MASTER OF TECHNOLOGY

INCORPORATION OF CONCENTRATED WHEY IN THE PRODUCTION OF MULTIGRAIN BREAD THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF TECHNOLOGY IN DAIRY TECHNOLOGY By SAYANTAN PAUL B. Tech. (Dairy Technology) DAIRY TECHNOLOGY DIVISION NATIONAL DAIRY RESEARCH INSTITUTE (ICAR) BANGALORE- 560 030, INDIA 2014 Reg. No. 2031210

DEDICATED TO MY BELOVED PARENTS & MY RESPECTED GUIDE

INCORPORATION OF CONCENTRATED WHEY IN THE PRODUCTION OF MULTIGRAIN BREAD By SAYANTAN PAUL Thesis Submitted to the National Dairy Research Institute (DeemeD University) in partial fulfillment of the requirements for the degree of MASTER OF TECHNOLOGY IN DAIRY TECHNOLOGY Approved By (External Examiner) (Dr. SATISH KULKARNI) Major Advisor (Guide) Members of Advisory Committee 1. Dr. K.Jayaraj Rao (Principal Scientist, Dairy Technology) 2. Dr. B.V. Balasubramanyam (Principal Scientist, Dairy Technology) 3. Dr. P. Heartwin Amaladhas (Senior Scientist, Dairy Engineering) 4. Dr. B. Surendra Nath (Principal Scientist, Dairy Chemistry)

DIVISION OF DAIRY TECHNOLOGY NATIONAL DAIRY RESEARCH INSTITUTE (Deemed University) (Indian Council of Agricultural Research) BANGALORE- 560030 (KARNATAKA), INDIA Dr. SATISH KULKARNI, Ph.D. Head & Principal Scientist Dairy Technology Section National Dairy Research Institute Southern Campus Bangalore-560 030 Dated: / /2014 CERTIFICATE This is to certify that the thesis entitled, INCORPORATION OF CONCENTRATED WHEY IN THE PRODUCTION OF MULTIGRAIN BREAD, submitted by SAYANTAN PAUL towards the partial fulfillment for the award of the degree of MASTER OF TECHNOLOGY in DAIRY TECHNOLOGY of the NATIONAL DAIRY RESEARCH INSTITUTE (Deemed University), Karnal (Haryana), India, is a bonafide research work carried out by him under my guidance, and no part of the thesis has been submitted for any other degree or diploma. (Dr. SATISH KULKARNI) Major Advisor

ACKNOWLEDGEMENT The present investigation entitled, INCORPORATION OF CONCENTRATED WHEY IN THE PRODUCTION OF MULTIGRAIN BREAD, has been conducted during 2013-2014 at National dairy Research Institute, Southern Campus, Bangalore. At the onset, I bow in gratitude before the Almighty who gave me enough patience and strength to complete my study and learn something new everyday during my project and to get rid of the few difficulties, which came across in my way during accomplishment of this endeavor. I would like to extend my sincere appreciation and immense gratitude to Dr. Satish Kulkarni. His constant support and understanding as a Major Advisor (Guide) and Chairman Advisory Committee,was the strongest support during my period of my dissertation. I am now, and will remain, extremely grateful for the invaluable guidance he has provided and the wisdom he has imparted and recognize how fortunate I am to have been able to work under such an exceptional man with such excellent leadership abilities. I admire his accuracy in planning the projects and immense ability of reasoning, and appreciate his cool demeanor, unperturbed composure and perseverance in tackling research problems. He understood me thoroughly and has been very kind and supportive to me all through the period we worked together. It has been an absolute privilege to work under sir and getting to learn so much more from him than just what is limited to my research problem. I sincerely thank my Advisory Committee members; who have provided valuable positive advise throughout the course of my study Dr. K. Jayaraj Rao Principal Scientist; Dr. B. V. Balsubramanyam, Principal Scientist; Dr. B. Surendra Nath, Principal Scientist; Dr. P.Heartwin Amaladhas, Senior Scientist; who have provided valuable positive advise throughout the course of my study and for their cordial treatment, valuable suggestions and practical help throughout my research Work. I am sure they will continue to give me able advise whenever I would consult them in the future. I wish to endow my thanks to Dr. A.K. Srivastava, Director, NDRI, Karnal, Dr. G.R. Patil, Joint Director, Dr. Satish Kulkarni, Head, SRS, NDRI, Bangalore and Dr. C.N. Pagote, Incharge, Dairy Technology Section for providing the required infrastructural facilities. I also acknowledge the scholarship provided by NDRI. I also take this opportunity to special thanks to Dr. K. Jayaraj Rao for their kind support, valuable suggestion, affection and help throughout my research work. I would also thank all the scientists at NDRI

who have always rendered their support to me in any situation and have always showered me with their affection which made NDRI-SRS feel like a home away from home to me. I take this opportunity to especially thank Mr. P. Aravindakshan who always kept motivating and freshening up the work environment with his witty nature. He was more like a friend who was never short of encouraging us at times when we were down. I would also express my gratitude to Vedavathi ma am, Sarwar sir Vimala ma am for all the support provided in doing analysis of my product. I would also take this opportunity to thank all members of Bakery Unit, UAS, Hebbal and Pratimbha Iyengers Bakery, Adugodi for providing my the required expertise in bread making. I would like to thank Mrs. Thivija Kumari for their kind support in academic matters and concern shown for me throughout the course work. I whole-heartedly thank Mr. P Muruganantham, Mr. Nanjundanswamy & Shiv Kumar, for their assistance in library matters and kind support in academic matters and concern shown to me throughout the course work. I am also thankful to all my PhD seniors Jatin, Kirti, Anjani, Arun, Kiran, Vikram and Wasnik sir for all the support and affection they showered on me. I had some wonderful times spent and some valuable knowledge gained from my M.Tech seniors- Shantanu, Yogesh, Harin, Srujan, Raghavendra, Anil, Bidyutava, akash, Rachit, Sukanta sir and Swapna and Annu madam during the first year of my stay at Bangalore. They were a wonderful bunch of people and the memories that I have with them will be there for the rest of my life. I acknowledge the help provided by the trainees from MAFSU during the course of my research. I acknowledge the support of everyone in my B.Tech college. My juniors were always full of respect and affection for me and I am grateful for the love they extended during my stay-sudin, Rakesh, Ravi Chauhan, Ravi Prajapati, Piyush, Swapnil, Roshan, Sandip, Christopher, Darshan and Arpita. I will forever cherish the moments spent with them. I always go by the philosophy that the best is saved for the last and the best memories and times that I have in the wonderful Garden City are with my classmates- Preeti, Madhav, Sanjeev, Prajeesha, Gursharan, Ketan, Mayank, Pavan, Braj, Asha, Trishul, Arun and Kiran. I will cherish the memorable times spent with them over the course of two years and their company made this two year phase one of the best in my life. I gratefully acknowledge the valuable help and all possible co-operation extended by all staff members of the Dairy Technology Section with a special mention for Chandrakantji, Kumarji, Jayramji, and Vasanthamma.

A special thanks to Ramaswamiji, Sanjay, Vijay, Jitendra, Manoj, Manjunatha and Goverdhanji for their constant endeavour in making our stay comfortable in the hostel and never made us feel away from home. It would certainly be incomplete if I don t offer my gratitude to my family and my dearest childhood friends who always have and will support me at all times. Words are not sufficient to express my devotion and gratitude to my dearest Mother and Father for their motivation, possessiveness, nurture and blessings in counteracting every obstacle coming in the way of my evolution which ensured to have a sensible head on my shoulders. They are and will remain my whole and sole inspiration behind each and every achievement of my life. Without their invaluable sacrifices and moral support, it would have not been possible for me to reach this landmark. Date: Sayantan Paul. Reg. No. 2031210

AbstrAct Whey is one of the major byproducts of the dairy industry all over the world. It contains about 40% of the total solids present in milk and is rich in organic matter that commands high Biological Oxygen Demand (BOD) of about 30,000 to 50,000 mg/liter, for which it is considered as a serious pollutant and entails expensive treatment before its disposal. Even though many attempts have been made to utilize whey successfully in food products, it has still not been utilized effectively in items of mass consumption. An attempt has been made in the present project to utilize paneer whey to optimize the conditions of production of multigrain bread. Multigrain bread is one of the fastest rising items of consumption throughout the world as changing lifestyles make people to look for multiple nutrients from a single food item. Whey concentrated to 15, 20 and 25% total solids was incorporated into multigrain bread dough containing 5% each oat, maize, sorghum and flaxseed flour to supplement with 80% wheat flour and the dough prepared was proofed and subjected to baking temperatures of 160, 185 and 210 o C. The effects of TS level of concentrated whey and baking temperature on the sensory, textural and physicochemical (reflectance) aspects of the bread were studied in the single experimental model. The studies revealed that whey concentrated upto 15%TS can be used to replace the dough water without adversely affecting the sensory properties and baking temperature of 185 o C provided the bread with best overall acceptability scores. Improvers permitted by the FSSAI- namely CaCO 3, Ca 3 (PO 4 ) 2 and ammonium persulphate were also used in the whey incorporated bread at their maximum permissible limits to improve the overall characteristics of the experimental sample. After extensive sensory studies it was decided to use Ca 3 (PO 4 ) 2 as the preferred improver at a level of 1500 ppm. The optimized multigrain bread made by incorporation of 15% concentrated whey contained 73.87% TS, 12.25% fat, 11.24% protein, 1.84% ash and 8.43% lactose. The physico-chemical characteristics of the experimental sample differed slightly from that of the control sample. The shelf life studies indicated that the experimental sample had better microbial keeping quality than the control samples at both 30 o and 5 o C but sensory scores showed acceptability upto 4 th day and 12 th day at 30 o and 5 o C respectively for both samples.

स र श म प र द नय म ड यर उ य ग क म ख byproducts म स एक ह. यह द ध म म ज द क ल ठ स क ब र म 40% ह त ह और यह एक ग भ र द षक क प म म न ज त ह, जसक लए लगभग 30,000 स 50,000 मल म / ल टर क उ च ज वक आ स जन डम ड (ब ओड ) आद श और पहल मह ग इल ज पर ज र द त ह क क ब नक पद थ म सम ह इसक नपट न. कई य स क ख य उ प द म सफलत प व क म क उपय ग करन क लए बन य गय ह, ह ल क यह अभ भ बड़ प म न पर उपभ ग क व त ओ म भ व ढ ग स उपय ग नह कय गय ह. एक य स multigrain र ट क उ प दन क थ त अन क लन करन क लए पन र म क उपय ग करन क लए वत म न प रय जन म कय गय ह. बदलत ज वन श ल ल ग क एक भ भ जन मद स कई प षक त व क लए द खन क लए कर क प म multigrain र ट भर म खपत क सबस त ज स बढ़त व त ओ म स एक ह. म 15, 20 क लए य न क त कय ह और 25% क ल ठ स 80% ग ह क आट क स थ प रक करन क लए य क जई, म क, व र और flaxseed आट 5% स य त multigrain र ट आट म श मल थ, और त य र आट proofed और 160, 185 और 210 o C क प क त पम न क अध न कय गय थ. र ट क स व द textural और भ तक र स य नक (reflectance) पहल ओ पर क त म और प क क त पम न क ट एस तर क भ व एक य ग मक म डल म अ ययन कय गय. पढ़ ई म 15% ट एस तक क त म तक ल स व द ग ण और 185 o C क प क त पम न सबस अ छ सम व क य त क र क स थ र ट उपल ध कर ई भ वत कए बन आट प न क जगह इ त म ल कय ज सकत ह क पत चल. FSSAI अथ त CaCO 3, Ca 3 (PO 4 ) 2 और अम नयम Persulphate व र अन म त Improvers भ य ग मक नम न क सम वश षत ओ क ब हतर बन न क लए उनक अ धकतम म य स म पर म श मल र ट म इ त म ल कय गय. य पक स व द अ ययन क ब द यह 1500 प प एम क तर पर वर य स धरन व ल क प म Ca 3 (PO 4 ) 2 क उपय ग करन क नण य लय गय थ. 15% क त म क सम व श स बन अन क लत multigrain र ट 73.87% ट एस, 12.25% वस, 11.24% ट न, 1.84% ऐश और 8.43% ल ट ज न हत. य ग मक नम न क र स य नक भ तक वश षत ओ नय ण नम न क उस स थ ड़ भ न ह. श फ ज वन क अ ययन क य ग मक नम न 30 o C और 5 o C द न पर नय ण क नम न स ब हतर म इ बयल रखत ह ए ग णव त स क त दय क ल कन स व द क र क द न नम न क लए मश 30 o C और 5 o C म 4 दन और 12 दन acceptability दख य.

CONTENTS Chapter Title Page no. 1.0 Introduction 1-3 2.0 Review of literature 4-40 2.1 Whey: Production, Composition and Nutritional Significance 4-8 2.1.1 Whey: Production and Composition 4 2.1.2 Nutritional, biological and therapeutic significance of whey 5 2.1.3 Functional properties of whey 8 2.2 Environmental implications of whey disposal 8-9 2.3 Benefits of whey utilization 9 2.4 Utilization of whey 9-22 2.4.1 Utilization of whey in dairy rations 10 2.4.2 Utilization of whey in lactose production 11 2.4.3 Whey as a fermentation media 11 2.4.4 Utilization of whey for human foods 12 2.4.4.1 Whey based soups 12 2.4.4.2 Whey based beverages 13 2.4.4.3 Whey in chocolates and confectionaries 16 2.4.4.4 Whey in dairy desserts 16 2.4.4.5 Whey in dairy products 17 2.4.4.6 Use of whey in other food products 18 2.4.4.7 Use of whey in fruit juices 19 2.4.4.8 Lactic acid production 19 2.4.4.9 As preservative in vegetables 20 2.4.4.10 Functionally modified whey 21 2.4.4.11 Whey based lassi 21 2.5 Utilization of Whey in Bakery products 22-25 2.5.1 Utilization of whey in biscuits and cakes 22 2.6 Utilization of Whey in bread 25-31

2.7 Multigrain - evoluation, benefits and applications. 32-39 2.7.1 History of Multigrain 32 2.7.2 Health benefits and Nutritional advantages 32 2.7.3 Novel applications of multigrain in foods 37 2.7.4 Use of multigrain in the bakery industry 38 2.8 Justification of the present project 39-40 3.0 Materials and Methods 41-68 3.1 Materials 41-42 3.1.1 Ingredients 41 3.1.2 Chemicals 41 3.1.3 Media 41 3.1.4 Glassware 41 3.1.5 Equipments 41 3.1.5.1 Planetary mixture: 42 3.1.5.2 Baking Oven 42 3.1.5.3 ph meter 42 3.1.5.4 Texture Analyzer 42 3.1.5.5 Water activity meter 42 3.1.5.6 Reflectance meter 42 3.1.5.7 Gerhardt digestion & distillation assembly 43 3.1.5.8 Weighing Balances 43 3.1.5.9 Single effect evaporator 43 3.2 Methods 43-68 3.2.1 Preparation of paneer whey 43 3.2.2 Concentration of whey 44 3.2.3 Preparation of bread 44 3.2.4 Standardization of bread manufacture by incorporation of 45 concentrated whey 3.2.4.1 Study of Dough characteristics of multigrain bread by 45 incorporating concentrated whey. 3.2.4.1.1 Determination of proofing time of multigrain 47

bread by incorporating concentrated whey 3.2.4.1.2 TPA measurements of the multigrain bread 47 dough 3.2.4.1.3 Viscoelastic characterization of the 49 multigrain bread dough 3.2.4.2 Optimization of concentration of whey 53 3.2.4.3 Proofing time reduction 54 3.2.4.4 Addition of Improvers to improve Bread 54 Characteristics 3.2.5 Storage studies 56 3.3 Analyses 57-68 3.3.1 Fat 57 3.3.2 Lactose 58 3.3.3 Physico-chemical analysis of bread 59 3.3.3.1 Sample preparation 59 3.3.2.2 Total solids 59 3.3.2.3 Fat 59 3.3.2.4 Total protein 60 3.3.2.5 Ash 61 3.3.2.6 Acid Insoluble Ash 61 3.3.2.7 Crude Fibre 62 3.3.2.8 Alcoholic Acidity 62 3.3.2.9 ph 63 3.3.2.10 Loaf volume 63 3.3.2.11 Reflectance 64 3.3.2.12 Water activity 64 3.3.3 Microbiological analysis of bread 65 3.3.4 Sensory evaluation of bread 65 3.3.5 Rheological characteristics of bread 66 3.4 Statistical Analysis 67-68 4.0 Results and Discussion 69-176

4.1 Proximate composition of paneer whey and concentrated paneer 69-71 whey 4.2 Effect of incorporation of paneer whey and baking temperature on 71-109 the sensory quality of multigrain bread. 4.2.1 Effect of incorporation of concentrated paneer whey and 71 baking temperature on colour and appearance of multigrain bread. 4.2.2 Effect of incorporation of concentrated paneer whey and 75 baking temperature on Body and Texture of multigrain bread. 4.2.3 Effect of incorporation of concentrated paneer whey and 78 baking temperature on Flavour of multigrain bread. 4.2.4 Effect of incorporation of concentrated paneer whey and 81 baking temperature on Overall Acceptability of multigrain bread. 4.2.5 Effect of whey incorporation and baking temperature on body 87 and texture characteristics. 4.2.5.1 Effect of incorporation of concentrated paneer whey 87 and baking temperature on the hardness values of multigrain bread. 4.2.5.2 Effect of incorporation of concentrated paneer whey 91 and baking temperature on the Cohesiveness values of multigrain bread. 4.2.5.3 Effect of incorporation of concentrated paneer whey 94 and baking temperature on the Springiness values of multigrain bread. 4.2.5.4 Effect of incorporation of concentrated paneer whey 98 and baking temperature on the Stress relaxation time of multigrain bread 4.2.5 Effect of incorporation of concentrated paneer whey and 101 baking temperature on the reflectance of multigrain bread. 4.2.6 Effect of incorporation of concentrated whey on the loaf 106 weight of multigrain bread 4.3 Optimization of proofing time in the preparation concentrated 107-120

whey incorporated multigrain bread 4.3.1 Effect of incorporation of concentrated whey of different TS 107 on the proofing rate of multigrain bread dough 4.3.2 Effect of enhanced Yeast level on the proofing rate of the 112 multigrain bread dough and the sensory scores of the bread. 4.3.3 Effect of temperature on the proofing of multigrain bread 117 dough 4.4 Effect of incorporation of improvers on the quality of multigrain 120-135 bread. 4.4.1 Effect of incorporation of improvers on the sensory quality 120 of multigrain bread. 4.4.2 Effect of incorporation of improvers on the texture profile 126 characteristic of multigrain bread 4.4.3 Optimization of levels of calcium phosphate 131 4.5 Physico chemical characteristic of control bread and optimize 136-138 bread made using incorporation of concentrated whey 4.6 Storage studies of whey incorporated multigrain bread. 139-176 4.6.1 Effect on sensory attributes 139 4.6.2 Effect on textural parameter 151 4.6.3 Changes in physico chemical properties during storage 167 4.6.3.1 Effect of storage on water activity(a w ) 167 4.6.3.2 Effect of storage on the crust colour of bread 169 4.6.3.3. Effect of storage on the TS content of the bread 170 4.6.3.4 Effect of storage on ph of bread 172 4.6.4 Effect of storage on yeast and mold count of bread 173 5.0 Summary and Conclusion 177-183 6.0 Bibliography i-xviii 7.0 Appendix i ii

LIST OF TABLES Serial No. Title of Tables Page no. 2.1 Composition of whey 5 2.2 Commercially available whey foods 13 2.3 Major constituents of breads (per 100 g) 26 2.4 Nutritional profile and health benefits of cereal grains 33 2.5 Multigrain used in Bakery formulations 38 4.1 Proximate composition of Paneer whey and concentrated paneer 70 whey 4.2 Effect of incorporation of concentrated paneer whey and baking 71 temperature on colour and appearance scores of multigrain bread. 4.3 ANOVA: Effect of incorporation of concentrated paneer whey and 72 baking temperature on colour and appearance scores of multigrain bread 4.4 TUKEY: Due to difference in TS of concentrated whey 73 4.5 TUKEY: Due to difference in baking temperature 73 4.6 Effect of incorporation of concentrated paneer whey and baking 75 temperature on body and texture scores of multigrain bread. 4.7 ANOVA: Effect of incorporation of concentrated paneer whey and 76 baking temperature on body and texture scores of multigrain bread 4.8 TUKEY: Due to difference in TS of concentrated whey 76 4.9 TUKEY: Due to difference in baking temperature 76 4.10 Effect of incorporation of concentrated paneer whey and baking 78 temperature on flavour scores of multigrain bread. 4.11 ANOVA: Effect of incorporation of concentrated paneer whey and 79 baking temperature on flavour scores of multigrain bread 4.12 TUKEY: Due to difference in TS of concentrated whey 79 4.13 TUKEY: Due to difference in baking temperature 80

4.14 Effect of incorporation of concentrated paneer whey and baking 82 temperature on Overall Acceptability scores of multigrain bread. 4.15 ANOVA: Effect of incorporation of concentrated paneer whey and 83 baking temperature on Overall Acceptability scores of multigrain bread 4.16 TUKEY: Due to difference in TS of concentrated whey 83 4.17 TUKEY: Due to difference in baking temperature 83 4.18 Effect of incorporation of concentrated paneer whey and baking 87 temperature on hardness values (N) of multigrain bread. 4.19 ANOVA: Effect of incorporation of concentrated paneer whey and 88 baking temperature on hardness values (N) of multigrain bread 4.20 TUKEY: Due to difference in TS of concentrated whey 89 4.21 TUKEY: Due to difference in baking temperature 89 4.22 Effect of incorporation of concentrated paneer whey and baking 91 temperature on cohesiveness values of multigrain bread. 4.23 ANOVA: Effect of incorporation of concentrated paneer whey and 92 baking temperature on cohesiveness values of multigrain bread 4.24 TUKEY: Due to difference in TS of concentrated whey 92 4.25 TUKEY: Due to difference in baking temperature 93 4.26 Effect of incorporation of concentrated paneer whey and baking 95 temperature on springiness values of multigrain bread. 4.27 ANOVA: Effect of incorporation of concentrated paneer whey and 95 baking temperature on springiness values of multigrain bread 4.28 TUKEY: Due to difference in TS of concentrated whey 96 4.29 TUKEY: Due to difference in baking temperature 96 4.30 Effect of incorporation of concentrated paneer whey and baking 98 temperature on SRT(s) of multigrain bread. 4.31 ANOVA: Effect of incorporation of concentrated paneer whey and 99 baking temperature on SRT(s) of multigrain bread 4.32 TUKEY: Due to difference in TS of concentrated whey 99

4.33 TUKEY: Due to difference in baking temperature 99 4.34 Effect of incorporation of concentrated paneer whey and baking 102 temperature on reflectance of multigrain bread. 4.35 ANOVA: Effect of incorporation of concentrated paneer whey and 103 baking temperature on reflectance of multigrain bread 4.36 TUKEY: Due to difference in TS of concentrated whey 103 4.37 TUKEY: Due to difference in baking temperature 103 4.38 Variation of Loaf weight due to incorporation of whey of different 106 TS% in multigrain bread dough 4.39 Effect of incorporation of concentrated paneer whey of different TS 108 on the proofing rate of multigrain bread dough 4.40 Effect of concentrated paneer whey incorporation on TPA 111 characteristics of multigrain bread dough 4.41 Effect of concentrated paneer whey incorporation on viscoelastic 112 characteristics of multigrain bread dough 4.42 Effect on proofing rate due to different levels of yeast 113 4.43 Effect of Increasing level of yeast in the dough on the sensory scores of bread 114 4.44 ANOVA: Effect of Increasing level of yeast in the dough on the C&A 115 scores of bread 4.45 TUKEY: Effect of Increasing level of yeast in the dough on the C&A 115 scores of bread 4.46 ANOVA: Effect of Increasing level of yeast in the dough on the 115 B&T scores of bread 4.47 TUKEY: Effect of Increasing level of yeast in the dough on the B&T 116 scores of bread 4.48 ANOVA: Effect of Increasing level of yeast in the dough on the 116 Flavour scores of bread 4.49 TUKEY: Effect of Increasing level of yeast in the dough on the Flavour scores of bread 116

4.50 ANOVA: Effect of Increasing level of yeast in the dough on the OA scores of bread 4.51 TUKEY: Effect of Increasing level of yeast in the dough on the OA scores of bread 4.52 Effect of variation of temperature on rate of proofing of multigrain bread dough 4.53 Effect of Increased temperature of proofing the dough on the Sensory scores of bread 4.54 Effect of incorporation of improvers on the sensory quality of multigrain bread 4.55 ANOVA: Effect of incorporation of improvers on the C & A of multigrain bread 4.56 TUKEY: Effect of incorporation of improvers on the C & A of multigrain bread 4.57 ANOVA: Effect of incorporation of improvers on the sensory perception of hardness of multigrain bread 4.58 TUKEY: Effect of incorporation of improvers on the sensory perception of hardness of multigrain bread 4.59 ANOVA: Effect of incorporation of improvers on the sensory perception of springiness of multigrain bread 4.60 TUKEY: Effect of incorporation of improvers on the sensory perception of springiness of multigrain bread 4.61 ANOVA: Effect of incorporation of improvers on the sensory perception of gumminess of multigrain bread 4.62 TUKEY: Effect of incorporation of improvers on the sensory perception of gumminess of multigrain bread 4.63 ANOVA: Effect of incorporation of improvers on the sensory perception of chewiness of multigrain bread 4.64 TUKEY: Effect of incorporation of improvers on the sensory perception of chewiness of multigrain bread 116 117 118 119 121 122 122 122 123 123 123 124 124 124 125

4.65 ANOVA: Effect of incorporation of improvers on the flavour of multigrain bread 4.66 TUKEY: Effect of incorporation of improvers on the flavour of multigrain bread 4.67 ANOVA: Effect of incorporation of improvers on the Overall Accpetability of multigrain bread 4.68 TUKEY: Effect of incorporation of improvers on the Overall Accpetability of multigrain bread 4.69 Effect of incorporation of improvers on the Texture profile characteristics of multigrain bread 4.70 ANOVA: Effect of incorporation of improvers on the Hardness (N) of multigrain bread 4.71 ANOVA: Effect of incorporation of improvers on the Springiness of multigrain bread 4.72 TUKEY: Effect of incorporation of improvers on the Springiness of multigrain bread 4.73 ANOVA: Effect of incorporation of improvers on the Cohesiveness of multigrain bread 4.74 TUKEY: Effect of incorporation of improvers on the Cohesiveness of multigrain bread 4.75 ANOVA: Effect of incorporation of improvers on the Gumminess of multigrain bread 4.76 TUKEY: Effect of incorporation of improvers on the Gumminess of multigrain bread 4.77 ANOVA: Effect of incorporation of improvers on the Chewiness of multigrain bread 4.78 TUKEY: Effect of incorporation of improvers on the Chewiness of multigrain bread 4.79 Effect of different levels of calcium phosphate on the sensory attributes of bread 125 125 126 126 126 128 128 128 128 129 129 129 130 130 131

4.80 ANOVA: Effect of different levels of calcium phosphate on the C&A scores of bread. 4.81 TUKEY: Effect of different levels of calcium phosphate on the C&A scores of bread 4.82 ANOVA: Effect of different levels of calcium phosphate on the B&T scores of bread 4.83 TUKEY: Effect of different levels of calcium phosphate on B&T scores of bread 4.84 ANOVA: Effect of different levels of calcium phosphate on Flavour scores of bread 4.85 ANOVA: Effect of different levels of calcium phosphate on OA scores of bread 4.86 TUKEY: Effect of different levels of calcium phosphate on OA scores of bread 4.87 Physicochemical composition of control and whey incorporated multigrain bread 4.88 Effect on sensory attributes of multigrain bread due to storage at 30 o C 4.89 ANOVA: Effect on C & A scores of control multigrain bread due to storage at 30 o C 4.90 TUKEY: Effect on C & A scores of control multigrain bread due to storage at 30 o C 4.91 ANOVA: Effect on C & A scores of Experimental multigrain bread due to storage at 30 o C 4.92 TUKEY: Effect on C & A scores of Experimental multigrain bread due to storage at 30 o C 4.93 ANOVA: Effect on B & T scores of Control multigrain bread due to storage at 30 o C 4.94 TUKEY: Effect on B & T scores of Control multigrain bread due to storage at 30 o C 131 132 132 132 132 133 133 136 139 140 140 140 141 141 141

4.95 ANOVA: Effect on B & T scores of Experimental multigrain bread due to storage at 30 o C 4.96 TUKEY: Effect on B & T scores of Experimental multigrain bread due to storage at 30 o C 4.97 ANOVA: Effect on Flavour scores of Control multigrain bread due to storage at 30 o C 4.98 TUKEY: Effect on Flavour scores of Control multigrain bread due to storage at 30 o C 4.99 ANOVA: Effect on Flavour scores of Experimental multigrain bread due to storage at 30 o C 4.100 TUKEY: Effect on Flavour scores of Experimental multigrain bread due to storage at 30 o C 4.101 ANOVA: Effect on Overall acceptability scores of Control multigrain bread due to storage at 30 o C 4.102 TUKEY: Effect on Overall acceptability scores of Control multigrain bread due to storage at 30 o C 4.103 ANOVA: Effect on Overall acceptability scores of Experimental multigrain bread due to storage at 30 o C 4.104 TUKEY: Effect on Overall acceptability scores of Experimental multigrain bread due to storage at 30 o C 4.105 Effect on sensory attributes of multigrain bread due to storage at 5 o C 4.106 ANOVA: Effect on C & A scores of control multigrain bread due to storage at 5 o C 4.107 ANOVA: Effect on C & A scores of experimental multigrain bread due to storage at 5 o C 4.108 ANOVA: Effect on B & T scores of control multigrain bread due to storage at 5 o C 4.109 TUKEY: Effect on B & T scores of control multigrain bread due to storage at 5 o C 141 142 142 142 142 143 143 143 143 144 145 146 146 147 147

4.110 ANOVA: Effect on B & T scores of experimental multigrain bread due to storage at 5 o C 4.111 TUKEY: Effect on B & T scores of experimental multigrain bread due to storage at 5 o C 4.112 Effect on Flavour scores of control multigrain bread due to storage at 5 o C 4.113 TUKEY: Effect on Flavour scores of control multigrain bread due to storage at 5 o C 4.114 ANOVA: Effect on Flavour scores of experimental multigrain bread due to storage at 5 o C 4.115 TUKEY: Effect on Flavour scores of experimental multigrain bread due to storage at 5 o C 4.116 ANOVA: Effect on OA scores of control multigrain bread due to storage at 5 o C 4.117 TUKEY: Effect on OA scores of control multigrain bread due to storage at 5 o C 4.118 ANOVA: Effect on OA scores of experimental multigrain bread due to storage at 5 o C 4.119 TUKEY: Effect on OA scores of experimental multigrain bread due to storage at 5 o C 4.120 Effect on textural parameters of multigrain bread due to storage at 30 o C 4.121 ANOVA: Effect of storage at 30 o C on the hardness of control bread. 4.122 TUKEY: Effect of storage at 30 o C on the hardness of control bread. 4.123 ANOVA: Effect of storage at 30 o C on the hardness of experimental bread. 4.124 TUKEY: Effect of storage at 30 o C on the hardness of experimental bread. 147 148 148 148 149 149 149 150 150 150 152 152 152 153 153

4.125 ANOVA: Effect of storage at 30 o C on the cohesiveness of control 154 bread. 4.126 TUKEY: Effect of storage at 30 o C on the cohesiveness of control 154 bread. 4.127 ANOVA: Effect of storage at 30 o C on the cohesiveness of 154 experimental bread. 4.128 TUKEY: Effect of storage at 30 o C on the cohesiveness of 155 experimental bread. 4.129 Effect of storage at 30 o C on the springiness of control bread. 155 4.130 TUKEY: Effect of storage at 30 o C on the springiness of control 155 bread. 4.131 ANOVA: Effect of storage at 30 o C on the springiness of 156 experimental bread. 4.132 TUKEY: Effect of storage at 30 o C on the springiness of 156 experimental bread. 4.133 ANOVA: Effect of storage at 30 o C on the SRT of control bread. 157 4.134 TUKEY: Effect of storage at 30 o C on the SRT of control bread. 157 4.135 ANOVA: Effect of storage at 30 o C on the SRT of experimental 157 bread. 4.136 TUKEY: Effect of storage at 30 o C on the SRT of experimental 158 bread 4.137 Effect on textural parameters of multigrain bread due to storage at 159 5 o C 4.138 ANOVA: Effect of storage at 5 o C on the hardness of control bread. 159 4.139 TUKEY: Effect of storage at 5 o C on the hardness of control bread 160 4.140 ANOVA: Effect of storage at 5 o C on the hardness of experimental 160 bread 4.141 TUKEY: Effect of storage at 5 o C on the hardness of experimental bread 160 4.142 ANOVA: Effect of storage at 5 o C on the cohesiveness of control 161

bread 4.143 TUKEY: Effect of storage at 5 o C on the cohesiveness of control 162 bread 4.144 ANOVA: Effect of storage at 5 o C on the cohesiveness of 162 experimental bread 4.145 TUKEY: Effect of storage at 5 o C on the cohesiveness of 162 experimental bread 4.146 ANOVA: Effect of storage at 5 o C on the springiness of control 163 bread 4.147 TUKEY: Effect of storage at 5 o C on the springiness of control 163 bread 4.148 ANOVA: Effect of storage at 5 o C on the springiness of 164 experimental bread 4.149 TUKEY: Effect of storage at 5 o C on the springiness of experimental 164 bread 4.150 ANOVA: Effect of storage at 5 o C on the SRT of control bread 165 4.151 TUKEY: Effect of storage at 5 o C on the SRT of control bread 165 4.152 ANOVA: Effect of storage at 5 o C on the SRT of experimental bread 166 4.153 TUKEY: Effect of storage at 5 o C on the SRT of experimental 166 bread 4.154 Change in water activity (a w ) of multigrain bread during storage 168 4.155 Change in crust colour (reflectance %) of multigrain bread during 169 storage 4.156 Change in solids content of the bread during storage 170 4.157 Change in moisture content of the bread during storage 171 4.158 Change in ph of bread during storage 173 4.159 Change in Yeast and Mold count of bread during storage 174

