J Food Sci Technol (January 2020) 57(1):282–292
https://doi.org/10.1007/s13197-019-04058-0
ORIGINAL ARTICLE
Rainbow trout fillet biopreservation by edible chitosan-based
coating containing egg yolk antibody (IgY) and lycopene
Ali Ehsani1,2 • Mohammad Hashemi3,4 • Mojtaba Raeisi5,6 • Seyedeh Samane Naghibi7
Asma Afshari4
Revised: 30 July 2019 / Accepted: 21 August 2019 / Published online: 30 August 2019
Ó Association of Food Scientists & Technologists (India) 2019
Abstract The aim of the study was to investigate the effect
of the extracted egg yolk antibody along with lycopene on
the chemical quality of the rainbow trout fillet during
16 days of refrigeration storage. Chickens were immunized
against Pseudomonas fluorescens (P. fluorescens), She-
wanella putrefaciens (S. putrefaciens) and total spoilage
bacteria and their eggs were collected for the isolation of
egg yolk antibodies. Then fish fillets were immersed in
chitosan-based coating solutions, containing lycopene and
extracted antibodies, and analyzed for lipid oxidation
changes (peroxide, thiobarbituric acid, free fatty acid and
fatty acid profile), physico-chemical properties (pH and
water holding capacity), and sensory evaluation, during
16 days of refrigeration storage. Results showed that chi-
tosan solutions with lycopene or IgY could significantly
(p \ 0.05) increase the oxidative stability of lipids in fish
& Asma Afshari
Asmafshr@gmail.com; Afsharias@mums.ac.ir
1
Department of Food Science and Technology, Faculty of
Nutrition and Food Science, Tabriz University of Medical
Sciences, Tabriz, Iran
2
Food and Drug Safety Research Center, Tabriz University of
Medical Sciences, Tabriz, Iran
3
Medical Toxicology Research Center, Mashhad University of
Medical Sciences, Mashhad, Iran
4
Department of Nutrition, Faculty of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran
5
Infectious Diseases Research Center, Golestan University of
Medical Sciences, Gorgan, Iran
6
Food, Drug and Natural products Health Research Center,
Golestan University of Medical Sciences, Gorgan, Iran
7
Department of Food Hygiene and Quality Control, Faculty of
Veterinary Medicine, Urmia University, Urmia, Iran
123
•
fillets; although, combinational use of lycopene and IgY
showed a higher effect on delaying the rate of lipid oxi-
dation. Significant differences were also observed between
treatments contained combination of chitosan, antibody
and lycopene with the control group, regarding pH and
WHC. Saturated fatty acids increased in all treatments,
although the changes in the treatments containing lycopene
and antibody were significantly (p \ 0.05) lower than the
control group. Hence the addition of egg yolk antibody and
lycopene in coating solution are good bio-preservatives for
seafood products as it improves sensory attributes and
prevents lipid oxidation.
Keywords Rainbow trout  Egg yolk antibody  IgY 
Lycopene  Chitosan  Chemical quality
Introduction
Fish oil is recommended for consumption due to the
numerous health benefits. It is an important source of
polyunsaturated and omega-3 fatty acids. On the other
hand, the fish oil is very sensitive against oxidation and
corruption due to significant amounts of polyunsaturated
fatty acids (PUFA) (Ehsani et al. 2018).
Biodegradable coatings with natural ingredients, have
attracted the consumers’ attention. The main advantage of
such coatings is their application as a carrier of functional
ingredients, such as flavors, antimicrobials, antioxidants,
nutrients, enzymes and colors (Quirós-Sauceda et al. 2014).
One of the most widely used edible coatings is chitosan,
a poly cationic polysaccharide, produced by deacetylation
of chitin (Ehsani et al. 2018). The main commercial source
of chitin is shells of crustaceans. Chitosan has some
exclusive properties, such as biodegradability, non-toxicity
J Food Sci Technol (January 2020) 57(1):282–292
and compatibility with the environment. Anti-microbial
properties, antioxidant and film formation are also listed as
the functional properties of chitosan (Hassanzadeh et al.
2017). Regardless of microbial corruption, chemical and
physical factors have major roles in keeping the quality of
fish. Lipid oxidation occurs during storage and thermal
processing of raw materials, or during the long-term stor-
age of the final product. In addition to the changes in
organoleptic properties, oxidized lipids may have adverse
effects on human health (Raeisi et al. 2018).
Natural antioxidants are good alternatives for synthetic
antioxidants, because of less harmful effects (Raeisi et al.
2018). Lycopene is a carotenoid pigment that is found nat-
urally in fruits and vegetables, such as tomato (93% of car-
otenoids) (Østerlie and Lerfall 2005). Epidemiological
studies have confirmed that lycopene, as one of the strongest
known antioxidant, exerts beneficial effects on health.
Lycopene is twice stronger than beta-carotene and ten times
stronger than alpha-tocopherol (Østerlie and Lerfall 2005).
Chicken egg yolk immunoglobulin (IgY) is recognized
as an inexpensive antibody source, useful for therapeutic
applications by passive immunization therapy, through
preventing some intestinal infections (Lee et al. 2002). The
beneficial properties of IgY, compared to mammalian IgG,
such as higher physico-chemical stability, smaller molec-
ular size, more acidic isoelectric point and binding ability
with mammalian complements, have made it as an appro-
priate immunological tool. By far, some studies have been
conducted on egg yolk antibodies (Xu et al. 2012; Lee et al.
