carbohydrate based prebiotic effects on quality …
TRANSCRIPT
CARBOHYDRATE BASED PREBIOTIC EFFECTS ON QUALITY ATTRIBUTES OF YOGHURT AND ITS
PROBIOTIC ENDURANCE
By
MAJID HUSSAIN
2001-ag-1053
M.Sc. (Hons.) Food Technology
A Thesis 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 AGRICULTURAL ENGINEERING AND
TECHNOLOGY
UNIVERSITY OF AGRICULTURE, FAISALABAD
PAKISTAN
2014
i
DECLARATION
I hereby declare that the contents of the thesis "Carbohydrate based prebiotic effects on
quality attributes of yoghurt and its probiotic endurance" are creation of my own
research and no part has been copied from any published source (except the references,
standard mathematical or geometrical models/equations/formulae/protocols etc.). I further
declare that this work has not been submitted for award of any other diploma/degree. The
University may take action if information provided us found inaccurate at any stage. (In case
of default the scholar will be proceeded against as per HEC plagiarism policy).
MAJID HUSSAIN
2001-ag-1053
ii
To,
The Controller of Examinations,
University of Agriculture,
Faisalabad-Pakistan
“We, the Supervisory Committee, certify that the contents and form of thesis
submitted by Majid Hussain, Regd. No. 2001-ag-1053, have been found satisfactory and
recommend that it be processed for evaluation, by the External Examiner(s) for the award of
degree”
SUPERVISORY COMMITTEE
1) Chairman ________________________________
Prof. Dr. Tahir Zahoor
2) Member _________________________________
Prof. Dr. Faqir Muhammad Anjum
3) Member _________________________________
Dr. Muhammad Shahid
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THIS HUMBLE EFFORT IS DEDICATED TO
TABEEB-E-ROOHAN, JAN-E-SAALKAN, SHEIK-UL-ULAMA, AFTAB-E-
WALAYAT, ABU-UL-HASAN
(DAMAT BARAKATAHUM-UL-ALIA)
Whose hands are always raised in prayer for me and who are with me to feel the bud of their
wishes and prayers, blooming into a flower
SYED AFZAAL HUSSAIN SHAH
HANFI SAIFI
iv
LIST OF ABBREVIATIONS
OS Oligosaccharides
CGG Crude guar gum
PGG Purified guar gum
BHGG Base Hydrolyzed Guar Gum
AHGG Acid Hydrolyzed Guar Gum
EHGG Enzyme hydrolyzed Guar Gum
PHGG Partial Hydrolyzed Guar Gum
PBS Phosphate Buffer Saline
SIM Small Intestinal Model
RBC’s Red Blood Cells
GM ratio Galactose and Mannose ratio
SEM Scanning Electron Microscopy
XRD X-Ray Diffraction
FTIR Fourier Infra-red Spectrophotometry
TGA Thermo Gravimetric Analysis
Cryo-SEM Cryogenic Scanning Electron Microscopy
WHC Water Holding Capacity
cfu Colony Forming Unit
CRD Completely Randomized Design
SOV Source of variation
df Degree of freedom
SD Standard deviation
v
LIST OF SYMBOLS AND UNITS
Gʹ Elastic or storage modulus
Gʺ Viscous or loss modulus
σ Shear stress
ϒ Shear rate
Ƞ Viscosity
f Frequency
Hz Hertz
Pa Pascal
Pa.s Pascal × second
cps centipoise
vi
ACKNOWLEDGMENT
Knowledge is limited and time is short to express the dignity of Almighty ALLAH,
the Propitious, the Benevolent and Sovereignty, the entire source of all the knowledge and
wisdom endowed to the mankind. Lips are trembling and eyes are wet to pray for the Holy
Prophet HAZRAT MUHAMMAD (PBUH); the bacon of enlightment, the fountain of
knowledge and the messenger of peace and forever torch of guidance for humanity.
This thesis arose in part out of a period of research. By that time, I have worked with
a number of people whose contribution in assorted ways to the research and the making of
the thesis deserved special mention. It is a pleasure to convey my gratitude to them all in my
humble acknowledgment. I deem it utmost pleasure to avail the opportunity to express the
heartiest gratitude and deep sense of devotion to my esteemed supervisor, Prof. Dr. Tahir
Zahoor for his kind guidance, assistance and endorsement from the very early stage of this
research as well as giving me extraordinary experiences throughout the research work. This
thesis would not have been possible unless his untiring support and sponsorship. His truly
scientist intuition exceptionally inspires and enriches my growth as a student, a researcher
and a scientist want to be. Above all and the most needed, he provided me unflinching
encouragement and support in various ways.
I wish to record my sincere appreciation to the members of my supervisory
committee; Prof. Dr. Faqir Muhammad Anjum and Dr. Muhammad Shahid for their
keen interest, incentive teaching, dynamic supervision, and valuable comments, scholastic
and constructive suggestions throughout my research work. I am also thankful to Dr.
Serafim Bakalis and Dr. Ourania Gouseti, School of Chemical Engineering, University of
Birmingham, UK for providing me opportunity to work in UK and their kindness,
sympathies, guidelines and valuable suggestions are always with me.
I would like to pay my special thanks to my younger sisters Nazia Khalid and
Samreen Ahsan for their help, motivation and dedication. They made it possible for me to
complete my project within the time limits unless their useful assistance and support. I pay
ineffable gratitude and deepest thanks to my all research fellows for their cooperation, well
wishes and moral support from time to time during the course of study. No acknowledgments
could ever adequately express my obligations to my affectionate father and dearly loved
mother and all family members especially my beloved spouse and children; Muhammad
Anas, Uswa Fatima, Muhammad Hasan and Muhammad Hashim who always raise their
hands in prayers for me and I can only say what I am today is just because of their prayers.
Financial support from Higher Education Commission, Government of Pakistan is
thankfully acknowledged.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 7
3 MATERIAL AND METHODS 33
4 RESULTS AND DISCUSSION 55
5 SUMMARY 170
LITERATURE CITED 178
APPENDICES 205
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CONTENTS
Chapter Contents Page
1 INTRODUCTION…………………………………………………….. 1
2 REVIEW OF LITERATURE………………………………………… 7
2.1. Oligosaccharides (OS)……………………………………………... 8
2.1.1. Introduction …………………………………………….... 8
2.1.2. Types of Oligosaccharides……………………………….. 9
2.1.3. Applications of Oligosaccharides………………………... 9
2.2. Guar Galactomannans ……………………………………………... 11
2.2.1. Concept and History……………………………………... 12
2.2.2. Structural Composition…………………………………... 12
2.2.3. Applications of Guar Galactomannans…………………... 13
2.2.3.1. Food Claims……………………………………. 13
2.2.3.2. Pharmaceutical Claims………………………… 14
2.2.3.3. Other Applications……………………………... 14
2.2.4. Health Benefits of Guar Galactomannans……….. 15
2.2.4.1. Gastrointestinal Effects………………… 15
2.2.4.2. Other Health Benefits………………….. 15
2.3. Hydrolysis of Guar Gum…………………………………………... 17
2.4. Prebiotics Effect of Guar Gum…………………………………….. 17
2.5. Guar Gum: Characterization……………………………………….. 18
2.5.1. Proximate Composition………………………………….. 18
2.5.2. Rheological properties…………………………………… 18
2.5.3. Galactose-Mannose Ratio………………………………... 19
2.5.4. Microstructural analysis………………………………….. 19
2.5.5. Haemolysis Study…………...…………………………… 20
2.6. Probiotics…………………………………………………………... 21
2.6.1. Probiotic Selection……………………………………….. 22
2.6.2. Health benefits of probiotics……………………………... 22
2.7. Synbiotic Food……………………………………………………... 22
2.7.1. Yogurt History…………………………………………… 23
2.7.2. Yogurt as a Synbiotic Food……………………………… 24
2.7.3. Nutritional and Therapeutic Aspects of Yogurt………….. 26
2.7.4. Yogurt composition…………………………………….. 27
2.7.4.1. Milk Proteins…………………………………... 27
2.7.4.2. Vitamin B………………………………………. 28
2.7.4.3. Lactose…………………………………………. 28
2.7.4.4. Fats or Lipids…………………………………... 28
2.7.4.5. Minerals………………………………………... 28
2.7.5. Chemistry of Yogurt (Whey-casein interactions)………... 29
ix
2.7.6. Rheological Properties of Yogurt………………………... 29
2.7.7. Physico-Chemical and Sensory Attributes of Yogurt……. 31
3 MATERIALS AND METHODS……………………………………... 33
3.1. Procurement of Raw Material……………………………………… 33
3.2. Purification of Guar Gum………………………………………….. 33
i) Reagents……………………………………………………… 33
ii) Procedure…………………………………………………….. 33
3.3. Hydrolysis of Guar Gum…………………………………………... 34
3.3.1. Enzymatic Hydrolysis……………………………………. 34
3.3.1.1. Reagents………………………………………... 34
(a) 1.0N HCl……………………………………. 34
(b) 0.1M Acetate Buffer………………………… 34
3.3.1.2. Guar Solution…………………………………... 34
3.3.1.3. Enzyme Buffer Solution……………………….. 34
3.3.1.4. Enzymatic Degradation………………………... 35
3.3.2. Acidic Hydrolysis………………………………………... 35
3.3.2.1. Reagents………………………………………... 35
(a) Methanol (99%)……………………………... 35
(b) Hydrochloric Acid…………………………... 35
3.3.2.2. Procedure………………………………………. 35
3.3.3. Basic Hydrolysis…………………………………………. 35
3.3.3.1. Reagents………………………………………... 35
(a) Barium Hydroxide…………………………... 35
(b) 1.0 M Sulfuric Acid………………………… 35
3.3.3.2. Procedure………………………………………. 36
3.4. Guar Gum Characterization………………………………………... 36
3.4.1. Chemical Analysis……………………………………….. 36
3.4.1.1. Moisture………………………………………... 36
3.4.1.2. Crude Protein…………………………………... 36
3.4.1.3. Total Ash………………………………………. 36
3.4.1.4. Crude Fat………………………………………. 37
3.4.1.5. Crude Fiber…………………………………….. 37
3.4.2. Rheological Properties of Aqueous Guar Solution………. 37
3.4.2.1. Shear Stress and Viscosity……………………... 37
3.4.2.2. Oscillatory Properties…………………...……... 38
3.4.2.3. Glucose Absorption in SIM……………………. 38
i) Preparation of solution……………………….. 39
ii) Effect of Mixing and Flow Rate on Glucose
Absorption………….......................................
39
iii) Glucose determination……………..……….. 39
x
3.4.3. Measurement of Galactose-Mannose Ratio……………… 40
3.4.3.1. Reagents………………………………………... 40
Trifluoroacetic Acid…………………………….. 40
3.4.3.2. Procedure………………………………………. 40
3.4.4. Scanning Electron Microscope (SEM)…………………... 40
3.4.5. X-ray Diffraction………………………………………… 41
3.4.6. FTIR Analysis……………………………………………. 42
3.4.7. Thermo Gravimetric Analysis…………………………… 42
3.4.8. Haemolysis Study……..………………………………… 43
3.4.8.1. Reagents………………………………………... 43
i) Phosphate Buffer Saline ……………………...
ii) 0.1% Triton X-100…………………………...
43
43
3.4.8.2. Procedure………………………………………. 43
3.5. Yogurt Manufacturing Process…………………………………… 44
3.5.1. Standardized Milk………………………………………... 46
3.5.2. Addition of Guar Gum…………………………………… 46
3.5.3. Pasteurization…………………………………………….. 46
3.5.4. Homogenization………………………………………….. 46
3.5.5. Cooling…………………………………………………... 46
3.5.6. Inoculation and Mixing…………………………………... 47
3.5.7. Packaging………………………………………………… 47
3.5.8. Incubation………………………………………………... 47
3.5.9. Storage………………………………………………….. 47
3.6. Yogurt Analysis…………………………………………………... 47
3.6.1. Physico-Chemical Analysis……………………………… 47
3.6.1.1. Compositional Analysis………………………... 47
i) Lactose……………………………………….. 47
ii) Total solids…………………………………... 48
iii) Fat…………………………………………… 48
iv) Protein………………………………............. 49
v) Ash…………………………………………… 49
3.6.1.2. pH……………………………………………… 50
3.6.1.3. Titrateable Acidity……………………............... 50
(a) Phenolphthalein Indicator…………... 50
(b) N/10 NaOH…………………………. 50
3.6.1.4. Viscosity……………………………………….. 50
3.6.1.5. Syneresis……………………………………….. 51
3.6.1.6. Water Holding Capacity (WHC)…………….... 52
3.6.1.7. Organic Acids………………………………….. 52
i) Reagents……………………………… 52
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ii) Extraction and Analysis of Organic
Acid……………………………………...
52
3.6.1.8. Cryo-Scanning Electron Microscopy (C-SEM)... 52
3.6.2. Sensory Evaluation………………………………………. 52
3.6.3. Microbial Analysis…………………………..................... 53
3.6.3.1. Viability of Lactic Acid Bacteria……………… 53
3.6.3.2. Bacterial Strains……………………….............. 53
i) Bifidobacterium………………………………. 53
ii) Yogurt Culture………………………………. 53
3.6.3.3. Reagents ……………………………………….. 53
3.7. Statistical Analysis…………………………………………………. 54
4 RESULTS AND DISCUSSION………………………………………. 55
(A) Guar Gum And its Hydrolyzed Forms……………………………... 55
4.1. Purification and Hydrolysis of guar gum…………………………... 55
4.2. Guar gum Characterization………………………………………… 58
4.2.1. Chemical Composition…………………………………... 58
4.2.2. Rheological properties of Guar Gum…………………….. 61
4.2.2.1. Steady shear properties………………………… 61
4.2.2.2. Oscillatory properties…………………………... 64
4.2.2.3. Glucose absorption (Small Intestinal Model)….. 69
4.2.3. Measurement of Galactose-Mannose Ratio……………… 72
4.2.4. Scanning Electron Microscopy (SEM)…………………... 74
4.2.5. X-ray Diffraction Analysis………………………………. 81
4.2.6. Fourier Transform Infra-red Spectrophotometric
analysis (FTIR)…………………………………………...
85
4.2.7. Thermo-gravimetric Analysis (TGA)……………………. 91
4.2.8. Haemolysis Study……...………………………………… 98
(B) Product Development………………………………………………. 102
4.3. Yogurt Analysis……………………………………………………. 105
4.3.1. Physico-chemical Analysis………………………………. 105
4.3.1.1. Compositional analysis………………………… 105
i) Fat………….…………………………………. 105
ii) Protein …..…………………...……………… 108
iii) Ash………………………………………….. 108
iv) Lactose……………………………………… 109
v) Total Solids…………………………………... 110
4.3.1.2. pH……………………………………………… 111
4.3.1.3. Acidity…………………………………………. 115
4.3.1.4. Viscosity……………………………………….. 118
4.3.1.5. Syneresis……………………………………….. 121
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4.3.1.6. Water Holding Capacity……………………….. 124
4.3.1.7. Organic Acids………………………………….. 127
i) Acetic acid………………..…………... 127
ii) Lactic acid…………………………… 127
iii) Citric acid…………………………… 130
iv) Butyric acid…………………………. 130
v) Pyruvic acid………………………….. 131
4.3.1.8. Cryo-Scanning Electron Microscopy………….. 132
4.3.2. Sensory evaluation……………………………………….. 143
4.3.2.1. Color…………………………………………… 143
4.3.2.2. Appearance…………………………………….. 147
4.3.2.3. Flavor…………………………………………. 149
4.3.2.4. Body and Texture……………………………… 151
4.3.2.5. Mouth feel…………………………………….... 153
4.3.2.6. Overall acceptability…………………………… 155
4.3.3. Microbial Analysis……………………………………….. 161
4.3.3.1. Bifidobacterium bifidum……………………….. 161
4.3.3.2. Streptococcus thermophilus……………………. 165
4.3.3.3. Lactobacillus bulgaricus………………………. 167
5 SUMMARY……………………………………………………………. 170
5.1 Conclusions…………………………………………………. 173
5.2 Importance of Results and Major Findings of this Study…... 174
5.3 Economic Impact on Yogurt Industry……………………… 174
5.4 Recommendations…………………………………………... 175
5.5 Limitations of Study………………………………………... 176
5.6 Future Research Directions…………………………………. 176
5.7 Future Studies………………………………………………. 177
LITERATURE CITED……………………………………………….. 178
APPENDICES…………………………………………………………. 205
Appendix I……………………………………………………… 205
Appendix II……………………………………………………... 207
Appendix III…………………………………………………….. 208
Appendix IV……………………………………………………. 209
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LIST OF TABLES
Table # Title Page No.
2.1 Functional properties of fermented foodstuffs 25
3.1 Composition of phosphate buffered saline (PBS) 43
3.2 Experiment plan for the preparation of yogurt 46
4.1 Mean squares for chemical composition of crude and hydrolyzed guar
gum
59
4.2 Means values for chemical composition (%) for the crude and
hydrolyzed guar gum
59
4.3 Mean squares for galactose-mannose of crude and hydrolyzed guar
gum
73
4.4 Mean values for galactose-mannose (%) of crude and hydrolyzed guar
gum
73
4.5 Functional groups evaluation of various guar gums at specific wave
number (cm-1
) in Infra-red spectral region
86
4.6 Haemolytic activity of controls (Triton-X 100, PBS solution) and five
guar samples at various concentrations (mg/mL)
99
4.7 Mean squares for compositional profile of probiotic yogurt during
refrigerated storage
106
4.8 Means storage values showing effect of crude and hydrolyzed guar
gum on fat, protein, ash, lactose and total solids (%) of probiotic
yogurt
106
4.9 Means treatment values showing effect of crude and hydrolyzed guar
gum on fat, protein, ash, lactose and total solids (%) of probiotic
yogurt
107
4.10 Mean squares for physico-chemical analysis of probiotic yogurt
during refrigerated storage
112
4.11 Means values showing effect of guar gum and storage time on pH of
probiotic yogurt
113
4.12 Means values showing effect of guar gum and storage time on acidity
(%) of probiotic yogurt
116
4.13 Means values showing effect of guar gum and storage time on
viscosity (cps) of probiotic yogurt
119
4.14 Means values showing effect of guar gum and storage time on
syneresis (%) of probiotic yogurt
122
4.15 Means values showing effect of guar gum and storage time on water
holding capacity (%) of probiotic yogurt
125
4.16 Mean squares for organic acids content of probiotic yogurt during
refrigerated storage
128
4.17 Means storage values showing effect of crude and hydrolyzed guar 128
xiv
gum on organic acids (mg/L) of probiotic yogurt
4.18 Means treatment values showing effect of crude and hydrolyzed guar
gum on organic acids (mg/L) of probiotic yogurt
129
4.19 Mean squares for sensory properties for probiotic yogurt 144
4.20 Mean values showing the color attributes of yogurt during storage 145
4.21 Mean values showing the appearance attributes of yogurt during
storage
148
4.22 Mean values showing the flavor attributes of probiotic yogurt during
storage
150
4.23 Mean values showing the body and texture attributes of probiotic
yogurt during storage
152
4.24 Mean values showing the mouth feel attributes of probiotic yogurt
during storage
154
4.25 Mean values showing the overall acceptability attributes of probiotic
yogurt during storage
156
4.26 Mean squares showing microbial analysis of probiotic yogurt during
storage
162
4.27 Viability of Bifidobacterium bifidum (cfu/g) during refrigerated
storage of probiotic yogurts
163
4.28 Viability of Streptococcus thermophilus (cfu/g) during refrigerated
storage of probiotic yogurts
166
4.29 Viability of Lactobacillus bulgaricus (cfu/g) during refrigerated
storage of probiotic yogurts
168
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LIST OF FIGURES
Fig # Title Page No.
2.1 Processing of guar gum; (a) guar pod (b) guar seed (c) dehusked
splits (d) guar gum powder
11
2.2 Chemical structure of guar galactomannan 13
2.3 The repeating unit of guar gum showing mannose galactose ratio 19
2.4 Formation of yogurt gel due to acidification of Milk 30
3.1 Rheometer 38
3.2 In vitro- Small Intestinal Model 38
3.3 Scanning Electron Microscope 41
3.4 X-Ray Diffractometer 41
3.5 Fourier Transform Infra-red Spectrophotometer 42
3.6 Thermo-Gravimetric Analyser 43
3.7 Flow diagram of yogurt preparation 45
3.8 Cryo-scanning Electron Microscope 52
4.1 Figure showing the effect of purification and hydrolysis of guar gum
on color appearance
57
4.2 (a) Flow behaviour (b) viscosity of aqueous solution (1%) of crude
and hydrolyzed guar gum
62
4.3 Viscoelastic properties of aqueous solution of (a) SCGG (b) CGG at
25°C
65
4.4 Viscoelastic properties of aqueous solution of (a) PGG (b) BHGG at
25°C
66
4.5 Viscoelastic properties of aqueous solution of (a) AHGG (b) EHGG
at 25°C
67
4.6 (a) Glucose calibration curve in a range from 0.0 to 10 mM
concentration (b) Absorption of glucose in 1% (w/v) guar solutions in
small intestinal model (SIM)
70
4.7 Scanning electron micrographs of crude guar gum (a) X1,000 (b)
X2,000
75
4.8 Scanning electron micrographs of purified guar gum (a) X1,000 (b)
X2,000
76
4.9 Scanning electron micrographs of base hydrolyzed guar gum (a)
X1,000 (b) X2,000
77
4.10 Scanning electron micrographs of acid hydrolyzed guar gum (a)
X1,000 (b) X2,000
78
4.11 Scanning electron micrographs of enzyme hydrolyzed guar gum (a)
X1,000 (b) X1,500
79
4.12 X-ray diffraction patterns of (a) CGG (b) PGG 82
4.13 X-ray diffraction patterns of (a) BHGG (b) AHGG 83
xvi
4.14 X-ray diffraction patterns of EHGG 84
4.15 FTIR spectra of (a) CGG (b) PGG 88
4.16 FTIR spectra of (a) BHGG and (b) AHGG 89
4.17 FTIR spectra of enzyme hydrolyzed guar gum (EHGG) 90
4.18 Thermo gravimetric analysis (TGA) curve of crude guar gum (CGG) 92
4.19 Thermo gravimetric analysis (TGA) curve of purified guar gum
(PGG)
93
4.20 Thermo gravimetric analysis (TGA) curve of base hydrolyzed guar
gum (BHGG)
94
4.21 Thermo gravimetric analysis (TGA) curve of acid hydrolyzed guar
gum (AHGG)
95
4.22 Thermo gravimetric analysis (TGA) curve of enzyme hydrolyzed guar
gum (EHGG)
96
4.23 Haemolytic activity, as a percentage of haemolysis caused by 0.1%
triton X-100, for 2.5, 5.0, 25, 50, 75, 100, 150, 200, 250 mg mL-1
of
guar gum fractions and controls
100
4.24 Probiotic yogurts produced (a) without guar gum (b) crude guar gum,
CGG and (c) purified guar gum, PGG with levels (0.1, 0.5, 1%)
103
4.25 Probiotic yogurts produced with (a) base hydrolyzed guar gum,
BHGG (b) acid hydrolyzed guar gum, AHGG and (c) enzyme
hydrolyzed guar gum, EHGG with levels (0.1, 0.5, 1%)
104
4.26 Cryo-scanning electron micrographs of control yogurt at (a) 0 day (b)
21st day of storage
133
4.27 Cryo-scanning electron micrographs of yogurt produced with 0.2%
CGG at (a) 0 day (b) 21st day of storage
134
4.28 Cryo-scanning electron micrographs of yogurt produced with 0.1%
PGG at (a) 0 day (b) 21st day of storage
135
4.29 Cryo-scanning electron micrographs of yogurt produced with 0.1%
BHGG at (a) 0 day (b) 21st day of storage
136
4.30 Cryo-scanning electron micrographs of yogurt produced with 0.3%
AHGG at (a) 0 day (b) 21st day of storage
137
4.31 Cryo-scanning electron micrographs of yogurt produced with 1%
AHGG at (a) 0 day (b) 21st day of storage
138
4.32 Cryo-scanning electron micrographs of yogurt produced with 0.3%
EHGG at (a) 0 day (b) 21st day of storage
139
4.33 Cryo-scanning electron micrographs of yogurt produced with 1%
EHGG at (a) 0 day (b) 21st day of storage
140
4.34 Sensory attributes of control yogurt To, Toʹ (without guar gum)
during storage
158
4.35 Sensory attributes of probiotic yogurt T1, T2, T3 (CGG) during storage 158
xvii
4.36 Sensory attributes of probiotic yogurt T4, T5,T6 (PGG) during storage 159
4.37 Sensory attributes of probiotic yogurt T7,T8,T9 (AHGG) during
storage
159
4.38 Sensory attributes of probiotic yogurt T10,T11,T12 (BHGG) during
storage
160
4.39 Sensory attributes of probiotic yogurt T13,T14,T15 (EHGG) during
storage
160
xviii
LIST OF APPENDICES
Appendix # Title Page No.
I Sensory evaluation score card of yogurt 205
II Viscosity for guar gum fractions at specific shear stress and
shear rate with passage of time
207
III Oscillatory properties of aqueous solution of guar gum fractions 208
IV Absorption of glucose in 1% (w/v) guar solutions in small
intestinal model (SIM)
209
xix
ABSTRACT
The research work was done to evaluate suitability of guar gum (CGG, PGG, AHGG,
BHGG and EHGG) utilization as prebiotic on probiotic (BB) behavior in yogurt. CGG was
purified (ethanol, 95:5v/v) and hydrolyzed using HCl, [Ba(OH)2] and mannanase enzyme.
SEM characterization exhibited rough surface morphology in CGG, PGG and BHGG,
powdery and fluffy appearance in AHGG and well defined porous structure in EHGG. The
consequences of XRD displayed amorphous structure and less crystallinity in CGG, PGG
and AHGG while EHGG slightly increased in crystallinity but lower than BHGG. FTIR
analysis revealed no major transformation of functional groups after hydrolysis of guar gum.
TGA evaluation of hydrolyzed guar gums indicated more heat stability than the crude ones.
Rheological studies revealed shear thinning non-Newtonian behavior at high shear rate and
Newtonian flow at low shear rate [high viscosity CGG (1.34 Pa.s); low viscosity AHGG
(0.15 Pa.s), EHGG (0.22 Pa.s)] while, in oscillatory inferences (viscous modulus, Gʺ; elastic
modulus, Gʹ), guar gums exhibited typical characteristics of weak viscoelastic gel. The small
intestine model (SIM) evaluation exposed non-significant difference in physiological
behavior of guar gums in reducing the glucose level. Haemolysis study showed non-toxic
effect (haemolysis, 1.9±0.03% to 7.24±0.02%) of guar gums to human RBC’s. Guar gum
treatments were evaluated for their prebiotic and probiotic endurance on yogurt quality
attributes. Periodic physico-chemical, sensory characteristics and bacterial viability of the
functional yogurt (Lactobacillus bulgaricus, Streptococcus thermophilus and
Bifidobacterium bifidum + 0.1%, 0.5%, 1.0% of AHGG and EHGG) at 4-6°C revealed
significant syneresis reduction, increase in WHC and viscosity exhibiting higher sensory
acceptability of prebiotic and probiotic endurance. Cryo-scanning electron microscopy
proved well embedded continuous casein network imparted by AHGG and EHGG granules.
In the whole, acidity and syneresis increased but pH, viscosity, WHC, bacterial viability and
consumer’s acceptability decreased with passage of time. The presence of guar gum as
prebiotic also caused a significant effect on organic acids contents in yogurt due to metabolic
activities of probiotic. The data obtained was analyzed using two-factor factorial design
under CRD in Statistix 8.1 software.
Key words: Guar gum hydrolysis, Characterization, Prebiotic, Probiotic, Functional Yogurt,
Health benefits
1
Chapter 1
I N T R OD U C TI ON
Carbohydrates (saccharides) are among the most abundant classes of biomolecules.
According to the number of sugar units, carbohydrates are classified as monosaccharides,
disaccharides and polysaccharides. Polysaccharides are ubiquitous can be either homo
polysaccharides or hetero polysaccharide and occur in algae (alginate), plants (pectin, guar
gum, fiber), microorganisms (dextran, xanthan gum), and animals (chitosan, chondroitin)
(Shahid et al., 2013).
Fibers are the type of carbohydrates derived from cell walls of plant. The dietary
fibers are non-digestible carbohydrates whereas; lignins are not degraded in the upper gut
and generally categorized into three groups: insoluble fibers (cellulose), soluble fibers
(pectin; gums), mixed type fibers (bran) that are not hydrolyzed by human gastro-intestinal
enzymes. It is, therefore, considered as the best food for bacteria in lower Gastro-Intestinal
Tract (GIT). Soluble fiber is an imperative source for bacterial fermentation and is
considered as the only food ingredient for which persuasive substantiation in support of a
prebiotic impact has been testified (Bengmark, 2001; Bouhnik et al., 2004; Weickert and
Pfeiffer, 2008). By definition prebiotics are non-digestible food component having beneficial
effect on host organism through exciting activity and growth of microorganisms (bacteria) in
lower part of GIT (Waitzberg et al., 2012).
Partially hydrolyzed guar gum (PHGG), xanthan gum, xylo-oligosaccharides, inulin,
lactulose, glucomannan and pectin are the prebiotic compounds which have been widely used
in industries. The reduction in cholesterol and glucose levels by guar gum aids in controlling
the weight loss / obesity. An improved satiation is achieved by gel forming capability of guar
gum that result in slow emptying of stomach. Diets supplemented with guar gum decrease
desire for eating, hunger and appetite (Mudgil et al., 2011).
Crude guar gum has beneficial physiologic effects; however its incorporation in
enteral solutions and food products is difficult because of its highly viscous interference with
digestion and absorption of nutrients. Furthermore, due to high molecular weight, it is not
easily available to the beneficial bacteria as food. Therefore, moderate hydrolysis processing
techniques have been developed by several workers to selectively reduce molecular weight
2
resulting in altered flow attributes in solution and enhanced prebiotics effect, without
disturbing the chemical nature of the gum (Cheng et al., 2002; Yoon et al., 2006). Partially
hydrolyzed guar gum (PHGG) can be achieved through acid and enzyme hydrolysis,
irradiation, microwave and ultra-sonication techniques (Singh and Tiwari, 2009).
PHGG is a natural dietary fiber having excellent water-solubility. It is stable and
soluble at various pH levels, heat, pressure and temperature exhibiting same physiological
functions as native guar gum (Yoon et al., 2008). In colon and cecum, fatty acids with short
chain length are formed by the fermentation of soluble dietary fibers, such as PHGG results
in decreased pH in gut that may enhance absorption of nutrients (Scholz-Ahrens et al., 2001).
PHGG has been observed to have a prebiotic effect when taken with fructo-oligosaccharides
(Tuohy et al., 2001) and also enhances the growth (population) of Bifidobacterium in human
gut (Slavin and Greenberg, 2003).
The thought of prebiotics is relatively new in the present era and has been well-
known because of its non-digestible behaviour as food component (so called) that help to
enhance and modify selectively the viability of bacteria, known to have progressive effect on
physiology of gut. Prebiotics (guar gum) are sources of energy for the micro flora and can
help to enhance lipid metabolism (Tomasik and Tomasik, 2003).
Guar gum is comprised of mannose and galactose units and it’s better soluble in cold
water. The composition of guar gum includes moisture contents 9.55%, fat 0.78%, crude
protein 2.16%, ash 0.54%, soluble fiber 75% and insoluble fibers 7.6% (Frias and Sgarbieri,
1998). Total dietary fiber present in guar gum in soluble form is 80-85% that may assist in
lowering cholesterol and glucose levels (Pszczola, 2003).
Guar gum is soluble, non-digestible polysaccharides and is main ingredient in the
formation of functional foods that can positively influence several desired functions suitable
for body growth (Senok et al., 2005) with efficient behaviour of possible reduction in
cardiovascular disease and plasma cholesterol level. This is an edible, viscous fiber having
possible effects on lowering cholesterol and blood sugar. Guar gum is widely used in food
industry (EU food additive code E412) as a thickener to increase the viscosity and to bind
available free water in ice cream, sauces and dressings, meat sausage, instant noodles, bread
improvers, beverages and even in pet foods (Butt et al., 2007).
3
The current customer’s information about the beneficial bacteria are guided in the
direction of inventive fermented milk products, and better nutrition leading to enriched
yogurt and other healthy worldwide demand for approval of fermented milk products. Yogurt
as food is praised for its therapeutic and beneficial role (Sarkar and Misra, 2002a).
Probiotics (living microbial dietary supplements) play a vital role in fermented dairy
products for their therapeutic effects and functional behaviour by providing positive effects
on the host health and improving microflora balance of the intestine (Aryana and McGrew,
2007). Bifidobacterium and Lactobacillus species are well-known used probiotics,
predominantly in fermented dairy products (Cardarelli et al., 2008).
It is suggested by various reporters that probiotics bestow a number of benefits to
human’s health. They have been shown in different levels to generate antimicrobial
compound, inhibit Helicobacter pylori, adjust host immune system, prevent autoimmunity,
assimilate cholesterol, alleviate lactose intolerance, exhibits antimutagenic properties,
prevention of respiratory tract disorders, hypo-cholesterolaemic influence, stool
normalization and consistency in case of constipation (Shah, 2007; Elizabeth et al., 2011).
Bifidobacteria are supposed to yield vitamin B, blood cholesterol levels, lessen blood
ammonia, hinder pathogens growth and help to boost up the immune system (Swennen et al.,
2006).
The addition of probiotics fruitfully in the food commodities must tolerate the food
processing and storage practices (e.g., fermented food products). Lactic acid bacteria (LAB)
have a great potential in the development of aroma and texture improvement in addition to
microbial safety (Grattepanche et al., 2007).
As people are gaining more awareness related to nutrition of food for promotion of
good health and wellbeing, functional foods (probiotic products) have become imperative to
the consumers (Cardarelli et al., 2008). Because functional foods beneficially affect target
functions in the body to an improved state of health, dairy products containing synbiotics
(probiotics and prebiotics containing food) attain a high popularity in this category of foods
(Tomasik and Tomasik, 2003; Allgeyer et al., 2010).
Probiotic and prebiotic yogurt are amongst all the mounting food sub-divisions during
the period of 2009 (CDC, 2009). Experiments have provided evidences that synbiotics
4
perform better than either probiotics or prebiotics alone in affecting the blood lipid profile
and protecting from colorectal cancer (Tuohy et al., 2003) but require further investigations.
Yogurt is considered as the supreme and famous food standard for the carriage of
beneficial probiotic microorganisms (Adolfsson et al., 2004). The microbes that are generally
used as probiotics are species of Bifidobacteria and the species of lactic acid bacteria (LAB)
in several foods but some varieties of yeasts and Bacilli are also helpful in maintaining life of
the organisms. Yogurt contains approximately at least 106 live cells of Lactobacillus and 10
9
for Bifidobacterium (FAO/WHO, 2003).
Yogurt is a complex colloidal system that is produced by extended weak framework
of protein entrapping the water molecules (Lucey, 2001). The word “yogurt” is derived from
“jugurt” which is a Turkish word. According to FAO and WHO (2001) yogurt is a viscous
dairy food that is obtained in the process of milk fermentation by the action of Streptococcus
thermophiles and Lactobacillus bulgaricus (Aswal et al., 2012).
Like many other fermented food products, yogurt has been part of the human diet
since human domesticated mammals and used milk for food (Chandan, 2006). The first
yogurt was likely fermented spontaneously by adventitious bacteria in the milk or in storage
containers used to transport milk (Karagul et al., 2004). It is estimated that there are more
than 400 different types of dairy products around the world, and yogurt is among the most
popular of the fermented dairy products. It is an integral diet constituent for the people living
all around the world and is used in many different ways. It can be consumed as such or as an
ingredient in other foods. A nutritious food product with recognized health benefits, yogurt
has a distinct acidic taste and flavor that makes it popular around the world.
Yogurt is fermented milk invention and used by huge sectors of our community either
as share of diet or a stimulating beverage. It is a nutritiously balanced food material
comprising nearly all nutrients present in milk but in a more available form. It is developed
by milk fermentation by the action of starter culture bacteria enclosing Lactobacillus
bulgaricus and Streptococcus thermophilus (Adolfsson et al., 2004).
The demand for fermented milks and yogurt has astonishingly extended in
consequence of new techniques e.g. minimally processed treatments and the addition of
functional constituents such as dietary fibre, probiotics (beneficial bacteria for human gut),
omega fatty acids etc., owing to their enhanced nutritional value, distinctive biological and
5
physiological effects (Tsevdou and Taoukis, 2008). The indigenous yogurts are mainly
manufactured in Middle East and in other several countries around the eastern part of the
Mediterranean. They are known as Mast (Iran), Leban (Iraq), Dahi (Pakistan: Traditionally
prepared through slope back procedure) and Libanan (Egypt). Yogurt is also popular because
it has assumed different forms e.g. stirred, set and frozen yogurt. Of all the varieties of
yogurts, fruit stirred yogurt is more familiar due to its nutritive digestibility, therapeutic
against immune related diseases as compare to milk from which it is prepared (Meydani and
Woel, 2000).
In addition to the health promoting effects of yogurt due to its nutritional composition
and existence of probiotic bacteria (EFSA, 2010), the technical features like physical
properties including appearance, taste, aroma and texture are imperative for customer
adequacy. PHGG has possible beneficial effects on the sensory, textural and rheological
properties of yogurt. It reduces syneresis, improves yield, firmness and overall sensory
characteristics (Brennan and Tudorica, 2008). The probiotics and prebiotics effects on human
health impart an impression on the yogurt market very strongly and proper addition of
probiotics and prebiotics can increase preference of consumer for the products (Allgeyer et
al., 2010).
Hypothesis and Importance of Research:
Carbohydrate based prebiotics are good source for probiotics survival in yogurt.
Carbohydrate based prebiotics in various forms is treated to formulate yogurt for the best
suited to the probiotics (LAB, Bifidobacteria) in order to get their best, maximum rationale
and valuable combination for health benefits which is lacking at the moment in time.
Prebiotics are being used for several treatments including constipation and hepatic
encephalopathy treatment. Therefore, in reality, the importance of this research is to provide
a food with prolonged survival of good bacteria to the consumers for better immunity,
improvement of functions of intestine and maintenance of the integrity of intestinal lining,
and to get the beneficial effects of lactic acid bacteria while rationalizing with food for them
(prebiotics modified form) which is need of the day and replaces medicines in several cases.
