Probiotics capable of resisting acute hepatopancreatic necrosis of prawns as well as extracellular product and application thereof

Abstract

The invention relates to the research on the capacity of three lactic acid bacteria with beneficial activity, namely, Weissella paramesenteroides, Lactococcus lactis and Lactococcus garview for resisting V.p AHPND (adenosine monophosphate-adenosine monophosphate-adenosine monophosphate), and particularly relates to the research on the capacity of the three lactic acid bacteria with beneficial activity, namely, Weissella paramesenteroides, Lactococcus lactis and Lactococcus garview for resisting V.p AHPND (adenosine monophosphate-adenosine monophosphate-adenosine monophosphate-adenosine monophosphate-adenosine monophosphate-adenosine monophosphate-adenosine monophosphate). Although the three lactic acid bacteria have no hemolytic activity, are sensitive to various antibiotics, and can tolerate various intestinal conditions (including pH 4-6, NaCl 1-2.5% and bile salt concentration 0.2-1.0%). However, compared with other two strains of lactic acid bacteria, the Weissella mesenteroides WP01 has the highest biological safety evaluation; the diameter of an inhibition zone can be obviously increased, the inhibition capability on other aquatic pathogenic bacteria is stronger, and the in-vitro AHPND inhibition effect of a single strain is superior to that of mixed bacteria. Besides, the abundance of AHPND in prawns can be remarkably reduced, the survival rate of prawns infected by AHPND can be remarkably increased, and it is found that the extracellular product IAA also has the capacity of inhibiting AHPND. Therefore, the Weissella mesenteroides WP01 is preserved in the China Center for Type Culture Collection, and the preservation number of the Weissella mesenteroides WP01 is CCTCC (China Center for Type Culture Collection) NO.M2025382.

Description

Technical Field

[0001] The present invention relates to the field of probiotics, and in particular to a probiotic capable of resisting acute hepatopancreatic necrosis of shrimp, and an extracellular product and application thereof. Background Art

[0002] As a major shrimp farming country, my country is facing many challenges, including frequent diseases (such as white spot syndrome, acute hepatopancreatic necrosis disease, and white feces syndrome), declining seed quality, and deteriorating aquaculture environments. Among them, acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease that poses a serious threat to the global shrimp farming industry. Shrimp infected with this disease usually show symptoms such as whitening of the hepatopancreas, weak swimming ability, and reduced feeding, which eventually lead to large-scale shrimp deaths and huge economic losses. Currently, the use of antibiotics is the main means of controlling AHPND, but long-term use of antibiotics not only leads to drug resistance in pathogens, but also causes drug residues, which endangers human health. Therefore, finding safe and effective alternative strategies has become the focus of current research.

[0003] Research has shown that the occurrence of acute hepatopancreatic necrosis disease (AHPND) is closely related to an imbalance in the shrimp aquaculture ecosystem. Currently, the use of probiotics in aquaculture has demonstrated significant benefits in promoting growth, improving the environment, preventing disease, and enhancing disease resistance. Extracellular μLar Products (ECPs) can also enhance probiotic properties. Lactic acid bacteria (LAB), as an important probiotic, have attracted considerable attention due to their ability to modulate the host immune system, inhibit pathogen growth, and improve intestinal health. Reports indicate that LAB can enhance disease resistance and growth performance in shrimp. However, most research has focused on LAB fermentation broths. The application of fermentation broths in aquaculture faces numerous challenges, including the high initial investment cost of fermentation equipment (e.g., fermentation tanks and control systems), the significant energy consumption (e.g., electricity and steam) during the fermentation process, and the technical complexity of precisely controlling parameters such as temperature, pH, dissolved oxygen, and agitation speed. In contrast, directly cultivating LAB in an aerobic environment eliminates the need for anaerobic reactor modification, reducing equipment investment costs. Therefore, it is of great significance to develop new probiotics and their extracellular products to resist pathogens, provide sustainable and environmentally friendly alternatives to antibiotic treatment and solve disease problems in shrimp farming. Summary of the Invention

[0004] The present invention aims to provide a probiotic strain of Weissella mesenteroides WP01 and its extracellular products to effectively protect against acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus. This invention aims to address the high mortality rates of shrimp caused by AHPND in aquaculture, the development of antibiotic resistance due to overuse of antibiotics, and the limited effectiveness of traditional control methods, thereby providing a safe, efficient, and sustainable biological control solution for the aquaculture industry.

