Bacillus Velez Bv-con2 and its application in turbot farming

Abstract

The present invention discloses Bacillus velezinis Bv-con2 and its application in turbot farming, belonging to the field of microbial technology. The strain is deposited in the China Center for Type Culture Collection, with a deposit address of Wuhan, China, a deposit date of December 27, 2024, and a deposit number of CCTCC NO: M 20242926. The present invention is beneficial in that: the strain is non-pathogenic to turbot, has good probiotic potential, and after being fermented into a microbial agent and mixed with and added to turbot, it can not only promote the growth of farmed turbot, increase the activity of trypsin and superoxide dismutase of turbot, improve the structure of turbot intestinal flora, and enhance the level of microbial diversity in the intestinal tract of turbot, but also delay the morbidity and death of turbot caused by infection with Aeromonas salmonicida BHAS-1, Edwardsiella piscicidalis H4-S18, Streptococcus paramammary BH2-4, and Pseudomonas putida PPD2.

Description

Technical Field

[0001] The invention relates to a Bacillus Velez and an application thereof, in particular to Bacillus Velez Bv-con2 and an application thereof in turbot farming, belonging to the technical field of microorganisms. Background Art

[0002] turbot Scophthalmus maximus Turbot, commonly known as turbot, is a coldwater, benthic marine fish and an important marine aquaculture species in my country's northern coastal areas. However, with the expansion of aquaculture, factors such as genetic degradation, deteriorating water quality, and the feeding of fresh, chilled fish have led to frequent diseases in farmed turbot. The indiscriminate use of drugs not only causes drug resistance in pathogens but also leads to high levels of fishery drug residues in fish, compromising the quality and safety of aquatic products and seriously hindering the healthy and green development of the industry. Reducing the use of antibiotic fishery drugs and pursuing green and healthy disease prevention methods are the best solutions to address the current disease problem.

[0003] The use of probiotics can prevent and control diseases at the source, and has played a positive role in reducing the invasion of pathogens, strengthening the nutritional structure of feed, improving digestion performance, enhancing the body's resistance to stress, and improving the aquaculture water environment. It has become one of the effective strategies for improving the health of farmed organisms in the aquaculture industry.

[0004] Bacillus ( Bacillus ) is a type of rod-shaped, spore-forming Gram-positive bacteria with strong stress resistance, high enzyme production ability, and fast growth. Because it is widely present in soil, on the surface and inside of plants, it is non-toxic to humans and animals and has broad-spectrum antibacterial activity, which gives it significant advantages in biological disease prevention.

[0005] Bacillus velez ( Bacillus velezensis ) As a new species of Bacillus, it can produce a variety of active antibacterial substances during its growth and reproduction. Its metabolites have broad-spectrum antibacterial activity and are often used in the field of disease and pest control in crops, livestock and poultry, with obvious application effects. Summary of the Invention

[0006] The purpose of the present invention is to provide a strain of Bacillus velez Bv-con2 derived from the environment (sea cucumber culture pond), which can promote the growth and development of turbot, improve the intestinal flora structure of turbot, and enhance the immunity of turbot, and its use as a turbot probiotic in turbot culture.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] Bacillus Velez Bv-con2, isolated from sea cucumber aquaculture pond, was classified and named Bacillus Velez Bv-con2 Bacillus velezensis Bv-con2, deposited in the China Center for Type Culture Collection, Wuhan, China, on December 27, 2024, with the deposit number CCTCC NO: M 20242926.

[0009] The application of the aforementioned Bacillus Velez Bv-con2 in turbot farming includes:

[0010] (1) Use of the microbial agent in the preparation of a microbial agent for improving the growth performance of turbot, wherein the growth performance includes body length and weight;

[0011] (2) Application in the preparation of bacterial agents for promoting the digestion and absorption of nutrients by turbot;

[0012] (3) Application in the preparation of microbial agents for improving the intestinal flora structure of turbot;

[0013] (4) Use of the present invention in the preparation of a bacterial agent for resisting infection by any one or more pathogens selected from Aeromonas salmonicida BHAS-1, Edwardsiella piscicidalis H4-S18, Streptococcus parauberis BH2-4 and Pseudomonas putida PPD2.