Keeping in view the significance of carbohydrate based prebiotics (PHGG) in yogurt,
with impact on probiotic growth and to provide as a natural curative measure in human, the
current project was planned to explicit the following objectives:
6
To compare the guar gum fractions hydrolysed with various methods
Microstructural characterization of guar galactomannans and its derivatives
To study the effect on sensory, physico-chemical and microbiological characters of
yogurt associated with prebiotic and probiotic mechanism (synbiotic)
To evaluate the effect of carbohydrate based prebiotics on probiotic behaviour in
yogurt so developed
7
Chapter 2
R E V I E W OF L I TE R A T U RE
Guar gum is a dietary fiber soluble in water obtained from leguminous plant
Cyamopsis tetragonolobus, has been cultivated in Indo-Pak for centuries. Extensive research
work is going on to explore its dietary and physiological roles. It is therefore guar gum under
the study hydrolyzed by appropriate methods and was utilized in synbiotic yogurt to evaluate
longevity of probiotics. Review of literature as a foundation of current research eliminates
the chances of unnecessary work done in this field of study. A lot of work has been done by
various scientists in this regard; hence the literature has been reviewed under the following
sub-headings:
2.1. Oligosaccharides (OS)
2.1.1. Introduction
2.1.2. Types of Oligosaccharides
2.1.3. Applications of Oligosaccharides
2.2. Guar Galactomannans
2.2.1. Concept and History
2.2.2. Structural Composition
2.2.3. Applications of Guar Galactomannans
2.2.3.1. Food Claims
2.2.3.2. Pharmaceutical Claims
2.2.3.3. Other Applications
2.2.4. Health benefits of Guar Galactomannans
2.2.4.1. Gastrointestinal effects
2.2.4.2. Other health benefits
2.3. Hydrolysis of guar gum
2.4. Prebiotics Effect of Guar Gum
2.5. Guar gum: Characterization
2.5.1. Proximate Composition
2.5.2. Rheological properties
2.5.3. Galactose-Mannose Ratio
8
2.5.4. Microstructural analysis
2.5.5. Toxicological Study
2.6. Probiotics
2.6.1. Probiotic selection
2.6.2. Health benefits of probiotics
2.7. Synbiotic Food
2.7.1. Yogurt History
2.7.2. Yogurt as a Synbiotic Food
2.7.3. Nutritional and Therapeutic Aspects of Yogurt
2.7.4. Yoghurt composition
2.7.4.1. Milk Proteins
2.7.4.2. Vitamin B
2.7.4.3. Lactose
2.7.4.4. Fats or Lipids
2.7.4.5. Minerals
2.7.5. Chemistry of Yogurt (Whey-casein interactions)
2.7.6. Rheological Properties of Yogurt
2.7.7. Physico-Chemical and Sensory Attributes of Yogurt
2.1. OLIGOSACCHARIDES (OS)
2.1.1. Introduction
Oligosaccharides (OS) are essential part of polymeric carbohydrates that are existed
either in combined or free forms in all living organisms. According to IUB-IUPAC
nomenclature, oligosaccharides may be defined as oligomers which are comprised of 2-10
monosaccharide units structurally connected through glycosidic linkages and readily
hydrolyzed to yield monosaccharides constituents either through specific enzyme or acids
(Nakakuki, 1993). Oligosaccharides are naturally present in fruits, vegetables, milk and
honey. Most of the oligosaccharides impart mild sweet taste and the mouth-feel to food that
has created interest for food industry to use oligosaccharides as partial substitute for sugars
and fats in food products. Moreover, oligosaccharides can be used as functional food
ingredients with good prospective to enhance various food‟s quality. Several investigations
9
have been reported to evaluate the physiological functionalities of this compound (Nakakuki,
2002).
2.1.2. Types of Oligosaccharides
Extensive research on production of oligosaccharides for its usage in food industry
has been started. Industrial production of oligosaccharides has been started at a larger scale
with the advancement in the field of enzymology and assessment of their functional attributes
all over the world. Many lactose, starch and sucrose-related oligosaccharides have been
established after which xylo-oligosaccharides, chitin/chitosan-oligosaccharides, manno-
oligosaccharides and agaro-oligosaccharides have been produced from various
polysaccharides such as xylan, chitin and chitosan, mannan and agar as the raw materials
(Murphy, 2001).
On the basis of physiological properties, the oligosaccharides are categorized into
digestible or non-digestible. The basic idea of non-digestible oligosaccharides (NDOs) in
their study was to configure monosaccharide units of certain dietary oligosaccharides on
anomeric C atom (C1 or C2) is such a way that makes osidic linkages non-digestible against
the hydrolytic activity of digestive enzymes of human beings. The major classes of NDOs
presently exist in the process of food development ingredients comprising carbohydrates that
include monosaccharide units of galactose, fructose, xylose and /or glucose (Roberfroid and
Slavin, 2000).
2.1.3. Applications of Oligosaccharides
In the early 1990s, Government of Japan legislated foods for specified health use
(FOSHU) by include more than 200 food items incorporating oligosaccharides as the
functional ingredient (Kalra, 2003). NDOs are fermentable substances which have significant
effect on the large intestinal flora to contribute to the human health the prebiotics (Mussatto
and Mancilha, 2007).
The oligosaccaharides (prebiotic) as a result yields formation of vitamins, short chain
fatty acids (SCFA), show antimicrobial effect and enhance immune system when utilized by
probiotics (saccharolytic microflora in stomach) e.g., bifidobacteria (Perrin et al., 2002).
Such types of prebiotics are also used to enhance the colonization and longevity of probiotics
in gut when incorporated food comodities (Ziemmer and Gibson, 1998). Since few decades,
10
several NDOs have been launched as functional food ingredients; therefore there is a
continuous increase in their industrial applications.
Among the main uses of OS, center of attention is in soft beverages including health
drinks, tea, fruit drinks, coffee, soda, cocoa and alcoholic beverages and milk products e.g.
ice cream, powdered milk, instant powders, fermented milks and probiotic yogurts containing
microorganisms that employ beneficial influences on host organism. The effects are imparted
due to enhancement of microbial population in intestine.
The products comprising of prebiotics and probiotics exhibit symbiotic influence on
the host organism by enhancing endurance and implanting active microbial dietary
supplements in human gastrointestinal tract (Gibson and Roberfroid, 1995). It has been
determined that certain oligosaccharides without being utilized, exert beneficial effects on
human health potentially by increasing bifidobacteria in the colon (Akalin et al., 2004).
Moreover, currently NDOs are being used in the food industry as an ingredient in the in
desserts comprising puddings, jellies, syrups, confectionary products (sweets, candy,
chocolate, cookies, biscuits), table spreads (jams, marmalades), pastries, breads, breakfast
cereals, and meat products (fish paste, tofu) (Voragen, 1998).
Physico-chemical and physiological characteristics of the products containing NDOs,
vary with kind of mixture prepared, the selection of most suitable oligosaccharide for a
specific food preparation fluctuates as well. Galacto-oligosaccharide, for instance, is a
suitable OS for incorporating in bread because of the reason that it is not broken down during
yeast fermentation and baking consequently imparting good taste and texture to the product.
Industrial and pharmaceutical uses of oligosaccharides include feed, drug delivery agent,
mouth washes and cosmetics products (Crittenden and Playne, 1996). Lactulose is being used
mostly in the production of pharmaceutical products that facilitates in preventing
portosystemic encephalopathy and constipation (Villamiel et al., 2002).
A number of oligosaccharides have been studied by various workers through in-vitro
methods, animal models and clinical trials on humans. The most useful ones reported are the
fructo-oligosaccharides (FOS), lactulose, galactooligosaccharides, xylo-oligoaccharides and
inulin. These OS are being produced and added into various products in the food industry at
large scale. Research trials has also been made in minor proportion about newly developed
11
OS like soybean-oligosaccharides, lacto-sucrose, isomalto-oligosaccharides, gluco-
oligosaccharides and xylooligosaccharides and found all these promoting the increase of
intestinal microflora. Xylo-oligosaccharides have shown intestinal improvment,
hypolipidemic activities and antimicrobial activity against some bacteria during studies
conducted by Christakopoulos et al. (2003).
2.2. Guar Galactomannans
Galactomannan is the neutral polysaccharides, comprised of a linear backbone of
mannose units having a single galactose unit on side chains that is acquired from the
endosperm of leguminous plant seeds (Azero and Andrade, 2002). Locust bean gum (LBG,
Ceratonia siliqua, M/G ratio: 3.5:1), tara gum (TG, Caesalpinia spinosa, M/G ratio: 3:1) and
guar gum (GG, Cyamopsis tetragonolobo, M/G ratio: 2:1) are considered as the major three
galactomannans of commercial importance in food industries (Gidley and Reid, 2006; Dakia
et al., 2008). Guar gum (guaran) is a polysaccharide and primarily the ground endosperm of
guar beans, comprised of mannose and galactose sub-units of sugars. Guar gum is obtained
by passing the guar seeds through the different processes of dehusking, milling and
screening. The typical characteristics of guar gum powder includes: coarse to fine ground,
off-white colored, pale and free flowing powder. Guar gum is highly viscous, soluble dietary
fiber with better emulsifying, thickening and stabilizing properties that can prevent the
growth of ice crystals in frozen foods (Chaplin, 2009).
Fig 2.1 Processing of guar gum; (a) guar pod (b) guar seed (c) dehusked splits (d) guar gum
powder
(a) (b) (c) (d)
12
2.2.1. Concept and History
Initially guar plant C. tetragonoloba was domesticated from a wild species C. senegalensis
found in Africa, which was taken to south Asian subcontinent by Arabs between 9th
and 13th
centuries A.D. The native varieties are usually related to Pakistan and India, where the guar
gum plant has been under cultivation for centuries while in USA, industry of guar gum
flourished in 1940s and 1950s (Whistler and Hymowitz, 1979; BeMiller, 2009; Mudgil et al.,
2011)
2.2.2. Structural Composition
The guar kernel is comprised of numerous layers, namely the outer husk (16-18%),
the germ (43-46%) and the endosperm (34-40%). The endosperm part of guar seed is mainly
galactomannan and the germ portion mainly protein. Guar gum predominately contains high
molecular weight (50,000 to 8,000,000 Da) polysaccharides which is linear chain of (1→4)-
linked β-D-mannopyranosyl units with (1→6)-linked α-D galactopyranosyl residues as
side/branched chains (Kawamura, 2008). The seed endosperm is being constituted by
mannose and galactose groups of galactomannan portion (Whistler and Hymowitz 1979).
The galactomannan contains mannose and galactose units in the ratio of 2:1 (Garti and Leser,
2001) which is supported by various other researchers (Mathur and Mathur, 2005) as in the
range of 1.6:1 to 1.8:1. The higher branching of guar gum is believed to be responsible for its
easier hydration features in addition to its better activity of hydrogen bonding. Similarly, it is
described that aggregates are apparent in aqueous solution of guar gum and may have
distinctive behavior in viscoelastic features of solution depending on how they are interlinked
(Gittings et al., 2000). The polysaccharide (guar gum) is also considered as one of the
naturally existing water soluble polymer. The variations in the viscosity of commercial grade
guar gum exist immensely depending on the molecular weight of galactomannans. Early
workers are also in accordance of the similar view about this character/property and that vary
extremely depending on method used. The various absolute procedures have also been
adopted to determine molecular weight, including light scattering techniques that are very
helpful in giving structural information about the polysaccharide (Ross-Murphy et al. 1998).
The reliable and relatively simple method to estimate molecular weight is to measure the
intrinsic viscosity, or the Mark-Houwink equation. On the other hand, latest results are
13
acquired through low angle laser light scattering and size exclusion chromatography that
presented the average molecular weight in the range of 106 to 2 × 10
6 (Mudgil et al., 2011).
Fig 2.2 Chemical structure of guar galactomannan
2.2.3. Applications of Guar Galactomannans
2.2.3.1. Food Claims
Guar gum is utilized as a fiber source and an innovative food additive to enhance the
stabilization of various products in food processing industry (Morris, 2010). Guar gum has
been very beneficial in various products as food additive for its structural, textural,
rheological and other advantages for making food more acceptable to the consumers. Guar
gum is soluble in cold water that makes it possible utilization in beverage processing
industry. It aids to increase the shelf life stability of beverages whereas, in cheese formation,
prevents the syneresis phenomena by managing the water phase and ultimately enhances the
body and texture of the finished product. It also improves the consistency of tomato ketchup
more conspicuously than any other hydrocolloids like sodium alginate, carboxy methyl
cellulose (CMC), pectin and gum acacia. Adding guar gum causes decreased flow values and
serum loss of tomato ketchup that makes it a unique thickening agent for tomato ketchup
(Gujral et al., 2002). Guar gum also enhances the chewiness, body and texture, and heat
shock resistance in ice cream. Addition of PHGG at a level of 2-6% concentration lowers
syneresis and develops the rheological and textural features of low fat yoghurt as good as
with full-fat yoghurt (Brennan and Tudorica, 2008).
Guar gum along with xanthan gum delay staling process in gluten-free rice cakes by
reducing the weight loss and retrogradation enthalpy (Sumnu et al., 2010). Likewise, guar
gum also hinders staling in chapati (flat bread) at refrigerated and room temperature through
by regulating the process of retro-gradation of starch (Shaikh et al., 2008). Guar gum is
14
efficiently utilized as a lubricant and binder in the production of stuffed meat and sausage
products due to high water holding capacity in cold and hot water. It executes particular
functionalities in processed meat products like control of syneresis and viscosity of liquid
phase during processing and control of fat migration and accumulation of the water in the can
during storage. Guar gum also develops the creaming stability and regulates rheological traits
of egg yolk emulsion (Ercelebi and Ibanoglu, 2010).
2.2.3.2. Pharmaceutical Claims
Guar gum and its derivatives forms are safe, stable and biodegradable. Therefore,
they are extensively utilized as potential target-specific drug delivery carriers due to its
advantageous features. GG can be used as a colon specific drug carrier in the form of matrix
and compression-coated tablets. Subsequently GG and its derivatized forms have excellent
film forming and controlled drug release capabilities, they have potential to be used as
transdermal drug delivery devices (Prabaharan, 2011) and to control the drug release in the
gastrointestinal tract, as a carrier for colon targeted drugs (Chourasia, and Jain, 2004), for
oral rehydration solutions in the treatment of cholera in adults (Alam et al., 2008) and for
anticancer drugs in the treatment of colorectal cancer (Chaurasia et al., 2006). The gum is
also used in transdermal drug delivery systems (Murthy et al., 2004) as synthetic cervical
mucus and as a visco-supplementation agent in osteoarthritis treatment (Cunha et al., 2005)
2.2.3.3. Other Applications
During last few decades, the formation of various guar gum derivatives resulted in
increased demand of guar gum to be used. Guar gum is presently used in gas and oil
particularly in hydraulic fracturing where high pressure is used to crack rock. Guar gum
increases the viscosity of fracturing fluid so that it can carry sand into fractured rock. Guar
derivatives for use in fracturing fluids are carboxymethyl hydroxypropyl guar (CMHPG) and
hydroxypropyl guar (HPG).
Guar gum is extensively used to thicken dye solutions in textile and carpet printing
that allows more sharp printing patterns. Adding little quantity of guar gum to wood pulp
enhance the production of paper. It also offers thicker surface to the paper utilized in printing
(Mudgil et al., 2011). Studies had also declared that guar gum is a valuable paper maker's
15
adjunct in attaining temporary wet strength in sheets, for instance paper toweling and this
gum assists hydration throughout the beating of different pulps.
2.2.4. Health Benefits of Guar Galactomannans
2.2.4.1. Gastrointestinal Effects
Microbial population of human gut plays a vital role in maintaining health of an
individual by improving GIT health through systemic absorption of metabolites. When guar
gum was ingested with fructo-oligisaccharides in the form of biscuits, the prebiotic effect
was experienced (Tuohy et al, 2001) that assist in maintaining good health. The interest has
been increasing in the human colonic micro biota and in the way its metabolic activities
influence the host health and well-being. It acts as a bulk forming laxative in GIT system. It
is claimed to be efficient in endorsing regular bowel movements and give relieve in
constipation and chronic associated functional bowel ailments for example irritable bowel
syndrome, colitis, diverticulosis and Crohn's disease as studied by Kandeepan and Sangma
(2010).
PHGG was also evaluated as curative agent against irritable bowel syndrome by
Giaccari et al. (2001). The investigators observed reduction in frequency of irritable bowel
symptoms for instance abdominal spasms and tension, and flatulence. They declared that
PHGG is simple to use due to its non-gelling properties and works well when altering
intestinal motility. Guar-gum enriched enteral nutrition reduces diarrheal episodes in the
patients with pre-existing diarrhea and maintaining a healthy microflora (Rushdi et al.,
2004).
Several workers indicated that PHGG can be beneficial for curing the constipation by
the fact that it lowered the laxative dependence in a nursing home population when adding to
the enteral nutrition diet. It also controlled the pervasiveness of diarrhea in septic patients
which could be due to the formation of SCFA related with the soluble fiber (Patrick et al.
1998; Spapen et al, 2001; Slavin and Greenberg 2003).
2.2.4.2. Other Health Benefits
Nandini et al. (2003) assessed the effects of wheat bran and guar gum against diabetic
rats. And guar gum was found to be more effective in controlling the glycemic index than
16
wheat bran but both of them were efficient in regulating renal enlargement, the symptoms of
which appears during initial stages of diabetes.
Chow et al. (2007) studied that nutrition bars containing guar gum, encouraged better
postprandial satiety and can be beneficial to manage the weight of people with type II
diabetes. This satiety-promoting influence of bar could be due to the existence of sugar
alcohols and fermentable carbohydrates through the production of fermentation products in
the colon.
Guar gum has been also used to control hypercholesterolemia and diabetes with
decreased risk for coronary heart disease (CHD). Numerous mechanisms have been proposed
to illuminate the beneficial effects of viscous gums/fibers: An increased excretion of bile
acids; the reduction of lipid emulsification, gastric and lipase activity; a reduction of the
hepatic synthesis, an impairment of cholesterol absorption (Favier et al., 1997a,b).
The ingesting of guar gum revealed a lessening in postprandial glucose and insulin
levels in various clinical studies, wherein the mechanism is the delaying of meal in the
stomach and small intestine due to high viscosity, preventing glucose access to the
epithelium. They also reported that gel forming properties of guar gum caused lowering
glucose and cholesterol effects and better satiation is attributed to the slow gastric emptying.
Its consumption also lowers the risk of heart diseases and helps in controlling obesity and
weight loss. Dietary supplements containing guar gum has reduced the hunger, appetite, and
desire to eat (Cameron-Smith et al., 1997; Butt et al., 2007).
In earlier studies, guar gum was found to have ability to bind toxins and remove them
out of the body, and significantly reduce the amount of blood sugar, triglycerides, cholesterol
and lipids in normal and diabetic rodents (Frias and Sgarbier, 1998). Ingestion of guar gum in
suitable amount as dietary fiber relieves in control of diabetes and digestive problems,
enhancement of mineral absorption and significant reductions in LDL-cholesterol (Yoon et
al., 2008). The mechanism of cholesterol reduction through guar gum is attributed to the
higher production of bile acids from cholesterol and hepatic free cholesterol concentration is
depressed (Rideout et al., 2008).
17
2.3. Hydrolysis of Guar Gum
PHGG is generally consumed as water soluble dietary fiber since its ingestion
displays physiological effects including, lowering pH of feces and lowering glucose
concentration, free fatty acids and serum cholesterol in humans (Miyazawa and Funazukuri,
2006). For this purpose, guar gum needs to be hydrolyzed in a controlled environment to
shorten its chain length and ultimately reduction in molecular weight fractions. PHGG can be
produced by various techniques such as acid hydrolysis, enzyme hydrolysis, microwave,
irradiation, and ultra-sonication methods (Singh and Tiwari, 2009).
PHGG produced in enzyme hydrolysis was found to have the same chemical structure
as the crude guar gum, but with apparent reduction in molecular weight of around 20,000 Da.
The crude guar gum aqueous solution (1%) was highly viscous having viscosity of 2000 to
3000 mPa.s while the modifications reduced the chain length of PHGG and its aqueous
solution with 5% concentration exhibited viscosity less than 10 mPa.s. The commercial brand
of PHGG modified through enzyme treatment is “Sunfibre” exhibiting similar physiological
functions as compared to the crude guar gum. The enzymatic treatment reflected the process
of digestion and the product formed termed as pre-digested guar gum (Yoon et al., 2008).
PHGG produced through enzyme like endo-β-D-mannanase was preferred to use for
food applications (Yoon et al., 2008). PHGG produced through endo-β-mannanase do not
have only less viscosity but also have numerous health assistances with better conditions of
constipation and improved intestinal microflora equilibrium.
2.4. Prebiotics Effect of Guar Gum
Microbial population of human gut plays a vital role in maintaining health of an
individual by improving GIT health and through systemic absorption of metabolites. It is
generally believed that certain bacterial species among gastrointestinal flora advantageously
affect the health such as Bifidobacterium and Lactobacillus spp. These bacteria have been the
focus of attention as increased population of them is an indication of health microbes. And
PHGG was found to enhance the population of Bifidobacterium in the gut (Slavin and
Greenberg, 2003).
PHGG has been used as a prebiotic in biscuits and the exact prebiotic effect of PHGG
in human volunteers was studied in comparison to the fructooligisaccharides (FOS). PHGG
18
and FOS were added in biscuits mainly to obtain increased counts of Bifidobacterium spp.
and Lactobacillus spp. The study was conducted on 31 human volunteers selected on the
basis of selected diet (daily consumption of 3 biscuits providing a total of 7g/day FOS and 3
g/day of PHGG). It was observed that after 7 days, the effect of PHGG and FOS in human
volunteers led to increase in the counts of Bifidobacteria. Thus, maintenance of FOS and
PHGG in final food product was suggested (Tuohy et al., 2001).
A lot of food commodities with rich amount of dietary fibers include fruits,
vegetables, legumes and cereal grains. Dietary fibers exhibit different extent of solubility,
some are easily soluble in water e.g. hemicellulose, pectin, inulin and guar gum helping in
gel formation in the gastrointestinal tract. This leads to their fermentability by the gut
microflora while increasing surface area for enzymatic attack thus imparting prebiotic effects
(Woods and Gorbach, 2001).
2.5. Guar Gum: Characterization
2.5.1. Proximate Composition
The final milled endosperm separated from germ is commercial gum which contains
about 10-15% of moisture, 0.5-0.8% of ash, 5-6% of protein and 2.5% of crude fiber (Cunha
et al., 2007). The chemical compositions of six genotypes of guar gum ranged in: 83.3-87.5
% carbohydrates, 0.5-0.9% fat, 3.5-5.5% protein, 4.8-8.7% moisture, 0.5-1.3% ash and 1.4-
2.0% fiber. The total available carbohydrates as galactose and mannose were ranged 28-33%
and 67-73% respectively. The mannose to galactose ratio is 2:1 (Sabahelkheir et al., 2012).
2.5.2. Rheological properties
Rheology is the study of the flow and deformation behavior of materials. Newtonian
fluids have a viscosity that is dependent of the stress rate applied. On dissolving, the
galactomannans lose their ordered structure and go into disordered solution i.e., “random
coils” (Sittikijyothin et al., 2005)
The unique benefit of galactomannans is their tendency to produce highly viscous
solutions even at low levels that are slightly influenced by heat processing, pH and ionic
strength (Sittikijyothin et al., 2010). By increasing shear rate and shear stress, distraction
prevails over establishment of new entanglements; molecules align in the direction of flow
and ultimately viscosity drops (Dakia et al., 2008).
19
In dynamic measurements, frequency sweeps of loss moduli (Gʺ) and storage (Gʹ)
served as a tool to discriminate between polymer gels and solutions. Aqueous solutions of
guar gum showed the typical shape for disordered random-coil polysaccharides. The
macromolecular solutions exhibited Gʺ>Gʹ at low frequencies whereas this behavior was
revered at higher frequencies i.e. Gʹ was predominant (Bourbon et al., 2010).
2.5.3. Galactose-Mannose Ratio
Basically galactomannans are comprised of mannose units as backbone or main
chain, with galactose units or side chains. So, with respect to guar gum, there is one galactose
side chain for every two mannose units; hence mannose to galactose ratio is 2:1. (Butt et al.,
2007)
Fig 2.3 The repeating unit of guar gum showing mannose galactose ratio
2.5.4. Microstructural analysis:
Microstructural analysis including SEM, TGA, XRD and FTIR is used to characterize
various biopolymers at microscopic level. SEM can be performed to give information about
the surface morphology. It is powerful technique extensively performed to analyze the
„network‟ characteristic structure in polymers (El Fray et al., 2007). SEM is performed to
explore the granular morphology of original and depolymerized guar gum. It was obvious
that after hydrolysis process, the granular morphology of original gum is lost and converted
into fibrillar morphology (Wang et al., 2006).
TGA is used to evaluate heat stability of polymers. It was revealed that hydrolyzed
guar gum was more heat stable as compare to the native one (Yoon et al., 2008). X-ray
diffraction (XRD) configuration of GG and PHGG indicated amorphous structure showing
low overall crystallinity at angle (2Ɵ) 20.2 and 72.5 respectively. PHGG hydrolyzed through
enzyme declared negligible change in XRD curve but increased the crystallinity index
20
slightly which may be due to short chain length and lower degree of polymerization of
molecule (Mudgil et al., 2012).
Infrared (IR) and Raman spectroscopy are imperative techniques that offer additional
information about molecular structure. By uniting microscopy and spectroscopy, molecular
information can be attained with great spatial resolution at the microscopic level. A number
of functional groups and organic compounds can be recognized by their distinctive pattern of
absorption (Wetzel and LeVine, 1999).
The peak in the spectral region around 3300 cm−1
was due to O-H stretching vibration
of polymer and water involved in hydrogen bonding. The spectra between 2800 and 3000
cm−1
shows C-H stretching modes. Interrelated water molecule gave rise to the band near
1650 cm−1
in the spectra. In PHGG, sharpening of absorption band around 1648 cm−1
shows
its better association with water molecule, which could be a confirmation of its increased
solubility as compared to crude guar gum. The region around 1400 cm−1
due to CH2
deformation was also observed. The peaks observed in the spectra between 800 and 1200
cm−1
represented the highly coupled C-C-O, C-OH and C-O-C stretching modes of polymer
backbone. It is assumed that crystallinity of polymer is signified by the spectral region
between 700 and 500 cm−1
and conformational changes are attributed to any modification in
spectra of this region. Hence this technique has been used to study the functional group
transformation during hydrolysis (Mudgil et al., 2012).
2.5.5. Heamolysis Study
Toxicological evaluations of PHGG has exposed that up-to dose level of 2500
mg/day, it is not mutagenic (Takahashi et al., 1994). In human clinical study non-insulin
dependent diabetic patients were examined to evaluate the effects of feeding 30g of guar gum
in daily dose dependent manner for a period of 16 weeks. No changes were observed in
hepatic, haematologic, or renal functions. Serologic screening also did not divulge any
changes in protein, mineral or lipid metabolism (McIvor et al., 1985).
The outcome of a 90-day study in rats supported the safety of PHGG prepared by
alkaline oxidation. No adverse effects were revealed up to the dose level of 50 g/kg diet
estimated to amount to 2500 mg/kg body weight per day (EFSA, 2004). European Food
Safety Authority (EFSA) has documented the safety of partially depolymerized (heat
21
treatment, acid hydrolysis or alkaline oxidation) guar gum as a stabilizer, emulsifier and
thickener in food (EFSA, 2007).
2.6. Probiotics
The literature regarding the introduction of probiotics indicated the origin since 1965
when the term “probiotics” was introduced for the first time by Lilly and Stillwell. Probiotics
are the live microorganisms that confer a beneficial health effects on the host organism when
administered in suitable extent. These are microbial derived factors that accelerate the growth
of other organisms. These are selected from the strains most beneficial for intestinal health
i.e. bacteria from the genera Bifidobacterium, Lactobacillus and yeast. Mostly Lactobacillus
and Bifidobacterium species are utilized as probiotics, but the species, Saccharomyces
cerevisiae and Bacillus are also known as a probiotics (Tomasik and Tomasik, 2003).
Bifidobacteria are important part of intestinal microflora of human and play vital role
in maintaining health (Akalin et al., 2004). The potential health benefits attributed to
bifidobacteria include calcium absorption and vitamin synthesis, stimulation of the immune
response, inhibition of bacterial pathogens, improvement of lactose tolerance and reduction
of serum cholesterol levels and colon cancer risks (Orrahg and Nord, 2000).
The viable numbers of bifidobacteria >106 cells per gram of yogurt has been
recommended to provide optimum therapeutic benefits to the host. Viewing the importance
of viability of bifidobacteria, Shin et al. (2000a) conducted surveys on poor viability of in
yoghurt preparations. Several factors have been proposed to ensure the viability of
bifidobacteria in yoghurt, including pH, acidity, oxygen content, hydrogen peroxide, storage
temperature and amount of acetic and lactic acids, etc. during manufacture and storage of
yoghurt (Akalin et al., 2004). Food processors are of great concern to deliver the products to
the consumer containing viable number of bifidobacteria until its use.
Besides desired health functionalities of these microorganisms, probiotics should
fulfill several basic requirements for the establishment of marketable probiotic products. The
main factors that influence viability of probiotics in the product include stability during
storage of the product. Additionally probiotics should not badly affect the taste or aroma of
the yoghurt or it should not have any effect on increase or decrease in the acidity of the
product during the shelf life (Heller, 2001).
22
Probiotic bacteria are present in different food commodities that include conventional
food products, dietary supplements and in foods that have medical value. Foods that contain
probiotic bacteria mostly include the dairy food products like yoghurt, the fluid milk contain
these culture such as “Acidophilus Milk”. Dairy foods especially fermented milk products
naturally contain probiotics. And consumers from all over the world obtain benefits from
fermented dairy products with live friendly starter culture (Sanders, 2000).
2.6.1. Probiotic Selection
Probiotic organisms should meet requirements for their viability that includes
resistance to gastric acidity, pancreatic and bile enzymes; colonization capacity; adherence to
intestinal mucosa cells; keeping viability for a long time storage and transportation, so they
can efficiently colonize the host; production of antimicrobial substances against pathogenic
bacteria and absence of translocation has been recognized by Tomasik and Tomasik (2003).
2.6.2. Health benefits of probiotics
The main function of probiotics is to assist in controlling irritable bowel syndrome
and diarrhea (McFarland, 2006), improve immune system, lactose digestion, decrease level
of blood cholesterol and pressure, enhances usage of nutrients and increase nutritional value
of food, cause an alteration of intestinal microbiota whereas and helps body to tolerate the
harmful pathogen„s attack and also maintain major body metabolisms. Prebiotics serve as an
additional source of fiber and stimulate functionality of probiotics. In the conditions, when
bowel function is poor, yogurt supplemented with probiotics specifically enhances the
effectiveness of the digestive tract (Agrawal, 2009; Ranadheera et al., 2009; Routray and
Mishra, 2011).
Probiotics have been declared to be helpful in the treatment of eczema, allergies,
psoriasis, acne, irritable bowel syndrome, gout, migraine, cystitis, colitis, candidiasis,
rheumatic and arthritic conditions and some forms of cancer (Shah, 2001). Hence efficiency
of a probiotic is directly related to the number of active cells consumed, it is of significance
to specify potency (cfu) of the culture per unit weight or volume of the product.
2.7. Synbiotic Food
The foods that potentially contain probiotics and prebiotics are regarded as
“synbiotic” and influence the host organism by stimulating selective growth of health
23
promoting bacteria when embedding with dietary supplements in the GIT tract (DiRienzo,
2000).
Yoghurt is a famous milk product which has a lot of therapeutic, nutritional and
sensory properties to further enhance these functions there is a need to develop yoghurt that
contain prebiotics, probiotics or both together assymbiotic activity (Nancy, 2010).
2.7.1. Yogurt History
Fermentation of dairy products such as cheese and yogurt has very old history
(Mckinley, 2005). Lactose digestion by the Fermented dairy products provide numerous
health benefits such as conversion of lactose into lactic acid that helps lactose digestion,
protection against colon cancer, improvement of immune-modulatory system, inhibition of
diarrhoea, lower the blood cholesterol level (Wollowski et al., 2001).
Yogurt is a very widespread food in most of countries among the consumers of all
ages (Sodini et al. 2005). It is believed that it is one of the oldest fermented products in
human history, initiating in the Asia and Middle East (Chandan, 2006). And it did thousands
of years ago when yogurt was made perhaps with the taming of sheep, goats and cow. In
recent decades, due to innovations and developments in the science of fermentation the
process to make yogurt has more organized. But it is a complex combination of science and
art to make yogurt (Tamime and Robison, 1999).
Yoghurt is a very delicious product that has the good nutrient medium for the
probiotic cultures and is highly preferred by the consumers as probiotic food (Siegrist et al.,
2008; Hailu et al., 2009). This leads to the formation of a lot of probiotic dairy products in all
over the world (Arayana et al., 2007; Almeida et al., 2008; Ramasubramanian et al., 2008).
There is a dire need to determine the relation and stability of the different probiotic strains
(Korbekandi et al., 2009; Sacarro et al., 2009) and of different quality parameters
(Mortazavian et al., 2007) during the product formation.
In the whole world, the eating habits of probiotic dairy foods have been enhanced
throughout the World in the recent decades. In Europe probiotic foods involve a total of 1.4
billion dollars, and its gross income from the products like yoghurt and dessert fulfill
approximately 72% of the total income (Saxelin, 2008). In the year 2008 in Brazil, the
24
probiotic dairy food products contribute about 2.65 billion dollars (Rocha and Madureira,
2009; Adriano, 2010).
Now a day a lot of consumers have established an interest to get know how about the
relationship between nutrition and food. Consumers require naturally healthy foods that are
easy to prepare and consume. Functional foods have different forms, conventional foods such
as yoghurt that have bioactive component (Sloan, 2005).
Yoghurt is particularly a well-known dairy food stuff that is familiar for the
application of Probiotic microorganisms and the marketing of these products rapidly
increasing day by day. In Australia there is a sudden increase from 5.3-6.8 kg per capita
consumption of yoghurt in 2007 (Dairy Australia, 2007). The yoghurt that contains friendly
bacteria only contributes about 82% of whole market of probiotic dairy foods (Anon, 2003).
The study was conducted by eating the probiotic fermented food products, and
conclusions were made for pH of colon (decreases or lowers). The low pH helps to control
the growth of pathogenic bacteria and encourage the growth of good bacteria. By the
lowering in the pH of the colon, the carcinogens which are bind with dietary fibers are easily
excreted as studied by Rowland (1995).
Cryo-protectants probiotics may be added in yoghurt mixture before fermentation to
encourage the survival probiotic organisms, good adoptability during refrigerated storage.
Cryo-protectants inserted within the cell helps to decrease the osmotic difference with their
external environment (Kets et al., 1996).
2.7.2. Yogurt as a Synbiotic Food
Fermented dairy products, having the tradition as healthy foods, are a natural choice
for their makeover as functional foodstuffs. Yogurt is derivative of „Jugurt‟ a Turkish word.
A vast array of yogurts is now available in the market to be fit for all enjoyments and feasts.
Different varieties of yogurt are available in texture (smooth, set and stirred), content of fat
(fat free and low fat) and flavors (natural, fruit and cereal). Various types of low-fat yogurt
offer a significant amount of a range of important nutrients with respect to calories and fat
content, so making yogurt a food dense in nutrients (McKinley, 2005; Shah, 2003). The
healthy image of yogurt is further endorsed by addition of various fruit preparations in yogurt
to include the health benefits of fruits such as providing fibre and antioxidants (O‟Rell and
25
Chandan, 2006). In recent years, further, inclusion of plant extracts to enhance yogurt
functionality, such as tea catechins for antioxidative and antimicrobial properties (Jaziri et
al., 2009) has also been considered.
Yogurt may be defined as a dairy product made by milk fermentation through starter
culture bacteria including Lactobacillus bulgaricus and Streptococcus thermophilus. Some
other countries like Australia, the starter cultures of other appropriate LAB are allowed to
use. Hence L. jugurti and L. helveticus are used to make yogurt by some manufacture.
However, US standards do not allow using other types of starter culture than Streptococcus
thermophilus and Lactobacillus bulgaricus (Shah, 2003).
Starter culture organism may be well-defined as „ preparation of microbes cells in a
large number at least of one microorganism that are added in raw material for the production
of fermented product by directing and speeding up the process of fermentation (Leroy and De
Vuyst, 2004). During fermentation, lactic acid is produced from lactose by starter culture
bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus. These LAB also produce
bacteriocins, ethanol, acetic acid, exopolysaccharides, aroma compounds, and numerous
enzymes. As a result they increase microbial safety and shelf life, add to the pleasing sensory
profile of the yogurt and improve texture (Leroy and De Vuyst, 2004). The functional
properties of LAB improve the functionality of fermented foodstuffs such as yogurt Table
2.1. Of the various functionalities, the review focuses on the health benefits that ensure from
yogurts as a consequence of fermentation
Table 2.1. Functional Properties of Fermented Foodstuffs
(Source: Leroy and De Vuyst, 2004)
The sources from where the probiotics are mainly obtained are the fermented milk
products like yoghurt. Yoghurt is made by the fermentation process which can be done by the
26
species of LAB like S. thermophilus and L. bulgaricus (Lourens-Hattingh and Viljoen, 2001).
In addition yoghurts can also be produced with the help of probiotic cultures that have
different viability level over a broad variety of shelf lives.
In the processing of yoghurt the incubation time is very important criteria. Incubation
time directly affects the viability of probiotics in yoghurt. With long incubation period to
yoghurt lead to decreased viability of probiotic due to involvement of oxygen contents.
Instead of incubation time, other factors like incubation temperature and storage time also
affects the cell viability as studied by AkIn et al. (2007).
2.7.3. Nutritional and Therapeutic Aspects of Yogurt
Yogurt provides different nutrients for humans such as vitamins, minerals and
proteins (whey and caseins). The culture used for fermentation breakdown the milk sugar i.e.
lactose, which is helpful for people who are lactose-intolerant (Chandan, 2006). The bacteria
present in yogurt are aid tolerant that can withstand acidic environment of stomach and lactic
acid is secreted by them in intestine of human beings (Shah, 2007). S. thermophilus and L.
bulgaricus make themselves as prevailing bacteria in the gut, reduces the number of harmful
bacteria and a variety of therapeutic bioactive compounds are produced by them e.g.
casooxins, lactoferroxins and casein phosphopeptides) that deliver different characteristics
such as immunostimulants, anticarcinogenic, antihypertensive, mineral transportation and
antistress (Chandan, 2006; Zsivkovits et al. 2003).
Being a good source of protein (casein and whey), minerals and vitamins for humans
a yogurt is considered as good food. Acid tolerant bacteria present in yogurt survive in the
human stomach that also secretes lactic acid which is beneficial for pathogens and GIT
system in several ways (Shah, 2007). Gut dominant bacteria are L. bulgaricus and S.
thermophilus, enhancing the number of therapeutic compounds (bio active peptides: casein
phophopeptides, casooxins, lactoferroxins) while decreasing the number of putrefactive
organisms, antistress features, antihypertensive, anticarcinogenic, immunostimulants, mineral
transportation and antithrombotic (Zsivkovits et al., 2003). Bioavailability of phosphorus,
magnesium and calcium is enhanced by casein phosphopeptides which prevent dental caries
and add to the ideal bone health (Chandan, 2006).