[0005] The following technical solutions are adopted: A strain of Weissella mesenteroides WP01 was deposited in the China Center for Type Culture Collection (CCTCC, located in Wuhan University, Wuhan, China), with the deposit number being CCTCC NO.M2025382 and the deposit date being March 5, 2025.

[0006] A strain of Weissella mesenteroides WP01 was deposited in the China Center for Type Culture Collection, and its deposit number is CCTCCNO.M2025382.

[0007] The intestinal contents of whiteleg shrimp (Penaeus vannamei) were cultured in MRS medium in vitro, and single colonies were isolated and purified. Subsequently, the agar punch method was used to screen for beneficial bacteria that inhibited the growth of V.pAHPND. Three strains of lactic acid bacteria with antibacterial effects were obtained. After 24 hours of in vitro culture, the beneficial bacteria were sent to Shenzhen BGI Genomics Co., Ltd. for 16S rRNA gene sequencing and identified as Weissella paramesenteroides, Lactococcus lactis, and Lactococeus garvieae. Among them, agar punch experiments revealed that among the three beneficial bacteria, W. paramesenteroides had significantly stronger antibacterial ability than Lactococcus lactis and Lactococcus lactis. W. paramesenteroides also exhibited the strongest resistance to acute hepatopancreatic necrosis disease (AHPND). Based on this characteristic, the present invention has deposited W. paramesenteroides WP01 with the China Center for Type Culture Collection under the accession number CCTCCNO.M2025382. The other two lactic acid bacteria strains were stored in this laboratory at -80°C using 50% glycerol as a preservative.

[0008] The method for culturing Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis with beneficial activities uses MRS medium. Its ingredients include: 10.0g of casein digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of triammonium citrate (C6H5O7(NH4)3), 5.0g of sodium acetate (CH3COONa), 0.2g of magnesium sulfate (MgSO4·7H2O), 0.05g of manganese sulfate (MnSO4·4H2O), 2.0g of dipotassium hydrogen phosphate (K2HPO4), and 1.0g of glucose (C6H 12 Prepare 20.0 g of O6 and 1.0 g of Tween-80. Adjust the pH to 5.7 ± 0.2. Weigh 54 g of this product and add 1 L of distilled or deionized water. Stir and boil until completely dissolved. Dispense into test tubes or Erlenmeyer flasks and autoclave at 121°C for 15 min.

[0009] An application of the above-mentioned Weissella mesenteroides WP01 is used to inhibit aquatic pathogens.

[0010] Preferably, the aquatic pathogens include one or more of Photobacterium mermanii, Staphylococcus aureus, Streptococcus dolphinii, Shewanella basaltii, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio cannelli.

[0011] The experiment found that Lactococcus, Lactococcus lactis, and Weissella mesenteroides can tolerate a wide range of intestinal conditions, including growth in a pH range of 4-6, NaCl concentrations (1-2.5%), and bile salt concentrations (0.2-1.0%). When comparing the probiotic activities of Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis, the Weissella mesenteroides group had the highest biosafety rating (survival rate of 83.33%) compared to the control group (survival rate of 86.67%), with no significant difference between the two groups (P < 0.05). Compared with the other two lactic acid bacteria strains, Weissella mesenteroides WP01 significantly increased the diameter of the inhibition zone (17.29±0.31 mm). The in vitro antibacterial activity of Weissella mesenteroides WP01 was superior to that of the mixed bacteria, with the latter group having a maximum inhibition zone of 14.54±0.32 mm. Furthermore, Weissella mesenteroides WP01 exhibited stronger inhibitory activity against aquatic pathogens such as Photobacterium damselae, Staphylococcus aureus, Streptococcus iniae, Shewanella basaltis, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio campbellii than Lactococcus and Lactococcus lactis. Furthermore, Weissella mesenteroides WP01 significantly reduced the abundance of Vp AHPND in shrimp and significantly increased the survival rate of whiteleg shrimp infected with Vp AHPND (63.33%, P < 0.05), compared to a survival rate of only 23.33% in the Vp AHPND-infected group. In addition, experiments have found that the extracellular metabolite indole-3-acetic acid (3-Indoleacetic acid: IAA) of Weissella mesenteroides WP01 also has the ability to resist Vp AHPND.

