A constrained brown rod bacterium isolated from the intestine of the leopard coral grouper and application thereof

Abstract

The application provides a constraint brown rod bacterium isolated from the intestine of Cephalopholis leopardus and an application thereof, and the preservation number is CGMCC No. 36569. The strain of the application has significant antagonistic effect on Vibrio harveyi, and can be used for preparing products for preventing and controlling aquatic Vibrio diseases. The strain is derived from fish intestine, has good environmental adaptability and host colonization capacity, is high in safety, has no hemolytic activity and carries no drug resistance gene. Feeding experiments show that the strain can promote fish growth, improve feed utilization rate, and enhance the immunity of the body. The application also provides an application of the strain in feed additives or bacterial liquid products, and the application has good popularization value.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial screening and application technology, and particularly relates to a constipated brown bacillus isolated from the intestine of leopard gill spiny perch and its application. Background Technology

[0002] Leopard-gill spiny perch ( Plectropomus leopardus The grouper, commonly known as the East Star Grouper, belongs to the order Perciformes, family Grouperidae, and genus *Scoidea*. It is mainly distributed in the tropical waters of the Indian and Pacific Oceans, and also has a natural distribution in the South my country Sea. The East Star Grouper is characterized by its tender flesh, delicious taste, high nutritional value, and vibrant color, making it highly popular with consumers and possessing significant market economic value and promising prospects for aquaculture development. In recent years, with the continuous development of seedling breeding technology, feed nutrition technology, and factory farming models, the scale of the East Star Grouper aquaculture industry has continued to expand, becoming one of the important species in marine aquaculture in southern my country.

[0003] However, with the increasing intensification and density of aquaculture, the disease problems faced by grouper during the farming process are becoming increasingly prominent. Due to the relatively enclosed farming environment and limited water exchange, the fish are subjected to complex conditions such as high density, stress, and fluctuating water quality, making them more susceptible to the proliferation, spread, and outbreak of pathogens. This leads to fish infections, decreased feed intake, slow growth, and weakened immunity, and in severe cases, even large-scale mortality, resulting in significant economic losses for aquaculture production. Existing research indicates that common diseases in grouper farming mainly include viral and bacterial diseases. Viral diseases include neuronecrosis virus disease and iridovirus disease, while vibriosis is the most prominent bacterial disease. Vibrio harveyi (Vibrio harveyi) Vibrio harveyi It is one of the important pathogens causing disease and high mortality in grouper. It is characterized by rapid spread, strong pathogenicity and wide range of damage, and has become one of the key factors restricting the healthy development of grouper farming.

[0004] For a long time, disease control in aquaculture has relied heavily on antibiotics and chemical drugs. While these measures can have a certain effect on inhibiting bacteria and controlling disease in the short term, long-term or improper use often leads to many problems, such as increased drug resistance in pathogens, imbalance of the aquaculture environment's microecology, increased drug residues, and increased risks to the quality and safety of aquatic products. This is not only detrimental to the stability of the aquaculture ecosystem but also incompatible with the current industry demands for green, healthy, and sustainable aquaculture. Therefore, developing safe, efficient, residue-free, and antibiotic-alternative biological control technologies has become an important research direction in modern aquaculture.

[0005] Among numerous alternative technologies, probiotics have gradually become an important means of disease control in aquaculture due to their multiple advantages, including regulating intestinal microecology, inhibiting the colonization of harmful bacteria, enhancing the body's immune function, improving digestion and absorption, and promoting host growth. Compared with traditional drug control, probiotics have advantages such as safety, environmental friendliness, and no obvious drug residues. They can reduce antibiotic dependence to a certain extent, improve the disease resistance of farmed organisms, and increase farming efficiency, thus showing broad application prospects in aquaculture.

[0006] However, the screening, identification, and application of probiotics are not simple tasks. Many commercially available probiotics on the market are derived from terrestrial animals, soil, or general environmental microorganisms. After entering aquatic animals, they often face problems such as insufficient salinity adaptation, limited temperature adaptability, weak colonization ability, and unstable interactions with the host, resulting in less than ideal practical effects. For aquatic animals, probiotics that can truly exert their beneficial effects typically need to possess strong environmental adaptability, good intestinal colonization ability, significant antibacterial activity, and sufficient safety to function stably in the complex aquaculture environment.

