DETECTION OF ADULTERATION OF FAT IN MILK USING … · content. Therefore, the present project was undertaken to detect of adulteration of fat in milk using specially designed dual

DETECTION OF ADULTERATION OF FAT IN MILK USING SPECIALLY DESIGNED DUAL PURPOSE

GERBER BUTYROMETER

THESIS SUBMITTED TO THE

NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL

(DEEMED UNIVERSITY)

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE

MASTER OF TECHNOLOGY IN

DAIRY CHEMISTRY

BY

RATHOD GOPAL HARISHCHANDRA B.Tech (DT)

DAIRY CHEMISTRY DIVISION

NATIONAL DAIRY RESEARCH INSTITUTE (I.C. A. R.)

KARNAL-132001 (HARYANA), INDIA

2012

Regn. No. 2021001

Dedicated

To

My Beloved Parents

&My Guide

ACKNOWLEDGEMENT

I take this opportunity to express my profound sense of gratitude to my major advisor Dr. Darshan Lal, Principal Scientist in DC Division for his novel ideas, valuable advice, able guidance and encouragement during the course of this investigation. His faith in my knowledge and hard work made this work a challenging and enjoyable experience. Every niche of this investigation is a beautiful conglomeration of ideas, appreciation and amalgamation of rich concepts borrowed from the members of advisory committee Dr. Raman Seth, Dr. Vivek Sharma, Dr. S. K. Kanawjia and Dr. D. K. Thompkinson.

I feel honored while extending my gratefulness to Dr. A. K. Shrivastava, Director, NDRI, Dr. G. R. Patil, Joint Director (Academics) and Dr. S. L. Goswami, Joint Director (Research), NDRI for providing the necessary infrastructural facilities to conduct this study. I am deeply indebted to all the Scientists of the Dairy Chemistry Division, for their valuable suggestions, good wishes and cordial assistance during my study and research at NDRI.

I am immensely grateful to my lab mates Neelam mam, Anil sir, and Prashant for their valuable support during my entire research work. I would like to elicit the gesture of indebtedness to Nilu sir, Aakash bhai, Santosh sir, Rahul sir, Sonam mam, Meenakshi mam, Ankit sir, and Prabhakar Sir, for helping me affectionately from time to time with their kind and humble thoughts.

My deep sense of gratitude and warm regards to Jagatji, Kulvinderji, Balwantji, Ingleji, Rajivji, Yadavji, Deepakji and Shakuntala mam for their help and co-operation extended during the course of this study. I thank all my friends Samriddhi, Vaibhao, Somnath, Ambar, Vivek, Shashank, Prashant, Pranoti, Prachi, Snehal, Siddhartha,

Shreyash, and Seniors who always let me an empathetic ear and was more than willing to help me and the good times spent with all of them remain unforgettable.

I shall cherish my association and support rendered by my juniors Saurabh, Suvartan, Pankaj, Jagdish, Shailesh and Mukesh for their Unending support and moral boosting is always remembered.

My Aai, Baba, Jijaji & my big brother are the earthy Gods in my life who deserve much more than what I can weigh in words. Their silent prayers, aesthetic love and affection, support and steel belief in my capabilities have enabled me to make this endeavor a successful one. Support and love rendered by my sisters Megha, Sindhu, Pratibha and Seema can’t be forgotten.

Above all, I wish to acknowledge the almighty GOD without whose blessing; this would have never a success.

Date: June, 2011

Place: Karnal Gopal H. Rathod

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ABSTRACT

Milk is considered as a nutritionally complete food and it is a natural source that contains high quality nutrients needed for good health. A rapid growth in demand for milk and milk products has led to the adulteration of milk. Earlier, mostly ghee used to be adulterated with foreign oils and fats, but there are some reports of addition of foreign fats and oils directly into milk, as generally milk is sold on the basis of its fat content. Therefore, the present project was undertaken to detect of adulteration of fat in milk using specially designed dual purpose Gerber butyrometer, using three vegetable oils (groundnut, soybean and sunflower oil) and three body fats (goat, buffalo and sheep body fat) as adulterants. For this study, four tests such as Apparent Solidification Time (AST) test, Complete Liquefaction Time (CLT) test, Butyro–refractometer (BR) reading at 40°C and thin layer chromatography of unsaponifiable matter were planned to be undertaken. While standardizing the AST test to be applied on Gerber butyrometer fat column, it was observed that there were problems of very large variations in the AST values among the samples as well as among the butyrometers for the same sample. Therefore, this part of the study on AST test was not continued further for its application. During CLT test also large variations among the samples were observed. Moreover, it was observed that 70-90% of all the milk samples adulterated with the three vegetable oils and three body fats individually (@ 20% level on fat basis) were within the limits of CLT values of pure cow and buffalo milks and failed to be detected. Therefore, it was concluded that this test cannot be recommended to be used for screening the milk for milk fat purity. The results on the BR readings obtained by Gerber butyrometer method were found to be lower than those obtained by heat clarification method. Therefore, in order to directly get the correct BR reading using Gerber butyrometer method, a correction factor of 1.083 was developed. After correcting the BR reading of fat isolated from milk by Gerber butyrometer method, adulteration of cow milk with vegetable oils was detectable at all the adulteration levels studied, except groundnut oil and sunflower oil at 5% level of adulteration (on fat basis). However, adulteration of cow milk with animal body fats was not detectable below 20 % level of adulteration (on fat basis), except pig body fat which was detectable even at 15% level. Adulteration of buffalo milk with vegetable oils was not detectable below 20 % level of adulteration (on fat basis) except soy bean oil which was detectable even at 10% level. Adulteration of buffalo milk with animal body fats was not detectable at all the levels studied. TLC of unsaponifiable matter extracted from heat clarified fat obtained from pure buffalo milk and buffalo milk adulterated with vegetable oils (5 and 10%) revealed that on the basis of additional bands matching with the tocopherols, as low as 5% adulteration of milk (on fat basis) with soy bean oil and 10% adulteration of milk (on fat basis) with groundnut oil and sunflower oil could be detected easily. The study on the effect of addition of formalin on BR reading of milk fat revealed that there was not much change in the BR reading of the fat whether obtained by Gerber butyrometer method or heat clarification method for the pure as well as adulterated milk samples. When the formalin preserved milk samples were stored for three months, it was observed that there was no change in the BR reading of heat clarified fats. However, in case of Gerber butyrometer method, upto one month of storage no change in the BR reading was observed, but after two months of storage, difficulty in the fat isolation was encountered. Therefore, for formalin preserved milk samples application of Gerber butyrometer method for recording the BR reading of isolated fat cannot be recommended.

Chapter

No.

Title Page No.

1.0 INTRODUCTION 1 – 3

2.0 REVIEW OF LITERATURE 4-29

2.1 METHODS OF DETECTION OF ADULTERATION APPLICABLE TO MILK FAT

5

2.1.1 METHODS BASED ON PHYSICAL PROPERTIES

5

2.1.1.1 Melting Point 5

2.1.1.2 Apparent solidification time (AST) test 6

2.1.1.3 Complete liquefaction (CLT) test 7

2.1.1.4 Crystallization time test 8

2.1.1.5 Bomer value 9

2.1.1.6 Butyro Refractometer (BR) Reading 9

2.1.1.7 Opacity Test 10

2.1.1.8 Critical Temperature of Dissolution (CTD)

11

2.1.1.9 Fractionation of Milk Fat 11

2.1.1.10 Spectroscopic Methods 13

2.1.1.11 Dilatation Behavior 15

2.1.1.12 Microscopic Examination of Fat 16

2.1.1.12 Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC)

16

2.1.2 METHODS BASED ON CHEMICAL PROPERTIES

17

2.1.2.1 Tests based on fatty acids 17

2.1.2.2 Tests based on the nature and content of unsaponifiable constituents

20

2.1.2.3 Tests based on whole fat/triglycerides 22

2.1.3 METHODS BASED ON TRACER COMPONENTS OF FATS AND OILS

25

2.1.4 MISCELLANEOUS METHODS 26

2.1.4.1 Tests for mineral oils 26

2.1.4.2 Tests for cottonseed oils 26

2.1.4.3 Color based platform test for detection of vegetable oils/fats in ghee

27

2.2 METHODS OF DETECTION OF ADULTERATION APPLICABLE DIRECTLY TO MILK

28

CONTENTS

3.0 MATERIALS AND METHODS 30- 40

3.1 Collection of milk sample 30

3.2 Collection and Preparation of Adulterant Fats/ Oils 30

3.3 Preparation of Adulterated Milk Samples 31

3.4 Qualitative analysis of milk fat using specially designed dual purpose Gerber butyrometer

32

3.5 Extraction of fat from pure and adulterated milk samples using heat clarification method for determination of Butyro-Refractometer reading:

33

3.6 Apparent Solidification Time (AST) test 34

3.7 Complete Liquefaction Time (CLT) test 35

3.8 Determination of Butyro-Refractometer (BR) Reading at 40°C

36

3.9 Thin layer chromatography of unsaponifiable matter 37

3.10 Effect of addition of formalin to milk on BR reading of milk fat obtained by isolation form Gerber butyrometer and by heat clarification method

39

3.11 Effect of storage of formalin preserved milk samples on BR reading of milk fat obtained by isolation form Gerber butyrometer and by heat clarification method

39

4.0 RESULTS AND DISCUSSION 41-73

4.1 Apparent Solidification Time (AST) test 41

4.2 Complete liquefaction time (CLT) test 44

4.3 Butyro-refractometer (BR) readings of fat isolated from pure cow and buffalo milks by heat clarification method and Gerber butyrometer method

46

4.4 Development of a correction factor for converting BR reading of fat isolated by Gerber butyrometer method to BR reading of fat obtained by heat clarification method

50

4.5 BR readings of pure milk fats and adulterant oils and fats

52

4.6 BR Reading of Gerber butyrometer fat and heat clarified fat obtained from pure cow milk and cow milk adulterated with vegetable oils and animal body fats.

53

4.7 BR Reading of Gerber butyrometer fat and heat clarified fat obtained from pure buffalo milk and buffalo milk adulterated with vegetable oils and animal body fats

57

4.8 Thin layer chromatography of unsaponifiable matter extracted from milk fat and adulterant oils and fats

64

4.9 Effect of addition of formalin to milk on BR reading of milk fat obtained by isolation form Gerber butyrometer and by heat clarification method

68

4.10 Effect of storage (at 37°C) of formalin preserved milk samples on BR reading of milk fat obtained by isolation form Gerber butyrometer and by heat clarification method

71

5.0 SUMMARY AND CONCLUSION 74-76

6.0 BIBLIOGRAPHY i – x

Table

No. Title

Page

No.

4.1 AST value (min-sec) of fat column in butyrometer for cow milk

samples at different temperatures. 41

4.2 AST value (min-sec) of fat column in butyrometer for buffalo milk

samples at different temperatures 41

4.3 AST value (min-sec) of fat column in butyrometers of same make

for cow milk samples at 18°C 42

4.4 AST value (min-sec) of fat column in butyrometers of same make

for buffalo milk samples at 18°C 43

4.5 CLT value (sec) of fat column in butyrometer for pure and

adulterated cow milk samples at 41°C 44

4.6 CLT value (sec) of fat column in butyrometer for pure and

adulterated buffalo milk samples at 41°C 44

4.7 Butyro-refractometer (BR) readings of fat isolated from cow milk

by heat clarification method and Gerber butyrometer method. 45

4.8 Butyro-refractometer (BR) readings of fat isolated from buffalo milk

by heat clarification method and Gerber butyrometer method 47

4.9

Correction factor for converting BR reading of fat obtained by

Gerber butyrometer method to BR reading of fat obtained by heat

clarification method.

49

4.10 BR readings of pure milk fats and adulterant oils and fats 51

4.11

BR Reading of Gerber butyrometer fat and heat clarified fat

obtained from pure cow milk and cow milk adulterated with

vegetable oils.

53

4.12 BR Reading of butyrometer fat and heat clarified fat obtained from

pure cow milk and cow milk adulterated with animal body fats. 55

LIST OF TABLES

4.13 BR Reading of butyrometer fat and heat clarified fat obtained from

pure buffalo milk and buffalo milk adulterated with vegetable oils 57

4.14

BR Reading of butyrometer fat and heat clarified fat obtained from

pure buffalo milk and buffalo milk adulterated with animal body

fats.

59

4.15

Effect of addition of formalin to cow milk on BR reading of milk fat

obtained by isolation from Gerber butyrometer and by heat


68

4.16

Effect of addition of formalin to buffalo milk on BR reading of milk

fat obtained by isolation from Gerber butyrometer and by heat


69

4.17

Effect of storage (at 37°C) of formalin preserved cow milk samples

on BR reading of milk fat obtained by isolation from Gerber

butyrometer and by heat clarification method

71

4.18

Effect of storage (at 37°C) of formalin preserved buffalo milk

samples on BR reading of milk fat obtained by isolation form

Gerber butyrometer and by heat clarification method.

72

Fig.

No. Title Page No.

3.1 Modified Gerber butyrometer 31

4.1 TLC of unsaponifiable matter of pure milk fat and adulterants

along with standards. 64

4.2 TLC of unsaponifiable matter of pure milk fat and vegetable

oils along with standards

65

4.3 TLC of unsaponifiable matter of pure milk fat and 5%

adulterated milk fats along with standards 66

4.4

TLC of unsaponifiable matter of pure milk fat and 10% adulterated

milk fats along with standards 66

LIST OF FIGURES

AR Analytical Reagent

AST Apparent Solidification Time

ATR-MIR Attenuated Reflectance Mid Infra Red

BBF Buffalo Body Fat

BR Butyro-refractometer

CLT Complete Liquefaction Time

CTD Critical Temperature of Dissolution

DEGS Diethylene Glycol Succinate

DSC Differential Scanning Calorimetry

DTA Differential Thermal Analysis

EU European Union

FTIR Fourier Transform Infra-Red

GBF Goat Body Fat

GLC Gas Liquid Chromatography

GNO Groundnut Oil

GS3 Trisaturated Glycerides

HCl Hydrochloric Acid

I.D. Internal Diameter

IR Infra-Red

ISI Indian Standards Institution

KOH Potassium Hydroxide

L.R. Laboratory Reagent

NDDB National Dairy Development Board

O.D. Outer Diameter

PBF Pig Body Fat

PBMF Pure Buffalo Milk Fat

PFA Prevention of Food Adulteration Act

PUFA Poly Unsaturated Fatty Acids

RM Reichert Meissl

S.E. Standard Error

SBO Soyabean Oil

SFO Sunflower Oil

LIST OF ABBREVIATIONS AND SYMBOLS

STG Saturated Triglycerides

TLC Thin Layer Chromatography

USM Unsaponifiable matter

UV Ultraviolet

% Percent

Infinite

< Less than

> Greater than and equal to

°C Degree centigrade

µ Micron

µl Microlitre

µm Micrometer

b.p. Boiling Point

C4:0 fatty acid Fatty acid with 4 carbon and no double bond

cm Centimeter

Cm-1 Wave Number

Fig. Figure

g Gram

L18 Liquid fraction obtained at 18°C

mµ Milli micron

Min Minute

ml Millilitre

mm Millimeter

N Normal

nm Nanometer

Rf Resolution factor

v/v Volume by volume

w/v Weight by volume

CHAPTER - 1

__________________

Introduction

───────────────

1 Introduction

1. INTRODUCTION

Milk and honey are the only diets whose sole function in nature is food. Milk

has been a significant source as a food for humans since the dawn of history. It is

considered as a nutritionally complete food and is a natural source that contains

high quality nutrients needed for good health. The nutrients present in milk are

proteins, lipids, lactose, minerals and vitamins. All the constituents of milk have

their own importance in providing energy and promoting human health and

growth. The natural function of milk proteins is to supply young mammals with the

essential amino acids required for the development of muscular and other

protein-containing tissues, and with a number of biologically active proteins, e.g.

immunoglobulins, vitamin-binding, metal-binding proteins and various protein

hormones. Lipids, a concentrated form of energy also serves as a source of

essential fatty acids and fat soluble vitamins (A, D, E, K). Lactose aids in brain

development, while minerals are important for enzyme activity, bone formation,

osmoregulation. Milk also contains water soluble vitamins (B complex vitamins

and ascorbic acid) which perform vital functions such as energy transfer and

regulation of metabolic activities in the body.

Today, India is ‘The Oyster’ of the global dairy industry. From a milk deficient

country in the early 1960s, India has emerged today as the largest milk producer

in the world with an annual production of 121.8 million tonnes according the latest

estimates for 2010-2011 (www.nddb.org/statistics/milkproduction.html). Milk

production in India serves three main sectors, viz., household sector, un-

organized sector and organized sector. The first two sectors account over 80% of

milk produced in India and remaining 20% is handled by the organized sector.

Almost 50% of the total milk produced is consumed in the form of liquid milk and

45% is converted into traditional products like butter, ghee, paneer, khoa, curd,

malai etc., and only 5% of the milk goes into the production of western products

like milk powder, cheese etc. The Indian dairy industry is rapidly growing and

trying to keep pace with the galloping progress around the world.

A rapid growth in demand for milk and milk products has led to the

adulteration of milk. The menace of adulteration has reached to an alarming

stage in recent years. Adulterants like water, salt, sugar, urea, hydrogen

http://www.nddb.org/statistics/milkproduction.html

2 Introduction

peroxide, pond water, wheat flour, baking soda, formalin and different vegetable

oils and animal body fats are used by unsocial elements. Some adulterants are

used for increasing the volume of milk, while others are used for increasing the

shelf life of milk. Adulteration is mostly motivated by economic greed. Either a

more expensive ingredient is substituted with cheaper one, or a valued

component is partially or fully removed in the hope that the consumer does not

notice the difference. One of such components of milk is milk fat. Milk fat being

the costliest edible fat increasingly catches the attention of the fraudulent people

for an easy adulteration with far cheaper oils and fats of vegetable and animal

origin. By adulterating the milk fat with animal body fats, the unscrupulous traders

are not only robbing the people of their money, but also playing with the religious

sentiments, especially of the vegetarian section of the society. Earlier mostly

ghee used to be adulterated with foreign oils and fats and accordingly several

methods were developed for the detection of adulterants in ghee. But there are

some reports of addition of foreign fats and oils directly into milk, as generally

milk is sold on the basis of its fat content.