27
Anti-carcinogenic activities of yogurt have been reported by Shah (2006). After
conducting a study on rats and mice, yogurt possesses anti-tumor effects and inhibited certain
types of tumors. On the other hand L. bulgaricus considered more effective than S.
thermophilus towards the inhibition of tumor. It has been reported that breast cancer and
exocrine pancreatic cancer can also be reduced by utilizing yogurt. During in-vitro studies it
has been reported that strains of L. bulgaricus and S. thermophilus reduces cholesterol levels
(Dilmi-Bouras, 2006) and reduction enhances by the addition of probiotic strains (Sarkar,
2008).
Even if the microorganisms present in the yogurt culture are not naturally present in
the human intestine yet it is suggested by the current theories for being providing a number
of fitness entities including alleviated lactose-intolerance, enhanced mineral absorption,
greater immunity, improved protein digestibility and controlled intestinal health (Shah,
2007).
2.7.4. Yoghurt composition
2.7.4.1. Milk Proteins
Ratio of available protein that is utilized and absorbed in the human body to the
amount of protein consumed (Accessibility of protein) is different for different types of
proteins (Bilsborough and Mann, 2006). In milk there are two major groups of proteins i.e.
casein and whey proteins.
One of the greater sources of calcium in the human diet is casein. Three types of
casein i.e. αs1-, αs2- and β-casein are affected by calcium while κ-casein is unaffected by the
calcium because calcium is part of its miceller structure. κ-casein covers the casein micelle
and has a hydrophilic tail and it prevent the precipitation of remaining caseins (Horne, 2006).
Whey proteins are globular, water soluble and grouped into 5 portions α-lactalbumin,
bovine serum albumin, β-lactoglobulin, proteose peptone fractions and immunoglobulins
(IGs) (de Wit, 1998). Whey proteins have an important amount of amino acids that contain
Sulpher (Cysteine) exhibiting higher availability of protein (>90%). Branched chain amino
acids e.g. leucine are present in whey proteins which are significant for the synthesis of
muscle protein (Ha and Zemel, 2003). Whey proteins are hurriedly digested as compare to
28
caseins offering larger amount of essential amino acids e.g. leucine, lysine and cystein (Haug
et al., 2007; Hoffman and Flavo, 2004).
2.7.4.2. Vitamin B
Lactic acid bacteria requires Vitamin for their optimum growth, while some can also
synthesize vitamin B themselves (Buttress, 1997). Lactic acid bacteria mainly utilize vitamin
B12 while some species can synthesize folate (Crittenden et al., 2003). 5-methyl-
tetrahydrofolate is a folate form that is present in milk. Folate in dairy products is produced
by streptococcus thermophillus and bifido bacteria, while lacto bacilli species mainly uses
folate. Greater concentration of folate was observed when combination of both starter
cultures was used while studying functional cultures and their health benefits by Shah (2007).
2.7.4.3. Lactose
Lactose is naturally occurring sugar in milk and yogurt is major source of lactose in
diet of humans (Voet and Voet, 2004). It is not directly absorbed or used as energy source by
human body. Therefore, it is hydrolyzed by lactase into galactose and glucose that absorbed
through human body easily. During fermentation 70-80% of lactose is hydrolyzed into
glucose and galactose (Bourlioux and Pochart, 1998) which is useful for the lactose intolerant
people who can consume yogurt (Vesa et al., 2000).
2.7.4.4. Fats or Lipids
Yogurt fats are biologically more available then milk fats due to biochemical changes
which occur during fermentation process. Free fatty acids are also released in minute
quantity. Higher concentration of linoleic acid has been observed in yogurt when studies
conducted by Shantha et al. (1995).
2.7.4.5. Minerals
Yogurt is a major source of calcium and phosphorous in diet. Milk and milk products
including yogurt and cheese contain highly available calcium. In yogurt calcium and
magnesium are present in their ionic forms because of lower pH of yogurt then milk.
Phosphorous and calcium have key role for body maintenance, growth and development.
Body requirements of calcium about 40% and phosphorous about 30-35% are fulfilled by
yogurt. Calcium has key role in bone formations and increases their strength it also helps to
improve immune system and decrease cholesterol level. Absorption of magnesium is also
29
increased because of the presence of lactic acid bacteria that helps to boost up the mineral
absorption (Shah, 2007).
2.7.5. Chemistry of Yogurt (Whey-casein interactions)
Whey proteins have the ability to directly attach with casein micelles and casein-
denatured whey protein complexes through cross-linkings, in a yogurt mix. The resultant
linkage of whey proteins to the casein micelles is attributed to changes in the size of casein
micelle that is induced by heating of the skim milk. And this level of association was
markedly affected by little change in pH of milk. (Puvanenthiran et al., 2002; Anema and Li,
2003) stated the size of casein micelle enlarged up to ~700 nm. Gel formation is changed by
these attachments in the course of acidification process by loosening casein-casein
association and increasing voids. Hence in a yogurt if whey protein is ≥ 1% then it may result
in self-aggregation of whey protein and distinct gel arrangement is developed that is
entrenched among the gel structure of casein and whey (Aziznia et al., 2008). The schematic
picture of yogurt formation is shown in figure 2.1, where on heating whey protein attach to
casein micelle and gel formation occurred due to neutralization of charge of casein micelle.
2.7.6. Rheological Properties of Yogurt
Jumah et al. (2001) examined influence of milk source on rheological attributes of
yoghurt in course of the gelation process. It was elaborated that highest viscosity value was
shown by the ovine milk then comes caprine, bovine and camel milks. No major variation in
viscosity was observed in camel milk during gelation. Mainly the rheological properties of
yogurt are affected by the chemical composition of milk specially protein content and total
solids. Pandya et al. (2004) reported that with increasing fat content sensory properties and
rheology of buffalo yogurt significantly improved. By increasing fat contents (1.5 to 4.5%),
viscosity was increased by 22.5%, curd tension improved by 10% and wheying off decreased
by 31%.
Yogurt is made from different heat treated milk samples and viscosity was measured
through viscometer with spindle No. 3 at speed of 20 rpm. Average viscosity of all types of
yoghurt was decreased by increasing in the heat treatment to milk samples. The entire yogurt
from milk samples formed at 90°C/10 min. is less noticeable to decline in viscosity occurring
throughout storage (Djurdjevic et al., 2002). The microstructural and rheological features of
30
yogurt were evaluated by Lee and Lucey (2003) and reported that elastic modulus of yogurts
was decreased with incubation temperature and increased with the increasing of heating
temperature. A decline in heating temperature and an increase in incubation temperature
resulted in continuous removal of whey from yogurt.
Fig 2.4 Formation of yogurt gel due to acidification of milk
Ares et al. (2006) measured the firmness in stirred yogurt results showed that yield
stress was with-in range of 250 Pa from manufacturing plants. During fermentation when
final acidity was varied then it gives the more pronounced results and it shows the value of a
standardized production for obtaining uniform texture. The yogurts made from processing
unit showed expressively more yield stress as compared with samples from the different
retail stores. During handling and distribution, syneresis was also occurred due to mechanical
damage.
31
2.7.7. Physico-Chemical and Sensory Attributes of Yogurt
Yogurt is a kind of gel, soft solids so it is sensitive to structural changes. Various
procedures are outlined to investigate structural and physical features of yoghurt. Processing
variables affect the structural features of the yogurt e.g. solid contents, incubation
temperatures and heat treatment. By better understanding of those factors that lead to
structural and physical properties, could improve the yogurt quality (Lee and Lucey, 2010).
An increase in hydrophobic interactions with the rise in incubation temperature has
been observed which resulted in shrinkage of casein particles and a more compact
conformation during the study of physical properties and structure of yogurt gel: effect of
incubation temperature and inoculation rate conducted by Lee and Lucey (2004). It is
publicized by this study that incubation temperature and inoculation rate are important
processing factors that influence physical and microstructural traits of yogurt gels.
Skimmed probiotic yogurt was stored at 7°C. A group of qualified judges analyzed
the cooled traditional dahi‟s quality. pH decreased apparently, with storage time, showing
that much acidity was not developed by dahi samples under the storage environment. After
giving 8 days for storage, panel concluded that slight bitterness in samples is common. Based
on the consequences, it was concluded that dahi is accepted by consumer up to 8 days during
storage (Yadav et al., 2007). It had also been found that the use of various kinds and
quantities of hydrocolloids in yogurt formation, did not influence the cholesterol, fat, water,
protein, ash, salt, pH and acid strength values of samples (Seckin et al., 2009).
Tarakci and Kucukoner (2003) studied different properties like sensory, physico-
chemical and microbiological properties of fruit-flavored yogurt and reported that there were
significantly differences in the fat, ash, protein, total solids content and titratable acidity for
all samples during storage. There were marked differences in the protein and dry matter due
to different flavor additives. Syneresis and titratable acidity increased over the storage period.
Hardi and Slacanac (2000) investigated that rheological properties are important
factors in the quality of yogurt. Texture depends upon a number of factors including starter
culture, milk composition, milk viscosity, heat treatments, fermentation kinetics and
homogenization. The effects of three factors (milk fat, starter culture, addition of inulin) on
coagulation and rheological properties were examined in yogurt, the result showed that
32
starter culture had greatest effect; inulin addition caused an increase in consistency of
probiotic vs non probiotic yogurt.
Ahmed (1999) prepared the fortified yogurt by incorporating the sugar and mango
fruit pieces noted its shelf life when kept at 6 degree centigrade for 40 days. He observed that
there was a slight decrease in the acidity of the yogurt samples. The addition of sugar caused
syneresis whereas, substantial increase in total solids was observed. The incorporation of
mango fruit pieces in the yogurt also increased the rate of syneresis. It was observed during
storage of the yogurt samples at 6 degree centigrade that there was substantial increase in the
amount of acidity, total solids and the rate of syneresis. The pH of yogurt samples decreased
significantly.
Chee et al. (2005) studied sensory and chemical properties in strawberry flavored
yogurt emulsified with algae oil, by hydro-peroxide measurements the oxidative deterioration
was reported after sensory evaluation by skilled panel consumer. In this supplemented
yogurts the hydro-peroxide content was increased with the passage of time prior to stage of
addition. After completion of 22 days storage sensory evaluation the product was
distinguished with a stronger fishy flavor and voted as „moderately liked‟ for both.
Damin and Oliveira (2003) studied sucrose and total solid content on firmness,
acidity, feasibility and viability of pro biotic bacteria in fermented milk and reported that
milk samples containing higher levels of total solids showed higher acidity and increasing the
amounts of sucrose and total solids in milk resulted in higher firmness. Anema et al. (2004)
reported variation in viscosity as dependent on milk pH and heating. During initial steps of
heating, viscosity increased remarkably at pH 6.5 and platitude on extended heating. But as
pH value of milk amplified, minute variations in viscosity examined at 6.7 pH value. There
was a linear correlation between change in viscosity and change in particle volume.
33
Chapter 3
M A T E RI A L S A N D M E T HOD S
The current research work was conducted in the Food Microbiology & Biotechnology
Labortary National Institute of Food Science and Technology (NIFSAT), Department of
Chemistry & Biochemistry, University of Agriculture, Pakistan and School of Chemical
Engineering, University of Birmingham, UK. Microstructural analysis was performed from
Centralized Resource Laboratory at University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
The research work was carried out to evaluate the effect of guar gum and its modified forms
on various characteristics of yogurt and on the viability of probiotic organisms.
3.1. PROCUREMENT OF RAW MATERIAL
Analytical grade guar gum was purchased from local the market of Faisalabad
Pakistan. Commercial freeze dried yogurt cultures (Streptococcus thermophilus and
Lactobacillus bulgaricus) and Bifidobacterium bifidum were purchased from Dansico Co.
(France) and Sacco (Italy). Standardized pasteurized milk for production of yogurt was
procured form Nestle Pakistan, (Pvt.) Ltd. Chemicals, reagents and media were purchased
from Sigma Aldrich (USA), Oxoid (UK) and Merck (Germany).
3.2. PURIFICATION OF GUAR GUM
Crude guar gum was subjected to purification by using different solvents (ethanol and
di-ethyl ether) according to the method of Oforikwakye et al. (2010).
i) Reagents
Ethanol (99%)
Di-ethyl ether (99%)
ii) Procedure
CGG (100 g) was dissolved in 200 mL of distilled water and allowed to stand for 24 h
with intermittent stirring. The gum mucilage was strained with muslin clothe to remove any
insoluble debris or impurities and precipitated with 350 mL of 99 % ethanol. The precipitated
gum was refiltered, washed with diethyl ether and dried in freeze dryer at -55°C (CHRIST,
Alpha 1-4 LD Plus, Version 1.26, Germany). The dried purified gum was milled and
screened through 180 μm sieve. Subsequently the powdered gum was used in further tests
and analyses as purified guar gum (PGG).
34
3.3. HYDROLYSIS OF GUAR GUM
3.3.1. Enzymatic Hydrolysis
Guar gum powder was hydrolyzed with enzyme mannanase following method as
described by Cheng and Prud’homme (2000).
3.3.1.1. Reagents
(a) 1N HCl (Riedel de haen, France) (37% pure, M = 36.46g/mol, Density, 1L = 1.19 Kg)
HCl (20.72 mL) was taken and made the volume with distilled water up to 250 mL in
volumetric flask.
(b) 0.1M Acetate Buffer (Merck, Germany)
Sodium acetate (M = 136 g/L)
Acetic acid
Sodium acetate (13.6 g) was taken in volumetric flask distilled water was added to it and
final volume was made upto 1000 mL to get 1L 0.1N sodium acetate solution. Then pH of
this solution was adjusted to 6 with of acetic acid adding slowly.
3.3.1.2. Guar Solution
A 1000 mL wide mouth jar containing 150 mL of deionized water was stirred using a
mixing impeller with speed adjusted to 1000 rpm to form a deep vortex. Then 1 g of guar
powder was sprinkled slowly onto the liquid-free surface over a 3-min interval to produce a
uniform dispersion and was stirred continuesly for 5 min. Another 49 mL of deionized water
was added to wash all the residual powder in the beaker walls into the solution. The mixing
speed was then reduced to 500 rpm for an next 60 min. The solution pH was adjusted to 7.0
using HCl (Riedel de haen, France). Finally, the polymer solution was transferred to a
container and placed for approximately 20-24 h at 25°C to get complete hydration. The
mixture was stirred through magnetic stirrer during the reaction. The final polymer
concentration was measured by dry weight analysis. Guar and enzyme mixture were
immediately heated to 100°C for 20 minutes to denature the enzyme and stop the reaction.
Mixture was filtered and residues were freeze dried at -55°C and ground to fine powder.
3.3.1.3. Enzyme Buffer Solution (Novozyme, EC 3.2.1.78)
The mannanase was obtained from Novozyme Inc., UK. Mannanase enzyme 0.04 mg
was diluted in 2 mL of 0.1M sodium acetate/acetic acid (Merck, Germany) buffer solution
and pH was adjusted to 6.
35
3.3.1.4. Enzymatic Degradation
The enzymatic degradation reaction was performed in a sealed jar at room
temperature (25°C) for 24 hrs. The pH of the guar solution was adjusted to 7.0. The enzyme
buffer solution was injected into 200 mL of guar solution using a syringe. The mixture was
stirred on magnetic stirrer during the reaction. After the specified time the mixture was
immediately heated to 100°C for 20 min to denature the enzyme and stop the reaction.
3.3.2. Acidic Hydrolysis
Hydrolysis of guar powder with hydrochloric acid (HCl) was performed following
method of Chauhan et al. (2009).
3.3.2.1. Reagents
(a) Methanol (Panreac, E.U. ) (99%)
(b) Hydrochloric Acid (Riedel de haen, France) (37% pure, M = 36.46 g/mol,
Density, 1L = 1.19 Kg) Pure methanol 160 mL was mixed with 40 mL of distilled
water and then 22.70 mL of HCl was dissolved slowly in it.
3.3.2.2 .Procedure
Liquefaction of guar gum (GG) was carried out by acidic hydrolysis. GG (10 g) was
taken in 80% aqueous methanol (200 mL) containing 5% w/v HCl in a round bottom flask
fitted with condenser and mercury pit. The reaction mixture was heated for 2.5 h at 65°C.
The depolymerized guar gum (GGH) was filtered under vacuum suction, washed with
methanol and freeze dried.
3.3.3. Basic Hydrolysis
Guar gum was basically hydrolyzed by barium hydroxide [Ba(OH)2] according to the
method of Beltran et al. (2008).
3.3.3.1. Reagents
(a) Barium Hydroxide (Winlab, USA)
(b) 1.0 M Sulfuric Acid (Riedel de haen, France) (98% pure, Density, 1L = 1.84 Kg)
Sulfuric acid 5.55 mL was poured in volumetric flask and volume was made with
distilled water up to 100 mL to get the desired molarity of 1.0 M.
36
3.3.3.2. Procedure
The crude gum (5g) was hydrolyzed with a saturated barium hydroxide solution (200
mL) at 95°C for 8 h. The hydrolyzed gum was neutralized with 1 M sulfuric acid (H2SO4),
filtered, and freeze-dried.
3.4. GUAR GUM CHARACTERIZATION
3.4.1. Chemical Analysis
Guar gum and its hydrolytic forms were analyzed for crude protein, crude fat, crude
fiber, moisture ash, and total soluble according to their respective methods stated below in
detail (AACC, 2000).
3.4.1.1. Moisture
The moisture content in each guar gum sample was determined according to AACC
(2000) method No. 44-15A by taking 5 g sample and drying it in an air forced draft oven at a
temperature of 105±5°C till a constant weight of the dried material was attained. The
moisture content was calculated according to the following formula:
Wt. of original guar gum sample - Wt. of dried guar gum sample
Moisture (%) = ------------------------------------------------------------------------------- × 100
Wt. of original gaur sample
3.4.1.2. Crude Protein
The crude protein content in each guar sample was estimated according to the
Kjeldahl’s method as described in AACC (2000) method No. 46-10. Two grams sample was
weighed and put into the digestion tube. Twenty milliliters of concentrated sulphuric acid
(98%) and 2 tablets of digestion mixture as catalyst were added into the digestion tube. The
digestion was carried out for 3-4 h (till the digested contents attained transparent color). The
digested material was allowed to cool at room temperature and diluted to a final volume of
50 mL. The ammonia trapped in H2SO4 was liberated by adding 40% NaOH solution through
distillation and collected in a flask containing 4% boric acid solution, possessing methyl
indicator and titrated against standard 0.1 N H2SO4 solution. The factor 5.7 was used for the
conversion of percent nitrogen into crude protein contents.
3.4.1.3. Total Ash
The guar gum samples were tested for total ash content by taking 3 g sample in tarred
crucibles and charred on a flame until it turned black and put into a muffle furnace
37
maintained at a temperature of 550°C for 5 hours or till a grey color of ash was obtained. The
details described in AACC (2000) method No. 08-01 were followed for the estimation of
total ash contents. The ash content was calculated according to the formula given below:
Wt. of ash
Ash (%) = ------------------------------------------- × 100
Wt. of guar gum sample
3.4.1.4. Crude Fat
The crude fat content in each guar gum sample was determined by taking 3g dried
gum sample and running through Soxhlet apparatus for 2-3 hours using petroleum ether as a
solvent by following the procedure described in AACC (2000) method No. 30-10.
Wt. of fat
Crude fat (%) = ---------------------------- × 100
Wt. of gum sample
3.4.1.5. Crude Fiber
The crude fiber was estimated according to the procedure as outlined in AACC
(2000) method No. 32-10. It was carried out by taking 3g of each fat free guar gum sample
and digested first with 1.25% H2SO4, washed with distilled water and filtered, then again
digested with 1.25% NaOH solution, washed with distilled water and filtered. Then ignited
the sample residue by placing the digested samples in a muffle furnace maintained for 3-5
hours at temperature of 550-650°C till grey or white ash was obtained. The percentage of
crude fiber was calculated after igniting the samples according to the expression given below
Weight loss on ignition
Crude fiber (%) = ------------------------------------ × 100
Weight of gum sample
3.4.2. Rheological Properties of Aqueous Guar Solution
3.4.2.1. Shear Stress and Viscosity
Shear stress and viscosity measurements were performed at 25°C using a controlled stress
rheometer (Bohlin CVO, UK) fitted with a cone and plate geometry (4° cone angle, 40 mm
diameter, 125 µm gap) with controlled shear rate (1/s) by following method stated by
Sittikijyothin et al. (2010).
38
Fig 3.1. Rheometer
3.4.2.2.. Oscillatory Properties
Dynamic measurements and frequency sweeps measurements were performed at
25°C using a controlled stress rheometer (Bohlin CVO, UK) fitted with a cone and plate
geometry (4° cone angle, 40mm diameter, 54 µm gap) with controlled shear stress in the 0.1–
100 rads-1
range (Sittikijyothin et al., 2010).
3.4.2.3. Glucose Absorption in Small Intestine Model (SIM)
Glucose absorption with respect to various guar gums through in vitro small intestinal
model was assessed following the method stated by Tharakan et al. (2010).
Fig 3.2 In vitro- Small Intestinal Model
39
i) Preparation of Solution
To prepare the guar-glucose (1% w/v) solutions, a known quantity of guar powder
and glucose was slowly added to the distilled deionized water in a beaker and continuously
stirred using a magnetic stirrer. Once the material had been added, the container was weighed
and the solution was heated to 80°C for 10 minutes before cooling to room temperature,
stirring was continued throughout during the process. Stirring continued for 12 hours to
ensure complete hydration of the guar gum-glucose solution; the final concentration was
calculated after finding the weight loss through evaporation. The formulations were used
within 24 hours of preparation as microbial growth after this time changes the properties of
the formulation.
ii) Effect of Mixing and Flow Rate on Glucose Absorption
The biopolymer-glucose (500 mL) solution was placed in the dialysis membrane and
in the outside tubing the diffused glucose was collected. Distilled deionized water (500 mL)
was used in the outer tube. The experiment was run at room temperature upto 90 min taking
samples every 5 minutes to determine glucose absorption. Two processing conditions were
investigated to study the effect of mixing on the glucose absorption:
(i) Mixing induced by segmentation contractions. In this case local flow was induced with
peristaltic pump 3rpm (12mL/min) by segmentation contractions through various jaws.
(ii) No mixing or segmentation (stationary flow). No segmentation movements. Absorption
of glucose was done by facilitated diffusion through the interfacial membrane as a result of
the concentration gradient.
The overall flow in the recipient side (outside of the membrane) was constant at 120 rpm
(480mL/min) during experiments and was generated from a variable speed peristaltic pump.
Each experiment was carried out in triplicate.
iii) Glucose determination
Diffusion across the membrane was monitored at time intervals during one hour by
measuring glucose concentration in the recipient side using the method of 3,5-dinitrosalicylic
acid (DNS) reagent for reducing sugars (Fonseca et al., 2011). This method tests for the
presence of the free carbonyl group (C=O) in monosaccharides. Simultaneously, the DNS
reagent is reduced to 3-amino-5-nitrosalicylic acid. For the determination of reducing sugars
a UV-VIS spectrophotometer Libra S12 (Biochrom, Cambridge, UK) was used for
40
measuring the absorbance of the standards and samples at 540 nm. All absorbance
measurements were performed in triplicate.
For glucose calibration curve standards covering the range 0-10.0 mM in the final assay
solution were used.
3.4.3. Measurement of Galactose-Mannose Ratio
Galactose and mannose content of guar gum samples was estimated by the following
method described by Jahanbin et al. (2012) with some modifications.
3.4.3.1. Reagents
Trifluoroacetic Acid 2 M (TFA, CF3COOH) (M = 12.98 g/L; Density, 1L =
1.485 Kg), NaBH4
3.4.3.2. Procedure
A sample of 10 mg of pure freeze-dried gum was hydrolyzed by heating at 120°C
(Memmert 100 universal bench, max temperature 220 ºC) for 3 h with 1 mL of 2 M
trifluoroacetic acid (TFA, CF3COOH) in a sealed tube. Excess acid was removed by flash
evaporation on a water bath at a temperature of 40°C and co-distilled with three times of
water. The hydrolyzed products were reduced with NaBH4 (50 mg) and filtered through a
0.45 µm filter, and 20 µL of the sample was injected into the HPLC column. An Perkin
Elmer Shimadzu HPLC unit and Rezex RCM-Monosaccharide Ca+2
, Phenomenex column
was used to carry out the analysis. HPLC grade water was used as mobile phase (isocratic) at
a flow rate of 0.6 mL/min. A refractive-index detector (Gradient LC) was used and the
column oven temperature was 80°C. Monosaccharides were identified by comparing their
retention times with the standard sugars. They were quantified according to their percentage
area, obtained by integration of the peaks.
3.4.4. Scanning Electron Microscope (SEM)
Guar gum and its hydrolytic forms were examined by scanning electron microscope
to provide information about structure size and shape of the respective particles.
Photographic images were recorded following the method of Sen et al. (2010). 30KV
Scanning Electron Microscope (JSM5910, JEOL, Japan) with SEI and EDX detectors
(INCA200/Oxford Instruments, UK) was used for purpose.
41
Fig 3.3 Scanning Electron Microscope
3.4.5. X-ray Diffraction
X-ray configurations of guar gum samples were examined by means of an X-ray
diffractometer (Xu et al., 2005). X-ray Diffractometer (JDX 3532, JEOL, Japan), CuKα
source. Measurements were carried out with a diffraction angle range of 5-60° and resolution
of 0.02° at room temperature.
Fig 3.4 X-Ray Diffractometer
42
3.4.6. FTIR Analysis
Infra-red spectral studies were performed on spectrometer under dry air at room
temperature. Spectra were taken between 4000 and 400 cm-1
using Bruker Tensor 27 FT-IR
following method stated by Gupta et al. (2009). The results obtained were compared with
library for detection of peaks.
Fig 3.5 Fourier Transform Infra-red Spectrophotometer
3.4.7. Thermo Gravimetric Analysis
Thermal analysis of crude, purified and partially hydrolyzed guar gum was carried
out with Thermo-Gravimetric and Differential Thermal Analyzer (TG/DTA) (Diamond
Series TG/DTA Perkin Elmer, USA), Max. Temperature 1200°C as stated by Mudgil et al.
(2012). TGA measurements of guar gum and hydrolyzed guar gum were carried out in
nitrogen atmosphere , at a heating rate of 10°C/min over a temperature range of 30-1200°C.
1) Held for 1.0 min at 30°C
2) Heated from 30°C to 1200°C at 10°C/min
43
Fig 3.6 Thermo-gravimeteric Analyser
3.4.8. Haemolysis Study
Hemolytic activity of the guar gum samples was assessed through the method
followed by Shahid et al. (2013).
3.4.8.1. Reagents
i) Phosphate Buffer Saline
ii) 0.1% Triton X-100
Table 3.1 Composition of Phosphate Buffered Saline (PBS)
Ingredients Quantity g/L
NaCl 8
KH2PO4 0.2
Na2HPO4 1.2
KCl 0.2
Note: Adjusted to pH 7.4, mixed for 60 min to stabilize pH
3.4.8.2. Procedure
Three millilitres of human blood cells were gently mixed, poured into a sterile 15mL
polystyrene screw-cap tube and centrifuged at 850×g for 5 min. The supernatant was poured
off and the viscous pellet was washed three times with 5 mL of chilled (4°C) sterile isotonic
44
phosphate-buffered saline (PBS) solution. The washed cells were suspended in a final
volume of 20 mL chilled, sterile PBS and the cells counted on a haemacytometer
(Marienfeld, Neubauer improved, Germany). The blood cell suspension was maintained on
wet ice and diluted with sterile PBS to 7.068 × 108 cells mL
-1 for each assay. Aliquots of 20
µmL of crude, purified and hydrolyzed guar galactomannans were aseptically placed into 2.0
mL microfuge tubes. For each assay, 0.1% Triton X-100 was the positive, 100% lytic control
and PBS was the negative, background (0% lysis) control. Aliquots of 180 µmL diluted
blood cell suspension were aseptically placed into each 2-mL tube and gently mixed three
times with a wide mouth pipette tip. The guar glactomannans concentration tested was 250
mmol L-1
. Tubes were incubated for 35 min at 37°C with agitation (80 rev min-1
).
Immediately following incubation, the tubes were placed on ice for 5 min then centrifuged
for 5 min at 1300×g. Aliquots of 100 mL of supernatant were carefully collected, placed into
a sterile 1.5 mL microfuge tube, and diluted with 900 mL chilled, sterile PBS. All tubes were
maintained on wet ice after dilution. Absorbance at 576 nm was measured using a Micro
Quant (Biotek, USA) using a 96 well plate. The readings were taken as in triplicates for each
samples.
3.5. YOGHURT MANUFACTURING PROCESS
Yoghurt was processed through the procedure according to the standard operating
procedure as adopted by Akalin et al. (2004). The flow diagram indicated in Fig 3.7.
45
Fig 3.7 FLOW DIAGRAM OF YOGURT PREPRATION
Standardized milk (3.5% Fat, 8.5% SNF)
Addition of Guar Gum (crude, purified and hydrolyzed)
Pasteurization
80-85°C for 30 min
Homogenization
55-65°C
Cooling to inoculation temperature (40-43°C)
Addition of starter culture (0.01g/100 L) + B. bifidum (0.001%)
Packing (250 mL cup)
Incubation (40°C, 4.5 h)
Cooling storage (at 4-6°C)
46
Table 3.2 Experiment plan for the preparation of probiotic yogurt
GROUPS Control
GUAR GUM B. bifidum
(%) CGG
(%)
PGG
(%)
AHGG
(%)
BHGG
(%)
EHGG
(%)
To No GG - - - - - -
Toʹ No GG - - - - - 0.001
T1 - 0.1 - - - - 0.001
T2 - 0.5 - - - - 0.001
T3 - 1 - - - - 0.001
T4 - - 0.1 - - - 0.001
T5 - - 0.5 - - - 0.001
T6 - - 1 - - - 0.001
T7 - - - 0.1 - - 0.001
T8 - - - 0.5 - - 0.001
T9 - - - 1 - - 0.001
T10 - - - - 0.1 - 0.001
T11 - - - - 0.5 - 0.001
T12 - - - - 1 - 0.001
T13 - - - - - 0.1 0.001
T14 - - - - - 0.5 0.001
T15 - - - - - 1 0.001
GG: Guar Gum; CGG: Crude Guar Gum; PGG: Purified Guar Gum; AHGG: Acidic Hydrolyzed Guar Gum;
BHGG: Basic Hydrolyzed Guar Gum; EHGG: Enzymatic Hydrolyzed Guar Gum
3.5.1. Standardized Milk
Standardized milk was procured from Nestle (Pvt.) Ltd. Pakistan with 3.5% fat.
3.5.2. Addition of Guar Gum
Then native, purified and hydrolyzed guar gum was added at a concentration of 0.1,
0.5 and 1% in milk as per treatment plane mentioned in table 3.2.
3.5.3. Pasteurization
After mixing, milk was pasteurized at 80-85ºC temperature for a period of 30 min.
3.5.4. Homogenization
Milk was homogenized in a homogenizer at 65ºC at 2500 psi to improve texture.
3.5.5. Cooling
Milk was cooled to a temperature of 40-43º C.
47
3.5.6. Inoculation and Mixing
Inoculation of yogurt culture and bifidobacterium bifidum was done at the rate of
0.0022% and 0.001 % respectively. The cultures were in freeze dried direct vat set (DVS)
form and used according to the manufacturer’s instructions.
3.5.7. Packaging
The inoculated milk was poured in cups of 250 mL volume and properly labeled.
3.5.8. Incubation
The inoculated milk was incubated at 40°C for about 4.5 hours resulting in 0.8-0.9 %
lactic acid.
3.5.9. Storage
The yoghurt was cooled to a temperature of 4-6°C to check further fermentation and
was subjected to sensory, physicochemical and microbial evaluation.
3.6. YOGHURT ANALYSIS
3.6.1. Physico-Chemical Analysis
3.6.1.1. Compositional Analysis
i) Lactose
Lactose was estimated by the method as given in AOAC (2000).
Preparation of Reagents
Fehling solution was prepared by mixing equal volumes of Fehling A and Fehling B
solution immediately before use.
(a) Copper Sulfate Solution (Fehling’s Solution A)
In one liter of distilled water 69.28 g of CuSO4.5H2O was dissolved and filtered
through whatman No. 4 filter paper.
(b) Alkaline Tartarate Solution (Fehling’s Solution B)
Rochelle salt (Potassium Sodium Tartarate) 346 g and sodium hydroxide (NaOH) 100
g were dissolved in distilled water made volume up to 1 liter.
(c) Methylene Blue, indicator
A solution of 1% methylene blue was prepared by dissolving one gram methylene
blue in 100 mL of ethyl alcohol.
48
Procedure
A well-mixed and homogeneous representative sample (10 mL) was taken in china
dish. It was heated to 65ºC and 5-8 drops of concentrated acetic acid were added, then it was
filtered through Whatman filter paper No. 4 into a 250 mL volumetric flask and filled up to
mark by adding distilled water. A freshly prepared 10 mL of Fehling’s solution was taken in
an Erlenmeyer flask, while boiling 2 drops of methylene blue were added and titrated against
lactose solution till a brick red color obtained.
Calculation
The total volume of lactose solution used was multiplied by the factor 0.0676 (as 10
mL of Fehling solution was equal to 0.0676 of lactose) to obtain the quantity of lactose ion
the sample.
ii) Total solids
Total solids were determined according to the method no. 952.23 described in AOAC
(2000).
Procedure
A sample (5 g) was taken in a clean dried weighed china dish. Then it was heated in a
water bath for 15 min. It was kept in a hot air oven for 3 h at 100º C and cooled in desiccators
for half an hour and weighed.
Calculations
Wt. of empty dish = W1
Wt. of dish + sample = W2
Wt. of sample = W2 - W1 = W3
Wt. of dish + residues = W4
Wt. of residues = W4- W1 = W5
Total solids (%) = W5/W3 × 100
iii) Fat
Fat was determined by Gerber method as described as described in AOAC (2000)
followed by Kirk and Sawyer (1991).
Reagents
Sulphuric Acid: An analytical grade sulphuric acid with density of 1.815 ± 0.002
g/mL at 20ºC was used.
49
Amyl Alcohol: Amyl alcohol of standard grade was obtained from Merck Darmstadt,
West Germany.
Procedure
Sample (11.3 g) after stirring and mixing was taken in a butyrometer containing 10
mL H2SO4 of specific gravity 1.835 and 1mL amyl alcohol. It was centrifuged for 5 minutes
at 1100 rpm at 65°C and then noted the reading.
iv) Protein
Protein contents in yoghurt sample was determined by using (Model: D-40599, Behr
Labor Technik GmbH, Germany) based on Kjeldhal’s method (991.20) of AOAC (2000).
Procedure
One gram yoghurt sample was digested in digestion tubes using two digestion tablets
and 20 mL concentrated sulphuric acid for 5-6 hours or till the digestion mixture attained
light green or transparent color. Digested sample was diluted and distillation was done by
taking 10 mL of diluted material and 10 mL of 40% NaOH solution in the distillation
apparatus. The ammonia thus liberated was collected in 4% boric acid solution containing
methyl red as an indicator finally the distillate was titrated against 0.1N HCl solution till
golden brown end point. The crude protein percentage was calculated by multiplying
nitrogen with a factor 6.38 as given below:
Vol. (sample-blank) HCl × Normality of HCl × 0.014
Nitrogen (%) = ----------------------------------------------------------------------- × 100
Weight of sample (g)
Crude Protein = Nitrogen (%) × 6.38
v) Ash
Ash was estimated by the method no. 942.05 as given in AOAC (2000).
Procedure
A well-mixed and homogenized yogurt sample (5 g) was taken in a crucible and
moisture was evaporated to dryness of sample on steam bath. Then crucible was placed in
muffle furnace at 550ºC until ash is carbon free. The crucible containing ash was placed in
desiccator for 30 minutes and weigh and calculate % ash.
50
3.6.1.2. pH
Electronic digital type of pH meter was used for pH determination according to
method given in AOAC (2000). A sufficient quantity of representative sample of yogurt was
taken in a beaker in which electrodes of pH meter were immersed and readings were
recorded after calibrating the instrument.
3.6.1.3. Titrateable Acidity
Acidity was determined by direct titration method no. 947.05 of AOAC (2000).
Reagents
(a) Phenolphthalein Indicator
Phenolphthalein indicator was made by dissolving one g phenolphthalein in ethyl
alcohol (95% v/v) to final volume 100 mL.
(b) N/10 NaOH
The N/10 NaOH was made by dissolving 4 g of sodium-hydroxide in distilled water
and makes the volume up to 1000 mL.
Procedure
A well-mixed homogeneous yoghurt sample (9 mL) was taken in a small beaker.
Then 1-2 drops of phenolphthalein solution were added as an indicator. After that it was
titrated against N/10 NaOH until a slight pink color appeared as an end point which persists
for 30 seconds. The percent acidity (as lactic acid) was calculated as under
0.009 × 0.1N NaOH used (mL)
Acidity % = ----------------------------------------- × 100
Wt. of sample (g)
3.6.1.4. Viscosity
The viscosity of yoghurt was estimated by the method elaborated by (Djurdjevic et
al., 2002). By using a Brookfield LVDVE-230 (MA, USA) viscometer. Apparent viscosity
was determined on yogurt at 10 to 15º C temperature; yogurt was stirred for 40 seconds
before viscosity measurement. Spindle number 4 was used for this measurement with a
rotation of 10 rpm. Viscometer reading was noted in centipoises (CPS) units and percent
torque.
51
3.6.1.5. Syneresis
The whey released by the yogurt samples was analyzed by the method of Rodarte et
al. (2004). Five ml of yoghurt was centrifuged at 5000 rpm for 20 min at 4°C and separated
whey was measured after 1 min. Amount of whey separation was expressed as volume of
separated whey per 100 mL of yoghurt.
3.6.1.6. Water Holding Capacity (WHC)
WHC was determined by the method as described by Singh and Muthukumarappan,
(2008). A sample of about 20 g of yogurt (Y) was centrifuged for 10 min at 669 × g and 20°C
in a model 3K-30 laboratory centrifuge (Sigma, Germany). The whey expelled (WE) was
removed and weighed. The WHC expressed in% was defined as
Y - WE
WHC (%) = ---------------- × 100
Y
Y= yogurt sample
WE= whey expelled
WHC= water holding capacity
3.6.1.7. Organic Acids
Organic acids of yoghurt was assessed by the method given by Seckin and Ozkilincs,
(2011) using HPLC (Perkin Elmer, USA) equipped with UV-visible detector.
i) Reagents
Phosphoric acid (0.1%)
ii) Extraction and Analysis of Organic Acids
Procedure
Seven gram of yoghurt sample was taken and then 40 mL mobile phase (0.1%
H3PO4) was added and mixed by ultra-turrax for 1 min. Mixture was held in water bath at
40°C for 1 h then centrifuged at 6000 rpm for 5 min. Upper phase was filtered through filter
paper (Whatman No: 1) and then through 0.45 µm membrane filter. A Perkin Elmer
Shimadzu HPLC apparatus equipped with a UV absorbance detector set at 214 nm was used.
