USE OF CRYOPROTECTANTS, MICROENCAPSULATION OF BACTERIAL CELLS AND ACID AND LOW TEMPERATURE ADAPTATIONS IN IMPROVING VIABILITY OF PROBIOTIC BACTERIA IN FERMENTED FROZEN DESSERTS

A thesis submitted for the degree of Doctor of Philosophy

By

Ramakanth Rao Ravula

2000

School of Life Sciences and Technology Victoria University of Technology W^erribee Campus, Victoria Australia DEDICATED TO MY PARENTS SAKKUBAI AND JANARDHAN RAO WER THESIS 63 7.4 RAV 30001006991194 Ravula, Ramakanth Rao Use of cryoprotact ants, microencapsulation of bacterial cells _ind acid and ABSTRACT

The main aim of this study was to improve the viability of probiotic bacteria

(Lactobacillus acidophilus and Bifidobacterium spp.) in fermented frozen dairy desserts. In order to improve the viability of probiotic bacteria, initial studies were undertaken to determine the existing levels of probiotic bacteria present in fennented frozen dairy desserts. Samples of frozen on stick (with 18% overmn) and in cups (with 100% overmn) were directly received from a manufacturer and were analysed for the viable populations of L. acidophilus,

Bifidobacterium spp, S. thermophillus and L. delbrueckii ssp. bulgaricus. The populations of the two probiotic organisms were very low (<10 cfu/g) even in the freshly frozen products.

Viability of yogurt bacteria (5". thermophilus and L. delbruekii ssp. bulgaricus) and probiotic bacteria (L. acidophilus and Bifidobacterium spp.) were assessed in several fennented frozen dairy dessert preparations obtained directly from commercial manufacturers. The counts of the four groups of organisms were monitored for 12 weeks storage at -18°C. The counts were found to be poor, especially in the case of L. acidophilus and bifidobacteria. The counts of S. thermophilus were better than those of I. delbrueckii ssp. bulgaricus.

Forty-one strains of probiotic and yogurt bacteria were screened for their ability to survive under frozen conditions, in presence of sugar (0, 4, 8, 12 and 16%)) and under acidic conditions. Five batches (one litre size) of probiotic were made. Yogurt made without any added sugar was used as a control (batch 1).

Yogurts were also made with 4, 8, 12 and 16% sugar (sucrose) levels (batches 2,

3, 4, and 5 respectively). The femientation process was monitored by measuring

111 pH, acidity, and the incubation time taken to reach pH 4.5. Initial and final counts of yogurt bacteria and probiotic bacteria in yogurts were also assessed. The water activity decreased as the percentage of sugar increased. Higher levels of sugar (>

8%) prolonged the fermentation time taken to reach the pH of 4.5. There was no significant difference in the initial coimts of yogurt and probiotic bacteria in all the five batches of yogurt. Final counts of S. thermophilus increased significantly in batches 1, 2, and 3 when compared to batches 4 and 5. The final viable counts of L. delbrueckii ssp. bulgaricus in batches 1 and 2 increased by about 2 log cycles and decreased in batches 3, 4 and 5. The final viable counts L. acidophilus in the batches 1, 2 and 3 were almost similar to initial counts, whereas in batches

4 and 5 the counts decreased by about 2 and 5 log cycles. The final viable counts of bifidobacteria in the batches 1, 2 and 3 increased by about one log cycle and the counts decreased in batches 4 and 5 by 2 to 3 log cycles.

Five one-litre batches of probiotic yogurts were made using reconstituted skim supplemented with 0, 4, 8, 12 or 16% sucrose. The incubation time to decrease the pH to 4.5 and changes in organic acids production and counts of yogurt bacteria (S. thermophilus and L. delbrueckii ssp. bulgaricus) and probiotic bacteria (L. acidophilus and Bifidobacterium spp.) in yogurt were assessed for up to 30 days. The organisms produced pymvic acid, acetic acid, lactic acid and propionic acid at varying levels. There were no major differences in the counts of yogurt and probiotic bacteria in all the five batches. However, the counts of all the four groups of organisms declined during storage, in particular in the batches containing 12 and 16% sugar. Thus sugar addition appeared deleterious to the

IV growth of yogurt and probiotic bacteria, especially in the products such as fermented frozen dairy desserts, which contain approximately 16% sugar.

Three batches of yogurts were prepared using three selected strains of each of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and Bifidobacterium spp. mixed with a base mix and liquid sugar (67% brix) and the mix frozen to prepare fermented frozen dairy desserts. In a separate experiment, three batches of yogurt were made with yogurt bacteria only and probiotic bacteria were added just before freezing. The viability of yogurt and probiotic organisms in the fermented frozen dairy desserts was monitored over a 12-week storage at -18°C.

Further, two 1000 L batches of fermented dairy desserts were made using selected strains of yogurt and probiotic bacteria using a continuous freezer to stimulate industrial setting and the viability of both organisms was monitored over a 12 week storage at -18°C. Most of the organisms survived freezing, acidic conditions and the presence of high levels of sugar. Thus the survival of these organisms appeared to be strain dependent.

Three batches of fermented frozen dairy desserts were made with milk supplemented with acid casein hydrolysate or cysteine. The control batch was made with milk supplemented with 2% skim milk powder. Viable counts of yogurt bacteria and probiotic bacteria were enumerated and pH, titratable acidity and p-D-galactosidase activity measured at fortnightly intervals over a 12-week storage at -18 C. S. thermophilus was most stable in all the samples of fermented frozen dairy desserts with counts > 10^ cfii/g throughout the storage period. The counts of L. delbrueckii ssp. bulgaricus were higher in products supplemented with acid casein hydrolysate and cysteine. The counts of L. acidophilus and bifidobacteria were >10^ cfu/g in the batches supplemented with acid casein hydrolysate or cysteine. The p-D-gaiactosidase activity decreased rapidly in the control sample during 12 weeks storage at -18''C as compared with those prepared with acid casein hydrolysate and cysteine.

A selective medium (which will be referred to as LC agar) was developed in this study for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain sttains of yogurt bacteria (S. thermophilus and L. delbrueckii ssp. bulgaricus), probiotic bacteria (L. acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pom- plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. The growth of S. thermophilus was prevented by adjusting the pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar.

Survival of probiotic bacteria was studied in fermented frozen dairy desserts made with or without cryoprotectants using a bench top and a commercial freezer. Cryoprotective agents were added at a rate of 0.2% to yogurt mix at a temperature of 30-35*^C before pasteurising at 85°C for 30 min. Various cryoprotective agents were initially evaluated for their effect on textural quality and the best resulting cryoprotectants were used in this study. The cryoprotectants that were used included RS 150 (pectin), glucose and GCF 639

VI (alginate). These cryoprotectants were of food grade standard and are suitable for the manufacture of dairy products. The final product was made by blending 45% yogurt, 45% of base mix and 10% of 65% syrup (liquid sucrose) followed by freezing through an ice freezer. The conttol batch and the batches containing RS 150, glucose and GCF 639 were made using a bench top freezer (1 L batch; -18% overrun). Two batches of fermented frozen dairy desserts were made with cryoprotectant RS 150 and without cryoprotectant

(control) using a commercial freezer (20 L batch; -100% overrun). The product was filled in plastic containers and stored at -18°C for 12 weeks.

The initial pH of milk (6.51-6.67 at 0 h) decreased to pH 4.52-4.59 during yogurt making. In general, there was a gradual decrease in the pH in all the products during storage for 12 weeks. The pH by the end of storage period was 4.41-4.46.

The titratable acidity of yogurt varied between 0.71 to 0.78% after fermentation and the acidity of fermented frozen dairy desserts varied between 0.79 to 0.86% at the end of the storage period. The counts of L. acidophilus and bifidobacteria at the end of 12 weeks storage period was >10^ cfii/g in all the products except the product containing glucose as the cryoprotectant which had a viable count of

<10'' cfu/g. The counts L. acidophilus and bifidobacteria decreased rapidly in

0 "X both products and the viable counts were <10 and<10 cfii/g, respectively by the end of the 12-week storage period. Loss of viability of L. acidophilus and bifidobacteria in the products made using the commercial freezer was possibly due to the presence of oxygen. The products containing cryoprotectants performed better in improving viability of probiotic bacteria. Glucose did not help improve their survival. Overall, there was a slight improvement in the viable

Vll counts of L. acidophilus and Bifidobacterium spp. in the presence of the cryoprotectant RS 150.

Viability of microencapsulated L. acidophilus and bifidobacteria during a storage period of 12 week was assessed in fermented frozen dairy desserts. Two batches of fermented frozen dairy desserts were made with or without encapsulated probiotic bacteria. Viable counts of probiotic bacteria were enumerated and pH, titratable acidity and p-D-galactosidase activity measured at fortnightly intervals for 12 weeks. The counts of Z. acidophilus and bifidobacteria decreased to <10^ cfu/g in the control batch, whereas the counts were >10^ cfu/g in the batches in which microencapsulated probiotic bacteria were used. The P-D-galactosidase activity decreased rapidly in the control during 12 weeks storage at -18 C as compared with those prepared with microencapsulated cells.

Selected strains of Z. acidophilus and bifidobacteria were adapted to acid and low temperature conditions. The organisms were stressed at lethal pH (pH 3.5) or sub-lethal pH (pH 5.5) for various times. The organisms survived better in the environment where sub-lethal stress was given. The organisms stressed in acid and at -18°C for 24 h were used for incorporation into fermented dairy desserts.

Viability of stress adapted probiotic bacteria in fermented frozen dairy desserts was assessed during a storage period of 12 week. Six 1 L batches of fermented frozen dairy desserts were made on a bench scale with or without acid and low temperature stress adapted probiotic bacteria. The counts of L. acidophilus and bifidobacteria decreased to <10^ cfu/g in the control batch, whereas the counts were >10^ cfii/g in the products made with the bacteria which were adapted to acid and low temperature.

VIU ACKNOWLEDGEMENT

I am fortunate to have a supervisor and guide like Dr. Nagendra Shah, Associate Professor, School of Life Sciences and Technology, to whom I wish to express my sincere gratitude. Dr. Shah was always there whenever there was a need for his knowledge and guidance and in preparing and editing all publications relevant to my thesis. I wish to acknowledge Australian Research Council for providing an Australian Postgraduate Research Award (Industry) scholarship for this study. 1 also appreciate the contribution offered by Regal Ltd. as a co-sponsor of the above scholarship. My special thanks are due to Mr. Ian Downey, Technical Director and Mr. Justin Downey Quality Assurance Manager for their support during the trial runs at the industry.

I express my gratitude to Prof Bob Fairclough, Prof Stirk Kyle, Mr. Dale Tomlinson, Stacey Llyod and other laboratory staff for their constant support in procuring of chemicals and equipments required for successfril operation of my research work. 1 express my sincere thanks and appreciation to my colleagues Kiran Gummadi, Raman Kumar, Hisham, Aslam, Goran Gambirosa, Amal Shihta and Lata Masih. I am highly indebted and I appreciate the friendship of Dr. Rajiv Dave, Harshul Vora, and Shiva Prasad.

1 would like to appreciate the love and inspiration of my parents, my sister and my in-laws and other family members and friends. 1 cannot forget the reconnaissance love and affection of my beloved wife Veena and son Vishnu who supported and inspired me throughout my research project and tolerated my long hours of work at times when they needed me the most. Above all I am thankful to the almighty without whose grace this study would not have been a reality.

Werribee, Australia

Date: RAMAKANTH RAO RAVULA PRESENTATIONS/PUBLICATIONS:

REFEREED PAPERS

• Ravula R.R and Shah N.P. 1998. Viability of yogurt and probiotic bacteria in fermented frozen dairy desserts. Food Australia 50(3): 136-139.

• Ravula R.R and Shah N.P. 1998. Effect of acid casein hydrolysate and cysteine on viability of yogurt and probiotic bacteria in fermented frozen dairy desserts. Australian Journal of Dairy Technology 53:175-179.

• Ravula R.R and Shah N.P. 1998. Selective enumeration of Lactobacillus casei from yogurt and fermented milk drinks. Biotechnology Techniques (UK) 12(11) 819-822.

• Ravula R.R., Shah N.P. and Ali. J. 2000. Populations of L. acidophilus. Bifidobacterium spp., and L. casei in commercial fermented milk products. Bioscience Microflora 19(1): 35-39.

• Ravula R.R and Shah N.P. 2000. Influence of water activity on fermentation, organic acids production and viability of yogurt and probiotic bacteria. Australian Journal of Dairy Technology. 55 (3): 127-131.

• Ravula R.R and Shah N.P. 2000. Survival of microencapsulated probiotic bacteria in fermented frozen dairy desserts. Australian Journal of Dairy Technology. 55(3): 139-144.

CONFERENCE PRESENTATIONS

• Ravula R.R and Shah N.P. 1997. Improving Viability of probiotic bacteria in fermented frozen dairdair y desserts, a poster presented at 30* Convention of AIFST Perth, 4-8 May

Ravula R.R and Shah N.P. 1998. Effect of acid casein hydrolysate and cysteine on viability of probiotic bacteria in fermented frozen dairy desserts, a paper presented (oral presentation) at 31^^ Annual convention of AIFST held in Melbourne, 26-29 April.

Ravula R.R and Shah N.P. 1998. Effect of cryoprotectants on viability of probiotic bacteria in fermented frozen dairy desserts, a poster presented at 25* International Dairy Congress in Arhus, Denmark, 22-24 September.

Ravula R.R and Shah N.P. 1999. Encapsulation of probiotic bacteria in fermented frozen dairy desserts. ADSA, Memphis, TN, USA. TABLE OF CONTENTS

Chapter Page 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 4 2.1 Yogurt 4 2.1.1 Definition 4 2.1.2 Types of yogurt 4 2.1.3 Consumption pattem 5 2.2 Functional foods 8 2.3 Fermented frozen dairy desserts 10 2.3.1 Definition 10 3.2 Types of fermented frozen dairy desserts 10 2.3.3 Consumption pattem 10 2.4 Yogurt bacteria 12 2.5 Prebiotics 13 2.6 Probiotics 14 2.7 L. acidophilus 15 2.8 Bifidobacterium spp. 19 2.9 L. casei 23 2.10 Enumeration of 5. thermophilus, L. delbruekii ssp. bulgaricus, L. acidophilus. Bifidobacterium spp. 27 2.11 Factors affecting viability of probiotic bacteria in frozen fermented dairy desserts 30 2.11.1 Sugar 32 2.11.2 Freeze injury 33 2.11.3 pH and acidity 34 2.12 Approaches taken to improve the viability 3 7 2.12.1 Use of cryoprotective agents 3 7 2.12.2 Use of microencapsulation 38 2.12.3 Use of micronutrients 41

111 2.12.4 Acid and low temperature adaptations 42 3.0 MATERIALS AND METHODS 48 3.1 Sources of chemicals, media and equipment 48 3.1.1 Chemicals 48 3.2 Equipments 48 3.2.1 Pasteuriser 48 3.2.2 Ice cream freezers 48 3.2.3 Laminar flow bench 49 3.2.4 Anaerobic jars 49 3.2.5 Centrifuge and microcentrifuge 49 3.2.6 Freeze drier and freezing drying of bacteria 49 3.2.7 Compound microscope and examination of beads 50 3.2.8 Scanning electron microscope and microscopy 50 3.3 Bacterial culture and maintenance 51 3.3.1 Lactic acid bacteria and probiotic bacteria 51 3.4 Microbiological analyses 51 3.5 Commercial products used for enumeration of Z. cai'e/ 52 3.6 Screening of strains of yogurt and probiotic bacteria 53 3.7 Analysis of samples to determine viability of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria in commercial fermented dairy products 53 3.8 Effects of various sugar levels on survival of probiotic bacteria 54 3.9 Effects of acidic and freezing conditions on survival of yogurt and probiotic bacteria 5 5 3.10 Influence of water activity on fermentation of probiotic bacteria and on viability of yogurt and probiotic bacteria 55

IV 3.11 Survival of yogurt and probiotic bacteria in fermented frozen dairy desserts made using a bench top ice cream freezer 56 3.12 Survival of yogurt and probiotic bacteria in fermented frozen dairy desserts made using a continuous ice cream freezer 56 3.13 Use of acid casein hydrolysate and cysteine in manufacture of fermented frozen dairy desserts 57 3.14 Assessment of effects of cryoprotectants for improving viability of L. acidophilus and Bifidobacterium spp. 58 3.15 Compositional analysis 5 8 3.15.1 Detemination of pH and titratable acidity 5 8 3.15.2 Determination of sugar 59 3.15.3 Determination of protein 5 9 3.15.4 Determination of total solids 60 3.15.5 Determinationof water activity 61 3.15.6 Determination of oxygen content and redox potential 61 3.15.7 Determination of P-D-galactosidase activity 61 3.15.8 Determination of growth curve 62 3.15.9 Determination of content 62 3.15.10 Detection and quantification of organic acids with HPLC 62 3.15.10.1 Extraction of organic acids 62 3.15.10.2 HPLC of organic acids 63 3.16 Media preparation 63 3.16.1 Peptone and water diluent 63 3.16.2 Preparation of serial dilution for pour plating 64 3.16.3 Streptococcus thermophilus agai 64 3.16.4 M17agar 64 3.16.5 MRS-basal, MRS-salicin, MRS-ribose, MRS-gluconate agars 65 3.16.6 MRS pH modified agar (pH5.2) 65 3.16.7 MRS-NNLPagar 65 3.16.8 LCagar 66 3.17 Preparation of fermented frozen dairy desserts 67 3.17.1 Preparation of yogurt 67 3.17.2 Preparation of base mix 67 3.18 Microencapsulation of probiotic bacteria and their incorporation into fermented frozen dairy desserts 67 3.18.1 Preparation of cell pellet 68 3.18.2 Entrapment of the microorganisms in beads 68 3.18.3 Effects of surface tension and emulsifier on bead size 69 3.18.4 Effect of freeze-drying on beads and viability of cells 69 3.18.5 Solubilisation of beads 69 3.19 Acid and low temperature adaptations of probiotic bacteria and their incorporation into fermented frozen dairy desserts 70 3.19.1 Preparation of buffers and reagents 71 3.19.1.1 Tris-HCl buffer (1.875 and pH 8.8) 71 3.19.1.2 Tris-HCl buffer (1.25 M and pH 6.6) 71 3.19.1.3 SDS (sodium dodecyl sulphate) (10%) Solution 71 3.19.1.4 Poly-acrylamide gel electrophoresis (PAGE), staining of gels, and preparation of mnning gel 72 3.19.1.5 Preparation of stacking gel 72 3.19.1.6 Staining the gels with coumassie blue 72

VI 3.19.1.7 Staining the gels with silver stain 73 3.19.1.8 Gel preparation and electophoresis 74

4.0 SELECTIVE ENUMERATION OF LACTOBACILLUS CASEI "EKOU YOGURTS AND FERMENTED MILK DRINKS 75 4.1 Introduction 75 4.2 Material and methods 76 4.3 Results and discussion 80 4.4 Conclusions 81 5.0 POPULATIONS OF 5". THERMOPHILUS, L. DELBRUEKII SSP. BULGARICUS, L ACIDOPHILUS, BIFIDOBACTERIUM SPP. AND L CASEI IN COMMERCIAL FERMENTED MILK PRODUCTS 85 5.1 Introduction 85 5.2 Material and methods 87 5.3 Results and discussion 89 5.4 Conclusions 92 6.0 SELECTION OF STRAINS OF YOGURT AND PROBIOTIC BACTERIA FOR INCORPORATAION INTO FERMENTED FROZEN DAIRY DESSERTS 97 6.1 Introduction 97 6.2 Material and methods 99 6.3 Results and discussion 101 6.4 Conclusions 103 7.0 INFLUENCE OF WATER ACTIVITY ON PRODUCTION PROBIOTIC YOGURT 126 7.1 Introduction 126 7.2 Material and methods 127 7.3 Results and discussion 127 7.4 Conclusion 130

Vll 8.0 INFLUENCE OF WATER ACTIVITY ON FERMENTATION OF PROBIOTIC YOGURT, ORGANIC ACIDS PRODUCTION AND VIABILITY OF YOGURT AND PROBIOTIC BACTERIA 13 6 8.1 Introduction 136 8.2 Material and methods 138 8.3 Results and discussion 141 8.4 Conclusions 144 9.0 ASSESMENT OF VIABILITY OF YOGURT AND PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS MADE USING A BENCH TOP ICE CREAM FREEZER 15 8 9.1 Introduction 158 9.2 Material and methods 159 9.3 Results and discussion 160 9.3 Conclusions 161 10.0 ASSESMENT OF VIABILITY OF YOGURT AND PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS MADE USING A COMMERCIAL ICE CREAM FREEZER 168 10.1 Introduction 168 10.2 Material and methods 170 10.3 Results and discussion 171 10.4 Conclusions 173 11.0 EFFECT OF ACID CASEIN HYDROLYSATE AND CYSTEINE ON VIABILITY OF YOGURT AND PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS 178 11.1 Introduction 178 11.2 Material and methods 180 11.3 Results and discussion 183

Vlll 11.4 Conclusions 187 12.0 EFFECT OF CRYOPROTECTANTS ON L ACIDOPHILUS AND BIFIDOBACTERIUM SPP. IN FERMENTED FROZEN DAIRY DESSERTS 194 12.1 Introduction 194 12.2 Material and methods 195 12.3 Results and discussion 197 12.4 Conclusions 199 13.0 SURVIVAL OF MICROENCAPSULATED PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS 204 13.1 Introduction 204 13.2 Material and methods 206 13.3 Results and discussion 212 13.4 Conclusions 218 14.0 ACID AND LOW TEMPERATURE ADAPTATION OF PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS 252 14.1 Introduction 252 14.2 Material and methods 254 14.3 Results and discussion 257 14.4 Conclusions 260 15.0 OVERALL CONCLUSIONS 279 16.0 FUTURE RESEARCH DIRECTION 284 17.0 REFERENCES 286

IX LIST OF TABLES

Serial Table Title Page No. No. No. I 2.1 Types of yogurts and their nutritional values that are 7 available in Australian markets 2 2.2 Species of genus Lactobacillus 18 3 2.3 Chronological order of the development of taxonomy of 21 bifidobacteria 4 2.4 Species of genus Bifidobacterium 22 5 2.5 Dairy products available in Australia with L. 23 acidophilus, bifidobacteria and L. casei 6 2.6 Therapeutic properties of probiotic bacteria 29 7 4.1 Counts of iS*. thermophilus, L. delbrueckii ssp. 82 bulgaricus, L. acidophilus, bifidobacteria, and L. casei on various bacteriological media 7 4.2 Counts of Z. casei from different commercial products 83 selectively enumerated on LC agar 8 5.1 Initial and final pH and viable counts of Z. (3c/Wop/2z7M5 93 9 5.2 Initial and final pH and viable counts of Z. acz(io/7/z//w5 94 and bifidobacteria 10 5.3 Initial and final pH and viable counts of Z. adJopMw^, 95 bifidobacteria and Z. casei II 5.4 Initial and Final pH and viable counts of Z. Casei 96 12 8.1 Effects of various concentrations of sugar on water 145 activity, pH and fermentation time LIST OF FIGURES

Serial Figure Title Page No. No. No. 1 4.1 Colonies formed by Z. ca^er on LC agar 84 2 6.1 Effects of freezing on survival of 5"./'/zerwop/2z7M5' 104 before and after freezing at -25°C for 4h 3 6.2 Effects of freezing on survival of Z. c/e/6rMecfe7 ssp. 105 bulgaricus before and after freezing at -25°C for 4h 4 6.3 Effects of freezing on survival of Z. ad£fo/?MM5 106 before and after freezing at -25^C for 4h 5 6.4 Effects of freezing on survival of bifidobacteria before 107 and after freezing at -25°C for 4h 6 6.5 Effects of freezing on survival of Z. ad^/opMwj 108 before and after freezing at -25°C for 4h at 8 and 16% sugar levels 7 6.6 Effects of freezing on survival of bifidobacteria before 109 and after freezing at -25°C for 4h at 8 and 16% sugar levels 8 6.7 Viability of 5*.//2erwc»/'Mw5 at pH 6.2 before and after 110 freezing at -25°C 9 6.8 Viability of-S.//zer/wo/7/z/7«5 at pH 5.0 before and after 111 freezing at -25°C 10 6.9 Viability ofS. thermophilus at pH 4.5 before and after 112 freezing at -25 C 11 6.10 Viability of-S. thermophilus at pH 4.0 before and after 113 freezing at -25°C 12 6.11 Viability of Z. delbrueckii ssp. bulgaricus at pH 6.2 114 before and after freezing at -25°C 13 6.12 Viability of Z. delbrueckii ssp. bulgaricus at pH 5.0 115 before and after freezing at -25 C 14 6.13 Viability of Z. delbrueckii ssp. bulgaricus at pH 4.5 116

XI before and after freezing at -25°C 15 6.14 Viability of Z. delbrueckii ssp. bulgaricus at pH 4.0 117 before and after freezing at -25°C 16 6.15 Viability of Z. acidophilus at pH 6.2 before and after 118 freezing at -25°C 17 6.16 Viability of Z. acidophilus at pH 5.0 before and after 119 freezing at -25''C 18 6.17 Viability of Z. acidophilus at pH 4.5 before and after 120 freezing at -25*'C 19 6.18 Viability of Z. aczWopMwi'at pH 4.0 before and after 121 freezing at -25 C 20 6.19 Viability of bifidobacteria at pH 6.2 before and after 122 freezing at -25°C 21 6.20 Viability ofbifidobacteria at pH 5.0 before and after 123 freezing at -25°C 22 6.21 Viability of bifidobacteria at pH 4.5 before and after 124 freezing at -25 °C 23 6.22 Viability of bifidobacteria at pH 4.0 before and after 125 freezing at -25°C 24 7.1 Flow diagram for making yogurt 131 25 7.2 Total solids and moisture contents of samples after 132 incorporation with various levels of sugar 26 7.3 Effects of various concentrations of sugars on water 133 activity 27 7.4 Effects of addition of sugars on changes in pH and 134 fermentation time 28 7.5 Initial and final viable counts of ^. r/zermo/7MM5,Z. 135 delbrueckii ssp. bulgaricus, L. acidophilus, and bifidobacteria in the products made with various levels of sugars 29 8.1 Levels of pyruvic acid in inoculated milk, after 146

Xll fermentation, and during 30 days storage at various concentrations of sugar in measurement of organic acids 30 8.2 Levels of acetic acid in inoculated milk, after 147 fermentation, and during 30 days storage at various concentrations of sugar in measurement of organic acids 31 8.3 Levels of lactic acid in inoculated milk, after 148 fermentation, and during 30 days storage at various concentrations of sugar in measurement of organic acids 32 8.4 Levels of propionic acid in inoculated milk, after 149 fermentation, and during 30 days storage at various concentrations of sugar in measurement of organic acids 33 8.5 Levels of hippuric acid in inoculated milk, after 150 fermentation, and during 30 days storage at various concentrations of sugar in measurement of organic acids 34 8.6 Effects of sugar concentration on pH over 30 day 151 storage period 35 8.7 Effects of addition of sugars on changes in pH and 152 fermentation time 36 8.8. Water activity at various levels of sugar 153 37 8.9 Counts of 5. ^/zeA'/wo/7Mw5 after inoculation, at 24 h 154 and during storage in the products made with various levels of sugar 38 8.10 Counts oiL. delbrueckii ssip. bulgaricus after 155 inoculation, at 24 h and during storage in the products made with various levels of sugar 39 8.11 Counts of Z. ac/

Xlll during storage in the products made with various levels of sugar 40 8.12 Counts ofbifidobacteria after inoculation, at 24 hand 157 during storage in the products made with various levels of sugar 41 9.1 Viable counts ofyogurt and probiotic bacteria during 162 12 weeks storage at -IS'^C probiotic bacteria were added along with yogurt bacteria 42 9.2 Viable counts ofyogurt and probiotic bacteria during 163 12 weeks storage at -18°C probiotic bacteria were added along with yogurt bacteria 43 9.3 Viable counts of yogurt and probiotic bacteria during 164 12 weeks storage at -18°C probiotic bacteria were added along with yogurt bacteria 44 9.4 Viable counts ofyogurt and probiotic bacteria during 165 12 weeks storage at -18'^C probiotic bacteria were added just before freezing 45 9.5 Viable counts ofyogurt and probiotic bacteria during 166 12 weeks storage at -18°C probiotic bacteria were added just before freezing 46 9.6 Viable counts of yogurt and probiotic bacteria during 167 12 weeks storage at -18°C probiotic bacteria were added just before freezing 47 10.1 Viable counts of 5. r/2ermop/z?7M5 during 12 weeks 174 storage at -18 C 48 10.2 Wiahle counts of L. delbrueckii ssp. bulgaricus during 175 12 weeks storage at-18 C 49 10.3 Viable counts of Z. aczWop/2r7M5 during 12 weeks 176 storage at -18 C 50 10.4 Viable counts ofbifidobacteria during 12 weeks 177 storage at -18 C

XIV 51 11.1 Changes in pH in fermented frozen dairy desserts 189 during storage for 12 weeks at -18*^C 52 11.2 Changes in titratable acidity in fermented frozen dairy 190 desserts during storage for 12 weeks at -18°C 53 11.3 Changes in P-D galactosidase in fermented frozen 191 dairy desserts during storage for 12 weeks at -18'^C 54 11.4 Viable counts of yogurt bacteria made with or without 192 acid casein hydrolysate and cysteine during 12 weeks storage at-18°C 55 11.5 Viable counts of probiotic bacteria made with or 193 without acid casein hydrolysate and cysteine during 12 weeks storage at -18°C 56 12.1 Viable counts of Z. aczWopMw^ with or without 200 cryoprotectants in fermented frozen dairy desserts made using a bench top ice cream freezer 57 12.2 Viablecountsofbifidobacteria wither without 201 cryoprotectants in fermented frozen dairy desserts made using a bench top ice cream freezer 58 12.3 Viable counts of Z. acz^iop/ziVw^ with or without 202 cryoprotectants in fermented frozen dairy desserts made using a continuous ice cream freezer 59 12.4 Viable counts of bifidobacteria wither without 203 cryoprotectants in fermented frozen dairy desserts made using a continuous ice cream freezer 60 13.1 Preparation of fermented frozen dairy desserts 221 61 13.2 Relationship between mean diameter of calcium 222 alginate beads and viscosity of sodium alginate 61 13.3 Release of enttapped bacteria from microencapsulated 223 gel beads 62 13.4 Scanning electron micrograph of LA 2415 224 encapsulated in calcium alginate bead

XV 63 13.5 Scanning electron micrograph of LA 2415 225 encapsulated in calcium alginate bead (cross section) 64 13.6 Scanning electron micrograph of LA 2415 226 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead) 65 13.7 Scanning electron micrograph of LA 2401 227 encapsulated in calcium alginate bead 66 13.8 Scanning electron micrograph of LA 2401 228 encapsulated in calcium alginate bead (cross section) 67 13.9 Scanning electron micrograph of LA 2401 229 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead) 68 13.10 Scanning electron micrograph of LA MIL A1 23 0 encapsulated in calcium alginate bead 69 13.11 Scanning electron micrograph of LA MJLAl 231 encapsulated in calcium alginate bead (cross section) 70 13.12 Scanning electron micrograph of LA MJLA1 23 2 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead 71 13.13 Scanning electron micrograph of BB1900 233 encapsulated in calcium alginate bead 72 13.14 Scanning electron micrograph of BB 1900 234 encapsulated in calcium alginate bead (cross section) 73 13.15 Scanning electron micrograph of BB 1900 235 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead) 74 13.16 Scanning electton micrograph of BB BDBB2 236 encapsulated in calcium alginate bead 75 13.17 Scanning electron micrograph of BB BDBB2 237 encapsulated in calcium alginate bead (cross section) 76 13.18 Scanning electron micrograph of BBBDBB2 238

