PHYSICOCHEMICAL AND FUNCTIONAL ASSESSMENT OF FOR VALUE ADDITION THROUGH FERMENTATION By Samreen Ahsan 2007-ag-2108

M.Sc. (Hons.) Food Technology (UAF)

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY IN FOOD TECHNOLOGY

National Institute of Food Science and Technology Faculty of Food, Nutrition and Home Sciences University of Agriculture, Faisalabad Pakistan 2019

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List of Abbreviations

(LAB) Lactic Acid Bacteria

(GRAS) Generally Recognized As Safe

(WHC) Water Holding Capacity

(US) United States

(USDA) United States Department of Agriculture

(T2DM) Type 2 Diabetes Mellitus

(LDL) Low Density Lipoprotein

(HDL) High Density Lipoprotein

(TSH) Thyroid Stimulating Hormone

(SNC) Nano Crystals

(MAP) Modified Atmosphere Packaging

(SPI) Isolate

(FOS) Fruto Oligo Saccharide

(SWP) Soy Whey Proteins

(MAPK) Mitogen-Activated Protein Kinase

(MITF) Microphthalmia-Associated Transcription Factor

(FSP) Fermented Soy Product

(MFCs) Microbial Food Cultures

(ACE) Angiotensin Converting Enzyme

(VLDL) Very Low-Density Lipoprotein

(TFC) Total Flavonoid Content

(TPC) Total Phenolic Content

(EPS) Exopolysaccharide

(SP) Soy Protein

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(OA) Osteo Arthritus

(MP) Milk Protein

(SP) Supplemental

(SHR) Pontaneously Hypertensive Rats

(ANFs) Anti-Nutritional Factors

(AARI) Ayub Agricultural Research Institute

(NARC) National Agricultural Research Centre

(NFE) Nitrogen Free Extract

(GC) Gas Chromatograph

(FID) Flame Ionizing Detector

(LOX) Lipoxygenase

(ELISA) Enzyme-Linked Immunosorbent Assay

(AAS) Atomic Absorption Spectrophotometer

(FC) Folin-Ciocalteu

(DPPH) 2,2-diphenyl-1-picrylhydrazyl

(FRAP) Ferric-reducing antioxidant power

(mgTE/g) miligram Trolox equivalent/gram

(ABTS) 2, 29-Azinobis (3-ethylene benzothiazoline) 6- Sulphonicacid)

(TPTZ) Fe3+- (2, 4, 6-Tri (2-pyridyl)-s-triazine)

(ATCC) American Type Culture Collection

(HPLC) High Pressure Liquid Chromatography

(SEM) Scanning Electron Microscopy

(FSM) Fermented Soy Milk

(NFSM) Non-Fermented Soy Milk

(NIFSAT) National Institute of Food Science and Technology

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(STZ) Streptozotocin

(IP) Intraperitoneally

(ALT) Alanine Amino Transferase

(ALP) Alkaline Phosphatase

(AST) Aspartate Aminotransferase

(BUN) Blood Urea Nitrogen

(RBC) Red Blood Cells

(WBC) White Blood Cells

(GAE) Gallic Acid Equivalent

(CAE) Catechin Equivalent

(AC) Antioxidant Capacity

(SNF) Solid Not Fat

(TSS) Total Soluble Solids

(TA) Titratable Acidity

(EPS) Exopolysaccharide

(PLC) Platelets Count

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ACKNOWLEDGEMENTS

I would like to give my sincere thanks to Dr. Tahir Zahoor for being my advisor. Especially for his patience guiding me through my research problems; he has always been a good mentor. He always opens my mind for any kind of research conversation, and always being a good listener.

I am also grateful to my committee members, Dr. Aamir Shehzad, and Dr. Muhammad Anjum Zia for guidance on many research problems and giving me very valuable comments.I would like to express my thanks to my foreign supervisor Dr. Arezoo Motavalizadeh Ardekani and her lab members for their kindness to provide research facilities on and details of soybeans to support my research. I would also like to thank to Dr. Nuzhat Huma you are a wonderful teacher and friend. You are everything one could look for in a good mentor. You groomed me to be sound professionals and made working with you an interesting and memorable experience. I will always be grateful to you for your support and kindness.

I want to say thank you to Dr. Bruce Cooper and Dr. Christopher J. Gilpin, Robert Seiler and Laurie Mueller for their patience and kindness to help me working in the laboratory and solving the problems for research analyses.

I would like to express my thanks to Adib Ahmad Zadegan thank you for your patience and always giving me correct answers for all kinds of questions. I am very thankful to the valuable friendships with all my friends Nazia Khalid, Dr. Muhammad Farhan Jahangir Chughtai, Dr. Saira Tanweer, Dr. Tariq Mehmood, Saima Naz, Iqra Yasmin, Atif Liaqat, and Rabia Ramzan. Thank you for encouraging and solving my problems.

Last but not least, I would like to express my thanks to my parents and my loving and caring husband Adnan Khaliq I could not finish my degree without your support and love. Your loves always make me have a faith in me.

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Table of Contents

CHAPTER 1 INTRODUCTION ...... 1 CHAPTER 2 REVIEW OF LITERATURE ...... 6 2.1 ...... 6 2.2 Climatic conditions ...... 7 2.3. Chemical composition of soybean ...... 7 2.3.1. Carbohydrate ...... 7 2.3.2. Protein ...... 8 2.3.3. Lipids ...... 9 2.4. Bioactive moieties in soybean ...... 10 2.4.1. Flavonoids ...... 11 2.4.2. Soy isoflavone ...... 12 2.4.3. Soybean antioxidants and their bioavailability ...... 14 2.5. Health benefits of soybean ...... 16 2.5.1. Soy intake and protective effect against age-related bone loss ...... 16 2.5.2. Soy intake and as an alternate form of hormonal replacement therapy (HRT) ..... 17 2.5.3. Soy intake as an anti-cancer ...... 17 2.5.4. Soy intake and diabetes ...... 18 2.5.5. Soy intake and heart health ...... 19 2.5.6. Soy intake and its role in obesity ...... 20 2.5.7. Soy intake and its effect on thyroid function ...... 21 2.6. Application of soy in food processing ...... 21 2.6.1. Soy bran as a fat replacer ...... 22 2.6.2. Soy protein isolate (SPI) and soy protein coatings ...... 22 2.6.3. Soy milk ...... 22 2.6.4. Soy in baking products ...... 23 2.6.5. Soy in desserts ...... 23 2.6.6. Soy yoghurt...... 24 2.6.7. Soy whey proteins (SWP) ...... 25 2.7. Fermentation with lactic acid bacteria (LAB) ...... 25

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2.7.1. Lactic acid bacteria in food applications ...... 26 2.7.2. Lactic acid bacteria benefiting health ...... 27 2.7.3. Lactobacillus acidophilus ...... 28 2.7.5. Fermentation of soybean ...... 30 2.7.6. Fermented soy products ...... 30 2.8. Bioactive peptides in fermented soybean ...... 31 2.9. Bio-functional properties of fermented soy milk ...... 32 2.9.1. Angio-tension converting enzyme (ACE) inhibitory peptides ...... 34 2.9.2. Antioxidant peptides ...... 36 2.9.3. Antidiabetic peptides ...... 36 2.9.4. Anticancer peptides ...... 36 2.10. Lactose intolerance ...... 38 2.11. Soy ...... 38 2.12. Conclusion ...... 39 Chapter 3 MATERIALS AND METHODS ...... 40 3.1. Procurement of raw materials ...... 40 Phase-I “Characterization of Soybean” ...... 40 3.2. Proximate analysis ...... 40 3.2.1. Moisture content ...... 41 3.2.2. Crude protein ...... 41 3.2.3 Crude fat ...... 42 3.2.4. Ash contents ...... 42 3.2.5. Crude fiber ...... 42 3.2.6. Nitrogen free extracts (NFE) ...... 43 3.3. Fatty acids compositional analysis ...... 43 3.4. Analysis of lipoxygenase (LOX) activity ...... 43 3.5. Mineral estimation ...... 44 3.6. screening test ...... 44 3.6.1. Total phenolic content (TPC) ...... 44 3.6.2. Total flavonoid content (TFC) ...... 44

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3.7. Antioxidant potential in soy flour samples ...... 45 3.7.1. 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay (DPPH) .... 45 3.7.2. Ferric-reducing antioxidant power (FRAP) assay ...... 45 3.7.3. (2, 29-Azinobis (3-ethylene benzothiazoline) 6-Sulphonicacid) ABTS ...... 45 Phase II “Value addition through fermentation of soy milk” ...... 46 3.8. Preparation of fermented soy milk ...... 46 3.8.1. Preparation of inocula and fermented soy milk ...... 46 3.9. Sensory evaluation of soy milk ...... 50 3.10. Analysis of value added fermented soy milk ...... 50 3.10.1. Compositional analyses of soy milk ...... 50 3.10.1.1. Total solids ...... 50 3.10.1.2. Solid Not Fat ...... 51 3.10.1.3. Protein ...... 51 i- ...... 51 ii- Distillation ...... 51 iii- Titration ...... 51 3.10.1.4. Fat ...... 52 3.10.1.5. Ash ...... 52 3.10.2. Viable cell counts ...... 52 3.10.2. Determination of pH ...... 52 3.10.3. Acidity ...... 53 3.10.4. Determination of water holding capacity (WHC) ...... 53 3.10.5. Anti-oxidative activities of fermented soy milk ...... 53 3.10.5.1. Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity assay ...... 53 3.10.5.2. Ferric reducing antioxidant power (FRAP) assay ...... 53 3.10.5.3. 2,2-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS) ...... 53 3.10.6. Rheological analysis ...... 54 3.10.7. Microstructure examination by scanning electron microscopy (SEM) ...... 54 3.10.7.1. Sample preparation ...... 54 3.10.7.2. Sputter coating ...... 55

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3.10.7.3. Scanning electron microscopy ...... 55 3.10.8. Quantification for isoflovone by high pressure liquid chromatography (HPLC) ..... 56 3.10.9 Selection of best treatments ...... 56 Phase III “In vivo study to check the hypoglycemic and hypocholesterolemic effect of Soy milk in Sprague Dawley rats” ...... 56 3.11. Efficacy trials ...... 57 3.11.1. Serum lipid profile analysis ...... 59 3.11.2. Serum glucose and insulin levels...... 59 3.11.3. Liver function probability ...... 59 3.11.4. Renal function tests ...... 59 3.11.5. Hematological analysis ...... 59 3.12. Statistical analysis...... 59 CHAPTER 4 RESULTS AND DISCUSSION ...... 60 4.1. Proximate analysis ...... 60 4.1.1. Moisture ...... 60 4.1.2. Crude protein ...... 61 4.1.3. Crude fat ...... 62 4.1.4. Crude fiber ...... 62 4.1.5. Ash ...... 63 4.1.6. Nitrogen free extract (NFE) ...... 66 4.1.2. Mineral content of soybean ...... 66 4.1.2.1. Potassium (K) ...... 66 4.1.2.2. Magnesium (Mg) ...... 67 4.1.2.3. Calcium (Ca)...... 68 4.1.2.4. Iron (Fe) ...... 69 4.1.2.5. Zinc (Zn) ...... 69 4.1.2.6. Copper (Cu) ...... 70 4.1.2.7. Sodium (Na) ...... 70 4.1.2.7. Manganee (Mn) ...... 71 4.1.3. Soybean fatty acids profile ...... 73 4.1.4. Lipoxygenase (LOX) activity ...... 74

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4.1.4.1. LOX-1 ...... 76 4.1.4.2. LOX-2 ...... 76 4.1.4.3. LOX-3 ...... 77 4.1.5. Phytochemical screening test ...... 80 4.1.6. Antioxidant potential of soybean ...... 82 4.1.6.1. DPPH ...... 82 4.1.6.2. FRAP ...... 83 4.1.6.3. ABTS ...... 83 4.2. Analysis of value added fermented soy milk ...... 85 4.2.1. Compositional analyses of soy milk ...... 85 4.2.1.1. Total soluble solids (TSS) ...... 86 4.2.1.2. Solid not fat (SNF) ...... 90 4.2.1.3. Protein ...... 93 4.2.1.4. Fat ...... 96 4.2.1.5. Ash ...... 100 4.2.2. Viable cell counts ...... 103 4.2.3. pH and acidity ...... 109 4.2.5. Determination of water holding capacity (WHC) ...... 116 4.2.6. Rheology (viscosity) ...... 120 4.2.7. Anti-oxidative activities of fermented soy milk ...... 126 4.2.7.1. 1,1-diphenyl-2-picrylhydrazyl (DPPH) ...... 126 4.2.7.2. Ferric Reducing Anti-oxidant Power (FRAP) ...... 131 4.2.7.3. 2, 2-azinobis- 3-ethylbenzothiazoline-6-sulphonate (ABTS) ...... 134 4.2.8. Sensory evaluation of soy milk ...... 137 4.2.8.1. Color ...... 138 4.2.7.2. Aroma ...... 141 4.2.7.3. ...... 142 4.2.7.4. Texture ...... 146 4.2.7.5. Overall acceptability ...... 147 4.2.9. Scanning electron microscopy ...... 153

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4.2.10. Quantification for isoflovone by high pressure liquid chromatography (HPLC) ... 160 4.2.10.1. ...... 160 4.2.10.2. ...... 166 4.3.1. Feed intake ...... 171 4.3.2. Drink intake ...... 173 4.3.3. Body weight ...... 175 4.3.4. Serum profile analysis ...... 178 4.3.4.1. Glucose ...... 178 4.3.4.2. Insulin ...... 181 4.3.4.3. Cholesterol ...... 184 4.3.4.4. Low Density Lipoprotein (LDL) ...... 186 4.3.4.5. High Density Lipoprotein (HDL) ...... 189 4.3.4.6. Triglycerides ...... 192 4.3.5. Liver functions tests ...... 194 4.3.5.1. Aspartate aminotransferase (AST) ...... 194 4.3.5.2. Alanine transaminase (ALT) ...... 196 4.3.5.3. Alkaline phosphatase (ALP) ...... 198 4.3.6. Renal function tests...... 200 4.3.6.1. Biological Urea Nitrogen (BUN) ...... 200 4.3.6.2. Creatinine...... 202 4.3.7. Hematological analysis ...... 205 4.3.7.1. Red blood cell (RBC) ...... 205 4.3.7.2. White blood cells count (WBCs) ...... 206 4.3.7.3. Platelets count (PLC) ...... 206 CHAPTER 5 SUMMARY ...... 210 Conclusions...... 213 Recommendations ...... 214 Literature Cited ...... 216 Appendix-I ...... 250 Appendix-II ...... 251

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Appendix-III ...... 253 Appendix-IV ...... 257

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List of Figures

Figure 2.1: Chemical structure of genistein, , daidzein and 17β estradiol and a representation of genistein metabolism ...... 13 Figure 2.2: (I) Inflammatory process associated with atherosclerosis. II) Soy/isoflavone diet blocks endothelial and monocytes activation start ...... 20 Figure 2.3: Formation and biological activity of bioactive peptides in fermented soybean .. 32 Figure 3.1: Flow sheet for preparation of Inocula of Lactobacillus acidophilus and Lactobacillus casei in Broth ...... 48 Figure 3.2: Flow sheet for preparation of fermented soy milk ...... 49 Figure 3.3: Fermented soy milk in Incubator ...... 50 Figure 3.4: Sample preparation for SEM ...... 54 Figure 3.5: Soy milk samples on stubs and Cressington 208HR sputter coater ...... 55 Figure 4.1: Proximate characteristics of soybean varieties...... 65 Figure 4.2: A chromatogram of fatty acids peaks in Ajmeri through GC-MS ...... 75 Figure 4.3: Comparison of inhibition potential of lipoxygenase-1 ...... 78 Figure 4.4: Comparison of inhibition potential of lipoxygenase-2 ...... 78 Figure 4.5: Comparison of inhibition potential of lipoxygenase-3 ...... 79 Figure 4.6: Overall comparison of inhibition potential of lipoxygenase ...... 79 Figure 4.7: Variation in viscosity of NARC-II soy milk at different shear rate at 0, 8th, 16th and 24th day ...... 123 Figure 4.8: Variation in viscosity of Willium-82 soy milk at different shear rate at 0, 8th, 16th and 24th day ...... 124 Figure 4.9: Variation in viscosity of Ajmeri soy milk at different shear rate at 0, 8th, 16th and 24th day ...... 125 Figure 4.10a: Interactive effect of treatmets*varieties*storage on the color of fermented soy milk ...... 140 Figure 4.10b: Interactive effect of treatmets*varieties on the color of fermented soy milk141 Figure 4.11a: Interactive effect of treatmets*varieties*storage on the aroma of fermented soy milk ...... 144 Figure 4.11b: Interactive effect of varieties*storage on the aroma of fermented soy milk..145 Figure 4.12a: Interactive effect of treatmets*varieties*storage on the flavor of fermented soy milk ...... 145 Figure 4.12b: Interactive effect of treatmets*varieties on the aroma of fermented soy milk ..146 Figure 4.13a: Interactive effect of treatmets*varieties*storage on the texture of fermented soy milk ...... 148 Figure 4.13b: Interactive effect of treatmets*storage on the texture of fermented soy milk ..149

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Figure 4.13c: Interaction of varieties*days for texture of fermented soy milk…………..150

Figure 4.13d: Interaction of treatment*varieties for texture of fermented soy milk……..150

Figure 4.14a: Interactive effect of treatmets*varieties*storage on the overall acceptability of fermented soy milk ...... 150 Figure 4.14b: Interactive effect of treatmets*varieties*on the overall acceptability of fermented soy milk ………………………………………………………………………..151

Figure 4.14c: Interactive effect of treatmets*storage on the overall acceptability of fermented soy milk ……………………………………………………………………….152

Figure 4.15: Scanning Electron micrograph of NARC-II L. acidophilus fermented soy milk a (100µm), b (10µm) and c (5µm) ...... 155 Figure 4.16: Scanning electron micrograph of NARC-II L. casei fermented soy milk a (300µm), b (30µm) and c (10µm) ...... 155 Figure 4.17: Scanning electron micrograph of NARC-II L. acidophilus + L. casei fermented soy milk a (50µm), b (30µm) and c (10µm) ...... 156 Figure 4.18: Scanning eElectron micrograph of Willium-82 L. acidophilus fermented soy milk a (300µm), b (10µm) and c (5µm) ...... 156 Figure 4.19: Scanning electron micrograph of Willium-82 L. casei fermented soy milk a (40µm), b (10µm) and c (5µm) ...... 157 Figure 4.20: Scanning electron micrograph of Willium-82 L. acidophilus + L. casei fermented soy milk a (X40µm), b (X10µm) and c (10µm) ...... 157 Figure 4.21: Scanning electron micrograph of Ajmeri L. acidophilus fermented soy milk a (100µm), b(10µm) and c (4µm) ...... 158 Figure 4.22: Scanning electron micrograph of Ajmeri L. casei fermented soy milk a (100µm), b(10µm) and c(5µm) ...... 158 Figure 4.23: Scanning electron micrograph of Ajmeri L. acidophilus+L. casei fermented soy milk a (300µm), b (40µm) and c (10µm) ...... 159 Figure 4.24: Calibration curve of “daidzein” a standard ...... 161 Figure 4. 25: Calibration curve of “genistein” a standard ...... 161 Figure 4.26: The typical HPLC chromatogram of the isoflavone standards (genistein and daidzein) ...... 162 Figure 4.27: A chromatogram of L.Casei Ajmeri soy milk ...... 162 Figure 4.28: Feed intake of rats g/rat/day a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats) ...... 172 Figure 4.29: Drink intake of rats mL/rat/day a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats) ...... 174 Figure 4.30: Effect on body weight of rats g/rat a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats) ...... 176 Figure 4. 31: Percent decrease in level of glucose ...... 180

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Figure 4.32: Percent increase in level of insulin ...... 182 Figure 4. 33: Percent decrease in level of cholesterol ...... 185 Figure 4.34: Percent decrease in level of low density lipoprotein (LDL) ...... 187 Figure 4.35: Percent increase in level of high density lippoprotein (HDL) ...... 191 Figure 4.36: Percent decrease in level of triglycerides ...... 193 Figure 4.37: Percent decrease in level of aspartate aminotransferase (AST) ...... 195 Figure 4.38: Percent decrease in level of alanine transaminase (ALT) ...... 197 Figure 4.39: Percent decrease in level of alkaline phosphatase (ALP) ...... 199 Figure 4.40: Percent decrease in level of biological urea nitrogen (BUN) ...... 201 Figure 4.41: Percent decrease in level of creatinine ...... 203 Figure 4.42: Percent increase in level of red blood cells (RBCs) ...... 204 Figure 4.43: Percent decrease in level of white blood cells (WBC) ...... 207 Figure 4.44: Percent increase in level of platelets count ...... 209

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List of Tables

Table 2.1. Physiologically functional substances in soybeans ...... 37 Table 3.1. Treatments used for the production of soy milk ...... 47 Table 3.2 Treatment plan for efficacy trials...... 58 Table 4. 1. Mean squares of proximate composition for soybean varieties ...... 64 Table 4. 2. Proximate composition of soybean varieties ...... 64 Table 4.3.Mean squares of minerals for soybean varieties ...... 72 Table 4.4. Effect of soybean varieties on mineral contents (mg/100g) ...... 72 Table 4.5: Fatty acid (%) profiling of soybean varieties ...... 75 Table 4.6: ANOVA for Mean squares of Lipoxygenase (LOX) ...... 78 Table 4.7 Analysis of variance for TPC and TFC ...... 81 Table 4.8 Effect of soybean varieties on TPC and TFC ...... 81 Table 4.9 Analysis of variance for antioxidants ...... 84 Table 4.10 Effect of soybean varieties on antioxidants (mg TE/g) ...... 84 Table 4.11 Mean Squares for composition of fermented soy milk ...... 87 Table 4.12a: Effect of treatment, varieties and days on the total soluble solids (TSS) (%) of soy milk ...... 88 Table 4.12b Effect of treatment and varieties on TSS (%) of soy milk …………………….. 90

Table 4.13a: Effect of treatment, varieties and days on the solid not fat (%) of soy milk 91

Table 4.13b: Effect of treatment and varieties on SNF (%) of soy milk….. ………………. 93 Table 4.14a: Effect of varieties, treatment and storage on protein (%) of soy milk ...... 94 Table 4.14b Effect of treatment and varieties on protein (%) of soy milk ………………… 96

Table 4.15a Effect of varieties, treatment and storage days on fat (%) of soy milk ...... 98 Table 4.15b: Effect of treatment and varieties on fat (%) of soy milk……………………. 100 Table 4.16a Effect of varieties, treatment and storage days on ash (%) of soy milk ...... 101 Table 4.16b: Effect of treatment and varieties on ash (%) of soy milk……………………. 103 Table 4.17 Mean squares for viable cell count and physico-chemical analysis of soy milk . 106

Table 4.18a: Effect of varieties, treatment and storage days on viable cell count (log10 CFU/mL) of soy milk ...... 107

Table 4.18b: Effect of treatment and varieties on viable cell count (log10 CFU/mL) of soy milk.. 109

Table 4.18c: Effect of treatment and days on viable cell count (log10 CFU/mL) of soy milk109 Table 4.19a: Effect of varieties, treatment and storage days on pH of soy milk ...... 112 Table 4.19b: Effect of treatment and varieties on pH of soy milk………………….. 114 Table 4.19c Mean values showing effect of treatment and days on pH of soy milk.. 114

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Table 4.19d: Effect of varieties and days on pH of soy milk………………………… 114 Table 4.20a Effect of varieties, treatment and storage days on acidity (%) of soy milk ...... 114 Table 4.20b: Effect of treatment and varieties on acidity (%) of soy milk………….. 116 Table 4.20c: Effect of treatment and days on acidity (%) of soy milk………………. 116 Table 4.20d: Effect of varieties and days on acidity (%) of soy milk………………. 116 Table 4.21a: Effect of varieties, treatment and storage time on water holding capacity (WHC) (%) of soy milk ...... 117 Table 4.21b Effect of treatment and varieties on water holding capacity (WHC) (%) of soy milk…………………………………………………………………………………….. 119

Table 4.21c Effect of treatment and days on water holding capacity (WHC) (%) of soy milk.119

Table 4.22: Mean Squares for antioxidant potential of soy milk ...... 128 Table 4.23a Effect of varieties, treatment and storage days on DPPH (%) of soy milk ...... 129 Table 4.23b: Effect of treatment and varieties on DPPH (%) of soy milk………… …131

Table 4.23c: Effect of treatments and days on DPPH (%) of soy milk……………… 131

Table 4.24a: Effect of varieties, treatment and storage days on FRAP (mmolFe2+/L) of soy milk ...... 132 Table 4.24b: Effect of treatment and varieties on FRAP (mmolFe2+/L) of soy milk.. 134

Table 4.25a: Effect of varieties, treatment and storage days on ABTS (%) of soy milk ...... 135 Table 4.25b Effect of treatment and varieties on ABTS (%) of soy milk …………….137

Table 4.26: Mean squares for sensory evaluation of fermented soy milk ...... 139 Table 4.27 Mean squares for genistein and daidzein of soy milk...... 164 Table 4.28a Effect of varieties, treatment and storage days on genistein (ppm) of soy milk .. 164 Table 4.28b Effect of treatments and varieties on genistein (ppm) of soy milk… …..166 Table 4.28c Effect of treatment and days on genistein (ppm) of soy milk………….. 166 Table 4.28d Effect of varieties and days on genistein (ppm) of soy milk…………… 166 Table 4.29a Effect of varieties, treatment and storage days on daidzein (ppm) of soy milk .. 169 Table 4.29b: Effect of treatment and varieties on daidzein (ppm) of soy milk…….. 170 Table 4.29c: Effect of treatment and days on daidzein (ppm) of soy milk………… 170 Table 4.29d: Effect of varieties and days on daidzein concentration (ppm) of soy milk. .171

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Table 4.30 Combined effect of treatment on (geinstenin+ daidzein) of soy milk ...... 170 Table 4.31Combined effect of soybean varieties on (geinstenin+ daidzein) of soy milk..... 170 Table 4.32 Mean squares for effect of drinks and time intervals on feed, water intake & body weight of rats in different studies...... 177 Table 4.33 Effect of fermented soy milk on glucose level (mg/dL) ...... 180 Table 4.34 Effect of fermented soy milk on insulin level (µU/mL) ...... 182 Table 4.35 Effect of fermented soy milk on cholesterol level (mg/dL) ...... 185 Table 4.36 Effect of fermented soy milk on low density lipoprotein (LDL) level (mg/dL) . 187 Table 4.37 Effect of fermented soy milk on high density lipoprotein (HDL) level (mg/dL) 191 Table 4.38 Effect of fermented soy milk on triglycerides level (mg/dL) ...... 193 Table 4.39 Effect of fermented soy milk on aspartate aminotransferase (AST) level (IU/L) ...... 195 Table 4.40 Effect of fermented soy milk on alanine transaminase (ALT) level (IU/L) ...... 197 Table 4.41 Effect of value added soy milk on alanine phosphatase (ALP) level (IU/L) ...... 199 Table 4.42 Effect of fermented soy milk on biological urea nitrogen (BUN) level (mg/dL) 201 Table 4.43: Effect of fermented soy milk on creatinine level (mg/dL) ...... 203 Table 4.44 Effect of fermented soy milk on red blood cell (RBC) cells/pL ...... 204 Table 4.45 Effect of fermented soy milk on white blood cells count (WBCs) cells/nL ...... 207

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ABSTRACT

The current research work was carried out to characterize the local (Faisal, NARC-II, Willium-82, Ajmeri and Rawal-I) promising soybean varieties. The physicochemical analysis like protein, fat, ash, minerals and fatty acids profile showed the significant variation. Phytochemical test like TPC, TFC, antioxidant activity (DPPH, ABTS and FRAP) showed substantial variation among them. Lipoxygenase (LOX-1, LOX-2 and LOX-3 that cause oxidation and off- flavor were lower in Ajmeri. The initially three secreened varieties (Ajmeri, Willium-82 and NARC-II) were used to prepare fermented soy milk fermented with Lactobacillus acidophilus and Lactobacillus casei to evaluate their single and coculture effect. The periodically physicochemical, functional, microbial, antioxidant potential and organoleptic evaluation of soy milk showed significant differences among three varieties and treatments of soy milk. The viscosity versus shear rate displayed higher viscosity in co- culture fermented soy milk especially in Ajmeri. Scanning Electron microscopy depicted that Ajmeri soy milk fermented with L. casei showed relatively more uniform texture and smaller pore size in comparison to L. acidophilus however, it was elucidated that combination of cultures depicting constant and precise pore formation with stronger cross linking of soybean protein that had modified structure stability. Isoflavones quantified through HPLC displayed were higher in comparison to genistein in Ajmeri soy milk fermented with co-culture. Afterwards Ajmeri soy milk fermented by co- culture and non-fermented was used to conduct bio-efficacy trial on Sprague dawley rats for Hyperglycemia and Hypercholesterolemia. The studies were intended as study I (Normal rats) study II (Hyperglycemic rats and study III (Hypercholestrolemic rats). The results showed percent decrease in glucose level from 3.61 to 17.11% and inclining insulin level from 1.8 to 8.3%. The value added drink was most effective in lowering cholesterol level from 2.62 to 13.51% likewise the LDL reduction and elevation of HDL was increased. The safety test on liver, kidney and blood biochemistry suggested it as a safe and non-toxic product. The research explored that soybeans are a good source of nutritional food and Ajmeri, Willium-82 and NARC- II have food grade value than other varieties. Furthermore, combination of L. acidophilus and L. casei in the fermentation of Ajmeri soy milk has a valuable effect against hyperglycemia and hypercholesterolemia is effective in comparison to non- fermented soy milk. Present study colclueded that soy based functional foods should be encouraged in diet to curb from various health remadies and protein requirement can be fulfilled in an inexpensive way by administrating fermented soy milk.

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CHAPTER 1 INTRODUCTION Soybean (Glycine max.) is such a famous oleaginous legumes considered as the golden bean for the 20th century because of its wealth for health benefits. Soybean is the novel and economical source of plant proteins and contributes the highest gross output as on oil seed crop throughout the world. Due to this fact, it is also known as the cradle of protein and oil being the most endorsed mean of plant protein which contains all essential amino acids. Other than proteins, soybean comprises primary nutritive components that are vitamins, minerals, lipids, free sugar and bioactive components like isoflavones, saponins and flavonoids. All these constituents boost the therapeutic values of soybean (Li et al., 2014; Sanjukta and Rai, 2016).

Therefore, it is essential to meet the required protein levels and also provide alternative supplies of protein. In this regard, legume seeds are of great interest contributing significantly to fulfill protein requirements and also helpful in tackling protein related malnutrition (Joelle and Dent, 2011). At the same time, industry is also designing food products by using novel ingredients to address lactose intolerance and to minimize the cholesterol level. All these approaches are appealing to the consumers because they are more concerned about the nutritional values of a product that are also helpful to mitigate life related disorders (Schmidt et al., 2016).

Considering the compositional profile, soybean comprises 36-40% protein that varies in different varieties, 20% oil contents, 15% soluble carbohydrates (raffinose, succrose and stachyose) and 15% insoluble carbohydrates (dietary fiber) (Kim et al., 2005). Soybean also contains several that are phenolic contents containing flavonoids, iso- flavonoids and phenolic acids with putative health benefits, thus making it as a momentous “functional food” (Faraj and Vasanthan, 2004). Functional foods are those foods that can promote health benefits beyond that of nutritional value. These foods may contain active compounds such as probiotics, prebiotics and sterols (Tootoonchi et al., 2015). Probiotics are live bacteria that live in human gut and improve natural microbiota overthere. Any food can be recommended as probiotic food if it cantain probiotics at least between 106 and 107 bacteria/g or per mL (Tootoonchi et al., 2015).

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Soy has an important influence for the growth of probiotics due to high eminence proteins, oligosaccharides, vitamins, dietary fiber and trace minerals which could meet prebiotic standards. In addition, soy is low in saturated fats and a superb source of complete plant protein that contains all essential amino acids to meet the protein requirements for vegetarians (Joelle and Dent, 2011).

Lactose deficiency has been reported in many countries like 55% in Mexico, 28% in Brazil and 18% in Finland with respect to their population. Soybean is cholesterol and lactose free that make it a plenteous and economical cradle of nourishment for vegetarians, lactose- intolerant persons, and patients who are milk-allergic (Liu and Lin, 2000). This is the main reason that demand of non-dairy soy foods has been increase especially for people who are intolerant to lactose or vegetarians. The major reason for inadequacy of consumption of dairy products made from milk and milk ingredients is lactose and cholesterol (Daniel et al., 2010).

Soy milk is the beverage that is extract of soybeans in water, which is a colloidal solution with full of nutrients to nourish the body. These nutrients in soy milk make it favorable medium for growth of lactic acid bacteria (Farnworth et al., 2007; Tran and Rousseau, 2013). Food industry seeks to employ new alternative ingredients to supplement consumption for all dimensions of consumers based upon a general acceptability for lactose intolerance, cholesterol free products whilst still maintaining a substantial nutritional value (Schmidt et al., 2016).

Soy milk shows therapeutically potential associated with antidiabetic, antiobesity (Sartang et al., 2015) anticancer (breast and prostate), osteoporosis risk reduction, cardiovascular diseases (Xu et al., 2015) and to reduce inflammation (Nurliyani, 2015). It has been reported by several scientists that bioactive moieties including isoflavones in soybean can act as anti- cancer and hormone-altering activities. It is also free from cholesterol with special concern to saturated fat contents that make it favorable for patients suffering from cardiovascular diseases (Joelle and Dent, 2011). The soy milk has more thickness and solid contents that are essential requirements to make yogurt from any beverage (Lopez-Lazaro and Akiyama, 2002; Kim et al., 2005).

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Alternate use of milk with vegetal food matrix like soy milk having high protein quality is an economical way to produce probiotic beverages in South Asia and South America (Tou et al., 2007). Probiotics not only provide health benefits but they can also be used for fermentation in soy milk that produces such peptides that prevent from heart diseases and known as antihypertensive (Donkor et al., 2005), promoting gut ecosystem equal balance (Cheng et al., 2005) and decreasing fiber contents that may cause flatulence (Wang et al., 2008; do Espirito-Santo et al., 2014).

Soybean is consumed in both fermented and unfermented forms; fried, roasted, powder, sauce, butter, yoghurt, cheese and soy pickles. In ancient ages, fermentation has been used as a process to preserve food, particularly in regions/areas having scarceness of perishable foods. In the current scenario, fermentation is a pragmatic way to enhance bioactive moieties (Rai and Jeyaram, 2015; Sourabh et al., 2015) and to eliminate anti-nutritional components (Difo et al., 2014; Rai and Appaiah, 2014). Soybean has a distinctive beany flavor and aroma and undigested dietery fiber can also cause flatulence so, in order to evade these issues and to amplify its acceptability fermentation of soy milk is a best way (Blagden and Gilliland, 2005).

Fermentation is a process in which microorganisms break complex organic compounds in simple molecules that play a physiological role other than nutritive functions. Fermentation with microbes helps to boost bio-functional properties of soy by enhancing peptides and isoflavones that can be easily absorb (Zhang et al., 2006; Sanjukta et al., 2015). Fermentation is also useful because it reduces anti-nutritional components i.e. , proteinase- inhibitors, oxalic acids, urease and other components causing immunoreactivity (Song et al., 2008). During fermentation of soybean, microbes are responsible to release proteases for proteolysis to convert proteins into peptides (Rai and Jeyaram, 2015; Sanjukta et al., 2015).

Soy fermented foods are full of bioactive peptides and they can efficiently exploit for preparation of functional foods, food supplements as well as pharmaceuticals. These products can be served as a protective approach to prevent from supernumerary side effects caused from synthetic drugs (Sanjukta and Rai, 2016).

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Soy foods comprises of isoflavones as glucoside form, but infrequently in the aglycone form. Although, fermentation does not change concentration of isoflavones in soy foods but it just changes glucosides or glycones into aglycone form due to bacterial hydrolysis of sugar (da Silva et al., 2011). In healthy grown persons, glucosidase enzyme converts non absorbable form of glycones to aglycones that can be easily absorbed. The process of fermentation with probiotics produces high levels of β-glucosidase in soy milk that can amend nutrition and bioactivity of soy milk (Otieno et al., 2006).

Lactic Acid Bacteria (LAB) show an imperative effect in processing of different products in food industries due to their flavor enhancing property because of acetic and lactic acid production that ultimately improves natural preservative property to relish its consumption. LAB is highly accepted because of its support in motivating host‟s immunity, acti as anti- carcinogenic (Axelsson, 2004; Castellano et al., 2010). Their usual existence in foods joined with their long ancient consumption subsidizes their approval as GRAS (Generally Recognized as Safe) for human intake. Fermentation with LAB can be done alone or as a coculture if they are used more than one (Farnworth et al., 2007; Tran and Rousseau, 2013).

The frequently used genus is “Lactobacillus” in fermented soy milk. Lactobacillus rhamnosus (Farnworth et al., 2007), Lactobacillus acidophilus (Liong et al., 2009) Lactobacillus casei (Wang et al., 2010) and Lactobacillus paracasei (Chiang and Pan, 2012) have also been proved to grow well in soy milk. Soy milk fermented by LAB not only enhance its flavor and aroma but also modulates and improves its textural properties such as apparent viscosity by modifying its water holding capacity (WHC) (Champagne et al., 2009).

Fermentation of soy offers chance to modify sensory features of soy-based foods. Peculiar smell is produced from cultures of lactic and other probiotic bacteria, which dramatically contributes to increased flavor of products (Chumchuere et al., 2000; Vuyst, 2000) by lowering levels of volatile compounds that causes natural beany flavor in soy products (Blagden and Gilliland, 2005). Probiotic LAB causes beneficial health effects, which include lowering level of hypercholesterolemia, relief from inflammatory bowel disease, and several infectious diseases caused by Helicobacter pylori that damages protective stomach layer and caused gastric ulcers and sometimes it causes inflammation in stomach known as and some times infection of urinary tract (Warensjo et al., 2008).

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It has been also studied that probiotic bacteria have various immune-modulatory characteristics. A soy product made by using probiotic bacteria would comprise the benefits both from soy and probiotics when used solely. Fermentation of soy, especially with precise reference to nutritious diet could augment these advantages;

 Improves bone metabolism and amends nourishing value  Improves  Modify flavor and texture  Combat difficulties associated with flatulence  Improves shelf life (Pyo et al., 2007)

Keeping in view the importance of soybean and fermented soy milk, having positive impact on human health; current project was planned to explicit following goals:

 To characterize different soybean varieties for their functional assessment  To develop value added soy milk through fermentation by using Lactobacilus acidophilus and Lactobacilus casei  Effect of fermented soy milk against hyperglycemia and hypercholesterolemia through in vivo study

The above mentioned goals of the present study suggested that hypothesis of the study is fermented soy milk can be effective against hyperglycemia and hypercholesterolemia.

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CHAPTER 2 REVIEW OF LITERATURE Soybean is prominent global agricultural crop due to its alluring application in food and feed products along with its economic significance on national and international markets.

Worldwide soybean production 2016/17 is estimated as United States 116.9 Argentina 57.8, Brazil, 114.1, Paraguay 10.7, Canada 6.6, India 11.5 and China 12.9 Million Tons. World soybean production in 2017/18 is forecast as in United States 119.5, Argentina 54.0, Brazil 112.0, Paraguay 9.2, Canada 8.0, India 9.5 and China 14.2 Million Tons. World soybean supply is estimated in 2016/17 was 429.3 Million Tons and in 2017/18 it is forecast as 443.1Million Tons. However world production is estimated as 351.3 Million Tons and forecast for 2017/18 is 346.9 Million Tons.

(World Agricultural Supply and Demand Estimates Report of February 8, 2018 https://www.usda.gov/oce/commodity/wasde/Secretary_Briefing.pdf). The USDA, 2017 has ranked Pakistan at 9th position in terms of production which was recorded as 2,000 Metric tons).

The lot of research work has been conducted to explore the soybean, and hence a brief review has been presented under various categories.

2.1 Soybean

Soybean is known as Glycine max in North America and Soybean in Britain. It is rich of protein, essential fatty acid and no cholesterol. Research studies back from 30-40 years; have suggested that soybean have health putative effect against various health disorders (Felberg et al., 2015).

The United States of America (U.S.A.) is the first country that cultivated soybean crops in small amount during late 18th century; however, its cultivation was expanded when Asians migrated from Europe to North America. In 1920‟s, consumption of began in U.S.A. and soy flour became popular as a protein source and an alternative of meat in Europe as well. Soy flour was used in great amount during World War I and II to help equipoise meat dearth. Now vast global development in cultivation, processing and application of soybeans has been

6 initiated. Brazil, US, Argentina and India are major producers to compete with the world‟s demand of soy for food as well as for feed consumption (Riaz, 2006).

Soybean meal, derived after mashing or after extracting of soy milk is used as a protein source and poultry feed. Soybean and soy derived products causing morethan 10 percent of global agricultural trade and being the 4th most cultivated crop, with more than 85% of soybeans being used for oil and meal. According to USDA, agricultural projections till 2025, soybean projects and their trade will increase within coming ten years. The top producer of soybean are U.S.A. and Brazil and more than 80% of global soybean exports is from both of these countries (USDA, 2016). Brazil‟s soybean export is projected to increase by 35% to 76.4 million tons and US export of soybean is predicted to grow 6% to 52.4 million tons in between 2016/17 and 2025/26.. However, soybean crop is expected to expand in the new areas to increase its production and oil export gradually (USDA, 2016).

2.2 Climatic conditions

The term „Climate‟ encompasses temperature, rainfall, relative humidity and solar radiation (Jaetzold et al., 2005). Temperature influences may physiological processes and biochemical processes such as germination, flowering and pod filling. Temperatures below 21oC and above 32oC can reduce floral initiation and pod set, whereas, extreme temperatures found to be unfavorable for seed production. Most critical stage is during flowering and pod filling. Moisture stress often resulting from mid- season drought significantly reduces the yield as it causes flower abortion and early senescence (Vandamme et al., 2013). Consequently, high relative humidity accelerates the disease prevalence especially the soybean rust. Soybean; being a hardy plant and able to adapt a variety of soils and climatic conditions is relatively less effected by climatic conditions mentioned above, making its cultivation and stable growth a good probability for its consumption as an alternative source.

2.3. Chemical composition of soybean

2.3.1. Carbohydrate

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Carbohydrates masquerades the sugars, and fiber contents in any food items. During the fermentation process in soybean and its related commodities the major effected carbohydrates are sucrose, stachyose and raffinose. The presences of carbohydrates act as prebiotics for lactic-acid bacteria so they can grow well in soy milk and produce organic acid especially lactic acid in soy milk (Liu and Lin, 2000). Total sugar concentration of soybean is approximately 14% of dry matter, comprising 40-45% of total carbohydrate. The principal quantity of each sugars is given as disaccharide sucrose 5.0%, trisaccharide raffinose 1.1% and tetra saccharide stachyose 3.8% along with 15% hemicellulose, 4% cellulose and 5.1% other carbohydrates (such as glucose, arabinose, and verbascose) account for total carbohydrate contents of 34% of whole bean (Grieshop et al., 2003). Glucose or other reducing sugars are present in green or immature beans in substantial amounts but they disappear as beans approach maturity; starch is seldom found in mature beans but reported to be found in immature beans (Karr-Lilienthal et al., 2005). Soybean fiber was characterized by high content of galactose, including hemicellulose and cellulose, in soybean hull. Raffinose and stachyose are oligosaccharides and non-reducing sugars that give no energy. They are similar in structure except one more galactose residue is present in stachyose (Chen et al., 2014) and these indigestible carbohydrates can be converted to digestible mono- or disaccharides with microbial action.

2.3.2. Protein

Soy proteomics as a functional and nutraceutical and food has been reported from many years. Soy is considered as cheapest source of protein to meet the deficiency of animal source of meat which is relatively much expensive. Among all the others proteins sources soybean provides more options for food variety through value addition (Adams et al., 2004; Ferreira et al., 2015)

Protein storage particles called protein bodies or aleurone grains vary from 15 to 20 µm in diameter with most of them falling in the narrower range of about 5 to 10 µm. At least 60- 70% of total protein in soybean is stored in protein bodies. Minimum solubility of these proteins occurs at pH 4.2, corresponding to apparent isoelectrical point of major proteins. Adjustment of aqueous or dilute sodium hydroxide extracts of defatted meal to pH 4.0-4.2 precipitates about 90% of extracted protein (Lampart-Sczapa, 2001).

