Archives of

1. Introduction
ncorhynchus mykiss is the scientific name
of rainbow trout. It is a favorable and common
edible fish in the world. Therefore,
recent studies have attempted to promote
its processing and production quality. Fish
fillets are very sensitive to bacterial spoilage and lipid
oxidation that can be related to the high moisture, pH,
protein, and presence of numerous unsaturated fatty acids
in it [1]. As fish products are very perishable, developing
preservation approaches for increasing their shelf
life is quite necessary [2]. Cold and freezing storage are
major and common approaches for preserving aquaculture
foods, although they cannot entirely prevent their
chemical reactions.
Additionally, freezing, chilling, and super chilling for
the drop marketing of these products can decrease their
nutritional value by destructing their nutritious ingredients
like vitamins, minerals, and proteins [1]. Other
methods such as drying, canning, salting, smoking, and
irradiation cannot fully protect seafood from oxidative
spoilage. This failure may be related to the existence of
different unsaturated fatty acids in the fish body, which
become rapidly oxidized in the presence of oxygen [3,
4]. The other option is using chemical preservatives to
enhance the shelf life of meat products. Consumers are
now complaining about synthetic preservatives, which
in their beliefs, might be detrimental to their health.
On the one hand, food industries are trying to find new
methods to extend food shelf life, such as various chemical
preservative applications. However, the consumers
find these compounds unpleasant as they have adverse effects.
Considering these problems, food researchers are
searching to find novel methods to solve this problem.
Natural antioxidant compounds such as different herbal
extracts are the new approach of the food industries for
enhancing meat foods’ shelf life [5]. In other words, one
of the new approaches for shelf life enhancement of
perishable products such as fish is natural preservative
applications instead of chemical ones. Natural food additives
are used as a natural antioxidant and antimicrobial
in foods, and they can elevate their nutritional value as
they have high health benefit properties [6].
The Apis mellifera L - bees produce a resinous propolis
or bee glue that protects the hive against various pests
such as arthropods and microorganisms. The polyphenolic
compounds of propolis are responsible for the
well-known biological functions of bee glue, such as
its pharmacological, anti-inflammatory, antitumor, antiviral,
antifungal, antibacterial, and antioxidant properties
[7, 8]. Propolis is mainly made of resin (60%) and
its remaining (40%) comprised vitamins, essential oils,
waxes, and microelements. Bees collect the resinous and
balsamic ingredients of pollen, flours, leaves, and foliage,
and after the aggregation of them in salivary secretions
and enzymes, propolis is made. Because of the unique
properties of propolis, it has been the subject of many
studies. According to the previous studies, this product
is a potent antioxidant linked to the flavonoids, cinnamic,
benzoic, and caffeic acids individually or in the
synergisms of these compounds [9, 10]. Recently, natural
preservatives' effects have been promoted by implanting
them into various edible films and coatings in foods. On
the one hand, incorporating natural preservatives (antioxidants
and antimicrobials) into films or coatings can
enhance the films’ antimicrobial and antioxidant features.
On the other hand, it delays the chemical spoilages
in different ways, like oxidation of lipid and protein by
oxygen or inhibition of water vapor leakage in the coated
food [5, 11]. The propolis extract is a stable, potent antioxidant,
antimicrobial, and odorless natural compound,
which is easily found in each honey bee hive. Besides,
it has beneficial effects on human health [12]. Reports
have revealed the antimicrobial properties of propolis
against various microorganisms, including Staphylococcus
aureus, Enterococcus faecalis, and Candida utilis.
These effects are mostly resulted from the polyphenolic
compounds [7, 10]. Kalogeropoulos et al. assessed the
antibacterial activities of Propolis extracts (Pes) from
Greece and Cyprus against Listeria monocytogenes,
Staphylococcus aureus, and Bacillus cereus [7].
Various polysaccharides, proteins, and lipid polymers
can compose different edible films and coatings with
specific characteristics. Because of their special filmforming
features, polysaccharides have been commonly
used to build coatings and films in food backgrounds.
Carboxymethyl Cellulose (CMC) is a material with cellulose
origin with many functions in the food industry,
such as stabilizer, thickener, and mouth-taste enhancer. It
is made of glucose units with β (1-4) links, methyl, and
carboxyl groups [13, 14]. CMC, a hydrophilic polymer,
can be a good choice for food coatings and films. There
are also examples of biopolymer-based coating and film
applications in improving the foods' shelf lives through
creating water and oxygen barrier, inhibiting microbial
growth, and reducing weight loss. They also protect the
foods' organoleptic features by preventing and retarding
protein and lipid oxidation [2, 15]. Combining hydrophobic
substances such as fatty acids, vegetable oils,
O
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resins, surfactants, and waxes in the hydrocolloid-based
films is one approach to boost the moisture barrier effects
[13]. Many research studies showed the antimicrobial
activity of the CMC film incorporated with natural
preservatives against various microorganisms [13, 14].
