production and bioevaluation of functional broiler...
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PRODUCTION AND BIOEVALUATION OF FUNCTIONAL
BROILER MEAT
BY
Muhammad Sohaib
M.Sc. (Hons.) Food Technology
A dissertation submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
National Institute of Food Science & Technology
FACULTY OF FOOD, NUTRITION & HOME SCIENCES
UNIVERSITY OF AGRICULTURE, FAISALABAD
PAKISTAN
2015
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Declaration
I hereby declare that the contents of the thesis “Production and bioevaluation of functional
broiler meat” are product of my own research and no part has been copied from any
published source (except the references, standard mathematical and genetic models/
equations/ formulae/ protocols etc.). I further declare that this work has not been submitted
for award of any other diploma/ degree. The University may take action if the information
provided is found inaccurate at any stage.
Signature of student
3
The Controller of Examinations,
University of Agriculture,
Faisalabad.
‘‘We, the Supervisory Committee, certify that the contents and form of thesis submitted by Mr.
Muhammad Sohaib, Regd. No. 2005-ag-1570, have been found satisfactory and recommend
that it be processed for evaluation by External Examiner(s) for the award of degree.’’
SUPERVISORY COMMITTEE:
Chairman _________________________________
(Prof. Dr. Masood Sadiq Butt)
Member _________________________________
(Dr. Muhammad Asim Shabbir)
Member _________________________________
(Dr. Muhammad Shahid)
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DEDICATED
TO
HOLY PROPHET MUHAMMAD
(Peace Be Upon Him)
ACKNOWLEDGEMENTS
I feel inept to regard the Highness of Almighty Allah, my words have lost their expressions,
knowledge is lacking and lexis scarce to express gratitude in the rightful manner to the
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blessings and support to Allah who flourished my ambitious and helped me to attain goals. I
present my humble gratitude from the deep sense of heart to the Holy Prophet Muhammad
(SAW) that without him the life would have been worthless.
I want to pay my gratitude to higher education commission (HEC), Pakistan for providing me
financial assistance in the form of Indigenous scholarship and an esteemed opportunity of six
month foreign training (IRSIP) to expand me erudite exposure.
I have no words to express my gratitude to my honorable supervisor Prof. Dr. Masood
Sadiq Butt, National Institute of Food Science and Technology, University of Agriculture
Faisalabad for his diligent cooperation and support during the entire degree program. I
expand my deepest appreciation to Dr. Muhammad Asim Shabbir, Assistant professor,
National Institute of Food Science and Technology, University of Agriculture Faisalabad for
his help. I also express my appreciation to Dr. Muhammad Shahid, Associate professor,
Department of Biochemistry, University of Agriculture Faisalabad for his compassionate
attitude and valued suggestions. I also want to convey my deepest gratitude to Prof. Dr.
Faqir Muhammad Anjum(T.I), Institute of Home and Food Sciences, Government College
University, Faisalabad for his kind guidance and moral support.
I am also obliged to Prof. Dr. Dong Uk Ahn, Department of Animal Science, Iowa State
University, USA for his tremendous support to carry out in vitro antioxidant research study
in his laboratories at Iowa State University, Ames IA, USA. Lastly, I am also grateful to all
my family members and friends for their consistent care and encouragement.
LIST OF ABBERIVATION
CLA Conjugated linoleic acid
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
TBHQ Tert-butylhydroquinone
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GIT Gastrointestinal tract
TC Total cholesterol
LDL Low density lipoproteins
HDL High density lipoproteins
TG Triglycerides
PUFA Polyunsaturated fatty acids
ROS Reactive oxygen species
OH- Hydroxyl
O-2 Superoxide
NO- Nitric oxide
LOO- Lipid peroxyl
ROOH Hydroperoxides
EPA Eicosapentaenoic acid
DHA Docosahexaenoic acids
SFA Saturated fatty acids
HOASS High oleic acid sunflower
TBARS Thiobarbituric acid reactive substance
MDA Malondialdehyde
VLDL Very low density lipoprotein
DEN Diethylnitrosamine
GC Gas chromatography
DPPH 1,1-diphenyl-2-picrylhydrazyl
FRAP Ferric reducing antioxidant power
MPO Myeloperoxidase oxidation
DEN Diethylnitrosamine
AST Aspartate transaminase
GHS Aspartate transaminase
ALT Alanine transaminase
SBP Systolic blood pressure
DDGS Distillers dried grains
LDLC Low density lipoprotein cholesterol
ACE Angiotensin converting enzyme
HUVEC Human umbilical vein endothelial cell
CEOOH Cholesterol ester hydroperoxide
CVD Cardiovascular diseases
COP Cholesterol oxidation products
FCR Feed conversion ratio
TPC Total phenolic contents
HPLC High performance liquid chromatography
SOD Superoxide dismutase
CAT Catalase
GRs glutathione reductase
Q3G Quercetin-3-O-glucoside
FOS Fructooligosaccharide
MUFA Mono unsaturated fatty acids
CM Chylomicron
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HCD High cholesterol diet
CONTENTS
Sr. No. Title Page
I INTRODUCTION 1
8
LIST OF CONTENTS
Acknowledgement v
List of abbreviations vi
Contents viii
List of contents ix
List of tables xiv
II REVIEW OF LITERATURE 6
III MATERIALS AND METHODS 32
IV RESULTS AND DISCUSSION 47
V SUMMARY 135
RECOMMENDATIONS 143
LITERATURE CITED 144
9
List of figures xvii
Appendices xviii
Abstract xix
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 6
2.1. Meat as a functional food 7
2.2. Poultry meat quality 8
2.3. Lipid oxidation and meat quality 9
2.4. Estimation of antioxidant potential 13
2.4.1. Reduction by Folin ciocalteu reagent to measure phenolic
contents…………………………………………………………….14
2.4.2. Free radical scavenging by DPPH 14
2.4.3. Colorimetric determination with iron salts 15
2.5. Antioxidants and broiler meat quality 15
2.6. Quercetin and alpha tocopherol as antioxidants 16
2.7. Storage stability of broiler meat products 28
3. MATERIAL AND METHODS 32
3.1. Procurement of raw materials 32
3.2 Experimental plan 32
3.3. Experimental site and bird’s management 32
3.3.1. Chicks vaccination 33
3.3.2. Duration of trial 33
3.3.3. Growth parameters 34
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3.3.4. Slaughtering of the experimental birds 34
3.3.5. Homogenization and sample preparation 34
3.4. Analysis of meat samples 34
3.4.1. Antioxidant potential of functional broiler meat 34
3.4.1.1. Total phenolic contents (TPC) 34
3.4.1.2. Free radical scavenging activity (DPPH assay) 35
3.4.1.3. Ferric reducing antioxidant power (FRAP) 35
3.4.2. TBARS assay 35
3.5. Quantification of quercetin and alpha tocopherol 36
3.5.1. Quercetin content 36
3.5.1.1. Sample preparation 36
3.5.1.2. Mobile phase 36
3.5.1.3. Standard preparation 36
3.5.1.4. HPLC quantification 37
3.5.2. Alpha tocopherol content 37
3.5.2.1. Sample preparation 37
3.5.2.2. Mobile phase 38
3.5.2.3. Standard preparation 38
3.5.2.4. HPLC quantification 38
3.6. Fatty acids profile 38
3.7. Antioxidant enzymes assay 39
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3.8. Serum biochemical analysis 39
3.8.1. Total cholesterol 39
3.8.2. High density lipoprotein (HDL) 40
3.8.3. Low density lipoprotein (LDL) 40
3.8.4. Triglycerides 40
3.8.5. Total protein 40
3.9. Product development 40
3.10. Nuggets analysis 40
3.10.1. Color 41
3.10.2. pH 41
3.10.3. Texture 41
3.10.4. TBARS assay 41
3.11. Sensory evaluation of nuggets 41
3.12. Selection of best treatment 42
3.12.1. Bioevaluation of functional broiler meat 42
3.12.2. Provision and distribution of the functional meat nuggets
43
3.12.3. Blood Sampling and Determinations 43
3.13. In vitro antioxidants supplementation in meat 44
3.13.1 TBARS assay of patties 44
3.13.2. Color 45
3.13.3. Volatile compounds 45
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3.14. Statistical Analysis 46
4. RESULTS AND DISCUSSION 47
4.1. Growth parameters 47
4.1.1. Body weight gain 47
4.1.2. Feed intake 48
4.1.3. Feed conversion ratio 49
4.2. Results of raw meat 54
4.2.1. Total phenolic contents of breast and leg meat 54
4.2.2. Free radical scavenging activity (DPPH assay) of breast and leg meat 55
4.2.3. Ferric reducing antioxidant power of breast and leg meat 56
4.2.4. Thiobarbituric acid reactive substances (TBARS) assay of meat 61
4.2.5. Quercetin content of breast and leg meat 62
4.2.6. Alpha tocopherol content of breast and leg meat 63
4.2.7. Fatty acid profile of breast and leg meat 70
4.3. Antioxidant enzymes 76
4.3.1. Superoxide dismutase (SOD) 76
4.3.2. Glutathione reductase (GRs) 78
4.3.3. Catalase 80
4.4. Serum Bio-chemical profile of birds 80
4.5. Results of antioxidant enriched product 82
4.5.1. Nuggets color 82
4.5.2. pH of nuggets 87
4.5.3. Texture of nuggets 87
4.5.4. TBARS of nuggets 88
4.6. Sensory evaluation of nuggets 100
4.7. Bio-evaluation study 112
4.7.1. Total cholesterol 112
4.7.2. LDL 116
4.7.3. Triglycerides 119
4.7.4. HDL 120
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4.7.5. Total protein 123
4.8. In vitro study 123
4.8.1. Oxidative stability of patties 123
4.8.2. Color of patties 124
4.8.3. Volatile compounds 128
5. SUMMARY 135
RECOMMENDATIONS 143
LITERATURE CITED 144
APPENDICES 172
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LIST OF TABLES
Sr.No. Title Page
1 Treatment plan 33
2 Recipe of the nuggets 41
3 Bioevaluation study plan 43
4 In vitro study plan 44
5 Mean squares for weight gain (WG), Feed intake (FI) and feed
conversion ratio (FCR) of broilers
50
6 Body weight gain of broilers 51
7 Feed intake of broiler birds 52
8 Feed conversion ratio of broiler birds 53
9 Mean squares for TPC, DPPH and FRAP assay of broiler meat 57
10 Total phenolic contents (mg GAE/100g meat) of broiler meat 58
11 Free radical scavenging activity (%) of broiler breast and leg meat 59
12 Ferric reducing antioxidant power of broiler meat 60
13 Mean squares for thiobarbituric acid reactive substances (TBARS) of
broiler meat
64
14 Mean squares for quercetin and alpha tocopherol contents of broiler
meat
67
15 Quercetin contents of broiler meat 68
16 Alpha tocopherol contents (mg/kg meat) of broiler meat 69
17 Fatty acids composition of broiler breast meat 72
18 SFA, MUFA, PUFA and PUFA: SFA ratio of broiler breast meat
73
19 Fatty acids composition of broiler leg meat 74
20 SFA, MUFA, PUFA and PUFA: SFA ratio of broiler leg meat
75
21 Mean squares for Superoxide dismutase (SOD), glutathione reductase
(GR) and catalase of broiler blood serum
78
15
22 Superoxide dismutase (SOD), glutathione reductase and catalase of
broiler blood serum
79
23 Mean squares for total cholesterol, HDL, LDL, triglycerides and
protein of broilers blood serum
83
24 Total cholesterol (TQ), HDL and LDL of broilers blood serum 84
25 Triglyceride and total protein of broiler blood serum 85
26 Mean squares for color and pH of broiler meat nuggets 89
27 Effect of treatments and storage on color of breast meat nuggets 90
28 Effect of treatments and storage on color of leg meat nuggets 91
29 Effect of treatments and storage on pH of breast meat nuggets 92
30 Effect of treatments and storage on pH of leg meat nuggets 93
31 Mean squares for texture and TBARS of broiler meat nuggets 95
32 Effect of treatments and storage on texture of breast meat nuggets 96
33 Effect of treatments and storage on texture of leg meat nuggets 97
34 Effect of treatments and storage on TBARS of breast meat nuggets 98
35 Effect of treatments and storage on TBARS of leg meat nuggets 99
36 Mean squares for appearance and flavor of nuggets 102
37 Effect of treatments and storage on appearance of breast meat
nuggets
103
38 Effect of treatments and storage on appearance of leg meat nuggets 104
39 Effect of treatments and storage on flavor of breast meat nuggets 105
40 Effect of treatments and storage on flavor of broiler leg meat nuggets 106
41 Mean squares for taste and overall acceptability of breast and leg
meat nuggets
107
42 Effect of treatments and storage on taste of broiler breast meat
nuggets
108
43 Effect of treatments and storage on taste of broiler leg meat nuggets 109
44 Effect of treatments and storage on overall acceptability of breast
meat nuggets
110
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45 Effect of treatments and storage on overall acceptability of broiler leg
meat nuggets
111
46 Means for total cholesterol, HDL, LDL cholesterol, triglycerides and
total protein of human subjects
115
47 Mean squares for TBARS and color values of patties 125
48 Means for TBARS of cooked breast meat patties stored at 4 ᵒC 126
49 Color values of cooked breast meat patties stored at 4 ᵒC 127
50
Volatiles flavor compounds (ion counts × 104) in cooked patties on
1st day of storage
130
51
Volatiles flavor compounds (ion counts × 104) in cooked patties on
3rd day of storage
131
52
Volatiles flavor compounds (ion counts × 104) in cooked patties on
7th day of storage
132
LIST OF FIGURES
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Sr. No. Title Page
1 TBARS of breast meat of broiler birds fed on quercetin and α-
tocopherol supplemented feed 62
2 TBARS of breast meat of broiler birds fed on quercetin and α-
tocopherol supplemented feed 63
3 Percent reduction in cholesterol as compared to control 118
4 Percent reduction in LDL cholesterol as compared to control 118
5 Percent reduction in triglyceride as compared to control 121
6 Percent increase HDL cholesterol as compared to control 121
7 Percent increase total protein as compared to control 122
LIST OF APPENDICES
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Appendix TITLE PAGE
1 Composition of basal diet 171
2 Performa for sensory evaluation of antioxidant enriched broiler meat
nuggets
172
ABSTRACT Globally, the consumption of functional foods is gaining popularity owing to their health
promoting prospectives. Purposely, the instant investigation was carried out to enhance the
functional worth of broiler meat and its products through quercetin and α-tocopherol based
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dietary supplementation. Accordingly, three levels of quercetin @100, 200 and 300 mg/kg feed
in combination with α-tocopherol @150, 225 and 300 mg/kg feed were given to the birds. The resultant meat was subjected to antioxidant assay, lipid stability and quantification of
antioxidants followed by product development and bioevaluation to assess its therapeutic
potential with special reference to hyperlipidemia. Feed imparted significant effect on weight
gain and feed conversion ratio (FCR) of the birds though, non-momentous differences were noticed for feed intake. The highest weight gain was recorded in T9 2374.67 & 2388 g/bird
followed by T8 & T6 2350 & 2353.33 and 2293.33 & 2307 g/bird, respectively whilst the lowest
in T0 as 1992.67 & 1999 g/bird during the experimental year 2013 and 2014. Similarly, birds fed
on α-tocopherol and quercetin enriched diets exhibited FCR values in T9, T8 and T6 by 1.68, 1.70 & 1.72, correspondingly compared to T0 by 1.94. Among treatments, T9 exhibited highest
values for TPC, DPPH & FRAP i.e. 158.70±0.84 & 156.05±1.02 mg GAE/100g, 82.40±0.93 &
77.02±0.98% and 682±2.11 & 673.33±1.82 µmol/Fe+2, respectively as compared to T0
104.27±1.64 & 107.23±1.22 mg GAE/100g, 54.71±0.64 & 52.31±0.91% and 542.67±1.74 & 541.67±1.96 µmol/Fe+2 of broiler breast and leg meat, correspondingly. Besides, production of
malondialdehydes in meat increased during storage as indicated by TBARS however,
antioxidants deposition varied significantly among treatments. HPLC quantification indicated maximum level of quercetin and α-tocopherol in T9 amongst leg and breast meat. In addition,
serum lipid profile of broiler birds explicated substantial declining trend in treatment T9 on total
cholesterol, LDL & triglycerides by 107.28±0.48, 25.04±0.33 and 48.81±0.15 mg/dL as
compared to control. The resultant functional broiler meat was used further for nuggets preparation and subjected to physicochemical analysis. It has been revealed that treatments had
momentous effect on product color, pH, texture and TBARS during storage. Nonetheless,
hedonic response showed decreased consumer acceptability as a function of storage. On the basis
of antioxidant potential, physicochemical analysis, serum biomarkers and sensory attributes, four best treatments were selected along with control for bioefficacy study. The efficacy trial was
carried out in human subjects through two consecutive trials for the validity of results. The
findings suggested that G1(meat containing 300 mg of quercetin + 300mg of α-tocopherol)
exerted maximum decline in cholesterol, LDL and triglycerides levels by 8.17 & 7.59, 7.42 & 7.72, 10.38 & 7.93% whilst HDL was affected non significantly in the experimental years.
Moreover, serum protein level of human were also affected momentously by the functional meat
nuggets. Furthermore, at Iowa State University, USA, in vitro antioxidant potential of patties was estimated which showed that treatments substantially affected TBARS, lightness (L*), redness
(a*), yellowness (b*) and volatile compounds of the product. The increasing trend in TBARS
was observed during storage however, the highest TBARS were found in T0 (1.93 mg of
MDA/kg meat) and the lowest in T6 (0.37mg/kg of meat) on 1st day that were increased to 3.47 and 0.90 mg MDA /kg meat in respective treatments at the termination of storage. The principal
volatile compounds found in patties samples include aldehydes, hydrocarbons, ketones, alcohol
and sulfur that varied significantly due to treatments and storage. From the current exploration, it
is inferred that supplementation of quercetin and α-tocopherol is a pragmatic approach to enhance the functional value of meat and allied products that is effectual to manage serum lipid
profile.
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Chapter 1 INTRODUCTION
Functional foods are one of the core elements of diet based therapy that contribute towards
health promotion and disease management beyond the provision of basic nutrition. These
foods may be raw, processed or modified to ensure the optimal bioavailability of active
ingredients (Hernández et al., 2008). In this context, designer foods have gained immense
attention of the health care personals and diet conscious consumers in the global nutrition
market (Ares et al., 2009). During the last few decades, scientific investigations have proven
their disease ameliorative potential against various metabolic ailments including
hypercholesterolemia, hyperglycemia, obesity and oncogenesis (Shahidi, 2009).
Phenolic compounds are vital for in the human diet by the virtue of their chemical structure
that signify numerous health promoting benefits. These compounds have the ability to
perform various biological functions, reducing oxidative stress and degenerative diseases
owing to their antioxidant potential. Besides, they provide protection by activating
endogenous defense system by promoting cell modulating processes (Mei et al., 2011). In
recent regime, nutritionists are striving to improve public health and life expectancy by
curtailing lifestyle related disorders. Accordingly, antioxidant enriched functional foods are
becoming popular not only in the developing but also in the developed world due to their
high biological activity and safe status (Potawale et al., 2008).
The poultry industry in Pakistan is one of the vibrant and organized segments of agriculture
sector. The total poultry meat production in yesteryear has been documented as 987,000 tons
(GOP, 2014). This economic segment generates employment and income about 1.5 million
people directly or indirectly. In Pakistan, the contribution of poultry in agriculture and
livestock is 6.1 and 10.8%, respectively whilst 28% towards total meat production (GOP,
2014). Meat and its products play an imperative role in human nutrition being an excellent
source of protein, essential amino acids, minerals and vitamins (Weiss et al., 2010).
There are various strategies for introducing modifications in meat to enhance its value. These
approaches focus on limiting the concentration of compounds with adverse physiological
21
effects and increasing the level of those considered beneficial (Decker & Park, 2010). The
functional worth of meat can be enhanced mainly by three ways; by adding functional
compounds like conjugated linoleic acid (CLA), antioxidants including vitamin E, ω-3 fatty
acids and selenium to animal diet followed by incorporating ingredients such as vegetable
proteins, fibers, herbs & spices, probiotics and lactic acid bacteria in meat products during
processing and thirdly favoring the production of bioactive peptides during processing by
enzymatic hydrolysis (Zhang et al., 2010). Dietary supplementation/fortification of
functional ingredients to broiler is an ideal vehicle for delivering of target nutrient because it
increases the concentration of respective compounds in muscles through deposition thereby
enhances the functional value of resultant product.
The broiler meat contains higher levels of polyunsaturated fatty acids (PUFA) that trigger
oxidative deterioration, resultantly decrease the quality of meat products (Luna et al., 2010).
The lipid oxidation is one of the major route for quality degradation in poultry meat, apart
from microbial spoilage. The mechanistic approach involves generation of reactive oxygen
species and formation of free radicals, which produce rancid odor, off-flavor and surface
discoloration of meat and meat products. The rate of lipid oxidation in fresh and cooked meat
products depends on various internal factors such as fat content, fatty acid composition, level
of antioxidants, heme pigment and iron contents (Min et al., 2008). The external conditions
like processing, packaging and storage factors play important role in controlling the rate of
lipid oxidation (Ahn et al., 2009). This reaction may occur through auto, photo, and
enzymatic oxidation processes. Nevertheless, autoxidation is considered as the major route
towards lipid deterioration that is initiated by reactive oxygen species (Min and Ahn, 2005).
Antioxidants are the substances that at low concentrations retard oxidative problems of
oxidizable biomolecules such as lipids and proteins in meat products thus improving their
shelf stability and quality (Karre et al., 2013). The addition of antioxidants rich formulations
in various fresh and cooked meat products have tendency to reduce oxidation problems by
hindering the free radical formation. Dietary supplementation of antioxidants into animal
feed has proven a convenient strategy to uniformly distribute them in inner and outer layers
of phospholipid membranes (Nieto et al., 2010). The incorporation of antioxidants in blends
performed better as compared to a single one in living tissues and meat based products
22
(McCarthy et al., 2000). The oxidative problems in fresh and cooked meat can be controlled
by using antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT) and tert-butylhydroquinone (TBHQ) however, synthetic antioxidants may cause
adverse effects. Purposely, the industry and consumers prefer food with natural counterparts.
Recently, researchers are involved in exploiting the role of natural antioxidants from
different herbs and plant extracts like oregano, sage, rosemary, grape seed to replace
synthetic forms in meat and meat based products (Goliomytis et al., 2014).
In animal nutrition, the use of plants and their bioactive derivatives including extracts,
essential oils, and secondary metabolites for the development of functional meat is a novel
trend. The administration of herbs and botanicals to animals regulate their feed intake,
maintain a balanced microflora in the gastrointestinal tract (GIT) and exhibit antimicrobial
properties thereby enhance immunomodulatory and anti-inflammatory properties (Wenk,
2003). The bioextracts from plants like ascorbic acid, α-tocopherol, β-carotene, flavonoids
and other phenolic compounds have tendency to improve the antioxidant potential of living
system as well as processed meat products (Pennington and Fisher, 2009).
The quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy- 4H-chromen-4-one] is one of the
flavonol belongs to class of flavonoids, ubiquitously present in fruits and vegetables specially
red onion, caper, apple and in some medicinal & aromatic plants (Bhagwat et al., 2013).
Several in vitro and in vivo studies conducted on human and experimental animals have
revealed that quercetin possesses antioxidant and anti-inflammatory prospectives (Chang et
al., 2005). The antioxidant activity of quercetin is attributed to its ability to scavenge free
radicals, donate hydrogen atoms or electrons or chelate metal cations (Bodas et al., 2011).
The pharmacokinetics of quercetin have proven that it ameliorates the absorption and
metabolism of nutrients when enter the physiological system (Manach et al., 2005). The
quercetin and its glycosidic metabolites possess antioxidant properties and modulate
biological processes such as cell signaling pathways and reduction of oxidative DNA damage
(Wilms et al., 2005).
The quecetin is a potent antioxidant having ability to reduce lipid peroxidation in rodents as
well as controls blood pressure of hypertensive human subjects. The rats fed on quercetin
based diet showed a decline in hypertension and cardiac hypertrophy. The other benefits of
23
quercetin including vasodilation and reduction in blood pressure were also documented in
rodents. These potential health enhancing claims of quercetin are attributed to its antioxidant
potential that can minimize oxidative damages to the body of animals (Jalili et al., 2006). The
oxidative deterioration of low density lipoproteins (LDL) can be prevented by dieatary
quercetin as it possesses ability to scavenge free radicals and chelate transition metal ions
generated through the oxidation of lipids. Accordingly, quercetin play a role in the
prevention of certain metabolic disorders like cardiovascular disease, atherosclerosis and
chronic inflammation in living body. It also minimizes the inflammation caused by free
radicals generated through the process of oxidation. The resultant free radicals generated may
involve in the activation of transcription factors responsible for pro-inflammatory cytokines
(Boots et al., 2008). The absorption mechanism of flavonoids have indicated their entry in
the colon where they are hydrolyzed by enterobacteria to aglycones followed by their
absorption in the large intestine owing to their lipophilic nature. The major processes of
flavonoid aglycones metabolism are O-methylation, glucuronidation and sulfation occuring
in liver (Molina et al., 2003).
There are eight different forms of vitamin E including four tocopherols and four tocotrienols.
Amongst, α-tocopherol is the most abundant form of vitamin E and possesses high biological
activity. It is absorbed in the gastrointestinal tract in an efficient manner, thus α-tocopherol is
frequently referred as vitamin E. The main sources of vitamin E are sunflower, corn, soybean
and wheat germ, however, it is rapidly destroyed by cooking, coagulation and commercial
processing (Blatt et al., 2001). The α-tocopherol is the natural antioxidant that protects cells
and tissues from oxidative damage induced by free radicals (Miller et al., 2005). Later
studies have reported that α-tocopherol is not deposited to a toxic level unlike other fat
soluble antioxidants. Its accumulation at tissue level is strongly regulated through increasing
hepatic metabolism that also regulates environmental toxins and speed of drug metabolism
(Mustacich et al., 2010).
The supplementation of α-tocopherol in poultry feed has been found to be one of pragmatic
choice to improve oxidative stability of meat lipids and inhibiting oxidation of cholesterol
(Morrissey et al., 1998). The inclusion of α-tocopherol in feed protects the broiler birds
against stress-induced increase in thiobarbituric acid reactive substances (TBARS) by
24
limiting oxidation as well as preserving animal health (Young et al., 2003). It also enhances
the lipid stability and quality of Beijing-you chicken muscles when supplemented through
diet (Li et al., 2009). The supplementation of α-tocopherol at a level of 200 IU is considered
effective to increase the oxidative stability of meat and its concentration in broiler muscles
gradually raise in a dose dependent manner (Kim et al., 2010).
Keeping in view the above facts, present study was designed to develop the functional broiler
meat by dietary supplementation of quercetin and alpha tocopherol. The resultant meat and
meat based products were evaluated for storage stability and their role against lifestyle
related disorders with special reference to hypercholesterolemia. The instant study was
planned to assess the following objectives;
1) Elucidate the role of dietary quercetin and α-tocopherol on growth performance of
broilers.
2) Explore the potential of dietary antioxidants on biochemical parameters of broiler
meat.
3) Examine the quality characteristics and storage stability of experimental broiler meat
and meat based product.
4) Bioevaluation of functional broiler meat on serum lipid profile of human subjects.
25
Chapter 2 REVIEW OF LITERATURE
Novel health care strategies have illuminated the functional foods as one of the promising
therapeutic tools to combat various lifestyle related disparities. In this context, antioxidants
enriched diets with functional ingredients are effective to curtail specific maladies in targeted
groups. According to the current nutritional guidelines, diet and health nexuses has motivated
the consumers to choose food with some additional health benefits beyond the basic
nutrition. The escalating consumption of antioxidants rich foods is considered a promising
strategy towards nutrient optimization and food synergy in the living system. Extensive
studies have suggested that dietary antioxidant supplementation can enhance the functional
value of meat by improving the oxidative stability of allied proteins and lipids (Delles et al.,
2013). Earlier, Li et al. (2009) reported that dietary supplementation with α-tocopherol
improved meat tenderness and reduced lipid oxidation in broiler meat and meat based
products. One of the researcher groups, Bermejo et al., (2014) indicated that consumption of
functional meat containing Ω-3-fatty acids and rosemary extract assuage the oxidative and
inflammatory response of the people at risk. Considering the facts, the present study was
intended to develop functional meat by providing quercetin and α-tocopherol through feed
supplementation to broilers. A comprehensive debate regarding various aspects of the present
research study has been gathered herein:
2.1. Meat as a functional food
2.2. Poultry meat quality
2.3. Lipid oxidation and meat quality
2.4. Estimation of antioxidant potential
2.5. Antioxidants and broiler meat quality
2.6. Quercetin and alpha tocopherol as antioxidants
2.7. Storage stability of broiler meat products
26
2.1. Meat as a functional food
Globally, the diversity in human diet depends on various socioeconomic, regional & ethnic
customs and environmental factors. In developing countries, the increase in health care cost
and desire to maintain good health have focused the attention of nutritionists towards
functional foods (Gula et al., 2015). In this context, array of strategies are in practice to
develop foods with improved antioxidant potential and their utilization against lifestyle
related maladies (Barboza et al., 2012). Antioxidants are the substances that at low
concentrations possess ability to retard the oxidative damage of the biomolecules like lipids
and proteins. They are connected with the modulation of various metabolic pathways as free
radical scavenger, anti-microbial and provide shield against malignancy (Karre et al., 2013).
Considering the history, Japan was the first country that has introduced the idea and
regulations of functional foods (Kwak and Jukes, 2001). These foods are essential component
of Asian cuisine however, USA and Canada are the leading markets owing to the consumer’s
aptitude towards consumption of health promoting products (Verschuren, 2002).
Meat and meat based products can serve as excellent carriers for health boosting food
ingredients due to their versatility from minimally processed to fully cooked products as well
as their intrinsic high quality nutrients like protein, vitamins and minerals (Grasso et al.,
2014). Moreover, they are source of micronutrients with special reference to vitamins &
minerals and omega-3 fatty acids that are considered vibrant for human health (DHHS,
2010). Nevertheless, meat products may contain some nutrients that are considered harmful
including high fat content, saturated fatty acids (SFA), sodium level and cholesterol; linked
with obesity, cardiovascular diseases and hypertension. These nutrients can be reduced at
different stages of meat production and processing by using strategies like feed manipulation
or postmortem technological processes of carcass (Jiménez-Colmenero et al., 2010).
There are several ways to introduce qualitative and quantitative modifications in meat and
allied products to enhance their functional value. These ways are directed to limit the
concentration of compounds that exert adverse physiological effects and enhance the
concentration of those considered beneficial for human health (Decker and Park, 2010). The
nutritive worth of meat may be enhanced mainly by three ways; by adding functional
27
ingredients such as conjugated linoleic acid (CLA), antioxidants including vitamin C & E, Ω-
3 fatty acids and selenium through animal diet followed by adding constituents like vegetable
protein, fiber, herbs & spices, probiotics and lactic acid bacteria in meat products during
processing and lastly favoring the production of bioactive peptides during processing
involving enzymatic hydrolysis (Zhang et al., 2010). The dietary
supplementation/fortification of functional ingredients to broiler is an ideal vehicle because it
increases the concentration of respective compound in the muscles through deposition that
enhance the functional worth of resultant product.
2.2. Poultry meat quality
Globally, the consumption of poultry meat is attaining attention of the consumers due to high
quality protein, balanced amount of fatty acids as well as essential vitamins and minerals.
Poultry meat is among the most popular meats in the world owing to its low price, short
production time and ease of preparation (Chouliara et al., 2007). Meat and meat based
products fulfill major portion of daily dietary protein requirement of the individuals in
industrialized economies. The consumption of these products is affected by different factors
including product characteristics, consumer’s choice and the keeping environment (Mielnick
et al., 2006). The polyunsaturated fatty acids considered beneficial for human health are
prone to oxidation that can deteriorate the flavor and nutritional quality of meat based
products (Bañón et al., 2012). In stored foods, oxidation of lipids is the major process
responsible for quality degradation. This mechanism results changes that can deteriorate
flavor, color and nutritive value of food (Ripoll et al., 2013).
Besides, due to enhanced consumer’s knowledge regarding safe food, there is increasing
demand for quality poultry meat products (Bihan-Duval et al., 2008). Technologically,
quality refers to water holding capacity, color intensity, firmness and processing yield of
meat based products (Barbut, 2000). Relatively abundant amount of polyunsaturated fatty
acids (PUFA) are present in poultry meat, particularly omega-3 fatty acids due to PUFA rich
feed of fast growing broilers. Owing to enhanced PUFA content, broiler meat is more
susceptible towards oxidation that can adversely affect color, flavor and storage stability
(Gobert et al., 2010).
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2.3. Lipid oxidation and meat quality
Oxygen is one of the essential elements mandatory for oxidative metabolic reaction involved
in energy production for living organisms. Besides, it also participates in various reactions
generating toxic compounds such as reactive oxygen species (ROS) that are considered
harmful for physiological system (Halliwell and Gutteridge, 1989). The natural defense
system in living beings is prone towards the harm produced by ROS. Numerous evidences
suggested that singlet oxygen may induce damage to body tissues by producing free radicals
that accumulate in the tissues (Zorov et al., 2014). This damage is associated with oxidative
stress that has been documented to be involved in many chronic diseases like cancer,
cardiovascular disease, immune dysfunction and ageing (Grasso et al., 2014). The harmful
effects of oxygen are due to its ability to generate ROS that act as free radical or pro-oxidant.
These free radicals are unstable due to their reactive nature (Kim et al., 2010). A free radical
is a chemical entity that possesses one or more unpaired electrons. These radicals include
hydroxyl (OH-), superoxide (O-2), nitric oxide (NO-) and lipid peroxyl (LOO-) that degrade
cellular constituents including protein, lipids and nucleic acid (Dalle-Donne et al., 2003). The
technological processes like restructuring and grinding of meat may enhance the exposure of
lipid bilayers to the air that triggers the oxidation (Xiao et al., 2013).
Lipid oxidation represents one of the most important causes of deterioration of meat and
meat products and it affects unsaturated fatty acids particularly PUFA in membrane
phospholipids as well as cholesterol, mainly LDL cholesterol. The end-products of this
process impair color, aroma, flavor and texture of meat and allied products, hence reduce the
nutritive value (Mohamed et al., 2008). Besides nutritional deterioration, lipid oxidation
generates cytotoxic and genotoxic compounds which are deleterious for humans health
(Muselík et al., 2007).
Specifically, lipid hydroperoxides that are primary product of lipid oxidation have higher
polarity than normal fatty acids thus disrupt the integral structure and function of the
membrane that results detrimental effects to the tissues (Min and Ahn, 2005). The aldehyde
4-hydroxynonenal generated during lipid peroxidation possesses cytotoxic properties for
29
human and animals as it binds to protein, thereby inhibiting their function (Gheisari and
Motamedi, 2010). Lipids are subjected to oxidation when catalytic system such as light, heat,
enzymes, metals, metalloproteins and microorganisms are present. The presence of
intermediate reactive species and free radicals as well as these conditions lead to
autoxidation, photooxidation, thermal or enzymatic oxidation. However, oxidation of lipid is
mainly caused by autoxidation that is a spontaneous reaction of lipids with oxygen through a
chain reaction of free radicals that further cascade (Shahidi and Zhong, 2010). Lipid
deterioration is slow in initial stage that increases rapidly after an induction period. The
process consists of three phases: initiation, propagation and termination.
The mechanistic approach regarding lipid oxidation indicate that in the presence of pro-
oxidants, a hydrogen atom is abstracted from methylene group from the hydrocarbon chain of
lipid molecule (RH), especially from unsaturated lipids thus produce free radicals (R·). The
lipid radical tends to stabilize through rearrangement of methylene interrupted double bond
in PUFA that generate conjugated dienes. Under aerobic conditions, conjugated dienes react
with oxygen to form lipid peroxyl radicals (ROO·). Conversely, in low oxygen conditions,
conjugated dienes react with each other in membrane of protein and cholesterol (Min and
Ahn, 2005). Once peroxyl radicals are formed, they are unstable and attack new lipid
molecules thus engaged in rapid progression of reaction. During propagation stage, peroxyl
radicals remove hydrogen atom (H·) from lipid molecule to form lipid hydroperoxides
(ROOH) which are primary products of oxidation process (Brewer, 2011). Secondary
oxidation products that are originated from lipid hydroperoxides include aldehydes (hexanal,
4-hydroxynonenal, malondialdheyde), ketones, alcohols, hydrocarbons, volatile organic acids
and epoxy compounds depending on the fatty acid substrates and reaction conditions. Some
of the secondary oxidation products have undesirable odor that can be detected at low
threshold level (Luciano et al., 2012).
The oxidative damage to meat based products results in problems like tissues damaging,
putrification & loss of nutrients, enhanced free radical generation and malonaldehydes
production that reduce the antioxidant capacity of products (Tres et al., 2009). Lipid stability
of meat mainly depends on the balance of antioxidants, oxidation substrate, cholesterol and
heam pigment (Luna et al., 2010). In commercial poultry production, oxidative stress is
30
mainly due to imbalance between antioxidants and pro-oxidant resulting in deterioration of
physiological functions and immunity.
The effect of oxidation products on human health has been explored in three main areas; lipid
peroxides, malonaldehyde and oxysterols. Generally, oxidation products are considered as
inducer of cardiovascular diseases and atherogenesis. The observation that oxidized low-
density lipoproteins (LDL) triggers early atherogenesis (Perez et al., 2010) influence the
progression of coronary diseases. The oxidation of LDL decreases PUFA in meat and
enhance the level of lipid peroxides, aldehydes and oxysterols (Cortinas et al., 2005).
In recent years, oxysterols an oxidation product of cholesterol is of key concern owing to its
associated health hazards. Accordingly, meat and meat based products low in oxysterols are
more demanding in various consumers segments. The ingestion of oxysterol may induce
cytotoxic, atherogenic, mutagenic and carcinogenic effects by modifying cholesterol
concentration in cell membrane. Among oxysterols, Yin et al. (2000) found that 7-
ketocholesterol is the strongest inhibitor of cell proliferation and 25-hydroxycholesterol is an
effective inducer of apoptosis.
Omega-3 polyunsaturated fatty acids (ω-3 PUFA) play vital role in prevention and treatment
of cardiovascular diseases, diabetes, inflammation and cancer (Yashodhara et al., 2009). As a
consequence, nutritionists are advising people to consume ω-3 fatty acids specifically
eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. An effective approach for
raising ω-3 PUFA content of meat is increasing their level through feed fortified with
antioxidants (Rymer and Givens, 2005).
The fatty acid profile of meat is modified to improve the sensory attributes of the resultant
product (Milicevic et al., 2014). Diets high in saturated fatty acids (SFA) increase LDL
cholesterol linked with incidence of coronary heart diseases. Nonetheless, monounsaturated
fatty acids (MUFA) and particularly PUFA have beneficial effect on human health.
