Kamal Gandhi, Rajan Sharma, Priyae Brath Gautam, Bimlesh Mann - Chemical Quality Assurance of Milk and Milk Products-Springer (2020)

Kamal
Gandhi
Rajan Sharma
Priyae Brath Gautam
Bimlesh Mann
Chemical Quality
Assurance
of Milk and Milk
Products
Chemical Quality Assurance of Milk
and Milk Products
Kamal Gandhi • Rajan Sharma •
Priyae Brath Gautam • Bimlesh Mann
Chemical Quality
Assurance of Milk
and Milk Products
Kamal Gandhi Rajan Sharma
Dairy Chemistry Division Dairy Chemistry Division
National Dairy Research Institute National Dairy Research Institute
Karnal, Haryana, India Karnal, Haryana, India
Priyae Brath Gautam Bimlesh Mann

Dairy Chemistry Division Dairy Chemistry Division
National Dairy Research Institute National Dairy Research Institute
Karnal, Haryana, India Karnal, Haryana, India
ISBN 978-981-15-4166-7 ISBN 978-981-15-4167-4 (eBook)

https://doi.org/10.1007/978-981-15-4167-4
# Springer Nature Singapore Pte Ltd. 2020

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Preface
In recent years, the Indian dairy industry has grown substantially. Dairy contributes
most to the agricultural sector’s overall output. But the dairy sector is still not in a
position to make a dent on the international market given all these facts. Indian
exports are still far below than those of the countries with less milk production. One
of the reasons for this is the quality of raw milk and thereby the quality of the
finished products. Most of the dairy plants are still sticking to the old system of
maintaining their quality, without considering the developments made in the analyt-
ical tools to attain a consistent quality of any food product. There is an absolute
necessity to assure the quality of milk right from the udder of the cattle until its
consumption. In chemical quality assurance, the quality of analysis is very much
dependent on the following factors: (1) accuracy of glassware and apparatus used,
(2) accuracy of the method used, and (3) purity and standardization of chemical
reagents. All the above three very important factors have been taken into consider-
ation while writing this book. This book has been written to cater to the needs of the
B.Sc./B.Tech./M.Sc./M.Tech. students of Dairy Science, people involved in quality
assurance activities in dairy industry, and for the food safety officials. The book
covers detailed information on several procedures including necessary reagents and
apparatus essential for checking milk quality in addition to theoretical principles and
information on them. Evidently, this book is a compilation of different well-
established methods of analysis recorded in different official publications, textbooks,
FSSAI, BIS, ISO, IDF documents, etc. Working and functioning of International
organizations like WTO, Codex Alimentarius Commission, ISO, IDF, USFDA, and
national organizations like FSSAI, Agmark, and BIS have also been covered.
Chapter 1 provides an overview of the dairy industry in India and the world.
Chapter 2 discusses the sampling plan for different milk products meant for chemical
analysis. Chapters 3–5 describe the detailed procedures for checking the quality of
raw, processed milk, and milk products. Chapters 6–8 discuss the methods to check
the accuracy of glasswares, standardization of reagents, and specifications of some
v
vi Preface
of the chemicals being used in the dairy industry. Chapters 9 and 10 discusses the
quality concepts and the safety and regulatory aspects of the dairy industry.
Hopefully, this book will respond to the queries of our esteemed readers related to
the quality of milk and milk products and encourage them to further study these
subjects.
Karnal, Haryana, India Kamal Gandhi

24 February 2020 Rajan Sharma
Priyae Brath Gautam
Bimlesh Mann
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Status of Milk Production and Consumption in India . . . . . . . . . 1
1.2 Status of Milk Production and Consumption Throughout
the World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3 Challenges Faced by Dairy Industry in Quality Assessment
of Raw Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2 Sampling Plan for Milk and Milk Products . . . . . . . . . . . . . . . . . . . 17
2.1 Scope of Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2 Sampling Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2.1 Samples Drawn for Regulatory Purposes . . . . . . . . . . 18
2.2.2 Samples Drawn for Monitoring Purpose
at the Factory Level . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3 Sampling Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4 Collection of the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Sampling Plan for Raw Milk . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5.1 Sampling from an Individual Container . . . . . . . . . . . 19
2.5.2 Sampling from Several Containers . . . . . . . . . . . . . . . 20
2.5.3 Sampling from Bulk Units . . . . . . . . . . . . . . . . . . . . 20
2.5.4 Sampling from Storage Tanks and Rail and Road
Milk Tankers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.6 Sampling Plan for Processed Milk . . . . . . . . . . . . . . . . . . . . . . 21
2.6.1 Sampling from a Storage Tank or Silo . . . . . . . . . . . . 21
2.6.2 Sampling of the Packed Milk . . . . . . . . . . . . . . . . . . 22
2.7 Sampling Procedure for Channa/Paneer/Cheese . . . . . . . . . . . . 22
2.7.1 Sampling by Cutting a Sector . . . . . . . . . . . . . . . . . . 22
2.7.2 Sampling Using a Trier . . . . . . . . . . . . . . . . . . . . . . . 22
2.7.3 Sampling by Taking the Whole Product . . . . . . . . . . . 22
2.7.4 Preparation of Paneer/Cheese/Channa Samples
for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.7.5 Sampling by Cutting Using a Knife with a Pointed
Blade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.7.6 Sampling Scale for Cheese . . . . . . . . . . . . . . . . . . . . 24
vii
viii Contents
2.8 Sampling Procedure for Khoa . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.9 Sampling Procedure for Sterilized Milk/Flavored Milk . . . . . . . 24
2.9.1 Scale of Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.9.2 Preparation of Samples for Chemical Analysis . . . . . . 25
2.9.3 Preparation of Samples for Microbiological
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.10 Sampling Procedure for Dahi, Yoghurt, and Srikhand . . . . . . . . 25
2.10.1 Preparation of Samples for Chemical Analysis . . . . . . 25
2.10.2 Preparation of Samples for Microbiological
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.11 Sampling Procedure for Ice Cream . . . . . . . . . . . . . . . . . . . . . . 26
2.11.2 Preparation of Sample of Ice Cream . . . . . . . . . . . . . . 27
2.12 Sampling Procedure for Condensed Milk . . . . . . . . . . . . . . . . . 27
2.12.2 Preparation of Sample of Condensed Milk
for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.13 Sampling Procedure for Milk Powders . . . . . . . . . . . . . . . . . . . 28
2.13.2 Preparation of Sample for Analysis . . . . . . . . . . . . . . 28
2.14 Sampling Procedure for Butter . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.14.2 Sampling Technique for Butter . . . . . . . . . . . . . . . . . 29
2.15 Sampling Procedure for Ghee (Anhydrous Milk Fat)/Butter Oil . . 31
2.15.2 Sampling Technique . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.16 Labeling of the Samples for Analysis . . . . . . . . . . . . . . . . . . . . 31
3 Quality Assessment of Raw Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1 Visual and Organoleptic Tests . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Sediment Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3 Clot-on-Boiling Test (COB) . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.4 Alcohol Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.5 Alcohol–Alizarin Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.6 Heat Stability Assay for Milk . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.7 Detection of Preservatives in Milk . . . . . . . . . . . . . . . . . . . . . . 36
3.7.1 Neutralizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.7.2 Boric Acid and Borates . . . . . . . . . . . . . . . . . . . . . . . 37
3.7.3 Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.7.4 Hydrogen Peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.7.5 Benzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.7.6 Salicylic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.7.7 Hypochlorites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Contents ix
3.8 Detection of Adulterants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.8.1 Sucrose or Cane Sugar . . . . . . . . . . . . . . . . . . . . . . . 40
3.8.2 Starch or Other Cereal Flours . . . . . . . . . . . . . . . . . . 40
3.8.3 Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.8.4 Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.8.5 Maltodextrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.8.6 Pond Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.8.7 Vegetable Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.8.8 Baudouin Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.8.9 Mineral Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.8.10 Animal Body Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.8.11 Mastitic/Abnormal Milk . . . . . . . . . . . . . . . . . . . . . . 45
3.8.12 Addition of Skim Milk Powder . . . . . . . . . . . . . . . . . 48
3.8.13 Detergent in Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.8.14 Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.8.15 Ammonia Compounds . . . . . . . . . . . . . . . . . . . . . . . 49
3.8.16 Soya Powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.8.17 Sulfates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.8.18 Detection of Adulteration in Milk Using Biosensor
and Immunological Techniques . . . . . . . . . . . . . . . . . 50
3.9 Compositional Analysis of Raw Milk . . . . . . . . . . . . . . . . . . . . 51
3.9.1 Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.9.2 Solids-Not-Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.9.3 Titratable Acidity . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.9.4 Protein Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.9.5 Lactose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.9.6 Ash Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.9.7 Lactate Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4 Quality Assessment of Processed Milk . . . . . . . . . . . . . . . . . . . . . . . 69
4.1 Chemical Tests for Processed Milk . . . . . . . . . . . . . . . . . . . . . . 69
4.1.1 Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.1.2 Solids-Not-Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.1.3 Total Solids/Moisture Content . . . . . . . . . . . . . . . . . . 73
4.1.4 Protein Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.1.5 Lactose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.1.6 Ash Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.1.8 Phosphatase Test . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1.9 Turbidity Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.1.10 Homogenization Efficiency . . . . . . . . . . . . . . . . . . . . 82
4.1.11 Creaming Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
x Contents
5 Quality Assessment of Milk Products . . . . . . . . . . . . . . . . . . . . . . . 85

5.1 Analysis of Cheese, Paneer, Channa, and Khoa . . . . . . . . . . . . . 85
5.1.1 Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.1.2 Moisture and Total Solids Content . . . . . . . . . . . . . . . 88
5.1.3 Total Protein Content . . . . . . . . . . . . . . . . . . . . . . . . 88
5.1.4 Soluble Protein Content . . . . . . . . . . . . . . . . . . . . . . 89
5.1.5 Total Ash Content . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.1.7 Salt Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5.1.8 Total Volatile Fatty Acids . . . . . . . . . . . . . . . . . . . . . 92
5.1.9 Ripening Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.2 Sweetened/Sterilized Flavored Milk . . . . . . . . . . . . . . . . . . . . . 93
5.2.1 Fat Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.2.2 Total Solids Content . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.2.4 Turbidity Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5.2.5 Sucrose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.3 Dahi, Yoghurt, and Srikhand . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.3.2 Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
5.3.5 Diacetyl Content . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5.3.6 Sucrose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.4 Ice Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.4.1 Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.4.4 Sucrose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
5.4.5 Overrun in Ice Cream . . . . . . . . . . . . . . . . . . . . . . . . 103
5.4.6 Melting Test and Shape Retention of Ice Cream . . . . . 104
5.4.7 Alcohol Test for Protein Stability of Ice Cream Mix . . . 104
5.5 Evaporated and Sweetened Condensed Milk . . . . . . . . . . . . . . . 105
5.5.1 Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.5.3 Sucrose Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.5.5 Protein Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.6 Ghee, Butter-Oil, and Anhydrous Butter-Oil . . . . . . . . . . . . . . . 108
5.6.1 Moisture Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.6.2 Free Fatty Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.6.3 Acid Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.6.4 Reichert-Meissel (RM) Value . . . . . . . . . . . . . . . . . . 110
5.6.5 Polenske Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Contents xi
5.6.6 Peroxide Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5.6.7 Saponification Number . . . . . . . . . . . . . . . . . . . . . . . 117
5.6.8 Iodine Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.6.9 Butyro Refractometer (BR) Reading . . . . . . . . . . . . . 119
5.6.10Unsaponifiable Matter and Cholesterol Content . . . . . 122
5.6.11Antioxidants in Ghee . . . . . . . . . . . . . . . . . . . . . . . . 124
5.6.12Adulterants in Ghee . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.6.13Fatty Acid Analysis of Ghee/Butter Oil/Anhydrous
Butter Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
5.7 Unsalted Butter and Table Butter . . . . . . . . . . . . . . . . . . . . . . . 131
5.7.1 Moisture Content . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
5.7.2 Fat and Curd Content . . . . . . . . . . . . . . . . . . . . . . . . 132
5.7.3 Salt Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
5.7.4 Test to Distinguish Between Annatto (Natural Color)
and Azo Dye (Synthetic Color) in Butter (IS:3507) . . . 134
5.8 Milk Powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.8.1 Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.8.4 Total Ash Content . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5.8.5 Acid Insoluble Ash . . . . . . . . . . . . . . . . . . . . . . . . . . 137
5.8.6 Total Carbohydrate (in Infant and Weaning Foods) . . . 137
5.8.8 Reconstitution Properties . . . . . . . . . . . . . . . . . . . . . 138
5.8.9 Hydroxymethylfurfural . . . . . . . . . . . . . . . . . . . . . . . 144
6 Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.1 Calibration of Milk Pipette . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
6.2 Calibration of Butyrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
6.3 Calibration of Lactometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6.4 Calibration of Other Pipettes . . . . . . . . . . . . . . . . . . . . . . . . . . 157
6.5 Calibration of Volumetric Flask/Measuring Cylinder/Beaker . . . 158
6.6 Calibration of Thermometer . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
6.7 Calibration of Weighing Balance . . . . . . . . . . . . . . . . . . . . . . . 160
6.8 Standardization of Acids, Bases, and Other Chemical
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
7 Tests to Ensure Quality of Dairy Products . . . . . . . . . . . . . . . . . . . 175
7.1 Determining Strength of Washing Solution . . . . . . . . . . . . . . . . 175
7.2 Determination of Available Chlorine in Hypochlorite Solution . . . 177
7.3 Determination of Iodine in Chemical Sanitizer . . . . . . . . . . . . . 179
7.4 Assessing Sterility of Dairy Equipment . . . . . . . . . . . . . . . . . . . 180
xii Contents
7.5 Hardness Determination of Water . . . . . . . . . . . . . . . . . . . . . . . 181

7.6 Biological Oxygen Demand of Dairy Effluent . . . . . . . . . . . . . . 184
7.7 Chemical Oxygen Demand of Dairy Effluent . . . . . . . . . . . . . . 185
7.8 Detection of Aflatoxin M1 in Milk . . . . . . . . . . . . . . . . . . . . . . 186
7.9 Determination of Melamine in Milk and Infant Formula . . . . . . 193
7.10 Determination of Pesticides in Milk . . . . . . . . . . . . . . . . . . . . . 196
7.11 Determination of Antibiotic Residues in Milk . . . . . . . . . . . . . . 201
8 Specifications of Chemicals Used in Dairy Industry . . . . . . . . . . . . . 213
8.1 Specifications of Gerber Sulfuric Acid (IS: 1224, Part-II) . . . . . . 213
8.1.1 Preparation of Gerber Sulfuric Acid . . . . . . . . . . . . . . 213
8.1.2 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
8.2 Specification of Amyl Alcohol (IS: 360) . . . . . . . . . . . . . . . . . . 214
8.2.1 Test for Furfural and Other Organic Impurities . . . . . . 214
8.2.2 Test of Amyl Alcohol for Suitability for Milk
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
8.2.3 Hydrochloric Acid Test . . . . . . . . . . . . . . . . . . . . . . . 215
8.2.4 Packing and Marking . . . . . . . . . . . . . . . . . . . . . . . . 215
8.2.5 Sampling Instructions . . . . . . . . . . . . . . . . . . . . . . . . 215
8.3 Specification of Common Salt (Edible) (IS: 253) . . . . . . . . . . . . 216
8.3.1 Preparation of the Sample for Chemical Tests . . . . . . . 216
8.3.2 Determination of Water Insoluble Matter . . . . . . . . . . 217
8.3.3 Determination of Chloride Content . . . . . . . . . . . . . . 217
8.3.4 Determination of Sulfate . . . . . . . . . . . . . . . . . . . . . . 217
8.3.5 Determination of Alkalinity . . . . . . . . . . . . . . . . . . . . 218
8.3.6 Determination of Water-Soluble Ca and Mg . . . . . . . . 218
8.3.7 Test for Lead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
8.4 Specification of Sodium Citrate (Food Grade) (IS: 5058) . . . . . . 221
8.4.1 Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
8.4.2 Packing, Storage, and Marking . . . . . . . . . . . . . . . . . 222
8.5 Specifications of Nitric Acid (IS: 264) . . . . . . . . . . . . . . . . . . . 223
8.5.1 Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
8.5.2 Determination of Total Acidity . . . . . . . . . . . . . . . . . 223
8.5.3 Determination of Sulfates . . . . . . . . . . . . . . . . . . . . . 225
8.5.4 Test for Heavy Metals . . . . . . . . . . . . . . . . . . . . . . . 225
8.6 Specification of Caustic Lye (IS: 252) . . . . . . . . . . . . . . . . . . . 226
8.6.1 Form and Description . . . . . . . . . . . . . . . . . . . . . . . . 226
8.6.2 Preparation of Sample for Testing . . . . . . . . . . . . . . . 227
8.6.3 Determination of Carbonates . . . . . . . . . . . . . . . . . . . 227
8.6.4 Determination of Sodium Hydroxide . . . . . . . . . . . . . 227
8.6.5 Measurement of Total Strength . . . . . . . . . . . . . . . . . 228
8.6.7 Sampling of Caustic Lye . . . . . . . . . . . . . . . . . . . . . . 230
Contents xiii
8.7 Specifications for Sodium Thiosulfate (IS: 14781) . . . . . . . . . . . 230

8.7.1 Test for Heavy Metals . . . . . . . . . . . . . . . . . . . . . . . 233
8.8 Specification for Reagent Grade Water (IS: 1070) . . . . . . . . . . . 234
9 Quality Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
9.1 What Is Quality? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
9.2 What Is Quality Control? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
9.2.1 Implementation of Effective Quality Control . . . . . . . 240
9.2.2 Responsibilities of Quality Control Department . . . . . 240
9.3 What Is Quality Assurance? . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
9.4 Objectives and Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
9.5 Responsibility of the Quality Assurance Department . . . . . . . . . 242
9.6 Principles of Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . 243
9.7 Deming’s Philosophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
9.8 The Deming Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
9.8.1 Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
9.8.2 Do . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
9.8.3 Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
9.8.4 Act . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
9.9 The Juran’s Philosophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
9.9.1 Quality Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
9.9.2 Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
9.9.3 Quality Improvement . . . . . . . . . . . . . . . . . . . . . . . . 249
9.10 Three Steps to Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
9.11 Ten Steps to Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
9.12 Pareto Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
9.13 Crosby’s Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
9.13.1 Crosby’s Absolutes . . . . . . . . . . . . . . . . . . . . . . . . . . 251
9.13.2 Zero Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
9.14 Ishikawa’s Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
9.15 Total Quality Management . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
9.15.1 Basic Principles of TQM . . . . . . . . . . . . . . . . . . . . . . 254
9.15.2 Elements of TQM . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
9.15.3 Approaches to TQM . . . . . . . . . . . . . . . . . . . . . . . . . 255
9.15.4 The Concept of Continuous Improvement
to Be Achieved by TQM . . . . . . . . . . . . . . . . . . . . . . 256
9.16 Pillars of TQM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
9.16.1 Obstacles Encountered During Implementation
of TQM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
9.17 Hazard Analysis and Critical Control Point (HACCP) . . . . . . . . 258
9.17.1 History of HACCP . . . . . . . . . . . . . . . . . . . . . . . . . . 259
9.17.2 Prerequisites in a HACCP Plan . . . . . . . . . . . . . . . . . 259
xiv Contents
9.18 Principles of HACCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

9.19 Housekeeping (5S) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
9.20 Meaning of 5S? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
9.20.1 Kaizen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
9.20.2 Hoshin Kanri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
9.20.3 Six Sigma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
9.21 DMAIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
9.22 DMADV or DFSS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
9.22.1 Key Roles in Six Sigma . . . . . . . . . . . . . . . . . . . . . . 270
10 Safety and Regulatory Aspects of Dairy Industry . . . . . . . . . . . . . . 273
10.1 WTO Agreements and SPS Measures . . . . . . . . . . . . . . . . . . . . 273
10.1.1 Most Favored Nations . . . . . . . . . . . . . . . . . . . . . . . . 276
10.1.2 Sanitary and Phytosanitary Measures . . . . . . . . . . . . . 277
10.2 Codex Alimentarius Commission . . . . . . . . . . . . . . . . . . . . . . . 277
10.3 International Organization for Standards (ISO) . . . . . . . . . . . . . 278
10.3.1 ISO Governance Structure . . . . . . . . . . . . . . . . . . . . . 279
10.4 Hazard Analysis and Critical Control Point . . . . . . . . . . . . . . . . 283
10.5 Regulatory Institutions of India . . . . . . . . . . . . . . . . . . . . . . . . 285
10.5.1 Legal and Quality Standards . . . . . . . . . . . . . . . . . . . 285
10.5.2 Quality Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
10.5.3 Requirement of Legal and Quality Standards . . . . . . . 287
10.6 Roles and Functions of FSSAI . . . . . . . . . . . . . . . . . . . . . . . . . 288
10.6.1 FSSAI Registration and Important Terminology . . . . . 288
10.6.2 Types of Licenses/Registration . . . . . . . . . . . . . . . . . 289
10.6.3 Penalty and Punishments . . . . . . . . . . . . . . . . . . . . . 289
10.6.4 Initiatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
10.6.5 Food Traceability . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
10.6.6 Food Recall Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
10.6.7 Composition of Food Authority (FSSAI) . . . . . . . . . . 298
10.6.8 Enforcement of the Act . . . . . . . . . . . . . . . . . . . . . . . 299
10.6.9 Central Advisory Committee . . . . . . . . . . . . . . . . . . . 300
10.6.10 Commissioners of Food Safety . . . . . . . . . . . . . . . . . 300
10.6.11 Procedure for Collection and Analysis of FSSAI
Food Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
10.6.12 Preservative Permitted to Be Added to Samples . . . . . 301
10.7 Agmark and BIS Standards for Milk Products . . . . . . . . . . . . . . 301
10.7.1 Objectives of Agmark Scheme . . . . . . . . . . . . . . . . . 302
10.7.2 AGMARK Standards for Butter . . . . . . . . . . . . . . . . 302
10.7.3 Specifications for Butter Under AGMARK . . . . . . . . 302
10.7.4 Grading of Ghee Under AGMARK . . . . . . . . . . . . . . 303
10.7.5 Bureau of Indian Standards . . . . . . . . . . . . . . . . . . . . 305
10.7.6 Structure of BIS/Members of BIS . . . . . . . . . . . . . . . 305
10.7.7 Objectives and Functions of BIS . . . . . . . . . . . . . . . . 306
10.7.8 How to Get Authority to Use ISI Mark? . . . . . . . . . . . 306
Contents xv
10.8 International Dairy Federation . . . . . . . . . . . . . . . . . . . . . . . . . 307

10.9 Food and Drug Administration . . . . . . . . . . . . . . . . . . . . . . . . . 308
10.9.1 Organizations of FDA . . . . . . . . . . . . . . . . . . . . . . . . 308
10.9.2 What Does FDA Regulates? . . . . . . . . . . . . . . . . . . . 308
10.9.3 Regulatory Programs of FDA . . . . . . . . . . . . . . . . . . 309
10.9.4 Food and Dietary Supplements . . . . . . . . . . . . . . . . . 309
10.9.5 Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
10.9.6 Vaccines, Blood and Tissue Products, and
Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
10.9.7 Medical and Radiation-Emitting Devices . . . . . . . . . . 310
10.9.8 Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
10.9.9 Veterinary Products . . . . . . . . . . . . . . . . . . . . . . . . . 310
10.9.10 Tobacco Products . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
10.9.11 What Does FDA not Regulate? . . . . . . . . . . . . . . . . . 311
10.9.12 FDA and India . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Appendix 1 FSSAI Standards for Milk and Milk Products . . . . . . . . . 313

Appendix 2 Scheme for Testing and Inspection for Milk
to be Adopted by Dairy Processing Units . . . . . . . . . . . . . . . . . . . . . . . . 335
About the Authors
Kamal Gandhi has been a Scientist at the Department of Dairy Chemistry,

National Dairy Research Institute, Karnal since 2016. He has received his PhD
degree in Dairy Chemistry from National Dairy Research Institute University in
2014, and worked at the Gujarat Cooperative Milk Marketing Federation
(GCMMF), Amul, for one and half years. His areas of expertise include milk and
milk products adulteration detection, functional foods, and milk lipids. He is the
author of 20 research, review and popular science articles in national and interna-
tional journals, and compendiums and recipient of best paper and poster awards. He
is a life member of the Indian Science Congress Association, Association of Food
Scientists and Technologists, India (AFSTI), and the Indian Dairy Association
(IDA).
Rajan Sharma is currently working as the Principal Scientist at the Department of

Dairy Chemistry, National Dairy Research Institute, Karnal. He has 21 years of
experience in the field of milk quality and analytical dairy chemistry. He has been
associated with the Food Safety and Standards Authority of India since 2009, as a
member of the Scientific Panels on Milk and Milk Products as well as on Methods of
Sampling and Analysis. He has worked as an assessor for the National Accreditation
Board for Testing and Calibration Laboratories (NABL) since 2003. Many of the
rapid methods for assessing milk quality developed by his group have been
commercialized for the dairy industry. He is a recipient of the NRDC Meritorious
Invention Award—2013, and is a Fellow of the National Academy of Agricultural
Sciences and National Academy of Dairy Science.
Priyae Brath Gautam is currently pursuing his PhD in Dairy Chemistry at the
National Dairy Research Institute, Karnal. He was the Deputy Manager (Quality
Assurance) of the Punjab State Co-operative Milk Producers' Federation Limited for
2 years. He was awarded the University Gold Medal for his academic excellence in
his undergraduate course in Dairy Technology.
xvii
xviii About the Authors
Bimlesh Mann is currently the Head and Principal Scientist at the Dairy Chemistry
Division, ICAR-National Dairy Research Institute, Karnal. Her research over the last
28 years has focused on the chemistry of milk and milk products, with a focus on
bioactive milk proteins and peptides, functional dairy foods and nanoencapsulation
of bioactive components for dairy foods. She is also involved in research related to
quality assurance of dairy products. She is the recipient of the Best Teacher Award
from the Indian Council of Agricultural Research.
List of Figures
Fig. 1.1 Milk production and yield in some regions of the world.
Source: OECD/FAO (2019), “OECD-FAO Agricultural Outlook,”
OECD Agriculture statistics (database),
https://doi.org/10.1787/agr-outl-data-e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Fig. 1.2 Per capita consumption of milk solids in fresh and processed
dairy products. Note: The milk solids are calculated by adding
the fat and solids-not-fat for a product. The processed dairy
products include butter, cheese, skim milk powder, and whole
milk powder. Source: OECD/FAO (2019), “OECD-FAO
Agricultural Outlook,” OECD Agriculture statistics (database),
https://doi.org/10.1787/agr-outl-data-en . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 11
Fig. 1.3 Prices of dairy products in the international market. Note: Butter
FOB export price, butter, 82% butterfat, Oceania, skim milk
powder, FOB export price, SMP, 1.25% butterfat, Oceania;
whole milk powder, FOB export price, 26% butterfat, Oceania;
cheese, FOB export price, cheddar cheese, 39% moisture,
Oceania. Real prices are nominal world prices deflated by the
US GDP deflator (2010 ¼ 1). Source: OECD/FAO (2019),
“OECD-FAO Agricultural Outlook,” OECD Agriculture statistics
(database), https://doi.org/10.1787/agr-outl-data-en . . .. . . . . . . . .. . . . . . 12
Fig. 1.4 Major exporters of dairy products. Source: OECD/FAO (2019),
“OECD-FAO Agricultural Outlook,” OECD Agriculture statistics
(database), https://doi.org/10.1787/agr-outl-data-en . . .. . . . . . . . .. . . . . . 13
Fig. 2.1 Plunger .. . . . . . . . .. . . . . . . . . .. . . . . . . . .. . . . . . . . . .. . . . . . . . .. . . . . . . . . .. . . . . . . . . 21
Fig. 2.2 Trier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Fig. 3.1 Somascope .. . . . .. . . . . .. . . . .. . . . .. . . . . .. . . . .. . . . .. . . . .. . . . . .. . . . .. . . . .. . . . 47
Fig. 3.2 Bactoscope .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . . 47
Fig. 3.3 Semiautomatic protein digestion unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Fig. 3.4 Automatic protein distillation unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Fig. 5.1 RM apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Fig. 5.2 Still head used for RM estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Fig. 5.3 Butyro refractometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
xix
xx List of Figures
Fig. 7.1 Structures of different aflatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

Fig. 7.2 Structure of organochlorine pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Fig. 7.3 Structure of some organophosphate pesticides . . . . . . . . . . . . . . . . . . . . . . 198
Fig. 7.4 Structure of some carbamate pesticides . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . 198
Fig. 7.5 General structure of penicillin and cephalosporin . . . . . . . . . . . . . . . . . . 202
Fig. 7.6 Structure backbone of carbapenem .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . 203
Fig. 7.7 Structure backbone of monobactam . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . 203
Fig. 7.8 Structure backbone of tetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Fig. 7.9 Structure of streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Fig. 7.10 Structure of the functional group in sulfonamide . . . . . . . . . . . . . . . . . . . 204
Fig. 7.11 Structure of erythromycin . . . . . .. . . . . . .. . . . . . .. . . . . .. . . . . . .. . . . . . .. . . . . 205
Fig. 10.1 Areas of IDF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
List of Tables
Table 1.1 State wise milk production (MT) in India . .. . . .. . .. . . .. . .. . .. . . .. . 2

Table 1.2 Milk production and per capita availability of milk in India . . . . 4
Table 1.3 State wise per capita consumption of milk . .. . . . . .. . . . . .. . . . . .. . . . 5
Table 1.4 Contribution of livestock and agriculture sector to GDP . . . . . . . . 7
Table 1.5 Milk production in different countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Table 1.6 Global dairy trade at a glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Table 2.1 Number of samples to be drawn randomly from
cans/containers (IS: 1479, Part-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Table 2.2 Sampling scale for cheese (IS: 2785) .. . . . .. . . . .. . . . .. . . . . .. . . . .. . . 24
Table 2.3 Sampling scale for sterilized milk/flavored milk (IS: 4238) . . . . . 24
Table 2.4 Sampling scale for dahi, yoghurt, and srikhand (IS: 4238) . . . . . . 25
Table 2.5 Selection of bulk containers on a random basis (IS: 2802) . . . . . . 26
Table 2.6 Selection of retail containers randomly (IS: 2802) . . . . . . . . . . . . . . . 26
Table 2.7 Selection of random samples for containers
of 400 g to 5 kg (IS: 1166) . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 27
Table 2.8 Selection of random samples for containers
of more than 5–20 kg (IS: 1166) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Table 2.9 Sampling for containers of 500 g and up to 5 kg (IS: 1165) . . . . 28
Table 2.10 Sampling for containers of more than 5 kg (IS: 1165) . . . . . . . . . . . 28
Table 2.11 Sampling scale for butter when packed in bulk containers
(IS: 3507) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Table 2.12 Sampling scale for butter when packed in packets or tin
(IS: 3507) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Table 2.13 Samples to be collected for ghee/butter oil/anhydrous
milk fat (IS: 3508) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Table 3.1 BIS standards for sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Table 3.2 Observation chart for alcohol–alizarin test . . . . . . . . . . . . . . . . . . . . . . . . 35
Table 3.3 Limit of detection (LOD) for different adulterants . . . . . . . . . . . . . . . 50
Table 3.4 Detection of some common adulterants/contaminants
by immunological and biosensor methods . . . . . . . . . . . . . . . . . . . . . . . . 52
xxi
xxii List of Tables
Table 3.5 Correction to be applied in the lactometer reading taken

at a temperature other than 27 C, to obtain the lactometer
reading of milk at 27 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Table 4.1 Conversion factor for various proteins to be used in the
estimation of total protein by Kjeldahl method . . . . . . . . . . . . . . . . . . . 76
Table 4.2 Interpretation of results for alkaline phosphatase test . . . . . . . . . . . . 81
Table 4.3 Relationship between creaming index and homogenization
efficiency for milk . . .. . .. . .. . .. .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. . 83
Table 5.1 Dimensions of the flat bottom flask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Table 5.2 Dimension of the still head . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 112
Table 5.3 Dimensions of the condenser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Table 5.4 Classification of ghee according to peroxide value
(IS: 3508) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Table 5.5 Butyro refractometer readings and their corresponding
refractive indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Table 5.6 Opacity time for pure ghee and adulterated ghee . .. .. . .. . .. .. . .. . 127
Table 5.7 Analytical conditions for HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Table 6.1 Classification of butyrometer as per BIS . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Table 6.2 Specification of lactometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Table 6.3 Specific gravity and lactometer reading of pure sodium
carbonate solution . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 156
Table 6.4 Specific gravity and lactometer reading of pure sodium
chloride solution .. . .. . .. . .. .. . .. . .. . .. .. . .. . .. . .. .. . .. . .. .. . .. . .. . .. . 157
Table 6.5 Recording of results for eccentric test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Table 6.6 Recording of results for repeatability tests . . . . . . . . . . . . . . . . . . . . . . . . 160
Table 6.7 Amount of chemical required to make 0.1 N primary
standard solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Table 6.8 Some pH-based indicators . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . 164
Table 6.9 Change in the indicator color at certain base–acid
combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Table 6.10 Approximate strength of commercially available
concentrated acids .. . . . .. . . . . .. . . . . .. . . . . .. . . . .. . . . . .. . . . . .. . . . .. . . . . 168
Table 6.11 Tolerance of various types of pipettes . . . . . . . . . .. . . . . . . . . . . .. . . . . . . 171
Table 6.12 Density of water and mercury at various temperatures . . . . . . . . . . 171
Table 6.13 Capacities, scales, and tolerance for butyrometers
(IS 1223:2001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Table 7.1 The sanitary condition of the can . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Table 7.2 Sanitary conditions of the area under test . . . . . . . . . . . . . . . . . . . . . . . . . 181
Table 7.3 Hardness of water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Table 7.4 List of mycotoxins produced by various fungi . . . . . . . . . . . . . . . . . . . 187
Table 7.5 Food regulatory standards for aflatoxin M1 in milk . . . . . . . . . . . . . 188
Table 7.6 Standards for melamine in milk and milk products . . . . . . . . . . . . . . 193
Table 7.7 Classification of pesticide based on the extent of hazard . . . . . . . . 197
List of Tables xxiii
Table 7.8 Classification of pesticides based on the target pest . . . .. . . .. . .. . . 197

Table 7.9 Differentiation between organochlorine, organophosphate,
and carbamate pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Table 7.10 HPLC operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Table 7.11 LC conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Table 8.1 Requirements for amyl alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Table 8.2 Specifications for dairy salt (IS: 253) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Table 8.3 Number of packages to be selected for sampling . . . . . . . . . . . . . . . . 220
Table 8.4 Requirements for sodium citrate (IS: 5058) . . .. . . .. . .. . . .. . .. . . .. . 221
Table 8.5 Selection of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Table 8.6 Specifications of Nitric acid (IS: 264) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Table 8.7 Selection of containers . . . .. . . . . .. . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . .. . . . 226
Table 8.8 Specifications of caustic Lye (IS: 252) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Table 8.10 Specifications of sodium thiosulfate (IS: 14781) . . . . . . . . . . . . . . . . . 231
Table 8.11 Requirements of reagent grade water . . . . .. . . . . .. . . . . . .. . . . . . .. . . . . 235
Table 9.1 Quality control vis-à-vis quality assurance . . . . . . . . . . . . . . . . . . . . . . . . 243
Table 9.2 HACCP analysis for market milk . . . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . 264
Table 10.1 Agmark standards for ghee . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . 303
List of Abbreviations
ADI Acceptable Daily Intake

AFB Aflatoxin B
AFM Aflatoxin M
AGMARK Agricultural Produce (Grading and Marking)
AMA Agricultural Marketing Advisor
AOAC Association of Official Analytical Chemists
APEDA The Agricultural and Processed Food Products Export Development
Authority
AR grade Analytical Reagent
ASTM American Society for Testing and Materials
ATCC American Type Culture Collection
BHA Butylated Hydroxy anisole
BHOG Blissful Hygienic Offering to God
BHT Butylated Hydroxy toluene
BIS Bureau of Indian Standard
BOD Biological Oxygen Demand
BR Butyro refractometer Reading
CA Certificate of Authorization
CAC Codex Alimentarius Commission
CAGR Compound Annual Growth Rate
CASCO Commerce and Administrative Student Charity Organization
CCFS Central Committee for Food Standards
CCP Critical Control Point
CDER Center for Drug Evaluation and Research
CDRH Centre for Devices and Radiological Health
CETA Comprehensive Economic and Trade Agreement
CFL Central Food Laboratory
CFSAN Centre for Food Safety and Applied Nutrition
CIBRC Central Insecticides Board & Registration Committee
CIP Cleaning in Place
CLR Corrected Lactometer Reading
CMO Chief Medical Officer
COB Clot-on-boiling
xxv
xxvi List of Abbreviations
COD Chemical Oxygen Demand

COPOLCO Consumer Policy Committee of the International Organization for
Standardization
CPTPP Comprehensive and Progressive Agreement for Trans-Pacific
Partnership
CQP Critical Quality Point
CVD Cardio Vascular Disease
CVM Centre for Veterinary Medicine
DAHD&F Department of Animal Husbandry Dairying and Fisheries
DART Daily Average Revenue Trade
DDT 1,1'-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene)
DEVCO ISO Committee on Developing Country Matters
DGHS Directorate General of Health Services
DMADV Define, Measure, Analyze, Design, and Verify
DMAIC Define, Measure, Analyze, Improve, and Control
DMI Directorate of Marketing & Inspection
DNA Deoxy-ribonucleic acid
EAMS Electronic Adjudication Management System
EBT Eriochrome black t
EDTA Ethylenediaminetetraacetic acid
EEE Embrace, Extend, and Extinguish
EFQM European Foundation for Quality Management
ELISA Enzyme-Linked Immunosorbent Assay
EMS Environmental Management Systems
EPA Environment Protection Act
FAO Food and Agriculture Organization
FBO Food Business Operators
FDA Food and Drug Administration
FFA Free fatty acid
FFRC Food Fortification Resource Centre
FID Flame Ionization Detector
FOB Free on Board
FoSTaC Food Safety Training and Certification
FSMS Food Safety Management System
FSO Food Safety Objective
FSSAI Food Safety and Standards Authority of India
FTAs Free Trade Agreements
GATT General Agreement on Tariffs and Trade
GDP Gross Domestic Product
GHP Good Hygienic Practice
GMP Good Manufacturing Practice
GOI Government of India
GVA Gross Value Added
H.Q. Headquarter
HACCP Hazard Analysis Critical Control Point
List of Abbreviations xxvii
HILIC Hydrophilic Interaction Liquid Chromatography

HMF Hydroxy-Methyl-Furfural
HPLC-MS High-Pressure Liquid Chromatography
HPTLC High-Pressure Thin Layer Chromatography
IARC International Agency for Research on Cancer
ICAR Indian Council of Agriculture Research
ICSAS Informatics and Computational Safety Analysis Staff
IDF International Dairy Federation
IEC International Electro-technical Commission
IFSA Indian Food Sharing Alliance
IMARC Information Management Research Centre
INR Indian Rupee
IS Indian Standards
ISI Indian Standard Institute
ISO International Organization of Standardization
ISO-CS International Organization for Standardization Central Secretariat
LB Liebermann Burchard
LC grade Laboratory Reagents Grade
LD50 Lethal Dose, 50%
LDH Lactate Dehydrogenase
LOD Limit of Detection
MBRT Methylene Blue Reduction Time
MFR Melamine Formaldehyde Resins
MRL Maximum Residual Level
NABL National Accreditation Board for Testing and Calibration
of Laboratories
NAS National Academy of Sciences
NASA National Aeronautics and Space Administration
NDGA Nordihydroguaiaretic acid
NFL National Food Laboratory
NGOs Non-Governmental Organization
NLT Not less than
NMT Not more than
NOEL No Observable Effect Level
OECD Organisation for Economic Co-operation and Development
OPDCA Observe Plan Do Check Act
PDCA Plan Do Check Act
PFA Prevention of Food Adulteration
PSA Primary secondary amine
PV Polenske Value
QA Quality Assurance
QAC Quaternary ammonium compound
QC Quality Control
QMS Quality Management System
rcf Relative Centrifugal Force
xxviii List of Abbreviations
RFL Referral Food Laboratories

RI Refractive Index
RM Reichert–Meissl Value
RP-HPLC Reverse-Phase High-Pressure Liquid Chromatography
RPM Revolution per minute
RUCO Repurpose Used Cooking Oil
SCM Sweetened Condensed Milk
SLST Sodium Lauryl Sulfate Test
SMP Skim Milk Powder
SNF Solid-Not-Fat
SOP Standard Operating Procedure
SPS Sanitary and Phytosanitary Standards
SQF Safe Quality Food
TFA Trifluoroacetic Acid
TI Test Instrument
TLC Thin Layer Chromatography
TMB Technical Management Board
TPA Third-Party Administrator
TQM Total Quality Management
TS Total Solid
UHT Ultra-High Temperature
USDA United States Department of Agriculture
USM Unsaponifiable Matter
UUT Unit Under Test
UV Ultra-violet
WBC White Blood cells
WMP Whole Milk Powder
WTO World Trade Organization
Introduction
1
1.1 Status of Milk Production and Consumption in India
Currently, India ranks first in the milk production throughout the world, which has
increased from 20.8 million tonnes in 1970–1971 to 187.75 million tonnes in
2017–2018 with an increase of 88.9%. Among the states of the Indian subcontinent,
Uttar Pradesh is the largest milk producer followed by Rajasthan and Madhya
Pradesh (Table 1.1). The per capita availability in India increased from 178 gm/
day in 1991–1992 to 394 gm/day in 2018–2019 (Table 1.2). Among the states, the
per capita consumption of milk is highest for Punjab, i.e., 1181 g/day, followed by
Haryana which is around 1087 g/day, while Mizoram has the least per capita
availability which was recorded to be 64 g/day (Table 1.3). The agriculture and
allied sectors contribute to around 17% to the total GDP while the livestock sector
contributes to 4.9% as recorded in 2017–2018 (Table 1.4). The Indian milk
processing industry is estimated to grow at a compound annual growth rate
(CAGR) of ~14.8% between FY 2018 and FY 2023, and will reach INR 2458.7
billion in FY 2023. The India Dairy Industry is worth Rs. 5.4 trillion by value,
having grown at a CAGR of 15% during 2010–2016. The major reason for the
increase in the demand for milk in the country is owed to the increasing population
and growing middle class. The majority of the milk produced in India is processed
and marketed by the unorganized sector, which accounts for around ~81.1% of the
Indian dairy and milk processing market in the FY 2018. The milk produced by the
unorganized sector is generally produced in unhygienic environment that reduces the
overall quality and nutrition levels of the milk produced. Apart from milk, value-
added products such as butter, curd, paneer, ghee, whey, flavored milk, ultra-high
temperature (UHT) milk, cheese, and yogurt also contribute to the revenue to the
Indian dairy and milk processing industry. The market size of butter, curd, paneer,
and ghee has grown by 14.5, 14.4, 14.1, and 14.4%, respectively, between 2016 and
2020. About 7% of the annual milk production is converted into Dahi. The market
size for Dahi was INR 896 crores in 2008 while in 2015 it was INR 5038 crores with
an annual growth rate of 28%. The current average growth rate of buttermilk
# Springer Nature Singapore Pte Ltd. 2020 1

K. Gandhi et al., Chemical Quality Assurance of Milk and Milk Products,
https://doi.org/10.1007/978-981-15-4167-4_1
2
Table 1.1 State wise milk production (MT) in India

States 01–02 02–03 03–04 04–05 05–06 06–07 07–08 08–09 09–10 10–11 11–12 12–13 13–14 14–15 15–16 16–17 17–18 18–19
All India 84,406 86,159 88,082 92,484 97,066 102,580 107,934 112,183 116,425 121,848 127,904 132,431 137,685 146,314 155,491 165,404 176,347 187,749
Andhra 5814 6584 6959 7257 7624 7938 8925 9570 10,429 11,203 12,088 12,762 13,007 9656 10,817 12,178 13,725 15,044
Pradesh
Arunachal 42 46 46 48 48 49 32 24 26 28 22 23 43 46 50 53 54 55
Pradesh
Assam 682 705 727 739 747 750 752 753 756 790 796 800 815 829 843 861 872 882
Bihar 2664 2869 3180 4743 5060 5451 5783 5934 6124 6517 6643 6845 7197 7775 8288 8711 9242 9818
Chhattisgarh 795 804 812 831 839 849 866 908 956 1029 1119 1164 1209 1232 1277 1374 1469 1567
Goa 45 46 48 57 56 57 58 59 59 60 60 61 68 67 54 51 55 57
Gujarat 5862 6089 6421 6745 6960 7533 7911 8386 8844 9321 9817 10,315 11,112 11,691 12,262 12,784 13,569 14,493
Haryana 4978 5124 5221 5222 5299 5366 5442 5745 6006 6267 6661 7040 7442 7901 8381 8975 9809 10,726
Himachal 756 773 786 870 869 933 1007 1026 971 1102 1120 1139 1151 1172 1283 1329 1392 1460
Pradesh
Jammu and 1360 1389 1414 1422 1400 1400 1519 1565 1592 1609 1614 1631 1615 1951 2273 2376 2460 2540
Kashmir
Jharkhand 940 952 954 1330 1335 1401 1442 1466 1463 1555 1745 1679 1700 1734 1812 1894 2016 2183
Karnataka 4797 4539 3857 3917 4022 4124 4244 4538 4822 5114 5447 5718 5997 6121 6344 6562 7137 7901
Kerala 2718 2419 2111 2025 2063 2119 2253 2441 2509 2645 2716 2791 2655 2711 2650 2520 2576 2548
Madhya 5283 5343 5388 5506 6283 6374 6572 6855 7167 7514 8149 8838 9599 10,779 12,148 13,445 14,713 15,911
Pradesh
Maharashtra 6094 6238 6379 6567 6769 6978 7210 7455 7679 8044 8469 8734 9089 9542 10,153 10,402 11,102 11,655
Manipur 68 69 71 75 77 77 78 78 78 78 79 80 82 82 79 79 82 86
Meghalaya 66 68 69 71 73 74 77 77 78 79 80 81 82 83 84 84 85 87
Mizoram 14 15 15 16 15 16 17 17 11 11 14 14 15 20 22 24 25 26
Nagaland 57 58 63 69 74 67 45 53 78 76 78 79 81 76 77 79 74 73
Orissa 929 941 997 1283 1342 1431 1625 1598 1651 1671 1721 1724 1861 1903 1930 2003 2088 2311
Punjab 7932 8173 8391 8554 8909 9168 9282 9387 9389 9423 9551 9724 10,011 10,351 10,774 11,282 11,855 12,599
Rajasthan 7758 7789 8054 8310 8713 10,309 11,377 11,931 12,330 13,234 13,512 13,946 14,573 16,934 18,500 20,850 22,427 23,668
Sikkim 37 45 48 46 48 49 42 42 44 43 45 42 46 50 67 54 59 61
Tamil Nadu 4988 4622 4752 4784 5474 6277 6540 6651 6787 6831 6968 7005 7049 7132 7244 7556 7742 8362
Telangana – – – – – – – – – – – – – 4207 4442 4681 4965 5416
Tripura 90 79 84 86 87 89 91 96 100 104 111 118 130 141 152 160 174 185
1 Introduction
Uttar Pradesh 14,648 15,288 15,943 16,512 17,356 18,094 18,861 19,537 20,203 21,031 22,556 23,330 24,194 25,198 26,387 27,770 29,052 30,519
Uttarakhand 1066 1079 1188 1195 1206 1213 1221 1230 1377 1383 1417 1478 1550 1565 1656 1692 1742 1792
West Bengal 3515 3600 3686 3790 3891 3983 4087 4176 4300 4471 4672 4859 4906 4961 5038 5183 5389 5607
A&N Islands 23 26 25 24 20 23 24 26 24 25 26 21 14 16 15 16 17 18
Chandigarh 43 43 44 43 46 46 47 47 46 45 45 44 44 44 43 36 42 45
Dadra and 8 8 8 4 5 5 10 10 10 11 11 11 11 9 9 8 8
Nagar Haveli
Daman and 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Diu
Delhi 294 296 299 303 310 288 445 450 466 480 502 287 284 280 281 279 279
Lakshadweep 2 2 1 1 2 2 2 2 2 2 2 2 6 4 3 3 3 4
Puducherry 37 37 40 41 43 45 46 46 46 47 45 47 47 48 48 48 49 49
Source: Department of Animal Husbandry, Dairying & Fisheries, Ministry of Agriculture and Farmers Welfare, Government of India
3
4 1 Introduction
Table 1.2 Milk production and per capita availability of milk in India
Year Production (Mt) Per capita availability (g/day)
1991–1992 55.6 178
1992–1993 58.0 182
1993–1994 60.6 186
1994–1995 63.8 192
1995–1996 66.2 195
1996–1997 69.1 200
1997–1998 72.1 205
1998–1999 75.4 210
1999–2000 78.3 214
2000–2001 80.6 217
2001–2002 84.4 222
2002–2003 86.2 224
2003–2004 88.1 225
2004–2005 92.5 233
2005–2006 97.1 241
2006–2007 102.6 251
2007–2008 107.9 260
2008–2009 112.2 266
2009–2010 116.4 273
2010–2011 121.8 281
2011–2012 127.9 290
2012–2013 132.4 299
2013–2014 137.7 307
2014–2015 146.3 322
2015–2016 155.5 337
2016–2017 165.4 355
2017–2018 176.3 375
2018–2019 187.7 384
Source: Basic Animal Husbandry Statistics, DAHD&F, Government of India
worldwide is about 2–3%, the production recorded in 2011 was 1429 KMT
which was increased to 1684 KMT in 2016. The forecasted CAGR of buttermilk
is expected to be about 3.5% from 2016 to 2024 because of the increase in the
demand of buttermilk powder. According to a recent report “Dairy Industry in India:
2013–2019,” the market of buttermilk has grown at a CAGR of approximately
21.4% in the period 2013–2019. The cheese industry in India is in its nascent
stage, accounting to just around 1% of the total milk products and is consumed by
the urban population. Cheese was first marketed in India under the brand name
“Amul” in 1970s. The cheese industry is estimated to grow at about 10–12% per year
in terms of volume and 16–17% per year in value terms. The per capita consumption
of cheese in India is too low, i.e., 2.4 kg/annum as compared to that in the United
States which is 20 kg/annum. The major variants of cheese in India are processed
Table 1.3 State wise per capita consumption of milk
State 01–02 02–03 03–04 04–05 05–06 06–07 07–08 08–09 09–10 10–11 11–12 12–13 13–14 14–15 15–16 16–17 17–18 18–19
All India 225 230 231 233 241 251 260 266 273 281 290 299 307 322 337 355 375 394
Andhra 209 231 238 250 260 268 298 316 342 364 391 409 413 436 475 522 574 623
Pradesh
Arunachal 105 112 109 114 113 114 73 55 59 63 44 49 93 98 105 109 111 112
Pradesh
Assam 70 71 71 72 72 71 70 70 69 71 70 69 69 70 70 71 71 71
Bihar 88 92 100 147 154 163 170 172 175 184 175 188 195 208 219 228 239 251
Chhattisgarh 105 103 102 103 103 102 103 106 110 117 120 127 130 130 133 141 149 157
Goa 91 91 93 110 105 104 102 99 96 93 113 92 98 94 74 68 70 71
Gujarat 317 321 330 344 349 372 385 402 418 435 445 476 506 527 545 563 592 626
Haryana 645 647 643 631 628 624 621 644 662 679 720 767 800 839 877 930 1005 1087
Himachal 341 339 337 378 373 393 420 424 397 446 447 460 461 466 505 521 542 565
Pradesh
Jammu and 367 365 363 364 353 348 372 378 379 378 352 316 302 352 395 400 401 401
Kashmir
Jharkhand 96 94 92 127 126 130 132 132 130 136 145 146 146 147 152 157 165 177
Karnataka 249 229 190 194 197 199 203 215 226 237 244 262 272 276 282 291 313 344
Kerala 234 203 173 169 171 174 183 197 201 210 223 216 203 206 200 189 192 189
Madhya 240 236 233 233 262 260 264 271 278 287 308 327 349 386 428 468 505 538
Pradesh
Maharashtra 172 172 172 176 178 181 184 188 190 197 206 213 219 228 239 243 256 266
Manipur 86 85 85 90 92 91 91 90 88 88 80 80 80 80 76 75 77 80
Meghalaya 78 78 78 81 82 81 83 83 83 83 74 83 84 84 83 83 83 84
Mizoram 43 45 44 46 43 46 47 47 29 31 35 36 40 53 57 62 63 64
Nagaland 78 78 83 90 96 86 58 67 96 93 108 94 95 88 89 91 84 81
Orissa 69 68 71 92 95 100 113 110 112 113 112 114 122 124 124 128 132 145
Punjab 892 895 898 917 943 957 956 955 944 937 945 961 980 1003 1032 1075 1120 1181
Rajasthan 376 368 371 376 387 449 486 501 509 538 539 555 572 655 704 785 834 870
(continued)
5
6
Table 1.3 (continued)

State 01–02 02–03 03–04 04–05 05–06 06–07 07–08 08–09 09–10 10–11 11–12 12–13 13–14 14–15 15–16 16–17 17–18 18–19
Sikkim 187 222 231 221 232 231 195 194 200 194 202 186 200 215 282 228 244 251
Tamil Nadu 219 198 198 204 231 263 272 274 278 278 265 541 280 282 283 294 300 322
Tripura 77 66 68 70 70 71 72 74 77 80 83 88 95 103 109 114 123 129
Uttar Pradesh 241 245 250 254 262 267 274 278 283 289 310 312 318 326 335 348 359 371
Uttarakhand 344 339 365 364 361 357 354 351 387 383 384 403 418 416 434 440 447 455
West Bengal 120 120 120 124 126 127 129 131 133 137 140 145 145 145 145 148 153 158
A&N Islands 177 195 183 165 135 148 146 154 137 142 187 131 84 90 87 89 92 96
Chandigarh 131 127 127 115 116 112 106 101 95 87 117 103 101 97 93 76 86 90
Dadra and 100 97 95 45 53 50 94 91 86 83 89 101 98 74 72 62 62 62
Nagar Haveli
Daman and 17 17 16 10 11 12 15 15 15 14 11 13 10 10 10 5 9 11
Diu
Delhi 58 57 56 54 54 48 73 72 72 72 82 41 39 37 36 35 35 35
Lakshadweep 90 87 43 45 64 79 79 84 84 71 9 82 219 147 113 110 120 119
Pondicherry 104 101 107 108 108 110 108 101 96 94 99 113 111 110 108 107 106 106
Source: Department of Animal Husbandry, Dairying & Fisheries, Ministry of Agriculture and Farmers Welfare, GoI
1 Introduction
1.1 Status of Milk Production and Consumption in India 7
Table 1.4 Contribution of livestock and agriculture sector to GDP

At current prices (values in INR crores)
Agriculture and allied sector Livestock sector
Year GVA Amount % Share to GVA Amount % Share to GVA
2011–2012 8,106,946 1,501,947 18.5 327,334 4.0
2012–2013 9,202,692 1,675,107 18.2 368,823 4.0
2013–2014 10,363,153 1,926,372 18.6 422,733 4.1
2014–2015 11,504,279 2,093,612 18.2 510,411 4.4
2015–2016 12,574,499 2,227,533 17.7 582,410 4.6
2016–2017 13,935,917 2,496,358 17.9 672,829 4.8
2017–2018 15,482,715 2,670,147 17.2 758,417 4.9
GVA Gross value added
Source: National Accounts Statistics-2019, Central Statistical Organization, Government of India
cheese, mozzarella, cheese spreads, flavored, and spiced cheese having a market
value of ~Rs. 4.5 billion. Processed cheese has the maximum market share at 60%
worth Rs. 2.7 billion, followed by cheese spread which accounts for 30% of market
share. Currently, the market for cheese in India is worth Rs. 1250 crores and it is
expected to grow at a CAGR of 18% during 2015–2020. The market for UHT milk
has reached around INR 50.3 billion in 2018 with a CAGR of approximately 25%
(2011–2018). This market is expected to grow and reach a value of INR 100 billion
in 2021, which will grow further and reach up to INR 193.3 billion by 2024.
Karnataka represents the largest market for UHT, followed by Maharashtra and
Tamil Nadu. The major players involved in the market for UHT are the Gujarat
Cooperative Milk Marketing Federation, Karnataka Milk Federation, Nestle, Tamil
Nadu Cooperative, and Mother Dairy (Report by IMARC).
The price of SMP, ghee, and butter in India ranged from Rs. 225 to Rs. 335/kg,
Rs. 480 to Rs. 510/kg, Rs. 430 to Rs. 450/kg during 2019. Around 16% and 15% of
the total monthly expenditure on food is on dairy in the urban and rural sectors,
respectively, followed by 16% and 20% for cereals in the urban and rural sectors,
respectively. The expenditure on fish, meat, and eggs is 9% in both rural and urban
areas, for pulses it is 5% while the remaining accounts for other food commodities.
The per capita consumption of fresh dairy products in India increased from 52.1 kg/
year in 1997 to 84.4 kg/year in 2017 and is estimated to increase to 116 kg/year by
2027. The dairy products are the second biggest source of protein for the Indian
population followed by cereals (53% of total protein consumed/day) accounting for
12%, followed by pulses (11%), eggs, fish, meat (2%), and other food products
(18%).
8 1 Introduction
1.2 Status of Milk Production and Consumption Throughout

the World
Cow contributes 81% to the total milk production followed by buffalo which is
around 15% while goat, camel, and sheep contribute to remaining 4% in the world.
The world’s milk production was recorded to be 838 million tonnes in 2018, 1.6%
more than the preceding year. The major exporters of dairy products, viz. European
Union, the United States, and New Zealand recorded the increase in milk production
by 0.8, 11, and 3.2%, respectively (milk production of some of the countries is given
in Table 1.5) in 2018. The milk production of China, which is the largest importer of
dairy products, recorded an increase in its milk production by 1.1% over the last 4
years in 2018. Among all the agricultural commodities, the production of milk
throughout the world is expected to rise at 1.7% per annum up to 981 Mt by 2028.
The major milk producers like India and Pakistan are expected to contribute to more
than half of the overall increase in the milk production in upcoming 10 years and
one-third of the entire world’s milk production will be contributed by them. The milk
production in the European Union (the second largest milk producer) is expected to
grow at a much slower pace as compared to the world’s average due to the small
increase in the demand of products like butter, cream, and cheese in the domestic
market along with increase in the demand of the dairy products globally. The milk
produced in the European Union is expected to be organic as more than 10% of the
dairy cows at present in Austria, Greece, Denmark, Sweden, and Latvia are in the
organic system. Presently around 3% of the milk production in the European Union
comes from organic farms having lower yield but the price of milk is higher. The
increase in the milk production in all parts of the world, the contribution of yield
growth towards production is expected to be higher than the herd growth. In
countries like India, the increase in production will occur in small herds composed
of fewer cows or buffaloes and it has been expected that the yield of milk will grow
at a faster rate than the herd growth and will thus contribute more toward the
production growth. The highest yield of milk per cow is expected to be highest for
North America (Fig. 1.1). The majority of the production in countries like India and
Pakistan will be consumed domestically as fresh products while the dairy products
will be traded internationally. The world capita consumption of fresh milk products
is expected to grow at 1% per annum in the coming decade as compared to the
previous decade due to the higher per capita income growth in countries like India.
Another reason for the increase in the demand for fresh milk products is due to the
increase in the population of the developing countries. The overall per capita demand
for fresh milk products in Europe and North America is expected to decline as the
consumption of dairy products is mainly restricted to cheese, which is the second
most important milk product with regard to the milk solids. The level of the
consumption of milk in terms of milk solids varies across the world (Fig. 1.2).
This is generally due to the regional preferences of the consumer and the per capita
income, for example, the per capita consumption of milk solids is high in India but
low in China. Considering the consumption patterns in Europe and North America,
the per capita demand for the fresh dairy products is on a decline, as the trend is
Table 1.5 Milk production in different countries
Country 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2017
India 20.80 25.60 31.56 44.02 53.68 65.37 79.66 95.62 121.85 155.69 176.27
Afghanistan 0.75 0.85 0.84 0.72 0.82 1.36 1.65 1.73 1.72 2.20 2.12
Argentina 4.19 5.65 5.31 5.64 6.28 8.77 10.12 9.91 10.63 12.06 10.10
Australia 7.76 6.70 5.57 6.23 6.46 8.46 10.85 10.13 9.02 9.49 8.80
Bangladesh 1.07 1.18 1.16 1.31 1.59 1.99 2.14 2.62 2.02 2.10 2.01
Brazil 7.42 10.05 12.06 12.57 15.08 17.13 20.53 25.53 30.96 34.86 33.74
Canada 8.31 7.75 7.41 7.48 7.98 7.92 8.16 7.81 8.24 8.14 8.10
Chile 1.12 1.00 1.12 1.05 1.39 1.90 2.00 2.31 2.54 2.04 2.00
China 1.96 2.37 2.93 4.76 7.04 9.46 12.37 32.02 41.16 36.28 34.87
Denmark 4.48 4.92 5.12 5.10 4.74 4.68 4.72 4.58 4.91 5.36 5.56
Finland 3.31 3.16 3.28 3.08 2.82 2.47 2.45 2.43 2.34 2.44 2.41
France 22.85 24.72 27.89 28.40 26.81 26.09 25.74 25.71 24.21 25.93 25.26
Germany 28.18 28.75 32.10 33.63 31.34 28.63 28.35 28.48 29.65 32.71 32.69
Indonesia 0.17 0.19 0.25 0.40 0.60 0.73 0.79 0.85 1.48 1.46 1.54
Ireland 3.08 3.59 4.72 5.83 5.40 5.35 5.16 5.38 5.33 6.59 7.48
Mauritania 0.24 0.18 0.23 0.22 0.27 0.28 0.32 0.37 0.69 0.78 0.77
Mexico 4.11 6.24 7.23 7.47 6.27 7.54 9.44 10.03 10.89 11.61 11.99
Nepal 0.63 0.71 0.75 0.81 0.92 1.01 1.17 1.35 1.62 1.86 2.05
Netherlands 8.24 10.22 11.79 12.53 11.23 11.29 11.16 10.85 11.81 13.55 14.54
1.2 Status of Milk Production and Consumption Throughout the World
New Zealand 5.99 6.10 6.70 7.88 7.51 9.29 12.24 14.64 17.01 21.94 21.37
Norway 1.73 1.84 1.97 1.98 1.99 1.93 1.74 1.59 1.58 1.61 1.57
Pakistan 7.45 8.19 9.01 10.86 14.72 19.01 25.57 29.44 35.49 41.59 44.29
Poland 14.96 16.38 16.49 16.44 15.84 11.64 11.89 11.95 12.30 13.25 13.70
Romania 3.12 3.81 4.34 4.32 3.81 5.02 4.62 5.55 4.62 4.68 4.33
Russian Federation 0.00 0.00 0.00 0.00 0.00 39.31 32.28 31.15 31.84 30.79 31.18
(continued)
9
Table 1.5 (continued)
10
Country 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2017
South Africa 2.91 2.50 2.50 2.20 2.48 2.79 2.54 2.87 3.12 3.54 3.20
Sri Lanka 0.14 0.19 0.24 0.29 0.25 0.29 0.16 0.17 0.23 0.30 0.41
Sweden 2.93 3.17 3.47 3.67 3.51 3.30 3.35 3.21 2.90 2.93 2.82
Switzerland 3.20 3.40 3.68 3.87 3.88 3.93 3.91 3.96 4.11 4.07 3.92
Thailand 0.00 0.01 0.03 0.06 0.13 0.31 0.52 0.89 0.91 1.00 0.42
United Kingdom 12.97 13.93 15.97 16.02 15.25 14.84 14.49 14.47 14.07 15.32 15.26
United States 53.07 52.34 58.24 64.93 67.01 70.44 76.02 80.25 87.52 94.64 97.76
Vietnam 0.02 0.03 0.04 0.05 0.06 0.06 0.08 0.23 0.34 0.75 0.91
World 391.95 424.73 465.82 512.98 542.53 540.07 579.31 648.22 724.45 801.13 827.88
Source: FAO Stat Report, 2019
1 Introduction
1.2 Status of Milk Production and Consumption Throughout the World 11
2016–18 2028 Yield 2016–18(t/animal)

Mt t/animal
250 12.5
200 10.0
150 7.5
100 5.0
50 2.5
0 0.0
Africa India Pakistan Latin China Russia New Australia European Canada United
America Zealand Union States
Fig. 1.1 Milk production and yield in some regions of the world. Source: OECD/FAO (2019),
“OECD-FAO Agricultural Outlook,” OECD Agriculture statistics (database), https://doi.org/10.
1787/agr-outl-data-e
Processed dairy products Fresh dairy products

kg/captial/year
60
50
40
30
20
10
0
2016–18 2028 2016–18 2028 2016–18 2028 2016–18 2028 2016–18 2028 2016–18 2028 2016–18 2028
European Union United States India Pakistan China Sub-Saharan Africa Latin America
Fig. 1.2 Per capita consumption of milk solids in fresh and processed dairy products. Note: The
milk solids are calculated by adding the fat and solids-not-fat for a product. The processed dairy
products include butter, cheese, skim milk powder, and whole milk powder. Source: OECD/FAO
(2019), “OECD-FAO Agricultural Outlook,” OECD Agriculture statistics (database), https://doi.
org/10.1787/agr-outl-data-en
shifting toward the consumption of milk fat like full-fat milk, cream, etc. This
transition may be due to the recent studies that have shed more light on the health
benefits of dairy fat consumption and the consumer preference for less processed
food. The per capita consumption of cheese is expected to increase in Europe, North
America, Oceania, which are the major producers of cheese. SMP and WMP will be
used in bakery, confectionary, and in infant formula. In regions like Africa, South-
east Asia, and Middle East, which are dependent on the import of the dairy products,
the rate of consumption will grow faster than the production due to the increase in the
12 1 Introduction
Fig. 1.3 Prices of dairy products in the international market. Note: Butter FOB export price, butter,
82% butterfat, Oceania, skim milk powder, FOB export price, SMP, 1.25% butterfat, Oceania;
whole milk powder, FOB export price, 26% butterfat, Oceania; cheese, FOB export price, cheddar
cheese, 39% moisture, Oceania. Real prices are nominal world prices deflated by the US GDP
deflator (2010 ¼ 1). Source: OECD/FAO (2019), “OECD-FAO Agricultural Outlook,” OECD
Agriculture statistics (database), https://doi.org/10.1787/agr-outl-data-en
import. The trading of liquid milk is more expensive, so the additional demand
growth is expected to be met with the milk powders.
The price of the milk in the international trade refers to the price of butter and
skim milk powder, as the unprocessed milk is not being traded. The reference for
milk fat is taken as butter while for other milk solids it is skim milk powder. The
reference price of the dairy solids (milk fat and skim milk powder) at the interna-
tional level refers to the processed products of the main exporters in Europe and
Oceania. The prices of butter, when compared with that of SMP, is considerably
higher since 2015, due to the higher demand for milk fat compared to the other milk
solids, the same trend is expected to be followed in the upcoming decade (Fig. 1.3).
The prices of SMP, which are at a lower level in comparison to the milk fat, are
projected to increase over the next decade. This can be mainly due to the higher stock
of SMP maintained in the European Union. The price of butter reached a new high in
2017, but has been on a downward slide since then. The prices of butter along with
the other agricultural produce is projected to decline in the coming decade, while the
prices for WMP, cheese will depend on the market price of butter and SMP. Milk is
traded in the processed form as dairy products in the international market. The per
capita consumption of dairy products in China is very less, but it is the largest
importer of whole milk powder, the other notable importers of dairy products are
Japan, Russia, Mexico, Middle East, and North Africa. The trade between these
countries is generally done as per certain international trade agreements like CPTPP,
CETA, the trade agreement between Japan and European Union, these trade
agreements create opportunities for further growth in trade. The bigger dairy con-
suming players like India and Pakistan are not involved in much of the International
dairy market but their involvement in the trade can have a substantial effect on the
world market. The production of butter is expected to grow at 1.9% per annum than
1.2 Status of Milk Production and Consumption Throughout the World 13
Rest of the World Argentina Australia European Union New Zealand United States
Mt
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2016–18 2028 2016–18 2028 2016–18 2028 2016–18 2028
Butter Cheese Skim milk powder Whole milk powder
Fig. 1.4 Major exporters of dairy products. Source: OECD/FAO (2019), “OECD-FAO Agricul-
tural Outlook,” OECD Agriculture statistics (database), https://doi.org/10.1787/agr-outl-data-en
the world milk production while that for SMP will be 1.3% during the next decade.
The production of cheese, WMP during the same period will grow at 1.2% per
annum. The slower growth in the production of cheese may be attributed to the high
importance of the slow-growing markets in continents like Europe and North
America. As the domestic markets are getting saturated and with the increase in
the demand for the milk fat, the exports from the United States will mainly be in the
form of SMP.
Approximately 8% of the total milk production is traded internationally. The
trade share of WMP and SMP accounts to be more than 40% of the total world trade,
but they are stored for longer periods. The major exporters of milk products like New
Zealand, European Union, the United States, and Australia will hold around 75% of
cheese, 79% butter, 78% WMP, and 81% SMP export in the coming decade (Fig.
1.4). Argentina is also a major exporter of WMP and will account to be around 5% of
the total world export in 2028. Belarus has also emerged as an important exporter of
the Russian markets. The primary source for the butter, WMP in the international
market will be dominated by New Zealand accounting for around 39% and 53%,
respectively, of the total world share by 2028. European Union will be the leading
cheese exporter in the international market, followed by the United States and New
Zealand. The European Union share in the world cheese production is likely to be
around 48% till 2028. The Middle East and North Africa are expected to import the
milk products from the European Union while, the United States and Oceania will be
the major suppliers of SMP to Southeast Asia. China will remain on the top of the
table for the dairy importers mainly for WMP. The export of butter and SMP to
China is mainly contributed by Oceania but the European Union has also emerged as
a main exporter for China during recent years. The net import of China is around 0.7
Mt which will increase at around 2.7% per annum in the coming decade. In 2016–
2018, around 55% and 39% of cheese and butter, respectively, were imported (of the
total world import) by the developed countries the same pattern for the import is
expected by 2028. The United Kingdom, Russia, Japan, European Union, and China
will be the top (major) five importers of cheese by 2028, i.e., majority of the cheese
14 1 Introduction
Table 1.6 Global dairy trade at a glance

World dairy export 1.8% New Zealand, Mexico, EU, Argentina, India,
Canada
Major importer(s) – China, Mexico, Malaysia, Egypt, Brazil
WMP world export Increase by 2.3% to Major importers: China, Brazil, Bangladesh,
2.5 Mt (2019) Singapore, Vietnam
Major exporters: New Zealand, Argentina,
Uruguay, Belarus
SMP world exports Increase by 2.8% to Major importers: China, Philippines, Malaysia
2.7 Mt (2019)
Butter world Increase by 2% Major importers: China, Egypt, Saudi Arabia,
export Malaysia, UAE
Cheese exports Increase by 1.7% to Major importers: Australia, Japan, Republic of
2.6 Mt (2019) Korea, Canada, USA, Mexico
Major exporters: New Zealand, EU, Argentina,
Belarus
will be imported by the developed countries. With the increase in the consumers’
choice for cheese, the export and international trade for cheese are expected to
increase (Table 1.6).
1.3 Challenges Faced by Dairy Industry in Quality Assessment

of Raw Milk
The quality of raw milk is of the utmost importance to ensure food safety to the
customers, manufacture of good quality dairy products. Fresh milk, when drawn
from a healthy animal, contains a low number of bacterial load, which may increase
by 100 fold within few hours of storage if the milk is not chilled or stored at a lower
temperature, i.e., below 5 C. The storage of raw milk at low temperatures is the
prerequisite to control the growth of spoilage causing bacteria and maintaining the
quality of the milk. This problem generally arises when milk is collected from
vendors, who transport milk in cans. The milk is held at ambient temperature for
longer periods in the cans before getting chilled. The hygienic conditions maintained
at the farm level and the containers or cans in which the milk is stored, also plays an
important role in the quality of raw milk. Once the quality of the raw milk gets
deteriorated it cannot be improved. Though bulk milk coolers, chilling centers have
now been installed to maintain the quality of raw milk, before processing, still it has
not achieved much success in maintaining the quality of milk. This is because of lack
of potable water, inadequate cleaning, and sanitation procedures followed, the
workers not following the standard operating procedures for the maintenance and
operation of the chilling plants. The health of the animals from which the milk is
produced also plays an important role in ascertaining the quality of raw milk. Milk
drawn from an unhealthy udder like in case of mastitis, has a poor keeping quality
and a high number of somatic cells which makes the milk not suitable for processing.
1.3 Challenges Faced by Dairy Industry in Quality Assessment of Raw Milk 15
These somatic cells when occur in larger numbers in milk, they also increase the
amount of certain enzymes in milk which lead to the breakdown of certain milk
components like fat, protein, leading to the development of various flavor defects
like bitterness, rancidity, etc.
Following good animal husbandry practices will certainly help in producing raw
milk of good quality. The incidence of the occurrence of antibiotics, pesticides,
aflatoxins, and heavy metals can be reduced by following certain practices, like in
case of antibiotics observing the withdrawal period, for aflatoxins the feed should be
stored properly as per the manufacturer’s guidelines or avoiding the storage of the
feed-in hot and humid conditions, in case of pesticides, the use of pesticides should
be done judiciously so that they do not get reflected in milk above their maximum
residual limits, when feed treated with pesticides is fed. To avoid the occurrence of
such types of contaminants in milk, the farmers or the milk producers should be
educated and trained to follow good agricultural practices, the manufacturing firms
of the cattle feed should be advised to ensure that the quality of the feed produced is
safe and free from contaminants. The veterinarian drugs or medicines should be used
under the supervision of the technical or veterinarians. This is helpful in avoiding the
occurrence of antibiotics in milk.
The chemical tests should be done at the bulk milk coolers or the chilling centers
to check for any adulteration in the milk being procured. Generally, abnormal milk
like colostrum or milk with developed acidity is also added to raw milk, which
reduces the quality of good quality milk and thus reduces its suitability to be
processed (such milk has low heat stability and reduced alcohol test). Occurrence
of high amount of sediment also indicates the hygienic quality of milk. Sediments
enter into milk when milking is done improperly leading to the entry of soil or other
extraneous matter into the milk. The sediments in milk can be reduced by cleaning
the teat and hindquarters of the animal before milking. Thus, all such factors that
affect or degrade the quality of raw milk can be addressed by training the farmers,
educating them on the importance of clean milk production and good hygienic
practices. Training should be provided to the personnel working in the milk collec-
tion centers like bulk milk coolers and chilling centers. The standard operating
procedures of cleaning, sanitation, milk reception, and adulteration testing should
be displayed and recorded. Equipment like water bath for testing the MBRT of the
received raw milk should also be done along with other tests carried for milk and the
producers supplying milk of low microbial quality should be identified and proper
guidance about the clean milk production practices should be given to them. The
milk collection unit installed at the milk collection centers should be automated with
a data collection unit. All the data generated during the procurement of milk like
quantity of raw cow milk and buffalo milk purchased, sale–purchase transactions,
payment summary, milk dispatch, and calibration data of the milk analyzer should be
directly connected with the server of the main dairy unit to eliminate the malpractices
done by the middlemen. Overall, all the factors that directly or indirectly affect the
quality of the raw milk should be followed and strictly monitored by the quality
personnel for quality improvement.
16 1 Introduction
Suggested Readings
Annual Report (2018–2019) Department of animal husbandry, dairying and fisheries ministry of
agriculture and farmers welfare, Government of India
Dairy and Milk Processing Market in India (2018–2023)
OECD/FAO (2018) OECD-FAO agricultural outlook 2018–2027. OECD Publishing, Paris/Food
and Agriculture Organization of the United Nations, Rome
OECD/FAO (2019) OECD-FAO agricultural outlook 2019–2028. OECD Publishing, Paris/Food
and Agriculture Organization of the United Nations, Rome
UHT milk market in India: industry trends, share, size, growth, opportunity and forecast 2019–
2024. Report by IMARC group. https://www.nddb.coop/
Sampling Plan for Milk and Milk Products
2
The regulations laid down for food and food products aim at the protection or
safeguarding the health of the consumer along with ensuring fair practices are
maintained during the production of food products. Sampling of milk and milk
products is done for chemical or bacteriological analysis or redressal of consumer
complaints. The sampling procedures are laid down to avoid or prevent the
difficulties that are faced to meet the regulatory, administrative, or technical
standards involved in the sampling and during the interpretation of the results. The
sampling should be done by an experienced person familiar with the sampling
techniques and should be well versed with the knowledge of the subject. The
sampling procedure for every product is different which depends on the nature of
the material and the type of analysis to be done. So, it is not possible to strictly
adhere to a single sampling plan for different products. Samples should be drawn to
prevent contamination and adulteration so that the inherent composition of the
sample remains preserved. For chemical analysis, sample should be drawn with
clean and dry sampling equipment and for microbiological analysis, all the
equipments like plunger, sampling bottles, corks or rubber stoppers should be sterile
and handeled under aseptic conditions. The drawn sample should be a true represen-
tative of the bulk to prevent false-positive results. Proper mixing of milk before
drawing of the sample should be done so as to ensure that the sample is drawn from a
homogenous mixture. In the case of liquid milk, thorough mixing using a suitable
instrument that reaches the entire depth of the liquid should be used while for
powdered or solid products, the sample should be drawn from different portions.
The temperature at which the sample is drawn plays an important role in maintaining
the characteristics of the sample, as chemical changes are susceptible to change in
temperature. In this chapter, we will discuss the sampling procedures for different
milk and milk products.

https://doi.org/10.1007/978-981-15-4167-4_2
18 2 Sampling Plan for Milk and Milk Products
2.1 Scope of Sampling
The guidelines mentioned for the sampling of milk and milk products are applicable
for the quality assurance personnel, food safety officers, and others related to the
sampling of the products to ensure their safety and quality requirements.
2.2 Sampling Purpose
The samples drawn should contain the labels containing the information like purpose
of sampling/drawing the sample, i.e., whether it is intended for in-house testing at the
factory level or for regulatory purposes.
2.2.1 Samples Drawn for Regulatory Purposes
Sampling guidelines for the government or food safety officials are documented in
the respective food regulations and should be followed.
2.2.2 Samples Drawn for Monitoring Purpose at the Factory Level
The sampling of the products while being manufactured is done to monitor the
ongoing process of production and the number of samples to be picked should be
sufficient in number so that they constitute the representative sample. The samples
drawn should range from 5 to 8 samples per location or product, e.g., for processed
milk, the sample should be drawn at the outlet of the pasteurizer, from the pasteurized
milk storage tank, packaging machine, cold storage, and at the time of dispatch while
in the case of milk products sample should be drawn from the outlet of the heat
exchanger, product filling tank, packaging machine, cold store, and at the time of
dispatch. The reports thus generated from the testing of the samples gives an idea
about the product quality, safety, and the requirement to review the manufacturing
process. The label on the sample should specify the type of the analysis to be done,
i.e., Quantitative, Chemical, Qualitative, or Microbiological (Manual on General
Guidelines on Sampling-FSSAI 2015).
2.3 Sampling Procedure
Sampling procedure for a specific commodity should be followed as per the standard
procedures laid down by food regulations like ISO 707 or BIS.
The sample drawn should be the representative of the whole lot and this is done
by random sampling that involves the collection of x number of items from a lot
containing N number of items and it should satisfy all the possibilities that the
sample collected for testing is x. The random sampling ensures that no dispute
2.5 Sampling Plan for Raw Milk 19
occurs when the samples are drawn. The plan for random sampling can be generated
either through a computer software alone (Manual on General Guidelines on
Sampling-FSSAI 2015) or by the combination of other sampling techniques as
mentioned in IS 4905.
2.4 Collection of the Samples
The samples to be drawn for chemical analysis should be done carefully; like the
person involved in sampling should use gloves, clean sample bottles or bags. The
sampling equipment should be clean and dry to prevent the contamination from lot to
lot. Sterilized equipment, containers should be used for samples taken for microbial
testing. The drawn samples should be handled carefully and be the true representa-
tive of the lot because the interpretation of the tests carried at the laboratory for a lot
depends on the sample homogeneity and trueness. The number of the samples drawn
should be statistically significant with respect to many of the food products.
The samples should be submitted to the laboratory in the original unopened form.
Containers that are leakproof, dry, wide mouthed, and sufficient in size to contain the
required quantity of the sample should be used. The containers should be airtight and
should not contaminate the sample. The storage conditions of the samples should be
maintained as underlined for a product and an additional sample should be drawn for
liquid samples to check the temperature of the actual product. The frozen samples
should be stored in pre-chilled containers or under cold conditions like using
insulated containers. The record containing all the details of the sample along with
the results of the tests performed should be thoroughly maintained. The time delay
between the receiving of the samples by the laboratory personnel and testing should
be minimized as possible (Manual on General Guidelines on Sampling-FSSAI
2015) .
2.5 Sampling Plan for Raw Milk
2.5.1 Sampling from an Individual Container
The milk to be sampled is poured into another container, three to four times or mixed
using a plunger. During mixing the milk, the plunger should be inserted up to the
bottom of the container followed by pulling it upwards as quickly as possible, follow
the same procedure of plunging for about 10 times. The plunger should be held in a
slanting position and its position in the container should be changed to ensure that
the milk in the vessel is properly mixed and agitated. The agitation/mixing of milk
should not be done at 26.5–29.5 C as the milk gets churned at this temperature.
Once thorough mixing is done, draw the sample, and store it at refrigeration before
analysis. The drawn sample should be properly labeled and must contain information
like the type of milk, society code, and route number.
Table 2.1 Number of samples to be drawn randomly from cans/containers (IS: 1479, Part-1)
Total number of cans/containers Number of sample(s) to be selected randomly
1 1
2–5 2
6–20 3
21–60 4
61–100 5
100 or more 5 plus one for each additional 100 units
2.5.2 Sampling from Several Containers
The sample from different containers is taken by pouring milk into a vat and mixing
it. If vat is not available then a composite sample is taken from the individual
containers by thoroughly mixing their contents. Proper labeling of the drawn sample
should be done and it should contain information like type of milk, society code, and
route number.
2.5.3 Sampling from Bulk Units
When milk having uniform quality is supplied in bulk, i.e., in cans or containers
filled from tanks, the samples should be drawn randomly as per Table 2.1.
2.5.4 Sampling from Storage Tanks and Rail and Road Milk Tankers
Milk that is meant to be sampled from tank or tanker should be mixed with a large
plunger (Fig. 2.1) sufficient in size or using a mechanical agitator or by means of a
compressed air. The plunger used for mixing milk in rail or road milk tankers should
be inserted into the manhole with the person being in a sitting or standing astride
position with the legs being apart from each other on the top of the tanker. The
plunger is thrust forward and pulled back, thrust downward and pulled back and
thrust backward and back. This procedure should be repeated for 15 min.
Once the plunging is completed, the sample may be taken from the top of the tank
or from the stopcock in the tank door or by opening the valve on the discharge line
connected to the tank. The drawn sample should be properly labeled and should
contain information like storage tank number/road or rail tanker number, and
temperature of milk recorded.
2.6 Sampling Plan for Processed Milk 21
Fig. 2.1 Plunger
2.6 Sampling Plan for Processed Milk
The sampling of processed milk can be done directly from the storage tank/silo in
which it is stored after processing or can be done at the time of packaging from the
packaging machine at a certain time interval as fixed by the manufacturer.
2.6.1 Sampling from a Storage Tank or Silo
The sampling of processed milk from a storage tank or silo is done by starting the
agitator at least 15 min before the sampling. The person responsible for drawing a
sample from the tank should carry all the sampling equipment like bottle, thermom-
eter, and marker with him. After 15 min of starting the agitator, the stopcock from the
storage tank is opened and about 2–3 L of milk is allowed to flow. Then in a
sampling container let 1 L of milk be collected in it and close the stopcock. Record
the temperature of milk; it should be below 4 C. Then fill four sampling bottles with
at least 300 ml of milk and mark the storage tank from which the milk sample is
taken.
Sampling from a silo is done similarly by turning the agitator on for 15 min and
then taking the sample by reaching its top. The top of the silo surrounding the
manhole should be covered by so as to prevent the bird droppings falling on its top or
manhole or even in a silo at the time of drawing the sampling. The cover should be
built such that the sampling person can be easily accommodated in it for drawing the
sample. Dip the dipper in the silo and fill the sampling container with 2–3 L of milk.
Then again, dip the dipper and observe the temperature of the milk and record it. Fill
four sample bottles with around 300 ml of milk and label it.
2.6.2 Sampling of the Packed Milk
Once the packaging of milk has been started, it is important to check the quality of
the milk being packed, so as to ensure that whether the quality of packed milk is as
per the legal requirements. The sampling can be done by taking four consecutive
packets of milk from each head of the packaging machine (generally a prepack
machine has two heads, i.e., two filling points). The temperature of the milk at the
time of filling should be recorded (filling temperature should be 3 C) along with its
batch number, use-by date, weight of the milk contained in the packet, etc.
2.7 Sampling Procedure for Channa/Paneer/Cheese
The sampling of paneer/channa/cheese is generally done by one of the following

three methods.
2.7.1 Sampling by Cutting a Sector
With the help of a knife or a sharp blade, two random cuts are made radially starting
from the center to its edge.
2.7.2 Sampling Using a Trier
The trier (Fig. 2.2) is driven at an angle onto the surface of cheese or channa or
paneer toward the center and also from the edges of the cheese at least 10–20 cm
from it. Take around 2 cm of the boring(s) obtained and close the hole with it. The
remaining portion of the boring(s) forms the sample. In case, if the product is
delivered in drums or in bulk packing, the sampling may be done by driving the
trier diagonally through the product starting from the top to its bottom. The trier can
be inserted perpendicular to one face of the product and then passed through the
center to the opposite face. The trier can be inserted horizontally into the vertical face
of the product at its center between the two plain faces.
2.7.3 Sampling by Taking the Whole Product
This method of sampling is followed when the product is packed in small containers
or packs. The whole product is used as a sample for analysis.
2.7 Sampling Procedure for Channa/Paneer/Cheese 23
Fig. 2.2 Trier
2.7.4 Preparation of Paneer/Cheese/Channa Samples for Analysis
The drawn samples should be grated using a grater, grinding the sample in a mortar,
or by cutting the samples into small pieces using a sharp knife. For microbiological
analysis, the preparation of samples should be done aseptically.
2.7.5 Sampling by Cutting Using a Knife with a Pointed Blade
When the product is packed in a circular container, two cuts radiating from the center
are made and the inedible portions are removed. The minimum quantity of the drawn
sample should be 150 g. If the product is packed in a rectangle-shaped container then
the sample should be drawn by making parallel cuts to the sides of it and the
minimum quantity after removing the inedible portion should be around 150 g.
Note: Remove any rind, smear, or moldy layer on the surface of the cheese prior
to taking the sample. The sample drawn from the product (cheese/channa/paneer)
should be stored in airtight containers below 10 C before analysis.
Table 2.2 Sampling scale for cheese (IS: 2785)

Sampling from bulk units Sampling from retail units
Number of units Sample(s) to be selected Number of units Samples to be selected
1 1 0–25 3
2–8 2 26–100 5
9–25 3 101–500 8
26–50 4 501–1000 10
51–100 5 1001–5000 13
Above 100 8 Above 5000 20
Table 2.3 Sampling scale for sterilized milk/flavored milk (IS: 4238)
Number of sample(s) to be selected randomly
Number of containers Chemical analysis Microbiological analysis
Up to 25 1 1
26–100 5 2
101–500 7 3
501–1000 9 3
1001–5000 11 4
5001 and above 13 4
2.7.6 Sampling Scale for Cheese
The samples to be drawn for cheese should be done as mentioned in Table 2.2.
2.8 Sampling Procedure for Khoa
The procedure for sampling khoa is the same as that discussed for cheese/channa/
paneer, except that the sample being drawn using a clean dry stainless steel knife
having a sharp pointed blade to cut the khoa. The sample of khoa is grated or ground
and mixed thoroughly. If the sample cannot be ground or grated, mix it by kneading
it thoroughly. The prepared sample is then stored in an airtight container and at a
temperature below 10 C prior to analysis.
2.9 Sampling Procedure for Sterilized Milk/Flavored Milk
2.9.1 Scale of Sampling
The number of samples to be drawn should be as per the Table 2.3.

