lycopene as a natural antioxidant extending the shelf-life of ghee

196 Pages·2016·4.01 MB·English

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DDOOCCTTOORR OOFF PHILOSOPHY
IN
DDAAIIRRYY CCHHEEMMIISSTTRRYY
BY
RUBY SIWACH
((MM..SScc.. FFoooodd TTeecchhnnoollooggyy))

DDAAIIRRYY CCHHEEMMIISSTTRRYY DDIIVVIISSIIOONN
NNAATTIIOONNAALL DDAAIIRRYY RREESSEEAARRCCHH IINNSSTTIITTUUTTEE
(I.C.A.R.)
KKAARRNNAALL-132001 (HARYANA), INDIA

2014
Regn Noo.. 11002211000055
DDDDeeeeddddiiiiccccaaaatttteeeedddd
To
MMMMyyyy gggguuuuiiiiddddeeee
For his benevolent guidance
MMMMyyyy hhhhuuuussssbbbbaaaannnndddd,,,, AAAAkkkkhhhhiiiillll SSSSaaaannnnggggwwwwaaaannnn
Whose friendship, love, care and humor gave me
strength and made me believe that “tough times
don’t last but tough people do.”
TTTToooo mmmmyyyy mmmmooootttthhhheeeerrrr
A strong and gentle soul who taught me
patience and perseverance
TTTToooo mmmmyyyy ffffaaaatttthhhheeeerrrr
WWhhoossee ppaasstt mmeemmoorriieess aanndd eenntthhuussiiaassmm ssttiillll
encourage me to believe in myself
TTTToooo mmmmyyyy mmmmooootttthhhheeeerrrr----iiiinnnn----llllaaaawwww
In whom I found a new mother who is caring
and adorable
TTTToooo mmmmyyyy ffffaaaatttthhhheeeerrrr----iiiinnnn----llllaaaawwww
Whose silent support always motivates me
TTTToooo mmmmyyyy bbbbrrrrooootttthhhheeeerrrr aaaannnndddd bbbbhhhhaaaabbbbhhhhiiii
for supporting me all the way and for filling my
life with little - little surprises
TTTToooo rrrreeeessssppppeeeecccctttteeeedddd DDDDiiiirrrreeeeccccttttoooorrrr SSSSiiiirrrr,,,, AAAA....KKKK.... SSSSrrrriiiivvvvaaaassssttttaaaavvvvaaaa
whose help, support and kindness is unforgettable

DIVISION OF DAIRY CHEMISTRY
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL-132001 (HARYANA), INDIA

____________________________________________

Dr. Raman Seth
Principal Scientist

CERTIFICATE

This is to certify that the thesis entitled “USE OF LYCOPENE AS A
NATURAL ANTIOXIDANT IN EXTENDING THE SHELF-LIFE OF
GHEE” submitted by Ms. Ruby Siwach towards the partial fulfillment of
the requirements for the award of the degree of DOCTOR OF
PHILOSOPHY in DAIRY CHEMISTRY of the NATIONAL DAIRY
RESEARCH INSTITUTE (DEEMED UNIVERSITY), Karnal (Haryana),
India, is a bonafide research work carried out by her under my
supervision and guidance and no part of the thesis has been submitted for
any other degree or diploma.

Dated:

