validation of methods for detection of …...validation of methods for detection of ghee...

VALIDATION OF METHODS FOR DETECTION OF

GHEE ADULTERATION WITH ANIMAL BODY FAT

THESIS SUBMITTED TO THE

NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL

(DEEMED UNIVERSITY)

IN PARTIAL FULFILMENT OF THE REQUIREMENT

FOR THE DEGREE OF

MASTER OF TECHNOLOGY

IN

DAIRYING

(DAIRY CHEMISTRY)

BY

PATEL AKASHAMRUT M. B. Tech. (Dairy Technology)

DIVISION OF DAIRY CHEMISTRY

NATIONAL DAIRY RESEARCH INSTITUTE

(I.C.A.R.)

KARNAL – 132001 (HARYANA), INDIA

2011

Regn. No. 2020903

Dedicated

to

My Guide

It is my privilege to convey my profound regards and feeling of gratitude to

my guide Dr. Vivek Sharma for his expert guidance, constructive criticism and

pertinent suggestions in planning and execution of this investigation.

I express my sincere thanks to members of Advisory Committee, Dr. (Mrs.)

B.K. Wadhwa, Head & Principal Scientist, DC Division; Dr. Darshan Lal,

Principal Scientist, DC Division; Dr. Sumit Arora, Senior Scientist, DC Division;

Dr. Ravinder Kumar Malhotra, Principal Scientist, AS Division; Dr. A.K. Singh,

Senior Scientist, DT Division and also to Dr. Suman Kapila, Senior Scientist, ABC

Division, for their valuable suggestions, encouragement and endless co-operation

from time to time.

My sincere thanks are due to Dr. A.K Shrivastva, Director and vice-

chancellor, NDRI, Karnal and Dr. B.K Wadhwa, Principal scientist & Head, DC

Division, for providing all the necessary facilities for conducting this study.

My deep sense of gratitude and warm regards to Mr. S.L. Ingle, Mrs.

Krishna and Mr. Kulvinder Singh for their sincere in-time help, affections and

moral support during the course of this study.

I am deeply indebted to all the Scientists, Technical Officers and Staff of

the Dairy Chemistry Division, for their necessary help as and when required.

I express heartfelt thanks to my senior Manvesh Sir for not only his help

and company, but also for all the refreshing moments we shared together.

Financial support received in the form of Fellowship from National Dairy

Research Institute, Karnal, is duly acknowledged.

All are not mentioned but no one is forgotten.

Date: 20 June, 2011 (PATEL AKASHAMRUT M.)

ACKNOWLEDGEMENT

Milk fat is the costliest fat among all edible fats available in the market

and so prone to adulteration with cheaper body fats and vegetable oils or their

mixtures. In recent years, several reports have appeared in the newspapers

indicating rampant malpractices of ghee adulteration in different parts of the

country. To counter this various detection tests were developed. These tests

need validation and so present study has been proposed to validate detection

tests in ghee.

Among various tests crystallization time test, apparent solidification time

test (AST) and complete liquification time test (CLT) were selected for

validation as they are suitable as platform tests. Sheep body fat, goat body fat,

buffalo body fat and pig body fat were chosen as adulterants among body fats.

Special kind of high RM oil was selected as vegetable oil to allow maximum

adulteration. In present study it was found that physico-chemical constants like

RM value, PV and BR reading was not effective in detection of adulteration

especially, mixtures of body fat and vegetable oil was able to escape detection

up to 75% in cow ghee and 50% in buffalo ghee respectively. Among detection

tests AST and CLT were more reliable than crystallization time for detection of

adulterants but were not very successful in adulteration detection.

From the present study, it can be concluded that in the present era of

rampant adulteration of milk and milk products no test is able and valid to

detect the adulteration of ghee with foreign fats. All the listed tests are generally

based on the physico-chemical properties of triglycerides hence being

manipulated by the unscrupulous persons involved in the trade. Therefore, there

is a need to develop such tests, which are based on the tracer components,

which are altogether absent in ghee.

ABSTRACT

भारे्कट भें उप्रफध्ध सफ खाने मोग्म पेट भें दधू र्का पेट सफस ेज्मादा भहॉगा है। इसी लरए उस

भें सस्ते फॉडी पेट औय वनस्ऩतत तेर औय उनरे्क लभक्सचसस र्की लभरावट र्की जाती है। सभाचाय ऩत्रों भें अबी अबी रे्क भें फहुत सायी रयऩोटसस देखी गई है जो ददखाती है र्की घी भें देश रे्क सबी बागो भें फेयोर्कटोर्क लभरावट र्की जाती है। इसे योर्कने रे्क लरए फहुत साये टेस््स ववक्क्सत कर्कमे गए है। इन

टेस््स र्को प्रभाणित र्कयने र्की जरुयत है औय इसी लरए मह अभ्मास भें उनर्को प्रभाणित कर्कमा जाना प्रस्ताववत कर्कमा जाता है।

,

औ , , औ आ

आ , औ आ औ 75% औ 50% औ

औ

आ आ

साराांश

1.0 INTRODUCTION ......................................................... 1

2.0 REVIEW OF LITERATURE ............................................ 3

2.1 METHODS BASED ON PHYSICAL PROPERTIES ........................... 3

2.1.1 MELTING POINT ......................................................................... 4

2.1.2 APPARENT SOLIDIFICATION TIME (AST) TEST ............................. 5

2.1.3 COMPLETE LIQUIFICATION TIME (CLT) TEST ............................... 5

2.1.4 CRYSTALLIZATION TIME TEST ..................................................... 6

2.1.5 SOLIDIFICATION POINT............................................................... 8

2.1.6 TITRE VALUE ............................................................................... 8

2.1.7 DENSITY AND SPECIFIC GRAVITY ................................................ 9

2.1.8 BÖMER VALUE (BV) .................................................................... 9

2.1.9 REFRACTIVE INDEX AND BUTYRO REFRACTOMETER

READING ................................................................................. 10

2.1.10 OPACITY TEST ......................................................................... 11

2.1.11 CRITICAL TEMPERATURE OF DISSOLUTION (CTD) ................... 13

2.1.12 FRACTIONATION OF MILK FAT................................................ 13

2.1.13 SPECTROSCOPIC METHODS .................................................... 16

2.1.13.1 Tests Based on Visible Spectroscopy ......................... 16

2.1.13.2 Tests Based on Ultraviolet (UV) Spectroscopy ........... 16

2.1.13.3 Tests Based on Infra-Red (IR) Spectroscopy .............. 17

2.1.14 DILATATION BEHAVIOUR ....................................................... 19

2.1.15 MICROSCOPIC EXAMINATION OF FAT .................................... 20

2.1.16 DIFFERENTIAL THERMAL ANALYSIS (DTA) AND

DIFFERENTIAL SCANNING CALORIMETRY (DSC) ...................... 21

2.2 METHODS BASED ON CHEMICAL PROPERTIES ....................... 21

2.2.1 TESTS BASED ON FATTY ACIDS ................................................. 22

2.2.1.1 Tests Based on Physico-Chemical Constants ............... 22

2.2.1.1.1 Reichert-Meissl (RM) value ............................. 22

2.2.1.1.2 Polenske value ................................................ 23

2.2.1.1.3 Iodine value .................................................... 23

2.2.1.1.4 Saponification value ....................................... 24

CONTENTS

2.2.1.2 Tests Based on Gas Liquid Chromatography (GLC)

of Fatty Acids .............................................................. 25

2.2.2 TESTS BASED ON THE NATURE AND CONTENT OF

UNSAPONIFIABLE CONSTITUENTS ........................................... 26

2.2.2.1 Tests Based on Sterols ................................................. 27

2.2.2.2 Tests Based on Tocochromanols ................................. 29

2.2.3 TESTS BASED ON WHOLE FAT/TRIGLYCERIDES ......................... 30

2.2.3.1 Tests based on paper chromatography of

triglycerides ................................................................ 30

2.2.3.2 Tests Based on Thin Layer Chromatography of

Whole Fat ................................................................... 31

2.2.3.3 Tests Based on Gas Liquid Chromatography of

Triglycerides ................................................................ 32

2.2.4 TESTS BASED ON RESIDUAL PROTEIN CONTENT IN

PURIFIED FAT .......................................................................... 34

2.3 METHODS BASED ON TRACER COMPONENTS OF FATS

AND OILS ................................................................................ 35

2.4 MISCELLANEOUS METHODS ................................................... 36

2.4.1 TESTS FOR MINERAL OILS ......................................................... 36

2.4.2 TESTS BASED ON THE CONTENT OF SPECIFIC FATTY ACIDS ...... 37

2.4.2.1 TESTS FOR COTTONSEED OILS ..................................... 37

2.4.2.1.1 Halphen Test .................................................. 37

2.4.2.1.2 Methylene Blue Reduction Test ...................... 38

2.4.3 HYDROXAMIC ACID TEST .......................................................... 38

2.4.4 MODIFIED BIEBER’S TEST ......................................................... 38

2.4.5 COLOUR BASED PLATFORM TEST FOR THE DETECTION OF

VEGETABLE OILS/FATS IN GHEE. ............................................. 39

2.4.6 TESTS BASED ON ENZYMATIC HYDROLYSIS .............................. 39

3.0 MATERIALS AND METHODS ..................................... 41

3.1 CHMICALS AND REAGENTS ..................................................... 41

3.1.1 CHEMICALS .............................................................................. 41

3.1.1.1 Salts ............................................................................ 41

3.1.1.2 Solvents....................................................................... 41

3.1.2 REAGENTS ................................................................................ 41

3.2 EQUIPMENTS .......................................................................... 42

3.3 COLLECTION AND PREPARATION OF SAMPLES ...................... 42

3.3.1 PREPARATION OF PURE SAMPLES ............................................ 42

3.3.1.1 Ghee ........................................................................... 42

3.3.1.2 Body Fats .................................................................... 43

3.3.1.3 Vegetable Oil ............................................................... 43

3.3.2 PREPARATION OF ADULTERATED SAMPLES ............................. 43

3.3.3 MARKET SAMPLES .................................................................... 44

3.4 MEHODS OF ANALYSIS ........................................................... 44

3.4.1 PHYSICO-CHEMICAL CONSTANTS ............................................. 44

3.4.1.1 Determination of Reichert-Meissl (R.M.) and

Polenske Value ............................................................ 44

3.4.1.2 Determination of Butyro-Refractometer (BR)

Reading at 40 °C .......................................................... 45

3.4.1.3 Determination of Moisture Content ............................ 45

3.4.1.4 Determination of Free Fatty Acids (FFA)...................... 46

3.4.2 TESTS TO BE VALIDATED .......................................................... 46

3.4.2.1 Crystallization Time Test ............................................. 47

3.4.2.2 Apparent Solidification Time (AST) Test ...................... 47

3.4.2.3 Complete Liquification Time (CLT) Test ....................... 47

4.0 RESULTS AND DISCUSSION ...................................... 49

4.1 ANALYSIS OF PURE AND ADULTERATED GHEE SAMPLES ........ 50

4.1.1 PHYSICO-CHEMICAL CONSTANTS OF PURE AND

ADULTERATED SAMPLES ......................................................... 51

4.1.1.1 Reichert-Meissl (RM) Value ......................................... 52

4.1.1.2 Polenske Value (PV) .................................................... 56

4.1.1.3 Butyro- Refractometer (BR) Reading at 40°C ............... 59

4.1.1.4 Moisture Content ........................................................ 62

4.1.1.5 Free Fatty Acid Content ............................................... 65

4.1.2 TESTS OTHER THAN PFA PARAMETERS (APPARENT

SOLIDIFICATION TIME (AST), COMPLETE LIQUEFACTION

TIME (CLT) AND CRYSTALLIZATION TIME TESTS) ..................... 67

4.1.2.1 Crystallization Time, Apparent Solidification Time

(AST), and Complete Liquefaction Time (CLT) of

Pure Fats/Oil ............................................................... 67

4.1.2.2 Crystallization Time Test at 17°C For Adulterated

Samples ...................................................................... 69

4.1.2.3 Apparent Solidification Time (AST) Test at 18°C

for Adulterated Samples ............................................. 72

4.1.2.4 Complete Liquification Time (CLT) Test at 46°C

for Adulterated Samples ............................................. 75

4.2 ANALYSIS OF MARKET GHEE SAMPLES ................................... 78

5.0 SUMMARY AND CONCLUSIONS ............................... 81

BIBLIOGRAPHY .............................................................. 85

Table 4.1 Physico-chemical constants of pure samples ............................... 51

Table 4.2 RM value of adulterated cow ghee samples ................................ 53

Table 4.3 RM value of adulterated buffalo ghee samples ........................... 55

Table 4.4 Polenske value of adulterated cow ghee samples ....................... 56

Table 4.5 Polenske value of adulterated buffalo ghee samples ................... 58

Table 4.6 BR reading of adulterated cow ghee samples .............................. 60

Table 4.7 BR reading of adulterated buffalo ghee samples ......................... 61

Table 4.8 Moisture of adulterated cow ghee samples ................................ 62

Table 4.9 Moisture of adulterated buffalo ghee samples ............................ 64

Table 4.10 FFA content of adulterated cow ghee samples .......................... 65

Table 4.11 FFA content of adulterated buffalo ghee samples ..................... 66

Table 4.12 Crystallization time test, AST and CLT time of pure samples ...... 68

Table 4.13 Crystallization time of adulterated cow ghee samples ............... 69

Table 4.14 Crystallization time of adulterated buffalo ghee samples .......... 71

Table 4.15 Apparent solidification time of adulterated cow ghee

samples ...................................................................................... 73

Table 4.16 Apparent solidification time of adulterated buffalo ghee

samples ...................................................................................... 74

Table 4.17 Complete liquification time of adulterated cow ghee

samples ...................................................................................... 76

Table 4.18 Complete Liquification time of adulterated buffalo ghee

samples ...................................................................................... 77

Table 4.19 Physico-chemical constants of market samples ......................... 78

Table 4.20 Crystallization, AST and CLT time of market samples ................. 78

LIST OF TABLES

AGMARK ......... Agricultural Marking

AR ..................... Analytical Reagent

AST ................... Apparent Solidification Time

BBF .................. Buffalo Body Fat

BG ..................... Buffalo Ghee

BIS .................... Bureau of Indian Standards

BR .................... Butyro-Refractometer

BV .................... Bomer Value

CG ..................... Cow Ghee

CLT .................. Complete Liquification Time

CTD ................. Critical Temperature of Dissolution

DSC .................. Differential Scanning Calorimetry

DTA ................. Differential Thermal Analysis

EU .................... European Union

GBF .................. Goat Body Fat

GLC ................. Gas Liquid Chromatography

GR ..................... Guaranteed Reagent

HPLC ............... High Performance Liquid Chromatography

HPTLC ............. High Performance Thin Layer Chromatography

IDF .................... International Dairy Federation

IR ...................... Infra-Red

LC-GC ............. Liquid Chromatography-Gas Chromatography

LR ..................... Laboratory Reagent

Min .................... Minute

MX1 .................. Mixture of VOL: BBF :: 10:6.5

MX2 .................. Mixture of VOL: BBF :: 10:15

ng ...................... Nanogram oC....................... Degree Centigrade

OD ..................... Optical Density

PBF ................... Pig Body Fat

LIST OF ABBREVIATIONS

PFA .................. Prevention of Food Adulteration

PUFA ............... Polyunsaturated Fatty Acids

PV .................... Polenske Value

RI ...................... Refractive Index

RM ................... Reichert- Meissl

RP-HPLC ......... Reverse Phase- High Performance Liquid Chromatography

SBF ................... Sheep Body Fat

SE ...................... Standard Error

sec ..................... Second

TLC ................... Thin Layer Chromatography

TMS ................. Trimethyl Silyl Ether

USM .................. Unsaponifiable Matter

UV ..................... Ultraviolet

VOL ................... Vegetable Oil

μg ...................... Microgram

CHAPTER – 1

Introduction

INTRODUCTION

1

Lipids, being the most important constituent of milk, play a significant

role in the economics, nutrition, flavour and physico-chemical properties of

milk and milk products. In South Asian countries, especially in India, milk

fat is mostly consumed in the form of ghee (clarified butterfat). Ghee is

regarded as good for having grace, enhancing memory power, grasping

power, power to control senses and to make these stronger. Even in

Ayurvedic and Unani systems of medicine, ghee is generally used as the

base. For culinary purposes, it is the only animal fat universally acceptable

by both ‘vegetarian’ and ‘non-vegetarian’ populations of India. Ghee is

preferred over other fats mainly because of it being a valuable source of fat

soluble vitamins and essential fatty acids, apart from having rich and

pleasant sensory attributes. The pleasing flavour of milk fat cannot be easily

duplicated by other fats. Milk fat is also uniquely distinct from other fats,

being the only fat containing short chain fatty acids.

India produces approximately 1.65 million tons of ghee, annually,

valued at Rs. 231 billion (Alam, 2005). However, in India, a very complex

situation arises especially during lean season in the summer months, when

the supply of milk and ghee falls short of the demand. Unscrupulous people

in the trade take undue advantage of such situations and indulge in the

malpractice of adulteration of milk and ghee with different types of

adulterants. Milk fat being a costly constituent, has attracted the attention of

the fraudulent traders to adulterate it with cheaper fats such as vegetable oils,

animal body fats, hydrogenated fats, interesterified fats and even inedible

mineral oils, like, liquid paraffin, etc.