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Soy storage proteins mainly compose of two proteins; globulins including β-conglycinin (7S) and glycinin (11S) account for approximately 80% of total proteins. The 7S is a quaternary trimeric glycoprotein composed of three subunits associated via hydrophobic interactions. The β-conglycinin possesses approximately two intermolecular disulfide bonds. Thus, it may possess amphiphilic properties for good surface activity and flavor binding. Glycinin consists of acidic and basic subunits associated by two pairs of disulfide linkage and 6 hydrophobic interactions. Glycinin has limited solubility limit at it isoelectric point was around pH 6.0. It also contains two free thiol groups which apparently are located on surface and can engage in thiol-disulfide interchange (Jiang et al., 2011). These proteins are mainly responsible for functional characteristics of soy protein ingredients (Lukaczer et al., 2006; Poysa et al., 2006; Garcia et al., 2009; Kanauchi et al., 2015).

The content of glycinin and β-conglycinin are different among soybean varieties and it depends on genetics and growing environment. Glycinin is a hexamer molecule and has six subunits each of which have basic and acidic polypeptides linked through eachother by disulphide bonds (Saeed et al., 2016). Beta conglycinin included in 7S globulin is a trimer having three subunits α, α‟, and β and they exist in the form of several combinations (da Silva et al., 2011).

Other proteins present in soybean are trypsin inhibitor, hemagglutinin, and lipoxygenases. Trypsin inhibitor possesses a single polypeptide chain and is compact, low in asymmetry and rigid in structure. Hemagglutinin is a glycoprotein. Absence of cystine crosslinks in this glycoprotein suggests that molecule may be fairly flexible and subjected to conformational changes very easily (Mandal, 2014).

2.3.3. Lipids

Lipid deposit or spherosomes are interspersed between protein bodies and are about 0.2-0.5 µm in diameter. Primary fatty acids present in are unsaturated fatty acids such as oleic acid 22.8%, linoleic and linolenic acids 57.6% and 14% of palmitic and stearic acids, while rest are 0.3% saturated fatty acids with a carbon number less than 14. These fatty acids and glycerol consist of 16.5% lipids in mature dry soybean (Tzen, 2012).

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The average composition of refined bleached and deodorized commercial soybean oil is 54% linoleic acid, 24% oleic acid, 11% palmitic acid, 7% linolenic acid and 4% stearic acid. Due to a high content of polyunsaturated fatty acids, particularly (linoleic omega-6 fatty acid) and linolenic acids (omega-3 fatty acid), soybean lipids are prone to oxidative instability problems (Slavin et al., 2009). Carbohydrates make remaining 30% of the bean and include 15% of soluble carbohydrates such as sucrose, raffinose, and stachyose, as well as 15% of insoluble carbohydrates such as dietary fiber (Cederroth and Nef, 2009).

Soybean contains 40% of biologically active proteins having all essential amino acids. It also comprises many micronutrients and phytochemical i.e. isoflavones, phytate, soyponins, phytosterol, vitamins and minerals. The six isoflavone in soybean are , genistin, , daidzein, genistein, and and three major groups of isoflavones found in soybeans were genistein, daidzein, and glycitein (Cederroth and Nef, 2009; Adie et al., 2015).

Soybean is famous for the production of oil; around 20% of the bean is composed of lipids (61% polyunsaturated fat, 24% monounsaturated fat and about 15% saturated fat). Lipids in soybeans are importance because it is cholesterol-free, low in saturated fatty acids, and high in unsaturated fatty acids (Cederroth and Nef, 2009).

2.4. Bioactive moieties in soybean

Foods encompassing bioactive components are getting great attention due to their functionality in deterrence and treatment of disease. Soybean and its products comprehend surplus bioactive phytochemicals such as isoflavones, phytic acids, phytosterols, saponins and inhibitors of trypsin. Soybean‟s phytochemicals are helpful in contraction of cholesterol and ultimate abatement of cardiovascular diseases, diabetes manifestation, bone loss and cancer preclusion (Isanga and Zhang, 2008).

Phenolic acids have been identified as embodying 28% to 72% of total phenolic contents in soybean (Chung et al., 2011). Phenolic acids in soybeans are primarily caffeic acid, coumaric acid, vanillic acid, ferulic acid, protocatechuic acid and predominantly. However, flavonoids are quercetin and glycosylated that are the isoforms of isoflavones namely genistin, daidzin,

10 and glycitin. Those are acclaimed for their “anti-inflammatory”, “anti-oxidant” and “anti- carcinogenic” exertions (Duenas et al., 2012).

In soybean, bioactive components other than phenolic acids and flavonoids are carotenoids that are hydrophilic and lipophilic which is in the oil part. Carotenoids retain antioxidant and radical scavenging activities; considering their pro-vitamin A having the potential to cure carcinogenic and coronary ailments (Morales-De La Pena et al., 2011). Correspondingly, have radical scavenging features to avert carcinogenesis, cardiac diseases and neurodegenerative maladies, such as Alzheimer‟s disease and Parkinson‟s disease (Dajanta et al., 2013; Ziegler et al., 2016). Soybean contains a lot of phytochemicals which included isoflavones, phytate, saponins and phytosterol along with other minor constituents such as vitamins and minerals. Nonetheless, beneficial properties of phytochemicals or phenolics are because of availability of saponins and phytosterols that are effective against cholesterol level in human body (Lukaczer et al., 2006). In soybean the quantity of phytochemicals may varied due to difference in grain genetic make up that includes seed coat, color, size and environmental conditions including growing region, temperature, precipitation and postharvest processing that includes heating, grinding, drying temperature, storage period and hydrothermal treatment (Chung et al., 2011; Ziegler et al., 2016).

There is huge versatility of isoflavones in soybeans because of variability in growth and environmental conditions. Fluctuations of environment conditions in the form of abiotic and biotic stresses as temperature change causes pest or insect infestation. Drought or extra rain fall can cause an adverse effect on composition and nutritional status of soybean. As an outcome of all these external factors, isoflavone content varies up to three fold of its actual value within the same cultivation in diverse geographical area in different time period of germination (Caldwell et al., 2005).

2.4.1. Flavonoids

Plant secondary metabolite that does not help in plant growth but are essential for their survivability and production of phenolics is one of these metabolites. Phenolic in plants are produced through phenylpropanoid pathway from aromatic amino acids and its two major

11 types are flavonoids and non-flavonoids. The flavonoids are compounds with 2- phenylchromen-4-one backbone while “isoflavonoids” are compounds with 3- phenylchromen-4-one backbone (Li et al., 2016).

Plant species harmonize an array of flavonoid that plays a role in flower pigmentation to captivate pollinators, in espouse plants verses pathogens, act as signal molecules in plant and microbe interactions and in safeguarding plants from ultra violet rays. Flavonoids also have momentous actions when ingested by animals, due to their marvelous effects. There is a structural similarity with preeminent estrogen, 17β-estradiol, isoflavonoids i.e. daidzein and genistein called as and they show estrogenic activity that is presumed to play imperative roles in human health (Iwashina et al., 2006). Soybean composed of phenolic composites, especially isoflavonoids (Malencic et al., 2008). These compounds show far reaching consequences as anticancer and has investigated on cancer cells inclusively as colorectal, breast, hepatocellular leukemia, ovarian, gastric and prostate cancers. Soybeans have comprehensive propitious effects on cancer inhibition, cardiovascular diseases, and osteoporosis risk reduction (Xu et al., 2010; Xu and Chang, 2011, 2012; Xu et al., 2015).

2.4.2. Soy isoflavone

When soy is ingested, genistein is released from glucoside and it is digested in stomach by acid hydrolysis or in intestine by microflora hydrolysis. When genistein is digested it converts into aglycone that can be absorbed or either further metabolized into different molecules e.g. dihydrogenistein and 5-hydroxy- (Matthies et al., 2012). Genistein causes multiple actions among which some are related to exterminate postmenopausal disorders and preventing breast cancer, bone decay, dropping risk of cardiovascular ailments and also effects on action of insulin (Messina and Wood, 2008). Besides isoflavones, soybeans have also been investigated as a food source of polyphenols, tannins, proanthocyanidins, flavonoids (majority of isoflavone), and phenolic compounds such as chlorogenic, ferulic, caffeic and p-coumaric acids (Malencic et al., 2008). Soybean genotypes are rich in isoflavones are desiderating in nutraceutical industry. Contrarily, soybean genotypes with low-isoflavones are favored in designing soy-based infant formula and in developing soy food products with less astringent taste (Vineet et al., 2015).

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Numerous in vivo and in vitro researches have shown that antioxidant extracts from soybean seed and soy products may increase the antioxidant enzymes comprising superoxide dismutase and catalase and decreases oxidation of low density lipoprotein (Takahashi et al., 2005). Soybean isoflavones and antioxidants varied in quantities in various soy based foods and concentrations can also be influenced by cultivars, extraction methods, and food processing methods (Xu, et al., 2007; Chung et al., 2008).

Figure 2.1: Chemical structure of genistein, genistin, daidzein and 17β estradiol and a representation of genistein metabolism Although soybeans have relatively high antioxidant activities, antioxidants should be retained in gut after release from food following digestion, and should be able to act as antioxidants in the body after absorption. Amount of bioactive compounds released from a food matrix before absorption through intestinal wall refers to bio accessibility. Bioavailability implies that bio accessible compounds are used for their bioactivity in target cells after absorption. Many studies have done to know the bioavailability of isoflavones in soy food, supplements or pure compounds using both in vivo and in vitro methodologies (Williamson and Manach, 2005).

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Bioaccessibility and bioavailability of isoflavones are affected by food processing methods (Bolling et al., 2009) however there is less studies have conducted to know the effect of food processing on bioaccessibility and bioavailability of soy total antioxidants. Few studies have evaluated bioaccessibility of isoflavones from soy isoflavone-enriched food products (Sanz and Luyten, 2007) and these studies did not account for soy total antioxidants. It is important to understand bioaccessibility of soybean antioxidants ingested in diet and their bioavailability to function in biological roles in human body.

Soybeans are leguminous plant and they form root nodules in a symbiotic relationship by Rhizobia that are nitrogen-fixing bacteria that lives in soil. “Isoflavonoids” also known as isoflavones are chemical signals that are released by soybean to attract nitrogen fixing bacteria. Phenylpropanoid pathway produces flavonoids and Isoflavonoids. Two isoflavones, genistein and daidzein are formed in this pathway instigates with naringenin and phenylalanine transformed into genistein by action of dehydratase and synthase. Another intermediate Naringenin chalcone, changed into daidzein due to action of chalcone reductase and type II chalcone isomerase as well as isoflavone synthase which are present solitary in legumes (Deavours and Dixon, 2005). There is no conclusive patient available for trials related to health orientation to check outcomes of diet enriched with soy or isoflavones. Although, researchers performed their tryouts on animals and human being although, an accurate food composition database is crucial for such studies.

2.4.3. Soybean antioxidants and their bioavailability

Different toxic and highly reactive molecules are generated during metabolism of cell e.g. reactive oxygen species that is single oxygen, hydroxyl ion, superoxide and hydrogen peroxide ions. These species damages the cell via oxidative damage of proteins, lipids, enzymes and DNA through covalent binding and lipid peroxidation which ultimately resulted in tissue injury. Natural antioxidant shows mop like affect to scavenge all these free radicals that severely damage the cells and tissues and may be resulted into cancer, neuro degeneration and inflammation (Young and Woodside, 2001).

The increase in production of oxygen simulated from free radicals is responsible to intricate in inception of numerous diseases i.e. cancer, , degenerative process

14 allied with aging, Alzheimer's and Parkinson's affliction. However, it has been recommended by many therapeutically investigations that adding natural antioxidant in diet (polyphenol) could be beneficial to cure these diseases. Soybean has partially been credited as a rich source of antioxidants and full of multifarious components as flavonoids, vitamins, phenolics and anthocyanins (Almeida et al., 2011).

Bioactive compounds naturally occur in less extent in plant and food products and there are three main factors when considering potential antioxidant activities of dietary bioactive compounds. The first concern is that after food consumption, bioactive compounds in food become bio accessible and then are absorbed by intestinal cells. The second concern is that antioxidant activities of compounds are stable during digestion process and persist in plasma after being absorbed into the body. The third concern is that derivatives of compounds after metabolic pathways still have an antioxidant capacity (Martins et al., 2012).

There are numerous studies that have investigated bioavailability of polyphenols as in vitro and in vivo studies (Williamson and Manach, 2005; Parada, and Aguilera, 2007), but few reports have examined bio accessibility of polyphenols. Many studies have examined bioavailability of soy antioxidants (isoflavones) in vitro and in vivo but bioaccessibility of soy antioxidants has not been widely studied. Most studies have focused on bioavailability of soy isoflavone aglycone forms such as genistein and daidzein in human subjects, since these two aglycones are found in relatively high concentration in plasma (Williamson and Manach, 2005).

These studies have supported the idea that absorption of soy isoflavone aglycones is faster than their glycoside forms. When comparing aglycones, plasma concentration of genistein is higher than daidzein after soy intake. For instance, it has been found that plasma concentration of aglycones was >100% higher than of glycosides after soy product ingestion. Within aglycones, concentration of genistein was also five times higher than daidzein in plasma at similar levels of intake (Tsuchihashi et al., 2008). They also reported that bioavailability of soy isoflavones in humans was influenced by factors such as physiological relevant intakes, gender, and age-related differences. Absorption of daidzein was faster in women than men, and absorption of daidzein and genistein from soy milk was faster than from or textured vegetable protein (Tsuchihashi et al., 2008). Isoflavones

15 concentrations in plasma is generally >10 µmol/L (Williamson and Manach, 2005). Isoflavone aglycone forms are generated by microbial activity in large intestinal where aglycones are absorbed. Recent reports have suggested that small intestinal tissues of rats may be involved in conversion of isoflavone glycosides to aglycones (Nemitz et al., 2015; Hirose et al., 2016).

2.5. Health benefits of soybean

Health benedictions of soybean and its related products have strongly recorded. Studies have reported that their consumption may prohibit assertive cancers and it is also effective in limiting risk of hyperlipidemia, cardiac endemic and osteoporosis chronic renal disease lower plasma cholesterol and act as an antiatherosclerotic (GolKhoo et al., 2008). Different health prospects of soybean are discussed here in detail;

2.5.1. Soy intake and protective effect against age-related bone loss

Soy based products have gained much popularity throughout the world and are being used as an alternate to dairy. Soy milk has non-steroidal phytoestrogen that is effective mainly against bone loss especially related to age and many chronic maladies (Cassidy et al., 2006).

Soy foods and isoflavones in them have a potential role in bone health, particularly in postmenopausal women (Reinwald and Weaver, 2005). Several studies have verified a positive relationship between incidence of osteoporosis and chronic intake of soy product or isoflavones. Effect of isoflavones and of soy protein with isoflavones on bone mass was evaluated and suggested that soy intake for more than six months gives best effects by attenuating loss of bone in postmenopausal women (Huang et al., 2006).

It has been indicated that daily supplementation of isoflavones for 6 months has decreased bone loss in postmenopausal women with a dose dependent effect (Ye et al., 2006). Recent studies showed the outcome of soy product consumption on bone health in menopausal or young women. If menopausal women daily consume isoflavones (>90 mg) for 6 months then it may be enough to provide beneficial effects on spine bones (Ma et al., 2009). Soybean and isoflavone consumption also showed positive impact on bone mass density in young women (Song et al., 2008). However, the positive effect of isoflavone on bone strength is

16 controversial because favorable impact of soy isoflavones on bone health is depends on estrogen hormone acceptor numbers in individuals and their certain age period. Further studies in humans are required to strengthen favorable influence of soy, soy products, or soy isoflavone consumption on bone health in women (Reinwald and Weaver, 2005).

2.5.2. Soy intake and as an alternate form of hormonal replacement therapy (HRT)

Menopause is considered as an altered hormones system effects on quality of life as a result of uncomfortable manifestation. One-third of women lifespan may passed in menopause. Considerate menopause-associated pathophysiology and emerging new strategies to improve treatment of menopausal-associated symptoms is a main topic to be discussed. Soy phytoestrogen has also been marketed as an alternate form of hormonal replacement therapy to prevent postmenopausal syndrome or osteoporosis in postmenopausal years (Matthews et al., 2011). These compounds have resemblance of structure with estradiol and they interact with cell proteins and organelle and resulting traditional HRT in situation of menopause (Moreira et al., 2014).

2.5.3. Soy intake as an anti-cancer

Consumption of soybean, a dietary source of (isoflavones), has also been associated with lower cancer risks (Scalbert et al., 2005). Soy isoflavones are dietary compounds and are effective to act as anticancer agent they obstruct transcription factors and genes that are imperative for proliferation of tumor cell, neovascularization and invasion. Bioactive components of soy like isoflavones could be an adequate harmonizing treatment replacing traditional therapies by effecting cancer cell and their mode of action. They act on those genes that are essential for persistence of tumor cell and angiogenesis that makes such cells more delicate for radiotherapy. A research have been done on prostate cancer patients and during their treatment with radiotherapy they were also provided soy isoflavones and they reported that isoflavones were beneficial to improve adversative radiation effects and that maybe by acting as antioxidant. These studies open novel promenade for exploring soy isoflavones as complements to traditional remedies (Hillman and Gupta, 2011).

Epidemiological researches proved that soy products, rich in isoflavones, have a protective role against hormone-related cancers including breast and prostate cancers (Messina and Wu,

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2009; Yan and Spitznagel, 2009), however, a reduced risk was observed only in patients with specific tumor lines, and indicated that protective role of soy against breast cancer depends on the behavior of breast cancer with different estrogen receptor status (Suzuki et al., 2008).

The relation between soy consumption and prostate cancer has been investigated by many researchers. A research was done on the feeding of soy foodstuffs rich in genistein, an isoflavone aglycone, showed a slight protective effect on prostate cancer, however, a meta- analysis was conducted and concluded that consumption of soy food is associated with a reduction of prostate cancer in men. They also suggested that quality and quantity of soy food intake may be related to protect effects against prostate cancer (Yan and Spitznagel, 2009) however; epidemiological studies on association between soybean intake and incidence of cancer are inconclusive (Vemuri et al., 2008).

2.5.4. Soy intake and diabetes

According to global report of world health organization (WHO) on diabetes an estimated prevalence of diabetes in the adult population has approximately raised doubled since 1980 from 4.7% to 8.5%. Globally much of the people are patients of Type 2 diabetes mellitus (T2DM) of all diabetes cases which is an austere metabolic syndrome and alarming situation is number of cases proliferating every year (WHO, 2016). T2DM is an increment for onset of multitudinal ailments such as cancer, myocardial infarction, atherosclerosis, and neurological infection. There are different varieties of soybean and among them black soybean has been reported to have best antidiabetes effect among all. People are using extract of black soybean in Japan as their traditional tea called “Kuromame-cha” which is being consumed for medicinal purposes for diseases is related to inflammation as well as for obesity (Matsukawa et al., 2015).

The relationship was established among soy food or soy protein consumption in women for the extent of type 2 diabetes. Soybean products change bioactive components, such as isoflavonoids and peptides, in ways which may alter their efficacy in treatment of type 2 diabetes. Intake of soybeans and fermented soybeans in Asians may be associated with antecedently lower percentage of type 2 diabetes (Villegas et al., 2008). Momentous decline

18 of plasma glucose has been observed in diabetic rats after consumption of soybean milk kefir (Tyas and Kristian, 2015).

2.5.5. Soy intake and heart health

Soybean is free from cholesterol and also it contains fewer quantity of saturated fat that makes it favorable in reducing cardiovascular diseases (Joelle and Dent, 2011). The effect of soy protein containing enriched or depleted isoflavones on the lipid profiles was determined, it was reported that both soy proteins with or without isoflavones significantly lowered low density lipoprotein (LDL) cholesterol, and soy protein containing isoflavones increased high density lipoprotein (HDL) cholesterol (Taku et al., 2007). In spite of recent results from meta analysis indicating more modest effects than initial reports on effects of soybean on heart diseases, Menissa and Lane concluded that soy foods can help decrease mean serum cholesterol levels of populations and may reduce vascular reactivity because of isoflavones (Messina and Lane, 2007). Soy isoflavones like estrogen act as cardio protector because they act on walls of blood vessels and encourage vascular reactivity. They cause oxidation of low density lipoprotein (LDL) cholesterol its existence in blood vessel walls confer development of atherosclerotic plaques and epidemiological investigations suggested that endothelial function and relaxation of arteries is boost up by soy isoflavones. They may impede effect of endothelial cell in atherosclerosis by hindering provocation of inflammatory cells and adherence to vascular endothelium (Nagarajan, 2010). It has been clinching that atherosclerotic conservation via soy isoflavones is arbitrated by the activation parameter of monocyte (Figure 2.2). Another contrivance that is linked to soy isoflavones about attrition of vascular contraction is through reticence of RhoA/Rho kinase signaling pathway, which is foremost for contraction of muscle contraction (Seok et al., 2008).

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Figure 2.2: (I) Inflammatory process associated with atherosclerosis. II) Soy/isoflavone diet blocks endothelial and monocytes activation start (Nagarajan, 2010)

Isoflavones showed interdict properties on the adipose tissue and battering the plasma lipid profile that will assist in prevention of obesity and its related diseases, however, in vivo research, particularly in humans resulted that soy isoflavones action depends upon gut microbiota and soy proteins (Orgaard and Jensen, 2008).

2.5.6. Soy intake and its role in obesity

Obesity is closely related to metabolic disorders in which extraneous fat augmented in the body, particularly in abdominal adipose tissue. The excess of fat is associated with many diseases which include cardiovascular disorders, diabetes, dyslipidemia, nonalcoholic fatty liver and fitness dilemma (Despres et al., 2008).

Soy proteins are amazing in improving resistance of insulin and to reduce body fat and blood lipids. It also has a wide range of biochemical and molecular actions that positively affect the cholesterol homeostasis by fat or lipid metabolism. Soy protein also effective to reduce obesity chances and main components of soy protein are conglycinin, soy saponins, phospholipids, and isoflavones that are related to obesity (Velasquez, and Bhathena, 2007).

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Another invivo study was done on mice by giving them fermented soybean paste and it showed significant results on obesity and its related parameters (Choi et al., 2016).

2.5.7. Soy intake and its effect on thyroid function

It has been discussed in literature that intake of soy isoflavone intake badly effects on functionality of thyroid glands. Thyroid glands release two important hormones called thyroxine (T4) and trio iodothyronine (T3) in about 80:20 ratio after signaling of thyroid stimulating hormone (TSH). Soy isoflavones interacted with thyroid gland functions by stimulating potent and beta cells in immunity and resulted in structural changes in thyroid peroxidase (Messina and Redmond, 2006).

Another study was done on postmenopausal women for six months by giving those isoflavones and it showed rise in T4 by using 56mg of isoflavones per day and in T3 by using 90 mg isoflavones per day in comparision from control group but these modifications were clinically reflected as non-cogent (Borchers et al., 2008).

The actual reason reported deviations is still not clear due to different factors like concentration of isoflavones and time of treatment may all be considered important factors. It has been narrated in a study that was conducted on children and resulted as beneficial effect of genistein that could impact on thyroid hprmonal function (Milerova et al., 2006)

Different other epidemiologic studies revealed that higher consumption of soy based food causes low occurrence of hormone related ailments. It has been revealed that isoflavones overcome the hepatic thyroid hormone receptor‟s binding ability to the thyroid hormone of specific gene (Adlercreutz et al., 2007).

2.6. Application of soy in food processing

Soybean has performed excellent functionality in the diversification of crops as concluded by different studies. Nowadays, soybean being the protuberant ingredient of numerous food products, endows a concise attention of research owing to its health boosting perspectives. Hence a new line to boost up innovative food products with the addition of soybean must be

21 developed. Contemporary, the nutritional profile of soybean is a new motivation for the industrialists to use their technologies for further processing and utilization.

2.6.1. Soy bran as a fat replacer

Innovations and value addition in food processing industries are attaining huge importance due to changing eating behaviors of the masses in present era. They are more concerned about the food ingredients and preferably used fat and sugar substitutes to fulfill their energy needs without compromising organoleptic properties of the food stuff. Psodorov et al. (2015) used fractionated soy bran as fat replacer in production of cookies and well documented elevated amount of dietary such as pentosans, lignins, inulin, hemicellulose and cellulose in the finished product. They also concluded that soy can be potentially used as fat substitute in industrial confectionary and bakery products.

2.6.2. Soy protein isolate (SPI) and soy protein coatings

SPI is composed of more than 90% protein on dry basis and is comprised on soy storage proteins like glycinin mainly 11S and β-conglycinin mainly 7S that make up to 70% of the protein in SPI (Taski-Ajdukovic et al., 2010). SPI has some extraordinary properties of film formation moreover its economical features for extractions from oil industry waste are remarkable. This type of proteins produces softer, more transparent and stretchy films in comparison to those derived from alternative sources. It acts as a barrier under intermediary moisture environments to oxygen, aromas and lipids. SPI films have developed augmented with starch nanocrystals (SNC) by using simple casting method. Although films were transparent but their opacity and degree of crystallinity was more elevated with amounts of nanocrystals and fundamental results were obtained on different parameters (González and Igarzabal, 2015). The soy protein coating application was found to be effective in procrastinating lipid oxidation and depreciation of beef patty quality throughout chilled storage. These coatings showed more significant results as compared to control and also on hedonic scale by different consumers saying that it did not affect taste as well as prolong shelf life of patties stored in modified atmosphere packaging (MAP) (Guerrero et al., 2015).

2.6.3. Soy milk

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Soy milk is used in ample of products processing like tofu, yoghurt, cheese and many more. Different food items made by using soy milk are a rich in protein, dietary fiber, low in saturated fat free from cholesterol and lactose. It is also rich in iron, and vitamin E. As it is free from galactose, it can safely use an alternate of breast milk in children suffering from galactosemia as well as for lactose intolerant or patients. It can suitably replace weaning formula of cow‟s milk for nourishment of infants (Vij et al., 2011).

2.6.4. Soy in baking products

In baking industry soy flour fortification for diversified baked products has been well documented. Sana et al. (2012) used different soybean mixtures (7%, 12%, 16% and 22%) with wheat flour to increase the nutritional and biological balanced values of bread. The study exhibited that incorporation of soybean mixtures increased water absorption and improved dough weakening while mixing time remains the same for less level of soy flour contents. They concluded that that soybean fortification in bakery products enhances the protein and fiber contents and also elevated certain inorganic constituents with higher biological values. Likewise, Rani et al. (2017) used different concentration of soy and black gram flour for the production of gluten free products and studied the organoleptic and nutritional values along with shelf life of the gluten free products. They testified that mixed flour can be magnificently used for the development of gluten free products (biscuits, macaroni, snacks) deprived of altering the sensory attributes. Soy also has been reported for cracker‟s fortification with significant results. In another study, soy flour fortified graham crackers and non-fortified peanut butter crackers prepared to compare their results. By its fortification there was no change in the moisture contents of all graham crackers. However, during comparison of graham crackers that were fortified by different levels of soy flour, it has been noted that high level of substitution like 100% w/w show less acceptability for desirable flavor as well as aftertaste, as compared to low level of soy flour (Joelle and Dent, 2011). It has also been studied that the soy protein could be used as an effective emulsifier for different product development (Puppo et al., 2008; Garcia et al., 2009).

2.6.5. Soy in desserts

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Soy can be used to develop the products like dessert ice-cream and flavored drink and juices with magnificent taste and flavor. Teh et al. (2005) prepared frozen blueberry-soy dessert with four different formulations of soy protein isolates and blueberry concentrates to appraise the overall acceptability of the product by using 9-point hedonic scale. Study exhibited that dessert containing 8.6% blueberry concentrates attained the highest score as compared to other formulations (10% and 17.2% blueberry concentrate). Similarly, Arancibia et al. (2015) studied the rheological attributes of high proteins soy dessert and concluded that inclusion of soy protein as hydrocolloids may improve the rheological behavior and thickening effect of dessert with acceptable taste. In another study, soy protein was used in guava juice at the level of 22, 27 and 32%. Results showed that guava juice containing 32% soy protein in guava juice is the best proportion for acceptability on the basis of sensory evaluation and other analyses. By the addition of soy these product become a source of vitamin C, fibers, iron and copper (Daniel et al., 2010). Soy protein can also be used in mayonnaise which is used in salad dressing (Puppo et al., 2008). Another research was done by Ahsan et al., (2015) they have used soy milk in preparation of soy ice cream and they stated its potential to commercialize as it was healthy product for vegetarians, lactose intolerant and can be cherished to all age groups.

2.6.6. Soy yoghurt

Soy based products are developed by using probiotics act as functional foods. Soy foodstuffs related to alternate of dairy products are used to overcome limitations associated with dairy products and act as a novel approach in food science. It has been reported that soy milk yoghurt would offer various nutritional benefits in comparison to animal milk yoghurt to consumers. These advantages include reduced levels of saturated fat, cholesterol and lactose (Mishra and Mishra, 2013). Soy milk contains such proteins that promote growth of different probiotics e.g. Lactobacillus acidophilus, Lactobacillus casei and Streptococcus thermophiles during the process of fermentation (Donkor et al., 2005).

In another research, symbiotic soy yoghurt was developed with the aim to improve product quality and consumer acceptability. Results showed that optimized well set yoghurt with 1.14% whey separation was best among all with acceptable textural, nutritional and sensory features (Pandey and Mishra, 2015).

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Another research for soy yoghurt was done by using different combinations of soy milk and cow milk and stored for 20 days in refrigerator. Results showed that 1:1 and 2:1 soy milk: cow milk give intermediate position. It was also found that storage of yoghurt for 10 days gives acceptable results (Osman and Razig, 2010).

2.6.7. Soy whey proteins (SWP)

During preparation of soy protein, isolate and cheese whey is separated out as a waste product but it also has marvelous nutritious components. Soy whey proteins are an efficient source in food applications as long as a superb emulsion activity, solubility, stability, and foaming capacity. SWP contains nutraceutical value because of its anti-hypertensive quality. In western countries, these proteins are used as a substitute source of animal based proteins. SWP shows properties even more than soy protein, SWP comprises of high molecular weight and hydrophobic in nature and effective in cholesterol lowering and binding bile acids (Lassissi et al., 2014).

2.7. Fermentation with lactic acid bacteria (LAB)

The term of fermentation is derived from “fervere” a Latin word which means “to boil”. The word boiling was used because of bubbles produced from carbon dioxide in the absence of oxygen and sugar catabolism in the extract (Stanbury et al., 2013). Fermentation is a metabolic process in which organic material or sugars are converted into gaseous products” like carbon dioxide and liquid e.g. different acids lactic acid, acetic acids, citric acid and different others (Fernández et al., 2011).

It is an economical, low energy used in processing and high efficiency rate with improved aspects of final products such as aroma, shelf life and safety terms. LAB are not new in the field of food technology since a long time in fermentation it is a natural bioprocessing technology used in food processing as well as preservation. Among LAB, main group is genus Lactobacillus, which encompasses more than 150 diverse species (Siezen et al., 2010). LAB has been reported many times to boost host‟s health. Different LAB have different sources of isolation. L. plantarum has sharp commercial application prospects and it is isolated from dairy and fermented products such as wines, cheeses, sauerkraut, olives,

25 sourdough, sausages, pickled vegetables and many more environmental sources (Parente et al., 2010; Bosch et al., 2011).

All LAB are typically Gram positive, catalase-negative, non-sporing, anaerobic as well as aerotolerant cocci or rods that ferment carbohydrates into lactic acid and energy. They are acid-tolerant and strictly fermentative producing lactic acid as main resultant component of fermentation. LAB can be alienated into homo and hetero-fermentative species. Homofermentative LAB converts sucrose in to lactic acid, whereas in heterofermentative e.g. acideuconostoc convert carbohydrates into ethanol, carbon dioxide, and acetic acids (Leroi, 2010).

LAB can grew well in soy milk that means soy milk is a good medium and act as a substrate for their growth. This suggested that LAB (Probiotics) hydrolyzed raffinose which is an α- galactoside sugar in soy milk so it can be suggested that these microbes can show their optimum growth level in soybean and its related fermented products. Donkor et al. (2007) who studied on fermented soy milk and stated that soy milk fermented with lactic acid bacteria having α-galactosidase activity that decreased non digestible carbohydrates contents. At that moment, Osundahunsi et al. (2007) also reported a decrease in raffinose and stachyose content of soy-yoghurt. After that, Amanze and Amanze, (2011) has reported carbohydrate (%) in soy yoghurt was 4.79± 0.06%. Chiang and Pan (2012) also reported that total carbohydrate contents of fermented milks were significantly lower than those of non- fermented milk. Likewise, Obadina, et al., 2013 have reported that carbohydrate in their investigation trial reduced significantly (P<0.05) in non-fermented milk than of fermented soy milk. However, the increase in time of fermentation can be due to variation in growth phase of microbes. Over all LAB change inactive form ofcarbohydrates, proteins, organic acids into active components and also release of aromatic and flavoring compounds as well as of antimicrobial. Beside food products, these bacteria are widespread in nature, as well as genital, intestinal and oral cavities of animal and human (Leroi, 2010).

2.7.1. Lactic acid bacteria in food applications

LAB act as bio-preservative and have status of GRAS playing a remarkable role in variety of food fermentations. LAB are also helpful to remove beany flavor of soy based products

26 hence increase overall flavor acceptability along with anti-cholesterolemic, anti-carcinogenic, and anti-oxidant properties (Gilliland, 2005). LAB are mostly used as probiotics in different fermented dairy and soy milk based food products some of them are also used to get benefits from their expo poly saccharides(Ahmed et al., 2013; Mende et al., 2013). LAB are used as primary starters in cheese or in fermented milks mostly are fortuitous heterofermentative (Ibrahim, 2016).

2.7.2. Lactic acid bacteria benefiting health

LAB are commonly used in the fermentation of different dairy products and beverages. Nutritious aspects of LAB have been known to many years. Some of the benefit includes:

 Lactose intolerance promoted  Shield from gastroenteritis  Deterrence of coronary heart disease  Deterrence of colon cancer, irritable bowel syndrome  Helps in digestion and gut function  Increase mineral bioavailability (Lara et al., 2014).

A research was done on fermented soy milk and its effect on melanogenesis. It was suggested tha beta glucosidase enzyme pointedly increase aglycone contents of isoflavone. Lactobacillus bacterial strains also inhibit antinutritional tyrosinase enzymatic activity and also the production of melanin. Fermented soy milk also repressed expression and activity of tyrosinase. Factors remained un-affected by this pathway were degradation of transcription factors and extracellular regulated kinase activation. So, fermented soy milk prevents melanogenesis and these findings can be helpful in developing novel healthy soy based commodities (Chen et al., 2013).

In another study, a functional product was made from peanut soy milk with inoculation of six different LAB strains. Fermentation was done both as co-culture and single culture. Best results were achieved from co-cultured because all strains were efficient for consumption of available carbohydrates and metabolite extraction as compared to fermentation with single culture. Average results were 78% carbohydrates used from co-culture and 58% from single

27 culture fermentation. Ethanol contents from final product were evaluated as 0.03% (v/v) or might be low from this so it is classified as non-alcoholic soy beverage (Santos and Schwan, 2014).

A study was done on juvenile rats to explore results of soy product fermented with Lactobacillus jugurti and Enterococcus faecium, moreover, complemented with isoflavones on blood lipid, adipose tissue, and glucose levels. Resulted product was offering new tactics to prevent obesity (Marla et al., 2005). Another study was done to investigate activity of LAB against obesity by using twp products of fermented soy milk. Findings showed that both soy milk commendably up-graded lipocyte activity and stifled heparin-releasable lipoprotein lipase activity for reduction of free acid accumulation LAB was responsible for conversion of isoflavones into daidzein and genistein causes improvement in dyslipidemia and decreasing HFD-persuaded obesity (Lee et al., 2013).

2.7.3. Lactobacillus acidophilus

Lactobacillus acidophilus genius Lactobacillus of LAB group are Gram-positive, non-spore- forming bacteria with round ends that occur single or in pairs with short chains of 0.6- 0.9 × 1.5-6 μm dimension. They are classified as obligatory homofermentative and habitually found in GIT, human and animal‟s vagina where environment is quite acidic (Ozogul and Hamed, 2016).

The Lactobacillus strains are well acknowledging as human probiotics owing to massive health promoting prospective. They resist in bile concentration and have ability to adhere with Gut flora and replaced pathogenic bacteria, ultimately deliver positive health benefits to the host. L. acidophilus delivers pronounced organoleptic properties like flavor and aroma to fermented products with extended shelf life. The contemporary marketplace owns variety of dairy and non-dairy products (yogurt, kefir, meat, vegetables/fruits juices) that are inoculated with L. acidophilus with certain health perspectives These products may possibly be used to curb GI ailments like constipation and diarrhea, fight against pathogenic bacteria and reduces cholesterol biosynthesis. It has also ability to inhibit certain intestinal enzymes that converts pre-carcinogens into active carcinogens (Ozogul and Hamed, 2016).

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In another study effects of inulin ( flour) was investigated. Its effect was invetigated throughout 28 days of storage at 4oC on Bifidobacterium animalis Bb- 12 and L. acidophilus La-5 viability in a fermented soy product (FSP). It was also studied for survival of probiotic under in vitro imitation gastrointestinal conditions. During 28 storage days viabilities of probiotic ranged from 8-9 log CFU/g, and inulin flour showed not any affect on viability of these strains. The resistance of Bb-12 was higher to artificial gastrointestinal juices than for La-5, since Bb-12 reduced about 0.6 log CFU/g and La-5 populations 3.8 log CFU/g, during the storage span (Bedani et al., 2013).

2.7.4. Lactobacillus casei

Lactobacillus casei is mesophilic, rod shaped, non-motile, non-sporing, and anaerobic. L. casei can be in assorted environments e.g. in raw and fermented plant and dairy products, intestinal tracts and reproductive systems of animals and humans. L. casei work best at 5.5 pH and produces lactic acid after fermentation that is why it is used to prepare cheeses and yoghurts and many other products. It also decreases cholesterol levels, improve immune response, regulate diarrhea, improve lactose intolerance, prevent intestinal pathogens and act as probiotics (Mishra and Prasad, 2005).

L. casei isolated from different environmental adaptations therefore molecular type is done to know evolution of its different species from various ecological zones (Cai et al., 2007). Genome of L. casei is sequenced to know phylogenetic relationships among numerous groups of bacteria in Lactobacillus. Species boundaries and their naming can be drawn if genome is sequenced (Desai et al., 2006). L. casei genome is also has a specified feature that is csp-A gene 66 amino acid residues. This is a cold shock protein gene that consents bacteria to acclimatize in low temperatures. Study has also revealed that CSP is required not only for cold shock response but for optimal growth in normal, unstressed cells (Sauvageot et al., 2006). L. casei is a facultative anaerobic and it can ferment glucose, galactose, mannose, mannitol, fructose, N-acetylglucosamine, and tagatose and gets energy from fermentation (Cai et al., 2007). Source of isolation also affects properties of bacteria like L.casei isolated from cheese and human gastrointestinal tracts having capability to ferment lactose in contrast to those isolated from plants (Cai et al., 2007).

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The other factors e.g. pH, temperature, oxygen and media play a significant role in growth rate plus activity of L. casei. Main production of fermentation is lactic acid that is obtained by fermenting lactate and glucose. L. casei can adapt to different environmental niches, one of which is gastrointestinal (GI) tract and act as probiotic (Mishra and Prasad, 2005). Probiotics live cells should not be less than 106-107 CFU/g in order to perform their functions (Nebesny et al., 2007).

L. casei is also helpful in maintenance of immune system in GI tract. It stimulates lymphoid tissues in gut and binds itself to luminal surface and provides immunity to the body. It also competes for nutrients against pathogens (Marcos et al., 2006). They can also prevent the growth of pathogens by producing organic acids and hence decreasing pH (Millette et al., 2007). Moreover, L. casei can conceal antimicrobial peptides of cationic, bacteriocins and amphiphilic molecules against pathogens (Sauvageot et al., 2006).

2.7.5. Fermentation of soybean

Fermented foods are well-known for their benefits to human gastro intestinal tract and metabolic fitness (Ouwehand and Röytiö, 2015). Microbes specially known as Microbial Food Cultures (MFCs) are added in different food products to increase physic chemical, nutritional and medicinal properties (Bourdichon et al., 2012). Soybean is mostly fermented by using bacterial cultures e.g. Lactic Acid Bacteria Bacillus spp. and different fungi to make soybean products (Donkor et al., 2005; Kwon et al., 2010). Fermentation in soybean enhances bioavailability of soy isoflavones. A research was conducted in Japan on postmenopausal women and results verified that bioavailability of isoflavones was much higher in fermented soy products enriched with aglycones in comparison to those women who consumed glucoside-rich non-fermented soybeans products (Okabe et al., 2011).

2.7.6. Fermented soy products

Soybean products that are fermented with Bacillus as a solely bacterial culture are: “natto, kinema and chungkookjang” while some are fermented only with specific fungal culture are: “sufu, tempeh and douche” and to modify features in some cases both cultures used in combination e.g. in “” it is a Korean old-style fermented soybean paste.

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Fermentation enhances efficiency of bioactive peptides that has reported by different studies in fermented soy commodities (Rai and Jeyaram, 2015).

Fermentation increases bioavailability of isoflavones and soluble calcium, moreover, it also helps in protein digestion and to boost immune system. 0.1 to 5 mg/g of isoflavones is as glycosidic form that is inactive form which cannot be absorb and conjugate in both fermented and unfermented soy foods that accounts for 80 to 90 percent of isoflavones. Glucoside form in unfermented soy food does not absorb in small intestine because of its high molecular weight and hydrophilicity as compared with fermented soy foods in which glucoside is converted into aglycones and this compound has high permeability. Lactic acid bacteria act as a probiotic in fermented soy milk and it converts glucoside isoflavones into their respective aglycones with no need to add any additional nutrient (Sanjukta et al., 2015). Fermented soy milk by using lactic acid bacteria is a rich source of bioactive peptides and aglycones (Kitawaki et al., 2009).

2.8. Bioactive peptides in fermented soybean

During fermentation of soybean, protein is hydrolyzed into small sized peptides due to action of microbial proteases and release bioactive peptides that can be easily digested (Figure 2.3) (Sanjukta and Rai, 2016). The bioactive peptide can be varying in sequence size from 2-20 amino acids and some may be 43 amino acids “”. Mostly bioactive peptides shows specificity for bio-functional attributes; lunasin has anticancer properties, and it also exhibit other therapeutic properties such as anti-oxidant, anti-hypertensive, antidiabetic, hypocholesterolemic, chemopreventive (Singh et al., 2014).

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Figure 2.3: Formation and biological activity of bioactive peptides in fermented soybean (Sanjukta and Rai, 2016) Soy allergy is very common due to its protein contents but soy milk fermentation degraded protein into di, tri-peptides and oligopeptides protein that does not cause allergy issues. Soy milk fermented with probiotics is an immunogenic agent and enhances immune system of the body. Fermentation at low pH also enhances calcium availability by using its complex carbohydrates. Low pH can have lethal effect on harmful or pathogenic bacteria in the intestine (Vij et al., 2011).

2.9. Bio-functional properties of fermented soy milk

Soy milk is prepared by soaking beans at least for 14 hours in water soaking to enhance malonyl glucoside isoflavone and then blending them with water in definite quantity after that slury is filtered and pasteurized. After that, fermentation produces lactic acid and acetic

32 acids that can affect metabolism of isoflavones and calcium absorption. Another study was done on rats consuming Bifidobacterium soy milk and reported that low density lipoprotein was reduced significantly (Champagne et al., 2009). Similarly, another in vivo study resulted in more absorption of aglycone Isoflavones in soy yoghurt as compared to unfermented soy that was enriched with glucoside (María Landete et al., 2015).

Although soybeans have a great nutritious value but just boiling and then reparation of raw soybeans showed diminutive bioavailability. To overcome its loss of bioavailability, “fermentation” has been extensively pragmatic in food industry to modify mouth feel and texture and . It could be achieved with soaking and heat treatments combined in some products with adding food spices. With enhancement of organoleptic properties and nutrition profile, fermentation has become a safe and valuable tool in value added product development to enhance nutritious compounds in them. There is a huge range of commercially available fermented products in markets. For example, China is popular for its tofu and douche while daily necessities for Japanese families are natto, sufu, and . Indonesians consumed tempeh and Koreans consuming Doenjang and . These are well known and renowned fermented commercially soybean products widely prevalent flavorings in Asian diets (Kwon et al., 2010).

