Food Chemistry 450 (2024) 139345

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The inhibition mechanism of PostbioYDFF-3 on quality deterioration of

refrigerated grass carp fillets from the perspective of endogenous enzyme
and microorganisms changes
Zhesheng Zhang a, b, c, Jinshan Zhao b, c, d, Jinhong Zang a, b, c, *, Chuantao Peng a, b, c,
Liangtao Lv a, b, Zhaojie Li a, b
a
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
b
Shandong Technology Innovation Center of Special Food, Qingdao 266109, China
c
Qingdao Special Food Research Institute, Qingdao 266109, China
d
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China

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

Keywords: The protective mode of PostbioYDFF-3 (referred to as postbiotics) on the quality stability of refrigerated fillets
Grass carp was explored from the aspects of endogenous enzyme activity and the abundance of spoilage microorganisms.
Postbiotics Compared to the control group, the samples soaked in postbiotics showed significant reductions in TVC, TVB-N
Refrigerated preservation
and TBARS values by 39.6%, 58.6% and 25.5% on day 5, respectively. In addition, the color changes, biogenic
Microbiological analysis
Endogenous enzyme
amine accumulation and texture softening of the fish fillets soaked in postbiotics were effectively suppressed.
Furthermore, the activity of endogenous enzyme activities was detected. The calpain activities were significantly
inhibited (p < 0.05) after soaking in postbiotics, which declined by 23%. Meanwhile, high throughput
sequencing analysis further indicated that the growth of spoilage microorganism such as Acinetobacter and
Pseudomonas were suppressed. Overall, the PostbioYDFF-3 was suitable for preserving fish meat.

1. Introduction processing, including dead bacteria and by-products (Vinderola,

Freshwater fish have a high yield and are rich in protein and essential
trace elements, which are deeply loved by the public. The global pro­
duction of grass carp reached 7.13 million tons in 2022, and it therefore
has strong economic benefits (FAO, 2022). Nevertheless, processed grass
carp is prone to spoilage during transportation and sales, resulting in
softening, discoloration, unpleasant odors and muscle dehydration (Pyz-
Lukasik & Paszkiewicz, 2019; Yu, Xu, Regenstein, et al., 2018). Zhuang,
Tian, Liu, et al. (2023) studied that it might be related to the changes in
microbial, oxidation and endogenous enzyme activity. Currently, the
most common preservation techniques for aquatic products are low
temperature refrigeration and freezing.
Many explorations have been made to extend the shelf life of fish
fillets, such as the preservation by chitosan coating (Chang, Li, Bai, et al.,
2023; Yu, Zhao, Dong, et al., 2022) and plant extraction, etc. (Xue, Jing,
Pan, et al., 2023; Zhou, He, Yu, et al., 2023). Practical innovative
refrigeration technology also received widespread attention. Postbiotics
refers to a series of metabolites produced by a variety of probiotics after
Sanders, & Salminen, 2022). Postbiotics has the advantages of
combating obesity, improving enteritis, regulating skin flora, degrading
pesticides, antibacterial and antioxidant activity (Cuevas-gonzález,
Liceaga, & Aguilar-Toalá, 2020). Current research focuses on the effects
of postbiotics on human health. In fact, it also contains a variety of
antibacterial agents, such as organic acids, bacteriocins, extracellular
polysaccharides and bioactive peptides (Sharafi, Divsalar, Rezaei, et al.,
2023). Therefore, the focus on antibacterial and antioxidant activity is a
novel direction. For example, the intracellular substances and metabo­
lites of 7 species of Bifidobacterium, 11 species of Lactobacillus, 6 species
of Lactococcus and 10 species of Streptococcus thermophilus showed high
antioxidant activity (Amaretti, Di Nunzio, Pompei, et al., 2013). Post­
biotics from Lactobacillus acidophilus LA5 showed antibacterial activity
against ground meat and whole milk (Moradi, Mardani, & Tajik, 2019).
In addition, postbiotics has good stability and environmental adapt­
ability, hence, which can be easily integrated into food formulations and
packaging materials in both dry and liquid forms (Sharafi et al., 2023).
Postbiotics ingredients can effectively improve the preservation of

* Corresponding author at: College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
E-mail address: 15190273775@163.com (J. Zang).

