Food Control 130 (2021) 108317

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Improvement of the microbial quality, antioxidant activity, phenolic and

flavonoid contents, and shelf life of smoked herring (Clupea harengus)
during frozen storage by using chitosan edible coating
Heba H.S. Abdel-Naeem a, Khalid Ibrahim Sallam b, *, Nermeen M.L. Malak a
a
Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt
b
Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Smoked herring is a very popular traditional fish product widely consumed in Egypt. Nonetheless, rapid mould
Smoked fish growth and lipid oxidation, with consequent short shelf life and loss of the essential characteristics, including the
Sensory attributes smoking color and flavor, are emerged as adverse problems during storage of this product. This study was aimed
Physicochemical quality
to improve the quality and lipid stability of smoked herring using chitosan coating. To achieve this goal, smoked
DPPH%
herrings were collected from local producers and divided into 5 groups, including two controls (water-dipped
and 0.5% lactic acid-dipped samples) and three treated groups (2%, 3% and 4% chitosan-coated samples) fol­
lowed by storage at − 18 ◦ C for 3 months during which microbiological, physicochemical, and sensorial analyses,
besides determination of phenolics, flavonoids, and the antioxidant activity were carried out. The results
revealed that chitosan-coated (3% and 4%) samples exhibited more than 4 log10 CFU/g reduction in aerobic plate
count, along with complete suppression of psychrotrophic, Enterobacteriaceae, yeast and mould counts, which
were all under the detectable levels (2 log10 CFU/g). Chitosan improved and maintained the phenolic and
flavonoid compounds along with the free radical scavenging activity in chitosan-coated smoked fish during
frozen storage. Moreover, chitosan coating induced significant improvement of the physiochemical quality
including; pH, total volatile base nitrogen, trimethylamine, thiobarbituric acid values, free fatty acid %, acid
number, together with the sensory attributes, compared to control uncoated ones. The study concluded that
chitosan coating can be a good choice for fish industry to overcome the problems that arise in smoked fish.

1. Introduction antioxidant activities of smoking compounds. Additionally, smoking

process is used to provide a smoke flavor and mahogany color, which is
Herring fish (Clupea harengus) is among the main fatty fish species in very attractive for consumers (Tenyang, Tiencheu, & Womeni, 2018).
domestic consumption due to it is rich in protein, vitamins (β-complex), The quality of smoked fish is affected by many factors such as the
minerals like iron, calcium and iodine (Stroud, 2001), besides the microbial load and lipid value of raw material, smoking process,
polyunsaturated fatty acids especially n-3 fatty acids, which have posi­ sawdust type, brine concentration and storage temperature (Goulas &
tive effects against cardiovascular disease and cancer (Breslow, 2006). Kontominas, 2005). Smoked fish, however, are usually more perishable
This fatty fish is used for production of many delicacy products such as than other dried, salted and marinated fish products due to improper
salted, marinated, dried, smoked and canned herring. Smoked salted smoking processing (Bremmer, 2002), where most of smoked fish
herring fish is very popular traditional fish products in Egypt, also products industry are used to add smoke flavor as an additives and
known as “Renga”, which is commercially produced by salting followed artificial coloring material as a replacement step for smoking process.
by smoking (Atef, 2013). This smoked fish is usually eaten as a seasonal Moreover, smoked herring fish are more susceptible to spoilage and lipid
diet among Egyptians, particularly in the Easter Feast (Sham EI-Nessim). oxidation and has short shelf life as it is rich in polyunsaturated fatty
Smoking is a natural traditional preservation method for herring fish as a acids and contains a high level of catalytic haeme-proteins therefore;
result of the combined effects of dehydration, antimicrobial and produce a relatively low pH, which activates haeme as a catalyst of lipid

* Corresponding author.
E-mail addresses: [email protected], [email protected] (K.I. Sallam).

https://doi.org/10.1016/j.foodcont.2021.108317
Received 29 March 2021; Received in revised form 17 May 2021; Accepted 4 June 2021
Available online 8 June 2021
0956-7135/© 2021 Elsevier Ltd. All rights reserved.
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