Great attention has sparked in fermented soy products for the development of antioxidant profile (Ping et al., 2012). Fermentation could also be used to prolong shelf life of fermented products as compared to raw soybean materials. In recent study, overall antioxidant capacities were measured by evaluating bioactivity and chemical analyses in different soy products that were commercially available. The results showed that both TFC (total flavonoid content) and TPC (total phenolic content) values and essential amino acids, amplified predominantly after fermentation in contrast of raw soybeans. Profiles of Isoflavone were also altered into aglycone form by activity of β-glycosidase. Fermentation is still an inexpensive method that can produce nutritious substances like isoflavones in aglycone form, free amino acids and antioxidant substances were also increased. This study also suggested a chance for consumers to select commercially fermented soybean products for consideration of food (Xu et al., 2015).

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The study has been done to investigate type of culture suitable for production of fermented soy milk. It was observed that fermented (Lactobacillus plantarum) soy milk had considerably higher water holding capacity, viable cell count, apparent viscosity and exopolysaccharide (EPS) in soy milk. It was suggested that EPS protein develop network of macromolecules of protein that could modify textural feaures of fermented soy milk. In comparison to original soy milk base, characteristic concentrations of flavor compounds were increased in fermented soy milk whereas off flavoring compounds; 2-pentanone, hexanal, and 2-pentylfuran were decreased. Moreover during storage period for 21 days microbial count and viscosity was remained upto significant level (Chengcheng et al., 2015).

Astronauts during long duration space missions face numerous health challenges, including bone decay, diminished immunity and radiations harmful effecr. These problems may be due to changes in their intestinal flora. To help alleviate these challenges, soy foods serve as a nutritious source for space travellers (Buckley et al., 2011).

The study was done to gauge efficiency of soy protein (SP) supplementation to get rid of discomfort and pain associated with Osteo Arthritus (OA). For this study, 135 free living individuals (71 women and 64 men) suffering from OA were selected. These designated persons were not attributed to rheumatoid arthritis or injury was conscripted for placebo- controlled, double-blind parallel research. Overall; SP modified not only OA-associated bone pain symptoms but it also improve quality of life as compared to animal milk protein. More profound results were seen in men as compared to women. Results of cartilage metabolism also strengthen the positive potential of soy protein in comparison to milk protein (Arjmandia et al., 2004).

2.9.1. Angio-tension converting enzyme (ACE) inhibitory peptides

ACE changes angiotensin I into angiotensin II and deactivates bradykinin which is a strong vasodilator therefore, it raises blood pressure and ultimately consequences of heart disease. High blood pressure and hypertension can be decreased by using ACE inhibitory peptides that are normally extracted from protein rich food. Fermented foods that are enriched with protein are satisfactory source of antihypertensive peptides and ACE inhibitory. Hydrophobic amino acids e.g. Ala, , Ile, Try, Trp and Phe or potentially charged amino acids for example arginine,

34 lysine and proline in ACE inhibitory peptide particularly at C terminal position (Haque and Chand, 2008; Rai et al., 2015). During the process of fermentation in soybean protein macro molecules (glycinin and beta conglycinin) break down into ACE inhibition peptide molecules (Kuba et al., 2005). Those can be categorized into 3 groups of e ACE inhibitory peptides.

(1) True inhibitor type: gastrointestinal digestion does not effects on them

(2) Substrate type: gastrointestinal digestion effects on them and converts the peptides with lower activity

(3) Pro-drug type: gastrointestinal digestion converts them into true inhibitors

Results showed that ACE inhibitory activity was increased from 11.55% to 37.61% during process of fermentation and ripening. Antihypertensive peptides and ACE inhibitory peptides have been found in fermented product famous in Japan known as natto fermented by using Bacillus subtilis as a bacterial culture (Ibe et al., 2009).

Another research was done to check antihypertensive activity of the peptide in spontaneously hypertensive rats (SHR) by giving them ACE peptides.The rate of dose administration varied as 1, 10 and 100 mg/kg of their body weight and within 5 h it decreases systolic blood pressure, even at lowest dose (Kuba et al., 2005; Martinez-Villauenga et al., 2012).

In another study, soy milk was fermented by using five different Lactic acid bacteria and results showed that ACE were produced (Tsai et al., 2006). Likewise, ACE inhibitory peptides were also produced when soy milk was fermented by using E. faecium (Martinez-Villauenga et al., 2012).

Soy yoghurt prepared with probiotic strain has also shown results for production of ACE inhibitory activity. Soy yoghurt was made by using cocktail of starter culture S. thermophilus, L. delbruecki and L. Bulgaricus. To get more efficient results probiotic B. lactis, L. paracasei were also used and significant results showed higher ACE activity along with its stability for 28 days at 4°C (Donkor et al., 2007). Hence it can be suggested that soybean products fermented by using different starter cultures and probiotics are potential source of ACE and these peptides can be efficiently show inhibitory ctivities and to cure hypertension.

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2.9.2. Antioxidant peptides

Human bodies have different biochemical processes that lead to oxidation of biomolecules which can damages the tissues or may be death of cells. Antioxidants can imprison the free radicals and prevent the body from onset of diabetes, cancers, arthritis, arteriosclerosis and various neurological issues. The process of fermentation can enhance antioxidant components including isoflavonides, phenolic and flavonoides (Ren et al., 2006; Moktan et al., 2008).

A research was done on soy fermented product and scientists reported that after fermentation when the protein degraded then free amino acids increases which increases bioavailability of antioxidants (Fan et al., 2009). In another study B. subtilis was used to ferment black and yellow soybean varieties and they reported that their antioxidant potential was increased after fermentation (Sanjukta et al., 2015).

2.9.3. Antidiabetic peptides

Diabetes mellitus is metabolic syndrome categorized by abnormal elevation of blood sugar level (glucose) and characterized as type I (T1DM)which is insulin dependent and type II (T2DM) does not depends on insulin (Hamid et al., 2015). Antidiabetic properties of fermented soybean against the type II diabetes mellitus has been well documented by different researchers around the globe (Kwon et al., 2010).

2.9.4. Anticancer peptides

Fermented soybean products (tempeh, tofu, sujae, soy milk and soy infant formula) holds lunasin a bioactive peptide which owns marvelous anticancer properties. Lunasin cancer preventive ability has been well documented by various scientists while carrying different cancer studies (Galvez et al., 2001; Hernandez-Ledesma et al., 2013; Lule et al., 2015). Fernandez-Tome et al. (2014) also enlightened the chemo-protective of lunasin during cell line experiments Lule et al. (2015) also summaries that lunasin owns intrinsic effect against oxidation, inflammation and cancer and it also play a fundamental role for cholesterol synthesis in the body. Even though soybean is opulent complete plant protein source obscured by several nutritional inhibitors. To overcome this problematic factor fermentation may be a great tool which can not only diminish toxins but it also plays a magnificent role in improvement of

36 nutritional quality and bioactivity of fermented soybean products. Peptides produced during fermentation process exhibited bioactive properties such as antidiabetic, antioxidant, antitumor, ACE and antimicrobial property (Chi and Cho, 2016). Bioactive peptides in different soybean fermented products affirmed its features to make its use as a functional food. The type and function of bioactive peptides relies on specific starter and strain with various pharmaceutical and functional attributes and may potentially use as substitutes of synthetic drugs (Sanjukta and Rai 2016).

Table 2.1. Physiologically functional substances in soybeans

Nutrients in Physiological roles soybeans

Proteins Decreases level of cholesterol in serum, prevention of cardiac disorders, reduces total body fat and improves insulin for diabetes

Peptide Aangiotensin-converting enzyme (ACE) inhibitor peptide, boost up immunity mechanism by phagosytosis, poduces collagen and antioxidant peptides

Effective against hypercholesterolemia, against bone loss , act as antioxidant, alleviation of menopausal symptoms, anti wrinkle, anti- Isoflavones cancer

Saponins Anticarcinogenic activities, cardiovascular effects, inhibition of platelet accumulation, HIV inhibiting role

Anthocyanin Anti melanogenesis, anti-inflammatory and photo protection

T activation role and aggregation of tumor cells (Hemagglutinin)

Nicotianamine ACE inhibiton role

(Jooyandeh, 2011)

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2.10. Lactose intolerance

Lactose intolerance occurs in people that lack β-galactosidase in their small intestine so they cannot digest lactose; a milk sugar. Lactose in undigested form reaches the colon where colon microbes ferment it and produce methane and hydrogen gas along with lactate. Person suffering from lactose intolerance shows symptoms of intestinal pain, flatulence and diarrhea (Mlichova and Rosenberg, 2006; Schaafsma, 2008). Customers with such characteristics try to avoid lactose containing products leading to an increase in demand for lactose free products around the world (Harju et al., 2012).

Most of the people normally stop to secrete lactase after weaning and considered as lactose intolerant. Therefore among adults 75% of the world‟s populace is lactose intolerant and they cannot digest lactose. However some of the populations have developed lactose perseverance, in which production of lactase lasts into adulthood (Silanikove et al., 2015). Different food industries are processing lactose hydrolyzed products by using β-galactosidase and they are extracting this enzyme from animals, plants, yeasts, fungi and bacteria (Harju et al., 2012; Husain, 2010). Hydrolyzed products are sweeter because of high monosaccharide content (Novalin et al., 2005; Adhikari et al., 2010; Harju et al., 2012) and contributes in decreasing sensory prospects as well as the physicochemical aspects like syneresis; same was seen when viscosity of yoghurt was effected by using hydrolyzed milk in a test (Ibarra et al., 2012). Another economical way to hydrolyse lactose is to ferment dairy products through co- hydrolyzing (Schmidt et al., 2016).

2.11. Soy allergy

Food allergy is a cogent public health issue which is affecting 3-6% of global population (Sicherer, 2011). symptoms can be gauged from mild to severe; sometimes being life-threatening (Boye et al., 2012). A drug for food is not yet been developed and the only way is to avoid such an allergy is to avoid foods that may cause allergy (Burks et al., 2012). Soybean allergy is one of the most common food allergy especially among children reported about 0.4% of young children (Barni et al., 2015). The soy proteins are very specific and almost 28 soy proteins are to bind with IgE in people who are allersoy-

38 allergic patients, however, only a few of these proteins are considered magic to soy (Kattan et al., 2011).

2.12. Conclusion

Soybean is a bumper crop with magnificent nutrient profile and therapeutic effects. Soy has a diverse range of usage in food, feed and for production of biodiesel. It must be grown and promoted all over the world because of its extensive array of products. A crop rich in protein, no cholesterol, no lactose and little or no saturated fat; beneficent and economic tool to combat food security.

Though its ubiquitousness in the diet, soybean has some limits including unpleasant beany flavor and un-digested fiber by human beings that can cause flatulence. In an effort to overwhelm these limitations and to produce a probiotic novel diet, the fermentation of soy milk with the probiotic cultures of lactic acid bacteria is a great approach. Soy milk has prodigious potential to be used as a supernumerary of animal milk due to its analogous health reimbursement.

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Chapter 3 MATERIALS AND METHODS The recent research was done at Food Microbiology & Biotechnology Lab National Institute of Food Science and Technology, Department of Biochemistry and Biotechnology, University of Agriculture, Faisalabad and in School of Mechanical Engineering, Bindley Bioscience and Whistler Hall of Agriculture at Purdue University, USA.

3.1. Procurement of raw materials

Soybeans were procured from Ayub Agricultural Research Institute (AARI), Faisalabad and National Agricultural Research Centre (NARC), Islamabad Pakistan. Following varieties of soybeans were procured for experimental purpose from the above mentioned research institutes:

Soybean varieties

i. Faisal ii. NARC-II iii. Willium-82 iv. Ajmeri v. Rawal-I

All reagents and standards were purchased from Oxoid (UK), Merck (Germany) and Sigma Aldrich (USA).

Phase-I “Characterization of Soybean”

3.2. Proximate analysis

Soy samples were analyzed for moisture, crude protein, crude fat, crude fiber, ash, and nitrogen free extract (NFE) according to their respective methods as described in AOAC, (2016). Soybeans were ground to fine flour and subjected to further analyses. All the tests were done in triplicates for repeatability and accuracy. Detailed protocols for these methods are given below:

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3.2.1. Moisture content

The moisture content of soybeans was determined by following the method as stated in AOAC (2016). Accordingly, 5 g sample was dried in air forced draft oven (Memmert Germany) and kept at a temperature of 105±5oC until the samples were dried completely. The samples were removed from oven and placed in desiccator to cool down and then weighed the dried samples. The moisture content was determined by following the expression: ( ) ( ) ( ) ( )

3.2.2. Crude protein

Crude protein (%) in soy flour was measured by following kjeldahl method which quantitatively calculates nitrogen in sample. There are three main steps of this method digestion, distillation and titration.

I- Digestion II - Distillation III - Titration  0.1 g soy flour, 3 g  20 mL diluted sample  The distillate was digestion mixture and was distilled with then titrated with 20 mg conc. sulphuric 40% sodium 0.1N Hydrochloric acid was heated that hydroxide (NaOH) acid (HCl) causes oxidation of  The color of the acid  The end point was organic substances changed from purple black to purple color and reduction of to greenish from greenish. nitrogen  Ammonium salt is  End point was black converted into to clear solution ammonia  Mixture was cooled at room temperature and diluted with distilled water up to 100 mL

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After that total Nitrogen % and crude protein was calculated by following the formulae. ( ) ( )

( ) ( )

3.2.3 Crude fat

The soy flour samples were analyzed for crude fat according to AOAC (2016). Oven dried 5 g soy flour sample was placed in thimble of soxhlet apparatus by using n-hexane as an extraction solvent. After three to four washing when the fat was clearly observed in solvent due to change its color, then the sample was removed from apparatus and kept in hot air oven to constant weight and re-weighed to obtain the weight of lipid.

( ) ( ) ( ) ( )

3.2.4. Ash contents

The ash content of soy flour was estimated by following method of AOAC (2016) for which, 5 g sample was directly charred on flame in crucible until there were no fumes coming out. Afterwards sample was ignited in muffle furnace (MF-102, PCSIR, Pakistan) at 550ºC for 5- 6 hrs or until grayish white residues were obtained.

( ) ( ) ( )

3.2.5. Crude fiber

Soybean flour crude fiber was calculated by the method as described in AOAC (2016). The fat-free sample was digested with 1.25% H2SO4 and then with 1.25% NaOH solution in a Labconco Fibertech apparatus. After those samples were filtered and washed with distilled water, the remaining residues were weighed and ignited in a muffle furnace at 550-650oC until gray or white ash was obtained.

The percentage of crude fiber was estimated from the expression given below.

( ) ( )

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3.2.6. Nitrogen free extracts (NFE)

The nitrogen free extract (NFE) of soybean was done by following the equation: ( )

3.3. Fatty acids compositional analysis

Oil was extracted from soybean flour by using hexane as a solvent in soxhlet apparatus and afterwards oil was purified in rotary evaporator. Then Fatty acid Methyl Esters (FAME) were prepared as; the samples of oil were merged in n-hexane (5 mL) by using vortex mixer. After that 250 µL sodium methoxide was added and again vortex for 90 seconds. Then 5 mL of saturated NaCl was added and vortex for 15 seconds and then let it to be stand for 10 min. Then hexane layer was transferred to a vial containing a small volume of sodium sulphate and used for analysis through gas chromatograph (GC) (Agilent 6890) by using flame ionizing detector (FID), Fused silica capillary column at an oven temperature of 90oC for 7 min then raised to 240oC at a rate 5oC/min. and then kept at this temperature for 15 min. However, the injector and detector temperature was 260oC. The flow rate of carrier nitrogen gas was 1.51 mL/min. and the split ratio was 1/50 µL/min. (Ozcan and Juhaimi, 2014).

3.4. Analysis of lipoxygenase (LOX) activity

There are three isozymes in soybean named as LOX-1, LOX-2 and LOX-3. This assay determined the LOX activity on UV-VIS spectrophotometer of LOX-1 and LOX- 2 at 234nm while LOX-3 at 280 nm in soy flour by using substrate (linoleic acid). Soy flour samples were defatted so that fat in sample do not interfere during analysis. According to this method, 1g defatted sample was extracted with 50 mL sodium phosphate buffer (0.2 M, pH 6.8) for 2 hrs in orbital shaker at 25oC after that it was centrifuged at 15,000 rpm for 10 min. The supernatant was used to ascertain the lipoxygenase activities. Enzyme caused an increase in absorbance of 1.0/min. Samples were loaded on ELISA plates and immediately readings were noted at the interval of 0, 1, 2, 3, 4 and 5 min. Lipooxygenase in soybean samples starts the lipid oxidation of Linoleic acid and produces H2O2 and it was recorded on the basis of time. The LOX activity was expressed as a change in absorbance per min (ΔA/min) (Mandal et al., 2014).

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3.5. Mineral estimation

Mineral profile of soybean for iron, zinc, magnesium and manganese concentrations was determined by using atomic absorption spectrophotometer (AAS) (AA240, Varian) whereas calcium, sodium and potassium was quantified using Flame Photo meter (Sherwood Scientific Ltd., Cambridge, Model 410) by following method of wet digestion as dried and ground samples (0.5 g) were digested using 5 mL of 65% HNO3 and 2 mL 35% H2O2 on hot plate until the clear solution appeared. After that the samples were filtered and diluted to make up the volume up to 50 mL with distilled water for further analysis on flame photometer and atomic absorption spectrophotometer (AOAC, 2016).

3.6. Phytochemical screening test

Sample preparation

Soy flour (5 g) each sample was weighed in centrifuge tubes and extraction solvent acetone/ water (50:50 v/v) was added in it and placed in orbital shaker: 300 rpm, 25oC for 3 hrs. After that it was placed for 14 h under dark environment. Later on the extracts were centrifuged for 15 min at 3000 rpm. After filtration the retentate was re extracted by using the same assay and then both extracts were combined and preserved in the dark conditions at 4oC for further analysis of phytochemicals and antioxidants. The extractions were conducted in triplicates for each (Xu et al., 2007).

3.6.1. Total phenolic content (TPC)

This is a quantitative assay to quantify total phenolic contents which is also called as folin- ciocalteu (FC) reducing assay. The phenolic compounds in the samples are oxidized, thus reducing the FC reagent in the solution resulting in blue color as end point which is later on measured on UV-visible spectrophotometer at 765 nm by using gallic acid as a standard (Xu and Chang, 2007; Xu et al., 2015).

3.6.2. Total flavonoid content (TFC)

Estimation of total flavonoid contents in samples were measured on UV-Vis spectrophotometer by using catechin as an external standard. The sample was mixed with

44

1250 µL distilled water and then 75 µL of 5% NaNO3 solution was added in it and kept aside for 5 min. at room temperature. Later on 150 µL of 10% AlCl3.6H2O was added in it and waited for another 5 min. 500µL 1 M NaOH and 275 µL distilled water were added to the mixture. Then the sample mixtures were instantaneously detected at 510 nm (Xu et al., 2015).

3.7. Antioxidant potential in soy flour samples

3.7.1. 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay (DPPH)

DPPH radical scavenging activity is estimation of non-enzymatic antioxidant activity. The stable and non-biological radicals are used in this assay for the estimation of total reducing capacity. Sample extracts/ Trolox standard were mixed with 3.8 mL ethanol solution of DPPH. The mixture kept in the dark for 30 min at room temperature. Thereafter, the absorbance was measured on UV-visible spectrophotometer at 517 nm against an ethanol blank. The free radical scavenging activity of the extracts was expressed as miligram Trolox equivalent (mgTE/g) of samples (Prvulovic et al., 2017).

3.7.2. Ferric-reducing antioxidant power (FRAP) assay

A colorimetric reaction during which Fe3+- (2, 4, 6-Tri (2-pyridyl)-s-triazine) TPTZ complex reduced to the ferrous ion (Fe2+) and end point is blue in color. The anti-oxidant activity of sample is measured by measuring change in absorbance at 595 nm on UV-Vis spectrophotometer. 0.3 M acetate buffer at pH 3.6, TPTZ solution and 20 mM ferric solution were used in this assay. The final working FRAP reagent was prepared freshly by mixing acetate buffer, TPTZ and ferric solutions at a ratio of 10:1:1. The ferric reducing ability of the extracts was expressed as milligram of Trolox equivalents per gram. The results were expressed in mg trolox equivalent per gram (mg TE/g) (Lee et al., 2005; Xu et al., 2015).

3.7.3. (2, 29-Azinobis (3-ethylene benzothiazoline) 6-Sulphonicacid) ABTS

ABTS is an assay in which ABTS is scavenges within 10 min. The ABTS solution was prepared by adding 88 µL from 140 mM potassium per sulphate K2S2O8 and 5 mL of 7 mM ABTS stock solution and it was left overnight in dark bottles for liberation of radicals. After

45 that phosphate buffer saline (PBS) was used to dilute the ABTS solution so that its absorbance can be adjusted at 734 nm. 10 µL samples and 100 µL diluted ABTS solution were coated in 96 wells micro plates. The decrease in absorbance at 734 nm was recorded on UV-Vis spectrophotometer for 10 min at 10 sec interval. Ethanolic solutions containing known amounts of Trolox were used for calibration. The free radical scavenging activity of the extracts was expressed as milligram of Trolox equivalents per gram. The results were expressed as mg trolox equivalent per gram (mg TE/g) (Prvulovic et al., 2017).

Phase II “Value addition through fermentation of soy milk”

3.8. Preparation of fermented soy milk

3.8.1. Preparation of inocula and fermented soy milk

The culture of Lactobacillus acidophilus (ATCC® 4356™) and Lactobacillus casei (ATCC® 393™) were procured from American type culture collection (ATCC). The cultures were inoculated into MRS broth for 72 h at 37oC. Then, it was centrifuged at 5000 rpm for 15 min at 24oC. The supernatant was removed and more MRS broth was added in it and kept under same incubation conditions for second time growth of culture. After two consecutive transfers of the activated cultures they were kept in 1mL of buffer peptone water. The soy milk was prepared by blending soaked soybeans with distilled water ten times of their weight for three min. The resultant slurry was filtered through double-layered cheesecloth to obtain soy milk and then sterilized by heating for 15 min at 121oC in an autoclave. Afterwards 100 mL of sterile soy milk was inoculated with 0.1 mL of inocula. Inoculated soy milk was incubated without shaking at 37oC for 12h. After that it was store in refrigeration temperature 4oC for further analysis (Wang et al., 2006; Ahsan et al., 2015).

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Table 3.1. Treatments used for the production of soy milk

Varieties Treatments Starter culture

NARC-II To Non-fermented soy milk

NARC-II T1 Lactobacillus acidophilus

NARC-II T2 Lactobacillus casei

Lactobacillus acidophilus+ NARC-II T3 Lactobacillus casei

Willium-82 To Non-fermented soy milk

Willium-82 T1 Lactobacillus acidophilus

Willium-82 T2 Lactobacillus casei

Lactobacillus acidophilus+ Willium-82 T3 Lactobacillus casei

Ajmeri To Non-fermented soy milk

Ajmeri T1 Lactobacillus acidophilus

Ajmeri T2 Lactobacillus casei

Lactobacillus acidophilus+ Ajmeri T3 Lactobacillus casei

47

Flow sheet for preparation of Inocula

MRS Broth+Bacteria and Incubation for 72h

Centrifuge after 3days @5000rpm for 15mins 24oC

Remove Supernatent, Add more MRS Broth, Kept for Incubation for 72 h

Centrifuge @5000rpm for 15mins 24oC

Remove supernatant and Add Buffer Peptone water 1mL

Figure 3.1: Flow sheet for preparation of Inocula of Lactobacillus acidophilus and Lactobacillus casei in Broth

48

Flow sheet of fermented soy milk preparation

Washing (100 g soybeans with deionized water)

Soaking (1 kg water for 14 h at 4°C)

Draining and dehuling

Blending (blending with 1000 mL water, 3 min)

Filtering (Cheese cloth)

Autoclave (121oC, 15 min)

Inoculation (0.1mL/100 mL soy milk at 37oC)

Incubation (12 h)

Storage at 4oC

Figure 3.2: Flow sheet for preparation of fermented soy milk

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3.9. Sensory evaluation of soy milk

The sensory evaluation of soy milk was done for colour, aroma, flavor, texture and overall acceptability by following nine point hedonic scale system. The panel of 9 trained judges at National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad evaluated all samples at different periods during storage study. The details are given in Appendix-I (Icier et al., 2015).

Figure 3.3: Fermented soy milk in Incubator

3.10. Analysis of value added fermented soy milk

3.10.1. Compositional analyses of soy milk

Soy milk was analyzed for its composition (fat, protein, ash, SNF and total solids) by following methods of AOAC (2016).

3.10.1.1. Total solids

The total solids in soy milk samples were calculated by following the method as described in AOAC (2016) protocols. According to which 5g soy milk sample in china dish was first kept in water bath at 65ºC for about 15 min. Then in hot air oven at 100ºC until the sample completely dried. Dried sample was cooled in desiccator and then weighed.

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( ) ( ) ( )

3.10.1.2. Solid Not Fat

The total solids-not-fat was determined as described by AOAC (2016). It was obtained by taking the difference between % total solids and % fat content.

( )

3.10.1.3. Protein

Total protein in the soy milk was determined by the method as described in AOAC (2016) protocols. The process for crude protein in soy milk completed in three steps as described below;

i- Digestion

For the estimation of nitrogen content in sample with 3 g digestion mixture and 20 mg conc. sulphuric acid was heated. The heating process stimulates oxidation of organic substances and reduction of nitrogen. The process was completed when the colour of mixture changed from black to clear solution. Mixture was cooled at room temperature and diluted with distilled water up to 100 mL.

ii- Distillation

After digestion, 20 mL diluted sample was distilled with 40% sodium hydroxide (NaOH). The color of the acid changed from purple to greenish and ammonium salt is converted into ammonia.

iii- Titration

The distillate was then titrated with 0.1N Hydrochloric acid (HCl) and the end point was black to purple color from greenish. After that total nitrogen% and crude protein was calculated by following the formulae.

( ) ( )

51

( ) ( )

3.10.1.4. Fat

The fat contents in soy milk were analyzed by following the Gerber method (AOAC, 2016).

10 mL 90% H2SO4 was taken in butyrometer and 11 g of sample was poured with help of pipette after its melting and then finally 1 mL of isoamlyl alcohol wad added and cork was placed on its neck. After proper mixing, butyrometer was placed in centrifuge machine for 5 min at 11000 rpm and 65 ºC. Layer of separated fat was noted on the scale of butyro meter which was expressed as percentage.

3.10.1.5. Ash

Ash content in milk was analyzed by following reference method of AOAC (2016). In this method, 2 g each of the samples was measured into a china dish of known weight, the sample was burnt to ash in a muffle furnace for charring at 550oC. It was then cooled in a desiccator and the weight of the ash was finally determined. The % Ash content was calculated as;

( ) ( ) ( )

3.10.2. Viable cell counts

To enumerate viable cell, 1 mL of each samples was homogenized aseptically with 9 mL of sterile physiological saline (0.85%, w/v) and suitable dilutions were plated on MRS agar (pH 6.2 ± 0.2, Oxoid- Ltd. Basingstoke, Hampshire, England). Confirmed colonies were o counted after anaerobic culture under 5% CO2 at 37 C for 48-72 h. Results were expressed as

Log10 CFU/mL of viable cell count (Chengcheng et al., 2014).

3.10.2. Determination of pH

The pH of milk was measured through electronic digital pH meter (Schott Lab-150) by immersing electrodes of pH meter in soy milk after calibrating the instrument AOAC (2016).

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3.10.3. Acidity

Acidity of samples was determined by titration method as given in AOAC (2016).

3.10.4. Determination of water holding capacity (WHC)

WHC can be defined as the capability of soy milk to hold firmly all or few part of its peculiar water. WHC of the samples was measured by the centrifugation method as detailed by (Isanga and Zhang, 2009; Bian et al., 2016).

Where, Y= weight of sample, WE= Whey expelled after centrifugation

3.10.5. Anti-oxidative activities of fermented soy milk

Soybean varieties were investigated for antioxidants activity including DPPH, FRAP and ABTS assay. The fermented soy milk was centrifuged (5000×g, 10 min) and the clear supernatant was used to check antioxidant potential.

3.10.5.1. Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity assay

DPPH in soy milk was calculated according to the protocol described by Pyo et al. (2005) and Subrota et al. (2013) and details of method were same as explained in section 3.7.

3.10.5.2. Ferric reducing antioxidant power (FRAP) assay

FRAP was calculated in soy milk by following the protocol described by Subrota et al. (2013) and details of method were same as explained in section 3.7.

3.10.5.3. 2,2-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS)

ABTS in soy milk was calculated according to the protocol described by Pyo et al. (2005) and Subrota et al. (2013) and details of method were same as described in section 3.7.

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3.10.6. Rheological analysis

The apparent viscosity of samples was measured using a HR-2 Discovery Hybrid Rheometer and performing small to large amplitude shear rate tests at 25°C. The geometery of instrument was fitted as Cone set at 40MM, 2DEG, cone angle (deg: min: sec) (1:57:47), cone diameter (40 mm) and Truncation (66 micron), gap was 66.0 µm and sweep flow method was used (Maftei et al., 2012).

3.10.7. Microstructure examination by scanning electron microscopy (SEM)

SEM was performed in Agricultural Research at Purdue, according to protocol of Ghosh et al. (2013) with some modifications. The details are given below:

3.10.7.1. Sample preparation

The small quantity of fermented soy milk was taken eppendorf tubes and kept in orbital shaker and then filtered to remove excess of water. The retentate on filter paper was placed on aluminium foil and then dried in hot air oven for 30 min at 65oC. The dried samples were fixed in 7% glutaraldehyde for 4h at 25oC. After that samples were rehydrated with deionised water and dehydrated sequentially in 20, 40, 60, 80 and 95% absolute alcohol, defatted two times in chloroform and preserved in absolute alcohol. The aluminium foil containing specimens was again dried in hot air oven. The dried samples were fractured to reveal the internal structure, fixed in an aluminium stub coated with mica plate of 100 oA thickness.

Figure 3.4: Sample preparation for SEM

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Microscopy was carried out on a FEI NOVA nano SEM Field Emission SEM, (Hillsborough, Oregon, USA); High vacuum imaging, Everhart Thornley detector (ETD) spot size 3, 5KV as noted on each image.

3.10.7.2. Sputter coating

A Cressington 208HR sputter coater with a platinum target sputtered for 30 sec at a 45 degree tilt (no rotation) and again at the opposing 45 degree angle for 30 sec more. The Cressington 208HR High Resolution Sputter coater is sold by (TED Pella, Inc., Redding, California).

Figure 3.5: Soy milk samples on stubs and Cressington 208HR sputter coater 3.10.7.3. Scanning electron microscopy

A platinum target was used 30 sec at a 45 degree tilt, and again in tilted in the opposing direction for optimal coating without rotation. Specimens were mounted vertically for imaging the fracture plane double sided carbon tape with fracture lines parallel to the top of the vertical portion of Ted Pella Large 45 degree/90 degree aluminum stubs for fracture plane imaging.

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3.10.8. Quantification for isoflovone by high pressure liquid chromatography (HPLC)

Standards of genistein and daidzein were purchased from Sigma Aldrich and their solutions were prepared by diluting them in HPLC grade methanol. The purity of the standards was based on the percentage peak area and final concentrations were adjusted on the basis of these purities. Standard curve was obtained by dissolving the standards in mobile phase 85% A (0.1% Acetic acid in water) and 15% B (0.1% acetic acid in acetonitrile). Stock solution was 2.5mg/mL and 5 point standard curve was prepared between 15.6 and 250 µg/mL (Pyo et al., 2005). 2mL of soy milk sample was used for isoflavones extraction by using 8mL of methanol as a solven and then orbital shaking was done at 25 oC for two hours. Then these samples were centrifuged for 25 minutes at 7197 rcf. After centrifugation clear supernatant was shifted to filter device (0.2 µm polyethersulfone memberane with polypropylene housing 50 units- 25 mm diameter) into eppendorf tubes (GolKhoo et al., 2008).

The HPLC system Agilent 1100 series (Agilent Technologies, Hewlett-Packard-Strasse 8, 76337 Waldbronn Germany) a binary system capability is of up to a 10 mL/min flow rate and 400 bar of pressure. The system was equipped with UV-vis detector (254 nm), column C18 with dimensions of 4.6 mm x 15cm, in-line degasser and 100 vial auto samplers. The mobile phase was flow at the rate of 1 mL / min and injection volume of 20 µL. The column temperature was constant at 25oC and the run time was 43 min. Quantitative data for daidzein and genistein aglycones were obtained through comparison with known standards. Data acquisition and analysis is performed using chem station software

3.10.9 Selection of best treatments

On the basis of Physicochemical analysis, antioxidant assay, rheological properties, strucural profile through scanning electron microscopy, bioactive componets and overall acceptability the best treatments among fermented soy milk (FSM) and non-fermented soy milk (NFSM) were selected for efficacy trials.

Phase III “In vivo study to check the hypoglycemic and hypocholesterolemic effect of Soy milk in Sprague Dawley rats”

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3.11. Efficacy trials

The selected best treatments were evaluated using sprague dawley rats as experimental model. For the purpose, male Sprague dawley rats (weighing 200-250 g) were purchased from national institute of health (NIH), Islamabad followed by acclimatizing in the animal room of national institute of food science and technology (NIFSAT). The rats were aclimatized to the laboratory by giving them normal chow diet for one week. The environmental conditions of rom as temperature (21-25ºC) and relative humidity was (50±5%) along with 12 h light-dark cycle were controlled throughout the trial period. The experiment models three different types of studies were conducted by categorizing them as normal, hyperglycemic and hypercholesterolemic group of rats. At the start of bioefficacy trial the rats were sacrificed randomly from each group to get the baseline. The rats were given soy milk at the rate of 8 mL/ (kg.d). Further, the feed and water were provided ad libitum for rats in all groups. The restorative potential of fermented soy milk (FSM) and non- fermented soy milk (NFSM) was evaluated during eight-week trial in all groups. During this trial period the feed and drink intakes was recorded on daily basis however, body weight gain or loss was monitered on weekly basis. Overnight fastened rats were decapitated at the end of the efficacy trials. Blood samples were collected and then for sera collection the blood samples were centrifuged at 4000 rpm for 6 min to collect the sera and it was stored at -80oC in microcentrifuge tubes. Sera samples were evaluated for numerous biochemical assays via Microlab 300, Merck, Germany (Lee et al., 2013; Sartang et al., 2015). The resultant data has expressed as mean ± standard error however graphical representation is on the percent change for each variable calculated by following the formula;

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Table 3.2 Treatment plan for efficacy trials

Study Group Diet

Study I I Non fermented soy milk

Normal Rats II Fermented soy milk

Study II I Non fermented soy milk

Hyperglycemic Rats II Fermented soy milk

Study III I Non fermented soy milk

Hypercholesterolemic Rats II Fermented soy milk

Study I: Normal rats

In study I, rats were divided homogeneously into two groups fed on normal diet along with provision of fermented soy milk or non-fermented soy milk as a drink.

Study II: Hyperglycemic rats

In study II, hyperglycemia was induced intraperitoneally (IP) in overnight fasted rats by using fresh prepared streptozotocin (STZ) injection. The glucometer was used to check glucose level in the blood and if the blood glucose level is above 150 mg/dL then it is indication of diabetes. The rats become diabetic within seven days of injection. The drink fermented soy milk or non-fermented soy milk was given via oral gavage simultaneously to synchronize their effect in respective group.

Study III: Hypercholesterolemic rats

In study III, cholesterol was given along with normal diet in order to induce hypercholesterolemia in rats. In addition to this fermented soy milk or non-fermented soy milk were also given to check their impact concurrently against HDL, LDL, cholesterol and triglycerides.

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3.11.1. Serum lipid profile analysis

Serum lipid profile of rats including total cholesterol, high density lipoproteins (HDL) and triglycerides was measured by using specific commercial kits. All values are expressed in mg/dL (Nikam et al., 2013; Jeong et al., 2010).

3.11.2. Serum glucose and insulin levels

Determination of glucose in plasma was done by using commercial kit and following method stated by (Nurliyani, 2015). Insulin concentration was determined by following chemiluminescent immune assay as stated by (Ejtahed et al., 2012).

3.11.3. Liver function probability

Liver functioning or hepato-protective tests like ALT (alanine aminotransferase), ALP (alkaline phosphatase) and AST (aspartate aminotransferase) were determined according to their respective protocols supported by Lin et al. (2005) and Chiang and Pan (2011).

3.11.4. Renal function tests

The renal functioning test like creatinine and blood urea nitrogen (BUN) concentration in serum was measured using the commercial kit. The results were expressed in mg/dL (Chiang and Pan 2011).

3.11.5. Hematological analysis

Hematological parameters including red and white blood cells (RBC and WBC) and platelets count were assessed by adopting the protocols of Niamah et al. (2017).

3.12. Statistical analysis

The data of each parameter was subjected to statistical analysis i.e. complete randomized design and three factors factorial design under complete randomized design (CRD) was used to analyse product analysis by using statistix 8.1 software to determine results by following guidelines of (Montgomery, 2008).

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CHAPTER 4 RESULTS AND DISCUSSION This research work was designed to screen the soy varieties on the basis of nutritional difference. Top three varieties were selected for the preparation of non-fermented and fermented soy milk by using Lactobacillus acidophilus and Lactobacillus casei solely and in combination. The last module of the research was to check the bio efficacy and therapeutic worth of both non-fermented and fermented soy milk on hypercholesterolemic and hyperglycemic rats. The results of the above mentioned research work is presented and discussed under different categories.

4.1. Proximate analysis

The statistical analysis of the results for proximate compositional profile of five soybean varieties is given in Table 4.1, while the mean values for moisture, crude protein, crude fat, crude fiber, ash and nitrogen free extract (NFE) have been explicated in Table 4.2. The results depicted the significant (P<0.05) difference in ash and moisture contents while highly significant (P<0.01) differences were observed in crude protein, crude fat, crude fiber and NFE contents.

4.1.1. Moisture

Moisture content has significant role due to many technical reasons. Higher moisture in the flour lowers its shelf life. It also exerts strong influence on quality of food products. The soy flour has low moisture content and higher proportion of total dry matter with higher emulsifying properties (Banureka and Mahendran, 2011).

The mean values for moisture contents mentioned in Table 4.2 show the moisture for Faisal, NARC- II, Willium-82, Ajmeri and Rawal-I were 9.15± 0.43, 9.68± 0.47, 9.95±0.48, 9.45±0.46 and 10.49±0.49% respectively. The variation in moisture contents among each other may be due to the difference in the agronomic growing conditions of the lines, the level of moisture at the time of harvest and the processing conditions.

The results of present findings were in accordance with Anwar et al. (2016). They studied the three different soybean varieties and reported that moisture in seeds of different varieties of soybean (Bovender special, Foster and F-8827) was found to be varied in the range of 8.4- 60

10.2%. Previously, Barros et al. (2014) have assessed the chemical composition in soybean treated with gamma irradiation in three different cultivars of soybean 213, 258 and 48 they found that moisture content in them was 9.68, 8.24, and 8.77% respectively. The recent outcomes are also in line with the findings of Lee et al. (2013) who conducted the research on soybeans and that moisture was 9.19% which was very close to Faisal variety 9.15% as in present finding). Another scientist Ozioko, (2012) stated that moisture content in soybean seed as 7.95%. Formerly, Thingom and Chhetry, (2011) estimated the nutrition profile of soybean and reported that moisture content of raw soybean (9.97%).

4.1.2. Crude protein

Soybean seeds contain high quality of protein that can be easily digested from infant to elder. The soy protein is a blessing for vegetarians because it contains all essential amin acids to fulfill their body requirements. However, the poor aminoacid in soybean is methionine but it is an excellent source of lysine which is deficient in wheat (Sanjukta and Rai, 2016).

The mean values of crude protein contents are represented in Table 4.2. The results showed that maximum protein was recorded in Ajmeri (37.65±1.84%) followed by Willium-82 (35.76±1.68%), NARC- II (34.45±1.69%), Rawal-I (32.73±1.60%) and Faisal (30.61± 1.44%).

In the past researchers have done studies on soybean composition and said that it can not be justified if we consider any other plant material as a complete protein source otherthan the soybean. A group of scientists, Anwar et al. (2016) has worked on soybean varieties and reported (41.67- 45.64%) higher crude protein in comparison to recent findings.Likewise, Ozcan and Al Juhaimi, (2014) studied the physicochemical properties of soybean seed and estimated maximum protein contents in the range of 44.81- 47.46% which is higher as compared to present study. However, Barros et al. (2014) also conducted study on chemical composition of soybean. Their finding for protein was in accordance to current results and they reported that the significant variations in different cultivar of soybean varied in between 35.10 to 38.82. Likewise, Lee et al. (2013) has reported crude protein in soybean as 34.19%. The existing results are also lower in protein contents than reported by Sharma et al. (2014)

61 during the study of different soybean genotypes and reported that soybean contained crude protein in the range of 39.4 to 44.4 %.

4.1.3. Crude fat

Soybean oil is a extracted from the seeds of the soybean. Oil contents in soybean seed varied in between 83 g/kg to 279 g/kg with a mean of 195 g/kg that‟s why it is considered as an oil seed crop (Wilson, 2004).

The crude fat estimated from soy bean flour is mentioned in (Table 4.2) for Faisal, NARC- II, Willium-82, Ajmeri and Rawal-I varieties. The values represented substantial variation in fat contents as maximum fat was recorded in Ajmeri (22.20±1.02%) trailed by Willium-82 (21.3 ±0.94%), NARC-II (19.2 ±0.83%), Rawal-I (18.03 ±0.74%) and Faisal (16.27±0.67%).

The results of present finding were supported by Anwar et al. (2016) who have determined the soybean oil content by using hexane as a solvent and stated that oil contents of soybean seed varied in the range of 15.85-19.49%. Later on, Barros et al. (2014) researched on three soybean cultivars they reported crude fat was in the range of 19.63 to 24.00%, Likewise, in the same year Abubakar et al. (2014) also reported the fat in soybean varied in the range of (17.0 -21.0%). The current outcomes are also in line with the outcomes of Lee et al. (2013) who reported crude oil in soybean was 14.45%. The existing results are also comparable with Sharma et al. (2014) they evaluated physical appearances and nutritional contents of new soybean genotypes and reported that oil contents varied in between (14.0-18.7%). Another scientist Ozioko, (2012) determined the percentage oil content of soybean seed was found to be 18.25% while studying on extraction and characterization of soybean oil based bio- lubricant.

4.1.4. Crude fiber

Dietary fibers mainly consist of lignin, polysaccharides and related constituents that cannot digest and absorb in the small intestine and undergoes fermentation in the large intestine of . The foods with higher fiber contents are considered as a functional food because they help to reduce cholesterol level and prevent from cardiovascular diseases certain types of cancers beyond their nutritional values (Poutanen et al., 2014).

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The results of recent findings for crude fiber are represented in Table 4.2. The results in the form of mean values are showing that crude fiber has a highly momentous difference among varieties as maximum fiber was estimated in Rawal-I (17.67 ±0.87%) and minimum in Ajmeri as 13.33 ±0.65%.

The results of recent findings are in accordance with Ferreira et al. (2015) they have studied on the varieties of Brazilian soybean for estimation of dietary fiber contents in them. The dietary fiber components of soybean samples measured by an enzymatic-gravimetric method were in the range of 10.64-19.50%. Moreover, Barros et al. (2014) analyzed the crude fiber while doing research on chemical composition in three different cultivars of soybeans and the fiber contents reported were 17.68, 16.89 and 16.01%. However, the outcomes of Anwar et al. (2016) were contradictory to present findings. They studied on three soybean varieties and reported lower fiber contents than present findings as (Bovender 6.60 ± 0.24%, Foster have7.10 ± 0.25% and F-882 7.60 ± 0.23%).

4.1.5. Ash

The ash is inorganic residue leftover when heat is applied and organic components such as moisture, fat and protein are removed by burning. It helps to determine the mineral makeup of the food, nutritional value and quality. Ash is composed of minerals and because they are elements they will always exist in the exact form as they are. Once our body digests the carbohydrates, proteins, vitamins and fats are burn out physiologically. The ash is what is left over, and more specifically what is left over are the minerals. In food industry, amount of ash or particular minerals is very important; Table 4.2 is depicting mean values for ash contents among different soybean varieties. The mean values for ash in Faisal showed ash contents as 4.00±0.19%, NARC- II (5.13±0.25%), Willium-82 (5.37±0.24%), Ajmeri (5.63±0.28%) and Rawal-I (4.56±0.22 %). The findings of current study are in agreement with Anwar et al. (2016) who stated that ash contents in soybean varied in the range of 5.50 ± 0.36 to 6.90 ± 0.26%. Likewise, Barros et al. (2014) reported ash contents 4.67, 4.92 and 5.46% in three different soybean cultivars. Similarly, Lee et al. (2013) has reported crude ash in soybean was 5.24%. Previously, Porter and Jones (2003) calculated mean value of ash in soybean (6.31%) while studying on soy flour and their mean value was little bit higher than present highest findings (5.63% in Ajmeri).