Shavisi et al. observed that during all storage times, fortified
Polylactic Acid (PLA) film with PE could hinder
oxidation reaction in the chilled minced meat [14]. The
edible coating could lower the speed of the oxidation reactions
and moisture retention and act as a barrier against
oxygen and water permeability. In other words, various
coatings such as chitosan, alginate, and CMC can increase
the quality and storage life of the foods by this
mechanism [4, 13, 14].
Therefore, this research planned to introduce propolis
as a potent natural food preservative in fortifying CMC
edible coating for elongating the shelf-life of the trout
flesh under refrigeration conditions (4°C).
2. Materials and Methods
Fillet samples' preparation
The fresh trout fish (approximately 1700±100g) was
purchased from a fish culture farm of Hamedan City,
Iran, and was immediately taken to the laboratory in insulated
boxes containing ice packs. The filleted fish were
washed by sterile distilled water and sliced into 10 g pieces
and covered in the coating solutions. All experiments
were carried out in the Department of Food Hygiene and
Quality Control, Faculty of Veterinary Science, Bu-Ali
Sina University, Hamedan City, Iran, in 2019.
PE preparation
Propolis was prepared from the present beehives in the
around of Kermanshah City, western Iran. The harvested
propolis was air-dried at environment temperature for
two weeks. Next, it was blended in a blender and mixed
to ethanol with a ratio of 30:100 g/mL, agitated at 250
rpm by an Earlene shaker for 24 h. Then, the obtained
solution was filtered and evaporated by a rotary evaporator
(Milan, Italy) at 40ºC. Finally, the concentrated extract
was dried under a vacuum at 50°C. The resulting
extract was preserved at -18ºC for subsequent use [10].
The coatings and treatments preparation
The solution of coatings was made with 1% CMC (w/v)
(average MW of 41 kDa, Cara gum Parsian Co. Tehran,
Iran), using sterile distilled water. Glycerol at 0.5 % (v/v)
was selected as a plasticizer compound into the CMC
coating solution. After heating the solution at 85°C for 5
min, it was cooled at room temperature. PE (1% and 2%)
was mixed with CMC coating dispersion. The prepared
coating solution consisted of 1% CMC, 1.5% glycerol,
alone or along with 1% and 2% PE. Four experimental
groups were designated as the following groups: control
(coated samples in sterile distilled water), CMC, CMCPE
1 %, and CMC-PE 2 %. The samples were immersed
in the obtained solution (sterile distilled water, CMC,
CMC-PE 1%, and CMC-PE 2%) for 2 min. The coated
fillets were packaged under aerobic conditions in polyethylene
zip packs, and then the packs were stored in a
refrigerator (4°C). Microbial, biochemical, and sensory
factors of the samples were assessed at 3-day intervals
for 15 days [13].
Microbiological evaluation
The 10 g sample was mixed with 90 mL of 0.1% peptone
water in a special stomacher pouch and homogenized
in the stomacher at 200 rpm for 60 seconds. Subsequent
dilutions were prepared in tubes containing 0.1%
peptone water and cultured on plates containing culture
medium. Microbial tests included total viable count,
psychrotrophic bacteria, lactic acid bacteria, Pseudomonas
spp., Enterobacteriaceae, and yeast-molds [16].
Biochemical analysis
pH value measurement
The pH value was measured according to Brannan
[17]. Five grams of the samples were homogenized in 25
mL of distilled water for 30 s. The pH values of resulting
homogenates were read by a pH meter (Jenway, UK).
Peroxide value measurement
After fat extraction of the samples, their Peroxide
Values (PV) were measured according to International
Dairy Federation [18]. PV was expressed as mEq of O2
per kg of fat.
Thiobarbituric Acid Reactive Substance (TBARS)
measurement
This index was identified as mg of malonaldehyde
equivalents per kg of the fillet. Also, 1, 1, 3, 3-Tetraethoxypropane
(TEP) was considered for preparing a standard
calibration curve [19].
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Total Volatile Basic Nitrogen (TVB-N) measurement
This experiment was carried out by distillation technique
using a Kjeldahl apparatus (Simax, Pyrexfan, Tehran,
Iran). The results were identified in mg of Nitrogen
(N) per 100 g of the trout fillets [20].
K value measurement
K value (the ratio of inosine and hypoxanthine to the
ATP and its decomposed products) was computed according
to the modified method of Fan et al. [21].
Adenosine-Triphosphate (ATP) and its breakdown
constituents were identified by HPLC (Knauer, Berlin,
Germany), along with Capcell, pack ODS C18 column
(4.0 × 100 mm, 3 μm). ATP and its decomposed compounds
were detected and then computed by Equation 1:
Equation 1
K value (%) =
[(HXR)+(HX)]
[(ATP)+(ADP)+(AMP)+(IMP)+ (HXR)+(HX)] ×100
Sensory analysis
Ten Master’s degree students (four females and six
males between 24 and 35 years old) of the Food Hygiene
and Quality Control Department performed the organoleptic
analysis of the treatments. The fillet samples were
cooked by steaming method for 20 min at 100±1°C after
salting (1.5%) them. The sensory analysis was carried
out using a 5-point hedonic scale to estimate the taste
and odor scores (1: poor, 5: excellent) [4, 22].
