evaluation of chromatographic methods for the detection of milk fat adulteration with animal body

124 Pages·2012·2.72 MB·English

EVALUATION OF CHROMATOGRAPHIC METHODS FOR
THE DETECTION OF MILK FAT ADULTERATION WITH
ANIMAL BODY FATS

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE

MASTER OF TECHNOLOGY
IN
DAIRY CHEMISTRY

BY

BHORE PRANOTI PRAMOD
B. Tech. (Dairy Technology)

DIVISION OF DAIRY CHEMISTRY
NATIONAL DAIRY RESEARCH INSTITUTE
(I.C.A.R.)
KARNAL-132001 (HARYANA), INDIA
2012

Regn. No. 2021006
EVALUATION OF CHROMATOGRAPHICMETHODS FOR
THE DETECTION OF MILK FAT ADULTERATION WITH
ANIMAL BODY FATS
By
BHOREPRANOTIPRAMOD
THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE
MASTER OFTECHNOLOGY
IN
DAIRY CHEMISTRY
~.
Approved by:
(~ ) (Dr. VIVEK SHARMA)
MAJOR ADVISOR & CHAIRMAN
EXTERNALEXAMI~~ I
['Dr. ~~dho v R. (GUIDE)
\~OIJItfj
MEMBERS OF ADVISORY COMMITTEE
1. Dr. Darshan Lal
Principal Scientist, Dairy chemistry Division
2. Dr.Sumit Arora
SeniorScientist,DairyChemistryDivision
3. Dr. S. K. Atreja
Principal Scientist, Animal Biochemistry Division
4. Dr. S.K. Kanwajia
Principal Scientist, Dairy Technology Division
DIVISION OF DAIRY CHEMISTRY
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL-132001 (HARYANA), INDIA
Dr.Vivek Sharma
Senior Scientist
CERTIFICATE
This is to certify that the thesis entitled "EVALUATION OF
CHROMATOGRAPHIC METHODS FOR THE DETECTION OF MILK FAT
.
ADULTERATION WITH ANIMAL BODY FATS" submitted by Miss. BHORE
PRANOTI PRAMOD in partial fulfilment of the requirement for award of the
I
;
degreeof MASTER OF TECHNOLOGY in DAIRY CHEMISTRY of the National
.
Dairy Research Institute (Deemed University), Kamal (Haryana), India, is a
bonafide research work carried out by her under my supervision and guidance
andno part of the thesis has been submitted for any other degree or diploma.
tJ1r~91IV
Dated: 20thJuly, 2012 (Dr. VIVEK SHARMA)
MAJOR ADVISOR & CHAIRMAN
(GUIDE)
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TTTTOOOO MMMMYYYY
BBBBEEEELLLLOOOOVVVVEEEEDDDD FFFFAAAAMMMMIIIILLLLYYYY
ACKNOWLEDGEMENTS
It gives me an immense pleasure and proud privilege to express my deep sense
of gratitude and heartfelt thanks to my guide Dr. Vivek Sharma, Senior Scientist,
Dairy Chemistry Division, National Dairy Research Institute, Karnal, for his
unstinted encouragement, benevolent guidance, morale boosting words, love, care
and affections not only towards academic work, but towards life as a whole. An
inspirational figure who has always made me realizes that ‘there is a home away
from home’.
I express my sincere thanks to Dr. (Mrs.) B.K. Wadhwa, Principal scientist
& Head, DC Division and members of Advisory Committee, Dr. Darshan Lal,
Principal Scientist, DC Division; Dr. Sumit Arora, Senior Scientist, DC Division,;
Dr. S.K. Atreja, Principal Scientist, ABC Division; and Dr. S.K.Kanwajia,
Principal Scientist, DT Division for their valuable suggestions, encouragement and
endless co-operation from time to time.
I express my deep sense of gratitude to Dr. A.K. Srivastava, Director,
NDRI, Karnal, Dr. G.R. Patil, Joint Director (Academics) and Dr. S. L.
Goswami, Joint Director (Research) for providing all the necessary facilities to
carry out the present investigation.
My deep sense of gratitude and warm regards to Mr. P.C. Singh, Mr. S.L.
Ingle and Mr. Kulvindar 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.
Sweet memories sliarea witli Praclii) Pritee) Prasliant) qopaC Samricflii)
Siacfartli)C}?jta()j)inesli) CRgnvir,jItliira) (j)ivya) Snelia) Sowmya) Inaumatlii,
Slireyesli)1(anif«1ana nice amGianceproviaea Gymy faGseniors:Manvesli sir,jInkft
sir,:Nicfliimam.jIni[ sir, :Naveen sir,jInamif«1 mam) illliawana mam) 1(avinara sir,
Seemamam are unforgettaGCe.I tliank. tliemfor tlieir ineffaGCerove) care ana
affection sliowerea upon me a[[ tliese crays. I afso tliank. to a[[ my Gatcli-mates
Jaydeep) o/aiGliao)Sliasliankt jImit) Somnatli) jImGer, SneliaC jInkfta for tlieir
caringana enjoyaGCemoments I spent witli tliem. I am tlianlifu[ to my room-mate
(]3u{6um[ amfor ey;J;enmngconstant lielp witliout any reservation tlirougliout tlie
study.
I e~ress my aeepestsenseof gratituae to myjIai) illaGa)SanaliyajItya ana
myyounger Grotlier :Nik.lii[for tlieir rove)affections) encouragements ana GCessings.
Pinancia[support receivea in tlieform of institutiona[ Pe[fowsliipfrom
IndianCounci[ofjIgncu[tura[ ~searCh):New(j)e[hi)isaufy ack.nowCecfgea.
Pina[fy) my liumGCeana aGso[uteoGeisanceto qoa jI[miglity) who have
a{ways Geenan invisiGCe mvine force Geliina my accomp{isliments.
}l{{arenot mentionea Gutno one isforgotten.
~~
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(fJate:Jury 2.0)2012 (illliorePranoti Pramod)
ABSTRACT