LIST OF FIGURES Serial Title Page no: no: 3.1 Flow diagram for the production of bread 46 3.2 Typical texture profile analysis curve 49 3.3 Typical curve for stress relaxation time 51 3.4 Typical creep curve 52 4.1 SPSS plots showing estmated marginal means of colour 74 and appearance scores with whey of different TS as separate lines 4.2 SPSS plots showing estmated marginal means of colour 74 and appearance scores with baking temperature as separate lines 4.3 SPSS plots showing estmated marginal means of body and 77 texture scores with whey of different TS as separate lines 4.4 SPSS plots showing estmated marginal means of body and 77 texture scores with temperature of baking as separate lines 4.5 SPSS plots showing estmated marginal means of flavour 80 scores with whey of different TS as separate lines 4.6 SPSS plots showing estmated marginal means of flavour 81 scores with temperature of baking as separate lines 4.7 SPSS plots showing estmated marginal means of Overall 84 Acceptability scores with whey of different TS as separate lines 4.8 SPSS plots showing estmated marginal means of Overall 84 Acceptability scores with temperature of baking as separate lines 4.9 Graphical representation of variation in sensory attributes of 85 multigrain bread at 160 o C 4.10 Graphical representation of variation in sensory attributes of 85

multigrain bread at 185 o C 4.11 Graphical representation of variation in sensory attributes of 86 multigrain bread at 210 o C 4.12 SPSS plots showing estmated marginal means of hardness 90 (N) with whey of different TS as separate lines 4.13 SPSS plots showing estmated marginal means of hardness 90 (N) with temperature of baking as separate lines 4.14 SPSS plots showing estmated marginal means of 93 cohesiveness with whey of different TS as separate lines 4.15 SPSS plots showing estmated marginal means of 94 cohesiveness with temperature of baking as separate lines 4.16 SPSS plots showing estmated marginal means of 97 springiness with whey of different TS as separate lines 4.17 SPSS plots showing estmated marginal means of 97 springiness with temperature of baking as separate lines 4.18 SPSS plots showing estmated marginal means of SRT with 100 whey of different TS as separate lines 4.19 SPSS plots showing estmated marginal means of SRT with 100 temperature of baking as separate lines 4.20 SPSS plots showing estmated marginal means of 105 reflectance with whey of different TS as separate lines 4.21 SPSS plots showing estmated marginal means of 105 reflectance with temperature of baking as separate lines 4.22 Effect of incorporation of concentrated whey of different TS 107 on the proofing time of multigrain bread dough 4.23 Effect of incorporating a higher level of yeast on the proofing 113 time of multigrain bread dough 4.24 Graphical representation of change in ph of multigrain 114 bread doughs due to variation in yeast level 4.25 Effect of variation of temperature on rate of proofing of 117

multigrain bread dough 4.26 Effect on ph of multigrain bread doughs due to variation in 118 proofing temperature 4.27 Effect of elevated proofing temperature on the sensory 119 charcteristics of multigrain bread 4.28 Comparison of effects of yeast level variation and elevated 120 proofing temperature on the sensory characteristics of whey based multigrain bread 4.29 Optimized flowchart ofbreadproduction using concentrated 135 whey 4.30 Effect on sensory attributes of control multigrain bread due 144 to storage at30 o C 4.31 Effect on sensory attributes of whey incorporated multigrain 145 bread due to storage at 30 o C 4.32 Effect on sensory attributes of control multigrain bread due 151 to storage at 5 o C 4.33 Effect on sensory attributes of control multigrain bread due 151 to storage at 5 o C 4.34 Effect of storage at 30 o C on the hardness of bread. 153 4.35 Effect of storage at 30 o C on the cohesiveness and 156 springiness of bread 4.36 Effect of storage at 30 o C on the Stress Relaxation Time 158 (SRT) of bread 4.37 Effect of storage at 5 o C on the hardness of bread 161 4.38 Effect of storage at 5 o C on the cohesiveness and 164 springiness of bread. 4.39 Effect of storage at 5 o C on the Stress Relaxation Time 166 (SRT) of bread 4.40 Effect of storage on the water activity of bread 168 4.41 Effect of storage on reflectance (%) of bread 170

4.42 Effect of storage on the % TS of bread 171 4.43 Effectof storage on % moisture of bread 172 4.44 Effect of storage on ph of bread 173 4.45 Effect of storage under room temperature on the Yeast and Mould count of bread 175

List Of AbbreviAtiOns $ American dollar ANOVA Analysis of Variance @ At the rate of Avg. Average BOD Biological oxygen demand B&T Body and Texture BM Buffalo milk CMC Carboxy Methyl Cellulose cfu Colony forming units C&A Colour and Appearance CPW Concentrated Paneer whey CM Cow milk CRT Creep retardation time o C Df DM EWP FSSAI GMP g HSD hr IU Kcal kg kj lit LDPE µ Microns Degree Celsius Degree of freedom Dry matter Egg white proteins Food Safety and Standards Authority of India Good Manufacturing Practices Gram (s) Honest significant difference Hour (s) International Units Kilocalories Kilogram Kilo-joule Litre Low density polyethylene

List Of Abbreviations mg Milligram ml Millilitre (s) mm Millimetre min Minute (s) MPM Malleable protein matrix mwpc Modified whey protein concentrate OA Overall acceptability PW Paneer whey ppm Parts per million % Per cent PWP Precipitated whey proteins PER Protein Efficiency Ratio RSM Response surface Methodology RO Reverse Osmosis sec Second (s) SPC Standard plate count SRT Stress relaxation time TA Texture analyzer TPA Texture profile analysis TS Total solids UF Ultrafiltration v/v Volume by volume w/v Weight by volume WP Whey Protein WPC Whey protein concentrate WPI Whey protein isolate Y&M Yeast and Mould

Chapter- 1 Introduction

1. IntroductIon Whey is one of the major byproducts of the dairy industry all over the world. A large part of whey however, is left unutilized and disposed off which results in significant loss of potential nutrients. Whey contains about 40% of the total solids present in milk and is rich in organic matter that commands high Biological Oxygen Demand (BOD) of about 30,000 to 50,000 mg/liter. In order to treat whey to the statutory pollution control norms, about 0.4 kw of energy per liter is required. Hence, the disposal of whey not only causes loss of nutritionally rich milk solids but also puts an additional financial burden on the dairies. Qualitatively cheese whey is categorized under sweet whey while casein whey is categorized under acid whey. The composition of whey varies marginally depending on the type of milk coagulation. (Khamrui and Rajorhia, 1998). In India large quantities of whey is produced during the production of chhana and paneer in the traditional dairy sector. It is estimated that about 2% of milk produced in India is converted to paneer and chhana and production of whey due to this can be estimated at around 4.84 million tonnes per annum which contains about 290 million kg of valuable milk solids (Aneja et al., 2002). In the traditional sector a part of these valuable solids is being utilized for animal feeding. However, data on extent of utilization is not well documented. Some technologies have been developed to utilize whey solids which include extraction of whey proteins, manufacture of whey soups, whey beverages, coffee drink, cream yoghurt etc. (Jelen, 2002). However, none of the products listed are of mass consumption type and hence large quantities of whey produced in the country by the organized sector is not being utilized to the desired level at present. Development of appropriate processes for the production of mass consumption products by utilizing whey solids will be in the larger interest of the dairy industry and also enhancing the nutritional profile of the products prepared. The bakery industry in India is the third largest sector of food processing industry accounting for over 3.2 billion Euros (Rs.210 billion) and is growing at a rate Page 1

Introduction of 13-15% per annum (Anon, 2013). The production of bakery products is increasing steadily, bread and biscuits accounting for nearly 85 % of the total bakery products produced in India (Rao, 2005); other major products are buns, soup sticks, rusks, cakes, pizza base etc. There is an immense potential for the utilization of whey solids in the above listed bakery products as a means of enhanced nutritive value, functional value, reduction of cost of production etc. Bread is one of the most widely consumed processed foods all over the world. The total market size of the bread industry is approximately 4.00 million tonnes in India. The bread industry consists of organized and unorganized sectors which contribute about 45 and 55 percent of the total bread production respectively. The organized sector consists of around 1800 small bread manufacturers, 25 medium manufacturers and two large industries. The unorganized sector consists of about 75,000 bread bakers mostly located in residential areas of cities and towns. Because of changing lifestyles a large number of consumers, especially professionals are adopting bread as a regular food and are willing to pay a price for the quality. As a result many new food chains have sprung up specializing just in bread production and offer up to 100 varieties for sale at one time (Rao, 2005). In this context, bread would provide tremendous potential for effective utilization of whey solids for desirable enhancement of nutritional and functional value. Multigrain bread can be made out of different grains either in whole or ground form. It is nutritionally superior to normal bread because the source of nutrients is from multiple sources rather than a single source. A multigrain product will always provide a bundle of nutrients which may not be possible to get sufficiently through consumption of single grain products. With an increase in urbanization and industrialization there is an increased demand for convenience as well as nutritious products, available in a short time period. Even though people have become health conscious, but due to the changing lifestyles people do not have the time to consume every single grain one at a time. Multigrain bread will provide all the nutritional benefits of all the grains used in its formulation. The multigrain bread will Page 2

Introduction further aid the consumers with different health benefits derived out of different grains. In India the traditional and organized bakery industry has grown considerably. These businesses are operating on small and medium scales and these processors are constantly on the lookout for newer products, cost reduction and value addition. The use of whey solids in appropriate manner is likely to provide low cost solids to the bakery product manufacturers besides resulting in newer, high nutritional value products. As already indicated paneer whey is available in large quantities as a byproduct of the Indian dairy industry. Since most of it is presently being drained out causing a burden on the effluent treatment plant, it is worthwhile to study its application in bakery products. So far few attempts have been made on the utilization of paneer whey in bakery products. It is in this direction, the present project is an additional attempt designed to utilize the paneer/cheese whey from the organized dairy sector in the production of multigrain bread, and the objectives of the present study are: 1) Process optimization for the production of multigrain bread by incorporating concentrated whey 2) Study the effect of whey incorporation on the physico-chemical and sensory properties of bread. 3) Assess the impact of concentrated whey on the shelf life of bread. Page 3

Chapter- 2 Review of Literature

2. Review of literature The existing prior art pertaining to the topic of present investigation has been reviewed and presented under following heads: 1. Whey: production, composition and nutritional significance. 2. Environmental implications of whey disposal. 3. Benefits of whey utilisation. 4. Utilization of whey. 5. Utilization of whey in bakery products 6. Utilization of whey in bread 7. Multigrain-evolution, benefits and applications 2.1 Whey: Production, Composition and Nutritional Significance 2.1.1 Whey: Production and Composition Whey is a by-product of Cheese, Paneer and coagulated dairy products and is a major source of lactose, good source of valuable protein and minerals and water soluble vitamins (Bande, 2011). Whey retains around 40-50% of total milk solids including approximately 80-90% lactose, 20% protein, 70% minerals and large part of water soluble vitamins. In India there has been a substantial increase in the production of Paneer in organised sector resulting in increased availability of whey. It is estimated that about 5% of milk produced in India is converted to Paneer (ICMR 2000, Chandan 2007), the figure being 4,493 Metric Tonnes in the year 2003-04 ( Joshi 2007, Srivastava and Goyal 2007) and production of whey due to this is estimated at around 4.84 million tonnes per annum, consisting 290 million kgs of valuable milk nutrients. (Nair and Thompkinson, 2007).Even though, whey contains valuable milk solids it is seldom used for human consumption and wasted in large quantities adding to environmental pollution (Jindal et al., 2004).Whey disposal is a serious problem and in order to reduce pollution load whey needs to be treated to obtain commercially viable products. (Gupte and Nair, 2010). The composition of different types of whey is tabulated below. Page 4

Review of literature Table 2.1 Composition of whey (Khamrui and Rajorhia, 1998). Sweet whey Medium acid whey Acid whey Type of Whey Composition CM cheddar BM cheddar Paneer Whey CM chhana BM chhana Casein whey Shrikhand Whey cheese whey cheese whey whey whey (HCl) Solids % 6.35 6.87 6.06 6.25 6.40 5.95 6.96 ph 6.10 6.40 5.60 5.50 5.70 4.60 NA Lactose % 5.00 5.01 5.03 4.86 4.90 4.30 NA Protein % 0.90 0.98 0.30 0.72 0.89 0.70 0.38 Fat % 0.06 0.34 0.13 0.10 0.10 NA 0.43 Ash % 0.59 0.54 0.60 0.57 0.44 0.72 NA Lactic acid % 0.13 0.14 0.21 0.19 0.20 0.25 0.24 Calcium ppm 448 501.5 710.65 526 446.3 1,298 NA Phosphorus 486 441.5 560.5 493.7 533.3 1,770 NA Ppm CM : Cow milk BM : Buffalo milk NA : Not available 2.1.2 Nutritional, biological and therapeutic significance of Whey. Whey is rich in lactose, proteins, minerals and vitamins. The nutrients are immensely valuable with regard to human dietary requirement. (Prendergast 1985, Mathur et al., 1988). It contains around 6 to 7 % of total solids. More than 75 % of the dry matter in whey is lactose, while whey proteins account for another 12%. Whey contains about 40% of the total solids present in milk. It is also rich in micronutrients like milk salts, water-soluble vitamins, peptides, immunoglobulins and lactoferrin (IDF, 2003). It also contains enzymes, hormones and growth factors. (De Wit, 2001). Whey consist of lactoferrin, which has antibacterial properties; lactoperoxidase enzyme that has anticarcinogenic activity apart from immunoglobulins, active peptides and growth factors, which stimulate cell growth.(durham et al., 1997) Each component of whey has its own Page 5

Review of literature nutritional significance. Milk components not only provide nutritional security but are also capable of providing potential health benefits in various forms. Many dairy ingredients are finding large-scale application in nutraceutical products, which possibly is the hottest trend in the food industry. Some dairy ingredients viz. whey carbohydrates and proteins are also considered as prebiotics which affect the host by selectively stimulating the growth and activity of one or more naturally present or administered bacterial species in the colon. The energy value of whey is around 210 Kcal/lit (Swaminathan, 1982). Whey contains highly nutritious constituents. The most valuable components of whey are whey proteins. Whey proteins have been regarded superior to most of the other proteins such as egg, beef, casein and soya proteins in nutritive value. The amino acid profile of whey protein shows that they contain all essential amino acids in excess of FAO standards. Biological value of whey protein is higher (104) as against whole egg (100), rice (74), soya (59), wheat proteins (54) and casein (77) (Poonam, 2007). They also have higher Protein Efficiency Ratio (PER) and Net Protein Utilization (NPU) than casein. The PER of whey protein is 3.6 as against 3.8 for egg protein and 2.9 for casein, whereas NPU is 95 for whey protein as against 93 for whole egg and 76 for casein (Renner and Abd-El-Salam, 1991).Gupta et al. (1992) reported that whey proteins are generally regarded as safe for food applications (Hugunin, 1987; Morr, 1991) because of their excellent nutritional and functional properties. Whey proteins are next to egg protein in terms of nutritive value. Whey Protein Concentrate (WPC) has been considered a major nutritional ingredient to improve nutritional profile of varieties of foods as recorded by many workers (Jelen et al., 1979; Huffman, 1998). Lactose, often referred to as milk sugar, is the primary milk constituent in whey which contributes around 80-90% of whey solids. Lactose commercially is available in different qualities, of which major distinguishable categories are food-grade and pharmaceutical-grade. The food sector remains the largest user of lactose, where in many products it is partially a functional replacer of more expensive dairy ingredients and it is also widely used in infant foods. Its specific functional properties have made lactose the favoured choice of the pharmaceutical industry as a substance for pills and capsules as well as an inert carrier for inhalation medicines (Zadow, 1991). The major mineral Page 6

Review of literature components in liquid whey contain the mineral cations like sodium, potassium, calcium and magnesium as well as anions like chlorine, citrate and phosphate (IDF bulletin, 2003). About 40% of calcium and 43% of phosphorus in milk are found in whey derived during Cheddar cheese making. In view of excellent nutrient and biological value, whey is being used since 460 B.C for treating an assortment of ailments. In middle ages, whey was recommended by many doctors for treating varied diseases and by mid 19 th century, whey cures reached a high point with the establishment of over 400 whey houses in Western Europe (Poonam, 2007). In Central Europe dyspepsia, uraemia, arthritis, gout, liver disease, anaemia and even tuberculosis were treated with the whey injection (Holsinger et al., 1974). Delaney (1976), has reported that whey protein concentrates are receiving considerable attention as base material for preparation of a range of dietetic/therapeutic products, e.g. electro dialysed whey based foods for use in the treatment of chronic uraemia, application of whey protein concentrate in a number of clinical disorders, use of whey protein concentrates and whey protein fractions in the manufacture of nonresidue preparations designed for prophylactic cardiac dietary regimes. Bounous and Gold (1991) was first to demonstrate the role of dietary whey protein concentrate in the successful treatment of colon cancer. Bounous also suggested that dietary milk products may exert an inhibitory effect on development of several kinds of tumours. Recent experiments have suggested that antitumor activity of milk is in the protein fraction and more specifically in the whey protein component of milk. Shahani et al. (1978) reported that whey and whey protein concentrates could be extensively used for a number of formulations in human foods because of their high nutritive value and especially for their therapeutic value. Whey could be used in infant food formulae e.g. whey protein concentrates and specialized diets for invalids, geriatrics and infants suffering from cardiac malfunction and convalescent patients. In a French patent, a method for preparing a partial hydrolysate of whey proteins and free amino acid content less than 10 percent, considered to be suitable as an ingredient in hypo allergic dietetic milk has been registered. (Mann, 1991). Beaulieu et al. (2010) reported that a malleable protein matrix (MPM) composed of whey fermented by a proprietary Lactobacillus kefirofaciens strain has immuno-modulatory and antiinflammatory properties. MPM consumption leads to a considerable reduction in Page 7

Review of literature cytokine and chemokine production (tumour necrosis factor alpha, interleukine-1 beta, interleukine-6), thus lowering chronic inflammation and metaflammation. Inhibition of metaflammation should provide positive impact with regard to dyslipidemia, insulin resistance and hypertension. Mcintosh et al. (1995) reported that whey proteins have anticarcinogenic activity. Walzem et al. (2002) reported that whey proteins have a considerable usage in infant s nutrition as whey predominant formulas and whey protein hydrolysates for infants with an intolerance to cow s milk protein. 2.1.3 Functional Properties of Whey Whey Proteins also exhibit excellent functional properties such as solubility, foaming, emulsifying, gelling and water binding etc. apart from their nutritional and therapeutic value. (Mathews, 1984; Patel and Kilara, 1990). A wide range of functional properties is exhibited by whey contributing to colour, flavour, texture and overall acceptability of dairy products. (Marshall et al., 1988; Mcintosh et al., 1998). 2.2 Environmental implications of whey disposal. Whey obtained as a by product retains almost about 40-50 % of the total milk solids containing milk sugar, protein, traces of fat, minerals and vitamins. Because of the presence of large part of organic constituents, the biological oxygen demand (BOD) of whey is very high (40,000-50,000 mg/kg) constituting a major burden of disposal as waste through the regular channel. Thus the disposal practices including drainage into sewage or spraying on the fields as organic manure are currently seldom practiced (Dabur and Brahm, 2007). The practice of whey disposal results in loss of large quantities of valuable milk solids. In recent times, the environmental regulations have become stringent across the world necessitating the treatment of whey prior to disposal through sewage system. A recent study indicates that treating 5 lakh liters of whey in the sewage would cost $ 10,000 per day for primary treatment or $ 145,000 for tertiary treatment (Khamrui and Rajorhia, 1998). In India it is estimated that the cost of treatment of each litre of Paneer whey is around 50-60 paisa which is considered expensive. Page 8

Review of literature Because of problems involved with the whey disposal, since long time, efforts are being made towards product diversification using whey components without much change in the existing infrastructure. This will be quite feasible and will be in the direction of reducing the pollution. 2.3 Benefits of whey utilization. Since whey contains almost all the nutrients of milk except casein and fat, utilization of whey results in reducing the wastage of valuable nutrients which can be diverted for the beneficial use in the form of food. There is increased awareness all over the world on potential benefits of whey utilization, primarily because of pollution control regulations, economic needs and future needs to ease food shortage (Horton, 1995). The various nutrients present in whey can supplement the energy value of a variety of foods consumed by the lower income group of population across the world. Whey utilization helps in generating new avenues for export promotion by the production of long life nutritious products like WPC by the use of membrane technology. A variety of functional foods, geriatric and pediatric products are produced by beneficially utilizing the valuable proteins and minerals constituents present in whey (Puranik et al., 1997). Whey can effectively be used as one of the ingredients in the animal feed ration (Schingoethe, 1976) and the effective utilisation will considerably reduce the pollution for the Cheese and Paneer factories. Whey can even be used in irrigation to add nutrients to soil after proper neutralization treatment. Thus utilisation of whey solids is in the larger interest of the society and thus attempts are continuing for developing appropriate processes for using the whey solids beneficially. 2.4 Utilization of whey Whey can be used in food products in different forms like liquid whey, condensed whey(40 to 50 percent solids), whey powder, demineralized whey powder, whey protein concentrates, protein hydrolysates like Whey Protein Isolates, individual proteins and their derivatives ( Bund and Pandit, 2007). A variety of approaches have been made to upgrade the utilisation of whey: The whey can be treated with the enzyme lactase and concentrated to syrups for use as sweeteners in various food products. Page 9

Review of literature Whey can be dried to a powder form and used to form gels to bind fat to water in products such as canned meats patties and sausages. Alcohol can be made from the whey - microorganisms such as Kluveromyces lactis or Kluveromyces fragilis will ferment the lactose to produce alcohol. This alcohol is now produced in Ireland and is eventually sold in products such as Baileys Irish Cream. The whey can be treated with lactase and it is then suitable to be used as a substrate for high alcohol yielding strains of Saccharomyces cerevisiae. Tests have shown that whey can be used to replace around 20% of the nonmalt sources of yeast nutrients in the brewing of beer. 2.4.1 Utilization of whey in dairy rations Whey can serve as a dietary component in dairy feeding regimen, because of it being a good source of energy, proteins, minerals and water-soluble vitamins. Whey has been used as liquid whey, condensed whey or dried whey products for feeding of calves and swine. Dried whey can be used in calf starter or as a pallet binder. 10 % dried whey in calf starter increases consumption of pallets, however, at 30 % the palatability decreases. Liquid whey can be used as a satisfactory need for steers and heifers. At first feeding whey protein concentrate (WPC) can be an effective substitute for Dam s colostrums. Because of their high lactose content whey and whey products can be used as milk replacer which is a good carbohydrate source for young calves. De-lactosed whey has been shown to increase microbial protein synthesis and amino acid flow to the abomasum; liquid whey feeding to high yielding cows overcomes the problem of milk fat depression. The nutrient composition of partially de-lactosed, de-proteinated dried whey contains about 55 to 60 % lactose and 10 to 20 % protein, and can be effectively used to replace the grain component of the dairy ration to a considerable extent. Also liquid whey feeding reduces the cost of raising calves by saving milk for human consumption or its conversion to value added dairy products such as Butter and Cheese (Gnanasekar and Balaraman, 2001). Page 10

Review of literature 2.4.2 Utilization of whey in lactose production Lactose and lactose-derived products are playing an increasingly important role in various branches of food industry. Dry matter of whey is about 6 to 7 %, of which lactose is about 75 % (Gonzalez siso, 1996). The disposal of whey is a great environmental problem because of its very high Biological oxygen demand (BOD). Since, lactose alone is responsible for 80 % of BOD (Mawson, 1994); attempts have been made by different researchers to solve this problem by isolation of lactose or by its conversion to single cell protein, lactic acid, ethanol and methane. Lactose has been isolated from whey by four different procedures: a) conventional concentration procedure, b) reverse osmosis and ultra filtration, c) precipitation with alkaline earth metal chlorides and d) precipitation by different alcohols. The yield, i.e. percent of lactose isolated varies from 60.37 to 90.27 %, depending upon the procedure adopted. Best quality lactose with 99.99 % purity was obtained by ethanol extraction preceded by deproteinization and demineralization of whey and it contains only 0.04 % ash and protein and fat below detectable limit, which makes this method the most suitable for use in pharmaceutical industries (Paul et al., 2002). Lactose has various applications in food and pharmaceutical industries due to its multiple functional properties (Kapil et al., 1991). 2.4.3 Whey as a fermentation media Whey contains about half the milk solids most of lactose, about one fifth of the protein and most of vitamins and minerals as well as essential amino acids. Thus, it can be potentially used as a fermentation media for the production of single cell protein, ethanol, baker s yeast, methane, lactic acid, etc. Various lactose-fermenting yeasts are capable of forming ethanol. De-proteinated Cheese or acid Casein whey are used as the substrate and may be concentrated by Reverse osmosis or supplemented by other lactose enriching streams to increase the lactose concentration to 10-13 % and yield obtained is around 75-85%. Whey is also used for the production of single cell proteins and one of the most popular processes is Bel-process of France. In this process strains of K. marxianus var. lactis or marxianus are commonly grown for this purpose. Product obtained contains about 50 % protein, 30 % carbohydrate, 6 % lipid and 8 % minerals on dry matter basis. Whey seems to provide the best medium for production of Page 11

Review of literature enzyme β-galactosidase using Kluyveromyces species grown on diluted whey. β- galactosidase is used to hydrolyze lactose to overcome the problem of lactose intolerance and to generate syrups, which are sweeter than lactose and which do not crystallize as readily. Baker s yeast or yeast autolysate can also be obtained by whey fermentation. The yeast autolysate may be used to replace yeast extract in microbiological media. Lactic acid commonly used in food industry; ammonium lactate, used as animal feed as well as propionic acid, fungi static agent, can be produced using whey as medium. Whey can also be fermented to obtain methane (biogas), which offers the major benefits of an efficient waste treatment process coupled with the production of a convenient energy source (Murdia et al., 2005). Koutinas et al. (2007), carried out industrial scale up of whey fermentation, promoted by raisin extracts and by using kefir cells. The fermented whey would be used as raw material to produce kefir like whey based drinks, potable and fuel alcohol as well as kefir-yeast biomass for use as baker s yeast. 6-fold promotion of whey fermentation by the use of 1% black raisin extracts supported this investigation. Pescuma et al. (2008) stated that whey is a source of biological and functional proteins. He worked to evaluate the potentiality of three Lactic Acid Bacteria strains to design a starter culture for developing functional whey based drinks. The studied strains were found to grow well (2-3 cfu/ml) independent of temperature and to degrade major whey proteins. 2.4.4 Utilization of whey for human foods In order to utilize the nutrient rich whey several attempts have been made worldwide and some of the products so developed are commercially available in the market. Some of the whey solids based foods with their specific role are summarized in table 2.2. 2.4.4.1 Whey based soups Soups are served as appetizers before meals all over the world. Kamat et al. (1999) found that paneer whey could be successfully utilized for the preparation of nutritious and delicious soup by blending it with tomato pulp at 25 % level and beetroot pulp at 20 % level. Sudhir and Singh (2001) developed Page 12

Review of literature shelf-stable tomato whey soup. Arora (2002) reported that whey could be utilized up to 80 % in tomato soup along with stabilizers like guar gum, carrageenan, and sodium alginate and carboxy methyl cellulose at the level of 0.5, 0.1, 0.3 and 0.3 % respectively. Singh and Mathur (1994) utilized Paneer whey and Cheese whey for the development of whey based mushroom, tomato and spinach soups. Cheese whey was preferred for the manufacture of vegetable soups than Paneer whey. Instant mushroom whey soup powder was obtained by cooking mushrooms in concentrated Cheese whey (28-30 % total solids) followed by spray drying with the addition of gelatinized corn flour (Ghosh and Singh, 1997). Chidanandaiah and Sanyal (2001) recommended addition of 20 % chicken heads for making a good quality chicken whey soup using heads of slaughtered spent hens and Paneer whey. 2.4.4.2 Whey based beverages Conversion of whey into beverages on a commercial scale has an economic advantage, as the whole sample is being used and there are no problems with leftover residues. Beverages in general provide energy, water to digest food, regulate body temperature, prevent dehydration, quench thirst and remove psychological tensions (Shaikh, 2001). Whey can also be used with the addition of fruit juices. Studies have shown that beverages with citric flavours have high consumer acceptability (Holsinger et al., 1974, Zadow, 1984). Table 2.2: Commercially available whey foods: - (Vyas et al., 1980) Food %Whey solids Contribution Used Dried infant food 25-40 Nutritional, biological Beverages 6 Flavour, body Dried soups and gravy 50-75 Flavour, body bases Dried culture media 85-97 Nutritional Bakery products 3-10 Flavour, texture, keeping quality Page 13

Review of literature Confectionary products 4-10 Flavour, moisture, whipping property Frozen desserts 3-4 Flavour, fruit stability Cheese foods 10 Flavour, body Dry mixes 10 Tenderizing, colour carrier for fats and Oils Whey could be used in the formulation of nutritive soft drinks (alcoholic and non-alcoholic) or high protein beverages and might be used with the addition of fruit juices. A variety of beverages consisting plain carbonated, alcoholic and fruit flavoured have been successfully developed and marketed all over the world and they hold great potential for utilizing whey solids (Saravanakumar, 2005). Prendergast (1985) reported that whey is a genuine thirst quencher, light and refreshing and has good potential profit margins. Whey drinks can be made using various technologies-using natural whey or whey permeates or refined whey or ultra-filtered whey retentate or whey concentrate. The advantage of fermenting whey into various types of beverages was highlighted by Gandhi (1989). A number of whey drinks and beverages have been developed including Wheyvit, acidowhey, whey-based fruit beverages. Nair et al. (2008), developed direct acidified whey based Lassi like beverage using optimization of ingredients. Magalhaes et al. (2010) investigated cheese whey (CW) and deproteinized cheese whey (DCW) for their suitability as novel substrates for producing kefir like beverages. Whey protein because of their solubility at low ph, can be used to formulate fruit beverages that are high in protein like weight management products, healthy children s drinks or fruit based sports drink with the ph range of 3.0-4.0 (Giese, 1994). Improved whey based fruit juices can be prepared by UHT processing and aseptic packaging (Mathur et al., 1988).Whey banana beverages were made from acid whey and over ripe steamed bananas at a ratio of 3:2 to get whey banana shake (Shekilango et al., 1997). Sterilized Chhana whey beverage could be prepared by the addition of 10 % sugar, 1.5 % citric acid, 3 % lemon juice and 0.06 % sorbic acid to deproteinized Chhana whey (Mandal et al., 1997). Whey based fruit beverages have also been developed by using clear juice of Page 14

Review of literature cherry, apple, grapes, strawberry and orange; 20 to 40% whey permeate sweetened with sucrose, @ 7-10% and acidified with sorbic acid @ 0.2 or 0.4% (Vojnovic et al., 1993). Apple juice (20%) and litchi juice (30%) in the whey based beverage with 0.1% sorbic acid was found to be quite satisfactory for about 2 months and 4 months of storage at room temperature (Shukla et al., 2000). Addition of Potassium metabisulphate @ 350 ppm was found to preserve the sensory qualities of whey based kinnow juice concentrate for at least 9 months at refrigerated conditions and 4 months at ambient conditions (Khamrui et al., 2000). Paneer whey was used to prepare whey-based Lassi and similar Lassi type cultured beverages from a mixture of cheese whey and skim milk in the ratio of 95:5. Gandhi (1989) developed a low cost, therapeutic, refreshing and palatable beverage named as Acido whey. Wheyvit is a fruit flavoured alcoholic beverage developed from fresh, separated, de-proteinized whey. Anotovskii and Yoroskenko (1950) prepared sparkling beverage from sweet whey inoculated for 24 hours with Lactobacillus acidophilus then Streptococcus lactis, then yeast and sugar (8-10%) added and then fermented in a sealed vessel at 8 o C for 3-4 days. Other alcoholic beverages include Brodost- a beer like beverage from deproteinized whey; Rivella- a whey beverage prepared by fermenting deproteinized whey with Lactic acid bacteria and condensing to 7:1 concentrate with added sugar and flavouring; whey champagne- a non-alcoholic whey beverage. Whey beverages with low alcohol content are produced from whey permeate by fermenting the lactose with Kluveromyces fragilis or Saccharomyces lactis. A pleasantly tart flavour is produced when acid whey permeate is used. A Polish product is produced by the inoculation of acid whey permeate with kefir fungi (30% addition, 5 hr incubation at 77 o F). The resultant beverage contains 0.6-0.7% lactic acid and 0.8-0.95% alcohol (Sienkiewicz and Riedel, 1990). Koumiss like drinks can be prepared by mixing milk and whey for fermentation from mixtures of whey and buttermilk. The koumiss like product can be stored for 4 weeks at 40 o F (Guan and Brunner, 1987). Whey based alcoholic drinks named mjoed and red wine is in use in Scandivian countries. The production of whey wine was reported after deproteinization of the whey by heating at 82 0 C for 5 minutes, approximately 22% dextrose was added depending upon the amount of alcohol desired in wine and then fermentation for seven days at room temperature (22-25 0 C) using yeast Saccharomyces Page 15

Review of literature cerevisae var. ellipsoidus (Delaney and Roxy, 1981). Pescuma et al. (2010) reported that WPC fermentation by rationally selected lactic acid bacteria might be used for developing functional beverages with improved characteristics such as reduced β-lactoglobulin content and increased branched-chain essential amino acids. Whey based sports beverages were developed keeping in mind that sports beverage is one of the most promising segments in the functional food category. A lactose hydrolysed whey based sports beverage was developed using paneer whey and 20% pineapple juice, 7.5% sugar and stabilizer and salt mixes. 2.4.4.3 Whey in chocolates and confectionaries Use of sweetened condensed whey in the manufacture of candies has been detailed by Webb et al.(1940). Whey is used to manufacture a confectionery product called Dulce de Leche in Latin American countries (Kosikowski, 1997). 10 parts of whey and 20 parts of sugar are boiled to more than 65% total solids. Whey syrups in which lactose has been hydrolyzed with an immobilized enzyme can replace up to 100% of condensed milk in enrobed fudges and caramels. Caramel made with hydrolyzed whey syrup in place of usual condensed skim milk (28.2%) exhibited less brittleness and showed less retraction after cutting and browned more rapidly and more intensely (Mann, 1991). In the United States, a special, neutralized whey powder has been developed for the chocolate and sugar confectionary industry where a low acid powder is required (Mann, 1982). Lactose could be introduced to prepare fondant, which resulted in control and reduction of sweetness, intensification of whiteness and economies of operation (Anon, 1984). Mixture of lactose and sucrose are used in sugar coating of cores in chocolates. 2.4.4.4 Whey in dairy desserts Egyptian workers have used dried whey to make (a) jelly to which fruit flavouring (strawberry, orange or mango) or chocolate powder added, or (b) custard flavoured with chocolate or vanilla. Cooled samples of flavoured jelly and custard indicated high acceptability. Another promising whey product developed was Empruv (Anon, 1989), a fermented product developed to impart improved palatability and texture to a variety of foods, including puddings and mousses, Page 16

Review of literature whilst a French patent describes a whey based milk product suitable for the preparation of dairy gels. β-galactosidase-hydrolyzed whey is concentrated to 60% total solids and the concentrate, at greater than 70 0 C, is blended with a hot solution of 4% pectin in a ratio of 7:1 and held at 70 0 C for 10 to 15 minutes before adding preservatives, potassium thiosulphate and vanilla flavouring, the mixture gelled at around 50 o C (Mann, 1990). According to a US patent, up to 75% of whole egg in baked custard can be replaced by a mixture of 40-60% blend of whey solids product with 0.5-5.0% carboxy methyl cellulose and 60-40% of acetylated shortening (Mann, 1982). Whey protein concentrate (WPC), dried skim milk and fat globules with modified membranes were used to manufacture mixed and filled dairy gels. With increasing WPC content, the syneresis of gels and their appearance changed from high and clear to low and turbid respectively (Aguilera and Kessler, 1989). There are other reports for manufacturing of jelly desserts based on deproteinized whey with added fruit juice, sugar colourings and flavourings (Mann, 1991). 2.4.4.5 Whey in dairy products Whey products are finding increased application in dairy products especially Ice cream, Yoghurts and dairy beverages. Studies on substitution of dried skim milk in Ice cream by dried whey, demineralised dried whey and ultra filtered dried whey, indicate that it can replace up to 25% level of dried skim milk (Mann, 1982) have been reported. The addition of whey solids above 25% results in decrease in viscosity, overrun, freezing point, melting resistance, protein content, body and texture scores and acceptability of Ice cream. However, there will be increase in specific gravity, acidity and lactose content of Ice cream (Vyas et al., 1980). Sherbets made using direct-acid-set whey had slight differences in acidity, total solids content and flavour score with no detected defect attributable to whey addition (Mann, 1982). Patel et al., (1993) studied the effect of addition of WPC to cow milk for preparation of khoa and reported that addition of WPC at 5% level produced a grainy texture in khoa, with additional browning, which partly masked the undesirable yellowish colour of cow milk khoa. Frozen yoghurt prepared by replacing skim milk solids by WPC to the extent of 50% level, appreciably decreased whey separation (Jayaprakasha et al., 2000). Whey protein concentrate obtained from direct-acid-set Cottage cheese whey when Page 17

