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Evaluation of Physicochemical and Mineral Analysis in
Chicken Egg and Honey with the Special Implication of
Health Risk Assessment in Haryana, India
THESIS SUBMITTED TO BABA MASTNATH UNIVERSITY ASTHAL BOHAR, ROHTAK
(HARYANA), INDIA
FOR PARTIAL FULFILLMENT OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ZOOLOGY
By
NAMRATA
Regn No.: 20-BMU-6860
Co-Supervisor
Supervisor
(Dr. Arun Kumar)
Scientist- I & Principal Investigator
Mahavir Cancer Institute and Research Centre
Phulwarisharif, Patna - 801505
Bihar, India
(Dr. Arup Giri)
Associate Professor
Department of Zoology,
Baba Mastnath University,
Asthal Bohar, Rohtak, Haryana
DEPARTMENT OF ZOOLOGY
FACULTY OF SCIENCES
BABA MASTHNATH UNIVERSITY
ASTHAL BOHAR ROHTAK- 124021 HARYANA (INDIA)
September 2024
CERTIFICATE
This is to certify that the thesis entitled “Evaluation of Physicochemical and Mineral Analysis
in Chicken Egg and Honey with the Special Implication of Health Risk Assessment in Haryana,
India” submitted to the Baba Masthnath University, in partial fulfillment of the requirements for the
award of the Degree of Doctor of Philosophy in Zoology is a record of original research work done by
Ms. Namrata during the period of 11th February 2022 to 11th September 2024 of her study in the
Department of Zoology, Faculty of Sciences, Baba Masthnath University, Asthal Bohar, Rohtak- 124021,
Haryana (India), under my supervision and guidance and the thesis has not formed the basis for the award
of any Degree / Diploma / Associateship / Fellowship or other similar title to any candidate of any
University.
Co- Supervisor Supervisor
(Dr. Arun Kumar)
Scientist- I & Principal Investigator
Mahavir Cancer Institute and Research Centre
Phulwarisharif, Patna - 801505
Bihar, India
(Dr. Arup Giri)
Associate Professor
Department of Zoology,
Baba Mastnath University,
Asthal Bohar, Rohtak, Haryana
DECLARATION
I Namrata hereby declare that the thesis, entitled “Evaluation of Physicochemical and
Mineral Analysis in Chicken Egg and Honey with the Special Implication of Health Risk
Assessment in Haryana, India” submitted to the Baba Masthnath University, in partial fulfillment
of the requirements for the award of the Degree of Doctor of Philosophy in Zoology is a record
of original and independent research work done by me during 11th February 2022 to 11th
September 2024 under the supervision and guidance of
Dr. Arup Giri in the Department of Zoology, Faculty of Sciences, Baba Masthnath University,
Asthal Bohar, Rohtak- 124021, Haryana (India), and it has not formed the basis for the award of
any Degree / Diploma / Associate ship / Fellowship or other similar title to any candidate in any
University. The work done in the thesis is plagiarism free as per the rules and regulations of
Baba Mastnath University.
(Namrata)
Ph.D. Candidate
Baba Mastnath University
Regn. No.: 20-BMU-6860
Place: Asthal Bohar, Rohtak, Haryana
Date:
This is verified that the above statement given by the student is right to the best of my
knowledge.
Co- Supervisor Supervisor Head of Department
(Dr. Arun Kumar)
Scientist- I & Principal Investigator
Mahavir Cancer Institute and
Research Centre
Phulwarisharif, Patna - 801505
Bihar, India
(Dr. Arup Giri) (Dr. Vikash Bhardwaj)
Associate Professor Associate Professor
Department of Zoology, Department of Zoology,
Faculty of Sciences, Faculty of Sciences,
Baba Mastnath University, Baba Mastnath University,
Asthal Bohar, Rohtak, Asthal Bohar, Rohtak,
Haryana Haryana
ACKNOWLEDGEMENTS
As with any piece of research that results in the production of a thesis, on the cover there
should be not only the name of the researcher, but also the names of all those unsung heroes,
those who to varying degrees provided assistance, encouragement and guidance. This thesis
would not have been possible without the guidance and the help of several individuals who in
one way or another contributed and extended their valuable assistance in the preparation and
completion of this study. It is a pleasure to thank those who made it a possibility.
Foremost, I would like to express my deep sense of gratitude and earnest thanks to my
esteemed supervisor, Dr. Arup Giri, Associate professor in Department of Zoology, Baba
Mastnath University, Rohtak, whose guidance, support and expertise have enabled me to
complete my PhD. His dedication and keen interest above all his overwhelming attitude to help
his students had been solely and mainly responsible for completing my work. From the initial
stages of refining my research proposal to the final submission of my thesis, his unwavering
presence and wealth of wisdom have been instrumental in shaping my academic growth. I highly
valued the biweekly meetings, which not only served as crucial checkpoints to keep me on track
academically, but also provided me with plenty of encouragement. Your insights have greatly
enriched the quality of my work. I am profoundly grateful for the immeasurable contributions he
made to my development and cannot envision a better PhD supervisor than him. I aspire to
follow in his footsteps one day.
I owe a deep sense of gratitude to my Co-Supervisor, Dr Arun Kumar, Senior Scientist
at Mahavir Cancer Institute and Research Centre, Patna, for his invaluable guidance, helpful
timely suggestions.
I am thankful to Baba Balak Nath Ji. (Chancellor) of Baba Mastnath University and Dr.
H.L. Verma (Vice Chancellor), Dr. Naveen Kapil (Dean Academic Affair), Dr. Ravi Kumar
Rana (Dean, faculty of Science), Dr. Manoj Kumar Verma, (Registrar) for allowing me to
complete this research work and for providing the facilities.
I am extremely grateful to Dr. Sunil, Assistant Professor and Kamlesh Kumar,
Research Scholar, University of Rajasthan, Jaipur and Poonam Mundlia, Research Scholar,
Baba Mastnath University for their assistance during sample analysis.
I am extremely grateful to Dr. Indu Sharma, Assistant Professor, Department of
Zoology, Panjab University for providing the necessary laboratory facilities and for their helpful
timely suggestions.
I am thankful to Dr. Sonu Saini, Dr. Anil Kumar and Dr. Vicky for their kind help
during sample analysis.
I am also thankful to our HOD, Dr. Vikash Bhardwaj, Associate Professor and Dr.
Parveen Kumar, Department of Zoology, Baba Mastnath University.
I am heartily thankful to Dr. Neha Rani Bhagat and Mr. Shardulya Shukla, Research
Scholar DRDO, DIHAR for their help during sample analysis.
I am thankful to my labmates Neeru Yadav, Geetika, Manisha Yadav, Dr. Sharda
Rani, Dr. Manjeet, Ruby, Dimple, Manju, Deepak Attri, Ajay and Manisha for their timely
help and motivation. Our collaborative writing sessions and informal chats provided a lifeline
during the challenging times. I also want to pay thanks to all the non teaching staff including
Miss Meena Ma’am, Miss Kavita Lab att. Sh.Subash sir for their regular help during the course
of study.
Words are insufficient to express my indebtedness to my family who supported me all the
way. I want to express my deepest gratitude to my family for their belief and support.Your
encouragement played an important role in my accomplishments. All the blessings and
inspiration from my parents Mr. Manoj and Mrs. Sarita made it possible to complete the
present work. I thank you for always being my rock and accompanying me through the highs and
lows of this academic journey. I hope you are aware of and proud of the significant role you have
played in shaping my path. To my father: Thankyou for everything! I dedicate this PhD thesis to
you.
To my brother Vikrant and my serotonin booster Rumi, thank you for your love. My
friends Twinkle, Aamna, Anjali, Shikha, Karuna and Pooja you have been my biggest
cheerleader throughout this journey and I cannot thank you enough for your love and support.
Finally, I would like to thank God, for letting me through all the difficulties.
Dated: (Namrata)
LIST OF TABLES
Table
No.
Title
Page
No.
2.1
Reported values of physicochemical parameters of honey in various
studies.
8-9
2.2
Reported antioxidant activity of honey in previous studies.
11
2.3
Reported ion and mineral contents in honey samples from different
studies conducted worldwide.
17-18
2.4
Reported values of physicochemical parameters of egg in various
studies.
24-25
2.5
Reported antioxidant activity of egg in previous studies.
25-26
2.6
Reported ion and mineral contents in egg samples from different
studies conducted worldwide.
31
4.1
Mean values of physicochemical parameters of honey samples
collected from various districts of Haryana.
54
4.2
Mean values of physicochemical parameters of egg samples collected
from various districts of Haryana in summer.
56-57
4.3
Mean values of physicochemical parameters of egg samples collected
from various districts of Haryana in winter.
57-58
4.4
Variation of physicochemical parameters in hen egg in different
seasons in Haryana
59-60
4.5
Mineral and heavy metal contents in honey samples collected from
different districts of Haryana.
61-62
4.6
Mineral and heavy metal contents in egg samples collected from
different districts of Haryana in summer.
63-64
4.7
Mineral and heavy metal contents in egg samples collected from
different districts of Haryana in winter.
65
4.8
Variation of trace metals in chicken egg in different seasons in
Haryana.
66-67
4.9
Antioxidant activity of honey samples collected from different
locations.
68
4.10
Antioxidant activity of egg samples collected from different locations
during summer season.
70
Table
No.
Title
Page
No.
4.11
Antioxidant activity of egg samples collected from different locations
during winter season.
70
4.12
Calculated EDI and HQ for metals in honey in both adults and children
in Haryana.
85
4.13
Calculated EDI and HQ for metals in egg samples in both adults and
children in summer season in Haryana.
89
4.14
Calculated EDI and HQ for metals in egg samples in both adults and
children in winter season in Haryana.
89-90
LIST OF FIGURES
Figure
No.
Title
Page
No.
2.1
Beneficial properties of honey.
13
2.2
Heavy metal contamination in honey through food chain.
16
2.3
Effects of heavy metals on human health.
19
2.4
Benefits of chicken egg to human health.
29
2.5
Heavy metal contamination in hen egg through food chain.
32
3.1
All honey and egg samples were collected from four 4 districts of Haryana
viz. Rohtak, Hisar, Gurgaon and Panipat.
34
3.2
Samples were collected from local apiaries, beehives and poultry farms.
36
3.3
Egg sample and data collection from poultry farms.
37
3.4
Egg samples collected during summer season.
37
3.5
Egg samples collected during winter season.
38
3.6
Honey samples collected from different locations.
38
3.7
Analysis of physicochemical parameters of honey and egg samples.
40
3.8
Sample processing for analysis of antioxidant content in honey and egg
samples.
42
3.9
UV-VIS spectrophotometer used for analysis of antioxidant content in
honey and egg samples.
43
3.10
Sample processing for metabolomic analysis of honey samples.
44
3.11
Sample processing for metabolomic analysis of egg samples.
46
3.12
GC-MS system used to analyze the metabolites present in honey and egg
samples.
47
3.13
Honey sample digestion for mineral analysis.
48
3.14
Egg sample digestion for mineral analysis.
49
4.1
Variation in physicochemical parameters of honey in different regions of
Haryana.
55
Figure
No.
Title
Page
No.
4.2
Variation in Physicochemical parameters of egg samples in different
regions of Haryana during summer.
58
4.3
Variation in Physicochemical parameters of egg samples in different
regions of Haryana during winter.
59
4.4
Seasonal variation in physicochemical parameters of egg samples.
60
4.5
Variation in mineral content of honey in different regions of Haryana.
62
4.6
Variation in mineral content of egg samples during summer.
64
4.7
Variation in mineral content of egg samples during winter.
66
4.8
Seasonal variation in mineral content of egg samples.
67
4.9
Seasonal variation in antioxidant capacity of honey samples
69
4.10
Seasonal variation in antioxidant capacity of egg samples
71
4.11
GC-MS Chromatogram of honey samples from Rohtak.
72
4.12
GC-MS Chromatogram of honey samples from Gurgaon.
73
4.13
GC-MS Chromatogram of honey samples from Hisar.
74
4.14
GC-MS Chromatogram of honey samples from Panipat.
75
4.15
GC-MS Chromatogram of egg samples from Rohtak during summer.
76
4.16
GC-MS Chromatogram of egg samples from Gurgaon during summer.
77
4.17
GC-MS Chromatogram of egg samples from Hisar during summer.
78
4.18
GC-MS Chromatogram of egg samples from Panipat during summer.
79
4.19
GC-MS Chromatogram of egg samples from Rohtak during winter.
80
4.20
GC-MS Chromatogram of egg samples from Gurgaon during winter.
81
4.21
GC-MS Chromatogram of egg samples from Hisar during winter.
82
4.22
GC-MS Chromatogram of egg samples from Panipat during winter.
83
4.23
Calculated EDI of honey in different regions of Haryana.
84
4.24
Calculated HQ of honey in different regions of Haryana.
85
Figure
No.
Title
Page
No.
4.25
Calculated EDI of egg in different regions of Haryana during summer.
86
4.26
Calculated EDI of egg in different regions of Haryana during winter.
87
4.27
Calculated HQ of egg in different regions of Haryana during summer.
88
4.28
Calculated HQ of egg in different regions of Haryana during winter.
89
LIST OF ABBREVIATIONS
%
Percentage
&
And
@
At the rate
<
Less than
≥
Greater than
°C
Degree centigrade
°E
Degree East
°N
Degree North
µg/L
Microgram per liter
µmol/L
Micromole per liter
approx.
Approximate
As
Arsenic
BIS
Bureau of Indian Standard
BLQ
Below the limit of quantification
Ca
Calcium
Cd
Cadmium
cm
Centimeter
Co
Cobalt
CO3
Carbonate
Cu
Copper
dL
Deciliter
DPPH
2, 2-diphenyl-1-picryl-hydrazyl
DRDO
Defence Research and Development Organization
eg.
Example
EPA
Environmental Protection Agency
et al.
Et alibi
Fe
Iron
FeCl3
Ferric chloride
FeSO4
Ferrous sulfate
Fig.
Figure
FRAP
FSSAI
Ferric reducing ability of plasma
Food safety and standards of authority India
g
Grams
g/dL
Gram per deciliter
g/L
GC-MS
Gram per liter
Gas chromatography/Mass Spectrometry
H2O
Water
H2O2
Hydrogen peroxide
HCl
Hydrochloric acid
HClO4
Perchloric acid
HCO3-
Bi-carbonate
HNO3
Nitric acid
hrs
Hours
i.e.
That is
ICP-OES
IR
Inductively coupled plasma optical emission of
spectrometry
Ingestion Rate
ISI
ISO
Indian Standards Institute
International Organization for Standardization
K
Potassium
kg
Kilogram
L or l
Liter
m
Meter
meq/L
Milliequivalents per liter
Mg
Magnesium
mg/dL
Milligram per deciliter
min
Minute
ml
Milli liter
mM
Millimoles
mm
Millimmeter
mmol/L
Millimole per liter
Mn
MSTFA
Manganese
N-Methyl-N-Trimethylsilyl Trifluoroacetamide
Na
Sodium
ng/mL
Nanogram per miliiliter
nm
Nano meter
No.
Number
ºC
Degree Celsius
OD
P
Optical density
Phosphorus
P < 0.0001
Significance at 1% level
P<0.05
Significance at 5% level
Pb
PC
pH
Lead
Protein Content
Potential of hydrogen
Ppm
QC
Rb
Parts per million
Quality control
Rubidium
Rpm
RfD
S
Sb
SD
Rotation per minute
Reference Dose
Sulphur
Antimony
Standard deviation
Se
SE
Selenium
Standard error
sec
Seconds
SEM
SI
Standard error of mean
Shape Index
Sl.No.
Serial number
SPSS
Statistical packages for social science
TPC
Total protein content
Type-II
UV-VIS
Type two
Ultraviolet–visible spectrophotometer
U.S.
United States
UN
United Nations
UNESCO
United Nations Educational, Scientific and Cultural
Organization
UV rays
Ultra violet rays
viz.
Namely
WHO
World Health Organization
Zn
Zinc
μg
Microgram
μL
Microliter
μM
Micromole
CONTENTS
Chapter
No.
Title
Page
No.
LIST OF TABLES
i
LIST OF FIGURES
iv
LIST of ABBREVIATIONS
vi
ABSTRACT
1
INTRODUCTION
1
2
REVIEW OF LITERATURE
6
2.1 Dairy development in Leh-Ladakh
6
2.2 Problems of Dairy Farming at Leh
12
2.3 Dairy cattle health in Leh
13
2.4 Impact of water quality on dairy cattle and human health
2.4.1 Important physico-chemical and microbial parameters of water
2.4.1.1 Groundwater resource
2.4.1.2 River water resource
2.4.2 Water Quality at High Altitude
21
24
25
25
26
2.4 The Role of minerals and heavy metals in dairy cattle health
2.4.1 Sodium
2.4.2 Potassium
2.4.3 Calcium
2.4.4 Magnesium
2.4.5 Manganese
2.4.6 Phosphorus
2.4.7 Sulfur
2.4.8 Iron
2.4.9 Zinc
2.4.10 Boron
2.4.11 Selenium
2.4.12 Silicon
42
42
42
42
43
43
43
43
43
44
44
44
44
Chapter
No.
Title
Page
No.
2.4.13 Aluminum
2.4.14 Copper
2.4.15 Arsenic
2.4.16 Lead
2.4.17 Cadmium
2.4.18 Cobalt
45
45
45
45
45
46
2.5 Role of cattle milk in human health
50
3
MATERIALS AND METHODS
56
3.1 Experimental Design
56
3.2 Study Area
56
Exclusion and Inclusion Criteria to Select the Study Area
Inclusion Criteria
Exclusion criteria
60
60
61
3.3 Sample Collection
3.3.1 Water
3.3.2 Blood
3.3.3 Milk
63
63
63
64
3.4 Sample Processing
3.4.1 Water
3.4.2 Blood
3.4.3 Milk
65
65
65
67
3.5 Analytical Procedure
3.5.1 Water
3.5.1.1 Physico-chemical parameters
3.5.1.2 Microbiological parameters
3.5.1.3 Minerals and Heavy Metals
3.5.2 Blood
3.5.2.1 Hematological parameters
3.5.2.2 Biochemical parameters
67
67
67
68
69
72
72
72
Chapter
No.
Title
Page
No.
3.5.2.3 Antioxidant related parameters
3.5.2.3.1 FRAP assay
3.5.2.3.2 DPPH assay
3.5.2.3.3 ABTS assay
3.5.2.3.4 LPO assay
3.5.2.3.5 Catalase assay
3.5.2.4 Minerals and Heavy Metals
3.5.3 Milk
3.5.3.1 Biochemical
3.5.3.2 Minerals and Heavy Metals
74
74
74
75
75
76
76
76
76
76
3.6 Statistical Analysis
3.6.1 Data of water samples
3.6.2 Data of Blood and Milk samples
77
77
77
4
3 RESULTS
78
4.1 General Information
78
4.2 Seasonal variation of Physico-chemical and microbiological
characteristics of ground water
78
4.3 Seasonal variation of physico-chemical and microbiological
characteristics of river water
82
4.4 Major factors governing ground water quality in summer
season
86
4.5 Major factors governing ground water quality in winter
season
90
4.6 Major factors governing river water quality in summer
season
94
4.7 Major factors governing river water quality in winter season
98
4.8 Seasonal variation in minerals and heavy metals level in
ground water
102
4.9 Seasonal variation in minerals and heavy metals level in
river water
106
Chapter
No.
Title
Page
No.
4.10 Seasonal variation in blood minerals and heavy metals level
of Jersey crossbred cattle
109
4.11 Seasonal variation of minerals and heavy metals level in
cattle milk
112
4.12 Evaluation of serum mineral’s interrelationship with
hematological and biochemical profile of dairy cows
115
5
4 DISCUSSION
119
5.1 Evaluation of seasonal variation in physico-chemical and
microbiological parameters of different sources of water at
high altitude
119
5.2 Identification of major contributing factors governing
groundwater and surface water quality at high altitude
120
5.3 Assessment of seasonal variation in trace minerals and
heavy metals in different sources of water at high altitude
125
5.4 Evaluation of bioavailability of trace minerals and heavy
metals in cattle blood and milk at high altitude
126
5.5 Evaluation of serum mineral’s interrelationship with
hematological and biochemical profile of dairy cows
127
6
SUMMARY AND CONCLUSION
133
7
RESEARCH OUTCOME
136
8
RECOMMENDATIONS
137
BIBLIOGRAPHY
138
5 PUBLICATIONS
177
6 CREDENTIALS
179
Abstract
The present research experimentally investigated the physicochemical parameters and
mineral content in honey and chicken eggs from different regions of Haryana. The egg samples
were evaluated for pH, protein content, length, width, shape index and weight, whereas honey
samples were evaluated for pH, moisture content, acidity, optical density, protein content and
electrical conductivity. By investigating these parameters, the study aims to evaluate the
nutritional value and safe consumption of these food products. Analyzing the mineral content in
honey and egg samples along with the seasonal variations is crucial to comprehend their role in
human health. In addition, this study also focuses on the metabolic parameters and antioxidant
content in these samples. A comprehensive examination of all these parameters was done during
summer and winter season to assess their quality. The samples were collected from four different
districts of Haryana- Rohtak, Gurgaon, Hisar and Panipat. Samples were collected during the
summer and winter season in the year 2022-2023. Hen eggs were collected from poultry farms
and honey samples were collected from bee hives and apiaries. A total number of 5 honey
samples were collected from each district whereas a total number of 10 eggs were collected from
each district during summer (May-June) and winter (Dec-Jan) season. Analysis of samples was
done using standard methods and the data obtained was executed with SPSS.