Accordingly, the key goal of current nutritional strategies is to focus on meat production with
lower level of saturated fatty acids and cholesterol (Wood et al., 2008).
In this context, Lu et al. (2014) conducted a study to determine the effect of antioxidant
blend and vitamin E on fatty acid composition, inflammatory status and liver function of
broiler birds. Purposely, 500 male broilers were distributed into 6 treatments including (1)
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high-oxidant diet with vitamin E @10 IU/kg, 3% oxidized oil, 3% polyunsaturated fatty acids
(PUFA) source (HO); (2) HO diet and vitamin E at 200 IU/kg (VE); (3) HO diet with
antioxidant blend at 135 mg/kg (AOX) (4) HO diet with vitamin E at 200 IU/kg and
antioxidant blend at 135 mg/kg (VE+AOX) (5) standard control (SC) (6) positive control
containing antioxidant blend 135 mg/kg. The results indicated that Ω-3 fatty acids and
concentrations of 20:4, 20:5, 22:5, 22:6 fatty acids are increased in abdominal fat of HO, VE,
AOX, and VE+AOX groups compared to standard and positive control on 21 and 42 days of
growth period. Higher PUFA content are found in HO, AOX and VE+AOX feed groups. On
21st day, the value for inflammation for AOX and VE+AOX birds are lower compared to
remaining groups. Conclusively, AOX and AOX+VE diets are effective in preserving PUFA
content of abdominal fat, moderately improved liver function and decreased the incidence of
inflammation.
Dietary supplementation of flavonoids (genistein and hesperidin) positively influence the
fatty acid profile of broiler meat. Earlier, a study was conducted by Kamboh and Zhu (2013),
investigated the effect of genistein and hesperidin as an alternative to synthetic antioxidant
on fatty acid composition, lipid metabolites and antioxidant status of broiler birds. One day
old, 360 birds were fed on control (basal diet), G5 (5 mg of genistein/kg), H20 (20 mg
hesperidin/kg), GH5 (genistein & hesperidin @5 mg/kg), GH10 (genistein & hesperidin @10
mg/kg) GH20 (genistein & hesperidin @20 mg/kg), respectively. The results revealed that
antioxidant potential of broiler meat was improved in a dose dependent manner (p<0.05) as
indicated by lower MDA and higher superoxide dismutase activity. Total cholesterol and
triglyceride levels were also decreased. However, PUFA content of breast meat was
significantly enhanced with increasing level of dietary flavonoids. Conclusively, flavonoids
are considered feasible alternative to synthetic additives for healthy chicken meat production.
The dietary supplementation of α-tocopherol acetate through feed protects the broiler meat
against lipid oxidation. Previously, Rebolé et al. (2006) evaluated the impact of addition of
two fat sources including high oleic acid sunflower (HOASS; 0, 50, 100, 150 and 200 g/kg
diet) and palm oil (PO) in combination with α-tocopherol acetat (200 mg/kg feed) on growth
performance, fatty acid profile and oxidative stability of meat under refrigerated storage. The
results suggested that weight gain and feed conversion ratio of birds are decreased with
32
supplementation of highest proportions of HOASS (150 and 200 g/kg). The results also
elucidated that addition of HOASS through diet decreased the PUFA content of broiler meat.
However, incorporation of α-tocopherol acetate amplified the PUFA content by hindering
their oxidation. Similarly, addition of α-tocopherol acetate via feed to broilers resulted in
lower TBARS that enhanced the shelf stability of meat under refrigerated storage. In nut
shell, increasing fatty acids contents of broiler meat by replacing dietary PO with HOASS
(up to 100 g/kg) do not affect the broiler growth performance and enhance the storage
stability.
2.4. Estimation of antioxidant potential
The comparison of different antioxidants in various food items remained a challenge owing
to diversified mechanisms that induce oxidation (photo, enzymatic and autoxidation, etc.)
and complexity of the matrix. In this context, different model studies are carried out in food
system to estimate the antioxidant potential. However, these models are costly, labor
intensive and rarely retain applicability across different food system. There are multiple
assays documented in the literature that are used to measure the in vitro total antioxidant
capacity. Nonetheless, scientists are trying to explore different methods for direct estimation
of antioxidants in foods. Such methods discussed herein are comparatively convenient,
accurate and reliable that can be used effectively in food and cellular matrixes.
The methods assess the oxidative status of biological systems can be classified as; detection
of primary or secondary commounds i.e. malonaldehyde & oxysterol and fluorescent
products, conjugated dienes assay, estimation of hydrocarbon gases and loss of PUFA.
However, sensory analysis may also be used to detect oxidative off-flavors by taste or smell
in order to decide that lipid containing food is suitable for consumption (Pokorny et al.,
2001). Primary products of lipid oxidation include lipid hydroperoxides that quantified by
using ferrous oxidation xylenol orange test. This technique is based on reaction between dye
xylenol orange and ferric ions which produces blue-purple complex with maximum
absorbance between 550-600 nm (Grau et al., 2000).
The measurement of secondary oxidation products are mainly quantified through cholesterol
oxidation products (oxysterol) or by determining thiobarbituric acid reactive substances
(TBARS). The quantification of oxysterol involves lipid extraction, purification,
33
saponification and column or thin layer chromatography fractionation (Frankel and Meyer,
2000). However, TBARS assay is a direct procedure to measure the quantity of
malondialdehyde (MDA), breakdown product mainly from oxidized PUFA. This assay is
based on the MDA’s ability to react with TBA at 530-537 nm (Veberg et al., 2006)
Another method to monitor oxidative deterioration is determination of conjugated dienes
(C=C-C=C-C). The formation of hydroperoxides from PUFA oxidation leads to conjugation
of the pentadiene structure. Conjugated dienes are measured because they showed an intense
absorption at 233 nm. Another technique is based on the quantification of volatile
compounds including pentane and ethane that are formed from -3 and -6 fatty acids,
respectively during oxidation. The quantification is done using gas chromatography (GC) and
is normally accompanied with sensory assessment (Huang et al. 2005).
2.4.1. Reduction by Folin ciocalteu reagent to measure phenolic contents
This method uses the folin ciocalteu reagent to estimate antioxidant capacity of different
foods that is based on reduction principle. The assay includes oxidation of mono- and vicinal
diphenols species and engage oxidant with high redox potential (0.7 V). Since, the reaction
involves molybdenum & tungsten and is assumed as following
M6+ + e- → M5+
In this reaction, phenolic species dissociate into phenolate anion, capable of reducing the
folic ciocalteu reagent which create a blue colored specie. The absorbance can be measured
between 730-760 nm. Depending on the nature of antioxidant, different variables are taken
into account such as presence of reducing sugar, amino acid, copper (I) complex, vitamin C
etc. Additionally, nature of the phenolate ion also makes pH an important factor.
2.4.2. Free radical scavenging by DPPH
The DPPH radical used to measure antioxidant activity of different foods is determined by
delocalized electron. The mechanistic approach involves bleaching of DPPH solution that
represents capacity of tested compound to scavenge free radicals after hydrogen donation.
This method is advantageous to estimate the antioxidant capacity because it occur over a
wide pH range and is sensitive to atmospheric oxygen and light. The major factors for the
assay are type of solvent and DPPH concentration. The extraction of antioxidant using
34
buffered methanol is preferred for non-polar or less polar compounds (over water and
acetone) although DPPH concentration of 25-70 μM gives accurate results for different
foods.
2.4.3. Colorimetric determination with iron salts
In meat and meat based products, heme-iron is a common reducing agent that assists in
oxidation of lipids and proteins. The ferric reducing antioxidant power (FRAP) assay
involves the reduction of ferric compounds (Fe3+) to ferrous (Fe2+) such as ferric
tripydriyltriazine [Fe(TPTZ)2] described as below
Fe(TPTZ)23+ + ArOH Fe(TPTZ)2
2+ + ArO∙ + H+
This method involves iron for determination of antioxidant potential thus useful for the
estimation of antioxidant activity of meat and meat based products (Benzie and Strain, 1999).
In FRAP, the redox reaction between ferric 2,4,6,-tripyridyl-s-triazine (Fe3+-TPTZ) to its
divalent form liberates an intense blue color that is measured at a wavelength of 593 nm. The
non-specificity of the assay is useful to compare multiple antioxidant but is problematic
regarding antioxidants of different stoichiometric nature e.g. vitamin C compared to
bilirubin.
2.5. Antioxidants and broiler meat quality
Antioxidants are the substances present in low concentrations compared to oxidizable
substrate that significantly delay or avoid substrate oxidation (Velasco and Williams, 2011).
An important defense system in biological functioning is the presence of antioxidant
enzymes such as glutathione peroxidase, superoxide dismutase and catalase that reduce the
concentration of harmful oxidants. The presence of nutrients like selenium, copper,
magnesium and zinc enhance the catalytic activity of antioxidant enzymes thereby play a
vital role to boost immunity of the living system against free radicals. Antioxidants scavenge
free radicals by reacting with substrates thus reduce their capacity of damaging. Antioxidants
are divided in two types; preventive antioxidants that protect target lipids from oxidation
initiators by blocking the formation of reactive oxygen species or scavenging species
responsible for oxidation initiation. Second type include chain breaking antioxidants that stop
35
the propagation phase intercepting radical oxidation propagators or indirectly stopping chain
propagation. They are also incorporated in the animal feed to increase the meat oxidative
stability either singly or in combination. However, Barroeta, (2007) suggested that
combination of antioxidants are more effective in retarding lipid oxidation. They are also
responsible to retard protein oxidation as well as the interaction of lipid derived compounds
with protein carbonyls that alter the functionality of protein in meat based products (Elias et
al., 2008).
There are different classes of antioxidant including synthetic such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertabutylhydroquinone
(TBHQ) and natural ones like vitamin E, C, β-carotene, lutein, carotenoids, α-lipoic acid,
quercetin etc. However synthetic counterpart may cause harmful effect on human health
(Tavarez et al., 2011 Several studies have been conducted to explore the natural antioxidants
like rosemary, sage, ginger, grape seed and green tea to reduce lipid peroxidation during
storage and found efficient to control the menace (Selim et al., 2013).
According to Staszewski et al. (2011), inclusion of rosemary and sage effectively reduced the
rancidity and improve shelf life of meat products. Similarly, Fassess et al. (2007) recorded
that addition of herbs like oregano, sage, thyme, basil, black and white pepper @0.5 to 2.5%
are effective in enhancing the antioxidant potential of meat products. Afterwards, Du and
Ahn (2009) reported that addition of vitamin E in irradiated sausages from turkey thigh meat
decreased redness and volatiles production. Likewise, addition of phenolic rich formulations
in meat products have potential to inhibit oxidation. The antioxidant properties of phenolic
acids and flavonoids are attributed to their redox potential and chemical structures that make
them feasible reducing agents and oxygen scavenger (Ganhao et al., 2010).
2.6. Quercetin and alpha tocopherol as antioxidants
Plants and their bioactive derivatives like extracts, essential oil and secondary metabolites
have attained immense attention of the researchers for the development of novel additives to
improve dietary nutrition. The quercetin is one of the flavonols present in fruits and
vegetables especially red onion, caper, apple as well as in some medicinal & aromatic plants
(Rupasinghe 2010). It is a powerful antioxidant holds ability to scavenge free radicals and
36
bind transitional metal ions. It is a powerful antioxidant that scavenges free radicals and
binds transitional metal ions (Soundararajan et al., 2008).
Antioxidants provide protection against oxidative stress that is linked to lifestyle oriented
disorders such as hypercholestermia, hyperlipidemia, cardiovascular dysfunction and cancer
insurgence. The hardening of arteries occurs under oxidative stress in atherosclerosis is
associated with oxidation of low density lipoproteins (LDL) that may be reduced by
increasing the consumption of antioxidant enriched foods. The oxidation products such as
isoprostans from arachidonic, eicosapentaenoic and docosahexaenoic acids, oxysterols from
cholesterol, hydroxyl fatty acids, lipid peroxides and aldehydes are decreased through
supplementation of antioxidants in living beings. The bioassay of lipid oxidation is
considered as a marker for risk assessment of cardiovascular disorders. These damages can
be healed by the intake of dietary antioxidants that act as a shield against free radicals (Zhang
et al., 2014).
The administration of quercetin to broilers enhanced the heart weight that potentially
contributes towards improved animal health. In a study, quercetin dietary supplementation at
a level of 0.5 and 1 g/kg of feed indicated that meat oxidative stability expressed as ng of
MDA/kg meat is improved (p< 0.05) for 3 to 9 days under refrigerated storage. Likewise,
Rupasinghe et al. (2010) also reported that when broiler are provided quercetin through diet
@0.5, 1.4, 2.8, and 5.6 g/kg feed is properly metabolized and absorbed by the birds,
Moreover, its metabolites are also detected in plasma and tissues.
The addition of brined onion extract in cooked turkey breast rolls improved the oxidative
stability under refrigeration storage. In this context, one of the researchers groups Tang et al.,
(2007) conducted a study in which they added brined onion extract in rolls that enhanced the
oxidative stability as well as cooking yield significantly (p<0.0001). The inclusion of dietary
quercetin improves water holding capacity and oxidative stability of pork products.
Accordingly, Kremer et al. (2013) elucidated the effect of feeding quercetin, a glycolytic
inhibitor on pork quality. Accordingly, Four hours before slaughter, pigs (111±7 kg) were
provided 547 g feed containing 0, 2.5 and 12.4 ppm of quercetin/ kg feed. The findings
indicated that dietary supplementation of quercetin is efficient to control pH fall in muscle
after postmortem this enhances cooking yield. They further elaborated the muscle pH was
37
0.08 to 0.12, greater in pigs fed on quercetin from 22 to 180 min after postmortem.
Moreover, 0.3 to 2.2 % less water loss was recorded in samples stored under freezing
conditions for 12 days.
In another study, one of the scientists groups Andrés et al. (2013) indicated that feed
supplementation with quercetin alone or in combination with flaxseed is helpful for lowering
discolouration in lamb meat. Accordingly, 32 lambs were fed on barley straw in combination
with concentrate formulated with palm oil, quercetin and flaxseed to determine their effect on
meat quality. The lambs were fed on concentrate for 5 weeks with normal experimental diet.
The results delineated that color variations of longissimus thoracis (LT) muscles were
affected among treatments however, meat samples of lambs fed on quercetin and flaxseed-
quercetin enriched diet groups resulted in less discoloration.
Dietary inclusion of quercetin reduces oxysterol content in fresh meat even after seven days
of refrigerated storage. In this context, lambs were fed on mixed ration formulated either with
palm oil (CTRL; 34 g palm oil kg-1) or whole flaxseed (+FS, 85 g flaxseed kg-1) alone and
in combination with quercetin (+QCT, 34 g palm oil plus 2 g quercetin kg-1). The results
indicated that flaxseed supplementation did not affect lipid peroxidation of meat. However,
quercetin supplementation significantly decreased oxysterol content of fresh meat after 7
days of storage. Sensorial response revealed that flaxseed imparted negative flavor in meat
owing to the modification of fatty acid profile (Andrés et al, 2014).
The oxidative stability of protein and lipids can be improved by the incorporation of
antioxidants thereby enhance the activity of cellular antioxidant enzymes. In this regard,
Delles et al. (2014) conducted a study to explicate the effect of antioxidants and oil quality
on oxidation and enzymatic properties of broiler breast meat stored under oxygen enriched
package (HiOx: 80% O2/20% CO2) in comparison with air permeable polyvinylchloride
(PVC) and skin packaging systems. The birds were provided diets containing low oxidized
(peroxide value 23 mEq of O2/kg) and high oxidized (peroxide value 121 mEq of O2/kg) oils
with and without antioxidants. It has been observed that lipid oxidation as measured by
TBARS is decreased 32.5% (p<0.05) by addition of antioxidants. They also documented that
enzymes i.e. glutathione peroxidase, catalase and superoxide dismutase activities are
significantly higher (p<0.05) in antioxidant supplemented group regardless of oil quality.
38
The fresh meat imparts bright red color due to muscle protein myoglobin (Mb) however,
color varies with redox state of heme. The red color of meat is imparted by oxymyoglobin
(MbO2) produced by the combination of oxygen and myoglobin. However, oxymyoglobin is
highly unstable and is converted to metmyoglobin resulting in brown color which decreases
the acceptability of fresh meat. Phenolic compounds of plant origin delay the conversion of
oxymyoglobim to metmyoglobin (MetMb). In this context, Inai et al. (2014) conducted a
study to explore the reduction activity of MbO2 to MetMb by three flavonols as kaempferol,
myricetin and quercetin, at 300 μmol/L against 60 μmol/L MetMb. The results elucidated
that quercetin reduces the conversion of MbO2 to MetMb whilst the mechanistic approach is
its capacity to stop redox reaction that rapidly convert the MbO2 to MetMb.
The flavonoids modify myeloperoxidase oxidation (MPO) and are considered useful in
protecting low density lipoproteins (LDL) from oxidative damage. According to Dhanya et
al., (2014) dietary quercetin is effective in protecting LDL against neutrophil mediated
modification at concentration of 1μM. In another study, David et al. (2011) reported that
thioacetamide induced hepatotoxicity caused by oxidative stress and apoptosis is reduced by
providing quercetin enriched diet. They further stated that quercetin treatment significantly
affected antioxidants enzyme such as superoxide dismutase, catalase and glutathione
peroxidase activity resultantly decreased the lipid peroxide level in thioacetamide treated rats
compared to the control.
The regular ingestion of flavonoids is linked with reduction of cardiovascular diseases.
Purposely, Benito et al. (2004) conducted a study to determine the impact of diet containing
quercetin and catechins (0.3% wt/wt) on oxidative biomarkers in healthy rats for 10 days.
The results documented similar level of α-tocopherol and retinol in all groups. However,
antioxidant status of rats fed on quercetin diet was improved. The glucuronide and sulfate
metabolities of quercetin are also found in the plasma of rats fed on quercetin enriched diet.
The quercetin plays an effective role against cardiovascular disorder due to its potential to
modulate dyslipidemia. The serum lipid level is mainly regulated by alteration in hepatic
lipid synthesis. In this context, Gnoni et al. (2009) investigated the potential of quercetin on
synthesis rate of fatty acids, cholesterol, neutral lipids, phospholipid and very low density
lipoproteins (VLDL) using rat experimental modeling. The results showed that incorporation
39
of quercetin in hepatocytes for 30 min resulted in the inhibition of fatty acid synthesis.
Nonetheless, highest reduction was reported in palmitic acid. The justification for this
reduction was the formation of VLDL and triacylglycerol complex resulting the
hypotriacylglycerolemic effect of quercetin.
The diethylnitrosamine (DEN) is a potent hepatocarcinogen mainly found in tobacco smoke
and some processed meat products. The administration of quercetin protects the animals
against DEN induced hepatotoxicity. In this reference, Gupta et al., (2010) conducted a study
to explore the effect of quercetin against DEN induced hepatotoxicity through rat
experimental modeling. The quercetin was administered @ 10, 30 and 100 mg/kg feed for 5
days after DEN (200 mg/kg) treatment and animals were killed 24 h after last dose
administration of quercetin. The DEN induced hepatotoxicity was evident by elevated MDA
level and decreased glutathione (GSH) contents of liver. A significant increase in plasma
aspartate transaminase (AST) and plasma alanine transaminase (ALT) was reported in DEN
treated group. The supplementation of quercetin restored AST, ALT and GSH levels at tested
dosage.
Dietary intake of flavonoids, carotenoids and antioxidants provides protection against
hypercholesterolemia. Recent evidences suggest that oxidative damage is involved in
atherogenesis thus diet containing antioxidants like α-tocopherol and quercetin may reduce
the insurgence of cardiovascular disease (Riccioni et al., 2012). It has been observed that
lipid soluble vitamin E acts as antioxidant that plays significant role in protection of cell
membrane against LDL cholesterol oxidation (Valko et al., 2007).
Hypercholesterolemia is a condition in which serum lipid level increases particularly total
cholesterol and low density lipoproteins (LDL). Besides, poor nutritional practices including
diet with high concentration of cholesterol and saturated fats adversely affect consumer’s
health. The resultant changes appear in the blood with altered concentration of total
cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL) and
triglycerides (TG). Estimation of these parameters is used as a diagnostic tool to assess the
degree of dysfunctioning. During hypercholesterolemic state, elevated serum LDL and
decreased HDL levels progressively induce atherosclerosis, inflammation and widen the
vascular lesions (Uchiyama et al., 2011)
40
Earlier, Egert et al. (2009) conducted a study to examine the effect of quercetin
supplementation for reducing systolic blood pressure and low density lipoprotein
concentration in overweight subjects. In this experimental, 93 obese people aged 25-26 were
used to elucidate the impact of quercetin on blood pressure, lipid metabolism, oxidative
stress and inflammation. The results indicated that administration of quercetin decreased
systolic blood pressure (SBP) by 2.6 mm Hg however, significant reduction in SBP (2.9 mm
Hg) was noticed in hypertensive and a reduction of 3.7 mm Hg in normal individuals.
Conclusively, quercetin inclusion through diet may reduce SBP and LDL concentrations in
overweight subjects.
Dietary antioxidants interact in a dynamic fashion to enhance their synergistic potential
against oxidative stress. In this context, Ameho et al. (2008) investigated the metabolism and
antioxidant capacity of quercetin in rats under vitamin E enriched (R-VE) and deficient diets
(D-VE). After 12 weeks of quercetin administration, samples were collected to estimate the
vitamin E contents, quercetin and its metabolite concentration, serum pyruvate kinase (PK),
protein carbonyls, and MDA level. The result expounded that diet without antioxidant
decreases α-tocopherol concentration and increases PK activity in time dependent manner.
However, this diet enhances protein carbonyl without affecting MDA level. Moreover,
dietary supplementation improved quercetin and its metabolities concentration in plasma and
liver of rats. Conclusively, quercetin did not reduce in vivo vitamin E synthesis that is
involved in the circulation and absorption of quercetin metabolites in muscles.
Flavonoids act synergistically with α-tocopherol in protecting low density lipoprotein
against oxidation. In this regard, Choi et al. (2003) reported that dietary administration of
quercetin increases α-tocopherol concentration in serum and liver of rat. Similarly, Frank et
al. (2006) also stated that dietary administration of quercetin and catechin improves α-
tocopherol level in serum and muscles. In this context, male sprague dawley rats were fed on
basal diet containing 2 g/kg cholesterol and diets fortified with quercetin, epicatechin and
catechin @2 g/kg feed. The results showed that flavonoid supplementation significantly
increases α-tocopherol concentration in plasma and liver. Several in vitro studies documented
that dietary quercetin possesses antioxidative, anti-inflammatory and immunomodulatory
properties thus induces positive impact on living organisms. Epidemiological studies also
41
confirmed that regular consumption of quercetin reduces incidence of lifestyle oriented
diseases and cancer.
Tocopherols are a group of naturally occurring compounds containing α, β, γ and δ
tocopherols and corresponding tocotrienols. They are derivatives of 6 chromanol with C16-
phytyl side chain while tocotrienols possess three fold unsaturated side chain. The
homologues tocopherol and tocotrienol differ in degree and position of methyl substitution at
C5, C7 and C8 of the chromanol ring (Golli and Azzi, 2010).
Accrding to Saaladino et al. (2008) tocopherols are the major lipid soluble antioxidants that
are not synthesized by animals so must be included in animal’s diet. The α-tocopherol is
among the lipid soluble antioxidants considered safe supplement for animals. It is the major
component of biological membranes that retards production of hydroperoxides by disrupting
the oxidation chain reaction (Morrissey et al., 2006). It is also an essential micronutrient for
maintaining the health and wellbeing of animals due to allied antioxidant properties
(Brigelius-Flohe and Galli, 2010). Alpha tocopherol is the active form of vitamin E that is
used in commercial formulations (Yang and Huffman, 2011).
The incorporation of α-tocopherol in combination with rosemary leaves enhances the
oxidative stability of turkey meat. In this context, Botsoglou et al. (2007) conducted a study
in which 36 birds were fed on diet containing 0, 0.5 and 1.0% dehydrated rosemary leaves in
combination with α-tocopherol acetate @10 and 300 mg/kg feed for 4 weeks. The meat
samples analyzed for malondialdehyde (MDA) and α-tocopherol content elucidated that α-
tocopherol acetate @300 mg/kg significantly (p<0.05) enhanced the α-tocopherol level. They
further stated that MDA production in meat was decreased linearly in a dose dependent
manner. Conclusively, providing diet enriched with 300 mg/kg α-tocopherol acetate is a
feasible strategy to improve enhance oxidative stability of turkey meat.
Alpha tocopherol in biological system improves tissue integrity and provides protection
against lifestyle oriented disorders due to its antioxidant prospective. The muscle contains α-
tocopherol however, its concentration is increased through fortification or supplementation.
The main reason for fortifying meat products with α-tocopherol is its ability to protect
against protein and lipid induced oxidation. Consequently, vitamin E supplementation
extends shelf life of meat and allied products without affecting sensory response. The dietary
42
supplementation of α-tocopherol improves water holding capacity that further decreases the
development of pale and soft exudative in poultry meat (Zhang et al., 2010).
Accordingly, Guo et al. (2001) carried out a study to determine the effect of dietary α-
tocopherol on growth performance and lipid stability of broiler thigh muscle. Purposely, 900
birds were distributed in five groups and fed on basal α-tocopherol level 13 mg/kg and
remaining on 0, 5, 10, 50 and 100 mg/kg feed. The results showed positive correlation
between the levels of α-tocopherl and hepatic α-tocopherol concentrations (p<0.05).
However, negative correlation with hepatic TBARS was recorded. The hepatic α-tocopherol
concentration was improved linearly in dose dependent manner (R2=0.98, p<0.001). The
TBARS value of meat was decreased with increasing level of α-tocopherol (p<0.05) under
storage. In the nut shell, α-tocopherol supplementation substantially improved the storage
stability of meat against oxidative deterioration.
Likewise, Zhang et al. (2013) investigated the effect of α-tocopherol acetate on growth
parameters and meat quality of broiler birds fed on diet containing maize distiller dried grains
with solubles (DDGS). Purposely, 360 birds were divided in treatments as DDGS (0, 10 and
20%) and α-tocopherol acetate at concentration of (0 & 200 mg/kg). The results elucidated
that addition of 20% DDGS in diet irrespective of α-tocopherol decreased the growth
performance of birds. Moreover, addition of 20% DDGS did not improve color, drip loss,
cooking characteristics and shear force of meat. However, dietary supplementation of α-
tocopherol @200 mg/kg feed significantly reduced cooking loss and shear force.
Conclusively, dietary supplementation of broilers with DDGS @10% in combination with α-
tocopherol acetate @200 mg/kg feed improved broiler growth performance and meat quality.
According to Tres et al. (2008), dietary supplementation of feed with 1.5% linseed oil and
1.5% beef tallow along with α-tocopherol acetate improves nutritional quality of rabbit meat.
The oxidative stability as measured by TBARS was also improved. However, incorporation
of linseed oil in combination with α-tocopherol acetate influenced fatty acid profile of meat.
The results suggested that lipid stability of rabbit meat is decreased by the addition of 3%
linseed oil. However, this problem is less apparent in raw meat compared to cooked meat.
43
In a study, Yesilbag et al. (2011) investigated the effect of supplementation of α-tocopherol
acetate, rosemary leaves and its oil on broilers growth performance and meat quality as
measured by TBARS, physicochemical analysis and sensorial attributes. Purposely, 800
chicks were divided in control and 7 experimental groups. The control group (VitE1) was
provided diet containing 50 mg/kg α-tocopherol acetate whilst experimental groups were
given 5.7 g/kg rosemary leaves (R1), 8.6 g/kg leaves (R2), 11.5 g/kg leaves (R3), 100 mg/kg
rosemary oil (RO1), 150 mg/kg rosemary oil (RO2), 200 mg/kg rosemary oil (RO3) and 200
mg/kg α-tocopherol acetate (VitE2). The results indicated non-significant differences for feed
intake, however, weight gain, feed efficiency and carcass yield were significantly improved.
The MDA level of broilers meat fed on diets containing rosemary leaves and its oil was
significantly lower compared to birds fed on α-tocopherol enriched diet. Additionally,
significant differences were reported for color, pH and sensory attributes of experimental
broiler meat compared to control.
The protection against deterioration of membrane lipids by α-tocopherol depends on its
concentration in the membrane. Highest accumulation of α-tocopherol occurs in
mitochondrial membrane followed by endoplasmic reticulum that protects the membrane
against generation of ROS (Dutta-Roy, 1999). Dietary supplementation of α-tocopherol
increases lipid stability of broiler thigh meat. In this context, Ruiz et al. (1999) conducted an
experiment to evaluate the impact of dietary fat (6% Lard, sunflower and olive oil) in
combination with α-tocopherol acetate on raw, cooked and chilled broiler meat. The results
revealed that oxidative stability of leg meat was decreased with the supplementation of
sunflower oil however, α-tocopherol acetate improved the oxidative stability as measured by
TBARS.
According to Rama Rao et al. (2011), use of α-tocopherol @50 and 100mg/kg feed improves
cell mediated immune response by enhancing the activity of antioxidant enzymes. Purposely,
3 types of oil i.e. sunflower(SFO), safflower (SAO) and palm oil (PO) were used in
combination with 3 levels of α-tocopherol @10, 50 and 100mg/kg feed. The results
elucidated that dietary α-tocopherol significantly improved total protein, globulin and
triglycerides of blood serum. The oxidative stability of meat was improved as indicated by
lower MDA level with increasing α-tocopherol concentration. Resultantly, diet
supplementation with α-tocopherol in combination with oil sources increased the activity of
44
antioxidant enzymes i.e. glutathione peroxidase and catalase which enhanced the cell
mediated immune response.
The presence of α-tocopherol in subcellular fraction of muscle increases the storage stability
of meat exposed to iron ascorbate induced oxidation. The inclusion of α-tocopherol acetate
@200 IU/kg feed reduced the rate of oxidation as indicated by lower TBARS level (Gavazza
and Cataala, 2006). Similarly, ((Barroeta, 2007) indicated that lipid stability of meat is
improved by providing 200 IU of α-tocopherol per kg feed. The increase in dietary α-
tocopherol concentration is a useful strategy to maintain cooking characteristics of broiler
meat. The vitamin E concentration of muscle is affected by several factors like duration of
supplementation, type of muscle fiber and metabolic characteristics of animal.
In another study, Gao et al. (2010) explored the effect of glucocorticoids and α-tocopherol
administration on lipid peroxidation of meat. Purposely, birds were provided diet with two
levels (20 & 200 mg/kg feed) of α-tocopherol. On 35th day, the birds were injected
dexamethasone (DEX, 2mg/kg of BW) for 6 days. The results indicated suppressed growth
by the administration of DEX. However, α-tocopherol supplementation improved survival
efficiency and growth performance of birds. Moreover, the DEX treatment resulted higher
TBARS compared to vitamin E supplemented group. Furthermore, superoxide dismutase
activity was increased by DEX treatment. Conclusively, vitamin E supplementation
improved broilers performance by alleviating oxidative stress induced by DEX treatment.
Dietary supplementation of vitamin E alone or in combination with selenium improves the
hematological parameters of broiler birds. In this context, Biswas et al., (2011) conducted a
study in which birds were fed on basil and antioxidant enriched diet containing 100 g (150 IU
vitamin E/kg + 0.5 mg Se/kg) and 200 g (300 IU vitamin E/kg + 0.5mg Se/kg) as a source of
vitamin E and selenium. It has been observed that antioxidants treated groups exhibited
higher weight gain than that of control. Moreover, total erythrocyte count (TEC), packed cell
volume (PCV) and hemoglobin (Hb) content were increased significantly (p<0.01) in treated
groups. However, erythrocyte sedimentation rate (ESR), glutamic oxaloacetic transaminase
(GOT) and glutamic pyruvic transaminase (GPT) were decreased substantially in
experimental groups.
45
The supplementation of long chain fatty acids and α-tocopherol acetate to broilers influence
meat quality. In this context, a study was conducted by Rymer and Givens (2010) who
explored the effect of long chain Ω-3 polyunsaturated fatty acids and α-toocpherol acetate on
fatty acid profile and sensory attributes. The birds were provided diet containing Ω-3 PUFA
(0, 9 and 18 g kg−1) and Vitamin E (100, 150 and 200 mg kg−1) feed for 42 days. The results
showed that nutritional profile of broiler meat was improved. However, α-tocopherol acetate
>100 mg/kg diet was needed to protect meat against oxidative deterioration.
Apple peels have ability to prevent low density lipoprotein cholesterol (LDLC) oxidation
being rich in antioxidants and bioactive compounds. The LDLC oxidation leads towards
initiation of atherosclerotic plaque formation. In this context, Surangi et al. (2013) used apple
peel quercetin enriched (QAE) and flavonoid rich (TAE) extract to inhibit in vitro LDLC
oxidation and documented these extracts efficiently controlled LDLC oxidation. The
IC50 values for QAE and TAE extracts were 0.06–8.29 mg/L and 29.58–95.49 mg/L,
respectively. They further stated that three in vivo quercetin metabolites; quercetin-3-
glucuronic acid, quercetin-3-sulfate and isorhamnetin-3-glucuronic acid have shown ability
to prevent LDLC oxidation.
Flavonoid and triterpene are major groups of bioactive moieties in apple peel. Accordingly.
Thilakarathna et al. (2012) conducted a study to determine the effect of flavonoid-rich (FRE)
and triterpene-rich (TTE) apple peel extract against cholesterol metabolism in diet induced
hypercholesterolemic male hamsters. The hamsters were provided FRE or TTE @50 mg
bioactives/kg/day. The results delineated that supplementation of FRE extract for 28 days
reduces serum total cholesterol (TC) and LDL cholesterol level however, TTE enhances
serum TC. They further stated that such extracts do not affect triacylglycerol and HDL
significantly. Moreover, FRE and TTE resulted increased absorption of cholesterol without
affecting its synthesis. In the nut shell, FRE and TTE affected cholesterol metabolism while
FRE effectively decreased serum cholesterol.
Hypertension is among the major public health problem rising across the globe. Inhibition of
angiotensin converting enzyme (ACE) is considered a main therapeutic target in controlling
high blood pressure. In this regard, Balasuriya and Rupasinghe (2012) investigated the ACE
inhibitory property of flavonoid-rich apple peel extract (FAE) and quercetin metabolites
46
using human umbilical vein endothelial cell (HUVEC) through biochemical assay of
inhibition of ACE. It has been recorded that FAE and tested flavonoids significantly inhibited
ACE.
Quercetin is a flavonoid that reduces Cu+2 induced oxidation of low density lipoprotein
(LDL). In this context, Janisch et al. (2004) determined the effect of quercetin conjugates
like isorhamnetin-3-glucuronide, quercetin-3-sulfate and quercetin-3-glucuronide to inhibit
Cu+2 induced oxidation of LDL. It has been noticed that lower level of quercetin-3-
glucuronide (<2 microM) increases LDL oxidation however, equivalent concentration
of isorhamnetin-3-glucuronide and quercetin-3-sulfate do not affect the phenomenon.
Similarly, Yamamoto et al. (1999) investigated quercetin and its metabolites capacity to
reduce copper induced lipid oxidation using rat experimental modeling. It has been observed
that cholesterol accumulation rate is lower in solution containing 0.4 mg/mL of quercetin
than that of control. Moreover, mixture containing antioxidants resulted lower
hydroperoxides concentrations after 2 hr of oxidation. Conclusively, quercetin and its
metabolites are effective against copper induced oxidation of low density lipoprotein.
The consumption of functional meat enriched with Ω-3 fatty acids and rosemary extract
improves oxidative and inflammatory status of people at risk of cardiovascular diseases. In
this reference, Bermejo et al. (2014) carried out study in which functional and conventional
meats are provided to 43 volunteers. It has been recorded that functional meat significantly
decreases PAI-1, fibrinogen and 8-iso-PGF2α after 12 weeks of consumption while FRAP is
significantly increased. Additionally, control meat significantly increases PAI-1 and reduces
FRAP. No significant differences are reported in anthropometric variables between tested
groups. In the nut shell, inclusion of functional meat in diet might be a healthy lifestyle
option.
The regular consumption of restructured meat products with or without added walnuts are
considered functional foods for subjects at risk for CVD. Accordingly, Olmedilla-Alonso et
al. (2013) conducted a study to assess the potential effect associated with consumption of
walnut enriched restructured meat products. The dietary intervention includes consumption
of meat products with or without walnuts five times/week for five weeks. The results
delineated that consumption of meat without walnuts decreases total cholesterol 6.8 mg/dL.
47
Compared to baseline, meat products with walnuts decreases total cholesterol 10.7 mg/dL,
LDL cholesterol 17.6 mg/dL and increases α-tocopherol level 8.9 mg/dL. Conclusively,
restructured meat product with added walnuts are considered functional food and its
consumption provokes reduction in total cholesterol 4.5% with respect to baseline and 3%
compared to restructured meat without walnuts.
The intake of phytosterol enriched ground beef lowers plasma total cholesterol (TC) and low
density lipoprotein (LDL) cholesterol in humans. In this reference, Matvienko et al. (2002)
conducted study to determine the effect of phytosterol supplemented ground beef on TC and
LDL cholesterol using hypercholesterolemic individuals. Purposely, triple-blind study
involving 34 male with elevated plasma TC (5.85 +/- 0.70 mmol/L), LDL cholesterol (4.02
+/- 0.60 mmol/L) and TC: HDL cholesterol ratio (5.5 +/- 1.2) are provided beef with or
without 2.7 g of phytosterols. The phytosterol mixture consists of β-sitosterol (48%),
campesterol (27%) and stigmasterol (23%). The results depicted that consumption of
phytosterol supplemented beef lowers TC, LDL-cholesterol and TC: HDL cholesterol ratio
by 9.3, 14.6 and 9.1%, respectively (p < 0.001). They further elaborated that LDL particle
size does not change indicating that diminish is primarily related to reduction of LDL
cholesterol particles.
2.7. Storage stability of broiler meat products
In processed meat products, fruit extracts and antioxidants are added to reduce oxidation that
also improves color & textural properties. In this regard, Ganhao, et al. (2010) determine the
influence of extracts like arbutus berry, rose, hawthorns as antioxidant on protein oxidation
of patties using dinitrophenylhydrazine (DNPH) assay. It is obvious from the findings that
patties of control meat resulted intense protein degradation due to oxidation that decreases
color of the product. Alongside, patties containing fruit extracts & antioxidants significantly
decreases the rate of carbonyls production which improve color and textural properties of
resultant product.
Nowadays, meat industries are striving for natural antioxidants to enhance the shelf stability
of meat based products against harmful effects of synthetic counterpart. In this reference, one
of researchers groups Kim et al. (2011) used hot water extracts of spices to investigate their
antioxidant potential. The results showed that oregano gave maximum extraction yield
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(41.33%) while mace (7.64%) the lowest. Moreover, DPPH free radical scavenging activity
of extracts was in following order; ascorbic acid > clove > thyme > rosemary > savory >
oregano. Furthermore, superoxide radical scavenging potential was as marjoram > rosemary
> oregano > cumin > savory > basil > thyme > fennel > coriander >ascorbic acid. The
flavonoid content of spices varied from 324.08 μg quercetin equivalent (QE)/g for thyme to
3.38 μg QE/g for coriander. They further elucidated that hot water extracts possess higher
antioxidant capacity due to the presence of higher flavonoids level.