XVI encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead) 77 13.19 Scaiming electron micrograph of BB 1941 239 encapsulated in calcium alginate bead 78 13.20 Scanning electron micrograph of BB 1941 240 encapsulated in calcium alginate bead (cross section) 79 13.21 Scanning electron micrograph of BB 1941 241 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead) 80 13.22 Releaseof beads in various pH levels using lactic acid 242 with or without phosphate buffer 81 13.23 Release of beads in various pH levels using bile with 243 or without phosphate buffer 82 13.24 Viability of Z. ac/Jop/zz/wj'with or without 244 encapsulation in fermented frozen dairy desserts 83 13.25 Viability of Z. acidophilus with or without 245 encapsulation in fermented frozen dairy desserts 84 13.26 Viability of Z. ad(ic>p/7z7M5 with or without 246 encapsulation in fermented frozen dairy desserts 85 13.27 Viability of Z. acidophilus with or without 247 encapsulation in fermented frozen dairy desserts 86 13.28 Viability of bifidobacteria with or without 248 encapsulation in fermented frozen dairy desserts 87 13.29 Viability of bifidobacteria with or without 249 encapsulation in fermented frozen dairy desserts 88 13.30 Viability of bifidobacteria with or without 250 encapsulation in fermented frozen dairy desserts 89 13.31 Viability of bifidobacteria with or without 251 encapsulation in fermented frozen dairy desserts 90 14.1 Growth curves of probiotic bacteria 261 91 14.2 Effects of acid (3.5 pH) and freezing at -18°C for 1 262

XVII hour on the viability of probiotic bacteria 92 14.3 Effects of acid (3.5 pH) and freezing at-18'^C for 2 263 hour on the viability of probiotic bacteria 93 14.4 Effects of acid (3.5 pH) and freezing at-18°C for 4 264 hour on the viability of probiotic bacteria 94 14.5 Effects ofacid (3.5 pH) and freezing at-18°C for 6 265 hour on the viability of probiotic bacteria 95 14.6 Effects of acid (3.5 pH) and freezing at-18°C for 12 266 hour on the viability of probiotic bacteria 96 14.7 Effects of acid (3.5 pH) and freezing at-18°C for 18 267 hour on the viability of probiotic bacteria 97 14.8 Effects of acid (3.5 pH) and freezing at-18°C for 24 268 hour on the viability of probiotic bacteria 98 14.9 Effects ofacid (3.5 pH) and freezing at-18°C for 1 269 hour on the viability of probiotic bacteria. (The organisms were directly stressed at lethal pH without using sub lethal pH) 99 14.10 Effects ofacid (3.5 pH) and freezing at-18°C for 2 270 hour on the viability of probiotic bacteria. (The organisms were directly stressed at lethal pH without using sub lethal pH) 100 14.11 Effects of acid (3.5 pH) and freezing at-18°C for 4 271 hour on the viability of probiotic bacteria. (The organisms were directly stressed at lethal pH without using sub lethal pH) 101 14.12 Effects of acid (3.5 pH) and freezing at-18°C for 6 272 hour on the viability of probiotic bacteria. (The organisms were directly sttessed at lethal pH without using sub lethal pH) 102 14.13 Effects of acid (3.5 pH) and freezing at-18°C for 12 273 hour on the viability of probiotic bacteria. (The

XVllI organisms were directiy stressed at lethal pH without using sub lethal pH) 103 14.14 Effects of acid (3.5 pH) and freezing at-18°C for 18 274 hour on the viability of probiotic bacteria. (The organisms were directly stressed at lethal pH without using sub lethal pH) 104 14.15 Effects of acid (3.5 pH) and freezing at-18°C for 24 275 hour on the viability of probiotic bacteria. (The organisms were directly stressed at lethal pH without using sub lethal pH) 105 14.16 Viable counts of stressed adapted probiotic bacteria 276 incorporated into fermented dairy desserts monitored for 12 weeks storage at-18°C 106 14.17 Viable counts of stressed adapted probiotic bacteria 277 incorporated into fermented dairy desserts monitored for 12 weeks storage at-lS'^C 107 14.18 Viable counts of stressed adapted probiotic bacteria 278 incorporated into fermented dairy desserts monitored for 12 weeks storage at -18°C

XIX 1.0 INTRODUCTION

Dairy products containing Z. acidophilus and Bifidobacterium spp. are rapidly gaining popularity in Australia. The shelf life of yogurts is approximately 3 weeks. As a result, it is not feasible to ship yogurts to overseas countries. Thus, the sales of such products are limited to local markets. With increasing popularity of frozen dairy desserts containing yogurt cultures, a market could be developed for dairy desserts similar to soft-serve and hard pack frozen yogurts but containing cultures selected for dietary benefits such as Z. acidophilus and

Bifidobacterium spp. Frozen dairy desserts have longer shelf life and thus the products could be shipped to overseas coimtries such as Japan.

It has been suggested that to have therapeutic effects, a minimum of one million viable cells of Z. acidophilus and Bifidobacterium spp. per gram of a product must be present in fermented frozen dairy desserts. However, introducing probiotic organisms into the frozen food chain can be a difficult task as the organisms are unstable in such products.

This study aimed at achieving improved viability of probiotic bacteria in fermented frozen dairy desserts. The specific objectives of this project were:

1 To assess the viability of yogurt and probiotic bacteria in commercial

products such as yogurts and fermented dairy desserts. 2. To evaluate several media in order to selectively enumerate Z. casei from

mixed populations ofyogurt bacteria and probiotic bacteria,

3. To select strains of yogurt bacteria and probiotic bacteria that survive

acidic conditions, low temperatures, high sugar concentrations and low

water activity conditions,

4. To investigate the use of micronutrients and cryoprotectants on improving

the survival of the probiotic bacteria,

5. To investigate the feasibility of encapsulating probiotic bacteria and of

incorporating them in fermented frozen dairy deserts, and

6. To examine the effects of low temperature and acid adaptations on

survival of probiotic bacteria.

Chapter 2 of this thesis contains the review of literature; chapter 3 contains materials and methods and chapter 4 deals with the viability of probiotic bacteria in commercial products. Chapters 5 and 6 studied the viability of the 5 groups of bacteria (S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei) in commercial dairy products. Chapters 7, 8, and 9

focus on selection of strains and effects of water activity on fermentation, organic

acid production and viability of probiotic bacteria. Chapters 10 and 11 contain

viability of probiotic bacteria in products made using a bench top and a continuous ice cream freezer. Chapters 12 and 13 deal with the effects of micronutrient and cryoprotectants on the viability of probiotic bacteria. Chapters

14 and 15 studied use of microencapsulation technique and acid and low temperature adaptations in improving viability of probiotic bacteria and finally

chapters 16, 17, and 18 give overall conclusions, future research directions and

references. 2.0 LITERATUIiE REVIEW

2.1 Yogurt

2.1.1 Definition

Yogurt is defined as a product resulting from milk by fermentation with the two organisms. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus growing together symbiotically. Carbon dioxide, acetic acid, di-acetyl, acetaldehyde and several other substances are formed in the conversion process, and these impart to the product its characteristic fresh taste and aroma. Yogurt varies considerably in composition, flavour and texture, according to the nature of fermentation and the type of organisms added, although some controversy still exists regarding the exact definition of yogurt in terms of its chemical composition and types of starter organisms used (Kosikowski, 1977; Tamime and

Deeth, 1981).

2.1.2 Types ofyogurt

Several types of yogurts are manufactured in Australia, however, the main types are: set, stirred and drinking yogurts. The minimum standards including composition and pH are fixed by the Austtalian standards H8. The set yogurt is prepared by heating the standardised milk to 85°C for 30 min or 95°C for 15 min and adding yogurt bacteria or a combination ofyogurt and probiotic bacteria after cooling to 42-45°C. The milk containing culture bacteria are filled in desired containers and incubated at the same temperature until a pH of 4.5 is attained.

Similar procedures of set yogurts are used in preparing stirred yogurts; however, the total solids are higher in this case and inoculated milk is set in bulk rather than in containers. The incubated mix after attaining pH 4.5 is stirred and chilled followed by addition of finits before packing into containers. Drinking yogurts are prepared using less total solids (5.5 - 11%)) and incubating at lower temperatures for long hours (37°C for 16-18h). Fruit puree, fhiit pulp and flavours are added before filling into bottles.

The post-incubation processing of yogurt may lead to various types of yogurts such as pasteurised, UHT, concentrated and dried yogurts. Pasteurised yogurt is heated after incubation using a shell tube pasteuriser to about 60-65°C leading to destruction of yogurt. In some cases probiotic bacteria are incorporated in order to avoid antagonism with yogurt bacteria and to improve the survival of probiotic bacteria. The dried yogurt can be produced either by sun drying, spray drying or

freeze-drying. The drying process transforms the junket into powder and also

causes loss of some flavour compounds and destruction of starter cultures. The

dried yogurt is mainly used in the Middle East. The dried yogurt is used in

making some traditional cooking. The manufacture of yogurt has teen reviewed

by several authors (Tamime and Robinson, 1985; Kurmaim, 1988; Rasic and

Kurmann, 1978).

2.1.3 Consumption pattern

Fermented milk products originated from the near east and subsequently gained

popularity in eastem and central Europe. The earliest example of cultured milk

was presumably produced accidentally by the nomads. This milk "turned sour"

and coagulated under the influence of certain infecting microorganisms. As luck would have it, the bacteria were of harmless, acidifying type and were not toxin- producing organisms. Among the fermented , yogurt is one of the common examples.

Yogurt is one of the most popular and best known in almost all comers of the world, particularly Mediterranean, Asia, and Central Europe, and is becoming more popular in America, Australia and Japan. The origin of yogurt is not very clear. However according to Tamime and Deeth (1980), the word yogurt is derived from the Turkish word 'jugurt'. The Slavs adopted the procedure of yogurt preparation and yogurt became their traditional food. Yogurt is prepared in Bulgaria from or boiled cows milk, inoculated at 40-45°C with a portion of previously . To keep the temperature constant, the containers containing inoculated milk are thoroughly wrapped in furs and placed for 8-10 h in a warm place until a smooth gel was formed. Table 2.1 shows the various types of yogurts and their composition, manufactured in Australia. Table 2.1 Types of yogurts and their nutritional values that are available in Australian markets (data shown is for 100 grams).

Type Protein Fat Total Energy Calcium Sodium Cholesterol (g) (g) Sugars (kJ) (mg) (mg) (mg) (g) Natural 4.7 3.4 4.7 281 171 55 16

Reduced fat 5.0 1.7 7.5 266 160 63 10

Low fat 5.9 0.2 5.8 200 209 70 5

Acidophilus 5.6 0.2 5.5 191 215 75 6

Vanilla skim 5.9 0.2 12.2 304 185 79 6

Strawberry skim 5.9 0.3 10.9 270 181 67 5

Fruit strawberry 4.9 2.8 12.4 381 171 60 12

Strawberry reduced fat 4.9 0.9 14.5 348 160 50 6

Strawberry low fat 5.2 0.2 12.8 299 179 61 3

Strawberry artificially 5.0 0.2 11.4 186 170 57 4 sweetened Strawberry A B 4.9 3.1 5.9 379 165 58 12

(Source: Australian yogurt and ice cream, Australian Dairy Corporation nutritional guide, 1998). 2.2 Functional foods

Functional foods are foods that convey nutritional/therapeutic benefits on human health. Functional foods are defined as "the foods similar in appearance to conventional foods and are intended to be consumed as part of a normal diet, but have been modified to subserve physiological roles beyond the provision of simple nutrient requirements" (National Food Authority, 1994). The interest in fijnctional foods continues to increase world wide and with the rapid growth of nutritional and medical sciences, it is hard to imagine that all the 'functional' components have been detected yet in the foods we commonly consume. The first international conference on East-west perspective on fimctional foods was held in

Singapore in 1995. The purpose of the conference was to discuss the many issues surrounding functional foods at an intemational level. The concept originated in

Japan because of two reasons, the highly competitive market and the great interest by consumers in health foods. This has now found a firm basis in the western world and is considered to be the most important development for the future.

Dairy products containing probiotic bacteria are a good example of functional foods. Yogurts and fermented frozen dairy desserts containing probiotic bacteria are popular dairy products. Japan has provided the technology for the introduction of probiotic fimctional foods and import probiotic functional foods into the country. The Japanese Food Authority has implemented minimum standards on the products before it is marketed to the consumers. Providing an affirmed legal product and specific labelling benefit, functional foods (Known in English as Foods for Specified Healtii Use, or FOSHU) are currently marketed in Japan, although many foods, which would be considered as fimctional foods, have not been submitted for formal FOSHU certification. As of June, 1997, 80

FOSHU foods have been approved (Bailey, 1997; Sanders, 1998). In the United

States, although consumer and corporate interest is high, functional foods have no legal definition. This has not stopped food companies from entering into functional food market (Sanders, 1998). In Europe the probiotic fimctional yogiut market offers considerable opportunities for Europe's dairy industry. Grijspaardt-

Vink (1996) identified health and convenience as the two major trends in the

European food market, citing keen awareness of European consumers to the relationship between diet and health. A leadership role in approaching the functional food area is being taken by Intemational Life Sciences Institute

(ILSL), which is managing a project started in 1995 to establish a science-based approach for targeted modification of food and food constituents. In Australia probiotic functional foods are popular and the products are exported to Japan and the middle-east countries. Fermented dairy products such as yogurt and fermented frozen dairy desserts have been the focus of many studies in terms of their alleged physiological fimctionality. In contrast to the biologically active components contained in the milk itself, the health-promoting effects of the fermented dairy products may be related to the biological activity of the bacteria used in the production of these foods and the metabolites generated during the fermentation process (Jelen and Lutz, 1998). 2.3 Fermented frozen dairy desserts

2.3.1 Definition

Frozen yogurt is a food product derived by blending of pasteurised ice-cream

base mix with plain yogurt followed by mixing before freezing. The proportion of

blending varies from manufacturer to manufacturer. The ice-cream mix may

contain whole milk, partially de-fatted milk, skim milk, cream, , butter oil,

sweeteners, stabilisers and emulsifiers. The yogurt mix may contain, milk,

sucrose, cream, butter, butter oil and lactic acid bacteria and/or probiotic bacteria.

2.3.2 Types of fermented frozen dairy desserts

There are two types of fermented frozen dairy desserts available in the market:

soft serve, hard pack and ice-poll type. Soft serve fermented frozen dairy desserts

may contain about 100% overrun and are packed in 200 mL, 500 mL, 1 L, 2 L

and 5 L containers. Soft serve fermented frozen dairy desserts are available in

different flavours and with fruits and/or fhiit pulp, whereas stick ice-poll may

have about 18% overrun and is moulded in fancy shapes and packed individually

in a box of 12 and 24.

2.3.3 Consumption pattern

Yogurt has proven to be an unacceptable vehicle to incorporate bifidobacteria

into human diet due to the high acidity of most products. Several commercial

products have been evaluated and only one low acid Japanese product was found

to contain bifidobacteria. The remaining samples were either negative for bifidobacteria or lacked fiiictose-6-phosphate phosphoketolase (F6PPK) activity.

F6PPK is indicative ofbifidobacteria (Scardovi, 1981; Modler and Garcia, 1993).

The ice cream industry has investigated several means of participating in the ever-growing market for acceptable yogurt products. The industty wanted to produce a desirable flavoured , which could be manufactured in an ice-cream plant and marketed through existing channels of distribution. Due to competitive reasons the quality of the new product might suffer without a published standard. Therefore it was decided that frozen yogurt was in fact a new frozen dessert and, therefore entitled to its own standard of identity. Frozen yogurt in its own right can be identified by several features.

In early 70s frozen yogurt was established in the market but was unsuccessful by the late 70s due to its sour taste and being gummy, icy and not very creamy. In

80s health consciousness of consumers have spurred the demand for the frozen yogurt. The acceptance and popularity have pushed frozen yogurt into full- service, year-round, any-time-of-day of product demand.

Dairy products containing Z. acidophilus and Bifidobacterium spp. are rapidly gaining popularity in Australia. The shelf life ofyogurt is approximately 3 weeks.

As a result, it is not feasible to ship yogurt to overseas countries. Thus, the sales of such products are limited to local markets. With increasing popularity of frozen dairy desserts containing yogurt cultures a market could be developed for dairy desserts similar to soft-serve and hard pack frozen yoghurts, but containing

11 culttires selected for health benefits such as Z. acidophilus and Bifidobacterium spp. Frozen dairy desserts have longer shelf life and thus tiie products could be shipped to overseas countries such as Japan.

It has been suggested tiiat to have therapeutic effects, a minimum of one million viable cells of Z. acidophilus and Bifidobacterium spp. per gram of a product must be present in fermented frozen dairy desserts. However, introducing probiotic organisms into the frozen food chain can be a difficult task as the organisms are unstable in such products.

2.4 Yogurt bacteria

In 1905, Grigoroff conducted several studies on yogurt bacteria. The classification of the lactic acid bacteria by Orla-Jensen is still recognised as the standard method for distinguishing these organisms, ie the sphere shape was

Streptococcus and the rod forms were Thermobacterium, Treptobacterium and

Betabacterium. According to Orla Jensen the yogurt starter organisms were thermophilic lactic acid bacteria capable of growing at 40-45°C.

According to the Garvie (1986), yogurt bacteria are classified into two separate families, the Streptococcaceae and Lactobacillaceae (Tamime and Robinson,

1985). The yogurt starter cultures are mixed strains of S. thermophilus and Z. delbrueckii spp. bulgaricus, which are normally propagated together at around

42°C. There is a symbiotic relationship between S. thermophilus and Z. delbrueckii spp. bulgaricus. Pette and Lolkema (1950) observed a rapid acid

12 development in mixed cultures, compared to single sttain cultures due to increase in numbers of streptococci. Dahiya and Speck (1968) also investigated the symbiosis theory, and supported the view that Z. delbrueckii ssp. bulgaricus stimulates S. thermophilus by releasing glycine and histidine into the growth medium. They concluded that histidine rather than valine was the most important requirement. Under anaerobic conditions, the former organism produces a stimulating factor for Z. delbrueckii ssp. bulgaricus that is equal to or can be replaced by formic acid.

Faster growth of streptococci at the beginning of fermentation brings about accumulation of moderate amounts of lactic and acetic acids, acetaldehyde, diacetyl and formic acid. Availability of formate and the changes in oxidation- reduction potential in the medium stimulate the growth of Z. delbrueckii spp.

bulgaricus. The proteolytic activity is more in Z. delbrueckii spp. bulgaricus than

S. thermophilus. The metabolic activity of yogurt bacteria results in a

considerable increase in cell numbers. The total numbers of yogurt bacteria

ranges from 10^ to 10^ and may decrease during the storage period (Ravula and

Shah, 1998b).

2.5 Prebiotics

The term "prebiotic" has been applied to compounds such as oligosaccharides,

which promote the growth of beneficial organisms. Oligosaccharides have been

recognised for health benefits in Japan, since tiie early 1990s many products,

especially drinks, have been developed and are being promoted for their oligosaccharide content. Some oligosaccharides, due to their chemical structure, are resistant to digestive enzymes and therefore pass into large intestines where they become available for fermentation by the saccharolytic bacteria (O'Sullivan,

1996).

2.6 Probiotics

The term probiotic is derived from the Greek word, which means for life.

Probiotic is defined as a viable mono-or mixed-culture of microorganisms, which when applied to animal or a man beneficially affects the host by improving the properties of the indigenous microflora. The term probiotic was first used by

Lilly and Stillwell (1965) to describe the growth promoting effect of one or more microorganisms against another. Soon afterwards, the term was applied primarily to animal feed supplements. Further changes in use of this term implied a live microbial feed supplement that improved the intestinal microbial balance of the host (Fuller, 1989). The term is now also being used with reference to human consumption of live microorganisms as food additives for nutritional health and well-being (Hull et al, 1992). The organisms that have been considered as probiotics are Z. acidophilus and Bifidobacterium spp., which must be capable of reaching and colonising the intestinal tract of the human. These organisms must be acid and bile tolerant and must provide a nutrient source, which selectively promotes growth of beneficial intestinal microflora (Lyons, 1988).

These bacteria are the most important group in intestinal organisms to be suggested for use as dietary adjuncts in dairy foods. The probiotic organisms are

14 predominant microflora of breast fed infants (Jao et al, 1978; Gilliland, 1979;

Rasic and Kurmann, 1978). As the person ages the number of intestinal bifidobacteria decreases and the number of Clostridia, streptococci and coliforms increases (Stark and Lee, 1982).

The basis of the probiotic concept is that these are the gastrointestinal tract microorganisms, which have a beneficial effect on the host. Modem life styles may have adverse affect on the delicate balance that exists between the many different species of microorganisms, which inhabit the gut. Our unnatural diet

(most of which is heat sterilised by cooking), antibiotic therapy and stress are all factors which can alter the composition of the gut microflora and nutritional and health consequences. Probiotics can reverse or prevent these consequences by restoring the flora to its natural state (Fuller, 1989)

2.7 Lactobacillus acidophilus

th

In the early 19 century Metchnikoff suggested that man should consume milk fermented with lactobacilli to prolong life (Metchnikoff, 1908). His theory was that the lactobacilli would displace the microorganisms normally occurring in the intestinal tract that produced "toxins", resulting in reduction of life span.

Continued worldwide interest in beneficial lactobacilli as dietary adjuncts has resulted in extensive research and has led to the development of foods including various dairy products. Several antibiotic-like substances such as acidolin

(Hamdan and Mikolajcik, 1974), acidophilin (Shahani et al., 1977), and lactocidin (Vincent et al., 1959) are produced by various strains of Z.

15 acidophilus. Mehta et al. (1984) reported a broad-spectrum inhibitor produced by

Z. acidophilus, which was active against a number of both gram-positive and gram-negative organisms. This inhibitory material was characterised as being a protein. This inhibitory system also was active against both gram-negative and gram-positive organisms. It was characterised as being resistant to proteolytic enzymes, heat resistant, and of low molecular weight. Z. acidophilus is capable of producing more than one inhibitory system and the antagonistic action they exert towards intestinal pathogens are the likely results from the involvement of more than one particular inhibitor.

Shahani et al. (1977) studied the influence of Z. acidophilus on tumour cells using rats as an animal model. The authors reported that the rats fed with milk containing Z. acidophilus had less tumour cells than the rats, which did not receive milk containing Z. acidophilus.

Goldin et al. (1980) showed that the consumption of milk containing Z. acidophilus could cause significant reduction in the activity of the P- glucuronidase, azoreductase, and nitoreductase enzymes. These enzymes can convert procarcinogens into carcinogens in the intestinal tract. Lactose maldigestion refers to the inability of the person to digest lactose adequately. The inability to digest lactose adequately is due to the absence of sufficient amounts of the enzyme P-galactosidase in the small intestines. The microorganisms in the gastrointestinal tract can influence the ability to digest lactose. A total of 56 of lactobacilli are shown in Table 2.2.

16 Table 2.2 Species of genus Lactobacillus 1 Lactobacillus acidophilus 29 Z. paracasei

2 L. amylophilus 30 L. rhamnosus

3 L. amylovorus 31 L. sake

4 L. crispatus 32 L. agilis

5 L. delbrueckii 33 L. pentosus

6 L. gallinarum 34 L.plantarium

7 L. gaserri 35 L. brevis

8 L. heliveticus 36 L. buchneri

9 L. gensenii 37 L. collinoides

10 L. johnsonii 38 L. fermentum

11 L. kefiranofacens 39 L. fructivorans

12 L. aviarius 40 L. hilgardii

13 L. farciminis 41 L.

14 L. salivarius 42 L. malofermentans

15 L. mali 43 L. oris

16 L. ruminis 44 L. parabuchnery

17 L. sharpeae 45 L. ruteri

18 L. acetotolerans 46 L. pontis

19 L. hamsteri 47 L. vaginalis

20 L. alimentarius 48 L. suebicus

21 L. bifermentans 49 L. vaccina St ercus

22 L. casei 50 L. sanfracisco

23 L. coryneformis 51 L. confusus 24 Z. curvatus 52 L.fructosus

25 Z. graminis 53 Z. halotolerans

26 Z. homohiochii 54 Z. viridescens

27 Z. intestinal is 55 Z. kandlery

28 Z. murinus 56 Z. minor

(Source: Wood and Holzapfel, 1995)

2.8 Bifidobacterium spp.

The isolation of bifidobacteria was made by Tissier in the year 1899 at the

Pasteur Institute in France. These organisms were isolated from infant stools and are most predominant component of normal intestinal microflora of infants and adults. Since then these organisms have been treated with a blend of endorsement and skepticism regarding the implied nutritional benefit derived from the ingestion of viable bifidobacteria or bifidogenic substances. The microbiology appears straightforward (Hughes and Hoover, 1991). Bifidobacteria are indigenous gut organisms well adapted for metabolism in the gastrointestinal tract of humans. Acetic, formic and lactic acids are produced in relatively high amount from the fermentation of sugars. The volatile fatty acids, particularly acetic acid is much more inhibitory toward gram-negative bacteria than is lactic acid (Hoover, 1993). Bifidobacteria are one of the first groups of organisms to establish in the intestinal tract and usually are the largest group represented in infants. Bottle fed babies normally have one log count less bifidobacteria present in faecal samples than breast fed babies (Braun, 1981) and bottle fed infants generally have higher levels of enterobacteriaceae, streptococci and anaerobes other than bifidobacteria (Yuhara et al, 1983). For breast fed babies, levels of

10 -10 bifidobacteria per gram of faeces are common (Modler et al, 1990).

The feces from breast-fed infants contained higher levels of acetic acid and acetate than did the faeces from bottle fed infants (Bullen et al, 1977). Enteric infection in children can be prevented or minimised when their intestinal tracts contain high levels of bifidobacteria (Bullen and WiUis, 1971). In Japan, successfril treatment of diarrhoeal diseases in children is accomplished by feeding dairy products containing large numbers ofbifidobacteria (Tojo et al, 1987).

Bifidobacteria can colonise the intestinal tract. Colonisation varies depending upon the specific bacterium and their host affinity. Bacteria are in competition for colonisation space. The original resident has an advantage over transient bacteria, however, the composition of the microflora is rather dynamic. If harmful bacteria proliferate, as in the case of diarrhoeal infections, the equilibrium is disturbed and it becomes difficult even for indigenous bacteria to maintain the necessary territory to colonise the intestine (Homma, 1988; Rasic, 1989).

With new technology available for providing anaerobic conditions, bifidobacteria are now being studied more extensively and are emerging as possibly the most important group of intestinal organisms with regard to human health. Among bifidobacteria species, B. bifidum is suggested for use as dietary adjuncts especially in dairy foods. Table 2.3 shows the chronological order of the development of taxonomy ofbifidobacteria.

19 Table 2.3 Chronological order of the development of taxonomy ofbifidobacteria Stage of development Author/reference Year 1 Bacillus bifidus Tissier 1900 2 Bacteroides bifidus Castellani 1919 3 Bacteroides bifidus Chalmers 1923-1934 4 Lactobacillus bifidus Bergey's Manual eds 1920 5 Bifidobacterium bifidum Holland 1924 6 Bacterium bifidum Orla-Jensen 1927 7 Tisser bifidia Lehmann and Neumaim 1929 8 Norcadia bifida Pribram 1931 9 Actynomyces bifidus Vuillemin 1934 10 Actinobacterium bifidum Nanni 1937 11 Lactobacillus acidophilus var. Weiss and Rettger 1938 bifidus 12 Lactobacillus parabifidus Weiss and Rettger 1938 13 Bifidobacterium bifidum Weiss and Rettger 1938 14 Lactobacillus bifidus Prevot 1939-1957 15 Cohnistreptothrix bifidus Bergey's Manual 1944 16 Corynebacterium bifidum Negrovi and Fisher 1949 17 Lactobacillus bifidus Olsen 1950 18 Lactobacillus bifidus var. Norris et al. 1953 pennsylvanicus 19 Description of human species Dehnert 1963 20 New animal species Reuter 1969 21 New animal species Mitsuoka 1969 22 New animal species Scardovi 1972 23 Creation of genus Bifidobacter ium Holdeman and Moore 1974 constituting 11 species 24 Inclusion of 24 species to genius Scardovi 1986 Bifidobacterium 25 Inclusion of 29 species to genus Sgorbati et al. 1995 Bifidobacterium Adapted from Sgorbati et al. (1995) According to the latest collection of species in tiie genus of Bifidobacterium, additional five species have been described (Table 2.4) making the total number of species as twenty nine (Biavati and Mattarelli, 1991; Biavati et al. 1991).

Table 2.4 Species of genus Bifidobacterium

1 B. bifidum 16 B. boum

2 B. longum 17 B. magnum

3 B. infantis 18 B. pullorum

3 B. breve 19 B. gallinarum

5 B. adolescentis 20 B. suis

6 B. angulatum 21 B. minimum

7 B. catenulatum 22 B. subtile

8 B. pseudocatenulatum 23 B. coryneformis

9 B. dentium 24 B. asteroids

10 B. globosum 25 B. indicum

11 B. pseudolongum 26 B. gallicum

12 B. cuniculi 27 B. ruminatium

13 B. choerinum 28 B. mericicum

14 B. animalis 29 B. saeculare

15 B. thermopilum

Adopted from Sgorbati et al (1995)

21 2.9 Lactobacillus casei

Due to viability problems associated with Z. acidophilus and bifidobacteria during storage (Anon, 1992; Shah et al, 1995; Dave and Shah, 1997 a, b), tiie recent trend is to add Lactobacillus casei as an adjunct bacteria to yogurt or probiotic cultures. Z. casei is claimed to be more stable than Z. acidophilus or bifidobacteria (McCaim et al, 1996). Several yogurts and fermented milk drinks currently available on markets already contain Z. casei in addition to Z. acidophilus and bifidobacteria. Z. casei, recently introduced to Australian yogurts, has shown good potential as a probiotic organism (Perdigon et al, 1995;

Wagner et al, 1997). Table 2.5 shows various dairy products available in

Australia with Z. acidophilus, bifidobacteria and Z. casei.

Table 2.5 Dairy products available in Australia with Z. acidophilus, bifidobacteria and Z. casei.

Type of Company Brand name Type of product culture Z. acidophilus Amarina Health Amarina Yogurt Foods Co. Pty. Ltd.

L. acidophilus Casa Casa Yogurt

L. acidophilus Kenilworth Country Kenilworth Yogurt Foods Pty. Ltd.

L. acidophilus Queensco-unity Bomhoffen Yogurt Dairy Foods

L. acidophilus Mundella Foods Pty. Mundella Yogurt Ltd.

Z. acidophilus Yoplait Austtalia Yoplait yoplus Yogurt

Z. acidophilus Attiki Pty. Ltd. Attiki Skim yogurt Z. acidophilus Burra Foods Gippsland Organic finit yogurt

Z. acidophilus Regal Cream Bulla lite Fruit yogurt Products Pty. Ltd. Reduced fat

L. acidophilus Hastings Co­ Hastings Fruit yogurt operative Limited Reduced fat

L. acidophilus Nestle Austtalia Pty. LCI Yogurt Ltd.

Z. acidophilus National Yo-Split Flavoured yogurt Limited

Z. acidophilus, Fonti Dairy Foods Fonti farm Yogurt Bifidum Pty. Ltd.

Z. acidophilus, Jalna Dairy Foods Jalna Yogurt Bifidum Pty. Ltd.

Z. acidophilus, Midcoast yogurt Mid Coast Yogurt Bifidum

Z. acidophilus, Jalna Dairy Foods Jalna Biodynamic Bifidum Pty. Ltd.

Z. acidophilus, Westhaven Weasthaven Yogurt Bifidum

Z. acidophilus. Regal Cream Bulla Drinking yogurt Bifidum Products Pty. Ltd.

Z. acidophilus, Jalna Dairy Foods Jalna Drinking yogurt Bifidum Pty. Ltd.

Z. acidophilus, Peter Farms Peter Yogurt Bifidum

Z. acidophilus, Nestie Austtalia Pty. Nestle Yogurt Bifidum Ltd.

Z. acidophilus, Q.U.F. Industries Danone Yogurt Bifidum Ltd.

Z. acidophilus, Regal Cream Bulla Frozen yogurt

23 Bifidum Products Pty. Ltd.

Z. acidophilus, Mundella Foods Pty. Mundella Yogurt Bifidum, Ltd. Z. casei

Z. acidophilus, Jalna Dairy Foods Jalna Yogurt Bifidum, Pty. Ltd. Z. casei

INA Australian Co­ Oak Frozen yogurt operative Foods Lid

INA Charioto Natural Ice Charioto Frozen yogurt cream frozen yogurt

INA Dairy Vale Eve Frozen yogurt

INA Norco Co-operative Norco Frozen yogurt Limited

INA Royal Copenhagen Royal Frozen yogurt Ice Cream Cone Co Copenhagen

Seendipity ice Seendipity Frozen yogurt Cream

INA Everest Colonial Frozen yogurt

INA Dairyfields Co­ That's Y Long Life yogurt operative Assoc Ltd.

