological safety cabinet. AU students/ staff wear appropriate protective clothing when haD.dling cultures or inoculated foods.

10. List the biol02ical safety cabinets to be used.

Building RoomNo. Manufacturer ModelNo. Seriai No. Date Certified 1-Jw ~"f;)? !Macdonald Stewart 1-055 Labconco 36205704 ~47196 11/09/01