The use of antioxidants with or without salt reduce lipid oxidation in chicken nuggets. In this
context, Sullivan et al. (2004) designed a study in which nuggets were prepared from
chicken meat fed on diet supplemented with α-tocopherol acetate. Additionally, rosemary,
sage and tea catechins @0.10% were added to control and vitamin E enriched groups. The
nuggets were tested for oxidative (TBARS) stability, color and cooking loss. The results
showed synergism between vitamin E and added antioxidants that substantially decreased
TBARS value of product. Moreover, antioxidants addition also improve color and cooking
loss. In conclusion, use of antioxidants retarded rate of lipid oxidation in nuggets with or
without salt. Similarly, Yesilbag et al. (2011) indicated that pH of broiler meat increases by
the addition of α-tocopherol @50 mg/kg feed than that of group rely on rosemary extract.
The results stated that addition of rosemary and α-tocopherol reduce the pH thereby delay
microbial degradation.
In another study Suresh et al. (2011) stated that addition of antioxidants in broiler meat
patties decreases the oxidation rate responsible for deterioration of product. The result
showed that lipid peroxidation of patties as measured by TBARS was considerably decreases
(p<0.05) during storage. According to Sanders et al., (1997) adding up of vitamin E in cattle
feed minimizes surface discoloration & lipid oxidation of meat thus improves overall
acceptability.
Later, Capitani et al. (2012) showed that malondialdehyde production in sausages reduces by
quercetin and rutin @0.05 g/100g meat without affecting sensorial attributes. Purposely,
sodium erythorbate (0.05 g/100 g) was replaced with mixture containing caffeic acid &
carnosic acid (47& 53%) and quercetin & rutin (67 & 33%). It has been reported that
phenolic mixture decreases oxidative stability of sausages during during 45 days of storage.
49
The addition of antioxidants increases storage stability of meat products. In this reference, a
study was tailored in which pomegranate rind powder @2.5 & 5% was used as natural
antioxidant in chicken meat balls. The result exhibited lower cooking loss, pH fall and
TBARS, however, higher value for emulsion stability was recorded. Moreover, cooking loss,
pH, TBARS and free fatty acid increased (p<0.05) with progression of storage whereas
emulsion stability decreased significantly (p<0.05). The microbial analysis depicted that
pomegranate rind powder extract @5% in meat balls significantly (p<0.05) reduced total
plate count. Likewise, organoleptic outcomes reported higher sensory score for antioxidants
enriched meat balls than that of control (Chandralekha et al., 2012).
The addition of antioxidants performs better in cooked meat products than fresh. In this
regard, Fasseas et al. (2007) conducted a study to investigate the antioxidant potential of
meat enriched with sage and oregano essential oil using 1,1-diphenylpicrylhydrazyl (DPPH)
and TBARS assay. The meat was ground and subjected to antioxidants followed by storage
at 4ºC. Afterwards, samples were cooked (85ºC for 30 min) and stored to carry out analysis
at 1st, 4th, 8th and 12th day. The results showed lower TBARS for antioxidants treated groups
however, antioxidant activity significantly decreased during cooking and storage.
Antioxidants are effective to control cholesterol oxidation products (COP) in meat based
products generated through high temperature processing or prolonged storage. Accordingly,
one of the scientists groups Wong et al. (2013) investigated the potential of antioxidants to
inhibit the formation of 7α-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol
in beef patties. The inhibitory activity measured at 0.4 mmol concentration showed that l-
ascorbic acid, retinoic acid, and α-tocopherol are effective to reduce the production of 7-
ketocholesteroland (50%), 7-α-hydroxycholesterol (20%) and 7-β-hydroxycholesterol (30%),
respectively.
Similarly, Mariutti et al., (2011) reported that incorporation of sage @0.1 g/100 g chicken
meat in the presence of pro-oxidants like salt, thermal treatment and frozen storage protect
against lipid and cholesterol oxidation. It has been recorded that unsaturated fatty acids are
not affected by the addition of sage however, volatile compound like hexanal and pentanal
were significantly reduced by addition of sage. The highest 7-ketocholesterol is found in raw
meat on 1st day while 7-α and β-hydroxycholesterol are reported in samples after 30 days of
50
storage. Alongside, cooking and storage significantly increased cholesterol oxides and
decreased α & γ-tocopherol. It is deduced that addition of sage is effective to retard lipid and
cholesterol oxidation that minimizes pro-oxidant activity of salt, heat processing and storage.
The addition of oleoresin alone or in combination with α-tocopherol reduces lipid oxidation,
maintains color stability and decreases volatiles generation in irradiated raw and cooked pork
loins under storage. The rosemary and α-tocopherol @0.05 and 0.02% were added
respectively. The results showed that antioxidant reduced the TBARS and volatile aldehydes
in samples however, this combination do not affect sulfur volatiles responsible for off odor in
meat products (Nam et al 2007).
The supplementation of broiler feed with α-tocopherol (200 mg/kg feed) in combination with
α-lipoic acid improves antioxidant potential, oxidative stability and quality of broiler breast
patties. The added antioxidants enhanced antioxidant potential as indicated by lower TBARS.
Nonetheless, sensory attributes are improved by the addition of antioxidants (Sohaib et al.,
2014).
51
Chapter 3 MATERIALS AND METHODS
The current study was conducted at the National Institute of Food Science and Technology
(NIFSAT) and Nutrition Research Center, University of Agriculture, Faisalabad, Pakistan.
Besides, the in vitro study of antioxidants supplementation in broiler meat was performed at
the Department of Animal Science, Iowa State University of Science and Technology, USA.
In present research, the influence of dietary quercetin and alpha tocopherol via feed
supplementation in broiler was explored to enhance the functional value of broiler meat and
meat products. Materials and protocols followed are described herein.
3.1. Procurement of raw materials
All reagents and chemicals required for the instant exploration were purchased from Sigma
Aldrich (Tokyo, Japan) and Merck (Merck KGaA, Darmstadt, Germany). The quercetin was
acquired from Shaanxi Jintai Biological Engineering Co. Ltd. China. Alongside, 300 one day
old broiler chicks (50±5 g body weight) were procured from Jadeed Chicks Pvt. Ltd.
Faisalabad, Pakistan. The study was conducted in duplicate to enhance the authenticity of
results.
3.2. Experimental plan
In the current study, 300 one day old broiler chicks were used as experimental animals for
the production of functional broiler meat. For the intention, they were weighed individually
and divided randomly into 10 groups each consisting of 30 birds reared for a period of six
weeks. The detail of the experimental feed given to the birds is mentioned in Table 1. The
composition of control feed provided to broiler is shown in Appendix 1.
3.3. Experimental site and bird’s management
Prior to research, the research area and all pens were thoroughly cleaned. For disinfection
purpose, bromosept and formalin aqueous solution with 1:12 ratio was used in the
experimental premises. Likewise, 2-3 inch thick layer of saw dust was spread in each pen as
a litter to keep the bed dry and soft. Moreover, all drinkers and feeders were thoroughly
washed and disinfected during the course of research trial. All the pens were tagged with
respective treatments and replication numbers. The temperature of the experimental room
was maintained at 95±2 °F during the first week of trial followed by a decrease of 5 °F till
52
reached to 75±2 °F. The light and proper ventilation were also maintained in the
experimental room. The experimental birds were provided free access to feed and fresh
water.
Table 1. Treatment plan of research
Treatments Description
T0 Control diet
T1 100 mg quercetin+ 150 mg α-tocopherol/kg feed
T2 100 mg quercetin+ 225 mg α-tocopherol/kg feed
T3 100 mg quercetin+ 300 mg α-tocopherol/kg feed
T4 200 mg quercetin+ 150 mg α-tocopherol/kg feed
T5 200 mg quercetin+ 225 mg α-tocopherol/kg feed
T6 200 mg quercetin+ 300 mg α-tocopherol/kg feed
T7 300 mg quercetin+ 150 mg α-tocopherol/kg feed
T8 300 mg quercetin+ 225 mg α-tocopherol/kg feed
T9 300 mg quercetin+ 300 mg α-tocopherol/kg feed
Dietary quercetin and α-tocopherol was added in feed by mixing with Palmolein oil @4%
3.3.1. Chicks vaccination
The glucose solution (50 g/5 L) was given to the chicks after 2 hr of distribution in pens for
waste removal. On 2nd day, cotrim-50 solution (1 g/5 L of water) was administrated to chicks
as an antibacterial agent. The chicks were vaccinated for new castle disease (N.D) and
infectious bronchitis (I.B) at 3rd and 4th day for the prevention from respective diseases.
Afterwards, on 10th and 18th day, birds were vaccinated against gamboro disease by
respective vaccine. Lastly, on 24th day, birds were vaccinated against new castle disease with
Lasuta vaccine (Tsiouris et al., 2014).
3.3.2. Duration of trial
The experimental birds were reared up to six weeks. For acclimatization, the chicks were fed
on control diet during the first two weeks of study. Afterwards, they were fed on diet
supplemented with quercetin and α-tocopherol as per treatment plan.
53
3.3.3. Growth parameters
The birds were weighed on weekly basis to calculate the weight gain. A measured quantity of
feed was provided to chicks during each week. At the termination of research trial, feed
conversion ratio of broilers (FCR) was calculated by dividing the feed consumed by weight
gained in the respective week using the following expression;
FCR = Feed consumed by the bird in one week/ Weight gain by the bird in respective week
3.3.4. Slaughtering of the experimental birds
At termination of growth period, the broiler birds were slaughtered by adopting Halal Islamic
Ethical Guidelines. Before slaughtering, blood samples were collected from the jugular vein
in the heparinzed blood tubes and stored. After slaughtering, breast and leg muscles of
broilers were separated, deboned, wrapped with aluminum foil and packed in polythene zip
lock bags followed by storage at -18 °C for further analysis.
3.3.5. Homogenization and sample preparation
For sample preparation, 5 g meat sample was taken in 50 mL capped polypropylene tube and
homogenized by using phosphate buffer and glycerol (20%) at pH 7.4 through homogenizer.
The tubes were placed in ice cold water to prevent the oxidation of muscle samples.
Afterwards, filtration of samples was carried out to remove connective tissues.
3.4. Analysis of meat samples
3.4.1. Antioxidant potential of functional broiler meat
Antioxidant potential of leg and breast broilers meat samples was estimated by using
respective analytical methods;
3.4.1.1. Total phenolic contents (TPC)
The total phenolic contents in leg and breast broiler meat samples were determined by
adopting the procedure as described by Senevirathne et al. (2006). The homogenized meat
sample (100 μL) was mixed with 500 μL (95% ethanol), distilled water (2.5 mL) and 250 μL
of 50% Folin-Ciocalteu reagent. After 5 min, 250 μL of 5% Na2CO3 was added to the
resultant mixture, vortex and placed in the dark room for 1 hr. Afterwards, absorbance of
54
samples was recorded at 725 nm through UV/Visible Spectrophotometer (CECIL-CE7200)
against control. The total phenolic contents of meat samples were estimated as gallic acid
equivalent (mg gallic acid/g).
3.4.1.2. Free radical scavenging activity (DPPH assay)
The free radical scavenging activity i.e. DPPH (1,1-diphenyl-2-picrylhydrazyl) of leg and
breast meat samples was measured using the protocol of Brand-Williams et al. (1995).
Purposely, 1 mL of DPPH solution was added to 4 mL of sample followed by incubation for
30 min at room temperature. The absorbance was measured at 520 nm using UV/Visible
Spectrophotometer. The DPPH free radical scavenging activity was calculated by the below
mentioned equation;
Inhibition (%) = 100 × (Ablank- Asample/ Ablank)
Ablank = absorbance of blank sample (t = 0 min)
Asample = absorbance of tested solution (t = 15 min)
3.4.1.3. Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant power of leg and breast broiler meat samples was estimated
by following the procedure of Rupasinghe et al. (2010). The homogenized sample (200 μL)
was mixed with 500 μL sodium phosphate buffer (0.2 M, pH 6.6) and 500 μL potassium
ferric cyanide (1%) followed by incubation at 50 °C in a water bath for 20 min. After
cooling, sample was mixed with 2.5 mL (10% TCA), dist illed water (1.25 mL) and 0.25
mL (0.1% ferric chloride) for 10 min. The absorbance was measured at 700 nm. During
the analysis, an increase in the absorbance of the reaction mixture indicated the higher
reducing power of the samples.
3.4.2. TBARS assay
The oxidative stability of broiler meat samples was measured by using thiobarbituric acid
reactive substances (TBARS) according to the guidelines of Liu et al. (2010). In this context,
5 g of ground broiler meat samples were weighed in a 50 mL test tube and homogenized with
50 μL of butylated hydroxytoluene (7.2%) and 15 mL of deionized distilled water using a
homogenizer for 15 sec. One mL of meat homogenate was transferred to a disposable test
tube (13×100 mm) and 2 mL of TBA/trichloroacetic acid (TCA; 15 mM TBA/15% TCA)
55
solution was added. The mixture was vortex and incubated in a boiling water bath for 15 min
to develop color. Afterwards, samples were cooled in ice water for 10 min, vortex again and
centrifuged for 15 min at 2,000×g at 4 °C. The absorbance of the resulting supernatant
solution was determined at 531 nm against a blank containing 1 mL of deionized distilled
water and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as
milligrams of malondialdehyde (MDA) per kilogram of meat. The amounts of TBARS were
calculated by using formula as described below
n-mole of malondialdehydes = (Sample absorbance – Blank)× Total sample volume
0.000156×1000
3.5. Quantification of quercetin and alpha tocopherol
3.5.1. Quercetin content
The quercetin content of broiler leg and breast meat sample was estimated through HPLC by
following the protocol of Yuangang et al. (2006). The preparatory steps are as follows;
3.5.1.1. Sample preparation
Purposely, 2 g meat was mixed with 50 mL of 70% v/v methanol/water. The mixture was
homogenized for 5 min with a blender. The solution was filtered through a Whatman No. 4
filter paper under reduced pressure. The residue was extracted again with 50 mL of 70% v/v
methanol/water for 5 min followed by filtration through a Whatman No. 4 filter paper and in
the resulting filtrate, methanol was added to make a volume of 100 mL. For HPLC analysis,
the solution was filtered through a 0.45 mL of nylon filter disc prior to analysis. Afterwards,
1 mL of 1000 mg/mL sorbic acid solution (internal standard) was added and the total volume
(25 mL) was made with methanol. The solution was filtered through a 0.45 mm nylon filter
before analysis
3.5.1.2. Mobile phase
The mobile phase comprised of 0.1% formic acid in water and methanol with gradient of
20:80%.
3.5.1.3. Standard preparation
The stock and working standards of quercetin were prepared in methanol.
56
3.5.1.4. HPLC quantification
The analyses were performed with an Agilent series 1100 quaternary solvent delivery system
with cooled autosampler (4 °C) and photodiode array detector (Agilent, Waldron Germany)
connected to a thermo electron ion trap mass spectrometer operating in negative ion
electrospray mode (Thermo Electron, San Jose, USA). The column was maintained at 30 °C
and mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in
methanol with the following gradient; 20:80% (0–10 min), 80% B (10–20 min), 80:20% (20–
20.5 min), 20% B (20.5–25 min) and flow rate of 0.2 mL/min. The HPLC column was fitted
with a C18 column maintained at 30 °C and a mobile phase of (A) 0.1% formic acid in water
and (B) 0.1% formic acid in acetonitrile with the following gradient: 5% B (0–5 min), 5:80%
(5–25 min), 80% B(25–30 min), 80–5% (30–31 min), 5% B(31–35 min) with a flow rate of
0.2 mL/min. All chromatograms were monitored at a wavelength of 280, 346, 364 and 370
nm.
3.5.2. Alpha tocopherol content
The alpha tocopherol content of meat samples was measured by the protocol of Asghar et al.
(1990).
3.5.2.1. Sample preparation
The homogenized meat sample (500 μL) was taken in a test tube followed by the addition of
1.5 mL of urea (6 M) to dissolve the meat tissue. Later, 0.5 mL of ascorbic acid (5%) was
added in the reaction mixture to prevent the oxidation of α-tocopherol in the meat samples
along with 1 mL of 6 M urea. The tubes were flushed with N2 and resultant mixture was
vortex for 12 min to extract the tocopherol components of samples. Next, 1 mL of 0.1 M
sodium dodecyl sulfate (SDS) solution was added and vortex for 1 min to disintegrate the
meat tissue. For deproteination and release of α-tocopherol, 4 mL of ethyl alcohol containing
1% pyrogallol was added in the resulting mixture. Thereafter, petroleum ether (10 mL) was
added and the resultant mixture was centrifuged at 5000×g for 5 min to facilitate the
separation of phases. The solvent layer containing α-tocopherol was separated in the vial and
the pooled solvent was evaporated under nitrogen. Alpha tocopherol content was dissolved in
the mobile phase (100% methanol) and then filtered through 0.45 μm microfilter, centrifuged
at 5000×g for 5 min to collect the filtrate and stored for HPLC analysis.
57
3.5.2.2. Mobile phase
The mobile phase comprised of methyl alcohol (HPLC grade); 100% methanol was prepared
by filtering through typhlon filter assembly and then adjusted according to requirements of
HPLC.
3.5.2.3. Standard preparation
The standard of α-tocopherol was prepared by using Sigma Aldrich packed standard 1
mg/mL of α-tocopherol as stock solution from which further dilutions (10, 20, 50 and 100
μg/mL of solutions) were prepared.
3.5.2.4. HPLC condition
The α-tocopherol was extracted and quantified by using HPLC (PerkinElmer, Series 200,
USA) chromatographic system at 290 nm with UV-Visible detector. The HPLC
chromatograms were obtained through C18 column (250mm×4.6mm, 5.0µm), system
controller SCL-10 A, water pump (LC-10 AT) and flow controller valve (FCV-10 AL) with a
mobile phase of 100% methanol at a flow rate of 1 mL/min.
3.6. Fatty acids profile
The fatty acid composition of functional broiler breast and leg meat samples was estimated in
the Department of Animal Science, Iowa State University, USA adopting the protocol of
Wang et al. (2000). Accordingly, 2 g meat samples were weighed into a test tube with 20 mL
of Folch solution (10 volumes, chloroform: methanol = 2:1, wt/vol) and homogenized using a
polytron for 10 sec. Moreover, 24 µL of butylated hydroxyanisole (BHA, 10% dissolved in
98% ethanol) was added to each sample prior to homogenization. The homogenate was
filtered through whatman no.1 filter paper into a 100 mL graduated cylinder and ¼ volume
(on the basis of Folch solution volume) of 0.88% NaCl solution was added. Afterwards, the
cylinder was capped with a glass stopper and the filtrate was mixed well. The cylinder was
washed twice with 10 mL of Folch solution (3:47:48/CHCl3:CH3OH:H2O) and the contents
were stored up to 6 hr until aqueous and organic layers were clearly separated. After
separation, upper layer containing methanol was siphoned and 0.5 mL of lower layer
(chloroform layer) was moved to a glass scintillation vial and dried at 70 °C under nitrogen
for 2-3 min. Moreover, 1 mL of BF3 in methanol was added as methylating agent to cut ester
58
bond to form fatty acids methyl esters and then heated for 50 min followed by cooling at
room temperature. Later, 3 mL of hexane and 5 mL of distilled water were added, mixed
thoroughly and left overnight for phase separation. The top (hexane) layer, containing
methylated fatty acids was used for gas chromatographic analysis. The fatty acid
compositional profiling was performed by using Gas Chromatograph (HP 6890) equipped
with an auto sampler and flame ionization detector. A capillary column (HP-5; 0.25 mm i.d.,
30 m, 0.25-µm film thickness) was used to inject samples (1 µL) into the capillary column.
The oven temperature conditions (180 °C for 2.5 min, increased to 230 °C at 2.5 C/min, then
held at 230 °C for 7.5 min) were maintained. The temperatures of the inlet and detector were
fixed at 280 °C. The helium was used as a carrier gas and a constant column flow of 1.1
mL/min was used. The flame ionization detector air, hydrogen (H2) and helium flows were
350, 35, and 43 mL/min, respectively. The identification of fatty acids was accomplished by
comparing mass spectral of fatty acids against their standards. The results of the fatty acid
were reported as percentage composition of total lipids and peak area was used to calculate
fatty acid composition of samples.
3.7. Antioxidant enzymes assay
The superoxide dismutase (SOD) activity was determined by measuring the ability of
enzyme to inhibit cytochrome ‘c’ oxidation (Sun et al., 1988). Whereas, the catalase (CAT)
activity of broiler blood serum was assessed by observing decomposition of hydrogen
peroxide (Block et al., 1980). The glutathione reductase (GRs) activity was examined by the
oxidation of NADPH (Paglia and Valentine, 1967).
3.8. Serum biochemical analysis
The blood samples were subjected to following analysis:
3.8.1. Total cholesterol
The total cholesterol concentration was estimated by liquid cholesterol method as described
by Desai et al. (2006). The distilled water, standard solution, or plasma samples (20 μL of
each) were taken into labeled test tubes. The absorbance of samples and standard against
blank was recorded at 505 nm.
59
3.8.2. High density lipoprotein (HDL)
The high density lipoprotein (HDL) was estimated by cholesterol precipitant method as
elaborated by Alshatwi et al. (2010). The blood serum sample (200 µL) was added to 500 µL
of diluted reagent followed by incubation for 10 min at a room temperature. The absorbance
of the samples and blank were recorded at a wavelength of 505.
3.8.3. Low density lipoprotein (LDL)
The low density lipoproteins (LDL) in sera samples were recorded by following the
guidelines of Kim et al. (2011).
3.8.4. Triglycerides
The triglycerides concentration was determined by the protocol of Anoni et al. (1982). The
standard and sample (10 µL each) were taken into test tubes along with one blank followed
by incubation at 37 °C for 5 min. The absorbance was recorded at a respective wavelength.
3.8.5. Total protein
The serum total protein concentration of broilers was measured by their respective kits
obtained from Sigma-Aldrich Chemicals Co. (Bradford, 1976).
3.9. Product development
The antioxidants enriched broiler meat was used for the preparation of nuggets by following
the protocol of Perlo et al. (2006). The control and antioxidant enriched functional broiler
meat was minced followed by blending of all ingredients to get a uniform mix. Next, the
mixture was extended in a thin layer and shaped (10±1 g/piece). The nuggets were placed in
flour and bread crumbs separately and then fried to attain golden brown nuggets. Afterwards,
they were packed in oxygen permeable bags. The nuggets were stored for 60 days at -18 °C
and subjected to quality assays like color, pH, texture and TBARS at storage intervals of 0,
15, 30, 45 and 60 days of storage. The recipe used for the preparation of nuggets is described
in Table 2.
3.10. Nuggets analysis
The analysis of nuggets was carried out at respective intervals according to their standard
methods.
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3.10.1. Color
The color of nuggets was measured by using hand held tristimulus colorimeter (Color Test
Meter II) by following the protocol of El-Gasim and Wesali (2000). The calibration of the
colorimeter was carried out by using standards (54 CTn for dark and 151 CTn for light). The
color values were determined by placing nuggets in petriplate under the photocell.
Table 2. Recipe of the nuggets
Sr.No. Ingredients Quantity
1 Chicken boneless 500 g
2 Oil As required for frying
3 Onion 100 g
4 Plain flour 120g
5 Bread crumbs 70g
6 Salt 15g
3.10.2. pH
The pH of nuggets was recorded through pH meter as described by Sallama et al. (2004).
3.10.3. Texture
The textural characteristics of antioxidant enriched nuggets were recorded using texture
analyzer (Mod. TA-XT2, Stable Microsystems, surrey, UK) following the procedure of
Carlos et al. (2009). The compression test was performed to check the texture of the nuggets
at regular storage intervals.
3.10.4. TBARS assay
The oxidative stability of broiler meat nuggets was measured through thiobarbituric acid
reactive substances (TBARS) assay by following the protocol of Liu et al. (2010) as
mentioned in the previous section.
3.11. Sensory evaluation of nuggets
The sensory evaluation of nuggets was carried out by a trained taste panel, employing 9-point
hedonic scale (9 = like extremely; 1 = dislike extremely) at different storage intervals by
following the guidelines of Meilgaard et al. (2007) as given in Appendix-II. Accordingly,
61
sensory response for various quality traits of nuggets like appearance, flavor, taste and
overall acceptability were recorded. All the evaluations were conducted by the panelists in
separate booths under clear white fluorescent light in the Sensory Evaluation Laboratory of
NIFSAT, University of Agriculture, Faisalabad. During evaluation process, they were
provided unsalted crackers and mineral water to neutralize and rinse their taste receptors for
rational assessment. The panelists were requested to rate the product quality by scoring for
the selected parameters.
3.12. Selection of best treatment
Considering the results of antioxidant potential, physico-chemical assay, serum biomarkers
and sensory attributes, four best treatments were selected along with control for efficacy
study.
3.12.1. Bioevaluation of functional broiler meat
Efficacy trial was conducted to evaluate the therapeutic potential of antioxidant enriched
functional meat against various metabolic disorder with special reference to hyperlipidemia.
The eligibility in the study required willingness and ability to adhere with the research
protocols. For the concern, 25 volunteers ranging between 20-40 years with no sign of
coronary complications were congregated and provided consent performa for participation in
the program. The participants were asked to complete a questionnaire about their dietary
intake, physical activity level, stress management and lifestyle oriented disorders that might
cause hindrance towards accurate physical and biochemical assessment. Before initiation,
detailed discussion was carried out with the subjects regarding the fate of project and their
role & importance of the study. The information, education and communication material
(IEC) about the functional broiler meat was also provided to the subjects. The volunteers
were divided randomly into five groups, each comprised of five participants. The first group
(G1) was provided control broiler meat whilst the remaining groups were given antioxidant
enriched functional meat. On protein requirement basis, the volunteers were provided baked
meat @ 130 g/volunteer/daily. The peoples were advised to avoid consumption of any other
meat and allied products during the course of study. The treatment plan followed for the
efficacy study is mentioned in Table 3.
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3.12.2. Provision and distribution of the functional meat nuggets
The selected treatments of the nuggets were prepared at the National Institute of Food
Science and Technology, University of Agriculture, Faisalabad. Five nuggets were packed in
oxygen impermeable polypropylene pack and 7 such packs (one week dose) were further
packed in specified color and printed box containing all the information about the research
project, functional meat importance, nutritional facts, date of manufacturing and best before
etc. Blue color was assigned to control group, whereas yellow for G2, red for G3, black for G4
and green for G5 group during the entire study. The nuggets were supplied to the volunteers
on weekly basis and advised to keep them in frozen. A diet Performa was handed over to the
each participant at every visit to record daily diet intake. This practice was started at 1st week
till the accomplishment of the study.
Table 3. Bioevaluation study plan
Groups Description
G0 Broiler meat
G1 Diet 1
G2 Diet 2
G3 Diet 3
G4 Diet 4
Diet 1 (Meat containing 300 mg quercetin + 300 mg α-tocopherol)
Diet 2 (Meat containing 300 mg quercetin + 225 mg α-tocopherol)
Diet 3 ((Meat containing 200 mg quercetin + 300 mg α-tocopherol)
Diet 4 (Meat containing 300 mg quercetin + 150 mg α-tocopherol)
3.12.3. Blood Sampling and Determinations
At the termination of bioevaluation study, blood samples were collected and subjected to
biochemical analysis. The total cholesterol concentration was estimated by following the
guidelines of Desai et al. (2006). The high density lipoprotein (HDL) was determined
through cholesterol precipitant method as elaborated by Alshatwi et al. (2010). The low
density lipoprotein (LDL) of sera was analyzed following protocol of Kim et al. (2011).
Likewise, triglycerides level was recorded through protocol of Anoni et al. (1982) and
protein concentration was determined through respective kits procured from Sigma-Aldrich
Chemicals Co. (Bradford, 1976).
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3.13. In vitro antioxidants supplementation in meat
The in vitro antioxidants supplementation study of broiler meat was performed in the
Department of Animal Science, Iowa State University, USA. For the intention, boiler meat
was purchased from a local store (Sams club, Ames IA, USA). The meat was ground twice
through 10 mm and 3 mm grinder plate (Kitchen Aid, Inc., St. Joseph, MI, USA) before the
application of antioxidants. The treatment plan for in vitro research study is shown as under;
Table 4. In vitro study plan
Treatments Description
T0 Control
T1 25 mg quercetin + 100 mg α-tocopherol /kg of meat
T2 25 mg quercetin + 200 mg α-tocopherol /kg of meat
T3 50 mg quercetin + 100 mg α-tocopherol /kg of meat
T4 50 mg quercetin + 200 mg α-tocopherol /kg of meat
T5 100 mg quercetin + 100 mg α-tocopherol /kg of meat
T6 100 mg quercetin + 200 mg α-tocopherol /kg of meat
The quercetin dihydrate was purchased from Alfa Aesar (Johnson Matthey Company,
Massachusetts, USA) and dissolved in alkaline water for complete solublization. The alpha
tocopherol was prepared by dissolving in corn oil before the initiation of experiment. The
aforementioned treatments were incorporated to the ground meat followed by mixing for 2
min using bowl mixer (Model KSM 90; Kitchen Aid, Inc., St. Joseph, MI, USA). Afterwards,
patties (100±3 g) were prepared, vacuum packaged in oxygen impermeable bags (O2
permeability, 9.3 mL O2/ m2/ 24 h at 0 oC, Koch, Kansas City, MO, USA) and cooked at 95
oC water bath (Isotemp, Fisher Scientific Inc., Pittsburgh, PA, USA) until the internal
temperature reached to 75 oC. The cooked patties were cooled and repacked in new oxygen
permeable bags (polyethylene, 4×6.2 mil, Association Bags Co., Milwaukee, WI, USA) and
stored at 4 oC. The analysis of patties were conducted at 1st, 3rd and 7th day of storage.
3.13.1 TBARS assay of patties
The lipid stability of patties containing quercetin and α-tocopherol was measured through
thiobarbituric acid reactive substances (TBARS) assay as mentioned in the previous section.
64
3.13.2. Color
The color of meat patties (surface color) containing quercetin and α-tocopherol was
measured using Hunter-Lab Mini Scan XE colorimeter (Hunter Laboratory Inc., Reston, VA)
with D65 illuminant and 10° standard observer. The instrument was calibrated against black
and white references before use for measuring L* (lightness), a* (redness), and b*
(yellowness).
3.13.3. Volatile compounds
The volatile compounds of cooked meat patties were measured through Solatek 72
Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann, Cincinnati,
OH, USA) connected to GC/MS (Model 6890/5973; Hewlett-Packard Co., Wilmington, DE,
USA) according to the method of Ahn et al. (2001). Purposely, 2 g meat sample was placed
in 40 mL sample vial, flushed with helium gas (40 psi) for 3 sec and capped airtight with a
Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA). The
samples of different treatment were randomly organized on the refrigerated (4 oC) holding
tray to minimize the variation of the oxidative changes in samples. The meat samples were
purged with helium (40 mL/min) for 14 min at 20 oC. The volatile compounds were trapped
using Tenax/charcoal/silica column (Tekmar-Dohrmann) and desorbed for 2 min at 225 °C,
maintained in a cryofocusing module (-70 oC) and then thermally desorbed into a capillary
column for 2 min at 225 oC. The HP-624 column (7.5 m, 0.25 mm i.d., 1.4 m), HP-1
column (52.5 m, 0.25 mm i.d., 0.25 m) and HP-Wax column (7.5 m, 0.250 mm i.d., 0.25
m) were connected through zero dead volume column connectors (J &W Scientific,
Folsom, CA, USA). Initially, the oven temperature was 25 oC for 5 min and increased to 85
oC @ 40 oC per min leading to 165 oC @ 20 oC per min and finally 230 oC @ 5 oC per min.
held at this temperature for 2.5 min. The constant column pressure 22.5 psi was maintained.
The ionization potential of mass spectrometer (MS) was 70 eV with scan range 20.1 to 350
m/z. The identification of volatiles was accomplished using the Wiley Library (Hewlett-
Packard Co.). The area of each peak was integrated using chemstation TM software
(Hewlett-Packard Co.) and peak area (total ion counts x 104) was calculated as an indicator of
volatiles generated from meat samples.
65
3.14. Statistical Analysis
The resultant data were analyzed through completely randomized design (CRD) using
Statistical Package (Statistic 8.1). Moreover, Analysis of variance (ANOVA) was performed
to measure the level of significance (Steel et al. (1997).
66
Chapter 4 RESULTS AND DISCUSSION
Present study was planned to develop functional broiler meat and meat based products by
providing quercetin and α-tocopherol through feed supplementation to broilers for
investigating the functional worth of meat nuggets against lifestyle oriented disorders with
special reference to hyperlipidemia in human subjects. For the intention, 300 broiler birds
were subjected to feed supplemented with quercetin and α-tocopherol. The project was
divided into three phases; firstly, the broiler birds were reared and provided feed containing
antioxidants i.e quercetin and α-tocopherol. Second phase encompassed product development
in which nuggets were prepared by using functional meat and tested for different parameters.
Finally, efficacy trial was conducted using human experimental modeling by providing
control and functional broiler meat to explicate its hypolipidemic potential. Collected data
was subjected to statistical analysis to estimate the level of significance. The debate
regarding examined attributes are herein.
4.1. Growth parameters
4.1.1. Body weight gain
The mean squares (Table 5) revealed that weight gain of broilers varied significantly among
treatments and study weeks, however, non-significant behavior was noticed for experimental
years. The type and level of different antioxidants i.e. quercetin and α-tocopherol imparted
momentous effect on weight gain of broilers. Alongside, interactions of variables also
exhibited significant effect on this trait.
The results (Table 6) depicted that body weight of different broiler groups T0, T1, T2, T3,
T4, T5, T6, T7, T8 and T9 were 133.3±2.33, 133±1.20, 135.6±2.73, 135.3±1.20, 136±2.08,
137±3.51, 137.7±3.18, 135.6±1.45, 134.7±2.33 and 135.6±0.88 g/bird, respectively during 1 st
week that subsequently increased to 1992.7±4.37 g/bird (T0), 2033.3±4.98 g/bird (T1),
2111.7±4.26 g/bird (T2), 2208.3±6 g/bird (T3), 2065±7.55 g/bird (T4), 2265.6±7.75 g/bird
(T5), 2293.3±3.48 g/bird (T6), 2157±6.24 g/bird (T7), 2350±6.93 g/bird (T8) and
2374.6±3.53 g/bird (T9) at the termination of trial.
Means of broiler birds fed on different combinations of α-tocopherol and quercetin
supplemented feed indicated highest weight gain in T9 (2374.6±3.53 & 2388±6.43) followed
67
by T8 and T6 as 2350±6.93 & 2353.3±4.0, 2293.3±3.48 & 2307±4.36 whilst lowest in T0
(1992.7±4.37 & 1999±6.81) g/bird, respectively in the year 2013 and 2014. Similar
increasing trend in weight gain (trial 2) with the passage of time was observed in
experimental groups. The body weight of broilers increased linearly with the progression of
growth period. The results elucidated maximum weight gain in broilers fed on higher level of
dietary quercetin and α-tocopherol.
The instant study is supported by the findings of Chae et al. (2006) and Rebole et al. (2006),
they noticed weight gain in broilers fed on α-tocopherol @200 mg/kg of feed. Likewise,
Malayoğlu et al. (2009) also found similar effect of α-tocopherol on weight gain. One of the
researchers groups, Yasin et al. (2012) indicated that higher concentration of α-tocopherol in
feed improved growth performance by yielding higher weight gain. Earlier, Biswas et al.
(2011) reported that utilization of natural antioxidants is an effective way to increase gain in
weight of broiler birds. One of the peers, Jang et al. (2010) stated that dietary
supplementation of quercetin enhanced the weight gain and growth efficiency in broilers at a
level of 200 ppm/kg via diet. The results of previous study by Lin et al. (1989) stated that
antioxidant supplementation in feed significantly improved survival efficiency and growth
performance of broilers. Recently, Goliomytis et al. (2014) reported that dietary
supplementation of quercetin at a concentration of 0.1 and 1g/kg feed enhanced heart weight
of chickens compared to control (p<0.05) which improved the growth performance of
broilers. Similarly, Jiang et al. (2007) found that inclusion of isoflavone in chicken diet at
concentrations of 10 to 80 mg/kg improved performance by higher weight gain and lower
feed conversion ratio. In present investigation, the treatments containing higher concentration
of α-tocopherol and quercetin performed better compared to rest of the treatments and the
current notions is supported by the aforementioned studies.
4.1.2. Feed intake
Mean squares (Table 5) indicated that feed intake of broiler birds varied non-significantly
among treatments and study years nevertheless, affected significantly due to weeks. The type
and level of different antioxidants i.e. quercetin and α-tocopherol imparted non-momentous
effect. The interactions of variables except week × treatment exhibited non- significant
variations. The results (Tables 7) showed that during first week, feed intake of birds ranged
68
between 157±3.38 to 176±2.33 and 153±11.4 to 165±1.20 g/bird during the year 2013 and
2014, respectively while the values for this trait during last week of growth period were
976±2 to 1035±10.5 and 977±1.53 to 1036±10.4 g/bird for respective years. Nevertheless,
feed intake of birds increased with progression of growth period and means during different
weeks W1, W2, and W3, W4, W5, and W6 were 162±0.90, 378±0.69, 543±0.73, 763±1.22,
932±1.38 and 1006±2.79, correspondingly.
The results of current study are supported by findings of Goliomytis et al. (2014) who
noticed non-significant differences for feed intake of broiler fed on dietary supplementation
of quercetin (p > 0.05). Similar findings were reported by Simitzis et al. (2011), observed
non-momentous effect of hesperidin (3 g/kg of feed) on this trait. Likewise, Peña et al.
(2008) also found non-significant differences for feed intake of broilers when ascorbic acid,
quercetin and rutin were provided to birds raised under heat stress conditions. They further
explained that quercetin and rutin did not affect feed consumption behavior. One of scientists
groups, Rezaeipour et al. (2011) delineated that feeding α-tocopherol enriched feed has non
momentous effect on feed intake of birds. The previous findings of Guo et al. (2001) have
also indicated that supplementation of α-tocopherol did not influence the feed intake of
broilers.
4.1.3. Feed conversion ratio
It is evident from the statistical analysis that feed conversion ratio (FCR) of broilers varied
significantly among treatments, weeks nevertheless, differed non-significantly due to
experimental years (Table 5). Besides, type and level of different antioxidants i.e. quercetin
and α-tocopherol imparted momentous effect. The interactions of variables except treatments
× weeks and treatments × weeks’ × years exhibited significant variations.
The results (Table 8) indicated that FCR in different broiler groups T0, T1, T2, T3, T4, T5,
T6, T7, T8 and T9 was 2.10±0.050, 2.12±0.044, 1.90±0.077, 1.84±0.012 and 1.85±0.02,
1.87±0.013, 1.85±0.079, 1.91±0.082, 1.88±0.068 and 1.88±0.03, respectively at 1st week.