[0007] Therefore, screening native strains from the intestines, mucous membranes, or living environment of healthy aquatic animals has become an important approach to obtaining highly effective aquatic probiotics. Compared to exogenous bacteria, native bacteria are generally more adaptable to the host's intestinal environment and more likely to form stable interactions with the host, thus exhibiting superior effects in inhibiting pathogens, maintaining intestinal microecological balance, and enhancing immune protection. However, truly valuable and superior strains are often not easy to obtain. The screening process typically involves multiple steps, including extensive sample collection, strain isolation and purification, in vitro antibacterial activity screening, evaluation of salt, acid, and bile salt tolerance, colonization ability analysis, and safety verification. This process is lengthy, labor-intensive, and technically demanding, and ultimately, only a limited number of strains can be retained. Even after obtaining strains with certain antibacterial capabilities from numerous candidate bacteria, further verification of their actual growth-promoting and disease-preventing effects in the target host is still required. Therefore, obtaining truly useful, stable, and applicable probiotic strains is not easy. Summary of the Invention

[0008] This invention relates to a type of *Bryophyte constrictionis* isolated from the intestine of the leopard-gill spiny perch and its application, which can be used to prevent and control diseases occurring in leopard-gill spiny perch farming.

[0009] This invention first provides a constraint on brown bacteria ( Phaeobacter inhibens Strain A6 was deposited on January 20, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36569; the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] In another aspect, the present invention also provides an use of the aforementioned restraining Pleurotus A6 strain in the preparation of an article for antagonizing pathogenic bacteria; The pathogenic bacterium mentioned above, as a specific example, is Vibrio harveyi; The present invention also provides another use of the aforementioned restrained brown bacillus A6 strain, which is the application in the reprocessing of products for improving the immunity of leopard gill spiny perch; The product can be a feed additive or a bacterial liquid product.

[0011] The present invention also provides an article for improving the immunity of leopard gill spiny perch, comprising live bacteria of the aforementioned restraint brown bacillus A6 strain.

[0012] The *Phaeobacterium* strain provided by this invention can effectively inhibit the growth of pathogenic bacteria, thereby reducing fish mortality caused by Vibrio infection. This strain originates from the gut of the red snapper and possesses good host adaptability and tolerance to marine environments, exhibiting strong colonization ability and application stability in marine aquaculture fish. The screening method established in this invention combines the chromogenic characteristics of inhibiting *Phaeobacterium* with antagonistic activity evaluation, enabling rapid and accurate screening of functional strains with probiotic potential from complex gut microbiota, improving screening efficiency and success rate. Simultaneously, this strain demonstrates good safety, exhibiting no hemolytic activity, sensitivity to commonly used antibiotics, and no drug resistance genes, resulting in low biosafety risks during application. Further feeding trials show that this strain can promote the growth of farmed fish, improve feed utilization efficiency, and enhance immune-related indicators, demonstrating significant application and promotion value. Attached Figure Description

[0013] Figure 1 Graph showing the antibacterial activity assay of the strains to be screened. Figure 2 Growth curves of the strains to be screened in 2216E medium. Figure 3 Phylogenetic tree of the tested strains constructed based on the 16S rDNA gene sequence. Figure 4 : Results of hemolysis and drug susceptibility testing of *Pseudomonas aeruginosa* strain A6. Figure 5 Biosafety testing diagram of Pleurotus A6 strain. Figure 6 The effect of adding *Phaeobacterium clavatum* strain A6 to feed on growth indicators of *Gnaphalium affine* is shown in the figure. Figure 7 The effect of adding *Phaeobacterium moniliforme* A6 to feed on immune indicators of *Gnaphalium affine*. Figure 8 Figure: Effect of constrained brown bacilli on the survival rate of Vibrio harveyi after infection with Steller's stellate. Detailed Implementation