2002); however, the combinational use of coatings and egg
yolk antibodies has been neglected in aquatics.
In this study, egg yolk antibodies formulated with
lycopene were evaluated in the fish fillet model, in order to
reduce oxidation, improving the shelf-life and sensory
characteristics of fish fillet for the first time.
Materials and methods
Sample preparation
Fresh fish samples were taken from the Artemia and Aquatic
Animals Research Institute farm of Urmia University (West
Azerbaijan, Iran), and were carefully filleted. Fillets were cut
into 10 g-pieces and randomly divided into ten groups, as fol-
lowed: T1: control, T2: chitosan coating, T3: chitosan coat-
ing ? lycopene level one (1.5%), T4: chitosan
coating ? lycopene level 2 (3%), T5: chitosan coating ? anti
Pseudomonas fluorescens antibody ? lycopene level 1 (1.5%),
T6: chitosan coating ? anti Pseudomonas fluorescens anti-
body ? lycopene level 2 (3%), T7: chitosan coating ? anti
Shewanella putrefaciens antibody ? lycopene level 1 (1.5%),
T8: chitosan coating ? anti Shewanella putrefaciens
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antibody ? lycopene level 2 (3%), T9: chitosan coat-
ing ? total antibody ? lycopene level 1 (1.5%), T10: chitosan
coating ? total antibody ? lycopene level 2 (3%).
Antigen production
S. putrefaciens and P. fluorescens were purchased from the
Iranian Biological Resource Center (IBRC 10667) and the
Persian Type Culture Collection (PTCC1711), respec-
tively. In order to prepare the total spoilage bacteria, fish
fillet samples were packed in polyethylene bags, preserved
in the refrigerator and the fillets spoilage was evaluated on
different days. Sensory evaluation of spoiled fish fillet
samples was carried out by 10 semi-trained panelists,
selected from scientific staff and PhD students of the Food
Hygiene and Quality control of the Artemia and Aquatic
Animal Research Institute of Urmia University.
In order to provide the desired concentrations of S.
putrefaciens and P. fluorescens (109 CFU mL-1) and total
spoilage bacteria, opacity adjustment, using standards
McFarland methods was performed and absorption at
600 nm was measured. After centrifugation (Hettich,
Germany) for 10 min at 3000 g, precipitated bacteria were
disabled with saline, containing formalin 1%, within 24 h.
The inactivated bacteria were washed three times with
0.9% normal saline, and used as antigen to immunize
Leghorn chickens (Ehsani et al. 2018).
Chickens immunization
Twelve White Leghorn layers, 28 weeks old, were pur-
chased from the commercial producers. The average
weights of the live birds were in the range of 1.7–1.8 kg.
Main ingredients of chicken feed were as followed; corn
bran, wheat, corn and imported fish meal.
In the first immunization step, 0.5 mL of adjuvant was
mixed with 0.5 mL of a suspension, containing 109 CFU of
desired bacteria, and 0.5 mL was injected on both sides of
the chicken’s chest. In order to increase antibody titer,
three more doses (109 CFU bacteria), were injected with an
interval of 2 weeks. For the confirmation of the chicken’s
immunization, blood samples were collected and were sent
to the University of Tabriz for serum separation and indi-
rect ELISA test (Ehsani et al. 2018).
Isolation of IgY from egg yolk
The eggs were collected 1 week after the final injection and
stored at 4 °C, until further processing. The antibodies were
extracted from egg yolk by dis-placement with poly-
ethylene glycol (PEG). Briefly, the egg yolk was separated
from white, washed with distilled water to remove albumin.
The membrane was punctured and the yolk was allowed to
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flow into a graduated cylinder. Phosphate buffered saline
(PBS) (10 mM phosphate, 100 mM NaCl, pH 7.5, con-
taining 0.01% sodium azide), equivalent to two volumes of
yolk, was added and thoroughly stirred. Polyethylene glycol
(PEG 6000) was added to a final concentration of 3.5%. The
mixture was stirred for 30 min at room temperature and
centrifuged (Hettich, Germany) at 14,000 g for 20 min at
4 °C. This operation caused the separation of three phases
in the centrifuge tubes, including a yellow fatty layer on the
surface of the fluid, a clear supernatant layer (SNF) and a
semi-solid pliable layer of casein-like vitelline, representing
about one-third of the total volume of a substance in the
centrifuge tubes. The precipitate was centrifuged at
14,000 g for 20 min. The tubes were centrifuged once more
and the residual PEG solution removed. The precipitate was
re-dissolved in 1.0 mL of phosphate buffer (without NaCl)
overnight. Then the content was de-salted by dialysis pro-
cess. The obtained crude fraction, IgY was further purified
by Diethylaminoethyl cellulose (DEAE-C) ion exchange
column chromatography. Finally IgY fraction was con-
centrated by poly vinyl pyrolidone (PVP), at room tem-
perature. The protein content of the purified IgY fraction
was determined by the method described by Polson et al.
(1980).
Preparation of the coating solutions
Chitosan solution was prepared with 2% (w/v) chitosan in
1% (v/v) acetic acid glacial. The solution was placed on a hot
plate/magnetic stirrer (Tehran-Iran) for 6 h. The resultant
chitosan-based coating solution was filtrated, through a
Whatman filter paper No: 2. Glycerol was added to the
solution at 0.75% (w/v), and stirred for more than 15 min.