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Table 4. 1. Mean squares of proximate composition for soybean varieties

Crude Crude Source DF Moisture Crude Fat Ash NFE Protein Fiber

Varieties 4 0.782* 39.94** 17.3817** 9.46** 1.293* 175.39**

Error 10 0.216 2.92 0.6873 0.539 0.5867 6.508

Total 14

* = Significant (P<0.05) **= Highly Significant (P<0.05) DF= Degree of freedom Table 4. 2. Proximate composition of soybean varieties

Varieties Moisture Crude Crude fat Crude fiber Ash (%) NFE (%) (%) Protein (%) (%) (%)

Faisal 9.15± 0.43b 30.61± 1.44d 16.27±0.67c 14.27± 0.67c 4.00±0.19d 25.60±1.20a

NARC-II 9.68±0.47ab 34.45±1.69bc 19.2 ±0.83b 15.87±0.78b 5.13±0.25b 10.06±4.27bc

Willium- 9.95±0.48ab 35.76±1.68ab 21.3 ±0.94a 16.76±0.79ab 5.37±0.24ab 14.10±3.43b 82

Ajmeri 9.45±0.46b 37.65±1.84a 22.20±1.02a 13.33 ±0.65c 5.63±0.28a 11.71±0.35c

Rawal-I 10.49±0.49a 32.73±1.60cd 18.03±0.74b 17.67 ±0.87a 4.56±0.22c 17.05±1.05a

Results are expressed as mean ± standard deviation of means; n = 3 sets abcd Means in column with different superscripts differ (P<0.01)

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Figure 4.1: Proximate characteristics of soybean varieties

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4.1.6. Nitrogen free extract (NFE)

NFE is the fraction that contains the sugars, starches and small quantity of other materials. The mean values for NFE given in Table 4.2 is depicting NFE were highest in Faisal as 25.60±1.20% and lowest in NARC-II as 10.06±4.27%.

The outcomes of recent research work are in line with Shurtleff and Aoyagi, (2016). They has reported nitrogen free extract in green soybean was 38.6% and in another study Barros et al. (2014) has reported NFE in different soy cultivars varied in the range of 16.41 to 22.65% .

The graphical representation (Figure 4.1) shows a clear picture of variation in composition among different varieties and it is showing that Ajmeri contains high amount of protein, fat, fiber and ash which signifies its nutritional attributes while Faisal was high in NFE contents and Rawal-I was high in moisture contents.

4.1.2. Mineral content of soybean

Minerals are essential for proper functioning of body and synthesis of hormones and enzymes (Costa et al., 2015). Soybean contains calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn). Statistical analysis of these minerals in soybean varieties is mentioned in Table 4.3. The results represented the highly momentous variation (P<0.01) in mineral contents of varieties and the mean values for mineral profile are illustrated in Table 4.4.

4.1.2.1. Potassium (K)

Potassium is one of those fantastic mineral that helps to control blood pressure and ultimately cardiovascular infirmities like heart stroke and coronary heart diseases and also to increase bones marrow density. The negative aspects of sodium that may be the cause of increase in blood pressure can be control by increasing intake of potassium in diet. World health organization (WHO) recommended that in adults its adequate level should be 3500 mg/day (Allen et al., 2006).

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The mean values presented in Table 4.4 showed that there was great variation in varieties of soybean regarding the potassium contents and it was at highest level than other minerals. The highest potassium value was noted in Ajmeri (1983.13±39.66 mg/100 g) followed by the NARC-11 (1894.37±37.89 mg/100 g), Willium-82 (1796.44±35.93 mg/100 g), Rawal-1 (1649.24±32.98 mg/100g) and Faisal (1098.54±21.97 mg/100g). The difference of potassium content between Ajmeri and NARC-II is non-significant.

The results of present research are in accordance with Porter and Jones (2003) who studied the composition of soy flour and reported K as 2333 mg/100g. Similarly, Shi et al. (2010) has documented the highest potassium concentration in the germinated soybean as 1900 mg/100 g. Later on Jiao et al. (2012) studied the composition of different varieties of soybean grown in US and Argentina and reported that potassium concentration varied from 1522.25 ± 40.00 to 1663.54 ± 24.45 mg/100g, which is close to the present finding. In the same year Ozcan and Al-Juhaimi (2014) worked on soybean composition and they also reported the highest level of potassium than the other minerals that varied at the level between 1637.5 mg/100g to 2035.7 mg/100g. After three years another study was conducted by Costa et al. (2015). The findings of their study (1363.42 mg/100 g) fall in the range of potassium content determined in the present work (1098.54 to 1983.13 mg/100g).

4.1.2.2. Magnesium (Mg)

Magnesium is essential for various cellular reactions e.g. to synthesize fatty acids and activate different amino acids for the formation of proteins, phosphorylation of glucose and in citrate and trans- ketolase reactions. Therefore the deficiency of magnesium may cause serious health disorders related to biochemical and symptomatic changes (Achanta et al., 2007).

Magnesium was the second highest mineral found in soybean varieties as indicated from results reported in Table 4.4. It is obvious from the results that mean values of magnesium for Faisal, NARC- II, Willium-82, Ajmeri and Rawal-I were 206.12 ± 4.12, 328.27 ± 6.57, 384.43 ± 7.69, 379.82 ± 7.60 and 267.39 ± 5.35 mg/100g respectively. The highest content of magnesium was in Willium-82 follwed by the Ajmeri, but the difference between these two

67 varieties was non-significant. Similar to the other mineral content, the minimum magnesium was recorded for Faisal.

The results of the present finding are in harmony with Costa et al. (2015), they reported that mean value for Mg content in soybean was 218.52 mg/100g. It is reported by vaious scientists (Ozcan and Al-Juhaimi, 2012; Jiao et al., 2012) that as the germination of soybean increases, the Mg content due to higher concentration of inorganic substances during formation of sprouts and germination. Shi et al. (2010) have documented the Mg concentration in the germinated soybean approximately of 33000 mg/100g. Moreover, Porter and Jones (2003) reported Mg as 320 mg/100g in soy flour.

4.1.2.3. Calcium (Ca)

Calcium is one of most crucial mineral for human health stratum. The 99% of calcium is found in bones and teeth while only 1% in sera. Calcium, vitamin D and phosphorous are fundamental elements for the bone formation and strengthening. Unfortunately, if its level is decrease below the required limit in serum, prone to the indication of bone weakness and teeth decay or loss (Beto, 2015).

The mean values for calcium in soybean varieties; Faisal, NARC- II, Willium-82, Ajmeri and Rawal-I, were 148.26 ± 2.97, 202.43 ± 4.05, 241.35 ± 4.83, 277.14 ± 5.54 and 189.45 ± 3.79 mg/100g respectively (Table 4.4). The result revealed the highest content in Ajmeri and lowest in Faisal, however the difference between Willium-82 and NARC-II was non- significant.

The outcomes of recent findings are in harmony with Porter and Jones (2003) who studied the soy flour composition and reported Ca as 321 mg/100g. Likewise, Costa et al. (2015) studied the mineral profile of soybean and recorded maximum level of Ca as 183.12 mg/100g. The results of present study are in harmony with Ozcan and Al-Juhaimi (2014) who reported calcium contents in soybean as 287.9 mg/100g. Likewise, the results of current research were supported by Jiao et al. (2012) who worked on the configuration of five different soybean varieties grown in US and Argentina and they reported that Ca varied in between 126.26 ± 7.11 and 228.05 ± 8.40 mg/100g. Contradictory to present finding

68 regarding the Ca content, a research reported by Shi et al. (2010) presented the lower content of calcium (80 mg/100g) in the germinated soybean.

4.1.2.4. Iron (Fe)

Iron is an essential mineral that is necessary for the blood production in body and also in various redox reactions. It is a major component of hemoglobin and muscle tissues. Its deficiency may lead to anemia, loss of control on body temperature and problems in the release of thyroid hormones. Insufficient iron in diet resulted in stunted growth and obesity in childhood (Achanta et al., 2007).

The results pertaining for iron concentration in soybean elucidated in Table 4.4, shows the highest iron content in Ajmeri (24.26±0.74 mg/100g) and lowest in Faisal (8.48±0.26 mg/100g). The results revealed the non- significant difference between NARC-II and Willium-82 (16.94±0.52 and 16.72±0.51 mg/100g respectively).

The results of current findings are in harmony with Costa et al. (2015) who has reported that iron contents were 8.84 mg/100g in soybean. Wan et al. (2010) also determined the mineral composition of soybean grown in different areas and reported the maximum content of Fe was 7.09 mg/100g. Similarly, Porter and Jones (2003) studied on soy flour composition and reported Fe contents as 7.44 mg/100g.

4.1.2.5. Zinc (Zn)

Zinc is a part of various proteins and enzymes. It is essential for binding of several transcription factors and stability of hormone receptors. It is important for intelligence development in human being. However, its scarcity may lead to retard growth, loss of immunity, night blindness, behaviour changes, hindered wound healing and decreased taste (Achanta et al., 2007). The mean values of Zn in different varieties of soybean have been given in Table 4.4. The highest content of Zn was noted for Ajmeri (5.89±1.42 mg/100g) and Willium-82 (5.24±1.42 mg/100g), while the lowest was found in Faisal (3.82±0.69 mg/100g). A non-significant difference was recorded among NARC- II, Faisal and Rawal-I.

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The results of present findings were supported by Porter and Jones (2003) who reported the Zn content in soy flour and reported as 5.15 mg/100g. Likewise, the zinc contents reported by Costa et al. (2015) were (5.32 mg/100g) and Lestienne et al. (2005) were (3.64±0.19mg/100g) supported the result of present investigation. The findings of Jiao et al. (2012) for Zn element varied from 2.21±0.08 to 3.88±0.24 mg/100g and were also lower than present findings.

4.1.2.6. Copper (Cu)

Copper help the body in the formation of red blood cells along with the iron. It also helps to keep the blood vessels, , immune system, and bones healthy (Achanta et al., 2007).

The mean values for copper are given in Table 4.4 for the soybean varieties like Faisal (0.72±0.02 mg/100g), NARC- II (1.44±0.04 mg/100g), Willium-82 (2.17±0.05 mg/100g), Ajmeri (5.23±0.13 mg/100g) and Rawal-I (1.08±0.03 mg/100g). The results depicted that there was a momentous variation in varieties for the copper, but similar to the other minerals elements, copper concentration was highest in Ajmeri and lowest in Faisal.

Porter and Jones (2003) analysed the soy flour and reported copper as 1.63 mg/100g. The level of copper (1.91 mg/100g) in soybean reported by Costa et al. (2015) was also in harmony to the current finding. It is concluded from the results of all mineral elements that maximum mineral contents were found in Ajmeri trailed by Willium-82, NARC-II, Rawal-I and Faisal.

4.1.2.7. Sodium (Na)

Sodium is most commonly used mineral in our daily diet in the form of table salt (NaCl). According to American Heart Association its level should not exceed from 2,300 mg/day. The daily recommended level should not exceed above 1,500 mg/day. No doubt it is an important mineral that plays a fundamental role to maintain normal physiological function. However, the sweating in body causes its dramatic loss that can be cover up through dietary intake (Valentine, 2007) particularly in hot weather.

The mean values of sodium depicted that soy milk is a good source of sodium. Its contents was recorded as 2.38 ± 0.10 mg/100g in Faisal, 3.69 ± 0.15 mg/100g in NARC- II, 3.23 ±

70

0.13 mg/100g in Willium-82, 4.4 ± 0.18 mg/100g in Ajmeri and 2.92 ± 0.12 mg/100g in Rawal-I (Table 4.4). It is obvious from the result that the difference in sodium content of Ajmeri and Willium-82 was non-significant. The sodium content of the other three varieties was lower than these varieties (2.38 to 3.69 mg/100g) but the difference was non-significant with each other. The sodium content determined by the Costa et al. (2015) was higher (12.08 mg/100g) than the present findings (2.38 to 4.4 mg/100 g) which could be due to difference of climate, soil, temperature and climate.

4.1.2.7. Manganee (Mn)

Manganese plays a role for activation of enzymes and is also an important component of many metalloenzymes. Its shortage causes decrease in growth, skeletal abnormalities, alteration in reproductive systems and also impairment in lipid and carbohydrate metabolism (Achanta et al., 2007).

The results of current investigations in Table 4.4 shows mean values of manganese in five varieties varies from 1.05±0.02 to 3.76±0.16 mg/100 g. Similar to the results of other mineral content, the highest value of manganese was recorded for Ajmeri followed by the NARC- II, Willium-82, Rawal-I and Faisal. The difference in Mn content of Rawal-I and Willium-82 was non-significant.

The results of present investigation are in accordance with the findings of Costa et al. (2015) who reported the 1.98 mg/100g manganese content in soybean. Likewise, Wan et al. (2010) reported the maximum 3.26 mg/100g content of Mn in Jiangxi soybean, which was significantly higher than the other varieties. Similarly, Porter and Jones (2003) reported Mn in soy flour as 3.11 mg/100g.

The contents of minerals were as K > Mg > Ca > Fe > Zn > Cu > Na > Mn. The significant difference in minerals could be due to the difference in soil chemistry and agronomic practices as reported by the Porter and Jones, 2003). This could be also due to rotation and continuous soybean cropping in the same field which could affect the seed composition (Bellaloui et al., 2010).

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Table 4.3.Mean squares of minerals for soybean varieties

Source D K Mg Ca Fe Zn Cu Na Mn F

Varietie 4 367761* 17477.8* 7325.3 112.02* 86.7 9.88 1.76* 3.38* s * * 6 ** * 1 ** ** * *

Error 10 1174 41.1 18.71 0.25 1.14 0.00 0.02 0.001 4 0

Total 14

** = Highly Significant (P<0.01) DF=Degree of freedom Table 4.4. Effect of soybean varieties on mineral contents (mg/100g)

Varieti K Mg Ca Fe Zn Cu Na Mn es

1098.54±2 206.12± 148.26± 8.48±0. 3.82±0 0.72±0 2.38±0 1.05±0 Faisal 1.97d 4.12d 2.97e 26d .69b .02e .10b .02d

NARC- 1894.37±3 328.27± 202.43± 16.94±0 4.64±0 1.44±0 3.69±0 1.66±0 II 7.89a 6.57b 4.05c .52b .81b .04c .15b .23b

Williu 1796.44±3 384.43± 241.35± 16.72±0 5.24±1 2.17±0 3.23±0 1.57±0 m-82 5.93b 7.69a 4.83b .51b .42a .05b .13a .14c

1983.13±3 379.82± 277.14± 24.26±0 5.89±1 5.23±0 4.4±0. 3.76±0 Ajmeri 9.66a 7.60a 5.54a .74a .42a .13a 18a .16a

Rawal- 1649.24±3 267.39± 189.45± 10.83±0 4.23±0 1.08±0 2.92 1.49±0 I 2.98c 5.35c 3.79d .33c .71b .03d ±0.12b .14c

Results are expressed as mean ± standard deviation of means; n = 3 sets a-e Means in column with different superscripts differ (P<0.01)

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4.1.3. Soybean fatty acids profile

Soybean oil is a major source of the essential fatty acids (linoleic, and linolenic) for humans. A lack of the daily requirements of these fatty acids can lead to serious health problems like diminished growth, depigmentation, fatty livers, kidney degeneration and the loss of muscle tone. Soybean oil consists of almost 16% saturated fatty acids, 24% monounsaturated fatty acids and 60% polyunsaturated fatty acids (linoleic and linolenic). The storage instability of soybean oil is just because of polyunsaturated fatty acids (Gillman et al., 2014).

The mean values of fatty acids detected in soybean oil are illustrated in Table 4.5 which showed that saturated fatty acids (SFAs) found were myristic, palmitic, stearic, behenic and arachdic. The mono unsaturated fatty acids were the oleic and eruic acids, while the poly unsaturated fatty acids determined were linoleic and linolenic fatty acids.

The saturated fatty acids are considered as bad for health because they are related to synthesis of cholesterol in human body. The maximum concentration (0.12±0.03%) of myristic acid was noted in Rawal-I and minimum was inWillium-82 (0.08±0.06%) but not detected in NARC-II and Faisal. Palmitic acid was the 3rd dominant fatty acid in soybean oil. Its highest concentration (12.47±0.31%) was found in Faisal and lowest (8.23±0.12%) in Ajmeri. The stearic fatty acid was at the 5th number regarding its strength in in the soybean oil. The highest concentration of stearic acid was in Rawal-I as 5.43±0.14% and lowest was in NARC-II as 2.72±0.07%. Arachdic acid was higher in Faisal variety (0.36±0.01%) and minimum in NARC-II (0.123±0.03%). Behanic acid was higher in Willium-82 (0.23±0.16%) and minimum in Rawal-I (0.13±0.04%) and not detected in Faisal.

The Oleic acid (monounsaturated fatty acid) is desirable for oil stability and is the 2nd major fatty acid of soybean oil. It was found highest in Ajmeri (34.56±0.52%) while the minimum level was noted in Faisal (22.19±0.55%) variety of soybean. The linoleic and linolenic are very important for the human health and must be obtain from diet. The quantity of linoleic was highest in soybean oil than other fatty acids, while linolenic was at the 4th position regarding the concentration of fatty acids in soybean oil. The maximum level of linoleic acid was in Ajmeri (56.34±0.84%) and minimum in Rawal-I as 49.25±1.23%. Likewise, linolenic acid was higher in Ajmeri as 8.23±0.12% and minimum in Faisal as 5.23±0.13%. Erucic acid

73 is monounsaturated omega-9 fatty acid, it was higher in Ajmeri as 0.42±0.006% and was not detected in Rawal-I, Faisal and NARC-II.

The results of current investigations are in line with findings of Wilson, (2004) who worked on the fatty acid composition of soybean oil and reported that average contents of palmitic, stearic, oleic, linoleic and linolenic acids were 11, 4, 23, 55 and 8% respectively.

The findings of another group of scientists, Jiao et al. (2012) have also supported the current research. They have reported fourteen fatty acids in different soybean varieties detected through GC-MS. The outcomes were reported as oleic acid 27%, linoleic acid 53%, palmitic acid 11% and linolenic acid 6%, which were the major fatty acids of soybean oil relative to their contents.

Later on, Ozcan and Al Juhaimi, (2014) studied the effect of sprouting and roasting processes of soybean oils‟fatty acid profile through GC-MS. They described that palmitic, oleic and linoleic acids were found as the major fatty acids of soybean genotypes. They reported a wide range of fatty acids in soybean oil i.e. oleic acid contents varied between 19.07% and 35.31%, linoleic from 42.17 to 54.76% while linolenic acid varied from 5.09 to 6.48% and stearic acid from 0.16 to 4.48%.

The results of current finding are also in harmony with the results of Bellaloui et al. (2010), who studied the influence of soybean corn rotation on the fatty acid profile of soybean oil. They found that oleic acid varied from 28.50% to 20.0%, stearic acid 3.47% to 3.43%, palmitic acid 11.12 to 11.40%, linoleic acid 61.24% to 61.18% and linolenic acid 5.27% to 5.03% with cropping rotation. They stated that variation in fatty acids composition due to cropping rotation could be due to maintenance of optimum nutrient concentrations in soil.

4.1.4. Lipoxygenase (LOX) activity

The off-flavor development in soybean based food and oil industry is considered as a serious problem. LOX acts as a good biocatalyst of cis poly unsaturated fatty acids. Normal mature soybeans contain three lipoxygenase (LOX) isozymes, LOX-1, LOX-2, and LOX-3.

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Table 4.5: Fatty acid (%) profiling of soybean varieties

Sr. Fatty Carbon No Acids Number Faisal NARC-II Willium-82 Ajmeri Rawal-I

1 Myristic C14:0 NDd NDd 0.08±0.06c 0.098±0.01b 0.12±0.03a

2 Palmitic C16:0 12.47±0.31a 9.35±0.37c 10.54±0.74b 8.23±0.12d 9.83±0.46c

3 Stearic C18:0 3.12±0.08d 2.72±0.07e 4.57±0.40b 4.01±0.06c 5.43±0.14a

4 Arachdic C20:0 0.36±0.01a 0.123±0.03e 0.16±0.02d 0.264±0.04c 0.34±0.09b

5 Behenic C22:0 NDd 0.20±0.05b 0.23±0.16a 0.18±0.03bc 0.13±0.04c

6 Oleic C18:1 22.19±0.55d 25.54±0.63c 32.75±2.29a 34.56±0.52a 29.23±0.73b

7 Linoleic C18:2 53.67±1.34ab 49.57±1.24c 52.45±3.67bc 56.34±0.84a 49.25±1.23c

8 Linolenic C18:3 5.23±0.13e 5.7±0.14d 6.43±0.45c 8.23±0.12a 6.92±0.17b

9 Erucic C22:1 NDd NDd 0.32±0.022b 0.42±0.006a NDd

Results are expressed as mean ± standard deviation of means; n = 3 sets abcd Different supersript in row vary significantly

Figure 4.2: A chromatogram of fatty acids peaks in Ajmeri through GC-MS

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The process involved the reaction between LOX isozymes that require substrates containing polyunsaturated fatty acids, including linoleic acid and linolenic acid which undergoes hydro peroxidation, thus generating physiological reactive species, including LOO˙, LO˙, HO˙ and O2. However, the antioxidants in soybean help out to reduce the rate of oxidation (King et al., 2001). The polyunsaturated fatty acids contribute to beany or grassy flavor in soy foods. The major flavoring component in soybean is n-hexanal, produced from peroxidation of linoleic acid by lipoxygenase (LOX), which decompose and form hydroperoxide (Barros et al., 2014). LOX promotes the destruction of free and esterified linoleic and linolenic acids.

4.1.4.1. LOX-1

The statistical analysis of the results regarding the LOX activities shows the significant difference (P<0.01) in LOX activities among varieties like Faisal, NARC-II, Willium-82, Ajmeri and Rawal-I. The LOX-1 activity of five varieties is presented in Figure 4.3. It is apparent from the graphical presentation that as the time period increased the mean values of absorbance also increased in all the varieties which showed the lipoxygenase 1 activity of the varieties. It is also noted that minimum absorbance was recorded at 0 min in Ajmeri as 0.24±0.006 and maximum at the same time was recorded in Faisal as 0.36±0.01. The highest value was recorded in Rawal-1 as 1.23±0.018 and minimum value was noted in Ajmeri as 0.87±0.013 at 5 min.

The Figure 4.3 elaborated the conventional shape of kinetic curve of all the varieties. The shape of the curve changed from conventional „rectangular hyperbola‟ to „plateau‟ shaped. The major reason behind this shape of curve is the initial burst phase rapidly decreased with increasing time and among the varieties. Ajmeri showed minimum absorption as compared to other varieties which means it contains less LOX 1 and low rate of hydro peroxidation followed by the NARC-II, Faisal, Willium-82 and Rawal-I (Figure 4.3).

4.1.4.2. LOX-2

The effect of varieties on the LOX-2 is highly significant (P<0.01). It is mentioned in Figure 4.4 that at 0 min the minimum value was recorded in Ajmeri (0.20±0.0031) and maximum in Faisal (0.29±0.0043). However, as time passed and hydroperoxidation increased the values

76 of absorbance also increased and maximum was recorded 0.49±0.0073 in Faisal and minimum in NARC-II 0.39±0.0058 at 5th min.

The graphical presentation of LOX-2 showed a nonconventional pattern having initial burst, a drifted lag phase and a plateau. But this was lifted towards the conventional shape containing an exponential burst phase. The maximum change in shape of the curve was observed in case of LOX-2 (Figure 4.4). Overall, the maximum activity of LOX-2 was recorded in Faisal (0.38) followed by Rawal-1 (0.36), Ajmeri (0.35), Willium-82 (0.33) and NARC-II (0.31).

4.1.4.3. LOX-3

A conventional shape of kinetic curve was obtained in case of LOX-3 in all the varieties of soybean. The isozyme LOX-3 showed least inhibition among all the isozymes. LOX-3 exhibited its maximal activity around pH 7.0 and displays a moderate preference for producing 9-HPOD but produces ketodiene. The Figure 4.5 is depicting the results for LOX- 3 which is in correspondence to other lipoxygenase activities because as the time span increases the hydroperoxidation also increased from 0 to 5 min. The values were varied in the range of 0.40±0.0062 (Ajmeri) to 0.53±0.0079 (Faisal) at zero min and 0.69±0.01 (Ajmeri) to 1.0±0.02 (Faisal) at 5 min. Overall, the lowest activity of LOX-3 was noted in Ajmeri, followed by the NARC-II, Willium-82, Rawal-I and Faisal.

The overall comparison of inhibition potential of lipoxygenase is illustrated from Figure 4.6. The figure is depicting that LOX-1 showed minimum inhibition potential followed by LOX-3 and Lox-2. Defatted flour is used in LOX analyses so that it may help in reduction of off- flavor generated. It was clearly evident in present work that LOX-2 isozyme was the predominant during storage, the most inhibiting prone isozyme for off flavor generation. The main factors, which were considered as modulators of LOX catalysis were the pH, the indicator of enzyme affinity (Chedea et al., 2008). Based on the observations, the LOX isozymes were extracted from defatted soybean according to their inhibition potential required for product formation. They suggested that future inhibition studies should be conducted on LOX-2 using natural antioxidant and bioactive compound to solve the issue of off-flavour in oil.

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Table 4.6: ANOVA for Mean squares of Lipoxygenase (LOX)

Source DF LOX-1 LOX-2 LOX-3 Time 5 0.99234** 0.07388** 0.26406** Varieties 4 0.15406** 0.01754** 0.17632** Time*Varieties 20 0.00951** 0.00129** 0.00429** Error 60 0.00016 0.00003 0.00010 Total 89 * = Significant (P<0.05) **= Highly Significant (P<0.05) DF= Degree of freedom

Figure 4.3: Comparison of inhibition potential of lipoxygenase-1

Figure 4.4: Comparison of inhibition potential of lipoxygenase-2

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Figure 4.5: Comparison of inhibition potential of lipoxygenase-3

Figure 4.6: Overall comparison of inhibition potential of lipoxygenase

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4.1.5. Phytochemical screening test

Phenolics are secondary metabolites and antioxidant that can mitigate oxidative damage and helps to improve health stratum (Wink, 2010; Zhao and Shah, 2014). Besides protein and oil contents, the phenolic contents should be also considered as an important characteristic of soybean seeds, on which the soybean variety can be selected.

The statistical analysis of the data regarding TPC and TFC mentioned in the Table 4.7 revealed the significant (P<0.01) influence of varieties on the phenolic contents of soybean. The concentration of the total phenolic content in different varieties is given in Table 4.8. It is demonstrated that maximum phenolic contents (TPC) and flavonoids contents (TFC) were recorded in Ajmeri as 2.76 mg GAE/g and 1.78 mg CE/g followed by the Willium-82, NARC-11, Raval-1 and Faisal.

The results of the current investigations are supported by various previous research works. Josipović et al. (2016) have conducted a study to evaluate the TPC and TFC of 33 soybean genotypes grown in 2010 and 2011. In their investigation significant differences were recorded between the genotypes and for the interaction between year and genotypes. The TPC in 2010 ranged from 2.330 to 3.227 mg/g and in 2011 it ranged from 2.121 to 3.164 mg/g of dry weight, expressed as gallic acid equivalents. Flavonoid content in 2010 varied from 0.433 to 0.659, and in 2011 from 0.428 to 0.580 mg/g of dry weight, expressed as catechin equivalents.

Likewise, Malencic et al. (2008) estimated the polyphenol in the seeds of 20 soybean hybrids and the TPC varied between 189.1 to 384.0 mg catechin/100 g. Similarly, the TFC determined were also in the range of 27.3 ± 1.6 to 88.7 ± 3.3 mg rutin/100 g. Later on, Malencic et al. (2012) worked on total polyphenols and total flavonoid contents in colored soybean seeds from central Europe and the result of their findings reported that TPC in yellow, ocher shine, ocher opaque, black 1, black 2, brown, green and reddish were 2.68±0.47, 3.57±0.32, 3.61±0.1, 6.13±0.12, 6.22±0.68, 4.94±0.10, 3.46 ±0.09 and 3.26±0.17 expressed as mg of gallic acid equivalents/g of dry material respectively. They also

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Table 4.7 Analysis of variance for TPC and TFC

Source DF TPC TFC

Varieties 4 1.57209** 1.1151**

Error 10 0.00113 0.00086

Total 14

**=Highly Significant (P<0.01)

Table 4.8 Effect of soybean varieties on TPC and TFC

Varieties TPC (mg GAE/g) TFC (mg CE/g)

d e Faisal 1.21±0.01 0.42±0.02

NARC-II 2.59 ±0.04b 1.12±0.03c

Willium-82 2.65±0.03b 1.64±0.04b

Ajmeri 2.76±0.05a 1.78±0.05a

Rawal-I 1.52±0.02c 0.58±0.02d

Results are expressed as mean ± standard deviation of means; n = 3 sets a-e Means in rows with similar superscripts do not differ (P>0.05).

81 determined the results for TFC in yellow, ocher shine, ocher opaque, black 1, black 2, brown and green soybean were 0.48±0.02, 0.47±0.10, 1.49 ±0.35, 2.19±0.46 , 1.16±0.298 , 1.16±0.29 and 1.07 ±0.40 expressed as mg of rutin/g of dry material.

Another group of scientists (Lee et al., 2011) analyzed that TPC and TFC in yellow soybean were 1.13±0.03 mg of gallic acid equivalent (GAE) and 0.41±0.03 mg of catechin equivalent (CAE) respectively.

4.1.6. Antioxidant potential of soybean

An antioxidant is the substance that when present in a food or body, it retards or hinders the oxidation rate considerably. Antioxidants can imprison free radicals with similar structure and prevent oxidation through chain breaking or delaying the oxidation (Nuutila et al., 2003; Georgetti et al., 2006). The Analysis of Variance showed that the antioxidant activities (DPPH, FRAP and ABTS) of soybean varied significantly (P<0.01) among varieties (Table 4.9). 4.1.6.1. DPPH

The mean values for the DPPH of the present exploration are given in Table 4.10. The result showed that DPPH values were generally found to be higher in Ajmeri (5.65±0.23 mg TE/g) followed by Willium-82 (5.32±0.21 mg TE/g), Rawal-I (4.56±0.18 mg TE g), NARC-II (4.02±0.16 mg TE/g) and Faisal (3.68±0.14 mg TE/g).

The outcome of present findings are in agreement with Prvulovic et al. (2016) who computed DPPH (mg TE/g) in five different soybean cultivars i.e. Merkur, Sava, Valjevka, Venera and Victoria. They took the extract of soybean in different solvents and then check the antioxidant potential and the results of their findings for DPPH in solvent acetone were 6.37±0.03, 5.10±0.05, 4.68± 0.05, 3.76± 0.03 and 5.42± 0.07 mg TE/g respectively.

The current results are also in harmony with the Chung (2009) who has studied antioxidant activities of soybeans and the result of his findings also showed variation in DPPH among different soybean varieties were such as the yellow soybean extracts (0.6- 2.0 mmoles TE /g) were significantly lower than that of the black soybean extracts (7.1-17.9 mmoles TE /g).

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Moreover, Handa et al. (2016) has investigated antioxidant activities on fermented soy flour and documented that DPPH values varied in between 4.00-10.08 µmol TE/g.

4.1.6.2. FRAP

The results (Table 4.9) regarding FRAP showed that soybean seeds have a significant oxidation reduction potential. The mean values of FRAP activity given in Table 4.10 showed that the trend among the soybean was the same as for the antioxidant potential DPPH except for Raval-I, which showed the higher potential of FRAP (11.34±0.79 mg TE/g) than NARC- II (11.23±0.78 mg TE/g).

It was obvious from current findings that the varieties which contained the higher phytochemicals also showed the higher antioxidant potential. Malenčić et al. in 2010 and Prvulovic et al. in 2012 stated that there was a positive correlation between concentration of phenolic compounds in samples of different plant origin and antioxidant capacity. Their finding also supported the present results as it is obvious from the results that higher the phenolic content, the higher is the antioxidant activity of the variety.

The results of present findings were in harmony with FRAP values as reported by Prvulovic et al. (2016). They analyzed FRAP of five soybean cultivars and the result of their finding for Merkur, Sava, Valjevka, Venera and Victoria were 12.85±0.65, 10.79±0.36, 11.41±0.47, 11.40±0.82 and 11.63 mg TE/g respectively. In the same year another group of researchers Handa et al. (2016) also investigated on antioxidant activities of fermented soy flour and reported that FRAP was varied in between 0.48 to 17.18 µmol TE/g.

4.1.6.3. ABTS

In the present exploration, the recorded values for ABTS were maximum in Ajmeri (29.56±2.06 mg TE/g) followed by Willium-82 (27.23±1.90 mg TE/g), NARC- II (26.54±1.86 mg TE/g), Rawal-I (23.36±1.63 mg TE/g) and Faisal (20.78±1.45 mg TE/g). However, there was non-substantial effect only between NARC-II and Willium-82, while Ajmeri contained the highest ABTS activity (Table 4.10).

The results of present outcome are supported with reported values of Prvulovic et al. (2016). They have studied ABTS scavenging potential of five soybean cultivars and the results of

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Table 4.9 Analysis of variance for antioxidants

Source DF DPPH FRAP ABTS

Varieties 4 2.09** 6.206** 35.56**

Error 10 0.035 6.669 3.231

Total 14

**=Highly Significant (P<0.01)

Table 4.10 Effect of soybean varieties on antioxidants (mg TE/g)

DPPH in five different soybean cultivars and the results of their findings varied in between 3.68±0.14 to 5.65±0.23 (mg TE/g) and FRAP values were 9.63±0.67c to13.48±0.94 TE/g while ABTS was 20.78±1.45 to 29.56±2.06 mg TE/g.

Varieties DPPH FRAP ABTS

Faisal 3.68±0.14c 9.63±0.67c 20.78±1.45c

NARC-II 4.02±0.16c 11.23±0.78bc 26.54±1.86ab

Willium-82 5.32±0.21a 12.41±0.86ab 27.23±1.90ab

Ajmeri 5.65±0.23a 13.48±0.94a 29.56±2.06a

Rawal-I 4.56±0.18b 11.34±0.79 abc 23.36±1.63bc

Results are expressed as mean ± standard deviation of means; n = 3 sets abcde Means in column with different superscripts differ (P<0.01)

84 their finding for Merkur, Sava, Valjevka, Venera and Victoria were 20.73±2.25, 27.09±2.89, 29.69±2.48, 31.17±2.18, and 26.54±2.03 mg TE/g respectively. In the same year another group of scientists Handa et al. (2016) also investigated antioxidant activities from fermented soy flour and reported that ABTS values varied in between 4.00 to 10.08 µmol TE/g).

Soybean has shown to possess the high antioxidant properties and other parameters that strengthen the hypothesis that varieties behave differently in terms of nutritional aspects. The results obtained suggest that the widespread use of the soybean varieties under study could be beneficial for human health. Soybean is an economically important crop whose consumption makes a significant contribution in promoting human health by providing dietary antioxidants. In the present study, varieties Ajmeri, Willium-82 and NARC-II have shown to possess high antioxidant potential as compared with other varieties. The nutritional quality oriented attributes in this study were competent as an index of their nutritional worth and can be recommend to farmers and consumers which may be graded as export quality soybean with good unique nutritional values in international market.

4.2. Analysis of value added fermented soy milk

The soy milk was prepared from top three selected varieties like NARC-II, Willium-82 and Ajmeri and fermented by using Lactobacillus acidophilus and Lactobacillus casei solely as well as in combination to check their co-culture fermentation effect. The soy milk is considered as a good carrier of Lactic acid bacteria (probiotic).

4.2.1. Compositional analyses of soy milk

Four types of soy milk treatments were prepared using starter culture as described To (Non- fermented soy milk), T1 (Soy milk made by using L. acidophilus), T2 (Soy milk made by using L. casei) and T3 (Soy milk made by using L. acidophilus and L. casei). The results regarding effect of different treatments and varieties (NARC-II, Willium-82 and Ajmeri) on the composition of soy milk are presented and discussed in this section.

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4.2.1.1. Total soluble solids (TSS)

Total soluble solids content is an important parameter for beverage evaluation in food industry. Its recovery from soybean is vital for the contents of lipids and proteins of soy milk and also for its nutritional value (Rinaldoni et al., 2012). Total soluble solids varied in different varieties and their higher contents are cherished by consumers. So, the soluble solids content was determined as a soy milk chemical character in the present research (Ma et al., 2015). The statistical analysis of TSS content in soy milk made from three different varieties is mentioned in Table 4.11. It is represented from the results that TSS content of soy milk highly significantly (P<0.01) effected by the treatment, varieties, and storage. It was also observed from the results that interactions between treatment and varieties showed highly momentous (P<0.01) effect on TSS content. However, a non-substantial effect was observed in the interaction of treatment and days, varieties and days and treatment, varieties and days.

The mean values specified in Table 4.12a showing the effect of treatment, varieties and storage days on TSS of soy milk. The overall effect of storage causes decline (10.76 to 10.3%) in total solids from zero to 24th day of storage. The maximum value in NARC-II soy milk recorded for To at the beginning of storage was 11.35±0.34% and minimum 10.62±0.47% in th T3 (24 day). The mean values for Willium-82 soy milk depicted that the maximum value was th recorded in To (11.40±0.35%) at zero day and minimum was in T3 (9.59±0.46%) on 24 day of storage. Likewise, mean values showing the effect of fermentation and storage time on TSS (%) of Ajmeri soy milk are also showing that fermentation causes reduction in total soluble solid contents such as 11.07±0.40% recorded in non-fermented soy milk whilst 9.64±0.39% was recorded in coculture fermented soy milk. The overall total solids decreased as a function of storage in all soy milk samples made from different varieties.

The mean values of TSS for the effect of interaction between treatment and varieties had given in Table 4.12b. The results are depicting the significant interaction among all treatments of NARC-II, Willium-82 and Ajmeri soy milk.The maximum total solids were recorded in non-fermented Ajmeri soy milk were11.05% and minimum in Ajmeri soy milk

T3 was 9.63% (Fermented soy milk with L. acidophilus and L. casei). However, overall TSS were recorded maximum in NARC-II (10.55%) and minimum in Ajmeri (10.32%). The

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Table 4.11: Mean squares for composition of fermented soy milk

Source DF TSS SNF Protein Fat Ash

Days 3 4.07666** 3.46456** 0.68916** 0.02650** 0.03667** Treatment 3 5.83719** 2.35199** 1.09600** 0.76310** 1.32432** Varieties 2 0.70742** 1.11642** 1.82342** 0.22689** 0.18087** Days*Treatment 9 0.07523NS 0.07512NS 0.01054NS 0.00119NS 0.00002NS Days*Varieties 6 0.21234 NS 0.2123 NS 0.00194 NS 0.00030NS 0.00019NS Treatment*Varieties 6 0.83280** 0.91025** 0.04061** 0.08092** 0.00709** Days*Treatment*Varieties 18 0.07376NS 0.077NS 0.00093 NS 0.00027NS 0.00017NS Error 96 0.19264 0.21750 0.01175 0.00176 0.00245 Total 143 **=Highly Significant (P<0.01) *=Significant (P <0.05) NS= non-significant (P >0.05)

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Table 4.12a: Effect of treatment, varieties and days on the total soluble solids (TSS) (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 11.35±0.34 11.3±0.49 11.26±0.48 11.22±0.46

T1 11.08±0.33 11.02±0.44 10.98±0.42 10.96±0.41 NARC-II

T2 10.92±0.33 10.89±0.49 10.82±0.48 10.78±0.46

T3 10.76±0.32 10.7±0.50 10.67±0.48 10.62±0.47

To 11.40±0.35 11.11±0.50 10.79±0.49 10.48±0.47

T1 10.71±0.33 10.42±0.43 10.10±0.41 9.78±0.40 Willium-82

T2 10.57±0.32 10.28±0.48 9.96±0.47 9.63±0.45

T3 10.52±0.32 10.23±0.49 9.90±0.48 9.59±0.46

To 11.53±0.35 11.24±0.50 10.92±0.49 10.6±0.47

T1 10.55±0.32 11.26±0.46 10.94±0.44 10.63±0.43 Ajmeri

T2 10.45±0.31 11.17±0.52 10.52±0.49 10.2±0.47

T3 10.09±0.31 9.81±0.47 9.50±0.45 9.18±0.44

Mean of storage days 10.76a 10.71a 10.36b 10.03c

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Table 4.12b: Effect of treatment and varieties on TSS (%) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

abc b b a To 10.87 10.93 11.05 10.95

bcd cd bc b T1 10.61 10.23 10.83 10.56

def de bcd b T2 10.42 10.09 10.57 10.36

d-g de e c T3 10.28 10.04 9.63 9.98

Mean 10.55a 10.52ab 10.32b

Results are expressed as mean ± standard deviation of means; n = 3 sets a-g Means in column with different superscripts differ substantially (P<0.01) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

89 fermentation with both cultures showed lower reduction of TSS (9.98%) in T3 and highest was (10.95%) in non-fermented soy milk (To).

The current findings of TSS were lower than reported by Mühlhansová et al. (2015). They studied on different parameters of soy milk and reported on total solids as 13.57 ± 0.04% which were higher than 11.53±0.35% recorded as highest in current findings. Previously Obadina et al. (2013) has studied the effect of natural fermentation on the chemical and nutritional composition of fermented soy milk. They reported the results at 24, 48 and 72 h of fermentation. The total solids were 6.90, 7.27 and 7.30 %, respectively which are lower than the current findings. Total soluble solids content shows a positive correlation with organoleptic properties of product particularly for the mouth feel, aroma and over acceptability of soy milk. This suggested a trend that superior soy milk varieties had higher total soluble solids content than the inferior, which was consistent with previous reports (Ma et al., 2015).

4.2.1.2. Solid not fat (SNF)

The constituents of soy milk other than fat are referred as SNF. The mean squares of SNF content in soy milk prepared from three different varieties are mentioned in Table 4.11. It is depicted from the results that SNF content of soy milk is effected highly significantly (P<0.01) by the treatment, varieties, and storage days. It was also noticed from the results that interactions between treatment and varieties has the significant (P<0.01) effect on SNF content. However, a non-significant effect was observed in the interaction of treatment*days, varieties*days and treatment*varieties*days.

In the current study, a substantial decrease for SNF was observed in soy milk throughout the storage span of 0 to 24th day from (9.09 to 8.41%). The highest SNF 9.45±0.30% for NARC-

II soy milk was recorded in To at 0 day and minimum was 8.23±0.50% in T2 soy milk fermented with L. casei at 24th day of storage. Likewise, the Willium-82 soy milk elucidated the effect of fermentation on SNF. The maximum SNF recorded in To was 9.45±0.30% on zero th day and minimum was 8.00±0.49 % in T2 on 24 day of storage. Similarly, the mean values for SNF of Ajmeri soy milk showed that fermentation process caused decrease in SNF such as

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Table 4.13a: Effect of treatment, varieties and days on the solid not fat (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 9.45±0.30 9.24±0.54 8.93±0.52 8.63±0.51

T1 9.22±0.29 9.02±0.48 8.72±0.47 8.43±0.45 NARC-II

T2 9.11±0.29 8.92±0.54 8.62±0.52 8.23±0.50

T3 9.07±0.28 8.87±0.54 8.59±0.52 8.29±0.51

To 9.45±0.30 9.26±0.54 8.97±0.53 8.67±0.51

T1 8.88±0.28 8.68±0.47 8.38±0.45 8.08±0.44 Willium-82

T2 8.79±0.28 8.61±0.52 8.31±0.51 8.00±0.49

T3 8.88±0.28 8.7±0.52 8.4±0.512 8.11±0.48

To 9.53±0.31 9.32±0.55 9.02±0.54 8.71±0.52

T1 8.89±0.28 9.69±0.50 9.39±0.48 9.12±0.47 Ajmeri

T2 8.87±0.36 9.68±0.56 9.06±0.52 8.78±0.51

T3 8.59±0.26 8.4±0.51 8.11±0.48 7.81±0.47

Mean of storage days 9.09a 9.03a 8.71b 8.41c

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Table 4.13b: Effect of treatment and varieties on SNF (%) of soy milk Varieties Treatments Means NARC-II Willium-82 Ajmeri

a-d abc ab a To 9.06 9.09 9.15 9.09

a-e cde a ab T1 8.85 8.51 9.28 8.87

a-e de abc bc T2 8.72 8.43 9.10 8.75

a-e b-e e c T3 8.71 8.53 8.22 8.49

Mean 8.84ab 8.64b 8.94a

Results are expressed as mean ± standard deviation of means; n = 3 sets abcde = Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

92 the highest value observed in To was 9.53±0.31% at 0day and least was 7.81±0.47% in T3 at 24th day (Table 4.13a).