All the physico-chemical parameters were estimated by standard methods. All the
minerals and heavy metals in honey samples were analyzed by Inductively Coupled Plasma-
Optic Emission Spectrophotometer (ICP-OES). Results showed that the pH, moisture content,
electrical conductivity, optical density, total protein content and acidity were 5.78, 20.89%, 0.81
mS/cm, 0.25, 0.00, and 0.21%, respectively. The mean values of minerals were 13.21 mg/100 g
for sodium; 177.52 mg/100 g for potassium; 20.26 mg/100 g for calcium and 71.10 mg/100 g for
magnesium. Among heavy metals, iron was most abundant with an average of 1.69 mg/100 g.
The mean concentrations of selenium and copper in investigated honey samples were 0.68
mg/100g and 1.50 mg/100 g, respectively. Non-carcinogenic risk related parameters like
estimated daily intake (EDI) and hazard quotient (HQ) were also analyzed. HQ level indicated
that there is a potential threat to children and adult population due to honey consumption in
future. On the other hand, the mean values of egg for width, length, weight, shape, pH, and
protein were 4.17 cm, 5.26 cm, 50.70 g, 22.22, 6.71, and 10.24 g, respectively, in summer, and
4.14 cm, 5.42 cm, 50.85 g, 22.93, 6.93, and 10.23 g, respectively, in winter. Regarding minerals
Abstract
and heavy metals, the mean values were recorded as follows in summer: sodium (123.51 mg/100
g), potassium (112.25 mg/100 g), calcium (71.47 mg/100 g), magnesium (18.96 mg/100 g),
copper (2.29 mg/100 g), and iron (1.55 mg/100 g). In winter, the values were sodium (123.53
mg/100 g), potassium (110.24 mg/100 g), calcium (70.87 mg/100 g), magnesium (18.04 mg/100
g), copper (2.08 mg/100 g), and iron (1.61 mg/100 g). Arsenic, lead, and selenium were below
the limit of quantification. The values recorded for estimated daily intake (EDI), Hazard
Quotient (HQ) indicate no potential health risk, as HQ for Cu and Fe was less than one, for both
adults and children. Thus, based on the results obtained from this study, there are currently no
apparent health risks to human health. However, owing to rapid urbanization and
industrialization, the likelihood of heavy metal pollution and toxicity in the near future is high.
Therefore, more research must be conducted in this regard, and new strategies should be
explored to combat heavy metal contamination.
Dietary antioxidants are known to be beneficial for reducing oxidative damage and
promoting human health. The antioxidant potential of all the samples was measured by DPPH
and FRAP assays. The FRAP values of the egg samples were reported to be significantly greater
in the summer season (25.80 mg GAE/g) than in the winter season (22.88 mg GAE/g). The
DPPH radical scavenging activity of poultry eggs exhibited a greater trend in winter (26.86%)
than in summer (24.53%). In contrast, the FRAP values of honey samples were reported to be
highest for Panipat (279.52 µM Fe(II)), followed by Gurgaon (141.19 µM Fe(II)), Rohtak (87.41
µM Fe(II)) and Hisar (87.19 µM Fe(II)) (the lowest). DPPH radical scavenging in honey samples
was greatest in Panipat (43.92%) and was similar in samples from Rohtak (17.79%), Gurgaon
(17.63%) and Hisar (17.02%). Various metabolites were identified in the analyzed honey. Most
of them had antibacterial, antifungal, antioxidant, analgesic and anti-inflammatory properties. To
date, little research has been conducted on this topic involving eggs and honey. For that reason,
more studies are required to determine the antioxidant properties of these food products and their
impact on human health.
Therefore, more research must be conducted in this regard, and new strategies should be
explored to combat heavy metal contamination. Our findings could lead to the need for future
research, emphasizing the importance of exploring sources of heavy metals and implementing
strategies to mitigate heavy metal contamination in honey.
Introduction
Page 1
Honey is a rich source of carbohydrates, naturally synthesized by the honey bees using
floral nectar. It has been widely utilized by all civilizations as a source of nutrients, food and
traditionally as a medicine. Honey is composed of sugar, moisture, minerals, polyphenols,
proteins, vitamins, enzymes and flavanoids (Manzoor et al., 2013). Sugars which are generally
abundant in honey are fructose and glucose. Phenolics, amino acids and organic acids are also
present in honey. In the various ancient civilizations of India, Greece and Egypt, honey has been
classified as a therapeutic agent and possesses therapeutic benefits (Sua´rez-Luque et al., 2002;
Pohl et al., 2012; Can et al., 2015).
Honey is said to be one of the most complex food product as it comprises of a minimum
of 181 components generally present in the form of a complex blend of sugars in concentrated
solution (Paul et al., 2017). However, the quality and composition of honey varies depending
upon several factors such as climate, geographical region, soil, processing techniques, its
botanical origin and storage conditions (Asif, 2002; White et al., 1964; Turhan et al., 2008).
According to international quality standards, mineral content and antioxidant potential
are the most significant criteria in order to determine the quality of honey. This is because both
of these properties can manipulate the color as well as taste of honey (Paul et al., 2017).
Hen eggs are known to be rich in nutrients, proteins, various chief vitamins and minerals,
an economical food product and eventually play a vital role as daily diet for humans worldwide
(Fakayode et al., 2003; Fu et al., 2014). Whole egg consists of 24.5% solid content, 12% protein
content, 1% carbohydrate, 1% ash and 10.9% lipid. Egg white contains of 12.1% solid content,
10.2% protein, 1% carbohydrate, 0.68% ash. Egg yolk comprises of 51.8% solid content, 16.1%
protein, 1% carbohydrate, 1.69% ash and 34.1% lipid content. Eggs are generally a composition
of 75% water, 12% of lipids, 12% proteins and approximately 1% carbohydrates and minerals
(Ashraf et al., 2017).
Due to the presence of abundant protein, the biological value of egg is known to be very
high. Also, the protein quality of eggs is also used as a standard in order to determine the protein
quality of other food products. Fat soluble vitamins, trace minerals, essential unsaturated fatty
acids such as linoleic and oleic acid are also present in egg (Stadelman and Newby, 2017).
Introduction
Page 2
Several factors such as diet, genotype, egg morphology, egg shape, weight, age of bird,
management system play a key role in determining the quality of eggs (Roberts, 2004; Kowalska
et al., 2020).
Honey has been a part of human life since over 4000 years and is considered to play an
effective role in maintaining the balance of three humors in human body. In Vedic civilization, it
has been known to be one of the most incredible gifts of nature to the mankind. The ancient
Egyptians utilized honey for the treatment of wounds (Liyanage and Mawatha, 2017).
In Ayurveda scriptures, honey is acknowledged as Madhu or Kshaudra and has
significant use in medicinal Ayurveda. Madhu, Makshika, Bhrungavantha, Madvika, Vantha,
Pushparasodbhava, Varati, Kshaudra and Saradha are the Sanskrit names of honey whereas in
Tamil it is known as Thein paani and Meepeni in Sinhala (Liyanage and Mawatha, 2017). Also,
it was believed by the ancient Greeks that eating honey would increase the lifespan of an
individual. In the Ayurvedic medicinal system, honey obtained from bees is classified into
various types. There are eight different types of honey as mentioned in Sushruta Samhita of
Ayurveda. Sushruta Samhita is the prehistoric text based on surgery and medicinal Ayurveda.
This text explains various theories regarding human body, therapeutics, etiology and symptoms
of various diseases. The eight different types of honey are Pauttika, Bhramara, Kshaudra,
Makshika, Chatra, Ardhya, Auddalaka and Dala. Pauttika is dry, hot and have effective
properties. This type of honey is produced from poisonous flowers and results in vitiation of
blood. Bhramara is known to be heavy, that is difficult to digest. It is extremely sweet and has
slimy properties. Kshaudra is cold, light honey with anti-obesive characteristics and can be easily
digested. Makshika is known to be the best honey and is specifically taken in use for cough and
asthma. Chatra honey is cold, sweet in taste, heavy and therefore not easily digested and also
slimy in nature. It is used as a remedy to treat leukoderma, worm infestations, bleeding disorders
and urethritic discharges. Ardhya honey, after digestion gives a pungent, bitter taste. This type of
honey is considered to be good for the eyes and also known to eliminate Kapha and Pitta Dosha.
Auddalaka honey is very effective for voice and is also used for the treatment of skin diseases.
Like Ardhya honey, post digestion Auddalaka also has a pungent taste. Dala honey is dry and is
used as a remedy to control diabetes mellitus and vomiting (Liyanage and Mawatha, 2017).
Introduction
Page 3
Honey is widely consumed due to its high nutrition and its efficacy towards human
health. Annual production of honey around the world is approximately 1.2 million tons.
Developed countries with good economy exhibit higher consumption of honey. Indian sub-
continent with a population greater than 1000 million people, itself is an enormous market of
honey (Kaur et al., 2016). Also, India is also a major exporter of honey across the globe (Mahnot
et al., 2019). Here, production of honey mainly depends on various local crops and flora like
eucalyptus, khair, mustard, vegetables, berries, karanz fruits and litchi. Honey produced from
different crops and flora species vary in terms of their color, flavor and even chemical
composition (European Union, 2002).
India is one of the top egg producing nations in the world with an approximate
production of 78.48 billion. In India, availability of eggs per person is 63 per year but according
to National Nutrition Institute it should be approximately 180 eggs (Kusum et al., 2018).
Generally, an egg comprises of 12% proteins, 11% fats and few other essential vitamins
and minerals (Panda, 1995). It is considered as a good source of nutrients and serves as a well
balanced diet to individuals of all ages, specifically children in growing phase and teenagers by
contributing nutrients which are required for speedy growth of the body (Stadelman and
Cotterill, 1995). Poultry eggs are an inexpensive nutrient-rich food product with proteins and
lipids that can be easily digested (Fisinin et al., 2008). Therefore, production of egg around the
globe has grown up to 203.2% because of its growing demand (Windhorst, 2007).
At present, egg is consumed as a staple food across the world and is classified by the
consumers as versatile and wholesome due to presence of all the essential nutrients (Watkins,
1995).
Apart from its nutritional values, honey is also an antifungal and antibacterial agent (El-
Haskoury et al., 2016). It also exhibit antioxidant properties which are provided by the
flavanoids, carotenoids, phenolic acids, proteins, ascorbic acid and few enzymes such as glucose
oxidase and catalase (Froschle et al., 2018; Saxena et al., 2010). However, according to some
studies it has been reported that temperature directly affects the quality of egg during the storage
period. Higher the temperature is, lower will be the quality of egg (Keener et al., 2006).
Introduction
Page 4
Both honey and egg are important food products and have multiple uses in our day to day
life. Earlier, in Ayurvedic science honey was utilized internally as well as externally for
therapeutic purpose. Externally it is applied for the treatment of cuts, burning wounds and eye
diseases. For internal use herbal mixtures are made to cure cough, respiratory problems like
asthma and phlegm, remedy for hiccups, thirst and vomiting, diabetes, diarrhea and obesity.
Moreover, it is taken in use as a natural preservative and as sweetener in various traditional and
Ayurvedic medicines like Navaratna kalka (Liyanage and Mawatha, 2017).
Eggs can be used in medicines prepared for human and animals because of their bioactive
properties (Banaszewska et al., 2019). They also act as antibacterial agents due to the presence of
a globular protein lysozyme (Adamski et al., 2016). Egg yolk is composed of phospholipids,
unsaturated and polyunsaturated fatty acids which increase its biological value (Banaszak et al.,
2020). In addition, eggs are also known to be an indicator of environmental contamination
(Esposito et al., 2014; Sparks et al., 2006). Primarily, they are major source of proteins,
vitamins, minerals, energy and are inexpensive and rich in nutrients.
Nevertheless, these might pose threat to health if they are contaminated with heavy
metals via the food chain. Elevated concentration of heavy metals in animal products can be due
to the diet, litter, water and environment (Korish and Attia 2020). Apart from these, domestic
waste, mining, industries and pesticides also play a significant role in heavy metal contamination
(Ahmed et al., 2003; Tsipoura et al., 2011). Since, heavy metals are nondegradable therefore
they keep accumulating in the food chain and eventually pass on to the eggs (Aslam et al., 2011;
Burger et al., 2009). Regular consumption of such eggs is very unsafe and it can lead to harmful
effects disrupting the biological functioning of the human body, specifically in children
(Balkhair and Ashraf, 2016; Jaishankar et al., 2014).
Cadmium, lead and arsenic are harmful as they are toxic to both humans and animals.
Lead acts as a neurotoxin and can result in metabolic disorders (Cunningham et al., 1997),
hemopoiesis and can negatively affect the functioning of gastrointestinal and nervous system.
Mercury, lead and cadmium are not essential for the functioning of the body (Ayar et al., 2009;
Qin et al., 2009), instead their excess intake can cause hypertension, kidney damage,
hepatocellular damage and pulmonary disorders. Arsenic toxicity can result headache, irritation
Introduction
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in gut and nausea. Likewise, elevated levels of copper can affect the functioning of brain, liver
and kidney and can also lead to haemolytic crisis (Korish and Attia 2020).
Health hazards due to heavy metal contamination that have been documented till date
include cancer, dermatitis, dysfunctioning of kidney and brain (Tripathi et al., 2019).
Similarly, environmental pollution has also increased the concentration of minerals in
honey. Nectar that is used to produce honey consists of metals which are absorbed by the plant
roots and water (Manzoor et al., 2013).
The concentration of minerals present in honey depends upon its geographical origin,
type of soil, floral origin and nutrients absorbed by the roots of plants. Toxic elements like
arsenic, lead and cadmium can also be found (Paul et al., 2017). Iron, copper, nickel, manganese
and zinc are the essential elements required for the proper development of human body.
However, if their concentration exceeds the permitted value then they can be toxic to human
health (Ali et al., 2019). In addition, nickel, cadmium and lead are known to possess
carcinogenic properties (Scripcă and Amariei, 2021). Heavy metals are a source of illness and
affects human health by damaging red blood cells, affecting the brain, kidney and nervous
disorders, respiratory disorders, vomiting, metabolic problems and nausea (Anonymous, 2002;
Garcai-Fernadez et al., 1996) (Manzoor et al., 2013).
Hence, heavy metal contamination in honey and egg is a matter of concern. Still, not
much study has been conducted on this aspect. Therefore, the present study examined the
physicochemical, metabolic parameters along with influence of seasons. It also investigated the
mineral content in honey and egg and health risks due to their consumption.
Following objectives were studied:
1. To evaluate physico-chemical and minerals level in chicken eggs and honey in Haryana.
2. To estimate the seasonal variation of physico-chemical and minerals level in chicken
eggs and honey in Haryana.
3. To analyze the seasonal variation of metabolic parameters in chicken eggs and honey.
Introduction
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4. To assess the health risk of local people associated with consumption of chicken eggs and
honey in Haryana.
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Physico-chemical properties of Honey
pH
Usually, honey is slightly acidic in nature having an average pH 3.9. This is because of
the presence of small amount of acid in honey. Organic acids and amino acids are chiefly
accountable for the distinctive taste of honey. Honey found in tropical countries is known to be
slightly acidic. The reason behind this is the water content which leads to increased fermentation
and drops the pH value. pH values less than 3.24 indicate that the honey samples were not stored
properly and may be impure (Aljohar et al., 2018). The variations in pH can be because of the
presence of different minerals and amino acids in honey or even due to its fermentation and
improper way of harvesting (Thakur et al., 2021).
Moisture Content
Naturally, the moisture content of honey is very low. This is an important property as it
does not allow the growth of most of the bacteria and other microorganisms (Geiling, 2013).
However, it is considered to be very hygroscopic in nature and its moisture content varies
depending upon the humidity and storage conditions. If the moisture content is higher then there
is a possibility for the fermentation of yeasts and this can change the flavor of honey. During the
process of fermentation, alcohol is formed. This alcohol, in presence of oxygen, breaks down
into acetic acid and water and eventually spoils the honey by changing its sweet taste to sour.
Moisture content is an important characteristic of honey for determining its stability and
resilience towards fermentation at the time of storage (Prica et al., 2014). Honey with high
moisture content tends to ferment easily and cannot be stored for a long time period (Singh and
Singh, 2018).
Electrical Conductivity
The electrical conductivity (EC) of honey varies depending upon the amount of organic
acids, minerals, proteins, inorganic salts and complex sugars (Lullah-Deh et al., 2018). If the
concentration of ions and organic acids is higher in honey, then its electrical conductivity will
also be higher. EC is generally used to distinguish between the blossom and honeydew honey
and also for classification of unifloral honey. Honey with EC > 0.8 mS/cm is classified ad
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honeydew honey and those with EC < 0.8 mS/cm are considered as blossom honey (Thakur et
al., 2021). This characteristic of honey is considered to be good criteria to determine its botanical
origin, purity and quality of honey. Bright colored honey usually indicates lower EC than dark
colored honey (International Honey Commission, 2009). Alongwith quality of honey, EC also
indicates the ash content in honey. The ashes in honey point towards pollution in environment
and its geographical origin (Živkov-Baloš et al., 2018).
Color Intensity
Color is a feature of honey that greatly influences the choice of consumers (Pascual-Mate
et al., 2018). It is generally used to judge the quality of food product and differentiate them on
the basis of geographical origin (Hutchings et al., 2012). Honey color varies greatly from being
clear, to amber, greenish, reddish, bright yellow and even black. Its visual analysis can help to
determine its botanical origin, purity of the product and also detect the presence of any fermented
products. In some countries, the cost of honey is correlated with its color. Light colored honey
cost more as compared to dark colored honey. However, in some regions dark honey is preferred
more (Tuberoso et al., 2014). Honey color not only depends upon its geographical origin but also
on some other factors like mineral and ash concentration, heat and storage conditions (Ahmed et
al., 2016).
Total Protein Content
Honey is known to contain very little amount of protein (around 0.1-0.5%) and few
enzymes like diastase, invertase and glucose oxidase. The content of protein varies in different
varieties of honey as it depends upon the type of pollen and origin of honey. Bees naturally
produce the protein in honey by enzymatic breakdown of nectar and pollen (El Sohaimy et al.,
2015; Chua et al., 2013).
Acidity
Honey has acidity ranging between pH 3.2 and pH 4.5 and do not allow the optimal
growth of most microorganisms like bacteria. This acidity is due to the presence of some organic
acids, particularly gluconic acid and formic acid. Some other acids include oxalic acid, lactic
acid, maleic acid, butyric acid, benzoic acid, citric acid, isobutyric acid, pyroglutamic acid,
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succinic acid, glycolic acid, pyruvic acid and α-ketoglutaric acid (Geiling, 2013; Prica et al.,
2014).
Acidity provides flavor to honey and some other properties like antioxidant and
antibacterial activities. Glycogenic acid produced as a result of oxidation of glucose is the most
efficient antibacterial agent (Almasaudi, 2021; Thakur et al., 2021). Table 2.1 depicts the
reported values for physicochemical parameters of honey in previous studies.
Table 2.1: Reported values of physicochemical parameters of honey in various studies.
Sl. No.
Parameters
Reported Levels
References
1.
pH
5.30-6.30
In present study
4.85-3.81
Kumar et al., 2018
3.52-3.78
Nayik and Nanda, 2015
3.01–4.35
Nayik et al., 2019
3.7-3.9
Shobham et al., 2017
2.
Moisture content
20.37-21.37%
In present study
19-25%
Gairola et al., 2013
17.5–19.1%
Nayik et al., 2019
18-24.5%
Kaur et al., 2016
3.
Electrical
conductivity
0.70-0.93 mS/cm
In present study
0.45-0.55 mS/cm
Shobham et al., 2017
0.351-1.447 mS/cm.
Ceylan et al., 2019
631.95-804.54 μS/cm
Kamal et al., 2019
4.
Optical density
0.23-0.29
In present study
0.29 -1.24
Parihar et al., 2020
0.786- 0.062
Thomas, 2021
0.513 - 2.977
Moulya and Jagadish, 2023
5.
Total protein content
Nil
In present study
0.27–0.64%
Habib et al., 2014
0.048-0.229%
Saxena et al., 2010
6.
Acidity
0.17-0.27%
In present study
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29.8-38.62 meq/kg
Parihar et al., 2020
14.83-40.17 meq/kg
Kumar et al., 2018
9.2-41.4 meq/kg
Shobham et al., 2017
Therapeutic Properties
Antimicrobial activity of Honey
Honey is known to produce an inhibitory effect against many species of bacteria (both
Gram-negative and Gram-positive) like Pseudomonas aeruginosa and Staphylococcus aureus.
Furthermore, honey has the ability to even kill bacteria with very resistant biofilm state. This
confirms that honey can be very effective like other antibiotics such as cefazolin, gentamicin,
rifampicin, oxacillin, etc. Besides, there is no proof supporting bacterial resistance to honey even
though chemical antibacterial drugs may result in microbial resistance and damaging effect on
the tissue of wound. Antimicrobial property of honey is accredited mainly to acidity, nectar,
pollen, osmolarity, hydrogen peroxide production and methylglyoxal (Wang et al., 2012; Grego
et al., 2016).
Anti-inflammatory activity of Honey
Honey is widely used as a medicine mostly to treat the infections. It has also been
reported to cure inflammations and has healing properties for the treatment of wounds. The
therapy used for the treatment of wounds primarily focuses to kill the infectious microorganisms
and removal dead tissue so that the microorganisms do not get a favorable environment for their
growth. Inflammation results in problems like painful wounds that are difficult to manage and
also interferes with the repairment and healing of wounds. Use of honey is beneficial in this case
as it cleans the infected area and wounds and preserves a hygienic condition by killing the
bacteria and removal of dead tissues. This reduces the inflammation and stimulates the growth of
different cells and tissues responsible for the generation of new cells for wound healing. A
number of studies have reported that application of honey on wounds not only reduces the
symptoms of inflammation but also provides a soothing effect when applied to burns and
wounds. Also, dressing of wounds with honey reduces the exudates which indicate the healing of
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inflamed wounds. Decreased scarring is also reported in some studies. Besides these clinical
observations, some animal studies indicate that application of honey reduces inflammation and
depicts anti-inflammatory action when compared to controls. Histological studies indicate that
use of honey reduces the inflammatory cells at the burned site and wounds. Such results clearly
suggest that components of honey except sugar produce an anti-inflammatory effect. The anti-
inflammatory characteristic of honey not only reduces the inflammation but also decreases the
bacteria or microorganisms present on the wound (Hadagali and Chua, 2014; Benhanifia et al.,
2011)
Antioxidant activity of Honey
Honey also exhibits antioxidant properties. In human body, it decreases the oxidative
reactions by scavenging of free radicals. It is also said that the anti-inflammatory activity of
honey may be due to its antioxidant properties as free radicals are a component of inflammation.