Two of the best concentrations, 60 and 90 mg mL-1
were prepared from freeze-dried IgY for S. putrefaciens
and P. fluorescens, respectively. After centrifugation at
10000 g for 30 min, the supernatant was collected and
sterilized by filtrating, through a 0.22 lm micro-filter.
Finally, IgY was added to chitosan-based coating solution.
The final coating solution was homogenized under aseptic
conditions, at 16000 g for 2 min.
Based on the antioxidant activity (the DPPH test) and
sensory evaluation of the lycopene in the fillets, two levels
of lycopene were chosen. For this purpose, appropriate
levels of lycopene were dissolved in 10 mL of acetone
(20%) and Tween 80 was added (0.2%) as an emulsifier.
The prepared lycopene solution was added to the coating
solution before glycerol addition (Ojagh et al. 2010).
Coating fish fillet
Fish fillets were immersed in 500 mL of different con-
centrations of solution for 120 s, using an interval of
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2 min. Treated fillets were dried at 4 °C for 5 h under
aseptic conditions and each 3 pieces were placed in sterile
polyethylene bags (Rasam Pack, 30*40 mm, 100 mic
thickness) and kept in the refrigerator for 16 days (Ojagh
et al. 2010). Total fat was extracted from fish fillet samples,
according to Bligh and Dyer (1959), on days 0, 4, 8, 12 and
16 of refrigeration storage.
Antioxidant evaluation of lycopene solutions
by DPPH method
Antioxidant evaluation of lycopene solution was performed
by DPPH method according to Raeisi et al. (2018). At first,
DPPH solution in methanol (24 mg mL-1) was prepared
and 2 mL of this solution was added to 50 lL of different
concentrations of lycopene. After 1-h incubation at room
temperature in the dark, absorbance at 517 nm was mea-
sured, using a spectrophotometer (Pharmacia LKB
Novaspec, Sweden). Scavenging capacity of lycopene was
calculated as follows:
ðRadical Scavenging ActivityÞ RSA%
¼ [(Ablank  ASample Þ=Ablank   100
Free fatty acid (FFA) measurement
FFA was measured by the method as described by Egan
et al. (1981). At first, 0.2 g of fat was dissolved in 50 mL
of the solvent (equal mixture of ethanol 96% with diethyl
ether). One to two drops of phenolphthalein were added, as
an indicator of the solution and were titrated with 0.1 N
sodium hydroxide. The constant pink color lasted about
30 s, indicated the end of the titration. The acid and free
fatty acids content were calculated by the following
equations:
56:1  N  V
Acid value ¼
W
FFA ð%Þ ¼ Acid value  1=2
N, normality of the sodium hydroxide; V, volume of
sodium hydroxide intake; W, weight of fat intake.
Peroxide value determination
Peroxide value (PV) of 0.1 g fat extracted from rainbow
trout fillet was determined by the method as described by
Egan et al. (1981). Fat was dissolved in 25 mL of the
solvent (a mixture of chloroform and acetic acid). Then,
1 mL of saturated solution of potassium iodine was added,
and kept in a dark environment for 10 min. After this time,
20 mL of distilled water and 1 mL of starch solution
(1.5%) were added.
J Food Sci Technol (January 2020) 57(1):282–292
The sample was titrated by 0.01 N Na2S2O3, until the
disappearance of the blue color. The amount of peroxide
was calculated, using the following equation.
PV ¼ ð1000 ðV1  V2ÞNÞ=W
V1–V2, consumed amount of sodium thiosulfate solution;
W, sample weight; N, normality of sodium thiosulfate
solution.
Thiobarbituric acid (TBA) measurement
TBA was measued by the method as described by Kirk and
Sawyer (1991). First, 0.2 g of fat extracted from rainbow
trout muscles was mixed with 1 mL of BHT and 35 mL of
C2HCl3O2. Then 100 mL of distilled water was added.
Fifty mililitre of distilled sample was collected to mix with
5 mL of TBA, and was incubated for 1 h in the water bath.
The absorbance of the samples was measured at 532 nm,
using a spectrophotometer.
TBA value ¼ (Asample  Ablank Þ  50=200
A, absorbance.
PH measurement
Fish fillet samples (20 g) were mixed evenly, with 100 mL
of distilled water, using a mixer. The mixture were then
filtered through a filter paper and pH was measured, using a
pH meter (crison GLP 22?) (Sallam 2007). Befor the
measurements, the instrument was frequently calibrated
using pH buffers (pH 4.01 and pH 7.00).
Water holding capacity (WHC) measurement
In order to measure WHC of fish fillet samples, 10 g of fish
and 15 mL of 6 M NaCl, were transferred into a 50 mL
falcon and were vortexed for 1 min. After the centrifuga-
tion (3000 rpm for 15 min at 4 °C), excessive moisture
released during centrifugation was absorbed with a single
sheet of WhatmanÒ 3 filter paper. The WHC of the muscle
was calculated as follows:
WHC ¼ 100  (Wfinal  Wprimary Þ=Wprimary
Wprimary, initial weight; Wfinal, weight of the sample after
centrifugation.
