Study of post-spraying technology on the effects of a multi-strain probiotic viability and growth performance of grass carp (Ctenopharyngodon idella)
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Study of post-spraying technology on the effects of
a multi-strain probiotic viability and growth
performance of grass carp (Ctenopharyngodon
idella)
Nan Luo
Shanghai Ocean University
Ling Wang
Shanghai Ocean University
Zhaoyue Wang
Shanghai Ocean University
Bingbing Xiao
Shanghai Ocean University
Nian Wang
Shanghai Ocean University
Xiaojuan Yu
Shanghai Ocean University
Denghui Wu
Yuehai Co., Ltd
Zengfu Song ( zfsong@shou.edu.cn )
Shanghai Ocean University
Research Article
Keywords: Post-spraying technology, Probiotics culture, Multi-strain probiotic, Viability, Grass carp, Growth
performance
Posted Date: June 13th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1710392/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 1/28Abstract
The aim of the present experiment was to study the feasibility of supplementing a multi-strain probiotic
(MSP, Bacillus subtilis and Lactobacillus plantarum) culture by the technology of post-spraying, the
viability of the MSP in the post-spraying and storage process, growth performance, digestive enzyme
activity, antioxidant capacity and intestinal microbiota of grass carp were assessed. Grass carp (41.4 ±
1.8 g) was fed with a basal diet (control group) and the MSP-basal diet containing 105 CFU/g (T1 group)
or 106 CFU/g (T2 group) probiotics supplemented by the post-spraying technology in 9 cages (100 fish/
cage) for 6 weeks. The population of the probiotics was appraised (Log CFU) for 30 days. The results
revealed that the viability of the B. subtilis and L. plantarum was 98.14% and 98.09% in the process of
post-spraying technology, 99.13% and 99.10% after 30 days of storage respectively. The diet
supplemented MSP significantly improved the percent weight gain (PWG), specific growth rate (SGR) and
survival rate (SR) of grass carp, and decreased the feed conversion ratio (FCR) (p < 0.05). Additionally, a
significant improvement on the digestive enzyme activity and antioxidant capacity was shown in the
MSP groups (p < 0.05). Meanwhile, the MSP beneficially modulated the intestinal microbiota of grass
carp. Overall, these results indicated that the post-spraying technology supplied a feasible and potential
way to supplement the liquid probiotics culture in the aquatic feedstuff industry with high viability to
promote the liquid probiotics application.
Introduction
Grass carp (Ctenopharyngodon idella), one of the major species cultured in freshwater aquaculture, is the
largest annual output worldwide and continues to increase, accounting for more than 10% of total global
aquaculture output [1]. However, the continued development of aquaculture causes a series of farmed
issues. For instance, the long-term abuse of antibiotic drugs has led to increasing resistance to the
aquatic pathogens, inflicting serious harm to aquatic products security and the ecological environment [2,
3]; degradation of germplasm resources causes a decline in the disease resistance of grass carp [4]; and
negligence in the water environment, leading to the deterioration of water quality and an increase in
disease outbreaks [5]. All these issues touch on the growth, immunity and mortality of grass carp, which
in turn affects food safety and aquaculture output and income.
Probiotics have been widely used as dietary additives in aquaculture, with the characteristics of high
security, improving growth, promoting nutrient digestion, enhancing host immunity, modulating the
balance of intestinal microbiota, and improving the aquaculture environment [6–8]. Probiotics are
considered a promising safe and non-polluting alternative to antibiotics [9]. Bacillus, Lactobacillus,
Enterococcus and Saccharomyces have been classified as the main probiotics [10–12]. Probiotics
operate on the host intestine to improve growth and digestion, due to self-secrete digestive enzymes or to
promote the production of digestive enzymes [13, 14]; to inhibit the adhesion and growth of pathogens,
maintain the intestinal microbiota structure and mucosal barrier, and modulate the immune response and
antioxidant levels to enhance the host resistance to disease [15–17]. Furthermore, the synergy in the
multi-strain probiotic (MSP) (mixed probiotics) is a more favorable option for the organism [18].
Page 2/28The effectiveness of probiotics added to the pellet feed is affected by the high temperatures and
pressures of pelleting, an extreme condition unsuitable for the survival of probiotics, posing a
functionality (dosage) issue for probiotics-supplemented feed [19, 20]. Currently, probiotics are protected
by pre-implementing measures such as coating and microencapsulation to reduce losses during the
pelleting process, or manually spraying probiotics after pellet feed processing to improve their viability
[21–23]. While the methods are effective in improving the survival of probiotics, entail additional
industrial steps and cost values. The post-spraying technology, in which heat-sensitive additives such as
enzymes, vitamins and probiotics are sprayed onto the surface of the pellets with atomized before the
feed is bagged and packed, effectively avoids losses during processing and safeguards the activity of the
additives [24, 25]. However, the post-spraying technology to supplement probiotic cultures was not
reported. Thus, this study was conducted to investigate the implemented effectiveness of the post-
spraying technology for supplementing probiotics, as well as the effects of the dietary supplementation
of a self-provisioned MSP on growth performance, digestive enzyme activity, antioxidant capacity, and
intestinal microbiota of grass carp, which will afford another simple supplement way for the use of the
probiotics in the aquatic feedstuff industry.
Material And Methods
Probiotics and diets preparation
The MSP including Bacillus subtilis and Lactobacillus plantarum were preserved in the refrigerator at -80
℃ in our laboratory. The B. subtilis and L. plantarum were incubated in Luria-Bertani broth (LB, Sangon,
China) and De Man Rogosa Sharpe broth (MRS, Thermo, United States) at 37 ℃ for 18 h at 180 rpm.
After incubation, the cultures were cultivated at 37 ℃ for 3 days at 150 rpm. Plate-spreading results
showed that the fermented cultures contained 1.24 × 108 CFU/mL B. subtilis and 2.28 × 108 CFU/mL L.
plantarum.
The preparation of MSP was sprayed onto the surface of the basal diet (Table 1) at 0.2 MPa (pressure)
and a ratio of 1/1000 (culture/ diet) through a post-spraying machine (Liyang Three-dimensional
Automatic Control Equipment Co., China). Two MSP diets were set up: treatment 1 (T1) group diet with
the final MSP concentration adjusted to 105 CFU/g, and treatment 2 (T2) group diet with 106 CFU/g. All
diets were prepared in the Yuehai Co., Ltd (Ningxia Hui Autonomous Region, China) and preserved at
room temperature till used.
Page 3/28Table 1
Ingredients of the basal diet*.
Ingredients Composition (g/ kg)
Fish meal 100
Soybean meal 120
Cottonseed meal 100
Soybean meal 250
Rice bran meal 130
Wheat bran meal 170
Soybean oil 50
Monocalcium phosphate 20
Protein powder 20
L-Lysine 20
Additive 20
Proximate composition
Ash 86.0
Crude protein 324.4
Crude lipid 54.0
* The basal diet was processed in the Yuehai Co., Ltd in Ning Xia Hui Autonomous Region, China
Fish and feeding trial
Grass carp were supplied from a local farm (Ningxia Hui Autonomous Region, China), and acclimatized in
the cage for 2 weeks before the experiments. After adaptation, a total of 900 fish (41.4 ± 1.8 g) were
arbitrarily divided into 9 cages (100 fish/ cage, 2.5m × 2m × 2m) and fed in triplicates with basal diet
(control), T1 and T2 diet for 6 weeks (42 days). The fish were maintained in a farmed pond and fed thrice
daily (10:00 am, 2:00 pm and 6:00 pm).
