modeling the antioxidant potential of the orange peel extract in ghee

118 Pages·2014·4.36 MB·English

MODELING THE ANTIOXIDANT
POTENTIAL OF THE ORANGE PEEL
EXTRACT IN GHEE

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

MASTER OF TECHNOLOGY
IN
DAIRY ENGINEERING

By
ASHA ASHOK
B. Tech. (Dairy Science & Technology)

SOUTHERN REGIONAL STATION
NATIONAL DAIRY RESEARCH INSTITUTE
(ICAR)
BANGALORE- 560 030, INDIA
2014

Reg. No. 2041202
DEDICTED TO MY BELOVED
PARENTS
MODELING THE ANTIOXIDANT
POTENTIAL OF THE ORANGE PEEL
EXTRACT IN GHEE

By
ASHA ASHOK
Thesis Submitted to the
National Dairy Research Institute
(Deemed University)
in partial fulfillment of the requirements for the degree of

MASTER OF TECHNOLOGY
IN
DAIRY ENGINEERING
Approv ed By

(External Examiner) (Dr. M. Manjunatha)
Major Advisor
(Guide)

Members of Advisory Committee
1. Dr. P. Heartwin Amaladhas
(Senior Scientist, Dairy Engineering)
2. Dr. Menon Rekha Ravindra
(Senior Scientist, Dairy Engineering)
3. Dr. B. Surendra Nath
(Principal Scientist, Dairy Chemistry)
4. Dr. K. Jayaraj Rao
(Principal Scientist, Dairy Technology)
SOUTHERN REGIONAL STATION
NATIONAL DAIRY RESEARCH INSTITUTE
(Deemed University)
(Indian Council of Agricultural Research)
BANGALORE- 560 030 (KARNATAKA), INDIA

Dated: 14/07/2014

Dr. M. Manjunatha, Ph.D.
Scientist
Dairy Engineering Section
National Dairy Research Institute
Southern Campus
Bangalore-560 030

CERTIFICATE

This is to certify that the thesis entitled, “MODELING THE ANTIOXIDANT
POTENTIAL OF THE ORANGE PEEL EXTRACT IN GHEE”, submitted by ASHA
ASHOK towards the partial fulfillment for the award of the degree of MASTER
OF TECHNOLOGY in DAIRY ENGINEERING of the NATIONAL DAIRY
RESEARCH INSTITUTE (Deemed University), Karnal (Haryana), India, is a
bonafide research work carried out by her under my guidance, and no part of the
thesis has been submitted for any other degree or diploma.

(Dr. M. MANJUNTHA)
Major Advisor
ACKNOWLEDGEMENT

The present investigation entitled, “Modeling the Antioxidant Potential of the Orange Peel
Extract in Ghee”, has been conducted during 2013-2014 at National Dairy Research
Institute, Southern Campus, Bangalore (India).

At the outset, I bow my head before the “Almighty God” who gave me enough patience and
strength to complete my study and to get rid of difficulties, which came across my way during
accomplishment of this endeavor.

I would like to extend my sincere appreciation to Dr. M. Manjunatha for his constant support
and understanding as a Major Advisor (Guide). I am now, and will remain, extremely
grateful to him for the invaluable guidance he has provided and recognize how fortunate I
am to have been able to work under such an exceptional man. I admire his acuity in planning
the projects and immense ability of reasoning, and appreciate his cool demeanor,
unperturbed composure and perseverance in tackling research problems. He understood me
thoroughly and has been very kind to me all through the period I worked with him.

I sincerely thank my Advisory Committee members; Dr. P. Heartwin Amaladhas, Senior
Scientist; Dr. Menon Rekha Ravindra, Senior Scientist; Dr. B. Surendra Nath, Principal
Scientist; Dr. K. Jayaraj Rao, Principal Scientist;, for their cordial treatment, valuable
suggestions and practical help throughout my research work.

I wish to endow my thanks to Dr. A.K. Srivastava, Director, NDRI, Karnal, Dr. G.R. Patil,
Joint Director, Dr. Satish Kulkarni, Head, SRS, NDRI, Bangalore for providing the required
infrastructural facilities. I also acknowledge the scholarship provided by ICAR.