Analysis of fatty acids by gas chromatography
Esterification of the fat was performed, according to the
method of Hassanzadazar et al. (2017), with minor modi-
fication in the reagent volume. For this purpose, 100 lL of
methanolic 2 M KOH and 1100 ll of n-heptane was added
to 0.1 g of fat. After centrifugation for 2 min, samples were
285
kept for 10 min in a 65 °C water bath. The samples were
centrifuged for 10 min at 4000 rpm, and the upper trans-
parent layer of methyl esters of n-Heptan was removed for
further analysis. To identify fatty acids in the sample, GC
(Gas Chromatography) apparatus (model Agilent 6890 N,
United States), equipped with capillary column DB-WAX-
type (30 m * 25 mm I.d., film thickness 25 lm) detector
type FID19 was used.
Sensory analysis
The panelist (10 semi-skilled members) were firstly trained
about the product characteristics; then they scored the
samples based on the attributes including color, odor,
texture and overal acceptability according to the following
scale: 1–3: bad or unacceptable, 4–6: good, 7–8: very good,
9: excellent.
Statistical analysis
Statistical analysis of the resulting data was performed,
using SPSS software Version 18. (SPSS Inc., Chicago, IL).
After analysis of the normality, using Kolmogorov–Smir-
nov, one-way variance ANOVA was performed, in a
completely randomized factorial design. Tukey test was
used to determine the significant difference between
treatments. It should be noted that in all stages of the
analysis, a p value of \ 0.05 was considered to be statis-
tically significant.
Results and discussion
Activity of antibodies in the serum of immunized
chickens
Binding activity of the obtained IgY to the corresponding
antigen (P. fluorescens and S. putrefaciens), was evaluated
by indirect ELISA. About 2 weeks after the first immu-
nization step, significant activity of anti-S. Putrefascience
antibody and anti-P. fluorescens antibody, was detected in
the test groups compared to the control. The highest value
of immunization was demonstrated in the highest dilution
(D: 0.001).
Antioxidant properties of lycopene by DPPH
method
Fish is a perishable food and its spoilage is assumed to be
faster than other meats so numerous studies have been
conducted to maintain fish product quality, especially with
the use of antioxidants (Heydari et al. 2015; Ehsani et al.
2014). In this study, the antioxidant activity of lycopene
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was measured by the DPPH method and showed accept-
able results, compared with BHT (Data not shown here).
According to the data reported by Selim et al. (2013), using
DPPH and TBARS assays, addition of tomato puree to the
broiler diets could improve meat quality, during 60 days of
frozen storage. Girao et al. (2012) suggested that lycopene
could exert an antioxidant role, by removing reactive
oxygen species (ROS), generated by cellular metabolism.
Sahin et al. (2014) also studied the use of lycopene in
rainbow trout exposed to high stocking density and con-
cluded that lycopene significantly improved antioxidant
status by activation of Nrf2/HO pathway (a major mecha-
nism in the cellular defense against oxidative or elec-
trophilic stress) in trout.
PH measurement
Data of pH value changes in different treatments, during
16 days of refrigeration storage are presented in Table 1.
Significant differences were observed between treat-
ments contained chitosan, antibody and lycopene and the
control group, regarding pH. Treatments containing anti-
P.fluorescens antibody were more effective than anti-S.
Putrefascience antibody in preventing the pH increase. It
seems that Pseudomonas can increase pH level, due to the
breakdown of proteins and the production of alkaline
compounds, such as ammonia or trimethylamine which all
of these could be postponed by the antibody treatment
(Ghaly et al. 2010). The pH of fresh fish fillets in this study
was in the range of 7. The lowest reported pH was 6.3,
following a gradual increase in the value which was due to
the decomposition of amine compounds by internal
enzymes (exchange of TMAO to TMA) (Sallam 2007).
The pH can be variable depending on the season, species
and other factors. pH values more than 7.1 can be con-
sidered, as an indicator of spoilage. In this study, the
control group was corrupted after 12 days, while no sig-
nificant changes were found in other treatments, during the
16 days of storage. There is no comprehensive study on the
effect of lycopene on the shelf-life of fish fillets at refrig-
eration temperature, but some studies had shown that
lycopene could reduce the pH of red meat (Candogan 2002;
Østerlie and Lerfall 2005), similar to the results of this
study. Serio et al. (2018) concluded that presence of acetic
acid in chitosan solutions could keep the pH lower in
treated samples until the end of the experimental period in
comparison with the control.
WHC measurement
According to the results presented in Table 1, the WHC
showed a downward trend in all treatments.