Milk adulteration adversely affects the consumer health and also the dairy

industry. Fraudulent malpractices create unfair competition. This leads to market

distortions, which in turn may impact the local or even the international trade.

Therefore, authentication of milk and milk products through quality testing is

important to both consumers as well as processors. Several tests have been

developed for detecting adulteration in milk and milk products such as ghee.

Unfortunately, the tests which are applicable for detecting adulteration in ghee

cannot be directly applied to milk because milk is not a single phase emulsion;

rather it is an oil- in-water type emulsion. Therefore, the fat phase of milk has to

be separated from its aqueous phase before applying any test for checking the

adulteration of fat. Keeping in view the need of rapid test which can be applied to

milk for detecting adulteration right at platform where milk is either accepted or

rejected, a novel approach was suggested by Lal et al (1998) which involves

isolation of fat from milk using specially designed dual purpose modified Gerber

butyrometer followed by determination of BR reading of isolated fat. This

approach was based on testing mustard oil added to milk (Arora et al, 1996).

Subsequently the same approach was used for detecting cottonseed oil added to

3 Introduction

milk (Boghra and Borkhatriya, 2004). However, there is a lack of information

regarding detection of other vegetable oils and body fats added to milk

Hence, keeping these aspects in mind, the present research work was

contemplated with the following objectives:

1) Detection of different vegetable oils (groundnut oil, soybean oil and

sunflower oil) added individually to milk at various levels.

2) Detection of different body fats (buffalo body fat, goat body fat and pig body

fat) added individually to milk at various levels.

CHAPTER - 2

─────────────────────────

Review of Literature ─────────────────────────

4 Review of literature

2. REVIEW OF LITERATURE

Lipids form one of the most important constituents of milk and milk

products. Major part of milk lipids consists of triglycerides (generally called fats).

Minor components of milk lipids include partial glycerides (mono- and di-

glycerides), phospholipids, fat soluble vitamins, cholesterol, squalene, waxes,

etc. Milk lipids play an important role in the economics, nutrition, flavour and

physico-chemical properties of milk and milk products. In most of the pricing of

milk and milk products, milk fat plays a significant role. It is also the richest

source of energy, carrier of fat soluble vitamins and essential fatty acids. It plays

an important role in the flavour problems which are closely related to body,

texture and consumer acceptability.

Role of milk fat in pricing of milk and milk products as well as its variable

chemical composition coupled with the intention of the traders to increase the

margin of profit, encourages the adulteration of milk fat with the far cheaper fats

and oils of vegetable and animal origin. The commonly used adulterants are

vegetable oils and fats, animal body fats, mineral oils, starchy material, etc.

Earlier mostly ghee used to be adulterated with foreign oils and fats and

accordingly several methods were developed for the detection of adulterants in

ghee. These methods were based on differences in the nature and contents of

major/minor components of ghee and adulterant fats/oils. Now-a-days, a new

trend of addition of foreign fats/oils directly into milk has been gaining

momentum, as generally milk is sold on the basis of its fat content. Unfortunately,

the tests, which are applicable for detecting adulteration in ghee, cannot be

directly applied to milk because milk is not a single-phase emulsion. Rather, it is

an oil-in-water type emulsion. Therefore, the fat phase of milk has to be

separated from its aqueous phase before applying any test for checking the

adulteration of milk fat.

This review delineates the current status of our knowledge with regard to

the various methods commonly used for the detection of adulteration of milk fat

with foreign fats. Most of the methods available are applicable to ghee, whereas


very few are applicable to milk. Therefore, the literature in this respect is

reviewed under the following two heads:

1) Methods of detection of adulteration applicable to milk fat

2) Methods of detection of adulteration applicable directly to milk

2.1 METHODS OF DETECTION OF ADULTERATION APPLICABLE TO MILK

FAT

The information under this head is divided into following four sub-heads: -

1) Methods based on physical properties, 2) Methods based on chemical

properties, 3) Methods based on tracer components of fats and oils, and 4)

Miscellaneous methods.

2.1.1 METHODS BASED ON PHYSICAL PROPERTIES

Physical properties of oils and fats are important criteria for judging their

quality and have also been used to determine their authenticity. Several methods,

which were used to check the authenticity of ghee on the basis of physical

properties, are as follows.

2.1.1.1 Melting Point

Melting point (slip point) of various oils and fats varies over a wide range

and this property has been employed for checking the adulteration of milk fat.

Body fats (36-51.3°C) and vanaspati (37.8-38°C) have slightly higher melting

point (Winton and Winton, 1999) while vegetable oils (20-30°C) have slightly

lower melting point than milk fat (28-41°C) as reviewed by Kumar et al. (2002).

Singhal (1973) reported that buffalo milk fat (33.4-34.2°C) has slightly higher

melting point than cow milk fat (30.6-31.2°C) and ghee from cotton tract area

showed considerably higher melting point (43.0-44.0°C) which resembled with


that of animal body fats (43.9-51.0°C). The study, however, concluded that

adulteration up to 20 percent level did not introduce significant changes in the

melting point of ghee and, therefore, the method was not found useful for the

detection of adulteration. Sharma and Singhal (1995) also confirmed these

observations using body fats and vanaspati.

2.1.1.2 Apparent solidification time (AST) test

The AST test was developed by Kumar et al. (2009a) to detect adulteration

of milk fat with vegetable oils and animal body fats. The apparent solidification

time is determined by taking three grams of melted fat sample in test tube (10.0 X

1.0 internal diameter) to apparently become solidified at 18°C.

Studies conducted by Kumar et al. (2009a, 2010) on the solidification

behavior of various oils and fats including milk fat in terms of AST at the selected

temperature (18°C) have revealed that the average AST values for buffalo and

cow milk fat were 2 min 40 sec and 3 min 10 sec, respectively. The average AST

values of pig body fat, goat body fat and vanaspati were 1 min 30 sec, 40 sec

and 1 min 50 sec, respectively. On the other hand, all the vegetable oils studied

remained liquid for an indefinite period. Addition of vegetable oils caused an

increase in the AST values of buffalo and cow pure milk fat, whereas the addition

of body fats and vanaspati (hydrogenated vegetable oils) resulted in the decrease

in the AST values of buffalo and cow pure milk fat, depending on the amount of

adulterant oils and fats added. Taking into account the overall range of AST

values at 18°C pertaining to fresh, stored and seasonal samples of both buffalo

and cow pure milk fat as the criteria for the detection of adulteration, it was found

that the technique could detect the addition of individual vegetable oils at all the

levels in case of cow milk fat but not in buffalo milk fat. Adulteration of buffalo milk

fat with vanaspati (hydrogenated vegetable fat) was detectable at levels ≥10%,

but not in cow milk fat. Addition of goat body fat to both cow and buffalo milk fat

was detectable at levels ≥ 10%, whereas pig body fat was detectable only in

buffalo milk fat at levels ≥ 10%.


2.1.1.3 Complete liquefaction time (CLT) test

This test was developed by Amit Kumar (2008) to detect adulteration of

milk fat with foreign fats and oils. The complete liquefaction time (CLT) of the fat

samples was recorded by observing the time taken by the solidified fat samples

to get melted completely at a 44oC.

At 44oC, the CLT values of pure cow ghee samples ranged from 2 min 12

sec to 3 min 15 sec with the mean of 2 min 52 sec, while that of pure buffalo

ghee ranged from 2 min 35 sec to 3 min 15 sec with the mean of 2 min 57 sec.

There were large variation between CLT values of ghee samples collected over a

period of whole year both for cows and buffaloes. Further, it was observed that

both (cow and buffalo) type of ghee samples showed higher CLT values in the

summer months (May to September) and lower CLT values in the winter months

(November to March).

Addition of vegetable oils (palm, rice bran and soybean) caused a

decrease, whereas addition of body fats (buffalo, goat and pig) resulted in an

increase in the CLT values of cow and buffalo pure ghee at 44°C. This decrease

or increase observed in CLT values caused by the addition of adulterant oils/ fats

to ghee depended upon the amount of adulterants added. Higher the quantity of

adulterant added, greater was the effect. However, in case of ghee samples

containing body fats, samples with pig body fat showed slightly lower increase in

CLT values than the samples containing buffalo and goat body fats.

Taking into account the overall range of CLT values at 44oC, i.e. from 2

min 12 sec to 3 min 15 sec for pure ghee (cow and buffalo) as the criteria for the

detection of adulteration, a perusal of the results on CLT values of adulterated

ghee samples revealed that addition of vegetable oils individually at the levels of

5, 10 and 15 percent to either cow ghee or buffalo ghee was not detectable.

However, among animal body fats, buffalo and goat body fats in either of the


ghee were detectable at 10% level while pig body fat was detected only at higher

(15%) level of adulteration.

2.1.1.4 Crystallization time test

The Crystallization time test is defined as the time taken for the onset of

crystallization of milk fat at 170C when dissolved in a solvent mixture consisting of

Acetone and Benzene (3.5:1.0). Panda and Bindal (1998b) studied the

crystallization behavior at 17°C of fat dissolved in a solvent mixture of acetone

and benzene (3.5:1) and reported that ghee, ghee adulterated with body fats

(10%), and ghee adulterated with vegetable oils and fats (10% level) took 19 min,

3 to 15 min and 22 to 23 min, respectively to crystallize. They concluded from the

study that even low level adulteration of animal body fats and vegetable oils and

fats could be detected in ghee.

Kumar (2008) applied this test on samples of both cow and buffalo pure

ghee pertaining to whole of the year collected on bimonthly basis, as well as the

ghee samples added with adulterants (animal body fats and vegetable oils)

individually at 5, 10 and 15% levels. Crystallization time for the pure cow ghee

and pure buffalo ghee samples ranged from 6 min 50 s to 16 min 20 s and from 6

min 30s to 12 min 30 s with the mean of 11 min 4s and 8 min 42 s, respectively.

The crystallization time of ghee samples increased when the samples were

adulterated with vegetable oils (palm, rice bran and soybean) while it was found

to decrease for ghee samples adulterated with animal (buffalo and goat) body

fats. The extent of increase and decrease was dependent on the level of

adulteration with the vegetable oil and animal body fat, respectively. Higher the

level of adulteration, greater was the effect. The crystallization time was highest

in the month of May while it was lowest in July. Generally, crystallization time is

expected to be higher if the fat has more unsaturated fatty acid i.e. crystallization

time will be increased with the increase in BR reading and iodine value of the fat.

They concluded that the crystallization time test is a useful tool to detect addition

of animal body fats particularly, buffalo and goat body fat to milk fat at 5% level.


However, pig body fat could not be detected by this test when added to milk fat at

any of the levels studied. Using the same test, recently Sofia et al. (2010) also

observed that crystallization time of ghee adulterated with coconut oil @ 5% or

palm oil @ 10% was higher (20-34 min) than that of pure cow ghee (16-20 min)

and can be employed for detection.

2.1.1.5 Bomer Value

Bomer value is defined as the sum of the melting point of saturated

triglycerides (isolated by diethyl ether method) and twice the difference between

this melting point and that of the fatty acids obtained after the saponification of

these triglycerides. The Bomer value of both cow and buffalo ghee ranges from

63 to 64, whereas those of animal body fats, e.g., goat, sheep and buffalo ranges

from 68 to 69 and that of pig body fat from 75 to 76. Singhal (1980, 1987)

reported that the Bomer value of ghee increased on adulteration with body fats

even in the presence of vegetable oils, but not when vegetable oils alone were

added. The method could be used as a confirmatory test for the detection of pig

body fat in ghee. However, genuine cotton tract ghee which behaved similar to

adulterated ghee samples could not be sorted out by this test and hence may be

mistaken as adulterated ghee. Sharma and Singhal (1996) also successfully

applied this test for the detection of body fats (buffalo, goat and pig) and

vanaspati added to buffalo ghee at 20 percent level, irrespective of mode of

adulteration either directly to ghee or through milk.

2.1.1.6 Butyro Refractometer (B.R.) Reading

The values for B.R. readings of milk fat (40-45) and vegetable oils and fats

(above 50) are so wide apart (Singhal, 1980; Gunstone et al., 1994) that this

property could be safely employed as an index for milk fat adulteration with

vegetable oils and fats, except coconut oil (35-39) and palm oil (39-40). Feeding

of cottonseed oil raises the B.R. by 5 units in case of ghee (Rangappa and

Achaya, 1974). The B.R. readings of animal body fats are in the range of 44 to 51


(Singhal, 1980). Adulteration of milk fat with animal body fats (Singhal, 1973;

Sharma and Singhal, 1995) and vanaspati (Sharma and Singhal, 1995) at a level

of 5 to 20 percent increased its B.R. readings. Recently, some workers (Arora et

al., 1996; Lal et al., 1998) have developed a simple platform test for the detection

of vegetable oil (refined mustard oil) added to milk at a level higher than 10

percent of the original fat on the basis of increase in B.R. reading of the fat. Arun

Kumar (2003) reported that using general limit of B.R. reading as 40-43,

adulteration of vegetable oil up to 5% in cow ghee and 15% in buffalo ghee can

be detected. Amit Kumar (2008) noticed that the adulteration of ghee with animal

body fat up to 15% level studied could not be detected by using B.R. reading.

2.1.1.7 Opacity Test

Singhal (1980) developed an opacity test to detect the adulteration of ghee

with animal body fats, based on the time taken by the melted fat sample to

become opaque (O.D. 0.5) at 23°C using 590 nm (yellow) filter and observed

that the normal ghee took more than 35 minutes, whereas animal body fats

(buffalo, goat and sheep) took only 10 to 20 seconds to become opaque. He

concluded that the adulteration with buffalo, goat and sheep body fats at 5

percent level and above could be safely detected by opacity test. However, the

limitations of this test are that the detection of pig body fat up to 10 percent level

is difficult and ghee from cotton tract area also cannot be distinguished. Test also

fails to detect the body fats in ghee in the presence of vegetable oils (Singhal,

1987). Sharma and Singhal (1996) carried out the opacity test at 25°C for pure

fats (ghee, body fats and vanaspati) and adulterated ghee samples (5, 10 and

20% level) and observed almost the similar results as reported earlier by Singhal

(1980). Panda and Bindal (1998a) also studied the opacity profile of pure ghee

and adulterants using the opacity test given by Singhal (1980), but with certain

modifications. They recorded the opacity time as the time required by a fat

sample at 23°C to acquire the O.D. in the range of 0.14 to 0.16 and consequent

transmittance of 68 to 72 showing the onset of solidification of fat. They reported

that the opacity time of pure ghee (14-15 min) was much higher than that of ghee

adulterated with animal body fats (2-9 min at 10% level and 3-11 min at 5% level


of adulteration) and much lower than that of ghee adulterated with vegetable oils

(21-25 min at 10% level and 19-21 min at 5% level of adulteration).

2.1.1.8 Critical Temperature of Dissolution (CTD)

Critical temperature of dissolution (temperature at which turbidity appears

on gradual cooling of the fat dissolved in a warm solvent or solvent mixture) is a

characteristic of a particular fat (Rangappa and Achaya, 1974; Boghra et al.,

1981). Bhide and Kane (1952) observed the CTD values for ghee and vanaspati

in the range of 39 to 45°C and 62 to 72°C, respectively, employing a 2:1 (v/v)

mixture of 95 percent ethanol and iso-amyl alcohol, and reported that gross

adulteration of ghee with vanaspati could easily be detected. Similarly, the

presence of body fats in ghee was detected by employing either a single solvent

such as absolute alcohol (Delforno, 1964) or a solvent mixture of 95 percent ethyl

alcohol and iso-amyl alcohol in 2:1 (Bhide and Kane, 1952). Likewise, CTD was

used for distinguishing oleo margarine from butter by Felman and Lepper (1950),

using 95 percent ethyl alcohol and iso-amyl alcohol (2:1) as solvent and detection

of mineral oils in milk fat by Kane and Ranadive (1951) using aniline. The CTD

test seems to be simple, but its efficacy is greatly affected by the free acidity

(FFA) and rancidity (peroxides). Arun kumar (2003), using solvent mixture (ethyl

alcohol and iso-amyl alcohol in 2:1), reported that adulteration of ghee up to 15%

level with vegetable oils, vanaspati and body fats could not be detected by CTD

value.

2.1.1.9 Fractionation of Milk Fat

Fractionation of fat with or without the use of solvent under suitable

conditions of time and temperature combinations, followed by examination of

fractions thus obtained has been exploited by some workers as a tool to detect

foreign fats in milk fat. Different solvents that have been used for fractionation

purpose include ethyl alcohol, acetone, hexane, isopropyl alcohol and 2-nitro

propane. Bhalerao and Kummerow (1954) separated the fat into solid (30%) and


liquid (70%) fractions after dissolving it in the hot absolute alcohol and

maintaining the same at 20°C for 2 hours. The insoluble fraction was further

fractionated using acetone at 0°C and keeping it overnight in order to increase

the concentration of adulterant in one of these fractions. The acetone soluble

fraction was iodinated and subsequently subjected to refractive index

measurement. Using this method, the presence of foreign fats at 10 percent level

could be detected. Arumughan and Narayanan (1979) fractionated the buffalo

and cow ghee at 29°C/3 days and reported that solid fraction of ghee differed

from the liquid fraction and from whole ghee in physico-chemical characteristics

and fatty acid composition. Similar observation was made by Arora and Rai

(1997) on goat ghee. Farag et al. (1983) carried out the fractional crystallization

of fat (pure and adulterated ghee samples) dissolved in silver nitrate-saturated

methanol/ acetone (70:30) in a ratio of 1:10 at 22, 7 and –8°C and the three

fractions obtained were subjected to GLC for their fatty acid profile. They reported

that for detecting adulteration, 18:0 and 18:1 fatty acids are of great importance in

first fraction, where as 22:0 is important in second and third fraction.

Arun Kumar (2003) reported that fractionation of milk fat into solid and

liquid fraction did not offer any extra help using BR. reading. However, using

apparent solidification time (AST) test, fractionation technique could extend help

in the detection of adulteration of cow ghee especially with the mixtures of goat

body fat with vegetable oils even at 5% level, which otherwise was not possible in

the un-fractionated ghee samples. According to Amit Kumar (2008), solvent

fractionation offered help in lowering the detection limit of adulteration in terms of

iodine value of last liquid fraction. He also reported that complete liquification time

(CLT) test can help considerably to detect the adulteration in ghee especially at

44°C without fractionation. However fractionation technique has offered further

advantage in lowering the detection limits particularly when the CLT is done at

46°C which otherwise could not be possible without fractionation, when mixture

(body fats and vegetable oils) is added.