[0012] Preferably, it is used to prepare preparations for preventing and treating acute hepatopancreatic necrosis.

[0013] These three strains of lactic acid bacteria (Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis) with beneficial activities can effectively help whiteleg shrimp (Litopenaeus vannamei) resist acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus, thereby maintaining the health of its hepatopancreas.

[0014] These three lactic acid bacteria with beneficial activities (Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis) showed sensitivity to multiple antibiotics, including: β-lactams (cefazolin, amoxicillin), aminoglycosides (gentamicin, neomycin), tetracyclines (doxycycline, florfenicol), quinolones (enrofloxacin) and peptides (polymyxin).

[0015] Preferably, the method is used to prepare a preparation for reducing the abundance of Vp AHPND in crustaceans.

[0016] Preferably, the invention is used for the preparation of a preparation for improving the survival rate of crustaceans infected with Vp AHPND.

[0017] Preferably, the shellfish comprises Penaeus vannamei.

[0018] A preparation obtained by the above application, comprising one or more of the Weissella mesenteroides WP01 and the extracellular products of the Weissella mesenteroides WP01.

[0019] Preferably, the extracellular product includes indole-3-acetic acid; in the preparation, the concentration of the extracellular product is 200 mM to 12.5 mM.

[0020] The extracellular product IAA of Weissella mesenteroides WP01 also exhibited the ability to inhibit Vp AHPND. The minimum inhibitory concentration graph showed that compared with the positive control (blank medium group), IAA had a highly significant antibacterial effect within the concentration range of 200mM to 12.5mM (P<0.05). The inhibition rates of IAA at concentrations of 100.00mM and 12.50mM were 98.17%±0.8% and 71.21%±2.87%, respectively, with significant differences (P<0.05).

[0021] Preferably, the preparation comprises one or more of medicine and feed.

[0022] The drug also includes pharmaceutically acceptable excipients or auxiliary ingredients.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Weissella mesenteroides WP01 and its extracellular products can significantly improve the survival rate of aquaculture animals infected with acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio spp. As a natural probiotic, Weissella mesenteroides WP01 and its extracellular products are environmentally friendly and do not cause antibiotic resistance, meeting the needs of green and healthy aquaculture. Furthermore, Weissella mesenteroides WP01 can tolerate certain salinity, bile salt, and pH variations, demonstrating good stability in complex aquaculture environments, ensuring its effectiveness in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Isolation and identification of probiotics: (A) Agar plate image of the three probiotic strains against Vp APNND in the inhibition zone experiment; (B) Determination of the diameter (mm) of the inhibition zone of the three probiotic strains against V.pAPNND; (C) Table of the strength of the three probiotic strains against V.pAHPND; (D) Neighbor-joining phylogenetic tree of the three probiotic strains and other species (accession numbers in the appendix) constructed using MEGA7.0 software; (E) Comparison with existing database sequences based on 16S rRNA gene sequences through BLASTN search.

[0026] Figure 2 The morphological characteristics and probiotic properties of probiotics: (AC) are the morphological characteristics of Lactococcus, Weissella mesenteroides, and Lactococcus lactis, respectively; (D) the growth curves of the three probiotic strains; (E) the bile salt tolerance curves of the three probiotic strains; (F) the pH levels of the three probiotic strains; (G) the NaCl tolerance curves of the three probiotic strains.

[0027] Figure 3 Hemolytic activity and antibiotic sensitivity of probiotics: (A) Hemolytic activity of three probiotic strains; (B) Antibiotic sensitivity of three probiotic strains; (C) Antimicrobial activity of three probiotic strains against other aquatic pathogens.

[0028] Figure 4 Safety test of probiotic immersion on whiteleg shrimp and the ability of co-cultured probiotics to resist V.pAHPND: (A) (B) (C) Safety tests of three probiotic strains in whiteleg shrimp; (D) Diameter (mm) of the inhibition zone of co-cultured probiotics against Vp APNND; (E) Table of inhibition zone values ​​of co-cultured probiotics against V.pAHPND.