In recent years, the problem of adulteration has assumed a very serious

dimension. Such a situation has tarnished the image of the dairy industry, not

only in India, but abroad also. In order to ensure a genuine product to the

consumer, the Government of India has prescribed the compositional

standards for butter and ghee under Prevention of Food Adulteration Act

(PFA, 2009) and Agmark Rules (De, 1996). However, these standards are

not comprehensive and can hardly establish the type and the level of added

1.0 INTRODUCTION

INTRODUCTION

2

adulterants. This may be because of wide variations in the physico-chemical

makeup of milk fat owing to different factors like animal species, feeding

practices, etc. Moreover, only a limited quantity of total ghee produced in

the country is graded and marketed under Agmark (Agmark, 1938).

If we go through the scientific literature, then various methods and

tests have been developed by the researchers to confirm the adulteration of

milk fat especially in ghee with foreign fats. However, there is no test or

analytical scheme is available which can be employed to ascertain the purity

of milk fat with authority in the present scenario. Almost each test or

analytical scheme has some or the other limitations or needs validation.

Therefore, the present study has been planned with the following objectives.

Objectives

1. Validation of existing methods (AST, Crystallization test, CLT)

2. To check effect of vegetable oil addition in adulterated samples on

detection.

3. Analysis of market ghee samples using validated tests.

CHAPTER – 2

Review of Literature

REVIEW OF LITERATURE

3

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

diglycerides), phospholipids, fat soluble vitamins, cholesterol, squalene,

waxes, etc. In India, milk fat is mostly consumed in the form of ghee

(clarified butterfat). However, due to its short supply, particularly in the lean

(summer) season and comparatively more demand, expensiveness (costing

three to four times as much as edible vegetable oils) and variable chemical

composition, ghee is prone to adulteration by the unscrupulous traders in the

market.

Common adulterants are vegetable oils and fats, animal body fats,

mineral oils, starchy material, etc. Extensive survey of the literature reveals

that several methods have been developed to detect the adulteration in ghee.

These methods are mostly based on chemical parameters like fatty acid

composition and the physico-chemical constants. However, very few

attempts have been made to detect the adulteration on the basis of minor

constituents such as cis-trans isomers, polyunsaturated fatty acids (PUFA),

sterols, tocochromanol isomers, natural and added tracer components, etc.

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. The literature is reviewed under the following four major

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 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 purity. Several

methods, which were used to check the purity of ghee on the basis of

physical properties, are as follows.

2.0 REVIEW OF LITERATURE


4

2.1.1 MELTING POINT

Among the various physical properties, melting point (slip point) has

been employed for checking the presence of foreign fats. Melting point of

the fats and oils is generally determined by placing a small quantity of the fat

in a capillary tube, hardening the fat under refrigeration for a specified

period of time, followed by gradually raising the temperature of the sample

suspended in a suitable melting point bath until a transparent condition of the

sample is achieved. Fats are generally heterogeneous mixture of glycerides

and therefore, do not have a sharp melting point. Melting point is also

sometimes referred as slip point.

Melting point (slip point) of various oils and fats covers 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). Although the average melting point of vanaspati ghee

is reported to fall between 31 and 37°C (Schwitzer, 1956; Sharma and

Singhal, 1995), Bolton (1999) reported that the melting point of vanaspati

(hydrogenated oils) varies between 30 to 65°C depending upon the extent of

hydrogenation. More the hydrogenation, higher is the melting point. 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

resembles with that of animal body fats (43.9-51.0°C). Among the animal

body fats, buffalo body fat showed the highest melting point (50.5-51.0°C),

while pig body fat showed the lowest (43.9-44.6°C). Addition of animal

body fats (buffalo, goat, pig and sheep) at 5 to 20% level increased the

melting point of pure buffalo or cow ghee. Buffalo body fat caused the

largest increase in melting point of ghee (5.7°C increase at 20% level), while

pig body fat caused the least increase (2.2°C at 20% level). The study,

however, concluded that adulteration up to 20% level does not make

significant change in the melting point of ghee and, therefore, the method is

not found to be useful for the detection of adulteration. Sharma and Singhal

(1995) also confirmed these observations using body fats (buffalo, goat and

pig) and vanaspati at 5 to 20% level of adulteration (irrespective of their


5

mode of addition, either directly to ghee or through milk) and noted that the

increase in melting point was more with body fats than vanaspati.

2.1.2 APPARENT SOLIDIFICATION TIME (AST) TEST

The AST test was developed by Kumar (2003) 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 (2003) 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 oil) 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 greater

than or equal to 10%, but not in cow milk fat. Addition of goat body fat to

both cow and buffalo milk fat was detectable at levels greater than or equal

to 10%, whereas pig body fat was detectable only in buffalo milk fat at

levels greater than or equal to 10%.

2.1.3 COMPLETE LIQUIFICATION TIME (CLT) TEST

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

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


6

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

fat samples to get melted completely at a 44°C.

At 44°C, 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 for both 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 44°C, 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.

2.1.4 CRYSTALLIZATION TIME TEST

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

crystallization of milk fat at 17°C when dissolved in a solvent mixture

consisting of Acetone and Benzene (3.5:1.0). Panda and Bindal (1998b)

studied the crystallization behaviour at 17°C of fat dissolved in a solvent

mixture of acetone and benzene (3.5:1) and reported that ghee, ghee


7

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 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. However, for

cotton tract ghee, Panda and Bindal (1998b) carried out crystallization test at

25°C instead of 17°C, but the test could not distinguish between cotton tract

ghee and the ghee adulterated with cow body fat at 5% level.

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 sec

to 16 min 20 sec and from 6 min 30 sec to 12 min 30 sec with the mean of 11

min 4 sec and 8 min 42 sec, 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 up on the level of adulteration with

vegetable oils and animal body fats, respectively. Higher the level of

adulterant oils/fats, greater was the effect. However, ghee samples

adulterated with pig body fat, initially showed a decrease in crystallization

time at 5% level of adulteration, which subsequently showed an increase as

the level of adulteration increased. At 15% level of pig body fat adulteration,

the values of crystallization time became closer to that of corresponding

average values for pure ghee samples. The same trend was observed for both

type of ghee (cow and buffalo).

The crystallization time was highest in the month of May while it was

lowest in July month. 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 a fat.

Kumar (2008) 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.


8

Recently Sofia et al., (2010) 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.

2.1.5 SOLIDIFICATION POINT

Solidifying point is defined as the temperature at which fat shows first

sign of appearance of solid phase on cooling. Solidification temperature of

milk fat depends very much on the procedure employed for cooling (Webb et

al., 1987). Rahn and Sharp (1928) reported solidification point of 19.7 and

23.6°C for samples of the same milk fat cooled by immersion at 14 and

20°C, respectively. The solidifying point of buffalo ghee (16.0-28.0°C) is

reported (Rangappa and Achaya, 1974) to be slightly higher than cow ghee

(15.0-23.5°C). The solidifying points for beef tallow and lard vary between

32 to 37°C and 25 to 30°C, respectively. Whereas, most of the vegetable

oils, such as, sunflower oil, maize oil, soya oil, groundnut oil, cottonseed oil

have very low solidification point in the range of -16 to 5°C, with the

exception of plam oil (22-40°C), palm kernel oil (24-26.5°C) and coconut oil

(22-23.5°C) whose values are close to that of animal fats reported above.

2.1.6 TITRE VALUE

The titre of ghee represents the highest temperature reached when the

liberated water insoluble fatty acids are crystallized under arbitrarily

controlled conditions. The titre is generally taken to represent the

solidification point of the fatty acids, although they actually solidify over a

range of temperature. For its determination, ghee (oils and fats) is saponified

with glycerol-potassium hydroxide solution. The resulting soap is

decomposed with sulphuric acid and the liberated water-insoluble fatty acids

are separated, washed free from mineral acid and dried. Titre is then

determined on these fatty acids (BIS, 1981). Titre value of butterfat lies

between 33 to 38°C (Hamilton and Rossell, 1986). Animal body fats such as

beef tallow, lard and mutton tallow generally have a higher range (32-48°C).

On the other hand, vegetable oils such as sunflower oil, safflower oil,

soybean oil, groundnut oil, plam kernel oil, etc. generally have lower titre

values (15-32°C), except cottonseed oil (30-37°C) and palm oil (40-47°C).

Doctor et al., (1940) reported the titre value of ghee as 33.5°C, whereas a


9

survey conducted by Directorate of Marketing and Inspection, Govt. of India

reported the titre value of ghee samples from cotton tract areas varying from

40.4 to 44.6°C (Singhal, 1973). At one time, the titre value was used for

judging the quality of ghee produced in cotton tract areas, but later dispensed

with as it was considered to be of not much use. Moreover, its use is limited

due to the introduction of more modern instrumental methods of analysis.

2.1.7 DENSITY AND SPECIFIC GRAVITY

Densities or specific gravities of the fatty oils have long been used in

connection with the analysis and identification of oils. The densities of fats

in their liquid form are commonly expressed as specific gravities rather than

in terms of absolute densities. The specific gravity of oils and fats depends

upon their chemical composition and the temperature (Karleskind, 1996) at

which density or specific gravity is measured.

Specific gravity values for cow ghee and buffalo ghee at 30°C are

reported to be 0.9358 to 0.9443 and 0.9340 to 0.9444, respectively

(Rangappa and Achaya, 1974). Walstra and Jenness (1984) reported that the

liquid milk fat at 20°C in general, has a density of about 0.915 g/ml. Most of

the edible vegetable oils and fats have a density in the range of 0.910 to

0.927 at 20°C. Animal body fats have slightly lower density values ranging

from 0.894 to 0.906 at 20°C (Martin, 1979). With a view to detect

adulteration, Singhal (1973) studied the density and specific gravity of ghee

(including cotton tract) and animal body fats (buffalo, goat, pig and sheep) at

40°C. Based on the narrow differences in the values for different type of oils

& fats, it was concluded that detection of adulteration may not be possible

using this property.

2.1.8 BÖMER VALUE (BV)

Bömer 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. This test, originally developed for the

detection of lard in tallow, depends upon the triglyceride structure of the fat

(Roos, 1963). The Bömer value of both cow and buffalo ghee ranges from

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


10

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

1987) reported that the Bömer 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% level,

irrespective of mode of adulteration either directly to ghee or through milk.

2.1.9 REFRACTIVE INDEX AND BUTYRO REFRACTOMETER READING

This property, which concerns the degree of bending of light waves

passing through a liquid or transparent solid, is a characteristic for the

particular liquid or solid. For oils and fats, it increases with the unsaturation

and decreases with rise in temperature. In the case of milk fat, the constant

may be determined readily with an Abbe refractometer at 40°C. The

instrument is calibrated in butyro-refractometer (BR) readings instead of

absolute refractive indices. The refractive index of milk fat generally, ranges

between 1.4538 and 1.4578 (Jenness and Patton, 1969; Walstra and Jenness,

1984). For animal fats at 40°C, it lies in the range of 1.4570 and 1.4630. For

most of the vegetable oils, it ranges from 1.4600 to 1.4750 but for some

vegetable oils like lauric fats (coconut oil, palm kernel oils and palm oil) it

lies in the range of 1.4480 to 1.4580 (Hamilton and Rossell, 1986;

Karleskind, 1996).

Refractive index and BR readings are inter-convertible (BIS, 1981;

Rangappa and Achaya, 1974; Bolton, 1999; Winton and Winton, 1999). 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 (38-39) and palm oil (39-40).

Feeding of cottonseed oil raises the B.R. reading by five units in case of ghee

(Rangappa and Achaya, 1974). Normally, BR reading or refractive index of

oils and fats increases with the increase in unsaturation and also chain length

of fatty acids. 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,


11

1973; Sharma and Singhal, 1995) and vanaspati (Sharma and Singhal, 1995)

at a level of 5 to 20% 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% of the original fat on the basis of increase in B.R.

reading of the fat. 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. Kumar (2008) concluded from his

study that adulteration of milk fat with vegetable oils (except palm oil) added

individually at 10 percent level in cow ghee and 15 percent level in buffalo

ghee, using the general limit of BR reading as 40 to 43 for Haryana state

(PFA, 2009), could be detected. However, adulteration of cow and buffalo

pure ghee with animal body fats could not be detected at any of the levels

studied.

Bhalerao and Kummerow (1954, 1956a) opined that the refractive

index of whole fat cannot give reliable results about adulteration and

suggested the use of alcohol fractionation (soluble and insoluble fractions) to

increase the concentration of adulterants in one of these fractions, causing

enough shift in the refractive index and thus making the detection of the

adulterants easier. They further improved the degree of accuracy of detection

by subjecting the above alcohol insoluble fraction to acetone fractionation,

and subsequent iodination of the acetone soluble fraction before determining

refractive index.

2.1.10 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

(5.0 g) in a test tube (8 cm × 1.5 cm) to become opaque (OD0.5) 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. In pure state, both cow and buffalo

ghee took almost similar time to become opaque, but adulterated cow ghee

took more time than the adulterated buffalo ghee to show a noticeable

opacity. The difference in opacity time between pure and adulterated ghee at

5, 10 and 20% level, respectively, was 7 to 8, 9 to 10 and 14 to 16 min in


12

case of adulteration with pig body fat; 18 to 20, 26 to 29 and 33 to 34 min in

case of adulteration with goat body fat; and 22 to 26, 31 to 32 and 35 to 36

min in case of adulteration with buffalo body fat. He concluded that the

adulteration with buffalo, goat and sheep body fats at 5% level and above

could be safely detected by opacity test. He further recommended that if the

sample exhibits opacity within 20 min, it is suspected to be adulterated with

animal body fats particularly; buffalo, goat or sheep body fats. However, the

limitations of this test are that the detection of pig body fat up to 10% 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). Their study revealed that ghee took more than 35 min to

become opaque, while vanaspati and lard (pig body fat) took 2 to 2.5 min

and 6 to 7 min, respectively. On the other hand, tallow (buffalo and goat

body fats) took very less time (<18 seconds) to become opaque. They did not

find any significant difference in the opacity time of pure ghee and ghee

samples adulterated with vanaspati and lard even up to 20% level. However,

in case of adulteration with buffalo and goat body fats, the opacity times

were significantly reduced, thereby making the detection possible. Here

also, mode of adulteration (whether added directly to ghee or through milk)

did not show marked differences.

In a modified procedure, Panda and Bindal (1998a) also studied the

opacity profile of pure ghee and adulterants and 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. 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). They also successfully employed the opacity test to detect the

presence of vegetable oils and animal body fats when added directly to milk.

These workers also observed the limitations of their test that the cotton tract


13

area ghee (opacity time; 11-12 min) resembled with ghee adulterated with

animal body fat such as pig body fat at 5% level (10-11 min), as reported

earlier by Singhal (1973).

2.1.11 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 which depends upon the nature

of the solvent, nature and amount of most insoluble glycerides (usually tri-

saturated glycerides) present in a fat as well as the mutual solubilizing power

exerted on these glycerides by the soluble glycerides (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% ethanol and iso-amyl

alcohol, and reported that gross adulteration of ghee with vanaspati could

easily be detected. Delforno (1964) reported the CTD values for butter as

50.5 to 57.5°C (average 54°C), for coconut oil as 28 to 41°C (average 33°C)

and for other oils and fats as 67 to 82.5°C (average 71-78°C). 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% ethyl alcohol and iso-amyl alcohol in the ratio of 2:1 (Bhide and Kane,

1952). Likewise, CTD was used for the detection of adulteration of ghee

with mineral oils by Kane and Ranadive (1951) using aniline as solvent. The

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

acidity (FFA) and rancidity (peroxides). Kumar (2003), using solvent

mixture (ethyl alcohol and iso-amyl alcohol, 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 and CTD value above 55 ºC indicates possible

adulteration.

2.1.12 FRACTIONATION OF MILK FAT

Fractionation is a thermally controlled process (with or without

solvent) in which the milk fat is subjected to a specific temperature/time

profile to allow a portion of milk fat to crystallize. The crystals are then

physically separated from the liquid fraction using vacuum filtration,


14

pressure filtration, centrifugation, an aqueous detergent solution, etc.

(Kayalegian et al., 1993). Besides these physical processes, chemical

(hydrogenation, inter-esterification) and biological processes are also used in

the industry for this purpose. Single step melt crystallization or dry

fractionation of milk fat into lower and higher melting fractions reveals that

the lower melting fractions of milk fat contain somewhat greater levels of

unsaturated acids and the fat constants for these fractions show a lower

saponification number, a higher iodine value, a higher refractive index and,

of course, a much lower melting range.

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,

isopropylalcohol and 2-nitropropane. Bhalerao and Kummerow (1954, 1956

a & b) 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% level could be detected. Arumughan and

Narayanan (1979) fractionated the buffalo and cow ghee at 29°C for 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. Some earlier workers (Tawde and Magar, 1957) demonstrated the

detection of vegetable fats in butterfat through fractionation of the urea

adducts prepared from 10 and 20% methanolic solution of urea followed by

determination of their refractive indices, iodine value, saponification

equivalents, melting point, etc. to ascertain adulteration.