2.1.1.10 Spectroscopic Methods

Spectroscopic methods using visible (400-800 mµ), ultraviolet (200-400

mµ) and infrared (2-15 µ) regions have been used by many workers for

characterizing fats and oils.

2.1.1.10.1 Tests Based on Visible Spectroscopy

Jha (1981) applied this technique for the detection of Cheuri (Madhuca

butyracea) fat in ghee, a common adulterant in Nepal. Pure ghee showed no

absorption band in visible range (600-700 nm), whereas Cheuri fat showed an

absorption band with maxima between 640 and 680 nm. Even 5 percent Cheuri

fat content added to ghee could be detected in this range.

2.1.1.10.2 Tests Based on Ultraviolet (UV) Spectroscopy

UV absorption spectroscopy has been applied for characterizing the

various oils and fats including milk fat. Singhal (1973) and Sharma (1989)

scanned the UV spectra of cow ghee, buffalo ghee and animal body fats (buffalo,

goat, pig and sheep) between 200 to 320 nm after dissolving the fats in n-hexane

and observed a maximum absorption between 220 to 230 nm. However, cow

ghee showed another small maximum at 270 nm. These workers also examined

the UV spectrum of unsaponifiable matter extracted from ghee and animal body

fats between 200 to 320 nm and observed an absorption maxima between 215 to

220 nm. The ghee samples showed a second maxima at 270 nm, which was

shifted to 260 nm (Singhal, 1973) or to 280 nm (Sharma, 1989) in case of animal

body fats. However on this basis, adulterated ghee could not be differentiated

from pure ghee (Sharma, 1989; Kumar, et al., 2010)


2.1.1.10.3 Tests Based on Infra-Red (IR) Spectroscopy

Infra-red absorption has been extensively used in the analysis of lipids

especially for cis- and trans- isomers. Unsaturated fatty acids of natural vegetable

oils and fats are in cis- configuration and are isolated (non-conjugated). Partial

hydrogenation or oxidation may result into formation of trans-isomers. Animal and

marine fats may also contain small amounts of natural trans-isomers (Akoh and

Min, 1998; Kirk and Sawyer, 1999). Bovine milk fat contains a low level (5%) of

trans fatty acids in comparison with hydrogenated vegetable oils, in which the

value may be as high as 50 percent due to non-stereospecific hydrogenation

(Fox and McSweeney, 1998). For demonstrating the presence of hydrogenated

fats in milk fat, some workers (Bartlett and Chapman, 1961) applied IR

spectrophotometry and observed that the absorption maxima at 10.36 µ gets

increased by the addition of hydrogenated fats containing iso-oleic acids (trans-

octadecenoic acids). Arun kumar (2003) reported that on the basis of increased

level of trans isomers in ghee, as low as 5% of vanaspati added to ghee could be

detected.

Konevets et al. (1987) studied the cis-trans configurations of individual fats

(milk fat, animal body fat, vegetable fat and hydrogenated fat) and their mixtures

using IR spectroscopy, and reported that the additions up to 10 percent of animal,

vegetable and hydrogenated fats to milk fat could be detected. Sato et al. (1990)

used near IR spectroscopic method for the detection of as little as 3 percent

foreign fat in milk fat. Sharma (1989) scanned the IR spectra of cow ghee, buffalo

ghee, animal body fats (Buffalo, goat, sheep and pig) and ghee adulterated with

body fats in the 4,000 to 600 cm-1 region and observed distinct differences

between body fats and ghee in the region of 1300 to 1180 cm-1 and 1120 to 1100

cm-1, respectively. Body fats showed the presence of 5 to 6 bands, while ghee

showed only two bands. Ghee samples adulterated with body fats also showed 3

to 6 extra bands. Unsaponifiable matter extracted from the ghee, body fats and

adulterated ghee samples also exhibited the similar pattern of bands as reported

above for the whole fat. He concluded that the differences in IR spectrum of ghee


and body fats could be used to detect ghee adulteration with body fats at 10

percent level.

Koca et al., (2010) studied temperature-controlled attenuated total

reflectance-mid-infrared (ATR-MIR) spectroscopy combined with multivariate

analysis as a simple and rapid method for the determination of butter adulteration

as a dairy food system. Commercial samples of butter fat were adulterated with

margarine fat at levels ranging from 0% to 100% (v/v). Partial least square

regression (PLSR) models gave standard error of cross-validation (SECV) of

<1.2% (v/v) and correlation coefficients (r) > 0.99. Excellent predicting capabilities

were obtained using an external validation set consisting of butter adulterated

with margarines at ratios of 2.5%, 13%, and 45%. Additionally, infrared

spectroscopy provided distinctive bands that allowed discrimination of butter and

margarine samples by forming well separated clusters for the different products

evaluated.

2.1.1.11 Dilatation Behavior

This property is based on the thermal expansion behavior of milk fat.

Using this property, Kalsi (1984) reported different solid and liquid fractions of

milk fat from different species in the temperature range of 10 to 80°C and

observed that solid and liquid fractions in equal proportions are obtained at 33, 30

and 24.5°C in case of buffalo, cow and goat milk fats, respectively. Kumar, et al.

(2007) applied this method for detection of adulteration in ghee. He studied the

proportion of solid and liquid fractions of pure and adulterated ghee at 5, 10 and

15% level and observed that the ratio of solid to liquid fraction for pure cow and

buffalo ghee was 2.37 and 3.10 respectively. On the basis of solid/liquid ratio, it

was found that vegetable oils could be detected in cow ghee while body fats and

vanaspati could be detected in buffalo ghee even at 5% level.


2.1.1.12 Microscopic Examination of Fat

Microscopic examination of the sterol crystals (Den Herder, 1955; IDF,

1965; BIS, 1981) has also been employed in the detection of adulteration of milk

fat with the foreign fats especially vegetable fats. If the sterol crystals only show

the form of a parallelogram with an obtuse angle of 100°, which is characteristic

for cholesterol, the fat sample is considered to be free from vegetable fat.

However, if the sterol crystals show the elongated hexagonal form with an apical

angle of 108°, which is characteristic for phytosterols, or if some of the sterol

crystals have a re-entry angle (Swallow’s tail), which is characteristic for mixtures

of cholesterol and phytosterols, the fat sample is considered to contain vegetable

fat. Arun kumar (2003) reported that adulteration of ghee samples with 15%

groundnut oil could be confirmed using this parameter.

2.1.1.13 Differential Thermal Analysis (DTA) and Differential Scanning

Calorimetry (DSC)

DTA and DSC are both closely related thermo-analytical techniques which

measure the physical properties such as phase transition and specific heats of

foods as a function of temperature. DTA measures the difference in temperature

(t) between a sample and an inert reference material as a function of

temperature. In DSC thermograms, the area delineated by the output curve is

directly proportional to the total amount of energy transferred in or out of the

sample.

Patel and Frede (1991) observed that the crystallization and melting

behaviour of buffalo milk fat was perceptibly different from that of cow milk fat, the

former generally beginning to solidify and melt at higher temperature, exhibiting a

higher solid fat content at a particular temperature in the melting range using

DSC technique. DTA and DSC have been used by different investigators for the

detection of foreign fats in milk fat. Antila et al. (1965) detected 5 percent coconut

fat, cocoa fat and hardened vegetable fat in butterfat by DSC based on the


differences in the shape of melting curves. However, tallow, lard and vegetable

oil added at 5 percent level in butterfat could not be detected by this method.

Roos and Tuinstra (1969) using DTA showed that addition of 5 to 10 percent of

beef tallow in butterfat changed the solidification curve as solidification started

earlier and showed two distinct minima in the curve. Lambelet et al. (1980)

detected goat body fat (more than 10%) in ghee by DTA technique on the basis

of differences in melting diagram and crystallization patterns of goat body fat and

ghee. Using DSC, detection of foreign fats like pig and buffalo body fats

(Lambelet and Ganguly, 1983) beef suet (Amelotti et al., 1983) and chicken fat

(Coni et al., 1994) in milk fat was reported. The method, however, failed to detect

coconut oil, cotton tract ghee and other animal body fats.

Aktas and Kaya (2001) carried out a study in which differential scanning

calorimetry (DSC) melting and crystallization curves of butterfat, beef body fat

(BBF) and margarine were formed by cooling gradually from 70 to 40°C. Then

margarine and BBF were added to butterfat at the rates of 5, 10 and 20% in order

to investigate their curves. When BBF or margarine was added to butterfat, 1.

and 2. peak areas increased in crystallization curves of butterfat with 2. peak

being more discernible. Results obtained show that DSC technique could be

used in order to determine adulteration of butterfat.

2.1.2 METHODS BASED ON CHEMICAL PROPERTIES

Several chemical methods based on fatty acids, triglycerides,

unsaponifiable matter, and specific tests using GLC, TLC, paper

chromatography, etc. have been used to characterize the various fats and oils

with a view to check the purity of milk fat.

2.1.2.1 TESTS BASED ON FATTY ACIDS

Before the advent of modern analytical techniques, like, GLC, TLC, paper

chromatography, etc., physico-chemical constants such as Reichert-Meissl,


Polenske, iodine, saponification values and BR reading were used as a measure

of fatty acids. However, these constants give information about the groups of

acids rather than individual fatty acids. Based on the differences in the fatty acids,

either as a group or individually, several tests have been developed for detecting

adulteration of milk fat with foreign fats, which are described below:

2.1.2.1.1 Tests Based on Physico-Chemical Constants

Several earlier workers (Achaya and Banerjee, 1946; Karim, 1953; Murthy,

1955; Ali and Tremazi, 1966; Velu, 1971) have reported that physico-chemical

constants failed to detect the adulteration of milk fat with beef tallow, refined

cottonseed oil, hydrogenated oils and even coconut oil separately or in mixture

even up to 10 percent level. Singhal (1973, 1980) also employed the physico-

chemical constants for detecting the animal body fats (buffalo, goat, sheep and

pig) added to buffalo and cow ghee and reported that the values for Reichert-

Meissl, Polenske, and BR indices remained within the legal limits for normal

ghee, when adulterated with animal body fats at 20 percent level. However, when

adulteration was done at 50 percent level, the values remained within the legal

limits set for cotton tract ghee. Sharma and Singhal (1995) also confirmed the

above findings using body fats (buffalo, goat and pig) and vanaspati ghee.

According to Arun Kumar (2003), by taking range of iodine values for buffalo and

cow ghee between 30.60 to 34.30, the detection of adulteration of cow ghee with

vegetable oils or vanaspati or body fats added individually or in combinations at

10 percent and above levels was possible. However in case of buffalo ghee, it

has not offered much help.

2.1.2.1.2 Tests Based on Gas Liquid Chromatography (GLC) of Fatty Acids

Milk fat derived from ruminant animals, contains an exceptional number

and variety of fatty acids from 4:0 to 26:0 (saturated) and from 10:1 to 22:5

(unsaturated). Body fats like tallow and lard contain mostly palmitic (16:0), stearic

(18:0) and oleic acid (18:1), while vegetable oils consist mainly of palmitic,


stearic, oleic and linoleic (18:2) acids. Coconut oil is the best known exception,

containing lauric (12:0) and myristic (14:0) acids in very large amount (Rangappa

and Achaya, 1974).

Some workers (Wolff, 1960; Francesco and Avancini, 1961; Boniforti,

1962) employed GLC technique and reported that the milk fat sample with a ratio

of C12:0 / C10:0 fatty acids >1.6 or C4 / C6+C8 fatty acids >1.8 was considered

to be adulterated with margarine, coconut oil or tallow or pig body fat trans-

esterified with butyric acid. Similarly, many other workers used the different fatty

acids ratios for checking the adulteration of milk fat with vegetable oils,

margarine, beef tallow, lard, goat body fat, substituted fats, synthetic fats, etc.

(Toppino et al., 1980; Ulberth, 1994; Sharma and Singhal, 1996; Panda and

Bindal, 1997; Arun kumar, 2003). Farag et al. (1983) determined the fatty acid

profile of 3 fractions separated by fractional crystallization from cow and buffalo

ghee adulterated with lard and margarine at various levels and reported that the

amounts of 16:0, 18:0 and 18:1 acids were significantly changed with different

adulteration levels and can be used as a marker to detect the admixture. Sharma

and Singhal (1996) analyzed the buffalo ghee samples adulterated with body fats

(buffalo, goat, pig) and vanaspati at 20 percent level and noted that short and

medium chain fatty acids decreased while long chain fatty acids increased on

adulteration. Panda and Bindal (1997) employed this technique for the detection

of adulteration in ghee with vegetable oils at level as low as 5 percent using

C18:2 or C22:1 as marker acid. Arun kumar (2003) found that the adulteration of

ghee with body fats or vanaspati at 15 percent level and above could be detected

using different fatty acid ratios like C14:0 / C16:0 and C18:0 / C18:1 whereas,

presence of even 5 percent vegetable oils in ghee could be detected easily, using

fatty acid ratios such as C14:0 / C18:2, C16:0 / C18:2, C18:0 / C18:2, as well as

linoleic acid (C18:2) as a marker fatty acid.


2.1.2.2 TESTS BASED ON THE NATURE AND CONTENT OF

UNSAPONIFIABLE CONSTITUENTS

The unsaponifiable matter (USM) which constitutes less than 2 percent by

weight of fat is a repository of so many valuable constituents, like, sterols

(cholesterol and phytosterols), fat soluble vitamins (A, D, E, K), hydrocarbons

such as squalene, pigments, etc. Mineral oil, if added to oils and fats, will appear

in USM (Kirk and Sawyer, 1999).

Sterols and tocopherols are the two most important constituents of USM,

which have been used to detect the vegetable fats in milk fat by using various

techniques like GLC, TLC, paper chromatography, etc.

2.1.2.2.1 Tests Based on Sterols

Sterols which represent maximum share of the USM range from 0.24 to

0.5 percent in butterfat, 0.03 to 0.14 percent in body fats and 0.03 to 0.5 percent

in vegetable oils (Arun Kumar, 2003). Cholesterol is the characteristic sterol of

animal fats, while sterols from plant sources consist of a mixture collectively

called as phytosterols and include ß-sitosterol, stigmasterol, campesterol,

brassicasterol, etc. Low concentration of cholesterol is also reported in the sterol

fractions of vegetable oils and fats (Kirk and Sawyer, 1999). In addition to

cholesterol, milk fat contains traces of lanosterol, dihydrolanosterol and ß-

sitosterol (Webb et al., 1987). The sterols can help to distinguish between fats of

animal and vegetable origin, since the melting point of cholesterol acetate

(112.76-116.40°C) is substantially lower than that of the acetates of any of the

phytosterols (126-137°C). Adulteration of milk fat with vegetable oils is confirmed

when melting point is more than 117°C (IDF, 1965; BIS, 1981).

A circular paper chromatographic method based on the difference in the

behaviour of USM isolated from fats in ghee using a solvent mixture of methyl

alcohol: petroleum ether: water (80:10:10; v/v) was developed by Ramachandra


and Dastur (1959) who reported that the spot of USM of ghee moved as a whole

along with the solvent front, while that of ghee adulterated with animal body fats

at 5 percent level or vanaspati at 10 percent level did not move at all. Sharma

(1989) applied paper chromatography to the USM of ghee, body fats and their

mixture, and observed that USM from ether soluble fractions of ghee adulterated

with 10 percent buffalo body fat exhibited two spots. However, pig body fat in

ghee could not be detected.

Ramamurthy et al. (1967) using thin layers of CaCO3 and soluble starch

(10 g + 4 g) impregnated with liquid paraffin and a solvent system consisting of

methanol:acetic acid:water (20:5:1; v/v) as a developer reported that the

presence of cottonseed oil, groundnut oil, sesame oil and hydrogenated fats at 10

to 13 percent level and coconut oil at 25 percent level in ghee could be detected

on the basis of Rf values of 0.53 and 0.44 for cholesterol and phytosterols,

respectively. Sharma (1989) carried out TLC of USM of ghee and animal body

fats using hexane : ether : glacial acetic acid : ethyl alcohol (25:20:5:1, v/v) as the

solvent system and reported that ghee samples adulterated with 10 percent body

fats resulted in the appearance of an extra spot due to dihydrocholesterol present

in body fats. Recently Kumar et al (2005a) also studied the TLC profile of USM

and reported the detection of groundnut oil and vanaspati in ghee on the basis of

appearance of additional bands in case of vegetable fat and their absence in milk

fat.

Using GLC technique, ß-sitosterol has been shown to be an index of

vegetable fat addition (Homberg and Bielefeld, 1979), however, by this method,

addition of body fats cannot be detected as body fats also have cholesterol.

2.1.2.2.2 Tests Based on Tocopherol

Vitamin E derivatives, consisting of 4 tocopherol and 4 tocotrienol isomers,

each designated as alpha, beta, gamma and delta on the basis of chromanol

ring, are now collectively known as tocochromanols (earlier commonly referred to


as tocopherols). The tocopherols have a saturated side chain, whereas

tocotrienols have an unsaturated side chain. Generally, seed oils are rich sources

of tocopherols whereas the tocotrienols are found predominantly in palm oil and

cereal oils such as barley and rice bran oil. They are the important constituents of

unsaponifiable matter of natural oils and fats which range from 0.002 to 0.005

percent in butterfat, 0.05 to 0.168 percent in vegetable oils and 0.0005 to 0.0029

percent in body fats (Kumar et al, 2002). Thus, tocopherol content of butterfat is

low as compared to most vegetable oils and fats, with the exception of coconut oil

(0.0083%). Therefore, addition of vegetable fats to butter will result in a

significant increase in tocopherol content of adulterated butterfat. Accordingly,

some workers (Keeney et al., 1971) have reported that vegetable fats and oils

added to ghee could be detected on the basis of tocopherol content. However,

body fats and coconut oil added to milk fat could not be detected.