Chromatographic separation was performed on a Shim-Pack CLC-ODS (C-18), 25cm ×
4.6cm, 5µm. The mobile phase was 0.1% (w/v) of phosphoric acid in distilled water (HPLC
grade) with a flow rate of 0.6 mL/min. 20 μL aliquots of individual standards were injected to
column and their retention time was determined. To obtain the calibration curves, a mixture
52
of standards of various concentrations was also injected into HPLC for their chromatograms
to be compared. After injection of the samples, chromatographic peaks were identified by
comparing retention time of samples to known standard. The quantity of organic acids was
estimated from peak areas of known amounts of standards automatically computed by the
data processor.
3.6.1.8. Cryo-Scanning Electron Microscopy (C-SEM)
Samples were prepared for Cryo-SEM by mounting them onto copper holders and
plunging into liquid nitrogen slush at -177°C. Frozen specimens were transferred under
vacuum into an attached preparation chamber where they were fractured with a cold scalpel
blade. The specimen were then etched at -85°C for 10 min and coated with 300Å of sputtered
gold. The specimen were transferred under vacuum onto the cold stage, maintained at -95°C
and imaged using SEM (XL 30 ESEM FEG Philips Electron Optics Eindhoven, The
Netherlands) at 5 kV. (Hassan et al., 2003)
Fig 3.8 Cryo-scanning Electron Microscope
3.6.2. Sensory Evaluation
Sensory evaluation of yoghurt samples was performed with the method of Farinde et
al. (2009). For assessment of acceptability of guar gum incorporated yoghurt was done by a
panel of 8 judges selected among the faculty members and research scholars at NIFSAT,
University of Agriculture, Faisalabad. The panel of judges was trained and familiar with
yogurt’s attributes The freshly prepared yogurts were subjected to organoleptic evaluation
53
using the 9-point hedonic scale where 9 = extremely liked and 1 = extremely disliked. The
yogurt samples and the commercial yogurt were presented to 10 yogurt consumers who
scored the samples for color, sourness, body texture, mouthfeel, flavor, aftertaste, and overall
acceptability. Total scores were obtained by adding the scores of all attributes. The yoghurt
samples were coded with numbers and presented to judges. Water was provided for rinsing
mouth after each sample
3.6.3. Microbial Analysis
3.6.3.1. Viability of Lactic Acid Bacteria
Viability of Lactobacillus delbrueckii subs. bulgaricus, Streptococcus thermophilus
and Bifidobacteria was enumerated according to the method of Saccaro et al. (2009). The
tests were performed during storage of the product prepared with guar gum (prebiotic) to
check the viability of probiotic added. The tests were performed during production and
storage period to judge the viability of probiotics added.
3.6.3.2. BACTERIAL STRAINS
i) Bifidobacterium bifidum
Bifidobacterium bifidum SP-9 (Sacco, Italy) was enumerated on media while
supplementing with 0.05% L-cysteine (Daejung, Korea) in MRS agar to promote good
growth specifically.
ii) Yoghurt Culture
Yoghurt culture [YO-MIXTM
300 LYO 100 DCU (Danisco, France)] containing
Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was used as
starter culture for yogurt preparation.
3.6.3.3. Reagents
i) MRS Agar (CM0361, Oxoid Ltd. Basingstoke, Hampshire, England)
ii) M-17 Agar (CM0785, Oxoid Ltd. Basingstoke, Hampshire, England)
iii) Peptone Water (CM0009, Oxoid Ltd. Basingstoke, Hampshire, England)
Procedure
The viable cell counts of starter cultures and additional culture of Bifidobacterium
bifidum (probiotic organisms) in all the products were analyzed. A samples of 1.0 mL was
added to 9.0 mL of sterile peptone diluents (0.1%; g/100 mL), and appropriate dilutions were
made. Subsequently, S. thermophilus and L. delbrueckii subsp. bulgaricus were enumerated
54
using M17 Agar (Oxoid, UK) and MRS Agar (Oxoid, UK) and incubated at 37°C during 48
h. B. bifidum was enumerated using MRS pH 6.2 supplemented with cysteine (0.5g/L) and
incubated under anaerobic conditions (AnaeroGen; Oxoid Ltd) at 37°C for 18-24 h. Plates
with number of colonies from 30 to 300 were enumerated.
3.7. STATISTICAL ANALYSIS
The significance of the results for the dietary treatments was analyzed statistically by
computing mean squares and F-values (ANOVA) at 5% probability (Steel et al., 1997). Two
factor factorial analysis with completely randomized design (CRD) was performed for
storage data using software Statistix 8.1.
55
Chapter 4
R E S U LT S A ND D I S C U S SION
Carbohydrate based prebiotic (guar gum) was studied to elaborate effects on quality attribute
of yogurt along with its impact as a prebiotic on probiotic endurance (synbiotic) at the
National Institute of Food Science and Technology, Department of Chemistry and
Biochemistry, University of Agriculture, Faisalabad-Pakistan and in the School of Chemical
Engineering, University of Birmingham, UK. The results obtained were expressed under
different headings and sub-headings as explained below:
(A) GUAR GUM AND ITS HYDROLYZED FORMS
Objective:
The main theme of current study was to purify and hydrolyse the guar gum and was
then subjected to characterization for various parameters to make its feasible use as an
effective prebiotic in yogurt formation as per following point:-
4.1. Purification and Hydrolysis of guar gum
4.2. Guar gum Characterization
4.2.1. Chemical Composition
4.2.2. Rheological properties of Guar Gum
4.2.2.1. Steady shear properties
4.2.2.2. Oscillatory properties
4.2.2.3. Glucose absorption (Small Intestinal Model)
4.2.3. Measurement of Galactose-Mannose Ratio
4.2.4. Scanning Electron Microscopy (SEM)
4.2.5. X-ray Diffraction Analysis
4.2.6. Fourier Transform Infra-red Spectrophotometric analysis (FTIR)
4.2.7. Thermo-gravimetric Analysis (TGA)
4.2.8. Haemolysis Study
4.1. Purification and Hydrolysis of guar gum
It is customary that crude guar gum is used in several foods as additive/ingredient for
various functionalities. The research under the title and plan makes it necessary to purify the
guar gum with ethanol and hydrolyse through enzyme, acid and base method in order to
56
check its impact on physico-chemical, microbiological and sensory evaluation on yogurt after
characterization. The simplest change is achieved by removing the impurities fractions in
purification procedures. The need of purification has been done by several scientists and they
found that the purified gum possesses good flow and compressional characteristics forming
good compacts at low compaction force. Guar gum possesses affirmative physiological
assistances; its higher viscosity makes it problematic to utilize into food products and
solutions. Crude guar gum can be hydrolyzed by both enzymatic and chemical methods to
broaden its range of applications. In this study the crude guar gum was allowed to swell in
water overnight, and ethanol was slowly added to obtain the white amorphous precipitates.
The precipitates were then dried in a freeze drier at -55°C as per following methods stated by
Sinha et al. (2011) and Oforikwakye et al. (2010) with suitable modifications. The gum was
then precipitated from the aqueous medium by adding ethanol (95%) slowly while stirring
and then dried (Shittu et al., 2010; Lubambo et al., 2013). Now there is huge shaft of
paradigm towards green technologies. So, in the current study, we also use the green solvent
for the precipitation of gum in the process of purification of gum from crude source.
The guar gum procured from Azeem Chemicals (Pvt.) Ltd. Faisalabad, was subjected
to purification and hydrolysis through enzymes, acid and base treatments. Partially
hydrolyzed guar gum produced in the plan was to provide a dietary fibre source and in order
to get possible benefits as prebiotic for developing functional food. After purification the
guar gum apparently showed difference in color and appearance as presented in Fig. 4.1.
57
Fig. 4.1 The effect of purification and hydrolysis of guar gum on color appearance (a) CGG,
crude guar gum; (b) PGG, purified guar gum; (c) AHGG, acid hydrolyzed guar gum; (d)
BHGG, base hydrolyzed guar gum; (e) EHGG, enzyme hydrolyzed guar gum
(a) (b)
(c) (d) (e)
58
4.2. Guar gum Characterization
After purification through ethanol treatment and hydrolysis through enzyme
(Mannanase), acid (HCl) and base (Barium Hydroxide) as per prescribed methods, guar gum
was subjected to characterization as detailed below:
4.2.1. Chemical Composition
Chemical composition (moisture, protein, ash, fat and fibre content) of crude, purified
and partially hydrolyzed (depolymerized) guar gum is precised in Table 4.1 (statistical
evaluation) and Table 4.2 that indicates values.
Statistical analysis of chemical composition of the crude guar gum followed by the
treatment for purification through ethanol (95% v/v) and hydrolysis through acid, base and
enzyme (treatments of guar gum) exhibited that the treatments were found to be highly
significant for ash and fiber, significant for protein whereas, non-significant for moisture and
fat contents.
The results from the table of means depicted that guar gum after partial hydrolysis
contained less moisture and fat content in comparison to the crude guar gum (native/crude
i.e., CGG). It is exposed that partial hydrolysis of guar gum although decreased the moisture
and fat content in the hydrolyzed treatments which were at par for the three CGG, PGG,
BHGG when compared with crude guar gum. However, there was marked increase or
equivalent to original value in protein except BHGG that was decreased in comparison to
native guar gum whereas ash contents were also increased in AHGG and BHGG except in
PGG and EHGG.
The values obtained in particular to moisture contents, decreased from 6.54% in CGG
to maximum in BHGG (5.09%) and fat 1.18% in CGG to 0.92% in AHGG accordingly. The
increase in ash was highly significant but lowest value was in PGG followed by second
lowest EHGG. Whereas, BHGG exhibited the highest value for ash content (1.88%) followed
by the second lowest in AHGG (1.49%), when compared to 0.42% ash in case of CGG.
The results regarding protein percent with the various treatments showed variability
in trend / results as the percent 9.48 in CGG was at par with AHGG whereas, significantly
increased in EHGG (14.96%) and in PGG (11.47%). Fiber contents of the CGG was
increased (highly significant) from 1.87% to maximum up-to 2.02% in AHGG and then to
2.00% in EHGG whereas, PGG and BHGG were at par.
59
Table 4.1 Mean squares for chemical composition of crude and hydrolyzed guar gum
Source Df Moisture Ash Fat Protein Fiber
Treatment 4 2.52NS 186** 5.59
NS 20.9* 32.7**
Error 5 1.3198 0.0326 0.0193 4.1876 0.0016
Total 9
**=Highly significant (P < 0.01)
*=significant (P < 0.05)
NS = non-significant (P > 0.05)
Table 4.2 Means values for chemical composition (%) for the crude and hydrolyzed guar
gum
Treatment Moisture Ash Fat Protein Fiber
CGG 6.54±0.57 0.42±0.16d 1.18±0.03 9.48±0.77
bc 1.87±0.04
b
PGG 5.37 ±0.55 0.22±0.70c 1.05±0.07 11.47±0.77
b 1.88±0.01
b
AHGG 6.03 ±0.04 1.49±0.06b 0.92±0.03 9.48±0.76
bc 2.02±0.02
a
BHGG 5.09 ±0.63 1.88±0.01a 0.96±0.08 7.98±0.11
c 1.91±0.03
b
EHGG 6.00 ±0.54 0.25±0.02c 0.95±0.07 14.96±1.51
a 2.00±0.02
a
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
guar fractions.
LSD Value: Moisture=1.3207, Ash=0.2078, Fat=0.1595, Protein=2.1453, Fiber=0.0845
CGG, crude guar gum; PGG, purified guar gum; AHGG, acid hydrolyzed guar gum; BHGG, base hydrolyzed
guar gum; EHGG, enzyme hydrolyzed guar gum
60
The results obtained in this study are comparable to the findings of Mudgil et al.
(2012c) who examined the decrease in moisture percent from 10.82 to 8.02 (difference of
2.80%) when compared the crude guar gum with hydrolyzed guar gum respectively. Their
results showed increase in ash contents which are in one way or the other supportive to
studies conducted in this manuscript particularly for ash increase AHGG and BHGG
whereas, in our case ash contents PGG and EHGG were decreased.
Increase in fiber contents in our case is also reinforced by the studies done by Mudgil
et al. (2012c) who obtained considerable quantity of total fibre and can be regarded as an
excellent source of total dietary fiber (TDF). The values for various chemical compositions
between our study vs López-Franco et al. (2013) when compared showed moisture contents
(6.54% vs 5.9%), protein (9.48% vs 5.1%), ash (0.66%) vs (0.42%) and fat (1.18% vs
0.005%) accordingly showing higher values than the data reported by them for crude guar
gum.
This also indicates that the varietal difference may exist in guar gum from different
sources showing variation in chemical composition. Other workers has also studied the
chemical composition with different results as Bourbon et al. (2010) reported higher
moisture (11.70%) and ash (0.72%) but lower protein (0.05%) content in guar gum when
compared to our results.
The overall results showed that chemical composition of guar gum analysed from
different sources and variety exhibited different values for moisture, ash, protein fat and fiber
and furthermore, it varies with the purification and hydrolysis when treated with acid/base
and enzymes. Variety, source and experimental protocols have definite effect on the outcome
of results. Other workers have also reported their results for the chemical composition: Gupta
et al. (2009b) reported the moisture (9-15%), protein (4.43%) and fiber (1.28%) contents for
the crude guar gum. The chemical composition of various guar gums on the basis of
genotype concluded that guar gum samples contained fat (0.33%) and ash (0.76%) contents
in the same range but moisture and protein contents varied from 7.35% to 12.31% and 0.5%
to 0.9% respectively (Gupta et al., 2009a). The protein contents were found to be in the range
of 3.2% to 4.0% and ash in range of 0.5% to 3.1% for various guar gums (Wang et al., 2003).
Moisture determined for different guar gum varied from 10.5% to 11.5% when studied by
Wang et al. (2006).
61
4.2.2. Rheological properties of Guar Gum
Rheology is the study of the flow and deformation behavior of materials. All
materials lie on a continuum between the ideal or Newtonian fluid and the ideal or Hookean
solid. Newtonian fluids have a viscosity that is dependent of the stress rate applied. On
dissolving, the galactomannans lose their ordered structure and go into disordered solution
i.e., “random coils” (Sittikijyothin et al., 2005)
4.2.2.1. Steady shear properties
The results for shear properties are presented in Fig. 4.2a and 4.2b. Under the heading
of shear properties, both shear rate and viscosity of crude guar gum and hydrolyzed guar gum
through acid, base and enzyme are studied. Fig. 4.2 shows the influence of shear rate on the
flow behavior curves of various aqueous guar solutions at 25°C. The experiments were
conducted for flow behavior, viscosity of aqueous solution (1%) of intact (CGG, PGG) and
hydrolyzed guar gum. (AHGG, BHGG, EHGG).
Intact CGG and hydrolyzed guar gums showed non Newtonian shear-thinning
behavior as it is evident from the results that when stress rate was applied to the solution of
respective guars, all of the guars showed decrease in viscosity. With 1% aqueous solution
concentration, SCGG (Sigma crude guar gum) exhibits the highest viscosity (18.59 Pa) while
AHGG (0.15 Pa) and EHGG (0.02 Pa) having the lowest viscosities (Fig 4.2b) showing the
effects of hydrolysis method. Purified guar gum (PGG) has the viscosity (0.22 Pa) lower than
the SCGG (18.59 Pa), CGG (1.35 Pa) and higher than the BHGG (0.06 Pa).
The values (see Appendix II) show that when the guar with different treatments was
subject to shear properties with 1% aqueous solution concentration, SCGG (sigma crude guar
gum) exhibited highest viscosity (18.59 Pa.s) whereas, AHGG (0.15 Pa.s) and EHGG (0.02
Pa.s) exhibited lowest viscosities (Fig 4.2b) due to hydrolysis of the guar gum. Purified guar
gum PGG showed the viscosity (0.22 Pa.s) which is again lower than the SCGG (18.59 Pa.s),
CGG (1.35 Pa.s), although higher than the obtained in BHGG (0.06 Pa.s).
Generally viscosity of all the tested guars was decreased with increase in shear stress
and rate applied. The values of viscosity at low shear rates is an indication of the consistent
behavior of the product (Morris and Taylor, 1982), while the values of viscosity at high
shear rates indicates viscosity of the product as a result of processing during industrial
operations (Dakia et al., 2008).
62
Fig 4.2 (a) Flow behaviour (b) viscosity of aqueous solution (1%) of crude and hydrolyzed guar gum
SCGG, sigma crude guar gum; CGG, crude guar gum; PGG, purified guar gum; AHGG, acidic
hydrolyzed guar gum; BHGG, basic hydrolyzed guar gum; EHGG, enzymatic hydrolyzed guar gum
63
The results of current study are supported by various researchers (Andrade et al.,
1999; Wientjes et al., 2000; Sittikijyothin et al., 2005; Bourbon et al., 2010). They stated that
shear thinning behavior arises from modifications in the macromolecular organization of the
solution as the shear rate changes. By increasing shear rate, disruption predominates over
formation of new entanglements; molecules align in the direction of flow and the viscosity
should be decreased (Sittikijyothin et al., 2005; Dakia et al., 2008).
Enzymatic degradation (β-mannanase) of guar galactomannan was studied using
steady shear viscometry. Under enzymatic hydrolysis, the solution (0.5% w/v) viscosity
decreased from 10 Pa.s to 0.01 Pa.s by over 2 orders of magnitude after 20 h showing a shear
thinning region at higher shear rates and Newtonian region at low shear rates. The
mechanism holds assurance for the controlled break down of electrolyte biopolymers in
industry, and provides a model for reviewing the enzyme-polymer interactions (Tayal et al.,
1999; Cheng and Prud‟homme, 2000). The trend of guar behavior for all the sample is also
buoyed by studies done by Bourbon et al. (2010) who stated that the viscosity decreases with
the increase in shear rate which is supportive to the findings in our studies.
Acid and enzymatic hydrolysis yielded guar galactomannans of different molecular
weights (MW). The Huggins coefficient for hydrolyzed guars is much lesser than crude guar,
signifying a weakening of intermolecular association in guar gum produced by enzymatic
and acid hydrolysis (Cheng et al., 2002). The weakening of intermolecular association would
help to solve the problem of phase separation in yogurt formation, increase concentration of
guar gum to be used and prebiotic effect enhance after hydrolysis. The findings of current
study may be in accordance with the studies of Mudgil et al. (2012c) who declared that
native and partially hydrolyzed guar gum exhibited a viscosity of 5500cps and 4cps with
molecular weight of 889742.06 Da and 7936.5 Da accordingly. The lessening in molecular
weight of PHGG was caused by the decrease in chain length size of guar gum.
In case of enzymatic hydrolysis of guar gum, rheological and physicochemical fea-
tures of guar gum significantly affected and resulted in to modify the guar gum from non-
Newtonian to Newtonian like behavior due to depolymerization as reported by Mudgil et al.
(2012a). They concluded that crude guar gum solution (1%) showed the viscosity (1.9 Pa.s)
at shear stress (55 Pa) while the enzymatically hydrolyzed guar found to be viscous with val-
ue 0.006 Pa.s at shear stress value of 6 Pa.
64
The rheological behavior of aqueous solutions of galactomannan with concentrations
(0.5% to 5%) was estimated at 25°C, using steady-shear and dynamic oscillatory measure-
ments. The solution with 0.5% concentration exhibited minimum value of viscosity
(0.002Pa.s) while 5% showed maximum value of viscosity (97.5 Pa.s) i.e. with increase in
solution percent the viscosity was increased and the aqueous solutions of gums exhibit shear
thinning Newtonian flow at low shear rate and non-Newtonian behavior at high shear rate
(Thombre and Gide, 2013). Whereas, molecular weight of gum was progressively reduced as
well as with the proceeding of the reaction when exposed to β-mannanase, confirming the
endo-action (i.e. depolymerization) of β-mannanase (Jian et al., 2013). It is observable that
the action of β-mannanase caused a reduction in viscosity of about 91.2% within the prelimi-
nary 10 min and chain length was declined by the breakage of the backbone of galactoman-
nan.
4.2.2.2. Oscillatory properties
Oscillatory measurements have been comprehensively used to study viscoelasticity of
polysaccharide solutions. Frequency sweeps of the storage (Gʹ) and loss moduli (Gʺ) in rheo-
logical measurement serve as a tool to differentiate between gels and polymer solutions. The
results of mechanical spectra of six guar fractions studied at a temperature of 25°C are shown
in Fig. 4.3, 4.4 and 4.5. It is evident from the results that values of moduli increased with in-
crease in frequency for all the six guar samples. The frequency sweeps for galactomannan
solutions showed the classic shape for macromolecular solutions at low frequencies (terminal
zone), the loss modulus, Gʺ, is higher than the storage one (Gʹ) whereas, at higher frequen-
cies, Gʹ is predominant. This kind of behavior was found for numerous galactomannans solu-
tions and other disordered random-coil polysaccharides (Oblonsek et al., 2003; Bourbon et
al., 2010).
The magnitude of Gʺ and Gʹ (see Appendix III) was in case of aqueous solution of
SCGG (0.858, 0.033 Pa), CGG (0.015, 0.0003 Pa), PGG (0.005, 0.00004 Pa), BHGG (0.002,
0.0003 Pa), AHGG (0.0007, 0.0002 Pa) and EHGG (0.0007, 0.0001 Pa) at low frequency.
The galactomannan solutions demonstrate a more pronounced viscous behavior, with higher
values of Gʺ and less elastic behavior Gʹ at lower frequency but at higher frequency gum so-
lution showed more value of Gʹ and less value of Gʺ. The values of Gʺ and Gʹ were at higher
65
Fig 4.3 Viscoelastic properties of aqueous solution of (a) SCGG (b) CGG at 25°C (SCGG, sigma crude guar gum; CGG, local crude guar gum)
66
Fig 4.4 Viscoelastic properties of aqueous solution of (a) PGG (b) BHGG at 25°C
(PGG, purified guar gum; BHGG, basic hydrolyzed guar gum)
67
Fig 4.5 Viscoelastic properties of aqueous solution of (a) AHGG (b) EHGG at 25°C (AHGG, acidic hydrolyzed guar gum; EHGG, enzymatic hydrolyzed guar gum)
68
frequency for SCGG (22880, 36290 Pa), CGG (25990, 32155 Pa), PGG (22190, 36240 Pa),
BHGG (23025, 33415 Pa), AHGG (13958, 37750 Pa), EHGG (22045, 34455 Pa).
The cross over frequency at which Gʹ and Gʺ become equal to each other was higher
in CGG (3.37 Hz), PGG (5.56 Hz) and BHGG (5.56 Hz) as compared to the SCGG (0.48
Hz), AHGG (0.12 Hz) and EHGG (1.28 Hz). The values of Gʺ and Gʹ were at cross-over fre-
quency for SCGG (15.55, 14.235Pa), CGG (4.816, 5.599 Pa), PGG (2.208, 2.546 Pa), BHGG
(2.32, 2.806 Pa), AHGG (0.0022, 0.0038 Pa), EHGG (0.106, 0.069 Pa) (Appendix III). The
cross over frequency steps towards the lower values as the concentration increases. This fea-
ture was described for several other random-coil polysaccharide solutions where Gʺ > Gʹ i.e.
the system exhibits a liquid-like behavior until a crossover frequency after which it is re-
versed and the elastic response prevails due to the highly structured nature of the polymer
(Brummer et al., 2003; Sittikijyothin et al., 2005).
It can be observed that the aqueous guar gum solution (0.1% to 4.0%) exhibits entan-
gled polymer solution behavior with the storage modulus Gʹ dominating over the loss modu-
lus Gʺ in the range of high frequency (Mao et al., 2012). Guar galactomannan solution dis-
played angular frequency dependence curves of the loss modulus and storage modulus that
were characteristic of an entangled polymer chains solution (Horinaka et al., 2012; Horinaka
et al., 2013). As expected, at the beginning, the guar aqueous solution served as a macromo-
lecular semi-dilute solution and the Gʺ (1.85 Pa) prevails over the Gʹ (0.65 Pa) presenting the
crossover frequency point of the moduli (27.5 Pa) at about 1 Hz. Moreover, both Gʹ and Gʺ
exhibit an apparent dependency on the frequency. At higher frequency, the magnitude of Gʺ
and Gʹ were 66 Pa and 78 Pa respectively (Sandolo et al., 2007).
The frequency sweeps for aqueous gum solutions (3%, 4%, 5%) presented typical shape of
macromolecular solutions (Gʺ > Gʹ at low frequency). Crossover of the moduli for gum
solution having 3%, 4%, 5% having values 0.03 Pa, 2.1 Pa and 112 Pa respectively was
perceived at the lowest concentration indicating the initiation of chain entanglement at low
concentrations (Thombre and Gide, 2013). The results acquired are in good settlement with
those described by Andrade et al. (1999) and Pollard et al. (2010).
69
4.2.2.3. Glucose absorption (Small Intestinal Model)
The influence of water soluble dietary fibres (guar gum, hydrolyzed guar gum) was
evaluated for glucose absorption in vitro in a small intestinal model (SIM) available in
School of Chemical Engineering, University of Birmingham, UK. Release of glucose from
the aqueous solution of crude and hydrolyzed guar gum was studied taking into consideration
the passage of time (90 min) as shown in Fig 4.6b. Standard calibration curve for various
glucose concentrations (0.0-10 mM) was drawn to measure the glucose concentration from
the tested samples has shown in Fig 4.6a (values are presented in Appendix IV).
The results obtained from the experiment declared that glucose absorption increased
from the lumen to annular side. The highest glucose concentration was measured at peak
time i.e. 90min. The glucose absorption study under the research was conducted on (CGG,
BHGG, AHGG and EHGG). The pattern of glucose release in the stomach model for 1%
glucose with segmentation and without segmentation indicated that there was highest release
of glucose 6.972 mM and 4.299 mM respectively after 90 min. The segmentation variations
of glucose release in SIM were purposely carried out for calculating the behavior of various
guar samples (crude and hydrolyzed) and differentiate with normal digestion process. In the
Fig 4.6a, standard curve for glucose was made to compare the concentration of glucose re-
lease at 540 nm absorbance whereas; the Fig 4.6b indicates the behavior of various hydro-
lyzed samples. The upper line (with segmentation) although shows higher glucose release
and lower line shows lower glucose release. Crude and hydrolyzed samples showed the simi-
lar pattern of glucose release (5.655 to 5.749 mM) in the anular side of SIM, indicating the
same physiological functions, and also attained in between the higher and lower glucose re-
lease. Resultantly, the pattern for all experimental samples and glucose alone indicated the
increasing trend with the passage of time (0-90 min).As the chain length and viscosity of hy-
drolyzed guar gum was reduced in the process of hydrolysis but the functional behavior of
guar gum in order to control the level of glucose, was not affected (Fig 4.6b).
The findings of current research are supported by Tharakan et al. (2010) who investi-
gated glucose absorption in guar solutions of varying viscosities. Incorporation of guar gum
gave rise to apparent reduction of glucose measured in the recipient side. It has been de-
scribed by Srichamroen et al. (2009) that galactomannan, a soluble fiber, reduce postprandial
blood glucose response. The uptake of low or high concentration of glucose was significantly
70
Fig 4.6 (a) Glucose calibration curve in a range from 0.0 to 10 mM concentration (b)
Absorption of glucose in 1% (w/v) guar solutions in small intestinal model (SIM); CGG,
crude guar gum; PGG, purified guar gum; AHGG, acidic hydrolyzed guar gum; BHGG,
basic hydrolyzed guar gum; EHGG, enzymatic hydrolyzed guar gum
71
and gradually minimized by increasing galactomannan concentrations in both obese and lean
rats. The findings propose that the galactomannan, due to its viscous property, has the ability
to decrease intestinal absorption of low or high glucose concentrations and hence for the ben-
efit of management of blood glucose.
A significant decrease in glycemic response was observed in 5% galactomannan
(GAL) group, when related to that of control and 2.5% GAL groups. The insulin plasma level
was as well apparently decreased (p < 0.001) in 5% GAL-fed rats (Srichamroen et al., 2008).
Guar gum reduced blood serum glucose only during the first month of the experiment, and no
variations were observed in the indices of protein utilization and absorption (Frias and Sgarb-
ieri, 1998). Thus the balanced diet containing fiber has acquired a certain role in the treat-
ment of obesity, hypercholesterolemia or hyperlipidemia and diabetes mellitus. It should be
given due significance in such therapies (Trivedi et al., 1998).
72
4.2.3. Measurement of Galactose-Mannose Ratio
Chemically, guar gum is a polysaccharide comprised of mannose and galactose
sugars units. The backbone is a linear chain of β 1,4-linked mannose residues to which
galactose residues are 1,6-linked at every second mannose, forming short side-branches. The
normal mannose and galactose ratio in guar gum is 2:1 as supported by Sabahelkheir et al.
(2012) during working on quality assessment of guar gum.
The crude (CGG), purified (PGG) and hydrolysed (BHGG, AHGG and EHGG)
samples of guar gum in our study were subjected to measurement of mannose and galactose
ratio through HPLC (Shimadzu 10 AL, Japan). The standards were consisted of 1% solutions
of galactose and mannose. The results are presented in Table 4.3 and 4.4. It is deduced from
the results that hydrolysis through acid, base and enzyme did not change the ratio of
galactose and mannose. However, the percentage of mannose and galactose was significantly
changed as depicted (Table 4.4). The effect of treatment was found to be highly significant.
CGG showed 73:35 which was the highest value for mannose and galactose followed by
PGG (71:33) and BHGG (67:33). The lowest values were obtained in EHGG (67:29) and
AHGG (66:30).
The findings of present study are supported by Cunah et al. (2007) who made purified
gaur gum by using different methods and found that mannose and galactose ratio remained
unchanged after process of purification. Kurakake et al. (2006) also reinforced the current
results while studying the production of galacto-manno-oligosaccharides from guar gum
through mannanase enzyme. Tapie et al. (2008) conducted study on natural galactomannans
and reported galactose and mannose ratio as 1:2 which supports results in this study. The
hydrolysis process expectedly reduce the chain length and molecular weight of the polymer
and finally the lower viscosity makes it an innovative soluble fiber that bear a resemblance to
the basic chemical structure with crude guar gum and possess a variety of applications in
clinical nutrition associated with dietary fiber ingestion (Slavin and Greenberg, 2003; Mudgil
et al., 2011).
73
Table 4.3 Mean squares for galactose-mannose of crude and hydrolyzed guar gum
Source Df Mannose Galactose
Treatment 4 1407** 94.2**
Error 5 0.0654 0.6368
Total 9
**=Highly significant (P < 0.01)
Table 4.4 Mean values for galactose-mannose (%) of crude and hydrolyzed guar gum
Treatment Mannose Galactose M/G Ratio
CGG 73 a 35 a 2:1
PGG 71 b 33 b 2:1
AHGG 66 d 30 c 2:1
BHGG 67 c 33 b 2:1
EHGG 67 c 29 d 2:1
Means with different letters are differ significantly at (P ≤ 0.05). Comparisons are made within the column for
each guar fractions to evaluate the sugar composition. CGG: Crude Guar Gum; PGG: Purified Guar Gum; AHGG: Acidic Hydrolyzed Guar Gum; BHGG: Basic
Hydrolyzed Guar Gum; EHGG: Enzymatic Hydrolyzed Guar Gum
74
4.2.4. Scanning Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM) can be performed to deliver information about
the sample's surface topography and composition. This is a powerful technique extensively
used to capture the characteristic „network‟ structure in polymers (El Fray et al., 2007).
SEM was carried out to observe the surface morphology of crude, purified, acid
hydrolyzed, basic hydrolyzed and mannanase hydrolyzed guar gum in order to verify visible
morphological modifications in material structure and to validate a final rearrangement of the
structure after hydrolysis process. The results of SEM are depicted in Fig 4.7 to 4.11 when
crude and hydrolyzed samples were checked at low magnification (X1000) and high
magnification (X2000) at 10µm for each sample. Crude guar particles exhibited small but
with rough surface morphology as revealed in the respective figure, which is helpful in
getting highly viscous aqueous solution. Guar gum existed in granular form without cross
linking network between the granules as shown in Fig 4.7.
A significant change (appearance) was observed in the surface morphology of the
guar gum after hydrolysis process. A clear difference between the crude, purified and
hydrolyzed guar gum was detected. A soft structure was formed when water molecules were
released during the lyophilisation of guar gum solution. Surface of hydrolyzed samples
indicated that morphological changes brought about by hydrolysis as deposits of the
hydrolyzed co-polymers were seen as compared to morphology of crude guar gum.
In PGG (Fig 4.8), it was observed that surface was rough and having compactness in
the molecular structure with high viscous solution. Viscosity is reduced when compared with
our previous results in comparison to CGG. BHGG (Fig 4.9) showed the agglomeration of
guar particles, compactness and rough surface morphology in their structure after hydrolysis.
Basic hydrolysis affected little to the structure of guar gum.
The extent of the effect to BHGG structure was less as compared to AHGG and
EHGG whereas; AHGG (Fig 4.10) showed the powdery and fluffy appearance after
hydrolysis. Acid hydrolysis of guar gum showed the maximum significant observable change
in its structure which might be due to higher metabolic rate yielding breakage of galactose
and mannose ratio, which is also confirmed due to reduction in viscosity of AHGG aqueous
solution as stated elsewhere. In EHGG, well defined porous structure was developed as it is
75
Fig 4.7 Scanning electron micrographs of crude guar gum (a) X1,000 (b) X2,000
(a)
(b)
76
Fig 4.8 Scanning electron micrographs of purified guar gum (a) X1,000 (b) X2,000
(b)
(a)
77
Fig 4.9 Scanning electron micrographs of base hydrolyzed guar gum (a) X1,000 (b) X2,000
(a)
(b)
78
Fig 4.10 Scanning electron micrographs of acid hydrolyzed guar gum (a) X1,000 (b) X2,000
(a)
(b)
79
Fig 4.11 Scanning electron micrographs of enzyme hydrolyzed guar gum (a) X1,000 (b)
X1,500
(a)
(b)
80
clearly indicated in Fig 4.11 which shows that excellent interconnected framework was
formed by the mannanase enzyme. However, the EHGG showed characteristics of
crosslinking, amorphous and porous structure. Although, the structure of EHGG was porous
and cross link but such type of structure has been known to provide health benefits by
increasing the calcium absorption that would be beneficial to the growth of bone cells when
added into the consumer‟s food as concluded by Hara et al. (1999) and Scholz-Ahrens et al.
(2007) while studying the effect of guar gum and various prebiotics on mineral absorption.
The average pore size, its distribution and interconnections are regarded as imperative
aspects of the hydrolyzed guar samples in promoting the physical characteristics of the
product particularly yogurt. Therefore, it is obvious that the actual granular morphology of
CGG is lost after acidic and enzymatic hydrolysis process and transformed into fine, fluffy
and well interconnected morphology, which is advantageous in working acceptable physical
behaviour of the product in which it would be added, along with imparting a similar rather
improved prebiotic endurance.
On the overall basis of SEM, guar gum particles were mostly ruptured on their
surface, although granules of crude guar gum have an asymmetrical but smooth surface and
are basically with no defects (Gong et al., 2012). In our study, results are supported in similar
manner with definite rupturing of granules and invariable forms of hydrolyzed morphological
structures with their respective functionalities.
Wang et al. (2010) also supported the idea of structural changes of guar gum
hydrolyzed in an alkaline environment when they conducted studies on swelling properties of
guar gum. Whereas Zheng et al. (2008) and Sen et al. (2010) were of the view that guar gum
generally found in granular structure and there was no cross linking between the granules. It
is obvious that the granular appearance of GG is lost after modification of guar and converted
into fibrillar morphology (Wang et al., 2006). It was experienced that a soft structure was
produced when water molecules were escaped from the guar gum solution during the
lyophilisation process (Cunah et al., 2005).
81
4.2.5. X-ray Diffraction Analysis
In XRD phenomena, constructive and destructive interference become visible when
molecular and crystalline structures are exposed to X-rays and solid matter can be described
as amorphous and crystalline (Birkholz, 2006).
X-ray diffraction (XRD) configuration of CGG, PGG, BHGG, AHGG and EHGG are
represented in Fig 4.12a, 4.12b, 4.13a, 4.13b and 4.14 respectively. Results demonstrated that
CGG, PGG and AHGG showed amorphous structure and exhibited low overall crystallinity
and peaks observed at diffraction angle (°2θ) of 20.2 as shown in Fig. 4.12a, 4.12b and
4.13b. Whereas, the crystalline regions of EHGG were slightly higher when seen at angle
(°2θ), peaks were observed at the diffraction of 20.4, 40.2 and 49.5 as shown in Fig. 4.14
which is indication of slight change in XRD curve. Basic hydrolysis augmented considerably
the crystallinity of the BHGG at angle (°2θ) seen at 20.5, 24.1, 26.0, 28.9, 31.4, 33.0, 34.3
and 42.8 as shown in Fig. 4.13a.
It is obvious from the results that crude guar gum exhibits a low crystallinity which is
supported by Gong et al. (2012) who concluded that a very small crystallinity of guar gum
was formed during the conducting experiment on characterization of guar gum and
rheological properties of its solution. Pal et al. (2007) reported the similar results while
working on characterization of cationic guar gum as flocculating agent. Zheng et al. (2008)
concluded that specific spectrum peak of guar gum near 2θ = 19.94° also found in the
spectrum, could be due to the weak crystallization or amorphous structure of guar gum.
Cunha et al. (2005) also declared that crude guar gum exhibited amorphous structure in the
range of 15-18 at diffraction angle (°2θ) suggested that the overall crystallinity in diffraction
band, even being low, increases after cross-linking in guar gum gel.
CGG and PHGG (enzymatic hydrolysis) presented amorphous structure. The former
is in line with our results whereas, the later showed a bit higher crystallinity in our study
which might be due to usage of enzyme, process, method or conditions adopted. GG and
PHGG presented less crystallinity at the angle (°2θ) in regions of 20.2 and 72.5. This means
that enzymatic hydrolysis of guar gum caused somewhat increased the crystallinity regions of
PHGG (Mudgil et al., 2012c). In another study, Mudgil et al. (2012a) reported crystallinity
index (%) measured for crude guar gum and PHGG was 3.86% and 13.2% accordingly. The
82
treatment of guar gum through enzymatic process caused an increase in crystallinity of
PHGG.
Fig 4.12 X-ray diffraction patterns of (a) CGG (b) PGG
(a)
(b)
83
Fig 4.13 X-ray diffraction patterns of (a) BHGG (b) AHGG
(b)
(a)
84
Fig 4.14 X-ray diffraction patterns of EHGG
85
4.2.6. Fourier Transform Infra-red Spectrophotometric analysis (FTIR)
Infrared (IR) spectroscopy is complementary technique that provides information on
molecular structure. Both quantitative and qualitative information can be attained by using
spectroscopy. Through unique pattern of absorption, a number of organic compounds and
functional groups can be identified, and the intensity of the absorption may be used for the
calculation of the relative concentration in the sampled entity (Wetzel and LeVine, 1999).