Subsequently, the FCR at the termination of study was 2.18±0.022 (T0), 2.07±0.038 (T1),
1.97±0.026 (T2), 1.79±0.045 (T3), 2.03±0.009 (T4), 1.65±0.032 (T5), 1.67±0.026 (T6),
69
Table 5. Mean squares for weight gain (WG), Feed intake (FI) and feed conversion ratio (FCR) of broilers
SOV df WG FI FCR
Treatments (A) 9 74461.3* 434.9 NS 0.27528**
Years (B) 1 1030.0NS 53.0NS 0.00005 NS
Weeks (C) 5 37145842.2** 6495188.6** 0.61941**
B × C 5 316.4** 66.0 NS 0.00943 NS
A × B 9 260.2** 15.7 NS 0.00296 NS
A × C 45 15172.8** 610.9** 0.08488**
A × B × C 45 186.2** 21.4 NS 0.00929**
Error 236 72.2 54.4 0.00487
Total 359
**=Highly significant
* = Significant
NS=Non-significant
70
Table 6. Body weight gain of broilers
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocpherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Year Weight gain (g/bird/week)
Means W1 W2 W3 W4 W5 W6
T0 2013 133.3±2.33 349.6±6.06 638.3±2.19 1049.4±5.24 1545.6±4.70 1992.7±4.37
952.8±111.04e 2014 133.7±1.76 349.0±4.58 635.4±4.06 1055.3±4.70 1553.0±3.61 1999.0±6.81
T1 2013 133.3±1.20 343.3±5.46 649±3.46 1073.5±1.86 1560.6±2.91 2033.3±4.98
966.6±113.28de 2014 136.7±2.60 344.3±2.85 646.3±5.49 1071.0±7.23d 1569.7±5.61 2039.0±10.10
T2 2013 135.6±2.73 348.3±7.97 656.6±1.76 1106.6±1.45 1607.7±6.39 2111.7±4.26
994.9±117.67d 2014 137.0±1.53 348.0±6.24 656.3±7.84 1108.7±4.67 1609.5±8.76 2112.3±6.69
T3 2013 135.3±1.20 348.2±3.84 685.3±3.18 1121.7±4.98 1645.6±5.30 2208.3±6.01
1026.3±122.99c 2014 135.3±1.76 348.3±3.28 686±5.29 1128.3±4.06 1656.3±7.31 2216.4±7.54
T4 2013 136±2.08 348.6±3.84 643.0±1.53 1095.0±2.65 1575.0±5.57 2065.0±7.55
986.2±115.40h 2014 135.6±2.03 349.0±3.00 647.3±6.96 1182.0±6.43 1584.0±7.57 2074.0±6.24
T5 2013 137.0±3.51 351.3±2.60 709.0±1.15 1142.0±4.73 1662.7±5.93 2265.6±7.75
1044.2±125.47bc 2014 139.0±2.08 348.5±1.45 706.4±3.18 1136.3±7.31 1661.6±6.96 2271.0±3.79
T6 2013 137.7±3.18 347.7±6.33 730.0±2.65 1152.7±5.93 1679.0±5.51 2293.3±3.48
1057.5±127.24b 2014 137.3±2.19 347.6±4.33 724.6±8.11 1154.3±3.53 1679.6±5.55 2307.0±4.36
T7 2013 135.6±1.45 347.6±4.98 669.7±1.76 1102.0±7.00 1628.3±5.04 2157.0±6.24
1007.6±120.18cd 2014 135.0±2.52 347.7±3.48 669.6±6.01 1108.3±5.46 1627.0±6.43 2163.3±4.4
T8 2013 134.7±2.33 349.0±2.65 745.7±2.91 1164.3±8.37 1695.0±4.36 2350.0±6.93
1073.6±129.86ab 2014 135.3±2.91 349.3±1.76 744.3±5.81 1169.0±6.66 1693.4±5.21 2353.3±4.06
T9 2013 135.6±0.88 348.6±4.33 762.0±4.62 1180.0±2.31 1713.3±2.40 2374.6±3.53
1086.5±131.45a 2014 135.3±3.18 349.0±3.79 763.7±6.94 1180.3±4.98 1708.0±5.29 2388.0±6.43
Means 135.7±0.46f 348.2±0.85e 688.4±5.62d 1124.1±5.40c 1632.8±7.0b 2188.7±16.74a
71
Table 7. Feed intake of broiler birds
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Year Feed intake (g/bird/week)
Means W1 W2 W3 W4 W5 W6
T0 2013 161±2.08 380±2.60 538±2.60 756±5.71 950±3.28 976±2.00
628.08±50.17 2014 165±1.20 381±3.76 538±3.18 759±5.70 953±3.71 977±1.53
T1 2013 176±2.33 378±2.89 542±2.73 756±8.95 919±4.67 978±1.20
624.14±49.10 2014 160±2.31 377±3.18 544±3.28 758±7.54 923±4.67 979±2.41
T2 2013 162±4.10 377±2.65 543±2.33 776±9.28 931±3.61 994±4.00
631.14±50.63 2014 163±2.73 373±2.08 545±2.60 778±9.54 934±3.46 998±3.06
T3 2013 158±3.38 380±2.31 538±2.08 762±3.67 933±1.23 1007±6.96
630.28±51.07 2014 157±1.76 380±0.88 538±3.93 764±3.84 936±4.84 1009±4.93
T4 2013 160±2.60 376±3.06 544±2.08 763±4.26 930±3.21 996±3.180
629.14±50.53 2014 164±2.03 372±3.51 545±2.08 767±3.61 934±4.16 999±1.76
T5 2013 162±2.60 378±3.21 542±3.21 763±4.33 933±4.51 996±3.46
630.03±50.57 2014 161±2.33 378±4.16 545±2.03 765±4.33 935±3.28 1001±2.03
T6 2013 161±2.91 376±2.89 542±2.31 763±2.65 939±4.33 1024±14.53
634.61±51.52 2014 163±3.61 377±1.76 546±3.93 766±2.65 940±2.85 1017±6.57
T7 2013 164±6.11 378±1.15 542±3.53 759±2.60 927±2.40 1015±4.67
630.39±51.14 2014 153±11.40 378±4.16 539±4.06 761±2.33 931±3.53 1017±3.79
T8 2013 160±3.46 374±2.03 545±2.08 765±2.85 932±3.21 1035±10.55
636.31±52.03 2014 160±2.96 377±4.73 545±1.86 768±3.00 937±2.45 1036±10.46
T9 2013 162±1.73 383±2.40 554±5.03 757±4.10 914±4.16 1033±4.81
633.67±51.19 2014 162±2.03 379±6.57 543±2.65 760±4.41 920±2.31 1035±3.71
Means 162±0.90f 378±0.69e 543±0.73d 763±1.22c 932±1.38b 1006±2.79a
72
Table 8. Feed conversion ratio of broiler birds
Means sharing similar Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Year Feed conversion ratio
Means W1 W2 W3 W4 W5 W6
T0 2013 2.10±0.05 1.76±0.05 1.86±0.02 1.84±0.02 1.91±0.01 2.18±0.02
1.94±0.03a 2014 2.02±0.04 1.77±0.02 1.87±0.01 1.81±0.03 1.92±0.02 2.19±0.04
T1 2013 2.12±0.04 1.80±0.06 1.77±0.02 1.78±0.04 1.89±0.01 2.07±0.04
1.89±0.03ab 2014 1.89±0.05 1.82±0.03 1.80±0.03 1.78±0.02 1.85±0.05 2.09±0.06
T2 2013 1.90±0.07 1.78±0.07 1.76±0.05 1.7230.01 1.86±0.01 1.97±0.03
1.84±0.02bc 2014 1.917±0.05 1.77±0.06 1.77±0.05 1.72±0.05 1.87±0.04 1.98±0.01
T3 2013 1.84±0.01 1.783±0.05 1.60±0.02 1.75±0.02 1.78±0.03 1.79±0.04
1.77±0.02cd 2014 1.89±0.02 1.787±0.03 1.60±0.04 1.73±0.02 1.77±0.04 1.81±0.05
T4 2013 1.85±0.02 1.77±0.04 1.85±0.03 1.69±0.01 1.94±0.02 2.03±0.01
1.87±0.04b 2014 1.96±0.06 1.74±0.02 1.83±0.06 1.44±0.04 2.33±0.09 2.04±0.03
T5 2013 1.87±0.01 1.77±0.06 1.51±0.01 1.76±0.03 1.79±0.03 1.65±0.03
1.73±0.02d 2014 1.85±0.02 1.80±0.03 1.52±0.01 1.79±0.04 1.78±0.01 1.64±0.02
T6 2013 1.85±0.08 1.79±0.06 1.42±0.02 1.80±0.03 1.78±0.01 1.67±0.02
1.72±0.03d 2014 1.91±0.07 1.79±0.05 1.45±0.02 1.78±0.02 1.78±0.01 1.62±0.01
T7 2013 1.91±0.08 1.79±0.03 1.68±0.02 1.75±0.03 1.76±0.01 1.92±0.05
1.80±0.02c 2014 1.84±0.08 1.78±0.03 1.68±0.06 1.73±0.01 1.79±0.02 1.90±0.05
T8 2013 1.88±0.06 1.75±0.02 1.37±0.01 1.83±0.04 1.75±0.01 1.58±0.01
1.70±0.03de 2014 1.93±0.05 1.76±0.04 1.38±0.01 1.81±0.04 1.78±0.04 1.57±0.02
T9 2013 1.88±0.03 1.80±0.05 1.34±0.01 1.81±0.02 1.71±0.01 1.56±0.01
1.68±0.04e 2014 1.94±0.07 1.78±0.08 1.31±0.02 1.82±0.02 1.74±0.04 1.53±0.02
Means 1.92±0.02a 1.78±0.01c 1.62±0.02d 1.76±0.02c 1.84±0.02b 1.84±0.03b
73
1.92±0.050 (T7), 1.58±0.012 (T8) and 1.56±0.006 (T9). The results further showed that birds fed
on combinations of α-tocopherol and quercetin enriched diet had lowest FCR in T9 (1.68±0.035)
followed by T8 and T6 as 1.70±0.031, 1.72±0.027 whilst, highest in T0 (1.94±0.026),
respectively.
Current results regarding FCR of broilers administrated quercetin and α-tocopherol
supplemented feed are concordant with the work of Lee et al. (2003), reported that inclusion of
carvacrol and quercetin @ 200 mg/kg feed improved FCR of broilers. Afterwards, Jiang et al.
(2007) recorded that addition of soybean isoflavone in chicken diet at 10 to 80 mg/kg feed
resulted lower FCR value. Likewise, Rezaeipour et al. (2011) observed improved FCR of broiler
birds linearly with increasing concentration of α-tocopherol. One of the researchers groups,
Goliomytis et al. (2014) noticed lower feed conversion ratio (p< 0.05) when birds were provided
feed containing higher level of quercetin. The results of previous findings of Villar-Patino et al.
(2002) also indicated that feed efficiency was increased significantly through α-tocopherol
supplementation. One of the scientists groups, Rebole et al. (2006) reported that feed conversion
efficiency of broilers was improved substantially when birds were given feed supplemented with
200 mg/kg of α-tocopherol.
4.2. Results of raw meat
4.2.1. Total phenolic contents of breast and leg meat
It is obvious from the statistical analysis that total phenolic contents (TPC) of breast and leg meat
of broilers varied significantly among treatments but differed non-significantly due to years
(Table 9). However, interaction of treatments × years exhibited non-momentous effect.
The results (Table 10) depicted that total phenolic contents of breast meat for different broiler
groups T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9 were 103.87±0.94, 119.73±1.04,
121.10±1.82, 124.73±0.49, 135.33±1.58, 137.47±1.30, 139.77±1.35, 153.13±0.61, 157.23±0.55,
158.70±0.84 (mg GAE/100g meat) in 1st trial while the value of this trait for 2nd trial were
104.67±3.52, 118.80±1.62, 119.70±0.67, 122.63±1.60, 134.10±0.55, 135.63±0.43, 139.13±0.61,
152.53±1.22, 156.27±0.50, 157.63±0.54 (mg GAE/100g meat), in respective treatments. The
highest TPC were reported in T9 (158.17±0.51) followed by T8 and T7 (156.75±0.39 and
152.83±0.63) whereas lowest in T0 (104.27±1.64) at completion of the study.
74
Similarly, TPC of leg meat varied from 108.20±1.01 to 156.77±0.94 and 106.27±2.34 to
155.33±1.98 mg GAE/100g meat during the year 2013 and 2014 (Table 9). The highest TPC for
leg meat was recorded in T9 (156.05±1.02) trailed by T8 and T7 (153.75±1.12 & 146.48±0.81)
whereas lowest in T0 (107.23±1.22), respectively. During both experimental years, identical
trend of TPC was observed in broilers fed on different combinations of quercetin and α-
tocopherol enriched diets.
The results of instant study are supported by the findings of Boots et al. (2011) who reported that
antioxidants provided through diet are deposited in muscles thereby alleviate oxidative stress of
animals by increasing concentration of phenolic compounds. They further stated that deposition
of phenolic compounds is higher in breast muscles than that of leg meat of birds. Similarly,
Sacchetti et al. (2008) indicated that broilers fed on α-tocopherol supplemented feed resulted
higher antiradical power due to the accumulation of phenolic compounds. Likewise, Serpen, et
al. (2012) also showed that feed containing exogenous antioxidants such as vitamin C & E,
carotenoids, ubiquinols and polyphenols enhance total phenolic potential of poultry meat when
birds are fed on antioxidant enriched feed. One of the researchers groups, Flis et al. (2010)
observed that total phenolic potential of pig meat was enhanced by increasing concentration of
phenolic compounds through feed. Recently, Kim et al. (2014) stated that increasing levels of α-
tocopherol alone or in combination with red ginseng increased total phenolic potential of chicken
meat. The enhanced total phenolic contents of poultry meat is attributed to antioxidant potential
of α-tocopherol.
4.2.2. Free radical scavenging activity (DPPH assay) of breast and leg meat
Mean squares indicted that free radical scavenging activity (DPPH) of broiler breast and leg meat
varied significantly among treatments whilst differed non-significantly for years (Table 9).
Furthermore, interaction of treatments × years exhibited non-momentous variations.
In treatments, the lowest value in free radical scavenging activity of breast meat was noticed in
T0 (control) 54.71±0.64% whereas, the highest in T9 (birds fed on 300mg quercetin & 300 mg
of α-tocpherol/kg feed) 82.40±0.93% followed by T8 (birds fed on 300mg quercetin & 225 mg
of α-tocpherol/kg feed) 80.49±0.79%, T6 (birds fed on 200mg quercetin and 300 mg of α-
tocpherol/kg feed) 77.22±0.58%, respectively. Likewise, the value of this trait for leg meat in
both study years for T0, T9, T8 and T6 were 52.78±1.78 & 51.84±0.88, 77.83±1.30 &
75
76.21±1.56, 75.27±1.49 & 73.80±1.18 and 73.30±1.18& 72.45±0.55%, correspondingly (Table
11). It is obvious from the results that free radical scavenging activity showed identical trend in
study years however, the activity in meat increased linearly in a dose dependent manner.
The results of present study are supported by the findings of Saw et al. (2014), observed free
radical scavenging activity of three flavonoids i.e. quercetin, kaempferol and pterostilbene. They
noticed the highest antiradical ability of quercetin followed by kaempferol and pterostilbene.
Likewise, Kim et al. (2014) also indicated that α-tocopherol alone or in combination with red
ginseng enhanced the DPPH radical scavenging capacity of meat. The mechanistic approach
elaborates that antioxidants directly combine with free radicals leading to their inactivation that
decreases the intracellular concentration of free radicals thus enhances oxidative stability of
meat. Similarly, Mielnik et al. (2003) and Fasseas et al. (2007) noticed that vitamin E and sage
extract increased the antiradical power of poultry meat than that of control. One of the scientists
groups, Warnakulasuriya et al. (2014) found that quercetin-3-O-glucoside (Q3G) in esterified
form exhibited 50 to 100% of primary and 30 to 75% secondary oxidation products in fish oil.
The Q3G showed significantly higher inhibition of Cu2+ and peroxyl radical induced oxidation.
The present study revealed higher free radical scavenging activity of broiler meat when birds
were provided diet containing α-tocopherol and quercetin that is supported by the findings of the
aforementioned studies.
4.2.3. Ferric reducing antioxidant power of breast and leg meat
Ferric reducing antioxidant power (FRAP) assay is an important indicator to estimate the
antioxidant potential of different foods based on chelating capacity of ferrous ion (Fe3+ to Fe2+).
Treatments imparted momentous effect on FRAP of breast and leg meat of broilers however,
years and interaction of treatments × years exhibited non-momentous effect (Table 9).
The results in Table 12 regarding FRAP of broiler meat in different groups depicted that amongst
treatments, the minimum FRAP value for breast meat was in T0 as 543.67±1.86 & 541.67±3.28
µmol/Fe+2/g meat while, the maximum in T9, T8 and T6 as 683.00±3.79 & 681.00±2.65,
675.33±3.48 & 674.67±2.03, 666.00±2.08& 667.00±2.08 µmol/Fe+2/g meat, respectively in the
year 2013 and 2014. Likewise, the highest value of this trait in leg meat was reported in T9 as
76
Table 9. Mean squares for TPC, DPPH and FRAP assay of broiler meat
SOV df TPC breast TPC leg DPPH breast DPPH leg FRAP breast FRAP leg
Treatments (A) 9 1921.222** 1605.878** 504.803* 419.923** 13917.333** 13463.667**
Years (B) 1 14.90NS 26.00NS 7.39 NS 6.22 NS 0.001NS 5.000 NS
A ×B 9 0.944NS 0.344NS 1.361 NS 0.750 NS 4.556 NS 3.444 NS
Error 36 3.95 4.828 3.876 4.538 32.194 37.028
Total 59
**=Highly significant
*=Significant
NS=Non-significant
77
Table 10. Total phenolic contents (mg GAE/100g meat) of broiler meat
Treatments Breast meat Leg meat
Year 1 Year 2 Mean Year 1 Year 2 Means
T0 103.87±0.94 104.67±3.52 104.27±1.64f 108.20±1.01 106.27±2.34 107.23±1.22f
T1 119.73±1.04 118.80±1.62 119.27±0.88e 117.97±1.28 116.80±1.30 117.38±0.86e
T2 121.10±1.82 119.70±0.67 120.40±0.92de 119.10±1.82 118.30±0.95 118.70±0.94de
T3 124.73±0.49 122.63±1.60 123.68±0.88cd 122.67±0.58 120.60±1.60 121.63±0.89cd
T4 135.33±1.58 134.10±0.55 134.72±0.80d 131.87±1.44 130.63±1.29 131.25±0.91d
T5 137.47±1.30 135.63±0.43 136.55±0.74c 135.87±1.05 134.33±1.76 135.10±0.98c
T6 139.77±1.35 139.13±0.61 139.45±0.68b 138.10±1.04 136.57±0.32 137.33±0.60b
T7 153.13±0.61 152.53±1.22 152.83±0.63bc 146.90±0.66 146.07±1.63 146.48±0.8bc
T8 157.23±0.55 156.27±0.50 156.75±0.39ab 154.07±1.40 153.43±2.07 153.75±1.12ab
T9 158.70±0.84 157.63±0.54 158.17±0.51a 156.77±0.90 155.33±1.98 156.05±1.02a
Means 135.11±3.18 134.11±3.16 133.15±2.88 131.83±2.93
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300mg quercetin and 300 mg of α-tocopherol/kg feed
78
Table 11. Free radical scavenging activity (%) of broiler breast and leg meat
Treatments Breast meat (%) Leg meat (%)
Year 1 Year 2 Mean Year 1 Year 2 Means
T0 54.21±0.68 55.20±1.15 54.71±0.64f 52.78±1.78 51.84±0.88 52.31±0.91f
T1 61.40±0.83 59.90±0.71 60.65±0.59ef 56.56±1.40 55.76±1.25 56.16±0.86ge
T2 62.15±0.90 61.47±0.58 61.81±0.50e 58.60±1.33 57.87±1.18 58.24±0.81de
T3 67.39±0.99 67.35±0.95 67.37±0.61d 63.25±1.25 64.04±1.08 63.65±0.76cd
T4 64.58±1.60 62.75±0.50 63.67±0.85de 60.54±0.97 59.92±0.66 60.23±0.55d
T5 71.58±1.45 71.08±1.28 71.33±0.87c 67.40±1.07 67.48±0.93 67.44±0.63c
T6 77.17±1.17 77.26±0.55 77.22±0.58b 73.30±1.18 72.45±0.55 72.87±0.61b
T7 73.24±1.01 72.92±1.75 73.08±0.91bc 69.30±1.19 69.03±0.86 69.17±0.66bc
T8 81.55±1.21 79.43±0.72 80.49±0.79ab 75.27±1.49 73.80±1.18 74.53±0.91ab
T9 82.95±1.41 81.85±1.44 82.40±0.93a 77.83±1.30 76.21±1.56 77.02±0.98a
Means 69.62±1.67 68.92±1.61 65.48±1.53 64.84±1.48
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300mg quercetin and 300 mg of α-tocopherol/kg feed
79
Table 12. Ferric reducing antioxidant power of broiler meat
Treatments Breast meat (µmol/Fe+2/g meat) Leg meat (µmol/Fe+2/g meat)
Year 1 Year 2 Mean Year 1 Year 2 Means
T0 543.67±1.86 541.67±3.28 542.67±1.74f 542.33±3.53 541.00±2.52 541.67±1.96f
T1 564.67±2.03 567.33±1.45 566.00±1.26e 556.33±2.03 555.00±2.65 555.67±1.52e
T2 581.67±4.91 580.33±3.48 581.00±2.71de 573.33±4.91 572.67±2.40 573.00±2.45de
T3 625.33±3.76 624.00±3.00 624.67±2.17cd 617.67±4.06 616.00±6.03 616.83±3.27cd
T4 597.33±3.48 599.00±5.20 598.17±2.82d 589.00±3.21 588.33±2.85 588.67±1.93d
T5 637.33±3.76 636.67±1.76 637.00±1.86c 629.67±3.48 630.00±5.86 629.83±3.05c
T6 666.00±2.08 667.00±2.08 666.50±1.34b 658.33±2.33 660.33±2.33 659.33±1.54b
T7 645.00±3.21 647.00±2.65 646.00±1.91bc 637.33±3.28 635.67±2.73 636.50±1.95bc
T8 675.33±3.48 674.67±2.03 675.00±1.81ab 667.00±3.21 668.67±3.53 667.83±2.17ab
T9 683.00±3.79 681.00±2.65 682.00±2.11a 674.67±3.53 672.00±1.53 673.33±1.82a
Means 621.93±8.54 621.87±8.51 614.57±8.3 613.97±8.4
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300mg quercetin and 300 mg of α-tocopherol/kg feed
80
673.33±1.82 (µmol/Fe+2/g meat) trailed by T8 and T6 as 667.83±2.17, 659.33±1.54 µmol/Fe+2/g
meat, whilst lowest in T0 (control) 541.67±1.96 µmol/Fe+2/g meat at termination of trial.
The current findings are concordant with work of Goni, et al. (2007), they indicated that birds
fed on vitamin E and grape pomacea supplemented diets exhibited higher antioxidant potential as
measured by FRAP. Likewise, Banerjee et al. (2012) also found higher ferric reducing power in
meat containing natural antioxidants compared to synthetic counterparts. They further elaborated
that antioxidants in meat decrease the reductants in meat that are involved in conversion of Fe3+
to Fe2+. One of the researchers groups, Jung et al. (2010) found higher reducing power in broiler
meat that were fed on 0.5 and 1.0% mixture of gallic acid and linoleic acid compared to control.
The enhanced ferric reducing power due to feeding of α-tocopherol and quercetin is attributed to
their antioxidant potential. Similarly, Rupasinghe et al. (2010) delineated that total antioxidant
capacity of broiler meat as measured by FRAP is significantly varied when birds are provided
feed enriched with 300 & 600 mg quercetin/kg body weight. They further stated that total
antioxidant capacity is approximately 2.9 and 7.7 times greater, respectively than that of control.
In current exploration, treatments containing higher concentration of quercetin and α-tocopherol
yielded higher ferric reducing antioxidant power as compared to other treatments and control.
4.2.4. Thiobarbituric acid reactive substances (TBARS) assay of meat
Mean squares regarding thiobarbituric acid reactive substances (TBARS) of breast and leg meat
of broiler showed significant differences due to treatments and storage (Table 13).
The results (Figure 1) explicated that TBARS of breast meat on initiation (1st min) of storage
varied from 0.181±0.0032 to 0.282±0.0023 and 0.229±0.0153 to 0.441±0.1594 mg of MDA/kg
meat whereas at 120th min from 0.283±0.0174 to 0.384±0.0175 and 0.314±0.0157 to
0.425±0.0289 mg of MDA/kg meat among treatments during the year 2013 and 2014,
respectively. The lowest TBARS of breast meat recorded in T9 as 0.298±0.0119 mg of MDA/kg
meat followed by T8 and T6 as 0.309±0.0120 and 0.315±0.0184 mg of MDA/kg meat whilst,
maximum in T0 0.405±0.0170 mg of MDA/ kg meat. Similarly, TBARS of leg meat in T9, T8,
T6 and T0 were 0.305±0.0130, 0.315±0.0125, 0.332±0.0128, 0.406±0.0128 mg of MDA/kg of
meat, respectively at the end of trial. Furthermore, there was a momentous increase in TBARS of
antioxidant enriched leg meat that varied from 0.268±0.0043 at initiation to 0.308±0.0042,
0.344±0.0041, 0.399±0.0043, 0.462±0.0044 at 30 th, 60th, 90th and 120th min of storage (Figure 2).
81
The TBARS of meat increased linearly with progression of storage. It is evident from
exploration that lower MDA production reported in breast meat compared to leg meat however,
TBARS increased as a function of storage.
Previous studies showed that quercetin and α-tocopherol have ability to attenuate the process of
lipid peroxidation. The results of current study are consistent with the findings of Goliomytis et
al. (2014), noticed the impact of dietary quercetin @ 0.5 and 1g/kg of feed to the birds. It has
been inferred that oxidative stability of broiler meat under refrigerated as measured by TBARS
was enhanced (p < 0.05) when birds were fed quercetin supplemented diet @ 1 g/kg feed. The
TBARS value in different groups at initiation of storage were 24.3 ng of MDA/g meat (control),
23.3 ng of MDA/g meat (0.5g/kg feed) and 20.2 ng of MDA/g meat (1g/kg feed) that
progressively increased to 95.5, 73.7 and 64.6 ng of MDA/g meat in respective treatments at
termination. One of the researchers groups, Luehring et al. (2011) observed concomitant increase
in lipid peroxidation in pig meat as indicated by higher (p<0.05) concentrations of TBARS and
8-iso-PGF2 nevertheless, it was ameliorated (p<0.05) by dietary fortification of quercetin.
Likewise, Jang et al. (2010) also found that supplementing poultry diet with quercetin @200
ppm/kg feed substantially reduced TBARS. Recently, Andrés et al. (2014) reported that
quercetin alone or in combination with flaxseed reduces the formation of oxysterol in meat
(p<0.05), an oxidation product of cholesterol and lipids after 7 days of refrigerated storage. The
result of previous studies by Nickander et al. (1996) and Morrissey et al. (1998) indicated that
dietary supplementation of antioxidants significantly suppresses the malondialdehyde (MDA)
production in meat. One of the researchers groups, Haak et al. (2009) expounded that α-
tocopherol alone or in combination with rosemary & green tea extract retards lipid oxidation in
meat.
4.2.5. Quercetin contents of breast and leg meat
The mean squares in Table 14 revealed momentous differences in quercetin content of
antioxidant enriched broiler breast and leg meat due to treatments. Contrary, years and
interaction of treatments × years were affected non-momentously. The results (Table 15)
presented that quercetin contents of breast meat in different groups T0, T1, T2, T3, T4, T5, T6,
T7, T8 and T9 were 0.00±0.00, 3.68±0.22, 4.20±0.24, 14.16±1.05, 8.74±0.54, 9.12±0.64,
9.92±0.51, 4.85±0.13, 14.73±1.04, 16.38±1.12 mg/kg meat in the 1st trial whereas value for 2nd
82
trial were 0.00±0.00, 3.57±0.15, 4.13±0.20, 14.11±1.12, 8.67±0.64, 9.01±0.75, 9.87±0.63,
4.74±0.15, 14.62±1.03, 16.34±1.07 mg/kg meat in respective treatments. The highest quercetin
were measured in T9 16.36±1.01 mg/kg meat followed T8 and T3 as 14.67±0.03 and 14.13±0.06
mg/kg meat, respectively nonetheless, qurcetin was not recorded in T0 (control) group.
Similarly, maximum turn down for quercetin content of leg meat was reported in T9 14.18±1.03
mg/kg meat trailed by T8 and T3 13.51±1.01 & 13.01±1.02 mg/kg meat, respectively.
The results of instant study are in harmony with Rupasinghe et al. (2010), reported that quercetin
and its metabolites such as quercetin glucuronides and sulfonates are deposited in liver, breast
and thigh muscles when birds are provided quercetin supplemented feed. Likewise, Tang and
Cronin, (2007) also found that quercetin level of turkey roll increased by the addition of onion
juice brine (OJB) directly in meat @25 (OJB25) and 50% (OJB50) solution. They further
reported quercetin content of turkey rolls 8.17 and 16.3 mg/kg meat in 25 and 50% brine
solution, respectively. However, cooking process depleted quercetin in resultant meat after
cooking (30 min), quercetin content was dropped by 24% for OJB25 and 38% for OJB50 group.
Present study findings indicated that deposition of quercetin was enhanced when broiler birds
were provided diet containing α-tocopherol and quercetin.
4.2.6. Alpha tocopherol content of breast and leg meat
Mean squares (Table 14) regarding α-tocopherol content of breast and leg meat of broiler birds
elucidated significant variations due to treatments however, non-substantial behavior was noticed
for years and interaction of treatments × years. In treatments, means explicated the lowest α-
tocopherol contents of breast meat in T0 (control) 10.98±0.21mg/kg meat whereas, the highest in
T9 (birds fed on 300mg quercetin & 300 mg of α-tocopherol/kg feed) 38.77±0.62 mg/kg meat
followed by T8 (birds fed on 300mg quercetin & 225 mg of α-tocopherol/kg feed) 31.77±0.55
mg/kg meat, T6 (birds fed on 200mg quercetin and 300 mg of α-tocopherol/kg feed) 29.89±0.25
mg/kg feed, respectively. Likewise, the value of this trait for leg meat in T9, T8, T6 and T0 were
35.21±0.67, 28.65±0.29, 27.33±0.33 and 9.96±0.20mg/ kg meat, respectively at the end of
experimental years (Table 16).
83
Table 13. Mean squares for thiobarbituric acid reactive substances (TBARS) of broiler
meat
SOV df Breast meat Leg meat
Treatments (A) 9 0.035327** 0.034190**
Years (B) 1 0.024756NS 0.003874NS
Storage intervals (C) 4 0.274151** 0.349495**
B × C 4 0.001779NS 0.001149**
A × B 9 0.003093NS 0.000014NS
A × C 36 0.001777** 0.000032**
A × B × C 36 0.001778NS 0.000013NS
Error 196 0.001804 0.000017
Total 299
**=Highly significant
*=Significant NS=Non-significant
84
Figure 1. TBARS of breast meat of broiler birds fed on quercetin and α-tocopherol
supplemented feed. A: TBARS of breast meat during study year 2013. B: TBARS of breast
meat during study year 2014.
85
Figure 2. TBARS of broiler leg meat fed on quercetin and α-tocopherol supplemented feed.
A: TBARS of leg meat of birds during study year 2013. B: TBARS of leg meat of birds during
study year 2014.
86
Table 14. Mean squares for quercetin and alpha tocopherol contents of broiler meat
SOV df Quercetin of breast Quercetin of leg α-tocopherol of
breast
α-tocopherol of
leg
Years (A) 1 0.0700 NS 0.0001 NS 3.720NS 0.060 NS
Treatments (B) 9 175.0267** 144.9311** 458.624** 352.750**
A × B 9 0.0022 NS 0.0989 NS 0.239 NS 0.244 NS
Error 36 0.0047 0.0022 0.792 2.298
Total 59
**=Highly significant
*=Significant
NS=Non-significant
87
Table 15. Quercetin contents of broiler meat
Treatments Breast meat (mg/kg meat) Leg meat (mg/kg meat)
Year 1 Year 2 Mean Year 1 Year 2 Means
T0 - - - - - -
T1 3.68±0.22 3.57±0.15 03.62±0.13e 2.91±0.15 2.80±0.13 2.86±0.14e
T2 4.20±0.24 4.13±0.20 04.16±0.17de 3.34±0.13 4.05±0.32 3.69±0.16de
T3 14.16±1.05 14.11±1.12 14.13±1.06b 13.07±1.02 12.94±1.02 13.01±1.02b
T4 8.74±0.54 8.67±0.64 08.70±0.63cd 6.35±0.52 6.28±0.45 6.31±0.52cd
T5 9.12±0.64 9.01±0.75 09.06±0.74c 6.88±0.60 6.74±0.54 6.81±0.64c
T6 9.92±0.51 9.87±0.63 09.89±0.72bc 7.39±0.51 7.32±0.62 7.35±0.62bc
T7 4.85±0.13 4.74±0.15 04.79±0.14d 3.86±0.14 3.84±0.12 3.85±0.12d
T8 14.73±1.04 14.62±1.03 14.67±1.03ab 13.58±1.02 13.45±1.02 13.51±1.01ab
T9 16.38±1.12 16.34±1.07 16.36±1.01a 14.23±1.04 14.12±1.01 14.18±1.03a
Means 8.58±0.95a 8.51±0.91a 7.16±0.87a 7.15±0.86a
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300mg quercetin and 300 mg of α-tocopherol/kg feed
88
Table 16. Alpha tocopherol contents (mg/kg meat) of broiler meat
Treatments Breast meat Leg meat
Year 1 Year 2 Mean Year 1 Year 2 Means
T0 10.98±0.21 10.42±0.12 10.70±0.17g 9.89±0.37 10.03±0.23 09.96±0.20f
T1 13.57±0.55 12.83±0.50 13.20±0.37f 13.45±2.46 13.97±0.79 13.71±1.16ef
T2 17.70±0.23 17.60±0.35 17.65±0.19ef 15.76±0.76 15.63±0.43 15.70±0.39e
T3 24.13±0.70 23.77±0.57 23.95±0.41de 21.47±1.54 21.93±1.20 21.70±0.88cd
T4 16.77±0.43 16.57±0.33 16.67±0.24e 16.38±1.18 16.66±0.65 16.52±0.61de
T5 24.35±0.96 24.13±0.67 24.24±0.53d 23.37±1.57 23.10±0.87 23.24±0.80c
T6 30.12±0.39 29.67±0.35 29.89±0.25c 27.46±0.53 27.20±0.49 27.33±0.33bc
T7 18.46±1.17 18.02±0.87 18.24±0.66e 20.72±0.58 20.02±0.47 20.37±0.37d
T8 31.97±0.89 31.57±0.82 31.77±0.55b 28.73±0.38 28.57±0.52 28.65±0.29b
T9 38.77±0.62 37.27±0.64 38.02±0.52a 35.46±1.33 34.97±0.63 35.21±0.67a
Means 22.68±1.57 22.18±1.53 21.27±1.41 21.21±1.34
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300mg quercetin and 300 mg of α-tocopherol/kg feed
89
The results of current study are supported by Kim et al. (2006) who indicated that α-tocopherol
concentration of breast and thigh muscles of birds was enhanced by increasing the level of
dietary α-tocopherol. They further stated that feed supplementation @200 or 400 IU of α-
tocopherol is effective in retarding the oxidative degradation of lipids and cholesterol. Likewise,
Arshad et al. (2011) also reported that supplementation of α-tocopherol via feed to broilers
increases the deposition of α-tocopherol in meat. One of the researchers groups, Gao et al.
(2010) listed that dietary supplementation of α-tocopherol acetate significantly amplified its level
in pectoralis muscles indicating its deposition in tissues. They also stated that α-tocopherol level
of muscles was not decreased with postmortem time. One of the scientists groups, Lu et al.
(2014) reported that vitamin E supplemented feed to the broiler birds increased vitamin E
concentration in breast muscle. Accordingly, they conducted a study in which birds were fed on
diets containing HO: high oxidant diet, vitamin E @10 IU/kg, 3% oxidized soybean oil, 3%
polyunsaturated fatty acid (PUFA), VE: the HO diet with vitamin E @200 IU/kg; AOX: the HO
diet with AB @135 mg/kg; VE+AOX: the HO diet with vitamin E @200 IU/kg & AB @135
mg/kg; SC: standard control; PC: positive control including SC diet with AB @135 mg/kg. The
results indicated that vitamin E concentration in breast muscle was highest in VE groups
followed by VE+AOX (p<0.001). Previously, Wen et al. (1997) also found highest α-tocopherol
concentration in mitochondrial fraction followed by microsomes and intact tissues. One of the
researchers groups, Parveen et al. (2013) stated that dietary supplementation of α-tocopherol
@200 mg/kg feed enhanced the vitamin E content of microsomal fraction than that of control.
4.2.7. Fatty acid profile of breast and leg meat
The results (Table 17) elucidated that fatty acid composition of broiler meat affected
significantly among treatments whilst years imparted non-momentously. It has been observed
eight fatty acids in meat samples mainly palmitic acid (C16:0), stearic acid (18:0), oleic acid
(18:1n-9) and linolieci acid (18:2n-9, 6). Nonetheless, α-linolenic acid (18:3) and arachidonic
acid (20:4) differed non-substantially among treatments in both years. The lowest amount of
palmitic acid (16:0) was recorded in T9 13.4 & 12.5% followed by T8 and T6 as 14.3 & 13.2,
14.85 & 13.9% whereas highest in T0 19.6 & 18.4% in respective years. Likewise, minimum
stearic acid (16:0) also found in T9 7.14 & 7.02% trailed by T8 and T6 7.56 & 7.01 and 7.96 &
7.04% though, highest amount was recorded in T0 10.06 & 9.84%. Furthermore, highest level of
90
oleic acid (18:1) was reported in T0 24.38 & 23.35% followed by T2 and T1 as 24.16 & 23.05,
23.76 & 22.65% whilst, lowest in T6 21.01 & 19.93%. The results expounded lowest saturated
fatty acid (SFA) in T9 21.69% whereas the values for this trait in T8, T6 and T0 were 23.01,
23.93 and 30.76%. Similarly, polyunsaturated fatty acids (PUFA) including oleic, linoleic and
linolenic acids in different groups ranged from 9.88 to 13.41% and 9.62 to 12.60% in respective
years (Table 18).