oxidation (Asbjorn, Hannes, Irek, & Valur, 2007). Furthermore, in the process in a smokehouse with the use of saw dust generator at 30 ◦ C for
Egyptian markets, most of the smoked fish are stored in wooden boxes at 24 h.
room temperature for long time, which give chance for bacterial and Forty kilograms of freshly prepared smoked herring fishes (Clupea
mould growth. Fungal contamination of smoked herring fish is consid­ harengus) were obtained from a local fish processing factory at the first
ered as the main cause of spoilage and it may constitute economic losses day of its processing and transferred directly to the Food Hygiene Lab­
and public health hazard due to formation of mycotoxins, which have oratory, Faculty of Veterinary Medicine, Cairo University. Food grade
carcinogenic, mutagenic, teratogenic, neurotoxic, nephrotoxic, immu­ chitosan, soluble in dilute aqueous acids, 95% deacetylation degree and
notoxic, and hepatotoxic effect (Dalie, Deschamps, & Richard-forget, of high molecular weight (batch NO. CHT- 010620-K) was purchased
2010). Unfortunately, the undesirable fungal growth on certain food from Chitosan Egypt Company. Lactic acid (98%) was purchased from
products can be eliminated by food suppliers through washing, me­ Sigma-Aldrich (St. Louis, MO). A solution of 0.5% lactic acid (v/v) in
chanical brushing or by cotton soaked with oil, nevertheless the myco­ sterile distilled water was prepared to be used for chitosan dissolving.
toxins still present inside such products (Iacumin et al., 2009). Different concentrations of chitosan solutions (2%, 3%, and 4%; w/v)
Consumers today set restrictive demands for their foods, which were prepared by adding the proper amounts of chitosan into 0.5%
should be healthy, natural, of good quality and safe. All these demands lactic acid solution, followed by stirring with a magnetic stirrer (IKA® C-
are forcing researchers and food industry to develop new food preser­ MAG HS hotplate stirrers; Merck KGaA, Darmstadt, Germany) at 50 ◦ C
vative strategies. Cooling and freezing are common preservation for 1 h until complete dissolving. Sterile glycerol (LobaChemie, Mumbai,
methods for herring fish although freezing could affect its sensory India), as a plasticizing agent, was then added at concentration of 1% (v/
quality during the storage (Ahmed, Shehata, Abd-Rabou, & v) to chitosan solutions, and the mixture was stirred for additional 10
El-Menshawy, 2019). Therefore, it is a great challenge for the fish in­ min, and the pH was adjusted to 5.6 using NaOH (1.0 M). The solutions
dustry to develop delicious, convenient and high quality smoked herring were allowed to cool down to the room temperature (⁓ 20 ◦ C) and were
fish product using natural preservatives. applied immediately for herrings coating.
Chitosan is mostly applied as a food additive or preservative and as a
food packaging material that able to retard microbial and fungal growth
2.3. Treatments application
(Devlieghere, Vermeulen, & Debevere, 2004), as well as oxidative
rancidity (Xie, Xu, & Liu, 2001) associated with smoked herring fish.
Smoked herring fish samples were divided into 5 groups (8 kg each)
Likewise, it acts as oxygen and moisture barriers, besides keeping the
to be dipped into sterile beakers containing the different control and
volatile aromas and smoked flavors inside the food (Miller & Krochta,
treatment solutions (⁓ 20 ◦ C). The first group was dipped into distilled
1997), and consequently, it can improve the sensory quality of smoked
water for 30 s as a negative control group, while the second group was
herring fish especially during frozen storage.
dipped into 0.5% lactic acid solution for 30 s as a positive control group.
Phenolics and flavonoids are well-known bioactive compounds
The third, fourth and fifth groups were coated with chitosan through
produced from smoking process that play an essential role in the pres­
dipping into 2%, 3% and 4% chitosan solutions for 30 s, respectively.
ervation and improvement of the organoleptic properties of smoked
Chitosan-coated fish were taken out from the dipping solutions and
products. Such compounds have been considered to be important con­
allowed to drain for 10 min onto a sterile stainless steel sieve, followed
tributors to smoke aroma and to the antioxidant and antimicrobial ac­
by drying in a hot air drying oven (Heraeus, D-63450 Hanau, Germany)
tivities in these foods (Sikorski, 2016). Although the application of
at 40 ◦ C for 30 min. All treated and control untreated samples were
chitosan in food has been widely reported, to the best of our knowledge,
packed in sterile polyethylene bags, kept in frozen storage at − 18 ◦ C for
no published data currently exist about the effect of chitosan treatment
3 months and examined periodically at the first day of treatments
on antioxidant activity and bioactive compounds content such as total
application and every month afterwards up to 3 months.
phenolics and flavonoids of smoked herring fish during storage. There­
fore, the main objective of the present work was to study the efficacy of
chitosan as a coating material in improving the microbial quality, 2.4. Examinations
antioxidant activity and sensory attributes as well as in the extension of
the shelf life of smoked herring fish during frozen storage at − 18 ◦ C for 3 2.4.1. Microbiological examination
months. Ten grams of smoked herring fish meat were taken under complete
aseptic condition and homogenized with 90 mL of sterile Ringer’s so­
2. Materials and methods lution (Oxoid BR0052G, Hampshire, England) in sterile homogenizing
bag using stomacher (Seward 80 Lab Blender, compact, 110VAC) for 2
2.1. Experimental design min to obtain a dilution of 10− 1, from which tenfold serial dilutions were
performed. Aerobic plate count (APC) was enumerated after spreading
The experiment was carried out in three independent replicates at 0.1 mL from the diluted tubes over the surface of double sets of Plate
different times to investigate the effect of using of different concentra­ Count Agar (PCA, Oxoid CM0325B, Hampshire, England), then incu­
tions (2%, 3% and 4%) of chitosan edible coating and lactic acid (0.5%) bated at 35 ◦ C for 48 h (Ryser & Schuman, 2015). Another set of the
on the microbial quality, physicochemical analysis, organoleptic char­ inoculated plate count agar was incubated at 7 ◦ C for 7–10 days to
acteristic, total phenolic and flavonoid contents and antioxidant activity enumerate the psychrotrophic bacteria (Vasavada & Critzer, 2015).
(DPPH%) of smoked herring fish. All treated and control untreated Enterobacteriaceae bacterial counts were enumerated after incubation
samples were stored at − 18 ◦ C for 3 months and their quality was of the inoculated Violet Red Bile Glucose agar plates (VRBG, Oxoid
assessed monthly. CM1082B, Hampshire, England) at 37 ◦ C for 24 h (Kornacki, Gurtler, &
Stawick, 2015). While, yeast and moulds were determined by incubation
2.2. Smoked herring sampling and preparation of treatment solutions of the inoculated Sabaroud Dextrose Agar plates (SDA, Oxoid CM0041,
Hampshire, England) at 25 ◦ C for 5 days (Ryu & Wolf-Hall, 2015).
Smoked herring is produced in Egypt from the imported (usually
from The Netherlands) frozen herring fishes (Clupea harengus), which 2.4.2. Physicochemical analyses
were thawed in a refrigerator for overnight at 4 ◦ C then salted (alter­
native layers of fish and salt) for 48 h, followed by washing to remove 2.4.2.1. Measurement of pH. The pH value was measured according to
the excess salt. Salted herrings were hanged in the smoking trays and left the method described by Lee and Shin (2019) using a digital pH meter
to dry at room temperature for 4 h; then subjected to cold smoking (Lovibond, Senso Direct) after homogenization of 5 g of smoked herring

