Screening and identification of Bacillus velezensis FLU-1 from the intestinal tract of largemouth bass and its use as a feed additive

Screening and identification of Bacillus velezensis FLU-1 from the intestinal tract of largemouth bass and its use as a feed additive | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening and identification of Bacillus velezensis FLU-1 from the intestinal tract of largemouth bass and its use as a feed additive Mingqi Yang, Sunan Wang, Lili Yun, Zhikun Liu, Xulu Chang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4515265/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Probiotics have been widely used in aquaculture and may serve as a potential alternative to antibiotics. Host-derived probiotics are widely used in aquaculture because they are able to adapt to the host intestinal environment more easily than other probiotics. This study was conducted to evaluate the probiotic potential of the bacteria isolated from the gut of largemouth bass. The actions of dietary supplementation with B. velezensis FLU-1 were investigated in the largemouth bass with respect to growth, the morphology of the intestine, digestive and immune functions, and antioxidant capacity, as well as intestinal microbiota. The results revealed that B. velezensis FLU-1 exhibited promising probiotic traits, including extracellular enzyme production, ability to withstand acidic conditions, high bile salt concentration, and elevated temperatures. Furthermore, it demonstrated high hydrophobicity and auto-aggregation cability, alongside being free from antibiotic resistance and displaying a non-hemolytic nature. A diet with host–derived B. velezensis FLU-1 supplementation improved the growth performance of the fish. It also increased the length of the intestinal villi and tight junction gene expression levels, including claudin-2, occludin , and ZO-1 . Host–derived B. velezensis FLU-1 supplementation enhanced the activities of protease, α-amylase, lipase, alkaline phosphatase, acid phosphatase, lysozyme, catalase, glutathione peroxidase, decreased the level of MDA, increased the level of the anti-inflammatory cytokine TGF-β, and decreased the level of the pro-inflammatory cytokine TNF-α. Furthermore, B. velezensis FLU-1 increased the levels of several probiotics, including Lactobacillus and Lactococcus , and bacteria that produce short-chain fatty acids, including Faecalibacterium , Bacteroides , and Clostridium . The results in vivo show that adding B. velezensis FLU-1 to the feed could reduce the mortality of largemouth bass after infection with A. hydrophila , as well as reduce the bacterial load in the spleen.The results indicated that further study is warranted concerning the use of B. velezensis FLU-1 combined with sodium gluconate as a diet supplement in other economically viable fish. Probiotics Intestinal microbiota Antioxidant capacity Immune function Digestive function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Fish protein is a valuable nutritional resource that plays a crucial role in meeting the dietary needs of populations worldwide[ 1 ]. With its high-quality amino acid profile, bioavailability, and numerous health benefits, fish protein serves as a cornerstone of human nutrition and food security [ 2 ]. In China, a variety of commercial fish were farmed to provide people with high-quality fish protein. Among them, the largemouth bass ( Micropterus salmoides ), also commonly known as the northern largemouth bass or the American black bass, has gained significant attention within the aquaculture industry [ 3 ]. Originally native to North America, the largemouth bass has been successfully introduced to Chinese waters, where it has become a prized species for both recreational fishing and commercial aquaculture. The largemouth bass is one of the most economically significant freshwater fish species in China. By 2022, annual largemouth bass production in China had soared to 0.8 million tons, marking a 14.3% increase from 2021. However, the industry faces various challenges, including disease outbreaks, environmental degradation, and the need for sustainable practices [ 4 ]. In addressing these challenges, probiotics have emerged as promising tools to enhance the health and productivity of aquaculture systems [ 5 ]. Among these probiotics, Bacillus velezensis has garnered attention for its potential applications in aquaculture [ 6 ]. One of the key challenges faced by largemouth bass aquaculture in China was the management of diseases and environmental stressors that can impact fish health and productivity [ 7 ]. In recent years, there has been a growing interest in utilizing probiotics as a sustainable and effective approach to mitigate these challenges. Among the probiotics under investigation, Bacillus velezensis has emerged as a promising candidate due to its potential to promote gastrointestinal health and enhance disease resistance in fish [ 8 ]. Bacillus velezensis , a Gram-positive bacterium, is renowned for its probiotic properties, including its ability to modulate the gut microbiota, enhance immune function, and improve disease resistance in aquatic organisms [ 6 ]. Its versatile nature and tolerance to harsh environmental conditions make it particularly well-suited for aquaculture applications [ 9 ]. Therefore, the aim of this work was to isolate potential Bacillus velezensis from the gut of largemouth bass. this study evaluated the effects of dietary with B. velezensis FLU-1 on fish growth and health of the intestinal tract in the largemouth bass. This investigation provided a theoretical basis to allow the use of B. velezensis FLU-1 in aquaculture, as well as increasing our understanding concerning the effective use of probiotics as dietary supplements in fish aquaculture. 2. Materials and methods 2.1. The source of B. velezensis FLU-1 B. velezensis FLU-1 was isolated from the gut of largemouth bass and identified using a polyphasic taxonomic approach. The B. velezensis FLU-1 was routinely cultured on freshwater fish agar (FWA) plates and stored by lyophilization. 2.2 Evaluation of probiotic properties in vitro 2.2.1 Analysis of enzyme production capacity The strain FLU-1 was incubated in FWA liquid at 28 ℃ for 24 h. Then, 2 µL of the bacterial suspension was inoculated onto media containing 1% sodium carboxymethyl cellulose (for cellulose), 1% skimmed milk powder (for protease), 0.5% konjac powder (for β-mannanase) and 0.2% soluble starch (for amylase), respectively. The hydrolysis circle's diameter was determined after incubation at 28 ℃ for 24 h. 2.2.2 Analysis of antibiotic sensitivity Antibiotic sensitivity was evaluated using the disc diffusion approach with discs impregnated with antibiotics (concentration/disc) as follows: gentamicin (10 µg), ampicillin (10 µg), erythromycin (15 µg), chloramphenicol (30 µg), penicillin G (10 U/IE), cefamezin (30 µg), sulfamethoxazole (1.25/23.75 µg), amikacin (30 µg), norfloxacin (10 µg) and ciprofloxacin (5 µg). Plates were incubated at 28 ℃ for 48 h under aerobic conditions, and inhibition zones were subsequently analyzed. 2.2.3 Acid, bile salt and high-temperature tolerance analysis For the assessment of acid tolerance, 500 µL of bacterial suspension (10 8 CFU/mL) was added to FWA broth with pH adjusted to 2, 3, 4 and 5. After incubation at 28 ℃ for 0, 1, 2, 3, and 4 h, viable bacteria were counted on FWA agar. To evaluate bile tolerance, 500 µL of bacterial suspension (10 8 CFU/mL) was introduced to FWA broth with 0.30% (w/v) bile salt (Sigma–Aldrich, USA), and incubated at 28 ℃ for 0, 1, 2, 3, and 4 h. Suspension was introduced into FWA broth to serve as a control. The strain's tolerance was determined as follows: Tolerance(%) = P1/P0×100%, where P1 and P0 represent the numbers of viable bacteria in FWA-bile salt broth and FWA broth, respectively. For the evaluation of high-temperature tolerance, 1-day-old cultures of strain FLU-1 were subjected to water baths at 60, 70 or 80 ℃ for 2, 5, or 10 min. Following cooling of the bacterial liquid to ambient temperature, viable bacteria were enumerated using the plate counting approach, allowing for the calculation of bacterial survival rates. Non-heated bacteria served as the control group for comparison. 2.2.4 Surface hydrophobicity analysis Hydrophobic organic solvents, including ethyl acetate, chloroform, and xylene, were employed to assess the surface hydrophobicity of strain FLU-1. The suspension of strain FLU-1 (10 8 CFU/mL) was obtained by centrifugation at 9,000 g for 3 min and rinsed twice with PBS buffer (pH = 7.4). The suspended pellets were reconstituted in the same buffer, and the optical density at 600 nm (OD 600 nm) was recorded as baseline (A0). The suspension and organic solvent were combined in a 1:1 ratio and incubated at 28 ℃ for 30 min. Subsequently, the absorbance of the aqueous phase was assessed at 600 nm (A1). The hydrophobicity of strain FLU-1 was determined as follows: hydrophobicity (%) = 1– (A1/A0) × 100%. 2.2.5 Auto-aggregation assay The suspension (10 8 CFU/mL) was prepared as described in section 2.2.4 . Absorbance readings were taken at 0, 1, 2, 3, and 24 hours using a spectrophotometer set at 600 nm. The percentage of auto-aggregation was computed as follows: auto-aggregation (%) = (1–[At/A0]) × 100%, where At denotes the absorbance at time t = 1,2,3,24 h, while A0 represents the absorbance at t = 0 h. 2.2.6 Haemolytic activity assay Strain FLU-1 was inoculated onto blood plates containing 5% fish blood. Following incubation at 28 ℃ for 24 h, the presence of α-hemolysis (ncomplete, green hemolysis), β-hemolysis (clear, complete lysis), or γ-hemolysis (no hemolysis) was evaluated. 2.3 Fish and rearing conditions Largemouth bass were obtained from a farm in Jiyuan City, Henan Province, China, and acclimated for two weeks at the Henan Normal University Aquaculture Base. During the acclimation period, the fish were fed with commercial pellet feed (Tongwei Co., Ltd., Henan Province, China) at a rate of 2% body weight every day. The breeding conditions were as follows: pH (7.2–7.6), water temperature (25 ± 1 ℃), NO 2 − -N level (< 0.05 mg/L), NH 4 + -N concentration (< 0.5 mg/L), dissolved oxygen concentration (6.1 ± 0.4 mg/L) and under a 12:12-h light/dark cycle. 2.4 Experimental diets and experimental design B. velezensis FLU-1 was inoculated in FWA medium at 28 ℃ for 48 h followed with centrifuged at 12,000 rpm for 10 min, then the pellets were collected and washed with phosphate-saline buffer (PBS) for twice. Finally, the cells of B. velezensis FLU-1 were collected by centrifugation as described above and resuspended in the same solution. Commercial pellet feed (Tongwei Co., Ltd., Henan Province, China) was used as the basic feed. B. velezensis FLU-1 was evenly sprayed into the basic feed at 0 (Control), 1×10 7 , 1×10 8 , and 1×10 9 CFU/g, respectively, and dried naturally for 24 h. The actual amounts of viable bacteria in the feed were calculated by plate counting method. After two weeks of acclimation, healthy largemouth bass with similar body weight (18.86 ± 0.47 g) were randomly divided into 4 groups, with 3 replicates in each group and 30 fish in each replicate. The fish in each group were feed with the corresponding experimental diet. The experimental breeding conditions were consistent with those during the acclimation period. The largemouth bass were fed three times a day (8: 00, 12: 00, 7: 00) at 3% of their body weight for ten weeks. 