Furthermore, the results showed lowest palmitic acid (16:0) for leg meat in T9 15.3 & 14.2%
followed by T8, T6 and T5 16.9 & 15.9 and 18.20 & 17.65, 18.8& 17.6% whereas highest in T0
21.8 & 20.6% in selected years. Likewise, stearic acid (18:0) in T9, T8, T6, T5 and T0 were
11.61 & 11.50, 12.12 & 11.21, 12.01 & 12.26, 13.55 & 12.41, 14.62 & 14.52%. Besides, oleic
acid in treatments T0, T1, T2, T3 and T9 were 35.06 & 33.16, 34.96 & 32.92, 34.48 & 32.68,
33.58 &31.48, 31.25 & 29.56%, respectively (Table 19). The results expounded lowest SFA for
leg meat in T9 30.21 & 31.99% trailed by T8, T6 and T5 31.52 & 28.51 and 32.61 & 31.06,
33.85& 34.55% whereas highest in T0 37.82 & 36.02% in the year 2013 and 2014 (Table 20).
The PUFA in leg meat ranged from 14.88 to 17.88 and 12.55 to 16.82% among different
treatments. Overall, fatty acid production decreased with increasing level of quercetin and α-
tocopherol in dose dependent manner depicted their lipid lowering potential however, reduction
in SFA fatty acids was more pronounced than that of PUFA in broiler meat.
The findings of instant study are in harmony with Oskoueian et al. (2013), they recorded that
dietary supplementation of quercetin @200 mg/kg feed to broiler birds affect fatty acid
composition of the resultant pectoralis muscle. They reported oleic (18:1n-9, 33-35.2%), palmitic
(16:0, 26.1-27.9%), linoleic (18:2n-6, 14.0-14.6%) and stearic acid (18:0, 11.8–13.7%) in
pectoralis muscle, respectively and further elaborated that quercetin addition significantly
(p<0.05) diminishes palmitic, oleic and linoleic acid production. Likewise, De Boer et al. (2006)
noticed that dietary administration of quercetin @1% in rat affected fatty acid synthesis rate and
diminished free fatty acids production. The mechanistic approach for reduction of SFA in broiler
meat due to antioxidants supplementation is their potential to inhibit activity of 9-desaturase
complex that converts SFA to MUFA thus inclusion of antioxidants in poultry feed is a practical
strategy to reduce SFA content in chicken meat. One of the scientists groups, Gnoni et al. (2009)
delineated
91
Table 17. Fatty acids composition of broiler breast meat
Treatments 2013 2014
114:0 216:0 316:1 418:0 518:1 618:2 718:3 820:4 114:0 216:0 316:1 418:0 518:1 618:2 718:3 820:4
T0 1.10c 19.6a 1.65d 10.06a 24.38ab 11.61a 0.86ab 0.94b 1.01c 18.40a 1.50b 9.84a 23.35ab 10.98a 0.76c 0.86b
T1 1.19a 18.4ab 1.78c 9.78ab 23.76bc 11.14ab 0.81bc 0.96ab 0.99c 17.26ab 1.60a 9.12ab 22.65bc 10.28ab 0.81ab 0.91a
T2 0.99d 17.6b 1.67cd 9.41b 24.16b 10.65b 0.84b 0.89c 1.14a 16.40b 1.52b 8.98b 23.05b 10.31ab 0.79b 0.87b
T3 1.20a 17.11bc 2.1a 8.98c 23.48c 9.96c 0.82bc 0.98a 1.16a 16.14c 1.51b 8.16c 22.18c 9.34bc 0.84a 0.82c
T4 1.17a 17.56b 1.84bc 9.11bc 24.64a 10.26bc 0.89a 0.94b 1.06bc 16.20bc 1.60a 8.64bc 23.71a 9.76b 0.80b 0.92a
T5 1.18a 16.8c 1.87b 8.63cd 21.07e 9.61cd 0.86ab 0.94b 1.08b 15.70cd 1.55ab 8.11cd 20.02e 9.10c 0.85a 0.89ab
T6 1.12b 14.85d 2.06a 7.96de 21.01e 8.56de 0.82bc 0.98a 1.04bc 13.90de 1.51b 7.04e 19.93f 8.09de 0.82ab 0.86b
T7 1.12b 16.3cd 1.85bc 8.26d 22.79cd 9.11d 0.78c 0.91bc 1.10b 15.23d 1.45c 7.86d 21.78cd 9.01cd 0.82ab 0.84bc
T8 1.15ab 14.3de 1.93ab 7.56e 21.45d 8.26e 0.87ab 0.91bc 1.08b 13.20e 1.56ab 7.01e 20.41de 8.21d 0.84a 0.91a
T9 1.15ab 13.4e 1.78c 7.14f 21.15de 8.08f 0.86ab 0.94b 1.12ab 12.50f 1.60a 7.02e 20.65d 8.01e 0.78bc 0.83c
Means sharing similar letters in a column do not differ significantly from one another (p˃0.05) 1Myristic acid, 2Palmitic, 3Palmitoleic, 4stearic, 5Oleic, 6Linoleic, 7α-Linolenic, 8Arachidonic
T0= control without antioxidants,
T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
92
Table 18. SFA, MUFA, PUFA and PUFA: SFA ratio of broiler breast meat
Treatments 2013 2014
ΣSFA ΣMUFA ΣPUFA PUFA:SFA ΣSFA ΣMUFA ΣPUFA PUFA:SFA
T0 30.76a 25.81b 13.41a 0.45 29.25a 24.55b 12.60a 0.43
T1 29.37ab 25.54bc 12.91ab 0.44 27.37b 24.25bc 12.10ab 0.44
T2 28.00b 23.12d 12.38b 0.44 26.52bc 24.87ab 11.97b 0.45
T3 27.29c 23.25cd 11.76c 0.44 25.46cd 23.69c 11.00c 0.43
T4 27.84bc 22.85de 12.09bc 0.43 25.90c 25.31a 11.48bc 0.44
T5 26.61cd 21.87e 11.41c 0.43 24.89d 21.57e 10.84cd 0.44
T6 23.93de 23.13d 10.36d 0.43 21.98e 21.44e 9.77e 0.44
T7 25.68d 24.64c 10.8cd 0.42 24.19de 23.23cd 10.67d 0.44
T8 23.01e 26.57a 10.04de 0.43 21.29ef 21.97de 9.96de 0.46
T9 21.69f 26.16ab 9.88e 0.45 20.64f 22.25d 9.62f 0.46
Means sharing similar letters in a column do not differ significantly from one another (p˃0.05) SFA = Saturated fatty acids MUFA = Mono unsaturated fatty acids PUFA = Poly unsaturated fatty acids PUFA/SFA = Ratio of polyunsaturated fatty acids and saturated fatty acids
T0= control without antioxidants, T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed,
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed, T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed, T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed, T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed, T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
93
Table 19. Fatty acids composition of broiler leg meat
Treatments 2013 2014
114:0 216:0 316:1 418:0 518:1 618:2 718:3 820:4 114:0 216:0 316:1 418:0 518:1 618:2 718:3 820:4
T0 1.40bc 21.80a 3.80a 14.62ab 35.06a 15.16a 1.11f 1.61b 0.90c 20.60a 3.50a 14.52a 33.16a 14.26a 0.96ab 1.60b
T1 1.30c 20.60ab 3.70ab 14.75a 34.96ab 14.36b 1.21e 1.63b 1.10b 19.45ab 3.35ab 13.71ab 32.92ab 13.45ab 0.98a 1.54bc
T2 1.40bc 21.90a 3.60b 14.81a 34.48b 13.71bc 1.26de 1.81a 1.00bc 18.75bc 3.32ab 13.78ab 32.68b 12.31b 0.98a 1.63ab
T3 1.60ab 19.10b 3.35c 13.22c 33.58c 12.63cd 1.47cd 1.71ab 1.30a 18.90b 3.21b 12.31c 31.48cd 11.45bc 1.01a 1.56bc
T4 1.60ab 19.50b 3.50bc 13.01cd 34.04bc 13.04c 1.31d 1.83a 1.31a 18.40c 3.10bc 12.03cd 32.08bc 12.06b 0.97ab 1.68ab
T5 1.50b 18.80bc 3.20cd 13.55bc 33.07cd 12.31cd 1.53c 1.76ab 1.10b 17.60d 2.90cd 12.41bc 31.67c 11.24bc 0.99a 1.55bc
T6 1.70a 18.20bc 2.80de 12.01de 32.06d 11.65e 1.71b 1.78ab 1.20ab 17.65cd 2.50d 12.26c 30.02de 10.54c 0.96ab 1.46c
T7 1.50b 18.30bc 3.10d 13.65b 32.98cd 12.08d 1.65bc 1.81a 1.20ab 17.20de 2.95c 12.54b 30.94d 11.21bc 0.94b 1.65ab
T8 1.50b 16.90c 2.60e 12.12d 31.65e 11.28e 1.76ab 1.84a 1.20ab 15.90e 2.35e 11.21d 29.68e 10.48c 0.95ab 1.78a
T9 1.30c 15.30d 2.50e 11.61e 31.25f 11.06e 1.80a 1.82a 1.21ab 14.20f 2.43de 11.5e 29.56e 10.08d 1.03a 1.44c
Means sharing similar letters in a column do not differ significantly from one another (p˃0.05) 1Myristic acid, 2Palmitic, 3Palmitoleic, 4stearic, 5Oleic, 6Linoleic, 7α-Linolenic, 8Arachidonic
T0= control without antioxidants,
T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
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Table 20. SFA, MUFA, PUFA and PUFA:SFA ratio of broiler leg meat
Treatments 2013 2014
ΣSFA ΣMUFA ΣPUFA PUFA:SFA ΣSFA ΣMUFA ΣPUFA PUFA:SFA
T0 37.82a 38.86a 17.88a 0.50 36.02a 36.66a 16.82a 0.47
T1 36.65b 38.66ab 17.20ab 0.50 32.26bc 36.27ab 15.97ab 0.50
T2 37.11ab 38.08b 16.78b 0.47 33.53b 36.00b 14.92b 0.47
T3 33.92c 36.93c 15.81c 0.47 31.51cd 34.69c 14.02c 0.45
T4 34.11bc 37.54bc 16.18bc 0.47 31.73c 35.18bc 14.71bc 0.46
T5 33.85c 36.27cd 15.6cd 0.47 31.11d 34.57cd 13.78cd 0.50
T6 32.61d 34.86de 15.14d 0.46 31.06d 32.52de 12.96de 0.47
T7 33.45cd 36.08d 15.54cd 0.47 33.94ab 33.89d 13.80cd 0.45
T8 31.52de 34.25e 14.88de 0.46 28.51e 32.03e 13.21d 0.45
T9 30.21e 33.75f 14.68e 0.47 29.5de 31.99f 12.55e 0.44
Means sharing similar letters in a column do not differ significantly from one another (p˃0.05) SFA = Saturated fatty acids MUFA = Mono unsaturated fatty acids PUFA = Poly unsaturated fatty acids PUFA/SFA = Ratio of polyunsaturated fatty acids and saturated fatty acids
T0= control without antioxidants, T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed, T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed, T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed, T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed, T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
95
that quercetin decreases the rate of fatty acid and triacylglycerol (TAG) synthesis in rats. One of
the researchers groups, Jung et al. (2010) documented a declining trend in the production of SFA
and MUFA in broilers breast meat through dietary supplementation of gallic acid in combination
with linoleic acid. One of the scientists groups, Kamboh and Zhu (2013) specified that dietary
inclusion of purified flavonoid i.e. genistein and hesperidin to broiler through diet decreased
SFA level in breast meat. They conducted a study in which one day old 360 birds were fed on
control (basal), G5 (5 mg of genistein/kg), H20 (20 mg hesperidin/kg), GH5 (genistein &
hesperidin @5 mg/kg), GH10 (genistein & hesperidin @10 mg/kg) GH20 (genistein &
hesperidin @20 mg/kg) diets, respectively. The results delineated that GH20 group reduced (P ≤
0.005) fatty acid proportions of 14:0 and 18:0 with maximum reduction of 30 and 8.7%,
respectively. Compared to control, genistein and hesperidin-supplemented group significantly
diminished fatty acid proportion of myristic & stearic acid in dose-dependent manner.
Furthermore, ratio of Ω-6 to Ω-3 fatty acid were also improved (P < 0.01) by genistein and
hesperidin supplementation (GH5, GH10, and GH20) with maximum effect 37% by GH20
group. The changes induced by quercetin and α-tocopherol in current study showed similar
pattern for reduction of fatty acid in muscles of the broiler birds.
4.3. Antioxidant enzymes
4.3.1. Superoxide dismutase (SOD)
The statistical analysis pertaining to superoxide dismutase (SOD) activity of broiler blood serum
delineated significant variations due to treatments however, years and interaction of treatments ×
years exhibited non-substantial differences (Table 21). The results regarding superoxide
dismutase activity (SOD) depicted that minimum value of SOD is in T0 as 136.68±0.79 U/mg of
protein while, its concentration in T9, T8, T6 T4 and T7 as 196.93±0.41, 193.29±0.87,
186.86±0.54, 174.39±1.13, 170.41±1.07 U/mg of protein, respectively (Table 22).
The results are in corroborates with the previous findings of Boa-Amponsem et al. (2000),
indicated that dietary supplementation of α-tocopherol increases SOD activity. Furthermore, they
were of the opinion that variations in enzyme activity are due to synergistic behavior of
antioxidants. Afterwards, Bahorun et al. (2006) documented that vitamin E inhibits oxidation
chain reaction by quenching free radicals thereby improves SOD performance. One of the
researchers groups, Saw et al. (2014) reported that berry constituents i.e. quercetin, kaempferol,
96
and pterostilbene improve the SOD activity by triggering Nrf2-ARE signaling pathway. One of
the scientists groups, Delles et al. (2014) carried out a study to explicate the influence of dietary
antioxidants and oils on oxidative and enzymatic prospects of chicken birds. The birds were fed
on diets with low-oxidized (peroxide value 23 mEq of O2/kg) or high-oxidized (peroxide value
121 mEq of O2/kg) oil supplemented with or without antioxidants for 42 days. It has been
observed that that ingestion of the high-oxidized oil diet reduced SOD activity compared to low-
oxidized counterpart. Similarly, Lu et al. (2014) reported a decline in SOD activity of broiler
birds fed on oxidant rich diet however, α-tocopherol supplemented diet improved SOD
performance. These finding supports results of current exploration that birds fed on higher level
of antioxidants i.e quercetin and α-tocopherol resulted higher superoxide dismutase activity.
4.3.2. Glutathione reductase (GRs)
The mean squares in Table 21 depicted that serum glutathione reductase (GRs) activity of
experimental birds are affected significantly in various groups because of treatments while years
and treatments × years demonstrated non-momentous variations. The results indicated that
glutathione reductase (GRs) activity among treatments varied from 33.44±0.52 to 54.09±0.81
and 33.13±1.02 to 53.32±1.04 U/mg of protein during the year 2013 and 2014, respectively
(Table 22). The maximum turn down of GRs activity in T9 (birds fed on 300mg quercetin & 300
mg of α-tocpherol/kg feed) 53.70±0.61 U/mg of protein trailed by T8 (birds fed on 300mg
quercetin & 225 mg of α-tocpherol/kg feed) and T6 (birds fed on 200mg quercetin and 300 mg
of α-tocpherol/kg feed) 52.34±0.59 and 50.90±0.48 U/mg of protein, respectively while, lowest
in T0 33.29±0.52 U/mg of protein at the termination of study.
The results of instant study are supported by the findings of Lu et al. (2014), they recorded
improvement in GRs activity of birds fed on diet enriched with α-tocopherol @200mg/kg feed.
Likewise, Delles et al. (2014) also documented improved glutathione reductase performance
(p<0.05) when birds were given antioxidant enriched diet. One of the researchers groups, Feng
et al. (2014) noticed that liver glutathione reductase activity of broiler decreases by feeding
oxidant rich diet. Similarly, Bansal et al. (2005) noticed a decrease GRs activity in liver of rats
that were fed on nitrosamine however, those pretreated with vitamin E resulted enhanced
97
Table 21. Mean squares for Superoxide dismutase (SOD), glutathione reductase (GR) and
catalase of broiler blood serum
SOV df SOD GR Catalase
Treatments (A) 9 2703.111** 319.4200** 652.711**
Years (B) 1 5.600NS 0.3300 NS 2.790 NS
A × B 9 0.444 NS 0.1944 NS 0.502 NS
Error 36 6.125 2.3119 1.81
Total 59
** = Highly significant
* = Significant
NS = Non-significant
98
Table 22. Superoxide dismutase (SOD), glutathione reductase and catalase of broiler blood serum
Treatments SOD (U mL-1) GR (U/mg protein) Catalase (U/g of protein)
Year 1 Year 2 Means Year 1 Year 2 Means Year 1 Year 2 Means
T0 137.05±1.26 136.30±1.18 136.68±0.79g 33.44±0.52 33.13±1.02 33.29±0.52f 119.54±0.86 117.70±0.38 118.62±0.59f
T1 143.71±1.26 143.54±1.67 143.63±0.94f 35.42±0.99 35.09±1.42 35.25±0.78ee 123.51±0.87 123.27±0.73 123.39±0.51e
T2 148.28±1.82 148.20±1.95 148.24±1.19ef 37.48±0.69 38.01±0.39 37.75±0.37de 127.43±0.91 126.77±0.78 127.10±0.56d
T3 153.62±1.41 153.07±0.81 153.35±0.74e 43.09±0.69 42.82±0.62 42.96±0.42cd 132.20±0.70 131.80±0.50 132.00±0.40cd
T4 175.12±1.57 173.67±1.85 174.39±1.13c 39.22±1.01 39.37±0.45 39.30±0.50d 125.46±0.88 125.91±0.94 125.68±0.58de
T5 165.55±1.81 164.80±1.88 165.17±1.18d 45.11±1.01 44.95±0.85 45.03±0.59c 135.24±0.77 134.93±0.13 135.09±0.36c
T6 187.04±0.87 186.67±0.81 186.86±0.54b 51.09±0.95 50.72±0.44 50.90±0.48b 144.42±1.02 144.17±0.61 144.29±0.53b
T7 170.82±1.94 170.00±1.33 170.41±1.07cd 47.72±0.77 47.61±0.62 47.66±0.45bc 137.19±0.81 136.80±0.32 137.00±0.40bc
T8 193.17±1.55 193.40±1.17 193.29±0.87ab 52.25±1.08 52.43±0.75 52.34±0.59ab 147.31±0.86 147.20±0.92 147.26±0.56ab
T9 197.62±0.60 196.23±0.09 196.93±0.41a 54.09±0.81 53.32±1.04 53.70±0.61a 149.02±0.66 148.47±0.59 148.74±0.41a
Means 167.20±3.77 166.59±3.75 43.89±1.3 43.74±1.2 134.13±1.83 133.70±1.86
Means sharing similar letters in a column do not differ significantly from one another (p˃0.05)
T0= control without antioxidants,
T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
99
activity. One of the scientists groups, Harset and Gulhan (2008) reported that α-tocopherol (50
IU/kg) supplementation to layer hens increases glutathione reductase and catalase activity.
4.3.3. Catalase
It is obvious from mean squares that serum catalase activity of birds varied significantly among
treatments but differed non-significantly for years (Table 21). Furthermore, interaction of
treatments × years exhibited non-momentous effect for this trait. The results (Table 22) revealed
that catalase performance in different groups T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9 were
119.54±0.86, 123.51±0.87, 127.43±0.91, 132.20±0.70, 125.46±0.88, 135.24±0.77, 144.42±1.02,
137.19±0.81, 147.31±0.86, 149.02±0.66 U/mg of protein in 1st trial while the value of this trait
during 2nd trial were 117.70±0.38, 123.27±0.73, 126.77±0.78, 131.80±0.50, 125.91±0.94,
134.93±0.13, 144.17±0.61, 136.80±0.32, 147.20±0.92, 148.47±0.59 U/mg of protein in
respective treatments. The results indicated maximum catalase activity in T9 as 148.74±0.41
U/mg of protein followed by T8 and T6 as 147.26±0.56, 144.29±0.53 U/mg of protein while
lowest in T0 118.62±0.59 U/mg of protein.
The results of present study are in agreement with Delles et al. (2014), noticed that catalase
activity was significantly higher (p < 0.05) in broiler birds fed on diet supplemented with
antioxidant. Likewise, Lu et al. (2014) also indicated that treatment containing blend of
antioxidants @200 mg/kg α-tocopherol resulted significantly (p < 0.05) higher catalase activity.
One of the scientists groups, Srivastava et al. (2010) observed a significant decline in tissue
catalase activity of rats fed on sunflower oil however, antioxidant supplemented group
containing α-tocopherol yielded higher performance.
4.4. Serum Bio-chemical profile of birds
The mean squares in Table 23 indicated that treatments imparted significant effect on total
cholesterol, LDL, HDL cholesterol, triglycerides and serum total protein of broiler birds
however, years and interaction of treatments×years exhibited non-momentous differences. The
means indicated lowest cholesterol in T9 (birds fed on 300 mg quercetin & 300 mg α-
tocopherol/kg feed) 107.28±0.48 mg/dL followed by T8 (birds fed on 300 mg quercetin & 225
mg α-tocpherol/kg feed) 109.72±0.20mg/dL, T6 (birds fed on 200 mg quercetin and 300 mg α-
tocpherol/kg feed) 110.57±0.45 mg/dL, T5(birds fed on 200 mg quercetin and 225 mg α-
tocpherol/kg feed) 115.76±0.66, T3 (birds fed on 100 mg quercetin along with 300 mg α-
100
tocpherol/kg feed)117.26±0.37 whereas, the highest in T0 (control) 133.37±0.59 mg/dL (Table
24).
Among groups, maximum LDL cholesterol was reported in T0 as 42.91±0.40mg/dL whilst,
minimum in T9 25.04±0.33mg/dL however, the value for LDL in T8, T6, T7 and T5 were
27.95±0.39, 28.86±0.36, 32.50±0.33 and 33.91±0.62 mg/dL, respectively (Table 24). Likewise,
triglycerides in Table 25 depicted highest value in T0 as 69.35±0.49 mg/dL whereas the lowest
48.83±0.23 in T9 nonetheless, 50.66±0.31, 51.27±0.64, 55.04±0.30 and 57.30±0.50 mg/dL in
T8, T6, T7 and T5, respectively.
Similarly, HDL cholesterol among treatments varied from 60.79±0.26 to 79.53±0.56 and
59.77±0.23 to 78.38±1.14 mg/dL during the study year 2013 and 2014, respectively (Table 25).
The maximum HDL was recorded in T9 78.96±0.62mg/dL trailed by T8 and T6 76.87±0.50 &
75.66±0.54 mg/dL while, the lowest in T0 as 60.28±0.28 mg/dL at termination of trial (Table
24).
Furthermore, serum protein of broiler birds in T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9 groups
were 2.62±0.02, 2.73±0.02, 2.90±0.02, 3.56±0.03, 3.04±0.03, 3.81±0.02, 4.42±0.02, 3.97±0.01,
4.90±0.36 and 4.94±0.02 g/dL during 1st trial while the values for 2nd trial were 2.56±0.03,
2.72±0.02, 2.89±0.02, 3.55±0.02, 3.02±0.02, 3.79±0.02, 4.39±0.02, 3.94±0.03, 4.52±0.02,
4.91±0.03 g/dL in respective treatments, correspondingly (Table 25).
The findings of instant exploration are consistent with Rama Rao et al. (2011), they found a
decline in sera cholesterol with dietary α-tocopherol supplementation irrespective of oil sources.
Likewise, Brenes et al. (2008) also reported reduced total cholesterol with increasing α-
tocopherol levels in feed. One of the researchers groups, Sikder et al. (2014) conducted research
in which rats were given high cholesterol diet 2% to induce hepatotoxicity afterwards, fed on
quercetin or rutin enriched diet (100 mg kg-1 body weight). The results demonstrated that
antioxidants decrease cholesterol through restoration and circulation of hepatic antioxidants. One
of their peers, Zahedi et al. (2013) observed that quercetin supplementation in women with type-
2 diabetes reduces blood pressure significantly (p < 0.05) however, reduction in systolic blood
pressure was more pronounced. One of the scientists groups, Phuwamongkolwiwat et al. (2013)
reported that quercetin-3-O-β-glucoside (Q3G) and fructooligosaccharide (FOS) enriched diet
101
improves glucose tolerance, insulin sensitivity and cholesterol through increasing blood
quercetin level.
The results of previous findings by Choi et al. (2010) delineated that increasing dietary
supplementation of α-tocopherol alone or in combination with garlic powder significantly
decreases total cholesterol and LDL (p < 0.05). Likewise, Graf et al. (2005) reported 21%
reduction in cardiovascular complications when the intake of quercetin is greater than 4 mg/day.
Earlier, Chopra et al. (2000) stated that LDL cholesterol in human decreases by dietary
supplementation of quercetin @30 mg/day in hyperlipidemic people nonetheless it also
minimizes LDL oxidation. Similarly, Zahedi et al. (2013) reported that dietary quercetin
supplementation enhances HDL among women with type-2 diabetes.
Previously, Brenes et al. (2008) established that dietary supplementation of α-tocopherol
increases sera protein concentration. The mechanistic approach for this high concentration is
attributed to improved digestion and absorption of dietary nutrients in the presence of α-
tocopherol. They also stated that broilers provided higher level of α-tocopherol triggered the
digestibility of dietary nutrients compared to those fed on control diet. These findings are further
supported by Rama Rao et al. (2011), who reported that total protein and globulin concentration
in sera increased significantly with dietary α-tocopherol fortification. Purposely, birds were
given 3 dietary concentrations of α-tocopherol (10, 50 and 100 mg/kg) along with three sources
of oil (sunflower-SFO, palm-PMO and safflower-SAO). It has been observed that total protein
and globulin concentrations in sera increased significantly with dietary α-tocopherol
supplementation irrespective of oil sources.
4.5. Results of antioxidant enriched product
4.5.1. Nuggets color
It is revealed from mean squares that color of breast and leg meat nuggets varied significantly
among treatments and storage but differed non-significantly due to years (Table 26). However,
interactions except treatments×days exhibited non-momentous differences. The results (Table
27) indicated that at initiation of storage, color values of breast meat nuggets in various groups
T0, T1, T2, T3, T4, T5, T6, T7, T8 and T9 were 102.33±0.882, 103.67±1.453, 105.33±0.882,
102
Table 23. Mean squares for total cholesterol, HDL, LDL, triglycerides and protein of
broilers blood serum
SOV df TQ HDL LDL Triglyceride Total protein
Treatments (A) 9 444.039** 236.157** 215.9522** 299.299** 4.2591**
Years (B) 1 24.940NS 20.500 NS 6.2000 NS 14.650NS 0.0487NS
A × B 9 0.629 NS 0.121 NS 0.3578 NS 0.459NS 0.0188NS
Error 36 0.612 0.673 0.9231 0.465 0.0183
Total 59
**=Highly significant
*=Significant
NS=Non-significant
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Table 24. Total cholesterol (TQ), HDL and LDL of broilers blood serum
Treatments Cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL)
Year 1 Year 2 Mean Year 1 Year 2 Means Year 1 Year 2 Means
T0 132.24±0.56 134.49±0.39 133.37±0.59a 60.79±0.26 59.77±0.23 60.28±0.28f 42.56±0.41 43.27±0.71 42.91±0.40a
T1 125.78±0.18 126.43±0.35 126.10±0.23ab 63.67±0.47 62.42±0.56 63.04±0.43e 41.26±0.57 41.39±0.53 41.32±0.35ab
T2 125.80±0.14 126.83±0.35 126.31±0.29ab 65.11±0.65 64.16±1.01 64.64±0.58de 38.87±0.37 40.33±0.50 39.60±0.43b
T3 116.50±0.32 118.01±0.18 117.26±0.37bc 70.24±0.55 69.41±0.62 69.83±0.41cd 35.47±0.36 36.41±0.44 35.94±0.33c
T4 124.23±0.36 125.50±0.47 124.86±0.39b 67.53±0.69 65.95±0.87 66.74±0.61d 37.36±0.51 37.91±0.97 37.64±0.50bc
T5 114.96±0.44 116.55±1.17 115.76±0.66c 72.33±0.49 71.04±0.80 71.69±0.51c 33.55±0.77 34.26±1.09 33.91±0.62cd
T6 109.86±0.38 111.28±0.62 110.57±0.45cd 76.32±0.47 74.99±0.89 75.66±0.54b 28.99±0.09 28.72±0.79 28.86±0.36de
T7 114.60±0.23 115.78±0.29 115.19±0.31c 73.50±0.73 72.78±0.97 73.14±0.57bc 32.34±0.45 32.66±0.57 32.50±0.33d
T8 109.72±0.32 109.72±0.32 109.72±0.20d 77.64±0.40 76.09±0.72 76.87±0.50ab 27.43±0.38 28.46±0.61 27.95±0.39e
T9 106.27±0.23 108.29±0.30 107.28±0.48e 79.53±0.56 78.38±1.14 78.96±0.62a 24.61±0.44 25.46±0.40 25.04±0.33f
Means 118.00±1.50 119.29±1.53 70.67±1.12 69.50±1.15 34.24±1.06 34.89±1.08
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
104
Table 25. Triglyceride and total protein of broiler blood serum
Treatments Triglycerides (mg/dL) Total protein (g/dL)
Year 1 Year 2 Means Year 1 Year 2 Means
T0 69.35±0.49 70.22±0.61 69.79±0.40a 2.62±0.02 2.56±0.03 2.59±0.02g
T1 66.24±0.53 67.44±0.41 66.84±0.40ab 2.73±0.02 2.72±0.02 2.72±0.01f
T2 63.87±0.27 64.74±0.49 64.30±0.32b 2.90±0.02 2.89±0.02 2.89±0.01ef
T3 58.57±0.53 59.32±0.53 58.95±0.37cd 3.56±0.03 3.55±0.02 3.56±0.02d
T4 61.06±0.31 62.76±0.44 61.91±0.45c 3.04±0.03 3.02±0.02 3.03±0.02e
T5 56.39±0.45 58.22±0.44 57.30±0.50cd 3.81±0.02 3.79±0.02 3.80±0.01cd
T6 51.27±0.64 52.64±0.41 51.96±0.46de 4.42±0.02 4.39±0.02 4.41±0.01b
T7 54.69±0.38 55.38±0.43 55.04±0.30d 3.97±0.01 3.94±0.03 3.95±0.01c
T8 50.66±0.31 51.31±0.59 50.98±0.33e 4.90±0.36 4.52±0.02 4.71±0.18ab
T9 48.83±0.23 48.79±0.25 48.81±0.15f 4.94±0.02 4.91±0.03 4.93±0.02a
Means 58.09±1.23 59.08±1.27 3.69±0.16 3.63±0.14
Means sharing similar letter in a row or in a column are statistically non-significant (p>0.05)
T0= control without antioxidants
T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed
T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed
T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed
T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed
T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
105
111.67±1.453, 108.33±0.882, 113.67±1.453, 121.67±0.882, 116.67±1.453, 123.67±0.667 and
124.67±1.453 CTn whereas at 60th day of storage decreased to 88.33±0.882 (T0), 89.67±1.453
(T1), 91±1.0 (T2), 98.67±1.453 (T3), 93.67±1.453 (T4), 99.67±1.453 (T5), 104.67±1.453 (T6),
101.67±1.453 (T7), 106±1 (T8) and 109.67±1.453 CTn (T9). The highest color value was
reported in T9 116.77±1.050 CTn followed by T8 and T6 114.6±1.065, 111.87±1.039 CTn
whilst, lowest 93.67±1.109 CTn in T0. Similarly, leg meat nuggets color in T9, T8, T6 and T0
were 107.67±0.685, 106.5±0.776, 103.63±0.657 and 84.8±0.742 CTn, respectively at the
termination of storage period. The results further expounded linear decrease in color values with
progression of storage.
The current study depicted higher color value with increasing levels of dietary quercetin and α-
tocopherol whilst, storage progression resulted darker color and these findings are supported by
Wu et al. (2000) who reported that meat products coated with antioxidants, color decreases as a
function of storage owing to release of moisture from coating. Similarly, Luciano et al. (2009)
found that dietary supplementation of antioxidants improve color of lamb minced meat which
also enhances shelf stability than that of control. Previously, Berri et al. (2005) stated that color
differences of nuggets are mainly due to myoglobin content, water holding capacity and fatty
acid profile of the meat. They further reported lighter color of nuggets at initiation of storage that
become darker with storage. Likewise, Naveena et al. (2007) also found a declining trend for
CTn color value of nuggets with storage advancement. The mechanistic approach for this
conversion of lighter color to darker is attributed to the conversion of myglobin to
metmyoglobin. One of the researchers groups, Muela et al. (2014) delineated that 200 and 300
ppm vitamin E addition improves the visual color of lamb meat product. Likewise, Chandralekha
et al. (2012) stated that chicken meat balls incorporated with pomegranate rind powder extract as
an antioxidant resulted significantly (p<0.05) higher color score compared to control however,
this value dwindled with advancement of storage. Recently, Goliomytis et al. (2014) observed a
linear dose response for L* and a* color for broiler meat (p< 0.05) through dietary
supplementation of quercetin. Likewise, Jiang et al. (2007) reported that isoflavone @40 mg/kg
feed supplementation resulted in lighter color compared to control.
106
4.5.2. pH of nuggets
The mean squares indicted that pH of breast and leg meat nuggets varied significantly among
treatments whilst, differed non-significantly for years (Table 26). Furthermore, interactions
except treatments×storage days exhibited non-momentous variations. The means for pH
indicated minimum value in T0 6.03±0.021 whereas, maximum in T9 6.57±0.009 followed by
T8 6.56±0.009, T6 6.52±0.011, T7 6.37±0.011 and T5 6.33±0.012, respectively (Table 30). The
results for pH demonstrated a substantial increase that ranged from 6.22±0.028 at initiation to
6.26±0.027, 6.3±0.025, 6.36±0.022 and 6.4±0.021 at 15 th, 30th, 45th and 60th day of storage,
respectively. Likewise, the value of this trait for leg meat nuggets in T0, T9, T8, T6, T7 and T5
were 5.92±0.023, 6.47±0.009, 6.45±0.009, 6.43±0.010, 6.26±0.013 and 6.21±0.012,
correspondingly. Furthermore, pH of leg meat nuggets on different storage intervals ranged from
6.1±0.028 at start to 6.17±0.025, 6.2±0.025, 6.27±0.021, 6.3±0.021 in respective storage
intervals (Table 29 & 30).
The results of instant exploration are supported by Jiang et al. (2007) who reported that
isoflavone supplementation @40 mg/kg feed resulted higher pH for breast fillets is attributed to
improved antioxidant status of broiler meat. One of the researchers groups, Kumar and Tanwar
(2011) delineated higher pH for nuggets prepared from meat containing antioxidants. They
further reported that degradation of meat proteins yielded higher pH for the product during
storage. Likewise, Bhat et al. (2011) reported increase in pH due to metabolites accumulation
that are generated through bacterial action and deamination of proteins. Likewise, increasing pH
was also recorded by Chidanandaiah et al. (2009) in buffalo meat patties, Sureshkumar et al.
(2010) in buffalo meat sausages and Bhat et al. (2010) in chevon Harrisa. These findings
supported the present results in which higher pH was found in nuggets prepared from meat
containing antioxidants (quercetin and α-tocopherol) in feed than that of control.
4.5.3. Texture of nuggets
The mean squares showed that treatments imparted momentous effects on texture of broiler
nuggets however, years and interactions exhibited non-momentous differences (Table 31). The
results in Table 32 regarding shear force of broiler meat nuggets in different groups depicted that
amongst treatments, the minimum shear force for breast meat nuggets was recorded in T0 as
4.14±0.026kg whereas the maximum in T9 5.47±0.026. Moreover, the value for this trait in T8,
107
T6 and T7 were 5.35±0.027, 5.25±0.026, 5.13±0.025kg, respectively. Similarly there was a
momentous increase ranging from 4.73±0.054kg at initiation to 4.82±0.055kg (15 th day),
4.93±0.054kg (30th day), 5.04±0.054kg (45th day) and 5.09±0.054kg (60th day) was also reported
in nuggets. Likewise, the highest value for this trait in leg meat nuggets was reported in T9
5.38±0.020kg trailed by T8 and T6 5.25±0.020 & 5.17±0.020kg, respectively whilst, lowest in
T0 (control) 4.05±0.020kg at the termination of storage (Table 33).
The results of present study are consistent with the findings of Ruiz-Ramırez et al. (2005); they
reported that shear force of meat balls varied significantly with the progression of storage period.
Likewise, Morán et al. (2012) also recorded lower shear force among treatments containing
antioxidants as compared to control. The decrease in shear force of meat was attributed to the
proteolytic changes occurring in myofibril protein architecture. The previous findings by Huff
Lonergan et al. (2010) and Estévez, (2011) explained the mechanism for toughening of meat,
which is related to oxidation of myofibrilar proteins that promotes aggregation and cross-linking
of muscles. One of their peers, Bodas et al. (2012) documented that carnosic acid (phenolic
compound) deposition in animal muscles modified the texture in positive way and also lowered
the oxysterol degradation. Similarly, Andrés et al. (2013) reported higher hardness for lamb meat
that were fed on quercetin and flaxseed enriched diet however, chewiness varied non-
substantially among treatment. One of the researchers groups, Lund et al., (2008) stated that
oxidation of myosin protein caused aggregation which produces free radicals due to exposure of
oxidized myoglobin that further enhances the clustering of myosin protein cross-links (both
disulfide and di-tyrosine). These cross-links are specific for oxidation catalyzed by iron and
metmyoglobin leads to the toughening of meat based products during storage (Xiong et al.,
2009).
4.5.4. TBARS of nuggets
The mean squares in Table 31 revealed momentous differences for TBARS of antioxidant
enriched meat nuggets due to treatments. On the other hand, years and interactions except
treatments×days differed non-momentously.