Cow milk contains exclusively α-tocopherol while human milk has 75% α-

tocopherol. The dominance of α-tocopherol is probably because of the fact that

mammals selectively absorb and deposit α-tocopherol in their tissues (Webb et

al, 1987 & Fox, 1995). However, Amit Kumar (2008) using HPLC analysis of

tocopherol isomers found appreciable proportion of all the three tocopherols

studied (α, γ, δ) in pure cow and buffalo ghee. He thus concluded that

adulteration of ghee with vegetable oils could not be detected on the basis of

tocopherol isomers, as vegetable oils also contain all the three tocopherols.

2.1.2.3 TESTS BASED ON WHOLE FAT/TRIGLYCERIDES

2.1.2.3.1 Tests based on thin layer chromatography (TLC) of whole fat

Sebastian and Rao (1974) detected the presence of vegetable oils and

fats in ghee (butterfat) to the extent of even 5 percent by employing TLC

technique using a self-coated silica gel glass plate and solvent mixture consisting

of cyclohexane, ethyl acetate and water (600:200:1) as developing solution on

the basis of appearance of bands more than the specific bands of pure ghee.


Paradkar et al. (2001) modified the method of Sebastian and Rao (1974) and

applied HPTLC on aluminium plates precoated with silica gel 60F254 using the

same solvent system. They reported that palm oil and groundnut oil adulteration

could be detected on the basis of appearance of some extra bands in adulterated

samples.

2.1.2.3.2 Tests based on gas liquid chromatography of triglycerides

Butterfat is composed predominantly of triglycerides with 26 to 52 carbon

number, while animal depot fats and common vegetable oils other than coconut

and palm kernel oil have mainly 50 to 54 carbon number. Coconut and palm

kernel oil contain short and medium chain length triglycerides with 30 to 52

carbon number, a range almost similar to butterfat (Parodi, 1969; Rangappa and

Achaya, 1974).

Using GLC, Kuksis and McCarthy (1964) detected the presence of

vegetable fat and lard in butterfat at 5 to 10 percent level based on the increase

in the content of high molecular weight triglycerides, C52 and C54 peaks,

respectively. Guyot (1978) found the ratio of C52/C50 was <1 in pure butter and

between 2 & 3-4, in case of beef tallow & lard, respectively. He concluded that

the C52 / C50 ratio together with C52 / C38 ratio gave a valuable indication of the

possible addition of tallow or lard to butter. Marjanovic et al. (1984) reported that

adulteration of the milk fat with margarine at 5 to 10% level could be detected on

the basis that margarine had more triglycerides with 48 to 54 acyl carbon atoms

than milk fat. Similarly, Luf et al. (1987) could also detect 5 to 10 percent of beef

tallow and lard as well as vegetable oils / fats in butter based on C52:0 / C40:0 and

C50:0 - C54:0 / C38:0 – C40:0 triglycerides, respectively.

Precht (1990, 1992a) designed a multiple linear regression equation on

the GLC profile of triglycerides by which foreign fats could be detected with

substantially improved sensitivity. Currently, European Union (EU) applies the

method of Precht (1992a) as an official method for evaluating the milk fat purity.


Povolo et al. (1999) applied the above said official method of EU coupled with the

determination of 3,5-cholestadiene content (Mariani et al, 1994) and reported that

the detection of beef tallow up to 0.5 to 1.0 percent using 3,5-cholestadiene

analysis and up to 2 percent using multivariate statistical techniques could be

done. Several investigators (Renterghem, 1997; Collomb et al, 1998; Banfi et al,

1999) carried out the triacylglycerol analysis by GLC and applied it to detect

adulteration of milk fat. Recently, Pinto et al (2002) did linear discriminant

analysis of triacylglycerides to determine the authenticity of pure milk fat. Naviglio

and Raja (2003) proposed a gas chromatographic analysis of butter using a

capillary column having 65% phenyl methyl silicone as stationary phase. This

method allows the detection of extraneous vegetable and animal fats in a simple,

rapid and precise way, even at lower levels.

Precht (1992b) carried out the gas chromatographic analysis of

triacylglycerol composition of 755 different milk fat samples and formulae were

derived from statistical evaluations. Any combination of different foreign fats

consisting e.g., of a mixture of medium- and long-chain triacylglycerols can be

detected by using this method. For the widely varying foreign fat mixtures, limits

of detection between 2–5% were established. For 23 different additions (3–15%)

of individual vegetable foreign fats or animal depot fats, as well as for 33 foreign

fat mixtures (4–7% addition) added to unknown milk fats from varying feeding

periods, absolute mean deviations of 0.7–0.8% were found.

However, all the methods based on glyceride analysis suffer from major

limitations that they require capillary columns with flow splitter which require

skilled hand for operation and maintenance. At present our laboratories are rarely

equipped with such sophisticated columns. Secondly, all these methods being

based on increase or decrease of glyceride ratio escape detection in most of the

cases (Parodi, 1973) and thus can’t ensure a foolproof detection.

Gutiérrez et al., (2009) utilized gas chromatography to determine

triacylglycerol profiles in milk and non-milk fat. The values of triacylglycerol were


subjected to linear discriminant analysis to detect and quantify non-milk fat in milk

fat. The samples of raw milk fat were adulterated with non-milk fats (Fish oil &

corn oil) in proportions of 0, 5, 10, 15, and 20%. The first function obtained from

the linear discriminant analysis allowed the correct classification of 94.4% of the

samples with levels <10% of adulteration. The triacylglycerol values of the

ultrapasteurized milk fats were evaluated with the discriminant function,

demonstrating that one industry added non-milk fat to its product in 80% of the

samples analyzed.

2.1.3 METHODS BASED ON TRACER COMPONENTS OF FATS AND OILS

Tracers are those compounds which are present in adulterant, either

naturally or by addition, but absent in pure ghee. Addition of some tracer

components has been suggested as a rapid and reliable tool to identify the

foreign fat in milk fat. Among tracers, in India, sesame oil is added (5% by weight)

to vanaspati according to food laws (PFA, 2010) for its detection in ghee by

Baudouin test. The method is based on the development of a permanent crimson

color due to the reaction between furfural and sesamol formed by the hydrolysis

of sesamolin (present in sesame oil) in the presence of concentrated HCl.

Another tracer is tannins which are assumed to be present as impurities in palm

oil. Ghee samples adulterated with palm oil gave purssian blue colour with

potassium ferricyanide and ferric chloride reagent. However, limitation of this

method is that the ghee samples having BHA as antioxidant also gave positive

test (Bector & Sharma, 2002).

Gamma oryzanol, a natural tracer, having antioxidant and cholesterol

lowering properties, is found to be present in the rice bran oil exclusively. It was

revealed to be a mixture of phytosteryl ferulates comprising cycloartenyl ferulate,

24- methylenecycloartenyl ferulate, and campesteryl ferulate as major

components (Iqbal et al, 2005; Chen et al, 2005). Crude rice bran oil can contain

≤ 2% (V/V) oryzanol (Norton, 1995). This compound has been indicated as a

marker of rice bran oil in other edible oils (Singhal et al, 1997). Recently Kumar et


al. (2008, 2009b) has developed a TLC and colorimetric methods for detection of

rice bran oil in ghee, qualitatively, even at 5 and 2% levels of adulteration

respectively.

2.1.4 MISCELLANEOUS METHODS

2.1.4.1 Tests for Mineral Oils

Adulteration of common edible oils with cheaper mineral oils, such as

paraffin oil, heavy and light fuel oil, petroleum jelly, etc. has become widespread

phenomenon because of the price difference. Unlike oils and fats, mineral oils are

not saponifiable by alkali. This characteristic behaviour of mineral oils has been

used as the basis for their detection in edible oils and fats. Venkatachalam and

Sundaram (1957) could detect the presence of even 1 percent of mineral oil in

ghee by saponifying the test sample (1 ml) with aqueous potash followed by

addition of alcohol (50%) and thorough shaking. Appearance of turbidity indicated

the presence of mineral oil. Kumar et al (2005b) have also reported the detection

of liquid paraffin added to ghee at the rate of 0.5% and above using Holde’s test

as described by Winton and Winton (1999).

2.1.4.2 Tests for Cottonseed Oils

Fatty acids containing cyclopropene ring, viz., malvalic (C18:-0) and

sterculic (C19:0) acids which are altogether absent in milk fat, but are

characteristic of cottonseed oil (Bailey et al., 1966; Pandey and Suri, 1982) have

been used as a tool by few workers (Shenstone and Vickery, 1959; Gunstone,

1969) for the detection of cottonseed oil in milk fat and also to distinguish cotton

tract ghee from normal ghee (Singhal, 1980) using Halphen test or methylene

blue reduction test.


Halphen test is based on the development of a crimson colour due to the

reaction between cyclopropenoic acids (constituents of cottonseed oil) and

Halphen reagents (1% sulphur solution in CS2 and equal volume of iso-amyl

alcohol) after incubation for an hour in a boiling bath of saturated sodium chloride

solution. Singhal (1980) developed a methylene blue reduction test for the

identification of cotton tract ghee and reported that the colour of methylene blue

dissolved in chloroform: methanol (1:1) was decolourised by cotton tract ghee or

ghee added with cottonseed oil due to the presence of cyclopropenoic acids.

2.1.4.3 Color based platform test for detection of vegetable oils/fats in

ghee

The Bieber’s test, hitherto employed for the detection of almond oil

adulteration with Kernel oil, was suitably modified by Sharma et al (2007) to

detect the adulteration of ghee with vegetable oils. Based on this rapid color

based test, their results showed the presence of orange brown color in case of

refined vegetable oils & fats, whereas in case of pure ghee samples no color was

observed. By this method adulteration of ghee with different vegetable oils to the

tune of 5-7% could be detected.

The above part of review reveals that although several methods based on

the physico-chemical characteristics of oils and fats have been developed to

detect the various types of adulterant fats such as animal body fats and

vegetable oils in milk fat, but most of the methods are quite tedious, time

consuming and have one or the other limitation. The detection methods available

till date are mainly based on the physico-chemical constants, fatty acid profile,

sterol analysis, partial solidification behavior, etc.


2.2 METHODS OF DETECTION OF ADULTERATION APPLICABLE

DIRECTLY TO MILK:

As reported above in the previous section (2.1), it can be noticed that most of

the methods of detection of adulteration of milk fat are developed for the clarified

milk fat / Ghee. A survey of literature reveals that very few methods have been

developed which can be directly applied to milk for detecting milk fat adulteration.

The detection of milk fat adulteration has to be done right at the reception dock

itself where milk is to be either accepted or rejected on the basis of its quality,

because very little can be done afterwards once the milk is accepted and

converted into milk products like butter, ghee etc. As the milk cannot be held at

the reception dock for longer time for its acceptance or rejection, it therefore

requires rapid tests for knowing the quantity and purity of milk fat. Moreover, it

appears that the fat has to be isolated from milk before subjecting it to testing for

authentication.

The technique for isolation of fat from milk for detecting adulteration of milk fat

at the platform is very difficult and time consuming and therefore, there is a great

demand for a rapid test to detect adulteration of milk with foreign fats and oils. A

rapid test based on Butyrorefractometer (BR) reading for detecting adulteration of

milk with refined mustard oil was developed by Arora et al (1996). This test

involves the use of a modified (specially designed dual purpose) Gerber

butyrometer having both ends open so that after reading the fat percentage in the

butyrometer column the fat can be isolated with the help of a syringe and can be

further used for determining the purity of milk fat using BR reading at 40°C, which

is the simplest test of fat that can be easily performed at the milk reception dock

itself. BR reading test, which requires only 2-3 drops of fat, is an important

indicator of adulteration in milk fat with foreign fats especially vegetable oils and

fats whose BR readings are much higher than milk fat, with the exception of

coconut oil and palm oil whose BR values are close to that of milk fat.


BR reading of the isolated fat as obtained in the above test was different from

that of normal heat clarified fat from the same milk sample, due to the inherent

effect of Gerber acid on milk fat. In order to account for this difference, a

correction factor was applied to the observed reading to get actual BR reading,

as follows:

Actual BR at 40°C = observed BR at 40°C + (0.08 Χ observed BR at 40°C)

This method which was developed at NDRI, has been also adopted by Bureau

of Indian Standards (BIS, 1960). But this method has one limitation that the

change in BR reading upto 10 % level of addition remained within the legal limit.

Same approach was used by Boghra and Borkhatriya (2004), for detecting

cottonseed oil in milk. Skim milk, milk with 2% fat and milk with 4% fat was

adulterated with cottonseed oil @ 1%, 2%, 4%, 6%. A different correction factor

(0.0909) was given by them, which they reported that the difference in their

correction factor as compared to that reported by Arora et al (1996) might be

attributed to variations in nature and types of fatty acids in milk specific to region,

breed and species of the animals as well as the feeding practices followed.

Apart from these, there are no reports on the detection of other vegetable oils

and also animal body fats added directly to milk. Therefore, in the present

investigation, a systematic study was undertaken on the detection of adulteration

of milk with some vegetable oils and body fats using specially designed dual

purpose Gerber butyrometer.

CHAPTER - 3

────────────────────────────

Materials and Methods ─────────────────────────────

30 Materials and methods

3. MATERIALS AND METHODS

For present investigation on detection of adulteration of fat in milk using

specially designed dual purpose Gerber butyrometer, the materials used and the

methodologies employed are dealt in this chapter.

3.1 Collection of milk sample:

Authentic pooled samples of raw cow and buffalo milk were separately

collected fresh from the cattle yard of the institute and used for all experiments.

Cow milk was a mixture of the milk obtained from the herd of Karan Swiss, Karan

Fries, Sahiwal and Tharparkar breeds. Buffalo milk used was the herd milk from

Murrah breed only.

3.2 Collection and Preparation of Adulterant Fats/ Oils:

Three body fats (buffalo, goat and pig) and three vegetable oils (soybean,

groundnut and sunflower) were used as the adulterant fats/ oils in the present

study.

Goat body fat and pig body fat were prepared separately from their respective

adipose tissues collected from the local slaughter house. The adipose tissues of

buffalo body fat were collected from slaughter house located at New Delhi (near

Sadar bazar). These adipose tissues after collection were washed thoroughly

under running tap water. After draining out the residual water, the adipose tissues

were heated at 130 to 150°C (buffalo-150°C, goat-140°C and pig-130°C) over

direct flame in a stainless steel vessel till a transparent liquefied animal body fat

was obtained. The liquid fat thus obtained was filtered through 6-8 folds of

muslin cloth followed by vacuum filtration using Whatman No. 4 filter paper. The

body fats thus obtained was filled in polypropylene bottle, cooled to room

temperature and kept in a refrigerator (6-8oC) for their subsequent use as the

adulterant fats.

The refined vegetable oils required for the study were collected from local

market. Refined soybean oil was of Fortune brand, manufactured by Adani


Wilmar Ltd., and refined groundnut oil was of Ginni brand, manufactured by Amrit

Banaspati Co. Ltd., while refined sunflower oil Ginni brand, manufactured Amrit

Banaspati Co. Ltd. After collection, all samples of refined vegetable oils were kept

in a refrigerator (6-8oC) till their use as adulterant oils.

3.3 Preparation of Adulterated Milk Samples:

For the preparation of adulterated milk samples, body fats were heated to

60-70°C for 10 minutes before mixing, while vegetable oils were heated at 60°C

for 5 minutes before mixing. The adulterant fats/oils were added to milk

individually at 5, 10, 15 and 20% levels on the basis of fat content of milk. The

mixing for one minute was done using blender after heating the milk at 65°C, to

get homogeneous mixture.

3.4 Qualitative analysis of milk fat using specially designed dual purpose

Gerber butyrometer:

3.4.1 Reagents and materials:

1. Gerber Sulphuric acid - density: 1.807 to 1.812 g/ml at 27°C

corresponding to a concentration of sulphuric acid from 90-91 % by

weight (AR Grade, s. d. Fine-chem, Ltd. Mumbai)

2. Amyl alcohol (AR Grade, s. d. Fine-chem, Ltd. Mumbai)

3. Modified Gerber butyrometers (ISI marked, Benny make,Jupitor Glass

Works Benny Impex Pvt. Ltd.)

4. Lock Stoppers and a key (ISI marked, Benny make)

5. Gerber centrifuge (benny impex pvt. Ltd)

6. Milk pipette, 10.75 ml (ISI marked, Borosil make,)

7. Water bath with temperature control (The Laboratory Glassware Co.,

Ambala Cantt., India)

8. An automatic system for delivering 1ml of amyl alcohol (Jain Scientific

Glass works., Ambala Cantt)

9. An automatic system for delivering 10ml of sulphuric acid (Jain Scientific

Glass works., Ambala Cantt)


3.4.2 Isolation of fat in pure and adulterated milk samples by Gerber

butyrometer method.

Isolation of fat in milk samples (pure and adulterated) was done by the

method as described by BIS (1960, 1981). The method followed in brief is given

as follows:

After closing the stem side of modified Gerber butyrometer with silicone

cork, 10 ml of Gerber sulphuric acid was added into it. Milk sample was warmed

to 40°C in a water bath, mixed gently and thoroughly, cooled to 26-28°C. 10.75

ml of milk was pipetted. Milk was then poured into the butyrometer along the side

wall without wetting the neck. Pipette was left to drain for three seconds and

pipette's tip was touched once against the base of the neck of the butyrometer.

Fig. 3.1 Modified Gerber butyrometer


Then, 1 ml of Amyl alcohol was added. Volume in the butyrometer was

maintained with distilled water whenever it was necessary. Butyrometers were

then closed with lock stopper, shaken and inverted until complete digestion of

casein particles was achieved. Butyrometers were then kept in a water bath for 5

minutes at 65 ± 2°C. The butyrometers were put in Gerber centrifuge, so as to

conform to radial symmetry, and as evenly spaced as possible, in order to protect

bearings of the centrifuge. Centrifugation was done for 5 minutes at 1100 rpm

Centrifuge was allowed to come to rest. Butyrometers were removed and placed

in water bath for 5 minutes at 65 ± 2°C.

Then a portion of the fat column was drawn out of the modified Gerber

butyrometer with the aid of pasture pipette for determination of Butyro-

Refractometer reading, as described in section 3.8.