2.5 Growth performance evaluation The weight of each fish was recorded at the beginning and end of the experiment. The growth performance of largemouth bass were evaluated according to the following formulas: Specific growth rate (SGR, %) = ln [final weight (g)] - ln [initial weight (g)]/ days × 100% Weight gain rate (WGR, %) = [final weight (g) - initial weight (g)] / initial weight (g) × 100% Feed conversion rate (FCR) = food intake (g) / [final body weight (g) - initial body weight (g)] Viscerosomatic index (VSI, %) = visceral weight (g)/whole body weight (g) × 100% Hepatosomatic index (HSI, %) = hepatopancreas weight (g)/whole body weight (g) × 100% 2.6 Sample collection At the end of the 10-weeks experimental period, samples were taken after 24 hours of fasting. Six fish were randomly selected from each tank and dissected by sterile surgical scissors, and the sample of gut, liver and head kidney were taken and stored in liquid nitrogen. Meanwhile, the intestinal contents were obtained under a sterile environment for gut microbiome analysis. Moreover, a portion of the midgut was fixed in Bouin's fluid for histomorphological analysis. 2.7 Gut morphological analysis The Bouin's fixed gut tissues were dehydrated using alcohol and then embedded in paraffin wax. The embedded tissues were cut into thin sections at 6-µm thickness and staining with hematoxylin and Eosin (H&E). The microstructure of the intestinal tissue was analyzed using a light microscope (Nikon Eclipse E400). 2.8 Determination of intestinal digestive enzyme activity The enzyme activities of lipase, α-amylase, and protease were detected using using commercial kits from Nanjing Jiancheng Bioengineering Institute. Briefly, 1 g of intestinal tissue was added to 9 mL of pre-cooled normal saline for homogenization. Then, the mixture was centrifuged at 3000 g for 10 minutes at 4 ℃, the supernatant was used for biochemical analyses. Lipase activity was determined using 1,2- o ‐dilauryl‐rac‐glycero‐3‐glutaric acid‐(6′‐methylresorufin) ester as substrate and detected with thiobarbituric acid. Amylase activity was measured using starch as the substrate with iodine-starch colorimetry. Protease activity was evaluated using casein as the substrate and reacting it with Folin phenol reagent. 2.9 Intestinal microbiota analysis The intestinal microbial DNA was extracted from intestinal contents of largemouth bass using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Hilden, Germany) according to manufacturer’s instructions. The DNA quality was detected using a NanoPhotometer spectrophotometer (Thermo Scientific NanoDrop 2000, USA). Then, the universal bacterial primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT) were used to amplify the bacterial V3-V4 hypervariable region of the 16S rRNA gene. The PCR products were purified using the AMPure® PB beads (Pacifc Biosciences, CA, USA) and quantified with Quantus™ Fluorometer (Promega, WI, USA). The purified PCR products were used to construct DNA library using the SMRTbell® Express Template Prep Kit 2.0 (Pacifc Biosciences, CA, USA) according to manufacturer’s instructions. DNA library sequencing was carried out with an Illumina MiSeq system (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Bioinformatic analysis was carried out using the Majorbio Cloud platform ( https://cloud.majorbio.com ). 2.10 Antioxidant activity analysis The antioxidant indexes of liver were measured by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) kit, the specific operation steps are as described in the kit instructions. Catalase (CAT) vitality was determined by ammonium molybdate method. Malondialdehyde (MDA) content was determined by thibabituric acid (TBA method). Total antioxidant capacity (T-AOC) was determined by ferric-reducing ability of plasma (FRAP) method. The content of reduced glutathione (GSH) was determined by microplate method. 2.11 Challenge with A. hydrophila The remaining largemouth bass of each group (n = 72) were used to determine the disease resistance against A. hydrophila . The fish in the control and three experimental groups were infected by intraperitoneal injection of 200 µL of A. hydrophila suspension at a semi-lethal concentration (7.5×10 7 CFU/mL). Another 72 fish fed with the basal diet were injected with 200 µL of PBS as a negative control. Then, 54 fish of each group were randomly selected for mortality rate calculation at 7 days postinfection. In addition, liver of the remaining fish of each group (n = 18) were sampled after 24 h of challenge. To quantify the bacterial loads in the liver, 0.1g sample was collected in a sterile environment and resuspended on 900 µL of sterile physiological saline, then homogenized with a homogenizer. Subsequently, serial dilutions of the homogenate solution were conducted. bacterial suspensions with appropriate dilution were selected and 100 µL of diluted solution was spread on LB agar. Bacterial load was determined as follows: Bacterial load = CFUs /the quality of liver. 2.12 Statistical analyses The data were analyzed using SPSS software (Version 22, Chicago, USA) and GraphPad Prism 8.0.2 (San Diego, USA). All data were presented as mean ± standard error of the mean (SEM). The normality and variance homogeneity of the data were confirmed before each parametric test. The significance of differences was analyzed using the Student’s t test, or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. A p value of < 0.05 was considered to represent statistical significance. 3. Results 3.1 Probiotic characteristics of B. velezensis FLU-1 in vitro The results of enzyme production capacity showed that B. velezensis FLU-1 generated α-amylase, protease, β-mannanase and cellulase (Fig. 1 A). Regarding bile salt resistance, the survival rates of strain FLU-1 after 0.3% bile salt exposure for 1, 2, 3 and 4 h were 79.66%, 51.96%, 35.00% and 26.07%, respectively (Fig. 1 B). No obvious difference ( P > 0.05) was found in percentage survival between 0 and 1 h of exposure to bile salts. However, a remarkable decrease ( P 0.05) were found in percentage survival among 1, 3, and 4 h of exposure to bile salts (Fig. 1 B). The pH tolerance experiment data indicated that although the survival rates of strain FLU-1 decreased after exposure to pH 2, 3, 4, and 5 for 1, 2, 3, and 4 h, more than 40% of the cells of strain FLU-1 survived following 4 h of exposure to the above mentioned pH conditions (Fig. 1 C). Moreover, the high-temperature resistance experiment results demonstrated no obvious change ( P > 0.05) in the survival rates of FLU-1 following exposure to 60, 70, 80, and 90°C for 5 minutes. However, after 10 minutes, the survival rates at each temperature were 93.56%, 81.97%, 73.80% and 65.77%, respectively (Fig. 1 D). In hydrophobicity experiments, strain FLU-1 exhibited the highest hydrophobicity towards chloroform (76.95%), followed by xylene (60.85%) and ethyl acetate (15.41%) (Fig. 1 E). The auto-aggregation experiment results showed that within the first 3 h, the auto-aggregation rates of strain FLU-1 were 31.40%, 35.07% and 37.18%, respectively. However, following 24 h, the auto-aggregation rates of strain FLU-1 markedly elevated ( P < 0.05) to 71.20% (Fig. 1 F). Hemolytic activity assay results indicated that B. velezensis FLU-1 exhibited γ-hemolysis (Fig. 1 G). Furthermore, the antibiotic susceptibility analysis revealed that strain FLU-1 was sensitive to 10 common antibiotics, including gentamicin, ampicillin, erythromycin, penicillin, chloramphenicol, cefamezin, amikacin, sulfamethoxazole, norfloxacin and ciprofloxacin (Table 1 ). Table 1 Effect of dietary B.velezensis FLU-1 on growth performance of largemouth bass. Index Groups Control 107 CFU/g group 107 CFU/g group 107 CFU/g group Initial weight(g) 18.18 ± 0.45 19.10 ± 0.39 18.90 ± 0.43 19.25 ± 0.31 Final weight(g) 72.77 ± 2.45 b 75.42 ± 2.20 b 88.22 ± 2.25 a 83.00 ± 2.40 a SGR (%) 1.98 ± 0.06 b 1.96 ± 0.04 b 2.20 ± 0.05 a 2.08 ± 0.04 ab WGR (%) 303.49 ± 18.37 b 295.85 ± 11.94 b 369.39 ± 15.93 a 331.30 ± 10.32 ab FCR 1.30 ± 0.06 a 1.21 ± 0.05 ab 1.09 ± 0.04 bc 1.06 ± 0.04 c VSI (%) 8.03 ± 0.16 a 7.06 ± 0.17 b 7.37 ± 0.27 b 7.18 ± 0.19 b HIS (%) 2.96 ± 0.09 a 1.81 ± 0.12 c 2.24 ± 0.14 b 2.22 ± 0.13 b 3.2 Effects of B. velezensis FLU-1 on the growth performance of the largemouth bass The effects of B. velezensis FLU-1 on the growth performance of largemouth bass were shown in Table 1 . There was no significant difference in the initial average body weight of largemouth bass among the four groups ( P > 0.05). After ten weeks, the final body weight of the 10 8 and 10 9 CFU/g groups was significantly higher than that of the control group (P 0.05). The specific growth rate and weight gain rate of the fish in 10 8 CFU/g group were significantly higher ( P < 0.05) than those of the control group. Similarly, feed conversion rate of the fish in 10 8 and 10 9 CFU/g groups was significantly lower than that of the control group ( P < 0.05). Moreover, viscerosomatic and hepatosomatic indexes of the fish in the 10 7 , 10 8 , and 10 9 CFU/g groups were significantly lower than those in the control group ( P < 0.05). 3.3 Effects of B. velezensis FLU-1 on the intestinal health of the largemouth bass The effects of B. velezensis FLU-1 on intestinal histomorphology of the largemouth bass were showed in Fig. 2 . The typical healthy intestinal morphology was observed in largemouth bass of the four groups (Fig. 2 A). The most noticeable histological findings were the lengthening of villi height and crypt depth in B. velezensis FLU-1 supplementation groups compared with control group (Fig. 2 B and C). The effects of B. velezensis FLU- on the mRNA expression levels of intestinal mucosa tight junction proteins ( occludin, zona occludens-1 (ZO-1) and claudin-2 ) were showed in Fig. 3 . Compared with the control group, supplementation with 10 8 and 10 9 CFU/g of B. velezensis FLU-1 significantly increased ( P 0.05). The effects of B. velezensis FLU-1 on the intestinal digestive enzyme activities of largemouth bass were shown in Fig. 4 .The lipase, α-amylase and protease activities in the 10 8 and 10 9 CFU/g B. velezensis FLU-1 groups were significantly higher ( P 0.05) was observed for lipase, α-amylase and protease activities between the control and 10 7 CFU/g B. velezensis FLU-1groups. High throughput sequencing was conducted to evaluate the effect of B. velezensis FLU-1 on the intestinal microbiome of largemouth bass. After filtering out the low-quality reads, 834, 020 reads (ranging from 58,232 to 105,781 per sample) were obtained from the 12 samples (Table S1 ). The rarefaction curves analysis based on Shannon index shows that as the sequencing volume increases, all curves tended to close to the saturation plateau (Fig. S1 ), indicating that the sequencing depth is sufficient to reflect the diversity of each sample. The alpha diversity of the intestinal microbiota was estimated by the Chao and ACE indexes for the four groups (Fig. 5 ). However, there were no significant differences in the alpha diversity indexes (Chao and Ace index) of the intestinal microbial community among the four groups, indicating that supplementation with B. velezensis FLU-1 did not affect the bacterial diversity of largemouth bass (Fig. 5 A and B). Additionally, the microbial communities in the groups supplementation with B. velezensis FLU-1 were structurally different from those in the control group in a two-dimensional PCoA plot (Fig. 5 C). These results indicate supplementation with B. velezensis FLU-1 could change gut microbial composition in largemouth bass. Then, the effects of B. velezensis FLU-1 on the intestinal microbial composition of largemouth bass was evaluated at the phylum and genus levels (Fig. 6 ). At the phylum level, the bacteria in the 12 intestinal samples were classified into 28 phyla, among which Actinobacteriota, Proteobacteria, and Firmicutes were the dominant phyla (Fig. 6 A). At the genus level, a total of 331 genera were detected in the gut of largemouth bass. Among them, norank_f__norank_o__PeM15, Achromobacte , norank_f__Rhizobiales_Incertae_Sedis and Gemmobacter , were the dominant genera (Fig. 6 B). Significant variations in the composition of gut microbiota of largemouth bass after feeding with B. velezensis FLU-1 were observed by using Kruskal–Wallis tests. At phylum level (Fig. 7 A), the relative abundance of Firmicutes in the gut of largemouth bass in 10 9 CFU/g B. velezensis FLU-1 group was significantly higher than that of largemouth bass in other three groups. At the genus level, the the abundance of Bacillus in the intestinal tracts of largemouth bass increased with increasing B. velezensis FLU-1 levels (Fig. 7 B). 