Review of literature used in Cottage cheese dressing; scored slightly lower sensory scores than the commercial Cottage cheese from same source (Mann, 1982). Prajapati (1979) concluded that Cheddar and Paneer whey yielded Ricotta cheese with desirable flavour characteristics whereas the cheese from casein whey was salty. There was significant increase in number of yeast and fungi in Prato cheese enriched with WPC. Whey proteins, when added before coagulation, increased cheese yields of Chanco cheese (Mann, 1991). The manufacture of whey product, which can be used in milk mixtures, or in dry form in the manufacture of dry or liquid infant or dietetic milk products, is covered by a Russian patent (Mann, 1982). For humanization of bovine milk whey predominant protein was preferable over casein predominant protein (Pedersen, 2003). In view of this a great potential exists in the application of WPC in infant food formulations. 2.4.4.6 Use of whey in other food products Whey and whey products are also used in several other food products like meat, traditional foods as well as in low-fat applications, sports nutrition etc because of its high functional and nutritive value (Raju et al., 2005). Simplesse derived from egg white, skim milk and whey protein, is a protein based fat substitute approved by the Food and Drugs Act (Sandrou and Aravanitoyannis, 2000). Whey Protein concentrates and whey protein isolates are used as fat replacers in meat products (Kumar et al., 2003). Concentrated whey was successfully used in the preparation of jaggery (Madariya and Rao, 2012), idli, dosa, rice and roti (Papinwar, 2009; Kumar, 2009). Ragi dosa was also prepared with incorporation of WPC up to 30% level was more acceptable than the one prepared without WPC (Tripathy et al., 2003). Whey protein based coatings over peanuts reduced its rancidity significantly (Lee et al., 2002). Whey proteins when added to the flour at 5% level in the preparation of noodles, little change was noticed apart from weakening of cooked noodles; addition of 10% WPC gave very sticky dough, noodles were significantly harder, with improved colour (Towler, 1982). Page 18

Review of literature 2.4.4.7 Use of whey in fruit juices Simple technologies to incorporate fruit juice to whey have been attempted by many researchers. Several experimental beverages with citrus flavor have been developed for which high consumer acceptability has been reported (Holsinger et al., 1974). Singh et al. (1994) utilized Paneer and Cheese whey for developing acceptable whey based mango, pineapple, lemon and banana beverages. Composition of an acceptable beverage formula varied with the type of fruit, with juice content ranging from 5% for lemon to 20% for pineapple or Banana with whey content ranging from 73 to 87%. TS, fat and protein content in different fruit beverages varied from 13.3 to 16.2%, 0.32-0.38% and 0.51 to 0.61 respectively. Among beverages, mango beverage was the most preferred. Shukla et al. (2000) developed ready to serve beverage from whey by the addition of 10% sugar and 30% litchi juice, While Sirohi et al. (2006) reported the preparation and storage of whey based mango beverage. Ghatak et al. (2009) reported the development of a beverage from deproteinized Channa whey by incorporation of mango pulp on a laboratory scale. Four varieties of mango viz. Himsagar, Amarapali, Langra and Fajli at four different levels (8, 12, 16 and 20%) were tried with different sugar levels (6, 8, 10, and 12%). The acidity of the beverage was adjusted to 0.4% by the addition of citric acid. The products were then heated to 80 0 C for 5 minutes, bottled and carbonated. Whey beverage from Amarapali variety of mango pulp at 12% with 8% sugar level scored the highest on the basis of sensory attributes. On an average, the finished product contained 17.60, 0.10, 0.21, 6.24, 8.74 and 0.48 % of TS, fat, protein, lactose, sucrose and ash respectively. 2.4.4.8 Lactic acid production Panesar et al. (2007) reported that whey disposal is a major problem for the dairy industry, which demands simple and economical solutions. The bioconversion of lactose present in whey to valuable products has been actively explored. Since whey and whey permeates contain significant quantities of lactose, an interesting way to upgrade this effluent could be as a substrate for fermentation. Production of lactic acid through lactic acid bacteria could be a processing route for whey lactose and various attempts have been made in this Page 19

Review of literature direction. Immobilized cell technology has also been applied to whey fermentation processes, to improve the economics of the process. A fermentative means of lactic acid production has advantages over chemical synthesis, as desirable optically pure lactic acid could be produced, and the demand for optically pure lactic acid has increased considerably because of its use in the production of a biodegradable polymer, and other industrial applications. This review focuses on the various biotechnological techniques that have used whey for the production of lactic acid. 2.4.4.9 As preservative in vegetables Martin-Diana et al. (2006) stated the whey permeate as a biopreservative for shelf life maintenance of fresh-cut vegetables. Whey permeate at different concentrations (0.5%, 1.5% and 3%) was used as natural sanitizing agent in the washing treatment of fresh-cut lettuce and carrots. These treatments were compared with chlorine at 120 ppm widely used in the industry. Microbiological, quality (colour changes, browning-related enzymes, headspace gas composition, textural changes and sensory analysis) and nutritional (ascorbic acid and carotenoids) markers were monitored over 10 days in fresh-cut lettuce and carrot packages stored at 4 0 C. Whey permeate at 3% resulted in equivalent or better microbial load reduction than chlorine. Although lower concentrations of whey permeate produced minor initial reduction, microbial counts at the end of the storage were below the recommended levels (108 Cfu/g) for safety of freshcut vegetables. Sensory analysis panel considered all the samples of fresh-cut lettuce acceptable. However, in the sensory results the sliced carrots treated with 3% whey permeate and chlorine scored lower acceptability due to higher surface whiteness, although these samples had lower microbial loads. Three percent WP controlled the browning-related enzymes better than 0.5%, 1.5% WP and chlorine and consequently the browning. However, this reduction in browning-related enzymes did not result in a lower browning appearance to visual observation during the 10 days storage. The use of high concentrations of WP accelerated the loss of ascorbic acid and carotenoids. These results suggest that whey permeate could be a promising alternative to chlorine for sanitizing fresh-cut vegetables. Page 20

Review of literature 2.4.4.10 Functionally modified whey Kester and Richardson (1984) reported that modification of whey proteins to enhance or alter their functional properties may increase food applications. Whey protein modification can be accomplished by chemical, enzymatic, or physical techniques. Acetylation, succinylation, esterification, amidation, phosphorylation, and thiolation are chemical modifications that can induce significant alterations of the structure and functional behavior of whey proteins. Enzymatic protein modification may entail partial proteolytic hydrolysis, incorporation of crosslinks within the protein conformation, or attachment of specific functional groups to the protein. Physical techniques to alter whey protein functionality include thermal treatment, biopolymer complexing and texturization. 2.4.4.11 Whey based lassi Kumar et al. (1987) developed a lassi type cultured beverage from cheese whey. Skim milk and cheese whey (5:95) were heated to 100 C and acidified with HCl (ph 4.5) to obtain the curd. Curd so obtained was added to separate whey to raise the protein level to 3%. At ph 7.0, the mixture was homogenised, pasteurized and cooled to 22 C and inoculated with LF 40 culture. After 16 h incubation, sugar @ 12.5% and synthetic pineapple flavour were added. Lassi packed in polyethylene pouches had a shelf life of 6 days at 5 C. The finished lassi contained 21 to 22% TS, 3.05% protein, 17.85% carbohydrate (including 12.5% added sugar) and 0.3% ash. Attempt for enhancing the shelf-life of Lassi apart from cold storage was made by Naresh and Prasad (1996). Addition of nisin @ 200 or 300 IU/ml and 400 or 500 IU/ml extended the acceptability to 24 and 32 h respectively, compared to 8 h for control at 30 C. Simultaneously at 20 C storage, the acceptability increased from 12 to 24 h and 48 h by adding nisin @ 100 or 200 IU/ml and 400 or 500 IU/ml, respectively. Nisin @ 500 IU/ml could extend acceptability up to 8 to 10 days at refrigerated storage. Lassi like beverage was developed using paneer whey and buffalo milk with pectin and CMC as stabilizers. After neutralization, paneer whey (ph 6.6) was mixed with standardized buffalo milk (6% fat), inoculated with NCDC167 Page 21

Review of literature culture @ 1% and incubated at 30 C for 14 to 16 h. Dahi was blended along with sugar syrup and flavour. The beverage had 1.35% protein, 19 to 36% TS, 1.9% fat and 4% lactose. The product with 70% whey and pectin, CMC in the ratio of 0.5:1.0, at 0.6 % level was adjudged most acceptable (Mittal, 2003). 2.5 Utilization of Whey in Bakery products Whey in the form of whole whey or concentrated or dried form has been widely used in the manufacture of several bakery products like varieties of bread, biscuits, cakes etc. Whey and whey products serve both as protein supplements and baking aids in baked products (Sienkiewicz and Riedel, 1990). The use of whey increases the dietetic value of baked products by introducing calcium, thiamin and riboflavin. Both whey and whey powder exert, moreover, a positive influence on the important properties of baked products such as - increase in product volume, increase in yield, improvement in crumb consistency, structure (texture, porosity), appearance (browning), fresh-keeping quality and the flavor. Their use also leads to a reduction in the quantity of flour which is necessary for the baking process. Owing to the high percentage of lactose, whey solids can be substituted for dextrose and sucrose in many baking formulae (Alesch, 1958). Three principal methods of application of whey in different products have been identified: (Bunnies and Timm, 1983; Seibel et al., 1984) - partial or full replacement of water used for the particular recipe, - partial or full replacement of the fat-free milk dry matter used, - replacement of eggs by whey proteins. 2.5.1 Utilization of whey in biscuits and cakes Dried form: A protein rich biscuit with a keeping quality of as long as one year and readily acceptable to children in South East Asia and New Zealand was reported by Chapman (1966). This biscuit was made from dried skim milk (46 percent); anhydrous butter fat (15 percent), cheese (15 percent), whey powder (10 percent), sucrose (10.5 percent) and water (3.5 percent), minerals, vitamins and flavoring substances were added before molding and vacuum drying. Page 22

Review of literature Concentrated form: Concentrated whey with 40 percent total solids was used at 2.5 and 10 percent levels, in manufacture of Banan a spiced biscuit with superior porosity, softness and good storage qualities (Shilovskaya, 1974). Concentrated whey was successfully used in the preparation of rusks and soup sticks (Jarita and Kulkarni, 2009). Poonam (2007) reported that concentrated whey can be successfully used in the production of buns with increased Protein Efficiency Ratio (PER). Demineralized and delactosed form: Ash and Colmey (1976) reported that bakery products like cakes, biscuits, muffins, pan-cakes, doughnuts and white bread can be fortified with partly demineralized and delactosed whey containing 32 percent lactose (by wt.) 54 percent protein, 10 percent minerals, 4 percent moisture and only trace amounts of fat to supplement dried skim milks. Egg substitute: In a review study conducted by Cocup and Sanderson (1987) it was found that whey protein concentrates with a protein content of 35 55% have found application in replacing whole egg as prebake glazes on biscuits and pastry. It was concluded that whey can be processed to promote properties with application in the shelf life extension of the baked products by retarding mold spoilage and replacing chemical additives. Arunepanlop et al., (1996) conducted a study to investigate the effects of partial replacement of egg white proteins (EWP) with whey protein isolate (WPI) on the appearance, structure, texture and sensory properties of angel food cakes. EWP replacement cakes were generally inferior to 100% EWP control cakes, whereas EWP replacement cakes with xanthan gum as additive were similar to 100% EWP control cakes with respect to appearance, texture and sensory properties. EWP replacement cakes with methyl cellulose exhibited air cell size distribution that was similar to that of control cakes. Puranik (1997) developed a technology for the manufacture of egg less cake mixes using milk byproducts, in lieu of egg in cake formulation.it was observed that the cakes had excellent physical and sensory characteristics, and the ready - to - use egg less cake mixes could be stored up to six months at ambient temperature. Page 23

Review of literature Sugar substitute: Studies have been conducted on the use of hydrolysed whey concentrates, in syrup form, as substitutes for sugar and other sweeteners in biscuits and cakes. These syrups are considered to be effective sugar substitutes with respect to their functional properties (sweetness, solubility and ability to be fermented by microorganisms) and are cheaper than beet sugar. Liquid whey: In a study conducted by Sharma and Khetarpaul (1996) rice and dehulled Bengal gram flour were mixed in ratios of 60:40, 70:30 and 80:20 (w/w). 100 g of the blend was then mixed with whey (105 ml) and fermented at 35 C for 18 h. The fermented slurries were used for the manufacture of biscuits and were evaluated for organoleptic characteristics. Biscuits with a blend of 70:30 were the most acceptable. As a result of incorporation of the fermented blend, the contents of antinutrients, such as phytic acid and polyphenols, were considerably reduced. Awasthi and Yadav (2004) studied the effect of incorporation of liquid dairy by products (Chhana whey and Skimmilk) on the chemical characteristics and then on sensory characteristics of soy fortified biscuits and reported that both skimmilk and whey increased the moisture, ash and crude fibre content of biscuits fortified with defatted soy flour. It also increased calcium, iron, phosphorus, sugar content and non enzymatic browning of soy fortified biscuits. Whey protein concentrate: Because of high functionality of whey proteins, WPC has been used in the production of functional bakery products like high protein products and fat replaced products (Kamaliya and Subhash, 2005). Mathur (1975) reported that WPC can also substitute egg white for the manufacture of meringues and macaroons. Arunepanlop et al. (1996) reported that whey protein isolate can replace up to 25% of egg white protein without adversely affecting physical and sensory properties of angel food cakes. In a study conducted by Raju (2004) high protein biscuits were developed by incorporating WPC up to 30% and was well accepted because of improved sensory attributes. The improved color in the product was attributed to the maillard reaction due to the interaction of the lactose and the proteins in WPC. The biscuits were found to have three times more protein content. High protein cake developed by incorporating WPC up to 30 % showed improved sensory characteristics and functional properties. Page 24

Review of literature In a study conducted by Sudhakara (2006), bakery products like milk cake were prepared by incorporating different levels i.e., 15, 20 and 25% of WPC gels and these products were subjected for sensory evaluation by panel of judges. The cake prepared by incorporating 20 % WPC gels had higher score than the control with respect to colour and appearance, flavour and mouth feel. The product also had better scores for colour of crumb and grain formation thus indicating that the added WPC gel improved the sensory attributes for better acceptability. Conforti and Lupano (2004) studied the functional properties of biscuits with WPC and honey. The presence of WPC with high protein content produced a decrease in the firmness and consistency and an increase in the cohesiveness of dough. Also the fracture stress of biscuits decreased with the incorporation of WPC. Gallaghar et al. (2005) investigated the effects of WPC and sodium caseinate on short dough biscuit formulation. Both protein powders increased the hardness of biscuits and hardness increased as the level of protein powder was increased. WPC resulted in biscuit shrinkage during baking, increased surface browning and also higher moisture and water activity was reported during storage period of 24 hours and 8 weeks. 2.6 Utilization of Whey in bread Bread is the food produced by baking a dough obtained by mixing wheat flour, salt, and potable water, leavened by specific microorganisms of bread fermentation such as Saccharomyces cerevisiae (Collado, 2003). There are many types of bread depending upon local demand like whole flour bread, brown bread, bread fortified with vitamins and minerals, milk bread and bread for diabetics (Rao, 2005). Bread also differs from one country to another such as rolls, pan bread, pita bread, French bread, toast bread, baguette, etc. (Collado, 2003). Since classical times, bread has continued to play an important role in the human diet. It is an important stable source of nutrients and energy and a source of complex carbohydrates. The major constituents of some breads have been illustrated in the table below: Page 25

Review of literature Table 2.3. Major constituents of breads (per 100 g): (Southgate, 1983) Bread type Whole meal Multigrain Bread Water Protein Fat Carbohydrate (g) (g) (g) (g) Dietary Energy Country fiber (kj) of (g) origin 38.3 9.2 2.5 41.6 5.8 914 UK 33 13 4 43 7 Egypt Brown 39.5 8.5 2 44.3 3.5 927 UK White 40.3 9.5 2 41.5 3.3 899 UK The principal attributes of bread are - loaf volume, crumb softness, grain uniformity, silkiness of texture, crust color, flavor and aroma, softness retention and nutritive value (Collado, 2003). Bread is a highly perishable product and has a shelf life of 3-5 days at room temperature, 1-2 weeks at refrigeration temperature and 3 months at freezer temperature. The three most common forms of bread deterioration are staling, moisture loss and microbial spoilage (Seiler, 1984). The reason why molds are important spoilage organisms in bread is that the food matrix has a relatively high moisture content and water activity (water activity= 0.94-0.97) with a ph of about 6. The bread most prone to spoilage by molds is sliced, prepacked and wrapped bread (Seiler, 1984). Liquid whey fermented or unfermented concentrated or unconcentrated - or whey solids or whey proteins could be used as one of the ingredients for bread manufacture for effective utilization of whey. Whole whey: Whole whey was utilized in liquid form successfully as a replacement of water (Imbs and Czerwinski, 1974). Fermented curd whey for usage as additive in bread manufacture was developed by Skudra et al. (1998). Curd whey fermented by using pure cultures of L. bulgaricus and L. acidophilus can be used as an additive in bread making to prevent the spoilage of wheat bread by Bacillus mesenthericus (Skudra et al., 1998). Whey fermented along Page 26

Review of literature with milk and wheat flour was prepared by Gelinas et al. (1995) for use in pan bread formulation. Concentrated form: Acidified and concentrated rennet whey (45 60% TS) was successfully used up to 5% level for production of bread of acceptable quality (Chramcov, 1977). Precipitated whey protein (PWP) upto 75% was successfully used in the formulation of Iranian Lavash flat bread. The increased supplementation of PWP resulted in increase in sensory scores of the samples (Jooyandeh, 2009). Divya (2007) reported that concentrated whey had a considerable positive impact on the loaf volume. Jayalakshmi (2011) reported that even lactose hydrolyzed whey had an influence in raising the loaf volume. Dried form: Whey in dried form along with other nutritive ingredients was utilized in production of protein enriched bread or buns for feeding elderly persons (Vukobratovic and Beleslin, 1991). Powdered whey was also utilized along with wheat germ etc. to prepare enriched bread (Vukobratovic and Beleslin, 1991). A preconcentrate of cottage cheese whey containing protein as high as 80 90 % prepared by UF vacuum evaporation and spray drying was developed for fortification of bread. Use of whey protein along with wheat, soya and yeast proteins was also reported by Titcomb and Juers (1976). Van Riemsdijk et al. (2011) reported the use of mesoscopically structured whey protein for the preparation of gluten free bread. Whey protein particles were prepared during phase separation by cold gelation of soluble whey protein aggregates. The addition of 2.4% whey protein particle suspension to wheat starch resulted in a dough that could be baked into leavened bread with specific volume upto 3.7 mg/l and a bubble size comparable with normal bread. Ultra-filtered cheese whey powder was reported to have the best effect on the sensory quality of Taftoon bread (Rostami et al., 2013). Paste form: Pashchenko et al. (1996) described a technological process for manufacturing a whey paste which contained on DM basis protein 10%, lactose 56 65%, salts 5.7 6% and water 30 35%. This paste was suitable not only for use in bread making but also in the manufacture of other bakery products. Page 27

Review of literature Demineralized and delactosed form : Ash and Colmey (1976) reported that bakery products such as white bread can be fortified with partly demineralized and delactosed whey containing 32% lactose, 54% protein, 10% minerals, and 4% moisture. Margarine substitute : Pashchenko (1991) has described the use of a combination of concentrates of whey or albumin milk (with a high alpha lactalbumin content) with a lipoprotein concentrate, prepared from protein bearing oilseeds such as rapeseed, as a substitute for margarine in the manufacture of bread and other bakery products. Advantages of this mixture compared with margarine included a higher proportion of polyunsaturated fatty acids, shorter processing times in bread making, and improved flavour, aroma and volume characteristics, as well as its lower cost. Effect on dough quality Whey solids because of good water binding quality are expected to affect the dough quality like stability, proofing time etc. Kadharmestan et al. (1998) reported that fortification of wheat flour with 10 % whey protein concentrate resulted in wet and sticky bread dough, but imparted improved handling properties. The stability and mechanical tolerance index of the French type bread dough were also found to improve by addition of 1% whey solids. Use of whey and whey solids also had an effect on the dough fermentation time or proofing. Imbs and Czerwinski (1974), reported that when water was replaced with liquid whey in bread formulation, it not only resulted in better kneading of dough but also improved yeast fermentation. Similarly use of 20 30% whey was found to reduce the total processing time by 12 13% (Silagadze and Lyushinskaya, 1980). Yousif et al. (1998) found that incorporation of liquid whey or acid whey or dried whey (@0.85 3.5% solids) improved the rheological properties of dough and also enhanced absorption and resulted in improved dough development time. Prior fermentation of whey before using in the formulation was also found to help leavening. Gelinas et al. (1995) reported that incorporation of cultured mixture of milk; whey and wheat flour in a pan bread formulation did not reduce proof time, but affected dough mixing stability. Sanina et al. (1996) after performing an analysis Page 28

Review of literature using a statistical tool generalized Lagrange multiplier method, established optimum whey concentration as 16.6% and moisture as 46.2% in dough for getting good quality of bread. Constandache (2005) reported that when untreated whey protein concentrate (WPC) and heat treated whey protein concentrate (WPCHT) were incorporated in the dough along with sodium casienate by the replacement into wheat flour, it decreased proofing time, increased loaf volume and improved the texture. WPCHT resulted in improved hanging properties of dough, bread volume and overall baking performance. Asghar et al. (2009) investigated the effect of modified whey protein concentrate on the TPA characteristics of frozen dough made from flour with different protein contents and found that values of instrumental texture parameters were significantly affected by the addition of modified whey protein concentrate (mwpc) and there was a significant decrease in hardness, cohesiveness, gumminess and springiness with its addition in dough samples. Ammar et al. (2011) reported the effect of addition of whey protein (i.e., acid whey, sweet whey, retentate of whey by Reverse Osmosis (R.O), the permeate, pasteurized acid whey, pasteurized sweet whey and whey protein concentrate) and soy protein (raw soy milk, sterilized soy milk, soy protein isolate) on the rheological properties of wheat dough using the studies of result conducted by a Farinograph. The addition of whey protein concentrated by R.O (retentate) exhibited the highest ability to increase water absorption, dough stability, dough development time and time required for breakdown of the dough (72.8%, 8.7, 7.7 and 10.0 min, respectively) followed by pasteurized sweet whey and whey protein concentrate. Meshkani et al. (2003) studied the effect of whey powder and carboxy methyl cellulose (CMC) on the rheological characteristics of bread dough by response surface methodology (RSM). The dough rheological properties (strength and elasticity) increased significantly on increasing the percentage of whey powder and CMC. Wail Alomari and Hadadin (2012) reported that hydrolyzed whey lactose syrup can be used as a sugar replacement for French type bread. The bread made by 25% sugar replacement was found to be better than the control bread on the basis of sensory scores. The bread dough made with 25% replacement of Page 29

Review of literature sugar by hydrolyzed whey lactose syrup was found to have better rheological properties than the control bread dough with regards to stability, rate of absorption and mechanical tolerance indication. Effect on bread quality Several studies indicated that use of whey has beneficial effects on bread quality like loaf volume, flavour, aroma, crumb quality etc. Replacement of 13 18% water in dough formulation with concentrated whey (20% TS) was found to improve bread volume (Preller, 1978). Similar effect was reported by Silagadze (1980) when whey was added @ 20 30%. Such a result was also observed by Erdogdu et al. (1996) on incorporation of commercial whey protein concentrate into dough. Similar observation was made by Kadharmestan et al. (1998) who reported increase in bread volume by 50 ml. Improvement in specific loaf volume was also reported by Yousif et al. (1998) as a result of addition of whey solids to formulation in the form of liquid whey or dried whey, and by Sudhakara (2006) using 20% WPC gels. But, according to Gelinas et al. (1995) use of cultured mixture of whey, milk and wheat flour reduced the specific volume of bread. Addition of whey was found to enhance the porosity of bread (Imbs and Czerwinski, 1974; Silagadze, 1980). It also resulted in lighter crumb color and improvement in crust quality (Imbs and Czerwinski, 1974). Use of 20 % WPC gels in dough also improved sensory scores with respect to crust color and character of bread (Sudhakara, 2006), but fermented whey either in dilute form or concentrated form was found to adversely affect the same (Venskutonis, 1995). With regard to crumb quality, use of 2% whey concentrate (30% TS) gave a more elastic crumb than usual (Liepin and Saara, 1984) and use of 20% WPC gel the crumb colour and also results in grain formation (Sudhakara, 2006). Use of powdered whey along with wheat germ, wheat bran and whole meal flour was found to result in excellent crumb quality (Vukobratovic and Beleslin, 1991). Cultured dairy ingredients including whey yielded a firmer crumb (Gelinas et al., 1995). Enhancement of flavour of bread as a result of whey incorporation was reported by Preller (1978). Similar result was reported by Liepin and Saara, (1984) with the addition of fermented whey concentrate, and the improved aroma Page 30

Review of literature was attributed to volatile fatty acids, alcohols and benzaldehyde (Venskutonis, 1995). Whey has also been utilized to prepare flavour enhancers for bread. Gelinas and Lachance (1995) described preparation of concentrated cultured dairy ingredients with enhanced aroma levels. This involves inoculation of equal mixture of milk and whey (20 % TS) with L. casei var. rhamnosus and incubation for 24 h. This could be used as such in bread manufacture or the same can be dried and used as flavour enhancer (@ 1 2 % dry basis) in bread making process. Effect on shelf life of bread Numerous studies aimed at prolonging bread s period of freshness and suitability reveal that the process of aging can be retarded by adding to the dough certain additives as antistaling agents, such as fat, milk, whey, enzymatic or other preparations (Rogers et al., 1988; Xu et al., 1992; Fik & Macura, 2001). Some studies have indicated that whey proteins have an influence on the rate of staling and thereby on shelf life. Erdogdu et al. (1996) utilized heat treated acid whey protein in bread and found that it lowered the rate of staling of bread as measured by universal testing machine and differential scanning calorimetry enthalpy changes. Similar results were reported by Yousif et al. (1998) who utilized unconcentrated and concentrated whey in French type bread and reported increased keeping quality by 2 days. Using whey proteins along with powdered whey, gelatin, potato flakes and acetic acid, it was reported that the bread made from the formulation remained wholesome and tasteful for eating even after seven days. In a review study conducted by Cocup and Sanderson (1987), it was concluded that whey could be processed to acquire properties helpful in the shelf life extension of bread and fermented baked products. Effect on nutritive value of bread Silagadze and Lyushinskaya (1980), noted that by addition of 20 30% whey to bread formulations, nutritive value of bread improved. Incorporation of whey protein concentrate at 10% levels was found to increase the protein content of bread up to 20.2% and also elevated the proportion of essential amino acids (Kadharmestan et al., 1998). Vukobratovic and Belesin, (1992) discussed the Page 31

Review of literature nutritional value of several products including bread enriched with wheat germs, whey powder, milk powder, soya flour, wheat bran and gluten flour. 2.7 Multigrain-evolution, benefits and applications: Multigrain products are obtained by mixing two or more grains together and processed to form a product with special health benefits. The basic ideology behind a multigrain product is that each grain has got its own nutritional profiles and by combining two or more grains additional benefits can be obtained. The source of nutrients from different grains is always better than the nutrients obtained from a single grain (Arya et al, 2013). 2.7.1 History of Multigrain: The history of multigrain can be traced back to Egypt when the Egyptians perfected the art of making bread with dough prepared by mixing water and unleavened grains. Although the exact date of origin of multigrain bread is not well documented yet indications point that bread made from a variety of grains were popular in most countries (Anon, 2011). The concept of commercially available multigrain bread dates back to 25 years. For example the process preparation of multigrain flaked cereals using oats, barley, wheat and corn was used by Nabisco Brands, Inc., in New Jersey (Karwowski and Ferraro, 1986). General Mills in 1991 launched a multigrain cereal named Cheerios Multgrain in 1991. Due to unawareness about health benefits and lesser sensory acceptability the products and researches related to multigrain slowed down thereafter. But in recent times there is an increasing trend among the consumers towards consumption of Multigrain products as they are more aware about the health benefits. 2.7.2 Health benefits and Nutritional advantages: Health benefits of multigrain products are due to the incorporation of whole grain forms and these benefits are particularly enhanced when different whole grains singly or in combination are used in food preparation (Arya et al, 2013). Research has shown that whole grain intake helps to lower the risk of cardiovascular diseases, ischaemic stroke, type II diabetes, metabolic syndrome Page 32

Review of literature and gastrointestinal cancers (Jones, 2006). At present there is significant scientific evidence highlighting the health benefits of consuming whole grains with regards to chronic disease prevention, particularly with regards to diabetes, cardiovascular disease and cancer (Cavazos and Gonzalos de Mejia, 2013). Diets high in wholegrains are associated with a 20-30 % reduction in the risk of developing diabetes (Type II Diabetes), which is attributed to a variety of wholegrain components notably dietary fibre, vitamins, minerals and phytochemicals (Belobrajdic and Bird, 2013). Multigrain products offer nutritional advantages being a rich source of mineral, vitamin, fibre and providing a rich source of energy. Whole grains are very rich sources of vitamin, particularly vitamin B complex (Thiamine, riboflavin, pyridoxine, niacin and folate) (Liebedzinska and Szefer, 2006). The following table highlights the nutritional profile and health benefits of some whole cereal grains: Table 2.4. Nutritional profile and health benefits of cereal grains Cereals Nutritional Profile Health Benefits Reference Wheat High: protein (11-13%), Dietary Fibres Zielinski et carbohydrates, insoluble prevent al., 2001 dietary fibres, potassium. constipation. Low: lysine, fats, sodium Also contains Fe, Zn, P, Se, Mn and Ca, phytochemicals including lignans, phenolic acids, phytic acids, plant sterols and saponins. Low-fat reduces risk of cardiovascular disorder. Phytoestrogens reduce hormone related cancer like breast cancer. Gani et al., 2012 Page 33

Review of literature Oats High: fat (7-8%), protein β-glucan helps to Zielinski et (14%), carbohydrates reduce cholesterol. al., 2001 mainly starch, soluble dietary fibres, K, gluten. Individuals with high dietary fibre Patel al.,2008 et Low: sodium, lysine Also contains vitamin B complexes and phytochemicals. intake appear to be at a significantly lower risk of developing coronary heart Anderson et al., 2009 Huttner and Arendt,2010 disease, stroke, hypertension, diabetes, obesity and certain gastrointestinal diseases. Sorghum High: carbohydrate, Polyphenols act as Abdel-Aal et potassium antioxidants and al., 2006 Low: Fat, lysine, Cu, Mn, Ca prevent diseases., Na Gluten free Moderate: Protein, dietary fibre, phytochemicals, Fe, formulation in celiac disease. Zn, Mg, P, Se, Vitamin B and Vitamin E Maize/Corn High: fat (4-5%), carbohydrates, mainly starch, dietary fibres, β- carotene, K, relatively high plant sterols than other cereals. Vitamin E is a well known antioxidant. Plant sterols reduce blood cholesterol. Phytoestrogens Ayatse et al., 1983. Page 34

Review of literature Low: Relatively lower protein than other grains (9%), lysine, tryptophan, Cu, Mn, Na Moderate: phytochemicals, trace elements, Vitamin-B, Vitamin-E reduce risk of hormone related cancers like breast cancer. Gluten free formulation in celiac disease. Gluten free Rice High: Insoluble dietary fibre, Lowering of blood Chotimarkorn carbohydrates cholesterol and Silalai, Relatively low: fat, protein (8%), Cu, Mn, Ca, Na Contains phytochemicals (greater in brown rice) Free from gluten Decreases the incidence of atherosclerosis disease, has laxative effect. 2008; Kahlon et al.,1994; Saunders, 1985; Saunders, 1990 Gluten free formulations in celiac disease. Barley High: carbohydrate, soluble β-glucan reduces Baik and fibre β-glucan, K blood cholesterol. Ullrich, 2008 Low: lysine, fat, Na Low glycemic index Zielinski et Moderate: protein (10%), vitamin E, trace-elements, lowers glucose. blood al., 2001 phyto-estrogens, vitamin Antioxidants and complexes phytoestrogens reduce risk of hormone related cancers like breast cancer. Page 35

Review of literature Rye High: protein (15%), lysine, Reduced heart Poutanen et dietary fibre, carbohydrates, attacks al., 2009 K Lower blood Zielinski et Low: fat, Na, Ca, Mn, Cu glucose levels. al., 2001 Moderate: Vitamin B, Phytoestrogens Vitamin E, Fe, Se, P, Mg, reduce risk of phytochemicals hormone related cancers like breast cancer. Flaxseed High: Protein (high in arginine, glutamic acid, aspartic acid), fat ( contains good amount of α-linoleic acid), Dietary fibre (liganan), Limiting amino acids are Lysine, methionine, cysteine ω-3 fatty acids reduce hypertension, triglyceride and cholesterol levels. Protein of flaxseed more effective in lowering triglyceride and plasma cholesterol levels compared to soy protein and casein protein. Being rich in dietary fibre, it can reduce heart disease, diabetes, colorectal cancer, obesity and inflammation. Oomah and Maza, 1998. Bhathena et al., 2002 Morris, 2003 Page 36

Review of literature Pseudocereals Protein is higher than wheat Very cheap, Arendt et al., (Amaranth, in all of them. Amaranth abundantly 2008 quinoa and buckwheat) leads in protein content followed by quinoa, buckwheat. available and can be used in composite flour Arendt and Bello,2008 Protein is generally in form formulations. of globulins & albumins. Dietary fibre: high in buckwheat, compared to grains in amaranth & quinoa. Excellent sources of vitamin E. High lipid content in buckwheat. Wheat provides 1670 kj energy /100g whereas, barley provides 1680 kj/100g, lower energy is provided by sorghum, brown rice and lowest energy is provided by rye because of its high fibre content, resistant starch (Patel et al., 2008). The factors to be considered for new formulations are: (Arya et al., 2013) - Physical and organoleptic characteristics - Significant health benefits - Physical properties of individual grains/their flours - Nutritional losses during processing - Price of the product 2.7.3 Novel applications of multigrain in foods: A large range of products have been developed commercially using multigrain which can be classified as bakery products, breakfast cereal, flour and flour products, extruded products, fermented products and coating (Panghal et Page 37