https://doi.org/10.1016/j.foodchem.2024.139345
Received 5 February 2024; Received in revised form 7 April 2024; Accepted 10 April 2024
Available online 15 April 2024
0308-8146/© 2024 Elsevier Ltd. All rights reserved.
Z. Zhang et al.
refrigerated meat (Mishra, Mishra, Mohanta, et al., 2024). However, the
mechanism of postbiotics in maintaining fish quality is still unclear.
The purpose of this study is to evaluate the inhibition mechanism of
PostbioYDFF-3 on quality deterioration of refrigerated grass carp fillets
from the perspective of endogenous enzyme and microorganisms
changes. This study contributed to better understanding of postbiotics
preservation effects and expansion of application in food preservation.
2. Materials and methods
2.1. Postbiotics and sample treatments
PostbioYDFF-3 was purchased from Qingdao Yuanda Biotechnology
Co., Ltd. (Qingdao, Shandong, China). The Limosilactobacillus fermentum
Postbio-Q7 and Lactobacillus paracasei Postbio-P6 were fermented at
37 ◦ C for 48 h and inactivated at 97 ◦ C for 2 h to obtain the PostbioYDFF-
3. PostbioYDFF-3 was dissolved in deionized water and centrifuged for
15 min using a Sigma 4 K15 centrifuge (Sigma Laboratory Centrifuge
GmbH, Ostrode, Germany) at 4 ◦ C and 4000 g to remove any insoluble
impurities. The fish fillets were soaked in different concentrations of
PostbioYDFF-3 (3%, 5% and 10% (m/v)) and deionized water respec­
tively. It was found that the concentration of postbiotics exceeded 3%,
the color of fish fillets became noticeably darker, resulting in unfavor­
able sensory acceptance (Fig. S1). Therefore, the concentration of 3%
was chosen for further experiments.
Fresh grass carp weighing approximately 2.2 ± 0.2 kg were pur­
chased from a local aquatic supermarket (Qingdao, Shandong, China).
After removing the head, scale and guts, the fish were rinsed and
covered with crushed ice for transportation to the laboratory within 20
min. The dorsal muscles of the fish were manually filleted into similar
cubes measuring (4 × 3 × 1.5 cm3), which were used for the subsequent
postbiotics and sterile water soaking treatment. The fish fillets were
randomly divided into two groups. One group was soaked in the post­
biotics solution (marked as the postbiotics group), while the other group
was soaked in deionized water (marked as the control group). Both
groups were subjected to the same conditions for 1 min, with a fillet-to-
solution ratio of 1:3 (m/v). The drained fillets were individually pack­
aged in sterile bags and stored at 4 ± 0.5 ◦ C in refrigerator for further
analysis.
2.2. Determination of physical and chemical indicators
2.2.1. Determination of sensory traits, color and pH
The sensory evaluation of fillets was conducted using a method of
Khazandi, Deo, Ferro, et al. (2017) with slight modification. A panel of 8
trained laboratory members, consisting of 4 men and 4 women aged
between 23 and 26 was invited to conduct sensory evaluation. Prior to
each sampling point, the panelists were provided with information
about the characteristics of fresh flesh on day 0, which was considered
the highest quality and given a rating of 5 according to the following
standard. The rating scale ranged from 1 to 5 with higher scores indi­
cating better quality. The weighted sensory index was calculated as
follows: preference scores = (2 × smell +2 × color +2 × texture)÷5. The
analysis was conducted in a specialized sensory laboratory. The pro­
vided samples were randomly labeled with unique three-digit codes to
prevent any interference between them. The average score from all
panelists represented the sensory preference, and scores below 3.5 were
deemed unacceptable. This study was conducted in strict accordance
with ethical guidelines and our institution has given permission to
conduct sensory panel research. We respected and protected the rights
and privacy of the participants and ensured the confidentiality of their
personal information. Participants had the right to know that their
participation was voluntary and that their informed consent had been
obtained, and they could withdraw from the study at any time.
Color and pH were measured according to the Romero, Sharp,
Dawson, et al. (2021) with slight modification. The color of the fish was
2
Food Chemistry 450 (2024) 139345
measured using a colorimeter (Chroma Meter CR-200, Konica Minolta,
Tokyo, Japan), which was calibrated on a standard white plate before
each measurement. The measurements include brightness (L*), redness
(a*) and yellowness (b*). The color of control group and postbiotics
group were measured six times. The sample (10 g) was mixed with
deionized water (90 mL) and homogenized for 1 min at 12,000 rpm. The
pH values of the samples were recorded using a digital pH meter (Orion
Star TM A211, Thermo Fisher Scientific Inc., Waltham, MA, United
States).
2.2.2. Determination of water loss and texture
Water loss and texture determination was carried out according to Yu
et al. (2022). The ratio of the weight difference between the sample on
the sampling day and the sample on the initial day to the initial weight
was used as a measure of water loss. The texture was evaluated using
instrumental measurement of shear force. It was achieved using TA.XT
Plus Texture analyzer equipped with an A/CKB blade (Stable Micro
Systems, Ltd., Shanghai, China). Samples were subjected to perpendic­
ular shearing, while maintaining a constant degree reached 50%. The
pre-test and post-test speeds were both set at 100 mm/min with a trigger
force of 5 g. The shear force was expressed by the pressure (g). The
texture of control group and postbiotics group were measured six times.
2.2.3. Determination of total viable count
Total viable count (TVC) were based on Roseiro, Santos, Gonçalves,
et al. (2017) with slight modification. Three grams of chopped samples
were aseptically transferred into 20 × 25 cm2 sterile homogenizing bag,