The effect of treatment and varieties on SNF (%) of soy milk (Table 4.13b) depicted the highest SNF noticed in non-fermented soy milk was 9.09% in To and minimum 8.49%. SNF was recorded in T3. Among the varieties maximum SNF contents were 8.94% in Ajmeri soy milk and minimum 8.84% in NARC-II. The results illustrated that effect of treatment and varieties also showed the variation in TSS of soy milk. The results explored that process of fermentation exhibited decrease in SNF contents. The SNF contents has been reported by Ahanian et al. (2014) who studied the ice cream made from soy milk and analyzed that SNF was 11.13% that was higher than present findings. 4.2.1.3. Protein

The presence of proteins in a product represents its quality index. By measuring the amount of nitrogen in a sample the quantity of crude protein is normally determined. Total nitrogen contributes to the flavor, body and texture to the finish product and also source of calories. It is also essential for the formation of small stable air cells and responsible for variation in solubility index as well as process variation.

The statistical analysis for protein in soy milk of different varieties and fermented with L. acidophilus and L. casei is given in Table 4.11. The results depicted the highly significant (P<0.01) variation in protein content of soy milk due to the effect of storage days, treatment, varieties and also due to interaction of treatment*varieties. However, no significant variation (P>0.05) was recorded for their interaction among days*treatment, days*varieties and days*treatment*varieties.

The mean values showing the effect of fermentation and storage time on protein of soy milk prepared from different varieties as given in Table 4.14a. The mean values depicted that protein contents varied significantly during storage of product and highest was 2.59% noted on zero day and then decreased periodically to 2.50% on 8th, 2.37% on 16th and 2.28% on 24th day of storage. Moreover, during 24 days of storage a decrease in protein (%) was observed because the process of fermentation by Lactic acid bacteria converts protein into oligopeptides.

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Table 4.14a: Effect of varieties, treatment and storage on protein (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 2.68±0.10 2.57±0.13 2.39±0.11 2.3±0.10

T1 2.53±0.11 2.48±0.10 2.4±0.12 2.31±0.31 NARC-II

T2 2.43±0.11 2.32±0.09 2.14±0.08 2.05±0.20

T3 2.33±0.10 2.22±0.10 2.04±0.08 1.95±0.10

To 2.82±0.08 2.71±0.12 2.54±0.11 2.45±0.11

T1 2.50±0.076 2.48±0.10 2.38±0.09 2.27±0.10 Willium-82

T2 2.37±0.072 2.25±0.11 2.07±0.09 1.99±0.09

T3 2.29±0.069 2.17±0.10 2.08±0.09 2.01±0.09

To 3.02±0.13 2.9±0.13 2.75±0.12 2.68±0.12

T1 2.8±0.11 2.76±0.11 2.66±0.10 2.57±0.10 Ajmeri

T2 2.78±0.13 2.69±0.12 2.52±0.12 2.44±0.11

T3 2.65±0.12 2.54±0.12 2.46±0.11 2.39±0.11

Mean of storage days 2.59a 2.50b 2.37c 2.28d

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Table 4.14b: Effect of treatment and varieties on protein (%) of soy milk Varieties Treatments Means NARC-II Willium-82 Ajmeri

cd bc a a To 2.48 2.62 2.83 2.64

d d ab b T1 2.43 2.39 2.69 2.51

e e bc c T2 2.23 2.16 2.61 2.34

e e cd d T3 2.13 2.13 2.51 2.26

Mean 2.32b 2.33b 2.66a

Results are expressed as mean ± standard deviation of means; n = 3 sets abcde= Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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NARC-II exhibited the maximum value of protein 2.68±0.10% in non-fermented soy milk on th the start of storage and minimum 1.95±0.10 % was noticed in T3 soy milk on 24 day of storage. The mean values of Willium-82 soy milk delineated in the same manner that fermentation causes decreased in protein contents as maximum protein contents were estimated th in To (2.82±0.08%) at 0 day and least (1.99±0.09%) was noted in T3 at 24 day of storage. Likewise, data expounded variation in treatments for protein (%) of Ajmeri soy milk as that the maximum recorded value was 3.02±0.13% in To and the minimum reported was 2.39±0.11% th on 24 day of storage in T3.

The effect of interaction among treatment and varieties on protein contents of soy milk varied significantly in all varieties. The maximum protein contents were noticed in Ajmeri (2.66%) followed by Willium-82 (2.33%) and NARC-II (2.32%). The effect of treatments showed variation as highest in non-fermented soy milk To (2.64%) trailed by 2.51, 2.34 and 2.26% in

T1, T2 and T3 respectively (Table 4.14b).

Protein content is an important factor that affects the quality of acid coagulation of protein gel products. The protein contents of soy milk were higher at the initial phase of storage and then declined as the storage progressed. The enhancement in protein content of fermenting soy milk in comparison to non fermented soy milk might be due to some anabolic processes leading to polymer build-up or due to microbial cell proliferation Obadina, et al. (2013). The results of recent findings are in harmony with Mühlhansová et al. (2015) who reported proteins 2.17±0.02% in soy milk. Likewise, Obadina et al. (2013) has studied the effect of natural fermentation on the chemical and nutritional composition of fermented soy milk. The results for protein contents after 24, 48 and 72 h of fermentation were such as 4.00, 4.72 and 5.09 %, respectively. Further, Amanze and Amanze, (2011) has reported 2.02±0.14% protein in soy yoghurt. Further, Yang and Li (2010) strengthen the results of recent findings by reporting the protein contents in germinated probiotic soy yoghurt that varied between 2.61 to 2.91%.

4.2.1.4. Fat

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Soy milk is considered as a healthy food as it is made from plant source that‟s why it is naturally free of cholesterol and low in saturated fat. It is a best choice for people suffering from heart diseases and other cholesterol related diseases.

The statistical results for fat contents revealed substantial differences (P<0.01) as a function of days, treatment, varieties and interaction of treatment*varieties. Whilst, non-substantial (P>0.05) difference was recorded for interaction in days*treatment, days*varieties and days*treatment*varieties (Table 4.11).

The mean values showing the effect of fermentation and storage time on fat contents of soy milk made from three different varieties are given in Table 4.15a. Overall variation during the storage period showed slightly decrease (1.62 to 1.69%) in fat contents from 0 to 24th day of storage. The fat contents in NARC-II soy milk varied from 1.83±0.09% in To (zero day) to th 1.54±0.038% in T3 (24 day). Similarly, the mean value explicated for willium-82 soy milk th varied from 1.87±0.075% in To (zero day) to 1.48±0.037% in T3 (24 day). Likewise, mean values for Ajmeri soy milk varied 1.92±0.048 at zero day in To and minimum was 1.37±0.034 th in T3 on 24 day.

The combined effect of treatment and varieties on soy milk fat contents varied significantly as given in Table 4.15b. The interaction effect showed the highest fat contents 1.90% in To and lowest 1.39 % in T3 of Ajmeri soy milk. Overall, the highest fat contents (1.71%) were observed in NARC-II soy milk and minimum was in Ajmeri (1.58%). The variation in fat showed 1.85% fat in non-fermented soy milk followed by 1.68% in L. acidophilus fermented soy milk, 1.61% in L.casei and 1.49% in coculture soy milk. During the process of fermentation the breakdown of fat contents occure due to action of lipolytic enzymes. The higher the fermentation time period higher will be the breakdown of triacylglycerol in fatty acid and glycerol. The another reason for fat reduction was reported by group of researchers Astuti et al. (2000) they stated that fatty acids were used as an energy source by LAB that may cause lowering fat content in soy milk at the completion of fermentation. The results of current study are in correspondence with Obadina et al. (2013) who has studied the effect of natural fermentation on the chemical and nutritional composition of fermented soy milk. They reported that fermentation showed the fat contents varied between 1.43 to 1.09%.

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Table 4.15a: Effect of varieties, treatment and storage days on fat (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 1.83±0.09 1.82±0.010 1.81±0.045 1.79±0.045

T1 1.78±0.044 1.77±0.044 1.75±0.043 1.73±0.043 NARC-II

T2 1.73±0.073 1.71±0.076 1.69±0.042 1.67±0.041

T3 1.61±0.040 1.59±0.039 1.56±0.039 1.54±0.038

To 1.87±0.075 1.85±0.046 1.82±0.078 1.81±0.045

T1 1.75±0.043 1.74±0.043 1.72±0.043 1.70±0.048 Willium-82

T2 1.7±0.0425 1.67±0.041 1.65±0.041 1.63±0.041

T3 1.56±0.039 1.53±0.038 1.5±0.037 1.48±0.037

To 1.92±0.048 1.92±0.048 1.90±0.092 1.89±0.047

T1 1.59±0.039 1.57± 0.039 1.55±0.038 1.51±0.037 Ajmeri

T2 1.52±0.050 1.49±0.037 1.46±0.036 1.42±0.035

T3 1.43±0.035 1.41±0.065 1.39±0.034 1.37±0.034

Mean of storage Days 1.69a 1.67ab 1.65bc 1.62c

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Table 4.15b: Effect of treatment and varieties on fat (%) of soy milk Varieties Treatments Means NARC-II Willium-82 Ajmeri

bc b a a To 1.81 1.82 1.90 1.84

cd de g b T1 1.75 1.73 1.55 1.68

ef f h c T2 1.70 1.66 1.46 1.61

g g i d T3 1.57 1.52 1.39 1.49

Mean 1.71a 1.69b 1.58c

Results are expressed as mean ± standard deviation of means; n = 3 sets a-i Means in column with different superscripts differ substantially (P<0.01) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

99

Previously, Amanze and Amanze (2011) have reported proximate composition of soy yoghurt and reported that crude fat was 1.3%. However, the findings of Mühlhansová et al. (2015) for fat contents in soy milk were higher than present findings as they reported the 3.75±0.35% of fat contents.

4.2.1.5. Ash

The inorganic material left after the burning for complete removal of water and organic matter in the food stuff is known as ash. Ash contents are also referred as inorganic material and signify the minerals level in food stuff low in caloric density. Soy milk is rich in calcium, iron, magnesium and zinc and all of these are important for human body because they are associated with many physiological and pathological processes such as activation of enzymes, contraction of muscles, immune response, cell differentiation, programmed cell death and neuronal activity (Pu et al., 2016).

The statistical analysis of ash content in soy milk made from three different varieties is given in Table 4.11. It is shown from the results that ash content of soy milk is effected highly significantly (P<0.01) by the treatment, varieties, and days. It is also noticed from the results that interactions between treatment*varieties was also varied highly significantly (P<0.01) for the effect on ash content. However, a non-significant effect was observed in the interaction of varieties*days, treatment*days and treatment*varieties*days.

The mean values showing effect of fermentation and storage time on ash contents of soy milk are displayed in Table 4.16a. The results illustrated that the significant increase in ash contents was observed in the range of 0.64 to 0.72% during 0 to 24th days of storage. The mean values demonstrated that ash contents for NARC-II soy milk varied and 0.26% was the lowest ash contents observed on start of storage in non-fermented soy milk (To) while the th maximum value 0.79% was observed in T3 on 24 day of storage. The outcomes showed that ash contents increased sharply after fermentation and T3 of NARC-II fermented soy milk with L. acidophilus and L. casei shows greatest increase of ash contents. Likewise, mean values of Willium- 82 soy milk showed the same trend like maximum value was recorded for

T3 (0.84%) and minimum was recorded for To (0.38%). The Table 4.16a, depicting that the mean values for Ajmeri soy milk varied during storage from (0.88%) T3 to 0.42% (To).

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Table 4.16a Effect of varieties, treatment and storage days on ash (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 0.26±0.018 0.28±0.02 0.3±0.02 0.32±0.022

T1 0.65±0.045 0.67±0.04 0.69±0.04 0.72±0.050 NARC-II

T2 0.68±0.047 0.7±0.049 0.73±0.05 0.76±0.053

T3 0.71±0.049 0.74±0.05 0.77±0.05 0.79±0.055

To 0.38±0.026 0.41±0.028 0.45±0.031 0.47±0.033

T1 0.69±0.048 0.72±0.050 0.74±0.052 0.75±0.052 Willium-82

T2 0.73±0.051 0.77±0.053 0.79±0.055 0.8±0.056

T3 0.78±0.054 0.81±0.056 0.82±0.057 0.84±0.058

To 0.42±0.34 0.44±0.031 0.47±0.033 0.49±0.034

T1 0.76±0.05 0.79±0.050 0.83±0.078 0.85±0.035 Ajmeri

T2 0.79±0.05 0.81±0.025 0.84±0.066 0.86±0.016

T3 0.8±0.045 0.83±0.015 0.85±0.061 0.88±0.026

Mean of storage days 0.64c 0.66bc 0.69ab 0.72a

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Table 4.16b: Effect of treatment and varieties on ash (%) of soy milk Varieties Treatments Means NARC-II Willium-82 Ajmeri

g f f d To 0.29 0.42 0.45 0.39

e de abc c T1 0.68 0.72 0.80 0.74

de bcd ab b T2 0.72 0.77 0.82 0.77

cd abc a a T3 0.75 0.81 0.84 0.80

Mean 0.61c 0.68b 0.73a

Results are expressed as mean ± standard deviation of means; n = 3 sets a-g = Means in column with different superscripts differ substantially (P<0.01) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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The mean values of ash contents between the interaction of treatment and varieties are given in Table 4.18b. The effect of interaction showed minimum value 0.29% in NARC-II soy milk and maximum 0.84% in T3 Ajmeri soy milk. The results also depicted that Ajmeri with 0.73% of ash contents was at the top whilst, NARC-II with 0.61% ash contents was the least.

The treatment effect showed maximum ash contents in T3 were 0.80%, in T2 0.77%, T1 0.73 and in To was 0.39%. In brief, it was observed that ash contents were more in soy milk fermented with combination of L. acidophilus and L. casei. The process of fermentation significantly increases the ash contents and it was also observed by Rekha and Vijayalakshmi, (2008) who studied the biomolecules and nutritional quality of soy milk fermented with probiotic yeast and bacteria and they reported that ash contents were significantly higher than those found in unfermented soy milk. Similarly, Obadina et al. (2013) has studied the effect of natural fermentation on the chemical and nutritional composition of fermented soy milk nono the results for 24, 48 and 72 h of fermentation revealed that the ash contents were 0.57, 0.67 and 0.74% they noticed that the effect of storage on ash contents displayed the increased during storage period and findings of current project are in harmony with them. They also suggested that the increase in ash content in fermented soy milk in comparison to non-fermented soy milk could be due to reduction of certain other compounds such as loss of moisture and breakdown of fat and carbohydrates. However, Odu et al. (2012) investigated on use of preservatives in soy milk and estimated ash was 0.22-0.41% and these results were in comparison of different unfermented soy milk in current study. Moreover, Amanze and Amanze (2011) has also reported proximate composition of soy yoghurt and reported that ash contents were 0.51± 0.23% and these values are also in association to current study.

4.2.2. Viable cell counts

Lactobacillus acidophilus and Lactobacillus casei are „probiotic‟. This word is evolved from the food industry to describe „live microbial food ingredients that are favorable to health of the host‟ by modifying its balance in gut microbiota. The processing of probiotics food must ensure good shelf life of live microbiota at the time of consumption and should be zero toxicity and pathogenicity. The action of microorganisms during the preparation of cultured foods or in the digestive tract has been shown to improve the quantity, availability and

103 digestibility of some dietary nutrients. So, higher the viable cell count is desirable as a functional food ingredient. However, the enumeration of probiotic bacteria in fermented foods indicates production of probiotic foods enriched with good bacteria that is demand of industry to develop a techno-functional soy product (Parvez et al., 2006; Vinderola, 2011).

The statistical results for viable cell count explicated in Table 4.17 represented that there was highly substantial (P<0.01) variation among treatment, varieties and storage days. The effect of their interaction was significant (P<0.05) among treatment*varieties and treatment *days whilst there was non-significant (P>0.05) impact for interaction of varieties*days and treatment*varieties*days on viability of bacteria under consideration.

The mean values of viable cell counts of fermented soy milk with two pure cultures of L. acidophilus and L. casei are presented in Table 4.18a. The storage period from 0 to 24th day showed increase in viable cell count from 5.93 log10 CFU/mL to 6.13 log10 CFU/mL. The results showed that soy milk facilitated the growth of both LAB tested. L. casei grew well in all samples as compared to L. acidophilus and their combination give more pronounced results. This difference may be attributed to the selectivity in the nutritional requirement and the raw material utilization rate. The mean values for viable cell count of NARC-II soy milk th in Table 4.18a showed the highest viable cell count was recorded in T3 on 8 day of storage and that was 9.03±0.38 log10 CFU/mL.

In the beginning of storage no microbial growth was observed in To because before starting storage study, soy milk was sterilized in autoclave. However, with the passage of time few colonies were recorded that might be during handling or analyzing a little contamination. The overall storage results were recorded for interval of 0, 8, 16 and 24 days and the growth was seems to be increased in first weekly interval but after that it gave declining trend. Mean values of Willium-82 soy milk given in Table 4.18a showed the effect of treatment is proving that soy milk is a good medium for the growth of L. acidophilus and L. casei and maximum th growth was 8.52±0.37 log10 CFU/mL in T3 on 8 day of storage. Similarly, in Ajmeri soy milk th the highest count was recorded in T3 (9.14±0.40 (log10 CFU/mL) on 8 day.

Mean values of viable cell count for the effect of treatment*varieties are given in Table

4.18b. Among the varieties, the maximum growth was observed in Ajmeri (6.26 log10

104

CFU/mL) and non-significant effect was noticed in NARC-II (6.05 log10 CFU/mL) and

Willium-82 (5.29 log10 CFU/mL). The effect of treatment showed that the momentous variation in T3 was 8.76 log10 CFU/mL, in T2 was 7.76 log10 CFU/mL, in T1 was 7.56 log10 CFU/mL and

To was without any culture but overall it showed 0.23 log10 CFU/mL.

The effect of treatment*days on viable cell count was also significant as given in Table 4.18c. The results depicted the effect of storage days and treatment with each other in such a manner that treatment T1 and T2 were not significantly varied with each other that is clearly depicted from identical superscripts on their mean values. The maximum growth was th recorded in T3 as 8.66, 8.81, 8.82 and 8.75 log10 CFU/mL on 0, 8, 16 and 24 day of storage respectively. The variation in storage time was such as at start logarithmically increase in growth was observed then after certain time it decline, that could be due to noteworthy variation in pH and ultimately acidity. However, both starter cultures reached the desired therapeutic level (108CFU/mL) likely due to their ability to metabolize oligosaccharides during fermentation in soy milk at 37ºC and alpha-glactosidase activity.

It has also been reported from other scientists that soy milk is a good substrate for probiotic bacteria and good base for fermentation process (Stijepic et al., 2013). The results of recent findings are in accordance with Ewe et al. (2010) who have investigated on viability and growth characteristics of Lactobacillus in soy milk and authors reported that L. acidophilus and L. gasseri grew best in soy milk.

Likewise, Chang et al. (2010) has studied on physicochemical and sensory characteristics of soy yoghurt fermented with Bifidobacterium breve, Streptococcus thermophilus, or Lactobacillus acidophilus Q509011 and reported the results at the interval of 0, 3, 6, 9, 12, 15 for L. acidophilus were such as 8.96±0.30 9.10±0.08, 9.22±0.21, 9.30±0.15, 9.44±0.19 and 9.12±0.04 log CFU/mL respectively. Similarly, Li et al. (2014) has studied on fermented soy milk by using strains of lactic acid bacteria and results of their findings showed that soy milk facilitated the growth of all LAB tested. L. plantarum 70810 grew better (8.57±0.04 log10 CFU/mL) than other two starter cultures (7.89±0.15 and 7.92±0.11 log10 CFU/mL).

105

Table 4.17: Mean squares for viable cell count and physicochemical analysis of soy milk

Source DF Viable Cell pH Acidity WHC Count Treatment 3 557.11** 56.7263** 0.61883** 31992.1** Varieties 2 1.43** 0.2903** 0.28988** 44.6** Days 3 0.357** 7.3389** 0.45288** 633.5** Treatment*Varieties 6 0.213* 0.9050** 0.16424** 84.8** Treatment*Days 9 0.28* 0.3347** 0.44226** 76.4** Varieties*Days 6 0.014 NS 0.3012** 0.19651** 9.6NS Treatment*Varieties*Days 18 0.028 NS 0.1446** 0.09102** 5.7 NS Error 96 0.090 0.0276 0.00030 4.8 Total 143 **=Highly Significant (P <0.01) *=Significant (P <0.05) NS= non-significant (P>0.05)

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Table 4.18a: Effect of varieties, treatment and storage days on viable cell count (log10 CFU/mL) of soy milk

Days of Storage Varieties Treatments 0 8 16 24

To 0±0 0.23±0.01 0.25±0.01 0.26±0.01

T1 7.39±0.30 7.58±0.32 6.7±0.29 6.39±0.29 NARC-II

T2 7.56±0.32 8.19±0.33 7.62±0.31 7.91±0.29

T3 8.8±0.38 9.03±0.38 8.56±0.36 8.75±0.36

To 0±0 0.29±0.02 0.31±0.013 0.34±0.04

T1 7.26±0.29 7.43±0.30 6.58±0.26 6.28±0.25 Willium-82

T2 7.52±0.32 8.14±0.35 7.59±0.32 7.25±0.31

T3 8.28±0.36 8.52±0.37 8.04±0.35 7.87±0.34

To 0±0 0.34±0.01 0.35±0.01 0.37±0.01

T1 7.69±0.32 7.87±0.32 7.03±0.28 6.73±0.27 Ajmeri

T2 7.77±0.33 8.41±0.36 7.86±0.33 7.52±0.32

T3 8.89±0.39 9.14±0.40 8.69±0.38 8.54±0.37

Mean of storage days 5.93b 6.09ab 6.15a 6.13a

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Table 4.18b: Effect of treatment and varieties on viable cell count (log10 CFU/mL) of soy milk Varieties Treatments Means NARC-II Willium-82 Ajmeri

f f f d To 0.19 0.24 0.27 0.23

de e cd c T1 7.46 7.36 7.86 7.56

cde cde c b T2 7.72 7.60 7.95 7.76

ab b a a T3 8.86 8.46 8.96 8.76

Mean 6.05b 5.92b 6.26a

Table 4.18c: Effect of treatment and days on viable cell count (log10 CFU/mL) of soy milk

Treatments Days of Storage

0 8 16 24

c c c c To 0.00 0.29 0.30 0.32

b b b b T1 7.45 7.57 7.65 7.58

b b b b T2 7.62 7.72 7.81 7.88

a a a a T3 8.66 8.81 8.82 8.75

Mean of storage days 5.93b 6.09ab 6.15a 6.13a

Results are expressed as mean ± standard deviation of means; n = 3 sets abcdef = Means in column with different superscripts differ substantially (P<0.05) T0= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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Furthermore, Kazemi et al. (2014) has studied the effect of addition of 20%, 40%, and 60% of soy milk in 1.5% fat milk and fermented with Lactobacillus acidophilus for 14 days. Results showed that all the samples possessed minimum effective dose of LA-5 on day 14, although a significant decrease in LA-5 was observed in the sample with 60% soy milk.

Later on, Falade et al. (2015) has researched on plain yoghurt of soybean and reported that the counts of LAB decreased by 0.6 log10 CFU/mL in the plain soy yoghurt samples after 9 days of cold storage at 7ºC. A significant increase in the LAB counts from 7.6 log10 CFU/mL to 8.8 log10 CFU/mL were recorded for soy yoghurts respectively after 9 days of storage at ambient temperature (27ºC).

4.2.3. pH and acidity pH is a negative logarithm of hydrogen ion concentration and it is a measure of acidity or basicity of that system. Acidity is measure of amount of acids in any food sample. The pH has direct influence on biochemical changes during storage and hence effects on flavour perception of the product. Acidity correlates with the pH, lower the pH results higher the acidity and vice versa.

The mean squares for the pH and acidity are depicted in Table 4.17. The results showed highly significant effect (P<0.01) of treatment, varieties and storage days on pH and acidity of soy milk. The interaction effect also showed highly significant (P<0.01) variation of treatment*varieties, treatment*days, varieties*days and also in treatment*varieties*days for both pH and acidity of soy milk as well. The mean values for pH of fermented and non- fermented NARC-II soy milk are presented in Table 4.19a. The pH value exhibited a significant decrease from 5.35 (0 day) to 4.35 (24th day).

The results showed that L. casei gives pH more towards acidic and it was desirable for the th shelf life of product. The lowest pH was recorded as 3.35±0.050 in T2 on 24 day of storage while maximum pH was recorded in non-fermented soy milk 6.97±0.004 at the start of product formation. The mean values of Willium-82 soy milk depicted substantial decline during storage as 6.61 pH was recorded in To on start of storage and minimum was 3.49±0.072 in T2 on 24th day of storage. Likewise, the findings for pH of Ajmeri soy milk showed the similar

109 trend. The maximum value of pH was 6.89±0.027 recorded in To and lowest was 3.4±0.14 in th T2 on 16 day of storage. Overall results for the average effect of storage showed decrease in pH throughout the storage span.

The interaction effect of treatment*varieties showed significant variation as depicted in Table

4.19b. It showed maximum pH in To of NARC-II soy milk as 6.82 and minimum was 3.74 in

T3 of NARC-II soy milk. Overall effect of treatment showed variation as 6.53, 4.54, 3.92 and

3.85 pH of To, T1, T2 and T3 respectively. The varieties effect showed maximum pH value 4.79 in Ajmeri and no any noteworthy variation was noticed in NARC-II and Willium-82 (4.64 and 4.70 pH respectively).

The interaction effect of treatment and days on pH of soy milk is given in Table 4.19c. It showed the significant variation on regular intervals of 0, 8, 16 and 24 days. The maximum pH was noticed in To in which no any LAB were used, so its pH was more towards neutral range and varied from 6.79 to 6.27. The treatment T1 (L. acidophilus) showed pH changes from 5.12 to 4.18. The T2 soy milk fermented with L. casei showed dramatic decrease in pH as 4.69 to 3.45.The soy milk fermented by using both starter cultures in T3 showed more variation in pH during storage period from 4.77 to 3.52. The mean values for the effect of interaction among varieties*days is given in Table 4.19d. The table is depicting significant variation during storage period in all varieties the maximum 5.65 pH was observed in Ajmeri on the start of storage that decreased to 4.25 on 24th day of storage.

Mean values regarding the acidity of soy milk (Table 4.20a) showing significant (P<0.01) effect of fermentation, storage time and varieties. The storage span of 24 days depicted significant decrease in acidity from 0.69% to 0.51%. The results of NARC-II soy milk showed the maximum pH and minimum acidity e.g. To (non-fermented soy milk) showed lowest acidity 0.39±0.015% at the start of product formation. The fermented soy milk was more acidic such th as the highest acidity (1.00±0.041%) was observed in T3 on 24 day of storage. The results of these treatments were reverse in case of pH. Likewise, in Willium-82 soy milk treatment effect was more pronounced as To (without any culture) gave lowest acidity results

(0.41±0.01%), T1 fermented with L. acidophilus showed higher 0.56±0.02% and L. casei showed smaller decrease in pH and higher acidity 0.91±0.03%. The combination of both cultures produces more acidity (0.94±0.02%) at the start of storage study. The results were same

110 for all varietal soy milk in terms of decrease in pH and acidity. Means for acidity of Ajmeri soy milk also showed minimum acidity in To was 0.42% and maximum was 1.01% in T3.

The mean values showing effect of interaction among treatment*varieties are given in Table 4.20b. The maximum acidity among varieties was noticed in Ajmeri as 0.72% followed by 0.61% in Willium-82 and 0.57% in NARC-II. The treatment effect showed non-significant variation among T1 and T2 as 0.72% and 0.71% respectively. Overall interaction effect showed maximum acidity of Ajmeri soy milk 0.89 in T3 and minimum of To NARC-II soy milk was 0.42%.

The mean values showing significant effect of interaction among treatment*days are displayed in Table 4.20c. The results depicting overall increase in acidity with increase in storage span.

The maximum acidity was observed in T3 0.93% on zero day that decreased gradually up to 0.36% on 24th day of storage.

The mean values showing the effect of interaction among varieties*days is given in Table 4.20d. The results showed increase in acidity with the storage time. The variation was observed from 0.72 to 0.41% in NARC-II soy milk, 0.70 to 0.40 in Willium-82 soy milk and 0.67 to 0.71% in Ajmeri soy milk.

The result of current investigation is supported by Ismail et al. (2016). They studied on soy milk yoghurt on fresh, 7 and 15 days and results of their findings for pH was 4.86, 4.72 and 4.61 respectively. In addition, Li et al. (2014) has also reported gradual decrease in pH values during storage of fermented soy milk at different storage temperatures. The pH of fermented soy milk appeared to be time-dependent. They observed significant decreases over 21 days of storage. By the end of storage period, the pH of fermented products were significantly (P<0.01) lower than that recorded at 0 day. Our observations are also consistent with the report of Wang et al. (2009), who found the significant variation in pH values in the fermented soy milk samples during storage using L. casei Zhang as starter culture and they reported that these differences may also be linked to different raw materials and types of products, as well as the fermentation properties and specific fermentation activities of the

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Table 4.19a: Effect of varieties, treatment and storage days on pH of soy milk

Storage days Varieties Treatments 0 8 16 24

a ab abc ab To 6.97±0.004 6.92±0.103 6.5±0.279 6.9±0.103

d-g h-l lmn lmn T1 4.89±0.002 4.23±0.055 3.72±0.152 3.86±0.798 NARC-II e-i lmn mn mn T2 4.59±0.183 3.79±0.052 3.45±0.148 3.35±0.050

i-l lmn mn mn T3 4.2±0.168 3.82±0.004 3.54±0.155 3.43±0.019

abc abc abc abc To 6.61±0.005 6.56±0.098 6.59±0.270 6.51±0.097

de e-i g-k i-l T1 5.11±0.001 4.61±0.001 4.43±0.190 4.189±0.006 Willium-82 f-i mn mn mn T2 4.52±0.181 3.61±0.003 3.56±0.156 3.49±0.072

e-h mn mn mn T3 4.77±0.191 3.69±0.008 3.52±0.161 3.51±0.005

d bc c d To 6.89±0.03 6.38±0.095 6.26±0.25 5.4±0.13

d def e-i f-j T1 5.38±0.01 4.99±0.007 4.57±0.19 4.49±0.05 Ajmeri d-g klm mn mn T2 4.95±0.05 3.91±0.025 3.4±0.14 3.50±0.02

d j-m mn mn T3 5.31±0.01 3.94±0.063 3.58±0.16 3.61±0.01

Mean of storage days 5.35a 4.71b 4.43c 4.35c

Results are expressed as mean ± standard deviation of means; n = 3 sets a-n = Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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Table 4.19b: Effect of treatment and varieties on pH of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

a b c a To 6.82 6.54 6.23 6.53

f e d b T1 4.17 4.59 4.86 4.54

h h gh c T2 3.79 3.80 3.94 3.92

h h fg c T3 3.74 3.87 4.13 3.85

Mean 4.64b 4.70b 4.79a

Table 4.19c Mean values showing effect of treatment and days on pH of soy milk

Treatments Storage days

0 8 16 24

a ab bc c To 6.79 6.62 6.45 6.27

d e f f T1 5.12 4.61 4.24 4.18

e gh i i T2 4.69 3.77 3.47 3.45

e g ghi hi T3 4.77 3.82 3.54 3.52

Table 4.19d: Effect of varieties and days on pH of soy milk

Varieties Storage days

0 8 16 24

NARC-II 5.16b 4.69 cd 4.30fg 4.38fg

Willium-82 5.23b 4.62cde 4.52def 4.42efg

Ajmeri 5.65a 4.81c 4.45efg 4.25g

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Table 4.20a: Effect of varieties, treatment and storage days on acidity (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

p op nop mno To 0.39±0.015 0.41±0.021 0.43±0.019 0.47±0.021

efg T1 0.63±0.027j 0.75±0.030i 0.88±0.045gh 0.95±0.043 NARC-II c-f ab r qr T2 0.89±0.039 0.97±0.041 0.94±0.038 0.93±0.048

ab a qr q T3 0.98±0.045 0.99±0.043 0.92±0.039 1.00±0.041

op nop mno lmn To 0.41±0.01 0.43±0.011 0.45±0.01 0.53±0.012

k j hi fg T1 0.56±0.014 0.64±0.016 0.78±0.02 0.84±0.021 Willium-82 cde ab a qr T2 0.91±0.022 0.96±0.024 0.99±0.02 0.99±0.003

abc ab r qr T3 0.94±0.023 0.98±0.024 0.98±0.003 0.98±0.004

nop mno lmn lm To 0.42±0.01 0.46±0.012 0.47±0.011 0.49±0.01

kl j i efg T1 0.52±0.013 0.62±0.015 0.73±0.018 0.87±0.02 Ajmeri efg c-f bcd i T2 0.85±0.021 0.89±0.022 0.93±0.023 0.98±0.01

def abc ab i T3 0.88±0.022 0.94±0.024 0.98±0.024 1±0.018

Mean of storage days 0.69a 0.75b 0.58c 0.51d

Results are expressed as mean ± standard deviation of means; n = 3 sets a-r = Means in column with different superscripts differ substantially (p<0.05) T0= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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Table 4.20b: Effect of treatment and varieties on acidity (%) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

0.42h 0.44gh 0.46g 0.44c T0

0.77c 0.71d 0.69d 0.72a T1

0.53f 0.75c 0.85b 0.713a T2

e f a b T3 0.58 0.55 0.89 0.67

0.57c 0.61b 0.72a Mean

Table 4.20c: Effect of treatment and days on acidity (%) of soy milk

Treatments Storage days

0 8 16 24

i hi gh g To 0.41 0.43 0.45 0.47

f e d c T1 0.57 0.67 0.78 0.86

c b e j T2 0.88 0.94 0.68 0.33

b a i j T3 0.93 0.97 0.42 0.36

Table 4.20d: Effect of varieties and days on acidity (%) of soy milk

Varieties Storage days

0 8 16 24

NARC-II 0.72cd 0.78a 0.38h 0.41g

Willium-82 0.70d 0.75b 0.59f 0.40gh

Ajmeri 0.67e 0.73c 0.78a 0.71cd

115 different strains studied. Likewise, the current results are also in conformity with Li et al. (2014). They have reported the dramatic increase in titratable acidity values of the fermented soy milk products during storage. The titratable acidity value of the fermented soy milk before storage was 36.02 °T, whereas the final titratable acidity values of the fermented product after the 21 days were 77.50°T at 4°C and 143.02 °T at 25°C, respectively. Likewise, Wang et al. (2009) has also reported the similar results for the titratable acidity values of fermented soy milk samples which varied in between 0.40 and 0.46%. They observed the dramatic increases in acidity values in the fermented soy milk samples during 28 days of storage.

4.2.5. Determination of water holding capacity (WHC)

WHC of fermented soy milk is an important parameter in manufacturing fermented soy milk. Water holding capacity is related to gel network of fermented soy milk and attributes to framework of protein. Physiochemical changes and microbial action during storage weakens network thus lowering WHC in the later stages (Bian et al., 2016).

The statistical analyses for water holding capacity mentioned in Table 4.17 represented that there was highly substantial (P<0.01) variation in WHC due to treatment, varieties and storage days. The effect of their interaction was also highly significant (P<0.01) treatment*varieties, treatment*days. The non-significant (P>0.05) influence was noticed for the interaction of varieties*days and treatment*varieties*days on water holding capacity of soy milk.

The mean values for the effect of fermentation, varieties and storage days on WHC of soy milk are given in Table 4.21a. The results showed significant decrease in WHC during storage of 24 days which varied in the range of 48.15 to 38.58%. The data represented that WHC was not detected in any of non-fermented samples due to its very thin and liquid in nature and not any structure stability. The mean values of NARC-II fermented soy milk showed appreciable higher WHC in T2 (71.32±3.06%) on 0 day and minimum was noticed in T1 (46.31±1.89%) on 24th day of storage. The water holding capacity of Willium-82 fermented soy milk also showed similar trend of WHC as in NARC-II. The highest average WHC was noticed in T2

(74.3±3.06%) fermented with L. casei than T3 (62.69±2.78%) and least was recorded in T1

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Table 4.21a: Effect of varieties, treatment and storage time on water holding capacity (WHC) (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 0±0 0±0 0±0 0±0

T1 59.01±2.41 58.27±2.38 54.33±2.23 46.31±1.89 NARC-II

T2 71.32±3.06 66.47±2.85 59.26±2.54 51.25±2.20

T3 63.21±2.78 61.15±2.69 56.17±2.47 50.15±2.20

To 0±0 0±0 0±0 0±0

Willium- T1 60.21±2.41 57.32±2.35 53.17±2.17 48.16±1.97 82 T2 74.3±3.06 73.15±3.15 70.25±3.02 60.28±2.59

T3 62.69±2.78 60.39±2.65 58.17±2.55 52.13±2.29

To 0±0 0±0 0±0 0±0

T1 62.59±2.56 60.17±2.46 59.76±2.45 53.16±2.17 Ajmeri

T2 69.54±2.99 65.39±2.81 62.15±2.67 54.87±2.35

T3 58.56±2.57 56.78±2.49 54.75±2.40 46.72±2.05

Mean of storage days 48.15a 46.59b 44.0c 38.58d

Results are expressed as mean ± standard deviation of means; n = 3 sets abcdefg Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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Table 4.21b Effect of treatment and varieties on water holding capacity (WHC) (%) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

e e e c To 0.00 0.00 0.00 0.00

d d c b T1 54.48 54.42 58.92 55.94

b a b a T2 62.07 68.75 62.98 64.60

c c d b T3 57.67 58.46 54.20 56.78

Mean 43.56b 45.41a 44.03b

Table 4.21c Effect of treatment and days on water holding capacity (WHC) (%) of soy milk

Treatments Storage days

0 8 16 24

g g g g T0 0.00 0.00 0.00 0.00

c cde e f T1 60.20 58.59 55.75 49.21

a a b e T2 70.73 68.34 63.89 55.47

bc cd de f T3 61.66 59.44 56.36 49.67

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(60.21±2.41%). The results for WHC (%) of Ajmeri soy milk showed that at initiation of storage period T2 with 69.54±2.99% was at top for value of WHC followed by 62.59±2.56% and 58.56±2.57% in T1 and T3 respectively. With the passage of time WHC goes on decreasing this effect was similar in all the treatments.

The effect of interaction between treatment*varieties in Table 4.21b showed that there is significant effect of both treatment and varieties with each other on WHC of fermented soy milk. Overall effect of varieties shown from mean values depicted that maximum WHC was recorded in Willium-82 was 45.41% and non-significant effect was noticed in Ajmeri

(44.03%) and NARC-II (43.56%). The effect of treatment was 64.60% in T2 soy milk fermented with L. casei lagged by 56.78% in T3 (L. acidophilus+ L. casei) and 55.94% in T1 (L. acidophilus).

The mean values for the effect of treatment*days given in Table 4.21c elucidated that there is a pronounced effect of treatment and storage time on quality of fermented soy milk. The th highest WHC was recorded on 0 day in T2 was 70.73% that decreased to 55.47% on 24 day of storage. The second highest effect was noticed in T3 which varied in between 61.66 to

56.36%. The variation in T1 fermented soy milk was in between 60.20 to 49.21% from 0 to 24th day of storage respectively.

WHC of a protein gel is an important parameter in manufacturing of fermented soy milk. The present research stated that water holding capacity (%) goes decreasing during storage and causes whey separation that partly may be due to the unstable gel network of fermented product when three dimensional networks of protein micelles cannot entrap water within its network and ultimate weak colloidal linkage is formed. The results of recent findings are supported by Maftei et al. (2012). They have studied the fermented beverage prepared from soy milk and sea buckthorn syrup. They find out that after 14 days of storage, water holding capacity varied between 74.39-72.44% for the sample fermented at 30ºC and between 74.74- 72.74% for the sample fermented at 37ºC. Likewise, Kovalenko and Briggs (2002) found 84.1-96% of water holding capacity in soy based desserts and Mocanu et al. (2009) reported that the water holding capacity of milk and sea buckthorn extract was 65.2%. Further, Yang and Li (2010) reported water holding capacity of probiotic soy yoghurt varied in between

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85.07 to 92.35%. Moreover, Stijepic et al. (2013) estimated the WHC (46.5-53%) in soy yoghurt made with using inulin.

4.2.6. Rheology (viscosity)

Rheology is a fundamental interdisciplinary science that has been gaining increasing importance in food product quality. Flow properties in foods, such as consistency, thickness, viscosity, viscoelasticity and yield stress, help to characterize macroscopic phenomena that occur before, during and after the deformation of materials. Rheology is an essential science for the chemico physical characterization of liquid systems and for the development of new products. Viscosity is an essential attribute for beverages and is usually measured to know the effect of gums, stabilizers or thickening agents added to improve their texture. Nowadays the trend is shifting towards usage of such cultures that plays a role in improving of functional features of fermented foods. LAB can act as a functional ingredient by modifying rheological features of siy milk (Vuyst et al., 2003). They can improve the flow behavior of fluids, gel stability, particle flocculation, encapsulation and emulsion formation (Charchoghlyan and Park, 2013). Therefore, soy milk fermented with LAB provides an approach for improving aroma and flavor. They also modulated the textural properties of soy milk which improved the water holding capacity (WHC) and apparent viscosity (Champagne et al., 2009; Yeo and Liong, 2010).

Rheology of all the soy milk samples were studied on 0, 8, 16 and 24th day of storage. It is obvious from graphical representations that soy milk is non-newtonian fluid because its viscosity did not remain constant when it was subjected under different shear rate applied at a constant temperature. No doubt, the soy milk prepared from three varieties was fermented with different cultures but all samples showed a non-linear relationship between viscosity and shear stress and behaved like a non-newtonian fluid. The rheology for soy milk suggested a momentous decline upon storage and maximum viscosity was observed in soy milk fermented with combinations of cultures. However, L. casei depicted more compact gelation properties in comparison to L. acidophilus. The process of fermentation showed a clear compact gel formation and a viscous fluid was formed after the fermentation as compared to non-fermented soy milk.

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The values for NARC-II soy milk suggested that on zero day, the highest viscosity was observed in T3 (mix culture) as 120.64 Pa.s trailed by T2 (containing L. casei) with 98.28

Pa.s, T1 (containing L. acidophilus) as 40.24 Pa.s and To (controlled) showed minimum 0.735

Pa.s. Rheology on 0.1 shear rate 1/s and at 2000 shear rate 1/s the values for To, T1, T2 and T3 were 0.005, 0.015, 0.007 and 0.008 Pa.s respectively. The storage study indicated a decline in viscosity as at 0.1 shear rate (1/s). The viscosity of soy milk for To, T1, T2 and T3 were 0.04, 21.07, 35.19 and 77.45 Pa.s respectively (Appendix-II).

The values for Willium-82 soy milk suggested that at zero day highest viscosity observed in

T3 was 449.93Pa.s trailed by T2 with 144.776 Pa.s, T1 as 60.129 Pa.s and To showed minimum as 0.97 Pa.s on 0.1 shear rate 1/s and on 2000 shear rate 1/s the values for To, T1,

T2 and T3 were 0.06, 0.009, 0.014 and 0.019 Pa.s respectively. The storage study indicated a decline in viscosity at 0.1 shear rate (1/s). The viscosity of soy milk for To, T1, T2 and T3 were 0.157, 16.680, 13.653 and 17.458 Pa.s respectively (Appendix-III).

The values calculated by rheometer for Ajmeri soy milk are given in Appendix- IV. The highest viscosity at 0.1 shear rate (1/s) recorded on zero day was in T3 (Ajmeri soy milk fermented with L. acidophilus + L. casei) was 891.69 Pa.s lagged by T2 (571.38 Pa.s), T1 th (425.31Pa.s) and To 0.08 Pa.s. The storage study caused decline in viscosity on 24 day of storage. The values for To, T1, T2 and T3 were 8.59E-03, 80.66, 98.28 and 252.56 Pa.s.

The graphical representation (Figure 4.7) exhibited variation in viscosity versus shear rate for NARC-II soy milk at 0, 8, 16 and 24th day. The graphs showed that soy milk indicated shear thinning behavior and pseudo plastic fluid because its viscosity decreased and it became thinner as higher shear strain rate was applied. When the shear rate was same, the viscosity of

T3 was higher than that of T1 and T2. However, To presented minimum viscosity as there was no gel or compact structure in non-fermented soy milk.