Study analysis
A total of 52 fish were placed in four groups (each 13
fish). Microbiological and chemical analyses were repeated
three times. The mean±Standard Deviations (SD)
values of the analyses were demonstrated. For statistical
analysis of the results, SPSS software (IBM SPSS statistics
21) was exploited. Analysis of Variance (ANOVA),
Tukey test, and Independent sample t test were utilized
for all data interpretation at P<0.05.
3. Results and Discussion
Microbiological analysis
Total viable count
Figure 1A–F displays the variations in Total Viable
Counts (TVC) , Pseudomonas spp., psychrotrophic
bacteria, Lactic Acid Bacteria (LAB), Enterobacteriaceae,
and yeasts-molds of the fillets. Figure 1A shows
the TVC during 15 days at refrigerator temperature. According
to the previous report, 4-6 log CFU/g, the total
bacterial counts of the fresh water fish species such as
rainbow trout are very changeable [23]. In this experiment,
all samples' primary level of TVC approximately
was 3 log CFU/g, which agrees with prior research on
the trout flesh [1, 4]. Figure 1A illustrates a significant difference
among all treatments during storage days. TVC
index of control samples reached 7.36 log CFU/g on the
sixth day. This value is above the maximum allowable
limit of 7 log CFU/g for TVC in the raw fish fillets in the
cold storage time [24]. On the 12th day, the TVC amount
of CMC treatment samples reached beyond the permissible
limit. In the treatment sample using 1% propolis,
the TVC value of the fish samples did not reach the standard
limit until the 12th day; while, 2% propolis could
maintain the TVC population of the fish samples in the
standard range until the end of the storage period. Thus,
for CMC, CMC-PE 1%, and CMC-PE 2% samples,
microbiological shelf-life elongation of 9, 12, and 15
days were respectively obtained in comparison to the
control sample. The shelf life increase for these groups
could be induced by the antimicrobial activity of CMC
and PE. According to the results, CMC-PE 2% had the
highest treatment impact for the total bacterial count of
the fillets, followed by CMC-PE 1% and CMC, respectively.
By creating a barrier against water and oxygen,
preventing microbial growth, and delaying fat oxidation,
biopolymer-based films, such as CMC, can maintain the
suitable quality and lengthen the shelf life of the food [2,
15]. Reports have revealed the antimicrobial properties
of propolis against various microorganisms, including
Staphylococcus aureus, Enterococcus faecalis, and Candida
utilis. These effects are mostly resulted from the
polyphenolic compounds [7, 25]. Besides, one study reported
the antibacterial activity of different percentages
of propolis water extract on the fresh Shubuta (Barbus
grypus) fillets during cold storage [9].
Psychrotrophic bacteria
The primitive series of bacteria that get blamed for
putrefying aerobically-stored fish meat at refrigerator
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temperatures are Gram-negative psychrotrophic bacteria
[26]. In this investigation, it was seen that in the
studied fillets, the psychrotrophic bacteria population
(day 0) was in the range of 2.63 log CFU/g to 2.82
log CFU/g. These results are consistent with previous
studies [24]. As seen in Figure 1B, during the storage
time, a rising trend was demonstrated in psychrotrophic
bacteria numeration. Furthermore, in all days of analyses,
control was the most populous group, followed by
CMC, CMC-PE 1%, and CMC-PE 2% samples. On the
sixth day, the psychrotrophic bacteria numeration of the
control fillets reached 7.83 log CFU/g that was more
than the acceptable level of 7 log CFU/g in the fresh
raw fish flesh [24]. This condition continued while on
the 9th and 15th days, psychrotrophic bacteria values of
CMC, CMC-PE 1%, and CMC-PE 2% went beyond the
allowed limit. Consistent with the results of TVC, the
potent treatment in decreasing psychrotrophic bacteria
population was CMC-PE 2%, followed by CMC-PE 1%
and CMC, respectively. Other researchers have argued
that coating can inhibit aerobic bacterial development,
which acts as an effective barrier against oxygen permeability
[2]. Based on Vargas-S Anchez et al., PE lowered
the microbial growth (mesophilic and psychrotrophic
enumerations) in the beef patties during eight storage
days in a refrigerator. This finding could be linked to the
polyphenolic compounds’ presence, demonstrating that
as a natural antimicrobial and antioxidant additive, i.e.,
PE has excellent potential in extending the beef patties’
shelf life [27].