[0014] Preferably, the preparation method of the bacterial agent is as follows:

[0015] Bacillus velezensis Bv-con2 was inoculated into the fermentation medium and fermented at 36-38 °C for 48 h to obtain a concentration of ≥1×10 8 CFU / mL of Bacillus velez Bv-con2 fermentation broth, wherein the fermentation medium has a formula of: glucose 6-15 g / L, corn flour 6-10 g / L, yeast extract 3-5 g / L, potassium dihydrogen phosphate 0.1-3 g / L, magnesium sulfate 0.5-1 g / L, and pH = 5.5-7.0.

[0016] The present invention is beneficial in that:

[0017] (1) The Bacillus Velezii Bv-con2 obtained by the present invention is non-pathogenic to turbot, and analysis at the genetic level shows that it has good probiotic potential;

[0018] (2) When the fermentation liquid (microbial agent) of Bacillus Velez Bv-con2 obtained by the present invention is mixed with the feed and fed to farmed turbot, it can promote the growth of farmed turbot, increase the activity of turbot digestion-related enzymes (trypsin) and antioxidant-related enzymes (superoxide dismutase), improve the structure of turbot intestinal flora, and comprehensively enhance the level of microbial diversity in the turbot intestine;

[0019] (3) After 60 days of mixed feeding, the immune system of turbot was greatly improved. In particular, after infection with common turbot pathogens (Aeromonas salmonicida BHAS-1, Edwardsiella piscicidalis H4-S18, Streptococcus paramammary BH2-4, and Pseudomonas putida PPD2), the mortality rate was significantly improved compared with the control group. The experimental results show that the exogenous addition of Bacillus Velezii Bv-con2 can effectively improve the disease resistance of turbot larvae. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the Gram staining result of strain Bv-con2. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to specific embodiments.

[0022] 1. Strain Screening

[0023] In February 2024, environmental sediment samples were collected from three different areas of a sea cucumber aquaculture pond at a sea cucumber aquaculture farm in Penglai District, Yantai City, Shandong Province: the water inlet, the center of the pond bottom, and the outlet. After removing the surface floating mud at the sampling points, 5-10 cm deep sediment was sampled. 100 g of sediment sample was collected from each site, placed in sterile sealed bags, and stored at -80°C for later use. 10 g of sediment sample from each site was added to 90 mL of sterile PBS buffer and shaken to mix. Sterilized glass beads with a diameter of 0.1-0.5 mm were then added and vortexed for 10 minutes. The sample was then centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected. The operation was repeated twice, and the supernatants were combined to prepare environmental microbial samples for the water inlet, the center of the pond bottom, and the outlet, respectively, for later use.

[0024] The above environmental microbial samples were subjected to 10 -1 to 10 -5 Gradient dilution was performed, and 10 μL of each gradient was spread on a nutrient agar plate supplemented with 2 wt% NaCl. The plate was inverted and cultured at 28°C for 48 h. Colonies of different morphologies (26 in total) were picked and purified and cultured separately until a pure culture was obtained. The culture was finally preserved with glycerol at a final volume fraction of 40% and stored in a -80°C refrigerator for future use.

[0025] The 26 purified strains were subjected to 16S rRNA sequence determination and comparison, and strains with potential beneficial functions were selected for subsequent experiments. A microbial sample from the drainage outlet of the sea cucumber aquaculture pond was initially screened out, 10 -3 The strain obtained after gradient dilution was used for subsequent experiments and was recorded as Bv-con2.

[0026] 2. Morphological characteristics and physiological and biochemical properties of strain Bv-con2

[0027] 1. Morphological characteristics

[0028] The strain Bv-con2 appeared milky white and opaque on the nutrient agar medium, and the colony surface was rough, with a bulge in the middle and wrinkles.