___________________________________________________________
Website: http://www.ndri.res.in

AAAACCCCKKKKNNNNOOOOWWWWLLLLEEEEDDDDGGGGEEEEMMMMEEEENNNNTTTT
It gives me immense pleasure and proud privilege to express my deep sense
of gratitude and heartfelt thanks to my guide DDDDrrrr.... RRRRaaaammmmaaaannnn SSSSeeeetttthhhh (Major
Advisor), principal scientist, Dairy Chemistry division 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 realized that ‘there is a home
away from home’. I express my sincere thanks to members of Advisory
Committee, Dr. Darshan Lal, Principal Scientist, DC Division; Dr. Rajan
Sharma, Senior Scientist, DC Division; Dr. S. K. Kanawjia, Principal
Scientist, DT Division; Dr. Suman Kapila, Principal Scientist, ABC Division;
Dr. Ravinder Malhotra, Principal Scientist, DESM Division of NDRI,
Karnal for their valuable suggestions, encouragement and endless co-
operation from time to time.
I feel honored while extending my gratefulness to Dr. A.K. Shrivastava,
Director and Vice-Chancellor, NDRI, Karnal; and Dr. G. R. Patil, Joint
Director (A), NDRI for providing all the necessary facilities for conducting
this study. I am deeply indebted to all the scientists of the Dairy Chemistry
Division, for their valuable suggestions, good wishes and cordial assistance
during my study and research at NDRI.
I am immensely grateful to my lab mates Sonam mam, Anamika mam,
Naveen Sir, Harish, Ekta, Sneha and Sandeep for their valuable support
during my entire research work. My deep sense of gratitude and warm
regards to Mr. P. C. Singh, Ingle sir, Jagatji, Mr. Kulvinder Singh,
Balawantji, Priya mam, Shakuntala Mam for their sincere in-time help
during the course of this study. I thank to all my friends and seniors who
always helped and encouraged me. My husband, maa, Pappa and my
brother are the earthy Gods in my life who deserve much more than what I
can weigh in words. Their silent prayers, aesthetic love and affection,
support and steel belief in my capabilities have enabled me to make this
endeavor a successful one.
Above all, I wish to acknowledge the almighty GOD without whose blessing;
this would never a success.

May 2014 Ruby Siwach
TABLE OF CONTENT

LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS

CHAPTER 1
INTRODUCTION……………………………………………………………………………….1

CHAPTER 2
REVIEW OF LITERATURE…………………………………………………………………...4
2.1 Ghee…………………………………………………………………………………………...4
2.1.1 Nutritive value of ghee………………………………………………………………….5
2.1.2 Rancidity in ghee………………………………………………………………………..6
2.1.3 Mechanism of production of secondary products of auto‒oxidation…………………...6
2.1.3.1 Initiation…………………………………………………………………………...6
2.1.3.2 Propagation………………………………………………………………………..6
2.1.4 Shelf‒life of ghee………………………………………………………………………..8
2.2 Lycopene……………………………………………………………………………………..10
2.2.1 Food Sources of lycopene………………………………………………………………….11
2.2.2 Lycopene and related carotenoid pigments…………………………………………….12
2.2.3 Physico‒chemical characteristics of lycopene…………………………………………12
2.2.4 Isomeric forms of lycopene……………………………………………………………14
2.2.5 Antioxidant properties of lycopene…………………………………………………….14
2.2.6 Synergistic effect of lycopene with other antioxidants………………………………...17
2.2.7 Lycopene in tomatoes………………………………………………………………….18
2.2.8 Effects of processing on lycopene……………………………………………………..19
2.2.8.1 Effect of heat……………………………………………………………………..20
2.2.8.2 Effect of light…………………………………………………………………….21
2.2.8.3 Effect of oxygen………………………………………………………………….21
2.2.9 Isomerization during processing……………………………………………………….22
2.2.10 Extraction of lycopene from tomatoes………………………………………………..24
2.2.10.1 Solvent extraction of lycopene………………………………………….............24
2.2.10.2 Microwave extraction of lycopene……………………………………………...25
2.2.10.3 Enzyme-aided extraction of lycopene…………………………………………..26
2.2.10.4 Using Naviglio extractor………………………………………………………..26
2.2.10.5 Supercritical fluid extraction of lycopene………………………………………26
2.2.11 Lycopene assay……………………………………………………………………….27
2.2.11.1 Spectrophotometric method…………………………………………………….28
2.2.11.2 High Performance Liquid Chromatography (HPLC) method………………….28
2.2.12 Lycopene in functional foods…………………………………………………………30
2.2.13 Future scope…………………………………………………………………………..31