Sample collection
After 42 days of growth trial, fish were fasted for 24 h priorly, 6 fish were randomly chosen from each
cage, and anesthetized in diluted tricaine methanesulfonate (MS-222, Sigma, United States) at the
concentration of 100 mg/L. Afterward, the flank and ventral surfaces of fish were disinfected using 75%
ethanol. Once the fish were aseptically dissected, the specimens of the total intestine and contents of 3
fish were harvested for microbial diversity detection. A portion of pyloric ceca and foregut were sampled
Page 4/28to assay the activities of digestive enzymes in the other 3 fish. Meanwhile, the remaining midgut was
sampled for antioxidant capacity analysis. All samples were kept at -80 ℃ for subsequent analysis.
Viability of the MSP between culture, post-spraying
machine and diet
The viability of the probiotics was appraised using the plate counting method. Briefly, the culture before
(CBS) and after being sprayed (CAS) by the nozzle of the post-spraying machine was sampled.
Subsequently, the culture was 10-fold serially diluted in sterile 0.9% saline, spread in triplicate onto the LB
and MRS media, and incubated at 37 ℃ for 24 h. Plates with a colony count ranging from 30 to 300 were
selected and used to calculate the colony forming units (CFU) of each probiotic.
The diet was preserved at room temperature after being sprayed with the MSP. Afterward, 10 g of the
MSP-supplemented diet (MPD) was accurately weighed every 3 days until day 30, added to 100 mL of
sterile saline with glass beads, soaked for 20 min, and cultivated at 200 r/min in the shaker machine for
30 min to prepare a suspension. The suspension was 10-fold serially diluted to calculate the CFU.
Growth performance
The fish were individually weighed and measured at the beginning and end of the trial (day 42) to
determine their growth performance. Several growth parameters like initial body weight (IBW), final body
weight (FBW), condition factor (CF), percent weight gain (PWG), survival rate (SR), specific growth rate
(SGR) and feed conversion ratio (FCR) were estimated using the following formula [26]:
weight
CF(\%) = 100 ×
(totallength) 3
FBW − IBW
PWG(\%) = 100 ×
IBW
finalfishnumber
SR(\%) = 100 ×
initialfishnumber
lnFBW − lnIBW
SGR(\%/days) = 100 ×
days
dryfeedconsumed
FCR =
weightgain
Digestive enzyme activity
The intestinal samples in triplicates for each cage were minced and homogenized in 0.9% saline at a ratio
of 1:9 (m/v), and then centrifuged at 3500 r/min for 15 min at 4°C to collect the supernatant, which was
preserved at -80°C for subsequent analyses. The activity of several digestive enzymes (total protease, α-
amylase, lipase and trypsin) was measured using kits (Jiancheng, China).
Page 5/28Antioxidant capacity analysis
According to somebody methods with a little modification [27]. The midgut samples were treated by
section 2.6. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and catalase
(CAT) activity were quantified by the kits (Jiancheng, China) according to the instructions of the
manufacturer.
DNA extraction, PCR amplification and high-throughput sequencing analysis of the intestinal microbiota
The genomic DNA of the intestine with content was extracted using a DNA extraction kit (TIANGEN,
China) following the instructions of the manufacturer, using 338F (5'-ACTCCTACGGGAGGCAGCAG-3')
and 806R (5'-GGACTACHVGGGTWTCTAAT-3') with barcode sequences as primers. The PCR amplification
was performed on the V3-V4 region sequence of the 16S rRNA gene to obtain a PCR product. After
purifying, quantifying and constructing MiSeq libraries, the samples were sequenced on the Illumina
MiSeq platform (Lianchuan Biotechnology, China).
After the sequences and reads were spliced, quality controlled and filtered for chimera, the high-quality
clean reads were obtained. Using the concept of ASVs (Amplicon Sequence Variants), an OTU
(Operational Taxonomic Units) -like the table was constructed to obtain the final feature table for
subsequent analysis [28].
The bioinformatic analysis of the samples was performed by alpha diversity analysis, beta diversity
analysis and species analysis. The richness and uniformity were reflected by the Chao1, Shannon and
Goods coverage indices of alpha diversity. The differences between samples were made by identifying
the distance between any two samples by the PCoA (Principal Co-ordinates Analysis) analysis of beta
diversity. The feature sequences were classified by SILVA
(https://www.arbsilva.de/documentation/release-132/) and NT-16S database, and the species
abundance table of different classification levels was obtained for different species composition and
difference analysis, parameter threshold: confidence > 0.7.
Statistical analysis
The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was
performed using the SPSS program version 23. A value of p < 0.05 was statistically significant and
followed Duncan's multiple comparisons was performed.
Results
Viability of the MSP between culture, post-spraying
machine and diet
Page 6/28During the process of the MSP was sprayed to the diet by the post-spraying machine, the samples of the
probiotics were taken from the culture, post-spraying nozzle and diet, and counted (Table 2). The results
showed the viability of the B. subtilis and L. plantarum before and after the spraying process which was
99.12% and 98.80% of the initial population respectively. Meanwhile, a small loss was also exhibited
between nozzle and diet, with probiotics viability of 99.02% and 99.27%, respectively. Eventually, the
viability of probiotics-supplemented was allowed to reach 98.14% and 98.09% during the entire post-
spraying technology.
Table 2
Effects of the post-spraying technology on the viability of probiotics
CBS (log CFU/mL) CAS (log CFU/mL) MPD (log 1000*×CFU/g)
B. subtilis 8.22 ± 0.04a 8.15 ± 0.05ab 8.07 ± 0.04b
L. plantarum 8.36 ± 0.03a 8.26 ± 0.03ab 8.20 ± 0.08b
Values are mean ± SD log CFU/g for 3 replicates, and values with different shoulder letters in the
same row indicate significant differences (p < 0.05). CBS, culture before being sprayed; CAS, culture
after being sprayed; MPD, MSP-supplemented diet.
*
The ratio of culture and diet was 1/1000.
The post-spraying MSP-supplemented diet was preserved at room temperature for 30 days, in which the
viability of B. subtilis and L. plantarum was in Fig. 1. As the results showed, the viability of the B. subtilis
revealed an increase of 1.76% in the first 3 days, then stabilized, decreased by 1.95% between the 15th
and 18th days, and hereafter stabilized until the 30th day. Eventually, the viability of the B. subtilis
decreased by 0.87% within 1 month. Meanwhile, a similar trend in the viability of L. plantarum was
observed, which increased by 0.38% in the first 3 days, and decreased by 2.68% between the 12th and
15th days, eventually plateaued until day 30. The viability of the L. plantarum decreased by 0.90%
compared to the initial population.
Growth performance
After the 6-week feeding trial, the growth performance of grass carp fed MSP-supplemented diets was
shown in Table 3. Results showed that in the MSP groups, the PWG and SGR were significantly higher
than that in the control group (p < 0.05), and the FCR of the MSP groups was significantly decreased (p <
0.05). Besides, SR was improved with increasing the dosage of probiotic supplementation. The SR of the
T2 group was significantly higher than that of the control group (p > 0.05). However, the CF showed no
significant differences among the different groups (p > 0.05).