INA Amarina Health Amarina Gourmet yogurt Food Co Pty Ltd.

INA Amarina Health Amarina Greek style yogurt Food Co Pty Ltd.

INA Delco Foods Delco Greek style yogurt Products

INA National Dairies Ltd. Yo-Fresh Greek style yogurt

Z. casei Australia Pty. Yakult Fermented milk Shirota Ltd. drink

24 Z. casei K.O.Z. World Wide Maeil Fermented milk drink

Z. casei GG Q.U.F Industries Vaalia Fermented milk drink

(Source: Australian yogurt and ice cream, Australian Dairy corporation nutritional guide 1998. INA: information not available)

25 2.10 Enumeration of 5. thermophilus, L. delbrueckii ssp bulgaricus, L. acidophilus. Bifidobacterium spp. and L. casei

Several media have been developed for differential enumeration of Z. acidophilus and Bifidobacterium ssp. Scardovi (1986) reviewed several complex media and media containing a wide variety of antibiotics to enumerate selectively

Bifidobacterium spp. and concluded that one selective medium is not appropriate for all species. For isolation and enumeration of bifidobacteria from aquatic environments, Munoa and Pares (1988) developed a selective medium, which consisted of reinforced clostridal agar (RCA) containing nalidixic acid, polymyxin B, kanamycin and iodoacetate. Iwana et al (1993) developed galactose agar containing lithium chloride and galactose as selective agents for enumeration of Bifidobacterium spp. Several other selective media have been reported, including one containing an oxygen reducing membrane fraction from

Escherichia coli (Burford, 1989), Rogosa modified selective medium and tryptone phytone yeast extract-S-agar (Samona and Robinson, 1991), X-gal- based medium (Chevalier et al, 1991) and dicloxacillin-based medium (Sozzi et al, 1990). Arroyo et al. (1994) evaluated brain heart infusion agar, modified

Columbia agar, RCA, modified deMan Rogosa Sharpe (MRS) agar and modified bile agar for enumeration of B. adolescentis, B. infantis and B. longum from pure cultures. However, these media may not be suitable for selective enumeration of

Bifidobacterium spp. in the presence of other lactic acid bacteria or from yogurt which typically contains Z. delbrueckii ssp. bulgaricus and S. thermophilus. Ml7 agar, developed by Terzaghi and Sandine (1975) from lactose yeast phosphate agar, has been found to support the growth of S. thermophilus and suppress the growth of Z. delbrueckii ssp. bulgaricus when the pH is 6.8 (Shankar and Davies,

1977; Terzaghi and Sandine, 1975). RCA has been found to be selective for Z. delbrueckii ssp bulgaricus by suppressing the growth of S. thermophilus when the pH of the agar was 5.5 (Johns et al, 1988). There is a growing concern that some media which contain antibiotics or bile may also restrict the growth of Z. acidophilus or bifidobacteria and that counts obtained are not necessarily representative of viable ceils which are in the product.

Selective enumeration of Z. casei in yogurt- type fermented milks containing probiotic bacteria based on 15 C incubation temperature and 14 days incubation time was studied by Champagne et al. (1997). However, an incubation time of 14 days may not be practical for the dairy industry if the results are required in a short time.

27 Table 2.6 Therapeutic properties of probiotic bacteria.

No Therapeutic properties Possible causes and mechanisms 1. Colonisation of gut and Survive gastric acid, resist lysozyme, tolerate inhibition of spoilage type high bile concenttation, adhere to intestinal organisms surface and production of inhibitory compounds i.e. acids, H2O2 and bacteriocins. 2. Improved digestibility and Partial breakdown of protein, fat, enhanced growth carbohydrates and improved bioavailability of nutrients. 3. Lactose tolerance Reduced lactose in the product and further availability of bacterial lactase enzymes. 4. Hypocholesterolaemic effect Production of inhibitors of cholesterol synthesis, deconjugation of biles and assimilation of cholesterol. Anticarcinogenic effect Inhibition of carcinogens and enzymes involved in converting procarcinogens to carcinogens, inhibition of growth of putrefying organisms and stimulation of host immune system. 6. Stimulation of the host Enhancement of macrophage formation, immunological system stimulation of suppressor cells and production of interferon.

7. Control of vaginal infections Inhibition of fungi and bacteria responsible for the infection. 8. Improved vitamin metabolism. Synthesis of group B vitamins. 9. Prevention of constipation Improvement in bowel movement and stabilisation of ecological balance in the intestinal ttact. 10. Recovery of damaged liver, Reduction in concentration of metabolites and effectiveness against aspects of enterotoxins produced by putrefying nausea, liver diseases, acne, etc organisms.

11. Prolongation of life Reduced intestinal putrefaction and auto­ intoxication.

(Source: Metchnikoff, 1908; Gilliland et al, 1975; Le et al, 1986; Van't Veer et al, 1989; Modler et al. 1990; Kurmann and Rasic, 1991; Huges and Hoover, 1991; Misra and Kuila, 1991; Kanbe, 1992; Mital and Garg, 1992; Ishibashi and Shimamura, 1993; Yamamoto etal, 1994).

28 2.11 Factors affecting the viability of probiotic bacteria in fermented frozen dairy desserts

Probiotic foods containing live microorganisms with beneficial effects on human health have been produced for many years. Lactobacillus acidophilus and

Bifidobacterium spp. are helpful in improving intestinal problems including bowel movements. These organisms can colonise the intestinal tract and offer antagonism against various pathogenic and spoilage organisms. Lactose intolerant people can exploit the lactase enzyme of the organisms, which could benefit in reducing lactose intolerance problem. Some workers have studied the effect of administering Z. acidophilus and B. bifidum to children suffering from leukaemia and children with obstructive jaundice.

The major factors affecting the healthy intestinal microflora of humans are diet, drugs, stress and ageing. As a result, use of probiotics or feeding of desirable types of bacteria to re-establish or maintain a healthy balance of gut microorganisms has gained considerable interest over the last several years (Hull et al, 1992). B. bifidum accounts for about 40% of the faecal flora in the large intestine of adults, therefore, the consumption of the minimal culture plays a beneficial role in maintaining a balanced intestinal flora (Rasic and Kurmann,

1978). Z. acidophilus and Bifidobacterium spp. have shown to correct intestinal disorders, lower serum cholesterol, posses anti-carcinogenic properties and improve lactose utilisation in lactose intolerant people (Gilliland, 1989; Shah and

Jelen, 1990; Shah, 1993; Shah, 1994). These organisms are inhibitory towards

Gram-negative bacteria (Hekmat and McMahon, 1992).

29 Poor survival of probiotic organisms in fermented frozen product is possibly due to pH and acidity, freeze injury and due to incorporation of air. Z. acidophilus and B. bifidum prefer to grow at 37°C and at a pH of 6.5 to 7.0 (Scardovi, 1986).

Gram positive bacteria are generally less susceptible to freeze injury than Gram negative bacteria (Speck and Ray, 1977). B. bifidum is not as acid tolerant as Z. acidophilus and the growth of this organisms is affected below pH. 5.0. Another factor affecting the viability of Z. acidophilus and B. bifidum is the incorporation of air due to whipping air into the product during freezing. As B. bifidum is anaerobic while Z. acidophilus is microaerophilic, the viability of B. bifidum is expected to be reduced more rapidly than that of Z. acidophilus.

Survival of Z. acidophilus and B. bifidum in ice cream for use as a probiotic food was studied by Hekmat and McMahon (1992). They have demonstrated that the beneficial microorganisms such as Z. acidophilus and B. bifidum can be grown to high numbers in ice cream mix and the organisms remained viable during frozen storage. Studies by Gilliland and Speck (1977), Schioppa et al. (1981), and Hull et al. (1984) have shown that Z. acidophilus and bifidobacteria are unstable in fermented dairy products. Z. acidophilus, when added to set yogurt after its manufacture, rapidly lost viability (90-99%) within three to five days and completely disappeared within seven days. When the organisms were added with yogurt cultures organisms during yogurt manufacture, loss of viability still occurred but the organisms survived better. In several studies (Anon, 1992; Shah et al, 1999) several brands of commercial yogurts and fermented milk drinks from Australian supermarkets were analysed for the presence of Z. acidophilus

30 and B. bifidum. All the products contained very low numbers of Z. acidophilus and bifidobacteria.

Several approaches have been taken to improve the viability of probiotic bacteria including selection of various strains on the basis of pH, effect of sugar, and freezing, use of various cryoprotectans, using acid casein hydrolysate and cysteine, encapsulating the bacteria and using acid and low temperature adaptations.

2.11.1 Effect of sugar

High concentrations of sugar or total solids added to milk before fermentation with yogurt starters may result in conditions inhibitory to bacterial growth, leading to long fermentation times and poor acidity development (Tramer, 1973;

Zourari et al, 1992). This is due to both adverse osmotic effects of the solutes in milk and low a^ (Larsen and Anon, 1989, 1990; Zourari et al, 1992). Influence of water activity on the fermentation of yogurt made with exttacellular polysaccharide producing or non-producing starters were studied. The authors reveal that glycerol reduced aw of the milk considerably, but it was less inhibitory to acid production than sucrose. The ropy strains did not show any advantage over non-ropy ones when they were used to produce yogurt in milk with reduced aw their performance was similar to non-producer strains in terms of lactic acid production.

31 Effect of water activity of milk on acid production by S. thermophilus and Z. delbrueckii ssp. bulgaricus was studied by Larsen and Anon (1989). The rate of acid production by Z. delbrueckii ssp. bulgaricus and S. thermophilus were measured at temperature between 30°C and 50°C in milks, which had been adjusted from 0.992 to 0.943 aw with glycerol. Optimal temperatures of acid production were unaffected by change of aw. Minimum aw for acid production was always lower when aw of milk was adjusted with glucose or sucrose than with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity although not related to the type of humectant, did depend on aw of the medium and was directly related to the extent of growth. Acid production appeared to be related to the presence and extent of growth at all of aw levels studied.

2.11.2 Freeze injury

Freezing is one of the main end points in manufacturing of fermented frozen dairy desserts. Intracellular ice formation during freezing has been described for many cellular systems, and the rate at which ice is formed determines the type of freezing damage suffered by cells (Mazur, 1970; Toner et al, 1993). Cells can be injured during freezing by physical factors such as ice crystal formation and dehydration. At high freezing rates, inttacellular freezing occurs, leading to cell damage mainly by ice crystal formation. However, at low freezing rates,

extracellular ice formation predominates, leading to intracellular dehydration

(Mazur, 1970). 2.11.3 pH and acidity

As the bacteria ferment lactose to lactic acid, tiie buffering-capacity of the growth medium is eventually overcome and the pH drops until acidic conditions become unfavourable for the bacteria and acid production stops usually around pH 4.6-4.7

(Thunell et al, 1984). According to Harvey (1960) starter growtii in an environment below pH 5.0 results in reduced enzyme activity. If bacteria remain at low pH for extended periods of time, a steady decrease in acid producing activity results and increase amounts of starter must be used to form comparable amounts of lactic acid over a given period of time (Thunell et al, 1984). The survival of microorganisms is effected by low pH of the environment (Shah and

Jelen, 1990). The populations of Z. acidophilus decreased rapidly at pH 2.0; however, there was no decrease in the number of viable cells at pH 4.0 (Hood and

Zottola, 1988). S. thermophilus is not as acid tolerant as Z. delbruekii ssp. bulgaricus.

The major inhibitory substances produced by yogurt bacteria are claimed to be acid and hydrogen peroxide (Lankaputhra and Shah, 1994; Shah et al, 1995).

The pH of yogurt may decrease to as low as 3.7 during storage (Kolars et al,

1984; Shah, 1994; Shah et al, 1995), which may be detrimental to bifidobacteria as the growth of bifidobacteria is significantiy retarded below pH 5.0

(Lankaputhra and Shah, 1995). Similarly hydrogen peroxide produced by yogurt organisms, especially by Z. delbruekii ssp. bulgaricus, may affect the viability of bifidobacteria and the presence of hydrogen peroxide in acidic conditions, such

33 as found in fermented dairy products, may cause synergistic inhibition of bifidobacteria.

Survival of bifidobacteria during refrigerated storage in the presence of acid and hydrogen peroxide was studied by Lankaputta et al. (1996 a, b). The autiiors concluded that the viability of 6 strains ofbifidobacteria (B. bifidum 1900 and

1901, B. adolescentis 1920, B. breve 1930, B. longum 20097 and B. thermophilum 20210) decreased with pH. The presence of acid and hydrogen peroxide resulted in further decrease in the viable counts of these 6 sttains of bifidobacteria, indicating a synergistic inhibition of bifidobacteria in the presence of acid and hydrogen peroxide. The remaining 3 strains of bifidobacteria (B. infantis 1912, 5. longum 1941 and B. pseudolongum 20099) survived well in the presence of acid and hydrogen peroxide.

The effect of pH on survival of Z. acidophilus and B. bifidum in frozen fermented dairy desserts was studied by Laroia and Martin (1991). Z. acidophilus and B. bifidum were incorporated into frozen fermented dairy desserts and their survival during frozen storage was determined before and just after freezing and at 2 weeks interval during 8 week storage at -29°C. Z. acidophilus and B. bifidum survived the freezing process and frozen storage in pH 5.6-5.8 in frozen fermented dairy products for 8 weeks. B. bifidum did not survive well in the fermented product at pH 3.9-4.6.

34 Frozen yogurt can have a widely variable level of acidity depending on manufacturers. The soft and hard frozen yogurts were observed to have mean acidities of 0.25-0.32%, respectively. These acidities are considerably higher than ice-cream mix and are about one-quarter of tiie acidity normally associated with fresh yogurts.

The viability of Z. acidophilus and B. bifidum in soft-serve frozen yogurt was studied by Holcomb et al. (1991) to determine if frozen yogurt would be an appropriate vehicle for these two organisms. Bile and acid tolerance of the two organisms, both before and after freezing in yogurt mix was determined. These organisms were able to survive and grow after freezing and both were found to grow in up to 0.45% bile salts before and after freezing. The two organisms were able to survive and grow at pH 5.4 before and after freezing in yogurt mix.

However, the authors found only Z. acidophilus to be able to survive and grow after exposure to O.OIN HCl for 2 h at 37°C.

35 2.12 Approaches taken to improve the viability

2.12.1 Use of cryoprotective agents in improving the viability of probiotic bacteria

Bacteria differ in their susceptibility to freezing and tiiawing. Cryoprotective agents provide protection against freeze-injury. Cellular damage is most likely due to elevated solute concenttation (which causes cell dehydration) and membrane destruction by inttacellular ice crystals. The addition of cryoprotectants results in greater survival rate during freezing and frozen storage by binding water and inhibiting either intracellular or extracellular ice crystal formation. The presence of most cryoprotectants results in formation of extracellular amorphous ice glass instead of crystalline ice upon freezing. These agents may include sorbitol, sucrose, sodium tripolyphosphate, glucose, xylitol,

glycerol, raffinose and maltodextrin (Thunnell et al, 1984).

The effect of various cryoprotectants on the viability of freeze-dried lactic acid bacteria has been studied by Valdez et al (1985). Adonitol produced the smallest

changes in water content at various times during drying and allowed the highest

rate of survival. Use of p-glycerophosphate as a cryoprotective agent gave

greatest protection to Z. acidophilus.

Kilara et al. (1976) reported that the extent of crvoprotection provided by any

aiven agent varied from culture to culture. Malt exttact and milk solids as the

cryoprotective agents resulted in tiie survival ratio of 58 and 80% for Z.

delbrueckii ssp. bulgaricus, respectively, while with Z. acidophilus the same two cryoprotectants provided 89% and 48% survival. powder improved viability of Z. delbrueckii ssp. bulgaricus and pectin that of 5. thermophilus. For improved viability of probiotic and yogurt organisms, a combination of cryopotectants may be required.

2.12.2 Use of microencapsulation

Microencapsulation is a process where the cells are retained witiiin the encapsulating membrane in order to reduce the cell injury or cell death.

Microencapsulation tends to stabilise cells, potentially enhancing viability and stability in the production, storage and handling of lactic cultures (Kim et al,

1988). In some food formulations, immobilised cells are more active than free cells (Kearney et al, 1990). Microencapsulation of lactic starter cultures is of interest to the dairy industry since it improves the control of the fermentation process (Champagne and Cote, 1987; Champagne, 1990).

Two-phase dispersion process for the production of biopolymer gel beads, the effect of various parameters on bead size and their distribution was studied by

Audet and Lacroix (1989). The authors concluded that the two-phase dispersion process could be effective in large-scale production of biocatalysts with high mechanical resistance and high productivit>' when used in fermentation process.

Covalent stabilisation of alginate gel for the enttapment of living whole cells was studied by Staffan et al. (1981) employing three methods. In the first method performed, alginate beads containing entrapped ceils were treated with

37 polyethyleneimine followed by glutaraldehyde. In the second metiiod alginate sol was treated with carbodiimide and n-hydroxysuccinunide and mixed with cells and extruded into calcium chloride solution. In the third method the alginate was treated with periodate. The beads were subsequently cross-linked with polyethyleneimine.

Lactococcus lactis released from calcium alginate beads was studied by

Champagne et al. (1988). The authors concluded that ethanol treatment or heating of beads appears to be one of the most promising methods for maintaining acidification activity while minimising viable cell release due to loosely

entrapped cells near the surface of the alginate beads.

Improving survival of culture bacteria in frozen desserts by microentrapment was

studied by Sheu et al. (1993). The authors concluded that the death of yogurt

bacteria in frozen desserts reduces the potential to produce health benefits.

Freezing with agitation kills a large portion of added lactobacilli. Loss of viable

cells was markedly decreased by microenttapment of cells in 3% calcium alginate

beads. Addition of 6% glycerol ftirther enhanced the viability of entrapped

lactobacilli and addition of glycerol did not affect overrun, texture, or body

characteristics of frozen ice milk. Therefore, addition of similar enttapped yogurt

bacteria to frozen dairy desserts would be advantageous to provide desirable

marketing and health benefits.

38 No reduction in cell umber was observed when lactic cultures were immobilised within alginate beads (Champagne and Cote, 1987; Kolot, 1988). Immobilisation prevents the cultures from phage contamination (Champagne et al, 1988;

Steenson et al, 1987). Immobilisation unproves culture stability (Champagne and Boyaval, 1986). Lactic cultures have been previously immobilised by k- carrageenan and locust bean gum beads. The advantage of immobilisation using natural polymers is that the reagents are non-toxic and the procedures are gentle to the microorganisms (Lacroix et al, 1990).

Lactococcus lactis ssp. cremoris was microencapsulated within alginate(alg)/poly-L-lysine (PLL), nylon or crosselinked polyethyleneimine (PEI) membranes. Toxic effects were observed with solvents and reagents used in nylon and PEI membrane formation. Alg/PLL encapsulation resulted in viable and active cell preparations, which acidified milk at a rate proportional to the cell

o concentration, but at rates less than that of free cell preparations. At 4 x 10 cfii per ml milk, encapsulated cells took 17% longer incubation time than free lactocci to reduce the pH of milk to 5.5. Similar activities of free and microencapsulated cells may be attained at higher cell concentrations. The authors observed the rate of lactic acid production of approximately 2 Mmol/h using an encapsulated cell concenttation of 4 x 10^ cfli/ ml (Larisch et al, 1994).

Viability and acidification activity of Lactococcus lactis ssp. lactis was demonstrated by Hyndman et al. (1993) by immobilisation within crosslinked gelatine microcapsules. Contact with the cross linking agent, toluene diisocyanate, was minimised by a reduction in tiie reaction agent concenttation to

34 mol dm" during capsule production and by minimising tiie surface to volume ratio of the microcapsules. A low viscosity oil (silicone) used in tiie dispersion step during microcapsule formation reduced the surface to volume ratio by 45%, yielding a 44% increase in activity over capsules produced in higher viscosity oil.

After encapsulation, activity and viability of the immobilised culture was increased by cell growth within the capsules.

2.12.3 Use of micronutrients

Probiotic bacteria grow slowly in milk due to lack of proteolytic activity. Dave and Shah (1997a, b; 1998) and Klaver (1993) have shown that milk supplemented with peptides and amino acids such as cysteine improved the survival of bifidobacteria. Cysteine is a sulfiir containing amino acid that is incorporated into agar media for the growth ofbifidobacteria (Laroia and Martin 1991). Cysteine is also known for lowering redox potential and addition of this amino acid could improve viability of anaerobic bifidobacteria. Similarly acid casein hydrolysate is a product that is made from hydrolysis of casein and is rich in amino acids and peptides. Thus, acid casein hydrolysate and cysteine may provide growth factors essential for the growth of probiotic bacteria and could improve their viability.

40 2.12.4 Acid and low temperature adaptations

Acid and low temperature adaptation is a novel method where the cells are exposed to a particular mild acidic environment followed by exposure to sub­ lethal acidic environment and frozen for various time and temperature combinations in order to determine whether any proteins are released during freezing. These proteins help the organisms cope under sttessful conditions.

While the physiology, biochemical and genetic mechanisms of many of these responses have not been delineated, what is clear is the following. When exposed to a mild dose of stress, microorganisms may adapt to the stress, thus developing tolerance or resistance to stronger doses of stress. Also it is becoming increasingly clear that the exposure and subsequent adaptation to one stress can confer resistance to other different stress. Most microorganisms must accommodate to a variety of changing conditions and stress in their environment in order to survive. Because of the impact of temperature on all reactions, cell adaptations to fluctuations in temperature are possibly the most common. In many organisms specific sets of cold shock proteins are induced upon abrupt shifts to colder temperatures. While this cold shock response has not been fully delineated, it appears to be adaptive and may function to promote the expression of genes involved in translation when cells are displaced to low temperamres.

The cold shock response oi Escherichia coli has been extensively studied and the major cold shock protein CspA appears to be involved in the regulation of response. Upon cold shock, the induction of CspA and its counterparts in most microorganisms studied is prominent, but transitory. Studies of this response in some psychotropic bacteria have reported constitutive synthesis and continued

41 synthesis during cold temperature growth of CspA homologous (Berry and

Foegeding, 1997).

Freezing is one of the main factors in manufacturing of fermented frozen dairy desserts. Intracellular ice formation during freezing has been described for many cellular systems, and the rate at which ice is formed determines the type of freezing damage suffered by cells (Mazur, 1990; Toner et al, 1993). Cells can be injured during freezing by physical factors such as ice crystal formation and dehydration. At high freezing rates, inttacellular freezing occurs, leading to cell damage mainly by ice crystal formation. However, at low freezing rates, extracellular ice formation predominates, leading to intracellular dehydration

(Mazur, 1990). The pretreatment of cells with a mild stress induces higher sttess tolerance and in some cases this confers cross protection to other, different types of stress (Mager and Ferreira, 1993). It has been assumed that different sttess conditions act through a variety of effects in cells, such as generation of abnormal or denatured proteins, internal acidification, alterations in the cytoskeleton or modulation of second-messenger levels, to produce signals that can provide coverage and stimulate more general stress-responding system (Chowdhury et al.

1992; Coote et al, 1991; Craig and Gross 1991). The freeze-thaw stress response of yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nuttitional state via the Ras-cyclic AMP signal transduction pathway as studied

by Jong et al (1997). The authors concluded that the ability of cells to survive

freezing and thawing is expected to depend on the physiological conditions

experienced prior to freezing. They examined factors affecting yeast ceil survival

42 during a freeze thaw stress, including those associated with growth phase, requirement for mitochondrial functions and prior sttess tteatment. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing was the main determinant of freeze-thaw damage.

The initial discovery of this response was made by Jones et al. (1987) in E. coli

growing at 37°C followed by a reduction in temperatin-e to 10°C. The growth was

halted for approximately 4 hours before exponential growth was resumed (Jones

et al. 1987; Ng et al, 1962). During this lag period, there is an induction of some

16 proteins, called the cold shock proteins (Jones et al. 1996; La Teana et al,

1991) Lelivelt and Kawula (1995) and Jones et al. (1992) further examined the

relationship of stringent control and the cold shock response and found that the

induction of the cold shock response is negatively regulated by (p) ppGpp

(guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-

diphosphate). The induction of the sttingent response, and therefore increased

(p)ppGpp, in wild-type E. coli prior to a temperature downshift resulted in

decreased synthesis of many transcriptional and translational proteins decreased

induction of many of the cold shock proteins. When a real spot mutant, which

cannot synthesize (p)ppGpp, was shifted from 24°C to 10°C, increased synthesis

of ttanscriptional and ttanslational proteins and greater induction of the cold

shock proteins was observed. In E. coli following a shift to 10°C cold shock

proteins are ttansiently synthesised at rates 2 to 10 fold greater than rates at 37°C

(Jones et al, 1987). However one cold shock protein CspA is more prominently

43 induced and because of this it has been designated the major cold shock protein

(Goldstein et al, 1990). Cold shock responses and cold shock proteins have been demonstrated in many other organisms, including psychrottophic and psychrophilic microorganisms. Cloutier et al. (1992) compared the cold and heat shock protein synthesis of three psychrotrophic and three mesophilic sttains of rhizobia. In their examination of cold shock protein synthesis the rhizobia were shocked from their respective optimal temperature to various cold temperatures according to their individual thermo-adaptation characteristics, such that the temperature ranges of the cold shocks were equivalent for different isolates.

These workers found that both psychrottophic and mesophilic rhizobia produced similar numbers of cold shock proteins, thus concluding different thermo- adaption levels were not reflected in the absolute number of cold shock proteins.

Julseth and Inniss (1990) examined the cold shock response of the psychrotrophic yeast Trichosporon pullulans and found that 26 cold shock proteins were induced upon a shift from 21 to 5°C. Only 6 of the 26 cold shock proteins were induced upon a shift from 15 to 5°C; these data are in line with observations in E. coli

(Jones et al, 1992). The magnitude of the induction is dependent upon the range of the temperature shift ie the greater the range of the temperature shift, the more pronounced the induction. Some cold adapted bacteria have been shown to produce both cold shock proteins and cold acclamation proteins (Berger et al,

1996; Roberts and Inniss, 1992). The results observed in yeast T. pullulans showed that the greater number of cold shock proteins were produced by a greater temperature shift. The induction of nine, seven and five cold shock

44 proteins was observed upon cold shocks from 20 to 0, 5 and 10°C (Julseth and

Inniss, 1990).

Acid tolerance in Listeria monocytogens such as the adaptive acid tolerance response (ATR) and growth-phase-dependent acid resistance was studied by

Davis et al. (1996). Z. monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress. Maximum acid resistance was seen when the organism was exposed to pH 5.0 for 1 hour prior to the challenge at pH 3.0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4.0 to 6.0. One hour adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of ATR required de novo protein synthesis. Chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Z. monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH dependent ATR seen during exponential growth.

Acid adapted survival of Bifidobacterium breve against environmental stress was studied by Lee et al. (1995). The authors concluded that acid adapted cells survived better than non-adapted cells. Acid tolerance in Leuconostoc oenos and isolation and characterisation of acid-resistance mutant was studied by Drici et al.

(1996) and found that the acid adapted cells survived better than non-adapted cells. Acid adaptation of Z. monocytogenes and their survival in acidic foods and during milk fermentation was studied by Gahn et al (1996). The authors

45 concluded that the ATR mechanism whether constimtive or induced could greatly influence the survival of Z. monocytogenes in low pH food environment.

Adaptive acidification tolerance response of Salmonella typhimurium was studied by John and Holly (1990). The authors concluded that logarithmically grown cells (pH 7.6) shifted to mild acid (pH 5.8) for one doubling as an adaptive procedure were 100 to 1000 times more resistant to subsequent sttong acid challenge (pH 3.3) than were unadapted cells shifted directly from pH 7.6 to 3.3.

This acidification tolerance response required protein synthesis and appears to be a specific defence mechanism for acid. No cross protection was observed for hydrogen peroxide, or heat shock. The Mg2-H--dependent proton-translocating

ATPase was also found to play an important role in acid tolerance.

Most of the dairy products in Japan, United States and Europe contain a mixed culture of bifidobacteria and Z. acidophilus. In spite of all the commercial products available, viability of probiotic microorganisms is still a major problem.

Hence, development of fermented frozen dairy dessert with high numbers of viable Z. acidophilus and bifidobacteria is needed in order to enhance the export potential of the product because of improved viability and shelf life.

46 3.0 MATERIALS AND METHOD

3.1 Sources of chemical, media and equipment

3.1.1 Chemicals and media

All chemicals were obtained from BDH Chemicals Company (Corporate Avenue,

Rowville, Australia) or from Sigma Chemicals Company (Castle Hill, New South

Wales, Australia).

3.2 EQUIPMENT

3.2.1 Pasteuriser

A pasteuriser (APV) was used in pastemrising milk for yogurt manufacture (95°C for 15 min) and for ice cream base mix (73°C for 15 sec).

3.2.2 Ice cream freezer

A continuous ice cream freezer (Crepaco Freezer, Model KM 240; USA) was used to make pilot scale (1000 L) fermented frozen dairy desserts with 100% overrun.

A bench top ice cream freezer (2 L size) (Kenwood Ice Cream Freezer (Model Im

300 PK 185/w, Kenwood Appliances, Italy) was used to make laboratory scale fermented frozen dairy desserts with 18% overrun.

47 3.2.3 Laminar flow

A laminar airflow (Gelman Sciences, HWS series Vic. Austtalia) was used as work-station for plating and inoculating culture bacteria.

3.2.4 Anaerobic jar

Anaerobic jars were used to enumerate Z. delbrueckii ssp bulgaricus, L. acidophilus, bifidobacteria and Z. casei. Anarobic jars having a capacity of sixteen and forty-eight plates were used. To create anaerobic conditions catalysts and gas generating kit (BR 38) were used according to the manufacturer instructions.

3.2.5 Centrifuge and microcentrifuge

Large volumes (100 to 1000 mL) of samples were centrifuged using Beckmann refrigerated J2-HS (Beckmann Instruments Inc., Palo Alto, CA, USA). For medium volumes (10 to 100 mL) of samples Bekmann refrigerated centrifuge and for small volumes microcentriflige (Beckmann) were used.

3.2.6 Freeze drier and freeze drying of bacteria

A freeze drier was used for freeze drying microencapsulated probiotic bacteria for incorporation into fermented frozen dairy desserts. Microencapsulated probiotic bacteria was placed in petridishes with lids and frozen at -25°C for 24 h using a freeze drier (Dynavac Engineering Pty. Ltd. Inc., Melboume, Australia).