Table 1 Changes in the pH and WHC values of treatments during 16 days of storage
Treatment/days 0 4 8 12 16

1 PH 6.36 ± 0.15a 6.13 ± 0.07a 6.32 ± 0.03a 7.26 ± 0.03a 7.53 ± 0.04a
a a d c
WHC 91.41 ± 1.07 79.23 ± 0.05 76.43 ± 2.13 72.68 ± 3.08 67.56 ± 4.21e
2 PH 6.32 ± 0.15a 6.1 ± 0.02ab 6.24 ± 0.02ab 6.52 ± 0.03b 6.7 ± 0.07b
a a cd bc
WHC 91.26 ± 1.05 80.37 ± 0.37 77.57 ± 2.04 73.08 ± 3.12 68.12 ± 4.13b
a ab abc c
3 PH 6.33 ± 0.1 6.09 ± 0.06 6.18 ± 0.04 6.38 ± 0.04 6.44 ± 0.02c
a a bcd abc
WHC 91.45 ± 1.09 82.12 ± 3.03 80.88 ± 2.09 75.71 ± 2.04 71.86 ± 3.06ab
a abc bcd c
4 PH 6.34 ± 0.02 6.03 ± 0.05 6.12 ± 0.02 6.3 ± 0.04 6.35 ± 0.03c
a a ab abc
WHC 91.66 ± 1.11 84.27 ± 3.11 83.54 ± 2.03 77.47 ± 2.05 74.28 ± 3.12ab
5 PH 6.38 ± 0.04a 5.74 ± 0.05d 5.76 ± 0.03e 5.9 ± 0.01e 5.94 ± 0.03e
WHC 90.88 ± 1.08a 83.31 ± 2.08a 82.51 ± 1.08ab 77.81 ± 1.31abc 75.04 ± 2.11ab
a e f f
6 PH 6.33 ± 0.09 5.51 ± 0.01 5.56 ± 0.1 5.78 ± 0.03 5.85 ± 0.07e
a a ab a
WHC 91.09 ± 1.14 85.87 ± 1.03 84.21 ± 1.12 80.05 ± 1.02 78.15 ± 1.08a
a bc cd d
7 PH 6.34 ± 0.1 5.98 ± 0 6.04 ± 0.7 6.08 ± 0.04 6.14 ± 0.01d
WHC 91.24 ± 1.03a 82.91 ± 2.04a 81.45 ± 1.05bc 76.18 ± 1.09abc 73.52 ± 2.07ab
a c d d
8 PH 6.35 ± 0.03 5.95 ± 0.06 6.02 ± 0.05 6.07 ± 0.08 6.10 ± 0.02d
a a ab ab
WHC 91.36 ± 1.08 85.07 ± 1.09 84.13 ± 1.04 78.11 ± 1.13 75.37 ± 1.04ab
a c d d
9 PH 6.31 ± 0.4 5.95 ± 0.03 6 ± 0.06 6.05 ± 0.02 6.12 ± 0.04d
a a bc abc
WHC 91.25 ± 1.11 82.42 ± 2.05 81.02 ± 1.07 75.78 ± 1.06 72.24 ± 2.08ab
10 PH 6.3 ± 0.01a 5.94 ± 0.0c 6 ± 0.02d 6.03 ± 0.07d 6.09 ± 0.03d
a a ab abc
WHC 91.31 ± 1.06 84.68 ± 1.08 83.76 ± 1.12 77.57 ± 1.03 74.88 ± 1.17ab

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Results showed that treatments, containing higher levels
of lycopene had a higher WHC (p \ 0.05), compared with
other treatments specially the control group, during
refrigeration storage. At the end of the preservation period,
significant differences were observed between samples,
containing higher levels of lycopene ? anti-P. fluorescens
antibody (T6), in comparison with the control and the
treatments, containing only chitosan (T1 and T2).
WHC is one of the most important quality control fac-
tors that directly impacts the quality of a product, including
its juiciness. Reduction in the WHC, is mainly due to the
changes in the functional properties of protein structure.
The production of formaldehyde and 2-dimethyl amine and
3-methyl-N-aminoxide can reduce protein solubility, and
thus reduces contraction and functional properties of pro-
teins. During spoilage, degenerated microbes (concentra-
tion of 108–109 CFU g-1) produce volatile metabolites,
such as alcohol, ketone, dihydrogen sulfide, dimethyl sul-
fide, trimethylamine, aldehydes and organic acids (Gram
and Dalgaard 2002). Thus, the presence of antibodies
against microorganisms, particularly spoilage microorgan-
ism, will prevent a sharp decline in WHC, by preventing
the production of these metabolites and protein changes.
Samples treated with lycopene, also showed lower rates
of reduction in this study. One possible reason for WHC
maintenance by lycopene might be the fat oxidation
reduction potential of this pigment (Candogan 2002). In
2002, Candogan showed that beef treatments, containing
high levels of lycopene had higher WHC, during 9 days of
storage in refrigerator, compared with the control group.
Evaluation of fatty acids oxidation
Results of PV, TBA and FFA changes, during 16 days of
storage are shown in Tables 2, 3 and 4.
The results showed that the level of peroxide, increased
significantly in all samples, during 4 days (p \ 0.05). But a
reduction was observed after 8 days which lasted until the
end of the storage period (p \ 0.05) (Table 2).
Addition of lycopene, separately or in combination with
antibodies, reduced the PV level in fish fillet samples. By
the way, fillets immersed in a solution of chitosan, anti-P.
fluorescence antibody and higher level of lycopene,
showed lower PV level. Acceptable level of peroxide index
is about 10–20 meq kg-1 (Khademi et al. 2016), and all
treatments in this study showed results within the accept-
able range, during refrigerated storage. A reduction was
observed on the 8th day which might be due to the oxi-
dation reactions and the production of carbonyle and
volatile compounds. This reaction also leads to the pro-
duction of volatile fatty acids, such as caproic acid, pro-
pionic acid and volatile gases (Ehsani et al. 2014).
Antioxidant and pro-oxidant efficiency of tomato powder,
added to the diet of quail were examined under normal and
thermal stress growing conditions, by Sahin et al. (2006).
