Bacillus subtilis q16 and application thereof

Abstract

The application discloses a bacillus subtilis Q16 and application thereof, the strain is preserved in the China typical culture preservation center, and the preservation number is CCTCC NO:M 20252232. The bacillus subtilis Q16 is isolated from a large yellow croaker, is added to large yellow croaker feed, and can significantly improve the growth performance, digestion, antioxidant and immune capacity of the large yellow croaker, and changes the intestinal flora composition structure of the large yellow croaker and the gene expression related to immunity and metabolism.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus subtilis Q16 and its applications. Background Technology

[0002] Large yellow croaker (Larimichthys crocea) is an important economic marine fish species along my country's coast, ranking second in aquaculture production among marine fish in 2023. However, due to factors such as unreasonable layout of aquaculture areas, high stocking density, and water pollution, diseases frequently occur in farmed large yellow croaker. At the same time, the overuse of antibiotics has further exacerbated the pollution of drug-resistant genes in fish and increased the risk to the ecological environment.

[0003] Probiotics, as a novel microecological preparation, are not only a tool for disease prevention and control, but also enhance aquaculture efficiency by promoting growth, strengthening immunity, and improving feed utilization, thus meeting the industry's complex demand for functional microbial agents. The gut microbiota plays a crucial role in nutrient absorption, immune regulation, and pathogen resistance in fish. The use of probiotics helps increase the number of beneficial bacteria in the host's gut, strengthens the barrier function of the intestinal mucosa, and promotes healthy host growth by adjusting the gut microbiota structure. By adding probiotics to aquaculture water or feed, they can regulate the gut microbiota after entering the fish's body, thereby affecting fish growth, development, and immune function. Notably, host-derived probiotics, due to their gut adaptability, have greater potential in targeted regulation of gut microbiota, immune activation, and growth metabolism. However, most existing commercially available microbial agents are derived from terrestrial or freshwater strains, lacking sufficient host adaptability for marine fish, and therefore unsuitable for the ecological aquaculture of large yellow croaker. Furthermore, existing research on gut probiotics for large yellow croaker is mostly at the screening stage and lacks application validation in large-scale aquaculture. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a Bacillus subtilis Q16 and its application. This invention obtains a compound-functional Bacillus subtilis (B. subtilis) Q16 through intestinal flora analysis of different populations of large yellow croaker and screening of probiotics in the intestines of all male large yellow croaker. Adding Bacillus subtilis Q16 to the feed significantly increases the abundance of short-chain fatty acid-producing bacteria in the large yellow croaker intestine, upregulates chemokine gene expression, and regulates metabolic pathways, significantly improving the growth performance, antioxidant stress resistance, and non-specific immune function of large yellow croaker.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of Bacillus subtilis Q16, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252232 and deposited on October 16, 2025.

[0006] Furthermore, the rDNA sequence of Bacillus subtilis Q16 is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides the use of the Bacillus subtilis Q16 in the preparation of an additive that promotes the growth of large yellow croaker.

[0008] In a third aspect, the present invention provides the application of the Bacillus subtilis Q16 in the preparation of a microecological preparation for improving the intestinal health and growth performance of large yellow croaker.

[0009] In a fourth aspect, the present invention provides a microecological preparation containing the aforementioned Bacillus subtilis Q16.

[0010] In a fifth aspect, the present invention provides the application of the Bacillus subtilis Q16 in the preparation of feed for large yellow croaker.

[0011] The present invention also provides a feed for raising large yellow croaker, containing the aforementioned Bacillus subtilis Q16.

[0012] Furthermore, in the feed for raising large yellow croaker, the viable count of Bacillus subtilis Q16 is 1×10⁻⁶. 5 ~1×10 9 CFU / g.

[0013] The present invention also provides the application of the above-described Bacillus subtilis Q16 in the preparation of a formulation for preventing and treating Vibrio harveyi infection in large yellow croaker.

[0014] The beneficial effects of this invention include at least the following: This invention extracts a novel Bacillus subtilis Q16 from large yellow croaker. This Bacillus subtilis Q16 can significantly improve the growth performance, digestion, antioxidant and immune capabilities of large yellow croaker, and change the composition and structure of the intestinal flora of large yellow croaker as well as the expression of genes related to immunity and metabolism.

[0015] The Bacillus subtilis Q16 strain screened in this invention can be used as a feed additive in actual aquaculture and has a positive impact. It not only provides a solution for the development of microecological preparations for large yellow croaker, but will also help the aquaculture industry shift from disease treatment to ecological prevention, further realizing the role of agricultural technological innovation in leading industrial development. Attached Figure Description

[0016] Figure 1 For the enzyme production status of strains, A. Number of enzyme-producing strains; B. Percentage of enzyme-producing strains; C. Enzyme production efficiency of some strains of amylase; D. Enzyme production efficiency of some strains of protease; E. Enzyme production efficiency of some strains of lipase.

[0017] Figure 2The results show the antibacterial activity of some strains: A. *Bacillus mermaidina*; B. *Vibrio harveyi*; C. *Staphylococcus aureus*; D. *Vibrio parahaemolyticus*.

[0018] Figure 3 Phylogenetic analysis of potential probiotics (NJ neighbor-joining method).

[0019] Figure 4 For the growth of potential probiotics, see: A. Growth curve of potential probiotics; B. Growth of potential probiotics at different pH values; C. Growth of strain Q16 at different salinities; D. Growth of strain H3 at different salinities.

[0020] Figure 5 The antibiotic susceptibility for Q16 is as follows: 1. Penicillin; 2. Ampicillin; 3. Cefazolin; 4. Ceftriaxone; 5. Cefotaxime; 6. Amikacin; 7. Gentamicin; 8. Norfloxacin; 9. Enrofloxacin; 10. Ciprofloxacin; 11. Tetracycline; 12. Doxycycline; 13. Chloramphenicol; 14. Florfenicol; 15. Erythromycin; 16. Trimethoprim-Sulfate.

[0021] Figure 6 Survival rates of different treatment groups after Vibrio harveyi infection.

[0022] Figure 7 To investigate the effects of feeding Bacillus subtilis Q16 on the diversity and composition of the gut microbiota in large yellow croaker, the following methods were used: A. Sample dilution curves; B. Sample OTU distribution; C. NMDS analysis based on Bray curtis distance; D. Cluster analysis based on Weighted Unifrac distance; E. Dominant flora at the phylum level; F. Dominant flora at the genus level.

[0023] Figure 8 To investigate the effects of feeding Bacillus subtilis Q16 on the differences in gut microbiota in large yellow croaker, the following diagrams were created: A. Heatmap of genus-level microbiota differences; B. LDA value distribution of BS0 and BS7 groups; C. LDA value distribution of BS0 and BS9 groups; D. LDA value distribution of BS7 and BS9 groups; E. Evolutionary clade diagram of BS0 and BS7 groups; F. Evolutionary clade diagram of BS0 and BS9 groups.

[0024] Figure 9 The effect of feeding Bacillus subtilis Q16 on the prediction of gut microbiota function in large yellow croaker (A. Level 1; B. Level 2).

[0025] Figure 10For the differential gene expression analysis between the Q16 treatment group and the control group, use: A. Venn diagram of co-expressed genes; B. Volcano diagram of differentially expressed genes; C. Heatmap of differentially expressed gene clusters; D. Bar chart of differentially expressed genes.