[0029] Figure 5 The basic characteristics of Weissella mesenteroides WP01: (A) Electron micrograph of Weissella mesenteroides WP01; (B) Gram staining of Weissella mesenteroides WP01; (C) Schematic diagram of the high temperature resistance of Weissella mesenteroides WP01; (D) Streak plate image of Weissella mesenteroides WP01 after high temperature treatment; (E) Survival rate of whiteleg shrimp against Vp AIIPND after probiotic soaking.

[0030] Figure 6 The effect of Weissella mesenteroides WP01 on the abundance of Vp AHPND in hepatopancreas samples at 24h, 48h, 72h, and 96h.

[0031] Figure 7Metabolomics analysis of Weissella mesenteroides WP01 metabolites: (A) QC sample analysis; (B) volcano plot analysis of differential metabolites; (C) analysis of changes in differential metabolite content; (D) analysis of changes in specific differential metabolites.

[0032] Figure 8 The effect of IAA, an extracellular product of Weissella mesenteroides WP01, against AHPND: (A) Minimum inhibitory concentration of IAA against Vp AHPND; (B) In vitro antibacterial experiment of IAA against Vp AHPND; (C) Inhibitory rate of IAA against Vp AHPND; (D) Effect of IAA on the survival rate of whiteleg shrimp. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Unless otherwise specified, the Weissella paramesenteroides described in the following examples refers to the deposited Weissella paramesenteroides WP01.

[0037] Example 1

[0038] Screening of beneficial bacteria

[0039] Thirty healthy Penaeus vannamei shrimp were purchased from Niutianyang Farm in Shantou, Guangdong Province, China and randomly divided into three groups. The shrimp were aseptically dissected on an ice plate in a clean bench and the intestines were aseptically collected. The intestines were placed in a sterile Petri dish (containing 0.9% saline) and one end of the intestine was fixed with sharp forceps. The other end of the intestine was gently cut open with dissecting scissors to facilitate scraping the intestinal contents. The intestinal contents were mixed in saline and 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 The solution was diluted 1 / 2 times and 30 μL was then plated on an MRS agar plate to isolate individual bacteria. The plate was incubated in a 30°C incubator for 48 hours, and the colony distribution was observed. Different colonies were numbered and recorded, and individual colonies were selected for isolation and purification.

[0040] First, using Vibrio parahaemolyticus as the indicator bacteria and the selected single colony as the test bacteria, the indicator bacteria and the test bacteria were activated and cultured in LB medium and MRS medium at 30°C and 180 rpm for 24 hours respectively. The cultured indicator bacteria were diluted to 1*10 5 CFU / mL, and spread 30 μL onto an LB agar plate. Then, spread 2.5 μL of the test bacteria solution onto a nutrient agar plate coated with indicator bacteria. Incubate the LB plate in a 30°C incubator for 24 hours, recording the strains that exhibit distinct transparent circles.

[0041] Then, the indicator bacteria were diluted to 1*10 5 CFU / mL, take 50 μL and apply it on the nutrient agar plate, use an agar puncher with an inner diameter of 7 mm to punch holes evenly on the nutrient agar plate, and add 80 μL of test bacteria solution to each hole. Place the plate in a constant temperature incubator at 30℃ for 24 hours, and use a vernier caliper to measure the diameter of the inhibition zone ( Figure 1 AC), determine the inhibitory effect of the test bacteria on the indicator bacteria. The beneficial bacteria that have an inhibitory effect on the indicator bacteria were morphologically identified. After the beneficial bacteria were activated, they were streaked and cultured on MRS solid medium. After 24-48 hours, the bacterial plate morphology was observed. It was found that Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis grew well on MRS medium. The surface colonies were round or slightly irregular, white, and the surface was moist and shiny ( Figure 2 AC).

[0042] Example 2

[0043] 16S rRNA identification of beneficial bacteria

[0044] The test bacteria with inhibitory effects were then sent to Sangon Biotech Co., Ltd. for sequencing. First, 1 mL of the bacterial suspension cultured for 16-20 hours was placed in a 1.5 mL sterile EP tube and centrifuged at 12,000 rpm for 5 minutes. After carefully aspirating the supernatant, 1 mL of RNase-free water was added and thoroughly pipetted to mix. The tube was then placed in a boiling water bath for 10 minutes and centrifuged again at 12,000 rpm for 5 minutes. The supernatant (DNA suspension) was used as a template for PCR amplification. PCR amplification was performed using the bacterial supernatant (DNA suspension) as a template using bacterial 16S rRNA universal primers. The primers were obtained from Shenzhen BGI Genomics Co., Ltd.:

[0045] Forward primer sequence (27F) 5'-AGAGTTTGATCCTGGCTCAG-3'

[0046] Reverse primer sequence (1492R) 3'-GGTTACCTTGTTACGACTT-5'

[0047] The PCR amplification system was 25 μL: 1 μL template DNA, 1 μL forward and reverse primers, 12.5 μL Premix ExTaq, and 9.5 μL ddH2O. The PCR amplification program was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 10 min. The PCR product fragment length was approximately 1500 bp, and the PCR product was detected by 1% agarose gel electrophoresis. Samples with target bands after detection were recovered from the gel and sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared for homology on the website http: / / ezgenome.ezbiocloud.net / ezg_BLAST to find out the category to which they belonged. The sequences were then analyzed for homology with the nucleotide sequences in the GenBank database. Multiple alignments were performed using Mega7.0 software, and a phylogenetic tree was constructed using the neighbor-joining method ( Figure 1 DE), and the identification results were Weissella mesenteroides, Lactococcus lactis, and Lactococcus.

[0048] Example 3

[0049] Probiotic properties of three probiotic strains

[0050] The growth curves of Weissella mesenteroides, Lactococcus lactis and Lactococcus lactis are shown in Figure 2 D. MRS medium was used to culture Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis. The results showed that after 42 hours, the OD values ​​of the three bacteria changed little, indicating that the bacteria had grown slowly and entered a stable period. In order to determine the tolerance of Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis to pH, NaCl, and bile salts, the bacteria were added to MRS broth at 30°C and cultured at a speed of 180 rpm for 24 hours. After 24 hours of culture, the bacterial density was adjusted to 10 7 CFU / mL, transferred to MRS broth adjusted to different pH values ​​(1-9), bile salts (0, 0.2%, 0.4%, 0.6%, 0.8% and 1%) or NaCl (0, 0.5%, 1%, 1.5%, 2%, 2.5% and 3%), the experiment was repeated three times; the density (OD) at 600nm absorbance was measured by microplate reader to determine the growth of bacteria. The results showed that Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis can grow in a wide range of pH 4 to 9, and can grow in NaCl concentrations (1% to 2.5%) and bile salt concentrations (0.2% to 1.0%). The results are shown in Figure 5. Figure 2 As shown in EG.

[0051] Example 4

[0052] Hemolysis and drug resistance analysis

[0053] The beneficial bacteria were activated and cultured for 24 hours, and 20 μL of the test bacteria solution was spotted on 5 different positions on the blood agar plate (Guangzhou Huankai Biological Co., Ltd.). After culturing in a constant temperature incubator at 28°C for 24 hours, the surrounding area was observed for hemolysis. The results showed that Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis were not hemolytic ( Figure 3 A).

[0054] Drug susceptibility testing was performed using the disc agar diffusion method (KB method). Eight common drug susceptibility discs (Hangzhou Microbiological Reagent Co., Ltd.) were selected: cefazolin, amoxicillin, gentamicin, neomycin, cefepime, florfenicol, enrofloxacin, and polymyxin. Beneficial bacteria were activated and cultured to a concentration of 1.0 × 10 7 CFU / mL, take 30μL of bacterial solution and spread it on the corresponding nutrient agar medium. Use sterile tweezers to stick the drug-sensitive paper on the surface of the culture medium. The distance between each paper is greater than 24mm, and the center distance of the paper should be more than 15mm from the edge of the plate. Place 2 drug-sensitive paper on each culture medium plate. After culturing in a constant temperature incubator at 28℃ for 24h, use a vernier caliper to measure the size of the inhibition zone and calculate the drug sensitivity. The results show that all three beneficial bacteria are sensitive to antibiotics ( Figure 3 B).

[0055] The agar diffusion method (Oxford cup method) in Example 1 was used to evaluate the antibacterial effects of the three strains of beneficial bacteria on eight aquatic pathogens, including Photobacterium damselae, Staphylococcus aureus, Streptococcus iniae, Shewanella basaltis, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio campbellii. The results showed that the antibacterial ability of Weissella mesenteroides WP01 was superior to that of Lactococcus and Lactococcus lactis. Figure 3 C).