[0029] The Gram staining results of strain Bv-con2 are shown in Figure 1 The staining results showed that the strain Bv-con2 was a Gram-positive short bacillus.

[0030] 2. Physiological and biochemical characteristics

[0031] The strain Bv-con2 was identified by IMVC biochemical system using Escherichia coli dry preparation biochemical identification kit. The identification results showed that the strain Bv-con2 was methyl red negative, indole positive, VP positive, and citrate positive.

[0032] 3. Identification and Whole Genome Information of Strain Bv-con2

[0033] 1. Strain identification

[0034] (1) 16S rRNA sequencing comparison

[0035] The 16S rRNA of strain Bv-con2 was amplified by PCR using bacterial 16S rRNA universal primers (27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No: 2), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No: 3)) to obtain the 16S rRNA nucleotide sequence of strain Bv-con2 (SEQ ID No: 1).

[0036] The results of NCBI online BLAST homology comparison showed that strain Bv-con2 and strain Bacillus velezensis CBMB205 (NR_116240.1) with a similarity of 97.68%.

[0037] The strain Bv-con2 was preliminarily identified as Bacillus velezensis.

[0038] (2) Whole genome sequencing comparison

[0039] strain Bv-con2 and strain Bacillus velezensis CBMB205 The average nucleotide identity (ANI) ranged from 97.87% to 99.82%, and the genomic digital DNA hybridization (dDDH) values ​​ranged from 78.22% to 86.41%. The ANI values ​​were lower than the classification threshold of 95% and the dDDH values ​​were lower than 70% with other Bacillus species.

[0040] Identification results: Combined with the results of 16S rRNA sequencing and physiological and biochemical characteristics, the strain Bv-con2 was identified as Bacillus Velezii and was recorded as Bacillus Velezii Bv-con2 (Latin name: Bacillus velezensis Bv- con2 ).

[0041] 2. Genomic information and functional prediction

[0042] To better understand the genome information of strain Bv-con2, we extracted the whole genome DNA of strain Bv-con2 using a kit and completed whole genome sequencing on the Illumina HISsq platform. FastQC was used to analyze the sequencing data of the sample, and the statistical results showed that:

[0043] (1) The full length of the genome of strain Bv-con2 is 3891358 bp, with a GC content of 46.39%. The gene prediction results showed that a total of 3840 coding genes were annotated, with a total length of 3473842 bp, and functional genes covered 89.27% ​​of the total length of the genome.

[0044] (2) In the prokaryotic COG database, 2939 coding genes were annotated in 21 categories, among which the coding genes annotated in the three functional categories of amino acid transport and metabolism, carbohydrate transport and metabolism, and transcription accounted for the largest number, accounting for 24.67%.

[0045] (3) 3,376 genes were annotated in the GO database of the gene function classification system, and 61.55%, 26.07% and 12.38% of the annotated coding genes were involved in biological processes, molecular functions and cellular composition, respectively.

[0046] (4) 1,630 genes were annotated in the KEGG biosystems database, among which the coding genes involved in metabolism accounted for the largest proportion, accounting for 77.67%.

[0047] (5) The gene set protein sequences were compared with the CAZy database, and 65 carbohydrate-active enzyme genes were annotated, including 6 oxidoreductases, 12 carbohydrate lipolytic enzymes, 27 glycoside hydrolases, 18 glycosyltransferases, and 2 polysaccharide lyases.

[0048] (6) The total number of strain proteins was 3749, of which 2793 were predicted CYT cytoplasmic proteins and 251 were predicted Signal P-TM signal peptide proteins.

[0049] 4. Safety Testing of Strain Bv-con2

[0050] The strain Bv-con2 stored in a -80℃ refrigerator was inoculated on a nutrient agar plate supplemented with 2wt% NaCl for activation. Then, a single colony in the exponential growth phase was picked and placed in nutrient broth. The culture was expanded at 28℃ with shaking. The culture medium was collected and finally prepared into a 10% PBS buffer solution. 9 The bacterial suspension with a concentration of CFU / mL is ready for use.