3.5 Effects of B. velezensis FLU-1 on the antioxidant capacity of the largemouth bass The effects of B. velezensis FLU-1 on the antioxidant capacity of largemouth bass were shown in Fig. 8 . The T-AOC levels in the 10 8 CFU/g group were significantly higher than those in the control, 10 7 and 10 9 groups ( P < 0.05) (Fig. 8 A). Dietary with 10 7 or 10 8 CFU/g B. velezensis FLU-1 significantly increased CAT activity compared to the control group ( P 0.05) (Fig. 8 B). The content of MDA was significantly decreased ( P < 0.05) in 10 8 and 10 9 CFU/g groups compared with control group (Fig. 8 C). Compared with the control group, the GSH levels were significantly increased ( P < 0.05) in probiotics supplement groups (Fig. 8 D). 3.6 Effects of B. velezensis FLU-1 on the anti-infection of the largemouth bass The cumulative survival of largemouth bass against A. hydrophila was shown in Fig. 9 A. The survival rate of the negative control group (NC), positive control group (PC) 10 7 , 10 8 , and 10 9 CFU/g groups were 100%, 46.67%, 56.67%, 73.33%, and 63.33%, respectively. Compared with the positive control group, the survival rate of the 10 7 , 10 8 , and 10 9 CFU/g groups increased 10%, 26.67%, 16.67%, respectively. Compared with the positive control group, the bacterial load in the spleen of the 10 7 , 10 8 , and 10 9 CFU/g B. velezensis FLU-1 supplemented groups decreased significantly ( P 0.05). 4. Discussion The largemouth bass stands as one of China's most economically significant freshwater fish. By 2022, annual largemouth bass production in China had soared to 0.8 million tons, marking a 14.3% increase from 2021. Nevertheless, the persistent threat of bacterial diseases has impeded the largemouth bass industry's progress [ 10 ]. Amidst efforts to reduce antibiotic usage and explore substitutes, the application of B. velezensis , renowned for its broad-spectrum antibacterial properties, brings renewed optimism to the largemouth bass industry. Subsequently, in vitro assessment of B. velezensis probiotic performance aimed to determine its suitability for largemouth bass aquaculture. Desirable probiotic characteristics include extracellular enzyme production, intestinal mucosa adhesion, heat and bile salt resistance, as well as safety [ 3 , 11 ]. Herein, B. velezensis FLU-1 exhibited promising probiotic traits, including extracellular enzyme production, ability to withstand acidic conditions, high bile salt concentration, and elevated temperatures. Furthermore, it demonstrated high hydrophobicity and auto-aggregation cability, alongside being free from antibiotic resistance and displaying a non-hemolytic nature. Overall, B. velezensis FLU-1 emerges as a safe and viable potential probiotic for largemouth bass aquaculture. Then, a ten-week culture trial was conducted to verify the probiotic and anti-infective effects of B. velezensis FLU-1 on largemouth bass in viv o. Previous studies have shown that B. velezensis could promote growth performance of fish [ 12 ]. Similarly, the results of this study indicated that the addition of B. velezensis FLU-1 to the diet could effectively increase the weight gain rate and specific growth rate of largemouth bass, and reduce feed conversion rate, viscerosomatic and hepatosomatic. The growth-promoting effects of B. velezensis FLU-1 may be related to it’s could produce various extracellular enzymes, which promotes the absorption of nutrients by largemouth bass [ 8 ]. Like terrestrial animals, fish on their intestines to absorb nutrients from their food [ 13 , 14 ].. A healthy intestine ensures efficient absorption of essential nutrients such as proteins, fats, carbohydrates, vitamins, and minerals, which are necessary for growth and overall health [ 9 ]. Previous studies have shown that a variety of probiotics, including B. velezensis , could regulate the intestinal health of fish. Intestinal villi are small, finger-like projections and essential for efficient nutrient absorption, immune function, and maintaining gut health in fish [ 15 ]. In the present study, the histological observations showed that the villi height and crypt depth increased with supplementation of B. velezensis FLU-1, indicating that that intestinal morphology was improved by B. velezensis FLU-1. The intestinal epithelial barrier is essential for protecting the body from harmful substances, facilitating nutrient absorption, regulating immune responses, and maintaining gut health and homeostasis [ 16 ]. Tight junction proteins, such as cludin-2, occludin, and ZO-1, play a crucial role in regulating the permeability of the epithelial barrier and maintaining tissue integrity [ 17 ]. Previous studies have found that the addition of probiotics in the diet could increase the expression mRNA levels of cludin-2 , occludin , and ZO-1 [ 18 , 19 ]. In support of this observation, the present study revealed that the addition of B. velezensis FLU-1 to the diet significantly increased cludin-2 , occludin , and ZO-1 expression levels in the gut of the largemouth bass, suggesting that B. velezensis FLU-1 could strengthen the intestinal epithelial barrier in these fish. The gut microbiota plays a crucial role in various aspects of health, including digestion, metabolism, immune function, and even mood regulation [ 20 ]. Factors such as diet, antibiotic use, stress, and genetics can influence the composition and diversity of the gut microbiota [ 21 ]. Previous studies have indicated that dietary with probiotics could maintain or restore the balance of the intestinal microbiota [ 22 ]. In this investigation, supplementing the diet with B. velezensis FLU-1 increased the numbers of several beneficial bacterial species without causing any apparent adverse effects on microbial diversity. Notably, the bacterial abundance of Bacillus increased significantly after the addition of B. velezensis FLU-1 in the feed. Bacillus is a genus of Gram-positive, rod-shaped bacteria that are commonly found in various environments, including soil, water, and the gastrointestinal tracts of humans and animals [ 23 ]. Some strains of Bacillus , such as Bacillus coagulans and Bacillus subtilis , have been studied for their potential health benefits as probiotics [ 24 ]. These bacteria are capable of surviving the acidic environment of the stomach and colonizing the intestine, where they may confer various health benefits, including supporting digestive health, modulating immune function, and enhancing nutrient absorption. In addition, dietary with B. velezensis FLU-1 could increase antioxidant capacity of largemouth bass. The antioxidant system plays a crucial role in protecting cells and tissues from damage caused by reactive oxygen species (ROS) and free radicals [ 25 ]. Dysregulation of the antioxidant system has been implicated in various pathological conditions, including metabolic disorders and decreased immunity. Therefore, feeding B. velezensis FLU-1 might improve the health of largemouth bass by increasing antioxidant capacity. The premise for probiotics to replace antibiotics in the prevention and control of bacterial diseases was that they also have the function of antagonizing pathogenic bacteria in vivo . The above results have shown that strain FLU-1 can inhibit a variety of pathogenic bacteria including A. hydrophila in vitro . The results in vivo show that adding B. velezensis FLU-1 to the feed could reduce the mortality of largemouth bass after infection with A. hydrophila , as well as reduce the bacterial load in the spleen. The results of our study are consistent with previous studies, which found that B. velezensis could antagonize pathogenic bacterial infections in Scophthalmus maximus and Ctenopharyngodon idella in vivo [ 12 ]. 5. Conclusion To conclude, the present study indicated that dietary host–derived B. velezensis FLU-1 supplementation in the largemouth bass improved growth performance. Furthermore, the intestinal digestive enzyme and antioxidant enzyme activities, intestinal immune function, as well as intestinal microbiome composition were all improved in the largemouth bass. These observations will hopefully stimulate researchers to carry out additional investigations concerning the addition of largemouth bass to the diets of other species of fish with commercial importance. Declarations Author Contribution Mingqi Yang: conceptualization, methodology, data curation, and original draft preparation; Sunan Wang: data curation and analysis; Lili Yun: methodology and data curation; Zhikun Liu: data curation and analysis;Xulu Chang: supervision, reviewing, and editing. Acknowledgement This work was supported by National Natural Science Foundation of China (32373155), Innovation and Entrepreneurship Training Program for Chinese College Students (202310476068). Data Availability Data is provided within the manuscript or supplementary information files References Qi XZ, Xue MY, Cui HB, Yang Kc, Song KG, Zha JW, Wang GX, Ling F (2020) Antimicrobial activity of Pseudomonas monteilii JK-1 isolated from fish gut and its major metabolite, 1-hydroxyphenazine, against Aeromonas hydrophila . Aquaculture 526 Ahmed N, Thompson S, Turchini GM (2020) Organic aquaculture productivity, environmental sustainability, and food security: insights from organic agriculture. Food Secur 12(6):1253–1267 Zhang Y, Qi XZ, Zhang ZY, Jin ZL, Wang GX, Ling F (2023) Effects of dietary Cetobacterium somerae on the intestinal health, immune parameters and resistance against of largemouth bass, Micropterus salmoides . Fish Shellfish Immun 135 Yu JJ, Mo HL, Li S, Zhong DB, Wang MM, Tao L, Zhang ZH, Wang LX (2024) Identification and evolution characteristics of galectins and their roles in largemouth bass ( Micropterus salmoides ) following external stress. Aquaculture 581 Amenyogbe E (2023) Application of probiotics for