They reported that higher tomato powder in the diet would
decrease the levels of malondialdehyde linearly in serum,
liver and muscle. Malondialdehyde levels (mg kg-1) of fat
varied between the range of 0.53–0.62 at the start of the
storage period, but increased during 8 days of storage
(Table 3).

Table 2 Changes in peroxide

Treatments/days 0 4 8 12 16
value (meq active oxygen kg-1
of fat) of treatments during 1 1.34 ± 0.11a 4.14 ± 0.07a 3.32 ± 0.03a 7.61 ± 0.09a 9.86 ± 0.04a
16 days of storage a b b b
2 1.31 ± 0.02 2.96 ± 0.05 2.02 ± 0.02 6.87 ± 0.13 8.96 ± 0.03b
a c b c
3 1.34 ± 0.07 2.71 ± 0.02 1.88 ± 0.18 5.36 ± 0.05 7.23 ± 0.08c
a c c d
4 1.31 ± 0.03 2.57 ± 0.03 1.62 ± 0.05 4.78 ± 0.02 6.52 ± 0.02d
5 1.33 ± 0.05a 1.93 ± 0.06d 0.98 ± 0.03d 3.97 ± 0.11e 6.19 ± 0.12e
a e d f
6 1.28 ± 0.02 1.7 ± 0.11 0.84 ± 0.12 3.48 ± 0.02 5.53 ± 0.07g
a d d e
7 1.23 ± 0.06 2.07 ± 0.14 1.06 ± 0.02 4.04 ± 0.03 6.43 ± 0.05d
a de d f
8 1.32 ± 0.04 1.88 ± 0.05 0.92 ± 0.09 3.66 ± 0.07 5.8 ± 0.02f
a d d e
9 1.27 ± 0.03 2.03 ± 0.09 1.03 ± 0.0 4.02 ± 0.03 6.41 ± 0.08d
10 1.24 ± 0.05a 1.88 ± 0.03de 0.87 ± 0.05d 3.61 ± 0.06f 5.77 ± 0.11f
Treatments 1: control, 2: chitosan coating, 3: chitosan coating ? lycopene level one, 4: chitosan coat-
ing ? lycopene level 2, 5: chitosan coating ? antibody anti Pseudomonas fluorescens ? lycopene level 1,
6: chitosan coating ? antibody anti Pseudomonas fluorescens ? lycopene level 2, 7: chitosan coat-
ing ? antibody anti Shewanella putrefaciens ? lycopene level 1, 8: chitosan coating ? antibody anti
Shewanella putrefaciens ? lycopene level 2, 9: chitosan coating ? total antibody ? lycopene level 1, 10:
chitosan coating ? total antibody ? lycopene level 2
Different letters indicate significant differences according to the Tukey test

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Table 3 TBA malondialdehyde

Treatments/days 0 4 8 12 16
levels (mg kg-1) of the
treatments during 16 days of 1 0.58 ± 0.03a 2.21 ± 0.11a 4.08 ± 0.13a 3.82 ± 0.05a 3.34 ± 0.08a
storage a b b b
2 0.6 ± 0.05 1.98 ± 0.07 3.83 ± 0.07 3.43 ± 0.06 3.06 ± 0.05b
a bc c c
3 0.57 ± 0.03 1.64 ± 0.04 3.5 ± 0.03 2.76 ± 0.03 2.57 ± 0.02c
a bc c d
4 0.62 ± 0.07 1.53 ± 0.01 3.31 ± 0.09 2.52 ± 0.07 2.21 ± 0.04c
a de de ef
5 0.61 ± 0.06 1.24 ± 0.03 2.83 ± 0.04 2.17 ± 0.11 2.09 ± 0.03de
6 0.58 ± 0.03a 1.01 ± 0.08e 2.62 ± 0.11ef 1.92 ± 0.04gh 1.72 ± 0.14ef
a cd def fg
7 0.57 ± 0.02 1.37 ± 0.1 2.72 ± 0.05 2.13 ± 0.09 1.96 ± 0.07def
a de f h
8 0.53 ± 0.05 1.25 ± 0.05 2.51 ± 0.09 1.88 ± 0.07 1.61 ± 0.02f
a de d cd
9 0.56 ± 0.08 1.37 ± 0.11 2.94 ± 0.03 2.57 ± 0.11 2.31 ± 0.06d
a de de de
10 0.59 ± 0.1 1.22 ± 0.09 2.77 ± 0.05 1.37 ± 0.06 2.02 ± 0.04de
Treatments 1: control, 2: chitosan coating, 3: chitosan coating ? lycopene level one, 4: chitosan coat-
ing ? lycopene level 2, 5: chitosan coating ? antibody anti Pseudomonas fluorescens ? lycopene level 1,
6: chitosan coating ? antibody anti Pseudomonas fluorescens ? lycopene level 2, 7: chitosan coat-
ing ? antibody anti Shewanella putrefaciens ? lycopene level 1, 8: chitosan coating ? antibody anti
Shewanella putrefaciens ? lycopene level 2, 9: chitosan coating ? total antibody ? lycopene level 1, 10:
chitosan coating ? total antibody ? lycopene level 2
Different letters indicate significant differences according to the Tukey test

Table 4 Changes in free fatty

Treatments/days 0 4 8 12 16
acids (%) of treatments during
16 days of storage 1 1.52 ± 0.22a 1.9 ± 0.23a 2.51 ± 0.25a 5.32 ± 0.3a 5.08 ± 0.27a
a a a a
2 1.5 ± 0.13 1.81 ± 0.24 2.32 ± 0.1 5.03 ± 0.05 4.86 ± 0.09a
3 1.56 ± 0.08a 1.68 ± 0.09a 1.83 ± 0.03b 4.37 ± 0.12b 4.23 ± 0.08b
a a b bc
4 1.53 ± 0.07 1.59 ± 0.03 1.72 ± 0.05 4.21 ± 0.09 4.08 ± 0.02bc
a a b de
5 1.53 ± 0.11 1.6 ± 0.05 1.71 ± 0.12 3.53 ± 0.05 3.38 ± 0.12ef
a a b e
6 1.52 ± 0.08 1.54 ± 0.12 1.62 ± 0.04 3.37 ± 0.14 3.07 ± 0.03f
a a b bc
7 1.48 ± 0.03 1.62 ± 0.07 1.78 ± 0.03 4.21 ± 0.09 4.06 ± 0.07bc
a a b bc
8 1.5 ± 0.08 1.53 ± 0.09 1.65 ± 0.11 4.02 ± 0.14 3.74 ± 0.03d
9 1.52 ± 0.12a 1.66 ± 0.11a 1.81 ± 0.09b 4.06 ± 0.03bc 3.89 ± 0.07cd