[0014] The method for screening Brownia strains provided by this invention includes the following steps: (1) Isolate and screen potential target strains; Take intestinal tissue from healthy spotted scorpion, grind it, add sterile phosphate-buffered saline (PBS), homogenize it, and then culture it in a shaker at 25–35℃ for 30–60 min. Take an appropriate amount of bacterial suspension and perform serial dilutions. Spread the solution onto 2216E agar medium and incubate upside down at 25–35℃ for 2–3 days. Brown single colonies were picked, purified by streak cleaning twice, and then inoculated into storage tubes containing 20–30% glycerol and stored at -80°C for later use. (2) Strain identification: To accurately determine the taxonomic position of the selected strains, their genomic DNA was extracted, and their 16S rDNA sequences were amplified using polymerase chain reaction (PCR). The sequencing results were compared with known sequences in the GenBank database for homology, and a phylogenetic tree was constructed based on the neighbor-joining method to clarify the phylogenetic relationships and species classification of the strains.

[0015] (3) Vibrio inhibition test: The test strain was inoculated into 2216E liquid medium at an inoculum of 1–5% and cultured at 25–35℃ for 24 h. The OD600 of the bacterial solution was adjusted to 0.6–0.8. The pathogen indicator bacteria (Vibrio harveyi) were cultured under the same conditions until OD600≈0.1. A plate containing the bacteria was prepared and incubated under aseptic conditions for 20–40 min. The test bacterial solution was spotted onto the plate containing the pathogen. After the droplets dried, the plate was cultured at 30℃ for 24 h. The strains that showed inhibition zones were selected.

[0016] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0017] Example 1: Screening and identification of *Pleurotus ostreatus* strain A6, which is native to the gut microbiota of *Pleurotus ostreatus*. 1. Isolation and purification of strains Intestinal tissue was collected from 10 healthy grouper. Sterile PBS (pH 7.2–7.4, 0.01 mol / L) was added at a ratio of 1:9 (w / v), and the mixture was homogenized using a tissue homogenizer (60 s working, 10 s rest, repeated twice). The homogenate was then placed on a shaker at 28°C and 180 rpm for 30 min. The bacterial culture was then subjected to 10⁻ 5 10⁻ 6Serial dilutions were performed, with 100 μL of each culture spread onto 2216E agar medium and incubated upside down at 30°C for 2–4 days. Brown single colonies were picked and streaked twice for purification. Pure cultures were then stored in 20% glycerol at -80°C for later use.

[0018] 2. Antibacterial test The test strain was inoculated at a 1% inoculum on 2216E liquid medium and cultured at 30℃ and 180 rpm for 24 h, adjusting the OD600 to 0.6–0.8. Simultaneously, *Vibrio harveyi* (isolated from diseased *Echinochloa crus-galli*, preserved in the laboratory) was cultured until OD600 ≈ 0.1, and bacterial plates were prepared and incubated aseptically for 30 min. 5 μL of the test bacterial suspension was spotted onto the plates, and the inhibition zone was observed after incubation at 30℃ for 24 h. The results showed that the test strain had a significant inhibitory effect on *Vibrio harveyi*. Figure 1 ).

[0019] The growth curve of this strain cultured in 2216E liquid medium at 30°C is shown below. Figure 2 As shown.

[0020] The above-mentioned 2216E liquid culture medium formula is as follows: 5.0g peptone, 1.0g yeast extract, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.1g ferric citrate, 1000mL distilled water, pH 7.6 ± 0.2; the 2216E agar medium is prepared by adding 15g of agar to the above-mentioned 2216E liquid culture medium.

[0021] 3. Strain identification Take 100 μL of bacterial culture, centrifuge at 12,000 rpm for 5 min, discard the supernatant, wash twice with PBS (pH 7.4, 10 mM), resuspend, boil in a water bath for 10 min, and cool in an ice bath to obtain crude DNA template.

[0022] PCR amplification was performed using universal 16S rDNA primers (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTACCTTGTACGCACTT-3′). The reaction mixture (50 μL) consisted of 18 μL ddH2O, 25 μL 2×SanTaq PCRMix, 1 μL each of forward and reverse primers, and 5 μL template DNA. PCR conditions were: 94℃ for 2 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, for 30 cycles; extension at 72℃ for 5 min.