In nature honey consists of several flavanoids like quercetin, chrysin, galangin, pinocembrin,
apigenin, kaempferol and hesperetin; products and peptides of Maillard rection; ascorbic acid;
tocopherols; phenolic acids like caffeic, ferulic, p-coumaric and ellagic acid; reduced
glutathione; catalase and superoxide dismutase, majority of them present a synergistic
antioxidant effect. Honey exerts the antioxidant action by repressing the formation of free
radicals and this reaction is generally catalyzed by few metal ions such as iron, copper etc. These
metal ions when form complexes they may be detained by some common components of honey
like flavanoids and other associated polyphenols, thus keeping the generation of free radicals in
first place. Different honey varieties also contain different phytochemicals as other substances
like enzymes, vitamins and organic acids. The type and quantity of antioxidants generally
depends on the variety of honey and the source of flora (Moniruzzaman et al., 2013; Chaikham
and Prangthip, 2015; Nweze et al., 2020). Table 2.2 represents the antioxidant activity of honey
in various studies.
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Table 2.2: Reported antioxidant activity of honey in previous studies
Sl. No.
Parameters
Reported Levels
References
1.
FRAP
337.77 ± 1.01 (µM Fe
(II)/100 g)
Khalil et al., 2012
1593.85 ± 567.98
(µmol TE/kg)
Dżugan et al., 2018
0.0048 ± 1.8 × 10−4
(mg/100 g honey)
Gül and Pehlivan et
al., 2018
23.34 mg AAE/ 100 g
Kek et al., 2017
4,933 µM Fe(II)/kg
Bundit et al., 2016
22.39 ± 12.86a (mg
TE/100 g ± SD)
Ibrahimi and Hajdari,
2020
2.
DPPH
44.57%
Khalil et al., 2012
42.59 ± 17.65%
Dżugan et al., 2018
8.79 mg/mL
Pontis et al., 2014
106.72 mg/mL
Liberato et al., 2011
109.0 mg/ml
Stagos et al., 2018
29.07 ± 1.42 mg/ml
Gül and Pehlivan et
al., 2018
99.06 mL/g
Kek et al., 2017
66.92 %
Ibrahimi and Hajdari,
2020
Honey as an immunity booster
Peptides and proteins present in honey may stimulate the immune system by generating
physiological response in the target cells through their particular receptors. For instance, the
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glycosylated proteins which initiate the TNF-α secretion from macrophages and major royal jelly
protein 1 (MRJP1), a glycoprotein are known to be responsible for the immune-modulatory
effects and wound healing mechanisms of honey. One more immune-stimulatory protein, the
type II arabinogalactan protein stimulates the monocytic cells to produce TNF-α. Nitric oxide
present in honey enhances the humoral immunity. Honey can also induce the production of
antibodies by decreasing the level of prostaglandins. Additionally, honey consists of probiotic
oligosaccharides which elevate the immune responses. Lately, Tonks and others reported that a
5.8 kDa component present in honey is responsible for the stimulation of immune function in
vitro (Majtan, 2014; Oryan et al., 2016).
Stimulation of wound regeneration and repair
In the process of wound healing formation of new tissues is the most important step.
Several studies have stated the efficiency of honey in the healing of wounds both acute wounds
like burns and lacerations and chronic wounds such as pressure ulcers and venous leg ulcers.
Honey enhances the process of wound healing by reducing the pain, edema and inflammation,
aid debridement and deodorization of wounds, formation of collagen, stimulation of fibroblast
and epithelial cell growth, formation of new blood vessels, prevention of scar tissue and
promotion of development of granulation tissue. It also diminishes the requirement for skin
grafting and no hypertrophication or extreme scarring is formed (Al-Waili et al., 2011; Maleki et
al., 2013; Oryan et al., 2016).
The process of wound healing involves three overlapping phases: inflammation,
proliferation and remodeling. The inflammatory phase includes stimulation of monocytes to
produce inflammatory cytokines like TNF-α, IL-1b and nitric oxide. These cytokines further
induce the synthesis of collagen by fibroblasts. They are also important for the stimulation and
magnification of inflammatory processes. Phenolic components present in honey are often
related to anti-inflammatory activity and are known to regulate the severity of infection.
Initiation of angiogenesis by honey in order to supply the obligatory oxygen in wound is an
important step in healing process. Honey removes oxygen from hemoglobin due to its acidic
nature. It increases wound contraction by stimulation of myofibroblasts, fibroblasts and
deposition of collagen. Further, it also advance re-epithelialization and keeps the edges of wound
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together because of high osmotic pressure. Re-epithelialization is considered as a major step in
cutaneous wound healing. In this process, the keratinocytes are proliferated and migrated to the
surface of skin. The enriched environment of honey facilitates the epithelial cells with enough
glucose so that they can migrate to the wound surface. Honey also consists of various trace
elements like Cu, Fe, Mg, Zn, Co and Mn which can aid in proliferation of keratinocyte by
altering the integrin expression at the time of re-epithelialization (Oryan et al., 2016; Nakajima et
al., 2013; Barui et al., 2013). Figure 2.1 depicts the useful properties of honey.
Figure 2.1: Beneficial properties of honey
Microbial characteristics of Honey:
Some microbes have also been reported to be present in honey. Fermentation in honey
because of inappropriate harvesting techniques like lack of proper hygiene at the time of
harvesting and storage, immature combs and broods, results in bacterial contamination. Lower
content of microbes, total viable bacteria, moulds and yeasts are an indicator of proper
management of beehives (Kamal et al., 2019). The microbial properties of honey are a result of
essential biota of bees, as honey is made from nectar of various flower species that is stored for
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varied time periods in their honey stomach without getting in touch with gut of the bee thus,
preventing any sort of contamination. However, contamination of honey may occur later at the
time of handling or extraction. Some of the common sources of microbial contagion in honey are
air, flowers, dust, digestive tract of honeybees and dirt. Secondary sources that result in
microbial contamination are most probably the same as for other food products (Vázquez-
Quiñones et al., 2018).
Honey based value added products
Honey is an outstanding food that is good in taste as well as has many health benefits. It
is well known to contain some bioactive substances which have antioxidant properties of their
own or when combined with other food products. Honey is the gift provided to the mankind by
nature. It is considered as a superfood with different qualities, good flavor and various health
benefits. The most well-known application of honey is its use for dehydration, allergies, skin and
hair issues, stomachaches, intercellular damage, also in antiseptic and cosmetic products. It
exhibit antibacterial and preservative actions in food and on people. Consumers in this modern
era choose honey for healthy and tastier products. They prefer food products in which sugar is
completely or partially replaced by honey. The beverage and confectionaries use honey widely in
order to manufacture healthier products. Since confectionary items are enjoyed by everyone and
are all time favourite therefore, many researchers have included honey to increase the nutrients
in food products like candies, toffees and other baked products (Babbar et al., 2022).
Value added lassi products have been successfully made using honey. Honey not only
improved sensory qualities of lassi but also enhanced its nutritional aspects. Apart from this, it
was also stated that these products had a shelf life of one week and above. This suggests that
honey can be used as an ingredient in industries in different food products (Shuwu et al., 2011).
Honey is widely utilized in beverages, jams, marmalades and spreads. Since its
nutritional value is high and also good texture and flavor, therefore it is highly accepted by the
consumers. Another study was conducted where honey was used as an ingredient in goat meat
spread to enhance its quality. Incorporation of 3% of honey in goat meat spread improved its
quality (Raziuddin et al., 2021).
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Antimicrobial properties play an important role in dermatological applications. Honey
can be used to dress wounds and burns and is also utilized in the treatment of dandruff, pityriasis,
hemorrhoids, tinea, psoriasis, seborrhea, anal fissure and diaper dermatitis. It is included in
different cosmetic formulations because it provides a humectant, soothing, conditioning and
emollient effects that makes the skin juvenile, slows the formation of wrinkles, maintains pH and
prevent pathogenic infections. There are several cosmetic products which are honey based like
cleansing milk, tonic lotions, lip ointments, shampoos, conditioners and hydrating creams. The
amount of honey used generally ranges between 1 to 10%. However, it may even reach 70%
when mixed in gel, polymer entrapment, emulsifiers and oils. Chemically modified, dried and
honey with intermediate moisture are also taken in use (Burlando and Cornara, 2013).
Minerals and heavy metals in Honey
Minerals are inorganic solid substances that occur naturally in the biosphere as a result of
biodegradation of animal and plant tissues. These are known to be a product of geological
processes and are crucial for regulating the metabolic pathways in living bodies. Minerals are
classified into 3 groups according to the requirement of body. These are: (a) major elements, (b)
trace elements and (c) ultra-trace elements. Major elements like calcium (Ca), sodium (Na),
magnesium (Mg), potassium (K), chlorine (Cl), phosphorus (P) and sulfur (S) must be present at
>50 mg/d. On the other hand, trace elements such as iron (Fe), zinc (Zn), iodine (I), copper (Cu),
fluorine (F), manganese (Mn), molybdenum (Mo), selenium (Se), nickel (Ni), chromium (Cr)
and cobalt (Co) should be present at <50 mg/d in humans. Ultratrace elements like lead (Pb),
mercury (Hg), barium (Ba), boron (Bo), cadmium (Cd), lithium (Li), bismuth (Bi), arsenic (As),
bromine (Br), cesium (Cs), aluminium (Al), ger-manium (Ge), silicon (Si), tin (Sn), thallium
(Tl), rubidium (Rb), tungsten (W), strontium (Sr), titanium (Ti), antimony (Sb) and samarium
(Sm) are generally below 1µg/g and mostly found at below 50 ng/g in diet (Soolayman et al.,
2016).
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Figure 2.2: Heavy metal contamination in honey through food chain
Trace minerals are very valuable for good health, particularly if their origin is from plant
or organic sources. In contrast, if their origin is from metallic or inorganic sources then their
specific gravity becomes approximately 5 times that of water and can be toxic (Figure 2.3). In
such case, these are considered as heavy metals (Ajibola et al., 2012).
The concentration of various heavy metals and other minerals in honey basically depends
upon the composition of soil and type of flora. This is because minerals are transported into
plants via roots, which are then transferred to the nectar and eventually to the honey obtained
from it. Moreover, some other factors like environmental pollution, processing of honey and
beekeeping practices also put in to the total mineral content found in different varieties of honey.
Several studies have reported that most of the macrominerals were usually found in honey from
all around the world, except Cl, as it was found only in honey samples of Spain (Soolayman et
al., 2016).
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Amongst all these major and minor elements found in honey, K is said to be found in
highest concentration (Fern´andez-Torres et al., 2005; Dag and others 2006), and then Na
(Nanda and others 2003; Mbiri and others 2011; Moniruzzaman and others 2014), whereas Fe,
Cu, Mg, Zn and Ca are reported in intermediate concentrations (Dag et al., 2006; Belouali et al.,
2008; Vit et al., 2010; Atanassova et al., 2012; Mondrag´on-Cortez et al., 2013). K contributes to
80% of the total because of it is secreted quickly by the nectar sources.
Overall, the amount of minerals in honey is usually low, between 0.02% to 0.3% in
blossom honey and about 1% in honeydew honeys. It is also affected by the climatic conditions
and nectar’s chemical composition that differs according to various botanical sources
contributing in the formation of honey. Variance in composition can also be due to harvesting
practices, beekeeping methods and the type of material collected by bee at the time of foraging
on flowers (Machado De-Melo et al., 2018). Table 2.3 represents the mineral content in honey
from different locations.
Table 2.3: Reported ion and mineral contents in honey samples from different studies conducted
worldwide.
Sl. No.
Parameters
Reported Levels
References
1.
Sodium
12.15-13.92 mg/100 g
In Present Study
19.37 mg/100 g
Angrau, 2022
0.30 mg/100 g
Ikegbunam and Okwu, 2021
153.0 ppm
Taha et al., 2016
2.
Potassium
176.20-178.51 mg/100 g
In Present Study
2.97 mg/100 g
Ikegbunam and Okwu, 2021
2018.0 ppm
Taha et al., 2016
3.
Calcium
19.25-21.92 mg/100 g
In Present Study
6.28 mg/100 g
Angrau, 2022
68.50 mg/100 g
Ikegbunam and Okwu, 2021
287.0 ppm
Taha et al., 2016
4.
Magnesium
6.23-7.85 mg/100 g
In Present Study
9.0-11.8 mg/kg
Shukla and Palaeobotanica,
Review of Literature
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2020
5.50 mg/100 g
Ikegbunam and Okwu, 2021
327.0 ppm
Taha et al., 2016
5.
Copper
1.08-2.11 mg/100 g
In Present Study
1.49 ppm
Taha et al., 2016
0.01–0.23 mg/kg
Sarker et al., 2015
0.01–0.09 μg/g
Tiwari et al., 2016
6.
Lead
BLQ
In Present Study
BLQ
Laaroussi et al., 2020
BLQ
Sarker et al., 2015
BLQ
Ikegbunam and Okwu, 2021
7.
Iron
1.28-1.88 mg/100g
In Present Study
3.29-4.56 µg/g
Kamboj et al., 2013
22.4 ppm
Taha et al., 2016
8.
Arsenic
BLQ
In Present Study
<0.01 mg/kg
Shukla and Palaeobotanica,
2020
BLQ
Singh, 2014
9.
Selenium
0.21-1.46 mg/100g
In Present Study
0.0003 mg/kg
Pehlivan and Gul, 2015
0.232- 0.810 mg/kg
Ahmed, 2015
Note: BLQ: Below limit of quantification
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Figure 2.3: Effects of heavy metals on human health
Poultry industry in India
Poultry practices is believed to be first started in Asia where Indian, Chinese and East
Asian farmers domesticated the poultry. Later, these practices were followed by farmers in
Europe and America. In Egypt, chickens have been bred under captivity since 1400 BC. The
initial records of poultry are dated to be in 3200 BC and it is supposed that the present chicken
breeds were originated in India itself (Pawar et al., 2016).
Currently, India is counted among the top producers of egg around the world. The value
of Indian poultry is approximately 35 thousand crore at present. Due to increased demand for
poultry products, the poultry sector employs workers around 1.6 million. The farmers directly
generate almost 80% of the employment in poultry sector whereas 20% comprises of
equipments, feed, pharmaceuticals and other services (Pawar et al., 2016).
India, being one of the most populated countries in the world is well known for its
agrarian and livestock practices. Poultry industry is among the sub sectors of agriculture that is
categorized under livestock. In recent years, there has been an increasing trend regarding poultry
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production and employment. Over the past three decades, the poultry industry is developing
quite significantly with each decade primarily focusing on different sectors. During 70’s a surge
was observed in egg production; in 80’s broiler production was accelerated; in the 90’s
advancement in poultry integration, production of feed and automation was observed. In the
present decade looking forward to develop value added products and explore trade globally
(Sridharan and Saravanan, 2013; Nisar and Kumar, 2021).
In India, among livestock based occupations poultry farming has a central position
because of its potential to promote economic growth with a very low investment. The Indian
poultry sector has observed one of the fastest 7.3% expansions in poultry population, 10% in
production of meat, 8.35% in broilers and 6% in egg production in the past decade. This sector
generates employment for almost five million people in the country (Pawariya and Jheeba,
2015).
Indian poultry sector depicts the success stories of the nation over the decade. India is
known to be the second largest producer of eggs and third largest in production of broiler
chicken with approximately 2.8 million tonnes of eggs and 3 million tones meat of broiler per
year. About 20 million farmers are engaged in poultry industry and hatcheries across India. The
production of poultry products has been increasing at the rate of 8-10 per cent per annum since
past two to three decades (Thirumalaisamy et al., 2016).
Poultry farming management
These days poultry farming is being managed by using new technologies and
implementation of sensors. The management system of livestock generally constitutes of three
functions: sensing, monitoring of livestock, analysis and decision making (Wolfert et al., 2017).
Precision livestock farming systems facilitate the mechanization of all these processes by
reducing the requirement for manual checking and making human judgement and thereby
reduces the efforts and time required to handle large amount of livestock. These systems
facilitate with the management and monitoring of livestock in real-time (Halachmi et al., 2019).
In this manner the producers can manage large number of animals with similar level of care to
each (Smith et al., 2015). Management of individual animal is not always possible on large
poultry farms having thousands of birds, though, some proportion of birds can be monitored
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through PLF methods and the inputs can be used for the assessment of flock health. Sensors are
used to measure various parameters and they assist PLF systems to monitor large numbers of
poultry. New technologies and sensors altogether focus on mainly three areas: environment of
the poultry house, precision feeding methods and poultry welfare (Astill et al., 2017).
Systems used for environmental monitoring: Poultry house environment is a major factor
for production and can be optimized or monitored. Environmental factors include
temperature, humidity, velocity of air, gas concentrations of gases like ammonia and carbon
dioxide, ventilation rate. PLF technologies monitor the environment of poultry house by
regulating the humidity using humidity sensors that alter the ventilation rate (Astill et al.,
2017). Relative humidity levels can affect the health of birds. In addition, levels of relative
humidity are also correlated with the levels of carbon dioxide and ammonia gas in poultry
houses. If the concentration of these gases increases then it can certainly affect the health of
birds and poultry production (David et al., 2015). At present, different technologies
required for the monitoring of environmental factors are available commercially and used in
poultry houses. Though, advanced systems are being researched by the scientists for
efficient monitoring of environment. Some multi-sensor systems have been proven to be
very effective in the regulation of ventilation systems. These sensors can track the
temperature, differential pressure and air velocity of broiler houses (Bustamante et al.,
2012).
Implementation of sensor technologies has positively affected the production of poultry
houses and helps the producers to reach their desired levels of production. For instance, a
prediction model was made (Jackman et al., 2015) using temperature, carbon dioxide and
ammonia gases, relative humidity sensor inputs which were combined with the data of bird
weight for the prediction of broiler weight 72 hour in advance. The accuracy of this model
was described as good to excellent by the authors. Such systems will allow the producers to
manage the weight of birds if it reported to be low. Since more non invasive surveillance
methods are being developed and used in poultry sectors, it will probably facilitate to
incorporate more data in predictive production models and increasing their prediction
potential.
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Precision feeding techniques: Feeding of poultry birds is an important factor for their
growth and PLF technologies has the most powerful effect on this area. These new
technologies optimize poultry feeding by regulating maximum growth, maintaining specific
conversion rates of feed and management of bird weight. Feeding strategies are more under
focus in broiler breeding farms where broiler chicks are produced by selective breeding.
This selective breeding results in a phenotype which facilitates the maximum production of
meat by increasing the weight of birds rapidly. Although broiler breeders have genes for
rapid growth but eggs are not laid proficiently by the breeders while gaining weight
quickly. Hence, controlled consumption of feed is highly required for the management of
broilers. Controlled intake of feed on farms leads to large variations in body weight and
also the production efficiency of broiler chick is reduced. Therefore, the producers should
constantly weigh birds and base feeding must be done according to the average weight
(Astill et al., 2017). However, this is a tedious process which requires consistent sampling
and if not done carefully, can lead to inappropriate management of feeding. A precision
feeding system has been made to experiment. It controls the release of feed to birds,
particularly an individual at a time on the basis of bird weight (Zuidhof et al., 2017).
Methods for welfare monitoring: The major concern of poultry sector is the maintenance
of proper standards for bird welfare. Some methods for improving the standards of bird
welfare have been reviewed (Sassi et al., 2016).
i) Digital imaging- In digital imaging the movement patterns of broilers are captured
which are then used to analyze their activity. Various experiments conducted on
poultry species using digital imaging demonstrated that it can be utilized for the
assessment of different factors associated with welfare. Some of these factors are bird
weight, response of bird towards varying environmental conditions, gait score and
stocking density in accordance to feeding patterns (Corkery et al., 2013).
ii) Vocal Analysis: It is another method which is being explored and it acts as an
indicator for well being of poultry. Here, bird sound is used to indicate their health
and well being. Birds produce a wide range of vocalizations. It has been reported that
high frequency calls linked with distress in broiler hens are produced when they are
deprived of nesting. In addition, a 38 days research on broilers concluded the peak
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frequency to be 600 Hz and it was observed that when broiler grew larger, their peak
frequency of sound is reduced (Fontana et al., 2015).
Rapid diagnosis: Poultry diseases or infections can be very harmful to both the as well
as the workers. Therefore, smart practices for poultry management should be done to
minimize the risk of infections and diseases by rapid and earlier detection of infectious
diseases. Point care diagnosis refers to the capability to carry out a medical diagnostic
test without a proper laboratory setting and in an area where the patient can receive care
and treatment. This practice is beneficial for poultry operations, since early and rapid
detection can probably lead to faster and better treatment and it can effectively decrease
or slow down the spreading of infections to other birds in the farm. Several new methods
are being currently explored that will make it feasible. Biosensors and wearable sensors
can potentially detect avian influenza in poultry birds (Astill et al., 2017).
Poultry birds can be infected by various pathogens like bacteria, parasites and
virus that can also infect human. The waste generated by the livestock in poultry houses
is a probable reservoir for different kinds of pathogens. Animal waste is generally
removed from the production houses by flushing it with water. The slurry is then
collected and stored in storage lagoons. Even though anaerobic digestion of waste that
takes place in storage lagoons can kill or efficiently decrease the pathogenic microbes,
but some might still be found at substantial densities in seepage of lagoons or spilling of
waste can cause contamination in groundwater and water bodies present at the surface.