I also take this opportunity to offer special thanks to Dr. Menon Rekha Ravindra for her kind
support, valuable suggestion, affection and help throughout my research work.

It gives me immense pleasure to thank Dr. C. N. Pagote, Principal Scientist, Dairy
Technology Section, Dr. B. V. Balasubramanium, Principal Scientist, Dairy Technology
Section, Dr. Dixit, Principal Scientist, Dairy Economics and Management Section, Dr.
Sivaram, Senior Scientist, Dairy Economics and Management Section, Dr B. C. Ghosh,
Principal Scientist, Dairy Technology Section, Mr. Chitranayak, Senior Scientist, Dairy
Engineering Section, Mrs. F. Magdaline E. E., Scientist, Dairy Engineering Section, Dr. S.
Varalakshmi, Scientist, Dairy Microbiology Section and all the scientists of SRS NDRI for
their kind support and co-operation.
I take this opportunity to thank Mr. P. Aravindakshan, Mrs. Vedavathi, and Mrs. Vimala for
all the support, encouragement and affection they have showered on me.

I would also like to thank all the dispensary staff especially Dr. S. Naik (Doctor) and Mr.
Louis for their kind support and help.

I would like to thank Mrs. Thivija Kumari for her kind support in academic matters and
concern shown for me throughout the course work. I am also thankful to all my seniors
especially Mr. P. G. Wasnik, Mr. Arun, Mrs. Neethu, Ms. Divya, Ms. Rahila, Mr. Shriyesh
Patel, Mr. Kiran and Mr. Mahesh Kumar, juniors and all my batchmates for their
cooperation and help whenever required. Their company was a great source of jubilation
and learning at NDRI, Bangalore.

Words are not enough to express my devotion and gratitude to my dearest “Mother and
father” for their motivation, possessiveness, nurture and blessings in counteracting every
obstacle coming in the way of my evolution which ensured to have a sensible head on my
shoulders. My loving sister Disha who was always there, supporting and encouraging me.
They are and will remain my whole and sole inspiration behind each and every achievement
of my life. Without their invaluable sacrifices and moral support, it would not have been
impossible for me to reach this landmark. It is none else but to them I dedicate this thesis.