2.10 Sampling Procedure for Dahi, Yoghurt, and Srikhand 25
2.9.2 Preparation of Samples for Chemical Analysis
Mix the contents of the container(s) selected. Take the representative sample about
200 g in quantity and transfer into a clean, dry, and properly labeled container. The
container in which the sample is transferred should contain information like name of
product, batch or code number, manufacturing date, time at which sample is drawn,
and temperature of the product during sampling.
2.9.3 Preparation of Samples for Microbiological Analysis
Sample size should be selected as mentioned in Table 2.3. Using a properly sterile
equipment draw 100 g of a sample under aseptic conditions. Transfer the sample into
sealed airtight sterile glass bottles/containers. The container in which the sample is
transferred should contain information like name of product, batch or code number,
manufacturing date, time at which sample is drawn, and temperature of the product
during sampling.
Note: The sample(s) should be stored below 10 C prior to analysis.
2.10 Sampling Procedure for Dahi, Yoghurt, and Srikhand
The samples to be drawn for chemical and microbiological examination should be

taken carefully preventing any contamination. The instrument to be used for sam-
pling should be dry and clean. For microbiological purposes, all the equipment used
to draw the sample should be sterilized by either heating them in a hot air oven for
2 h at 160 C or autoclaving for minimum 15 min at 120 C. The number of samples
to be selected should be done as mentioned in Table 2.4.
2.10.1 Preparation of Samples for Chemical Analysis
Mix the contents of the container(s) selected. Take the representative sample about
200 g in quantity and transfer into a clean, dry, and properly labeled container. The
Table 2.4 Sampling scale for dahi, yoghurt, and srikhand (IS: 4238)
Number of sample(s) to be selected randomly
Number of containers Chemical analysis Microbiological analysis
Up to 25 1 1
26–100 5 2
101–500 7 3
501–1000 9 3
1001–5000 11 4
5001 and above 13 4
container in which the sample is transferred should contain information like name of
product, batch or code number, manufacturing date, time at which sample is drawn,
and temperature of the product during sampling.
2.10.2 Preparation of Samples for Microbiological Analysis
Sample sizes should be selected as mentioned in Table 2.4. Using properly sterile
equipment, draw 100 g of the sample under aseptic conditions. Transfer the sample
into sealed airtight sterile glass bottles/containers. The container in which the sample
is transferred should contain information like name of product, batch or code
number, manufacturing date, time at which sample is drawn and temperature of
the product during sampling.
Note: The sample(s) should be stored below 10 C prior to analysis.
2.11 Sampling Procedure for Ice Cream
When the product is supplied in bulk packs, the sample size should be selected as
mentioned in Table 2.5.
When the product is supplied in retail units, the sample size should be selected as
If any possibility due to variation occurs between different units, sampling should
be done for every unit.
Table 2.5 Selection of Total number of units (N) No. of unit(s) to be selected (n)
bulk containers on a
1 1
random basis (IS: 2802)
2–5 2
6–20 3
21–60 4
61–100 5
Above 100 5 plus one for every 100 units
Table 2.6 Selection of Total number of units (N) No. of unit(s) to be selected (n)
retail containers randomly
1–100 1
(IS: 2802)
101–1000 2
1001–10,000 4
Above 10,000 4 plus one for every 2500 units
2.12 Sampling Procedure for Condensed Milk 27
2.11.2 Preparation of Sample of Ice Cream
The samples should be stored at less than 15 C. During transit, the temperature of
ice cream should not exceed more than 15 C.
The minimum quantity of the sample to be drawn should be 100 g. If the pack is
of a smaller size, several small packages should be taken to make up the required
sample quantity. Using suitable equipment like a spoon or a sharp knife, the ice
cream is scooped off and transferred into sterile jars (glass, wide-mouth containers
having dimensions of mouth as 4.5 cm). The jar should be closed tightly using a
screw cap or any suitable closure made from fat proof, nonabsorbent material. If a
multilayered ice cream is to be analyzed, the sample should contain the same amount
of each layer as present in the original product. The layers should not be separated
during sampling and a complete sample consisting of all layers should be placed in
the sample jar. When sampling from bulk containers, the upper layer is removed
followed by taking the required amount of sample from different portions of the
container using a sterile spoon or blade. For sampling chocolate or fruit-nut ice
cream, the product should be commuted finely using a mixer. 100–200 g of ice
cream is filled in the mixer and allowed to melt at 37 C (fruit ice cream should be
mixed for 3–5 min while nut ice cream should be mixed for up to 7 min to mix the
insoluble particles). The product can also be ground using mortar and pestle. In the
case of chocolate ice cream, the covering of chocolate should be removed and only
the ice cream portion should be taken for analysis.
2.12 Sampling Procedure for Condensed Milk
The number of containers to be selected from each lot should be selected on a

random basis and as mentioned in Tables 2.7 and 2.8.
Table 2.7 Selection of Lot size (N) No. of containers to be selected (n)
random samples for
Upto 300 3
containers of 400 g to 5 kg
(IS: 1166) 301–500 5
501–1000 7
Above 1000 10
Table 2.8 Selection of Lot size (N) No. of containers to be selected (n)
random samples for
Upto 100 2
containers of more than
5–20 kg (IS: 1166) 101–300 3
301–500 4
Above 500 5
The sampling of containers of 200 g and above 20 kg pack size should be decided
as per the manufacturer’s requirements.
2.12.2 Preparation of Sample of Condensed Milk for Analysis
The components of condensed milk like fat, protein, and lactose tend to get separated
on storage, thus it is necessary to mix the content of the container prior to analysis.
The container should be placed in a water bath at 40 C. When the temperature of the
container reaches around 40 C, open the container lid. Reincorporate the material
adhering to the lid back into the container. Using a spatula, mix the contents
thoroughly by stirring such that the top layer gets mixed with the lower layer and
vice versa. Repeat the stirring before drawing the sample for testing various
parameters.
2.13 Sampling Procedure for Milk Powders
The number of containers to be selected from each lot should be done as mentioned
in Tables 2.9 and 2.10.
2.13.2 Preparation of Sample for Analysis
Using a suitable sampling instrument, draw equal quantities of the material from
different parts of the container into a clean, dry, and airtight container. The minimum
quantity of the sample to be drawn should be 150 g.
Table 2.9 Sampling for Lot size (N) No. of containers to be selected (n)
containers of 500 g and up
Up to 100 3
to 5 kg (IS: 1165)
101–300 5
301–500 7
Above 501 9
Table 2.10 Sampling for Lot size (N) No. of containers to be selected (n)
containers of more than
Up to 50 2
5 kg (IS: 1165)
51–100 3
101–300 4
Above 301 5
2.14 Sampling Procedure for Butter 29
Table 2.11 Sampling scale for butter when packed in bulk containers (IS: 3507)
Total number of units No. of unit(s) to be selected
1 1
2–9 2
10–49 3
50–99 4
100–199 5
Over 200 Add 1 for every 250 units (i.e., 5 plus 1 and so on)
2.14 Sampling Procedure for Butter
The number of containers to be selected from each lot should be done as mentioned
in Table 2.11.
2.14.2 Sampling Technique for Butter
2.14.2.1 Hard and Semihard Butter Kept Under Cold Storage

(a) Sampling from churns
Samples should be drawn from four cores that should be equidistant from each other.
At least two should be from near the center of the churn.
(b) Sampling from trollies
Four cores (one each from the two ends and the other two from the sides) shall be
drawn with the help of a trier.
(c) Sampling from boxes
The trier should be inserted vertically into the block at three different places, one
at the center and the other two at the diagonally opposite corners.
(d) Sampling from casks
Three cores shall be drawn by inserting a trier at three points equidistant from the
circumference of one end of the block and directed through the center of the block.
(e) Sampling from small packets

Table 2.12 Sampling scale for butter when packed in packets or tin (IS: 3507)
Total number of units No. of unit(s) to be selected
1–100 1
101–1000 2
1001–10,000 4
Over 10,000 Add 1 for every 2500 units (i.e., 4 plus 1 and so on)
The packets should be taken randomly as mentioned in Table 2.12 and should be
further used for analysis.
(f) Sampling from Barrel
The trier is inserted diagonally from the edge of the barrel, the trier is rotated to
make a complete turn, then remove the trier and take out the plug of butter. Draw
another plug by inserting the trier at any point on the surface and push it vertically
downwards to the bottom of the butter, rotate the trier completely, then remove the
trier and takeout the plug of butter. The obtained plugs of butter are used for further
testing.
(g) Sampling from butter blocks
The trier should be inserted from one side of a top corner diagonally through the
center to its bottom. Rotate the trier completely and withdraw it. Repeat the same
process from the opposite top corner and use the drawn samples for analysis. If the
quantity of the butter packed is less than 500 g, then the whole pack should be
selected as a sample.
(h) Sampling of pasty butter kept under warm conditions
If the product is kept in small quantities, the sample is drawn from the deeper
layers at the center and from other two points that are equidistant from the center. If
the product is kept to form heaps or blocks, the sample is drawn by selecting three
points, one at center others about 2–3 cm away from the bottom while the third one
on the opposite side to the first hole.
Note: The trier used for sampling of butter, cheese/paneer/channa should have a
diameter of 30 mm (minimum) and should be sufficiently large so as to pass the base
of the container diagonally. Its stem and blade should be made of SS having suitable
hardness and it should withstand sterilization. The stem and blade may be one piece
while the transition from stem to blade shall be smooth. The stem should be circular
in cross section while the grooves on the blade should have sufficient depth while
edges of the blade should be sufficiently sharp so that sampling of hard butter can be
done. The surface of the blade should be smooth and well polished. The spatulas or
knives used for removing the portions of samples from the trier should be made
of SS.
2.16 Labeling of the Samples for Analysis 31
Table 2.13 Samples to be collected for ghee/butter oil/anhydrous milk fat (IS: 3508)
Total number of containers No. of sample(s) to be selected randomly
1 1
2–40 2
41–110 3
111–300 5
301–600 7
Above 601 10
2.15 Sampling Procedure for Ghee (Anhydrous Milk Fat)/

Butter Oil
The number of containers to be selected for sampling should be done as mentioned

in Table 2.13.
The samples should be placed in wide mouth jar/containers/bottles of 50, 100,
200, or 250 ml capacity. The bottles should be glass stoppered. For chemical
analysis, the bottles may be closed using rubber stoppers lined with butter paper if
sensory of the sample is not to be done.
2.15.2 Sampling Technique
The sampling instruments, containers should be clean and dry. The sample to be
drawn from the container should be sampled using a sampling instrument that is
inserted through an opening in the container. The samples drawn from a single batch/
code should be placed in a clean and dry glass container. The samples should be
placed in a cool place away from light and heat.
2.16 Labeling of the Samples for Analysis
The samples should be placed in airtight bottle or jars and should be properly labeled
with the following information marked on the container:
• Date and time of sampling

• Type of product
• Name of the person taking the sample
• Quantity of the sample
• Batch/code number of the product
• Type of preservative added, if any
• Storage temperature
• Temperature of the product at the time of filling

• Define the type of analysis to be done like chemical, microbial, and sensory
• Additional information (like food intended to be produced as per any certification
mark like ISI, Agmark, and Export)
• The sampling is done for microbiological, sensory, or chemical analysis and
should be properly labeled
Suggested Readings
Gupta V (ed) (2018) The food safety and standards act, 2006, 11th edn. Commercial Law
Publishers, New Delhi
IS 1165 (2002) Milk powder. Bureau of Indian Standards, New Delhi
IS 1166 (1986) Condensed milk, partly skimmed and skimmed condensed milk. Bureau of Indian
Standards, New Delhi
IS 1479 (Part I) (1960) Methods of test for dairy industry, rapid examination of milk. Bureau of
Indian Standards, New Delhi
IS 1699 (1995) Methods of sampling and test for food colors. Bureau of Indian Standards, New
Delhi
IS 2785 (1979) Natural cheese (hard variety), processed cheese, processed cheese spread and soft
cheese. Bureau of Indian Standards, New Delhi
IS 2802 (1964) Ice-cream. Bureau of Indian Standards, New Delhi
IS 3507 (1966) Method of sampling and test for butter. Bureau of Indian Standards, New Delhi
IS 3508 (1966) Method of sampling and test for ghee. Bureau of Indian Standards, New Delhi
IS 4238 (1967) Sterilized milk. Bureau of Indian Standards, New Delhi
IS 4905 (2014) Random sampling and randomization procedures. Bureau of Indian Standards, New
Delhi
IS 9617 (1980) Dahi. Bureau of Indian Standards, New Delhi
ISO 707 (IDF 50: 2008) Milk and milk products-guidance on sampling. International Organization
for Standardization, Geneva
Manual on General Guidelines on Sampling-FSSAI (2015)
Quality Assessment of Raw Milk
3
Milk is a food product obtained from animal origin and is widely consumed by
different age groups. Its primary function is to provide nourishment and immuno-
logical protection to the newborn. It plays an important role in human nutrition, as it
contains balanced quantities of nutrients like fat, protein, vitamins, carbohydrates,
and minerals. Apart from playing an important role in daily diet, milk is also a
suitable vehicle for various additives or adulterants without causing any significant
changes in its appearance. It is also prone to several post secretion changes, which
can be natural or manmade. The quality of the raw milk used further determines the
quality of milk products. In order to ensure the quality of raw milk, dairy plants
follow various chemical quality control tests (platform tests) so as to check the
suitability of raw milk for processing.
3.1 Visual and Organoleptic Tests
The milk received at the dairy plant is assessed for any objectionable flavor or color
or for the presence of any objectionable material in the milk. The milk failing to pass
such tests is rejected for further processing.
1. Remove the lid of the can or tanker and observe for the presence of any extrane-
ous matter.
2. Sniff the milk for any objectionable flavor that is unnatural to milk.
3. Put 20–30 ml of milk in the mouth and roll into the mouth cavity and the palate
for assessing taste and flavor of milk.
4. If the taste, flavor, or color of milk is found to be unnatural or objectionable, it
should be rejected.

https://doi.org/10.1007/978-981-15-4167-4_3
34 3 Quality Assessment of Raw Milk
Table 3.1 BIS standards Quantity of sediment (mg) Grade

for sediment
0.0 Excellent
0.2 Good
0.5 Fair
1.0 Poor
2.0 Very poor
3.2 Sediment Test
This test is done to assess the cleanliness of milk procured at the dairy plant and is
performed using a sediment tester.
1. The sediment disk is placed in the space provided and the tester is dipped in the
bottom of the can (make sure that the can remains undisturbed).
2. Milk is then collected from different parts of the can/tanker slowly by pulling the
plunger upward.
3. The sediment tester is then removed and the plunger is pressed down to empty the
tester.
4. The sediment disk is then dismantled carefully and compared with standard
sediment disk.
5. Alternatively, the disk can also be weighed and compared with the unused disk.
6. The milk is graded in accordance to the BIS standard (Table 3.1).
3.3 Clot-on-Boiling Test (COB)
COB test is done to check the suitability of milk for pasteurization or other heat
treatment. Development of clots or flakes on boiling indicates milk with a higher
developed acidity, thus not suitable for heat treatment like pasteurization. Milk
showing a positive COB test if pasteurized will lead to choking of the pasteurizer.
1. 5 ml milk sample is taken in a test tube and placed in a boiling water bath for
5 min (or hold it the over flame and boil).
2. Observe the formation of clots or flakes on the wall of the test tube.
3.4 Alcohol Test
Milk to be processed for high heat treatment such as condensing or UHT processing
needs to be highly heat stable. This test gives an indication of the quality of milk in
terms of its salt balance along with acidity.
3.6 Heat Stability Assay for Milk 35
Table 3.2 Observation chart for alcohol–alizarin test

Range of color Presence of flakes Approximate acidity (%)
Brown red Nil/no 0.16
Red to yellow – 0.18–0.36
Reddish-brown Small 0.20
Yellowish-brown Small 0.24
Brownish-yellow Large 0.28
Yellow Large 0.36
Violet – Alkaline
1. Take 5 ml of milk in a petri plate or test tube and add the same volume of ethanol
(75% for cow milk and 68% for buffalo milk) slowly.
2. Mix the contents properly and observe for the formation of flakes on the walls of
test tube or petri dish.
3. Appearance of flakes indicates a positive test.
3.5 Alcohol–Alizarin Test
This test is done to assess the suitability of milk for high heat treatment and also
gives an idea about the acidity of milk.
1. Take 5 ml of milk in a test tube and add an equal amount of 0.2% alcohol–alizarin
solution (0.2%).
2. Mix the contents of the tubes properly by inverting it for several times.
3. Observe the formation of flakes and color of the contents.
4. Match the results with Table 3.2.
3.6 Heat Stability Assay for Milk
Heat stability is defined as the time taken by milk to show any visible coagulation,
flocculation, or gelation when heated at 140 C. This test is done to assess the
suitability of milk to be processed at higher temperatures like UHT or in-bottle
sterilization. It may also be defined as the ability of the milk protein system,
especially the caseinate system, to remain in colloidal form when milk is heated.
The heat stability assay for milk to be used for UHT or in-bottle sterilization is done
at 140 C while for milk to be processed for evaporated or concentrated milk is done
at 120 C. The milk is sealed in a glass tube and placed in an oil bath maintained at
140 C or 120 C, the milk in the tube is rotated to see any visible coagulation.
Apparatus
(a) Thermostatically controlled oil-water bath with a shaker.
(b) Tubes made of corning glass of 10 cm length having an internal diameter of
8 cm and open at both ends.
(c) Corks of suitable size, made of silicon rubber, are not affected by oil or milk at
higher temperatures.
(d) Metal frame to hold the tubes.
(e) Stop watch.
(f) Table lamp.
Procedure
(a) Close one end of the tube with a silicon rubber cork.
(b) Add 1 ml of milk in it and close the other end with silicon rubber cork.
(c) Fix the tube in the metal frame and place it in the oil bath maintained at 140 C
(for concentrated milk temperature of the oil bath should be 120 C).
(d) Start the stopwatch and the shaker so that the tubes are shaken continuously.
(e) Observe the tubes under sufficient light and observe any visible coagulation
of milk.
(f) The time lapsed by milk sample to form visible clots are recorded as the heat
stability or heat coagulation time.
Note: To observe the maximum heat stability of a milk sample adjust the pH of
milk as 6.6, 6.7, 6.8, 6.9, and 7.0 and carry out the measurement of the heat
coagulation time as mentioned above. The pH at which the coagulation or clot
formation in the milk sample occurs, at last, is considered as the pH for maximum
heat stability of milk.
3.7 Detection of Preservatives in Milk
Milk and milk products should be free from any kind of additives that increase its
shelf life or retard bacterial growth. As per law or food regulation authorities, the
addition of any kind of preservative to milk is prohibited. Preservatives are generally
added to milk where the chilling or transportation facilities are not proper, especially
in tropical countries. The commonly used preservatives in milk are neutralizers,
boric acid, formalin, hydrogen peroxide, benzoic acid, or maybe from chlorine
sanitizers used for sterilization of milk contact surfaces/equipment.
3.7.1 Neutralizers
To neutralize the acidity produced in milk, neutralizers (NaOH, Na2CO3, and

NaHCO3) are added to it. The neutralizers in milk are detected by using rosolic
acid, which is a pH indicator. The other method for detecting the presence of
neutralizer in milk is by determining the alkalinity of ash.
3.7 Detection of Preservatives in Milk 37
(a) Rosolic acid test
Principle
Rosolic acid is an indicator that gives red color in alkaline condition while orange-
brown color under acidic condition. As this test is based on the change in pH of milk
following neutralization, the test may not work if the neutralization of milk has been
done carefully or milk develops acidity.
(a) Take 10 ml of milk in a test tube and add an equal volume of ethyl alcohol
(95%).
(b) Add a few drops of rosolic acid solution (0.1%—100 g rosolic acid in 30 ml
ethyl alcohol and make up the volume to 100 ml with distilled water). Mix the
contents.
(c) Development of rose-red color indicates the presence of neutralizer while brown
color appears in case of pure milk. Note: Rose-red color will appear on the
addition of NaOH, KOH, and Ca(OH)2 while pink color will appear on the
addition of NaHCO3, KCO3, and CaCO3.
(b) Alkalinity of ash test
Principle
When the added neutralizer is neutralized by the developed acidity, alkalinity of the
ash content is tested. Due to the presence of neutralizer in milk, the ash content of
milk will show more alkalinity as compared to control milk.
(a) Take 20 ml of milk in a silica crucible.

(b) First, evaporate the water to dryness, and then burn the content to ash in a muffle
furnace at 550 C.
(c) Add 10 ml of distilled water in the ash and titrate it against 0.1 N HCl using
phenolphthalein as an indicator.
(d) Milk containing neutralizer will consume more than 1.2 ml of 0.1 N HCl.
3.7.2 Boric Acid and Borates
Boric acid and its salts give a characteristic red color with turmeric paper.
(a) Add 5 ml of milk in a test tube and add 1 ml conc. HCl to it.
(b) Mix it well, then dip a strip of turmeric paper in acidified milk.
(c) Dry the paper strip and note the change in color.
(d) Red color on the paper indicates the presence of boric acid.
(e) Then add a drop of ammonium hydroxide solution.
(f) Change from red to dark-green color confirms the presence of boric acid.
3.7.3 Formalin
Formalin is a 40% solution of formaldehyde and gives violet color with ferric salts
and other oxidizing agents. Its presence is generally determined using Hehner,
Leech, and chromotropic acid test.
Hehner Test
(a) Take 10 ml of milk sample in a test tube.
(b) Add gently 0.5 ml of 10% ferric chloride solution.
(c) Add 5 ml of concentrated sulfuric acid through the sides of the test tube such that
it forms a separate layer at the bottom without mixing with milk.
(d) Formation of violet to purple colored ring at the junction of the two liquids
indicates the presence of formalin in the milk sample.
Note: The presence of sucrose interferes with the test. If sucrose is present in
milk, then 25 ml of milk sample is subjected to distillation. The test is then carried out
by taking 2–3 ml of distillate and adding 2 ml of formaldehyde-free milk to
it. Development of a purple ring at the junction of the filtrate and sulfuric acid
indicates the presence of formaldehyde.
Leech Test
(a) Take about 5.0 ml of milk in a test tube. Add to it equal volumes of concentrated
HCl containing 1 ml of 10% ferric chloride solution (500 ml HCl + 1 ml FeCl3).
(b) Heat for about 5 min over a flame.
(c) To break the curd, rotate the tube, and observe the color. The violet color
appearance indicates the presence of formaldehyde added as a preservative.
Chromotropic Acid Test

(a) Take 1 ml of milk in a test tube.
(b) Add 1 ml of chromotropic acid reagent (saturated solution of 1, 8-dihydroxy
naphthalene-3, 6-disulfonic acid in about 72% sulfuric acid (about 500 mg/
100 ml) results in a light straw-colored solution) and mix the contents well.
(c) Presence of formalin in milk is confirmed by the appearance of gray color;
whereas, control sample remains colorless.
3.7.4 Hydrogen Peroxide
Principle Hydrogen peroxide is determined on the basis of principle of

oxidation of para-phenylenediamine by it. Para-phenylenediamine also called as
1,4-diaminobenzene is yellow in color which after oxidation with hydrogen peroxide
gets converted into quinone diamine and imparts blue color.
3.7 Detection of Preservatives in Milk 39
(a) Take 2 ml of milk in a test tube and add an equal volume of alcohol.
(b) Add 5 drops of 2% para-phenylenediamine solution and shake it well.
(c) Blue color confirms that the milk contains hydrogen peroxide.
(d) Perform a blank test using milk sample that is free from hydrogen peroxide. In
this case, there will be absence of blue color.
3.7.5 Benzoic Acid
(a) Take 10 ml of milk in a test tube and add 5 ml of HCl (1:3) and mix well.
(b) Filter and extract the filtrate with 50–100 ml ethyl ether.
(c) Wash the ether layer with two 5 ml portions of water.
(d) Evaporate the ether in a porcelain dish on a water bath to dryness.
(e) Dissolve the residue in hot water. Add a few drops of 0.5% neutral ferric
chloride solution.
(f) Salmon colored precipitate indicates the presence of benzoic acid.
3.7.6 Salicylic Acid
(a) Transfer 10 ml of milk in a test tube and add 5 ml of HCl (1:3) and mix well.
(b) Filter and extract the filtrate with 50–100 ml ethyl ether.
(c) Wash the ether layer with two 5 ml portions of water.
(d) Evaporate the ether in a porcelain dish on a water bath to dryness.
(e) Dissolve the residue in hot water. Add 2 drops of 10% NaOH solution and
evaporate to dryness.
(f) Add 1–2 drops of 0.5% ferric chloride solution.
(g) A violet color indicates the presence of salicylic acid.
3.7.7 Hypochlorites

(b) In another tube add 3 ml of 0.025% stannous chloride solution.
(c) Place the tubes in a freezing mixture of ice and salt for 3–4 min.
(d) Take the mixture in a centrifuge tube and centrifuge at 2500 rpm for 3–5 min.
(e) Examine the tube for the development of yellow-green color.
(f)Alternatively, examine the tube under UV light of mercury vapor lamp fitted
with woods filter.
(g) Yellow fluorescence confirms the presence of hypochlorite.
3.8 Detection of Adulterants
Milk is a fluid in which any additive can be added without bringing any change to its
physical appearance. These additives are added such that they defy the lactometer
test that is done to check any deviation in specific gravity or solids-not-fat content of
milk. Additives like common salt, starch, sugar, wheat flour, baking soda, urea, and
washing soda are added to milk. However, these are not allowed to be added by law
and their detection is important. Many adulterants are used to increase the volume of
milk while some are added to preserve and improve the shelf life of milk.
3.8.1 Sucrose or Cane Sugar
Principle
The added cane sugar in milk is detected by Seliwanoff’s reagent (0.5% resorcinol
solution: 0.5 g resorcinol in 40 ml of distilled water, add 35 ml of concentrated HCl
and make the volume up to 100 ml using distilled water). On addition of reagent,
diluted hydrochloric acid hydrolyzes the sucrose into glucose and fructose. Boiling
will lead to the interaction of fructose with resorcinol to give red color. In case,
sucrose is not present, the sample will remain white.
(a) Take 1 ml of milk sample and add 1 ml of 0.5% resorcinol solution to it.
(b) Mix the contents and heat the test tube for 5 min in a boiling water bath.
If sugar is present in the sample, the rose-red color will be produced while if sugar
is absent in milk then the sample will remain white.
3.8.2 Starch or Other Cereal Flours
Starch being cheaper is added to the milk to raise its solid not fat content. Its
detection is based on the development of blue color in the presence of iodine.
Principle
Iodine solution in potassium iodide produces triiodide that interacts with starch to
form a violet-blue color complex.
Iodine Solution Dissolve 2.5 g potassium iodide and 1 g pure iodine crystal; in a
sufficient quantity of water and makeup to 100 ml.
(a) Take 5.0 ml of milk sample in a test tube and boil it.
(b) Cool the test tube to room temperature.
(c) Add 1–2 drops of iodine solution to the test tube. Blue color appearance in the
test tube indicates the presence of starch, which disappears when the sample is
boiled and reappears on cooling.
3.8 Detection of Adulterants 41
3.8.3 Urea
1. Urea detection using dimethyl aminobenzaldehyde reagent
Urea is a natural constituent of milk and is a major component of the nonprotein

nitrogen of milk. Urea content in natural milk ranges between 200 and 700 ppm.
However, urea content above 700 ppm indicates “added urea.” Urea is added to milk
to increase the nitrogen content and thereby corresponding protein content. The
admixing of urea to milk can be detected by using dimethylamino benzaldehyde
(DMAB). This method is based on the principle that urea forms a yellow complex
with DMAB in a low acidic solution at room temperature.
DMAB Reagent Dissolve 1.6 g DMAB in 100 ml ethyl alcohol containing 10 ml

conc. HCl.

(b) Add 5 ml of 1.6% DMAB reagent.
(c) The intense yellow color appearance indicates the presence of added urea
whereas the development of slightly yellow color is due to natural urea in milk.
2. Urea detection using urease enzyme
Principle The urease enzyme that acts on urea liberates ammonia, which turns the
color of the solution blue in the presence of a bromothymol blue indicator.
H2 O þ H2 NCONH2 þ urease ! 2NH3 þ CO2
• Urease (2%, w/v): Dissolve 2 g urease enzyme in water and make up the volume
to 100 ml.
• Bromothymol blue solution (0.5%): Dissolve 0.5 g of bromothymol blue in water
and make up the volume to 100 ml.

(b) Add 0.2 ml of urease enzyme solution and shake well at room temperature.
(c) Add 0.1 ml of bromothymol blue solution.
(d) The development of blue color after a period of 10–15 min indicates the
presence of added urea.
3.8.4 Glucose
Glucose being a reducing sugar poses many problems in its detection. Moreover, it is
easily available in commercial form as concentrated syrup.
Principle
Barfoed’s test is used to detect glucose adulteration in milk. Under the acidic
condition, glucose reduces cupric to cuprous form. In the presence of these cuprous
ions, phosphomolybdic acid (colorless) is converted into phosphomolybdous acid
(blue color). The time of heating after the addition of cupric acetate should be less
than 3 min, as longer heating time may convert disaccharides into monosaccharides.
Reagents
(a) Barfoed’s reagent: Prepared by dissolving 24 g of cupric acetate in 450 ml
boiling distilled water and immediately add 25 ml of 8.5% lactic acid to the hot
solution, cool and dilute the contents to 500 ml.
(b) Phosphomolybdic acid reagent: Dissolve 35 g of ammonium molybdate and 5 g
of sodium tungstate in 400 ml of 5% (w/v) sodium hydroxide solution. Boil the
contents vigorously for 20–40 min so that the reactants are dissolved properly.
During boiling, the ammonia is released. To check the ammonia escaping in the
vapors, put red litmus paper in the path of vapors, if it turns blue means reagent
is still not free from ammonia. No change in the color of litmus paper indicates
that the reagent is free from ammonia. As a result of boiling, water gets
evaporated, now cool the contents and dilute to about 350 ml and add 125 ml
of concentrated (85%) phosphoric acid. Finally, make up the volume to 500 ml
with distilled water.
Procedure
(a) Take 1 ml of adulterated milk sample in a test tube.
(b) Add 1 ml of Barfoed’s reagent.
(c) Heat the mixture for 3 min in a boiling water bath and cool for 3 min under tap
water.
(d) Add 1 ml of phosphomolybdic acid reagent and mix the contents.
(e) Formation of a blue color indicates the presence of glucose.
(f) In the case of pure milk, the development of only faint bluish color due to
diluted Barford’s reagent appears.
3.8.5 Maltodextrin
Maltodextrin is a polysaccharide having 3–20 glucose units linked with α(1 ! 4)

glycosidic bonds. It is produced by partial starch hydrolysis and usually used as a
food additive. Iodine when reacts with no or little amylase containing waxy starch
gives red-brown color.
(a) Take 5 ml of suspected milk sample in a test tube.

(b) Add 2 ml of iodine solution (0.05 N) and mix the contents.
(c) Development of chocolate red-brown color indicates the presence of dextrin/
maltodextrin.
Alternate Method
This test is based on the release of glucose after enzymatic reaction, which can be
detected with a simple diastic strip being specific for glucose.
Enzyme Solution Dissolve 0.2 g of alpha glucoamylase enzyme in 100 ml of

distilled water in a volumetric flask. Stopper and store under refrigeration. This
solution should not be more than 15 days old.
Lactic Acid Solution Take 10 ml of conc. lactic acid in 100 ml volumetric flask and
make the volume with distilled water.
Testing Strip Diastic strip for glucose. Store below 30 C but do not store under
refrigeration. Use before 6 months.
(a) Take 20 ml of milk sample in 100 ml beaker.

(b) Adjust pH to 4.0–4.5 using 0.8–1.5 ml of lactic acid solution.
(c) Add 1 ml of enzyme solution and incubate at 62 C for 5 min.
(d) Cool to room temperature.
(e) Dip the diastic strip for 30 s and remove the excess liquid by a single jerk.
(f) Compare the color change from green to brown on the strip with the color chart
on the testing strip bottle and record the result.
3.8.6 Pond Water
Pond water is heavier than the tap water; some unscrupulous persons usually prefer it
to adulterate milk. This method actually detects nitrates/nitrites present in the pond
water. Nitrates gain entry into the pond water from the fertilizers used in the fields.
(a) Rinse the test tube with the milk sample.

(b) Along the sides of the test tube add about 1 or 2 drops of 2% solution of
diphenylamine (2%, w/v, in sulfuric acid).
(c) The sides of the test tube will turn blue if the milk sample contains pond water.
3.8.7 Vegetable Fat
Vegetable fat is cheaper than milk fat, thus it is used as an adulterant in raw milk. Its
presence is determined by checking the Butyro-refractometer (BR) reading of milk
sample (BR of pure milk fat ranges from 40–43 at 40 C).
(a) Separate the cream from the milk sample using a cream separator or a centrifuge.
(b) Prepare ghee from the separated cream and apply a drop of the melted ghee
sample on the prism of butyro-refractometer maintained at 40 C.
(c) Note the BR reading.
Alternate Method
(a) Take 1–2 g of molten heat clarified ghee sample and dissolve in 2–3 ml of
hexane.
(b) Add 1.5–2.0 ml of color developing reagent (water, nitric acid, and sulfuric acid
in the ratio 20:14:6).
(c) Shake vigorously and stand undisturbed till the separation of two layers.
(d) Appearance of distinct orange color in the upper layer indicates the presence of
vegetable oils.
3.8.8 Baudouin Test
As per FSSA, 2006, it is mandatory to add 5% sesame (Til) oil in vanaspati for its
detection. Sesamolin a constituent of sesame oil is degraded by mineral acid, i.e.,
hydrochloric acid and sesamol are released in its free form. Some sesamol is also
liberated during hydrogenation of vanaspati containing sesame oil. The sesamol on
condensation with furfural produces a characteristic red color.
(a) Prepare ghee from the milk sample by heat clarification.

(b) In a test tube take about 5 g of melted fat.
(c) Add 5 ml of concentrated HCl (AR grade).
(d) Add 0.4 ml furfural solution (2% in alcohol) and vortex the tube for 2 min.
(e) Leave the tube undisturbed for separation.
(f) In presence of vanaspati, pink or red color will get developed in the test tube.
(g) Confirm by adding 5 ml distilled water and shake again.
(h) Persistence of the color in the acid layer, confirms presence of vanaspati. If the
color disappears, it is absent.
3.8.9 Mineral Oil
Ghee can be adulterated using cheaper mineral oils like petroleum jelly, paraffin oil,
liquid paraffin (heavy and light), fuel oils, etc. because of huge difference in the
price. Mineral oils are referred to as white oils that are nonedible and have different
viscosity and refractive indices in comparison with milk fat due to certain composi-
tional differences. Thus, as per food regulations, addition of such nonedible oils to
ghee or any type of edible oils and fats is not only an unlawful and unethical practice,
but can also pose a serious health hazard(s). The detection of mineral oil in edible fat
and oils is based on the fact that they resist saponification as ghee or other edible fats
and oils show. Mineral oils, which contain varying fractions of long-chain
hydrocarbons, occurring mainly in earth, do not get saponified by alkali and are
also non-utilizable as human food. This forms the basis for the appearance of
turbidity due to the presence of minerals oils in other saponified oils and fats.
(a) Take 1 g of clarified fat in a standard joint test tube.

(b) Add 5 ml of 0.5 N ethanolic KOH solution.
(c) Reflux the contents on boiling water bath for 10 min.
(d) Add 5 ml of distilled water to the saponified solution.
(e) Appearance of turbidity indicates the presence of mineral oil.
3.8.10 Animal Body Fat
(a) 5 g of heat clarified ghee sample is melted at 50 + 1 C in a test tube and

maintain for 3 min to equilibrate.
(b) Then transfer the test tube in a water bath maintained at 23 0.2 C and record
the opacity time (time taken by the fat sample to acquire absorbance between
0.14 and 0.16 at 570 nm or the Klett reading should be between 58 and 62 at
100% transmittance).
Buffalo ghee shows opacity after 14–15 min, cow ghee shows opacity after
18–19 min and ghee from cotton tract area has an opacity time of 11–12 min. Lesser
opacity time of ghee indicates presence of animal body fats while a higher opacity
time indicates presence of vegetable oils.
3.8.11 Mastitic/Abnormal Milk
The type of milk in which variation occurs in its composition and it becomes
physically, chemically, or microbiologically abnormal. Such type of milk poses
problems when processed and causes health hazard when consumed. Thus, it is
important to carry out tests that can detect abnormal milk. Mastitis is a mammary
gland-related inflammation, which causes many physical and chemical changes in
milk like increase in chloride and decrease in lactose content, redness in milk,
increase in the number of somatic cells, leucocytes, and epithelial cells. Mastitis is
caused by a number of microorganisms but Streptococcus agalactiae, Streptococcus
dysgalactiae, Staphylococcus aureus, and Escherichia coli are mostly responsible
for it. Increase in somatic cell count of milk is observed during the udder infection
due to the leaking of the blood components into the milk.
Strip Cup Test It is used to detect mucosa and milk clots in milk.
(a) Take first 2–3 strips of foremilk on a fine mesh cloth or over a cup.
(b) Observe for any clots, mucosa, or blood cells on the screen.
(c) The presence of clots, mucosa, or blood cells indicate infection.
Leukocyte Counts (Microscopic Test)

(a) Take 0.01 ml of milk sample on a slide and spread it uniformly.
(b) After drying, dip the slide in Newman stain for 30–60 s.
(c) Dry in air, mount and observe under oil immersion lens and observe for presence
of cells (WBC).
(d) The number of WBC’s is expressed as WBC/ml of sample. Range of WBC in
normal milk is 70,000–100,000 WBC/ml.
(e) Counts above 150,000 indicates infection.
Bromothymol Blue Test

(a) Take 5 ml of fresh milk in a test tube.
(b) Add 1 ml of 0.04% aqueous solution of bromothymol blue.
(c) Mix well and observe the color.
(d) Development of greenish-blue or blue color indicates mastitic infection.
Sodium Lauryl Sulphate Test (SLST)

(a) Take 2 ml of milk in a test tube and add 2 ml of sodium lauryl sulfate reagent
(4 g of sodium lauryl sulfate is dissolved in 15% teepol solution (pH 12.0)).
(b) Shake the test tube gently for 20 s and observe for the coagulation of leucocytes.
Interpretation
No precipitate: Negative for subclinical mastitis.
Slight precipitate: Doubtful for subclinical mastitis.
Distinct precipitation with little tendency to form gel: Considered as evidence for
clinical mastitis.
Immediate gel formation of persistent nature: Severe case of clinical mastitis.
Somatic Cell Count

Somatic cells are the cells which enter milk from the lining of the mammary gland
and due to the response of the mammary gland to any type of infection or injury. The
somatic cells in milk generally constitute leucocytes (75%), i.e., neutrophils,
lymphocytes, macrophages, erythrocytes, and 25% of epithelial cells. Normal
milk that has been produced from a healthy mammary gland has a somatic cell
count lower than 105 cells/ml while somatic cell count above 2 lac/ml indicates
mastitis infection. The somatic cell count in milk is determined by direct microscopic
count (DMC) as per the method of Tirkey (2016).
Procedure
(a) The milk samples are mixed properly to disperse the fat.
(b) 10 μL of milk sample is dispensed on a clean and grease-free glass slide over an
area of 1 cm2.
(c) The smear is air dried and then submerged in xylene for 10 min to dissolve
the fat.
(d) Immerse the smear in 95% methanol for 10 min to fix it, followed by draining
the excess of methanol and air dry it.
(e) Stain the slide using alcoholic methylene blue solution and keep for 3–5 min.
(f) Wash the excess of the stain under freshwater and then examine the slide under
the microscope using oil immersion lens.
(g) Count the number of somatic cells in each field.
Fig. 3.1 Somascope
Fig. 3.2 Bactoscope
Calculations
Area of the smear ¼ πr2
Microscopic factor ¼ Area of smear (1 cm2)/Area of microscopic field (πr2) vol-
ume of milk
Somatic cells/ml of milk ¼ Microscopic factor Average number of somatic
cells per field
Nowadays, somatic cells in milk are analyzed by using various instruments like
Lactoscan, SomaScope (Fig. 3.1), Bactoscope (Fig. 3.2), and Fossomatic 7. These
instruments work on the principle of flow cytometry. The time taken by these
instruments to analyze a milk sample ranges from a few seconds to a minute and
the data generated by the instrument can be saved for further use as the instrument
can be connected to a computer.
3.8.12 Addition of Skim Milk Powder
The addition of skimmed milk powder (SMP) is not legally permitted for adjustment
of solid not fat in the sale of cow/buffalo or mixed milk. The method to detect SMP
addition in whole milk is based on the reaction of the phosphomolybdic acid with
milk protein. Coagulation of reconstituted skim milk powder with acetic acid,
followed by boiling and addition of phosphomolybdic acid gives intense blue
color due to certain reducing groups present in the proteins of skimmed milk powder.
(a) Take 50 ml of milk in a 60 ml centrifuge tube.

(b) Centrifuge the tube at 5000 rpm for 15 min.
(c) Decant the supernatant carefully and add 0.5 ml of acetic acid solution (4%) for
coagulation.
(d) Again, centrifuge the tubes at 5000 rpm for 5 min and decant the supernatant.
(e) Wash the precipitate with distilled water twice.
(f) In the supernatant, add 2 ml of phosphomolybdic acid solution (1%). Mix the
contents thoroughly.
(g) Heat in a boiling water bath for 15 min and then cool.
(h) The color of the curd obtained from pure milk will be green in color while the
curd from the sample containing skimmed milk powder will develop blue color.
The intensity of blue color is proportional to the amount of the skim milk
powder present in the sample.
3.8.13 Detergent in Milk
Detergents are not present in milk but they gain entry into milk when synthetic milk
is added to it or when the milk contact surfaces are not rinsed or washed properly to
make them detergent free. Detergents in milk are detected by using methylene blue
indicator.
The principle of detection is that methylene blue is soluble in the aqueous phase
but forms a blue colored complex with anionic detergents. The blue colored complex
is soluble in chloroform.
Methylene Blue Solution Dissolve 12.5 mg of methylene blue in water and make
up the volume to 100 ml.
Chloroform AR Grade
(a) Take 1 ml of milk, add 1 ml of methylene blue solution followed by adding 2 ml

chloroform.
(b) Vortex for 15 s and then centrifuged at 1100 rpm for 3 min.
(c) Development of more intense blue color in the lower layer indicates the pres-
ence of detergent while a more intense color in the upper layer indicates absence
of detergent in milk.
3.8.14 Salt
The determination of salt is based on the reaction of silver nitrate with sodium
chloride in the presence of potassium chromate as an indicator.
AgNO3 þ NaCl ! AgCl þ NaNO3
2AgNO3 þ K2 CrO4 ! Ag2 CrO4 þ 2KNO3
(a) Take 5 ml of milk and add 2 ml of 0.1 N silver nitrate solution.

(b) Mix the contents and add 0.5 ml of 10% potassium chromate solution.
(c) Pure milk shows chocolate brown color that indicates absence of dissolved
chloride while yellow color indicates presence of dissolved chlorides.
3.8.15 Ammonia Compounds
The ammonia compounds are detected by Nessler’s reagent producing brown color
in milk containing ammonium compounds.
Neesler’s Reagent
Eight gram of mercuric chloride in 150 ml distilled water, 60 g of NaOH in 150 ml
distilled water, 16 g of KI in 150 ml of distilled water. Mix the reagents and make up
the volume to 500 ml using distilled water.
(a) Take 5 ml of milk in a test tube and add 1 ml of Nessler’s reagent.

(b) Mix the contents and observe the color.
(c) Milk containing ammonium compounds develop brown color while pure milk
shows yellow color.
3.8.16 Soya Powder
The urease enzyme present in the soybean powder converts the urea into carbon
dioxide and ammonia. The ammonia makes the medium alkaline that turns the color
of the solution to pink.
Soybean Powder Detection Solution Make 0.1% of phenol red solution in dis-
tilled water. Keep the solution overnight and filter it. Add 1 g of urea in 100 ml of the
phenol red solution. Store the solution in a cool and dry place. Make the solution
fresh after 1 week.
(a) Take 5 ml milk in a test tube and add 1 ml of soybean powder detection solution.
(b) Mix the contents and leave undisturbed for 10–15 min.
(c) Development of pink color indicates presence of soybean powder in milk.
Table 3.3 Limit of detection (LOD) for different adulterants

S. no. Adulterant Method of detection LOD (%)
1 Neutralizer Rosolic acid test 0.1 (Na2CO3, NaOH), 0.2
(NaHCO3)
2 Starch Iodine test 0.02
3 Cane sugar Resorcinol test 0.15
4 Hydrogen peroxide Para-phenylenediamine 0.025
test
5 Formaldehyde Chromotropic acid test 0.05
6 Formaldehyde Leech/Hehner test 0.1
7 Urea DMAB test 0.25
8 Detergent Methylene blue test 0.0125
9 Maltodextrin Iodine test 0.3
10 Maltodextrin Enzyme test 0.05
10 Salt Silver nitrate test 0.02
11 Ammonium Nessler’s reagent test 0.15
compounds
12 Glucose Modified Barfoed’s test 0.05
13 Pond water Nitrate reduction test 0.2 as KNO3
14 Sulfates Barium chloride test 0.05
3.8.17 Sulfates
The sulfates are detected by the reaction between the sulfates and barium chloride
producing barium sulfate which settles down as a white precipitate.
(a) In a beaker, take 20 ml of milk and add 1 ml of 5% lactic acid solution.

(b) Boil the contents and cool them.
(c) Filter the solution and collect the filtrate in a test tube.
(d) Add 3–4 drops of 1% barium chloride solution.
(e) Appearance of white precipitates indicates presence of sulfates while no
precipitates shows pure milk. Table 3.3 shows the limit of detection of various
methods to detect adulterants in milk.
3.8.18 Detection of Adulteration in Milk Using Biosensor

and Immunological Techniques
Various regulatory agencies have laid a lower degree of limit of detection for various
adulterants/contaminants in milk. Despite being sensitive, the conventional detection
techniques have many limitations like extensive sample preparation, trained person-
nel, infrastructure, and limited sample throughput, as a result, they are not used.
3.9 Compositional Analysis of Raw Milk 51
Thus, nowadays emphasis is laid on the development of rapid, sensitive methods like
biosensor or immunological based methods. A biosensor is a device which is capable
of detecting or sensing an analyte with the help of a biological recognition element.
The bio-recognition element generates a signal in response to the analyte, which
is detected qualitatively or quantitatively. Biosensors are generally classified as
electronic, piezoelectric, optical. The detection of some common adulterants/
contaminants is summarized in Table 3.4.
3.9 Compositional Analysis of Raw Milk
Preparation of Sample
Warm the milk sample to 37–40 C by transferring it to the beaker and holding it at a
temperature of 40–45 C in a water bath, with slow stirring ensuring its proper
mixing. Further, thoroughly mix the sample by pouring back into the bottle, stirring
to dislodge any residual fat that sticks to the sides and pouring it back into the beaker.
Do not shake the bottle vigorously during mixing. Allow the sample to cool to room
temperature (26–28 C) for immediate analysis.
3.9.1 Fat Test
(a) Gerber method (volumetric method)
Fat is the most important constituent of milk as it is used as a basis for determining
the buying and selling price of milk. It helps to detect the adulteration of water and
skimming. This method was discovered by Dr. N. Gerber of Zurich Switzerland in
1892–1895. H2SO4 is used in this test to increase the specific gravity of the skim
milk portion (or milk serum), which increases the difference between milk serum
and fat globules. It also destroys the stickiness of milk by dissolving all the SNF.
Under the influence of centrifugal force the free fat globules rise to the surface and
the heat produced by the mixture of acid and milk, keeps the fat in melted state,
enabling the fat particles to come to the surface freely. The specific gravity of fat is
0.9 and that of acid milk mixtures is 1.43. This situation facilitates the complete
separation of fat when adequate centrifugal force is applied. Application of centrifu-
gal force causes the lighter substance (butterfat), toward center and the remaining
heavier portion, i.e., the milk serum is thrown toward the periphery. The addition of
amyl alcohol helps the separation of fat from the milk–acid mixture and prevents
charring of fat and milk carbohydrate by H2SO4. The method is based on the
principle that when sulfuric acid (90–91%) is added to milk, it dissolves the proteins
of milk while the fat globules remain free and in liquid form due to the heat produced
by the acid. The two phases are separated by centrifugation with fat being lighter of
the two portions. Addition of amyl alcohol facilitates better separation between the
fat and nonfat portion.
52
Table 3.4 Detection of some common adulterants/contaminants by immunological and biosensor methods
Target adulterant Technique of detection Advantage Disadvantage References
Vegetable ELISA Large sample throughput, greater Antigen selectivity a major issue, Sanchez et al.
proteins sensitivity, helps in the detection of semiquantitative (2002)
wheat proteins and high heat milk
powders
Pea, wheat Optical biosensor Fast, reliable, and sensitive Expensive Abdulhalim
protein, and soya et al. (2007)
Rennet whey Lateral flow assay Detects rennet whey above 4% False-positive results due to presence of Martin-
(immunochromatographic) pseudo-c-GMP arising due to the action Hernandez et al.
of proteinases of bacterial origin (2009)
Melamine Molecularly imprinted High sensitivity, fast – Hu et al. (2015)
polymers and surface-enhanced LOD: 0.012 mmol/l
Raman spectroscopy
Melamine Fluorescence polarization LOD: 9.3 ng/ml – Wang et al.
immunoassay Results similar to HPLC-MS (2011)
Urea Enzyme-based piezoelectric High specificity, accurate, easy to – Renny et al.
biosensor handle (2005)
3
Soy protein Lateral flow assay Rapid, high sensitivity, ease of – Wang et al.
handling, and minimal sample (2015), Gautam
preparation et al. (2017)
Aflatoxin M1 Bioelectric recognition assay Rapid (3 min) – Larou et al.
LOD: 5 ppt (2013)
Estrogenic Bioluminescent cell biosensors High speed, low cost, and increased – Valimaa et al.
mycotoxins sensitivity (2010)
residue
Sweet whey in Immunoassay (antibody based) Fast, easy, and direct – Oancea (2009)
UHT and
condensed milk
Quality Assessment of Raw Milk
Procedure
1. Add 10 ml of sulfuric acid (Gerber acid) into the milk butyrometer (range 0–10%)
with an automatic tilt measure.
2. Mix the milk sample properly and pipette 10.75 ml of milk using milk pipette in
the butyrometer.
3. The milk should be added from the side of the butyrometer wall slowly to prevent
the charring.
4. Add 1 ml of amyl alcohol with an automatic tilt measure.
5. Stopper the butyrometer with a lock stopper and mix the contents properly.
6. Place the butyrometer in a water bath maintained at 65 C for 5 min (to keep the
fat in melted state).
7. Transfer the butyrometer in the Gerber centrifuge and centrifuge at
1100–1200 rpm for 5 min.
8. Place the butyrometer again in the water bath maintained at 65 C for 2 min.
9. Read the fat content with the help of the markings drawn on the butyrometer stem.
Note: The milk sample should be heated at 40 C and cooled to 27 C before

analysis of fat. Heating of milk at 40 C converts fat to melted state.
(b) Rose-Gottlieb method (gravimetric method)
Ammonia disrupts the fat globule membrane surrounding the milk fat. The
released milk fat is then transferred from the aqueous phase to the solvent phase
using diethyl and petroleum ether. Ethanol precipitates the proteins. This method is
regarded as a reference method for fat estimation.
Procedure
1. Weigh about 10 g of well-mixed milk sample into the mojonnier/Rose-Gottlieb
extraction tube.
2. Add 1.25 ml of concentrated ammonia (sp. gravity 0.8974) and mix well gently.
3. Then add the following reagents with proper mixing after adding each reagent:
10 ml of ethanol, 25 ml of peroxide-free diethyl ether. Stopper the flask and mix
well for a minute.
4. Then add 25 ml of petroleum ether having a boiling point of 40–60 C. Stopper
the flask and mix for a minute.
5. Allow the tube to stand until the ethereal layer gets separated from the aqueous
phase (in case of Rose-Gottlieb method).
6. Alternatively, for Mojonnier method centrifuge the flask at low speed.
7. Decant the ethereal layer into a previously weighed vessel containing 5–6 glass
beads (flask, aluminum dish, etc.).
8. Repeat the extraction twice using 15 ml of diethyl ether and petroleum ether, so
as to extract the fat completely from the sample.
9. Add the ethereal layer to the same previously weighed vessel containing the
previously extracted ethereal layer.
10. The ether is evaporated by placing the vessel on a hot plate or water bath at
60–65 C.
11. Place the vessel at 102 2 C for at least 2 h.
12. Cool the dish in a desiccator and weigh.
13. Heat the vessel again in the oven followed by cooling and weighing.
14. Repeat the steps 11–13 till the difference between the two successive weights
does not differ by more than 1 mg.
15. The difference in the weights before drying and after drying is the amount of fat
present in the sample.
16. Repeat the same process for blank also.
17. The difference between the sample and the blank should not exceed 0.5 mg.
Calculation
Fat%ðw=wÞ ¼ ðA=BÞ 100
A ¼ weight of extracted fat, i.e., difference in weight of vessel before drying and
after drying.
B ¼ weight of milk.
3.9.2 Solids-Not-Fat Test
Lactometers are used for the determination of solids-not-fat (SNF) content in milk.
The determination of SNF is based on the Archimedes principle of flotation. This
law states that whenever a solid is immersed partially or fully inside a liquid it is
acted upon by an upward thrust, which is equal to the weight of the liquid displaced
by it. Lactometers are a type of hydrometers and are calibrated beforehand with a
liquid of known specific gravity.
Procedure
1. Adjust the temperature of milk sample at the temperature prescribed for a
lactometer.
2. Mix the sample well. Avoid incorporation of air or foam formation.
3. Pour sufficient milk into the lactometer jar.
4. Dip the lactometer into the lactometer jar containing milk carefully so that the
lactometer does not touch the walls of the jar.
5. Wait until the lactometer attains a stable state and assumes a constant level.
6. Read the lactometer reading and check the temperature of milk. If the temperature
of milk is higher or lower than that prescribed for the lactometer, use the
correction from the standard table (Table 3.5) for the corresponding temperature.
This is called as corrected lactometer reading (CLR).
7. Calculate the SNF using the formula mentioned below.
Table 3.5 Correction to Fat percent of sample

be applied in the lactometer
Temperature 0 2 4 6 8
reading taken at a
temperature other than 19.0 2.2 2.4 2.6 2.7 2.9
27 C, to obtain the 19.5 2.1 2.3 2.4 2.6 2.7
lactometer reading of milk 20.0 2.0 2.1 2.2 2.4 2.5
at 27 C 20.5 1.8 2.0 2.1 2.2 2.3
21.0 1.7 1.8 1.9 2.0 2.2
21.5 1.5 1.7 1.7 1.9 2.0
22.0 1.4 1.5 1.6 1.7 1.8
22.5 1.3 1.4 1.4 1.5 1.6
23.0 1.1 1.2 1.3 1.4 1.4
23.5 1.0 1.1 1.1 1.2 1.3
24.0 0.8 0.9 1.0 1.0 1.1
24.5 0.7 0.8 0.8 0.9 0.9
25.0 0.6 0.6 0.6 0.7 0.7
25.5 0.4 0.5 0.5 0.5 0.5
26.0 0.3 0.3 0.3 0.3 0.4
26.5 0.1 0.2 0.2 0.2 0.2
27.0 0 0 0 0 0
27.5 0.1 0.2 0.2 0.2 0.2
28.0 0.3 0.3 0.2 0.3 0.4
28.5 0.4 0.5 0.5 0.5 0.5
29.0 0.6 0.6 0.6 0.7 0.7
29.5 0.7 0.8 0.8 0.9 0.9
30.0 0.8 0.9 1.0 1.0 1.1
30.5 1.0 1.1 1.1 1.2 1.3
31.0 1.1 1.2 1.3 1.4 1.4
31.5 1.3 1.4 1.4 1.5 1.6
32.0 1.4 1.5 1.6 1.7 1.8
32.5 1.5 1.7 1.7 1.9 2.0
33.0 1.7 1.8 1.9 2.0 2.2
33.5 1.8 2.0 2.1 2.2 2.3
34.0 2.0 2.1 2.2 2.4 2.5
34.5 2.1 2.3 2.4 2.6 2.7
35.0 2.2 2.4 2.6 2.7 2.9
Calculation
Specific gravity ¼ 1 þ ðCLR=1000Þ
Percent SNF ¼ ðCLR=4Þ þ ð0:2 fat ð%ÞÞ þ 0:1 ð0:1 is the factorÞ
Note: The factor to be added in the calculation of SNF varies according to the type
of lactometer used (due to different temperature for different lactometer).
3.9.3 Titratable Acidity
The natural/apparent acidity of milk is due to the presence of casein, citrates,

albumins, globulins phosphates, and carbon dioxide. Milk develops acidity due to
the action of lactic acid bacteria on lactose thus producing lactic acid. This is called
as developed or real acidity. The total titratable acidity of milk is the sum of natural
and developed acidity. It is measured by titration of a known volume of milk with a
standard alkali solution using an indicator (phenolphthalein) and expressed as
percentage lactic acid.
CH3 CHOHCOOH þ NaOH ! CH3 CHOHCOONa þ H2 O
Procedure
1. Transfer 10 ml of properly mixed milk sample into a white porcelain dish or glass
beaker.
2. Add a few drops of 1% phenolphthalein solution.
3. Titrate the contents with 0.1 N NaOH solution.
4. Observe the change in color to light pink. Mark it as the endpoint of the titration.
5. Note the amount of alkali consumed.
The titratable acidity is expressed as lactic acid equivalent per 100 ml of milk.
Titratable acidity ¼ 0:9 V 1 N
where
V1 ¼ Volume ml of the standard NaOH solution required for titration
N ¼ Actual normality of the NaOH solution
3.9.4 Protein Content
The most widely used method for determination of protein content is done by the
Kjeldahl method. In this method, the protein is first digested using concentrated
sulfuric acid in the presence of a catalyst mixture. This method is based on two
assumptions:
1. The dietary carbohydrates and fat do not contain nitrogen.

2. Nearly all of the nitrogen in the food is present as amino acids (aa) in proteins.
The Kjeldahl method involves the following steps:
(a) The protein nitrogen is converted into ammonium sulfate by oxidation.

(b) The ammonium sulfate is reacted with a strong alkali and undergoes decompo-
sition, resulting in the evolution of ammonia which is trapped in boric acid.
(c) Titration of the trapped ammonia with standard acid.

(d) The calculation of the percentage protein in the sample from its weight and the
volume of standard acid required to titrate the ammonia.
Sulfuric acid and the catalyst mixture facilitate the digestion of all the organic
matter except nitrogen. Carbon and hydrogen are converted to CO2 and H2O, sulfur
is converted into sulfur dioxide, and phosphorous is converted into phosphorous
pentaoxide.
The reduced form of the nitrogen is retained as ammonium sulfate. The neutral
salts such as potassium sulfate raise the boiling point of the reaction mixture during
the digestion process thus increasing the effectiveness of the digestion process.
Copper sulfate acts as a catalyst, fastens the digestion, and producing a clear reaction
mixture. Copper, selenium, and mercury in the form of their salts can also be used.
They act as oxidation catalysts (O2 carriers) because they readily pass from a higher
to a lower valence and vice versa. The solution is digested over moderate heat
(<420 C) so as to produce even, gentle boiling. Clearing of the digest is not a
criterion of completion of the digestion process. Digestion should be continued after
clearing. Overheating causes loss of volatile nitrogenous compounds and inconsis-
tent results.
The digest is then neutralized using an alkali thus liberating ammonia. Then the
liberated ammonia is distilled off and trapped in boric acid, which is then titrated
with standard acid. Boric acid does not get involved in the titration but forms
ammonium borate when reacts with ammonia. The formed ammonium–borate
complex is titrated with acid using methyl red-bromocresol green indicator till the
green distillate changes from colorless to pink.
Reaction
The volume of the acid consumed during the titration is equivalent to the
concentration of ammonia that is equivalent to the nitrogen content of the actual
protein present in the sample. This gives the crude protein as some of the nitrogen is
contributed from the nonprotein component present in milk.
Procedure
1. Weigh 5 g of milk in a clean and dry Kjeldahl flask.
2. Add 25 ml of concentrated sulfuric acid, 0.2 g of copper sulfate and 10 g of
Potassium sulfate (catalyst mixture).
3. Place the flask on a digestion heater (Fig. 3.3) and heat gently to boil until the
contents clear. Continue digestion for another 2 h.
4. Allow the liquid to cool and dilute with 300–500 ml of distilled water. Also,
wash the neck of the flask with distilled water.
5. Add 5–6 glass beads in the Kjeldahl flask.
6. Fit the digestion assembly (Fig. 3.4). Add 75 ml of 50% NaOH to the diluted
digest through the walls of the Kjeldahl flask. The interface between the two
liquids on mixing should be clear and clean.
7. Connect the flask to the distillation assembly with the tip of the condenser being
dipped in 50 ml of boric acid solution in an Erlenmeyer flask containing the
indicator (0.1 g of methyl red in 50 ml ethanol and 0.5 g of bromocresol blue in
250 ml of ethanol and mix them).
8. Start heating the flask. Make sure that the uninterrupted supply of cold water is
maintained through the condenser.
9. When the quantity of the distillate reaches around 150 ml stop the distillation.
10. Titrate the boric acid solution containing trapped ammonia with standard HCl
solution (0.1 N) till the appearance of pink color. Note the reading.
11. Simultaneously run a blank test following the same procedure as described
above by replacing milk with 5 ml water and 0.85 g of sucrose (nitrogen content
not more than 0.002% m/m).
Fig. 3.3 Semiautomatic protein digestion unit

Fig. 3.4 Automatic protein distillation unit
Calculation
Percent total nitrogen by weight ¼ 1:4007 ðV s V b Þ N=W
where,
Vs ¼ volume in ml of standard HCl used for sample
Vb ¼ volume in ml of standard HCl used for blank
N ¼ Normality of standard HCl
W ¼ mass of sample
To calculate crude protein content—Multiply the nitrogen content with a factor of
6.38 (in case of milk). Milk proteins on an average contain 15.65% nitrogen, thus the
total crude protein is calculated using a factor of 6.38 (100/15.65).
Where an exact factor is not known, 6.25 is most often used. It should be noted,
however, that such a figure for protein often involves some nonnitrogenous
compounds and also that nitrogen in certain forms (e.g., nitrites, nitrates, and nitroso
compounds) is not estimated in Kjeldahl process unless the procedure is modified.
For example, if nitrates are to be included, a chemical readily nitrated such as
salicylic acid or phenol must be added prior to sample digestion. The nitrated
compound then has N reduced to NH2 and subsequently to NH3.
3.9.5 Lactose Content
Lane–Eynon Method (Volumetric Method)

The estimation of lactose is based on the reduction properties of the lactose found in
milk. Reducing sugars have the ability to function as reduction agents as they have a
free aldehyde or ketone group in their structure thus capable of reducing Fehling
solution or Tollen’s reagent, etc. by reducing metal ions like copper, iron, or silver in
alkaline conditions. In this method, Fehling solution (a mixture of Fehling A

(CuSO4) and Fehling B solutions (alkaline sodium–potassium tartrate)) is used.
Mixing of both the solutions, i.e., Fehling A and Fehling B, results in an alkaline
solution. The CuSO4, gets converted to Cu(OH)2. This compound [Cu(OH)2] has the
ability to get precipitated but sodium–potassium tartrate prevents its precipitation by
keeping it in soluble form, by forming a complex with copper. The complex being
soluble, behaves/reacts as if it is alkaline Cu(OH)2. When the mixture of Fehling
solution is heated it gets converted to cupric oxide (CuO) which reacts with the
reducing sugar and to form cuprous oxide (Cu2O precipitates are brick red in color),
indicating the conversion of sugar to its corresponding acid by oxidation. The
estimation of lactose content in milk requires the treatment of milk samples with
acetic acid to precipitate fat and protein. The filtrate thus obtained is treated with
alkaline copper sulfate solution (Fehling reagent) with continuous heating. During
the reaction, Cu2O gets precipitated as brick red in color. The titration is carried out
using methylene blue as an indicator, which on reduction, changes color of the
solution from blue to colorless. At this stage, complete precipitation of Cu2O occurs
in the form of brick red precipitates.
Reaction
Procedure
(a) Preparation of milk filtrate:
1. Take 10 ml of well-mixed milk sample in a 100-ml volumetric flask.
2. Add 30–40 ml of distilled water and heated to 40–45 C, immediately add 1.5 ml
of acetic acid (10%). Mix well and adjust final volume up to the mark with
distilled water. Leave the flask undisturbed for 30 min.
3. After 30 min, filter the contents of the flask by using Whatman filter paper 42.
Discard the first few milliliters and collect the remainder of the filtrate in a clean,
dry Erlenmeyer flask fitted with stopper.
(b) Standardization of Fehling Solution:

1. Pipette 5 ml of Fehling solution A and 5 ml of Fehling solution B in a 250-ml
Erlenmeyer flask using two separate pipettes. Fill the burette with the standard
lactose solution.
2. Heat the Erlenmeyer flask over burner or heater and keep boiling it moderately for
2 min. Add some inert boiling chips to avoid the bumping. Add 3–4 drops of
methylene blue while boiling. Titrate the contents of the flask against standard
lactose solution from the burette till blue color disappears and the bright brick-red
color of precipitated Cu2O appears (at the endpoint the Cu2O suddenly settles
down providing a clear supernatant). Note the amount of lactose solution required
for the reduction of Fehling solution. After this preliminary titration, further
titration should be carried out, by adding practically all of the standard volume
of lactose solution (1 ml less than required as observed in the first titration)
required before commencing heating and continuing titration as before. The
titration must be done within 3 min from the commencement of the boiling. To
this experiment let X ml be the titer.
Note: The boiling should be maintained uniformly during titration.
(c) Determination of lactose in milk:
Step 1
1. Pipette 5 ml of Fehling A and 5 ml of Fehling B solution in a 250-ml Erlenmeyer

flask. Fill the burette with prepared milk filtrate.
2. Add 10 ml of milk filtrate from the burette into a flask containing Fehling solution
and heat to boiling. Boil for about 15 s and rapidly add further amount of lactose
filtrate until a faint perceptible blue color remains. Add 3–4 drops of methylene
blue indicator and complete the titration to the first disappearance of blue color by
adding lactose filtrate dropwise to red color.
Step 2
1. Repeat the steps as mentioned in step 1, add almost complete amount of milk
filtrate (1 ml less than determined in step 1).
2. Heat the mixture to boiling followed by the addition of 3–4 drops of methylene
blue indicator and titrate to brick red color end point.
3. Run a blank titration by replacing 0.5% standard lactose solution with milk filtrate
in the burette.
4. Note the volume of milk filtrate (Y ) and standard lactose solution consumed
during the titration.
Calculation
%lactose in milk ¼ 5 ðX=Y Þ
1 ml of standard lactose solution contain ¼ 0.005 g of lactose

Let X ml ¼ volume in ml of the standard lactose solution required to reduce 10 ml
of Fehling solution (5 ml each of Fehling A and B solutions)
Let Y ml ¼ volume in ml of the milk filtrate solution required to reduce 10 ml of
Fehling solution (5 ml each of Fehling 1 and 2 solutions)
10 ml of Fehling solution ¼ X ml of standard lactose solution

¼ Y ml of milk filtrate
So, Y ml of milk filtrate ¼ X ml of standard lactose solution ¼ X 0.005 g of

lactose
100 ml of milk filtrate ¼ ðX 0:005Þ=Y 100
Since 100 ml of milk filtrate is obtained from 10 ml of milk

Lactose present in 100 ml of milk ¼ X 0.005 100 100/(Y 10)
3.9.6 Ash Content
The salts of milk include those constituents except hydrogen ions and hydroxyl ions
that are present as ions or in equilibrium with ions. Milk contains various salts that
are present in soluble, ionic, or colloidal form like sodium, potassium, calcium,
magnesium, phosphorous, citrates, sulfates, bicarbonates, chlorides, carbonates, and
magnesium. Some of these minerals are often present or associated with casein
micelles (calcium and phosphorous) or as phospholipids. The ash content of milk is
prepared by heating the sample at 500–550 C for 4 h. Ashing or incineration of milk
decomposes its organic matter while the soluble inorganic salts (mineral
constituents) are left behind in the form of ash. But the determination of the ash
content of milk does not give the true picture of the mineral composition of milk.
This is due to the fact that (a) citrates and other organic radicals get destroyed during
ashing, (b) phosphorous or sulfur that is associated with proteins and lipids may also
get reflected in the ash, and (c) some alkalis like sodium or potassium may get
vaporized at the temperature employed for ashing. The ash content of normal milk
ranges between 0.7 and 0.8%. An increase in ash content can be attributed to late
lactation milk/udder infection or milk adulterated with neutralizers.
Procedure
1. Weigh an empty silica crucible and note its weight.
2. Weigh 5 g of milk sample into it and record the weight.
3. Evaporate the sample to dryness on a hot plate.
4. Place the crucible in a muffle furnace at 550 C for 4 h or till ash is free of carbon.
5. Cool the crucible by placing it in a desiccator.
6. Weigh the crucible and note its weight.
7. Repeat the steps 4–6 till the difference between two successive weights is not
more than 0.1 g.
Calculation
Weight of empty silica crucible ¼ W
Weight of silica crucible with milk sample ¼ W1
Weight of silica crucible after drying ¼ W2
Percent ash by weight ¼ ðW 2 W Þ=ðW 1 W Þ 100
3.9.7 Lactate Content
Principle
The quantification of D-lactic acid involves two enzymatic reactions. In the first
reaction, D-lactic acid (D-lactate) is oxidized to pyruvate in the presence of nicotin-
amide adenine dinucleotide (NAD+). This reaction is catalyzed by D-lactate dehy-
drogenase (D-LDH) (3.1).
ðDLDHÞ
D Lactate þ NADþ , pyruvate þ NADH þ Hþ ð3:1Þ
It is clear from the reaction (3.1) that the equilibrium of the reaction lies firmly in
the favor of D-lactic acid and NAD+, so it is important to trap the pyruvate produced
in the reaction. The enzyme D-glutamate–pyruvate transaminase (D-GPT) converts
the pyruvate to 2-oxoglutarate and D-alanine in the presence of excess of
D-glutamate (3.2).
ðDGPTÞ
Pyruvate þ D glutamate ! D alanine þ 2 oxoglutarate ð3:2Þ
In the above reaction, it is clear that the amount of the NADH is stoichiometric
with the amount of D-lactic acid. Finally, the amount of the lactate present is
proportional to the NADH formed which is measured by recording the increase in
absorbance at 340 nm.
Similarly for the estimation of L-lactic acid (L-lactate), the L-lactate is oxidized to
pyruvate which is catalyzed by the enzyme L-lactate dehydrogenase (L-LDH) (3.3)
in the presence of NAD+.
ðLLDHÞ
L Lactate þ NADþ ! pyruvate þ NADH þ Hþ ð3:3Þ
The pyruvate thus formed is again trapped using the enzyme D-GPT in
D-glutamate (3.2). For the assays involving the estimation of D- and L-lactic acid
can be sequentially, but in the standard format they are performed as the incubations
are to be performed simultaneously, thus reducing the total reaction time.
KIT Method and Reagents

The kits available for the estimation of D- and L-lactic acid contains the following
reagents:
Bottle 1: Buffer (25 ml, pH 10.0) plus D-glutamate and sodium azide (0.02% w/v)
as a preservative. Stable for >2 years at 4 C.
Bottle 2: NAD+. Stable for >5 years below 10 C.
Bottle 3: D-Glutamate–pyruvate transaminase suspension (2.2 ml). Stable for
>2 years at 4 C.
Bottle 4: L-Lactate dehydrogenase suspension (1.1 ml). Stable for >2 years at
4 C.
Bottle 5: D-Lactate dehydrogenase suspension (1.1 ml). Stable for >2 years at
4 C.
Bottle 6: D-/L-Lactic acid standard solution (5 ml, 0.15 mg/ml of each) in 0.02%
(w/v) sodium azide. Stable for >2 years at 4 C.
Preparation of Reagent Solutions

1. The reagent supplied in bottle 1 is used as such.
2. Dissolve the contents of bottle 2 in 5.5 ml distilled water. Stable for >1 year at
4 C or stable for >2 years below 10 C. Divide the reagent into aliquots to
avoid the repetitive freezing/thawing cycles. Store the reagent in polypropylene
tubes.
3. Use the contents of bottles 3, 4, and 5 as supplied. Mix the bottles properly before
use and store in the upright position.
4. Use the contents of bottle 6 as supplied.
Apparatus and Equipment

1. Volumetric flasks (50 ml and 100 ml).
2. Disposable plastic or glass cuvettes (1 cm light path, 3.0 ml).
3. Micropipettes (20 μl, 200 μl, and 1 ml).
4. Positive displacement pipettes (to dispense 0.5 ml aliquots of Buffer 1 and 0.1 ml
aliquots of NAD+ solution).
5. Analytical balance.
6. Spectrophotometer set at 340 nm.
7. Vortex mixer.
8. Whatman No. 1 (9 cm) filter papers.
Sample Preparation
1. Carrez I solution: Dissolve 3.60 g of potassium hexacyanoferrate (II) in 100 ml of
distilled water. Store at room temperature.
2. Carrez II solution: Dissolve 7.20 g of zinc sulfate in 100 ml of distilled water.
Store at room temperature.
3. Sodium hydroxide (NaOH, 100 mM): Dissolve 4 g of NaOH in 1 l of distilled
water. Store at room temperature.
Weigh 10 g of milk into a 100-ml volumetric flask containing 60 ml of distilled

water. Add the following solutions in a stepwise manner and mix the contents of the
volumetric flask after addition to each solution: 2 ml of Carrez I solution, 2 ml of
Carrez II solution and 4 ml of NaOH solution (100 mM). Make up the volume to
100 ml using distilled water, mix and filter. This filtered sample should be used for
further analysis.
Procedure
I. Sequential assay of D- and L-lactic acid
Wavelength 340 nm
Cuvette 1 cm light path (glass or plastic)
Temperature ~25 C
Final volume 1.12 ml (D-lactic acid) and 1.13 ml (L-lactic acid)
Sample solution 0.5–30 μg of L-lactic acid per cuvette (in 0.1–1.5 ml sample volume)
Read against air (Without cuvette in the light path) or against water
Pipette into cuvette Blank (μl) Sample (μl)

Distilled water (~25 C) 800 750
Sample – 50
Solution 1 (buffer) 250 250
Solution 2 (NAD+) 50 50
Suspension 3 (D-GPT) 10 10
Mixa, read the absorbance of the solutions (A1) after approx. 3 min and start the reactions by
addition of:
Suspension 5 (D-LDH) 10 μl 10 μl
Mixa, read the absorbance of the solutions (A2) at the end of the reaction (approx. 5 min). If the
reaction has not stopped after 5 min, continue to read the absorbance at 1 min intervals until the
absorbance either remain the same or increase constantly over 1 minb
Suspension 4 (L-LDH) 10 μl 10 μl
Mixa, read the absorbance of the solutions (A3) at the end of the reaction (approx. 10 min). If the
reaction has not stopped after 10 min, continue to read the absorbance at 5 min intervals until the
absorbance either remain the same, or increase constantly over 5 minb
a
For example, with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette
cap or Para film
b
If this “creep” rate is greater for the sample than for the blank, extrapolate the absorbance (sample
and blank) back to the time of addition of suspension 4 or 5
Calculation
Determine the absorbance difference (A2 A1) for both blank and sample. Subtract
the absorbance difference of the blank from the absorbance difference of the sample,
thereby obtaining ΔAD-lactic acid.
Determine the absorbance difference (A3 A2) for both blank and sample.
Subtract the absorbance difference of the blank from the absorbance difference of
the sample, thereby obtaining ΔAL-lactic acid.
The value of ΔAD-lactic acid should, as a rule, be at least 0.100 absorbance units to
achieve sufficiently accurate results.
The Concentration of D- and L-lactic Acids Can Be Calculated as Follows:

MW
C ðg=lÞ ¼ V ΔADlactic acid
εdv
where:
V ¼ final volume (ml)
MW ¼ molecular weight of lactic acid (g/mol)
ε ¼ extinction coefficient of NADH at 340 nm 6300 (l mol1 cm1)
d ¼ light path (cm)
v ¼ sample volume (ml)
For D-lactic Acid

C ðg=lÞ ¼ ð1:12 90:1=6300 1:0 0:05Þ ΔADlactic acid
C ðg=lÞ ¼ 0:3203 ΔADlactic acid
For L-lactic Acid

C ðg=lÞ ¼ ð1:13 90:1=6300 1:0 0:05Þ ΔALlactic acid
C ðg=lÞ ¼ 0:3232 ΔALlactic acid
If the sample has been diluted during preparation, the result must be multiplied by
the dilution factor, F.
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(6):1390–1397
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Quality Assessment of Processed Milk
4
Milk being food product of animal/biological origin is generally consumed after

being subjected to minimal processing so as to make it safe for human consumption.
The chilled raw milk received at the dairy factory is processed to various forms of
market milk as per the demand and marketing strategies. Such milk is subjected to
various processing conditions like pasteurization and sterilization keeping in view of
the prevailing legal standards as prescribed by the regulatory body of that country or
place. The types of processed (market) milk in India are generally categorized as full
cream milk, standardized milk, toned milk, double toned milk, and skim milk. The
basis of differentiation between them is the fat and the solids-not-fat content. The
raw milk after being processed is tested for various compositional (fat, solids-not-fat,
protein, lactose, ash, and total solids/moisture content) and chemical tests (titratable
acidity, phosphatase test, turbidity test, creaming index, homogenization efficiency,
etc.).
4.1 Chemical Tests for Processed Milk
4.1.1 Fat Test
(a) Gerber method (volumetric method)
The method is based on the principle that when sulfuric acid (90–91%) is added to
milk, it dissolves the proteins of milk while the fat globules remain free and in liquid
form due to the heat produced by the acid. The two phases are separated by
centrifugation with fat being lighter of the two portions. Addition of amyl alcohol
facilitates better separation between the fat and the nonfat portion. If the concentra-
tion of the Gerber acid is less than 90% (acid used is weak) then the fat column
obtained after centrifugation is light colored as the milk nonfat content does not gets
dissolved completely and if the concentration of the acid used is more than 91% (or a
concentrated acid is used) it may lead to the charring of milk proteins causing the

https://doi.org/10.1007/978-981-15-4167-4_4
70 4 Quality Assessment of Processed Milk
development of black specs in the fat and nonfat interface obtained in the
butyrometer stem.
Procedure
1. Add 10 ml of sulfuric acid (Gerber acid) into the milk butyrometer (range 0–10%)
with an automatic tilt measure.
2. Mix the milk sample properly and pipette 10.75 ml of milk using milk pipette in
the butyrometer.
3. The milk should be added from the side of the butyrometer wall slowly to prevent
the charring.
4. Add 1 ml of amyl alcohol with an automatic tilt measure.
5. Stopper the butyrometer with a lock stopper and mix the contents properly.
6. Place the butyrometer in a water bath maintained at 65 C for 5 min.
7. Transfer the butyrometer in the Gerber centrifuge and centrifuge at
1100–1200 rpm for 5 min.
8. Place the butyrometer again in the water bath maintained at 65 C for 2 min.
9. Read the fat content with the help of the markings drawn on the butyrometer stem.
Note: The milk sample should be heated at 40 C and cooled to 27 C before

analysis of fat. Heating of milk at 40 C converts fat to melted state.
Why 10.75 ml Milk Is Taken for Estimation of Fat in Gerber Method?