Another method which drastically decreases the pathogens in waste is composting (at
55°C). However, improper management can lead to inconsistent reduction of pathogens
(Heaney et al., 2015; Hu et al., 2017).
Chicken Egg properties
pH
The pH of albumen in a fresh egg is generally within the range 7.6 to 7.9. However, the
pH steadily increases upto 9.0-9.7 during storage. This is because the CO2 from an egg diffuses
into air via stomata (Li and Zhang, 2005; Stadelman et al., 2007). Long term storage periods lead
to elevated pH in egg (Scott and Silversides, 2000). Initially, the changes in pH values occur at a
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higher pace and eventually slow down later (Lapao et al., 1999; Silversides and Budgell, 2004).
Freshness of an egg is usually evaluated by the measurement of albumen pH using a pH meter
because the changes in yolk pH are not evident (Qi et al., 2020).
Protein Content
Eggs have been reported to be among the best sources of protein and consist of amino
acids that are essential for human diet all over the world. High content of protein makes egg an
excellent and desirable source of protein for application in food industry. Other than nutritional
benefits, egg proteins are also important for several functional properties such as gelation,
emulsification and foam formation (Kudre et al., 2018; Talansier et al., 2009). In general, both
yolk and egg white are rich in dietary protein (Zaheer, 2015; Bashir et al., 2015). Egg white or
albumen consists of approximately 56.9% of the whole egg mass and account of 10.6% of
protein (Belitz, 2009; Ahn, 2014). The plasma of yolk comprises of 85% of low density
lipoproteins (LDL) and 15% of globular glycoproteins known as as α-, β-, and γ-livetins. LDL
proteins are also known as lipovitellins, which is the major component in egg yolk. It
corresponds to about 68% of the overall dry matter. LDL also exhibit emulsifying properties
present in egg yolk. In contrast, the granular portion of yolk comprises of 70% of high density
lipoproteins (HDL), 12% LDL and 16% phosvitins (Kudre et al., 2018). Table 2.4 represents the
physicochemical parameters studied in previous investigations.
Table 2.4: Reported values of physicochemical parameters of egg in various studies
Sl. No.
Parameters
Reported Levels
References
1.
Width
4.09-4.16 cm
Present study
33.86 mm
(Bagh et al., 2018)
39.92 mm
(Rath et al., 2015)
4.32 cm
(Hanusova et al.,
2015)
2.
Length
5.11-5.66 cm
Present study
45.63 mm
(Bagh et al., 2018)
54.39 mm
(Rath et al., 2015)
5.72 cm
(Hanusova et al.,
2015)
3.
Shape
21.13-23.53
Present study
74.23
(Bagh et al., 2018)
82.68
(Abo El-Maaty et al.,
2021)
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73.53
(Rath et al., 2015)
4.
Weight
48.64-54.48 g
Present study
27.95 g
(Bagh et al., 2018)
57.78 g
(Rath et al., 2015)
41.84 g
(Biswas et al., 2010)
5.
pH
6.71-7.07
Present study
7.8
(Marzec et al., 2019)
7.74
(Dong et al., 2017)
8.09
(Jin et al., 2011)
6.
Protein content
10.14-10.40 g
Present study
5.80 g/dL
(Abo El-Maaty et al.,
2021)
11.09 %
(Lordelo et al., 2017)
Antioxidant Compounds in Eggs
There are various compounds in egg yolk and albumen that display antioxidant properties.
Several egg proteins like phosvitin, ovalbumin, and egg lipids like phospholipids, ovotransferrin,
some micronutrients, vitamin A, selenium, carotenoids and vitamin A are known to exhibit
antioxidant properties. Additionally, eggs can be made rich in antioxidants such as carotenoids,
selenium, iodine and vitamin E by manipulating the poultry feed (Nimalaratne and Wu, 2015).
Ovalbumin is a naturally occurring antioxidant found in egg. It is made up of 385 amino acids
and accounts for about 54% of total egg protein (Li-Chan and Kim, 2008; Huopalahti et al.,
2007). Ovotranferrin constitute 12 to13% of total egg protein and belongs to transferring family.
It is also called as conalbumin (Li-Chan and Kim, 2008; Superti et al., 2007). Ovotransferrin also
exhibit SOD like activity that is promoted by binding of metal (Ibrahim et al., 2007). The
antioxidant activity of egg in previous studies has been mentioned in Table 2.5.
Table 2.5: Reported antioxidant activity of egg in previous studies
Sl. No.
Parameters
Reported Levels
References
1.
FRAP
0.06%
Ali et al., 2019
0.879%
Nuningtyas, 2013
0.61 (TE/ mg)
Lee et al., 2017
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0.11 [µg Fe2+/mg]
Czelej et al., 2023
2.
DPPH
35.00 %
Ali et al., 2019
15.1610 %
Nwadi and Okonkwo,
2021
34.809 %
Nuningtyas, 2013
2.53%
Lee et al., 2017
0.92 [µmol
Trolox/mg]
Czelej et al., 2023
Chicken Egg production in India
India is known to be the third-largest producer of egg around the world, followed by
Japan (Zaheer, 2015). Poultry sector in India has achieved a remarkable growth since last few
decades and has effectively transformed itself into an active agri-based industry from the
backyard farming. Its progress is not only restricted in size but also in sophistication, quality and
productivity. In India, chicken is the dominating bird in poultry houses. They produce about 95%
of the total eggs. The poultry sector significantly contributes to nation’s GDP and provides
employment to almost 5 million people in the country. Poultry industries are concentrated in
some parts of the country. Andhra Pradesh is the leading state in production of poultry products
and is followed by West Bengal, Maharashtra, Tamil Nadu and Punjab. The accessibility of eggs
is highly variable in various parts of the country because of the wide range of production levels.
Egg consumption is much more by the urban population in comparison to rural and tribal people.
They have little access to meat and eggs produced by the industrial sector and are generally
available in low quantities (Panda and Samal, 2016).
In the year 1950-51, the total production of egg in country was just about 1.83 billion and
ever since that egg production has continuously risen over the time period. Of the total egg
produced, approximately 94% is contributed by the chicken and rest 6% is via partial
contribution of ducks and other poultry birds. There has been an increment in the per capita
accessibility of eggs in India. It was just 5 eggs per annum in the year 1950-1951 to about 58
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eggs in 2012-2013. Although, it is still very low as ICMR recommends 365 eggs per annum. The
per capita accessibility of eggs is highest in Tamil Nadu i.e., 336; 261 in Andhra Pradesh; 189 in
Lakshadweep, 168 in Haryana and 165 in Andaman & Nicobar Island (Islam et al., 2016).
Domestic egg production is anticipated to be 5.9 Mt in the year 2030. It appears that India can
probably meet the domestic requirement for eggs with marginal surplus (Kumar et al., 2016).
Health benefits of Chicken Egg
Nutritional aspects
Egg contains 6.5g of proteins with a balanced amount of 9 amino acids (leucine,
isoleucine, threonine, histidine, tryptophan, methionine, valine, lysine and phenylalanine) that
are essential for human health. The concentration of amino acids in protein determines the
quality of protein that is evaluation of the effectiveness of protein ingested in human body. These
amino acids also play key role in the synthesis of enzymes, components of DNA, few hormones,
hormone receptors and some other components that are required for the growth, maintenance of
tissues and to regulate metabolic functions.
Polyunsaturated fatty acids, linoleic acid (n-6) and α-linoleic acid are vital for better
human health. Eggs consist of almost 70 mg of omega-3 fatty acids (n-3). The linoleic acids are
converted into arachidonic acid whereas the α-linoleic acid is metabolized to eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA). These are long-chain essential fatty acids and are
constituents of phospholipids that provide flexibility to cell wall and decrease cholesterol levels
in plasma. EPA and DHA also tend to decrease cardiovascular risks, mental health disorders,
inflammation, immune infections and risks associated with central nervous system (Zaheer,
2015).
An egg also consists of various antioxidants that decrease the free radicals generated
during cellular metabolism. These include selenium that decreases the oxidative stress due to free
radicals eventually causing heart disease; carotenoids like lutein and zeaxanthin in egg yolk
prevent cataracts and age-linked macular degeneration; vitamin E which reduces fat oxidation of
low-density lipoproteins thereby improving the transport of cholesterol and decreases the risk of
heart attack and other heart diseases (Wong, 2010; Abdel-Aal et al., 2013).
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Hen’s egg is known to be a potent source of antibodies such as “IgY” which is considered
to be better than “IgG” immunoglobulin found in mammals. In a time period of 6 weeks, a hen
produces approximately 298 mg of IgY antibodies whereas a rabbit produces only about 17 mg.
These immunoglobulin antibodies are essential components of egg, being very nutritious they
might help to relieve human infections caused by virus and bacteria. Several proteins such as
ovotransferrin, avidin and lysozyme found in egg white are known to exhibit different biological
activities (Shin et al., 2002; Narahari et al., 2004; Abdou et al., 2013).
Antioxidant Activities
Oxidative stress for a longer period in gastrointestinal tract can result in chronic intestinal
disorders. Many antioxidant compounds are present in hen egg which include minerals, vitamins,
trace elements, carotenoids and egg-white proteins (Abeyrathne et al., 2018; Nimalaratne and
Wu, 2015; Nimalaratne et al., 2015) like ovomucoid and its hydrolysates (Chen et al., 2012;
Abeyrathne et al., 2015) ovomucin hydrolysates and some derived peptides (Chang et al., 2013)
proteins in egg yolk like phosvitin (Yousr and Howell, 2015).
The majority of such molecules have been synthesized in vitro however few assays
conducted in a porcine model have stated the valuable effect of proteins obtained from egg yolk
in decreasing the generation of pro-inflammatory cytokines (Young et al., 2010).
Anti-Cancerous Substances
There are some studies which suggest that proteins and peptides obtained from food can
be useful in prevention and to cure cancer (Hernández-Ledesma and Hsieh, 2017). It has been
also confirmed that lysozyme found in egg white exhibit tumor-inhibitory activities. Its effect
usually depends on immunopotentiation (Sava, 1989). Ovomucin and its peptides also
demonstrated anti-tumor activities through cytotoxic effects and by activating immune system
(Omana et al., 2010). The anti-cancerous activity of egg tripeptides (Liao et al., 2018) and
hydrolytic peptides of ovotransferrin (Ibrahim and Kiyono, 2009) were also reported. There is
not much data regarding this however studies are being conducted to explore these activities.
Some remarkable data might come up from studies focusing on egg protease inhibitors (Saxena
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and Tayyab, 1997) as many alike molecules found in food products have been depicted to be
potential chemopreventive agents (Clemente and Arques, 2014).
Antihypertensive Properties
Taking into account the prevalence of hypertension around the world (more than 1.2
billion individuals), various research are being conducted to find strategies to control such
multifactorial disease. The major factors involved in long term control of blood pressure are
potassium and sodium ingestion and the rennin-angiotensin-aldosterone system. Most peptides
obtained from egg possess anti-hypertensive activities and inhibitory activities against the
enzyme that converts angiotensin. This enzyme initiates the processing and activates angiotensin
I into an active vasoconstrictor that is angiotensin II. Some other peptides derived from yolk also
exhibit antihypertensive activities along with hydrolysates present in egg white and
ovotransferrin. Few of these peptides consist of only three amino acids and these tripeptides were
exhibited to be active in vivo. In a study conducted on rats, it was found that these peptides
significantly decrease the blood pressure and may aid in reducing the occurrence of
cardiovascular risks (Réhault-Godbert et al., 2019). The beneficial properties of egg have been
depicted in Figure 2.4.
Figure 2.4: Benefits of chicken egg to human health
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Minerals and heavy metals in Chicken Egg and their consequences in human health
Chicken eggs are an important component of human diet since they are composed of
essential nutrients, minerals such as zinc, iron and selenium, proteins, vitamins, essential amino
acids and fats. Even though, the components of egg are important for the normal development of
body, growth and tissue repairment but if contaminated with toxic heavy metals, they can result
in severe outcomes and affect the public health. Also, these can be passed on to the next
generation i.e., from parents to offspring and also from hen to their eggs through the food chain
and environmental factors such as mining, agricultural and waste disposal activities, industries
and burning of fossil fuels. Chickens under serious rearing systems are usually exposed to
various trace elements and heavy metal by inhaling the polluted air and consumption of
contaminated food that cause indigestion. Poultry feed consists of high amount of minerals that
is directly mixed with the ration in order to fulfill the requirements of the bird’s body; however,
in some cases it exceeds the required amount. Moreover, in many cases the poultry ration
includes bones and fish meals, which are known to be a key source of metal contamination in
feed. These metals can further cross the egg shell and can even pose a threat to the embryos of
chicken. Heavy metal contamination is threatening because it can lead to toxicity and tend to
accumulate causing biomagnification in food chain (Figure 2.5). Therefore, assessment of metal
concentrations in eggs have become an important topic as it is associated with safety of food,
poultry medicine and an alarming issue for environment (Saad Eldin and Raslan, 2017; Giri and
Singh, 2019).
Knowledge and evaluation of mineral concentration of eggs is quite significant for
various reasons that are linked with health and nutritional significance of eggs. Some heavy
metals such as Cr, Mn, Co, Fe, Zn and Cu are known as micronutrients. They are essential but
can also be toxic if consumed above permitted limits. On the other hand, heavy metals like As,
Cd, Hg and Pb are considered to be nonessential elements and they can toxic even if present in
trace levels (Basha et al., 2013). Table 2.6 represents the mineral content in egg reported in
previous studies.
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Table 2.6: Reported ion and mineral contents in egg samples from studies
Sl. No.
Minerals
Reported Levels
References
1.
Sodium
123.35-122.64
Present study
1633 mg/kg
(Michalak et al., 2011)
1410.3 µg/g
(Ieggli et al., 2010)
2.
Potassium
109.60-110.71
Present study
1074 mg/kg
(Michalak et al., 2011)
524 mg/100 g
(Heflin et al., 2018)
1185.8 µg/g
(Ieggli et al., 2010)
3.
Calcium
70.34-71.24
Present study
130 mg/kg
(Michalak et al., 2011)
230 mg/100 g
(Heflin et al., 2018)
525.2 µg/g
(Ieggli et al., 2010)
4.
Magnesium
17.50-18.58
Present study
116 mg/kg
(Michalak et al., 2011)
50.8 mg/100 g
(Heflin et al., 2018)
154.3 µg/g
(Ieggli et al., 2010)
5.
Copper
1.74-2.47
Present study
0.233 mg/kg
(Michalak et al., 2011)
2.70 µg/g
(Abdulkhaliq et al.,
2012)
1.17 ppm
(Korish and Attia,
2020)
6.
Iron
1.43-1.80
Present study
0.881 mg/kg
(Michalak et al., 2011)
25.96 µg/g
(Abdulkhaliq et al.,
2012)
11.83 ppm
(Korish and Attia,
2020)
7.
Arsenic
BLQ
Present study
259 µg/kg
(Michalak et al., 2011)
BLQ
(Korish and Attia,
2020)
8.
Lead
BLQ
Present study
BLQ
(Michalak et al., 2011)
0.27 µg/g
(Abdulkhaliq et al.,
2012)
BLQ
(Korish and Attia,
2020)
9.
Selenium
BLQ
Present study
BLQ
(Korish and Attia,
2020)
66 ng/g
(Giannenas et al., 2009)
Note: BLQ: Below limit of quantification
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Figure 2.5: Heavy metal contamination in hen egg through food chain
Exposure to arsenic can potentially lead to gastrointestinal disorders that can range from
mild abdominal cramps, diarrhea and even severe life risks like hemorrhagic gastroenteritis
linked with shock. Long term exposure can result in skin lesions, diabetes, cancer, neurological
symptoms, cardiovascular risks, pulmonary dysfunction and reproductive toxicity (Feng et al.,
2013).
Copper, an essential trace element is required as a cofactor for different enzymes like
cytochrome oxidase and superoxide dismutase. It is also needed for respiration process in cells
and defense against free radicals. Exposure to excess amount of Cu results in oxidative damage
to some macromolecules like DNA, enzymes with thiol and lipoproteins. In poultry sector,
copper sulfate is added in poultry rations as a growth promoter in United States (Darwish et al.,
2014; Pang and Applegate, 2007). Nickel exposure in humans can lead to serious ailments like
dermatitis and even cancer risk. In a study it was reported that adding 500 mg Ni/kg to diet of
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broilers increases the activity of enzymes in liver which indicates some sort of damage in
parenchyma of liver. The results were confirmed through a mild form of pathological fatty
infiltration of liver in poultry birds (Saad Eldin and Raslan, 2017).
Lead also affects normal functioning of human body. It tends to accumulate in the bones
and turns over with half-life of approximately 30 years, especially in children and development
of nervous system at fetal stage. Even at low concentration, children are prone to exposure and
can suffer from irreversible impacts on neurological functions, affects learning process,
educational attainment and overall conduct. In adults, long term exposure to lead can result in
increased blood pressure, damage to cardiovascular, advancement of cancers and
neurodegeneration. Moreover, Pb and Cd act as nephrotoxic agents, predominantly in renal
cortex region. It was also reported that the content of Pb was higher in cancerous tissues as
compared to healthy tissues in case of colorectal cancer. This suggests that Pb can play a major
role in development of colorectal cancer. Long term exposure to Cd may pose threat to liver,
cardiovascular system, kidneys, impariment of sight and hearing, skeletal system and cancers
associated with lungs, prostate, pancreas, breast, nasopharynx and urinary bladder. Besides, Cd
toxicity can also result in stimulating liver injury and inhibit development of early stage liver
cancer (Aendo et al., 2022). It can reduce fertility, cause hypertension, tumour, kidney failure,
endocrine disruption, oxidative damage, genotoxicity, ion regulatory disruption and hepatic
dysfunction (Ullah et al., 2022).
Chromium exposure can even result in severe cardiovascular, neurological, respiratory,
hematological, renal, gastrointestinal and hepatic effects which may eventually lead to death
(Ullah et al., 2017).
Therefore, health risk assessment, a systematic method is widely used to evaluate the
potential effect of health hazards on human population for a particular time period. This can
provide detailed information and data to risk managers and policy makers. There are different
models of health risk evaluation. Even some degree of approximation can be useful to determine
complex causes and effects and probably be efficient for addressing the associated health risks
(Hashemi et al., 2019).
Materials and Methods
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Experimental Design
Four different districts (Rohtak, Gurgaon, Hisar and Panipat) of Haryana, India were
selected for the collection of hen egg and honey samples (Figure 3.1). Samples were collected
during the summer and winter season in the year 2022-2023. Hen eggs were collected from
poultry farms and honey samples were collected from bee hives and apiaries (Figure 3.2; Figure
3.3). Analysis of samples was done using standard methods and the data obtained was executed
with SPSS.
Study Area
Haryana is a state located between 27°39' to 30°35' N latitude and between 74°28' and
77°36' E longitude in the northern part of India. Out of 1.4% of the geographical area of the
country, Haryana occupies a total of 4.42 m ha of geographical area. The altitude of the state
varies between 700 and 3600 ft (200 meters to 1200 meters) above the sea level. The climate of
Haryana is usually hot in summer with around 45 °C (113 °F) and mild in winter. Its hottest
temperature is reported to be in the months of May and June and coldest in December and
January. In general, the climate is arid to semi-arid with an average rainfall of 354.5 mm.
Figure 3.1: All honey and egg samples were collected from four 4 districts of Haryana viz.
Rohtak, Hisar, Gurgaon and Panipat
Sample Collection
Materials and Methods
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Honey: For the collection of honey samples four different districts (Rohtak, Gurgaon, Hisar and
Panipat) were chosen. A total number of 5 samples were collected from each district. Samples
were collected from the local apiaries and beehives. All the samples were collected in pre-
labeled falcon tubes and were ensured to be free from contamination. All the samples collected
from different locations were heated at 400°C for 30 min and then cooled for 24 hours. It was
then filtered using a cotton filter mesh and stored in falcon tubes at room temperature (25 to
35°C) for further use (Figure 3.6).
Hen egg: Same four districts (Rohtak, Gurgaon, Hisar and Panipat) were selected for the
collection of egg samples. A total number of 10 eggs were collected from each district during
summer (May-June) and winter (Dec-Jan) season. Eggs were cleaned using distilled water and
then drying was done using a towel to decontaminate the shell. Length and width of eggs were
measured with the help of a vernier caliper, while weight was measured using a weighing
balance. Shape index was calculated by using the following formula: Shape index (%) = Width
of egg/ Length of egg × 100. Eggs were then broken and the content was poured in sterilized
falcon tubes. These were then stored at 4°C for further use (Figure 3.4; Figure 3.5).
Materials and Methods
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Figure 3.2: Samples were collected from local apiaries, beehives and poultry farms
Materials and Methods
Page 37
Figure 3.3: Egg sample and data collection from poultry farms
Figure 3.4: Egg samples collected during summer season
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Figure 3.5: Egg samples collected during winter season
Figure 3.6: Honey samples collected from different locations
Analytical Procedures
Materials and Methods
Page 39
Physicochemical Parameters of Honey and Egg samples:
The moisture content of honey was determined using a refractometer (Labman Digital
Auto Refractometer Rfm 950). It was calibrated regularly using distilled water and was
thermostated at 20°C (Bogdanov, 2009). For this, five five g of homogenized sample was poured
in a flask and kept in water bath at 50°C. The solution was then cooled to room temperature,
mixed and then kept on the prism of refractometer. The refractive index was recorded after 2
minutes at 20°C. For each sample the values were recorded thrice and average value was
obtained.
10% (w/v) solution was prepared for the honey samples and pH was measured using a pH
meter (Deluxe pH meter ME-963P) (Bogdanov, 2009). Ten g of sample was mixed in 75 ml CO2
free water in a beaker. The solution was then stirred using a magnetic stirrer and pH was
recorded by pH meter.