[0026] Figure 11 Enrichment analysis of differentially expressed genes GO and KEGG pathways (A. GO; B. KEGG). Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] In this embodiment, the culture medium formulation includes: Protease production medium: 3 g beef extract, 10 g peptone, 5 g NaCl, 16 g agar, 10 mL 0.4% bromothymol blue solution, 500 mL distilled water; 25 g skim milk powder, 500 mL distilled water; sterilize separately, adjust pH to 7.2-7.4, sterilize at 121 ℃ for 15 min, cool and pour into plates for later use.

[0030] Amylase production medium: 3 g beef extract, 10 g peptone, 10 g soluble starch, 5 g NaCl, 16 g agar, 1000 mL distilled water, adjust pH to 7.2 ~ 7.4, sterilize at 121 ℃ for 15 min, cool and pour into plates for later use.

[0031] Lipase production medium: 10 g peptone, 0.1 g CaCl2 · H2O, 10 mL Tween-80, 5 g NaCl, 16 g agar, 1000 mL distilled water, adjust pH to 7.2 ~ 7.4, sterilize at 121 ℃ for 15 min, cool and pour into plates for later use.

[0032] LB liquid medium: 20 g powder (10 g tryptone, 5 g yeast extract, 10 g NaCl), 1000 mL distilled water, pH 7.2 ~ 7.4, sterilized at 121 ℃ for 20 min, and cooled for later use.

[0033] BHI liquid culture medium: 37 g powder (10 g peptone, 12.5 g dehydrated calf brain extract powder, 5 g dehydrated calf extract powder, 5 g NaCl, 2 g glucose, 2.5 g disodium hydrogen phosphate), pH 7.4 ± 0.2, sterilized at 121 ℃ for 20 min, cooled for later use.

[0034] LB and BHI agar medium: Add 15 g of agar to the liquid medium, sterilize at 121 °C for 20 min, cool and pour into plates for later use.

[0035] MRS agar medium: 69.2 g MRS agar powder (10 g peptone, 8 g beef extract, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 15 g agar, 1 g Tween-80, pH 6.5 ± 0.2), dissolved in 1000 mL distilled water, sterilized at 121 ℃ for 20 min, cooled and poured into plates for later use.

[0036] YPD medium: 49 g powder (10 g peptone, 20 g glucose, 5 g yeast extract, 14 g agar) dissolved in 1000 mL distilled water, sterilized at 121 °C for 20 min, cooled and poured into plates for later use.

[0037] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Isolation and Identification of Bacillus subtilis Q16 1. Materials and Methods (1) Equipment and reagents Major instruments: SW-CJ-2FD ultra-clean workbench and GZX-9240MBE electric heating drying oven were purchased from Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory (Shanghai, China); LDZX-75L vertical high-pressure steam sterilizer was purchased from Shanghai Shenan Medical Instrument Factory (Shanghai, China); LRH-500F biochemical incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd. (Shanghai, China); ZHTY-70E constant temperature shaker was purchased from Shanghai Zhichu Instrument Co., Ltd. (Shanghai, China); benchtop refrigerated high-speed centrifuge, Applied Biosystems PCR instrument, metal thermostat, and ultra-low temperature freezer were purchased from Thermo Fisher. Scientific (USA), DYY-60 gel electrophoresis apparatus purchased from Beijing Liuyi Biotechnology Co., Ltd. (Beijing, China), Tannon-1600 gel imaging system purchased from Shanghai Tianneng Co., Ltd. (Shanghai, China), V-5000 visible spectrophotometer purchased from Shanghai Yuanxi Technology Co., Ltd. (Shanghai, China), Oxford cup purchased from Shanghai Precision Instruments Co., Ltd. (Shanghai, China), pH meter and electronic balance purchased from Sartorius (Germany), vortex mixer purchased from Scilogex (USA).

[0038] Main reagents: Beef extract, peptone, and sodium chloride were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China); Tween-80, soluble starch, agar, LB medium, PBS buffer, and Lugol's iodine solution were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); BHI liquid medium, MRS agar medium, YPD medium, NA medium, 2216E medium, and blood agar plates were purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, Shandong, China); antimicrobial susceptibility testing discs were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, Zhejiang, China); glycerol, anhydrous ethanol, hydrochloric acid, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); agarose was purchased from Xiamen SunMed Biotechnology Co., Ltd. (Xiamen, Fujian, China); universal bacterial primers were purchased from Hangzhou Youkang Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China); PCR Master Mix was purchased from Beijing Bio-Rad Biotechnology Co., Ltd. (Beijing, China); and 0.9% physiological saline was purchased from Shandong Kelun Pharmaceutical Co., Ltd. (Binzhou, Shandong, China).

[0039] (2) Experimental fish and test strains Four healthy, all-male large yellow croakers with normal body size and no external injuries were selected from the Baishishan cage culture base of Ningbo Marine and Fisheries Research Institute for the isolation of intestinal strains. Their weight was (167.5 ± 6.3) g. For the artificial induction experiment, 210 healthy common large yellow croakers were cultured in a recirculating water tank. Their weight was (16.9 ± 2.6) g, the water temperature was 24 ± 1 ℃, and they were fed once daily using a micro-flow aeration system with continuous aeration. After one week of temporary rearing, they were used in the experiment.

[0040] The pathogenic bacteria of marine fish include P. damselae, P. plecoglossicida, Vibrio parahaemolyticus, Vibrio harveyi, and Staphylococcus aureus, all of which are strains preserved in the laboratory of Ningbo Aquatic Animal Disease Prevention and Quarantine Center.

[0041] (3) Isolation of intestinal bacteria from large yellow croaker Disinfect the fish's body surface with 75% alcohol. Under aseptic conditions, dissect and obtain the midgut. Wash with 0.9% saline 3-5 times to remove food residue. Then, scrape the intestinal lining microbial mucus with a disposable inoculation loop and add it to LB medium. Repeat the process several times and shake well. Take 100 µL of the suspension and add it to BHI, YPD, and MRS selective media, respectively. Inoculate 3 plates for each medium. Take another 100 µL of the suspension heated at 80 °C for 20 min and add it to BHI medium. Spread the suspension on plates and incubate at 28 °C for 24 h. Pick single colonies from the medium with clear colonies, appropriate density, and colony counts between 30 and 300 CFU / plate for bacterial isolation. Streak the bacteria onto BHI, YPD, and MRS selective media for isolation and purification. Inoculate the purified strains into BHI liquid medium and preserve them in a -80 °C ultra-low temperature freezer using the glycerol preservation method for subsequent experiments.

[0042] (4) Screening test for enzyme-producing strains The spot inoculation method was used to screen enzyme-producing strains. Bacterial suspension was inoculated onto enzyme-producing media using an inoculation loop. For protease and lipase-producing media, incubation was performed at 28 °C for 72 h. A clear ring around the colony indicated enzyme production (positive result), while the absence of a clear ring indicated a negative result. For amylase-producing media, after incubation at 28 °C for 72 h, the appearance of a colorless clear ring after adding Lugol's iodine solution indicated a positive result, while the absence of a clear ring indicated a negative result. Strains producing all three enzymes were used as the experimental strains. Bacterial suspension was inoculated onto protease, lipase, and amylase-producing media, respectively, and incubated at 28 °C for 48 h. Colony diameter and hydrolysis zone diameter were measured using calipers, and the ratio of hydrolysis zone diameter to colony diameter (Dh / Dc) was calculated to compare the enzyme production capacity of each strain.