Krienke (1953) fractionated the fat by selective solidification at 95,

80, 70 and 60°F into four solid fractions and one liquid fraction. On the basis

of R.M. values of these fractions from pure milk fat and adulterated milk fat,

as little as 2% foreign fat in milk fat could be detected. Latif and Mazloum


15

(1969 a & b) carried out fractionation by dry acetone at 20, 0 and -10°C,

followed by determination of the physical constants such as saponification

value, iodine value, Reichert-Meissl (RM) value, Polenske and Kirschner

value and reported that the addition of 10% vegetable fat increased the

iodine value, while animal body fat caused depression in the RM and

Kirschner value, thus enabling their detection. Farag et al., (1983) carried

out the fractional crystallization of fat (pure and adulterated ghee samples)

dissolved in silver nitrate-saturated methanol acetone mixture (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, whereas 22:0 is important in second and third fraction.

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

liquid fraction did not offer any added advantage in detecting adulteration of

milk fat with mixture of vegetable oils (sunflower oil, soyabean oil &

groundnut oil) or vanaspati with body fats (goat or pig) using B.R. 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 unfractionated ghee samples.

Kumar (2008) employed solvent fractionation approach using acetone

as a solvent to get different fractions. Three temperatures (16, 8 and 4°C)

were selected for successive fractionation. The solid fractions obtained at 16

and 8°C were named as S16 and S8, respectively, while solid and liquid

fractions obtained at 4°C were named as S4 and L4, respectively. On

fractionation, animal body fats got concentrated in the first fraction (S16)

whereas vegetable oils got concentrated in the last fraction (L4). Hence, first

fraction was analyzed for complete liquification time (CLT) test performed

at 44 and 46°C, and last fraction was analyzed for BR reading and iodine

value. Results of CLT test at 44oC done for first fraction (S16) of cow and

buffalo pure ghee showed that the temperature of 44°C is not suitable for

application of CLT test on first fractions because it has failed in case of pure

cow ghee as fraction of pure cow ghee obtained in the months of summer

season (May to September) did not melt completely and some opacity

remained throughout in the body of the fat fraction. CLT of solid fractions


16

(S16) of pure cow ghee and pure buffalo ghee at 46oC ranged from 4 min 5

sec to 9 min and from 5 min 10 sec to 7 min 15 sec, respectively.

Considering the overall range of CLT at 46°C i.e. from 4 min 5 sec to 9 min

for both cow and buffalo pure ghee and also considering the melting

behaviour of samples at this temperature, it was observed that buffalo body

fat adulteration along with either of the vegetable oils could be detected even

at 5% level in case of cow ghee, while it was detected only at 15% level in

buffalo ghee. Goat body fat along with either of the vegetable oils was

detected at 10% level of adulteration in cow ghee, while it was detected only

at 15% level of adulteration in buffalo ghee. However, pig body fat was

detected at 15% level of adulteration with either of the vegetable oils in both

cow and buffalo ghee.

2.1.13 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

characterization of fats and oils.

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

2.1.13.2 Tests Based on Ultraviolet (UV) Spectroscopy

Ultraviolet (UV) spectroscopy has been applied for characterizing the

poly-unsaturated fatty acids (PUFA) content of various oils and fats

including milk fat and also for the detection of butterfat adulteration with

foreign fats. Conjugated dienoic acids show an absorption maximum at 233

µm, conjugated trienoic acids at 268 µm, conjugated tetraenoic acid at 315

mµ and conjugated pentaenoic acid at 346 µm (AOCS, 1959). Their non-

conjugated counterparts are estimated at the same wavelength after alkali-

isomerization to the conjugated form, thus permitting the total PUFA

measurement of oils and fats. Using UV spectrophotometry, Ramamurthy

and Narayanan (1972) reported that the average values for total dienoic,


17

trienoic, tetraenoic and pentaenoic acids for cow and buffalo milk fats

ranged between 1.667 to 1.921%, 0.544 to 0.565%, 0.134 to 0.202%, and

0.062 to 0.107%, respectively. Similar values for lard (Magidman et al.,

1963) averaged as 12.2, 0.87, 0.35 and 0.15%, respectively. Not much

information is available in the literature on the PUFA content of other body

fats as well as vegetable oils and fats. Morris et al., (1952), however,

reported that conjugated tetraenoic systems present in butterfat were

significantly absent in margarine fats and cottonseed oils. Accordingly,

Roos (1963) suggested that the presence of small amounts of conjugated

tetraenoic acid in milk fat can be used for distinguishing milk fat from

vegetable fat.

Rego et al., (1964) examined the UV spectra of the fats from butter,

margarine and their mixture after dissolving in hexane, and reported that all

the samples of genuine butter, but not the margarine, gave a peak with a

maximum at about 232 nm.

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 for both fats. However, 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. But on this basis, adulterated

ghee could not be differentiated from pure ghee (Sharma, 1989; Kumar,

2003).

2.1.13.3 Tests Based on Infra-Red (IR) Spectroscopy

The infra-red (IR) region, which extends from 0.8 to 1,000 µm

(14,000 to 20 cm-1

in wave number) is generally divided into three areas:

near infra-red (0.8-2.5 µm), mid-infra-red (2.5 to 15.38 µm) and far infra-red

(15-1,000 µm). 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-


18

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% 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; Firestone and Villadelmar, 1961) applied IR

spectrophotometry and observed that the absorption maxima at 10.36 µ had

been increased by the addition of hydrogenated fats containing iso-oleic

acids (trans-octadecenoic acids). DeRuig (1968) used differential IR

spectroscopy and reported that the addition of more than 5% hydrogenated

fats (palm oil and fish oil) in pure butterfat could be detected by getting the

values outside the ellipse obtained in a graph plotted between transmittances,

T10.85 to T10.33 on the abscissa and T10.85 to T10.55 on ordinate. Beef

tallow in butterfat could also be detected by studying the IR absorption

spectra using 10.85 µ (920 cm-1

) at 32.5°C, in which butterfat adulterated

with 10% tallow shows a band, but pure butterfat does not show any band.

Francesco et al., (1971) studied the compositional differences in the

unsaponifiable matter of butterfat and synthetic fat by IR spectroscopy of the

fractions obtained by thin layer chromatography on silica gel and observed

that unsaponifiable matter of butterfat contained a polyene fraction which

was absent from that of synthetic fat. They also carried out the fractionation

of the unsaponifiable matter from the two fats by column chromatography on

alumina and on the basis of differences in their IR absorption pattern; they

reported that adulteration of butterfat with synthetic fat at 10% level 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% 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% 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


19

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% level.

Recently, 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. Bellorini et al., (2005) stated that Fourier Transform IR

spectroscopy in conjugation with multivariable statistical analysis can be

safely utilized for identification of pure fat samples of cocoa butter, fish oil

and butter fat, lard, chicken body fat and tallow from each other. However,

the 10% tallow in lard cannot be detected reliably if other fats than lard and

tallow present in the mixture. If mixture contain only 10% tallow in pure lard

it can be detected using multivariate statistic but with not sufficient accuracy.

Using Temperature Controlled Attenuated Total Reflectance Mid Infrared

Spectroscopy (ATR-MIR) Koca N. et al., (2010) successfully detected

margarine adulteration in butter fat.

2.1.14 DILATATION BEHAVIOUR

This property is based on the thermal expansion behaviour 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 (2003) applied this property 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.


20

2.1.15 MICROSCOPIC EXAMINATION OF FAT

Microscopic examination of fats sometimes yields valuable

information. Microscopic examination is usually direct evidence, whereas

chemical examination is generally an indirect evidence requiring

interpretation. Microscopic examination can be used for checking milk fat

purity suspected with the presence of foreign fats. For this, fat dissolved in

methylated ether is allowed to stand for 30 minutes in ice water or at 20°C

for 24 hours. The crystals separated are observed under microscope. Beef fat

crystallizes in characteristic fan like tufts with ends more or less pointed, but

are never chisel shaped as in case of lard. Inferences, based on these

observations must be drawn from the results of first crystallization as

repeated recrystallization causes the loss of characteristic chisel ends. Palm

oil crystals, which melt rapidly when mounted on a slide, cannot be

examined by this test. Hydrogenated oil crystals simulate the fan like beef

crystals very closely, but the crystals are smaller and may arrange

themselves to give cellular appearance to the field.

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. Using this

parameter, Kumar (2003) reported that adulteration of ghee samples with

15% groundnut oil could be confirmed.

2.1.16 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


21

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% 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% level in butterfat could

not be detected by this method. Roos and Tuinstra (1969) using DTA

showed that addition of 5 to 10% 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.

2.2 METHODS BASED ON CHEMICAL PROPERTIES

Several chemical methods based on fatty acids, triglycerides,

unsaponifiable matter, and specific tests using gas liquid chromatography

(GLC), thin layer chromatography (TLC), paper chromatography, etc. have

been used to characterize the various fats and oils with a view to check the

purity of milk fat.

2.2.1 TESTS BASED ON FATTY ACIDS

Edible oils and fats including milk fat are consisted of 95 to 99% as

glycerides (Triglycerides, 95-96%; diglycerides, 1.26-1.59%; mono-

glycerides, 0.016-0.038%). Of the total weight of glycerides, 94 to 96% is


22

contributed by fatty acids. Thus, the chemistry of oils and fats is, to a very

large extent, the chemistry of their constituent fatty acids. Milk fat contains

more than 437 fatty acids of different chain length and unsaturation. 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:

2.2.1.1 Tests Based on Physico-Chemical Constants

Certain well-known physical and chemical constants have been

derived for the purpose of characterization of oils and fats. Among those

constants three determinations, the Reichert-Meissl value, the Polenske value

and the Iodine value, measure certain specific constituents of milk fats while

other two, the Saponification value and Butyro-refractometer reading, give

an overall average nature of the constituent fatty acids present (Rangappa

and Achaya, 1974). These physico-chemical constants are described briefly

in the following sections:

2.2.1.1.1 Reichert-Meissl (RM) value

RM value is the number of milliliter of 0.1 N alkali solution required

to neutralize the steam volatile, water soluble fatty acids distilled from 5 g of

fat under specified conditions. This constant for milk fat is quite significant

since it is primarily a measure of butyric (C4:0) and caproic (C6:0) acid.

RM value for milk fat ranges from 17 to 35, which is well above the value

(generally 1) for all other fats and oils except coconut oil and palm kernel oil

for which the value ranges between 4 to 8 (Singhal, 1973 & 1980). Feeding

of cottonseed to milch animals lowers the RM value of ghee by 5 to 6 units

(Rangappa and Achaya, 1974). Singhal et al., (1973) in their study on the

physico-chemical properties of different layers of ghee obtained after one

month storage at 30 to 35°C reported that the liquid layer exhibited a higher

RM value than solid layer. The study conducted by Kumar (2008) revealed

that adulteration of pure cow ghee with animal body fats body (buffalo body


23

fat, goat body fat and goat body fat) and all vegetable oils (palm oil, rice

bran oil and soybean oil) individually at 10 percent level, whereas in case of

pure buffalo ghee at 15 percent level of adulteration could be detected using

RM value as a base.

2.2.1.1.2 Polenske value

Polenske value denotes the number of milliliters of 0.1 N alkali

solution required to neutralize the steam volatile and water insoluble fatty

acids distilled from 5 g of fat under specified conditions. This value is

substantially a measure of caprylic (C8:0) and capric (C10:0) acid. The

Polenske value for milk fat ranges from 1.2 to 2.4. This value for other oils

and fats (Singhal, 1980; Winton and Winton, 1999) is also low (less than 1)

except the coconut oil (15-20) and palm kernel oil (6-12). Feeding of

cottonseeds to milch animals reduces the Polenske value of ghee by 0.3 to

0.7 units (Rangappa and Achaya, 1974). Singhal et al., (1973) reported that

the liquid portion of ghee obtained on storage at 30 to 35°C for one month

showed higher Polenske value than the solid portion. Kumar (2008) revealed

that adulteration of cow and buffalo pure ghee with any of the adulterants

either animal body fats or vegetable oils could not be detected at any of the

levels studied, except adulteration of buffalo ghee with the palm and soybean

oils which could be detected at 15 percent level.

2.2.1.1.3 Iodine value

Number of grams of iodine absorbed by 100 g of fat under specified

conditions represents the iodine value. This constant is a measure of

unsaturated linkages present in a fat. The iodine value for milk fat ranges

from 26 to 35, which is low in comparison to most of the other fats & oils

(Singhal, 1973). Animal body fats show slightly higher iodine value ranging

from 36 to 49. Whereas, for vegetable oils, the value is very high (74-145)

except coconut oil (6-10) and palm kernel oil (10-18). For hydrogenated

fats, it lies in the range of 70 to 79. Feeding of cottonseed raises the iodine

value of ghee up to 10 units (Rangappa and Achaya, 1974). Liquid fraction

obtained as a result of ghee storage at 30 to 35°C for one month showed

higher iodine value than solid fraction (Singhal et al., 1973). Kumar (2008)

reported that iodine value can be a useful parameter in detecting adulteration


24

of cow ghee as well as buffalo ghee with various vegetable oils (except palm

oil) at the level of more than 10 percent.

2.2.1.1.4 Saponification value

Saponification value, which denotes the number of milligrams of

KOH required to saponify one gram of fat, gives an indication of average

molecular weight of fatty acids present. For milk fat, animal body fats,

vegetable oils and hydrogenated fats, the value ranges from 210 to 233, 192

to 203, 170 to 197 and 197 to 199, respectively. Coconut oil and palm kernel

oil show higher saponification value ranging from 243 to 262 (Jenness and

Patton, 1969; Singhal, 1980). Feeding of cottonseeds to milch animals

lowers this value by 7 units (Rangappa and Achaya, 1974). Liquid portion of

ghee separated on storage at 30 to 35°C for one month showed higher

saponification value than solid portion (Singhal et al., 1973).

Kumar (2008) concluded from his study that based on saponification

value, adulteration of pure cow ghee with either animal body fats or

vegetable oils at 10 percent levels of adulteration could be detected except

buffalo body fat which could be detected even at 5 percent level of

adulteration. Whereas, in case of buffalo ghee, only 15 percent level of

adulteration either with animal body fats (except pig body fat) or with

vegetable oils (except palm oil) could be detected. However, when the data

on cow and buffalo ghee was pooled, the study revealed that adulterants

(body fats and vegetable oils) studied could be detected at 10 and 15 percent

level of adulteration, except palm oil and pig body fats which could be

detected only at 15 percent level of adulteration.

Several earlier workers (Achaya and Banerjee, 1946; 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% 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% level.

However, when adulteration was done at 50% level, the values remained


25

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 irrespective of mode of adulteration whether added directly to

ghee or through milk.

2.2.1.2 Tests Based on Gas Liquid Chromatography (GLC) of Fatty

Acids

The technique of GLC, which utilizes retention time, a characteristic

of a particular component under specified conditions, gives fatty acid profile

of oils and fats obtained following the conversion of the triglycerides into

more volatile methyl esters of their component 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 (14:0) and myristic (16:0) acids in very large amount

(Rangappa and Achaya, 1974).

The GLC technique was employed by several workers for the

detection of milk fat adulteration through determining the ratios of different

fatty acids. 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 C4:0/C6:0+C8:0 fatty

acids >1.8 was considered to be adulterated with margarine, coconut oil or

tallow or pig body fat trans-esterified with butyric acid, while other workers

(Provvedi and Cialella, 1961) suggested ratios of other fatty acids such as

C4:0/C12:0 > 3, C18:1/C18:0 > 2 and C12:0/C10:0 >1 for the detection of

vegetable oils in butterfat. 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., 1982; Ulberth, 1994; Sharma and Singhal, 1996; Panda

and Bindal, 1997; Kumar, 2003). Farag et al., (1983) determined the fatty

acid profile of three 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


26

changed with different adulteration levels and can be used as a marker to

detect the admixture. Singhal (1987) reported that on GLC analysis of

saturated triglycerides (STGs), ratio of fatty acids, viz., C15:0/C17:0 and

C16:0/C18:0 was lower in ghee samples adulterated with body fats even in

the presence of vegetable oils. Sharma and Singhal (1996) analyzed the

buffalo ghee samples adulterated with body fats (buffalo, goat, pig) and

vanaspati at 20% 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% using C18:2 or C22:1 as

marker acid. Kumar (2003) found that the adulteration of ghee with body

fats or vanaspati at 15% 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% vegetable oils (sunflower oil, soybean oil and groundnut oil) 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.

Using GC-MS and using Principle Component Analysis (PCA) Bellorini et

al., (2005) concluded that pure samples of vegetable fats and oils and fish

oils are very well identified among other pure samples. Tallow and lard can

be identified if samples are purely composed of tallow or lard respectively

but only greater than 10% addition of tallow in lard is detectable. However,

method is not suitable to detect mixtures of fats and works only for

identification of pure samples.