3.5 Extraction of fat from pure and adulterated milk samples using heat

clarification method for determination of Butyro-Refractometer reading:

3.5.1 Materials:

1) Electric heater (Vikrant, Jain Enterprises, India)

2) Spatula (stainless steel)

3) Funnel

4) Filter paper: Whatman no.4, England

5) Centrifuge (REMI laboratory centrifuge)

6) Beaker

3.5.2 Method of extraction of fat from milk:

Pure and adulterated milk samples were warmed to 40°C and then centrifuged

at 3000 RPM for 5 minutes in a centrifuge. The cream plug from the centrifuge

tubes was removed with the help of spatula. The cream plug thus obtained was

then subjected to heat clarification in a beaker over electric heater till the

transparent fat was obtained and the residue was slightly brown. The


temperature at this stage was around 110-115°C. The contents were then filtered

through Whatman no.4 filter paper so as to obtain the clear fat.

3.6 Apparent Solidification Time (AST) test

Apparent Solidification Time (AST) test was previously developed by Kumar

et al., (2009a) for the detection of adulteration of clarified milk fat (Ghee) with

foreign fats and oils. This test was performed in the test tube of the specific

dimensions at a particular temperature and was based on the principle of partial

solidification behaviour of milk fat. The time taken by the melted fat sample to

become apparently solidified at a given temperature was taken as the basis of

this test for detecting the presence of foreign oils and fats in milk fat. In the

present study, this test was modified slightly so as to be performed on the fat

column in the milk butyrometer itself. The AST test in the milk butyrometer was

carried out as described below:

3.6.1 Materials:

1) Water bath maintained at 60±2°C

2) Refrigerated water bath (J Lab Tech, Daihan Labtech Co. Ltd., Korea)

3) Stopwatch

3.6.2 Methodology for AST test:

For this test, after completion of Gerber fat test (section 3.4.2), the

butyrometers were kept in water bath maintained at 60±2°C for 10 minutes for

pre-equilibration. The butyrometers were then transferred to refrigerated water

bath maintained at different temperatures (21, 20, 19, 18 and 17°C). The

butyrometers were observed constantly until the apparent solidification of fat

column in Gerber butyrometer took place, which was confirmed by non-

movement of fat column on tilting the butyrometer. At this stage, when the fat

column was apparently solidified, the time taken for the same was recorded as

Apparent Solidification Time (AST) using stopwatch.


3.7 Complete Liquefaction Time (CLT) test

This test was previously developed by Amit Kumar (2008) for the detection of

adulteration of clarified milk fat (Ghee) with foreign fats. This test was performed

in the test tube of the specific dimensions at a particular temperature. In CLT test,

the time taken by the solidified milk fat to completely liquefy forms the basis of

this test. At particular temperature solidified milk fat takes characteristic time to

liquefy, whereas addition of foreign fat or oil to milk fat may increase or decrease

this time, depending upon the type of adulterant. In the present study, the CLT

test was modified slightly so as to be performed on the fat column in the

butyrometer itself. The CLT test in the milk butyrometer was carried out as

described below:

3.7.1 Materials:

1) Water bath maintained at 60±2°C

2) Water bath maintained at 41±0.2°C

3) Refrigerator (6-8°C)

4) Stopwatch

3.7.2 Methodology for CLT test:

After completing the fat test (see section 3.4.2), the butyrometers were kept in

the water bath maintained at 60±2°C for 10 minutes for pre-equilibration. These

butyrometers were then placed in refrigerator (at 6-8°) for 45 minutes for

apparent solidification. Then butyrometers were transferred to water bath

maintained at 41±0.2°C and observed constantly until the complete liquefaction

of fat column in Gerber butyrometer took place, which was confirmed by

movement of fat column on tilting the butyrometer. At this stage, when the fat

column was completely liquefied, the time taken for the same was recorded as

Complete Liquefaction Time (CLT) using stopwatch.


3.8 Determination of Butyro-Refractometer (BR) Reading at 40°C

Butyro-Refractometer (BR) reading of fat sample (isolated from

butyrometer and heat clarified fat) was determined by the method as described

by BIS (1981).

3.8.1 Materials:

1) Butyro-Refractometer (PERFIT, India)

2) Circulatory water bath (PERFIT, India)

2) Glass rod

3) Tissue paper

4) Diethyl ether (s.d. Fine-chem Ltd., Mumbai, India)

3.8.1 Methodology for determination of Butyro-Refractometer (BR) Reading at

40°C of fat isolated from butyrometer:

Before determining the BR reading of a sample, the temperature of the

Butyro-Refractometer was adjusted to 40.0 ± 0.1°C using circulatory water bath

and the prisms were cleaned with diethyl ether and dried completely. The Butyro-

Refractometer was calibrated with the standard provided by the company before

taking the reading of different samples. A drop of the fat was drawn out from the

modified Gerber butyrometer using pasture pipette and was placed on the lower

prism of the Butyro-Refractometer and the prisms were closed and held for 2

minutes. After adjusting the instrument and light to get the most distinct reading

possible and bringing the temperature to 40°C, the BR reading of the fat was

recorded.

3.8.2 Methodology for determination of Butyro-Refractometer (BR) Reading at

40°C of heat clarified fat:

The Butyro-Refractometer was conditioned as mentioned above and then

a drop of the molten heat clarified fat (see section 3.5) was placed on the lower


prism of the Butyro-Refractometer and the prisms were closed and held for 2

minutes. After adjusting the instrument and light to get the most distinct reading

possible and bringing the temperature to 40°C, the BR reading of the fat was

recorded.

3.9 Thin layer chromatography of unsaponifiable matter

In order to check the presence of vegetable oils in milk samples, technique

of thin layer chromatography (TLC) was applied on the heat clarified fat using the

method of Arun Kumar (2005).

3.9.1 Reagents and materials:

1) TLC plates (Silica Gel 60, F254, MERCK specialities private Ltd, Mumbai,

India)

2) Ethyl alcohol (s.d. Fine-chem Ltd., Mumbai, India)

3) 50 percent (w/v) potassium hydroxide solution

4) Distilled water

5) Separating funnel

6) Peroxide-free diethyl ether (s.d. Fine-chem Ltd., Mumbai, India)

7) Sodium sulphate (anhydrous) (LR grade, Qualigens fine chemicals,

Mumbai, India)

8) Water bath

9) Chloroform (GR grade, MERCK specialities private Ltd, Mumbai, India)

10) Chromatographic vessel

11) Cyclohexane:ethyl acetate:water in the proportion of 600:200:1

12) Oven maintained at 100oC (Narang Scientific Works Pvt. Limited, New

Delhi, India)

13) Phosphomolybdic acid in ethanol (98%, v/v)

14) Reference solution consisting of pure cholesterol ester, cholesterol, β-

sitosterol, tocopherol mixture (ɑ, δ, γ- tocopherol), vitamin A, Vitamin D,

and carotene.

15) Chromatographic sprayer


3.9.2 Method:

3.9.2.1 Extraction of unsaponifiable matter from the samples

Accurately, 5.0 g of heat clarified fat wae weighed and saponified by

refluxing with 50 ml of ethyl alcohol and 7 ml of 50 percent (w/v) potassium

hydroxide solution for 30 minutes. After cooling, 150 ml of distilled water were

added and the contents were transferred to a separating funnel. The

unsaponifiable matter was extracted three times with 50 ml of peroxide-free

diethyl ether each time. All the three ether extract were combined together and

washed with water to make it alkali free. The alkali-free ether extract was dried

over sodium sulphate (anhydrous) and finally the ether was evaporated on water

bath under reduced pressure.

3.9.2.2 Application of unsaponifiable matter on TLC plates and its development

By means of a micropipette,3 µl of unsaponifiable matter extracted from

the heat clarified fat (cow and buffalo) after dissolving in chloroform, were spotted

on the TLC plates at a distance of about 2 cm from the bottom.

These plates were introduced into chromatographic vessel having solvent

mixture consisting of Cyclohexane: Ethyl acetate: Water in the proportion of

600:200:1 by volume, as developing solution. The plates were developed

according to ascending chromatographic technique. The development was

discontinued when the solvent (developer) front had travelled over height of

about 1 cm below the other end of the plate. The developed plates were dried in

the air for 5 minutes to remove excess of the solvent followed by drying in an

oven maintained at 100oC for 3 minutes. The plates were then taken out and

allowed to cool at room temperature and sprayed uniformly with 10 percent

phosphomolybdic acid in ethanol (98%, v/v). The plates were kept back in drying

oven till distinct bluish bands were developed which took 3 to 5 minutes.

Simultaneously, reference solution consisting of pure cholesterol ester,

cholesterol, and phytosterols (ergosterol and stigmasterol) were also run under

similar conditions.


3.10 Effect of addition of formalin to milk on BR reading of milk fat obtained

by isolation from Gerber butyrometer and by heat clarification method.

For preservation purposes, formalin (40% formaldehyde, Qualigens fine

chemicals, Mumbai, India) is the only preservative permitted to be added at the

rate of 0.4% to the milk samples meant for chemical analysis (PFA, 2010).

Therefore, in order to see whether addition of formalin has any effect on the BR

reading of the fat isolated from milk either by modified Gerber method or heat

clarification method, formalin at a level of 0.4% was added to pure and

adulterated milk samples (at 20% level of adulteration with vegetable oils and

body fats separately). Immediately after the addition of formalin to these milk

samples, the fat was isolated for determining the BR reading as described below.

In case of isolation of fat from formalin treated milk samples using Gerber

butyrometer, the modified method of fat estimation in formalin treated samples as

given by Raj and Singhal (1987) was followed, in which concentration of sulphuric

acid was increased to 95% instead of routine 90-91% as the addition of formalin

interferes with the fat test when less concentrated acid is used. In the present

study, specially designed dual purpose Gerber butyrometers were used in place

of normal butyrometers.

In case of isolation of fat by heat clarification method from formalin treated

samples, the normal method of obtaining heat clarified milk fat as described

earlier (see section 3.5) was followed. Isolated fats were then used for

determining BR reading in the same way as described earlier (see section 3.8).

3.11 Effect of storage of formalin preserved milk samples on BR reading of

milk fat obtained by isolation from Gerber butyrometer and by heat


In order to study the effect of storage of formalin treated milk samples on

BR reading of milk fat obtained either by isolation form Gerber butyrometer or by

heat clarification method, formalin at a level of 0.4% was added only to pure milk

samples (separately pooled cows and buffaloes milk) in glass stoppered bottles

of 250 ml capacity and the milk samples containing formalin were stored at 37°C

for three months. Fresh milk samples immediately before and after the addition of


formalin were analysed for fat content and BR reading of the isolated fat. During

storage also, the samples were drawn at one month interval and analysed for fat

content and BR reading of isolated fat. Nine bottles each of formalin treated milk

samples were separately stored for cow and buffalo milks. At the interval of

storage of one month, every time a new set of three bottles were drawn each for

cow and buffalo milks added with formalin for the analysis of fat content and BR

reading of the isolated fat. For this part of study, three trials were conducted each

for cow and buffalo milk.

For fat estimation in formalin preserved samples by Gerber butyrometer,

the modified method given by Raj and Singhal (1987) was followed, in which

concentration of sulphuric acid was increased to 95% instead of routine 90-91%,

with the exception that in this study, specially designed dual purpose Gerber

butyrometer having both ends open was used in place of normal butyrometer.

After reading the fat percentage in the butyrometer, a portion of the fat

from the butyrometer column was drawn out with the help of pasture pipette and

analysed for BR reading as described earlier (see section 3.8.1). In case of

isolation of fat by heat clarification method from formalin treated samples, the

normal method of obtaining heat clarified milk fat as described earlier (see

section 3.5) was followed. Isolated fats were then used for determining BR

reading in the same way as described earlier (see section 3.8.2).

CHAPTER - 4

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Results and Discussion ───────────────────────────

41 Results and discussion

4. RESULTS AND DISCUSSION

A study on the detection of adulteration of fat in milk using specially designed

dual purpose Gerber butyrometer was carried out in the present investigation. The

first objective was aimed to detect different vegetable oils added to milk at various

levels. The second objective was to detect different body fats added to milk at

various levels. To meet the objectives of the study, three vegetable oils (groundnut,

soybean, sunflower), three body fats (goat, buffalo and pig) were procured from the

local market, whereas pooled cow and buffalo milk samples were collected from the

Institute’s Cattle Yard. Adulterated milk samples were prepared by adding the

preheated adulterant oils and fats individually to milk at 5, 10, 15 and 20 percent

levels on the basis of fat content in milk, followed by mixing with a blender after

preheating milk to about 60-65°C.

For checking the adulteration of milk with foreign fats, four tests such as

Apparent Solidification Time (AST) test, Complete Liquefaction Time (CLT) test,

Butyro–refractometer (BR) reading at 40°C of milk fat obtained by isolation form

Gerber butyrometer and by heat clarification method and thin layer chromatography

of unsaponifiable matter were planned to be undertaken. In addition to these

experiments, effect of addition of formalin to milk on BR reading of milk fat obtained

by isolation form Gerber butyrometer and by heat clarification method, effect of

storage of formalin preserved milk samples on BR reading of milk fat obtained by

isolation from Gerber butyrometer and by heat clarification method and thin layer

chromatography of unsaponifiable matter obtained from pure as well as adulterated

milk samples were also studied.

In whole of the study, unless otherwise mentioned, ten trials were conducted.

The results obtained in the present investigation for the above mentioned

parameters are presented in the Tables 4.1 to 4.18 and Figures 4.1 to 4.4.

4.1 Apparent Solidification Time (AST) test:

For conducting the AST test on the fat column in the Gerber butyrometer,

attempts were made to select the temperature at which the fat column takes

reasonably less time to get solidified. For this, experiments were conducted at 17 to

21°C (in a refrigerated water bath) on ten different pooled samples of cow and


buffalo milks and the results obtained on the time taken by the fat column at different

temperatures are given in Tables 4.1 and 4.2.

Table 4.1 AST value (min-sec) of fat column in butyrometer for cow milk samples at

different temperatures

Table 4.2 AST value (min-sec) of fat column in butyrometer for buffalo milk samples

at different temperatures

It can be seen from the Table 4.1 that the time taken by the fat column at

temperatures of 21, 20, 19, 18 and 17°C in case of cow milk samples ranged from

Sample AST value (min-sec)

21°C 20°C 19°C 18°C 17°C

1 32-12 30-05 12-30 01-16 00-52

2 15-29 07-28 01-40 01-20 01-03

3 30-00 08-35 07-15 05-13 02-10

4 17-00 10-57 14-02 13-40 03-40

5 18-50 12-30 06-30 08-09 01-35

6 30-20 20-03 12-30 05-10 02-26

7 42-00 34-03 17-03 09-15 03-35

8 41-23 30-33 03-02 02-00 01-02

9 42-25 32-43 07-49 01-59 00-59

10 27-00 25-12 01-28 00-45 00-40

Mean±S.E. 29-56±03-20 21-21±03-35 08-38±02-12 05-27±01-36 02-20±00-21

Sample AST value (min-sec)

21°C 20°C 19°C 18°C 17°C

1 09-02 01-35 01-09 01-01 00-59

2 22-50 12-01 10-30 03-35 01-43

3 40-00 08-32 03-44 02-23 01-50

4 12-35 03-59 02-35 01-53 00-59

5 25-15 15-30 04-32 03-50 01-50

6 19-50 09-40 05-20 02-53 01-10

7 27-12 13-02 08-04 04-57 01-02

8 17-35 08-42 04-55 03-23 00-59

9 08-52 01-32 09-43 04-22 00-51

10 21-04 11-10 06-21 02-43 01-02

Mean±S.E. 18-55±03-21 08-57±01-52 06-09±00-57 03-01±00-22 01-24±00-07


15-29 to 42-25, 07-28 to 34-03, 01-40 to 17-03, 00-45 to 13-40 and 00-40 to 03-40,

respectively with an average of 29-56±03-20, 21-21±03-35, 08-38±02-12, 05-27±01-

36 and 02-20±00-21, respectively. The corresponding values for buffalo milk

samples were found as 08-52 to 40-00, 01-32 to 15-30, 01-09 to 10-30, 01-01 to 04-

57 and 00-51 to 01-50, respectively with an average of 18-55±03-21, 08-57±01-52,

06-09±00-57, 03-01±00-22 and 01-24±00-07 respectively. Temperatures above 21

and 17°C were not taken up for the study because at 21°C the samples took

sufficient large, while at 17°C the samples took very less time to get apparently

solidified.

The study revealed that there are large variations in the AST values

among the samples at all the temperatures studied both for cow and buffalo milk

samples. Therefore, in order to understand the large variations in the AST values

observed for individual samples experiments were conducted on recording the AST

values for the same milk sample using different butyrometers of the same make.

These experiments, however, were carried out only at 18°C and the results so

obtained are given in Tables 4.3 and 4.4. It can be seen from these tables that for

the same milk samples there were large variations in the AST values, which may

possibly be due to the reason that the wall thickness of the butyrometers used in the

experiments may not be the same.

Table 4.3 AST value (min-sec) of fat column in butyrometers of same make for cow

milk samples at 18°C

Cow milk sample

AST value (min-sec) for different Gerber butyrometers

1 2 3 4 5 6 7 8 9 10

1 01-57 01-56 02-14 01-27 01-54 01-34 01-45 02-59 02-50 01-27

2 05-18 07-29 05-00 05-27 05-59 03-10 03-50 04-43 04-20 03-39

3 30-00 33-00 40-00 59-00 25-00 27-00 50-00 25-00 35-00 57-00

4 09-00 20-00 28-00 22-00 21-00 27-00 08-22 17-00 32-00 40-00

5 01-20 01-02 01-36 56-00 01-00 01-03 01-01 01-18 01-30 57-00

6 01-55 01-59 03-70 05-27 01-59 01-20 01-37 02-10 02-50 09-50


Table 4.4 AST value (min-sec) of fat column in butyrometers of same make for

buffalo milk samples at 18°C

Therefore, keeping in view the very large variations observed for AST values

among the samples as well as among the butyrometers for the same sample, this

part of the study on AST test was not continued further for its application in detecting

the adulteration of milk with foreign oils and fats.