[0043] (5) Hemolysis test for screening strains Hemolysis tests were performed on strains producing the three enzymes. Single colonies cultured overnight on BHI solid medium were inoculated onto blood agar plates and incubated at 28 °C for 24–48 h. The presence of a hemolytic zone around the colony was observed. Hemolysis types include α-hemolysis (incomplete hemolysis), β-hemolysis (complete hemolysis), and γ-hemolysis (non-hemolysis). α-hemolysis and β-hemolysis produce a hemolytic zone around the colony, indicating that the bacteria are hemolytic and potentially pathogenic. γ-hemolysis indicates that the bacteria do not produce a hemolytic zone and are not hemolytic.

[0044] (6) Antimicrobial test of the selected strains The indicator bacteria were inoculated onto BHI solid medium for activation and culture. The culture was carried out at 28 °C and 180 r / min in a shaker for 12 h. The bacterial suspension was then diluted with PBS to adjust the concentration to 10. 6 CFU / mL, on a clean bench, take 100 µL of bacterial suspension and spread it evenly on 2216E agar medium. Then, using sterile forceps, place the Oxford cup on the medium and gently press it down. Add 0.25 mL of a 10 CFU / mL solution to the Oxford cup. 5 The probiotic suspension of CFU / mL was placed in a 4 ℃ refrigerator for diffusion for 2 h, and then transferred to a 28 ℃ biochemical incubator for constant temperature incubation for 24 ~ 48 h. The presence of a clear inhibition zone around the Oxford cup was observed and the diameter of the inhibition zone was measured. Each treatment group was repeated 3 times.

[0045] (7) Molecular biological identification of potential probiotic strains a. Preparation of bacterial DNA template Strains stored at -80 ℃ were streaked onto BHI solid medium and incubated overnight at 28 ℃. Single colonies were then inoculated into BHI liquid medium and incubated at 28 ℃ and 180 r / min for 24 h. 1 mL of the bacterial suspension was used to prepare DNA using a DNA extraction kit, which was then used as a template for PCR amplification.

[0046] b. PCR amplification and sequencing of bacterial 16S rRNA sequence The extracted DNA was used as a template for PCR amplification. The primers used were the universal primers 24F / 1492R for 16S rRNA sequence amplification: upstream primer 24F: 5'-AGAGTTTGATCCTGGCTCAG - 3'; downstream primer 1492R: 5'-TACGGTTACCTTGTTACGACTT - 3', synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The PCR reaction system consisted of (25 µL): 12 µL PCR Master Mix, 0.5 µL each of upstream and downstream primers 24F / 1492R, 1 µL template DNA, and 11 µL ddH2O. The reaction program was: pre-denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C (30 s), 55 °C (30 s), and 72 °C (45 s), and finally held at 72 °C for 5 min. The amplified products were detected by 1% agarose gel electrophoresis to observe the target gene band.

[0047] c. 16S rRNA sequence analysis and phylogenetic tree construction The PCR amplification products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The 16S rRNA of the tested strain was searched and analyzed by BLAST on NCBI. The Clustal W method was used to compare the homology sequence with the known 16S rRNA gene sequences of bacterial genera. The phylogenetic tree was constructed by repeating the neighbor-joining (NJ) method in MEGA 11.0 software 1000 times.

[0048] (8) Determination of the growth capacity of potential probiotics Growth curve determination: 2 mL of fresh bacterial culture was inoculated into 200 mL of LB liquid medium and cultured at 29 ℃ and 180 r / min for 28 h. OD was measured every 2 h using a visible spectrophotometer. 600 The values ​​were compared with those obtained using LB medium as a control.

[0049] Salinity tolerance test: 2 mL of fresh bacterial culture was inoculated into 200 mL of LB liquid medium, and NaCl concentrations of 0 g / L, 25 g / L, 50 g / L, and 75 g / L were added. The culture was carried out at 29 ℃ and 180 r / min for 24 h. The OD of the solution was measured every 6 h using a visible spectrophotometer. 600 The values ​​were calculated using LB medium as a blank control.

[0050] pH tolerance test: 2 mL of fresh bacterial culture was inoculated into 200 mL of LB liquid medium, and the pH of the medium was adjusted to 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0, respectively. The culture was incubated at 29 ℃ and 180 r / min in a shaker. After 24 h, the OD was measured using a visible spectrophotometer. 600 The values ​​were calculated using LB medium as a blank control.

[0051] (9) Artificial retrospective testing of potential probiotics The selected Q16 and H3 strains were inoculated onto LB solid medium and activated overnight at 28 ℃. Single colonies were picked and cultured in LB liquid medium. The strains were then cultured at 30 ℃ and 180 r / min with shaking until the exponential phase, followed by centrifugation at 4000 r / min for 5 min and resuspending in sterile PBS to the appropriate concentration. 210 healthy large yellow croakers (weight 16.9 ± 2.6 g) were divided into 7 groups (6 experimental groups and 1 control group), with 30 fish in each group, divided into 3 replicates. After one week of temporary rearing, they were used in the experiment. Safety was tested using the intraperitoneal injection method. The experimental groups were injected with 10... 7 10 8 10 9 Each large yellow croaker was injected with 50 µL of bacterial strains Q16 and H3 at a concentration of CFU / mL, while the control group was injected with an equal volume of sterile PBS solution. The experimental fish were cultured in a recirculating aquarium at a water temperature of 24 ± 1℃ using a micro-flow aquarium with continuous aeration and no feeding. The fish were observed for 10 days after injection, and the growth and mortality of each group were recorded. On day 10, randomly selected fish were dissected to observe the normality of internal organs. The liver and spleen were streaked onto BHI medium to isolate bacteria and cultured at 28℃ for 48 h.

[0052] (10) Antimicrobial susceptibility test of strain Q16 Q16 was selected as the candidate strain. The susceptibility of Bacillus subtilis Q16 to 17 commonly used antibiotics was determined using the disk diffusion method. After overnight incubation and activation, the Q16 strain was centrifuged at 3000 r / min, the supernatant was removed, and the bacterial concentration of Bacillus subtilis Q16 was adjusted to 10⁻⁶ using sterile PBS. 8CFU / mL, 100 µL of bacterial suspension was evenly spread onto LB agar medium. After the medium was allowed to cool and dry naturally, 17 antimicrobial susceptibility test discs were evenly affixed to the surface of the medium, with 4 discs on each agar plate and 3 replicates for each antimicrobial susceptibility test disc. After incubation at 29 ℃ for 24 h, the diameter of the inhibition zone (mm) of each antimicrobial susceptibility test disc was measured using calipers. The experimental results were used to determine the resistant strains (R), the toxic and sensitive strains (I), and the sensitive strains (S) according to the CLSI international standard for antimicrobial susceptibility testing.

[0053] (11) Data statistics and analysis One-way analysis of variance (ANOVA) was performed using IBM SPSS 27.0 to compare differences between different treatment groups. If a significant difference was found overall, multiple comparisons were performed using either the LSD or Tamhane's T2 test, depending on the homogeneity of variance. Experimental data are expressed as mean ± standard deviation (Mean ± SD), with P < 0.05 considered statistically significant. Different letters indicate statistically significant differences.

[0054] 2. Experimental Results (1) Intestinal strains obtained by isolation A total of 340 bacterial strains were isolated from the intestine of large yellow croaker using BHI agar medium, yeast medium and MRS selective medium.

[0055] (2) Enzyme production characteristics of intestinal strains The enzyme production of 340 bacterial strains isolated from the intestines of healthy large yellow croaker was analyzed. The results are as follows: Figure 1 As shown in Figure A, 168 strains of bacteria produced protease, accounting for 49.4%; 98 strains produced amylase, accounting for 28.8%; and 124 strains produced lipase, accounting for 36.5%. 99 strains did not produce enzymes, accounting for 29.1%; and 241 strains produced enzymes, accounting for 70.9%. Among these, 133 strains produced only one enzyme, accounting for 39.1%; 65 strains produced two enzymes, accounting for 19.1%; and 43 strains produced three enzymes, accounting for 12.6%. Figure 1 B). Demonstrating the amylase production of some strains ( Figure 1 C), protease ( Figure 1 D) and lipase ( Figure 1 E) Result.