sustainable and environment-friendly aquaculture management-A review. Cogent Food Agr 9(1) Yi YL, Zhang ZH, Zhao F, Li H, Yu LJ, Zha JW, Wang GX (2018) Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus . Fish Shellfish Immun 78:322–330 Yuan XY, Zhang XT, Xia YT, Zhang YQ, Wang B, Ye WW, Ye ZF, Qian SC, Huang MM, Yang S, Fei H (2021) Transcriptome and 16S rRNA analyses revealed differences in the responses of largemouth bass ( Micropterus salmoides ) to early Aeromonas hydrophila infection and immunization. Aquaculture 541 Kang MR, Su X, Yun LL, Shen YH, Feng JC, Yang GK, Meng XL, Zhang JX, Chang XL (2022) Evaluation of probiotic characteristics and whole genome analysis of Bacillus velezensis R-71003 isolated from the intestine of common carp ( Cyprinus carpio L.) for its use as a probiotic in aquaculture. Aquacult Rep 25 Chang XL, Kang MR, Yun LL, Shen YH, Feng JC, Yang GK, Zhang JX, Meng XL (2023) Sodium gluconate increases Bacillus velezensis R-71003 growth to improve the health of the intestinal tract and growth performance in the common carp ( Cyprinus carpio L). Aquaculture 563 Hou DQ, Li M, Li PJ, Chen B, Huang W, Guo H, Cao JM, Zhao HX (2023) Effects of sodium butyrate on growth performance, antioxidant status, inflammatory response and resistance to hypoxic stress in juvenile largemouth bass ( Micropterus salmoides ). Front Immunol 14 Jang WJ, Lee SJ, Jeon MH, Kim TY, Lee JM, Hasan MT, Lee HT, Park JH, Lee BJ, Hur SW, Lee S, Kim KW, Lee EW (2021) Characterization of a sp. KRF-7 isolated from the intestine of rockfish and effects of dietary supplementation with mannan oligosaccharide in rockfish aquaculture. Fish Shellfish Immun 119:182–192 Wu ZB, Qi XZ, Qu SY, Ling F, Wang GX (2021) Dietary supplementation of B8 enhances immune response and resistance against in grass carp. Fish Shellfish Immun 115:14–21 Wang JS, Zhu ZY, Li R, Wang XC, Leng XJ, Chen LM (2021) Impact of supplementary Lactobacillus casei K17 on growth and gut health of largemouth bass Micropterus salmoides . Aquaculture Rep 20 Poolsawat L, Li XQ, He M, Ji D, Leng XJ (2020) Clostridium butyricum as probiotic for promoting growth performance, feed utilization, gut health and microbiota community of tilapia ( Oreochromis niloticus x O. aureus ). Aquacult Nutr 26(3):657–670 Xia Y, Wang M, Gao F, Lu M, Chen G (2020) Effects of dietary probiotic supplementation on the growth, gut health and disease resistance of juvenile Nile tilapia ( Oreochromis niloticus ). Anim Nutr 6(1):69–79 Gelmez E, Jeron A, Bruder D (2022) Negative elongation factor: a key factor in the maintenance of intestinal epithelial barrier integrity. Cell Mol Immunol 19(4):453–455 Chang X, Kang M, Shen Y, Yun L, Yang G, Zhu L, Meng X, Zhang J, Su X (2021) Bacillus coagulans SCC-19 maintains intestinal health in cadmium-exposed common carp ( Cyprinus carpio L.) by strengthening the gut barriers, relieving oxidative stress and modulating the intestinal microflora, Ecotoxicol Environ Saf 228 112977 Zhou W, Xie M, Xie Y, Liang H, Li M, Ran C, Zhou Z (2022) Effect of dietary supplementation of Cetobacterium somerae XMX-1 fermentation product on gut and liver health and resistance against bacterial infection of the genetically improved farmed tilapia (GIFT, Oreochromis niloticus ). Fish Shellfish Immunol 124:332–342 Xie MX, Hao Q, Olsen RE, Ringo E, Yang YL, Zhang Z, Ran C, Zhou ZG (2022) Growth performance, hepatic enzymes, and gut health status of common carp ( Cyprinus carpio ) in response to dietary Cetobacterium somerae fermentation product. Aquaculture Rep 23 Zhang YP, Liang XF, He S, Feng HX, Li L (2022) Dietary supplementation of exogenous probiotics affects growth performance and gut health by regulating gut microbiota in Chinese Perch ( Siniperca chuatsi ). Aquaculture 547 Wang AR, Ran C, Wang YB, Zhang Z, Ding QW, Yang YL, Olsen RE, Ringo E, Bindelle J, Zhou ZG (2019) Use of probiotics in aquaculture of China-a review of the past decade. Fish Shellfish Immun 86:734–755 Morrison DJ, Preston T (2016) Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes 7(3):189–200 Zhang YR, Wu ZC, Yu MN, Zhang DM, Wang QJ, Lin LL, Wang GQ, Elsadek MM, Yao Q, Chen YK, Guo ZX (2022) Evaluating the probiotic potential and adhesion characteristics of Bacillus spp. isolated from the intestine of Rhynchocypris lagowskii Dybowski . Aquacult Int 30(2):747–772 Padeniya U, Davis DA, Liles MR, LaFrentz SA, LaFrentz BR, Shoemaker CA, Beck BH, Wells DE, Bruce TJ (2023) Probiotics enhance resistance to in Nile tilapia reared in biofloc systems. J Fish Dis 46(10):1137–1149 Ding ZL, Kong YQ, Shao XP, Zhang YX, Ren CC, Zhao XM, Yu WS, Jiang TQ, Ye JY (2019) Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn ( Macrobrachium nipponense )to chronic lead exposure. Chemosphere 217:289–297 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 04 Jun, 2024 Submission checks completed at journal 03 Jun, 2024 First submitted to journal 01 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4515265","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310298610,"identity":"aa72149a-d693-4601-b042-175f3217b50e","order_by":0,"name":"Mingqi Yang","email":"","orcid":"","institution":"Henan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mingqi","middleName":"","lastName":"Yang","suffix":""},{"id":310298611,"identity":"e7b02123-d07e-41a8-8274-c333257726c0","order_by":1,"name":"Sunan Wang","email":"","orcid":"","institution":"Henan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Sunan","middleName":"","lastName":"Wang","suffix":""},{"id":310298612,"identity":"277ddc09-95e4-411a-a8f2-165a097fa04e","order_by":2,"name":"Lili Yun","email":"","orcid":"","institution":"Henan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Yun","suffix":""},{"id":310298613,"identity":"ba63d7d8-45df-44ef-8f32-ff83721e1a61","order_by":3,"name":"Zhikun Liu","email":"","orcid":"","institution":"Henan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhikun","middleName":"","lastName":"Liu","suffix":""},{"id":310298614,"identity":"2faa8960-80c1-4eff-8595-cffd40720764","order_by":4,"name":"Xulu Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACCQglw8DewAZiMDYQq4WHgecAyVokEojUwj+7+dnDr22HeQxuvjF7zMNgI7vhAPOzB3gtuXPM3FjmDFDL7RxzYx6GNOMNB9jMDfBpMZBIMJOWqABpyd0mzcNwOHHDAR42Cfxa0r9JSxiAHHYWpOU/MVpyzCQ/gGy5wQvScoCwFokbOWXSDGfSeSTP5H+TnGOQbDzzMJsZXi38M9K3Sf5ss5bjO34sTeJNhZ1s3/HmZ3i1gAAzD8KdIC4h9UDA+IMIRaNgFIyCUTCCAQCglEQaxIQC3QAAAABJRU5ErkJggg==","orcid":"","institution":"Henan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Xulu","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-06-02 02:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4515265/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4515265/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58475785,"identity":"8ba245b6-dc29-4970-91e6-62346e81defe","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466739,"visible":true,"origin":"","legend":"\u003cp\u003eProbiotic properties of \u003cem\u003eB.velezensis\u003c/em\u003e FLU-1 \u003cem\u003ein vitro\u003c/em\u003e. (A) Enzyme-producing properties, amylase (a), protease (b), β-mannanase (c), cellulase (d); (B) 0.3 % bile salt tolerance; (C) acid resistance; (D) high temperature resistance; (E) the Auto-aggregation ability; (F) cell surface hydrophobicity; (G) hemolytic activity.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/57e1923e98454f4187f81521.png"},{"id":58475784,"identity":"92160512-cbfd-401e-b232-7b5118acb63a","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":892846,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis\u003c/em\u003eFLU-1 on intestinal tissue morphology of largemouth bass. (A) Haematoxylin-eosin staining in intestinal sections; villus height (B) and muscle thickness (C). Values in bars that have the different letter are significantly different among treatments (\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/eee53b5bb49ccbe2b1447d79.png"},{"id":58476906,"identity":"d1b697c8-bc30-43d8-841a-1750896a81b4","added_by":"auto","created_at":"2024-06-17 07:12:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39955,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis\u003c/em\u003eFLU-1 on mRNA levels of tight junction genes of largemouth bass. (A) \u003cem\u003eClaudin\u003c/em\u003e; (B) \u003cem\u003eOccludin\u003c/em\u003e; (C) \u003cem\u003eZO-1.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/bb99dca9d774a31f4fc9a7d8.png"},{"id":58475792,"identity":"381934f0-43e2-43f6-81ec-a48e20d9c3db","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38832,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis \u003c/em\u003eFLU-1 on intestinal digestive enzymes of largemouth bass. (A) Lipase; (B) Pepsin; (C) Amylase.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/d99812833ef7871a2c842773.png"},{"id":58476400,"identity":"5568be5b-9a30-44f7-96ac-c668945494e5","added_by":"auto","created_at":"2024-06-17 07:04:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100244,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis \u003c/em\u003eFLU-1 on the gut microbial community of largemouth bass. (A) Ace index; (B) Chao index; (C) Principal coordinates analysis of the unweighted UniFrac scores of the microbial communities.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/5c675052d46355b467a40227.png"},{"id":58475787,"identity":"d4d3d2fb-be76-41ce-a01a-767b418a21e7","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100992,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial composition of the different communities at the phylum level (A) and genus level (B). Taxa with abundances \u0026lt;1% are included in “others”.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/929a46ec46af2f0b9d7bcc97.png"},{"id":58475794,"identity":"cab844f1-92c2-4d8b-a5b8-b7957493d973","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":85441,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis \u003c/em\u003eFLU-1 on the gut microbial community composition at phylum and genus levels. Differential analysis of phylum (A) and genus levels (B). (*: P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/64bc65bfaf2a587c303d041f.png"},{"id":58475790,"identity":"2d8439ed-f0c8-4a7a-a952-57935142b45d","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":38513,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis\u003c/em\u003eFLU-1 on antioxidant activities of largemouth bass. (A) T-AOC level; (B) CAT activity; (C), MDA level; (D) GSH content; (E) SOD activity. Data are presented as means ± SEM. Values in bars that have the different letter are significantly different among treatments (\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/2cab7072f280531f5d9ce0be.png"},{"id":58475791,"identity":"d6b89063-5f4a-48ab-9c7e-e81522789315","added_by":"auto","created_at":"2024-06-17 06:56:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":38647,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effects of \u003cem\u003eB. velezensis \u003c/em\u003eFLU-1 on survival of largemouth bass after infection with \u003cem\u003eA hydrophila\u003c/em\u003e; (B) Effects of \u003cem\u003eB. velezensis B. velezensis \u003c/em\u003eFLU-1 on the bacterial load in largemouth bass liver after infection with \u003cem\u003eA. hydrophila\u003c/em\u003e. Data are presented as means ± SEM. Values in bars that have the different letter are significantly different among treatments (\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/083b796fd3d041ef80276af9.png"},{"id":58477459,"identity":"27d700c1-8d10-4b1a-a85f-f179fdeb32a9","added_by":"auto","created_at":"2024-06-17 07:20:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2904581,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/5624527a-3b65-4798-902a-5b360596e483.pdf"},{"id":58476402,"identity":"ff459aea-2109-4c00-b0fa-69fa13d52d12","added_by":"auto","created_at":"2024-06-17 07:04:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":126023,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4515265/v1/0f69e9561b391ab3a5cd2027.