Review of literature al., 2009). Certain products may fit in more than one category. The products in the food industry where multigrain find application are bakery products, breakfast cereals, snacks, extruded products, beverages, meat and meat products. 2.7.4 Use of multigrain in the bakery industry: Bakery industry offers a huge area for application of multigrain in formulation of products such bread, biscuits, bagels and muffins. Some of the multigrain used in bakery product formulations is mentioned in the table below: Table 2.5. Multigrain used in Bakery formulations (Arya et al., 2013) PRODUCTS GRAINS USED OTHER INGREDIENTS REFERENCES Bread Flour: Durum wheat, Extract of olive Varvello and malted wheat, soft pulp, water, salts, Varvello, 2010. wheat, whole oat, α-amylase, flax, wheat germ antioxidants Biscuits/Cookies Flour: Wheat, barley, rye, millet or sorghum Milk solids, honey, sugar, salt emulsifier and raising agents Abdel-Aal et al., 2006 Muffins Flour: Wheat, Barley Baik and Ullrich, 2008 The bakery industry in India is the third largest revenue earner among the processed food industries and biscuits contributed most significantly to the sector s growth. The industry was worth $ 4.7 billion in 2010 and is expected to touch $ 7.6 billion mark by 2015. (Anon, 2013). Hence, this is an area which presents huge opportunities for improving the health profile of its products using multigrain. Page 38

Review of literature Even though for nutritional importance multigrain has been used in several products there is not much of information on the use of whey solids in multigrain products. In order to fill this gap the present investigation is undertaken and the justification is initiated. 2.8 Justification of the present Project : Whey is one of the major by-products of the dairy industry all over the world. A large part of this whey however, is left unutilized and disposed off which results in significant loss of potential nutrients. Whey contains almost 40% of solids present in milk and is rich in organic matter and commands high biological oxygen demand of about 30,000-50,000 mg/litre. In order to treat this whey to the statutory pollution control norms, about 0.4 kw of energy per litre at a cost of 55-60 paise is incurred. Thus, disposal of whey not only causes the loss of nutritionally rich milk solids but also puts additional financial burden on the dairies. Bread is one of the staple foods of the world population and is made from wheat and serves as a major source of carbohydrates. About 55% of the body s daily energy requirement comes from carbohydrates. Bread and biscuits account for nearly 85% of the total bakery products in India. Due to changing lifestyles people are adopting bread as a regular food and are ready to pay the price for quality and variety. Multigrain bread can made out of different grains and is nutritionally superior because the carbohydrates come from different sources rather than from a single cereal. The grains may be used as such in the preparation or in the form of flour. Multigrain bread is becoming increasingly popular as consumers are growing more and more health conscious. The cost incurred by dairy plant for the treatment of the whey can be minimized by utilizing whey appropriately with minimal process operation. There are several ways by which whey can effectively be used and one such possibility is to utilize it for the preparation of dough in bread manufacture. The addition of whey will also render the bread nutritionally superior to the other breads. Multigrain bread would in comparison, offer tremendous potential for effective utilization of whey proteins for desirable enhancement of nutritional and functional value of the bread. The protein content of the bread is expected to increase as Page 39

Review of literature well as the quality of the protein is expected to get better by use of potentially low cost whey solids. Based on the above hypothesis, present project is proposed for detailed investigation with following objectives: 1. Process optimization for the production of multigrain bread by incorporating concentrated whey. 2. Study the effect of whey incorporation on the physico-chemical and sensory characteristics of bread. 3. Assess the impact of addition of concentrated whey on the shelf life of bread. The following chapter deals with the materials used and the methods adopted in carrying out the investigation pertaining to this project. Page 40

Chapter- 3 Materials and Methods

3.MATERIALS AND METHODS This chapter deals with various materials, equipments and instruments used and methods employed during the investigation relating to the sample preparation & collection, quality evaluation and final analysis. 3.1 Materials 3.1.1 Ingredients Paneer whey and cane sugar was collected from the Institute s Experimental Dairy. Wheat flour, Maize flour, Sorghum flour, Oats (Quaker) and Flaxseed were procured from M/S-Sivananda General stores (Bangalore). Common salt, refined sunflower oil and yeast (PRESTIGE brand with ISI mark) used for bread manufacture were procured from local market. 3.1.2 Chemicals All the chemicals used in the study were of LR / AR grade and procured from Nice Chemicals, Chennai. 3.1.3 Media Potato Dextrose Agar obtained from Himedia, Bombay was used for microbiological analysis. 3.1.4 Glassware Glasswares used were of Schott & Duran and Borosil brand and were thoroughly cleaned by soap solution, dried & used. For microbiological analysis the glasswares were sterilized as per standard practice before use. 3.1.5 Equipments The following equipments were used in the study : Page 41

Materials and methods 3.1.5.1 Planetary mixer: A bakery model planetary mixer supplied by Lalith Industries, Bangalore was used in the preparation of dough for the preparation of multigrain bread. 3.1.5.2 Baking Oven: A baking oven, supplied by M/S-Dolar equipment Pvt Ltd., Bangalore, which is electrically operated with thermostatically operated control device for both top and bottom coils, was used for baking the multigrain dough to bread. 3.1.5.3 ph meter : A ph meter, Digisun Electronics, DI-707, supplied by Servewell instruments pvt Ltd., Bangalore, was used to measure the ph of concentrated whey, multigrain dough and multigrain bread. 3.1.5.4 Texture Analyzer A TA.XT plus (Stable Micro System, England) was used for measuring TPA characteristics and viscoelastic characteristics of the dough and the multigrain bread during the period of investigation. 3.1.5.5 Water activity meter : Rotronic Hygroskop, BT-RS1 Ag Switzerland was used for measuring the water activity of multigrain bread. 3.1.5.6 Reflectance meter : A reflectance meter supplied by Elico., Hyderabad, was used for measuring the crust colour of the multigrain bread samples. Page 42

Materials and methods 3.1.5.7 Gerhardt digestion & distillation assembly A digestion and distillation assembly of Gerhardt make was used in the determination of protein content of whey and the samples. 3.1.5.8 Weighing Balances: A weighing balance supplied by Spark instruments, Bangalore was used for weighing the ingredients for bread making. Another weighing balance of Sartorius make, Bangalore was used for weighing the samples taken for gravimetric tests. 3.1.5.9 Single effect evaporator : A single effect evaporator of APV instruments, Calcutta was used for concentrating the paneer whey obtained from the Experimental Dairy of NDRI to the desired level of solids content for incorporation during dough preparation of multigrain bread. 3.2 Methods 3.2.1 Preparation of paneer whey Cow milk containing 4.2 % fat and 12-13 % total solids obtained from the Experimental Dairy of the Institute was used for obtaining paneer whey. Thirty liters of milk was heated to boiling and then allowed to cool to about 85 0 C with occasional stirring to avoid skin formation. A solution of 2 % citric acid was added for coagulation of milk. The coagulum obtained was removed by filtering the whey using a clean filter cloth. The whey was held for another 2 min. at 85 0 C before drawing it into a clean container for further processing. About 25 litres of whey was thus obtained in each batch. Page 43

Materials and methods 3.2.2 Concentration of whey Concentration of whey was done by a pilot scale batch type APV vacuum evaporator of 40 lit capacity of A.P.V. make. The condenser was first cleaned using a 1 % solution of sodium hydroxide at 70 0 C for 15-20 min. followed by circulation of a 0.25 percent solution of nitric acid at 55-60 0 C for 15 min., after which the evaporator was thoroughly rinsed with potable water. Twenty liters of paneer whey was cooled to 55-60 0 C, and fed into the evaporator; care was taken to avoid foaming of whey in the evaporator and its escape along with the condensate. The whey was condensed at 55 0 C under vacuum (24 of mercury) for approximately 30 min. Steam under 10 lb pressure was passed through the calandria to heat the whey and maintain a temperature of 55 0 C. A product with approximately 25 % total solids was obtained, which was roughly estimated by refractometer. The condensed whey was drawn into a clean container and TS % (total solids) was adjusted to desired level by addition of distilled water. The final TS content of the concentrated whey was accurately assessed by gravimetric method. 3.2.3 Preparation of bread In the preparation of bread, seven and a half grams of baker s yeast was dissolved in 50 g of lukewarm water (15%,20% and 25% TS concentrated whey in case of experimental samples) with about 2 g of sugar and was allowed to set for 10 min. Later 23 g of sugar was mixed to the water containing yeast. Two hundred grams of wheat flour was sieved and supplemented with 12.5 g each of maize, flaxseed, oat and sorghum flour. 2g of salt was added to the multigrain flour mixture and it was thoroughly mixed before adding the water containing yeast and sugar. A good mixing of salt was ensured to avoid the deactivation of yeast during fermentation. Thirty-seven and a half grams of refined oil was used for kneading the dough into a smooth texture. The ingredients were taken in a Hobart mixer and kneaded for 4-5 minutes into a smooth dough during which the remaining amount of warm water (15%,20% and 25% TS concentrated whey in case of experimental samples) was slowly added to the dough. The dough was divided in equal portions of 400 g. The divided dough was rounded to realign gluten fibrils and to facilitate Page 44

Materials and methods CO 2 retention and to give it a smooth surface. The rounded doughs were kept for intermediate proofing for 10-15 minutes for better machinability. The dough was then molded by the operations of sheeting, curling, seaming to redistribute gas cells and give it a final crumb structure. The molded doughs were then placed in rectangular tin molds of size seven by three inches. The molds were maintained at 28 o C-30 o C, covered with a cloth until the dough rose to full size of molds and then baked at a certain temperature. The loaves were then cooled slowly to room temperature. The flow diagram for the preparation is detailed in the Fig 3.1. 3.2.4 Standardization of bread manufacture by incorporation of concentrated whey Concentrated paneer whey of 15, 20 and 25 % TS was incorporated into the bread formulation and its effect on dough proofing time and sensory, physical, chemical and rheological quality of the product was studied and optimized. 3.2.4.1 Study of Dough characteristics of multigrain bread by incorporating concentrated whey. Water portion of the bread formulation was replaced completely by three varying levels of concentrated whey of TS 15, 20 and 25%. The dough was studied for proofing time and rheological characteristics after molding. Rheological parameters studied: a) TPA characteristics i. Hardness ii. Cohesiveness iii. Springiness iv. Adhesiveness v. Adhesive Force vi. Gumminess vii. Resilience Page 45

Materials and methods Flow Diagram for the production of Bread Wheat flour : 200 g Oat Flour : 12.5 g Maize Flour : 12.5 g Sorghum Flour : 12.5 g Flaxseed Flour : 12.5 g Oil : 37.5 g Sugar : 25 g Water/Whey : 160g Salt : 5 g Yeast : 7.5 g INGREDIENTS MIXING KNEADING IN HOBART MIXER (5-6 minutes) PORTIONING (400 g) Addition of the other 100 ml of warm water/whey slowly during kneading ROUNDING Activation of yeast in 50 ml warm water/whey with 2 g sugar and adding it after proper mixing of all the ingredients except the remaining amount of water. INTERMEDIATE PROOFING (10-15 MINUTES) MOLDING PANNING FERMENTATION BAKING COOLING (Room Temperature / 40 minutes) SLICING OR CUTTING OF BREAD PACKAGING (LDPE POUCH) Figure-3.1 Flowchart for preparation of multigrain bread Page 46

Materials and methods b) Viscoelastic characteristics i. Stress Relaxation time ii. Co-efficient of viscosity iii. Modulus of elasticity iv. Creep retardation time v. Compliance. 3.2.4.1.1 Determination of proofing time of multigrain bread by incorporating concentrated whey: A study was conducted to determine the proofing time of multigrain bread dough with the incorporation of concentrated whey. 50 g of prepared dough was weighed and taken in a 200ml measuring cylinder after intermediate proofing and molding. The height of the dough was adjusted to a desired volume by tapping with a glass rod. Then the measuring cylinder containing the dough was kept at 30 o C and the volume attained by the dough during proofing was recorded at intervals of every 15 minutes. The time taken by the dough to attain the maximum volume was recorded as the proofing time of the multigrain bread dough. 3.2.4.1.2 TPA measurements of the multigrain bread dough : The TA-XT plus texture analyzer was switched on and the computer was linked to it. The Texture exponent 32 program was opened. In the Texture exponent 32 program TA settings were selected and library option was selected in the TA settings. In the library option TPA was selected under special tests. The project settings were entered as follows: Pre-test speed- 1mm/sec Test speed-0.5 mm/sec Post-test speed-10.00 mm/sec Target mode- distance Page 47

Materials and methods Distance-7.5mm Time-5 sec Trigger type-auto (force) Trigger force-5.0g Break mode- off Tare mode-auto Advanced options- on Control oven-disabled Frame deflection mode-off (XT2 compatibility) Accessory: P/75 plunger probe The probe was calibrated to a distance of 50mm, above the top of the container or the sample surface. The sample of dough tempered to about 25 C was cut into pieces of 40mm x 40mm x 15 mm size. The sample was positioned centrally over the platform and the computer was allowed to execute the program by activating run a test option, then the sample was compressed by the plunger twice in a gap of 5 sec to yield a force time curve. The height of the force peak on the first compression cycle (first bite) is the value of hardness (F). The ratio of the positive force under the second and first compressions (A 2 /A 1 ) is cohesiveness while the ratio of the time difference between D-E to the time difference between A-B is springiness as shown in the curve (Figure 3.2). The negative peak gives us the adhesive force and the area of the negative peak gives us the adhesive force. Resilience is the ratio of the area between B-C and A-B. Gumminess and Chewiness are derived parameters calculated as follows: Gumminess: Hardness x cohesiveness Chewiness: Gumminess x springiness= hardness x cohesiveness x springiness. Page 48

Materials and methods A-B A1 B-C D-E A2 Figure 3.2. Typical texture profile analysis curve 3.2.4.1.3 Viscoelastic charcacterization of the multigrain bread dough: Stress relaxation test of the doughs was determined using Texture Analyzer (TA-XT plus, Stable Micro Systems, England) under the following test conditions : Test mode : Compression Pre-Test Speed : 1.0 mm/s Test Speed : 0.5 mm/s Post-Test Speed : 10.0 mm/s Target mode : Distance Distance : 5 mm Hold time : 180 seconds Trigger Type : Auto Trigger force : 0.5 g Page 49

Materials and methods Accessory: P/ 75 plunger probe Procedure: The probe was calibrated to a distance of 50mm, above the top of the container or the sample surface. The dough was cut into pieces of 40mm x 40mm x 15 mm size. The sample was positioned centrally over the platform and the computer was allowed to execute the program by activating run a test option, then the sample was compressed by the plunger to about 33.3 % of its original height and the compression held for a period of 180 sec to yield a force time curve. The difference between the maximum value obtained on the force time curve (F 0 ) and the value obtained at the end of 180 seconds (F r ) was multiplied with a factor 0.367 to obtain F m /e. The value of (F m /e+ F r ) was calculated, the corresponding value of which on the time axis yields the stress relaxation time (Ƭ) of the bread. The curve for stress relaxation time (SRT) is given in figure 3.3. Modulus of elasticity is calculated from the formula: E o = (σ0- σr)/0.33 Where, σ0=f o /A where A is the area of the sample in contact with the probe in m 2 σr=f r /A where A is the area of the sample in m 2 Modulus of elasticity of the residual portion is calculated as follows: E 1 = σr/0.33 Total modulus of elasticity is: E(t)= E o e -t/ Ƭ + E 1 The coefficient of viscosity is calculated by the formula: ɧ=Ƭ x E o Page 50

Materials and methods F 0 F m /e + F r F r SRT Figure 3.3. Typical curve for stress relaxation time Creep retardation test of the doughs was determined using Texture Analyzer (TA-XT plus, Stable Micro Systems, England) under the following test conditions : Test mode : Compression Pre-Test Speed : 1.0 mm/s Test Speed : 0.5 mm/s Post-Test Speed : 10.0 mm/s Target mode : Force Force : 500 g Hold time : 180 seconds Trigger Type : Auto Trigger force : 0.5 g Accessory: p/ 75 plunger probe Procedure: The probe was calibrated to a distance of 50mm, above the top of the container or the sample surface. The dough was cut into pieces of 40mm x 40mm x 15 mm size. The sample was positioned centrally over the platform and the computer was allowed to execute the program by activating run a test option, then the sample was compressed by the plunger by a force of 500 g and the compression Page 51

Materials and methods was held for a period of 180 sec to yield a strain time curve. The difference between the maximum value obtained on the strain time curve (Ɛ 1 ) and initial strain (Ɛ 0) was divided with a factor 0.63 to obtain a value, the corresponding value of which on the time axis yields the creep retardation time (Ƭ) of the bread. The curve for creep retardation time (CRT) is given in figure 3.4. Compilance is obtained by the following formula: D t =D 1 (1-e -t/ Ƭ )+D 0 Where, D 1 = Ɛ 1 /σ D 0 = Ɛ 0 /σ Figure 3.4.Typical creep curve Page 52

Materials and methods 3.2.4.2 Optimization of concentration of whey Water portion of the bread formulation was completely replaced with concentrated whey, i.e. dough was prepared using concentrated whey instead of water. The concentrated paneer whey was used at three levels of total solids viz. 15, 20 and 25 % for manufacture of bread. The doughs made using 15,20 and 25% TS whey were baked at three different temperatures for 30 minutes-160 o C,185 o C and 210 o C. The bread samples were served to a panel of judges for sensory evaluation. Based on the judge s scores and remarks, an optimum level of TS% in bread and the best temperature for baking were selected for further studies. Parameters studied: a) Sensory Color and appearance. Flavor. Body and texture. Overall acceptability. b) Rheological: Texture profile analysis: - Hardness, N. - Springiness. - Cohesiveness. Stress relaxation time (SRT), seconds. c) Physical: Loaf weight d) Reflectance Page 53

Materials and methods 3.2.4.3 Proofing time reduction In order to achieve a proofing period same as in control, the following variables were tried: (1) Addition of yeast at two levels viz. 3 and 6 %. (2) Fermentation of the dough at a higher temperature of 40 C. The bread samples obtained from the dough samples as leavened under above conditions were served to the panel of judges for sensory evaluation. Based on the judge s scores and remarks, an optimum level of proofing time in bread was selected for further studies. Parameters studied: a) Proofing time. b) Dough ph c) Dough volume d) Sensory Color and appearance. Flavor. Body and texture. Overall acceptability. 3.2.4.4 Addition of Improvers to improve Bread Characteristics: In order to further improve the characteristics of the optimized multigrain bread, three different improvers were tried at maximum levels as per regulations of FSSAI. The improvers were Calcium phosphate (5000 ppm on flour basis), Calcium Carbonate (2500ppm on flour basis) and Ammonium Persulphate (2500 ppm on flour basis). Page 54

Materials and methods The improvers were added to the flour mixture before adding the oil and yeast and mixing in a planetary mixture. The bread made by incorporating concentrated whey with the addition of improvers was evaluated for the following characteristics. 1) Sensory a) Colour and Appearance b) Body and Texture i) Hardness ii) Springiness iii) Gumminess iv) Chewiness c) Flavour d) Overall Acceptability 2) Rheology: TPA i) Hardness ii) Cohesiveness iii) Springiness iv) Gumminess v) Chewiness The improver giving bread with highest overall acceptability was selected for use and was tried at three different levels and optimization of improver level was done based on the sensory scores. The whey incorporated multigrain bread prepared by adding different levels of the same improver was evaluated for the following sensory characteristics: 1. Colour and Appearance 2. Body and Texture 3. Flavour 4. Overall Appearance. Page 55

Materials and methods 3.2.5 Storage studies Bread loaves prepared by the standardized formulation using concentrated paneer whey were packaged in LDPE pouches, which were measured for gauge thickness and stored at temperatures of 30 and 5 o C and evaluated at regular intervals of 2 days during which it was analyzed for its sensory, rheological and physico-chemical changes. Parameters studied: a) Sensory: Colour and appearance. Flavour. Body and texture. Overall acceptability. b) Rheological: Texture profile analysis: - Hardness, N. - Springiness. - Cohesiveness. Stress relaxation time (SRT), seconds. c) Physico-chemical: ph. Moisture. a w Reflectance d) Microbiological: Yeast and mold count. Page 56

Materials and methods 3.3 Analyses 3.3.1 Analysis of whey Fat (Mojonnier method), total solids (gravimetric method), protein (Kjeldahl method) and ash (gravimetric method) of the whey samples were determined as per (ISI, 1981). 3.3.1 Fat About 10-11 g of whey is accurately weighed into a Mojonnier fat extraction tube. 1 ml of ammonia solution (specific gravity=0.88) was added to the contents of the tube and mixed thoroughly. 10 ml of ethyl alcohol (95% by volume) was added and the contents of the flask were shaken vigorously for about half a minute by closing the flask with a cork. After that the flask was shaken vigorously each time after addition of 25 ml of di-ethyl ether (specific gravity=0.720) and petroleum ether (boiling point=40-60 o C) respectively. The tube was then allowed to stand for 30 minutes. A pre-weighed beaker is taken and the ether layer is decanted upto the maximum extent. The extraction is repeated using 5ml ethyl alcohol and 15 ml each of diethyl ether and petroleum ether. The flask contents are again allowed to stand for 10 minutes and the ether layer is again decanted in the same pre-weighed beaker. The extraction is repeated once again. The ether layer of the flask was evaporated in boiling water bath or hot plate and then the beaker contents were oven dried for one hour at 110 o C followed by cooling in the desiccators. The procedure was repeated again until two successive weights do not differ by more than 1 mg. Fat % is computed by the following formula. Weight in g of beaker after drying Weight in g of empty beaker Fat%= Weight in g of the sample Page 57

Materials and methods 3.3.2 Lactose Lactose content of whey sample was determined as per the BIS procedure (ISI, 1981). 25 ml of milk was measured and taken in a 250 ml conical flask. The contents in the flask were diluted to about 150 ml with distilled water. 3.75 ml of 10 % acetic acid was added to the solution in the conical flask and the resulting mixture was boiled. The contents of the flask were cooled and transferred to a 250 ml volumetric flask and the volume was made upto the mark with distilled water. The contents of the volumetric flask were filtered through a Whatman No 42 filter paper and the first few ml of filtrate was discarded. The rest of the filtrate was collected in a dry conical flask. Fehling s solution to be used was standardized against a standard lactose solution of known strength. A burette was taken and filled up with this filtrate. 5 ml each of Fehling-A and Fehling-B solution respectively were pipetted out with a safety bulb pipette into a 250 ml conical flask. A pilot titration was carried out by adding the filtrate containing the lactose from the burette 1 ml at a time, to the mixed Fehling solution which is kept boiling. Once the Fehling solution starts to boil 5 drops of Methylene blue was added and the solution from the burette was kept running till the colour of the solution in the conical flask permanently turns to brick red. The experiment should ideally be completed 2 minutes from the initiation of boiling in the conical flask. Now, another 10 ml of mixed Fehling s solution is taken in another conical flask and the filtrate is run from the burette 1 ml less than the reading obtained in the pilot titration so that not more than 0.5 to 1 ml will be required to be added dropwise to complete the titration. The Fehling s mixture in the conical flask was boiled and 5 drops of Methylene Blue was added at the start of boiling. The filtrate is run dropwise from the burette and the titration is completed within 2 minutes from the start of boiling which is indicated by the change of colour to permanent brick red. The titration is repeated until we obtain at least two concordant readings. Lactose % is computed by the following formula: Lactose % in the standard lactose solution Lactose %= Titre value obtained during titration Page 58

Materials and methods 3.3.3 Physico-chemical analysis of bread 3.3.3.1 Sample preparation Sample slices of the bread to be analyzed were powdered in the mixer along with the crust (AOAC, 2005). This was taken for all further analysis. 3.3.2.2 Total solids In the determination of the total solids content of bread (AOAC, 2005), about 2 g of well mixed sample was weighed accurately and dried in a hot air oven at 110ºC ± 3ºC for 3 hours or till a constant weight was obtained and the percentage of total solids in the product was calculated. %Total Solids = W eig ht of sam p le a fter d rying x W eigh t of sam ple taken 10 0 3.3.2.3 Fat Fat in bread was determined by the acid hydrolysis method (AOAC, 2005). About 2 g of well-mixed sample was accurately weighed into a 50ml beaker and 2 ml of alcohol was added and stirred to moisten all the particles and to prevent lumping when acid is added. The above contents were then added with 10 ml HCl (25 parts of conc HCl + 11 parts of water), mixed well and set on water bath held at 70-80 C and stirred at frequent intervals during 30-40min.Following this 10 ml of alcohol was added, cooled and mixture was transferred to a Mojonnier fat extraction apparatus. Diethyl ether (25 ml) was added and the apparatus was shaken vigorously for one minute. Twenty-five ml of petroleum ether (40-60ºC) was added with repeated vigorous shaking and allowed to rest for half an hour. The ethereal layer was then decanted into a preweighed dish along with a few pumice stones. The extraction of aqueous layer was repeated twice using 15 ml ether mixture (diethyl ether and petroleum ether in 1:1 ratio). The solvent in the dish was dried on steam bath and the residual fat was further dried in the oven at 100 ±1ºC for one hour followed by cooling in desiccator and then weighed. Fat percentage was calculated as follows: Page 59

Materials and methods Fat percent = (Weight of Fat X 100)/Weight of the sample taken (g) 3.3.2.4 Total protein The percent protein in bread was determined by standard Micro Kjeldahl method described in AOAC (2005). The procedure in brief is as follows: Approximately 1 g sample was weighed accurately and transferred carefully to 300 ml Kjeldahl flask using butter paper. Five grams of digestion mixture (K 2 SO 4 and CuSO 4 ) and 12.5 ml of concentrated sulphuric acid (AR) were added to the flask. The contents were digested in a digestion assembly until clear and colourless residue was obtained. After cooling, the Kjeldahl flask was washed with10-15 ml of distilled water and added to the Kjeldahl distillation tube. Twenty ml of 50% NaOH solution was added to make the solution alkaline. The contents were steam distilled and the liberated ammonia was collected in 25 ml of saturated boric acid solution containing 2-3 drops of the mixed indicator (methyl red and methylene blue). The distillation was continued until about 65-75 ml of distillate was collected. The distillate was titrated against N/35 H 2 SO 4 until purple color end point appeared. A blank test was carried out simultaneously using all the reagents except the test material and the percent protein was calculated as follows: % Protein = 2 4 Wt. of the sample g F X 1.4 X Sample Reading Blank Reading x Normality of H SO where, F = factor for conversion of % nitrogen into % protein = 6.25 for concentrated whey, 5.7 for control samples and 5.78 (derived) for experimental samples. 3.3.2.5 Ash The ash content was determined as per (AOAC, 2005). Approximately 3 g of bread sample was accurately weighed into a silica crucible and heated in a muffle Page 60

Materials and methods furnace at 550ºC ±10ºC until light grey ash resulted and until constant weight was obtained. 3.3.2.6 Acid Insoluble Ash Acid insoluble ash was determined according to the method described in IS 12711:1989. Ash previously obtained in crucible is added with 25 ml dil. HCl (5 N) and the dish is covered with watch glass. The solution containing the ash in the crucible is heated for 10 minutes. Then the contents in the crucible is allowed to cool and then filtered through a Whatman no. 42 filter paper or its equivalent. The filter paper is washed until the washings are acid free. The filter paper and the residue were returned to the dish and the dish was kept in an electric air oven (110±3 o C) till it gets dried. The contents of the dish were ignited over a burner till it got completely charred. The ignition was completed by transferring the dish to a muffle furnace maintained at 550±100 o C until light grey ash is obtained. The dish is cooled in the desiccator and weighed. The dish was again heated in the muffle furnace for 30 minutes and then cooled in a desiccator and weighed. The process of heating for 30 minutes, cooling and subsequent weighing is repeated until the difference between successive weights is less than 1 milligram. The lowest mass is recorded. Acid insoluble ash, percentage by weight, A= W 1 -W x100 W 2 W 1 = Weight in gm of dish containing acid insoluble ash W = Weight in gm of empty dish in which sample is taken for ashing W 2 = weight in gram of the sample 3.3.2.7 Crude Fibre Crude fibre was determined by the procedure described in I.S. 1155:1968. About 2.5g dried sample is weighed and transferred to a Soxhlet extraction apparatus. The sample is extracted with petroleum ether, air dried and transferred to a dry one litre conical flask. Full defatting should be ensured as any trace of fat Page 61

Materials and methods remaining may affect the end result. The defatted material is boiled with boiling sulphuric acid (1.25% (w/v)). To prevent bumping some drops of octanol is added after addition of acid. The contents in the flask are boiled for 30 minutes and the flask is rotated frequently. The flask is removed and the acid is then filtered through a cloth and the cloth is washed till the washings are acid free. The residue on the filter cloth is now added to boiling Sodium Hydroxide solution (1.25% (w/v)). The boiling is again carried out for 30 minutes. The contents of the flask are immediately filtered after boiling in Sodium Hydroxide solution for 30 minutes. The residue is collected in a pre-weighed Gooch crucible after repeated washings. The Gooch crucible and contents is then dried in a hot air oven at 105±2 o C until constant weight is achieved. Then crucible is weighed after cooling it by keeping it in a desiccator. The contents of the Gooch crucible are then incinerated in a muffle furnace until all carbonaceous matter is burnt. After cooling down the crucible by keeping it in a desiccator the weight of the Gooch crucible is taken. W1 W2 Crude fibre present, by weight= x100 W W 1 = Weight of Gooch crucible and contents before ashing W 2 = Weight of Gooch Crucible after ashing W=Weight in gm of dried material taken for the test. 3.3.2.8 Alcoholic Acidity Alcoholic acidity of bread was determined by the procedure described in I.S 11271:1989. 5.0 g of dried sample is weighed into a stoppered conical flask and 50 ml of 90% neutral alcohol, previously neutralized against phenolphthalein is added to it. The flask is stoppered, shaken and allowed to stand for 24 hours with additional shaking. The alcoholic extract is filtered through a dry filter paper and the extract is titrated against 0.05N NaOH solution using phenolphthalein as indicator. Page 62

Materials and methods No of ml of 1N NaOH require for titration of 100g of the sample= T i t r e x N o r m a l i t y o f N a O H x W e i g h t o f s a m p l e t a k e n 1 0 0 3.3.2.9 ph The electrode assembly of a digital ph meter A ph meter, Digisun Electronics, DI-707, supplied by Servewell instruments pvt Ltd., was calibrated against standard buffer of ph 7.0 and 4.0 (Qualigens Fine Chemicals). Then about 10 g of bread sample was mixed with 20 ml of distilled water taken in a 50ml beaker, mixed well and the ph of the bread was determined using the calibrated digital ph meter. For measurement of ph of dough, a sufficient quantity of dough was taken in a 50 ml beaker and the electrode was dipped directly into the dough. The ph value was noted down after the value was stabilized on the display unit. 3.3.2.10 Loaf volume The volume of loaf was determined as per Rape Seed Method(AOAC, 2005). The procedure was modified by using Ragi seeds instead of rape seeds. Determination of density of seeds: A 500 ml graduated cylinder was weighed on the weighing scale and filled up to 500 ml mark with ragi seeds and reweighed. The average of three readings were taken. Density( g/ml)= (B-A)/500 where, B = Avg mass in g of the cylinder filled with ragi seeds up to 500 ml level. A = Mass in g of cylinder. Determination of loaf volume : The loaf was weighed after it was cooled to room temperature and recorded for its mass. The mold was filled with seeds and the top surface of the seeds was levelled with a wooden plate and weighed. Two such readings were taken and the average was recorded. The mold was then emptied leaving a thin layer of the seeds Page 63

Materials and methods at bottom following which the loaf was placed and filling the rest of the space within the box with the seeds. The top surface was levelled by a wooden plate.the mold was weighed again and two such readings were taken. Vol. of loaf (ml) = ( C-D)/ E where, C = Average mass in g of the mold filled with the seeds plus mass of loaf. D = Average mass in g of the mold filled with the seeds in the residual space. E = Density of ragi seeds. 3.3.2.11 Reflectance The reflectance meter was switched on and allowed for stabilization of about 10 min. The reading on the reflectance scale was adjusted to zero value using completely opaque plate (black) under colour mode. Thereafter, percent reflectance values were adjusted to the specified values using standard colour plates under 450 nm filter plate. It means the colour of the samples was measured by measuring the percent reflectance of light of 450 nm wave length. The lamp of the reflectance meter was placed on different portions of the crust and the percent reflectance as shown by the pointer was recorded. 3.3.2.12 Water activity Water activity of the samples was measured using the water activity meter (Rotronic, Switzerland). Procedure: The equipment was switched on and 15 minutes warm up time was allowed before measurement of water activity. Powdered sample was filled in sample cups up to half mark and placed in the cup holder. The sensor probe assembly was placed on the cup holder and the fan button put on for hastening the equilibration process. When the sample attained equilibration, the same was Page 64

Materials and methods indicated by the simultaneous appearance of two trend arrow signals on the display unit. At this stage, water activity value displayed was noted down. 3.3.3 Microbiological analysis of bread The polythene pouch containing the sample was opened and 11 g of the product was weighed and transferred to 99 ml of the sterile phosphate buffer aseptically. Further dilution to desired level was carried out by serially transferring 1 ml of diluted sample to 9 ml sterile saline blanks. Yeast & mould count Yeast & mould count was determined by plating 1, 2, 3 dilution of bread suspension using potato dextrose agar (Hi Media). The ph of the medium was adjusted to around 5.4 by adding 1-2 drops of sterile tartaric acid solution (10%) to each plate, before pouring the medium. The count was taken after 3 5 days of incubation at 30ºC. 3.3.4 Sensory evaluation of bread The organoleptic quality of bread was evaluated at regular intervals by an expert panel of judges on a 9-point hedonic scale wherein a score of 1 represented dislike extremely and score of 9 represented like extremely. The samples for evaluation were coded appropriately before serving the samples to the judges for sensory evaluation. Sensory evaluation of the samples was carried out in the sensory evaluation room. The panelists were asked to score for the following parameters : a) Colour and appearance b) Flavour c) Body and texture d) Overall acceptability The sensory scores for some rheological parameters also were taken based on 9-point hedonic scale for bread samples incorporated with different improvers. Page 65

Materials and methods The parameters studied were: 1) Hardness 2) Springiness 3) Gumminess 4) Chewiness 3.3.5 Rheological characteristics of bread a) Texture profile analysis (TPA): Texture profile analysis of the samples of bread was carried out using Texture Analyzer (TA-XT plus, Stable Micro Systems, England) under the following test conditions : Mode : Measure Force in Compression Pre-Test Speed : 1.0 mm/s Test Speed : 0.5 mm/s Post-Test Speed : 10.0 mm/s Target mode : Distance Distance : 5 mm Time : 5 seconds Trigger Type : Auto 5g Accessory: p/ 75 plunger probe Procedure: The probe was calibrated to a distance of 50mm, above the top of the container or the sample surface. The sample of bread tempered to about 25 C was cut into pieces of 40mm x 40mm x 10 mm size. The sample was positioned centrally over the platform and the computer was allowed to execute the program by activating run a test option, then the sample was compressed by the plunger twice in a gap of 5 sec to yield a force time curve. The height of the force peak on the first compression cycle (first bite) is the value of hardness (F). The ratio of the positive force under the second and first compressions (A 2 /A 1 ) is cohesiveness while Page 66