Milk fat plays an important role in the economics, nutrition, flavour and physico-
chemical properties of milk and milk products. Ghee (milk fat) is also regarded as good
for having grace, enhancing memory and grasping power, and power to control senses
and to make these stronger. Adulteration of milk fat has always been a serious problem
because of the economic advantages by partly replacing the high priced milk fat by low
priced oils and fats without labeling the product accordingly. In the past, several
techniques have been developed to detect milk fat adulteration. In continuation of the
work, chromatographic methods have been tried to detect animal body fat adulteration
in milk fat. In present study, it was observed that physico-chemical constant like
Polenske value was not helpful for the detection of adulteration of ghee with animal
body fat. However, Reichert-Meissl value could detect animal body fat adulteration up
to 10 percent level in cow ghee and 20 percent level in buffalo and mixed ghee. Butyro-
refractometer (BR) reading could detect adulteration of cow ghee with pig body fat at
20% level but in case of buffalo and mixed ghee values were within the range given by
FSSAI (2011). Various solvent systems were tried to detect the adulteration on the
basis of amino acids and triglycerides components. When water/ methanolic extracts of
ghee and body fats were used for TLC/ paper chromatographic analysis, they showed
similar pattern of bands in both ghee as well animal body fats. HPLC chromatograms
revealed that number of peaks was more in pure ghee than body fats and adulterated
samples but still no marker peak was observed in the chromatograms of body fats and
adulterated samples. Therefore, it can be concluded that TLC, paper chromatography
and HPLC did not prove useful for the detection of adulteration on the basis of amino
acids of ghee and animal body fats. Paper chromatographic analysis for triglyceride
separation was also performed for the detection of adulteration. In most of the solvent
systems, spots of ghee samples showed the clear movement from the origin of
application and adulterated samples also followed the similar trend. In solvent system
acetone: benzene (3.5:1, v/v) adulteration of ghee with body fats up to 5% level was
detectable. However, this solvent system failed to detect adulteration with admixture of
buffalo body fat: vegetable oil (2.5:7.5) when added to the tune of 5% in ghee. The
reproducibility of this method was also found to be low. In another attempt i.e. solvent
fractionation coupled with paper chromatography, the admixture of buffalo body fat:
vegetable oil (2.5:7.5) was fractionated and then lower fractions were applied on paper
chromatogram. In this case solvent system acetone: benzene (3.5:1, v/v) could detect
5% level of adulteration. Solvent fractionation method showed potential to detect
adulteration of ghee with body fats. However the reproducibility of the method remained
questionable. Hence the method needs to be validated in more and more adulterated
samples prepared in lab before the application in the field. Therefore, on the basis of
above results, it can be concluded that paper chromatography of triglycerides may be a
useful technique to ascertain the purity of milk fat especially ghee.
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CONTENTS

