Aquatic Bacillus Velez P12 and its use

Abstract

The present invention relates to the technical field of microbial application, and specifically discloses a strain of aquatic Bacillus Velez P12 and a use thereof, wherein the Bacillus Velez P12 has a deposit number of CCTCC M 20221921 and a deposit date of December 12, 2022. The Bacillus Velez P12 isolated and obtained by the present invention can inhibit Vibrio and is suitable for being used as feed for raising grass carp.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to an aquatic Bacillus Velez P12 and a use thereof. Background Art

[0002] In the past, there was a widespread use of antibiotics to ensure the development of intensive aquaculture. However, the use of antibiotics has led to a series of problems related to food safety and the environment, such as the destruction of the original microbial community structure in the aquaculture environment, the pollution of water sources, the accumulation of antibiotics in food, and the rising cost of disease treatment. Due to the limitations of aquatic animal vaccine development, it is difficult to obtain aquatic animal vaccines at this stage and for quite some time in the future. In the absence of vaccines, many alternative solutions have emerged to reduce the abuse of antibiotics in aquaculture. These include antimicrobial peptides, Chinese herbal extracts, probiotics, prebiotics and synbiotics, among which probiotics have attracted much attention due to their high abundance, low cost and convenient application. However, sometimes commercially available probiotics are relatively ineffective because they cannot maintain optimal concentrations or remain active in the intestines of aquatic animals. Many probiotics used by aquatic animals come from other animals and plants. In recent years, studies have reported that host-associated probiotics, that is, bacteria originally isolated from aquaculture water or the gastrointestinal tract of the host that can promote host growth and improve health levels, have better probiotic effects than terrestrial probiotics, which may be related to their better performance in the native environment. Currently, there are a large number of candidate probiotics in aquaculture, but biotechnological issues and limited reports on actual effects limit their large-scale application, and there are still many research gaps to be filled. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a strain of aquatic-derived Bacillus Velez P12, which can be used for preparing feed in aquaculture and can inhibit a variety of Vibrio.

[0004] The present invention provides an aquatic Bacillus Velez subtilis P12, the preservation number of the Bacillus Velez subtilis P12 is: CCTCC M 20221921, and the preservation date is December 12, 2022.

[0005] The invention also provides use of Bacillus Velez P12 in aquaculture.

[0006] Furthermore, the aquaculture includes but is not limited to whiteleg shrimp farming and grass carp farming.

[0007] Furthermore, the Bacillus Velez P12 can inhibit Vibrio.

[0008] Furthermore, the Vibrio include Vibrio anguillarum, Vibrio cholerae, Vibrio fluvii, Vibrio owensii, Vibrio rotifera, Photobacterium mermaidense, Vibrio campbellii, Vibrio vulnificus and Vibrio parahaemolyticus.

[0009] Furthermore, the Bacillus Velez P12 can be used to prepare feed for raising grass carp.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] 1. The Bacillus velezensis P12 provided by the present invention has good antibacterial properties, can secrete some beneficial extracellular enzymes, has no hemolytic property, no drug resistance, and has the potential to be used as a probiotic.

[0012] 2. The Velez Bacillus P12 of the present invention has good tolerance and high biosafety, and can be used as an additive in preparing aquatic animal feed. The P12 strain is mixed with a granular expanded material to prepare a feed, and the white shrimp is fed for 4 consecutive weeks, and then artificially infected with pathogens. The results show that the cumulative mortality rate of the experimental group with the addition of Velez Bacillus P12 is 35%, and the cumulative mortality rate of the control group is 76%, indicating that P12 can be used as a feed additive to reduce the mortality rate caused by pathogen infection to white shrimp. Adding Velez Bacillus to the feed of grass carp can significantly improve the intestinal morphology of grass carp.