The graduations marked (0–10) on the tube or stem of the butyrometer correspond to
an internal volume of 0.125 ml for each 1% mark.
So, the mass of the fat contained in 1% of the stem ¼ Volume Density, i.e.,
0.125 0.9, this value comes out to be 0.1125 g (weight of 1% of milk fat).
If 1% of this corresponds to 0.1125 g, then 100% corresponds to
0.1125 100 ¼ 11.25 g.
This indicates that the amount of milk to be pipetted into the butyrometer should
be 11.25 g, but as the isoamyl alcohol has certain impurities that lead to overestima-
tion of the fat by 2.5–3% (average ¼ 2.667%).
So, the fat is 11.25 (11.25 2.667/100) ¼ 10.95 g.
Thus, we should pipette 10.95 g of milk, which corresponds to a volume of
10.65 ml (10.65/1.028).
Since approximately 0.1 ml of milk remains stuck to the walls and at the tip of the
pipette, so we should pipette out 10.75 ml of milk.
(b) Rose–Gottlieb method (gravimetric method)
Ammonia disrupts the fat globule membrane surrounding the milk fat and also
dissolves the protein. The released milk fat is then transferred from the aqueous
phase to the solvent phase using diethyl and petroleum ether. Ethanol precipitates the
proteins. This method is regarded as a reference method for fat estimation.
4.1 Chemical Tests for Processed Milk 71
Procedure
1. Weigh about 10 g of well-mixed milk sample into the Mojonnier/Rose–Gottlieb
extraction tube.
2. Add 1.25 ml of concentrated ammonia (sp. gravity 0.8974) and mix well gently.
3. Then add the following reagents with proper mixing after adding each reagent:
well for a minute.
4. Then add 25 ml of petroleum ether having a boiling point of 40–60 C. Stopper
5. Leave the tube undisturbed so that the ethereal layer gets separated from the
aqueous phase (in case of Rose–Gottlieb method).
6. Alternatively for the Mojonnier method centrifuge the flask at low speed.
7. Decant the ethereal layer into a previously weighed vessel containing 5–6 glass
8. Repeat the extraction twice using 15 ml of diethyl ether and petroleum ether, so
as to extract the fat completely from the sample.
9. Add the ethereal layer to the same previously weighed vessel containing the
10. The ether is evaporated by placing the vessel on a hot plate or water bath at
60–65 C.
11. Place the vessel at 102 2 C for at least 2 h.
12. Cool the dish in a desiccator and weigh.
13. Heat the vessel again in the oven followed by cooling and weighing.
14. Repeat steps 11–13 till the difference between the two successive weights does
not differ by more than 1 mg.
15. The difference in the weights before drying and after drying is the amount of fat
present in the sample.
16. Repeat the same process for blank also.
17. The difference between the sample and blank should not exceed 0.5 mg.
Calculation
after drying.
4.1.2 Solids-Not-Fat Test
Lactometers are used for the determination of solids-not-fat (SNF) content in milk.
The determination of SNF is based on the Archimedes principle of flotation. This
law states that whenever a solid is immersed partially or fully inside a liquid it is
acted upon by an upward thrust, which is equal to the weight of the liquid displaced
by it. Lactometers are type of hydrometers and are calibrated beforehand with a
liquid of known specific gravity.
Procedure
1. Adjust the temperature of milk sample at the temperature prescribed for a
lactometer.
2. Mix the sample well. Avoid incorporation of air or foam formation.
3. Pour sufficient milk into the lactometer jar.
4. Dip the lactometer into the lactometer jar containing milk carefully so that
lactometer should not touch the walls of the jar.
5. Wait until the lactometer attains a stable state and assumes a constant level.
6. Read the lactometer reading and check the temperature of milk. If the temperature
of milk is higher or lower than that prescribed for the lactometer, use the
correction from the standard table for corresponding temperature (refer to
Chap. 3). This is called as Corrected Lactometer Reading (CLR).
7. Calculate the SNF using the formula mentioned below.
Calculation
Specific gravity ¼ 1 þ ðCLR=1000Þ
Percent SNF ¼ ðCLR=4Þ þ ð0:2 fat ð%ÞÞ þ 0:1 ð0:1 is the factorÞ
Note: The factor to be added in the calculation of SNF varies according to the type
of lactometer used (due to different temperatures for different lactometer).
Richmond formula to determine percent total
solids ¼ (CLR/4) + (1.25 fat) + 0.44
Richmond formula to determine percent solids not
fat ¼ (CLR/4) + (0.25 fat) + 0.44
The factor 0.44 varies from condition to condition, i.e., lactometer to lactometer
or region to region. The factor should be calculated by determining the total solids in
the milk by gravimetric method and comparing it to the solids not fat determined by
the lactometer.
For example: The SNF of a milk sample calculated by lactometer ¼ x
The fat content of the milk sample ¼ y
Total solid of the milk ¼ x + y
Total solids of milk calculated by gravimetric method ¼ z
SNF of the same milk sample ¼ z y
Compare the SNF content determined by the lactometer and by gravimetric
method for the milk sample and calculate the factor.
4.1.3 Total Solids/Moisture Content
The total solids of processed milk varies from the variant packed ranging from
10.5% (min) for double toned milk to 15% (min) for full-cream milk (Indian
conditions, this range of total solids may also vary from region to region or from
country to country). The total solids of milk can be calculated by evaporating the
moisture/water from a known quantity of sample to dryness at 102 2 C.
Procedure
1. Warm the milk sample homogeneously to 35–40 C so as to mix the cream
adhering to the sample.
2. Weigh a clean, dry empty aluminum moisture dish.
3. Cool the milk sample and add 5 ml of it in the dish and record the weight.
4. Place the dish on the boiling water bath or hot plate such that the bottom of the
dish is directly in contact with the steam or the heat.
5. Continue the boiling of milk till almost all of the water is evaporated.
6. Remove the dish and wipe its bottom.
7. Transfer the dish into the hot air oven maintained at 102 2 C for 2 h.
8. Transfer the dish to a desiccator and allow it to cool.
9. Weigh the dish and record its weight.
10. Repeat the steps from 7 to 9 till the difference between two successive weights
does not exceed more than 1 mg.
Calculation
Weight of empty aluminum dish ¼ W
Weight of aluminum dish with milk sample ¼ W1
Weight of aluminum dish after drying ¼ W2
Total solid content ¼ ðW 2 W Þ=ðW 1 W Þ 100
Moisture content ¼ 100 %TS
The most widely used method for the determination of protein content is done by
Kjeldahl method. In this method, the protein is first digested using concentrated
sulfuric acid in the presence of a catalyst mixture. This method is based on two
assumptions:
1. The dietary carbohydrates and fat do not contain nitrogen.

2. Nearly all of the nitrogen in the food is present as amino acids (aa) in proteins.
The Kjeldahl method involves the following steps:
(a) The protein nitrogen is converted into ammonium sulfate by oxidation.

(b) The ammonium sulfate is reacted with a strong alkali and undergoes decompo-
sition, resulting in the evolution of ammonia, which is trapped in boric acid.
(c) Titration of the trapped ammonia with standard acid.
(d) The calculation of the percentage protein in the sample from its weight and the
volume of standard acid required to titrate the ammonia.
Sulfuric acid and the catalyst mixture facilitate the digestion of all the organic
matter except nitrogen. Carbon and hydrogen are converted to CO2 and H2O, sulfur
is converted into sulfur dioxide, and phosphorous is converted into phosphorous
pentaoxide.
The reduced form of the nitrogen is retained as ammonium sulfate. The neutral
salts such as potassium sulfate raise the boiling point of the reaction mixture during
the digestion process thus increasing the effectiveness of digestion process. Copper
sulfate acts as a catalyst, fastens the digestion, producing a clear reaction mixture.
Copper, selenium, and mercury in the form of their salts can also be used. They act as
oxidation catalysts (O2 carriers) because they readily pass from a higher to a lower
valence and vice versa. The solution is digested over a moderate heat (<420 C) so
as to produce even, gentle boiling. Clearing of the digest is not a criterion of
completion of the digestion process. Digestion should be continued after clearing.
Overheating causes loss of volatile nitrogenous compounds and inconsistent results.
The digest is then neutralized using an alkali thus liberating ammonia. Then the
liberated ammonia is distilled off and trapped in boric acid, which is then titrated
with standard acid. Boric acid does not get involved in the titration but forms
ammonium borate when reacts with ammonia. The formed ammonium–borate
complex is titrated with an acid using methyl red-bromocresol green indicator till
the green distillate changes from colorless to pink.
Reaction
The volume of the acid consumed during the titration is equivalent to the
concentration of ammonia, which is equivalent to the nitrogen content of the actual
protein present in the sample. This gives the crude protein as some of the nitrogen is
contributed from the nonprotein component present in milk.
Procedure
1. Weigh 5 g of milk in a clean and dry Kjeldahl flask.
2. Add 25 ml of concentrated sulfuric acid, 0.2 g of copper sulfate, and 10 g of
potassium sulfate (catalyst mixture).
3. Place the flask on a digestion heater and heat gently to boil until the contents are
clear. Continue digestion for another 2 h.
4. Allow the liquid to cool and dilute with 300–500 ml of distilled water. Also,
wash the neck of the flask with distilled water.
5. Add 5–6 glass beads in the Kjeldahl flask.
6. Fit the digestion assembly. Add 75 ml of 50% NaOH to the diluted digest
through the walls of the Kjeldahl flask. The interface between the two liquids on
mixing should be clear and clean.
7. Connect the flask to the distillation assembly with the tip of the condenser being
8. Start heating the flask. Make sure that uninterrupted supply of cold water is
9. Stop the distillation when the quantity of the distillate reaches around 150 ml.
10. Titrate the boric acid solution containing trapped ammonia with std. HCl
11. Simultaneously run a blank test following the same procedure as described
above by replacing milk with 5 ml water and 0.85 g of sucrose (nitrogen content
Calculation
where,
W ¼ mass of sample
total crude protein is calculated using a factor of 6.38 (100/15.65). The conversion
factor for different types of protein is mentioned in Table 4.1.
Where an exact factor is not known, 6.25 is most often used. It should be noted,
however, that such a figure for protein often includes some non-nitrogenous
Table 4.1 Conversion Proteins Conversion factors

factor for various proteins
Meat and eggs 6.25
to be used in the estimation
of total protein by Kjeldahl Milk and dairy products 6.38
method Rice 5.95
Rye, barley, wheat, and oats 5.83
Wheat flour 5.70
Wheat bran 6.31
Soybean 5.71
compounds and also that nitrogen in certain forms (e.g., nitrates, nitrites, and nitroso
compounds) is not estimated in Kjeldahl process unless the procedure is modified.
For example, if nitrates are to be included, a chemical readily nitrated such as
salicylic acid or phenol must be added prior to sample digestion. The nitrated
compounds then have N reduced to NH2 and subsequently to NH3.
4.1.5 Lactose Content
Lane-Eynon Method (Volumetric Method)

The estimation of lactose is based on the reduction properties of the lactose found in
milk. Reducing sugars have the ability to function as reduction agents as they have a
free aldehyde or ketone group in their structure thus capable of reducing Fehling’s
solution or Tollen’s reagent, etc., by reducing metal ions like copper, iron, or silver
in alkaline conditions. In this method, Fehling’s solution (a mixture of Fehling’s A
(CuSO4) and Fehling’s solution B (alkaline sodium-potassium tartrate)) is used.
Mixing of both the solutions, i.e., Fehling A and Fehling B, results in an alkaline
solution. The CuSO4, gets converted to Cu(OH)2. This compound [Cu(OH)2] has the
ability to get precipitated but sodium potassium tartrate prevents its precipitation by
keeping it in soluble form, by forming a complex with copper. The complex being
soluble, behaves/reacts as if it is alkaline Cu(OH)2. When the mixture of Fehling
solution is heated it gets converted to cupric oxide (CuO) that reacts with the
reducing sugar and to form cuprous oxide (Cu2O precipitates are brick red in
color), indicating the conversion of sugar to its corresponding acid by oxidation.
The estimation of lactose content in milk requires the treatment of milk samples with
acetic acid to precipitate fat and protein. The filtrate thus obtained is treated with
alkaline copper sulfate solution (Fehling’s reagent) with continuous heating. During
the reaction, Cu2O gets precipitated as brick red in color. The titration is carried out
using methylene blue as an indicator, which on reduction, changes color of the
solution from blue to colorless. At this stage, complete precipitation of Cu2O occurs
in the form of brick-red precipitates.
Reaction
Procedure
(a) Preparation of milk filtrate:
1. Take 10 ml of well-mixed milk sample in a 100-ml volumetric flask.
2. Add 30–40 ml of distilled water and heated to 40–45 C, immediately add 1.5 ml
of acetic acid (10%). Mix well and adjust final volume up to the mark with
distilled water. Leave the flask undisturbed for 30 min.
3. After 30 min by using Whatman filter paper 42 filter the contents of the flask.
Discard the first few ml and collect the remainder of the filtrate in a clean, dry
Erlenmeyer flask fitted with a stopper.
(b) Standardization of Fehling’s Solution:
1. Pipette 5 ml of Fehling’s solution A and 5 ml of Fehling’s solution B in a 250-ml
Erlenmeyer flask using two separate pipettes. Fill the burette with the standard
lactose solution.
2. Heat the Erlenmeyer flask over burner or heater and keep boiling it moderately for
2 min. Add some inert boiling chips to avoid the bumping. Add 3–4 drops of
methylene blue while boiling. Titrate the contents of the flask against standard
lactose solution from the burette till blue color disappears and the bright brick-red
color of precipitated Cu2O appears (at the end point, the Cu2O suddenly settles
down providing a clear supernatant). Note the amount of lactose solution required
for the reduction of Fehling’s solution. After this preliminary titration, further
titration should be carried out, by adding practically all of the standard volume of
lactose solution (1 ml less than required as observed in the first titration) required
before commencing heating and continuing titration as before. The titration must
be done within 3 min from the commencement of the boiling. Let X ml be the
titer to this experiment.
Note: The boiling should be maintained uniformly during titration.

(c) Determination of lactose in milk:
Step 1
1. Pipette 5 ml of Fehling’s A and 5 ml of Fehling’s B solution in a 250-ml

Erlenmeyer flask. Fill the burette with prepared milk filtrate.
2. Add 10 ml of milk filtrate from the burette into a flask containing Fehling’s
solution and heat to boiling. Boil for about 15 s and rapidly add further amount of
lactose filtrate until a faint perceptible blue color remains. Add 3–4 drops of
methylene blue indicator and complete the titration to the first disappearance of
blue color by adding lactose filtrate dropwise to red color.
Step 2
1. Repeat the steps as mentioned in step 1, add almost complete amount of milk
filtrate (1 ml less than determined in step 1).
2. Heat the mixture to boiling followed by the addition of 3–4 drops of methylene
blue indicator and titrate to brick red color end point.
3. Run a blank titration by replacing 0.5% standard lactose solution with milk filtrate
in the burette.
4. Note the volume of milk filtrate (Y ) and standard lactose solution consumed
during the titration.
Calculation
1 ml of standard lactose solution contain ¼ 0.005 g of lactose

Let X ml ¼ volume in ml of the standard lactose solution required to reduce 10 ml
of Fehling’s solution (5 ml each of Fehling’s A and B solution)
Let Y ml ¼ volume in ml of the milk filtrate solution required to reduce 10 ml of
Fehling’s solution (5 ml each of Fehling’s 1 and 2 solutions)
10 ml of Fehling’ s solution ¼ X ml of standard lactose solution

¼ Y ml of milk filtrate
So, Y ml of milk filtrate ¼ X ml of standard lactose solution ¼ X 0.005 g of

lactose
100 ml of milk filtrate ¼ ðX 0:005Þ=Y 100
Since 100 ml of milk filtrate is obtained from 10 ml of milk

Lactose present in 100 ml of milk ¼ X 0.005 100 100/(Y 10)

4.1.6 Ash Content
The salts of milk include those constituents except hydrogen ions and hydroxyl ions
that are present as ions or in equilibrium with ions. Milk contains various salts that
are present in soluble, ionic, or colloidal form like sodium, potassium, calcium,
magnesium, phosphorous, citrates, sulfates, bicarbonates, chlorides, carbonates, and
magnesium. Some of these minerals are often present or associated with casein
micelles (calcium and phosphorous) or as phospholipids. The ash content of milk is
prepared by heating the sample at 500–550 C for 4 h. Ashing or incineration of milk
decomposes its organic matter while the soluble inorganic salts (mineral
constituents) are left behind in the form of ash. But the determination of the ash
content of milk does not give the true picture of the mineral composition of milk.
This is due to the fact that (a) citrates and other organic radicals get destroyed during
ashing (b) phosphorous or sulfur that is associated with proteins and lipids may also
get reflected in the ash (c) some alkalis like sodium or potassium may get vaporized
at the temperature employed for ashing. The ash content of normal milk ranges
between 0.7 and 0.8%. An increase in ash content can be attributed to late lactation
milk/udder infection or milk adulterated with neutralizers.
Procedure
1. Weigh an empty silica crucible and note its weight.
2. Weigh 5 g of milk sample into it and record the weight.
3. The sample is evaporated to dryness on a hot plate.
4. Place the crucible in a muffle furnace at 550 C for 4 h or till the ash is free of
carbon.
5. Transfer the crucible into the desiccator and cool at room temperature.
6. Weigh the crucible and note its weight.
7. Repeat the steps 4–6 until the difference between two successive weights is not
more than 0.1 g.
Calculation
Weight of silica crucible with milk sample ¼ W1
Percent ash by wt ¼ ðW 2 W Þ=ðW 1 W Þ 100
The natural/apparent acidity of milk is due to the presence of casein, citrates,

albumins, globulins phosphates, and carbon dioxide. Milk develops acidity due to
the action of lactic acid bacteria on lactose thus producing lactic acid. This is called
as developed or real acidity. The total titratable acidity of milk is the sum of natural
and developed acidity. It is measured by titration of a known volume of milk with a
standard alkali solution using an indicator (phenolphthalein) and expressed as %

lactic acid.
CH3 CHOHCOOH þ NaOH ! CH3 CHOHCOONa þ H2 O
From this reaction, it is clear that 40 g of NaOH neutralizes 90 g of lactic acid.

Or
One molecular weight of sodium hydroxide neutralizes one molecular weight of
lactic acid.
Thus, 1 l of 1 N NaOH neutralizes 90 g of lactic acid
Or 1 ml of 1 N NaOH neutralizes 0.09 g lactic acid. Thus if V ml of 1 N NaOH is
required by 1 ml of milk/milk product, then V ml of 1 N NaOH neutralizes 0.09V g of
lactic acid (assuming the acidity is contributed by lactic acid). Therefore, 100 ml or
100 g of milk or milk product would require 100 V ml of 1 N NaOH
i.e., 100 V 0.09, so for W ml or W g of milk 1 N NaOH required is
100 0.09 V/W.
Percent Titratable acidity of milk using 0.1 N NaOH ¼ 9 V N/W (N ¼ nor-
mality of NaOH used, i.e., 0.1, W ¼ amount of milk taken, V ¼ volume of NaOH
consumed in the titration).
Procedure
1. Transfer 10 ml of properly mixed milk sample into a white porcelain dish or glass
beaker.
2. Add a few drops of 1% phenolphthalein solution.
3. Titrate the contents with 0.1 N NaOH solution.
4. Observe the change in color to light pink. Mark it as the end point of the titration.
5. Note the amount of alkali consumed.
6. The titratable acidity is expressed as lactic acid equivalent per 100 ml of milk.
Calculation
where
V1 ¼ Volume, ml of the standard NaOH solution required for titration.
N ¼ Actual normality of the NaOH solution (0.1).
4.1.8 Phosphatase Test
Alkaline phosphatase is naturally found in milk. It loses its activity when milk is
subjected to temperatures equivalent to pasteurization temperature or higher. Thus,
this enzyme is adopted as an index of the efficiency of pasteurization. The activity of
alkaline phosphatase is measured by incubating the sample of milk with disodium
p-nitrophenyl phosphate at alkaline pH. Alkaline phosphatase hydrolyzes the
Table 4.2 Interpretation Disk reading Interpretation

of results for alkaline
0–3 Properly pasteurized
phosphatase test
2–6 Doubtful
10 and above Under pasteurized
disodium salt of nitrophenyl phosphate liberating p-nitrophenol, which is a yellow-

colored compound at alkaline conditions. Properly pasteurized milk does not
develop yellow color. The test cannot be applied to sour milk or preservative
added milk.
Procedure
1. Add 5 ml of buffer substrate (0.15% in bicarbonate buffer of pH 9.5) solution in a
test tube.
2. Stopper the tube and place it in a water bath maintained at 37 C.
3. Add 1 ml of milk sample into the tube and incubate at 37 C for 2 h.
4. Simultaneously prepare a blank of boiled/pasteurized milk sample and repeat
steps 1–3.
5. Place the blank and the test sample in the Lovibond comparator stand.
6. Revolve the disk and compare the color of both the test tubes.
7. Record the test reading.
8. Interpret the result as mentioned in Table 4.2.
Precautions
1. Fresh pipette should be used for every milk sample. The pipettes should not be
contaminated with saliva.
2. The milk sample should be tested immediately after pasteurization or stored at
temperatures between 3 and 5 C. This enzyme has the ability to renature itself
after 6 h at 30 C.
3. Generally, the suspected milk samples develop yellow color within 30 min after
incubation at 37 C (generally employed at industrial level).
4.1.9 Turbidity Test
This test is generally performed to test the efficiency of sterilization. Heat labile milk
proteins like albumins are denatured after sterilization. The albumin present in milk
gets separated from casein on the addition of inorganic acids or salts. In this test, salts
like ammonium sulfate are first added to milk followed by its filtration. The filtrate
thus obtained is heated, cooled, and observed for any signs of visible turbidity.
Insufficient heat-treated milk will develop turbidity due to the presence of
undenatured albumin. Properly sterilized milk will not show any turbidity due to
the denaturation of the albumin.
Procedure
1. Measure 20 ml of milk into a conical flask and add 4 g of ammonium sulfate to it.
2. Shake the flask so as to ensure that ammonium sulfate is dissolved completely.
3. Allow the flask to stand for 5 min.
4. Filter the contents of the flask using Whatman No. 12 filter paper in a test tube.
5. Place the obtained filtrate on a boiling water bath for 5 min.
6. Cool the test tube in cold water and examine the contents for any visible turbidity.
Interpretation: Properly sterilized milk will not show any turbidity.
4.1.10 Homogenization Efficiency
Milk is subjected to homogenization to prevent the formation of a cream layer on the

surface of the milk. Homogenization breaks the fat globules into smaller size of not
more than 2 μ. As a result, the fat globules do not cluster and their rise is prevented.
This test gives the measure of the stability of fat emulsion in homogenized milk.
Procedure
1. Take a sample of homogenized milk into a 250-ml glass bottle.
2. Allow it to stand undisturbed for 48 h in a refrigerator.
3. Carefully remove 100 ml of the upper layer, mix it well, and test its fat content.
4. Mix the remaining sample and test its fat content separately.
5. Calculate the difference in the fat content of the two layers.
6. The difference between fat contents should not be more than 10%.
7. Difference between 7 and 8% indicates efficient homogenization.
4.1.11 Creaming Index
The principle for this test is the same as that of homogenization efficiency.
Procedure
1. Take 50 ml of homogenized milk in three different centrifuge tubes.
2. Centrifuge the contents for 15 min at 1000 rpm.
3. Transfer 5 ml of the upper portion from each tube into the beaker and determine
the fat content. Record it as “A.”
4. Empty the remaining portion of the tubes into a separate beaker and test its fat
content. Record it as “B.”
Calculation
Creaming index ¼ ðA BÞ 100=B
Interpret the results as per Table 4.3.

Table 4.3 Relationship Creaming index Homogenization quality

between creaming index
Up to 10 Excellent
and homogenization
efficiency for milk 11–20 Good
21–30 Fair
Over 30 Bad
Suggested Readings
IS 1224-1 (1977) Determination of fat by the Gerber method, part I milk. Bureau of Indian
Manual of Methods of Analysis of Foods-Milk and Milk Products-FSSAI (2015) Lab manual 1
Srivastava MK (2010) Handbook of milk analysis chemical and microbiological analysis of liquid
milk. Idbc Publishers, Lucknow
Agency, New Delhi
Quality Assessment of Milk Products
5
The role of milk and milk products has been increasing in human health, along with
an increase in the demand for safe and hygienically produced products. The compet-
itiveness among the dairy industries is also increasing every day. The biggest
challenge faced by the dairy industry nowadays is to produce good quality products
that are safe and as per the legal standards laid by the food regulating bodies. These
legal standards are made to have a quality control check on the products being
marketed, so as to safeguard the consumers from any health-related issue. Thus, it is
now imperative to check and determine the different parameters that define the
product safety, quality, and its composition as per the standards. This chapter covers
the various parameters that are tested for a product to maintain the prescribed
standards.
5.1 Analysis of Cheese, Paneer, Channa, and Khoa
5.1.1 Fat Test
The estimation of fat content in cheese, paneer, channa, and khoa (free from added
sugar) is done by acid digestion and is called Schmid-Bondzynski-Ratzlaff (SBR)
method. The principle for the estimation of fat by this method is that the milk
proteins are digested by using concentrated hydrochloric acid. Alcohol, diethyl
ether, and petroleum ether are added to the acid digested sample for extraction of
fat. The ethers are then evaporated and the left-over residue is weighed. In case if the
khoa shows presence of sugar, the fat content is tested by using Rose-Gottlieb/
Mojonnier method.
Procedure
• Take 1–2 g of the sample (cheese, channa, and paneer) in 100 ml beaker.
• Add 10 ml of concentrated HCl to the sample, followed by heating on the burner
with constant stirring so as to dissolve the solid particles.

https://doi.org/10.1007/978-981-15-4167-4_5
86 5 Quality Assessment of Milk Products
• Cool the contents to room temperature.

• Add 10 ml of ethanol and transfer the contents to the Mojonnier flask.
• To the Mojonnier flask, add 25 ml of diethyl ether. Stopper the flask with cork and
shake the contents vigorously for 1 min. Add 25 ml of petroleum ether and shake
the flask vigorously.
• Centrifuge the flask at 600 rpm for 10 min.
• Transfer the ethereal layer into a previously weighed conical flask containing
glass beads.
• Repeat the extraction protocol twice using 15 ml of diethyl ether and petroleum
ether and decant the ethereal layer into a conical flask.
• Evaporate the ether from the conical flask by placing it on hot plate at 60–65 C.
• Once the residual fat is obtained, place the conical flask in the oven maintained at
102 2 C.
• Remove the flask and cool in a desiccator. Then record the weight after drying so
as to get a constant weight, i.e., difference between two successive weights after
drying should not be more than 1 mg.
Calculation
after drying.
B ¼ weight of the sample.
Fat Content in Khoa (Having Added Sugar)

Ammonia is added to disrupt the fat globule membrane surrounding the milk fat and
also to dissolve the protein. The released milk fat is then transferred from the
aqueous phase to the solvent phase using diethyl and petroleum ether. Ethanol
facilitates the precipitation of the proteins. This method is regarded as a reference
method for fat estimation.
Procedure
• In a beaker, weigh 2–3 g of the sample and mix it with 5 ml of warm water to
make a homogenous suspension.
• Transfer the contents into the Mojonnier flask and rinse the contents of the beaker
with warm water so that no part of the suspension is left in the beaker. Transfer the
washings of the beaker to the Mojonnier flask.
• Add 1.25 ml of concentrated ammonia (sp. gravity 0.8974) and mix well gently.
• Then add the following reagents with proper mixing after adding each reagent:
well for a minute.
• Then add 25 ml of petroleum ether having a boiling point of 40–60 C. Stopper
5.1 Analysis of Cheese, Paneer, Channa, and Khoa 87
• Leave the tube undisturbed for the separation of the aqueous and ethereal layers
(in case of the Rose-Gottlieb method).
• Alternatively, for Mojonnier method centrifuge the flask at low speed.
• Decant the ethereal layer into a previously weighed vessel (flask, aluminum dish,
etc.) containing 5–6 glass beads.
• Repeat the extraction of fat twice using 15 ml of diethyl ether and petroleum
ether, to ensure the complete extraction of fat.
• Add the ethereal layer to the same previously weighed vessel containing the
• The ether is evaporated by placing the vessel on a hot plate at 60–65 C.
• Place the vessel at 102 2 C for at least 2 h.
• Cool the dish in a desiccator and record its weight.
• Heat the vessel again in the oven followed by cooling and weighing.
• Repeat the process to a constant weight, i.e., difference between two successive
weights should not be more than 1 mg.
The quantity of the residue left after drying is the amount of fat present in the
sample.
Calculation
after drying.
B ¼ weight of sample.
The estimation of fat by Werner Schmidt or Rose-Gottlieb method is time
consuming and tedious, also the sample throughput is less. So to eliminate the
shortcomings at industrial level, fat content in cheese, paneer, channa, or khoa can
be done by Gerber method. The principle for estimation of fat by Gerber method has
been discussed in Chap. 4 and its procedure is as follows:
• Grate the sample properly using a grater and transfer 3 g of it to the cheese
butyrometer (fat scale 0–40%).
• Add 5 ml of lukewarm water to it and mix properly. Add 10 ml of Gerber sulfuric
acid using automatic tilt measure.
• Using the automatic tilt measure, add 1 ml of amyl alcohol and stopper the
butyrometer using a lock stopper.
• Shake the contents of the butyrometer properly followed by transferring the
butyrometer into the water bath at 65 C for 5 min.
• Centrifuge the butyrometer by placing it in a Gerber centrifuge at 1200 rpm for
5 min.
• Place the butyrometer again in the water bath at 65 C for 5 min.
• Read the fat content with the help of the markings drawn on the butyrometer stem.
5.1.2 Moisture and Total Solids Content
The total solids can be calculated by evaporating the moisture/water from a known
quantity of sample to dryness at 102 2 C.
Procedure
• Weigh a clean, dry empty aluminum moisture dish containing sand, and a glass
rod. Heat it in hot air oven for 1 h. Then cool it in a desiccator at room
temperature.
• Weigh 3 g of the sample in the previously dried dish containing sand and glass
rod and record the weight.
• Add a few drops of distilled water in the sand to saturate it and mix the sample
with sand using a glass rod. The sample should be mixed and spread homo-
geneously on the surface of the dish.
• Place the dish on a hot plate or boiling water bath to evaporate the water.
• Wipe the bottom of the dish and place it in a hot air oven at 102 2 C for 4 h.
• Transfer the dish in the desiccator and let it cool to room temperature.
• Weigh the dish and note the weight.
• Repeat the heating and cooling to a constant weight differing not more than
0.5 mg. Record the final weight.
Calculation
Weight of aluminum dish with sample ¼ W1
5.1.3 Total Protein Content
The principle involved in the estimation of protein content by the Kjeldahl method
has been discussed in Sect. 4.1.4. The procedure for the determination of protein is
discussed below:
Procedure
• Weigh 0.2 g of sample in a clean and dry Kjeldahl flask.
• Add 25 ml of concentrated sulfuric acid, 0.2 g of copper sulfate and 10 g of
• Place the flask on a digestion heater and heat gently to boil until the contents clear.
Continue digestion for another 2 h.
• Allow the liquid to cool and dilute with 300–500 ml of distilled water. Also, wash
the neck of the flask with distilled water.
• Add 5–6 glass beads in the Kjeldahl flask.

• Fit the digestion assembly. Add 75 ml of 50% NaOH to the diluted digest through
the walls of the Kjeldahl flask. The interface between the two liquids on mixing
should be clear and clean.
• Connect the flask to the distillation assembly with the tip of the condenser being
• Start heating the flask. Make sure that uninterrupted supply of cold water is
• When the quantity of the distillate reaches around 150 ml stop the distillation.
• Titrate the boric acid solution containing trapped ammonia with std. HCl solution
(0.1 N) till the appearance of pink color. Note the reading.
• Simultaneously run a blank test following the same procedure as described above
by replacing the sample with 5 ml water and 0.85 g of sucrose (nitrogen content
Calculation
where
W ¼ mass of sample
5.1.4 Soluble Protein Content (Mamo 2017)
Determination of soluble protein is done by using Sharpe’s extraction solution,

which extracts the soluble protein. The extracted protein is then determined by the
Kjeldahl method. Proteins get solubilized during the ripening of cheese due to the
proteolytic action of enzymes and microorganisms.
Sharpe’s Extraction Solution Weigh 136.1 g sodium acetate, 8.9 g of calcium

chloride, 47 g of sodium chloride, and dissolve in 57.5 ml glacial acetic acid. Mix the
contents and make up the volume to 1000 ml using distilled water. The working
solution of Sharpe’s extraction solution is prepared by diluting the stock solution
four times with distilled water.
Procedure
• Weigh 3 g of cheese in a mortar and add a small amount of Sharpe’s extraction
solution (previously tempered to 50 C) to make a paste.
• Transfer the paste in a 100-ml volumetric flask and make up the volume to 100 ml
using Sharpe’s solution.
• Heat the flask on the water bath at 50–55 C for 1 h accompanied with intermittent
shaking.
• Filter the contents using Whatman No. 42 filter paper and collect the filtrate.
• Take 10 ml of this filtrate and transferred it to the Kjeldahl flask.
• The filtrate was then digested, distilled and titrated using a standard acid solution,
as to be done for the estimation of nitrogen or total protein content.
• Simultaneously run a blank test.
Calculation
%Soluble nitrogen ¼ ðA BÞ 1:4007 N 10 100=W
%Soluble protein ¼ %soluble nitrogen 6:38
A ¼ ml of acid consumed for titration of the sample

B ¼ ml of acid consumed for the titration of blank
W ¼ weight of the sample
N ¼ Normality of standard acid used
5.1.5 Total Ash Content
The ash content of cheese, channa, paneer, and khoa is determined by incinerating
the sample at 500–550 C for 4 h. Ashing or incineration decomposes the organic
matter present in the sample while the soluble inorganic salts (mineral constituents)
are left behind in the form of ash.
Procedure
• Weigh an empty silica crucible and note its weight.
• Weigh 1 g of the sample into it and record the weight.
• The sample is evaporated to dryness on a hot plate.
• Place the crucible in a muffle furnace at 550 C for 4 h or till the ash is free of
carbon.
• Transfer the crucible into the desiccator and cool at room temperature.
• Weigh the crucible and note its weight.
• Repeat the steps of heating and cooling to a constant weight (difference between
two successive weights should not be more than 0.1 g).
Calculation
Weight of silica crucible with sample ¼ W1

The total titratable acidity of cheese, channa, paneer, or khoa is measured by

neutralizing the developed acidity of a known quantity of sample with a standard
alkali solution using an indicator (phenolphthalein) and expressed as % lactic acid.
Procedure
• Take 2 g of the sample in a mortar and make a fine paste by mixing it with 20 ml
of distilled water.
• Transfer the paste into a white porcelain dish or glass beaker.
• Add a few drops of 1% phenolphthalein solution.
• Titrate the contents with 0.1 N NaOH solution till the appearance of slight pink
color (end point).
• Note the amount of the alkali consumed.
The titratable acidity is expressed in terms of as lactic acid equivalent per 100 g of
the product.
Calculation
Titratable Acidity ¼ 9 V 1 N=W
where
V1 ¼ Volume in ml of the standard NaOH solution required for titration
N ¼ Actual normality of the NaOH solution (0.1 N)
W ¼ Amount of sample taken in g
5.1.7 Salt Content
Salt is added to cheese to enhance taste and as a preservative. The determination of

salt is based on the reaction of silver nitrate with sodium chloride in the presence of
potassium chromate as an indicator. The formation of brick red color precipitates of
silver chromate indicates the end point.
Reaction
Procedure
• Weigh 2 g of cheese in a mortar and make a fine paste by mixing it with 20 ml of
distilled water.
• Transfer the contents into a conical flask.
• Add 1 ml of potassium chromate indicator (5%).
• Titrate it against N/10 silver nitrate solution till chocolate red color persists.
Calculation
Percent salt as NaCl ¼ V 5:844 N=M
V ¼ Volume in ml of silver nitrate used

W ¼ weight in g of cheese taken
N ¼ normality of silver nitrate
Derivation of the Formula

1 l of 1 N silver nitrate ¼ 1 l of 1 N sodium chloride ¼ 58.44 g of sodium chloride
1 ml of 1 N silver nitrate ¼ 1 ml of 1 N sodium chloride ¼ 0.05844 g of sodium
chloride
So, 1 ml of 1 N silver nitrate is required to react with 1 ml of sodium chloride
(0.05844 g)
Say, V ml of 1 N silver nitrate is used for M g of the sample
Then, V ml of 1 N silver nitrate neutralizes V 0.05844 g of sodium chloride.
M g of the sample contains ¼ 0.05844 V g of sodium chloride
1 g of the sample contains ¼ (0.05844 V )/M g of sodium chloride
100 g of sample contains ¼ (0.05844 V/M) 100 g of sodium chlo-
ride ¼ (5.844 V )/M
This is when the silver nitrate is of 1 N, if its normality is N, then the equation
becomes as
% salt (as NaCl) ¼ (5.844 V N )/M
5.1.8 Total Volatile Fatty Acids
The total volatile fatty acids in cheese are determined by the distillation of the sample
containing sulfuric acid and magnesium sulfate. The distillate is then titrated against
0.1 N NaOH to phenolphthalein end point.
Procedure
• Take 10 g cheese in a mortar and add 25 ml of 10% sulfuric acid. Mix the contents
to make a paste.
• Transfer the contents to a Kjeldahl flask and add 25 ml of 10% sulfuric acid in the
mortar and transfer it again to the Kjeldahl flask.
5.2 Sweetened/Sterilized Flavored Milk 93
• Add 35 g of MgSO4 along with 5–6 glass beads. Mix the flask thoroughly by
shaking.
• Add 250 ml of distilled water to it and subject the flask to steam distillation.
• Collect the distillate and take 280 ml of it. Titrate the obtained distillate with 0.1 N
NaOH using phenolphthalein indicator (A).
• Wash the condenser with 25 ml of neutralized ethyl alcohol and titrate the
washing with 0.1 N NaOH using phenolphthalein indicator (B).
• Add both the readings A and B.
Calculation
Volatile fatty acids ¼ ml of 0.1 N NaOH (A + B) consumed per 10 g of cheese.
5.1.9 Ripening Index
The ripening index of the cheese is calculated by calculating the total protein as
discussed in Sect. 5.1.3 and subtracting total soluble protein (discussed in Sect.
5.1.4) from it.
Ripening index ¼ Total protein content Total soluble protein
5.2 Sweetened/Sterilized Flavored Milk
The methodology for drawing the sample of sterilized or sweetened flavored milk is
discussed in Chap. 2 Sect. 2.9. The scale of sampling should also be followed in the
same manner as mentioned.
5.2.1 Fat Test
The fat content in sterilized or sweetened flavored milk is determined by Gerber or

Mojonnier/Rose-Gottlieb method. The principle and procedure for both the tests
have been discussed earlier in the Sect. 4.1.1.
5.2.2 Total Solids Content
For determination of total solids content of sweetened/sterilized milk refer Sect.

4.1.3.
The titratable acidity of flavored milk is difficult to determine as it contains added

color (milk free from added color, determination of titratable acidity is the same as
that for processed milk), so perception of a distinct end point is difficult. So, to
determine the acidity of sweetened flavored milk, potentiometric titration is carried
using a pH meter.
Procedure
• Calibrate the pH meter using buffer solutions of known pH or by standard buffer
solutions. The calibration should be done at atleast three different pH values.
• Pipette out 20 ml of the well-mixed sample in a beaker containing magnetic
stirrer bar.
• Place the beaker on the magnetic stirrer and start the titration using 0.1 N NaOH.
• Simultaneously dip the pH electrode in the milk. Continue the titration till the pH
of the sample reaches between 8.35 and 8.40.
• Record the ml of 0.1 N NaOH consumed.
Calculation
Titratable acidity ¼ 9 V 1 N=W
where
N ¼ Actual normality of the NaOH solution (0.1)
W ¼ volume of the sample taken
5.2.4 Turbidity Test
The efficiency of sterilization is checked by turbidity test. Heat labile milk proteins
like albumins are denatured after sterilization. The albumin present in milk gets
separated from casein on the addition of inorganic acids or salts. In this test, salts like
ammonium sulfate are first added to milk followed by its filtration. The filtrate thus
obtained is heated, cooled and observed for any signs of visible turbidity. Insufficient
heat-treated milk will develop turbidity due to the presence of undenatured albumin.
Properly sterilized milk will not show any turbidity due to the denaturation of the
albumin.
Procedure
• Measure 20 ml of milk into a conical flask and add 4 g of ammonium sulfate to it.
• Mix the contents of the flask properly to dissolve the ammonium sulfate.
• Leave the flask undisturbed for 5 min.
• Filter the contents of the flask using Whatman No. 12 filter paper in a test tube.
• Place the obtained filtrate on a boiling water bath for 5 min.
5.2 Sweetened/Sterilized Flavored Milk 95
• Cool the test tube in cold water and examine the contents for any visible turbidity.
Interpretation: Properly sterilized milk will not show any turbidity.
5.2.5 Sucrose Content
The estimation of sucrose content in sweetened flavored milk is estimated by

clarifying the sample with Carrez-I (zinc acetate solution) and Carrez-II (potassium
ferrocyanide solution). One part of the clarified sample is then treated with
hydrochloric acid for inversion and is used for estimation of the reducing sugars in
both inverted and non-inverted samples. The content of reducing sugar is estimated
by determining the volume of the unknown sugar solution required to completely
reduce the known volume of Fehling’s solution to form the red insoluble precipitates
of cuprous oxide.
Reagents Used
• Sodium hydroxide solution: 0.1 N.
• Stock solution of invert sugar: Weigh 9.5 g of pure sucrose and transfer it to a 1 l
volumetric flask by dissolving it in 100 ml of distilled water. Add 5 ml of
concentrated hydrochloric acid for inversion. Leave the solution undisturbed at
20–25 C for 3 days and then makeup to volume with water (This solution is
stable for several months).
• Standard solution of invert sugar: 40 ml of the stock solution of invert sugar is
neutralized with sodium hydroxide solution and diluted with water to 100 ml
volume.
• Methylene blue indicator solution: 0.2%.
• Fehling’s solution: Prepared by mixing equal volumes of Fehling’s solution A
and Fehling’s solution B, immediately before use (Fehling’s A and B are avail-
able as ready-made solutions).
• Standardization of Fehling’s Solution: Pipette 10 ml of both the Fehling’s
solutions (solution A and B) into a 250 ml Erlenmeyer flask. Titrate it with a
standard solution of invert sugar by running almost whole of the invert sugar
solution from the burette required to reduce the copper present in the Fehling
solution mixture so that not more than 1 ml is required to complete the titration.
Heating should be continued during the titration. Gently boil the contents for
2 min. Add about 1 ml of methylene blue indicator solution. As the contents of the
flask begin to boil again, add the standard invert sugar solution till the disappear-
ance of the blue color. The complete titration should be completed within 3 min
with continuous boiling.
• Carrez-I (Zinc acetate solution): Dissolve 21.9 g of crystalline zinc acetate (Zn
(CHCOO)22H2O) in water and add 3 ml of glacial acetic acid. Makeup to 100 ml.
• Carrez-II (Potassium ferrocyanide solution): 10.6%.
• Concentrated Hydrochloric acid solution: (Sp.gr. 1.16).
• Concentrated ammonia solution: (Sp.gr. 0.88).
• Dilute ammonia solution: 10%.

• Dilute acetic acid solution: 10%.
Procedure
1. Weigh 40 g of the thoroughly mixed sample in a 100 ml beaker. Add 50 ml of hot
water (80–90 C) to the sample. Mix it well and transfer it quantitatively into a
250-ml volumetric flask, add the successive washing in the flask with distilled
water (60 C) to a volume of 120–150 ml. Mix and cool to room temperature and
then add 5 ml of the dilute ammonia solution. Mix and leave undisturbed for
15 min. Neutralize the added ammonia by adding 5 ml of dilute acetic acid. Mix
well and add 12.5 ml of Carrez-I solution followed by 12.5 ml of Carrez-II
solution. Mix the contents and adjust the volume to 250 ml. Allow to settle and
filter. Mark this solution B-I.
2. Take 50 ml of the B-I solution into a 100-ml volumetric flask, add 5 ml of
concentrated hydrochloric acid and keep it in water bath at 65–68 C for 5 min
with mixing the flask for the first 3 min.
3. Cool the solution and neutralize with sodium hydroxide solution and adjust the
volume to 100 ml. Mark this solution A-I.
4. Dilute the solutions B-I (50 ml of B-I solution is diluted to 100 ml) and A-I (15 ml
of A-I solution is diluted to 100 ml). The solutions are diluted so that the volume
of the solution required to reduce the copper in 10 ml Fehling’s solution should be
between 15 and 50 ml. Mark them B-II and A-II, respectively.
Titration of the Diluted Samples

1. Take 50 ml of the solution (BII) in a burette. Add 5 ml of Fehling’s A and 5 ml of
Fehling’s B into a 250-ml conical flask and titrate with the solution from the
burette till the disappearance of the blue color (all the copper gets reduced at this
point).
2. Boil the contents of the flask for 2 min. Then add 1 ml of methylene blue indicator
solution. Again titrate the contents of the flask, by adding the prepared solution
from the burette until the disappearance of the blue color. The complete titration
should be completed within 3 min with continuous boiling.
3. Repeat the steps (1 and 2) using solution AII.
Calculation
Sucrose, percent by weight ¼ ½25 W 1 =W 2 ½2f2 =V 2 f1 =V 1
Original reducing sugar, percent by weight

¼ ½W 1 =V 1 250 ½100=W 2 ½1=1000 f1
Total reducing sugar ¼ sucrose þ original reducing sugar
where,
W1 ¼ weight in mg of sucrose required to reduce 10 ml of Fehling’s solution
W2 ¼ weight in g of the sample taken for analysis
5.3 Dahi, Yoghurt, and Srikhand 97
f2 ¼ dilution factor for solution A-II from A-I

V2 ¼ volume in ml of solution A-II required to reduce 10 ml of Fehling’s solution
f1 ¼ dilution factor for the solution B-II from B-I
V1 ¼ volume in ml of solution B-II required to reduce 10 ml of Fehling’s solution
5.3 Dahi, Yoghurt, and Srikhand
The sampling plan for dahi, yoghurt, or Srikhand has been discussed in Sect. 2.10.
The representative sample for the product to be analyzed should be taken by mixing
the product thoroughly using a spoon or spatula. The product should be mixed
properly so that the drawn sample attains required homogeneity.
The moisture or total solids content in fermented products like dahi, yoghurt, or
srikhand is determined by neutralization of the developed acidity followed by
evaporation of the moisture at 102 C.
Neutralization with Sodium Hydroxide (Method 1)
Procedure
• Weigh a clean and dry empty dish and record its weight.
• Add 4–5 g of the sample and weigh it.
• Add 1–2 drops of phenolphthalein indicator and titrate with 0.1 N NaOH till
phenolphthalein end point.
• Note the volume of NaOH consumed.
• Place the dish on a water bath and evaporate the water content.
• Clean the bottom of the dish and place it in a hot air oven at 102 C for 3 h.
• Remove the dish and cool it in a desiccator. Record its weight.
• Repeat the heating and cooling of the dish to a constant mass (difference not more
than 0.5 mg).
• Deduct half the volume of 0.1 N NaOH required to neutralize the developed
acidity from the weight of the residue left after drying.
Calculation
A ¼ N V 40=1000 2
Total solids ð%Þ ¼ 100ðW AÞ=w
N ¼ Normality of NaOH
W ¼ weight in g of the residue left after drying
w ¼ weight in g of the sample taken
A ¼ half the volume of 0.1 N NaOH added
Determination of Total Solids Using Zinc Oxide (Method 2)

The determination of total solids in the test sample involves the evaporation of the
water from the sample containing zinc oxide at 102 C. The lactic acid developed is
determined separately in other samples and is added to the moisture content deter-
mined after the drying of the sample.
Procedure
• The sample is drawn homogeneously from the container.
• Weigh the aluminum dish previously dried at 102 C containing 2 g of zinc oxide
and a stirring rod.
• Tilt the dish so that the zinc oxide moves to one side of it and add 1 g of the
sample to it. Weigh and record its weight.
• Add 5 ml of distilled water to the dish and mix the contents of the dish
thoroughly. The mixture is then spread evenly on the surface of the dish.
• The dish is then heated on a boiling water bath for 30 min, with continuous
stirring so as to facilitate the maximum evaporation of the water.
• Wipe the bottom of the dish and transfer it into the hot air oven for 3 h.
• Then remove the dish and cool it in a desiccator. Record the weight of the dish.
• Repeat the heating and cooling to a constant mass.
• Estimate the developed acidity (lactic acid) by titration in another sample by zinc
oxide.
Calculation
Total solids ð%Þ ¼ ½ðM 2 M Þ=ðM 1 M Þ 100 þ 0:1A
M ¼ mass of dish, containing zinc oxide and stirring rod (g)

M1 ¼ mass of dish containing sample, zinc oxide, and stirring rod (g)
M2 ¼ mass of the recorded after drying (g)
A ¼ mass of the lactic acid obtained in method 1
5.3.2 Fat Content
The fat content of the fermented products like dahi, yoghurt, and srikhand is
determined by Rose-Gottlieb or Mojonnier method. The principle of fat estimation
by this method is discussed earlier in Sect. 4.1.1 (the quantity of the sample to be
taken for the test should be 1–2 g).
The principle involved and the procedure for the estimation of protein content by the
Kjeldahl method have been discussed in Sect. 4.1.4.
5.3 Dahi, Yoghurt, and Srikhand 99
Titratable acidity of dahi, yoghurt, or srikhand plays an important role in determin-

ing the quality of the final product. The final acidity of sweet dahi should not be more
than 0.8% while that for sour dahi should not be more than 1.0%. The acidity of
yoghurt is kept slightly on a higher side when compared with sweet dahi which is
0.9%. The acidity of dahi or yoghurt attained after the incubation period/at the time
of transfer to cold store should be monitored closely. The acidity at which dahi is
transferred to cold storage is generally 0.6% while that for yoghurt is 0.75%. Any
fluctuation in the acidity at the time of shifting to the cold storage affects the product
quality. Transfer at a lower acidity than required leads to a product having a weak
body, while transfer at a higher acidity produces a product of higher acidity with sour
taste, wheying-off. The acidity is determined by titration of the developed acidity
(lactic acid) against a standard alkali using phenolphthalein as an indicator.
Procedure
• Weigh or pipette out 1 g of the sample in a beaker or conical flask.
• Add 10 ml of distilled water to make a paste.
• Add a few drops of 0.5% phenolphthalein indicator and titrate against 0.1 N
NaOH till the appearance of slight pink color.
Calculation
%Titratable acidity ðas lactic acidÞ ¼ 9 A N=W
A ¼ ml of 0.1 N NaOH required to neutralize the lactic acid

W ¼ weight in g of the sample taken
5.3.5 Diacetyl Content
Diacetyl is a typical flavor compound, which is produced during the fermentation

carried by lactic acid bacteria like Lactococcus lactis subsp. Diacetylactis. It is
produced from citric acid by the action of enzyme citratase that cleaves citrate ion
as follows:
Reaction
Citratase
Citrate ! Acetic acid þ oxalo acetic acid
Oxaloacetic decarboxylase
Oxaloacetic acid ! Pyruvic acid þ CO2
The pyruvic acid is then converted into diacetyl. The mother compound of
diacetyl is acetyl methyl carbinol. The diacetyl content in dahi or curd is measured
spectrophotometrically at 570 nm.
Procedure
• Take 1 g dahi sample in a stoppered test tube.
• 0.5 ml of 10% chromotropic acid is added followed by 5 ml of 98% sulfuric acid
in a drop wise manner.
• Vortex the tubes and heat them in a water bath at 100 C for 1 h.
• Cool the test tube and transfer 50 ml of the solution in a volumetric flask.
• The volume is adjusted using distilled water and filtered the solution through
Whatman No. 41 filter paper.
• Take 1 ml of the filtrate and transfer to 25 ml volumetric flask. Make up the
volume using distilled water.
• Prepare the standard curve for diacetyl by making the standard solutions in
distilled water (0.05, 0.1, 0.15, 0.2, and 0.25 mg/g). Give the same treatment to
the standard solutions as given to the dahi sample except for the dilution step.
• The absorbance of the sample and the standard solutions is measured at 570 nm.
• Interpolate the diacetyl content in the sample from the standard curve and apply
the sample dilution factor.
For estimation of the sucrose content in srikhand or yoghurt refer to Sect. 5.2.5.
5.4 Ice Cream
Ice cream is a frozen dairy product, which is a blend of milk solids with flavors,
color, and sugar with or without stabilizer. The incorporation of air in the ice cream
makes it different from other milk products.
The sampling procedure for ice cream has been discussed in Chap. 2, Sect. 2.11
and the sample for chemical analysis should be drawn accordingly based on the type
of packing.
5.4.1 Fat Content
The fat content in ice cream is determined with Rose-Gottlieb method, the principle
for estimation of fat by this method has been described earlier while the procedure is
discussed below:
Procedure
• Weigh 4–5 g of the drawn sample into the Mojonnier tube or fat extraction flask.
5.4 Ice Cream 101
• Add 10 ml of distilled water and mix the contents properly.

• Add 2 ml of ammonia, mix and heat in water bath at 60 C for 20 min with
shaking occasionally.
• Cool the contents and add the following reagents with proper mixing after adding
each reagent: 10 ml of ethanol, 25 ml of peroxide-free diethyl ether. Stopper the
flask and mix well for a minute.
• Leave the tube undisturbed so that the ethereal layer gets separated from aqueous
• Alternatively for Mojonnier method centrifuge the flask at low speed.
• Decant the ethereal layer into a previously weighed vessel containing 5–6 glass
• Repeat the extraction twice using 15 ml of diethyl ether and petroleum ether, so as
to extract the fat completely from the sample.
• The ether is evaporated by placing the vessel on a hot plate or water bath at
60–65 C.
• Place the vessel at 102 2 C for 3 h.
• Cool the dish in a desiccator and weigh.
• Repeat the steps of heating and cooling the vessel to a constant weight.
Calculation
after drying.
The total solids content in ice cream is determined by evaporating the water from the
sample at 102 C.
Procedure
• Weigh a clean and dry moisture dish containing sand and stirring rod, note the
recorded weight.
• Transfer 5 g of sample in the dish and add a few drops of distilled water. Mix well
the sample with the sand using the stirring rod.
• Place the dish on a boiling water bath or a hot plate to evaporate moisture from the
sample.
• Wipe the bottom of the dish and place it in hot air oven for 3 h maintained at
102 C.
• After 3 h remove the dish and cool in a desiccator. Record its weight.
• The heating and cooling of the dish should be repeated to a constant weight.
Calculation
Total Solid Content ¼ ðW 2 W Þ=ðW 1 W Þ 100
The principle for the estimation of protein content is described in Sect. 4.1.4.
Procedure
• Weigh 5–8 g of ice cream sample in a clean and dry Kjeldahl flask.
• Place the flask on a digestion heater and heat gently on a low flame till the initial
frothing ceases.
• During heating rotate the flask and continue the digestion for 1 h till the contents
become pale blue.
• Allow the liquid to cool and dilute with 200 ml of distilled water. Also, wash the
neck of the flask with distilled water.
• When the quantity of the distillate reaches around 150 ml stop the distillation.
• Titrate the boric acid solution containing trapped ammonia with standard HCl
by replacing ice cream with 5 ml water and 0.85 g of sucrose (nitrogen content not
more than 0.002% m/m).
5.4 Ice Cream 103
Calculation
Percent total nitrogen by weight ¼ ½1:4007 ðV s V b Þ N=W 100
where,
W ¼ mass of sample
6.38 (in case of ice cream or kulfi). For frozen dessert calculate the protein content as
N 6.25.
For estimation of sucrose in ice cream refer to Sect. 5.2.5.
5.4.5 Overrun in Ice Cream
Overrun is defined as the volume of the ice cream that is obtained in excess to the
volume of the mix. Overrun in ice cream is mainly due to the incorporation of air
during the freezing of the ice-cream mix. The amount of the air incorporated depends
on the composition of the mix and the way the mix is processed. The regulation of
the incorporation of air, in turn, regulates the body, texture, and palatability. If air is
incorporated in excess, it results in the snowy, fluffy, and unpalatable product while
too low air leads to a heavy and soggy product. For the determination of overrun in
ice cream, the volume of water and alcohol used corresponds to the volume of the air
contained in the ice cream.
The overrun is generally determined using two basic techniques, i.e., by weight
and by volume
%overrun ¼ ½ðvolume of ice creamÞ ðvolume of mixÞ=volume of mix 100
%overrun ¼ ½ðweight of unit volume of mixÞ ðweight of unit volume of

ice creamÞ=weight of unit volume of ice cream 100
Procedure
• Weigh the sample (i.e., cup or carton) of ice cream.
• Weigh and record the weight of a beaker.
• Add the frozen ice cream into the beaker and place the beaker on a water bath at
45 C to melt it.
• To it add 1–2 drops of amyl alcohol (anti-foaming agent) to remove the trapped
air. Mix the contents properly.
• Fill the cup or the carton with the melted ice cream free from air in the carton
or cup.
• Record the weight of the cup or carton (record the weight of empty cup or carton).
Calculation
%overrun ¼ ½ðW 3 W 2 Þ=ðW 2 W 1 Þ 100
W1 ¼ wt of empty cup or carton

W2 ¼ wt of carton or cup containing ice cream
W3 ¼ wt of empty carton or cup containing ice-cream mix free from air
5.4.6 Melting Test and Shape Retention of Ice Cream

(Arbuckle 2013)
Procedure
• The block of ice cream stored at 4 F is placed on a wire gauge (10 wires/inch)
and kept at 60 F. A Petri plate should be kept below the wire gauge to collect the
melted ice cream.
• The time for the collection of first 10 ml of the ice-cream liquid is noted.
• At every 10 min interval collect and note the volume of the liquid collected.
• Plot a graph between volume collected and time. Calculate the slope as meltdown
milliliters per hour.
• When the sample gets melted, i.e., after 4 h, the leftover material is examined for
the shape it has retained and to which degree, its appearance.
• The attributes are scored as excellent, good, fair, poor, or bad.
• In case when the melting liquid is being collected contains excess amount of
foam, the weight of the residue left should be recorded at the intervals.
• Taking the photographs of the residue mat also helps in assessing its melt
characteristics.
5.4.7 Alcohol Test for Protein Stability of Ice Cream Mix

(Arbuckle 2013)
This test indicates the protein stability of an ice cream mix.
Procedure
• Take 5 ml of the mix in a test tube.
• Add distilled water to it followed by alcohol (95%). The addition of water and
alcohol should be done starting with 9 ml water and 1 ml alcohol, then 8 ml water
and 2 ml alcohol in the next test tube (Volume of the water and alcohol added to
the mix should be 10 ml always).
• Mix the tube containing the lowest amount of alcohol by slowly inverting it three
times.
5.5 Evaporated and Sweetened Condensed Milk 105
• Continue the same process of mixing for each tube till visible coagulation is
visible on the wall of the test tube.
• Record the amount of alcohol required for the formation of slight precipitate.
• The stability of the protein is assessed as the amount of alcohol required to
precipitate the proteins, i.e., 2–3 ml poor, 4–5 fair, 6–7 good, over 7 excellent.
5.5 Evaporated and Sweetened Condensed Milk
The sample preparation of evaporated and sweetened condensed milk (SCM) for
chemical analysis is done by placing the container of the product in a water bath at
40 C. When the temperature of the container reaches around 40 C mix the contents
of the container by shaking it frequently by inverting it. Once the thorough mixing
has been achieved open the container’s lid and reincorporate the material that had
adhered to the lid while mixing back into the container. Using a spatula, mix the
contents thoroughly by stirring such that the top layer gets mixed with the lower
layer and vice versa. The mixing should be repeated every time a sample is drawn for
a chemical test.
5.5.1 Fat Content
The fat content in evaporated and SCM is determined by Rose-Gottlieb or Majonnier

method (please refer to the Sect. 4.1.1 for principle of the estimation). The procedure
for determination of fat is discussed below:
Procedure
• Weigh 2–2.5 g of a thoroughly mixed sample in a beaker and add 1 ml of distilled
water to make a paste by stirring with a glass rod.
• Add 9 ml of distilled water again and 1.25 ml of concentrated ammonia
(sp. gravity: 0.8974) and warm the contents on a water bath at 45 C.
• Transfer the contents to a fat extraction flask and mix properly.
• Cool the contents and add the following reagents with proper mixing after adding
each reagent: 10 ml of ethanol, 25 ml of peroxide free diethyl ether. Stopper the
flask and mix well for a minute.
• Alternatively for Mojonnier method centrifuge the flask at low speed.
60–65 C.
• Place the vessel at 102 2 C for 3 h.
• Repeat the steps of heating and cooling the vessel to a constant weight.
Calculation
A ¼ weight of extracted fat i.e. difference in weight of vessel before drying and
after drying.
The total solids content in evaporated or SCM is determined by evaporating the

water from the sample at 102 C.
Procedure
• Weigh a clean and dry moisture dish containing sand and stirring rod, note the
recorded weight.
• Transfer 5 g of sample in the dish and add a few drops of distilled water. Mix well
the sample with the sand using the stirring rod.
• Place the dish on a boiling water bath or a hot plate to evaporate moisture from the
sample.
• Wipe the bottom of the dish and place it in hot air oven for 3 h maintained at
102 C.
• After 3 h remove the dish and cool it to the room temperature in a desiccator.
Record its weight.
• The heating and cooling of the dish should be repeated to a constant mass (two
successive weights should not differ by more than 0.5 mg).
Calculation

5.5 Evaporated and Sweetened Condensed Milk 107
The sucrose content in evaporated or SCM is estimated as per the procedure

mentioned in Sect. 5.2.5.
Determination of titratable acidity of evaporated/SCM is based on neutralization

reaction between an acid, i.e., lactic acid and an alkali like sodium hydroxide.
Procedure
• Weigh 10 g of well-mixed sample in a conical flask or beaker.
• Add 30 ml of warm distilled water to it so as to dilute the concentrated milk.
• Add few drops of phenolphthalein indicator and titrate it against 0.1 N NaOH to
Calculation
%titratable acidity ðlactic acidÞ ¼ 9 A N=W
A ¼ ml of 0.1 N NaOH required to neutralize the lactic acid

W ¼ weight in g of the sample taken
The protein content in evaporated/SCM is determined by Kjeldahl method and is

expressed as the mass fraction of protein to the mass fraction of solids not fat. The
principle for protein estimation by Kjeldahl method is discussed in Sect. 4.1.4, while
the detailed procedure is discussed below:
Procedure
• Weigh 1–2 g of milk in a clean and dry Kjeldahl flask.
• Place the flask on a digestion heater and heat gently to boil until the contents clear.
Continue digestion for another 2 h.
• Allow the liquid to cool and dilute with 300–500 ml of distilled water. Also, wash
the neck of the flask with distilled water.
• Stop the distillation when the quantity of the distillate reaches around 150 ml.
• Titrate the boric acid solution containing trapped ammonia with standard HCl
by replacing milk with 5 ml water and 0.85 g of sucrose (nitrogen content not
more than 0.002% m/m).
Calculation
where,
W ¼ mass of sample
To express the protein content on the basis of milk solids not fat divide the protein
content (%) with solids not fat (%).
5.6 Ghee, Butter-Oil, and Anhydrous Butter-Oil
5.6.1 Moisture Test
• Weigh 10 g of the sample in a moisture dish previously dried and weighed.

• Place the dish in a hot air oven maintained at 105 C for 1 h.
• Remove the dish from the oven, cool it in the desiccator to room temperature.
• Record the weight. Repeat the heating and cooling till a constant weight (two
successive weights should not differ by 1 mg) is obtained.
Calculation
5.6 Ghee, Butter-Oil, and Anhydrous Butter-Oil 109
Weight of aluminum dish with sample after drying ¼ W2
Moisture content ¼ ðW 1 W 2 Þ=ðW 1 W Þ 100
5.6.2 Free Fatty Acid
The acidity of a fat rich product like ghee is measured as the extent of hydrolysis
liberating the free fatty acids from the triglyceride molecule and is expressed as %
oleic acid. The free fatty acid (FFA) content of fresh ghee ranges from 0.09 to 0.28%.
The sensory acceptability of ghee deteriorates as the free fatty acid content increases.
Ghee which is made from cream held at higher temperatures for a longer time or
from butter having higher curd content or stored at cold storage for a period of more
than 1 year shows a higher value of free fatty acid content. This is because of the
hydrolytic rancidity occurring in the base material, i.e., butter or cream thus releasing
free fatty acids due to lipase action (indigenous or microbial).
Ghee with a higher value of free fatty acid indicates faster rate of oxidation, thus
having a low keeping quality. The free fatty acid in ghee is estimated by titrating the
fatty acids with a standard alkali using phenolphthalein as an indicator.
RCOOH þ NaOH ! RCOONa þ H2 O
• Pipette out 10 g of ghee in a 250-ml conical flask.

• Boil 50 ml of neutralized ethanol (titrated with 0.1 N NaOH using phenolphtha-
lein as an indicator) in another conical flask.
• 50 ml of the boiled and neutralized ethanol is added to the flask containing ghee
sample and mix properly.
• Heat the contents to boil and titrate while hot with 0.1 N NaOH.
• Stop the titration till the pink color persists for 15 s.
Calculation
Free fatty acid ð%oleic acidÞ ¼ ðT N=M Þ 28:2
T ¼ volume in ml of 0.1 N NaOH required for titration

M ¼ mass in g of ghee sample taken
N ¼ normality of NaOH used

1 l of 1 N NaOH ¼ 1 l of 1 N oleic acid ¼ 282 g of oleic acid
1 ml of 1 N NaOH ¼ 1 ml of 1 N oleic acid ¼ 0.282 g of oleic acid
So, 1 ml of 1 N NaOH is required for neutralization of 1 ml of oleic acid (0.282 g)
Say, T ml of 1 N NaOH is used for M g of the sample
Then T ml of 1 N NaOH neutralizes T 0.282 g of oleic acid
M g of the sample contains ¼ 0.282 T g of oleic acid
1 g of the sample contains ¼ (0.282 T)/M g of oleic acid

100 g of sample contains ¼ (0.282 T/M ) 100 g of oleic acid ¼ (28.2 T )/M
This is when the NaOH is of 1 N, if its normality is N, then the equation becomes
as
% FFA (oleic acid) ¼ (28.2 T N )/M
5.6.3 Acid Value
The acid value is equivalent to the number of ml of KOH required to neutralize the
free fatty acids present in 1 g of ghee sample. It is estimated in a similar way as free
fatty acids except using 0.1 N KOH instead of 0.1 N NaOH.
Calculation
Acid value ¼ ðT=M Þ 5:61
T ¼ volume in ml of 0.1 N KOH required for titration

M ¼ mass in g of ghee sample taken
5.6.4 Reichert-Meissel (RM) Value
The RM value is equivalent to the ml of 0.1 N alkali (NaOH) required to neutralize

the steam volatile and water soluble fatty acids that have been distilled from 5 g of
ghee under precise conditions.
The RM value is a measure of the short-chain fatty acids like butyric acid (C4:0)
and caproic acid (C6:0). Butyric acid contributes predominantly to the RM value, as
it contributes to about three-fourth of the RM while caproic acid contributes to the
other one-fourth. As the short-chain fatty acids are predominantly found in milk fat,
hence RM value helps to indicate the purity of the ghee. The RM value for milk fat or
ghee must be minimum 28, which is highest among all types of fats or oils because of
presence of butyric acid (which is principal fatty acid to milk fat). The reduction in
the RM value of ghee from 28 indicates the adulteration of ghee with extraneous fat.
This value becomes less than 28 when animals are fed on cottonseed. The effects of
the seasonal variation have not shown any deviation in the RM value for pure ghee.
The principle for RM value estimation is that ghee is saponified using glycerol
and sodium hydroxide. Once the saponification is completed, the mixture is diluted
with water and acidified with dilute sulfuric acid followed by steam distillation. The
conditions during the steam distillation should be adjusted such that the quantity of
the distillate collected after 19–21 min should be 110 ml. 100 ml of the distillate is
then titrated against 0.1 N NaOH using phenolphthalein indicator.
Saponification Reaction
Addition of Sulfuric Acid After Saponification

RCOONa þ H2 SO4 ! Na2 SO4 þ RCOOH
Titration of the Distillate with Alkali

RCOOH þ NaOH ! RCOONa þ H2 O
Apparatus
1. Measuring cylinders
2. Pipette: 50 ml
3. Flat bottom boiling flask (Polenske): The glass used for making the flask should
be heat resistant, having the dimensions mentioned in Table 5.1, see Fig. 5.1.
4. Still head: It should be made of glass tubing having a wall thickness of
1.25 0.25 mm and should have the dimensions as mentioned in Table 5.2,
also see Fig. 5.2.
A rubber stopper should be fitted below the bulb of the longer arm of the
still head.
5. Condenser: It should be made of glass and should have the dimensions as
6. The receiving volumetric flask should have two graduation marks, one at 100 ml
and the other at 110 ml.
7. Gas burner.
8. Asbestos board.
Table 5.1 Dimensions of Total volume till bottom of the neck 310 10 ml
the flat bottom flask
Internal diameter of the neck 75 5 mm
Overall height 21 1 mm
Diameter of the base 45 5 mm
Fig. 5.1 RM apparatus
Table 5.2 Dimension of the still head

A 180 5 mm
B 107.5 2.5 mm
C 80 5 mm
D 70 5 mm
E 20 2 mm
F 4 1 mm
G (outer diameter of the bulb) 37.5 2.5 mm
Internal diameter of the tubing 8 0.5 mm
Angle made between the slope of the still head and the vertical 60 2
Fig. 5.2 Still head used for RM estimation
Table 5.3 Dimensions of the condenser

Overall length 520 5 mm
Length of water jacket 300 5 mm
Length of widened part above water jacket 70 10 mm
Wall thickness of widened part 1.25 0.25 mm
Internal diameter of widened part 20 1 mm
External diameter of inner tube within water jacket 12 0.5 mm
Wall thickness of inner tube 1.0 0.2 mm
Wall thickness of outer jacket 1.25 0.25 mm
External diameter of water jacket 30 2 mm
Reagents
1. Dilute Sulfuric acid: 25 ml of concentrated sulfuric acid is diluted to 1:1 with
distilled water. 40 ml of the dilute sulfuric acid solution should neutralize 2 ml of
50% sodium hydroxide solution.
2. Glycerol—98% (w/w).
3. Standard NaOH—0.1 N.
4. Phenolphthalein indicator—0.5% solution in 95% (v/v) ethyl alcohol.

5. Ethyl alcohol—95% (v/v) neutralized to phenolphthalein immediately before use.
Procedure
• Weigh exactly 5.00 0.01 g of ghee in round bottom flask.
• Add 20 g of glycerol (by weight) and 2 ml of 50% NaOH (w/w) solution into
round bottom flask containing ghee.
• Immediately heat the flask on the flame for saponification to get a clear liquid. The
mixture should be mixed continuously. The solution should not be overheated.
• Measure 93 ml of boiled, cooled distilled water (boiled for 15 min), and then add
to the saponified solution of ghee, before it gets solidified. The water should be
added slowly from the sides of the flask.
• The saponification should be repeated, if the solution is not transparent or darker
than light yellow.
• Add two glass beads, followed by 50 ml of dil. sulfuric acid. Attach the distilla-
tion apparatus to the round bottom flask.
• The flask is heated gently till all the insoluble acids get melted. Then increase the
flame and distill 110 ml in volumetric flask in between 19 and 21 min maintaining
the temperature of distillate between 18 and 21 C.
• Remove the flame, when 110 ml of the distillate gets collected. Then place the
volumetric flask in cold water.
• Once the contents of the volumetric flask get cooled, dry the flask from outside
after removing it from cold water.
• Invert the flask 4–5 times without violent shaking and filter the distillate with
Whatman No. 4 filter paper.
• Collect 100 ml of the filtrate in a dry volumetric flask, cork it and retain it for
titration. Pour 100 ml of filtrate into a titration flask, add few drops of phenol-
phthalein indicator and titrate against 0.1 N standardized NaOH solution for light
pink color end point.
• Carry a blank test without using ghee.
Calculation
RM value ¼ 1:1 ðA BÞ
A ¼ volume in ml of 0.1 N NaOH required for titration of the sample

B ¼ volume in ml of 0.1 N NaOH required for titration of the blank
The steam volatile and water soluble fatty acids are present in the 110 ml of the
filtrate. We take 100 ml of this filtrate to calculate the water soluble and steam
volatile fatty acids, so the amount of these fatty acids in 100 ml of the filtrate will be
110/100, i.e., 1.1.
5.6.5 Polenske Value
The Polenske value is equivalent to the ml of 0.1 N alkali (NaOH) required to

neutralize the steam volatile and water-insoluble fatty acids that have been distilled
from 5 g of ghee under precise conditions.
It is a measure of caprylic (C8:0) and capric (C10:0) fatty acids in the milk fat.
Out of the total Polenske value, C8:0 contributes to one-fourth of it while C10:0
contributes three-fourth of it. The Polenske value for milk fat or ghee ranges from 1.2
to 2.4. Increase in Polenske value indicates adulteration of milk fat with coconut oil
or an oil rich in capric or caprylic fatty acids. The determination of Polenske value of
ghee is done after the distillate obtained during the RM value determination.
Procedure
• The condenser, still head should be washed with 15 ml of cold distilled water for
three times. Each washing should pass into the 110 ml flask through the cylinder,
the filter, and the funnel. Discard the washings.
• Now wash the condenser, filter paper, cylinder three times with 15 ml of
neutralized ethanol in 100 ml volumetric flask. The collection of the washings
in the 110-ml volumetric flask should be used for titration for estimation of
Polenske value, as the washings contained the insoluble volatile fatty acids.
• Titration is carried using sodium hydroxide to the phenolphthalein end point.
Calculation
Polenske value ¼ C D
C ¼ volume in ml of 0.1 N NaOH required for titration of the alcoholic washings

D ¼ volume in ml of 0.1 N NaOH required for titration of the blank
Note: It has been observed that pressure affects the RM and Polenske values.
When the analysis is done at low barometric pressures like at high altitudes, the RM
and Polenske values are found to be less. In such conditions the correction should be
applied as follows:
Corrected RM value ¼ ½ðObserved value 10Þ log 760= log P þ 10
Corrected Polenske value ¼ Observed value ½760 45=P 45
P ¼ barometric pressure in mm of mercury at the place and time of analysis

The maximum deviation between duplicate readings of a sample should not
exceed more than 0.5 units for RM and 0.3 units for Polenske value.
5.6.6 Peroxide Value
Peroxide value is the measure of the oxidative rancidity in ghee and is equivalent to
the ml of 0.002 N sodium thiosulfate per gram of the sample or as milliequivalents of
peroxide oxygen per kg of the sample.
Fats and oils get deteriorated mostly due to rancidity that develops due to the
oxidation of the fat. Developed rancidity in a ghee sample affects its flavor and color.
The oxidative stability of ghee gets deteriorated when the unsaturated fatty acids get
oxidized by the oxygen molecule. Generally, the singlet oxygen molecule being the
more reactive species attacks the α-carbon to the double bond, i.e., addition of
oxygen molecule on the methylene group adjacent to the double bond leading to
the formation of peroxides and hydroperoxides which are flavourless, tasteless and
odourless. These hydroperoxides being unstable in nature get converted into
aldehydes, ketones, and alcohols (secondary oxidation products), which impart
flavor to the ghee. Peroxide value is the indicator of the primary oxidation products,
i.e., the peroxides and hydroperoxides.
These hydroperoxides are determined iodometrically, as under acidic conditions
the peroxides liberate free iodine from the potassium iodide. The amount of the
liberated iodine is proportionate to the amount of the peroxides present in the sample.
The peroxide value determines the developed peroxides but if even the peroxides
produced are lower than the detection limit of this test, at that level also they can greatly
affect the flavor of the oil or fat. As the oxidation of the fat continues, the peroxide
value increases and then decreases after a certain period of time. This is because,
peroxide value determines the hydroperoxides formed, once all the double bonds get
saturated with oxygen to form hydroperoxides, the formed hydroperoxides being
unstable get decomposed thus giving a lesser amount of peroxide value. So to interpret
the results of this test, the ghee (fat/oil) sample must be tested on sensory basis.
Fresh ghee should have a peroxide value of zero. Table 5.4 shows the peroxide
values of different grades of ghee as per IS:3508.
Reaction
Table 5.4 Classification Peroxide value Grade

of ghee according to
<1.5 Very good
peroxide value (IS: 3508)
1.6–2.0 Good
2.1–2.5 Fair
2.6–3.5 Poor
3.6–4.0 Unacceptable
• Weigh 5 g of fat/ghee in a 250-ml glass stoppered Erlenmeyer flask and add 1 g of

powdered potassium iodide.
• Add 20 ml of solvent mixture (2 parts of glacial acetic acid and 1 part of
chloroform). Swirl the flask to dissolve the sample.
• Boil the flask for 30 s in boiling water bath.
• Close the opening using a glass rod when the vapors begin to escape.
• Cool immediately under the tap water and add 20 ml of 5% aqueous potassium
iodide solution and 25–30 ml of distilled water twice.
• Titrate the solution with sodium thiosulfate using starch indicator. Perform a
blank test. The amount of sodium thiosulfate consumed for bank should not
exceed 0.1 ml.
Calculation
Peroxide value ðmilliequivalent per 1000 g of fat=oilÞ ¼ 8 1000 TV N=W
TV ¼ ml of sodium thiosulfate consumed (subtracted from blank)

N ¼ normality of sodium thiosulfate (0.002 N)
W ¼ weight of ghee
The peroxide value is expressed in terms of milliequivalents of oxygen per kg of
the sample, so the factor of 8 is used, as the equivalent weight of oxygen is 8.
5.6.7 Saponification Number
The saponification number is the number of milligrams of potassium hydroxide

required to saponify 1 g of fat. This number indicates the average molecular weight
of the fatty acid present. For milk fat this value ranges between 210 and 233 which is
more than other fats or oils except than that of coconut and palm kernel oils. This
value helps in the detection of mineral oils like paraffin oil as they do not form soap
when acted upon by alkali. The higher the number of short-chain fatty acids per gm
of fat the higher will be the saponification value and the higher the number of long-
chain fatty acids per gm of fat lower will be the saponification value. The saponifi-
cation equivalent is calculated by dividing 56,100 with saponification value.
Procedure
• Weigh accurately 2 0.001 g ghee into a conical flask.
• Add 25 ml solution of alcoholic KOH.
• Add 1 or 2 glass beads and reflux condenser for 30–60 min, with stirring the
contents of the flask at regular intervals.
• The hot solution is titrated with 0.5 N HCl, using 0.5 ml of 1% phenolphthalein
indicator to determine the excess of alkali.
Calculation
Saponification value ¼ 28:05ðB AÞ=W
A ¼ volume of 0.5 N HCl consumed in titration of the sample

B ¼ volume of 0.5 N HCl consumed in titration of the blank
W ¼ weight of the sample taken for analysis
To calculate saponification number divide 56,100 by saponification value.
5.6.8 Iodine Number
It is the number of grams of iodine absorbed by 100 g of fat under the specified
conditions. Iodine number indicates the degree of unsaturation present in the fat. For
milk fat this value ranges from 26 to 35 that is lower than other fats or oils. The
iodine value of free acids is higher than the triglyceride molecule.
This is generally determined by IBr (Hanus method) or ICl (Wij method). One
molecule of the halogen is absorbed by each unsaturated bond present.
The organic solvent like carbon tetrachloride or chloroform is added to solubilize
the fat while addition of water facilitates the transfer of the fat into the organic
solvent while iodine monochloride gets solubilized with water getting converted into
I2. The potassium iodide converts the excess of iodine monochloride into free iodine
(blue in color), which when titrated with sodium thiosulfate becomes colorless.
Reagents
1. Potassium iodide solution—10% freshly prepared.
2. Starch solution—Dissolve 5 g of starch in 30 ml cold water and add it slowly into
the 1 l of boiling water. Boil the solution for 3 min. Cool the solution and decant
off the supernatant clear liquid.
3. Standard sodium thiosulfate solution—0.1 N. Standardize it against potassium
dichromate before use.
4. Iodine Crystals—resublimed.
5. Acetic acid—glacial 99%.
6. Potassium dichromate—Weigh about 5 g of potassium dichromate (previously
dried at 105 2 C) into a 1 l volumetric flask and store in a dark place.
7. Wijs’ iodine monochloride solution: Dissolve 10 ml of iodine monochloride in
about 1.8 l of glacial acetic acid and mix vigorously. Take 5 ml of this solution
and add 10 ml of potassium iodide solution and titrate with 0.1 N standard sodium
thiosulfate solution, using starch as an indicator. Adjust the volume till the
solution is 0.2 N.
8. Carbon tetrachloride (CCl4) or chloroform—inert to Wijs’ solution.
Procedure
• Weigh 0.4–0.45 g of clear ghee in a clean dried conical flask.
• The fat is dissolved in 15 ml of CCl4. Then add 25 ml of Wijs’ reagent. Stopper

the flask and mix properly. Leave the flask undisturbed for 1 h in the dark.
• Add 20 ml of potassium iodide solution, immediately add 150 ml of distilled
water and swirl slowly to mix the contents of the flask.
• Titrate with 0.1 N sodium thiosulfate solution till the disappearance of yellow-
brown color.
• Add 1 ml of the starch solution and titrate until the blue color disappears. Carry
out a blank test, using the same quantities of the reagents.
Calculation
Iodine value ¼ 12:69ðX Y Þ N=W
where,
X ¼ volume in ml of 0.1 N sodium thiosulfate consumed for blank
Y ¼ volume in ml of 0.1 N sodium thiosulfate consumed for sample
W ¼ weight of ghee taken
N ¼ normality of sodium thiosulfate

1 l of 1 N sodium thiosulfate ¼ 127 g of iodine (the equivalent weight of iodine is
126.9)
1 ml of 1 N sodium thiosulfate ¼ 0.1269 g of iodine
Say Y is the ml of 1 N sodium thiosulfate is used for W g of the sample
Then Y ml of 1 N sodium thiosulfate ¼ 0.1269 Y g of iodine
W g of the sample contains ¼ 0.1269 Y g of iodine
1 g of the sample contains ¼ (0.1269 Y)/W g of iodine
100 g of the sample contains ¼ (0.1269 Y/W ) 100 g of iodine ¼ (12.69 Y/
W) g of iodine
This is when the sodium thiosulfate is of 1 N, if its normality is N, then the
equation becomes as: Iodine value ¼ 12.69 Y N/W
5.6.9 Butyro Refractometer (BR) Reading
When a ray or beam of light travels from one medium to another, there occurs
bending in its normal path. This bending occurs due to the difference in the speed of
light in different mediums. The bending of the light ray can be towards or away from
the normal path (when a light ray travels from rarer to denser medium it bends
toward the normal, while it bends away from it when travels from denser to rarer).
Refractive index (RI) is defined as the ratio of the velocity of light in vacuum to the
velocity of light in the medium. It is generally given by Snell’s law, i.e., the ratio of
sine of angle of incidence to the sine of angle of refraction. This property of
refraction is used to detect the purity of ghee. When a light ray of known wavelength
(generally 589.3 mμ of the D-sodium line is used) travels from air into ghee, it gives
Fig. 5.3 Butyro

refractometer
a certain value of refraction for ghee. This value is called RI and is measured using
Abbe refractometer or can be expressed as butyro refractometer (BR) reading
measured by a butyro refractometer (Fig. 5.3). The RI for milk fat falls between
1.4527 and 1.4566 while in terms of BR reading it ranges between 40 and 43. The RI
for milk fat is low in comparison to other fats and oils because of the presence of a
higher number of saturated and short-chain fatty acids. A direct relation exists
between RI and the degree of unsaturation and the chain length of the fatty acids.
The BR reading for vegetable fat or oils is more than 50 except for coconut (38–39)
and palm oil (39–40) which have short-chain fatty acids. Animal body fats have a
BR in the range 44–51. The BR measurement for ghee is done at 40 C so that the
ghee is sufficiently clear and transparent for light to pass through it.
Procedure
• Start on the butyro refractometer and set the temperature of the water bath at
40 C.
• When the temperature reaches 40 C, place a drop of oil of known BR value on
the prism and calibrate the instrument if any deviation in the BR reading is
observed.
• Melt and filter the ghee with Whatman No. 4 filter paper.
• Clean the prism of the instrument with ethyl alcohol and then place a drop of the
filtered ghee sample on the prism. Make sure that the drop covers the prism
completely.
• Close the prism and let the temperature of the water bath to reach 40 C.
• Observe the line on the scale from the lens of the instrument. The line coinciding
with the value on the scale of the instrument is the BR reading.
Conversion of RI into BR reading
BR reading ¼ 42 þ Factor ðObserved RI 1:4538Þ
Observed RI Factor
1.45–1.4515 1400
1.4515–1.4530 1410
1.4530–1.4545 1420
1.4545–1.4560 1430
1.4560–1.4575 1440
Conversion of BR reading into RI
RI ¼ 1:4538 þ Factor ðObserved BR 42Þ
Observed BR reading Factor