[0056] Example 5

[0057] Biosafety evaluation of beneficial bacteria and antagonistic ability of mixed culture of probiotics against Vp AHPND

[0058] The beneficial bacteria and Vp AHPND were activated and cultured to a concentration of 10 8CFU / mL (culture conditions are the same as those in Example 3). For the beneficial bacteria group and the Vp AHPND group, 100 mL of the test bacterial suspension of the corresponding group was added into a 10 L natural seawater culture tank, so that the concentration of the test bacteria in the water was 10 7 CFU / mL; the control group added 100mL of physiological saline to a 10L natural seawater culture bucket. 30 whiteleg shrimp were placed in each culture bucket, and the water was replaced daily for 96 hours. During this period, they were fed and managed normally, and feces were cleaned daily. The survival rate of each bucket was calculated. The results showed that among the three lactic acid bacteria, Weissella mesenteroides WP01 had the highest safety assessment (83.33%), with no significant difference from the control group (86.67%) ( Figure 4 AC). The calculation formula is as follows:

[0059] Survival rate (%) = (number of shrimps at the beginning of the experiment - number of dead shrimps) / number of shrimps at the beginning of the experiment × 100

[0060] According to the agar punch method of Example 1 and the culture conditions of Example 3, Weissella mesenteroides, Lactococcus lactis, and Lactococcus lactis were mixed and cultured, and their ability to resist Vp AHPND was tested. The results showed that the mixed culture did not enhance the ability of beneficial bacteria to resist Vp AHPND (the highest inhibition zone of the mixed bacteria was: 14.54±0.32mm). In comparison, the use of Weissella mesenteroides WP01 alone was more effective and could significantly increase the diameter of the inhibition zone (17.29±0.31mm) ( Figure 4 DE).

[0061] Example 6

[0062] Electron microscopy analysis

[0063] 1. Sampling: Remove the bacteria to be activated from -80℃ and quickly dissolve them in a 37℃ water bath. In a sterile operating table, aspirate 50μL of bacterial solution and inoculate it into 5mL of MRS liquid medium. Incubate on a shaker at 180rpm for 12h. Centrifuge at 12000×g for 10min at 4℃ to collect the visible bacterial precipitate (sesame to mung bean size). Discard the medium and gently rinse the precipitate with PBS. Repeat twice. Finally, centrifuge and discard the PBS. Add electron microscopy fixative and blow off the bacteria to suspend them in the fixative. Fix at room temperature in the dark for 30min.

[0064] 2. Dehydration: Centrifuge 1 mL of the fixed bacterial suspension at 5000 × g for 10 min at 4°C. Discard the supernatant and collect the precipitate. Dehydrate the suspension using a gradient of ethanol solutions (30% → 50% → 70% → 80% → 90% → 95% → 100% → 100%) for 15 min per step. Centrifuge at 5000 × g for 1 min at 4°C.

[0065] 3. Fixation: Aspirate 100% ethanol together with the precipitate and place 1-2 drops on a glass slide.

[0066] 4. Drying: Place the slides in a critical point dryer (Quorum (K850)) and dry for 2-3 hours.

[0067] 5. Conductive treatment of samples: Place the glass slide tightly on the conductive carbon film double-sided tape, place it on the sample stage of the ion sputtering instrument (HITACHI (MC1000)), and spray gold for about 30 seconds. By spraying a gold layer on the sample surface, the conductivity and thermal conductivity of the sample are significantly increased, and the damage to the sample by the electron beam is reduced, thereby obtaining a clearer image and improving image quality.

[0068] 6. Microscopic examination: Using a panoramic scanning electron microscope HITACHI (SU8100), we took photos and saved them. The results showed that the morphology of Weissella mesenteroides WP01 under the electron microscope was spherical. Figure 5 A).

[0069] Example 7

[0070] Gram stain

[0071] The following Gram staining steps were used to stain Weissella mesenteroides WP01. It was found that it appeared purple under the microscope, which confirmed that Weissella mesenteroides WP01 was a Gram-positive bacterium ( Figure 5 B), the steps are as follows:

[0072] 1. Smear Preparation: Prepare the bacterial solution as described in Example 3. Place a small drop of saline in the center of a clean glass slide. Using an inoculating loop, scoop a small amount of Weissella mesenteroides WP01 bacterial solution into the water droplet in the center of the slide. Smear the solution evenly and allow to dry naturally.