The freeze dried microencapsulated probiotic bacteria were kept in the same petridishes and sealed with parafilm and stored in a freezer at -18°C until used.

48 3.2.7 Compound microscope and examination of beads

Microencapsulated calcium alginate beads were stained with safranin and placed on a glass slide. Diameters of calcium alginate beads were measured with an eyepiece micrometer using an optical microscope (CHT, Olympus Optical Co.

Ltd., Japan) at a magnification of 100. At least 80 randomly selected beads were measured for each sample.

3.2.8 Scanning electron microscope and scanning electron microscopy

Scanning electron microscopy was employed to examine the extemal and intemal appearance of calcium alginate beads. The beads were freeze-dried using a freeze drier. For extemal structure intact beads were attached to the aluminium stubs using conductive carbon paint (CCP) (Probing and Structure, now Pro Science

Tech., Melboume). For intemal stmcture the freeze-dried beads were mechanically broken using fine tweezers and mounted using CCP as described above.

The specimens were coated with 10 |im gold using Edwards Sputter Coater.

Extemal and intemal structures were observed using scanning electron microscopy (SEM) (Philip 500 at 20 kv, Holland).

49 3.3 Bacterial culture and maintenance

3.3.1 Lactic acid bacteria and probiotic bacteria

Pure strains (yogurt and probiotic organisms) were collected from the Division of

Food Science and Technology, Commonwealth Scientific and Industrial Research

Organisation (CSIRO), Highett, Victoria, Austtalia; Mauri Laboratory (now.

Gist-brocades) and Chr. Hansen. The organisms were maintained at -20°C in

12% reconstituted skim milk (RSM) supplemented with 1.0% glucose and 0.5%

yeast extract. L-cysteine hydrochloride (0.05%) was added for the maintenance

of bifidobacteria. Working cultures were propagated using frozen stock cultures.

Sterile RSM (10 mL) portions were propagated by inoculating 1% of each culture

and incubated at 37°C for 18 h. All cultures were activated by propagating into

sterile RSM on a weekly basis for a maximum of twenty propagations. After

which new working cultures were propagated using frozen stock culture. This

was done to minimize the loss of any plasmid associated inhibitory activities.

3.4 Microbiological analyses

S. thermophilus was enumerated on S. thermophilus agar (ST agar; 10.0 g of

tryptone, 10.0 g of sucrose, 5.0 g of yeast extract and 2.0 g K2HPO4 in 1 L of

distilled water) by incubating the plates aerobically at 37°C for 24 + 3h (Dave

and Shah, 1996). Counts of Z. delbrueckii ssp. bulgaricus were determined on

MRS agar (Oxoid, Basingstoke, Hampshire, UK) adjusted to pH 5.2 and

incubated anaerobically at 43°C for 72h. MRS-salicin and MRS-sorbitoI agars

were used for enumeration of Z. acidophilus (Dave and Shah, 1996; Lankaputra

50 et al, 1996a, b). Counts of bifidobacteria were estimated on MRS-NNLP

(nalidixic acid, neomycin sulfate, lithium chloride, paromomycin sulfate) agar

(Laroia and Martin, 1991). The enumeration was carried out using the pour plate

technique. Plates containing 25 to 250 colonies were enumerated and recorded as

colony forming (cfu) units per gram of culture. All the experiments and analyses

were repeated at least twice. The results presented are averages of two replicates.

3.5 Commercial products used for enumeration of Z. casei

Nine samples of commercial yogurts and fermented milk drinks claimed to

contain S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus,

bifidobacteria and/or Z. casei were analyzed to validate the selective medium (LC

agar) in composite culture conditions. The products analyzed were: Mundella

(Mundella Foods Private Limited, Westem Austtalia, Austtalia) claimed to

contain S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus,

bifidobacteria and Z. casei; Jalna skim milk yogurt and Jalna mild continental

yogurt (Jalna Dairy Foods Private Limited, Thomastown, Australia) claimed to

contain S. thermophilus, L delbrueckii ssp. bulgaricus, L. acidophilus,

bifidobacteria and Z. casei; Yoplait yoplus natural yogurt (National Foods

Limited, Morewell, Australia) claimed to contain S. thermophilus, L. acidophilus,

and bifidobacteria; Nestle LCI (Nestle (Australia) Private Limited, Mulgrave,

Austtalia) claimed to contain S. thermophilus, L. delbrueckii ssp. bulgaricus, and

Z. acidophilus; Yakult (Yakult (Austtalia) Limited, Dandenong, Australia)

claimed to contain Z. casei Shirota sttain; Maeil (imported by KOZ Worldwide,

Queensland, Australia) claimed to contain Z. casei; Bulla drinking yogurt (Regal

51 Cream Products Private Limited, North Melboume, Austtalia) claimed to contain

S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria;

Vaalia (QUF Industries Limited, South Brisbane, Austtalia) claimed to contain S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei ssp. GG. All experiments and analyses were carried out in duplicate. The results presented are averages of duplicate experiments.

3.6 Screening of strains of yogurt and probiotic bacteria for selecting strains that survive freezing

The organisms were propagated three times before using for freeze tolerance studies. Ten millilitre of sterile RSM was inoculated with 2% culture followed by

incubation at 37°C till the pH 4.5 was attained. The samples were transferred to a refrigerator till the temperature dropped to 4°C followed by freezing for 4 h at -

25°C using a glycol bath. The temperature dropped to -25°C in 15 min. Samples were thawed using tap water (~20°C). Bacterial counts before and after freezing were determined. The organisms resistant to freeze injury were used for making

fermented frozen dairy desserts and the viable counts of yogurt and probiotic organisms were monitored at weekly intervals for 12 weeks.

3.7 Analysis of samples to determine viability of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and/or L. casei in commercial fermented dairy products

Twenty-six commercial yogurts and fermented milk drinks claimed to contain S.

thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and/or

Z. casei were analysed for their population levels. The products that were

52 analysed included Mundella containing 5. thermophilus, L delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei. (Mundella Foods Private

Limited, Westem Australia, Austtalia); Jalna skim milk yogurt and Jalna mild continental yogurt containing S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei. (Jalna Dairy Foods Private Limited,

Thomastown, Australia); Yoplait yoplus, natural yogurt containing S. thermophilus, L. acidophilus, and bifidobacteria (National Foods Limited,

Morewell, Australia); Nestle LCI containing S. thermophilus, L. delbrueckii ssp. bulgaricus, and Z. acidophilus (Nestle (Austtalia) Private Limited, Mulgrave,

Australia); Yakult containing Z. casei Shirota strain (Yakult (Australia) Limited,

Dandenong, Australia); Maeil containing Z. casei (imported by KOZ Worldwide,

Queensland, Australia); Bulla drinking yogurt containing S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria (Regal Cream

Products Private Limited, North Melboume, Australia). Vaalia, containing S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei GG (QUF Industries Limited, South Brisbane, Austtalia). All experiments and analyses were carried out in duplicate. The results presented are averages of duplicate experiments.

3.8 Effects of various sugar levels on survival of probiotic bacteria.

Ten millilitre of RSM containing 8 and 16% sucrose were inoculated with 2% each of Z. acidophilus and bifidobacteria and incubated at 37°C till the pH of 4.5 was attained. Samples were ttansferred to a refrigerator till the temperature dropped to 4°C and then frozen at -25°C for 4 h using a glycol bath. The

53 temperattire dropped to -25°C in 18 min. Samples were thawed usmg tap water

(~20°C). Bacterial counts before and after freezing were determined as before.

3.9 Effects of acidic and freezing conditions on survival of yogurt and probiotic bacteria.

Samples of RSM were adjusted to pH 6.2 (conttol), 5.0, 4.5 and 4.0 with lactic acid (BDH Laboratory Supplies, Poole, England). One millilitte of each culture of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, and

Bifidobacterium spp. was added to 9 mL of the pH adjusted RSM in separate test tubes. The test tubes were kept in a refrigerator at 4°C for 5 h followed by freezing at -25°C for 4 h using a glycol bath. The temperature dropped to -25°C in 15 min. Samples were thawed using tap water (-20°C). Bacterial counts before and after freezing were determined as before.

3.10 Influence of water activity on fermentation of probiotic yogurt and on viability ofyogurt and probiotic bacteria

Five batches ofyogurt (1 L) batch size were made using 12% RSM containing 0,

4, 8, 12 and 16% sugar levels. Yogurt made without added sugar was used as control (batch 1). The mix was heated at 85°C for 30 min, cooled to 42 C and yogurt bacteria (S. thermophilus ST WJ7 and Z. delbrueckii ssp. bulgaricus LB

WJ7) and probiotic bacteria (Z. acidophilus LA 2415 and bifidobacteria BB

1941) were added at the rate 0.5% of each, mixed for 5 min using a magnetic stirrer and filled in 100 mL plastic containers and incubated at 42*^C till the pH

4.5 was attained. The yogurt was ttansferred to a walk-in-cooler and kept over night at 4°C.

54 3.11 Survival ofyogurt and probiotic bacteria in fermented frozen dairy desserts made using a bench top ice cream freezer

Six different batches (Bl-6) of fermented frozen dairy desserts were made as follows:

For batch Bl, yogurt was made by using ST 2010, LB 2501, LA 2415 and BB

20099. Similarly, B2 was made with ST 2008, LB 2515, LA 2401, BB 1912 and

B3 with ST/LB Wj7, MjLAl, BDBB2. B4, B5 and B6 were made with ST 2010,

2008, ST WJ7 and LB 2501, 2515, LB WJ7 only and probiotic strains LA 2415,

2401, MJLAl, BB 20099, 1912, BDBB2 were incorporated just before freezuig.

Yogurt and base mix were added at a ratio of 45:45 and 10% of liquid sucrose

syrup (65%) was added, followed by freezing the contents using a bench top

Kenwood ice chef ice cream freezer (Model Im 300 PK i85/w, Kenwood

Appliances, Italy). The pH of the final product ranged between 4.51 and 4.59.

3.12 Survival of yogurt and probiotic bacteria in fermented frozen dairy desserts made using a continuous freezer

Two different batches of fermented frozen dairy desserts each of 1000 L were made on a pilot scale using an ice cream freezer. For batch 1, yogurt was made by using ST 2008, LB 2501, LA 2401, and BB 20099. Similarly, batch 2 was made with ST/LB Wj7, MjLal, and BDBB2. Yogurt and base mix were added at a ratio of 45:45 and 10% of sucrose syrup (65%) was added, followed by freezing the contents using a continuous ice cream freezer (Crepaco Freezer, Model KM

240, U.S.A). The pH of the final product ranged between 4.51 and 4.59.

55 3.13 Use of acid casein hydrolysate and cysteine in manufacture of fermented frozen dairy desserts

This study investigated the use of acid casein hydrolysate and cysteine in improving the viability of Z. acidophilus and B. bifidum noting that

Bifidobacterium spp is a strict anaerobe and Z. acidophilus is considered to be microaerophilic, although the latter organism grows well under anaerobic conditions. Effect of cysteine as an oxygen scavenging agent was investigated. L-

Cysteine has been found to be essential for the growth of bifidobacteria (Laroia and Martin, 1991).

Fermented frozen dairy desserts were made according to the method of Ravula and Shah (1998b). Yogurt and base mix were prepared separately and blended at a rate of 45% of each and 10% sucrose syrup was added. The blend was then frozen using a bench top ice cream freezer (Model Im 300 PK 185/w, Kenwood

Appliances, Italy).

Three batches of yogurt (3L) were made with homogenised and pasteurised milk

(1% fat) supplemented with 2% skim milk powder. The mix was heated at 85°C for 30 min, cooled to 42°C and divided into three lots. Acid casein hydrolysate

(0.5g/L) (Sigma Chemical Co. St. Louis, MO) and L-cysteine (0.05g/L) (Sigma) were added to the two batches of heated and cooled yogurt mix to study the effect of these growth factors on viability of probiotic bacteria in fermented frozen dairy desserts. The third batch was made with milk supplemented with 2% skim milk powder only (control). Yogurt bacteria (S. thermophilus STWJ7 and Z.

56 delbrueckii ssp. bulgaricus LBWJ7), and probiotic bacteria (Z. acidophilus LA

MJLAl and bifidobacteria BB BDBB2) were added at the rate of 0.5% each and the inoculated mix incubated at 42°C till pH 4.5 was attained. The yogurt was transferred to a cold room till the temperature decreased to 4^C. The yogurt and base mix were mixed at a rate of 45% of each and 10% of liquid sucrose (65%) was added. All the three batches of fermented frozen dairy desserts were made using a bench top ice cream freezer with an overrun of 18%.

3.14 Assessment of effects of cryoprotectants for improving viability of Z. acidophilus and Bifidobacterium spp.

Effects of cryoprotectants such as fiiictose, glucose, com symp solids, vegetable gum, glycerol and pectin etc. for improving the viability of Z. acidophilus and

Bifidobacterium spp. was assessed. Products were made from fermenting a mix with organisms selected from previous studies, containing variable amounts of cryoprotectants and mixed with a pasteurised base containing 16% sugar and

10% milk solids not fat and whipped air while freezing to achieve 90 to 100% overrun. Viable numbers of probiotic and yogurt organisms were enumerated at weekly intervals for 12 weeks

3.15 Compositional analysis

3.15.1 Determination of pH and titratable acidity

The pH values of yogurt, fermented frozen dairy desserts, milk samples, broth samples, and agar samples were measured at 22-25°C using a pH meter (Model

420 A, Orion Research Inc. Boston, U.S.A.). The pH meter was calibrated between pH 7.0 and 4.0 using a standard buffer solution according to the

57 manufacturer instructions. The titratable acidity was determined after mixing a sample with 10 mL of hot distilled water and titrating with O.IN NaOH using

0.5% phenolphthalein indicator to the end point of faint pink colour that persisted for at least 30 seconds.

3.15.2 Determination of sugar

Two milliliters of sugar solution containing between 10 and 70 ^1 of sugar is pipetted into a colorimetric tube, and 0.05 ml of 80% phenol is added followed by rapid addition of 5 ml of concentrated sulfiiric acid. The stream of acid being directed against the liquid surface rather than against the side of the test tube in order to obtain good mixing. The tubes were allowed to stand for 10 minutes, mixed well and placed for 10 -20 minutes in a water bath at 25 to 30° C and the readings are taken. The absorbance values were measured at 490 nm (Dubois et al, 1956).

3.15.3 Determination of protein

Protein content of fermented frozen dairy desserts was determined by the

Kjeldahl method using Kjeltec system and 1002 distillation unit (Tecator ab,

Hoganas, Sweden). One gram of well mixed fermented frozen dairy desserts was measured carefully (avoiding the sample touching the sides of the tube) into digestion tubes and catalyst tablets were added according to the manufacturer instructions into the digestion tube containing the samples. To the tubes, sulfuric acid (12.5 mL) was carefully added using a dispenser and the content was mixed by swirling the tubes with hand (protected with heavy duty gloves) and the tubes

58 were placed in the preheated digester, preset at (420°C) with maximum air flow through the exhaust for 3-5 min. The digestion was continued imtil all samples were digested as indicated by a clear solution and the lack of black residue in the tubes. The digestion time for the most samples ranged between 45 to 55 min. The tubes containing digested samples were removed from the digester and were placed in the cooling stand. After cooling, 50 mL of nitrogen free tap water was added for dilution. The distillation was carried out and the distillates were collected into a receiver flask containing 25 mL boric acid solution. After complete distillation, the content was tittated against 0.05 M H2SO4 until the solution in the receiver flask tum to natural grey.

3.15.4 Determination of total solids

The total solids contents were determined by standard gravimetric method using an oven at 100°C. One gram of well mixed sample was weighed into moisture dish and dried for one hour. The dish was cooled using a desiccator jar containing silica gel. The cooled dish was measured and dried again for another one hour

and weighed and the drying process continued for ftirther one hour. The

difference in the weight of the sample before and after drying was calculated and

percent total solids were obtained.

TS = initial wt. - final vA. x 100 initial wt.

59 3.15.5 Determination of water activity

Water activity was measured using a water activity meter. Three milliliters of the

sample were measured and placed in the chamber at 25°C according to the

instructions of the manufacturer and the results were obtained. Three

determinations were performed and results were presented as average of three

replicates.

3.15.6 Determination of oxygen content and redox potential

The dissolved oxygen in parts per million (ppm) was measured using LC82

Oxygen meter (TPS Pty. Ltd., Brisbane, Australia), after calibrating each time

before use. The redox potential was measured with a platinum electrode (Model P

14805-SC-DPAS-K85/325, Ingold (Mettler Toledo), Urdorf, Switzeriand)

connected to pH meter (model H 18418, Hanna Instruments, Padova, Italy).

3.15.7 Determination of fi-D galactosidase

The P-D-galactosidase activity was determined according to the method of Shah

and Jelen (1990). Ten grams of each product were mixed with distilled water,

blended for 1 min and the final volume made up to 100 mL in a volumetric flask.

One millilitre of the solution was used in the assay. A solution of 0.005M O-

nitrophenyl-P-D-galactopyranoside (ONGP) was prepared in O.IM phosphate

buffer (pH 7.0), and 1 mL aliquots of the diluted samples were incubated with 5

mL ONGP solution for 15 min at 37°C. The reaction was stopped by adding 2.5

mL IM cold sodium carbonate solution. The amount of O-nitrophenol released

was measured with a spectrophotometer at 420 nm. One unit of P-D-

60 galactosidase activity was defined as the amount of enzyme, which liberated one lamole O-nitrophenol from ONGP per min per gram sample at 37°C.

3.15.8 Determination of the growth curve

To achieve maximum number of viable bacteria, it was felt necessary to study the log phase of the organisms. The growth curve was determined using a

spectrophotometer. Probiotic bacteria, Z. acidophilus and Bifidobacterium spp., were propagated three times before inoculating in MRS broth (L-cysteine was

added for bifidobacteria). The inoculated MRS broth was incubated at 37°C and hourly readings of absorbance (OD) were measured by weighing the samples in

cuvettes, using a spectrometer at 450 nm. The pH of the broth was also measured

at hourly basis using a pH meter. A growth curve was plotted to identify the log phase of the organisms to have maximum survival.

3.15.9 Determination of fat content

The total fat content of the samples were estimated using Babcock method

(Atherton and Newlander, 1977).

3.15.10 Detection and quantification of organic acids with HPLC

3.15.10.1 Extraction of organic acids

Four grams of yogurt samples were diluted with 25 mL of O.OIN H2SO4, and

filtered through 0.02-|j.m Teflon filters (Schleicher and Schuell, Dassel,

Germany). The extraction of organic acids was carried out in duplicates.

61 3.15.10.2 HPLC of organic acids

The standards of organic acids were pipetted into HPLC vials through 0.02 ^m millipore filters (Millipore Australia, Lane Cove, Austtalia). Similarly, diluted

samples of yogurts were pipetted into HPLC vials through 0.02 fim filters. The

vials were used in an auto sampler for analysis of organic acids. The

concentration of each acid was determined with Varian HPLC (Varian,

Mulgrave, Australia) using an UV-Vis detector. An Aminex HPX-87H ion

exclusion column (300 x 7.8 mm) (BioRad, North Ryde, Australia) and a mobile

phase of 0.0075 M H2SO4 were used for the analysis. Quantification of organic

acids was carried out by the extemal standard method. Linear regression curves

based on peak heights were calculated for individual organic acids after duplicate

injections. Identifications of acids were based on matching retention times of

standards.

3.16 Media preparation

16.1 Peptone and water diluent

Diluent of peptone and water was prepared by dissolving 15 g of peptone and

water medium (Oxoide, West Heidelberg, Austtalia) in IL of distilled water, the

pH adjusted to 7.0 ± 0.2, followed by autoclaving 9 mL portions at 121°C for 15

min. After autoclaving, peptone and water diluent in McCartney bottles were

stored at room temperature for up to 15 days.

62 3.16.2 Preparation of serial dilutions for pour plating

One gram of well-stirred sample was weighed accurately added to 9 mL of 1.5%) peptone water diluent. The sample and peptone water diluent were vortexed and 1 mL of the dilution was transferred into 9 ml of peptone water diluent using standard sterile pipettes (1 mL) and a series of ten-fold dilutions were prepared.

One millilitre of aliquot was placed in an empty sterile petridish. Melted agar medium (45^C) was poured into the plates followed by gentle mixing and the agar was allowed to solidify.

3.16.3 Streptococcus thermophilus agar (ST agar)

The ST agar was prepared according to the method of Dave and Shah (1996). The ingredients were dissolved in 1 L of distilled water, warmed to 25°C while stirring the medium, using a hot plate and a magnetic stirrer. The pH was adjusted to 6.8 ±0.1, and 6 mL of 0.5% bromocresol purple was added to the medium. Twelve grams of bacteriological agar was added to IL of the medium.

The medium was autoclaved at 121°C for 15 min. Plates in duplicate were incubated aerobically at 37 C for 24 h.

3.16.4 Ml?agar

The Ml7 agar (Amyl Media, Dandenong, Austtalia) was prepared as recommended by the manufacturer. The medium was autoclaved at 121 C for 15 min. Plates in duplicate were incubated aerobically at 37°C for 24 h.

63 3.16.5 MRS-basal, MRS-salicin, MRS-sorbitol, MRS-ribose, MRS-gluconate agars

MRS-basal medium was prepared according to Lankaputhra and Shah (1996) without any sugars and sterilised at Ul'^C for 15 min. The ingredients were dissolved in 1 L of distilled water, warmed to 25°C while stirring the medium, using a hot plate and a magnetic stirrer. Ten millilitres of membrane-filtered sterile solutions of 10%) salicin, sorbitol, ribose or glauconite were added per 90 ml of the basal medium (1% final concenttation) just before pouring the agar medium. Plates in duplicate were incubated anaerobically using anaerobic jars

(Oxoid) at 37°C for 72 h (Dave and Shah, 1996).

3jl6Co MRS-pH modified agar

MRS broth (Oxoid) was prepared according to the manufacturer instructions. The pH was adjusted to 5.2 using 1.0 M HCl. After the broth was prepared, the agar powder was added at the rate of 1.2%, and the medium was autoclaved at 121°C for 15 min. Plates in duplicate were incubated anaerobically at 43°C for 72 h

(Dave and Shah, 1996)

3.16.7 MRS-NNLPagar

The MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate (all from Sigma Chemical Co., Castle Hill, Australia) agar was prepared according to the method described by Laroia and Martin (1991).

The basal medium was MRS agar. Fitter-sterilised NNLP was added to the autoclaved MRS basal medium just before pouring. Filter-sterilised L-cysteine

64 HCl (0.05% final concentration) was also added at the same time to lower the oxidation-reduction potential of the medium and to enhance the growth of bifidobacteria. Plates in duplicate were incubated anaerobically at 37°C for 72 h.

3.16.8 LCagar

The ingredients of LC agar were dissolved in 1 L of distilled water. The pH was adjusted to 5.1 ± 0.1, and 6 ml of bromocresol green and 12 g of agar were added to the medium. The medium was steriHsed at 121°C for 15 min. Ten millilitte of membrane-filtered sterile solution of 10% ribose was added per 90 ml of the medium (1% final concentration) just before pouring the agar medium. Plates in duplicate were incubated anaerobically at 27 C for 72 to 96 h.

65 3.17 Preparation of fermented frozen dairy desserts

3.17.1 Preparation ofyogurt

Pasteurized skim milk was warmed to 40-45 °C in a saucepan using a hot plate and magnetic stirrer and skim milk powder (2%) was added. The milk was heated to 85°C for 30 min and cooled to 43 °C. Yogurt bacteria and/or probiotic bacteria were added at 0.5% level and the mix was incubated at 43°C till the pH reached to 4.5 and the yogurt stored overnight before adding into the base mix.

3.17.2 Preparation of base mix

Pasteurized full cream milk was warmed to 40-45°C in a saucepan using a hot plate and a magnetic stirrer. Sugar (16%) and gelatin (0.3%) were added to milk slowly while stirring. The mix was heated to 85°C for 15 min and cooled to 4°C.

Fresh pasteurised cream was added to make up the fat percentage to 10.5%. The base mix was left overnight for maturing in the cold room before blending with yogurt.

3.18 Preparation of microencapsulation of probiotic bacteria and their incorporation into fermented frozen dairy desserts

Immobilisation of cells were carried out as follows: cultures grown in deMan

Rogosa and Sharpe (MRS) broth were harvested by centrifugation in a refrigerated centtifuge, suspended in broth and the cell slurry added to an equal weight of 3% k-carrageenan solution in sterile distilled water. The mixed solution, with the help a syringe, was added drop-wise into a solution of 3M KCl

66 at 20°C in a 500 mL Buchner flask connected to a supply of nittogen to help maintain anaerobic atmosphere and develop a positive pressure in the flask. The mixture of k-carrageenan and cell slurry was gelled into small beads on contact with the cold KCl solution, the gelled beads were separated by decantation and

lyophilised. The resulting freeze-dried organisms were incorporated into the base

mix followed by mixing and freezing.

3.18.1 Preparation of cell pellet

Microorganisms were propagated three times in sterile reconstituted skim milk

and the fermentation terminated at the log phase. The organisms (2%) from the

third propagation were inoculated in MRS broth and incubated for 18 h and the

broth was cooled to 4°C. The cooled broth was centrifuged using a refrigerated

centrifuge at 4000 RPM (4°C) for 25 min. The supernatant was suspended and

the cell pellet was collected. The cell pellet was washed and resuspended in

Q saline to give 10 cfti/gram.

3.18.2 Entrapment of the microorganisms in beads

Various concenttations of sodium alginate were analysed using a viscometer.

Sodium alginate (3%) was prepared using a volumetric flask. Cells were

microentraped by mixing 50 ml of culture concentrate with 200 ml of 3% (w/v)

sodium alginate. The mixture was added dropwise to 5 parts of vegetable oil

containing Tween 80 (0.2%), stirring at 200 rpm without any evidence of free

aqueous phase. Calcium alginate beads were formed by addition of calcium

chloride quickly but gently down the side of the beaker until water/oil emulsion

67 was broken. The beads were separated and washed by using sterile distilled water by gentle centrifugation (500 rpm).

3.18.3 Effect of surface tension and emulsifier on bead size

Effect of surface tension and emulsifier on bead size were studied. Gel sttength was studied using a viscometer. The surface tension of 3% sodium alginate was varied by addition of various levels of sodium lauryl sulfate (0, 0.1 and 0.5%) and three different concentrations of Tween 80 (0, 0.1 and 0.2%) were added to the

oil phase.

3.18.4 Effect of freeze-drying on beads and viability of cells

The entrapped cells were freeze-dried using a freeze dryer. The beads were

placed in sterile petri-dishes with lids on and freeze-dried for 24 hrs. The beads

were collected and plates were sealed with parafilm and stored for further testing.

Viable counts before and after freeze-drying were enumerated. The size of the

bead was studied.

3.18.5 Solubilization of beads

The entrapped bacteria were released from the gel according to the method of

Ohlson et al. (1979). One millilitte of the bead suspension (Ig in 59 mL water)

was added to 9 mL phosphate solution (concentrations from 0.001 to O.IM)

followed by gentle shaking for 30 min. The pH of the phosphate solutions was

varied from 4.8 to 8.5 by adjusting the ratio of monosodium phosphate and

68 disodium phosphate buffer solutions. The number of released cells was determined by plate count using MRS agar.

3.19 Acid and low temperature adaptations of probiotic bacteria and their incorporation into fermented dairy desserts

Two adaptation conditions were used: one with sublethal pH of 5.5 and the other with lethal pH 3.5. For adaptation studies, the organisms were propagated three time in MRS broth (0.05% cysteine was added for bifidobacteria) at 2% level and incubated at 37^C for 18 h. The fermentation was slowed down by transferring the samples into a refrigerator and the temperature was decreased to 4°C. The initial pH of the sample was assessed with a pH meter and the sample plated for enumeration for viable counts. Ten millilitres of the samples were stored in a freezer for gel-electrophoresis. The pH of the broth (100 ml) was increased to 6.5 using 10% NaOH and the broth kept in a refrigerator for one hour. After 1 h of refrigeration (temperature adaptation), the broth was divided into two portions (A

& B) of 50 ml each and decreased to lethal pH of 3.5. The sample B was kept at

sublethal pH of 5.5 for one hour before dropping the pH to the lethal pH of 3.5.

The samples A and B were maintained at this pH (3.5) for 3 h, followed by

freeze stressing at -20°C for 1 hour. The samples were thawed and the pH

increased to 6.5 with 35% NaOH and allowed to recover the stress at this pH for

one hour and the pH of the samples was decreased to 4.5. to observe any

increased survival in the counts.

69 Similarly bacterial cells were stressed at pH 3.5 as before and freeze sttessed for

2h, 4h, 8h, 12h, 16h or 24h. Studies showed that sample A when adapted to sublethal pH survived better. On this basis, 3 strains each of Z. acdophilus and bifidobacteria were selected and incorporated into fermented frozen dairy desserts. The viable counts were monitored for 12 weeks.

3.19.1 Preparation of buffers and reagents

3.19.1.1 Tris-HCl buffer (1.875M and pH 8.8)

Tris-HCL buffer (1.875M and pH 8.8) was prepared by dissolving 56.8 g of Tris

in 150 mL distilled water. The solution was made up to 250 mL and adjusted to

pH 8.8 with 5N HCl and stored at 4°C for up to four weeks.

3.19.1.2 Tris-HCl buffer (1.25 M and pH 6.6)

Tris-HCl buffer (1.25 M and pH 6.6) was prepared by dissolving 37.8 g of Tris in

150 mL of distilled water. The pH was adjusted to 6.8 with 5 N HCl and the

solution was made up to 250 mL with distilled water.

3.19.1.3 SDS (Sodium dodecyl sulphate) (10%) solution

Ten gram of SDS was dissolved in 85 mL distilled water and the solution was

made up to 100 mL with distilled water. SDS was stored at room temperature up

to four months.

70 Similarly bacterial cells were stressed at pH 3.5 as before and freeze sttessed for

2h, 4h, 8h, 12h, 16h or 24h. Studies showed that sample A when adapted to sublethal pH survived better. On this basis, 3 strams each of Z. acdophilus and bifidobacteria were selected and mcorporated into fermented frozen dairy desserts. The viable counts were monitored for 12 weeks.

3.19.1 Preparation of buffers and reagents

3.19.1.1 Tris-Hcl buffer (1.875M and pH 8.8)

Tris-HCL buffer (1.875M and pH 8.8) was prepared by dissolving 56.8 g of Tris in 150 mL distilled water. The solution was made up to 250 mL and adjusted to pH 8.8 with 5N HCl and stored at 4°C for up to four weeks.

3.19.1.2 Tris-HCl buffer (1.25 M and pH 6.6)

Tris-HCl buffer (1.25 M and pH 6.6) was prepared by dissolving 37.8 g of Tris in

150 mL of distilled water. The pH was adjusted to 6.8 with 5 N HCl and the solution was made up to 250 mL with distilled water.

3.19.1.3 SDS (Sodium dodecyl sulphate) (10%) solution

Ten gram of SDS was dissolved in 85 mL distilled water and the solution was made up to 100 mL with distilled water. SDS was stored at room temperature up to four months.

71 3.19.1.4 Poly-acrylamide gel electrophoresis (PAGE), staimng of the gels, and preparation of running gel

Aliquots of 3.33 mL of stock (30%) acrylamide, 2.00 mL of 1.875 M ttis -HCl

(pH 8.8) and 4.70 mL distilled water, 50 ^l 10% APS, 150 ^il 10% SDS were mixed and degassed for 15 min. Ten microlitre of TEMED was added to the degassed mix quickly. A 3.5 mL quantity was added per gel.

3.19.1.5 Preparation of stacking gel

Aliquots of 0.66 mL of acrylamide (30%) 0.50 mL of 1.25 M tris HCL (pH 6.8) and 3.80 mL distilled water, 10 ^il 10% APS and 50 ^1 10% SDS were degassed for 15 min under vacuum. Five microlitre of TEMED was added to the degassed mix and mixed quickly. The stacking mix was applied on to the top of the running gel and combs were inserted.