37.5–40 0.00072
40–42.5 0.00071
42.5–45 0.00070
45–47.5 0.00069
If the temperature at the time of analysis is not 40 C, then a value “A” is added or
subtracted from the observed BR reading. If temperature is more, then A is added for
each degree more than 40 C while on the other hand A is subtracted for each degree
below 40 C. Table 5.5 shows the BR reading and their corresponding RI values
calculated using the above formula.
A ¼ 0:55
Table 5.5 Butyro refractometer readings and their corresponding refractive indices
BR reading RI BR reading RI BR reading RI
35.0 1.4488 40.5 1.4527 46.0 1.4565
35.5 1.4491 41.0 1.4531 46.5 1.4569
36.0 1.4495 41.5 1.4534 47.0 1.4572
36.5 1.4499 42.0 1.4538 47.5 1.4576
37.0 1.4502 42.5 1.4541 48.0 1.4579
37.5 1.4506 43.0 1.4545 48.5 1.4583
38.0 1.4509 43.5 1.4548 49.0 1.4586
38.5 1.4513 44.0 1.4552 49.5 1.4590
39.0 1.4517 44.5 1.4555 50.0 1.4593
39.5 1.4520 45.0 1.4558
40.0 1.4524 45.5 1.4562
5.6.10 Unsaponifiable Matter and Cholesterol Content
The unsaponifiable matter (USM) includes lipids substances naturally occurring like
sterols, higher aliphatic alcohols, vitamins, and hydrocarbons. The USM is not able
to form soap when treated by an alkali, i.e., USM does not undergo saponification
reaction. In milk, the principal USM is cholesterol, while vitamins A, D, E, and K,
traces of squalene and n-alkyl methyl ketones containing odd numbers of carbon
from C3 to C15 also forms the part of USM.
The USM is determined by reacting the triglycerides with a strong alkali to
produce glycerol, free fatty acids, and USM. Since USM does not get saponified
so they are extracted using organic solvent. The organic solvent containing the
extracted USM may contain traces of the alkali, which is removed by washing it
with water and then drying the washed solvent to a constant mass to get the USM.
Procedure
• Take 5 g of ghee or fat in a flat bottom flask and add 50 ml of alcoholic potassium
hydroxide solution (0.5 N) along with 3–4 glass beads and mix the contents.
• Reflux the contents of the flask with swirling the contents in between. The heating
should be continued for at least 1 h from the initiation of the boiling.
• On the completion of saponification, transfer the contents in a separating flask or
funnel containing 150 ml of distilled water.
• Add 100 ml of diethyl ether, stopper the flask and mix the contents vigorously
with the intermittent release of the vapors.
• Leave the funnel undisturbed in its stand for the separation of two layers.
• The soapy layer is collected in a beaker while the ethereal layer is transferred to
the other separating funnel containing 20 ml water.
• Extract the alcoholic soap solution with 50 ml of diethyl ether twice and collect
the ethereal layers in the separating funnel containing 20 ml distilled water.
• Add 20 ml of water in the ethereal layer and shake vigorously.
• The ethereal layer is washed repeatedly with 0.5 N alcoholic KOH, 20 ml distilled
water to remove the soap (the presence of soap in the wash water is checked by
adding few drops of phenolphthalein indicator. Development of pink color
indicates the presence of soap).
• Once the complete washing is accomplished, transfer the washed ethereal layer in
a pre-weighed conical flask and evaporate the solvent at 60 C on a water bath or
hot plate.
• Transfer the conical flask in the oven at 80 C for 1 h, cool the flask in a desiccator
and record its weight.
• Repeat the heating and cooling steps to a constant weight.
Calculation
USM in ghee ð%Þ ¼ ðW 2 W Þ=ðW 1 W Þ 100
Weight of empty conical flask ¼ W

Weight of conical flask with sample ¼ W1

Weight of conical flask with sample after drying ¼ W2
Cholesterol Content
The cholesterol content is determined by treating it with sulfuric acid. Cholesterol
gets converted into persulfate cholesterol which being unstable gets converted into
its dehydrated product in the presence of sulfuric acid to form cholesterol
3,5-dienoid or cholesterol 2,4-dienoid that is purple in color. The intensity of the
purple color produced is proportional to the cholesterol present and is determined at
650 nm. Acetic acid is used to stabilize the color formed as it starts to fade as the time
progresses. The starting material for cholesterol estimation is the residue left after
drying while determining the USM content.
Liebermann Burchard Reagent 1 ml of concentrated sulfuric acid, add 20 ml

acetic anhydride (AR Grade) and mix the contents in cold conditions. Keep the
solution at 0 C for 30 min.
Procedure
• Dissolve the residue obtained after drying in acetic acid and transfer it in a 25-ml
volumetric flask.
• Make up the volume to 25 ml and filter the solution with Whatman No. 1 filter
paper.
• Take 3 ml of the filtrate in a 25-ml volumetric flask and add 4 ml of Liebermann
Burchard Reagent (LB reagent).
• Leave the flask for 35 min at 25 C till the development of the color.
• Record the absorbance at 650 nm against a blank made from 3 ml acetic acid and
4 ml LB reagent.
• Also prepare a standard curve for different concentrations of cholesterol by
making a stock solution of cholesterol (0.5%) in acetic acid.
• The preparation of the working solution of cholesterol is made by taking 1 ml of
the stock solution in a 25-ml volumetric flask and volume made up of acetic acid.
• The cholesterol solution of different concentrations should be taken as mentioned
below:
Blank Tube number

Reagents (ml) 1 2 3 4 5 6 7 Sample
Cholesterol working solution 0 0.5 1.0 1.5 2.0 2.5 3.0 –
Diluted USM solution – – – – – – – 3
Acetic acid 3 2.5 2 1.5 1 0.5 0 –
LB reagent 4 4 4 4 4 4 4 4
Keep at 25 C for 35 min
Record OD at 650 nm
Calculation
Standard stock solution is 0.5% cholesterol in acetic acid (500 mg cholesterol/100 ml
acetic acid or 5 mg/1 ml or 5000 μg/1 ml acetic acid).
As we have 1 ml of the stock solution which is diluted to 25 ml with acetic acid,
So, 25 ml of working solution contains 5000 μg cholesterol
1 ml of working solution contains 200 μg cholesterol
0.5 ml of working solution contains 100 μg cholesterol
From the standard curve, 3 ml of diluted USM solution contains ¼ X μg
cholesterol
25 ml of diluted USM contains ¼ 25 X/3 μg cholesterol
Amount of ghee sample taken ¼ W g
W g ghee contains ¼ 25 X/3 W μg cholesterol
So, 100 g of ghee contains ¼ (25 X/3 W ) 100 μg cholesterol
5.6.11 Antioxidants in Ghee
Antioxidants are compounds which can donate hydrogen atom to any free radical
like fatty acid radical, fatty peroxy radical. They break or terminate the free radical
involving reactions by donating the hydrogen molecule. The natural antioxidants
which are present in milk are vitamin E, vitamin C, superoxidase dismutase,
carotenoids, and thiol groups while some synthetic antioxidants like BHA, BHT,
TBHQ, and propyl gallates are added to ghee to prevent the autoxidation of fat and
maintain the quality of the product. They are classified as primary antioxidants,
chelating agents, scavengers, or antioxidant regenerators. The synthetic antioxidants
are compounds generally contain a benzene ring in their structure along with a
hydroxyl group, i.e., they are ortho- or para-substituted phenols. Their use in dairy
products is prohibited in most countries, so their detection is important. The easiest
method to detect antioxidants is by using Ehrlich reagent.
Ehrlich reagent: 0.5% solution of sodium nitrite in distilled water and 0.5%
solution of sulphanilic acid in distilled water containing 50% conc. HCl. The nitrate
and the sulphanilic solution are added in 1:100 ratio on each working day to perform
the test.
Procedure
• Take 1 ml of melted ghee or fat sample in a test tube and add 2 ml of 72% ethyl
alcohol.
• Shake the test tube and add 1 ml of Ehrlich reagent followed by the addition of
1 ml of 1 N NaOH solution.
• Observe the color developed in the test tube.
Interpretation
Red-purple color indicates the presence of BHA and shows maximum absorption at
535 nm.
Salmon pink color indicates the presence of BHT and gives maximum absor-
bance at 505 nm.
Propyl gallates show yellow color that fades rapidly and show no maxima in the
visible region. Other classes of gallates show the same behavior.
Wine red color that changes rapidly to brown and brown yellow shows the
presence of NDGA.
5.6.12 Adulterants in Ghee
Ghee being a costlier product is generally adulterated with cheaper fat or oil from
other sources. Generally, vegetable fat, animal body fat, or mineral oils are added to
ghee which makes their detection important. The general basis on which these
adulterants are detected in ghee relies on certain specific differences in the chemical
composition or certain properties of milk fat and the adulterant.
5.6.12.1 Vanaspati in Ghee (Baudouin Test)

As per FSSA, 2006, it is mandatory to add 5% sesame (Til) oil in vanaspati for its
detection. Sesamolin, a constituent of sesame oil is degraded by mineral acid, i.e.,
hydrochloric acid and sesamol are released in its free form. Some sesamol is also
liberated during hydrogenation of vanaspati containing sesame oil. The sesamol on
condensation with furfural produces a characteristic red color.
Procedure
• In a test tube take about 5 g of melted fat.
• Add 5 ml of concentrated HCl (AR grade).
• Add 0.4 ml furfural solution (2% in alcohol) and vortex the tube for 2 min.
• Leave the tube undisturbed for separation.
• In presence of vanaspati, pink or red color will get developed in the test tube.
• Confirm by adding 5 ml distilled water and shake again.
• Persistence of the color in the acid layer, confirms presence of vanaspati. If the
color disappears, vanaspati it is absent.
5.6.12.2 Mineral Oil

Ghee can be adulterated using cheaper mineral oils like petroleum jelly, paraffin oil,
liquid paraffin (heavy and light), and fuel oils because of the huge difference in the
price. Mineral oils are referred to as white oils, which are nonedible and have
different viscosity and refractive indices in comparison with milk fat due to certain
compositional differences. Thus, as per food regulations, addition of such nonedible
oils to ghee or any type of edible oils and fats is not only an unlawful and unethical
practice, but can also pose a serious health hazard(s). The detection of mineral oil in
edible fat and oils is based on the fact that they resist saponification as with ghee or
other edible fats and oils. Mineral oils, which contain varying fractions of long-chain
hydrocarbons, occurring mainly in earth, do not get saponified by alkali and are also
non-utilizable as human food. This forms the basis for the appearance of turbidity
due to the presence of minerals oils in other saponified oils and fats.
Table 5.6 Opacity time for pure ghee and adulterated ghee
Type of ghee Opacity time (min)
Pure buffalo ghee 14–15
Pure cow ghee 18–19
Ghee from cotton tract area 11–12
Buffalo ghee containing 10% vanaspati 10–11
Buffalo ghee containing 10% pig body fat 8–9
Buffalo ghee containing 10% buffalo body fat 2–3
Buffalo ghee containing 10% cow body fat 3–4
Buffalo ghee containing refined oil 20–25
Procedure
• Pipette out 1 ml of ghee or clear fat in a test tube.
• Add 25 ml of 0.5 N alcoholic KOH solution and reflux the contents until the
solution becomes clear.
• Transfer the solution into the tube cylinder and add 25 ml of boiled distilled water
and mix.
• Appearance of turbidity indicates adulteration with mineral oil while for pure
ghee the developed turbidity will disappear after some time.
5.6.12.3 Animal Body Fat

The presence of animal or vegetable oils in ghee can be done by the opacity test.
Procedure
• 5 g of heat clarified ghee sample is melted at 50 + 1 C in a test tube and maintain
for 3 min to equilibrate.
• Then transfer the test tube in a water bath maintained at 23 + 0.2 C and record the
opacity time (Time taken by the fat sample to acquire absorbance between 0.14
and 0.16 at 570 nm or the Klett reading should be between 58 and 62 at 100%
transmittance).
• Lesser opacity time indicates the presence of animal body fat and a higher value
of opacity test indicates adulteration with vegetable oils (Table 5.6).
Vegetable Oils Can Also Be Detected by Modified Bieber Test

• Take 1 ml of melted ghee and dissolve in 1.5 ml of hexane.
• Add 1 ml of Bieber reagent (distilled water, sulfuric acid, and nitric acid in
20:6:14 ratio).
• Shake vigorously and leave undisturbed till the two layers get separated.
• The appearance of distinct orange-yellow color in the upper layer is taken as the
criteria for a positive Bieber test.
• This method can detect vegetable oils up to 5–7% in ghee.
5.6.12.4 Phytosterol Test

This test is done to detect the presence of vegetable fat in ghee. Vegetable fat
contains phytosterol as a major sterol and the sterol content in the sample is
determined gravimetrically after saponifying the fat and precipitating the sterols by
alcoholic digitonin solution.
Procedure
• Weigh 10 g of the ghee in a conical flask and add 10 ml of KOH solution to it
followed by addition of 20 ml of 95% ethyl alcohol.
• Add 2–3 glass beads, connect the air-cooled condenser to the conical flask and
heat the contents on the boiling water bath till the solution becomes clear,
continue boiling for 30 min.
• Add 60 ml of water and 180 ml of 95% ethyl alcohol and maintain the tempera-
ture at 40 C.
• Add 30 ml of alcoholic digitonin solution (1%) and shake well.
• Cool the contents in a refrigerator for 12 h.
• Filter the contents of the conical flask using Whatman No. 1 filter paper in a
Buchner funnel.
• Wash the precipitate with cold water (5 C) until the foaming stops.
• Again wash with 25 ml of ethyl alcohol and 25 ml of diethyl ether.
• Dry the filter paper containing the precipitate in an oven at 102 C for 10–15 min.
• Collect the precipitate and note its weight.
Calculation
Sterol content ð%Þ ¼ ½ð0:25 AÞ=W 100
A ¼ weight of the residue (precipitate)

B ¼ weight of the sample
5.6.12.5 Detection of Vegetable Oil in Ghee by Reversed Phase

High-Performance Liquid Chromatography (RP-HPLC)
(Rani et al. 2016)
The detection of vegetable oil in ghee is based on the difference in the type of sterol
present in ghee and vegetable oil. Ghee is obtained from animal source so it contains
cholesterol as a major sterol while vegetable oils contain β-sitosterol as a major
sterol. These two sterols have different retention times when analyzed using
RP-HPLC, which forms the basis of this test (the marker sterol to differentiate
between ghee and vegetable oil is β-sitosterol). Cholesterol elutes at
17.7 0.2 min while β-sitosterol elutes at 22.7 0.2 min.
Extraction of USM from Ghee

• One gram of ghee sample is weighed in a screw-capped test tube.
• Add 25 ml of 5% methanolic KOH to it.
• Place the test tube at 90 C for 50 min with regular vigorous shaking.
• Add 5 ml of water and 15 ml of hexane to the test tube and vortex for 1 min.
Table 5.7 Analytical Column C 18

conditions for HPLC
Column temperature 30 C
Mobile phase Acetonitrile: Isopropanol (9:1, v/v)
Flow rate 1.5 ml/min
Elution type Isocratic
Run time of analysis 30 min
Detector wavelength 205 nm
• Centrifuge the tube at 3000 rpm for 5 min.

• The supernatant was taken and dried to obtain the USM.
• Dissolve the dried USM in 0.3 ml of chloroform and make up the volume to
0.5 ml with methanol.
• Filter the sample using 0.22 μm filter paper (Millipore) and use it for RP-HPLC
analysis.
• Also, prepare the standard for cholesterol and β-sitosterol of 1 mg/ml concentra-
tion (Table 5.7).
Procedure
• Inject 20 μl of the prepared USM sample and compare it with the retention time of
the sterols with the reference standards of cholesterol and β-sitosterol.
• Appearance of the peak of β-sitosterol at 22.7 0.2 min in the sample indicates
the presence of vegetable oil in ghee.
• This method can detect vegetable oil at 1–5% level in ghee (5% for coconut oil,
1% refined soyabean oil, 2% refined sunflower, and groundnut oil in ghee).
5.6.13 Fatty Acid Analysis of Ghee/Butter Oil/Anhydrous Butter Oil
Principle
The milk fat is a complex mixture of around 400 different fatty acids, which makes it
one of the most complex natural fat. The fatty acid composition of milk fat is
determined as the methyl esters of fatty acids by gas liquid chromatography. The
fatty acid methyl esters to be analyzed using the gas chromatography are prepared by
base catalysis reaction in which the fatty acids in the glyceride molecule undergo
methanolysis in a nonalcoholic solution. The reaction mixture is then neutralized to
stop the methanolysis by addition of crystalline sodium hydrogen sulfate. This
neutralization is done to prevent the saponification of the formed esters. The fatty
acid composition of milk fat (as grams of individual free acids) per 100 g of total
fatty acids (free acids) is expressed as a mass fraction in percent.
Reagents
• Methanol: Should not contain more than 0.5% moisture by mass.
• Solvent: n-hexane.
• Sodium hydrogen sulfate monohydrate.
• Potassium hydroxide or sodium methoxide (2 M): They act as transesterification

reagent. Dissolve 10.8 g of sodium methoxide or 11.2 g of potassium hydroxide
in methanol and mix well. Alternatively, 4.6 g of sodium can be dissolved in
methanol to form the methanolic solution of sodium methoxide.
Procedure for Preparation of Fatty Acid Methyl Esters (ISO 15884: 2002)
• Filter the melted fat using Whatman No. 1 filter paper to remove any particle or
debris from it.
• Take a 15-ml centrifuge tube and weigh to the nearest 5 mg, 100 mg of the
prepared sample.
• Add 5 ml of hexane to the centrifuge tube and mix the contents well.
• Add 0.2 ml of the KOH or sodium methoxide reagent and cap the centrifuge tube.
• Vortex the contents of the tube for 1 min.
• Add 0.5 g of sodium hydrogen sulfate after 5 min from vortexing.
• Mix the contents well and centrifuge the tubes at 350 g for 3 min at room
temperature.
• Take 1.5 ml of the supernatant obtained after centrifugation in the vials for GC
analysis.
• The prepared aliquot can also be stored in a deep freezer for several days or also at
room temperature by decanting the ester solution. Care must be taken to avoid the
loss of the methyl esters as they are volatile in nature.
Procedure for Analysis by GC (ISO 15885: 2002)
Reagents
• Reference milk fat for quantification: Use the milk fat whose fatty acid composi-
tion is known.
• Solvent: n-hexane.
• Carrier gas: Hydrogen, Nitrogen or helium having a purity of 99.999%, the
oxygen content should be less than 2 106.
Apparatus
• The gas liquid chromatography consists of the following:
(a) Injector: The injectors should be of the vaporizing type and be maintained at a
temperature of 220 C (minimum). If the column is a cold on-column injector
type, the injector should be maintained at a temperature few degrees below the
boiling point of the solvent while if the injector is a programmed temperature
injector (PTV) type, the final temperature should be at least 220 C.
(b) Column oven: Capable of operating at temperatures ranging from ambient to
260 C.
(c) Column: The column should be made of narrow or large bore glass or silica
capillary column.
5.7 Unsalted Butter and Table Butter 131
(d) Flame ionization detector (FID): The detector should be able to withstand the
operating temperature maintained up to 20 C above the column oven
temperature.
(e) Carrier gas pneumatics: It should be able to maintain the column head pressure
to maintain a linear gas velocity, oxygen-diffusion proof and should contain
flow control to provide the required flow rates. When the vaporizing split
injector is used, the split vent flow should be controlled to get a split ratio of
1:50 to 1:100.
(f) Injection syringe: Capacity of 10 μl or auto-injector type as required.
Procedure
1. Switch on the instrument and set the injection port temperature at least at 240 C.
2. Set the FID detector temperature at 20 C over the column temperature.
3. Set the oven temperature to 100 C followed by a gradual rise in the temperature
per min to 240 C.
4. Switch on the carrier gas supply and set its flow rate.
5. As the temperature of the FID is reached, switch on the fuel gas supply.
6. Run n-hexane through the column for cleaning it till a stable baseline is
obtained.
7. Now inject the sample (fatty acid methyl ester) and start the analysis.
8. Similarly, run the standards for the known fatty acid methyl esters.
9. The peaks obtained for the sample is compared with the peak obtained for the
standard.
10. Calculate the area of the peak and express the relative percentage of fatty acid in
the sample.
5.7 Unsalted Butter and Table Butter
The procedure and technique for drawing the sample of butter for chemical analysis
are discussed in Sect. 2.14.
5.7.1 Moisture Content
• Weigh a clean and dry moisture dish containing a stirring rod, note the weight.
• Transfer 5 g of sample in the dish and place the dish on a boiling water bath or a
hot plate to evaporate moisture from the sample.
• Wipe the bottom of the dish and place it in a hot air oven for 1 h maintained at
102 C.
• After 1 h remove the dish and cool it in a desiccator.
• Record the weight.
• The heating and cooling of the dish should be repeated to a constant mass
(difference between two successive weights not more than 0.5 mg).
Calculation
Weight of aluminum dish with sample after drying ¼ W2
5.7.2 Fat and Curd Content
The fat content in butter is determined by separating the by petroleum ether and
evaporating the ether while the residue left after separating the fat is used for
determination of curd content while in case of table butter, the salt content is also
determined separately to calculate the curd content.
Procedure
• Take 10 g of well-mixed butter sample in the moisture dish and add 25 ml of
petroleum ether to it.
• Mix the contents well and transfer the ethereal layer into a previously weighed
conical flask containing 5–6 glass beads (to prevent bumping).
• Repeat the extraction with petroleum ether twice and decant the ethereal layer into
the flask.
• Keep the flask on the hot plate or water bath at 55–60 C to evaporate the ether.
• Then transfer the flask into a hot air oven at 100 for 30 min.
• Cool the flask in the desiccator and record its weight.
• Repeat the heating and cooling to a constant weight differing not more than
0.1 mg.
• Preserve the residue for the estimation of salt.
Calculation
Fat% ¼ ½ðW wÞ=m 100
w ¼ empty weight of conical flask with glass beads

W ¼ weight of flask containing residue after drying
m ¼ weight of sample
Determination of Curd Content

• The residue left in the moisture dish (after decanting the ethereal layer) during the
estimation of fat in butter is used for the estimation of curd content.
• The moisture dish is placed in a hot air oven at 100 C for 30 min, cooled to room
temperature in a desiccator and weighed to a constant weight.
5.7 Unsalted Butter and Table Butter 133
Calculation
%curd ¼ ½ðW wÞ=m 100
w ¼ weight of empty moisture dish

W ¼ weight of moisture dish + residue after drying
m ¼ weight of sample
5.7.3 Salt Content
Salt is added to table butter to enhance its taste and has a preservative effect. Salt
content in butter is determined by Mohr’s method.
Mohr’s Method
The determination of salt is based on the reaction of silver nitrate with sodium
chloride in the presence of potassium chromate as an indicator. The formation of
brick red color precipitates of silver chromate indicates the end point.
Procedure
• Weigh 5 g of cheese in a conical flask and add 100 ml of boiling distilled water to
it. Mix the contents and leave it undisturbed for 5–10 min with swirling in
between.
• Cool the contents to 50–55 C and add 2 ml of potassium chromate indicator (5%)
and mix well.
• Add 0.25 g of calcium carbonate and mix.
• Titrate it against N/10 silver nitrate solution till brown-red color persists.
• Also, carry out a blank test without the butter sample.
Calculation
Percent salt as NaCl ¼ ðV vÞ 5:844 N=M
V ¼ Volume in ml of silver nitrate used for sample

v ¼ volume in ml of silver nitrate used for blank
W ¼ weight in g of butter taken
N ¼ normality of silver nitrate
Note: The curd content for table butter can also be calculated as % curd ¼ 100 (%
fat + % salt + % moisture).
5.7.4 Test to Distinguish Between Annatto (Natural Color) and Azo

Dye (Synthetic Color) in Butter (IS:3507)
FSSAI has permitted the use of natural color like annatto in table butter, which gives
a faint yellow color to the product. The color annatto is extracted from the seeds of
the plant called as Bixa orellana. The color that is extracted from the seeds by
dissolving them in neutral oil is yellowish red in color. But instead of using this
natural color, people add harmful and cheap synthetic artificial azo dye to the
product (examples of some azo dyes: Methyl Red, Methyl Blue, Orange G,
Tartrazine, Sudan yellow 3G, Sudan yellow FCF, Ponceau S, etc.). The test for the
detection of the presence of azo dye in butter is based on the principle that annatto
being a conjugated diene (alternate double bond), develops different color with
different reagents, while the azo dye that has the functional group R – N ¼ N R1
(where R and R1 are alkyl or aryl groups) develop a different color with different
reagents, i.e., the azo dye in the acidic medium gives pink to wine red color while
under basic conditions it shows no color. This development of color in the acidic
medium is the basis of the differentiation between annatto and azo dye.
Reagents
• 2% NaOH solution
• 10% NaOH solution
• 40% stannous chloride solution 40%, containing a sufficient amount of HCl to
make the solution acidic and a small piece of tin to keep it in reduced form
• Hydrochloric acid (1:1)
Procedure
Method 1
• Filter the melted butterfat and take 2 ml of it in two different test tubes.
• Add 2 ml diethyl ether in both the test tubes separately.
• Mix the tubes well.
• Add 2 ml of 10% NaOH solution to one test tube and 2 ml of HCl (1:1) in the
other test tube.
• If the azo dye is present, the acid solution will show pink to wine red color while
the alkaline solution develops no color.
• While annatto or any vegetable color does not develop any pink or wine red color
in acid solution and the basic solution remains yellow.
Method 2
• Mix the butter with 5 ml of warm 2% NaOH solution and pour it on the moistened
filter paper.
• On washing the filter paper with water, if it remains dyed straw yellow, it shows
the presence of annatto.
5.8 Milk Powder 135
• Dry the filter paper and add two drops of stannous chloride to it.
• Dry it again, development of purple color confirms the presence of annatto.
5.8 Milk Powder
Milk powder is classified as whole milk powder and skim milk powder. The powder
is generally prepared in the flush season when the quantity of milk is surplus. The
moisture content of the milk is evaporated by spray drying and the powder is stored.
The sampling for powder has been discussed in Chap. 2 Sect. 2.13.
5.8.1 Fat Content
The fat content of milk powder is determined by Rose-Gottlieb method.
Procedure
• Weigh 1 g of the sample in a Mojonnier flask and mix it with 10 ml of warm water
(65 C) so as to dissolve the powder to get milky consistency.
• Add 1.25 ml of concentrated ammonia (sp. gravity 0.8974) and mix well.
• Then add the following reagents with proper mixing after adding each reagent:
well for a minute.
• Alternatively for Mojonnier method, centrifuge the flask at low speed.
60–65 C.
• Place the vessel at 102 2 C for at least 2 h.
• Repeat the steps of heating and cooling to a constant weight.
Calculation
after drying.
The total solids in the product can be calculated by evaporating the moisture/water
from a known quantity of sample to dryness at 102 2 C.
Procedure
• Weigh 3 g of the sample in the previously dried and weighed dish and record the
weight.
• Place the dish in a hot air oven maintained at 102 2 C for 3 h.
• Cool the dish in the desiccator at room temperature.
• Weigh the dish and note the weight.
• Repeat the heating and cooling till the difference between two successive weights
is not more than by 0.5 mg. Record the weight as final weight.
Calculation
The protein content in milk powder is determined by the Kjeldahl method and is
done in a similar way as that for estimation of protein content in milk except for the
amount of sample taken which is 0.5 g.
5.8.4 Total Ash Content
• Weigh an empty silica crucible and note its weight.

• Weigh 3 g of milk powder sample into it and record the weight.
• The crucible is heated on a burner or a hot plate first till the contents of the
crucible turn black.
• Place the crucible in a muffle furnace at 550 C for 3 h or till the ash is free of
carbon.
• Transfer the crucible into the desiccator and cool at room temperature.
5.8 Milk Powder 137
• Weigh the crucible and note its weight.

• Repeat the above steps until the difference between two successive weights is not
more than 0.1 g.
Calculation
Weight of silica crucible with milk powder sample ¼ W1
5.8.5 Acid Insoluble Ash
• The acid insoluble ash is measured by treating the residue left after ashing with a
dilute acid. It is a measure of the silicates present in the product.
• Add 25 ml of dilute HCl (5 N) to the residue obtained after ashing and cover the
crucible with a watch glass.
• Heat the crucible on a water bath for 10 min.
• Cool and filter the contents of the crucible using Whatman No. 42 filter paper.
• Wash the filter paper with distilled water until the washings are free from acid
(check using 0.1 N silver nitrate solution, washings do not form white ppt with
silver nitrate).
• Keep the washed filter paper in the crucible and place it in a hot air oven
maintained at 102 C for 3 h.
• Then transfer it to the muffle furnace for 1 h.
• Cool the crucible in a desiccator and record its weight.
• Repeat the heating and cooling to a constant weight (difference between two
successive weights should not exceed 1 mg).
Calculation
Acid insoluble ash ð%by wtÞ ¼ ½ðM 2 M Þ=ðM 1 M Þ 100
M ¼ weight in g, of the empty crucible

M1 ¼ weight in g, of the crucible with the residue before drying
M2 ¼ weight in g, of the crucible after drying containing acid insoluble ash
5.8.6 Total Carbohydrate (in Infant and Weaning Foods)
The total carbohydrate in infant foods is calculated by difference. The fat, moisture,
protein, and ash are determined as per the procedure discussed and are subtracted
from 100, to get the total carbohydrates like sucrose, dextrose, lactose, and maltose.
Calculation
Total carbohydrate ð%Þ ¼ 100 ða þ b þ c þ dÞ
a ¼ percent fat
b ¼ percent protein
c ¼ percent ash
d ¼ percent moisture
It is measured by titration of a known amount of sample with a standard alkali

solution using an indicator (phenolphthalein) and is expressed as % lactic acid.
Procedure
• Reconstitute 10 g of SMP or 13 g of WMP in distilled water (24 C) and adjust the
volume to 100 ml.
• Mix well to dissolve the powder and leave it undisturbed for 15 min for the foam
to settle.
• Pipette 10 ml of the reconstituted sample into a white porcelain dish or glass
beaker.
• Add 1% phenolphthalein indicator solution and titrate with N/10 NaOH solution
till appearance of light pink color (end point).
• Note the amount of alkali consumed.
The titratable acidity is expressed as lactic acid equivalent per 100 ml of milk.
where
N ¼ Actual normality of the NaOH solution (0.1 N)
5.8.8 Reconstitution Properties
5.8.8.1 Bulk Density

It is expressed as weight per unit volume of the powder (g/ml), it is the mass of the
milk powder occupying a known volume. The bulk density depends on particle
density, arrangement of particles in the container, particle internal porosity, size and
shape of the particles, the interstitial air pockets in the powder, and within the
powder particles (occluded air). Bulk density of the powder can be controlled by
amount of total solids in the feed, viscosity of the concentrate, drying temperature,
particle size distribution, agglomeration of the powder, concentrate feed flow rate,
5.8 Milk Powder 139
atomization pressure, and inlet air temperature. As the concentration of the total
solids in the feed increases, the bulk density of the powders also increases because of
a decrease in the entrapped air, same is the effect of viscosity and the feed flow rate.
Bulk density is classified into four types.
Compact Density The density obtained after compressing the powder bulk mass by
mechanical forces like vibration and impact.
Tap Density The density of the powder obtained after tapping a known quantity of
powder.
Loose Bulk Density It is the density which is measured when the powder is poured
freely into the container.
Aerated bulk density It is done for agglomerated or fluidized powder. The bulk
density of such aerated powders is atleast about 40–60% less than the normal
powders.
Bulk density is an important factor which determines the handling, transportation,
and packing cost for a powder. Bulk density of skim milk powder ranges from 0.3 to
0.6 g/ml (for a powder of good quality it should be 0.5–0.6 g/ml) while bulk density
for whole milk powder is slightly less than SMP.
The true densities of SMP and WMP (whole milk powder) are 1.44–1.48 and
1.26–1.32 g/ml, respectively.
• In a measuring cylinder weigh 25 g of milk powder and note the volume.

• Tap the cylinder 50 times so as to allow the packing of powder.
• Note the volume of the powder again.
• Calculate the loose and Bulk (packed) densities of powder sample.
Calculation
Loose density ¼ weight of powder=volume of powder
Bulk density ¼ mass of powder after tapping=volume of powder after tapping
5.8.8.2 Solubility Index

It is the amount of powder that is insoluble in water and is expressed as the volume of
the sediment obtained in milliliters. The solubility index of spray-dried powder
should not be more than 2 ml while roller dried milk should be no more than
15 ml. It depends on the chemical composition of powder and its physical state.
Factors such as the presence of lactic acid, heat treatment of milk, type of drying,
level of salts and concentration of solids in the feed affect the solubility index. The
insolubility of powder is due to the unfolding of beta-lactoglobulin and its complex-
ation with casein especially κ-casein. Reduction of the outlet temperature improves
solubility of powder. The outlet temperature should not be more than 90 C while a
higher inlet temperature increases the solubility index. Increasing the concentrate
feed flow rate and atomization pressure also reduce the solubility index.
The relationship between the speed of the centrifuge and the diameter of the cup it
accommodates is mentioned below:
Diameter (cm) Speed (rpm)

25.4 1074
30.5 980
35.5 909
40.6 848
45.7 800
50.8 750
55.9 724
61.0 695
The diameter of head, is the distance between the inside bottoms of opposite cups
measured through the center of rotation of the centrifuge head while the cups are
horizontally extended.
Procedure
• Reconstitute 13 g of whole milk powder or 10 g of skim milk powder in distilled
water to 100 ml.
• Leave the reconstituted milk for removal of the foam.
• Transfer the milk into 50 ml graduated centrifuge tubes and centrifuge at
3000 rpm for 5 min.
• Remove the supernatant and add water up to the mark.
• Again centrifuge the contents for 5 min.
• Note the volume of the sediment settled below.
5.8.8.3 Scorched Particles

These are the burnt powder particles that are produced when the excessive heat
treatment is done during the drying stage. Also, if the product is left in the drying
chamber for longer periods or incorporation of the sweeping (powder particles stuck
to the walls of the drier) leads to scorched particles.
Procedure
• Take 25 g of skim milk powder or 32.5 g of whole milk powder and mix in 250 ml
of warm water.
• Mix well and ensure the powder gets dissolved.
• Filter the reconstituted milk through cotton disc using an aspirator. Rinse the
blender with 50 ml of water and pass through the filter disk.
• Remove the disc and dry it at 35–45 C.
• Compare the disk with the standard print provided.
5.8 Milk Powder 141
• The disk that shows more scorched particles than the standard disk A but less than
the disk B should be assigned grade B and should be interpreted similarly for
other disks also.
5.8.8.4 Dispersibility
The ability of powder to be distributed in water by separating into individual
particles. It is processed in which the lumps and aggregates fall apart in water.
International Dairy Federation considers dispersibility as the single best criteria to
access the reconstitution ability of powder. It is expressed as the percentage of the
solids dissolved in water. The dispersibility of SMP is more than that of WMP which
is 90% vis-à-vis 85%. To get a powder of good dispersibility the manufacturing
conditions should be adjusted such that the powder particles should have a size of
30–50 micron. Large particles or agglomerated powder show better dispersibility
while small particles show clumping and flocculate. A free fat content of less than
20% shows good dispersibility which increases with a decrease in free fat content.
Holding of the concentrate at higher temperatures before drying, high protein
content of the dried milk powder, decrease particle size, and packaging at
temperatures more than 76 C affects dispersibility adversely (Mathur et al.
2008; De 2009).
Procedure
• Take 400 ml of distilled water (40 C) and add 52 g of powder in it.
• Mix the contents using a stirrer for 20 s.
• Pass the milk through a 72-mesh screen and collect it in a beaker.
• Make up the volume to 500 ml.
• Take 10 ml of this sample in a pre-weighed dish and evaporate the moisture in a
hot air oven.
• Cool the dish in a desiccator and weigh the leftover solids.
• Multiply the weight of the leftover solids with 50 and express the dispersibility
in gm.
5.8.8.5 Flowability
It is the ability of the powder particle to flow freely with respect to each other and
forms an angle along with the base diameter. It is expressed as an angle of repose and
is determined as the tangent angle between the height of pile to its radius (Mathur
et al. 2008; De 2009). Flowability is the measure of free flow of a powder and is
important from the manufacturer’s point of view with respect to handling, proper
packaging, and measurement. It is an important factor in designing the machinery so
as to ensure the proper flow of powder and avoiding formation of clogs. For a
powder to have good flowability it should have large agglomerates and fewer fine
particles. Spherical, smooth, and high-density particles tend to show better flow
characteristics. Powders having a high-fat content have less flowability than powders
having less fat because fat has the tendency to break the motion of the particles by
melting them into an adhesive, rubbery, and viscous liquid generally at high
temperatures employed during powder manufacturing (Sharma et al. 2012). Free-
flowing agents like silicates of sodium or calcium salts can also be added at 0.5–2%
to improve the flowability of powder.
Procedure
• Take 100 g of powder and pass it through a glass funnel placed 2 cm above a
40 40 cm plain paper sheet. The powder should be poured in a fine stream.
• Note the height of the cone, once all the powder passes.
• Draw a boundary line around the radius of the cone.
• Weigh the paper and calculate the radius from the weight per unit area of paper
and the area of circular base.
• Divide the radius of the cone by the height of the pile.
• Express the flowability as the cotangent angle of the angle of repose determined
through the tangent.
• Flowability of powder is directly proportional to the angle of repose made by it.
Cotangent angle ¼ cone radius=cone height
5.8.8.6 Wettability
The ability of powder particles to absorb water on its surface, to be wetted and to
penetrate the water into its particles. It takes place when the water gets penetrated by
capillary action into the powder particles. As the wettability is influenced by the
particle size of the powder, sampling must be done carefully to minimize the particle
breakdown. The sample containers must be completely filled and agitation of the
powder during sampling or transport must be avoided (Sharma et al. 2012).
Density, particle porosity, surface charge, surface area, and the presence of
amphipathic substances affects the wettability of powder. A powder having large
and irregularly shaped particles wets faster than the small and symmetrical particles
as they have close packing which does not allow the water to rise by capillary action
efficiently. Lactose addition to powder increases (up to a total conc. of 40–50%) the
hygroscopicity of powder and improves the wettability while fat has a negative
impact on the wettability. Lecithinization of powder also improves the wettability.
Reducing the nozzle pressure/increasing the orifice size, increasing the viscosity or
total solids (30–40% solids concentration) in the feed, homogenization of the feed
before spray drying, decreasing the outlet temperature or reducing the speed of the
atomizer improves the wettability of powder. Agglomeration largely plays an
important part in improving the wettability of powder. SMP wetted in less than
15 s is referred to as instant powder while WMP should get wetted in 30–60 s.
Procedure
• Weigh 10 g of powder sample and take 250 ml of distilled water (25 C) into a
500 ml beaker.
• Ensure that inner walls of the beaker above the water level remain dry.
• Place a steel plate on the top of the beaker such that one edge of the plate is close
to the rim of the beaker.
• Spread the powder sample evenly on the surface of the glass plate.
5.8 Milk Powder 143
• Start the stopwatch and withdraw the glass plate with one hand such that the
powder sample falls freely and progressively within 2.5 s on the surface of the
water.
• Record the time from the beginning of withdrawal of the glass plate from the top
of the beaker until all the particles of the powder get wet.
• Carry out the measurements in duplicate.
5.8.8.7 Sinkability
It is the ability of the powder particles to penetrate into the surface tension of the
water. It is closely related to the wettability. Agglomerated powder has better
sinkability than normal spray-dried powder as agglomeration increases the weight
of the powder particles. Agglomerated SMP has better sinkability than agglomerated
WMP. Sinkability is expressed as the milligram of powder sinking per minute per
cm2 (Mathur et al. 2008; De 2009).
Procedure
• Weigh 10 mg of powder in a weighing boat.
• Add 3.5 ml of distilled water in a spectrophotometer cuvette.
• Adjust the wavelength of the spectrophotometer at 760 nm.
• Place the cuvette in the spectrophotometer.
• Dust the powder sample on the surface of distilled water contained in the cuvette.
• Measure the percent transmission for 6 min at an interval of 2 min.
• The curve obtained gives the sinkability characteristics of the powder.
5.8.8.8 Particle Size

It is related to the appearance, reconstitution property and the flow behavior of the
powder. Processing conditions, quality of initial milk, type of drying treatment
employed to influence the particle size. The spray-dried particles are spherical in
shape having size of 10–250 μm while the particles of agglomerated powder are
large and irregular. Powder having larger particles show better reconstitution
properties like dispersibility (Mathur et al. 2008; De 2009; Sharma et al. 2012).
Procedure
• Weigh 100 g of powder in 150-micron sieve.
• Place it on 75-micron sieve having a receiving container.
• Place the sieve nest on the sieve shaker and shake for 15 min.
• Transfer the residue of each of the sieve into two tared containers.
• Calculate the amount of the material retained in each sieve.
Calculation
Material retained on the 75 micron sieve ð%massÞ ¼ ðA þ B=W Þ 100
Material retained on the 150 micron sieve ð%massÞ ¼ ðB=W Þ 100
A ¼ weight of the material left on 75-micron sieve

B ¼ weight of the material left on 150-micron sieve
W ¼ weight of the sample taken
5.8.9 Hydroxymethylfurfural (Sokolinska et al. 2005)
Hydroxymethylfurfural (HMF) is the indicator to assess the heat damage or the

intensity of heat treatment given to milk or milk products. It is absent in raw milk and
is formed when milk is exposed to higher temperatures by degradation of lactose or
as an intermediate product of Maillard reaction. HMF is majorly a product found in
dried milk, UHT milk or in-bottle sterilized milk. The HMF level is directly related
to the time and temperature of storage.
• Take 10 ml of the reconstituted milk in a test tube and add 5 ml of 0.5 N oxalic
acid (prepare fresh).
• Place the test tubes by covering them in a boiling water bath for 1 h.
• Cool the tubes and add 5 ml of 40% trichloroacetic acid and mix the contents.
• Filter through Whatman No. 42 filter paper and take 4 ml of the filtrate in a 10-ml
stoppered test tube.
• Add 1 ml of 0.05 M solution of thiobarbituric acid, stopper the tubes and mix
properly.
• Place them on a water bath at 40 C for 40 min.
• Cool the tubes and measure the absorbance at 443 nm.
• Carry out a blank test by taking distilled water instead of the sample.
• Also, prepare a stock solution of HMF (100 μM) by dissolving 12.6 mg HMF and
making the volume to 100 ml with HPLC grade water. The stock solution is
diluted to prepare standards of HMF so as to get a standard curve.
Suggested Readings
Arbuckle WS (2013) Ice cream, 4th edn. Avi Publication Company, Westport, p 362, 364
De SK (2009) Outlines of dairy technology. Oxford University Press, New Delhi
IS 1166 (1986) Condensed milk, partly skimmed and skimmed condensed milk. Bureau of Indian
IS 1224-2 (1977) Determination of fat by the Gerber method, part 2 milk products. Bureau of Indian
IS 3507 (1966) Method of sampling and test for butter. Bureau of Indian Standards, New Delhi
IS 3508 (1966) Method of sampling and test for ghee. Bureau of Indian Standards, New Delhi
IS 9617 (1980) Dahi. Bureau of Indian Standards, New Delhi
ISO 15884 (2002) Milk fat - preparation of fatty acid methyl esters. International Organization for
Standardization, Geneva
ISO 15885 (2002) Milk fat - determination of the fatty acid composition by gas-liquid chromatog-
raphy. International Organization for Standardization, Geneva
Mamo A (2017) Cheddar cheese characterization and its biochemical change during ripening. Int J
Adv Sci Res Manag 2:53–59
Manual of Methods of Analysis of Foods-Milk and Milk Products-FSSAI (2015). Lab manual 1
Oh HI, Shin TS, Chang EJ (2001) Determination of cholesterol in milk and dairy products by high-
performance liquid chromatography. Asian-Australas J Anim Sci 14(10):1465–1469
Rani A, Sharma V, Arora S, Ghai DL (2016) Comparison of rapid reversed phase high-performance
liquid chromatography (RP-HPLC) method with rapid reversed phase thin layer chromatogra-
phy method for detecting vegetable oils in ghee (clarified milk fat). Int J Food Prop 19
(5):1154–1162
Sharma A, Jana AH, Chavan RS (2012) Functionality of milk powders and milk based powders for
end use applications—a review. Compr Rev Food Sci Food Saf 11(5):518–528
Sokolinska D, Pikul J, Dankow R, Wojtowski J (2005) Changes in some selected physico-chemical
and sensory parameters of UHT milk during storage at different temperatures.
Milchwissenschaft 60:37–41
Agency, New Delhi
Calibration and Standardization
6
Calibration may be defined as establishing and recording the measurement uncer-

tainty of the measuring equipment. It does not involve adjustment but may show/
demonstrate the necessity for adjustment. In simpler words, calibration is a tech-
nique in which a comparison between measurements of known magnitude or
correctness is made or set with one device and another measurement made in a
similar way with a second device. The device which is used as a reference generally
has a known or assigned degree of correctness and is referred as standard. The
second device which is to be calibrated is generally referred as the unit under test
(UUT), test instrument (TI), etc.
In a dairy plant, milk is critically analyzed for a number of factors like its
composition and quality during reception, processing, and during dispatch for sale.
This is required to ensure optimum price of the product, checking its suitability for
processing, and to know whether it meets the legal standards. In order to make sure
that all the results generated during the analysis of milk and milk products are
accurate and to prevent the generation of erroneous results, the calibration of
glassware is a must. Therefore, calibration of glassware before being used for
analysis is of utmost importance.
The glasswares that are commonly used during analysis in a dairy plant labora-
tory are butyrometer, lactometer, milk pipette and other pipettes, volumetric flask,
measuring cylinder, beaker, thermometer, etc.
The reliability of the obtained results not only depends on the calibration of the
glasswares involved in the testing but also on the chemicals involved in various tests.
So in order to ensure that the prepared solutions are accurate, we need to go for their
standardization using some primary standard substances like oxalic acid, sodium
carbonate, sodium hydrogen carbonate, etc. Such chemicals are referred as primary
substances. To ensure proper standardization of chemicals, one must have some
basic knowledge of terms like normality, molarity, and molality. These terms are
discussed below.

https://doi.org/10.1007/978-981-15-4167-4_6
148 6 Calibration and Standardization
Reagent A substance that can react with another substance or an agent and brings
about the chemical change/reaction.
Solution A solution is a homogeneous mixture of two or more substances

(or components). It consists of two components namely solute and solvent.
The component present in higher proportions is called solvent.
The component present in smaller proportions is called solute.
In general terms when a solute is dissolved in a solvent, the resulting mixture is
referred as solution.
A solution in which a small amount of solute is present is called a dilute solution.
A solution in which a large amount of solute is present is called a concentrated
solution.
A solution in which no more solute can be dissolved in a given amount of solvent
at a particular temperature is called a saturated solution.
Standard Solution A solution whose strength or concentration is known is called a

Standard solution.
Concentration or strength of solution can be expressed in different ways:
• Normal solution
• Molar solution
• Molal solution
Normal Solution (Normality) It is the number of gram equivalents of a solute/

molecule present in 1 L of a solution (denoted by N ). N ¼ gram equivalent of solute/
volume of solution in liter.
Gram equivalent weight is equal to the molecular weight of the compound
divided by its valance or reactive ion or by the number of replaceable hydrogen
(for acids) or hydroxyl ions (for base).
Number of moles of solute is equal to the weight of the solute (g) divided by its
molecular weight.
Gram equivalent moles is equal to the weight of the solute (g) divided by its gram
equivalent weight.
For instance, equivalent weight of NaOH is 40 as it has one replaceable hydroxyl
ion (Na+ OH). Equivalent weight of HCl is 36.5 as it has one replaceable hydrogen
ion (H+Cl).
Equivalent weight of Na2CO3 is 53 as the number of reactive ion or metal ion is
2, i.e., 2 molecules of sodium (2 Na+ CO32).
Molar Solution A solution which contains 1 mol (Mol. wt. if the substance is a
molecule or At. wt. if the substance is an atom) of a solute dissolved in 1 L of the
solution is called a molar solution.
6.1 Calibration of Milk Pipette 149
Molarity The number of moles of solute dissolved per liter of the solution. It is
denoted by M
M ¼ mole of solute/volume of solution in liter
Molal Solution A solution which contains 1 mol solute dissolved in a kilogram of

solvent. The total quantity of the resulting solution may or may not be equal to 1 L,
depending upon the density of a solvent.
Molality The number of moles of solute dissolved per 1000 g of solvent. It is

denoted by “m”
m ¼ No. of moles of solute/wt. of solvent (kg)
Note Normality and molarity are temperature dependent because volume depends
on temperature while molality is temperature independent.
Other ways of expressing the strength of a solution are w/w, v/v, and %.
6.1 Calibration of Milk Pipette
Pipettes are classified into two groups namely volumetric and graduated.
1. Volumetric Pipette: The volumetric pipette has a single graduation mark. It is

used to deliver one specific volume accurately. They are available in different
sizes with respect to the volume to be dispensed like 1, 5, 10, 25, 50, and 100 ml.
Milk pipette (10.75 ml) is also a type of volumetric pipette.
2. Graduated pipette: Graduated pipette is used to deliver a variable amount of
volume. It is further classified into two types:
• Mohr Pipette: A Mohr pipette is a graduated pipette that dispenses water from
top graduation mark to lowest graduation mark.
• Serological Pipette: The serological pipette is a graduated pipette that
dispenses water from top graduation mark to tip of the pipette with the last
drop blown out.
Milk pipette is used for fat estimation and has a measuring capacity of 10.75 ml.
The capacity of the milk pipette is equivalent to the volume of water (ml) delivered
by it at 27 C when fully emptied. The capacity shall be within the tolerance limit of
10.75 0.03 ml. The time required for emptying the pipette should be 7 2 s
(IS 1223: 2001). Milk pipette is calibrated by the below-mentioned methods:
(a) Comparison Method By comparison method, we mean that the fat content of a
milk sample estimated by the newly purchased pipette using Gerber method is
compared with the fat content estimated by using a well-calibrated pipette of the
previous batch (ensuring the butyrometer(s) is/are calibrated) of the same sample. If
the result of the milk fat content using new pipettes is same as that of the old ones,
the new pipettes are accepted, otherwise in case of any positive or negative
deviation, it is rejected. Although it is not an accurate method, but is still widely used
at industrial level.
Limitation: The previous pipettes used for comparison may not be accurate or due
to the breakage of their tips, their internal volume might have been changed.
(b) BIS Method This method is based upon the definition of milk pipette as given
by BIS. According to BIS, the milk pipette is defined as a pipette that dispenses
10.75 0.03 ml of distilled water at 27 C when held for 7 s.
1. Thoroughly clean and rinse the pipette with water to remove any dust or extrane-
ous matter adhering to its inner walls.
2. Suck the distilled water maintained at 27 C up to a short distance above the
graduation mark. Release the extra volume of the water by releasing the pressure
of the finger and make sure that the water is at level with the graduation mark.
3. Wipe the bottom outside end of the pipette so as to remove any extra drop of
water adhering to it with clean filter paper.
4. Take an empty beaker and weigh it. Note its weight.
5. Allow the water to run out slowly into the beaker by releasing the pressure of the
finger. Collect the dispensed liquid in the beaker weighed previously.
6. Allow the pipette to drain for 15 s until the visible outflow gets ceased. Make sure
that the tip of the jet of the pipette remains in contact with the inside wall of the
beaker during the whole process of emptying.
7. Remove the receiving beaker at the end of draining time from contact with the jet
of the pipette. Determine the weight of the water delivered.
8. Calculate the volume of water by using the density formula. Density of water at
27 C is 0.99654.

Volume of water ¼ Mass of water dispensed=density of water at 27 C ð0:99654Þ:
If the calculated volume of water dispensed by the pipette is equal to

10.75 0.03 ml, then the pipette is accepted, otherwise rejected.
Note In this method while emptying the pipette a small quantity of water remains in
the jet of the pipette. One should not use any method of emptying the pipette like
blowing the pipette forcefully that helps in expelling the liquid completely from the
jet or changes the natural rate of delivery. Carry out the calibration at room
temperatures. Always observe the temperature of the water to calculate the volume
of water delivered by the pipette at 27 C. If any variation in temperature exists,
apply the appropriate corrections for the corresponding density of water
(Table 6.12).
(c) Mathematical Method of Calibration and Graduation For checking the

marks/points corresponding to 10.75 ml capacity of milk pipettes, the following
procedure can be applied:
6.2 Calibration of Butyrometer 151
As the stem of the pipette is of uniform cross section, therefore, internal volume
per unit length of the stem at any point is constant. Fill the pipette with distilled water
to a temporarily marked point “A” on the upper stem of the pipette and dispense in a
tared beaker and weigh. Knowing the density of water, calculate the volume of
pipette up to the mark A. Similarly again fill up the pipette with the water up to
another point “B” (above A) and find the volume of water dispensed.
Let volume of water up to point A ¼ Va ml

Volume of water up to point B ¼ Vb ml
Therefore, volume between A and B ¼ (Vb – Va) ml
Now note the distance between A and B points, let it be ¼ d cm
Therefore, the stem of the pipette has volume ¼ (Vb – Va)/d ml per cm length.
Now, let Va is less than 10.75 ml
Therefore, difference of volume ¼ (10.75 – Va) ml
Now as we know that volume (Vb – Va)/d ml occupies length ¼ 1 cm
And (10.75 – Va) ml occupies length ¼ d (10.75 – Va)/(Vb – Va) cm
Therefore, mark a point above A at a distance ¼ d (10.75 – Va)/(Vb – Va) cm,
which will correspond to 10.75 ml mark.
6.2 Calibration of Butyrometer
The types of butyrometer as specified by BIS (1223: 2001) are mentioned in

Table 6.1. The classification is based on the amount of fat to be tested for a specific
type of product. Capacities, scales, and tolerance for different types of butyrometers
as per IS 1223:2001 are depicted in Table 6.13.
The butyrometer should be made from well-annealed glass. It should be resistant
to chemicals, thermal shocks exposed during the analysis, and free from any visible
defects.
Each butyrometer shall have permanently and legibly marked on the body, the
following points, i.e.,
Table 6.1 Classification of butyrometer as per BIS

S. No. Scale (%) Product
1 0–0.5 Skim milk
2 0–4 Partly skimmed milk, buttermilk
3 0–10 Whole milk, evaporated milk (unsweetened)
4 0–20 Dry milk powder
5 0–40 Ice cream, condensed milk, cheese
6 0–70 Cream
7 0–90 Butter
(a) The quantity of sample required for analysis, i.e., 10.75 ml (whole milk) or
21.5 ml (skim milk) or 5 g cream or 3 g cheese, etc.
(b) Temperature, i.e., 65 C
(c) Production unit’s name
(d) Identification number.
The graduation marks on the stem of the butyrometer should be fine, clearly
etched, and permanent. The thickness of the lines should be uniform lines with even
space between the lines.
Principle
The graduations in the milk butyrometer are etched in such a manner that each 1%
mark corresponds to an internal volume of 0.125 ml (for cream and cheese
butyrometer, the internal volume corresponds to 0.0568 and 0.0338 ml, respec-
tively). So, for the milk butyrometer to be accurate, the internal volume of each
1% mark is determined and should be equal to 0.125 0.001 ml. A minimum of
3 points on the butyrometer scale should be checked to obtain proper results. Mostly
mercury is used for calibrating the butyrometer.
Procedure to Purify Mercury

1. The mercury should be free from grease. In order to render the mercury free from
grease, it should be washed thoroughly using a 10% solution of potassium
hydroxide.
2. It should be free from metallic contamination. For this, the mercury is mixed with
an equal volume of 3 N nitric acid for several hours. This treatment will remove
most of the metallic contamination.
3. It is then washed with distilled water, followed by drying it by heat at 110 C in a
porcelain dish. The contents are then filtered by forcing it with compressed air
through the chamois skin, or are passed through a pinhole in the apex of a glazed
paper funnel.
4. The obtained mercury is then distilled using the method as suggested by Hulett
and Minchin (Phys Rev 21, 388, 1905). In this method, air is bubbled through the
mercury in the still during distillation, which causes the oxidation of the volatile
foreign metals.
5. The mercury is passed through a filter of a glass tube with one of its end drawn
into a capillary opening. This process should be repeated until all the traces of
oxide disappear from the surface of the mercury.
Why Mercury Is the Choice of Material?

(a) Mercury does not wet the walls of the container, i.e., it does not stick to the sides
of the container. Thus, error due to the sticking of the liquid at the unwanted area
or sides of the butyrometer gets eliminated.
(b) It has a very high density, thus a small change in volume will correspond to a
great change in weight.
6.2 Calibration of Butyrometer 153
Methods of Calibration
The butyrometer is calibrated using three methods:
1. Method I (Comparison Method)

In this method, the newly purchased butyrometers are compared for their accuracy
using the calibrated butyrometers from the previous batch by estimating the fat of
one milk sample by Gerber method (the milk pipette used to pipette milk should be
calibrated). If the readings of the fat values of new butyrometers match the readings
of the old ones, then the new butyrometers are accepted otherwise rejected. How-
ever, this method has a limitation that the previously used butyrometers may not be
accurate/properly calibrated or a change in their internal volume might have
occurred due to corrosion by acid. It is not an accurate method for its use in dairy
plant.
2. Method II (BIS Method (IS 1223:2001))

In this method, the butyrometer is calibrated using a special type of mercury pipette.
The procedure of calibration is based on the principle that the internal volume of the
butyrometer stem (graduated tube) is 0.125 ml, which corresponds to each 1% fat
range. Thus, the full scale of the graduated tube from 0 to 10% fat marks, has an
internal volume of 1.25 ml. An automatic mercury pipette is one which is designed in
such a manner so as to dispense 0.3125 ml mercury in one shot exactly filling the
tube corresponding to 2.5% graduation marks. To calibrate the full scale of the
butyrometer from 0 to 10%, the bulb of the butyrometer is first filled up to 10%
graduation mark as the base point (consider it as a zero mark). Add the mercury in
the butyrometer using the mercury pipette dispensing 0.3125 ml of mercury thus
filling the butyrometer column by 2.5% fat scale. Add mercury again for three more
times and if the graduated column of the butyrometer gets filled from 0 to 10% fat
scale in four deliveries, then it can be concluded that the butyrometer is accepted,
otherwise rejected.
3. Method III
1. Place a clean and dry milk butyrometer in a flask in vertical position.
2. Fill the mercury up to 10% in milk butyrometer using a spatula or a micro burette.
The meniscus of the mercury should be adjusted exactly at 10%. Record the
weight of the butyrometer.
3. Then fill the butyrometer so that it fills by 1% graduation mark (10–9%, 8–7%,
6–5% as the case may be) and note the weight.
4. Then using the density formula (D ¼ M/V ) the volume required to fill 1 % gradu-
ation marks of butyrometer can be calculated (weight of mercury used and density
of mercury both being known (Density of mercury at different temperatures is
shown in Table 6.12)). Each 1% graduation corresponds to a volume of
0.125 0.001 ml. Any butyrometer having greater deviation than 0.001 is
discarded.
For cream butyrometer, calibration may be checked at each 10% graduation

(at any three points). Here the internal volume of each 10% graduation is checked
which should be 0.568 0.004 ml.
For cheese butyrometer, calibration may be checked at each 5% graduation
(at any three points). Here the internal volume of each 5% graduation is checked
which should be 0.169 0.002 ml.
6.3 Calibration of Lactometer
Lactometers are specially designed hydrometers which are used to determine the
specific gravity of milk. It is based on the Archimedes law of floatation. The most
commonly used thermometers in dairy industry are of three types: Quevenne, BIS,
and Zeal. These lactometers are classified on the basis of their calibration
temperatures.
1. Quevenne’s lactometer—calibrated at 15.5 C

2. BIS lactometer—calibrated at 27 C
3. Zeal lactometer—calibrated at 29.4 C
The marking on the lactometer stem varies from lactometer to lactometer. The
BIS lactometer has a graduation range 20–35 while the quevenne type has a range
15–40.
The lactometer shall have permanently and legibly marked on the paper bearing
the scale, or on a label fixed inside the bulb:
• Basis of the scale, namely, specific gravity at 27 C.

• Inscription “50 mN/m” (lactometer should be calibrated by a liquid which has a
surface tension of 50 mN/m measured by Drop Method).
• Manufacturer’s name, or recognized trade mark.
• Identification number.
The specification of lactometer as per IS 9585: 1985 is mentioned in Table 6.2
Size
The lactometer should be such that it is capable of being used with about 200 ml
of milk.
Scale
• The graduation lines should be marked in black having uniform thickness on a
paper that is fixed securely inside the stem.
• The lines should be regularly spaced.
6.3 Calibration of Lactometer 155
Table 6.2 Specification of lactometer

Sr. Dimension/
no. Characteristic tolerance/division
1 Range covered by scale, specific gravity 1.020–1.035
2 Specific gravity equivalent for each subdivision 0.0005
3 Permissible error at any point
0.0005
4 Number of subdivision on scale 30
5 Number of subdivision beyond nominal scale of the top One or nil
graduation
6 Length in mm of stem above top graduation mark 20 5
7 Scale length in mm 41 5
8 Distance in mm below the lowest graduation mark, where the stem 10
has to remain uniform in diameter, Min.
9 External diameter in mm of stem containing scale (approx.) 4.0
10 External diameter in mm of bulb 22 1
11 Length in mm of uniform stem, Max. 80
12 Volume in ml below bottom graduation line:
(a) Not more than 37
(b) Not less than 31
13 Length in mm of the bulb 105 5
• The scale shall be straight, wrinkle free, and without twist. The graduation lines
shall be at 90 to the axis of the lactometer.
• The graduation lines should have a thickness between 0.10 and 0.20 mm.
• The shortest graduation line should be at least 2 mm in length while the alternate
line shall extend beyond the shortest lines.
• The numbers shall be on the left of the scale, immediately above the graduation
marks to which they relate and they shall not encroach upon the space occupied
by the shortest graduation marks. The numbers shall not touch or intersect any
graduation mark.
Material of Construction
• Well annealed, clear glass, free from visible defects should be used for making the
bulb and stem.
• In the base of the lactometer bulb, lead shots must be filled or any other type of
suitable loading material embedded in a suitable cementing material.
Lactometer Jars
The jar used for pouring milk and dipping the lactometer should be cylindrical in
shape, made of glass or metal with an internal diameter of 32 2 mm and
185 5 mm in height.
Lactometer can be calibrated by the following methods:

1. Comparison Method
In this method, the reading obtained by a newly purchased lactometer when dipped
in a liquid (say milk) is compared with the reading of a previously calibrated or
standard lactometer in the same liquid. Both the lactometers should be dipped at the
same time in the same liquid (temperature of the liquid in both of the lactometer jars
should be the same). If the lactometer reading of the newly purchased lactometer
matches with that of the standard lactometer, then the new lactometers should be
accepted otherwise rejected.
2. BIS Method for Testing BIS Lactometer (IS 9585:1985)

BIS Lactometer is calibrated at 27 C. The lactometer is immersed in a solution of
known specific gravity. Anhydrous sodium carbonate solution is used to calibrate
the lactometer. Known quantity of anhydrous sodium carbonate is dissolved in
water. Ethyl alcohol is used to adjust the surface tension of the liquid to that of the
surface tension of milk.
1. The solutions used should have a specific gravity of 1.025, 1.030, and 1.034 at
27 C. The amount of anhydrous sodium carbonate required to make 300 ml of
the solution is mentioned in Table 6.3.
2. Then add 50 ml of industrial ethanol (92%) to each of the 300 ml solution and
makeup to the total volume of 500 ml with distilled water.
3. Insert the lactometer in the jar containing these solutions and leave it undisturbed
until it gets stabilized.
4. Observe the reading of the lactometer at 27 C.
5. Calculate the specific gravity using the formula:

Specific gravity of Na2 CO3 at 27 C ¼ 1 þ Lactometer Reading=1000
The difference in the calculated specific gravity should not differ from that in the
Table 6.3 by more than 0.0005.
If the error in the lactometer is negative then mention the error with a positive sign
indicating that the error is to be added in the lactometer reading. If the error in the
lactometer reading is positive then indicate the error with a negative sign indicating
that the error is to be subtracted from the lactometer reading.
Table 6.3 Specific gravity and lactometer reading of pure sodium carbonate solution
Wt. of anhydrous Na2CO3 Specific gravity at 27 C Lactometer reading at
S. no. (g/500 ml) (g/ml) 27 C
1 19.2 1.025 25
2 21.3 1.030 30
3 24.0 1.034 34
6.4 Calibration of Other Pipettes 157
Table 6.4 Specific gravity and lactometer reading of pure sodium chloride solution
S. no. Pure sodium chloride solution (%) Sp. Gravity at 15.5 C Lactometer reading
1 3.863 1.026 26
2 4.415 1.032 32
3. Method of Testing Quevenne Lactometer

This can be done by checking the specific gravity of sodium chloride solution at
15.5 C.
1. A known amount of anhydrous sodium chloride is dissolved in 300 ml distilled

water (Table 6.4).
2. Add 50 ml of ethyl alcohol and make up the volume to 500 ml.
3. Fill the lactometer jar with NaCl solution and dip the lactometer in it. Maintain the
temperature to 15.5 C.
Note down the lactometer reading when the lactometer attains a stabilized
position and check whether the specific gravity calculated using lactometer matches
the specific gravity mentioned in Table 6.4.
The specific gravity of such solutions should be checked using a pycnometer.
The difference in the calculated specific gravity should not differ from that in the
Table 6.4 by more than 0.0005. The error in the reading of the lactometer should be
mentioned on the lactometer as discussed for BIS lactometer.
6.4 Calibration of Other Pipettes
The calibration of other pipettes is done following the same procedure as mentioned
for milk pipette. The two types of pipettes that are commonly used in laboratories are
categorized on the basis of their tolerance level (See Appendix, Table 6.11).
Type A
These pipettes are used when a higher degree of accuracy is required. It complies
with the requirements of ASTM E694 standard. The glassware has the letter A and is
calibrated to half the tolerance level of Class B type glassware. A calibration
certificate is issued along with it which has a unique serial number for traceability.
Type A glassware does not require any further calibration.
Type B
These are calibrated at double the tolerance limit of Class A glassware. They are
used for routine work and should be calibrated before use.
6.5 Calibration of Volumetric Flask/Measuring Cylinder/

Beaker
The principle involved in the calibration of volumetric flask/measuring cylinder/

beaker is the same as that of the pipette. The volume of these glasswares is equal to
the volume of water contained in these glasswares at 27 C (volume depends on their
mentioned capacity).
Procedure
1. An empty volumetric flask/measuring cylinder/beaker is weighed (W1).
2. It is then filled with distilled water up to the graduation mark mentioned over it.
3. Then the weight of the filled glassware (W2) is taken and the temperature of water
is kept constant say 27 C.
4. The net capacity (volume) of the glassware is calculated by using the density
formula as the quantity of water is known (W2–W1) and the corresponding
density of water at a specified temperature (Density of water at 27 C is
0.99654). Table 6.12 shows the density of water at different temperatures.
5. If the tolerance level is within the limits then accept otherwise reject.
6.6 Calibration of Thermometer
Liquid expansion thermometer is one of the most common types of thermometer.

These types of thermometers contain an expansion liquid mercury or alcohol, filled
in a glass bulb that is attached to a long stem having a uniformly bored expansion
column. As the bulb of the thermometer is heated, the liquid expands and rises up the
bore until it reaches a stable level as it reaches a thermal equilibrium with the
material whose temperature is being measured. The expansion of the liquid causes
it to rise which in turn is linear with the temperature change. The lines marked on
these thermometers indicate the temperature and are equally spaced. They are
generally calibrated at two different “fixed point” temperatures (usually at higher
end and lower end of the scale). Alcohol thermometers, although less accurate than
mercury thermometers, are commonly used because mercury is toxic. The alcohol
thermometer is less accurate due to two reasons: (1) alcohol is more volatile as
compared to mercury, thus can vaporize into the space above the liquid in the stem;
(2) alcohol, unlike mercury, can “wet” the sides of the stem’s bore resulting in a part
of liquid to be adhered as the drop in temperature occurs. In a milk testing laboratory,
mainly two types of liquid expansion thermometers are used—(a) thermometer
(range 0–100 C) and (b) freezing point depression thermometer (range 0.5 to
0 C).
6.6 Calibration of Thermometer 159
1. Calibration of 0–100 C Thermometer
Calibration at the Ice Point of Water

1. Fill a container with crushed Ice.
2. To the container add pre-cooled distilled water so that it covers the ice (make sure
the quantity of added water does not cause the ice to float).
3. Thoroughly mix the ice–water mixture.
4. Insert the thermometer in the container.
5. Leave the thermometer undisturbed so that the temperature shown by it gets
stabilized.
6. Mark the point at which the reading gets stabilized. The ice point of water is stable
at 0.00 C.
Calibration at the Boiling Point of Water

1. Place a round bottom flask containing distilled water (half filled) on a water bath
and fix a rubber cork having a hole.
2. Fix the thermometer in the hole such that its bulb is immersed in distilled water.
3. Heat the water to boiling and note the temperature at which water starts boiling
completely.
4. When the temperature attains a stable reading, observe the temperature carefully.
Water boils at 100 C at atmospheric pressure.
Note: The boiling point of water is extremely sensitive to the atmospheric

pressure. If the atmospheric pressure is not 760 mm, then the appropriate correction
factor is applied. Generally, 0.038 C is added or deducted from the observed boiling
point per mm pressure of mercury lower or higher than 760 mm, respectively.
2. Calibration of Freezing Point Depression Thermometer

We know that:
1 Molar solution
It is defined as a solution containing 1 mol of a solute dissolved in a solvent so as

to make 1000 ml solution.
1 Molal solution
It is defined as a solution containing one-mole solute dissolved in 1000 g of

solvent.
Depression in freezing point is a colligative property. Such properties depend on
the number of moles of solute present in the solution. Thus, the soluble ions or
molecules play a very important role in depression of the freezing point of any
solvent such as water. Milk is also a solution containing water as a solvent and
certain solutes like lactose, some minerals in dissolved phase (true solution). These
dissolved solutes lower the freezing point of milk below that of water, i.e., below
0 C. One molal solution of any ion or unionized molecule depresses the freezing
point of water by 1.86 C. It means one molal solution of non-ionized substances like
sugar (360 g in 1000 g of water), and glucose (180 g in 1000 g of water) depress the
freezing point by 1.86 C. However, one molal solution of ionized molecules
depresses the freezing point by (1.86 numbers of ions per molecules) C. For
example, one molal solution of sodium chloride (Na+Cl) will depress the freezing
point by 1.86 2 ¼ 3.72 C.
Now, 1.86 C is depressed by a solution ¼ 1 molal
1 C is depressed by a solution ¼ 1/1.86 ¼ 0.538 molal
0.5 C is depressed by a solution ¼ 0.269 molal
Therefore, –0.5 C and 1 C points on the freezing point depression thermome-
ter are checked by using the 0.269 and 0.538 molal solutions, respectively.
For pure milk 0.54 C ¼ 0.29 molal or 0.55 C ¼ 0.3 molal.
6.7 Calibration of Weighing Balance
The weighing balance or instruments are widely used in quality assurance laboratory
and their accuracy plays a major role in performing various chemical assurance tests
successfully. The chemicals which are used in the testing of milk and milk products
or other routine tests are prepared by weighing their known amount. So, it is essential
that the amount of the weighed chemicals should be accurate. Generally, the body
that takes care of the calibration of weighing instruments is the metrological dept.
and comes under Weight and Measurement Act (in India).
The calibration of a weighing scale is done by three methods, i.e., eccentric
test (Table 6.5), repeatability test (Table 6.6), and linearity test. Before starting the
calibration following these three terminologies needs to be understood:
Table 6.5 Recording of results for eccentric test

Location on the weighing Weight to be Recorded
scale tested weight Mean SD RSD
1
2
3
4
5
Table 6.6 Recording of results for repeatability tests

S No Weight to be tested Recorded weight Mean SD Uncertainty
1
2
3
4
5
6.7 Calibration of Weighing Balance 161
• Eccentric test: During the normal weighing the material to be weighed (load) is
not always placed at the center of the weighing platform as a result the value of
the recorded weight can vary when the load is placed at the different locations.
Thus to minimize the variation in the weight recorded with respect to the location
on the weighing platform eccentricity tests are performed. In this test, the standard
weights are placed at different locations like at center, at four different corners.
The test load to be used in this test should be at least one-third of the maximum
load of the weighing instrument. In case the weighing instrument has multiple
ranges the eccentric test can be done using the highest and the lowest weight.
• Repeatability test: The weighing instrument can also suffer from the repeatability
issues, which means that when a known amount of weight is weighed on the
weighing scale the recorded weight varies. To resolve this issue, repeatability test
is carried out by weighing a known weight at the same place repeatedly say ten
times and then observed for the variation. This test is done by using a single load,
but can be performed by using loads of different values.
• Linearity test: This test is done by selecting multiple points in the entire measur-
ing range of the instrument. The objective of this test is to check whether the
instrument measures accurately in its measuring range or not. Even if the zero and
the maximum value recorded are correct, but still chances may exist that certain
error would exist in the middle range. This type of error is referred to as linearity
error or nonlinearity.
Nonlinearity, if persists in the instrument a hysteresis is observed when plotting a

graph by increasing the weight progressively to maximum and then reducing the
weight progressively to minimum. Care should be taken that the increase or decrease
in weight should not be to the maximum or minimum value, otherwise the hysteresis
information cannot be drawn, rather the weights should be increased or decreased
progressively.
General Procedure to Be Followed for Calibration of the Weighing Balance

• Clean the weighing balance platform.
• Switch on the mains supply to the electronic balance.
• Press the power button of the balance.
• Wait for the initialization process to complete.
• Allow the instrument to be stable and should be zero. The base at which the
weighing balance is placed should be vibration free.
• Place the standard weight on the weighing platform and record the weight.
• Repeat the measurement as per the test to be carried out like eccentric, linearity, or
repeatability.
• Once the calibration is done switch off the power button of the balance and then
the main supply.
• The calibration schedule for weighing balance is generally done on daily basis
(placing the maximum and minimum weight) while the monthly check is done by
checking the weights in its entire range along with the eccentric and
repeatability test.
6.8 Standardization of Acids, Bases, and Other Chemical

Solutions
In order to carry out certain tests like titratable acidity, strength of caustic soda or
acid used for cleaning, analysis of minerals, peroxide value, free fatty acids, RM
value, etc. a number of chemicals are required. To ensure the accuracy of results, the
standardization of chemicals is very important. A solution whose strength is known
is called a standard solution. Two types of substances are used for preparing standard
solutions:
• Primary Standard substances

• Secondary Standard substances
Primary Standard Substance Substances that can easily be obtained in pure and
crystalline form, e.g., oxalic acid, sodium carbonate, potassium dichromate, potas-
sium hydrogen phthalate, potassium iodate, and sodium chloride.
Characteristics of Primary Standard Substances

• It should be easy to obtain, to purify, and to preserve.
• It must not be hygroscopic.
• It should not decompose at ordinary temperatures.
• It should be readily soluble under the conditions in which it is used.
• Its reaction with other reagents should be quantitative and practically quick.
• It should have high equivalent weight so that any error due to weighing is
minimized.
• It should be fairly cheap.
Preparation of Primary Standard Solutions

These solutions are prepared by using pure standard substances in distilled water
(IS 1070: 2008). These substances have a constant weight, high purity and are
non-hygroscopic as a result, the resulting solution has a known and definite
concentration.
The preparation of some primary standard solutions is discussed below:
Preparation of 0.1 N Oxalic Acid Solution

Oxalic acid is used to find the strength of alkali solutions like NaOH, KOH
(Secondary standards) whose standard solutions cannot be made by direct weighing.
The molecular weight of oxalic acid [(COOH)2.2H2O] is 126. As it has two
replaceable hydrogen atoms thus its equivalent weight is 126/2 ¼ 63.
• Weigh 6.3 g oxalic acid (hydrated salt) in a clean and dry weighing boat.
• Dissolve it in distilled water.
• Transfer the contents into a volumetric flask and make up the volume to 1 L in
volumetric flask.
6.8 Standardization of Acids, Bases, and Other Chemical Solutions 163
Table 6.7 Amount of chemical required to make 0.1 N primary standard solution
Molecular Equivalent Amount (g) required to
S. No. Primary standard weight weight make 0.1 N solution
1 Potassium dichromate 294.185 49.03 4.903
2 Potassium bromate 167 27.83 2.783
3 Sodium chloride 58.5 58.44 5.844
4 Sodium bicarbonate 84.007 84.007 8.400
5 EDTA 292.24 292.24 29.224
Preparation of 0.1 N Sodium Carbonate Solution

Sodium carbonate is used for finding strength of acids like HCl and H2SO4 whose
standard solutions cannot be prepared directly. The molecular weight of sodium
carbonate (Na2CO3) is 106 while its equivalent weight is 53 (the number of reactive
atoms is 2, i.e., Na).
• Weigh 5.3 g of pure anhydrous salt in a clean and dry weighing boat.
• Dissolve it in distilled water.
• Transfer the contents into a volumetric flask and make up the volume to 1 L in
volumetric flask.
Preparation of 0.1 N Potassium Hydrogen Phthalate Solution

Potassium hydrogen phthalate (C8H5KO4) is also used to determine the strength of
alkali solutions like NaOH, KOH. Its molecular weight is 204.22. For preparing its
0.1 N solution, take 20.42 g (as it has one reactive potassium atom, so its equivalent
weight is 204.22) and dissolve in 1 L distilled water. Table 6.7 shows the amount of
different primary standards that is required to be dissolved in 1 L distilled water
to make their 0.1 N solution.
Secondary Standard Substance

Those substances which cannot be obtained in pure state and do not possess the
properties of primary standard, e.g., standard alkalis, standard acids, silver nitrate,
and sodium thiosulfate. The strength of solutions prepared by using these chemicals
can be determined by using a primary standard solution of known strength. Before
learning about the standardization of secondary standard substances, let us have a
look at some common terms:
• Direct Titration
A type of titrimetric reaction in which one of the analytes is treated with titrant
and the volume of titrant required to complete reaction is measured.
• Back Titration
This is another type of titrimetric reaction in which an excess of standard reagent

is made to react with an analyte. Then the excess reagent is titrated with a second
reagent or with a standard solution of analyte. This type of titration is done when the
molar concentration of the excess reactant is known and there is a need to determine
the strength of the analyte. This is done when the acid or base is insoluble in nature
or where the endpoint of a direct titration is difficult to ascertain or when the reaction
proceeds or occurs slowly. Back titration is also called as indirect titration.
In this type of titration two steps are carried out, first the analyte is reacted with
the excess reagent and second the titration is conducted on the remaining quantity of
the known solution.
• Equivalence point
It is the point in a titration when the amount of added standard reagent is exactly
equivalent to the amount of analyte.
• End point
It is the point in a titration at which an observable physical change (like color

change) signals the equivalence point. It is slightly higher than the equivalence point.
• Indicator
It is often added to the analyte solution to produce an observable physical change

(the end point) at or near the equivalence point. Table 6.8 presents the pH range of
different indicators as well as the color they produce in acidic and basic
solutions. While the Table 6.9 shows the types of indicators which are to be used
for different cases of acid-base titrations.
Standardization of 0.1 N Sodium Hydroxide Solution

Sodium hydroxide is not a primary standard substance because of its non availabil-
ity in a sufficiently pure state. So to prepare its standard solution, it has to
be standardized against the primary standards such as oxalic acid or potassium
hydrogen phthalate in order to accurately determine its normality. The principle
Table 6.8 Some pH-based Color change

indicators
Indictor pH range Acid Base
Methyl orange 3.0–4.4 Red Yellow
Methyl red 4.4–6.3 Red Yellow
Bromothymol blue 6.6–7.6 Yellow Blue
Litmus 6.0–8.0 Red Blue
Phenolphthalein 8.2–10 Colorless Pink
Table 6.9 Change in the indicator color at certain base–acid combinations

Base–acid Indicator color change at pH range
Strong base–strong acid Any indicator changing color (pH 3–11)
Strong base–weak acid Any indicator changing color (pH ~ 9.5)
Weak base–strong acid Any indicator changing color (pH 3–6)
Weak base–weak acid No suitable indicator
involved in standardization is simply a neutralization reaction that occurs when an

acid reacts with an alkali resulting in the formation of salt and water. An indicator is
used in this reaction, which does not participate in the reaction but indicates the end
point by undergoing a characteristic color change. When phenolphthalein is used as
an indicator the color changes from colorless in acidic medium to pink in basic
medium, i.e., at pH 8.3.
Reaction
2NaOH þ ðCOOHÞ2 ! Na2 C2 O4 þ 2H2 O
ðSodium hydroxideÞ ðOxalic acidÞ ðSodium OxalateÞ
Procedure
1. Preparation of 100 ml of 0.1 N NaOH solution
(a) Weigh approximately 0.42 g of NaOH pellets and dissolve it in 50 ml of
distilled water in a clean dry beaker.
(b) Then transfer the above 50 ml of solution into a 100-ml volumetric flask.
(c) Make up the volume to 100 ml using distilled water. The solution is approxi-
mately 0.1 N (equivalent weight of NaOH ¼ 40.01).
2. Standardization of 0.1 N NaOH
(a) Fill the burette with prepared 0.1 N NaOH up to a known mark.
(b) Pipette out 10 ml of 0.1 N oxalic acid solution (as discussed earlier) in a
100 ml conical flask.
(c) To it add two to three drops of phenolphthalein indicator.
(d) Note down the initial reading on the scale of burette and then titrate with
NaOH solution until the color changes from colorless to pink.
(e) Note down the final reading on the burette, repeat the titration till at least
three concordant readings are obtained.
(f) Determine the actual normality of the prepared NaOH solution using the
volume of NaOH solution consumed to neutralize 10 ml of oxalic acid.
(g) If the Normality of the NaOH calculated is not same as intended, then dilute it
accordingly to have required normality (0.1 N NaOH Solution).
(h) To check the normality of the final NaOH solution, again titrate it and if the
volume of oxalic acid consumed is equal to the volume of NaOH taken, then
the solution is standardized one.
(i) Record the results in tabular form and take concordant readings for
calculation.
Calculation
Burette reading Titration 1 Titration 2 Titration 3
Initial reading
Final reading
Volume consumed
Calculate the normality of the NaOH solution using equation
N1V 1 ¼ N2V 2 ð6:1Þ
where
N1 ¼ normality of standard oxalic acid (0.1)

V1 ¼ volume in ml of oxalic acid used for titration (10 ml)
N2 ¼ normality of prepared NaOH (to be determined)
V2 ¼ volume in ml of prepared NaOH consumed in the titration
Note: A slightly concentrated solution of NaOH is prepared and then it is diluted to
actual normality after titration
Now,
NaVa ¼ N2Vb
Na ¼ required final normality (0.1 N) of NaOH solution
Va ¼ required volume of solution
N2 ¼ Normality of prepared NaOH solution determined from Eq. (6.1)
Vb ¼ Volume of prepared solution to be dilute
Let V ml of prepared NaOH solution having normality N2 is to be diluted to get

0.1 N solution ¼ (100 0.1)/N2 (100 is the total volume of the final solution).
So add V ml of prepared NaOH solution having normality N2 and make up the
volume to 100 ml using distilled water so as to get a 0.1 N NaOH solution.
Standardization of 0.1 N HCl

As is the case with NaOH, HCl solutions cannot be used directly for analysis. They
require standardization so as to ascertain their strength. The standardization of HCl is
done using sodium carbonate, which is a primary standard. The reaction is a
neutralization type of reaction resulting in the formation of a salt with the evolution
of carbon dioxide. For the titration of strong acids against weak alkali, we used
methyl orange as an indicator (its color changes from yellow to red which indicates
the end point).
Reaction
Na2 CO3 þ 2HCl ! 2 NaCl þ H2 O þ CO2
Procedure
1. Preparation of 100 ml of 0.1 N HCl solution
(a) Directly take 0.85 ml of 12 N HCl into a volumetric flask or calculate using
the formula1 to determine the quantity of HCl required to make 0.1 N
solution.
(b) Then make up the volume up to 100 ml using distilled water.
2. Standardization of 0.1 N HCl
(a) Fill the burette with prepared 0.1 N HCl solution.
(b) To a 250-ml conical flask pipette out 10 ml of 0.1 N sodium carbonate
solution (preparation discussed earlier).
(c) Add one to two drops of methyl orange indicator.
(d) Run down HCl solution from the burette slowly and mix the contents of the
flask.
(e) Note the volume of the HCl solution required to change the color of the
solution into a permanent orange (pink) color from pale yellow.
(f) Repeat the titration for at least three concordant readings.
(g) If the Normality of the HCl solution is greater than 0.1 N, then it is to be
diluted and vice versa.
Calculation
Burette reading Titration 1 Titration 2 Titration 3
Initial reading
Final reading
Volume consumed
Calculate the normality of the HCl solution using equation
N1V 1 ¼ N2V 2 ð6:2Þ
where
N1 ¼ normality of standard sodium carbonate (0.1)

V1 ¼ volume in ml of sodium carbonate used for titration (10 ml)
N2 ¼ normality of prepared HCl (to be determined)
V2 ¼ volume in ml of prepared HCl consumed in the titration
Note: A slightly concentrated solution of HCl is prepared and then it is diluted to

actual normality after titration
Now,
N aV a ¼ N 2V b
1
Formula used is Specific gravity/equivalent weight 1000 % assay.
Na ¼ required final normality (0.1 N) of HCl solution