2.2.2 TESTS BASED ON THE NATURE AND CONTENT OF UNSAPONIFIABLE

CONSTITUENTS

The unsaponifiable matter (USM) which can be obtained from oils and

fats after saponification with alkali and subsequent extraction by a suitable

organic solvent constitutes less than 2% by weight of fat. It is a repository of

so many valuable constituents, like sterols (cholesterol and phytosterols), fat-

soluble vitamin (A, D, E and K), hydrocarbons such as squalene, pigments,

etc. Mineral oil, if added to oils and fats, will appear in USM (Kirk and

Sawyer, 1999). Milk fat contains USM in the range of 0.30 to 0.45% by

weight (Jenness and Patton, 1969) chiefly consisting of cholesterol (0.25 to

0.40% by weight of fat). Vegetable oils and animal body fats like lard and


27

tallow have USM in the range of 0 to 2% and 0 to 1%, respectively

(Hamilton and Rossell, 1986).

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.

2.2.2.1 Tests Based on Sterols

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

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

vegetable oils (Kumar et al., 2002). Cholesterol is the characteristic sterol of

animal fats, while sterols from vegetable 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). Vegetable fats

contain the sterols mainly in the ester form, while animal body fats contain

mostly the free form (Rangappa and Achaya, 1974). Milk fat contains

cholesterol in free and ester form in the ratio of 10:1 (Fox, 1995).

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 of sterol acetate fraction is more than 117°C

(IDF, 1965; Rangappa and Achaya, 1974; 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% level or vanaspati at 10% level did not move at

all when observed under UV light or when exposed to iodine vapors.

However, ghee from cottonseed fed animals behaved like adulterated ghee.

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


28

and their mixture, and observed that USM from ether soluble fractions of

ghee adulterated with 10% buffalo body fat exhibited two spots. However,

pig body fat in ghee could not be detected. IDF (1966) recommended a TLC

method for the detection of vegetable fats in milk fat based on the

appearance of a small band of ß-sitosterol acetate in addition to the major

band of cholesterol acetate using reversed phase system consisting of

undecane/acetic acid-acetonitrile saturated with undecane. 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% level and coconut oil at 25% level in ghee could be detected on

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

respectively. Parodi (1972) differentiated inter-esterified fat (a mixture of

tributyrin, beef tallow and coconut oil) from milk fat by examining the free

and esterified sterols using solvent mixtures of hexane: di-isopropyl ether in

the ratio of 80:20 and 93:7 (v/v), respectively, on glass plates coated with

silica gel G. Inter-esterified fats contained mainly sterol esters of high

molecular weight fatty acids, whereas milk fat contained cholesterol mainly

(90-95%) in the free form. Mathew and Kamath (1978) could detect

vegetable oil/vanaspati in ghee even up to 2% level employing reverse phase

TLC on Kieselguhr G coated glass plates which were impregnated in a

solvent mixture of undecane (pre-equilibrated with acetic acid and

acetonitrile) and petroleum ether before applying the fat samples. Sterol

acetates of the fat samples (obtained by treating the fat dissolved in

chloroform, with digitonin and acetic anhydride) were spotted on the TLC

plates and developed twice in a solvent mixture of acetic acid and

acetonitrile (1:3, v/v) and bromine (0.5%) followed by spraying with 10%

phosphomolybdic acid in ethanol. Cholesterol acetate and phytosterol acetate

appeared as blue spots with Rf values of 0.48 and 0.39, respectively. A

simple TLC method was developed by Chourasia et al., (1994) by spotting

USM on Silica gel G coated plates using chloroform: acetone: acetic acid

(90:10:1; v/v) as the developing solvent. Based on the appearance of a

characteristic violet spot in adulterated ghee observed at Rf 0.55 to 0.60,

Kokum (Garcinia indica) fat in ghee up to a level of 5% could be detected.

Sharma (1989) carried out TLC of USM of ghee and animal body fats using


29

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% body

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

present in body fats. TLC of free and esterified sterol digitonides could

detect adulteration of ghee with 10% body fats, while TLC of trimethyl silyl

ethers (TMS) from USM failed to detect ghee adulteration. 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 (Colombini et al., 1978; Homberg and Bielefeld,

1979), however, by this method, addition of body fats cannot be detected as

body fats also have cholesterol. Kamm et al., (2002) applied on-line liquid

chromatography-gas chromatography (LC-GC) for the detection of vegetable

oils in milk fat using -sitosterol as marker. The method involved trans-

esterification of the fat, pre-separation of the sterol fraction from other lipid

constituents and on-line transfer to the capillary GC system. This method

allowed the detection of adulterations at low levels.

2.2.2.2 Tests Based on Tocochromanols

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% in butterfat, 0.05 to 0.168% in

vegetable oils except coconut oil, which contains only 0.0083% and 0.0005

to 0.0029% in body fats (Bailey, 1951 and Kumar et al., 2002). Thus,

tocopherol content of butterfat is low as compared to most vegetable oils and

fats. Therefore, addition of vegetable fats to butter will result in a significant

increase in tocopherol content of adulterated butterfat. Accordingly, some

workers (Mahon and Chapman, 1954; Nazir and Magar, 1959; Markuze-


30

Zofia, 1962; 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). This differential status of isomers of

tocochromanols in milk fat as against vegetable oils has not been used as a

tool for their differentiation.

2.2.3 TESTS BASED ON WHOLE FAT/TRIGLYCERIDES

Differences in the chemical composition of triglycerides have also

been extensively used as a basis for the detection of adulteration of milk fat

with foreign fats. Based on their fatty acid composition, the possible number

of triglycerides in milk fat is calculated to be more than 1,300 (Barron et al.,

1990). Several methods based on paper chromatography, thin layer

chromatography (TLC) and gas liquid chromatography of triglycerides,

employed to detect the adulteration of milk fat, have been described in the

following sections.

2.2.3.1 Tests based on paper chromatography of triglycerides

Ramachandra and Dastur (1960) standardized a circular paper

chromatographic technique for the detection of common ghee adulterants,

like vanaspati and animal body fats by spotting 50% solution of fats in CCl4

on the paper chromatogram using a solvent system of ethyl alcohol: iso-amyl

alcohol: CCl4 (35:65:10; v/v) and observed that normal ghee sample moved

well away from the origin, whereas cottonseeds fed buffalo ghee did not

move and behaved like adulterated ghee. Adulteration of ghee with vanaspati

(10%) and body fat (5-10%) could be detected using this technique.

Rego and Garcia-Olmedo (1963) employed one dimensional paper

chromatography for the butter, margarine and their mixtures and reported

that adulteration of butter with margarine could be detected from the size of

spot due to butyric acid of hydrolyzed fat. Singhal (1973) employed circular

paper chromatography using ethyl alcohol (95%) and iso-amyl alcohol in 1:1

ratio as solvent mixture for the detection of ghee adulteration with body fats


31

(goat, sheep, pig and buffalo). Samples dissolved in CCl4 solution were

spotted on filter paper. Pure cow or buffalo ghee moved with the solvent

giving a single spot at the solvent front, whereas ghee samples adulterated

with any of the body fats (buffalo, goat and sheep) at 5% level showed

streaking from the point of origin. Adulteration with pig body fat (10%

level) could not be detected by this method. Cotton tract ghee behaved

similar to body fat samples. Singhal (1987) reported that body fats and ghee

samples adulterated with body fats at 10% level showed streaking even in

the presence of vegetable oils, but the method failed when vegetable oils

alone were added. Sharma (1989) employed ascending paper

chromatography using dichloromethane: isopropanol: glacial acetic acid

(80:30:20; v/v) as solvent system to ghee and body fats (goat, sheep and pig)

and reported that adulteration of ghee with buffalo and goat body fats below

10% level and pig body fat above 10% level could be detected even in the

presence of cotton tract ghee.

2.2.3.2 Tests Based on Thin Layer Chromatography of Whole Fat

Chakrabarty et al., (1968, 1969 & 1980) developed a thin layer

chromatographic technique for the detection of hydrogenated groundnut oil,

tallow and mohua oil in butterfat (ghee) by first separating the trisaturated

glycerides (GS3) by argentation-TLC on Silica Gel-G and then resolving

them into individual glyceride components on paraffin impregnated

Kieselguhr G plates. After microsaponification, fatty acids were isolated on

paraffin coated Kieselguhr G plates to indicate the component fatty acids of

particular glyceride spots. The solvent systems used were: (i) acetone-

methanol-acetic acid (60:40:0.5) for the triglycerides and (ii) 90% acetic acid

for fatty acids. Based on the difference in triglycerides and their fatty acids,

especially C12:0 to C16:0, detection of adulteration up to 5% level was

possible by this method. Hendrickx and Huyghebaert (1968, 1970)

employed TLC on Silica gel plate, using petroleum ether: diethyl ether:

formic acid (60:40:1.5% v/v), for the detection of substitution fats

(containing inter-esterified fats) in butterfat by identifying the

monoglycerides formed during the preparation of substitution fats. As little

as 2.5% substitution fats could be detected by the appearance of a clear band

of monoglycerides. Singhal (1973) applied TLC on Silica Gel-G plate using

acetone: methanol: glacial acetic acid (70:30:2; v/v) as the solvent system


32

and reported that ghee and body fats behaved in a similar way exhibiting

streaking. Adulterated samples even at 10% level did not show any

differential behaviour.

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

fats in ghee (butterfat) to the extent of even 5% 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. Singhal (1987) reported that saturated triglycerides (STG)

obtained from body fats and ghee samples adulterated with body fat showed

streaking even in the presence of vegetable oils. Adulteration with vegetable

oils alone could not be detected. 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.

2.2.3.3 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% level based on the increase in

the content of high molecular weight triglycerides, C52:0 and C54:0 peaks,

respectively. Parodi and Dunstan (1969) reported that the trisaturated

glycerides (GS3) analysis could be used for the detection of beef tallow in

Australian butterfat at 5 to 10% level. Parodi (1971, 1973) carried out GLC

analysis of butterfat adulterated at various levels with beef tallow and

reported that out of the total samples analyzed, 59.8% at 10% level, 35.7% at

15% level, 9% at 20% level and 2.7% at 25% level of adulteration were not

detected on the basis of triglycerides and fatty acid ratios. However,

subsequently in 1973, the same author using the similar technique observed


33

that adulteration of butterfat samples with beef tallow (20% level) and

vegetable oils (5% level) was detectable. Guyot (1978) using GLC of

commercial butter found that triglycerides were in the order of C36:0 >

C38:0 > C40:0 > C42:0 > C44:0 > C50:0 > C52:0. Beef tallow and lard

triglycerides ranged mainly from C44:0 to C54:0, with C52:0 as main

triglyceride. He found the ratio of C52:0/C50:0 was less than 1 in pure

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

concluded that the C52:0/C50:0 ratio together with C52:0/C38:0 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% 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.

Some workers (Precht, 1990 & 1992; Lipp, 1996a & b) compared the

triacylglycerol composition of different fats as analyzed by GLC and

designed a multiple linear regression equation by which foreign fats could be

detected with substantially improved sensitivity. However, the method was

suitable only when a single foreign fat was added to milk fat. Currently,

European Union (EU) applies the method of Precht (1992) for triglyceride

analysis 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% using 3,5-

cholestadiene analysis and up to 2% using multivariate statistical techniques

could be done. Several investigators (Contarini et al., 1993; Renterghem,

1997; Collomb, 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. The objective

of the study was to propose a methodology for the detection of adulteration

of 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


34

vegetable and animal fats in a simple, rapid and precise way, even at lower

levels.

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 cannot ensure a

foolproof detection.

2.2.4 TESTS BASED ON RESIDUAL PROTEIN CONTENT IN PURIFIED FAT

Bellorini et al., (2005) used immunoassay and PCR technique and

applying PCA succeeded in detection of tallow in lard. For the purpose of

immunoassay and PCR the fat samples are classified according to Residual

Insoluble Impurities (RII) i.e. RII concentration above 0.15%, between 0.15

and 0.02 and below 0.02.

Immunoassay was capable of detecting ruminant protein even in

material that was heat treated up to 138°C. As RII concentration went down

detection became difficult. At RII below 0.02% in tallow such tallow cannot

be detected in lard added in any proportion. In RII concentration range

between 0.15 and 0.02 in tallow such tallow if added at the rate of even at

5% it can be detected but not at 2%.

PCR technique proved more sensitive than immunoassay and in some

cases, it can detect even 2% adulteration of lard with tallow containing

0.02% RII but not in all cases. However, when analyzing tallow of the

highest quality (“permier jus”), the immunoassay and PCR technique was

not applicable as residual protein or DNA in the sample was too low.

2.3 METHODS BASED ON TRACER COMPONENTS OF FATS AND

OILS

Tracer components can be defined as those compounds, which are

present in adulterant oils and fats, either naturally or by addition, but absent

in pure ghee. Addition of some tracer component in the likely adulterant of

ghee has been suggested as a rapid and reliable tool to identify them in milk


35

fat. A tracer can be a latent colour, which is not detectable visually, but get

identified by its colour reaction with certain chemicals or a colouring matter

(natural or synthetic) which may impart direct colouration distinct from that

of the natural colour of butterfat.

Among tracers, in India, sesame oil is added (5% by weight) to

vanaspati according to food laws (PFA, 2009) for its detection in ghee by

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

crimson colour due to the reaction between furfural and sesamol formed by

the hydrolysis of sesamolin (present in sesame oil) in the presence of

concentrated HCl. Use of hydrofuramide (Kapur et al., 1960) or P-hydroxy

benzaldehyde (Sharma, 1989) has also been suggested in place of furfural for

this test. Another tracer is tannins which are assumed to be naturally present

as impurities in palm oil. Ghee samples adulterated with palm oil give

purssian blue colour with potassium ferricyanide and ferric chloride reagent

and based on this reaction; Bector & Sharma (2002) have reported that palm

oil added to ghee can be detected at the level of 5%. However, limitation of

this method is that the ghee samples having BHA as antioxidant also give

positive test.

Gamma oryzanol, a natural tracer, having antioxidant and cholesterol

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

revealed to be a mixture of phytosteryl ferulates comprising cycloartenyl

ferulate, 24- methylenecycloartenyl ferulate, and campesteryl ferulate as

major components (Xu & Godber, 1999; Chen and Bergman, 2005; Iqbal et

al., 2005). Crude rice bran oil contains less than or equal to 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). Apart from rice bran

oil, gamma oryzanol is also present as natural component of corn and barley

oils (http://www.cncahealth .com/health-notes.htm?ContentID=2850009).

On the basis of presence of gamma oryzanol, some methods have been

developed recently to detect it in other edible oils. Nasirullah et al., (1992)

reported a thin layer chromatographic method while Shukla et al., (2004,

2005) reported a colorimetric method for detection of rice bran oil in other

vegetable oils. However, no such study has been done on the detection of

rice bran oil in ghee. Rapid colorimetric test to detect even 2% rice bran oil

based on gamma oryzanol is described by Kumar et al., (2009).

http://www.cncahealth.com/health-notes.htm?org=cnca&ContentID=3596001


36

2.4 MISCELLANEOUS METHODS

In this section, methods such as to detect mineral oil and cotton seed

oil, and methods based on enzymatic hydrolysis, etc., are described in brief.

2.4.1 TESTS FOR MINERAL OILS

Adulteration of common edible oils and fats including milk fat with

cheaper mineral oils, such as paraffin oil, heavy and light fuel oil, petroleum

jelly, etc., has become a widespread phenomenon because of the price

difference. Mineral oils also referred as white oils are of different thickness

and refractive indices. 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% 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. Silverberg (1962) isolated the mineral oil from

adulterated ghee sample by adsorbing the latter on alkaline alumina column

followed by elution with light petroleum. Presence of mineral oil in eluted

material was confirmed by infra-red peaks at 3.40, 6.82 and 7.25 µ.

Besides the above reports of mineral oil detection in ghee, few reports

have also appeared for their detection in vegetable oils and fats using

turbidity test. Using Holde’s test, the presence of as little as 0.3% of mineral

oil in a fat can be detected by saponifying 10 drops of test sample (1 ml)

with 5 ml of 0.5 N ethanolic potassium hydroxide solution and adding 5 ml

of water to the hot soap solution and noting the appearance of turbidity

(Winton and Winton, 1999). 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).

2.4.2 TESTS BASED ON THE CONTENT OF SPECIFIC FATTY ACIDS

Certain specific fatty acids such as butyric acid, erucic acid, iso-

valeric acid, iso-oleic acid, cyclopropenoic acids, etc. which are either

characteristic or absent in milk fat or present in less quantity as compared to

adulterant fats have been used as an index for the detection of foreign fats in


37

milk fat. Unusual fatty acids like iso-oleic acid, iso-valeric acid, erucic acid,

cyclopropenoic acid, etc. are altogether absent in milk fat, but are found in

vegetable fats.

Butyric acid is found only in milk fat, but not in adulterant fats. Some

workers (Harper and Armstrong, 1954; Eckizen and Deki, 1976) reported

that any decrease in butyric acid content of milk fat below 9.6 mole

percentage would indicate its adulteration with foreign fat. The presence of

iso-valeric acid in dolphin oil has been used as the basis for its detection in

milk fat by several workers (Tappi and Menziani, 1952; Antoniani and

Cerutti, 1954; Parrozzano and Mancinelli, 1954; D’Arrigo, 1955; Bottini and

Campanello, 1955; Priori, 1955) using ascending paper chromatography.