[0056] Strains producing all three enzymes were selected and inoculated onto selection media for protease, amylase, and lipase production using the spot inoculation method. The enzyme production capacity of each strain was determined by the ratio of the diameter of the hydrolysis zone (Dh) to the colony diameter (Dc) (Dh / Dc). Strains producing all three enzymes were selected as candidate probiotics, and their enzyme production capacities are shown in Table 1. The strain with the strongest amylase production capacity was B182, with a Dh / Dc ratio of 2.36; the strain with the strongest protease production capacity was B12, with a Dh / Dc ratio of 3.48; and the strain with the strongest lipase production capacity was B43, with a Dh / Dc ratio of 2.78.

[0057] Table 1. Enzyme production capacity of strains producing three enzymes

[0058] (3) Hemolysis of the three enzyme-producing strains Hemolysis tests were performed on the strains producing the three enzymes, and the results are shown in Table 2. No hemolytic zones were observed around strains B43, Q5, Q14, Q16, Q21, Q22, Q23, Q24, Q26, Q29, Q31, QM2, QM4, and H3. This indicates that these strains do not exhibit hemolytic activity but rather γ-hemolysis, and may be potential probiotics.

[0059] Table 2 Hemolysis status of strains producing the three enzymes

[0060] (4) Antibacterial ability of the three enzyme-producing strains For strains producing three enzymes that are non-hemolytic, an antibacterial test was conducted. The antibacterial ability of the enzyme-producing strains was expressed as the diameter of the inhibition zone (mm) (Table 3). The presence of a distinct inhibition zone around the Oxford cup was observed, and its diameter was measured. Figure 2 The results are from the antibacterial activity test.

[0061] Table 3. Antibacterial activity of the three enzyme-producing strains

[0062] Note: "0" indicates no antibacterial ability.

[0063] Based on a comprehensive analysis of the results of enzyme production, hemolysis, and antibacterial experiments, strains Q16 and H3 were selected for the next stage of experiments in this embodiment.

[0064] (5) Identification of 16S rRNA and construction of phylogenetic tree of the selected strains The 16S rRNA sequences of candidate strains were searched on NCBI BLAST. Clustal W algorithm was used for homology sequence analysis and comparison with known 16S rRNA gene sequences of bacterial genera, and further analysis was performed from a phylogenetic tree (…). Figure 3As can be seen from the data, strain Q16 clusters with Bacillus subtilis subsp. inaquosorum in the same minimal branch, indicating that strain Q16 is most closely related to Bacillus subtilis and can be identified as Bacillus subtilis. Similarly, strain H3 clusters with Bacillus cereus in the same minimal branch, indicating that strain H3 is most closely related to Bacillus cereus and can be identified as Bacillus cereus.

[0065] (6) Growth capacity of potential probiotics Potential probiotics entered the logarithmic growth phase after approximately 2 hours of continuous shaking at 29 ℃ and 180 r / min, and entered the stationary phase at approximately 12 h and 14 h, respectively, subsequently stabilizing until 28 h. OD 600 The value stabilized between 1.3 and 1.4. Figure 4 A). Potential probiotics can grow under different pH conditions. With increasing pH, the growth of both potential probiotic strains showed a trend of first increasing and then decreasing, and the differences were significant under different pH conditions (P<0.05). Based on the results, the optimal growth pH for the strains is between 6.0 and 8.0. Figure 4 B). The growth of potential probiotics varied significantly under different salinity conditions. As the NaCl concentration increased, the growth of all strains was inhibited to varying degrees, with poor growth observed in media prepared with NaCl concentrations above 50 g / L (5%). Figure 4 C; Figure 4 D).

[0066] (7) Safety of potential probiotics The experimental results (Table 4) showed that after injection of strain Q16 into the fish, the large yellow croaker exhibited good vitality, with no disease or mortality; 10 9 The H3 strain (CFU / mL) caused fish mortality in parallel experiments 1 and 3, indicating that the H3 strain carries a certain risk. After the experiment, randomly sampled fish were dissected and infected with the Q16 strain. No abnormalities were observed in their internal organs, and no bacteria were isolated, indicating that the Q16 strain has high safety for large yellow croaker. Based on the above experimental results, Q16 was selected as a candidate strain for probiotic testing in this example, and the drug susceptibility of the Q16 strain was determined.

[0067] Table 4. Results of artificial retro-infection of the strains

[0068] (8) The sensitivity of strain Q16 to different antibiotics The results of susceptibility testing on 17 antibiotics showed that Q16 was sensitive to 13 antibiotics, including penicillin and ampicillin, moderately sensitive to ceftriaxone, tetracycline, and erythromycin, and developed resistance to cefazolin (Table 5). Figure 5 ).

[0069] Table 5. Antibiotic susceptibility in Q16

[0070] In summary, this experiment, combining morphological observation and 16S rRNA phylogenetic analysis, showed that strain Q16 has the highest similarity to Bacillus subtilis desert subsp. tumefacilitator, based on the phylogenetic tree, strain Q16 can be identified as Bacillus subtilis. It has been deposited at the China Center for Type Culture Collection on October 16, 2025.

[0071] In this embodiment, the potential probiotic Q16 showed inhibitory effects against five common pathogens in marine fish. The growth capacity of the strain and its tolerance to environmental fluctuations such as temperature, pH, and salinity were also important. Furthermore, the strain needed to demonstrate host safety. Artificial re-sensing tests were one of the evaluation criteria. In this embodiment, the potential probiotic Q16 did not cause disease or death in fish during artificial re-sensing tests at three concentration gradients, indicating that the Q16 strain is safe for the host.

[0072] Example 2 Evaluation of the beneficial effects of Bacillus subtilis Q16 on large yellow croaker 1. Materials and Methods (1) Main instruments and reagents Major instruments: SW-CJ-2FD ultra-clean workbench, GZX-9240MBE electric heating drying oven, and DK-8D water bath were purchased from Shanghai Boxun Industrial Co., Ltd. (Shanghai, China); LDZX-75L vertical high-pressure steam sterilizer was purchased from Shanghai Shenan Medical Instrument Factory (Shanghai, China); LRH-500F biochemical incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd. (Shanghai, China); ZHTY-70E constant temperature shaker was purchased from Shanghai Zhichu Instrument Co., Ltd. (Shanghai, China); JXFSTPRP-24 fully automatic sample rapid grinding machine was purchased from Shanghai Jingxin Industrial Development Co., Ltd. (Shanghai, China); full-wavelength microplate reader, benchtop refrigerated high-speed centrifuge, Applied Biosystems PCR instrument, metal thermostat, and ultra-low temperature freezer were purchased from Thermo Fisher. The DYY-60 gel electrophoresis system was purchased from Beijing Liuyi Biotechnology Co., Ltd. (Beijing, China), the Tannon-1600 gel imaging system was purchased from Shanghai Tianneng Co., Ltd. (Shanghai, China), and the V-5000 visible spectrophotometer was purchased from Shanghai Yuanxi Technology Co., Ltd. (Shanghai, China).