FTIR spectra of GG and PHGG were verified to compare the changes in their chemi-
cal structure and their characteristic IR wave number analysed is summarized in Table 4.5.
FTIR spectra of crude (CGG), purified (PGG) and partially hydrolyzed guar gum (BHGG,
AHGG and EHGG) are shown in Fig 4.15, 4.16 and 4.17.
In the IR spectra of guar gum, peaks were appeared in different regions. In hydro-
lyzed guar fractions peaks appeared in the range from 827.1060 cm-1
to 852.8397cm-1
indi-
cating the presence of alkyl halides (C-Cl stretch) which were not present in CGG and PGG.
Peaks appeared in all the guar fractions except BHGG in the range from 1038.6944cm-1
to
1198.8154cm-1
, 1627.7108cm-1
to 1647.7259cm-1
, 2313.9431cm-1
to 2339.6772cm-1
and
2685.6528cm-1
to 2802.8842cm-1
is due to aliphatic amines (C-N stretch), 1° amines (N-H
bend), nitriles (C≡N stretch) and aldehydes (H-C=O: C-H stretch) respectively. The sharp
peaks for nitro compounds (N-O symmetric stretch) in spectra of all the guar fractions ap-
peared was ranging from 1301.7503cm-1
to 1367.5142cm-1
while the peaks for aromatics (C-
C stretch in ring) only appeared in CGG (1559.0875cm-1
) and BHGG (1521.9166cm-1
). The
absorbance of nitro compounds (N-O asymmetric stretch) and for carboxylic acids appeared
in the range from 1501.9015cm-1
to 1507.6201cm-1
and 2611.3110cm-1
to 2694.3551cm-1
respectively in all the guar fractions except EHGG. In spectral array of CGG, PGG and
EHGG, peaks were observed in the range from 1719.2085cm-1
to 1782.1130cm-1
and
2851.4924cm-1
to 2911.5377cm-1
that were assigned to ketones (C=O stretch) and alkanes
(C-H stretch) respectively. The characteristics absorbance for alkynes was observed in PGG,
AHGG and EHGG in the range 2082.3399cm-1
to 2236.7423cm-1
. Another peak around
2356.8330 to 2379.7074 was observed in all the spectra except PGG which is possibly due to
ammonium ions (N-H).
86
Table 4.5 Functional groups evaluation of various guar gums at specific wave number (cm-1
) in Infra-
red spectral region
Compound Functional
group CGG PGG AHGG BHGG EHGG
C-Cl stretch Alkyl halides ----- ----- 852.8397 847.1211 827.1060
C-N stretch Aliphatic
amines 1041.5537 1147.3479 1038.6944 ----- 1198.8154
N-O symmetric
stretch
Nitro
compounds 1341.7805 1301.7503 1301.7503 1367.5142 1359.7391
N-O
asymmetric
stretch
Nitro
compounds 1504.7608 1507.6201 1505.7042 1501.9015 -----
C–C stretch
(in–ring) Aromatics 1559.0875 ----- ----- 1521.9166 -----
N–H bend 1° amines 1629.5945 1636.2887 1647.7259 ----- 1627.7108
C=O stretch Ketones 1782. 1130 1833.5806 ----- ----- 1719.2085
–C≡C– stretch Alkynes ----- 2236.7423 2156.6818 ----- 2082.3399
C≡N stretch Nitriles 2313.9435 2325.3807 2313.9431 ----- 2339.6772
N-H Ammonium
ions 2356.8330 ----- 2379.7074 2359.6923 2365.4109
O-H stretch Carboxylic
acids 2619.8889 2611.3110 2625.6075 2694.3551 -----
H-C=O: C-H
stretch Aldehydes 2685.6528 2780.0098 2802.8842 ----- 2788.5877
C-H stretch Alkanes 2911.5377 2851.4924 ----- ----- 2894.3819
CGG, crude guar gum; PGG, purified guar gum; AHGG, acid hydrolyzed guar gum; BHGG, base
hydrolyzed guar gum; EHGG, enzyme hydrolyzed guar gum
87
The findings of current study are supported by Mudgil et al. (2012c) who found the
spectral regions at various wave lengths. The region of FTIR spectra between 2800 and
3000cm-1
present C-H stretching modes. The peak in the spectra around 2600 cm-1
was due
to OH stretching vibration of carboxylic acid of polymer and water involved in hydrogen
bonding and spectra around 1700-1850cm-1
was C=O stretching vibrations of ketone group
(Prasad et al., 2012). In PHGG, sharpening of absorption band around 1627 cm-1
shows its
increased association with water molecule, which could be a description about its better solu-
bility compared to crude guar gum (Zheng et al., 2008). The protein content of the samples
could cause the presence of the absorption band at 1650cm-1
that is characteristic of the N-H
bending (amide bond) (López-Franco et al., 2013).
Additional characteristic absorption bands of gaur gum appears at 1607cm-1
and
1534cm-1
due to C=C stretching vibrations and N-H bending vibrations (Prasad et al., 2012).
Associated water molecule resulted in the band near 1650cm-1
in the spectra. The region
around 1400cm-1
due to CH2 bending vibration was also detected (Sun et al., 2004; Shobha et
al., 2005; Dodi et al., 2011; Gong et al., 2012; Mudgil et al., 2012c). The other key features
experienced were the spectral region between 800 and 1200cm-1
, which is due to highly cou-
pled C-C-O, C-OH and C-O-C stretching modes of polymer backbone (Kacurakova et al.,
2000; Kacurakova and Wilson, 2001; Sen et al., 2010).
The region between 500 and 700cm-1
is supposed to be sensitive to change in crystal-
linity that was indicative of conformational changes. Crystallinity index for depolymerized
guar galactomannan was higher than native, representing more crystallinity of the product,
which could be possible due to its smaller size (Shobha et al., 2005; Wang and So-
masundaran, 2007).
Crude, purified and chemical modified guar gum were characterized by FTIR spec-
troscopy (Dodi et al., 2011). FTIR spectra confirmed that there was no change in the func-
tional group or the basic molecular structure of guar gum with partially hydrolyzed guar gum
(Mudgil et al., 2012a; Mudgil et al., 2012c).
88
Fig 4.15 FTIR spectra of (a) CGG (b) PGG
(b)
(a)
89
Fig 4.16 FTIR spectra of (a) BHGG and (b) AHGG
(b)
(a)
90
Fig 4.17 FTIR spectra of enzyme hydrolysed guar gum (EHGG)
91
4.2.7. Thermo-gravimetric Analysis (TGA)
Thermo-gravimetric Analysis (TGA) is a simple and precise method for analysing the
decomposition pattern and the thermal stability of polymers. Thermal stability of the polymer
is an essential characteristic that could make the material suitable for food applications where
material is processed thermally using unit operations for instance baking and sterilization etc.
as studied by Mudgil et al. (2012c).
The results for TGA curves of CGG as shown in Fig 4.18 essentially indicate
involvement of three distinct zones of weight reduction. The initial weight loss was 11.715%
at 30-120°C which was due to the presence of moisture contents traces in the samples. The
second zone of weight loss ranged from 230-345°C showing the loss of 56.041% that could
be attributed to degradation of secondary alcohol -CHOH whereas, the third zone of weight
loss occurred at 345-760°C resulting in mass reduction of 32.243% which could be due to
degradation of backbone of polymer (primary alcohol -CH2OH). The results from are in line
with Sen et al. (2010) who declared that the mass loss of CGG occurs with the rise in
temperature in three distinct zones which is in coordination to results for CGG in our studies.
In case of PGG, weight loss occurred in four zones of temperature ranges as shown in
Fig 4.19. The first, second and third zones were in the range of 30-120°C, 255-405°C and
405-910°C exhibiting the weight loss of 9.825%, 69.509% and 11.271% accordingly. In
addition to the previous zones of weight loss, PGG had an extra zone which was in the range
of 910-1200°C displaying the weight reduction of 3.757%. The fourth extra zone of weight
loss as compared to CGG, might be due to the amide group (-CONH2) of the synthesized
polymer. Therefore, the existence of this additional zone is a clear indication that some
functional groups have been attached onto the backbone of guar gum.
TGA curve for BHGG presented in Fig 4.20, declared weight loss in four distinct
zones. Initial weight loss started at zone 30-110°C. This zone indicated the major loss about
40.282%. The second zone of weight loss involves the range of 220-390°C showing the
weight loss of 24.972%. Weight reduction of about 5.971% occurred in the third zone of
mass loss at 390-700°C whereas, fourth zone (700-980°C) contributing the weight loss of
polymer as about 10.857%. In Fig 4.21, TGA profile of AHGG shows the weight loss in five
zones. The first zone occurs at 30-110°C contributing to loss of moisture about 9.742%. The
92
Fig 4.18 Thermo gravimetric analysis (TGA) curve of crude guar gum (CGG)
Start = 30 End = 120 Weight loss = 0.763 mg (11.715%)
Start = 230 End = 345 Weight loss = 3.650 mg (56.041%)
Start = 345 End = 760 Weight loss = 2.10 mg (32.243%)
93
Fig 4.19 Thermo gravimetric analysis (TGA) curve of purified guar gum (PGG)
Start = 30 End = 120 Weight loss = 0.523 mg (9.825%)
Start = 255 End = 405 Weight loss = 3.70 mg (69.509%)
Start = 405 End = 910 Weight loss = 0.60 mg (11.271%)
Start = 910 End = 1200 Weight loss = 0.20 mg (3.757%)
94
Fig 4.20 Thermo gravimetric analysis (TGA) curve of base hydrolyzed guar gum (BHGG)
Start = 30 End = 110 Weight loss = 3.710 mg (40.282%)
Start = 220 End = 390 Weight loss = 2.30 mg (24.972%)
Start = 390 End = 700 Weight loss = 0.55 mg (5.971%)
Start = 700 End = 980 Weight loss = 1.0 mg (10.857%)
95
Fig 4.21 Thermo gravimetric analysis (TGA) curve of acid hydrolyzed guar gum (AHGG)
Start = 30 End = 110 Weight loss = 0.68 mg (9.742%)
Start = 215 End = 400 Weight loss = 4.0 mg (57.306%)
Start = 400 End = 710 Weight loss = 0.45 mg (6.446%)
Start = 710 End = 940 Weight loss = 0.90 mg (12.893%)
Start = 940 End = 1200 Weight loss = 0.80 mg (11.461%)
96
Fig 4.22 Thermo gravimetric analysis (TGA) curve of enzyme hydrolyzed guar gum(EHGG)
Start = 30 End = 105 Weight loss = 0.627 mg (10.233%)
Start = 210 End = 460 Weight loss = 4.20 mg (68.549%)
Start = 460 End = 900 Weight loss = 0.5 mg (8.160%)
Start = 900 End = 1200 Weight loss = 0.4 mg (6.528%)
97
second zone ranges in 215-400°C and resulted in distinct mass loss of polymer (57.306%).
The third zone (400-700°C) gave rise to relatively less weight loss of about 6.446%. The
fourth (710-940°C) and fifth (940-1200°C) zone contributed towards mass loss of 12.893%
and 11.461% respectively.
In EHGG, TGA thermo-gram as presented in Fig 4.22, thermal degradation comprises
of four major zones. Initial thermal degradation starts in the range of 30-105°C giving rise to
10.233% of moisture loss. The second zone (210-460°C) contributed distinct mass loss of
68.549%. The third (460-900°C) and fourth (900-1200°C) zones showed weight loss of
8.160% and 6.528% respectively.
The findings from the current studies are in line with those of Prasad et al. (2012)
who declared that thermal degradation of CGG was completed in two steps, 180-365°C and
390-504°C resulting in weight loss of about 60% and 18% respectively. While thermal
degradation of modified polymer was completed in three steps, at 150-300°C, 320-410°C and
420-500°C with the weight loss of about 30%, 20% and 27% in the first, second and third
steps of degradation accordingly. An extra zone of weight reduction in modified guar gums
declared the increased heat stability as compared to CGG. In another study, thermo-
gravimetric investigation exhibited reduction of weight in two phases. In the first phase,
minor weight loss in the samples may be attributed to the loss of adsorbed and structural
water of biopolymers or due to desorption of moisture as hydrogen bounded water to the
saccharide structure. The second weight loss event may be attributed to the decomposition of
polysaccharide as conducted by Bothara and Singh (2012).
An initial weight loss (8-12%) in the range 80-120°C was experienced by Iqbal et al.
(2011). They found that the first major phase of decomposition is characterized by the initial
decomposition temperature (IDT) in the range 220-270°C and final decomposition
temperature (FDT) in the range 310-375°C. The initial stage caused a weight loss of about
39-56% while in the second main decomposition stage, the IDT and FDT range was 415-
450°C and 490-550°C. The loss in weight was about 20% that is attributed to the complete
degradation of hydrogels.
TGA profiles of residual mass demonstrated increased stability of PHGG at higher
temperature range than crude guar gum. TGA results also exposed that no major change was
observed in the chemical structure of PHGG (Mudgil et al., 2012c). According to Zohuriaan
98
and Shokrolahi (2004), the integral procedural decomposition temperature (IPDT) values
calculated based on the TGA thermo-grams showed that modified gums were recognized to
be more thermal stable than other polysaccharides.
4.2.8. Haemolysis Study
The classical haemolysis of human RBCs is due to saponins, which are glycosides of
steroids, tri-terpenoids and sugars (Haralampidis et al., 2002; Woldemichael and Wink,
2001).
The results regarding haemolytic bioassays of CGG, PGG, BHGG, AHGG and
EHGG at various levels (2.5 to 250 mg/100mL), executed on human RBCs are shown in
Table 4.6. Standard references used were phosphate buffer saline (PBS) as negative control
(0% haemolysis) and Triton X-100 as positive control (100% haemolysis). All guar gum
fractions exhibited the absorbance in the range of 0.036 to 0.136 (Table 4.6), checked at 576
nm wave length, showing lower values of haemolysis when compared to positive and
negative control showing the absorbance value of 1.876 and 0.005 respectively at 576 nm. In
Fig 4.23, it is depicted that haemolysis percentage was increased with increasing
concentration for all the guar gum fractions. The highest and lowest percentage value of
haemolysis observed was 7.24% and 1.9% in AHGG (250 mg/mL) and EHGG (2.5 mg/mL)
respectively.
The comparative percent haemolysis of treatments with reference to Fig 4.23, the
findings of current study are supported by Hassan et al. (2010) who declared that 100%
methanolic soluble extract of guar meal was found to haemolyse red blood cells. Guar meal
(GM) is a by-product of guar gum processing, composed of hull and germ fractions
containing 13% DM crude saponin (Curl et al., 1986). In human clinical studies, conducted
by McIvor et al. (1985), non-insulin dependent diabetic patients were examined to evaluate
the effects of feeding 30g daily dose of guar gum for a period of 16 weeks. No changes were
observed in hepatic, haematologic, or renal functions. Serologic screening also did not
divulge any changes in protein, mineral or lipid metabolism.
The outcome of a 90-day study in rats also supported the safety of the partially
depolymerized guar gum prepared by alkaline oxidation. No adverse effects were revealed up
to the dose level of 50 g/kg diet estimated to amount to 2500 mg/kg body weight per day
(EFSA, 2004). In an acute and sub-chronic oral toxicity study, mice and rats were treated
99
Table 4.6 Haemolytic activity of controls (Triton-X 100, PBS solution) and five guar samples at various concentrations (mg/mL)
Optical absorbance at 576nm
Conc.
mg/mL
Positive control Negative control CGG PGG AHGG BHGG EHGG
2.5 1.876 ± 0.012a 0.005 ± 0.007
f 0.044 ± 0.022
d 0.044 ± 0.022
d 0.072 ± 0.012
b 0.065 ± 0.031
c 0.036 ± 0.022
e
5.0 1.876 ± 0.012a 0.005 ± 0.007
g 0.059 ± 0.032
e 0.065 ± 0.021
d 0.082 ± 0.021
b 0.069 ± 0.021
c 0.039 ± 0.028
f
25 1.876 ± 0.012a 0.005 ± 0.007
f 0.067 ± 0.042
d 0.068 ± 0.012
d 0.091 ± 0.018
b 0.072 ± 0.022
c 0.046 ± 0.022
e
50 1.876 ± 0.012a 0.005 ± 0.007
f 0.074 ± 0.021
d 0.073 ± 0.012
d 0.098 ± 0.022
b 0.079 ± 0.012
c 0.052 ± 0.034
e
75 1.876 ± 0.012a 0.005 ± 0.007
f 0.088 ± 0.024
c 0.081 ± 0.021
d 0.106 ± 0.018
b 0.086 ± 0.021
c 0.061 ± 0.022
e
100 1.876 ± 0.012a 0.005 ± 0.007
f 0.089 ± 0.021
d 0.089 ± 0.028
d 0.109 ± 0.023
b 0.091 ± 0.011
cd 0.065 ± 0.031
e
150 1.876 ± 0.012a 0.005 ± 0.007
e 0.096 ± 0.023
c 0.096 ± 0.012
c 0.117 ± 0.021
b 0.097 ± 0.023
c 0.073 ± 0.012
d
200 1.876 ± 0.012a 0.005 ± 0.007
f 0.100 ± 0.022
d 0.102 ± 0.013
cd 0.129 ± 0.012
b 0.104 ± 0.012
c 0.078 ± 0.021
e
250 1.876 ± 0.012a 0.005 ± 0.007
f 0.103 ± 0.059
d 0.106 ± 0.022
c 0.136 ± 0.032
b 0.106 ± 0.032
c 0.085 ± 0.018
e
The values are mean ± SD (n = 3)
Means with different superscripts differ significantly at (P ≤ 0.05). Comparisons are made within the row for each concentration of guar frac-
tions to evaluate the effect on haemolysis activity
CGG, crude guar gum; PGG, purified guar gum; AHGG, acid hydrolyzed guar gum; BHGG, base hydrolyzed guar gum; EHGG, enzyme hy-
drolyzed guar gum.
0.1% Triton-X 100, as positive control; PBS, phosphate buffer saline solution, as negative control.
100
Fig 4.23 Haemolytic activity, as a percentage of haemolysis caused by 0.1% triton X-100, for 2.5, 5.0, 25, 50, 75, 100, 150, 200, 250 mg mL-1
of
guar gum fractions and controls. CGG, crude guar gum; PGG, purified guar gum; AHGG, acid hydrolyzed guar gum; BHGG, base hydrolyzed
guar gum; EHGG, enzyme hydrolyzed guar gum. 0.1% Triton-X 100, as positive control (100% haemolysis); phosphate buffer saline, as
negative control (0% haemolysis)
101
with partially hydrolyzed guar gum at a dose of 6000mg/kg and as dietary admixture at
concentrations of 0.2, 1.0 and 5.0% for 13 weeks respectively (Koujitani et al., 1997) and 0,
500, and 2500mg/kg/day for 28 days (Takahashi et al., 1994). There was no lethal effect
attributable to PHGG in any examinations and proved to have no mutagenic potential.
Similarly another study was conducted in which group of male and female rats and
mice were served with diets containing 2.5 or 5.0% agar, guar gum, locust-bean gum, tara
gum, or gum arabic for 103 week. The administration of the test materials did not reveal any
histo-pathological effects. None of these polysaccharides showed carcinogenic effects for rats
or mice of each sex (Melnick et al., 1983). Acid-hydrolyzed guar gum is identical in
composition to the native guar gum. The main difference lies in chain length. Hydrolyzed
galactomannan polymers are of shorter chain length, resulting in changed viscosity
properties. The comparatively high salt content of the final product, resulting from the
neutralization of the acid, meets the specifications of Food Chemical Codex (FCC) (FCC,
2003).
Healthy female students were administered at 12.5 g/day of partially hydrolyzed guar
gum (PHGG, purity 80%, equivalent to 10 g of dietary fiber) in their meal. There was no
adverse reaction to the treatment reported (Sakata and Shimpo, 2006). The European Food
Safety Authority (EFSA) has documented the safety of partially depolymerized guar gum as a
stabilizer, emulsifier and thickener in food (EFSA, 2007).
102
(B) PRODUCT DEVELOPMENT
Objective:
The other main theme of this study was to assess the influence of crude, purified and hydro-
lyzed guar gum incorporation (at various levels) on the physicochemical, microbial viability
and sensory quality of yogurt. The yogurt produced by adding different guar gum as prebiotic
is presented in Fig 4.24 and 4.25. The yogurt is considered as a good carrier of probiotic.
Therefore, keeping in view the objectives of the study based on prebiotic (guar gum) and
probiotic (Bifidobacterium bifidum) endurance, the product (yogurt) was made and tested for
the following analysis.
4.3. Yogurt Analysis
4.3.1. Physico-chemical Analysis
4.3.1.1. Compositional analysis
i) Fat, ii) Total solids, iii) Lactose, iv) Ash, v) Protein
4.3.1.2. pH
4.3.1.3. Acidity
4.3.1.4. Viscosity
4.3.1.5. Syneresis
4.3.1.6. Water Holding Capacity (WHC)
4.3.1.7. Organic Acids
i) Acetic acid, ii) Lactic acid, iii) Citric acid, iv) Butyric acid, v) Pyruvic acid
4.3.1.8. Cryo-Scanning Electron Microscopy
4.3.2. Sensory evaluation
4.3.2.1. Color
4.3.2.2. Appearance,
4.3.2.3. Flavour
4.3.2.4. Body and Texture
4.3.2.5. Mouth feel
4.3.2.6. Overall acceptability
4.3.3. Microbial Analysis
4.3.3.1. Bifidobacterium bifidum (as probiotic)
4.3.3.2. Streptococcus thermophilus (as yogurt starter culture)
4.3.3.3. Lactobacillus bulgaricus (as yogurt starter culture)
103
(a)
(b)
(c)
Fig 4.24 Probiotic yogurts produced (a) without guar gum (b) crude guar gum, CGG and (c)
purified guar gum, PGG with levels (0.1, 0.5, 1%)
104
(a)
(b)
(c)
Fig 4.25 Probiotic yogurts produced with (a) base hydrolyzed guar gum, BHGG (b) acid
hydrolyzed guar gum, AHGG and (c) enzyme hydrolyzed guar gum, EHGG with levels (0.1,
0.5, 1%)
105
4.3. Yogurt Analysis
4.3.1. Physico-chemical Analysis
4.3.1.1. Compositional Analysis
i) Fat
Fat is important milk constituent. It helps to improve flavor, appearance and texture
of the product. Fat contents of milk vary from animal to animal and even from species to
species of same animal due to difference in their fatty acid profile and feed etc. Fat contents
of milk must be standardized for the processing of yogurt in order to achieve uniformity in
product at commercial levels. Normally it is standardized at 3.5% for yogurt production.
The statistical results presented in the Table 4.7 indicates the fat content in probiotic
yogurt treated with different guar fractions varied significantly during storage. The effect of
treatments and their interaction with storage days was found to be non-significant on fat
contents in various treatments. The results exhibited that initial fat content 3.50% was
reduced to 3.41% (Table 4.8) with a significant effect during storage whereas treatment
means showed non-significant values (Table 4.9) for fat in all yogurt samples treated with
guar gum fractions. The reduction in fat contents with the passage of time could be attributed
to the lipolytic activity of enzymes. The lipolytic effect of LAB in yogurt may also decrease
fat contents with the passage of time.
The findings of current study are in accordance with Huma et al. (2003) who
conducted studies on apple yogurt and reported decrease in fat contents from 3.38% to 3.34%
during 15 days of storage. Likewise, Dublin-Green and Ibe (2005) also stated reduction in fat
contents while studying pineapple fruit yogurt during 16 days of storage. Hussain (2004)
conducted studies on influence of various protein sources on keeping quality of yogurt and
reported a decreasing trend in the fat contents during storage of 21 days. The decreasing
trend of fat contents has also been reported by Multag and Hassan (2008) in concentrated
yogurt during storage of 21 days. Although some workers have quoted that there was no
change in fat contents during storage (Ayar et al., 2006; Janhoj and Michael, 2006; Anjum et
al., 2007; Eissa et al., 2011). They also elucidated that there was no effect of type and
amount of culture on fat contents of yogurt.
106
Table 4.7 Mean squares for compositional profile of probiotic yogurt during refrigerated
storage
SOV Df Fat Protein Ash Lactose Total
Solids
Days 4 18.11* 236.59** 1.16NS
1996.66** 241.34**
Treatments 16 0.81NS
157.24** 10.61* 27.71** 324.16**
C1 vs C2 1 0.38 NS
153.97** 0.02 NS
16.96** 41.46**
C vs others 1 0.18 NS
71.64** 1.69 NS
59.57** 244.81**
Gums 4 0.62 NS
222.32** 17.16** 41.83** 169.57**
Levels 2 0.16 NS
122.652** 7.42* 13.42** 1476.76**
Gums × Levels 8 1.20 NS
144.45** 10.56** 21.58** 158.56**
Days × Treatments 64 0.71NS 133.60** 7.63* 5.77* 231.31**
Error 170 0.593 1.246 0.158 1.072 1.811
Total 254
**=Highly significant (P< 0.01) *=significant (P< 0.05)
NS= non-significant (P> 0.05)
Table 4.8 Means storage values showing effect of crude and hydrolyzed guar gum on fat,
protein, ash, lactose and total solids (%) of probiotic yogurt
Parameters (%) 0 day 7 day 14 day 21 day 28 day
Fat 3.50a 3.47
b 3.46
bc 3.44
c 3.41
d
Protein 3.40d 3.69
a 3.45
c 3.24
e 3.64
b
Ash 1.03ab 1.03
ab 1.04a 1.04
a 1.02b
Lactose 5.14a 4.75
b 4.42c 4.16
d 3.87e
Total solids 12.38a 12.15
b 12.09c 11.98
d 11.77e
*Means with different letters are significantly differ from each other (P ≤ 0.05). Comparisons are made within
the row for each week of storage to evaluate the compositional effects. LSD Value: Fat=0.0231, Protein=0.0335, Ash=0.0119, Lactose=0.0310, Total solids=0.0403
107
Table 4.9 Means treatment values showing effect of crude and hydrolyzed guar gum on fat,
protein, ash, lactose and total solids (%) of probiotic yogurt
Treatments Fat % Protein % Ash % Lactose % Total solids %
To 3.47 3.80b 1.04
cde 4.42
g 11.92
f
Toʹ 3.45 3.41g 1.05
bc 4.30hi 11.67
i
T1 3.45 3.80b 1.05
bc 4.46
fg 11.70
hi
T2 3.45 3.65cd
1.04c 4.53
cde 11.82
g
T3 3.45 3.50ef 1.11
a 4.28i 12.22
e
T4 3.46 3.26h 1.04
cd 4.55
bcd 11.53
j
T5 3.43 3.52e 0.98
g 4.32
hi 13.06
a
T6 3.45 3.61d 0.96
h 4.42g 12.75
b
T7 3.46 3.26h 1.02
ef 4.36h 11.70
hi
T8 3.44 3.09i 1.02
def 4.49ef 11.97
f
T9 3.49 3.89a 0.99
g 4.47
fg 12.60
c
T10 3.46 3.22h 1.01
f 4.61
ab 11.54
j
T11 3.47 2.87j 1.07
b 4.57abc 11.75
gh
T12 3.43 3.45fg
1.11a 4.62
a 12.77
b
T13 3.45 3.76b 1.00
fg 4.61ab 11.69
hi
T14 3.47 3.68c 1.02
def 4.50def 12.16
e
T15 3.46 3.45fg 1.10
a 4.46fg 12.40
d
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions to evaluate the compositional effects.
LSD Value: Fat=0.0426, Protein=0.0617, Ash=0.0220, Lactose=0.0572, Total solids=0.0744
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
108
ii) Protein
In milk there are two major groups of proteins i.e., caseins and whey proteins.
Caseins proteins are one of the major sources of calcium phosphate in human diet and whey
proteins that offer large amount of essential amino acids. Whey proteins attach to casein
micelle leading to gel formation, due to neutralization of charge of casein micelle (Haug et
al., 2007; Aziznia et al., 2008). In yogurt formation, protein is essential ingredient to control
the syneresis and give assurance of solid consistency (Bake, 2003).
The protein contents in probiotic yogurt treated with different guar fractions was
highly significant (P<0.01) for storage days, treatments and their interactions. Data presented
in Table 4.8 depicted means of protein (%) as an effect of treatments. Protein contents varied
from 3.69% to 3.24% randomly during storage period of 28 days. Variation in protein
contents may be attributed to the proteolytic activity of enzymes. Results specified in Table
4.9, showed that overall means for treatment exhibited highest protein contents 3.89% in T9
(1% AHGG) followed by 3.80% in T1 (1% CGG), 3.76% in T13 (0.1% EHGG) and 3.68% in
T14 (0.5% EHGG) whereas, the lowest value was 2.87% in T11 (0.5% BHGG).
The findings of the current study are in accordance with El-Owni and Mahgoub
(2012) who concluded that there was a higher variation in protein content showing haphazard
trend during storage, while working on effect of storage on different characteristics of yogurt.
The results are also supported by Koestanti and Romziah (2008) who reported that during the
fermentation process, microbe biomass were increased, thus the sum of microbe protein was
increased, that automatically increasing protein inside the yogurt. Serra et al. (2009) worked
on proteolysis of yogurts during 28 days storage and reported that due to hydrolysis of
caseins, increase in production of hydrophobic peptides was observed during storage, which
might be due to the increase in soluble nitrogen at the end of the storage.
iii) Ash
Ash comprises minerals part of food commodities. After burning of organic matter,
the remaining contents generally called as ash. Calcium and phosphorus are among the major
minerals present in yogurt. Calcium has key role in bone formations and increases their
strength. It also helps to improve immune system and decrease cholesterol level (Shah,
2007).
109
The statistical results presented in the Table 4.7 indicated that the ash contents in
probiotic yogurt treated with different guar fractions varied significantly (P<0.05) among
treatments and their interactions whereas the effect of storage period was found to be non-
significant (P>0.05). Ash contents varied from 1.02% to 1.04% as presented in Table 4.8,
during storage of 28 days. Results specified in Table 4.9, showed that overall means for
treatments showed highest ash contents 1.11% in T3 (1%CGG) and T12(1% BHGG) followed
by 1.10% in T15 (1% EHGG), 1.07% in T11 (0.5% BHGG) and1.05% in Toʹ and T1 (0.1%
CGG) whereas, the lowest value was 0.96% in T6(1% PGG).
The findings of current research are in accordance with Bano et al. (2011) who
reported that ash contents showed non-significance throughout the storage of 28 days while
working on preparation of functional yogurt. The results are also in agreemet with those of El
Owni and Mahgoub (2012) who studied effect of storage on chemical, microbial and sensory
characteristics of yogurt. They declared that ash content was not significantly affected during
12 days of storage.
iv) Lactose
Lactose is natural milk sugar composed of glucose and galactose. LAB breakdown
lactose and convert it into lactic acid resulting in droping pH causing milk coagulation in
yogurt formation. As it is main sugar is utilized by LAB therefore, amount of lactose
continues to decrease after milk coagulation (Bourlioux and Pochart, 1998; Voet and Voet,
2004)
The statistical results presented in the Table 4.7 indicated that the lactose contents in
probiotic yogurt treated with different guar fractions varied highly significantly during
storage period and among treatments whereas, their interaction was found to be significant.
Decrease in lactose contents was from 5.14% to 3.87% as presented in Table 4.8, during
storage period. Decrease in lactose contents may be attributed to the utilization of lactose by
LAB (Anjum et al. 2007). Results specified in Table 4.9, showed that overall means for
treatment exhibited highest lactose contents 4.62% in T12 (1% BHGG) followed by 4.61% in
T10 (0.1% BHGG) and T13(0.1% EHGG), 4.57 in T11 (0.5% BHGG) and T4 (0.1% PGG)
whereas, the lowest value was 4.28% in T3(1% CGG).
Findings of the study are in accordance with those of Saccaro et al. (2009) who
observed a decrease in lactose contents of yogurt during storage. Egwaikhide and Faremi
110
(2010) reported that LAB present in the yogurt consumes milk lactose during fermentation
with varying rate resulting in pronounced differences in the acidity due to more lactic acid
production.
v) Total Solids
The percent residue after drying is called as the total solids. Total solids are
considered as all constituents of yogurt other than water. Thus all other constituents like fat,
protein, ash, lactose etc. whenever effected; they give a pronounced change in contents of
total solids.
The statistical results presented in the Table 4.7 indicated that the total solid contents
in probiotic yogurt treated with different guar fractions varied highly significantly (P<0.01)
during storage days, treatments and their interactions. Data regarding total solids depicted
that total solid contents showed a highly significant variation throughout storage. Total solid
contents varied from 11.77% to 12.38% as presented in Table 4.8, during the storage period.
Results specified in Table 4.9, showed that overall means for treatment exhibited highest
total solid contents 13.06% in T5 (0.5% PGG) followed by 12.77% in T12 (1% BHGG),
12.75% in T6 (1% PGG) and 12.60% in T9 (1% AHGG) whereas, the lowest value was
11.53% in T4(1% PGG).It is also evident from the Table 4.9 that total slid contents were
increased by increasing the level of gum added.
The current findings are supported by Anjum et al. (2007), who declared that total
solids decreased gradually during storage period. In another study, Abdel Moneim et al.
(2011) stated that total solid contents of yogurt decreased from 8.62% to 6.0% during 15
days of storage at 6ºC. Khalifa et al. (2011) and Kavas et al. (2003) specified that the total
solids increased with the passage of time during storage which is in contradiction to current
findings.
111
4.3.1.2. pH
The pH of yogurt is of great importance for many technical and scientific reasons as it
identifies shelf life of the product. It is highly influenced by biochemical changes during
production, fermentation and storage of the product.
The results pertaining to the statistical analysis of variance (ANOVA) for the pH of
probiotic yogurt is presented in Table 4.10 and mean values are presented in Table 4.11.The
statistical results indicated that pH of yogurt samples differed highly significantly (P<0.01)
for storage days and treatments whereas their interaction (days × treatments) was found to be
significant (P<0.05).
Data regarding pH depicted that the storage interval exhibited a decreasing trend. The
mean value for pH at 0 day of storage was 4.46 and it was reduced to 4.14 after 28th
day of
storage on overall basis (Table 4.11). The decreased throughout the storage is due to activity
of lactic acid bacteria that convert lactose into lactic acid that add acidity in the product
which inversely decrease pH. Therefore, decreased in pH is an indicative to increase in
acidity as a function of lactose conversion into lactic acid (Salvador and Fiszman, 2004).
The results given in Table 4.11 indicates that overall mean for treatment showed
maximum pH value 4.324 in To followed by 4.312 in Toʹ and 4.292 in T2 (0.5% CGG)
whereas, the lowest value was observed in T14 (0.5% EHGG) as 4.256.The control samples
showed higher value of pH, but these are comparable with the experimental treatments
showing highly significant difference as presented herein. In the results, values with same
letters indicate non-significant difference whereas different letters are indicating the
significant effectiveness of treatments on pH. It is apparent from the results that AHGG (1%)
and EHGG (0.5%) showed less pH as comparatively because of the increased activity of
bacteria and due to increased prebiotic effect of guar gum after hydrolysis (Yoon et al.,
2006). Acid and enzyme hydrolysis of guar gum with reduced chain length and viscosity
appears more acceptable for yogurt formulation (Yoon et al., 2008; Mudgil et al. 2011).
In case of interaction highest mean value of pH observed was 4.53 in To (control) at 0
day of storage which was changed to 4.14 at 28th
day of storage whereas, the lowest pH
(4.10) was found in T14(0.5% EHGG) at 28th
day of storage.
112
Table 4.10 Mean squares for physico-chemical analysis of probiotic yogurt during refrigerated storage
SOV Df pH Acidity Viscosity Syneresis WHC
Days 4 2900.67** 528.77** 1801.38** 1931.14** 49400.8**
Treatments 16 27.05** 32.24** 758.47** 96.94** 6186.55**
C1 vs C2 1 4.03* 5.98* 6.49** 2.73** 59.2**
C vs others 1 95.11** 48.34** 85.08** 606.00** 1935.70**
Gums 4 60.68** 24.10** 302.77** 99.26** 6758.3**
Levels 2 19.24** 87.11** 3241.20** 60.41** 15214.1**
Gums × Levels 8 6.55** 23.85** 543.81** 53.06** 4941.1**
Days × Treatments 64 5.20* 8.99* 39.50* 9.92* 1110.99**
Error 170 0.09740 0.12273 2830176 673.0 29.4
Total 254
**=Highly significant (P< 0.01) *=significant (P< 0.05)
113
Table 4.11 Means values showing effect of guar gum and storage time on pH of probiotic
yogurt
Days of Storage
Treatments 0 7 14 21 28 Mean
To 4.53±0.01 4.42±0.01 4.32±0.01 4.21±0.02 4.14±0.01 4.324a
Toʹ 4.51±0.02 4.38±0.01 4.29±0.02 4.22±0.01 4.16±0.01 4.312b
T1 4.48±0.01 4.37±0.01 4.26±0.02 4.19±0.01 4.13±0.01 4.286cd
T2 4.46±0.01 4.33±0.02 4.28±0.02 4.21±0.01 4.18±0.02 4.292c
T3 4.44±0.02 4.36±0.02 4.27±0.01 4.18±0.02 4.12±0.01 4.275def
T4 4.48±0.06 4.37±0.01 4.32±0.01 4.26±0.02 4.17±0.01 4.320ab
T5 4.45±0.01 4.35±0.01 4.31±0.02 4.25±0.01 4.19±0.01 4.311b
T6 4.44±0.03 4.36±0.02 4.33±0.01 4.27±0.01 4.20±0.01 4.320ab
T7 4.44±0.03 4.35±0.02 4.29±0.01 4.20±0.01 4.11±0.02 4.278de
T8 4.43±0.01 4.33±0.01 4.28±0.02 4.21±0.01 4.12±0.02 4.274ef
T9 4.40±0.02 4.32±0.01 4.28±0.01 4.22±0.02 4.14±0.01 4.272ef
T10 4.51±0.02 4.34±0.01 4.30±0.01 4.24±0.01 4.19±0.01 4.316ab
T11 4.47±0.01 4.35±0.02 4.28±0.01 4.19±0.01 4.11±0.01 4.280de
T12 4.46±0.01 4.34±0.01 4.27±0.02 4.18±0.02 4.12±0.01 4.274ef
T13 4.47±0.02 4.32±0.01 4.29±0.01 4.20±0.01 4.13±0.02 4.282cde
T14 4.44±0.02 4.33±0.02 4.25±0.01 4.16±0.01 4.10±0.02 4.256g
T15 4.43±0.02 4.32±0.01 4.28±0.01 4.19±0.02 4.11±0.01 4.266fg
Mean 4.46a 4.35
b 4.29
c 4.21
d 4.14
e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions and in row for storage to evaluate the pH effects. (Overall treatment mean; Max.
value = 4.32, Min. value = 4.25) LSD value Days=0.0063, LSD value Treatments =0.0118, LSD value Interactions (D×T) =0.0264
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
114
The pH values obtained in this manuscript are in accordance with the findings of Cruz
et al. (2013) who reported that the storage time has significant effect on pH. They
documented that decrease in pH during storage of yogurt was as result of formation of lactic
acid by the activity of lactic acid bacteria. The current result is also in accordance with
findings of Mazloomi et al. (2011) who conducted a study to examine the attributes of
synbiotic yogurt up to 14 days. They observed a substantial decrease in pH (6.61 to 4.48)
during storage as a function of increase in acidity.