2
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

fish samples with 20 mL distilled water and calibration of pH meter with their experience in sensory evaluation of fish and fish products. Addi­
7.0 and 4.0 buffers solution. tionally, they received a preparatory session related to descriptive
analysis of smoked herring fish according to ISO-11035 (1994).
2.4.2.2. Total volatile base nitrogen (TVBN). Total volatile base nitrogen Retraining was performed continuously during the evaluation period of
(TVBN) was determined using steam-distillation method (Malle & Tao, 3 months. In the sensory profiling, a series of words for attributes were
1987). Fish extracts from smoked herring fish samples were prepared developed to describe the sensory characteristics comprising appear­
through homogenization of 100 g from the sample with 200 mL aqueous ance, color, flavor, tenderness and overall-acceptability. The samples
solution of 7.5% trichloroacetic acid (TCA, v/v) in a laboratory ho­ were served in random order and the sensory attributes were assessed
mogenizer at high speed for 1 min. The prepared homogenate was using nine-point hedonic scale with 9 corresponding to extremely like
centrifuged for 5 min at 3000 rpm then the supernatant liquid was and 1 corresponding to extremely dislike. The evaluations were per­
filtered through Whatman No. 1 filter paper. Twenty-five milliliters of formed in separated booths with controlled temperature, free from odor
the filtrate and 5 mL aqueous solution of 10% NaOH (w/v) were added with adequate lightning and the assessors used water between each
into a Kjeldahl-type distillation tube. Steam-distillation was performed sample to clean their palates.
and a 40 mL of the distillate were collected into a receiving beaker
containing 10 mL aqueous solution of 4% boric acid (v/v) and 0.04 mL 2.4.4. Determination of total phenolic contents
of methyl red and bromocresol green indicator. The boric acid solution Total phenolics content of the samples was measured using the Folin-
turned from pink to green color when alkalinized by the distilled TVBN. Ciocalteau reagent according to the procedure recommended by
Finally, the collected distillate was titrated with sulfuric acid solution Singleton, Orthofer, and Lamuela-Raventos (1999) with some modifi­
(0.1 N) till complete neutralization and the color turned pink. TVBN cations. To prepare the sample extract, 2 g of sample were homogenized
values were calculated from the volume of sulfuric acid (0.1 N) used for in an aqueous solution of methanol (80%) and centrifuged in cooling
titration multiplied by 16.8 and the result was expressed in mg nitro­ centrifuge (Jouan, MR 18–12, USA) at 10,000 rpm for 15 min. The su­
gen/100 g of sample. pernatant was taken and the sediment was re-extracted again with
methanol (80%). All the supernatant was collected, transferred into
2.4.2.3. Trimethylamine acid (TMA). Measurement of TMA was carried evaporating dishes, left to dry at room temperature and the residue was
out with the same procedure of TVBN, while the only difference was the dissolved in 5 mL of distilled water. A 0.5 mL of this extract was added to
addition of 20 mL of 16% formaldehyde (v/v) into the distillation tube 0.5 mL of Folin–Ciocalteu solution, and after 3 min, 1 mL of sodium
to block both of the primary and secondary amines and allowing to react carbonate (20%) was added. The total volume of the mixture was
only with the tertiary amines. completed up to 2.5 mL with distilled water and then kept in darkness
for 60 min. The absorbance of the developed blue color was measured
2.4.2.4. Fat oxidation parameters. Thiobarbituric Acid (TBA) was with a spectrophotometer (Unico 1200, USA) against the blank at 725
determined according to the method proposed by Tarladgis, Watts, and nm using gallic acid (GA) as a standard and the result was expressed as
Yonathan (1960). Ten grams of smoked herring fish sample were mg GA/100 g.
blended with 2.5 mL of HCl solution (4 N) and 97.5 mL of distilled water
for 2 min. The blend was loaded into distillation tube and 2.4.5. Determination of total flavonoid contents
steam-distillation was completed until a volume of 50 mL of distillate Total flavonoid contents of the samples was measured using
was obtained. Five milliliters of the distillate were transferred into a test aluminum chloride colorimetric method according to Jia, Tang, and Wu
tube containing 5 mL of 0.02 M TBA reagent in acetic acid (90%) fol­ (1999) with some modifications by Dewanto, Wu, Adom, and Liu
lowed by heating for 35 min in a boiling water bath to obtain the (2002). Two hundred and 50 μL of the sample extract were added to
TBA-MDA chromogen. The test tubes were cooled for 10 min under 1.25 mL of distilled water in a test tube followed by addition of 75 μL of
running tap water and the absorbance was measured by spectropho­ sodium nitrite solution (5%). After 6 min, 150 μL of aluminum chloride
tometer (Unico 1200, USA) at 538 nm against a blank containing 5 mL of solution (10%) and 0.5 mL of NaOH (1 mol/L) were added and allowed
distilled water and 5 mL of TBA reagent. The malonaldehyde (MDA) to stand for another 5 min. The mixture was brought to 2.5 mL with
amount was calculated based on the standard calibration curve using distilled water and mixed well. The absorbance was measured imme­
different dilutions of 1,1,3,3-tetraethoxypropane. A conversion factor of diately with a spectrophotometer (Unico 1200, USA) against the blank
7.8 was adopted to convert the TBA-MDA absorbance readings to TBA at 510 nm using rutin as standard (1 mg/mL) and the result was
value, which expressed as mg MDA/kg. expressed as mg rutin/100 g.
Measurement of free fatty acids content (FFAs% as Oleic acid) and
acid number (AN mg NaOH/g) in smoked herring samples were carried 2.4.6. Measurement of antioxidant activity using DPPH assay
out according to the standard method outlined by AOCS (1997). Fifteen The antioxidant activity of the samples was studied through the
grams of sample were homogenized in 60 mL of chloroform and 60 mL measurement of the free radical scavenging ability towards the
of methanol solvents. After 24 h, 48 mL of water were added, and oil was stable1,1-diphenyl-2-picrylhydrazyl (DPPH) radical according to
collected. The oil extracted in the presence of phenolphthalein (1%) was Brand-Williams, Cuvelier, and Berset (1995). One-hundred microliters
titrated by sodium hydroxide (0.1 N). FFAs (% as Oleic acid) and AN (mg of sample extract were mixed with 3.9 mL aliquot of DPPH solution
NaOH/g) were calculated using the following equations: (0.0634 mM) in methanol (95%) for 5–10 s and kept in darkness for 30
min. The absorbance was measured with a spectrophotometer (Unico
FFAs (%) =
mL of alkali x N x 28.2 1200, USA) at 515 nm against a blank of methanol without DPPH. The
W percentage of DPPH radical scavenging activity was calculated from the
following equation
where: N = Normality of NaOH solution; W = Weight of oil (g).
(AD – AS)
Acid number (mg NaOH/g) = 1.99 × FFAs (%) DPPH(%) = × 100
AD
.
where AD is the absorbance of the methanolic solution of DPPH (without
sample) and AS is the absorbance of the sample.
2.4.3. Sensory examination
Sensory analysis of smoked herring fish samples was carried out by
trained fifteen panelists. These panelists were selected on the basis of