with a fillet-to-solution ratio of 1:9 (m/v). The samples were homoge­
nized with saline for 6 min. The samples were continuously diluted ten
times after homogenization. The diluted sample was applied to the plate
to determine the total viable count. The plates were subsequently
incubated at 37 ◦ C for a period of 2 days. The TVC was expressed as log
colony-forming units per gram (lg cfu/g).
2.2.4. Determination of TBARS
The TBARS values were analyzed using the colorimetric method (Yu
et al., 2022). The chopped sample was homogenized with 10% TCA
solution (m/v) at a ratio of 4:1. Resulting mixture was filtered to obtain a
clarified solution. Thereafter, transfer the filtrate to a test tube with a
screw cap, which was pre-filled with an equal amount of 0.02 mol/L
TBARS solution. The test tube was heated for 20 min in boiling water.
The absorbance of the reaction mixture at 532 nm was determined by
UV-1000 spectrophotometer (Techcomp Co., Ltd., Shanghai, China)
after cooling, and the reagent blank was used as the reference. The
TBARS value was determined by comparing the absorbance reading to a
standard curve, and result was expressed as mg MDA eq/kg flesh.
2.2.5. Determination of TVB-N
The TVB-N determination was carried out according to the method of
Li, Hao, Chu, et al. (2022) with slight modification. The samples were
homogenized with pre-cooled deionized water for 3 min initially. After
centrifugation at 4000g for 15 min at 4 ◦ C, the certain amount of Mgo
was added to the supernatant and the mixture was decanted into the
digestive tube. The Kjeldahl nitrogen analyzer Kjeltec 9 (Beijing Fuhua
Instrument Co., Ltd., Beijing, China) was used for determination. The
TVB-N value was expressed as mg N/100 g of flesh.
2.2.6. Determination of biogenic amines
The extraction and pre-column derivatization of biogenic amines
(BAs) were carried out in accordance with the method described by Yu,
Zhao, Yang, et al. (2021). The quantification of biogenic amines was
conducted using a High-Performance Liquid Chromatography (HPLC)
system (Beijing Huapu Instrument Co., Ltd., Beijing, China). The
gradient elution program involved starting with 50% mobile phase A
(acetonitrile), which was increased to 90% in 0 to 35 min. Subsequently,
the proportion of mobile phase A was returned to 50% in 35–45 min and
Z. Zhang et al.
maintained for an additional 10 min. The remaining mobile phase B
consisted of 0.1 mol/L ammonium acetate solution. The flow rate was
set at 1.0 mL/min and detection wavelength was set at 254 nm. The
quantification of biogenic amine contents was determined by comparing
the peak area of the sample and the biogenic amine standard (Sigma-
Aldrich Co., Shanghai, China).
2.2.7. Determination of Illumina-MiSeq high throughput sequencing
The extraction and amplification of microbial DNA, along with
Illumina-MiSeq high throughput sequencing were conducted following
Yu et al. (2021). The V3 - V4 region of bacterial 16S rRNA gene was
amplified with 338F (5’-ACTCCTACGGGAGGCAGCA-3′) & 806R (5’-
GGACTACHVGGGTWTCTAAT − 3′). According to the Kit procedure,
purification was performed with DNA Clean beds (Vazyme, Qingdao,
Shandong, China) and quantification was performed with the Quantit™
dsDNA HS Assay Kit (Thermo Fisher Science, Waltham, MA, USA).
Finally, equimolar amplified sequence analysis was performed on the
Illumina HiSeq 2500 platform (Beijing Biomarker Technology Co., LTD.,
China).
2.2.8. Determination of endogenous enzyme activity
The extraction of crude endogenous enzyme solution and determi­
nation of enzyme activity refered to Ge, Xu, and Xia (2015). The sample
was mixed with 20 mmol/L Tris-HCl solution with neutral pH. The
mixture was homogenized in an ice bath for 1 min and centrifuged at
12000g for 20 min at temperature of 4 ◦ C. The supernatant was collected
as a crude enzyme solution. The specific fluorescent substrates Z-Arg-
Arg-AMC, Z-Phe-Arg-AMC, and N-Suc-Leu-Tyr-AMC were added to the
crude enzyme solution. The substrate reacts with the protease in the
solution, releasing 7-amino-4-methylcoumarin (AMC). For the cathepsin
D activity assay, the peptides released when acidified bovine serum
protein reacted with protease were detected by fluorescence. The fluo­
rescence values were determined using F-4500 fluorescence spectro­
photometer (Techcomp Co., Ltd., Shanghai, China), with excitation and
emission wavelengths of 380 and 485 nm, respectively. The enzyme
activity was defined as the amount of enzyme required to release 1
μmol/L AMC within 1 min at 37 ◦ C.
2.2.9. Determination of K value
The K value (ATP-related compound) was determined according to
the method of Yu et al. (2021) with slight modification. The sample (2 g)
was homogenized with 7.5 mL cold perchloric acid solution (0.6 mol/L,
4 ◦ C) for 2 min and centrifuged at 4000 g for 10 min at 4 ◦ C to obtain the
supernatant. The above procedure was repeated once to merge the su­
pernatant. The pH of supernatant was adjusted to 6.5–6.8 with 1 mol/L
KOH solution and volumed to 25 mL via deionized water. A HITACHI
5610 HPLC system equipped a photodiode array (PDA) detector (Hitachi
high-tech International Trade Co., LTD, Shanghai, China) and ODS C18
column (4 μm, 4.0 mm id × 200 mm, Nanologica AB, Stockholm,
Sweden) was adopted. The mobile phase for isocratic elution consisted
of 98% potassium phosphate buffer (0.05 mol/L, pH 6.8) and 2%
methanol. ATP-related compounds were valued according to the reten­
tion time and peak area of the standards (Sigma-Aldrich Co., Beijing,
China). The sample (2 μL) was flowed at 1.0 mL/min for 10 min and the
peak was observed at 254 nm. K value was calculated by the following
equation:
[ ]
WHxR + WHx
K(%) = × 100%
WATP + WADP + WAMP + WIMP + WHxR + WHx
(WATP, adenosine triphosphate; WADP, adenosine diphosphate; WAMP,
adenosine monophosphate; WIMP, inosine monophosphate; WHxR, hy­
poxanthine ribonucleoside; WHx, hypoxanthine).
3
Food Chemistry 450 (2024) 139345