2.4.2.1 TESTS FOR COTTONSEED OILS

Fatty acids containing cyclopropene ring, viz., malvalic (C18:1) and

sterculic (C19:1) 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, 1961;

Christie, 1970) for the detection of cottonseed oil in milk fat and also to

distinguish cotton tract ghee from normal ghee using Halphen test or

methylene blue reduction test.

2.4.2.1.1 Halphen Test

This 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. This test finds its application for differentiating the cotton

tract ghee from normal ghee (Singhal, 1980) as well as for the detection of

cottonseed oil in milk fat.

2.4.2.1.2 Methylene Blue Reduction Test

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


38

cyclopropenoic acids, while normal pure ghee did not reduce the colour of

methylene blue dye.

2.4.3 HYDROXAMIC ACID TEST

It is a colorimetric test, which is used to distinguish between butterfat

and other vegetable and animal fats (Nelson, 1954) based on the fact that fats

derived from milk (cow, goat, sheep, etc.), will form water soluble

hydroxamic acid-iron complexes. These complexes appear as a pink to

purple colour in water layer. The hydroxamic acid-iron complexes formed

from fatty acid esters in vegetable fats except coconut oil and of animal fats

are insoluble in water and do not contribute a distinctive pink to purple

colour to the water layer. Shipe (1955 a & b) modified the above test by

separating the water soluble and water insoluble hydroxamates. The soluble

fraction containing the complexes of butyric, caproic, caprylic and capric

acids is extracted with a butanol-ethanol mixture which removes the caprylic

and capric acids and leaves the other two in the water. The relative

proportion of these two pairs of acids estimated by comparing the colour

intensity in the aqueous phase before and after extraction could be used to

distinguish butterfat from other fats.

2.4.4 MODIFIED BIEBER’S TEST

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

adulteration with Kernel oil, was suitably modified by Sharma et al., (2005)

to detect the adulteration of ghee with vegetable oils. Their results showed

the presence of orange brown colour in case of refined vegetable oils & fats,

whereas in case of pure ghee samples no colour was observed. By this

method, adulteration of ghee with different vegetable oils to the tune of 5-

7% could be detected.

2.4.5 COLOUR BASED PLATFORM TEST FOR THE DETECTION OF VEGETABLE

OILS/FATS IN GHEE.

Sharam et al., (2007) have shown that minimum 7.5% level of

vegetable oil in ghee can be easily detected using rapid color based test. In

this test, one milliliter of clear molten fat was dissolved with 1.5 ml of

hexane in a tightly capped test tube. To this was added 1.0 ml of color


39

developing reagent (H2O:H2SO4:HNO3 :: 20:6:14), shaken vigorously and

kept undisturbed till it is separated into two layers. The appearance of a

distinct orange tinge in the upper layer indicates the presence of vegetable

oils/fats. This test was not affected by presence of animal body fats in

samples adulterated with vegetable oils.

2.4.6 TESTS BASED ON ENZYMATIC HYDROLYSIS

Lipases (triacylglycerol acyl hydrolases) are the enzymes that catalyze

the reversible hydrolysis of triacyl glycerols under natural conditions.

Pancreatic lipase which specifically hydrolyzes the primary hydroxyl

positions of glycerides can catalyze the complete breakdown of

triacylglycerols to free fatty acids and glycerol (Akoh and Min, 1998)

involving at least one isomerization step, in addition to the three hydrolytic

steps (Coleman, 1963), as follows:

Triglyceride 1,2 Diglyceride 2 Monoglyceride 1 Monoglyceride Glycerol

Pancreatic lipase digests some classes of milk triglycerides more

rapidly than others (de Man, 1961; Weihe, 1961; Raghuveer and Hammond,

1967; Soliman and Younes, 1986) due to difference in their glyceride

structure especially with regard to fatty acids distribution. Further,

triglycerides containing unsaturated fatty acids in 1 and 3 positions are

hydrolyzed faster as compared to those having saturated fatty acids in these

positions (Coleman, 1963). Fats such as buffalo milk fat with higher melting

triglycerides and long chain saturated fatty acids (C16:0 and C18:0) were

hydrolyzed slowly by pancreatic lipase as compared to cow milk fat

(Ramamurthy and Narayanan, 1974). Also, triglycerides with low melting

points greatly contribute to the faster rate of hydrolysis (Lakshminarayana

and Ramamurthy, 1986).

It is expected that other fats such as body fats and vegetable fats,

which have the preponderance of long chain fatty acids, will also have

different rates of hydrolysis vis-à-vis milk fat. Information on this aspect is

obscure. Moreover, it is also not known whether the rate of hydrolysis of fat

by lipase can be used for detecting milk fat adulteration.

The above review reveals that the detection of foreign fats in milk fat

is a very complex phenomenon, almost comparable with the detection of


40

Pacific water in a sample of Atlantic water. 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. Literature also reveals that

pig body fat (lard) among the animal body fats, and coconut and palm oils

among the vegetable oils, pose lot of difficulties owing to their resemblance

with milk fat in many respects. The detection methods available till date are

mainly based on the physico-chemical constants, fatty acid profile, sterol

analysis, partial solidification behaviour, etc. However, most of these

methods fail when a mixture of body fats and vegetable oils & fats is added

to milk fat.. On the basis of above preamble, a systematic study has been

planned to validate available tests.

CHAPTER – 3

Materials and Methods

MATERIALS AND METHODS

41

This chapter deals with the materials and methodologies used in the

present study on detection of different adulterants in ghee.

3.1 CHMICALS AND REAGENTS

3.1.1 CHEMICALS

3.1.1.1 Salts

Sodium Hydroxide (LR grade, S.D. Fine Chem Ltd., Mumbai, India),

Phenolphthalein Indicator (AR grade, Sisco Research Laboratories Pvt. Ltd.,

Mumbai, India)

3.1.1.2 Solvents

Glycerol (LR grade, Qualigens Fine Chemicals Ltd., Mumbai, India),

Sulphuric Acid, Ethyl Alcohol (GR, Merck Specialities Pvt. Ltd., Mumbai,

India), Acetone, Benzene (AR grade, S.D. Fine Chem Ltd., Mumbai, India)

3.1.2 REAGENTS

3.1.2.1 Sodium Hydroxide 50% (w/w) solution: Sodium hydroxide is

dissolved in an equal weight of water and the solution is stored in a bottle

protected from carbon dioxide. The clear portion free from deposit is used.

3.1.2.2 Dilute Sulphuric Acid: Approximately 25 ml of concentrated

sulphuric acid is diluted to 1:1 and adjusted until 40 ml neutralize 2 ml of

50% sodium hydroxide solution.

3.1.2.3 Phenolphthalein Indicator: 0.5% solution in 95% (v/v) ethyl

alcohol or rectified spirit.

3.1.2.4 Ethyl Alcohol: 95% (v/v) neutralized to faint pink end point using

phenolphthalein as indicator immediately before use.

3.1.2.5 Sodium Hydroxide Solution (0.1N): Dissolve approximately 6g

sodium hydroxide pallets in distilled water and make up volume to 1 liter.

Keep this solution for overnight to precipitate impurities. Decant clear

3.0 MATERIALS AND METHODS


42

supernatant and discard impurities. Adjust strength of supernatant solution to

0.1N using oxalic acid as primary standard.

3.1.2.6 Solvent Mixture for Crystallization Test: Mix acetone and benzene

in 3.5:1 ratio.

3.2 EQUIPMENTS

3.2.1 Butyro Refrectometer: Naveen Scientific Industries, Ambala, India.

3.2.2 Cream Seprator: Kamdhenu, KD-60E, Benny Implex, New Delhi,

India.

3.2.3 Electronic Balance: Precisa

3.2.4 Hot Air Oven with Thermostat: Matrix

3.2.5 Waterbath with Thermostat: The Laboratory Glassware Co.,

Ambala, India.

3.3 COLLECTION AND PREPARATION OF SAMPLES

3.3.1 PREPARATION OF PURE SAMPLES

3.3.1.1 Ghee

Cow and buffalo milk used for the preparation of respective ghee

samples were collected from the Cattle Yard of the Institute. Cow milk was a

bulk milk consisting of mixture of the milk obtained from the herd of Karan

Swiss, Karan Fries, Sahiwal and Tharparkar breeds. Buffalo milk used was

also bulk milk obtained from Murrah breed only.

Samples of cow/buffalo ghee were prepared by direct cream heating

method (De, 1996). Soon after the collection of milk samples, these were

warmed to 40°C and separated into cream using mechanical cream separator.

The cream was heated on direct flame in stainless steel vessel to evaporate

all water with continuous stirring and it was converted into gee at

temperature of 120 °C. Ghee was then filtered through 6-8 fold muslin cloth

followed by Whatman No. 4 filter paper. Filtered ghee was filled in

containers and stored at -18°C till further analysis.

The flow chart for the preparation of control ghee samples was as

follows:


43

Collection of cow/buffalo milk

Cream separation (at 40 °C)

Clarification at 120 °C

Filtration (muslin cloth and W.N. 4 filter paper)

3.3.1.2 Body Fats

Four body fats (sheep, goat, buffalo and pig) were used as the

adulterant fats/oils in the present study. Goat, sheep and pig body fat were

prepared separately from their respective adipose tissues collected from the

local slaughterhouse. The adipose tissues of buffalo body fat were collected

from slaughterhouse located at New Delhi (near Sadar bazar). These adipose

tissues after collection were washed thoroughly under running water. After

draining out the residual water, the adipose tissues were heated at 130 to

150°C (buffalo-150°C, goat and sheep-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 fold

muslin cloth followed by Whatman No. 4 filter paper, filled in polyethylene

bottle, cooled to room temperature and kept in a refrigerator (-18 °C) for

their further use as the adulterant fats.

3.3.1.3 Vegetable Oil

The refined vegetable oil with high RM used in the study was

collected from local oil trader.

3.3.2 PREPARATION OF ADULTERATED SAMPLES

For the preparation of adulterated ghee samples, ghee, body fats and

vegetable oil were heated to 60-70°C for 10 min before mixing. The

adulterant fats/oils were added to ghee individually as well as in their

combinations. At individual levels, these adulterants were separately added

to ghee from 2 to 25 percent levels. In case of vegetable oil and body fat

combined mixtures, the ghee was added with 2 to 75 percent levels of

adulterants.


44

3.3.3 MARKET SAMPLES

Branded and non-branded samples of ghee were collected from local

market and stored at -18°C till analysis

3.4 MEHODS OF ANALYSIS

In this section, various methods followed to analyze the samples are

described briefly.

3.4.1 PHYSICO-CHEMICAL CONSTANTS

The physico-chemical constants such as Reichert-Meissl (RM) value,

Polenske value (PV), Butyro-refractometer reading, moisture content and

free fatty acid content of the pure, adulterated and market ghee samples were

determined by the methods as described in SP: 18 (Part XI) – 1981 (BIS,

1981). The details of the procedures for these physico-chemical constants

are, in brief, as follows:

3.4.1.1 Determination of Reichert-Meissl (R.M.) and Polenske Value

Accurately 5.0 g of sample were weighed in a Polenske flask and then

saponified with 20.0 g of glycerol and 2.0 ml of 50% (w/w) sodium

hydroxide solution on a direct flame. Then 93 ml of freshly boiled distilled

water were added followed by 50 ml of dilute sulphuric acid. The flask was

immediately connected with the distillation apparatus and 110 ml of the

distillate was collected within 21 minutes. The flask was replaced with 25 ml

cylinder and the flame was removed. The distillate was cooled in a water

bath maintained at 15°C for 10 min. Then it was filtered through a dry 9 cm

Whatman No.4 filter paper and 100 ml of the filtered distillate were titrated

against 0.1 N sodium hydroxide solution using phenolphthalein as an

indicator. Similarly, a blank test was also done by using all reagents in

similar fashion except fat sample. From this, the RM value was calculated as

follows:

Reichert-Meissl Value = 1.10 (T1-T2).

Where;

T1 = Volume (in ml) of 0.1 N NaOH solution used for sample titration


45

T2 = Volume (in ml) of 0.1 N NaOH solution used for blank titration

For Polenske value (PV), the condenser, 25 ml cylinder, 110 ml flask

and the filter paper were washed with three successive washings of 15 ml

portions of cold water followed by neutralized alcohol. The washings with

neutralized alcohol were collected and then titrated against 0.1 N sodium

hydroxide solution using phenolphthalein as an indicator. Similarly, a blank

was also done. From this, the polenske value was calculated as follows:

Polenske Value = T3-T4

Where;

T3 = Volume (in ml) of 0.1 N NaOH solution used for sample titration

T4 = Volume (in ml) of 0.1 N NaOH solution used for blank titration

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

Before determining the BR reading of a sample, the temperature of the

refractometer was adjusted to 40.0 ± 0.1°C using circulatory water bath and

the prisms were cleaned and dried completely. The refractometer was

calibrated with the standard provided by the company before taking the

reading of the different samples. A drop of the molten fat sample was placed

on the lower prism of the 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.

3.4.1.3 Determination of Moisture Content

Moisture dishes used had been dried and cooled in the desiccator and

then weighed (W1). About 10 g of the sample was weighed into a moisture

dish (W2). Dish with sample was placed in the hot air-oven for

approximately one hour at 105±1°C. Dish was removed from the oven,

cooled in the desiccator to room temperature and weighed. This procedure

was repeated but keeping the dish in the oven only for half an hour each time

until the difference between the two successive weighings did not exceed 1

mg (W3).


46

Moisture percent by weight

Where;

W1 = weight of dry empty moisture dish

W2 = weight of sample plus moisture dish before drying

W3 = weight of sample plus moisture dish after drying

3.4.1.4 Determination of Free Fatty Acids (FFA)

10 g of the sample was weighed in a 250-ml conical flask. In a second

flask 50 ml of alcohol was brought to the boiling point and while still above

70°C it is neutralized to phenolphthalein (using 0.5 ml) with 0.1 N sodium

hydroxide. The neutralized alcohol was poured on ghee in the flask and the

content of the flask was mixed. Flask content was brought to boil and while

it was still hot, titrated with 0.1N sodium hydroxide, shaking vigorously

during the titration. The end point of the titration was reached when the

addition of a single drop produced a slight but definite colour change

persisting for at least 15 seconds.

FFA percent by weight

Where;

T = amount of 0.1N NaOH consumed during titration

W= weight of sample

3.4.2 TESTS TO BE VALIDATED

For validation, three tests were chosen. Crystallization test (Panda,

1996), Apparent Solidification Time test (Kumar, 2003) and Complete

Liquification Time test (Kumar, 2008). The brief details of protocols are as

follows:


47

3.4.2.1 Crystallization Time Test

Crystallization test was done according to the method of Panda and

Bindal (1998b) as follows:

0.8 ml of clear melted fat sample was transferred separately to the

glass tube (specifications: Length- 10 ± 0.1 cm, Internal diameter- 1.0 ± 0.02

cm, External diameter- 1.2 ± 0.02 cm) with the help of 1 ml pipette. To this,

2.5 ml of the solvent mixture (acetone: benzene :: 3.5:1) was added. The

contents in the glass tube were mixed thoroughly and placed in a water bath

maintained at 20°C/5 min for temperature equilibration. Thereafter, the tube

was placed in a water bath essentially maintained at 17±0.2°C till the onset

of crystallization of fat in the tube and the time of onset of crystallization

was noted down.

3.4.2.2 Apparent Solidification Time (AST) Test

The AST of the fat samples was recorded by studying the time taken

by the melted fat samples to become apparently solidified at 18±0.2°C as

described Kumar et al. (2009). The method, in brief, was as follows:

In this test, three gram of the completely melted fat samples were

placed in test tubes (10 x 1.0 cm ID) and kept in water bath maintained at

60°C for a period of 5 min. The test tubes were then kept in a refrigerated

water bath maintained at 18±0.2°C. The test tubes were observed constantly

until the apparent solidification of fat samples took place, which was

confirmed by non-movement of fat samples on tilting the test tube. At this

stage, when the fat sample was apparently solidified, the time taken for the

same was recorded as AST using a stopwatch.

3.4.2.3 Complete Liquification Time (CLT) Test

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 46oC as described by Kumar (2008). The method, in brief, was

as follows:

Three gram of the completely melted fat sample was taken into a test

tube (12 x 100 mm) and kept in an oven maintained at 60°C for a period of 5

minutes. Thereafter, the test tube, containing fat sample, was kept in a


48

refrigerator (6-8 °C) for 45 min for solidification of the melted fat sample.

After that the solidified sample was subjected to liquefaction process at 46°C

for complete melting of the sample. The time for the sample to liquefy

completely was recorded as CLT using stopwatch.

CHAPTER – 4

Results and Discussion

RESULTS AND DISCUSSION

49

A study for the validation of tests to detect adulteration in ghee was

undertaken in the present investigation. For this purpose Crystallization

test, Apparent Solidification Time test (AST), and Complete

Liquification Time test (CLT) which seemed to be easy and rapid to adopt

in any test laboratory, were selected. Various adulterants were added to pure

cow and buffalo ghee samples in such a fashion that the RM value, BR

reading, moisture and FFA content of the adulterated samples fulfill PFA

standards.