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H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

2.5. Statistical analysis Table 1

Microbial counts (Log10 CFU/g) of smoked herring fish coated with chitosan
All measurements were carried out in triplicates, and all data were edible coat during frozen storage at − 18 ◦ C for 3 months.
analyzed using SPSS statistics 27.0 for windows, expressed as mean ± SE Microbial category Treatments Storage period (months)
and compared using one-way analysis of variance (ANOVA). The sig­ 0-time 1st 2nd 3rd
nificance was determined using the least square difference test (LSD) month month month
procedure and the main effects were considered significant either at the
APC Control 4.93c,w 5.74c,x 6.36d,y 6.86d,z
P < 0.05 or P < 0.01 levels. ± 0.08 ± 0.14 ± 0.07 ± 0.13
Lactic acid 4.90c,w 5.50c,x 6.00c,y 6.20c,y
3. Results and discussion 0.5% ± 0.06 ± 0.10 ± 0.06 ± 0.20
Chitosan 2.89b,w 3.55b,wx 3.86b,xy 4.20b,y
2% ± 0.09 ± 0.25 ± 0.13 ± 0.12
3.1. Microbiological quality Chitosan <2a,w <2a,w ± 2.47a,x 2.52a,x
3% ± 0.00 0.00 ± 0.09 ± 0.23
Table 1 summarized the results of microbial counts (Log10 CFU/g) of Chitosan <2a,w <2a,w ± 2.30a,x 2.49a,x
smoked herring fish coated with chitosan edible coat during frozen 4% ± 0.00 0.00 ± 0.17 ± 0.13
Psychrotrophs Control 2.68b,w 3.50c,x 3.97c,x 4.93c,y
storage at − 18 ◦ C for 3 months. The aerobic plate counts (APC), psy­
± 0.14 ± 0.25 ± 0.20 ± 0.23
chrotrophs, Enterobacteriaceae, yeast and mould counts of chitosan Lactic acid <2a,w 2.27b,x 3.40b,y 4.20b,z
(2%, 3% and 4%)-coated samples were significantly (P < 0.01) lower 0.5% ± 0.00 ± 0.15 ± 0.21 ± 0.20
than those of the control uncoated and 0.5% lactic acid-treated samples Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
2% ± 0.00 0.00 0.00 ± 0.00
at the day 0 of storage and thereafter until the end of the storage period
Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
(3 months). Conversely, 0.5% lactic acid-treated samples exhibited non- 3% ± 0.00 0.00 0.00 ± 0.00
significant (P > 0.05) reduction of APC in comparison with control Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
uncoated samples during the first month of frozen storage and only 4% ± 0.00 0.00 0.00 ± 0.00
expressed less than 1 log10 CFU/g reduction at the 2nd and 3rd month of Enterobacteriaceae Control 3.90b,w 5.04b,x 5.28c,x 6.23c,y
± 0.15 ± 0.27 ± 0.31 ± 0.14
the storage. Nevertheless, chitosan coating (2%) resulted in more than 2
Lactic acid <2a,w <2a,w ± 2.47b,x 3.27b,y
log10 CFU/g reduction in the counts of APC when compared with the 0.5% ± 0.00 0.00 ± 0.24 ± 0.15
control one. Interestingly, APC count was under the detectable level (<2 Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
Log10 CFU/g) in samples coated with 3% and 4% chitosan during the 2% ± 0.00 0.00 0.00 ± 0.00
Chitosan <2a,w <2a,w ± <2a,w ± <2a,w ±
first month of storage and started to increase gradually with the storage
3% ± 0.00 0.00 0.00 0.00
but still lower (>4 log10 CFU/g reduction) than the control uncoated Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
samples by the end of storage time. Surprisingly, psychrotrophs, 4% ± 0.00 0.00 0.00 ± 0.00
Enterobacteriaceae counts as well as yeast and mould counts were under Yeast Control 2.80c,w 5.05c,x 5.30c, 6.10c,y
x
the detectable level (<2 Log10 CFU/g) in samples coated with different ± 0.12 ± 0.13 ±0.15 ± 0.06
Lactic acid 2.26b,w 3.87b,x 4.58b, 5.07b,z
concentrations of chitosan (2%, 3% and 4%) during the entire storage y
0.5% ± 0.14 ± 0.09 ±0.19 ± 0.07
period. Chitosan <2a,w <2a,w ± <2a,w ± <2a,w ±
Unfortunately, the APC of control uncoated and 0.5% lactic acid- 2% ± 0.00 0.00 0.00 0.00
treated samples exceeded the recommended limit (5 Log10 CFU/g) Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
3% ± 0.00 0.00 0.00 ± 0.00
regulated by ES-288 (2005) for smoked herring fish by the end of the
Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
first month of examination. Additionally, yeast and mould counts of 4% ± 0.00 0.00 0.00 ± 0.00
these samples exceeded the recommend limit (should be free) set by Mould Control 3.80c,w 4.20c,x 4.97c,y 5.87c,z
ES-288 (2005) starting from the zero time of examination. Conversely, ± 0.12 ± 0.10 ± 0.15 ± 0.09
chitosan-coated samples with different concentrations (2%, 3% and Lactic acid 2.17b,w 3.30b,x 4.07b,y 4.70b,z
0.5% ± 0.09 ± 0.15 ± 0.07 ± 0.12
4%), till the end of 3rd month of the frozen storage, revealed APC within
Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
the permissible limit and they were yeast- and mould-free. These results 2% ± 0.00 0.00 0.00 ± 0.00
clearly demonstrated that chitosan coat especially 3% and 4% exerted a Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
strong inhibitory effect on the microbial growth of smoked herrings and 3% ± 0.00 0.00 0.00 ± 0.00
extended their shelf life. Similar to this finding, Bonilla, Atares, and Chitosan <2a,w <2a,w ± <2a,w ± <2a,w
4% ± 0.00 0.00 0.00 ± 0.00
Vargas (2013) noticed that the microbial counts decreased with
a–d
increasing the concentration of chitosan solution and they attributed Means with different superscripts within the same column for each parameter
such effect to the thicker film, which constitute higher barrier and me­ are significantly (P < 0.05 or P < 0.01) different.
w-z
chanical properties and consequently, offers more effective antimicro­ Means with different superscripts within the same row are significantly(P <
0.05 or P < 0.01) different.
bial ability.
Values represent the mean of 3 independent replicates± SE.
The results obtained concerning the significant (P < 0.01) reduction
of APC and psychrotrophs of chitosan-coated samples were in agreement
with those reported by Jeon, Kamil, and Shahidi (2002); Santos et al. sausages did not significantly reduce the yeast and moulds counts during
(2017) and Alboghbeish and Khodanazary (2018), although, lower storage at 4 ◦ C, and this may be due to using of low concentration of
bacterial reductions (2–3 log10 CFU/g; less than 1 log10 CFU/g and 1–2 chitosan.
log10 CFU/g) were recorded by these authors, respectively in compari­ Several hypotheses for the antimicrobial activity of chitosan have
son to our result (>4 logs reduction), and this may be due to using of low been suggested. The most commonly established antibacterial mecha­
chitosan concentration in their studies. Moreover, the undetected nism is based on the interaction between the positive charge of the
Enterobacteriaceae, yeast and mould count in chitosan-coated sample in chitosan molecule and the negative charge of the microbial cell mem­
this study was in agreement with the result noticed by Kücükkaya, brane with chelation of certain ions from the lipopolysaccharide layer of
Arslan, Soncu, Ertürk, and Soyer (2020) in chitosan-coated fermented bacterial cell membrane, resulting in changes to the membrane perme­
sausage. Additionally, Yilmaz Atay (2019) indicated that chitosan has ability and leakage of intracellular constituents (Chen, Liau, & Tsai,
higher inhibitory effects on fungi than bacteria. On the contrary, Roller 1998). In this context, Arshad and Batool (2017) reported that chitosan
et al. (2002) found that incorporation of chitosan (0.6%) into the coatings acted as an oxygen barrier and can inhibit the growth of aerobic