Date: 14/7/14 Asha Ashok
Reg. No. 2041202
ABSTRACT
Butylated hydroxyl anisole (BHA) is the commonly used ‘synthetic
antioxidant’ in ghee. Use of synthetic antioxidants has been decreased due to their
suspected health issues. In recent years, few researchers have been demonstrated
to use of natural source like tulsi, shatavari, coriander extracts etc in ghee. These
commodities are costly and not easily available in all the seasons. Therefore, the
present work was carried for modelling the antioxidant potential of orange peel
extract in ghee at different storage temperatures for 21 days. The optimized
extraction parameters for orange peel powder extract were: 1 g orange peel
powder; 10 ml ethanol; 30 °C temperature; 6 h time. The total phenol content
(TPC) and radical scavenging activity (RSA) of the extract were 98.17 mg of
caffeic acid equivalent (CAE)/g and 93.27 % inhibition at the optimized extraction
process. The regression models describing the effect of extraction parameters on
TPC and RSA of orange peel extract were found to be linear model and quadratic
model, respectively. The colour and flavour scores of ghee incorporated with
orange peel powder extract (OPPE) @ 0.5 to 1% were comparable with the
sensory scores of the control. Significantly (p<0.01) higher radical scavenging
activity and significantly (p<0.01) lower peroxides, conjugated dienses,
thiobarbituric acid values and free fatty acids were observed in the ghee samples
incorporated with 1% orange peel powder extract followed by 0.5% OPPE and
BHA (0.02%) treated ghee samples than control. Modeling results revealed that
most of the treatments followed first order and fractional conversion first order
kinetic models for describing the antioxidant activities. Experimental results of the
present study revealed that orange peel powder extract has got antioxidant
properties and could be utilized as natural antioxidant for shelf-life extension and
maintaining quality of fat rich food products like ghee.
साराांश
Butylated हाइड्रॉक्सिल Anisole (BHA) घी में आमतौर पर इस्तेमाल किया 'सिथिं ेटिि
एिंिीऑसिीडेंि' है. सिथिं ेटिि एिंिीऑक्सिडेंि िा उपयोग उनिे ििंटिग्ध स्वास््य िे मुद्दों
िे िारण िम हो गया है. हाल िे वर्षों में, िुछ शोधितााओिं घी में तुलिी, shatavari,
धननया अिा आटि जैिी प्रािृनति स्रोत िा उपयोग िरने िे सलए प्रिशना किया गया
है. इन वस्तुओिं महिंगा और िभी मौिमों में आिानी िे उपलब्ध नहीिं हैं. इिसलए,
वतमा ान िाय ा 21 टिनों िे सलए अलग भिंडारण तापमान पर घी में ितिं रे िे नछलिे
ननिालने िी एिंिीऑसिीडेंि क्षमता मॉडसलगिं िे सलए किया गया था. नारिंगी िे नछलिे
िा पाउडर ननिालने िे सलए अनुिूसलत ननिािी मापििंडों थे: 1 ग्राम नारिंगी िे नछलिे
िा पाउडर; 10 समलीलीिर इथेनॉल; 30 डडग्री िेक्सियि तापमान; 6 घिंिे िा िमय है.
ननिालने िी िुल किनोल िामग्री (िीपीिी) और िट्टरपिंथी ििाई गनतववधध
(आरएिए) अनुिूसलत ननिालने िी प्रकिया में caffeic एसिड बराबर (िीएई) / जी और
93.27% ननर्षेध िे 98.17 समलीग्राम थे. िीपीिी और नारिंगी छील ननिालने िी
आरएिए पर ननिािी मापििंडों िे प्रभाव िा वणना प्रनतगमन मॉडल िमशः रेखीय
मॉडल और द्ववघात मॉडल होना पाया गया. 0.5 िे 1% @ नारिंगी िे नछलिे िा
पाउडर ननिालने (OPPE) िे िाथ शासमल घी िा रिंग और स्वाि स्िोर ननयिंत्रण िा
ििंवेिी स्िोर िे िाथ तुलनीय थे. िािी गौरतलब (पी <0.01) उच्च िट्टरपिंथी ििाई
गनतववधध और (पी <0.01) िम peroxides, ििंयुक्ग्मत dienses, thiobarbituric एसिड
मूसयों और मुसत िैिी एसिड 0.5% OPPE द्वारा पीछा 1% नारिंगी िे नछलिे िा पाउडर
ननिालने िे िाथ शासमल घी िे नमूने में मनाया गया और BHA (0.02%) ननयिंत्रण िे
इलाज घी िे नमून.े मॉडसलगिं पररणाम उपचार िा िबिे पहला आिेश और
एिंिीऑसिीडेंि गनतववधधयों वणना िरने िे सलए आिंसशि रूपािंतरण पहले िे आिेश
गनतज मॉडल है कि बाि में पता चला. वतमा ान अध्ययन िी प्रयोगात्मि पररणाम
नारिंगी िे नछलिे िा पाउडर ननिालने एिंिीऑसिीडेंि गुण हो गया है और शसे ि
जीवन ववस्तार िे सलए प्रािृनति एिंिीऑसिीडेंि और घी िी तरह विा युसत भोजन
उत्पािों िे बनाए रखने िे गुणवत्ता िे रूप में उपयोग किया जा ििता है कि पता
चला.
CONTENTS