[0073] 2. Drying and fixing: After the smear is dry, quickly pass it through the flame 2-3 times to fix it.

[0074] 3. Staining steps:

[0075] a) Primary staining: Add crystal violet staining solution to the coated area, stain for 1 minute, and then rinse with water.

[0076] b) Mordanting: Rinse with Gram's iodine solution to remove residual water, cover for 1 minute, and then rinse with water.

[0077] c) Decolorization: Shake off the water on the slide, tilt the slide and place it on a white paper as the backing, add 95% ethanol dropwise until no purple color fades (about 20-30 seconds), and immediately rinse slowly with water.

[0078] d) Restaining: Add safranin staining solution, stain for 3-5 minutes, and then wash with water.

[0079] 4. Wait for it to dry naturally and then examine it under a microscope.

[0080] Example 8

[0081] Is Weissella mesenteroides WP01 resistant to high temperatures and its effects on in vivo experiments in whiteleg shrimp

[0082] In order to test the tolerance of Weissella mesenteroides WP01 to high temperature, the concentration of 10 7 The bacterial suspension containing 100 CFU / mL was placed in a boiling water bath at 100°C for 10 minutes. Subsequently, agar punching was used to conduct a plate test to test the resistance to V.pAHPND. The results showed that after high-temperature treatment, the bacterial suspension no longer had the ability to resist Vp AHPND. When the high-temperature treated bacterial suspension was streaked onto an MRS plate, no colony growth was observed, indicating that high temperature inactivated Weissella mesenteroides WP01. Figure 5 CD). The ability of Weissella paramesenteroides WP01 to resist Vp AHPND in whiteleg shrimp was further studied. First, shrimp were soaked in W. paramesenteroides solution and then injected with Vp AHPND (1.5×10 5 Over time, W. paramesenteroides immersion significantly reduced the abundance of Vp AHPND in the hepatopancreas and the intestine at 96 h ( Figure 6 At the same time, the survival rate of shrimp in the Weissella mesenteroides group (63.33%) was significantly higher than that in the AHPND group (23.33%) ( Figure 5 E), indicating that W. paramesenteroides has a significant antibacterial effect in shrimp.

[0083] Example 9

[0084] Metabolomic analysis of the supernatant of Weissella mesenteroides WP01

[0085] After the sample was slowly thawed at 4°C, an appropriate amount of sample was added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortexed, sonicated at low temperature for 30 min, allowed to stand at -20°C for 10 min, centrifuged at 14000g at 4°C for 20 min, and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) was added for re-dissolution, vortexed, and centrifuged at 14000g at 4°C for 15 min. The supernatant was taken for sampling and analysis. Samples were separated using a Vanquish LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column. The column temperature was 25°C, the flow rate was 0.5 mL / min, and the injection volume was 2 μL. The mobile phase composition was A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile. The gradient elution program was as follows: 95% B from 0 to 0.5 min; linear gradient from 95% to 65% from 0.5 to 7 min; linear gradient from 65% to 40% B from 7 to 8 min; hold at 40% from 8 to 9 min; linear gradient from 40% to 95% B from 9 to 9.1 min; hold at 95% from 9.1 to 12 min. Samples were maintained in an autosampler at 4°C throughout the analysis. To minimize the influence of instrument signal fluctuations, samples were analyzed sequentially in random order. QC samples were inserted into the sample queue to monitor and evaluate system stability and the reliability of the experimental data. The samples were separated by Vanquish LC ultra-high performance liquid chromatography (UHPLC) system and analyzed by Orbitrap Exploris TM Mass spectrometry analysis was performed using a 480 mass spectrometer (Thermo) using electrospray ionization (ESI) in positive and negative ion modes. Figure 7 As shown in AD.