10 1.52 ± 0.09a 1.56 ± 0.07a 1.67 ± 0.04b 3.87 ± 0.01cd 3.65 ± 0.07de
Treatments 1: control, 2: chitosan coating, 3: chitosan coating ? lycopene level one, 4: chitosan coat-
ing ? lycopene level 2, 5: chitosan coating ? antibody anti pseudomonas ? lycopene level 1, 6: chitosan
coating ? antibody anti Pseudomonas fluorescens ? lycopene level 2, 7: chitosan coating ? antibody anti
Shewanella putrefaciens ? lycopene level 1, 8: chitosan coating ? antibody anti Shewanella putrefa-
ciens ? lycopene level 2, 9: chitosan coating ? total antibody ? lycopene level 1, 10: chitosan coat-
ing ? total antibody ? lycopene level 2
Different letters indicate significant differences according to the Tukey test

TBA is widely used as a secondary indicator of fat
oxidation. The reaction of reactive substances with TBA,
demonstrates the second phase auto-oxidation of this index
(Fernández et al. 1997). TBA values increased in all
treatments until 8th days of storage, and then a sudden
decline was observed in all treatments. After decomposi-
tion of primary oxidation products to secondary oxidation
substances, the decrease in TBA is due to the destruction of
volatile secondary oxidation products to organic alcohols
and acids and also due to the interaction between TBA
substances and protein molecules in fish fillet samples
(Maqsood and Benjakul 2010). This result is in agreement
with Amiza and kang (2013), who evaluated the effect of
chitosan on gelling properties, lipid oxidation, and micro-
bial load of surimi gel made from African catfish (Clarias
gariepinus).
The results showed that although the treatments con-
taining lycopene, significantly reduced the amount of TBA,
compared to the control group, but treatments containing
lycopene in combination with antibodies showed better
performance. The antibodies against S. Putrefaciens
revealed more effective role, in reducing the amount of
TBA. According to other studies, the TBA value should not
exceed more than 3 and 5 mg of malondialdehyde per kg of

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J Food Sci Technol (January 2020) 57(1):282–292
Fig. 1 Chromatographic analysis of total fatty acids in fish samples by GC. a Fatty acids at the first day; b fatty acids at the 8th day; c: Fatty
acids at the 16th day (%)
fat in high and good quality products (Kykkidou et al.
2009). In this study, samples contained lycopene and
antibodies were acceptable, in terms of TBA values, during
16 days of storage.
In 2002, Candogan reported that lycopene is effective in
reducing pH, TBA and visual acceptability, compared with
the control group.
FFA evaluation
In general, the FFA amounts did not show any significant
differences at the start of the storage, but increased until
12th day, following a slight decrease to the end of the
storage period (Table 4).
Increased amount of FFA during 12 days of storage,
following a minor reduction until 16 days of storage,
indicated that lycopene efficiently reduced the amount of
FFA in all treatments groups, compared with the control
group. In addition, treatments containing lycopene, in
combination with antibody, specially anti-P.fluorescens
antibody (T5, T6), were more efficient compared to the
treatments without antibody. Also, there was an inverse
289
relationship between the amount of lycopene and FFA in
the fillets. These results are consistent with the results of
the Naghibi et al. (2016) that reported fewer changes of
FFA in chitosan and lycopene-chitosan treatments in
regard to the control during 16 days of refrigeration stor-
age. In another study Ehsani et al. (2017) investigated the
effect of alginate coating without or with lycopene on the
quality of rainbow trout fillets. Treatments coated with
alginate with or without lycopene showed significantly
lower free fatty acid compared to the control.
FFA formation during short-term storage is due to the
catalytic effect of endogenous enzymes (mainly lipase and
phospholipase). Pseudomonas is responsible for FFA for-
mation in food, by lipase and phospholipase production
(Kykkidou et al. 2009).