3.19.1.6 Staining the gels with coumassie blue

The protein gels were transferred from the gel plates into a fixing solution prepared by adding 4 parts of ethanol, 1 part of acetic acid and 5 parts of H2O.

The gels were left in the fixing solution for 30 min and transferred into a staining solution prepared by mixing 1.5 g of Coomassie blue, 105 ml of 95% ethanol, 30 mL of acetic acid and 165 mL of distilled water. After staining for 30 min in coomassie blue, the gels were transferred into a de-staining solution, which was similar in composition to the fixing solution (4 parts of ethanol, 1 part of acetic acid, and 5 parts of H2O) during de-staining, the gels were transferred into fresh

72 de-staining solutions three times. After de-staining, the gels were ttansferred into a preserving solution prepared by mixing 50 mL of glycerol, 175 ml of 95%) ethanol, 50 mL of acetic acid and the volume made up to 500 mL with distilled water.

3.19.1.7 Staining the gels with silver stain

The protein gels were transferred from the gel plates into fixing solution prepared by adding 4 parts of ethanol, 1 part of acetic acid and 5 parts of dH20. The gels were left in the fixing solution for 30 min and transferred into an incubation solution prepared by mixing 75 mL ethanol, 17 g of sodium acetate, 3 mL dH20,

1.3 ml glutaraldehyde (25% w/v), 0.5g sodiumthiosulphate (pentahydrate) and the volume made up to 250 mL with distilled water. After incubation, the gels were washed with distilled water 3 times and placed in a silver solution prepared by mixing 0.25 g of silver nitrate and 50 |il of formaldehyde in 250 mL distilled water. The gels were transferred into developing solution (made with 6.25 g

Na2C03 and 25|.il of formaldehyde in 250 mL of distilled water) after 40 min of silver reaction. The gels were then placed in stop solution prepared by dissolving

3.65 g of EDTA-Nai dehydrate in 250 mL distilled water. The gels were then placed in preserving solution prepared with 20 ml of glycerol made up to 200 ml of distilled water.

3.19.1.8 Gel preparation and electrophoresis

The frozen cells extract was thawed at room temperature and the protein contents were determined using Lowrey assay. The extracts were diluted with double

73 distilled water to achieve a final concentration of 10 p,l/niL, and the samples were mixed with loading buffer and boiled for 5 min in Eppendorf tubes. The boiled samples were briefly centrifuged and loaded in to the gel along with the molecular size standards (in the first and the last well). A standard was also loaded alongside the samples into the gel and electrophoresis was carried out at

30 MAmp using a power-pack and the gel stained with Coomassie blue. Stained gels were photographed with a Polaroid camera and dried using a gel drier.

74 4.0 SELECTIVE ENUMERATION OF LACTOBACILLUS CASEI FROM YOGURTS AND FERMENTED MILK DRINKS

4.1 Introduction

Yogurts are made from the symbiotic growth of the two bacteria: Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. These yogurt bacteria do not survive the gastric passage or colonise in the gut (Shah and Jelen,

1990). Dairy products containing probiotic bacteria such as Lactobacillus acidophilus and bifidobacteria are becoming popular because of their potential therapeutic benefits. However, due to viability problems associated with Z. acidophilus and bifidobacteria during storage (Anon, 1992; Shah et al, 1995;

Dave and Shah, 1997a, b, c), the recent ttend is to add Z. casei as an adjunct bacteria to yogurt or probiotic cultures. Z. casei is claimed to be more stable than

Z. acz"<:/op/2z7w>S'or bifidobacteria (McCann er cr/., 1996).

In order to achieve health benefits, a minimum of one million viable cells per gram of a product must be present. It is important that the cells must remain viable throughout the projected shelf life of a product so that when consumed the product contains sufficient viable cells.

A version of this chapter has been published. Ravula, R.R and Shah, N.P. Biotechnology Techniques (UK) 12(11): 819-822 (1998). Several media have been developed for selective enumeration of Z. acidophilus and bifidobacteria in yogurts and fermented milks (Kneifel and Pacher, 1993; Int.

Dairy Federation, 1997; Dave and Shah, 1996; Lankaputhra and Shah, 1996).

However, there is little information on selective enumeration of Z. casei in yogurt and fermented milk drinks, which may contain yogurt bacteria and probiotic bacteria.

Selective enumeration of Z. casei in yogurt-type fermented milks containing

o probiotic bacteria based on 15 C incubation temperature and 14 day incubation time was studied by Champagne et al. (1997). However, an incubation time of 14 days may not be practical for the dairy industry if the population of Z. casei is to be known in a short time.

In this study, the ability of Z. casei to ferment ribose was used as an approach to develop a medium for selective enumeration of this organism in fermented products containing yogurt bacteria, probiotic bacteria and Z. casei. The efficacy of the medium for selective enumeration of Z. casei was assessed using nine commercial yogurts and fermented milk drinks obtained from local supermarkets.

4.2 Materials and methods

4.2.1 Bacteria

Two strains each of S. thermophilus, L delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria were used as reference for the validation of the medium developed in this study. S. thermophilus ST WJ7, Z. delbrueckii ssp. bulgaricus LB Wj7, Z. acidophilus LA MJLAl and bifidobacteria BB BDBB2 were obtained from Gist-brocades (Moorebank, Australia). S. thermophilus ST

2008, Z. delbrueckii ssp. bulgaricus LB 2501, Z. acidophilus LA 2415, and

bifidobacteria BB 20099 (B. pseudolongum) were obtained from the Division of

Food Science and Technology, Commonwealth Scientific and Industrial Research

Organisation (CSIRO), Highett, Australia. Z. casei was obtained from Chr.

Hansen Pty Ltd. (Bayswater, Austtalia).

4.2.2 Microbiological analysis

4.2.2.1 Streptococcus thermophilus agar (ST agar)

The ST agar was prepared according to the method described by Dave and Shah

o (1996). Plates in duplicate were incubated aerobically at 37 C for 24 h.

4.2.2.2 M17agar

The Ml7 agar (Amyl Media, Dandenong, Australia) was prepared as

o recommended by the manufacturer. The medium was autoclaved at 121 C for 15

o min. Plates in duplicate were incubated aerobically at 37 C for 24 h.

4.2.2.3 MRS-basal, MRS-salicin, MRS-sorbitol, MRS-ribose, MRS-gluconate

agars

deMan, Rogosa, and Sharpe (MRS)-basal medium was prepared according to

Lankaputiira and Shah (1996) witiiout any sugars and the medium sterilised at

\2\Q for 15 min. Ten millilitres of membrane-filtered sterile solutions of 10%

77 salicin, sorbitol, ribose or gluconate were added per 90 ml of the basal medium

(1% final concenttation) just before pouring the agar medium. Plates in duplicate were incubated anaerobically using anaerobic jars (Oxoid) at 37°C for 72 h (Dave and Shah, 1996).

4.2.2.4 MRS-pH modified agar

MRS broth (Oxoid) was prepared according to the manufacturer instructions. The pH of the broth was adjusted to 5.2 using 1.0 M HCl, agar powder added at the rate of 1.2%, and the medium was autoclaved at 121 C for 15 min. Plates in duplicate were incubated anaerobically at 43 C for 72 h (Dave and Shah, 1996).

4.2.2.5 MRS-NNLP agar

The MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride, and paromomycin sulfate (all from Sigma Chemical Co., Castle Hill, Australia) agar was prepared according to the method described by Laroia and Martin (1991).

The basal medium was MRS agar. Filter-sterilised NNLP was added to the autoclaved MRS basal medium just before pouring. Filter-steriKsed L- cysteine-HCl (0.05% final concentration) was also added. Plates in duplicate were

o incubated anaerobically at 37 C for 72 h.

4.2.2.6 LC agar

The ingredients of LC agar (10.0 g bacteriological peptone; 1.0 g yeast extract;

4.0 g Lab Lemco (Oxoid); 2.0 g KH2PO4; 3.0 g sodium acetate; 1.0 g tri-

78 ammonium citrate; 0.2 g MgS04; 0.05 g MnS04 (BDH Chemicals Pty. Ltd.,

Kilsyth, Australia); 1.0 g acid casein hydrolysate; 1.0 g tween 80 (Sigma

Chemicals Co. St. Louis, USA) were dissolved in 1 L of distilled water. The pH

was adjusted to 5.1 ± 0.1, and 6 ml of bromocresol green and 12 g of

bacteriological agar (Amyl Media, Dandenong, Australia) were added to the

o

medium. The medium was sterilised at 121 C for 15 min. Ten millilitte of

membrane-filtered sterile solution of 10% D (-)ribose (Sigma) was added per 90

ml of the medium (1% final concentration) just before pouring the agar medium. o Plates in duplicate were incubated anaerobically at 27 C for 72 to 96 h.

4.2.3 Enumeration of bacteria

Pure strains were given three successive transfers before enumeration. One gram

of each culture was 10-fold serially diluted (lO'^ to 10^) in 0.15% sterile peptone

and water diluent. The enumeration was carried out using the pour plate

technique. Anaerobic jars and gas generating kits (Anaerobic System BR 38,

Oxoid Ltd., Hampshire, England) were used for creating anaerobic conditions.

Plates with 25-250 colonies were enumerated and recorded as colony forming

units (cfli) per gram of the product or culture.

4.2.4 Commercial products

Nine commercial yogurts and fermented milk drinks claiming to contain single or

mixed sttains of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus,

bifidobacteria and Z. casei were analysed to validate the efficacy of the method.

The products analysed were: Product 1- yogurt; Product 2-skim milk yogurt;

79 Product 3- mild continental yogurt; Product 4-natural yogurt; Product 5-yogurt;

Product 6- fermented milk drink; Product 7-fermented milk drink; Product 8- drinking yogurt; and Product 9- dairy fmit drink. The organisms that were claimed in these products are shovm in Table 2.

All experiments and analyses were carried out in duplicate. The results presented are averages of duplicate experiments.

4.3 Results and Discussion

Table 4.1 shows the viable counts of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei on various bacteriological media. ST agar and Ml7 agar were selective for S. thermophilus and MRS-pH modified agar was selective for Z. delbrueckii ssp. bulgaricus. L acidophilus and

Z. casei formed colonies on MRS-salicin, MRS-sorbitol agar, MRS-ribose agar and MRS-gluconate agar. Thus these media cannot be used for selective enumeration of Z. casei. MRS-salicin agar was reported to be selective for Z.

acidophilus (Dave and Shah, 1996; Lankaputhara and Shah, 1996). However, as

shovm in Table 4.1, Z. casei also grew in this medium. Hence, MRS-salicin

medium cannot be used for selective enumeration of Z. casei in products

containing Z. acidophilus and Z. casei. MRS-NNLP medium (Laroia and Martin,

1991) was selective for enumeration of bifidobacteria. LC agar inhibited the

growth of S. thermophilus, L. delbrueckii ssp. bulgaricus, L acidophilus and

bifidobacteria, and thus the medium was selective for Z. casei.

80 The efficacy of the medium was validated using nine commercial yogurts and fermented milk drinks claiming to contain single or mixed sttains of S. thermophilus, L. delbrueckii ssp. bulgaricus, L acidophilus, bifidobacteria and Z. casei. Table 4.2 shows the counts of Z. casei in various commercial products.

Products 4, 5 and 8 did not contain Z. casei and this organism was not detected in these products. Products 1, 2, 3 and 9 claimed to contain yogurt bacteria, probiotic bacteria and Z. casei and Z. casei was detected in these products.

However, the population of Z. casei in both Products 2 and 3 were low. An

earlier report (Anon, 1992) on Australian dairy products also showed low counts

of Z. acidophilus and bifidobacteria in several products. Products 6 and 7 claimed

to contain strains of Z. casei and Z. casei was detected in these products. Thus, it

appears that LC agar was selective for Z. casei. Figure 4.1 shows colonies formed

by Z. casei on LC agar, whereas no other organisms formed colonies on the LC

agar confirming that LC agar can be selective for enumerating Z. casei

4.4 Conclusion

LC agar developed in this study was found to be selective for Z. casei from pure

cultures. The efficacy of the medium was verified using several commercial

products containing single or mixed population of yogurt bacteria, probiotic

bacteria and Z. casei. LC agar could be used for selective enumeration of Z. casei

in yogurts and fermented milk drinks containing mixed population of S.

thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria.

81 o o o o O o o o o o o o o o o 00 o I, en o o V V V V V V V V

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?3 o o o O o o o p p vo ^rt p p p p p r^ r»i vo 0\ rn rn rn rn en V V t-^ t^ V V V V V

o

• S o o o o o c> o o in p p o o (Nl =3 O IT) «n rn en 00 rn <» OO CO C V V V 00 V V on C S "o00i a V 00 00

O .in C/2 CO . o o o O o o c a? 00 o O 00 IT) o p ^-1 :2 t>0 en o o rn rn u ^^ V rn rn V 00 00 V V 00 "^ .2 V V "

o > I C o O O o O o -4—» o p p o m 00 p p .a "^ c3 op rn VO rn m 00 V V V V 00 00 V V 00 3 ^ CO CO § CO O O o o o o O O o o u O o o P^ CO cd O o o o o o bCT^3 rn m m en V V V V V V V V V o

o o o o o o o T3 5^ OS p p p p -Si tr> o en en en en '^ OO p p 0-, Or, .2 o so m m m V V V V V V V ..—I ...... o o o o « 00 o o o p p o p H 00 m •o m p p en en o ^ 00 00 V V t>0 is V en rn V V V II a O CQ CO OH ^ C < OS OO OS X2 CQ 's Q ^ U H CQ < a ^—> H o H PQ D CQ c/: J J CQ C/3 00 (N C/O I—1 -5 Table 4.2 Counts of Z. casei from different commercial products selectively enumerated on LC agar

Products Organisms claimed to LC agar contained in the product

Product 1 - yogurt S. thermophilus, L. 7.49 delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria andZ. casei

Product 2- skim milk S. thermophilus, L. 3.41 yogurt delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria and Z. casei

Product 3- mild S. thermophilus, L. 3.72 continental yogurt delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria andZ. casei

Product 4- yogurt S. thermophilus, L. <3.00 acidophilus and bifidobacteria

Product 5- yogurt S. thermophilus, L. <3.00 delbrueckii ssp. bulgaricus, L. acidophilus

Product 6- fermented L. casei 8.22 milk drink

Product 7- fermented L. casei 6.98 milk drink

Product 8- drinking S. thermophilus, L. <3.00 yogurt delbrueckii ssp. bulgaricus, L acidophilus, and bifidobacteria

Product 9- dairy fruit S. thermophilus, L. 8.08 drink delbrueckii ssp. bulgaricus, L. acidophilus, bifidobacteria andZ. casei. Figure 4.1. Colonies formed by Z. casei on LC agar 5.0 Populations of Lactobacillus acidophilus. Bifidobacterium spp., and Lactobacillus casei in commercial fermented milk products

5.1 Introduction

Over the past decade, the per capita consumption of yogurt in Austtalia has increased by 112% (IDF, 1997). Yogurt has been considered a highly nutritious food and a useful therapeutic agent (Tamime and Deeth, 1981). There is a growing consumer demand for 'bio-yogurts' and other milk products that contain live probiotic cultures. Japan is the world leader in probiotics. In Europe, probiotic containing food and beverages remains one of the fastest growing segments of the cultured dairy products (Hughes and Hoover, 1991).

Probiotic bacteria are claimed to prevent the growth of intestinal pathogens such as Salmonella, and Shigella. Bifidobacteria are useful for the maintenance of human health and the reduction or disappearance of bifidobacteria in the human intestine would imply an 'unhealthy' state (Mitsuoka, 1990; Shah et al, 1995).

Probiotic organisms are also claimed to stimulate immune response and to prevent cancer. In addition, these organisms improve digestibility of lactose in lactose intolerant people (Shahani and Chandan, 1979).

A version of this section has been accepted for publication. Bifidobacteria

Microflora 19(1): 35-39 (2000).

85 In order to obtain health benefits associated with probiotics, it is necessary to

ingest a minimum of one million organisms per gram of a product (Sellars, 1991).

Studies (Hamann and Marth, 1984; Rohm et al, 1990) have shown that yogurt

bacteria (Streptococcus thermophilus and Lactobacillus delbrueckeii spp.

bulgaricus) survive well in yogurt until the use-by-date. However, probiotic

bacteria (Z. acidophillus and Bifidobacterium spp.) showed a rapid decline after

manufacture (Gilliland and Speck, 1977; Medina and Jordano 1994; Shah et al,

1995; Rybka and Fleet, 1997). Thus, it is misleading to describe a probiotic

product as having health promoting properties unless the minimum level of viable

cells are present at the time of consumption (Kailaspathy and Rybka, 1997).

Lactobacillus casei, recently introduced to Australian yogurts, has shown good

potential as a probiotic organism (Perdigon et al, 1995; Hudault et al, 1997;

Wagner et al, 1997). Additionally, this organism is claimed to have good

viability in yogurt products. However, the population of Z. casei in Australian

yogurt has not been reported.

This paper reports the populations of Z. acidophillus, Bifidobacterium spp. and Z.

casei in 26 fermented milk products representing 14 companies. Enumeration of

bacterial populations was carried out immediately after purchase and at the expiry

date. 5.2 Materials and Methods 5.2.1 Commercial products

Twenty-six commercial yogurts and fermented milk drinks representing 14

companies were purchased from local super markets in the Melboume

metropolitan area. The types of product that were analysed included fermented

liquid milk drink, set yogurt, drinking yogurt, fruit yogurt and soy yogurt. All

products claimed to contain probiotic bacteria. Six products (products A-F)

contained Z. acidophilus only, 12 products (G-R) contained Z. acidophilus and

bifidobacteria, 6 products (S-X) claimed Z. acidophilus, bifidobacteria and Z.

casei, and 2 products (Y-Z) contained Z. casei only. Relevant information was

recorded from the package including the expiry date, flavour, composition, type

of probiotic bacteria, and the address. The products were stored at 5°C during the

stated shelf life. To protect reputations of the companies, the details of the

manufacturers are not given.

5.2.2 Measurement ofpH

The pH was measured with a pH meter (Model 420 A; Orion Research Inc.,

Boston, MA).

5.2.3 Microbiological analyses

MRS-salicin agar and MRS-sorbitol agar were used for enumeration of Z.

acidophilus (Dave and Shah 1996; Lankaputhra et al, 1996a, b). Counts of

bifidobacteria were enumerated on MRS-NNLP (nalidixic acid, meomycin

sulfate, lithium chloride, and paromomycin sulfate) agar (Laroia and Martin

1991; Dave and Shah 1996). Counts of Z. casei were enumerated on LC agar

87 (Ravula and Shah 1998). Enumeration was carried out using the pour plate technique and plates were incubated at 27°C for 72 to 96 h under anaerobic conditions. Plates containing 25 to 250 colonies were enumerated and recorded as colony forming units (cfu) per gram of the product.

The determination of pH and microbiological analyses were carried out in duplicate. The results presented are averages of duplicate experiments.

The products that were analysed included: (A) banana flavoured yogurt containing Z. acidophilus; (B) vanilla flavoured yogurt containing Z. acidophilus; (C) fruit yogurt containing Z. acidophilus; (D) vanilla flavoured yogurt containing Z. acidophilus; (E) black cherry fruit yogurt containing Z. acidophilus; (F) low fat black cherry yogurt containing Z. acidophilus; (G) vanilla flavoured yogurt with fruit and nuts containing Z. acidophilus and bifidobacteria; (H) passion fruit reducted fat yogurt, d'lite containing Z. acidophilus and bifidobacteria; (I) peach 'n' mango reduced fat and fmit yogurt containing Z. acidophilus; and bifidobacteria; (J) strawberry organic soy yogurt containing Z. acidophilus and bifidobacteria; (K) so natural non-dairy soy strawberry yogurt containing Z. acidophilus and bifidobacteria; (L) non-dairy soy wild-berry yogurt containing Z. acidophilus and bifidobacteria; (M) reduced fat raspberry drinking yogurt containing Z. acidophilus and bifidobacteria; (N) reduced fat fruit salad fhiit yogurt containing Z. acidophilus and bifidobacteria;

(O) yo-split reduced fat vanilla yogurt with museli 'n' fruit containing Z. acidophilus and bifidobacteria; (P) diet lite, boysenberry low fat yogurt

88 containing Z. acidophilus and bifidobacteria; (Q) fhiit salad yogurt with honey containing Z. acidophilus and bifidobacteria; (R) apricot yogurt with honey containing Z acidophilus and bifidobacteria; (S) apricot mango, reduced-fat yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (T) sttawberry sundae, reduced-fat set yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (U) skim milk berry fmit yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (V) premium vanilla creamy yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (W) banana custard yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (X) Swiss vanilla reduced-fat fiiiit drinking yogurt containing Z. acidophilus, bifidobacteria and Z. casei; (Y) probiotic fermented milk drink containing Z. ca^er; (Z) fermented milk drink containing Z. casei

Shirota strain.

5.3 Results and Discussion

The populations of Lactobacillus acidophilus. Bifidobacterium spp. and

Lactobacillus casei were determined in 26 commercial dairy products and the results grouped together on the basis of the type of organisms contained in the products.

Table 5.1 shows the initial and final pH and viable counts in the products claimed to contain Z. acidophilus (products A-F). There was a slight decrease in pH (.04 to .07 units) in all the products. Three of the 6 products showed the initial viable counts of Z. acidophilus above 10^ cfu/g, while the viable counts and at the

89 expiry date decreased below 10^ cfu/g. In the product C, tiie initial counts were

10'* cfii/g and at the expiry date the counts decreased below 10^ cfu/g.

Table 5.2 shows the initial and final pH and viable counts of Z. acidophilus and bifidobacteria in the AB products (products G-R). The initial pH ranged between

3.69 to 4.55 and the final between pH 3.64 to 4.26. The drop in pH ranged between .04 to .32. The initial counts of Z. acidophilus in only 50% of the products (products I, L, O, P, Q, R) were above 10^ cfii/g, while only 33% of the products (products O, P, Q, R) showed counts above 10^ cfio/g. The initial counts of Z. acidophilus in 25% of the products (products G, H, K) were very low (<10^ cfli/g). The product L showed a decline of 6 log in the count of Z. acidophilus.

Only one product (product P) of 12 products (80%) showed initial and final counts ofbifidobacteria of >10^ cfu/g.

Table 5.3 shows the initial and final pH and viable counts of Z. acidophilus, bifidobacteria and Z. casei. Overall, there was a slight decrease in pH (.02 to .07) in all the products. The initial counts of Z. acidophilus in 50% of the products

(products S, T, V) were above 10^ cfii/g. The initial counts ofbifidobacteria in only one product (product S) was above 10^ cfu/g. All the products showed bifidobacteria count of <10^ at the expiry date. Four of 6 products (products S, T,

V, X) had initial counts of Z. casei above 10^ cfii/g, while 3 of these 4 products maintained viability of above 10^ at the expiry date. The counts of Z. casei in the product X dropped below <10 cfii/g at the expiry date.

90 Table 5.4 shows the pH and viable counts in the products claimed to contain Z. casei only. There was a slight decrease in pH in both products. The initial coimts in both products were >10^ cfii/g, however, at the expiry date the counts ui the product Y decreased below 10^ cfu/g.

Initially, Z. acidophilus was added to fermented product as a probiotic organism.

Then AB products containing Z. acidophilus and bifidobacteria were introduced.

The recent trend is to incorporate all the three groups of bacteria (Z. acidophilus,

bifidobacteria and Z. casei) called ABC products. Z. casei is claimed to be hardy,

as a result, this organism is incorporated in fermented products. However, results

suggest that 50% of the ABC products had <10^ cfii/g viable counts.

It is recommended that, to have therapeutic benefits, the product must contain

viable probiotic population of 10^ cfii/g. However, the viable populations of

probiotic bacteria decline after manufacturing. Several factors have been found

to affect the viability of probiotic bacteria including pH of the product, post-

acidification due to yogurt bacteria, production of hydrogen peroxide by Z.

delbrueckii ssp. bulgaricus (Dave & Shah, 1997 a, b), and antagonism among

yogurt and probiotic bacteria (Joseph et al., 1998). Previous reports by Shah et

al, (1995) and Rybka and Fleet (1997) have also found variable levels of these

organisms in the probiotic products. Fourteen to 24% of the samples in the study

of Rybka and Fleet (1997) had viable population of Z. acidophilus and

Bifidobacterium spp. above 10^ cfti/g. The viable population of Z. casei in

commercial products has not been reported.

91 5.4 Conclusion

Five of the AB and two ABC products contained Z. acidophilus counts of above

10^ cfu/g, whereas only one of AB and ABC products contained bifidobacteria counts of above 10^ cfio/g at the expiry date. Three of the ABC products and only one LC product contained Z. casei counts of > 10^ cfu/g at the expiry date. To have therapeutic effects a minimum of one million viable cells per gram of the product has been recommended. The products containing low viable counts may not provide any health benefits. Advances in the understanding of the problems associated with the viability and survival of probiotic organisms in fermented milk products are yet to reach the commercial market.

92 Table 5.1 Initial and final pH and viable counts of Lactobacillus acidophilus

Products Remaining shelf pH pH LA LA life (days) initial final initial final A. Banana flavoured yogurt 25 4.17 4.10 3.7 X 10* 3.0x10^

B. Vanilla flavoured yogurt 25 4.18 4.13 9.1x10^ 9.8x10^

C. Fruit yogurt 26 4.18 4.11 4.0x10^ <10^

D. Vanilla flavoured yogurt 24 4.15 4.10 1.9x10^ 1.9x10'

E. Black cherry fmit yogurt 23 4.15 4.11 2.5x10* 3.7x10'

F. Low fat black cherry yogurt 28 4.24 4.17 3.0x10^ 3.0x10'

LA = Lactobacillus acidophilus

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Pr o N _1 6.0 SELECTION OF STRAINS OF YOGURT AND PROBIOTIC BACTERIA TO INCORPORATE IN FERMENTED FROZEN DAIRY DESSERTS

6.1 Introduction

Probiotic organisms such as Lactobacillus acidophilus and Bifidobacterium spp. are claimed to correct intestinal disorders, lower serum cholesterol, posses anti- carcinogenic properties and improve lactose utilisation in lactose intolerant people

(Gilliland, 1989; Shah and Jelen, 1990; Shah, 1993).

Dairy products containing L. acidophilus and Bifidobacterium spp. are rapidly gaining popularity in Australia. The shelf life of yogurt is approximately 3 weeks.

As a result, it may not be feasible to ship yogurt to overseas countries and the sales of such product are limited to local markets. With an increasing popularity of frozen dairy desserts containing yogurt cultures, a market could develop for dairy desserts similar to soft-serve frozen yogurt but containing L. acidophilus and

Bifidobacterium spp. Frozen dairy desserts have longer shelf life and thus the products could be shipped to overseas countries. However, introducing probiotic organisms into the fi-ozen food chain can be a difficult task as the organisms are unstable in such products. The loss of viability of probiotic organisms in frozen yogurt may be due to acidity of the product, freeze injury, sugar concentration and oxygen toxicity.

A version of section 6 has been published Ravula, R.R. and Shah, N.P. 1998. Food Australia 50(3): 136-139.

97 Using ice cream as a mechanism to incorporate bifidobacteria and finuctooligosaccharides into the human diet was studied by Modler et al. (1990). In the presence of neosugars and Jerusalem artichoke flour, 90% survival of bifidobacteria (Bifidobacterium adolescentis. Bifidobacterium infantis and

Bifidobacterium longum) was observed at the end of 70 days. The 10% loss in viable counts occurred at the time of freezing due to incorporation of air to achieve 100% overrun. The initial counts were 7.5, 6.9 and 6.6 logio cfii/ml and the final counts were 7.3, 6.8 and 6.3 logio cfii/ml respectively in the presence of neosugars. In the presence of Jerusalem artichoke flour the initial counts were 7.3, 7.4 and 6.9 and the final counts were 7.3, 7.2 and 6.4, respectively. The effect of pH on survival of L. acidophilus and Bifidobacterium bifidum in frozen fermented dairy desserts was studied by Laroia and Martin (1991). L. acidophilus and B. bifidum survived the freezing process and frozen storage of 8 weeks in pH 5.6-5.8 frozen fermented dairy desserts. The initial counts of B. bifidum were 5.7 cfu/ml and the final counts were

4.0 cfii/ml and the initial counts of L. acidophilus were 11.4 cfu/ml and the final counts were 11.4 cfu/ml.

Viability of L. acidophilus and B. bifidum in soft serve frozen yogurt was studied by

Holcomb et al (1991). L acidophilus was able to survive when exposed to O.OIN

HCl for 2 h at 37°C. The initial counts of Z. acidophilus before freezing were 5.90 log cfii and the final counts after freezing were 5.86 log cfu. Survival of L. acidophilus and B. bifidum in ice cream was also studied by Hekmat and McMahon

(1992). Initial coimts of the organisms were 1.5 x 10^ cfli/ml for L. acidophilus and

98 2.5 X 10^ cfu/ml for B. bifidum. After freezing the fermented mix at -29*^C and storing for 17 weeks the bacterial counts decreased to 4 x 10^ and 1x10^ cfu/ml, respectively. Survival of L acidophilus and Bifidobacterium spp. in the presence of acid and bile salts was studied by Lankaputra and Shah (1995). Of several strains L. acidophilus, of 2401, 2409 and 2415 survived best under acidic condition, while B. longum and B. pseudolongum showed best survival under acidic conditions.

In this study, several yogurt and probiotic bacteria were screened for their ability to survive under freezing and acidic conditions and in the presence of high levels of sugar. Frozen fermented dairy desserts were made with selected yogurt and probiotic organisms and survival of these organisms was monitored for 12 weeks during frozen storage at -18°C. Further, fermented frozen dairy desserts were made on a pilot scale (1000 L) and survival ofyogurt and probiotic organisms were monitored for 12 weeks during frozen storage at -18*^0.

6.2 Materials and method

6.2.1 Propagation ofyogurt and probiotic cultures

Yogurt and probiotic organisms were obtained from the Division of Food Science and Technology, Commonwealth Scientific and Industrial Research Organisation

(CSIRO), Highett, Victoria, Australia; Mauri Laboratory (now. Gist-brocades Pty

Ltd) and Chr. Hansen Pty. Ltd. A total of 41 strains were used for initial study including 10 strains of Streptococcus thermophlius (ST 1, ST 2, ST 2000, ST 2002,

ST 2008, ST 2010, ST 2013, ST 2014, Tsl, ST WJ7), 7 strains of Lactobacillus delbrueckii ssp. bulgaricus (LB 4, LB 2501, LB 2505, LB 2515, LB 2517, LB

99 2519,LB Wj7), 13 strains of Lactobacillus acidophilus (LA 2400, LA 2401, LA

2404, LA 2405, LA 2406, LA 2411, LA 2412, LA 2414, LA 2415, M0LA2, PLA2,

BDLA2, MJLAl) and 11 strains ofbifidobacteria (BB 1912, BB 1920, BB 1941,

BB 1978, BB 5089, BB 5092, BB 5094, BB 5095, BB 20099, BB 20210, BDBB2).

6.2.2 Screening of strains ofyogurt and probiotic bacteria for selecting strains that survive freezing

The organisms were propagated three times before using for freeze tolerance studies.

Ten millilitre of sterile RSM was taken in Pyrex test tubes and inoculated with 2% culture followed by incubation at 37°C till a pH 4.5 was attained. The samples were transferred to a refrigerator till the temperature dropped to 4°C followed by freezing for 4 h at -25°C using a glycol bath. The temperature dropped to -25°C in 15 min.

Samples were thawed using tap water (~20^C). Bacterial counts before and after freezing were determined.