Panesar and Shinde (2011) strengthened this instant exploration that drop in pH of
yogurt is due to utilization of residual carbohydrates mainly lactose by microorganisms for
production of lactic acid, CO2 and formic acid. However, some researcher stated that this
phenomenon is due to production of residual enzymes produced by starters during
fermentation (Christopher et al., 2009). Recently Prasanna et al. (2013) also reported
decrease in pH with the passage of time which supports results obtained in this manuscript.
It is therefore, presented through the results that pH in the study reduces with the passage of
time as well as it was altered due to treatments of guar gum applied for the production of
yogurt as prebiotic. Among treatments EHGG and AHGG showed good relation to the
stability of the product indicating good combination of guar gum as prebiotic with
bifidobacterium as probiotic combination and the steady change in pH seems more stable
product.
115
4.3.1.3. Acidity
Acidity expresses the total percentage of lactic acid production. LAB in yogurt acts
on milk sugar (lactose) and convert it to lactic acid that produce acidic flavor. Acidity is
directly affected by a number of bacteria and storage period. Increase in the acidity has a
negative impact on yogurt quality leading to deterioration of product resulting with syneresis
and instable character of yogurt.
The results concerning analysis of variance (ANOVA) for the acidity of probiotic
yogurt is presented in Table 4.10 and for mean values is presented in Table 4.12. The
statistical results exhibited highly significant (P<0.01) effect on acidity due to storage days
and treatments whereas their interaction (days × treatments) was found to be significant
(P<0.05).
Data illustrated that the storage time presented highly significant influence on acidity
with an increasing trend. The mean value for acidity at 0 day of storage was 0.944% and it
was increased to 1.171% after 28th
day of storage on overall basis (Table 4.12). The increase
in acidity during storage period is an effect of lactic acid bacteria that convert lactose into
lactic acid (Salvador and Fiszman, 2004).
The overall means for treatment showed highest acidity value 1.091% in T9 (1%
AHGG) followed by 1.085% in To and 1.067% in T1 (0.1% CGG) and T15 (1% EHGG)
whereas, the lowest value was observed in T2 (0.5% CGG) as 0.913%. The control samples
showed relatively higher value of acidity, but these are comparable with the experimental
treatments showing highly significant difference as presented herein. In the results, values
with same letters indicate non-significant difference whereas different letters are indicating
the significant effectiveness of treatments on acidity. It is apparent from the results that
AHGG (1%) and EHGG (0.5%) showed more acidity comparatively because of the increased
activity of bacteria and due to increased prebiotic effect of guar gum after hydrolysis (Yoon
et al., 2006). As far as interaction is concerned highest mean value of acidity observed was
1.280% in T10 (0.1% BHGG) at 28th
day of whereas, the lowest acidity (0.770%) was found in
T2 (0.5% CGG) at 0 day of storage. The findings of current study are in accordance with the
outcomes of Shaghagi et al. (2013), they reported an increase in acidity during storage when
116
Table 4.12 Means values showing effect of guar gum and storage time on acidity (%) of
probiotic yogurt
Days of Storage
Treatments 0 7 14 21 28 Mean
To 1.040±0.010 1.047±0.006 1.077±0.035 1.110±0.050 1.150±0.070 1.085ab
Toʹ 0.980±0.040 1.043±0.005 1.067±0.015 1.090±0.050 1.123±0.050 1.061cd
T1 0.940±0.010 0.993±0.005 1.067±0.050 1.083±0.010 1.250±0.015 1.067bc
T2 0.770±0.030 0.773±0.030 0.933±0.035 1.043±0.025 1.047±0.025 0.913i
T3 0.940±0.010 1.020±0.010 1.037±0.015 1.043±0.005 1.147±0.046 1.037ef
T4 0.993±0.015 1.010±0.010 1.060±0.040 1.060±0.040 1.093±0.006 1.043def
T5 0.950±0.036 0.980±0.030 1.020±0.020 1.047±0.006 1.090±0.040 1.028fg
T6 0.897±0.045 1.013±0.025 1.013±0.015 1.050±0.020 1.243±0.015 1.043def
T7 0.990±0.010 1.000±0.010 1.020±0.020 1.087±0.015 1.133±0.025 1.046def
T8 0.910±0.010 0.973±0.035 1.033±0.015 1.030±0.030 1.193±0.015 1.028fg
T9 0.990±0.020 1.040±0.010 1.097±0.005 1.123±0.020 1.207±0.035 1.091a
T10 0.897±0.015 0.987±0.005 1.040±0.010 1.093±0.035 1.280±0.036 1.057cde
T11 0.933±0.057 0.980±0.005 1.010±0.010 1.040±0.010 1.060±0.020 1.006h
T12 0.960±0.020 0.993±0.015 0.997±0.005 1.010±0.020 1.237±0.045 1.039ef
T13 0.960±0.010 0.977±0.015 0.990±0.010 0.997±0.015 1.270±0.010 1.039ef
T14 0.987±0.012 1.007±0.005 1.030±0.010 1.080±0.010 1.153±0.005 1.057cde
T15 0.930±0.026 0.973±0.020 1.007±0.015 1.193±0.045 1.233±0.015 1.067bc
Mean 0.944e 0.989
d 1.029
c 1.069
b 1.171
a
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions and in row for storage to evaluate the acidity effects. (Overall treatment mean;
Max. value = 1.28%, Min. value = 0.770%) LSD Value Days=0.0105, LSD Value Treatments =0.0194, LSD Value Interactions (D × T) =0.0433
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
117
when studying the effect of prebiotics incorporation on the quality of synbiotic yogurt.
Similarly, in another study Khalifa et al. (2011) found that the acidity increases with the
increase of storage interval while evaluating the application of stabilizers in yogurt
production during 10 days of storage. Chougrani et al. (2008) also reported the similar
finding while conducting studies on use of lactic acid strains in the yogurt manufacture.
Likewise, Gueimonde et al. (2003) also concluded that the acidity of yogurt increases with
the increased storage period due to microbial activity and lactose change into lactic acid. As
acidity is reverse of pH so some researchers correlate effect of pH with acidity as pH of
sample decreases, acidity will increase resulting in more bitter taste and increased whey
separation (Ahmad et al., 2008). Karaca (2013) studied the effect of different prebiotic
stabilizers and types of molasses on different characteristics of probiotic set yogurt and
reported an increase in acidity with the passage of time.
118
4.3.1.4. Viscosity
Viscosity is a measure of internal resistance or thickness of fluid of any substance
expressed in centipoises (cps). In case of yogurt viscosity can be increased by adding
stabilizers like gums, pectin, CMC etc. If conditions become unfavourable during prolonged
storage time, syneresis starts to increase, resulting in decrease in viscosity of the yogurt.
The analysis of variance (ANOVA) for the viscosity of probiotic yogurt is presented
in Table 4.10 and means values are presented in Table 4.13. The statistical data indicated that
viscosity of yogurt samples differed highly significantly among storage days and treatments
whereas their interaction (days × treatments) was found to be significant. Data regarding
viscosity showed highly significant effect of storage time exhibiting a decreasing trend. The
mean value for viscosity at 0 day of storage was 2681.4cps and it was reduced to 787.3cps
after 28th
day of storage on overall basis (Table 4.13). The decrease in viscosity may be as a
result of increased syneresis with the passage of time. This could be due to increased acidity
or increase in lactic acid.
The results (Table 4.13) indicated that overall mean for treatment showed maximum
viscosity 3000.0cps in T6 (1% PGG) followed by 2869.3cps in T3 (1% CGG) and 2363.2cps
in T15 (1% EHGG) whereas, the lowest value was observed in To as 1514.7cps. The control
samples showed lower value of viscosity, but these are comparable with the experimental
treatments showing highly significant difference. The results showed that 0.5% concentration
of CGG, PGG and BHGG exhibited ineffective use and showed no resistance towards the
spindle movement in DV-E viscometer. Inappropriate gel formation due to phase separation
could be one of the reasons for lower viscosity. results obtained in this study are in line with
those of Rohart and Michon (2013) who concluded that at low guar gum concentrations a
denser network was formed whereas higher guar gum concentrations lead to phase separation
(filamentous or protein-rich droplets) during their work on designing microstructure into acid
skim milk/guar gum gels. At higher level (1%), the results presented in Table 4.13, declared
the different behvior was exhibited with all guar gum fractions towards the yogurt formation
but which guar gum is suitable as far as viscosity of the product is concerned. In case of
CGG, PGG and BHGG, although yogurt gel was not formed but experimental treatments
comparatively showed higher viscosity. The findings are supported by Yoon et al. (2008)
who reported that 1 % solution of CGG showed viscosity 2000-3000 Pa.s in comparison to
119
Table 4.13 Means values showing effect of guar gum and storage time on viscosity (cps) of
probiotic yogurt
Days of Storage
Treatments 0 7 14 21 28 Mean
To 2173.3±70.2 1966.7±61.1 1786.7±100.7 946.7±100.7 700.0±34.6 1514.7j
Toʹ 2380.0±242.5 2073.3±189.3 1500.0±103.9 1246.7±130.1 973.3±98.6 1634.7i
T1 2306.7±102.6 2140.0±249.8 2220±60 1840±125 1193.3±133.2 1940.0g
T2 360±60 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000 72.000k
T3 5060.0±192.8 3820.0±202.9 2240±120 1793.3±70.3 1433.3±61.01 2869.3b
T4 3253.3±64.3 2800±125 2240.0±183.3 1513.3±13.2 1200.0±72.1 2201.3e
T5 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000 0.0000k
T6 4540.0±150.9 3940.0±295.9 3120±80 2340.0±111.3 1060.0±111.3 3000.0a
T7 2586.7±110.5 2386.7±133.2 1920.0±121.6 1386.7±70.2 820.0±121.6 1820.0h
T8 2686.7±133.7 2486.7±83.3 2053.3±213.8 1440.0±111.3 770±36 1887.3gh
T9 3180±60 2753.3±202.3 2346.7±110.5 1133.3±117.2 763.3±68.1 2035.3f
T10 3160.0±91.6 2666.7±122.2 2240.0±341.2 1506.7±133.2 853.3±94.5 2085.3f
T11 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000 0.0000k
T12 3353.3±147.4 2940.0±158.7 2460±80 1413.3±50.3 953.3±70.2 2224.0e
T13 3360±159 3100±308 2260.0±150.9 1640.0±144.2 900.0±52.9 2252.0de
T14 3573.3±30.5 2986.7±172.4 2760±140 1540.0±91.6 840.0±72.1 2340.0cd
T15 3610.0±149.3 3160.0±249.8 2440±140 1682.0±153.1 923±105 2363.2c
Mean 2681.4a 2307.1
b 1858.0
c 1260.2
d 787.3
e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions and in row for storage to evaluate the viscosity effects. (Overall treatment mean;
Max. value = 5060, Min. value = 770) LSD Value Days=50.439, LSD Value Treatments =93.004, LSD Value Interactions (D × T) =207.96 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
120
1% solution of PHGG that showed viscosity below 10 Pa.s as hydrolysis of guar gum reduces
its viscosity.
Interaction among the treatments and storage days was found to be significant.
Highest mean value of viscosity observed was 5060.0cps in T3 (1% CGG) at 0 day of storage
which was changed to 1433.3cps at 28th
day of storage whereas, the lowest viscosity
(700.0cps) was found in To at 28th
day of storage.
In current study, the difference in viscosity owing to storage period is related to
findings reported by Ramasubramanian et al. (2008) who observed decrease in viscosity due
to increased syneresis during storage of probiotic yogurt. In another study on quality features
of yogurt containing probiotic and different prebiotics, decrease in viscosity was observed
during 5 weeks storage. This reduction may be explained by the action of bacterial enzyme
action on the casein micelle matrix over time (Kosikowski, 1997; Aryana and MacGrew,
2007).
121
4.3.1.5. Syneresis
Whey separation (wheying-off) is defined as the removal of watery portion from the
network which then becomes visible as surface whey. Wheying-off affects negatively to
consumer’s perception of yogurt. Yogurt producers use stabilizers such as gelatin, pectin and
starch in order to prevent wheying-off. The typical reason of whey separation is spontaneous
syneresis that is attributed to the contraction of gel without application of any external force
(Amatayakul et al., 2006; Lee and Lucey, 2010).
The results concerning analysis of variance (ANOVA) for the syneresis of probiotic
yogurt are presented in Table 4.10 and mean values are presented in Table 4.14. It is
indicated that syneresis differed highly significantly (P<0.01) among storage days and
treatments whereas their interaction (days × treatments) was found to be significant (P<0.05).
Data depicted (Table 4.14) that the storage time had a highly significant influence on
syneresis with an increasing trend with the passage of time. The mean value for syneresis at 0
day of storage was 47.41% that and it was increased to 79.06% at 28th
day of storage on
overall basis. The increase in syneresis may be due to the activity of the lactic acid bacteria
(LAB) and Bifidobacterium bifidum. Increased whey separation is attributed to an unstable
and excessive rearrangement of weak network of gel (Lucey, 2001).
The results (Table 4.14) indicated that overall mean for treatment showed maximum
syneresis 74.2% in T2 (0.5% CGG) followed by 72.8% in T5 (0.5% PGG), 72.6% in T11
(0.5% BHGG) and 69.4% in T3 (1% CGG) whereas, the lowest value was observed in T14
(0.5% EHGG) as 59.8%. The results on overall basis indicated that syneresis increased in
controlled as well as treated sample, particularly discussing T7, T8, T13, T14 and T15. This
indicates the comparative quality of yogurt texture and body formation but additionally with
positive trends for the objectives taken into considerations e.g., acceptable synbiotic
relationing of probiotics and prebiotics that will ultimately increase probiotic benefits to the
consumer (Yoon et al., 2008; Mudgil et al. 2011).
Highest mean value of syneresis for interaction (days × treatments) observed was
85% in T2 (0.5% CGG) at 28th
day of storage whereas, the lowest syneresis (40%) was found
in T1 (0.1% CGG), T10 (0.1% BHGG), and T14 (0.5% EHGG) at the start of storage.
The results of current research are in line with the findings of Athar et al. (2000) who
122
Table 4.14 Means values showing effect of guar gum and storage time on syneresis (%) of
probiotic yogurt
Days of Storage
Treatments 0 7 14 21 28 Mean
To 42±0.02 56±0.03 70±0.02 73±0.02 80±0.03 64.2de
Toʹ 46±0.02 48±0.02 60±0.02 65±0.21 70±0.05 57.8j
T1 40±0.03 60±0.02 66±0.14 70±0.02 72±0.02 63efg
T2 60±0.01 70±0.02 75±0.24 82±0.02 85±0.03 74.2a
T3 58±0.02 64±0.02 65±0.07 78±0.15 80±0.01 69.4c
T4 50±0.05 58±0.73 60±0.15 65±0.15 82±0.02 61.6gh
T5 55±0.05 68±0.02 78±0.09 80±0.5 83±0.01 72.8ab
T6 56±0.04 70±0.04 73±0.03 78±0.02 80±0.02 71.4b
T7 44±0.02 52±0.02 60±0.02 65±0.08 80±0.03 60.2hi
T8 43±0.01 50±0.01 60±0.02 70±0.15 80±0.03 60.6hi
T9 48±0.01 58±0.02 65±0.01 70±0.24 74±0.07 63efg
T10 40±0.02 54±0.25 66±0.02 70±0.03 80±0.01 62.0fgh
T11 52±0.02 70±0.12 77±0.02 80±0.10 84±0.02 72.6ab
T12 50±0.02 60±0.03 66±0.01 70±0.5 80±0.12 65.2d
T13 44±0.02 56±0.15 67±0.01 70±0.11 80±0.12 63.4ef
T14 40±0.01 52±0.02 62±0.02 65±0.09 80±0.02 59.8i
T15 42±0.02 60±0.10 68±0.01 70±0.02 74±0.01 62.8efg
Mean 47.41e 59.18
d 66.94c 72.41
b 79.06a
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions and in row for storage to evaluate the syneresis effects. (Overall treatment mean;
Max. value = 85, Min. value = 40) LSD Value Days=0.7778, LSD Value Treatments =1.4342, LSD Value Interactions (D×T) =3.2069
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
123
described that yogurt without any stabilizer showed a higher increase in syneresis as
compared to the yogurt treated with various stabilizers. The mean for syneresis in case of all
yogurt samples were increased during 0 to 15 days of storage. Aryana and McGrew (2007)
when studied the quality characteristics of yogurt with probiotics and prebiotics also found
the steady increase in syneresis with the increase in storage time. In another study, Brennan
and Tudorica (2008) stated that various samples of yogurt containing PHGG exhibited a
significant reduction in syneresis as compared to with the control yogurt having low-fat
(P<0.001). Whereas, they calculated that by increasing the levels of PHGG in the yogurt
preparations gave rise to reduction in syneresis of low-fat yogurt, bringing it at the levels
comparable to the full-fat control yogurt specifically when the levels of addition was used
above 2%. The incorporation of thickeners significantly (P < 0.001) decreased the syneresis
as compared to the control yogurt.
Moreover, yogurt produced with increased level of gelatin exhibited the lowest
syneresis values (Goncalvez et al., 2005). In different study, syneresis of yogurt samples was
measured at 4°C. The results showed that samples with gums had less syneresis during
storage. Samples containing xanthan gum at a level of 0.01% demonstrated high resistance to
syneresis throughout storage (Hematyar et al., 2012).
124
4.3.1.6. Water holding capacity (WHC)
Water holding capacity is related to gel network of yogurt and attributes to
framework of protein. Linkage of colloidal calcium phosphate (CCP) between casein
micelles make stronger network of yogurt gels. Physiochemical changes during storage
weakens network thus lowering WHC of yogurt in the later stages.
The statistical evaluation for the WHC of probiotic yogurt is presented in Table 4.10.
Their mean values are presented in Table 4.15. The statistical results indicates that WHC
differed highly significantly (P<0.01) among storage days and treatments whereas, their
interaction (days × treatments) was found to be significant (P<0.05).
The results depicted that with the passage of storage time WHC decreased. The mean
value for WHC at 0 day of storage was 69.07%, later on it was reduced to 38.15% at 28th
day
of storage on overall basis. The decrease in WHC may be due to the activity of the lactic acid
bacteria (LAB) and Bifidobacterium bifidum and as an effect of increased acidity during
storage.
The results given in Table 4.15 showed that overall mean for treatment showed
maximum WHC 66.12% in T9 (1% AHGG) followed by 65.61% in T14 (0.5% EHGG),
63.94% in T8 (0.5% AHGG) and 60.08% in T13 (0.1% EHGG) whereas, the lowest value was
observed in T2 (0.5% CGG) as 39.22%. The results indicated that controlled as well as
treated samples exhibited the decreasing trend in WHC. It is apparent from the results that
AHGG (1%) and EHGG (0.5%) showed less WHC comparatively. Lower WHC is related to
unstable and excessive rearrangements of a weak network of gel (Lucey, 2001). The acid and
enzyme hydrolyzed guar gum are suitable for yogurt development as these guar gum have
less viscosity (Yoon et al., 2008; Mudgil et al., 2011).
Highest mean value of WHC observed was 83.07% in T9 (1% AHGG) at 0 day of
storage whereas, the lowest WHC (27.17%) was found in T12 (1% BHGG) at 28th
day of
storage as far as interaction among the treatments and storage days is concerned.
The findings of current study are supported by Bahrami et al. (2013) who evaluated
that syneresis and water-holding capacity (WHC) in the yogurt samples were influenced by
kind and level of stabilizer. WHC in samples containing 0.1% of guar gum had significant
125
Table 4.15 Means values showing effect of guar gum and storage time on water holding
capacity (%) of probiotic yogurt
Days of Storage
Treatments 0 7 14 21 28 Mean
To 68.67±0.76 68.11±0.11 64.33±0.61 51±0.5 43.62±0.38 59.15f
Toʹ 71.03±1.27 66.75±0.25 60.6±0.6 50.9±0.45 40.57±0.33 57.98h
T1 73.98±0.49 72.5±0.5 58.05±0.05 46.33±0.15 42.4±0.4 58.65g
T2 32.5±0.5 38.14±0.144 68.52±0.66 28.52±0.49 28.4±0.4 39.22o
T3 74.5±0.35 68.05±0.05 46.5±0.5 38.27±0.27 34.67±0.21 52.40l
T4 73.91±0.41 72.73±0.11 71.98±0.40 43.28±0.06 36.04±0.14 59.59e
T5 42.3±0.15 44.69±0.1 44.65±0.15 26.8±0.4 39.9±0.45 39.67n
T6 72.8±0.36 65.02±0.02 63.36±0.12 40.3±0.1 30.48±0.15 54.39j
T7 80±2 70.18±0.18 50.87±0.175 46.69±1.04 30.14±0.03 55.58i
T8 82.33±0.38 69.03±0.03 62.68±0.18 57.84±0.34 47.8±0.4 63.94c
T9 83.07±0.07 69.65±0.05 66±0.25 63.37±0.07 48.49±0.49 66.12a
T10 73.5±0.5 68.68±0.18 62.21±0.1 52.34±0.34 38.96±0.46 59.14f
T11 39.48±0.1 40.1±0.1 54.94±0.05 35.29±0.06 30.87±0.30 40.14m
T12 68.41±0.02 64.34±0.04 58.56±0.06 45.29±0.21 27.17±0.17 52.76k
T13 75.5±0.16 73.3±0.3 58.77±0.09 47.34±0.34 45.5±0.25 60.08d
T14 80.8±0.4 75.04±0.06 74.21±0.21 53.4±0.4 44.6±0.3 65.61b
T15 74.46±0.21 67.5±0.15 62.84±0.32 45.21±0.02 38.96±0.31 57.80h
Mean 69.07a 64.34
b 61.12
c 45.42
d 38.15
e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
concentration of guar fractions and in row for storage to evaluate the WHC effects. (Overall treatment mean;
Max. value =83.07, Min. value = 27.17) LSD Value Days=0.1626, LSD Value Treatments =0.2998, LSD Value Interactions (D×T) =0.6704 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
126
variation (p<0.05) compared to the control sample. With an increased concentration of guar
gum, there was an incremental reduction of WHC, so that the minimum WHC was perceived
in the sample containing guar gum 0.3%. Milanovic et al. (2007) is in accordance with that
of current study that WHC of yogurt samples decreased during storage if no water binding
substance is added in it.
Similar findings were testified by Galal et al. (2003) who assured that in all yogurt
samples, both syneresis and curd tension increased with the increase of storage period
resulting in decreasing the WHC of yogurt. Singh and Muthukumarappan (2008) declared
that fortified yogurt apparently (P<0.005) enhanced the water holding capacity (WHC) by
2.99% on 1st day of storage that was higher than control fruit yogurt on 7
th and 14
th day of
storage.
127
4.3.1.7. Organic Acids
Organic acids play a vital role as natural preservatives in fermented dairy products
and also give excellent sensory characteristics to the product. Organic acids are produced due
to the hydrolysis of butterfat (fatty acids), bacterial metabolism or biochemical metabolic
processes. They are key indicators for the metabolic activity of bacteria in fermented dairy
products like yogurt and cheese (Guzel-Seydim et al., 2000; Adhikari et al., 2002).
The analysis of variance for the organic acids of probiotic yogurt is shown in Table
4.16, whereas, effect of storage and treatment on organic acids is shown in Table 4.17 and
4.18 respectively.
i) Acetic acid
Results concluded that the effect of storage time and treatment was found to be highly
significant (P < 0.01) whereas their interaction was found to be significant (P < 0.05).
Acetic acid increased with the passage of time and at initial stage the increase was
higher but with the prolonged storage interval the increase was declined from 21st day. Acetic
acid contents varied from 229.74mg/L to 332.99mg/L during storage period of 28 days.
Overall means for treatment exhibited highest acetic acid contents 328.56mg/L in T13 (0.1%
EHGG) followed by 326.94mg/L in T14 (0.5% EHGG), 325.46mg/L in T15 (1% EHGG) and
320.42mg/L in T7 (0.1% AHGG) whereas, the lowest value was 157.00mg/L in To (Table
4.18). It is evident that treatments containing AHGG and EHGG attained highest contents of
acetic acid due to fermentation.
The findings are in agreement with those of Adhikari et al. (2002) who worked on
organic acid profile changes on set, stirred and plain yogurt during 30 days of storage
particularly discussing about an increase in acetic acid content with the passage of time in
yogurt due to lactic acid bacteria.
ii) Lactic acid
Statistical data concluded that the effect of storage time and treatment effect was
significant (P < 0.05) and highly significant (P < 0.01) respectively whereas their interaction
was non-significant (P > 0.05).
The results predicted increase in lactic acid during the prescribed storage from
6524.0mg/L to 6622.4mg/L. The increase in this lactic acid is due to fermentation process of
converting lactose to lactic acid through the cultures used (Salvador and Fiszman, 2004).
128
Table 4.16 Mean squares for organic acids content of probiotic yogurt during refrigerated
storage
SOV Df Acetic
acid
Lactic
acid
Citric
acid
Butyric
acid
Pyruvic
acid
Days 4 5102.28** 4.66* 0.09NS
10.47** 2053.83**
Treatments 16 1189.92** 432.55** 128.79** 477.50** 728.12**
C1 vs C2 1 6986.14** 4.80* 18.14** 3006.52** 72.21**
C vs others 1 7712.55** 877.57** 2038.96** 3819.12** 1676.91**
Gums 4 867.75** 1490.0** 0.55 NS
171.60** 1917.40**
Levels 2 289.54** 21.19** 0.53 NS
7.02* 378.07**
Gums × Levels 8 36.24** 4.50* 0.03 NS
14.24** 184.38**
Days × Treatments 64 7.46* 0.02 NS
0.05NS
0.76NS
1.56*
Error 170 20 17513 6674 739 1.06
Total 254
**=Highly significant (P< 0.01) *=significant (P< 0.05)
NS= non-significant (P> 0.05)
Table 4.17 Means storage values showing effect of crude and hydrolysed guar gum on
organic acids (mg/L) of probiotic yogurt
Parameters (%) 0 day 7 day 14 day 21 day 28 day
Acetic acid 229.74d 265.24
c 301.89
b 332.11
a 332.99
a
Lactic acid 6524.0c 6557.7
bc 6589.2
ab 6609.8
a 6622.4
a
Citric acid 5445.7 5442.7 5439.9 5438.3 5437.3
Butyric acid 1823.6a 1813.5
ab 1805.1bc
1797.7cd
1792.8d
Pyruvic acid 35.62d 30.32
e 40.22
c 42.52
b 47.42
a
*Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the row to evaluate
the storage effect
LSD Value: acetic acid=1.7455, lactic acid =51.732, citric acid =10.935, butyric acid =10.629, pyruvic acid
=0.4032
129
Table 4.18 Means treatment values showing effect of crude and hydrolysed guar gum on
organic acids (mg/L) of probiotic yogurt
Treatments Acetic acid Lactic acid Citric acid Butyric acid Pyruvic
acid
To 157.00k 5954.9
e 6136.9
a 2366.9
a 30.38
mn
Toʹ 293.28f 5961.2
de 6009.9
b 1822.5
bcd 33.58
k
T1 298.90e 6234.2
b 5360.0
d 1784.5
ef 37.68
h
T2 281.10h 6055.4
cd 5366.0
d 1840.1
b 29.78
n
T3 276.20j 5855.4
f 5379.0
c 1821.1
bcd 32.88
kl
T4 299.30e 6254.0
b 5355.0
e 1778.5
f 40.58
g
T5 284.42g 6077.8
c 5359.9
e 1830.1
b 30.78
m
T6 277.72ij 6051.9
cd 5370.8
c 1810.1
cd 34.88
j
T7 320.42b 7485.6
a 5346.1
f 1737.5
g 45.98
d
T8 317.84bc
7503.2a 5344.0
f 1697.1
ij 48.78
b
T9 315.80c 7497.5
a 5356.0
e 1709.1
hi 45.08
e
T10 309.40d 6267.2
b 5351.1
e 1767.5
f 42.68
f
T11 279.68hi
6079.8c 5355.8
e 1824.1
bc 32.78
l
T12 278.64hij
6054.2cd
5362.8d 1803.1
de 36.48
i
T13 328.56a 7506.5
a 5348.1
f 1727.5
gh 47.38
c
T14 326.94a 7514.6
a 5338.8
f 1686.1
j 51.68
a
T15 325.46a 7517.4
a 5353.0
e 1705.1
ij 45.41
de
Means with different letters are significantly different (P ≤ 0.05). Comparisons are made within the column for
each concentration of guar fractions
LSD Value: acetic acid=3.2186, lactic acid =95.388, citric acid =13.884, butyric acid =19.599, pyruvic acid
=0.7434
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
130
Overall means for treatment exhibited highest lactic acid contents 7517.4mg/L in T15 (1%
EHGG) followed by 7514.6mg/L in T14 (0.5% EHGG), 7506.5mg/L in T13 (0.1% EHGG)
and 7503.2mg/L in T8 (0.5% AHGG) whereas, the lowest value was 5855.4mg/L in T3 (1%
CGG).
The results are supported by Seckin et al. (2009), who reported that using
polysaccharides in yogurt formation apparently influenced the lactic acid contents while
studying the effects of prebiotics on quality traits of dried yogurt. In another study, Guler
(2013) reported significant increase in lactic acid contents during storage while studying on
organic acid and carbohydrate changes in fortified set-type yogurts during refrigerated
storage.
iii) Citric acid
The statistical data concluded that the treatment effect was found to be highly
significant (P < 0.01) whereas storage time and the interaction of days and treatment were
found to be non-significant (P > 0.05). Citric acid contents varied from 5445.7mg/L to
5437.3mg/L during storage period of 28 days. The interactions as overall means for treatment
exhibited highest citric acid contents 6136.9mg/L in To followed by 6009.9mg/L in Toʹ,
5379.0mg/L in T3 (1% CGG) and 5370.8mg/L in T6 (1% PGG) whereas, the lowest value
was 5338.8mg/L in T14 (0.5% EHGG).
Seckin and Ozkilincs (2011) reported that changes in the amount of citric acid during
storage were found to be irregular. This situation may be because of storage; some organic
acids may be metabolized and converted into other products. According to Guzel-Seydim et
al. (2000), lactic acid bacteria prefer the citric acid as substrate for the formation of acetoin
and diacetyl. This indicates that the lower contents of acetic acid in yogurt containing AHGG
and EHGG may be due to the higher viability of the bacteria that utilized citric acid.
iv) Butyric acid
The statistical evaluation declared that the effect of storage time and treatment was
found to be highly significant (P < 0.01) whereas their interaction was non-significant (P >
0.05). The decrease in butyric acid content was observed with the passage of time. Butyric
acid contents reduced from 1823.6mg/L to 1792.8mg/L during storage period of 28 days.
Overall means for treatment exhibited highest butyric acid contents 2366.9mg/L in To
131
followed by 1840.1mg/L in T2 (0.5% CGG), 1830.1mg/L in T5 (0.5% PGG) and 1822.5mg/L
in Toʹ whereas, the lowest value was 1686.1mg/L in T14 (0.5% EHGG).
The results obtained in this manuscript were in line with Adhikari et al. (2002) who
worked on changes in organic acid profile of probiotic set yogurt. They declared that the
butyric acid contents reduced from 195.4 to 172.1mg/100g at 0 and 30 days of storage
respectively. Vaseji et al. (2012) also reported decrease in butyric acid concentration during
20 days of storage while working on comparison of butyric acid concentrations in ordinary
and probiotic yogurt.
v) Pyruvic acid
The statistical results deduced that the effect of storage time and treatment was found
to be highly significant (P < 0.01) whereas their interaction was found to be significant (P <
0.05). Pyruvic acid contents varied from 36.62mg/L to 47.42mg/L during storage period of
28 days. All treatments exhibited a trend in which the pyruvic acid content decreased until 7th
day then gradually increased up-to 28th
day of storage. The results might be due to
involvement of pyruvic acid as an intermediary or starting metabolites in metabolic pathways
as stated by Adhikari et al. (2002).
Pyruvic acid contents 51.68mg/L in T14 (0.5% EHGG) followed by 48.78mg/L in T8
(0.5% AHGG), 47.38mg/L in T13 (0.1% EHGG) and 45.98mg/L in T7 (0.1% AHGG) was
observed whereas, the lowest value was 29.78mg/L in T2 (0.5% CGG).
The findings are supported by Fernandez-Garcia and McGregor (1994) was perceived
a decreasing trend in pyruvic acid contents in plain yogurt during storage at 4°C for 4 weeks.
In another study, Kaminarides et al. (2007) declared that the little amount of pyruvic acid
detected in yogurt samples, is an intermediary compound resulting from the transformation
of glucose and citric acid by the lactic acid bacteria.
132
4.3.1.8. Cryo-Scanning Electron Microscopy (Cryo-SEM)
Cryo-SEM is a rapid, effective and reliable technique; widely used for observing
complex microstructure of dairy products (cheese, yogurt). It is rapid process that can take as
little as five minutes for the viewing of a frozen, fractured and sputter coated sample and
there is slight or no mechanical damage to the sample and generally no exposure to toxic
reagents (Hassan et al., 2003).
The results of Cryo-SEM are depicted in Fig 4.26 to 4.33 when probiotic yogurt made
with crude, purified and hydrolyzed guar gum was checked at different magnifications. There
was a significant change (appearance) in microstructure of probiotic yogurt and a clear
difference between the yogurts was observed.
Fig 4.26 showed the microstructure of probiotic yogurt made without prebiotic. At 0
day of storage (Fig 4.26a), good gel structure of casein network was produced but at 21st day
(Fig 4.26b) the casein network was disturbed, weakened and broken. The disrupted network
may be due to the increase in acidity, syneresis and decrease in pH, water holding capacity
with the increasing days of storage.
The resultant micrographs of probiotic yogurt incorporated with 0.2% CGG at 0 day
and 21st are shown in Fig 4.27a and Fig 4.27b respectively. The micrographs depicted that
CGG disturbed and ruptured casein network structure of yogurt and hence proper gel
structure was not developed. And during storage, it led to gradual destruction of gel structure
due to various other physico-chemical attributes. The micrographs for probiotic yogurt
containing 0.1% PGG and BHGG at 0 and 21st day are shown in Fig 4.28 and 4.29. At 0 day
of storage, both the yogurt showed little damage to the casein structure due to depletion
flocculation mechanism. The damage was accentuated with the passage of time up-to 21st day
leading to more unstable structure of casein-guar gum mixtures.
Cryo-SEM micrographs for probiotic yogurt containing AHGG and EHGG with 0.3%
and 1% concentration each at 0 and 21st day of storage are presented in Fig 4.30 to 4.33.
AHGG and EHGG granules embedded in a continuous network principally comprised of
milk proteins giving rise to strengthening the framework with both levels (0.3% and 1%)
even during the last days of storage. AHGG and EHGG have low osmotic potential due to
less viscosity as made by HCl and mannanase treatment. So it can be easily incorporated into
133
Fig 4.26 Cryo-scanning electron micrographs of control yogurt at (a) 0 day (b) 21st day of-
storage
(a)
(b)
134
Fig 4.27 Cryo-scanning electron micrographs of yogurt produced with 0.2% CGG at (a) 0
day (b) 21st day of storage
(a)
(b)
135
Fig 4.28 Cryo-scanning electron micrographs of yogurt produced with 0.1% PGG at (a) 0
day (b) 21st day of storage
(a)
(b)
136
Fig 4.29 Cryo-scanning electron micrographs of yogurt produced with 0.1% BHGG at (a) 0
day (b) 21st day of storage
(a)
(b)
137
Fig 4.30 Cryo-scanning electron micrographs of yogurt produced with 0.3% AHGG at (a) 0
day (b) 21st day of storage
(a)
(b)
138
Fig 4.31 Cryo-scanning electron micrographs of yogurt produced with 1% AHGG at (a) 0
day (b) 21st day of storage
(a)
(b)
139
Fig 4.32 Cryo-scanning electron micrographs of yogurt produced with 0.3% EHGG at (a) 0
day (b) 21st day of storage
(a)
(b)
140
Fig 4.33 Cryo-scanning electron micrographs of yogurt produced with 1% EHGG at (a) 0
day (b) 21st day of storage
(a)
(b)
141
the casein network not creating any phase separation between hydrocolloid and milk
proteins. Acid and enzyme hydrolyzed guar gums with short chain length and low viscosity
seem more acceptable as far as yogurt formulation is concerned (Yoon et al., 2008; Mudgil et
al. 2011). The results acquired in this study are in line with those of Tuinier et al. (2000) who
concluded that phase separation occur in casein and guar gum interaction but by decreasing
the chain length of guar gum through depolymerization higher amounts of gum can be used
as compared to native guar gum. They conducted studies on the influence of depolymerized
guar gum on skim milk stability.
The results regarding the phase separation in CGG, PGG and BHGG are also
supported by the findings of Thaiudom and Goff (2003). They studied the effect of k-
carrageenan (0, 0.025, 0.05%) on phase separation between polysaccharides (0.36% of locust
bean gum (LBG), guar gum, or xanthan gum and milk proteins (from 10.5% skim milk
powder) in solution. Xanthan gum was seen to be the most incompatible with milk proteins,
followed by guar gum and LBG. Casein micelles were more incompatible with all
polysaccharides than whey proteins.
The issue of some non-settling of the yogurt treatments is reproducibly due to
depletion flocculation mechanism in case of crude and purified gums (up to 0.2%) which
showed even a thin layer of separation although subsequent measurements (rheological and
textural properties) are dependent on the yogurt base Whereas, higher dose of (>0.2%)
resulted in phase separation mechanism. Anyhow acid and enzymatically hydrolyzed gums
(up to 1%) exhibited good rheological and better textural properties.
Bourriot et al. (1999a,b) supported the current results and declared that in a mixture
containing 3% casein and 0.2% guar, upon separation the casein-enriched phase contained
6.5% casein and 0.03% guar gum while the guar-enriched phase was composed of 0.3% guar
gum and 0.02% casein. In another study, mixtures were prepared with 6.5% β-lactoglobulin
concentration and 0.31-0.82% LBG or 0.23-0.71% TG concentration. All mixed systems
were of two phases. The microstructure was clearly dependent on the concentration of the
galactomannan in the mixture (Sittikijyothin et al., 2010). Arltoft et al. (2007) found that gels
were produced using carrageenan with low (0.2-0.25%) and high (0.7-1.0%) quantity in skim
milk. The low dosage of carrageenan gave rise to fine stranded gels with a carrageenan-
protein microstructure, while the high dosages caused intensely flocculated microstructures.