For the determination of electrical conductivity 20% (w/v) solution was prepared in milli-
Q water and EC was measured through a conductivity meter (Bogdanov et al., 1999).
Optical density of honey was measured using a UV-VIS spectrophotometer (LABINDIA
UV 3092). For this 1 g honey samples were diluted using 9 ml of distilled water and then
centrifuged at 3000g for 10 min. The absorbance was measured at 530 nm against distilled water
which was taken as blank (Sohaimy et al., 2015).
The total protein content of honey samples was determined by Kjeldahl method as
mentioned in (AOAC, 2005), by converting organic nitrogen into (NH4)2SO4. 1g of sample was
dried and then subjected to digestion and distillation. A selenium catalyst and sulphuric acid
(15ml, 95-98%) was then mixed into it. This solution was distilled after addition of NaOH and
this distillate was taken in a flask with H3BO3 (4%) and a mixed indicator. This mixture was
further titrated with 0.1 N HCl. The percentage of quantified nitrogen was turned into protein
content by multiplying it with conversion factor of 6.25 (Sohaimy et al., 2015).
Acidity of honey was measured using volumetric method. Ten gram honey was mixed
with 75 ml distilled water and this solution was titrated to pH 8.30 with 0.1 M NaOH. Acidity of
Materials and Methods
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honey samples is represented in milliequivalents/kg honey (mEq/kg) (International Honey
Commission Method, 2009).
The pH of the egg samples was measured using a pH meter (Deluxe pH meter ME-963P)
(Chaiyasit et al., 2019) (Figure 3.7).
Protein content of the egg samples was determined using Kjeldahl method involving the
process of digestion and distillation of samples (AOAC, 1995; Gultemirian et al., 2009).
Figure 3.7: Analysis of physicochemical parameters of honey and egg samples
Antioxidant Analysis of Honey samples:
Total Antioxidant Capacity
Ferric Reducing Antioxidant Potential (FRAP)
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Page 41
For determination of FRAP, Benzie and Strain (1996) method was used with slight
modifications. O.2ml of 50% (w/v) honey solution was dissolved in 2.8ml of FRAP reagent
containing 2.5ml of 10mM 2,4,6-tripyridyl-s-triazine solution in 40mM HCl, 2.5ml of 20mM
FeCl3, 25ml 0.3 M acetate buffer with pH 3.6 (Figure 3.8). It was then incubated for 15 min at
37°C. The absorbance of the reaction mixture was measured spectrophotometrically at 593nm
(LABINDIA UV 3092). For the calibration curve aqueous standard solution of FeSO4.7H2O
(100-1000µM) was used and the results were expressed as FRAP value (µM Fe(II)) of 50%
honey solution (Zarei et al., 2019).
DPPH assay for honey and egg samples
Radical scavenging activity of honey was measured with 2, 2-diphenyl-1-picrylhydrazyl
(DPPH), a synthetic free radical. This assay was previously illustrated by Blois, 1958. This
method was used for present study along with slight modifications. For this, 2 g of honey
samples were mixed in 10 mL of distilled water to make solution. Then 0.2 ml of this honey
solution was dissolved in 1.8 mL of 0.1 mM DPPH solution with methanol and was kept at room
temperature in dark for 60 min. The UV-VIS spectrophotometer (LABINDIA UV 3092) at 517
nm was used to measure the decrease in absorbance. The concentration of trolox and quercetin
was 0.1-100 µg/mL in methanol was taken as positive control. For analysis of egg samples, 100
µL of egg sample was dissolved in 1000 µL of methanol (Maisto et al., 2021; Babbar et al.,
2011). All calculations were in triplicate. The radical scavenging activity was determined using
following equation:
Antioxidant Analysis of Egg samples:
Ferric Reducing Antioxidant Potential (FRAP)
The ferric reducing property of egg samples was determined using assay described in
(Omri et al., 2019; Benzie and Szeto, 1999; Lim and Murtijaya, 2007) with some modifications.
Aqueous solution of egg samples was prepared. 0.15ml of egg yolk extract was dissolved in
2.4ml distilled water, 0.75ml HCl, 0.75ml 1% potassium ferricyanide (C6N6FeK3), 0.45ml
ethanol, 0.25ml 1% sodium dodecyl sulfate (NaC12H25SO4) and 0.25 mL of 0.2% ferric chloride
(FeCl3). The tubes with samples were then capped and incubated for 20 min at 50°C. It was then
Radical scavenging activity (%) =
Control-Sample
Control
× 100
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Page 42
allowed to cool at room temperature. Absorbance was taken at 750nm (Figure 3.9). The
antioxidant activity was expressed as mg equivalents gallic acid (standard) per g of sample
(Muhammad et al., 2021).
Figure 3.8: Sample processing for analysis of antioxidant content in honey and egg samples
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Page 43
Figure 3.9: UV-VIS spectrophotometer used for analysis of antioxidant content in honey and
egg samples
Metabolomics of Honey samples:
Extraction of Honey samples for metabolomic analysis
For extraction, honey samples were tempered at room temperature prior to the analysis. 3
g of honey was weighed in a 20 mL vial. After that 100 µL of MeOH solution and 30 µL of
internal standard (toluene and p-xylene) solution were added at 10 mg/L. This mixture was then
shaked for 1 min with the help of a vortex at 1500-2000 rpm and homogenized. Further, the vial
with the sample was kept for incubation at 750 rpm for 20 minutes at 90°C (Figure 3.10).
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Figure 3.10: Sample processing for metabolomic analysis of honey samples
GC-MS Analysis
Out of the extracted sample 1.5 mL was injected in the GC machine. The injection
temperature of the system was set to 100°C at 10:1 ratio (split mode). Helium was used as carrier
gas and flow was set at 1mL/min. The GC oven was initially started at 40°C for 5 min, then
increased to 130°C at the rate of 5°C/min and then further raised to 200 °C at the rate of 35
°C/min, consequently leading to a total runtime of 25 minutes. The ion source temperature was
set at 230°C, transfer line at 300°C and quadrupole temperature at 150°C.
The MS was carried out in electron impact (EI) mode at 70 eV and experiments were
conducted using scan mode within the range of 35-500 m/z. The compounds were quantified by
selected ion monitoring (SIM) mode and ion chromatograms of targeted ions were extracted
(Castell et al., 2023).
Metabolomics of Egg samples:
Materials and Methods
Page 45
Extraction of Egg samples for analysis of metabolites
The egg sample extraction for separation of metabolites was done using the Bligh and
Dyer extraction method (Bligh & Dyer, 1959). In this process, lyophilized egg white (20 mg)
was poured into eppendorf tubes. Then 250 mL methanol and 15 mL of chloroform was added in
these tubes. The samples were then vigorously vortexed at 15 min intervals for a total number of
4 cycles. Consequently, 380 mL chloroform and 0.2 M potassium chloride (90 mL) were added
in the sample. It was then vortexed for 30 sec. The resultant suspension was then centrifuged at
13,572 rpm at 4°C for 10 min. After centrifugation, the upper layer (aqueous phase) was
transferred to a glass vial and then lyophilized. To increase the suitability of volatile metabolites
for GC-MS analysis, the extracts were then derivatized using MSTFA and methoxamine. 50 mL
of pyridine with 10 mg/mL was added to all the dessicated extract. The samples were then
incubated for 17 hours. Subsequently, the samples were then diluted with 600 mL hexane before
the GCMS analysis (Figure 3.11).
Materials and Methods
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Figure 3.11: Sample processing for metabolomic analysis of egg samples
Materials and Methods
Page 47
GC-MS Analysis
One mL of extracted egg sample was injected into splitless mode in the gas
chromatograph coupled with mass spectrophotometer. The injecting temperature was kept 200°C
and flow of gas through the column was 1mL/min. Fused silica capillary column with a thickness
of 0.25 mm was used. The temperature of the system was initially set at 5°C for isothermal
heating for 3 min. It was then raised to 250°C at 3°C/min and then held for 25 min at 250°C. The
ion source temperature was set at 180°C and transfer line at 280°C. The ions were produced in
electron impact ionization with electron beam energy of 70eV and were recorded at 1.6 scans per
sec over mass range of 50-550 m/z. Thermo Scientific Trace1300 GC coupled with ISQ 7000
Single Quadrupole MS system (Figure 3.12) was used to determine the metabolites in samples.
Metabolites were identified by comparison of their mass spectra with those of NIST08 MS
database (Dermane et al., 2024).
Figure 3.12: GC-MS system used to analyze the metabolites present in honey and egg samples
Ion and Mineral Analysis of Honey and Egg samples:
Sample Processing for mineral analysis
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Honey: The honey samples were digested using a microwave assisted sample digestion method.
0.5g of honey was digested using 4ml of HNO3 and 2ml of H2O2. The digestion process began at
500W, 1 min ramped and then it was kept for 4 min. Second step was done at 1000W; 5 min
ramped and was then kept for 5 min. The third step was carried out at 1400W; 5min ramped and
kept for 10 min (Figure 3.13).The digested samples were then diluted using double deionized
water and the volume was made up to 50ml for mineral analysis (Moniruzzaman et al., 2014).
Figure 3.13: Honey sample digestion for mineral analysis
Hen Egg: Di-acid digestion method was used to digest the egg samples. 1g of egg sample was
taken in a Kjedahl digestion tube. 7ml of HNO3 was then added to this tube and was kept for 30
Materials and Methods
Page 49
min. Further, 3ml HCLO4 was also added to the tube and it was heated at 150°C for 30 min on
the digestion block and then at 250°C. Vine green or clear water color indicated the end of
digestion process (Figure 3.14). For analysis of heavy metals, the volume of egg samples was
made up to about 25 ml (Kabeer et al., 2021).
Figure 3.14: Egg sample digestion for mineral analysis
For detection of heavy metals, inductively coupled plasma- optic emission spectrometer
(PerkinElmer Aveo 2000) was used. All the honey samples were first digested using a
microwave (Metalab Hot Air Oven) oven. Microwave setting for sample digestion were 15
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Page 50
min/600 W at 120°C, 20min/600 W at 180°C and 20 min of venting. Double-deionized water
was used for all dilutions. Concentrated nitric acid (65%) and hydrogen peroxide (30%) were
used for digestion of honey samples. To determine the heavy metals, 1 gram of honey samples
was weighed and dissolved in 10 milliliter of concentrated nitric acid. Then, sample digestion
was done using a microwave oven. Blank solutions were made with the help of nitric acid. Since,
ICP-OES method gives results with high accuracy and precision therefore this technique was
used for the elemental analysis. The precision of this technique was evaluated in terms of
repeatability of results and was represented as standard deviation (S.D). For verification of
accuracy, calibration was done (Aghamirlou et al., 2015).
The ions in honey and egg samples were analyzed using ion chromatography technique.
For this, the samples were dissolved in deionized water and vortexed for 5 minutes. It was then
filtered using a 0.45 mm filter membrane. These sample solutions were then analyzed for
different ions. Working standard solutions were made by diluting them with stock solutions
(Fermo et al., 2013).
The trace elements of egg samples were determined by inductively coupled plasma optic
emission spectrometry (ICP-OES). For this, all the dilutions and aqueous solutions were made
using double deionized water. Five gram of egg-white sample was weighed using a digital
analytical balance and then dissolved in 5 ml of nitric acid (65%) and 2 ml hydrogen peroxide.
This mixture was then heated and the volume was decreased upto 3-5 ml by evaporation. To this
solution, 10-15 ml of deionized water was added and then passed through a paper filter. It was
further diluted with deionized water to make up the volume upto 50 ml. Heavy metals were then
analyzed by ICP-OES and blank solutions were prepared similarly without egg-white (Farahani
et al., 2015).
Health Risk Assessment
EDI, HQs and CR levels will be calculated in order to assess health risks (cancer and
non-cancer) in human associated with consumption of chicken egg and honey.
Non Carcinogenic Parameters
EDI (Estimated Daily Intake)
Oral exposure to harmful non-carcinogenic substances will be quantified by the EDI,
using Eq. 1
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(1)
Here, C represents metal content, IR (ingestion rate), ED (exposure duration), EF
(exposure frequency), BW (body mass) and AT (average time) (Duru and Duru, 2021; Yaqub et
al., 2020).
HQ (Hazard Quotient)
It is the measure of the ratio of the estimated daily intake (ADI) and reference dose
(RfD), which is calculated using Eq. 2 (Duru and Duru, 2021).
(2)
Carcinogenic Parameters
CR (Cancer Risk)
It may be defined as the possibility that an individual might develop over a lifespan when
exposed to contaminants and can be measured using Eq. 3 (Giri et al., 2021a).
(3)
Here, Efr represents the exposure frequency, ED (exposure duration), AT (average time)
and CSF₀ is the carcinogenic slope factor.
Quality control and assurance for controlled methods
Quality control during analysis was the primary concern. The environmental conditions
of the experimental areas were maintained according to the ISO standards. The glassware used
during the experiments was kept in 20% HNO3 (6 M) for one day and then washed carefully with
deionized water. All the samples were analysed in triplicate. In every analysis, quality control
(QC) was carried out at regular intervals. A control chart was constructed at the time of analysis
to check for deviations from the QC standard (Giri et al., 2021).
Statistical Analysis
BW×AT
C×IR×ED×EF
×EF
EDI=
===
ADI
RfD
HQ =
×10-3
Efr×ED×EDI×CSF₀
AT
CR =
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Data of Honey samples:
The obtained data for honey samples was analyzed for the mean and standard
error (SE). The significance level among different districts (Rohtak, Gurgaon, Panipat and Hisar)
was calculated by one-way ANOVA and t tests using SPSS statistical software.
Data of Hen Egg samples:
The obtained data for egg samples was analyzed for the mean and standard error
(SE). The significance level among different districts (Rohtak, Gurgaon, Panipat and Hisar) was
calculated by one-way ANOVA and t test using SPSS software.
Materials and Methods
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Results
54
Results of objective 1: To evaluate physicochemical and mineral levels in chicken eggs and
honey in Haryana
4.1 Physico-chemical parameters of honey:
4.1.1 pH
The pH values obtained from the honey samples ranged from 5.31 to 6.13. All the
samples had acidic pH values, and the lowest value was recorded in the Panipat district
(5.31±0.01), while the highest was recorded in the Hisar district (6.13±0.09) (Table 4.1; Figure
4.1). Rohtak and Gurgaon samples recorded the pH values as 5.7 and 5.9, respectively. The
analysis of variance suggested no significant difference (p < 0.05) in the pH of the honey
collected from the different locations.
4.1.2 Moisture Content
The moisture content of the analyzed honey samples ranged between 20.37 and 21.37%
and was under the maximum acceptable limit set by the FSSAI (< 25%). The high value for
moisture content was recorded in Hisar (21.32%), followed by Gurgaon (21.14%), Panipat
(20.73%) and lowest in Rohtak (20.40%).
4.1.3 Electrical Conductivity
The EC values from the honey samples collected from different districts ranged between
0.70 and 0.93 mS/cm. The mean EC values of honey from Rohtak and Hisar (0.80±0.01 and
0.71±0.01, respectively) (Table 4.1; Figure 4.1) were below the permitted limits of 0.800 mS/cm,
whereas their values exceeded those of samples from Gurgaon and Panipat, i.e. 0.91±0.01 and
0.83±0.03, respectively. The highest value for EC was recorded in honey samples from Gurgaon
(0.91 mS/cm) and the lowest in honey samples collected from Hisar (0.71 mS/cm).
4.1.4 Optical Density
The optical density varied from 0.23-0.29 and was lowest (0.25) in honey samples from
Gurgaon. All the samples were light-dark in colour. In this study, the mean optical density was
similar in samples collected from Rohtak, Hisar and Panipat (0.26). Among the samples
collected from various districts, the OD of the honey samples ranged from 0.23-0.29 in Rohtak,
0.23-0.27 in Gurgaon, 0.24-0.27 in Hisar and 0.23-0.29 in Panipat (Table 4.1; Figure 4.1). The
OD values of Rohtak and Panipat were statistically similar.
4.1.5 Total Protein Content
Results
55
The total protein content of the studied honey samples was found to be nil.
4.1.6 Acidity
The acidity of the samples from different locations ranged from 0.17-0.27%. The mean
values for acidity were recorded to be highest in the Gurgaon and Hisar honey samples (0.23%
each) honey samples and lowest (0.20%) in samples from Panipat (Table 4.1; Figure 4.1).
Therefore, none of the samples exceeded the permitted value of 50 meq/kg. All the samples were
consistent with the established standards, which suggest that the honey was fresh and not
fermented.
Table 4.1: Mean values of physicochemical parameters of honey samples collected from various
districts of Haryana
Rohtak
Gurgaon
Hisar
Panipat
pH
5.70±0.01b
5.90±0.03b
6.13±0.09c
5.31±0.01a
Range
5.68-5.72
5.85-5.94
6.00-6.30
5.30-5.32
MC
20.40±0.02a
21.14±0.02b
21.32±0.03b
20.73±0.15a
Range
20.37-20.43
21.10-21.17
21.29-21.37
20.43-20.90a
EC
0.80±0.01b
0.91±0.01c
0.71±0.01a
0.83±0.03b
Range
0.77-0.82
0.90-0.93
0.70-0.73
0.80-0.88
OD
0.26±0.02a
0.25±0.01a
0.26±0.01a
0.26±0.02a
Range
0.23-0.29
0.23-0.27
0.24-0.27
0.23-0.29
Acidity
0.21±0.02a
0.23±0.02a
0.23±0.01a
0.20±0.01a
Range
0.17-0.24
0.21-0.27
0.21-0.25
0.19-0.22
Note: MC: moisture content; EC: electrical conductivity; OD: optical density
Results
56
Figure 4.1: Variation in physicochemical parameters of honey in different regions of Haryana
4.2 Physico-chemical parameters of chicken egg:
4.2.1 Length
The length of the egg samples ranged between 4.90-5.72 cm during summer and between
5.11-5.66 cm during winter. During the summer season, the length of egg samples from Hisar
(5.72 cm) was longer, followed by Rohtak (5.36 cm), Panipat (5.05 cm) and the smallest in
Gurgaon (Table 4.2; Figure 4.2). In winter, the length was longest in samples from Rohtak (5.66
cm), followed by Hisar (5.59 cm), Gurgaon (5.32 cm) and the shortest in Panipat (5.11 cm)
(Table 4.3; Figure 4.3).
4.2.2 Width
The width of the egg samples ranged from 4.07-4.28 cm during summer and 4.09-4.16
cm during winter. In summer, eggs collected from Hisar (4.28 cm) had more width, followed by
Rohtak (4.23 cm), Panipat (4.11 cm) and Gurgaon (4.07 cm) (Table 4.2; Figure 4.2). During
winter, the average width of egg samples was 4.16 cm in Hisar, 4.15 cm in Rohtak, 4.14 cm in
Panipat and 4.09 cm in Gurgaon (Table 4.3; Figure 4.3).
Results
57
4.2.3 Weight
All the egg samples weighed within the range of 47.58-55.12 g. During summer, the
highest value for weight was recorded in egg samples from Hisar (55.12 g), followed by Rohtak
(50.41 g), Panipat (47.71 g) and lowest in Gurgaon (47.58 g) (Table 4.2; Figure 4.2). In winter,
the highest value for weight was recorded in Hisar (54.48 g), followed by Gurgaon (49.17 g),
Panipat (49.14 g) and lowest in Rohtak (48.64 g) (Table 4.3; Figure 4.3).
4.2.4 Shape
The analyzed egg samples' shape index (SI) was 19.97-24.47. In summer, the highest
value for SI was observed in samples from Hisar (24.47), followed by Rohtak (22.65), Panipat
(20.79) and Gurgaon (19.97) (Table 4.2; Figure 4.2). Likewise, in winter, the SI values were
recorded to be highest in Rohtak (23.53) and then in Hisar (23.28), followed by Gurgaon (21.78)
and lowest in Panipat (21.13) (Table 4.3; Figure 4.3).
4.2.5 pH
In summer and winter, the pH of egg samples was found in the range of 6.61-6.90 and
6.71-7.07. All the egg samples had acidic pH during the summer and acidic to neutral in the
winter. The highest pH value was reported in Gurgaon (6.90), followed by Rohtak (6.70),
Panipat (6.62) and lowest in Hisar (6.61) during summer (Table 4.2; Figure 4.2). In winter, the
average pH values were recorded to be highest in Rohtak (7.07) and subsequently in Gurgaon
(7.03), Panipat (6.91) and Hisar (6.71) (Table 4.3; Figure 4.3).
4.2.6 Protein Content (PC)
The protein content in whole egg was reported to be within the range of 10.10-10.33 g in
summer and 10.14-10.40 g in winter season. During summer, the highest amount was recorded in
Hisar (10.33 g), followed by Rohtak (10.30 g), Panipat (10.22 g) and lowest in Gurgaon (10.10
g) (Table 4.2; Figure 4.2). In winter, Hisar (10.4 g) egg samples had the highest value for protein
content and subsequently in Panipat (10.2 g), Rohtak (10.17 g) and lowest in Gurgaon (10.14 g)
(Table 4.3; Figure 4.3).