6.2.3 Survival of probiotic bacteria at various sugar levels

Ten millilitre of RSM containing 8 and 16% sucrose were inoculated with 2%) each of L. acidophilus and bifidobacteria and incubated at 37°C till a pH of 4.5 was attained. Samples were transferred to a refrigerator till the temperature dropped to

4°C and then frozen at -25°C for 4 h using a glycol bath. The temperature dropped to

-25°C in 18 min. Samples were thawed using tap water (~20°C). Bacterial counts before and after freezing were determined as before.

100 6.2.4 Survival of yogurt and probiotic bacteria under acidic and frozen conditions

Samples of RSM were adjusted to pH 6.2 (control), 5.0, 4.5 and 4.0 with lactic acid

(BDH Laboratory Supplies, Poole, England). One millilitre of each culture of S. thermophilus, L. delbrueckii ssp. bulgaricus, L acidophilus, and Bifidobacterium spp. was added to 9 mL of the pH adjusted RSM in separate test tubes. The test tubes were kept in a cold room (4*^C) for 5 h followed by freezing at -25°C for 4 h using a glycol bath. The temperature dropped to -25°C in 15 min. Samples were thawed using tap water (~20°C). Bacterial counts before and after freezing were determined as before.

6.3 Results and discussion

6.3.1 Composition

The protein content of the fermented frozen dairy dessert was -3.75%. The total solids contents were in the range of 32.5-32.8%. The titratable acidity ranged between 0.14% and 0.76%. The fat content was 10.3%, while the sugar content was

16.8%). The pH ranged between 4.51 and 4.59. The overrun of the product made with a bench top freezer was 18% and that made with Crepaco freezer was 100%.

6.3.2 Effect of freezing on survival ofyogurt and probiotic bacteria

The effect of freezing on survival of yogurt and probiotic bacteria before and after freezing is shown in Tables 6.1 and 6.2. There was no major difference in the viable counts of 5". thermophilus (Table 6.1), L. delbrueckii ssp. bulgaricus (Table 6.2), L

101 acidophilus (Table 6.3) and bifidobacteria (Table 6.4) before and after freezing at -

25°C for 4 h.

6.3.3 Effect of sugar levels on survival of probiotic bacteria before and after freezing

The effect of sucrose levels on survival of yogurt and probiotic bacteria before and after freezing is shown in Tables (6.3 - 6.6). At 8 and 16% sucrose levels, L. acidophilus showed minor differences after freezing at -25°C for 4 h. Counts of L. acidophilus 2414 were reduced by about 1 log cycle. L. acidophilus strains 2404 and

2411 were reduced by about 2 log cycles at both sugar levels. Counts of bifidobacteria 5092 were reduced by about 1 log cycle after freezing both at 8 and

16% sucrose levels. However, other strains did not show any decrease in cell counts.

6.3.4 Survival of yogurt and probiotic bacteria under acidic and frozen conditions

The survival of yogurt and probiotic bacteria before and after freezing under acidic and frozen conditions is shown in Tables (6.7 - 6.22). Counts of S. thermophilus

2013 were reduced by about 1 log cycle after freezing at pH 4.5 and by about 2 log cycles at pH 4.0. Counts of Z,. delbrueckii ssp. bulgaricus 2515 were reduced by about 1 log cycle after freezing at pH 4.0. Counts of I. acidophilus MJLAl were reduced by about 1 log cycle at pH 4 and pH 4.5. Conversely, all five strains of bifidobacteria (1912, 1920, 1978, 20099 and BDBB2) did not show any decrease in counts at various pH levels.

102 6.4 Conclusion

All strains screened for freeze tolerance remained viable before and after freezing and at 8 and \6% sugar levels. Of the various pH levels studied, pH 4.5 and 4.0 were found to be detrimental for S. thermophilus 2013, L. delbrueckii ssp. bulgaricus

2515 and L. acidophilus MJLAl, while rest of the organisms were not affected by pH.

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Sjunoo o^qBiA Soq 7.0 INFLUENCE OF WATER ACTIVITY ON PRODUCTION OF PROBIOTIC YOGURT

7.1 Introduction

High concentrations of sugar added to milk before fermentation with yogurt starters may result in conditions inhibitory to bacterial growth, leading to long fermentation times and poor acidity development (Tramer, 1973; Zourari et al,

1992). This is due to both adverse osmotic effects of the solutes in milk and low aw (Larsen and Anon, 1989, 1990; Zourari et al, 1992).

Effect of water activity on acid production by S. thermophilus and L delbrueckii ssp. bulgaricus was studied by Larsen and Anon (1989. A decrease in aw inhibited acid production by both organisms. Minimum aw for acid production was always lower when aw of milk was adjusted with glucose or sucrose than with glycerol. Moisture requirements for the growth and metabolite production by lactic acid bacteria were studied by Troller and Stinson (1981). Media adjusted with sucrose generally were inhibitory than those in which glycerol was used as the humectant. Tifratable acidity although not related to the type of humectant, did depend on aw of the medium and was directly related to the extent of growth. Acid production appeared to be related to the presence and extent of growth at all of aw levels studied. 7.2 MATERIALS AND METHODS

7.2.1 Yogurt bacteria and probiotic bacteria

7.2.2 Manufacture ofyogurt Yogurt was manufactured as outlined in Fig 7.1. Five batches ofyogurt (1 L size) were made using 12% reconstituted skim milk (RSM).

Experiments were carried each using four levels of sugar (4, 8, 12 and 16%) added to RSM. Yogurt made without added sugar was used as a control (batch 1).

The mix was heated at 85^C for 30 min, cooled to 42°C, and yogurt bacteria (S. thermophilus ST WJ7 and L. delbrueckii ssp. bulgaricus LB WJ7) and probiotic bacteria (L. acidophilus LA 2415 and bifidobacteria BB 1941) were added at the rate of 0.5% each. The organisms were allowed to mix for 5 min using a sterile magnetic stirrer. The inoculated mix was filled in 100 millilitre plastic containers and incubated at 42°C till pH 4.5 was attained. The yogurts were transferred to a cold room and kept overnight at 4°C. Microbiological and chemical analysis were monitored for 24 hrs and then for 30 days at 10-day intervals.

7.3 RESULTS AND DISCUSSION

7.3.1 pH and titratable acidity

The initial pH of reconstituted skim milk ranged between 6.25 to 6.36 and the

final pH after fermentation ranged from 4.45 to 4.58. The fermentation time to

decrease pH to 4.5 ranged between 3.25 h to 22.5 h. As the sugar concentration

increased, the fermentation time increased. For the batch containing 16% sugar. the fermentation time was 22.5h. The final pH by the end of storage period was

4.50-4.44 (Figure 7.4).

High concentrations of sugar added to milk before fermentation with yogurt starters may result in conditions inhibitory to bacterial growth, leading to long fermentation times and poor acidity development (Tramer, 1973; Zourari et al,

1992). This is due to both adverse osmotic effects of the solutes in milk and low aw (Larsen and Anon, 1989, 1990; Zourari et al, 1992).

Figure 7.3 shows the water activity of all the five batches of yogurt. The water activity decreased as the percentage of sugar increased. Fajardo-Lira et al. (1997) studied the effect of water activity on exopolysaccharide producing or non- producing starters and the authors concluded that sucrose had an inhibitory effect on fermentation process. Effect of water activity of milk on acid production by S. thermophilus and L delbrueckii ssp. bulgaricus was studied by Larsen and Anon

(1989). Decrease of aw inhibited acid production by both organisms. Minimum aw for acid production was always lower when aw of milk that was adjusted with glucose or sucrose than with glycerol.

Figure 7.2 shows the total solids content in all the five batches of yogurt. The increase in total solid showed increased fermentation time. The total solids contents ranged between 12.53 to 27.56%).

128 7.3.2 Viable counts ofyogurt and probiotic bacteria

Figure 7.5 shows the viable counts ofyogurt bacteria and probiotic bacteria in the five batches of yogurt. In all the five batches, the initial counts (before incubation) of yogurt bacteria and probiotic bacteria were almost similar. The final counts (at pH 4.5) of 5. thermophilus in batches containing 0, 4 or 8%) sugar increased approximately by 3 log cycles, whereas in the batches with 12 and 16% sugar, the counts increased by 2 and 1 log cycle, respectively. There was approximately 2 log cycle increase in the counts of i. delbrueckii ssp. bulgaricus in the batches containing 0 and 4% sugar. The viable counts decreased in the other batches. The viable counts of L. acidophilus in the batches containing 0, 4 and 8% sugar were almost similar as the initial counts. The viable counts in the batches containing 12 and 16% sugar decreased by 2 and 5 log cycles. The viable counts of bifidobacteria in batches made with 0, 4 and 8% sugar increased by almost 1 log cycle, however, the counts decreased by 2 and 3 log cycles in the batches made with 12 and 16% sugar. The decrease in the coimts of yogurt and probiotic bacteria clearly indicates the role of low water activity caused by addition of sugar.

Lankaputhra and Shah (1996) studied the survival of probiofic bacteria at various pH levels. Of the various strains studied, over 50% of the strains lost viability after 6 days of storage at pH <4.3. The minimum pH for survival of L. acidophilus and bifidobacteria is 5.5 (Holt, 1986). Thus a decline in their counts is expected to occur. However, the authors concluded that the strain La 2415 and

BB 1941 survived pH 4.5. However our study revels that the strains, which

129 survive low pH and high acidity does not necessarily survive high levels of sugar

and total solids.

High concentrations of sugar added to milk before fermentation with yogurt

starters may result in conditions inhibitory to bacterial growth, leading to long

fermentation times and poor acidity development (Tramer, 1973; Zourari et al,

1992). This is due to both adverse osmotic effects of the solutes in milk and low

aw (Larsen and Anon, 1989, 1990; Zourari etal, 1992).

7.4 Conclusion

Influence of water activity on fermentation of fimctional probiotic yogurt used in

the manufacture of fermented frozen dairy desserts was studied. Higher levels of

sugars (sucrose) lead to longer fermentation time and poor survival of yogurt and

probiotic bacteria. Fermented frozen dairy desserts require incorporation of high

levels of sugar, thus it may not be feasible to make such products.

130 Reconstituted skim milk (12%) i

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(0, 4, 8,12 or 16%) i

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Cooling to incubation temperature (42°C) i

Addition ofyogurt bacteria and probiotic bacteria, mixing i

Filling in containers

Incubation at 42"C till the pH reached 4.5 i

Storage at 4"C

Fig 7.1 A Flow diagram for making yogurt.

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thermophilus; LA = Lactobacillus

135 8.0 INFLUENCE OF WATER ACTTV^TY ON FERMENTATION, ORGANIC ACIDS PRODUCTION AND VIABILITY OF YOGURT AlVD PROBIOTIC BACTERIA

8.1 Introduction

A number of health benefits associated with the consumption of probiotic organisms such as Lactobacillus acidophilus and Bifidobacterium spp. have been claimed

(Hughes and Hoover, 1991; Kanbe, 1992; Mital and Garg, 1992). It has been suggested that, to produce health benefits, a minimum level of probiotic bacteria in fermented dairy products should be 10^ viable cells per gram of a product. However, several studies (Anon, 1992; Shah et al, 1995, 1999) report that the probiotic organisms do not survive well in fermented products.

Several factors have been claimed to affect the viability of probiotic bacteria in fermented milk products. According to the Australian Food Standards H8, the pH of yogurt must be <4.5, which affects the viability of probiotic bacteria. Similarly, oxygen is incorporated in fermented frozen dairy desserts and has been identified to affect their viability during manufacture and storage (Lankaputhra and Shah, 1994,

1995; Lankaputhra and Shah 1996; Ravula and Shah, 1998b).

A version of section 8 has been accepted for publication. Ravula, R.R and Shah, N.P. Australian Journal of Dairy Technology (2000).

136 Fermented frozen dairy desserts typically contain 16% sugar (Ravula and Shah,

1998b) High concentrations of sugar added to milk before fermentation may result

in conditions inhibitory to yogurt starter growth, leading to long fermentation times

and poor acidity development (Tramer, 1973; Zourari et al, 1992). This is due to

both adverse osmotic effects of the solutes in milk and low aw (Larsen and Anon,

1989, 1990; Zourari et al, 1992).

Organic acids are particularly important for the final properties of fermented dairy

products such as yogurt. These acids help preserve the sensory characteristics of the

product. Lactic acid has been found to inhibit the growth of certain pathogenic

bacteria in yogurt (Zourari et al, 1992)

Effects of water activity on acid production by S. thermophilus and L. delbrueckii

ssp. bulgaricus were studied by Larsen and Anon (1989). A decrease in aw inhibited

acid production by both organisms. The minimum aw for acid production was always

lower when aw of milk was adjusted with glucose or sucrose than with glycerol.

The final pH of yogurt can affect the growth and viability of L acidophilus and

Bifidobacterium spp., most notably of the latter organism (Laroia and Martin 1991;

Hekmat and McMahon, 1992). Acid production by lactic acid bacteria, especially

post-acidification, affected cell viability of L acidophilus and B. bifidum (Ishibashi

and Shimamura, 1993). This may be due to residual activity of lactic bacteria that

produce lactic or acetic acid.

137 The aims of this study were: (i) to investigate the influence of water activity due to addition of sucrose on incubation time and viability of yogurt and probiotic bacteria, and (ii) to assess changes in organic acids during a 30-day storage period.

8.2 Materials and Methods

8.2.1 Yogurt bacteria and probiotic bacteria

Yogurt bacteria (Streptococcus thermophilus STWJ7 and Lactobacillus delbrueckii ssp. bulgaricus LBWJ7) and probiotic bacteria (Lactobacillus acidophilus MJLAl and Bifidobacterium BDBB2) were obtained from Gist-brocades (formerly Mauri

Laboratories Pty. Ltd., Moorebank, Australia). All the strains were tested for purity by Gram stain and propagated every week in sterile 12% reconstituted skim milk

(RSM) supplemented with 2% glucose and l%o yeast extract. All the cultures were grown for 18 h at 37°C using 1% inoculum. For propagation of Bifidobacterium spp., sterile RSM was supplemented with 0.05% L-cysteine hydrochloride in order to provide anaerobic condition and to stimulate their growth. The cultures were maintained in the same medium at 4°C. Before yogurt manufacture, the cultures were transferred successively three times for activation.

8.2.2 Manufacture ofyogurt

Five batches ofyogurt (1 litre size) were made using 12% RSM with four levels of sugar (4, 8, 12 and 16%). A control batch was made without added sucrose. The mix was heated at 85°C for 30 min, cooled to 42°C and yogurt bacteria (S. thermophilus

138 STWJ7 and L delbrueckii ssp. bulgaricus LBWJ7) and probiotic bacteria (L. acidophilus MJLAl and Bifidobacterium spp. BDBB2) were added at the rate of

0.5%) of each. The inoculated mix was filled in 100 millilitre plastic containers and incubated at 42°C until the pH of 4.5 ± 1 was attained. The yogurts were stored in a cold room (4°C) and microbiological and chemical analyses were monitored at 24 h and at 10-day intervals for 30 days.

8.2.3 Detection and quantification of organic acids with HPLC

8.2.3.1 Extraction of organic acids

Four grams ofyogurt samples were diluted with 25 mL of O.OIN H2SO4, and filtered through 0.02-|im Teflon filters (Schleicher and Schuell, Kassel, Germany). The extraction of organic acids was carried out in duplicates.

8.2.3.2 HPLC of organic acids

The standards of organic acids were pipetted into HPLC vials through 0.02 \xm millipore filters (Millipore Australia, Lane Cove, Australia). Similarly, diluted samples of yogurts were pipetted into HPLC vials through 0.02 \x.m filters.Th e vials were used in an auto sampler for analysis of organic acids. The concentration of each acid was determined with Varian HPLC (Varian, Mulgrave, Australia) using an

UV-Vis detector. An Aminex HPX-87H ion exclusion column (300 x 7.8 mm)

(BioRad, North Ryde, Australia) and a mobile phase of 0.0075 M H2SO4 were used for the analysis. Quantification of organic acids was carried out by the extemal standard method. Linear regression curves based on peak heights were calculated for

139 individual organic acids after duplicate injections. Identifications of acids were based on matching retention times of standards.

8.2.4 Microbiological analysis

8.2.4.1 Peptone and water diluent

Peptone and water diluent were prepared by dissolving 15 g of peptone and water

medium (Oxoid, West Heidelberg, Australia) in IL of distilled water, the pH

adjusted to 7.0 ± 0.2, followed by autoclaving 9 mL portions at 121°C for 15 min.

8.2.4.2 Enumeration of bacteria

S. thermophilus was enumerated on ST agar (composition: 10.Og tryptone; 10.Og

sucrose; 5.0g yeast extract; 2.0g K2HPO4 and 6 mL bromo-cresol purple (0.05%)) per

litre of water, pH adjusted to 6.8) and by incubating plates aerobically at 37°C for 24

± 3 h (Dave and Shah, 1996; Lankaputhra and Shah, 1996b). MRS agar (Oxoid)

adjusted to pH 5.2 and anaerobic incubation at 43°C for 72 h were used for selective

enumeration of L. delbrueckii ssp. bulgaricus. MRS-salicin and MRS-sorbitol agars

were used for the selective enumeration of L. acidophilus (Dave and Shah, 1996).

The populations of bifidobacteria were enumerated on MRS-NNLP (nalidixic acid,

neomycin sulfate, lithium chloride, and paromomycin sulfate) agar (Laroia and

Martin, 1991). The initial counts (counts after inoculation but before incubation) and

those after reaching pH of 4.5 (final counts) of both yogurt and probiotic bacteria

were enumerated. The populations of the four types of organisms were monitored at

24 hour and then at 10-day intervals for 30 days.

140 8.2.5 Analyses

The water activity (aw) was measured at 25°C with a water activity meter (Deacon

CX-1; Decagon Devices Inc., Pullman, WA). The pH was measured with a pH meter

(Model 420A, Orion Research Inc., Boston, MA). All the measurements were carried out in triplicates. The results presented are averages of the three replicates.

8.3. Results and Discussion

8.3.1 pH, sugar concentration and incubation time

Initial pH of RSM ranged between 6.25 to 6.36 and the final pH 4.45 to 4.58 (Table

8.1). The incubation time to decrease the pH ofyogurt mix to 4.5 ranged between 4 h to 24 h (Figure 8.7). In general, the incubation time increased as the sugar concentration increased, in particular in batches containing 12 or 16%) sugar.

8.3.2 Water activity

Figure 8.8 shows the water activity of all the five batches of yogurt. The water activity decreased as the percentage of sugar increased. Fajardo-Lira et al. (1997) studied the effects of water activity on exopolysaccharide producing or non- producing starters and found that sucrose had an inhibitory effect on fermentation process. Effects of water activity of milk on acid production by S. thermophilus and

L. delbrueckii ssp. bulgaricus were studied by Larsen and Anon (1989). A decrease in aw inhibited acid production by both organisms. The minimum aw for acid production was always lower when aw of milk was adjusted with glucose or sucrose than with glycerol.

141 8.3.3 Production of organic acids

Figures (8.1 - 8.5) show the percent organic acids present in the inoculated milk, after fermentation and during the storage period of 30 days. Pyruvic acid, acetic acid, lactic acid, propionic acid, and hippuric acid were identified and quantified in inoculated yogurt mix, after fermentation and during storage. The levels of pyruvic

acid after fermentation increased with the level of sugar, but decreased during

storage.

In general, the level of acetic acid increased during fermentation. There was a slight

increase in the level of acetic acid during storage. The decrease was inversely

proportional to the level of sugar. The production of lactic acid followed a similar

pattem except that there was no production of lactic acid in inoculated but

unfermented products.

Low levels of propionic acid were detected in inoculated but unfermented batches

(Figure 8.4). The propionic acid level at 16% sugar level was lower than at 12%, but

similar to the 4%) and 8%o sugar levels and higher than the zero sugar control. Low

levels of hippuric acid were detected in inoculated milk not after fermentation or

storage.

8.3.4 Viable counts ofyogurt and probiotic bacteria

Figures (8.6 - 8.9) show the viable counts ofyogurt bacteria and probiotic bacteria

in the five batches of yogurt. In general, the initial counts after inoculation but

142 before incubation of yogurt bacteria and probiotic bacteria were similar. The coimts of 5. thermophilus at 16%) sugar level was lower than at 12%, but similar to the 0%,

4% and 8%) sugar levels after 24 hour storage in batches containing 0, 4 or 8%o sugar increased by approximately 2 log cycles (Figure 8.6). There was approximately 1 log cycle increase in the counts of L. delbrueckii ssp. bulgaricus in the batches containing 0, 4 and 8%) sugar (Figure 8.7). The viable counts decreased as with S. thermophilus in the two batches containing 12 and 16% sugar. The initial viable counts of L. acidophilus in the batches containing 0, 4 and 8% sugar were similar

(Figure 8.8). There was a sharp decline in the viable counts during storage in the batches containing 12 and 16%) sugar. The viable counts ofbifidobacteria showed a similar trend (Figure 8.9). The decrease in the counts of yogurt and probiotic bacteria indicates the adverse effects of lowering water activity due to addition of sugar. The viability of L. acidophilus and Bifidobacterium spp. was most adversely affected followed by that of yogurt bacteria, L. delbrueckii. ssp. bulgaricus, and S. thermophilus.

Lankaputhra and Shah (1996) studied the survival of probiotic bacteria at low pH conditions. Of the various strains studied, over 50%) of the strains lost viability after

6 days of storage at pH 4.3. The minimum pH for survival of Z,. acidophilus and bifidobacteria is 5.5 (Holt, 1986). Thus a decline in their counts at pH 4.3 is expected to occur.

143 Frozen fermented dairy products typically contain 16%o sugar. Thus such a high concentration of sugar is likely to inhibit the growth of probiotic bacteria and production of organic acids. In this study, only one set of four bacterial strains was tested. However, results from these experiments are indicative of issues that might need to be considered in understanding the effects of sugar in yogurt, but other

strains must be evaluated on a case-by-case basis.

8.4 Conclusions

The influence of water activity due to addition of sucrose on fermentation of

probiotic yogurt and viable coimts of yogurt and probiotic bacteria were smdied.

High levels of sugar, especially 12%o and 16%) led to longer fermentation time,

decreased levels of organic acids and poor survival of yogurt and probiotic bacteria.

The growth of L. acidophilus and Bifidobacterium spp. was most adversely affected

at sugar concentrations of 16% followed by that ofyogurt bacteria, L. delbrueckii

ssp. bulgaricus and S. thermophilus. Fermented frozen dairy desserts typically

contain 16%) sugar, thus it may not be feasible to achieve a satisfactory count of

probiotic bacteria in such products if sugar is added prior to fermentation.

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9.1 Introduction

The term probiotic was first used by Lilly and Stillwell (1965) to describe the growth promoting effect of one or more microorganisms against another. Soon afterwards, the term was applied primarily to animal feed supplements. Further changes in use of this term implied a live microbial feed supplement that improved the intestinal microbial balance of the host (Fuller, 1989). The term is now also being used with reference to human consumption of live microorganisms as food additives for nutritional health and well-being (Hull et al, 1992).

The basis of the probiotic concept is that these are the gastrointestinal tract microorganisms, which have a beneficial effect on the host. Modem life styles may have adverse effect on the delicate balance that exists between the many different species of microorganisms, which inhabit the gut.

A version of section 9 lias been published. Ravula, R.R. and Shah, N.P. 50 (3) 136-139. Food Australia (1998).

158 Our unnatural diet (most of which is heat sterilised by cooking), antibiotic therapy and stress are all factors which can alter the composition of the gut microflora and have nutritional and health consequences. Probiotics can reverse or prevent these

consequences by restoring the flora to its natural state (Fuller, 1989)

However, introducing probiotic organisms into the frozen food chain can be a

difficult task as the organisms are unstable in such products. The loss of viability of probiotic organisms in frozen yogurt may be due to acidity of the product, freeze

injury, sugar concentration and oxygen toxicity.

In this study, fermented frozen dairy desserts were made with selected yogurt and

probiotic organisms and survival of these organisms was monitored for 12 weeks

during frozen storage at -18°C.

9.2 Materials and Method

9.2.1 Yogurt and probiotic bacteria in fermented frozen dairy desserts made using a bench top ice cream Freezer

Six different batches (Bl-6) of fermented frozen dairy desserts were made as

follows:

For batch Bl, yogurt was made by using ST 2010, LB 2501, LA 2415 and BB

20099. Similarly, B2 was made with ST 2008, LB 2515, LA 2401, BB 1912 and B3

with ST/LB Wj7, MjLAl, BDBB2. B4, B5 and B6 were made with ST 2010, 2008,

ST WJ7 and LB 2501, 2515, LB WJ7 only and probiotic strains LA 2415, 2401,

159 MJLAl, BB 20099, 1912, BDBB2 were incorporated just before freezing. Yogurt and base mix were added at a ratio of 45:45 and 10% of liquid sucrose syrup (65%) was added, followed by freezing the contents using a bench top Kenwood ice chef ice cream freezer (Model Im 300 PK 185/w, Kenwood Appliances, Italy). The pH of the final product ranged between 4.51 and 4.59.

9.3 Results and discussion

9.3.1 Viability ofyogurt and probiotic bacteria in fermented frozen dairy dessert made using a bench top freezer during frozen storage for 12 weeks

Counts of 5". thermophilus (2010, 2008 and STWJ7) were reduced by about 1 log cycle in the first 3 to 5 weeks of storage and there was no ftirther decrease during the rest of the storage period (Figures 9.1-9.3). There was no marked difference in the viable counts when the organisms were added just before freezing (Figures 9.4-9.6).

Similar trends were observed with L. delbrueckii ssp. bulgaricus (2501, 2515 and

LBWJ7) (Figures 9.1-9.6).

Counts of I. acidophilus 2415 were reduced by about 6 log cycles when added along

with S. thermophilus, L. delbrueckii ssp. bulgaricus and bifidobacteria (Figures 9.1-

9.3). The viable count at the end of the storage period was 10^ cfu/g. Counts of L.

acidophilus MJLAl were reduced by about 2 log cycles when the organisms were

added along with other bacteria, whereas when the organisms were added just before

freezing the counts were reduced by about 4 log cycles by the end of storage period

(Figures 9.4- 9.6).

160 Counts ofbifidobacteria 20099 reduced by about 1 log cycle when added along with other bacteria, or added just before freezing. Counts of strain 1912 decreased by 5 log cycles at the end of the storage period when the organisms were added along with other bacteria and also just before freezing (Figures 9.1-9.6). Counts of bifidobacteria BDBB2 were reduced by about 1 log cycles when added along with other bacteria, whereas by about 2 log cycles when added just before freezing.

9.4 Conclusion

All strains screened for freeze tolerance remained viable before and after freezing and at 8 and 16% sugar levels. Of the various pH levels studied, pH 4.5 and 4.0 were found to be detrimental for S. thermophilus 2013, L. delbrueckii ssp. bulgaricus

2515 and L. acidophilus MJLAl, while rest of the organisms were not affected by pH.

Yogurt bacteria reduced by about 1 log cycle in the products made using a bench top freezer during 12 weeks storage. L. acidophilus 2415 was adversely affected and the counts declined by 6 log cycles within 8 weeks of storage at -18^C. Amongst bifidobacteria strain 1912 decreased by 5 log cycles at the end of 12 weeks storage.

L. acidophilus MJLAl and bifidobacteria BDBB2 reduced by about 2 log cycles in the products made on a pilot scale after 12 weeks storage.

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OS OO r-- so >n ^ m (N L o/sjunoo sjqBiA §on[ ux 10.0 ASSESSMENT OF VIABILITY OF YOGURT AND PROBIOTIC BACTERIA IN FERMENTED FROZEN DAIRY DESSERTS MADE USING COMMERCIAL ICE CREAM FREEZER

10.1 INTRODUCTION

Probiotic organisms such as Lactobacillus acidophilus and Bifidobacterium spp. are claimed to correct intestinal disorders, lower serum cholesterol, posses anti- carcinogenic properties and improve lactose utilisation in lactose intolerant people

(Gilliland, 1989; Shah and Jelen, 1990; Shah, 1993).

Dairy products containing L acidophilus and Bifidobacterium spp. are rapidly gaining popularity in Australia. The shelf life of yogurt is approximately 3 weeks.

As a result, it may not be feasible to ship yogurt to overseas countries and the sales of such product are limited to local markets. With an increasing popularity of frozen dairy desserts containing yogurt cultures, a market could develop for dairy desserts similar to soft-serve frozen yogurt but containing L. acidophilus and

Bifidobacterium spp. Frozen dairy desserts have longer shelf life and thus the products could be shipped to overseas countries. However, introducing probiotic organisms into the frozen food chain can be a difficult task as the organisms are unstable in such products.

• A version of section 10 has been published Ravula, R.R and Shah, N.P. 50 (3) 136 - 139). Food Australia (1998).

168 The loss of viability of probiotic organisms in frozen yogurt may be due to acidity of the product, freeze injury, sugar concenfration and oxygen toxicity. Using ice cream

as a mechanism to incorporate bifidobacteria and fructooligosaccharides into the

human diet was studied by Modler et al. (1990). In the presence of neosugars and

Jerusalem artichoke flour, 90% survival of bifidobacteria (Bifidobacterium

adolescentis. Bifidobacterium infantis and Bifidobacterium longum) was observed at

the end of 70 days. The 10% loss in viable counts occurred at the time of freezing

due to incorporation of air to achieve 100% overrun. The initial counts were 7.5, 6.9

and 6.6 logio cfii/ml and the final counts were 7.3, 6.8 and 6.3 logio cfu/ml

respectively in the presence of neosugars. In the presence of Jerusalem artichoke

flour the initial counts were 7.3, 7.4 and 6.9 and the final counts were 7.3, 7.2 and

6.4, respectively. The effect of pH on survival of Z. acidophilus and Bifidobacterium

bifidum in frozen fermented dairy desserts was studied by Laroia and Martin (1991).

L acidophilus and B. bifidum survived the freezing process and frozen storage of 8

weeks in pH 5.6-5.8 frozen fermented dairy desserts. The initial counts of 5. bifidum

were 5.7 cfii/ml and the final counts were 4.0 cfu/ml and the initial counts of L.

acidophilus were 11.4 cfii/ml and the final counts were 11.4 cfu/ml.

Viability of L. acidophilus and B. bifidum in soft serve frozen yogurt was studied by

Holcomb et al (1991). L. acidophilus was able to survive when exposed to O.OIN

HCl for 2 h at 37°C. The initial counts of L acidophilus before freezing were 5.90

log cfu and the final counts after freezing were 5.86 log cfu. Survival of L.

acidophilus and B. bifidum in ice cream was also studied by Hekmat and McMahon

169 (1992). Initial counts of the organisms were 1.5 x 10^ cfu/ml for L acidophilus and

2.5 X 10^ cfu/ml for B. bifidum. After freezing the fermented mix at -29°C and storing for 17 weeks the bacterial counts were decreased to 4 x 10^ and 1 x lO' cfu/ml, respectively. Survival of L. acidophilus and Bifidobacterium spp. in the presence of acid and bile salts was studied by Lankaputra and Shah (1995). Of several sfrains L. acidophilus, of 2401, 2409 and 2415 survived best under acidic condition, while B. longum and B. pseudolongum showed best survival under acidic conditions.

In this study, several yogurt and probiotic bacteria were screened for their ability to survive under freezing and acidic conditions and in the presence of high levels of sugar. Frozen fermented dairy desserts were made with selected yogurt and probiotic organisms and survival of these organisms was monitored for 12 weeks during frozen storage at -18^C. Further, fermented frozen dairy desserts were made on a pilot scale (1000 L) and survival ofyogurt and probiotic organisms were monitored for 12 weeks during frozen storage at -18°C.