[0073] Main reagents: Eugenol, sodium alginate, and sodium chloride were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China); double-distilled water was purchased from Beijing Cooler Master Technology Co., Ltd. (Beijing, China); PBS buffer, agar, and LB medium were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China); BHI liquid medium was purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, Shandong, China); 0.9% physiological saline was purchased from Shandong Kelun Pharmaceutical Co., Ltd. (Binzhou, Shandong, China); kits for α-amylase (AMS), trypsin, lipase (LPS), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), total protein (TP), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (ALP) were all purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, Jiangsu, China).

[0074] (2) Experimental fish, bacterial strains and basic feed The common yellow croaker used in the experiment came from the Baishishan sea area cage culture base of Ningbo Marine and Fisheries Research Institute. The average weight was (36.95 ± 4.29) g and the average body length was (12.74 ± 1.23) cm.

[0075] The experimental strain was Bacillus subtilis Q16, which was screened in Example 1. The Vibrio harveyi strain used in the challenge infection test was a strain preserved in the laboratory of Ningbo Aquatic Animal Disease Prevention and Quarantine Center.

[0076] The basic feed used in this experiment was a refined floating feed for large yellow croaker, with a particle size of (3.0 ± 0.2) mm. The main nutritional components were as follows: crude protein ≤ 49.0%, crude fat ≥ 10.0%, moisture ≤ 12.0%, crude fiber ≤ 10.0%, crude ash ≤ 17.0%, lysine ≥ 2.5%, and total phosphorus ≥ 0.8%.

[0077] (3) Preparation of experimental bacterial solution and feed The additive used in this experiment was Bacillus subtilis desert subsp. Q16 strain. The experimental setup was to add Bacillus subtilis Q16 strain at concentrations of 0 (BS0) and 10. 7 CFU / g (BS7) and 10 9 CFU / g (BS9). The bacterial solution was diluted to the specified concentration according to the set dosage. Different concentrations of bacterial solution were sprayed onto the surface of the base feed, dried at 30 ℃, and then coated with sodium alginate at 3% of the feed's dry weight. The mixture was stirred thoroughly to ensure the probiotics were fully encapsulated within the feed. To ensure the activity of Bacillus subtilis, the experimental feed was prepared every 5 days and temporarily stored at 4 ℃.

[0078] (4) Experiment on the effect of adding Q16 bacterial solution to feed on the farming effect of large yellow croaker The experiment was conducted at the Ningbo Marine Fisheries Science and Technology Innovation Base. The experimental fish came from the Baishishan sea area cage culture base of the Ningbo Marine and Fisheries Research Institute. First, 1500 experimental fish were evenly distributed into three 5-ton tanks for temporary rearing, during which time they were fed a basic large yellow croaker feed to allow the fish to adapt to the feed and feeding time. After one month, 900 uniformly sized and robust large yellow croakers were selected and randomly assigned to nine tanks, 100 fish per tank, with three replicates per group, for a total of three groups (two experimental groups and one control group). A Bacillus subtilis Q16 strain concentration of 0 (BS0) was set as the control group, and 10... 7 CFU / g (BS7) and 10 9 The experimental group consisted of individuals with CFU / g (BS9) (Table 6). A 60-day culture experiment was conducted, during which the fish were fed diets supplemented with Q16 bacterial solution at 7:00 and 17:00 daily, with the amount fed until the fish were satiated. Water temperature was controlled at 17.1–22.4 °C, salinity at 26–30, dissolved oxygen at 6.2–7.5 mg / L, and ammonia nitrogen concentration <0.1 g / mL. Feeding and mortality of the large yellow croaker were observed and recorded, and any dead individuals were promptly removed.

[0079] Table 6 Feeding Scheme

[0080] (5) Sample collection and processing Feeding was stopped for 1 day before sampling. Then, 15 fish from each group were taken and anesthetized with eugenol solution (1:1000). Weight, body length, and total length were measured. 12 fish were taken, and venous blood was drawn from the tail using a disposable sterile syringe. The blood was allowed to stand at 4 ℃ for 4 h and then centrifuged (3500 r / min, 4 ℃) for 10 min. The supernatant serum was aspirated with a pipette and placed in a -80 ℃ freezer for subsequent determination of serum immune indicators. 15 fish were dissected (the body surface was disinfected with 75% alcohol) to obtain their livers and visceral masses, and they were weighed separately. The livers of 6 fish were used for the determination of antioxidant indicators, and the livers of 9 fish were placed in sterile, enzyme-free cryovials for transcriptome analysis. Subsequently, the intestines of the fish were taken under sterile conditions. The intestines of 6 fish from each group were used for the determination of intestinal digestive enzyme activity, and the intestines of 9 fish from each group were placed in sterile, enzyme-free cryovials for intestinal flora analysis. All samples were first stored in liquid nitrogen for later use, and then transferred to a -80 ℃ freezer.

[0081] (6) Calculation of growth performance The body length and weight of each fish were measured and recorded on day 0 and day 60, respectively. The specific formulas for calculating growth performance indicators are as follows: Survival ratio (SR, %) = 100 × N t / N 0 ; Weight gain rate (WGR, %) = 100 × ( W t - W 0 ) / W 0 ; Feed conversion ratio (FCR, %) = I / ( W t - W 0 + W d ); Specific growth rate (SGR, % / d) = 100 × (ln W t - ln W 0 ) / t ; Hepatosomatic index (HSI, %) = 100 × W L / W t ; Visceral ratio (viscerosomatic index, VSI, %) = 100 × W V / W t ; Condition factor (CF, %) = 100 × ( W t / L 3 ).

[0082] in: I This indicates the quality of the feed fed into the bucket; N 0 and N t These represent the number of large yellow croakers in the bucket at the beginning and end of the experiment, respectively. W 0 (g) and W t (g) represent the initial and final weights of the experimental fish, respectively; t (d) represents the experimental time;W d (g) represents the mass of the dead fish in the bucket; W L (g) and W V (g) represents the liver and viscera mass of the experimental fish in the barrel, respectively; L (cm) represents the length of the experimental fish.

[0083] (7) Measurement of intestinal digestive indicators Intestinal digestion assays included α-amylase (AMS), trypsin, and lipase (LPS). The assays were performed using kits purchased from Nanjing Jiancheng Biotechnology Research Institute, following the instructions provided. Three samples were collected from each group, and each sample was analyzed in triplicate.

[0084] (8) Measurement of liver antioxidant indicators The liver antioxidant indicators included: total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA). The assays were performed using kits purchased from Nanjing Jiancheng Biotechnology Research Institute, following the instructions provided. Three samples were collected from each group, and each sample was tested in triplicate.

[0085] (9) Serum immune marker measurement Serum immunoassay indicators included: total protein (TP), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (ALP). The assays were performed using kits purchased from Nanjing Jiancheng Biotechnology Research Institute, following the instructions provided in the manufacturer's manual. Three samples were collected from each group, and each sample was analyzed in triplicate.

[0086] (10) Vibrio harveyi infection test Vibrio harveyi was cultured overnight in BHI liquid medium at 28 °C and 200 r / min. The bacterial culture was then centrifuged at 3000 r / min and 4 °C for 10 min in a high-speed refrigerated centrifuge, followed by resuspending in sterile PBS solution and adjusting the concentration to 10. 7 CFU / mL. On day 60 of the experiment, 80 large yellow croakers were randomly selected and divided into 3 experimental groups (BS9+V. harveyi group, BS7+V. harveyi group, BS0+V. harveyi group) and 1 control group (PBS group). Each large yellow croaker in the experimental group was injected with 200 μL of PBS at a concentration of 10 CFU / mL using a single sterile intraperitoneal injection method. 7 The experimental fish were injected with a CFU / mL Vibrio harveyi suspension, while the control group was injected with an equal volume of sterile PBS solution. The fish were cultured in a recirculating aeration tank at 20 ± 2 ℃ with continuous aeration and no feeding. They were observed for 60 hours after injection, and the growth and mortality of the large yellow croaker in each group were recorded.