108
Table 26. Mean squares for color and pH of broiler meat nuggets
SOV df Breast nuggets
Color
Leg nuggets
Color
Breast nuggets
pH
Leg nuggets
pH
Treatments (A) 9 2051.389** 2010.944** 1.16404** 1.11740**
Years (B) 1 354.30NS 843.40 NS 0.0102NS 0.0163NS
Days (C) 4 2025.275** 899.475** 0.32340** 0.37535**
B × C 4 6.225NS 19.375 NS 0.0019NS 0.00148NS
A× B 9 10.967 NS 12.433 NS 0.00730 NS 0.00234 NS
A × C 36 1.592** 1.847** 0.00380** 0.00408**
A ×B × C 36 2.311NS 1.472NS 0.00090NS 0.00075NS
Error 196 3.079 2.233 0.00077 0.00061
Total 299
**=Highly significant
*=Significant
NS=Non-significant
109
Table 27. Effect of treatments and storage on color of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 102.33±0.882 99.33±0.333 96.33±0.667 91.67±1.453 88.33±0.882
93.67±1.109f 2014 100.33±0.882 95.67±0.882 91.67±1.202 87.33±0.882 83.67±0.882
T1 2013 103.67±1.453 100.33±0.882 96.33±0.882 93.67±1.453 89.67±1.453 95.10±1.057e
2014 102.25±0.882 96.67±0.882 92.67±0.882 88.67±0.882 87.00±0.577
T2 2013 105.33±0.882 101.67±0.333 97.33±0.882 95.67±1.453 91.00±1.0 97.10±1.019de
2014 104.13±0.882 99.33±0.882 95.67±0.882 92.00±0.577 88.67±0.882
T3 2013 111.67±1.453 109.33±0.882 105.67±1.453 102.33±0.882 98.67±1.453 103.40±1.008cd
2014 109.00±1.155 104.00±0.577 100.33±0.882 97.67±0.667 95.33±0.882
T4 2013 108.33±0.882 105.33±0.667 100.67±0.667 95.67±0.882 93.67±1.453 99.90±0.993d
2014 106.33±0.882 101.67±0.667 98.33±0.882 96.33±0.882 92.67±0.882
T5 2013 113.67±1.453 111.33±0.882 106.67±0.882 102.00±1.00 99.67±1.453 106.13±0.967c
2014 112.00±1.155 108.67±0.882 105.67±0.882 102.33±1.202 99.33±1.453
T6 2013 121.67±0.882 116.33±0.882 113.00±1.155 107.33±0.333 104.67±1.453 111.87±1.039b
2014 118.33±0.882 114.00±0.577 110.33±0.882 107.33±0.333 105.67±0.882
T7 2013 116.67±1.453 115.33±0.667 109.67±1.453 105.67±1.453 101.67±1.453 109.40±1.047bc
2014 116.67±0.882 112.33±0.882 108.10±0.882 105.33±0.882 102.33±1.202
T8 2013 123.67±0.667 119.67±0.667 115.33±1.202 111.67±1.453 106.00±1.00 114.60±1.065ab
2014 120.33±0.882 117.33±0.882 114.20±0.882 110.10±0.882 106.10±0.882
T9 2013 124.67±1.453 121.67±0.667 117.67±1.453 114.67±0.667 109.67±1.453 116.77±1.050a
2014 123.33±0.882 120.00±1.155 116.33±0.882 111.33±0.882 108.25±0.882
Means 112.23±1.037a 108.5±1.073b 104.62±1.074c 101±1.062d 97.62±1.028e
110
Table 28. Effect of treatments and storage on color of leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 91.10±0.882 91.15±0.882 91.10±0.882 91.30±0.882 91.30±0.882 84.8±0.742f
2014 88.67±0.886 88.65±0.882 88.60±0.882 88.67±0.882 88.67±0.882
T1 2013 93.20±0.667 93.20±0.667 93.30±0.667 93.15±0.667 93.20±0.667 87.43±0.770e
2014 93.30±0.667 93.35±0.667 93.30±0.667 93.33±0.667 93.33±0.667
T2 2013 96.30±0.882 94.20±0.667 90.33±0.882 90.20±1.202 86.15±0.882 88.83±0.831de
2014 91.20±1.453 87.00±0.577 85.65±1.202 84.00±1.155 82.67±1.202
T3 2013 103.33±0.333 101.33±0.882 99.20±0.882 95.67±0.882 92.33±0.882 95.97±0.845cd
2014 99.00±0.577 95.30±0.882 93.00±1.155 90.33±0.882 90.00±0.577
T4 2013 98.15±0.333 95.67±0.333 93.05±0.882 90.00±0.577 88.00±1.00 91.37±0.762d
2014 94.35±0.882 91.35±0.882 90.70±1.202 87.33±0.882 84.67±0.882
T5 2013 106.00±0.577 102.15±0.333 99.67±0.882 96.60±0.333 92.60±1.453 98.47±0.771c
2014 101.65±0.882 99.25±0.882 97.35±0.882 95.67±0.882 93.33±1.202
T6 2013 109.33±0.667 107.00±0.577 105.33±1.453 101.55±0.882 98.70±1.202 103.63±0.657b
2014 106.30±0.882 104.40±0.882 102.30±0.882 101.60±1.202 99.67±1.202
T7 2013 108.67±0.667 106.67±0.667 102.67±1.453 99.65±0.882 94.65±1.453 100.9±0.921bc
2014 104.70±1.202 102.00±1.528 98.66±0.333 96.65±1.453 94.67±2.028
T8 2013 114.00±0.577 111.33±0.882 108.15±0.882 105.55±0.882 101.55±0.882 106.5±0.776ab
2014 109.35±0.333 106.30±0.882 104.30±0.882 102.70±0.882 101.33±0.882
T9 2013 113.33±0.333 111.00±0.577 109.00±1.000 106.60±0.667 104.60±0.882 107.67±0.685a
2014 111.65±1.202 108.20±0.882 106.15±0.882 104.00±0.577 101.67±0.882
Means 101.48±1.072a 98.92±1.071b 96.58±1.058c 94.2±1.048d 91.6±1.036e
111
Table 29. Effect of treatments and storage on pH of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 5.89±0.047 5.89±0.015 5.98±0.015 6.14±0.020 6.19±0.020
6.03±0.021e 2014 5.93±0.009 5.96±0.015 6.03±0.009 6.1±0.006 6.16±0.012
T1 2013 5.96±0.015 6.09±0.012 6.11±0.020 6.16±0.015 6.24±0.006
6.10±0.017d 2014 5.99±0.009 6.02±0.009 6.09±0.009 6.16±0.009 6.22±0.021
T2 2013 6.04±0.023 6.13±0.023 6.17±0.026 6.23±0.012 6.29±0.012
6.14±0.017cd 2014 6.01±0.009 6.03±0.009 6.09±0.006 6.15±0.009 6.23±0.012
T3 2013 6.18±0.026 6.23±0.015 6.27±0.020 6.32±0.017 6.36±0.020
6.27±0.011bc 2014 6.21±0.009 6.24±0.006 6.28±0.009 6.29±0.009 6.34±0.009
T4 2013 6.09±0.017 6.16±0.020 6.21±0.026 6.28±0.015 6.32±0.020
6.18±0.017c 2014 6.05±0.020 6.09±0.012 6.15±0.006 6.22±0.012 6.27±0.009
T5 2013 6.23±0.020 6.28±0.020 6.32±0.020 6.36±0.020 6.41±0.020
6.33±0.012b 2014 6.29±0.009 6.31±0.012 6.34±0.006 6.37±0.006 6.44±0.009
T6 2013 6.48±0.020 6.49±0.020 6.53±0.020 6.57±0.012 6.61±0.015
6.52±0.011ab 2014 6.44±0.015 6.46±0.012 6.53±0.012 6.55±0.009 6.59±0.012
T7 2013 6.27±0.020 6.32±0.017 6.36±0.020 6.43±0.009 6.45±0.020
6.37±0.011b 2014 6.32±0.015 6.34±0.003 6.38±0.009 6.42±0.009 6.44±0.009
T8 2013 6.51±0.018 6.52±0.020 6.55±0.020 6.58±0.020 6.63±0.015
6.56±0.009a 2014 6.48±0.018 6.53±0.010 6.55±0.007 6.58±0.009 6.62±0.012
T9 2013 6.53±0.023 6.56±0.020 6.58±0.020 6.61±0.020 6.65±0.020
6.57±0.009a 2014 6.49±0.009 6.54±0.012 6.56±0.018 6.59±0.009 6.62±0.012
Means 6.22±0.028e 6.26±0.027d 6.30±0.025c 6.36±0.022b 6.40±0.021a
112
Table 30. Effect of treatments and storage on pH of leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 5.72±0.009 5.78±0.015 5.86±0.015 6.04±0.020 6.07±0.015
5.92±0.023e 2014 5.80±0.012 5.91±0.012 5.94±0.015 6.02±0.015 6.09±0.012
T1 2013 5.84±0.015 5.97±0.020 5.99±0.020 6.06±0.020 6.11±0.017
6.00±0.018d 2014 5.86±0.009 5.95±0.015 6.01±0.012 6.09±0.009 6.13±0.009
T2 2013 5.91±0.017 6.02±0.009 6.05±0.020 6.12±0.026 6.16±0.020
6.06±0.016cd 2014 5.94±0.009 6.04±0.009 6.07±0.009 6.14±0.012 6.17±0.012
T3 2013 6.06±0.020 6.13±0.023 6.15±0.020 6.22±0.020 6.24±0.017
6.17±0.013bc 2014 6.09±0.009 6.14±0.009 6.18±0.009 6.24±0.009 6.27±0.006
T4 2013 5.97±0.020 6.05±0.023 6.09±0.015 6.18±0.020 6.20±0.020
6.10±0.016c 2014 5.99±0.009 6.06±0.009 6.11±0.009 6.17±0.009 6.23±0.009
T5 2013 6.11±0.017 6.17±0.009 6.20±0.020 6.27±0.010 6.30±0.015
6.21±0.012b 2014 6.14±0.009 6.17±0.012 6.19±0.012 6.24±0.007 6.29±0.009
T6 2013 6.36±0.020 6.39±0.020 6.41±0.015 6.46±0.007 6.50±0.012
6.43±0.010a 2014 6.36±0.009 6.39±0.009 6.42±0.012 6.46±0.007 6.50±0.007
T7 2013 6.14±0.012 6.21±0.015 6.24±0.026 6.31±0.023 6.33±0.023
6.26±0.013b 2014 6.19±0.009 6.22±0.012 6.25±0.009 6.31±0.012 6.36±0.009
T8 2013 6.38±0.020 6.41±0.015 6.44±0.018 6.48±0.026 6.51±0.020
6.45±0.009a 2014 6.39±0.003 6.42±0.009 6.46±0.006 6.49±0.006 6.51±0.006
T9 2013 6.40±0.010 6.44±0.006 6.49±0.009 6.51±0.009 6.55±0.012
6.47±0.009a 2014 6.41±0.012 6.45±0.009 6.47±0.009 6.49±0.009 6.53±0.012
Means 6.1±0.028e 6.17±0.025d 6.20±0.025c 6.27±0.021b 6.30±0.021a
113
At initiation, means for TBARS among treatments varied from 0.32±0.012 to 1.30±0.021 and
0.45±0.021 to 1.42±0.018 mg MDA/kg meat however, at termination varied from 1.24±0.029 to
2.51±0.054 and 1.36±0.015 to 2.54±0.029 mg of MDA/kg meat in the year 2013 and 2014,
respectively. The means reported lowest TBARS value in T9 0.77±0.062 mg of MDA/kg meat
followed by T8 and T6 0.86±0.065 & 0.89±0.066 mg of MDA/kg, respectively whilst, highest in
T0 as 1.83±0.079 mg of MDA/kg meat (Table 34). Likewise, TBARS for leg meat nuggets on
start of storage varied from 0.63±0.012 to 1.64±0.015 and 0.67±0.009 to 1.67±0.015 that were
also increased at termination of storage from 1.61±0.018 to 2.83±0.023 and 1.64±0.020 to
2.88±0.017 mg of MDA/kg meat in respective experimental years (Table 35). The means of
TBARS for leg meat nuggets in T9, T8, T6 and T0 were 1.09±0.064, 1.18±0.066, 1.22±0.062,
2.13±0.083 mg of MDA/kg. The production of malonaldehydes (MDA) of tested product
increased linearly as a function of storage. It is evident from current exploration that MDA
generation was lower in breast meat nuggets compared to the leg meat however, its production
increased significantly with storage advancement.
The results regarding oxidative stability of nuggets are in accordance with Muthukumar et al.
(2012) who determined the TBARS value of patties made from antioxidants enriched broiler
meat. They further reported that dietary antioxidants deposited in the muscles which retarded the
rate of free radical formation in resultant product with storage. Likewise, Capitani et al. (2012)
also elucidated that antioxidant mixture containing quercetin and rutin @ 0.05 g/ 100 g meat
impart significant effect on inhibiting the MDA compounds formation during shelf stability
study of sausages in which sodium erythrorbate was replaced with natural antioxidants. They
further reported an increasing trend for malonaldehydes production as a function of storage. One
of the researchers groups, Kumar and Tanwar (2011) documented that chicken meat nuggets
containing antioxidants decrease the production of MDA however, the value of TBARS
increased significantly with storage. Additionally, TBARS in tested nuggets ranged from
0.347±0.003 to 0.889±0.226. The instant results regarding lipid stability of meat product by
antioxidants are equally supported by the views of Chandralekha et al. (2012); they observed
lower TBARS in chicken meat balls during refrigerated storage study in which pomegranate rind
powder extract was added as an antioxidant source. Likewise, Sampaio et al. (2012) also found
that combination of antioxidants exhibits better antiradical activity and reduces the TBARS &
114
Table 31. Mean squares for texture and TBARS of broiler meat nuggets
SOV df Breast nuggets
texture
Leg nuggets
texture
Breast nuggets
TBARS
Leg nuggets
TBARS
Treatments (A) 9 5.75046** 5.76469** 3.69257** 3.38786**
Years (B) 1 0.27000NS 0.12080NS 0.92840NS 0.25190NS
Days (C) 4 1.32955** 0.81183** 10.95520** 10.55325**
B × C 4 0.01275NS 0.00145NS 0.00095NS 0.00263NS
A× B 9 0.00000NS 0.00000NS 0.00468NS 0.00073NS
A × C 36 0.00000 NS 0.00000 NS 0.01918** 0.03576**
A ×B × C 36 0.00000NS 0.00000NS 0.00112NS 0.00074NS
Error 196 0.00099 0.00097 0.00112 0.00079
Total 299
**=Highly significant
*=Significant
NS=Non-significant
115
Table 32. Effect of treatments and storage on texture of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 3.97±0.012 4.09±0.012 4.17±0.012 4.28±0.012 4.34±0.012
4.14±0.026f 2014 3.92±0.012 3.98±0.012 4.13±0.012 4.24±0.012 4.28±0.012
T1 2013 4.17±0.015 4.29±0.015 4.37±0.015 4.48±0.017 4.54±0.017
4.34±0.026e 2014 4.12±0.015 4.18±0.015 4.33±0.015 4.44±0.017 4.48±0.017
T2 2013 4.45±0.023 4.57±0.020 4.65±0.027 4.76±0.022 4.82±0.022
4.62±0.026de 2014 4.40±0.021 4.46±0.020 4.61±0.023 4.72±0.022 4.76±0.022
T3 2013 4.84±0.012 4.96±0.021 5.04±0.022 5.14±0.021 5.20±0.021
5.00±0.027cd 2014 4.79±0.024 4.85±0.022 5.00±0.020 5.10±0.021 5.14±0.021
T4 2013 4.64±0.015 4.76±0.015 4.84±0.015 4.94±0.021 5.00±0.021
4.80±0.026d 2014 4.59±0.015 4.65±0.015 4.8±0.015 4.90±0.021 4.94±0.021
T5 2013 4.91±0.022 5.03±0.022 5.11±0.024 5.21±0.021 5.27±0.021
5.07±0.026c 2014 4.86±0.025 4.92±0.021 5.07±0.021 5.17±0.021 5.21±0.021
T6 2013 5.09±0.026 5.21±0.020 5.29±0.020 5.39±0.021 5.45±0.021
5.25±0.026b 2014 5.04±0.021 5.10±0.022 5.25±0.020 5.35±0.021 5.39±0.021
T7 2013 4.96±0.018 5.08±0.018 5.16±0.018 5.27±0.018 5.33±0.018
5.13±0.025bc 2014 4.91±0.018 4.97±0.018 5.12±0.018 5.23±0.018 5.27±0.018
T8 2013 5.18±0.017 5.30±0.025 5.38±0.025 5.49±0.022 5.55±0.022
5.35±0.027ab 2014 5.13±0.024 5.19±0.022 5.34±0.022 5.45±0.022 5.49±0.022
T9 2013 5.31±0.015 5.43±0.015 5.51±0.015 5.61±0.015 5.67±0.015
5.47±0.026a 2014 5.26±0.015 5.32±0.015 5.47±0.015 5.57±0.015 5.61±0.015
Means 4.73±0.054e 4.82±0.055d 4.93±0.054c 5.04±0.054b 5.09±0.054a
116
Table 33. Effect of treatments and storage on texture of leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 3.92±0.017 4.01±0.017 4.07±0.017 4.14±0.017 4.23±0.017
4.05±0.020f 2014 3.89±0.017 3.96±0.017 4.04±0.017 4.10±0.017 4.18±0.017
T1 2013 4.12±0.023 4.21±0.023 4.27±0.023 4.34±0.023 4.43±0.023
4.25±0.021e 2014 4.09±0.023 4.16±0.023 4.24±0.023 4.30±0.023 4.38±0.023
T2 2013 4.37±0.012 4.46±0.012 4.52±0.012 4.59±0.014 4.68±0.012
4.50±0.020de 2014 4.34±0.012 4.41±0.012 4.49±0.012 4.55±0.012 4.63±0.012
T3 2013 4.78±0.017 4.87±0.017 4.93±0.017 5.00±0.017 5.09±0.017
4.91±0.020cd 2014 4.75±0.017 4.82±0.017 4.90±0.017 4.96±0.017 5.04±0.017
T4 2013 4.55±0.021 4.64±0.019 4.70±0.018 4.77±0.012 4.86±0.025
4.68±0.020d 2014 4.52±0.022 4.59±0.024 4.67±0.022 4.73±0.020 4.81±0.026
T5 2013 4.86±0.029 4.95±0.029 5.01±0.029 5.08±0.029 5.17±0.025
4.99±0.021c 2014 4.83±0.029 4.9±0.029 4.98±0.029 5.04±0.029 5.12±0.021
T6 2013 5.03±0.015 5.12±0.015 5.18±0.015 5.25±0.015 5.34±0.015
5.17±0.020b 2014 5.00±0.015 5.07±0.015 5.15±0.015 5.21±0.015 5.29±0.015
T7 2013 4.90±0.012 4.99±0.012 5.05±0.012 5.12±0.012 5.21±0.012
5.04±0.020bc 2014 4.87±0.012 4.94±0.012 5.02±0.012 5.08±0.012 5.16±0.012
T8 2013 5.11±0.018 5.20±0.018 5.26±0.018 5.33±0.018 5.42±0.018
5.25±0.020ab 2014 5.08±0.018 5.15±0.018 5.23±0.018 5.29±0.018 5.37±0.018
T9 2013 5.24±0.018 5.33±0.018 5.39±0.018 5.46±0.018 5.55±0.018
5.38±0.020a 2014 5.21±0.018 5.28±0.018 5.36±0.018 5.42±0.018 5.5±0.018
Means 4.67±0.054e 4.75±0.054d 4.82±0.054c 4.89±0.054b 4.97±0.054a
117
Table 34. TBARS of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 1.30±0.021 1.44±0.032 1.72±0.018 2.03±0.015 2.51±0.054
1.83±0.079a 2014 1.42±0.018 1.52±0.026 1.75±0.021 2.11±0.017 2.54±0.029
T1 2013 0.98±0.015 1.14±0.015 1.50±0.023 1.73±0.018 2.26±0.026
1.56±0.084b 2014 1.07±0.018 1.23±0.023 1.54±0.026 1.81±0.017 2.34±0.017
T2 2013 0.89±0.015 1.04±0.018 1.34±0.012 1.63±0.023 2.11±0.023
1.47±0.083bc 2014 1.01±0.015 1.12±0.017 1.55±0.021 1.78±0.012 2.22±0.009
T3 2013 0.64±0.015 0.76±0.022 0.98±0.015 1.22±0.021 1.63±0.023
1.1±0.067b 2014 0.75±0.012 0.88±0.018 1.11±0.022 1.32±0.022 1.75±0.017
T4 2013 0.85±0.009 0.99±0.012 1.24±0.018 1.54±0.020 2.04±0.021
1.39±0.080c 2014 0.96±0.015 1.09±0.018 1.38±0.017 1.64±0.032 2.16±0.022
T5 2013 0.59±0.021 0.73±0.015 0.96±0.012 1.18±0.018 1.59±0.012
1.06±0.067de 2014 0.68±0.022 0.85±0.021 1.06±0.021 1.26±0.026 1.72±0.018
T6 2013 0.44±0.015 0.59±0.012 0.75±0.012 1.02±0.021 1.41±0.020
0.89±0.066ef 2014 0.51±0.015 0.69±0.015 0.85±0.026 1.12±0.020 1.54±0.015
T7 2013 0.54±0.018 0.66±0.012 0.78±0.018 1.06±0.023 1.52±0.023
0.98±0.067e 2014 0.65±0.023 0.75±0.017 0.96±0.015 1.24±0.015 1.65±0.023
T8 2013 0.40±0.007 0.54±0.015 0.72±0.012 0.99±0.015 1.35±0.018
0.86±0.065f 2014 0.52±0.023 0.64±0.022 0.86±0.017 1.13±0.009 1.49±0.012
T9 2013 0.32±0.012 0.44±0.018 0.66±0.009 0.86±0.017 1.24±0.029
0.77±0.062g 2014 0.45±0.021 0.57±0.026 0.84±0.02 0.99±0.021 1.36±0.015
Means 0.75±0.039e 0.88±0.039d 1.13±0.044c 1.38±0.047b 1.82±0.052a
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Table 35. TBARS of leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 1.64±0.015 1.69±0.023 1.98±0.017 2.36±0.024 2.83±0.023
2.13±0.083a 2014 1.67±0.015 1.78±0.021 2.04±0.015 2.42±0.018 2.88±0.017
T1 2013 1.27±0.012 1.45±0.012 1.71±0.018 2.08±0.009 2.55±0.020
1.84±0.085b 2014 1.32±0.018 1.51±0.015 1.75±0.021 2.12±0.025 2.61±0.017
T2 2013 1.15±0.018 1.26±0.012 1.73±0.023 1.93±0.023 2.45±0.018
1.75±0.086bc 2014 1.23±0.009 1.45±0.020 1.78±0.009 1.99±0.012 2.51±0.018
T3 2013 0.92±0.015 1.06±0.017 1.28±0.018 1.54±0.020 1.94±0.012
1.37±0.068d 2014 0.96±0.015 1.12±0.015 1.35±0.015 1.58±0.017 1.99±0.015
T4 2013 1.15±0.018 1.26±0.009 1.55±0.017 1.85±0.012 1.92±0.017
1.57±0.058c 2014 1.20±0.018 1.31±0.024 1.60±0.020 1.88±0.017 1.99±0.015
T5 2013 0.89±0.012 1.07±0.009 1.29±0.015 1.47±0.012 1.93±0.011
1.36±0.068d 2014 0.93±0.009 1.13±0.012 1.34±0.015 1.54±0.022 1.99±0.012
T6 2013 0.76±0.009 0.96±0.009 1.16±0.020 1.32±0.023 1.76±0.015
1.22±0.062e 2014 0.84±0.015 1.02±0.015 1.22±0.015 1.35±0.015 1.78±0.018
T7 2013 0.85±0.009 0.91±0.012 1.14±0.015 1.40±0.015 1.81±0.009
1.25±0.065de 2014 0.89±0.02 0.99±0.015 1.22±0.018 1.46±0.009 1.85±0.023
T8 2013 0.72±0.02 0.87±0.015 1.09±0.015 1.35±0.021 1.72±0.009
1.18±0.066f 2014 0.78±0.015 0.94±0.015 1.15±0.017 1.43±0.018 1.78±0.018
T9 2013 0.63±0.012 0.77±0.012 1.05±0.017 1.24±0.015 1.61±0.018
1.09±0.064g 2014 0.67±0.009 0.85±0.023 1.12±0.012 1.31±0.021 1.64±0.024
Means 1.02±0.038e 1.17±0.036d 1.43±0.040c 1.68±0.047b 2.08±0.051a
119
hexanal generation in cooked chicken meat. Similarly, Juntachote et al. (2006) narrated that
antioxidants are effective in minimizing lipid oxidation in meat products by limiting the
generation of MDA. One of the peers, Naveena et al. (2007) showed that dietary α-tocpherol
administration to broiler birds increased the storage stability of chicken patties by diminishing
the generation of MDA compounds. One of the scientists groups, Sullivan et al. (2004) found
that antioxidant enriched broiler meat nuggets showed lower (p<0.01) TBARS than that of
control meat nuggets.
4.6. Sensory evaluation of nuggets
The functional meat nuggets were assessed for various sensory attributes including appearance,
flavor, taste and overall acceptability. The mean squares pertaining to appearance, flavor, taste
and overall acceptability of antioxidant enriched broiler breast and leg meat nuggets showed
significant variations due to treatments and storage intervals however, non-substantial
differences were noticed for years and interactions except treatments×storage (Table 36 & 41). It
is evident from the results in Table 37 that maximum appearance score for breast meat nuggets
was assigned to T9 (meat of birds fed on 300mg quercetin & 300 mg of α-tocopherol/kg feed)
7.7±0.079 followed by T8 (meat of birds fed on 300mg quercetin & 225 mg of α-tocpherol/kg
feed) 7.61±0.080 and T6 (meat of birds fed on 200 mg quercetin and 300 mg of α-tocpherol/kg
feed) 7.58±0.080 whilst, minimum score to T0 (control) 6.78±0.093 at the termination of storage
study. Likewise, the appearance score for leg meat nuggets in T9, T8, T6 and T0 were
7.35±0.081, 7.28±0.079, 7.23±0.079, 6.48±0.092, correspondingly (Table 38). Additionally,
storage led to a decline in appearance scores i.e. 7.51±0.039 for leg meat nuggets at start to
7.34±0.033, 7.03±0.032, 6.58±0.039, 6.38±0.043 at 15 th, 30th, 45th and 60th day, respectively.
Similarly, flavor of breast meat nuggets depicted that amongst treatments the lowest score was
recorded in T0 as 6.64±0.103 while, the maximum 7.51±0.093 in T9 however, the value for this
trait in T8, T6,T7 and T5 were 7.43±0.089, 7.33±0.087, 7.26±0.089 and 7.15±0.089, respectively
(Table 39). Likewise, the highest flavor score for leg meat nuggets was also reported in T9
7.21±0.080 trailed by T8, T6, T7 and T5 as 7.11±0.082, 7±0.084, 6.88±0.082 &6.76±0.084,
respectively whilst, lowest in T0 (control) 6.18±0.107 at termination of storage. Furthermore,
storage resulted a decline in flavor rating from initiation to termination of study for breast and
120
leg meat nuggets as 8.1±0.009 to 5.82±0.017 and 7.72±0.044 to 5.25±0.079, correspondingly
(Table 40).
In treatments, the minimum taste score for breast meat nuggets was noticed in T0 (control)
6.78±0.091whereas, the maximum in T9 as 7.8±0.089 trailed by T8, T6 and T7 as 7.74±0.085,
7.65±0.086 & 7.54±0.080, respectively. The results further demonstrated that taste of breast meat
nuggets differed during storage from 7.95±0.044 at start to 7.64±0.044, 7.45±0.051, 6.97±0.037,
6.69±0.051 on 15th, 30th, 45thand 60th day of storage (Table 42). Likewise, the value for this trait
in leg meat nuggets for T9, T8, T6, T7 and T0 groups were 7.53±0.090, 7.45±0.086, 7.37±0.087,
7.25±0.089 and 6.67±0.100, correspondingly. Additionally, nuggets taste during storage varied
from 7.72±0.038 at beginning to 7.51±0.030 (15th day), 7.2±0.037 (30th day), 6.71±0.038 (45th
day) and 6.43±0.042 (60th day) of storage (Table 43).
At initiation, overall acceptability scores for breast meat nuggets among treatments varied from
7.35±0.029 to 8.30±0.029 and 7.25±0.027 to 8.20 ±0.034 however, at termination ranged from
5.95±0.018 to 7.02±0.017 and 5.85±0.034 to 6.92±0.017 in the experimental years 2013 and
2014. The means for overall acceptability score showed the highest response in T9 7.64±0.090
followed by T8 and T6 7.56±0.091 & 7.48±0.098, whilst lowest in T0 6.7±0.096. The findings
further showed that overall acceptability for breast meat nuggets differed from 7.81±0.041 at
initiation to 7.58±0.043, 7.34±0.033, 6.84±0.036 and 6.41±0.046 on 15 th, 30th, 45th and 60th day
of storage (Table 44). Similarly, overall acceptability score for leg meat nuggets in T9, T8, T6
and T0 were 7.39±0.082, 7.29±0.082, 7.24±0.083 and 6.47±0.096. Furthermore, overall
acceptability ranged from 77.55±0.036 at beginning to 7.31±0.037 (15th day), 6.96±0.041 (30th
day), 6.54±0.03745th day) and 6.36±0.046 (60th day) of storage (Table 45). The overall
acceptability of nuggets decreased with advancement of storage period. The results further
showed that dietary supplementation of antioxidants enhanced the storage stability of tested
nuggets resulted higher consumer acceptability.