Chapter Title Page
No.
1.0 INTRODUCTION 1-2
2.0 REVIEW OF LITERATURE 3-40
2.1 Methods Based on Physical Properties 3
2.1.1 Melting Point 4
2.1.2 Solidification Point 5
2.1.3 Titre Value 5
2.1.4 Apparent Solidification Time (AST) Test 6
2.1.5 Complete Liquification Time (CLT) Test 7
2.1.6 Crystallization Time Test 8
2.1.7 Opacity Test 9
2.1.8 Refractive Index and Butyro - 11
Refractometer Reading
2.1.9 Differential Thermal Analysis (DTA) and 12
Differential Scanning Calorimetry
2.110 Fractionation of Milk Fat 13
2.1.11 Critical Temperature of Dissolution(CTD) 15
2.1.12 Bomer Value 16
2.1.13 Microscopic Examination of Fat 16
2.1.14 Spectroscopic Methods 17
2.1.14.1 Tests Based on Visible 17
Spectroscopy
2.1.14.2 Tests Based on Ultraviolet 17
Spectroscopy
2.1.14.3 Tests Based on Infra-Red 18
Spectroscopy
2.1.15 Dilation Behaviour 20
2.1.16 Cryoscopic Method and Heat of 21
Dissolution
2.2 Methods Based on Chemical Properties 21
2.2.1 Tests Based on Fatty Acids 21
2.2.1.1 Tests Based on Physico-Chemical 22
Constants
2.2.1.1.1 Reichert- Meissel(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
2.2.1.2 Tests Based on Gas Liquid 24
Chromatography (GLC) of Fatty Acids
2.2.2 Tests Based on Nature and Content of 26
Triglycerides
2.2.2.1 Tests Based on Gas Liquid 26
Chromatography of Triglycerides
2.2.2.2 Tests Based on Thin Layer 28
Chromatography of Whole Fat
2.2.2.3 Tests Based on Paper Chromatography 30
of Triglycerides
2.2.3 Tests Based on the Nature and Content 31
of Unsaponifiable Constituents (Natural
or Extraneous)
2.2.3.1 Tests Based on Naturel Unsaponifiable 31
Constituents
2.2.3.1.1 Tests Based on Sterols 31
2.2.3.1.2 Tests Based on Tocopherols 34
2.2.3.2 Tests Based on Extraneous 34
Unsaponifiable Constituents
2.2.3.2.1 Tests Based on Mineral Oils 34
2.2.4 Tests Based on the Content of Specific 35
Fatty Acids
2.2.4.1 Butyric Acid 36
2.2.4.2 Iso- Valeric Acid 36
2.2.4.3 Cyclopropenoic Acid 36
2.2.5 Tests Based on Specific Constituents of 36
Fats
2.2.5.1 Baudouin Test 36
2.2.5.2 Phytosterol Acetate Test 37
2.2.5.3 Halphen Test 37
2.2.5.4 Methylene Blue Reduction 37
Test
2.2.5.5 Hydroxamic Acid Test 37
2.2.6 Tests Based on Nature and Content of 38
Tracers
2.2.7 Tests Based on Enzymatic Hydrolysis 38