Page 7/28Table 3
Effects of dietary post-spraying MSP on the growth performance of grass carp.
Control T1 T2
IBW (g) 41.63 ± 2.22 41.27 ± 0.97 41.27 ± 2.68
FBW (g) 58.57 ± 3.76 62.37 ± 1.55 63.93 ± 4.60
SR (%) 73.67 ± 4.93b 76.33 ± 4.51ab 84.00 ± 4.58 a
PWG (%) 40.63 ± 2.34c 51.09 ± 0.78b 54.88 ± 1.08a
SGR (%/d) 0.81 ± 0.04c 0.98 ± 0.01b 1.04 ± 0.02a
CF (g× cm− 3) 1.45 ± 0.05 1.46 ± 0.02 1.42 ± 0.05
FCR (g / g) 2.03 ± 0.19a 1.62 ± 0.05b 1.52 ± 0.12b
Values are mean ± SD for 3 replicates, and values with different shoulder letters in the same row
indicate significant differences (p < 0.05). IBW, initial body weight; FBW, final body weight; CF,
condition factor; PWG, percent weight gain; SR, survival rate; SGR, specific growth rate; FCR, feed
conversion ratio.
Digestive enzyme activity
The results were shown that the TP (Fig. 2A) and the activities of α-AMS (Fig. 2B), LPS (Fig. 2C) and TPS
(Fig. 2D) in the MSP-supplemented groups were significantly increased in comparison with the control
group (p < 0.05). Meanwhile, the TP and the activities of LPS and TPS in the T2 group were significantly
increased in comparison with that in the T1 group (p < 0.05). However, there were no significant
differences in the α-AMS between the T1 group and the T2 group (p > 0.05).
Antioxidant capacity
The grass carp was fed two dosages of the post-spraying MSP-supplemented diet, the results of
antioxidant capacity were shown in Fig. 3. The activities of SOD and CAT were significantly increased,
and MDA content was significantly decreased in the MSP groups relative to the control group (p < 0.05).
The CAT of the T2 group was significantly higher than that of the T1 group (p < 0.05). However, there were
no significant changes in SOD and MAD between the two MSP groups (p > 0.05).
Intestine microbial diversity
After the high-quality screening, each of the samples obtained valid tags of 71066–81308, and the
effective ratio was 86.30% − 94.84%. The alpha diversity of intestinal samples was reflected by Chao1
(Fig. 4A), Shannon (Fig. 4B) and Goods coverage (Fig. 4C) indices. Compared with the control group, the
Chao1 and the Shannon index reflected species richness and evenness were reduced in the two treatment
groups. Conversely, the Goods coverage index representing microbial coverage was higher in the MSP
Page 8/28groups than that in the control group. Furthermore, the PCoA (Fig. 4D) executed to represent the similarity
between microbial communities was used for the analysis of beta diversity. Except for one sample in the
T2 group, there were obvious distances between the remaining samples in the two treatment groups and
the samples in the control group.
Intestine microbial composition
To examine the composition of gut microbiota at the phylum and genus levels in grass carp in Control, T1
and T2 groups, the percentage of relative abundance of intestinal flora was presented in Fig. 5. At the top
30 most abundant phyla level, the dominant phyla in all samples were Bacteroidetes, Firmicutes,
Fusobacteria and Proteobacteria. Compared with the control group, the abundance of Bacteroidetes
(Control, 7.21%; T1, 40.87%; and T2, 40.11%) and Firmicutes (7.15%, 24.39%, 19.95%) in the two MSP
groups was significantly increased (p < 0.05). However, the abundance of Proteobacteria (28.87%, 9.94%,
10.90%), Actinobacteria (15.18%, 1.43%, 6.03%), Chloroflexi (6.54%, 0.32%, 1.63%), Cyanobacteria (4.08%,
0.32%, 1.30%) and Patescibacteria (2.10%, 0.25%, 0.56%) in the two MSP groups was obviously
decreased in comparison with the control group (p < 0.05).
At the top 30 most abundant of genus level, Bacteroides and Cetobacterium were the dominant genus in
all groups. Two MSP groups compared with the control group, the relative abundance of Bacteroides
(5.92%, 40.07%, 39.11%), Lachnospiraceae_unclassified (1.66%, 8.57%, 7.22%), Erysipelatoclostridium
(0.93%, 4.88%, 3.30%), ZOR0006 (0.41%, 3.86%, 1.42%) and Clostridium (0.25%, 2.10%, 2.48%) was
significantly increased (p < 0.05). Meanwhile, the relative abundance of Cetobacterium (9.99%, 15.69%,
11.78%) in the T1 group was significantly increased in comparison with the T2 group and control group
(p < 0.05). However, the PeM15_unclassified (8.08%, 0.64%, 2.79%), Pirellulaceae_unclassified (5.52%,
0.53%, 1.91%) and Caldilineaceae_unclassified (4.63%, 0.22%, 1.23%) in two MSP groups were
significantly decreased compared with that in control group (p < 0.05). In addition, the abundance of
Fusobacterium (3.08%, 2.52%, 0.66%) was obviously decreased in only the T2 group (p < 0.05).
The community composition data of the 30 highest relative abundances at the class level were clustered
according to the abundance distribution of taxa or the similarity between samples and presented by a
heat map in Fig. 5C. At the class level, differences in gut microbiota were shown between control and
treatment groups. For instance, Chlamydiae, Alphaproteobacteria and Gammaproteobacteria were more
abundant in the control group, while Clostridia, Bacterodia and Deltaproteobacteria were more abundant
in the treatment group. Additionally, Spirochaetia, Fusobacteriia and Thermoanaerobaculia were the only
most abundant in the T1 group.
Discussion
Probiotics, one of the beneficial feed additives in aquaculture, have a significant impact on disease
resistance, growth performance, immune response, as well as other positive effects on cultured hosts
[29]. In the present study, B. subtilis and L. plantarum were the most widely used probiotics with the most
Page 9/28comprehensive effects in aquaculture, nowadays [6]. On the single-strain, B. subtilis or L. plantarum and
their intracellular products showed significant improvements in growth and health (survival, digestion,
immunity, antioxidant capacity, and intestine) of aquatic animals [30–32]. The supplementation of MSP
to feeds was more beneficial to the host in some respects than single-strain probiotics (SSP) [33, 34].
However, the MSP on B. subtilis and L. plantarum only was reported once on Nile tilapia (Oreochromis
niloticus), and not was studied on grass carp [35]. Others were combinations between Lactobacillus and
Bacillus except for B. subtilis or L. plantarum [36–38]. It was found that Bacillus could promote the
growth of Lactobacillus, including a study that demonstrated that B. subtilis could improve the survival of
L. plantarum [39–41]. The present study smoothly used their synergy, which also was the reason for
higher viability in the initial culture of L. plantarum in the MSP (Table 2).