2.3. Statistical analysis
All data were analyzed three times except for shear force and color
analysis, which were performed six times. All the experimental and
control groups were compared at the same time point. The data differ­
ences among the mean values of each sampling point were analyzed by
one-way ANOVA and Duncan’s multiple range test of SPSS 19.0 software
(SPSS Inc., Chicago, IL, USA), and the significance level set at p < 0.05.
The analysis for Illumina sequencing data was performed on Biomarker
Cloud Platform (Biomarker Technologies Co., Ltd., Beijing, China).
3. Results and discussion
3.1. Sensory, color and pH analysis
The sensory evaluation of samples was shown in Fig. 1A. With the
extension of storage time, the sensory scores from both groups showed a
decreasing trend but with different degrees. The sensory scores of each
postbiotics group were higher than those of the control group until day
5, and the postbiotics group was better than the control group especially
in terms of odor at day 5 (p < 0.05). Bazargani-Gilani and Pajohi-
Alamoti (2019) also showed that fillets were unacceptable on day 6.
Higher scores in treated group showed that postbiotics treatment could
significantly improve the quality of refrigerated fish.
The color of the samples was shown in Fig. 1B-D. The redness values
of the control group and postbiotics group was gradually increased,
while the brightness value was showed a trend of first increasing and
then decreasing with the extension of the refrigeration period. The
brightness of the postbiotics group remained at a high level of 61.5,
while the control group was dropped to 58.5 by day 3. As shown in
Fig. 1, the postbiotics treatment could significantly improve the
brightness of refrigerated fish compared to the control. It was found that
the migration rate of water loss in fish body was slowed down after
slaughter, resulting in an increase in water content and a decrease in
brightness value (He, Huang, Li, et al., 2017). In addition, the redness of
the postbiotics group reached 5.8 increased by 30% compared to the
control group on day 2. The decreased in redness value on the 2nd day
might be due to the loss of myoglobin as a result of water loss. The
increased in the redness value after the second day might be due to the
large loss of heme caused by myoglobin interacting with peroxidase
(Desai, Joseph, Suman, et al., 2019). The yellowness values of the
control group and the postbiotics group was showed a gradually
increasing trend, while the reasons for the elevations might be as fol­
lows, on the one hand, it might be due to oxidative breakdown of
autolystic lipids of fish, on the other hand, myoglobin binding to
hydrogen sulfide also caused the fillets to appear yellow (Olafsdottir,
Nesvadba, Di Natale, et al., 2019). However, there was no significant
difference in yellowness between the control group and the postbiotics
group.
The pH evaluation of the sample was shown in Fig. 1E. The pH value
of the control group and postbiotics group showed a slow rising trend.
The pH of the control group and the postbiotics group were reached 6.93
and 6.81 on day 4 respectively, while the postbiotics group was signif­
icantly decreased. The pH initial decrease might be attributed to the
production of lactic acid and phosphate through anaerobic glycolysis
and ATP degradation (Liu, Liang, Xia, et al., 2013). The increased in pH
might be due to volatile bases (such as ammonia and trimethylamine)
produced by proteolysis. The results showed that postbiotics might
effectly inhibit the production of amines by microorganisms.
3.2. Texture and water loss analysis
The texture and water loss of fish flesh were crucial physical prop­
erties that greatly impacted consumer acceptance. As shown in Fig. 2A,
the initial shear force value for the fresh sample was approximately 350
g, and gradually decline was observed in both groups. The shear force of
Z. Zhang et al.
Fig. 1. Changes in sensory (A), color (B to D) and pH (E) of refrigerated grass carp fillets throughout the storage.
Fig. 2. Changes in shear force (A) and water loss (B) of refrigerated grass carp fillets throughout the storage.
all postbiotics groups was higher than that of the control group, while
the postbiotics group showed a more stable texture with the extension of
storage time. The softening process could be roughly divided into two
stages. The first stage occurred during the initial three days of storage,
while the postbiotics group increased by 42% to maintain 230 g
compared to the control group on day 3. Yu, Zhao, Wan, et al. (2023)
also proposed a cliff like decrease in shear force in the first three days.
Subsequently, the second stage occurred during the after three days of
storage, the rate of softening was further slowed in both groups, how­
ever, the postbiotics group still increased by 15% to attain 190 g on day
5 compared with the control group. It was generally believed that the
early softening of fish was mainly due to the action of endogenous en­
zymes (Singh & Benjakul, 2018). Postbiotics might penetrate into fish
tissue to maintain texture. Hua, Wong, and Li (2022) also reported that
postbiotics penetrated into salmon fillets from coating to improve the
quality of fillets. Therefore, postbiotics might improve the softening of
4
Food Chemistry 450 (2024) 139345
fish meat.
As shown in Fig. 2B, the water loss of the control and postbiotics
samples gradually increased during storage period. The difference be­
tween the control and postbiotics groups was significant after the 2nd
day, while the postbiotics group had a 2.5% lower rate of water loss than
the control group on the 5th day. Water loss might be due to more
changes in water evaporation and protein structure, resulting in
decreased muscle hydration ability (Roiha, Tveit, Backi, et al., 2020).
Postbiotics might ameliorate changes in protein structure.
3.3. TVB-N, K value and TBARS analysis