Materials and methods the ratio of the time difference between C-D to the time difference between E-F is springiness as shown in the figure 3.2. b) Stress relaxation test: Stress relaxation test of the samples of bread was determined using Texture Analyzer (TA-XT plus, Stable Micro Systems, England) under the following test conditions : Mode : Measure Force in Compression Pre-Test Speed : 1.0 mm/s Test Speed : 0.5 mm/s Post-Test Speed : 10.0 mm/s Target mode : Distance Distance : 8 mm Hold time : 180 seconds Trigger Type : Auto 5g Accessory: p/ 75 plunger probe Procedure: The probe was calibrated to a distance of 50mm, above the top of the container or the sample surface. The sample of bread was cut into pieces of 40mm x 40mm x 10 mm size. The sample was positioned centrally over the platform and the computer was allowed to execute the program by activating run a test option, then the sample was compressed by the plunger to about 80 % of its original height and the compression held for a period of 80 sec to yield a force time curve. The maximum value obtained on the force time curve (F) was multiplied with a factor 0.367 to obtain F/e the corresponding value of which on the time axis yields the stress relaxation time of the bread. The curve for stress relaxation time (SRT) is given in figure 3.3. 3.4 Statistical analysis Data obtained during the present project work were subjected to statistical analysis as described by Snedecor and Cochran (1967) and employing MS-EXCEL and SPSS computer packages. Page 67

Materials and methods The results obtained during the course of the investigation are tabulated and discussed in the subsequent chapter. Page 68

Chapter- 4 Results and Discussion

4. Results and discussion The results obtained during the present investigation have been compiled, analyzed and discussed in the order given below: 1. Composition of paneer whey used in the trials. 2. Effect of the level of incorporation of concentrated paneer whey on the sensory quality, textural and physical characteristics of multigrain bread baked at different temperatures. 3. Optimization of proofing time in the preparation of concentrated whey incorporated multigrain bread. 4. Effect of addition of different improvers on the sensory and textural characteristics of whey incorporated multigrain bread and optimization of the level of addition of selected improver. 5. Effect of incorporation of concentrated whey on the physico-chemical characteristics of multigrain bread. 6. Storage studies of whey incorporated multigrain bread. 4.1 Proximate Composition of Paneer Whey The paneer whey procured from the experimental dairy was analyzed for various parameters and the results are presented in table 4.1. The paneer whey was observed to have a mean total solids (TS) content of 6.61±0.028% and that of the concentrated whey was 15.08±.026%. The fat content was observed to be 0.57 ± 0.071% and 1.21±0.113% for paneer whey and concentrated paneer whey respectively. The mean protein content of paneer whey was observed to be 0.38±0.008% while that of concentrated whey was 0.81±0.047%. The mean lactose content of paneer whey ranged from 4.98-5.13% while that of concentrated paneer whey ranged from 12.03-12.13%. The mean ash content was 0.64± 0.074% and 1.46±0.062% respectively for paneer whey and concentrated paneer whey respectively. The ph of paneer whey was 5.83±1.03 while that of concentrated paneer whey was 5.15±0.73. Page 69

4. Results and Discussion Table 4.1. Proximate composition of Paneer whey and concentrated paneer whey Composition (%) Paneer whey Concentrated Paneer Whey Total Solids 6.61± 0.028 15.08±.026 Fat 0.57 ± 0.071 1.21±.113 Protein 0.38 ± 0.008 0.81±0.047 Lactose 5.05 ± 0.085 12.08±0.049 Ash 0.64 ± 0.074 1.46±0.062 ph 5.83±0.103 5.15±0.073 The compositional variation of paneer whey observed in the present investigation was in agreement with the earlier observations by Khamrui and Rajorhia (1998) and Jarita and Kulkarni (2009). During paneer manufacture however, large part of whey proteins co-precipitate with casein, so lesser amount go along with whey during drainage, resulting in lower protein content in the whey. Also because of acidic nature of coagulum formation, most of the minerals are solubilized and escape into whey, resulting in higher ash content of paneer whey. These observations are corroborated by other workers like Jelen (2002). Because of the easy availability and higher contents of minerals and lactose it was decided to use paneer whey in the present project. The concentration of whey was restricted to 25% TS based on earlier observations of Jarita and Kulkarni (2009) where it was reported that beyond 25% TS concentration the sensory characteristics of bakery products were adversely affected. Hence in the present investigation the paneer whey concentrated between 15-25% levels were incorporated in the dough for the preparation of multigrain bread and its effect on sensory and textural parameters was evaluated. The concentration of whey was exactly adjusted to the desired TS level by addition of calculated quantity of potable water to the concentrated whey. In Page 70

4. Results and Discussion this the variation in composition of the concentrated whey to be utilized in the experiments was minimized to the lowest possible level. 4.2 Effect of incorporation of concentrated paneer whey and baking temperatures on the sensory quality of multigrain bread. The effect of incorporation of concentrated whey and also the temperature of baking on the physico-chemical and sensory characteristics of multigrain bread was studied in a single experimental design and the sensory scores and the statistical analysis results are presented in tables 4.2 to 4.17. The method of incorporation of whey and also the variation in temperature of baking is detailed in flow diagram (Figure 3.1). 4.2.1 Effect of incorporation of concentrated paneer whey and baking temperatures on colour and appearance scores of multigrain bread. Table 4.2 Effect of incorporation of concentrated paneer whey and baking temperature on colour and appearance scores of multigrain bread. Temperature of Baking( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 7.2 a A ±0.33 7.38 ba ±0.37 7.87 c B ±0.29 7.93 c B ±0.34 7.6 B ±0.31 185 7.27 a B ±0.23 7.82 b C ±.19 7.83 b B ±0.21 7.84 b B ±0.24 7.69 C ±0.24 210 7.5 b B ±0.14 7.55 b B ±.22 7.51 b A ±0.23 7.23 a A ±0.25 7.48 A ±0.13 Mean scores due to Whey TS 7.32 a ±0.28 7.58 b ±0.33 7.74 c ±0.3 7.67 b,c ±0.42 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. ** Scores on 9 point hedonic scale It is observed from table 4.2 that mean colour and appearance (C&A) score was observed to increase gradually from 7.32±0.28 for control to 7.58±0.33 and 7.74±0.3 when 15% TS whey and 20% TS whey was used respectively. However, the score was observed to decrease to 7.67±0.42 when whey of 25%TS was used for replacing water. Page 71

4. Results and Discussion The effect of baking temperature on the colour and appearance score indicated that the mean colour and appearance scores were 7.6±0.31, 7.69±0.24 and 7.48±0.13 at 160 o C, 185 o C and 210 o C respectively. The mean score was observed to decrease with increase in temperature beyond 185 o C. Table 4.3 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on colour and appearance scores of multigrain bread. Factors affecting Colour Sum of squares Df Mean Square F-value Significance and Appearance Temperature of baking 2.878 2 1.439089 20.271 *.000 Whey solids Level 7.118 3 2.372624 33.42 *.000 Interaction 9.423 6 1.570524 22.122 *.000 (Temperature * Whey solids level) Within Groups 19.593 276 0.070992 Total 39.013 287 * Significant p<0.05 The statistical analysis of the data indicated that the colour and appearance scores differed significantly with TS content of whey used as diluent and also due to temperature of baking (F-value 33.42 and 20.27). The mean C&A score of bread prepared with 15% TS whey was observed to be significantly higher than control and significantly lower than bread prepared using 20% TS whey as diluent. The mean C&A scores of bread prepared with 25% TS whey was significantly higher than control only. The results indicated suitability of 20% TS whey incorporation when C&A score was the index of quality. The statistical analysis of the results due to the effect of temperature of baking indicated that there was significant difference between all the temperatures of baking of 160 o C, 185 o C and 210 o C (F value=20.27). However, the highest mean score was observed to be at 185 o C at 7.69±0.24 which was significantly higher than the other temperatures. Page 72

4. Results and Discussion TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on colour and appearance scores of multigrain bread. Table 4.4 Due to difference in TS of concentrated whey whey solids level (% TS) N Subset 1 2 3 control 72 7.3222 15 72 7.5840 25 72 7.6660 7.6660 20 72 7.7382 Sig. 1.000.254.365 Table 4.5 Due to difference in Baking temperature Temperature of baking ( O C) N Subset 1 2 3 210 96 7.4469 160 96 7.5964 185 96 7.6896 Sig. 1.000 1.000 1.000 The statistical analysis further indicated that even though the mean C&A score decreased when whey with 25%TS was used, the difference was observed to be non-significant in comparison to the other experimental samples. However, the score was significantly higher than the control samples (Tables 4.2 to 4.5). The effect of incorporation of concentrated whey and baking temperatures on C&A scores is also presented in figures 4.1 and 4.2. Page 73

4. Results and Discussion Figure 4.1. SPSS plots showing estmated marginal means of colour and appearance scores with whey of different TS as separate lines Figure 4.2. SPSS plots showing estmated marginal means of colour and appearance scores with baking temperature as separate lines Page 74

4. Results and Discussion 4.2.2 Effect of incorporation of concentrated paneer whey and baking temperature on Body and Texture scores of multigrain bread. The effect of incorporation of concentrated whey on the body and texture scores is detailed in tables 4.6 to 4.9 and figures 4.3 and 4.4. Table 4.6 Effect of incorporation of concentrated paneer whey and baking temperature on Body and Texture scores of multigrain bread. Temperature of baking( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 7.45 c B ±.42 7.7 b B ±.323 6.9 a B ±.303 6.85 a B ±.369 7.22 B ±.36 185 7.7 b C ±.2 7.71 b B ±.189 7.13 a C ±.271 6.94 a B ±.272 7.37 C ±.34 210 6.93 b A ±.39 6.84 b A ±.306 6.3 a A ±.349 6.23 a A ±.42 6.57 A ±.31 Mean scores due to Whey TS 7.36 b ±.48 7.41 b ±.5 6.77 a ±.47 6.67 a ±.48 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. ** scores on 9 point hedonic scale It is observed from table 4.6 that the body and texture score of bread of bread improved with the increase in solids content of whey used as diluent upto 15%. Subsequent increase in whey solids content was observed to decrease the body and texture score. The scores were 7.36±0.48, 7.41±0.5, 6.77±0.47 and 6.67±0.48 for control and experimental samples with 15, 20 and 25% TS in whey respectively. The scores differed significantly as reflected by statistical analysis (F value=97.29, table 4.7). The temperature of baking was observed to have significant effect on the body and texture scores (F=155.60, table 4.7). The scores were 7.22±0.36, 7.37±0.34 and 6.57±0.31 which were observed to differ significantly between each other. However it was noted that the score at 210 o C was significantly lower than the other two temperatures and the score for bread baked at 185 o C was highest at 7.37±0.34. Page 75

4. Results and Discussion Table 4.7 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on Body and Texture scores of multigrain bread. Factors affecting Body Sum of Df Mean F-value Significance and Texture squares Square Temperature of baking 34.449 2 17.22449 155.6026 *.000 Whey solids Level 32.309 3 10.76966 97.29096 *.000 Interaction (Temperature* 0.932 6 0.155252 1.402513.214 Whey solids level) Within Groups 30.552 276 0.110695 Total 98.241 287 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on Body and Texture scores of multigrain bread. Table 4.8 Due to difference in TS of concentrated whey Whey solids level(% TS) N Subset 1 2 25 72 6.67153 20 72 6.77361 control 72 7.36111 15 72 7.41389 Sig..256.777 Table 4.9 Due to different baking temperatures Temperature of baking ( O C) N Subset 1 2 3 210 96 6.57344 160 96 7.22188 185 96 7.36979 Sig. 1.000 1.000 1.000 Page 76

4. Results and Discussion Figure 4.3. SPSS plots showing estmated marginal means of body and texture scores with whey of different TS as separate lines Figure 4.4. SPSS plots showing estmated marginal means of body and texture scores with temperature of baking as separate lines Page 77

4. Results and Discussion 4.2.3 Effect of incorporation of concentrated paneer whey and baking temperature on Flavour scores of multigrain bread. The effect of incorporation of concentrated whey and temperature of baking on flavour scores is detailed in the tables 4.10 to 4.13 and figures 4.5 to 4.6. It is observed from table 4.10 that the mean flavour score was 7.53±0.28 for control and 7.56±0.27, 7.32±0.35 and 7.18±0.38 for breads prepared with 15, 20 and 25% TS whey respectively. The scores were observed to decrease with increase in TS content of whey. The statistical analysis (F value=23.58, table 4.11) also indicated a significant effect on the flavour scores of bread due to increase in the level of TS in whey. The scores however were significantly lower in case of bread prepared with 20 and 25% TS in whey. The baking temperature was also observed to have a significant effect on the flavour scores. The flavour scores were 7.38±0.14, 7.50±0.14 and 7.31±0.2 at 160, 185 and 210 o C respectively. The mean score of 7.50±0.14 for the bread baked at 185 O C was the highest and significantly higher than the other two treatments. The bread baked at 210 o C showed the least sensory score as it was accompanied by a distinct burnt flavour. Table 4.10 Effect of incorporation of concentrated paneer whey and baking temperature on Flavour scores of multigrain bread. Temperature of baking( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 7.44 b A ±.37 7.55 b A ±.29 7.34 a,b B ±.26 7.18 a A,B ±.28 7.38 A ±.14 185 7.61 b A ±.22 7.64 b A ±.22 7.47 b B ±.22 7.28 a B ±.31 7.50 B ±.14 210 7.53 b A ±.17 7.49 b A ±.28 7.15 a A ±.45 7.08 a A ±.47 7.31 A ±.2 Mean scores due to Whey TS 7.53 c ±.28 7.56 c ±.27 7.32 b ±.35 7.18 a ±.38 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. ** scores on 9 point hedonic scale Page 78

4. Results and Discussion The statistical analysis also reflected a significant effect due to temperature and also due to the level of solids in the whey (F=9.25 and 23.58, table 4.11). The interaction effect was observed to be non-significant indicating independent nature of the influence on the flavour score of the bread. The statistical analysis (tables 4.11 to 4.13) also further indicated there was no significant difference between the flavour score of control and the bread prepared with 15% TS whey. Table 4.11 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on Flavour scores of multigrain bread. Factors affecting Flavour Sum of squares Df Mean Square F-value Significance Temperature of baking 1.840 2 0.920 9.249 *.000 Whey solids Level 7.035 3 2.345 23.575 *.000 Interaction (Temperature * Whey solids level) 0.572 6 0.095 0.958.454 Within Groups 27.454 276 0.099 Total 36.901 287 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on Flavour scores of multigrain bread. Table 4.12 Due to difference in TS of concentrated whey whey solids level (% TS) N Subset 1 2 3 25 72 7.1806 20 72 7.3181 control 72 7.5292 15 72 7.5618 Sig. 1.000 1.000.925 Page 79

4. Results and Discussion Table 4.13 Due to difference in temperature of baking Temperature of baking( O C) N Subset 1 2 210 96 7.3099 160 96 7.3792 185 96 7.5031 Sig..282 1.000 Since flavour is an important sensory characteristic of the bread this observation is important in the final selection of the levels of TS content in whey which can be used as diluent. The temperature of baking was however observed to be optimum at 185 O C since flavour was observed to be highest with a score of 7.50±0.14. The mean flavour scores observed for the control and the treatment samples are depicted in figures 4.5 and 4.6. Figure 4.5. SPSS plots showing estmated marginal means of flavour scores with whey of different TS as separate lines Page 80

4. Results and Discussion Figure 4. 6. SPSS plots showing estmated marginal means of flavour scores with temperature of baking as separate lines 4.2.4 Effect of incorporation of concentrated paneer whey and baking temperature on Overall Acceptability scores of multigrain bread. The overall acceptability (OA) scores due to incorporation of concentrated whey and baking temperature are presented in tables 4.14 to 4.17. The mean OA scores of control samples without the use of whey in the preparation of bread was 7.37±0.39 while that of experimental samples were 7.47±0.51, 6.88±0.46 and 6.78±0.51 for bread samples with 15%, 20% and 25% TS whey respectively. The results indicated that the OA score decreased with increase in solids content of whey (Table 4.14). Similarly the OA scores were observed to be highest at 7.35±0.30 when the bread was baked at 185 o C while the next highest score was for the bread baked at 160 o C with a score of 7.29±0.33. The OA score was least for the sample baked at 210 o C. Page 81

4. Results and Discussion Table 4.14 Effect of incorporation of concentrated paneer whey and baking temperature on Overall Acceptability scores of multigrain bread. Temperature of baking( O C) Mean scores due to baking temperature Control 15% TS whey 20% TS whey 25% TS whey 160 7.52 b B ±.39 7.7 b B ±.32 7.06 a B ±.36 6.89 a B ±.43 7.29 B ±.33 185 7.50 c B ±.31 7.769 b B ±.26 7.1 a B ±.22 7.03 a B ±.29 7.35 B ±.30 210 7.1 b A ±.3 6.95 b A ±.43 6.49 a A ±.47 6.4 a A ±.52 6.73 A ±.3 Mean scores due to Whey TS 7.37 b ±.39 7.47 b ±.51 6.88 a ±.46 6.78 a ±.51 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. ** scores on 9 point hedonic scale The statistical analysis of data (table 4.15) indicated that the scores were affected significantly both due to the solids content in whey and also due to the baking temperature (F value=61.89 and 79.40, table 4.15). The interaction effect was however, observed to result in non-significant impact on the overall OA score. The effects further indicated that there was no significant difference between control and the experimental samples prepared with 15% TS whey while the OA scores of samples prepared with 20 and 25% TS whey were observed to be significantly lower (tables 4.16 to 4.17). The trends of OA scores of samples prepared with different levels of whey solids and also baked at different temperatures are also presented in figures 4.7 and 4.8. Page 82

4. Results and Discussion Table 4.15 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on Overall Acceptability scores of multigrain bread. Factors affecting overall acceptability Sum of squares Df Mean Square F-value Significance Temperature of baking 22.35429 2 11.17714 79.4003 *.000 Whey solids Level 26.14065 3 8.71355 61.8994 *.000 Interaction 1.312448 6 0.218741 1.553897.161 (Temperature * Whey solids level) Within Groups 38.8524 276 0.14077 Total 88.65978 287 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on Overall Acceptability scores of multigrain bread. Table 4.16 Due to difference in TS of concentrated whey whey solids level(% TS) N Subset 1 2 25 72 6.7750 20 72 6.8833 control 72 7.3722 15 72 7.4729 Sig..309.374 Table 4.17 Due to difference in Baking Temperatures Temperature of baking( O C) N Subset 1 2 210 96 6.7333 160 96 7.2927 185 96 7.3516 Sig. 1.000.523 Page 83

4. Results and Discussion Figure 4.7. SPSS plots showing estmated marginal means of Overall Acceptability scores with whey of different TS as separate lines Figure 4.8. SPSS plots showing estmated marginal means of Overall Acceptability scores with temperature of baking as separate lines Page 84

4. Results and Discussion It can be observed from the results of sensory evaluation that based on the OA scores, whey concentrated to 15%TS can effectively be used as a diluent in the preparation of multigrain bread (Figures 4.9-4.11). SENSORY SCORES ON HEDONIC SCALE 8.2 8 7.8 7.6 7.4 7.2 7 6.8 6.6 6.4 6.2 Figure 4. 9. Graphical representation of variation in sensory attributes of multigrain bread at 160 o C. SENSORY SCORES ON HEDONIC SCALE 8 7.8 7.6 7.4 7.2 7 6.8 6.6 6.4 Figure 4. 10. Graphical representation of variation in sensory attributes of multigrain bread at 185 o C. Colour and Appearance Body and Texture Flavour Overall Acceptability SENSORY CHARACTERISTICS Colour and Appearance Body and Texture Flavour Overall Acceptability SENSORY CHARACTERISTICS CONTROL MULTIGRAIN BREAD MULTIGRAIN BREAD WITH 15% TS WHEY MULTIGRAIN BREAD WITH 20% TS WHEY MULTIGRAIN BREAD WITH 25% TS WHEY CONTROL MULTIGRAIN BREAD MULTIGRAIN BREAD WITH 15%TS WHEY MULTIGRAIN BREAD WITH 20%TS WHEY MULTIGRAIN BREAD WITH 25%TS WHEY Page 85

4. Results and Discussion SENSORY SCORES ON HEDONIC SCALE 7.9 7.7 7.5 7.3 7.1 6.9 6.7 6.5 6.3 6.1 Colour and Appearance Body and Texture Flavour Overall Acceptability SENSORY CHARACTERISTICS CONTROL MULTIGRAIN BREAD MULTIGRAIN BREAD WITH 15%TS WHEY MULTIGRAIN BREAD WITH 20%TS WHEY MULTIGRAIN BREAD WITH 25%TS WHEY Figure 4. 11. Graphical representation of variation in sensory attributes of multigrain bread at 210 o C. The results further indicated that the optimum baking temperature is 185 o C while using whey as diluent. Incorporation of whey to certain extent contributed for better colour and appearance scores which could be ascribed to the enhanced Maillard browning in the product due to the increased lactose level. It was further observed that there was significant interaction between the level of whey solids incorporation and the baking temperature on the colour and appearance scores of the bread. At higher temperatures of baking however, increase in level of whey solids incorporation resulted in decreased colour and appearance scores. The enhanced Maillard browning in bakery products due to whey solids incorporation has also been reported earlier by Jarita and Kulkarni(2009) and Divya and Rao (2010). The results in the present study are also in agreement with the earlier observations. The incorporation of whey solids in the form of concentrated whey resulted in increased water binding characteristics of multigrain bread dough. Upon baking it was observed that the bread acquired crumbly textural characteristics. This could be ascribed to the water binding capacities of the whey protein contributing for the crumbly characteristics as reported earlier by Hutton and Campbell (1981). The results obtained did not show any significant difference in score between control and the multigrain bread prepared with 15%TS whey incorporation. Page 86

4. Results and Discussion Higher level of incorporation of whey solids contributed to slight sour taste and marked flavour of whey in the bread. A similar observation was reported earlier by Poonam (2007), during the preparation of buns and soupsticks indicating that the whey solids could be incorporated upto a certain level and in the present investigation beyond 15% TS whey contributed for negative acceptability with decreased sensory scores. 4.2.5 Effect of incorporation of concentrated paneer whey and baking temperature on body and texture characteristics. During the course of optimization the breads were also subjected to objective analysis of the select body and texture characteristics as detailed in 3.2.4.2. 4.2.5.1 Effect of incorporation of concentrated paneer whey and baking temperature on the hardness values (N) of multigrain bread. It is observed from table 4.18 that the mean hardness value of bread increased gradually with the increase in the level of solids in whey used in the preparation of dough. Table 4.18 Effect of incorporation of concentrated paneer whey and baking temperature on Hardness values (N) of multigrain bread. Temperature of baking ( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 19.93 a,a ±2.73 18.09 a,a ±3.91 34.55 b,a ±6.19 40.93 c,a ±2.64 28.38 A ±9.65 185 22.10 a,a ±2.93 22.02 a,b ±3.99 35.07 b,a ±3.55 45.03 c,b ±1.81 31.06 B ±9.66 210 24.91 a,b ±3.57 24.94 a,b ±2.69 42.05 b,b ±3.41 49.11 c,c ±2.24 32.26 C ±10.6 Mean scores due Whey TS 22.31 a ±3.78 21.69 a ±4.64 37.22 b ±5.82 45.02 c ±4.11 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. The mean hardness value of control bread was 22.317±3.782 while that of samples was 21.687±4.636, 37.223±4.636 and 45.024±4.109 for the breads with 15, Page 87

4. Results and Discussion 20 and 25% TS whey respectively. The mean hardness values to a large extent differed significantly except that there was no significant difference between the control and the use of whey with 15% TS. The baking temperature was observed to have statistically significant effect on the hardness value with positive correlation (Table 4.18). The hardness of bread was observed to be 28.377±9.651 at a baking temperature of 160 o C, 31.056±9.657 at 185 O C and 32.255±10.626 at 210 o C (table 4.18). The estimated mean hardness values are also graphically represented in figures 4.12 and 4.13. Table 4.19 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on Hardness values (N) of multigrain bread. Factors affecting Sum of squares Df Mean Square F-value Significance Hardness Temperature of baking 865.3468 2 432.6734 * 31.77559 *.000 Whey solids Level 10698.4 3 3566.135 * 261.8974 *.000 Interaction 76.73527 6 12.78921 0.939241.471 (Temperature * Whey solids level) Within Groups 1307.187 96 13.61654 Total 12947.67 107 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on Hardness values (N) of multigrain bread. Table 4.20 Due to difference in TS of concentrated whey Whey solids level(% TS) N Subset 1 2 3 15 27 21.6871 control 27 22.3171 20 27 37.2227 25 27 45.0235 Sig..923 1.000 1.000 Page 88

4. Results and Discussion Table 4.21 Difference due to difference baking temperatures Temperature of baking( O C) N Subset 1 2 3 160 36 28.37685 185 36 31.05622 210 36 35.25475 Sig. 1.000 1.000 1.000 Figure 4. 12. SPSS plots showing estmated marginal means of hardness (N) with whey of different TS as separate lines Page 89

4. Results and Discussion Figure 4. 13. SPSS plots showing estmated marginal means of hardness(n) with temperature of baking as separate lines 4.2.5.2 Effect of incorporation of concentrated paneer whey and baking temperature on the Cohesiveness values of multigrain bread. The cohesive characteristics of the control and the experimental samples of bread are tabulated in table 4.22 and the statistical analysis of the data is presented in tables 4.23-4.24. It is observed from table 4.22 that cohesiveness decreased with the addition of whey as diluent. The results were negatively correlated indicating that as the TS content in whey increased the cohesiveness decreased and the decrease at all levels was statistically significant. Page 90

4. Results and Discussion Table 4.22 Effect of incorporation of concentrated paneer whey and baking temperature on the Cohesiveness values of multigrain bread. Temperature of baking ( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160.519 c A ±.062.477 b,c B ±.077.442 b B ±.029 377 a A,B ±.062.454 B ±.052 185.574 c B ±.063.533 b,c C ±.024.484 b B ±.06.425 a B ±.057.504 C ±.056 210.475 c A ±.045.41 b A ±.049.361 a,b A ±.040.335 a A ±.09.395 A ±.053 Mean scores due Whey TS.523 d ±.071.473 c ±.076.429 b ±.069.379 a ±.066 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. The mean cohesiveness values were 0.523±0.071 for control sample and 0.473±0.076, 0.429±0.069 and 0.379±0.066 for breads with 15, 20 and 25% TS concentrated whey respectively. The estimated mean cohesiveness due to whey incorporation is depicted in figure 4.14. The baking temperature was also observed to have significant effect on the cohesiveness (table 4.22) but however, the trend in variation was observed to be non-uniform. The cohesiveness values were 0.454±0.052, 0.504±0.056 and 0.395±0.057 at 160, 185 and 210 o C temperature of baking respectively. The results indicated that cohesiveness increased with increase in the temperature upto 185 o C and subsequent increase in baking temperature resulted in decrease in cohesiveness. Page 91

4. Results and Discussion Table 4.23 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on the Cohesiveness values of multigrain bread. Factors affecting Sum of squares Df Mean Square F-value Significance cohesiveness Temperature 0.214067 2 0.107034 34.09307 *.000 Whey solids Level 0.305179 3 0.101726 32.40261 *.000 Interaction 0.006258 6 0.001043 0.332234.918 (Temperature * Whey solids level) Within Groups 0.301387 96 0.003139 Total 0.826892 107 * Significant p<0.05 The differences in cohesiveness were statistically significant as per the statistical analysis of the data (Tables 4.23 to 4.25). The estimated mean cohesiveness due to variation in temperature of baking is depicted in figure 4.15. TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on the Cohesiveness values of multigrain bread. Table 4.24 Due to difference in TS of concentrated whey Whey solids level (% TS) N Subset 1 2 3 4 25 27.37914 20 27.42922 15 27.47342 control 27.52285 Sig. 1.000 1.000 1.000 1.000 Page 92

4. Results and Discussion Table 4.25. Due to difference in baking temperature Temperature of baking( O C) N Subset 1 2 3 210 36.39528 160 36.45397 185 36.50422 Sig. 1.000 1.000 1.000 Figure 4. 14. SPSS plots showing estmated marginal means of cohesiveness with whey of different TS as separate lines Page 93

4. Results and Discussion Figure 4. 15. SPSS plots showing estmated marginal means of cohesiveness with temperature of baking as separate lines 4.2.5.3 Effect of incorporation of concentrated paneer whey and baking temperature on the Springiness values of multigrain bread. The effect of whey incorporation on the springiness of the bread was evaluated and the results along with the statistical analysis are presented in tables 4.26 to 4.29. Springiness is an important characteristic of the bread which reflects the softness and also its ability to get back to its original condition when subjected to different handling conditions. The springiness values for control samples was 0.702±0.074 and for the experimental samples the values were 0.667±0.131, 0.568±0.09 and 0.541±0.084 for bread with 15, 20 and 25% TS whey respectively. The decrease in springiness values was gradual and significant (Tables 4.26 & 4.28). The values were observed to be non significant between control and the 1 st level of treatment (15% TS whey) and also between 2 nd and 3 rd levels of treatments (Tables 4.26 & 4.28). Page 94

4. Results and Discussion Similar to cohesiveness the change in springiness due to baking temperature was observed to be non-uniform. The springiness value at 160 o C baking was 0.659±0.052, which increased to 0.674±0.076 at 185 o C and decreased to 0.525±0.084 at 210 o C. The springiness values were observed to be non-significant between the baking temperatures of 160 and 185 o C and subsequently decreased significantly at 210 o C baking. Table 4.26 Effect of incorporation of concentrated paneer whey and baking temperature on the Springiness values of multigrain bread. Temperature of baking ( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160.701 b B ±.020.718 b B ±.067.63 a,b B ±.031.59 a B ±.009.659 B ±.052 185.756 b B ±.094.746 b B ±.116.605 a B ±.065.592 a B ±.044.674 B ±.076 210.649 c C ±.031.539 b A ±.084.471 a A ±.061.441 a A ±.059.525 A ±.08 Mean scores due Whey TS.702 b ±.074.667 b ±.131.568 a ±.090.541 a ±.084 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids. Table 4.27 ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on the Springiness values of multigrain bread. Factors affecting Sum of squares Df Mean Square F-value Significance Springiness Temperature of 0.488038 2 0.244019 53.04043 *.000 baking Whey solids Level 0.482897 3 0.160966 34.98777 *.000 Interaction 0.055806 6 0.009301 2.0217.070 (Temperature * Whey solids level) Within Groups 0.44166 96 0.004601 Total 1.468401 107 * Significant p<0.05 Page 95

4. Results and Discussion The significant decrease in springiness indicated that in the case of multigrain bread there exists an optimum temperature range to obtain better sensory characteristics as observed for any other varieties of breads (Indrani et al., 2007). The estimated marginal means of springiness due to baking temperatures and variations in TS contents in whey are also graphically represented in figures 4.16 and 4.17. TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on the Springiness values of multigrain bread. Table 4.28. Due to difference in TS of concentrated whey Whey solids level(% TS) N Subset 1 2 25 27.54085 20 27.56831 15 27.66744 control 27.70184 Sig..449.251 Table 4.29. Due to difference in baking temperature Temperature N Subset 1 2 210 36.52494 160 36.65942 185 36.67448 Sig. 1.000.615 Page 96

4. Results and Discussion Figure 4. 16. SPSS plots showing estmated marginal means of springiness with whey of different TS as separate lines Figure 4. 17. SPSS plots showing estmated marginal means of springiness with temperature of baking as separate lines Page 97

4. Results and Discussion 4.2.5.4 Effect of incorporation of concentrated paneer whey and baking temperature on the Stress relaxation time of multigrain bread: The stress relaxation time (SRT) is another important parameter which indirectly reflects the softness of the bread or non-crumbly characteristics. Generally stored bread becomes crumbly and SRT values can be used as an index of freshness of the bread. The mean SRT values are presented in table 4.30 and statistical analysis of the data are presented in tables 4.31 to 4.33. The SRT values were observed to increase with the replacement of water by concentrated whey as a diluent. It is further observed that as the TS content of whey increased, the SRT values also increased indicating a positive correlation. Table 4.30. Effect of incorporation of concentrated paneer whey and baking temperature on the Stress relaxation time (sec) of multigrain bread. Temperature of baking ( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 20.68 a A ±.57 22.32 a A ±.61 25.47 b A ±1.12 29.06 c A ±2.14 24.381 A ±3.20 185 23.83 a B ±1.89 27.50 b B ±4.16 31.96 c B ±3.95 36.47 d B ±4.89 29.941 B ±4.74 210 27.99 a C ±1.72 30.98 b C ±2.03 36.92 c C ±1.74 40.59 d C ±2.04 34.122 C ±4.98 Mean scores due Whey TS 24.169 a ±3.42 26.933 b ±4.55 31.451 c ±5.45 35.372 d ±5.91 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids The SRT values were 24.169±3.42 for control while the values were 26.933±4.55. 31.451±5.45 and 35.372±5.91 for bread prepared by incorporating 15, 20 and 25 % TS whey respectively (Table 4.30). Similarly the baking temperature was also observed to have a positive correlation with increase in values of SRT with increase in temperature of baking. The SRT of bread baked at 160 o C was 24.381±3.2, 29.941±4.74 for bread baked at 185 o C and 34.122±4.98 for bread baked at 210 o C. The statistical analysis of the data indicated that the SRT values varied significantly both due to the TS content of whey and also due to the temperature of baking (tables 4.31 to 4.33). The values were observed to vary significantly for all Page 98

4. Results and Discussion the levels of treatments. The estimated means of SRT are also presented graphically in figures 4.18 and 4.19. Table 4.31. ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on the Stress relaxation time of multigrain bread. Factors affecting Sum of Df Mean F-value Significance Hardness squares Square Temperature of baking 1719.269 2 859.6344 113.0148 *.000 Whey solids Level 1979.028 3 659.676 86.72658 *.000 Interaction (Temperature 73.69773 6 12.28295 1.614821.151 * Whey solids level) Within Groups 730.2133 96 7.606388 Total 4502.208 107 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on the Stress relaxation time of multigrain bread. Table 4.32. Due to difference in TS of concentrated whey Whey solids level (% TS) N Subset 1 2 3 4 control 27 24.168 15 27 26.933 20 27 31.450 25 27 35.372 Sig. 1.000 1.000 1.000 1.000 Table 4.33. Due to difference in baking temperatures Temperature of baking( O C) N Subset 1 2 3 160 36 24.381 185 36 29.940 210 36 34.122 Sig. 1.000 1.000 1.000 Page 99

4. Results and Discussion Figure 4.18. SPSS plots showing estmated marginal means of SRT with whey of different TS as separate lines Figure 4. 19. SPSS plots showing estmated marginal means of SRT with temperature of baking as separate lines Page 100

4. Results and Discussion The results indicated that the use of concentrated whey as diluent affected the textural characteristics of multigrain bread significantly. It is also observed from the results discussed that even the baking temperature had a significant effect on the textural characteristics. The hardness of the bread was observed to increase due to the use of concentrated whey as a diluent which could be ascribed to the slightly increased TS content in the bread. Further this also could be attributed to increased water binding properties especially due to whey protein denaturation which generally results in better water binding properties as reported by Hutton and Campbell, 1981. The water binding property of denatured whey protein was also reported earlier by Kulkarni et al. (1990). Bread, being foamy in structure, its firmness to a large extent depends on the number of air cells which depends on the baking characteristics of the dough (Walstra et al., 1999). It is also observed that as the water binding properties increased the hardness of bread increased but at the same time resulted in a decrease in cohesiveness implying that the product was becoming crumbly in body & texture. This was also reflected in decreased body and texture scores at higher levels of incorporation of whey solids. The variations in springiness and SRT also could be attributed basically to the textural characteristic variations due to the denaturation of whey proteins (Collado, 2003) and also marginal increase in the total solids contents in the final product (Divya and Rao, 2010). 4.2.5 Effect of incorporation of concentrated paneer whey and baking temperature on the reflectance of multigrain bread. Reflectance is another important property which provides an indication of the brownness of the crust. Lower value of reflectance indicates browner crust. The reflectance values measured by the reflectance meter are presented in table 4.34 and the statistical analysis is illustrated in tables 4.35 to 4.37. Page 101