[0013] Information on the deposit of biological materials

[0014] P12, referred to as aquatic Bacillus velezensis P12 in this application, was deposited in the China Center for Type Culture Collection on December 12, 2022, with the deposit number CCTCC M 20221921. The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code: 430072, and the classification name is Bacillus velezensis P12. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The colony morphology and scanning electron microscope image of Bacillus Velez P12 in the present invention;

[0017] Among them, Figure A shows the P12 streak inoculated colony on TSB solid plate;

[0018] Figure B is a single colony morphology of P12;

[0019] Figure C is the Gram staining of P12;

[0020] Figure D shows P12 spore staining;

[0021] Figures E, F, and G are SEM images of P12;

[0022] Figure 2 The P12 drug sensitivity test result of Bacillus Velez P12 in the present invention;

[0023] AJ stands for: A: β-lactams; B: macrolides; C: aminoglycosides; D: tetracyclines; E: chloramphenicol; F: peptides; G: rifamycins; H: coumarins; I: quinolones; J: sulfonamides.

[0024] Figure 3 It is the detection result of the extracellular product of Bacillus Velez P12 in the present invention;

[0025] Among them, Figure a, Figure b, Figure c, Figure d and Figure e respectively represent the detection of extracellular products phospholipase, gelatinase, protease, amylase and hemolysis of P12;

[0026] Figure 4 This is a tolerance experiment (high temperature, acidity, bile salts, simulated gastric juice, simulated intestinal juice) of the Bacillus Velez P12 of the present invention;

[0027] Among them, Figure a shows the tolerance of P12 in a high temperature 60-100℃ water bath;

[0028] Figure b shows the tolerance of P12 in TSB liquid medium with pH values ​​of 2.0, 2.5, and 3.0;

[0029] Figure c shows the tolerance of P12 in simulated gastric fluid, intestinal fluid and bile salt;

[0030] Figure 5 is the whole genome map of Bacillus Velez P12 in the present invention;

[0031] Figure 6 It is a statistical diagram of the prediction classification of virulence genes and drug resistance genes of Bacillus Velez P12 in the present invention;

[0032] Among them, Figure A is the statistical diagram of the predicted classification of virulence genes of P12;

[0033] Figure B is a statistical diagram of the predicted classification of P12 drug resistance genes;

[0034] Figure 7 The results of the biosafety experiment of Bacillus Velez P12 on white shrimp and grass carp in the present invention;

[0035] Figure 8 These are longitudinal sections of grass carp intestines, where Figure A is the control group and Figure B is the experimental group. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0037] Example 1

[0038] A strain of Bacillus Velezii P12 and its isolation and application

[0039] 1. Separation and screening

[0040] The shrimp used in the experiment were healthy whiteleg shrimp from the Litopenaeus vannamei farm in Lianyungang City, Jiangsu Province. The shrimp was about 1 cm long, with uniform size and vigor. The separation process was as follows: take the whole shrimp in 5 mL of sterile saline, grind it with an electric pestle, and pause and wait for a moment every 10 seconds before grinding to prevent the temperature from being too high during grinding. After standing for 30 minutes, take the supernatant and dilute it 10, 100 and 1000 times. Each gradient was aspirated and spread on a tryptone soy broth (TSB) agar plate, and inverted at 28°C for 24-48 hours. Select a plate with different bacterial colonies and good distribution, mark the well-spaced colonies for separation, and streak the separated colonies on the TSB plate and TCBS agar plate for pure culture. Culture at 28°C for 24-48 hours, and observe the growth of the colonies continuously. Pick strains of different morphologies for seed preservation and store them in a -80°C refrigerator.

[0041] The pathogen indicator bacteria was Vibrio anguillarum, which was cultured in TSB medium to the logarithmic growth phase and diluted to 10 with physiological saline. 8CFU / mL, 100 μL of bacterial solution was spread on TSB solid plate, and holes were punched with a hole puncher (6 mm in diameter, 4 holes were punched on each plate). After drying for 10 minutes, the above-screened bacterial solution (50 μL) was added to the small wells (3 replicates), and cultured at 28°C for 24 hours. The size of the inhibition zone of each well was measured, and the antagonistic strain with the largest inhibition zone was screened and named: P12. An endogenous strain P12 was obtained by the above separation method.