Current results regarding hedonic response of nuggets are in conformity with the findings of Biswas
et al. (2004) who determined the effect of antioxidant coatings and storage on sensory profile of
patties. They observed patties coated with antioxidants exhibited higher sensory score than that
of patties from control. One of the scientists groups, Wu et al. (2000) reported that meat products
containing antioxidant resulted higher sensory response compared of control. Likewise, Kumar
121
Table 36. Mean squares for appearance and flavor of nuggets
SOV df Breast nuggets
appearance
Leg nuggets
appearance
Breast nuggets
flavor Leg nuggets flavor
Treatments (A) 9 2.76292** 2.58189** 2.68226** 3.53864**
Years (B) 1 0.18900NS 0.36470NS 0.48000NS 0.48000NS
Days (C) 4 14.78545** 14.05663** 18.49450** 16.46875**
B × C 4 0.00223NS 0.01095NS 0.01125NS 0.01125NS
A× B 9 0.00252NS 0.0000NS 0.00000NS 0.00000NS
A × C 36 0.03244** 0.02034** 0.01235** 0.02641**
A ×B × C 36 0.00084NS 0.00001NS 0.00000NS 0.00000NS
Error 196 0.00434 0.00178 0.00346 0.00324
Total 299
**=Highly significant
*=Significant
NS=Non-significant
122
Table 37. Effect of treatments and storage on appearance of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Days
Means Day 0 Day 15 Day 30 Day 45 Day 60
T0 2013 7.35±0.029 7.25±0.029 6.95±0.029 6.42±0.017 6.05±0.029
6.78±0.093e 2014 7.30±0.029 7.18±0.060 6.90±0.058 6.38±0.060 6.00±0.029
T1 2013 7.50±0.029 7.45±0.029 7.13±0.009 6.45±0.140 6.19±0.007
6.92±0.099de 2014 7.45±0.029 7.40±0.029 7.10±0.029 6.40±0.029 6.15±0.029
T2 2013 7.60±0.029 7.45±0.029 7.27±0.017 6.69±0.021 6.37±0.015
7.05±0.086d 2014 7.53±0.017 7.38±0.017 7.23±0.033 6.65±0.029 6.35±0.029
T3 2013 7.90±0.032 7.72±0.044 7.45±0.029 6.90±0.058 6.70±0.003
7.29±0.085c 2014 7.75±0.126 7.60±0.029 7.43±0.033 6.85±0.029 6.62±0.044
T4 2013 7.60±0.058 7.45±0.029 7.40±0.026 6.84±0.019 6.70±0.012
7.19±0.069cd 2014 7.62±0.060 7.45±0.050 7.37±0.044 6.82±0.060 6.65±0.029
T5 2013 7.95±0.050 7.75±0.015 7.51±0.035 6.95±0.029 6.75±0.015
7.36±0.086bc 2014 7.90±0.050 7.72±0.017 7.47±0.044 6.88±0.060 6.72±0.060
T6 2013 8.20±0.029 7.95±0.029 7.63±0.044 7.18±0.044 7.05±0.029
7.58±0.080b 2014 8.10±0.029 7.90±0.029 7.60±0.029 7.13±0.017 7.02±0.044
T7 2013 7.95±0.029 7.75±0.029 7.55±0.020 6.95±0.050 6.87±0.017
7.39±0.080bc 2014 7.88±0.044 7.68±0.017 7.53±0.060 6.92±0.060 6.85±0.050
T8 2013 8.27±0.033 7.95±0.029 7.67±0.033 7.25±0.017 7.10±0.029
7.61±0.080ab 2014 8.15±0.029 7.90±0.029 7.60±0.029 7.20±0.029 7.02±0.044
T9 2013 8.30±0.029 8.00±0.029 7.80±0.029 7.35±0.029 7.15±0.029
7.70±0.079a 2014 8.25±0.050 7.95±0.029 7.75±0.076 7.30±0.029 7.12±0.060
Means 7.83±0.042a 7.64±0.033b 7.42±0.033c 6.88±0.039d 6.67±0.047e
123
Table 38. Effect of treatments and storage on appearance of leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 7.15±0.029 6.90±0.029 6.60±0.029 6.11±0.029 5.80±0.029
6.48±0.092e 2014 7.07±0.033 6.85±0.029 6.50±0.029 6.06±0.029 5.75±0.029
T1 2013 7.18±0.017 7.10±0.009 6.82±0.017 6.14±0.007 5.96±0.009
6.61±0.091de 2014 7.08±0.017 7.05±0.009 6.72±0.017 6.09±0.007 5.91±0.009
T2 2013 7.25±0.029 7.13±0.015 6.90±0.029 6.40±0.006 6.10±0.029
6.72±0.081d 2014 7.15±0.029 7.08±0.015 6.80±0.029 6.35±0.006 6.05±0.029
T3 2013 7.60±0.009 7.38±0.017 7.07±0.033 6.65±0.029 6.47±0.017
7.00±0.078c 2014 7.50±0.009 7.33±0.017 6.97±0.033 6.6±0.029 6.42±0.017
T4 2013 7.40±0.009 7.30±0.015 7.03±0.017 6.55±0.003 6.40±0.029
6.90±0.073cd 2014 7.30±0.009 7.25±0.015 6.93±0.017 6.50±0.003 6.35±0.029
T5 2013 7.60±0.015 7.42±0.017 7.13±0.017 6.60±0.029 6.51±0.007
7.02±0.080c 2014 7.50±0.015 7.37±0.017 7.03±0.017 6.55±0.029 6.46±0.007
T6 2013 7.85±0.029 7.60±0.029 7.30±0.015 6.90±0.029 6.68±0.044
7.23±0.079b 2014 7.75±0.029 7.55±0.029 7.20±0.015 6.85±0.029 6.63±0.044
T7 2013 7.70±0.029 7.47±0.033 7.22±0.015 6.69±0.007 6.60±0.029
7.10±0.079bc 2014 7.60±0.029 7.42±0.033 7.12±0.015 6.64±0.007 6.55±0.029
T8 2013 7.90±0.058 7.65±0.029 7.33±0.017 6.95±0.029 6.75±0.029
7.28±0.079ab 2014 7.80±0.058 7.60±0.029 7.23±0.017 6.90±0.029 6.70±0.029
T9 2013 8.00±0.026 7.72±0.017 7.40±0.043 7.02±0.019 6.80±0.029
7.35±0.081a 2014 7.90±0.026 7.67±0.017 7.30±0.043 6.97±0.019 6.75±0.029
Means 7.51±0.039a 7.34±0.033b 7.03±0.032c 6.58±0.039d 6.38±0.043e
124
Table 39. Effect of treatments and storage on flavor of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 7.33±0.033 7.08±0.044 6.85±0.029 6.30±0.058 5.82±0.017
6.64±0.103f 2014 7.23±0.033 7.03±0.044 6.75±0.029 6.25±0.058 5.72±0.017
T1 2013 7.43±0.033 7.20±0.029 6.95±0.029 6.40±0.029 5.95±0.029
6.75±0.101e 2014 7.33±0.033 7.15±0.029 6.85±0.029 6.35±0.029 5.85±0.029
T2 2013 7.50±0.029 7.25±0.029 7.00±0.029 6.53±0.017 6.07±0.033
6.83±0.096de 2014 7.40±0.029 7.20±0.029 6.90±0.029 6.48±0.017 5.97±0.033
T3 2013 7.80±0.029 7.43±0.033 7.23±0.044 6.72±0.017 6.40±0.058
7.08±0.094cd 2014 7.70±0.029 7.38±0.033 7.13±0.044 6.67±0.017 6.30±0.058
T4 2013 7.60±0.029 7.30±0.029 7.10±0.058 6.63±0.017 6.23±0.033
6.93±0.091d 2014 7.50±0.029 7.25±0.029 7.00±0.058 6.58±0.017 6.13±0.033
T5 2013 7.80±0.023 7.53±0.044 7.30±0.029 6.8±0.029 6.50±0.058
7.15±0.089c 2014 7.70±0.023 7.48±0.044 7.20±0.029 6.75±0.029 6.40±0.058
T6 2013 7.97±0.017 7.73±0.033 7.48±0.017 6.95±0.05 6.73±0.044
7.33±0.087b 2014 7.87±0.017 7.68±0.033 7.38±0.017 6.90±0.05 6.63±0.044
T7 2013 7.91±0.012 7.65±0.003 7.45±0.003 6.90±0.003 6.62±0.044
7.26±0.089bc 2014 7.81±0.012 7.60±0.003 7.35±0.003 6.85±0.003 6.52±0.044
T8 2013 8.07±0.018 7.82±0.017 7.61±0.007 7.10±0.029 6.77±0.033
7.43±0.089ab 2014 7.97±0.018 7.77±0.017 7.51±0.007 7.05±0.029 6.67±0.033
T9 2013 8.20±0.009 7.90±0.029 7.65±0.003 7.20±0.026 6.82±0.109
7.51±0.093a 2014 8.10±0.009 7.85±0.029 7.55±0.003 7.15±0.026 6.72±0.109
Means 7.71±0.036a 7.46±0.035b 7.21±0.036c 6.73±0.037d 6.34±0.046e
125
Table 40. Effect of treatments and storage on flavor of broiler leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 6.83±0.033 6.70±0.029 6.37±0.044 5.94±0.047 5.25±0.079
6.18±0.107f 2014 6.73±0.033 6.65±0.029 6.27±0.044 5.84±0.047 5.20±0.079
T1 2013 6.93±0.044 6.80±0.029 6.50±0.029 6.10±0.029 5.53±0.044
6.33±0.094ef 2014 6.83±0.044 6.75±0.029 6.40±0.029 6.00±0.029 5.48±0.044
T2 2013 7.05±0.029 6.88±0.017 6.63±0.033 6.20±0.029 5.70±0.029
6.45±0.091e 2014 6.95±0.029 6.83±0.017 6.53±0.033 6.10±0.029 5.65±0.029
T3 2013 7.20±0.029 7.05±0.029 6.82±0.033 6.40±0.029 5.90±0.029
6.63±0.088d 2014 7.10±0.029 7.00±0.029 6.72±0.033 6.30±0.029 5.85±0.029
T4 2013 7.13±0.017 6.95±0.029 6.70±0.029 6.32±0.044 5.80±0.029
6.54±0.088de 2014 7.03±0.017 6.90±0.029 6.60±0.029 6.22±0.044 5.75±0.029
T5 2013 7.32±0.017 7.20±0.029 6.90±0.029 6.48±0.017 6.10±0.058
6.76±0.084cd 2014 7.22±0.017 7.15±0.029 6.80±0.029 6.38±0.017 6.05±0.058
T6 2013 7.62±0.017 7.42±0.017 7.10±0.029 6.72±0.017 6.37±0.017
7.00±0.084b 2014 7.52±0.017 7.37±0.017 7.00±0.029 6.62±0.017 6.32±0.017
T7 2013 7.43±0.033 7.32±0.017 7.02±0.044 6.60±0.003 6.25±0.029
6.88±0.082c 2014 7.33±0.033 7.27±0.017 6.92±0.044 6.50±0.003 6.20±0.029
T8 2013 7.72±0.06 7.52±0.015 7.20±0.029 6.80±0.029 6.53±0.017
7.11±0.082ab 2014 7.62±0.06 7.47±0.015 7.10±0.029 6.70±0.029 6.48±0.017
T9 2013 7.82±0.044 7.58±0.015 7.32±0.044 6.91±0.007 6.65±0.029
7.21±0.080a 2014 7.72±0.044 7.53±0.015 7.22±0.044 6.81±0.007 6.60±0.029
Means 7.26±0.042a 7.12±0.039b 6.81±0.039c 6.40±0.040d 5.98±0.056e
126
Table 41. Mean squares for taste and overall acceptability of breast and leg meat nuggets
SOV df Breast nuggets
taste
Leg nuggets
taste
Breast nuggets overall
acceptability
Leg nuggets overall
acceptability
Treatments (A) 9 3.82759** 2.51086** 2.96033** 2.87733**
Years (B) 1 0.48000NS 0.60750 NS 0.48000NS 0.48000NS
Days (C) 4 15.42165** 17.46655** 19.42100** 15.12555**
B × C 4 0.01125NS 0.02625 NS 0.01125NS 0.01125NS
A× B 9 0.00000NS 0.00000 NS 0.00000NS 0.00000NS
A × C 36 0.02703** 0.01484** 0.01967** 0.01969**
A ×B × C 36 0.00000NS 0.00000 NS 0.00000NS 0.00000NS
Error 196 0.00323 0.00288 0.00335 0.00202
Total 299
**=Highly significant
*=Significant
NS=Non-significant
127
Table 42. Effect of treatments and storage on taste of broiler breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 7.45±0.029 7.15±0.029 6.92±0.044 6.48±0.017 6.08±0.044
6.78±0.091f 2014 7.35±0.029 7.10±0.029 6.82±0.044 6.43±0.017 5.98±0.044
T1 2013 7.63±0.033 7.25±0.029 7.00±0.029 6.60±0.029 6.27±0.06
6.91±0.090e 2014 7.53±0.033 7.20±0.029 6.90±0.029 6.55±0.029 6.17±0.06
T2 2013 7.68±0.017 7.40±0.05 7.08±0.044 6.85±0.029 6.40±0.058
7.04±0.084de 2014 7.58±0.017 7.35±0.05 6.98±0.044 6.80±0.029 6.30±0.058
T3 2013 8.00±0.029 7.62±0.06 7.62±0.017 7.00±0.029 6.72±0.044
7.35±0.087cd 2014 7.90±0.029 7.57±0.06 7.52±0.017 6.95±0.029 6.62±0.044
T4 2013 7.80±0.029 7.48±0.06 7.20±0.029 6.88±0.017 6.55±0.029
7.14±0.082d 2014 7.70±0.029 7.43±0.06 7.10±0.029 6.83±0.017 6.45±0.029
T5 2013 8.05±0.029 7.72±0.044 7.68±0.017 7.07±0.017 6.85±0.029
7.43±0.083c 2014 7.95±0.029 7.67±0.044 7.58±0.017 7.02±0.017 6.75±0.029
T6 2013 8.30±0.029 8.00±0.029 7.83±0.017 7.20±0.029 7.12±0.044
7.65±0.086b 2014 8.20±0.029 7.95±0.029 7.73±0.017 7.15±0.029 7.02±0.044
T7 2013 8.15±0.029 7.85±0.029 7.75±0.003 7.14±0.013 7.03±0.018
7.54±0.080bc 2014 8.05±0.029 7.80±0.029 7.65±0.003 7.09±0.013 6.93±0.018
T8 2013 8.40±0.003 8.07±0.033 7.90±0.029 7.32±0.044 7.15±0.029
7.74±0.085ab 2014 8.30±0.003 8.02±0.033 7.80±0.029 7.27±0.044 7.10±0.029
T9 2013 8.48±0.017 8.15±0.029 7.98±0.017 7.40±0.029 7.20±0.029
7.80±0.089a 2014 8.38±0.017 8.10±0.029 7.88±0.017 7.35±0.029 7.10±0.029
Means 7.95±0.044a 7.64±0.044b 7.45±0.051c 6.97±0.037d 6.69±0.051e
128
Table 43. Effect of treatments and storage on taste of broiler leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Days
Means Day 0 Day 15 Day 30 Day 45 Day 60
T0 2013 7.40±0.058 7.13±0.033 6.82±0.044 6.27±0.017 5.95±0.029
6.67±0.100f 2014 7.30±0.058 7.08±0.033 6.67±0.044 6.22±0.017 5.85±0.039
T1 2013 7.45±0.029 7.28±0.017 6.95±0.029 6.32±0.017 6.12±0.044
6.78±0.098e 2014 7.35±0.029 7.23±0.017 6.80±0.029 6.27±0.017 6.02±0.044
T2 2013 7.49±0.019 7.35±0.016 7.05±0.029 6.55±0.029 6.23±0.017
6.89±0.089de 2014 7.39±0.019 7.30±0.018 6.90±0.029 6.50±0.029 6.13±0.017
T3 2013 7.67±0.017 7.52±0.044 7.25±0.029 6.72±0.06 6.43±0.073
7.07±0.089cd 2014 7.57±0.017 7.47±0.044 7.10±0.029 6.67±0.06 6.33±0.073
T4 2013 7.60±0.029 7.45±0.029 7.15±0.029 6.65±0.029 6.30±0.029
6.99±0.091d 2014 7.50±0.029 7.40±0.029 7.00±0.029 6.60±0.029 6.20±0.029
T5 2013 7.75±0.029 7.60±0.029 7.30±0.029 6.77±0.017 6.53±0.06
7.15±0.088c 2014 7.65±0.029 7.55±0.029 7.15±0.029 6.72±0.017 6.43±0.06
T6 2013 8.05±0.029 7.70±0.029 7.53±0.033 7.03±0.015 6.75±0.029
7.37±0.087b 2014 7.95±0.029 7.65±0.029 7.38±0.033 6.98±0.015 6.65±0.029
T7 2013 7.90±0.029 7.65±0.029 7.45±0.029 6.85±0.003 6.65±0.029
7.25±0.089bc 2014 7.80±0.029 7.60±0.029 7.30±0.029 6.80±0.003 6.55±0.029
T8 2013 8.13±0.017 7.80±0.029 7.58±0.017 7.10±0.029 6.87±0.044
7.45±0.086ab 2014 8.03±0.017 7.75±0.029 7.43±0.017 7.05±0.029 6.77±0.044
T9 2013 8.25±0.029 7.90±0.029 7.65±0.029 7.14±0.007 6.94±0.013
7.53±0.090a 2014 8.15±0.029 7.85±0.029 7.50±0.029 7.09±0.007 6.84±0.016
Means 7.72±0.038a 7.51±0.030b 7.20±0.037c 6.71±0.038d 6.43±0.042e
129
Table 44. Overall acceptability of breast meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Storage days
Means 0 15 30 45 60
T0 2013 7.35±0.029 7.10±0.029 6.95±0.029 6.37±0.017 5.95±0.018
6.7±0.096f 2014 7.25±0.027 7.05±0.029 6.85±0.029 6.32±0.017 5.85±0.034
T1 2013 7.50±0.076 7.15±0.076 7.14±0.007 6.48±0.017 6.05±0.029
6.83±0.097e 2014 7.40±0.076 7.10±0.076 7.04±0.007 6.43±0.017 5.95±0.029
T2 2013 7.58±0.017 7.35±0.029 7.20±0.029 6.73±0.017 6.15±0.029
6.96±0.095de 2014 7.48±0.017 7.30±0.029 7.10±0.029 6.68±0.017 6.05±0.029
T3 2013 7.85±0.019 7.55±0.029 7.35±0.058 6.85±0.029 6.35±0.029
7.15±0.100cd 2014 7.75±0.029 7.50±0.029 7.25±0.058 6.8±0.029 6.25±0.029
T4 2013 7.70±0.029 7.45±0.029 7.30±0.029 6.75±0.029 6.25±0.029
7.05±0.099d 2014 7.60±0.029 7.40±0.029 7.20±0.029 6.70±0.029 6.15±0.029
T5 2013 7.90±0.029 7.70±0.029 7.40±0.029 6.93±0.017 6.55±0.029
7.26±0.093c 2014 7.80±0.029 7.65±0.029 7.30±0.029 6.88±0.017 6.45±0.029
T6 2013 8.15±0.076 7.93±0.033 7.66±0.007 7.10±0.058 6.75±0.024
7.48±0.098b 2014 8.05±0.076 7.88±0.033 7.56±0.007 7.05±0.058 6.65±0.034
T7 2013 8.05±0.029 7.80±0.029 7.45±0.029 6.97±0.017 6.62±0.017
7.34±0.098bc 2014 7.95±0.029 7.75±0.029 7.35±0.029 6.92±0.017 6.52±0.017
T8 2013 8.20±0.029 8.00±0.029 7.70±0.029 7.20±0.029 6.90±0.026
7.56±0.091ab 2014 8.10±0.031 7.95±0.029 7.60±0.029 7.15±0.029 6.80±0.031
T9 2013 8.30±0.029 8.05±0.029 7.77±0.012 7.25±0.029 7.02±0.017
7.64±0.090a 2014 8.20±0.034 8.00±0.029 7.67±0.012 7.20±0.029 6.92±0.017
Means 7.81±0.041a 7.58±0.043b 7.34±0.033c 6.84±0.036d 6.41±0.046e
130
Table 45. Overall acceptability of broiler leg meat nuggets
Means sharing similar letter in a column or row do not differ significantly (p > 0.05)
T0= control without antioxidants; T1= 100 mg quercetin and 150 mg of α-tocopherol/kg feed
T2= 100 mg quercetin and 225 mg of α-tocopherol/kg feed T3= 100 mg quercetin and 300 mg of α-tocopherol/kg feed
T4= 200 mg quercetin and 150 mg of α-tocopherol/kg feed T5= 200 mg quercetin and 225 mg of α-tocopherol/kg feed
T6= 200 mg quercetin and 300 mg of α-tocopherol/kg feed T7= 300 mg quercetin and 150 mg of α-tocopherol/kg feed
T8= 300 mg quercetin and 225 mg of α-tocopherol/kg feed T9= 300 mg quercetin and 300 mg of α-tocopherol/kg feed
Treatments Years Days
Means Day 0 Day 15 Day 30 Day 45 Day 60
T0 2013 7.23±0.033 6.9±0.029 6.48±0.044 6.15±0.029 5.79±0.019
6.47±0.096f 2014 7.13±0.035 6.85±0.029 6.38±0.044 6.10±0.029 5.69±0.016
T1 2013 7.30±0.003 7.05±0.029 6.65±0.029 6.13±0.044 5.97±0.017
6.58±0.096e 2014 7.20±0.003 7.00±0.029 6.55±0.029 6.08±0.044 5.87±0.017
T2 2013 7.35±0.029 7.10±0.029 6.75±0.029 6.42±0.017 6.13±0.017
6.71±0.083de 2014 7.25±0.029 7.05±0.029 6.65±0.029 6.37±0.017 6.03±0.017
T3 2013 7.55±0.029 7.25±0.029 6.95±0.029 6.5±0.029 6.40±0.029
6.89±0.082cd 2014 7.45±0.029 7.20±0.029 6.85±0.029 6.45±0.029 6.30±0.029
T4 2013 7.41±0.007 7.15±0.029 6.85±0.029 6.45±0.029 6.35±0.029
6.80±0.075d 2014 7.31±0.007 7.10±0.029 6.75±0.029 6.40±0.029 6.25±0.029
T5 2013 7.61±0.007 7.35±0.029 7.05±0.029 6.60±0.029 6.47±0.017
6.97±0.081c 2014 7.51±0.007 7.30±0.029 6.95±0.029 6.55±0.029 6.37±0.017
T6 2013 7.90±0.029 7.63±0.017 7.30±0.029 6.8±0.029 6.77±0.037
7.24±0.083b 2014 7.80±0.029 7.58±0.017 7.20±0.029 6.75±0.029 6.67±0.037
T7 2013 7.72±0.017 7.46±0.009 7.22±0.017 6.67±0.035 6.58±0.044
7.09±0.082bc 2014 7.62±0.017 7.41±0.009 7.12±0.017 6.62±0.035 6.48±0.044
T8 2013 7.92±0.017 7.70±0.029 7.38±0.017 6.85±0.029 6.82±0.015
7.29±0.082ab 2014 7.82±0.017 7.65±0.029 7.28±0.017 6.80±0.029 6.72±0.015
T9 2013 8.05±0.029 7.75±0.029 7.43±0.017 7.05±0.029 6.85±0.029
7.39±0.082a 2014 7.95±0.029 7.70±0.029 7.33±0.017 7.00±0.029 6.75±0.029
Means 7.55±0.036a 7.31±0.037b 6.96±0.041c 6.54±0.037d 6.36±0.046e
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and Tanwar, (2011) also stated that sensory response including appearance, flavor, taste and
overall acceptability of the nuggets decrease significantly with progression of storage. One of the
researchers groups, Capitani et al. (2012) examined the effect of hydrophilic antioxidants
(caffeic and carnosic acids) on sensory attributes of sausages. They found a momentous increase
in sensory acceptability for those treatments containing antioxidants however, an inverse
association was reported between scores and storage.
One of the researchers groups, Kala et al. (2007) documented higher flavor score for chicken
meat patties containing antioxidants nevertheless, decreases with passage of storage. Likewise,
Pereira et al. (2010) indicated that flavor of meat patties decreases with storage. The mechanistic
approach behind this reduction was peroxidation of PUFA that generates rancid odor and off
flavor compounds. Similarly, Chandralekha et al. (2012) showed that incorporation of
pomegranate rind powder extract @2.5 and 5% imparts momentous effect on flavor profile of
chicken meat balls nonetheless, score decreases significantly (p<0.05) as a function of storage.
The mechanistic approach behind this reduction in flavor of nuggets was generation of volatile
compounds which decrease the product quality. One of the researchers groups, Pereira et al.
(2010) observed a decline in overall acceptability of nuggets with passage of storage that is
attributed to generation of off flavors volatiles. Earlier, Carvalho et al. (2005) measured the
impact of antioxidants on the sensory and shelf life study of meat products and found effective in
enhancing the overall acceptability of the products. One of the scientists groups, Hwang et al.
(2013) observed a better response in the keeping quality of fried chicken nuggets by the
application of antioxidants. They conducted a study in which deep fried chicken nuggets
containing various levels of ganghwayakssuk ethanolic extract in combination with ascorbic acid
were evaluated for shelf-life during refrigerated storage (4°C). It was inferred that addition of
antioxidants improves the color characteristics of tested nuggets however, the acceptability
decreases with storage.
4.7. Bio-evaluation study
4.7.1. Total cholesterol
The mean squares in Table 46 showed that treatments imparted significant variations on cholesterol
of human subjects. The results indicated that maximum cholesterol was observed in G0
(volunteers fed on broiler meat without antioxidants) as 154.2±5.4 mg/dL followed by G4
(volunteers fed on meat containing 300 mg quercetin + 150 mg α-tocopherol), G3 (meat
132
containing 200 mg quercetin + 300 mg α-tocopherol), G2 (volunteers fed on meat containing 300
mg quercetin + 225 mg α-tocopherol) as 149.2±3.4, 147.5±5.3, 145±4.8 mg/dL whilst, minimum
in G1 (volunteers fed on meat containing 300 mg quercetin + 300 mg α-tocopherol) 141.6±4.3
mg/dL. Similarly, means for cholesterol in trial II indicated highest value in G0 (155.4±4.7
mg/dL) that momentously reduced to 143.6±5.1, 147.5±4.8, 149.6±3.9 and 151.2±5.4 mg/dL in
G1, G2, G3 and G4, respectively (Table 46). It is obvious from the Figure 1 that G1 resulted
maximum reduction in cholesterol trailed by G2, G3 and G4. The percent diminish of cholesterol
in various groups G1, G2, G3 and G4 during 1st trial was as 8.17, 5.97, 4.35 and 3.24 %
respectively. Likewise, in trial II highest reduction was exhibited by G1 (7.59%) trailed by G2
(5.08%), G3 (3.73%) and G4 (2.70%), respectively (Table 3).
Cholesterol is lipophilic compound required for lipoproteins transportation in the blood. In this
context, chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL)
and high density lipoprotein (HDL) are the notable carriers that facilitate its circulation however,
HDL and LDL are the most promising (Yang et al., 2012). The growing evidences indicated a
correlation between imbalance of LDL and HDL for development of coronary complications
(Michos et al., 2012). The findings of instant exploration are consistent with Matvienko et al.
(2002); they noticed an inverse association between phytosterol supplemented meat consumption
and cholesterol level in humans. They carried out a triple-blind study involving 34 male with
elevated plasma cholesterol (5.85 +/- 0.70 mmol/L), LDL (4.02 +/- 0.60 mmol/L) and TC: HDL
ratio (5.5 +/- 1.2) and were given control meat and meat with 2.7 g of phytosterols. It has been
observed that the provision of phytosterol supplemented meat decreases the cholesterol up to 9.3%
compared to control at termination of study. They suggested that phytosterols have ability to inhibit
the LDL particles and that is a key reason behind this reduction. One of the scientists groups,
Bermejo et al. (2014) stated that consumption of functional meat containing Ω-3 fatty acids and
rosemary extract improves oxidative and inflammatory status of people at risk of cardiovascular
diseases (CVD). The results indicated that functional meat consumption decreased the cholesterol
in human subjects. One of their peers, Olmedilla-Alonso et al. (2013) also observed reduction in
the lipid profile of human subjects that were at CVD risk. They conducted a randomized crossover
research involving 25 men, provided functional meat with or without walnuts five times /week for 5
weeks. The results indicated that consumption of meat products without walnuts decreased
cholesterol upto 6.8 mg/dL. Compared to baseline, meat products with walnuts decreased total
133
cholesterol 10.7 mg/dL, LDL 17.6 mg/dL and also increased α-tocopherol level as 8.9 mg/dL. It
has been inferred that consumption of functional meat with or without added walnuts resulted
reduction in cholesterol 4.5 & 3% with respect to baseline. One of the scientists groups, Scott et
al. (1994) reported that consumption of poultry and beef lowers the cholesterol and LDL in
hypercholesterolemic subjects. In this context, they conducted a study in which 38 men were
provided dietary intervention for 13 weeks including normal diet for 3 weeks followed by 5
weeks stabilization diet. The meat fed to the subjects includes either 85 g cooked beef of 8% fat
or 85 g chicken containing 7% fat. The results showed that provision of beef and chicken
significantly decreased plasma cholesterol 7.6%, 10.2% and LDL 9%, 11% for beef and chicken,
respectively.
One of the researchers groups Auger et al. (2006) stated that provision of catechin, quercetin and
resveratrol phenolic compounds prevent the development of atherosclerosis in hamsters. They
conducted a study in which 32 hamsters were fed on catechin, quercetin and resveratrol
supplementation diets to explore their effect on early atherosclerosis development. The hamsters
were given force-feeding 7.14 mL/kg body weight catechin, quercetin and resveratrol in water. It
has been observed that cholesterol concentration decreased in treated groups compared to
control. Moreover, apolipoprotein (Apo) A1 concentration was also enhanced in plasma of tested
animals by 26, 22 and 19% in catechin, quercetin and resveratrol feeding groups, respectively
however, Apo-B concentration was not affected. Similarly, Sikder et al. (2014) stated that
supplementation of quercetin and rutin protected albino mice against high cholesterol diet
induced hepatotoxicity. They further elaborated that cholesterol in the rats was mainly decreased
through restoration of hepatic antioxidants. Likewise, Rama Rao et al. (2011) reported that
cholesterol (TC) concentration in sera was decreased significantly with dietary α-tocopherol
supplementation in broiler birds. One of the researchers groups, Brenes et al. (2008) also found a
reduction in cholesterol with increasing dietary α-tocopherol supplementation in poultry birds.
Similarly, Zahedi et al. (2013) noticed that quercetin provision to women with type-2 diabetes
reduced systolic blood pressure significantly (p<0.04) that is involved in the cardio vascular
complications. Furthermore, Phuwamongkolwiwat et al. (2013) found that quercetin-3-O-β-
glucoside (Q3G) and fructooligosaccharide (FOS) enriched diets improved glucose tolerance,
insulin sensitivity and cholesterol.
134
Table 46. Means for total cholesterol, HDL, LDL cholesterol, triglycerides and total protein
of human subjects
**=Highly significant
*=Significant NS=Non-significant
G0= Volunteers fed on control meat without antioxidants
G1= Volunteers fed on meat containing 300 mg quercetin + 300 mg α-tocopherol
G2= volunteers fed on meat containing 300 mg quercetin + 225 mg α-tocopherol
G3= volunteers fed on meat containing 200 mg quercetin + 300 mg α-tocopherol
G4= volunteers fed on meat containing 300 mg quercetin + 150 mg α-tocopherol
Cholesterol Treatments
MS value G0 G1 G2 G3 G4
Trial 1 154.2±5.45 141.6±4.36 145±4.83 147.5±5.36 149.2±3.45 125.680**
Trial 2 155.4±4.71 143.6±5.15 147.5±4.54 149.6±3.94 151.2±5.39 93.2416**
LDL
Trial 1 79.5±3.24 73.6±3.64 75.1±2.96 75.9±3.76 76.8±4.13 23.0544**
Trial 2 80.3±2.36 74.1±3.75 76.2±4.84 76.7±4.71 77.2±2.45 25.8325**
Triglycerides
Trial 1 144.5±5.31 129.5±6.45 132.6±5.81 134.1±7.56 136.5±8.62 180.615**
Trial 2 149.3±4.92 134.11±5.34 137.4±5.16 139.2±4.92 141.5±4.87 158.051**
HDL
Trial 1 49.1±3.23 51.3±3.55 51.2±4.26 50.75±3.91 50.44±4.75 3.12931NS
Trial 2 49.88±4.16 51.56±3.24 51.31±3.74 50.9±4.39 50.63±3.62 1.99904NS
Sera protein
Trial 1 3.98±0.26 4.60±0.31 4.45±0.27 4.25±0.18 4.12±0.19 0.31023*
Trial 2 4.06±0.25 4.66±0.14 4.51±0.21 4.32±0.12 4.21±0.31 0.28395*
135
4.7.2. LDL
The mean squares regarding LDL of human subjects showed significant differences due to
treatments (Table 46). The means for volunteers (trial 1) presented diminishing trend for LDL in
G1 (73.6±3.6 mg/dL) trailed by G2 (75.1±2.9), G3 (75.9±3.7 mg/dL) and G4 (76.8±4.1 mg/dL).
Likewise in trial 2, G0 showed highest LDL (80.3±2.3 mg/dL) that gradually reduced in G4
(77.2±2.4), G3 (76.7±4.7) G2 (76.2±4.8) and G1 (74.1±3.7), respectively (Table 46). The Figure
4 exposed percent decline in LDL of human subjects that were provided functional meat. It has
been revealed substantial decline for LDL in G1 (volunteers fed on meat containing 300 mg
quercetin and 300 mg α-tocopherol) i.e. 7.42% followed by G2, G3 & G4 by 5.53, 4.53, 3.40 %
(trial 1). In the subsequent trial, similar LDL lowering tendency was noticed for G1, G2, G3 and
G4 as 7.72, 5.11, 4.48 and 3.86%, respectively.
The findings of Matvienko et al. (2002) are in accordance with the current results; they noticed
an inverse association between consumption of phytosterol supplemented beef and LDL.
Accordingly, they carried out a triple blind study involving 34 male with elevated LDL (4.02 +/-
0.60 mmol/L) and provided meat with or without 2.7 g of phytosterols. At the termination of
study, LDL was reduced up to 14.6% compared to control. One of the researchers groups,
Olmedilla-Alonso et al. (2013) observed reduction in lipid profile of human subjects that were at
risk of CVD. They conducted a study involving 25 men, given functional meat with or without
walnuts five times /week for five weeks. It has been observed that meat products with walnuts
decreased LDL 17.6 mg/dL compared to baseline. Previously, findings by Choi et al. (2010)
delineated that dietary supplementation of α-tocopherol alone or in combination with garlic
powder significantly decreased cholesterol and LDL. Likewise, Graf et al. (2005) recorded 21%
reduction in cardiovascular diseases when intake of quercetin was greater than 4 mg/day.
Similarly, Chopra et al. (2000) stated that consumption of quercetin @ 30mg/day decreased LDL
cholesterol in hyperlipidemic subjects. Afterwards, Sikder et al. (2014) recorded that quercetin
and rutin protect albino mice against high cholesterol diet (HCD) induced hepatotoxicity. They
noticed 76% increases for LDL in HCD group, however, LDL burden was reduced by 30 and
25% respectively for quercetin and rutin groups compared to HCD. One of their peers,
Phuwamongkolwiwat et al. (2013) documented that quercetin-3-O-β-glucoside (Q3G) and
fructooligosaccharide (FOS) enriched diet decreased LDL cholesterol. One of the scientists
groups, Murota et al. (2007) reported that quercetin metabolites accumulate in the plasma after
136
the consumption of quercetin rich diet in albumin bound conjugates form that enhance plasma
antioxidant capacity against LDL oxidation in dose dependent manner. They provided cooked
fried onions equivalent to 150 mg quercetin aglycone to healthy male volunteers and blood
samples were collected after 1.5 hr of onion consumption. The results revealed that more than
80% quercetin metabolites were localized in plasma fraction containing concentrated albumin
however, other lipoprotein fractions contain little quercetin metabolites. It has been observed that
quercetin metabolites delay the clearance of albumin from plasma thereby its circulation
increases antioxidant capacity. Similarly. Mariee et al. (2012) indicated that quercetin has ability
to ameliorate high cholesterol diet (HCD) induced oxidative injury in rats through its antioxidant
potential. Accordingly, they investigated the effect of quercetin against hepatic oxidative injury
using rats modeling that were divided into three groups as control, HCD (1 mL/100 g), HCD diet
enriched with quercetin (15 mg/kg) group. It has been observed that quercetin administration
significantly decreased liver triglycerides, cholesterol and LDL by 24, 20 and 31%, respectively.
Likewise, Talirevic and Jelena (2012) investigated the effect of quercetin consumption on lipid
variables among healthy peoples. Their results revealed that tested group showed diminishing
trend in cholesterol and LDL as 5.09 and 2.91 mmol/L, respectively. One of the scientists
groups, Egert et al. (2009) stated that quercetin supplementation provides protection against
CVD. In this context, they conducted a research study in which quercetin @ 150mg /d for 6
weeks was given to 93 obese subjects and its effect on lipid metabolism, oxidative stress
biomarkers and inflammation was recorded. It has been observed that supplementation increased
plasma quercetin contents from 71 to 269 nmol/L (p<0.001) nonetheless, its provision also
decreased plasma cholesterol and LDL concentrations in overweight subjects.
137
Figure 3. Percent reduction in cholesterol as compared to control
Figure 4. Percent reduction in LDL cholesterol as compared to control
8.177.59
5.975.08
4.353.73
3.242.70
0.00
2.00
4.00
6.00
8.00
10.00
Trial 1 Trial 2
% D
ecre
ase
G1 G2 G3 G4
7.427.72
5.535.11
4.53 4.48
3.403.86
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Trial 1 Trial 2
% D
ecre
ase
G1 G2 G3 G4
138
4.7.3. Triglycerides
The mean squares in Table 46 revealed momentous differences in triglycerides of volunteers due
to treatments. The means (trial 1) presented a gradual decline in triglycerides from 144.5±5.3
(G0) to 129.5±6.4 (G1) mg/dL. Likewise, the second trial showed similar decreasing trend for
triglycerides 134.11±5.3, 137.4±5.1, 139.2±4.9 and 141.5±4.8 mg/dL for G1, G2, G3 and G4,
respectively (Table 46). The Figure 5 depicted percent decline for triglycerides as 10.38, 8.24,
7.20 and 5.54% for G1, G2, G3 and G4, respectively (trial 1). Moreover, trial 2 showed alike
decreasing tendency from G1 to G4 as 10.18 to 5.22%.
There are a number of scientific evidences that illuminated a linear correlation between high fat
diets and increase in cholesterol, triglycerides and LDL due to the production of free fatty acids
(FFA) thus trigger the lipogenesis (Kim et al., 2012). Besides, antioxidants and polyphenols
enriched diets have ability to normalize lipid abnormalities by inhibiting intestinal lipid
absorption, increasing fecal excretion of fat, suppressing the activity of fat synthesis enzymes
and preventing lipogenesis (Delíndice, et al., 2011). The exploration of Beauchesne-Rondeau et
al. (2003) supported the current finding for the effect of antioxidant enriched broiler meat on
triglycerides reduction in the human subjects; they observed 25% decline in triacylglycerol in
hypercholesterolemic men fed on lean poultry meat. Similarly, VLDL triacylglycerols and
cholesterol were also reduced by 28–29% after consumption of the poultry meat. One of the
scientists groups, Bermejo et al. (2014) reported that consumption of functional meat containing
Ω-3 fatty acids and rosemary extract improves oxidative status by decreasing triglycerides.
Similarly, Lopez-Huertas et al. (2010) showed that consumption of functional foods containing
EPA and DHA impart positive effect on lipid profile, especially the reduction of triglycerides. In
this regard, a meta-analysis involving 36 controlled, crossover trials in which 3-4 g of EPA +
DHA were provided to human volunteers that showed reduction in triglycerides however,
reduction was more pronounced when baseline value of triglycerides was >177 mg/dL. One of
the researchers groups, Odbayar et al. (2006) reported that supplementation of quercetin
dihydrate significantly lowered serum cholesterol, phospholipids and triacylglycerol levels in
mice. In this context, they carried out a research in which mice were fed on experimental diet
containing quercetin @ 1% for 15 days. It has been observed that provision of quercetin
dihydrate significantly reduced the activity of enzymes such as fatty acid synthase, ATP-citrate
lyase & glucose 6-phosphate dehydrogenase and mRNA levels involved in hepatic fatty acids
139
synthesis. They noticed hepatic lipogensis as a key mechanism behind the hypolipidemic
potential of quercetin dihydrate. Likewise, Juzwiak et al. (2005) indicated that quercetin
possesses hypolipemic and antiatherogenic properties. In this regard, they determined the
influence of quercetin on hyperlipidemia and atherosclerotic lesions developments through rabbit
animal modeling that were fed on high fat diet for 12 weeks. The results revealed that quercitin
was effective in reducing triglycerides and cholesterol elevated by high fat diet. Additionally,
triglycerides were comparable among study groups and the value for this trait in control was 1.28
± 0.24 mmol/L that momentously decreased to 0.77 ± 0.16 mmol/L in quercetin supplemented
group.
4.7.4. HDL
The mean squares revealed that treatments imparted non momentous effect on HDL cholesterol
of human subjects (Table 46). Means expounded that HDL slightly varied from 49.1±3.1 to
51.3±3.5 and 49.88±4.1 to 50.63±3.6 mg/dL during trial I and II, respectively (Table 46).
The slogan “good cholesterol” is attributed to HDL mainly due to its capacity to reverse the
cholesterol transport, removes the excess cholesterol from arteries back to liver. It predominantly
acts on sub-endothelial space in caliber artery which is the main place for cholesterol deposition.
Currently, various epidemiological studies estimated the role of HDL for the management of
cardiovascular complications (Gadi et al., 2012). The results of this study are in harmony with
Bermejo et al. (2014), who reported non-significant effect on HDL cholesterol of human subjects
by the consumption of functional meat. They carried out a study in which functional broiler meat
containing Ω-3 fatty acids and rosemary extract was fed to human and observed 0.2±5.9 mg/dL
change for this trait at the termination of study. Similarly, Olmedilla-Alonsn et al. (2013)
observed non-significant differences for HDL in subjects that were at risk for cardiovascular
disorder by the consumption of functional meat. One of the researchers groups, Matvienko et al.
(2002) also found non-significant variations for HDL cholesterol in young, mild
hypercholesterolemic men fed on soybean phytosterols enriched ground beef for 4 weeks.
140
Figure 5. Percent reduction in triglyceride as compared to control
Figure 6. Percent increase HDL cholesterol as compared to control
10.38 10.18
8.24 7.977.20
6.76
5.54 5.22
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Trial 1 Trial 2
% D
ecre
ase
G1 G2 G3 G4
3.203.37
3.002.87
2.09 2.04
1.47 1.50
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Trial 1 Trial 2
Perc
en
t in
cre
ase
G1 G2 G3 G4
141
Figure 7. Percent increase total protein as compared to control
15.5814.78
11.8111.08
6.78 6.40
3.52 3.69
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Trial 1 Trial 2
% i
ncr
ease
G1 G2 G3 G4
142
4.7.5. Total protein
The mean squares in Table 46 regarding sera protein elucidated significant variations due to
treatments. The results (Table 46) indicated highest protein in G1 4.60±0.31g/dL trailed by G2,
G3 and G4 as 4.45±0.27g/dL, 4.25±0.18 & 4.12±0.19, respectively whilst, lowest in G0
3.98±0.26 g/dL. Likewise, trial II also indicated minimum sear protein in G0 4.06±0.25 g/dL that
momentously increased to 4.66±0.14, 4.51±0.21, 4.32±0.12 and 4.21±0.31g/dL in G1, G2, G3
and G4, respectively. It is obvious from Figure 5 that G1 resulted maximum increase trailed by
G2, G3 and G4, correspondingly however, all the values for this trait were within the normal
range. The percent increase for protein in G1, G2, G3 and G4 groups during both trials were
15.58 & 14.78, 11.81 & 11.80, 6.78 & 6.40, 3.52 & 3.69%, respectively (Table 7).
The findings of instant study are concordant with Brenes et al. (2008) who reported that dietary
supplementation of α-tocopherol increase protein concentration in blood sera. They suggested
that increased protein concentration is attributed to improved digestion and absorption of dietary
nutrients in the presence of α-tocopherol. Likewise, Roberts et al. (2007) stated that higher
concentration of α-tocopherol triggered the digestibility of nutrients in birds compared to those
fed on diet lacking in antioxidants. These findings are further supported by Rama Rao et al.
(2011), they documented that total protein and globulin concentration in sera increased
substantially with dietary α-tocopherol fortification.
4.8. In vitro study
4.8.1. Oxidative stability of patties
It is obvious from mean squares that oxidative stability as measured by TBARS of quercetin and
α-tocopherol enriched patties varied significantly due to treatments and storage (Table 47). The
results showed that at initiation of storage TBARS in T0, T1, T2, T3, T4, T5 and T6 groups were
1.93±0.02, 0.80±0.01, 0.66±0.06, 0.63±0.05, 0.58±0.02, 0.38±0.03 and 0.37±.01 that
subsequently increased to 3.47±0.14 (T0), 2.97±0.09 (T1), 2.56±0.01 (T2), 2.43±0.04 (T3),
2.32±0.03 (T4), 0.94±0.34 (T5), 0.90±0.05 (T6) mg of MDA/kg meat, correspondingly at
termination of storage. The TBARS also demonstrated a substantial increase ranging from 0.76
at start to 1.47 and 2.23 mg of MDA/kg meat, respectively on 3rd and 7th day of storage (Table
48).
143
Several studies indicated that antioxidants have ability to attenuate the process of lipid
peroxidation (Fellenberg and Speisky, 2006) thus incorporated in meat based products to
enhance storage stability. The findings of current study are in agreement with Goliomytis et al.,
2014); they documented that oxidative stability of meat products improves significantly (p <
0.05) when birds were fed on quercetin @1 g/kg feed supplementation. Similarly, Jang et al.
(2010) reported that quercetin supplementation @ 200 ppm reduces the MDA production in
resultant broiler thigh muscles compared to control. One of the researchers groups, Capitani et
al. (2012) also elucidated that antioxidant mixture containing quercetin and rutin @ 0.05 g/ 100 g
meat impart significant effect on inhibiting the formation of MDA compounds during shelf
stability study of sausages in which sodium erythrorbate was replaced with natural antioxidants.
They further reported an increasing trend for malonaldehydes production as a function of
storage. One of the scientists groups, Kumar and Tanwar, (2011) documented that chicken meat
nuggets containing antioxidants indicated less production of MDA nevertheless, the value of
TBARS increased progressively with storage that ranged from 0.347±0.003 to 0.889±0.226
among treatments. Similarly. Avila-Ramos et al. (2013) evaluated the effect of added
antioxidants on lipid stability of cooked chicken patties. Accordingly, birds were fed on 2 levels
of vitamin E (10 or 100 mg/kg feed) and resultant meat was used for patties preparation followed
by storage at 4ᵒC for 9 days. It has been observed that cooked meat containing 10mg vitamin
E/kg feed showed higher MDA values compared to other antioxidant combination during
storage. Likewise, Sampaio et al. (2012) also found that combination of antioxidants exhibits
better antiradical activity and reduces TBARS & hexanal generation in chicken meat product.
Similarly, findings of Juntachote et al. (2006) explicated that antioxidants are effective in
minimizing the oxidation of lipids and protein in meat products by limiting the generation of
MDA. One of the scientists groups, Sullivan et al. (2004) found that antioxidant enriched broiler
meat nuggets showed lower (p<0.01) TBARS than that of control.