4. Results and Discussion Table 4.34. Effect of incorporation of concentrated paneer whey and baking temperature on the reflectance of multigrain bread. Temperature of baking( O C) Control 15% TS whey 20% TS whey 25% TS whey Mean scores due to baking temperature 160 56.97 d C ±.9 55.37 c C ±1.41 53.13 b C ±1.03 51.88 a C ±2.01 54.34 C ±2.41 185 55.67 d B ±.98 50.99 c B ±1.23 48.27 b B ±1.19 47.23 a B ±1.78 50.54 B ±3.54 210 50.67 c A ±1.47 44.47 b A ±1.37 42.85 a A ±.98 41.88 a A ±1.86 44.97 A ±3.73 Mean scores due Whey TS 54.43 d ±2.96 50.27 c ±2.71 48.08 b ±4.38 47.0 a ±4.52 *Capital superscripts show variations due baking temperature and smaller superscripts show variations due to variation in level of whey solids The reflectance was observed to decrease on use of concentrated whey as diluent and the values further decreased with increase in TS of the concentrated paneer whey. The reflectance values were 54.43±2.96% for control bread while it was 50.27±2.71, 48.08±4.38 and 47.0±4.52% for bread made with whey having 15, 20 and 25% TS respectively (table 4.34). Similarly, the mean reflectance value showed a negative correlation with increase in baking temperature indicating that higher temperature leads to browner colour of the crust. The mean reflectance at 160 o C baking was 54.34±2.41%, 50.54±3.54% at 185 o C baking and 44.97±3.73% at 210 o C baking (table 4.34). The statistical analysis of the values showed that reflectance values differed significantly due to both the solids content of the concentrated whey used as diluent and also the temperature used for baking (F value=486.14 and 1333.18 respectively). The mean values of all the treatments using whey as diluent were significantly different from each other. The temperature of baking was also shown to have significant effect on the reflectance of the bread. The reflectance values at all the three temperatures of baking are observed to differ significantly from each other as seen from tables 4.34 and 4.37. It is also evident from table 4.35 that the interaction between temperature of baking and the TS of whey used as diluent (F value=18.95) had a significant effect, indicating that the browning of the crust was a result of the synergistic effect of both the TS of whey as well as the temperature of baking. Page 102

4. Results and Discussion Table 4.35. ANOVA: Effect of incorporation of concentrated paneer whey and baking temperature on the reflectance of multigrain bread. Factors affecting Sum of Df Mean F-value Significance Reflectance squares Square Temperature of baking 2666.25 2 1333.175 * 684.203 *.000 Whey solids Level 1458.404 3 486.135 * 249.491 *.000 Interaction (Temperature * Whey solids level) 113.704 6 18.951 * 9.726 *.000 Within Groups 327.349 168 1.949 Total 4565.808 179 * Significant p<0.05 TUKEY: Effect of incorporation of concentrated paneer whey and baking temperature on the reflectance of multigrain bread. Table 4.36. Due to difference in TS of concentrated whey whey solids level(% TS) N Subset 1 2 3 4 25 45 46.998 20 45 48.082 15 45 50.273 control 45 54.433 Sig. 1.000 1.000 1.000 1.000 Table 4.37. Due to difference in baking temperature Temperature of baking( O C) N Subset 1 2 3 210 60 44.9650 185 60 50.5383 160 60 54.3367 Sig. 1.000 1.000 1.000 Page 103

4. Results and Discussion The trend of variation of reflectance values could be used to explain the variation shown in colour and appearance scores. The mean colour and appearance score was maximum for bread made using 20% TS whey as diluent. Reflectance values indicated that the bread crust became browner as the TS in the whey used as diluent increased. This could be attributed to the presence of lactose in the whey which becomes available in a more concentrated state as TS of whey goes on increasing. Lactose contributes to Maillard browning and thus adding to the colour of the crust. The Maillard browning in bakery products due to enhanced solids level of the whey incorporated as diluent was earlier reported by Jarita and Kulkarni (2009) and Divya and Rao (2010). Increase in temperature of baking led to more browning of crust and hence the reflectance scores showed a negative trend with rise in temperature. The bread crust appeared to be most brown at a baking temperature of 210 o C. However subjective analysis indicated that the bread baked at 185 o C had the highest sensory scores for colour and appearance. This clearly indicates that intense brown colour of the bread crust is not preferred by judges. Optimum development of brown colour of the crust was observed at a temperature of 185 o C and on using whey having TS of 20% as diluent. Figures 4.20 and 4.21 illustrate graphically the variation in values of reflectance with variation of temperature of baking as well as variation of whey solids. Page 104

4. Results and Discussion Figure 4. 20. SPSS plots showing estmated marginal means of reflectance with whey of different TS as separate lines Figure 4. 21. SPSS plots showing estmated marginal means of reflectance with temperature of baking as separate lines. Page 105

4. Results and Discussion 4.2.6 Effect of incorporation of concentrated whey on the loaf weight of multigrain bread: The effect of incorporation whey on the loaf weight is presented in table 4.38 Table 4.38. Variation in Loaf weight due to incorporation of concentrated whey in multigrain bread dough: Bread Loaf weight (g) Control multigrain bread 359.70 a ±0.909 Multigrain bread with 15%TS whey 367.46 b ±0.566 Multigrain bread with 20%TS whey 371.67 c ±0.432 Multigrain bread with 25%TS whey 374.23 d ±0.921 * Significant p<0.05 The loaf weight was observed to increase on replacement of the water of the dough by concentrated whey. Further, the loaf weight was seen to increase progressively with increase in TS of the whey used as diluent. The loaf weight was 359.70±0.909 g for control and 367.46±0.566, 371.67±0.432 and 374.23±0.921g for multigrain bread incorporated with 15, 20 and 25% TS whey respectively. The loaf weight of the control was the least and it was significantly lower than the breads made with use of concentrated whey as diluent. The loaf weight of the experimental breads increased significantly with increase in TS content of whey used as diluent. The increase in loaf weight due to incorporation of whey in bread has also been reported earlier by Divya and Rao (2010) and Jayalakshmi (2010). The milk solids present in the concentrated whey contribute to the increase in weight of the loaf. All the above results indicated that without affecting the sensory and the rheological parameters of the bread it is possible to replace water with concentrated paneer whey of 15 % TS during dough preparation and thus economically utilize whey for the production of multigrain bread. Page 106

4. Results and Discussion 4.3. Optimization of proofing time in the preparation concentrated whey incorporated multigrain bread: The effect of incorporation of concentrated whey on the proofing time is depicted in figure 4.22. The effects of increasing the levels of yeast in dough on the sensory characteristics of bread and the related statistical analysis of the data are presented in subsequent tables. 4.3.1 Effect of incorporation of concentrated paneer whey of different TS on the proofing rate of multigrain bread dough The proofing rate of dough and the total time required for proofing is an important parameter in the preparation of bread and other bakery products. This refers to the increase in volume of dough with time caused by gas production due to the activity of yeasts. The yeast produces carbon-dioxide (CO2) in the dough which gets entrapped in the dough forming vacuoles which ultimately is responsible for the typical structure of the bread (Wieser, 2003). 140 Volume, ml 120 100 80 60 40 0 25 control 50 15%TS whey 75 100 20% TS whey 125 150 175 25% TS whey Fig4.22. Effect of incorporation of concentrated whey of different TS on the proofing time of multigrain bread dough Page 107

4. Results and Discussion It is observed during trials that the rate of proofing was slower (fig 4.22) in whey incorporated bread than the control. The proofing rate was slower at higher level of whey solids incorporation. Table 4.39 Effect of incorporation of concentrated paneer whey on the proofing rate of multigrain bread dough: Bread dough Dough of control multigrain bread 2.144 Dough of multigrain bread with 15% TS whey 1.833 Dough of multigrain bread with 20% TS whey 1.711 Dough of multigrain bread with 15% TS whey 0.655 Rate of proofing as obtained by slope values (upto linear region) This was confirmed by the decreasing slope values of the curves which were 2.144, 1.833, 1.711 and 0.655 for control, 15, 20 and 25% TS whey incorporated dough, respectively(table-4.39). The results indicated that whey incorporation led to retardation of proofing rate which can be taken as a hindrance to ultimate utilization of whey in bread making. Similar observation was made by Divya and Rao (2010) who reported that concentrated whey incorporation decreased the proofing rate of wheat bread dough. They attributed it to retarded yeast activity in presence of whey proteins. In the present investigation the whey contained 0.83% proteins which might have acted as retarding agent for the growth of yeasts. This was also reported by Collado (2003). However, Yousif et al. (1998) found that incorporation of liquid whey or acid whey or dried whey (@ 0.85 3.5% solids) improved dough development time. In their work, prior fermentation of whey before using in the formulation was also found to help leavening. Gelinas et al. (1995) reported that incorporation of cultured mixture of milk, whey and wheat flour in a pan bread formulation did not reduce proofing time, but affected dough mixing stability. Sanina et al. (1996) after performing an analysis using a statistical tool generalized Lagrange multiplier method, established optimum whey concentration as 16.6% and moisture as 46.2% in dough for getting good quality of bread. Page 108

4. Results and Discussion It has been shown from the results that use of concentrated whey of 15% TS did not significantly affect the dough quality and offered the advantage of complete replacement of water with the concentrated whey. However, there was an adverse influence on the dough proofing time. Incorporation of the whey was found to enormously delay the proofing time, sometimes leading to stagnation in dough rising after a certain time. This was considered as disadvantage if concentrated whey has to be effectively recommended as a replacement to water in bread formulations. So, different means were thought of for enhancement of proofing rate of whey- containing dough so that bakers can easily adopt the process for bread preparation using whey. Two procedures were planned with an aim to reduce the dough proofing time of experimental samples to that of control sample: 1) Enhanced yeast level: Addition of yeast at double the amount added in control i.e. at 6 % level and 2) Enhanced fermentation temperature: Fermentation of the dough at a higher temperature of 40 o C. Effect of incorporation of whey on textural characteristics of multigrain bread dough: Incorporation of concentrated whey during dough preparation did not affect the process of dough making as such, because the whey provided as much liquidness as water used in dough preparation. However, there were variations in textural quality of dough prepared using concentrated whey as shown by instrumental values (Tables 4.40 and 4.41). The hardness of dough increased as the TS content in whey incorporated increased. However, there was not much influence on springiness and resilience. The dough became less cohesive, but more adhesive. The gumminess of dough also increased. The cohesiveness of dough was sustained almost up to that of the original dough because of gluten and carbohydrates in grain flours. In this regard, the role of gluten in dough formation is well understood and documented (Cauvain, 1998). During mixing, water is absorbed by the gluten as well as starch resulting ultimately cohesive dough. When the dough is rested for some time after kneading, hydration of proteins take place and the dough develops a softer texture. When concentrated whey was incorporated, in presence of whey Page 109

4. Results and Discussion proteins more water might have been absorbed as whey proteins have good water binding and hydration ability (Hutton and Campbell, 1981). Further, increased solids in whey used for dough preparation enhanced the solids content in dough. This has resulted in increased hardness, adhesiveness and gumminess. These effects were evident by the addition of 15% TS whey, but use of higher TS whey did not further affect these attributes; it means that whey proteins affected the dough properties significantly when used at or below 15% TS level. These observations were further corroborated by modulus of elasticity (ME) which is indicative of solid character of any material (Rao and Steffe, 1992). The ME increased with the increase in TS level in whey, which also led to considerable enhancement in coefficient of viscosity (Table-4.41). SRT of multigrain dough, which is defined as the time taken for the initial applied stress to decay to 36.7% or 1/e of its original value (Rao and Steffe, 1992), also increased with increase in TS content of whey. Creep retardation time, which was 12.2 sec for control dough increased to 73.3 sec for 25%TS whey incorporated dough (Table- 4.41). The compliance of dough decreased as the TS in whey increased. These results showed that use of whey significantly affected viscoelastic properties of dough (P<0.05). This is attributed to the water absorbing ability of the whey proteins and the slight overall rise in solids during dough making. Creep retardation time refers to the time taken for the strain to reach 63% of the total deformation on the Kelvin portion of creep curve (Rao and Steffe, 1992). This has significance in subsequent proofing and loaf making. The viscosity of dough has a great influence on the subsequent proofing rate because it controls the lamellar strength in bread foam. TPA and viscoelastic parameters data showed significant influence of concentrated whey incorporation on almost all the textural attributes of dough (Table-4.40 & 4.41). These observations are strengthened by Kadharmestan et al. (1998) who reported that fortification of wheat flour with 10 % whey protein concentrate resulted in wet and sticky bread dough, but imparted improved handling properties. Asghar et al., (2009) investigated the effect of modified whey protein concentrate on the TPA characteristics of frozen dough made from flour with different protein contents and found that values of instrumental texture parameters Page 110

4. Results and Discussion were significantly affected by the addition of modified whey protein concentrate (mwpc) and there was a significant decrease in hardness, cohesiveness, gumminess and springiness with its addition in dough samples. The stability and mechanical tolerance index of the French type bread dough were also found to improve by addition of 1% whey solids. Yousif et al. (1998) found that incorporation of liquid whey or acid whey or dried whey (@0.85 3.5% solids) improved the rheological properties of dough. Gelinas et al. (1995) reported that incorporation of cultured mixture of milk, whey and wheat flour in a pan bread formulation affected dough mixing stability. Owing to the limited influence of whey incorporation on multigrain dough quality, the 15% TS whey was used for further trials as textural attributes were least affected. Table 4.40 Effect of concentrated paneer whey incorporation on TPA characteristics of multigrain bread dough TPA attributes %TS in Whey 0 15 20 25 Hardness (N) 6.26±3.6 a 14.28±1.12 b 17.49±2.12 b 19.11±.85 b Cohesiveness 0.76±0.06 b 0.72±0.03 a,b 0.69±0.02 a,b 0.62±0.08 a Springiness 0.91±0.02 b 0.92±0.01 b 0.91±0.02 b 0.77±0.06 a Adhesive -0.98±0.43 c -2.45±0.14 b -3.33±0.45 a -3.24±0.26 a Force (N) Adhesiveness 1185.10±805.81 b -3191.42±119.7 a -4670.86±813.5 a -4080.36±645.2 a (N-sec) Gumminess (N) 4.51±2.59 a 9.40±1.32 b 11.09±2.05 b 9.18±1.7 b Resilience 0.15±.04 a 0.12±.001 a 0.08±.05 a 0.08±.05 a Page 111

4. Results and Discussion Table 4.41. Effect of concentrated paneer whey incorporation on viscoelastic characteristics of multigrain bread dough Attributes %TS in whey 0 15 20 25 Stress relaxation 12.00± 0.48 a 12.26 ± 0.40 b 12.48 ± 0.37 b 14.23 ± 0.08 b time, sec Coefficient of 1.01 ± 0.30 a 150.98±27.5 a,b 193.97 ± 37.3 b 160.6± 118.0 a,b viscosity (x 10-5 ), Pa.sec Modulus of elasticity 3.53 ± 0.4 a 658.0 ±11.2 b 921.55± 86.0 b 809.0± 226.3 b (x 10-3 ), Pa Creep retardation 12.2 ± 1.5 a 13.6 ± 3.5 a 18.9 ± 2.6 a 73.3 ± 24.7 b time, sec Compliance, Pa 17.5 ± 1.4 d 15.0 ± 0.96 c 11.5 ± 0.72 b 8.48 ± 0.53 a 4.3.2 Effect of enhanced Yeast level on the proofing rate of the multigrain bread dough and the sensory scores of the bread. Enhancing the yeast level increased the proofing rate; the rate increased from 1.477 to 1.722 by using 6% yeast level (Table 4.42, Fig. 4.23). It may be seen that the proofing curve using 6% yeast is near to that of control. This happened as expected because more number of yeasts means more production of gas and so enhanced proofing rate. Divya and Rao (2010) reported that use of 6% yeast resulted in enhancement in proofing rate. This could be attributed more to presence of more yeast cells producing more carbon dioxide. Page 112

4. Results and Discussion Volume of dough, ml 160 140 120 100 80 60 40 20 0 15 30 control 45 60 75 Time, min 3% yeast 90 105 120 135 6% yeast Fig 4.23.Effect of incorporating a higher level of yeast on the proofing time of multigrain bread dough Table 4.42. Effect on proofing rate due to different levels of yeast Experimental particulars Rate of proofing as obtained by slope values(upto linear region) Control multigrain bread dough 2.255 Multigrain bread dough with 15%TS whey having 3% yeast 1.477 Multigrain bread dough with 15%TS whey having 6% yeast 1.755 It is well known that during fermentation, ph of dough decreases, principally owing to the formation of lactic acid. Yeast cells multiply fermenting sugar into carbon dioxide and ethanol. Both of these dissolve in aqueous phase of dough. When the aqueous phase is saturated with CO2, the gas evaporates from there to the gas cells. As CO2 is dissolved in aqueous phase the ph decreases. The ph reduction can also be attributed to the growth of lactic acid bacteria in flour (Wieser, 2003). The added lactose through whey might have been responsible for production of lactic acid, but it was recorded that undissociated organic acids are also produced during this process. These acids act as inhibitors of yeast activity (Collado, 2003). So, in the present study, it was observed that whey incorporation slowed down the rate of proofing as reflected by changes in volume, indicating that fermentation was retarded by incorporation of whey solids. This has also prolonged the proofing Page 113

4. Results and Discussion periods. With this background of slow yeast activity, yeast level was enhanced to 6% level to accelerate gas production and the proofing process. This yeast level resulted in proofing times closer to control values. On the contrary, Imbs and Czerwinski (1974) reported that when water in bread formulation was replaced with liquid whey, it not only resulted in better kneading of dough but also improved yeast fermentation. Similarly use of 20 30% whey was found to reduce the total processing time by 12 13% (Silagadze and Lyushinskaya, 1980). However there was a marked decrease in sensory scores because incorporation of higher level of yeast resulted in bread with unusually soft body and unpleasantly sour taste. 5.4 5.3 5.2 p H 5.1 5 4.9 0 20 Control 40 60 80 Time (in mins) 100 Whey Bread with 3% yeast 120 140 160 Whey bread with 6% yeast Fig 4.24.Graphical representation of change in ph of multigrain bread doughs due to variation in yeast level Table 4.43 Effect of increasing level of yeast in the dough on the sensory scores of bread Sensory attributes Control 3% Yeast sample 6% Yeast Sample colour and appearance 6.813a±.347 7.31b±.166 7.313b±.211 body and texture 7.263a±.235 7.473b±.126 6.516c±.261 Flavour 7.247b±.208 7.626c±.272 5.597a±.499 Overall Acceptability 6.983b±0.286 7.613c±.126 5.691a±.492 ** Scores on 9-point hedonic scale Page 114

4. Results and Discussion The results revealed that use of increased yeast content resulted in significant decrease in the overall acceptability score (Table 4.43). The OA score of control samples was 6.983±0.286 while that of the 3% yeast incorporated experimental sample was 7.613±0.126 and at 6% yeast incorporation into the experimental sample the score decreased to 5.691±0.492. The scores differed significantly (table 4.44 to 4.51) Table 4.44 ANOVA: Effect of increasing level of yeast in the dough on the C&A scores of bread Sum of squares Df Mean square F Sig Between Groups 4.966889 2 2.483444 37.40429 *.000 Within Groups 5.776333 87 0.066395 Total 10.74322 89 * Significant p<0.05 Table 4.45 TUKEY: Effect of increasing level of yeast in the dough on the C&A scores of bread Multigrain breads N Subset 1 2 control multigrain bread 30 6.8133 control multigrain whey bread (3% yeast) 30 7.3100 multigrain whey bread with 6% yeast 30 7.3133 Sig. 1.000.999 Table 4.46 ANOVA: Effect of increasing level of yeast in the dough on the B&T scores of bread Sum of Squares Df Mean Square F Sig. Between Groups 15.16822 2 7.584111 155.9853 *.000 Within Groups 4.23 87 0.048621 Total 19.39822 89 * Significant p<0.05 Page 115

4. Results and Discussion Table 4.47 TUKEY: Effect of increasing level of yeast in the dough on the B&T scores of bread Multigrain breads N Subset for alpha =.05 1 2 3 multigrain whey bread with 6% yeast 30 6.5167 control multigrain bread 30 7.2633 multigrain whey bread (3% yeast) 30 7.4733 Sig. 1.000 1.000 1.000 Table 4.48 ANOVA: Effect of increasing level of yeast in the dough on the Flavour scores of bread Sum of Squares Df Mean Square F Sig. Between Groups 69.878 2 34.939 276.763 *.000 Within Groups 10.983 87.126 Total 80.861 89 * Significant p<0.05 Table 4.49 TUKEY: Effect of increasing level of yeast in the dough on the Flavour scores of bread Multigrain breads N Subset for alpha =.05 1 2 3 multigrain whey bread with 6% yeast 30 5.5967 control multigrain bread 30 7.2467 multigrain whey bread (3% yeast) 30 7.6267 Sig. 1.000 1.000 1.000. Table 4.50 ANOVA: Effect of increasing level of yeast in the dough on the OA scores of bread Sum of Squares Df Mean Square F Sig. Between Groups 57.474 2 28.737 245.230 *.000 Within Groups 10.195 87.117 Total 67.669 89 * Significant p<0.05 Page 116

4. Results and Discussion Table 4.51 TUKEY: Effect of increasing level of yeast in the dough on the OA scores of bread Multigrain breads N Subset for alpha =.05 1 multigrain whey bread with 6% yeast 30 control multigrain bread 30 multigrain whey bread (3% yeast) 30 Sig. 2 3 5.6933 6.9833 7.6133 1.000 1.000 1.000 4.3.2 Effect of temperature on the rate of proofing of multigrain bread dough. The temperature at which proofing of dough is carried out is a critical parameter in the production of bread. In order to increase the rate of proofing another treatment variable adopted was to alter the temperature of proofing and the results are presented in tables 4.52 to 4.53 and figures 4.25 and 4.26. Effect of Temperature on Proofing Rate volume of dough (in ml) 160 140 control 120 100 whey bread room temp 80 whey bread 40 deg 60 40 0 25 50 75 100 125 150 time in minutes Fig 4.25. Effect of variation of temperature on rate of proofing of multigrain bread dough Page 117

4. Results and Discussion Table 4.52. Effect of variation of temperature on rate of proofing of multigrain bread dough Experimental particulars Rate of proofing as obtained by slope values (upto linear region) Control multigrain bread dough 1.777 Multigrain bread dough with 15%TS whey proofed at 28OC 1.544 Multigrain bread dough with 15%TS whey proofed at 40o C. 2.311 Proofing is highly dependent on the temperature at which it is carried out. The proofing temperatures of bread varies from 26oC to 28oC (Wang, 2007) and higher the proofing temperature, higher will be the proofing rate (Collado, 2003). Higher temperatures enhance the yeast activity (Collado, 2003) and enhance gas production as reflected in figure 4.25. The proofing rates were 1.777, 1.544 and 2.311 for control, 30oC and 40oC, respectively (Table-4.52), which could be attributed to the faster growth of yeast at higher temperatures, the fermentation being very active at 400C (Collado, 2003). However, further increase in temperatures resulted in decreased activity. It was earlier reported by Cauvain (2012) that yeast activity increases from 35 C to 43 C and then decreases subsequently up to 55 C, and thereafter the activity ceases. 5.4 5.35 5.3 5.25 5.2 5.15 5.1 5.05 0 Control 20 40 60 80 100 Whey Bread Fermented at room temperature 120 140 160 Whey bread fermented at 40 deg C Fig 4.26.Effect on ph of multigrain bread doughs due to variation in proofing temperature Page 118

4. Results and Discussion sensory scores on hedonic scale 7.6 7.4 7.2 7 6.8 6.6 Effect of elevated proofing temperature on sensory characteristics of multigrain bread Colour and appearance Body and Texture Flavour sensory characteristics OA Multigrain bread with 15% whey proofed at 40 degree C Control multigrain bread Multigrain bread with 15% whey proofed at room temperature Fig 4.27. Effect of elevated proofing temperature on the sensory charcteristics of multigrain bread. Table 4.53. Effect of Increased temperature of proofing the dough on the Sensory scores of bread Sensory attributes CONTROL Whey Bread-28 o C Whey Bread-40 O C Colour and appearance 7.091 a ±.446 7.273 c ±.102 7.228 b ±.661 Body and Texture 7.024 a ±.111 7.525 b ±.139 7.518 b ±.686 Flavour 7.099 a ±.711 7.513 b ±.417 7.519 b ±.475 OA * Scores on 9-point hedonic scale 7.06 a ±.589 7.538 c ±.081 7.397 b ±.079 The effect of proofing temperature on the sensory scores is tabulated in Table 4.53. The results indicated that increasing the proofing temperature to 40 o C resulted in significant increase in the overall acceptability from that of the control. The OA scores were 7.06±0.589, 7.538±0.081 and 7.397±0.079 for the control, whey incorporated breads proofed at 28 o and 40 o C respectively. The results differed significantly. The body and texture and flavour scores did not differ significantly between the concentrated whey incorporated multigrain breads proofed at 28 and 40 o C. The data is also presented graphically in figures 4.27. Page 119

4. Results and Discussion The results, based on OA score indicated that the proofing time of dough can be effectively controlled by enhancing the temperature of proofing to 40 o C (Figure 4.28). sensory scores on hedonic scale 8 6 4 2 0 comparison of effects of elevated temperature & higher level of yeast on the sensory characteristics of multigrain bread Colour and appearancebody and Texture Flavour OA sensory characteristics Fig 4.28. Comparison of effects of yeast level variation and elevated proofing temperature on the sensory characteristics of whey based multigrain bread. multigrain bread with 15%TS whey proofed at 40 degree C multigrain bread with 15%TS whey containing 6% yeast 4.4. Effect of incorporation of improvers on the quality of multigrain bread: The Food Safety and Standards Authority of India (FSSAI) regulations permit certain improvers to be incorporated in the manufacture of bread. The permitted improvers are Calcium Carbonate (CaCO 3 ), Calcium Phosphate ( Ca 3 (PO 4 ) 2 ) and Ammonium persulphate. These improvers were incorporated at the maximum permitted levels in the manufacture of multigrain bread and the sensory characteristics were evaluated. 4.4.1. Effect of incorporation of improvers on the sensory quality of multigrain bread It is observed from table 4.54 that the incorporation of improvers caused significant increase in the OA score. The control bread without any improver scored the least with 7.06±0.59, which was significantly lower than all other treatments. The improver Calcium Phosphate incorporated at 2500ppm resulted in the highest score of 7.71±0.38, which was significantly higher than all other treatments except the Page 120

4. Results and Discussion treatment with Calcium Carbonate. Whey optimized bread without improvers had the minimum OA score amongst all the treatments with a score of 7.32±0.58 and the addition of CaCO 3 improved the score to 7.61±0.45 but was not statistically significant. Amongst the improvers ammonium persulphate incorporated resulted in no significant improvement with the score being 7.45±0.26. The variations in the sensory scores of hardness, gumminess, springiness and chewiness, colour and appearance and flavour are also presented in table 4.54 and their statistical analysis is presented in tables 4.55 to 4.68. Table 4.54. Effect of incorporation of improvers on the sensory quality of multigrain bread Attributes Control Whey Optimized CaCO 3 Ca 3 (PO 4 ) 2 Ammonium Per sulphate Colour and 7.09 a ±.45 7.22 a ±.66 7.65 b ±.38 7.67 b ±.313 7.59 b ±.46 Appearance Body and Texture Hardness 6.96 a ±.49 7.13 a ±.39 7.45 b ±.53 7.71 b ±.35 7.6 b ±.37 Gumminess 7.12 a ±.55 7.14 a ±.53 7.47 a ±.49 7.47 a ±.73 7.41 a ±.33 Springiness 6.87 a ±.59 6.89 a ±.69 7.7 b ±.44 7.75 b ±.41 7.52 b ±.29 Chewiness 7.14 a ±.54 7.14 a ±.56 7.44 a,b ±.44 7.51 b ±.36 7.3 a,b ±.42 Flavour 7.1 a ±.71 7.38 a,b ±.48 7.58 b,c ±.42 7.77 c ±.30 7.5 b,c ±.30 Overall Acceptability 7.06 a ±.59 7.32 a,b ±.58 7.61 b,c ±.45 7.71 c ±.38 7.45 b,c ±.26 * Scores on 9-point hedonic scale It is observed from table 4.54 that addition of improvers resulted in significant improvement in colour and appearance, significant improvement in hardness score, springiness score, chewiness score and also the flavour score. No significant variation in the gumminess score was observed. Page 121

4. Results and Discussion Table 4.55. ANOVA: Effect of incorporation of improvers on the C & A scores of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 9.622 4 2.405 10.659 *.000 Within Groups 37.234 165.226 Total 46.855 169 * Significant p<0.05 Table 4.56. TUKEY: Effect of incorporation of improvers on the C&A scores of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 control multigrain bread 34 7.0912 whey optimized control multigrain bread 34 7.2235 multigrain whey bread with ammonium persulphate as 34 7.5853 improver multigrain whey bread with Ca-carbonate as improver 34 7.6471 multigrain whey bread with Ca-phosphate as improver 34 7.6647 Sig..780.959 Table 4.57. ANOVA: Effect of incorporation of improvers on the sensory perception of hardness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 13.657 4 3.414 17.840 *.000 Within Groups 31.578 165.191 Total 45.235 169 * Significant p<0.05 Page 122

4. Results and Discussion Table 4.58. TUKEY: Effect of incorporation of improvers on the sensory perception of hardness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 control multigrain bread 34 6.96176 whey optimized control multigrain bread 34 7.12500 multigrain whey bread with Ca-carbonate as improver 34 7.44706 multigrain whey bread with ammonium persulphate as improver 34 7.60294 multigrain whey bread with Ca-phosphate as improver 34 7.70882 Sig..539.103 Table 4.59. ANOVA: Effect of incorporation of improvers on the sensory perception of springiness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 25.617 4 6.404 24.712 *.000 Within Groups 42.762 165.259 Total 68.379 169 * Significant p<0.05 Table 4.60 TUKEY: Effect of incorporation of improvers on the sensory perception of springiness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 control multigrain bread 34 6.87353 whey optimized control multigrain bread 34 6.88529 multigrain whey bread with ammonium persulphate as improver 34 7.52059 multigrain whey bread with Ca-carbonate as improver 34 7.69853 multigrain whey bread with Ca-phosphate as improver 34 7.75000 Sig. 1.000.344 Page 123

4. Results and Discussion Table 4.61 ANOVA: Effect of incorporation of improvers on the sensory perception of gumminess of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 4.253 4 1.063 3.516 *.009 Within Groups 49.897 165.302 Total 54.150 169 * Significant p<0.05 Table 4.62 TUKEY: Effect of incorporation of improvers on the sensory perception of gumminess of multigrain bread Multigrain breads N Subset for alpha =.05 1 control multigrain bread 34 7.1191 whey optimized control multigrain bread 34 7.1426 multigrain whey bread with ammonium persulphate as improver 34 7.4138 multigrain whey bread with Ca-carbonate as improver 34 7.4647 multigrain whey bread with Ca-phosphate as improver 34 7.4735 Sig..065 Table 4.63 ANOVA: Effect of incorporation of improvers on the sensory perception of chewiness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 4.274 4 1.068 4.684 *.001 Within Groups 37.634 165.228 Total 41.907 169 * Significant p<0.05 Page 124

4. Results and Discussion Table 4.64 TUKEY: Effect of incorporation of improvers on the sensory perception of chewiness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 control multigrain bread 34 7.14118 whey optimized control multigrain bread 34 7.14176 multigrain whey bread with ammonium persulphate as improver 34 7.42941 7.42941 multigrain whey bread with Ca-carbonate as improver 34 7.43824 7.43824 multigrain whey bread with Ca-phosphate as improver 34 7.51176 Sig..082.954 Table 4.65 ANOVA: Effect of incorporation of improvers on the flavour scores of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 8.275 4 2.069 9.229 *.000 Within Groups 36.988 165.224 Total 45.263 169 * Significant p<0.05 Table 4.66 TUKEY: Effect of incorporation of improvers on the flavour scores of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 3 control multigrain bread 34 7.099 whey optimized control multigrain bread 34 7.382 7.382 multigrain whey bread with ammonium persulphate as improver 34 7.459 7.459 multigrain whey bread with Ca-carbonate as improver 34 7.578 7.578 multigrain whey bread with Ca-phosphate as improver 34 7.765 Sig..102.435.064 Page 125

4. Results and Discussion Table 4.67 ANOVA: Effect of incorporation of improvers on the Overall Acceptability scores of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 8.831 4 2.208 9.786 *.000 Within Groups 37.222 165.226 Total 46.052 169 * Significant p<0.05 Table 4.68 TUKEY: Effect of incorporation of improvers on the Overall Acceptability scores of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 3 control multigrain bread 34 7.06 whey optimized control multigrain bread 34 7.321 7.321 multigrain whey bread with ammonium persulphate as 34 7.445 7.445 improver multigrain whey bread with Ca-carbonate as improver 34 7.612 7.612 multigrain whey bread with Ca-phosphate as improver 34 7.709 Sig..164.089.159 4.4.2. Effect of incorporation of improvers on the Texture profile characteristics of multigrain bread Table 4.69 Effect of incorporation of improvers on the Texture profile characteristics of multigrain bread ATTRIBUTES Control Control Whey Multigrain Bread MG Whey bread with improver CaCO 3 MG Whey bread with improver Ca 3 (PO 4 ) 2 MG Whey bread with improver ammonium persulphate Hardness 22.65 a ±4.17 26.80 a ±5.8 20.91 a ±1.7 19.16 a ±3.98 22.57 a. ±9.94 Cohesiveness 0.51 b ±12 0.56 b ±0.11 0.48 a,b ±0.09 0.48 a,b ±0.07 0.38 a ±0.03 Springiness 0.76 a,b ±0.06 0.75 a,b ±0.07 0.78 b ±0.05 0.77 b ±0.08 0.67 a ±0.74 Gumminess 11.76 a,b ±4.12 14.60 b ±2.76 9.98 a ±1.9 9.16 a ±2.34 8.48 a ±3.82 Chewiness 9.02 b ±3.22 11.07 a,b ±2.72 7.82 a,b ±1.64 7.11 a ±2.12 5.8 a ±2.83 Page 126