4
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

bacteria, which explained the significant reduction of APC of Table 2

chitosan-coated samples in this study. The antifungal mechanism of Protein deterioration and fat oxidation parameters of smoked herring fish coated
chitosan was elucidated by its polycationic nature and its ability to with chitosan edible coat during frozen storage at − 18 ◦ C for 3 months.
suppress the sporulation and spore germination moreover; chitosan Physicochemical Treatments Storage period (months)
oligomers diffuse inside the hyphae, and interfere with the enzymes parameters
0-time 1st 2nd 3rd
activities, which are responsible for the fungus growth (Hernánde­ month month month
z-Lauzardo et al., 2008). The degree of the antimicrobial efficacy of
pH Control 6.38d,w 6.47c,x 6.53c,y 6.63c,z
chitosan depends on numerous factors such as molecular weight, ± 0.01 ± 0.01 ± 0.01 ± 0.01
deacetylation degree, concentration, water-solubility, pH, and temper­ Lactic acid 5.76a,w 5.86a,x 6.10a,y 6.21a,z
ature (Holappa et al., 2006). The degree of deacetylation of chitosan 0.5% ± 0.01 ± 0.03 ± 0.05 ± 0.01
(about 95%) used in this study and the high molecular weight explained Chitosan 6.17c,w 6.27b,x 6.33b,y 6.35b,y
2% ± 0.02 ± 0.01 ± 0.01 ± 0.01
its high antimicrobial activity. Chitosan 6.05bc, 6.22b,x 6.27a,y 6.29a,y
w
3% ± ± 0.01 ± 0.01 ± 0.01
3.2. Physicochemical parameters 0.02
Chitosan 6.02b,w 6.20b,x 6.25a,y 6.28a,y
4% ± 0.01 ± 0.01 ± 0.01 ± 0.00
3.2.1. pH, total volatile base nitrogen (TVBN) and trimethylamine (TMA)
TVBN Control 25.70d, 33.04d,x 35.56d,y 38.25d,
Total volatile base nitrogen is a group of biogenic amines include w
± ± 0.11 ± 0.10 z
± 0.20
ammonia (NH3), dimethylamine (DMA) and trimethylamine (TMA). 0.19
TMA is produced by the decomposition of trimethylamine oxide Lactic acid 22.12c, 24.92c,x 30.60c,y 35.24c,z
w
0.5% ± 0.03 ± 0.05 ± 0.07
(TMAO) through bacterial spoilage and enzymatic activity, which con­ ±
0.05
tributes to the characteristic ammonia-like off-odor and “fishy” off- Chitosan 18.20b, 19.09b, 19.66b, 20.21b,x
flavours. Both TVBN and TMA are commonly used as an index for 2% w
± wx
± wx
± ± 0.22
assessment of the degree of spoilage in seafood (Dalgaard, 2000). 0.34 0.39 0.25
Our results revealed that lactic acid (0.5%) and chitosan (2%, 3%, Chitosan 13.83a, 14.61a,x 15.29a,y 15.63a,
w y
3% ± 0.17 ± 0.16 ± 0.11
4%) coatings induced a significant (P < 0.05) reduction of pH value of
±
0.24
smoked herring fish in comparison with control uncoated one Chitosan 11.96a, 12.30a,w 14.08a,x 14.43a,
throughout the storage period (Table 2). Likewise, there were significant 4% w
± ± 0.15 ± 0.22 x
± 0.26
(P < 0.01) reductions in TVBN and TMA value of all treated samples 0.15
TMA Control 18.48c,w 21.84d,x 24.08d,y 27.44d,
during the entire storage period as compared to control uncoated sam­ z
± 0.09 ± 0.07 ± 0.04 ± 0.10
ples especially in those coated with chitosan 3% and 4% (Table 2). In Lactic acid 9.91b,w 12.99c,x 13.67c,y 14.49c,z
addition, pH, TVBN and TMA values were increased significantly (P < 0.5% ± 0.35 ± 0.24 ± 0.17 ± 0.46
0.05) with the storage in control uncoated and 0.5% lactic acid-treated Chitosan 8.00b,w 9.02b,x 9.13b,x 9.30b,x
samples and non-significantly (P > 0.05) in chitosan (2%, 3% and 4%)- 2% ± 0.39 ± 0.14 ± 0.11 ± 0.00
Chitosan 4.37a,w 5.45a,x 5.49a,x 5.83a,x
coated samples. Interestingly, TVBN and TMA values exceeded the limit
3% ± 0.09 ± 0.05 ± 0.15 ± 0.23