Table 4.2: Mean values of physicochemical parameters of egg samples collected from various
districts of Haryana in the summer
Mean
Rohtak
Gurgaon
Hisar
Panipat
Width (cm)
4.23±0.05ab
4.07±0.06a
4.28±0.04b
4.11±0.06ab
Results
58
Range
3.85-4.38
3.84-4.31
3.96-4.41
3.84-4.42
Length (cm)
5.36±0.08b
4.90±0.08a
5.72±0.07c
5.05±0.11ab
Range
5.08-5.81
4.60-5.23
5.37-6.04
4.53-5.67
Shape
22.65±0.55bc
19.97±0.55a
24.47±0.49c
20.79±0.70ab
Range
19.94-25.39
17.66-22.54
22.02-26.64
17.53-23.98
Weight (g)
50.41±1.46ab
47.58±1.66a
55.12±0.60b
47.71±1.60 a
Range
43.46-56.80
42.35-56.39
52.87-57.14
41.92-56.71
pH
6.70±0.01b
6.90±0.01c
6.61±0.01a
6.62±0.02a
Range
6.69-6.71
6.88-6.92
6.60-6.62
6.60-6.65
PC (g)
10.30±0.03ab
10.10±0.01a
10.33±0.02c
10.22±0.02a
Range
10.25-10.34
10.08-10.13
10.30-10.37
10.20-10.26
Table 4.3: Mean values of physicochemical parameters of egg samples collected from various
districts of Haryana in winter
Mean
Rohtak
Gurgaon
Hisar
Panipat
Width (cm)
4.15±0.06a
4.09±0.07a
4.16±0.06a
4.14±0.07a
Range
3.87-4.36
3.77-4.42
3.85-4.48
3.83-4.41
Length (cm)
5.66±0.01b
5.32±0.08a
5.59±0.06b
5.11±0.05a
Range
5.59-5.73
4.91-5.62
5.38-5.92
4.83-5.29
Shape
23.53±0.38b
21.78±0.57ab
23.28±0.55b
21.13±0.50a
Range
21.63-24.90
19.44-24.84
21.06-26.52
18.93-23.24
Weight (g)
48.64±0.96a
49.17±1.49a
54.48±0.64b
49.14±1.35a
Range
44.79-52.84
42.87-55.36
51.04-57.32
43.66-55.37
pH
7.07±0.03b
7.03±0.09b
6.71±0.01a
6.91±0.02ab
Range
7.00-7.10
6.90-7.20
6.69-6.73
6.89-6.94
Results
59
PC (g)
10.17±0.09ab
10.14±0.06a
10.40±0.01b
10.20±0.01ab
Range
10.00-10.30
10.07-10.26
10.38-10.42
10.17-10.22
Figure 4.2: Variation in Physicochemical parameters of egg samples in different regions of
Haryana during summer
Results
60
Figure 4.3: Variation in Physicochemical parameters of egg samples in different regions of
Haryana during winter
4.3 Seasonal variation in physicochemical parameters of egg:
No significant variation was observed in the physicochemical properties of egg samples
during summer and winter. The average width of the egg samples was reported to be 4.13 cm in
winter and 4.17 cm in summer. The length of the samples was found to be 5.42 cm in winter and
5.26 cm in summer. The average shape index was reported to be 22.43 in winter and was slightly
lower (21.97) in summer. pH of the egg samples was found to be weakly acidic in winter (6.93)
as well as in summer (6.71). Likewise, the protein content was reported to be almost similar in
both winter and summer seasons (Table 4.4; Figure 4.4).
Table 4.4: Variation of physicochemical parameters in hen eggs in different seasons in Haryana
Parameter
Season
Mean
Width (cm)
Winter
4.13±0.03
Results
61
Summer
4.17±0.03
Length (cm)
Winter
5.42±0.05
Summer
5.26±0.07
Shape
Winter
22.43±0.29
Summer
21.97±0.39
Weight (g)
Winter
50.36±0.68
Summer
50.20±0.83
pH
Winter
6.93±0.05
Summer
6.71±0.04
Protein Content (g)
Winter
10.23±0.04
Summer
10.24±0.03
Figure 4.4: Seasonal variation in physicochemical parameters of egg samples
Results
62
4.4 Mineral content in honey:
The mean value of minerals found in the analyzed honey samples from all four districts is
shown in Table 4.5 and Figure 4.5. The minerals found in the samples were sodium (Na),
potassium (K), calcium (Ca) and magnesium (Mg), which ranged from 12.15-13.92 mg/100 g,
176.20-178.51 mg/100 g, 19.25-21.92 mg/100 g and 6.23-7.85 mg/100 g, respectively. K was the
most abundant mineral found in honey. The average Na levels in honey were reported to be
13.20 mg/100 g in Rohtak, 12.60 mg/100 g in Gurgaon, 13.60 mg/100 g in Hisar and 13.47
mg/100g in Panipat. The concentration of K was found to be highest in Panipat (178.31 mg/100
g), followed by Gurgaon (177.26 mg/100 g), Hisar (177.13 mg/100 g) and Rohtak (176.38
mg/100 g). Ca level was highest in Panipat (21.83 mg/100 g), followed by Hisar (21.19 mg/100
g), Rohtak (20.74 mg/100 g) and Gurgaon (19.29 mg/100 g). Mg levels were reported to be
highest in Panipat (7.34 mg/100 g) and Gurgaon (7.33 mg/100g), followed by Hisar (7.18
mg/100 g) and lowest in Rohtak (6.58 mg/100 g). Among heavy metals, Arsenic (As) and Lead
(Pb) were below the level of quantification. Cu was found to be highest in Gurgaon (1.78 mg/100
g), followed by Panipat (1.59 mg/100 g) and lowest in Hisar (1.34 mg/100 g) and Rohtak (1.30
mg/100 g). The average Se concentration was reported to be highest in Panipat (1.20 mg/100 g),
followed by Hisar (0.67 mg/100 g), Rohtak (0.52 mg/100 g) and Gurgaon (0.33 mg/100 g). Fe
concentration was highest in Rohtak (1.78 mg/100 g) and Gurgaon (1.78 mg/100 g), followed by
Hisar (1.70 mg/100 g) and Panipat (1.49 mg/100 g).
Table 4.5: Mineral and heavy metal contents in honey samples collected from different districts
of Haryana.
Attributes
(mg/100 g)
Rohtak
Gurgaon
Hisar
Panipat
Sodium
13.20±0.01ab
12.60±0.23 a
13.60±0.17 b
13.47±0.25 b
Range
13.19-13.22
12.15±12.91
13.27±13.83
13.05-13.92
Potassium
176.38±0.12 a
177.26±0.26 b
177.13±0.19ab
178.31±0.12 c
Range
176.20-176.61
176.78±177.69
176.81-177.48
178.09-178.51
Calcium
20.74±0.17 b
19.29±0.02a
21.19±0.36bc
21.83±0.05 c
Range
20.41-20.97
19.25-19.33
20.56-21.79
19.25-21.92
Magnesium
6.58±0.21 a
7.33±0.07 a
7.18±0.41 a
7.34±0.10 a
Range
6.23-6.96
7.21-7.44
6.43-7.85
7.18-7.53
Copper
1.30±0.13 a
1.78±0.25 a
1.34±0.05 a
1.59±0.18 a
Range
1.08-1.54
1.29-2.11
1.27-1.44
1.25-1.87
Results
63
Selenium
0.52±0.08 a
0.33±0.07 a
0.67±0.23ab
1.20±0.13 b
Range
0.37-0.62
0.21-0.46
0.22-0.93
1.02-1.46
Iron
1.78±0.06 a
1.78±0.07 a
1.70±0.08 a
1.49±0.15 a
Range
1.66-1.85
1.65-1.88
1.59-1.86
1.28-1.77
Figure 4.5: Variation in mineral content of honey in different regions of Haryana
4.5 Mineral content in chicken eggs:
The mean value of minerals found in the analyzed egg samples from all four districts during the
summer and winter seasons is shown in Tables 1 and 2, respectively. The egg samples were
analyzed for the following minerals: sodium (Na), potassium (K), calcium (Ca) and magnesium
(Mg). During the summer season, Na, K, Ca and Mg were found to be within the range 122.56-
124.42 mg/100 g, 109.62-116.23 mg/100 g, 70.58-73.45 mg/100 g, and 18.41-19.46 mg/100 g,
respectively. In the winter season, Na, K, Ca and Mg were found to be within the range 122.64-
124.62 mg/100 g, 109.6-110.71 mg/100 g, 70.34-71.24 mg/100 g, and 17.5-18.58 mg/100 g,
respectively. Na was the most abundant of all the minerals, followed by K, Ca and Mg. Our
results for Na and K were lower than those reported in other studies (Table 7). Mg was the least
abundant mineral (17.50-18.58 mg/100 g). Trace mineral analysis indicated Cu and Fe presence,
Results
64
whereas As Pb and Se were below the detection level. Cu was most abundant in Gurgaon during
the summer season. The Cu content in hen egg ranged between 1.61-2.69 mg/100 g. The highest
value (2.69 mg/100 g) was recorded in Gurgaon during the summer, and the lowest value (1.61
mg/100 g) was recorded in Hisar during the summer. The Fe concentration in the present study
ranged from 1.43- 1.80 mg/100 g. The highest value (1.80 mg/100 g) for Fe was reported in
Rohtak during winter (Table 4.7; Figure 4.7). In summer, the lowest value (1.43 mg/100 g) was
recorded in Hisar and the highest value (1.68 mg/100 g) was recorded in Panipat (Table 4.6;
Figure 4.6). In the present study, As, Se and Pb were not detected in any of the egg samples and
were below the level of quantification. No significant variation in mineral content was observed
in the summer and winter seasons(Table 4.8; Figure 4.8).
Table 4.6: Mineral and heavy metal contents in egg samples collected from different districts of
Haryana in the summer
Mean
Rohtak
Gurugram
Hisar
Panipat
Sodium
122.56±0.16a
124.25±0.12b
124.42±0.16b
122.81±0.12a
Range
122.24-122.77
124.08-124.47
124.19-124.72
122.57-122.94
Potassium
111.77±0.08b
111.37±0.08b
109.62±0.18a
116.23±0.09c
Range
111.65-111.93
111.27-111.52
109.26-109.82
116.06-116.37
Calcium
71.14±0.03a
70.58±0.15a
70.71±0.12a
73.45±0.16b
Range
71.09-71.20
70.29-70.81
70.52-70.93
73.15-73.71
Magnesium
19.39±0.09b
18.41±0.12a
18.58±0.18a
19.46±0.13b
Range
19.26-19.55
18.25-18.64
18.24-18.83
19.23-19.66
Copper
2.60±0.11b
2.69±0.12b
1.61±0.18a
2.29±0.10b
Results
65
Range
2.39-2.78
2.44-2.83
1.36-1.97
2.11-2.45
Iron
1.59±0.22a
1.51±0.23a
1.43±0.16a
1.68±0.13a
Range
1.16-1.88
1.07-1.85
1.16-1.71
1.43-1.88
Figure 4.6: Variation in mineral content of egg samples during summer
Table 4.7: Mineral and heavy metal contents in egg samples collected from different districts of
Haryana in winter
Mean
Rohtak
Gurugram
Hisar
Panipat
Sodium
123.35±0.15b
123.52±0.14b
124.62±0.17c
122.64±0.10a
Results
66
Range
123.15-123.64
123.25-123.70
124.33-124.92
122.49-122.83
Potassium
110.71±0.08b
110.47±0.23b
109.60±0.14a
110.16±0.09ab
Range
110.55-110.82
110.17-110.92
109.36-109.84
110.05-110.33
Calcium
70.38±0.16a
70.34±0.13a
71.24±0.11 b
70.52±0.22ab
Range
70.09-70.64
70.18-70.60
71.06-71.43
70.28-70.96
Magnesium
18.51±0.18b
17.50±0.11a
18.58±0.14b
18.55±0.26b
Range
18.22-18.83
17.30-17.66
18.32-18.78
18.07-18.95
Copper
2.34±0.17bc
1.78±0.11ab
1.74±0.13a
2.47±0.07c
Range
2.04-2.62
1.57-1.93
1.49-1.91
2.37-2.61
Iron
1.80±0.05a
1.43±0.21a
1.50±0.15a
1.69±0.11a
Range
1.73-1.91
1.06-1.78
1.22-1.75
1.49-1.88
Results
67
Figure 4.7: Variation in mineral content of egg samples during winter
Table 4.8: Variation of trace metals in chicken eggs in different seasons in Haryana
Element
Season
Mean
Sodium
Summer
123.51±0.26
Winter
123.53±0.22
Potassium
Summer
112.25±0.74
Winter
110.23±0.14
Calcium
Summer
71.47±0.35
Winter
70.62±0.13
Magnesium
Summer
18.96±0.15
Winter
18.29±0.16
Copper
Summer
2.30±0.14
Winter
2.08±0.11
Results
68
Iron
Summer
1.55±0.09
Winter
1.61±0.08
Figure 4.8: Seasonal variation in mineral content of egg samples
Results of objective 2: To evaluate the antioxidant properties of chicken eggs and honey in
Haryana
4.6 Antioxidant Activity of Honey
The DPPH radical scavenging assay was used to determine the radical scavenging
activity of the collected honey samples. DPPH is known to be a stable radical with nitrogen in
the centre. It has been widely utilized to measure different samples' free radical scavenging
potential. DPPH is used for this evaluation because the antioxidant potential of honey is known
to be directly linked with its concentration of phenols and flavonoids (Beretta et al., 2005). In the
present study, the radical scavenging activity of honey was expressed in terms of % DPPH
inhibition. The higher scavenging activity of DPPH refers to the high antioxidant potential of the
sample. The samples from Panipat exhibited the highest inhibition percentage (43.92%),
Results
69
consequently signifying their high antioxidant activity. In contrast, the DPPH scavenging activity
of samples from Rohtak, Gurgaon and Hisar were 17.79%, 17.63% and 17.02%, respectively.
Honey samples from Hisar exhibited the lowest DPPH scavenging activity.
The antioxidant capacity of honey samples was determined via FRAP assay. It is an
uncomplicated test generally carried out to analyze the antioxidant activity of various samples.
Among all the samples, the honey samples from Panipat had the highest antioxidant capacity
(279.52 µM Fe(II)), which indicates their higher antioxidant properties. Higher FRAP values
suggest an increased reduction of ferric ions into ferrous ions (Khalil et al., 2012). The
antioxidant capacity of Rohtak, Gurgaon and Hisar samples was reported to be 87.41 µM Fe(II),
141.19 µM Fe(II) and 87.19 µM Fe(II), respectively. Rohtak and Hisar's Honey had similar
antioxidant capacities (Table 4.9; Figure 4.9).
Table 4.9: Antioxidant activity of honey samples collected from different locations
Parameters
Rohtak
Gurgaon
Hisar
Panipat
FRAP (µM
Fe(II))
87.41±0.73a
141.19±1.06 b
87.19±1.60 a
279.52±3.29c
Range
85.97-88.30
139.30-142.97
84.97-90.30
272.97-283.30
DPPH (%)
17.79±0.50 a
17.63±0.53 a
17.02±0.62 a
43.92±1.47b
Range
17.13-18.78
16.80-18.62
15.97-18.12
41.99-46.80
Mean± standard Error bearing superscripts (a-d) signifies a significant difference (p< 0.05)
Results
70
Figure 4.9: Seasonal variation in antioxidant capacity of honey samples
4.7 Antioxidant activity of chicken egg
During the summer season, DPPH scavenging activity was recorded to be highest in honey
samples of Gurgaon (24.86%), followed by Rohtak (24.78%), Hisar (24.53%) and lowest in
Panipat (23.93%). However, there was no significant difference between the values obtained
after analyzing honey samples from all four districts (Table 4.10). The antioxidant capacity
analyzed using FRAP assay was found to be highest in Panipat (31.64 mg GAE/g) followed by
Gurgaon (26.19 mg GAE/g), Hisar (23.30 mg GAE/g) and was lowest in Rohtak (22.08 mg
GAE/g). In winter, the samples collected from Hisar recorded the highest radical scavenging
activity (27.94%), followed by Panipat (27.05%), Rohtak (26.89%) and lowest in Gurgaon
(25.58%) (Table 4.11). The average concentration of FRAP was found to be highest in Panipat
(26.30 mg GAE/g), followed by Hisar (25.97 mg GAE/g), Gurgaon (20.08 mg GAE/g) and
lowest in Rohtak (19.19 mg GAE/g). Overall, the FRAP values of analyzed honey samples were
reported higher during summer than in winter. Correspondingly, the DPPH radical scavenging
activity was higher in the winter than summer (Figure 4.10).
Results
71
Table 4.10: Antioxidant activity of egg samples collected from different locations during the
summer season
Parameters
Rohtak
Gurgaon
Hisar
Panipat
FRAP (mg
GAE/g)
22.08±0.45 a
26.19±1.79 a
23.30±0.67 a
31.64±1.45 b
Range
21.63-22.97
22.63-28.30
22.63-24.63
28.97-33.97
DPPH (%)
24.78±0.80 a
24.86±0.30 a
24.53±0.15 a
23.93±0.04 a
Range
23.39-26.17
24.27-25.16
24.27-24.78
23.89-24.02
Mean± standard Error bearing superscripts (a-d) signifies a significant difference (p< 0.05)
Table 4.11: Antioxidant activity of egg samples collected from different locations during the
winter season
Parameters
Rohtak
Gurgaon
Hisar
Panipat
FRAP (mg
GAE/g)
19.19±0.67 a
20.08±1.44 a
25.97±1.50 b
26.30±0.69 b
Range
17.97-20.30
17.63-22.63
24.30-28.97
24.97-27.30
DPPH (%)
26.89±0.60 b
25.58±0.11 a
27.94±0.39 b
27.05±0.00 b
Range
26.17-28.07
25.41-25.79
27.43-28.70
27.05-27.05
Mean± standard Error bearing superscripts (a-d) signifies a significant difference (p< 0.05)
Results
72
Figure 4.10: Seasonal variation in antioxidant capacity of egg samples
Results of objective 3: To analyze the seasonal variation of metabolic parameters in
chicken eggs and honey
4.8 Metabolites in honey
The samples were analyzed for metabolites by GC-MS, and several peaks were obtained
for different compounds in the chromatogram. The honey samples from Rohtak were reported to
have many metabolites such as 7-epi-cis-sesquisabinene hydrate, 3-
Trifluoroacetoxypentadecane, 5-Cyclopropylcarbonyloxypentadecane; 2-Dodecene, (E)-; 2-
Undecene, (E)-; 1-Hexadecanol, 2-methyl, Octadecane, 3-ethyl-5-(2-ethylbutyl)-, Dodecane, 5,8-
diethyl-; Tetradecane, 2,6,10-trimethyl-; Octadecanal, 2-bromo-; Decane, 2,4,6-trimethyl-,
Ethanol, 2-(octadecyloxy)-; Hexadecane, 1,1-bis(dodecyloxy)-; 4-Hepten-3-one, 4-methyl-; 4-
Hexen-3-one, 4,5-dimethyl-; Octadecane, 3-ethyl-5-(2-ethylbutyl); Octadecane, 1,1'-[(1-methyl-
1,2-ethanediyl)bis(oxy)]bis-; Ethanol, 2-(octadecyloxy)-; Octadecane, 3-ethyl-5-(2-ethylbutyl)-;
Results
73
Octadecane, 1,1'-[(1-methyl-1,2-ethanediyl)bis(oxy)]bis-; Tetrapentacontane, 1,54-dibromo-;
Octadecane, 3-ethyl-5-(2-ethylbutyl)- (Figure 4.11).
Figure 4.11: GC-MS Chromatogram of honey samples from Rohtak
Compounds identified in honey samples from Gurgaon were 7-epi-cis-sesquisabinene
hydrate; Carabrol; TMS, Hexane; 2,3,4-trimethyl-; Hexane, 3-ethyl-; Hexane, 2,3,5-trimethyl-;
2-Decene, (Z)-; 2-Dodecene, (E)-; trans-3-Decene; 3-Hydroxyhex-4-enethioic acid, S-t-butyl
ester; 4-Hydroxy-4-methylhex-5-enoic acid; tert.-butyl ester; Octadecane, 3-ethyl-5-(2-
ethylbutyl)-Undecane, 4,7-dimethyl-; Decane, 2,4,6-trimethyl-; 1-Hexadecanol, 2-methyl-, 2-
Trifluoroacetoxypentadecane, Decane, 2,4,6-trimethyl-; Octane, 6-ethyl-2-methyl-; 2,3-
Dimethyldecane; Undecane; 4,7-dimethyl-; Dodecane; 2,7,10-trimethyl-; 2-Bromo dodecane,
Eicosane, 2-methyl-; Tetradecane, 2,6,10-trimethyl-; Heptadecane, 2,6-dimethyl- (Figure 4.12).
Results
74
Figure 4.12: GC-MS Chromatogram of honey samples from Gurgaon
The following compounds were detected in honey samples from Hisar: 5-Octadecene,
(E)-; Octadecane, 3-ethyl-5-(2-ethylbutyl)-; Octadecane, 6-methyl-; Ethanol, 2-(octadecyloxy)-;
Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1R-(1R*,4Z,9S*)]; Caryophyllene;
Eicosane, 2-methyl-; Heptadecane, 2,6,10,15-tetramethyl-; Decane, 2,3,5,8-tetramethyl-; Oleic
acid; 3-(octadecyloxy)propyl ester; Octadecane, 1,1'-[(1-methyl-1,2-ethanediyl)bis(oxy)]bis-,
2,4-Di-tert-butylphenol; Phenol, 2,6-bis(1,1-dimethylethyl)-; Phenol, 3,5-bis(1,1-dimethylethyl)-
; Spirost-8-en-11-one, 3-hydroxy-; (3ß,5a,14ß,20ß,22ß,25R)-; Ingenol, 3TMS; 3-
(octadecyloxy)propyl ester; Octadecane, 3-ethyl-5-(2-ethylbutyl)- (Figure 4.13).
Results
75
Figure 4.13: GC-MS Chromatogram of honey samples from Hisar
The following compounds were detected in honey samples from Panipat: Decane, 2-
methyl-; Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1R-(1R*,4Z,9S*)]-;
Caryophyllene; Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-; Quinoline, 1,2-
dihydro-2,2,4-trimethyl-; 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3ß,5Z,7E)-;
Eicosane, 2-methyl-; Heptadecane, 2,6,10,15-tetramethyl-; Decane, 2,3,5,8-tetramethyl-;
Octadecane, 3-ethyl-5-(2-ethylbutyl)-; Tetradecane, 2,6,10-trimethyl-; Ethanol, 2-
(octadecyloxy)-; Dodecane, 2,7,10-trimethyl-; Dodecane, 2,6,11-trimethyl-; Octadecane, 6-
methyl- (Figure 4.14).