Probiotics were added to pellet feeds in a variety of forms. Traditionally, probiotics were pelleted together
with feed ingredients, which have been eliminated due to the losses of high temperature, high pressure
and mechanization. Currently, the most commonly used form of probiotics was to artificially spray
suspensions or mix frozen-dry powder before using finished pellet feed, which avoided damage from the
production process [42, 43]. Not optimistically, the form, low yield, was only suitable for small batch
feeding or experimental studies on the performance of probiotics [44]. With the in-depth studies on
probiotics, microencapsulation technology was verified to be effective in improving the viability of
probiotics, with the advantages of resistance to preservation, high temperature, high pressure and acid,
but the particular requirements and higher price of embedding materials increased the cost in the mass
supplementation of feed [21, 45, 46]. The emergence of heat-killed probiotics, potentially with little effect
on high temperature and pressure in feed processing, was less well studied for certain therapeutic yet
[47]. Nowadays, the issues including feed raw material scarcity, high supply and food safety restricted the
cost of feed processing [48–50]. The post-spray technology to supplement probiotics in the present paper
effectively addressed issues of scale, commercialization, stability and security. Moreover, the technology
was well-established, with a straightforward sprayed operation, a diverse selection of probiotic cultures,
and more commercial value.
Pressure and temperature were implicated in microbial growth and survival [51–53]. Bacillus, which
produced spores with superior thermal stability and viability [54, 55]. Lactobacillus was labeled with a
characteristic ability to adhere but was poorly resistant to high temperatures [56, 57]. In the present study,
the viability of MSP (B. subtilis and L. plantarum) in the culture among the nozzle was 99.12% and
98.80% respectively (Table 2). Spraying pressure was probably the primary factor of probiotic loss. B.
subtilis with spores exhibited higher viability. The temperature (< 50°C) showed minimal impact on the
probiotics as the proximity of the spraying process to the feeds packaging machine and the similar
viability of both probiotics. The viability of B. subtilis and L. plantarum between nozzle and feed was
99.02% and 99.27% separately. After the culture was sprayed-out, the angle and velocity at which the
pellet feeds fell was the principal cause of probiotics wastage [58]. During the spraying process of
probiotic culture, the feed velocity was rapid, which caused a decrease in the capability of the sprayed
culture to adhere to the surface of the pellet feeds [59, 60]. Moreover, the droplets drifted due to airflow,
thus reducing the amount of contact with the feed surface [61]. The adhesion of L. plantarum allowed for
Page 10/28higher viability. Three commercial B. subtilis and feed were extruded into the diet at three temperatures
(75°C, 85°C and 90°C) and the probiotics recovery was over 90% (95.3%, 96.5% and 92.9%) in all cases
[62]. In the present study, B. subtilis and L. plantarum were obtained at excellent viability of 98.14% and
98.09% respectively after the completion. Certainly, the post-spraying technology carried the equally
vigorous probiotics reduced the probiotics-added species restriction. In addition, the metabolites in the
culture-supplemented were potential merits for host growth and immunity [63–65].
Microorganisms survive on a surface with a little moisture and some nutrients [66]. However, individuals
in a population of a single or multiple bacterial species competed with each other when nutrients were
limited [67]. In addition, microbial populations adapt rapidly to new environments, even under constant
conditions [68]. The pellet feed contained nutrients such as protein, amino acids, fat and a small amount
of water [69]. In the present study, B. subtilis and L. plantarum showed an increasing trend in viability on
the diet surface for the first 3 days, with slight fluctuations from the 3rd day to the 18th day and
subsequent equalization until the 30th day (Fig. 1). The variations in viability were due to the constant
adaptation, competition and multiplication of probiotics in the new environment. Notably, the viability of
B. subtilis decreased by 0.87% when preserved at room temperature for 30 days, due to the greater
viability of Bacillus. Likewise, the decrease of L. plantarum was 0.90%. Similarly, the probiotics diet was
obtained by mixing feed with 5% (w/w) water and 1% (w/w) freeze-dried pellets of probiotics and left at
25°C for 15 days with no significant decrease in cell survival [70]. The microencapsulated probiotics
(Bacillus spp. and S. cerevisiae) with commercial feed were sent for extrusion processing at 90°C
(4.20×107 CFU/g, 3.18×108 CFU/g) and showed probiotics counts (Log CFU) decreased by 4.6% and 2.2%
compared to the 45th day (1.80×107 CFU/g, 2.05×108 CFU/g), and improved the zootechnical and health
aspects of juvenile Nile tilapia [71]. Obviously, the post-spraying MSP feed performed better in terms of
survival. Meanwhile, most aquaculture farmers consumed a batch of diet within 30 days, which was
more conducive to the viability and influence of probiotics in the present study.
Previous studies have demonstrated that the supplementation of MSP in feed improved the growth
performance of aquatic animals, for instance, Nile tilapia, catfish (Pangasianodon hypophthalmus),
common carp (Cyprinus carpio), Rohu (Labeo rohita), Asian seabass (Lates calcarifer), Litopenaeus
vannamei, Chinese mitten crab (Eriocheir sinensis) [72–78]. The present study showed that diet
supplemented 105 and 106 CFU/g MSP improved the PWG, SGR, FCR and SR of grass carp, with the 106
CFU/g group performing better on them (Table 3). The present study was consistent with a report which
found the MP1.68 (Bacillus spp. 5×106 CFU/g and Lactobacillus 1.26×106 CFU/g) fed to grass carp
significantly increased WG and SGR as well as decreased FCR[79]. Analogously, diets supplemented with
probiotics (L. plantarum NIOFSD018 and B. subtilis NIOFSD017, 1×107 CFU/g) fed to Nile tilapia,
obviously improved SGR and FCR [35]. Nevertheless, the improvements in the growth performance of
tilapia fed with some combination of probiotics (B. subtilis, L. plantarum, L. rhamnosus, L. acidophilus
and L. delbrueckii, 2×105 CFU/g) were insignificant [80]. Growth performance was improved due to
probiotics enhancing the attractiveness and palatability of the diet, which was observed in a study to be
more dynamic for fish to consume [81]. At the same time, lower FCR values indicated that fish absorbed
Page 11/28nutrients from diet more efficiently [82]. Grass carp survival in cultured ponds was restricted by multiple
factors, such as water conditions, weather and disease (enteritis, significant cause) [83, 84]. Both B.
subtilis and L. plantarum were found to significantly increase the survival of Japanese eel (Anguilla
japonica) [85]. In the present study, the improvement of the SR in the MSP groups presumably
strengthened the adaptability and resistance of grass carp in the cages.
Digestion and absorption of nutrients in the intestine were influential factors of growth. Bacillus and
Lactobacillus self-secreted digestive enzymes, including proteases, lipases, amylases and cellulases,
which were regarded as the major enzymes for nutrient digestion and absorption in the intestine, thereby
promoting growth and feed utilization[86, 87]. In the present study, grass carp were fed 105 or 106 CFU/g
of MSP, accompanied by effectively increasing intestinal protease, lipase and amylase activities, which
provided favorably for growth (Fig. 2). Moreover, consistent with the studies on aquatic animals such as
common carp, roach (Rutilus rutilus caspicus) and Caspian white fish (Rutilus frisii kutum), growth and
digestion were improved to varying degrees[88–90].
In the antioxidant defense of the host, SOD regulated the levels of superoxide and hydrogen peroxide
produced by cells to maintain a healthy cellular state [91]. CAT scavenged free radicals to curtail harmful
impacts and supported appropriate immune function [92]. MAD, an indicator of lipid peroxidation in cell
membranes, excessive levels of MAD accelerated cell and tissue damage [93]. Probiotics presented
antioxidant activity and reduced damages caused by oxidation[94]. Previous studies demonstrated that
probiotics increased the activity of antioxidant enzymes in serum and liver, as well as up-regulated the
expression of intestinal antioxidant-related parameters in aquatic animals [95–98]. However, there were a
few assessments of probiotics that modulated the intestinal antioxidant capacity of the aquatic animal.