(30–35 mg% and 10 mg%, respectively) recommended by ES-288 Chitosan 3.63a,w 4.47a,x 4.95a,x 5.36a,x
(2005) in smoked herring fish in control and 0.5% lactic acid-treated 4% ± 0.20 ± 0.11 ± 0.11 ± 0.25
sample, while in chitosan-coated samples (2%, 3% and 4%) these TBA Control 4.00c,w 4.33c,w 5.77b,x 6.27b,x
± 0.13 ± 0.02 ± 0.02 ± 0.22
values were within the permissible limits throughout the storage period
Lactic acid 3.89c,w 4.07c,w 5.42b,x 5.60b,x
of 3 months (Table 2). 0.5% ± 0.08 ± 0.01 ± 0.06 ± 0.06
The lower pH values of chitosan-coated samples when compared Chitosan 3.22b,w 3.32b,w 3.36a,w 3.38a,w
with control uncoated ones in this study were in harmony with that 2% ± 0.01 ± 0.06 ± 0.30 ± 0.26
reported in chitosan-coated fish fillets during refrigerated storage Chitosan 2.44a,w 2.53a,w 2.90a,x 3.34a,y
3% ± 0.09 ± 0.19 ± 0.06 ± 0.03
(Alboghbeish & Khodanazary, 2018). This may be due to the inhibitory
Chitosan 2.42a,w 2.52a,w 2.87a,x 3.32a,y
effect of chitosan coat against internal enzymes activities and microbial 4% ± 0.04 ± 0.07 ± 0.04 ± 0.09
growth (Chamanara, Shabanpour, Khomeiri, & Gorgin, 2013) or the FFAs Control 0.56c,w 0.87c,w 1.43b,x 2.32b,y
release of lactic acid from chitosan matrix. Conversely, the increase in ± 0.02 ± 0.04 ± 0.16 ± 0.01
pH value of control untreated sample may be due to the growth of Lactic acid 0.51c,w 0.80c,wx 1.25b,x 2.18b,y
0.5% ± 0.01 ± 0.03 ± 0.09 ± 0.02
spoilage bacteria that utilize the amino acids present in fish muscle with Chitosan 0.22b,w 0.26b,w 0.36a,x 0.43a,y
the accumulation of alkaline component such as ammonia (Volpe et al., 2% ± 0.01 ± 0.02 ± 0.01 ± 0.01
2015), which elevate the pH towards the alkalinity, with a subsequent Chitosan 0.15a,w 0.17a,w 0.24a,x 0.31a,y
negative affect on fish quality during the storage especially in terms of 3% ± 0.00 ± 0.01 ± 0.01 ± 0.01
Chitosan 0.14a,w 0.16a,w 0.24a,x 0.31a,y
sensorial characteristics such as color, flavor and texture.
4% ± 0.00 ± 0.00 ± 0.01 ± 0.02
The significant (P < 0.01) decrease in TVBN values of chitosan- AN Control 1.11c,w 1.73c,w 2.85b,x 4.62b,y
coated smoked herring fish was in agreement with the findings recor­ ± 0.04 ± 0.08 ± 0.32 ± 0.33
ded in different fish species by Jeon et al. (2002); Souza et al. (2010), Lactic acid 1.00c,w 1.59c,wx 2.49b,x 4.34b,y
Chamanara et al. (2013), and Santos et al. (2017). Similarly, Albogh­ 0.5% ± 0.02 ± 0.05 ± 0.17 ± 0.40
Chitosan 0.44b,w 0.52b,w 0.72a,x 0.86a,y
beish and Khodanazary (2018) indicated that 2% chitosan coated fish 2% ± 0.02 ± 0.03 ± 0.03 ± 0.03
fillets (Carangoides coeruleopinnatus) exhibited significant lower TMA Chitosan 0.30a,w 0.34a,w 0.48a,x 0.62a,y
values in comparison with the control fillets (2.83 vs 6.53 mg%) by the 3% ± 0.02 ± 0.02 ± 0.02 ± 0.02
end of the storage at 4 ◦ C for 12 day. On the other hand, Li, Mei, Shen, Chitosan 0.28a,w 0.32a,w 0.48a,x 0.62a,y
4% ± 0.02 ± 0.02 ± 0.01 ± 0.04
and Xie (2018) recorded non-significant change in TMA values (3.7 vs 3
mg%) between control uncoated and 1% chitosan-coated fish fillet TVBN: total volatile base nitrogen (mg %); TMA: trimethylamine (mg %); TBA:
(Cynoglossus Semilaevis), respectively by the end of 18-day storage at thiobarbituric acid (mg malonaldehyde/kg); FFA: free fatty acid % as Oleic acid;
4 ◦ C. AN: Acid number (mg NaOH/g).
a–d
The reduction of both TVBN and TMA values of chitosan-coated Means with different superscripts within the same column for each parameter
are significantly (P < 0.05 or P < 0.01) different.
samples may be clarified by its ability to suppress the bacterial and