TVB-N and TBARS were two crucial indicators of freshness in fishery
products, which were determined nitrogen decomposition products and
lipid oxidation. As shown in Fig. 3A, the initial content of TVB-N in fresh
grass carp flesh was approximately 5.8 mg N/100 g. The patterns of
Z. Zhang et al.
Fig. 3. Changes in TVB-N (A) and TBARS (B) of refrigerated grass carp fillets throughout the storage.
TVB-N in control group and postbiotics group gradually increased dur­
ing storage, obviously, the difference between the two groups began on
the 2nd day. The postbiotics group decreased by 64% to 8.8 mg N/100 g
compared to the control group until day 5. The results based on Yu,
Regenstein, Zang, et al. (2018) set the critical value of grass carp dete­
rioration as 13 mg N/100 g. The postbiotics group was significantly
differenced from the critical value by 4.2 mg N/100 g. Commonly, the
TVB-N was produced by nitrogen-containing compounds that break
down proteins by microorganisms (Li, Jia, Hong, et al., 2020). Conse­
quently, we were speculated that postbiotics might interfere with the
normal growth of spoilage microorganisms, thereby further prevent
protein breakdown.
The changes of TBARS were shown in Fig. 3B. The initial value of
fresh flesh was approximately 0.17 mg MDA eq/kg. The TBARS value in
the control group and the postbiotics group were showed different linear
growth. The TBARS value of all postbiotics groups were lower than that
of control group, the control group achieved 1.05 mg MDA eq/k while
the postbiotics group decreased by 28.6% to 0.75 mg MDA eq/k on the
5th day. The TBARS value serves as an indicator of the degree of sec­
ondary oxidation of fatty acids, while the peroxide was further decom­
posed into volatile products during the oxidation process (Hua et al.,
2022). It was speculated that the low TBARS value of postbiotics group
might be due to the hidden protective layer formed by soaking post­
biotics, which enhanced the blocking effect on oxygen. Additionally,
postbiotics had excellent free radical scavenging ability in vitro, and
might also help protect the enzyme antioxidant system in fish muscle,
thereby blocking the lipid oxidation reaction (Khani, Shkouhian, Kafil,
et al., 2023).
Nucleotide degradation in muscle tissue was the corresponding re­
action before bacterial proliferation (Li, Zhang, Song, et al., 2017).
Previous studies reported that fish with a K value <20% were very fresh,
the value of 20–50% was considered relatively fresh and values above
60% were concerned as undesirable (Bazargani-Gilani & Pajohi-
Alamoti, 2019). As shown in Fig. S2, with the extension of storage
time, the K value from both groups showed an increasing trend, while
the postbiotics group increased more slowly. There were significant
differences (p < 0.05) between the two groups from the 2nd day to the
5th day. The postbiotics group reached 39.8% and remained relatively
fresh on day 5, while the control group reached unacceptable levels. He,
Li, and Jiao (2021) also showed that K values grew rapidly from 0 to 6
days. The 5-nucleotidase could decompose IMP into inosine and hypo­
xanthine. Therefore, the lower K value in postbiotics group might be
related to the inhibitory effect of postbiotics on 5-nucleotidase activity.
5
Food Chemistry 450 (2024) 139345
3.4. Biogenic amines analysis
Biogenic amines were commonly used as reliable indicators to
monitor fish spoilage. As shown in Table 1, the initial levels of putres­
cine, cadaverine and histamine were relatively low in fresh fish flesh,
while spermidine and spermine might initially be present in fish at
higher levels and gradually decreased during refrigeration. Similar
result was also reported by Wang, Yu, Han, et al. (2018) for refrigerated
grass carp flesh. However, putrescine, cadaverine and histamine in the
control group were showed significant and rapid increases between day
3 and day 5 (p < 0.05). It was consistented with the research results of
Zhuang, Liu, Li, et al. (2021). The three types of BAs were reached 4.24,
0.48 and 0.81 mg/kg in the postbiotics group by the 5th day respec­
tively, and significantly decreased by 72.2%, 61.9% and 49.7%
compared with the control group respectively. The production of BAs
might be due to microorganisms inducing amino acid decarboxylation
(Wang, Liu, Zhang, et al., 2017). Postbiotics might have positive effect
on inhibiting the growth of microorganisms.
3.5. TVC analysis
TVC measurement was a traditional method for fish freshness eval­
uation. The initial TVC for fresh fish was usually 2–4 log cfu/g whereas
the maximum acceptable threshold for fish was set at 6 log cfu/g
(Rathod, Nirmal, Pagarkar, et al., 2022). As shown in Fig. 4B, the control
group and the postbiotics group were below 2.6 log cfu/g on day 0. The
control group sharply increased to 5.98 log cfu/g on the 3rd day, while
the postbiotics group remained within the maximum acceptable
threshold range only reaching 5.92 log cfu/g on day 5. The two groups
were shown significantly different (p < 0.05). The results showed that
the microbe was sensitive to postbiotics. Therefore, we speculated that
postbiotics might effectively inhibit the growth of microorganisms and
show antibacterial effects, which was similar to the result of Huang,
Xiong, Zou, et al. (2019).
3.6. Microbial diversity analysis
High-throughput sequencing was used to gain a deeper under­
standing of the changes in microbial communities on grass carp fillets in
refrigerated storage. As shown in Fig. 4A, the B0 to B5 and A0 to A5 were
represented control samples and postbiotics samples at 0 to 5 days
respectively, while the Fresh was refered to fresh grass carp fillets before
soaking treatment. A total of 26,447 feature reads were obtained. Most
microbial species were detected in all samples due to all coverage levels
≥0.999. Alpha diversity indexes, such as Chao/Ace and Shannon/
Z. Zhang et al. Food Chemistry 450 (2024) 139345