4.8.2. Color of patties
The mean squares depicted that lightness (L∗-values), redness (a*) and yellowness (b*) color
values of patties affected significantly due to treatments and storage (Table 47). Among
treatments, the maximum L* value was recorded in T0 (control) 80.77 whereas, minimum 73.90
in T6 nonetheless values were 74.58, 76.59 and 76.39 for T5, T4 and T3, respectively. The
results also demonstrated that L* value of the product varied during storage from 78.06 at start to
144
Table 47. Mean squares for TBARS and color values of patties
SOV df TBARS L* value a* value b* value
Treatment 6 4.9652** 53.6999** 6.37901** 5.79580**
Day 2 11.2583** 19.0770** 2.71482** 6.63974**
Treatment*day 12 0.3381** 0.7046NS 0.1781** 0.8438*
Error 42 0.0176 0.6362 0.06644 0.34654
Total 62
**=Highly significant
*=Significant
NS=Non-significant
145
Table 48. Means for TBARS of cooked breast meat patties stored at 4 ᵒC
Storage days T0 T1 T2 T3 T4 T5 T6 Means
1 1.93±0.02 0.80±0.01 0.66±0.06 0.63±0.05 0.58±0.02 0.38±0.03 0.37±0.01 0.76±0.21c
3 2.76±0.11 2.10±0.12 1.48±0.13 1.47±0.06 1.48±0.11 0.53±0.09 0.51±0.02 1.484±0.34b
7 3.47±0.14 2.97±0.09 2.56±0.01 2.43±0.04 2.32±0.03 0.94±0.34 0.90±0.05 2.23±0.42a
Means 2.72±0.15a 1.96±0.06b 1.57±0.09c 1.51±0.06c 1.46±0.05c 0.62±0.13d 0.59±0.06d
Means sharing similar letter within a row or column are statistically non-significant (p > 0.05)
T0=Control without antioxidants
T1= 25 mg quercetin dihydrate + 100 mg α-tocopherol/kg meat
T2= 25 mg quercetin dihydrate +200 mg alpha tocopherol/kg meat
T3= 50 mg quercetin dihydrate +100 mg α-tocopherol/kg meat
T4= 50 mg quercetin dihydrate +200 mg α-tocopherol/kg meat
T5= 100 mg quercetin dihydrate +100 mg α-tocopherol/kg meat
T6= 100 mg quercetin dihydrate + 200 mg α-tocopherol/kg meat
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Table 49. Color values of cooked breast meat patties stored at 4 ᵒC
Storage days T0 T1 T2 T3 T4 T5 T6 Means
L* value
1 81.14±0.37 79.24±0.73 79.77±0.94 77.89±0.73 78.03±0.65 75.65±0.37 74.67±0.62 78.06±0.65a
3 81.22±0.28 78.85±1.12 79.12±0.5 76.48±0.43 75.92±1.51 74.31±0.70 73.38±0.12 77.04±0.71b
7 79.96±1.56 77.62±0.97 77.44±0.70 75.38±0.24 75.24±0.65 73.77±0.89 73.66±0.80 76.15±0.55c
Means 80.77±0.98a 78.78±0.87b 78.57±0.76b 76.39±0.85c 76.59±0.84c 74.58±0.65d 73.90±0.77d
a* value
1 -3.50±0.15 -3.50±0.26 -3.32±0.06 -3.16±0.06 -2.95±0.14 -2.08±0.23 -2.17±0.29 -2.955±0.11a
3 -4.39±0.19 -4.38±0.12 -3.63±0.28 -3.63±0.12 -3.77±0.06 -2.17±0.13 -1.95±0.16 -3.42±0.13b
7 -4.65±0.31 -4.54±0.19 -3.99±0.21 -3.99±0.02 -3.81±0.07 -2.54±0.20 -2.14±0.08 -3.66±0.17c
Means -4.181±0.17c -4.139±0.21c -3.647±0.16b -3.59±0.16b -3.51±0.09b -2.27±0.19a -2.09±0.19a
b* value
1 18.18±0.23 17.03±0.14 17.75±0.19 17.56±0.25 16.98±0.20 17.87±0.76 16.23±1.28 17.37±0.65a
3 18.53±0.08 16.37±0.67 17.80±0.36 16.65±0.58 16.07±0.22 16.87±0.10 16.83±0.19 17.02±0.54a
7 18.34±0.33 16.37±0.33 16.77±0.26 15.58±0.39 14.61±0.57 16.05±0.12 16.16±0.28 16.27±0.45b
Means 18.35±0.17a 16.58±0.37c 17.44±0.34b 16.59±0.41c 15.88±0.31d 16.93±0.44bc 16.41±0.23cd
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77.04 & 76.15, respectively on 3rd and 7th day of storage. Likewise, a* value in T0, T6, T5, T4
and T3 were -4.181, -2.09, -2.27, -3.51 and -3.59, correspondingly. Similarly b* values in
various groups ranged from 16.23±1.28 to 18.18±0.23 nevertheless, the value for this trait was
17.37 at the beginning whilst, 17.01 and 16.269 at 3rd and 7th day of storage (Table 49).
The findings of current exploration are in harmony with Mancini and Hunt (2005); they noticed a
declining trend for color of product with storage progression due to generation and accumulation
of metmyoglobin (MetMb). There is correlation between MetMb production and discoloration of
meat products however, factors like pH, storage conditions & duration and lipid peroxidation
influence the conversion of oxymyglobin to metmyoglobin (Bekhit and Faustman, 2005). The
added antioxidants retard the oxidative degradation in meat products thereby stabilize color
during storage. One of the researchers groups, Wu et al. (2000), reported that color of meat
product coated with antioxidants decreases as a function of storage owing to release of moisture
from antioxidants coating. Similarly, Muela et al. (2014) indicated that addition of vitamin E
@200 & 300 ppm improves color in lamb meat product. One of the scientists groups,
Chandralekha et al. (2012) stated that chicken meat balls containing pomegranate rind powder
extract as an antioxidant source resulted significantly (p<0.05) higher color scores compared to
control however, the value decreased with storage. Additionally, Andrés et al. (2013) also
recorded lower discoloration (p<0.05) in longissimus lumborum muscles of lamb fed on
quercetin alone or in combination with flaxseed. Likewise, Kumar and Tanwar (2011) also
reported higher color values for nuggets prepared from meat containing antioxidants however,
the value decreased with advancement of storage.
4.8.3. Volatile compounds
The generation of off flavor is a critical problem that affect storage stability of cooked meat
products (Jo et al., 2006). Primarily, volatiles are generated through oxidation that further
degraded into secondary components, considered major reason for the development of rancid
flavor. The direct addition of alpha tocopherol and quercetin in meat product formulations
diminish the generation of off odor volatiles. Major categories of volatiles identified in patties
are hydrocarbons, aldehydes, ketones, alcohols and sulfur containing compounds (Huan, et al.
2005).
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Hydrocarbons are among the major volatiles compounds that affect the flavor of meat products
during storage. The hydrocarbons recoded in samples are pentane, heptane, 1-octene, octane, 2-
octene and 1-heptene. It has been observed that generation of hydrocarbon increased
progressively during storage. At initiation, pentane, heptane, 1-octene, octane and 2-octene
ranged from 3013±188 to 104062±1824, 349±29 to 3808±129, 352±31 to 2239±109, 6439±213
to 32775±976, 191±11 to 2533±109 (ion counts×104), respectively. Heptane and octane
production decreased with increasing level of quercetin and α-tocopherol however, progression
of storage increased their generations in samples. 1-Heptane was not found on 1st day however,
2-octene varied non-significantly among various groups. Likewise, highest pentane was recorded
in T6 78607±319 trailed by T5, T2 and T3 71928±317, 48133±182 & 31390±197 (ion
counts×104) whilst, lowest in T0 as 14677±177 (ion counts×104).
Aldehydes are considered important for flavor development in cooked meat products and showed
linear correlation with TBARS. The results indicated that aldehydes such as propanal, butanal,
pentanal, hexanal, heptanal, octanal and nonanal were found in the patties samples at different
storage intervals nonetheless, hexanal and pentanal are major contributor for the estimation of
oxidative storability in meat products. At initiation, hexanal in T1, T2, T3, T4, T5 and T6 groups
were 54768±431, 11568±283, 8902±383, 7367±318, 3701±111, 3759±34 & 2488±103 (ion
counts×104) that significantly increased to 359826±1285, 279277±1048, 286879±1048,
239163±1104, 244203±999, 112550±897 and 9569±607 (ions counts×104), respectively at the
termination of storage. Similarly, penatal on 7th day of storage in different groups were
96422±304 (T0), 65251±384 (T1), 69319±335 (T2), 56315±3432 (T3), 59438±314 (T4),
21064±247 (T5) and 13840±186 (T6), respectively (Table 50-52). Additionally, heptanal,
octanal and nonanal were not determined in the treated groups on start of storage however, their
production increased as a function of storage. The values of heptanal, octanal and nonanal in T0
groups on initiation of storage were 1737±66, 97±7, 806±30 (ion counts×104) that significantly
increased to 7313±53, 933±52 and 1092±65 (ion counts×104), respectively at termination.
Among ketones, 2-propanone and 2-butanone were noticed in patties samples at different storage
intervals. At initiation, the lowest 2-propanone was recorded in T6 6408±163 trailed by T5 and
T4 6598±201 & 7081±237 (ion counts×104), respectively whereas, the highest in T0 12309±289
(ion counts×104). Moreover, the value for this trait in T6, T5, T4 and T0 were increased to
33102±228, 32990±221, 27303±163 and 15113±104 (ion counts×104) at termination. Similarly,
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Table 50. Volatiles flavor compounds (ion counts × 104) in cooked patties on 1st day of storage
Compounds T0 T1 T2 T3 T4 T5 T6
Hydrocarbons
Pentane 3013±188e 33820±1549cd 49361±1669c 85936±1099ab 30246±1667d 71611±1969b 104062±1824a
Heptane 3808±129a 2405±198b 573±49d 866±56c 771±65cd 349±29e 986±83c
1-octene 352±31d 2239±109a 927±52bc 441±37c 1029±81b 1012±92b 1810±152ab
Octane 32775±976a 27636±1058b 11151±406c 6439±413e 8238±377cd 6745±164de 7861±202d
2-octene 191±11e 2044±103b 1020±85d 1233±64c 1222±70c 1823±71bc 2533±219a
1-heptene
Ketones
2-propanone 12309±289a 12328±266a 8047±224b 7990±207b 7081±237c 6598±201d 6408±163d
2-Butanone - - 1562±121 1485±88 - - -
Aldehydes
Propanal - - - - - - -
Butanal 3618±98a 3257±199ab 1475±121b 703±52b 3399±152b 3253±179c 3462±91d
Pentanal 11927±380a 3391±104b 2431±96bc 1874±45c 1626±69c 939±63d 1222±45e
Hexanal 54768±431a 11568±283b 8902±383bc 7367±318c 3701±111d 3759±34d 2488±103e
Heptanal 1737±66a - - - - - -
Octanal 97±7a - - - - - -
Nonanal 806±30a - - - - - -
Alcohol - - - - - -
Cyclopentanol - - - - - - -
Others
Dimethyldisulfide 4037±133b 5907±182a 2596±216d 2081±239e 3410±217c 2215±105de 3918±175bc
Benzoic acid 364±26 513±44 - - - - -
1,3-octadiene - - - - - - -
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Table 51. Volatiles flavor compounds (ion counts × 104) in cooked patties on 3rd day of storage
Compounds T0 T1 T2 T3 T4 T5 T6
Hydrocarbons
Pentane 12877±79f 28958±128d 83941±145a 68506±149b 22902±132e 76007±1710ab 40153±106c
Heptane 1037±53d 3244±81a 2302±26c 2865±36b 1875±26bc 998±34d 1584±31c
1-octene - 1337±61 1276±56 1237±84 1505±75 1198±46 1459±35
Octane 10753±149bc 16792±183a 13030±107b 16181±123a 14604±104ab 7444±407c 10071±98bc
2-octene 729±36e 3690±116b 1586±66d 1970±83cd 1835±78cd 2412±86c 4666±116a
1-heptene 109±9 94±7 - - - - -
Ketones
2-propanone 18196±166c 16417±124d 24778±253a 20207±136b 23353±139ab 19291±132bc 17417±109cd
2-Butanone 5650±62c 7274±88ab 6263±85b 7534±97ab 9198±170a 6257±107b -
Aldehydes
Propanal 6830±189a 3726±104c 5078±111b 5971±115ab 4244±121b 1541±91d 1092±83e
Butanal 8148±274a 6656±60b 7201±145ab 8099±201a 7220±174ab 4223±140c 5229±110c
Pentanal 61228±1134a 39879±936b 33392±531b 38633±596b 31788±477b 4467±92c 7550±391c
Hexanal 263841±1239a 203916±2406ab 184930±1765b 181390±1446bc 161374±1088c 18259±427d 9136±307e
Heptanal 7313±53a 3892±79b 3909±60b 3379±89bc 2896±75c - -
Octanal 933±52a 345±29c 461±37b 371±24bc 285±17d - -
Nonanal 1092±65a 738±37b 731±43b 664±46c 633±42c - -
Alcohol
Cyclopentanol - - - - - - -
Others
Dimethyldisulfide 4968±126c 2633±143d 7229±194ab 8168±190a 6123±154b 2524±148d 588±42e
Benzoic acid 743±34a 660±24ab 352±21b 271±12c - - -
1,3-octadiene - 462±23a 363±29bc 418±12b 349±28c - -
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Table 52. Volatiles flavor compounds (ion counts × 104) in cooked patties on 7th day of storage
Compounds T0 T1 T2 T3 T4 T5 T6
Hydrocarbons
Pentane 14677±177f 27436±128de 48133±182c 31390±197d 23013±101e 71928±317b 78607±319a
Heptane 5114±168a 3916±114b 3531±124bc 1985±140d 2604±109c 2002±72cd 1557±63e
1-octene - 1200±31c 822±47d 355±28e 593±26de 1789±38b 2337±41a
Octane 13920±181b 16972±174a 12739±176bc 9029±180cd 11830±153c 11574±168c 8083±107d
2-octene 1075±44e 3327±48b 2111±26c 1383±24d 1841±37cd 3368±57b 4505±35a
1-heptene 291±19ab 547±22a 210±15ab 129±11b 120±9b - -
Ketones
2-propanone 15113±104c 17980±114c 21044±195bc 20689±107bc 27303±163ab 32990±221a 33102±228a
2-Butanone 5661±96e 7826±82d 9716±87c 9680±85c 11751±101b 14666±107a 10120±84bc
Aldehydes
Propanal 8421±142ab 6060±169c 7241±189b 7241±133b 9038±154a 6115±157c 2948±91d
Butanal 10892±131a 8833±126bc 9592±145b 8321±229c 9387±152ab 7037±121d 6153±134e
Pentanal 96422±304a 65251±384bc 69319±335b 56315±3432c 59438±314c 21064±247b 13840±186e
Hexanal 359826±1285a 279277±1048bc 286879±1048b 239163±1104c 244203±999c 112550±897d 9569±607e
Heptanal 13046±118a 7073±81bc 8339±73b 5764±65cd 6409±67c 1744±46d 330±23e
Octanal 2455±46a 856±34bc 1726±32b 633±26bc 949±37bc - -
Nonanal 1974±21a 1530±27ab 1406±23b 983±16c 1082±39bc 313±18d -
Alcohol
Cyclopentanol 1682±33a 788±23b 658±26bc 247±17c 161±9d - -
Others
Dimethyldisulfide 4159±146bc 3843±132c 5492±154a 3427±147cd 4454±113b 3263±101b 2916±75e
Benzoic acid 889±24ab 916±28a 480±14c 263±15d 678±31b - -
1,3-octadiene 193±11c 421±21b 847±26a 88±7d 813±19ab 352±22bc 412±14b
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2-butanone on 1st day was found in T2 and T3 groups however, at the end of storage highest 2-
butanone was reported in T5 14666±107 (ion counts×104) trailed by T4 and T6 11751±101 &
10120±84 (ion counts×104), respectively whilst, the lowest in T0 5661±96 (ion counts×104).
Among treatments, cyclopentanol was the only alcohol detected in antioxidant enriched patties.
Cyclopental was not observed in the samples at start nonetheless, the value for this trait at
termination in T0, T1, T2, T3 and T4 groups were 1682±33, 788±23, 658±26, 247±17 & 161±9
(ion counts×104), respectively. Similarly, dimethyl disulfide at the beginning of storage varied
from 2081±239 to 4037±133 (ion counts×104) while, at termination ranged from 2916±75 to
4159±146. Additionally, benzoic acid and 1,3-octadiene were not found consistent among
treatments and storage.
It has been proven that incorporation of antioxidants significantly affect the volatiles production
in meat based products. The results of current exploration are in agreement with Vieira et al.
(2012); they reported positive correlation between aldehyde compounds and TBARS in poultry
meat products. Likewise, Sivadier et al. (2008) also stated that aldehydes production in meat
products is attributed to lipid oxidation/degradation which is further increased with cooking and
storage. One of the researchers groups, Ismail et al. (2009) reported that addition of antioxidants
especially sesamol, ascorbic acid and α-tocopherol in ground beef are effective in reducing the
production rate of aldehydes during storage. They further reported that aldehydes generation
increased as a function of storage however, most significant increase was reported in control
group. Similarly, Lee et al. (2003) indicated that sulfur compounds are considered vital for off
flavor development in cooked meat products however, they escape with storage due to high
volatility. One of the scientists groups, Du et al. (2003) reported that hexanal and pentanal are
major indicators of lipid oxidation and generally their values increased rapidly with storage,
reflecting relationship between aldehydes and lipid oxidation. Furthermore, Nam et al. (2003)
indicated that addition of antioxidants is effective in controlling oxidative deterioration
especially in ground meat products such as patties and sausages. Likewise, (Ahn et al. (2001)
stated that 2-propanone mainly arise from the autoxidation or β-oxidation of fatty acids that are
considered as precursors to contribute fatty aroma in cooked meat (Pham et al., 2008). Similarly,
Veberg et al. (2006) indicated that hexanal, a grassy aroma compound is the most abundant
aldehyde recorded in cooked meat products. It has also been observed that hexanal in ground
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meat products arises from oxidative decomposition of fatty acids as poultry meat is a rich source
of polyunsaturated fatty acids.
From the current debate, it is concluded that supplementation of quercetin and α-tocopherol to
the poultry birds enhanced the antioxidant status of meat by diminishing the peroxidation thereby
increased the oxidative stability of resultant meat product. Similarly, addition of antioxidants is
also effective to improve the flavor profile of patties. Decisively, antioxidants i.e. quercetin and α-
tocopherol are effectual to enhance the functional worth and quality of meat based products.
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Chapter 5
Summary
Novel dietary guidelines have established a strong relationship between functional foods and
human health indicating the affirmative role of antioxidant enriched foods in diet based
therapies. Scientific investigations regarding functional foods as a preventive approach are
rapidly growing owing to their safe status. The current study was an attempt to develop
functional broiler meat and its products through quercetin and α-tocopherol dietary
supplementation to the broiler birds. For the purpose, 300 day old birds were used as an
experimental animal that were given three levels of quercetin @100, 200 and 300 mg/kg feed in
combination with α-tocopherol @150, 225 and 300 mg/kg feed. To explicate the effect of
antioxidants on growth performance, various parameters like weight gain, feed intake and feed
conversion ratio (FCR) of the birds were measured. The resultant meat was subjected to
antioxidant assay, lipid stability and quantification of antioxidants followed by product
development phase. Finally, the antioxidant enriched functional meat nuggets were provided to
the human subjects to assess their therapeutic potential against hyperlipidemia. The data obtained
from various parameters were subjected to statistical analysis and results are summarized below.
The results indicated that feed imparted momentous effect on weight gain and FCR of birds
however, non-momentous differences were noticed for feed intake. The highest weight gain was
recorded in T9 2374.67±3.53 & 2388±6.43 g/bird followed by T8 and T6 as 2350±6.93 &
2353.33±4.0 and 2293.33±3.48 & 2307±4.36 g/bird, respectively whilst lowest in T0 (control) as
1992.67±4.37 & 1999±6.81 g/bird) during the experimental year 2013 and 2014. Similarly, birds
fed on α-tocopherol and quercetin enriched diets revealed minimum FCR in T9 1.686±0.035
trailed by T8 and T6 as 1.701±0.031 &1.722±0.027, correspondingly whereas maximum in T0
(1.938±0.026) in respective years. Moreover, feed intake of birds at the 1st week varied from
157.67±3.38 to 176.33±2.33 and 153.00±11.4 to 165.33±1.20 that increased to 976.00±2.00 to
1035.33±10.5 and 977.00±1.53 to 1036.00±10.4 g/bird at the termination of trial.
The antioxidant potential of broiler meat was estimated through total phenolic contents (TPC)
along with DPPH and FRAP assay. The TPC of breast meat ranged from 103.87±0.94 to
158.70±0.84 and 104.67±3.52 to 157.63±0.54 mg GAE/100g whilst for leg meat 108.20±1.01 to
156.77±0.94 and 106.27±2.34 to 155.33±1.98 mg GAE/100g meat during the respective years.
155
Likewise, DPPH assay of breast meat indicated significant scavenging activity by T9
(82.40±0.93%) followed by T8, T6 as 80.49±0.79, 77.22±0.58%, respectively comparative to T0
(54.71±0.64%) whilst leg meat depicted DPPH value for this trait 77.02±0.98, 74.53±0.91,
72.87±0.61 and 52.31±0.91% in respective treatments. The ferric reducing power of breast meat
varied from 543.67±1.86 to 683.00±3.79 and 541.67±3.28 to 681.00±2.65 however, in leg meat
ranged from 542.33±3.53 to 674.67±3.53 and 541.00±2.52 to 672.00±1.53 µmol/Fe+2/g meat
among treatments in study year I and II, respectively.
The oxidative stability of broiler meat as measured by TBARS assay in both years indicated
values for breast meat on 1st min 0.181±0.0032 to 0.282±0.0023 and 0.229±0.0153 to
0.441±0.1594 mg of MDA/kg meat that subsequently increased to 0.283±0.0174 to
0.384±0.0175 and 0.314±0.0157 to 0.425±0.0289 mg of MDA/kg meat at the end of storage. The
lowest value for TBARS were noticed in T9 0.298± 0.0119 trailed by T8 and T6 0.309±0.0120
& 0.315±0.0184 compared to T0 0.405±0.0170. Likewise, TBARS of leg meat observed in T9,
T8, T6 and T0 as 0.305±0.0130, 0.315±0.0125, 0.332±0.0128 and 0.406±0.0128 mg MDA/kg
meat, respectively. The values of TBARS in broiler meat elucidated linear increase with
progression of storage.
The deposition of supplements i.e. α-tocoperol and quercetin in broiler meat varied significantly
among treatments. HPLC quantification of α-tocopherol in breast meat showed maximum α-
tocoperol in T9 (38.02±0.52) followed by T8 and T6 (31.77±0.55 & 29.89±0.25),
correspondingly whereas minimum by T0 (10.70±0.17mg/kg meat). Similarly, highest α-
tocpherol for leg meat was noticed as 35.21±0.67 in T9 trailed by 28.65±0.29 and 27.33±0.33 in
T8 and T6, respectively compared to T0 (9.96±0.20 mg/kg meat). The maximum quercetin level
for breast meat was recorded in T9 (16.36±0.01) followed by T8 and T3 (14.67±0.03 and
14.13±0.06), respectively although it was not detected in T0 (control) samples. Likewise,
quercetin contents in leg meat were between 0 to 14.23±0.04 and 0 to 14.12±0.01 mg/kg meat
during the respective experimental years.
The dietary supplementation of quercetin and α-tocopherol to the birds resulted momentous
effect on fatty acid composition of broiler meat. It has been observed 8 fatty acids in meat
samples mainly include palmitic acid (C16:0), stearic acid (18:0), oleic acid (18:1n-9) and
linoleic acid (18:2n-9, 6). Nonetheless, α-linoleic acid (18:3) and arachidonic acid (20:4) were
156
not differed substantially with respect to treatments in both years. The results expounded lowest
saturated fatty acids (SFA) in breast meat by T9 (21.69) followed by T8 & T6 (23.01 and 23.93),
respectively whereas highest in T0 (30.76). Similarly, T9 showed lowest PUFA as 9.88 & 9.62
trailed by T8 & T6 (10.04 & 9.96 and 10.36 & 9.97), correspondingly as compared to T0 (13.41
& 12.60) in experimental years. Likewise, leg meat exhibited minimum SFA as 30.21 & 31.99 in
T9 followed by 31.52 & 28.51 and 32.61 & 31.06 in T8 & T6, respectively as compared to T0
(37.82 & 36.02). Overall, fatty acid production was decreased with increasing quercetin and α-
tocopherol levels that depicted their lipid lowering potential though, reduction in SFA was more
obvious than that of PUFA in meat.
The results further elucidated that treatments imparted momentous effect on the activity of
antioxidant enzymes i.e. superoxide dismutase (SOD), glutathione reductase (GRs) and catalase
of broiler blood serum. The SOD activity was varied between 3.44±0.52 to 54.09±0.81 and
33.13±1.02 to 53.32±1.04 U/mg of protein during the year I and II. Likewise, maximum SOD
activity recorded in T9 (196.93±0.41) trailed by T8 and T6 that were 193.29±0.87, 186.86±0.54
whereas lowest in T0 (136.68±0.79) U/mg of protein. Similarly, performance of GRs and
catalase was increased with dietary supplementation of antioxidants. The maximum values for
GRs and catalase were 53.70±0.61 &148.74±0.41 U/mg of protein in T9 followed by T8 and T6
(52.34±0.59 & 147.26±0.56), (50.90±0.48 & 144.29±0.53) whereas minimum in T0 133.29±0.52
& 118.62±0.59 U/mg of protein in respective years.
The lipid profile of broiler birds fed on quercetin and α-tocopherol enriched diets varied
substantially among the treatments. The results specified total cholesterol (106.27±0.23 to
132.24±0.56 and 108.29±0.30 to 134.49±0.39 mg/dL), HDL (60.79±0.26 to 79.53±0.56 and
59.77±0.23 to 78.38±1.14 mg/dL), LDL (24.61±0.44 to 42.56±0.41 and 25.46±0.40 to
43.27±0.7), triglycerides (48.83±0.23 to 69.35±0.49 and 48.79±0.25 to 70.22±0.61 mg/dL) and
serum protein (2.62±0.02 to 4.94±0.02 and 2.56±0.03 to 4.91±0.03 g/dL) among treatments. The
resulted further suggested a declining trend for cholesterol, LDL and triglycerides with
supplementation of quercetin and α-tocopherol in a dose dependent manners. Minimum
cholesterol was recorded in T9 (107.28±0.48 mg/dL) followed by T8 and T6 (109.72±0.20,
110.57±0.45 mg/dL) whereas highest value of this trait in T0 (133.37±0.59 mg/dL). Similarly,
lowest LDL and triglycerides were also recorded in T9 (25.04±0.33 & 48.81±0.15mg/dL)
followed by T8 and T6 (27.95±0.39 & 50.98±0.33 mg/dL) and (28.86±0.36 & 55.04±0.30
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mg/dL) whereas highest in T0 (42.91±0.40& 69.79±0.40 mg/dL) in respective study years.
Additionally, highest serum protein was exhibited by T9 (4.93±0.02 g/dL) trailed by T8 and T6
(4.71±0.18 & 4.41±0.01 g/dL) however, lowest value of the trait was observed in T0 (2.59±0.02
g/dL).
The resultant functional broiler meat was used to prepare functional meat nuggets stored at
freezing condition for 60 days. During storage, nuggets were subjected to physicochemical
analysis which showed that treatments and storage imparted momentous differences on the color,
pH, texture and TBARS of the product tested at 1st, 15th, 30th, 45th and 60th day. The results
indicated that at initiation of storage, color of breast meat nuggets for different groups T0, T1,
T2, T3, T4, T5, T6, T7, T8 and T9 were 102.33±0.882, 103.67±1.453, 105.33±0.882,
111.67±1.453 and 108.33±0.882, 113.67±1.453, 121.67±0.882, 116.67±1.453, 123.67±0.667 and
124.67±1.453 CTn that subsequently decreased to 88.33±0.882 (T0), 89.67±1.453 (T1), 91±1.0
(T2), 98.67±1.453 (T3), 93.67±1.453 (T4), 99.67±1.453 (T5), 104.67±1.453 (T6), 101.67±1.453
(T7), 106±1 (T8) and 109.67±1.453 CTn (T9) at termination of storage. Similarly, the value of
this trait for leg meat nuggets on 1st day varied from 91.33±0.882 to 113.33±0.333 and
88.67±0.882 to 111.67±1.202 CTn that significantly dwindled to 80.33±0.333 to 104.67±0.882
and 79.33±0.882 to 101.67±0.882 CTn at the completion of storage. The results showed a linear
decline in color as a function of storage.
Additionally, pH of nuggets on 1st day varied from 5.89±0.047 to 6.53±0.023 and 5.93±0.009 to
6.49±0.009 that subsequently increased to 6.19±0.020 to 6.65±0.020 and 6.16±0.012 to
6.62±0.012. Likewise, the value for this trait in leg meat nuggets exhibited identical trend with
highest pH in T9 (6.47±0.009) followed by T8 and T6 (6.45±0.009 & 6.43±0.010) whilst lowest
in T0 (5.92±0.023) at the end of storage. It has been documented that treatments impart
momentous effect on the shear force of nuggets however, its value increased linearly with
progression of storage.
The oxidative stability of nuggets as measured by TBARS indicated that oxidative stability of
nuggets decreased as a function of storage. The TBARS of breast meat nuggets on initation of
storage varied from 0.32±0.012 to 1.30±0.021 and 0.45±0.021 to 1.42±0.018 mg of MDA/kg
meat that subsequently increased from 1.24±0.029 to 2.51±0.054 and 1.36±0.015 to 2.54±0.029
mg of MDA/kg meat at the termination of storage in both years. The means depicted lowest
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TBARS by T9 (0.77±0.062) followed by T8 and T6 (0.86±0.065 and 0.89±0.066) whilst , highest
by T0 (1.83±0.079). Similarly, TBARS of leg meat nuggets on 1st day varied from 0.63±0.012
to 1.64±0.015 and 0.67±0.009 to 1.67±0.015 that were subsequently increased from 1.61±0.018
to 2.83±0.023 and 1.64±0.020 to 2.88±0.017 mg of MDA/kg meat during respective
experimental years. The results further suggested that leg meat nuggets exhibited higher TBARS
than that of breast meat nuggets however, the value of this trait showed linear increase with
progression of storage.
Sensory response was also assessed using 9-point hedonic scale by assigning score for
appearance, flavor, taste and overall acceptability of the antioxidant enriched meat nuggets.
Mean squares for sensory evaluation expounded significant differences due to treatments and
storage however, their interaction except treatments × storage showed non momentous
variations. The results indicated that on 1st day, appearance of breast meat nuggets varied from
7.35±0.029 to 8.30±0.029 and 7.30±0.029 to 8.25±0.050 that subsequently decreased from
6.05±0.029 to 7.15±0.029 and 6.00±0.029 to 7.12±0.060 in the respective experimental years.
Likewise, the value of this trait for leg meat nuggets on 1st day ranged between 7.15±0.029 to
8.00±0.026 and 7.07±0.033 to 7.90±0.026 that subsequently reduced from 5.80±0.029 to
6.80±0.029 and 5.75±0.029 to 6.75±0.029 at termination. The results further suggested that taste,
flavor and overall acceptability score declined as a function of storage. The score for overall
acceptability at initiation varied from 7.35±0.029 to 8.30±0.029 and 7.25±0.027 to 8.20 ±0.034
in the year 2013 and 2014 that diminished from 5.95±0.018 to 7.02±0.017 and 5.85±0.034 to
6.92±0.017 in respective years. Moreover, means for overall acceptability of leg meat nuggets
recorded maximum value for this trait in T9 (7.39±0.082) trailed by T8 and T6 that were
(7.29±0.08, 7.24±0.083) whereas minimum in T0 (6.47±0.096) group.
On the basis of antioxidant potential, physico-chemical analysis, serum biomarkers & sensory
attributes, four best treatments were selected along with control for bioefficacy study. Purposely,
25 volunteers were divided into 5 groups in which G0 group rely on control broiler meat whereas
remaining groups G1 (300 mg of quercetin + 300 mg of α-tocopherol), G2 (200 mg of quercetin
+ 300 mg of α-tocpherol), G3 (200 mg of quercetin + 300 mg of α-tocopherol) and G4 (300 mg
of quercetin + 150 mg of α-tocopherol) were provided antioxidant enriched functional meat
@130 g/volunteer/daily. Mean squares showed momentous effect on lipid profile of human
subjects through consumption of antioxidant enriched functional meat. The results delineated
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maximum cholesterol in G0 (154.2±5.45 mg/dL) followed by G4 (149.2±3.45 mg/dL), G3
(147.5±5.36 mg/dL), G2 (145.0±4.8) groups however, minimum value (141.6±4.3 mg/dL) in G1.
The percent reduction indicated that G1 resulted maximum decline 8.17 &7.59% followed by G2
(5.97 & 5.08%), G3 (4.35 & 3.73%) and G4 (3.24 & 2.70%) in respective trials. Likewise,
maximum LDL cholesterol was recorded in G0 (79.5±3.24 mg/dL) trailed by G4 (76.8±4.13
mg/dL), G3 (75.9±3.76 mg/dL) and G2 (75.1±2.96) groups however, minimum LDL was
recorded in G1 (73.6±3.64 mg/dL). Considering percent reduction in LDL, G1 caused maximum
reduction (7.42 &7.72%) trailed by G2 (5.53 & 5.11%), G3 (4.53 & 4.48%) and G4 (3.40 &
3.86%) in trial I and II, respectively. Moreover, lowest HDL cholesterol was reported in G0
(49.1±3.1 mg/dL) followed by G4 (50.44±4.75 mg/dL), G3 (50.75±3.91 mg/dL), G2 (51.2±4.26
mg/dL) groups however, highest level was noticed in G1 (51.3±3.55 mg/dL).
Treatments showed substantial differences on triglycerides and serum total protein in human
subjects those provided functional meat. The highest triglycerides level was found in G0
(144.5±5.3 mg/dL) while the value of this trait in G1, G2, G3 and G4 were 129.5±6.4,
132.6±5.8, 134.1±7.5 and 136.5±8.6 mg/dL, respectively. The percent diminish in triglycerides
10.38, 8.24, 7.20 and 5.54% were reported in G1, G2, G3 and G4, correspondingly. Besides,
lowest protein was documented in G0 (3.98±0.26 g/dL) followed by G4 (4.12±0.19 g/dL), G3
(4.25±0.18 g/dL) and G2 (4.45±0.27 g/dL) groups although, maximum in G1 (4.60±0.31g/dL).
The percent increase in total protein was 15.58, 11.81, 6.78 and 3.52% in G1, G2, G3 and G4,
correspondingly.
Furthermore, at Iowa State University, USA, in vitro antioxidant study of patties was carried out
in which treatments and storage depicted momentous differences on TBARS, lightness (L*),
redness (a*) and yellowness (b*) of quercetin and α-tocopherol enriched product. At initiation,
TBARS value in T0, T1, T2, T3, T4, T5, T6 groups were 1.93±0.02, 0.80±0.01, 0.66±0.06,
0.63±0.05, 0.58±0.02, 0.38±0.03, 0.37±.01 mg of MDA/kg meat that subsequently increased to
3.47±0.14 MDA/kg meat (T0), 2.97±0.09 MDA/kg meat (T1), 2.56±0.01 MDA/kg meat (T2),
2.43±0.04 MDA/kg meat (T3), 2.32±0.03 MDA/kg meat (T4), 0.94±0.34 MDA/kg meat (T5),
0.90±0.05 MDA/kg meat (T6) at the termination of storage.
The color analysis showed L* of patties on initiation of storage varied from 74.67±0.62 to
81.14±0.37 that decreased upto 73.66±0.80 to 79.96±1.56 as a function of storage. The
160
maximum L* reported in T0 (80.77±0.98) followed by T1 and T2 (78.778±0.87 & 78.57±0.76)
whilst, lowest in T6 (73.90±0.77). Similarly, at the beginning a* value varied from -3.50±0.15 to
-2.17±0.29. The results expounded highest a* value in T6 (-2.09±0.19) trailed by T5 and T4 (-
2.27±0.19 & -3.51±0.09) whereas lowest value of this trait was noticed in T0 (-4.181±0.17).
Likewise, b* value ranged from 16.23±1.28 to 18.18±0.23 that were diminished with progression
of storage.
The off flavor generation is a critical problem that affect storability of cooked meat products.
Hydrocarbons are among the major class of volatile compounds that contribute flavor in meat
based products. Among hydrocarbons, pentane, heptane, 1-octene, octane and 2-octene were
found in samples at storage. On 1st day, pentane, heptane, 1-octene, octane and 2-octene were
varied from 3013±188 to 104062±1824, 349±29 to 3808±129, 352±31 to 2239±109, 6439±413 to
32775±976, 191±11 to 2533±219, correspondingly in different treatments. Heptane and octane
were diminished with increasing levels of quercetin and α-tocopherol.
Aldehydes play imperative role in flavor development of ground meat products like patties,
sausages etc. On 1st day, highest hexanal was reported in T0 (control) and lowest in T5 and T6
containing 100 ppm quercetin dehydrate in combination with 200 ppm of α-tocopherol that were
significantly increased at termination of study. Additionally, propanol, heptanal, octanal and
nonanal also determined in treatments that showed a positive association with storage.
Among ketones, 2-propanone and 2-butanone were found in the patties samples. At initiation,
lowest 2-propanone was recorded in T6 6408±163 trailed by T5 and T4 6598±201 & 7081±237
whilst, highest in T0 12309±289 that increased to 33102±228, 32990±221, 27303±163 and
15113±104 in respective treatment at the end of study. Cyclopentanol was observed in samples
only on 7th day of storage however, T6 (100ppm of quercetin and 200 ppm of α-tocopherol)
combination inhibited its production. Additionally, dimethyldisulfide, benzoic acid and 1,3 -
octadiene were also recorded in the samples. The dimethyldisulfide diminished with storage
nonetheless, benzoic acid and 1,3-octadiene were not consistent among treatments.
In the nutshell, supplementation of quercetin and α-tocopherol to the broiler birds enhanced the
antioxidant status of meat by diminishing the peroxidation thus increased the storage stability of
resultant product. The in vitro study also indicated the potential of quercetin and α-tocopherol to
mitigate oxidation of cooked meat patties. From the current exploration, it is inferred that
161
supplementation of quercetin and α-tocopherol is a practical approach to increase the functional
value of meat and allied products.
162
RECOMMENDATIONS
Use of functional meat products should be encouraged for their significance in improving
human health.
Antioxidant enriched functional meat should be promoted in the daily diet as shield
against various lifestyle oriented disorders.
Healthy ingredients like quercetin and α-tocopherol can be added in feed to improve
growth performance, lipid stability and nutritional quality of broiler meat.
Broiler breast meat containing functional ingredients could be used for the development
of healthier meat product with improved volatile profile.
Researchers/academia and industrialists should collaborate and tailor projects to address
meat industry oriented problems.
Dietitians should recommend functional meat products to manage lipid profile and to
cope with protein energy malnutrition.
Community based programs ought to be launched to enlighten the consumers towards the
use of fortified meat based products for better nutrition & health.
163
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Appendix I: Composition of basal diet
Ingredients Quantity (g/kg feed)
Corn 490
Rice broken 20.7
Rice polishing 56.0
Cotton seed meal 22.0
Canola meal 20.0
Corn gluten 60% 23.0
Sunflower meal 124.0
soybean meal 150.0
Fish meal 66.0
L-lysine 1.50
DL-methionine 00.8
Dicalcium phosphate 12.0
Limestone 12.0
Premix 02.0
Nutrient composition (calculated)
Metabolized energy (Kcal /kg) 2934
Crude protein (%) 21.03
Lysine (%) 1.10
Methionine (%) 0.52
191
APPENDIX II
Performa for sensory evaluation of antioxidant enriched broiler meat nuggets Name of the judge……………………………….. Date……………..........................
Parameters
Treatments
T0 T1 T2 T3 T4 T5 T6 T7 T8 T9
Appearance
Flavor
Taste
Overall
acceptability
Signature……………………..
INSTRUCTIONS
Eat antioxidant enriched broiler meat nuggets and score for appearance, flavor, taste and overall
acceptability using the following 9-point Hedonic Scale:
Extremely poor 1
Very poor 2
Poor 3
Below fair above poor 4
Fair 5
Below good above fair 6
Good 7
Very good 8
Excellent 9
Note:
1. Eat Functional broiler meat nuggets and score for appearance and flavor etc.
2. Before proceeding to the next sample, rinse mouth with water.
3. Make inter comparison of the sample and record the score.
4. Don't disturb the order of samples.