[0042] 2. Identification

[0043] The screened strain Bacillus velezensis P12 was streaked onto a TSB solid plate, cultured at 28°C for 18 hours, and then observed for colony morphology. P12 was subjected to Gram staining, and a single colony was transferred to TSB liquid culture medium, cultured at 28°C and 120rpm for 8 hours, and then subjected to Gram staining to observe bacterial morphology. P12 was subjected to spore staining, and a single colony was transferred to TSB liquid culture medium, cultured at 28°C and 120rpm for 36 hours, and then subjected to malachite green spore staining to observe spore and bacterial morphology. P12 was observed under a scanning electron microscope, and a single colony of P12 was picked and transferred to a 2.5% glutaraldehyde fixative, fixed for more than 12 hours, and observed under a scanning electron microscope.

[0044] The results are as follows Figure 1 As shown, Figure 1 As shown in Figure B, the colony of P12 is light yellow, with a rough surface and irregular edges, a convex feel, opaque, and sticky. After Gram staining, the bacteria were found to be stained purple and short rod-shaped, which is a Gram-positive bacterium ( Figure 1 Figure C). Spore staining shows that the bacteria are stained red and the spores are stained blue-green ( Figure 1 Figure D). Figure 1 The scanning electron microscopy results of Figures E, F, and G show that for strain P12, most of the fungi are arranged in pairs or chains, a few are arranged singly, and have a rough surface and hyphae.

[0045] 3. Antibacterial effect

[0046] For example, Vibrio anguillarum, Vibrio cholerae, Vibrio cholerae, Vibrio owensii, Vibrio cholerae, Photobacterium damselae, Vibrio campbellii, Vibrio vulnificus and Vibrio parahaemolyticus were used as indicator bacteria to conduct in vitro antibacterial experiments of P12. Using the Oxford cup method, Vibrio was evenly spread on the TSB plate, 100 μL of P12 bacterial solution was added to the Oxford cup wells, and the plate was cultured at 28°C for 24 hours, and the diameter of the inhibition zone was observed and measured.

[0047] The results are shown in Table 1. The P12 strain was able to inhibit the growth of nine Vibrio species, and the diameters of the inhibition zones were all above 14 mm.

[0048] Table 1 P12 antibacterial test results

[0049]

[0050]

[0051] The results show that the Bacillus velezensis P12 isolated and obtained by the present invention has a strong antibacterial effect on Vibrio anguillarum, Vibrio cholerae, Vibrio cholerae, Vibrio owensii, Vibrio cholerae, Photobacterium damselae, Vibrio campbellii, Vibrio vulnificus and Vibrio parahaemolyticus, and the diameter of the inhibition zone is all above 14 mm.

[0052] IV. Drug sensitivity test

[0053] The agar diffusion method was used to detect the inhibitory effects of 27 antimicrobial drugs (penicillin GP, ​​oxacillin OX, ampicillin AM, cefradine RAD, ceftriaxone CTR, ceftazidime CAZ, erythromycin E, azithromycin AZM, gentamicin GM, streptomycin S, kanamycin K, neomycin N, tetracycline TE, doxycycline DX, minocycline MI, chloramphenicol C, florfenicol FON, polymyxin B PB, rifampicin RA, neomycin NV, nalidixic acid NA, pipemidic acid PPA, enrofloxacin ENR, ciprofloxacin CIP, ofloxacin OFX, norfloxacin NOR and co-trimoxazole SXT) on P12. After culturing P12 for 16-18 h, a single colony was picked and rinsed in 1 mL of sterile saline to adjust the concentration to 1×10 8 CFU / mL. Take 100μL of bacterial suspension and spread it evenly on TSB plate, and stick the drug-sensitive paper on the surface of culture medium. Culture at 28℃ for 24h, measure the diameter of inhibition zone to judge the sensitivity of strain to antimicrobial drugs. The test results are judged according to "WS / T 639-2018 Technical Requirements for Antimicrobial Susceptibility Test".