A study proved that the SOD and CAT in the intestine of crucian carp (Carassius auratus) were
significantly increased under high-density culture using the compound probiotics (106-108 CFU/mL) [99].
Exposed to the luminal environment and exogenous food, the intestine was susceptible to oxidative
damage from oxidative inducers in the lumen [100]. The present study found that the midgut SOD and
CAT were significantly increased while MDA was significantly decreased in the treated group, indicating
that the MSP enhanced the intestinal antioxidant level and physiological state of grass carp (Fig. 3).
The fish intestinal microbiota, which performed essential roles in protective, nutritional, endocrine,
neurological and physiological functions, along with the intestinal epithelium, formed the main barrier to
host nutrient absorption and immune protection [101–103]. Alpha diversity refers to the diversity within a
particular environment or ecosystem, which was primarily used to reflect species richness and evenness
[104]. The alpha diversity (Chao1 and Shannon) of the group fed MSP in the present study was lower
than that of the control group, but not significantly different, suggesting that probiotics had little effect on
the diversity of grass carp intestinal microbiota (Fig. 4). Equally, the Chao1 index in gut microorganisms
of Chinese Perch (Siniperca chuatsi) fed B. subtilis was lower but not dramatically disparity from the
control [105]. On the one hand, the colonization of probiotics in the intestine inhibited the growth of
pathogenic; on the other hand, the dosage of the supplemented probiotics was excessive, affecting the
diversity of intestinal microbiota [76]. Beta diversity was a better process-level understanding of
Page 12/28biodiversity change and its consequences for ecosystems [106]. PCoA plot was used to further assess
and visualization of beta diversity. The present study observed that the treatment group was separated
from the control group in the PCoA plot, which demonstrated that grass carp fed MSP diet altered the
structure of the intestinal microbial composition.
The intestinal microbial dates of all groups in the present exploration were consistent with the studies
which have confirmed that Proteobacteria, Firmicutes and Bacteroidetes were the main dominant
bacterial phyla in the intestine of grass carp [107–109]. The diet supplemented probiotics fed to aquatic
animals provided varying degrees of changes in the relative abundance of intestinal microbiota [110,
111]. Bacteroidetes, the cornerstone of a stable healthy intestinal environment, was associated with
nutrient digestion and metabolic function, as well as participated in the immune modulation and the
regulation of the gut-brain-axis [112]. Furthermore, Bacteroidetes played an important role in host
development, which was often accompanied by an increase [113]. Butyrate, produced by Bacteroidetes,
was the main microbiota-derived intestinal mucosal immunity regulator and the best functional marker of
the healthy mature anaerobic intestinal microbiota [114]. Firmicutes, a positive indicator of intestinal
health, contained a variety of probiotics such as Bacillus, Streptococcus, Lactobacillus, Leuconostoc, and
others that were used as beneficial aquatic animal feed additives [115, 116]. An increased incidence of
the Proteobacteria was a sign of an intestine microbial dysbiosis and a potential diagnostic criterion for
disease [117]. Cyanobacteria endangered the digestive health of aquatic animals through the production
of cyanotoxins [118]. Notably, compared to the control group, the treatment group in the present study
showed a substantial increase in the abundance of Bacteroidetes and Firmicutes and a significant
decrease in Proteobacteria and Cyanobacteria at the phylum taxonomic level, which showed that grass
carp fed MSP exhibited a more balanced intestinal microbial community structure, lower prevalence of
disease and better intestinal health and growth trends (Fig. 5). Actinobacteria and Chloroflexi were
regarded as beneficial phyla in the intestinal microbiota [119, 120]. The possible reasons for the reduced
abundance of Actinobacteria and Chloroflexi were uncertain in the present study, but there were no visible
negative effects in terms of the results. At the genus taxonomic level, Clostridium generated butyric acid
for regeneration and repair of the intestinal epithelium, alleviated inflammation, and regulated a healthy
microecological environment in the intestine [121]. Cetobacterium produced large amounts of vitamin
B12, which promoted carbohydrate metabolism [122]. The increased abundance of Cetobacterium and
Clostridium in the MSP groups improved the intestinal self-repair and metabolic capacity of grass carp in
the present study.
In conclusion, the present study demonstrated the feasibility of supplementing probiotics by the post-
spraying technology and the beneficial effects of MSP. The results indicated that the post-spraying
technology showed a little influence on the viability of the feed supplemented probiotics and subsequent
storage. The dietary supplemented MSP improved the growth performance, digestive enzyme activity and
antioxidant capacity of grass carp as well as modulated the host intestinal microbiota. The MSP showed
the potential to strengthen the survivability of fish. Thus, these findings present a promising technology
and combination for feed-supplemented probiotics in aquaculture.
Page 13/28Declarations
Acknowledgments
This work was supported by Shanghai Aquatic Collaborative Innovation Center of Breeding and Genetics
(ZF1206), and Yuehai Co., Ltd in Ning Xia Hui Autonomous Region, China.
Author’s Contribution
Nan Luo and Ling Wang contributed equally to this work. All authors contributed to the study
methodology. Nan Luo and Ling Wang prepared the material, and were a major contributor in writing the
manuscript. Nian Wang and Bingbing Xiao analyzed and visualized the data. Zhaoyue Wang and
Xiaojuan Yu investigated and collected resources. Denghui Wu and Zengfu Song performed the
supervision and project administration. All authors reviewed, edited commented and approved the
manuscript.
Conflict of Interest
The authors declare no competing interests.
Funding
This work was partially (or fully) sponsored by Shanghai Aquatic Collaborative Innovation Center of
Breeding and Genetics (ZF1206), China, and Yuehai Co., Ltd in Ning Xia Hui Autonomous Region, China.
References
1. FAO (2020).The State of World Fisheries and Aquaculture 2020: Sustainability in action. FAO. Rome.
https://doi.org/10.4060/ca9229en
2. Santos L, Ramos F (2018) Antimicrobial resistance in aquaculture: Current knowledge and
alternatives to tackle the problem. Int J Antimicrob Agents 52: 135-143.
https://doi.org/10.1016/j.ijantimicag.2018.03.010.
3. Reverter M, Sarter S, Caruso D et al (2020) Aquaculture at the crossroads of global warming and
antimicrobial resistance. Nat Commun 11. https://doi.org/10.1038/s41467-020-15735-6.
4. Magnadottir B (2010) Immunological control of fish diseases. Mar Biotechnol (NY) 12: 361-379.
https://doi.org/10.1007/s10126-010-9279-x.
5. Ahmad A, Abdullah SRS, Abu Hasan H et al (2021) Aquaculture industry: Supply and demand, best
practices, effluent and its current issues and treatment technology. J Environ Manage 287.
https://doi.org/10.1016/j.jenvman.2021.112271.
6. El-Saadony MT, Alagawany M, Patra AK et al (2021) The functionality of probiotics in aquaculture:
An overview. Fish Shellfish Immun 117: 36-52. https://doi.org/10.1016/j.fsi.2021.07.007.