Enterobacteriaceae
According to the findings of Gui et al., Enterobacteriaceae
were the crucial components of the spoilage microflora
in the flesh, stored at 4°C [28]. Initial numeration of
Enterobacteriaceae ranged from 1.75 log CFU/g to 2.005
log CFU/g in accordance with Volpe et al. on the coated
trout fillets with carrageenan under refrigerator temperature
[1]. After 15 storage days, Enterobacteriaceae counts
reached 6.015, 6.89, 7.615, and 8.69 log CFU/g in the
CMC-PE 2%, CMC-PE1%, CMC, and control fillets, respectively
(Figure 1C). Also, such a steady growth in Enterobacteriaceae,
according to Volpe et al., could be for
the stored trout flesh in the refrigerator [1]. Such a finding
was likely due to the gradual growth rate exhibited by
these bacteria compared to other Gram-negative psychrotrophic
spoilers. CMC and PE reduced the Enterobacteriaceae
growth rate in the fillets more significantly than
the control during refrigeration. Throughout the study
period, the lowest Enterobacteriaceae population was
found for the CMC-PE 2% containing fillets, followed by
CMC-PE 1% and CMC samples. Other researchers displayed
the antibacterial influence of carboxymethyl cellulose/
sodium alginate coating against the major member
of Enterobacteriaceae, i.e., Escherichia coli O157:H7,
inoculated in the chilled silver carp flesh [29]. Based on
the results of one study, the propolis extracts that could
be considered effective natural food preservers are likely
to prevent E.coli’s growth in vitro successfully. They reported
that most propolis components, mainly flavonoids,
have a phenolic origin. It is notified that polyphenols are
known as strong antimicrobial substances. Similarly,
Gallic acid derivatives of the propolis indicated inhibitory
activities against bacteria. In response to microbial
infections, plants produce flavonoids, resulting from
which they are identified with antimicrobial impacts on
a variety of microorganisms [30].
Pseudomonas spp
As observations suggest, Pseudomonas spp. can be
a critical member of the bacterial spoilage of the meat
kept in a refrigerator. Proteolysis is a determinant and
effective phenomenon in meat spoilage. When the Pseudomonas
spp. populations reach 107-108 CFU/g in meat
products, they cause proteolysis reaction followed by
slime production [31]. According to the primary count
of about 3.18-3.25 log CFU/g for Pseudomonas spp.
(day 0) of the trout samples (Figure 1D), similar initial
numeration (day 0) related to the rainbow trout was also
found by other studies [32, 33]. During the storage time,
Pseudomonas spp. count raised to the final numeration
of 11.12 log CFU/g (control fillets) while the counts of
CMC, CMC-PE 1%, and CMC-PE 2% reached 9.98,
8.82, and 7.7 log CFU/g at the last interval, being less
than the control fillets. Pseudomonas spp. count in all
groups was significantly (P<0.05) less than the control,
showing that PE-containing treatments were the strongest
concerning the inhibition treatments of Pseudomonas
spp. The mentioned finding may be linked to the
added antibacterial effects of CMC and PE. In their research,
Raeisi et al. showed that CMC-containing coatings
combined with Zataria multiflora Boiss essential oil
and grape seed extract could lower the growth of Pseudomonas
spp. in the rainbow trout flesh at refrigerated
storage [13]. De Marco et al. demonstrated that as the
adjuvants in treating P. aeruginosa chronic putrefaction,
the usage of propolis extracts could be effective not only
for their anti-biofilm features but also for their low toxicity
and biological (anti-inflammatory and antioxidant)
activities [25]. Some prior research also confirmed the
antibacterial characteristics of several variations of propolis
against Pseudomonas aeruginosa [8].
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Lactic acid bacteria
As a well-known facultative anaerobic bacterium, lactic
acid bacteria are a part of the original microflora of
the trout flesh; it can increase under aerobic and anaerobic
conditions [1]. Primary Lactic Acid Bacteria (LAB)
count ranged from 1.405 to 1.52 log CFU/g and did not
exceed 4.01 log CFU/g in the control fillets until the 15th
day of storage (Figure 1E). This condition holds while
compared to the control fillets, significantly (P<0.05)
lower LAB enumeration was found for the CMC and
CMC-PE samples, which were kept during the refrigeration
time. The most potent treatment was CMC-PE 2%
in preventing the replication of LAB in the fillet samples
among other groups of the researchbecause it induced a
1.22 log cycle reduction at the end of the refrigeration.
Such a finding may be reported for the synergistic antimicrobial
effect of CMC and PE. LAB is the most capable
Gram-positive bacteria against antimicrobial treatments
based on some research [34]. While compared to
other spoilage bacteria, LAB was more resistant versus
incorporated CMC with PE. In this research, the lowered
LAB count by CMC-PE was less than other bacterial
groups. One paper also investigated the impact of
aqueous PE during storage of shibuta (Barbus grypus)
flesh at 4°C. The conclusion was that 0.5% aqueous PE
remarkably lowered the shibuta lactic acid bacteria count
at all storage times compared to the control samples. As
suggested, such a decrease may be related to the phenolic
compounds’ presence in the PE [9]. Kalogeropoulos
et al. assessed the antibacterial activities of PEs from
Figure 1. Variation trends of the population in TVC (a), psychrotrophic bacteria (b), Enterobacteriaceae (c), Pseudomonas spp.