Va ¼ required volume of solution
N2 ¼ Normality of prepared HCl solution determined from Eq. (6.2)
Vb ¼ Volume of the prepared solution to be dilute
Let V ml of prepared HCl solution having normality N2 is to be diluted to get

0.1 N solution ¼ (100 0.1)/N2 (100 is the total volume of the final solution).
So, add V ml of prepared HCl solution having normality N2 so as to get a 0.1 N
HCl solution.
Standardization of Sulfuric Acid and Nitric Acid

The principle involved in the standardization of sulfuric acid and nitric acid is same
as that for hydrochloric acid using methyl orange as an indicator. The amount of
sulfuric acid and nitric acid required to make their 0.1 N solutions is mentioned in
Table 6.10. The titration is similar as in case of HCl. The final corrected strength can
be obtained by using the same calculations as that for 0.1 N HCl.
Standardization of Sodium Thiosulfate Solution

The strength of sodium thiosulfate solution is determined using standard potassium
dichromate (K2Cr2O7) solution. The reaction between sodium thiosulfate and potas-
sium dichromate is an oxidation–reduction reaction. In this reaction, during the
titration iodine is liberated from potassium iodide, hence this chemical reaction is
referred to as iodometric titration. On addition of K2Cr2O7 to an acidic solution of
KI, dichromate gets reduced liberating an equivalent amount of Iodine. In acidic
conditions, the dichromate ion is a strong reducing agent as compared in alkaline
conditions, so the medium is made acidic by the addition of hydrochloric acid.
K2 Cr2 O7 þ 14 HCl þ 6 KI ! 8 KCl þ 2 CrCl3 þ 7 H2 O þ 3 I2
The liberated iodine reacts with Na2S2O3.
2 Na2 S2 O3 ðsodium thiosulfateÞ þ I2 ! 2NaI þ Na2 S4 O6
In this reaction, iodine acts as an oxidizing agent while dichromate ion acts as a
reducing agent.
In the second step of the experiment, starch is used as an indicator, which in
contact with free iodine produces a deep blue color.
Table 6.10 Approximate strength of commercially available concentrated acids

Specific Approx. Quantity in ml required to make
S. No. Acids Gravity strength 100 ml of 0.1 N solutiona
1 Glacial acetic acid 1.05 16 N 0.625
2 Hydrochloric acid 1.16 12 N 0.833
3 Nitric acid 1.42 16 N 0.625
4 Sulfuric acid 1.84 36 N 0.278
a
Normality of stock solution ¼ (Sp. gravity % purity 1000)/(equivalent weight 100)
Starch þ I2 ! Starch iodine complex ðblueÞ
Starch Iodine þ 6 S2 O3 ! Starch þ 6 I þ 3 S4 O6
Procedure
Preparation of sodium thiosulfate solution

1. Boil a small amount of distilled water and cool it.
2. Add 24.8 g of sodium thiosulfate crystals to the cooled distilled water.
3. The volume of the solution was made up to 1 L. Store the solution in a cool and
dark-colored bottle.
Standardization of sodium thiosulfate solution

1. Weigh exactly about 5 g of potassium dichromate (Eq. Wt. ¼ 49.04) previously
dried to a constant weight at 105 2 C and dissolve in a small quantity of
distilled water.
2. Transfer the contents quantitatively to a 1000-ml volumetric flask and make up
the volume to 1000 ml with distilled water.
3. Into a 250 ml conical flask, 25 ml of potassium dichromate solution is added and
to it about 2 g of solid KI is added followed by addition of about 10 ml of
2 N HCl.
4. Mix the contents of the conical flask thoroughly and allow it to stand for a while,
preferably in the dark. Fill the burette with sodium thiosulfate solution.
5. Slowly add sodium thiosulfate solution from the burette, when the solution
becomes yellowish in color, add 1 ml of starch solution resulting in the develop-
ment of blue color.
6. Continue adding thiosulfate solution from the burette drop by drop, with constant
shaking of the flask, until the blue color changes to green.
7. Repeat the experiment till at least two concordant values are obtained.
Calculation
Volume of K2Cr2O7 pipetted out ¼ V1 ml ¼ 25 ml
Normality of K2Cr2O7 solution ¼ N1
Volume of Na2S2O3 run down from the burette ¼ V2 ml
Therefore, Normality of Na2S2O3 ¼ N2
Normality of Na2S2O3 solution ¼ 25 W/49.03 V
where
W ¼ weight in g of K2Cr2O7
V ¼ volume in ml of Na2S2O3 required for titration
Standardization of 0.1 N Silver Nitrate Solution

Silver nitrate solution to be standardized is titrated against sodium chloride of known
strength using potassium chromate as an indicator. Sodium chloride converts silver
nitrate to silver chloride. When all the chloride ions are precipitated in the form of
silver chloride, the excess of silver nitrate reacts with potassium chromate to form a
reddish-brown precipitate of silver chromate (Ag2CrO4).
2 AgNO3 þ K2 CrO4 ! Ag2 CrO4 þ 2 KNO3
Procedure
Preparation of 0.1 N AgNO3 solution
Dissolve 1.7 g of silver nitrate and make up the volume to 100 ml using halogen-free
distilled water. Store the solution in dark or an amber-colored bottle.
Standardization of 0.1 N silver nitrate solution

1. Dissolve 1.5 g of sodium chloride in 250 ml volumetric flask and make up the
volume with distilled water. This solution is 0.1 N NaCl.
2. Fill the burette with 0.1 N silver nitrate solution.
3. Pipette out 10 ml of 0.1 N sodium chloride in a 100 ml conical flask.
4. Add a few drops of potassium chromate (5% solution) and mix well.
5. Rundown silver nitrate from the burette till appearance of reddish-brown color
marking it as the end point.
6. Repeat the experiment to get concordant readings.
7. Carry a blank experiment using distilled water and potassium chromate in the
conical flask and titrate with silver nitrate.
8. Deduct the blank reading from the previous reading obtained for standard NaCl
solution.
Calculations
Volume of NaCl pipetted out ¼ V1 ml
Normality of NaCl (by calculation) ¼ N1
Volume of AgNO3 ¼ V2 ml
Normality of AgNO3 (N2) ¼ V1 (N1)/V2
Result: The strength of the given AgNO3 solution ¼ _____ N
Appendix 171
Appendix
See Tables 6.11, 6.12, and 6.13
Table 6.11 Tolerance of various types of pipettes

Tolerance ml
One mark type (Volumetric Serological type
pipette) (graduated) Mohr type
Capacity (ml) Class A Class B Class A Class B Class A Class B
0.1 – – 0.006 0.01 0.006 0.01
0.2 – – 0.006 0.01 0.006 0.01
0.5 – – 0.006 0.01 0.006 0.01
1 0.006 0.015 0.006 0.01 0.006 0.01
2 0.010 0.020 0.010 0.02 0.010 0.02
5 0.015 0.030 0.030 0.05 0.030 0.05
10 0.020 0.040 0.050 0.10 0.050 0.10
20 0.030 0.060 – – – –
25 0.030 0.060 0.100 0.20 0.100 0.20
50 0.050 0.100 – – – –
100 0.080 1.160 – – – –
Source: IS 1223:2001
Table 6.12 Density of Density g/cm3

water and mercury at
Temp. ( C) Water Mercury
various temperatures
20 0.99823 13.5461
21 0.99802 13.5437
22 0.99780 13.5412
23 0.99756 13.5388
24 0.99732 13.5363
25 0.99707 13.5339
26 0.99681 13.5314
27 0.99654 13.5290
28 0.99626 13.5265
29 0.99597 13.5241
30 0.99567 13.5216
31 0.99537 13.5191
32 0.99505 13.5167
33 0.99473 13.5142
34 0.99440 13.5118
35 0.99406 13.5094
172
Table 6.13 Capacities, scales, and tolerance for butyrometers (IS 1223:2001)
Basis of graduation
% Qty of (corresponding to 1% fat) Capacity of Length of Graduated at Graduations of intermediate Numbered Error
scale sample (ml) body (ml) scale (mm) each percent length at each percent at each % %
6 10.75 ml 0.125 20.5 0.5 65 0.1 0.5 1 0.05
8 10.75 ml 0.125 20.5 0.5 70 0.1 0.5 1 0.05
10 10.75 ml 0.125 20.5 0.5 75 0.1 0.5 1 0.05
4 10.75 ml 0.125 20.5 0.5 60 0.05 0.5 1 0.025
0.5 21.5 ml 0.125 43.5 0.5 20 0.02 0.1 0.1 0.01
6
70 5g 0.0568 21.0 0.5 65 1 5.0 10 0.5

40 3g 0.0338 21.0 0.5 60 1 5.0 10 0.5
Calibration and Standardization
Suggested Readings
BiPM IEC, IFCC I, IUPAC I, ISO O (2012) The international vocabulary of metrology—basic and
general concepts and associated terms (VIM 3rd edition). JCGM 200:2012
Hulett GA (1911) The distillation of amalgams and the purification of mercury. Phys Rev (Series I)
33(4):307
IS 1223 (2001) Indian standard: apparatus for determination of milk fat by Gerber method-
specification. Bureau of Indian Standards, New Delhi
IS 7659 (1996) Indian standard: reagents and standard solutions for use in chemical analysis of
metals, ores and minerals: part 1-volumetric solutions. Bureau of Indian Standards, New Delhi
IS 8897 (1978) Indian standard: calibration and method of verification of volumetric glassware.
Bureau of Indian Standards, New Delhi
IS 9585 (1985) Indian standard: specification for lactometer. Bureau of Indian Standards, New
Delhi
Qualification of Balances Annex 8 to the OMCL Network Guideline “Qualification of Equipment”
PA/PH/OMCL (12) 77 7R
Weast RC (ed) (1972) Handbook of chemistry and physics 53rd edition. CRC, Boca Raton, FL,
p 54
Tests to Ensure Quality of Dairy Products
7
Now-a-days the use of chemicals in the dairy industry is growing at a rapid rate. The
dairy industry uses a number of chemicals to compete with their competitors to
ensure quality of manufactured milk and milk products. Thus, a number of chemical
compounds enter the milk and related products either intentionally or unintention-
ally. Such contamination can occur during milk production, processing or packaging
and can pose health-related issues when consumed. Contaminants like veterinary
drugs, aflatoxin M1, pesticides, heavy metals get introduced into milk due to feeding
poor quality feed, irresponsible use of veterinary drugs (or failure to observe
withdrawal period for antibiotics) and due to poor farm management or practices.
Such chemicals get excreted into milk as residues and may give rise to food safety
problems. Apart from contamination occurring at production stage, chemical
residues may enter milk during processing also. These residues are generally the
leftover chemical/sanitizer used for cleaning or sterilization, mixing of CIP
chemicals/water accidentally into milk storage tanks or by use of hard water during
processing or water contaminated with heavy metals like arsenic, mercury, etc.
Contaminants like bisphenol A, tin, lead, antimony, benzophenone, and
perfluoroctanoic acid can also enter during packaging stage through packaging
materials or by packaging machines (lubricants). The quality assurance personnel
should ensure that the entry of such contaminants in milk should be prevented or if
present should be below an acceptable limit. Considering the above-mentioned
points, this chapter will cover various tests which will help in determining the
presence of such contaminants to prevent the occurrence of any food safety hazard.
7.1 Determining Strength of Washing Solution
Milk is a biological fluid, which is a good source of nutrients like proteins, salts,
lactose, and vitamins, these nutrients make milk as an excellent material for the
growth of microorganisms. The deposition of milk constituents on heating can also
cause the scaling of heat exchangers, pipelines and other milk contact surfaces. The

https://doi.org/10.1007/978-981-15-4167-4_7
176 7 Tests to Ensure Quality of Dairy Products
deposition of traces of milk in the pipes and valves deteriorates the quality of milk. In
order to prevent this, the cleaning and sanitation of all milk contact surfaces like
pipelines, storage tanks, silos, heat exchanger, and packaging machines are of
utmost importance to meet the sanitary and phytosanitary requirements of the
industry. The strength of the detergents or washing solution and sanitizers is affected
by the strength of the detergents and sanitizers used.
The cleaning detergents are classified into two classes: alkaline and acid, which
are often formulated with surfactants, chelators, and emulsifiers to improve the
effectiveness of the detergent. For detergent to be most effective, it should be
an alkaline solution containing chelators and surfactants along with a wetting
agent, which helps them to float off. The emulsifier breaks the oil film by reducing
the interfacial tension.
Alkaline Detergents
It is a mixture of alkali, polyphosphates, and wetting agents, none of the single
substance, when used alone, can meet the requirements of a good cleaning agent.
Basic alkalis like soda ash, sodium metasilicate, caustic soda, and trisodium
phosphate make the bulk of most of the dairy cleaners. A combination of two or
more types of such substances overcome the weakness of a single compound and
increases the cleaning power of a detergent synergistically. Caustic soda is used for
the dissolution of milk proteins and has a germicidal action. Soda ash is an inexpen-
sive form of alkali having good emulsifying and flocculating property but it cannot
be used with hard water as it may lead to development of milk stones by precipitation
of calcium carbonate. Trisodium phosphates or other higher phosphates are easily
soluble having excellent emulsifying and flocculating powers. Phosphates and
metasilicates are good water softening agents and are capable of holding the milk
soil in suspension thus providing a complete cleaning effect.
Acid Detergents
The acid detergents are used to remove the milk stone, scaling due to the deposition
of calcium and magnesium carbonates. They are more effective against bacteria than
alkaline detergents. The most common type of acid detergents are nitric acid,
phosphoric acid. Nitric acid removes milk stones by attacking proteins. It has the
ability to form chromium oxide on the surface of equipment made of stainless steel
(contains chromium) thus preventing the leaching of iron into the milk. It is
generally used at a concentration of 1–1.5%. Phosphoric acid also removes milk
stones and is used at a concentration of 2–3%. Addition of an acid-stable surfactant
improves its cleaning performance. Generally, commercial aqueous blends of acid
detergents contains nitric acid (5–30%) and phosphoric acid (15–40%). Nowadays
enzymes like lipases and proteases are also added to the detergent mixture which
enhances the removal of milk deposits of protein, lactose, or minerals.
The strength of a washing or cleaning solution is expressed in terms of NaOH.
The strength of the alkali solution used for cleaning is generally maintained between
0.5 and 2% depending on the type of cleaning surface. The repeated use of washing
solutions in automatic machines gets diluted or before the use of a new cleaning
7.2 Determination of Available Chlorine in Hypochlorite Solution 177
solution, checking of its strength is important. The strength of the cleaning solution
in terms of its alkalinity or acidity is determined by titrating a known amount of the
cleaning solution with an acid or a base.
Alkalinity Test of Cleaning Solution

Reaction
NaOH þ HCl ! NaCl þ H2 O
Take 10 ml of the washing solution in a 100-ml volumetric flask and make up the
volume with distilled water. Transfer 10 ml of this solution into a 100 ml conical
flask. Add a few drops of phenolphthalein indicator and titrate with 2.5 N
hydrochloric acid to form a colorless solution (pink color disappears). Record the
amount of acid consumed as A.
Calculation: % caustic (NaOH) ¼ A 10.
Acidity of Cleaning Solution

Transfer 10 ml of the cleaning solution (nitric acid) in 100 ml volumetric flask and
dilute to 100 ml with distilled water. Pipette out 10 ml of this solution in a conical
flask and add few drops of phenolphthalein indicator. Titrate against 0.1 N NaOH till
Calculation: % nitric acid ¼ amount of NaOH consumed 6.3
Also, to check the strength of the caustic solution or acid solution the specific
gravity can be checked using hydrometer at 27 C and matching it with the specific
gravity as mentioned by the supplier on the certificate of analysis.
7.2 Determination of Available Chlorine in Hypochlorite

Solution
According to the United States Environmental Protection Agency (EPA)

specifications, sanitizers are the compounds which possess antimicrobial activity
against bacteria, fungi, and virus. A sanitizer can kill or irreversibly inactivate at least
99.9% of biolife present on a surface. The sanitizers are generally toxic in nature, the
common sanitizers used are chlorine, iodine, phenol, or quaternary ammonium
compounds. A sanitizer to be used in a dairy industry should posses properties
like noncorrosive, inexpensive, nontoxic, quick acting, and water soluble, should not
lose its activity at high or low temperature.
Chlorine-Based Sanitizers
Chlorine-based sanitizers have a broad-spectrum antimicrobial activity, which act on
the microbial membranes by inhibiting the enzymes participating in glucose metab-
olism which causes a lethal effect on the DNA leading to the oxidation of the cellular
protein. The examples of chlorine-based sanitizers are liquid chlorine, hypochlorites,
inorganic chloramines, and organic chloramines. These sanitizers form
hypochlorous acid (HOCl, the most active form) in solution which gives it the
germicidal property. The active chlorine shows activity at low temperature and
leaves a minimal residual effect on the contact surface.
Iodine-Based Sanitizers
The iodine-based sanitizers are called as iodophors, as they contain free iodine as an
active molecule. These sanitizers contain a surfactant as a carrier agent. Iodophor
solutions are most potent at low pH and have broad-spectrum activity like the
chlorine bases sanitizers. These compounds show germicidal activity against bacte-
ria (especially nonspore-forming bacteria), viruses, yeasts, molds, fungi, and
protozoans.
Quaternary Ammonium Compounds

These compounds have a general formula as [R4N]+X (R is alkyl group). Quater-
nary Ammonium Compounds (QACs) are positively charged, hence they act by
attracting the negatively charged bacterial proteins. QACs are found to be active and
stable over a broad temperature range. They also possess the properties of a
detergent. Thus, they are less affected by light soil than are other sanitizers.
Peroxyacetic Acid
This compound is known for its germicidal action, which remains for a long period.
Peroxyacetic acid is relatively stable when used with a strength of 100–200 ppm.
This compound is free from phosphates and does not form foam when used. Another
advantage of this compound is that they are less corrosive, can tolerate hard water
and are biodegradable. Peroxyacetic acid solutions are found to be useful in remov-
ing biofilms.
The strength of the chlorine solution decreases with time thus making it necessary
to check its strength before use. The determination of chlorine in the hypochlorite
solution is based on the reaction between the available chlorine in the hypochlorite
solution and iodine liberated from an acidic solution of potassium iodide. The
liberated iodine is titrated against sodium thiosulfate using starch as an indicator.
The amount of available chlorine is calculated from the volume of sodium thiosulfate
consumed.
If sodium hypochlorite (NaOCl) is used as chlorine sanitizer
(a) [CH3COOH + KI ! CH3COOK + HI] 2

(b) NaOCl + 2HI ! NaCl + H2O + I2
By adding a and b
(c) NaOCl + 2KI + 2CH3COOH ! NaCl + 2CH3COOK + H2O + I2

(d) 2Na2S2O3 + I2 ! Na2S4O6 + 2NaI
If bleaching powder, i.e., calcium hypochlorite [Ca(OCl)2] is used as chlorine

sanitizer
7.3 Determination of Iodine in Chemical Sanitizer 179
(e) [CH3COOH + KI ! CH3COOK + HI] 2

(f) Ca(OCl)2 + 2HI ! CaCl2 + H2O + I2
By adding e and f
(g) Ca(OCl)2 + 2KI + 2CH3COOH ! CaCl2 + 2CH3COOK + H2O + I2

(h) 2Na2S2O3 + I2 ! Na2S4O6 + 2NaI
Pipette 5 ml of the sodium hypochlorite sample into a 250-ml volumetric flask.

Make up the volume with distilled water and mix well. Pipette 50 ml of this diluted
solution in a 250-ml conical flask. Add 2 g potassium iodide crystals to the solution
and dissolve potassium iodide then add 10-ml glacial acetic acid. Available chlorine
that is liberated from hypochlorite by the action of acid liberates an equivalent
amount of iodine from potassium iodide which produces a yellowish-brown color.
Immediately titrate the mixture against N/10 sodium thiosulfate until the brown
color changes to light straw yellow. Immediately add 1% of the freshly prepared
starch solution and titrate till the color disappears. Note the reading of N/10 sodium
thiosulfate as V ml. Make the blank determination using the same reagents and
deduct from V ml. Record the values of both the titration in the appropriate Table.
Calculation
1 ml of N/10 sodium thiosulfate ¼ 0.003546 g available chlorine
V ml of N/10 sodium thiosulfate ¼ (V ) (0.003546) g available chlorine
(in 50 ml diluted solution)
Therefore, available chlorine in 250 ml diluted solution ¼ V0:003546250
50 g
available chlorine ¼ available chlorine in 5 ml of stock solution
1 ml of stock solution will contain ¼ V0:003546250
505 g available chlorine
Percent chlorine ¼ V0:003546250100
505 :
7.3 Determination of Iodine in Chemical Sanitizer
Pipette 100 ml of sanitizer test solution in a 250-ml conical flask and titrate against
0.1 N sodium thiosulfate till disappearance of yellow color.
Calculation
1 l of 1 N sodium thiosulfate ¼ 127 g/l of iodine (the equivalent weight of iodine is
127)
1 l of 0.1 N sodium thiosulfate ¼ 12.7 g/l of iodine
1 ml of 0.1 N sodium thiosulfate ¼ 0.0127 g/ml of iodine
1 ml of 0.1 N sodium thiosulfate ¼ 12.7 mg/l or 12.7 ppm of iodine
1 ml of 0.1 N sodium thiosulfate ¼ 12.7 mg/l of iodine
Iodine concentration (mg/l) ¼ Volume of sodium thiosulfate used 12.7
Table 7.1 The sanitary condition of the can

Counts/lit capacity Sterility
<1000 Satisfactory
1000–5000 Fair
>5000 Unsatisfactory
7.4 Assessing Sterility of Dairy Equipment
Cleaning and sanitization are some of the important dairy operations. Improper
cleaning and sterilization may lead to cross contamination of milk and milk products.
So, in order to ensure that the milk contact surfaces are cleaned and sterilized
properly the microbial load on their surface must be checked periodically. The
sanitary conditions can be checked either by rinse or by swab tests. Milk cans,
pails, containers, or other small equipment can be tested by rinse method while silos,
storage tanks, vats, pipelines, heat exchangers, and other large equipment can be
tested by swab test.
Rinse Test
Add 500 ml of phosphate buffer or 0.85% saline in the container say milk can or
container to be tested. Rotate for 10–12 times gently such that whole of the internal
surface gets wet. Transfer the sample to a sterile sample bottle. Take 1 ml of this
sample into a Petri dish and pour melted agar (45 C). Leave the Petri plate
undisturbed for the agar to set. Incubate the Petri plate at 37 C for 48 h. Count
the number of colonies developed and express the result as cfu (colony forming
unit)/ml. To express the results in terms of counts per can ¼ cfu/ml 500.
Colony count per liter capacity ¼ count per can/capacity of a can (Table 7.1).
Swab Test
Swab preparation: Take a piece of metal wire or a glass rod long enough to wind the
cotton wool at one end over a length of 50 mm and empty space to hold the swab
comfortably. Generally, the length of a swab is 35-cm long. The cotton wool should
be secured with a thread.
Place a swab in a test tube containing phosphate buffer or 0.85% saline. Plug the
mouth of the tube with a cotton plug and autoclave for 15 min at 121 C. Take the
swab and remove excess liquid by rolling it against the walls of the tube. Rub the
swab back and forth with pressure and rotating action over an area of 10 10 cm2
from different parts of the surface. Dip the swab in the test tube and mix the swab
properly before use. Transfer 1 ml of the well-mixed swab sample into Petri plate
and pour agar media and allow it to set. A higher dilution can also be prepared.
Incubate the Petri plate at 37 C for 48 h and count the colonies developed in the
Petri plate and express as cfu/ml. The sanitary conditions can be judged from
Table 7.2.
7.5 Hardness Determination of Water 181
Table 7.2 Sanitary No. of colonies/1000 cm2 area Sterility

conditions of the area
<5000 Satisfactory
under test
5000–25,000 Fair
>25,000 Unsatisfactory
7.5 Hardness Determination of Water
The hardness of water is due to the presence of bicarbonates, chlorides, and sulfates
of calcium and magnesium. When such water is treated with soap, it gets precipitated
due to the formation of the insoluble salts of calcium and magnesium. Thus, the hard
water does not form lather with soap. Hardness of water is measured in terms of
calcium carbonate (due to the presence of calcium and magnesium ions). It is of two
types:
1. Temporary hardness—The hardness which is contributed by the bicarbonates of

calcium and magnesium and can be easily removed by boiling.
2. Permanent hardness—Chlorides and sulfates of calcium and magnesium are
responsible for this type of hardness which cannot be removed by boiling.
Hardness of water is done to check its quality. It is defined as calcium and

magnesium ion content and is expressed as parts per million (ppm) of calcium
carbonate (by weight) as most analyses do not distinguish between Ca2+ and Mg2+
and majorly hardness is caused by carbonate mineral deposits.
Water hardness is usually noticed because of the inability to form lather with soap
as the divalent calcium and magnesium ions cause the precipitation of the soap scum
due to the formation of their insoluble salts. Hard water also results in the formation
of the scaling in the boiler tubes or in the plate heat exchangers. The scaling due to
hard water is formed when the water comes in contact with the dissolved carbonates
resulting in the formation of insoluble calcium carbonate. The scaling can be as
severe that it can block the pipelines from inside and can also reduce the heat transfer
rates in the boiler and heat exchangers (Table 7.3).
Principle
A water sample is buffered to pH 10.1 and taken into a conical flask. Addition of
Erichrome Black-T (EBT) indicator to a solution containing calcium and magnesium
ions, there occurs color change to wine red. Ethylenediaminetetraacetic acid
(EDTA), is used as a titrant, forms complex with magnesium and calcium ions,
which leads to the dissociation of the ionic complex formed with the indicator.
EDTA binds the metal ions as it can lose four H+ on complete neutralization thus the
four acid oxygen sites and the two nitrogen atoms contain unpaired electrons,
resulting in the formation of coordination bonds with the metal ions. The complex
formed is stable and the conditions of its formation can be controlled for a particular
Table 7.3 Hardness of Calcium carbonate (ppm) Types of water

water
0–43 Soft
43–150 Slightly hard
150–300 Moderately hard
300–450 Hard
450 and above Very hard
type of ion metal ion. The complex formed between EDTA and the Mg and Ca ions
is blue in color, which signifies the end point of the titration.
Role of Indicator
The EDTA solution is used to determine the hardness of water. The magnesium ion
forms a wine red complex with Erichrome Black T indicator. A small amount of this
complex is present in the solution during the titration which forms complex with
EDTA. As the EDTA–calcium ion complexation increases, it displaces the magne-
sium ion from the indicator–magnesium ion complex formed previously leaving the
indicator to its original uncombined state which is blue in color, establishing the end
point of the titration.
Role of Buffer
The pH of the solution during the estimation of hardness is adjusted at 10 using the
ammonia/ammonium hydroxide buffer. At this pH the NH3/NH4+ buffer, keeps
EDTA (H4B) in the form of HB3, making it highly specific for EDTA to complex
with divalent cations.
Reaction
Taking H4B, H3In, and MgIn as the formulas for EDTA, Erichrome Black T, and
magnesium indicator complex respectively, the following reactions take place dur-
ing the titration:
A. Titration reaction:

HB3 ðaqÞ þ Ca2þ ðaqÞ!CaB2 ðaqÞ þ Hþ ðaqÞ also for Mg2þ
B. End point reaction:
HB3 ðaqÞ þ MgIn ðaqÞ!MgB2 ðaqÞ þ HIn2 ðaqÞ

wine red ! sky blue
Since the indicator requires a trace of Mg2+ to operate properly, a little magne-
sium ion can be added to each solution and the effect of the added Mg2+ can be
subtracted by titrating a blank.
7.5 Hardness Determination of Water 183
Reagents
(a) Buffer Solution: Dissolve 1.179 g of EDTA in 50 ml of distilled water and add
16.9 g of ammonium chloride to it. Further, add 780 mg of magnesium sulfates
and 143 ml of ammonium hydroxide solution using a measuring cylinder and
mix thoroughly. Make the volume up to 250 ml mark by adding distilled water.
(b) EBT indicator: Dissolve 0.5 g of Erichrome black T in distilled water and make
the volume exactly. Prepare the reagent freshly.
(c) Standard EDTA solution (0.02 M): Dissolve 3.723 g of EDTA sodium salt in
distilled water and make up the volume up to 1000 ml mark.
Procedure
1. Determination of Total Hardness
(a) Pipette 20 ml of the water sample in a clean 250 ml conical flask.
(b) Add 2 ml of Ammonia buffer to maintain the pH between 9 and 10.
(c) Add few drops of EBT indicator which turns the sample color to wine red in
color.
(d) Rinse the burette with EDTA and then fill it with 0.02 M EDTA solution to
zero level.
(e) Titrate the sample against the EDTA solution till the appearance of a blue
color indicating the end point of the titration (at this point all Ca and Mg ions
are complexed with EDTA). Note down the burette reading.
(f) Repeat the titration for concordant values.
2. Determination of permanent hardness
(a) Take 250 ml of water sample in 500 ml capacity volumetric flask and boil it
gently for 30 min, cool it and filter it using ordinary filter paper.
(b) Mix the filtered water sample and take 20 ml of water sample.
(c) Further, treat the sample as done for Total hardness.
(d) Note down the burette reading.
(e) Repeat the titration for concordant values.
Calculation Total hardness as ppm of CaCO3
50 Normality of EDTA Volume of EDTA used in titration 1000

Total Hardness ¼
Volume of water sample taken
50 Normality of EDTA Volume of EDTA used in titration 1000

Permanent Hardness ¼
Volume of boiled water sample taken
Temporary Hardness ¼ Total Hardness Permanent Hardness
Result
Total Hardness of the given sample of water ¼ . . .. . .. . .. . .. . .. . .ppm
Temporary Hardness of the given sample of water ¼ . . .. . .. . .. . .. . .. . .ppm
Permanent Hardness of the given sample of water ¼ . . .. . .. . .. . .. . .. . .ppm
7.6 Biological Oxygen Demand of Dairy Effluent
Biological oxygen demand (BOD) is a measure of the degradable organic matter

present in water. It is a measure of the amount of oxygen required by the
microorganisms for degradation of the biologically degradable organic matter
under aerobic conditions.
Reagents
1. Phosphate buffer: Weigh 8.5 g potassium dihydrogen phosphate, 21.75 g
dipotassium hydrogen phosphate, 33.4 g disodium hydrogen phosphate
heptahydrate, and 1.7 g ammonium chloride and dissolve in 500 ml distilled
water followed by adjusting the volume up to 1000 ml with distilled water. The
buffer solution should have a pH of 7.2.
2. Magnesium sulfate solution: Dissolve 22.5 g magnesium sulfate in distilled
water and make the volume up to 1 L.
3. Calcium chloride solution: Dissolve 27.5 g anhydrous calcium chloride in
distilled water and dilute to 1 L.
4. Ferric chloride solution: Dissolve and make up the volume of 0.25 g ferric
chloride to 1 L in distilled water.
5. Manganese sulfate solution: Dissolve 480 g of manganese sulfate (tetrahydrate
salt) in distilled water. Filter the solution and make the volume to 1 L. The
prepared solution should not give color with starch when added to acidified KI
solution.
6. Alkali azide iodide reagent: Dissolve 500 g sodium hydroxide and 135 g sodium
iodide, respectively, in distilled water and dilute to 1 L. Dissolve 10 g sodium
azide in 40 ml water separately and add it to 1 L of prepared solution. This
solution should not give color with starch solution when diluted and acidified.
7. Concentrated sulfuric acid.
8. Sodium thiosulfate (0.025 N)
9. Starch solution: Dissolve 5 g starch in 800 ml of boiling water with continuous
stirring. Dilute to 1 L and boil again for few minutes. Leave undisturbed
overnight. Use clear supernatant.
10. Seeding material: The purpose of seeding is to introduce the biological matter to
oxidize the organic matter in the wastewater. The seed material is generally a
settled domestic sewage or cow dung stored at 20 C for 24–36 h. The concen-
tration of seed solution is 1–2 ml/L of distilled water.
Procedure
• To a stainless steel vessel of 5 L capacity, add 2 L of distilled water (distilled
using glass distillation apparatus). The distilled water should be saturated with
oxygen before use (maintain the temperature around 20 C). Add 1 ml of
phosphate buffer, manganese sulfate solution, calcium chloride solution, and
ferric chloride solution for each liter of this water. Mark this as dilution water.
• Take 15 ml of effluent and 975 ml of dilution water. Add the seeding material at
0.1–1%.
7.7 Chemical Oxygen Demand of Dairy Effluent 185
• Fill this seeded material in three separate bottles (BOD bottles). Incubate two
bottles at 20 C for 5 days.
• To one bottle add 1.5 ml of manganese sulfate and 1.5 ml of alkaline iodide
solution (place the tip of the pipette in each case below the surface of the liquid).
• Replace the stopper without inclusion of air and mix the contents thoroughly.
• Allow the precipitate to settle. When at least 100 ml of clear supernatant, add 2 ml
of concentrated sulfuric acid by the side of the bottle and stopper it. Mix gently by
inversion to ensure uniform distribution of iodine in the bottle.
• Take 200 ml of solution and titrate immediately against sodium thiosulfate
solution using 1 ml of starch indicator, when the color becomes pale yellow
and completing the titration to the disappearance of blue color.
• 1 ml of 0.025 N sodium thiosulfate solution is equal to 0.2 mg of dissolved
oxygen.
• After incubation for 5 days; determine the dissolved oxygen in two bottles.
• Prepare the blank, in the same manner, using distilled water in place of sample
and incubate for 5 days.
Calculation
BOD ðmg=lÞ ¼ ðD1 D2 Þ ðC1 C2 Þ=V
D1 ¼ ml of 0.025 N sodium thiosulfate used for sample

D2 ¼ ml of 0.025 N sodium thiosulfate used for sample after incubation
C1 ¼ ml of 0.025 N sodium thiosulfate used for blank
C2 ¼ ml of 0.025 N sodium thiosulfate used for blank after incubation
V ¼ volume of the sample taken
7.7 Chemical Oxygen Demand of Dairy Effluent
Chemical oxygen demand (COD) is the measure of the oxygen required for oxida-
tion of the organic matter. The organic matter gets oxidized by potassium dichromate
in the presence of sulfuric acid releasing carbon dioxide. The excess of potassium
dichromate is titrated against ferrous ammonium sulfate. The potassium dichromate
consumed (i.e., the amount of ferrous ammonium sulfate consumed in blank and
sample titrations) gives the amount of oxygen required for the oxidation of the
organic matter.
The chlorides, straight-chain aliphatic compounds, nitrites, aromatic
hydrocarbons, fatty acids, and iron are the major interfering radicals. Mercuric
sulfate is added to remove the interferences due to chlorides (0.4 g mercuric sulfate
complexes 40 mg chloride in form of poorly ionized mercuric chloride) while
interference caused by nitrite is removed by addition of sulphamic acid to potassium
dichromate solution (nitrite has a COD of 1.1 mg/mg N). Silver sulfate acts as a
catalyst (accelerates the oxidation of straight-chain aliphatic and aromatic
compounds). A final concentration of sulfuric acid at 18 N is required to be

maintained to ensure complete oxidation of organic matter.
Reagents
1. Standard potassium dichromate: Dissolve 12.259 g of potassium dichromate
dried at 105 C for 24 h in distilled water. Add 120 mg of sulphamic acid to it
and dilute to 1 L.
2. Sulfuric acid concentrate: Add 22 g of silver sulfate to 4 kg of concentrated
sulfuric acid. Keep overnight for dissolution.
3. Standard ferrous ammonium sulfate (0.1 N): Dissolve 39 g of ferrous ammonium
sulfate in distilled water. Add 20 ml of concentrated sulfuric acid, cool, and dilute
to 1 L.
4. Ferroin indicator: Dissolve 1.485 g 1, 10-phenanthroline monohydrate and
695 mg ferrous sulfate in water and dilute to 100 ml. (Ready-made indicator is
also available in the market).
5. Mercuric sulfate crystals.
Procedure
Take 0.4 g mercuric sulfate in a COD flask and add 20 ml of sample. Mix properly so
as that all the chlorides are converted into poorly ionized mercuric chloride. Add
glass beads to the flask along with 10 ml of standard potassium dichromate solution.
Add 30 ml concentrated sulfuric acid and mix well. If the color turns green, add more
potassium dichromate and sulfuric acid (or take a fresh sample with smaller quan-
tity). Reflux for 2 h at 150 C then cool and wash the condenser with 60 ml of
distilled water. Cool and titrate with standard ferrous ammonium sulfate solution
(0.1 N) using ferroin indicator till the color turns from green blue to wine red/reddish
brown. Similarly, carry a blank titration using distilled water instead of effluent
sample. The COD is expressed as mg/l of O2.
Calculation
COD ðmg=lÞ ¼ ðA BÞ 8000 N=ml of sample
where, A ¼ ml of ferrous ammonium sulfate consumed for blank, B ¼ ml of ferrous

ammonium sulfate consumed for sample, N ¼ Normality of ferrous ammonium
sulfate used.
7.8 Detection of Aflatoxin M1 in Milk
Mycotoxins are the secondary metabolites produced by fungi. Mycotoxin is made

from two Greek words “mukos” and “toxikon” which means “fungus” and “poison,”
respectively. The class of fungi which produce mycotoxins are Alternaria, Aspergil-
lus, Trichoderma, Acremonium, and Penicillium. The various types of mycotoxins
produced by several fungi are listed in Table 7.4.
7.8 Detection of Aflatoxin M1 in Milk 187
Table 7.4 List of mycotoxins produced by various fungi

Fungi Mycotoxin Source
Aspergillus flavus Aflatoxin B1, Corns, peanuts, barley, and
B2 oats
Metabolites of B1 and B2 Aflatoxin M1, Milk and milk products
M2
Penicillium verrucusum, Aspergillus Ochratoxin A Corn, wheat, barley, beer, and
ochraceus wine
Penicillium, Aspergillus Patulin Apples
Fusarium trichothecenes Trichothecenes Corn, barley, oats, and wheat
Fusarium roseum Zearalenone Corn, barley, oats, and wheat
Fusarium moniliforme Fumonisins Corn and other cereal grains
Pennicilium camemberti Cyclopiazonic Grains, legumes, milk, meat,
acid and cheese
Aflatoxin is mainly produced by Aspergillus species like Aspergillus flavus,

Aspergillus parasiticus, and Aspergillus nominus under hot and humid conditions
mainly in tropical countries in the south and southeast area. The mycotoxins are not
proteins thus they are not detected by the immune system of humans and animals.
Aflatoxins are heterocyclic compounds that are closely related to each other due to
similarity in their structure. The term aflatoxin was first coined in 1960 due to the
outbreak of the famous turkey “X” disease in London which led to the death of large
number of livestock that were fed with contaminated groundnut feed. The optimum
temperature for the growth of Aspergillus flavus is 30 C while it can grow between
10 and 45 C at a relative humidity of 80%. The proper storage of the feed can
prevent its contamination by Aspergillus species. These fungi can also contaminate
the food commodity at different stages like production, processing, transportation,
and storage.
Aflatoxin is found in various forms in nature like B1, B2, G1, G2, M1, and M2,
which differ slightly in their structures (Fig. 7.1). Aflatoxin M1 and M2 are the
hydroxylated form of aflatoxin B1 and B2, respectively, produced in the milk of the
lactating animals, when they are fed on contaminated feed containing aflatoxin B1
and B2. The aflatoxins are grouped on the basis of fluorescence like B-type aflatoxin
give blue fluorescence in the UV range, G group gives yellow-green fluorescence
while the M group shows blue fluorescence. Aflatoxins of G group have lactone ring
in their structure while B and M type have cyclopentane ring. The furan moiety is
saturated in aflatoxin B2, G2, M2, and unsaturated in aflatoxin B1, G1, and M1, the
double bond in the furan moiety makes the aflatoxin B1 and G1 more toxic than B2
and G2 and the order of toxicity of aflatoxins is AFB1 > AFG1 > AFB2 > AFG2.
AFM1 is less carcinogenic (around one-tenth) than its precursor AFB1 and
genotoxicity is around one-third. International Agency of Research on Cancer
(IARC) classified aflatoxin B1 as group 1 carcinogen (carcinogenic to humans)
and aflatoxin M1 as Group 2A carcinogen (probably carcinogenic to humans). The
conversion of AFB1 to AFM1 occurs in the liver by the enzyme cytochrome P450
and is secreted in milk, about 5% of the ingested AFB1 is converted into AFM1 by
Fig. 7.1 Structures of different aflatoxins
Table 7.5 Food regulatory FSSAI 0.5 ppb (milk), 30 ppb (other food items)
standards for aflatoxin M1
Codex 0.5 ppb
in milk
USFDA 0.5 ppb
EU 0.05 ppb (milk), 0.025 ppb (infant foods)
the animal. The level of AFM1 in milk decreases to undetectable levels after 72 h of
the ingestion of AFB1. The epoxide form of AFB1, i.e., AFB1-8,9-epoxide that is
formed by cytochrome P450 in the liver, binds with the DNA causing mutations,
hepatotoxicity, teratogenicity, and carcinogenicity in humans and animals. Aflatoxin
also affects the animals by causing liver disease, a high level of aflatoxin causes
aflatoxicosis like liver lesions, weight loss, reduction in milk production, reduced
feed consumption while chronic effects of the low-level consumption of aflatoxin in
cattle lead to immunosuppression, reduced reproductivity, and reduced feed effi-
ciency. AFM1 is soluble in mild polar solvents like acetonitrile, chloroform, metha-
nol, dimethylsulfoxide, and its solubility in water is about 10–30 mg/l. Food
regulatory standards for aflatoxin M1 in milk are presented in the Table 7.5.
Determination of Aflatoxin M1 in Milk, Cheese, and Khoa

Apparatus
Separating funnel 250 ml, centrifuge, TLC/HPTLC plates, Whatman
No. 1, borosilicate vials with Al screw capping.
Reagents
1. Solvents like acetic acid, acetone, acetonitrile, chloroform, ether, hexane, ethanol,
isopropanol, and toluene.
2. Sodium chloride solution (40%).
3. Silica gel for column chromatography: Stir the silica gel in methanol for 1 h and
filter. Then treat it again with chloroform in a similar manner and wash. The silica
gel is then activated by drying at 105 C for 1 h. Add water at 1 ml/100 g, shake to
mix well. Store in airtight container for 15 h.
4. Sodium sulfate anhydrous.
5. Celite.
6. AFM1 standard.
7. Densitometer.
Sample Preparation
1. In a 250-ml separating funnel, add 50 ml milk, 10 ml saturated sodium chloride
solution (40%), 120 ml chloroform at 30 C, shake it well and leave undisturbed
for 2 min to facilitate the separation. In case of milk powder, reconstitute 5 g of
powder in 50 ml water, for solid products like khoa and cheese, blend 15 g of
sample with 1 ml of saturated salt solution, 5 g celite, and 100 ml of chloroform,
mix well for 1 min in a blender.
2. Remove the chloroform layer in a 125 ml Erlenmeyer flask. To the separated
chloroform layer, add 10 g of anhydrous sodium sulfate. Filter the contents into
the 100 ml graduated cylinder and save the filtrate for further use in column
chromatography.
Column Chromatography
1. Fill the column with chloroform to half of its capacity.
2. Add 2 g of the silica gel slurry prepared with chloroform.
3. Add 2 g of sodium sulfate to the gel.
4. Then drain the excess of chloroform and rinse the silica column sides with
chloroform.
5. Add the sample in the column and let it drain completely through the column.
6. Collect the washings of the sample from the column into a cylinder previously
rinsed by chloroform.
7. Wash the column with 25 ml of toluene–acetic acid solution mixed in 9:1 ratio.
The colored compounds get removed in this step.
8. Again wash the column with 25 ml of hexane–ether–acetonitrile solution (5:3:2)
to remove the fat.
9. Elute the aflatoxin M1 using 40 ml chloroform–acetone mixture (4:1).
10. Evaporate the eluate to dryness and use for TLC/HPTLC.
Thin Layer Chromatography

1. Dissolve the dried sample residue in 0.1 ml of acetonitrile–benzene mixture (1:9)
and mix well.
2. Apply 20 μl spot of the test sample on the TLC or HPTLC plate. Also apply 2, 4,
6, 8, 10 μl spots of AFM1 standard (0.25 μg/ml).
3. Develop the plate in the solvent mixture containing chloroform–acetone–
isopropanol (87,10:3).
4. Calculate the concentration of AFM1 as follows:
Aflatoxin M1 ðppb=μg=kgÞ ¼ ðS A BÞ=ðW X Þ
Where,
A ¼ concentration of the aflatoxin M1 standard in μg/ml.

B ¼ volume of final dilution of the sample extract in μl.
S ¼ volume of aflatoxin M1 standard which matches with the sample.
X ¼ volume of the sample extract applied on the plate which matches with the
fluorescence intensity equivalent to the corresponding aflatoxin M1 standard.
W ¼ volume of the sample contained in the final extract (weight or volume of test
portion filtrate volume)/120
Determination of Aflatoxin M1 and M2 in Liquid Milk by Using Liquid

Chromatography
Principle
The aflatoxins M1 and M2 in the milk sample are extracted using C18 cartridge
and eluted with ether into the silica column. Then it is again eluted with a solution
containing dichloromethane and alcohol, then aflatoxin M1 is derivatized using
trifluoracetic acid (TFA). The peaks obtained for the derivatized aflatoxin is then
detected using fluorescence detector.
Reagents
1. Acetonitrile, dichloromethane, isopropyl alcohol, ethanol (reagent grade), hex-
ane, methanol, TFA, ether (0.01% ethyl alcohol preservative), and water
(deionized, filtered through 0.45 μm filter).
2. Acetonitrile wash solution (5%).
3. Methylene chloride–alcohol elution solution (95:5).
4. Mobile phase: Water: Isopropyl alcohol: acetonitrile (80:12:8). Sonicate the
solution to degas it.
5. Aflatoxin standard solution: Aflatoxin M1 and M2 from sigma. Prepare the stock
solution (200 μg/ml of aflatoxin M1 and 100 μg/ml of aflatoxin M2) in acetoni-
trile. Make the working standard solutions of 0.5 μg/ml of aflatoxin M1 and
0.1 μg/ml of aflatoxin M2 in benzene: acetonitrile solution (9:1). To be used in
preparing aflatoxin M1-TFA derivative.
6. Dichlorodimethylsilane (DDS): 5% solution in toluene. Stopper the volumetric
flask and store in cold.
Apparatus
1. Silica gel cleanup columns—0.8 4 cm polypropylene column with Luer tip,
35 μg porous polypropylene bed support disk and 10 ml reservoir.
2. Silica gel cleanup columns packing and preparation: Dry the silica gel 60, having
a particle size of 0.4–0.063 mm in oven at 105 C for 1 h. Cool and add 1% water
on weight basis of silica gel. Transfer to a container, seal it, shake the contents
properly and leave the container for equilibration. Assemble the polypropylene
column assembly and 25 ml vacuum flask. Fill the column to 2 ml with silica gel.
Apply the gentle vacuum so as to pack the bed, then add 1 g anhydrous sodium
sulfate on the top of silica gel bed.
3. Extraction cartridges C18.
4. Fluorescene detector: Adjusting the excitation wavelength at 365 nm and emis-
sion wavelength at 400 nm.
5. LC analytical column: The particle size of the column should be 5 μm C18 silica
bonded gel, having dimensions of 0.4 25 cm.
6. Vacuum regulator to control and maintain full or partial vacuum.
7. Silylated vials for aflatoxin standard solution: Fill the glass vials with 5% DDS
(4,40 -disuccinoylaminodiphenyl sulfone) and heat it at 45–55 C for 40 min.
Discard the solution, rinse the vials with toluene three times and with methanol
three times. Transfer the vials to the oven at 75 C for 20–30 min or till
evaporation of methanol. Cap the vials and store it.
Extraction
1. Attach the longer stem of the C18 cartridge with the luer tip of 30–50 ml syringe.
2. Attach the cartridge and syringe to the vacuum flask and maintain a vacuum of
5 mm Hg pressure.
3. The solvent will pass and be collected into the flask.
4. Prime the cartridge with 5 ml methanol and then 5 ml of water in the stem.
5. Stop the vacuum and move the cartridge–syringe assembly from the stopper to
prevent the loss of the priming solution.
6. Mix the milk sample maintained at room temperature at least ten times.
7. Mix the milk sample with 20 ml of hot water (80 C), to reduce the viscosity
of milk.
8. Replace the cartridge syringe assembly in stopper.
9. Pour the warm milk sample 40 ml in the syringe and pull the liquid gently from
the cartridge at a flow rate of 30 ml/min (the flow should be adjusted such that
aflatoxin gets adsorbed in the column).
10. Add 10 ml of water–acetonitrile wash solution to the syringe and pull it with
vacuum. The wash solution should get removed completely from the column
packing.
11. Again reprime the cartridge with 150 μl of acetonitrile to the inlet bed support
disk and let the solvent to soak in the packing for 30 s.
12. Attach the cartridge to a dry glass or plastic 10 ml luer tip syringe, the same stem
should be retained as the inlet.
13. Insert the silica gel cleanup column in a 250-ml vacuum flask fitted to a rubber
stopper having a single hole.
14. Wash the column with 5 ml ether, then add 7 ml ether to syringe cartridge placed
above the silica gel cleanup column.
15. With the plunger of the syringe, slowly apply the force on the cartridge, to
collect the eluate in the column reservoir.
16. Using vacuum remove the ether from the silica cleanup column at a flow rate of
10 ml/min. Rinse it further with 2 ml of ether, discard the wash solution.
17. Remove the column and stopper from the flask and add 7 ml of
dichloromethane–alcohol solution to the column reservoir.
18. Pull the solvent from the column in the collecting tube at a flow rate of 10 ml/
min using vacuum.
19. Now evaporate the eluate to dryness under nitrogen at room temperature or
under vacuum at less than 35 C.
20. Transfer the residue to the vial with dichloromethane and evaporate to dryness
on steam bath or heating block at less than 50 C under nitrogen. Save the
residue for derivatization.
Liquid Chromatography
1. Prepare the derivative from the residue by adding 200 μl hexane and TFA to dry
the residue in the vial.
2. Shake the contents on the vortex and leave it for 10 min at 40 C in a water bath.
Then evaporate to dryness under nitrogen in a water bath maintained (<50 C).
3. Add 2 ml of acetonitrile (25%) to the vial for the residue to dissolve. Shake well in
vortex for LC analysis.
4. For the derivatization of standard of aflatoxin M1, add 200 μl of hexane and 50 μl
TFA to the silylated vial and mix well.
5. Add 50 μl M1–M2 working standard solution into the hexane–TFA mixture and
vortex for 5–10 s. This is the test solution.
6. Adjust the flow rate of the instrument at 1 ml/min and run water–isopropanol–
acetonitrile solution (80:12:8) for stabilization.
7. Adjust the detector so that 50–100 μl of standard (0.625–1.25 ng of M1,
0.125–0.25 ng of M2) gives 50–70% full-scale reading for aflatoxin M1.
8. Inject the standard 2–3 times till the peak heights become constant, then prepare
the standard curve.
9. Inject the test solution (50–100 μl) and run the analysis. The retention time for
TFA derivative of aflatoxin M1 is 4–5 min while that of aflatoxin M2 is 7 min.
Calculations
M1 or M2 ðppbÞ ¼ H A V Y=H1 V1 W
Where,
H, H1 ¼ peak height of the test solution and standard, respectively.

A ¼ concentration of standard (ng/μl).
V, V1 ¼ volume injected of standard and test solution, respectively.
W ¼ volume of milk taken (20 ml).
Y ¼ final total test solution volume.
7.9 Determination of Melamine in Milk and Infant Formula 193
7.9 Determination of Melamine in Milk and Infant Formula
Melamine (C3H6N6; MW: 126.12) is a polar molecular, heterocyclic triazine

containing three amino groups in its structure. It is a white colored crystalline
powder and has very high nitrogen content of approximately 66% by mass. Since
1950s, it has been used in production of paper finishers, commercial filters, wrinkle-
free textile and nanomaterials by converting it to melamine formaldehyde resins
(MFR). But nowadays it has been illegally added to dairy products by unscrupulous
milk producers, owing to its high nitrogen content to obtain incorrectly higher
protein content, as nitrogen content of milk is used as a metric for milk payment.
The tests such as Kjeldahl estimate the protein nitrogen in milk, which is
overestimated when milk contains melamine. This has led to several health
outbreaks due to the consumption of melamine adulterated food/milk. One of them
was reported in the Republic of China in 2008, where around 294,000 children
became victims of melamine adulterated milk, while six died. Melamine is reported
to have low toxicity, but when it combines with cyanuric acid, it forms insoluble
crystals which can cause kidney stones or kidney toxicity. Observing the food safety
concerns with regard to melamine, the maximum permissible limit for melamine has
been prescribed in some of the food regulations as mentioned in Table 7.6.
Melamine is detected by liquid chromatography (LC-MS/MS), melamine is first
extracted by 2.5% formic acid followed by filtration, centrifugation, and dilution.
The HILIC (hydrophilic interaction liquid chromatography) LC column is used for
the detection of melamine and cyanuric acid (a structural analogue of melamine).
Positive ion mode is used to detect melamine while cyanuric acid is detected using
negative ion mode.
Reagents
1. Melamine.
2. Acetonitrile (LC Grade).
3. Formic acid (purity >95%).
4. Water (LC Grade, Resistivity >18 M-ohm).
5. Ammonium formate (Purity >97%).
6. Mobile Phase A: 0.1% Formic acid in acetonitrile (5:95, v/v). Mix 50 ml of 0.1%
formic acid in water with 950 ml of acetonitrile in 1 L volumetric flask.
7. Mobile Phase B: 20 mM ammonium formate in acetonitrile (50:50). Mix 500 ml
of this solution with 500 ml of acetonitrile in 1 L volumetric flask.
8. 2.5% Formic acid in water.
9. 20 mM ammonium formate in water.
Table 7.6 Standards for melamine in milk and milk products

Regulatory
body Melamine content (max, ppb)
FSSAI 2500 (raw milk), 1000 (powdered infant formula), 150 (liquid infant formula)
EU 2500 (raw milk), 1000 (infant formula and milk powder)
Equipment
1. Liquid chromatography and liquid chromatography column (HILIC,
2.1 150 mm, 5 μm).
2. Mass spectrometer with a triple quadrupole.
3. Centrifuge and microcentrifuge.
4. Ultrasonic bat with heater and timer.
5. Syringe filters made of polyvinylidene fluoride, 13 mm, 0.22 μm.
6. Syringes.
Standard Preparation
1. Stock solution: 10 mg of melamine standard is weighed and transferred to a
100 ml volumetric flask. Add 70 ml of 0.1% formic acid in water and sonicate the
contents of the volumetric flask for 10 min. Make up the volume to 100 ml with
0.1% formic acid in water solution. Use this stock solution for making standard
mixture dilution of 50 μg/ml in a glass scintillation vial.
2. Standard mixture dilutions: 3.6 and 1.4 μg/ml are used to prepare post fortified
control extracts to calculate the matrix effects. Take 720 or 280 μl of 50 μg/ml
standard mix to make 3.6 μg/ml \and 1.4 μg/ml of standard mixture dilution,
respectively. Make up the volume to 10 ml using 0.1% formic acid in water.
3. Dilute mix standards are prepared by diluting 3.6 and 1.4 μg/ml standards ten
times with 0.1% formic acid.
Sample Preparation
1. Take 2 g of the sample powder in 50 ml polypropylene centrifuge tube.
2. To the sample add 14 ml of 2.5% formic acid in water and seal the tube. Dissolve
the sample by vortexing for 15–30 s.
3. Sonicate the tube in ultrasonic bath and mix on multi-vortex for 30 min.
4. Centrifuge the contents of the tube at 4000 rpm for 10 min.
5. Transfer 1.4 ml of the supernatant to 1.5 ml microcentrifuge tube and centrifuge
at 13,200 rpm for 30 min.
6. Transfer the supernatant into a 3-ml plastic syringe and force through a 13 mm,
0.22 μm PVDF filter in microcentrifuge tube (In some cases, some formulations
need more force or twice filtration to obtain a clear solution).
7. Vortex again for 30 s and centrifuge at 13,200 rpm for 30 min.
8. Transfer the supernatant to the 2 ml autosampler vial, avoid the formation of
precipitate.
Instrumental Analysis
1. The instrument operating parameters should be maintained as:
Inlet LC method
(a) Mobile phase A: ACN (Acetonitrile)/0.1% formic acid in water (95:5).
(b) Mobile phase B: Ammonium formate 20 mM/acetonitrile (50/50).
(c) Column temperature 30 C.
(d) Injection volume: 10 μl.
(e) Run time: 14 min.
7.9 Determination of Melamine in Milk and Infant Formula 195
(f) Retention time Melamine: 5.5 min and for cyanuric acid: 3.1 min.
(g) Backpressure: 400 psi and 2000 psi maximum.
2. The column should be equilibrated with Mobile Phase A with a flow rate of
0.4 ml/min for 30–60 min.
3. The sample and standards should be injected using the following sequence:
Mobile phase, extracted matrix standards from 0.25 to 5 μg/g, solvent blank,
control extracts, post-fortified extracts, solvent standards to calculate recovery
and matrix effect, solvent blank, and samples.
Calculation
Use the external standard calibration, the calibration curve should not include the
origin but the matrix blank having a concentration of zero.
Recovery % ¼ calculated from extracted calibration curve.

Matrix effect % ¼ 100 post-fortified sample/solvent standard (same conc.).
Note
Negative control samples: Samples that show signal less than or equal to 10 of the
0.25 μg/g equivalent standard when tested.
Extracted matrix calibration standard: Prepared at 0.25, 0.5, 1, 2.5, 5 μg/g by
adding 10, 20, 40, 100, 200 μl of the 50 μg/ml standard mix in 2 g of the sample.
Pre fortified control standard: Prepared at the level of interest, i.e., 0.5 or 1 μg/g
by adding calculated amount of 50 μg/ml standard to 2 g of the sample.
Post-fortified control samples are the negative extracts to which 50 μl of standard
mixture solutions, i.e., 360 or 140 ng/ml is added at the final step, this sample has a
final concentration of 2.5 and 1 μg/g, respectively. The samples are used to calculate
matrix effects and percent recovery.
Solvent standards: These are used to calculate the matrix effects equivalent to 2.5
and 1 μg/g. The solvent standards are prepared by adding 50 μl of standard mixture
solutions, i.e., 360 and 140 ng/ml to 950 μl of acetonitrile. The solutions will have a
concentration of 18 and 7 ng/ml. This 7 ng/ml solution can be used to determine the
initial instrument suitability.
Determination of Melamine in Milk by TLC

It is a reliable, inexpensive and rapid method of detecting melamine in milk.
Apparatus
1. Potassium permanganate solution (8%).
2. Hydrochloric acid solution: Mix 25 ml of 32% HCl in 50 ml distilled water.
3. Wuster’s reagent: Mix 500 mg, tetramethyl-1,4-phenylammonium chloride in
100 ml acetone.
4. Starch iodine solution: Dissolve 800 mg of KI in 20 ml distilled water and
separately 800 mg starch in 20 ml distilled water. Mix both the solutions in
10 ml ethanol.
5. Mobile phase: 2-Propanol, dichloromethane, and water in 3:1:1. This is the

developing reagent.
Procedure
1. On the glass plates, coat K60 silica gel and allow it to dry.
2. Apply 1 μl of sample using degas sample applicator.
3. Dip the TLC plates in a chamber containing mobile phase (the chamber should
not be saturated with mobile phase) for 35 min or to a distance of 55 mm.
4. Dry the plates and place them in a chamber in a stream of air.
5. Place the plates in potassium permanganate–HCl for 5 min.
6. Dip the plates in wuster’s reagent for 2 s for the formation of deep blue zones.
7. Observe the plates under 550–615 nm using a diode array scanner.
8. If the starch iodine solution is used to develop the zones, the chlorinated plates
(dipped in potassium permanganate–HCl solution) are dipped for 1 s and scanned
under 490–610 nm.
9. Measure the retention factor values (Melamine peak appears at a distance of
around 18.7 mm with the Rf value 0.30).
7.10 Determination of Pesticides in Milk
As per FAO, pesticide may be defined as “Any substance intended for preventing,
destroying, attracting, repelling, or controlling any pest including unwanted species
of plants or animals during the production, storage, transport, distribution and
processing of food, agricultural commodities, or animal feeds or which may be
administered to animals for the control of ectoparasites. The term includes
substances intended for use as a plant growth regulator, defoliant, desiccant, fruit
thinning agent, or sprouting inhibitor and substances applied to crops either before or
after harvest to protect the commodity from deterioration during storage and trans-
port. The term normally excludes fertilizers, plant and animal nutrients, food
additives and animal drugs.” Pesticide residues may be defined as a specified
substance in a food matrix, agricultural commodity, or animal feed which may result
from the use of pesticide(s). This may include a derivative of a pesticide, their
conversion products, metabolites, impurities, reaction products, which are consid-
ered to be of toxicological significance.
Classification of Pesticides
The World Health Organization has classified pesticides on the basis of the risk they
can pose on the health as extremely hazardous, highly hazardous, moderately
hazardous, slightly hazardous, and unlikely to cause acute hazards. They have
been described in Table 7.7.
Pesticides can also be classified on the basis of the target pests and according to
their chemical nature.
Based on the target pest: The classification of the pesticides on the basis of the
target pest is mentioned in Table 7.8.
7.10 Determination of Pesticides in Milk 197
Table 7.7 Classification of pesticide based on the extent of hazard

LD50 value for the rat
Classes of pesticides by WHO Oral Dermal
Іa Extremely hazardous <5 <50
Іb Highly hazardous 5–50 50–200
ІІ Moderately hazardous 50–2000 200–2000
ІІІ Slightly hazardous >2000 >2000
U Unlikely to present acute hazard 5000 or higher
Table 7.8 Classification of pesticides based on the target pest

S. No. Classes Targeted pest/ Use Common example
1 Insecticides To control insects DDT, Malathion
2 Fungicides To control fungi Mancozeb,
Carbendazim
3 Acaricides To control mites Dicofol,
chlorobenzilate
4 Rodenticides To control rats, mice, and rodents Zinc phosphide,
warfarin
5 Fumigants To control insects/other pests by Methyl bromide and
fumigation ethylene bromide
6 Molluscicides To control slugs and snails Metaldehyde, Fentin
acetate
7 Nematicides To control nematodes Aldicarb
8 Plant growth To regulate/enhance the growth and Ethephon, Gibberellic
regulators developments of plants acid
9 Herbicides To control weeds 2,4-D, Paraquat
Classification Based on Chemical Structure The pesticides are classified as

organochlorine, organophosphorous, carbamates, and pyrethroid. This classification
is based on the principle of chemical component or molecule present in it.
Organochlorine These are the chlorinated hydrocarbons that have at least one
covalently bonded atom of chlorine. These pesticides are lipophilic in nature, so
they have the tendency to accumulate in the adipose tissue. They disrupt the sodium/
potassium balance in the nerve fiber, causing the nerve to transmit continuously.
Their use has been phased out as they tend to bioaccumulate and are persistent
organic pollutants. The examples of some organochloro pesticides are DDT, chlor-
dane, heptachlor, aldrin, lindane, and 2,4-D. DDT was first ever synthesized in
187 (Fig. 7.2).
Organophosphate They are the esters of phosphoric acid and are soluble in water.
They operate by inhibition of the acetylcholinesterase, as a result acetylcholine
transfers nerve impulses indefinitely, thus causing weakness or paralysis.
Organophosphates were developed during the early nineteenth century. Examples
Fig. 7.2 Structure of organochlorine pesticides
Fig. 7.3 Structure of some organophosphate pesticides
Fig. 7.4 Structure of some carbamate pesticides
of organophosphorus are malathion, parathion, phorate, methyl parathion, etc.

(Fig. 7.3).
Carbamates These are the esters of dimethyl N-methyl carbamic acid and are less
persistent than the organophosphate and organochlorine pesticides. Their mode of
action is similar to organophosphates as they also inhibit the acetylcholinesterase by
attaching the electrophilic steric carbamoyl sites of the enzymes through
carbamylation. Examples of carbamates are carbaryl, aldicarb, carbofuran, etc.
(Fig. 7.4).
Pyrethroids These are chemical compounds similar to natural pyrethrins that are
synthetically produced. They are also less persistent than organochlorine and organ-
ophosphate pesticides (Table 7.9).
Pesticides gain entry into milk when the animals are fed with the feed
contaminated by pesticides due to their indiscriminate and inadequate use, through
7.10 Determination of Pesticides in Milk 199
Table 7.9 Differentiation Organochlorine Organophosphorus Carbamate

between organochlorine,
Nonpolar Moderately polar Polar
organophosphate, and
Most persistent Less persistent Less persistent
carbamate pesticides
Toxic Most toxic More toxic
water (as an adulterant or when an animal drinks pesticide-contaminated water), use

of chemicals to control ectoparasites, cross contamination, etc.
International and National Regulations That Monitor the Pesticide Residue

in Food Commodities
The application of pesticides is regulated by various national and international
bodies. They are given approval to be used only after checking its safety on the
health of consumer and its impact on the environment. These regulatory bodies
establish the standards for the pesticide residue in food products. There may be
differences in the national and international standards for pesticide residues in food
products. Regulatory bodies in India that monitor the level of pesticide residues in
food are the Central Insecticides Board and Registration Committee (CIBRC), Food
Safety and Standards Authority of India (FSSAI), Agricultural, and Processed Food
Products and Export Development Authority (APEDA). CIBRC approves for the
introduction of a new pesticide and issues guidelines for using and manufacturing
the pesticide. Till date, 275 pesticides have been registered under CIBRIC. FSSAI
has laid the Maximum Residual Level (MRL) for the pesticides that have been
registered by CIBRIC. The international authorities like Codex Alimentarius Com-
mission (CAC) and European Union (EU) have specified the MRL for a wide range
of pesticides.
Acceptable Daily Intake Acceptable daily intake (ADI) is the measure of the
amount of a specific substance in a food or drinking water that can be ingested
(orally) on a daily basis for a lifetime without an appreciable health risk. It is
expressed as milligrams per kilograms of body weight per day. ADI is determined
by dividing NOEL (no observed adverse effect) by 100.
MRL It is the maximum amount of a substance that is expected to remain in the

food commodity which does not pose a food safety concern.
The estimation of pesticide residues is done by QuEChERS method, which is a
quick, safe, cheap, effective and rugged method. In this, the pesticides are extracted
using acetonitrile and salting out, i.e., by using magnesium sulfate and sodium
acetate. This technique is useful for foods having a high-fat content like milk. The
extracted pesticides are then subjected to clean up process by dispersive solid phase
extraction using various chemicals to eliminate the matrix interference which is then
used for GC or LC analysis.
The basic form of QuEChERS method includes liquid microextraction, solid
phase cleanup, LC/MS/MS or GC/MS/MS. QuEChERS method has advantages
like increase in the sample throughput and reducing the costs, minimize the
degradation of the susceptible compounds like acid and base sensitive pesticides, a
wide range of matrices can be tested for pesticide residues.
Extraction Reagents Used in QuEChERS Method and Their Function

1. Magnesium sulfate, anhydrous: This facilitates solvent partitioning and improves
the recovery of polar analytes.
2. Acetic acid: Adjustment in the pH.
3. Acetonitrile: The organic solvent facilitates the extraction of a wide range of
pesticides with least number of co-extractables and can be used in both GC and
LC analyses.
4. Buffers: This prevents the degradation of pH-sensitive analytes by maintaining
the required optimal pH.
5. Sodium chloride: It is added to reduce the polar interferences.
Clean up Reagents and Their Functions

1. Aminopropyl: It removes the sugars and fatty acids. The function of adding
aminopropyl is the same as that of primary secondary amine (PSA), but is less
likely to cause the degradation of base-sensitive analytes. Its capacity for cleanup
is less than PSA.
2. C18: This helps in the removal of long-chain fatty compounds, sterols, and other
non-polar interferences.
3. Magnesium sulfate anhydrous: Removes water from organic phase.
4. Primary secondary amine (PSA): Removes sugars, fatty acids, organic acids,
lipids, and some pigments. In combination with C18 column, additional lipids
and sterols can be removed.
5. Graphitized carbon black: It is a strong sorbent used for removing the pigments,
polyphenols, and other polar compounds.
The detection of pesticides in milk by using QuEChERS method employs the

AOAC prescribed method.
Procedure for the Detection of Pesticide by QuEChERS Method

1. Take 15 ml of milk in a 50 ml centrifuge tube and add to it, 15 ml acetonitrile with
1% acetic acid.
2. Add 30 μl of 50 ppm triphenyl phosphate (internal standard).
3. Then add 6 g of magnesium sulfate, 1.5 g of sodium acetate and mix well (vortex
for 10 s) to enhance the phase separation.
4. Centrifuge the contents of the tube at >1500 rcf for 1 min.
5. Transfer 1 ml of the supernatant to a 2 ml dispersive solid phase cleanup tube
containing 150 mg magnesium sulfate, 50 mg PSA, 50 mg C18.
6. Shake for 30 s and centrifuge at >1500 rcf for 1 min.
7. Preserve the supernatant with toluene for GC/MS or with 6.7 mM formic acid for
LC/MS/MS.
7.11 Determination of Antibiotic Residues in Milk 201
Parameters for LC-MS/MS

Column temperature: 40 C
Injection volume: 10 μl
Flow rate: 0.2 ml/min
Autosampler temperature: 10 C
Mobile Phase A: 0.3% formic acid and 0.1% ammonia formate in water
Mobile Phase B: 0.1% formic acid in methanol
Time (min) Mobile phase A (%) Mobile phase B (%)

0 99 1
1.5 99 1
3.5 20 80
10 10 90
12 0 100
15 0 100
15.2 99 1
20 99 1
Divert the mobile phase to waste between 0 and 0.5 min and from 15 to 20 min to
prevent ion source contamination.
MS Parameters
Polarity ESI +
Collision gas and pressure Ar at 1.5 mTorr
Scan type SRM
Cycle time 1s
Vaporizer temperature 300 C
Ion transfer capillary 200 C
7.11 Determination of Antibiotic Residues in Milk
Antibiotic word has been derived from the Greek word “anti” which means against
and “bios” means life. Antibiotics are the molecules that can kill or inhibit or stop the
growth of microorganisms like bacteria and fungi. The type of antibiotics that can
kill the microorganisms (bacteria) are called as bactericidal and which can stop their
growth are termed as bacteriostatic. The first antibiotic was discovered by Ernest
Duchesne in 1896 and was rediscovered by Alexander Fleming from the fungi
Penicillium notatum in 1928. This antibiotic was called penicillin. The antibiotics
are generally classified on the basis of their chemical structure, mode of action, or
spectrum of activity.
Classification on basis of chemical structure: Antibiotics are classified as

β-lactam, tetracycline, sulfonamides, macrolides, aminoglycosides, quinolones,
and miscellaneous.
β-Lactam Antibiotics These are broad-spectrum antibiotics containing a β-lactam

ring in their structure. A lactam ring is a four-membered cyclic amide in which the
nitrogen atom is attached to the β-carbon with respect to the carbonyl group. The
simplest form of β-lactam is 2-azetidinone. They work by inhibiting the cell wall
biosynthesis in the target organism and are the most widely used antibiotic. It has
been found that the bacteria develop resistance to beta-lactam antibiotics as they
synthesize β-lactamase enzyme. This resistance can be overcome by using
β-lactamase inhibitors like clavulanic acid. The different types of β-lactam
antibiotics are Penicillin, Cephalosporins, Carbapenems, and Monobactams.
Penicillins These are the type of antibiotics which are derived from moulds like
Penicillium. These antibiotics were the first type of medicine, which were effective
against streptococcal and staphylococcal infections. They are widely used
antibiotics, though many bacteria have developed resistance to it. Some types of
antibiotics that falls under this class are penicillin V (administered orally) and
penicillin G (intravenous use, procaine, benzathine (subject intramuscularly)).
Cephalosporins They have the same mode of action as that of the other β-lactam
antibiotics but are less susceptible to the β-lactamases. These antibiotics disrupt the
synthesis of the peptidoglycan layer of the bacterial cell wall, so they are bactericidal
type of antibiotics. The cephalosporins have been evolved and grouped into various
generations on the basis of their antimicrobial activity. Different generations of
cephalosporins are first generation (ampicillin, Cephalexin, cephalothin, etc.), sec-
ond generation (cefazolin, cefotetan, cefamandole, etc.), third generation
(ceftizoxime, ceftazidime, ceftriaxone, etc.), fourth, and fifth generation (fosamil,
cefepime, cefquinome, etc.). The difference between the antibiotics of one genera-
tion with the other is that the newer generation has a higher antimicrobial activity
against the Gram-negative bacteria than the antibiotic of the preceding generation,
mostly the activity of the antibiotic against Gram-positive bacteria decreases. The
fourth-generation cephalosporins have broad-spectrum activity. Common difference
in the structure of penicillin (1) and cephalosporin (2) (Fig. 7.5).
Fig. 7.5 General structure of

penicillin and cephalosporin
Fig. 7.6 Structure backbone

of carbapenem

of monobactam

of tetracycline
Carbapenem These are a class of highly effective antibiotics administered for the
treatment of severe and high-risk infections caused by bacteria. These antibiotics are
given for known or suspected multidrug-resistant bacterial infections. Their mode of
action is similar to that of the penicillins, cephalosporins as they kill the bacteria by
binding to the penicillin-binding proteins that results in the inhibition of the bacterial
cell wall synthesis. These antibiotics have a broad spectrum of antibiotic activity as
compared to penicillins or cephalosporins. Carbapenems are unaffected by the
antibiotic resistance. Carbapenems were developed by Merck & Co from the product
thienamycin which was derived from Streptomyces cattleya. Bacteria like Klebsiella
pneumonia and Enterobacteriaceae are resistant to the antibacterial action of
carbapenems, hence not used in the treatment of the infections caused by these
organisms. Their structure is similar to penicillin but the sulfur atom has been
introduced at position 1 replacing the carbon atom and a double bond is present in
its structure (Fig. 7.6). Examples for carbapenem are biapenem, ertapenem,
doripenem, panipenem, etc.
Monobactams These are the class of β-lactam antibiotics in which the β-lactam
ring is alone and not fused with other rings as in case of other classes for β-lactam
antibiotics. The commercially available monobactam is aztreonam. Structure of
monobactam is depicted in Fig. 7.7.
Tetracycline These antibiotics are called so because they have four hydrocarbon
rings in their structure (Fig. 7.8). They work by inhibiting the protein synthesis by
inhibiting the binding of aminoacyl-tRNA to the mRNA–ribosome complex. They
Fig. 7.9 Structure of

streptomycin
Fig. 7.10 Structure of the

functional group in
sulfonamide
do it by binding to the 30S and 50S ribosomal subunits in the mRNA translation
complex, thus preventing the introduction of the new amino acids to the peptide
chain. These antibiotics are used for the treatment of malaria, plague, acne, cholera
and is taken orally. The World Health Organization’s List of Essential Medicines
marks tetracycline as the safest and most effective medicines required in a health
system. This antibiotic was made from Streptomyces species. It has a broad spectrum
of action except for Pseudomonas aeruginosa and Proteus spp., which are resistant
to its action. Examples of tetracycline are tetracycline, chlortetracycline, doxycy-
cline, etc.
Aminoglycosides The antibiotics that contain an amino-modified glycoside in its

structure are termed as aminoglycoside. They act against Gram-negative bacteria
(aerobes), some anaerobic bacilli by inhibition of the protein synthesis but do not
work against Gram-positive and anaerobic Gram-negative bacteria. These antibiotics
require short contact time and act mostly on the rapidly multiplying bacterial
populations. Streptomycin (Fig. 7.9) is an example of aminoglycoside which was
derived from Streptomyces griseus. Other examples of aminoglycosides are
gentamycin, neomycin, kanamycin, lincomycin, etc.
Sulfonamides These antibiotics contain sulfonamide group and are also called as
sulfa drugs. Some of the antibiotics of this group do not have any antibacterial
activity like anticonvulsant sultiame. These antibiotics are seldomly prescribed by
doctors in developed countries but are still used in developing countries because of
their low price. These antibiotics are competitive inhibitors of the enzyme
dihydropteroate synthase. This enzyme is involved in folate synthesis and thus the
antibiotic acts as a bacteriostatic agent for the bacteria. Examples for sulfonamides
are sulfabenzamide, sulfamethazine, sulfadoxine, sulfathiazole, etc. (Fig. 7.10).
Macrolides These are a class of antibiotics that contain a macrocyclic lactone ring
to which the deoxy sugar(s) like desosamine and cladinose are attached. The
attached lactone ring(s) are generally 14, 15, or 16 membered. Their mode of action
is by inhibition of the protein synthesis by preventing the peptidyltransferase from
Fig. 7.11 Structure of

erythromycin
adding the peptide chain to the tRNA to the next amino acid and also by inhibiting
the ribosomal translation. They also work by immunomodulation and have been
employed in the treatment of diffuse panbronchiolitis by reducing the bronchial
inflammation by suppressing the neutrophil granulocyte proliferation, lymphocyte
activity, and obstructive secretions that occur in the airways. Examples of
macrolides are azithromycin, fidaxomicin, erythromycin, etc. These antibiotics are
used to treat the infections caused by Gram-positive bacteria like Streptococcus
pneumoniae and to some extent infections caused by Gram-negative bacteria like
Bordetella pertussis and Haemophilus influenzae. The antimicrobial activity of
macrolides is wider than the penicillins and thus macrolides are used as a substitute
for the patients suffering from penicillin allergy (Fig. 7.11).
Entry of Antibiotics in Milk and Its Technological Significance

The dairy animals are treated with antibiotics to cure various diseases like mastitis,
foot and mouth disease, the risk of contamination with antibiotics is mainly when the
treatment is done by using intramammary drugs. These drugs are applied to treat
mastitis or during the drying off which form the major reason for contamination of
secreted milk.
1. Misapplication of the injection: When the antibiotic is given into the muscle, the
withdrawal time for the antibiotic is lower, but when it is given into the udder, the
withdrawal time is higher.
2. Inter-uterine infusions and bolus (sulfa drugs and tetracycline used to flush the
placenta).
3. Administration of the medicine intramuscularly or intravenously.
4. Unaware about the treatment of the newly purchased animal.
5. Failure to observe the withdrawal period in lactating animals. If this period is not
taken care of, the contaminated milk when mixed with the bulk milk, it will tend
to contaminate the whole lot of the milk.
Withdrawal period is defined as the time required after the administration of the
drug to the dairy animal so that the amount of the drug residue in the milk is below
the maximum residual limit for that particular drug. If the antibiotic residues are
present in the milk or if the proper withdrawal time is not observed it may lead to
microbial drug resistance, can cause allergic responses, affect the processing of
fermented milk products due to starter failure, etc.
Detection of Antibiotics in Milk Using Microbial Inhibitor Test

This is a reference method for determination of the antibiotic residues in raw and
processed milk given by the International Dairy Federation (IDF 1991). This test
used Bacillus stearothermophilus var calidolactis, ATCC 10149. The organism has
a high sensitivity to inhibitory substances like antibiotics. This test involves the
incubation of the milk sample with the bacterial spores in the medium. The change in
color on the incubation of the milk with the bacterial spores indicates the growth of
microorganisms indicating absence of antibiotics while no change in color indicates
the presence of antibiotics due to inhibition of the growth of the organism.
Determination of Tetracycline
Reagents
1. Citric acid
2. Disodium hydrogen phosphate
3. Phosphoric acid
4. Oxalic acid
5. Acetonitrile (LC Grade)
6. Methanol (LC Grade)
7. Water (LC Grade)
• McIlvaine Buffer (pH 4): 0.1 M Citric acid monohydrate, 0.2 M disodium
hydrogen phosphate. Mix 61.45 ml of 0.1 M citric acid solution and 38.55 ml
of 0.2 M disodium hydrogen phosphate. Adjust the pH to 4 using dilute
phosphoric acid.
• 0.01 M oxalic acid (Dissolve 1.26 g of oxalic acid in 1 L methanol).
• Standard tetracycline hydrochloride: 108 mg tetracycline hydrochloride is
dissolved in methanol and adjusted to 100 ml with it, to get a 1000 ppm stock
solution.
• Working solution: Dilute 1 ml of the stock solution to 1000 ml with methanol.
Prepare 100, 200, 300, 400, and 500 ppb of working solution from it. Table 7.10
and 7.11 shows the operating conditions of the HPLC required for the determina-
tion of tetracycline in milk.
Table 7.10 HPLC operating conditions

Detection 350 nm (UV detector)
Flow rate 1 ml/ min
Mobile phase Methanol: 0.01 M oxalic acid in water: Acetonitrile (73:10:17)
Table 7.11 LC conditions

Column C8, 2.1x 150 mm
Temperature 30 C
Flow rate 0.3 ml/min
Injection 5 μl
Mobile Water/0.1% formic acid
phase A
Mobile Methanol
phase B
Gradient 0–10 min B from 5–30%, 10–12 min B from 30–40%, 12.5–18 min B 65%,
18.5–25 min B 95%, 25.5 min B 5%
Total run 28 min
time
MS Settings Source ESI (Positive ion polarity)
MRM Settings
Name of fragment Precursor ion Product ions
4-Epitetracycline 445 410,427
4-Epioxytetracycline 461 426,444
Tetracycline 445 410,427
Oxytetracycline 461 426,443
4-Epichlortetracycline 479 444,462
Chlortetracycline 479 444,462
Procedure
• Weigh 5 g of milk sample and dissolve in McIlvaine buffer. Adjust the volume to
50 ml in a colorimetric tube.
• Vortex it for 1 min and then extract the contents ultrasonically on an ice water
bath for 10 min.
• Transfer to 50 ml polypropylene centrifugal tube and cool to 0–4 C.
• Centrifuge the tube at 5000 g for 10 min (below 15 C) and filter using fast filter
paper.
Column Chromatography Separation

• Attach the SPE extractor with the C18 cartridge.
• Run 20 ml of methanol followed by 20 ml of buffer to condition the tube.
• Collect the sample filtrate in a tube (ensure the flow rate not to be more than 4 ml/
min).
• Rinse with 20 ml of distilled water.
• Elute the contents of the column twice with 5 ml of 0.1 M oxalic acid.
• Collect the elute and evaporate under nitrogen to get a final quantity of 1 ml.
• Adjust the volume to 1 ml using methanol.
• Before injecting the sample, filter it using 0.2 μm filter.
LC Analysis
• Inject 60 μl of the filtered sample into the LC system, maintained at a flow rate of
1 ml/min of the mobile phase (isocratic).
• Similarly, inject 60 μl of the working standards to obtain a standard curve.
Sequence of Injection
The injection should be done in the following order: calibration standard, recovery
sample, blank sample (to verify absence of analytes), and sample extract.
Calculation
• Review the chromatogram and verify the analyte peaks are within the required
retention time.
• Calculate the normalized peak for each component of interest.
Normalized response component ¼ Response of component=Response of internal standard
• Prepare a standard curve of each analyte using the normalized response to

concentration.
• The response for the blank should be less than 5% of the recovery used.
Determination of Chloramphenicol (HPLC-MS/MS Method)

Reagents
• Carbon tetrachloride (AR/HPLC)

• Acetonitrile (HPLC Grade)
• Hexane (AR)
• Reference standard chloramphenicol base
• Internal Standard Deuterated Chloramphenicol (CAP-d5)
• Ethyl acetate (HPLC Grade)
• Standard Chloramphenicol solution: Prepare a stock solution of chloramphenicol
having a concentration of 1000 ppm in acetonitrile (store at 18 C, stable for
1 year). Use this solution to prepare the working standard solution, on the day of
analysis.
• Internal standard solution: Make an internal standard solution of CAP-d5 of
concentration 20 ppb in water (stable for 3 months) from intermediate solution
(of 1 ppm prepared in 50:50 v/v ACN: water) that is prepared from 100 ppm stock
solution.
• Instrument Condition
HPLC conditions MS/MS conditions

Mobile Water and ES Negative
phase acetonitrile
Flow 0.3–1.0 ml/ MRM 321 > 152 (for quantitation) and 321–257
rate min (for confirmation)
Run time 6–12 min MRM of internal 326–156
standard
Procedure
• Take 1–5 g of defatted milk in a centrifuge tube and add 200 μl of CAP-d5 (20 μg/
kg) to it.
• Add 10 ml ethyl acetate and mix thoroughly for 10 min on a vortex.
• Centrifuge at 5000 g for 15 min.
• Transfer the upper ethyl acetate layer in a clean turbovap tube concentrator under
Nitrogen.
• Repeat the extraction again with 5 ml of ethyl acetate.
• Collect the ethyl acetate layers in the same turbovap and dry under nitrogen at
45 C.
• Mix the dried residue in 1 ml of Hexane: Carbon tetrachloride mixture (1:1) and
vortex it.
• Add 1 ml water and vortex again.
• Centrifuge the contents at 5000 g for 15 min.
• Quantitatively transfer the supernatant into the LC vial for further analysis.
Injection Sequence for HPLC-MS-MS Analysis

Calculation
retention time.

concentration.
Determination of Quinolones (HPLC-MS-MS Method)

Reagents
• Flumequine, Oxolinic acid, Nalidixic acid, Cinoxacin, Piromidic acid, Pipemidic

acid, Marbofloxacin, Norfloxacin, Ciprofloxacin, Lomefloxacin, Danofloxacin,
Enrofloxacin, Sarfloxacin, Difloxacin, Ofloxacin, Enoxacin, Orbifloxacin,
Desethylene ciprofloxacin, Sodium hydroxide, ultrapure sodium sulfate
anhydrate, formic acid (HPLC Grade), and acetonitrile (HPLC Grade).
• Instrument Conditions
Column type Analytical (4.6 mm, 5 μm)

ESI Positive mode
Injection volume 20 μl
Mobile phase A 20 mM ammonium formate in 0.1% formic acid
Mobile phase B Acetonitrile
Flow rate 0.7 ml/ min
Gradient pattern 0 min A: 85%, B: 15%, 7 min A: 30%, B: 70%, 8 min A: 5%, B: 95%,
9 min A: 5%, B: 95%, 10 min A: 85%, B: 15%, 12 min A: 85%, B: 15%.
Triple quadrupole Coupled to HPLC
MSMS
• Standard solution: Prepare a 200-ppm standard stock solution of all the

18 quinolones mentioned above in 505 acetonitrile containing 2% 0.1 N NaOH.
• Working standard solution: Prepare a serial dilution of the standard stock solution
using 10% acetonitrile. Preserve the stock and working standard solution at 4 C
in brown-colored volumetric flask (stable for 3 months). Prepare 5 working
solutions ranging between 0.5 and 10 ng/ml, for external standard calibration.
Sample Preparation
• Pipette 2 ml of milk sample in a 50-ml polypropylene centrifuge tube and add
20 ml acetonitrile containing 0.1% formic acid.
• Mix for 5 min and leave undisturbed.
• Add 2 g of sodium sulfate and mix vigorously for 5 min followed by centrifuga-
tion at 6000 rpm for 10 min at 4 C.
• Evaporate the acetonitrile extract at 40 C and resuspend the leftover residue in
2 ml of 10% acetonitrile containing 0.1% formic acid.
• De-fat the sample by extracting the fat by adding 4 ml hexane and vortex for 15 s.
• Centrifuge the mixture at 4000 rpm for 5 min at 4 C and filter the supernatant
through 0.22 μm nylon membrane. Store the sample for further analysis.
Injection Sequence for LC-MS-MS Analysis

Calculation
retention time.

concentration.
Now-a-days newer methods have been developed for the detection of the antibi-
otic residues, pesticide residues, aflatoxin M1, and melamine in milk which are
available commercially in the form of ready to use kits. These kit have the advantage
that the screening of milk for the presence of such contaminants can be done rapidly.
The reproducibility, sensitivity, and repeatability of these tests are much better than
the analytical methods. Moreover, these methods can be used in the on-field testing
of milk because they do not require testing instruments, sample preparation, can be
performed by anyone and the interpretation of results is very easy as the presence or
absence can be detected by just observing for the change in color. ICAR- National
Dairy Research Institute, India has developed certain kits for the detection of
adulterants, pesticides, and antibiotics in milk and the cost of each test is very less
with a high degree of sensitivity and reproducibility.
Suggested Readings
AOAC Official Method 2007.01 (2007) Pesticide residues in foods by acetonitrile extraction and
partitioning with magnesium sulfate.
AOAC Official Methods of Analysis (2005a) Ch 23.1.17, Method 995.09. pp 22–36
AOAC Official Methods of Analysis (2005b) Ch 49.3.06 Method 986.16
Eldridge BF (2008) Pesticide application and safety training for applicators of public health
pesticides. California Department of Public Health
Fagnani R, Beloit V, Battaglini APP, Dunga KS, Tamanini R (2011) Organophosphorus and
carbamates residues in milk and feedstuff supplied to dairy cattle. Pesqui Vet Bras 31:598–602
FAO (1989) Food and agricultural organization of the United Nation. International Code of
Conduct on the distribution and use of pesticides, Rome, Italy
Garcia FP, Ascencio SYC, Oyarzun JCG, Hernandez AC, Alavarado PV (2012) Pesticides:
classification, uses and toxicity. Measures of exposure and genotoxic risks. J Res Environ Sci
Toxicol 1(11):279–293
IDF (1991) Detection and confirmation of inhibitors in milk and milk products. Bulletin of the
International Dairy Federation No. 258, International Dairy Federation, 41 Square Vergote,
Brussels
Iftikhar B, Siddiqui S, Rehman S (2014) Assessment of the dietary transfer of pesticides to dairy
milk and its effect on human health. Afr J Biotechnol 13:476–485
IS 2488 Part 1–5 (1998) Methods of sampling and test for industrial effluents. Bureau of Indian
IS 11673 (1992) Indian standard: sodium hypochlorite solution-specification. Bureau of Indian

Jeong IS, Kwak BM, Ahn JH, Jeong SH (2012) Determination of pesticide residues in milk using a
QuEChERS-based method developed by response surface methodology. Food Chem 133
(2):473–481
King DE, Malone R, Lilley SH (2000) New classification and update on the quinolone antibiotics.
Am Fam Physician 61(9):2741–2748
Lehotay S (2007) AOAC official method 2007.01 pesticide residues in foods by acetonitrile
extraction and partitioning with magnesium sulfate. J AOAC Int 90(2):485–520
Liu Y, Todd EED, Zhang Q, Shi J, Liu X (2012) Recent developments in the detection of melamine.
J Zhejiang Univ Sci B 13:525–532
MacLachlan DJ, Bhula R (2007) Estimating the residue transfer of pesticides in animal feedstuffs to
livestock tissues, milk and eggs: a review. Aust J Exp Agric 48:589–598
Manual of Methods of Analysis of Foods Antibiotics and Hormone Residues-FSSAI (2012) Lab
manual 15
Manual of Methods of Analysis of Foods Mycotoxin-FSSAI (2015) Lab manual 7
Schultz MJ (2004) Macrolide activities beyond their antimicrobial effects: macrolides in diffuse
panbronchiolitis and cystic fibrosis. J Antimicrob Chemoth 54(1):21–28
Stine CB, Reimschuessel R, Keltner Z, Nochetto CB, Black T, Olejnik N, Scott M, Bandele O,
Nemser SM, Tkachenko A et al (2014) Reproductive toxicity in rats with crystal nephropathy
following high doses of oral melamine or cyanuric acid. Food Chem Toxicol 68:142–153
Tenson T, Lovmar M, Ehrenberg M (2003) The mechanism of action of macrolides, lincosamides
and streptogramin B reveals the nascent peptide exit path in the ribosome. J Mol Biol 330
(5):1005–1014
Agency, New Delhi
Tsiplakou E, Anagnostopoulos CJ, Liapis K, Haroutounian SA, Zervas G (2010) Pesticides residues
in milks and feedstuff of farm animals drawn from Greece. Chemosphere 80:504–512
Turnipseed S, Casey C, Nochetto C, Heller DN (2008) Determination of melamine and cyanuric
acid residues in infant formula using LC-MS/MS. US FDA Laboratory Information Bulletin
24:4421
USFDA (2009) Action levels for aflatoxins in animal feeds. In (Vol. 07/15/2012): United States
Food and Drug Administration. Sec. 683.100 (CPG 7126.33). Available at: http://www.fda.gov/
ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm074703.htm
Vaz A, Cabral Silva AC, Rodrigues P, Venancio A (2020) Detection methods for aflatoxin M1 in
dairy products. Microorganisms 8(2):246
World Health Organization (2010) The WHO recommended classification of pesticides by hazard
and guidelines to classification 2009
www.cibrc.nic.in
www.fssai.gov.in
www.apeda.gov.in
www.codexalimentarius.org
www.ec.europa.eu
Specifications of Chemicals Used in Dairy
Industry 8
Chemicals play an important role in every phase of the dairy industry. A wide range
of chemicals like acid, base, and salt are being used in the industry. All these
chemicals have various uses like in testing, cleaning, sanitation, and as additive.
The specifications of some of the chemicals are tailor-made and vary from one dairy
industry to another whereas specification of some others is laid down by a regulatory
authority. In this chapter, we will discuss the specifications of various chemicals in
detail.
8.1 Specifications of Gerber Sulfuric Acid (IS: 1224, Part-II)
For estimation of fat in milk by Gerber method, sulfuric acid is used to dissolve
casein in the milk. The strength of sulfuric acid is adjusted such that it is just strong
enough to dissolve the casein without charring the fat. In case, the sulfuric acid is
diluted than the desired concentration it may lead to the precipitation of casein,
whereas too concentrated sulfuric acid can cause charring of the organic matter.
Further, it produces the adequate amount of heat to keep the fat in the liquid state.
8.1.1 Preparation of Gerber Sulfuric Acid
1. In a 1-liter volumetric flask, fill 100 ml of distilled water in it.