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening and identification of Bacillus velezensis FLU-1 from the intestinal tract of largemouth bass and its use as a feed additive","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFish protein is a valuable nutritional resource that plays a crucial role in meeting the dietary needs of populations worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With its high-quality amino acid profile, bioavailability, and numerous health benefits, fish protein serves as a cornerstone of human nutrition and food security [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In China, a variety of commercial fish were farmed to provide people with high-quality fish protein. Among them, the largemouth bass (\u003cem\u003eMicropterus salmoides\u003c/em\u003e), also commonly known as the northern largemouth bass or the American black bass, has gained significant attention within the aquaculture industry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Originally native to North America, the largemouth bass has been successfully introduced to Chinese waters, where it has become a prized species for both recreational fishing and commercial aquaculture.\u003c/p\u003e \u003cp\u003eThe largemouth bass is one of the most economically significant freshwater fish species in China. By 2022, annual largemouth bass production in China had soared to 0.8\u0026nbsp;million tons, marking a 14.3% increase from 2021. However, the industry faces various challenges, including disease outbreaks, environmental degradation, and the need for sustainable practices [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addressing these challenges, probiotics have emerged as promising tools to enhance the health and productivity of aquaculture systems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these probiotics, \u003cem\u003eBacillus velezensis\u003c/em\u003e has garnered attention for its potential applications in aquaculture [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the key challenges faced by largemouth bass aquaculture in China was the management of diseases and environmental stressors that can impact fish health and productivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In recent years, there has been a growing interest in utilizing probiotics as a sustainable and effective approach to mitigate these challenges. Among the probiotics under investigation, \u003cem\u003eBacillus velezensis\u003c/em\u003e has emerged as a promising candidate due to its potential to promote gastrointestinal health and enhance disease resistance in fish [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eBacillus velezensis\u003c/em\u003e, a Gram-positive bacterium, is renowned for its probiotic properties, including its ability to modulate the gut microbiota, enhance immune function, and improve disease resistance in aquatic organisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Its versatile nature and tolerance to harsh environmental conditions make it particularly well-suited for aquaculture applications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the aim of this work was to isolate potential \u003cem\u003eBacillus velezensis\u003c/em\u003e from the gut of largemouth bass. this study evaluated the effects of dietary with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on fish growth and health of the intestinal tract in the largemouth bass. This investigation provided a theoretical basis to allow the use of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 in aquaculture, as well as increasing our understanding concerning the effective use of probiotics as dietary supplements in fish aquaculture.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1. The source of B. velezensis FLU-1\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. velezensis FLU-1\u003c/em\u003e was isolated from the gut of largemouth bass and identified using a polyphasic taxonomic approach. The \u003cem\u003eB. velezensis FLU-1\u003c/em\u003e was routinely cultured on freshwater fish agar (FWA) plates and stored by lyophilization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Evaluation of probiotic properties in vitro\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Analysis of enzyme production capacity\u003c/h2\u003e \u003cp\u003eThe strain FLU-1 was incubated in FWA liquid at 28 ℃ for 24 h. Then, 2 \u0026micro;L of the bacterial suspension was inoculated onto media containing 1% sodium carboxymethyl cellulose (for cellulose), 1% skimmed milk powder (for protease), 0.5% konjac powder (for β-mannanase) and 0.2% soluble starch (for amylase), respectively. The hydrolysis circle's diameter was determined after incubation at 28 ℃ for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Analysis of antibiotic sensitivity\u003c/h2\u003e \u003cp\u003eAntibiotic sensitivity was evaluated using the disc diffusion approach with discs impregnated with antibiotics (concentration/disc) as follows: gentamicin (10 \u0026micro;g), ampicillin (10 \u0026micro;g), erythromycin (15 \u0026micro;g), chloramphenicol (30 \u0026micro;g), penicillin G (10 U/IE), cefamezin (30 \u0026micro;g), sulfamethoxazole (1.25/23.75 \u0026micro;g), amikacin (30 \u0026micro;g), norfloxacin (10 \u0026micro;g) and ciprofloxacin (5 \u0026micro;g). Plates were incubated at 28 ℃ for 48 h under aerobic conditions, and inhibition zones were subsequently analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Acid, bile salt and high-temperature tolerance analysis\u003c/h2\u003e \u003cp\u003eFor the assessment of acid tolerance, 500 \u0026micro;L of bacterial suspension (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was added to FWA broth with pH adjusted to 2, 3, 4 and 5. After incubation at 28 ℃ for 0, 1, 2, 3, and 4 h, viable bacteria were counted on FWA agar.\u003c/p\u003e \u003cp\u003eTo evaluate bile tolerance, 500 \u0026micro;L of bacterial suspension (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was introduced to FWA broth with 0.30% (w/v) bile salt (Sigma\u0026ndash;Aldrich, USA), and incubated at 28 ℃ for 0, 1, 2, 3, and 4 h. Suspension was introduced into FWA broth to serve as a control. The strain's tolerance was determined as follows: Tolerance(%)\u0026thinsp;=\u0026thinsp;P1/P0\u0026times;100%, where P1 and P0 represent the numbers of viable bacteria in FWA-bile salt broth and FWA broth, respectively.\u003c/p\u003e \u003cp\u003eFor the evaluation of high-temperature tolerance, 1-day-old cultures of strain FLU-1 were subjected to water baths at 60, 70 or 80 ℃ for 2, 5, or 10 min. Following cooling of the bacterial liquid to ambient temperature, viable bacteria were enumerated using the plate counting approach, allowing for the calculation of bacterial survival rates. Non-heated bacteria served as the control group for comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Surface hydrophobicity analysis\u003c/h2\u003e \u003cp\u003eHydrophobic organic solvents, including ethyl acetate, chloroform, and xylene, were employed to assess the surface hydrophobicity of strain FLU-1. The suspension of strain FLU-1 (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was obtained by centrifugation at 9,000 g for 3 min and rinsed twice with PBS buffer (pH\u0026thinsp;=\u0026thinsp;7.4). The suspended pellets were reconstituted in the same buffer, and the optical density at 600 nm (OD 600 nm) was recorded as baseline (A0). The suspension and organic solvent were combined in a 1:1 ratio and incubated at 28 ℃ for 30 min. Subsequently, the absorbance of the aqueous phase was assessed at 600 nm (A1). The hydrophobicity of strain FLU-1 was determined as follows: hydrophobicity (%)\u0026thinsp;=\u0026thinsp;1\u0026ndash; (A1/A0) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Auto-aggregation assay\u003c/h2\u003e \u003cp\u003eThe suspension (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was prepared as described in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e2.2.4\u003c/span\u003e. Absorbance readings were taken at 0, 1, 2, 3, and 24 hours using a spectrophotometer set at 600 nm. The percentage of auto-aggregation was computed as follows: auto-aggregation (%) = (1\u0026ndash;[At/A0]) \u0026times; 100%, where At denotes the absorbance at time t\u0026thinsp;=\u0026thinsp;1,2,3,24 h, while A0 represents the absorbance at t\u0026thinsp;=\u0026thinsp;0 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6 Haemolytic activity assay\u003c/h2\u003e \u003cp\u003eStrain FLU-1 was inoculated onto blood plates containing 5% fish blood. Following incubation at 28 ℃ for 24 h, the presence of α-hemolysis (ncomplete, green hemolysis), β-hemolysis (clear, complete lysis), or γ-hemolysis (no hemolysis) was evaluated.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fish and rearing conditions\u003c/h2\u003e \u003cp\u003eLargemouth bass were obtained from a farm in Jiyuan City, Henan Province, China, and acclimated for two weeks at the Henan Normal University Aquaculture Base. During the acclimation period, the fish were fed with commercial pellet feed (Tongwei Co., Ltd., Henan Province, China) at a rate of 2% body weight every day. The breeding conditions were as follows: pH (7.2\u0026ndash;7.6), water temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃), NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N level (\u0026lt;\u0026thinsp;0.05 mg/L), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration (\u0026lt;\u0026thinsp;0.5 mg/L), dissolved oxygen concentration (6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/L) and under a 12:12-h light/dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental diets and experimental design\u003c/h2\u003e \u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 was inoculated in FWA medium at 28 ℃ for 48 h followed with centrifuged at 12,000 rpm for 10 min, then the pellets were collected and washed with phosphate-saline buffer (PBS) for twice. Finally, the cells of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 were collected by centrifugation as described above and resuspended in the same solution. Commercial pellet feed (Tongwei Co., Ltd., Henan Province, China) was used as the basic feed. \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 was evenly sprayed into the basic feed at 0 (Control), 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e, 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e, and 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g, respectively, and dried naturally for 24 h. The actual amounts of viable bacteria in the feed were calculated by plate counting method.