Table 1
Changes in biogenic amines (BAs) of refrigerated grass carp fillets throughout the storage.
Bioamine (mg/kg) group Storage time (days)

0 1 2 3 4 5

Control 0.42 ± 0.03a 0.47 ± 0.02a 0.59 ± 0.02a 0.74 ± 0.03b 0.86 ± 0.02b 0.94 ± 0.03c
TRY
Postbiotics 0.42 ± 0.03a 0.49 ± 0.02a 0.53 ± 0.02a 0.62 ± 0.02a 0.68 ± 0.03b 0.75 ± 0.03b
Control 1.21 ± 0.14a 2.26 ± 0.08b 3.05 ± 0.04c 3.11 ± 0.06c 3.85 ± 0.13c 4.06 ± 0.06d
PHE
Postbiotics 1.21 ± 0.14a 1.75 ± 0.06a 2.34 ± 0.07b 2.89 ± 0.15b 3.45 ± 0.03c 3.61 ± 0.18c
Control 1.23 ± 0.25a 2.42 ± 0.08a 3.27 ± 0.09a 5.55 ± 0.06b 6.73 ± 0.04b 15.24 ± 0.89c
PUT
Postbiotics 1.23 ± 0.25a 1.31 ± 0.08a 1.41 ± 0.13a 2.01 ± 0.33a 2.51 ± 0.05a 4.24 ± 0.22b
Control ND ND 0.17 ± 0.07a 0.28 ± 0.02a 0.38 ± 0.01b 1.26 ± 0.01c
CAD
Postbiotics ND ND ND ND 0.13 ± 0.03a 0.29 ± 0.02a
Control 0.19 ± 0.02a 0.32 ± 0.09a 0.40 ± 0.03a 0.74 ± 0.03b 1.09 ± 0.25b 1.61 ± 0.07c
HIS
Postbiotics 0.19 ± 0.02a 0.23 ± 0.06a 0.26 ± 0.10a 0.33 ± 0.10a 0.50 ± 0.10b 0.81 ± 0.13b
Control 1.97 ± 0.07a 2.55 ± 0.56a 3.11 ± 0.07a 4.68 ± 0.55b 6.27 ± 0.57b 10.92 ± 0.47c
TYR
Postbiotics 1.97 ± 0.07a 2.29 ± 0.45a 2.62 ± 0.05a 2.74 ± 0.41a 2.91 ± 0.43a 4.36 ± 0.30b
Control 3.32 ± 0.25a 2.86 ± 0.08b 2.61 ± 0.15b 2.34 ± 0.08b 1.87 ± 0.06c 1.31 ± 0.08c
SPD
Postbiotics 3.32 ± 0.25a 2.94 ± 0.09b 2.82 ± 0.05b 2.59 ± 0.03b 2.23 ± 0.02b 1.77 ± 0.48c
Control 19.54 ± 0.64a 16.69 ± 0.55b 15.08 ± 0.68b 13.86 ± 0.35c 13.71 ± 0.20c 13.11 ± 0.06c
SPM
Postbiotics 19.54 ± 0.64a 18.55 ± 0.25b 17.05 ± 0.11b 16.52 ± 0.40b 15.88 ± 0.25b 14.25 ± 0.16c