It is shown in Figure 4.8 that soy milk samples of Willium-82 variety exhibit non-newtonian behavior because its texture was also damaged after shearing. The increasing rate of shear caused decrease in viscosity. The treatments T1 and T2 showed low viscosity because they contain solely culture as compared to T3 which have combination of L. acidophilus and L. casei. The Figure 4.9 indicated the effect of shear rate on viscosity of Ajmeri soy milk in

121 such a manner that as shear rate increased from 0.1 to 200-1/s. The deformation in texture of soy milk showed a significant decrease in viscosity. However, T3 displayed the maximum viscosity in comparison to T2 and T1. The non-fermented soy milk To showed very low viscosity as there was no culture used in that can modify its textural properties. Among the cultures, L. casei samples showed great potential to sustain higher viscosity in comparison of

L. acidophilus but their combination in T3 exhibited a best gel formation among all.

In a nut shell, the results for the viscosity of three fermented soy milk products (Willium-82, NARC-II and Ajmeri) decreased sharply with the increase of shear rate, which indicated that these fermented products were shear thinning fluid and the product is non-Newtonian. The viscosity of soy milk increase dut to coagulation of soy protein molecules and development of well strengthened structure. The increase in shear rate effects on dispersion of water molecules and ability of gel to retain water molecules within cells effects badly. That causes increase in shear rate and decrease of viscosity.

The results of current findings are in harmony with Li et al. (2014) who studied on rheological properties of fermented soy milk with exopolysaccharide (EPS) producing lactic acid bacterial strains and they also reported a decline in apparent viscosity of soy milk during 21 days of storage and also that the higher shear rate showed decrease in viscosity of fermented soy milk. Likewise, do Espirito-Santo et al. (2014) support the results of recent findings. They studied on soy milk and reported that the apparent viscosity increased significantly as the result of fermentation. They studied on rice, soy and passion fruit fiber gruels containing L. plantarum, L. acidophilus and B. lactis. The cultures promoted the lowest apparent viscosity which was not different between them, in spite of the significant differences in the fermentation time. The soy milk fermented by L. casei and L. acidophilus presented the area of thixotropic loop which pointed out that these bacteria either produced exopolysaccharides that stabilized the protein gel network or promoted proteolysis.

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Figure 4.7: Variation in viscosity of NARC-II soy milk at different shear rate at 0, 8th, 16th and 24th day

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Figure 4.8: Variation in viscosity of Willium-82 soy milk at different shear rate at 0, 8th, 16th and 24th day

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Figure 4.9: Variation in viscosity of Ajmeri soy milk at different shear rate at 0, 8th, 16th and 24th day

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It can be stated that remarkable proteolytic activity of the probiotic strains L. acidophilus and L. casei can be one of the responsible factors for the production of antihypertensive peptides in soy milk at acidic pH around 4.5. So, the lowest apparent viscosity of soy milks fermented by L. acidophilus can be attributed to a more extensive proteolytic activity by these probiotics.

Moreover, Kailasapathy and Masondole (2005); Kailasapathy, (2006) also reported that exopolysaccharides can be produced by strains of L. acidophilus and L. casei and are used in food industry as thickeners, stabilizing or emulsifying agents. So, it can be also stated that other than the composition of ingredients of a fermented or soy based food product, the viscosity of the final product depends on several factors such as the pH, proteolytic activities of the fermenting bacteria and the exopolysaccharides produced by them, the degree of soy protein gelation and protein content (Nguyen et al., 2007; Grygorczyk and Corredig, 2013).

4.2.7. Anti-oxidative activities of fermented soy milk

The mean square for antioxidant potential of soy milk prepared from three different varieties was checked by DPPH, ABTS and FRAP‟activities depicted in Table 4.22. It is elucidated from the results that effect of treatment, varieties and storage days was highly significant (P<0.01) for DPPH, ABTS and FRAP activities of soy milk. It is also noticed from the results that interactions between treatment*varieties has the significant (P<0.01) effect on DPPH, FRAP and ABTS and treatment*days had significant (P<0.05) on DPPH but non- significant (P>0.05) on FRAP and ABTS potential of soy milk. However, a non-significant (P>0.05) effect was also observed due to interaction of varieties*days and treatment*varieties*days.

4.2.7.1. 1,1-diphenyl-2-picrylhydrazyl (DPPH)

DPPH is a stable free radical with a maximum absorbance at 517 nm in ethanol as a solvent. When DPPH encounters a proton-donating substance such as an antioxidant, the radical is scavenged and the absorbance is reduced (Yang et al., 2008) and this radical scavenging activity is visually noticeable as a change in the color of DPPH, i.e. from purple to yellow, in the presence of an antioxidant (Liu et al., 2005; Zhang et al., 2011).

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The effect of treatment, storage days and varieties on the free radical scavenging activity of soy milk is mentioned in Table 4.23a. The non-significant interaction was recorded for the effect of treatment*days*varieties. The DPPH values showed decrease in antioxidant activities from 44.06% to 37.33% during 24 days of storage. The results for NARC-II soy milk show that the mean values of DPPH content fall in the range of 19.15±0.38% to 56.25±1.12%. The minimum scavenging activities were recorded in non-fermented soy milk of NARC-II (To was 25.40±0.51 to19.15±0.38%) and highest recorded in T3 was (56.25±1.12 to 50.79±1.01%) at the time of start to 24th day of storage. The effect of fermentation and storage of Willium-82 soy milk is depicting non-significant interaction among th treatment*days. The minimum DPPH activity recorded in To was 18.13±0.36% on 24 day while the maximum noticed in T3 (zero day) was 54.37±1.08%. The free radical scavenging ability of Ajmeri soy milk showed variation due to fermentation in different treatments. The th scavenging ability for To was from 20.09±0.40% (24 day) to 60.23±1.20% in T3 (zero day). Overall the study evaluated that antioxidant potential of fermented soy milk was augmented in comparison to non-fermented soy milk. After 24 days of storage the antioxidant activity was decreased but it still remained higher than non-fermented soy milk.

The effect of fermentation by different treatments and varieties on free radical scavenging activity of soy milk is mentioned in Table 4.23b. The effect of fermentation gives higher DPPH values in Ajmeri soy milk (43.38%) as compared to NARC-II (40.59%) and Willium-82 (39.02%). The findings are correlated to growth of cultures in media as viable cell count was higher in samples with combined cultures followed by L. casei and L. acidophilus indicated ultimately higher antioxidant activity as determined by DPPH. The mean values for T3 showed maximum activity (53.97%) followed by T2 (45.53%), T1 (41.98%) and To (22.51%) for DPPH. The antioxidant activity was higher in soy milk fermented with combination of both cultures and among them L. casei effect was more reflective as compared to L. acidophilus. The overall antioxidant activity was higher in fermented samples than that of non-fermented.

The present results are in conformity with Abubakr et al. (2012). They have studied on the antioxidant activity of lactic acid bacteria fermented skim milk and reported DPPH for seven isolates was ranging from 14.7 to 50.8% after 24 to 72 h fermentation, respectively.

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Table 4.22: Mean Squares for antioxidant potential of soy milk

Source DF DPPH FRAP ABTS Treatment 3 6381.79** 1.44101** 5472.95** Varieties 2 233.97** 0.048** 322.63** Days 3 304.77** 0.022** 644.58** Treatment*Varieties 6 10.31** 0.0021** 20.68** Treatment*Days 9 1.76* 0.00014 NS 9.91NS Varieties*Days 6 1.05NS 0.00044 NS 12.61 NS Treatment*Varieties*Days 18 0.86 NS 0.00027 NS 2.59 NS Error 96 0.73 0.00028 9.08

Total 143 **=Highly Significant (P<0.01) *=Significant (P <0.05) NS= non-significant (P >0.05)

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Table 4.23a Effect of varieties, treatment and storage days on DPPH (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 25.40±0.51 23.42±0.46 21.37±0.43 19.15±0.38

T1 44.53±0.89 43.77±0.87 40.16±0.80 38.25±0.76 NARC-II

T2 49.06±0.98 46.21±0.92 44.79±0.89 40.56±0.81

T3 56.25±1.12 53.56±1.07 52.15±1.04 50.79±1.01

To 24.36±0.48 23.76±0.47 20.23±0.41 18.13±0.36

Willium- T1 42.29±0.84 41.56±0.83 39.72±0.79 36.59±0.73 82 T2 45.96±0.91 43.76±0.87 42.15±0.84 38.72±0.77

T3 54.37±1.08 52.83±1.05 51.76±1.03 48.15±0.96

To 26.23±0.52 25.56±0.51 22.37±0.44 20.09±0.40

T1 48.17±0.96 45.39±0.91 43.17±0.86 40.17±0.80 Ajmeri

T2 51.87±1.03 50.71±1.01 48.36±0.96 44.16±0.88

T3 60.23±1.20 58.12±1.16 56.31±1.12 53.17±1.06

Mean of storage days 44.06a 42.38b 40.21c 37.33d

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Table 4.23b: Effect of treatment and varieties on DPPH (%) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

i i h d To 22.34 21.62 23.56 22.51

f g e c T1 41.68 40.04 44.23 41.98

e f d b T2 45.16 42.65 48.77 45.53

b c a a T3 53.19 51.77 56.957 53.97

Mean 40.59b 39.021c 43.380a

Table 4.23c: Effect of treatments and days on DPPH (%) of soy milk

Storage days Treatments 0 8 16 24

k k l m To 25.33 24.25 21.32 19.12

g h i j T1 44.99 43.57 41.02 38.34

e f g i T2 48.96 46.89 45.10 41.15

a b c d T3 56.95 54.84 53.41 50.70

Results are expressed as mean ± standard deviation of means; n = 3 sets a-m =Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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In the same year another group of researchers Marazza et al. (2012) has strengthened the present findings. They reported enhancement of the antioxidant capacity of soy milk by fermentation with L. rhamnosus. They noted that the DPPH radical scavenging was 29.5% than the unfermented soy milk used as control whose value for DPPH was reported as 9.2%. The current research is in assenting with findings of Subrota et al. (2013), who have studied the antioxidative activity in fermented soy milk supplemented with WPC-70 by probiotic lactobacilli and results for DPPH % reduction was 42.72-50.09 in fermented soy milk while no any reduction was detected in non-fermented soy milk. Later on, Zhao et al. (2014) studied the changes in antioxidant capacity of soy milk during extended fermentation and the results for DPPH radical scavenging ability (%) for Lactic acid bacteria were 16.9 to 19.6% at 4.55 pH and 28.4-31.6 at 4.15 pH and 30.8 to 36.5 at pH 3.85.

Likewise, Ma and Huang, (2014) have conducted research on antioxidant activity of non- fermented soy milk samples and stated that antioxidant DPPH varied in between 1.36-1.83 (µmol TE g-1). After that, Embiriekah et al. (2016) studied the selection of lactobacillus strains for improvement of antioxidant activity of different soy, whey and milk protein drinks. Their results showed that strain Lb. acidophilus exhibited higher antioxidant activity 64.9% of all tested substrates during 24 h of fermentation. It is therefore concluded that lactic acid fermentation may constitute a promising route to improve the antioxidants ability and nutritional qualities of processed soy milk.

4.2.7.2. Ferric Reducing Anti-oxidant Power (FRAP)

FRAP measures the ferric reducing ability of the samples, in acidic medium forming an intense blue color as the ferric tripyridyltriazine (Fe3+-TPTZ) complex is reduced to the ferrous (Fe2+) form.

The mean values for the effect of treatment, varieties and storage time on FRAP (mmolFe2+/L) of soy milk is illustrated in Table 4.24a. The significant effect of storage showed variation in reducing power from 0.56 to 0.51 mmolFe2+/L during 24th days of storage period. The FRAP values were recorded higher in fermented soy milk in comparison to non-fermented. In

NARC-II soy milk the maximum FRAP recorded in in T3 (0 day) was 0.69±0.0172 2+ th 2+ mmolFe /L. In To (24 day) was 0.18±0.004 mmolFe /L. The mean values for the effect of

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Table 4.24a: Effect of varieties, treatment and storage days on FRAP (mmolFe2+/L) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 0.23±0.006 0.21±0.005 0.23±0.005 0.18±0.004

T1 0.56±0.088 0.55±0.013 0.54±0.013 0.52±0.013 NARC-II

T2 0.64±0.016 0.62±0.015 0.61±0.015 0.59±0.014

T3 0.69±0.0172 0.65±0.016 0.64±0.016 0.64±0.016

To 0.26±0.006 0.25±0.006 0.23±0.009 0.21±0.01

Willium- T1 0.62±0.015 0.58±0.014 0.56±0.014 0.53±0.01 82 T2 0.67±0.016 0.65±0.016 0.63±0.016 0.61±0.02

T3 0.71±0.017 0.69±0.017 0.67±0.017 0.65±0.06

To 0.31±0.007 0.28±0.007 0.26±0.007 0.25±0.006

T1 0.67±0.016 0.65±0.016 0.63±0.016 0.61±0.015 Ajmeri

T2 0.69±0.017 0.67±0.016 0.65±0.016 0.63±0.015

T3 0.73±0.018 0.72±0.018 0.69±0.017 0.67±0.016

Mean of storage days 0.56a 0.54b 0.53c 0.51d

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Table 4.24b: Effect of treatment and varieties on FRAP (mmolFe2+/L) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

j i h d To 0.21 0.24 0.28 0.24

g f d c T1 0.54 0.58 0.64 0.59

e d c b T2 0.62 0.64 0.66 0.64

c b a a T3 0.65 0.68 0.70 0.68

Mean 0.51 c 0.53 b 0.57 a

Results are expressed as mean ± standard deviation of means; n = 3 sets a-j =Means in column with different superscripts differ substantially (P<0.05) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

133 fermentation and storage time on FRAP of Willium-82 soy milk is mentioned in Table 4.24a and results showed that LAB has promising potential in enriching antioxidant potential of soy milk. Likewise, reducing potential of non-fermented soy milk (To 0.31±0.007 2+ 2+ mmolFe /L) and T3 the coculture fermented soy milk was 0.73±0.018 mmolFe /L at the start of product storage.

The mean values for the effect of treatments on fermentation and varieties on reducing ability of soy milk are given in Table 4.24b. The effect of treatment was highly positive in 2+ 2+ fermented soy milk such as in T3, T2 and T1 were 0.68 mmolFe /L, 0.64 mmolFe /L and 0.59 mmolFe2+/L and in non-fermented soy milk showed 0.24 mmolFe2+/L. The maximum 2+ 2+ value recorded in Ajmeri T3 was 0.70 mmolFe /L and minimum was 0.21 mmolFe /L in To. This showed that LAB may have potential to enrich soy milk by enhancing antioxidant values. The overall effect of varieties showed the maximum reducing power in Ajmeri (0.57 mmolFe2+/L) followed by 0.53 mmolFe2+/L in Willium-82 and 0.51 mmolFe2+/L in NARC- II.

The current research is in assenting with findings of Subrota et al. (2013) who have studied the anti-oxidative activity in fermented soy milk supplemented with WPC-70 by probiotic lactobacilli. Their results for FRAP varied in between 719.79 to 801.25µM3 in fermented soy milk and for non-fermented soy milk it was 722.57 µM3. Likewise, Ma and Huang, (2014) studied on characterization and comparison of phenols, flavonoids and isoflavones of soy milk and they correlated these with antioxidant activity and reported that FRAP was 1.83 to 4.20 mmol Fe2+/100g in non-fermented soy milk. The value of ferric reducing antioxidant power were synchronized with Embiriekah et al. (2016) who studied the effect of lactobacillus strains for improvement of antioxidant activity of different soy, whey and milk protein substrate. They reported that L. acidophilus increase the antioxidant activity of all tested substrates during 24 h of fermentation and reported that FRAP was 0.6127 mmol Fe2+/L.

4.2.7.3. 2, 2-azinobis- 3-ethylbenzothiazoline-6-sulphonate (ABTS)

The mean values pertaining effect of treatments, varieties and storage time on ABTS (%) of soy milk is presented in Table 4.25a.

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Table 4.25a: Effect of varieties, treatment and storage days on ABTS (%) of soy milk

Storage days Varieties Treatments 0 8 16 24

To 52.37±1.59 50.63±1.54 45.37±1.38 39.32±1.20

T1 74.12±2.26 71.39±2.18 69.15±2.11 62.16±1.89 NARC-II

T2 78.23±2.38 75.13±2.29 71.89±2.19 68.37±2.08

T3 83.17±2.54 80.13±2.44 77.16±2.35 71.42±2.18

To 58.23±1.77 54.83±1.67 52.10±1.59 47.16±1.44

Willium- T1 76.72±2.34 73.76±2.25 71.67±2.18 67.37±2.05 82 T2 79.73±2.43 74.53±2.27 73.05±2.23 70.69±2.16

T3 85.46±2.61 82.31±14.28 78.37±2.39 74.56±2.27

To 59.81±1.82 56.17±1.716 53.31±1.62 48.57±1.48

T1 78.37±2.39 75.68±2.31 73.17±2.23 69.07±2.11 Ajmeri

T2 80.37±2.45 78.16±2.38 76.37±2.33 72.14±4.47

T3 87.69±2.67 83.12±2.53 80.31±2.45 77.39±2.36

Mean of storage days 74.03a 70.27b 68.05c 63.90d

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Table 4.25b: Effect of treatment and varieties on ABTS (%) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

g f f d To 46.61 52.78 54.10 51.165

e de cd c T1 68.74 71.89 73.58 71.406

d bcd bc b T2 72.91 74.00 77.19 74.702

bc ab a a T3 77.45 77.93 81.58 78.985

Mean 66.43c 69.15b 71.61a

Results are expressed as mean ± standard deviation of means; n = 3 sets a-g = Means in column with different superscripts differ substantially (P<0.05) T0= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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The results showed that soy milk pocesses greater antioxidant activity. The storage effect showed a decrease in ABTS (74.03 to 63.90%) from 0 to 24th day of storage. The maximum

ABTS was observed in T3 (83.17±2.54%) on zero day and minimum was noticed in To (39.32±1.20%) on 24th day of storage. The storage time showed a decline in ABTS (%) from 71.97±13.58% (on zero day) to 60.31±14.51% (on 24th day). Mean values showing the effect of fermentation and storage time on ABTS (%) of NARC-II, Willium-82 and Ajmeri soy milk is mentioned in Table 4.25a. The results followed the same trend as in NARC-II soy milk. The overall antioxidant potential was seems to be higher in soy milk fermented with L. acidophilus and L. casei at the same time and reduced activity was noticed in non-fermented soy milk. The highest value was observed in T3 (85.46±2.61%) of Willium-82 and (87.69±2.67%) of

Ajmeri soy milk on zero day, while least was in To (47.16±1.44%) Willium-82 soy milk and (59.81±1.82%) in Ajmeri soy milk at 24th day of storage.

The mean values showing combined effect of treatment*varieties on ABTS (%) of soy milk are given in Table 4.25b. The treatment effect showed the clear picture that highest ABTS activity was noticed in T3 (78.99%) followed by T2 (74.70%), T1 (71.41%) and To (51.16%). Among the varieties pronounced effect of Ajmeri was 71.61% and 69.15% in Willium-82 and least was 66.43% in NARC-II.

The results for higher antioxidant values of fermented soy milk in all samples are suggesting that strain L. acidophilus and L. casei is capable to produce high amount of antioxidant compounds on soy protein substrate, probably due to its higher proteolytic activity on protein source. It could be said that enzymatic system of L. casei and L. acidophilus much efficiently hydrolyzes the soy protein sources leading to the production of significantly higher antioxidant activity.

The current research is in affirmative with findings of Subrota et al. (2013) who have studied antioxidative activity in fermented soy milk and results for ABTS% inhibition were in the range of 88.05-97.05% while in non-fermented soy milk it was 71.65%.

4.2.8. Sensory evaluation of soy milk

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The demands of food industry are increasing globally due to stimulation of great competitions, high quality and enhanced shelf life with low cost and more options with no trade barrier throughout the world. In this challenging framework the success of industry is based on its new functional product innovation and consumer awareness about its health benefits and its best implementation and measurement. The sensory evaluation is a critical key tool to process this all. Sensory evaluation of soy milk is an important step to know the consumer perception about value added product. Soy milk was evaluated by using 9 point hedonic scale at the four intervals for parameters i.e. color, appearance, aroma, flavor, taste and overall acceptability.

4.2.8.1. Color

In any of food products, color and appearance are the main attributes that influence the consumer‟s opinion and perceptions of taste, flavor, and acceptance. These are the two foremost factors that motivate the consumers to the long-lasting purchase of such foods (Granato et al., 2010). Generally speaking, consumers of soy milk expect the product to have a pale yellow color. Therefore, commercial manufacturers tend to maintain this natural color of soybean without adding color agent. In this research, natural soybeans were used and no color agent was added.

The statistical analysis of variance for color of fermented soy milk is given in Table 4.26. The outcomes showed highly significant (P<0.01) effect of color on days, varieties and treatments. The interactive effect was also highly significantly varied (P<0.01) for treatment*varieties while non-significant (P>0.05) effect on treatment*days, varieties*days and days*varieties*treatment for color of soy milk.

The mean values for effect of fermentation and storage time on color of NARC-II soy milk are illustrated in Figure 4.10a. The results showed the maximum score for color of To

(8.12±0.32) and minimum for T3 (7.1±0.50) on 0 day. Likewise, in Willium-82 soy milk the highest color score were (8.12±0.32) awarded to T2 and minimum to T1 (6.43±0.45). Similarly, the mean values for Ajmeri soy milk also showed significant variations in color such as maximum score was recorded in T3 (8.67±0.34) and minimum for T1 (7.03±0.49). The findings for color scores showed negative impact of storage on color of soy milk (data is

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Table 4.26: Mean squares for sensory evaluation of fermented soy milk

Source DF Color Aroma Flavor Texture Overall acceptabilit y Days 3 1.88380* 2.0504** 1.5879** 22.953** 1.6003** * Varieties 2 3.43340* 2.3002** 20.6280* 40.595** 26.3009** * * Treatment 3 2.72231* 65.4245* 56.9382* 172.147* 41.9408** * * * * Days*Varieties 6 0.29914N 1.0075** 0.1236NS 10.432 ** 0.0597NS S Days* Treatment 9 0.23719N 1.0567** 0.0421 NS 8.862 ** 0.1706* S Varieties* Treatment 6 1.30627* 4.6397** 1.8498** 0.361NS 0.9875** * Days*Treatment* 18 0.16819N 1.0688** 0.0699NS 2.967 ** 0.0374NS Varieties S Error 384 0.18514 0.2534 0.0576 0.368 0.0835 Total 431 **= Highly significant (P<0.01) *= Significant (P<0.05) NS= Non-significant

139

12 Color 10

8

6

Hedonicscale 4

2

0 To T1 T2 T3 To T1 T2 T3 To T1 T2 T3 NARC-II Willium-82 Ajmeri

0 Day 8 Days 16 Days 24 Days

Figure 4.10a: Interactive effect of treatmets*varieties*storage on the color of fermented soy milk

Figure 4.10b: Interactive effect of treatmets*varieties on the color of fermented soy milk

140 not shown). The Figure 4.10a is clearly representing that the highest score was given to Ajmeri T3 (Soy milk fermented with L. acidophilus and L. casei).The influence of treatments

(To, T1, T2 and T3) and varieties (NARC-II, Willium-82 and Ajmeri) on the color of fermented soy milk is represented in Figure 4.10b. It is illustrated in figure that treatment and varieties effect significantly with each other on color of fermented soy milk that each variety has different effect on treatments.

The present findings were comparable with Ashaye et al. (2001) who also reported continuous decrease in scoring of color which varied from 3.44 to 2.24 for soy yoghurt fermented with different starter cultures during 0 to 8 days of storage. The results of recent findings were consistent with Ma et al. (2015) who reported the highly significant differences among various soybean genotypes for soy milk color. They signify that the sensory property mainly determined by genotypic factor. It is concluded that different varieties and storage time affects the products organoleptic properties.

4.2.7.2. Aroma

Fermentation of soy milk offers chance to vary sensory features of soy-based foods. It gives peculiar aroma due to lactic acid production which dramatically contributes to flavor of products.

Statistical analysis of variance for sensory evaluation of aroma is given in Table 4.26. The results showed highly significant (P<0.01) variation among storage period, treatment, varieties and also pronounced effect was noticed among interaction of treatment*varieties, treatment*days, varieties*days along with treatment*days*varieties effect on aroma of soy milk.

Mean values for the effect of fermentation and storage time on aroma of soy milk given in Figure 4.11a depicted that NARC-II soy milk has noteworthy effect of fermentation on treatments such as minimum score was assigned to non-fermented To (3±0.12) while maximum to T1 (7.4±0.29). The score of aroma in Willium-82 soy milk, was maximum assigned to T3

(7.55±0.302) lagged by T2 (7.43±0.29), T1 (6.53±0.261) and To (4.12±0.164) on start of storage period while effect of storage showed drop in aroma scores. The mean values showing effect of fermentation and storage time on Aroma of Ajmeri soy milk have

141 explicated the maximum value was assigned to T3 (7.82±0.31) trailed by T2 (7.54±0.30), T1

(7.32±0.29) and To (3.50±0.14). The score below 4 was considered as poor quality of product that was given to non-fermented soy milk.

The interaction effect of varieties and storage days at the interval of (0, 8th, 16th and 24th day) shows that scores decreased momentously with increasing the storage days (Figure 4.11b). The varieties affected differently on different days of storage. The scores were significantly varied on each day and overall maximum scores were obtained by Ajmeri as 6.53, 6.28, 6.59 and 5.95 on regular intervals.

The scoring rate of present research work for fermented soy milk was of considerable quality and can enhance consumer‟s perception. The findings were also consistent with Ashaye et al. (2001) who reported consistent decrease in scoring of organoleptic properties of soy yoghurt fermented with different starter cultures. The results of recent findings are consistent with Falade et al. (2015) who also probed the mean scores of sensory attributes of soy and bambara plain yoghurts and reported that there was no significant difference between the aromas of both yoghurts. However, the aroma of plain soy yoghurt got aroma score of 4.40. They also reported a decline in scoring as storage period increased. Likewise, Ma et al. (2015) studied the soy milk and reported that soy milk aroma parameter had significant variances showing that environmental conditions plays a vital role. The genetic factors play a significant role in organoleptic properties of soy milk (Min et al., 2005; Poysa and Woodrow, 2002).

4.2.7.3. Flavor

Flavor may influence food market habits, and consumer‟s judgement.The product acception is mainly depends on flavor of any product than consumer will goes toward health benefits. The soy natural flavor is beany and astringent that did not liked by consumers. So, soy functional food is mainly targeting for flavor development. Hence, the improvement of products sensory features by combining functional ingredient can upgrade the consumption level of soy based products. Fermentation of soy offers chance to vary sensory features of soy-based foods. It gives peculiar aroma from lactic acid bacteria which dramatically produces flavor of products. Further, process of fermentation with lactobacilli assists in

142 flavor modification. It will decrease levels of volatile that causes natural beany flavor in soy products (Blagden and Gilliland, 2005).

The mean squares values of flavor for soy milk is given in Table 4.26. The findings were highly compelling (P<0.01) for the function of days, treatment, varieties and their interaction (treatment*varieties), whilst non-significant (P>0.05) effect of interaction was recorded for Ajmeri soy milk.

The mean values for effect of fermentation and storage time on flavor of soy milk have been explained graphically in Figure 4.12. The effect of treatment showed fermentation was helpful in increasing flavor scores of all treatments as compared to non-fermented soy milk.

The highest score for NARC-II soy milk flavor was recorded in T3 that varied in between

5.84±0.23 to 5.56±0.14, followed by T2 (5.54±0.22 to 5.00±0.12), T1 (5.43±0.21 th to5.32±0.13) and To (4.01±0.16 to 3.5±0.08) from 0 to 24 day of storage. Likewise, in Willium-82 soy milk score for flavor were decreased throughout storage span and maximum score was assigned to T3 that varied in between 6.84±0.27 to 6.14±0.15 straggled by T2

(6.56±0.26 to 6.37±0.16), T1 (6.34±0.25 to 6.22±0.16) and To (4.52±0.18 to 3.98±0.09).

Similarly, in Ajmeri soy milk the expert panelist gave higher score to T3 lagged by T2, T1 and

To i.e. 7.03±0.32, 6.76±0.29, 6.72±0.22 and 4.14±0.17 at initiation of storage days and after that it showed decrease in flavor scores.

The effect of treatment interaction with varieties is given in Figure 4.12b. The figure is illustrating that the significant effect was recorded in all treatments except that non- fermented soy milk To. The maximum score for flavor was observed in the interaction of T3 the fermented soy milk (L. acidophilus and L. casei) with all varieties as 5.67 to NARC-II. 7.83 Willium-82 and 6.82 to Ajmeri. Overall, the results showed that panelist liked the flavor of fermented soy milk.

The findings were also consistent with Ashaye et al. (2001) who reported consistent decrease in scoring of flavor that varied from 3.37 to 2.24 of soy yoghurt fermented with different starter cultures during 8 days of storage. The results of recent findings were in harmony with Ma et al. (2015) who also found highly significant variations among various soybean

143

Figure 4.11a: Interactive effect of treatmets*varieties*storage on the aroma of fermented soy milk

Figure 4.11b: Interactive effect of varieties*storage on the aroma of fermented soy milk

144

Figure 4.12a: Interactive effect of treatmets*varieties*storage on the flavor of fermented soy milk

Figure 4.12b: Interactive effect of treatmets*varieties on the aroma of fermented soy milk

145 genotypes for soy milk flavor attributes, suggesting that the sensory property was mainly determined by genotypic factor. Some authors stated that the activity of the lactic acid bacteria during the storage period is the reason of chemical alterations such as it causes an increase of acidity that affects the flavor attributes of soy milk. It has been stated that lactic acid increases the nutritional value of fermented products by engendering flavor and structure (Kun et al., 2008). Another group of scientists Khiralla et al. (2009) has studied on probiotic fermented soy milk and found significant enhancements in the odor and flavor due to using the probiotics in fermentation of soy milk comparing with the unfermented soy milk. Ara et al. (2002) stated that this may be due to organic acids and flavoring agents produced by probiotic bacteria in soy milk. LAB can influence on the metabolism of carbohydrates and proteins which improve the nutritional and final sensory quality of the fermented products. Moreover, fermentation in soy milk improves the sensory quality of final product by metabolizing n-hexanal which causes beany flavor in soy milk. Fermentation also decreases the activity of galacto-oligosaccharides that improves the digestibility of fermented soy milk (Mühlhans et al., 2015).

4.2.7.4. Texture

A statistical result for mean squares of sensory evaluation for texture in fermented soy milk is given in Table 4.26. The results illustrated that there was highly significant (P<0.01) impact of storage days, treatment, varieties and interaction among varieties*days and treatment*days on texture of soy milk. Conversely non-significant (P>0.05) variation was determined among interaction between varieties*treatments and treatment*days*varieties.

The mean values for the consequence of fermentation and storage time on texture of NARC-II soy milk has been represented in Figure 4.13. The outcomes were such as To get 2.45±0.06 score due to absence of culture. There was no texture development in it then T1 (6.74±1.54) followed by T2 (6.92±0.17) and T3 (7.45±0.19), which was at top of all. The storage period due to microbial activity showed decrease in texture score. Texture of Willium-82 soy milk has also showed that effect of fermentation improves the texture stability such as T3 get highest score (8.24±0.21) trailed by T2 (7.87±0.19), T1 (7.65±0.14) and To (2.33±0.52) at start of storage study. The outcomes for Ajmeri soy milk treatments were such as the product fermented with combined culture obtained highest score 8.89±0.22 in T3 followed by T2

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(8.45±0.21), T1 (7.53±0.18) and To (2.56±0.6) at the start of storage. Overall storage period showed debility in texture from start to end of storage. The scores of non-fermented soy milk were assigned as poor quality parameter because there was no texture development in To due to absence of LAB.

The interactive effect of varieties with storage days was varied significantly with each other for texture of fermented soy milk as represented in Figure 4.13b. The maximum scores were recorded in Ajmeri soy milk at 0 day was 7.47 and non -significant change was observed on 8th day (6.85) and 16th day (6.89) and 5.91 score was noticed on 24th day.

The treatment affected significantly (Figure 4.13c) the texture of fermented soy milk. The

Figure is illustrating that maximum score for texture was assigned to T3 that decreased gradually during storage days as 8.19, 8.13, 7.76 and 7.29 scores were recorded at 0, 8th, 16th and 24th day of storage. The interactive influence of treatment with varieties is illustrated in

Figure 4.13d for texture of fermented soy milk. The effect of T1and T2 was non-significant in

Ajmeri and Willium-82. However, the maximum score observed in T3 Ajmeri was 8.62 and least was of To non-fermented soy milk due to absence of culture.

The findings were also in agreement with Ashaye et al. (2001) who reported consistent decrease in scoring of texture that varied from 3.39 to 2.24 for soy yoghurt fermented with different starter cultures during 8 days of storage at refrigerated temperature. Later on, Chang et al. (2010) studied on soy yoghurt by mixing B. breve with S. thermophilus and L. acidophilus. Their sensory findings suggested that the mixed culture may be an ideal starter for the preparation of soy yoghurt.

4.2.7.5. Overall acceptability

Overall acceptability of any product plays a significant role in product accpetion rate. The quality of any product is mainly based on sensory properties and among which overall acceptability is top of all. The industry always develops any of the products after testing their color, aroma, texture, flavor and overall acceptability. So, the fermented soy milk was also passed from all these features for its commercialization and consumer acceptance (Granato et al., 2010).

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Figure 4.13a: Interactive effect of treatmets*varieties*storage on the texture of fermented soy milk

Figure 4.13b: Interactive effect of treatmets*storage on the texture of fermented soy milk

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Figure 4.13c: Interaction of varieties*days for texture of fermented soy milk

Figure 4.13d: Interaction of treatment*varieties for texture of fermented soy milk

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Figure 4.14a: Interactive effect of treatmets*varieties*storage on the overall acceptability of fermented soy milk

Figure 4.14b: Interactive effect of treatmets*varieties*on the overall acceptability of fermented soy milk

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Figure 4.14c: Interactive effect of treatmets*storage on the overall acceptability of fermented soy milk

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Statistical results for mean squares depicted in Table 4.26 showed highly significant (P<0.01) effect of days, treatments and varieties on the acceptability of soy milk. The interaction among varieties*treatment was also significant (P<0.01). The interaction between treatment*days was significant (P<0.05), while non-significant (P>0.05) variation among interaction of varieties*days and treatment*days*varieties on the acceptability of soy milk.

Figure 4.14a is elucidating that the results of fermentation and storage affected the overall acceptability of NARC-II soy milk. The significant variation among score of treatments was recorded as minimum in To (5.04±0.20 to 4±0.16) and maximum in T3 (6.9±0.27 to 6.57±0.26). Likewise, the influence of fermentation and storage time on overall acceptability of Willium-82 soy milk also exhibited substantial effect of treatments as 5.6±0.22, 7.23±0.28,

7.53±0.30 and 7.87±0.31 in To, T1, T2 and T3 respectively on start of storage. The outcomes of Ajmeri soy milk also showed similar trend as the highest score was recorded for T3

(8.55±0.34) that was very close to T2 (8.45±0.33) followed by T1 (8.23±0.32) and To (5.65±0.22) on 0 day.

The interaction of treatments with different varieties also varied significantly as given in

Figure 4.14b. The Figure is representing that panelist like mostly Ajmeri T3 (L.acidophilus and L.casei) and T2 (L.casei) fermented soy milk. The non-fermnted soy milk was below fair quality scores for soy milk.

The interactive influence regarding treatment with storage days varied as significantly and a decline trend was reported from start of product storage up to 24th day. The highest effect of th treatment with storage was observed in T3 which showed 7.79 scores at 0 day, 7.42 at 8 day, 7.46 at 16th day and 7.28 at 24th day of storage.

The group of other scientists Ashaye et al. (2001) has reported constant decrease in scoring of general acceptability as varied from 3.49 to 2.24 of soy yoghurt fermented with different starter cultures during 0 to 8 days of storage. However, Opara et al. (2013) worked on soy yoghurt made by using LAB and they reported the sensory properties of the soy-yoghurt evaluated by expert panelist. They reported that there were no observed differences in terms of overall acceptability for the sample except for the fact that there is high level of deterioration in soy milk based products during storage.

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Moreover, Ma et al. (2015) studied on soy milk made from seeds of different genotypes and reported that noticeably, the overall acceptability was merely affected by genotypes and independent of two environments in this study, which implied that it could be a stable parameter in soy milk sensory evaluation among soybean genotypes. They studied correlation coefficient (r) that showed that the overall acceptability was significantly and positively linked with other soy milk sensory parameters. This suggested that overall acceptability as an important sensory attribute.

4.2.9. Scanning electron microscopy

The microstructures of fermented soy milk of all three varieties were investigated to visualize texture and microstructure by using FEI NOVA nano SEM Field Emission SEM, Everhart Thornley detector (ETD) spot size 3 with 5KV. The observed specimens visualized that there was a pronounced microstructural differences between the networks developed by L. acidophilus and L. casei or with their combination in fermented soy milk samples.

Figure 4.15 is showing micrograph of NARC-II L. acidophilus soy milk. As shown in micrograph at 100 µm, the texture is looking uniform and well defined but a porous structure was more pronounced in 5 and 10 µm. Figure 4.16 is depicting micrograph of NARC-II soy milk fermented with L. casei. The results at 300 µm were looked like in correspondence with Figure 4.15, a but as focused on 30 and 10 µm, the micrographs exhibited more uniform and less porous structure in comparison to L. acidophilus. However in Figure 4.17 (50 µm, 30 µm and 10µm) is elucidating micrograph of NARC-II soy milk fermented with combination of L. acidophilus and L. casei it was evident from micrographs that a more compact network with more fine and uniform pore size were developed by using cultures in their combination.

Micrograph in Figure 4.18 comprises of Willium-82 L. acidophilus soy milk at 300, 10 and 5 µm showing well developed textural features however quite open protein micelles were observed. Figure 4.19 is depicting micrograph (40, 10 and 5 µm) of Willium-82 soy milk fermented with L. casei in which soy protein macro aggregates has developed more uniform and precise pore size as compared to L. acidophilus while notably in Figure 4.20 is elucidating micrograph of at 40 and 10 µm of Willium-82 soy milk fermented with combination of L. acidophilus and L. casei that has intermingled in soy protein network and

153 soy protein has remarkably developed a more rigid and aggregated gel by making an organized texture. Overall micrographs of Willium-82 showed more uniform and well developed structure as compared to NARC-II fermented soy milk.

The Figure 4.21 is showing micrograph (100, 10 and 4 µm) of Ajmeri L. acidophilus soy milk it showed more interspaces because soybean storage protein has showed weak linkage and larger pore dimensions but embedded bacteria was also seen in its texture as among L. acidophilus in all varieties its higher viable growth was recorded in Ajmeri soy milk. Figure 4.22 is depicting a micrograph at (100, 10 and 5 µm) of Ajmeri soy milk fermented with L. casei. It is showing relatively more uniform texture and smaller pore size in comparison to L. acidophilus sample. Figure 4.23 is elucidating micrograph (300, 40 and 10 µm) of Ajmeri soy milk fermented with combination of L. acidophilus and L. casei depicting constant and precise pore formation and stronger cross linking of soybean protein that have modified structure stability. The L. casei showed significant interaction with soy protein in all samples as compared to L. acidophilus that showed consistent looseness in structure stability.

The results regarding structure development in fermented soy milk are supported by do Espirito-Santo et al. (2014) who explored influence of co-fermentation by amylolytic Lactobacillus strains and probiotic bacteria on the fermentation process. The viscosity and microstructure of probiotic yogurt made of rice, soy milk and passion fruit fiber showed extraordinary differences in between the microstructures of non-fermented and fermented probiotic yogurt observed through SEM. The non-fermented gruel was characterized by a coarser and irregular structure with large and heterogeneous fragments while fermented probiotic yogurt made more homogeneous and smoother structure with smaller fragments.

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Figure 4.15: Scanning Electron micrograph of NARC-II L. acidophilus fermented soy milk a (100µm), b (10µm) and c (5µm)

a b c

Figure 4.16: Scanning electron micrograph of NARC-II L. casei fermented soy milk a (300µm), b (30µm) and c (10µm)

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b c a

Figure 4.17: Scanning electron micrograph of NARC-II L. acidophilus + L. casei fermented soy milk a (50µm), b (30µm) and c (10µm) b c a

Figure 4.18: Scanning eElectron micrograph of Willium-82 L. acidophilus fermented soy milk a (300µm), b (10µm) and c (5µm)

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a b c

Figure 4.19: Scanning electron micrograph of Willium-82 L. casei fermented soy milk a (40µm), b (10µm) and c (5µm)

a b c

Figure 4.20: Scanning electron micrograph of Willium-82 L. acidophilus + L. casei fermented soy milk a (X40µm), b (X10µm) and c (10µm)

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a b c

Figure 4.21: Scanning electron micrograph of Ajmeri L. acidophilus fermented soy milk a (100µm), b(10µm) and c (4µm)

a b c

Figure 4.22: Scanning electron micrograph of Ajmeri L. casei fermented soy milk a (100µm), b(10µm) and c(5µm)

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a b

c

Figure 4.23: Scanning electron micrograph of Ajmeri L. acidophilus+L. casei fermented soy milk a (300µm), b (40µm) and c (10µm)

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4.2.10. Quantification for isoflovone by high pressure liquid chromatography (HPLC)

Bioactive peptides are inactive chains of 2-20 amino acids and their activity depends on amino acid composition, sequence of amino acids and chain length. Isoflavones are also bioactive peptides that can improve functional properties of fermented food, having different therapeutic values and act as an antioxidant (Sanjukta et al., 2015), antihypertensive (Zhang et al., 2006), anti-tumor, antidiabetic (Kwon et al., 2011) and are also being recognized to preclude atherosclerosis. This allows them to act as an alternate to synthetic drugs.

The statistical analysis of the data regarding the genistein and daidzein value of fermented soy milk is mentioned in Table 4.27. The results showed the highly substantial (P<0.01) variation for the impact of days, treatment and varieties. It is obvious from the results that interaction of all the factors is also significant (P<0.01) on level of isoflavones.

The calibration curve of genistein area is mentioned in Figure 4.24 was y=- 0.2539x2+142.45x+1136.5 with R2 = 0.978 showed it near linear was obtained by running different concentrations of genistein standard. Where x is the absorbance and y is the concentration of standard solution expressed as µg/mL (ppm). The calibration curve of daidzein area is mentioned in Figure 4.25 was y=-0.0795x2+34.49x+166.52 and R2 = 0.99 showed it that calibration curve was near to linear obtained by running different concentrations of daidzein standard. Where x is the absorbance and y is the concentration of standard solution expressed as µg/mL (ppm).

The typical HPLC chromatogram of the isoflavone standards (genistein and daidzein) is mentioned in Figure 4.26 and as a template achromatogram of L. casei Ajmeri soy milk is mentioned in Figure 4.27.

4.2.10.1. Genistein

Among isoflavones and specially the aglycones in soybean genistein has remarkable biological activities like it effectively inhibit protein tyrosine kinase and topoisomerase II, and also potential to inhibit the activity of phytoestrogenic and angiogenesis. It has been

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Daidzein Area 4000

3500

3000

A 2500 y = -0.0795x2 + 34.489x + 166.52 r R² = 0.9866 e 2000 a 1500 Area

1000

500

0 0 50 100 150 200 250 Conc.µg/mL

Figure 4.24: Calibration curve of “daidzein” a standard

Genistein Area

25000

20000

y = -0.2539x2 + 142.45x + 1136.5 15000 R² = 0.9785

10000

5000

0 0 50 100 150 200 250 300

Figure 4. 25: Calibration curve of “genistein” a standard

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Figure 4.26: The typical HPLC chromatogram of the isoflavone standards (genistein and daidzein)

Figure 4.27: A chromatogram of L.Casei Ajmeri soy milk

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reported that it also showed a great potential to inhibit LNCaP growth cell line which is human prostate cancer cell line and also effective to control the PhIP mammary tumors Ohta et al. (2000). The administration of fermentation in soy milk is effective to enhance the aglycones isoflavones in soy milk that include genistein and daidzein that can absorb more efficiently in gastrointestinal tract.

The interaction of treatment, varieties and storage days illustrated in Table 4.28a indicated that as the storage proceeded, the concentration of genistein decreased in all the treatment and varieties. The overall mean of genistein was 5.71 ppm at the zero day which decreased to 4.9 ppm at the end of 24 days. The outcomes for treatment effect were significantly varied in such a way that maximum concentration was recorded in T3 (6.92±0.32 ppm) trailed by T2

(5.47±0.24 ppm), T1 (4.29±0.18 ppm) and To (4.06±0.16 ppm) on zero day of storage in NARC-II. Likewise in Willium-82 soy milk the fermentation improved concentration of genistein such as maximum concentration was recorded in T3 was (5.65±0.24 ppm) was on top followed by T2 (4.36±0.18 ppm), T1 (3.47±0.14 ppm) and least was observed in non- fermented soy milk To (0.26±0.07 ppm). The similar trend was recorded for Ajmeri soy milk.