[0054] The results are as follows Figure 2 As shown, P12 was sensitive to most antimicrobial drugs, but was resistant to polymyxin B and pipemidic acid, and had intermediate resistance to ceftazidime, azithromycin, tetracycline, and rifampicin.

[0055] 5. Determination of extracellular products

[0056] (1) Prepare egg yolk plate to measure phospholipase activity: Add 100 mL of 50% egg yolk emulsion to every 1000 mL of TSB agar medium. Pipette 5 μL of bacterial suspension and drop it on the egg yolk plate, with at least three drops for each strain. Incubate at 28°C for 48 hours. Bacteria containing phospholipase can decompose phosphatidylcholine, forming an insoluble, opaque turbid band around the culture medium.

[0057] (2) Prepare gelatin plates to measure gelatinase activity: Add 0.5% gelatin (w / v) to TSB agar medium. Pipette 5 μL of bacterial suspension and transfer to gelatin plates. After culturing at 28°C for 48 hours, inject saturated ammonium sulfate solution. After incubation at room temperature for 5 minutes, take photos to record the gelatinase decomposition ability of the strain. A transparent circle will form around the colonies with gelatinase activity.

[0058] (3) Prepare oil plate to measure lipase activity: TSB agar medium, add 10mL Tween 80 per 1000mL, adjust pH to 7.2, add 1mL 1.6% neutral red aqueous solution, sterilize, and pour the plate. Pipette 5μL of bacterial suspension and transfer to oil plate, incubate at 28℃ for 48h. Colonies with lipase activity are red. No red colonies appeared in P12 colonies.

[0059] (4) Use casein agar to prepare a casein plate to measure protease activity. Pipette 5 μL of bacterial suspension and transfer to the casein plate. After constant temperature culture at 28°C for 48 hours, take a photo to record the casein decomposition ability of the strain. A transparent ring appears around the bacterial colonies that can secrete protease. A transparent ring appears around the P12 bacterial colony.

[0060] (5) Add 10g of starch to every 1000mL of TSB agar medium to measure the activity of starch hydrolyzing enzymes. Pipette 5μL of bacterial suspension and transfer to starch plate, incubate at 28℃ for 48h, drip Lugol's iodine solution around the colonies and incubate for 5min. Colonies without amylase activity will not change color, while colonies with amylase activity will have brown rings around them. A brown ring will appear around P12 colonies.

[0061] (6) Prepare blood plate to test hemolysis: Add 5% sterile defibrinated sheep blood to TSB agar medium. Take 5 μL of bacterial solution and drop it on the blood plate. Incubate at 28°C for 48 hours and observe the colony diameter and hemolysis zone size. Hemolysis zone appears at P12.

[0062] The results are as follows Figure 3 As shown, the Bacillus velezensis P12 isolated and obtained in the present invention has the ability to secrete extracellular phospholipase, gelatinase, protease, and amylase, but cannot secrete lipase and is hemolytic.

[0063] 6. Virulence gene detection

[0064] PCR was used to detect the hemolytic enterotoxin gene hbl, the non-hemolytic enterotoxin gene nhe, the enterotoxin FM gene (entFM), the enterotoxin T gene (bceT), the potential enterotoxin hly II, the cytotoxin K (cytK), and the vomiting toxin ces. The primers used are shown in Table 2.

[0065] Table 2 Primers for detecting virulence genes of candidate probiotics

[0066]

[0067]

[0068] The results show that the Bacillus velezensis P12 isolated by the present invention does not carry virulence genes such as the hemolytic enterotoxin gene hbl, the non-hemolytic enterotoxin gene nhe, the enterotoxin FM gene (entFM), the enterotoxin T gene (bceT), the potential enterotoxin hly II, the cytotoxin K (cytK) and the vomiting toxin ces.