[0051] Healthy turbot larvae with an average body weight of 0.91 g ± 0.06 g were purchased (healthy manifestations include: individuals are energetic, no obvious lesions on the body surface, and no parasitic infection on the body surface and gills observed under a microscope), temporarily raised for one week, and then randomly divided into a low-concentration injection group, a medium-concentration injection group, a high-concentration injection group, an injection control group, and a blank control group, with 10 turbot larvae in each group.

[0052] The low concentration injection group, medium concentration injection group and high concentration injection group were injected with 10 7 CFU / mL, 10 8 CFU / mL, 10 9 The control group was injected with 100 μL of a suspension of strain Bv-con2 containing 100 CFU / mL of bacteria, while the blank control group received no treatment. The mortality of turbot larvae in each group was then closely observed.

[0053] After observation, 14 days later, there were 0, 1 and 0 turbot fry deaths in the low concentration injection group, medium concentration injection group and high concentration injection group respectively, 1 turbot fry died in the injection control group and blank control group respectively, and the turbot fry that did not die showed no abnormalities. 7 CFU / mL-10 9 CFU / mL) of the strain Bv-con2 is relatively safe to healthy turbot.

[0054] Furthermore, the gene annotations did not include virulence genes commonly found in pathogenic Bacillus species, such as the hemolysin BL and enterotoxin NHE. Furthermore, only 5.87% of strain Bv-con2 had virulence factor annotations, indicating that strain Bv-con2 was essentially non-virulent.

[0055] 5. Preparation of microbial agents

[0056] In order to obtain a large amount of high-density bacterial liquid, the culture medium formula and fermentation conditions were optimized based on the carbon source and nitrogen source of the universal culture medium. The optimized culture medium formula and fermentation conditions are as follows:

[0057] Culture medium formula: glucose 6-15g / L, corn flour 6-10g / L, yeast extract 3-5g / L, potassium dihydrogen phosphate 0.1-3g / L, magnesium sulfate 0.5-1g / L, pH=5.5-7.0.

[0058] Fermentation conditions: seed liquid addition ratio 1%, fermentation temperature 36-38℃, fermentation time 48h.

[0059] In this embodiment, the culture medium formula is as follows: glucose 8g / L, corn flour 5g / L, yeast extract 3g / L, potassium dihydrogen phosphate 0.1g / L, magnesium sulfate 0.05g / L, pH=6.0. After fermentation at 37.5℃ for 48h, the concentration of the fermentation liquid (i.e., the inoculum) reached 2.6×10 9 CFU / mL.

[0060] The concentration of the fermentation broth was adjusted to 1×10 8 CFU / mL, which was used for subsequent probiotic addition experiments, and the fermentation broth was recorded as bacterial agent Bv-con2.

[0061] 6. Probiotics Addition Experiment

[0062] Turbot fry with an initial body weight of 0.82±0.01g and a body length of 2.81±0.02cm were randomly divided into two groups: a supplemented group and a control group, with 3 replicates in each group and 100 fish in each replicate, to carry out a comparative experiment on the effect of probiotic addition.

[0063] The control group was fed with flounder pellet feed purchased from the market, once in the morning and evening every day, at a rate of 3% of body weight; the supplemented group was fed with the same feed supplemented with the bacterial agent Bv-con2, once in the morning and evening every day, at a rate of 3% of body weight.

[0064] The preparation method of the same feed supplemented with the microbial agent Bv-con2 in the supplementation group is as follows: the microbial agent Bv-con2 (concentration 1×10 8 Spray the 100g (25mL) CFU / mL (0.5% Fibre, 0.05%) nutrient solution evenly onto commercially available flatfish pellets at a ratio of 25mL:100g. Mix thoroughly, air-dry, and then feed to the fish. To ensure viable bacterial counts, the feed supplemented with the microbial agent Bv-con2 was freshly prepared daily in the supplemented group.