4. Results and Discussion The effect of improvers on the texture profile analysis is presented in table 4.69 and their related statistical analysis in tables 4.70 to 4.78. It is observed from table 4.69 that most of the attributes of texture profile differed significantly due to the addition of improvers. The hardness of the bread was observed to increase due to whey incorporation as the hardness value increased from 22.65±4.17 for control samples to 26.80±5.8 for the whey incorporated bread. The addition of improvers resulted in a significant decrease in the hardness value and addition of calcium phosphate caused the maximum reduction. The cohesiveness also established a similar trend with initial increase from 0.51±.0.12 for control samples to 0.56±0.11 for whey incorporated bread. These values decreased to 0.48±0.09, 0.48±0.07 and 0.38±0.03 for CaCO 3, Ca 3 (PO 4 ) 2 and ammonium persulphate added bread respectively. The decrease in cohesiveness was significant except between the addition of CaCO 3 and Ca 3 (PO 4 ) 2 which exhibited similar results. The trends in springiness were however different as the addition of whey resulted in decreased springiness. The springiness value for the control sample was 0.76±0.06 which decreased to 0.75±0.07 in the whey incorporated bread. The addition of improvers increased the values to 0.78±0.05 for the CaCO 3 incorporated bread, 0.77±0.08 for Ca 3 (PO 4 ) 2 incorporated bread and 0.67±0.07 for the ammonium persulphate incorporated bread. The values differed significantly due to the treatment. However the difference was non-significant between control and whey incorporated bread, between CaCO 3 and Ca 3 (PO 4 ) 2 improvers. The gumminess values showed a trend similar to the springiness with the value for control 11.75±4.12 increasing to 14.60±2.76 for the whey incorporated bread. Addition of improvers reduced the gumminess significantly with the values being 9.98±1.99, 9.16±2.34 and 8.48±3.82 for CaCO 3, Ca 3 (PO 4 ) 2 and Ammonium Persulphate incorporated breads respectively. The values differed significantly between all the treatments. The chewiness values also showed a similar trend (Table 4.69). Page 127

4. Results and Discussion Table 4.70 ANOVA: Effect of incorporation of improvers on the Hardness, (N) of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 289.465 4 72.366 1.910.128 Within Groups 1515.552 40 37.889 Total 1805.018 44 Table 4.71 ANOVA: Effect of incorporation of improvers on the Springiness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups.072 4.018 3.538 *.015 Within Groups.204 40.005 Total.277 44 * Significant p<0.05 Table 4.72 TUKEY: Effect of incorporation of improvers on the Springiness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 multigrain whey bread with ammonium persulphate as improver 9.67056 whey optimized control multigrain bread 9.75178.75178 control multigrain bread 9.76056.76056 multigrain whey bread with Ca-phosphate as improver 9.77200 multigrain whey bread with Ca-carbonate as improver 9.78389 Sig..077.874 Table 4.73 ANOVA: Effect of incorporation of improvers on the Cohesiveness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups.157 4.039 4.447 *.005 Within Groups.354 40.009 Total.511 44 * Significant p<0.05 Page 128

4. Results and Discussion Table 4.74 TUKEY: Effect of incorporation of improvers on the Cohesiveness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 multigrain whey bread with ammonium persulphate as 9.37768 improver multigrain whey bread with Ca-carbonate as improver 9.47556.47556 multigrain whey bread with Ca-phosphate as improver 9.47567.47567 control multigrain bread 9.50544 whey optimized control multigrain bread 9.55956 Sig..197.337 Table 4.75 ANOVA: Effect of incorporation of improvers on the Gumminess of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 217.314 4 54.328 4.973 *.002 Within Groups 436.991 40 10.925 Total 654.305 44 * Significant p<0.05 Table 4.76 TUKEY: Effect of incorporation of improvers on the Gumminess of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 multigrain whey bread with ammonium persulphate as 9 8.47697 improver multigrain whey bread with Ca-phosphate as improver 9 9.15789 multigrain whey bread with Ca-carbonate as improver 9 9.97612 control multigrain bread 9 11.75225 11.75225 whey optimized control multigrain bread 9 14.60364 Sig..060.075 Page 129

4. Results and Discussion Table 4.77 ANOVA: Effect of incorporation of improvers on the Chewiness of multigrain bread Sum of Squares Df Mean Square F Sig. Between Groups 144.140 4 36.035 4.856 *.003 Within Groups 296.831 40 7.421 Total 440.971 44 * Significant p<0.05 Table 4.78 TUKEY: Effect of incorporation of improvers on the Chewiness of multigrain bread Multigrain breads N Subset for alpha =.05 1 2 multigrain whey bread with ammonium persulphate as improver 9 5.79538 multigrain whey bread with Ca-phosphate as improver 9 7.10468 multigrain whey bread with Ca-carbonate as improver 9 7.81936 7.81936 control multigrain bread 9 9.01901 9.01901 whey optimized control multigrain bread 9 11.06801 Sig..109.104 The results revealed that the improvers did result in a significant improvement in the quality of the bread assessed by both subjective and objective assessments. The role of improvers in the improvement of the quality of bakery products has also been reported earlier by Jarita and Kulkarni (2009) for rusks and soupsticks. Amongst the three improvers Ca-phosphate showed better quality parameters during sensory evaluation and also during TPA analysis. Hence Ca-phosphate was selected for further studies to optimize the levels in the product of whey incorporated bread. In the earlier studies all the improvers were incorporated at their maximum permitted levels as per the FSSAI standards. However, it was hypothesized that addition of improvers may not be required at the highest level and hence further optimization trials were conducted and the results are presented subsequently. Page 130

4. Results and Discussion 4.4.3. Optimization of levels of Calcium phosphate: Based on the evaluation of the improvers as discussed in the preceding chapter, calcium phosphate was selected for further optimization. During the comparative evaluation the improvers were incorporated in the dough at the maximum permitted levels of FSSAI regulations. However it is hypothesized that the maximum permitted levels may not be necessary in the preparation of bread. In order to test this hypothesis bread was prepared by incorporating Ca-phosphate at 3 levels: 1500ppm, 2000ppm and the maximum permitted level of 2500ppm. Table 4.79. Effect of different levels of calcium phosphate on the sensory attributes of bread. Sensory attributes Control Ca-phosphate 2500ppm Ca-phosphate 2000ppm Ca-phosphate 1500ppm Colour and 7.59±0.205 a 7.8±0.15 b 7.79±0.14 b 7.8±0.14 b Appearance Body and Texture 7.55±0.25 a 7.66±0.2 a 7.65±0.2 a 7.66±0.21 a Flavour 7.56±0.25 a 7.64±0.27 a 7.66±0.25 a 7.64±0.26 a Overall Acceptability 7.5±0.19 a 7.72±0.18 b 7.73±0.16 b 7.73±0.18 b * Scores on 9-point hedonic scale The results are presented in table 4.79 statistical analysis in tables 4.80 to 4.86. Table 4.80 ANOVA: Effect of different levels of calcium phosphate on the C&A scores of bread. Sum of Squares Df Mean Square F Sig. Between Groups 0.376 3.125 4.301 *.010 Within Groups 1.281 44.029 Total 1.656 47 * Significant p<0.05 Page 131

4. Results and Discussion Table 4.81 TUKEY: Effect of different levels of calcium phosphate on the C&A scores of bread Level of Ca-phosphate (ppm) N Subset for alpha =.05 1 2 Control 12 7.5917 2000 12 7.7917 1500 12 7.7958 2500 12 7.8000 Sig. 1.000.999 Table 4.82 ANOVA: Effect of different levels of calcium phosphate on B&T scores of bread. Sum of Squares Df Mean Square F Sig. Between Groups.667 3.222 5.880 *.002 Within Groups 1.665 44.038 Total 2.332 47 * Significant p<0.05 Table 4.83 TUKEY: Effect of different levels of calcium phosphate on B&T scores of bread. Level of Ca-phosphate (ppm) N Subset for alpha =.05 1 2 Control 12 7.3833 2000 12 7.6500 2500 12 7.6583 1500 12 7.6583 Sig. 1.000 1.000 Page 132

4. Results and Discussion Table 4.84 ANOVA: Effect of different levels of calcium phosphate on Flavour scores of bread. Sum of Squares Df Mean Square F Sig. Between Groups.073 3.024.339.798 Within Groups 3.177 44.072 Total 3.250 47 Table 4.85 ANOVA: Effect of different levels of calcium phosphate on OA scores of bread. Sum of Squares Df Mean Square F Sig. Between Groups.467 3.156 4.480 *.008 Within Groups 1.528 44.035 Total 1.994 47 * Significant p<0.05 Table 4.86 TUKEY: Effect of different levels of calcium phosphate on OA scores of bread. Level of Ca-phosphate (ppm) N Subset for alpha =.05 1 2 control 12 7.5000 2500 12 7.7167 2000 12 7.7317 1500 12 7.7333 Sig. 1.000.996 The results revealed that the addition of Calcium phosphate resulted in significant improvement in the OA score with F-value being 4.48. However it was also observed that there was no significant difference in OA score between the three levels of Ca-phosphate incorporation. It was also observed that there was no significant difference between any of the treatments with respect to any of the sensory attributes (table 4.80). Page 133

4. Results and Discussion The results indicated that the addition of Ca-phosphate is not required at the highest level permitted by FSSAI and the level can be reduced to 1500ppm without compromising with quality. Similar observations have earlier been reported by Jarita and Kulkarni (2009) and the results in the present investigation are in agreement with the earlier observations. Thus based on all the optimized conditions as detailed in the preceding chapters bread was prepared for physicochemical evaluation and study of shelf life. The optimized conditions of the bread production are detailed in flow diagram (Fig 4.29). Page 134

4. Results and Discussion OPTIMIZED BREAD PREPARATION FLOWCHART Wheat flour : 200 g Oat Flour : 12.5 g Maize Flour : 12.5 g Sorghum Flour : 12.5 g Flaxseed Flour : 12.5 g Oil : 37.5 g Sugar : 25 g Whey (15%TS): 160g Salt : 5 g Yeast : 7.5 g Ca-Phosphate :1500ppm INGREDIENTS MIXING KNEADING IN HOBART MIXER (5-6 minutes) DIVIDING (400 g) Addition of the other 100 ml of warm whey slowly during kneading ROUNDING INTERMEDIATE PROOFING (10-15 MINUTES) Activation of yeast in 50 ml warm whey with 5 g sugar and adding it after proper mixing of all the ingredients except the remaining amount of whey. MOLDING PANNING FERMENTATION (75 MIN @40 O C) BAKING (185 0 C / 30 MIN) COOLING (Room Temperature / 40 minutes) SLICING OR CUTTING OF BREAD PACKAGING (LDPE POUCHES) Fig4.29 Optimized flowchart of bread production using concentrated whey Page 135

4. Results and Discussion 4.5 Physico-chemical characteristics of control bread and optimized bread made using incorporation of concentrated whey. The multigrain bread with and without the addition of whey was analyzed for different physico-chemical characteristics and the results are tabulated in the table 4.87. Table 4.87. Physicochemical analysis of control and whey incorporated multigrain bread Control multigrain Optimized experimental Constituents bread multigrain bread Mean* ± SD Mean* ± SD Volume, ml Total solids, % Fat, % Protein, % Ash, % Acid Insoluble Ash Lactose, % Crude Fiber,% ph Water activity Alcoholic Acidity, ml 1N NaOH Reflectance,% * Mean of three trials 1300.88±8.39 71.85 ± 0.18 11.37 ± 0.12 10.96 ± 0.04 1.72 ± 0.025.081±.031 ------ 7.25±0.05 5.21 ± 0.05 0.87 ± 0.001 0.5±.01 51.3 1394.62±12.02 73.87 ± 0.18 12.25 ± 0.11 11.24 ± 0.1 1.84 ± 0.033.078±.02 8.43±.434 7.23±0.12 5.17 ± 0.03 0.84 ± 0.001 0.3±.01 45.5 It is observed from the table that the baked volume of bread increased due to the addition of concentrated whey replacing water. The volume of the control bread samples was 1300.88±8.79 ml, while the experimental sample had a volume of 1394±12.02 ml. The TS content of the experimental sample was also higher at 73.87±0.18 percent compared to 71.85±0.18 percent for the control. Similar increasing trend was also observed with respect to the fat content which was 12.25±0.11 percent compared to 11.37±0.12 percent in case of the control bread. Page 136

4. Results and Discussion The protein content of the whey incorporated bread was 11.24±0.1 percent while that of control was 10.96±0.04 percent. Similarly the ash content was also observed to be higher at 1.84±0.033 percent compared to 1.72±0.025 percent for the control sample. The lactose content of the experimental bread was observed to be 8.43±0.434 which was found to be absent in the control sample. The crude fibre content ranged from 7.20-7.30 for control samples while the range was 7.11-7.35 percent for the experimental samples. The ph of control bread was 5.21±0.05 while that of the experimental bread was 5.17±0.03. The water activity was higher in the control sample at 0.87±0.001 compared to 0.84±0.001 in the experimental sample. The alcoholic acidity was 0.5±0.01 for the control sample which was higher than the value of 0.3±0.01 for the experimental samples. The reflectance value of the control sample was 51.3% which was higher than 45.5% for the experimental sample. Based on the above results it is inferred that the incorporation of whey in the preparation of multigrain bread caused marginal variations in the physico-chemical properties. The volume increased by about 7% which could be ascribed to the increased holding of the air cells in the bread. The increase in the holding of air cells could be due to the increased fibrous network due to the denaturation of the whey proteins and also due to protein-protein interaction in the multigrain bread. The replacement of dough water by whey resulted in increased bread volume was also reported by other workers (Preller, 1978 ; Silagadze and Lyushinskaya, 1980). But Gelinas et al. (1995) reported that use of a cultured mixture of whey, milk and wheat flour reduced the specific volume of bread. The increase in TS content of the experimental bread was due to the solids content in whey which is expected. Similar marginal increase in TS content of bakery products due to whey incorporation was reported earlier (Jarita and Kulkarni,2009; Divya and Rao,2010; Poonam, 2007). As the TS increased it is imperative that the fat, protein and ash contents increased as described in the previous paragraph. The results observed in the present study are also in agreement with the earlier observations by Silagadze and Lyushinskaya(1980). The acid insoluble ash was marginally lower in the experimental sample which could be attributed to the fact that the increased solids content from whey which did not contain any acid insoluble ash had a dilution effect. Page 137

4. Results and Discussion The lactose content of the experimental sample was mainly due to addition of concentrated whey which contained about 12.5% lactose. The crude fibre which was contributed mainly by cereals was almost identical. Similar to ph the slight decrease in water activity of the experimental samples could be ascribed to the increased TS content and better binding of moisture in the denatured protein matrix. Divya and Rao (2010) also reported decrease in water activity due to the use of concentrated whey in the preparation of bread. The decrease in alcoholic acidity was also as per hypothesis since the alcoholic acidity is contributed mainly by the cereal carbohydrates. The use of whey as a diluent resulted in increased soluble solids in the dough which lowered water activity (Jarita and Kulkarni, 2009). Effect of increased soluble solids in decreasing water activity was hypothesized by Rockland and Stewart (1981). The reflectance value of control bread was higher at 51.3% indicating a lighter shade whereas the addition of whey made the bread brown due to the increased Maillard Browning reaction (Divya and Rao, 2010). Thus it is observed from the above results that the replacement of water by concentrated whey as diluent influences the physico-chemical characteristics of multigrain bread. Page 138

4. Results and Discussion 4.6 Storage Studies: The bread samples prepared with optimized process was evaluated for the shelf life by storing at 30 and 5 o C after packing in LDPE film of 25 micron thickness. The bread samples were evaluated at regular intervals of two days for sensory, rheological, chemical and microbial quality. The results are tabulated in tables 4.88-4.159. 4.6.1 Effect on sensory attributes: The sensory evaluation of bread stored at 30 o C indicated that the overall acceptability score decreased with the increase in storage period. Table 4.88. Effect on sensory attributes of multigrain bread due to storage at 30 o C Sensory attributes Day 0 Day 2 Day 4 Control Sample Control Sample Control Sample Colour and Appearance 7.75 b ±0.29 7.88 B ±0.26 7.71 b ±0.23 7.78 B ±0.23 6.47 a ±0.4 7.38 A ±0.3 Body and Texture 7.65 b ±0.23 7.71 C ±0.18 7.55 b ±0.22 7.3 B ±0.25 7.11 a ±0.21 6.71 A ±0.22 Flavour 7.58 b ±0.18 7.74 B ±0.17 7.56 b ±0.21 7.68 B ±0.21 6.85 a ±0.18 6.4 A ±0.24 Overall acceptability 7.73 b ±0.18 7.81 C ±0.15 7.55 b ±0.16 7.6 B ±0.22 6.9 a ±0.27 6.7 A ±0.19 * small superscripts denote variations in control and capital denotes variation in sample. ** scores on 9 point hedonic scale In general the shelf life was about 4 days for both the control and experimental samples. The deterioration in colour and appearance was the least but significant while decrease in flavour score was steep and significant. The change in body and texture score was also significant but less marked than the flavour score. The OA score for the control sample was 7.73±0.18, 7.55±0.16 and 6.9±0.27 on day 0,2 and 4 respectively and the decrease was significant. In the case of experimental Page 139

4. Results and Discussion samples the OA score was observed to be 7.81±0.15, 7.6±0.22 and 6.7±0.19 on day 0, 2 and 4 respectively (Table 4.88) and the decrease was statistically significant. Table 4.89. ANOVA: Effect on C & A scores of control multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 12.594 2 6.297 57.695 *.000 Within Groups 3.602 33.109 Total 16.196 35 * Significant p<0.05 Table 4.90. TUKEY: Effect on C & A scores of control multigrain bread due to storage at 30 o C Day N Subset for alpha =.05 1 2 4 12 6.4750 2 12 7.7083 0 12 7.7500 Sig. 1.000.949 Table 4.91. ANOVA: Effect on C & A scores of Experimental multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 1.721 2.860 11.170 *.000 Within Groups 2.542 33.077 Total 4.262 35 * Significant p<0.05 Page 140

4. Results and Discussion Table 4.92. TUKEY: Effect on C & A scores of Experimental multigrain bread due to storage at 30 o C Day N Subset 1 2 4 12 7.3750 2 12 7.7750 0 12 7.8833 Sig. 1.000.609 Table 4.93. ANOVA: Effect on B & T scores of Control multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 1.994 2.997 19.249 *.000 Within Groups 1.709 33.052 Total 3.703 35 * Significant p<0.05 Table 4.94. TUKEY: Effect on B & T scores of Control multigrain bread due to storage at 30 o C Day N Subset 1 2 4 12 7.1083 2 12 7.5500 0 12 7.6500 Sig. 1.000.535 Table 4.95. ANOVA: Effect on B & T scores of Experimental multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 6.067 2 3.034 58.946 *.000 Within Groups 1.698 33.051 Total 7.766 35 * Significant p<0.05 Page 141

4. Results and Discussion Table 4.96. TUKEY: Effect on B & T scores of Experimental multigrain bread due to storage at 30 o C Day N Subset 1 2 3 4 12 6.7083 2 12 7.3000 0 12 7.7083 Sig. 1.000 1.000 1.000 Table 4.97. ANOVA: Effect on Flavour scores of Control multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 4.161 2 2.080 52.977 *.000 Within Groups 1.296 33.039 Total 5.456 35 * Significant p<0.05 Table 4.98. TUKEY: Effect on Flavour scores of Control multigrain bread due to storage at 30 o C Day N Subset 1 2 4 12 6.8500 2 12 7.5583 0 12 7.5833 Sig. 1.000.949. Table 4.99. ANOVA: Effect on Flavour scores of Experimental multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 12.772 2 6.386 137.211 *.000 Within Groups 1.536 33.047 Total 14.307 35 * Significant p<0.05 Page 142

4. Results and Discussion Table 4.100. TUKEY: Effect on Flavour scores of Experimental multigrain bread due to storage at 30 o C Day N Subset 1 2 4 12 6.4500 2 12 7.6833 0 12 7.7417 Sig. 1.000.787 Table 4.101. ANOVA: Effect on Overall acceptability scores of Control multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 4.602 2 2.301 46.397 *.000 Within Groups 1.637 33.050 Total 6.239 35 * Significant p<0.05 Table 4.102. TUKEY: Effect on Overall acceptability scores of Control multigrain bread due to storage at 30 o C Day N Subset 1 2 4 12 6.9000 2 12 7.5500 0 12 7.7333 Sig. 1.000.124 Table 4.103. ANOVA: Effect on Overall acceptability scores of Experimental multigrain bread due to storage at 30 o C Sum of Squares Df Mean Square F Sig. Between Groups 8.371 2 4.186 107.858 *.000 Within Groups 1.281 33.039 Total 9.652 35 * Significant p<0.05 Page 143

4. Results and Discussion Table 4.104. TUKEY: Effect on Overall acceptability scores of Experimental multigrain bread due to storage at 30 o C Day N Subset 1 2 3 4 12 6.7000 2 12 7.6000 0 12 7.8125 Sig. 1.000 1.000 1.000 The statistical analysis of the OA scores also reflected a significant decrease in score in both control and experimental samples (F value=46.397 and 107.858, Tables 4.101 and 4.103). The variations in sensory characteristics of control and experimental samples due to storage at 30 O C are graphically presented in figures 4.30 and 4.31. Sensory Scores 9 8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 Variation of sensory characteristics of control multigrain bread on storage at 30 o C Colour and Appearance Body and Texture Flavour Overall acceptability Sensory Attributes Day 0 Day 2 Day 4 Figure 4.30 Effect on sensory attributes of control multigrain bread due to storage at 30 o C temperature Page 144

4. Results and Discussion Sensory Scores 9 8 7 6 5 4 3 2 1 0 Variation of sensory characteristics of whey incorporated multigrain bread on storage at 30 o C Colour and Appearance Body and Texture Flavour Overall acceptability Sensory Attributes day 0 day 2 Day 4 Figure 4.31 Effect on sensory attributes of whey incorporated multigrain bread due to storage at 30 o C temperature The sensory score of bread samples stored at 5 o C is presented in table 4.105 and the statistical analysis of the data is presented in tables 4.106 to 4.119. The graphical presentation is given in tables 4.32 and 4.33. Table 4.105. Effect on sensory attributes of multigrain bread due to storage at 5 o C Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 C S C S C S C S C S C S C S C &A B& T 7.63 a ±.4 5 7.58 d ±.4 5 8.16 A ±.37 7.57 C ±.3 2 7.63 a ±.13 7.37 c,d ±.25 7.87 A ±.37 7.37 B,C ±. 27 7.53 a ±.49 7 b,c,d ±.42 7.75 A ±.42 7.13 A,B,C ±.32 7.55 a ±.42 6.83 a,b,c,d ±.42 7.62 A ±.41 7.05 A,B,C ±.35 7.35 a ±.55 6.63 a,b,c ±.42 Fl 7.55 7.68 7.3 c 7.38 7.07 7.05 6.87 6.65 6.6 c ±.5 E D,E,c C,D ±.4 ±.36 b,c ±.3 B,C b,c ±. 2 5 ±.19 ±.4 ±.3 6 35 ±.22 OA 7.28 7.6 E 7.22 7.37 6.98 7.08 6.75 b 6.65 6.5 a, c ± ± c ± E ± b,c ± D,E ±.,c ±.4 C,D ±. b,c ±..68.32.61.25.46 33 8 33 42 4 * small superscripts denote variations in control and capital denotes variation in sample. 7.43 A ±.51 6.95 A,B ±.32 6.4 B ±.2 6.32 B,C ±. 7.33 a ±.62 6.42 a,b ±.36 6.12 a,b. ±.31 6.1 a,b ±.23 7.3 A ±.5 8 6.8 1 A,B ±.2 5 6.0 B ±. 38 5.9 8 A,B ±.4 7.2 7 a ±.7 6 6.1 8 a ±. 37 5.4 7 a ±. 29 5.6 7 a ±. 33 7.2 2 A ±.6 6 6.5 8 A ±.2 9 5.4 A ±. 25 5.4 5 A ±.28 **scores on 9 point hedonic scale Page 145

4. Results and Discussion It is observed from table 4.105 that shelf life of bread increased upto 10 days when stored at 5 o C. As observed during storage at 30 o C, the deterioration was minimum with respect to colour and appearance but significant; quite marked with respect to body and texture and significant.the decrease in flavour score was steep and significant. Consequently the OA score also decreased significantly with increase in storage period. The OA score of control sample was observed to decrease from 7.28±0.68 on the day of production to 7.22±0.6, 6.98±0.46, 6.75±0.48, 6.5±0.42, 6.1±0.23 and 5.67±0.33 on days 2, 4, 6, 8, 10 and 12 respectively. For experimental samples the scores for the corresponding periods were 7.6±0.32, 7.37±0.25, 7.08±0.33, 6.65±0.33, 6.32±0.4, 5.98±0.4 and 5.45±0.28. Based on the flavour and OA scores it can be inferred that the breads can be stored at 5 o C and used upto 10 days period since the critical score for a good and acceptable product is considered to be 6 (Kulkarni et al., 1990). The statistical analysis of the data is presented in tables 4.106 to 4.119. Table 4.106. ANOVA: Effect on C & A scores of control multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups.829 6.138.391.880 Within Groups 12.377 35.354 Total 13.206 41 Table 4.107. ANOVA: Effect on C & A scores of experimental multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 4.059 6.677 2.389.049 Within Groups 9.912 35.283 Total 13.971 41 Page 146

4. Results and Discussion Table 4.108. ANOVA: Effect on B & T scores of control multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 9.036 6 1.506 8.255 *.000 Within Groups 6.385 35.182 Total 15.421 41 * Significant p<0.05 Table 4.109. TUKEY: Effect on B & T scores of control multigrain bread due to storage at 5 o C Day N Subset 1 2 3 4 12 6 6.1833 10 6 6.4167 6.4167 8 6 6.6333 6.6333 6.6333 6 6 6.8333 6.8333 6.8333 6.8333 4 6 7.0000 7.0000 7.0000 2 6 7.3667 7.3667 0 6 7.5833 Sig..146.243.071.061 Table 4.110. ANOVA: Effect on B & T scores of experimental multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 3.927 6.654 5.863 *.000 Within Groups 3.907 35.112 Total 7.833 41 * Significant p<0.05 Page 147

4. Results and Discussion Table 4.111. TUKEY: Effect on B & T scores of experimental multigrain bread due to storage at 5 o C Day N Subset 1 2 3 12 6 6.5833 10 6 6.8167 6.8167 8 6 6.9500 6.9500 6 6 7.0500 7.0500 7.0500 4 6 7.1333 7.1333 7.1333 2 6 7.3667 7.3667 0 6 7.5667 Sig..093.093.134 Table 4.112. ANOVA: Effect on Flavour scores of control multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 16.959 6 2.827 12.018 *.000 Within Groups 8.232 35.235 Total 25.191 41 * Significant p<0.05 Table 4.113. TUKEY: Effect on Flavour scores of control multigrain bread due to storage at 5 o C Day N Subset 1 2 3 12 6 5.4667 10 6 6.1167 6.1167 8 6 6.6000 6.6000 6 6 6.8667 6.8667 4 6 7.0667 2 6 7.1833 0 6 7.4500 Sig..263.134.062 Page 148

4. Results and Discussion Table 4.114. ANOVA: Effect on Flavour scores of experimental multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 22.249 6 3.708 30.975 *.000 Within Groups 4.190 35.120 Total 26.439 41 * Significant p<0.05 Table 4.115. TUKEY: Effect on Flavour scores of experimental multigrain bread due to storage at 5 o C Day N Subset 1 2 3 4 5 12 6 5.4167 10 6 6.0500 8 6 6.4000 6 6 6.6500 6.6500 4 6 7.0500 7.0500 2 6 7.3833 7.3833 0 6 7.6833 Sig. 1.000.066.431.640.742 Table 4.116. ANOVA: Effect on OA scores of control multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig. Between Groups 12.810 6 2.135 7.677 *.000 Within Groups 9.733 35.278 Total 22.543 41 * Significant p<0.05 Page 149

4. Results and Discussion Table 4.117. TUKEY: Effect on OA scores of control multigrain bread due to storage at 5 o C Day N Subset 1 2 3 12 6 5.6667 10 6 6.1000 6.1000 8 6 6.5000 6.5000 6.5000 6 6 6.7500 6.7500 4 6 6.9833 6.9833 2 6 7.2167 0 6 7.2833 Sig..119.084.166 Table 4.118. ANOVA: Effect on OA scores of experimental multigrain bread due to storage at 5 o C Sum of Squares Df Mean Square F Sig Between Groups 18.83543 6 3.139238 30.39373 *.000 Within Groups 2.892 28 0.103286 Total 21.72743 34 * Significant p<0.05 Table 4.119. TUKEY: Effect on OA scores of experimental multigrain bread due to storage at 5 o C Day N Subset 1 2 3 4 5 12 6 5.4500 10 6 5.9833 5.9833 8 6 6.3167 6.3167 6 6 6.6500 6.6500 4 6 7.0833 7.0833 2 6 7.3667 0 6 7.6000 Sig..182.697.697.403.211 Page 150

4. Results and Discussion Variation of sensory characteristics of control multigrain bread on storage at 5 o C Sensory scores 9 8 7 6 5 4 3 2 1 0 Colour and Appearance Body and Texture Flavour Overall acceptability Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Sensory attributes Figure 4.32 Effect on sensory attributes of control multigrain bread due to storage at 5 o C Variation of sensory characteristics of whey incorporated multigrain bread on storage at 5 o C Sensory Scores 9 8 7 6 5 4 3 2 1 0 Day 0 Day 2 Day 4 day 6 Day 8 Day 10 Colour and Appearance Body and Texture Flavour Overall acceptability Sensory attributes Day 12 Figure 4.33 Effect on sensory attributes of control multigrain bread due to storage at 5 o C 4.6.2 Effect on textural parameters. The effect of storage on the textural characteristics of the breads stored at 30 o C and 5 o C and the relevant statistical analysis are presented in tables 4.120 to 4.136 and the relevant graphical presentations are presented in figures 4.34 to 4.39. Page 151

4. Results and Discussion Table 4.120. Effect on textural parameters of multigrain bread due to storage at 30 o C Parameter Control Sample Day 0 Day 2 Day 4 Day 0 Day 2 Day 4 Hardness,(N) 5.25 a 9.59 b 21.93 c 5.45 A 12.72 B 41.74 C Cohesiveness 0.43 c 0.33 b 0.26 a 0.58 C 0.43 B 0.40 A Springiness 0.67 c 0.56 b 0.49 a 0.72 C 0.59 B 0.41 A SRT, (sec) 27.49 a 31.7 a 77.93 b 34.57 A 51.45 B 85.75 C * small superscripts denote variations in control and capital denotes variation in sample. It is observed from table 4.120 that the hardness value of bread in both the control and experimental samples increased with increase in storage period. The hardness value in control and experimental samples were 5.25 and 5.45 N respectively on the day of production. The hardness in both the varieties increased on day 2 and 4 when stored at room temperature (Table 4.120). The variations in hardness were observed to be statistically significant (Tables 4.121-4.124). Table 4.121. ANOVA: Effect of storage at 30 o C on the hardness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups 449.590 2 224.795 271.170 *.000 Within Groups 4.974 6.829 Total 454.564 8 * Significant p<0.05 Table 4.122. TUKEY: Effect of storage at 30 o C on the hardness of control bread. Day N Subset for alpha =.05 1 2 3 0 3 5.25033 2 3 9.59000 4 3 21.93467 Sig. 1.000 1.000 1.000 Page 152

4. Results and Discussion Table 4.123. ANOVA: Effect of storage at 30 o C on the hardness of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups 2211.702 2 1105.851 1610.417 *.000 Within Groups 4.120 6.687 Total 2215.822 8 * Significant p<0.05 Table 4.124. TUKEY: Effect of storage at 30 o C on the hardness of experimental bread. Day N Subset for alpha =.05 1 2 3 0 3 5.44767 2 3 12.71800 4 3 41.73567 Sig. 1.000 1.000 1.000 Hardness, N 50 40 30 20 10 0 Changes in hardness of multigrain bread due storage at 30 o C day0 day 2 day 4 storage days control sample Figure 4.34. Effect of storage at 30 o C on the hardness of bread. The cohesiveness of bread samples was observed to decrease with increase in storage period in both the control and experimental samples. The cohesiveness in control was 0.43 on the day of production and this decreased to 0.33 and 0.26 on Page 153

4. Results and Discussion day 2 and 4 respectively. In case of experimental samples the value decreased from 0.58 on the day of production to 0.43 and 0.40 on day 2 and 4 respectively. The differences were observed to be statistically significant (Tables 4.125 to 4.128). Table 4.125. ANOVA: Effect of storage at 30 o C on the cohesiveness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups.046 2.023 25.901 *.001 Within Groups.005 6.001 Total.052 8 * Significant p<0.05 Table 4.126. TUKEY: Effect of storage at 30 o C on the cohesiveness of control bread. Day N Subset for alpha =.05 1 2 3 4 3.25600 2 3.33367 0 3.43167 Sig. 1.000 1.000 1.000 Table 4.127. ANOVA: Effect of storage at 30 o C on the cohesiveness of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups.058 2.029 235.616 *.000 Within Groups.001 6.000 Total.059 8 * Significant p<0.05 Page 154

4. Results and Discussion Table 4.128. TUKEY: Effect of storage at 30 o C on the cohesiveness of experimental bread. Day N Subset for alpha =.05 1 2 3 4 3.39500 2 3.43400 0 3.58167 Sig. 1.000 1.000 1.000 The springiness is an important characteristic in case of bread. The springiness values were observed to decrease with increase in storage period at 30 o C in both control and experimental samples. The springiness for control and experimental samples on the day of production was 0.67 and 0.72 respectively and these decreased to 0.49 and 0.41 on the 4 th day of storage at 30 o C. The decrease was observed to be statistically significant (Tables 4.129 to 4.132). Table 4.129. ANOVA: Effect of storage at 30 o C on the springiness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups.050 2.025 32.612 *.001 Within Groups.005 6.001 Total.054 8 * Significant p<0.05 Table 4.130. TUKEY: Effect of storage at 30 o C on the springiness of control bread. N Subset for alpha =.05 Day 1 2 3 4 3.48500 2 3.55767 0 3.66567 Sig. 1.000 1.000 1.000 Page 155

4. Results and Discussion Table 4.131. ANOVA: Effect of storage at 30 o C on the springiness of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups.145 2.073 412.439 *.000 Within Groups.001 6.000 Total.146 8 * Significant p<0.05 Table 4.132. TUKEY: Effect of storage at 30 o C on the springiness of experimental bread. N Subset for alpha =.05 Day 1 2 3 4 3.41200 2 3.59067 0 3.72200 Sig. 1.000 1.000 1.000 0.8 Changes in cohesiveness and springiness of multigrain bread due storage at 30 o C TPA characetristics 0.7 0.6 0.5 0.4 0.3 0.2 day0 day 2 day 4 Storage days cohesiveness control multigrain bread cohesiveness sample multigrain bread springiness control multigrain bread springiness sample multigrain bread Figure 4.35. Effect of storage at 30 o C on the cohesiveness and springiness of bread. Page 156

4. Results and Discussion The SRT was also observed to increase with increase in storage period at 30 o C. The SRT on the day of production was 27.49 and 34.57 for control and experimental samples respectively and these values increased gradually to 31.7 and 77.93 on day 2 and 4 for control samples. The SRT values for experimental bread samples were 51.45 and 85.75 respectively on the 2 nd and 4 th days of storage at 30 o C. The variations were observed to be statistically significant (Table 4.133 to 4.136 ). Table 4.133. ANOVA: Effect of storage at 30 o C on the SRT of control bread. Sum of Squares Df Mean Square F Sig. Between Groups 4702.059 2 2351.029 267.980 *.000 Within Groups 52.639 6 8.773 Total 4754.697 8 * Significant p<0.05 Table 4.134. TUKEY: Effect of storage at 30 o C on the SRT of control bread. Day N Subset for alpha =.05 1 2 0 3 27.49433 2 3 31.66033 4 3 77.93033 Sig..272 1.000 Table 4.135. ANOVA: Effect of storage at 30 o C on the SRT of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups 4079.965 2 2039.983 293.344 *.000 Within Groups 41.725 6 6.954 Total 4121.691 8 * Significant p<0.05 Page 157