4.2 Complete liquefaction time (CLT) test:

After having encountered a problem of variations among the samples as well as

among the butyrometers for the same sample in case of AST test, only a limited

study restricted to one temperature (41°C) at which the milk fat is supposed to be

completely liquid was carried out. Only the pure cow and buffalo milk samples along

with milk samples adulterated at highest level of adulteration (20% on fat basis)

studied were taken up for this part of the study. The results thus obtained on the time

taken by fat column of the Gerber butyrometer for complete liquefaction for above

said samples are given in Table 4.5 and 4.6.

It can be clearly seen from the Tables 4.5 and 4.6 that although the average

values reveal a visible trend of decreasing and increasing CLT values with the

addition of vegetable oils and animal body fats respectively, but as noticed in case of

AST test here also it was observed that there were large variations among the

samples which again may possibly be due to the variations in the wall thickness of

the butyrometers used in the experiment.

Buffalo milk sample

AST value (min-sec) for different Gerber butyrometers

1 2 3 4 5 6 7 8 9 10

1 02-51 02-50 03-50 04-22 02-30 02-40 02-50 02-01 04-20 05-27

2 02-47 03-10 03-20 02-57 02-10 02-59 02-49 03-20 04-10 02-42

3 05-00 01-55 01-29 01-34 01-54 01-27 01-25 01-59 02-10 01-27

4 25-00 23-00 38-00 21-00 22-00 39-00 17-00 59-00 27-00 23-00

5 01-57 02-10 03-01 02-59 01-50 01-30 01-59 02-10 03-10 02-57

6 07-08 09-10 16-56 08-50 04-44 02-02 16-02 02-50 03-50 01-59


Table 4.5 CLT value (sec) of fat column in butyrometer for pure and

adulterated cow milk samples at 41°C

Table 4.6 CLT value (sec) of fat column in butyrometer for pure and

adulterated buffalo milk samples at 41°C

Moreover, it can be seen from the CLT values in case of milk samples of cows

and buffaloes (Table 4.5 and 4.6), considering the overall range of CLT as 17 to 32

seconds of pure milk samples, 70-90% of all the milk samples adulterated with the

three vegetable oils and three body fats individually (@ 20% level on fat basis) were

within the limits of CLT values of pure milk samples and hence could not be

detected.

Level of adulteration

CLT value (sec) of different Cow milk samples

1 2 3 4 5 6 7 8 9 10 Mean±S.E.

0% 22 19 23 23 32 24 17 18 21 24 22.3±1.33

Ground nut oil (20%) 20 18 18 17 21 21 17 17 15 20 18.4±0.64

Soy bean oil (20%) 22 20 20 19 27 22 16 18 15 17 19.6±1.11

Sunflower oil 20% 21 19 21 17 24 24 14 11 21 21 19.3±1.32

Goat body fat (20%) 31 25 25 25 34 29 25 38 27 27 28.6±1.42

Buffalo body fat (20%) 27 29 34 32 40 32 25 20 27 24 29±1.81

Pig body fat (20%) 30 25 32 24 37 34 36 28 23 36 30.5±1.67

Level of adulteration CLT value (sec) of different Buffalo milk samples

Mean±S.E. 1 2 3 4 5 6 7 8 9 10

0% 19 24 29 30 21 23 24 24 19 25 23.8±1.16

Ground nut oil (20%) 16 19 17 18 18 17 17 16 17 19 17.4±0.34

Soy bean oil (20%) 18 19 22 12 22 17 21 18 16 15 18±1.01

Sunflower oil 20% 19 17 23 15 16 21 18 16 19 21 18.5±0.82

Goat body fat (20%) 23 38 29 27 35 24 24 34 24 32 29±1.72

Buffalo body fat (20%) 27 27 32 32 38 27 26 30 24 29 29.2±1.27

Pig body fat (20%) 21 32 30 42 34 20 27 30 40 28 30.4±2.25


Therefore, it can be concluded from the above part of the study that the large

percentage of adulterated milk samples were failed to be detected by the CLT test

performed in a Gerber butyrometer, and hence this test cannot be recommended to

be used as a platform test for screening the milk for milk fat purity.

4.3 Butyro-refractometer (BR) readings of fat isolated from pure cow and

buffalo milks by heat clarification method and Gerber butyrometer method.

The results obtained on the Butyro-refractometer (BR) readings at 40°C of fat

isolated from pure cow and buffalo milks by heat clarification method and Gerber

butyrometer method are given in Table 4.7 and 4.8. It can be seen from these Tables

that the BR readings obtained by Gerber butyrometer method were lower as

compared to those obtained by heat clarification method. The BR readings in case of

Gerber butyrometer method are supressed probably due to the inherent hydrolytic

effect of Gerber sulphuric acid on the fat.

Table 4.7 shows that based on 60 cow milk samples, the BR reading of cow

milk fat isolated by heat clarification method ranged from 41.4 to 42.8 with an

average of 42.11±0.06. While the BR reading of cow milk fat isolated by Gerber

butyrometer method from the same 60 cow milk samples ranged from 38.1 to 39.8

with an average of 38.85±0.06.

Table 4.7 Butyro-refractometer (BR) readings of fat isolated from cow milk by

heat clarification method and Gerber butyrometer method.

Sample No.

BR Reading at 40°C

Heat Clarification Method

Gerber Butyrometer Method

1 41.8 38.6 2 41.9 38.9 3 42.2 38.4

4 42.8 39.4 5 41.8 38.5 6 41.9 39.3 7 42.2 38.7 8 42.7 39

9 41.9 38.2 10 42.2 39 11 41.9 38.9


12 41.4 38.8

13 42.6 39.6 14 41.6 38.4 15 41.5 38.6

16 42.4 38.4

17 41.8 38.4

18 41.5 38.7

19 41.6 38.6 20 42.8 39.6 21 42.2 39.1 22 42.7 39.6

23 42.4 39 24 42.5 39 25 41.4 38.2

26 42.3 38.6

27 42.6 39

28 41.7 38.1 29 41.9 39.5 30 42.8 39.6

31 42.6 39.8 32 42.2 39

33 41.6 38.4

34 41.8 38.4 35 41.4 38.3

36 42.8 39 37 42.7 39.2 38 41.8 38.4

39 41.6 38.4

40 42.7 39.8 41 42.8 39.2

42 41.6 38.2

43 41.8 38.9 44 42 38.6 45 41.8 38.8

46 41.7 38.5 47 42.2 39

48 42.6 39.6

49 41.6 38.3 50 41.9 38.9 51 42.8 39.7 52 42.6 39 53 42.6 39.2

54 41.9 39 55 42.8 39

56 41.6 38.2 57 41.8 38.6 58 42 39

59 41.6 38.4 60 42.6 38.8

Mean±SE 42.11±0.06 38.85±0.06


It can be seen from Table 4.8 that based on the 60 buffalo milk samples, the BR

reading of buffalo milk fat isolated by heat clarification method ranged from 40.00 to

42.4 with an average of 40.72±0.07. While the BR reading of buffalo milk fat isolated

by Gerber butyrometer method from the same 60 buffalo milk samples ranged from

36.4 to 39.2 with an average of 37.64±0.08.

Table 4.8 Butyro-refractometer (BR) readings of fat isolated from buffalo milk

by heat clarification method and Gerber butyrometer method.

Sample No.

BR Reading at 40°C Heat Clarification

Method Gerber Butyrometer

Method

1 40.6 38

2 40.3 37.8 3 41 37.5

4 40.1 37.4

5 40.4 38 6 41.3 37.8

7 40.2 37 8 42.1 39.2

9 40.2 36.8 10 40.8 38 11 42 39.1

12 40.5 37

13 40.8 37.6 14 40.4 37.2

15 40 37.2 16 40.3 38 17 40.6 38

18 41 37.8

19 40.4 37 20 41 38

21 40.7 37.8 22 40.1 37 23 42.2 39.1

24 40.2 37.1 25 40 36.5

26 40.4 37.2 27 40.4 37.4 28 40.2 37 29 41.8 38.8 30 40.3 36.4 31 40.8 37.4 32 40.3 37 33 40.2 37


34 41.1 37.8

35 41.8 38.4 36 40.7 37.4 37 40.3 38

38 40.4 37 39 40 37

40 41 38

41 40.8 38 42 40.3 37.2 43 41.4 37.9 44 40.8 37.6

45 40.2 37.6 46 41.2 38.2 47 41 37.6

48 40.4 36.4

49 40.2 37.2

50 40.6 38 51 41.2 37.9 52 40.3 37

53 40.4 37.2 54 40.6 38

55 40.8 38

56 41 38 57 40.6 38.4

58 42.4 39.1 59 41.1 37.6 60 41.2 37.6

Mean±SE 40.72±0.07 37.64±0.08

When the data obtained on the BR readings of fat obtained from cow and buffalo

milks was combined together, it was observed that based on 120 milk samples (60

cow milk samples and 60 buffalo milk samples) the BR reading of fat isolated by heat

clarification method ranged from 40.0 to 42.8 (Tables 4.7& 4.8) with an average of

41.42 ± 0.08 (Table 4.9). Whereas, the BR reading of fat isolated by Gerber

butyrometer method ranged from 36.4 to 39.8 (Tables 4.7& 4.8) with an average of

(Table 4.9) 38.25 ± 0.07.


4.4 Development of a correction factor for converting BR reading of fat

isolated by Gerber butyrometer method to BR reading of fat obtained by heat


As reported above, the BR readings obtained by Gerber butyrometer method

were found to be lower as compared to those obtained by heat clarification method.

Therefore, in order to directly get the correct BR reading of milk fat at 40°C after

obtaining the BR reading of fat isolated by Gerber butyrometer method without

actually observing the BR reading of heat clarified fat, a correction factor was

developed taking into consideration the ratio of BR reading of fat isolated by heat

clarification method and fat isolated by Gerber butyrometer method for cow and

buffalo milk fats, by using the formula, as follows:

Corrected BR Reading at 40°C = Observed BR Reading at 40°C Χ Correction factor

Where,

Corrected BR Reading at 40°C = BR reading of milk fat obtained by heat

clarification method,

Observed BR Reading at 40°C = BR reading of milk fat obtained by Gerber

butyrometer method.

In the present study, as given in Table 4.9, the correction factors for cow and

buffalo milk fats were observed to be 1.084 and 1.082, respectively, as described

below:

Table 4.9 Correction factor for converting BR reading of fat obtained by Gerber

butyrometer method to BR reading of fat obtained by heat clarification method.

* Average of 60 samples

** Average of 120 samples

Type of milk BR reading at 40°C

Correction factor (A/B)

Heat Clarification method (A)

Gerber butyrometer method (B)

Cow milk 42.11* 38.85* 1.084

Buffalo milk 40.72* 37.64* 1.082

Cow & Buffalo milks Combined together

41.42 ** 38.25** 1.083


Cow milk fat

Corrected BR Reading at 40°C (42.11) = Observed BR Reading at 40°C (38.85) Χ

Correction factor (1.084)

Buffalo milk fat

Corrected BR Reading at 40°C (40.72) = Observed BR Reading at 40°C (37.62) Χ


Since in the market, generally the supply of liquid milk is comprised of mixture of

cow and buffalo milks, therefore it was felt appropriate to work out a correction factor

which can be applied to individual cow or buffalo milk or mixture thereof for

converting the observed Gerber butyrometer fat BR Reading to that of heat clarified

fat. Therefore, the formula based on total of 120 Cow & Buffalo milk samples

Combined together is given below:

Cow & Buffalo milks Combined together: Corrected BR Reading at 40°C (41.42) = Observed BR Reading at 40°C (38.25) Χ


In an earlier study, Arora et al (1996) and Lal et al (1998) have also developed a

similar correction factor of 1.08 for converting the observed BR Reading at 40°C to

actual BR Reading at 40°C. However, Boghra and Borkhatriya (2004) have reported

a correction factor of 1.0909 and reported that such a deviation in correction factor in

their study might be attributed to variations in nature and types of fatty acids in milk

specific to region, breed, and species of the animals as well as the feeding practices

followed. The results obtained in present investigation are in general agreement with

the findings of above workers.

In the present study, the BR reading of the milk fat, whether obtained by heat

clarification method or by Gerber butyrometer method, was observed to be higher in

case of cow milk than buffalo milk. This difference may be attributed to the species

characteristics as the milk samples were collected from cows and buffaloes

maintained at the institute under identical conditions of feeding and management.

Similar differences between the BR readings of the milk fats of the two species have

also been reported by earlier workers (Bector and Narayanan, 1974; Lal and

Narayanan, 1984; Ashvinkumar, 2010 ; Kumar et al, 2011 )


4.5 BR readings of pure milk fats and adulterant oils and fats:

Table 4.10 shows the BR readings of pure milk fats and adulterant oils and

fats. As mentioned earlier the BR readings of heat clarified pure cow milk fat and

buffalo milk fat ranged between 41.4 to 42.8 (mean 42.1) and 40.0 to 42.4 (mean

40.7), respectively. The BR readings for the adulterant vegetable oils such as

groundnut oil, soy bean oil and sunflower oil ranged between 55.3 to 57.9 (mean

56.8), 62.1 to 65.1 (mean 63.9) and 59.2 to 63.7 (mean 61.4), respectively. The

corresponding values for adulterant body fats such as goat body fat, buffalo body fat

and pig body fat ranged between 43.6 to 47.5 (mean 45.6), 44.9 to 48.2 (mean 46.7),

48.6 to 53.1 (mean 51.3), respectively.

Table 4.10 BR readings of pure milk fats and adulterant oils and fats

Cow milk fat (heat clarified) 42.1±0.06*

Buffalo milk fat (heat clarified) 40.7±0.07*

Groundnut oil 56.8±0.40**

Soybean oil 63.9±0.43**

Sunflower oil 61.4±0.62**

Goat body fat 45.6±0.67**

Buffalo body fat 46.7±0.58**

Pig body fat 51.3±0.78**

* Data represent Mean±S.E. of 60 observations

** Data represent Mean±S.E. of 6 observations

It can be observed from the Table 4.10 that pure milk fats (cow and buffalo)

and adulterant oils and fats differed markedly from each other with respect to BR

reading. Among the various oils and fats including milk fat examined, pure milk fats

(cow and buffalo) showed the lowest BR readings as compared to adulterant oils and

fats. Among the vegetable oils, soy bean oil showed the highest BR reading at 40°C,

whereas among the animal body fats, pig body fat showed the highest BR reading.

The results obtained in the present study on the BR readings of various

oils and fats including milk fat are similar to those reported by earlier workers on

ghee (Bector and Narayanan, 1974; Lal and Narayanan, 1984; Ashvinkumar, 2010 ;

Kumar et al, 2011), vegetable oils (Singhal, 1980; Rangappa and Achaya, 1974;

Gunstone et al., 1994; Kumar et al, 2011) and body fats (Singhal, 1980; Sharma and

Singhal, 1995; Kumar et al, 2011).


4.6 BR Reading of Gerber butyrometer fat and heat clarified fat obtained from

pure cow milk and cow milk adulterated with vegetable oils and animal body

fats.

Tables 4.11 and 4.12 show the BR readings at 40°C of Gerber butyrometer fat

and heat clarified fat isolated from pure cow milk and cow milk adulterated with three

vegetable oils (groundnut oil, soybean oil and sunflower oil) and three animal body

fats (Goat body fat, buffalo body fat and pig body fat) at 5, 10, 15 and 20 per cent

levels on the basis of fat content.

It can be seen from Table 4.11 that the BR readings of fat obtained by Gerber

butyrometer method in case of groundnut oil added to cow milk at 0, 5, 10 , 15, and

20 per cent levels (on fat basis) was observed to be 38.8, 39.44, 40.22, 40.84 and

41.79, respectively. After applying a general correction factor of 1.083 (a correction

factor based on the observations of BR readings of cow and buffalo milk fats

obtained by Gerber butyrometer method and heat clarification method, as described

earlier in section 4.4), the corresponding corrected BR readings of these samples

were found to be 42.02, 42.71, 43.56, 44.23 and 45.26, respectively. Whereas, the

BR readings of these samples obtained by heat clarification method were found to

be 42.14, 42.81, 43.75, 44.24, 45.36, respectively. This indicated that BR readings of

the milk fat samples were increased with increasing level of adulteration of

groundnut oil.

The BR readings of fat obtained by Gerber butyrometer method in case of soy

bean oil added to cow milk at 0, 5, 10 , 15, and 20 per cent levels (on fat basis) was

observed to be 38.8, 39.8, 40.79, 41.82 and 43.04, respectively. After applying a

general correction factor of 1.083, the corresponding corrected BR readings of these

samples were found to be 42.02, 43.10, 44.17, 45.29 and 46.61, respectively.

Whereas, the BR readings of these samples obtained by heat clarification method

were found to be 41.91, 43.32, 44.53, 45.4, 46.8, respectively (Table 4.4). Here also,

it was observed that higher the level of adulteration of milk fat with soy bean oil,

higher were the BR readings.

The BR readings of fat obtained by Gerber butyrometer method in case of

sunflower oil added to cow milk on the basis of fat at 0, 5, 10 , 15, and 20 per cent


Table 4.11 BR Reading of Gerber butyrometer fat and heat clarified fat obtained from pure cow milk and cow milk adulterated with

vegetable oils.

Level Of Adulteration

BR Reading at 40°C

Groundnut oil Soybean oil Sunflower oil

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

0% 38.8±0.13 42.02±0.13 42.14±0.12 38.8±0.14 42.02±0.16 41.91±0.16 38.97±0.17 42.20±0.18 42.25±0.15

5% 39.44±0.15 42.71±0.12 42.81±0.13 39.8±0.12 43.10±0.14 43.32±0.17 39.68±0.16 42.97±0.18 43.10±0.21

10% 40.22±0.16 43.56±0.18 43.75±0.07 40.79.±0.08 44.17±0.10 44.53±0.17 40.35±0.19 43.70±0.21 43.83±0.17

15% 40.84±0.20 44.23±0.23 44.24±0.18 41.82±0.15 45.29±0.17 45.4±0.18 41.20±0.09 44.62±0.10 44.76±0.11

20% 41.79±0.11 45.26±0.12 45.36±0.14 43.04±0.06 46.61±0.08 46.8±0.20 41.91±0.15 45.39±0.17 45.6±0.21

The data represents Mean ± S.E. of 10 observations


levels was observed to be 38.97, 39.68, 40.35, 41.20 and 41.91, respectively. After

applying a general correction factor of 1.083, the corresponding corrected BR

readings of these samples were found to be 42.20, 42.97, 43.70, 44.62 and 45.39,

respectively. Whereas, the BR readings of these samples obtained by heat

clarification method were found to be 42.25, 43.10, 43.83, 44.76 and 45.6,

respectively (Table 4.11). As noted earlier for groundnut oil and soy bean oil, the

same pattern of increase in BR reading with increased level of adulteration was

observed in case of sunflower oil also.