[0087] (11) Extraction of gut microbiota DNA, 16S rRNA amplification and high-throughput sequencing of healthy control and Q16 treatment group fish The steps are the same as those in Example 1.

[0088] (12) RNA extraction, cDNA library construction and sequencing from the livers of healthy controls and Q16-treated fish The samples were collected using a pooled sampling method. The control group was the BS0 group (CL group), and the experimental group was the BS7 group (LL group), which showed better growth performance. Nine livers from each of the healthy control group (CL) and the Q16 treatment group (LL) were collected, with three livers pooled together to form three biological replicates, named CL1, CL2, CL3 (control group) and LL1, LL2, LL3 (experimental group), respectively. Total RNA was extracted using the TRIzol Reagent Kit (Invitrogen, USA). Purity and integrity were assessed using agarose gel electrophoresis, and RNA integrity and total amount were accurately measured using Qubit 2.0 and Agilent 2100. Subsequently, mRNA was enriched using Oligo(dT) magnetic beads to construct a cDNA library. The constructed library was analyzed using an Agilent 2100, and the effective concentration of the library was accurately quantified by qRT-PCR. After quality control, transcriptome sequencing was performed using the NovaSeq 6000 sequencing platform (Beijing Novogene).

[0089] (13) Differentially expressed gene analysis and functional annotation After removing adapter sequences, sequences with indeterminate base information, and low-quality sequences from the raw data, clean data was obtained for subsequent analysis. A reference genome was constructed using HISAT2 (v2.0.5) and genome alignment was performed. Gene expression levels in the control group (CL) and experimental group (LL) were quantified based on FPKM values. Differential expression analysis between the two groups was performed using DESeq2 (1.20.0) software. The difference in gene expression levels between the large yellow croaker control group and the Q16 treatment group was screened using an adjusted p-value Padj < 0.05 and |log2(FoldChange)| > 1. GO and KEGG pathway enrichment analysis was performed on differentially expressed genes using clusterProfiler (3.8.1) software.

[0090] (14) Data processing and statistical analysis Refer to Example 1.

[0091] 2. Experimental Results (1) Effects of Bacillus subtilis Q16 on the growth performance of large yellow croaker Compared with the BS0 group, the weight gain rate (WGR) and specific growth rate (SGR) of the BS7 group were significantly improved (P<0.05). The hepatobiliary-to-body ratio (HSI) and visceral-to-body ratio (VSI) of the BS7 and BS9 groups were significantly higher than those of the BS0 group (P<0.05), but there was no significant difference between the BS7 and BS9 groups (P>0.05). There were no significant differences in initial body weight (IBW), final body weight (FBW), survival rate (SR), feed conversion ratio (FCR), and condition factor (CF) among the groups (P>0.05). This indicates that the addition of Bacillus subtilis Q16 to the diet can significantly improve the growth performance of large yellow croaker (Table 7).

[0092] Table 7. Effects of feeding Bacillus subtilis Q16 on the growth performance of large yellow croaker.

[0093] Note: Different lowercase letters in the superscript indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0094] (2) Effects of Bacillus subtilis Q16 on intestinal digestive indicators of large yellow croaker Compared with group BS0, trypsin activity was significantly increased in both groups BS7 and BS9, with significant differences between groups (P<0.05). Meanwhile, lipase and α-amylase activities were higher in group BS9 than in groups BS7 and BS0, but the differences between groups were not significant (P>0.05). This indicates that adding Bacillus subtilis Q16 to the feed helps to improve the activity of intestinal digestive enzymes in large yellow croaker (Table 8).

[0095] Table 8. Effects of feeding Bacillus subtilis Q16 on intestinal digestion in large yellow croaker.

[0096] Note: Different lowercase letters in the superscript indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0097] (3) Effects of Bacillus subtilis Q16 on antioxidant indices in the liver of large yellow croaker Compared with group BS0, group BS7 showed a significant increase in total antioxidant capacity (T-AOC) (P<0.01), and catalase (CAT) levels were significantly increased in both groups BS7 and BS9 (P<0.001), although there was no significant difference between groups BS7 and BS9 (P>0.05). Superoxide dismutase (SOD) levels showed no significant difference among groups (P>0.05). Meanwhile, malondialdehyde (MDA) concentrations showed significant differences among groups (P<0.05), with group BS7 showing the lowest concentration, followed by group BS9, and group BS0 showing the highest concentration. This indicates that adding Bacillus subtilis Q16 to the feed helps improve the antioxidant capacity of large yellow croaker liver (Table 9).

[0098] Table 9. Effects of feeding Bacillus subtilis Q16 on the antioxidant capacity of large yellow croaker liver.

[0099] Note: Different lowercase letters in the superscript indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0100] (4) Effects of Bacillus subtilis Q16 on serum immune indicators of large yellow croaker There were no significant differences in serum total protein (TP) content among the different groups (P>0.05). The concentration of acid phosphatase (ACP) in group BS9 was significantly higher than that in groups BS0 and BS7 (P<0.05). The concentration of alkaline phosphatase (ALP) in group BS7 was significantly higher than that in group BS0 (P<0.05), but there was no significant difference between group BS9 and the two other groups (P>0.05). Lysozyme (LZM) showed significant differences among all groups (P<0.01), with the highest concentration in group BS7, followed by group BS9, and the lowest concentration in group BS0. This indicates that adding Bacillus subtilis Q16 to the feed helps improve the serum immunity of large yellow croaker (Table 10).

[0101] Table 10 Effects of feeding Bacillus subtilis Q16 on serum immunity in large yellow croaker

[0102] Note: Different lowercase letters in the superscript indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0103] (5) Bacillus subtilis Q16 improves the survival rate of large yellow croaker against Vibrio harveyi. Following infection with *Vibrio harveyi*, the survival rate of large yellow croaker in the experimental groups decreased over time, while the control group (PBS) maintained a 100% survival rate. Specifically, the survival rate of the BS7 group was consistently higher than that of the BS9 and BS0 groups at different time points. After 60 hours, the BS7 group still maintained a 50% survival rate, while the BS0 group only achieved 25%. These results indicate that *Bacillus subtilis* Q16 improved the survival rate of large yellow croaker infected with *Vibrio harveyi*. Figure 6 ).

[0104] (6) Effects of Bacillus subtilis Q16 on the intestinal flora of large yellow croaker A. Effects of Bacillus subtilis Q16 on the diversity and composition of gut microbiota in large yellow croaker 16S rRNA was amplified by PCR and then sequenced using high-throughput sequencing. A total of 1,444,168 optimized sequences were obtained from all samples, achieving 100% sequencing coverage. (From the dilution curves...) Figure 7A) As can be seen, the curves of all three groups of samples tend to be stable, indicating that the number of species does not increase significantly with the increase of sequencing depth, suggesting that the sequencing results have basically reached saturation and the sequencing depth has met the requirements for data analysis. Based on the Venn diagram of OTU distribution for different samples (…),… Figure 7 B) It can be seen that, after 97% similarity classification, BS0 and BS7 groups shared 51 OTUs, BS0 and BS9 groups shared 86 OTUs, and BS7 and BS9 groups shared 41 OTUs, for a total of 74 shared OTUs across the three groups. Most OTUs were specific to different groups, with BS0, BS7, and BS9 groups having 987, 583, and 366 OTUs specific, respectively. Alpha diversity analysis of gut microbiota in the three groups showed (Table 11) that the richness and diversity of the microbiota increased with increasing Bacillus subtilis concentration in the feed, but feeding with Bacillus subtilis Q16 did not significantly affect the Alpha diversity index of gut microbiota in large yellow croaker (P>0.05).