2. Add 900 ml of concentrated H2SO4 (say 900 ml) in small quantities through the
sides of the flask at a time. Ensure the temperature of the container is maintained
to avoid heating of the flask due to acid addition (keep the flask in ice-cold water).
3. Mix the contents gently.
4. After cooling check the specific gravity of Gerber acid with a hydrometer (having
a range 1800–2000) and if necessary adjust the Gerber acid to the correct specific
gravity with the addition of water or acid taking the same precaution as before till

https://doi.org/10.1007/978-981-15-4167-4_8
214 8 Specifications of Chemicals Used in Dairy Industry
specific gravity is in the range 1.807–1.812 at 27 C corresponding to a concen-

tration of 90–91%.
5. Transfer the acid in a glass stoppered bottle to avoid absorption of water.
8.1.2 Precautions
1. Sulfuric acid is very corrosive. Handle it carefully.

2. Always add acid to water. Slowly add small quantities of acid to water by swirling
the flask and cool it. Never add water to acid as the dilution of acid is an
exothermic reaction. It can fume, spatter, or even boil, resulting in corrosive
fumes and droplets. If you add acid to the water, the water absorbs the heat that
only warms slightly but is not reactive. The explanation is the hydrogen bond in
water, which requires a great deal of energy to boil it—the heat from a dilution is
usually inadequate to do it. Use heat-resistant flask for dilution.
8.2 Specification of Amyl Alcohol (IS: 360)
It has been discussed earlier that amyl alcohol is used during the estimation of fat in
milk and dairy products by Gerber method. For the results of the test to be satisfac-
tory, the quality of the amyl alcohol used during the test plays a very important role
and it should be conforming to certain requirements. Amyl alcohol is generally
divided into 2 grades based on its intended end use.
Grade 1: For use in testing of milk.
Grade 2: As a solvent for industrial use generally as a thinner and in paints as a
solvent.
The detailed specification of Grade 1 alcohol is given below in Table 8.1.
8.2.1 Test for Furfural and Other Organic Impurities
This test is based on the principle of development of yellow or brown color in the
presence of H2SO4.
Table 8.1 Requirements for amyl alcohol

S. No. Characteristic Requirement
1 Color Clear and colorless
2 Specific gravity (27 C) 0.807–0.809
3 Distillation range Not less than 95 ml within 2 degrees in the temperature
range 128–132 C at 760 mm of Hg
4 Furfural and other Shall pass the test (discussed below)
organic impurities
5 Hydrochloric acid test Shall pass the test (discussed below)
8.2 Specification of Amyl Alcohol (IS: 360) 215
Add 5 ml of concentrated H2SO4 to 5 ml of amyl alcohol and mix well.

Development of brown or yellow color indicates the presence of furfural and organic
impurities.
8.2.2 Test of Amyl Alcohol for Suitability for Milk Analysis
This test is done to ensure that amyl alcohol is free of fat. This is done by carrying
Gerber test using water instead of milk. Amyl alcohol is said to pass this test if no fat
layer is visible in the butyrometer scale on the top of sulfuric acid after
centrifugation.
8.2.3 Hydrochloric Acid Test
In this test, 10 ml of concentrated HCl is added to 10 ml of amyl alcohol and mixed

well. The test is said to be passed if both the liquids mix well to form a homogenous
solution and get separated into two layers on the addition of 1 ml of water.
8.2.4 Packing and Marking
Packing The material should be packed in galvanized drums or any other container
as agreed between the purchaser and the supplier or as per the regulatory
requirements. The containers should be dry and clean ensuring that no impurity
which may affect the end use of the material is present.
Marking The material should be supplied in the container marked with the caution
label “FLAMMABLE” and with a symbol indicating dangerous material. Each
container should have information indicating the name of the material,
manufacturer’s name, trademark if any, net, gross and tare weight, month and year
of manufacture. The material can also be supplied as per the marking and delivery
instructions laid down by the purchaser.
8.2.5 Sampling Instructions
The sample(s) should be drawn such that they represent the complete lot or consign-
ment. When all the containers in a given consignment are of the same grade and size,
the drawn samples should be categorized as a single lot whereas if a consignment
consists of different batches or grades or containers of different sizes, the sample
drawn from the same grade/batch/ container size should be grouped as a single lot
while that from other sets of grade/container size/lot should be grouped into another
lot. Then from each lot, 5 containers should be selected randomly so as to carry out
the tests to ensure its conformity to the requirements. A composite sample of about
500 ml should be taken from these randomly selected containers and be divided into
3 equal parts (one for supplier, second for purchaser, and the third kept for future
purpose, in case of any dispute). The composite sample should be marked properly
(as discussed under marking section) and sealed while that for future purposes
should contain the seal of both the supplier and the end user.
8.3 Specification of Common Salt (Edible) (IS: 253)
Common salt is used as an additive in butter and cheese and is also called as dairy
salt. It is permitted to be used in butter and cheese to max 3% by weight. It is used as
a flavor enhancer and preserver in these dairy products. The specification of dairy
salt is given below (Table 8.2).
Description: The material should be free from visible impurities and appear as a
crystalline white solid.
Particle size: 99% by weight of the material should pass through 850-micron
sieve.
Moisture: The material should not contain more than 0.5% by wt of moisture
when tested gravimetrically.
The material may contain 15 ppm (maximum) of a suitable anticaking additive
like potassium or sodium ferrocyanide or ammonium ferric citrate.
8.3.1 Preparation of the Sample for Chemical Tests
Weigh 80–100 g of the sample in a Petri dish, spread it uniformly on it and dry at
140–150 C for 4 h (for moisture analysis). This sample (S) should be used in tests
whereever indicated.
Table 8.2 Specifications for dairy salt (IS: 253)

S. No. Characteristic Dairy salt
1 Water insoluble matter, % by mass (max) 0.03
2 Chloride content (as NaCl), % by mass (min) 99.6
3 Calcium (as Ca), water soluble, % by mass (max) 0.01
4 Magnesium (as Mg), water soluble, % by mass (max) 0.01
5 Sulfates (as SO4), % by mass (max) 0.30
6 Alkalinity (as Na2CO3), % by mass (max) 0.10
7 Lead (as Pb), ppm (max) 2.00
8 Iron (as Fe), ppm (max) 10.00
9 Arsenic (as As), ppm (max) 1.00
10 Copper (as Cu), ppm (max) 2.00
8.3 Specification of Common Salt (Edible) (IS: 253) 217
8.3.2 Determination of Water Insoluble Matter
Weigh 50 g of the dried sample and dissolve in 200 ml of water in a beaker and boil.
After cooling, filter it through a sintered glass crucible followed by washing the
residue until free from soluble salts. Collect the filtrate and washings in a one-liter
graduated flask and dilute to mark. Mark this solution as S1, to be used in further
tests. Dry the crucible containing the leftover insoluble residue to a constant mass.
Matter insoluble in water ¼ 100 (M/m)
where, M ¼ mass of the residue obtained after drying and m ¼ mass of the dried
sample.
8.3.3 Determination of Chloride Content
Take 10 ml of the sample (S1) into a conical flask and add 1 ml of potassium
chromate indicator (5% solution). Titrate the contents of the conical flask against
standard silver nitrate solution till the appearance of reddish-brown tinge (should
persist on shaking). Carry out a similar experiment using water as a sample (blank
titration).
Sodium chloride (as NaCl), % by mass ¼ 584.5 VN/M
where V ¼ volume in ml of standard silver nitrate used in the titration with the
material, corrected to blank. N ¼ normality of standard silver nitrate solution.
M ¼ mass in g of the dried sample taken in 1000 ml of the solution taken for the test.
8.3.4 Determination of Sulfate
Mix 20 g of dried dairy salt (S) in about 400 ml of water. Filter the contents and wash
the leftover residue in the filter paper to remove the soluble salts. Transfer the filtrate
and the washings in a conical flask. Add 10 ml of dilute HCl (4 N) and add one drop
of methyl orange till pink color develops and then boil. Add 10–12 ml barium
chloride solution (10%) dropwise to the boiling solution and boil for 4 min till
appearance of granular precipitate. Leave the mixture undisturbed for 4 h, followed
by filtering through a sintered glass crucible (G 4) or Gooch crucible previously
weighed. Wash the precipitate to remove the traces of chloride if any. Dry it to a
constant mass at 105–110 C. Record the weight after drying.
Calculation
Sulfate (as SO4), percent by mass ¼ 41.13 M1/M2
Where, M1 ¼ mass in g of barium sulfate. M2 ¼ mass in g of dried sample
(S) taken.
8.3.5 Determination of Alkalinity
Take 20 g of the sample (S) in a 100-ml measuring flask. Fill carbon dioxide-free
distilled water up to the mark of 100 ml. Shake the contents well so that all salt gets
dissolved in water while magnesium carbonate remained in suspension. Filter the
solution using Whatman filter No. 1, discard the first 10 to 15 ml of the filtrate.
Collect the remaining filtrate and titrate 50 ml of the solution against 0.1 N HCl using
methyl orange (0.1% solution in water) as an indicator.
Na2CO3 + HCl ! NaHCO3 + NaCl
NaHCO3 + HCl ! NaCl + H2O + CO2
Calculation
Alkalinity (as Na2CO3) ¼ 1.06 V/M (% by mass)
where V ¼ volume of standard HCl used for titration. M ¼ mass in g of the
prepared sample taken for the test.
Derivation
1 l of 1 N HCl ¼ 1 l of 1 N sodium carbonate ¼ 53 g of sodium carbonate.
1 ml of 1 N HCl ¼ 0.053 g of sodium carbonate.
So, 1 ml of 1 N HCl is required for neutralization of 1 ml of sodium carbonate,
then V ml of 1 N HCl neutralizes V0.053 g of sodium carbonate or for 100 ml
sodium carbonate, volume of 1 N HCl consumed ¼ V5.3.
If the HCl used is of N normality then the volume of HCl consumed ¼ VN5.3.
As from the reaction, it is clear that two molecules of HCl neutralizes one
molecule of sodium carbonate.
So, the volume of HCl consumed ¼ VN10.6 ¼ The alkalinity in terms of
sodium carbonate.
8.3.6 Determination of Water-Soluble Ca and Mg
Reagents
Standard calcium solution: Weigh 1 g of calcium carbonate and dissolve in mini-
mum amount of dilute HCl (1:1). Dilute it to 1000 ml with distilled water. One ml of
this solution contains 0.4008 mg of calcium.
Standard EDTA solution: Dissolve 3.72 g of disodium ethylene diamine
tetraacetate dihydrate in water and dilute to 1 l.
Erichrome black T indicator solution: Dissolve 0.1 g of the dye in 20 ml of
rectified spirit. Always prepare it fresh.
Dilute sodium hydroxide solution (10%).
Murexide indicator solution: Grind 0.2 g of murexide with 10 g of NaCl to make
a homogenous mixture.
Calcein indicator: Grind 0.1 g of calcein and 0.06 g of thymolphthalein with 10 g
of KCl.
8.3 Specification of Common Salt (Edible) (IS: 253) 219
Ammonium chloride: Ammonium hydroxide buffer solution—Take 57 ml of

ammonium hydroxide and add 67.5 g of ammonium chloride to it. Add 250 ml of
water. Dissolve separately a mixture of 0.931 g of disodium ethylene diamine
tetraacetate dihydrate and 0.616 g magnesium sulfate in 50 ml of water. Mix the
two solutions and make up the volume to 1 l.
Procedure
Standardization of EDTA solution:
1. Pipette out 25 ml of standard calcium solution in a conical flask, add 25 ml of

water, 10 ml of buffer solution, 5 drops of Erichrome Black T indicator. Titrate
using standard EDTA solution till the appearance of blue color (end point).
Record the ml consumed (A).
2. Titrate 25 ml of buffer solution with EDTA solution using Erichrome Black T
indicator. Subtract the amount of standard EDTA solution consumed for the
buffer from A and note the final reading. Calculate the calcium equivalent to
1 ml of EDTA solution (let B).
Determination of Calcium and Magnesium

1. Take 100 ml of sample S1 in 250 ml conical flask and add 10 ml of buffer
solution.
2. Add 5 drops of Erichrome back T indicator and titrate it against standard EDTA
solution till wine red color changes to blue end point.
3. Note the reading of EDTA consumed (V1).
4. Take 100 ml of sample S1 in 250 ml conical flask and add 5 ml of NaOH solution,
mix well.
5. Add 0.2 g murexide (or 100 mg calcein mixed indicator).
6. Titrate against standard EDTA solution till appearance of blue end point with
murexide (or green to purple with mixed calcein indicator).
7. Note the volume of standard EDTA solution consumed (V2).
Calculation
Calcium (as Ca) % by mass (on dry matter) ¼ B V2/M.
Magnesium (as Mg) % by mass (on dry matter) ¼ 0.6068 B (V1–V2)/M.
Where B ¼ calcium equivalent in mg of 1 ml of EDTA solution. M ¼ mass in g of
the dried sample (S) taken.
8.3.7 Test for Lead
Apparatus
Nessler Cylinders (50 ml).
Reagents
Acetic acid: 33% (v/v).
Ammonium hydroxide (dilute): 4 N.

Potassium cyanide solution: Dissolve 10 g of KCN in 90 ml water and add 2 ml of
hydrogen peroxide. Allow to stand for 24 h and make up the volume to 100 ml with
distilled water.
Sodium sulfide solution: 10%.
Standard Lead solution: Dissolve 0.16 g of lead nitrate in 5 ml of concentrated
nitric acid and dilute to 100 ml. Dilute this solution 100 times, so that one ml of this
solution contains 0.01 mg of lead.
Procedure
1. Take 5 g of sample (S) and dissolve in water.
2. To it add 5 ml of acetic acid followed by addition of dilute ammonium hydroxide
to make the solution alkaline and add 1 ml of potassium cyanide solution.
3. If turbidity appears filter it.
4. Add 2 drops of sodium sulfide and mix well. Make up the volume to 50 ml.
5. Carry out a blank test using 1 ml of standard lead solution instead of sample (S).
6. Compare the color produced in both the cylinders against a white background.
The intensity of the sample should not be more than that for the control test.
Packing Dairy salt should be packed in moisture-proof packing material like

polyethylene or a material non-permeable to moisture or any other material as per
the specifications of the purchaser.
Marking The package should be securely closed and should have manufacturer
name, net contents in kg, batch number, and date of packing.
Sampling
The selection of the samples of dairy salt should be done on a random basis as per
Table 8.3.
Table 8.3 Number of Lot size (N) No. of packages to be selected (n)
packages to be selected for
4–50 3
sampling
51–100 4
101–150 5
151–300 7
301 and above 10
8.4 Specification of Sodium Citrate (Food Grade) (IS: 5058) 221
8.4 Specification of Sodium Citrate (Food Grade) (IS: 5058)
With the increase in the demand of processed foods, manufacturers have started
addition of large number of substances, in minute quantities, so as to improve the
sensory attributes like appearance, flavor, texture as well as in some cases to enhance
the nutritional score and storage stability of the processed foods. These substances
may contain some impurities which can be harmful, thus a strict quality control of
these food additives is necessary. Sodium citrate is a similar type of food additive
which is used as an emulsifying and stabilizing agent and is permitted to be used as a
food additive. The standards, sampling plan, etc. will be discussed below.
Description
1. It should be in the form of colorless crystals or white crystalline powder.
2. It may be available in anhydrous form or may contain two molecules of water of
crystallization.
3. 1 g of sodium citrate dihydrate should dissolve in 1.5 ml of water at 25 C and in
0.6 ml of boiling water. It is insoluble in alcohol.
8.4.1 Requirements
The requirements for sodium citrate of food-grade quality are mentioned in

Table 8.4.
Test for Oxalate

1. Dissolve 1 g of sodium citrate in a mixture of 1 ml water and 3 ml dilute HCl
(1:3).
2. To it add 4 ml of 90% alcohol and 4 drops of solution of calcium chloride.
3. Leave it undisturbed for 1 h. A clear solution indicates absence of oxalate.
Table 8.4 Requirements for sodium citrate (IS: 5058)

1 Purity on dry matter basis, % by mass (min) 99
2 Moisture (%) max Anhydrous: 1
Dihydrate: 13
3 Alkalinity To pass the test
4 Arsenic (dry matter basis) mg/kg (max) 3
5 Heavy metals (as Pb), mg/kg (max) 10
6 Oxalate Shall pass the test
7 Readily carbonizable substances Shall pass the test
Readily Carbonizable Substances

1. In a test tube, take 10 ml of sulfuric acid (94.5–95.5% strength).
2. Add 1 g of sodium citrate. Heat it on a boiling water bath for a period of 60 min.
3. Not more than pale brown color should be produced.
Determination of Purity
1. Take 2 g of sample and heat till carbonized and then cool it.
2. Add 50 ml water, 50 ml sulfuric acid (0.5 N) to the residue and boil it.
3. Filter and wash the filtrate obtained with water.
4. The excess of acid in the filtrate is titrated with sodium hydroxide (0.5 N) using
methyl orange as an indicator.
5. Each ml of 0.5 N sulfuric acid is equivalent to 0.04902 g of sodium citrate
(C6H5O7Na32H2O).
Determination of Moisture
1. Weigh 2 g of sample in aluminum dish and distribute it evenly to a depth of 5 mm.
2. Place it in the oven maintained at 180 1 C for 18 h.
3. Then remove the dish and cool it in a desiccator.
4. Record the weight after drying.
5. Calculate the loss in weight due to drying and express it in percentage.
Test for Alkalinity

1. Prepare a 5% solution of sodium citrate in water; this solution is alkaline in nature
(confirm by litmus paper).
2. Add 0.2 ml of sulfuric acid (0.1 N) to it and no pink color should be developed on
addition of one drop of phenolphthalein.
8.4.2 Packing, Storage, and Marking
Packing The material should be packed in containers or any other type of packing
material that provides protection from light and moisture. The container should be
such that it may preclude contamination of the contents with metals or any other
impurity. The material should be stored in a cool and dry place. The material should
be stacked on plastic crates.
Marking Each container should have the following information mentioned on it

like name of the material, food grade, type of material (anhydrous or dihydrate),
name and address of the manufacturer, net content, batch number, and date of expiry.
Sampling
The samples should be collected on a random basis as mentioned in Table 8.5.
8.5 Specifications of Nitric Acid (IS: 264) 223
Table 8.5 Selection of samples

Lot size (number of containers) Sample size (containers to be selected)
2–15 2
16–50 3
51–150 5
150 and above 8
Table 8.6 Specifications of Nitric acid (IS: 264)

1 Total acidity (as HNO3), min 52.0
2 Residue on ignition (% by mass), max 0.1
3 Chlorides (as Cl) (% by mass), max 0.03
4 Sulfates (as H2SO4) (% by mass), max 0.2
5 Heavy metals (as Pb) (% by mass) Shall pass the test
6 Ammonium salts (as NH3) (% by mass), max 5 ppm
8.5 Specifications of Nitric Acid (IS: 264)
8.5.1 Description
It should not be darker than pale brown in color and shall be free from sediment and
other visible impurities. The material should comply with the specifications as listed
in Table 8.6.
8.5.2 Determination of Total Acidity
The total acidity of nitric acid is determined by treating the acid with excess of
sodium hydroxide solution and back titration of the excess alkali with sulfuric acid.
1. Weigh 2 g of nitric acid and transfer it to a stoppered conical flask containing

100 ml of distilled water and 50 ml sodium hydroxide solution (1 N).
2. Stopper the flask and mix the contents of the flask properly, make sure that
mixing is carried out in cold conditions.
3. Add few drops of methyl orange indicator (0.05% w/v) in the conical flask and
titrate the excess of sodium hydroxide with sulfuric acid (1 N).
4. Carry out a blank test replacing nitric acid with water.
Calculation
Total acidity (as nitric acid, % by mass) ¼ (V–Y) 6.3 N/M.
Where, V ¼ volume in ml of sulfuric acid consumed in blank titration. Y ¼ vol-
ume in ml of sulfuric acid consumed for a sample. N ¼ normality of sulfuric acid
(1 N). M ¼ mass in g, of the sample.

1 l of 1 N sulfuric acid ¼ 1 l of 1 N nitric acid ¼ 63 g of nitric acid.
1 ml of 1 N sulfuric acid ¼ 1 ml of 1 N nitric acid ¼ 0.063 g of nitric acid.
So, 1 ml of 1 N sulfuric acid is required for neutralization of 1 ml of nitric acid,
then V ml of 1 N sulfuric acid neutralizes V0.063 g of nitric acid or for 100 ml nitric
acid, volume of 1 N sulfuric acid consumed ¼ V6.3.
If the sulfuric acid used is of N normality then the volume of sulfuric acid
consumed ¼ VN6.3 ¼ the amount of nitric acid (% acidity).
Determination of Residue on Ignition

1. Transfer 100 g of sample in a weighed sillica dish.
2. Heat the dish carefully, so that most of nitric acid gets evaporated to a final
volume of around 5–10 ml.
3. Allow the dish to cool at room temperature.
4. Add 1 ml of concentrated sulfuric acid and heat the dish to dryness.
5. Place the dish on an electric furnace maintained at 800 25 C for around
15 min.
6. Remove the dish and cool it in a desiccator.
7. Weigh the dish and repeat the heating and cooling to a constant weight.
Calculation
Residue on ignition ¼ M 100/m (% by mass).
Where, M ¼ mass in g of the residue after drying. m ¼ mass in g of the sample.
Determination of Chlorides
The opalescence produced by a known quantity of the sample with silver nitrate is
compared with the opalescence produced in the control sample containing a known
amount of chloride.
Reagents
Silver nitrate solution: 0.1 N.
Dilute Nitric acid: 4 N.
Standard Chloride solution A: Dissolve 1.648 g NaCl (dry it at 110 C) in
1000 ml of water. Take 100 ml of this solution and dilute it 10 times using distilled
water. One ml of this solution has 0.1 mg of chloride (Cl).
Procedure
1. Measure 1 ml of the sample in Nessler cylinder (50 ml capacity) and add 1 ml of
silver nitrate solution.
8.5 Specifications of Nitric Acid (IS: 264) 225
2. Add water up to 50 ml mark and observe the opalescence.

3. Carry out a control test using 10 ml of dilute nitric acid (4 N) and 4.2 ml of
standard chloride solution A.
4. The opalescence produced by the sample should not be more than that produced
in the control test for the sample to be accepted.
8.5.3 Determination of Sulfates
The sulfates are determined by precipitation of the sulfates in the form of barium
sulfate and then weighing it (gravimetric method).
1. Weigh 50 g of the acid in a porcelain dish.

2. Add 0.5 g sodium chloride (AR grade) and evaporate the contents on a water bath
to dryness.
3. Add 5 ml of HCl and transfer to 250 ml beaker using 100 ml of water.
4. Boil the contents at low flame and add 5 ml of barium chloride (slowly) under hot
conditions with constant stirring.
5. Boil it for 2 min and leave undisturbed for 4 h.
6. Filter the supernatant through a tared sintered glass crucible or gooch crucible, G
No. 4.
7. Transfer the precipitate thus obtained in the crucible and wash it with distilled
water to remove chlorides.
8. Heat the crucible to a constant mass at 105–110 C.
Calculations
Sulfate as H2SO4 (% by mass) ¼ M 42.02/m.
Where, M ¼ mass in g of the precipitate m ¼ mass in g of the sample taken.
8.5.4 Test for Heavy Metals
Ammonia is added to neutralize the acid and the iron oxide is removed if
precipitated. Hydrogen sulfide is passed from the solution, which is examined for
formation of lead sulfide.
1. Take 100 ml of water in a 250-ml conical flask and add 10 ml of acid to it.
2. Add ammonium hydroxide (20% m/v) slowly by swirling the flask till the
characteristic smell of ammonia is obtained.
3. Leave the flask for 10 min.
4. Filter if iron oxide precipitates settle at the bottom.
5. Bubble H2S gas from Kipp’s generator into the solution for 5 min.
The solution should not become darker than light brown color or no deposition of
precipitate should be observed.
Table 8.7 Selection of S. No. Lot size (N) No. of containers to be selected (n)
containers
1 0–15 2
2 16–25 3
3 26–50 4
4 51–100 5
5 101–300 6
6 301–500 7
7 501–800 8
8 801–1000 9
9 Above 1000 10
Packing Nitric acid should be supplied in screw stoppered stone bottles or glass
carboys. The containers should have leak-tight stoppers. Pent top packing cases
should be used for packing the bottles. Expanded polystyrene or expanded polyeth-
ylene containers may also be used before placing the bottles in corrugated fiber board
boxes in an upright position. Carboys should be packed in iron hampers or wooden
crates and the interspace between containers should be well packed to prevent
movement (material used should be noncombustible).
Marking The containers should be marked in red letters showing clearly items like
name of material, manufacturer’s name, grade and mass of material, and year of
manufacture. A cautionary warning “CORROSIVE, HANDLE WITH CARE”
should be displayed and must be visible.
Sampling
The containers should be selected randomly from a lot as per Table 8.7.
8.6 Specification of Caustic Lye (IS: 252)
Lye is a metal hydroxide or a strong alkali which when dissolved in water produces
basic solution. Lye is generally referred to as sodium hydroxide. Caustic lye is used
in the cleaning of dairy equipment, machines, pipelines, storage tanks, silos,
pre-pack machines, etc. So, it is important to ensure that the quality of the caustic
lye to attain its maximal effect in terms of cleaning. The specification of caustic lye is
given in Table 8.8.
8.6.1 Form and Description
The material should be in the form of lye and free from foreign matter, dirt, or other
impurities. The density of the caustic lye should be as per agreement between the
8.6 Specification of Caustic Lye (IS: 252) 227
Table 8.8 Specifications of caustic Lye (IS: 252)

S. No. Characteristic Specification on dry basis
1 Sodium carbonate (% by mass), max 2
2 Sodium hydroxide (% by mass), max 95
3 Sulfates (as Na2SO4) (% by mass), max 3.5
4 Iron (as Fe), ppm, max 350
5 Matter insoluble in water (% by mass), max 0.02
purchaser and the supplier. The other specification for caustic lye is mentioned in
Table 8.8.
8.6.2 Preparation of Sample for Testing
Weigh 0.01 g of the material and dissolve in 200 ml of water. The solution should be
transferred quantitatively in a 500-ml volumetric flask and made up the volume. This
sample (S) should be used for further analysis.
8.6.3 Determination of Carbonates
Double Indicator Method

1. Pipette 25 ml of the sample (S) in a conical flask and titrate using 1 N HCl using
phenolphthalein as an indicator.
2. Record the reading as A just before the disappearance of the pink color.
3. Titrate it again using 0.1 N HCl till the disappearance of pink color.
4. Record this reading as B.
5. Add 2–3 drops of methyl orange indicator (0.1%) and titrate using 0.1 N HCl.
6. Record this reading as C.
Calculation
Carbonates (%by mass) ¼ 212 (C B) N/M
Where, N ¼ normality of HCl (0.1 N). M ¼ mass of NaOH taken.
8.6.4 Determination of Sodium Hydroxide
1. Transfer 20 ml sample (S) in a conical flask and add 80 ml of water.

2. Add few drops of methyl orange indicator and titrate with HCl (1 N) till the color
changes from yellow to orange.
Calculation
Sodium hydroxide (as NaOH) percent by mass ¼ B (A 40/53)
Where, B ¼ total alkalinity (as NaOH). A ¼ carbonate content.
(V ¼ volume of 1 N HCl consumed, N ¼ normality of HCl used)

Here, the alkalinity of caustic lye is contributed by sodium carbonate and sodium
hydroxide. So for the determination of sodium hydroxide by percent mass, the
equivalent weight of NaOH is divided by the equivalent weight of sodium carbonate,
to get the mass fraction of the total alkalinity contributed by sodium hydroxide.
8.6.5 Measurement of Total Strength
The determination of the total strength is determined by titration of the sample. In

this method, phenolphthalein is used as an indicator for first half of the reaction
and gives the volume of acid required to neutralize all the sodium hydroxide present
and half of the sodium carbonate. The remaining half of sodium carbonate is
determined by using methyl orange as an indicator.
Reaction
When phenolphthalein is used as an indicator.
2NaOH þ H2 SO4 ! Na2 SO4 þ 2H2 O
2Na2 CO3 þ H2 SO4 ! 2NaHCO3 þ Na2 SO4
When methyl red is used as an indicator.
2NaHCO3 + H2SO4 ! Na2SO4 + 2H2O + 2CO2
Procedure
1. Rinse a clean burette and fill with 0.1 N sulfuric acid.
2. Take 25 ml of the lye in the conical flask.
3. Add 1–2 drops of phenolphthalein indicator and titrate with 0.1 N sulfuric acid till
disappearance of pink color to colorless.
4. Then, add a drop of methyl orange indicator and titrate with 0.1 N sulfuric acid till
yellow color changes to light orange color.
5. Volume of the acid consumed to phenolphthalein indicator gives the amount of
NaOH and half of sodium carbonate.
6. Volume of acid used using methyl orange gives the other half of the amount of
sodium carbonate.
7. The volume of the acid used for the first titration minus the volume of acid from
the second titration gives the amount of NaOH.
Calculation
Volume of 0.1 N H2SO4 used using phenolphthalein indicator ¼ y ml (gives all
NaOH and half sodium carbonate).
Volume of 0.1 N H2SO4 used when methyl orange indicator ¼ V ml (gives
remaining half of sodium carbonate).
So, volume of 0.1 N H2SO4 required for all the sodium carbonate ¼ 2V ml.
Volume of 0.1 N H2SO4 required for sodium hydroxide ¼ y–v ml.
8.6 Specification of Caustic Lye (IS: 252) 229
For sodium carbonate

N1 V1 ¼ N2 V2
0.1 * 2V ¼ N2 * 25 Strength of sodium carbonate ¼ Normality equivalent
weight ¼ N2 53.
Where, N1 ¼ normality of sulfuric acid, N2 ¼ normality of sodium carbonate.
For Sodium Hydroxide

N3 V3 ¼ N4 V4
0.1 * (y v) ¼ N4 * 25 Strength of NaOH ¼ Normality equivalent
weight ¼ N4 40.
Where, N3 ¼ normality of sulfuric acid, N4 ¼ normality of sodium hydroxide.
Determination of Sulfates
1. Dissolve 10 g of sample in 100 ml water.
2. Neutralize the alkali with concentrated HCl and leave an excess of acid in the
solution.
3. Boil the contents for decomposition of the carbonates.
4. Filter the solution and collect the filtrate along with the washings in a 500-ml
beaker.
5. Dilute to 250 ml, boil it and add 10 ml of 10% hot barium chloride solution.
6. Boil for 2 min and leave it undisturbed for 4 h.
7. Filter the contents using a tared sintered glass crucible (G No 4).
8. Wash the precipitates so as to remove chloride. Dry the crucible to a
constant mass.
Barium chloride in excess is added to lower the solubility of barium sulfate.

Precipitation by addition of hot solution accompanied by slow stirring reduces the
mechanical occlusion of barium chloride which produces a coarse precipitate that
has low solubility in acids.
Calculation
Sulfates (as Na2SO4) ¼ 60.84 M/m.
Where, M ¼ mass of precipitate. m ¼ mass of sample taken.
Determination of Water Insoluble Matter

1. Weigh 50 g of sample and transfer to a 1 l beaker.
2. Add 300 ml of water and mix well.
3. Add concentrated HCl till the solution is just alkaline to phenolphthalein. Boil the
solution and leave it undisturbed for 15 min on the hot plate.
4. Filter through tared sintered glass crucible and wash with hot water to remove the
alkali (ensure water gets drained completely).
5. Dry it at 105–110 C, cool and weigh in desiccator.
Calculations
Matter insoluble in water ¼ 100 M/m % by mass.
Where, M ¼ mass of insoluble matter, m ¼ mass of sample taken.
Packing
The solution can be supplied in tanks or steel drums or as agreed by the supplier and
customer.
Marking
Each container should contain information like name of material, source of manu-
facture, net weight, batch number and should be marked with “CORROSIVE –
HANDLE with CARE” clearly.
8.6.7 Sampling of Caustic Lye
From Tank
Take sample from each tank using a suitable sampling instrument. Mix it well and
take about 1 l of sample and transfer it to a glass bottle.
From Containers
The representative sample should be drawn from each selected container after
thorough mixing. Take 150–200 ml of sample from each container. The selection
of containers for sample collection must be as per Table 8.9.
8.7 Specifications for Sodium Thiosulfate (IS: 14781)
In the dairy industry, sodium thiosulfate is used in iodometric titrations. The type of
titration that involves iodine or deals with reactions in which iodine is liberated is
called iodometric titration. Peroxide value estimation of ghee or butter involves the
use of sodium thiosulfate. In the previous chapter, we have discussed about the
standardization procedure of sodium thiosulfate. In this chapter, we will discuss the
specifications of analytical grade of sodium thiosulfate.
Table 8.9 Selection of Lot size No. of containers to be selected randomly

samples
3–50 3
51–200 4
201–400 5
401–650 6
651–1000 7
8.7 Specifications for Sodium Thiosulfate (IS: 14781) 231
Table 8.10 Specifications of sodium thiosulfate (IS: 14781)

1 Sodium thiosulfate (as Na2S2O3.5H2O) (% by weight) 99.0–101.0
2 pH of 10% solution 6.0–7.5
3 Insoluble matter and metals other than alkali metals (% by weight), Nil
max
4 Sodium sulfide (as Na2S) (% by weight), max 0.001
5 Heavy metals (as Pb) (% by weight), max 0.001
6 Iron (as Fe) (% by weight), max 0.0005
7 Calcium (as Ca) (% by weight), max 0.005
8 Sulfate and sulfite (as SO4) (% by weight), max 0.01
9 Arsenic (as As2O3) (% by weight), max 0.001
The material should be in the form of colorless crystals or in the form of a

crystalline powder, free from extraneous impurities.
The freshly prepared 40% solution of sodium thiosulfate prepared in carbon
dioxide-free water should be clear from sediment apart from showing slight
flocculence.
The material should meet the specifications as mentioned in Table 8.10.
Determination of Sodium Thiosulfate

The sodium thiosulfate is determined by titrating the solution of sodium thiosulfate
with standard iodine solution using starch as an indicator.
Reagents
Starch solution: Mix 5 g of starch and 0.01 g mercuric iodide with 30 ml of water.
Pour the resulting paste in 1 l of boiling water and boil for 3 min. Cool the solution
and decant off the clear liquid.
Iodine Solution (0.1 N)

1. Weigh 1 g of the material and mix in 50 ml water.
2. Titrate this solution with iodine solution using starch solution as an indicator till
the appearance of blue color.
Calculation
Sodium thiosulfate (% by weight) ¼ 24.82 VN/M.
Where, V ¼ volume of standard iodine solution used in titration. N ¼ normality
of standard iodine solution. M ¼ mass in g of material taken.

1 l of 1 N Iodine ¼ 1 l of 1 N Sodium thiosulfate ¼ 248.2 g.
1 ml of 1 N Iodine solution ¼ 1 ml of 1 N sodium thiosulfate solution ¼ 0.248 g.
So, 1 ml of 1 Iodine solution is required to react with 1 ml of 1 N sodium
thiosulfate, then V ml of 1 N iodine solution reacts with V0.248 g of sodium
thiosulfate or for 100 ml sodium thiosulfate, volume of 1 N iodine solution

consumed ¼ V24.82.
If the iodine solution used is of N normality then the volume of iodine solution
consumed ¼ VN24.82.
Determination of pH of 10% Solution

Dissolve 5 g of the material in freshly boiled and cooled water, dilute to 50 ml and
mix well. Determine the pH using a pH meter.
Determination of Insoluble Matter and Metals Other Than Alkali Metals

This test is carried out by mixing sodium thiosulfate of known quantity with water
followed by addition of ammonium oxalate, ammonium phosphate, and ammonium
hydroxide. No turbidity should be observed in the solution.
1. Weigh 10 g of sample and dissolve in 75 ml of water.

2. Add 5 ml of ammonium oxalate solution, 2 ml of ammonium phosphate, and
10 ml of dilute ammonium hydroxide (1:9).
3. Leave it for 4 h and observe for any turbidity in the solution.
Determination of Calcium
Calcium if present in the sample reacts with ammonium oxalate to form calcium
oxalate, which produces turbidity.
1. Dissolve 2 g of the sample in 20 ml water.

2. Add 2 ml of dilute ammonium hydroxide (5 N) and 2 ml of ammonium oxalate
solution (2.5%).
3. Shake well and leave for 10 min.
4. Observe for any turbidity in the solution.
Test for Sulfate and Sulfite

Iodine when added to the sample oxidizes sulfite to sulfate. The formation of barium
sulfate produces turbidity in the solution and thus is taken as the indicator for sulfites
and sulfate.
1. Weigh 1 g of material accurately into a Nessler tube of 50 ml capacity.

2. Dissolve it in water and add iodine solution (0.1 N) till the solution turns to faint
yellow.
3. Add 1 ml of barium chloride solution.
4. The solution should not develop any turbidity.
Test for Iron

Ferric iron on reaction with potassium thiocyanate produces red color. The color thus
produced by the sample after oxidation is compared with that of the color produced
by standard iron solution.
8.7 Specifications for Sodium Thiosulfate (IS: 14781) 233
Reagents
Butanolic potassium thiocyanate solution: Weigh 10 g of potassium thiocyanate and
dissolve in 10 ml of water. Add a sufficient amount of n-butanol and make up the
volume to 100 ml. Mix the solution vigorously till the solution is clear.
Standard iron solution: In 10 ml dilute sulfuric acid (10% v/v) dissolve 0.702 g of
ferrous ammonium sulfate and dilute with water to 1 l. Pipette out 10 ml of the
solution and dilute with water to make up the volume to 100 ml. This solution
contains 0.01 mg/ml of iron (as Fe).
Procedure
1. Dissolve 0.2 g of the sample in 20 ml of water.
2. Add 5 ml dilute ammonium hydroxide (5 N) and then add slowly 40 ml of 15%
hydrogen peroxide. Leave for 10 min.
3. Evaporate the solution to dryness on a steam bath.
4. Add 5 ml of HCl (5 N) and 10 ml water. Boil the solution and then dilute to
100 ml with distilled water (mark it as sample S).
5. Add 20 ml of the sample S in a Nessler tube (50 ml).
6. To it add 1 ml concentrated HCl, 30 mg potassium persulfate and 15 ml of
butanolic potassium thiocyanate solution.
7. Perform a blank test using 0.5 ml of standard iodine solution in place of sample S.
8. Shake both the tubes for 30 s and leave for the liquid to get separated.
9. The intensity of the red color produced in the butanolic layer of the sample should
not be more than that of the control test.
8.7.1 Test for Heavy Metals
Reagents
p-nitrophenol indicator: Mix 0.2 g of p-nitrophenol indicator in hot water and dilute
to 100 ml with distilled water.
Standard lead solution: Dissolve 1.6 g of lead nitrate in distilled water, add 1 ml
of concentrated nitric acid and make up the volume to 1 l. Dilute this solution ten
times, so that this solution contains 0.01 mg/ml of lead (as Pb).
Procedure
1. Take 20 ml of sample S in a Nessler tube and neutralize it with 5 N ammonium
hydroxide using p-nitrophenol as an indicator.
2. Add HCl (1: 99) dropwise until neutral to the indicator and then add 1 ml of acid
in excess.
3. Add 10 ml of hydrogen sulfide and dilute to 50 ml mark. Shake the tube
vigorously.
4. Perform a blank test with 1 ml of standard lead solution and compare the color
produced in both the tubes (color produced by sample should be less than the
color produced in the control test to pass the test).
Packing
The material should be packed in hard-glass bottles with air-tight stoppers.
Marking
The container should be marked with information like source of manufacture, net
weight, name of the manufacturer, batch number, and month or year of manufacture.
Sampling
The quantity of the sample drawn from each container to carry out all the tests should
not be less than 200 g. The samples should be stored in dry opaque glass bottles or
other suitable containers and sealed airtight.
The samples (r) can be drawn using the formula ¼ Number of containers in the
lot/5 (rounded to the nearest integral part of the value).
8.8 Specification for Reagent Grade Water (IS: 1070)
Water is used for making chemicals used for analysis. Generally, the water used in
the laboratory should fulfill certain requirements so that its use may not alter the
efficiency, reproducibility of chemical analysis. The requirements and methods of
test for reagent grade water to be used in laboratory will be discussed.
Grades: The reagent grade water is categorized into three groups on the basis of
its purity.
Grade 1: When the test method requires maximum precision with minimum
interference and accuracy being the top priority, Grade 1 water is recommended. It
may be prepared by distillation of feed water having a conductivity of 20 μohm1/
cm (max) when measured at 25 C. It should be then polished with mixed bed
deionizers and passed through 0.45 μm membrane filter.
Grade 2: When the general laboratory tests are to be done in which freedom from
organic impurities is required. Such type of water is not used for biological or
medical analysis and organic trace analysis.
Grade 3: For washing glasswares/pre-rinsing of glasswares/use of feed water for
producing higher grade water or when a large quantity of water of low purity is
required for making synthetic test solutions.
Requirements
The water should be prepared by treating with thermal distillation or by ion exchange
method and subsequently purified when necessary. It should be clear, odorless,
colorless, and tasteless. Table 8.11 contains the specifications of reagent grade water.
8.8 Specification for Reagent Grade Water (IS: 1070) 235
Table 8.11 Requirements of reagent grade water

Grade
S. No. Characteristic 1 2 3
1 Specific conductivity μohm1/cm at 25 (Max) 0.1 1.0 5.0
2 pH at 25 C NS NS 5.0–8.0
3 Total solids or nonvolatile residue at 105 C, mg/l (max) NS 1.0 2.0
4 Silica (as SiO2) mg/l (max) 0.01 0.1 1.0
5 Color retention of KMnO4(minutes) 60 10 10
Table 8.12 Selection of Consignment size (N) Sample size (n)

samples
Up to 15 3
16–25 4
26–50 5
51–100 7
101–150 8
151 and above 10
Color Retention Time (Oxidizable Matter)

1. Take 500 ml of water sample, add 1 ml of concentrated sulfuric acid to it followed
by addition of 0.2 ml potassium permanganate solution (0.316% w/v).
2. Stopper the flask and leave it undisturbed at room temperature.
3. The color of permanganate should not disappear completely for the indicated
period of time.
4. A blank test should be carried using water free from organic matter.
Packing
The reagent grade water should be packed in clean glass, polyethylene or any
suitable plastic container. The material of the container should be inert to water
and should not affect the quality of water. The container should be well and tightly
closed.
Marking
The container should contain the name of the material, name of the manufacturer,
volume in liters, date of manufacturer, and batch number.
Sampling
The samples should be drawn randomly as per Table 8.12.
Suggested Readings
IS 360 (1964) Amyl alcohol. Bureau of Indian Standards, New Delhi
IS 4238 (1967) Sterilized milk. Bureau of Indian Standards, New Delhi
IS 5058 (1996) Sodium citrate. Food Grade. Bureau of Indian Standards, New Delhi
IS 252 (1991) Caustic soda, pure and technical. Bureau of Indian Standards, New Delhi
IS 253 (1985) Edible common salt. Bureau of Indian Standards, New Delhi
IS 264 (2005) Nitric acid. Bureau of Indian Standards, New Delhi
IS 14781 (2000) Sodium thiosulphate. Bureau of Indian Standards, New Delhi
IS 1070 (1992 Reaffirmed 2008) Reagent grade water-specification (3rd revision). Bureau of Indian
Quality Concepts
9
With the advent of food safety concepts due to the liberalization of the food industry,
the quality of the food products being produced is of utmost importance. The
integration of the food supply chains now requires newer approaches to ensure
food safety. The dairy industry nowadays is also adopting some of these approaches
to ensure the safety of the milk and milk products being produced. Food regulatory
bodies have also formulated strict laws and standards for milk and milk products.
Food safety is now not only limited to the products being exported but also to the
domestic market and consumers. So in response to the food safety concept, the
public and the private sector have now modified their production process with
adoption of newer technologies and have also applied stringent laws to ensure
product safety. Safety and quality of a product must be ensured in the entire chain
involved in the production, i.e., from the udder to the consumer or from farm to fork.
Milk being a highly perishable commodity and being at a higher risk to get
contaminated by either environmental factors or due to poor farm practices demands
high care from milk producers and the industrial persons to ensure its safety and
quality, especially where the infrastructure is not proper. Deterioration due to
environmental factors means failure or time lapse in chilling milk to refrigeration
temperature especially in hot and humid or tropical regions while contamination by
poor farm practices means that entry of pesticide, heavy metal, aflatoxin, or antibi-
otic residues to milk. The pesticides, aflatoxins, and heavy metals get reflected into
the milk due to the ingestion of feed which is either contaminated or which contains
a higher amount of residues of these chemicals while the antibiotic residues enter
milk due to the failure to observe withdrawal period after the animal is given the
antibiotic for the treatment of udder infections like in mastitis or other diseases like
foot and mouth disease. These contaminants not only adversely affect the health of
the consumer but can also hamper the quality of milk products like starter culture
failure in fermented foods due to the inhibitory action of the antibiotics. Apart from
contamination, the quality of milk is also challenged by adulteration which is done in
order to earn more profits or to preserve milk for longer periods which again can also
affect the health of consumers. Thus, the personnel engaged in the dairy sector

https://doi.org/10.1007/978-981-15-4167-4_9
238 9 Quality Concepts
should be more careful and proactive in order to maintain the quality and safety.
These food safety and quality requirements can be achieved by application of certain
quality management systems like Hazard Analysis and Critical Control Point
(HACCP), ISO 22000-Food Safety and Management System, Total Quality Man-
agement, Six Sigma, and other quality concepts like PDCA, and Quality Assurance.
Considering all these challenges faced by dairy industry, the adoption of quality
assurance and certain quality concepts will help the dairy industry in the following
ways:
• Curbing the menace of adulteration which will be subsequently reflected in the

quality of milk and milk products.
• It will help in developing the confidence and faith of domestic as well as
international consumers toward the quality of milk and milk products produced.
• It will help in generating more revenue to the dairy industry which will, in turn,
help the employees and the farmers.
9.1 What Is Quality?
According to ISO 9001:2005, quality is degree to which a set of inherent

characteristics of an object fulfils requirements. It is the totality of the features and
characteristics of a product or a service that bears on its ability to satisfy stated or
implied needs.
1
Quality /
variability
Quality has an inverse relation with variability. As the variability in the
characteristics or features of a product or a service is reduced the quality of the
product or the service given gets increased. Note: The term “quality” can be used
with adjectives such as poor, good or excellent, “Inherent”, as opposed to
“assigned”, means existing in the object.
When the parameters or specifications (information/data concerning the chemi-
cal, microbiological, and physical characteristics of the product) as per the
customer’s requirements are stated/mentioned.
These are the implied needs, i.e., understood needs embedded in the transaction
between the customer and the supplier.
Quality embraces many characteristics:
• Physical
• Chemical
• Technological
• Bacteriological
• Nutritional
• Aesthetic (Appearance)
9.2 What Is Quality Control? 239
Quality is the combination of characteristics like physical, technological,

nutritional, chemical, microbiological, and aesthetic which determine the degree of
acceptability for a product by the consumer.
Quality of a product is judged by subjective and objective tests.
Subjective tests are based on one’s perception and include mainly sensory tests.
Objective tests are based on scientific or logical evidence like in Chemical,
Microbiological, and Nutritional tests (performed in laboratory).
A consumer mainly focuses on the specifications of a product or service and
compares the same product available from different manufacturers. The definition of
quality has evolved with time and is defined in different ways:
• American Society for Quality: Quality can have two meanings, i.e., the
characteristics of a product or a service that bear on its ability to satisfy the stated
or the implied needs and a product or a service delivered, which should be free of
shortcomings or deficiencies.
• Philip b Crosby: “Quality is defined as the conformance to requirements.”
• W. Edwards Deming: According to Deming, quality is defined as “the efficient
production of the quality that the market expects.”
• Peter Drucker: Drucker defined quality as “Quality in a product or service is not
what the supplier puts in. It is what the customer gets out and is willing to pay
for.”
• Noriaki Kano and others: Gave a two-dimensional model of quality: must-be
quality and attractive quality. “Must be quality” is similar to “fitness to use” while
attractive quality means what the customer has thought but has not yet thought
about.
• Joseph M Juran: “Fitness for use.” This means that the perception of quality of
any product or service can vary from one person to another like a person having
lactose intolerance may not consume milk even if it is of best quality.
• Six sigma: It is a term which states that “Number of defects per million
opportunities.”
9.2 What Is Quality Control?
It is defined as the set of activities which ensure that the products and services meet/
fulfill requirements for quality. It was categorized as a laboratory function which
aims at end point testing of the finished goods or products by analyzing the samples
and making decision to accept or reject them.
As per American society for quality “Quality control is the observation technique
and activities used to fulfill requirements of quality”. Quality control is a failure
detection system that uses a testing technique to identify the flaws and the errors in
the products by drawing random samples from a specified lot at regular intervals.
It aims at ensuring that all the products being manufactured are as per the
standards that have been specified by the regulating body, so that the health and
legal rights of the consumers remain protected. It is a technique which aims at
detection rather than prevention. The number of samples recalled is more in a system
following quality control because it focuses on end-product testing. This finally
affects the reputation of the company due to chances of a higher failure.
9.2.1 Implementation of Effective Quality Control
1. Decide which specific standards the product must meet: The specifications of the
products with respect to its chemical, microbiological standards. The standards
are fixed so that the end product should meet the legal requirements as laid down
by the food regulatory body(s).
2. The extent of quality control actions must be determined: Once the standards are
laid, the production parameters, the type of tests to be conducted, the sampling
plan for a product need to be decided.
3. Real-world data must be collected and the results to be reported to the manage-
ment personnel: The results of various chemical, microbial, sensory, shelf-life
tests should be recorded and submission of the report to the concerned official
taking care of the quality control activities. The decision on whether the product is
to be accepted/dispatched to the market or rejected is based on the test reports.
4. Corrective action must be decided upon and taken: In case of product failure, i.e.,
if the product fails to meet any of the specified standards, then the corrective
action must be taken so that the product failure should not take place in the future.
5. If failures occur repeatedly, the process must be revised to improve the produc-
tion planning so that the quality of the product or service gets improved. The
revised or improved plan must be put into action as soon as possible.
6. At last, the quality control process must be followed on a regular basis to find out
any flaw in the process and take necessary action, when required.
9.2.2 Responsibilities of Quality Control Department
1. Inspection of the supplies, materials, and raw products: The quality parameters
of the raw material, supplies like additives, packaging material, flavoring, or
coloring matter are checked and the results are matched with the specifications as
per the norms of the company or the purchaser.
2. Scheduling of the operations: All the unit operations to be involved in the
production of any product need to be verified for any flaw or error in it. Also,
the equipment or machines should be checked for proper functioning.
3. Measurement of production and equipment efficiency: The production done in a
shift or the amount of the milk handled or the product produced per hour gives the
production efficiency while the quantity of milk handled and processed by the
machine per shift gives equipment efficiency (in this the idle time or machine
downtime is also considered).
9.3 What Is Quality Assurance? 241
4. Inspection of the finished product: The chemical, microbial, and sensory analysis
of the finished product needs to be done on a regular basis to analyze the quality
of the product being manufactured.
5. Shipping and storage control: The storage of the produced product should be
finalized like whether to store in cold storage or to store in a dry and dehumidified
environment. The mode of transportation like in refrigerated vehicle or insulated
vehicle, of the finished goods to be underlined and regular monitoring to be done
to check whether any of the things are in order or not.
6. Preparation of standard operating procedures (SOP) and specifications in writ-
ten form: The standard operating procedure(s) for the production of a product(s),
SOP(s) for various chemical, microbial, and sensory analysis, also the corrective
measure or plan to be taken in case of any failure should be well documented. The
record keeping for all the activities that come under quality control needs to be
done (as records speak better than men).
7. Sanitation inspections: Regular inspections with regard to the sanitary conditions
should be done.
8. Conformance to local and federal regulations: The products manufactured
should be checked for conformance according to the standards laid down by
regulatory bodies.
9. Waste disposal control: The generation of waste from a dairy plant has always
been a concern. Waste generated from dairy plant composed of effluent, gases,
and ash released from the boiler, etc., a proper procedure and a tolerance limit for
these types of waste are to be framed and also measures to bring it under control
in case of any failure in the waste treatment or disposal must be developed.
9.3 What Is Quality Assurance?
It is defined as a set of activities which ensure that the quality levels of products and
services are properly maintained and that the supplier and customer quality issues are
properly resolved. Quality assurance gives confidence to the manufacturer and the
customer that the product or service will satisfy the requirements of quality. It is a
method to prevent the occurrence of mistakes and defects in the manufactured
products or services rendered when delivered to the customer. ISO defines quality
assurance as “part of quality management focused on providing confidence that
quality requirements will be fulfilled”. It is a systematic way of comparing the
product or service with the standard, monitoring of the process, and an associated
feedback system that confers error prevention.
Quality assurance concept has two principles: “Fit for purpose,” which means that
the product produced should be suitable for the intended use and “right first time”
which aims at preventing the occurrence of mistakes or their elimination. It gives
confidence to the management within an organization (internal purpose) and also
provides confidence to the consumers or end users outside the organization (external
purpose). In contrast to quality control, quality assurance covers a wider sense as it
aims at full control in terms of quality ranging from the quality of raw materials,
controlling the process at every stage as well as the distribution system, etc. Quality
assurance is a prevention rather than a detection system, i.e., it is a proactive
approach not a reactive approach. The product to be manufactured is monitored at
each step so that the product made fulfills the legal as well as the customer
requirements.
9.4 Objectives and Importance
1. Maintaining the legal standards and requirements.

2. To fulfill customer requirements for attributes like body, color, texture, chemical
composition, microbiological, sensory, safety, and packaging.
3. Checking for the presence of any adulterants in the raw material to maintain the
quality of the final product and to minimize the occurrence of any hazard or
process failure.
4. Analyzing the efficiency of the process involved in the production, waste dis-
posal, utility services, etc.
5. Ensuring the cleanliness and sanitary conditions.
6. Improvement in the production of efficiency and minimize the rejection.
7. Reducing the variable costs like cost of production, labor cost, electricity cost,
water consumption, etc. to increase the revenue generation.
8. Reduction of customer complaints.
9. Increasing the morale and confidence of the employee.
9.5 Responsibility of the Quality Assurance Department
1. Good Manufacturing practices.

2. Good hygienic practices.
3. Pest control.
4. Sanitation standard operating procedure.
5. Documentation.
6. Hazard control.
7. Calibration and Standardization.
8. Quality control.
9. Sensory analysis.
10. Waste management and disposal.
11. Preventive maintenance.
12. Labeling and packaging as per regulations.
13. Redressal of customer complaints.
14. Internal auditing.
15. Framing a procedure for product traceability.
16. Product recall.
17. Storage of raw material, packaging material, additives, etc.
18. Storage of finished products.
19. Apply for certifications like ISO 22000, ISO 14000, Jewelry policy, FSSAI
certification.
9.7 Deming’s Philosophy 243
Table 9.1 Quality control vis-à-vis quality assurance

Quality control Quality assurance
Product oriented Process oriented
Reactive approach Proactive approach
Corrective action Preventive action
Focuses on testing quality Focuses on building quality
Detect defects Prevent defects
Makes sure that the results of what you have Makes sure that you are doing the right thing in
done are what you expect the right way
Meant for implementing the process Meant for developing and organizing the best
developed by a team, Line function quality process, Staff function
20. Employee training.

21. Cleaning and sanitation of the equipment.
9.6 Principles of Quality Assurance
In a dairy industry, the quality assurance program can be implemented keeping in

view of three things:
1. Raw material control: The quality of all the materials involved in the production
of a product like raw milk, sweeteners, flavors, colors, packaging material, and
sanitizer should be checked on procurement and any substandard product should
be rejected.
2. Process control: Time–temperature combination for heat treatment, type of heat
treatment to be given, amount or level of the preservative, stabilizers/emulsifiers,
and antioxidants to be added should be strictly monitored.
3. Finished product inspection: The testing of the finished product to be done to
check whether the product is as per the legal standards specified for a product.
By following these three principles, the number of nonconfirming products can be

reduced appreciably because quality check at every step is done which will subse-
quently reduce the product recall rate from the market and finally will help in
building a better relationship between the producer and the consumer.
Table 9.1 shows the difference between Quality control and Quality assurance.
9.7 Deming’s Philosophy
William Edwards Deming was an American professor, statistician, lecturer, author,

engineer, and management consultant. He shifted to Japan after the second world
war to help them with the census. He also taught process control using statistics to
some of the leading business leaders of Japan and gave them the message that “by
improving the quality, companies can decrease their expenses along with an increase
in their productivity and market share. These techniques of Deming helped the
businesses like Toyota, Sony, and Fuji to achieve huge amount of success. The
quality of the products manufactured by them was far superior from their
counterparts as well as the costs were lower. The ideas taught by Deming helped
Japan to become the world’s largest economy. Deming influenced on better design-
ing of the products for improving the service, products manufactured should have a
high degree of uniformity in terms of their quality, the product to be tested at the
workplace and the research centers to ensure the quality and increasing the sales by
targeting the global markets.
Dr. Deming taught that by adopting the appropriate principles of management,
organizations can increase quality simultaneously reducing the cost of production by
reduction in waste generation, rework, and increasing the customer loyalty. This can
be achieved by improving the system continuously and thinking about it as a whole.
9.8 The Deming Cycle
Deming proposed a cycle that is often called as PDCA cycle and is also known as
Shewhart cycle. PDCA is a four-step management technique, which stands for Plan-
Do-Check-Act. This concept focuses on monitoring of the process and eliminating
the root cause of the failure or substandard performance at each step. It is an ongoing
and never-ending process. The PDCA cycle shows the importance of continuous
improvement in maintaining the quality rather than making the changes after the
failure. The other name of PDCA cycle is OPDCA in which O stands for observation
or observing the current condition.
9.8.1 Plan
• The goals of the company need to be established.

• Once the goals are fixed, the strategy toward achieving those goals is to be made
like standardization of the working procedure, formulation of the product, etc.
• The employees should be trained and informed about the standardized procedure,
formulation of the product, testing of the raw as well as finished product and other
ingredients. Employees should also be trained and informed about the regulations
concerning about food safety.
9.8.2 Do
• As the planning is done, the work should be carried out in accordance to the
decided plan like for manufacturing a product, the type of raw material to be used
should be as per the specifications decided, the type and quality of the additives,
the packaging material, production planning, etc.
9.8 The Deming Cycle 245
9.8.3 Check
• Regular checks and inspection should be done to verify that whether all the
protocols are being followed according to the plan, in case any deviation occurs
it should be corrected. Inspection at regular intervals helps to find the cause of
failure of the product and steps to be taken to rectify it. The records of testing and
those related to the production procedures should be documented and matched
with the specifications laid down. The data from the records should be placed in
the form of a chart so as to compare the actual results with the expected outcomes.
9.8.4 Act
• It is also called as adjust. When any noncompliance is found or recorded, its root
cause should be identified and the plan should be made to remove the root cause.
The performance of the process after rectification of the root cause should be
documented and checked regularly to analyze the working of the new procedure.
If the specifications of the product are as per the desired then the rectified
procedure should be followed otherwise further rectification should be done
and the performance of the procedure should be recorded, analyzed, and
documented.
Deming also gave 14 points for management which will help in transforming the
organization are discussed below:
1. Create constancy of purpose: Create constancy of purpose toward improvement

of product and service, with the aim to become competitive and to stay in
business and to provide jobs. Constant improvement should be the ultimate
goal of an organization if it has to compete and grow. The process of improve-
ment should be carried out on the whole process and not only be selective at the
end product. The required adjustments should be made while scrutinizing the
whole process while the adjustments made at the end, i.e., after the product has
been manufactured, should be avoided. The corrections or amendments which
have been recognized should be incorporated with immediate effect.
2. The new philosophy: The organization’s working should be flexible such that
any new idea which will help in further improving the process and product
quality, can be incorporated without changing the working structure. Resistance
to change should be avoided as without changing an organization cannot sustain
for longer periods, as it is said that innovation occurs every day. Companies like
Kodak, Nokia, and Ambassador resisted to change according to the market
demands, as a result, they were taken over by their competitors.
3. Discontinue the dependence of inspection: Eliminate the need for inspection on
a mass basis by building quality into the product in the first place. The overde-
pendence on the final checking and inspection to ensure the quality should be
avoided. The quality inspections should be done as the process is going on so
that the improvements if any, can be made earlier. The approach of an organiza-
tion should be preventive and proactive instead of being reactive. The quality
building process should start from the beginning. Statistical control methods
along with the physical inspections can help in improving the process of
working.
4. End lowest tendercontract: End the practice of awarding business on the basis of
price tag. Instead, minimize total cost. Move toward a single supplier for any
one item, on a longterm relationship of loyalty and trust. Generally, the procure-
ment of raw materials, machinery, and equipment is done by selecting the
supplier who offers the lowest price. This idea should be dropped down because
if the organization has to build a long-term relationship with its suppliers, then it
should ascertain that the item should be procured from a single supplier which
should be fixed. This approach will help in building trust and loyalty between
the company and the supplier. The company must rely and have faith in the
suppliers, as they are the first link for the development of a high-quality product.
5. Continually seek out problems: Improve constantly and forever the system of
production and service, to improve quality and productivity, and thus constantly
decrease costs. Improvement should be done on a daily basis and forever.
Continuous improvement in production and service leads to improvement in
quality and productivity, which finally leads to increase in the efficiency of the
process subsequently decreasing the cost of production. The concept of PDCA
should be followed, also Kaizen can be used as an effective tool to reduce waste
and improve the productivity. This point is similar to the concept explained in
first and third points. With the improvement in the quality at each step, the
generation of waste and idle time will be reduced.
6. Introduce novel and modern methods of training on job: The employees should
be given training regarding the responsibilities or the work assigned to them.
Training helps in improving the skills of an employee and their overall devel-
opment. The training employed to the employees should come under their
personal development plan. The upgradation of the manpower is very important
because it is the manpower only who will deal with the problems that occur
during the production and other related activities. With regular training, the
employee will become competent in his respective field which will indirectly
help the entire organization. Training helps in reducing the variation and allows
the workers to understand their roles in a big picture.
7. Supervision: The aim of supervision should be to help people and machines and
gadgets to do a better job. Leadership plays an important part in carrying the
organization forward. The managers lead by example and also supervise guide
the employees and suggest them new ideas or ways to improve productivity.
They can visualize and observe everything which happens in the workplace. The
importance of participative management and transformational leadership should
be emphasized. The goal of a leader must be to find the ways that the employee
should work to its full potential rather than just focusing on meeting the targets.
Good supervisors are coaches, not policemen.
8. Remove the fear of fear: Drive out fear, so that everyone may work effectively
for the company. The fear of failure, top management or anything must be
9.8 The Deming Cycle 247
eliminated at the workplace. The employee should be given the freedom to bring
out the best in him. Everyone should feel safe, motivated, should respect each
other, the communication should be two way and transparent. The employees
should not only get restricted to their area of work rather should show interest in
the work of co-workers also. The workers should be made to feel that they are a
valuable asset to the organization.
9. Remove the barriers: Break down barriers between departments. People in
research, design, sales, and production must work as a team to foresee problems
of production and use that may be encountered with the product or service. All
the departments in the organization should be integrated with each other and no
form of boundary should separate them. The best results are achieved through
cooperation and by regular communication with each other. This can be
achieved by creating a multifunctional team or a team composed of employees
from each department to accomplish the work and achieve the laid targets, this
will help in sharing ideas with each other and will help in building a healthy
working environment in the organization.
10. Eliminate Numerical Goals, Posters and Slogan: Eliminate slogans,
exhortations, and targets for the work force asking for zero defects and new
levels of productivity. The focus should be on how the process is carried out and
not only on the targets. Deming said that though the targets encourage high
output but may also reduce the quality of the final product. Rushing through the
production activities or processes can increase the probability of errors. Focus
should be on quality instead of quantity. The resources and the support should
be provided by the top management to the employees to improve the production
quantity and quality.
11. Unclear slogans should be eliminated: The people should be assured about what
they should do and what the top management expects. Stimulating slogans,
warnings, and exhortations should be removed. The quality and production
problems do not arise due to an individual but due to the system itself. The
employee should be encouraged and praised for doing good work.
12. Remove the barriers to pride of workmanship: Everyone should take pride in
what they do and should not be rated or compared to the working style of
everyone is different. All the workers should be treated equally and not made to
compete with each other for monetary or other rewards.
13. Institute education: Institute a vigorous program of education and
selfimprovement. Encourage the workers to learn new skills and improve on
their current skills so as to prepare for the future challenges and changes. This
point is similar to the sixth point.
14. Everyone should consider transformation as his job: Transformation with time
should be the ultimate target for an organization to survive and grow. Every
small step should be analyzed and should take a step closer to improve the
quality. Put everybody in the company to work to accomplish the transforma-
tion. The transformation is everybody’s job.
The seven deadly diseases as said by Deming should be avoided in an organiza-

tion. These diseases are lack of constancy of purpose, emphasis on short-term profits,
evaluation by performance, mobility of management, operating the company on

visible figures and not using the factual data, excess of medical costs, and excessive
costs of liabilities.
9.9 The Juran’s Philosophy
Joseph Juran was born in 1904 and was an American engineer and management
consultant. His quality philosophy also took root in Japan as was with Deming. He
laid emphasis on the importance of a wide organizational level approach to achieve
quality. He stated that the quality management begins from the top management and
continues to the bottom. Juran gave an approach to quality which was called as Juran
trilogy concept. This quality concept consists of Quality Planning, Quality control,
and quality improvement. During Juran’s time, the primary focus of a business was
to maintain the quality of the final product as is stressed by Deming too. Juran
changed the concept and added the human dimension of quality management. He
stressed on the value of educating and training the managers. According to Juran, the
main cause for the quality issues of a product was due to the resistance to change and
human relations problems. His theory was focused on the nonmanufacturing units,
especially the service providers or the service-related processes.
9.9.1 Quality Planning
It is a phase in which the products and processes are developed to meet the
consumers’ demand. It involves creating awareness of the customer needs, setting
goals, need for improvement, and planning to achieve the goals. The quality
planning begins with the management, as it has to develop a vision that commits
to make a change and plan for the same. It requires skilled, trained, and qualified
staff. The steps to be followed in the quality planning process are as follows:
• Establish the goals that are to be met and targeted.

• Identify the end users or the customers who will be impacted directly by the
efforts made to reach the goals and finally with the quality of the product.
• Determine what the consumer needs.
• Develop the process that can produce the right quality of the product that is
desired by the customer.
• Establishing the process in real-time situation at the workplace and make sure that
the next lot or batch is manufactured by the new or modified process.
9.9.2 Quality Control
This step aims at executing the plans in a right manner to a right place (as the plans
made are specific for a process). It involves monitoring the entire process at every
9.11 Ten Steps to Quality 249
step to analyze the differences between the set goals and the actual performance.
This process is done to develop or standardize the methods that will help in testing
the quality of the product. In case of any deviation from the standard, it will lead to
modifying or changing the process with a motive to improve. This process is
summarized in three steps:
• Evaluate the quality performance.

• Compare the actual performance with the stated fixed targets or goals.
• Make a specific action plan in case of any deviation occurred.
9.9.3 Quality Improvement
It is a continuous process that aims to gain perfection in terms of the quality of the
product and efficiency of the process. The management compares the actual data
with the set targets and praises when the things are done correctly. The problems
occurring must be diagnosed and should be eliminated from its roots. This process is
summarized in the following steps:
• Establish resources like infrastructure, money required to attain the annual quality
improvement.
• Identify the needs required to improve the project and product.
• Form the teams which will lead and will carry out all the activities with full
responsibility to bring the project to success.
• Provide the training and educate the employees which are a part of the team so
that they become capable enough to find out the flaws and rectify them and
establish control over the process once again.
9.10 Three Steps to Progress
Juran also gave the three basic steps to progress, which the organization must
implement to achieve high quality.
1. Accomplish the improvements that are well structured with commitment and with
a sense of urgency.
2. Build a training program.
3. The seeds of commitment and leadership should be cultivated in the top manage-
ment as they are the driving force that will carry the organization toward success.
9.11 Ten Steps to Quality
Juran also proposed the following ten steps for an organization to inculcate better
quality in their product:
1. Establish the awareness for the need to improve, the necessity of improvement,
and the opportunities available for improvement.
2. Identify and freeze the goals for improvement.
3. Plan should be made that how to reach the goals.
4. Train the personnel involved.
5. Carry out projects to solve problems. Implement the process framed to solve the
existing problems.
6. Record and report the progress and the level of improvement achieved.
7. Praise when things are going right, do not forget to give recognition.
8. Communicate the results among all the employees.
9. Maintain a scorecard to analyze and compare the progress achieved and bench-
mark with the competitors.
10. Maintain the pace by carrying on the process of improvement.
9.12 Pareto Principle
Juran in 1941 started to apply the Pareto principle, which was given by Vilfredo
Pareto. The Pareto principle states that 80% of the problem is caused by 20% of the
causes. This principle is also called as “the vital few and the trivial many.” Juran
reformed it to “the vital few and the useful many” to lay emphasis on that the
remaining 80% of the causes should not be overlooked. Juran told that the
organizations having knowledgeable persons, should focus less on the meaningless
issues and lay stress on identifying and correcting the remaining 20%, which means
the cause of the defects need to be eliminated. On applying the Pareto’s in food
safety, it can be said that hazard prioritization should be of the utmost importance. If
one assumes that 20% of the hazards are the cause for 80% of the food and health-
related issues, so by categorizing the hazards the quality assurance personnel can
target those 20% hazards, which affect the health of the consumer significantly. On
the other hand, if the hazards are categorized on a random basis, the quality
assurance personnel should fix one of the 80% of the hazard that may account for
some of the remaining 20% of the injuries. Like Deming, he pointed that the money
spent on ensuring the quality, is the money that is well spent and is worthful. He gave
four absolutes of quality management and also 14 steps for quality improvement.
9.13 Crosby’s Theory
Philip B Crosby was an author and businessman, who was recognized for its
management and quality management theories. He started his work in quality
management much later than Deming and Juran. He initiated the zero-defect pro-
gram at the Martin company. He was credited for a 25% reduction in the rejection
rate and a 30% reduction in the scrap costs while working as a quality control
manager of the Pershing missile program. “Doing it right the first time” was his
mantra and the answer to the quality crisis. His philosophy was expressed in
9.13 Crosby’s Theory 251
absolutes of Quality Management and the Basic elements of Improvement. He was

the author of best selling books like “Quality is free” and “Quality without fears”.
9.13.1 Crosby’s Absolutes
1. Define quality as the conformance or adherence to the requirements: Each

product or service should have it stated requirements describing what the cus-
tomer demands. A product is said to be of quality when it attains those stated
requirements. Zero defect will be attained when these requirements are met.
2. Prevention is better than cure, i.e., the best way to ensure quality is by prevention. It
is important for a process to be defect-free that the cause of a defect should be
recognized and eliminated. The time and money should be spent on fixing the
process rather than testing or analyzing the product for defects, which makes no use.
3. The level of performance should be compared with a standard that is “zero
defects” with respect to the requirements. In simple words, a product that does
not meet the final requirements of the customer is deemed to be rejected by him.
Even if the product only satisfies the customer’s need even though it does not
meets all the requirements, the set of the requirements fixed for a product should
be reviewed and changed accordingly.
4. Quality is measured as the price of nonconformance. Every defect has a hidden
cost that is invested in terms of inspection time, rework, waste generation, labor,
utilities, and most important the customer dissatisfaction. When the defect is
identified, the cost can be measured and can provide the justification for spending
the money in eliminating the defect and thus improve the quality. This is a way to
measure the zero-defect directly in terms of monetary value.
9.13.2 Zero Defects
According to Crosby, zero defect is a performance evaluation method that states that
the person should commit oneself to closely monitor the details and avoid errors. By
following this principle they can move a step closer to the zero-defect goal.
According to him, the term zero defect is not just a manufacturing principle but
was a theory that affects each and every decision that we make.
Crosby gave the 14 steps for quality improvement, which are as follows:
1. The entire management should be committed toward quality improvement.

2. A team should be constituted with their main goal being quality improvement.
3. Define the standards for each quality improvement activity.
4. Train the supervisors regularly.
5. Determine the cost of quality and show how the improvement will benefit in
monetary terms.
6. The employees should be encouraged to fix the defects and to keep a record of
what they have done and how.
7. Create a zero-defect analyzing committee.
8. The employees and supervisors should understand the steps to be taken to

achieve a certain level of quality.
9. Celebrate a zero-defect day, which will reflect the company’s commitment
toward quality management and improvement.
10. Set the time limit for the accomplishment of the goals.
11. Evaluate the root cause of failure or errors and eradicate them from the process.
12. The employees should be given incentives for the work they have done.
13. Form a quality council and hold regular meetings.
14. Repeat again from the first step.
The other theories which are of importance for improvement in the quality of the
product or the process are EFQM framework and Ishikawa theory. These two
theories are explained in brief.
EFQM Framework The acronym EFQM stands for The European Foundation for
Quality Management. It is a model that is based on the nine criteria for quality
management. The five criteria focus on what the company has to do (like strategy,
partnership, leadership, resources, and processes), which are also called as enablers,
and the other four criteria include what a company achieves (like customer, business
results, society, and people) and are referred to as results. This methodology refrains
from focusing on one methodology or technique, but focuses on the use of different
quality management methodologies. The eight core values to drive a sustainable
success as defined by this model are:
1. Focus on the results.