[0065] 1. Effects of Bv-con2 in feed on the growth performance of turbot larvae

[0066] After 60 days of feeding, 15 fish were randomly sampled from each parallel group of the supplemented group and the control group, and their body length and weight were measured. The growth indicators such as weight gain rate and specific growth rate of the supplemented group and the control group were analyzed.

[0067] Weight gain rate (WGR, %) = [(W t -W0) / W0]×100%

[0068] Specific growth rate (SGR, % / d) = [(lnW t-lnW0) / t]×100%

[0069] Where W t W0 is the final body weight and W0 is the initial body weight.

[0070] The statistical results of the average body length and average weight of turbot larvae in the supplemented group and the control group, as well as the calculated results of weight gain rate and specific growth rate are shown in Table 1.

[0071] Table 1 Changes in growth indicators of turbot larvae after adding the microbial agent Bv-con2 to feed

[0072]

[0073] Note: The same superscript letters in the same column indicate no significant differences between groups ( p >0.05), different superscript letters indicate significant differences between groups ( p <0.05).

[0074] As shown in Table 1, the average weight, weight gain rate and specific growth rate of turbot larvae in the supplementation group were significantly different from those in the control group ( p <0.05).

[0075] The above results show that adding the bacterial agent Bv-con2 to the feed can effectively promote the growth of turbot fry.

[0076] 2. Effects of Bv-con2 in feed on the activities of digestive and antioxidant enzymes in turbot larvae

[0077] After 60 days of feeding, nine random samples were taken from each of the supplementation and control groups. The livers were rapidly dissected and isolated on an ice tray, rinsed with pre-chilled sterile PBS, and dried with filter paper. The liver samples were homogenized with 9 volumes of sterile saline in an ice-water bath and then centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was collected to obtain a 10% liver homogenate.

[0078] The activities of digestive enzymes such as trypsin (TPS), lipase (LPS), and amylase (AMS), and the activities of oxidases such as superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (ALP) in the liver homogenate were determined using kits.

[0079] The results of the activity determination of various digestive enzymes and oxidases in the liver of turbot larvae in the supplementation group and the control group are shown in Table 2.

[0080] Table 2 Changes in enzyme activities in the liver of turbot larvae after the addition of the microbial agent Bv-con2 to feed

[0081]

[0082] Note: The same superscript letters in the same column indicate no significant differences between groups ( p >0.05), different superscript letters indicate significant differences between groups ( p <0.05).

[0083] As shown in Table 2, except for amylase (AMS) and catalase (CAT), the activities of various digestive enzymes and oxidases in the liver of turbot fry in the supplemented group were higher than those in the control group. Among them, the differences in trypsin (TPS) and superoxide dismutase (SOD) were significant, while the differences in lipase (LPS) and alkaline phosphatase (ALP) were not significant.

[0084] These results are consistent with the predicted genome information of strain Bv-con2. Whole-genome sequencing data suggests that strain Bv-con2 may be able to degrade cellulose, chitin, and starch, and could be used as a feed additive or organic waste degrader.

[0085] The above results indicate that the addition of the bacterial agent Bv-con2 can promote the digestion and absorption of nutrients by the host turbot fry to a certain extent, and enhance the immune capacity.

[0086] 3. Effect of Bv-con2 in feed on intestinal flora diversity of turbot larvae

[0087] After 60 days of feeding, 9 turbot larvae were randomly sampled from each of the supplementation group and the control group, and the intestinal flora diversity of the turbot larvae in the supplementation group and the control group was tested. The test results are shown in Table 3.

[0088] Table 3 Changes in intestinal microbial diversity of turbot larvae after addition of the microbial agent Bv-con2 to feed

[0089]

[0090] As shown in Table 3, among the five indicators for evaluating bacterial diversity, namely Shannon index, Simpson index, ACE index, Chao index and PD index, the values ​​of the supplemented group were higher than those of the control group (the difference was not significant), and the intestinal bacterial abundance of turbot larvae in the supplemented group was higher.