142
The incorporation of oligo-fructose concentration with increasing levels of has
exhibited a variable effect on quality parameters of the yogurt. Keeping in view the
rheological features, the yogurt produced with oligofructose has exposed a weak gel behavior
(Cruz et al., 2013)
143
4.3.2. Sensory evaluation
To access the rating of liking and disliking by judges towards the product, sensory
evaluation was carried out. The panellists assigned score for sensory attributes to probiotic
yogurt. Normal drinking water was provided for mouth washing in between different treated
samples to counteract the effect of freshly tasted sample. The parameters taken for sensory
attributes were color, appearance, flavor, body and texture, mouth feel and overall
acceptability, following the 9 point hedonic scales. Evaluation performa (Appendix-I) was
presented to the panellists for recording scores. Statistical results for various sensory
characteristics are presented in Tables 4.19 to 4.25 and values with same letters specified
non-significance among the treatments whereas; different letters are presenting the
significant effectiveness at P<0.01. The graphical representation for sensory attributes of
probiotic yogurts are presented in Fig 4.34 to 4.39.
4.3.2.1. Color
The color of the product is an important factor in relation to consumer’s perception
that gives an aesthetic appeal at first instance. The results pertaining to the statistical analysis
of variance (ANOVA) for the color of probiotic yogurt is presented in Table 4.19 whereas
mean values are presented in Table 4.20. The results indicated that the effect of storage days
and treatments was found to be highly significant (P<0.01) whereas, their interaction (days ×
treatments) was found to be significant (P<0.05).
The storage time had shown negative effect on the color of yogurt because scoring of
color decreased with the passage of storage period. On overall basis, color during storage was
changed from 5.27 (max.) to 2.33 (min.) from 0 day to 28th
day (Table 4.20).
The effect of treatments indicated that sensory scores for yogurt color ranged from
7.67 to 1 among the treatments during storage intervals. The overall treatment means for
maximum scores of color 7.20 in T15 (1% EHGG) followed by 6.60 in T14 (0.5% EHGG),
6.33 in T13 (0.1 EHGG) and 6.00 in T9 (1% AHGG). The lowest value was observed in T11
(0.5% BHGG) and T5 (0.5% PGG) as 1. It is clear from the results that acid and enzyme
hydrolyzed guar gum showed better scores for sensory acceptability of color as compared to
144
Table 4.19 Mean squares for sensory properties for probiotic yogurt
SOV Df Color Appearance Flavor Body and
Texture
Mouth
feel
Overall
acceptability
Days 4 194.19** 223.29** 169.73** 127.04** 214.41** 230.29**
Treatments 16 189.96** 238.53** 176.67** 165.80** 139.70** 220.56**
C1 vs C2 1 0.08NS
1.68NS
3.06NS
2.73NS
2.73NS
1.68NS
C vs others 1 128.29** 198.20** 177.63** 138.43** 118.12** 151.12**
Gums 4 455.57** 606.63** 478.44** 421.60** 340.18** 589.46**
Levels 2 276.53** 297.45** 214.28** 236.31** 191.00** 248.36**
Gums × Levels 8 66.95** 74.38** 37.98** 44.07** 46.46** 65.19**
Days × Treatments 64 7.37* 6.99* 6.98* 5.19* 6.12* 6.84*
Error 170 64.67 54.00 66.67 74.67 74.67 54.00
Total 254
**=Highly significant (P< 0.01) *=significant (P< 0.05)
145
Table 4.20 Mean values showing the color attributes of probiotic yogurt during storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 6.33±0.57 7.33±1.15 6.33±0.58 6.00±0.00 3.00±0.00 5.53ef
Toʹ 6.33±0.58 7.00±0.00 7.33±0.57 5.00±0.00 1.00±0.00 5.46ef
T1 6.00±1.0 5.67±0.59 6.67±0.57 4.33±0.58 3.00±0.00 5.13f
T2 1.66±0.56 1.33±0.58 1.00±0.00 1.00±0.00 1.00±0.00 1.20jk
T3 4.33±1.15 2.33±0.57 2.67±2.08 2.33±0.58 1.33±0.59 2.60h
T4 6.67±1.15 6.33±0.61 5.67±0.58 2.33±0.63 2.00±0.00 4.60g
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00k
T6 2.67±0.58 2.33±0.52 2.00±0.00 1.67±0.65 1.00±0.00 2.00i
T7 7.00±0.00 6.33±0.61 6.33±0.67 5.00±0.00 3.33±0.58 5.60de
T8 7.00±0.00 6.00±1.73 6.67±0.54 5.33±0.61 4.00±0.00 5.80de
T9 7.66±0.62 7.00±0.00 6.33±0.59 6.00±0.00 3.00±0.00 6.00cd
T10 7.00±1.00 7.67±0.62 7.33±0.52 4.33±0.64 1.33±0.67 5.53ef
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00k
T12 2.33±0.66 1.67±0.62 1.33±0.58 1.33±0.57 1.00±0.00 1.53j
T13 7.33±0.61 6.67±1.15 7.67±0.67 6.67±0.57 3.33±0.54 6.33bc
T14 7.67±0.59 7.00±1.00 7.00±1.00 7.33±0.55 4.00±0.00 6.60b
T15 7.67±0.61 7.33±0.59 7.66±0.58 7.33±0.62 6.00±0.00 7.20a
Mean 5.27a 4.94
b 4.98
b 3.98
c 2.33
d
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the color effects
LSD Value Days=0.2411, LSD Value Treatments =0.4446, LSD Value Interactions (D×T) =0.9941
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
146
other treatments. Acid and enzyme hydrolysis exhibited more acceptability by the judges as
per color of the product is concerned.
Highest mean value for color observed was 7.67 in T14 and T15 at 0 day of storage
which was changed to 4 and 6 after 28th
days of storage accordingly. Lowest scores for color
were observed in the treatments containing 0.5 and 1% of CGG, PGG and BHGG throughout
the storage as far as interaction is considered.
The results are in comparison to those of Brennan and Tudorica (2008) who
conducted studies on carbohydrate based fat replacers (PHGG and inulin) in yogurt
production and concluded that perception for yogurt color was improved with increasing
concentration (2-6%) of fat replacers. Likewise, Bano et al. (2011) reported that color of
functional yogurt was significantly affected during storage period. The initial sensory score
for yogurt color was 7.50 at 0 day that was reduced to 4.88 at the end of 28 day storage.
Although some workers are in contradiction to our results (Aryana and McGrew, 2007) who
found non-significant influence of treatment and storage time (upto 5 weeks) on color
attribute of yogurt produced with Lactobacillus casei and various prebiotics the reason could
be else.
147
4.3.2.2. Appearance
The appearance is one of the main characteristics in yogurt that attract the consumers
and enhance the perceiving value of the food products. The result pertaining to the statistical
analysis of variance (ANOVA) for the appearance is presented in Table 4.19 and for mean
values is shown in Table 4.21. It is indicated from the results that storage days and treatments
were found to be highly significant (P<0.01) whereas their interaction (days × treatments)
was found to be significant (P<0.05).
Similar to that of color, scores for appearance of probiotic yogurt decreased during
the storage period. The change in the sensory score for appearance was 5.29 to 2.37 from 0 to
28th
day when collective effect of storage days was observed (Table 4.21). Cumulative means
for the effect of treatments indicated that sensory scores for yogurt appearance ranged from
8.33 to 1. The overall treatment means for maximum scores of appearance was 6.93 in T15
(1% EHGG) followed by 6.80 in T14 (0.5% EHGG), 6.47 in T13 (0.1% EHGG) and 6.33 in T9
(1% AHGG). The lowest value was observed in T5 (0.5% PGG) and T11 (0.5% BHGG) as 1.
It is observed from the results that yogurt containing AHGG and EHGG were at the top for
appearance.
Highest scores for appearance observed was 8.33 in T13 (0.1% EHGG) at 0 day of
storage which was reduced to 3.67 at 28th
days of storage. Treatments containing 0.5 and 1%
of CGG, PGG and BHGG attained the lowest scores during the storage. Improper gel
formation due to phase separation could be one of the reasons for change in appearance
which reflected unacceptability by the judges.
Tarakci and Kucukoner (2003), reported similar results with decreased scores for
appearance of yogurt during storage when studied different characteristics of fruit flavoured
yogurt. The results are in line too with Salwa et al. (2004) who studied different properties
and consumer acceptance of carrot yogurt and declared a reduced score for yogurt
appearance during storage period. In another study, Yadav et al. (2007) declared that sensory
scores for appearance parameter decreased slightly during the storage of one week.
148
Table 4.21 Mean values showing the appearance attributes of probiotic yogurt during storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 6.33±0.60 7.00±0.00 6.66±0.57 6.00±0.00 3.00±0.00 5.80ef
Toʹ 7.00±1.00 7.33±0.61 6.66±0.56 4.33±0.57 1.00±0.00 5.53f
T1 5.66±0.57 4.67±0.59 4.67±0.61 4.00±0.00 2.00±0.00 5.07g
T2 1.00±0.00 3.00±1.72 1.33± 0.60 1.00±0.00 1.00±0.00 1.20k
T3 3.33±1.15 2.67±0.56 1.67±0.58 1.00±0.00 1.00±0.00 2.40i
T4 3.67±0.63 4.67±1.52 5.33±0.57 4.33±0.57 2.00±0.00 4.60h
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00k
T6 2.33±0.57 1.67±0.58 1.67±0.61 1.00±0.00 1.00±0.00 1.93j
T7 7.33±0.62 7.33±0.57 5.67±0.63 4.67±0.61 3.33±0.57 5.67ef
T8 7.33±0.55 7.33±0.54 6.00±0.00 6.00±0.00 3.33±0.61 6.00de
T9 7.33±0.66 6.00±2.00 5.33±0.59 6.00±0.00 4.00±0.00 6.33cd
T10 6.33±0.61 7.00±0.00 6.33±0.55 4.00±0.00 1.00±0.00 5.07g
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00k
T12 1.67±0.59 1.33±0.51 1.67±0.61 1.00±0.00 1.00±0.00 1.40k
T13 8.33±0.60 7.67±0.57 7.33±0.63 5.00±0.00 3.67±0.62 6.47bc
T14 7.33±0.60 7.00±1.00 7.00±0.00 6.33±0.57 4.33±0.59 6.80ab
T15 8.00±0.00 7.33±0.56 7.67±0.63 6.00±0.00 5.00±0.00 6.93a
Mean 5.29a 5.06
b 4.76
c 4.04
d 2.37
e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the appearance effects
LSD Value Days=0.2203, LSD Value Treatments =0.4062, LSD Value Interactions (D×T) =0.9084 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
149
4.3.2.3. Flavor
Flavor is the most important quality aspect in acceptance after color and appearance
of the dairy based products. The characteristic yogurt flavor is attributed to acetaldehyde and
described as a combined perception of taste, aroma and mouth feel (Ott et al., 2000).
The results pertaining to the statistical analysis of variance (ANOVA) for the flavor
of probiotic yogurt is shown in Table 4.19 and for mean values is presented in Table 4.22.
The statistical results declared that the effect of storage days and treatments were found to be
highly significant (P<0.01) whereas their interaction (days × treatments) was found to be
significant (P<0.05).
On overall basis flavor changed from 5.00 to 2.22 during storage of 28 days (Table
4.22). It is indicated that treatments (effect) mean values for sensory scores of yogurt flavor
was ranged from 8.33 to 1 during different storage interval. Maximum overall scores of
flavor was 6.80 in T15 (1% EHGG) followed by 6.40 in T14 (0.5% EHGG) and T13 (0.1%
EHGG), 5.73 in T9 (1% AHGG) whereas the lowest value was observed in T5 (0.5% PGG)
and T11 (0.5% BHGG) as 1. It is clear from the results that acid and enzyme hydrolyzed guar
gum showed better scores for sensory acceptability of flavor in comparison to other
treatments.
Interaction showed the significant effect of highest mean value for flavor was 8.33 in
T13 (0.1% EHGG) at 0 day of storage which was changed to 3.33 at the end of storage.
Lowest scores for flavor were observed in the treatments containing 0.5 and 1% of CGG,
PGG and BHGG during the storage.
The findings of current study are in agreement with Hoppert et al. (2013) who studied
the consumer acceptance of regular and reduced-sugar yogurt supplemented with various
kinds of dietary fiber and reported a significant effect of fiber on characteristic flavor of
yogurt. Tarakci and Kucukoner (2003) also found a decreased flavor perception of yogurt
during 10 days of storage at 5°C while studying different characteristics of fruit flavoured
yogurt. The other workers also noticed the decrease in flavor in their yogurt samples due to
increased storage periods which supports our study (Yadav et al., 2007; Bano et al., 2011).
150
Table 4.22 Mean values showing the flavor attributes of probiotic yogurt during storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 6.67±0.57 7.00±0.00 7.33±0.60 5.33±0.57 2.00±0.00 5.67bc
Toʹ 7.00±1.00 7.33±0.56 6.67±0.57 4.33±0.56 1.00±0.00 5.27cd
T1 5.66±0.56 4.67±0.61 4.67±0.59 4.00±0.00 2.00±0.00 4.20e
T2 1.00±0.00 3.00±1.73 1.33±0.61 1.00±0.00 1.00±0.00 1.47g
T3 3.33±1.15 2.67±0.57 1.67±0.60 1.00±0.00 1.00±0.00 1.93f
T4 3.67±0.58 4.67±1.52 5.33±0.57 4.33±0.57 2.00±0.00 4.00e
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00h
T6 2.33±0.61 1.67±0.58 1.67±0.58 1.00±0.00 1.00±0.00 1.53fg
T7 6.67±1.15 6.33±1.15 6.00±1.00 5.67±0.61 3.66±0.57 5.67bc
T8 7.67±0.60 6.33±2.10 4.33±0.62 5.00±0.00 3.00±0.00 5.27cd
T9 7.33±0.57 6.00±2.00 5.33±0.63 6.00±0.00 4.00±0.00 5.73b
T10 6.33±0.59 7.00±0.00 6.33±0.57 4.00±0.00 1.00±0.00 4.93d
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00h
T12 1.67±0.61 1.33±0.61 1.67±0.57 1.00±0.00 1.00±0.00 1.33gh
T13 8.33±0.63 7.67±0.60 7.00±0.00 6.33±0.57 3.33±0.61 6.40a
T14 7.33±0.56 7.00±1.00 7.00±0.00 6.33±0.59 4.33±0.60 6.40a
T15 8.00±0.00 7.33±0.63 7.67±0.59 6.00±0.00 5.00±0.00 6.80a
Mean 5.00a 4.82
a 4.49
b 3.65
c 2.22
d
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the flavor effects
LSD Value Days=0.2448, LSD Value Treatments =0.4514, LSD Value Interactions (D×T) =1.0093 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
151
4.3.2.4. Body and Texture
Texture defines the quality of the yogurt and influences its mouth feel, appearance
and overall acceptability (Yoon and MacCarthy, 2002).
The statistical analysis of variance (ANOVA) for the texture of probiotic yogurt is
displayed in Table 4.19 along with their mean values in Table 4.23. The statistical results
depicted that the storage days and treatments effect was highly significant (P<0.01) whereas
their interaction (days × treatments) was found to be significant (P<0.05).
The hedonic scale for yogurt texture was 5.27 when freshly prepared but declined to
2.75 when studied at the end of storage (Table 4.23). The results indicated that yogurt texture
ranged from 8.33 to 1 scores among the different treatments during storage. Overall
treatment means for maximum scores of texture was 7.27 in T15 (1% EHGG) followed by
6.93 in T13 (0.1% EHGG) and T14 (0.5% EHGG), 6.07 in T9 (1% AHGG) and 5.93 in To
whereas the lowest was observed in T5 (0.5% PGG) and T11 (0.5% BHGG) as 1. The effect
of acid and enzyme hydrolysis exhibited more acceptability by the judges.
The significant effect of interaction among the storage days and treatments showed
highest mean value for texture was 8.33 in T15 (1% EHGG) at 0 day of storage and 6.00 after
28th
days of storage. Treatments containing 0.5 and 1% of CGG, PGG and BHGG exhibited
lower scores for body and texture attributed to weaker gels formation due to phase separation
(Thaiudom and Goff, 2003).
The results in current study are in line with the outcomes of Taracki and Kucukoner
(2003) who studied fruit flavoured yogurts scored 4.21 and 3.99 at 0 and 10th
day of storage
respectively. Brennan and Tudorica (2008) reported improved body and texture of probiotic
yogurt with increasing in firmness and reduction in whey separation as perceived by
scooping when inulin and PHGG were added in the product. The results are in line with
Aryana and McGrew (2007), they examined quality features of yogurt produced with
probiotics and different prebiotics resulted in improved body and texture of yogurt similar to
those of Yi et al. (2010). They stated that yogurts containing inulin and hydrolyzate had
apparently higher scores for body and texture (P < 0.05). Comparable results have been
declared by Moeenfard and Tehrani (2008) who found that increases in concentration of
stabilizers resulted in increase in hedonic score for texture.
152
Table 4.23 Mean values showing the body and texture attributes of probiotic yogurt during
storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 6.00±0.00 8.00±1.73 7.00±0.00 5.67±0.57 2.00±0.00 5.93bc
Toʹ 7.33±0.57 7.00±1.00 7.33±1.15 5.00±0.00 1.00±0.00 5.53c
T1 5.33±0.57 5.33±1.52 5.33±0.57 3±0 3.33±0.57 4.47d
T2 1.00±0.00 1.00±0.00 1.33±0.6 1.00±0.00 1.00±0.00 1.07g
T3 4.00±0.00 3.33±1.52 2.67±2.08 2.33±0.57 2.33±0.57 2.93e
T4 6.00±0.00 6.33±0.57 6.33±0.57 3.00±0.00 2.00±0.00 4.73d
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00g
T6 3.33±1.15 2.67±0.57 2.33±0.57 2.00±0.00 1.00±0.00 2.27f
T7 6.33±0.57 7.00±1.00 6.33±0.57 4.67±0.57 3.67±0.57 5.60bc
T8 7.00±0.00 5.33±1.52 5.67±0.57 5.33±0.57 4.00±0.00 5.47c
T9 7.67±0.57 6.00±1.00 6.67±0.57 6.67±0.57 3.33±0.57 6.07b
T10 6.67±0.57 6.67±0.57 6.33±0.57 5.33±0.57 2.33±0.57 5.47c
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00g
T12 3.33±1.15 2.33±0.57 2.00±0.00 1.67±0.57 1.00±0.00 2.07f
T13 8.00±0.00 7.33±0.57 7.33±0.57 6.00±1.00 3.33±0.57 6.93a
T14 7.33±0.57 7.67±0.57 7.67±0.57 6.67±0.57 4.67±0.57 6.93a
T15 8.33±0.57 7.00±1.00 8.00±1.00 7.00±0.00 6.00±0.00 7.27a
Mean 5.27a 5.00
b 4.98
b 3.98
c 2.75
d
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the body and texture effects
LSD Value Days=0.2591, LSD Value Treatments =0.4777, LSD Value Interactions (D×T) =1.0682 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
153
4.3.2.5. Mouth feel
Mouth feel is a category of sensations that occurs in the oral cavity and it is an
important sensory property of yogurt (Lawless and Heyman, 1999). Mouth feel can be
influenced by the microstructure and variations in viscosity due to application of shear stress
in the mouth (Kok, 2009). It is how a food product will behave when taken in to the mouth. It
shows either a product is crystalline, sticky, coarse or fine and hard. This property is affected
by the crystallization, fat content and water holding capacity of the protein and other
ingredients of the product.
The statistical analysis of variance (ANOVA) for the mouth feel of probiotic yogurt is
presented in Table 4.19 mean values are shown in Table 4.24. The statistical results showed
highly significant (P<0.01) effect of storage days and treatments whereas their interaction
(days × treatments) was found to be significant (P<0.05).
Mouth feel sensory effect of probiotic yogurt decreased with length of storage period.
On overall basis the change in the sensory scores for this character was 5.37 to 2.04 from 0 to
28th
day respectively (Table 4.24). The results indicated that mouth feel scoring ranged from
8 to 1 among the cumulative treatments effect during different storage period. Overall
treatment means for highest scores of mouth feel was 6.67 in T14 (0.5% EHGG) followed by
6.53 in T15 (1% EHGG), 6.13 in T13 (0.1% EHGG) and 5.60 in To whereas, the lowest value
was observed in T5 (0.5% PGG) and T11 (0.5% BHGG) as 1. The hydrolysis treatment of
guar gum imparted better scores for sensory acceptability of mouth feel as compared to other
treatments. The significant effect of interaction (mean values) exhibited highest mouth feel
effect as 8 in T13 (0.1% EHGG) and T14 (0.5% EHGG) at 0 day of storage which was changed
to 2.33 and 3.33 at the end of storage accordingly. Lowest scores for mouth feel were
observed in the treatments containing 0.5 and 1% of CGG, PGG and BHGG throughout the
storage. Improper gel formation due to phase separation led to filamentous or protein-rich
droplets in a guar gum continuous phase that affected negatively the smoothness of gel which
is an indicator of mouth feel (Rohart and Michon, 2013; Meyer et al., 2011 ). The results of
current research are in line with the results of Brennan and Todurica (2008). They worked on
quality features of yogurt with the addition of various prebiotics and found that the PHGG
gave smoother mouth feel. Likewise in another study, it has been reported that addition of
prebiotics in dairy products caused an improve consistency and mouth-feel of product (Golob
154
Table 4.24 Mean values showing the mouth feel attributes of probiotic yogurt during storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 7.33±0.57 6.67±0.57 7.33±0.57 4.66±0.57 2.00±0.00 5.60c
Toʹ 7.00±0.00 7.00±1.00 6.67±0.57 4.33±0.57 1.00±0.00 5.20cde
T1 5.67±1.15 6.00±1.00 6.33±0.57 3.67±0.57 2.00±0.00 4.73ef
T2 1.33±0.57 2.33±1.15 1.33±0.57 1.00±0.00 1.00±0.00 1.20i
T3 4.33±1.15 4.00±1.00 2.67±0.57 2.33±0.57 1.67±0.57 3.00g
T4 6.67±0.57 6.00±1.00 5.00±1.00 4.00±0.00 1.33±0.57 4.60f
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00i
T6 3.00±1.00 2.67±0.57 2.33±0.57 1.67±0.57 1.00±0.00 2.13h
T7 7.33±0.57 6.00±1.00 6.00±0.00 4.33±0.57 3.33±0.57 5.40cd
T8 7.67±0.57 6.00±2.00 6.33±0.57 4.33±0.57 3.33±0.57 5.53c
T9 7.00±0.00 6.00±1.73 5.33±0.57 5.33±0.57 3.33±0.57 5.40cd
T10 6.67±0.57 6.00±1.00 7.33±0.57 3.33±0.57 1.67±0.57 5.00def
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00i
T12 2.00±1.00 1.67±0.57 1.33±0.57 1.33±0.57 1.00±0.00 1.47i
T13 8.00±1.00 7.67±0.57 7.00±0.00 5.67±0.57 2.33±0.57 6.13b
T14 8.00±0.00 7.33±0.57 7.67±0.57 7.00±0.00 3.33±0.57 6.67a
T15 7.33±0.57 7.33±1.15 7.33±0.57 6.33±0.57 4.33±0.57 6.53ab
Mean 5.37a 4.98
b 4.82
b 3.61
c 2.04
d
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the mouth feel effects
LSD Value Days=0.2591, LSD Value Treatments =0.4777, LSD Value Interactions (D×T) =1.0682
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
155
et al., 2004). Deis (2001) also stated that inulin improves mouth-feel in most food systems.
In other studies, the panelists specified the improved perceived creaminess in samples with
increasing level of guar gum addition (P < 0.05) (Kok, 2009).
4.3.2.6. Overall acceptability
The overall acceptability of the yogurt is a quality indicator of the product. Color,
flavor, appearance, texture and mouth feel are vital traits of food quality (Devereux et al.
2003).
The results regarding the statistical analysis of variance (ANOVA) for the overall
acceptability of probiotic yogurt is shown in Table 4.19 and for mean values is presented in
Table 4.25. The results indicated highly significant (P<0.01) effect of storage days and
treatments whereas their interaction (days × treatments) was found to be significant (P<0.05).
The storage time does affect undesirability after some days on overall acceptability of
probiotic yogurt which decreased during prolonged storage. The change in the sensory score
for overall acceptability during storage was 5.18 to 2.29 from 0 to 28th
day (Table 4.25) when
collective effect was evaluated.
The effect of treatments indicated ranking from 8 to 1 among the treatments. The
overall treatment means for maximum scores of overall acceptability was 6.93 in T15 (1%
EHGG) followed by 6.87 in T14 (0.5% EHGG), 6.40 in T13 (0.1% EHGG) and 6.00 in T9 (1%
AHGG) whereas, the lowest value was observed in T5 (0.5% PGG) and T11 (0.5% BHGG) as
1. Acid and enzyme hydrolyzed guar gum exhibited better scores for overall sensory
acceptability as compared to other treatments which might be due to reasons mentioned
under various characteristics.
The effect of interaction showed highest mean value for overall acceptability
observed as 8 in T13 (0.1% EHGG) and T15 (1% EHGG) at 0 day of storage which was
changed to 3.33 and 5.33 after 28th
days of storage respectively. The lower overall
acceptability was observed in the treatments containing 0.5 and 1% of CGG, PGG and
BHGG throughout the storage. Improper gel formation due to phase separation could be one
of the reasons for unacceptability by the judges.
The results are supported by Brennan and Tudorica (2008) who worked on
carbohydrate-based fat replacers for the modification of sensory and rheological quality of
156
Table 4.25 Mean values showing the overall acceptability attributes of probiotic yogurt
during storage
Days of Storage
Treatments 0 7 14 21 28 Mean
To 6.00±0.00 7.33±0.15 6.67±0.57 5.33±0.57 2.33±0.57 5.53de
Toʹ 7.00±1.00 7.33±0.57 6.67±0.57 4.33±0.57 1.00±0.00 5.27e
T1 5.67±0..57 5.33±0.57 5.33±0.57 4.00±0.00 2.33±0.57 4.53f
T2 1.00±0.00 1.67±0.57 1.00±0.00 1.00±0.00 1.00±0.00 1.13i
T3 3.33±0.57 2.67±0.57 2.33±0.57 2.00±0.00 1.00±0.00 2.27g
T4 5.67±0.57 5.00±1.00 4.67±0.57 3.33±0.57 2.33±0.57 4.20f
T5 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00i
T6 3.00±1.00 2.67±0.57 2.33±0.57 2.00±0.00 1.00±0.00 2.20g
T7 7.00±0.00 6.67±0.57 6.00±0.00 4.67±0.57 3.33±0.57 5.53de
T8 7.33±0.57 7.33±0.57 5.33±0.57 5.33±0.57 3.67±0.57 5.80cd
T9 7.33±0.57 7.33±0.57 5.67±0.57 6.33±0.57 3.33±0.57 6.00bc
T10 6.67±0.57 7.33±1.52 6.33±0.57 4.33±0.57 1.33±0.57 5.20e
T11 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00i
T12 2.33±0.57 2.00±0.00 1.67±0.57 1.67±0.57 1.00±0.00 1.73h
T13 8.00±0.00 7.33±0.57 7.33±0.57 6.00±1.00 3.33±0.57 6.40b
T14 7.67±1.15 7.67±0.57 7.67±0.57 6.67±0.57 4.67±0.57 6.87a
T15 8.00±0.00 7.33±1.15 7.67±0.57 6.33±0.57 5.33±0.57 6.93a
Mean 5.18a 5.12
a 4.63
b 3.84
c 2.29
d
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the overall acceptability effects
LSD Value Days=0.2203, LSD Value Treatments =0.4062, LSD Value Interactions (D×T) =0.9084
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
157
yogurt. They found that overall acceptability of probiotic yogurt was good, the panelists ex-
posing no particular preference for a type of product (P > 0.05), although the yogurt contain-
ing PHGG at 6% addition, received the higher rankings for acceptability. In another study,
Irvine and Hekmat (2011) evaluated sensory properties of probiotic yogurt containing prebi-
otic fibres and stated that probiotic yogurt maintained a smooth, creamy, homogenous texture
and received good sensory acceptability. Rezaei et al. (2011) reported that guar gum and ara-
bic gum at a concentration of 0.2% and 0.5% respectively exhibited the most favorable sen-
sory assessments. Milani and Koocheki, (2011) studied the effects of date syrup and guar
gum on rheological and sensory characteristics of low fat frozen yogurt dessert and declared
that guar gum incorporation up to 0.3% enhanced the samples quality and hence the score
given by panelists also increased.
158
Fig 4.34 Sensory attributes of control yogurt To, Toʹ (without guar gum) during storage
Fig 4.35 Sensory attributes of probiotic yogurt T1, T2, T3 (CGG) during storage
159
Fig 4.36 Sensory attributes of probiotic yogurt T4, T5,T6 (PGG) during storage
Fig 4.37 Sensory attributes of probiotic yogurt T7,T8,T9 (AHGG) during storage
160
Fig 4.38 Sensory attributes of probiotic yogurt T10,T11,T12 (BHGG) during storage
Fig 4.39 Sensory attributes of probiotic yogurt T13,T14,T15 (EHGG) during storage
161
4.3.3. Microbial Analysis
Probiotics are beneficial microorganism, constituting a major part of intestinal
microflora; confer health assistances on the host organism when ingested in sufficient
amount. Species of Lactobacillus and Bifidobacterium are most commonly used probiotics
(Tomasik and Tomasik, 2003).
The statistical results regarding microbial analysis (Bifidobacterium bifidum, S.
thermophilus and L. bulgaricus) are shown in the Table 4.26. The data revealed that storage
interval, treatments and their interaction (storage days × treatment) impart a highly
significant effect on viability of bacteria under consideration.
4.3.3.1. Bifidobacterium bifidum (BB)
Bifidobacteria constitute a major part of the human intestinal microflora and play an
important role in maintaining good health. At present, bifidobacteria are increasingly
incorporated into fermented dairy products. Bifidobacteria grow slowly in milk and the usual
practice is to add yoghurt starter bacteria to enhance the fermentation process for making
probiotic yoghurt (Samona and Robinson, 1994; Akalin et al., 2004).
It is evident from the results (Table 4.27) that storage time showed highly significant
effect on the viability of BB. The average viable count of BB at the time of yogurt formation
was 2.02×106
that increased to 2.17×106
cfu/g at 14th
day of storage. After 14 days, viability
of BB started decreasing till 28th
day (1.53×105
cfu/g) which might me due to significant
variation in pH and ultimately acidity. Shin et al. (2000a) studied that at low pH values,
fermentation acids like lactic and acetic are powerful antimicrobial agents and may have a
role in modulating survival of bifidobacteria Interaction among the storage days and
treatments was also highly significant. Maximum mean value for viable count of BB
observed was (4.00×106cfu/g) in T14 at 14
th day of storage. The lowest viable count observed
was (2.15×104cfu/g) in T2 at 28
th day of storage.
Overall mean (Table 4.27) for treatment exhibited maximum count of 2.97×106 cfu/g
in T14 (0.5% EHGG) followed by 2.88×106 cfu/g in T13 (0.1% EHGG), 2.87×10
6 cfu/g in T15
(1% EHGG) and 2.76×106
cfu/g in T8 (0.5% AHGG). The lowest count was observed in T2
(1.72×105 cfu/g) and T3 (1.75×10
5 cfu/g It is obvious from the results that EHGG and AHGG
retained the highest BB count during the storage days whereas, CGG, PGG and BHGG did
not retain stability in BB count. As the guar percent used in case of CGG, PGG and BHGG
162
Table 4.26 Mean squares showing microbial analysis of probiotic yogurt during storage
SOV Df B. Bifidum L. bulgaricus S. thermophilus
Days 4 493211** 33596.5** 662.19**
Treatments 16 334450** 19189.7** 370.21**
C1 vs C2 1 592874** 190.8 2.87NS
C vs others 1 83992** 2183.9 184.44**
Gums 4 657888** 47889.1 917.20**
Levels 2 598413** 31128.8 573.31**
Gums × Levels 8 105745** 6355.8 115.078**
Days × Treatments 64 17889.8** 1005.54** 19.87**
Error 170 0.012 0.308 23.89
Total 254
**=Highly significant (P< 0.01)
163
Table 4.27 Viability of Bifidobacterium bifidum (cfu/g) during refrigerated storage of
probiotic yogurts
Treatments
Days of Storage
Mean 0 7 14 21 28
To 0 0 0 0 0 0
Toʹ 2.92×106 2.98×10
6 2.99×10
6 2.91×10
6 2.45×10
5 2.41×10
6 h
T1 2.81×106 2.88×10
6 2.86×10
6 2.45×10
6 2.20×10
5 2.24×10
6 i
T2 2.75×105 2.79×10
5 2.65×10
5 2.39×10
4 2.15×10
4 1.72×10
5 l
T3 2.63×105 2.68×10
5 3.0×10
5 2.35×10
4 2.23×10
4 1.75×10
5 l
T4 2.99×106 3.13×10
6 3.33×10
6 2.92×10
6 2.53×10
5 2.52×10
6 f
T5 3.00×105 2.98×10
5 3.00×10
5 2.91×10
4 2.45×10
4 1.90×10
5 k
T6 2.9×105 2.98×10
5 3.0×10
5 2.91×10
4 2.35×10
4 1.88×10
5 k
T7 3.39×106 3.43×10
6 3.62×10
6 2.90×10
6 2.25×10
5 2.71×10
6 e
T8 3.45×106 3.54×10
6 3.68×10
6 2.89×10
6 2.55×10
5 2.76×10
6 d
T9 3.30×106 3.48×10
6 3.56×10
6 3.00×10
6 2.23×10
5 2.71×10
6 e
T10 2.92×106 3.14×10
6 3.20×10
6 2.98×10
6 2.34×10
5 2.49×10
6 g
T11 3.09×105 3.12×10
5 3.32×10
5 2.93×10
4 2.39×10
4 2.01×10
5 j
T12 3.15×105 3.11×10
5 3.29×10
5 2.91×10
4 2.45×10
4 2.02×10
5 j
T13 3.59×106 3.63×10
6 3.92×10
6 3.00×10
6 2.56×10
5 2.88×10
6 b
T14 3.75×106 3.84×10
6 4.00×10
6 2.99×10
6 2.85×10
5 2.97×10
6 a
T15 3.50×106 3.58×10
6 3.91×10
6 3.11×10
6 2.65×10
5 2.87×10
6 c
Mean 2.02×106
c 2.08×106
b 2.17×106
a 1.73×106 d 1.53×10
5 e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the bifidobacteria viability
LSD Value Days=0.0033, LSD Value Treatments =0.0062, LSD Value Interactions (D×T) =0.0138 Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
164
was 0.1, 0.5 and 1%. But results have shown the ineffectiveness of using higher percentages
i.e. 0.5 and 1% for the three types. Predominantly, 0.1% retained higher level of BB count
although this was not more than AHGG and EHGG with same percent guar gum used. The
results may be supported with those of Rohart and Michon (2013) who concluded that at low
guar gum concentrations a denser network was formed whereas higher guar gum
concentrations lead to phase separation (filamentous or protein-rich droplets) during their
work on designing microstructure into acid skim milk/guar gum gels.
Such an effect of guar galactomannans is consistent with the observations of various
researchers during their studies. Capela et al. (2006) observed a clear beneficial action of
guar gum as prebiotic on the viability of L. rhamnosus, L. acidophilus and Bifidobacterium
spp. in yogurt. In another study, it was confirmed that complex carbohydrates components
are excellent source of potential prebiotic (Wang and Daun, 2004). A similar observation was
reported by Akalin et al. (2004) who declared that viability of B. longum and B. animalis in
regular yogurt incorporated with prebiotic was higher than that of control yogurt (no
supplement). According to Oliveira et al. (2009) probiotic counts were stimulated by the
addition of different prebiotics in non-fat synbiotic fermented milk. The results from current
study are also satisfied with Srisuvor et al. (2013) who reported that viable count of probiotic
bacteria fell into recommended limit (106cfu/g) up-to 21
st day of storage, while studying the
effects of prebiotic on quality features of low-fat probiotic set type yogurt. Shin et al. (2000a)
also stated the bifidobacteria to provide their therapeutic effects should be viable and
consumed in numbers ≥ 106 cells per gram.
165
4.3.3.2. Streptococcus thermophilus
Streptococcus thermophilus is a Gram-positive, catalase negative, non-motile,
facultative anaerobe homo-fermentative, ovoid or spherical shaped cell and is used in yogurt
formation in combination with Lactobacillus bulgaricus (Frank and Hassan, 1998).Through
symbiotic relationship between the two species, there is rapid acid development and texture
formation than in the single strain culture (Tamime and Robinson, 2000).
The results (Table 4.28) showed storage time as highly significant effect on the
viability of S. thermophilus. The average viable count of S. thermophilus at 0 day was
3.36×106
that increased to 3.42×106
cfu/g till 14th
day of storage. After 14 days, viability of S.
thermophilus started decreasing till 28th
day of storage which was 2.72×105
cfu/g.
Overall means for treatment showed maximum count 4.51×106 cfu/g in T14 (0.5%
EHGG) followed by 4.46×106 cfu/g in T15 (1% EHGG), 4.36×10
6 cfu/g in T13 (0.1% EHGG)
and 4.41×106 cfu/g in T8 (0.5% AHGG). The lowest count was observed in T3 (1% CGG) as
2.39×105 cfu/g. In general, the highest viable count of S. thermophiles was computed in
experimental treatment with EHGG and AHGG. Lowest count for S. thermophilus was
observed in the treatments containing 0.5 and 1% with CGG, PGG and BHGG throughout
the storage period. Higher concentrations (0.5% and 1%) of CGG, PGG and BHGG exhibited
ineffective usage when confound to 0.1%. Bourriot et al. (1999a,b) concluded that phase
separation occur in casein guar gum systems when level of gum exceeds certain limit (>
0.1%) while working on phase separation, rheology and microstructure of micellar casein-
guar gum mixtures.
Interaction among the storage days and treatments was also found to be highly
significant. Maximum mean value for S. thermophilus count observed was (5.89×106cfu/g) in
T14 (0.5% EHGG) at 14th
day of storage whereas, the lowest viable count observed was
(2.45×104cfu/g) in T5 (0.5% PGG) at 28
th day of storage.