\u003c/p\u003e \u003cp\u003eAfter two weeks of acclimation, healthy largemouth bass with similar body weight (18.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 g) were randomly divided into 4 groups, with 3 replicates in each group and 30 fish in each replicate. The fish in each group were feed with the corresponding experimental diet. The experimental breeding conditions were consistent with those during the acclimation period. The largemouth bass were fed three times a day (8: 00, 12: 00, 7: 00) at 3% of their body weight for ten weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Growth performance evaluation\u003c/h2\u003e \u003cp\u003eThe weight of each fish was recorded at the beginning and end of the experiment. The growth performance of largemouth bass were evaluated according to the following formulas:\u003c/p\u003e \u003cp\u003eSpecific growth rate (SGR, %)\u0026thinsp;=\u0026thinsp;ln [final weight (g)] - ln [initial weight (g)]/ days \u0026times; 100%\u003c/p\u003e \u003cp\u003eWeight gain rate (WGR, %) = [final weight (g) - initial weight (g)] / initial weight (g) \u0026times; 100%\u003c/p\u003e \u003cp\u003eFeed conversion rate (FCR)\u0026thinsp;=\u0026thinsp;food intake (g) / [final body weight (g) - initial body weight (g)]\u003c/p\u003e \u003cp\u003eViscerosomatic index (VSI, %)\u0026thinsp;=\u0026thinsp;visceral weight (g)/whole body weight (g) \u0026times; 100%\u003c/p\u003e \u003cp\u003eHepatosomatic index (HSI, %)\u0026thinsp;=\u0026thinsp;hepatopancreas weight (g)/whole body weight (g) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sample collection\u003c/h2\u003e \u003cp\u003eAt the end of the 10-weeks experimental period, samples were taken after 24 hours of fasting. Six fish were randomly selected from each tank and dissected by sterile surgical scissors, and the sample of gut, liver and head kidney were taken and stored in liquid nitrogen. Meanwhile, the intestinal contents were obtained under a sterile environment for gut microbiome analysis. Moreover, a portion of the midgut was fixed in Bouin's fluid for histomorphological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Gut morphological analysis\u003c/h2\u003e \u003cp\u003eThe Bouin's fixed gut tissues were dehydrated using alcohol and then embedded in paraffin wax. The embedded tissues were cut into thin sections at 6-\u0026micro;m thickness and staining with hematoxylin and Eosin (H\u0026amp;E). The microstructure of the intestinal tissue was analyzed using a light microscope (Nikon Eclipse E400).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of intestinal digestive enzyme activity\u003c/h2\u003e \u003cp\u003eThe enzyme activities of lipase, α-amylase, and protease were detected using using commercial kits from Nanjing Jiancheng Bioengineering Institute. Briefly, 1 g of intestinal tissue was added to 9 mL of pre-cooled normal saline for homogenization. Then, the mixture was centrifuged at 3000 g for 10 minutes at 4 ℃, the supernatant was used for biochemical analyses. Lipase activity was determined using 1,2-\u003cem\u003eo\u003c/em\u003e‐dilauryl‐rac‐glycero‐3‐glutaric acid‐(6\u0026prime;‐methylresorufin) ester as substrate and detected with thiobarbituric acid. Amylase activity was measured using starch as the substrate with iodine-starch colorimetry. Protease activity was evaluated using casein as the substrate and reacting it with Folin phenol reagent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Intestinal microbiota analysis\u003c/h2\u003e \u003cp\u003eThe intestinal microbial DNA was extracted from intestinal contents of largemouth bass using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Hilden, Germany) according to manufacturer\u0026rsquo;s instructions. The DNA quality was detected using a NanoPhotometer spectrophotometer (Thermo Scientific NanoDrop 2000, USA). Then, the universal bacterial primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT) were used to amplify the bacterial V3-V4 hypervariable region of the 16S rRNA gene. The PCR products were purified using the AMPure\u0026reg; PB beads (Pacifc Biosciences, CA, USA) and quantified with Quantus\u0026trade; Fluorometer (Promega, WI, USA). The purified PCR products were used to construct DNA library using the SMRTbell\u0026reg; Express Template Prep Kit 2.0 (Pacifc Biosciences, CA, USA) according to manufacturer\u0026rsquo;s instructions. DNA library sequencing was carried out with an Illumina MiSeq system (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Bioinformatic analysis was carried out using the Majorbio Cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Antioxidant activity analysis\u003c/h2\u003e \u003cp\u003eThe antioxidant indexes of liver were measured by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) kit, the specific operation steps are as described in the kit instructions. Catalase (CAT) vitality was determined by ammonium molybdate method. Malondialdehyde (MDA) content was determined by thibabituric acid (TBA method). Total antioxidant capacity (T-AOC) was determined by ferric-reducing ability of plasma (FRAP) method. The content of reduced glutathione (GSH) was determined by microplate method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Challenge with A. hydrophila\u003c/h2\u003e \u003cp\u003eThe remaining largemouth bass of each group (n\u0026thinsp;=\u0026thinsp;72) were used to determine the disease resistance against \u003cem\u003eA. hydrophila\u003c/em\u003e. The fish in the control and three experimental groups were infected by intraperitoneal injection of 200 \u0026micro;L of \u003cem\u003eA. hydrophila\u003c/em\u003e suspension at a semi-lethal concentration (7.5\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU/mL). Another 72 fish fed with the basal diet were injected with 200 \u0026micro;L of PBS as a negative control. Then, 54 fish of each group were randomly selected for mortality rate calculation at 7 days postinfection. In addition, liver of the remaining fish of each group (n\u0026thinsp;=\u0026thinsp;18) were sampled after 24 h of challenge. To quantify the bacterial loads in the liver, 0.1g sample was collected in a sterile environment and resuspended on 900 \u0026micro;L of sterile physiological saline, then homogenized with a homogenizer. Subsequently, serial dilutions of the homogenate solution were conducted. bacterial suspensions with appropriate dilution were selected and 100 \u0026micro;L of diluted solution was spread on LB agar. Bacterial load was determined as follows: Bacterial load\u0026thinsp;=\u0026thinsp;CFUs /the quality of liver.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analyses\u003c/h2\u003e \u003cp\u003eThe data were analyzed using SPSS software (Version 22, Chicago, USA) and GraphPad Prism 8.0.2 (San Diego, USA). All data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The normality and variance homogeneity of the data were confirmed before each parametric test. The significance of differences was analyzed using the Student\u0026rsquo;s t test, or one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparisons test. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered to represent statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Probiotic characteristics of B. velezensis FLU-1 in vitro\u003c/h2\u003e \u003cp\u003eThe results of enzyme production capacity showed that \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 generated α-amylase, protease, β-mannanase and cellulase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Regarding bile salt resistance, the survival rates of strain FLU-1 after 0.3% bile salt exposure for 1, 2, 3 and 4 h were 79.66%, 51.96%, 35.00% and 26.07%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). No obvious difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was found in percentage survival between 0 and 1 h of exposure to bile salts. However, a remarkable decrease (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in percentage survival occurred following 2 h of bile salt exposure. Interestingly, no obvious differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were found in percentage survival among 1, 3, and 4 h of exposure to bile salts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The pH tolerance experiment data indicated that although the survival rates of strain FLU-1 decreased after exposure to pH 2, 3, 4, and 5 for 1, 2, 3, and 4 h, more than 40% of the cells of strain FLU-1 survived following 4 h of exposure to the above mentioned pH conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, the high-temperature resistance experiment results demonstrated no obvious change (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the survival rates of FLU-1 following exposure to 60, 70, 80, and 90\u0026deg;C for 5 minutes. However, after 10 minutes, the survival rates at each temperature were 93.56%, 81.97%, 73.80% and 65.77%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn hydrophobicity experiments, strain FLU-1 exhibited the highest hydrophobicity towards chloroform (76.95%), followed by xylene (60.85%) and ethyl acetate (15.41%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The auto-aggregation experiment results showed that within the first 3 h, the auto-aggregation rates of strain FLU-1 were 31.40%, 35.07% and 37.18%, respectively. However, following 24 h, the auto-aggregation rates of strain FLU-1 markedly elevated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to 71.20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Hemolytic activity assay results indicated that \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 exhibited γ-hemolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Furthermore, the antibiotic susceptibility analysis revealed that strain FLU-1 was sensitive to 10 common antibiotics, including gentamicin, ampicillin, erythromycin, penicillin, chloramphenicol, cefamezin, amikacin, sulfamethoxazole, norfloxacin and ciprofloxacin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of dietary \u003cem\u003eB.velezensis\u003c/em\u003e FLU-1 on growth performance of largemouth bass.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIndex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107 CFU/g group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e107 CFU/g group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e107 CFU/g group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial weight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal weight(g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSGR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWGR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e303.49\u0026thinsp;\u0026plusmn;\u0026thinsp;18.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e295.85\u0026thinsp;\u0026plusmn;\u0026thinsp;11.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e369.39\u0026thinsp;\u0026plusmn;\u0026thinsp;15.