5
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

w-z
Means with different superscripts within the same row are significantly (P < 3.3. Sensory examination
0.05 or P < 0.01) different.
Values represent the mean of 3 independent replicates± SE. Coating of smoked herring fish with chitosan (2%, 3% and 4%)
induced significant (P < 0.05) improvement of appearance, color, flavor,
endogenous enzymes activities with subsequent retardation of the tenderness and overall-acceptability scores during frozen storage in
spoilage (Rasulu, Praseptiangga, Joni, & Ramelan, 2019). However, the comparison with control uncoated samples and 0.5% lactic acid-treated
increase of TVBN and TMA values in control uncoated samples may be samples (Figs. 1–3). In addition, treatment of smoked herring fish with
attributed to their higher microbial load (Table 1), which could break­ lactic acid (0.5%) resulted in non-significant (P > 0.05) difference in all
down the peptides, amino acids and trimethylamine oxide (TMAO) sensory attributes except the flavor and tenderness at 2nd and 3rd
compounds and resulted in an increase in the basic nitrogen fraction. month of frozen storage compared to control uncoated samples
(Figs. 1–3). Similar to our findings, Jeon et al. (2002), Fan et al. (2009),
3.2.2. Fat oxidation parameters and Alboghbeish and Khodanazary (2018) reported significant
The TBA content is widely used as an indicator of the quality of improvement for all sensory attributes of chitosan-coated fish samples
seafood products as it represents the degree of secondary lipid oxidation compared to control uncoated ones. Conversely, Wu et al. (2016) re­
of the fish flesh (Fan et al., 2009). Moreover, FFAs are produced by ported that chitosan-coated samples achieved lower sensory scores than
degradation of phospholipids and triglycerides and can be used to the control samples at the first day of storage due to the acidity of the
evaluate the degree of lipolysis in fish that caused by endogenous en­ coating solution (1% acetic acid) although they noticed significant
zymes and microbial activities. FFAs undergo further oxidation to pro­ improvement of the sensory scores at the end of the storage period and
duce low molecular weight compounds that are responsible for they attributed such effect to the off-odor, which occurs faster in the
off-flavor and undesirable taste of fish and fish products (Oucif et al., control samples than those of coated samples.
2017). Chitosan coating act as water vapor barrier, and consequently, pre­
Coating of smoked herring fish with the different concentrations of vent dehydration and improve the tenderness of smoked herring fish.
chitosan significantly (P < 0.01) reduced TBA, FFAs and AN values This explanation was also confirmed by Chamanara et al. (2013), who
throughout the storage period in comparison with control uncoated and noticed that chitosan-coated samples significantly decreased hardness
0.5% lactic acid-treated samples (Table 2). Nonetheless, no significant
changes were noticed in TBA, FFAs and AN values between 0.5% lactic
acid-treated samples and control untreated samples during the storage
period. The TBA value of smoked herring fish should be not more than
4.5 mg MAD/kg (ES-288, 2005). Accordingly, TBA values of control
uncoated samples and 0.5% lactic acid-treated samples exceeded this
limit starting from the 2nd month of frozen storage while, chitosan coat
keep this value within the recommended level until the end of the 3rd
month of frozen storage (Table 2).
Similar to our findings, Jeon et al. (2002); Souza et al. (2010) and
Santos et al. (2017) recorded a significant reduction in TBA value of
chitosan-coated fish samples compared to control uncoated samples.
Moreover, Alboghbeish and Khodanazary (2018) and Haghighi and
Yazdanpanah (2020) reported a significant reduction in FFAs value of
chitosan-coated fish samples in comparison with the control ones.
Controlling of lipid oxidation of fish meat is closely related to the
oxygen-barrier property of the chitosan coating and its antioxidant ac­
tivity through chelation ability with ferrous ions in fish proteins and
binding capacity with lipid (Santos et al., 2017). This antioxidant ac­
tivity of chitosan was magnified with the higher chitosan concentrations
(3% and 4%) and this observation was also reported in a previous study
on chitosan-coated tilapia fillets (Lee, Jahurul, Pua, Shapawi, & Chan,
2019). Such effect was elucidated by the presence of a large number of
ionic functional groups in chitosan, which create strong polymer in­
teractions that restrict the chain motion and results in good oxygen
barrier properties (Jeon et al., 2002).
The increase of TBA value in control uncoated smoked herring fish
was in agreement with that of Jeon et al. (2002), who observed that
herring fillet was generally more prone to lipid oxidation due to its high
proportion of dark muscles, total lipids, pro-oxidant heme pigments and
metal ions. Additionally, the relevant higher TBA, FFAs and AN values,
in the present study in control uncoated and lactic acid-treated samples
during the storage compared to chitosan-coated samples (2%, 3% and
4%) would correspond to the action of the higher counts of psychro­
trophic bacteria (Table 1), which has been reported to possess phos­
pholipase activity (Koka & Weimer, 2001). In another study,
Fig. 1. Appearance and color scores of smoked herring fish coated with chi­
Kilincceker, Dogan, and Kucukoner (2009) referred the increase of lipid
tosan edible coat during frozen storage at − 18 ◦ C for 3 months. C: Control; LA
oxidation of control uncoated samples during storage to the partial
0.5%: Lactic acid 0.5%; CH 2%: Chitosan 2%; CH 3%: Chitosan 3%; CH 4%:
dehydration of fish and interacting lipids with air oxygen.
Chitosan 4%. Columns with different letters within the same month of storage
are significantly different at P < 0.05 or P < 0.01. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web
version of this article.)

6
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

and chewiness due to its high water absorbance ability. Likewise,

Nawaz, Fatima, Shah, and Afzal (2020) reported that chitosan could
maintain the texture and firmness of coated fish throughout the storage
period of 24 days at 4 ◦ C. In this regard, Li et al. (2012) indicated that the
retardation of the microbial and autolytic processes induced by natural
preservatives could reduce the myofibrillar protein degradation, which
in turn maintains the texture and firmness of food. Furthermore, chi­
tosan could prevent the loss of marination ingredients and smoke flavor
and subsequently maintain the flavor especially during the storage. In
another study, Fan et al. (2009) explained the improvement of sensory
attributes of chitosan-coated samples by chitosan’s functional properties
such as antimicrobial, antioxidant and oxygen barrier. Lactic acid
(0.5%) improved only the tenderness of smoked fish due to lowering pH
value (Table 2), which is responsible for meat tenderization, while it
adversely affect the appearance and color. Chitosan coat especially in
concentrations of 3% and 4% could maintain the storage stability of
smoked herring fish based on the results of microbiological, physico­
chemical and sensory examination.