Note: Values are expressed as mean ± standard deviation; ND indicates not detected; different lowercase letters in the same row indicate statistically significant
differences (p < 0.05).

Simpson, which were used to assess species richness and diversity
respectively. Compared with fresh samples, the richness and diversity of
microbial communities in control and postbiotics groups decreased to
different degrees with the passage of time. As shown in Fig. 4C, the top
three microbial communities in fresh fillets (Fresh) were composed of
Unclassified_Bacteria genera, Pseudomonas genera and Unclassified_G­
emmatimonadaceae genera, accounting for 8.8%, 7.6% and 5.1%
respectively, while other species accounted for <1%. Moreover, the
proportion of the three major microbial communities in the postbiotics
group was reduced compared to the fresh sample on day 0. The results
showed that initial soaking of postbiotics reduced bacterial attachment
on the surface of the fillets. In the control group, Acinetobacter rapidly
became the dominant genus, accounting for 47.2% of total microbial
community on the 2nd day. Interestingly, the microbial community in
the postbiotics group remained consistent with fresh fillets by the 2nd
day. The results showed that the postbiotics group effectively influenced
the growth advantage of Acinetobacter. Similarly, Costa, Silva, Vicente,
et al. (2017) also observed that Acinetobacter baumannii could be effec­
tively inhibited by substances with antibacterial properties (bacteriocin,
chitosan, etc). In addition, the numbers of Acinetobacter and Lactococcus
continued to increase in the control group, while that in the postbiotics
group increased slowly throughout the storage period. Finally, the top
six microbial communities of the control group reached 82.5% while the
postbiotics group reached 61.4% on the 5th day. In fact, the spoilage
capacity of Acinetobacter was relatively low compared to the Pseudo­
monas and Shewanella. However, Acinetobacter might be another key
factor in fish spoilage. Because Acinetobacter could produce the group
sensing signals of acyl homoserine lactones (AHLs), which promoting
the growth of Pseudomonas and Shewanella (Huang, Xiong, Shi, et al.,
2021; Zhu, Wu, Zhang, et al., 2018). Therefore, Acinetobacter might be
the specific spoilage microorganism (SSOs) that caused the decay of fish
fillets.
The difference of microbial community composition was visually
demonstrated by principal component analysis (PCA) and heat maps
during storage period. As shown in Fig. 4D, the PCA analysis revealed
two major variations PC1 and PC2, which accounted for 66.31% and
23.42% respectively. The points A0 to A4 and points B0 to B1 were
closed to the point of Fresh samples (mark with red circles), indicating a
close relationship between microbial community composition. It might
be that postbiotics impeded spoilage bacteria growth, resulting in the
postbiotics group being highly similar to fresh samples. Nevertheless,
the significant differences between the samples except for the proximity
of B3 and A5 were due to the bacterial community differences. As shown
in Fig. 4E, the heat-map displayed the relative abundance of the genera,
and the depth of the red/blue color was indicated abundance level. The
similarity between genera was reflected by the cluster trees presented on
the left and top of the heat-map. Additionally, the composition of mi­
crobial communities in fresh samples showed the highest abundance.
Only a few yellow-red stripes representing Shewanella and Acinetobacter
were remained in the control samples. The postbiotics group showed
patterns similar to fresh samples in the early stage from the color of the
cube and top clustering tree. However, there were significant differences
in the relative abundance of A0/3/4 samples compared to other sam­
ples. Based on the above microbial analysis results, we speculated that
postbiotics were effective inhibition in SSOs (especially Acinetobacter
and Pseudomonas).
3.7. Endogenous enzyme activities analysis
Endogenous enzymes played an important role in the decomposition
of fish structural proteins (Wang, Vang, Pedersen, et al., 2011). The
changes in activities of cathepsin B, cathepsin B + L, cathepsin D and
calpain in each group of fish fillets during refrigeration were shown in
Fig. 5. The activities of cathepsin B and calpain showed the tendency
increasing at the begining and declining in late, while cathepsin B + L
and cathepsin D showed an increasing trend. The cathepsin B activity
reached a high of 8.3 mU/g in the control group whereas was only 6.2
mU/g in the postbiotics group on day 1. Moreover, the calpain activity of
all postbiotics groups were lower than that of control group, especially
on the 2nd day, the value of calpain in the postbiotics group reached
0.72 mU/g and decreased by 31.4% compared with the control group. It
appeared that cathepsin B was slightly less sensitive to postbiotics than
calpain. Furthermore, the value of cathepsin B + L and cathepsin D in
the postbiotics group was declined 10.7% and 5.4% compared with
control group by day 5 respectively, however, the postbiotics group
cathepsin B + L was 5.3% higher than cathepsin D on day 5. The value of
cathepsin B + L was slightly higher than cathepsin D during storage,
which was consistented with the research report by Ge et al. (2015).
Based on the above results, the postbiotics group was able to more
effectively inhibited calpain activity, the inhibition mechanism might be
due to the interaction between the cysteine sulfhydryl group of calpain
and the carbonyl group of postbiotics, which led to the inhibition of
calpain and ultimately led to the weakening of calpain activity. Zhang,
Yu, Xu, et al. (2023) also expressed similar inhibitory effect on calpain.
Calpain was one of the potential targets of action to improve proteolytic
induced softening of fillets during storage (Ge, Xu, Jiang, et al., 2016).
Therefore, the decreases in calpain enzyme activity further explained
the better maintenance of texture in the postbiotics group, especially on