The results are in accordance with Akalin et al. (2004,2007) who indicated a decrease
in the viable count of S. thermophilus during 28 days of storage. They declared that viable
count was initially increased up-to middle of storage days of 28 and then started decreasing
gradually till the end of storage. In another study, Cakmakci et al. (2012) worked on sensory
features and storage stability of probiotic yogurts and reported a decrease in viable counts of
S. thermophilus after 7 days. Likewise, Zare et al. (2011) also declared a decrease in viable
166
Table 4.28 Viability of Streptococcus thermophilus (cfu/g) during refrigerated storage of
probiotic yogurts
Treatments
Days of Storage
Mean 0 7 14 21 28
To 4.12×106 4.28×10
6 4.36×10
6 3.92×10
6 3.35×10
5 3.40×10
6 de
Toʹ 3.94×106 3.98×10
6 4.00×10
6 3.91×10
6 3.45×10
5 3.63×10
6 cd
T1 4.06×106 4.13×10
6 4.29×10
6 3.89×10
6 3.30×10
5 3.34×10
6 e
T2 3.75×105 3.79×10
5 3.75×10
5 3.39×10
4 3.15×10
4 2.40×10
5 g
T3 3.63×105 3.68×10
5 4.00×10
5 3.35×10
4 3.23×10
4 2.39×10
5 g
T4 3.78×106 3.86×10
6 3.94×10
6 2.96×10
6 2.63×10
5 2.96×10
6 f
T5 3.70×105 3.98×10
5 3.90×10
5 2.97×10
4 2.45×10
4 2.43×10
5 g
T6 4.65×105 4.92×10
5 4.97×10
5 3.91×10
4 3.35×10
4 3.05×10
5 g
T7 5.22×106 5.29×10
6 5.35×10
6 4.67×10
6 4.53×10
5 4.19×10
6 b
T8 5.25×106 5.64×10
6 5.79×10
6 4.89×10
6 4.64×10
5 4.41×10
6ab
T9 5.28×106 5.59×10
6 5.61×10
6 4.71×10
6 4.74×10
5 4.33×10
6ab
T10 4.82×106 4.94×10
6 4.99×10
6 3.79×10
6 3.65×10
5 3.77×10
6 c
T11 4.39×105 4.46×10
5 4.62×10
5 3.93×10
4 3.39×10
4 2.84×10
5 g
T12 4.45×105 4.61×10
5 4.29×10
5 3.91×10
4 3.63×10
4 2.82×10
5 g
T13 5.38×106 5.49×10
6 5.65×10
6 4.85×10
6 4.45×10
5 4.36×10
6ab
T14 5.46×106 5.74×10
6 5.89×10
6 4.99×10
6 4.85×10
5 4.51×10
6ab
T15 5.45×106 5.69×10
6 5.81×10
6 4.91×10
6 4.65×10
5 4.46×10
6ab
Mean 3.36×106 a 3.36×10
6 a 3.42×10
6 a 2.81×10
6 b 2.72×10
5 c
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the S. thermophilus viability.
LSD Value Days=0.1465, LSD Value Treatments =0.2702, LSD Value Interactions (D×T) =0.6042
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
167
count of S. thermophilus during 28days storage interval while conducting study on physical,
microbial and sensory characteristics of yogurt supplemented with lentil flour. So there was
in general decrease in viability of S. thermophilus as studied by various scientists but in one
case the viability firstly increased up-to 14 days when tested followed by decrease up-to 28th
day data.
4.3.3.3. Lactobacillus bulgaricus
Lactobacillus bulgaricus is a Gram-positive, rod shaped, non-motile, catalase
negative, anaerobic and homo-fermentative organisms (Chandan and Rell, 2006). Starter
culture bacteria (L. bulgaricus and S. thermophilus) show their symbiotic behavior during
fermentation of yogurt and produce lager quantity of flavoring compounds and acid. S.
thermophilus produce lactic acid and minute quantity of formic acid, which promotes the
growth of L. bulgaricus. On the other hand L. bulgaricus produce amino acids that stimulate
the growth of S. thermophilus (Aswal et al., 2012).
It is evident from Table 4.29 that storage time showed highly significant effect on the
viability of L. bulgaricus. The average viable count of L. bulgaricus at the time of yogurt
formation was 2.63×106
cfu/g that increased to 2.79×106cfu/g upto 14
th day of storage. After
14 days, viability of L. bulgaricus started decreasing till 28th
day of storage which was
2.09×105
cfu/g. Interaction among the storage days and treatments exhibited maximum mean
value for viable count of L. bulgaricus observed was (4.91×106cfu/g) in T15 (1% EHGG) at
14th
day of storage whereas, the lowest was (2.15×104cfu/g) in T2 (0.5% CGG) at 28
th day of
storage.
Overall mean (Table 4.29) for treatment showed highest count 3.78×106 cfu/g in T15
(1% EHGG) followed by 3.75×106 cfu/g in T14 (0.5% EHGG), 3.59×10
6 cfu/g in T13 (0.1%
EHGG) and 2.55×106 cfu/g in T8 (0.5% AHGG). The lowest count observed in T3 (1% CGG)
was 1.74×105 cfu/g. In general, the highest viable counts of L. bulgaricus were counted in
experimental treatments containing EHGG and AHGG. Lowest count for L. bulgaricus was
observed in the treatments containing 0.5% and 1% of CGG, PGG and BHGG throughout the
storage exhibiting ineffective usage of higher concentration. The results are in line with
Tuinier et al. (2000) who studied the influence of depolymerized guar gum on skim milk
stability. They concluded that phase separation can be controlled by depolymerizing guar
gum and higher amounts can be used as compared to native guar gum.
168
Table 4.29 Viability of Lactobacillus bulgaricus (cfu/g) during refrigerated storage of probi-
otic yogurts
Treatments
Days of Storage
Mean 0 7 14 21 28
To 3.12×106 3.28×10
6 3.36×10
6 2.92×10
6 2.35×10
5 2.58×10
6 g
Toʹ 2.94×106 2.98×10
6 3.00×10
6 2.91×10
6 2.45×10
5 2.37×10
6 i
T1 3.06×106 3.13×10
6 3.29×10
6 2.89×10
6 2.30×10
5 2.52×10
6 h
T2 2.75×105 2.79×10
5 2.75×10
5 2.39×10
4 2.15×10
4 1.75×10
5 l
T3 2.63×105 2.68×10
5 3.0×10
5 2.35×10
4 2.23×10
4 1.74×10
5 l
T4 3.78×106 3.86×10
6 3.94×10
6 2.96×10
6 2.63×10
5 2.96×10
6 f
T5 3.70×105 3.98×10
5 3.90×10
5 2.97×10
4 2.45×10
4 2.42×10
5 j
T6 3.65×105 3.92×10
5 3.97×10
5 2.91×10
4 2.35×10
4 2.41×10
5 j
T7 4.22×106 4.29×10
6 4.35×10
6 3.87×10
6 3.53×10
5 3.42×10
6 e
T8 4.25×106 4.64×10
6 4.49×10
6 3.99×10
6 3.64×10
5 2.55×10
6 d
T9 4.28×106 4.59×10
6 4.61×10
6 3.83×10
6 3.74×10
5 2.54×10
6 d
T10 3.82×106 3.94×10
6 3.99×10
6 2.79×10
6 2.65×10
5 2.96×10
6 f
T11 3.39×105 3.46×10
5 3.62×10
5 2.93×10
4 2.39×10
4 2.20×10
5jk
T12 3.45×105 3.11×10
5 3.29×10
5 2.91×10
4 2.63×10
4 2.08×10
5 k
T13 4.38×106 4.49×10
6 4.65×10
6 4.10×10
6 3.45×10
5 3.59×10
6 c
T14 4.46×106 4.74×10
6 4.89×10
6 4.29×10
6 3.85×10
5 3.75×10
6 b
T15 4.45×106 4.69×10
6 4.91×10
6 4.51×10
6 3.65×10
5 3.78×10
6 a
Mean 2.63×106
c 2.73×106 b 2.79×10
6 a 2.31×10
6 d 2.09×10
5 e
The values are mean ± SD (n = 3)
Means with different letters differ significantly at (P ≤ 0.05). Comparisons are made within the column for each
probiotic yogurt and in row for storage to evaluate the L. bulgaricus viability
LSD Value Days=0.0166, LSD Value Treatments =0.0307, LSD Value Interactions (D×T) =0.0686
Control: (To, Toʹ; without guar gum)
CGG: Crude Guar Gum; (T1, 0.1%; T2, 0.5%; T3, 1%)
PGG: Purified Guar Gum; (T4, 0.1%; T5, 0.5%; T6, 1%)
AHGG: Acid Hydrolyzed Guar Gum; (T7, 0.1%; T8, 0.5%; T9, 1%)
BHGG: Base Hydrolyzed Guar Gum; (T10, 0.1%; T11, 0.5%; T12, 1%)
EHGG: Enzyme Hydrolyzed Guar Gum; (T13, 0.1%; T14, 0.5%; T15, 1%)
169
Normally, L. bulgaricus during storage period as a whole decrease, although for some
initial periods (1-14 days) the viability increased and then due to certain reasons decreased as
pointed out by several workers. During their working on different parameters, it has been
concluded that L. bulgaricus count increases and then starts decline which supports the result
stated here in (Akalin et al., 2004,2007; Zare et al., 2011). In another study, Cakmakci et al.
(2012) evaluated quality attributes of probiotic yogurts and reported a decrease in viable
counts of L. bulgaricus after 7 days.
It is confirmed in this manuscript that the enzyme and acid hydrolyzed guar gum with
reduced chain length show better prebiotic activity as compare to the crude, purified and base
hydrolyzed guar gum. The viability of probiotics remains stable up-to certain time period and
then starts decreasing that may be due to accumulation of toxic metabolites secreted by the
bacteria themselves that hinder their growth and number of viable cells decline (Stanbury et
al., 2003).
170
Chapter 5
S U M M A R Y
The research work was carried out to assess the suitability of guar gum (crude guar
gum, CGG; purified guar gum PGG; acidic hydrolysed guar gum, AHGG; basic hydrolysed
guar gum, BHGG; enzymatic hydrolysed guar gum, EHGG) utilization as prebiotic on
probiotics behavior in synbiotic food (yogurt) a functional food. Guar galactomannans, a
water soluble dietary fiber obtained from leguminous plant Cyamopsis tetragonoloba (L) was
purified with the organic solvent (ethanol : water 95:5% v/v) and partially depolymerized by
controlled acid (HCl), alkali [Ba(OH)2] and enzyme (mannanase) actions. All guar fractions
were characterized using basic chemical composition and advanced analytical techniques
including scanning electron microscopy (SEM), X-ray diffraction (XRD), fourier transform
infrared spectroscopy (FTIR), thermo-gravimetric analysis (TGA). The chemical
composition analysis exposed that partial hydrolysis of guar gum decreased significantly the
moisture and fat content, although CGG, PGG, BHGG were at par for the later, when
compared with native guar gum. However, there was marked increase in protein (except
BHGG) and ash (except PGG and EHGG) content in comparison to native guar gum.
SEM revealed that after the process of hydrolysis, a significant change was observed
in the surface morphology of the guar gum. CGG, PGG and BHGG showed the differences
in their shape, size and structure having rough surface morphology and highly viscous
aqueous solution. AHGG showed the powdery and fluffy appearance. HCl strongly affected
the morphology of hydrolysed guar gum. In EHGG, well defined porous structure was
developed. Excellent interconnected framework was formed by the mannanase enzyme.
XRD-pattern of CGG, PGG and AHGG demonstrated amorphous structure and exhibited low
overall crystallinity while EHGG and BHGG resulted in slightly increased the crystallinity
regions. Increased crystallinity of EHGG was seen at angle (°2θ) 20.4, 40.2 and 49.5. Basic
hydrolysis increased considerably the crystallinity of the BHGG at angle (°2θ) seen at 20.5,
24.1, 26.0, 28.9, 31.4, 33.0, 34.3 and 42.8.
FTIR spectral analysis suggested that there was no major transformation of functional
groups after hydrolysis of guar gum. TGA analysis declared that hydrolyzed guar gum was
more heat stable than the native ones. In CGG, thermal degradation occurs in three zones
171
while in PGG, BHGG, AHGG and EHGG, decomposition of weight comprising 4-5 major
zones of temperature occurred. It was experienced that partial hydrolysis of guar gum could
be achieved by inexpensive methods and utilized as a functional soluble dietary fiber as
thickening, stabilizing and therapeutic agents for food industry particularly having better
prebiotic activity which is major factor in the current study.
Rheological studies were performed to explore physiological behavior of guar gum
and its hydrolytic forms. The rheological behavior of aqueous solutions of guar fractions was
studied at 25°C, using steady-shear and dynamic oscillatory measurements performed with a
controlled stress rheometer Bohlin CVO (Malvern Instruments) fitted with cone-and-plate
geometry. The guar fractions exhibited shear thinning non Newtonian behavior at high shear
rate and Newtonian flow at low shear rate. At low shear rate, sigma crude guar gum (SCGG)
and LCGG exhibited the maximum viscosity 18.59 and 1.34 Pa.s respectively while AHGG
and EHGG exhibited minimum 0.15 and 0.22 Pa.s respectively.
Oscillatory measurement results were characteristic of a random coil polysaccharide.
At low frequencies, viscous modulus (Gʺ) was above elastic modulus (Gʹ); while as
frequency increased, Gʹ surpassed Gʺ exhibited characteristics typical of weak viscoelastic
gel. All guar fractions decreased the glucose absorption in simulated small intestinal model
(SIM) which revealed that all guar fractions with different viscosities had the non-significant
difference in physiological functions.
Haemolysis studies were performed to explore toxic behavior of guar gum and its
hydrolytic forms. In this study, CGG, PGG, BHGG, AHGG and EHGG (2.5 to 250 mg mL-1
)
were subjected to haemolysis using 96-well plates to evaluate the toxic effects during the
process of hydrolysis as compared to phosphate buffer saline (PBS; negative control, 0%
haemolysis) and 0.1% Triton-X 100 (positive control, 100% haemolysis). The guar fractions
exhibited minor haemolytic activity (1.9±0.03% to 7.24±0.02%) showing no toxic effects to
the human RBC‟s.
Furthermore, guar gum incorporation in a synbiotic food (product development) was
done with the objective of appraising suitability of the prebiotic ingredient in food products
with respect to consumer‟s acceptance, good sensory qualities and positive health benefits
through functional food (yogurt) under studies. Yogurt has been reported as an appropriate
vehicle for carrying prebiotic or probiotic ingredients; however, in this study yogurt‟s
172
indigenous starter cultures (Lactobacillus bulgaricus, Streptococcus thermophilus) and
Bifidobacterium bifidum as probiotic bacteria were used, whereas, guar gum served as the
prebiotic portion. Prebiotics are authenticated as non-digestible food component that have the
beneficial effect on the host by stimulating the growth and activity of
bacteria/microorganisms in one or a restricted number in lower part of GIT which has been
proved in this study too. Probiotics (living microbial dietary supplements) play a vital role in
fermented dairy products for their therapeutic effects and functional behaviour by providing
positive effects on the host health and improving microflora balance of their intestinal
Yogurts produced were stored at 4-6°C for 28 days and were analyzed for chemical
composition, physico-chemical and sensory characteristics as well as viability of yogurt
culture (Lactobacillus bulgaricus, Streptococcus thermophilus) and Bifidobacterium bifidum
bacteria at 0, 7, 14, 21 and 28 days of storage.
It was evident from the results that addition of guar gum fractions and storage intervals
had significant effect on qualitative attributes of yogurt produced. Addition of AHGG and
EHGG (0.1%, 0.5% and 1.0%) significantly reduced syneresis, increased WHC and viscosity
of yogurt however in case of CGG, PGG and BHGG; yogurt was only produced with the
0.1% levels of concentration because with 0.5% and 1.0% levels, depletion-flocculation
mechanism between casein and guar gum occurred due to high osmotic pressure. The cryo-
scanning electron microscopy proved and further defined the ruptured casein network of
yogurt with guar gum flocculants of CGG, PGG and BHGG, whereas, AHGG and EHGG
granules were well embedded in a continuous casein network.
On overall basis in treated samples acidity and syneresis increased while pH,
viscosity and WHC decreased with the passage of time. The organic acids (acetic, lactic,
citric, butyric and pyruvic acid) were affected significantly during refrigeration due to
metabolic activities of probiotics.
The sensory attributes of yogurt based on judges opinion differed significantly by the
level and type of guar gum as well as storage intervals. It was evident from the scores given
by judges that yogurt prepared with 0.5 and 1% addition of EHGG and AHGG got the higher
acceptability with respect to sensory attributes and least scores were given to 0.5 and 1%
addition of CGG, PGG and BHGG due to phase separation. Storage effect reduced
acceptability of all yogurt treatments.
173
The results depicted that AHGG and EHGG exhibited higher prebiotic effect or
synbiotic relationship with probiotic. Different guar fractions significantly influenced the
viability of probiotic bacteria as well as yogurt culture bacteria. EHGG (0.5%) showed
maximum viability of Bifidobacterium bifidum, Streptococcus thermophiles and
Lactobacillus bulgaricus. Storage significantly reduced the viability of these bacteria.
Good quality yogurt sustains the strong curd integrity without any sign of shrinkage
and disintegration into lumps and whey-off. It also possesses pleasant flavor and odor,
especially with the set yogurt, the defect of syneresis which can adversely affect
acceptability or preference of consumers. For that reason, proper dose of prebiotics is of
great concern for the production of probiotic yogurt. It is evident that hydrolyzed guar gum
could improve physico-chemical, microbial viability and sensory characteristics of the
product if defined so.
5.1. CONCLUSIONS
On the basis of findings following conclusions has been made:
Crude, purified and acid hydrolyzed guar gum exhibits amorphous structure, whereas
guar gum hydrolyzed with enzyme raises the crystalline structure but lower than the
gum when hydrolyzed with base.
Guar gum hydrolyzed with acid and enzyme reduces viscosity give better prebiotic
activity and stability against temperature and pH when characterized through
rheological evaluation and TGA respectively in contrast to other fractions, although
all fractions have same physiological pattern i.e., glucose absorption level.
All guar gum fractionations does not exhibit toxic effects to human RBC‟s which is
1.9 ± 0.03% to 7.24 ± 0.02% which is far lower than the toxic levels elsewhere.
Therefore, hydrolyzed form (AHGG, EHGG) of guar gum can be used in the product
development as an additive (prebiotic) qualitatively adding 0.1 and 0.5% dosage, the
later the best, along with probiotic endurance of bifidobacterium having the best
acceptability.
174
5.2. IMPORTANCE OF RESULTS AND MAJOR FINDINGS OF THIS STUDY:
The thermal behavior of guar gums revealed that hydrolyzed guar gum is more heat
stable / resistant and stable for pH during the processing. The conditions helped to
develop a novel food product with good quality features.
AHGG and EHGG with reduced viscosity (de-polymerization) exhibited shear
thinning non-Newtonian flow behavior similar to that of crude guar gum.
In a simulated small intestinal model, addition of guar gum in glucose solutions
resulted in a 20% reduction in mass transfer and also decreased the glucose release.
The similar pattern of glucose release for all guar gums showed that process of
hydrolysis without disturbing the physiological functioning of guar gum solutions.
Rheological and physiological features of hydrolyzed guar gum established its use as
a soluble dietary fiber in food as well as in the pharmaceutical industry to provide
good texture and structure. This helps in formulating better food product and in
curing certain human diseases.
Hemolytic effects indicated that intact and hydrolyzed guar galactomannans exhibit
negligible haemolytic activity with no toxic effects to the human RBC‟s but can be
used as thickening, stabilizing and therapeutic agents with reduced viscosity.
Prebiotic activity of hydrolyzed gums is enhances with non-toxic effect to the bacteria
(probiotic).
The utilization of guar gum for yogurt development is case sensitive. CCG, BHGG
and PGG is not effective at the rate of more than 0.2% concentration which is due to
phenomenon of phase separation. In contrast, hydrolyzed gum (AGHH and EHGG)
can be used up to 1% concentration due to their shorter chain length providing better
results of physico-chemical and textural properties. Additionally, the increase in
concentration also provides longevity to the beneficial bacteria (probiotic) during
their storage period. This ultimately reaches at their destined of consumer to give a
better life in terms of good health.
5.3. ECONOMIC IMPACT ON YOGURT INDUSTRY:
The finding of the present study is beneficiary from industrial point of view in order
to develop the versatile dairy products having valuable influence on human health that
provide healthy gut functions with a balance between „bad bacteria‟ and „good bacteria‟.
175
Disturbance in balance occurs due to certain diseases of infections and antibiotics intake in
human life style. The symbiotic product thus formed contains probiotic and prebiotics in their
composition and are helpful in maintaining this balance and thus stimulates the health of
human beings in a positive way due to:
Enhanced endurance and stimulation of higher count of good bacteria (endogenous,
exogenous) in yogurt with extended probiotic benefits throughout the gut
Improvement in metabolic activity of the bacteria, antagonistic toward pathogenic
bacteria acting as antimicrobial agent while producing bacteriocidal substances like,
hydrogen peroxide, bacteriocins, organic acids etc.
Production of anti-inflammatory, anti-mutagenic, anti-carcinogenic compounds
Production of biologically active compounds such as peptides, enzymes, vaccines etc.
contributing in the product for human benefits.
The study will have a good economic impact when executed in the form of
commercial synbiotic food products. This will replace/reduce the cost of
medicine/treatment of the individual‟s disease that might have caused due to not
utilization of the synbiotic foods.
5.4. RECOMMENDATIONS
Awareness about the pro and prebiotics products should be launched at national and
international level through government, non-government organizations and industry
to deliver the knowledge about the beneficiary effects of commodities.
Novel food products with functional properties may be developed by incorporation of
hydrolysed guar gum and probiotics (Bifidobacteria, LAB). Incorporation of
hydrolysed guar gum in synbiotic foods should be evaluated with human in-vivo
studies as well.
Domestic dairy products may be taken in practice to support the economy and allied
stakeholders
Consumers should be motivated to include pro and prebiotic dairy products in their
daily diet to tackle various health related divergences in a natural way. Furthermore,
hydrolysed guar gum can further be used as substrates for the production of short
chain fatty acids.
176
5.5. LIMITATIONS OF STUDY:
Although from the present research, physico-chemical, rheological and structural
characterization have been performed but the promising potential health effects of
synbiotics are not being observed. More standardized and verifiable clinical studies
are needed to demonstrate the safety, efficacy, and limitations of a putative
synbiotics, to determine effects on the immune system in healthy and diseased
individuals and effects of long-term consumption, and to resolve whether it is
superior to existing therapies.
The facility of “In vitro stomach and small intestinal model” is lacking at University
of Agriculture, Faisalabad. This facility is available in University of Birmingham,
UK. So there is need to develop it to conduct future studies on different aspects
related to human health.
To conduct microbiological analysis, there is need to develop rapid and quick
methods for checking the viability of micro-organisms. In this regard, flow
cytometric approach is the latest technique for observing the viable cells of micro-
organism in no time. This facility is also lacking at NIFSAT, UAF, Pakistan.
Collaboration with the industry or any foreign research institute is needed to get their
valuable inputs for further success of this project.
5.6. FUTURE RESEARCH DIRECTIONS:
The use of synbiotics enriched fermented food products provide carriage in terms of
beneficial health effects. Functional foods (symbiotic food) should be developed with
specific functional characteristics targeting specific groups of individuals of consumers
(infant, children, and adults). Probiotics, prebiotics, and their combinations have been found
to be clinically effective for a large number of disorders. In recent times, diabetes and
cholesterol reduction, cancer prevention, and immunology are the major areas of research on
probiotics and prebiotics. Genetic engineering and other approaches are being used to
enhance the advantageous effects of probiotic microbes. Much work has already been
accomplished to help us to understand synbiotics and the manner in which they function.
However, some issues like dosage and viability of probiotic strains, industrial
standardization, and safety aspects are needed to be studied well.
177
New and cheaper sources of prebiotics and probiotics should be generated from waste
agriculture and biomass (i.e. residues from plant, animal and microbial processing). Better
analytical methods should be developed, which can identify novel prebiotic in foods; ideally,
these should have good reproducibility, reliability and should not involve expensive,
specialist analytical equipment. Technology now seems to be available for the expansion of
new ranges of foods and drinks containing synbiotics that can provide better prospects and an
opportunity to the industry for growth. The use of synbiotics in food in critical care is
promising; however, they should be administered carefully and cautiously, and only on the
basis of strong scientific evidence.
5.7. FUTURE STUDIES:
Although there is growing stage of functional foods with synbiotics, but the concept
of synbiotics has been studied in present work to a limited extent, however it needs further
investigations. Very few studies uptill now have been carried out on human model for the
effectiveness of synbiotics hence should be promoted to get the knowhow about actual status
to be executed for food.
In vitro digestibility of synbiotic yogurt in simulated stomach and small intestinal
model
Lethality and chronic toxicological studies on guar gum and its hydrolyzed form in
vivo models
Effect of synbiotic foods on human health and metabolic profile
In-vitro studies on effect of probiotics on production of short chain fatty acids in the
presence of various prebiotics
Influence of guar galactomannans on multiplication of lactic acid bacteria under in
vitro conditions
178
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APPENDIX-I
Sensory Evaluation Score Card of Yogurt
Name of judge: ----------------------------
Signature: ---------------------------
Date: ---------------------------
Treatments Color Appearance Flavor Mouth
Feel
Body and
Texture
Overall
Acceptability
To
To´
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
Remarks___________________________________________________________________
___________________________________________________________________________
__________________________________________________________________
206
Hedonic Score System
9-points hedonic scale was follow for rating the sample
Quality description Hedonic scale
Dislike extremely 1
Dislike very much 2
Dislike moderately 3
Dislike slightly 4
Neither dislike or like 5
Like Slightly 6
Like moderately 7
Like very much 8
Like extremely 9
Score Sheet:
Name of judge: ------------------
Signature: ------------------
Date: ------------------
207
APPENDIX-II
Viscosity for guar gum fractions at specific shear stress and shear rate with passage of time
Time Shear Rate SCGG CGG PGG BHGG AHGG EHGG
(sec) ϒ 1/s Ƞ Pa.s σ Pa Ƞ Pa.s σ Pa Ƞ Pa.s σ Pa Ƞ Pa.s σ Pa Ƞ Pa.s σ Pa Ƞ Pa.s σ Pa
10.175 0.07506 18.59±0.07 1.252±0.016 1.346±0.085 0.096±0.007 0.217±0.051 0.015±0.003 0.056±0.041 0.004±0.003 0.149±0.184 0.952±1.349 0.022±0.011 0.001±0.0007
20.665 0.1062 19.535±0.502 2.118±0.012 1.453±0.118 0.157±0.011 0.238±0.028 0.026±0.002 0.055±0.031 0.006±0.003 0.128±0.172 1.648±2.331 0.031±0.006 0.003±0.0006
31.175 0.16298333 18.165±1.378 3.295±0.279 1.472±0.119 0.243±0.020 0.236±0.017 0.039±0.003 0.049±0.028 0.008±0.004 0.076±0.105 1.704±2.410 0.030±0.007 0.005±0.001
41.69 0.24921667 17.12±1.032 4.228±0.317 1.453±0.119 0.362±0.029 0.225±0.009 0.057±0.002 0.039±0.021 0.010±0.005 0.138±0.195 0.080±0.113 0.025±0.010 0.006±0.002
52.18 0.37701667 15.32±0.692 5.774±0.176 1.398±0.098 0.530±0.037 0.222±0.005 0.085±0.003 0.029±0.014 0.011±0.006 0.288±0.401 1.867±2.637 0.019±0.005 0.007±0.002
62.665 0.57236667 13.19±0.68 7.572±0.365 1.316±0.074 0.759±0.043 0.219±0.003 0.126±0.001 0.030±0.007 0.017±0.003 0.204±0.279 0.263±0.366 0.014±0.002 0.008±0.001
72.88 1.31121667 11.17±0.42 9.738±0.427 1.216±0.060 1.067±0.053 0.215±0.002 0.188±0.003 0.028±0.004 0.025±0.004 0.275±0.335 0.207±0.478 0.009±0.0006 0.008±0.0005
83.365 1.323 9.145±0.707 12.2±0.933 1.096±0.046 1.454±0.065 0.207±0.0001 0.276±0.071 0.026±0.005 0.035±0.007 0.015±0.021 -0.049±0.070 0.009±0.001 0.011±0.002
93.73 1.90683333 7.423±0.327 15.025±0.742 0.966±0.041 1.95±0.077 0.203±0.002 0.334±0.102 0.024±0.002 0.048±0.004 0.017±0.021 0.099±0.147 0.006±0.0008 0.012±0.001
104.2 3.05533333 5.743±0.204 17.645±0.700 0.837±0.042 2.563±0.136 0.191±0.0003 0.587±0.001 0.025±0.001 0.076±0.003 0.012±0.0002 -0.0006±0.0007 0.004±0.0007 0.007±0.002
114.7 4.75 4.445±0.175 20.715±0.784 0.714±0.032 3.311±0.155 0.182±0.0002 0.849±0.003 0.025±0.001 0.116±0.006 0.019±0.012 0.032±0.125 0.004±0.0003 0.017±0.001
125.2 7.0385 3.506±0.123 24.72±0.947 0.592±0.027 4.18±0.197 0.170±0.0004 1.199±0.0007 0.024±0.0004 0.174±0.003 0.002±0.002 0.010±0.023 0.003±0.0002 0.020±0.001
135.55 10.6683333 2.616±0.087 28.11±0.961 0.482±0.021 5.166±0.248 0.156±0.00007 1.672±0 0.024±0.0005 0.259±0.007 0.002±0.0001 0.025±0.001 0.003±0.00001 0.028±0.0002
146.05 16.1133333 1.934±0.062 31.545±0.982 0.386±0.015 6.265±0.255 0.140±0.00007 2.288±0.001 0.023±0.0008 0.388±0.012 0.002±0.0006 0.039±0.009 0.002±0.00004 0.047±0.0004
156.25 24.5883333 1.417±0.041 35.095±0.997 0.301±0.011 7.470±0.282 0.124±0.0004 3.083±0.010 0.023±0.0002 0.571±0.002 0.001±0.0005 0.038±0.013 0.002±0.0002 0.066±0.0003
166.75 37.23 1.03±0.029 38.74±1.159 0.233±0.008 8.785±0.333 0.109±0.0002 4.091±0.012 0.022±0.0003 0.850±0.012 0.001±0.0004 0.048±0.017 0.002±0.0003 0.097±0.009
177.1 56.5416667 0.741±0.023 42.39±1.371 0.178±0.006 10.177±0.357 0.093±0.0006 5.348±0.031 0.022±0.0004 1.251±0.022 0.001±0.00007 0.083±0.004 0.002±0.0003 0.135±0.018
187.45 85.8866667 0.531±0.017 46.07±1.513 0.135±0.005 11.755±0.473 0.079±0.00009 6.874±0.012 0.021±0.0002 1.835±0.028 0.001±0.0005 0.096±0.048 0.002±0.0002 0.206±0.015
197.95 130.216667 0.382±0.014 50.295±1.902 0.102±0.003 13.57±0.494 0.066±0.00004 8.686±0.038 0.021±0.0003 2.678±0.041 0.001±0.0006 0.148±0.080 0.002±0.0002 0.311±0.024
208.3 197.35 0.275±0.011 54.94±2.220 0.078±0.003 15.785±0.629 0.056±0.0005 10.955±0.106 0.019±0.0002 3.820±0.016 0.001±0.0003 0.289±0.073 0.002±0.0003 0.491±0.048
208
APPENDIX-III
Oscillatory properties of aqueous solution of guar gum fractions
SCGG CGG PGG BHGG AHGG EHGG
Time
(sec)
Freque
ncy
(Hz)
Gʹ (Pa) Gʺ(Pa) Gʹ (Pa) Gʺ(Pa) Gʹ (Pa) Gʺ(Pa) Gʹ (Pa) Gʺ(Pa) Gʹ (Pa) Gʺ(Pa) Gʹ (Pa) Gʺ(Pa)
102.2 0.01 0.033±0.015 0.858±0.1465 0.0003±0.00003 0.015±0.008 0.00004±0.00001 0.005±0.0009 0.0003±0.0004 0.002±0.001 0.0002±0.00003 0.0007±0.00004 0.0002±0.00004 0.0008±0.0000
8
166.1 0.01624 0.088±0.029 1.389±0.249 0.0006±0.00001 0.024±0.012 0.0002±0.000005 0.008±0.001 0.000006±0.000004 0.002±0.0003 0.0003±0.00002 0.0008±0.00001 0.0001±0.00001 0.0008±0.0000
2
206.3 0.02637 0.242±0.086 2.239±0.398 0.0015±0.00009 0.040±0.019 0.0005±0.0003 0.013±0.002 0.00002±0.00002 0.003±0.0005 0.00008±0.000005 0.0008±0.00001 0.00009±0.00007 0.001±0.00006
231.8 0.04281 0.645±0.210 3.541±0.604 0.003±0.00002 0.066±0.032 0.0006±0.00004 0.022±0.004 0.0001±0.00005 0.005±0.0007 0.0002±0.0001 0.0011±0.0001 0.0003±0.000005 0.001±0.0002
248.4 0.06953 1.548±0.428 5.362±0.839 0.007±0.0008 0.109±0.051 0.0008±0.0002 0.037±0.006 0.0006±0.0003 0.009±0.001 0.0002±0.0002 0.0009±0.0006 0.0008±0.0007 0.002±0.0008
259.4 0.1129 3.247±0.778 7.63±1.08 0.011±0.003 0.187±0.083 0.001±0.00005 0.064±0.009 0.0005±0.0002 0.015±0.003 0.004±0.001 0.002±0.001 0.002±0.003 0.003±0.002
267 0.1834 5.881±1.082 10.238±1.290 0.013±0.0005 0.326±0.137 0.0002±0.00008 0.115±0.015 0.008±0.0004 0.028±0.0007 0.003±0.001 0.010±0.002 0.004±0.0003 0.010±0.001
272.6 0.2979 9.604±1.564 12.93±1.51 0.008±0.006 0.574±0.218 0.005±0.002 0.214±0.026 0.001±0.0009 0.047±0.001 0.005±0.0007 0.007±0.002 0.002±0.002 0.009±0.001
276.8 0.4839 14.235±2.085 15.55±1.69 0.096±0.046 1.009±0.324 0.017±0.003 0.386±0.053 0.016±0.10 0.049±0.019 0.009±0.004 0.010±0.005 0.0044±0.004 0.008±0.009
280.2 0.7853 19.81±2.672 18.075±1.873 0.396±0.137 1.756±0.451 0.085±0.011 0.737±0.130 0.038±0.042 0.188±0.044 0.015±0.020 0.007±0.009 0.028±0.033 0.038±0.029
283.85 1.277 25.985±3.316 20.34±1.98 1.305±0.301 2.866±0.615 0.264±0.015 1.389±0.404 0.049±0.038 0.194±0.019 0.033±0.008 0.094±0.045 0.069±0.066 0.106±0.009
287.5 2.083 32.055±3.853 22.225±2.269 3.292±0.635 4.078±0.833 0.769±0.105 1.81±0.197 0.079±0.074 0.285±0.029 0.048±0.041 0.161±0.059 0.163±0.115 0.076±0.024
291 3.371 37.64±4.864 23.46±2.42 5.599±1.065 4.816±1.074 1.335±0.167 2.288±0.312 0.070±0.023 0.279±0.057 0.556±0.156 0.070±0.074 0.485±0.201 0.199±0.129
294.35 5.556 51.63±2.757 24.625±1.647 13.59±0.66 9.479±5.982 2.546±3.464 2.208±1.452 2.806±2.100 2.32±0.521 0.906±0.570 8.572±9.302 1.510±1.241 7.008±5.971
297.75 9.091 67.48±7.537 21.1±2.432 19.565±3.599 1.594±0.389 8.310±5.798 0.580±0.429 6.883±2.052 3.595±0.296 7.282±8.623 0.607±0.074 9.924±8.493 1.279±0.436
301.05 15 116.7±18.667 2.012±1.556 63.64±0.961 14.5±2.6 41.31±10.60 19.395±10.359 45.43±8.259 21.8±2.856 76.005±17.783 106.69±68,74 61.32±57.24 51.38±42.99
304 25 312.7±16.546 463.3±433.7 396.3±103.1 113.66±43.75 236±61.37 197±56 252.95±17.46 144.65±1.626 365.4±165.1 328.59±421.018 1008.49±1376.74 188.97±159.42
307.9 42.86 1232.8±1148.7 727.75±328.02 1871±131.5 967.95±17.46 1571±123.03 1059.5±10.6 1728.5±51.62 1066.5±10.60 1666.5±159.0 1123.3±527.1 1710±643.4 354±48.22
311.3 75 14050±1442.4 9503±1664.5 12980±353.5 8022.5±303.3 13830±254.5 9140±8.48 12455±799 8265±253.1 15865±4857 8530.5±949.64 12640±141.4 7690.5±494.2
314.6 100 36290±565.6 22880±933.3 32155±2227.4 25990±3111.3 36240±636.39 22190±2375 33415±1661 23025±304.05 37750±24395 13958.5±9010.6 34455±1986.9 22045±1704.1
209
APPENDIX-IV
Absorption of glucose in 1% (w/v) guar solutions in small intestinal model (SIM)
Time
(min)
1%Glucose (no
segmentation)
1%Glucose
(segmentation)
1%CGG+Glucose
(segmentation)
1%EHGG+Glucose
(segmentation)
1%AHGG+Glucose
(segmentation)
1%BHGG+Glucose
(segmentation)
0 0.003±0.003 0.005±0.069 0.036±0.060 0.000±0.000 0.003±0.003 0.049±0.004
5 0.010±0.006 0.091±0.004 0.066±0.053 0.038±0.002 0.068±0.004 0.121±0.004
10 0.034±0.005 0.173±0.001 0.133±0.041 0.109±0.004 0.138±0.006 0.193±0.006
15 0.090±0.004 0.232±0.002 0.192±0.044 0.178±0.005 0.204±0.006 0.288±0.005
20 0.143±0.008 0.321±0.002 0.264±0.070 0.258±0.006 0.278±0.006 0.289±0.004
25 0.215±0.004 0.415±0.040 0.329±0.074 0.321±0.004 0.344±0.006 0.352±0.009
30 0.258±0.002 0.483±0.050 0.409±0.079 0.390±0.004 0.425±0.006 0.412±0.006
35 0.322±0.004 0.567±0.009 0.475±0.098 0.463±0.003 0.509±0.004 0.496±0.004
40 0.386±0.069 0.660±0.002 0.539±0.080 0.515±0.006 0.563±0.008 0.544±0.003
45 0.436±0.080 0.738±0.001 0.616±0.101 0.627±0.005 0.612±0.004 0.595±0.007
50 0.487±0.045 0.818±0.010 0.681±0.096 0.687±0.006 0.676±0.005 0.683±0.006
55 0.547±0.089 0.893±0.022 0.729±0.088 0.761±0.002 0.735±0.006 0.693±0.004
60 0.596±0.006 0.969±0.007 0.808±0.096 0.790±0.002 0.786±0.006 0.777±0.008
65 0.634±0.005 1.076±0.005 0.875±0.085 0.860±0.006 0.846±0.008 0.877±0.005
70 0.715±0.005 1.165±0.004 0.951±0.087 0.887±0.001 0.933±0.005 0.902±0.005
75 0.752±0.001 1.220±0.008 0.993±0.092 0.943±0.001 0.973±0.004 0.942±0.003
80 0.803±0.007 1.281±0.032 1.045±0.098 1.019±0.004 1.025±0.008 1.029±0.032
85 0.856±0.005 1.371±0.011 1.121±0.096 1.069±0.002 1.101±0.004 1.092±0.006
90 0.872±0.002 1.414±0.009 1.166±0.085 1.147±0.004 1.154±0.005 1.166±0.001
The values are mean ± SD (n = 3) and observed at 540nm in spectrophotometer