[0069] 7. Resistance test

[0070] 1. High temperature tolerance

[0071] The bacterial solution cultured for 24 hours was treated in a water bath at 60℃, 80℃, and 100℃ for 20 minutes, and the strain not subjected to high temperature treatment was used as the control group. The solution was diluted 10 times in a continuous gradient and diluted to an appropriate concentration for plating and counting.

[0072] 2. Acid tolerance

[0073] Bacteria cultured for 15 h were collected by centrifugation at 8000 rpm for 3 min, washed twice with sterile saline, resuspended in TSB liquid culture medium with pH values ​​of 2.0, 2.5, and 3.0, and cultured at 37°C for 0, 1 h, 2 h, and 3 h, then serially diluted in 10-fold gradients and appropriate concentrations were used for smearing and counting.

[0074] 3. Simulated gastric fluid tolerance

[0075] Prepare simulated gastric fluid. Take the bacterial solution cultured for 15 hours, centrifuge it at 8000rpm for 3 minutes, wash it twice with sterile saline, and resuspend it in an equal volume of simulated gastric fluid. Take samples at 0h, 1h, and 3h to detect the absorbance at 620nm.

[0076] 4. Simulated intestinal fluid tolerance

[0077] Prepare simulated intestinal fluid. Take the bacterial solution cultured for 15 hours and centrifuge at 8000rpm for 3 minutes to collect the bacteria, wash twice with sterile saline, and resuspend in an equal volume of simulated intestinal fluid. Take samples at 0h, 1h, and 3h to detect the absorbance at 620nm.

[0078] 5. Bile salt tolerance

[0079] Prepare TSB medium containing 0.3% bile salts. Take the bacterial solution cultured for 15 hours and centrifuge at 8000 rpm for 3 minutes to collect the bacteria, wash twice with sterile saline, and resuspend in TSB medium containing bile salts. Take samples at 0h, 1h, and 3h to measure the absorbance at 620nm.

[0080] The results are as follows Figure 4 As shown, the Bacillus velezensis P12 isolated and obtained in the present invention can tolerate high temperature (80°C), acid (pH 2.0), bile salt (2%) and simulated intestinal environment, indicating that the bacteria can withstand digestion by intestinal fluid if mixed with feed.

[0081] 8. Whole genome sequencing analysis

[0082] After the strain P12 was cultured to the logarithmic growth phase, the bacteria were collected, the genomic DNA was extracted, and the whole genome sequencing analysis was performed.

[0083] The results showed that the genome sequencing results showed that the length of the whole genome sequence of the P12 strain was 3929473bp, its G+C content was 46.50%, there were 3752 coding genes, a total of 27 rRNAs, 9 for 16S, 23S and 5S respectively. Figure 5 ). Strain P12 was predicted to have 430 virulence genes and 209 drug resistance genes ( Figure 6 ). Two point mutations in parY of Streptomyces caused resistance to aminocoumarins (streptomycin belongs to aminoglycosides). The drug sensitivity test results of P12 showed sensitivity to neomycin in the coumarin class. There is a beta-lactamase gene Laribacter hongkongensis ampC beta-lactamase. Among the beta-lactam drugs, P12 was only sensitive to CAZ and was insensitive to the rest.

[0084] IX. Biosafety Assessment

[0085] 80 healthy whiteleg shrimp (Litopenaeus vannamei) were randomly divided into two groups and injected intraperitoneally. Each shrimp in one group was injected with 1.0×10 8 CFU / mL bacterial suspension 10μL, the control group shrimp injected with the same amount of sterile saline. After injection, the shrimp were returned to the original water environment for continued breeding. The number of deaths and health status of each group of shrimp were recorded every day, and the observation was continued for 14 days. Finally, the dissection was performed to observe whether there were any lesions.