[0091] The above results indicate that the addition of the bacterial agent Bv-con2 enriched the intestinal microbial species of turbot larvae to a certain extent and increased the diversity of intestinal microorganisms of turbot larvae.

[0092] After analysis, it was found that after 60 days of feeding, the supplementation group increased the abundance of Proteobacteria and Actinobacteriota, the core bacterial flora in the intestine of turbot larvae, especially the abundance of bacteria such as Rhodobacterium, Verrucomicrobia and Lactobacillus.

[0093] In summary, the addition of the bacterial agent Bv-con2 can not only increase the number of core flora in the intestine of turbot larvae, but also increase the abundance and diversity of intestinal microorganisms in turbot larvae, which has a positive effect on turbot larvae's resistance to adverse external environment.

[0094] VII. Effect of Bv-con2 in feed on disease resistance of turbot larvae

[0095] 1. Preparation of challenge strains

[0096] Aeromonas salmonicida BHAS-1, Edwardsiella piscicida H4-S18, Streptococcus parauberis BH2-4, and Pseudomonas putida PPD2 were selected for artificial infection. These strains are all common pathogens of turbot.

[0097] Aeromonas salmonicida BHAS-1, Edwardsiella piscicida H4-S18, Streptococcus parauberis BH2-4, and Pseudomonas putida PPD2 were activated, eluted with sterile PBS, and the concentration was adjusted to 10 8 CFU / mL, and placed in a 4°C refrigerator for subsequent artificial infection.

[0098] In addition, the pathogens with adjusted concentrations are mixed at a volume ratio of 1:1:1:1 to prepare a mixed pathogen for later use.

[0099] 2. Artificial infection

[0100] After the probiotics supplementation experiment, the turbot fry in the supplementation group and the control group were temporarily reared for one week. During the period, they were fed with a basic diet and the culture conditions remained unchanged. One week later, they were artificially infected.

[0101] Sixty fish were randomly sampled from each of the supplemented and control groups. The supplemented and control groups (180 fish) were then divided into six injection groups (five pathogen-injected groups and one control group), each with 30 fish. The five pathogen-injected groups received intraperitoneal injections of 100 μL of the above-mentioned adjusted concentrations of Aeromonas salmonicida BHAS-1, Edwardsiella piscicidalis H4-S18, Streptococcus parauberis BH2-4, Pseudomonas putida PPD2, and a mixed pathogen. The injection control group received an intraperitoneal injection of 100 μL of sterile PBS.

[0102] After infection, the number of dead turbot larvae in each injection group was observed and recorded every day. The experiment was stopped 10 days after artificial infection, and the number of surviving turbot larvae in each injection group was counted.

[0103] It has been observed that after artificial infection with pathogens:

[0104] (1) Control group: After the injection of pathogens, deaths began to occur on the second day, and the death rate continued to increase on the fourth and fifth days, with a peak in deaths. The number of deaths gradually decreased from the sixth day.

[0105] (2) Added groups: After the injection of pathogens, the turbot fry in each injection group performed well, with only a few deaths.

[0106] The statistical results of the survival number of turbot larvae in each injection group are shown in Table 4.

[0107] Table 4 Statistical results of the survival number of turbot larvae in each injection group

[0108]

[0109] From Table 4 we can see that:

[0110] (1) Control group: After injection of different pathogens, the survival rates of infected turbot larvae were 26.67% (injected with Aeromonas salmonicida BHAS-1), 33.33% (injected with Edwardsiella piscicidalis H4-S18), 6.67% (injected with Streptococcus parauberis BH2-4), 16.67% (injected with Pseudomonas putida PPD2), and 10.00% (injected with mixed pathogens). The average survival rate after infection was 18.67%;

[0111] (2) Added groups: After injection of different pathogens, the survival rates of infected turbot fry were 93.34% (injected with Aeromonas salmonicida BHAS-1), 83.34% (injected with Edwardsiella piscicidalis H4-S18), 90.00% (injected with Streptococcus parauberis BH2-4), 76.67% (injected with Pseudomonas putida PPD2), and 66.67% (injected with mixed pathogens). The average survival rate after infection was 82.00%.