Page 14/287. Mondal S, Mondal D, Mondal T et al (2022) Application of probiotic bacteria for the management of
fish health in aquaculture. Academic Press, pp. 351-378. https://doi.org/10.1016/B978-0-323-85624-
9.00024-5.
8. Ringø E (2020) Probiotics in shellfish aquaculture. Aquaculture and Fisheries 5: 1-27.
https://doi.org/10.1016/j.aaf.2019.12.001.
9. Assefa A, Abunna F (2018) Maintenance of Fish Health in Aquaculture: Review of Epidemiological
Approaches for Prevention and Control of Infectious Disease of Fish. Vet Med Int 2018: 5432497.
https://doi.org/10.1155/2018/5432497.
10. Anokyewaa MA, Amoah K, Li Y et al (2021) Prevalence of virulence genes and antibiotic
susceptibility of Bacillus used in commercial aquaculture probiotics in China. Aquacult Rep 21.
https://doi.org/10.1016/j.aqrep.2021.100784.
11. Martinez Cruz P, Ibanez AL, Monroy Hermosillo OA et al (2012) Use of probiotics in aquaculture. ISRN
microbiology 2012: 916845. https://doi.org/10.5402/2012/916845.
12. Wang AR, Ran C, Wang YB et al (2019) Use of probiotics in aquaculture of China-a review of the past
decade. Fish Shellfish Immun 86: 734-755. https://doi.org/10.1016/j.fsi.2018.12.026.
13. Olmos Soto J (2017) Bacillus Probiotic Enzymes: External Auxiliary Apparatus to Avoid Digestive
Deficiencies, Water Pollution, Diseases, and Economic Problems in Marine Cultivated Animals.
Advances in food and nutrition research 80: 15-35. https://doi.org/10.1016/bs.afnr.2016.11.001.
14. Nunes CS (2018) Probiotics and enzymes in the gastrointestinal tract. Academic Press, pp. 413-427.
https://doi.org/10.1016/B978-0-12-805419-2.00021-6.
15. De BC, Meena DK, Behera BK et al (2014) Probiotics in fish and shellfish culture: immunomodulatory
and ecophysiological responses. Fish Physiol Biochem 40: 921-971.
https://doi.org/10.1007/s10695-013-9897-0.
16. Kuebutornye FKA, Abarike ED, Lu YS et al (2020) Mechanisms and the role of probiotic Bacillus in
mitigating fish pathogens in aquaculture. Fish Physiol Biochem 46: 819-841.
https://doi.org/10.1007/s10695-019-00754-y.
17. Amoah K, Huang QC, Tan BP et al (2019) Dietary supplementation of probiotic Bacillus coagulans
ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response,
and disease confrontation of Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immun 87:
796-808. https://doi.org/10.1016/j.fsi.2019.02.029.
18. Kwoji ID, Aiyegoro OA, Okpeku M et al (2021) Multi-Strain Probiotics: Synergy among Isolates
Enhances Biological Activities. Biology-Basel 10. https://doi.org/10.3390/biology10040322.
19. Kumar P, Libchaber A (2013) Pressure and Temperature Dependence of Growth and Morphology of
Escherichia coli: Experiments and Stochastic Model. Biophys J 105: 783-793.
https://doi.org/10.1016/j.bpj.2013.06.029.
20. Kechagia M, Basoulis D, Konstantopoulou S et al (2013) Health benefits of probiotics: a review. ISRN
Nutr 2013: 481651. https://doi.org/10.5402/2013/481651.
Page 15/2821. Yin M, Yuan Y, Chen M et al (2022) The dual effect of shellac on survival of spray-dried Lactobacillus
rhamnosus GG microcapsules. Food Chemistry: 132999.
https://doi.org/10.1016/j.foodchem.2022.132999.
22. Apiwattanasiri P, Charoen R, Rittisak S et al (2022) Co-encapsulation efficiency of silk sericin-
alginate-prebiotics and the effectiveness of silk sericin coating layer on the survival of probiotic
Lactobacillus casei. Food Biosci 46. https://doi.org/10.1016/j.fbio.2022.101576.
23. Wang AY, Lin J, Zhong QX (2021) Spray-coating as a novel strategy to supplement broiler feed pellets
with probiotic Lactobacillus salivarius NRRL B-30514. Lwt-Food Sci Technol 137.
https://doi.org/10.1016/j.lwt.2020.110419.
24. Fernandes P (2010) Enzymes in food processing: a condensed overview on strategies for better
biocatalysts. Enzyme Res 2010: 862537. https://doi.org/10.4061/2010/862537.
25. Encarnação P (2016) Functional feed additives in aquaculture feeds. Academic Press, San Diego, pp.
217-237. https://doi.org/10.1016/B978-0-12-800873-7.00005-1.
26. Ma LL, Zhang JM, Kaneko G et al (2022) Growth performance, intestinal microbiota and immune
response of grass carp fed isonitrogenous and isoenergetic diets containing faba bean extracts.
Aquacult Rep 22. https://doi.org/10.1016/j.aqrep.2021.100924.
27. Zhang FL, Hao Q, Zhang QS et al (2022) Influences of dietary Eucommia ulmoides leaf extract on the
hepatic lipid metabolism, inflammation response, intestinal antioxidant capacity, intestinal
microbiota, and disease resistance of the channel catfish (Ictalurus punctatus). Fish Shellfish
Immunol 123: 75-84. https://doi.org/10.1016/j.fsi.2022.02.053.
28. Blaxter M, Mann J, Chapman T et al (2005) Defining operational taxonomic units using DNA barcode
data. Philos T R Soc B 360: 1935-1943. https://doi.org/10.1098/rstb.2005.1725.
29. Puvanasundram P, Chong CM, Sabri S et al (2021) Multi-strain probiotics: Functions, effectiveness
and formulations for aquaculture applications. Aquacult Rep 21.
https://doi.org/10.1016/j.aqrep.2021.100905.
30. Li YZ, Yang YM, Song LM et al (2021) Effects of dietary supplementation of Lactobacillus plantarum
and Bacillus subtilis on growth performance, survival, immune response, antioxidant capacity and
digestive enzyme activity in olive flounder (Paralichthys olivaceus). Aquaculture and Fisheries 6: 283-
288. https://doi.org/10.1016/j.aaf.2020.10.006.
31. Xue JJ, Shen KK, Hu Y et al (2020) Effects of dietary Bacillus cereus, B. subtilis, Paracoccus
marcusii, and Lactobacillus plantarum supplementation on the growth, immune response,
antioxidant capacity, and intestinal health of juvenile grass carp (Ctenopharyngodon idellus).
Aquacult Rep 17. https://doi.org/10.1016/j.aqrep.2020.100387.
32. Giri SS, Sen SS, Chi C et al (2015) Effects of intracellular products of Bacillus subtilis VSG1 and
Lactobacillus plantarum VSG3 on cytokine responses in the head kidney macrophages of Labeo
rohita. Fish Shellfish Immun 47: 954-961. https://doi.org/10.1016/j.fsi.2015.10.033.
33. Wang YC, Hu SY, Chiu CS et al (2019) Multiple-strain probiotics appear to be more effective in
improving the growth performance and health status of white shrimp, Litopenaeus vannamei, than
Page 16/28single probiotic strains. Fish Shellfish Immun 84: 1050-1058.
https://doi.org/10.1016/j.fsi.2018.11.017.