[0105] Table 11 Effects of feeding Bacillus subtilis Q16 on the α-diversity of gut microbiota in large yellow croaker

[0106] Note: Different lowercase letters in the superscript indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0107] Sample NMDS analysis and species cluster analysis were used to determine the similarity and differences between and within groups of samples. NMDS analysis based on Bray curtis distance (…) Figure 7 C; Stress = 0.050) and cluster analysis based on Weighted Unifrac distance ( Figure 7 D) shows that groups BS7 and BS9 showed better clustering, indicating a high similarity in species composition between the two groups. Group BS0, however, was significantly dispersed from groups BS7 and BS9, indicating a certain difference in bacterial composition between group BS0 and the other two groups. Sequence alignment and annotation of the gut microbiota of the three groups of fish samples were performed, and the bacterial groups with the largest proportions at the phylum and genus levels in each group were selected to generate relative abundance bar charts. Figure 7 E; Figure 7(F) This allows for a visual examination of the species with relatively high abundance and proportion at the phylum and genus levels in each group. The results showed that the dominant phyla in the gut microbiota of the three large yellow croaker groups were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Compared to group BS0, the relative abundance of Firmicutes in group BS7 increased significantly (P<0.05). Genera such as *Cupriavidus* and *Faecalibacterium* were the dominant genera in the gut microbiota of the three large yellow croaker groups.

[0108] B. Effects of Bacillus subtilis Q16 on the differential gut microbiota of large yellow croaker Heatmaps can more intuitively reflect the differences in gut microbiota at the genus level among different groups of large yellow croaker. Figure 8 A).

[0109] LEfSe analysis was performed on the gut microbiota of the three groups of large yellow croaker using LDA>3.5 as the threshold to identify gut microbiota with significant differences at the genus and species levels among the different groups (P<0.05). The results showed that, compared with the BS7 group, *Streptococcus*, *Blautia*, and *Blautia glucerasea* were found to be specific gut microbiota in the BS7 group, while *Brevundimonas* was a specific gut microbiota in the BS0 group. Figure 8 B, E); In the comparison between BS0 and BS9 groups, *Faecalibacterium*, *Bacteroides*, and *Oscillibacter* were found to be specific bacterial groups in the intestines of large yellow croaker in group BS9, while no significantly different specific genera or species were found in group BS0. Figure 8 C, F); In the comparison between BS7 and BS9 groups, *Clostridium* and *Bacteroides* were found to be specific bacterial groups in the intestines of large yellow croaker in BS9 group, while UCG 005 was a specific bacterial group in the intestines of large yellow croaker in BS7 group. Figure 8 D).

[0110] C. Effects of Bacillus subtilis Q16 on the potential function of gut microbiota in large yellow croaker Based on gut microbiota sequencing results, the metabolic pathways of three groups of large yellow croaker were statistically analyzed and KEGG function was predicted. Figure 9The results showed that gut microbiota function was distributed across all six major metabolic pathways, with the BS7 and BS9 groups exhibiting more similar microbial functions. Specifically, the BS0 group had the highest proportion of microbes associated with metabolism and human diseases, while the BS9 group had the highest proportion of microbes associated with organismal systems. Furthermore, the expression of pathways such as environmental information processing, gene information processing, and cellular processes was higher in the BS7 and BS9 groups than in the BS0 group.

[0111] D. Effects of Bacillus subtilis Q16 on the liver transcriptome of large yellow croaker a. mRNA sequencing results Liver transcriptome sequencing was performed on the large yellow croaker control group (CL) and Q16 treatment group (LL). Illumina sequencing results showed that the control group and Q16 treatment group yielded 22.53 G and 20.73 G of raw data, respectively. After removing sequences with adapters, sequences with indeterminate base information, and low-quality sequences, 21.81 G and 20.32 G of clean data were obtained, respectively. The proportion of clean bases was not less than 96.54% in both groups, and the overall sequencing error rate was 0.01%. Base composition showed an average GC content of 50.03%, and base quality showed that the proportion of Q30 in each group was not less than 96.28% (Table 12).

[0112] Table 12 Statistical summary of mRNA sequencing data

[0113] Note: samples: sample name; raw reads: number of reads in the raw data; raw bases: number of bases in the raw data; clean reads: number of reads after filtering the raw data; clean bases: number of bases after filtering the raw data; error rate: overall sequencing error rate; Q30: percentage of bases with a Phred value greater than 30; GCcontent: percentage of G and C bases in the clean reads.

[0114] b. Differential gene expression analysis The healthy control group (CL) and the Q16 treatment group (LL) had a total of 12,224 co-expressed genes. Figure 10 A). The differences in gene expression levels between the large yellow croaker control group and the Q16 treatment group were screened using Padj < 0.05 and |log2(FoldChange)| > 1 as criteria. Figure 10B). The graph is plotted with log2(FoldChange) on the x-axis and -lg(Padj) on the y-axis; the larger the absolute value, the greater the difference. Each dot in the graph represents a gene: green dots represent downregulated genes, red dots represent upregulated genes, and gray dots represent genes with no significant expression difference. The results showed that, compared to the healthy control (CL), daily probiotic administration led to significant changes in 172 genes (DEGs) in the probiotic-treated group (LL) fish, including 64 upregulated and 108 downregulated differentially expressed genes. Figure 10 D).

[0115] The differentially expressed genes between the control group and the Q16 treatment group were merged to form the differentially expressed gene set. Hierarchical clustering was then used to perform cluster analysis on the FPKM values ​​of the genes to compare the expression of the same gene in different samples. In the heatmap, genes or samples with similar expression patterns are clustered together, with red representing genes with high expression levels and green representing genes with low expression levels. Figure 10 C). The results showed that, compared with the intragroup differences, there were significant differences in gene expression between the Q16 treatment group (LL) and the control group (CL); after adding the Q16 strain to the feed, the expression levels of a large number of genes were significantly upregulated and downregulated, and these genes may be involved in the interaction pathways related to the large yellow croaker microbiota.

[0116] c. GO and KEGG annotation and enrichment analysis of differentially expressed genes To further investigate the biological functions of 172 differentially expressed genes, this experiment used clusterProfiler software to perform GO (Gene Ontology) enrichment analysis of the differentially expressed genes. A total of 723 GO terms were enriched, including 471 biological processes (BP), 59 cellular components (CC), and 193 molecular functions (MF). This experiment selected the 30 most significantly enriched GO terms... Figure 11A). Among molecular functional types, differentially expressed genes for chemokine activity, chemokine receptor binding, cytokine activity, G protein-coupled receptor binding, and cytokine receptor binding were highly abundant. Significant enrichment of differentially expressed genes for phospholipid transporter activity, organic hydroxyl compound transmembrane transporter activity, amide transmembrane transporter activity, ligase activity, and anion transmembrane transporter activity was also observed. Among cellular component functional types, differentially expressed genes for extracellular regions, organelle membranes, membranes, organelle membranes, mitochondrial membranes, mitochondrial envelopes, and mitochondrial portions were highly abundant. The main biological processes were carbohydrate metabolism and immune responses, characterized by upregulated genes. Genes related to intrailiac transport, estrogen-stimulated cellular responses, estrogen responses, glucose metabolism, microtubule transport, microtubule transport, localization of protein complexes, and regulation of protein kinase activity were highly enriched. These GO terms are rich in metabolic-related gene information, indicating that the liver of large yellow croaker underwent metabolic regulation processes after Q16 administration.