Cow and buffalo ghee samples were prepared in the laboratory using

direct cream heating method, respectively from cow and buffalo milk

collected from the Cattle Yard of the Institute. Animal body fats namely goat

body fat (GBF), sheep body fat (SBF), buffalo body fat (BBF), and pig body

fat (PBF) were prepared by heating raw fat tissues purchased from local

market. Vegetable oil with high R.M. value and moderate B.R. reading was

acquired from local oil trader. For the preparation of adulterated samples the

above mentioned body fats were added to pure cow and buffalo ghee from 2

to 25 percent levels. To check effect of presence of vegetable oils on

detection rates, vegetable oil and body fat mixtures were prepared in such a

fashion that the PFA parameters for ghee are not disturbed very much and

added @ 2 to 75 percent levels in cow and buffalo ghee samples. In case of

mix fat addition the ratio of vegetable oil to body fat was also optimized to

allow maximum level of adulteration with minimum change in PV.

The pattern of adulterated samples was as follows:

Pure Ghee Samples

Cow ghee (CG)

Buffalo Ghee (BG)

Adulterants

Sheep Body Fat (SBF)

Goat Body Fat (GBF)

Buffalo Body Fat (BBF)

Pig Body Fat (PBF)

4.0 RESULTS AND DISCUSSION


50

Vegetable Oil (VOL)

Mix Fat One (MX1) mixture of VOL and BBF in 10:6.5

ratio

Mix Fat Two (MX2) mixture of VOL and BBF in 10:15

ratio

Adulterated Ghee Samples

Cow Ghee added with

2%, 5% Sheep Body Fat

2%, 5% Goat Body Fat

2%, 5% Buffalo Body Fat

2%, 5% Pig Body fat

5%, 10%, 15%, 20%, 25%, 50%, 75% MX1

Buffalo Ghee added with

2%, 5%, 10%, 15%, 20%, 25% Sheep Body Fat

2%, 5%, 10%, 15%, 20%, 25% Goat Body Fat

2%, 5%, 10%, 15%, 20%, 25% Buffalo Body Fat

2%, 5%, 10%, 15%, 20%, 25% Pig Body Fat

2%, 5%, 10%, 15%, 20%, 25%, 50%, 75% MX2

In the present study, the following physico-chemical parameters were

investigated for both pure cow and buffalo ghee, adulterant oil and fats as

well as ghee samples adulterated with various oils and fats at different levels.

1. Physico-chemical constants:

a. RM value

b. PV

c. Butyro-refractometer reading at 40°C

d. Free Fatty Acid (FFA) content

e. Moisture content

2. Crystallization Time test at 17°C

3. Apparent Solidification Time test at 18°C

4. Complete Liquification Time test 46°C

4.1 ANALYSIS OF PURE AND ADULTERATED GHEE SAMPLES

In this section it will be discussed that samples of pure ghee (cow and

buffalo), pure animal body fats (sheep, goat, buffalo and pig), vegetable oil

(VOL), and mixtures (MX1 and MX2) were analyzed for physico-chemical


51

constants, crystallization time test, complete liquification time (CLT) test

and apparent solidification time (AST) test. These tests were also then

applied in the adulterated samples prepared as mentioned above.

4.1.1 PHYSICO-CHEMICAL CONSTANTS OF PURE AND ADULTERATED

SAMPLES

Physico-chemical constants such as RM value, Polenske value, BR

reading (at 40°C), Free Fatty Acid content and Moisture content of pure cow

and buffalo ghee, and adulterant oil and fats are presented in table 4.1.

Similarly, physico-chemical constants of adulterated samples are presented

in table 4.2 to table 4.11.

Table 4.1 Physico-chemical constants of pure samples

Sample RM PV BR Moisture FFA

CG 28.67±0.03 1.70±0.09 42.2±0.49 0.0663±0.0017 0.42±0.02

BG 36.56±0.09 1.53±0.07 41.1±0.55 0.0219±0.0042 0.20±0.05

SBF 0.11±0.04 0.10±0.02 Solid at 40°C 0.0260±0.0013 0.25±0.06

GBF 0.04±0.02 0.10±0.04 Solid at 40°C 0.0962±0.0186 0.23±0.03

BBF 0.09±0.04 0.03±0.01 49.7±0.49 0.0362±0.0003 1.58±0.02

PBF 0.07±0.03 0.10±0.04 51.8±0.30 0.0760±0.0001 2.23±0.02

VOL 46.68±0.06 12.23±0.03 31.1±0.42 0.1162±0.0027 0.23±0.09

MX1 28.27±0.05 7.50±0.08 38.5±0.17 0.0851±0.0010 0.40±0.04

MX2 18.55±0.08 4.87±0.07 42.1±0.36 0.0681±0.0016 0.76±0.02

Data represent Mean ± S.E. of three determinations.

It is evident from table 4.1 that cow and buffalo ghee had all the

physico-chemical parameters with in the values prescribed in PFA standards,

indicating that ghee samples prepared for the present study were meeting all

the quality standards and were of good quality. It can also be seen in data

that RM value and PV for animal body fats are very low. RM values were in

the range of 0.04±0.02 to 0.11±0.04. Similarly, polenske values were in the


52

range of 0.03±0.01 to 0.10±0.04 for different body fats. On the contrary,

vegetable oils had RM and Polenske values to the tune of 46.68±0.06 and

12.23±0.03, respectively. Butyro- refractometer reading of BBF and PBF

were more than that of ghee, whereas VOL had BR less than the BR of ghee.

Moisture and FFA contents were very low in all the cases indicating that all

the fats had been processed properly. From the table it can be seen that the

vegetable oil (VOL) had very high RM value, which is unusual in regular

vegetable oils and fats. Hence, the VOL was selected as a best candidate to

act as adulterant for ghee along with animal body fats. These types of

vegetable oils may be designed specifically to cater to the needs of specific

uses or misuses in the industry. Our results for the physico-chemical

parameters of body fats are in accordance with the results of earlier workers

(Singhal, 1973; Rangappa and Achaya, 1974; Sharma and Singhal, 1995;

Kumar, 2003; Sofia, 2005; Kumar, 2008). However, the results obtained in

this study on vegetable oil with regard to high RM value and moderate BR

reading are not in agreement with earlier workers (Singhal, 1973; Rangappa

and Achaya, 1974; Sharma and Singhal, 1995; Kumar, 2003; Sofia; 2005;

Kumar, 2008). This indicates that this type of vegetable oils (VOL) are the

new entrant in the market and a happy choice for the unscrupulous persons

involved in the trade of milk and milk products.

4.1.1.1 Reichert-Meissl (RM) Value

RM value, which primarily measures steam volatile and water soluble

fatty acids such as butyric (C4:0) and caproic (C6:0) acids, is a significant

physico-chemical constant, as these short chain fatty acids are found only in

milk fat and not in animal body fats and vegetable oils. Therefore, this

parameter has been often employed as a criterion to differentiate milk fat

from other oils and fats and also to establish the purity of milk fat. RM value

is one of the quality parameter covered under PFA Rules (2009) for ghee,

which varies from region to region including cotton tract area (Appendix I).

The average RM value for pure cow ghee and pure buffalo ghee was

28.67 and 36.56 respectively (Table 4.2 & Table 4.3). The average RM

values for sheep body fat, goat body fat, buffalo body fat and pig body fat

were observed to be 0.11, 0.04, 0.09 and 0.07 respectively (Table 4.1).

Vegetable oil had shown very high RM value, which is not characteristic of

vegetable oils because, it was probably tailor made fat as mentioned in


53

section 4.1.1 (Table 4.1). Mix fat one and two had respectively 28.27 and

18.55 RM value, which was in intermediate range (Table 4.1).

Table 4.2 RM value of adulterated cow ghee samples

Pure Sample

Adulterant (RM Value)

Level of Adulteration %

RM Value

Cow Ghee

Nil 0.00 28.67±0.03

Sheep Body Fat (0.11)

2.00 28.16±0.08 5.00 27.06±0.05

Goat Body Fat (0.04)

2.00 28.27±0.08 5.00 27.02±0.11

Buffalo Body Fat (0.09)

2.00 28.38±0.16 5.00 27.13±0.18

Pig Body Fat (0.07)

2.00 28.27±0.03

5.00 27.02±0.11

MX1 (28.27)

2.00 28.71±0.10

5.00 28.60±0.08 10.00 28.44±0.12

15.00 28.56±0.12

20.00 28.64±0.23 25.00 28.59±0.09

50.00 28.38±0.05 75.00 28.17±0.11


It is evident from table 4.2 that RM value of cow ghee falls upon

addition of body fats because body fats have RM value ranging from 0.04 to

0.11 while cow ghee has average RM value 28.67. Data clearly showed that

in the present investigation, addition of individual body fats @ 5% to cow

ghee could be detected using RM value parameter. However, when a

cleverly designed mixture of body fat and vegetable oil (MX1) was added to

the same ghee, the adulteration even @ 75% remained undetected. It is also

clear from the data that the level of detection depends upon the RM value of

pure ghee. In the present investigation, the RM value of pure cow ghee was

slightly above the minimum RM value listed in the PFA standards of pure

ghee so the level of detection of animal body fats was 5%. This level of

detection can vary from sample to sample depending upon the initial RM

value of pure ghee.


54

In case of Buffalo ghee (Table 4.3), where RM value was 36.56, the

level of detection of animal body fats increased to the tune of 20-25%

depending upon the animal body fat source. Perusal of data also showed that

the addition of pig body @ 25% remained undetected. In this case different

mixture of body fat and vegetable oil MX2 was used instead of MX1 to

imitate the Polenske value as well. Here the Detection level of MX2 was

50% unlike 75% in cow ghee. However, the RM value on addition of 50% of

MX2 was decreased marginally and sample seemed to be a marginal case of

adulteration.

Our findings in case of buffalo ghee adulteration are in general

agreement with the findings of earlier workers (Singhal, 1973 & 1980;

Sharma, 1992; Sofia, 2005), who reported that adulteration of ghee with

body fats at 20 percent level is not detectable. However, in case of cow ghee

the detection level of individual animal body fats was about 5%. This may be

possible because the RM values of ghee observed in the present study were

close to the minimum value of RM as specified by PFA (2009) standards for

Haryana region.

The results in the present investigation are also slightly different from

the findings of Kumar (2008), where it was reported that 15 percent level of

adulteration with animal body fats and vegetable oils is detectable using RM

value as a base.

Perusal of data revealed that RM value is not a very good indicator of

ghee adulteration in the present context, where adulteration of milk and milk

product has become rampant.


55

Table 4.3 RM value of adulterated buffalo ghee samples

Pure Sample

Adulterant (RM Value)


RM Value

Buffalo Ghee

Nil 0.00 36.56±0.09


2.00 36.15±0.08 5.00 35.30±0.07

10.00 33.33±0.08 15.00 31.74±0.19

20.00 29.37±0.08

25.00 27.50±0.10


2.00 36.08±0.09

5.00 35.09±0.08 10.00 32.84±0.04

15.00 31.35±0.16

20.00 29.43±0.12 25.00 27.54±0.13


2.00 35.57±0.03 5.00 35.48±0.04

10.00 34.27±0.04 15.00 31.24±0.09

20.00 28.93±0.08

25.00 27.27±0.12

Pig Body Fat (0.07)

2.00 35.42±0.05

5.00 35.31±0.16 10.00 33.06±0.04

15.00 31.46±0.06 20.00 30.54±0.08

25.00 28.27±0.05

MX2 (18.55)

2.00 36.23±0.16 5.00 36.08±0.20

10.00 35.64±0.39 15.00 33.42±0.62

20.00 32.43±0.24

25.00 32.13±0.42 50.00 27.84±0.16

75.00 22.27±0.19



56

4.1.1.2 Polenske Value (PV)

Polenske value is substantially a measure of steam volatile and water

insoluble fatty acids such as caprylic (C8:0) and capric (C10:0) acids which

contribute one fourth and three fourth, respectively, to this value. Again, like

butyric acid and caproic acid (C6:0), caprylic (C8:0) and capric (C10:0)

acids are also mainly present in milk fat and they are not found in animal

body fats and vegetable oils except palm kernel oil and coconut oil. These

two exceptional oils are reported to show the average polenske value of 6.12

and 15.20 (Winton and Winton, 1999), respectively, which are much higher

than the average value of 1.7 and 2.0 reported for buffalo and cow ghee

(Rangappa and Achaya, 1974; Sofia, 2005).

Table 4.4 Polenske value of adulterated cow ghee samples

Pure Sample

Adulterant (PV)


PV

Cow Ghee

Nil 0.00 1.70±0.09


2.00 1.60±0.06

5.00 1.53±0.12


2.00 1.65±0.15

5.00 1.57±0.04


2.00 1.58±0.07

5.00 1.49±0.11

Pig Body Fat (0.10)

2.00 1.52±0.08

5.00 1.45±0.09

MX1 (7.50)

2.00 1.68±0.05 5.00 1.79±0.12

10.00 1.83±0.07 15.00 2.00±0.12

20.00 2.16±0.07 25.00 3.03±0.18

50.00 4.48±0.04

75.00 6.18±0.13


Average polenske value for pure cow ghee was 1.70, whereas that for

pure buffalo ghee was 1.53 (Table 4.4 & Table 4.5). The average polenske

values for sheep, goat, buffalo and pig body fats, MX1 and MX2 were

observed to be 0.10, 0.10 and 0.03 and 0.10, 7.50 and 4.87, respectively


57

(Table 4.1). The Polenske value can be safely employed to classify pure

samples into three broad classes i.e. milk fats, body fats and vegetable oils as

values fall wide apart between classes.

Cow ghee has generally a slightly higher content of caprylic acid and

capric acid (Ramamurthy and Narayanan, 1971; Bector and Narayanan,

1974a; Arumughan and Narayanan, 1979; Lal and Narayanan, 1984; Kumar,

2003) which could be responsible for the higher polenske value of cow ghee

than buffalo ghee observed in the present study.

Cow ghee had Polenske value 1.7. Addition of adulterants like sheep,

goat, pig and buffalo body fat decreased Polenske value, while mix fat

increased Polenske value (Table 4.4). On the basis of PV detection of body

fats could be done at more than 5% level in cow ghee but in this study, we

restricted the addition to 5%, because above 5%, RM value of the adulterated

samples decreased below 28 (Table 4.2). Wherever, in case of the addition of

MX1 the level of detection based on PV was 20%, indication that concoction

of animal body fat and vegetable oil remained undetected at higher level.

It is also evident from the data that Buffalo ghee had Polenske value

1.53, which had been reduced upon addition of animal body fats. However, a

reverse trend was observed on the addition of admixture i.e. MX2 (Table

4.5). The level of detection of individual animal body fats in buffalo ghee

was to the tune of 20 – 25 %, depending upon the source of animal body fat.

Similarly, based on PV the level of detection of mixture of body fat and

vegetable oil MX2 was 15%. It can be concluded from the results that even

PV is not offering any help in detecting lower levels of adulteration in ghee.

Earlier workers (Singhal, 1980; Sharma, 1992; Sofia, 2005; Kumar, 2008)

have also made similar observations that the polenske value is not a good

characteristic to detect adulteration in ghee.


58

Table 4.5 Polenske value of adulterated buffalo ghee samples

Pure Sample

Adulterant (PV)


PV

Buffalo Ghee

Nil 0.00 1.53±0.07


2.00 1.50±0.03 5.00 1.42±0.12

10.00 1.34±0.09 15.00 1.37±0.15

20.00 1.20±0.10

25.00 1.08±0.04


2.00 1.57±0.06

5.00 1.50±0.14 10.00 1.46±0.18

15.00 1.33±0.11

20.00 1.25±0.04 25.00 1.12±0.09


2.00 1.44±0.03 5.00 1.40±0.07

10.00 1.28±0.05 15.00 1.17±0.04

20.00 1.04±0.11

25.00 0.81±0.05

Pig Body Fat (0.10)

2.00 1.54±0.02

5.00 1.50±0.07 10.00 1.41±0.04

15.00 1.45±0.07 20.00 1.34±0.10

25.00 1.40±0.05

MX2 (4.87)

2.00 1.45±0.03 5.00 1.55±0.18

10.00 1.75±0.04 15.00 2.05±0.04

20.00 2.22±0.12

25.00 2.40±0.17 50.00 3.43±0.20

75.00 4.08±0.35



59

4.1.1.3 Butyro- Refractometer (BR) Reading at 40°C

Butyro-refractometer (BR) reading or refractive index which measures

the index of refraction between air and the liquid fat, and vary with the

nature of the fat, is usually determined at 40oC. Refractive index is a

characteristic for the particular liquid or solid, and is often considered as an

important criterion for the evaluation of milk fat purity. BR reading is one of

the quality parameter covered under PFA rules (2009) for ghee and it varies

from region to region, including cotton tract area (Appendix I). As per PFA

rules (2009), BR reading of pure ghee varies from 40.0 to 43.0 for most of

the areas, whereas the values may lie between 41.5 and 45.0 for some

notified cotton tract areas.

The average BR readings for fresh pure cow ghee and buffalo ghee

were 42.2 and 41.1 respectively (Table 4.1). The mean values of BR reading

for buffalo body fat and pig body fat were observed to be 49.7, 51.8,

respectively, however, sheep body fat and goat body fat became solid at

40°C, hence BR for these fats was not possible to record (Table 4.1). BR of

vegetable oil and admixture of vegetable oil and animal body fat (MX1 &

MX2) had reading 38.5 and 42.1, respectively (Table 4.1). Using 40.0 to

43.0, a range of BR readings of milk fat, as prescribed for Haryana under

PFA Rules (2009), it was noticed that the adulteration of cow ghee with

animal body fats to the tune of 5% could not be detected and the data

showed that even the adulteration more than 5% is not possible to detect.