34. Lin HL, Shiu YL, Chiu CS et al (2017) Screening probiotic candidates for a mixture of probiotics to
enhance the growth performance, immunity, and disease resistance of Asian seabass, Lates
calcarifer (Bloch), against Aeromonas hydrophila. Fish Shellfish Immun 60: 474-482.
https://doi.org/10.1016/j.fsi.2016.11.026.
35. Essa MA, El-Serafy SS, El-Ezabi MM et al (2010) Effect of Different Dietary Probiotics on Growth,
Feed Utilization and Digestive Enzymes Activities of Nile Tilapia, Oreochromis niloticus. The Arabian
Aquaculture Society 5: 143-162
36. Aly SM, Ahmed YAG, Ghareeb AAA et al (2008) Studies on Bacillus subtilis and Lactobacillus
acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica
(Oreochromis niloticus) to challenge infections. Fish Shellfish Immun 25: 128-136.
https://doi.org/10.1016/j.fsi.2008.03.013.
37. Seenivasan C, Radhakrishnan S, Shanthi R et al (2014) Effect of Lactobacillus sporogenes on
survival, growth, biochemical constituents and energy utilization of freshwater prawn
Macrobrachium rosenbergii post larvae. The Journal of Basic & Applied Zoology 67: 19-24.
https://doi.org/10.1016/j.jobaz.2013.12.002.
38. Parthasarathy R, Ravi D (2011) Probiotic bacteria as growth promoter and biocontrol agent against
Aeromonas hydrophila in Catla catla (Hamilton, 1822). Indian J Fish 58: 87-93
39. Zhang YR, Xiong HR, Guo XH (2014) Enhanced viability of Lactobacillus reuteri for probiotics
production in mixed solid-state fermentation in the presence of Bacillus subtilis. Folia Microbiol 59:
31-36. https://doi.org/10.1007/s12223-013-0264-4.
40. Horie M, Koike T, Sugino S et al (2018) Evaluation of probiotic and prebiotic-like effects of Bacillus
subtilis BN on growth of lactobacilli. J Gen Appl Microbiol 64: 26-33.
https://doi.org/10.2323/jgam.2017.03.002.
41. Yu T, Kong J, Zhang L et al (2019) New crosstalk between probiotics Lactobacillus plantarum and
Bacillus subtilis. Sci Rep-Uk 9. https://doi.org/10.1038/s41598-019-49688-8.
42. Guo GX, Li KX, Zhu QH et al (2022) Improvements of immune genes and intestinal microbiota
composition of turbot (Scophthalmus maximus) with dietary oregano oil and probiotics. Aquaculture
547. https://doi.org/10.1016/j.aquaculture.2021.737442.
43. Kolanchinathan P, Kumari PR, Raja K et al (2022) Analysis of feed composition and growth
parameters of Penaeus monodon supplemented with two probiotic species and formulated diet.
Aquaculture 549. https://doi.org/10.1016/j.aquaculture.2021.737740.
44. Tang XQ, Ma S, Sun LL et al (2022) Isolation, identification, and positive effects of potential
probiotics on Carassius auratus. Aquaculture 548.
https://doi.org/10.1016/j.aquaculture.2021.737668.
45. Olivares A, Silva P (2019) Viability dataset on microencapsulated probiotics: Sodium alginate
viscosity effect. Data Brief 27: 104735. https://doi.org/10.1016/j.dib.2019.104735.
Page 17/2846. Rokka S, Rantamaki P (2010) Protecting probiotic bacteria by microencapsulation: challenges for
industrial applications. Eur Food Res Technol 231: 1-12. https://doi.org/10.1007/s00217-010-1246-2.
47. Tran NT, Yang W, Nguyen XT et al (2022) Application of heat-killed probiotics in aquaculture.
Aquaculture 548. https://doi.org/10.1016/j.aquaculture.2021.737700.
48. Behnke KC (1996) Feed manufacturing technology: Current issues and challenges. Anim Feed Sci
Tech 62: 49-57. https://doi.org/10.1016/S0377-8401(96)01005-X.
49. Béné C, Arthur R, Norbury H et al (2016) Contribution of Fisheries and Aquaculture to Food Security
and Poverty Reduction: Assessing the Current Evidence. World Development 79: 177-196.
https://doi.org/10.1016/j.worlddev.2015.11.007.
50. Boyd CE (2015) Overview of aquaculture feeds: Global impacts of ingredient use. Woodhead
Publishing, Oxford, pp. 3-25. https://doi.org/10.1016/B978-0-08-100506-4.00001-5.
51. Chai HE, Sheen S (2021) Effect of high pressure processing, allyl isothiocyanate, and acetic acid
stresses on Salmonella survivals, storage, and appearance color in raw ground chicken meat. Food
Control 123. https://doi.org/10.1016/j.foodcont.2020.107784.
52. Giannoglou M, Katsaros G, Moatsou G et al (2019) Effect of high hydrostatic pressure treatment on
the viability and acidification ability of lactic acid bacteria. Int Dairy J 96: 50-57.
https://doi.org/10.1016/j.idairyj.2019.04.012.
53. Ostlie HM, Treimo J, Narvhus JA (2005) Effect of temperature on growth and metabolism of
probiotic bacteria in milk. Int Dairy J 15: 989-997. https://doi.org/10.1016/j.idairyj.2004.08.015.
54. Ramlucken U, Lalloo R, Roets Y et al (2020) Advantages of Bacillus-based probiotics in poultry
production. Livest Sci 241. https://doi.org/10.1016/j.livsci.2020.104215.
55. Ohkubo Y, Uchida K, Motoshima H et al (2019) Microbiological safety of UHT milk treated at 120
degrees C for 2 s, as estimated from the distribution of high-heat-resistant Bacillus cereus in dairy
environments. Int Dairy J 91: 36-40. https://doi.org/10.1016/j.idairyj.2018.12.011.
56. Tropcheva R, Georgieva R, Danova S (2011) Adhesion Ability of Lactobacillus Plantarum Ac131.
Biotechnol Biotec Eq 25: 121-124. https://doi.org/10.5504/bbeq.2011.0123.
57. Matejcekova Z, Spodniakova S, Dujmic E et al (2019) Modelling growth of Lactobacillus plantarum
as a function of temperature: Effects of media. J Food Nutr Res-Slov 58: 125-134
58. Klinkov SV, Kosarev VF, Shikalov VS (2019) Influence of nozzle velocity and powder feed rate on the
coating mass and deposition efficiency in cold spraying. Surf Coat Tech 367: 231-243.
https://doi.org/10.1016/j.surfcoat.2019.04.004.
59. Schmidt T, Assadi H, Gartner F et al (2009) From Particle Acceleration to Impact and Bonding in Cold
Spraying. J Therm Spray Techn 18: 794-808. https://doi.org/10.1007/s11666-009-9357-7.
60. Schmidt T, Gartner F, Assadi H et al (2006) Development of a generalized parameter window for cold
spray deposition. Acta Mater 54: 729-742. https://doi.org/10.1016/j.actamat.2005.10.005.
61. Al Heidary M, Douzals JP, Sinfort C et al (2014) Influence of spray characteristics on potential spray
drift of field crop sprayers: A literature review. Crop Protection 63: 120-130.
Page 18/28https://doi.org/10.1016/j.cropro.2014.05.006.