4. Results and Discussion Table 4.136. TUKEY: Effect of storage at 30 o C on the SRT of experimental bread N Subset for alpha =.05 Day 1 2 3 0 3 34.57933 2 3 51.45433 4 3 85.75333 Sig. 1.000 1.000 1.000 SRT,s 90 80 70 60 50 40 30 20 Changes in SRT due to storage at 30 o C day0 day 2 day 4 storage days control sample Figure 4.36. Effect of storage at 30 o C on the Stress Relaxation Time (SRT) of bread. Textural changes in bread stored at 5 O C: The changes occurring in the textural parameters of bread during storage at 5 o C were evaluated and the results are presented in table 4.137. The statistical analysis is presented in tables 4.138 to 4.153 and the graphical presentation is depicted in figures 4.37 to 4.39. Page 158

4. Results and Discussion Table 4.137 Effect on textural parameters of multigrain bread due to storage at 5 o C Control multigrain bread Whey incorporated multigrain bread Day Day Day Day Day Day Day Day Day Day Day Day Day Day 0 2 4 6 8 10 12 0 2 4 6 8 10 12 H, N 6.27 12.2 17.5 39 c 45 c 59.6 70.8 16.4 25.4 31.2 57.7 63.5 E 79.9 86.9 a a, b b d e A B C D F G Co 0.65 0.61 0.59 0.48 c 0.4 b 0.32 0.29 0.47 0.36 0.3 B 0.3 B 0.29 0.28 0.21 e d,e d a a D C B B A Sp 0.89 0.84 0.79 0.61 c 0.60 0.49 0.41 0.8 D 0.74 0.7 C 0.55 0.53 B 0.44 0.4 A e d,e d c b a C B A SRT, 14.5 16.8 17.3 18.7 a, 23.6 25.7 27.4 15.5 15.6 19.3 23.1 24.9 27.4 29.3 sec a a,b a,b b b,c c c A A A,B B,C C,D C,D D * small superscripts denote variations in control and capital denotes variation in sample. The results revealed that the hardness value of bread stored at 5 O C increased with increase in storage period in both control and experimental samples which is similar to the results observed in case of the bread stored at 30 O C. The trend in change was observed to be similar in both control and experimental samples (Figure 4.37). The change in hardness value was observed to be statistically significant in most of the cases (Tables 4.138-4.141). Table 4.138. ANOVA: Effect of storage at 5 o C on the hardness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups 10956.595 6 1826.099 314.632 *.000 Within Groups 81.255 14 5.804 Total 11037.850 20 * Significant p<0.05 Page 159

4. Results and Discussion Table 4.139. TUKEY: Effect of storage at 5 o C on the hardness of control bread N Subset for alpha =.05 Day 1 2 3 4 5 0 3 6.26667 2 3 12.16000 12.16000 4 3 17.53333 6 3 39.00333 8 3 45.03117 10 3 59.62133 12 3 70.79333 Sig..104.161.092 1.000 1.000 Table 4.140. ANOVA: Effect of storage at 5 o C on the hardness of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups 13719.540 6 2286.590 580.922 *.000 Within Groups 55.106 14 3.936 Total 13774.646 20 * Significant p<0.05 Table 4.141. TUKEY: Effect of storage at 5 o C on the hardness of experimental bread. Day N Subset for alpha =.05 1 2 3 4 5 6 7 0 3 16.4126 2 3 25.3666 4 3 31.1585 6 3 57.658 8 3 63.54 10 3 79.89 12 3 86.944 Sig. 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Page 160

4. Results and Discussion Changes in hardness of multigrain bread due storage at 5 o C 100 Hardness, N 80 60 40 20 Hardness of control Hardness of sample 0 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Storage Days Figure 4.37. Effect of storage at 5 o C on the hardness of bread. The cohesiveness was observed to decrease with increase in storage period in both control and experimental samples. Similar decrease was observed for bread samples stored at 30 o C. The trend of decrease was similar in both the control and experimental samples. The change in cohesiveness was marked and significant between the day of production and the 12 th day of storage in both the control and experimental samples. In case of control samples the decline in cohesiveness was gradual with increase in storage period, while the decrease was marked between day of production and the 4 th day of storage in case of experimental samples. The change in cohesiveness was observed to be non-significant between 4 th and 10 th of day of storage, but significant between 10 th and 12 th day of storage (Tables 4.142 to 4.145 ). Table 4.142. ANOVA: Effect of storage at 5 o C on the cohesiveness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups.372 6.062 171.116 *.000 Within Groups.005 14.000 Total.377 20 * Significant p<0.05 Page 161

4. Results and Discussion Table 4.143. TUKEY: Effect of storage at 5 o C on the cohesiveness of control bread. N Subset for alpha =.05 Day 1 2 3 4 5 12 3.29267 10 3.32100 8 3.40133 6 3.48033 4 3.58767 2 3.61033.61033 0 3.64967 Sig..554 1.000 1.000.763.220 Table 4.144. ANOVA: Effect of storage at 5 o C on the cohesiveness of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups.117 6.020 131.828 *.000 Within Groups.002 14.000 Total.119 20 * Significant p<0.05 Table 4.145. TUKEY: Effect of storage at 5 o C on the cohesiveness of experimental bread. N Subset for alpha =.05 Day 1 2 3 4 12 3.21000 10 3.27667 8 3.29033 6 3.29967 4 3.30167 2 3.36033 0 3.46817 Sig. 1.000.225 1.000 1.000 Page 162

4. Results and Discussion The springiness values of bread were observed to decrease with storage period in both control and the experimental samples. The trend in change was similar to the changes observed when the bread was stored at 30 o C. The springiness of control on the day of production was 0.89 which gradually reduced to 0.42 on the 12 th day of storage. The decrease was significant (Tables 4.146 to 4.149). Similarly in the case of experimental samples the springiness value was observed to decrease from 0.8 on the day of production to 0.4 on the 12 th day of storage which was statistically significant. The changes have been presented in table 4.148 and in figure 4.38. Table 4.146. ANOVA: Effect of storage at 5 o C on the springiness of control bread. Sum of Squares Df Mean Square F Sig. Between Groups.587 6.098 115.635 *.000 Within Groups.012 14.001 Total.599 20 * Significant p<0.05 Table 4.147. TUKEY: Effect of storage at 5 o C on the springiness of control bread. Day N Subset for alpha =.05 1 2 3 4 12 3.4133 10 3.4900 8 3.5967 6 3.6067 4 3.7867 2 3.8367.8367 0 3.8877 Sig..070.999.400.379 Page 163

4. Results and Discussion Table 4.148. ANOVA: Effect of storage at 5oC on the springiness of experimental bread. Sum of Squares * Df Mean Square Between Groups.578 6.096 Within Groups.006 14.000 Total.584 20 F Sig. 231.665 *.000 Significant p<0.05 Table 4.149. TUKEY: Effect of storage at 5oC on the springiness of experimental bread. Day N Subset for alpha =.05 1 2 3 4 5 12 3 10 3 8 3.60733 6 3.61233 4 3.79333 2 3.83667 0 3 Sig..41867.49333.83667.89000 1.000 1.000 1.000.197.073 TPA characteristics Changes in cohesiveness and springiness of multigrain bread due storage at 5o C 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 Cohesiveness of control Cohesiveness of sample Springiness of control springiness of sample Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Storage days Figure 4.38. Effect of storage at 5oC on the cohesiveness and springiness of bread. Page 164

4. Results and Discussion The SRT was observed to increase with increase in storage period in both the control and the experimental samples. Similar results were observed in bread samples stored at 30 o C. The SRT for control samples increased from 14.5 seconds on the day of production to 16.8, 17.3, 18.7, 23.6, 25.7 and 27.4 seconds on 2 nd, 4 th,6 th,8 th,10 th and 12 th days of storage respectively. Similarly for the experimental bread under similar storage conditions the SRT was 15.5, 15.6, 19.3, 23.1, 24.9, 27.4 and 29.3 seconds respectively. The experimental bread samples stored at 30 o C also exhibited similar increase in SRT. The change observed in both the control and experimental samples were observed to be statistically significant (Tables 4.150 to 4.153). Table 4.150. ANOVA: Effect of storage at 5 o C on the SRT of control bread. Sum of Squares Df Mean Square F Sig. Between Groups 444.646 6 74.108 12.430.000 Within Groups 83.471 14 5.962 Total 528.117 20 * Significant p<0.05 Table 4.151. TUKEY: Effect of storage at 5 o C on the SRT of control bread. N Subset for alpha =.05 Day 1 2 3 0 3 14.45000 2 3 16.82900 16.82900 4 3 17.25767 17.25767 6 3 18.73333 18.73333 8 3 23.55633 23.55633 10 3 25.65533 12 3 27.47233 Sig..378.054.475. Page 165

4. Results and Discussion Table 4.152. ANOVA: Effect of storage at 5 o C on the SRT of experimental bread. Sum of Squares Df Mean Square F Sig. Between Groups 548.716 6 91.453 23.546.000 Within Groups 54.377 14 3.884 Total 603.093 20 * Significant p<0.05 Table 4.153. TUKEY: bread. Effect of storage at 5 o C on the SRT of experimental Days N Subset for alpha =.05 1 2 3 4 0 3 15.57833 2 3 15.60200 4 3 19.33933 19.33933 6 3 23.11167 23.11167 8 3 24.90667 24.90667 10 3 27.43733 27.43733 12 3 29.39410 Sig..293.290.172.146 35 Changes in SRT of multigrain bread due storage at 5 o C SRT, in sec 30 25 20 15 SRT control SRT sample 10 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Storage Days Figure 4.39. Effect of storage at 5 o C on the Stress Relaxation Time (SRT) of bread. Page 166

4. Results and Discussion The results of the objective tests reflected that with increase in storage period the softness of bread decreased in both the control and experimental samples. This was reflected by increase in hardness value and stress relaxation time(srt). Staling of bread during storage is well known (Seiler, 1984) during which changes take place in the structural elements of the bread foam. The decrease in cohesiveness and springiness values indicated that the binding network of bread was negatively affected due to the storage of bread. Similar observations have been reported earlier by Divya and Rao (2010) in bread, Poonam (2007) in buns and Kumar (2010) in chapatis. The change can be ascribed to the changes in carbohydrate structure occurring during the storage of bread. The changes are reflected both in values assessed by objective tests and also through sensory evaluation. 4.6.3 Changes in physico-chemical properties during storage The physico-chemical properties are important characteristics to assess the quality of bread. The changes in physico-chemical properties will bring about variations in sensory evaluation mainly with respect to flavour and body and texture characteristics. Kulkarni et al.(2001) reported that changes in sensory characteristics of foods during storage has a direct relation to physico-chemical and microbial changes during storage. During storage period the changes in major physico-chemical characteristics were monitored at regular intervals in both the control and experimental samples stored at 30 and 5 o C. The results are presented in tables 4.154 to 4.159 and are also graphically represented in figures 4.40 to 4.44. 4.6.3.1 Effect of storage on Water activity (a w ) It is observed from table 4.154 that the water activity increased gradually in both the control and the experimental samples with increase in storage period. The a w for control samples on the day of production was 0.847 and it increased gradually to 0.862 and 0.878 on the 2 nd and 4 th day respectively. The a w for experimental samples was 0.806, 0.809 and 0.819 on the day of production, 2 nd and 4 th day respectively. At 5 O C the a w for both the control and experimental bread Page 167

4. Results and Discussion decreased upto the 4th day of storage and then increased upto the 12th day of storage. The increase was similar to the values of samples stored at 30OC. However, the magnitude of change was lower in both control and experimental samples in comparison to the samples stored at 30OC. The aw was 0.847 on the day of production for control sample and it declined upto 0.773 on the 4th day and then increased gradually to 0.831 on the 12th day. Similarly, the aw was 0.806 on the day of production for experimental sample and decreased to 0.721 on the 4th day of storage and thereafter gradually increased to 0.778 on the 12th day of storage. Table 4.154 Change in water activity (aw) of multigrain bread during storage Treatment 30oC 5 oc Days Days 0 2 4 0 Control 0.847 0.862 0.878 0.847 15% TS PW 0.806 0.809 0.819 0.806 2 4 6 8 10 12 0.777 0.773 0.781 0.796 0.822 0.831 0.727 0.721 0.73 0.753 0.775 0.778 water activity Change in water activity of bread samples during storage 0.9 0.88 0.86 0.84 0.82 0.8 0.78 0.76 0.74 0.72 0.7 control 30 deg C. sample 30 deg C control 5 deg C sample 5 deg C day 0 day 2 day 4 day 6 day 8 day 10 day 12 water activity Figure 4.40 Effect of storage on the water activity of bread Page 168

4. Results and Discussion 4.6.3.2 Effect of storage on the crust colour of bread. Reflectance is a measure of colour of bread and was also observed to decrease in both the control and experimental samples at both the temperatures of storage (Table 4.155). Table 4.155. Change in crust colour (reflectance %) of multigrain bread during storage 30 o C 5 o C Treatment Days Days 0 2 4 0 2 4 6 8 10 12 Control 54.33 50.5 50.17 55.23 52.17 47 46.33 43 42.67 42.17 15% TS PW 52.17 41.67 41 51.76 43.33 42.83 40.33 40.67 39 37.67 The reflectance value on the day of production was 54.33% for control sample while the same was 52.17% for the experimental sample. The reflectance of control sample decreased gradually to 50.5% on the 2 nd day and 50.1% on the 4 th day of storage when stored at 30 O C. Similarly the values decreased from 52.17% on the day of production to 41.67 and 41.0% on the 2 nd and 4 th day of storage respectively in the case of experimental sample. At 5 O C storage the reflectance values decreased gradually from 55.23% on the day of production to 42.17% on the 12 th day of storage in case of control samples. In the case of experimental samples the values decreased from 51.76% on the day of production to 37.67% on the 12 th day of storage. Page 169

4. Results and Discussion Changes in crust colour of bread during storage 60 % reflectance 55 control 30 deg C 50 sample 30 deg C 45 control 5 deg C 40 sample 5 deg C 35 day 0 day 2 day 4 day 6 day 8 day 10 day 12 Storage days Figure 4.41 Effect of storage on the reflectance(%) of bread 4.6.3.3 Effect of storage on the TS content of the bread The variation in TS content due to moisture evaporation during storage is presented in tables 4.156 and 4.157 and figure 4.42 and 4.43. Table 4.156. Change in solids content of the bread during storage Treatment 30oC 5 oc Days Days 0 2 4 0 Control 71.83 73.07 73.98 71.87 15% TS 73.79 74.57 75.36 73.94 2 4 6 8 10 12 73.41 74.85 75.93 77.48 79.47 81.07 75.68 76.69 78.47 79.68 81.38 82.18 PW Page 170

4. Results and Discussion Table 4.157. Change in moisture content of the bread during storage Treatment 30oC 5 oc Days Days 0 2 4 0 Control 28.17 26.93 26.02 28.13 15% TS 26.21 25.43 24.64 26.06 2 4 6 8 10 12 26.59 25.15 24.07 22.52 20.53 18.93 24.32 23.31 21.53 20.32 18.62 17.82 PW It is observed from table 4.156 and 4.157 that at both 30 and 5OC and in both the control and the experimental samples the moisture content decreased gradually with concurrent increase in the TS content. The moisture content of control samples stored at 30OC was 28.17% on the day of production and decreased to 26.02% on the 4th day of storage. For similar storage conditions the moisture content decreased from 26.21 to 24.64% in the case of experimental samples. 84 Change in solids content of the bread during storage 82 %TS 80 control 30 deg C 78 sample 30 deg C 76 74 control 5 deg C 72 sample 5 deg C 70 Day 0 Day 2 Day 4 Day 6 Day 8 Storage days Day 10 Day 12 Figure 4.42 Effect of storage on the %TS of bread In the case of samples stored at 5OC the variation was marked over a period of 12 days of storage. In control and experimental samples the moisture content on the day of production was 28.13 and 26.06% respectively. During storage the Page 171

4. Results and Discussion moisture decreased from the 2nd day of storage in both samples and on the 12th day the moisture content was 18.93 and 17.82 % respectively. The decrease in moisture content was gradual throughout the storage period. The total moisture reduction in case of control samples was 9.20% and in the case of experimental samples the drop was 8.24%. The gradual decrease in moisture content is presented graphically in figure 4.43. Change in moisture content of the bread during storage 29 % moisture 27 control 30 deg C 25 sample 30 deg C 23 21 control 5 deg C 19 sample 5 deg C 17 Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 storage days Figure 4.43. Effect of storage on the %moisture of bread 4.6.3.4 Effect of storage on ph of bread The ph of bread was also observed to decrease with increase in storage period at both 30 and 5oC and the results are presented in table 4.158 and graphical representation is given in figure 4.44. It is observed from table 4.158 that ph reduced gradually in both control and experimental samples stored at 30 and 5OC. The ph of control sample on the day of production was 5.23 and reduced to 5.18 and 5.11 at the end of the 2nd and 4th day of storage at 30OC. Under similar conditions of storage, the ph of experimental samples reduced gradually to 5.13 and 5.05. In the control samples stored at 5OC, the ph reduced gradually from 5.21 on the day of production to 5.19, 5.16, 5.11, 5.03 and 4.93 on 2nd, 4th, 6th, 8th, 10th and 12th day of storage respectively. The Page 172

4. Results and Discussion decrease in ph was steeper than the decrease observed at 30OC storage. Under similar conditions of storage the decrease in ph of experimental samples was also observed to be gradual and the value reduced from 5.16 on the day of production to 5.14, 5.07, 5.01, 4.91, 4.78 and 4.71 on the 2nd, 4th, 6th, 8th, 10th and 12th day of storage respectively. The decrease in ph was observed to be steeper than the change observed when the samples were stored at 30OC. Table 4.158. Change in ph of bread during storage Treatment 30oC 5 oc Days Days 0 2 4 0 Control 5.23 5.18 5.11 5.21 15% TS PW 5.18 5.13 5.05 5.16 5.3 2 4 6 8 10 12 5.19 5.16 5.11 5.03 4.93 4.81 5.14 5.07 5.01 4.91 4.78 4.71 Changes in ph of multigrain bread during storage 5.2 control 30 deg C sample 30 deg C control 5 deg C sample 5 deg C ph 5.1 5 4.9 4.8 4.7 4.6 day 0 day 2 day 4 day 6 day 8 day 10 day 12 storage days Figure 4.44 Effect of storage on the ph of bread 4.6.4 Effect of storage on the Yeast and Mold count of bread. The spoilage of bread generally occurs with the growth of visible yeasts and molds (Srilakshmi, 2003). Hence the Yeast and Mold (Y&M) count of control and Page 173

4. Results and Discussion experimental samples were monitored during storage period. The results are tabulated in table 4.159 and graphically represented in figure 4.45 It is observed that at both the temperatures of storage the yeast and mold count was observed to increase gradually with storage period. At 30 O C storage the increase in control sample was marked with the count increasing from 10 on the day of production to 200 on the second day of storage and 900 on the 4 th day of storage. However at 5 O C the increase in count was slow and gradual as detailed in table 4.159. On the 12 th day of storage the count reached 150 in the case of control samples. In the case of experimental samples the count increased from 10 to 240 at the end of 4 days at 30 O C storage while the count reached 130 at the end of 12 days at 5 O C storage. It was noticed that for the products stored at 5 0 C the yeast and mold count was far less than those stored at 30 0 C, which is due to the reason that yeast grows well at higher temperatures (> 20 0 C) and the growth is slow at lower temperatures, there being no activity at 4 0 C (Collado, 2003). The breads at 5 o C are stored well upto 12 days without much deterioration. Table 4.159. Change in Yeast and Mold count of bread during storage Treatment 30 o C 5 o C Days Days 0 2 4 0 2 4 6 8 10 12 Control 10 200 900 10 10 30 40 60 100 150 (Cfu/g) 15% TS PW (Cfu/g) 10 40 240 0 10 10 20 30 70 130 Page 174

4. Results and Discussion YEAST AND MOULD (cfu/g) Changes in Yeast & Mold count of bread during storage 1000 800 Control 30 deg C 600 Sample30 deg C 400 Control 5 deg C 200 Sample5 deg C 0 Day 0 Day 2 Day 4 Day 6 Day 8 Storage days Day 10 Day 12 Figure 4.45 Effect of storage under room temperature on the Yeast and Mould count of bread Thus it is observed that the quality of bread gets affected with increase in storage period. Similar results have been earlier reported by Divya (2007), Jayalakshmi (2010) and Poonam (2007). The changes could be ascribed mainly to the loss of moisture during storage at both the temperatures of storage. The loss of moisture will make the bread dry with increase in storage period and due to this the hardness value increases and these results are in tune with the earlier observations made by Divya and Rao (2010). As the hardness value increases the cohesiveness and springiness decrease concurrently which could be ascribed to the weak structure network due to the moisture evaporation. The springiness is an indicator of the softness of bread and generally it decreases with storage period. Hence it is a common practice to feel the softness of bread to assess its freshness. Now the sensual routine test is corroborated with the decrease in springiness. The decrease in springiness has also been reported earlier by Divya and Rao (2010). The increase in SRT is due to the slow recovery which could be due to loss of moisture with concurrent decrease in softness (Divya, 2007). The increase in water activity of bread during storage may be ascribed to the increase in availability of free water during storage period. Page 175

4. Results and Discussion The darkening of crust colour as reflected by the reflectance values at both temperatures of storage could be ascribed to the increase in TS content during storage due to the loss of moisture. These results are in agreement with the earlier observations of Swapna (2013). The change in ph during storage is similar to the earlier observations (Poonam, 2007) and could be ascribed to the increase in TS content due to the moisture evaporation during storage. The spoilage of bread is caused predominantly through the increase in yeast and mold count. In the present study also it was observed in case of control and experimental samples that the first sign of spoilage occurred through visible yeast and mold count as explained in preceding paragraphs. The increase in Y&M count has also been observed earlier by Divya and Rao (2010), Jayalakshmi (2010). Thus based on the above results it was inferred that the experimental samples had significantly higher shelf life in comparison to control under similar storage conditions which could be due to increased solids content and also due to the increased Maillard reaction during the course of baking. This increase in shelf life of bread added with whey was also observed by other workers. (Cocup and Sanderson, 1987; Yousif et al., 1998). Thus, the results of the study indicated that it is possible to use concentrated whey in the production of multigrain bread. The use of whey resulted in increased yield, better shelf life and nutrition besides reducing load on effluent treatment. Page 176

Chapter- 5 Summary and Conclusion

Summary and conclusion Whey is one of the most common byproducts of the dairy industry. It is a storehouse of valuable milk solids, 40% of milk solids being contained in it. In India paneer whey forms one of the major by-products. The paneer whey is rich in minerals and lactose as compared to cheese whey. Whey also is a rich storehouse of proteins containing all the essential amino acids. Whey solids are in fact considered as a treasure of nutritional and functional ingredients and are used beneficially in various areas of dietetics, sports foods, baby foods, specialty foods, etc. Whey, with its high solids content has a high Biological Oxygen Demand (BOD). So disposal of whey is a serious problem causing environmental pollution. On the other hand treatment of whey before disposal is a costly affair. So, whey can be used up as an ingredient for formulation of products of mass consumption such as bread, cutting out the costs of its treatment as well as increasing the functionality and the nutritional quality of the product. Bread is one of the staple food items of the world population. It is an item of mass consumption and hence it was targeted as a product in which whey can be utilized in. Moreover, bread is a rich source of carbohydrates and fibres. As modern day consumers are becoming more and more health conscious multigrain bread is growing in popularity. They are more inclined towards one product which offers numerous health benefits because of the busy nature of the world. Multigrain bread is one such product that offers numerous benefits from a single product, the benefits being derived from variety of grains present in the bread. The quality of nutrients from different sources is much better than the quality of nutrients from a single source. A rising trend has been observed in the consumption of multigrain bread in recent years and hence it has been targeted as a product in which whey can be utilized to enhance its nutritional benefits. Multigrain bread can be made by using different kinds of cereals and pseudocereals using wheat flour as the main base material. All these cereals and the pseudo-cereals possess their own nutritional benefits. In this present project we have chosen oats, flaxseed, maize and sorghum (in flour form) based on their health benefits to supplement wheat flour. The formulation of bread flour consisted of 80% Page 177

Summary and conclusion wheat flour supplemented by 5% each of oat, flaxseed, maize and sorghum flour. Based on the flour mixture 10% of sugar, 2% of salt, 15% of oil, 3% of yeast and 60% of water were added to make dough which was finally baked into bread. Paneer whey was concentrated in a single effect calandria in the experimental dairy of National Dairy Research Institute, Bangalore. Paneer whey was analyzed prior to concentrating it in the evaporator. The paneer whey obtained as a byproduct of paneer manufacture contained TS of 6.61±0.028. The fat and protein contents were 0.57±0.071 and 0.38±0.008 respectively. The ph of freshly prepared paneer whey ranged between 5.83 and 5.87. Paneer whey was concentrated at three levels for utilization in preparation of multigrain bread. The level at which paneer whey was concentrated for incorporation into multigrain bread were 15%, 20% and 25% TS. The doughs made by the incorporation of the concentrated whey were baked at 160, 185 and 210 o C for 30 minutes. The sensory evaluation of experimental samples indicated that a combination of 15%TS whey used as diluent with a baking temperature of 185 o C resulted in most acceptable product based on flavour, B&T and OA scores. The scores obtained for multigrain bread by incorporation of 15% whey solids for B&T, flavour and OA were 7.41±0.5, 7.56±0.27 and 7.47±0.51 respectively. The scores for the same attributes decreased progressively with further increase in solids level of concentrated whey. The effect of incorporation of concentrated whey having different solids level in the dough was studied and it was observed that proofing rates decreased progressively with increase in level of concentration of whey. The rate of proofing of control multigrain bread dough was 2.144. The rate of proofing of multigrain bread dough with incorporation of 15, 20 and 25% TS of whey were 1.833, 1.711 and 0.655 respectively as obtained by slope values upto the linear region. It was found that hardness of control multigrain bread dough (6.26±3.6) was significantly different from that of the dough made by incorporating concentrated whey of different TS. The hardness values progressively increased with increase in TS level of concentrated whey. The cohesiveness value was highest for the dough of control multigrain bread (0.76±0.06) whereas the springiness values were almost Page 178

Summary and conclusion similar for all the four doughs. The stress relaxation time of control multigrain bread dough (12±0.48 sec) differed significantly from the doughs made by incorporating concentrated whey of different solids level. Stress relaxation time (SRT) increased progressively with increase in TS of concentrated whey. Creep retardation time(crt) also showed a similar trend but the CRT of dough containing concentrated whey of 25% TS (73.3±24.7 sec) was significantly different from the other three multigrain bread doughs. It was observed that all the four doughs differed significantly from one another with respect to compliance values. However compliances computed were 17.5±1.4, 15±0.96, 11.5±0.72 and 8.48±0.53 Pa for control, 15%,20% and 25% TS whey incorporated multigrain bread dough respectively, showing a trend opposite to that of creep retardation time and stress relaxation time. As the proofing rate of multigrain bread dough made by incorporation of 15% concentrated whey was slower than that of the control, trials were conducted to achieve a proofing rate almost similar to that of the control multigrain bread dough. The measures included 1) Enhancement of yeast level in the experimental sample from 3% to 6% and 2) Increase in proofing temperature from the normal 28 o C to 40 o C. Incorporation of 6% yeast in the concentrated whey incorporated multigrain bread dough revealed that the proofing rate increased considerably but was still lower than that of the control. The slope values calculated up to the linear region of proofing curves were 2.255, 1.477 and 1.755 for control, 15%TS concentrated whey incorporated multigrain bread dough with 3 and 6% yeast respectively. However, yeast level enhancement resulted in an adverse impact on the sensory characteristics of the multigrain bread. The OA scores for control multigrain bread, 15%TS concentrated whey incorporated multigrain bread with 3 and 6% yeast were 6.983±0.286, 7.613±0.126 and 5.691±0.492 respectively. Elevation in temperature of proofing (40 o C) of the multigrain bread dough made by using 15% whey solids resulted in a sharp increase in proofing rate, even higher than that of the control. The slope values obtained upto the linear region of Page 179

Summary and conclusion the proofing curve were 1.777, 1.544 and 2.311 for control multigrain bread dough, multigrain bread dough containing 15% whey solids proofed at 28 o C and 40 o C respectively. The optimized conditions for preparation of whey incorporated multigrain bread were- addition of concentrated whey having 15% TS to the dough to fully replace the dough water, proofing at 40 0 C and baking at a temperature of 185 o C for about 30 minutes. However, to further improve upon sensory and textural attributes of the optimized bread closer to that of normal wheat bread three chemical improvers were tried-calcium carbonate, calcium phosphate and ammonium persulphate, as permitted by Food Safety and Standard Authority of India. The calcium improvers resulted in significant increase in B&T characteristics of the multigrain bread having 15% whey solids. The OA scores were 7.06±.59, 7.32±.58, 7.61±.45, 7.71±.38 and 7.45±.26 for control, multigrain bread having 15% whey solids made without using improver, multigrain bread having 15% whey solids made using calcium carbonate, calcium phosphate and ammonium persulphate as improver respectively. The hardness values were observed to considerably reduce with the addition of calcium improvers. The chewiness and gumminess values of the optimized bread made by adding improvers were much less than that of the control multigrain bread and the whey incorporated multigrain bread made without addition of improvers. Based on the sensory scores it was concluded that calcium improvers helped in enhancing the sensory attributes of multigrain bread. Calcium phosphate was selected as the improver to be used in bread making because of its cost effectiveness. Calcium phosphate was added at different levels-(1500, 2000 and 2500 ppm) on flour basis in the multigrain bread dough and its effect on the sensory characteristics was studied. There was no significant difference in scores of any of the sensory attributes between the concentrated whey incorporated multigrain bread made by using different levels of calcium phosphate as improver. Hence it was decided to use the lowest level of calcium phosphate in bread formulation. Page 180

Summary and conclusion The procedure for preparation of multigrain bread by replacing dough water fully with concentrated whey having 15% TS was optimized. In the optimized process the proofing was carried out at 40 O C, yeast was incorporated at 3% and calcium phosphate was used as improver at the rate of 1500 ppm on flour basis. The dough was baked at 185 o C for about half an hour. Physico-chemical analysis of the experimental multigrain bread revealed that it had higher total solids content than that of the control. The solids content was 71.85±0.18 and 73.87±0.18 for control and the experimental multigrain bread respectively. The fat content of the experimental multigrain bread was also greater than that of the control. The ph of control and experimental samples were 5.21±0.05, 5.17 ± 0.03 while the water activity was 0.87 ± 0.001 for control and 0.84 ± 0.001 for experimental sample. The multigrain bread was stored under two conditions (30 o C and 5 o C) in LDPE pouches having thickness of 25 micron as measured by digital callipers. The sensory scores of both the control and experimental samples decreased with increase in storage period. The decrease in score was marked for the experimental sample. The OA scores decreased from 7.73±0.18 to 6.9±0.27 for control at the end of 4 days while for the experimental sample it decreased from 7.81±0.15 to 6.7±0.19 after 4 days of storage at 30 o C. At 5 O C, the sample could be kept upto 12 days after which it was found to be unacceptable. The sensory scores decreased progressively with increase in days of storage. The OA scores for control sample at 5 o C storage decreased from 7.28±0.68 to 5.67±0.33 and for experimental sample from 7.6±0.32 to 5.45±0.28 from the day of production to the 12 th day of storage. Hardness and SRT was found to increase with increase with storage period for both control and experimental samples stored at both 30 and 5 o C. The cohesiveness and springiness of all the samples was observed to decrease with increase in storage period. Page 181

Summary and conclusion Water activity of the samples increased during storage at 30 O C but the increase was less for the experimental sample in comparison to the control sample. The water activity increased from 0.847 to 0.878 and from 0.806 to 0.819 from the day of production to the 4 th day of storage for control and experimental samples respectively. At 5 o C storage the water activity for both the control and experimental samples was observed to decrease in the initial few days and then increase during subsequent storage period. The water activity of the control was more than that of the experimental sample at the end of 12 th day of storage. The crust of the bread became darker in colour with increase in storage period for both control and experimental samples under both the storage conditions as reflected by reflectance values. The reflectance values decreased progressively with increase in storage period indicating that the crust became browner with progress in storage period. The ph of both the multigrain bread samples decreased with increase in storage period indicating increased development of acidity. The experimental sample always showed lower ph value in comparison to control. Study of yeast and mold count revealed that the experimental sample had a higher microbial shelf life than that of control sample under similar conditions. The counts obtained for yeast and mold were 900 and 240 for the control and experimental samples respectively after storage for 4 days storage at 30 O C. At 5 O C, the two samples did not show any marked difference in yeast and mold counts. The counts at 5 o C storage at the end of 12 days were 150 and 130 for control and experimental samples respectively. Page 182

Summary and conclusion CONCLUSION: Paneer whey concentrated to 15% TS can effectively be used as a diluent, replacing water in the production of multigrain bread. In addition to improving the nutritional attributes of the bread, this replacement will also contribute to the economy of operation of dairy plants by reducing the cost of effluent treatment. The use of concentrated whey however, resulted in an increase in production time cycle due to decrease in rate of proofing during manufacture. In order to achieve a proofing rate almost equal to that of the control, proofing temperature was raised to 40 o C, which aided in catalyzing the activity of yeast and in turn the fermentation process. In order to improve the body and texture characteristics of multigrain bread to almost the same level as that of normal wheat bread, permitted improvers were incorporated into the dough mix. Based on subjective tests, Calcium phosphate was selected as the improver to be added and its level of addition was optimized at 1500ppm. The composition of breads were- 71.85 ± 0.18 and 73.87 ± 0.18 % TS, 11.37 ± 0.12 and 12.25 ± 0.11% fat, 10.96 ± 0.04 and 11.24 ± 0.1% protein, 1.72 ± 0.025 and 1.84 ± 0.033 % ash, 7.25±0.05 and 7.23±0.12 % crude fibre in control and experimental samples respectively. The products stored at 30 o C remained upto 4 days in acceptable sensory condition whereas the products stored at 5 o C could be stored upto 12 days. The investigation indicated that concentrated paneer whey upto 15% TS can be successfully used to replace water as diluent in the production of multigrain bread without affecting the sensory attributes. Page 183

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Appendices

APPENDIX l Sensory Evaluation on 9-point Hedonic Scale DAIRY TECHNOLOGY SECTION Name of Judge: - Time: Date: Batch no: ----------------------------------------------------------------------------- is /are served to you for organoleptic evaluation. Please judge the product on 9- point hedonic scale and write your valuable comments below. Attributes A B C D Colour & Appearance Flavour Body & Texture Overall acceptability Comments on A: Comments on B: Comments on C: Comments on D: Signature:- 9-Like extremely; 8-like very much; 7-like moderately; 6-like slightly; 5 neither like nor dislike; 4-dislike slightly; 3-dislike moderately; 2-dislike very much; 1-dislike extremely

APPENDIX li Photographs Fig- (From left) Experimental and control multigrain bread. Fig -Baking Oven