[0086] Example 10

[0087] IAA's ability to defend against AHPND

[0088] First, add blank culture medium to the first well of rows A, B, and C of a 96-well plate to determine the drug MIC in triplicate (samples in rows A, B, and C). Subsequently, add 100 μL of the prepared stock solution (200 mM concentration) to the first well of rows A, B, and C. Next, perform a two-fold dilution of the drug: After adding the drug solution to well 1, pipette vigorously (at least three times) to mix the drug and broth thoroughly. Then, pipette 100 μL into well 2, mix thoroughly again by pipetting, and pipette 100 μL into well 3. Repeat this operation until the last well, and finally draw 100 μL and discard; at this time, the drug concentrations in each well from left to right are 200, 100, 50, 25, 12.5, 6.25, 6.25, 3.125, 1.56, 0.78, 0.39, normal saline (negative control group), blank culture medium (positive control group), and then add the diluted bacterial solution (1*10 5 CFU / ml) 10μL. After culturing the 96-well culture plate in a 37℃ constant temperature incubator for 16-20h, the results were observed using a microplate reader at OD600. The results showed that when the concentration of IAA was between 200mM and 12.5mM, the antibacterial effect was extremely significant (P<0.05) (such as Figure 8 AB).

[0089] First, prepare a bacterial suspension, dilute the bacteria with physiological saline (0.65%), and adjust the concentration of Vibrio parahaemolyticus to 10 5 CFU / mL. Next, prepare the IAA dilution solution and set the maximum concentration of IAA according to 4 times the dose, that is, use normal saline (0.65%) to prepare 12.50, 15.00, 50.00 and 100.00mM IAA solutions. Take 60μL of the bacterial suspension prepared above and mix it with an equal volume of IAA solution. Use normal saline (0.65%) and bacteria as a negative control, and incubate it in a 37°C incubator for 2 hours. Take 30μL of the incubated solution and spread it on the LB plate (Vibrio parahaemolyticus), with 3 replicates for each group. Place the plate in a 37°C incubator and culture for 12-16 hours, take pictures and record the number of colonies on the plate (such as Figure 8 C). The bacterial inhibition rate was calculated according to the following formula. The results showed that the inhibition rates of IAA at concentrations of 12.5 mM and 100.00 mM against bacteria were 98.17% ± 0.8% and 71.21% ± 2.87%, respectively. There were significant (P < 0.05) or extremely significant (P < 0.01) differences (e.g. Figure 8 D).

[0090] Bacterial inhibition rate = (number of colonies in the negative control - number of colonies in the experimental group) / number of colonies in the negative control × 100%.

[0091] To investigate the effect of indoleacetic acid (IAA) on the survival rate of whiteleg shrimp, 60 healthy shrimps of uniform size were selected and randomly divided into three groups, with 20 shrimps in each group. The experimental groups were injected with 100 μL of IAA solution (12.5 mM, 6.25 mM), and the control group was injected with an equal amount of normal saline (0.65%). After 24 hours, all shrimps were injected with Vibrio parahaemolyticus Vp AHPND (105 CFU / mL). The number of deaths was recorded at 24 hours, 48 ​​hours and 72 hours respectively. The experimental results showed that compared with the 12.5 mM IAA group (survival rate 10%) and the 3.125 mM IAA group (survival rate 15%), the 6.25 mM IAA group exhibited better in vivo antibacterial effect (survival rate 30%), while the survival rate of the Vp AHPND control group was 0% (as shown in Figure 2). Figure 8 E).

[0092] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A strain of Weissella mesenteroides WP01, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO.M2025382.

2. A use of Weissella mesenteroides WP01 as claimed in claim 1, characterized in that: Used to inhibit aquatic pathogens.

3. The use according to claim 2, characterized in that The aquatic pathogens include one or more of Photobacterium mermanii, Staphylococcus aureus, Streptococcus dolphinii, Shewanella basaltii, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Vibrio cannulatus.

4. The use according to claim 2, characterized in that Used for preparing preparations for preventing and treating acute hepatopancreatic necrosis.

5. The use according to claim 4, characterized in that Used for preparing a preparation for reducing the abundance of V.pAHPND in crustaceans.

6. The use according to claim 4, characterized in that Used for preparing a preparation for improving the survival rate of crustaceans infected with Vp AHPND.

7. The use according to claim 5 or 6, characterized in that The crustacean includes whiteleg shrimp.

8. A preparation obtained by the application according to claim 2, characterized in that The preparation comprises one or more of the Weissella mesenteroides WP01 and the extracellular products of the Weissella mesenteroides WP01.

9. The preparation according to claim 8, wherein The extracellular product includes indole-3-acetic acid; in the preparation, the concentration of the extracellular product includes 200mM to 12.5mM.

10. The preparation according to claim 8, wherein The preparation comprises one or more of medicine and feed.

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