(d), lactic acid bacteria (LAB) (e) and yeasts–molds (f), during storage of trout fillets at 4°C
Treatments: Control (C), Carboxymethyl Cellulose (CMC); Carboxymethyl Propolis Extract 1 % (CMC-PE 1%); Carboxymethyl
Cellulose containing Propolis Extract 2% (CMC-PE 2%); Different lowercases exhibit significant differences (P<0.05) in each
interval.
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Greece and Cyprus [7]. They concluded that compared
with lactic acid bacteria, the Minimum inhibitory concentration
(MIC) of all studied propolis ethanolic extracts
were higher regarding the Listeria monocytogenes,
Staphylococcus aureus, and Bacillus cereus.
Yeasts/molds species
The yeast and mold species are common agents of microbial
spoilage in refrigerated meat. Like prior investigations,
the primary count (day 0) of yeast/mold of trout
fillets was 2.775-2.96 log CFU/g (Figure 1F) [26]. All
used treatments in the present study induced significantly
lower (P<0.05) counts of the yeasts/molds in CMC,
CMC-PE 1%, and CMC-PE 2% fillet samples in comparison
to the control under refrigeration condition (Figure
1F). Antifungal activity of the PE on the shibuta meat
in a refrigerator was confirmed by Duman and Ozpolat
[9]. Another study from Cyprus and Greece reported the
antifungal feature of propolis extracts against Candida
tropicalis and Candida albicans [7]. Mohammadzadeh
et al. also represented in vitro antifungal effect of the Iranian
propolis against Aspergillus niger and Candida albicans
[8]. Noshirvani et al. showed the antifungal activity
of the chitosan-CMC film incorporated with natural
preservatives against Aspergillus niger [35].
Figure 2. Variation trends of pH value during storage of trout fillets at 4°C
Treatments: Control (C); Carboxymethyl Cellulose (CMC); Carboxymethyl Cellulose containing Propolis Extract 1% (CMC-PE
1%); Carboxymethyl Cellulose containing Propolis Extract 2% (CMC-PE 2%); Different lowercases exhibit significant differences
(P<0.05) in each interval.
Figure 3. Variation trends of peroxide value during storage of trout fillets at 4°C
Treatments: Control (C), Carboxymethyl Cellulose (CMC), Carboxymethyl Cellulose containing Propolis Extract 1% (CMC-PE
1%), Carboxymethyl Cellulose containing Propolis Extract 2% (CMC-PE 2%). Different lowercases exhibit significant differences
(P<0.05) in each interval.
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Physicochemical analysis
pH value
Figure 2 illustrates pH index variations in trout fillets
during 4°C storage. The primary pH of the fresh fish
flesh (pH 6.27-6.69) agreed with the reports of prior
scholars [1, 36], but the obtained values of the present
research were slightly higher than the reported quantities
by other research studies [37]. Such discrepancies
in the findings may result from the dissolution and dissociation
of CO2 in the fillet samples. The pH indexes of
all samples escalated in the postmortem time; this may
be because of the activity of the endogenous or microbial
enzymes, like lipase and protease, in the presence
of oxygen, causing the enhancement of volatile bases
(trimethylamine and ammonia) [21]. Degradation of nitrogenous
substances led to pH enhancement, affecting
the freshness of the product during storage time. Following
that, the sensory features of the sample, such as taste,
odor, color, texture, and acceptability, were also negatively
affected [1]. In this study, after the storage time,
the pH of control samples increased from 6.69 to 7.61,
while the pH ranged from 7.39, 7.21 to 6.97, respectively
Figure 4. Variation trends of TBARS value during storage of trout fillets at 4°C
Treatments: control (C), Carboxymethyl Cellulose (CMC), Carboxymethyl Cellulose containing Propolis Extract 1% (CMC-PE
1 %), Carboxymethyl Cellulose containing Propolis Extract 2% (CMC-PE 2%). Different lowercases exhibit significant differences
(P<0.05) in each interval.
Figure 5. Variation trends of TVB-N value during storage of trout fillets at 4°C
Treatments: Control (C), Carboxymethyl Cellulose (CMC), Carboxymethyl Ccellulose containing Propolis Extract 1% (CMCPE
1%), carboxymethyl cellulose containing Propolis Extract 2% (CMC-PE 2%). Different lowercases exhibit significant differences
(P<0.05) in each interval.
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for the CMC, CMC-PE 1%, and CMC-PE 2% samples
in 15 days. These findings were exhibited for the protective
action of CMC edible coating against spoiling
reaction of the oxygen, significantly (P<0.05) increased
by propolis, especially in the group with a higher dose.