Results
76
Figure 4.14: GC-MS Chromatogram of honey samples from Panipat
4.9 Metabolites in egg during summer
Following metabolites were reported in the egg samples of Rohtak during summer:
(5ß)Pregnane-3,20ß-diol, 14a,18a-[4-methyl-3-oxo-(1-oxa-4-aza; psi.,.psi.-Carotene, 1,1',2,2'-
tetrahydro-1,1'-dimethoxy; 1,2-Propanediol, 3-(octadecyloxy)-, diacetate; 10-Heneicosene (c,t);
13-Docosenamide, (Z); 17-Pentatriacontene; 1-Hexadecanol;1-Hexadecanol, 2-methyl; 1-
Octanol, 2,2-dimethyl; 1-Octene, 3,7-dimethyl; 2-(tert.-Butyldimethylsilyl)oxybenzylidene
acetophenone; 2,3-Dimethyldecane; 2,4-Di-tert-butylphenol; 2,7-Diphenyl-1,6-
dioxopyridazino[4,5:2',3']pyrrolo[4',5'-d]pyridazin; 2-Bromo dodecane; 4-
Trifluoroacetoxytridecane; 5,6,7,8,9,10-Hexahydro-9-methyl-spiro[2H-1,3-benzoxazine-4,1; 5-
Ethyl-1-nonene; 7,8-Epoxylanostan-11-ol, 3-acetoxy; 7-Hexadecene (Figure 4.15).
Results
77
Figure 4.15: GC-MS Chromatogram of egg samples from Rohtak during summer
During summer, the egg samples from Gurgaon were found to have Heptadecane,
2,6,10,15-tetramethyl; Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene;
Caryophyllene; Ergosta-5,22-dien-3-ol, acetate, (3ß,22E); 5,6,7,8,9,10-Hexahydro-9-methyl-
spiro[2H-1,3-benzoxazine-4,1'-cyclohexane]-; 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol,
(3ß,5Z,7E); Eicosane, 2-methyl; Decane, 2,3,5,8-tetramethyl; Octadecane, 3-ethyl-5-(2-
ethylbutyl); Tetrapentacontane, 1,54-dibromo; Oleic acid, 3-(octadecyloxy)propyl ester (Figure
4.16).
Results
78
Figure 4.16: GC-MS Chromatogram of egg samples from Gurgaon during summer
The following compounds were determined in egg samples from Hisar: Decane, 2,3,5,8-
tetramethyl; Tetradecane, 2,6,10-trimethyl; Octadecane, 3-ethyl-5-(2-ethylbutyl); Octadecane,
1,1'-[(1-methyl-1,2-ethanediyl)bis(oxy)]bis; 1,2-Propanediol, 3-(hexadecyloxy)-, diacetate; Oleic
acid, 3-(octadecyloxy)propyl ester; 1-Hexadecanol; 7-Hexadecene, (Z); Cetene; Octadecane, 6-
methyl; Caryophyllene; Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene; Oleic acid,
3-(octadecyloxy)propyl ester; 4a,7b-Dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-9a-((2-
methylpropanoyl)oxy)-5-oxo-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro; 1,25-Dihydroxyvitamin D3,
TMS derivative (Figure 4.17).
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79
Figure 4.17: GC-MS Chromatogram of egg samples from Hisar during summer
The metabolites identified in egg samples of Panipat in summer were: Cholest-22-ene-
21-ol, 3,5-dehydro-6-methoxy-, pivalate; Cycloheptasiloxane, tetradecamethyl; Decane, 2,3,5,8-
tetramethyl; Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1R; Caryophyllene;
Cetene; 9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester,; Androstane-11,17-
dione, 3-[(trimethylsilyl)oxy]-, 17-[O-(phenyl; Benzene, (1-methyldodecyl); 7,9-Di-tert-butyl-1-
oxaspiro(4,5)deca-6,9-diene-2,8-dione; 7-Hexadecene, (Z); 9-(Acetyloxy)-4a,7b-dihydroxy-3-
(hydroxymethyl)-1,1,6,8-tetram (Figure 4.18).
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Figure 4.18: GC-MS Chromatogram of egg samples from Panipat during summer
4.10 Metabolites in egg during winter
During winter following compounds were identified in the egg samples from Rohtak:
Phenol; Carbamic acid, phenyl ester; Phosphonic acid, (p-hydroxyphenyl)-; 5-Undecene, (Z)-;
Hexadecane, 1,1-bis(dodecyloxy)-; 9-octadecenoic acid, 2,2,2-trifluoroethyl ester; Calealactone
B, TMS derivative; 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3ß,5Z,7E); Octadecane,
1,1'-[1,3-propanediylbis(oxy)]bis-; Decane, 2,4,6-trimethyl-; Decane; Undecane; Octadecane, 3-
ethyl-5-(2-ethylbutyl)-; Ethanol, 2-(octadecyloxy)-; 2-Myristynoyl pantetheine.; Tetradecane,
2,6,10-trimethyl-; Octadecane, 3-ethyl-5-(2-ethylbutyl)- (Figure 4.19).
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Figure 4.19: GC-MS Chromatogram of egg samples from Rohtak during winter
In winter, the following metabolites were reported in the egg samples of Gurgaon; 7-epi-
trans-sesquisabinene hydrate; Hexane, 2,3,4-trimethyl-; Octadecane, 3-ethyl-5-(2-ethylbutyl)-;
Heptane, 2,4-dimethyl-; 2-Dodecene, (E)-; 3-Undecene, (Z)-; 3-Hydroxyhex-4-enethioic acid, S-
t-butyl ester; 4-Hydroxy-4-methylhex-5-enoic acid, tert.-butyl ester; Ethanol, 2-(octadecyloxy)-;
Undecane; Undecane, 4,7-dimethyl-; Decane, 3,7-dimethyl-; 2-Trifluoroacetoxypentadecane;
Decane, 3-methyl-; 2,3-Dimethyldecane; Decane, 2,4,6-trimethyl-; 2-Bromo dodecane;
Eicosane, 10-methyl-; Dodecane, 2,6,11-trimethyl-; Eicosane, 2-methyl- (Figure 4.20).
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Figure 4.20: GC-MS Chromatogram of egg samples from Gurgaon during winter
The following metabolites were discovered in the egg samples of Hisar: Heptadecane,
2,6,10,14-tetramethyl-; Dodecane, 2,7,10-trimethyl-; 2-Bromo dodecane; Disulfide, di-tert-
dodecyl; Eicosane, 10-methyl-; Octadecane, 6-methyl-; Sulfurous acid, dodecyl pentyl ester;
Heptadecane, 2,6,10,15-tetramethyl; Decane, 2,3,5,8-tetramethyl-; Decane, 3,7-dimethyl-; 7-
Tetradecene, (Z)-; 1-Hexadecanol; 3-Tetradecene, (Z)-; Decane, 2-methyl-; Octadecane, 6-
methyl-; 7-epi-cis-sesquisabinene hydrate; 2H-Pyran, 2-(7-heptadecynyloxy)tetrahydro-; Z,Z,Z-
1,4,6,9-Nonadecatetraene; 1,2-Dimethyltryptamine (Figure 4.21).
Results
83
Figure 4.21: GC-MS Chromatogram of egg samples from Hisar during winter
The analyzed egg samples from Panipat reported the presence of 1-Hexadecanol; 3-
Trifluoroacetoxytetradecane; 1-Hexadecanol, 2-methyl-; Oleic acid, 3-(octadecyloxy)propyl
ester; Cholest-22-ene-21-ol, 3,5-dehydro-6-methoxy-, pivalate; Octadecane, 3-ethyl-5-(2-
ethylbutyl)-; Caryophyllene oxide; Oleic acid, 3-(octadecyloxy)propyl ester; 1,25-
Dihydroxyvitamin D3, TMS derivative; Octadecane, 3-ethyl-5-(2-ethylbutyl)-; 4a,7b-Dihydroxy-
3-(hydroxymethyl)-1,1,6,8-tetramethyl-9a-((2-methylpropanoyl); Octadecane, 3-ethyl-5-(2-
ethylbutyl)-; 2,4-Di-tert-butylphenol; Phenol, 3,5-bis(1,1-dimethylethyl)-; Phenol, 2,6-bis(1,1-
dimethylethyl)- (Figure 4.22).
Results
84
Figure 4.22: GC-MS Chromatogram of egg samples from Panipat during winter
Results of objective 4: To assess the health risk of local people associated with the
consumption of chicken eggs and honey in Haryana
4.11 Health risk assessment of Honey:
4.11.1 Estimated Daily Intake
The calculated values of the estimated daily intake (EDI) of each metal for both human
adults and children are shown in Table 5. The obtained EDIs of Cu, Se and Fe for adults were
0.15, 0.06 and 0.20 mg/kg/day, respectively (adults) and 0.04, 0.02 and 0.05 mg/kg/day,
respectively (children) in Rohtak; 0.20, 0.04 and 0.20 mg/kg/day, respectively (adults) and 0.05,
0.01, and 0.05 mg/kg/day, respectively (children) in Gurgaon; 0.15, 0.08 and 0.20 mg/kg/day,
respectively (adults); and 0.04, 0.02 and 0.05 mg/kg/day, respectively (children) in Hisar; 0.18,
0.14 and 0.17 mg/kg/day, respectively (adults); and 0.05, 0.04 and 0.04 mg/kg/day, respectively
(children) in Panipat. The decreasing pattern of the EDI values of the metals was Fe > Cu > Se
(Figure 4.23).
Results
85
Figure 4.23: Calculated EDI of honey in different regions of Haryana
4.11.2 Hazard Quotient
The HQ data are presented in Table 5. The HQs for Cu, Se and Fe in adults were 3.75,
12.00 and 0.28, respectively, in Rohtak; 5.00, 8.00 and 0.28, respectively, in Gurgaon; and 3.75,
16.00, and 0.28, respectively, in Hisar; and 4.50, 28.00, and 0.24, respectively, in Panipat. The
HQs for Cu, Se and Fe in children were 1.00, 4.00, and 0.07, respectively, in Rohtak; 1.25, 2.00,
and 0.07, respectively, in Gurgaon; 1.00, 4.00, and 0.07, respectively, in Hisar; and 1.25, 8.00,
and 0.06, respectively, in Panipat (Figure 4.24; Table 4.12).
Results
86
Figure 4.24: Calculated HQ of honey in different regions of Haryana
Table 4.12: Calculated EDI and HQ for metals in honey in both adults and children in Haryana
Location
Element
EDI (Adult)
HQ (Adult)
EDI (Children)
HQ (Children)
Rohtak
Copper
0.15
3.75
0.04
1.00
Selenium
0.06
12.00
0.02
4.00
Iron
0.20
0.28
0.05
0.07
Gurgaon
Copper
0.20
5.00
0.05
1.25
Selenium
0.04
8.00
0.01
2.00
Iron
0.20
0.28
0.05
0.07
Hisar
Copper
0.15
3.75
0.04
1.00
Selenium
0.08
16.00
0.02
4.00
Iron
0.20
0.28
0.05
0.07
Panipat
Copper
0.18
4.50
0.05
1.25
Selenium
0.14
28.00
0.04
8.00
Iron
0.17
0.24
0.04
0.06
Results
87
4.12 Health risk assessment of Egg samples:
4.12.1 Estimated Daily Intake
The values of estimated daily intake (EDI) for humans (adults and children) were
calculated for both the summer and winter seasons (Tables 9 and 10). The EDIs of Cu and Fe for
adults and children during summer were 0.003 and 0.002 mg/kg/day, respectively (adults) and
0.012 and 0.007 mg/kg/day, respectively (children) in Rohtak; 0.003 and 0.002 mg/kg/day
(adults) and, 0.013 and 0.007 mg/kg/day, respectively (children) in Gurgaon; 0.002 and 0.002
mg/kg/day, respectively (adults) and, 0.008 and 0.007 mg/kg/day, respectively (children) in
Hisar; 0.003 and 0.002 mg/kg/day, respectively (adults) and, 0.012 and 0.008 mg/kg/day,
respectively (children) in Panipat (Figure 4.25; Table 4.13).
Figure 4.25: Calculated EDI of eggs in different regions of Haryana during the summer
During the winter season, EDIs were recorded as 0.003 and 0.002 mg/kg/day,
respectively (adults) and, 0.011 and 0.008 mg/kg/day, respectively (children) in Rohtak; 0.002
and 0.002 mg/kg/day (adults) and, 0.008 and 0.007 mg/kg/day, respectively (children) in
Gurgaon; 0.002 and 0.002 mg/kg/day, respectively (adults) and, 0.008 and 0.007 mg/kg/day,
Results
88
respectively (children) in Hisar; 0.003 and 0.002 mg/kg/day, respectively (adults) and, 0.012 and
0.008 mg/kg/day, respectively (children) in Panipat (Figure 4.26; Table 4.14).
Figure 4.26: Calculated EDI of eggs in different regions of Haryana during winter
4.12.2 Hazard quotient
The values for hazard quotient (HQ) in adults during the summer season for Cu and Fe
were recorded to be 0.08 and 0.00, respectively; in Rohtak, 0.08 and 0.00, respectively; in
Gurgaon; 0.05 and 0.00, respectively; in Hisar; 0.07 and 0.00 respectively, in Panipat. The HOs
for Cu and Fe in children were 0.3 and 0.01, respectively, in Rohtak; 0.31 and 0.01, respectively,
in Gurgaon; 0.19 and 0.01, respectively, in Hisar; 0.27 and 0.01, respectively; in Panipat, (Figure
4.27).
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89
Figure 4.27: Calculated HQ of eggs in different regions of Haryana during the summer
HQs for adults during the winter season for Cu and Fe were recorded to be 0.07 and 0.00
respectively, in Rohtak; 0.05 and 0.00 respectively, in Gurgaon; 0.05 and 0.00 respectively, in
Hisar; 0.07 and 0.00 respectively, in Panipat (Table 10). The HQs for Cu and Fe in children were
0.27 and 0.01, respectively; in Rohtak, 0.21 and 0.01, respectively; in Gurgaon, 0.20 and 0.01,
respectively; in Hisar, 0.29 and 0.01, respectively; in Panipat, (Figure 4.28).
Results
90
Figure 4.28: Calculated HQ of eggs in different regions of Haryana during winter
Table 4.13: Calculated EDI and HQ for metals in egg samples in both adults and children in the
summer season in Haryana
Location
Elements
EDIT
(Adult)
HQ
(Adult)
EDI
(Children)
HQ
(Children)
Rohtak
Copper
0.003
0.08
0.012
0.30
Iron
0.002
0.00
0.007
0.01
Gurgaon
Copper
0.003
0.08
0.013
0.31
Iron
0.002
0.00
0.007
0.01
Hisar
Copper
0.002
0.05
0.008
0.19
Iron
0.002
0.00
0.007
0.01
Panipat
Copper
0.003
0.07
0.011
0.27
Iron
0.002
0.00
0.008
0.01
Table 4.14: Calculated EDI and HQ for metals in egg samples in both adults and children in the
winter season in Haryana
Location
Elements
EDIT
(Adult)
HQ
(Adult)
EDI
(Children)
HQ
(Children)
Rohtak
Copper
0.003
0.07
0.011
0.27
Iron
0.002
0.00
0.008
0.01
Results
91
Gurgaon
Copper
0.002
0.05
0.008
0.21
Iron
0.002
0.00
0.007
0.01
Hisar
Copper
0.002
0.05
0.008
0.20
Iron
0.002
0.00
0.007
0.01
Panipat
Copper
0.003
0.07
0.012
0.29
Iron
0.002
0.00
0.008
0.01
Results
50
Discussion
92
5.1 Discussion of objective 1: To evaluate physicochemical and mineral levels in chicken
eggs and honey in Haryana
5.1.1 Physico-chemical parameters of honey:
pH is associated with the storage of honey and the growth of microorganisms that can
alter the texture and stability of honey (Feas et al., 2010). According to the Iranian National
Standards Organization (INSO), the lowest tolerable pH value for honey is 3.5 (Zarei et al.,
2019). Food safety organizations have not described the pH limits of honey (Chefrour et al.,
2009); however, our results indicate that honey is acidic.
Our values for pH were higher than those obtained from other parts of India (Kumar et
al., 2018; Nayik & Nanda, 2015; Nayik et al., 2019; Shobham & Nayar, 2017). Kumar et al.
(2018) investigated the honey samples obtained from cotton flora and rosewood floral sources
and found the pH to be 4.85 and 3.81, respectively.
Honey is usually acidic, and its variations in values can be attributed to its source,
enzymatic process, or conversion of raw material by fermentation and salivary secretions from
honey bees (Abselami et al., 2018).
Moisture is a significant factor determining honey's potential to stay fresh and prevent
fermentation (Ferreira et al., 2009). In 2011, BISs were grouped with honey based on their
moisture content. According to these criteria, honey samples with moisture levels less than 20%
were categorized as "Special Grade", those with < 22% moisture as "Grade A", and those with
moisture < 25% as "Standard Grade". In our study, all the samples were found to have moisture
levels < 22%; hence, they can be categorized as Grade A honey. No significant difference was
observed in the moisture content of honey samples collected from different locations (Kumar et
al., 2018).
Our results were consistent with those of a study by Gairola et al. (2013). They reported
the average value for moisture content in honey to be between 19 to 25%. Our values were more
significant than Nayik et al. (2019) observed. Their study reported that the average moisture
content was 17.5-19.1%. Kaur et al. (2016) found the moisture percentage in analyzed honey
samples to be 18-24.5%. The higher values can be attributed to removing unripe honey from the
bee hives.
This variation can also be attributed to the source of origin, climatic conditions, grade of
maturity in hives, processing, season in which harvesting is performed, and storage conditions;
Discussion
93
thus, the variation can differ from time to time. A high amount of water can cause unwanted
fermentation of honey at the time of storage, eventually resulting in a bitter taste. A greater
humidity during the removal of honey from hives is more likely to elevate the moisture content
(Imtara et al., 2018).
The EC of honey is directly associated with the concentration of organic acids and
minerals in honey (Habib et al., 2014). The distribution of honey is highly variable depending
upon the source of nectar; hence, nectar intake is considered a significant factor in the
classification of honey (Kumar et al., 2018; Terrab et al., 2004). Kamal et al. (2019) reported the
EC range in investigated honey samples to be 631.95-804.54 μS/cm. Our results were
comparable to those of Kamal et al. (2019) and were higher than those reported by other
researchers (Shobham et al., 2017; Ceylan et al., 2019).
Optical density is a critical factor in determining the colour and freshness of honey.
Overall, OD values can provide helpful information about the colour and freshness of honey
samples (Moulya et al., 2023). Our study produced similar results to those reported by (Parihar et
al., 2020), and the values were lower than those in the studies of Thomas and Kharnaior (2023).
The total protein content of honey strongly depends upon floral sources and may be
promoted by enzymes produced either by bees or derived from nectar (Alvarez-Suarez et al.,
2010). Usually, the protein content of honey is less than 5.00 g/kg (Saxena et al., 2020). The
amount of pollen and nectar in honey likely determines its protein content. In our study, protein
content was below the detection level in the analyzed honey samples. However, in some other
studies, proteins were present in honey (Habib et al., 2014; Saxena et al., 2020).
Acidity plays a vital role in contributing to the flavour of honey, increasing chemical
reactions, antioxidant and antibacterial activity and stability against microbes (Gheldof et al.,
2002). The acidity of honey is due to different gluconic acids, particularly gluconic acid, lactones
and inorganic ions such as chlorides, sulfates and phosphates. A high amount of acidity indicates
sugar fermentation, which is transformed into organic acids, as mentioned by Gomes et al.
(2010) and Habib et al. (2014), whereas low acidity is an indicator of the freshness of honey
(Shobham et al., 2017). In the present study, all samples were within the permitted value, i.e. 50
meq/kg. All the samples were consistent with the established standards, which suggest that the
honey was fresh and not fermented.
Discussion
94
5.1.2 Mineral content in honey:
Various trace minerals and heavy metals were analyzed in the honey samples in order to
determine their quality. The minerals found in the samples were sodium (Na), potassium (K),
calcium (Ca) and magnesium (Mg), which ranged from 12.15-13.92 mg/100 g, 176.20-178.51
mg/100 g, 19.25-21.92 mg/100 g and 6.23-7.85 mg/100 g, respectively. Of these, K was the most
abundant mineral found in honey, and Ca was the second most abundant mineral, with the
highest amount in samples collected from Panipat and the lowest in those collected from
Gurgaon. Our results for Na and Ca were lower than the values reported by Ikegbunam and
Okwu (2021).
The least abundant mineral was magnesium (6.23-7.85 mg/100 g). Among the trace
minerals analyzed in the honey, Cu was most abundant in Panipat honey (1.25-1.87 mg/100 g)
and least abundant in Rohtak honey samples (1.08-1.54 mg/100 g). The selenium (Se) content
was highest in Panipat, between 1.02 mg/100 g and 1.46 mg/100 g, and lowest in Gurgaon (0.21-
0.46 mg/100 g). Similarly, iron (Fe) was most abundant in honey samples from Gurgaon (1.65-
1.88 mg/100 g) and least abundant in those from Panipat (1.28-1.77 mg/100 g). Lead (Pb) and
arsenic (As) concentrations were below the limit of quantification (0.05 mg/kg).
The obtained values for Cu ranged from 1.08-2.11 mg/100 g and were below the
permitted value of Cu (30 mg/kg). However, the average values were much more significant than
those reported in previous studies in New Zealand (0.25 mg/kg; (Vanhanen et al., 2011),
Slovenia (3.22 mg/kg); (Golob et al., 2005), Croatia (1074 µg/kg); (Bilandžić et al., 2011),
Turkey (0.23–2.41 and 0.25–1.10 mg/kg); (Tizen et al., 2007; Tuzen & Soylak, 2005), Italy (890
and 960 µg/kg); (Buldini et al., 2001; Pisani et al., 2008), and the Black Sea area of Turkey
(9.75–35.8 µg/kg) (Silici et al., 2008).