[0112] Comparing the mortality of the added group and the control group, it can be seen that the exogenous bacterial agent Bv-con2 can effectively resist the disease and death of turbot caused by infection with Aeromonas salmonicida BHAS-1, Edwardsiella piscicidal H4-S18, Streptococcus paramammaries BH2-4, Pseudomonas putida PPD2 and mixed pathogens, and significantly reduce the mortality rate of turbot.

[0113] 3. Changes in the expression of immune-related genes after artificial infection

[0114] After the artificial infection experiment, all individuals that survived the injection of pathogens in the supplementation group and the control group were quickly dissected and the livers separated in an ice tray. The livers were rinsed with pre-cooled sterile PBS solution and dried with filter paper to obtain two mixed liver samples: the mixed liver sample of the supplementation group and the mixed liver sample of the control group.

[0115] Weigh 20 mg of mixed liver samples from the spiked group and the control group, grind each sample in liquid nitrogen, extract total RNA using an RNA extraction kit, and reverse transcribe the RNA into cDNA using a reverse transcription kit. Real-time fluorescence quantitative polymerase chain reaction (PCR) was performed in a real-time fluorescence quantitative PCR instrument.

[0116] Reaction system (25 μL): SYBR-Premix ExTaqII 12.5 μL, double-distilled water 10.5 μL, cDNA 1 μL, and upstream and downstream primers 0.5 μL each.

[0117] Reaction conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 s, annealing at 59°C for 10 s, extension at 72°C for 20 s, 39 cycles.

[0118] Primers for the tumor necrosis factor α (TNF-α) gene, interleukin-1β (IL-1β) gene, and transforming growth factor β (TGF-β) gene were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequences of the primers for each gene are shown in Table 5 .

[0119] Table 5 Gene primer sequences

[0120]

[0121] The relative expression levels of various immune-related genes in the supplementation group and the control group are shown in Table 6.

[0122] Table 6 Relative expression detection results of various immune-related genes

[0123]

[0124] As shown in Table 6, the relative expression levels of TNF-α, IL-1β and TGF-β genes in the supplementation group were higher than those in the control group, but the differences were not significant.

[0125] Research has shown that turbot immunity is closely linked to inflammatory responses. Proinflammatory cytokines TNF-α and IL-1β are widely believed to contribute to inflammatory responses, while TGF-β, as an anti-inflammatory cytokine, can inhibit the abnormal expression of proinflammatory cytokines and other immune response factors. The relative expression levels of immune-related genes were similar to those observed in artificial infection, indicating that the addition of the microbial agent Bv-con2 stimulated the expression of immune-related genes in turbot, thereby reducing mortality.

[0126] 8. Strain Preservation

[0127] From previous studies, it can be seen that the Bacillus Velezii Bv-con2 screened by the present invention can be used as an exogenous probiotic for farmed turbot, and can improve growth performance, increase the activity of digestion-related enzymes (trypsin) and antioxidant-related enzymes (superoxide dismutase), improve intestinal flora structure, and enhance resistance to infection by any one or more pathogens including Aeromonas salmonicida BHAS-1, Edwardsiella piscicidal H4-S18, Streptococcus parauberis BH2-4, and Pseudomonas putida PPD2.

[0128] The Bacillus velezensis Bv-con2 screened by the present invention was deposited in the China Center for Type Culture Collection on December 27, 2024, with a deposit address in Wuhan, China, and a deposit number of CCTCC NO: M 20242926.

[0129] It should be noted that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Bacillus Velez Bv-con2, isolated from sea cucumber aquaculture pond, classified and named Bacillus Velez Bv-con2 Bacillus velezensis Bv-con2 , deposited in the China Center for Type Culture Collection, Wuhan, China, on December 27, 2024, with the deposit number CCTCC NO: M 20242926.

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