10.2 Materials and Method

10.2.1 Survival ofyogurt and probiotic bacteria in fermented frozen dairy desserts made on a pilot scale

Two different batches of fermented frozen dairy desserts each of 1000 L were made on a pilot scale using an ice cream freezer. For batch 1, yogurt was made by using

ST 2008, LB 2501, LA 2401, and BB 20099. Similarly, batch 2 was made with

170 ST/LB Wj7, MjLal, and BDBB2. Yogurt and base mix were added at a ratio of

45:45 and 10% of sucrose syrup (65%) was added, followed by freezuig the contents using a continuous ice cream freezer (Crepaco Freezer, Model KM 240, U.S.A). The pH of the final product ranged between 4.51 and 4.59.

10.2.2 Microbiological analyses

Counts of S. thermophilus were enumerated on Streptococcus thermophilus agar.

Counts of L. delbrueckii ssp. bulgaricus were enumerated on MRS agar. MRS-

salicin and MRS-sorbitol agars were used for enumeration of L. acidophilus. Counts of bifidobacteria were enumerated on MRS-NNLP. Enumeration was carried out using the pour plate technique. Plates containing 25 to 250 colonies were enumerated and recorded as colony forming (cfii) units per gram of culture. All the

experiments and analyses were repeated at least twice. The results presented are

averages of two replicates.

10.3 Results and Discussion

10.3.1 Composition

The protein content of the fermented frozen dairy dessert was -3.75%. The total

solids contents were in the range of 32.5-32.8%. The titratable acidity ranged between 0.74% and 0.76%. The fat content was 10.3%, while the sugar content was

16.8%. The pH ranged between 4.51 and 4.59. The overrun of the product made with a bench top freezer was 18% and that made with Crepaco freezer was 100%.

171 10.3.2 Viability ofyogurt and probiotic bacteria in fermented frozen dairy dessert made using a pilot scale ice cream freezer during frozen storage for 12 weeks

Figs (10.1-10.4) shows the viability of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria in fermented frozen dair>' dessert made using a continuous ice cream freezer. The counts of S. thermophilus WJ7 decreased by about 1 log cycle after 6 weeks and those of I. delbrueckii ssp. bulgaricus 2501

and WJ7 reduced by about 1 log cycle after 3 and 8 weeks. The counts of L

acidophilus MJLAl were reduced by about 1 log cycles after 1 week and 2 log

cycles after 8 and 9 weeks. There was no decrease in the population ofbifidobacteria

20099 for up to 8 weeks of storage; however, there was 1 log cycle decrease during

the rest the of storage period. At the end of 12 weeks storage period, the counts of Z.

acidophilus and bifidobacteria were ~ 10^ cfu/g.

Several factors affect the viability of probiotic bacteria in frozen fermented dairy

products. Survival of these organisms is affected by low pH of the environment. The

standard requires the pH of the product to be < 4.5, which affects their viability. In the manufacture of fermented frozen dairy desserts air is incorporated to achieve

100% overrun. Bifidobacteria are anaerobic and L acidophilus is microaerphilic and

these organisms prefer a low oxidation-reduction potential for growth. Addition of

sugar increases the osmotic pressure which may have effect on the growth and

viability of probiotic bacteria. The optimum temperature for the growth of L.

acidophilus and bifidobacteria is 37°C. Thus, when the product is frozen to -18 C or

below, low temperature affects the viability of these organisms. The survival of

172 these organisms appeared to be sfrain dependent, thus selection of sfrains will be an important factor for the survival.

10.4 Conclusion

All strains screened for freeze tolerance remained viable before and after freezing and at 8 and 16%) sugar levels. Of the various pH levels studied, pH 4.5 and 4.0 were found to be defrimental for S. thermophilus 2013, L delbrueckii ssp. bulgaricus

2515 and L. acidophilus MJLAl, while rest of the organisms were not affected by pH.

Counts of yogurt bacteria were reduced by about 1 log cycle in the products made using a bench top freezer during 12 weeks storage. L. acidophilus 2415 was adversely affected and the counts declined by 6 log cycles within 8 weeks of storage at -18°C. Amongst bifidobacteria strain 1912 decreased by 5 log cycles at the end of

12 weeks storage. L. acidophilus MJLAl and bifidobacteria BDBB2 reduced by about 2 log cycles in the products made on a pilot scale after 12 weeks storage.

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11.1 Introduction

A number of health benefits have been claimed for probiotic organisms such as Lactobacillus acidophilus and Bifidobacterium spp. (Hughes and Hoover

1991; Kanbe 1992; Mital and Garg 1992). However, probiotic organisms are

unstable in such products. The loss of viability of probiotic organisms in a

frozen yogurt may be due to acidity, freeze injury and oxygen toxicity.

According to the Food Standards Code H8, the pH of the product must be

<4.5, which affects the viability of probiotic bacteria. Air is incorporated

during freezing process which affects the growth of microaerophlic L.

acidophilus and anaerobic bifidobacteria.

Survival of L. acidophilus and Bifidobacterium bifidum in ice cream was

studied by Hekmat and McMahon (1992). They reported that the coimts of I.

acidophilus and B. bifidum and their P-D-galactosidase (lactase) activity

decreased after freezing at -29°C and during frozen storage for 17 weeks.

A version of section 11 has been published. Ravula, R.R., and Shah, N.P. Australian Journal of Dairy Technology 53:175-179 (1998)

178 The survival of i. acidophilus cells during freezing at -196°C and subsequent refrigerated storage was studied by Brashears and Gilliland (1995). Organisms that assimilated cholesterol were also affected due to freezing (Brashears and

Gilliland, 1995).The authors concluded that the number of lactobacilli declined over time as did the p-D-galactosidase activity when the organisms were suspended in milk and stored at 7°C. Using ice cream as a medium to incorporate bifidobacteria and fructo-oligosaccharides into human diet was studied by Modler et al. (1990). In the presence of neosugars and Jerusalem artichoke flour, 90%) survival of bifidobacteria was observed at the end of 70 days of frozen storage. A 10%) loss in viable counts occurred at the time of freezing due to incorporation of air to achieve 100% overrun.

The effect of pH on survival of L. acidophilus and B. bifidum in frozen fermented dairy desserts was studied by Laroia and Martin (1991). L. acidophilus and B. bifidum survived the freezing process and frozen storage of

8 weeks in fermented frozen dairy desserts having pH of 5.6-5.8.

Probiotic bacteria grow slowly in milk due to lack of proteolytic activity. Dave and Shah (1997b, 1998) and Klaver (1993) have shown that milk supplemented with peptides and amino acids such as cysteine improved the survival of bifidobacteria. Cysteine is a sulfur containing amino acid that is incorporated into agar media for the growth of bifidobacteria (Laroia and

Martin, 1991). Cysteine is also known for lowering redox potential and addition of this amino acid could improve viability of anaerobic

179 bifidobacteria. Similarly acid casein hydrolysate is a product that is made from hydrolysis of casein and is rich in amino acids and peptides. Thus, acid casein hydrolysate and cysteine may provide growth factors essential for the growth of probiotic bacteria and could improve their viability.

The aim of this study was to investigate the effect of acid casein hydrolysate and cysteine on the viability of I. acidophilus and bifidobacteria in fermented frozen dairy desserts.

11.2 Materials and Methods

11.2.1 Propagation ofyogurt bacteria and probiotic bacteria

Yogurt bacteria (Streptococcus salivarius ssp. thermophilus ST WJ7 and

Lactobacillus delbrueckii ssp. bulgaricus LB Wj7) and probiotic bacteria

(Lactobacillus acidophilus LA MJLAl and bifidobacteria BB BDBB2) were obtained from Gist-brocades (formerly Mauri Laboratories Pty. Ltd.,

Moorebank, Sydney). All the strains were tested for purity by Gram stain and were propagated every week in sterile 12% reconstituted skim milk (RSM) supplemented with 2% glucose and 1% yeast extract. All the cultures were grown for 18 h at 37°C using 1% inoculum. For propagation ofbifidobacteria,

sterile RSM was supplemented with 0.05% L-cysteine. hydrochloride in order to provide anaerobic condition and to stimulate their growth. The cultures were maintained in the same medium at 4^C. Before enumeration, the cultures were fransferred successively three times for activation.

11.2.2 Manufacture of fermented frozen dairy desserts

180 Fermented frozen dairy desserts were made according to the method of Ravula and Shah (1998b). Yogurt and base mix were prepared separately and blended at a rate of 45% of each and 10% sucrose syrup was added. The blend was then frozen using a bench top ice cream freezer (Model Im 300 PK 185/w,

Kenwood Appliances, Italy).

Three batches of yogurt (3L) were made with homogenised and pasteurised

milk (1% fat) supplemented with 2% skim milk powder. The mix was heated

at 85°C for 30 min, cooled to 42°C and divided into three lots. Acid casein

hydrolysate (0.5g/L) (Sigma Chemical Co. St. Louis, MO) and cysteine

(0.05g/L) (Sigma) were added to the two batches of heated and cooled yogurt

mix to study the effect of these growth factors on viability of probiotic

bacteria in fermented frozen dairy desserts. The third batch was made with

milk supplemented with 2% skim milk powder only (control). Yogurt bacteria

(S. salivarius ssp. thermophilus STWJ7 and L. delbrueckii ssp. bulgaricus

LBWJ7), and probiotic bacteria (L. acidophilus LA MJLAl and bifidobacteria

BB BDBB2) were added at the rate of 0.5% each and the inoculated mix

incubated at 42°C till pH 4.5 was attained. The yogurt was transferred to a

cold room till the temperature decreased to 4°C. The yogurt and base mix were

mixed at a rate of 45% of each and 10% of liquid sucrose (65%) was added.

All the three batches of fermented frozen dairy desserts were made using a

bench top ice cream freezer with an overrun of 18%.

11.2.3 Microbiological analysis-

181 Microbiological analysis described as per material and method section 3.4

11.2.4 /3-D-galactosidase activity

The P-D-galactosidase activity was determined according to the method of

Shah and Jelen (1990). Ten grams of each product were mixed with distilled water, blended for 1 min and the final volume made up to 100 mL in a volumetric flask. One millilitre of the solution was used in the assay. A solution of 0.005M 0-nitrophenyl-P-D-galactopyranoside (ONGP) was prepared in O.IM phosphate buffer (pH 7.0), and 1 mL aUquots of the diluted samples were incubated with 5 mL ONGP solution for 15 min at 37°C. The reaction was stopped by adding 2.5 mL IM cold sodium carbonate. The amount of O-nitrophenol released was measured with a spectrophotometer at

420 nm. One unit of P-D-galactosidase activity was defined as the amount of enzyme which liberated one )j,mole O-nitrophenol from ONGP per min per gram sample at 37°C.

11.2.4 Compositional analyses

The protein content of the product was determined by the Kjeldahl's method using the Kjeltec digestion system and Tecator Kjeltec distillation unit. The total solids contents were determined by drying samples at 101 °C for 2 h using an air oven (Model 500, Memmert, GmbH+ Co., Schwabach) (Atherton and

Newlander, 1977). The titratable acidity was measured by tifrating 9 g of sample with O.OIN NaOH using phenolphthalein indicator (Atherton and

Newlander, 1977). The fat content was determined by the Mojonnier method

182 (Atherton and Newlander, 1977). The pH was measured witii a pH meter

(Model 420A, Orion Research Inc., Boston, U.S.A). The sugar content was determined by the colorimetric method (Dubois et al, 1956).

11.3 Results and Discussion

11.3.1 Composition

The average protein content of the product was 3.82%. The total solids contents were in the range of 31.4-32.3%. The tifratable acidity ranged between 0.77% and 0.79% . The average fat content was 10.3%, while the average sugar content was 16.9%. The pH ranged between 4.51 and 4.53. The overrun of the product was 18%.

The overrun of the commercial frozen desserts is usually 100%. However, an overrun of 100% cannot be achieved with a bench top freezer. Incorporation of air may influence the viability of microaerophlic L acidophilus and anaerobic bifidobacteria.

11.3.2 pH and titratable acidity

The initial pH of milk (6.56-6.64 at 0 h) decreased to pH 4.51-4.53 during yogurt making. In general, there was a gradual decrease in the pH in all 3 products during storage for 12 weeks. The final pH by the end of storage period was 4.45 for confrol, 4.48 for the product supplemented with acid casein hydrolysate and 4.51 for that supplemented with cysteine (Figure 11.1).

183 The final pH of yogurt or of frozen fermented dairy desserts can affect the growth and viability of L. acidophilus and B. bifidum, most notably that of bifidobacteria (Laroia and Martin, 1991; Hekmat and McMahon, 1992). It has been found that acid production by lactic acid bacteria, especially post- acidification, affected cell viability of i. acidophilus and B. bifidum (Ishibashi and Shimamura, 1993). This may be due to residual activity of lactic bacteria that produce lactic or acetic acid that decreases the pH. It is interesting to note that the products containing acid casein hydrolysate and cysteine had higher pH than the control and the two batches also showed better survival of yogurt bacteria and probiotic bacteria. Acid casein hydrolysate and cysteine may have increased buffering capacity of the product (Kailasapathy et al, 1996) and resulted in less decrease in pH.

Lankaputhra and Shah (1995) studied the survival of probiotic bacteria at various pH levels. Of the various sfrains studied, over 50% of the strains lost viability after 6 days of storage at pH <4.3. The minimum pH for survival of

L. acidophilus and bifidobacteria is 5.5 (Holt, 1986). Thus a decline in their counts is expected to occur.

The changes in tifratable acidity (TA) in fermented frozen dairy desserts is illusfrated in Figure 11.2. The initial TA of milk 0.14 increased to 0.77-0.79% after fermentation and the final acidity at the end of storage period further

184 increased to 0.82-0.89%>. The increase in TA of the confrol sample was the highest (0.89%).

11.3.3 Enzyme activity

The P-D-galactosidase activity in the three samples is shown in Figure 11.3.

The p-D-galactosidase activity declined by 34% in the control sample, whereas in the sample supplemented with acid casein hydrolysate and cysteine the enzyme activity declined only by 10% and 5.5%, respectively. This may be due to buffering action of acid casein hydrolysate and of cysteine, which may have provided protection to organisms resulting in increased enzyme activity.

Hekmat and McMahon (1992) observed that P-D-galactosidase activity was lost more rapidly in a refrigerated yogurt than in a frozen yogurt. Mashayekh and Brown (1992) observed that 20% of P-D-galactosidase activity in the yogurt was lost after 30 days of refhgerated storage, whereas in a frozen ice cream only 11% activity was lost during the same storage period. Speck and

Geoffrion (1980) also found about 50% reduction in the P-D-galactosidase activity of unfrozen yogurt during a 20 days storage period, but there was no ftirther decrease in the P-D-galactosidase activity of the frozen yogurt.

185 11.3.4 Viable counts ofyogurt bacteria and probiotic bacteria

Figure 11.4 shows the viable counts ofyogurt bacteria in the three batches of fermented frozen dairy desserts. In all the three batches, the counts of S. thermophilus remained high until the end of the storage period. There was approximately 2 log cycle decrease in the counts of L delbrueckii ssp. bulgaricus and the counts decreased to 10^ cfu/g by the end of 12 week storage period. However, for the product supplemented with acid casein hydrolysate and cysteine, the counts of L delbrueckii ssp. bulgaricus decreased by 1 log cycle only. The viable counts of probiotic bacteria are shown in Figure 11.5. The counts of L. acidophilus and bifidobacteria decreased rapidly in the control sample and the viable counts were <10 cfu/g by the end of 12 weeks storage period. The decrease in the counts of L. acidophilus and bifidobacteria in samples supplemented with acid casein hydrolysate or cysteine was less than those with the control sample and the viable counts at the end of 12 weeks storage period was >10^ cfu/g. Improved survival and viability of L. acidophilus and bifidobacteria are possibly due to nufrients such as peptides and amino acids supplied through acid casein hydrolysate and cysteine and due to decreased redox potential. Improved viability could have also been due to buffering action of acid casein hydrolysate and cysteine, which resulted in less drop in pH (Figure lA).

Cysteine is a redox-potential reducing agent and is an essential amino acid required for the growth of bifidobacteria. Thus improved survival of probiotic bacteria, in particular bifidobacteria in fermented frozen dairy desserts could also due to redox potential reducing property of cysteine.

186 Effect of cysteine, whey protein concenfrate, acid casern hydrolysate and tryptone on viability of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and bifidobacteria was studied by

Dave and Shah (1998). They reported that the addition of these mgredients improved viability of L. acidophilus and bifidobacteria by providing growth factors as these probiotic bacteria lack proteolytic activity.

Air is incorporated in fermented frozen dairy desserts and air may affect survival of microaerophilic L. acidophilus and anaerobic bifidobacteria. This study was carried out using a bench top ice cream freezer and the level of overrun was only 18%, whereas the level of overrun could be 100% for commercial ice cream freezers. Therefore, in commercial practice, the level of acid casein hydrolysate or cysteine may need to be increased in order to provide sufficient peptides and amino acids, and to decrease redox potential for improved viability of probiotic bacteria.

11.4 Conclusion

Survival of yogurt and probiotic bacteria was studied in fermented frozen dairy desserts, made from milk with or without supplementary acid casein hydrolysate and cysteine, at fortnightly intervals for 12 weeks. Initial counts of the bacteria and their subsequent survival were better in the supplemented products due to nutrients such as peptides and amino acids and due to reduced redox potential. The P-D-galactosidase activity declined more rapidly in the

187 confrol sample than in the samples supplemented with acid casein hydrolysate or cysteine. The results suggested that the acid casein hydrolysate and cysteine stimulated the growth of L. acidophilus and bifidobacteria which resulted in improved viability of these organisms. CN

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12.1 Introduction

Bacteria differ in their susceptibiUty to freezuig and thawing.

Cryoprotective agents provide protection against freeze-injury. Cellular damage is most likely due to elevated solute concentration (which causes cell dehydration) and membrane destruction by intracellular ice crystals.

The addition of cryoprotectants results in greater survival rate during freezing and frozen storage by binding water and inhibiting either intracellular or extracellular ice crystal formation. The presence of most cryoprotectants results in formation of extracellular amorphous ice glass instead of crystalline ice upon freezing. These agents include sorbitol, sucrose, sodium tripolyphosphate, glucose, xylitol, glycerol, raffinose and maltodextrin (Thunnell et al, 1984).

The effect of various cryoprotectants on the viability of freeze-dried lactic acid bacteria has been studied by Valdez et al (1985). Adonitol produced

smallest changes in water content at various times during freeze drying and

allowed the highest rate of survival. Use of P-glycerophosphate as a

cryoprotective agent gave greatest protection to L. acidophilus.

Kilara et al (1976) reported that the extent of cryoprotection afforded by

any given agent varied from culture to culture. Malt extract and milk solids

194 as the cryoprotective agents resulted in the survival ratio of 58 and 80% for

I. delbrueckii ssp. bulgaricus, respectively, while v^th L. acidophilus the same two cryoprotectants provided 89 and 48% survival. Whey powder improved viability of L. delbrueckii ssp. bulgaricus and pectin that of S. thermophilus. For improved viability of probiotic and yogurt organisms, a combination of cryopotectants may be required.

The objective of this study was to determine the effect of cryoprotective agents on improving the viability of probiotic bacteria in fermented frozen dairy desserts.

12.2 Materials and Methods

13.2.1 Propagation ofyogurt bacteria and probiotic bacteria

Yogurt bacteria (Streptococcus thermophilus STWJ7 and Lactobacillus delbreuckii ssp. bulgaricus LBWJ7) and probiotic bacteria (Lactobacillus acidophilus LA MJLAl and bifidobacteria BB BDBB2) were selected and propogated as described previously (Ravula and Shah, 1998a).

13.2.2 Preparation of cryoprotectedprobiotic bacteria

The organisms were transferred three times successively in MRS broth and added to sterile MRS broth. L-cysteine was added to the broth for bifidobacteria. The fermentation was terminated after 16h. The broth was cooled to 4°C and transferred to centrifuge tubes and centrifuged at 4000 rpm for 25 min. The supernatant was discarded and the cell pellets were

195 washed with cold O.IM phosphate buffer (pH 6.8). The washed cell pellet

(10%) was re-suspended into 50 ml of O.IM phosphate buffer containing

2.0% level (w/v) of cryoprotectants (pectin, sodium alginate, xanthan gum, guar gum, glucose, locust bean gum, fructose, glucose and glycerol). The cell pellet was allowed to mix xmiformly in the phosphate buffer and the mixture was transferred into petri-plates. The organisms were frozen before freeze drying. Viable counts were monitored after inoculation, in the MRS broth, after 18 hrs of fermentation, and in cell pellets before and after freeze-drying. The freeze dried cryoprotected probiotic bacteria was added to blend mix of yogurt and base mix and fermented fi-ozen dairy desserts were made using an ice cream freezer. Viable counts were monitored at fortnightly interval for 12 weeks. A similar procedure was used to prepare a control batch except that no the cryoprotective agent was used. All experiments were carried out in duplicate.

12.2.3 Fermented frozen dairy desserts

Fermented frozen dairy dessert were made according to the method of

Ravula and Shah (1998b). Cryoprotective agents were added at a rate of

0.5% to yogurt mix at a temperature of 30-35°C before pasteurising at

85 C for 30 min. Various cryoprotective agents were initially evaluated for their effect on texture and the best resulting cryoprotectants were used in this study. The cryoprotectants that were used includ: RS 150 (pectin).

Glucose and GCF 639 (alginate). These cryoprotectants were of food grade standard and are suitable for the manufacture of dairy products.

196 The final product was made by blending 45%) yogurt, 45%) of base mix and

10% of 65%) syrup (liquid sucrose) followed by passing through an ice cream freezer. The control batch and the batches containing RS 150,

Glucose and GCF 639 were made using a bench top ice cream freezer (1 L batch; 18%) overrun). Two batches of fermented frozen dairy desserts were made with cryoprotectant RS 150 and without cryoprotectant (control) using a commercial freezer (20 L batch; 100% overrun). The fermented frozen dairy dessert was filled in plastic containers and stored at -18°C for

12 weeks

12.2.4 Enumeration of probiotic bacteria

MRS-salicin and MRS-sorbitol agars were used for the selective enumeration of L. acidophilus. The population of bifidobacteria was enumerated on MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride, paromomycin sulfate) agar.

12.3 Results

12.3.1 pH and titratable acidity

The initial pH of milk (6.51-6.67 at 0 h) decreased to pH 4.52-4.59 during yogurt making. In general, there was a gradual decrease in the pH in all the products during storage for 12 weeks. The pH by the end of storage period was 4.41-4.46. The TA of yogurt varied between 0.71 to 0.78%) after fermentation and the acidity varied between 0.79 to 0.86%) at the end of the storage period.

197 12.3.2 Viability

Figures 12.1 -12.4 show the viable counts of probiotic bacteria in the 4 batches of fermented dairy desserts made using a bench top freezer. The counts of L acidophilus (Figure 12.1) and bifidobacteria (Figure 12.2) at the end of 12 weeks storage period was >10^ cfii/g in all the products except the product containing glucose as the cryoprotectants, which had a viable count of <10'* cfu/g.

Figures 12.3 and 12.4 shows the viable counts of probiofic bacteria in the two batches of fermented frozen dairy desserts made using a continuous freezer on a pilot scale. The counts of Z. acidophilus (Figure 12.3) and bifidobacteria (Figure 12.4) decreased rapidly in both products and the viable counts were <10 and <10 cfu/g, respectively by the end of the 12 week storage period. Loss of viability of L. acidophilus and bifidobacteria in the products made using the commercial freezer was possibly due to presence of air. Air is incorporated during the manufacture of fermented frozen dairy desserts to achieve 100% overrun, which may affect the growth of microaerophilic L. acidophilus and anaerobic bifidobacteria. The products containing cryoprotectants performed better than without cryoprotectants for improving viability of probiotic bacteria. Glucose did not help improve their survival. Overall, there was a slight improvement in the viable counts of L. acidophilus and Bifidobacterium spp. in the presence of the cryoprotectant RS 150.

198 12.4 Conclusions

Survival of probiotic bacteria was studied in fermented frozen dairy desserts made with or without cryoprotectants using a bench top and a

commercial freezer. The organisms lost their viability faster in the products

made using a commercial freezer. Overall, the viability improved with

cryoprotectants RS 150 (pectin) and GCF 639 (alginate) The results suggest

that use of RS 150 and GCF 639 cryoprotectants in fermented frozen dairy

desserts might improve the viability of probiotic bacteria.

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13.1 Introduction

Several health benefits associated with the consumption of probiotic organisms such as Lactobacillus acidophilus and Bifidobacterium spp. have been claimed

(Kearney et al, 1990; Hughes and Hoover 1991; Kanbe 1992; Mital and Garg,

1992). However, these organisms do not survive in frozen yogurt or fermented frozen dairy desserts (Hekmat and McMahon, 1992; Ravula and Shah, 1998b).

The loss in viability of probiotic organisms in frozen yogurt or fermented frozen dairy desserts was claimed to be due to acidity of the product, freeze injury and oxygen toxicity (Lankaputhra and Shah, 1996a). According to the Australian

Food Standards Code H8, the pH ofyogurt must be <4.5; such low pH affects the viability of probiotic bacteria. Air is incorporated during the freezing process in production of fermented frozen dairy desserts to achieve desired overrun and oxygen is found to affect the growth of microaerophilic L. acidophilus and anaerobic bifidobacteria (Ishibashi and Shimamura, 1993; Dave and Shah, 1997a, b).

A revised manuscript of this chapter has been submitted. Int. Dairy J. (2000). Ravula, R. R., and

Shah, N.P.

204 Methods to preserve probiotic bacteria in ice cream and frozen fermented dauy desserts have been reported by Laroia and Martin (1991) and Hekmat and

McMahon (1992). The survival of probiofic bacteria was reported to be unsatisfactory.

Microencapsulation is a process where the cells are retained within the

encapsulating material in order to reduce the cell injury or cell loss.

Encapsulation of lactic starter cultures is of interest to the dairy industry since it

improves the control of the fermentation process (Champagne and Cote, 1987;

Champagne, 1990).

In some food formulations, encapsulated cells are found to be more active than

free cells (Kearney et al, 1990). Encapsulation of bacterial cells in calcium

alginate beads is simple. The advantage of encapsulation using natural polymers

is that the reagents are non-toxic and the matrices formed are gentle to the

microorganisms (Lacroix et al, 1990). The other advantage is that calcium

alginate can be solubilized by sequestering calcium ions thus releasing the

entrapped cells.

Sheu and Marshall (1993) studied the survival of entrapped culture bacteria in

frozen ice milk. The viability of bacterial cells was markedly improved by micro-

entrapment of cells in calcium alginate gels and 40%) more lactobacilli survived

freezing of ice milk. Thus addition of entrapped probiotic bacteria to frozen dairy

205 desserts could be advantageous to provide desirable marketing and health benefits.

Viability and acid production activity of immobilised Lactococcus lactis ssp. lactis within cross-linked gelatin micro-capsules have been reported by Hyndman et al. (1993). After encapsulation, activity and viability of the immobilised culture was increased by cell growth within the capsules.

This study was focused on factors necessary for producing desirable bead size as well as on the effects of acid, bile, pH of phosphate buffer and stirring time on the release of the organisms from the beads. The influence of micro-entrapment on survival of probiotic organisms in fermented frozen dairy desserts was confirmed in an industrial setting.

13.2 Materials and Methods

13.2.1 Propagation of probiotic bacteria

Probiofic bacteria (Lactobacillus acidophilus LA MJLAl, LA 2415, LA 2400,

LA 2401 and Bifidobacterium spp. BB BDBB2, BB 20099, BB 1900, BB 1941) were selected based on our previous study (Ravula and Shah, 1998b). All the sfrains were tested for purity by Gram stain and sugar fermentation tests. The organisms were propagated every week in sterile \2% reconstituted skim milk

(RSM) supplemented with 2%o glucose and 1% yeast extract. All the cultures were grown for 18 h at 37°C using !%> inoculum. For propagafion of bifidobacteria, sterile RSM was supplemented with 0.05%) L-cysteine

206 hydrochloride in order to provide anaerobic condition and to stimulate thefr growth. The cultures were maintained in the same medium at 4°C. Before enumeration, the cultures were transferred successively three times for activation.

13.2.2 Preparation of encapsulated beads

Both probiotic bacteria were microencapsulated separately according to the flow diagram shown in Figure 13.1. The organisms were grown in sterile RSM (12%o) containing glucose (\%) and yeast extract (0.5%)). Each organism was then transferred twice successively in deMan, Rogosa, and Sharpe (MRS) broth for 18 h at 37°C. The organisms were harvested by centrifuging (3838 x g, 25 min,

4°C), washed, and suspended in saline to obtain 10'° cells mL'\ The cell suspension was mixed with equal amount of 3%) sodium alginate. The mixture was added drop-wise to 5 parts of vegetable oil containing Tween 80. A turbid emulsion was obtained. Calcium chloride (0.05%)) was gently added to the bottom of the flask with the help of a pipette until the emulsion was broken. The calcium alginate encapsulated beads formed were collected by decantation, freeze dried, and incorporated into a mix prior to freezing for manufacturing of fermented frozen dairy desserts as detailed under the manufacture of fermented frozen dairy desserts section.

13.2.3 Effects of surfactant and emulsifier on bead size

Effects of a surface tension reducing agent (sodium lauryl sulfate) and an emulsifier (Tween 80) on bead size were investigated. Several concentrafions (1 to 4%) of sodium alginate were examined for their effect on bead size, and a 3%)

207 concenfrafion of sodium alginate was found to be optimal. Surface tensions of 3%) sodium alginate solution were varied by adding 4 levels of sodium lauryl sulfate

(0, 0.1, 0.5 and 1.0%), while four concentrations of Tween 80 (0, 0.1, 0.2 and

0.3%)) were added to the oil phase.

The diameters of beads were measured vsdth an eyepiece micrometer using an optical microscope at a magnification of 400x or lOOOx after staining with safranin according to the method of Sheu and Marshall (1993). At least 80 randomly selected beads were measured for each sample.

13.2.4 Measurement of diameter of calcium alginate beads

Diameters of encapsulated beads were measured with an eyepiece micrometer on an optical microscope at a magnification of 400x or lOOOx. At least 80 randomly selected beads were measured for each sample.

13.2.5 Scanning electron microscopy

A scanning electron microscope was used to examine the extemal and intemal appearances of freeze-dried beads. For extemal structure, intact beads were attached to aluminium stubs using conductive carbon paint (CCP) (Probing and

Structure, now Pro Sci Tech., Thuringowa, Australia). For intemal structure, the freeze-dried beads were mechanically broken using a fine tweezer and mounted on aluminium stubs using CCP.

208 The specimens were coated with a thin layer of gold using an Edwards Sputter

Coater. The extemal and intemal structures were observed using a Philips

SEM515 scanning electron microscope at 20 kV.

13.2.6 Release of probiotic organisms from microencapsulated beads

One gram of encapsulated beads was added to 9 ml of phosphate buffer at various pH (4.5, 5.5, 6.5 and 7.5). One gram of organisms were further diluted with 59 mL of sterile distilled water. One gram of the mixture containing beads and distilled water were added to 9 mL of peptone water and counts were enumerated by using appropriate peptone water dilutions.

13.2.7 Release of entrapped bacteria from microencapsulated gel beads

The effects of pH of phosphate buffer, stirring time and of acid and bile on release of probiotic bacteria from freeze dried encapsulated beads were carried out. One gram of encapsulated beads was added to 59 mL of sterile distilled water and mixed. One millilitre aliquot from this mix was added to 9 mL of a phosphate buffer in test tubes adjusted to various pH levels (4.5, 5.5, 6.5, 7.5, and

8.5) with mono- or di-sodium phosphate followed by stirring at 200 rpm for 0, 5,

10 or 15 min. The released bacterial populations were enumerated using the pour plate technique as outlined above.