We chose the level of 5% in case of cow ghee because above this level RM

value was able to detect the adulteration (Table 4.2).

In case of buffalo pure ghee the addition of animal body fats could not

be detected at any of the levels studied. The admixtures of body fat and

vegetable oils (MX1 & MX2) were even more problematic and their level of

detection in cow ghee was 75%, whereas in buffalo ghee the admixture

(MX2) remained undetected even at 75% level. Therefore, it can be

concluded that even BR reading is not a very good parameter in the detection

of adulteration of ghee.

The results obtained in the present study on the BR readings are in

general agreement with those reported by earlier workers on ghee (Bector

and Narayanan, 1974b; Lal and Narayanan, 1984; Kumar, 2003) and

vegetable oils (Singhal, 1980; Rangappa and Achaya, 1974; Gunstone et al.,


60

1994;). Sharma and Singhal (1995) reported that 10 percent levels of

adulteration of body fats could be detected, whereas Kumar (2008) reported

that body fats even up to 15 percent level could not be detected. Our findings

are also in close agreement with the findings of above mentioned workers.

Table 4.6 BR reading of adulterated cow ghee samples

Pure Sample

Adulterant (BR Reading)


BR Reading

Cow Ghee

Nil 0.00 42.2±0.49

Sheep Body Fat (Solid at 40°C)

2.00 42.4±0.38

5.00 42.2±0.24

Goat Body Fat (Solid at 40°C)

2.00 42.0±0.49

5.00 42.5±0.31


2.00 42.4±0.67

5.00 42.4±0.53

Pig Body Fat (51.8)

2.00 42.7±0.51 5.00 42.3±0.21

MX1 (38.5)

2.00 42.3±0.64 5.00 41.8±0.47

10.00 41.2±0.68

15.00 40.5±0.23 20.00 40.4±0.48

25.00 40.4±0.23 50.00 40.6±0.56

75.00 39.3±0.48



61

Table 4.7 BR reading of adulterated buffalo ghee samples

Pure Sample

Adulterant (BR Reading)


BR Reading

Buffalo Ghee

Nil 0.00 41.1±0.55

Sheep Body Fat (Solid at 40°C)

2.00 41.1±0.27 5.00 41.2±0.41

10.00 41.5±0.46 15.00 41.4±0.37

20.00 41.6±0.48

25.00 41.4±0.32

Goat Body Fat (Solid at 40°C)

2.00 41.1±0.13

5.00 41.0±0.47 10.00 41.4±0.21

15.00 41.4±0.61

20.00 41.7±0.20 25.00 42.0±0.40


2.00 41.4±0.15 5.00 41.6±0.67

10.00 42.0±0.29 15.00 42.3±0.63

20.00 42.2±0.36

25.00 42.7±0.57

Pig Body Fat (51.8)

2.00 40.9±0.46

5.00 41.2±0.47 10.00 40.4±0.41

15.00 41.3±0.49 20.00 42.2±0.50

25.00 42.5±0.51

MX2 (42.1)

2.00 41.3±0.17 5.00 41.0±0.60

10.00 40.6±0.43 15.00 40.5±0.32

20.00 41.3±0.63

25.00 41.3±0.44 50.00 41.7±0.51

75.00 42.0±0.39



62

4.1.1.4 Moisture Content

PFA (2009) has specified the moisture of pure ghee should not be

more than 0.5%. Though moisture content is not the parameter to detect the

adulteration of milk fat, but a constant to judge the storage stability.

However, all the samples were analyzed to ensure that all the samples had

almost uniform moisture content to avoid any interference in crystallization,

AST and CLT tests.

It is evident from table 4.1 that pure cow and buffalo ghee had

moisture content 0.0663% and 0.0219%, respectively. Animal body fats like

sheep body fat, goat body fat, buffalo body fat and pig body fat had 0.0260,

0.0962, 0.0362 and 0.0760 percent moisture, respectively. Vegetable oil had

0.1162% moisture. Mix fats had intermediate moisture content (Table 4.1).

Table 4.8 Moisture of adulterated cow ghee samples

Pure Sample

Adulterant (Moisture)


Moisture

Cow Ghee

Nil 0.00 0.0663±0.0017


2.00 0.0692±0.0024 5.00 0.0696±0.0001


2.00 0.0688±0.0004 5.00 0.0702±0.0003


2.00 0.0690±0.0053 5.00 0.0712±0.0002

Pig Body Fat (0.0760)

2.00 0.0705±0.0001

5.00 0.0691±0.0002

MX1 (0.0851)

2.00 0.0723±0.0006

5.00 0.0428±0.0020 10.00 0.0575±0.0037

15.00 0.0751±0.0013

20.00 0.0774±0.0009 25.00 0.0754±0.0017

50.00 0.0787±0.0021 75.00 0.0810±0.0008


Moisture content of adulterated cow and buffalo ghee samples are

given in table 4.8 and table 4.9 respectively. As expected it can be seen from


63

the data that the addition of various adulterants had not affected the moisture

content of both cow and buffalo ghee samples.


64

Table 4.9 Moisture of adulterated buffalo ghee samples

Pure Sample

Adulterant (Moisture)


Moisture

Buffalo Ghee

Nil 0.00 0.0219±0.0042


2.00 0.0287±0.0006 5.00 0.0292±0.0014

10.00 0.0295±0.0004 15.00 0.0294±0.0006

20.00 0.0309±0.0004

25.00 0.0302±0.0005


2.00 0.0255±0.0007

5.00 0.0287±0.0003 10.00 0.0291±0.0005

15.00 0.0270±0.0003

20.00 0.0251±0.0002 25.00 0.0289±0.0007


2.00 0.0282±0.0010 5.00 0.0290±0.0013

10.00 0.0134±0.0002 15.00 0.0312±0.0005

20.00 0.0320±0.0002

25.00 0.0334±0.0005

Pig Body Fat (0.0760)

2.00 0.0308±0.0017

5.00 0.0327±0.0001 10.00 0.0358±0.0003

15.00 0.0376±0.0004 20.00 0.0393±0.0003

25.00 0.0407±0.0003

MX2 (0.0681)

2.00 0.0301±0.0006 5.00 0.0321±0.0007

10.00 0.0338±0.0004 15.00 0.0367±0.0005

20.00 0.0397±0.0006

25.00 0.0356±0.0002 50.00 0.0427±0.0008

75.00 0.0523±0.0013



65

4.1.1.5 Free Fatty Acid Content

Free fatty acid content is defined as percentage by weight oleic acid.

PFA has maximum limit of FFA 3%. To ensure that all samples fulfill this

requirement FFA content was analyzed.

FFA content of pure cow and buffalo ghee was 0.42% and 0.20%

respectively. Animal body fats like sheep body fat, goat body fat, buffalo

body fat and pig body fat had FFA content 0.25, 0.23, 1.58 and 2.23

percentage, respectively. Vegetable oil had FFA content 0.23% (Table 4.1).

Table 4.10 FFA content of adulterated cow ghee samples

Pure Sample

Adulterant (FFA Content)


FFA Content

Cow Ghee

Nil 0.00 0.42±0.02


2.00 0.59±0.03

5.00 0.59±0.01


2.00 0.65±0.03 5.00 0.65±0.01


2.00 0.59±0.05 5.00 0.62±0.02

Pig Body Fat (2.23)

2.00 0.59±0.02 5.00 0.67±0.03

MX1 (0.40)

2.00 0.42±0.01

5.00 0.42±0.02 10.00 0.42±0.06

15.00 0.42±0.02 20.00 0.42±0.01

25.00 0.43±0.02

50.00 0.42±0.03 75.00 0.41±0.04


Addition of various adulterants in pure cow and buffalo ghee had

altered FFA content of the adulterated samples as an effect of the initial FFA

content of adulterant fats/oil. However, in any case FFA content was not

altered beyond the PFA standards for ghee (Table 4.10 and Table 4.11).


66

Table 4.11 FFA content of adulterated buffalo ghee samples

Pure Sample

Adulterant (FFA Content)


FFA Content

Buffalo Ghee

Nil 0.00 0.20±0.05


2.00 0.40±0.02 5.00 0.44±0.01

10.00 0.42±0.02 15.00 0.42±0.02

20.00 0.42±0.03

25.00 0.38±0.03


2.00 0.51±0.01

5.00 0.51±0.08 10.00 0.49±0.02

15.00 0.45±0.03

20.00 0.45±0.02 25.00 0.45±0.07


2.00 0.42±0.02 5.00 0.42±0.01

10.00 0.43±0.06 15.00 0.42±0.03

20.00 0.49±0.02

25.00 0.57±0.03

Pig Body Fat (2.23)

2.00 0.46±0.02

5.00 0.51±0.04 10.00 0.63±0.01

15.00 0.73±0.03 20.00 0.82±0.04

25.00 0.93±0.07

MX2 (0.76)

2.00 0.59±0.03 5.00 0.62±0.02

10.00 0.59±0.07 15.00 0.56±0.03

20.00 0.66±0.02

25.00 0.62±0.05 50.00 0.79±0.02

75.00 0.92±0.03



67

4.1.2 TESTS OTHER THAN PFA PARAMETERS (APPARENT SOLIDIFICATION

TIME (AST), COMPLETE LIQUEFACTION TIME (CLT) AND

CRYSTALLIZATION TIME TESTS)

Detection of added animal body fats in milk fats has always been a

challenging task for dairy professionals. In the past, some physical methods,

which were based on the partial solidification behaviour of milk fat such as

opacity test (Singhal, 1980), crystallization test (Panda and Bindal, 1998b)

and apparent solidification time (AST) test (Kumar, 2003) and complete

liquification time (CLT) test (Kumar, 2008) were developed and applied to

detect the added animal body fats in milk fats (ghee). These tests reported to

be simple and handy in ensuring the quality of milk fat despite of their own

limitations. In the present investigation an attempt has been made to validate

these tests in adulterated samples of ghee, specifically prepared using body

fats and vegetable oil of high RM and moderate BR reading.

4.1.2.1 Crystallization Time, Apparent Solidification Time (AST), and

Complete Liquefaction Time (CLT) of Pure Fats/Oil

Data pertaining to Crystallization Time, Apparent Solidification Time

and Complete Liquification Time for pure ghee both cow and buffalo,

individual body fats, vegetable oil (VOL), MX1 and MX2, is presented in

table 4.12. It is evident from the table that both cow buffalo ghee had almost

similar values of AST and CLT but differs significantly in crystallization

time. From the table it can be seen that crystallization time of cow ghee

recorded in the present study is 8.35±1.13 min, which is close to the

observations in cow ghee prepared in the month of November (7-15 min) by

Kumar (2008). In case of buffalo ghee the value for crystallization time

recorded in the present study is 4.26±0.27 min, which is below than the

lowest crystallization time (6 to 30 min) recorded for buffalo ghee (Kumar,

2008). Based on this observation even genuine buffalo ghee sample seemed

to be adulterated. This test seems to be very subjective and variation in

observations from individual to individual is more. It was observed during

the study that crystal formation was rather continuous process and time

reported depends upon subject and illumination conditions. In powerful light

source, smaller crystals become easily visible and crystallization time tends

to decrease. These facts force one to raise doubts over applicability of this

test to detect adulteration. Moreover, the range of crystallization time as


68

recorded by Kumar (2008) was very wide. Therefore, the test could not be

very useful to check the detection of ghee adulteration.

In case of body fats it was observed that all body fats, except pig body

fat were crystallized during equilibrium period of three minute during

crystallization time test, therefore the crystallization time of these fats could

not be recorded and depicted as zero. Pig body fat had crystallization time

of 7 minute, which is intermediate between cow and buffalo ghee

crystallization times. Vegetable oil had not shown any crystals till 20

minutes of incubation at 17°C and so crystallization time reported as not

detectable (ND).

For AST it was observed that sheep body fat and goat body fat had

minimum AST time while, buffalo body fat and pig body fat had AST time

similar to that of cow and buffalo ghee. Perusal of data revealed that AST

can be one of the tools to ensure the purity of milk fat/ghee.

It can be noticed that AST time of pure sheep and goat body fat is

significantly different form other samples.

Table 4.12 Crystallization time test, AST and CLT time of pure samples

Sample Crystallization Time at 17°C

AST at 18°C CLT at 46°C

CG 8.35±1.13 3.18±0.03 2.76±0.04

BG 4.26±0.27 2.56±0.04 2.97±0.12

SBF 0.00±0.00 0.53±0.08 ND

GBF 0.00±0.00 0.56±0.04 ND

BBF 0.00±0.00 2.38±0.10 3.40±0.14

PBF 7.00±0.63 3.13±0.09 3.25±0.16

VOL ND ND 2.75±0.06

MX1 24.95±1.27 24.82±0.25 2.95±0.11

MX2 3.70±0.41 14.30±0.17 3.12±0.15



69

4.1.2.2 Crystallization Time Test at 17°C For Adulterated Samples

In the present investigation, it was found that genuine buffalo ghee

sample had crystallization time below the time reported by Kumar (2008).

This observation showed that test had its own limitation as mentioned in

section 4.1.2.1, even then the test was subjected to validation in adulterated

samples.

Table 4.13 Crystallization time of adulterated cow ghee samples

Pure Sample

Adulterant (Cryst. Time)


Crystallization Time at 17°C

Cow Ghee

Nil 0.00 8.35±1.13


2.00 7.45±0.56 5.00 6.69±0.84

Goat Body Fat (0.0)

2.00 7.03±0.97 5.00 6.50±0.46


2.00 8.17±1.53 5.00 7.52±0.61

Pig Body Fat (7.00)

2.00 8.00±1.18

5.00 7.77±0.67

MX1 (24.95)

2.00 8.55±0.43

5.00 8.75±0.14 10.00 8.86±1.03

15.00 9.78±1.59

20.00 11.20±1.32 25.00 12.53±0.35

50.00 13.36±0.74 75.00 19.74±0.19


Crystallization time of pure cow ghee was found to be 8.35±1.13

minute, which is within the range of crystallization time observed by Kumar

(2008). It is clear from table 4.13 that addition of body fats decreased the

crystallization time in cow ghee, which was maximum in case of 5%

addition of goat and sheep body fats. Effect on crystallization time was

minimum, when pig body fat was added @ 5% in cow ghee. Addition of

MX1increased crystallization time as it has longer crystallization time

compared to pure cow ghee (Table 4.13). Detection level in case of cow ghee


70

adulterated with MX1 was 75%, as the crystallization time was falling out of

the range (6-50 to 16-20 min) reported by Kumar (2008).

It is evident from table 4.14 that the crystallization time of buffalo

ghee observed in the study was far below the crystallization time (17.5 min)

observed by Panda (1996) and 6-30 to 12-30 min recorded by Kumar (2008).

On the basis of the crystallization time recorded for buffalo ghee in the

present investigation, the genuine buffalo ghee failed in the beginning itself.

However, the test was extended to the adulterated buffalo ghee samples. It

was found that addition of all the body fats as well as MX2 decreased the

crystallization time. However the initial value in control sample was below

the values recorded by earlier workers (Panda, 1996 and Kumar, 2008), so

test was of no use in the present study to detect the adulteration of ghee.

It can be concluded from the finding in the present study that

crystallization time test is very subjective and not easy to reproduce,

therefore this test is not useful in ensuring the purity of fat in the present

context.


71

Table 4.14 Crystallization time of adulterated buffalo ghee samples

Pure Sample

Adulterant (Cryst. Time)


Crystallization Time at 17°C

Buffalo Ghee

Nil 0.00 4.26±0.27


2.00 4.28±0.16 5.00 3.58±0.29

10.00 1.21±0.15 15.00 0.09±0.04

20.00 0.00±0.00

25.00 0.00±0.00


2.00 4.05±0.58

5.00 3.00±0.73 10.00 1.00±0.35

15.00 0.58±0.21

20.00 0.25±0.09 25.00 0.00±0.00


2.00 3.92±0.52 5.00 3.78±0.55

10.00 3.67±0.73 15.00 3.62±0.19

20.00 3.23±0.30

25.00 2.83±0.27

Pig Body Fat (7.00)

2.00 4.25±0.88

5.00 5.02±0.89 10.00 5.46±0.27

15.00 5.63±0.31 20.00 6.35±1.32

25.00 6.46±0.33

MX2 (3.70)

2.00 4.18±0.84 5.00 4.19±0.72

10.00 4.27±0.41 15.00 4.33±0.70

20.00 4.21±0.48

25.00 4.17±0.95 50.00 4.35±1.23

75.00 4.08±0.32



72

4.1.2.3 Apparent Solidification Time (AST) Test at 18°C for Adulterated

Samples

Time taken at 18°C for solidification of fat sample was reported as

AST time (Kumar et al., 2009). It was reported to be a rapid and simple test

to detect addition of animal body fat in ghee, hence it was selected for

validation.