2. Focus on the customer.
3. Constancy of purpose and consistent visionary leadership.
4. Process and facts form the management focus.
5. Training employees.
6. Learning should be a continuous process.
7. Develop partnerships.
8. Social responsibility of the corporation.
9.14 Ishikawa’s Theory
The last theory for quality management was given by Dr. Kaoru Ishikawa. Ishikawa
emphasized on quality from a human point. He outlined the seven basic tools for
quality improvement are as follows:
1. Pareto analysis: A bar chart where the bars are arranged in descending order of
magnitude. This helps to identify the big problems of a process. It helps to
prioritize actions needed to solve complex problems, to sort out the “vital few”
from the “trivial many and to separate important from unimportant causes
contributing to a problem.
9.15 Total Quality Management 253
2. Cause and effect diagrams: A tool used to graphically display the relationship
between an effect (e.g., a problem statement) and the its causes. Cause and effect
diagrams help to identify the root cause of problems. It is also known as Fish bone
diagram.
3. Stratification: Breaking down data into categories so you can make sense of
it. When data from a variety of sources or categories have been lumped together,
the meaning of the data can be impossible to see. This technique separates the
data so that patterns can be seen.
4. Check sheets: A data-collection form used to manually tally and record the
number of observations or occurrences of certain events during a specified time
period. The check sheet shows how frequently a problem occurs.
5. Histograms: A bar chart that displays the distribution of individual
measurements. It is also also called a frequency distribution. It helps to study
the possible relationship between one variable and another. A type of analytical
tool that shows the degree of variation in the data. It provides clues to reducing
variation and causes of problems.
6. Scatter charts: A plot of one measured variable against another. Paired
measurements are taken on each item and plotted on a standard X-Y graph.
They demonstrate the relation between various factors.
7. Process control charts: A line graph of the measurements of a product or process
over time that has statistically based control limits placed on it. It helps to display
and manage variation in process output over time, to identify when a process
changes, to distinguish special from common causes of variation and to tell the
operator when to take and when not to take action and just let the system run.
9.15 Total Quality Management
Total Quality Management (TQM) is the process of continuous improvement in the

operations or processes by detecting and reducing or eliminating the errors that occur
during the manufacturing process, improving the customer’s satisfaction by the
involvement of every member of the organization. TQM can also be defined as the
integrated organizational approach of delighting the consumer by meeting their
requirements on a regular basis by the involvement of every member of the organi-
zation through continuous improvement in all fields along with the problem-solving
methodology. TQM was originated in the 1950s and became popular in the early
1980s. TQM has been defined by several organizations:
British Standards Institution standard BS 7850-1:1992: “A management philosophy

and company practices that aim to harness the human and material resources of an
organization in the most effective way to achieve the objectives of the
organization.”
International Organization for Standard (ISO 8402: 1994): “A management
approach of an organization centered on quality, based on the participation of
all its members and aiming at long term success through customer satisfaction and
benefits to all members of the organization and society.”
The American Society for Quality: “A term first used to describe a management
approach to quality improvement.”
The Chartered Quality Institute: “TQM is a philosophy for managing an organiza-
tion in a way which enables it to meet stakeholder needs and expectations
efficiently and effectively, without compromising ethical values.”
Some of the organizations that have implemented the concept of TQM are Ford
motor company, SGL Carbon, Motorola, Philips Semiconductor, etc.
TQM activities must include:
• Commitment from the senior management and all the employees in the company.
• Satisfying the consumer’s requirements.
• Just in time manufacturing.
• Reduction of processing and service costs.
• Systems flexible to adapt the improvements incorporated.
• Employee empowerment.
• Challenging and meeting the targeted goals.
• Benchmarking.
• Incorporation of the strategic plans made by the top management.
9.15.1 Basic Principles of TQM
1. Focus on customer: The prime motive of TQM activity should be that the
consumer should be at the center of all the things we do. The needs or
requirements of the customer should be satisfied at any cost, if a company has
to thrive and attain its long-term goals.
2. Always do the things right the first time and every time: The focus of everyone
should be to do the things correct each and every time, this will help in reducing
the variability in the quality of the final product, thus gaining the customer’s faith
and trust.
3. Continuous improvement: The tools like PDCA cycle should be followed in each
process, which will help in finding the ways of improvement. Also tools like
Kaizen, Housekeeping, POKA YOKE should also be practiced.
4. Educate and communicate: All the persons should be educated about their
responsibilities that what they have to do and what is the right process to do it,
training should be given to improve their skills and improving their capability to
face any real-time problem or failure. The communication between the top
management and the employees should be done on regular basis, communication
gap makes the implementation of TQM difficult because of the message that what
the higher officials need will not be communicated to those working on the
manufacturing floor. Everyone should know what is going on.
9.15 Total Quality Management 255
5. Measure and record: While freezing and finalizing the goals, the quality
indicators should be finalized and the measures are taken to accomplish the
goals should be recorded, also the results or the progress should be documented.
Record keeping helps the company to make decisions based on the actual data.
6. Do it together: Teamwork makes everything possible and easy which a single
person cannot do, so everyone should be introduced in the TQM system. Form a
team that consists of TQM coordinators who will have a check on the ongoing
activities and will record the progress also.
7. Fighting the problems: The vision of all the members should be wide enough and
while solving the problems they should find ways (solutions to the problems)
keeping in view of its long-term significance.
The principles of TQM can be summarized as follows:
1. Management commitment
2. Employee empowerment
3. Fact-based decision-making
4. Continuous improvement
5. Customer focus
9.15.2 Elements of TQM
The three major elements of TQM are:
1. Total employee involvement.

2. Total waste elimination: The principles of segregation, arrangement, cleaning,
maintenance, i.e., housekeeping should be implemented. Follow just in time to
reduce the cost and the quantity of the inventory maintained.
3. Total quality control: Statistical tools like sampling plan, PDCA cycle, ISO 9000,
ISO 22000, ISO 14000, HACCP, control charts should be used.
9.15.3 Approaches to TQM
1. Analyze what the consumer wants: This can be done by organizing surveys of the
focused group. It involves both the internal (they include the company employees
who work in the actual work area and are the part of the process) and the external
consumers (the common people who are to use the product).
2. Design the product or service in such a way that all the traits can exceed or meet
the customer requirements/expectation.
3. Design a process that will do the job correctly for the first time and every time.
The process should be a foolproof to prevent or reduce waste.
4. Keep a note on all the activities which are going on and used them as a tool for
improvement.
5. Extend the same concept to the suppliers and the distributors because they also
form an integral part of the system and can have a direct or indirect impact on the
quality of the product or service.
9.15.4 The Concept of Continuous Improvement to Be Achieved

by TQM
TQM is related to the continuous improvement, which includes strategic planning,

decision-making at the top level to the complete execution of the plans and ideas in
the work area. Improvement can be done by understanding that the mistakes can be
avoided and the defects can be minimized or prevented. This will result in the
improvement of the process, technology, equipment performance, and overall
throughput. Continuous improvement is not only confined to get the best results
but it focuses on improving the process to produce and get better results in the future.
This can be achieved by finding the root cause of the mistakes and eliminating them.
The major ways to prevent the occurrence of mistakes are:
1. Poka-Yoke or mistake proofing (which means that the occurring of the mistakes
should be minimized): Poka-Yoke is a term which is originated in Japan and was
initially developed and implemented by Dr. Shigeo Shingo in Toyota Production
system. It is basically a tool which was originated in Japan and its main objective
is the reduction in waste, elimination of the wastage, increasing productivity, and
ultimately increasing the profit, which is the ultimate agenda in any
manufacturing system/production system. Poka stands for “errors” or we can
call it as a mistake, so to be more precise it is inadvertent errors and Yoke is
nothing but the proofing or as we can call it as the elimination. So, Poka-Yoke is
nothing but the mistake proofing, the elimination of the mistakes basically to
correct mistakes before they happen. Examples of Poka-Yoke can be found
everywhere in daily life. Poka-Yoke is a lean tool that can be applied to any
type of process. It is a mistake proofing, the process by changing the method or
equipment to ensure that a particular error cannot happen. The step that causes the
error is eliminated and replaced by a step that is error proof. Poka-Yoke involves
analyzing a process for all the ways wherein mistakes could potentially happen
and then designing a process to prevent those errors in analyzing processes for
improvement. Use data to help mistake proof. You can use a simple bar chart or
Pareto chart in help identifying the most common areas where errors can occur
and create systems to eliminate the likelihood of errors. Pareto charts are
extremely useful for analyzing what problems need attention because the taller
bars on the chart, which represent frequency clearly illustrate which variables
have the greatest effect on a given process. The Pareto chart provides a graphic
depiction of the Pareto principle, a theory maintaining that 80% of the output in a
given situation or system is produced by 20% of the input. Spellchecker, which is
used in our smart phones and computers, automatically corrects the spelling
mistake and prevents the mistakes from happening. So, it falls under the category
of prevention-based Poka-Yoke. Other examples of Poka-Yoke are the
9.16 Pillars of TQM 257
microwave oven does not works when the door is opened, a key can enter a
keyhole only in one way, needle cap that prevents needle pinpricks, the USB
cable or the pen drive can enter from only its one side, the magnets or metal
detector removes the metal pieces from the food commodity, before packaging,
gloves, masks, and other personal protective equipment are given to the workers
in a food industry to prevent the falling of hair and other foreign objects in
the food.
2. Areas where the mistakes cannot be completely prevented, they should be
detected and measures should be taken to prevent them so that they do not
carry forward to the next step or to the value-added chain.
3. Areas where mistakes occur regularly, the production should be shut down till the
process is not rectified.
9.16 Pillars of TQM
The three pillars of TQM is related to the change in the attitude. The following points
are important in achieving attitude change:
1. Continuous improvement: All the processes related to the manufacturing of the

products need to be improved as per the requirement, also the standards of the
finished product should be modified which meets the customer’s requirement.
2. Competitive benchmarking: The products manufactured by the company should
be compared with their competitors for the level of quality. Benchmarking gives
the ideas to improve the process so as to achieve the same order of quality as that
of the competitors.
3. Team work: Teams can have a synergistic effect on the accomplishment of work
or task, also the teamwork inculcates cooperation between the co-workers which
improves the working environment.
4. Knowledge of tools: The employees and the management should be well trained
about the use of the tools.
5. Suppliers quality management: The suppliers should also be included in the TQM
plan because the quality of the raw material supplied by them will have a direct
impact on the quality of the end product or the service. They should be made to
understand that the reputation of the company depends on the product quality.
The company should also make the suppliers feel that they are of importance to
them and should try to win their trust and faith.
6. TQM champion: Make a TQM champion or coordinator whose work is to carry
forward the TQM activities throughout the organization. A coordinator should
also keep a check on the ongoing processes, record maintenance, and convey the
same to the higher management.
7. Quality at the source: Every worker should be given the responsibility for his/her
work, as expectations from each employee will increase their zeal and commit-
ment to complete the task or duty assigned. This will also increase the faith of the
manager on his/her employees.
9.16.1 Obstacles Encountered During Implementation of TQM
1. Lack of company’s definition of quality: The lack of vision of quality in the

company can create fuss and miscoordination among the people. Also, using the
non-standardized procedures for production may lead to the production of sub-
standard products, which will affect the image of the company.
2. Lack of strategic plan: This reduces the chances for success as nobody is aware of
their responsibilities and the goals which they have to achieve. Lack of a strategic
plan will also lead to further complications during any failure, error, or break-
down, as no one will have a backup plan to remove or rectify the fault.
3. Lack of customer focus: This increases the risk of failure to meet the customer
requirements, as the needs of the consumer are not focused and no importance is
given to the quality of the product manufactured with respect to the consumer
demands.
4. Poor intraorganizational communication: This leads to frustration and confusion
as the complete message is not conveyed to the intended person. Thus, people
have no clear idea of the orders given and its purpose.
5. Quality being viewed as a quick fix: Quality is not a one-time process and it
cannot be achieved in a single day, rather it is a continuous process through which
the improvement in the product is achieved.
6. Focus on short-time financial returns: When the companies focus on short-term
results, they are not able to reach their tenth anniversary. A company should
emphasize on quality building first which will help in winning the customer faith
which will help the company to grow further.
7. Presence of internal politics and policies: This leads to interruption in the normal
working of the company, which causes delays, demotivation, and fall in the
performance of many motivated employees. Such politics also result in quitting
the job by employees.
8. Lack of leadership: This happens when the management shows no interest in
implementing the TQM policy in the organization.
9. Lack of motivation: This problem arises when all the members of the organization
do not have the same vision for the organization. They do not focus and bother
about the goals that are important to achieve a certain plan as decided.
So for the successful implementation of the TQM, the environment within the
organization should be supporting, the management should be committed toward
the goals, regular training to the employees should be given, the various quality
concepts like PDCA, Kaizen, 5S, HACCP, and ISO should be followed and most
important the resistance to change should be removed.
9.17 Hazard Analysis and Critical Control Point (HACCP)
Codex defines HACCP “as a system which identifies, evaluates and controls hazards
which are significant for food safety.” HACCP is a systematic system that uses
scientific evidence to assess the risk posed by hazards to human health. HACCP
9.17 Hazard Analysis and Critical Control Point (HACCP) 259
system identifies the specific hazards (biological, physical, chemical, and radiologi-
cal that are of significance to food safety), which are capable to affect human health,
the measures to be taken to control their occurrence in food. It relies on the concept
of preventing the occurrence of a hazard rather than focusing on end point product
testing. The process should be designed such that it is capable to accommodate
changes like change in equipment design, technological advancement, etc., which
are taken in HACCP. HACCP focuses on ensuring food safety from farm to fork,
from product to plate. It requires commitment and involvement of the entire man-
agement and workforce, a multidisciplinary approach that should be compatible with
the implementation of the food safety quality management systems. The HACCP
system can be applied at any stage of the food supply chain. Apart from applying to
the food industry, HACCP can be applied in cosmetics and pharmaceuticals.
9.17.1 History of HACCP
1951: In this year, NASA requested and collaborated with the Pillsbury to produce
food for the astronauts. The US Army laboratories also got involved in this
project. This was a preventive approach (end point testing) to make safe food
for space expeditions.
1971: HACCP was applied to the low-acid canned foods by the USDA and asked
Pillsbury to train the FDA inspectors for the examination of canned foods. This
step was taken by USDA because several cases due to botulism were reported
when processed low-acid canned foods were consumed.
1985: The National Academy of Sciences (NAS) recommended the implementation
of HACCP for controlling microbial hazards.
1992: The HACCP guidelines were framed by the Codex Committee for food
hygiene.
1995: Safe quality food (SQF) and quality code were released.
1997: The HACCP guidelines were once again reviewed and revised by Codex.
9.17.2 Prerequisites in a HACCP Plan
The prerequisites are the foundation of HACCP plan, they help in addressing the
food safety at every step in the food supply chain. The prerequisites that should be
followed while implementing the HACCP plan are transportation and storage,
equipment, training, sanitation, pest control, recall, allergen control, supplier quality
assurance, etc.
Equipment The surface of the equipment or storage units/tanks should be nontoxic,

made of inert material, corrosion resistant. Its surface should be smooth so that
cleaning can be done properly. It should be accessible to manual cleaning and should
be easy to dismantle.
Production and Process Control The raw material should be free from any of the
pests, antibiotic, or pesticide residue, its microbiological status should be of good
quality. The storage rooms, production space, and floor should be clean and spa-
cious. The circulation of air should be proper, undesirable opening should not be
present in the production hall, the humidity, temperature of the production space
should be maintained properly. Waste disposal should be done in a correct manner,
proper labeling should be done so as to quickly identify the things. The rejected or
defective product should be isolated and the decision for its reworking or disposal
should be taken immediately. Inventory management should be done using the
principles of 5S.
Transportation and Storage The storing facility for the raw material and the
finished product should be clean, the temperature and humidity conditions should
be maintained wherever necessary. The transportation of the finished products to the
customers should be done in good conditioned vehicles (refrigerated or insulated).
Training The workers should be trained and told about the aspects of personal
hygiene. They should be told about the importance of food safety and how it can be
achieved. Training to identify the hazards, role of CCPs in assuring food safety
should be imparted. The employees should be trained on how to carry out the tasks
assigned to them to ensure that the product manufactured is safe and does not pose
any food safety concerns. Training for record keeping and documentation should
also be given.
Sanitation and Pest Control The cleaning in place (CIP) of the product contact
surfaces like pipelines, machinery, and storage tank should be done. Any deposit of
the food material should be removed. Manual cleaning should be done wherever CIP
is not possible. The plate heat exchangers should be cleaned manually on a quarterly
basis. Air curtains or strip curtains should be installed on the entry door of each
section, also foot dip/wash containing sanitizer solution should be just before the
entry door of each section. All the area should be cleaned regularly, with proper
placement of labeled dustbins. The production area, product storage area should be
free from rodents, pests, flies, and mosquitoes. They can be prevented through pest
control system.
Allergen Control The allergen in a food product should be identified and men-
tioned on the package of the product, as a warning, to safeguard the health issues of
allergic persons.
Supplier Quality Assurance Suppliers are also an important part of the process as
we rely on them to supply us with safe ingredients and packaging material. A vendor
(or supplier) approval process should be made which makes sure that they supply the
products that should meet the buyer’s specifications. The incoming material should
be inspected to make sure that the material is of good quality, in good condition
(damaged or not), contains a lot or batch number, date of manufacture, best before or
date of expiry, etc., and decided upon whether it is to be accepted or rejected.
9.18 Principles of HACCP 261
9.18 Principles of HACCP
The Codex guidelines stated 12 steps and 7 principles of HACCP which are as
follows:
1. Assemble a HACCP team: The function of the HACCP team is to develop and
drive the company’s HACCP or food safety policy. It ensures that the project
should continue and move in a forward direction. The HACCP team collects the
data and reports the progress to the leader of the team. The HACCP team should
be a balanced mixture of technical and industrial experience along with an
expert who can advise when needed. The team members should have a detailed
knowledge of the raw material, ingredients, finished products, processing equip-
ment, process, procedure, prerequisite program, and the environmental
conditions in which the product is being manufactured. The specialist should
have the full knowledge of the type of all the possible hazards related to the
product, raw material, process, and the probability of their occurrence. He
should also possess the knowledge of the regulatory aspects of the food and
also should have the technical knowledge about HACCP.
2. Describe the product: The full description of the product should be drawn up
including the safety information like composition, physical, and chemical infor-
mation such as pH, water activity, etc. The method or the treatment given to the
food to preserve it like heat treatment, freezing, salting, smoking, irradiation,
chemical preservative, etc. should also be mentioned. The product description
should have the information like product name, inner and outer packaging,
storage condition, method of preservation, compositional characteristics,
shelflife, labeling, consumer preference, etc.
3. Identify the intended use of the product: The use of the product should be
identified like the target consumers who will consume the food with special
reference to the sensitive age groups like immune-compromised people, infants,
persons susceptible to allergy, pregnant ladies, sick, elder person, etc.
4. Construct a process flow diagram: The entire process flow diagram is the one
which contains all the details like type of processing given to food, storage
conditions, transportation facility, delays in transportation, inputs given during
manufacturing like raw materials, packaging material, additives, color, flavor,
water, etc., the output from the process like the finished product, waste
generated, rework, and rejected products.
5. Verify the process flow diagram: The HACCP team should verify the process
flow diagram properly by doing the onsite verification. It should also observe the
product flow, activities to be done during the production process, conduct
interviews and cover all the routine and nonroutine operations which are to be
done during the processing. All the members of the HACCP team should get
involved during the verification process, the leader of the team must sign and
date the process flow diagram after verifying it.
6. List all the potential hazards: A hazard is a physical, chemical, or biological
agent in a food, which has the potential to cause adverse health effect. All the
hazards like physical, biological, and chemical hazards should be identified and
listed. Hazard evaluation is done by conducting a hazard analysis and determin-
ing the significance of the identified hazard in relation to the food safety of the
product. The severity of the risk associated with prerequisite programs should
also be determined. Once the hazards and their severity is determined, the
control measures should be identified. Control measures are a set of actions or
activities that are taken to eliminate or control or reduce the hazard to an
acceptable level that it does not pose any food safety issue.
Control measures for biological hazards: Heat treatments like pasteurization,
sterilization, time–temperature combination of the heat treatment, fermentation,
acidification, pickling, drying or concentration or freezing (reduction in water
content), cooling, and preventing cross contamination are some of the measures
to control biological hazards.
Control measures for chemical hazards: Procurement of quality raw material,
which should meet the specifications of the buyer, certificate of analysis to be
supplied with the product, applying sanitation program, pest control, using food-
grade chemicals, pesticides, antibiotics and heavy metal residue checking, labels
on food material containing allergens, use of soft water for processing, and
following a correct CIP programme.
Control measures for physical hazards: Sieves (should not have metal wires),
magnets, filters, metal detectors, glass control policy, jewelry policy, use of
plastic pellets instead of wooden pellets, proper window panes etc.
7. Determine critical control points (CCPs): A CCP is a step in a process at which
the control can be applied and is essential to prevent, reduce or eliminate a food
safety hazard to an acceptable level. Control of CCP is a must in a HACCP
program.
Control point (CP): It is a step in a process at which if the control is lost it will
not lead to the expression of a significant food safety hazard or the hazard will
not reach the unacceptable level. For a single hazard identified during the hazard
analysis, there must be one or more CCP applied to control the hazard. As a CCP
can be used to control one or more hazards similarly more than one CCP can be
used to prevent or control a single hazard.
Critical quality point (CQP): A CQP is a step in the process where the
identified hazard should be controlled. The control can be applied to the opera-
tional, environmental, OHPS hazards, and is essential to prevent/eliminate or
reduce a quality hazard to acceptable level.
8. Establish critical limit: Critical limit is the maximum or a minimum value,
which is applied on a biological or physical or chemical parameter to control a
CCP for prevention, elimination or reduction of a food safety hazard to accept-
able level. Critical limits are the limits that are applied to separate the acceptable
product from the unacceptable one, safe from unsafe. These critical limits are the
tolerance limits for safety in terms of the product acceptance. Critical limit is
generally a demarcation line which helps in deciding whether a food is safe to
consume or not. The critical limits should be applied to all the CCPs and CQPs.
The critical limits can be applied to factors like temperature, pH, water activity,
9.18 Principles of HACCP 263
humidity, moisture level, salt concentration, and preservative level. The critical
limit in pasteurization of milk is maintaining a temperature of 72 C for 15 s to
destroy all the pathogenic microorganisms present in milk. The critical limits
should be validated and most important be a measurable quantity. For setting
critical limits the source of information should be published literature or data,
expert advice, experimental data, regulatory guidelines, mathematical, or statis-
tical modeling but the best way to set a critical limit is validation.
9. Establish a monitoring system: A monitoring system should be established for
every CCP/CQP. Monitoring may be defined as the act of conducting a planned
sequence of measurements to check whether the CCPs and CQPs are under
control and well below the specified limits. Monitoring can be done by
analyzing the recorded data obtained during the production process,
end-product testing, audits, etc.
10. Establish corrective action: A corrective action should be planned for each
CCP, CQP in a HACCP system in the case when the deviations occur. The
corrective actions must be planned in such a way that when they are applied, the
CCP and CQP are brought under control, also action should include the correct
disposal of the rejected product (the disposal procedure for the rejected products
should be well documented, signed, and verified by the HACCP team leader).
Corrective actions are taken when the control over the CCP or CQP is lost.
Corrective action is of two types, i.e., immediate or short term, which means the
adjustment of the process in order to regain the control and deal with the
suspected product, second type is preventive action which is a long-term control,
the root cause of the deviation is determined, someone is assigned the responsi-
bility to correct it and the details of the action is recorded.
11. Establish verification procedures: Verification is necessary to check whether the
HACCP system is working properly. The verification includes auditing,
analyzing the records, random sampling and analysis, reviewing the process,
parameters like temperature, and pressure recorded during the process. The
verification activities include like reviewing the HACCP system, its records,
reviewing the deviations, quantity of the product rejected and disposed, and
analyzing whether the CCPs and CQPs are under control.
12. Establish record keeping and documentation: Record keeping is essential for the
successful implementation of the HACCP system. All the operational
procedures, testing reports, product analysis reports, monitoring records, cor-
rective action records, establishment of critical limits, evidence of 12 codex
steps, HACCP plan, document related to the occurrence of any accident and its
cause should be documented.
The points from 1 to 6 form the first principle of HACCP plan while points 7, 8,
9, 10, 11, 12 form second, third, fourth, fifth, sixth, and seventh principle of HACCP
plan, respectively. Table 9.2 show the HACCP analysis applied to market milk.
264
Table 9.2 HACCP analysis for market milk

Processing Monitoring Monitoring
step Hazards Preventive measure Critical limits procedure frequency Corrective action
Raw milk Microbial, Maintain quality of raw Free from unacceptable Testing and applying Each lot Maintain cold chain,
(CCP 1) physical, or milk, proper cold chain material quality assurance educate the farmers
chemical to be maintained for clean milk
production
Pasteurization Survival of Check the time–temp. Set the temperature at Monitor the steam Routinely, Adjust the temp.,
(CCP 2) pathogenic combination 72 C for 15 s pressure, monitor the once in a maintain the steam
microbes temperature recorder shift pressure, cleaning of
in the instrument PHE
Packaging Microbial Maintain quality of Packaging material of Testing the Each lot of Change the supplier
(CCP 3) contamination packaging material, CIP good quality, proper packaging material, packaging if material not of
of packaging machine cleaning of pipelines to swab, or rinse test material good quality
and connecting lines prevent cross
contamination
Storage (CCP Microbial Maintain the cold store Temperature of the Checking the Hourly Increase the
4) growth, temp. below 3 C product not to be more temperature of the basis efficiency of
product than 5 C cold store, IBT tank, refrigeration unit,
deterioration and product install more chillers
in cold store
9 Quality Concepts
9.20 Meaning of 5S? 265
9.19 Housekeeping (5S)
The 5S’s are simple but effective methods to organise the workplace. It however,
goes beyond this simple concept, and is concerned with making orderly and
standardized operations the norm, rather than the exception. 5S or housekeeping is
a system that focuses on organizing spaces by putting everything in the right place to
which it belongs. This makes the workplace clean that enables the work to be
performed effectively, efficiently without wasting time and reducing the risk of
any injury. It inculcates the discipline in each individual so that a world-class
environment can be maintained at the workplace. 5S is one of the first fundamental
steps taken toward the implementation of the total quality management in an
organization. Henry Ford in 1972 started using this tool for the first time as
CANDO programme, i.e., Cleaning up, Arranging, Neatness, Discipline, and Ongo-
ing improvement. The 5S terminology was later popularized as a Japanese 5S
concept by Hiroyuki Hirano in 1980 and is generally called as workplace manage-
ment in Japan. Applying the 5S program in the system has benefitted many
organizations.
9.20 Meaning of 5S?
5S is the combination of five Japanese words: Seiri, Seiton, Seiso, Seiketsu, and
Shitsuke. These are the five steps, which should be followed while implementing the
housekeeping technique for organizing the workplace in a better way.
1. Seiri: Remove all of the clutter from the work place. This requires the classifying
of items into two categories, necessary and unnecessary, and storing or removing
the latter. Its meaning is to “sort.” By sorting the items that are of value or use and
eliminating or removing the unnecessary items we can save our time. While sort
asks the questions like what is the purpose of this item, who uses it, when it was
last used and how frequently, does it really belongs to this place? Questions like
these help to determine the value of an item as the workspace would contain a lot
of unnecessary items or which are not used frequently. One can also red tag an
item in which the person can fill the information of an item like location,
description to those items whose value is uncertain. If the red-tagged item is
not used for a long time then it can be removed.
2. Seiton: It means to “set in order.” Organize in an efficient and ergonomic manner.
Arrange items to minimise search time and effort. Each item should have a
designated place. A place for everything and everything in its place. The sorted
things that are of use should be allocated a place and that item should be placed in
the same place, so that it is easy to locate the item when to be used. Arrangement
of the items should be done in a logical manner like while arranging the items
sufficient space should be maintained between the shelves so that the people can
walk easily and also the type of arrangement in which the generation of waste is
minimum like waiting time, excess inventory, extra motion, etc.
3. Seiso: Seiso means “shine.” Clean up the entire area removing all dirt. It can help
in the spotting of potential problems as well as reducing the risk of fire/injury by
cleaning away the potential causes of accidents. The workplace should not look
messy, it should becleaned always. This means that the work area should be
cleaned, mopped, dusted, etc. along with the machinery and equipment.
4. Seiketsu: It means to “Standardize.” Ensure standard ways of working for the first
three stages. It can also be viewed as the continuation of the work carried out in
Seiri, Seiton, and Seison. The changes that have been made in the first three steps
should be monitored and maintained every time. Once the top 3S are accom-
plished, then after some time the things are taken back and the workplace
becomes again messy. In this step, regular tasks should be assigned, schedules
and instructions should be given regularly till these activities become a habit. A
standard operating procedure should be made so as to ensure that 5S does not get
wayward. A checklist can also be made which may be useful to monitor whether
5S is being followed or not.
5. Shitsuke: “Sustain,” the rule should be followed to keep the workplace 5S right all
thetime. Ensure that 5S principles are part of the culture. It means that the process
of following 5S must involve everyone in the organization. The managers should
also practice 5S along with the employees on the actual work area, i.e., the
manufacturing region. Sustain helps in converting 5S into a long-term process
and it should become the culture of the organization.The 5S’s may be viewed as a
philosophy, with employees following established and agreed upon rules at each
step. By the time they arrive at Shitsuke they will have developed the discipline to
follow the 5S’s in their daily work.
The 5S training should be imparted to everyone in the organization starting from

the top level to the bottom. The people take things seriously when they see their
superiors doing the same. The company should appoint a 5S coordinator who should
educate or guide other of its importance, keeping track of the 5S activities and
maintaining it. The training for 5S can be given by showing people visual aids
through DVD or other means, by doing hands-on-activities, a demonstration at the
workplace helps a lot. They should be told about why the company is starting 5S, its
importance and its benefits. The type of 5S activities carried out in one department of
the organization can be different from other like the 5S activity to be followed in the
production area will comprise of sorting, placing the things like packaging materials
according to its type say polyfilms are placed together, corrugated boxes, or cartons
are placed on one side, the cups are grouped and placed together while the same
activities carried in a laboratory will be different like arrangement of the chemicals in
alphabetical order, arranging like-like glasswares together, etc.
9.20.1 Kaizen
The word Kaizen is a Japanese term made from two words “Kai” means change and
“Zen” means good. So the term “Kaizen” means change for betterment, in Kaizen
the word improvement means, which can be one time or continuous, large or small.
The use of kaizen has helped a company named Toyota and now is followed by a
large number of companies throughout the world. It is a practice of making small
changes for building a more productive, safer and efficient workplace. All the
members in the organization from top to bottom should constantly find ways to
improve the working conditions, a process, which will have an impact on the quality.
Kaizen can be achieved by certain tools like PDCA cycle, Quality circles, and 5S.
Kaizen benefits an organization by making the processes effective and smoother,
lowering the costs, increasing the quality of the products or service, better customer
service, improvement in employee participation and boosting their morale (by giving
the best kaizen award every month), clean and safe workplaces. Kaizen has a dual
nature, it is partly an action plan and partly a philosophy. As an action plan, it is
about organizing events that focus on improving the specific areas in the company by
involving all the employees from all the levels with special emphasis on the
employees working in the actual work area. As a philosophy, it is about constructing
a culture in an organization that all the employees are involved actively in suggesting
the areas of improvement. Kaizen works best in a standardized process, as the areas
of improvement can be easily noticed.
Kaizen Events
A Kaizen event should be conducted in the following manner:
• Set the goals.

• Review the current situation and form a plan for improvement.
• Work to achieve improvements.
• Fix the problems that need improvement.
• Record and report the results.
The PDCA cycle plays an important role in implementing Kaizen wherever

required.
9.20.2 Hoshin Kanri
Hoshin Kanri is a Strategy Management method. Hoshin Kanri is a Japanese word.

The direct translation is “Policy Deployment.” Some people call it as “Compass
Management.” Hoshin Kanri as a method that was initiated by Toyota in the 1960s
and has spread worldwide. As per Toyota’s— “While clarifying as a ‘Company
Plan’ consisting of three parts: ‘Basic Plan’, ‘Long-term Plan’ and ‘Annual Plan’, we
established a system, ‘Hoshin Kanri’, to expand the Company Plan and follow up
with each department.” Toyota started using Hoshin Kanri company wide in 1963
and has continued using it.
The strategic objectives of the company are defined clearly by fixing the targets
for a period of 1 year or more. Once the goals are freezed the team focuses on the
objectives on a monthly basis by making plans on how to achieve them. The bigger
goals are divided into the smaller ones like on a weekly or monthly basis, as
discussed earlier. The progress toward achieving goals is generally reviewed on a
monthly basis and a complied data reviewed annually. The data of the current year
are compared with those of the previous year during the same time to access the
progress. Hoshin Kanri is an approach that involves persons from the top level to the
bottom level. The progress of all the activities needs to be communicated effectively
to the higher management and they tend to give suggestions to the employees and
their managers through interaction during review meetings. Hoshin Kanri also
involves the use of PDCA cycle because proper planning is required to set any
goal and as the goals are finalized, the team starts to act to reach them, which are then
checked by the higher authorities and then certain steps are taken in case of
underperformance. Also, Juran’s theory of quality control principles can also be
followed in Hoshin Kanri.
9.20.3 Six Sigma
It is a set of tools and techniques which aim at process improvement. Bill Smith, an
American engineer introduced this concept when working in Motorola in 1980. Six
Sigma is a process in which out of all the opportunities available to produce a
product or any part of it, 99.99966% times it is expected to be free from defects. The
most widely accepted definition of Six Sigma is a process that produces 3.4 defects
parts per million opportunities (DPMO). The strategy involved in Six Sigma process
aims to improve the quality of the process output by analyzing the process,
identifying the cause of the defect and removing it, thus minimizing the variability
in the quality of the product and manufacturing process. The six sigma concept
followed in an organization follows a set of defined steps to achieve certain defined
targets such as reduction of process run time, reduce the cost of production, increase
in the efficiency of machines, labor, utilities, etc., increase the loyalty to the
customer, revenue maximization, and increase in the profit earned. The purpose of
six sigma is to improve the performance of the organization. The limits for a six
sigma process depends on the organization that what is the maximum limit of the
defective parts per million opportunities. This value need not be 3.4 always, this
value can be determined by understanding the process followed, the type of the end
user or target customer, e.g., for a pharmaceutical company the six sigma limit can be
much less than 3.4 because the failure in the efficiency of the medicine can directly
affect the health status of the person, which may lead to some other health
complications. In simple words, the purpose of a Six Sigma concept is to evaluate
that whether a process is improving, stagnant, and deteriorating when compared with
the competitors. It helps to analyze that whether the business is heading at present
and how will it progress in the future. The International Organization for
Standardization has framed the standards “ISO 13053:2011” for a Six Sigma process
in 2011.
Six Sigma doctrine asserts

• Continuous improvement and application of efforts to achieve stable and
expected results (e.g., increase in efficiency of the machinery).
• The processes involved in manufacturing and business organizations have certain
characteristics that can be measured, defined, controlled, analyzed, and improved.
• To achieve a sustained level of improvement in the quality, the entire organization
should be involved and fully committed, especially the decision and policy
making top management.
How Six Sigma is different from other quality management tools?

• A clear focus on achieving the set targets and financial returns.
• Increased importance on strong and zealous administrative leadership and
support.
• Decision-making on the basis of the factual data and using statistical methods
instead of using assumptions and guesses.
Six Sigma follows two strategies, which is inspired by Deming’s PDCA cycle.
The two methodologies are DMAIC and DMADV.
Why is Six Sigma = 3.4 DPMO?

Historically processes were controlled in 3σ, and this was the basis of control
charts. When you have a process, which is centered around the mean, it will have
99.73% items within 3σ and will have 0.27% items outside the 3σ limits. Out of
this 0.27%, you will have half of the rejection (0.135%) on the lower end and another
half rejection on the upper end. Now instead of 3σ, you look at the Normal
Distribution curve with 6σ, you will see that the rejection area is 0.000000197%
(or 0.00197 DPMO) and not 3.4 DPMO. There is a 5σ shift allowed in the process
over a long time. A Six Sigma process is allowed to move 1.5σ on both sides from
the mean. If, for example, the process shifts 1.5σ to the right, then we will be left
with 4.5σ (6.0 1.5) as the acceptance area in the right and 7.5σ (6.0 1.5) on
the left. With this shift, the rejection rate will be 3.4 DPMO. Similarly taking the
other extreme case, where the process moves 1.5σ to the left then also the rejection
rate will be 3.4 DPMO. Both these cases are the worst case scenarios. Hence, Six
Sigma process has the maximum defects as 3.4 DPMO.
9.21 DMAIC
It is a five-step strategy in which D stands for “define the system,” M stands for
“Measure,” A stands for “Analyze,” I stands for “Improve,” and C stands for
“Control.”
9.22 DMADV or DFSS
D—Define, M—Measure, A—Analyze, D—Design, and V—Verify. The DMADV

methodology is called as DFSS (Design for Six Sigma).
Define the goals that should meet the customer’s needs and also should meet the
organizational strategy or work environment.
Measure and identify the critical characteristics or points which have a direct
impact on the quality. Also, the production process efficiency, labor capability,
extent of risks, etc. should be measured.
Analyze to develop and design the alternatives.
Design a much better alternative which can be integrated into the process in case
of any failure or when the number of failure increases.
Verify the actual design by taking trials and tests at pilot or at a smaller scale.
Once the whole process has been verified, the process should be implemented in a
real-time situation or conditions.
9.22.1 Key Roles in Six Sigma
Six Sigma is a combination of the production process and statistics. Earlier in other
quality management tools, both the production and the statistics were supervised
separately. In Six Sigma, the roles of all the persons involved are fixed and a certain
hierarchy is established. For the successful implementation of six sigma the roles to
be played are mentioned as under:
• Executive leadership: It includes the higher management that is involved in the

decision and policy making like the CEO and other top management. They set a
vision for the implementation of the six sigma program. They also ascertain a
certain degree of freedom to the other persons involved in the successful imple-
mentation of six sigma. They help the other members by providing resources to
explore new ideas so that continuous improvement is achieved and inculcate the
desire to change whenever required in the entire system.
• Champions: They take the responsibility for the implementation of Six Sigma in
integrating all the departments and the employees of the organization. The
leadership qualities in them are drawn from the upper management. Champions
also act as mentors for the black belts.
• Master black belts: They are selected and appointed by the champions and act as
the in-house coaches for Six Sigma. They devote all their time to Six Sigma and
assist the champions. They act as a bridge between the champions and the green
and black belts by guiding them in the accomplishment of their responsibilities
and the roles assigned to them. Along with the statistical tasks assigned to them,
they devote their time in ensuring the consistent application of Six Sigma across
various departments and in all the processes.
• Black Belts: They work under the guidance of master black belts to apply Six
Sigma in all the projects categorized under sig sigma. Like master black belts they
also devote all their time to six sigma. They focus on the Six Sigma execution and
are assigned special leadership for special tasks while the champions and master
black belts focus on the identification of the projects, process, or functions to be
involved in Six Sigma.
• Green belts: Apart from the assigned job responsibilities, they are also indulged in
the implementation of the six sigma under the guidance of black belts.
• Yellow belts: Yellow Belts participates on and supports the project teams, typi-
cally in the context of his or her existing responsibilities.
All the persons who are assigned these above-mentioned roles are given special
training so that they should follow the correct methodology involved in six sigma. In
general, the hierarchy of six sigma follows the order as top management, i.e.,
executive leadership, champions, master black belts, black belts, green belts, and
yellow belts. All these are inter-related to each other and assist in the successful
implementation of six sigma. Six Sigma is mostly applied in large organizations but
still the scope of Six Sigma varies depending on where it is implemented. It can be
implemented in manufacturing, construction, engineering, finance, supply chain,
food, and health care.
All these quality improvement concepts like quality control/assurance, HACCP,
TQM, six sigma, PDCA cycle, 5S, or the theories given by quality gurus like
Deming, Crosby, and Juran will help in modification of the production process as
per the customer needs or requirements. The successful implementation of these
concepts can work wonders towards improving the quality of the product and the
reputation of the company towards winning the faith of the consumer. These
concepts, when applied in combination, will have a better impact on the process,
product, and service.
Suggested Readings
Crosby PB (1996) Quality is still free: making quality certain in uncertain times, vol 111. McGraw-
Hill, New York
Gapp R, Fisher R, Kobayashi K (2008) Implementing 5S within a Japanese context: an integrated
management system. Manag Decis 46(4):565–579
Harry MJ, Mann PS, De Hodgins OC, Hulbert RL, Lacke CJ (2010) Practitioner’s guide to statistics
and lean six sigma for process improvements. Wiley, New York
Hendricks KB, Singhal VR (1996) Quality awards and the market value of the firm: an empirical
investigation. Manag Sci 42(3):415–436
Ishikawa K (1985) What is total quality control? The Japanese way. Englewood Cliffs, New Jersey,
Prentice-Hall
Jackson TL (2006) Hoshin Kanri for the lean enterprise: developing competitive capabilities and
managing profit. CRC Press, Florida
Juran JM (1995) A history of managing for quality: the evolution, trends, and future directions of
managing for quality. Asq Press, Milwaukee
Laraia AC, Moody PE, Hall RW (1999) The kaizen blitz: accelerating breakthroughs in productiv-
ity and performance. Wiley, New York
Martin L (1993) Total quality management in the public sector. Nat Prod Rev 10:195–213
Metri BA (2006) Total quality transportation through Deming’s 14 points. J Public Transport 9(4):3
Neave HR (1987) Deming’s 14 points for management: framework for success. J R Stat Soc Ser D
Stat 36(5):561–570
Orriss GD, Whitehead AJ (2000) Hazard analysis and critical control point (HACCP) as a part of an
overall quality assurance system in international food trade. Food Control 11(5):345–351
Tennant C, Roberts P (2001) Hoshin Kanri: a tool for strategic policy deployment. Knowl Process
Manag 8(4):262–269
Wallace C, Williams T (2001) Pre-requisites: a help or a hindrance to HACCP? Food Control 12
(4):235–240
Safety and Regulatory Aspects of Dairy
Industry 10
The perception of the consumer is changing significantly nowadays and has become
more oriented toward food safety and quality. The customers demand products that
are nutritious, healthy and cost effective, and processed under hygienic conditions.
The concept of food safety is now gaining importance and the demand of food safety
and quality assurance is increasing now. Trading of the dairy products in the
domestic market and across international borders requires food safety and quality.
Several government organizations/agencies work in ensuring the food quality assur-
ance and safety. Some of these organizations are World Health Organization, World
Trade Organization, Codex Alimentarius Commission, USFDA, FSSAI, and so on,
engaged in safeguarding the consumer’s interest and play an important role in
defining the standards for different food commodities. In this chapter, we will
discuss such regulatory bodies in detail.
10.1 WTO Agreements and SPS Measures
World Trade Organization commonly referred to as the WTO, is an international

agreement of member nations committed in principle to free multinational trade
through the reduction of trade barriers. As a multilateral organization, it covers
goods, services, and intellectual property rights established in the Uruguay round.
The organization replaced GATT or general agreement on tariffs and trade on
January 1st, 1995. The five main aims of the WTO is to increase the international
trade by lowering trade barriers, to be a forum at the international level for trade
negotiations, to handle the disputes among the member countries, to create economic
stability in the world through a multilateral system and lastly to cooperate with other
multinational corporations as for the structures and mechanisms of the organization.
Firstly, the WTO’s aim is to liberalize the international trade is considered success-
ful. Considering a hypothetical example two countries with comparative advantage,
uncertain commodities are able to benefit from improved trade conditions if they
were to specialize in products that yield a lower opportunity cost. Trade agreements

https://doi.org/10.1007/978-981-15-4167-4_10
274 10 Safety and Regulatory Aspects of Dairy Industry
can also reduce taxes on the exchange of goods and services that leads to freer access
to the foreign exchange market which in turn leads to economic growth however
critics claim that oftentimes less developed countries have little influence on
decision-making. Some countries, especially those in sub-Saharan Africa, have
faced lower real GDP per capita than they had 10 years ago. It is also argued that
WTO treaties are unfairly biased toward the interest of more developed countries and
multinational corporations. Lastly, critics have pointed out that free trade does not
necessarily make life better for the poor but has only resulted in widening the gap
between the rich and the poor. The WTO has encountered increased economic
instability mainly due to the current economic crisis that began with the collapse
of the mortgage market in the United States. It is currently involved in the Doha
Development round which commenced in November 2001 and continues till now. In
this trade negotiation round the WTO aimed to encourage global trade by reducing
protectionist measures. The major issues at hand include agriculture, industrial
tariffs, trade remedies, and nontariff barriers; however, the success of the Doha
round is debated because of two reasons, firstly the United States and the European
Union are asked to lower subsidies and agriculture commodities and secondly, the
newly industrialized countries such as India and Brazil are pushed by the most
developed countries to lower trade barriers or imported products as a final remark.
The World Trade Organization is considered very important, especially with the
globalization of this world because it encourages foreign direct investment that refers
to the investment by overseas firms in another country although disputes exist in, for
example, the Doha round negotiations.
The World Trade body was established on the first of January back in 1995 in
Geneva, Switzerland. Its creation marked the biggest reform in the international
trade since the end of the Second World War in less than 25 years of existence. The
WTO has produced close to 256 rulings addressing hosts of issues like taxes and
alcoholic beverages, subsidies given for civilian aircraft production, importation of
solar cells, antidumping duties on shrimps, packaging regulations for cigarettes,
regulation of gambling services, and measures affecting imports of beef on several
occasions. The WTO has passed certain judgments against powerful and developed
nations such as the United States both in terms of sheer volume of cases and the
wealth of jurisprudence that has been generated. The appellate body of the WTO has
worked better and outperformed most of the international courts and international
tribunals over the years. WTO is a main regulatory body which guides trade in
almost all the economies throughout the world. There are a lot of developments like
the advent of Free Trade Agreements (FTAs). Now, the economies are able to sign
FTAs and the multilateral agreements also and the only basis of the bilateral
agreements, the few economies are going fast though there are some issues in the
free trade agreements also. But overall the free trade agreements are very successful
for the growth of the economies and going forward with more and more
developments at the domestic level. Countries should also reform their domestic
trade facilitation by years and WTO is a good regulatory body to handle all these
issues however on its 25th anniversary, confidence in the global body has
plummeted to a historical low. The United States blocked the functioning of the
10.1 WTO Agreements and SPS Measures 275
appellate body which is the highest advocate adjudicating body for resolving global
trade disputes. Washington has repeatedly criticized the functioning of the
adjudicating body for allegedly straying away from the dispute settlement. WTO
is the only organization at the global level that deals or frames the rules of trade
between countries. The WTO agreements are negotiated and then signed by most of
the countries who are to get involved in trading across the international borders in
their parliament with a common goal, i.e., the trade between its 164 member
countries flows smoothly with minimum or no dispute. WTO is the successor to
the GATT, which was founded in 1948 with the objective to create rules for the
modern multilateral trading system. WTO works by looking at the trade agreements,
settlement of the trade disputes, acting as a platform for trade negotiations, reviewing
the national trade policies, building or increasing the trade capacity of the developing
economies, cooperation with other international organizations and so on. Till date
11 ministerial conferences had taken place since the foundation of WTO, the 12th
meeting is scheduled to take place in June 2020. These ministerial meetings or
conferences are conducted by the general council of the WTO. In Kazakhstan, after
the establishment of WTO, there is a lot of progress in the trade negotiations and the
graph for the trade has been toward a high, though there are slowdowns and
sometimes the headwinds because of the various developments in the global econ-
omy but overall WTO is a very successful body to settle the trade negotiations
between the economies. The WTO deals with international trade between nations.
So, if India is trading with the United States it has to then follow the disciplines of
WTO. It has to follow the commitments that it has made to the WTO in respect of all
other members of the WTO which is known as the Most Favored Nation. Similarly,
the United States will also have to abide by its commitments, disciplines on
subsidies, etc. The WTO is guiding principles and remained the pursuit of open
borders guarantee of the most favored nation, principled nondiscriminatory treat-
ment, and a commitment to transparency in the conduct of its activities. However,
critics argue that WTO owes an agenda that is driven by the business interests and
that its rules weaken the composure of its member states. Recently, due to the lack of
the talks between the countries, especially the developed countries have forced some
countries to look for trade agreements and relations among smaller groups.
The United States is also threatened to block its budget accusing it of
overstepping its money while China, Russia, EU, and several other members try to
cobble up efforts to save the global trade body for now. The international trade
disputes remain in a limb or stalemate between the United States and other members
is pushing the World Trade Organization to the verge of collapse. WTA’s appellate
body effectively stopped functioning as the United States blocked new appointments
to the panel accusing the court of serious overreach a minimum of three judges are
needed for the appellate body to fulfill its role as the arbiter of global trade disputes
with two judges completing their term—the powerful body became dysfunctional.
Robert as a way though the Director-General of the World Trade Organization said it
will take a few months to fix its main body for settling trade disputes. Settlement
mechanism is going to continue, members are going to continue to solve their
disputes through panels even the second stage so an appeal can happen. Their
members are going to continue to resolve their disputes, though arbitration is the
biggest challenge of the WTO. There are two very important challenges first
challenge is the WTO has three pillars on which it stands the first one is dispute
resolution among countries, the second one is negotiating for further market access
for the benefits, and the third one is the monitoring mechanism to see if countries are
abiding by their commitments. Now, as far as the first is concerned there is a
two-stage process like our High Court and Supreme Court. So, the first stage is the
panel process, the second stage is the appellate body till the appellate body disposes
of an appeal, the case is not finally disposed of. Now because of the United States
attitude in the past 2 years, they have not been allowing the nomination of fresh
members to the appellate body which has seven members and any three of which are
required to hear an appeal, so there is only one member. The appellate body has the
final say on global trade disputes that cover billions of dollars of international trade
and its decisions are considered to be binding. The current situation also signals the
potential demise of the 25-year-old WTO itself as settling disputes has been its most
important function. As a WTO dispute settlement body, the appellate body has seven
judges with staggered terms. Each judge has a 4-year term and can be reappointed for
an additional 4 years. Over the last couple of years, the membership of the appellate
body has dwindled to three from the required seven. The United States has been
blocking reappointment of the WTO’s Appeals judges since 2017, as a result, the
understaffed body has been unable to stick to its two to the three-month deadline for
appeals filed in the last few years due to lack of quorum, if any party in a dispute
appears. The dispute remains indefinitely unresolved, the first thing is that the
dispute settlement mechanism has taken a very big dent, so we will need to put it
back on the rails because without that no country would feel safe and secure,
especially smaller trading nations.
10.1.1 Most Favored Nations
WTO is a trade organization that functions under a set of rules, which regulate trade.
There are certain principles which in many of the situations are in-built in all
multilateral agreements. The most important principle is what is called as Most
Favored Nation. As per this principle, one member cannot discriminate against a
member of WTO when it deals with trade. If there are three countries say A, B, and C
and A is favoring B in trade and being C also the member of WTO must be also
favored. But there can be exceptions to this principle. The main exceptions are:
WTO itself has allowed that when a country goes for a preferential trade agreement,
then it can violate this rule. The preferential trade agreement can be between one
country that is bilateral preferential trade agreement or it can be within a group of
countries. It gives preference to a country or a group of countries. The logic of
preferential trade agreement is that the preferential group will be charging either zero
import duty or very less import duty so that the preferential country items can be
easily accessed by the preference country. Second exception is for least developed
countries where WTO has given the special provision of so-called Duty-free Quota
10.2 Codex Alimentarius Commission 277
free to them. Duty-free means no import duty and quota free that one cannot restrict
the import by setting a quota. It says that in a year, one can allow you to import only
this much. So, for developing countries or the least developing countries, the most
favored clause can be violated. The third exception is that in special circumstances, if
a country feels the imported items are unfairly traded items, then that country can
impose an extra import duty.
10.1.2 Sanitary and Phytosanitary Measures
The Sanitary and Phytosanitary Measures (SPS) agreement sets out the basic rules
for food safety, animal, and plant health. Its rules allow its members the right to take
the necessary sanitary and phytosanitary measures but also ensure that these rights
are not misused and do not cause unnecessary barriers in the international trade.
These measures can be applied at any part in the manufacturing stage like procure-
ment of the raw material or the product from a disease-free area, conducting safety
inspections or stating the maximum residual limits for contaminants like pesticides
and antibiotics. These rules are applicable not only to the imported goods but also on
the food products produced domestically or to local animal or plant disease. The SPS
committee in the WTO takes care of the SPS agreement between the trading
countries. The standards or regulations that are laid to ensure the food safety,
safeguarding the animal and plant health are based on the scientific data and by
assessing the extent of risk in real conditions but the member countries of the WTO
can apply the safety measures without scientific evidence if and only if these
evidences are not sufficient. Such applied measures are provisional and are subject
to various conditions which fall under the Article 5.7 of the SPS agreement. Another
condition is that the measures should not pose any unjustified discrimination
between the members and within their own territory where the conditions are similar
or identical. These measures should not pose a disguised restraint on global trade. It
is also true that the differences may arise due to the different existing conditions of
climate or existing pests or diseases or food safety conditions, so it is not always fair
to impose the same set of SPS measures between all the members. In addition, and to
conclude, the SPS agreement also includes other very important requirements
notably transparency obligations, these are related to the notification of SPS
measures, to the publication of these measures, to the setting up of an SPS notifica-
tion of authority and an SPS inquiry.
10.2 Codex Alimentarius Commission
Codex Alimentarius Commission (CAC) was established in 1962 jointly by the FAO
and WHO for the implementation of the joint FAO/WHO food standards. CAC aims
at safeguarding the health of the customers by ascertaining that fair practices are
being followed in the food trade. It acts as a bridge between the government and the
non-government organization indulged in the formulation of food standards. For the
implementation of the combined food standards of the FAO/WHO, CAC makes the
proposals that are consulted by the Directors-General of the Food and Agriculture
Organization (FAO) and the World Health Organization (WHO). Apart from the
food standards, CAC includes the general standards that are applicable in all the
foods and are not product specific. Such standards include food labeling, additives,
contaminants, sampling procedure, analysis, food hygiene code, guidelines for food
import, export, pesticide residues, and antibiotic residues.
Most of the standards take a number of years to develop. As the standards are
established and adopted by the commission, the standard is then added to the Codex
Alimentarius. The Codex Alimentarius now has such a well-established reputation
as an international reference that it has become customary for health authorities,
government food control officials, manufacturers, scientists, and consumer
advocates to ask first of all: What does the Codex Alimentarius has to say? The
HACCP (Hazard Analysis and Critical Control Point) guidelines have been
formulated by CAC under the sanitary and phytosanitary (SPC) measures.
HACCP has now become an important tool in ensuring food safety. It has become
mandatory for all the countries following the SPS measures to implement HACCP in
their food system.
10.3 International Organization for Standards (ISO)
International Organization for Standards (ISO) is an international standard-setting

body. The body has representatives from various national standards organizations.
ISO provides and promotes worldwide proprietary industrial and commercial
standards. It was founded on February 23, 1947, and has headquarters in Geneva,
Switzerland. The United Nations Economic and Social Council granted general
consultative status to the ISO, making it eligible for participation in the working of
the United Nations. English, French, and Russian are the three official languages of
the ISO. The organization’s name has different acronyms in different languages like
IOS in English and in French it adopted the short name ISO, derived from a Greek
word meaning “equal.” Both the name ISO and its logo are the registered trademarks
and their use is restricted. ISO began in 1926 as the International Federation of the
National standardizing associations. It was dissolved in 1942 during World War II in
October 1946. Thus, afterward International Organization for Standardization came
into existence and officially began its operations from February 1947. ISO is a
voluntary organization whose members are recognized authorities on standards.
There are 164 members in the ISO and each member represents a country, who
meets annually in the General Assembly to discuss the future strategies of ISO. The
central secretariat in Geneva coordinates and governs the organization. A Council
with a rotating membership of 20 member bodies provides guidance and governance
including setting the Central Secretariat’s annual budget. The ISO standards are
formulated by technical management. Apart from forming the international
standards, it also publishes technical reports, publicly available specifications,
10.3 International Organization for Standards (ISO) 279
technical specifications, technical core agenda, and guides sale of standards, which
are one of the funding sources for the organization.
ISO is a global network of the world’s leading standards publishing the interna-
tional standard. ISO does not match our name the International Organization for
Standardization. ISO is actually derived from the Greek word IOS meaning equal so
whatever the country, whatever the language, ISO is always equal. The motto is that
great things happen when the world agrees. Its core business is to bring around one
single table for international experts that tackle global challenges and find solutions
together through consensus. So, the consensus is an absolutely keyword because
essentially nothing moves forward at ISO without consensus and ISO being an
international standard body. International consensus is a big challenge but at the
same time when our technical committees and experts are able to achieve this
international consensus. It also means that the international standards that we publish
are globally relevant so when this core principle of consensus is achieved, great thing
happens such as safer toys for kids, worldwide banking cards working everywhere
when we travel, safer planes while traveling to our destinations so on and so forth.
There is a lot of examples out there of how standards are used everywhere so ISO
was founded just after the war when reconstruction and cooperation were obviously
most needed.
10.3.1 ISO Governance Structure
The General Assembly is the highest instance at ISO. It can compare it with the
shareholders of a company. It is composed of the principal officers of ISO and all the
country members. So, the ISO General Assembly meets once a year usually in
September and it meets to take key decisions, key high-level decisions such as
approving the annual report, the strategy and the finances. So, we talked about ISO
members being part of the GA. The ISO General Assembly could be compared to the
shareholders of a company. The ISO and council can somewhat be compared to the
board of directors of a company so it is composed of representatives from 20 ISO
members. Some are permanent and some are elected and rotated and as well as ISO
officers and chairs from our policy development committees. Their role is to appoint
the TMB, to appoint the secretary-general and to develop a proposal for the ISO
strategy, which is approved by the General Assembly. The president’s committee is
composed of the ISO principal officers. So we have the ISO president, the vice-
president policy the vice-president, technical management, the vice-president
finance, the ISO treasurer, and the ISO secretary-general. So, this group essentially
makes recommendations for the council’s and they also provide guidance to the
secretary-general of ISO. The ISO Council is also supported by several standing
committees and they are specialized, they are created and they are specialized in
specific domains like IT finance and so forth. There are also three policy develop-
ment committees as CASCO, DEVCO, and COPOLCO looks after conformity
assessment. The TMB stands for the technical management board. It is composed
of 15 members from ISO members and the TMB members are elected for 3-years
terms. So as the General Assembly as the highest instance at ISO, the technical
management board is the highest instance for the technical work at ISO and they
oversee the overall management of the technical work that is the work of the
technical committees. Some other tasks include (a) establishment and the dissolution
of technical committees (b) To approve the titles and scope of these technical
committee, (c) to appoint the Chairman of the technical committees, (d) to resolve
conflicts and appeals and (e) to review the ISO performance regarding standards
development. So, all eyes are on the technical committee’s report to the technical
management board. The technical committees are actually in charge of developing
standards. Last but not least we have the ISO central secretariat (CS) which coordi-
nate the day-to-day activities of the standard development process and also this is
where ultimately international standards get published. ISO CS is also there to
support the technical committees.
ISO 9000
This is the series based on the international quality management. It looks like a
system with minimum quality requirements. A company can organize and manage
its resources to achieve and improve quality economically by a quality system or
mechanism.
This series consists of:
• ISO 9000
• ISO 9001
• ISO 9002
• ISO 9003
• ISO 9004
These five standards are not specifically based on services or products. Out of
these, two are only guidelines.
STANDARDS
ISO 9001: This standard consists of 20 elements including development, design,
productions, and servicing.
ISO 9002: Consists of 18 elements including supplier capabilities in production
and installation. This is the same as 9001 without design service.
ISO 9003: Consists of only 12 elements, covering final inspection and testing for
lab, warehouses, etc.
GUIDELINES
ISO 9001: This is used for determining the three standards which apply.
ISO 9004: This provides assistance and guidelines for interpreting the standards
and contains suggestions that are not mandatory.
Importance of ISO 9000

This has become essential if you want to do business with the countries that make up
European companies (EC). To facilitate trade, the European companies are in
10.3 International Organization for Standards (ISO) 281
process of removing standard barriers. Since ISO 9000 is already recognized as an

international quality standard. It is expected that the organizations having ISO
registration are more preferred by a large number of companies.
Benefits of ISO 9000

• For identifying and planning tasks and their method of performance were enabled
by the user in this quality system.
• It helps in identification of the problems and measures to be taken to resolve them,
thus preventing their reoccurrence, thereby improving performance.
• Poor quality cost is also cut by this quality system.
• Training for all personnel involved which will upgrade or improve the quality and
performance of the product or service in the market.
• In different countries, ISO 9000 is a necessary prerequisite.
• It reduces the liability risks.
• Marketing advantages, publicity, and recognition.
• Betterment in job satisfaction and fire-fighting operations were reduced by this
standard.
Conclusion
It provides a mechanism for:
• Determining and fulfilling customer’s needs

• Preventing errors
• Correcting the errors
• Improving the process
• Consistency in quality of products and services
ISO 14000
This is a family of standards which is related to environmental management that exists
to help many organizations. This was evolved in early nineties with the main aim to
minimize the negative effects on the environment along with applicable laws,
regulations, and other environmentally oriented requirements. ISO 14000, is similar
to ISO 9000 quality management. The essentials of ISO are integral part of the
European Union’s Eco Management and Audit Scheme (EAMS). ISO 14000 is not a
performance standard; it is product and process oriented. This is the international
voluntary standards for providing common system for managing environmental issues.
ISO 14000:
• Reduces environmental liability
• Enhances public image and reputation
• Satisfies investor criteria
• Assures customers
• Improves government–industry relations
• Reduces your consumption of materials and energy
• Reduces the cost
• Facilitates and permits authorizations
Six Key Elements: ISO 14000

• Continual improvement
• Policy
• Management review
• Planning
• Checking and corrective action
• Implementation and operation
ISO 14001: Environmental management system specifications with guidance

for use.
ISO 14004: Guidelines on EMS principles, system, and supporting techniques.
ISO 14010: Environment auditing general principles.
ISO 14011: Audit procedures.
ISO 14012: Qualification criteria for environmental auditors.
EMS
An environmental management system (EMS) is that management structure that
allows an organization to check and control the environmental impacts of its
products, activities, or services. ISO 14000/EAMS/BS 7750:—all are standards of
implementation of EMS.
ISO 14001 Standard

ISO 14001 defines the criteria for an EMS. It does not states the requirements for
environmental performance but is a certain set of systems that a company or
organization should follow for effective implementation of EMS. The targets like
reduction in waste, cost reduction, improving resource and process efficiency can be
attained by this system. ISO 14001 is a generic management system standard, means
any organization can use these standards to improve and manage resources more
effectively.
ISO 22000:2005
It is a management system that aims at controlling the food safety hazards that may
occur in the entire food chain. ISO 22000 ensures that the food which is consumed is
safe for human consumption. The standard was developed by ISO/TC 34 and was
published on September 1, 2005. It is based on FSMS.
FSMS (Food Safety Management System)

This is required for intense farming and processing of food.
• Increase in ready-to-eat foods.

• Increased amount of exotic imported foods.
• More travelling across all over the world.
• Increase in number of susceptible people.
• Increased in meals consumed outside the home.
10.4 Hazard Analysis and Critical Control Point 283
ISO 22000:2005
• This is the first global food safety standard.
• A common understanding is enabled towhat a food safety management system is.
• Legal compliance checking is required.
• Integrates existing good practices.
• Harmonizes the voluntary international standard.
Benefits
• This is the family of standards.
• Integrated food chain approach.
• Continuous improvement.
• Act as a passport for exporting internationally.
• Enables a quantitative approach.
10.4 Hazard Analysis and Critical Control Point
India is an amazing country with a population close to 130 crores and we are the
biggest producer of agricultural raw material. Our ex-president Dr. APJ Abdul
Kalam has said that India will be the world power in Food and Agriculture by
2020 and that is going to be true because India has a diverse agroclimatic zones.
India is the only country where one can get around the year sunshine in one of the
corners. India is the only country where apricot to orange and pineapple to apple can
be produced. No other country in the world can produce such diverse agro fruits and
vegetables. We also have a vast pool of skilled manpower which is involved in
research and extension work. We are number one in milk production, pulses, and tea.
Every state has a lot of produce and they are producing in the agricultural community
but when it comes to the processing, we are very low. When it comes to the food
there are various sectors that include food, dairy, meat product, marine, agro-
processing, grain processing, oils, and beer and alcohol, consumer products like
biscuits, snacks, and health supplements. The total turnover of the food processing
industry per se is about 12 lakh crore. The problem in Indian agricultural processing
is that our processing numbers are far below like for the United States and Malaysia,
they are about 80 percent and we are merely below 5 percent so there is a huge
requirement and there is a huge scope for Indian food processing Industry. Food
safety management system is a continual activity, it is not a stationary event, it is a
continual journey. So, to comply with the basic food safety regulations, a license
holder has to comply with the basic regulation. It starts with the basic regulation,
then it goes into the GMP and GHP and Schedule 4, which are the prerequisite. Then
one can go for HACCP or ISO 22000 to ensure consumer safety.
Good hygienic practices abbreviated as a GHP have been derived by Codex
Alimentarius Commission. They had given a certain code of practices which the
industry should follow to ensure hygiene and is called as GHP. There are basic eight
requirements of GHP, viz. primary production, design and facility establishment,
control over all the existing operations, proper sanitation and maintenance, personal
hygiene, transportation, product description, and consumer awareness and training.
Every food business operator (FBO) needs to meet these GHP requirements. Four
key elements of FSMS on the certification site are HACCP, ISO 22000, and FSSC
22000 which includes prerequisites like GMP, GHP, quality management system
which is called QMS, structural requirement of the element like or act like FSSA and
communication of those. The FSS Act has defined a food safety management system
as the adaptation of good manufacturing practices, good hygienic practices, hazard
analysis critical control point and such other practices as may be specified by
regulation. So, these are the requirement of FSMS along with the Schedule 4, the
FSMS is a program based on the FSSA that includes Schedule 4 requirement and
FSMS plan which will give the critical control point. There is a checklist of
Scheduled 4 which is based on GMP and GHP and FSMS is a plan which includes
flowchart, hazard, and control points, critical limit for the monitoring corrective
actions and responsibilities. To summarize, FSMS plan includes Schedule 4 compli-
ance and FSMS plan you might be aware about digital flow requirements should info
requirements are based on GMP and GHP. There are 12 requirements which are
stated as:
1. Location and surrounding

2. Layout and design of food establishment premises
3. Equipment
4. Facilities
5. Food operations and control
6. Management and supervision
7. Food testing facilities
8. Audit, documentation, and records
9. Sanitation and maintenance of the establishment premises
10. Personal hygiene
11. Product information and consumer awareness
12. Training
Basically, these requirements of the Schedule 4 are taken as it is from GMP and
GHP. Indian food industries are divided on two basis, one is manufacturing and
other is the service industry. FSMS is applicable to both types of industries.
Manufacturing means the production units, it could be a petty manufacturer, small
manufacturer, and big manufacturer. In the service industry, it could be catering
industry, hotel industry, and transportation agency. The principles of the FSMS will
be the same but the requirements will be different for both the categories. The
prerequisite of primary production is that what is the condition of the environment,
location, or surroundings of the area where the food unit is placed. Then the design
of the unit has to be in accordance with the GMP and GHP. Like for a dairy industry,
all the surfaces that comes in contact with the milk should be nonabsorbing,
noncorrosive, free from crevices, etc., the flooring should be properly sloped to
prevent waterlogging. So, these requirements are called as design and facilities. The
facilities like air, water, steam, and lighting should be adequately available. People
should wear the head glows, apron, and there must a pesto-flash (which kills flies).
10.5 Regulatory Institutions of India 285
Control of operation is applicable to the raw material and the finished product. The
movement of the raw material and the finished product should be separate because
there should not be any cross contamination. The equipment should be maintained in
a clean condition. There must be a provision of pest/rodent control. Sanitation is the
most important activity in the food system. The workers in direct contact with the
food product should always sanitize their hands with 70% isopropyl alcohol. Estab-
lishment of personal hygiene is very important for all the people working in a
manufacturing unit. Like in the catering or dairy industry, it is an important criterion
to get medical checkup for each person. The medical checks include regular health
tests, examining for any type of communicable disease, etc. Apart from maintaining
the health records of the employees, the same should be maintained for the outsiders
or visitors entering the production unit. Transportation of the raw materials,
additives, food product is very important, as these things are very susceptible to
temperature change and can deteriorate at a faster rate. Like for transportation of ice
cream, the temperature of the vehicle should be to be maintained below 10 C to
prevent the melting of ice cream. While transporting onions they cannot be clubbed
with apples, so the containers should be loaded properly. Gap should be maintained
between the crates to ensure proper air circulation for maintaining the product
temperature. Information and consumer awareness is the most important part
because the customer should be aware of whatever the manufacturers say. There
should be a label and should be duly filled in all respects like customer care number,
manufacturing date, batch code, and address. Training has to be conducted for all the
employees engaged in food business operation and it should effective. These are the
detailed GHP requirements.
There are six requirements for GMP which are in line with the Schedule 4 of
FSMS which includes personal hygiene, plant and ground sanitary operation, equip-
ment and utensils process controls, warehousing, and distribution. These
requirements are the same as that of GHP which has been discussed above.
10.5 Regulatory Institutions of India
10.5.1 Legal and Quality Standards
Every country has its own set of legal and quality standards. At the international
level, we have Codex standards given by Codex Alimentarius Commission.
Harmonizing the national standards with Codex standards is helpful in the interna-
tional trade of foods. In India, there are two types of standards that are to be followed
for the marketing of food products including milk and milk products.
These are:
1. Legal standards
2. Quality standards
Legal standards are the standards that pertain to the law and are made by the
government to ensure that the finished food products should meet certain minimum
requirements in terms of chemical quality (i.e., composition), microbiological qual-

ity, labeling, and packaging requirements. In India, the legal standards are given
under Food Safety and Standards Act, 2006 (FSS Act, 2006) and Food Safety and
Standards Rules, 2011 (FSS Rules, 2011) under the authority known as Food Safety
and Standards Authority of India (FSSAI), whose headquarter is located at New
Delhi. It was previously known as the Prevention of Food Adulteration Act and
Rules (PFA) (PFA Act, 1954 and PFA Rules, 1955).
Legal standards or FSSAI standards recommend that all the food commodities
produced, processed or marketed under the Indian conditions should meet the
minimum requirements laid in the standard. The food which does not satisfy or
confirms these requirements is deemed to be called adulterated, irrespective of
whether anything has been added to or removed from the original food.
There are six major parameters that has been taken into consideration while fixing
the legal standards are as follows:
• Purity
• Composition
• Additives
• Efficiency of processing
• Bacteriological quality (hygienic quality)
• Packaging and labeling requirements
1. Purity
It should be clearly mentioned on the food product that whether anything is
removed or added during the processing of the product.
2. Composition
The composition and compositional standards generally vary from product to
product. By compositional standards, we mean that what is the minimum amount
of the components present in the food. For example, in case of milk products like
SMP, WMP, market milk, cream, cheese, etc., the minimum fat and solids-not-fat
content are specified in the standards and should be mentioned on the product.
3. Additives
The additives are added to a food product to enhance and improve its quality,
stability, flavor, shelf life, etc. If the additives have been added in the product,
they should be clearly mentioned as specified in the rules along with their
added levels. For example, additives like sodium citrate, nisin, and BHA are
permitted to be used in products like cheese, ghee, butter, and whole milk
powder.
4. Efficiency of processing
The type of processing treatments to which the food is subjected should be
mentioned. The tests to be performed to check the efficiency of such treatments as
specified in the rules should be specified. For instance, phosphatase test should be
negative for pasteurized milk while turbidity test should be negative for
sterilized milk.
10.5 Regulatory Institutions of India 287
5. Microbiological quality (Hygienic quality)

The microbial count like coliform count, total bacterial count, yeast, and mould
count should within the permissible limits as mentioned in the standards. The
microbiological standards also vary from product to product.
6. Packaging and labeling requirements
It is the package that first comes in contact with the consumer. Information like
composition, batch number, additives added if any, manufacturing date, best
before or use by date, net weight, and storage temperature should be mentioned
clearly and must in a readable form. Nowadays, nutritional facts are to be given
on the labels.
10.5.2 Quality Standards
Quality standards means those specifications which are laid down by the government
or some expert body constituted by the government for the purpose of producing
high-quality products. Unlike the legal standards which are compulsory to meet for a
food product, the quality standards are not compulsory. These standards are also
called as voluntary standards.
There are two types of quality standards in India:
• BIS standards
• Agmark standards
These standards are over and above the FSSAI standards, as these standards do
not aim at meeting the minimum quality standards. These standards are useful for
producing products of export quality. The Bureau of Indian Standards (BIS) are laid
for both the processed food products and nonfood products while the Agmark
standards are applicable to the raw agricultural produce like oil seeds, cereals,
eggs, pulses, butter, ghee, and spices. Considering the dairy products all the products
are covered under BIS except ghee and table butter which fall under Agmark. Before
1987, BIS was a voluntary standard but since 1987 these standards have been made
mandatory for some products which contain the ISI mark. For example, products like
milk powder, processed cheese, condensed milk, vanaspati, food colors, additives,
vanaspati, and containers for packing.
10.5.3 Requirement of Legal and Quality Standards
These standards aim at protecting the interest of the consumer, although the interest
of the manufacturer also gets protected, as a certified product containing some
certification marks like ISI, Agmark is expected to be consumed more than the
uncertified product, as the customer is assured about the quality of certified goods or
products. The customer does not mind to pay a somewhat higher amount in case of
certified products as a customer demands quality product. These standards safeguard
the consumer rights by not leaving them at the hands of the producers, who are
ignorant about the quality of the finished product. The consumer has every right to
consume a pure, safe product free from harmful microorganisms, contaminants like
pesticides, veterinary drugs, microbial toxins, and heavy metals. So, the food
authorities have framed the standards above the minimum standards in order to
improve the quality of the product to a higher degree.
Pre-2006, there were several acts and orders in force such as PFA, 1950, milk and
milk products order, 1992. In order to bring clarity in food legislation, the authorities
framed a single act that is Food Safety and Standards Act, 2006 that was enacted on
August 23, 2006. Accordingly, FSSAI, is a statutory body, established under the
Ministry of Health and Family Welfare in September 2008. The rules came in
existence with effect from August 05, 2011.
10.6 Roles and Functions of FSSAI
It is a central legislation body to lay down food standards based on science.