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e331.30\u0026thinsp;\u0026plusmn;\u0026thinsp;10.32\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVSI (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIS (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2 Effects of B. velezensis\u003c/em\u003e FLU-1 \u003cem\u003eon the growth performance of the largemouth bass\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on the growth performance of largemouth bass were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There was no significant difference in the initial average body weight of largemouth bass among the four groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After ten weeks, the final body weight of the 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups was significantly higher than that of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there was no significant difference in the final body weight between the 10\u003csup\u003e7\u003c/sup\u003e CFU/g and the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The specific growth rate and weight gain rate of the fish in 10\u003csup\u003e8\u003c/sup\u003e CFU/g group were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of the control group. Similarly, feed conversion rate of the fish in 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups was significantly lower than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, viscerosomatic and hepatosomatic indexes of the fish in the 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups were significantly lower than those in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 \u003cem\u003eEffects of B. velezensis\u003c/em\u003e FLU-1 \u003cem\u003eon the intestinal health of the largemouth bass\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on intestinal histomorphology of the largemouth bass were showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The typical healthy intestinal morphology was observed in largemouth bass of the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The most noticeable histological findings were the lengthening of villi height and crypt depth in \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplementation groups compared with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003eThe effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU- on the mRNA expression levels of intestinal mucosa tight junction proteins (\u003cem\u003eoccludin, zona occludens-1 (ZO-1)\u003c/em\u003e and \u003cem\u003eclaudin-2\u003c/em\u003e) were showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Compared with the control group, supplementation with 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/g of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the expression levels of \u003cem\u003eoccludin\u003c/em\u003e and \u003cem\u003eclaudin-2\u003c/em\u003e genes in largemouth bass. Feed supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 had no significant effect on the expression of \u003cem\u003eZO-1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on the intestinal digestive enzyme activities of largemouth bass were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.The lipase, α-amylase and protease activities in the 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/g \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 groups were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than in the control group. However, no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was observed for lipase, α-amylase and protease activities between the control and 10\u003csup\u003e7\u003c/sup\u003e CFU/g \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1groups.\u003c/p\u003e \u003cp\u003eHigh throughput sequencing was conducted to evaluate the effect of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on the intestinal microbiome of largemouth bass. After filtering out the low-quality reads, 834, 020 reads (ranging from 58,232 to 105,781 per sample) were obtained from the 12 samples (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The rarefaction curves analysis based on Shannon index shows that as the sequencing volume increases, all curves tended to close to the saturation plateau (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating that the sequencing depth is sufficient to reflect the diversity of each sample.\u003c/p\u003e \u003cp\u003eThe alpha diversity of the intestinal microbiota was estimated by the Chao and ACE indexes for the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, there were no significant differences in the alpha diversity indexes (Chao and Ace index) of the intestinal microbial community among the four groups, indicating that supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 did not affect the bacterial diversity of largemouth bass (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Additionally, the microbial communities in the groups supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 were structurally different from those in the control group in a two-dimensional PCoA plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 could change gut microbial composition in largemouth bass.\u003c/p\u003e \u003cp\u003eThen, the effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on the intestinal microbial composition of largemouth bass was evaluated at the phylum and genus levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). At the phylum level, the bacteria in the 12 intestinal samples were classified into 28 phyla, among which Actinobacteriota, Proteobacteria, and Firmicutes were the dominant phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). At the genus level, a total of 331 genera were detected in the gut of largemouth bass. Among them, norank_f__norank_o__PeM15, \u003cem\u003eAchromobacte\u003c/em\u003e, norank_f__Rhizobiales_Incertae_Sedis and \u003cem\u003eGemmobacter\u003c/em\u003e, were the dominant genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Significant variations in the composition of gut microbiota of largemouth bass after feeding with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 were observed by using Kruskal\u0026ndash;Wallis tests. At phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), the relative abundance of Firmicutes in the gut of largemouth bass in 10\u003csup\u003e9\u003c/sup\u003e CFU/g \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 group was significantly higher than that of largemouth bass in other three groups. At the genus level, the the abundance of \u003cem\u003eBacillus\u003c/em\u003e in the intestinal tracts of largemouth bass increased with increasing \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.5 Effects of B. velezensis\u003c/em\u003e FLU-1 \u003cem\u003eon the antioxidant capacity of the largemouth bass\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on the antioxidant capacity of largemouth bass were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The T-AOC levels in the 10\u003csup\u003e8\u003c/sup\u003e CFU/g group were significantly higher than those in the control, 10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Dietary with 10\u003csup\u003e7\u003c/sup\u003e or 10\u003csup\u003e8\u003c/sup\u003e CFU/g \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 significantly increased CAT activity compared to the control group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05), and there were no significant differences in CAT activity between the 10\u003csup\u003e9\u003c/sup\u003e CFU/g and control groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The content of MDA was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups compared with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Compared with the control group, the GSH levels were significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in probiotics supplement groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.6 Effects of B. velezensis\u003c/em\u003e FLU-1 \u003cem\u003eon the anti-infection of the largemouth bass\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe cumulative survival of largemouth bass against \u003cem\u003eA. hydrophila\u003c/em\u003e was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA. The survival rate of the negative control group (NC), positive control group (PC) 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups were 100%, 46.67%, 56.67%, 73.33%, and 63.33%, respectively. Compared with the positive control group, the survival rate of the 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e CFU/g groups increased 10%, 26.67%, 16.67%, respectively. Compared with the positive control group, the bacterial load in the spleen of the 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e CFU/g \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplemented groups decreased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). There were no significant differences among the three \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplemented groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe largemouth bass stands as one of China's most economically significant freshwater fish. By 2022, annual largemouth bass production in China had soared to 0.8\u0026nbsp;million tons, marking a 14.3% increase from 2021. Nevertheless, the persistent threat of bacterial diseases has impeded the largemouth bass industry's progress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Amidst efforts to reduce antibiotic usage and explore substitutes, the application of \u003cem\u003eB. velezensis\u003c/em\u003e, renowned for its broad-spectrum antibacterial properties, brings renewed optimism to the largemouth bass industry.\u003c/p\u003e \u003cp\u003eSubsequently, \u003cem\u003ein vitro\u003c/em\u003e assessment of \u003cem\u003eB. velezensis\u003c/em\u003e probiotic performance aimed to determine its suitability for largemouth bass aquaculture. Desirable probiotic characteristics include extracellular enzyme production, intestinal mucosa adhesion, heat and bile salt resistance, as well as safety [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Herein, \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 exhibited promising probiotic traits, including extracellular enzyme production, ability to withstand acidic conditions, high bile salt concentration, and elevated temperatures. Furthermore, it demonstrated high hydrophobicity and auto-aggregation cability, alongside being free from antibiotic resistance and displaying a non-hemolytic nature. Overall, \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 emerges as a safe and viable potential probiotic for largemouth bass aquaculture.\u003c/p\u003e \u003cp\u003eThen, a ten-week culture trial was conducted to verify the probiotic and anti-infective effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 on largemouth bass \u003cem\u003ein viv\u003c/em\u003eo. Previous studies have shown that \u003cem\u003eB. velezensis\u003c/em\u003e could promote growth performance of fish [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, the results of this study indicated that the addition of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 to the diet could effectively increase the weight gain rate and specific growth rate of largemouth bass, and reduce feed conversion rate, viscerosomatic and hepatosomatic. The growth-promoting effects of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 may be related to it\u0026rsquo;s could produce various extracellular enzymes, which promotes the absorption of nutrients by largemouth bass [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Like terrestrial animals, fish on their intestines to absorb nutrients from their food [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].. A healthy intestine ensures efficient absorption of essential nutrients such as proteins, fats, carbohydrates, vitamins, and minerals, which are necessary for growth and overall health [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Previous studies have shown that a variety of probiotics, including \u003cem\u003eB. velezensis\u003c/em\u003e, could regulate the intestinal health of fish. Intestinal villi are small, finger-like projections and essential for efficient nutrient absorption, immune function, and maintaining gut health in fish [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the present study, the histological observations showed that the villi height and crypt depth increased with supplementation of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1, indicating that that intestinal morphology was improved by \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1.