It is evident from table 4.12 that AST of pure cow and buffalo ghee

was 3.18 and 2.56 minutes, respectively. AST value for cow and buffalo

ghee was in accordance with the AST (2-56 to 3-26 min) reported for cow

ghee and (2.25-2.48) for buffalo ghee by Kumar et al. (2009). In case of

sheep body fat, goat body fat, buffalo body and pig body fat the AST values

were 0.53, 0.56, 2.38 and 3.13 minute, respectively. AST values for fats

other than pig body fat were in accordance with the AST values reported by

Kumar et al. (2009). In case of pig body fat the AST observed in the present

study was higher than the AST observed (1-30 min) by Kumar et al. (2009).

This variation may be due to the animal to animal variation in the

composition of pig body fat. Vegetable oil not solidified till 20 minute at

18°C and so AST for vegetable oil was reported as not detectable (ND).

MX1 and MX2 samples had AST of 24.82±0.25 and 14.30±0.17 minutes,

respectively, which was far more than cow and buffalo ghee AST time.

It was observed that on the addition of body fats AST time of cow

ghee was decreased, but the decrease was not very prominent up to the

addition of 5% of body fats (Table 4.15). On the contrary, MX1 addition

increased the AST of adulterated sample and AST value crossed the

maximum limit of 3-26 min at 10% level, providing indication that sample

was adulterated. AST value at 5% was slightly below to range provided by

Kumar et al. (2009) and so the level of detection can be declared at 10%.

However, AST failed to detect adulteration by pure body fats at 2 and 5

percent level in cow ghee (Table 4.15).

Similarly addition of pure body fats to buffalo ghee, decreased the

AST. The decrease in AST time was in proportion of added body fats. In the

case of MX2 addition AST increased like cow ghee adulteration with MX1

(Table 4.16). Considering normal range of AST (2-25 to 2-48 in traditional

minute-second or 2.41 to 2.80 in decimal system), it becomes evident that

sheep, goat, buffalo and pig body fats were detectable at 10, 15, 10 and 15


73

percent levels by AST in buffalo ghee. Similarly, MX2 was detected at even

5 percent level using this test.

Table 4.15 Apparent solidification time of adulterated cow ghee samples

Pure Sample

Adulterant (AST)


AST at 18°C

Cow Ghee

Nil 0.00 3.18±0.03


2.00 3.16±0.09 5.00 3.15±0.10


2.00 3.19±0.03 5.00 3.12±0.04


2.00 3.18±0.09 5.00 3.15±0.10

Pig Body Fat (3.13)

2.00 3.14±0.07

5.00 3.17±0.15

MX1 (24.82)

2.00 3.13±0.12

5.00 3.22±0.07 10.00 3.63±0.03

15.00 3.67±0.16

20.00 4.13±0.07 25.00 6.08±0.05

50.00 9.76±0.14 75.00 16.49±0.06



74

Table 4.16 Apparent solidification time of adulterated buffalo ghee

samples

Pure Sample

Adulterant (AST)


AST at 18°C

Buffalo Ghee

Nil 0.00 2.56±0.04


2.00 2.54±0.06

5.00 2.54±0.11

10.00 2.38±0.17 15.00 2.21±0.10

20.00 2.00±0.04

25.00 1.84±0.15


2.00 2.56±0.16

5.00 2.47±0.17 10.00 2.41±0.03

15.00 2.36±0.06 20.00 2.24±0.04

25.00 1.96±0.08


2.00 2.60±0.07

5.00 2.53±0.11

10.00 2.38±0.09 15.00 2.28±0.14

20.00 2.20±0.09 25.00 2.13±0.03

Pig Body Fat (3.13)

2.00 2.53±0.05

5.00 2.52±0.14 10.00 2.43±0.02

15.00 2.36±0.19 20.00 2.27±0.07

25.00 2.22±0.13

MX2 (14.30)

2.00 2.75±0.02

5.00 2.88±0.23

10.00 3.04±0.03 15.00 3.18±0.19

20.00 3.39±0.10 25.00 5.24±0.18

50.00 7.67±0.12

75.00 10.05±0.06



75

4.1.2.4 Complete Liquification Time (CLT) Test at 46°C for Adulterated

Samples

Milk fat melts over a wide range of temperature. It is anticipated to be

liquid above 40oC and completely solidified below -40

oC (Fox, 1995). At

7°C milk fats and adulterant fats of interest i.e. body fats and vegetable oils

got solidified and so complete liquification time can be used to detect

adulteration. On the basis of this principle Kumar (2008) developed a rapid

and simple test to detect adulteration of ghee. He reported CLT at 46°C for

cow ghee 1- 35 to 3- 05 min and for buffalo ghee 2-10 to 2-55min.

In the present study it was observed that CLT at 46°C for pure cow

and buffalo ghee was 2.76 and 2.97 minutes, respectively. Cow ghee CLT is

within the range but buffalo ghee has slightly higher CLT time as per ranges

(cow ghee 1-35 to 3-05 and buffalo ghee 2-10 to 2-55 minutes) reported by

Kumar (2008). In case of pure body fats, the CLT observed has been given

in table 4.12. Data revealed that sheep and goat body fat did not melt at 46°C

so their CLT times were not determined. CLT recorded for buffalo and pig

body fat was 3.40, and 3.25 minute, respectively. Vegetable oil, MX1 and

MX2 had CLT time 2.75 min 2.95 and 3.12 minute, respectively (Table

4.12).

Addition of body fats and MX1 in case of cow ghee increased the

CLT as evident in Table 4.17. However, in all the cases CLT never crossed

the upper limit of 3 min- 05 sec as reported by Kumar (2008). Perusal of data

revealed that CLT was not able to detect the adulteration of cow ghee even

when addition was to the extent of 75% of MX1.

Almost similar kind of observations were recorded in case of buffalo

ghee. In this case again addition of body fats increased CLT time of

adulterated buffalo ghee samples. The increase in CLT was in proportion of

level of additions. Mix fat two addition has decreased CLT time slightly

(Table 4.18). In the present study the CLT value of buffalo ghee was slightly

above the upper limit fixed by Kumar (2008), hence CLT could not be

employed to draw any conclusive observation to detect the adulteration of

buffalo ghee.


76

Table 4.17 Complete liquification time of adulterated cow ghee samples

Pure Sample

Adulterant (CLT)


CLT at 46°C

Cow Ghee

Nil 0.00 2.76±0.04

Sheep Body Fat (Not Detected)

2.00 2.87±0.15 5.00 2.82±0.06

Goat Body Fat (Not Detected)

2.00 2.78±0.09 5.00 2.84±0.10


2.00 2.77±0.11

5.00 2.81±0.03

Pig Body Fat (3.25)

2.00 2.75±0.06

5.00 2.78±0.02

MX1 (2.95)

2.00 2.69±0.08

5.00 2.72±0.09

10.00 2.77±0.04 15.00 2.79±0.13

20.00 2.81±0.05 25.00 2.83±0.07

50.00 2.86±0.13 75.00 2.88±0.20



77

Table 4.18 Complete Liquification time of adulterated buffalo ghee

samples

Pure Sample

Adulterant (CLT)


CLT at 46°C

Buffalo Ghee

Nil 0.00 2.97±0.12

Sheep Body Fat (Not Detected)

2.00 3.08±0.05

5.00 3.15±0.13

10.00 3.37±0.09 15.00 3.76±0.04

20.00 4.32±0.02

25.00 4.63±0.05

Goat Body Fat (Not Detected)

2.00 3.00±0.07

5.00 3.03±0.02 10.00 3.23±0.05

15.00 3.61±0.10 20.00 3.93±0.07

25.00 4.25±0.03


2.00 2.98±0.01

5.00 3.02±0.08

10.00 3.05±0.01 15.00 3.12±0.05

20.00 3.20±0.07 25.00 3.27±0.01

Pig Body Fat (3.25)

2.00 2.89±0.08

5.00 2.94±0.09 10.00 3.06±0.14

15.00 3.03±0.06 20.00 3.13±0.07

25.00 3.21±0.02

MX2 (3.12)

2.00 3.00±0.03

5.00 3.00±0.04

10.00 3.04±0.02 15.00 2.96±0.15

20.00 3.10±0.05 25.00 3.05±0.16

50.00 3.04±0.13

75.00 3.08±0.11



78

4.2 ANALYSIS OF MARKET GHEE SAMPLES

Physico-chemical constants of market ghee samples are reported in

table 4.19. All samples have passed PFA requirements in analyzed

parameters. Similarly crystallization, AST and CLT time was reported in

table 4.20.

Table 4.19 Physico-chemical constants of market samples

Sample RM PV BR Moisture FFA

Organized Sector

A 31.02 ±0.03

1.10 ±0.02

41.3 ±0.64

0.0863 ±0.0005

0.07 ±0.02

B 28.38 ±0.12

1.00 ±0.04

41.2 ±0.45

0.0329 ±0.0014

0.05 ±0.01

C 31.02 ±0.02

1.10 ±0.07

41.5 ±0.68

0.0797 ±0.0003

0.14 ±0.03

Unorganized Sector

D 31.79 ±1.06

1.00 ±0.09

41.2 ±0.41

0.2756 ±0.0009

1.8 ±0.02

E 32.23 ±0.15

1.00 ±0.16

41.3 ±0.47

0.1618 ±0.0008

0.9 ±0.05

F 31.02 ±0.07

1.20 ±0.08

41.2 ±0.56

0.0737 ±0.0045

1.4 ±0.03


The values of all three detection tests were within or near range

provided by earlier workers and It can be concluded that samples was pure

without any adulteration.

Table 4.20 Crystallization, AST and CLT time of market samples

Sample CRT AST CLT

Organized Sector

A 5.16±0.93 2.21±0.06 2.83±0.04 B 7.50±0.47 3.17±0.04 2.13±0.12

C 4.88±1.28 2.10±0.19 3.16±0.15

Unorganized Sector

D 9.23±0.42 3.27±0.15 2.19±0.10 E 9.72±0.68 2.54±0.03 2.24±0.19

F 4.10±0.49 2.19±0.11 3.26±0.03




detect the adulteration of ghee with foreign fats. All the listed tests are


79

generally based on the physico- chemical properties of triglycerides hence

being manipulated by the unscrupulous persons involved in the trade.

Therefore, there is a need to develop such tests, which are based on the tracer

components, which are altogether absent in ghee.

CHAPTER – 5

Summary and Conclusions

SUMMARY AND CONCLUSIONS

81

A study for validation of existing tests for detection of adulteration

was carried out. For this purpose, three tests were selected. First

crystallization time test at 17°C, second apparent solidification time test at

18°C and third complete liquification time test at 46°C. Pure samples were

procured form local market. Cow and buffalo ghee was adulterated with goat

body fat, sheep body fat, buffalo body fat and pig body fat from 2 to 25%

levels. Mixtures of high RM (RM 46.68) vegetable oil and body fat was

prepared and added from 2 to 75% levels in cow and buffalo ghee. Mix fat

MX1 was added in cow ghee while MX2 was added in buffalo ghee.

Maximum limits of adulteration was determined by the fact that all

adulterated samples must fulfill PFA requirements of RM, PV, BR, Moisture

and FFA content for Haryana state. All samples are analyzed for physico-

chemical constants and selected detection tests.

It was evident that RM value of cow ghee falls upon addition of body

fats because body fats have RM value ranging from 0.04 to 0.11 while cow

ghee and buffalo ghee has RM values 28.67 and 36.56 respectively. RM

value can detect more than 5% adulteration in cow ghee while 25% in case

of buffalo ghee depending on initial RM values of respective ghee samples.

However, in case of mix fat detection was not possible before 50% in buffalo

ghee and not up to and beyond 75% in cow ghee.

Cow ghee has shown slightly higher PV (1.7) than buffalo ghee has

(1.53). It was not possible to detect adulteration up to 5% in cow ghee with

animal body fats and up to 20% in case of mix fat MX1 by this test. Buffalo

ghee has also exhibited PV in range of 1 to 2 till 25% addition of body fats

except in case of addition of buffalo body fat where value fell below one at

25%. Mix fat MX2 was detectable in buffalo ghee at 15% level using PV. It

is revealed that in conjugation with RM if used PV can reduce detection

rates.

BR reading of cow ghee was 42.2 while of buffalo ghee was 41.1.

Body fats exhibited higher BR reading compared to ghee samples in general.

Using BR reading it was not possible to detect adulteration in cow ghee at

5.0 SUMMARY AND CONCLUSIONS


82

two and five percent level with body fats. However, mix fat MX1 was

detected at 75% level. Buffalo ghee exhibited normal range of BR reading

till 25% addition of body fats and 75% mix fat MX2. So in general it can be

concluded that BR reading is not very useful in detection of adulterants and

mix fats completely bypass this parameter till very high level (75%).

Moisture content of all analyzed samples was below 0.5% level

prescribed for ghee by PFA. The parameter was not of use in detection of

adulteration directly still it was supposed to affect detection tests selected i.e.

crystallization time, AST and CLT and so analyzed for all samples. It was

almost uniform in all samples. Similarly, FFA content of the samples, which

is measure of storage stability, was within 3% limit supplied by PFA and this

has helped in assuring that all samples were in good quality till analysis.

Crystallization time of cow ghee was 8.35 minute while that of buffalo

ghee was 4.26 minute. Addition of body fats had lowered the crystallization

time in both cow and buffalo ghee while, mix fat has increased

crystallization time in cow ghee and slightly decreased in buffalo ghee. Up

to 5% body fat addition in cow ghee was not detectable by this test while

mix fat MX1 was detected at 75% level. In case of buffalo ghee the

crystallization time of pure sample was lower than lower limit of

crystallization time recorded by Kumar (2008) and so it was not possible to

draw any conclusions from comparison of present data. It was observed

during the study that crystal formation was rather continuous process and

time reported depends upon subject and illumination conditions. In powerful

light source, smaller crystals become easily visible and crystallization time

tends to decrease. Therefore, the test could not be very useful to check the

detection of ghee adulteration.

Cow ghee and buffalo ghee has apparent solidification time 3.18 and

2.56 minute respectively. AST test failed to detect 2 and 5 percent body fats

in cow ghee. Mix fat MX1 was detected at 10% level in cow ghee. Buffalo

ghee exhibited detection rates of 10, 15, 10 and 15 percent for sheep, goat,

buffalo and pig body fats respectively using AST value. MX2 was detectable

at 5% level in buffalo ghee, which was lower in comparison to detection rate

in cow ghee.


83

It was observed that CLT at 46°C for pure cow and buffalo ghee was

2.76 and 2.97 minutes, respectively. Addition of body fats and MX1 in case

of cow ghee increased the CLT. However, in all the cases CLT never crossed

the upper limit of 3 min- 05 sec as reported by Kumar (2008). CLT was not

able to detect the adulteration of cow ghee even when addition was to the

extent of 75% of MX1. Like the case in crystallization time, the CLT values

of buffalo ghee was also slightly above the upper limit fixed by Kumar

(2008), hence CLT could not be employed to draw any conclusive

observation to detect the adulteration of buffalo ghee.



detect the adulteration of ghee with foreign fats. All the listed tests are

generally based on the physico- chemical properties of triglycerides hence

being manipulated by the unscrupulous persons involved in the trade.

Therefore, there is a need to develop such tests, which are based on the tracer

components, which are altogether absent in ghee.

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Appendix I: Standards of Ghee under PFA Rules

Sr. No.

Name of the State & U.T.

BR Reading at 40oC

RM (Reichert Meissl)

value (Min)

Percentage of

FFA (as Oleic acid)

(Max)

Moisture (Max)

1. Bihar, Chandigarh, Delhi, Punjab,

Haryana (Areas other than cotton

tract areas), West Bengal (Areas

other than Bishnupur sub-

division), Sikkim, Jharkhand.

40-43

28

3

0.5

2. Manipur, Meghalaya, Mizoram,

Arunachal Pradesh, Orissa,

Uttaranchal, Nagaland, Tripura,

Assam, Goa, Kerala, Himachal

Pradesh, U.P., J & K, Rajasthan

(Areas other than Jodhpur Divn),

Haryana (Cotton tract areas),

Lakshadweep, Maharashtra(Areas

other than cotton tract areas).

40-43

26

3

0.5

3. Karnataka (Belgaum district),

Madhya Pradesh (Areas other

than cotton tract areas),

Pondicherry, Chhatisgarh.

40-44

26

3

0.5

4. Andhra Pradesh, Daman & Diu,

Dadar & Nagar Haveli, Karnataka

(Areas other than Belgaum distt.)

40-43

24

3

0.5

5. Andaman & Nicobar Island, Tamil

Nadu.

41-44 24 3 0.5

6. Gujarat (areas other than cotton

tract).

40-43.5 24 3 0.5

7. Gujarat (cotton tract areas),

Madhya Pradesh (Cotton tract

areas), Maharashtra (cotton tract

areas), Rajasthan (Jodhpur sub

division), West Bengal (Bishnupur

sub division).

41.5-45

21

3.0

0.5

a) Baudouin test shall be negative

b) By cotton tract is meant the areas in the state where cotton seed is extensively fed to the cattle and so notified by the State Govt. concerned.

c) Usually such cotton tract areas ghee has low RM value and high BR reading compared to other areas

d) Ghee may contain BHA not more than 0.02% as antioxidant.

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