Transparency and consultation framing of regulations to regulate food businesses
in India. At present, there are 18 regulations such as licensing and registration, food
products standards, food additive, packaging and labeling, laboratory and sampling
analysis, import, alcoholic beverages, fortification of food, advertisement, and
claims. Laying down guidelines for accreditation of laboratories for food testing,
harmonizing the standards as per global norms and contributing to the development
of international and technical standard of foods. FSSAI coordinates with state food
safety authorities for enactment of the food safety and standard act in their area of
jurisdiction. FSSAI creates awareness about safe food, proper nutrition, and also
disseminates important information amongst the consumers. In India, the structure of
FSSAI is headed by a chairperson and administered by the Chief Executive Officer
who looks after day-to-day administration of the authority. Headquarters of FSSAI is
located in New Delhi. Its regional offices are located in Delhi, Mumbai, Kolkata,
Chennai, and Guwahati. It has two import offices situated at Cochin and Tuticorin.
Further, it has two labs, that is, National Food Laboratory, Ghaziabad (NFL) and
Central Food Laboratory, Kolkata (CFLK). In addition to this, FSSAI has
recognized NABL accredited labs for food testing.
10.6.1 FSSAI Registration and Important Terminology
Food: Food means any substance whether processed or partially processed or

unprocessed, which is intended for human consumption.
Food business operator: any person or entity carrying out food activities such as
manufacturing, production, storage, distribution, sales and imports, and transporta-
tion of food.
Petty food business operators: These are petty retailers, hawkers, vendors, and
temporary stall holders whose food business turnover is less than Rs. 12 lakh per
10.6 Roles and Functions of FSSAI 289
annum or having production capacity less than 100 kg per day or less than 500 liters
per day in case of milk.
Need for license/registration: FSSAI license/ registration is mandatory for
starting any food business in India. As per Section 31 of the FSS act (2006), no
person shall commence or carry out any food business except under a license/
registration.
10.6.2 Types of Licenses/Registration
For petty FBOs: With turnover less than Rs. 12 lakhs per annum.
State licenses: FBOs operating in one state and having a turnover less than
Rs. 20 crores per annum.
Central licenses: FBOs operating in more than one state or having turnover more
than Rs. 20 crores per annum or involved in import–export or having premises
located at central government establishments such as railways, airport, seaports, and
defense.
Responsibilities of FBOs: Every FBO shall ensure that any article of food should
comply with the rules and regulations of the FSS Act, 2006. Every FBO shall
comply with conditions of the license. Every FBO shall follow hygienic and sanitary
practices as laid down under schedule 4 of FSS (Licensing and registration of food
businesses) Regulations, 2011. No FBO shall himself or by any person on his behalf
manufacture, store, distribute, unsafe or misbranded, or substandard articles of food.
No FBO shall employ any person who is suffering from infectious contagious or
loathsome disease. No FBO shall sell or offer for sale any article of food without any
guarantee in writing as specified by regulations.
10.6.3 Penalty and Punishments
FBO can be penalized, when the food product that does not meets the minimum
requirements as per FSSAI standards is marketed. Such food can be substandard, not
as per the required quality, misbranded or mislabeled, unhygienic, adulterated, for
contraventions for which no specific penalty is provided. The penalty ranges from
Rs. 25,000 to 10 lakh or imprisonment up to term of life, depending upon the gravity
of the injury.
10.6.4 Initiatives
FSSAI is targeting both FBOs and consumers for creating awareness toward food
safety and standards. For FBO to become self-compliant, FSSAI has launched a
training program namely Food Safety Training and Certification (FoSTaC). For
consumers, various initiatives like Eat right India campaign, Aaj se Thoda Kam,
BHOG, and Clean Street Food Hub have been initiated.
FSSAI has recently released the full report of National Milk Safety and called it as
the survey 2019. The survey results have demolished the unfounded perception of
large-scale milk adulteration in the country. The survey has shown that over 93% of
the samples are absolutely safe for human consumption. Just 12 out of 6432 samples
were found adulterated rendering such milk unsafe for human consumption. A major
finding in the survey was presence of aflatoxin M1 residues beyond permissible
limits in 5.7% of the samples, 1.2% of the samples were found to have antibiotic
residues above permissible limits, only 1 raw milk sample contained pesticide
residue above the permissible level. No samples failed for other parameters namely
cellulose, glucose, starch, and vegetable oils. FSSAI is committed to zero tolerance
on adulteration and contamination in milk. This survey has helped in identifying the
areas which require more intensified efforts for surveillance and enforcement.
Quality issues in milk, however, persist for about 41% samples, though safe, they
fell short of fat or SNF or both. This included both raw and processed milk samples.
Worryingly presence of maltodextrin was found in 156 samples and sugar in
78 samples which were mostly in the processed milk. Maltodextrin and sugar are
added to milk to raise its SNF content. Even though safe, it is an undesired practice
that needs to be curbed. The survey has shown that contamination due to aflatoxin
M1 and antibiotic residues is a more serious problem than milk adulteration. Thus,
the outcome of the survey is a myth buster, it clearly indicates that the milk in India is
largely safe for consumption. This is contrary to the widespread perception of large-
scale milk adulteration in the country. Combating adulteration requires more vigilant
consumers, enforcement machinery, addressing the problem of contamination
requires systematic efforts in Animal Husbandry practices.
Safe and nutritious food is extremely important for a healthy being and that is
why FSSAI has taken this initiative to have a 360-degree approach and have a
nationwide campaign to help people eat safe, healthy and also very importantly what
should and how much you should eat. Eat right at all places including home, school,
and workplaces. This is because we are facing a huge burden of rising diseases with
the rising incidence of all these diet-related disorders like lifestyle disorders diabetes
mellitus and CVD’s. There is a dire need to start eating right and eat healthy. In order
to promote these particular initiatives, the FSSAI team has come up with a number of
ways and also resources. The purpose is to actually bring about a social and
behavioral change amongst the citizens and bring in the needs of food safety,
hygiene, and consumption of healthy diets. This should be done not just by sitting
at home and eating home-cooked food but also when you are going out to work. We
mindlessly eat whatever comes on our plate. It is absolutely important to also instill
these right practices very early in our lives and that is why initiative targeting for
school kids as well. The philosophy is to create informative content along with
simple messages to all the citizens so that they become aware of what are the right
healthy practices they should adopt voluntarily. The SNF resources are extremely
interesting and cover a wide range. The good hygiene practices have to be initiated
early in life and after that we practice everywhere at all times, be it at the time of
selecting the food processing, the food storing or even serving the food. The primary
objective over here is to inculcate safe eating practices and select the right kind of
food. Now for this, a three-pronged approach has been put forward in which you
have the first one which talks about the pink book which is basically a resource for all
the safe consuming, buying, selecting, eating, processing, and serving practices at
home. The second approach to disseminate knowledge of safe and nutritious food
through the community outreach that can be through the NGOs, the nutritional
groups and also through the art of awareness in various societies. It also provides
training and capacity building and that is done through FOSTOC. We need to be
aware of the foods, which are commonly adulterated. Detect adulteration with rapid
test (DART) book includes very easy handy methods of detecting adulteration with
qualitative or rapid tests, which can be very easily performed at home. For instance,
if you want to check for the presence of water in milk, a very simple drop test can be
performed at home. One can actually trickle a spoon of milk over a platter or over a
saucer and could find that the speed with which the milk flows will indicate
indirectly the quality of the milk. Supposing there are some additives such as starch
or formaldehyde that is added to the milk. In that case also the viscosity of milk
changes. There is another problem of today is the massive cause of obesity and
overweight amongst children. According to statistics, about 40% school going
students in this country are suffering from obesity and that is because of simply
wrong selection and consumption of foods for which there is a huge concern and that
is why we need to understand what best can be done.
So, SNF (Safe and nutritious food) at school is another very popular initiative
which propagates the need and consumption of safe and nutritious food at school.
Children are very powerful change agents and all habits die hard. So, inculcating
good and right eating practices early in life would have better-lasting impressions on
their brains. This project works by creating health and wellness coordinators in every
school. Now, these health and wellness coordinators build a team consisting of
students, teachers, and caregivers, probably caterers of people, or serving food in the
schools. They deliver strong messages around safe and nutritious food consumption
as well as delivery. The teachers are requested and they are also building on
curricular and cocurricular activities around that particular concept and there are
curricular changes that are also being worked on. Finally, it also ensures that you
have a regulatory framework that promotes the sale and delivery of only nutritious
and safe routes in the school, canteens, and the cafeteria. Use of mascots like “Miss
Sehat” and “Master Sehat” has been very instrumental in having a lasting impression
on students and the books have been created at two levels: level one and level two
that are actually age appropriate. The target of school students who are inducted in
this particular program are from ages six to fourteen and we find that curricular and
cocurricular engagement had a very lasting and engaging impression on the minds of
the students. The third resource is the orange book and this is the guide to eating
nutritious and safe food at the workplace. The employees are spending a lot of time
at their workplaces and they generally consume at least one or sometimes even more
than one meal during the working hours. Therefore, it is realized that it should be a
shared responsibility of not just the individual but also the employer, employee,
caterer, and also the food handler to serve and to provide the right ecosystem to
ensure that this food which is served, enjoyed, and eaten among the colleagues is not
only nutritious but also safe for their consumption. This particular significance of
food safety of the corporate cafeterias cannot be underestimated because this is
related to the economic development not just of the country but also the places of
work. It is very important to have a nutritious and safe food concept because it helps
in improving the health, well-being and eventually the productivity of the employee.
It is very important to invest in the health of the employees because this significantly
reduces the financial burden of the workplace and that is how the imperative cost on
the healthcare also comes down with absenteeism and low productivity also being
looked after. This is a very sustainable solution because ultimately human resources
are what they make the company what it is. So that is why it helps in improving the
profile and prestige of the company and the organization and certainly in supplies
trust in the long term.
All Indians love street food and in fact we have a heart associated wherever there
is tasty, palatable, enticing food served but the challenge is that the food has to be
absolutely clean, hygienic, and should not lead to what called as food-borne
illnesses. India has a rich tradition of street food and that is what is reflected in our
local culture. They are very popular and will remain popular because they are easily
available, have a wide variety and are freshly prepared in front of the customer and
comparatively low prices but often as we know that it is not served at the best of the
conditions. So, the other project which is called as Project clean Street food is one of
the initiatives of the FSSAI which has a 360-degree approach to food safety and
healthy nutrition. It also provides training and capacity building of the street food
vendors and ensures proper regulatory oversight over them under the FSSAI Act,
2006. Project clean street food not only ensures health, hygiene, and safety standard
of street food vendors but also the consumers. It is ensuring social and economic
upliftment of the street vendor because it helps them to improve their niche as the
product which they are selling would be better off and that is why it will attract more
consumers and naturally add to their economic upliftment. It also enhances the
popularity of street food by transforming it into a global brand by itself. So, this
has a four-pronged approach where the infrastructure is being looked into the
upgradation of the infrastructure of the food streets is also facilitated through certain
incentives and this recognition also given to those particular vendors who are
abiding by these particular principles. It is very important to focus on safety and to
develop safe eat out places which eventually could be also benchmarked for hygiene
and safety practices and this has to undergo through certain rigorous
implementations. It also encourages the consumer and the locals to perhaps propa-
gate the regional cuisines and to strategically get them and professionally manage
them so that this can also generate a lot of local revenue as well as trade and also
tourism. This will eventually enhance the popularity of the food streets by
transforming them into the global branch by itself and getting in the tourism.
Another very popular initiative and also the focus of the strategy is to manage
micronutrient deficiencies, which is one growing challenge in our country. In this
regard, a food fortification Research Center which is called as FFRC has been
launched in FSSAI and is designated as a resource hub to promote fortified foods
specifically for all these staples and thus ensure safe and wholesome food for all. The
function of the FFRC is also to provide strategic information, technical assistance on

standards, food safety and technology processes, premix, and equipment procure-
ment as well as quality assurance, and control through production of fortified foods.
This particular initiative comes with the “+f” logo that needs to be put on every
commodity which is qualified. So, the target is with cereals which are wheat, rice,
salt is now double fortified with iron as well as iodine, fats and oils, milk, etc. with
the important micronutrients.
Indian Food Sharing Alliance This is a social initiative of the Food Safety
Authority of India and it helps to solve India’s problem of food wastage and hunger.
This helps by integrating with various NGOs, organizations and recovery agencies
because we produce enough food to feed all but because of the postharvest losses
and wastages we find that a lot of people go to bed without having a square meal
also. One report reflects that about one-third of the world’s food is wasted each year
and which equals to about 1.3 billion tonnes. Now, this is sufficient to feed about
700 million people who are hungry. We find that most of the vegetables are lost
before they even hit the streets or are available to the consumers. So, if alone we can
curtail the food wastages and the losses, we will be able to help a lot of people who
do not get enough food to eat. The objectives of IFSA are very simple:
• It believes in caring by minimizing food wastage across the supply chain and by
redistributing the food which is left over to the poor and the needy. That is how it
aligns FSSAI with the NGOs who take this leftover food and feed those people
who have gone to bed without a meal.
• It is an awareness generation exercise where mobilization of people or generating
awareness is done amongst the consumers to minimize the food wastage on their
plate.
• It helps us to share and distribute surplus foods by connecting to trained food
recovery agencies with food chains. A lot of food which is produced is some time
wasted because it is nearing the deadline for the best before date and just before
that this can be handed over through these particular agencies. It can certainly be
distributed to a lot of people who deserve to have that particular meal.
• It helps in preparation that means it educates the food businesses to adopt best
practices and encourage them to adopt the same in order to prevent food losses
along the supply chain.
• Declaration is important because it provides strategic policy, regulatory frame-
work, and program support for food loss and waste reduction initiatives.
The Eat right movement is the umbrella for all the initiatives in the country which
are launched by the FSSAI. It is built on two broad pillars, which are eat healthy and
eat safe. It aims at engaging with the consumers and the citizens and exciting them to
become responsible citizens and adopt right eating practices which are not only
nutritious but also immensely important to improve their health and well-being. This
is no doubt a collective effort to make both the demand and supply interventions
through the engagement of key stakeholders.
Swast Bharat Yatra was a pan India cyclothon under the eat right movement
where the primary aim was basically to message the entire country to eat right and be
healthy and look after the health because that is the best wealth which one can equate
itself or we can gather to lead a productive life. India being the young nation in the
world, we have a huge responsibility to make sure that all those people under the age
of 35 years and even above lead a healthy and productive life. All the FSSAI
initiatives converge with different stakeholders. So, it is converging with some of
the very important key government programs like Ayushman Bharat, Poshan
Abhiyan-Jan Andolan under which you have all the safe and nutritious food be it
home, school, hospital, eating out, or at workplace. The other partners are in the civil
society organizations, NGOs, and cooperatives and in this case we have integrated it
with existing marketing channels and there are specific implementation projects as
well, which are looking at eat right movement and all the SNF initiatives of nutrition
courses. So safe and nutritious food at home, work, at school is the basis of the eat
right movement. The eat right tool kit has some key messages, it again reiterates the
fact that we must eat right and for that selecting healthy nutritious food which is also
hygienically prepared, packaged and served is extremely important. So, it must
encourage people to select the right foods, for instance, a balanced diet, avoid
foods which happen to be rich in fat, sugar, and salt as they considered unhealthy
and they are the leading causes of noncommunicable diseases like diabetes mellitus,
overweight, obesity, and cardiovascular disorders. Eating safe would include good
hygiene practices, good sanitation practices, safe food practices, and also to eat
healthy food, which is nonadulterated and absolutely safe for human consumption.
The objectives of the eat right toolkit include having a health and wellness coordi-
nator center under the Ayushman Bharat platform which is under the Ministry of
Health and Family Welfare. It serves a supplementary engagement resource that can
be mainstreamed into national nutrition and public health programs. The eat right
tool kit which is basically on two pillars of eat healthy and eat safe. It delivers clear,
precise, simple messages on eating healthy, well-balanced diets, which is fortified
and also paying special emphasis to the nutrition during the first thousand days of the
life of an infant and foods that should be avoided are the ones which are high in fat,
sugar, and salt containing food. The eat right tool kit also contains a handbook and
tools for the frontline health workers and it also has an engaging material for all the
citizens. FOSTAC which stands for Food Safety Training and Certification is one of
the most successful programs of the FSSAI. It has covered almost 1,50,000 people
who have been trained so far. This partnered with number of training partners which
are now training all over the country. This four-step program is divided into three
parts which is basic training that can be given to the street food vendors or small-time
caterer, advanced training, which includes manufacturing, storage, transportation,
retail, and distribution and specific special advanced training which is for specific
sectors like milk and milk products, meat, fish and poultry, packaged drinking water,
oils, and bakery. The FSSAI recommends that according to the rule, every food
business operator should have at least one trained and certified food safety supervi-
sor. It has about 17 types of competence-based certification programs and the
training partners could be organizations which have capacity for imparting training
and food safety. It could be domain experts from academia, industry, or

professionals who can get associated with this particular for a training program or
perhaps anyone in food business it could be a student or food professional who can
first get certified as food safety officer.
Another initiative is called as serve safe and this is dealing with the caterers. This
is a hygiene rating scheme that is done online which is absolutely objective,
transparent, and it is a rating method by which the consumer would come to know
when he is deciding a place to eat. It simply aims to allow consumers to make
informed choices about the places where they can eat out and through these choices
it helps in facilitating and encouraging business, improves, and helps in improving
their hygiene standards and definitely improves in reducing the incidence of food-
borne illnesses. The mandatory requirements of serve safe are:
• It should have qualified FBO’s, need to have FSSAI license or registration and
must follow the Schedule 4 requirements, which is good hygiene practices.
• It also aims to ensure that the food which is being served to the consumers is of
good quality and is absolutely safe for human consumption.
• It should train all food handlers and appoints a certified full safety supervisor.
• It must have food safety display boards prominently displayed in the premises and
get food samples and water tested periodically.
Enrolling in this hygiene rating scheme is very simple, it begins with logging
in. There is a self-assessment of food safety compliance that needs to be completed,
it can generate and display the hygienic ratings based on that and finally an
inspection and validation are done by the Food Safety Officer (FSO) or the Third-
Party Audit (TPA).
BHOG stands for blissful hygienic offering to God is an initiative that aims to
ensure that the food served and sold in places of worship is safe and hygienic. Now,
this is a very unique initiative that has been undertaken by the food safety authority
and it ensures the safety and hygienic preparation as well as dissemination of the
food to the people who are coming to the temples or the places of worship. This
particular program includes a BHOG manual that provides basic guidelines and
standard operating procedures to follow and implement all the requirements of
Schedule 4 of the Food Safety and Standards Act of 2006. This initiative is quite
popular and it is making a lot of difference to some very prominent places of
worship.
RUCO simply stands for repurpose used cooking oil. This is another initiative of
the FSSAI which talks about the correct practices of cooking and frying oils. This is
primarily collecting the used oil and repurposing it for the preparation of biodiesel.
The objectives are:
• Currently, the used cooking oil is either not discarded or disposed of in such a
manner that it chokes drains and sewerage systems. Sometimes it is sold off to the
petty vendors, which is not the right practice because it is very unhygienic as
primarily it is also not good for health.
• So, under this initiative, 64 companies at 101 locations have been identified that
enable the collection of the used cooking oil. Businesses that use more than
100 liters of oil for frying, to maintain a stocked register and ensure that used
cooking oil has handed over to only registered collecting agencies. The correct
method of using cooking oil after frying is only once but only in case where you
need to because oil is an expensive commodity you can use it the second time
also. Consuming that particular oil in two days is the ideal way otherwise again
rancidity of the oil takes place. So, repeated heating of vegetable oil leads to the
formation of total polar compounds (TPC) and this has an adverse effect on the
health. In order to safeguard Public Health, FSSAI has fixed that limit to 25%
beyond which the vegetable oil is unfit for human consumption. FSSAI is
employing the EEE strategy that stands for education, enforcement, and ecosys-
tem to ensure that the used cooking oil is discarded in an environment-friendly
manner and does not enter the food supply chain. TPC is the measure of the
quality of oil. The level of TPC increases every time the oil is reheated.
Aaj Se Thoda Kam is an initiative of the Food Safety Authority of India and
focuses on the reduction of high fat, sugar, and salt-containing foods. This does not
include only the packaged and the processed foods but also the popular snack food of
our country like samosas, chole bhature, and chowmein, which are high in fat and
salt content and we need to bring down the amount of consumption from these
particular commodities. Carbonated beverages are loaded with sugar so that is why
efforts are being made to bring down the total calorie or the sugar content of these
particular beverages as well and that is why we say that between fruit juices and
fruits, it is nice to have fruit juices, whole wheat flour is a better option than maida
and that is why we must cut down on all these particular foods which are leading to
overweight and obesity in our country.
Food means any substances whether processed, partially processed or unpro-
cessed, which is intended for human consumption and includes primary food to the
extent defined in the Act, genetically modified foods or food containing such
ingredients, infant food, packaged drinking water, chewing gum, and any other
substances including water used into the food preparation during its manufacturing,
preparation, or treatment but does not include any animal feed, live animals unless
they are prepared or process for human consumption, plants prior to the harvesting,
drugs, and medical products, cosmetics, necrotic, or psychotropic substances. Food
definition is so elaborative that everything that we eat is covered under the food
including nutraceutical. Food alert is the system for the Food Authority of letting
local authorities and the consumer to know about a problem associated with the food
and in some cases provide detail of specific action if or to be taken. In the food alert,
there are two kinds of communications: one is for information and one is for action.
Often these alerts are issued in conjunction with the product withdrawals or recall.
So, food alert is about alerting people about the food if there is any food safety
problem associated with any kind of imported or domestic food. One of the recent
food alerts issued by FSSAI authority was on recall of the ice cream and other frozen
products due to the contamination with Listeria issued on May 07, 2015.
10.6.5 Food Traceability
ISO 22000 has defined the traceability as the ability to follow the movement of feed
or food through specific stages of production, processing, and distribution. Trace-
ability system should be able to document the history of the product or to locate the
product in the food chain. It is important because if any outbreak occurs due to food
poisoning or any other food safety issue, the product recall should be possible.
Traceability has two components, backward traceability and forward traceability.
Tracing is basically its ability to identify what are the raw ingredients, packing
ingredients, etc., which goes onto produce the finished product. For example, the
tracing of a product can be done by its label which contains the batch number or
code, manufacturing date, details of the manufacturing unit, type of processing to
which the product is subjected. Tracking is the ability to trace the destination of the
product, where the product is being made, how it goes to the distributor, from the
distributor to the retailer. It helps in recalling the product at any destination. So, there
are two parts of tracing, one is tracing and other one is tracking with reference to the
traceability. Traceability is the only tool for the establishment of product authentic-
ity, reliability, identification of the problem, area of the purpose of tracking, and the
product recall. If some problem arises in one of the batches, then it should be
possible to identify its root cause. The food safety issue in a food product can
occur either through the raw material, the ingredients used or due to any failure in
the processing. Traceability makes the identification process easier which helps in
taking the specific corrective action. So, in the section 22 of the FSSAI standards, a
food recall procedure has been mentioned for a successful food recall. The traceabil-
ity of the entire process should be documented properly, in this the batch code which
is very important shows the manufacturing date and batch number. Section 28 of the
food recall talks about food recall; it says that if the food business operator considers
or has a reason to believe about any food safety issue, the product must be recalled
basically for the consumer safety. A notification by FSSAI was issued in 2009 for the
food business operator on how to establish a recall plan for food recall of a particular
batch. Once the product has been recalled, the follow-up action post recall should be
established in order to ensure the effectiveness of the recall and prevent its reoccur-
rence. Food recall should be done with the food poses a threat to public health. So, it
is very important to understand whenever there is a threat to the public health due to
the product quality or if the product does not meets the standards as per the FSSAI,
the recall of such a product is a must. All food business operators engaged in the
wholesale supplier, manufacturer, import, or any of the activity fall under the scope
of a food recall. It is interesting to note that retail is excluded, the food recall is
primarily the duty of the food business operator which is the main producing center.
The food business operator should maintain a record of all its distributors and
retailers. In case of the food recall, the food business operator should submit a recall
alert notification to the state authority and food authority immediately. Also, the
distribution of the product of that particular batch should be stopped with immediate
effect. Interestingly in last year, we have seen some kind of recall which are noodle
recalls issued by Food Authority and those recalls were based on the food safety
measures as there was a threat to the health of the consumers.
10.6.6 Food Recall Plan
The State Food Authority or Food Authority India reviews the adequacy of the
proposed recall plan and recommends the appropriate changes. Sometimes they
provide some more instructions to recall the product or they may actually restrict
the recall to only those batches. So, it depends on the gravity of the issues related to
food safety. So, for food recall, before starting of any recall it is very important to
understand, assemble the team and identify the responsibilities. Every food business
operator should have a team who is responsible for a food recall. It is a necessity, it
cannot be post operation when a food safety issue has occurred and the team is to be
selected. There should be a plan and a team in place with identified responsibilities
with reference to the food safety, which has a major impact on the consumer. An
immediate notification to the state and central authority must be provided. Identified
individual and business to whom the food may have been supplied should be tracked
(forward tracking). So, for forward tracking it is very important to identify where the
particular product has been supplied to and where the product recall has to be
implemented, i.e., at various locations like at the wholesaler, at the depots or the
factory store. The product should be taken back and sent for reworking or
reprocessing or disposed off. Then analyze the effectiveness of the recall and submit
the concerned authority with the interim report including action taken, results of the
recall and how the product has been disposed off. The state food authority after
analyzing that if the recall is successful, it will terminate the recall. These are the
stages of the food recall which should be taken by the food safety operator. So, when
it comes to the responsibility of the food recall there are three stakeholders: food
business operator, state food authority, and central food authority (FSSAI). So, the
food business operator needs to initiate the food recall when there is an issue food
safety issue and its termination is also dependent on the FBO once the recall has been
successfully implemented. The State Food Authority is responsible to supervise the
recall initiated by the food business operator. It is also responsible to inspect the food
business operator’s capability of the recall. The FSSAI is responsible to supervise
the state Food Authority whether the recall has been successfully implemented or
not, it checks the competency of the state food authorities. The State Food Authority
is the one who will be actually inspecting the food actually.
10.6.7 Composition of Food Authority (FSSAI)
FSSAI has one chairperson and 22 members.

Out of these 22 members, 1/3 (one-third) shall be women.

FSSAI—falls under the Directorate General of Health Services (DGHS), Ministry
of Health and Family Welfare.
22 Members (Details):
A. 7 members—Not below the rank of joint secretary to Govt. of India (to be

appointed by the Central Government to represent the ministries or departments
of Central Govt. dealing with):
• Agriculture
• Commerce
• Consumer Affairs
• Food Processing
• Health
• Legislative Affairs
• Small Scale Industries (government)
B. 2 representatives from the food industry of which one shall be from small-scale
industries (private)
C. 2 representatives from consumer organizations
D. 3 eminent food technologists or scientists
E. 5 members to be appointed by rotation every 3 years to represent states and
Union Territories
F. 2 persons to represent farmer’s organizations
G. 1 person to represent retailer’s organizations
FSSAI has one Chief Executive Officer (CEO), not below the rank of addi-
tional secretary to the Govt. of India, who acts as member secretary to the
authority.
Objectives of FSSAI
Two main objectives:
1. Forming the standards for food articles on the basis of scientific studies
and data.
2. To regulate the manufacture, storage, distribution, sale, and import of food
articles to ensure safe and wholesome food for human consumption.
10.6.8 Enforcement of the Act
FSSAI at the central government level and the State Food Safety Authorities enforce
the various provisions of the act.
10.6.9 Central Advisory Committee
FSSAI has a central advisory committee (CAC). The function of the central advisory
committee is to advise the FSSAI on all the matters that arise from the administration
of FSS Act, 2006. It is a very influential committee which is composed of the persons
from all the major ministries and departments. CEO (Chief Executive Officer) who is
the member-secretary of FSSAI, is the chairperson of CAC (Central Advisory
Committee). Earlier, in PFA, there was a CCFS (Central Committee for Food
Standards) for a similar purpose.
10.6.10 Commissioners of Food Safety
State Govt. appoints the commissioner of food safety for the state for the effective
implementation of food safety and standards along with the other requirements laid
down in this act and the rules and regulations. Commissioner of food safety has
powers to appoint various designated officers, food safety officers, and food
analysts.
10.6.11 Procedure for Collection and Analysis of FSSAI Food

Samples
Under FSSAI, food samples are collected by Food Safety Officer (earlier called food
inspector in PFA). The manufacturing unit from whom food sample is drawn is
called Food Business Operator.
After collecting the sample, the food safety officer divides the sample into 4 parts,
as follows:
1. One sample is sent for analysis to the Food Analyst (in food lab) under intimation
to the Designated Officer (usually the Chief Medical Officer).
2. Two parts are sent to the Designated Officer for safe custody.
3. The remaining sample is sent to an FSSAI accredited laboratory for analysis, if
requested by the Food Business Operator (within 24 hours) under intimation to
the designated officer.
The Food Analyst should send the analysis report within 14 days of the receipt
of the sample.
However, in case if the sample cannot be analyzed within the stipulated period,
the Food Analyst shall inform the Designated Officer and Commissioner of Food
Safety, giving reasons and also should specify the time to be taken for analysis. In
case the report by Food Analyst is alright, the matter ends. In case of an adverse
reports from the Food Analyst, the second part of the sample is sent to Referral Food
Laboratory (earlier called Central Food Laboratory) for analysis whose decision on
analysis is taken as final. No time limit is specified for the analysis of a sample at
RFL. The third part of the sample is kept to meet the exigencies like damage/
10.7 Agmark and BIS Standards for Milk Products 301
destruction/breakage on the way when the first part of the sample is sent to the Food
Analyst for analysis. The report of Food Analyst on the first part of the sample stands
superseded by the certificate issued by the Director of RFL on the analysis of second
part of the sample. The fourth part of the sample that is analyzed at the behest of the
Food Business Operator at his/her cost by the accredited lab is probably for the
satisfaction of Food Business operator, which can be used in case of controversy of
report of Food Analyst and this report given by accredited lab. There are several
Food laboratories in the country where the first part of the sample is sent for analysis.
These labs were earlier called Public Health Labs or Public Food Labs. Each district
has a lab that is controlled by the Designated Officer (CMO). But there are only
4 RFL (Referral Food Laboratories) in the country, which are located at Kolkata,
Mysore, Pune, and Ghaziabad. These labs fall under a certain zonal area and the
samples of a particular zone are sent to the concerned RFL.
10.6.12 Preservative Permitted to Be Added to Samples
When the Food Safety Officer (FSO) draws the sample of any food for analysis, there
occurs a time lag between the analysis and the sample drawn. So, in order to preserve
the food so that it remains in a suitable condition, preservative is added to it. The
common preservative used is called as “Formalin” which is a 40% solution of
Formaldehyde. It is added at the rate of 0.4% except for ice cream in which it is
added at 0.6%.
10.7 Agmark and BIS Standards for Milk Products
“Agmark” is Agricultural marking. In 1937, the Indian Legislature enacted the act
known as the Agriculture Produce (Grading and Marking) Act of 1937. This act was
created to ensure that the agricultural products should be marketed on the basis of
some established quality parameters. This is not a mandatory act. By fact, it is
permissive. It is an option to go for Agmark Grading only if one can follow their
requirements. The rules which have been established and are to be followed in
accordance with this act are termed as “General Grading and Marking Rules,
1937.” In 1988, the rules were updated and they are known as General Grading &
Marking Rules, 1988. The “Agmark Grading or Agmark certification” is the grading
of agricultural items according to these rules. Agmark is India Govt’s exclusive
property. It is not a trademark for private use. DMI is the Agmark authority whose
headquarters is in Faridabad and branched headquarters in Nagpur. The Agricultural
Produce Act, 1937 is enforced by the DMI. The standards for agricultural
commodities and services such as food, oil seeds, fats, creamery butter, ghee,
legumes, and eggs are laid down under the act. The agricultural goods are graded
into different grades like exceptional, excellent, fair, and ordinary on the basis of
their quality.
10.7.1 Objectives of Agmark Scheme
• To ensure that a safe and quality product is provided to the consumer.

• To make better returns to the producer of good quality products.
• Ensuring that the product has a uniform composition and is as per the quality
standards as prescribed in the act.
• To eliminate the adulteration from the entire food chain that commences from the
producer to the manufacturer and finally the customer.
Ghee and creamery butter (Table butter or salted butter) are the only dairy products
that can be graded under Agmark. Desi or cooking (unsalted or white) butter is not
graded under Agmark.
10.7.2 AGMARK Standards for Butter
Grading of Creamery butter, i.e., Table butter or Salted butter under AGMARK.
There are no AGMARK standards for Desi butter or Cooking or Unsalted or White
butter.
Grading of butter was started in 1941. Relevant rules are called “Creamery butter
grading and marking rules, 1941.” Creamery butter under AGMARK is graded as
“Pasteurized Table Butter.”
10.7.3 Specifications for Butter Under AGMARK
Grade designation: called Pasteurized Table Butter.

Special characteristic: Shall be made from pasteurized cream and the phosphatase
test shall be negative.
General characteristics: 10 characteristics
1. General: Shall be made from cream obtained from the milk of cows or buffalos
or both, with or without the addition of clean dairy salt (NMT 3% as per FSSAI).
The butter shall contain no other preservatives, except BHA at the level NMT
0.02%.
2. Flavor and aroma: shall be clean, pleasant and free from rancidity. No extrane-
ous flavor except Diacetyl shall be added, at a level NMT 4 ppm. It shall be
indicated in capital letters on packet “CONTAINS ADDED FLAVOUR-
DIACETYL.”
3. Body and texture: Shall be firm at 15 C and be neither greasy nor oily.
4. Color: Shall be uniform without any colored spots or signs of curd particles.
Only annatto or carotene may be used as a coloring matter (level not specified).
It shall be indicated in capital letters on the packet “CONTAINS PERMITTED
COLOR ANNATTO/CAROTENE.”
5. Moisture content: NMT 16% uniformly distributed in the body.
6. Acidity: NMT 0.15% (L.A.).

7. Milk fat content: NLT 80%.
8. Curd: NMT 1.5%.
9. Purity: Physicochemical constants of the fat obtained from butter shall resemble
(match) with the physicochemical constants prescribed for ghee for that area.
10. The product shall be free from other animal fats, vegetable oils or fats, mineral
oils, wax, or any other extraneous substances or impurities.
10.7.4 Grading of Ghee Under AGMARK
Grading of ghee was started in 1938. Relevant rules are called “Ghee grading and
marking rules, 1938.” Three grades of ghee are specified under AGMARK.
Grade Color of Agmark label

Special Grade Red Label
General Grade Green Label
Standard Grade Chocolate Label
Note: Specifications of standard grade ghee are equivalent or identical to that specified for ghee
under FSSAI standard
The major difference in these three grades of ghee is on the basis of free fatty acid
(FFA), i.e., acidity.
Grade Acidity (% oleic acid)

Special Grade NMT 1.4
General Grade NMT 2.5
Standard Grade NMT 3.0
There are two types of standards for both special and general grades:
All India standard (for areas other than a cotton tract of Saurashtra and Madhya
Pradesh).
Regional and seasonal standard (for cotton tract areas) (Table 10.1).
In summer, when RM value is between 19 and 21, Phytosterol acetate test must be
done and should be negative. In the case of Phytosterol acetate test, ghee is
saponified and the unsaponifiable matter is extracted and sterols present are isolated
Table 10.1 Agmark standards for ghee

Regional and seasonal
S. No. Specifications All India Winter (Sept–Feb) Summer (Mar–Aug)
1 Baudouin test Negative
2 BR at 40 C 40.0–43.0 41.5–44.0 42.5–45.0
3 RM value Not less than 28.0 Not less than 23.0 Not less than 21.0
4 Polenske value 1.0–2.0 0.5–1.2 0.5–1.0
5 Moisture Not more than 0.3
with the help of digitonin and then converted into acetates and then melting point is
determined.
Cholesterol acetate—melting point—114–115 C.
Phytosterol acetate—melting point—125–137 C.
If the melting point is more than 117 C, this indicates adulteration with
vegetable oil.
Process of Getting Agmark Certification The manufacturing or the production

unit which is interested to use the Agmark labels on their products, have to apply to
the Agricultural Marketing Advisor (AMA) to Govt. of India at Directorate of
Marketing & Inspection (DMI) whose H.Q. is at Faridabad (Branched H.Q. at
Nagpur, Chandigarh)
• The duly filled application form by the party concerned firm or party should be
submitted through the state marketing officer.
• The unit should satisfy certain requirements of Agmark like they should have
well-equipped laboratory with qualified staff for the testing of the manufactured
goods as per the Agmark requirements.
• The processing unit should have well trained, qualified, and experienced staff
engaged in the manufacturing of products like butter or ghee. The person who
will be given the authority of being the Agmark chemist of the unit should have a
degree from a recognized agricultural/dairy institute.
• Units that are to market pasteurized table butter should have the facilities like
pasteurization unit, cream separator, and cold storage facility.
• All the equipment like a cream separator, butter vat which is to be involved in the
manufacturing of the product should be hygienic and well equipped.
• Regular testing of the cream and raw materials should be done to assure the
quality of the end product, along with testing for the presence of adulterants like
foreign fat should be done.
• Containers used for packing ghee in tins should be rust free. They should be
properly lacquered.
• Vegetable fat or animal body fat or any foreign fat, artificial flavoring, or coloring
matter should not be seen near the factory and cream separating station.
• The temperature for ghee clarification should not be more than 110 C.
• One sample of butter/ghee should be sent at regular intervals to the Regional
Agmark Lab or any other specified lab as advised by AMA.
• The prerequisites and instructions from AMA should be followed strictly. For
example, method of sampling, sealing and marking of tins or cartons, mainte-
nance of records, labeling procedure as prescribed by AMA.
• The record for the labels should be updated every time, when the labels are issued
to the production section for labeling on the container. The labels should be
clearly pasted and should have the Certificate of Authorization (C.A.) number,
product description (pasteurized butter or ghee), serial number of the label, etc.
• Other information like the batch number, date of manufacture, net weight, name
and address of the packaging, and manufacturing unit should be clearly men-
tioned on the container.
If the manufacturing unit can fulfill the above-mentioned requirements, then they
can write to AMA at Faridabad/Nagpur through the state marketing officer. Then,
AMA or its authorized officials visit the premises for inspection of the production
facility, qualification of the technical staff, hygienic status of the unit, pollution
certificates from air and water department, boiler maintenance certificate, a certifi-
cate from weights and measurement department, etc. If the inspection officer
becomes satisfied, the AMA will issue the certificate of authority to use Agmark
Labels. The Certificate of Authorization is renewed generally after 5 years and also
on the basis of the past performance of the authorized packer.
In case of the dairy industry, as the Agmark authorities issue the certificate to use
the Agmark Labels, they employ their chemist called as Agmark Chemist in the
manufacturing unit. The Agmark chemist supervises all the operations taking place
right from the procurement of the raw material to the sealing and labeling of the final
product. The manufacturing unit can also depute their employee who is having a
degree in dairying or agriculture or chemistry to the Agmark office for getting
trained as an Agmark chemist.
10.7.5 Bureau of Indian Standards
BIS is a standard formulating body at the national level, which specifies and
formulates standards for a number of products or articles (edible and nonedible),
testing procedures, apparatus, etc. This organization was previously named as ISI
(Indian Standards Institution), established in 1947. ISI was changed to BIS on April
01, 1987, under the BIS Act 1986.
10.7.6 Structure of BIS/Members of BIS
The members of the BIS come from different fields, thus making it one of the most
diverse national organization. The members are elected in such a way that all the
interests and objectives of the BIS are addressed. The president of BIS is the
“Minister for food and civil supplies.”
The Members of BIS comprises of:
• Members of Parliament
• Ministers of State Government
• Nominees of central Govt. Ministries and departments
• Member representing the farmer community
• Member representing the consumer’s organizations
• Member from the academic and research institutions
• Member from the industry
• Professional associations
10.7.7 Objectives and Functions of BIS
1. Formulation of the standards for various types of articles, methods of test, codes
of practices, processes, etc. and to ensure that they are being properly
implemented. The standards formulated by BIS are referred to as Indian
Standards.
2. Formulating the concepts of standardization and quality control in industries.
3. To coordinate the efforts of producers and users for making improvements in the
materials, products, processes, and methods.
4. To provide the ISI certification to the manufacturing units.
5. Establishment of its testing laboratories to ensure that the products under the BIS
certification are as per the standards or not.
6. Providing BIS certification to other laboratories for testing the quality of the
finished products as per BIS.
7. Providing consultancy and technical assistance or consultancy within and outside
the country.
8. To have cooperation and coordination with the international standard making
bodies like ISO.
BIS is a member of both ISO (International organization for standardization) and
IEC (International electro-technical commission)
10.7.8 How to Get Authority to Use ISI Mark?
The manufacturers who are willing to use the IS mark on their products, have to
apply to the BIS for permission. The team of BIS officials will visit the
manufacturing unit or the factory for inspection and to verify whether the unit is
capable to produce the products as per IS standards or not. They also check the
quality of the raw material procured, entire operation, and process involved in the
production, quality of the end product, and other related facilities. Once the officials
get satisfied, they authorize and permit the unit to use ISI mark. BIS ensures that a
proper, well-defined production and quality control system is in place. It also aims at
exercising the control measures at not only the production stage but through the
entire chain like testing the raw material, verifying the process involved in the
production, final product testing, storage, and distribution of the final product.
The officials of the BIS visit the manufacturing premises regularly for inspections
and audits to keep a control and to ensure that the products using the ISI marks are as
per the requirements of the Indian standards. BIS also has a Quality Audit System
which draws samples of the finished product from the production unit during the
ongoing process or from the factory store or from the market and then tests the
samples in their own labs or from the other BIS recognized labs to check the
conformity with respect to the Indian standards. The drawn samples from the factory
store or production unit are drawn in triplicate (one sample is sealed and given to the
manufacturer, one sealed sample is used sent for testing to the BIS lab or a BIS
recognized lab while the other sealed sample is stored for future purpose when
10.8 International Dairy Federation 307
required). BIS also entertains and addresses the customer complaints and provides
them with the free replacements for the defective ISI-marked goods. It also provides
training in the area of statistical quality control to its licensees for improving their
technical skill.
10.8 International Dairy Federation
International Dairy Federation (IDF) is the preeminent body at the global level that
provides scientific and technical expertise for all the stakeholders associated with the
dairy chain. Currently, there are 57 members of IDF and the number is growing
every day. IDF aims at providing the best scientific expertise, knowledge, and skills
at the global level. It lays emphasis on the quality of the milk and milk products
delivered to the customers with respect to its nutrition and well-being. IDF has
published jointly with ISO various standard procedures for sampling and analysis of
milk and milk products. IDF is a unique forum where the stakeholders related to the
dairy industry whether they are from government organization or researchers or
primary producers, manufacturers, and laboratory experts. All those related share
their scientific and technical knowledge on various aspects of the dairy industry.
Figure 10.1 depicts the areas of working of IDF.
Work Areas of IDF
Economics and Marketing Dairy Farming
Science and Technology Food Standards
Hygiene and Safety Analytical Methods
Nutrition
Fig. 10.1 Areas of IDF

10.9 Food and Drug Administration
Food and Drug Administration (FDA or USFDA) is an agency of the United States
Department of Health and Human Services. The main aim of FDA is similar to that
of the other food regulatory bodies. It protects the customer interest, public health by
regulating the food produced. It covers food articles like tobacco products, dietary
supplements, prescription, and over-the-counter pharmaceutical drugs
(medications), vaccines, biopharmaceuticals, blood transfusions, medical devices,
electromagnetic radiation emitting devices (ERED), veterinary products, and
cosmetics. The FDA also enforces other laws, which include sanitation requirements
on interstate travel and control of disease on products ranging from certain house-
hold pets to sperm donation for assisted reproduction. FDA was founded in 1906 and
its headquarters is located at White Oak, Maryland. FDA has a wide network of
223 field offices and 13 laboratories located across 50 states. In 2008, FDA has
opened its offices in foreign lands like China, India, Costa Rica, Chile, Belgium, and
the United Kingdom.
10.9.1 Organizations of FDA
The major offices and centers of FDA are:
• Center for Biologics Evaluation and Research

• Center for Devices and Radiological Health (CDRH)
• Center for Drug Evaluation and Research (CDER)
• Division of Manufacturing and Product Quality
• Division of New Drugs and Labeling Compliance
• Division of Scientific Investigations
• Division of Drug Marketing, Advertising and Communications
• Informatics and Computational Safety Analysis Staff (ICSAS)
• Center for Food Safety and Applied Nutrition
• Center for Tobacco Products
• Center for Veterinary Medicine
• National Center for Toxicological Research
• Office of Regulatory Affairs
10.9.2 What Does FDA Regulates?
Approximately $1 trillion worth of consumer goods, 25% of consumer expenditures

are being regulated by the FDA in the United States, out of which food sales cost
worth $466 billion, drugs around $275 billion, cosmetics $60 billion and the vitamin
supplements of worth $18 billion. Majority of the expenditure is on the goods which
are imported by the United States; FDA monitors around one-third of the total
10.9 Food and Drug Administration 309
imports of the United States. Most federal laws concerning the FDA are part of the
Food, Drug and Cosmetic Act.
10.9.3 Regulatory Programs of FDA
FDA carries certain safety regulation programs that depend on the type of product,
the potential risks associated with it and the powers imparted to the agency. For
example, the FDA regulates the labeling and safety of cosmetics while on the other
hand, it controls all the steps involved in drug making which include testing,
manufacturing, labeling, storage, marketing, advertising, and safety. The FDA
regulates most products by following a certain set of standards along with
conducting various inspections. FDA regulates the following areas.
10.9.4 Food and Dietary Supplements
The Center for Food Safety and Applied Nutrition (CFSAN) which is a branch of the
FDA ensures that all the food products (except meat products which are from
domestic animals like chickens, cattle as it comes under the jurisdiction of the
United States Department of Agriculture Food Safety and Inspection Service) in
the United States are safe for consumption and properly labeled. CFSAN’s
establishes and maintains food standards, like the standards of product identity (for
example, what are the requirements for a product to be labeled as cheese), the
standards of maximum acceptable contamination, nutritional labeling. The food
standards and nutrition labeling requirements are part of the Code of Federal
Regulations.
10.9.5 Drugs
The Centre for Drug Evaluation and Research has a different approach for different
types of drugs. The three major types of drugs are new drugs, generic drugs, and
over-the-counter drugs. A drug is said to be “new” if it is made by a different
manufacturer by using excipients or inactive ingredients for its use for a different
purpose.
10.9.6 Vaccines, Blood and Tissue Products, and Biotechnology
The Centre for Biologics, Evaluation and Research ensures the safety and efficacy of
biological therapeutic agents such as gene therapy products, blood and blood
products, cell- and tissue-based products, and vaccines. The original authority for
government regulation of biological products was by the Biologics Control Act
(1902), with additional authority established by the Public Health Service Act
(1944). Along with these acts, the Federal Food, Drug, and Cosmetic Act applies to
all biological products, as well. Originally, the entity responsible for the regulation
of biological products resided under the National Institutes of Health; this authority
was transferred to the FDA in 1972.
10.9.7 Medical and Radiation-Emitting Devices
The Centre for Devices and Radiological Health (CDRH) regulates the production,
quality, safety of the medical devices. It also gives the pre-market approval for the
devices used in medical line. The medical devices are one of the most diverse groups
of articles regulated by CDRH, a medical device can be a simple toothbrush or a
complex implantable brain pacemakers. CDRH also accesses the safety performance
of nonmedical radiation emitting devices like cellular phones, laser products, airport
baggage screening equipment, television receivers, microwave ovens, and tanning
booths.
10.9.8 Cosmetics
The Centre for Food Safety and Applied Nutrition regulates the cosmetics. Such
products need not require premarket approval by the FDA until and unless they make
“structure or function claims,” which make them into drugs. Only the FDA approved
coloring agents that are added during the manufacture of the cosmetics should be
used. FDA also regulates the labeling of cosmetics and they have a warning clearly
mentioning that the cosmetics have not been subjected to thorough safety testing.
10.9.9 Veterinary Products
The Centre for Veterinary Medicine (CVM) regulates commodities like food, food
additives, animal food and drugs that are given to animals. The handling of the
vaccines for animals is done by the United States Department of Agriculture. CVM’s
focuses on the medications that are employed to the food animals keeping in view
that it does not affect the human supply. The FDA’s requirements to prevent the
spread of bovine spongiform encephalopathy are also administered by CVM through
inspections of feed manufacturers.
10.9.10 Tobacco Products
Since the Family Smoking Prevention and Tobacco Control Act 2009, FDA also
controls tobacco products. In 2009, the US Congress passed a law in which the
cigarettes packages and the printed advertisement should have a color warning,
along with written text warnings from the U.S. Surgeon General. FDA in June
10.9 Food and Drug Administration 311
2011 announced nine new graphic warning labels, which were to be displayed on the
packaging by September 2012.
10.9.11 What Does FDA not Regulate?
FDA does not regulate:
• Advertising (except for prescription drugs, medical devices, and tobacco

products)
• Alcoholic beverages
• Products such as paint, child-resistant packages, baby toys, and household
appliances (except for those that emit radiation)
• Illegal drugs of abuse, such as heroin and marijuana
• Health insurance
• Meat and poultry (except for game meats, such as venison, ostrich, and snake)
• Restaurants and grocery stores
FDA shares the responsibility for regulating these products with other govern-
ment agencies
10.9.12 FDA and India
In India, FDA ensures that the food and pharmaceutical products which are exported
to the United States from India are safe, effective and have good quality. They keep a
check on all the activities related to the procurement of the raw material, processing,
storage, and testing of the products to be exported. One of its activity also includes
reviewing the products for marketing authorization in the United States, those
products which are already available in the US market. To this end, FDA activities
in India include:
• Collection of the information from the Indian regulatory authorities related to the
clinical trials conducted for the products that are to be marketed in the United
States.
• Taking certain initiatives on the capacity building with their Indian counterparts.
• Working with industries which are to export their products to the United States,
making them aware of the expectations and the standards for the required
products.
• Working with the US government agencies in ensuring the product quality and its
safety-related issues, which can have an impact on the exported goods.
• Carrying out regular inspections especially of the high-risk facilities; and
collaborating with the private- and public-sector that wish to work with FDA
by getting third-party certifications related to the products being exported.
Suggested Readings
Bello JH (1996) The WTO dispute settlement understanding: less is more. Am J Int Law 90
(3):416–418
Gupta V (ed) (2018) The food safety and standards act, 2006, 11th edition. Commercial Law
Publishers, New Delhi
Henson S, Loader R (1999) Impact of sanitary and phytosanitary standards on developing countries
and the role of the SPS Agreement. Agribusiness Int J 15(3):355–369
Henson S, Loader R (2001) Barriers to agricultural exports from developing countries: the role of
sanitary and phytosanitary requirements. World Dev 29(1):85–102
Heras-Saizarbitoria I, Boiral O (2013) ISO 9001 and ISO 14001: towards a research agenda on
management system standards. Int J Manag Rev 15(1):47–65
Manual on General Guidelines on Sampling – FSSAI (2015)
Melo O, Engler A, Nahuehual L, Cofre G, Barrena J (2014) Do sanitary, phytosanitary, and quality-
related standards affect international trade? Evidence from Chilean fruit exports. World Dev
54:350–359
Papademas P, Bintsis T (2010) Food safety management systems (FSMS) in the dairy industry: a
review. Int J Dairy Technol 63(4):489–503
Randell AW, Whitehead AJ (1997) Codex alimentarius: food quality and safety standards for
international trade. Rev Sci Tech 16:313–318
Singh SP, Tripathi SC (2008) Food quality assurance: a concerted approach. G.B.Pant University of
Agriculture and Technology, Pantnagar, Uttarakhand
Surak JG (2007) A recipe for safe food: ISO 22000 and HACCP. Qual Prog 40(10):21
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millenium: [report by the Consultative Board to the Director-General Supachai Panitchpakdi.
WTO
www.codexalimentarius.org
https://www.fil-idf.org/
Appendix 1
FSSAI Standards for Milk and Milk Products

Standards for Milk
State or Union Minimum milk solids-not- Minimum milk
S. No. Class of milk Territory fat (SNF) (percent, m/m) fat (percent, m/m)
1. Buffalo milk Assam 9.0 6.0
Bihar
Chandigarh
Delhi
Gujarat
Haryana
Jharkhand
Maharashtra
Meghalaya
Punjab
Sikkim
Uttar Pradesh
Uttarakhand
West Bengal
Andaman and 9.0 5.0
Nicobar Islands
Andhra Pradesh
Arunachal
Pradesh
Chhattisgarh
Dadra and Nagar
Haveli Goa
Daman and Diu
Himachal
Pradesh
Jammu and
Kashmir
Karnataka
Kerala
Lakshadweep
Madhya Pradesh
Manipur
(continued)

https://doi.org/10.1007/978-981-15-4167-4
314 Appendix 1

Mizoram
Nagaland
Odisha
Puducherry
Rajasthan
Tamil Nadu
Telangana
Tripura
2. Cow milk All India 8.3 3.2
3. Goat or sheep Chandigarh 9.0 3.5
milk Haryana Kerala
Madhya Pradesh
Maharashtra
Punjab
Uttar Pradesh
Uttarakhand
Andaman and 9.0 3.0
Nicobar Islands
Andhra Pradesh
Arunachal
Pradesh
Chhattisgarh
Dadra and Nagar
Haveli Goa
Daman and Diu
Assam
Bihar
Delhi
Goa
Himachal
Pradesh
Jammu and
Kashmir
Karnataka
Gujarat
Jharkhand
Manipur
Mizoram
Nagaland
Odisha
Puducherry
Rajasthan
Meghalaya
Lakshadweep
Tamil Nadu
Telangana
Tripura
West Bengal
(continued)
Appendix 1 315

4. Camel milk All India 6.0 2.0
5. Mixed milk All India 8.5 4.5
6. Standardized All India 8.5 4.5
milk
7. Toned milk All India 8.5 3.0
8. Double toned All India 9.0 1.5
milk
9. Skimmed All India 8.7 Not more than 0.5
milk
10. Full-cream All India 9.0 6.0
milk
Standards for Evaporated or Concentrated Milk

Evaporated Evaporated Evaporated
Evaporated partly skimmed skimmed high-fat
Parameter milk milk milk milk
Milk fat, %, (m/m) 7.5 More than 1 and 1.0 15.0
(minimum) Less than 7.5 (maximum) (minimum)
Milk solids, minimum, %, 25.0 20.0 20.0 26.5
(m/m)
Milk protein in milk solids- 34.0 34.0 34.0 34.0
not-fat, minimum, %, (m/m)
Protein content is 6.38 multiplied by the total nitrogen determined
Standards for Sweetened Condensed Milk

Sweetened Sweetened Sweetened Sweetened
condensed condensed partly condensed condensed
Parameter milk skimmed milk skimmed milk high-fat milk
Milk fat, %, (m/m) 8.0 1–8 1.0 16.0
(minimum) (maximum) (minimum)
Milk solids, minimum, 28.0 24.0 24.0 –
%, (m/m)
Milk solids-not-fat – 20.0 – 14.0
minimum, %, (m/m)
Milk protein in milk 34.0 34.0 34.0 34.0
solids-not-fat,
minimum, %, (m/m)
316 Appendix 1
Standards for Khoa

Parameter Khoa
Total solids, minimum, %, (m/m) 55.0
Milk fat, minimum, %, (m/m), dry matter basis 30.0
Total ash, maximum, %, (m/m) 6.0
Titratable acidity (as % lactic acid), maximum, % 0.9
Standards for milk fat products

Milk fat, Anhydrous milk fat,
Parameter butter oil anhydrous Butter Oil Ghee
Moisture, maximum, %, (m/m) 0.4 0.1 0.5
Milk fat, minimum, %, (m/m) 99.6 99.8 99.5
BR at 40 C 40.0 to 44.0 40.0 to 44.0 As per table for
Reichert Meissl Value, minimum 28.0 28.0 Ghee given below
Polenske value 1.0–2.0 1.0–2.0 –
FFA as oleic acid, maximum, % 0.4 0.3 3.0
Peroxide value (Milli-equivalent 0.6 0.3 –
of oxygen/Kg fat), maximum
Baudouin Test Negative Negative Negative
Additionally, the Butyro refractometer reading and Reichert Meissl value of ghee produced in a
State or Union territory specified in column (1) of the table below shall be as specified against the
said State or Union Territory in the corresponding columns (2) and (3) of the said table
BR at Minimum Reichert Meissl Polenske
Name of State or Union Territory 40 C value Value
Andhra Pradesh/Telangana 40.0 to 24.0 –
43.0
Andaman and Nicobar Islands 41.0 to 24.0 –
44.0
Arunachal Pradesh 40.0 to 26.0 –
43.0
Assam 40.0 to 26.0 –
43.0
Bihar 40.0 to 28.0 –
43.0
Chandigarh 40.0 to 28.0 –
43.0
Chhattisgarh 40.0 to 26.0 –
44.0
Dadra and Nagar Haveli 40.0 to 24.0 –
43.0
(continued)
Appendix 1 317

Delhi 40.0 to 28.0 –
43.0
Goa 40.0 to 26.0 –
43.0
Daman and Diu 40.0 to 24.0 –
43.5
Gujarat 40 to 43.5 24.0
(a) Areas other than cotton tract 41.5 to 45 21.0
areas.
(b) Cotton tract areas.
Haryana 40.0 to 28.0 –
(a) Areas other than cotton tract 43.0 26.0
areas. 40.0 to
(b) Cotton tract areas. 43.0
Himachal Pradesh 40.0 to 26.0 –
43.0
Jammu and Kashmir 40.0 to 26.0 –
43.0
Jharkhand 40.0 to 28.0 –
43.0
Karnataka- 40.0 to 24.0 –
(a) Areas other than Belgaum 43.0 26.0
district. 40.0 to
(b) Belgaum district. 44.0
Kerala 40.0 to 26.0 –
43.0
Lakshadweep 40.0 to 26.0 –
43.0
Madhya Pradesh 40.0 to 26.0 –
areas. 41.5 to
Maharashtra 40.0 to 26.0 –
areas. 41.5 to
Manipur 40.0 to 26.0 –
43.0
Meghalaya 40.0 to 26.0 –
43.0
Mizoram 40.0 to 26.0 –
43.0
Nagaland 40.0 to 26.0 –
43.0
(continued)
318 Appendix 1

Odisha 40.0 to 26.0 –
43.0
Puducherry 40.0 to 26.0 –
44.0
Punjab 40.0 to 28.0 –
43.0
Rajasthan 40.0 to 26.0 –
(a) Areas other than Jodhpur district. 43.0 21.0
(b) Jodhpur district. 41.5 to
45.0
Tamil Nadu 41.0 to 24.0 –
44.0
Tripura 40.0 to 26.0 –
43.0
Uttar Pradesh 40.0 to 26.0 –
43.0
Uttarakhand 40.0 to 26.0 –
43.0
West Bengal- 40.0 to 28.0 –
(a) Areas other than Bishnupur 43.0 21.0
Subdivision. 41.5 to
(b) Bishnupur Subdivision. 45.0
Sikkim 40.0 to 28.0 –
43.0
Standards for Butter

Parameter Table butter White butter/cooking butter
Moisture, maximum, %, (m/m) 16.0 –
Milk fat, minimum, %, (m/m) 80.0 76.0
Milk solids-not-fat, maximum, %, (m/m) 2.0 –
Common salt, maximum, %, (m/m) 3.0 –
Appendix 1 319
Standards for Milk Powders and Cream Powder

Partly Skimmed
Whole milk skimmed milk Cream
Parameter powder Milk powder powder powder
Moisture, maximum, %, (m/m) 5.0 5.0 5.0 5.0
Milk fat, %, (m/m) Minimum More than 1.5 42.0
26.0 and less 1.5 and less (maximum) (minimum)
than 42.0 than 26.0
Milk protein in milk solids- 34.0 34.0 34.0 34.0
not-fat, minimum, %, (m/m)
18.0 18.0 18.0 –
Titratable acidity, maximum
(ml 0.1 NaOH for 10 g solids-
not-fat)
Insolubility index, maximum, 2.0 2.0 2.0 –
ml
Total ash, maximum, % (m/m), 9.3 9.3 9.3 –
on moisture and fat-free basis
Scorched particles, maximum Disc B Disc B Disc B Disc B
Standards for Dairy Whitener

Requirement
Skimmed Low-fat Medium- High-fat
milk dairy dairy fat dairy dairy
S. No. Characteristics whitener whitener whitener whitener
1. Moisture, maximum, %, 4.0 4.0 4.0 4.0
(m/m)
2. Milk fat, %, (m/m) 1.5 More than Minimum 20.0
(maximum) 1.5 and 10.0 and (minimum)
less than less than
10.0 20.0
3. Milk protein (in solids- 34.0 34.0 34.0 34.0
not-fat), minimum, %,
(m/m)
4. Insolubility Index, ml, 1.5 1.5 1.5 1.5
Maximum
5. 9.3 9.3 9.3 9.3
Total ash (on moisture,
added sugar and fat-free
basis), maximum, %, (m/m)
6. Acid insoluble ash, 0.1 0.1 0.1 0.1
maximum, %, (m/m)
7. Added sugar 18.0 18.0 18.0 18.0
(as sucrose), maximum, %,
(m/m)
(continued)
320 Appendix 1
Requirement
Skimmed Low-fat Medium- High-fat
milk dairy dairy fat dairy dairy
S. No. Characteristics whitener whitener whitener whitener
8. Titratable acidity, 1.5 1.5 1.5 1.2
maximum, % (as lactic acid)
9. Scorched particles, Disc B Disc B Disc B Disc B
Maximum
Standards for Whey Powder

Acid whey
Parameter Whey powder powder
Moisturea, maximum, %, (m/m) 5.0 4.5
Milk fat, maximum, %, (m/m) 2.0 2.0
Milk proteinb, minimum, %, (m/m) 10.0 7.0
Lactose contentc as anhydrous lactose, minimum, %, 61.0 61.0
(m/m)
pH (in 10% solution) more than 5.1e (max)
5.1d
Total ash, maximum, %, (m/m) (on dry basis) 9.5 15.0
a
The moisture does not include the water of crystallization of lactose
b
Protein content is multiplied by 6.38 with the total nitrogen content
c
Lactose content expressed as anhydrous lactose, 100 parts of lactose monohydrate contains
95 parts of anhydrous lactose
d
< 0.35% (as lactic acid)
e
0.35% (as lactic acid)
Standards for Fermented Milk Products

Partly skimmed yoghurt Skimmed yoghurt
Yoghurt and and flavored partly and lavored
Parameter flavored dahi skimmed dahi skimmed dahi
Milk Fat, %, (m/m) Not less than 3.0 More than 0.5 and Less 0.5 (maximum)
and not more than 3.0
than 15
Milk solids-not-fat, 8.5 8.5 8.5
minimum, %, (m/m)
Milk protein, 2.9 2.9 2.9
minimum, %, (m/m)
Titratable acidity, 0.6 0.6 0.6
minimum, %
(as lactic acid)
Appendix 1 321
Chakka shall conform to the following compositional specifications:

Skimmed milk Full-cream
S. No. Parameter Chakka chakka chakka
1 Total solids, minimum, %, (m/m) 30.0 20.0 28.0
2 Milk fat, %, (m/m), on dry basis 33.0 (min) 5.0 (max) 38.0 (min)
3 Milk protein, minimum, %, (m/m), 30.0 60.0 30.0
on dry basis
4 Titratable acidity, maximum, % 2.5 2.5 2.5
(as lactic acid)
5 Total ash, maximum, %, (m/m), on 3.5 5.0 3.5
dry basis
Shrikhand shall conform to the following compositional specifications:

Parameter Shrikhand Full-cream Fruit
Shrikhand Shrikhand
Total solids, minimum, %, (m/m) 58.0 58.0 58.0
Milk fat, minimum, %, (m/m), on dry basis 8.5 10.0 7.0
Milk protein, minimum, %, m/m, (on dry 9.0 7.0 6.0
basis)
Titratable acidity, maximum, % (as lactic 1.4 1.4 1.4
acid)
Sugar (sucrose), maximum %, m/m (on dry 72.5 72.5 72.5
basis)
Total ash, maximum, %, m/m (on dry basis) 0.9 0.9 0.9
Standards for Ice Cream, Kulfi, Chocolate Ice Cream, Softy Ice Cream, Milk Ice, Milk Lolly, and
Dried Ice Cream Mix
Low-fat ice cream or
Ice cream or Kulfi or Medium fat ice cream or Kulfi or chocolate ice
chocolate ice cream or Kulfi or chocolate ice cream or softy ice
Parameter softy ice cream cream or softy ice cream cream
(i) Ice cream, Kulfi, Chocolate Ice cream, and Softy Ice Cream
Total Solids, 36.0 30.0 26.0
minimum,
%, (m/m)
(continued)
322 Appendix 1
Low-fat ice cream or

Ice cream or Kulfi or Medium fat ice cream or Kulfi or chocolate ice
chocolate ice cream or Kulfi or chocolate ice cream or softy ice
Parameter softy ice cream cream or softy ice cream cream
Weight, 525.0 475.0 475.0
minimum,
g/l
Milk fat, %, 10.0 (minimum) 2.5–10.0 2.5 (maximum)
(m/m)
Milk 3.5 3.5 3.0
protein,
minimum,
%, (m/m)
Parameter Milk ice or Milk Lolly

(ii) Milk Ice or Milk Lolly
Total solids, minimum, %, (m/m) 20.0
Milk fat, maximum, %, (m/m) 2.0
Milk protein, minimum, %, (m/m) 3.5
Standards for Cheese and Cheese Products

Milk fat,
minimum, Lactose,
% (dry maximum,
Product Moisture, maximum, % (m/m) basis) % (m/m)
i. Cheese
a. Hard-pressed 39.0 48.0 –
cheese
b. Semi-hard 45.0 40.0 –
cheese
c. Semi-soft cheese 52.0 45.0 –
d. Soft cheese 80.0 20.0 –
e. Extra hard 36.0 32.0 –
cheese
f. Mozzarella 60.0 35.0 –
cheese
g. Pizza cheese 54.0 35.0 –
ii. Extra hard 36.0 32.0 –
grating Cheese
(continued)
Appendix 1 323
Milk fat,
minimum, Lactose,
% (dry maximum,
iii. Named variety
cheeses
a. Cheddar 39.0 48.0 –
b. Danbo 39.0 45.0 –
c. Edam 46.0 40.0 –
d. Gouda 43.0 48.0 –
e. Havarti
• Havarti. 48.0 45.0 –
• 30% Havarti. 53.0 30.0 –
• 60% Havarti. 60.0 60.0 –
f. Tilsiter
• Tilsiter. 47.0 45.0 –
• 30% Tilsiter. 53.0 30.0 –
• 60% Tilsiter. 39.0 60.0 –
g. Cottage cheese 80.0 –
and Creamed
cottage Cheese
h. Cream cheese 55.0 70.0 –
i. Coulommiers 56.0 46.0
j. Camembert
• 30% 62.0 30.0 –
Camembert.
• 40% 59.0 40.0 –
Camembert.
• 45% 57.0 45.0 –
Camembert.
• 55% 52.0 55.0 –
Camembert.
k. Brie 56.0 40.0 –
l. Saint Paulin 56.0 40.0 –
m. Samsoe
• Samsoe. 44.0 45.0 –
• 30% Samsoe. 50.0 30.0 –
n. Emmental 40.0 45.0 –
o. Provolone
• Smoked. 45.0 45.0 –
• Unsmoked. 47.0 45.0 –
(continued)
324 Appendix 1
Milk fat,
minimum, Lactose,
% (dry maximum,
iv. Cheese products
a. Processed cheese 47.0 (50% for chiplets (packed sliced 40.0 5.0
processed cheese), when sold in a
package other than tin
b. Processed cheese 60.0 40.0 5.0
spread
Standards for Edible Casein Products

Edible acid Edible rennet Edible
Parameter casein casein caseinate
Moisturea, maximum % (m/m) 12.0 12.0 8.0
Milk fat, maximum, %, (m/m) 2.0 2.0 2.0
Milk proteinb, minimum, %, (m/m), dry 90.0 84.0 88.0
matter basis
Casein in protein, minimum, %, (m/m) 95.0 95.0 95.0
Lactosec, maximum, %, (m/m) 1.0 1.0 1.0
Total ash including P2O5, %, (m/m) 2.5 7.5 (minimum) –
(maximum)
Free acid, maximum, ml of 0.1 N sodium 0.27 – –
hydroxide per g
pH (in 10% solution), maximum – – 8.0
a
The moisture does not include the water of crystallization of lactose
b
Protein content is multiplied by 6.38 with the total nitrogen content
c
Lactose content expressed as anhydrous lactose, 100 parts of lactose monohydrate contains
95 parts of anhydrous lactose
Standards for Frozen Desserts or Confections with added vegetable oil/fat or vegetable protein, or
both
Medium-fat frozen
Frozen dessert or dessert or frozen Low-fat frozen dessert
Parameter Frozen confection confection or frozen confection
(i) Frozen dessert or frozen confection
Total solids, 36.0 30.0 26.0
minimum, %,
(m/m)
Weight, 525.0 475.0 475.0
minimum, (g/l)
Total fat, %, 10 (minimum) More than 2.5 and less 2.5 (maximum)
(m/m) than 10.0
(continued)
Appendix 1 325
Medium-fat frozen
Frozen dessert or dessert or frozen Low-fat frozen dessert
Parameter Frozen confection confection or frozen confection
Protein, 3.5 3.5 3.0
minimum,%
(m/m)
Standards for Chhana and Paneer

Parameter Chhana or paneer Low fat chhana or paneer
Moisture, maximum, %, (m/m) 65.0 (for Chhana) 65.0 (for Chhana)
60.0 (for Paneer) 60.0 (for Paneer)
Milk fat, %, (m/m), dry matter basis 50.0 (minimum) 15.0 (maximum)
Standards for Infant Milk Mood

1. Moisture, percent. by weight (not more than) 4.5
2. Total milk protein, percent. by weight (not less than) 12.0
3. Milk fat, percent. by weight (not less than) 18.0
4. Total ash, percent. by weight (not more than) 8.5
5. Ash insoluble in dilute HCl, percent. by weight (not more than) 0.1
6 Solubility Index (ml), maximum 2.0
Solubility, percent. by weight (not less than) 98.5
7. Vitamin A (as retinol), μg per 100 g (not less than) 350
8. Added vitamin D (expressed as cholecalciferol or ergocalciferol) μg, per 100 g 4.5
(not less than)
9. Vitamin C, mg per 100 g (not less than) 35
10. Thiamine, μg per 100 g (not less than) 185
11. Riboflavin, μg per 100 g (not less than) 275
12. Niacin, μg per 100 g (not less than) 1160
13. Pyridoxine, μg per 100 g (not less than) 160
14. Folic acid, μg per 100 g (not less than) 20
15. Pantothenic acid, mg per 100 g (not less than) 1.4
16 Vitamin B12, μg per 100 g (not less than) 0.7
17 Choline, mg per 100 g (not less than) 32
18 Vitamin K, μg per 100 g (not less than) 18
19 Biotin, μg per 100 g (not less than) 7.0
20 Sodium, mg per 100 g (not less than) 90
21 Potassium, mg per 100 g (not less than) 370
22 Chloride, mg per 100 g (not less than) 250
(continued)
326 Appendix 1
23 Calcium, mg per 100 g (not less than) 230

24 Phosphorous, mg per 100 g (not less than) 115
25 Magnesium, mg per 100 g (not less than) 22
26 Iron, mg per 100 g (not less than) 5.0
27 Iodine, μg per 100 g (not less than) 20
28 Copper, μg per 100 g (not less than) 280
29 Zinc, mg per 100 g (not less than) and 2.5
not more than (mg) 5.0
30 Manganese, μg per 100 g (not less than) 20
31 Selenium, μg per 100 g (not less than) 14
32 Bacterial count, per g. (not more than) 10,000
33 Coliform count absent in 0.1 g
34 Yeast and mould count absent in 0.1 g
35 Salmonella and Shigella absent in 25 g
36 Escherichia coli absent in 0.1 g
37 Staphylococcus aureus absent in 0.1 g
Standards for Infant Formula

1. Carotenes Not less than 0.25 mg/L
2. Fluorine Not less than 0.107 mg/L
3. Amino acids Not less than 9 mg/L (only L forms of amino acids should be used)
4. Nonprotein Not less than 173 mg/L
nitrogen
5. Nucleotides Not less than 11.7 mg/L
6. Carnitine Not less than 11.27 μg/L
7. Lactalbumin Not less than 1.4 g/L
8. Lactoferrin Not less than 0.27 g/L
9. Lysozyme Not less than 0.8 g/L
10. Fucose Not less than 1.3 g/L
11. Glucosamine Not less than 0.7 g/L
12. Inositol Not less than 0.39 g/L
13. Citric acid Not less than 0.35 g/L
14. Cholesterol Not less than 88 mg/L
15. Lipid phosphorus Not less than 7 mg/L
16. Prostaglandins Not less than PGE 150 mg/L Not less than PGF 400 mg/L
Appendix 1 327
It may contain the following food additives

Food additives Maximum level in 100 ml of the ready-to-drink product
pH-adjusting agents Limited by Good Manufacturing Practice and within the
Sodium hydroxide limits for sodium and potassium in all types of infant
Sodium hydrogen carbonate formulae
Sodium carbonate
Potassium hydroxide
Potassium hydrogen carbonate
Potassium carbonate
Calcium hydroxide
Sodium citrate Limited by Good Manufacturing Practice in all types of
Potassium citrate infant formulae
L (+) Lactic acid-producing
cultures
Citric acid
Antioxidants 1 mg in all types of infant formulae
Mixed tocopherols concentrate
and L-ascorbyl palmitate
Mono- and diglycerides 0.4 g
It should confirm to following requirements:

2. Total milk protein, percent. by weight (not less than) and 10.0
not more than 16.0
3. Total fat, percent. by weight (not less than) 18.0
Milk Fat, percent. by weight (not less than) 12.0
Linoleate, g per 100 g (not less than) 1.398
5 Ash insoluble in dilute HCl, percent. by weight (not more than) 0.1
6 Solubility: 2.0
(a) Solubility Index (ml), maximum
(b) Solubility percent. by weight (not less than) 98.5
8. Added vitamin D (expressed as cholecalciferol or ergocalciferol), μg per 100 g 4.5
(not less than)
10. Thiamine, μg per 100 g (not less than) 185
13. Pyridoxine, μg per 100 g (not less than) 160
14. Folic acid, μg per 100 g. (not less than) 20
16. Vitamin B12, μg per 100 g (not less than) 0.7
17. Choline, mg per 100 g (not less than) 32
18. Vitamin K, μg per 100 g (not less than) 18
(continued)
328 Appendix 1
19. Biotin, μg per 100 g (not less than) 7.0

20. Vitamin E (as a-tocopherol compounds), IU per 100 g (not less than) 3.15
21. Sodium, mg per 100 g (not less than) 90
22. Potassium, mg per 100 g (not less than) 370
23. Chloride, mg per 100 g (not less than) 250
24. Calcium, mg per 100 g (not less than) 230
25. Phosphorous, mg per 100 g (not less than) 115
26. Magnesium, mg per 100 g (not less than) 22
27. Iron, mg per 100 g (not less than) 5.0
28. Iodine, μg per 100 g (not less than) 20
29. Copper, μg per 100 g (not less than) 280
30. Zinc, mg per 100 g (not less than) and 2.5
31 Manganese, μg per 100 g (not less than) 20
32. Selenium, μg per 100 g (not less than) 14
33. Bacterial count, per g (not more than) 10,000
34. Coliform count absent in 0.1 g
35. Yeast and mould count absent in 0.1 g
36. Salmonella and Shigella absent in 25 g
37. Escherichia coli absent in 0.1 g
38. Staphylococcus aureus absent in 0.1 g
Standards for Milk Cereal-based Complementary food

2. Total protein, percent. by weight (not less than) 15.0
3. Fat, percent. by weight (not less than) 7.5
4. Total carbohydrate, percent. by weight (not less than) 55.0
7. Crude fiber (on dry basis) percent. by weight (not more than) 1.0
8. Vitamin A (as retinol) μg per 100 g (not less than) 350
9. Added vitamin D, μg per 100 g (expressed as cholecalciferol or ergocalciferol 5
(not less than)
11. Thiamine (as hydrochloride), mg per 100 g (not less than) 0.5
12. Riboflavin, mg per 100 g (not less than) 0.3
13. Niacin, mg per 100 g (not less than) 3.0
14. Folic acid, μg per 100 g (not less than) 20
16. Zinc, mg per 100 g (not less than) 2.5
and not more than (mg) 5.0
(continued)
Appendix 1 329

It may contain the following additives

Emulsifiers Maximum level in 100 g of the product on a dry weight basis
Lecithin 1.5 g
pH-adjusting agents Limited by Good Manufacturing Practice within the limit for
Sodium hydrogen carbonate sodium
Sodium carbonate
Sodium citrate
Potassium hydrogen
carbonate
Potassium carbonate
Potassium citrate
Sodium hydroxide
Calcium hydroxide
Potassium hydroxide
L (+)Lactic acid
Citric acid
Antioxidants 300 mg/ kg fat, singly or in combination
Mixed tocopherols
concentrate
1-Tocopherol
L-Ascorbyl palmitate 200 mg/kg fat
Processed Cereal-based Complementary foods may contain the following additives

Maximum level in a 100 g of product on a dry weight
Name of the food additives basis
Emulsifiers
Lecithin 1.5 g
pH-adjusting agents
Sodium hydrogen carbonate Limited by Good Manufacturing Practice and within
the limits for sodium
Potassium hydrogen carbonate Limited by Good Manufacturing Practice
Calcium carbonate}
(continued)
330 Appendix 1
Maximum level in a 100 g of product on a dry weight

Name of the food additives basis
L(+) lactic acid 1.5 g
Citric acid 2.5 g
Antioxidants
Mixed tocopherols concentrate 300 mg/kg fat, singly or in combination
Alpha-tocopherol
L-Ascorbyl palmitate 200 mg/kg fat
L-Ascorbic acid and its sodium and 50 mg, expressed as ascorbic acid and within limits for
potassium salts sodium
Enzymes
Malt carbohydrates Limited by Good Manufacturing Practice
Leavening agents
Ammonium carbonate Limited by Good Manufacturing Practice
Ammonium hydrogen carbonate
It shall conform to the following requirements

2. Total protein, percent. by weight (not less than) 15.0
3. Total Carbohydrate, percent. by weight (not less than) 55.0
6. Crude fiber (on dry basis) percent. by weight (not more than) 1.0
8. Added vitamin D, μg per 100 g (expressed as cholecalciferol or ergocalciferol 5
(not less than)
10. Thiamine (as hydrochloride), mg per 100 g (not less than) 0.5
11. Riboflavin, mg per 100 g (not less than) 0.3
12. Niacin, mg per 100 g (not less than) 3.0
13. Folic acid, μg per 100 g (not less than) 20.0
15. Zinc, mg per 100 g (not less than) 2.5
and not more than (mg) 5.0
Appendix 1 331
Standards for Follow Up Formula Complementary food

Maximum level in 100 ml of product
ready-for-consumption
pH-adjusting agents Limited by Good Manufacturing
Sodium hydrogen carbonate Practice within the limit for sodium
Sodium carbonate
Sodium citrate, Potassium hydrogen carbonate,
potassium carbonate, potassium citrate, sodium
hydroxide, calcium hydroxide
Potassium hydroxide, L(+) Lactic acid
Citric acid
Antioxidants
Mixed tocopherols concentrate 3 mg singly or in combination
1-Tocopherol
L-Ascorbyl palmitate 5 mg singly or in combination.
It should conform to the following requirements

S.
No. Characteristics Requirements
2. Total milk protein, percent. by weight (not less than) and 13.5
(not more than) 24.75
3. Total fat, percent. by weight (not less than) and 18.0
(not more than) 27.0
Linoleate per 100 g (not less than) 1.398
6. Solubility:
Solubility Index (ml), maximum 2.0
Solubility percent. by weight (not less than) 98.5
8. Added Vitamin D (expressed as Cholecalciferol or Ergocalciferol),
μg per 100 g (not less than) 4.5
10. Thiamin, μg per 100 g (not less than) 180
13. Pyridoxine, μg per 100 g (not less than) 202.50
14. Folic acid, μg per 100 g (not less than) 20.0
16. Vitamin B12, μg per 100 g (not less than) 0.675
17. Choline, mg per 100 g (not less than) 32
(continued)
332 Appendix 1
S.
No. Characteristics Requirements
18. Vitamin K, μg per 100 g (not less than) 18
19. Biotin, μg per 100 g (not less than) 6.75
20. Vitamin E (as a-tocopherol compounds), I.U. per 100 g (not less than) 3.15
21. Sodium, mg per 100 g (not less than) 90
22. Potassium, mg per 100 g (not less than) 360
23. Chloride, mg per 100 g (not less than) 247.50
24. Calcium, mg per 100 g (not less than) 405
25. Phosphorous, mg per 100 g (not less than) 270
26. Magnesium, mg per 100 g (not less than) 27
27. Iron, mg per 100 g (not less than) 5
28. Iodine, μg per 100 g (not less than) 22.50
29. Copper, μg per 100 g (not less than) 280
30. Zinc, mg per 100 g (not less than) and 2.5
31. Manganese, μg per 100 g (not less than) 20
32. Selenium, μg per 100 g (not less than) 14
Standards for Edible Lactose

S. No. Parameters Limits
1. Total moisture, maximum, %, (m/m) 6.0
2. Lactose, minimum, %, (m/m), on dry basis 99.0
3. Sulfated ash, maximum, %, (m/m) 0.3
4. pH (10% solution) 4.5–7.0
5. Scorched particle, maximum Disc B
FSSAI regulations for Pesticide residues in milk and milk products

MRL
S. No. Name of insecticide (mg/kg)
1 2,4-Dichlorophenoxy acetic acid 0.05
2 Acephate (mixture of methamidophos and acephate) 0.02
3 Acetamiprid 0.02
4 Azoxystrobin 0.01
(continued)
Appendix 1 333
MRL
5 Sum of benomyl and carbendazim expressed as carbendazim 0.1 F
6 Bifenthrin 0.2
7 Bitertanol 0.05
8 Buprofezin 0.01
9 Carbaryl 0.05
10 Carbendazin 0.1 F
11 Carbofuran (sum of carbofuran and 3-hydroxy carbofuran expressed as 0.05 fat
carbofuran) basis
12 Chlorantraniliprole 0.05
13 Chlorothalonil 0.07
14 Chlorpyriphos 0.02
15 Chlothianidin 0.02
16 Cypermethrin (sum of isomers) (fat soluble residue) 0.05
17 Deltamethrin 0.05
18 Dichloros 0.01
19 Difenoconazole 0.02
20 Dimethoate 0.05
21 Dinotefuran 0.1
22 Mancozab (dithhiocarbamates) 0.05
23 Metiram as CS2 0.05
24 Difenoconazole 0.02
25 Edifenphos 0.01 F
26 Emamectin Benzoate 0.01
27 Ethion 0.5 F
28 Ethofenprox 0.02
29 Fenpropathrin 0.1
30 Fenvalerate 0.01 F
31 Fipronil 0.02
32 Flubendiamide 0.1
33 Flusilazole 0.05
34 Glufosinate ammonium 0.02
35 Imidacloprid 0.1
36 Indoxacarb 0.02
37 Kusoxium Methyl 0.01
38 Methomyl 0.02
39 Methyl chlorophenoxy acetic acid 0.04
40 Metolachlor 0.01
41 Monocrotophos 0.02
42 Oxydemeton-methyl 0.01
43 Paraquat dichloride 0.01
(continued)
334 Appendix 1
MRL
44 Penconazole 0.01
45 Phenthoate 0.01 F
46 Phorate 0.05 F
47 Primiphos methyl 0.05 F
48 Propiconazole 0.01
49 Pyraclostrobin 0.03
50 Tebuconazole 0.01
51 Thiacloprid 0.05
52 Thiamethoxam 0.05
53 Thiophanate-methyl 0.05
54 Trichlorfon 0.05
55 Tricontanol 0.01
56 Triadimefon 0.01
Maximum residue limit fixed at Limit of Quantification (LOQ)
F: Maximum residue limit calculation on fat basis
Appendix 2
Scheme for Testing and Inspection for Milk to be Adopted by

Dairy Processing Units
The Food Safety Standards Authority of India (FSSAI) has developed a new scheme
and plan to ensure the safety or quality of the milk which is supplied to the customer.
This plan is to be implemented by the units engaged in dairy processing. This system
is a self-monitoring type that will help in strengthening the internal quality control of
the dairy unit. The frequency of the implementation of this scheme is mentioned
below. The tests which are to be done by the dairy units are testing of raw milk,
processed, and finished products for microbial contamination, adulterants, hygiene
indicators, and contaminants like aflatoxin, pesticides, and antibiotics. The personnel
involved in the testing should have the proper knowledge of sampling of raw and
processed milk through the entire processing line. The points from which the
samples are to be drawn can be selected by the dairy unit on the basis of its
procurement, processing capacity and the risk or hazards associated with the product
or the process.
The dairy unit should have a well-equipped in-house laboratory for testing
chemical and microbiological parameters. The personnel involved in the testing
should be well qualified and trained. The tests which require advanced analytical
equipment can be carried out from an FSSAI notified lab. All the test records should
be maintained by the testing unit and should be made available to the Food Safety
official(s) when required.
In case of any noncompliance or deviation which can affect the quality or the
safety of milk, then it should be reprocessed and tested to ensure that whether the
reprocessed product is as per the requirements or not. If the noncompliance is due to
the presence of pesticide or adulterant, the whole batch should be rejected. The root
cause of the deviation or the failure should be analyzed and the necessary corrective
or preventive action should be taken accordingly. The records of such corrective
action and the product, which has been rejected or is substandard should be
maintained. Record for the method of disposal of the product and the reason for its
nonconformance to the requirements should also be maintained.
STI is a document that specifies the control over production process from raw
milk receipt till finished goods dispatch, which the manufacture is required to record,

https://doi.org/10.1007/978-981-15-4167-4
336 Appendix 2
maintain, and ensure compliance in terms of standards and safety parameters. This
document is to be maintained by the FBOs for liquid milk business.
Information required for testing of milk and milk products

S. No. Test parameters Chemical/equipment
1 Seal Visual
2 Appearance Visual
3 Taste and flavor Sensory
4 Foreign matter Visual
5 Temperature Thermometer
6 Fat Sulfuric acid, Isoamyl alcohol, Gerber centrifuge, milk
butyrometer/ rapid test apparatus—Milkoscreen
7 SNF Lactometer, thermometer/rapid test apparatus—Milkoscreen
8 SMP Acetic acid, Phosphomolybdic acid
9 Acidity Sodium hydroxide, phenolphthalein
10 Cellulose Iodine, zinc chloride (Qualitative)
11 Starch Iodine, potassium iodide (Qualitative)
Ethanol, sodium hydroxide, sodium carbonate (Quantitative)
12 Formalin Sulfuric acid
13 Hydrogen Vanadium pentaoxide, sulfuric acid (1st method) or Para-
Peroxide phenylenediamine (2nd method)
14 Boric acid Turmeric acid, hydrochloric acid, ammonium hydroxide
15 Detergent, Tetrachloroethane, citric acid, sodium hydroxide
caustic soda
16 Vegetable oil/ fat Sulfuric acid, isoamyl alcohol, Gerber centrifuge
17 Maltodextrin Potassium iodide, iodine, lactic acid/rapid test apparatus—
Milkoscreen
18 Dextrose or Modified Barford’s reagent, phosphomolybdic acid
glucose
19 Urea Para-dimethylaminobenzaldehyde (DMAB), ethyl alcohol,
hydrochloric acid/rapid test apparatus—Milkoscreen
20 Sucrose Resorcinol/rapid test apparatus—Milkoscreen
21 Salts Silver nitrate, potassium chromate
22 Neutralizers Rosolic acid, ethyl alcohol
23 Nitrate Diphenylamine, sulfuric acid
24 Added water Lactometer/rapid test apparatus—Milkoscreen
25 Pesticide residue Organophosphate, organochloride, and carbamate pesticide,
Rapid test kit/chromatographic techniques involving high-end
equipment
26 Antibiotic, Lateral flow assay, rapid test equipment, consumables-25
veterinary drug antibiotics/chromatographic techniques involving high-end
equipment
27 Aflatoxin M1 Lateral flow assay, rapid test equipment, consumables/
chromatographic techniques involving high-end equipment
Scheme of testing and inspection for milk (STI)
Inspection Inspection/test
S. No. characterstic method Specification/limits Inspection point Testing frequency
Physical/ Raw milk reception/ Standardization/ Filling Finished goods Raw milk Processed
Appendix 2
chemical/ release (raw milk pasteurization on (filling (processed packed milk milk
compositional can/ milk tanker) (silo/milk storage area) and dispatch milk
parameters tanks) tanker)
1 Seal of integrity Visual inspection Ok √ Every
tanker
2 Appearance Visual inspection White to cream color, odor √ √ Every Every batch
typical of fresh milk tanker/ or silo
container
3 Taste and flavor Sensory evaluation Satisfactory √ √ Every Every batch
(organoleptic tanker/ or silo
evaluation) container
4 Foreign matter Visual inspection/ Absent √ Every
filtration tanker/
container
5 Temperature Thermometer At max. 6 C √ (only raw chilled √ √ Every Every batch
milk) tanker/ or silo
container
6 Fat Chemical Specified as per FSSR √ √ Every Every batch
extraction, Gerber tanker/ or silo
method, electronic container
7 SNF Density (e.g., Specified as per FSSR √ √ Every Every batch
lactometer), tanker/ or silo
gravimetric, container
electronic
8 SMP (for species Chemical Negative √ Every batch
identified milk (species
and mixed milk) identified
products)
9 Acidity Titration Min. 0.10% √ √ √ Every batch
Max. 0.15% or silo
(as lactic acid) at 8.5% SNF
(continued)
337
338
Adulterants
10 Cellulose Chemical, Negative √ Quarterly
electronic approved per milk
strip/ rapid detection route
tests
11 Starch Chemical, Negative √ Every
electronic approved tanker
strip/ rapid detection
tests
12 Formalin, H2O2, Chemical, Negative √ Every
boric acid electronic approved tanker
tests
13 Detergent/caustic Chemical, Negative √ Every
soda electronic approved tanker
tests
14 Vegetable oil/ fat Chemical, Negative √ Every
tests
15 Maltodextrin Chemical, Negative √ Every
tests
16 Dextrose Chemical, Negative √ Every
(¼glucose) electronic approved tanker
tests
17 Urea Chemical, 700 mg/kg √ Every
tests
Appendix 2
18 Sucrose (Cane Chemical, Negative √ Every
sugar) electronic approved tanker
tests
Appendix 2
19 Salts (NaCl, KCl) Chemical, Negative √ Every

tests
20 Neutralizer Chemical, Negative √ √ Every Every batch
(Carbonate, electronic approved tanker or silo
bicarbonate, per strip/ rapid detection
carbonate) tests
21 Nitrates Chemical, Negative √ Every
tests
Chemical contaminants
22 Pesticides residue Chemical, Specified as per FSSR √ √ Quarterly Quarterly
(with isomers) electronic approved per milk
tests
23 Antibiotic/ Chemical, Specified as per FSSR √ √ Quarterly Quarterly
veterinary drugs electronic approved per milk
residues strip/ rapid detection route
tests
24 Aflatoxin M1, Chemical, 0.5 μg/kg √ √ Quarterly Quarterly
max. electronic approved per milk
tests
25 Melamine Chemical, Specified as per FSSR √ √ Every six
electronic approved months
tests
Microbiological contaminants
26 MBRT Dye reduction Min 30 minutes for raw chilled √ √ Every Every batch
milk and min. % hrs tanker or silo
30 minutes for pasteurized
milk
339
(continued)
340
27 Phosphatase Chemical, dye NA √ √ Every batch
reduction or silo
28 SPC/ ml Pour plate method, √ Every
electronic milk route
29 Coliform/ml# Pour plate method, √ √ Every Every batch
electronic milk route or silo
30 Water supply for dairy processing unit (As per is 10,500) to be used Every 6 months
Note: FSSAI manual of analysis for milk and milk products and any other appropriate method which includes, BIS test methods, AOAC test methods, FSSAI
approved rapid kit or test methods as applicable
Iit is only a hygiene indicator, # Desirable but the FBO could take decision on what best to be done for compliance and safety related to the marked parameter
Appendix 2
Appendix 2 341
Note: FSSAI Manual of Methods of Analysis for Milk and Milk Products and any other appropriate
method, which includes BIS test methods, AOAC test methods, FSSAI approved rapid kit or test
method as applicable
Format of the records to be maintained for STI by dairy unit(s)

Test Batch Action taken in
S. parameter Test Sampling no/silo no/ case of
No. Date as per STI method point tanker no Results noncompliance
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .

Kamal Gandhi, Rajan Sharma, Priyae Brath Gautam, Bimlesh Mann - Chemical Quality Assurance of Milk and Milk Products-Springer (2020)

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Kamal Gandhi, Rajan Sharma, Priyae Brath Gautam, Bimlesh Mann - Chemical Quality Assurance of Milk and Milk Products-Springer (2020)

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Kamal

Priyae Brath Gautam Bimlesh Mann

ISBN 978-981-15-4166-7 ISBN 978-981-15-4167-4 (eBook)

# Springer Nature Singapore Pte Ltd. 2020

Karnal, Haryana, India Kamal Gandhi

2.8 Sampling Procedure for Khoa . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.8 Detection of Adulterants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5 Quality Assessment of Milk Products . . . . . . . . . . . . . . . . . . . . . . . 85

5.6.6 Peroxide Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

7.5 Hardness Determination of Water . . . . . . . . . . . . . . . . . . . . . . . 181

8.7 Speciﬁcations for Sodium Thiosulfate (IS: 14781) . . . . . . . . . . . 230

9.18 Principles of HACCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

10.8 International Dairy Federation . . . . . . . . . . . . . . . . . . . . . . . . . 307

Appendix 1 FSSAI Standards for Milk and Milk Products . . . . . . . . . 313

Kamal Gandhi has been a Scientist at the Department of Dairy Chemistry,

Rajan Sharma is currently working as the Principal Scientist at the Department of

Fig. 7.1 Structures of different aﬂatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

Table 1.1 State wise milk production (MT) in India . .. . . .. . .. . . .. . .. . .. . . .. . 2

Table 3.5 Correction to be applied in the lactometer reading taken

Table 7.8 Classiﬁcation of pesticides based on the target pest . . . .. . . .. . .. . . 197

ADI Acceptable Daily Intake

COD Chemical Oxygen Demand

HILIC Hydrophilic Interaction Liquid Chromatography

RFL Referral Food Laboratories

1.1 Status of Milk Production and Consumption in India

# Springer Nature Singapore Pte Ltd. 2020 1

Table 1.1 State wise milk production (MT) in India

Table 1.3 (continued)

Table 1.4 Contribution of livestock and agriculture sector to GDP

1.2 Status of Milk Production and Consumption Throughout

2016–18 2028 Yield 2016–18(t/animal)

Processed dairy products Fresh dairy products

Table 1.6 Global dairy trade at a glance

1.3 Challenges Faced by Dairy Industry in Quality Assessment

# Springer Nature Singapore Pte Ltd. 2020 17

2.1 Scope of Sampling

2.2 Sampling Purpose

2.2.1 Samples Drawn for Regulatory Purposes

2.2.2 Samples Drawn for Monitoring Purpose at the Factory Level

2.3 Sampling Procedure

2.4 Collection of the Samples

2.5 Sampling Plan for Raw Milk

2.5.1 Sampling from an Individual Container

2.5.2 Sampling from Several Containers

2.5.3 Sampling from Bulk Units

Fig. 2.1 Plunger

2.6 Sampling Plan for Processed Milk

2.6.1 Sampling from a Storage Tank or Silo

2.6.2 Sampling of the Packed Milk

2.7 Sampling Procedure for Channa/Paneer/Cheese

The sampling of paneer/channa/cheese is generally done by one of the following

2.7.1 Sampling by Cutting a Sector

2.7.2 Sampling Using a Trier

2.7.3 Sampling by Taking the Whole Product

Fig. 2.2 Trier

2.7.4 Preparation of Paneer/Cheese/Channa Samples for Analysis

2.7.5 Sampling by Cutting Using a Knife with a Pointed Blade

Table 2.2 Sampling scale for cheese (IS: 2785)

2.7.6 Sampling Scale for Cheese

2.8 Sampling Procedure for Khoa

2.9 Sampling Procedure for Sterilized Milk/Flavored Milk

2.9.1 Scale of Sampling

The number of samples to be drawn should be as per the Table 2.3.

2.9.2 Preparation of Samples for Chemical Analysis