Table 4.12 shows the BR readings at 40°C of Gerber butyrometer fat and heat

clarified fat isolated from pure cow milk and cow milk adulterated with three animal

body fats (Goat body fat, buffalo body fat and pig body fat) at 5, 10, 15 and 20 per

cent levels.


butyrometer method in case of goat body fat added to cow milk at 0, 5, 10 , 15, and

20 per cent levels (on the basis of fat content) was observed to be 38.87, 38.89,

38.96, 39.38 and 39.91, respectively. After applying a general correction factor of

1.083, the corresponding corrected BR readings of these samples were found to be

42.10, 42.11, 42.20, 42.65 and 43.22, respectively. Whereas, the BR readings of

these samples obtained by heat clarification method were found to be 42.12, 42.24,

42.32, 42.78, 43.42, respectively. This indicated that BR readings of the milk fat

samples were only slightly increased with increasing level of adulteration goat body

fat.

The BR readings of fat obtained by Gerber butyrometer method in case of buffalo

body fat added to cow milk at 0, 5, 10 , 15, and 20 per cent levels was observed to

be 38.8, 38.82, 39.23, 39.31 and 40.26, respectively. After applying a general

correction factor of 1.083, the corresponding corrected BR readings of these



were found to be 42.00, 42.16, 42.54, 42.78, 43.73, respectively (Table 4.12). Here

also, it was observed that BR readings of the milk fat samples were only slightly

increased with increasing level of adulteration buffalo body fat.


Table 4.12 BR Reading of butyrometer fat and heat clarified fat obtained from pure cow milk and cow milk adulterated with animal

body fats.



BR Reading at 40°C

Goat body fat Buffalo body fat Pig body fat

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

0% 38.87±0.18 42.10±0.20 42.12±0.17 38.8±0.13 42.02±0.14 42.0±0.13 38.89±0.14 42.12±0.15 42.23±0.16

5% 38.89±0.12 42.11±o.13 42.24±0.11 38.82±0.17 42.04±0.19 42.16±0.16 39.18±0.07 42.44±0.08 42.51±0.18

10% 38.96±0.13 42.20±0.14 42.32±0.05 39.23±0.10 42.48±0.12 42.54±0.15 39.31±0.09 42.57±0.11 42.94±0.19

15% 39.38±0.11 42.65±0.13 42.78±0.11 39.31±0.10 42.57±0.12 42.78±0.20 40.06±-0.11 43.31±0.13 43.51±0.18

20% 39.91±0.10 43.22±012 43.42±0.10 40.26±0.12 43.60±0.14 43.73±0.17 40.61±0.09 43.98±0.11 44.1±0.14


The BR readings of fat obtained by Gerber butyrometer method in case of pig

body fat added to cow milk at 0, 5, 10 , 15, and 20 per cent levels was observed to





were found to be 42.23, 42.51, 42.94, 43.51, 44.1, respectively (Table 4.12). As

noted earlier for goat body fat and buffalo body fat, the same pattern of only a small

increase in BR reading with increased level of adulteration was observed in case of

pig body fat also.

4.7 BR Reading of Gerber butyrometer fat and heat clarified fat obtained from

pure buffalo milk and buffalo milk adulterated with vegetable oils and animal

body fats.

Table 4.13 shows the BR readings at 40°C of Gerber butyrometer fat and heat

clarified fat isolated from pure buffalo milk and buffalo milk adulterated with three

vegetable oils (groundnut oil, soybean oil and sunflower oil) and three animal body

fats (Goat body fat, buffalo body fat and pig body fat) at 5, 10, 15 and 20 per cent

levels on the basis of fat content.


butyrometer method in case of buffalo milk added with groundnut oil at 0, 5, 10 , 15,

and 20 per cent levels was observed to be 37.75, 37.92, 39.07, 39.52 and 40.6,

respectively. After applying a general correction factor of 1.083 (a correction factor

based on the observations of BR readings of cow and buffalo milk fats obtained by

Gerber butyrometer method and heat clarification method, as described earlier in

section 4.4), the corresponding corrected BR readings of these samples were found

to be 40.88, 41.06, 42.31, 42.80 and 43.97, respectively. Whereas, the BR readings

of these samples obtained by heat clarification method were found to be 40.7, 41.10,

42.38, 43.04, 44.1, respectively. This indicated that BR readings of the milk fat

samples were increased with increasing level of adulteration of groundnut oil.

Table 4.13 shows the BR readings of fat obtained by Gerber butyrometer method in

case of buffalo milk added with soy bean oil at 0, 5, 10 , 15, and 20 per cent levels

was observed to be 37.69, 38.52, 39.84, 40.36 and 41.21, respectively. After

applying a general correction factor of 1.083, the corresponding corrected BR


Table 4.13 BR Reading of butyrometer fat and heat clarified fat obtained from pure buffalo milk and buffalo milk adulterated with

vegetable oils



BR Reading at 40°C

Groundnut Oil Soybean Oil Sunflower Oil

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

0% 37.75±0.20 40.88±0.17 40.7±0.20 37.69±0.20 40.82±0.16 40.7±0.17 37.43±0.29 40.56±0.24 40.63 ±0.24

5% 37.92±0.17 41.06±0.13 41.10±0.11 38.52±0.13 41.72±0.18 41.78±0.12 37.92±0.18 41.06±0.11 41.13±0.22

10% 39.07±0.11 42.31±0.09 42.38±0.10 39.84±0.11 43.14±0.21 43.19±0.21 38.7±0.24 41.91±0.21 42.14±0.21

15% 39.52±0.17 42.80±0.21 43.04±0.09 40.36±0.24 43.71±0.08 43.93±0.18 39.61±0.22 42.90±0.18 42.94±0.26

20% 40.6±0.31 43.97±0.10 44.1±0.06 41.21±0.18 44.63±0.28 44.94±0.16 40.88±0.26 44.27±0.13 44.86±0.33


readings of these samples were found to be 40.82, 41.76, 43.14, 43.71 and 44.63,

respectively. Whereas, the BR readings of these samples obtained by heat

clarification method were found to be 40.7, 41.78, 43.19, 43.93, 44.94, respectively.

Here also, it was observed that higher the level of adulteration of milk fat with soy

bean oil, higher were the BR readings.

From Table 4.13, it can be seen that the BR readings of fat obtained by

Gerber butyrometer method in case of buffalo milk added with sunflower oil at 0, 5,

10 , 15, and 20 per cent levels was observed to be 37.43, 37.92, 38.7, 39.61 and

40.88, respectively. After applying a general correction factor of 1.083, the

corresponding corrected BR readings of these samples were found to be 40.56,

41.06, 41.91, 42.90 and 44.27, respectively. Whereas, the BR readings of these

samples obtained by heat clarification method were found to be 40.63, 41.13, 42.14,

42.94, 44.86, respectively. As noted earlier for groundnut oil and soy bean oil, the

same pattern of increase in BR reading with increased level of adulteration was

observed in case of sunflower oil also.

Table 4.14 shows the BR readings at 40 C of Gerber butyrometer fat and

heat clarified fat isolated from pure buffalo milk and buffalo milk adulterated with

three animal body fats (Goat body fat, buffalo body fat and pig body fat) at 5, 10, 15

and 20 per cent levels.

The BR readings of fat obtained by Gerber butyrometer method in case of buffalo

milk added with goat body fat at 0, 5, 10 , 15, and 20 per cent levels was observed to





were found to be 40.66, 40.82, 41.25, 41.59, 41.96, respectively (Table 4.14). This

indicated that BR readings of the milk fat samples were only slightly increased with

increasing level of adulteration goat body fat.

As shown in Table 4.14, the BR readings of fat obtained by Gerber

butyrometer method in case of buffalo milk added with buffalo body fat at 0, 5, 10 ,

15, and 20 per cent levels was observed to be 37.57, 37.58, 37.82, 38.19 and 38.7,

respectively. After applying a general correction factor of 1.083, the corresponding


Table 4.14 BR Reading of butyrometer fat and heat clarified fat obtained from pure buffalo milk and buffalo milk adulterated with

animal body fats.



BR Reading at 40°C

Goat Body Fat Buffalo Body Fat Pig Body Fat

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

0% 37.5±0.17 40.61±0.10 40.66±0.17 37.57±0.17 40.69±0.31 40.69±0.14 37.88±0.18 41.02±0.32 40.96±0.19

5% 37.61±0.08 40.73±0.16 40.82±0.15 37.58±0.12 40.70±0.20 40.74±0.15 37.92±0.20 41.07±0.07 41.12±0.21

10% 38.08±0.07 41.24±0.21 41.25±0.15 37.82±0.09 40.95±0.16 40.98±0.16 38.15±0.19 41.31±0.23 41.42±0.18

15% 38.39±0.10 41.57±0.17 41.59±0.15 38.19±0.09 41.35±0.29 41.47±0.16 38.59±0.17 41.80±0.16 41.83±0.18

20% 38.64±0.06 41.85±0.09 41.96±0.10 38.7±0.13 41.91±0.09 42.06±0.19 39.26±0.19 42.52±0.11 42.95±0.21


corrected BR readings of these samples were found to be 40.69, 40.70, 40.95,

41.35 and 41.91, respectively. Whereas, the BR readings of these samples

obtained by heat clarification method were found to be 40.69, 40.74, 40.98,

41.47, 42.06, respectively. Here also, it was observed that BR readings of the

milk fat samples were only slightly increased with increasing level of adulteration

buffalo body fat.

The BR readings of fat obtained by Gerber butyrometer method in case of

buffalo milk pig body fat at 0, 5, 10 , 15, and 20 per cent levels was observed to



samples were found to be 41.02, 41.07, 41.31 41.80 and 42.52, respectively.

Whereas, the BR readings of these samples obtained by heat clarification

method were found to be 40.96, 41.12, 41.42, 41.83, 42.95, respectively (Table

4.14). As noted earlier for goat body fat and buffalo body fat, the same pattern of

only a small increase in BR reading with increased level of adulteration was

observed in case of pig body fat also.

As the study was planned to detect adulteration of fat in milk using specially

designed dual purpose Gerber butyrometer, therefore, the corrected BR readings

using Gerber butyrometer method were used in the present investigation for

detecting adulteration of fat in milk with foreign fats and oils. However, the results

thus obtained were compared with those obtained by using heat clarification

method. Hence, keeping in view the general range of BR reading of milk fat as

40.0 to 43.0, it was revealed from the results obtained in the present study on the

corrected BR readings using Gerber butyrometer method (Tables 4.11 to 4.14)

that adulteration of cow milk with vegetable oils was detectable at all the

adulteration levels studied, except groundnut oil and sunflower oil at 5% level of

adulteration (on fat basis). Almost similar results were obtained by heat

clarification method except in case of sunflower oil for which 5% adulteration (on

fat basis) was not detectable by Gerber butyrometer method (corrected

BR=42.97) but was detectable by heat clarification method (BR=43.10).

However, adulteration of cow milk with animal body fats was not detectable below

20 % level of adulteration (on fat basis), except pig body fat which was detectable


even at 15% level. These observations were also supported by the results

obtained on the BR readings of corresponding heat clarified fat.

On the other hand, adulteration of buffalo milk with vegetable oils was not

detectable below 20 % level of adulteration (on fat basis) except soy bean oil

which was detectable even at 10% level. Almost similar results were obtained by

heat clarification method except in case of groundnut oil for which 15%

adulteration (on fat basis) was not detectable by Gerber butyrometer method

(corrected BR=42.8) but was detectable by heat clarification method (BR=43.04).

Similarly, adulteration of buffalo milk with animal body fats was not detectable at

all the levels studied. These results were also supported by BR readings of

corresponding heat clarified fat.

Very few studies have been carried out in the past on the detection of

adulteration of foreign fats added directly in milk using BR readings of fat isolated

by Gerber butyrometer method. Arora et al. (1996), in their study on the detection

of mustard oil added to milk using specially designed dual purpose Gerber

butyrometer, reported that adulteration at the level of 10 percent (on fat basis)

can be detected. Similarly, Boghra and Borkhatriya (2004), while studying the

detection of vegetable oils in milk and milk fat by a rapid method using Gerber

butyrometer, reported that adulteration of mixed milk or buffalo milk having 2

percent fat with 1 percent cottonseed oil (amounting to 50 percent adulteration on

fat basis) can be detected without suspicion. However, in the present study using

Gerber butyrometer method, adulteration of cow milk with vegetable oils was

detectable at all the adulteration levels studied, except groundnut oil and

sunflower oil at 5% level of adulteration (on fat basis). Whereas in case of buffalo

milk, adulteration with vegetable oils was not detectable below 20 % level of

adulteration (on fat basis) except soy bean oil which was detectable even at 10%

level. In the present study, using BR reading, it was possible to detect 5% soy

bean oil in case of cow milk and 10% soy bean oil in case of buffalo milk as

against other vegetable oils where higher than these levels were detectable.

Such differences could be ascribed to higher BR reading of soy bean oil (63.9) as

compared to lower BR reading of groundnut oil (56.8) and sunflower oil (61.4). As

such there are no reports available in the literature on the detection of animal


body fats added to milk using BR readings of fat isolated by Gerber butyrometer

method to compare the results obtained in the present study.

Several reports are available on the detection of adulteration using BR reading

when foreign fats are added to heat clarified fat (Ghee). Sofia (2005) observed

that 20 percent addition of palm oil and coconut oil was not detectable in case of

cow milk fat using BR reading. Amit Kumar (2008) reported that rice bran and

soybean oils added to cow ghee could easily be detected at 10 and 15 percent

levels, while addition of the same oils to buffalo ghee could be detected only at

15 percent level. Palm oil was not detectable when added to cow and buffalo

ghee at 5, 10 and 15 percent level studied. Kumar et al (2011) reported that as

low as 5 percent vegetable oils (groundnut oil, soy bean oil and sunflower oil)

added to cow milk fat can be detected using BR reading, whereas in case of

buffalo milk fat addition of 15 percent vegetable oils can be detected. For the

detection of animal body fats in ghee, Sharma and Singhal (1995) reported that

10 percent levels of adulteration could be detected. Amit Kumar (2008) and

Kumar et al (2011) mentioned that addition of animal body fats even up to 15

percent level could not be detected. The results obtained in the present study on

the BR reading of fat obtained by heat clarification method for the detection of

vegetable oils and animal body fats added to milk are in general agreement with

the above reports.

It was observed in the present study that the corrected BR readings of milk fat

(cows and buffaloes both) obtained by Gerber butyrometer method were, in

general, close to that of heat clarification method for all the adulterated milk

samples, irrespective of type of adulterant. This indicated that the correction

factor developed, based on comparison of BR readings of pure milk fats of cow

and buffalo milks obtained by Gerber butyrometer method and heat clarification

method, is working well in case of adulterated milk samples also.

It may be concluded from the above part of the study that using the corrected

BR reading of milk fat obtained by specially designed dual purpose Gerber

butyrometer, the adulteration of cow milk with vegetable oils was detectable at all

the adulteration levels studied, except groundnut oil and sunflower oil at 5% level

of adulteration (on fat basis), and adulteration of buffalo milk with vegetable oils


was not detectable below 20 % level of adulteration (on fat basis) except soy

bean oil which was detectable even at 10% level. On the other hand, adulteration

of cow milk with animal body fats was not detectable below 20 % level of

adulteration (on fat basis), except pig body fat which was detectable even at 15%

level, however, adulteration of buffalo milk with animal body fats was not

detectable at all the levels studied.

4.8 Thin layer chromatography of unsaponifiable matter extracted from

milk fat and adulterant oils and fats

Thin layer chromatography (TLC) of unsaponifiable matter extracted from

heat clarified milk fat (buffalo and cow), the vegetable oils (groundnut oil,

soybean oil and sunflower oil), animal body fats (goat body fat, buffalo body fat

and pig body fat) and heat clarified buffalo milk fat samples adulterated with

vegetable oils (5 and 10%) was carried out and the typical chromatograms

depicting the separation of unsaponifiable constituents of samples along with

standards is shown in Figure 4.1 to 4.4.

On comparing the bands of standard cholesterol, cholesterol ester,

stigmasterol, β-sitosterol, heat clarified cow milk fat, heat clarified buffalo milk fat,

pure vegetable oils and pure animal body fats (Fig 4.1), it can be seen that heat

clarified cow and buffalo milk fats and animal body fats showed a prominent band

with a similar distance moved from the spotting line as that of standard

cholesterol. Similarly, stigmasterol and β-sitosterol showed very little difference

with cholesterol in respect of their distance covered from the spotting line. Pure

vegetable oils showed more number of bands (in addition those of sterols) than

heat clarified cow and buffalo milk fats and animal body fats.

The cholesterol ester showed a separate band at a distance higher than that

of standard cholesterol, stigmasterol and β-sitosterol. Although milk fat and

animal body fats contain cholesterol ester also in addition to cholesterol, but the

heat clarified cow and buffalo milk fats and animal body fats have not shown a

separate band of cholesterol ester because during saponification of the sample,

the former got converted to its alcoholic form, i.e., cholesterol.

It can be concluded from Figure 4.1 that animal body fats did not show any

distinct band as compared milk fat; therefore, TLC of unsaponifiable matter


Fig. 4.1 TLC of unsaponifiable matter of pure milk fat and adulterants along with

standards.

cannot used to differentiate these two types of fats. Whereas, in case of

vegetable oils, in addition to sterols some additional bands were observed. Based

on these observations further experiments on TLC were confined to pure milk fat

samples and milk fat samples adulterated with vegetable oils. Moreover, in order

to understand the type of additional bands some more standards such as

tocopherols and fat soluble vitamins were also applied on TLC.