[0086] Healthy grass carp (Ctenopharyngodon idella) were temporarily acclimated in the laboratory for 7 days. During the acclimation period, the water temperature was 21±0.5℃, the light / dark cycle was natural, and the dissolved oxygen was 5.5-6.0mg / L. After fasting for two days, commercial feed (Tongwei 180 model) was fed twice a day (09:00 and 17:30). After the temporary acclimation, 80 fish were randomly divided into 2 groups. Each fish in one group was injected with 1.0×10 8 CFU / mL bacterial suspension 20μL, the control group fish injected with the same amount of sterile saline. After injection, the grass carp was returned to the original water environment for continued breeding. The number of deaths and health status of each group of fish were recorded every day, and the observation was continued for 14 days. Finally, the dissection was performed to observe whether there were any lesions.

[0087] The results are as follows Figure 7 As shown, after injection of P12, the survival rate of white shrimp was 90% and the survival rate of grass carp was 95% within two weeks.

[0088] In vitro biosafety experiments show that the Bacillus velezensis P12 isolated and obtained by the present invention has good safety to both white shrimp and grass carp.

[0089] 10. Applied Research

[0090] 180 healthy whiteleg shrimps were randomly divided into two groups, with three replicates in each group. The P12 strain was inoculated into LB liquid medium for overnight culture, and then the bacteria were collected by centrifugation and suspended in sterile PBS buffer. The bacteria were then added to the granular expanded feed to prepare 1.5×10 8 CFU / g of feed was fed to the experimental group, and the control group was fed with the same amount of untreated pelleted feed. After continuous feeding for 4 weeks, an artificial infection experiment was conducted to evaluate the biological control effect of feeding P12 strain. In the artificial infection experiment, shrimp-derived Vibrio anguillarum strains were used. The strains cultured to the logarithmic growth phase were collected and resuspended in sterile PBS to prepare 2×10 8 CFU / mL of bacterial suspension. Ten shrimps were randomly selected from each experimental group and control group for artificial infection experiments, and 0.1mL of bacterial suspension was injected into the peritoneal cavity of each shrimp. The death and morbidity of shrimps were recorded every day and observed for 2 weeks. The results showed that the cumulative mortality rate of shrimps in the feeding group was 35%, and the cumulative mortality rate of the control group was 76%, indicating that feeding P12 strain can significantly improve the disease resistance of white shrimp to Vibrio anguillarum.

[0091] Tongwei 180 feed was used as the basic feed, the treatment group without P12 added to the basic feed was used as the control group, and the treatment group with P12 added to the basic feed was used as the feed group. The amount of P12 added was about 3×10 7 CFU / g. After the 30-day breeding experiment, the grass carp intestine was sliced ​​to observe the intestinal growth and measure the number of mucus cells, intestinal muscle layer thickness, villus height and villus width.

[0092] The results show that Figure 8 ), compared with the control group, after feeding the feed supplemented with P12, the number of intestinal mucous cells of grass carp increased significantly, the thickness of the mucosal muscular layer increased, and the degree of villus folds increased, which greatly increased the intestinal absorption area.

[0093] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0094] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Bacillus Velezii from aquatic sources ( Bacillus velezensis ) P12, characterized in that, The deposit number of the Bacillus Velez P12 is: CCTCC M 20221921, and the deposit date is December 12, 2022.

2. Use of the Bacillus Velez P12 according to claim 1 in preparing aquatic animal feed, characterized in that: The aquatic animal is whiteleg shrimp or grass carp.

3. Use of the Bacillus Velez P12 according to claim 1 in preparing an inhibitor, characterized in that: The inhibitor is used to inhibit Vibrio anguillarum ( Vibrio anguillarum ), Vibrio owensii ( Vibrio owensii )、Vibrio rotifer( Vibrio cholerae ) and Photobacterium mermanii ( Photobacterium damselae ).

4. Use of the Bacillus Velez P12 according to claim 1 in the preparation of a drug for treating Vibrio anguillarum infection in white shrimp.

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