The Se values ranged between 0.21 and 1.46 mg/100 g. These values were much more
significant than those reported in other studies. Pehlivan and Gul (2015) reported that the Se
concentration was 0.0003 mg/kg. Similarly, in another study by Dhahir and Hemed (2015), the
Se concentration ranged from 0.232- 0.8100 mg/kg.
In the present study, the concentration of Fe ranged between 1.28 and 1.88 mg/100 g.
Thus, the obtained values are much lower than the permissible limit, i.e., 800 mg/kg. These
values were higher than those reported in previous studies. In Turkey, Fe concentrations were
Discussion
95
reported to be 268-1036 µg/kg (Kiliç Altun et al., 2017). Saghaei et al. (2009) reported a Fe
value between 0.70 ± 0.20 mg/kg, ranging from 0.37 - 1.98 mg/kg in honey samples; a study
conducted in Kahramanmaraş Province, Turkey, reported a Fe level in the honey of 0.36 mg/kg
(Erbilir & Erdoĝrul, 2005); and that in the Lazio region in Italy reported a Fe level of 4.51 ± 0.39
mg/kg.
5.1.3 Physico-chemical parameters of chicken egg:
There was no significant difference between the lengths of egg samples from different
seasons. The length of the egg samples ranged between 4.90-5.72 cm during summer and
between 5.11-5.66 cm during winter. The egg samples collected from Gurgaon during summer
had the most petite length. Egg samples collected from Hisar were the longest. Our results were
similar to those mentioned by Hanusova et al. (2015) and Rath et al. (2015). Hanusova et al.
(2015) reported the average length of egg samples to be 5.72 cm. Likewise, Rath et al. (2015), in
their study, reported the average length of eggs to be 54.39 mm (5.43 cm). However, Bagh et al.,
in 2018, investigated the average length of egg samples and found it to be 45.63 mm (4.56 cm),
which was lower than the values stated in our study.
The width of the egg samples ranged between 4.07-4.28 cm during summer and 4.09-
4.16 cm during winter. There was no significant difference in the width of the samples collected
from various districts. Samples collected from Gurgaon during summer had the shortest width,
and the longest width was recorded in samples collected from Hisar. The results obtained in the
present study were similar to those reported by Hanusova et al. (2015) and Rath et al. (2015).
They reported the average width of egg samples to be 4.32 cm and 39.92 mm (3.99 cm),
respectively. However, our values for width were higher than those stated by Bagh et al. (2018).
They investigated the egg samples; the average width was 33.86 mm (3.38 cm).
All the analyzed egg samples weighed within the range of 47.58 g-55.12 g. The lowest
value for weight was recorded in samples from Gurgaon during summer, and the highest value
was found in Hisar during summer. Our results were quite similar to those reported by Rath et al.
(2015). Their study stated the average weight of eggs to be 57.78 g. On the other hand, Biswas et
al. (2010) and Mohanta et al. (2018) found the average weight to be 41.84 g and 27.95 g,
respectively, which is lower than the present study.
Discussion
96
The values obtained for SI (21.13-23.53) in the present study were lower than those
obtained in previous studies. Rath et al. (2015), Bagh et al. (2018) and Abo El-Maaty et al.
(2012) investigated egg samples and reported the Si to be 73.53, 74.23 and 82.68, respectively.
In summer and winter, the pH of egg samples was found in the range of 6.61-6.90 and
6.71-7.07. All the egg samples had acidic pH during the summer and acidic to neutral in the
winter. The lowest value was reported in Hisar (6.61) during the summer season, and the highest
was reported in Rohtak (7.07) during the winter. Our values for pH were similar to those
reported by Marzec et al. (2019), i.e. 7.8 and Dong et al. (2017), i.e. 7.74. Jin et al. (2011)
analyzed the egg samples and reported their pH to be 8.09, higher than the values reported in the
present study.
The protein content in whole eggs was reported to be within the range of 10.10-10.33 g in
summer and 10.14-10.40 g in winter. The highest amount was recorded in Hisar during winter
and the lowest in Gurgaon during the summer. Our result was in the range detected in a study
conducted by Lordelo et al. (2017). El-Maaty et al. (2021) reported the protein content in hen
eggs to be 5.80 g/dL.
5.1.4 Mineral content in egg:
Na was the most abundant of all the minerals, followed by K, Ca and Mg. Our results for
Na and K were lower than those reported in other studies. Mg was the least abundant mineral
(17.50-18.58 mg/100 g). Analysis of trace minerals indicated the presence of Na, Ca, Mg, K, Cu
and Fe, whereas As Pb and Se were below the detection level. Cu was most abundant in Gurgaon
during the summer season. There was a slight seasonal variation in the distribution of mineral
content.
In the present study, Na was found to be 123.35-122.64 mg/100 g. However, in previous
studies conducted by Michalak et al. (2011) and Ieggli et al. (2010), Na values were reported to
be 1633 mg/kg and 1410.3 µg/g, respectively. K values in analyzed egg samples were within the
range of 109.60-110.71 mg/100 g. In their study, Heflin and others (2018) reported K content to
be 524 mg/100 g. These values were comparatively more than those presented in our study.
Michalak et al. (2011) and Ieggli et al. (2010) reported the values to be 1074 mg/kg and 1185.8
µg/g, respectively. Ca values (70.34-71.24 mg/100 g) were comparatively lower than K, whereas
Mg was the least abundant, ranging between 17.50-18.58 mg/100 g. Michalak and others (2011)
Discussion
97
indicated the K levels to be 116 mg/kg. In another study by Heflin et al. (2018), K values were
reported to be relatively higher (50.8 mg/100 g) than in the present study.
Copper (Cu) is essential for various body functions (NRC, 1994). However, increased Cu
concentration can lead to jaundice, liver and renal issues, nausea and diarrhoea. Also, excessive
deposition of Cu in the liver, brain, and eyes indicates Wilson's disease (Ogwok et al., 2014;
Elsharawy, 2018). The Cu content in hen egg ranged between 1.61-2.69 mg/100 g. The highest
value (2.69 mg/100 g) was recorded in Gurgaon during the summer, and the lowest value (1.61
mg/100 g) was recorded in Hisar during the summer. In previous studies, the Cu content was
reported to be 0.233 mg/kg (Michalak et al., 2011), 2.70 µg/g (Abdulkhaliq et al., 2012) ) and
1.17 ppm (Korish & Attia, 2020). The permissible limit for Cu in eggs has been reported to be 10
ppm (Roychowdhury et al., 2003).
Iron (Fe) is important as its deficiency may result in myocardial risks, gut infections and
nose bleeding (Jaishankar et al., 2014). Nevertheless, a higher concentration of Fe can cause
depression, respiratory disorder, coma and cardiac arrest. The permissible limit for Fe for adults
is 45 ppm per day and for children 40 ppm per day. The Fe concentration in the present study
ranged from 1.43- 1.80 mg/100 g. The highest value (1.80 mg/100 g) for Fe was reported in
Rohtak during winter. In summer, the lowest value (1.43 mg/100 g) was recorded in Hisar and
the highest (1.68 mg/100 g) was recorded in Panipat.
Arsenic (As) is an element stored in animal tissues based on their source of diet. If
present in excess amounts, toxicity can result in headaches, gut irritation and nausea (Korish &
Attia, 2020). In the present study, As was not detected in any of the egg samples. However, in
their study, Michalak et al. (2011) reported As to be 259 µg/kg. In another study by Korish and
Attia (2020), the As level was below the detection limit. The permissible limit for As in eggs is
0.1 ppm (Saad et al., 2018).
The selenium (Se) content in egg samples was found to be below the limit of detection in
both summer and winter. In another study conducted by Korish and Attia (2020), Se content was
not detected and was below the limit of quantification. However, 66 ng/g of Se was reported by
Giannenas et al. (2009).
Lead (Pb) is a metal that can act as a neurotoxin, resulting in metabolic impairment (Neal
& Guilarte, 2013; Virginia & Aschner, 2021). It can also affect renal functioning, nervous,
gastrointestinal, and hemopoiesis (Korish & Attia, 2020). The present study recorded the
Discussion
98
concentration of Pb in egg samples to be below the limit of quantification. Similarly, studies by
Korish and Attia (2020) and Michalak et al. (2011) reported Pb concentration below the
detection limit. However, in their study, Abdulkhaliq et al. (2012) reported Pb concentration to
be 0.27µg/g. The maximum tolerance level for Pb is 0.43 ppm (Khan et al., 2016).
5.2 Discussion of objective 2: To evaluate the antioxidant properties of chicken eggs and
honey in Haryana
The percentages of DPPH inhibition in the analyzed honey samples were similar to those
recorded in previous studies conducted by Khalil and others and Dżugan and others in 2018. The
values obtained for antioxidant capacity in honey were more significant than those reported by
Khalil and others in 2012 and Bundit and others in 2016. The antioxidant properties of honey are
usually contributed by pollen, nectar and substances that consist of enzymes, organic acids and
vitamins (Gheldof et al., 2002). Variation in these properties also depends on the floral source,
collection method (Jantakee & Tragoolpua, 2015), processing and handling, storage method,
season and other environmental factors (Bundit et al., 2016).
Analysing honey samples from four locations revealed a significant difference in the
DPPH radical scavenging and FRAP values. The overall antioxidant activity of honey tended to
increase in the summer season. This could be because of heat stress. The most favourable
temperature for laying hens is 20°-25°C (Tumova & Gous, 2012). Therefore, if the temperature
surpasses 30°C, environmental challenges linked to temperature start appearing, particularly heat
stress. Increased temperature conditions severely affect health and production in the poultry
sector (Lara & Rostagno, 2013). Antioxidant activities differ significantly due to increases in
heating temperature. This could be most likely due to alterations in the structure of proteins
present in eggs. High temperatures can damage food's antioxidant compounds (Nahariah &
Hikmah, 2021). The deficiency of naturally protecting substances and increased exposure
stimulates the generation of reactive oxygen metabolites (Miller et al., 1993), which results in
the progression of oxidative damage to some significant biological macromolecules, such as
DNA, proteins and lipids. This interferes with their normal functioning, resulting in decreased
performance and several diseases (Valko et al., 2007).
Discussion
99
5.3 Discussion of objective 3: To analyze the seasonal variation of metabolic parameters in
chicken eggs and honey
The analyzed honey and egg samples from different regions of Haryana indicated the
presence of different chemical compounds such as alcohols, phenols, aldehydes, carboxylic acids
and some other derivatives. So far, a total of more than 600 different volatile and semi-volatile
compounds of various chemical families, such as ketones, terpenes, aldehydes, phenols, alcohols,
carboxylic acids and derivatives of pyran and furan, have been detected in honey (Montenegro et
al., 2009). In our study, compounds like 17-pentatriacontene which show antibacterial,
anticancer, anti-arthritic and anti-inflammatory activities (Albratty et al., 2021); heptacosane, 1
chloro having antimicrobial activities (Hawar et al., 2023); eicosane 2-methyl having antifungal
activity (Ahsan et al., 2017); cis-11-eicossenamide having antimicrobial activity (Tareq et al.,
2020); 7-hexadecene, (Z) having anticancer properties (El Fakir et al., 2021) were determined.
Along with quality assessment, the profiling of volatile compounds also determines the botanical
source of honey. Their amount is directly related to the flora or source of origin (Tsagkaris et al.,
2021).
Some other compounds like Decane, 2,3,5,8-tetramethyl; Tetradecane, 2,6,10-trimethyl;
Octadecane, 3-ethyl-5-(2-ethylbutyl); Octadecane, 1,1'-[(1-methyl-1,2-ethanediyl)bis(oxy)]bis;
1,2-Propanediol, 3-(hexadecyloxy)-, diacetate; Oleic acid, 3-(octadecyloxy)propyl ester; 1-
Hexadecanol; 7-Hexadecene, (Z); Cetene; Octadecane, 6-methyl; Caryophyllene;
Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene; Oleic acid, 3-(octadecyloxy)propyl
ester; 4a,7b-Dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-9a-((2-methylpropanoyl)oxy)-5-
oxo-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro; 1,25-Dihydroxyvitamin D3, TMS derivative were also
reported in egg samples. The metabolites in summer and winter did not present any significant
variance. However, their concentrations can vary depending upon several factors such as
geographical origin, storage and stress conditions and the health status of animals (Muroya et al.,
2020). In another study on egg whites and yolk, 31 metabolites were determined, including
carbohydrates and amino acids. (Zhang et al., 2020; Cavanna et al., 2018).
5.4 Discussion of objective 4: To assess the health risks in local people associated with the
consumption of chicken eggs and honey in Haryana
Discussion
100
The presence of heavy metals in honey and eggs is now an issue of concern as it is a
threat to consumers' health. They can affect quality of life by accumulating to a level that could
be toxic (Aghamirlou et al., 2015). Therefore, a health risk assessment was performed to analyze
the risks associated with consuming honey and eggs. Risk assessment is a risk analysis process
that yields qualitative and quantitative explanations of the probability of hazards associated with
exposure to a harmful chemical. This process involves identifying and collecting data regarding
health risks due to exposure to toxic chemicals, integrating the collected data and analyzing the
relationships between the duration of exposure to a particular toxin, its dose, dose-response
analysis and the associated adverse health risks (Sobhanardakani & Kianpour, 2016).
The calculated EDI values of metals in honey exhibit the following pattern: Fe > Cu > Se.
A HQ < 1 indicates no probable adverse health effects; however, a HQ > 1 indicates probable
adverse effects on health (Fakhri et al., 2018; Dadar & Adel, 2017). All the HQ values for Fe
were less than 1 for adults and children, indicating that Fe is not a potential threat to human
health. However, an HQ > 1 for Cu and Se suggested potential human health risks. Heavy metals
enter the human body through the food chain. These heavy metals are very harmful and can lead
to various diseases such as anaemia (Zhang et al., 2020), cancer (Kim et al., 2015) and heart
failure (Lamas et al., 2016), disorders in the synthesis of haemoglobin, inflammation,
gastrointestinal bleeding, and renal and pulmonary infections (Skalny et al., 2020).
In the egg case, the hazard quotient (HQ) values of analysed metals in adults and
children were less than 1. This suggests that there are no potential health risks related to them
shortly.
SUMMARY AND CONCLUSION
101
In conclusion, our investigation into the quality of honey and eggs in Haryana has
revealed essential insights with implications for health and sustainability. No probable health risk
was observed due to the consumption of eggs. Currently, the study reports concentrations of
heavy metals within an acceptable range. However, elevated levels of copper and selenium in
honey raise concerns about potential health risks for consumers, underscoring the need for
ongoing monitoring and awareness. This study addresses a critical gap in understanding the
impact of industrialization on honey quality in Haryana. Our findings could lead to the need for
future research, emphasizing the importance of exploring sources of heavy metals and
implementing strategies to mitigate heavy metal contamination in honey.
Moving ahead, the integration of regular monitoring programs has become crucial,
aligning with the principles of human sustainability. By fostering consumer awareness and
implementing strategies to reduce pollutant levels in floral sources, we can contribute to a
healthier future for individuals and the ecosystem. Therefore, this study highlights the immediate
need for vigilance in honey quality and underscores the broader goal of fostering human
sustainability through responsible practices and a commitment to the well-being of both
consumers and the environment. Also, the escalating pollution and discharge of pollutants and
effluents into the environment may result in the bioaccumulation of chemicals and excessive
minerals in eggs. This could eventually lead to toxicity, potentially causing severe effects on
human health in the long term. Such repercussions not only impact health but also jeopardize the
sustainability of food sources. Therefore, researchers should emphasize the significance of
exploring various sources of metal contamination in the environment and developing effective
mitigation strategies. Future research efforts should focus on creating sustainable practices for
the poultry industry. This approach will enable industries to thrive without compromising the
health of consumers. Regular monitoring and awareness programs among consumers will
promote a healthier environment and safe food consumption.
Our study also provides insight into the antioxidant potential of honey and poultry eggs.
Moreover, it also depicts the effect of season on the antioxidant activity in eggs. The figure also
shows the variations at different selected locations. The FRAP values of poultry eggs tended to
increase in the summer. This could be due to increased temperature and heat stress conditions
during summer. The FRAP values of honey from different locations varied significantly and
were highest in Panipat and lowest in Hisar. This indicates that season is an essential factor for
SUMMARY AND CONCLUSION
102
antioxidant activity. However, other factors can also be considered. To date, the antioxidant
potential of honey and eggs has yet to be explored much.
Additionally, animal-derived antioxidants are scarce compared to data obtained from
plant sources. Therefore, further investigations and surveys must be conducted on this topic.
Such studies could unveil the significant antioxidant properties and components in food products
and their benefits in promoting human health.
The analyzed samples reported the presence of several different metabolites. Most have
antibacterial, antioxidant, anti-inflammatory, anti-cancer and analgesic properties, which benefit
human health. The metabolomic profiling is also a parameter that can determine the quality of
any sample. Overall, the findings can help beekeepers, poultry farmers, and local consumers
learn about the quality of honey and eggs in their region. As we navigate the complexities of
modern development, it is imperative to consider not only the immediate health risks posed by
heavy metal contamination but also the long-term sustainability of food sources. Sustainable
practices should be at the forefront of these efforts, ensuring that the honey industry continues to
thrive without compromising the health of consumers or the environment.
RESEARCH OUTCOME
103
1. Honey and Egg Quality: Elevated levels of heavy metals in honey and potential
pollutant bioaccumulation in eggs necessitate continuous monitoring. This
vigilance will help mitigate risks and ensure the safety of food sources.
2. Implications for Industrialization: The study highlights the impact of
industrialization on honey quality, revealing a critical gap in understanding how
these practices affect honey composition.
3. Future Research: The findings suggest a need for further research to explore the
sources of heavy metal contamination in honey and to develop strategies to
mitigate these risks. This will be crucial for ensuring the safety and sustainability
of honey production in Haryana.
4. Moving forward, integrating regular monitoring programs is essential to align
with the principles of human sustainability. By raising consumer awareness and
implementing strategies to reduce pollutant levels in floral sources, we can
advance toward a healthier future for individuals and the ecosystem. This study
highlights the immediate need for vigilance regarding honey quality and
underscores the broader goal of fostering human sustainability through
responsible practices and a commitment to consumer and environmental well-
being.
5. Honey and Egg Quality: Elevated levels of heavy metals in honey and potential
pollutant bioaccumulation in eggs necessitate continuous monitoring. This
vigilance will help mitigate risks and ensure the safety of food sources.
6. Pollution Impact: Escalating pollution and discharge of pollutants into the
environment may lead to the bioaccumulation of chemicals and excessive
minerals in eggs, potentially causing long-term health issues. Addressing these
concerns through research and sustainable practices is critical to preserving food
source sustainability.
7. Antioxidant Potential: Our study has shed light on the antioxidant potential of
honey and poultry eggs. It was observed that the FRAP (Ferric Reducing Ability
RESEARCH OUTCOME
104
of Plasma) values for poultry eggs increased during summer, possibly due to heat
stress. The FRAP values of honey varied by location, with the highest values in
Panipat and the lowest in Hisar, indicating that seasonal factors significantly
affect antioxidant activity. Further research is needed to explore these antioxidant
properties more deeply, especially since data on animal-derived antioxidants are
limited compared to plant sources.
8. Metabolomic Profiling: The analysis revealed several beneficial metabolites
with antibacterial, antioxidant, anti-inflammatory, anti-cancer, and analgesic
properties. This metabolomic profiling can provide insights into the quality and
health benefits of honey and eggs.
9. Sustainable Practice: To ensure that honey and poultry industries thrive without
compromising health or environmental integrity, future research should focus on
developing and implementing sustainable practices. These practices should
include monitoring pollutant sources, exploring mitigation strategies, and
promoting responsible industry standards.
Recommendations
105
Based on the findings of this research work, here are some recommendations:
1. Screening: A regular evaluation of physicochemical parameters, mineral content,
antioxidant content and metabolomic parameters must be done in order to assess
the quality of food products in a particular region.
2. Seasonal Variation: Evaluating the samples from different season will provide an
insight on the impacts of different seasons on a particular food product and
different strategies could be developed to cope up the effects of seasonal
differences.
3. Enhancing the Nutritional Value: The average protein content and antioxidant
content in egg can be enhanced further by developing strategies to improve the feed
of hen.
4. Sewage Treatment: The waste water from factories and industries must not be
allowed to seep directly into the agricultural fields. The water must be treated
appropriately before releasing it on the fields and direct sewage inflow into the
fields must be prevented. This will not hamper the mineral content in the crop
fields.
5. Minimize the Use of Pesticides and Fertilizers: Pesticides and fertilizers
deteriorate the quality of soil and this eventually impacts the quality of crops grown
on such fields.
6. Mitigation Strategies: Strategies must be developed to minimize the heavy metal
content in egg and honey. Heavy metals are very toxic to human health and lead to
severe diseases like cancer.
7. Health Risk Assessment: The health risk assessment must be done both for adults
and children to determine the carcinogenic and non-carcinogenic risks (if any)
associated with egg and honey.
Recommendations
106
8. Designer Eggs: Research must be directed towards formulation of designer eggs by
improving their overall quality content and decreasing the cholesterol concentration
in egg.
9. Education and Awareness: Awareness programmes must be initiated for farmers,
poultry workers. They must be given knowledge about feed and their impact on an
organism. Training on poultry management practices must be given to the poultry
farmers.
10. Future Research: The present study can serve as a basis for further research on the
quality parameters of egg and honey. Till date not much study has been conducted
in this aspect, therefore, this field requires more exploration. Further, a broad study
can be conducted on the impact of some other environmental factors on egg and
honey.
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