Fig. 4.2 depicts the bands of cholesterol, cholesterol ester, β-sitosterol,

vitamins A and D, tocopherols (mixture of ɑ, Ƴ, and δ), carotene, pure buffalo

milk fat, vegetable oils. It can be seen from the Figure 4.2 that there are some

bands in vegetable oils with same Rf values as that of tocopherols but there are

some additional bands which are very prominent in case vegetable oils but not

that prominent in case of buffalo milk fat and are also not matching with the

bands of any reference standards. Therefore, only those additional bands whose

Rf value matches with tocopherols can be used for the detection of vegetable oils

added to milk fat, as these bands are almost invisible in case of pure buffalo milk

fat. Kilcast and Subramaniam (2000) reported that milk fat (10–46 mg/kg)

1 2 3 4 5 6 7 8 9 10 11 12

1- Cholesterol 2- Cholesterol acetate 3- Stigmasterol 4- β-sitosterol 5- Pure cow milk fat 6- Pure buffalo milk fat

7- Soybean oil 8- Sunflower oil 9- Groundnut oil 10- Goat body fat 11- Buffalo body fat 12- Pig body fat


contains very less content of total tocopherols as compared to vegetable oils

such as soy bean oil (666-1259 mg/kg), groundnut oil (238-489 mg/kg) and

Fig. 4.2 TLC of unsaponifiable matter of pure milk fat and vegetable oils along with

standards.

sunflower oil (482-926 mg/kg). Schwartz et al (2008) have reported that butteroil

contains only traces of various fractions of tocopherols and tocotrienols as

compared to vegetable oils like sunflower oil.

Fig 4.3 and 4.4 depicts the bands of cholesterol, cholesterol ester, β-sitosterol,

vitamins A and D, tocopherols (mixture of ɑ, Ƴ, and δ), carotene, pure buffalo

milk fat, vegetable oils added to milk @ 5 and 10% (on fat basis). On the basis of

additional bands matching with the tocopherols, it may be concluded that as low

as 5% adulteration of milk (on fat basis) with soy bean oil and 10% adulteration of

milk (on fat basis) with groundnut oil and sunflower oil could be detected easily

using TLC of unsaponifiable matter. These adulteration levels were, however, not

detectable by corrected BR reading because the values of BR reading for pure

buffalo milk fat are on the lower side of the general range of 40.0 to 43.0.

A B C D E F G H I J K

A- Cholesterol B- Cholesterol acetate

C- β-sitosterol D- Vit-A

E- Vit-D

F- Tocopherol G- β-carotene

H- Pure buffalo milk fat

I- Soybean oil J- Groundnut oil K- Sunflower oil


Fig. 4.3 TLC of unsaponifiable matter of pure milk fat and 5% adulterated milk fats

along with standards.

Fig. 4.4 TLC of unsaponifiable matter of pure milk fat and 10% adulterated

milk fats along with standards.

Ramamurthy et al. (1967), using unsaponifiable matter as the spotting

material, have also reported the detection of milk fat adulteration with vegetable

oils at 10 to 13 percent level on the basis of the appearance of two spots, i.e.,

cholesterol with Rf value of 0.53 and phytosterol with Rf value of 0.44 using





E- Vit-D


H- Pure buffalo milk fat (PBMF)

I- PBMF+5% Soybean oil J- PBMF+5%Groundnut oil K- PBMF+5%Sunflower oil



E- Vit-D


H- Pure buffalo milk fat (PBMF)

I- PBMF+10% Soybean oil J- PBMF+10%Groundnut oil K- PBMF+10%Sunflower oil



reverse phase TLC involving the solvent system consisting of methanol : acetic

acid : water (20 : 5 : 1, v/v) as a developer.

Sebestian and Rao (1974) using the whole fat instead of unsaponifiable

matter, and Kumar et al (2005) under similar experimental conditions of

developing the chromatograms as followed in the present investigation, have also

reported the detection of as low as 5 percent vegetable oils on the basis of

appearance of additional number of bands in case of ghee adulterated with

vegetable oils.

4.9 Effect of addition of formalin to milk on BR reading of milk fat obtained

by isolation from Gerber butyrometer and by heat clarification method.

In order to study the effect of addition of formalin (the only preservative legally

permitted to be added to milk samples meant for chemical analysis) on BR

reading of milk fat obtained by isolation form Gerber butyrometer and by heat

clarification method, the cow and buffalo milk samples (pure and adulterated at

20% level on fat basis) were added with formalin at 0.4% level and the BR

readings of the fat isolated by Gerber butyrometer and by heat clarification

method were recorded and the results obtained are presented in Table 4.15 and

4.16. During the isolation of fat by Gerber butyrometer method it was observed

that although there was blackening in the contents of the Gerber butyrometer but

after centrifugation the fat column was clear and transparent and there was no

problem in the recording of BR readings. It can be seen from the Table 4.15 and

4.16 that there was not much change in the BR reading of the fat whether

obtained by Gerber butyrometer method or heat clarification method for the pure

as well as adulterated milk samples after the addition of formalin both in case of

cow and buffalo milks.


Table 4.15 Effect of addition of formalin to cow milk on BR reading of milk fat obtained by isolation from Gerber

butyrometer and by heat clarification method.


Cow milk

TYPE OF ADULTERANT

Level of adulteration

0% 20%

Control Formalin added Control Formalin added

Gerber Butyromete

r method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification method

Gerber Butyrometer method

(A)

Corrected value

( AΧ1.083)

Heat Clarification method

Gerber Butyrometer method

(A)

Corrected value

( AΧ1.083)

Heat Clarification

method

GNO 38.8±0.13 42.02±0.13 42.14±0.12 38.7±0.14 41.91±0.08 42.2±0.11 41.79±0.11 45.26±0.12 45.36±0.14 41.8±0.10 45.27±0.21 45.4±0.08

SOY 38.8±0.14 42.02±0.16 41.91±0.16 38.84±0.18 42.06±0.12 42.14±0.19 43.04±0.06 46.61±0.08 46.8±0.20 43.0±0.12 46.57±0.24 46.69±0.12

SFO 38.97±0.17 42.20±0.18 42.25±0.15 39.0±0.23 42.24±0.21 42.10±0.18 41.91±0.15 45.39±0.17 45.6±0.21 41.88±0.24 45.36±0.07 45.6±0.14

GBF 38.87±0.18 42.10±0.21 42.12±0.17 38.78±0.11 42.00±0.17 42.28±0.21 39.91±0.10 43.22±0.13 43.42±0.10 40.02±0.18 43.34±0.18 43.52±0.18

BBF 38.8±0.13 42.02±0.18 42±0.13 38.8±0.19 42.02±0.19 42.00±0.23 40.26±0.12 43.60±0.21 43.73±0.17 40.3±0.13 43.64±0.23 43.68±0.22

PBF 38.89±0.14 42.12±0.09 42.23±0.16 38.92±0.21 42.15±0.15 42.34±0.08 40.61±0.09 43.98±0.17 44.1±0.14 40.52±0.19 43.88±0.17 43.9±0.16


Table 4.16 Effect of addition of formalin to buffalo milk on BR reading of milk fat obtained by isolation from Gerber



Buffalo milk

TYPE OF

ADULTERANT

0% 20%

Control Formalin added Control Formalin added

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

Gerber Butyrometer

method (A)

Corrected value

( AΧ1.083)

Heat Clarification

method

GNO 37.75±0.20 40.88±0.12 40.7±0.20 37.5±0.21 40.61±0.21 40.62±0.12 40.6±0.31 43.97±0.17 44.1±0.06 40.71±0.16 44.09±0.30 44.23±0.06

SOY 37.69±0.20 40.82±0.21 40.7±0.17 37.71±0.12 40.83±0.22 40.84±0.15 41.21±0.18 44.63±0.21 44.94±0.16 41.35±0.26 44.78±0.26 45.12±0.19

SFO 37.43±0.29 40.56±0.21 40.63±0.24 37.51±0.18 40.62±0.09 40.68±0.27 40.88±0.26 44.27±0.30 44.86±0.33 40.93±0.12 44.32±0.19 44.91±0.12

GBF 37.5±0.17 40.61±0.19 40.66±0.17 37.38±0.17 40.48±0.12 40.58±0.21 38.64±0.06 41.85±0.24 41.96±0.10 38.67±0.22 41.88±0.18 41.96±0.21

BBF 37.57±0.17 40.69±0.09 40.69±0.14 37.68±0.08 40.8±0.18 40.84±0.17 38.7±0.13 41.91±0.18 42.06±0.19 38.65±0.16 41.86±0.22 42.00±0.20

PBF 37.88±0.18 41.02±0.14 40.96±0.19 37.47±0.18 40.58±0.15 40.7±0.13 39.26±0.19 42.52±0.13 42.9±0.21 39.32±0.15 42.58±0.09 42.92±0.33


4.10 Effect of storage (at 37°C) of formalin preserved milk samples on BR

reading of milk fat obtained by isolation from Gerber butyrometer and by heat


Cow and buffalo milk samples, in triplicates, were added with formalin @ 0.4%

and stored at 37°C in glass stoppered bottles for the period of three months and the

results obtained on the effect of storage of formalin preserved milk samples on BR

reading of milk fat obtained by isolation using Gerber butyrometer method and heat

clarification method are presented in Tables 4.17 and 4.18. Control samples got

spoiled spontaneously after 12 hours of storage. As reported above here also it was

observed that during the isolation of fat by Gerber butyrometer method, although

there was blackening in the contents of the Gerber butyrometer but after

centrifugation the fat column was clear and transparent and there was no problem in

the recording of BR readings. Moreover, there was no change in the BR reading of

milk fat immediately after the addition of formalin, both in case of fat isolated by

Gerber butyrometer method and by heat clarification method in cows as well as

buffalo milk samples as observed in previous section 4.8. After one month of storage

also, in general, no change was observed in the BR reading of milk fat, both in case

of fat isolated by Gerber butyrometer method and by heat clarification method in

cows as well as buffalo milk samples. However, after two months of storage, it was

observed that there was a problem of charring and blackening of fat column in the

Gerber butyrometer method even after centrifugation. Therefore, the BR readings

were not recorded for these samples. On the other hand, there was no problem in

recording of BR readings of fats in case of heat clarification method during the period

of three months storage. However, no change in the BR readings of fats isolated by

heat clarification method was noticed both in case of cow and buffalo milk samples

during the entire storage period. Therefore, for formalin preserved milk samples

application of Gerber butyrometer method for recording the BR reading of isolated fat

cannot be recommended.


Table 4.17 Effect of storage (at 37°C) of formalin preserved cow milk samples

on BR reading of milk fat obtained by isolation from Gerber butyrometer and

by heat clarification method

Sample 1 Sample 2

Sample 3 Mean±S.E.

Control

Gerber butyrometer method (A)

38.3 39.1 38.6 38.67±0.23

Corrected value (A Χ 1.083)

41.48 42.34 41.80 41.87±0.25

Heat clarification method

41.6 42.4 42 42±0.23

Formalin Added

0 day


38.2 39 38.6 38.6±0.23


41.37 42.24 41.80 41.8±0.25


41.4 42.4 42 41.93±0.29

1 month


38 39 38.2 38.4±0.30


41.15 42.24 41.37 41.59±0.33


41.2 42.4 42 41.87±0.35

2 month


---- ---- ----


---- ---- ----


41 42.4 42 41.8±0.42

3 month


---- ---- ----


---- ---- ----


41 42.4 42 41.8±0.42

---- indicates that the samples could not be analysed.


Table 4.18 Effect of storage (at 37°C) of formalin preserved buffalo milk

samples on BR reading of milk fat obtained by isolation from Gerber


---- indicates that the samples could not be analysed.

Sample 1

Sample 2

Sample 3

Mean±S.E.

Control


37.2 38 37.4 37.53±0.24


40.29 41.15 40.50 40.65±0.26


40.3 41.3 40.6 40.73±0.30

Formalin Added

0 day


37.2 38 37.6 37.6±0.23


40.29 41.15 40.72 40.72±0.25


40.4 41.4 40.6 40.8±0.30

1 month


37.2 38 37.4 37.53±0.24


40.29 41.15 40.50 40.65±0.26


40.4 41.4 40.4 40.73±0.33

2 month


---- ---- ----


---- ---- ----


40.4 41.4 40.6 40.8±0.31

3 month


---- ---- ----


---- ---- ----


40.4 41.4 40.6 40.8±0.31

CHAPTER - 5

───────────────────────────────

Summary and Conclusions ───────────────────────────────

74 Summary and conclusion

5. SUMMARY AND CONCLUSION

An investigation on the detection of adulteration of fat in milk using specially

designed dual purpose Gerber butyrometer was carried out. For this, pooled cow

and buffalo milk samples were collected from the Institute’s Cattle Yard.

Adulterant oils and fats such as vegetable oils (groundnut, soybean, sunflower)

and animal body fats (goat, buffalo and pig) were procured from the local market.

Adulterated milk samples were prepared by adding the preheated adulterant oils

and fats individually to preheated milk (60-65°C) at 5, 10, 15 and 20 percent

levels on the basis of fat content in milk, followed by mixing.

For checking the adulteration of milk with foreign fats, four tests such as

Apparent Solidification Time (AST) test, Complete Liquefaction Time (CLT) test,

Butyro–refractometer (BR) reading at 40°C and thin layer chromatography of

unsaponifiable matter were planned to be undertaken. Initially, the attempts were

made to standardize the conditions for AST test which is hitherto developed for

checking the ghee adulteration. While standardizing the AST test to be applied on

Gerber butyrometer fat column, it was observed that there were problems of very

large variations in the AST values among the samples as well as among the

butyrometers for the same sample. Therefore, this part of the study on AST test

was not continued further for its application with an aim of detecting the

adulteration of milk with foreign oils and fats. After having encountered a problem

of variations among the samples as well as among the butyrometers for the same

sample in case of AST test, only a limited study on the CLT test at 41°C was

carried out. For this, only the pure cow and buffalo milk samples along with milk

samples adulterated at highest level of adulteration (20% on fat basis) were

analyzed. Although, a visible trend of decreasing and increasing CLT values with

the addition of vegetable oils and animal body fats, respectively was observed,

but as noticed in case of AST test here also large variations among the samples

were observed. Moreover, it was observed that 70-90% of all the milk samples

adulterated with the three vegetable oils and three body fats individually (@ 20%

level on fat basis) were within the limits of CLT values of pure cow and buffalo

milks and failed to be detected. Therefore, it was concluded that this test cannot


be recommended to be used as a platform test for screening the milk for milk fat

purity.

For application of BR reading test in this study, pooled cow and buffalo milk

samples brought from the cattle yard of the institute were divided into two

portions. One portion was subjected to fat test using dual purpose Gerber

butyrometers and a small portion of fat column was drawn out with the aid of

pasture pipette for checking its BR reading. The remainder portion of milk

samples were subjected to cream separation and the cream thus obtained was

converted into clarified fat using heat clarification method. These heat clarified

fats were then analyzed for BR reading. The results on the BR readings obtained

by Gerber butyrometer method were found to be lower than those obtained by

heat clarification method. Therefore, in order to directly get the correct BR

reading using Gerber butyrometer method, a correction factor of 1.083 was

developed. Based on these results, the following formula is recommended for

converting BR reading obtained by Gerber butyrometer method to correct BR

reading.

Corrected BR Reading at 40°C = Observed BR Reading at 40°C Χ 1.083

Adulterated milk samples were also divided into two portions and analysed for

BR reading using Gerber butyrometer method and heat clarification method as

described above for pure milk samples. Using Gerber butyrometer method, the

corrected BR readings obtained by above formula were considered for detecting

adulteration of fat in milk with foreign fats and oils. Hence, keeping in view the

general range of BR reading of milk fat as 40.0 to 43.0, it was revealed that

adulteration of cow milk with vegetable oils was detectable at all the adulteration

levels studied, except groundnut oil and sunflower oil at 5% level of adulteration

(on fat basis). Almost similar results were obtained by heat clarification method

except in case of sunflower oil for which 5% adulteration (on fat basis) was not

detectable by Gerber butyrometer method (corrected BR=42.97) but was

detectable by heat clarification method (BR=43.10). However, adulteration of

cow milk with animal body fats was not detectable below 20 % level of

adulteration (on fat basis), except pig body fat which was detectable even at 15%

level. These observations were also supported by the results obtained on the BR

readings of corresponding heat clarified fat.


On the other hand, adulteration of buffalo milk with vegetable oils was not

detectable below 20 % level of adulteration (on fat basis) except soy bean oil

which was detectable even at 10% level. Almost similar results were obtained by

heat clarification method except in case of groundnut oil for which 15%

adulteration (on fat basis) was not detectable by Gerber butyrometer method

(corrected BR=42.8) but was detectable by heat clarification method (BR=43.04).

Similarly, adulteration of buffalo milk with animal body fats was not detectable at

all the levels studied. These results were also supported by BR readings of

corresponding heat clarified fat.

Thin layer chromatography (TLC) of unsaponifiable matter extracted from heat

clarified fat obtained from pure buffalo milk and buffalo milk adulterated with

vegetable oils (5 and 10%) was carried out. This study revealed that on the basis

of additional bands matching with the tocopherols, as low as 5% adulteration of

milk (on fat basis) with soy bean oil and 10% adulteration of milk (on fat basis)

with groundnut oil and sunflower oil could be detected easily using TLC of

unsaponifiable matter. These adulteration levels were, however, not detectable

by corrected BR reading because the values of BR reading for pure buffalo milk

fat are on the lower side of the general range of 40.0 to 43.0.

The study on the effect of addition of formalin on BR reading of milk fat

obtained by isolation form Gerber butyrometer and by heat clarification method

from cow and buffalo milk samples (pure and adulterated at 20% level on fat

basis) was also carried out. This study revealed that there was not much change

in the BR reading of the fat whether obtained by Gerber butyrometer method or

heat clarification method for the pure as well as adulterated milk samples. When

the formalin preserved milk samples were stored for three months, it was

observed that there was no change in the BR reading of heat clarified fats.

However, in case of Gerber butyrometer method, upto one month of storage no

change in the BR reading was observed, but after two months of storage,

difficulty in the fat isolation was encountered. Therefore, for formalin preserved

milk samples application of Gerber butyrometer method for recording the BR

reading of isolated fat cannot be recommended.

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DETECTION OF ADULTERATION OF FAT IN MILK USING … · content. Therefore, the present project was undertaken to detect of adulteration of fat in milk using specially designed dual