3.4. Total phenolics, total flavonoids and antioxidant activity

Smoking process resulted in emission of many volatile complexes,

including phenolic and flavonoid compounds, which are very important
for preserving and improving the sensory quality of smoked products,
nonetheless the concentration of these compounds in the smoked
products depends on smoking methods, time, temperature and types of
the wood used in the smoking process (Guillen & Manzanos, 1996). In
this respect, Sérot, Baron, Knockaert, and Vallet (2004) found that liquid
smoke vaporization at 24 ◦ C and 32 ◦ C allowed the deposition of a high
quantity of phenolic compounds than at 16 ◦ C, and they recorded that
the phenolic compounds content in smoked herring fillets smoked by the
electrostatic smoking process and friction was lower than that smoked
by traditional smoking process.
Fig. 2. Flavor and tenderness scores of smoked herring fish coated with chi­ In the present study, chitosan coating (2%, 3% and 4%) significantly
tosan edible coat during frozen storage at − 18 ◦ C for 3 months. C: Control; LA (P < 0.01) increased the total phenolic and total flavonoid contents
0.5%: Lactic acid 0.5%; CH 2%: Chitosan 2%; CH 3%: Chitosan 3%; CH 4%: along with DPPH% activity of smoked herring fish and maintained their
Chitosan 4%. Columns with different letters within the same month of storage high levels during the storage when compared with control uncoated
are significantly different at P < 0.05 or P < 0.01. and 0.5% lactic acid-treated samples (Fig. 4). On the contrary, the total
phenolic and total flavonoid contents in control uncoated and 0.5%
lactic acid-treated samples showed no significant changes within the
first 2 months of storage, although they expressed a drastic (P < 0.05)
decrease by the third month of storage, while the DPPH% showed a
gradual decrease as the storage period increased (Fig. 4).
The increased amounts of total phenolic and flavonoid contents in
chitosan-coated samples may be elucidated by the ability of chitosan to
stimulate the activities of phenylalanine ammonia-lyase and tyrosine
ammonia-lyase, which participate in the biosynthesis of these com­
pounds (Khan, Prithiviraj, & Smith, 2003). Moreover, Katiyar, Heman­
taranjan, and Singh (2015) explained the improving of free radical
scavenging activity of chitosan coat by inducing antioxidant enzyme
activities such as superoxide dismutase, catalase and peroxidase. Addi­
tionally, chitosan could maintain the higher values of phenolics, flavo­
noids and DPPH% in chitosan-coated herrings due to its barrier property
that preserving these compounds within the stored fish.
Phenolic compounds possess both antioxidant and antimicrobial
activities. The antioxidant activities of phenolics are equal or higher
than that of the synthetic antioxidants such as butylhydroxytoluene and
butylhydroxyanisole, and their antioxidant actions are mostly correlated
to pyrogallol, resorcinol, guaiacol and syringol compounds (Sikorski,
Fig. 3. Overall-acceptability scores of smoked herring fish coated with chitosan 2016). These phenolics could inhibit lipid oxidation even at low con­
edible coat during frozen storage at − 18 ◦ C for 3 months. C: Control; LA 0.5%: centrations through capturing the free radicals that initiate the chain
Lactic acid 0.5%; CH 2%: Chitosan 2%; CH 3%: Chitosan 3%; CH 4%: Chitosan reaction of lipid autoxidation and the secondary radicals with subse­
4%. Columns with different letters within the same month of storage are quent termination of the reaction (Sikorski, 2016). Additionally, the
significantly different at P < 0.05 or P < 0.01. aromatic ring of the phenolic compounds possesses a hydroxyl group
that enables them to scavenge the peroxyl radicals. The antioxidant
activity of phenolic compounds can be also related to their capability to

7
H.H.S. Abdel-Naeem et al. Food Control 130 (2021) 108317

coated food of animal origin during storage are very limited. Ruíz-­
Cruz et al. (2019) obtained a significant improvement of total phenolic
contents and DPPH% of chitosan-coated chicken fillets as compared
with control samples during storage at 4 ◦ C for 16 days. On the other
hand, the total phenolic contents in 1% chitosan coat itself was 1.2 mg
GAE/g (Casettari, Gennari, Angelino, Ninfali, & Castagnino, 2012),
while the DPPH% activity of 1% chitosan coat was 49% (Alparslan &
Baygar, 2017). However, Montaño-Sánchez et al. (2020) found that total
phenolic contents and free radical scavenging activity (FRSA %) of 1%
chitosan coat was 0.03 mg GAE/g and 0.16%, and these values could be
improved to reach 3.3 mg GAE/g and 92.5%, respectively by incorpo­
ration of 0.5% green tea extract into the coat.
The antimicrobial and antioxidant activities of phenolic compounds
that magnified and maintained by chitosan coating during herrings
storage could preserve the taste and color of smoked herrings from the
deteriorative changes, besides providing the smoke aroma, which are all
resulted in significant (P < 0.05) improvement of microbiological
quality (Table 1), physicochemical analysis (Table 2) and sensory scores
(Figs. 1–3) of chitosan-coated samples during the storage when
compared with control uncoated samples and 0.5% lactic acid-treated
samples.

4. Conclusions

In this study, commercial chitosan of low price was used to overcome

the major problems occurred in smoked herring, including the rapid
mould growth, lipid oxidation, short shelf life and loss of the essential
characteristics smoking color and flavor. Although the application of
chitosan in food has been widely reported, to the best of our knowledge,
this study is the first concerning the effect of chitosan coating on the
antioxidant activity and the bioactive compound contents such as total
phenolics and flavonoids of smoked herring fish during frozen storage.
The study explored that chitosan coating exerted dual effects, including
enzymes activation for production of phenolics and flavonoids, as well
as the maintenance of these compounds throughout the storage period,
resulting in improvement of the quality and lipid stability of smoked
herring fish during frozen storage at − 18 ◦ C for 3 months. Therefore,
chitosan is quite applicable in fish processing industries and is consid­
ered as a good choice to overcome the problems that arise in smoked
fish.

CRediT authorship contribution statement

Heba H.S. Abdel-Naeem: Conceptualization, Data curation, Inves­

tigation, Methodology, Resources, Writing – original draft. Khalid
Ibrahim Sallam: Conceptualization, Data curation, Investigation,
Formal analysis, Project administration, Writing – review & editing.
Nermeen M.L. Malak: Investigation, Methodology, Resources.

Fig. 4. Total phenolic contents, total flavonoid contents and DPPH% activity of Declaration of competing interest
smoked herring fish coated with chitosan edible coat during frozen storage at
− 18 ◦ C for 3 months. C: Control; LA 0.5%: Lactic acid 0.5%; CH 2%: Chitosan
None.
2%; CH 3%: Chitosan 3%; CH 4%: Chitosan 4%. Columns with different letters
within the same month of storage are significantly different at P < 0.05 or P
< 0.01. References

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