6
Z. Zhang et al. Food Chemistry 450 (2024) 139345

Fig. 4. Changes in alpha diversity estimation (A), TVC (B), composition and relative abundance of microbiota at the genus level (C), principal component analysis
(D) and community heat-map analysis at the genus-level (E) of grass carp fillets during storage (B0 to B5 represented control samples were collected at 0–5 days; A0
to A5 represented postbiotics samples were collected at 0–5 days; Fresh was refered to fresh grass carp fillets before soaking treatment).

7
Z. Zhang et al. Food Chemistry 450 (2024) 139345

Fig. 5. Changes in cathepsin B (A), cathepsin B + L (B), cathepsin D (C) and calpain (D) of refrigerated grass carp fillets throughout the storage.

days 2–3 (As shown in Fig. 2). We speculated that postbiotics might react CRediT authorship contribution statement
with enzymes in muscles by penetrating into fish tissues, thereby
inhibiting enzyme activity. Therefore, we concluded that postbiotics had Zhesheng Zhang: Writing – original draft, Methodology, Investi­
a broad spectrum inhibition effect on the main endogenous enzymes in gation, Funding acquisition, Formal analysis, Data curation, Conceptu­
the muscle of grass carp, while the inhibitory effect on calpain was alization. Jinshan Zhao: Writing – review & editing, Supervision,
particularly significant. Project administration, Funding acquisition. Jinhong Zang: Writing –
review & editing, Supervision, Project administration, Methodology,
4. Conclusions Funding acquisition. Chuantao Peng: Resources, Funding acquisition.
Liangtao Lv: Investigation, Formal analysis. Zhaojie Li: Investigation,
In summary, the effects of PostbioYDFF-3 on the quality stability of Formal analysis.
refrigerated fish fillets were investigated from the aspects of endogenous
enzyme activity and the abundance of spoilage microorganisms. The
Declaration of competing interest
postbiotics treatment effectively delayed the deterioration of refriger­
ated grass carp fillets, improved the texture of fish meat, and inhibited
The authors declare that they have no known competing financial
the accumulation of biogenic amines. The protective mode of
interests or personal relationships that could have appeared to influence
PostbioYDFF-3 on the texture stability of fillets was mainly attributed to
the work reported in this paper.
the reductions of the calpain activity and the inhibition of specific
Supplementary data to this article can be found online at https://doi.
spoilage microorganisms, such as Acinetobacter and Pseudomonas. This
org/10.1016/j.foodchem.2024.139345.
study provided preliminary insights into the antibacterial and antioxi­
dant of postbiotics. Further research was needed, for example, to explore
Data availability
the fillets of protein hydrolysis and the deterioration mechanism of fil­
lets caused by spoilage microorganisms, in order to more fully elucidate
Data will be made available on request.
protection mechanism.

Acknowledgements

This research was supported by the National Natural Science

8
Z. Zhang et al.
Foundation of China (Grant No. 32202077); the Breeding Plan of
Shandong Provincial Qingchuang Research Team-Innovation Team of
Functional Plant Protein-Based Food (2021).
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