SL. NO TITLE PAGE NO.
1.0 INTRODUCTION 1-3
2.0 REVIEW OF LITERATURE 4-18
2.1 OVERVIEW 4
2.2 OXIDATION 5
2.2.1 Lipid Oxidation 5
2.2.2 Ghee Oxidation 7
2.3 ANTIOXIDANTS 8
2.3.1 Inhibition Mechanism of Antioxidants 8
2.3.2 Synthetic Antioxidants 8
2.3.3 Natural Antioxidants 9
2.3.4 Orange Peel 10
2.3.5 Extraction of Antioxidants from Natural Source 12
2.4 GHEE PREPARATION AND EXTARCT
13
INCORPORATION
2.5 ACCELERATED STUDY 14
2.6 ANALYSIS OF ANTIOXIDANT PROPERTIES
15
AND GHEE QUALITY
2.6.1 Free Fatty Acids (FFA) 15
2.6.2 Peroxide Value 15
2.6.3 Conjugated Dienes 16
2.6.4 Diphenylpicrylhydrazyl (DPPH) 16
2.6.5 Thiobarbituric Acid Reactive Substances
16
(TBARS) Assay
2.7 ANTIOXIDANT PROPERTIES IN GHEE 16
2.8 RESPONSE SURFACE METHODOLOGY 18
3.0 MATERIALS AND METHODS 19-35
3.1 MATERIALS/INGREDIENTS 19
3.1.1 Orange Fruit 19
3.1.2 Cream 19
3.1.3 Butylated Hydroxyanisole 19
3.1.4 Apparatus and Glassware 19
3.1.5 Chemicals and Reagents 20
3.2 METHODS 20
3.2.1 Preparation and Standardization of 0.2N Sodium
thiosulphate pentahydrate (Na S O ) solution (Stock 20
2 2 3
Solution)
3.2.2 Preparation of 0.002N Solution Of Na S O 21
2 2 3
3.2.3 Preparation of N/10 NaOH 21
3.2.4 Starch Indicator (1%) 22
3.2.5 Mixed Solvent 22
3.2.6 Neutralized Ethanol 22
3.2.7 Standard aqueous Sodium Hydroxide Solution
22
(0.1N)
3.2.8 TBA Reagent 22
3.2.9 Trichloroacetic Acid (35%, w/v) 22
3.2.10 Folin Ciocalteu Reagent (0.2N) 22
3.2.11 Sodium Carbonate Solution (7.5%, w/v) 22
3.2.12 Caffeic Acid Stock Solution (1 mg/ml) 23
3.2.13 Preparation of 0.1mm 2,2-Diphenyl-1-
23
Picrylhydrazyl
3.2.14 2,2-Diphenyl-1-Picrylhydrazyl (6.09×10-5 mol/l) 23
3.2.3 ORANGE PEEL POWDER 23
3.2.3.1 Orange Fruit 23
3.2.3.2 Washing 23
3.2.3.3 Peeling and Slicing 23
3.2.3.4 Drying 23
3.2.3.5 Grinding 24
3.2.3.6 Packaging and Storage 24
3.2.4. EXTRACTION OF EXTRACT FROM
24
ORANGE PEEL POWDER
3.2.5 ANALYSIS OF ORANGE PEEL POWDER
25
EXTRACT
3.2.5.1 Total Phenolic Content of Orange Peel Powder
25
Extract
3.2.5.1a Standard Curve Preparation for TPC 26
3.2.5.1b Total Phenolic Content Calculation 27
3.2.5.2 Radical-Scavenging Activity By DPPH (2,2-
27
Diphenyl-1-Picrylhydrazyl) Model System
3.2.6 OPTIMIZATION OF EXTRACTION
PARAMETERS FOR ORANGE PEEL POWDER
27
EXTRACT USING RESPONSE SURFACE
METHODOLOGY (RSM)
3.2.6.1 Design of Experiment Used for Optimization of
28
Extraction Process
3.2.6.2 Optimization Technique 29
3.2.7 PREPARATION OF GHEE 29
3.2.8 ADDITION OF ORANGE PEEL POWDER
30
AND INCORPORATION OF ORANGE PEEL
30
POWDER EXTRACT
3.2.8a Addition of Orange Peel Powder into Ghee 30
3.2.8b Incorporation of Orange Peel Powder Extract
30
into Ghee
3.2.9 EVALUATION OF OXIDATIVE STABILITY
30
OF GHEE