The lower pH index of other treatments (CMC, CMCPE
1%, and CMC-PE 2%) may have occurred from preventing
endogenous and exogenous (microbial) proteases'
activities at various degrees of the trout fillet through
the study treatment [2]. Duman and Ozpolat showed that
the pH value of the fresh shibuta (Barbus grypus) fillets
that contained propolis extract was lower compared to
the control group at all storage times [9].
Peroxide value
The lipid oxidation reaction is the essential agent in
the chemical spoilage of seafood. As the primary product
of the auto-oxidation, the Peroxide Value (PV) estimates
hydroperoxides’ quantity [18]. Peroxide values
of the samples are reflected in Figure 3. The PV of the
control treatment raised more than other groups during
the storage period. After 15 days, the PV in the control
group reached from 1.99 to 17.97 mEq peroxides/kg
lipid, while, during this time, the PV of CMC, CMC-PE
1 %, and CMC-PE 2 % increased from 1.89 to 12.98,
1.81 to 10.00, and 1.95 to 8.02 mEq peroxides/kg lipid,
respectively. Compared to the control, all treatments
demonstrated significantly reduced peroxide production
during refrigeration (P<0.05). In their work, Rezaei and
Shahbazi indicated that at all storage times, the composite
film based on the Sodium Alginate-Carboxymethyl
Cellulose (SA-CMC) could lower the peroxide index in
the sauced silver carp fillet when compared to the control
group [38]. The edible coating could lower the speed
of the oxidation reactions and moisture retention and act
as a barrier against oxygen and water permeability. In
other words, various coatings such as chitosan, alginate,
and CMC can increase the quality and storage life of
the foods by this mechanism [4, 22, 29, 38, 39]. Utilizing
CMC-PE 2% treatment, a maximal decrease in the
peroxide formation was acquired, followed by CMC-PE
1% and CMC; this condition may result from the potent
antioxidant activity of PE 2%. Shavisi et al. found
that Polylactic Acid (PLA) film that contained propolis
ethanolic extract can lower more peroxide value of the
minced beef than the control samples during 11 days of
storage at the refrigerator [14].
TBARS value
TBA index has widespread uses in estimating the lipid
oxidation content of foods. Reactive substances of TBA
developed by peroxide oxidation to ketone and aldehyde
are remarkable products of auto-oxidation’s terminal
phase during food oxidation [19]. Findings of the TBA
index of the samples under refrigeration conditions
are illustrated in Figure 4. According to Figure 4, the
TBARS index of all samples rose continuously during
the storage period. The results of other researchers indicated
the same pattern as well [12, 21, 40, 41]. Higher
TBA value may have been induced from the enhanced
oxidation of polyunsaturated fatty acids. As Connell suggested,
a TBA index of 2 mg MDA/kg, regarded as the
TBA value’s maximal level, demonstrated a desirable
quality of the fish fillet (chilled, frozen, or ice-stored)
beyond which, the fish may reflect unfavorable sensory
properties in terms of taste and odor [42]. The primary
TBA index of the trout in the present research was 0.42-
Figure 6. Variation trends of K value during storage of trout fillets at 4°C
Treatments: Control (C), Carboxymethyl Cellulose (CMC), Carboxymethyl Cellulose containing Propolis Extract 1% (CMC-PE
1 %), Carboxymethyl Cellulose containing Propolis Extract 2% (CMC-PE 2%). Different lowercases exhibit significant differences
(P<0.05) in each interval.
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0.47 mg MDA/kg, which increased by 2 mg MDA/kg
on the 12th day of refrigeration for the control samples.
While, the TBA values of the CMC, CMC-PE 1%, and
CMC-PE 2% treatments were 2.145, 1.935, and 1.495
mg MDA/kg, respectively, on the last day of the storage.
All treated fillets (CMC, CMC- PE 1% and CMC-PE
2%) had significantly lower Malondialdehyde (MDA)
values (P<0.05) than the Control (C) sample. According
to Figure 4, except for day 0, significant differences
(P<0.05) existed among designated groups during refrigeration.
The TBA indexes of CMC, CMC-PE 1%, and
CMC-PE 2% were significantly (P<0.05) lower than the
control during refrigeration. This finding means that the
CMC coating had the potential to prevent the lipid oxidation
reaction effectively. Previous scholars acknowledged
the ability of the CMC edible coating in controlling
the lipid oxidation of various food models [13, 38,
43]. Lower TBA values were reflected in PE-containing
treatments compared to the others, likely due to the antioxidants’
presence (PE). Additionally, the best effect was
reported from CMC-PE 2%. The antioxidant activity of
the propolis has been reported by prior studies as well.
As observed during all storage times, PLA film that contained
PE could hinder oxidation reaction in the chilled
minced meat [14].
Figure 7. Variation trends of sensory characteristics (a-taste and b-odor) during storage of trout fillets at 4°C
Treatments: Control (C), Carboxymethyl Cellulose (CMC), Carboxymethyl Cellulose containing Propolis Extract 1% (CMC-PE
1%), Carboxymethyl Cellulose containing Propolis Extract 2% (CMC-PE 2%). Different lowercases exhibit significant differences
(P<0.05) in each interval.