Results of GC analysis of fatty acids
A total of 19 fatty acids, including monounsaturated fatty
acid (MUFA), polyunsaturated fatty acid (PUFA) and sat-
urated fatty acid (SFA), were identified. Major PUFAs
were C18:2n-6 and C22:6n-3 while the major SFAs were
123
290
Fig. 2 Sensory evaluation of fish fillet samples during 16 days of storage (M ± SD)
C16:0 and C18:0. At the beginning of the storage period,
Omega-3 and omega-6 unsaturated fatty acids were in the
range of 20.81–20.87% and 11.9–12.2%, respectively
(Fig. 1a). The identified fatty acids did not show any sig-
nificant differences in different treatments, at the beginning
of the storage period (p [ 0.05). SFA value increased in all
treatments, but the changes in treatments containing lyco-
pene and antibody (T5, T6, T7, T8, T9, T10) were signif-
icantly (p \ 0.05) far less than the control group (Fig. 1b,
c).
Fat content and fatty acid profile of fish fillet are vari-
able, not only in different species but also within a species.
The fatty acid composition of fish fillet can change under
the influence of metabolic factors, such as beta-oxidation
and lipogenic activities, as well as size, age, sex, species,
environmental factors, such as temperature, salinity, sea-
sonal changes and the proportion of dark and light muscles
(Kolakowska 1991). At the end of the refrigerated storage,
treatments containing combination of chitosan, lycopene
and anti-P. fluorescens antibody showed the least changes
in the amount of SFA, MUFA, n-3 PUFA and n-6 PUFA,
compared to other treatments (p \ 0.05) (T5, T6). This
indicates effective antioxdation potential of chitosan and
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J Food Sci Technol (January 2020) 57(1):282–292
lycopene, especially in combination with anti-P. fluo-
rescens antibody.
The SFA changes were inversely increased in treatments
containing different amounts of lycopene. Comparison
between the chitosan-based coated samples and the control
group, did not show any significant differences, during
refrigeration storage. The results of this study also showed
an increase in the amount of MUFA in all treatments
(Fig. 1b, c). Control and treatments with anti-P. fluores-
cence antibody ? high levels of lycopene (T6), had the
highest and lowest MUFA level, during the study period,
respectively, and increasing the lycopene level, decreased
the MUFA level. Treatments with high levels of lycopene
and anti-P. fluorescens antibody (T6), showed better
results, compared with the anti-S. Putrefascins antibody
treatments (T7, T8) (Fig. 1b, c).
Fatty acid composition analysis also showed that
omega-3 and omega-6 fatty acids reduced during storage in
all treatments. At the end of refrigerated storage, treatments
containing anti-P. fluorescens antibody and high levels of
lycopene showed lower n-3 and n-6 PUFA changes, than
other treatments (p \ 0.05). Aquatic foods are one of the
main sources of PUFAs, thereby humans obtain EPA and
J Food Sci Technol (January 2020) 57(1):282–292
DHA, mainly by consuming fish. Investigating new
preservation methods that could protect PUFAs against
oxidative changes is assumed critical. In Yazdan et al.
study (2009), breaded fillets had lower changes in the fatty
acid composition than non-breaded fillets. In contrast,
another study showed that addition of vitamin E to chi-
tosan-based coating, could not affect lipids and omega-3
fatty acid amounts (Duan et al. 2010).
Sensory evaluation
Sensory scores of all treatments reduced during storage,
especially in the control group (p \ 0.05). Treatments
containing antibody and treatments with high concentra-
tions of lycopene, showed better scores regarding odor
(p \ 0.05) (Fig. 2a). At the end of the storage period, the
color of treatments containing antibodies, particularly anti-
total antibody (T9), was significantly (p \ 0.05) accept-
able (Fig. 2b). Sensory score of treatments with different
concentrations of antibody did not show any significant
difference.
Although it has been reported that chitosan has a number
of functional properties such as antioxidant activity (Ojagh
et al. 2010) due to the formation of a stable fluorosphere
with volatile aldehydes, derived from the damage of fats
during oxidation (Li et al. 2012) but treatments containing
chitosan (T2), without antibodies, showed lesser scores in
all sensory features (odor, color, texture and overall
acceptability), in comparison with treatments, containing
antibody and lycopene (p \ 0.05) (Fig. 2c). The highest
overall acceptability score belonged to the treatment,
containing combination of total-antibody and lycopene
(T9) (Fig. 2d).
Appearance feature is one of the most common
approaches in determining the quality of marine products.
Autolytic decomposition of nucleotides and special activity
of spoilage bacteria (such as S. Putrefasciens and Photo-
bacterium phosphoreum) lead to the oxidation reactions of
fats and proteins, production of volatile compounds, such
as aldehydes, ketones, esters and sulphides, reducing sen-
sory and flavor scores (Gram and Dalgaard 2002).
Conclusion
In conclusion, this study suggests that chitosan coating
containing egg yolk antibody (anti Pseudomonas fluo-
rescens antibody and anti Shewanella putrefaciens anti-
body) in combination with lycopene, especially at higher
concentration, had the greatest effect on the rainbow trout
fillets. This biopreservative coating can be used in seafood
products improving their sensory and chemical (PV,TBA,
291
FFA, pH and WHC) attributes and prolong their shelflife
by 16 days.
Acknowledgements The authors would like to express their grati-
tude to the Urmia University and Iran National Science Foundation
(INSF),Tehran, Iran, (project code and ethical approval ID:
91003260), for financial support, and the Artemia and Aquatic
Research Institute for providing support and facilities.
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