[0023] After PCR products were detected by 1% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the NCBI database, and a phylogenetic tree was constructed using MEGA 12.0 software. Figure 3 The results showed that this strain was similar to the model strain. Phaeobacter inhibens The 16S rDNA similarity of DSM16374 reached 98.9%, and it was identified as *Bryophyte constrictionis*, named A6. It was deposited on January 20, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36569; the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] Example 2: Evaluation of strain safety and feeding application 1. Safety test Hemolysis test: The strain was inoculated on Columbia blood agar plates and incubated at 25°C for 24 h. No hemolysis zone was observed.

[0025] Antimicrobial susceptibility testing: The susceptibility of the strain to tetracycline (30 μg / tablet), ampicillin (10 μg / tablet), furazolidone (100 μg / tablet), and chloramphenicol (30 μg / tablet) was tested using the disk diffusion method. The results showed that the strain was sensitive to all of the above antibiotics. Figure 4 ).

[0026] In vivo safety: Healthy grouper were divided into a bathing group (10... 7 CFU / mL, soaking for 3 h), injection group (10) 8 CFU / mL, 100 μL intraperitoneally injected per fish) and a control group were cultured under the same conditions and observed continuously for 7 days. No disease or death was observed in any of the experimental fish, and no abnormalities were found in the liver, spleen, intestines, or other tissues upon dissection. Figure 5 ).

[0027] 2. Feeding experiment Three replicates were set up for the experimental group (containing *Pseudomonas aeruginosa* A6) and the control group, with 200 *Gymnocladus orientalis* fish released into each group (initial weight 114.53 ± 6.41 g). The experiment lasted for 8 weeks, and the experimental group was fed with a bacterial content of 1 × 10⁻⁶ per day. 8 The experimental group was fed a diet containing CFU / g of feed, while the control group was fed a normal commercial diet. Results showed that after 56 days, the experimental group had significantly higher weight gain and specific growth rate than the control group (p < 0.05), and significantly lower feed conversion ratio (p < 0.05). Figure 6 ).

[0028] Serum and intestinal immune marker tests showed that the activities of SOD, AKP, and LYS in the serum of the experimental group were significantly increased (p < 0.05), and ACP and C3 were extremely significantly increased (p < 0.01); the activities of SOD, AKP, C3, and IgG in the intestine were significantly increased (p < 0.05), and LYS and ACP were extremely significantly increased (p < 0.01). Figure 7 ).

[0029] Example 3: Anti-Vibrio activity against Brownia strain A6 After the feeding experiment, 120 grouper from each of the experimental and control groups (3 replicates per group, 40 grouper per replicate) were collected for Vibrio harveyi challenge experiment. The challenge experiment was conducted in six 200 L circulating water tanks. Vibrio harveyi suspension (200 μL per group, bacterial concentration 1.0 × 10⁸.4 CFU / mL) was injected intraperitoneally. Mortality was recorded at 6, 12, 24, 36, 48, 60, and 72 h after injection. The results showed that 72 h after challenge, the survival rate of the experimental group was significantly higher than that of the control group (p < 0.05). Figure 8 ).

[0030] In summary, the *Pseudomonas aeruginosa* A6 strain provided by this invention exhibits significant antagonistic effects against *Vibrio harveyi*, effectively enhancing the resistance of grouper to Vibrio infection after feeding, promoting grouper growth, improving feed utilization, and strengthening the body's immunity. This strain, isolated from the fish intestine, demonstrates high safety and has the potential for development and application as a highly efficient aquatic probiotic.

Claims

1. A type of restrained brown bacillus, characterized in that, The preservation number of the aforementioned *Bryophyte constrictionis* is CGMCC No. 36569.

2. The use of the method described in claim 1 for the preparation of products containing *Bryophyte constriction* for antagonizing pathogenic bacteria.

3. The application as described in claim 2, characterized in that, The pathogenic bacterium mentioned is Vibrio harveyi.

4. The use of the product prepared from the *Bryophyte constriction* according to claim 1 for improving the immunity of the leopard gill spiny perch.

5. The application as described in claim 4, characterized in that, The product in question is a feed additive.

6. A feed for feeding leopard-gill spiny perch, characterized in that, The feed contains the *Bryophyte constrictionis* as described in claim 1.

7. A product for enhancing the immunity of leopard-gill spiny perch, characterized in that, The product contains the *Bryophyte constrictionis* as described in claim 1.

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