Bazargani-Gilani B, et al. Propolis Extract and Carboxymethyl Cellulose in Trout. Arch Hyg Sci. 2021; 10(2):117-132.
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Spring 2021. Volume 10. Number 2
TVB-N value
Total Volatile-Based Nitrogen (TVB-N) is a standard
index for the evaluation of meat spoilage. TVB-N value
comprised nitrogenous substances like dimethylamine,
trimethylamine, and ammonia which are probably derived
from the breakdown of nitrogenous ingredients,
such as nucleic acids and proteins by the exogenous
(microbial) and endogenous enzymes. Increasing the
TVB-N index of the meat can be a sign of spoilage. Giménez
et al. reported 25 mg N/100 g of the trout fillet to
be the highest allowable limit for consumers [44]. Figure
5 reflects the impacts of the studied groups on the
TVB-N index of the fish flesh during refrigeration. The
amount of TVB-N of all fillets continuously increased
during storage. The Primary TVB-N index of the trout
was 8.06-8.92 mg N/100 g that was consistent with prior
findings [1, 2, 45]. Primary TVB-N indexes of large yellow
croaker, northern snakehead, rainbow trout, bream,
and trout were 11.502-11.893, 9.50-10.00, 9.33-12.13,
12.62, and 11.35 mg/100 g in the above-mentioned studies.
From the beginning to the final day of the refrigeration
period, the TVB-N indexes of C, CMC, CMC-PE
1 %, and CMC-PE 2 % were (8.15-49.91 mg N/100 g),
(8.06-40.02 mg N/100 g), (8.92-24.95 mg N/100 g) and
(8.38-23.07 mg N/100 g). Significant (P<0.05) differences
were found among the considered groups in all analysis
intervals. TVB-N indexes in the control and CMC
samples were higher than an acceptable limit on the sixth
and ninth days of refrigeration, respectively. However,
until the last day of analysis, the samples that contained
PE, such as CMC-PE 1% and CMC-PE 2%, remained
in the allowable level of TVB-N. The TVB-N values of
CMC-PE 1% and CMC-PE 2% increased by 24.95 and
23.07 mg N/100 g at the end of the refrigeration period,
respectively, indicating a significant difference (P<0.05).
Control fillets only were fresh until the sixth day of
storage based on the aforementioned standard limit,
while CMC, CMC-PE 1%, and CMC-PE 2% groups
retained the trout freshness for the 9th and 15th days of
the refrigeration time, respectively. Through lowering
gas permeability, especially oxygen, the edible coatings
could raise the shelf life of the tout fillet after blocking
the bacterial growth [2, 13, 29, 38]. As findings indicate,
PE, an antioxidant that retains fish freshness during the
refrigeration period, can rapidly reduce the bacterial
ability and population for the deamination reaction of
non-protein nitrogen substances [2, 4]. According to
Duman and Ozpolat observations, the lowest limits of
TVB-N belonged to the propolis-treated shibuta (Barbus
grypus) fillets compared to the other treatments. The
rationale for this result can be the antimicrobial effect of
the propolis and the reduction ability of the bacteria to
operate deamination reaction of Non-Protein Nitrogen
(NPN) ingredients [9].
K value
Initiating before the bacterial spoilage, a postmortem
reaction in the fish body in many respective reactions is
nucleotides’ decomposition in the meat until the hypoxanthine
generation by the endogenous enzymes in the
muscle tissues during-storage. After enhancing the bacterial
growth, exogenous enzymes started linking to the
endogenous ones. Estimating these resulted compounds
has been concerned as an indicator of the fish freshness or
K value, recognized as the ratio (×100) of the non-phosphorylated
ATP’s decomposed products to the total ATP’s
decomposed products [46]. K value variations of the fillets
during the storage period are reflected in Figure 6.
Based on the prior studies, trout fillet, whose K index was
<20%, was relatively fresh, while the indexes <50% were
known as moderately fresh, and the indexes >70% were
regarded as unacceptable [28]. In the present research,
the trout samples’ primary K value was 15.31%-16.82%.
Also, other researchers observed that their rainbow trout
fillets initiated with the K values of 19.67%-19.82 % [28,
47]. Accordingly, their result is in the same line with these
study’s findings. As displayed in Figure 6, all samples in
all groups, especially control samples, presented a rapid
growth of the K value during the storage days. A similar
model was found in the results of the previous studies on
the bream [2], rainbow trout [28, 47], and silver carp [21].
But in other edible fish, including large yellow croaker
and the black skipjack, we observed a gradually raising K
value during the storage times; no variation was reported
later, and the enhancement rate did not accelerate [47,
48]. Such differences may be derived from different edible
fish, primary microbial load, and storage properties.