The effect of interaction among treatment*varieties showed significant effect on concentration of genistein (ppm) is given in Table 4.28b. The overall genistein concentration was noticed maximum in T3 (7.09 ppm) followed by T2 (5.95 ppm), T1 (4.84 ppm) and lowest was measured in To (3.34 ppm). The interaction effect showed 8.51 maximum genistein concentrations in Ajmeri (T3) and least was (2.09 ppm) in To (Willium-82). The varieties showed significant variation on genistein concentration as maximum was observed in Ajmeri (6.57 ppm) then in NARC-II (4.49 ppm) and least was in Willium-82 (4.42 ppm).

The effect of storage and treatment interaction is elucidated in Table 4.28c. The data is showing that with the storage time genistein concentration gradually decrease. It can be stated that genistein is antioxidant and as previously described that antioxidant power was decreased during storage. The reduction was reported as 7.49 to 6.64 ppm in T3 followed by th T2 6.20 to 5.69 ppm, T1 5.16 to 4.60 ppm and To 3.98 to 2.62 ppm from 0 to 24 days of storage. The effect of storage with varieties also showed significant variation as given in Table 4.28d. The Table is showing that maximum concentration recorded in Ajmeri soy milk 163

Table 4.27 Mean squares for genistein and daidzein of soy milk

Source DF Genistein Daidzein Treatment 3 69.13** 590.09** Varieties 2 45.47** 706.45** Days 2 5.86** 62.99** Treatment*Varieties 6 1.61** 34.19** Treatment*Days 6 0.34** 2.32** Varieties*Days 4 0.50** 5.44** Treatment*Varieties*Days 12 0.28** 4.45** Error 72 0.06 0.70 Total 107 **= Highly significant (P<0.01)

Table 4.28a Effect of varieties, treatment and storage days on genistein (ppm) of soy milk

Varieties Treatments Days of storage 0 12 24 kl kl kl NARC-II To 4.06±0.16 4.04±0.17 3.00±0.12 jk kl kl T1 4.29±0.18 4.19±0.18 4.15±0.17 gh gh hi T2 5.47±0.24 5.44±0.24 5.10±0.22 bcd cd efg T3 6.92±0.32 6.63±0.30 e 5.93±0.27 lm jl kl Willium-82 To 0.26±0.07 0.25±0.06 0.23±0.09 ijk kl kl T1 3.47±0.14 1.78±0.07 1.02±0.04 fgh hij hij T2 4.36±0.18 3.87±0.16 3.85±0.16 bcd def fgh T3 5.65±0.24 5.08±0.22 5.03±0.22 ijk ijk kl Ajmeri To 4.42±0.18 4.33±0.17 3.96±0.16 bcd d-g fgh T1 6.82±0.29 6.2±0.266 5.8±0.24 b bc bcd T2 7.48±0.32 7.35±0.32 6.96±0.30 a a a T3 8.72±0.40 8.51±0.39 8.29±0.38 Mean of days 5.71a 5.31b 4.9c Results are expressed as mean ± standard deviation of means; n = 3 sets a-l =Means in column with different superscripts differ substantially (P<0.01)

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Table 4.28b Effect of treatments and varieties on genistein (ppm) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

f g e To 3.70 2.09 4.24 3.34d e ef c T1 4.21 4.03 6.27 4.84c d d b b T2 5.34 5.25 7.26 5.95

c c a a T3 6.49 6.28 8.51 7.09 4.94b 4.42c 6.57a Mean

Table 4.28c Effect of treatment and days on genistein (ppm) of soy milk

Treatments Days of Storage 0 12 24 g h i To 3.98 3.38 2.62 e f f T1 5.16 4.76 4.60 c cd d T2 6.20 5.96 5.69 a a b T3 7.49 7.15 6.64

Table 4.28d Effect of varieties and days on genistein (ppm) of soy milk

Varieties Days of Storage 0 12 24 NARC-II 5.18c 5.08c 4.55d Willium-82 5.08c 4.27d 3.90e Ajmeri 6.86a 6.59a 6.25b

Results are expressed as mean ± standard deviation of means; n = 3 sets a-i =Means in column with different superscripts differ substantially (P<0.01) To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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on 0 day was 6.86ppm that decreased to 6.25 on 24th day and minimum was in Willium-82 (5.08 ppm) on zero day that decreased up to 3.90 ppm on 24th day of storage.

The current data pertaining to daidzein concentration was in accordance with work of Cheng et al. (2011). They observed black soybean milk fermented for six days by using Rhizopus oligosporous as a starter culture and reported concentration of genistein was 18.9±0.6 aglycone/mM and upon complete fermentation it was 27.8±1.3 aglycone/mM.

A group of researchers Chien et al. (2013) strengthen the results of recent findings. They studied on stability of isoflavone isomers in lactic fermented soy milk powder and reported that genistein reduced from 26.62±0.27 to 23.99±0.15×102 μg due to degradation of product. Genistein and daidzein are much more stable isoflavones and have almost comparable degradation patterns, despite the differences in their concentrations in the fermented soy milk. However, it was observed that 4 °C was the most appropriate storage temperature in order to guarantee minimal degradation of bioactive isoflavone aglycones in any of fermented product. The increment in the concentration of bioactive isoflavone aglycones through microbial beta glucosidases is an important step in enhancing the potential clinical effectiveness of soy-based foods (Otieno et al., 2006).

The conclusion drawn from the present study is the increase in antioxidant activity of fermented soy milk is due to the significant bioconversion of the glucosidic form of isoflavones (genistin and daidzin) into their bioactive aglyconic form of isoflavones (genistein and daidzein) Rekha and Vijayalakshmi (2008).

4.2.10.2. Daidzein

Isoflavone glycosides in soy milk converted to isoflavone aglycones by lactic acid fermentation because of cleavage of glycosyl bond by microbial fermentation. Fermented soy milk is a superior functional food modulating lipid metabolism and many other benefits. Although soy milk isoflavones seem to be 85% degraded in the intestine, the bioavailability, especially of daidzein, may be sufficient to exert some health-protective effects Pyo et al. (2005).

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The mean values for the effect of fermentation (treatments) and storage time on concentration of daidzein (ppm) in soy milk made from three different varieties is given in Table 4.29a. The results represented that fermentation causes increase in concentration of isoflavones such as in NARC-II soy milk at start of storage in To (10.03±0.41 ppm), T1 (11.68±0.50 ppm), T2

(14.13±0.62 ppm) and T3 (16.86±0.76 ppm). Likewise, in Willium-82 soy milk the highest value was recorded in T3 (25.83±1.57 ppm), lagged by T2 (24.27±1.07 ppm), T1 (21.28±0.98 ppm) and To (14.58±0.59 ppm). Daidzein concentration in Willium-82 soy milk were noted th maximum in T3 (25.83±1.57 ppm) on 0 day and minimum (7.87±0.36 ppm) on 24 day of storage. Overall the concentration decreased with prolonged storage duration (Table 4.29b). Similarly, mean values of Ajmeri soy milk also showed that treatment having combination of both cultures exhibited highest concentration such as in T3 (27.83±1.28 ppm) lagged by Ajmeri soy milk fermented with L. casei in T2 (24.42±1.07 ppm) than in L. acidophilus milk T1

(21.58±0.93ppm) and the least was recorded in non-fermented soy milk To (14.86±0.61 ppm) at the start of product formation. The storage study showed decrease in concentration at regular intervals of 0, 12th and of 24th day.

The interaction effect of treatment*varieties showed the significant effect on daidzein concentration as shown in Table 4.29b. The interaction effect reduced maximum daidzein level in T3 of Ajmeri soy milk (25.86 ppm) and minimum in To of NARC-II soy milk as 9.57 ppm. Overall effect of treatment showed noteworth11.51, 17.19, 19.84 and 22.43 in To, T1,

T2 and T3 respectively. The varieties effect showed maximum level of daidzein in Ajmeri (20.87 ppm) lagged by Willium-82 (19.86 ppm) and in NARC-II (12.67 ppm).

The interaction effect of treatment*days on level of daidzein given in Table 4.29c is showing significant variation on regular intervals of 0, 12 and 24 days. The minimum level was noticed in To (13.16-10.06 ppm), because no LAB has been used in it that can convert primary metabolites to secondary and enhance their concentration. The treatment T1 (L. acidophilus) showed variation from 18.18 to 15.38 ppm, the T2 soy milk fermented with L. casei showed dramatic increase as 20.94 to 18.29 ppm. The soy milk fermented by using both starter cultures in T3 showed variation during storage period from (23.51 to 21.57 ppm).

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The mean values for the effect of interaction among varieties*days is given in Table 4.29d. The Table is depicting significant variation during storage period in all varieties and maximum concentration of 22.17ppm was observed in Ajmeri on start of storage decreased to 19.68 ppm on 24th day of storage followed by Willium-82 (21.49 to 17.57 ppm) and NARC-II varied in between 13.18 to 11.73 ppm. The current data pertaining to daidzein concentration was in accordance with the work of Cheng et al. (2011). They observed black soybean milk fermented for six days by using Rhizopus oligosporous as a culture and reported concentration of daidzein as 19.3±0.8 ppm on 0 day and after complete fermentation on 6th day it was recorded as 57.8±1.5 aglycone/mM. They documented that concentration of daidzein was higher than genistein.

Another group of researchers (Chien et al., 2013) studied on the stability of isoflavone isomers in lactic fermented soy milk powder and reported that daidzein at the initial was 29.96±0.47 μg/mL but after 10 weeks of storage reduced to 27.59×102 μg/mL. They also reported that it was major aglycones among isoflavones in soy milk.

Similarly, Otieno et al. (2006) observed that aglycones in the L. casei fermented soy milk had smaller degradation constants compared to glucoside isoflavones at lower temperature (−80 and 4ºC) than at higher temperature (24.8 and 37ºC) and indicated that storage temperature was an important factor affecting the stability of the isoflavone in soy milk.

The combined effect is clear from Table 4.30a depicting overall comparison of treatments for total determined isoflavones which is showing maximum concentration in T3 was 29.52 ppm, followed by T2 (25.79 ppm), T1 (22.03 ppm) and in To (14.85 ppm). So, it can be stated that bioactive moites of fermented soy milk can be enhanced if coculture fermentation is done and overall fermentation shows higher (genistein and daidzein) as compared to nonfermented soy milk. The effect of varieties was more obvious from Table 4.30b, showing that total determined isoflavones. The Table is showing that maximum concentration of two major isoflavone was found in Ajmeri (17.61 ppm) followed by Willium-82 (24.1 ppm) and NARC-II (27.44 ppm).

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Table 4.29a Effect of varieties, treatment and storage days on daidzein (ppm) of soy milk

Varieties Treatments Days of storage 0 12 24 qrs rs st NARC-II To 10.03±0.41 9.55±0.39 9.13±0.37 op op opq T1 11.68±0.50 11.41±0.49 11.24±0.48 lm ijk no T2 14.13±0.62 15.57±0.68 12.3±0.54 i ij klm T3 16.86±0.76 15.95±0.73 14.24±0.65 kl pqr t Willium-82 To 14.58±0.59 10.56±0.43 7.87±0.36 fg fg i T1 21.28±0.98 21.26±0.97 16.62±0.73 d ef g T2 24.27±1.07 22.460±1.08 20.10±0.98 b bc bc T3 25.83±1.57 25.75±1.27 25.67±1.23 jkl lm mn Ajmeri To 14.86±0.61 13.82±0.56 13.17±0.53 fg h T1 21.58±0.93ef 21.4±0.920 18.28±0.78 e ef T2 24.42±1.07cd 22.82±1.00 22.49±0.98 bcd bcd T3 27.83±1.28a 24.95±1.15 24.81±1.14 Days of storage 18.95a 17.96b 16.33c Results are expressed as mean ± standard deviation of means; n = 3 sets a-t =Means in column with different superscripts differ substantially (P<0.01)

Table 4.29b: Effect of treatment and varieties on daidzein (ppm) of soy milk

Varieties Treatments Means NARC-II Willium-82 Ajmeri

h g f d To 9.57 11.00 13.95 11.51

g d d c T1 11.44 19.72 20.42 17.19

f c b b T2 14.00 22.27 23.24 19.84

e a a a T3 15.68 25.75 25.86 22.43 Mean 12.67c 19.68b 20.87a

Table 4.29c: Effect of treatment and days on daidzein (ppm) of soy milk

Treatments Days of Storage 0 12 24 g h i To 13.16 11.31 10.06 e e f T1 18.18 18.02 15.38 cd d e T2 20.94 20.28 18.29 a b bc T3 23.51 22.22 21.57

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Table 4.29d: Effect of varieties and days on daidzein concentration (ppm) of soy milk

Varieties Days of Storage 0 12 24 NARC-II 13.18f 13.12f 11.73g Willium-82 21.49b 20.01d 17.57e Ajmeri 22.17a 20.75c 19.68d Results are expressed as mean ± standard deviation of means; n = 3 sets a-i=Means in column with different superscripts differ substantially (P<0.05)

Table 4.30: Combined effect of treatment on (geinstenin+ daidzein) of soy milk

Treatments Genistein (ppm) Daidzein (ppm) Genistein+ Daidzein (ppm) d d To 3.34 11.51 14.85 c c T1 4.84 17.19 22.03 b b T2 5.95 19.84 25.79 a a T3 7.09 22.43 29.52 abc Means in columns with similar superscripts do not differ (P>0.05)

Table 4.31: Combined effect of soybean varieties on (geinstenin+ daidzein) of soy milk

Treatments Genistein (ppm) Daidzein (ppm) Genistein+ Daidzein (ppm) NARC-II 4.94b 12.67c 17.61 Willium-82 4.42c 19.68b 24.1 Ajmeri 6.57a 20.87a 27.44 abc Means in columns with similar superscripts do not differ (P>0.05)

To= (Non-Fermented soy milk) T1= (Soy milk fermented with Lactobacillus acidophilus) T2= (Soy milk fermented with Lactobacillus casei) T3= (Soy milk fermented with Lactobacillus acidophilus+ Lactobacillus casei)

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On the whole, it was generally found that levels of isoflavone declined as the storage period was extended, regardless of storage conditions. Additionally, it was also noted that the rate of reduction in isoflavone content, generally, is higher during the initial weeks of storage than during the following period.

The present study demonstrated the fermentation characteristics of two pure starter cultures Lactobacillus acidophilus and Lactobacillus casei in soy milk made by using three varieties during storage. Being probiotic and inducing the intestinal health, these bacteria could be used to alter the biological activity of soy milk by transforming the predominant concentration of isoflavone glucosides to bioactive aglycones. Overall, Lactobacillus casei and L. acidophilus showed adequate technology characteristics and abundant potential for further possible application in the development of high viscosity fermented soy milk when used in combination. The Ajmeri soy milk was selected to check the therapeutical potential of non-fermented (To) and fermented soy milk (T3).

4.3 Efficacy study

The third objective of study was the evaluation of soy milks for the hypoglycemic and hypocholesterolemic potential through efficacy study. This part of study was carried out on Sprague dawley rats.

4.3.1. Feed intake

The statistical analysis regarding the feed intake by rats is displayed in Table 4.31. The mean squares elucidated that there was highly momentous (P<0.01) effect of feeding period (Weeks) and soy milk (group) on the feed intake of rats, while their interaction exhibited a non-significant (P>0.05) effect in study I, II and III.

The results of feed intake are mentioned in graphical representation in Figure 4.28. The Figure 4.28a clearly presents that as the feeding interval increased the feed intake also increased. The effect of groups and feeding intervals on the study I control group was from

28.01± 1.13 g/rat/day to 37.96± 0.75 g/rat/day and in T1 was from 29.26± 0.46 g/rat/day to 39.85± 1.33 g/rat/day.

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a

b

c

Figure 4.28: Feed intake of rats g/rat/day a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats) 172

It is obvious from the graph in Figure 4.28b that gradual increase in feed intake was recorded in To (29.02±0.54 g/rat/day) and T1 (29.79±0.78 g/rat/day) at the initiation of research and after that at the termination of studies it increased to 37.87±1.03 g/rat/day to 38.80±0.43 g/rat/day respectively. However, study III (hypercholesterolemic rats) showed higher intake in T0 group rather than of T1. The graph in Figure 4.28c clearly showed that the difference in

To and T1 was smaller (0.48) at the initial stage of study III, but as the time passed, the difference between these two (1.54) became wider at the end of study.

4.3.2. Drink intake

Mean squares for drink intake in Table 4.31 exhibited that there was highly significant (P<0.01) effect of treatments and time intervals (weeks) on drink intake whilst a non- significant (P>0.05) effect was recorded for their interaction (weeks*Treatments) in all studies. The graphical representation in Figure 4.29a depicted that drink intake of fermented soy milk (T1) was higher in all studies in comparison to non-fermented group. Mean values for drink intake in normal rats (study I) were ranged from 18.47±0.36 mL/rat/day to

25.76±0.94 mL/rat/day in To and for T1 it varied in between 19.87±0.68 mL/rat/day to 26.31±11.78 mL/rat/day from start to end of study. It is obvious from the Figure 4.29a that the effect of treatment was higher in T1 (23.42±2.27 mL/rat/day) drinking fermented soy milk than (22.83±2.35 mL/rat/day) in To group who received non-fermented soy milk.

The graphical representation in Figure 4.29b elucidated mean values in study II

(Hyperglycemic rats) that fermented soy milk T1 group showed higher drink intake

24.54±2.20 mL/rat/day as compared to 24.54±2.20 mL/rat/day in To group taking non- fermented soy milk. The results of current study reported that drink intake was increased from 21.10±0.58 mL/rat/day to 27.77±0.82 mL/rat/day and as in T1 it was 19.44±0.68 mL/rat/day to 25.83±0.33 mL/rat/day in To.

Graphical representation (Figure 4.29c) of mean values exhibited an increasing trend for To starting from 19.88±0.55 to 27.20±1.06 mL/rat/day and for T1 as started from 21.32±0.61 to

27.74±0.49 mL/rat/day. It is also derived from the study that difference between To and T1 was wider up to fourth week of study but afterwards it became narrow. The result of current 173

a

b

c

Figure 4.29: Drink intake of rats mL/rat/day a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats)

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findings are consistent with Sengupta et al. (2016) while studying on hypercholesterolemia in mice, they reported higher consumption of soy yoghurt 6.22 g/day in comparison to 5.22 g/day in control group.

4.3.3. Body weight

Mean squares for weight gain depicted in Table 4.31 showed great substantial (P<0.01) variation due to treatments and storage intervals whilst non-substantial (P>0.05) variation was reported with respect to their interaction (weeks*treatment). The graphical explanation in Figure 4.30a for study I (Normal rats) explored that T1 had higher body weight in comparison to To in all the studies. The mean values showed in graph for To were started from 225.98±12.87 g/rat and with increase in time span their weight was increased up to th 350.74±20.15 g/rat on 8 week of study. Likewise in T1 group their weight was recorded as 236.97±13.52 g/rat on first day of study and ended with 404.51±23.64 g/rat on 8th week of study. Overall mean value for T1 was 328.61±52.95 g/rat and To was 295.67±42.05 g/rat. The mean for weight of rats increased throughout time span from 231.47±7.77 g/rat to 377.65±38.02 g/rat respectively from 0 to 56th day of study.

The graphical representation in Figure 4.30b for study II (Hyperglycemic rats) explicated that initial body weight for To was 216.33±10.11 g/rat and T1 was 231.92±6.42 g/rat however, at the end of study their weight were reached to 297.65±10.15 in To group and to 333.51±7.09 g/rat in T1. Overall body weight increased throughout the trial was from 224.12±11.02 to 315.58±25.36 g/rat from initiation to till end of study. The graph (4.30b) depicted that overall

T1 group showed higher body weight 251.38 ± 31.66 g/rat as compared to To 287.54± 35.36 g/rat in non fermented soy milk feeding group. In case of hypercholesterolemic rats the graphical explanation (Figure 4.30c) depicted that augmentation in body weight of To group were 224.71±4.45 to 301.60±4.24 and for T1 was 221.12 ±6.21 to 329.92±4.63 g/rats.

However, overall treatment effect showed that T1 group had weight of 277.43±38.11 g/rat and To group was 246.51±32.74 g/rat. The recorded mean values for weight gain at commencement were 222.91±2.54 g/rat and 315.76±20.03 g/rat at 8th week of trial.

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a

b

c

Figure 4.30: Effect on body weight of rats g/rat a: Study-I (Normal Rats), b: Study II (Hyperglycemic rats), c: Study III (Hypercholesterolemic rats) 176

Table 4.32: Mean squares for effect of drinks and time intervals on feed, water intake & body weight of rats in different studies

Study 1 Study II Study III

(Hypercholesterolemic (Normal Rats) (Hyperglycemic rats) S.O.V Df rats) Body Body Body Feed Drink Feed Drink Feed Drink Weigh Weigh Weigh intake intake intake intake intake intake t t t Week 112.56 52.463 22494. 77.152 48.624 11069. 73.518 55.807 12131. 8 (A) 7** 3** 3** 0** 7** 3** 6** 0** 8** Treatme 53.855 7.5169 24413. 17.108 36.915 29425. 62.450 40.508 21516. 1 nts(B) ** ** 5** 6** 2** 9** 0** 3** 3** 1.134N 0.5634 371.6 0.7975 0.3983 195.2 0.9589 1.0912 489.9 A x B 8 S NS NS NS NS NS NS NS NS Error 72 1.083 0.5435 312.0 0.4782 0.3713 103.8 0.6812 0.6501 96.8 Total 89 ** = Highly significant (P < 0.01) * = Significant (P < 0.05) NS = Non-significant (P> 0.05)

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The results of current findings are in harmony with Niamah et al. (2017) who also found the increase in body weight during 0 to 40 days of trial. They reported that as on 0 day the weight of control group (Tc) was 143.66 g/day and fermented soy milk fed group (T1) was

142.0 g/day but after 40 days the weight was recorded as 190.0 g/day in TC and 208.1 (g/day) in T1. Likewise, Tsai et al. (2009) also reported increase in body weight while studying on atherosclerosis-preventing activity of lactic acid bacteria-fermented soy milk with Momordica charantia. The results explored that the body weight and daily intake of the hamsters increased routinely during 8 weeks of trial.

Similarly, Sengupta et al. (2016) worked on hypercholesterolemia mice in three groups as (group I: Soy Yoghurt + RBO-SO, group II: Soy Yoghurt, group III: without yoghurt (control) RBO: Rice Bran Oil; SO: Sesame Oil). The results for the group I fed on 1.25% cholesterol diet showed increased in body weight of 11.27 g as compared with group III (4.28 g). They reported the increase in body weight in all groups as compared to their initial weight as in group I. The weight was increased from 22.43±2.68 g to 33.71±2.01 g; group II from 23.29±1.10 g to 32.00±4.36 g while in group III from 19.74±0.87 g to 24.02±1.69 g.

Likewise, Sartang et al. (2015) delineated the effect of fermented soy milk on weight gain in diabetic rats and reported significant changes in the body weight of diabetic rats from the first week until the end of the experiments. They said that oral administration of three different products (soy milk, fermented soy milk and fermented soy milk + omega-3) for 28 days improved body weight significantly with the maximum weight gain seen in the FSM + omega-3 group as compared to other groups.

Moreover, Abd El-Gawad et al. (2005) reported increase in body weight during six week of study period as body weight gain was 48.8% in positive control group fed on cholesterol enriched died and 29.9 % in negative control fed on basal diet while NFSM group showed 37.1 % in comparison to 52.3% cholestrolic group fed on fermented soy milk.

4.3.4. Serum profile analysis

4.3.4.1. Glucose

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Glucose is also called as blood sugar that is most commonly used in our body for performing different functions but its higher level in blood is associated with biomarker of diabetes and healthy range of blood sugar is considered to be 70-100 mg/dL.

The statistical results regarding the blood glucose level presented in Table 4.32 showed that treatments (fermentation) have non-substantial (P>0.05) effect on glucose in study I, while the substantial (P<0.05) effect was noticed in study II and study III.

Mean values relating to glucose level in study I (normal group) for To was 88.61±4.1 mg/dL and in T1 it was 85.44±3.1 mg/dL. Whereas in study II (hyperglycemic) glucose level was momentously decline by using fermented soy milk T1 (258.57±11.2 mg/dL) in comparison to non-fermented soy milk To (311.97±15.3 mg/dL). Glucose level in study III (hypercholesterolemia) showed a significant effect in reducing glucose level by feeding fermented soy milk. The recorded glucose level in To was 271.79±13.3 mg/dL and in T1, it was 241.49±11.4 mg/dL.

The Figure 4.31 represented the percent decrease in glucose level in different rat‟s groups. The study I elucidated 3.61% decrease in glucose level. In study II hyperglycemic rats and it perceived highest decrease in glucose as 17.11% while in study III of hypercholestrolemic rats also showed substantial decrease of 7.46% glucose in blood.

The current findings for fermented soy milk (L. acidophilus+ L. casei) are supported by Sartang et al. (2015) who study the effects of probiotic fermented soy milk (Bifidobacterium lactis) fortified with Omega-3 on blood glucose in STZ induced diabetic rats.

They reported significant changes in the blood glucose levels of diabetic rats from the first week until the end of the experiment. Normal rats maintained a normal blood glucose level during the study while the diabetic rats significantly increased the level of blood sugar in comparison to normal rats. They reported increase in glucose level from 114.08 to 162.58 mg/dL.

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Table 4.33 Effect of fermented soy milk on glucose level (mg/dL)

Studies To T1 F value

Study I 88.61±4.12a 85.44±3.14a 1.13NS

Study II 311.97±15.3a 258.57±11.2b 23.8**

Study III 271.79±13.3a 241.49±11.4b 8.99*

**= Highly significant (P <0.01) *= Significant (P<0.05) NS= Non-significant abMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4. 31: Percent decrease in level of glucose

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At the end of experiment, blood glucose in all treated diabetic rats had been significantly reduced (P<0.05) as compared to the DC rats. Fermented soy milk (FSM) + omega-3, FSM and soy milk exhibited the reduction of 47.2%, 39.3% and 35.8%, respectively. Another research supported the present study that fermented soy milk is valuable to control the diabetes (Nurliyani, 2015). They studied the effect of soy milk kefir on diabetic rats and reported the reduction on plasma glucose level. The glucose recorded in diabetic rats before treatment was 391.24±146.57 and after feeding the soy milk kefir, reduced to 257.38±175.16 mg/dL and overall change in pre and post treatment was recorded as -133.85±172.37 as compared to normal control (43.18±4.95mg/dL) and in diabetic control (96.61±111.51mg/dL).

4.3.4.2. Insulin

Insulin is a hormone secreted by the pancreas which determines either a patient has diabetes or not. Insulin production is directly related with blood sugar because higher level of glucose in blood activates pancreas to secrete more insulin into the bloodstream. Insulin reaches in cells and let the glucose to come in that ultimately decreases glucose level in blood stream. When the glucose reaches in the cells they transform glucose into energy or store it in the form of glycogen that can be use later on.

The statistical analysis of data regarding the insulin in rats is presented in Table 4.33. The analysis revealed the non-significant (P>0.05) effect of treatment on insulin level whilst study II presented the highly significant (P<0.01) effect and study III depicted the significant effect (P>0.05) of fermented soy milk on insulin level in blood of rats.

The mean value for insulin in the study I (Normal rats) was reported as 8.73±0.4 µU/mL in group To fed on (non- fermented soy milk) and 8.89±0.7 µU/mL in group T1 fed on fermented

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Table 4.34 Effect of fermented soy milk on insulin level (µU/mL)

Studies To T1 F value

Study I 8.73±0.4a 8.89±0.7a 0.12NS

Study II 7.28±0.18b 7.89±0.15a 20.7**

Study III 6.57±0.14b 6.93±0.12a 11.7*

**= Highly significant (P<0.01) *= Significant (P<0.05) NS= Non-significant abMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4.32: Percent increase in level of insulin

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soy milk. Study II showed that, insulin level elucidated in To was 7.28±0.18µU/mL and in T1 was7.89±0.15 µU/mL. In hypercholesterolemic rats (study III), least level of insulin was detected as 6.57±0.14 µU/mL in To group and 6.93±0.12 µU/mL in T1 group. Figure 4.32 elucidated that fermented soy milk was effective in inclining insulin level, while vibrant effect documented in study II was 8.3% increase in insulin level trailed by 5.4% in study III and 1.8% in study I.

There are array of components that may support the benefits of soy milk consumption for type 2 diabetes management. Likewise, Wagner et al. (2008) studied on male monkeys to check the effects of soy protein and isoflavones (bioactive peptides) on insulin resistance. They reported that consumption of soy protein with its isoflavones augment the secretion of insulin that drops glucose level in blood. Kikuchi-Hayakaw et al. (2000) reported that probiotics bifidobacteria during fermentation yields organic acid (lactic and acetic acid) due to breakdown of carbohydrates. Lactic acid helps to delay the gastric emptying rate and led to drops in glucose absorption and plasma insulin in a glucose tolerance test in rats. Glucose and insulin regulate lipogenic enzyme activities and gene expression.

Likewise, Davis et al. (2005) have documented that Isoflavonoids and protein in soybean are associated in dropping insulin resistance and modifying glycaemic index due to lowering total cholesterol and triglycerides level in plasma.

Later on, Sartang et al. (2015) have highlighted that fermented soy milk played a role in decreasing insulin to glucagon ratio and finally decreased the lipogenic gene expression. From previous and present studies it is concluded that fermented soy milk decrease the plasma triglyceride and atherogenic index than soy milk. So, it can be suggested that including fermented soy milk may be included in daily diet which would help to dispose of the insulin efficiently by reducing insulin secretion in the body. In this way it can help type 2 diabetic patients by minimizing insulin resistance.

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4.3.4.3. Cholesterol

Cholesterol is essential to develop outer covering of cells, produces bile acids and vitamin D. Although it is essential but its higher level in blood causes formation of plaque that narrow down the blood arteries and becomes a primary factor of heart diseases (Ma, 2004).

The statistical analysis of the results regarding the cholesterol level is depicted in Table 4.34. The F value showed non- momentous (P>0.05) effect of soy milk in study I (Normal rats) however, substantial (P<0.05) variations were observed in study II (Diabetic rats) and most effective results were recorded in study III.

The mean values for study I showed a slight change (2.14 mg/dL) of cholesterol level after feeding with fermented and non-fermented soy milk as 81.25±4.14 mg/dL in To group and

79.11±4.43 mg/dL in T1 group. The study II showed an obvious change (10.2 mg/dL) in cholesterol level as 108.84±4.46 mg/dL was in To group and 98.64±4.24 mg/dL in T1 group. The maximum cholesterol lowering effect (20.75 mg/dL) was observed in study III

(153.55±6.29 mg/dL - 132.80±5.7 mg/dL) for T1.

It is obvious from Figure 4.33 that value added drink was most effective in lowering cholesterol level as 13.51% decrease was recorded in study III of high cholesterol rat group. Similarly, in study II 9.37% reduction was recorded in diabetic group while least reduction was reported in study I (2.62%) with control group.

The results of present study were in accordance with the results of recently reported by Niamah et al. (2017), who studied the effect of fermented soy milk feeding on rats. The results of their findings suggested that probiotics used to ferment the soy milk play a fundamental role in the reduction of total cholesterol level of rat‟s blood with in ten days of feeding FSM. They reported the cholesterol in group fed on fermented soy milk as 118.06 mg/dL in comparison to control group 124.67 mg/dL after 40 days of trial. Likewise, Sengupta et al. (2016) studied on diabetic rats. They stated significant decrease in group feeding was (155.58 mg/.dL) and 220mg/dL was in non-soy yoghurt control group.

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Table 4.35 Effect of fermented soy milk on cholesterol level (mg/dL)

Studies To T1 F value

Study I 81.25±4.14a 79.11±4.43a 0.37NS

Study II 108.84±4.46a 98.64±4.24b 8.23*

Study III 153.55±6.29a 132.80±5.7a 17.9**

**= Highly significant (P<0.01) *= Significant (P<0.05) NS= Non-significant abMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4. 33: Percent decrease in level of cholesterol

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Another research work on diabetic rats was reported by Sartang et al. (2015), who stated that fermented soy milk showed a more significantly decrease 20.8% in total cholesterol of diabetic control group. It is reported by scientists that fermented soy milk has positive effect on the cholesterol level of blood. It has been studied that probiotic bacteria L. acidophilus and Bifidobacterium has reduction effect on total cholesterol level of serum when feeding fermented soy milk (Anandharaj et al., 2015). A group of researchers (Svejstil et al., 2015) has reported that soy milk has many chain sugars (stachyose 4% and raffinose 1%) which can be used by Lactic acid bacteria as carbon source. Moreover, the lactic acid bacteria have the ability to absorb and bind cholesterol as well as bile acids with their bacterial cells. Further, Tomat et al. (2011) has also supported by reporting that probiotic bacteria binds the cholesterol and inhibit its absorption in the intestine. They also stated another reason that the undigested pepsin fraction of soybean protein may effects fecal excretion of steroids or bile acids, which may influence the cholesterol metabolism.

The El-Gawad et al. (2005) explained that decrease of plasma cholesterol in cholesterolemic rats was 195.1 mg 100 mL-1 in group of feeding fermented soy milk in comparison to 231 mg 100mL-1 in group taking non-fermented soy milk during six weeks of trial. Later on, Wang et al. (2012) assessed the effect of soy milk fermented with L. plantarum on lipid metabolism in hyperlipidemic rats. They reported decrease in total cholesterol as 1.95 mmol/L was reorded in FSM feeding group and 2.09 mmol/L in NFSM group.

4.3.4.4. Low Density Lipoprotein (LDL)

The main function of LDL is to transport cholesterol from the liver to tissues that incorporate it into cell membranes. The credence that (LDL) cholesterol is the cause of atherosclerosis and heart disease is a fundamental precept of modern medicine. The modern medicines developed with the aim to decrease plasma LDL cholesterol because LDL cholesterol may causes formation of fatty deposits in arterial walls, which developed into plaques that enlarge, break, and arouse the production of blood clots that may causes blockage in arteries and eventually heart related disorders (Colpo, 2005).

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Table 4.36 Effect of fermented soy milk on low density lipoprotein (LDL) level (mg/dL)

Studies To T1 F value

Study I 12.50±0.51a 11.75±0.50a 3.24NS

Study II 24.42±1.00a 22.15±0.95b 8.10*

Study III 39.21±1.68a 34.32±1.40b 14.9**

**=Highly Significant (P<0.01) *=Significant (P<0.05) NS= non-significant (P> 0.05) abMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4.34: Percent decrease in level of low density lipoprotein (LDL)

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The statistical results for the consequence of value added soy milk on LDL (mg/dL) is depicted in Table 4.35. The study I (normal rats) showed non-substantial effect (P>0.05) on LDL level, but study II (diabetic rats) showed significant (P<0.05), however the most obvious effect was recorded in study III (hypercholestrolemic rats) where LDL level varied highly significant (P<0.01).

The mean values for study I showed a diminishing effect on LDL level as 12.50±0.51mg/dL in To and 11.75±0.50 mg/dL in T1. However, in study II the LDL level was changed significantly as in To, LDL recorded was 24.42±1.00 mg/dL and in T1 it was 22.15±0.95 mg/dL. Whilst, in study III LDL for To was 39.21±1.68mg/dL and in T1 was 34.32±1.40 mg/dL. It is absolutely clear from the Figure 4.34 illustrating the LDL reduction% in serum level of rats. Higher reduction was recorded in study III (12.47% LDL) which supported that value added drink is effective in lowering LDL in hypercholesterolemia. However, LDL reduction was also observed during study II (9.29%) and (6%) in study I (normal rats).

The results of recent findings are supported by findings of Sartang et al. (2015). They observed that the LDL cholesterol (11.45 mg/dL) in non-fermented soy milk feeding group and in fermented soy milk feeding group11.77 mg/dL. The results of current study are consistent with the findings of Babashahi et al. (2015) who reported 3.30% reduction in LDL-C by feeding soy milk and 13.32 % by feeding probiotic soy milk in hyperglycemic Wistar rats. Likewise, Sengupta et al. (2016) explicated that consumption of soy yoghurt significantly decreases the LDL level 42.01 mg/dL as compared to 143mg/dL in control group while studying on hypercholesterolemia mice.

Fermentation helps to improve heart related disorders by decreasing the level of LDL in blood which is considered as biomarker of cardio vascular diseases but the use of soybean products and specially fermented soybean helps to improve human health. Likewise, results were explored by Hong et al. (2012) who found LDL was 5.30 mg/dL in group fed on black soybean pulp in comparison to fermented black soybean pulp group (4.55 mg/dL).

It has been reported by Tsai et al. (2009) that feeding the rats with a high-cholesterol diet and fermented soy milk with L. plantarum and L. plantarum + soy milk supplement with M.

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charantia showed significant (P<0.05) reduction in ratio of LDL-C/HDL-C, as compared to when only high cholesterol diet was given.

The recent results of LDL reduction are also in agreement with El-Gawad et al. (2005) who reported that diminution of plasma VLDL+LDL-cholesterol in hypercholesterolemia rats was 209.4 mg100mL-1 in non-fermented soy milk group however, it was decreased to 163.7 and 149.4 mg100mL-1 respectively by feeding soy milk fermented with B. lactis and other group with B. longum during six weeks of trial. Likewise, Wang et al. (2012) also reported decline in LDL-C by feeding soy milk fermented with L. plantarum to hyperlipidemic rats while studying on lipid metabolism in hyperlipidemic rats. They reported that LDL-C was higher in non-fermented soy milk group was 2.0 in comparison to 1.46 mmol/L group fed on fermented soy milk.

4.3.4.5. High Density Lipoprotein (HDL)

HDL is the slightest and thickest of the lipoproteins, comprising the highest fraction of protein to cholesterol. A healthy person contains 20 to 30% HDL of its total plasma cholesterol level. HDL-C is also considered as good cholesterol due to its high density it does not form plaque in arteries instead of it pass out and excreted through the body. The HDL is significant for steroid and remarkably known for positive effect on heart diseases as reported by Rader and Hovingh (2014) that the cholesterol enclosed within HDL is inversely linked with risk of coronary heart disease and is a key component of predicting cardiovascular risk. HDL is associated with reverse cholesterol transport from tissues and arteries and sends it back to the liver for its decomposition and ultimately excretion that decreases chances of atherosclerosis (hardening of arteries).

The statistical analysis of the results regarding HDL level in blood of rats is mention in Table 4.36. The analysis showed the non-substantially (P>0.05) effect in study I, while significant (P<0.05) in study II and highly significant (P<0.01) in study III.

The mean values in study I (Normal rats) increased gradually from 44.85±0.97 mg/dL in To group (Non- fermented soy milk) to 46.10±0.76 mg/dL in T1 group, whilst in diabetic rats the

HDL level increased from 38.05±0.53 (To) to 39.61±0.27 (T1) mg/dL. Similarly a greater 189

increase was observed in study III from 36.73±0.36 (To) to 40.23±0.72 (T1) mg/dL (Table- 4.36). It is visible from Figure 4.35 that value added soy milk was more helpful in increasing HDL level (9.52 %) in study III and 4.02 in study II and 2.78% in study I.

Sengupta et al. (2016) elucidated that consumption of soy yoghurt significantly improved the HDL-C level (96 mg/dL) as compared to (60 mg/dL) in control group while studying on hypercholesterolemia mice. The results of current findings are also in accordance with Sartang et al. (2015). They reported that in diabetic control group, the fermented soy milk consumption showed tendency to produce greater HDL-C concentrations (24.8%) in comparison to the soy milk (20.4%). Likewise Babashahi et al. (2015) who reported the serum HDL-C levels of probiotic soy milk group increased to 16.80% in comparison to 9.36% in plain soy milk of hyperglycemic rats group.

The increase in HDL level was reported by Hong et al. (2012) that after the feeding of fermented black soybean pulp (FBSP) (27.83 mg/dL) than that in the control group (22.98 mg/dL). Another research was concluded by Tsai et al. (2009) who supported the present findings. They feed high-cholesterol diet for 8 weeks with fermented milk-soy milk by L. plantarum NTU 102 which showed that increase in HDL level (70.2 mg/dL) in comparison to control group on normal diet (53.6 mg/dL). Likewise, Wang et al, (2012) also stated that level of HDL-C was enhanced upto 0.39 mmol/L by feeding soy milk fermented with L. plantarum p-8 as compared to 0.38mmol/L in non-fermented soy milk rats while studying on hyperlipidemia rats. The results of HDL augmentation are also in agreement with El-Gawad et al. (2005) who reported that increase of plasma HDL-cholesterol in hypercholesterolemia rats was 22.4 mg/100mL in non-fermented soy milk group. However, it was increased to 31.4 and 33.6 mg 100mL-1 respectively by feeding soy milk fermented with B. lactis and with B. longum during six weeks of trial.

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Table 4.37 Effect of fermented soy milk on high density lipoprotein (HDL) level (mg/dL)

Studies To T1 F value

Study I 44.85±0.97a 46.10±0.76a 3.09NS

Study II 38.05±0.53b 39.61±0.27a 20.6*

Study III 36.73±0.36b 40.23±0.72a 56.7**

**=Highly Significant (P<0.01) *=Significant (P<0.05) NS= non-significant (P>0.05) ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.35: Percent increase in level of high density lippoprotein (HDL)

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4.3.4.6. Triglycerides

Triglycerides are type of fat in the bloodstream whose higher level in body is associated with heart diseases. Its level varied by sex and age but diabetic and heart patients has higher level of triglycerides (Ma, 2004).

The analysis of variance of the results regarding the triglyceride mentioned in the Table 4.37 showed the non- significant (P>0.05) effect of treatments (To and T1) on study I, while their response was significant (P<0.01) during study II and study III.

The mean value of triglyceride in study I was higher (66.64±2.73 mg/dL) in To than T1

(64.59±2.77 mg/dL). In study II (diabetic rats), the triglyceride contents was also higher in To

(79.60±3.42 mg/dL) than T1 (72.48±2.97 mg/dL). The prominent variations were observed in study III (hypercholesterolemic rats) as non-fermented soy milk group To exhibited the higher 98.94±4.05 mg/dL triglycerides than fermented soy milk (T1) which had 86.73±3.73 mg/dL of triglycerides (Table 4.37). It is noticeable from Figure 4.36 that value added soy milk showed maximum triglycerides reduction in study III (12.34%) lagged by study II (8.94%) study I (3%).

The results of recent findings were also supported by Niamah et al. (2017) who reported that fermented soybean milk reduced the level of cholesterol and triglycerides as compared to the control sample. They reported that during 40 days of study the result showed decreasing rate of triglyceride in soy milk fermented group (74.5 mg/dL) as compared with control (124.67 mg/dL).

Sartang et al. (2015) stated that triglycerides concentrations of all treated diabetic rats were significantly decreased (푃<0.05), but maximum reduction was observed in the fermented soy milk + Ѡ-3 group (39.3%) lagged by fermented soy milk group (11.2%).

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Table 4.38 Effect of fermented soy milk on triglycerides level (mg/dL)

Studies To T1 F value

Study I 66.64±2.73a 64.59±2.77a 0.83 NS

Study II 79.60±3.42b 72.48±2.97a 7.40 *

Study III 98.94±4.05b 86.73±3.73a 14.7 **

**= Highly significant (P<0.01) *= Significant (P<0.05) NS= Non-significant ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.36: Percent decrease in level of triglycerides

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According to Hong et al. (2012) fermented black soybean pulp is helpful in decreasing the atherosclerosis. They reported triglycerides level 15.50±2.08 mg/dL in control group but in fermented black soy pulp group it was reported as 11.50±4.44 mg/dL and 12.25±1.89 mg/dL in non-fermented black soy pulp feeding group of rats. Likewise, another research work was done on antidiabetic potential of soy milk kefir made by using kefir grain by Nurliyani (2015). They reported that triglycerides level in diabetic rats feeding on soy milk kefir was 98.96 mg/dL at the start of study and reduced to 63.08 mg/dL at end of study as compared to normal control group it was 112.98 mg/dL at start of study and 174.97 mg/dL was at the end.