[0117] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database integrating genomic, chemical, and systemic functional information. This experiment enriched 87 KEGG pathways, and selected 20 KEGG pathways with significant differences for analysis. Figure 11 B). The six pathways most enriched in the differentially expressed KEGG genes include: the PPAR signaling pathway, glycine, serine, and threonine metabolism, valine, leucine, and isoleucine degradation, the insulin signaling pathway, carbon metabolism, and alanine, aspartic acid, and glutamate metabolism. These KEGG pathways may act as major factors in the gene regulation of amino acid and glycolipid metabolism in large yellow croaker by strain Q16. In addition, glycolysis / gluconeogenesis, glycerol lipid metabolism, amino acid biosynthesis, and glyoxylate and dicarboxylate metabolism contain a wealth of genetic information on carbohydrate, lipid, and amino acid metabolism, and also have certain research value.

[0118] 3. Discussion (1) Effects of Bacillus subtilis Q16 on growth performance and intestinal digestive activity of large yellow croaker Bacillus subtilis, as a probiotic for aquaculture, possesses characteristics such as safety, acid and alkali resistance, low salt tolerance, high temperature resistance, and strong enzyme activity. It can secrete various digestive enzymes required for fish growth, breaking down complex nutrients into short-chain fatty acids and essential amino acids, thus promoting fish growth and improving their nutritional and digestive functions. As a feed additive, it can significantly improve fish growth performance and intestinal digestive enzyme activity. Due to differences in host, environment, and diet, the optimal addition amount of Bacillus subtilis has not yet been determined by a unified standard; the recommended concentration is 10%. 7 CFU / g - 10 9CFU / g of bacterial agent is considered to be effective in exerting probiotic effects in most scenarios. In this example, compared to the control group, the added concentration was 10. 7 CFU / g of Bacillus subtilis Q16 significantly increased the weight gain and specific growth rate of large yellow croaker, at a concentration of 10. 9 While the concentration of CFU / g of Bacillus subtilis increased, it was not statistically significant, indicating that lower concentrations showed a better growth advantage. However, both groups significantly increased the liver-to-body ratio and viscera-to-body ratio. Simultaneously, both groups significantly increased intestinal protease activity, which may be related to the high protease activity of the selected strains. Furthermore, a concentration of 10... 9 The CFU / g concentration of Bacillus subtilis showed higher intestinal digestive enzyme activity than the other two groups, indicating that the high concentration of the bacterial agent exhibited superior digestive capacity.

[0119] (2) Effects of Bacillus subtilis Q16 on antioxidant and immune function of large yellow croaker Bacillus subtilis can scavenge excess free radicals in fish and resist oxidative stress. The activity level of antioxidant enzymes can reflect the body's antioxidant level to some extent. In this example, adding Bacillus subtilis Q16 to the feed significantly increased the total antioxidant capacity and catalase activity in the liver of large yellow croaker, and significantly reduced malondialdehyde concentration. The addition concentration was 10... 7 The CFU / g concentration showed better results. In this example, the addition of Q16 had no significant effect on the total serum protein of large yellow croaker, but it significantly increased the activities of serum acid phosphatase, alkaline phosphatase, and lysozyme, with the addition concentration being 10%. 7 The CFU / g concentration showed superior efficacy. Furthermore, Bacillus subtilis exhibits strong pathogen-inhibiting capabilities, effectively suppressing the growth of marine pathogens such as *Photobacterium damselae* and *Shewanella*. Feeding fish with probiotic-supplemented feed followed by pathogen challenge is an effective method for evaluating the protective potential of probiotics. The Bacillus subtilis Q16 screened in this example demonstrated inhibitory capabilities against five tested pathogens. Challenge experiments showed that the survival rates of groups with different concentrations of the bacterial agent after infection with *Vibrio harveyi* were all higher than the control group, verifying that Q16 helps improve the immunity of large yellow croaker against pathogens such as *Vibrio harveyi*.

[0120] (3) Effects of Bacillus subtilis Q16 on gut microbiota and differentially expressed genes in large yellow croaker The gut microbiota regulates host immune responses, metabolic regulation, and digestive processes through the gut-brain axis to maintain homeostasis. The types and quantities of bacteria have a significant impact on the gut and overall health of fish; changes in the level and composition of the microbiota may indicate host immune function and health levels. Adding probiotics to feed has been found to have the potential to regulate the gut microbiota of fish. Feeding carp (Cyprinus carpio) with Bacillus subtilis significantly altered its gut microbiota at both the phylum (Fusobacteria and Proteobacteria) and genus (Cetobacter). In this example, feeding large yellow croaker with Bacillus subtilis Q16 did not significantly affect the alpha diversity index of the gut microbiota, but it significantly increased the relative abundance of Firmicutes in group BS7 and the relative abundance of Clostridium plasmids in group BS9; specific bacteria producing short-chain fatty acids were significantly enriched in groups BS7 and BS9. The better clustering effect in groups BS7 and BS9 indicates that Q16 altered the composition of the large yellow croaker gut microbiota and made the composition of the groups with added bacteria more similar. Microbial function predictions indicated that the microbial functions of the Q16 treatment groups (BS7 and BS9 groups) were more similar.

[0121] In this embodiment, the addition of Bacillus subtilis Q16 to the feed caused significant changes in 172 genes (DEGs) in the experimental group fish. These genes were distributed in signaling pathways such as chemokines, chemokine receptor binding, cytokines, PPARs, and amino acid metabolism, and were mainly involved in immune and metabolic regulation-related activities.

[0122] In summary, Bacillus subtilis Q16 significantly improved the growth performance and intestinal protease activity of large yellow croaker; significantly enhanced the activity of liver antioxidant enzymes and serum immunoenzymes in large yellow croaker, and improved the ability of large yellow croaker to resist Vibrio harveyi; altered the composition of the intestinal flora of large yellow croaker, with a significant increase in the relative abundance of beneficial bacteria; the flora function of different concentrations of Q16-added groups was more similar to that of the control group; significantly upregulated the expression of genes related to pathways such as hepatic chemokines and chemokine receptor binding in large yellow croaker, and significantly enriched them in pathways such as PPAR signaling, glycine, serine, and threonine metabolism.

[0123] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0124] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0125] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Bacillus subtilis Q16 ( Bacillus subtilis ), characterized in that, It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 20252232, on October 16, 2025.

2. The Bacillus subtilis Q16 according to claim 1, characterized in that, The rDNA sequence of Bacillus subtilis Q16 is shown in SEQ ID NO.

1.

3. The use of Bacillus subtilis Q16 as described in claim 1 in the preparation of an additive to promote the growth of large yellow croaker.

4. The application of Bacillus subtilis Q16 as described in claim 1 in the preparation of a microecological preparation for improving the growth performance of large yellow croaker.

5. A microecological preparation containing Bacillus subtilis Q16 as described in claim 1.

6. The application of Bacillus subtilis Q16 as described in claim 1 in the preparation of large yellow croaker feed.

7. A feed for raising large yellow croaker, characterized in that, Contains Bacillus subtilis Q16 as described in claim 1.

8. The feed according to claim 7, characterized in that, The viable count of the Bacillus subtilis Q16 was 1×10⁻⁶. 5 ~1×10 9 CFU / g.

9. The use of Bacillus subtilis Q16 as described in claim 1 in the preparation of a formulation for preventing and treating Vibrio harveyi infection in large yellow croaker.

Images