\u003c/p\u003e \u003cp\u003eThe intestinal epithelial barrier is essential for protecting the body from harmful substances, facilitating nutrient absorption, regulating immune responses, and maintaining gut health and homeostasis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Tight junction proteins, such as cludin-2, occludin, and ZO-1, play a crucial role in regulating the permeability of the epithelial barrier and maintaining tissue integrity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies have found that the addition of probiotics in the diet could increase the expression mRNA levels of \u003cem\u003ecludin-2\u003c/em\u003e, \u003cem\u003eoccludin\u003c/em\u003e, and \u003cem\u003eZO-1\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In support of this observation, the present study revealed that the addition of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 to the diet significantly increased \u003cem\u003ecludin-2\u003c/em\u003e, \u003cem\u003eoccludin\u003c/em\u003e, and \u003cem\u003eZO-1\u003c/em\u003e expression levels in the gut of the largemouth bass, suggesting that \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 could strengthen the intestinal epithelial barrier in these fish.\u003c/p\u003e \u003cp\u003eThe gut microbiota plays a crucial role in various aspects of health, including digestion, metabolism, immune function, and even mood regulation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Factors such as diet, antibiotic use, stress, and genetics can influence the composition and diversity of the gut microbiota [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Previous studies have indicated that dietary with probiotics could maintain or restore the balance of the intestinal microbiota [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this investigation, supplementing the diet with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 increased the numbers of several beneficial bacterial species without causing any apparent adverse effects on microbial diversity. Notably, the bacterial abundance of Bacillus increased significantly after the addition of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 in the feed. \u003cem\u003eBacillus\u003c/em\u003e is a genus of Gram-positive, rod-shaped bacteria that are commonly found in various environments, including soil, water, and the gastrointestinal tracts of humans and animals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Some strains of \u003cem\u003eBacillus\u003c/em\u003e, such as \u003cem\u003eBacillus coagulans\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e, have been studied for their potential health benefits as probiotics [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These bacteria are capable of surviving the acidic environment of the stomach and colonizing the intestine, where they may confer various health benefits, including supporting digestive health, modulating immune function, and enhancing nutrient absorption.\u003c/p\u003e \u003cp\u003eIn addition, dietary with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 could increase antioxidant capacity of largemouth bass. The antioxidant system plays a crucial role in protecting cells and tissues from damage caused by reactive oxygen species (ROS) and free radicals [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Dysregulation of the antioxidant system has been implicated in various pathological conditions, including metabolic disorders and decreased immunity. Therefore, feeding \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 might improve the health of largemouth bass by increasing antioxidant capacity.\u003c/p\u003e \u003cp\u003eThe premise for probiotics to replace antibiotics in the prevention and control of bacterial diseases was that they also have the function of antagonizing pathogenic bacteria \u003cem\u003ein vivo\u003c/em\u003e. The above results have shown that strain FLU-1 can inhibit a variety of pathogenic bacteria including \u003cem\u003eA. hydrophila in vitro\u003c/em\u003e. The results \u003cem\u003ein vivo\u003c/em\u003e show that adding \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 to the feed could reduce the mortality of largemouth bass after infection with \u003cem\u003eA. hydrophila\u003c/em\u003e, as well as reduce the bacterial load in the spleen. The results of our study are consistent with previous studies, which found that \u003cem\u003eB. velezensis\u003c/em\u003e could antagonize pathogenic bacterial infections in \u003cem\u003eScophthalmus maximus\u003c/em\u003e and \u003cem\u003eCtenopharyngodon idella in vivo\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eTo conclude, the present study indicated that dietary host\u0026ndash;derived \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplementation in the largemouth bass improved growth performance. Furthermore, the intestinal digestive enzyme and antioxidant enzyme activities, intestinal immune function, as well as intestinal microbiome composition were all improved in the largemouth bass. These observations will hopefully stimulate researchers to carry out additional investigations concerning the addition of largemouth bass to the diets of other species of fish with commercial importance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMingqi Yang: conceptualization, methodology, data curation, and original draft preparation; Sunan Wang: data curation and analysis; Lili Yun: methodology and data curation; Zhikun Liu: data curation and analysis;Xulu Chang: supervision, reviewing, and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (32373155), Innovation and Entrepreneurship Training Program for Chinese College Students (202310476068).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQi XZ, Xue MY, Cui HB, Yang Kc, Song KG, Zha JW, Wang GX, Ling F (2020) Antimicrobial activity of \u003cem\u003ePseudomonas monteilii\u003c/em\u003e JK-1 isolated from fish gut and its major metabolite, 1-hydroxyphenazine, against \u003cem\u003eAeromonas hydrophila\u003c/em\u003e. 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J Fish Dis 46(10):1137\u0026ndash;1149\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing ZL, Kong YQ, Shao XP, Zhang YX, Ren CC, Zhao XM, Yu WS, Jiang TQ, Ye JY (2019) Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn (\u003cem\u003eMacrobrachium nipponense\u003c/em\u003e)to chronic lead exposure. Chemosphere 217:289\u0026ndash;297\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Probiotics, Intestinal microbiota, Antioxidant capacity, Immune function, Digestive function","lastPublishedDoi":"10.21203/rs.3.rs-4515265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4515265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotics have been widely used in aquaculture and may serve as a potential alternative to antibiotics. Host-derived probiotics are widely used in aquaculture because they are able to adapt to the host intestinal environment more easily than other probiotics. This study was conducted to evaluate the probiotic potential of the bacteria isolated from the gut of largemouth bass. The actions of dietary supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 were investigated in the largemouth bass with respect to growth, the morphology of the intestine, digestive and immune functions, and antioxidant capacity, as well as intestinal microbiota. The results revealed that \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 exhibited promising probiotic traits, including extracellular enzyme production, ability to withstand acidic conditions, high bile salt concentration, and elevated temperatures. Furthermore, it demonstrated high hydrophobicity and auto-aggregation cability, alongside being free from antibiotic resistance and displaying a non-hemolytic nature. A diet with host\u0026ndash;derived \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplementation improved the growth performance of the fish. It also increased the length of the intestinal villi and tight junction gene expression levels, including \u003cem\u003eclaudin-2, occludin\u003c/em\u003e, and \u003cem\u003eZO-1\u003c/em\u003e. Host\u0026ndash;derived \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 supplementation enhanced the activities of protease, α-amylase, lipase, alkaline phosphatase, acid phosphatase, lysozyme, catalase, glutathione peroxidase, decreased the level of MDA, increased the level of the anti-inflammatory cytokine TGF-β, and decreased the level of the pro-inflammatory cytokine TNF-α. Furthermore, \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 increased the levels of several probiotics, including \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eLactococcus\u003c/em\u003e, and bacteria that produce short-chain fatty acids, including \u003cem\u003eFaecalibacterium\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, and \u003cem\u003eClostridium\u003c/em\u003e. The results \u003cem\u003ein vivo\u003c/em\u003e show that adding \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 to the feed could reduce the mortality of largemouth bass after infection with \u003cem\u003eA. hydrophila\u003c/em\u003e, as well as reduce the bacterial load in the spleen.The results indicated that further study is warranted concerning the use of \u003cem\u003eB. velezensis\u003c/em\u003e FLU-1 combined with sodium gluconate as a diet supplement in other economically viable fish.\u003c/p\u003e","manuscriptTitle":"Screening and identification of Bacillus velezensis FLU-1 from the intestinal tract of largemouth bass and its use as a feed additive","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-17 06:56:23","doi":"10.21203/rs.3.rs-4515265/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-04T06:23:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-03T23:37:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2024-06-02T02:03:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"af1924a5-ae78-4d84-a415-16ca109b7899","owner":[],"postedDate":"June 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-17T06:56:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-17 06:56:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4515265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4515265","identity":"rs-4515265","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}