209 13.2.8 Effect ofacid on release of entrapped bacterial cells from encapsulated beads.

The effects of lactic acid on the release of probiotic bacterial cells from calcium alginate beads were studied by adding 0.1 %o of beads to sterile distilled H2O adjusted to pH 2.5, 3.5 and 4.5 using lactic acid, followed by incubation of the mixture for 3 h at 4°C. Samples were taken at hourly intervals for 3 h for enumeration of viable counts. The beads incubated in acid were then added to phosphate buffer (pH 7.5) and plated for enumeration after stirring for 5 minutes

at 200 rpm in order to verify the levels of cells released.

13.2.9 Effect of bile on bacterial cells from encapsulated beads in (bile)

The effects of bile on the release of probiotic bacterial cells from calcium alginate

beads were investigated by adding 0.1%) of beads to solutions of bile salts (0.1,

0.5 and 1%) prepared in sterile distilled water, followed by incubation of the

mixture for 3 h at 4°C. Samples were taken at hourly intervals for 3 hours for

enumeration of bacterial counts. The beads incubated in bile salts were also

incubated in phosphate buffer (pH 7.5) and plated for enumeration to verify the

levels of cells released.

13.2.10 Manufacture of fermented frozen dairy desserts

The industrial procedure for manufacture of fermented frozen dairy desserts

typically involves mixing of yogurt (45%)), base mix (45%) and liquid sugar

(10%) (Ravula and Shah, 1998b). The blend mix is then frozen while

210 incorporating air to achieve up to 100%) overrun, sknilar to the manufacture of ice

cream. Unlike ice cream, the pH of fermented frozen dairy desserts must typically

be <4.5.

Two batches ofyogurt (lOL) were made with homogenised and pasteurised milk

supplemented with 2% skim milk powder. The mix was heated at 85°C for 30

min, cooled to 42 C and commercial yogurt starter bacteria (Streptococcus

thermophilus STWJ7 and Lactobacillus delbrueckii ssp. bulgaricus LBWJ7)

were added at the rate 0.5%) of each and the inoculated mix incubated at 42°C.

The fermentation was terminated at pH 4.5 and the yogurt was stored in a cold

room (4°C) for 18h (Figure 1). Similarly the base mix was prepared as shown in

the flow diagram in Figure 1. The yogurt, base mix and liquid sugar were mixed

and two batches of fermented frozen dairy desserts were prepared using a

continuous ice cream freezer. One batch of the blend contained micro-entrapped

probiotic bacteria (L. acidophilus MJLAl and Bifidobacterium spp. BDBB2), and

for the other batch non-encapsulated cells were added; in both cases the rate of

addition was 0.5 g L''. The enumeration of I. acidophilus and Bifidobacterium

spp. was carried out at fortnightly intervals for 12 weeks.

13.2.11 Enumeration of bacteria

MRS-salicin agar and MRS-sorbitol agar were used for the selective enumeration

of L acidophilus (Dave and Shah, 1996; Lankaputhra and Shah, 1996). The

population of bifidobacteria was enumerated on MRS-NNLP (nalidixic acid.

211 neomycin sulfate, lithium chloride, and paromomycin sulfate) agar as reported by

Dave and Shah (1996).

All analyses were carried out in duplicate, and all the experiments were repeated at least twice. The results presented are the average of all data.

13.3 Results and Discussion

The use of calcium alginate to microencapsulate cells of lactobacilli was reported by several workers (Nilsson et al, 1983; Sheu and Marshall, 1993; Kim, 1988).

Rao et al. (1989) reported the survival of microencapsulated Bifidobacterium pseudolongum in simulated gastric and intestinal juices. However, details of gel

structure and survival of L. acidophilus and Bifidobacterium spp. in fermented

frozen dairy desserts were not reported. Similarly, the effects of acid and bile on

release of L acidophilus and Bifidobacterium spp. have not been previously

studied. To provide any health benefits, probiotic bacteria must survive in acidic

conditions encountered in the stomach and bile concentrations in the intestine

(Lankaputhra and Shah, 1995; Rao et al, 1989).

13.3.1 Composition

The protein content of the product was 3.82%o. The total solids contents were in

the range of 31.6-33.5%). The titratable acidity ranged between 0.76%o and 0.79%).

The fat content was 10.3%), while the sugar content was 16.7%o. The pH ranged

between 4.51 and 4.53

212 13.3.2 pH and titratable acidity

The inifial (at 0 h) pH of milk (6.56-6.64) decreased to pH 4.56-4.58 during yogurt making. In general, there was a gradual decrease in pH in all tiie products during storage for 12 weeks. The final pH by the end of storage period was 4.50-

4.44.

The initial TA of milk 0.14 increased to 0.72-0.76%) after fermentation and the final acidity further increased to 0.75-0.82%o at the end of storage period. The increase in TA of the control sample was the highest (0.79%).

13.3.4 Effect of surfactant and emulsifier on bead size

Emulsifier is necessary to effect a water/oil emulsion. Tween 80 was used as an emulsifier because it associated with the oil phase. With high association in the oil phase (having high hydrophilic and lipophilic balance value), the Tween 80 was easily removed from the surface of the spheres causing disruption of water/oil emulsion (Govin and Leeder, 1971; Nawar, 1985; Sheu and Marshall,

1993). Tween 80 was essential to prevent spheres from coalescing before the breaking of the emulsion.

For frozen fermented dairy desserts, the diameter of calcium alginate beads is an important aspect as too large a diameter may be detected in the mouth. The mean diameter of the beads without any surfactant or emulsifier was 1.17 mm (Figure

13.2). The diameter of the beads ranged from 0.82mm to 1.44mm.

213 A solufion having a low surface tension resulted in small spheres (Sheu and

Marshall, 1993). Sodium lauryl sulfate, a surfactant, was used to lower the surface tension in the alginate mix in order to reduce the size of the spheres. The solution having lower surface tension resulted in smaller beads (Figure 13.2). As the concentration of the sodium lauryl sulfate increased the viscosity decreased.

As the concentration of sodium lauryl sulfate and Tween 80 increased, the diameter of calcium alginate beads decreased. At 0.3% Tween 80 and 1% sodium lauryl sulfate, the mean diameter was 0.064 mm. The bead size in the absence of any emulsifier or surfactant of 1.17 mm decreased to 0.064 mm in the presence of

0.3%o of Tween 80 and 1%) of sodium lauryl sulfate. Thus, there was approximately 18 times reduction in the size of beads due to addition of Tween

80 and sodium lauryl sulfate.

Figures 13.4-13.21 show electron micrographs of a calcium alginate beads showing the extemal appearance and cross section of beads. The beads appear to be spherical and 1.3 to 1.4 mm in diameter. The microorganisms appear to be located in the matrices formed within the beads.

13.3.5 Effect of concentration of sodium alginate on bead size

214 Several concentrations (1 to 4%) of sodium alginate were examined for their effect on bead size. At a stirring speed of 200 rpm, the apparent viscosity increased with an increase in concentration of sodium alginate from 1 to 4%, making it difficult to form a smooth gel, especially at 4%> concentration. Higher concentrations of sodium alginate resulted in larger size bead formation, with the mean diameters approaching a plateau when the concentration of sodium alginate was 3%). Thus, a 3% concentration of sodium alginate was used for the rest of the experiments, imless otherwise indicated.

13.3.6 Release of entrapped bacteria from microencapsulated gel beads

In order to estimate the survival of probiotic bacteria in fermented frozen dairy desserts and to investigate the effects of freezing of encapsulated bacteria, it was important to determine optimum conditions including pH of the phosphate buffer and stirring time in the buffer for complete release of the entrapped bacteria.

Releasing was carried out after freeze drying of the encapsulated beads. The pH of the phosphate buffer, which ranged from 4.5 to 8.5, did not have any effect on the release of the organisms from the beads. A 5-minute stirring at 200 rpm resulted in complete release of bacterial cells from the encapsulated beads (Figure

13.3).

215 13.4.7 Release of entrapped bacterial cells from encapsulated beads acidic conditions

The encapsulated beads were inoculated for 3 h in lactic acid at various pH (2.5,

3.5 and 4.5) and the viable counts showed that lactic acid had no effect on the release of probiotic bacteria. This was confirmed by adding the acid treated beads to phosphate buffer (pH 7.5) and plating. The viable counts as determined in

MRS-salicin agar (for L. acidophilus) and MRS-NNLP agar (for bifidobacteria) varied from 6.62 to 6.72 log cfu g"'. The counts after release from beads in

phosphate buffer were similar. This may suggest that the organisms could survive

in acidic condifions generally encountered in the stomach (Figure 13.22).

13.3.8 Release of entrapped bacterial cells from encapsulated beads in bile

The encapsulated beads were incubated for 3 h in bile salts (0.1, 0.5 and l%o)

alone and bile and phosphate buffer. It was possible that tiie organisms may not

have been completely released from the beads in the presence of bile. Therefore,

the organisms from tiie beads were released using phosphate buffer to verify the

results. The levels of organisms released in the presence of bile were similar to

those released in the presence of bile and phosphate buffer (Figure 13.23),

suggesting that the organisms could be completely released in the intestine in the

presence of bile.

216 13.3.9 Effects of addition of beads on product manufacturing

The products made with encapsulated and non-encapsulated cells were analysed for overrun and mouth feel. It was observed that there were no much difference m overrun and in the taste of the both samples.

13.3.10 Viable counts of probiotic bacteria in fermented frozen dairy desserts made using continuous ice cream freezer on a pilot scale

Figures 13.24-13.31 show the viable counts of probiotic bacteria in eight batches of fermented frozen dairy desserts. The counts L. acidophilus and

Bifidobacterium spp decreased in the control samples and the viable counts ranged between 10 .10 cfu/mL by the end of 12 weeks storage period. The decrease in the counts of L. acidophilus and Bifidobacterium spp. in samples where encapsulated bacterial cells were used were less than those with the control sample and the viable counts at the end of 12 weeks storage period were >10^ cfu/g. Improved survival and viability of L acidophilus and bifidobacteria were possibly due to the protection given by entrapment in calcium alginate gels.

Studies have shovm that bifidobacteria did not survive well in fermented products at pH 4.6 and below (Laroia and Martin, 1991), whereas the studies by Rao et al

(1989) shown that micro-encapsulated bifidobacteria survived in large numbers than non-encapsulated bacteria in gastric and intestinal juices. Reduction of culture bacteria in frozen yogurt was observed after freezing (Mashayekh and

Brown, 1992). About 40%) more lactobacilli survived freezing of ice milk when they were entrapped in calcium alginate beads (Sheu and Marshall, 1993).

217 13.4 Conclusion

Fermented frozen dairy desserts typically have pH of 4.5. Such low pH affects the survival of probiotic organisms. Encapsulation of bacterial cells in sodium alginate provided protection to probiotic organisms in fermented frozen dairy desserts. The viable population decreased during storage, however, there was 2-3 log cycle improvement in the viable counts due to encapsulation. The

encapsulated probiotic bacteria also survived in acidic conditions (at pH 2.5) in

calcium alginate beads; however, the organisms were released in the presence of

bile. The study suggests that encapsulated probiotic bacteria incorporated in

fermented frozen dairy deserts could survive in low pH of the product and in

acidic conditions such as encountered in the human stomach and could be

delivered in the intestine.

218 Legends to figures (electron micrographs)

Figure 13.4 - Scanning election micrograph of LA 2415 encapsulated in calcium alginate bead

Figure 13.5 - Scanning electron micrograph of LA 2415 encapsulated in calcium alginate bead (cross section).

Figure 13.6 - Scanning electron micrograph of LA 2415 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

Figure 13.7 - Scanning electron micrograph of LA 2401 encapsulated in calcium alginate bead.

Figure 13.8 - Scanning electron micrograph of LA 2401 encapsulated in calcium alginate bead (cross section).

Figure 13.9 - Scanning electron micrograph of LA 2401 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

Figure 13.10 - Scanning electron micrograph of LA MJLAl encapsulated in calcium alginate bead.

Figure 13.11 - Scanning electron micrograph of LA MJLAl encapsulated in calcium alginate bead (cross section).

Figure 13.12 - Scaiming electron micrograph of LA MJLAl encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

Figure 13.13 - Scanning electron micrograph of BB 1900 encapsulated in calcium alginate bead.

Figure 13.14 - Scanning electron micrograph of BB 1900 encapsulated in calcium alginate bead (cross section).

Figure 13.15 - Scanning electron micrograph of BB 1900 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

Figure 13.16 - Scanning electron micrograph of BB BDBB2 encapsulated in calcium alginate bead.

219 Figure 13.17 - Scanning electron micrograph of BB BDBB2 encapsulated in calcium alginate bead (cross section).

Figure 13.18 - Scaiming election micrograph of BB BDBB2 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

Figure 13.19 - Scanning election micrograph of BB 1941 encapsulated in calcium alginate bead.

Figure 13.20 - Scanning electron micrograph of BB 1941 encapsulated in calcium alginate bead (cross section).

Figure 13.21 - Scanning electron micrograph of BB 1941 encapsulated in calcium alginate bead (showing the location of the organism in the matrix of the bead).

220 Method of vogurt base mix microencavsulation of probiotic bacteria

Probiotic bacteria Homogenised and Homogenised and (L. acidophilus or pasteurised milk pasteurised milk bifidobacteria) i i i Incubation 37°C/18h Tempering (40-45°C) Tempering (40-45°C) i i i Centrifugation Addition of skim milk Addition of ingredients (4000 rpm 25 min.) powder 2% (skim milk powder, cream, sugar and stabiliser) i i i Cell pellet Heat treatment (85°C/30 min) Heat treatment (85°C/30 mir i i i Mixing with Cooling to incubation Cooling(~4°C) sodium alginate (3%)) temperature (42°C) i i i Addition of mix into veg. oil Addition of selected strains Ageing of yogurt bacteria i i Addition of calcium chloride Filling in container i ^^ Collecting the beads Incubation at 42°C till the pH reaches to 4.5 i i Freeze drying Storage at 4-6°C for 12h

Blending yogurt and base mix i

.iVLlU-'VddJi tion of freeze dried encapsulated beads

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00 r-^ vo m Tj- en (N Sjunoo 9]qBiA So^ 14.0 ACID AND LOW TEMPERATURE ADAPTATION OF PROBIOTIC BACTERIA FOR INCORPORATION IN FERMENTED FROZEN DAIRY DESSERTS

14.1 Introduction

Acid and low temperature adaptations are a novel method where the cells are exposed to a particular mild acidic environment and followed by exposure to lethal acidic environment and the organism frozen for various time and temperature combinations in order to see any proteins released during freezing.

These proteins help the organisms cope under stressful conditions.

While the physiology, biochemical and genetic mechanisms of many of these responses have not been delineated, what is clear is the following: When exposed to a mild dose of stress, microorganisms may adapt to the stress, thus developing tolerance or resistance to stronger doses of stress. Also it is becoming increasingly clear that the exposure and subsequent adaptation to one stress can confer resistance to other different stress (Berry and Foegeding, 1997).

The initial discovery of this response was made by Jones et al. (1987) in E. coli growing at 37°C and shifted to 10°C. The growth halts for approximately 4 hours before exponential growth is resumed (Jones et al. 1987; Ng et al, 1962). During tills lag period, there is an induction of some 16 proteins, called the cold shock proteins (Jones et al. 1996; La Teana et al, 1991; Lelivelt and Kawula, 1995;

iones etal, 1992)

252 Most microorganisms must accommodate to a variety of changing conditions and stress in their environment in order to survive. Because of impact of temperature on all reactions of the cell, adaptations to fluctuations in temperature are possibly the most common. In many organisms specific sets of cold shock proteins are induced upon abrupt shifts to colder temperatures. While this cold shock response has not been fully delineated, it appears to be adaptive and may function to promote the expression of genes involved in translation when cells are displaced to low temperatures. The cold shock response of Escherichia coli has been extensively studied and the major cold shock protein CspA appears to be involved in the regulation of response. Upon cold shock, the induction of CspA and its counterparts in most microorganisms studied is prominent, but transit.

Studies of this response in some psychrotrophic bacteria have reported constitutive synthesis and continued synthesis during cold temperature growth of

CspA homologues (Berry and Foegeding, 1997).

Freezing is one of the main factors in manufacturing of fermented frozen dairy desserts. Intracellular ice formation during freezing has been described for many cellular systems, and the rate at which ice is formed determines the type of freezing damage suffered by cells (Mazur, 1970; Toner et al. 1993). Cells can be injured during freezing by physical factors such as ice crystal formation and dehydration. At high freezing rates, intiacellular freezing occurs, leading to cell damage mainly by ice crystal formation. However, at low freezing rates, extracellular ice formation predominates, leading to intracellular dehydration

(Mazur, 1970). The pretieatment of cells with a mild stress induces higher stress

253 tolerance and in some cases tiiis confers cross protection to otiier, different types of stress (Mager and Ferreira, 1993). It has been assumed tiiat different stress conditions act through a variety of effects in cells, such as generation of abnormal

or denatured proteins, intemal acidification, alterations in the cytoskeleton or

modulation of second-messenger levels, to produce signals tiiat can provide

coverage and stimulate more general stress-responding system (Chowdhury et al,

1993; Coote et al, 1994; Craig and Gross, 1994).

The freeze-thaw stress response of yeast Saccharomyces cerevisiae is growth

phase specific and is controlled by nutritional state via the Ras-cyclic AMP signal

transduction pathway as studied by Jong and Ahn (1997). The authors concluded

that the ability of cells to survive freezing and thawing is expected to depend on

the physiological conditions experienced prior to freezing. They examined factors

affecting yeast cell survival during freeze thaw stress, including those associated

with growth phase, requirement for mitochondrial functions and prior stress

tieatment. Supercooling occurred without reducing cell survival and was

followed by freezing. Loss of viability was proportional to the freezing duration,

indicating that freezing was the main determinant of freeze-thaw damage.

The aim of this study was to adapt L. acidophilus and bifidobacteria to acidic and

low temperature conditions so that they can survive in fermented frozen dairy

desserts.

254 14.2 Materials and Methods

14.2.1 Ice cream freezer

A bench top ice cream freezer (2 L size) was used to make laboratory scale fermented frozen dairy desserts as described in section 3,2.2.

14.2.2 Centrifuge and microcentrifuge

Large volumes (100 to 1000 mL) of samples were centriftiged using Beckmann

refrigerated J2-HS and for medium volumes (10 to 100 mL) of samples

Beckmann refrigerated centrifuge and for small volumes microcentrifuge

(Beckmann) were used as described in section 3.2.5

14.2.3 Bacterial cultures and maintenance

The bacterial cultures and maintenance are described in section 3.3.

14.2.4 Lactic acid bacteria and probiotic bacteria

Yogurt bacteria and probiotic bacteria were described in section 3.3.1

14.2.5 Microbiological analyses

Microbiological analyses were described in section 3.3.3

14.2.6 Determination of the growth curve

Probiotic bacteria, L. acidophilus and Bifidobacterium spp., were propagated three times before inoculating in MRS broth (L-cysteine was added for

bifidobacteria). The inoculated MRS broth was incubated at 37°C and hourly

absorbance readings were taken from cuvettes, using a spectrometer at 450 nm.

The pH of the broth was also measured at hourly intervals using a pH meter. A

growth curve was plotted to identify the log phase of the organisms.

255 14.2.9 Peptone and water diluent

As described in section 3.16.1

14.2.10 Preparation of fermented frozen dairy desserts

As described in section 3.17

14.2.11 Acid and low temperature adaptations of probiotic bacteria and their incorporation into fermented dairy desserts

Two adaptation conditions were used: one with sublethal pH of 5.5 and the other with lethal pH of 3.5. For adaptation studies, the organisms were propagated three time in MRS broth (0.05%o cysteine was added for bifidobacteria) at 2% level and incubated at 37°C for 18 h. The fermentation was slowed down by transferring the samples into a refrigerator and the temperature was decreased to

4°C. The initial pH of the sample was assessed with a pH meter and the sample plated for enumeration for viable counts. Ten millilitres of the samples were stored in a freezer for gel-electiophoresis. The pH of the broth (100 ml) was increased to 6.5 using 10%) NaOH and the broth kept in a refiigerator for one hour. After 1 h of refrigeration (temperature adaptation), the broth was divided into two portions (A & B) of 50 ml each and the pH in the sample A decreased to lethal pH of 3.5. The sample B was kept at sublethal pH of 5.5 for one hour before decreasing the pH to the lethal pH of 3.5. The samples A and B were maintained at this pH (3.5) for 3 h, followed by freeze stressing at -20°C for 1 hour. The samples were tiiawed and the pH increased to 6.5 with 35%) NaOH and allowed to recover the stress at this pH for one hour and tiie pH of the samples was decreased to 4.5. to observe any increased survival in the counts.

256 Similarly bacterial cells were stiessed at pH 3.5 as before and freeze-stressed for

2h, 4h, 8h, 12h, 16h or 24h. Studies showed that sample A when adapted to

sublethal pH survived better. On this basis, 3 strains each of L. acidophilus and

bifidobacteria were selected and incorporated into fermented frozen dairy

desserts. The viable counts were monitored for 12 weeks.

14.2.12 Preparation of buffers and reagents

As described in section 3.19.1 to 3.19.1.8

14.3 Results and Discussion

Most microorganisms must accommodate to a variety of changing conditions and

stress in their environment in order to survive and multiply (Berry and

Foegeding, 1997).

Figure 14.1 shows the growth curve of 3 strains each of L. acidophilus and

Bifidobacterium ssp. The organisms used in adaptation experiments were grown

to log phase before they were used in the experiment. Figures 14.2 - 14.8 shows

the results of the adaptation of probiotic bacteria where sub-lethal pH conditions

were used. Figure 14.2 shows that when tiie probiotic bacteria stressed at low

temperature for one hour there was no effect on the organisms. The results

suggest that the organisms can survive acid and low temperature for one hour.

Figures 14.3 show similar trend as with one hour exposure suggesting that the

257 organisms can survive 2 h of freeze stress. However, exposure time of 4 h affected the viability of the organisms (Figure 14.4)

Figure 14.5 shows that after stress at -18°C for 6 hour, there was a decrease in one log cycle. As shown in figure 14.6 as the exposure time increased, the viability decreased. The viability of the organisms was lost by about 3 log cycles after stressing for 12 hours.

Figures 14.7 and 14.8 shows more adverse effects on exposure for 18 and 24 hours. Thee organisms lost the viability by 5 log cycles after exposure to 24 hours. The viable counts of the organisms did not improve by increasing the pH suggesting that the effect on the organisms was lethal.

Figures 14.9 - 14.15 shows the results on the probiotic bacteria where the organisms were not exposed to sublethal pH. The organisms were exposed to

lethal pH (3.5) only.

The organisms lost tiie viability by about 1 log cycle even after stressing only for

one hour (Figure 14.9). As the exposure time increased, the loss in viability

became more obvious (Figure 14.10-14.14). There were no survivors after 24 h of

exposure (Figure 14.15).

258 Gel electrophoresis was used to detect any new protein that could be identified.

The results reveal that there was no new protein except that the protein concentration was higher in the band at pH 3.5 at -18°C.

14.3.1 Survival of acid and low temperature probiotic bacteria in frozen fermented dairy desserts

Figure 14.16 shows the survival of L acidophilus MJLAl and bifidobacteria

BDBB2 in fermented frozen dairy desserts. The counts of I. acidophilus MJLAl in the products incorporated with acid and stress adapted cells were higher at the end of 12 weeks storage period than those made with non-stress adapted cells.

The drop in the counts of!., acidophilus during storage was by almost 1 log cycle after 7 weeks and remained the same by the end of 12 week storage period.

Whereas the control L. acidophilus Mjlal lost the viability by one log cycle after one week and further decreased by one more log cycle after 7 weeks and by the end of 12 week storage period remained 4 log cycles. In the same experiment bifidobacteria lost viability by 1 log cycle after 1 week in both the control and

stress adopted organisms and by the end of 12 week storage period control bifidobacteria remained viable by 4 log cycles and stressed by 5 log cycles.

However stress adapted L. acidophilus showed better response than the other

counterparts.

Figure 14.17 shows the survival of I. acidophilus La 2409 and bifidobacteria BB

20210 incorporated into fermented frozen dairy desserts The L. acidophilus La

2409 lost viability by one log cycle after third week in the control sample and by

259 the end of 12 week storage period remained at 2 log cycles. A similar trend was observed in the control bifidobacteria. The stressed adapted L. acidophilus and bifidobacteria lost the viability after 5 weeks by one log cycle and remained at 5 log cycles at the end of storage period. These strains have been claimed to be poorly survival in acidic and freezing conditions. From the results it can be observed that stress adaptation improves the viability of the bacteria.

In the experiment (Figure 14.18) shows no much effect of acid and low temperature adaptations. There is a minimal decrease in the viable counts of L.

acidophilus LA 2401 and bifidobacteria BB 1941 when compared to the control

organisms. This might be the reason that the organisms are resistant to acid and

freezing conditions.

14.4 Conclusion

Survival of probiotic bacteria was studied in fermented frozen dairy desserts

made with or without acid and low temperature adapted cells at fortnightly

intervals for 12 weeks. The counts were higher in the products made with stress

adapted organisms. The results suggested that the use of acid and low

temperature adapted probiotic bacteria in fermented frozen dairy desserts survive

under low pH, and freezing conditions.

It was also observed that the probiotic bacteria survived better in the samples

where sub-letiial pH was used when compared to the bacteria where lethal stress

was directly used.

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LC agar developed in this study was found to be selective for L casei from pure cultures. The efficacy of the medium was verified using several commercial products containing single or mixed population of yogurt bacteria, probiotic bacteria and L. casei. LC agar could be used for selective enumeration of I. casei in yogurts and fermented milk drinks containing mixed population of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus and bifidobacteria.

Five of the AB (L. acidophilus and Bifidobacterium spp.) and two ABC (L. acidophilus and Bifidobacterium spp. and L. casei) products contained L. acidophilus counts of above 10^ cfii/g, whereas only one of AB and ABC products contained bifidobacteria coimts of above 10 cfu/g at the expiry date.

Three of the ABC products and only one LC product contained L. casei counts of

> 10^ cfu/g at the expiry date. To have therapeutic effects a minimum of one million viable cells per gram of the product has been recommended. The products that contained low viable counts may not provide any health benefits. Advances in the understanding of the problems associated -with the viability and survival of probiotic organisms in fermented milk products are yet to reach the commercial market.

All strains screened for freeze tolerance remained viable after freezing and at 8 and 16% sugar levels. Of the various pH levels studied, pH 4.5 and 4.0 were found to be detrimental for S. thermophilus 2013,1, delbrueckii ssp. bulgaricus

279 2515 and L. acidophilus MJLAl, while rest of the organisms were not affected by pH.

Influence of water activity on fermentation of functional probiotic yogurt used in the manufacture of fermented frozen dairy desserts was studied. High levels of sugars (>12%) lead to longer fermentation time and poor survival of yogurt and probiotic bacteria.

The growth of L. acidophilus was most adversely affected at sugar concentrations of 16% followed by that of bifidobacteria. The counts of both yogurt bacteria were also affected at 12 and 16% sugar levels. Fermented frozen dairy desserts typically contain 16% sugar, thus it may not be feasible to achieve a satisfactory count of probiotic bacteria in such products if sugar is added prior to fermentation.

The percent organic acids present in the inoculated milk, after fermentation and during the storage period of 30 days was determined. Pyruvic acid, acetic acid, lactic acid, propionic acid, and hippuric acid were identified and quantified in inoculated yogurt mix, after fermentation and during storage. The levels of pyruvic acid after fermentation increased with the level of sugar, but decreased during storage.

280 In general, the level of acetic acid increased during fermentation. There was a slight increase in the level of acetic acid during storage. The decrease was inversely proportional to the level of sugar. The production of lactic acid followed a similar trend except that there was no production of lactic acid in inoculated but unfermented products.

Low levels of propionic acid were detected in inoculated but unfermented batches. The propionic acid level at 16% sugar level was lower than at 12%, but similar to the 4% and 8% sugar levels and higher than the zero sugar control.

Low levels of hippuric acid were detected in inoculated milk not after fermentation or storage.

All strains screened for freeze tolerance remained viable after freezing and at 8 and 16% sugar levels. Of the various pH levels studied, pH 4.5 and 4.0 were found to be detrimental to 5". thermophilus 2013, L. delbrueckii ssp. bulgaricus

2515 and L. acidophilus MJLAl, while rest of the organisms were not affected by pH.

Yogurt bacteria counts reduced by about 1 log cycle in fermented frozen dairy desserts made using a bench top freezer during 12 weeks storage. L acidophilus

2415 were adversely affected and the counts declined by 6 log cycles within 8 weeks of storage at -18°C. For the bifidobacteria strains 1912 decreased by 5 log cycles at the end of 12 weeks storage. L. acidophilus MJLAl and bifidobacteria

281 BDBB2 counts reduced by about 2 log cycles in the products made on a pilot scale using a commercial freezer after 12 weeks storage.

Survival of yogurt and probiotic bacteria was studied in fermented frozen dairy desserts made from milk with or without supplementation with acid casein hydrolysate and cysteine. Samples were withdrawn at fortnightly intervals for 12 weeks to determine viable counts. Initial counts of the bacteria and their subsequent survival were better in the products supplemented due to nutrients

such as peptides and amino acids and with acid casein hydrolysates or cysteine due to reduced redox potential. The p-D-galactosidase activity declined more rapidly in the control sample than in the samples supplemented with acid casein hydrolysate or cysteine. The results suggested that the acid casein hydrolysate

and cysteine stimulated the growth of L. acidophilus and bifidobacteria, which resulted in improved viability of these organisms.

Survival of probiotic bacteria was studied for 12 weeks in fermented frozen dairy

desserts made with or 'without encapsulated bacteria. The counts were better in

the products made with encapsulated cells using a pilot scale and a continuous ice

cream freezer. The results suggested that the use of encapsulation of the bacterial

cells in fermented frozen dairy desserts protected the organisms against acid

stress. The beads did not release the bacterial cell in the presence of acid but

released in the presence of bile suggesting that the probiotic organisms can

survive in acidic conditions such as found in the stomach and can be delivered

into the intestine. Thus incorporating probiotic bacteria into fermented frozen

282 dairy desserts would be advantageous to provide improved health benefits, and a more marketable product.

283 16.0 FUTURE RESEARCH DIRECTIONS

Several types of dairy products are manufactured in Australia and fermented frozen dairy desserts are also gaining popularity in the market. The dairy manufacturers would benefit from longer shelf life of fermented frozen dairy desserts for exporting the products to overseas market. Due to an increase in shelf life and increased demand from local and overseas markets increasing number of dairy products manufacturers have started incorporating probiotic bacteria (L. acidophilus and bifidobacteria) into dairy products. The more recent trend is to incorporate L. casei in addition to L acidophilus and bifidobacteria. At present, there are several brands of fermented frozen dairy desserts available in the super market with yogurt bacteria and/or probiotic bacteria. Different manufacturers use various strains of probiotic bacteria and due to complexity of these mixed bacteria, there is reason to standardize the species of bacteria which would provide health benefits. There is a need to incorporate prebiotics into the dairy products to increase the viability of probiotic bacteria, especially in the colon. A combination of probiotics and prebiotics will give a symbiotic product.

Our studies demonstrated that incorporation of micronutrients, encapsulation and adapting bacteria to low acid and low temperature conditions improve the viability of the bacteria. However there is a need for extended studies especially in acid and low temperature stress adaptations.

The techniques developed in the encapsulation process provide both the consumers as well as starter culture manufacturers a means of enhancing the

284 viability of probiotic bacteria by protecting them from acid and low temperature exposures. If this is done then consumer 'will be more ensured that they are taking the recommended levels of live bacteria for improving their health. Further, human studies can be undertaken in human volimteers to confirm in vitro results.

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