62. Amerah AM, Quiles A, Medel P et al (2013) Effect of pelleting temperature and probiotic
supplementation on growth performance and immune function of broilers fed maize/soy-based
diets. Anim Feed Sci Tech 180: 55-63. https://doi.org/10.1016/j.anifeedsci.2013.01.002.
63. Kostelac D, Geric M, Gajski G et al (2021) Lactic acid bacteria isolated from equid milk and their
extracellular metabolites show great probiotic properties and anti-inflammatory potential. Int Dairy J
112. https://doi.org/10.1016/j.idairyj.2020.104828.
64. Kim YO, Mahboob S, Viayaraghavan P et al (2020) Growth promoting activity of Penaeus indicus by
secondary metabolite producing probiotic bacterium Bacillus subtilis isolated from the shrimp gut. J
King Saud Univ Sci 32: 1641-1646. https://doi.org/10.1016/j.jksus.2019.12.023.
65. Chávarri M, Diez-Gutiérrez L, Marañón I et al (2021) Secondary Metabolites From Probiotic
Metabolism. Academic Press, pp. 259-276. https://doi.org/10.1016/B978-0-12-822909-5.00017-4.
66. Kolter R, Greenberg EP (2006) Microbial sciences - The superficial life of microbes. Nature 441: 300-
302. https://doi.org/10.1038/441300a.
67. Hibbing ME, Fuqua C, Parsek MR et al (2010) Bacterial competition: surviving and thriving in the
microbial jungle. Nat Rev Microbiol 8: 15-25. https://doi.org/10.1038/nrmicro2259.
68. Elena SF, Lenski RE (2003) Evolution experiments with microorganisms: The dynamics and genetic
bases of adaptation. Nat Rev Genet 4: 457-469. https://doi.org/10.1038/nrg1088.
69. Hardy RW, Brezas A (2022) Diet formulation and manufacture. Academic Press, pp. 643-708.
https://doi.org/10.1016/B978-0-12-819587-1.00002-1.
70. Samedi L, Charles AL (2019) Isolation and characterization of potential probiotic Lactobacilli from
leaves of food plants for possible additives in pellet feeding. Ann Agr Sci-Cairo 64: 55-62.
https://doi.org/10.1016/j.aoas.2019.05.004.
71. de Moraes AV, Owatari MS, da Silva E et al (2022) Effects of microencapsulated probiotics-
supplemented diet on growth, non-specific immunity, intestinal health and resistance of juvenile Nile
tilapia challenged with Aeromonas hydrophila. Anim Feed Sci Tech 287: 115286.
https://doi.org/10.1016/j.anifeedsci.2022.115286.
72. Rodrigues Dias JA, Alves LL, Lima Barros FA et al (2022) Comparative effects of using a single strain
probiotic and multi-strain probiotic on the productive performance and disease resistance in
Oreochromis niloticus. Aquaculture 550. https://doi.org/10.1016/j.aquaculture.2021.737855.
73. Chowdhury MA, Roy NC (2020) Probiotic supplementation for enhanced growth of striped catfish
(Pangasianodon hypophthalmus) in cages. Aquacult Rep 18.
https://doi.org/10.1016/j.aqrep.2020.100504.
74. Mohammadian T, Monjezi N, Peyghan R et al (2022) Effects of dietary probiotic supplements on
growth, digestive enzymes activity, intestinal histomorphology and innate immunity of common carp
(Cyprinus carpio): a field study. Aquaculture 549.
https://doi.org/10.1016/j.aquaculture.2021.737787.
Page 19/2875. Salehi M, Bagheri D, Sotoudeh E et al (2022) The Combined Effects of Propionic Acid and a Mixture
of Bacillus spp. Probiotic in a Plant Protein-Rich Diet on Growth, Digestive Enzyme Activities,
Antioxidant Capacity, and Immune-Related Genes mRNA Transcript Abundance in Lates calcarifer
Fry. Probiotics Antimicro. https://doi.org/10.1007/s12602-021-09902-4.
76. Xie JJ, Liu QQ, Liao SY et al (2019) Effects of dietary mixed probiotics on growth, non-specific
immunity, intestinal morphology and microbiota of juvenile pacific white shrimp, Litopenaeus
vannamei. Fish Shellfish Immun 90: 456-465. https://doi.org/10.1016/j.fsi.2019.04.301.
77. Wan JJ, Pan JL, Shen MF et al (2022) Changes in the growth performance, antioxidant enzymes and
stress resistance caused by dietary administration of synbiotic (fructooligosaccharide and
probiotics) in juvenile Chinese Mitten Crab, Eriocheir sinensis. Aquacult Int 30: 467-481.
https://doi.org/10.1007/s10499-021-00811-5.
78. Saravanan K, Sivaramakrishnan T, Praveenraj J et al (2021) Effects of single and multi-strain
probiotics on the growth, hemato-immunological, enzymatic activity, gut morphology and disease
resistance in Rohu, Labeo rohita (vol 540, 736749, 2021). Aquaculture 543.
https://doi.org/10.1016/j.aquaculture.2021.737039.
79. Chen XQ, Xie JJ, Liu ZL et al (2020) Modulation of growth performance, non-specific immunity,
intestinal morphology, the response to hypoxia stress and resistance to Aeromonas hydrophila of
grass carp (Ctenopharyngodon idella) by dietary supplementation of a multi-strain probiotic. Comp
Biochem Phys C 231. https://doi.org/10872410.1016/j.cbpc.2020.108724.
80. Mohammadi G, Rafiee G, Tavabe KR et al (2021) The enrichment of diet with beneficial bacteria
(single- or multi- strain) in biofloc system enhanced the water quality, growth performance, immune
responses, and disease resistance of Nile tilapia (Oreochromis niloticus). Aquaculture 539.
https://doi.org/10.1016/j.aquaculture.2021.736640.
81. Hassani MHS, Jourdehi AY, Zelti AH et al (2020) Effects of commercial superzist probiotic on growth
performance and hematological and immune indices in fingerlings Acipenser baerii. Aquacult Int 28:
377-387. https://doi.org/10.1007/s10499-019-00468-1.
82. Giri SS, Sukumaran V, Sen SS et al (2014) Effects of dietary supplementation of potential probiotic
Bacillus subtilis VSGI singularly or in combination with Lactobacillus plantarum VSG3 or/and
Pseudomonas aeruginosa VSG2 on the growth, immunity and disease resistance of Labeo rohita.
Aquacult Nutr 20: 163-171. https://doi.org/10.1111/anu.12062.
83. Jia BB, Armstrong B, Stryhn H et al (2018) A case study of time-series regression modeling: Risk
factors for pond-level mortality of farmed grass carp (Ctenopharyngodon idella) on a southern
Chinese farm. Aquaculture 484: 58-64. https://doi.org/10.1016/j.aquaculture.2017.10.037.
84. Tran NT, Zhang J, Xiong F et al (2018) Altered gut microbiota associated with intestinal disease in
grass carp (Ctenopharyngodon idellus). World J Microbiol Biotechnol 34: 71.
https://doi.org/10.1007/s11274-018-2447-2.
85. Lee S, Katya K, Park Y et al (2017) Comparative evaluation of dietary probiotics Bacillus subtilis
WB60 and Lactobacillus plantarum KCTC3928 on the growth performance, immunological
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