CHAPTER 3
MATERIAL AND METHODS…………………………………………………………….….33
3.1 Raw materials………………………………………………………………………………..33
3.1.1 Chemicals……………………………………………………………………………....33
3.1.2 Solvents………………………………………………………………………………...33
3.1.3 Reagents………………………………………………………………………………..34
3.1.3.1 Potassium iodide solution (10%)………………………………………………...34
3.1.3.2 Potassium iodide solution (5%)………………………………………………….34
3.1.3.3 Standardizing nickel sulphate solution…………………………………………..34
3.1.3.4 Starch indicator…………………………………………………………………..34
3.1.3.5 Sodium thiosulphate solution (0.1N)…………………………………………….34
3.1.3.5.1 Standardization…………………………………………………………….34
3.1.3.6 Sodium thiosulphate solution (0.002N)………………………………………….35
3.1.3.7 Thiobarbituric acid (TBA) reagent………………………………………………35
3.1.3.8 Oxalic acid solution (0.1N)………………………………………………………35
3.1.3.9 Phenolphthalein indicator (1%)………………………………………………….36
3.1.3.10 Solvent mixture………………………………………………………………....36
3.1.3.11 Sodium hydroxide solution (0.1N)……………………………………………..36
3.1.3.12 Potassium hydroxide solution…………………………………………………..36
3.1.4 Collection of cream……………………………………………………………………36
3.1.5 Procurement of tomatoes ……………………………………………………………. .36
3.2 Methods…………………………………………………………………………………...…36
3.2.1 Processing of tomatoes……………………………………………………………….. 37
3.2.2 Extraction of lycopene…………………………………………………………………37
3.2.3 Quantification of lycopene…………………………………………………………….37
3.2.4 Purification of lycopene………………………………………………………………..38
3.2.4.1 Procedure ………………………………………………………………………..38
3.2.5 Chromatographic analysis of lycopene………………………………………………...39
3.2.6 Measurement of free radical scavenging activity……………………………………...39
3.2.6.1 ABTS [2,2’‒azinobis‒(3‒ethylbenzothiazoline‒6‒sulfonic acid)] method……..39
3.2.6.1.1 Reagents……………………………………………………………………39
3.2.6.1.1.1 Potassium persulphate solution (140mM)…………………………...39
3.2.6.1.1.2 ABTS [2,2’‒azinobis‒(3‒ethylbenzothiazoline‒6‒sulfonic acid)] stock
solution…………………………………………………………………………...39
3.2.6.1.1.3 Phosphate buffered saline…………………………………………...40
3.2.6.1.1.4 ABTS working solution……………………………………………...40
3.2.6.1.1.5 Trolox solution……………………………………………………….40
3.2.6.1.1.6 Preparation of standard curve………………………………………..40
3.2.6.1.1.7 Trolox equivalent antioxidant capacity (TEAC)…………………….41
3.2.7 Experimental design and statistical analysis for process optimization………………..42
3.2.8 Preparation of cow and buffalo ghee…………………………………………………..43
3.2.8.1 Ageing of cream………………………………………………………………….43
3.2.8.2 Churning of cream……………………………………………………………….43
3.2.8.3 Clarification of butter…………………………………………………………….43
3.2.9 Preparation of lycopene extract………………………………………………...……...44
3.2.10 Incorporation of lycopene extract in cow and buffalo ghee………………………….45
3.2.11 Incorporation of BHA in cow and buffalo ghee……………………………………...45
3.2.12 Packaging and storage………………………………………………………………...45
3.2.13 Physico-chemical analysis of cow and buffalo ghee…………………………………45
3.2.13.1 Determination of moisture……………………………………………………...45
3.2.13.2 Determination of Butyro-refractometer (B.R.) reading………………………...47
3.2.13.3 Determination of color………………………………………………………….47
3.2.13.3.1 Spectrophotometric method………………………………………………47
3.2.13.3.2 Colorimetric method……………………………………………………...48
3.2.13.4 Determination of Free Fatty Acids (FFA) in cow and buffalo ghee……………48
3.2.13.5 Determination of Peroxide value (PV) in cow and buffalo ghee……………….49
3.2.13.6 Determination of Thiobarbituric Acid Value (TBA) in cow and buffalo ghee...50