The treatments demonstrated remarkably lower K values
than the control sample at all storage times (P<0.05).
The K value of the control (77.89%), CMC (79.15%), and
CMC-PE 1% (71.74%) samples raised the allowed maximum
quantity (70%) on the 9th, 12th, and 15th days, respectively.
During the refrigeration time, however, the K value
of CMC-PE 2% did not increase by 70%. A significant
difference (P<0.05) was found among all samples after 0
days and until the final interval. For preventing nucleotide
degradation, the most potent treatments were CMC-PE
2%, followed by CMC-PE 1% and CMC. Findings confirmed
the treatments with PE such as CMC-PE 1% and,
specifically, CMC-PE 2% as more helpful in preventing
ATP decomposition and preserving the freshness of the
trout flesh.
Bazargani-Gilani B, et al. Propolis Extract and Carboxymethyl Cellulose in Trout. Arch Hyg Sci. 2021; 10(2):117-132.
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Spring 2021. Volume 10. Number 2
Sensory properties
Sensory properties’ differences in the steamed fillet
over the storage times are represented in Figures 7A and
B. Allowable fillet samples for consumption obtained
the sensory grade above 4 [4, 22]. Missed grades in the
taste feature, observed in Figure 7A, belong to the offflavor
and inedible samples. Taste and odor scores of
studied samples decreased with increasing the storage
time. According to the declaration of the panelists, the
taste and odor scores of the control fillets were undesirable
on the sixth day.
These observations were in line with the biochemical
results of the fillets, which may have been obtained from
lipid oxidation, protein degradation, and NPN decomposition
compounds, including aldehyde, ketone, hypoxanthine,
trimethylamine, dimethylamine, and ammonia.
They produce the off odor and off flavor products
that could be a rationale for these samples’ poor grades.
As illustrated in Figures 7 A and B, more significant differences
(P<0.05) are seen among all groups than the
control sample during the refrigeration time. Control,
CMC, CMC-PE 1%, and CMC-PE 2% groups achieved
admissible scores in the taste and odor after the 3rd, 6th,
12th, and 15th days of storage. Antimicrobial, antioxidant,
and gas barrier effects resulting from coating minimized
the spoilage reactions besides elongating the shelf life of
the samples and preserving their quality. A combination
of sodium alginate and CMC in another study extended
the shelf life of the silver carp fillet on the eighth day of
chilled condition [29].
Ojagh et al. [4] and Raeisi et al. [13] showed that the implementation
of the biopolymer coatings enriched with
antimicrobial substances induced a remarkable enhancement
in the overall acceptability of the fish fillets. Incorporating
PE into CMC coating significantly (P<0.05)
preserved the scores of the fresh sensory features of
taste and odor in the trout flesh until the last interval.
The sensory analyses revealed that a combination of PE
with CMC enhanced the shelf life of the samples on the
15th day; but, it did not show any additional effect on the
sensory properties in terms of taste and odor, which was
invisible on the surface of the rainbow trout fillet as well.
As Duman and Ozpolat suggested, the simultaneous usage
of the vacuum and aqueous PE increased the shelf
life of the shibuta (Barbus grypus) fillets for about 1-2
weeks [9]. This finding can be linked to the antimicrobial
effect of the PE that was derived from the phenolic
components' presence in this extract. Some prior studies
have also reported similar results [14].
4. Conclusion
Propolis, as a helpful product of honeybees, could
lower and slow the physicochemical reactions and microbial
growth. Also, its combination with CMC edible
coating prolonged the shelf life of the rainbow trout fillet
at the chilled storage. Concerning the point that natural
food substances such as propolis are safer and more costeffective
than synthetic ones, adding these compounds
into the food products may upgrade the consumers’
health and the food products’ preservation without any
unfavorable changes in their sensory properties at low
concentrations. However, to identify the effect of PE on
other meat products, further studies are required. Also,
the uses of other kinds of PE-containing packages or
coatings in increasing the shelf life of these new food
models should be tested in future works.
Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered
in this research.
Funding
This study was supported by the Faculty of Veterinary
Science, Bu-Ali Sina University, Hamedan City, Iran.
Authors' contributions
Conceptualization and supervision: Behnaz Bazargani-
Gilani, Mohammadreza Pajohi-Alamoti; Methodology:
Mojtaba Raeisi, Parviz Hassanzadeh; Investigation,
writing - original draft, and editing: All authors; Data
analysis: Parviz Hassanzadeh, Mojtaba Raeisi; Funding
acquisition and resources, data collection: Behnaz
Bazargani-Gilani.
Conflict of interest
The authors declared no conflict of interests.
Acknowledgments
The authors would like to appreciate all staf of the
Faculty of Veterinary Science, Bu-Ali Sina University,
Hamedan City.
Bazargani-Gilani B, et al. Propolis Extract and Carboxymethyl Cellulose in Trout. Arch Hyg Sci. 2021; 10(2):117-132.
130
Spring 2021. Volume 10. Number 2
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