According to El-Gawad (2005), fermented soy milk play a major role in reducing heart diseases as higher level of triglycerides are associated with metabolic syndrome that may increase the risk of heart disease and diabetes. They studied on hypercholesterolemia rats and reported that feeding non-fermented soy milk for 6 weeks trial showed effect on triglycerides was (69.7 mg/100mL) while soy milk fermented with B. lactis and other group with B. longum resulted decline in triglycerides (64.6 and 40.3 mg/100mL) respectively. Similarly, Wang et al. (2012) also stated that level of triglycerides reduced upto 1.73mmol/L by feeding soy milk fermented with L. plantarum p-8 as compared to 1.91mmol/L in NFSM rats while studying on hyperlipidemia rats.

4.3.5. Liver functions tests

During bio efficacy study, the effect of fermented soy milk was also evaluated for safety test on hepatic tissue. The major liver functioning tests was performed including aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP).

4.3.5.1. Aspartate aminotransferase (AST)

AST enzyme is one of the important liver functioning tests whose level above average usually mean the liver is injured.

The results presented in Table 4.38 is depicted the effect of value added soy milk on AST. The results revealed that treatment‟s effect were non-substantial (푃 >0.05) in study I and study II whilst, substantial (푃<0.05) effect was recorded in study III. 194

Table 4.39 Effect of fermented soy milk on aspartate aminotransferase (AST) level (IU/L)

Studies To T1 F value

Study I 104.25±4.27a 101.60±4.36a 0.56NS

Study II 121.18±4.96a 112.54±4.83a 4.66NS

Study III 138.28±5.66a 121.03±5.20b 15.1*

*=Significant (P<0.05) NS= non-significant (P>0.05) abMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4.37: Percent decrease in level of aspartate aminotransferase (AST)

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The Table 4.38 is showing the mean values of AST. It is obvious that AST values were lower in all studies in T1 fermented soy milk group as compared to To group, as in study I

104.25±4.27 IU/L was in To and 101.60±4.36 IU/L in T1, in study II it was 121.18±4.96 IU/L in To and 112.54±4.83 IU/L in T1 the same trend was noticed in study III and the value recorded was 138.28±5.66 IU/L in To and 121.03±5.20 IU/L in T1.

The difference in To and T1 in all the studies (I,II and III) is clear in Figure 4.37, that illustrated the highest reduction 12.74% in study III followed by 7.12% in study II and 2.54% in study I.

The results of present study are also in accordance with Sengupta et al. (2016) who reported AST in soy yoghurt feeding group was 143.71 IU/L while in yoghurt group it was 224.44 IU/L. Similarly, Shin et al. (2009) studied on improvement of experimentally induced hepatic disorders in rats using lactic acid bacteria-fermented (BF) soybean extract. They reported that BF-administered rat group showed lower concentration of AST level. Such as in the BF group, AST was significantly (푃<0.05) lowered to 1423±1857, 1009±1395 and 142±161 IU/L on day 1, 2 and 3 respectively, that was approximately 45% lower level than control group. These results recommend that fermented soy milk may play a fundamental role in hepatic related disorders and ultimately helpful for maintaining better health in humans as well. Similarly, the Hong et al. (2012) reported 159.63U/L AST in group fed on black soybean pulp in comparison to fermented black soybean pulp group (117.28 IU/L). In a nut shell it can be suggested that fermentation may help to decrease AST level in serum. It is deduced from findings of current research that soy milk fermented by L. acidophilus and L. casei was positive in averting hepatocellular damage.

4.3.5.2. Alanine transaminase (ALT)

The ALT enzyme helps to process proteins and its higher level in the body is the indication that liver is injured or swollen. Statistical results presented in Table 4.39 for ALT level in liver showed non-momentous effect of fermented and non- fermented (푃>0.05) soy milk in study I and II and momentous effect (푃<0.05) in study III.

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Table 4.40 Effect of fermented soy milk on alanine transaminase (ALT) level (IU/L)

Studies To T1 F value

Study I 45.54±1.86a 43.59±1.87a 1.63NS

Study II 53.42±2.19a 49.16±2.11a 5.88NS

Study III 58.78±2.41a 51.22±2.20b 16.1*

**=Highly Significant (P<0.01) *=Significant (P<0.05) NS= non-significant (P>0.05) ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.38: Percent decrease in level of alanine transaminase (ALT)

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The mean values for the effect of ALT presented that fermented soy milk showed small reduction in comparison to non-fermented soy milk. In study I, AST level in To group was

(45.54±1.86 IU/L) higher than T1 (43.59±1.87 IU/L). Similarly, in study II To was (53.42±2.2

IU/L) higher than T1 (49.16±2.11 IU/L). The highest reduction was noticed in study III from

58.78±2.41 IU/L (To) to 51.22±2.20 IU/L (T1).

The Figure 4.38 depicted the (58.78-51.22 IU/L) maximum ALT level decreased as 12.86% in study III trailed by (53.42-49.16 IU/L) study II as 7.97% and minimum ALT level (45.54- 43.59 IU/L) reduced as 4.26% in study I.

The findings in this project are in accordance with Sengupta et al. (2016) who also reported maximum decrease in ALT level in soy yoghurt group vs. non-soy yoghurt group resulted as 113.03 vs. 121.8 ALT (IU/L) on albino mice fed high cholesterol diet.

Another group of scientists, Shin et al. (2009) studied on improvement of experimentally induced hepatic disorders in rats using lactic acid bacteria-fermented soybean extract. They reported that in the case of ALT, the increased activity found in controls was also depressed in the bio- fermented soybean extract group with the value at 2 weeks significantly different (P<0.05). Fermentation helps to improve liver related disorders as increase in level of AST in serum is considered as liver disorder but the use of fermented soybean products helps to improve liver health. Likewise, results were explored by Hong et al. (2012) who found 43.40 IU/L in group fed on black soybean pulp in comparison to fermented black soybean pulp group it was 31.15 IU/L.

4.3.5.3. Alkaline phosphatase (ALP)

ALP is an important test to know the abnormal condition of liver. The higher level of the ALP enzyme is an indicator of liver disorder.

Statistical analysis (Table 4.43) of ALP data indicated that treatment‟s effect was non- significant (P>0.05) in study I, however, significant (P<0.05) effect was observed in study II and study III. The mean values revealed that fermented soy milk (T1) was more helpful in

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Table 4.41 Effect of value added soy milk on alanine phosphatase (ALP) level (IU/L)

Studies To T1 F value

Study I 139.39±5.71a 134.02±5.76a 1.31NS

Study II 220.84±9.05a 201.38±8.65a 7.24*

Study III 267.25±10.95a 232.25±9.98b 16.7*

**=Highly Significant (P<0.01) *=Significant (P<0.05) NS= non-significant (P>0.05) ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.39: Percent decrease in level of alkaline phosphatase (ALP)

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reduction of ALP level in comparison to non-fermented soy milk (To). The decrease in ALP level was recorded as 139.39±5.71 IU/L (To) to 134.02±5.76 IU/L (T1) in study I,

220.84±9.05 IU/L (To) to 201.38±8.65 IU/L (T1) in study II and 267.25±10.95 IU/L (To) to

232.25±9.98 IU/L (T1) in study III. It is profound from Figure 4.39 that maximum ALP reduction recorded in study III (267.25 - 232.25 IU/L) was 15.06 %, lagged by study II (220.84-201.38 IU/L) as 9.66% and in study I (220.84 to 201.38 IU/L) was 4%.

Shin et al. (2009) studied on improvement of experimentally induced hepatic and renal disorders in rats using lactic acid bacteria-fermented (BF) soybean extract. They reported that bacterial fermented feeding rat group showed lower concentrations of ALP level. It was 635 IU/L in bacterial fermented administrated feed group in comparison to control (694 IU/L). These results recommend that fermented soy milk may play a fundamental role in hepatic related disorders and ultimately helpful for maintaining better health in humans as well.

4.3.6. Renal function tests

Value added soy milk‟s effect was also checked for the safety status of kidney (renal).

4.3.6.1. Biological Urea Nitrogen (BUN)

BUN gives a rough estimation of the glomerular filtration rate (GFR), the speed at which blood is filtered in the kidneys. During metabolism of protein in the liver the final product is formed as urea which is excreted via kidneys from the body. If the kidney is not functioning properly then ultimately BUN level will be increased in the blood from normal range.

The statistical analysis (Table 4.41) is indicating effect of value added soy milk on BUN level of rats. The effect of treatments showed non-significant (P>0.05) variations on the level of BUN in (study I) whilst, highest significant (P<0.01) effect was noticed in study II and substantial (P<0.05) variations in hypercholesterolemia rats (study III).

The effect of treatment on the BUN level as indicated from mean values in Table 4.44 showed that fermented soy milk (T1) was more effective in the reducing of BUN level in blood as compared to non-fermented soy milk (To). BUN level decreased from 22.51±0.92

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Table 4.42 Effect of fermented soy milk on biological urea nitrogen (BUN) level (mg/dL)

Studies To T1 F value

Study I 22.51±0.92a 21.88±0.94a 0.69NS

Study II 40.06±0.43a 38.72±0.34b 17.9**

Study III 36.02±0.41a 35.23±0.18b 9.00*

**= Highly significant (P<0.01) *= Significant (P<0.05) NS= Non-significant ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.40: Percent decrease in level of biological urea nitrogen (BUN)

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to 21.88±0.94 mg/dL in study I, 40.06±0.43 mg/dL to 38.72±0.34 mg/dL in study II and 36.02±0.41 mg/dL to 35.23±0.18 mg/dL in study III.

The Figure 4.40 elucidated that maximum BUN reduction was noticed in study II as 3.45% followed by 2.87% in study I and 2.24% in study III. It has been revealed from study that fermented soy milk showed a profound effect in the reduction of BUN in blood.

The results of current findings were also supported by Shin et al. (2009) who studied the improvement in experimentally induced hepatic and renal disorders in rats using lactic acid bacteria-fermented soybean extract. They reported that bio fermented (BF)-administered rat group exhibited lesser concentrations of blood urea nitrogen as 26.2 mg/dL was recorded on 0 day and at the end of trial on 6th week BUN was 19.8 mg/dL. Two another researchers Chiang and Pan (2011) studied on ovariectomized (OVX) mice and documented that BUN level decreases by feeding fermented soy skim fermented with L. paracasei (24.4 mg/dL) and L. plantarm (25.5mg/dL). The non-fermented soy skim milk resulted in decline of creatinine level up to 24.2 mg/dL. These results suggest that BF test may play a role in renal functioning disorders and may also be valuable for maintaining health in human beings.

4.3.6.2. Creatinine

Craeatinine is expelled entirely by the kidneys so its level in the blood is checked to know kidney functioning. Creatinine is main muscle component which is formed due to breakdown of creatine. Creatinine level in the blood is proportional to the glomerular filtration rate. Kidney damage is mostly associated with higher level of creatinine so, it is the more efficient as compared to BUN.

Statistical analysis of the data to know the effect of value added soy milk on creatinine level (mg/dL) is given in Table 4.42. The results indicated that treatment‟s effect was non- significant in study I (Normal rats). While, treatments have showed significantly variation in creatinine contents (P<0.05) in study II and study III.

The mean of creatinine values in Table 4.42 showed variations in fermented and non- fermented group. The non-fermented soy milk group showed less efficient in the reduction of

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Table 4.43: Effect of fermented soy milk on creatinine level (mg/dL)

Studies To T1 F value

Study I 0.82±0.01a 0.80±0.02a 6.00NS

Study II 0.97±0.04a 0.90±0.01b 8.65*

Study III 0.87±0.01a 0.83±0.02b 9.60*

*=Significant (P< 0.05)

NS= non-significant (P> 0.05) ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.41: Percent decrease in level of creatinine

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Table 4.44 Effect of fermented soy milk on red blood cell (RBC) cells/pL

Studies To T1 F value

Study I 7.35±0.30a 7.51±0.32a 0.39NS

Study II 7.49±0.08b 7.85±0.13a 15.9*

Study III 7.15±0.18b 7.65±0.11a 16.9**

**=Highly Significant (P<0.01) *=Significant (P<0.05) NS= non-significant (P>0.05) ab Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.42: Percent increase in level of red blood cells (RBCs)

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creatinine level as compared to fermented soy milk group. The mean values for To and T1 were 0.82±0.01 mg/dL and 0.80±0.02 mg/dL respectively in study I, 0.97±0.04 mg/dL and 0.90±0.01 mg/dL in study II and 0.87±0.01 mg/dL and 0.83±0.02 mg/dL in study III. The results in Figure 4.41 illustrated that maximum 7.22% reduction of creatinine was noticed in study II lagged by 4.59 % in study III and 2.43 in study I.

The results of recent findings are in agreement with findings Chiang and Pan (2011) studied on ovariectomized (OVX) mice and documented decrease in creatinine level by feeding fermented soy skim with L. paracasei as (0.24 mg/dL) and L. plantarm (0.21 mg/dL) while non-fermented soy skim milk resulted in decline of creatinine level up to 0.18 mg/dL.

4.3.7. Hematological analysis

4.3.7.1. Red blood cell (RBC)

Statistical analysis given in Table 4.43 showed outcome of value added soy milk on RBC. The statistical analysis showed that consequence of treatment was non-significant (P>0.05) in study I but a significant variations (P<0.05) were recorded for study II and highly significant (P<0.01) in study III.

The mean values are showing increase on RBC in case of fermented soy milk group (T1) as compared to To (non-fermented soy milk) group. The RBC contents of To was 7.35±0.30 cells/pL and of T1 was 7.51±0.32 cells/pL during study I, similarly in To was 7.49±0.08 cells/pL and in T1 was 7.85±0.13 cells/pL in study II and 7.15±0.18 cells/pL (To) and

7.65±0.11 cells/pL (T1) in study III. It is obvious from graphical representation in Figure 4.42 that maximum increase in RBC was reported as 6.99% in study III, 4.81% in study II and 2.17% in study I. The results of recent study were also in harmony with Niamah et al. (2017) when studied the effect of feeding probiotic soy milk for 40 days on rats. They documented that percentage of RBC for group feeding on fermented soy milk was 6.4 cells/pL while the control sample was 6.0 cells/pL after 40 days of dosing. Further, Sartang et al. (2015) reported that RBC was 7.96 cells/pL in rats feeding on normal diet and 8.20 cells/pL was in fermented soy milk feeding group.

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4.3.7.2. White blood cells count (WBCs)

The statistical results for WBCs inferred that WBCs were not affected by the treatments in all the studies. The statistical results indicated non- momentous (P>0.05) effect of treatments in stydy I, II and III (Table 4.44).

The mean values for WBCs in study I (normal rats) were 6.99±0.94 cells/nL for To and

6.53±0.25 cells/nL for T1, whilst mean values in study II (diabetic rats) showed WBCs in To it was 7.86±0.21 cells/nL and in T1 it was 7.35±0.34 cells/nL similarly, in study III non- fermented soy milk group (To) result was 7.65±0.28 cells/nL and fermented soy milk group

(T1) was 6.93±0.45 cells/nL of WBCs. However the results were not as higher that significant variation could be noticed but overall it is noticeable in Figure 4.43 that value added soy milk showed maximum white blood cells reduction in study III as 9.41% lagged by 6.48% in study II and only 3.58% in study I. The decrease in WBC was non-significant for treatment effects in all studies. Sartang et al. (2015) also reported a slight increase in WBC as 6.78 cells/nL in normal control group, 5.60 cells/nL in diabetic control while Soy milk group 7.88 cells/nL and fermented soy milk group showed 9.13 WBCs cells/nL.

The results of recent study were also in agreement with Niamah et al. (2017) who studied the effect of feeding probiotic soy milk for 40 days on rats. They documented the percentage of 3 WBC for T1 treatment of probiotic soy milk was 18.96 (mm ) while the control sample, 12.6 (mm3) after 40 days of dosing.

4.3.7.3. Platelets count (PLC)

The statistical analysis of the results for the effect of value added soy milk on platelets count has depicted in Table 4.45 The results showed the non- momentous (P>0.05) effect of soy milk in study I (Normal rats) , study II (Diabetic rats) and study III (Hypercholesterolemic rats) as well. The mean values showed a slight change of platelets count after feeding with 9 fermented and non-fermented soy milk as 7.15±0.42 x 10 /L in To group and 7.26±0.38 x 9 9 10 /L in T1 group in normal rats. The study II showed change as6.65±0.3 x 10 /L in To group 9 and 6.74±0.39 x 10 /L in T1 group. The variations observed in study III was 6.17±0.34 x 9 9 10 /L for To and 6.27±0.39 x 10 /L for T1. 206

Table 4.45 Effect of fermented soy milk on white blood cells count (WBCs) cells/nL

Studies To T1 F value

Study I 6.99±0.94a 6.53±0.25a 0.68NS

Study II 7.86±0.21a 7.35±0.34a 4.89NS

Study III 7.65±0.28a 6.93±0.45a 5.54NS

NS= non-significant (P> 0.05) a Means in rows with similar superscripts do not differ (P>0.05)

Figure 4.43: Percent decrease in level of white blood cells (WBC)

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It is noticeable from Figure 4.44 that value added drink showed a minor increase in platelets count as 2.65% increase was recorded in study III (high cholesterol rat group). Similarly, in study II 2.53% the increase was recorded in diabetic group while least increase was reported in study I as 1.53% however, the increase was non-significant in all studies.

The results of present study were supported by findings of Niamah et al. (2017). They studied the effect of feeding fermented soy milk on experimental rats as the percentage of 3 3 PLT for T1 treatment was 246.3(mm ) while the control sample were 206.3 (mm ) after 40 days of dosing.

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Table 4.45- Effect of fermented soy milk on platelets count (PLC) x 109/L

Studies To T1 F value

Study I 7.15±0.42a 7.26±0.38a 0.12NS

Study II 6.65±0.32a 6.74±0.39a 0.10NS

Study III 6.17±0.34a 6.27±0.39a 0.12NS

NS= non-significant (P> 0.05) aMeans in rows with similar superscripts do not differ (P>0.05)

Figure 4.44: Percent increase in level of platelets count

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CHAPTER 5 SUMMARY Soybean is most endorsed source of plant proteins containing all essential amino acids and also comprises of nutritious ingredients such as minerals, vitamins, lipids and bioactive components e.g. isoflavones, flavanoids, saponins and peptides. All these constituents boost up the therapeutic value of soybean. The use of probiotics in functional foods is getting consideration of the scientists to restrain life style related infirmities. Functional foods that are prepared with "scientific intelligence" nourish the body with proteins, vitamins, fats, carbohydrates, etc., are needed to boost up healthy survival. In Pakistan there is no authentic research yet available on soy varietal characterization, moreover on fermented soy milk as a functional food and its efficacy study. Keeping in view the significance of soybean and fermented soy milk having positive impact on human health; current project was planned to select the suitable variety for the development of fermented soy milk by using Lactobacilus acidophilus and Lactobacilus casei. The soy milk was also evaluated for the hypoglycemic and hypocholesterolemic attributes through in vivo study.

The composition assessment of soybean varieties (Faisal, NARC-II, Willium-82, Ajmeri and Rawal-I) depicted the highly significant difference. It was observed that Ajmeri contains high amount of crude protein (37.65±1.84%), crude fat (22.20±1.02%), crude fiber (17.67±0.87%) and ash (5.63±0.28%) which signifies its nutritional attributes while Faisal was high in NFE contents (25.60±1.20%) and Rawal-I was high in moisture contents(10.49±0.49%).

Ajmeri also contain the high level of potassium, calcium, iron, zinc, copper and sodium while, magnesium was in Willium-82. The fatty acids myristic and stearic was found to be higher in Rawal-I, palmitic and arachdic fatty acid was higher in Faisal, behenic was in Willium-82 and oleic, linoleic, linolenic, erucic acid were higher in Ajmeri soybean oil. The lipoygenase were lower in Ajmeri among all other varieties.

The maximum concentration of total phenolic contents was (2.76±0.05 mg GAE/g) and total flavonoids TFC was (1.78±0.05 mg CE/g) in Ajmeri.The antioxidant checked by DPPH were higher (5.65±0.23 mg TE/g) value, reducing power FRAP was (13.48±0.94 mg TE/g) and ABTS was 29.56±2.06 mg TE/g in Ajmeri which was relatively higher than other varieties.

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The soy milk prepared from top three selected varieties (NARC-II, Willium-82 and Ajmeri) and fermented by using Lactobacillus acidophilus and Lactobacillus casei solely as well as in combination to check their coculture fermentation effect. Four types of soy milk treatments were prepared using starter culture as described To (non-fermented soy milk), T1

(Soy milk made by using L. acidophilus), T2 (soy milk made by using L. casei) and T3 (soy milk made by using L. acidophilus and L. casei).

The fermented soy milk during physicochemical analysis showed higher TSS in NARC-II (10.55%), SNF were maximum (8.94%), protein (2.66%) and ash contents were (0.73%) in Ajmeri soy milk, highest fat contents (1.71%) were in NARC-II soy milk. These components reduced during storage and fermentation in T3 followed by T2, T1 and To.

The survivability of viable cells showed that soy milk facilitated the growth of both LAB tested, L. casei grew well in all samples as compared to L. acidophilus. Ajmeri soy milk showed the higher count. The number of cells increases upto 16th day of storage and afterwards it started to decrease. The maximum pH was noticed in To (6.79 to 6.27) followed by T1 (5.12 to 4.18), T2 (4.69 to 3.45) and T3 (4.77 to 3.52). The maximum acidity was noticed in Ajmeri (0.72%) followed by Willium-82 (0.61%) and NARC-II (0.57%). Effect of varieties depicted that maximum WHC was recorded in Willium-82 (45.41%) followed by

Ajmeri (44.03%) and NARC-II (43.56%). The effect of treatment on WHC was higher in T2 followed by T3 and T1.

The effect of fermentation gives higher DPPH values in Ajmeri soy milk as compared to NARC-II and Willium-82. The FRAP and ABTS activities were higher in Ajmeri followed by

Willium-82 and NARC-II. Among the treatments T3 proved better regarding the antioxidant activities. The sensory evaluation of soy milk shows the more acceptances for Ajmeri variety and T3 treatment was highly appreciated than followed by T2, T1 and To.

The scanning electron microscopy of suggested that Ajmeri soy milk fermented with combination of L. acidophilus and L. casei has more and precise pore formation and stronger cross linking of soybean protein which modified structure stability. The L. casei showed

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significant interaction with soy protein in all samples as compared to L. acidophilus that showed consistent looseness in structure stability.

Isoflavons (genistein and daidzein) quantified in soy milk. The isoflavones concentration was higher in T3 followed by T2, T1 and lowest was in To. These both isoflavones are in higher concentration in Ajmeri followed by NARC-II and Willium-82.

During efficacy study it was proved that Ajmeri soy milk fermented with coculture fermentation was more effective for the study. The therapeuical effect of soy milk was checked on Sprague dawley rats against hyperglycemia and hypercholestrolemia. It was concluded that fermented soy milk has more reduction effect on cholesterol, LDL and triglycerides in blood however, HDL was increased. The reduction was higher in hyperchoolesterolemic group than the hyperglycemic and normal rats. The effect of fermented soy milk showed decrease in glucose level and increase in insulin level as 17.11% glucose was decreased with 8.3% increase in insulin in hyperglycemic group followed by 7.46% glucose reduction and 5.4% insulin increment in high cholesterol group and 3.61% glucose was decreased and 1.8% increase in insulin was reported in normal group.

During safety test on hepatic tissue, the level of Aspartate aminotransferase (AST), Alanine transaminase (ALT) and Alkaline phosphatase (ALP) was decreased. The reduction effect was more pronounced in hypercholesterolemic rats as AST was (12.74%), ALT (12.86%) ALP was reduced (15.06%). The safety status of kidney (renal) showed increase in RBC and platelets count and minor reduction in white blood cells.

Conclusively soybean has worth as functional food to address various life style nutritional disorders. The varieties tested exhibited wide variability in nutritional components, phenolic contents and a great free radical scavenging activity. Out of five tested varieties Ajmeri was found to contain highest nutritional quality which may be recommended for export quality. The current research suggested that soy milk fermented by L. acidophilus and L. casei has more beneficial effect on hyperlipidemia and hyperglycemia in rats.

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Conclusions

The novelty of this work was characterization of soybean varieties available in Pakistan based on their consumption and fermentation chracteristics. The best selected variety was also evaluated for bio-efficacy study.

NARC-II, Willium-82 and Ajmeri were selected due to:

 Nutritional perameters like highest fat, protein, minerals and fatty acid profiling  Highest phenolic contents, flavonoids and antioxidant activities  Lipo-oxygenase was lower in these varieties as compared to others

The results from this research indicated the potential of value added soy milk fermented with L. acidophilus and L. casei in Ajmeri characterized as chief among all due to:

 Physicochemical and compositional results were superior  Maximum antioxidant activity based on DPPH, ABTS and FRAP  Rheology showed high viscosity  Sensory evaluation showed more acceptability  SEM analysis showed more compact and rigid structure in it  Bioactive component (genistein and daidzein) were higher

Fermented Ajmeri soy milk with both L. acidophilus and L. casei showed great potential in controlling Hypercholesterolemia and Hyperglycemia in experimental rats as compared to Non-fermented soy milk due to:

 Reduction in glucose and increment in Insulin level  Cholesterol reduction and HDL increment with lowering LDL level  Liver functioning test showed decrease in AST, ALT and ALP  Kidney and renal functioning test also assure that fermented soy milk is safe to use

Therefore, Ajmeri soy milk fermented by using (L. acidophilus and L. casei) can be targeted as a part of complementary dietary strategies for type 2 diabetes and hypercholesterolemia and related diseases combined with traditional drug and nutritional therapies, including fruits, 213

vegetables and other legumes. Fermented soy milk prevented hypercholesterolemia by modulating cholesterol metabolism and hyperglycemia by decreasing glucose level and increment in insulin production.

Recommendations

 Soy based functional foods should be promoted in daily diet as shield against numerous health related disorders  Locally available soybean varieties must be explored to motivate farmers to upsurge its production in Pakistan  In a developing country like Pakistan protein requirement can be fulfilled in an inexpensive way by administrating soy protein  Locally prepared fermented soy milk must be prepared and sold at commercial level rather than to import it to support economy and allied stakeholders  Soybean oil and soy meal production must be encouraged from local varieties  Toxicological and safety perspectives of fermented soy milk should be explored further for lactose intolerant, diabetic and cholestrolemic patients  Community based research trails ought to be launched to enhance diligence regarding physiological threats  Advertisement about importance of fermented soy milk should be promoted among the public through media or mass communication  Dieticians should recommend fermented soy milk based in routine diet to mitigate many metabolic ailments

Limitations

The issues in in vivo studies on animals is that animal models oftenly do not gives actual results as that of human models due to difference in drug‟s rate of metabolism, transportation and absorption in human body. That‟s why there is need to develop in vitro models of human intestinal function, including cell culture systems. There is need to check the effect of these LAB on a mciro engineered model “gut on a chip” a microfluidic device, faster and to augment the effect of fermented foods in plummeting 214

inflammation or other disorders. Human Gut on Chip will be advanced to evaluate the beneficial effects of fermented products and their probiotic effects regarding the mitigation of gut residing microbes in order to combat against different diseases related to inflammation.

215

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249

Appendix-I

Sensory Evaluation Performa for Soy Milk

Name of Judge…………………. Designation…………………

Character Colour Aroma Flavour Texture Overall

acceptability

T0

NARC-II T1

T2

T3

T0

Willium- T1 82 T2

T3

T0

Ajmeri T1

T2

T3

Scale for Acceptability 1- Extremely Poor 2- Very Poor 3- Poor 4- Below fair above poor 5- Fair 6- Below good above fair 7- Good 8- Very Good 9- Excellent Signature………………………. Date……………………..

250

Appendix-II

VISCOSITY (Pa.s) Shear

rate NARC-II on 0 Day NARC-II on 8th Day NARC-II on 16th Day NARC-II on 24th Day

1 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3

2 1/s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s

0.73 40.24 98.28 120.64 0.71123 0.21 26.63 62.24 84.31 0.03 21.07 35.19 77.44 3 0.1 28.8494 65.8091 102.313 5 0 0 1 5 7 3 9 0 5 3 7 9

- 0.58 18.89 56.95 0.56898 0.26 16.70 34.18 48.01 14.73 21.71 42.72 4 0.1585 54.919 19.3427 30.4669 46.5872 0.01 0 3 4 5 2 2 5 7 2 6 5 6

0.40 10.10 35.46 0.18 11.16 21.34 30.11 0.01 12.22 13.86 24.27 5 0.25121 32.307 0.34793 14.0752 17.1584 26.5415 9 2 4 8 1 2 4 6 9 2 7

0.39807 0.29 21.06 0.17546 0.06 43.66 18.82 0.04 13.42 6 5.758 18.642 9.05976 9.73782 15.6349 7.272 9.196 8.312 2 7 2 5 8 4 4 3 3

0.21 12.02 0.02 51.00 11.05 0.00 7 0.63098 3.383 10.277 0.07612 5.07597 5.7691 8.54083 4.250 5.808 4.938 6.748 1 8 3 0 8 2

0.16 0.03849 0.01 0.00 8 1.00002 1.919 6.806 5.456 2.9863 3.47312 4.39307 2.208 4.416 6.457 3.536 2.743 3.830 0 1 4 7

0.12 0.02018 0.00 0.00 9 1.58501 1.139 3.780 3.078 1.81584 1.78131 2.08305 1.261 2.453 3.607 2.048 1.652 2.427 2 2 3 3

251

1 0.09 0.01244 0.97335 0.00 0.00 2.5119 0.666 2.147 1.830 1.12834 1.05631 0.724 1.377 2.085 1.256 0.948 1.465 0 5 8 4 6 3

1 0.07 0.60229 0.54759 0.58224 0.00 0.00 3.98112 0.395 1.266 0.878 5.20E-03 0.517 0.889 1.456 0.803 0.560 0.786 1 3 5 8 8 2 2

1 0.05 0.37034 0.26882 0.32004 0.00 0.00 6.30957 0.253 0.699 0.486 5.20E-03 0.333 0.513 1.071 0.496 0.418 0.326 2 8 8 3 5 3 3

1 0.04 0.23829 0.15335 0.21088 0.00 0.00 9.99999 0.165 0.420 0.267 4.65E-03 0.226 0.322 0.727 0.317 0.289 0.145 3 6 3 8 8 2 3

1 0.03 0.12804 0.12699 0.00 0.00 15.8489 0.113 0.204 0.146 4.21E-03 0.11926 0.180 0.213 0.451 0.172 0.208 0.133 4 8 1 3 2 3

1 0.03 0.07698 0.09388 0.00 0.00 25.1188 0.083 0.102 0.083 3.65E-03 0.06687 0.138 0.185 0.303 0.089 0.146 0.145 5 1 5 2 2 3

1 0.02 0.05067 0.06806 0.04296 0.00 0.00 39.8107 0.064 0.058 0.054 3.17E-03 0.105 0.120 0.227 0.054 0.101 0.108 6 6 3 8 8 2 3

1 0.02 0.05009 0.02976 0.00 0.00 63.0958 0.051 0.037 0.039 2.73E-03 0.03729 0.082 0.092 0.177 0.038 0.069 0.077 7 1 2 2 2 2

1 0.01 0.02933 0.03855 0.02283 0.00 0.00 99.9999 0.041 0.028 0.030 2.38E-03 0.063 0.074 0.137 0.030 0.047 0.056 8 8 1 7 7 2 2

1 0.01 0.02386 0.03002 0.01849 0.00 0.00 158.489 0.033 0.021 0.024 2.13E-03 0.049 0.061 0.104 0.025 0.033 0.040 9 5 7 2 5 2 2

251.189 0.026 0.016 0.020 1.95E-03 0.01983 0.038 0.050 0.081 0.020 0.024 0.029 2 0.01 0.02353 0.01518 0.00 0.00

252

0 3 6 6 2 2

2 0.01 0.01637 0.01861 0.01252 0.00 0.00 398.108 0.021 0.013 0.017 1.84E-03 0.029 0.040 0.061 0.017 0.018 0.022 1 0 2 2 3 2 2

2 0.00 0.01322 0.01486 0.00 0.00 630.956 0.017 0.010 0.014 1.77E-03 0.01042 0.023 0.031 0.047 0.014 0.013 0.016 2 9 2 9 2 2

2 0.00 0.01065 0.01167 0.00 0.00 1000 0.013 0.009 0.012 1.77E-03 8.54E-03 0.018 0.023 0.035 0.011 0.010 0.013 3 7 4 5 2 3

2 0.00 0.00 0.00 1584.89 0.011 0.007 0.009 1.90E-03 8.44E-03 9.16E-03 6.89E-03 0.014 0.017 0.026 0.009 0.008 0.010 4 6 2 3

2 0.00 0.00 0.00 2000 0.009 0.007 0.008 2.02E-03 7.27E-03 7.71E-03 6.05E-03 0.012 0.014 0.021 0.008 0.007 0.009 5 5 2 3

Appendix-III

VISCOSITY (Pa.s) Shear rate Willium-82 on 0 Day Willium-82 on 8th Day Willium-82 16th Day Willium-82 on 24th Day

1 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3

2 1/s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s

253

3 0.97 60.1 144.7 449.9 0.30 24.1 103.3 280.9 0.29 21.4 76.7 120.8 0.15 16.6 13.6 17.4 0.1 4 29 76 31 9 22 89 35 3 73 30 74 7 80 53 58

4 0.71 37.7 80.58 371.9 0.26 16.8 59.63 155.9 0.25 16.4 48.6 74.39 0.12 10.2 10.7 9.70 0.1585 3 52 4 45 4 09 5 37 7 60 76 5 0 43 63 4

5 0.2512 0.49 24.4 46.46 193.1 0.20 12.5 34.02 88.57 0.19 11.0 29.4 45.46 0.12 6.57 7.11 6.00 1 8 53 4 77 7 85 9 7 6 42 30 8 7 6 6 8

6 0.3980 0.38 15.4 27.24 106.1 0.13 8.80 19.21 53.84 0.14 7.22 18.2 28.05 0.11 3.98 4.53 3.81 72 5 18 6 04 3 3 4 0 7 8 53 4 5 9 9 8

7 0.6309 0.30 8.56 15.03 62.60 0.07 5.39 11.00 34.58 0.09 4.60 11.7 17.59 0.08 2.35 2.67 2.39 8 3 1 9 0 2 9 9 6 0 8 44 5 5 0 5 0

8 1.0000 0.20 4.87 35.22 0.06 3.28 21.95 0.08 2.84 6.70 10.90 0.05 1.60 1.57 1.60 9.135 6.338 2 4 3 6 9 9 0 0 6 6 4 4 2 8 1

9 1.5850 0.14 3.06 19.54 0.04 1.93 12.66 0.06 1.82 3.98 0.05 0.85 0.98 0.95 5.884 3.755 6.664 1 5 0 5 7 0 0 4 9 7 2 7 0 7

1 0.11 1.73 0.03 1.14 0.05 1.19 2.36 0.04 0.48 0.62 0.60 3.312 9.910 2.166 7.536 4.056 0 2.5119 1 4 4 0 1 4 8 0 7 7 2

1 2.007 5.013 1.330 4.185 2.434 3.9811 0.08 0.96 0.02 0.67 0.03 0.79 1.36 0.03 0.29 0.40 0.39

254

1 2 2 3 9 0 9 1 9 3 1 4 1

1 6.3095 0.06 0.57 0.02 0.42 0.03 0.53 0.82 0.02 0.18 0.26 0.25 1.014 2.755 0.824 2.259 1.475 2 7 5 5 3 5 1 9 0 7 6 6 1

1 9.9999 0.05 0.40 0.01 0.30 0.02 0.37 0.49 0.02 0.16 0.19 0.16 0.629 1.498 0.479 1.324 0.930 3 9 1 9 9 1 6 1 1 3 5 9 5

1 15.848 0.04 0.31 0.01 0.22 0.02 0.26 0.30 0.02 0.13 0.14 0.11 0.353 0.783 0.288 0.805 0.603 4 9 1 0 6 4 2 4 2 0 4 0 5

1 25.118 0.03 0.23 0.01 0.16 0.01 0.19 0.19 0.01 0.09 0.09 0.08 0.248 0.411 0.174 0.519 0.397 5 8 4 1 4 8 8 3 2 7 6 2 5

1 39.810 0.02 0.17 0.01 0.12 0.01 0.14 0.13 0.01 0.06 0.06 0.06 0.183 0.205 0.110 0.320 0.263 6 7 8 6 2 7 5 3 2 5 8 3 7

1 63.095 0.02 0.13 0.01 0.09 0.01 0.10 0.08 0.01 0.04 0.04 0.05 0.116 0.111 0.073 0.194 0.176 7 8 4 5 0 5 3 7 7 3 8 4 5

1 99.999 0.02 0.10 0.00 0.07 0.01 0.07 0.06 0.01 0.03 0.03 0.04 0.082 0.067 0.048 0.126 0.119 8 9 0 1 9 2 1 9 3 2 5 2 5

1 158.48 0.01 0.07 0.00 0.05 0.01 0.05 0.04 0.01 0.02 0.02 0.03 0.058 0.042 0.034 0.086 0.084 9 9 7 4 8 7 0 9 1 1 6 4 6

255

2 251.18 0.01 0.05 0.00 0.04 0.00 0.04 0.02 0.01 0.02 0.01 0.02 0.042 0.028 0.025 0.061 0.061 0 9 4 4 7 6 8 4 9 0 0 8 9

2 398.10 0.01 0.03 0.00 0.03 0.00 0.03 0.02 0.00 0.01 0.01 0.02 0.031 0.019 0.018 0.045 0.044 1 8 2 9 6 7 7 3 1 9 5 4 3

2 630.95 0.01 0.02 0.00 0.03 0.00 0.02 0.01 0.00 0.01 0.01 0.01 0.024 0.015 0.014 0.033 0.033 2 6 0 9 5 0 6 5 6 8 2 1 9

2 0.00 0.02 0.00 0.02 0.00 0.02 0.01 0.00 0.00 0.00 0.01 0.019 0.012 0.011 0.024 0.024 3 1000 8 3 4 4 5 0 2 7 9 8 5

2 1584.8 0.00 0.01 0.00 0.01 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.016 0.010 0.009 0.018 0.019 4 9 7 8 4 7 4 5 0 6 8 7 1

2 0.00 0.01 0.00 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.014 0.019 0.008 0.015 0.015 5 2000 6 5 3 2 4 2 8 6 7 6 9

256

Appendix-IV

VISCOSITY (Pa.s) Shear rate Ajmeri on 0 Day Ajmeri on 8 Day Ajmeri on 16 Day Ajmeri on 24 Day

1 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3 T0 T1 T2 T3

2 1/s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s Pa.s

3 - 0.088 425. 571. 891. 0.086 136.3 311.5 656.2 0.029 109.4 192.5 441.2 80.65 98.27 252.5 8.59 68 313 381 696 715 8 38 38 591 39 15 99 63 95 57 0.1 E-03

4 0.120 217. 318. 427. 0.098 80.41 164.6 346.0 0.096 51.41 109.7 193.5 0.015 47.89 56.95 111.1 0.1585 536 762 879 887 125 87 6 05 828 52 31 41 309 87 35 52

5 - 0.091 130. 187. 248. 0.116 49.87 94.48 196.5 0.102 27.43 67.28 107.7 30.35 35.46 63.03 0.2512 0.029 891 985 647 201 579 34 59 87 889 53 49 63 76 38 82 1 43

6 0.3980 0.066 82.0 121. 149. 0.101 32.00 52.84 116.4 0.039 14.17 41.09 63.58 0.018 18.98 21.06 35.05 72 161 236 12 374 269 42 91 8 474 93 05 73 202 52 24 49

7 0.6309 0.050 51.2 77.4 93.4 0.043 20.95 31.00 69.39 0.038 7.262 25.36 37.09 0.013 11.88 12.02 20.02

257

8 137 675 175 844 617 47 85 32 16 06 54 57 223 03 8 82

8 1.0000 0.037 31.7 44.9 59.5 0.026 11.42 17.91 39.29 0.024 3.817 15.41 21.26 0.021 7.141 6.806 12.15 2 864 477 125 039 328 32 78 01 547 6 99 38 065 69 19 18

9 1.5850 0.030 15.8 22.4 35.1 0.015 6.353 10.45 19.14 0.019 1.920 9.539 10.99 8.19 4.150 3.780 7.275 1 807 8 385 346 75 3 32 23 505 83 85 8 E-03 24 08 18

1 0.026 7.83 12.9 16.4 0.016 3.799 5.950 9.611 0.018 1.208 5.418 4.426 8.91 2.253 2.147 4.403 0 2.5119 012 732 867 035 647 73 52 3 184 9 01 73 E-03 81 07 24

1 3.9811 0.019 4.22 7.00 9.64 0.010 2.415 3.280 5.731 0.014 0.652 2.975 2.590 8.15 1.451 1.266 2.725 1 2 858 077 686 763 506 32 02 66 088 04 78 78 E-03 85 01 77

1 6.3095 0.015 2.90 3.81 5.65 0.010 1.664 1.801 3.412 0.013 0.361 1.808 1.243 7.62 0.822 0.698 1.367 2 7 738 709 488 145 177 42 95 15 417 928 15 9 E-03 233 841 62

1 9.9999 0.013 2.03 2.84 3.64 8.95 1.185 0.778 2.167 0.011 0.209 1.042 0.841 6.82 0.504 0.420 0.690 3 9 016 868 651 601 E-03 94 885 27 964 281 26 228 E-03 622 403 198

1 15.848 0.010 1.26 1.73 2.37 8.09 0.760 0.480 1.388 0.010 0.154 0.664 0.578 6.30 0.380 0.203 0.353 4 9 959 87 912 214 E-03 83 901 17 74 509 357 655 E-03 298 614 047

1 25.118 9.09E 0.58 0.94 1.39 7.26 0.561 0.346 0.958 9.69 0.084 0.332 0.389 5.82 0.284 0.102 0.189 5 8 -03 0155 253 418 E-03 328 218 4 E-03 767 34 869 E-03 957 303 306

258

4

1 0.53 7.94E 0.24 0.80 6.58 0.359 0.232 0.521 8.79 0.056 0.175 0.283 5.34 0.218 0.057 0.107 6 39.810 266 -03 7505 7545 E-03 568 551 756 E-03 638 978 57 E-03 135 593 071 7 7

1 0.33 7.10E 0.10 0.55 5.99 0.206 0.168 0.324 8.00 0.040 0.100 0.210 4.90 0.159 0.037 0.060 7 63.095 371 -03 9857 007 E-03 513 193 354 E-03 797 3 824 E-03 216 485 496 8 3

1 99.999 6.41E 0.06 0.23 0.38 5.48 0.145 0.124 0.227 7.29 0.030 0.064 0.162 4.51 0.119 0.027 0.041 8 9 -03 7368 924 4116 E-03 012 574 461 E-03 007 803 482 E-03 305 77 198

1 0.17 5.83E 0.04 0.26 5.02 0.110 0.095 0.161 6.66 0.023 0.047 0.124 4.14 0.090 0.020 0.030 9 158.48 240 -03 9015 4393 E-03 536 595 191 E-03 382 142 613 E-03 268 511 051 9 3

2 0.12 5.33E 0.03 0.18 4.59 0.084 0.073 0.116 6.10 0.018 0.037 0.095 3.83 0.069 0.015 0.024 0 251.18 315 -03 8221 2947 E-03 588 683 493 E-03 01 904 978 E-03 395 767 084 9 4

2 0.08 4.89E 0.03 0.12 4.24 0.064 0.056 0.084 5.61 0.014 0.030 0.074 3.55 0.053 0.012 0.019 1 398.10 954 -03 0686 7214 E-03 751 578 563 E-03 191 065 046 E-03 193 604 752 8 9

259

2 0.06 2 4.51E 0.02 0.08 3.92 0.049 0.042 0.061 5.17 0.011 0.024 0.055 3.28 0.040 0.010 0.016 592 -03 476 989 E-03 84 853 225 E-03 635 755 725 E-03 617 195 926 630.95 5 6

2 0.04 3 4.18E 0.02 0.06 3.62 0.037 0.032 0.044 4.81 9.71 0.020 0.042 3.03 0.030 8.60 0.014 858 -03 0194 4875 E-03 526 519 843 E-03 E-03 51 828 E-03 526 E-03 285 7 1000

2 0.03 4 3.95E 0.01 0.04 3.39 0.026 0.024 0.033 4.53 8.24 0.017 0.033 2.83 0.022 7.38 0.011 638 -03 62 7231 E-03 853 602 493 E-03 E-03 307 127 E-03 877 E-03 3 1584.8 5 9

2 0.02 5 3.88E 0.01 0.03 3.31 0.021 0.019 0.027 4.46 7.49 0.014 0.026 2.74 0.018 6.71 9.92 948 -03 393 7853 E-03 27 931 246 E-03 E-03 474 955 E-03 444 E-03 E-03 8 2000

260