CN117965340B - Bacillus subtilis for preventing and treating nocardia and regulating blood sugar and blood fat and application thereof - Google Patents

Abstract

The invention discloses a bacillus subtilis strain HS-B2, which has a strain preservation registration number of GDMCCNo:63663. the bacillus subtilis HS-B2 has the activities of protease, amylase and cellulase in vitro, has an inhibiting effect on common pathogenic bacteria of aeromonas schubertii and nocardia seriolae in the process of breeding the hybrid snakeheads, and passes safety and tolerance tests. In the breeding test, the bacillus subtilis HS-B2 effectively reduces the mortality rate of the hybrid snakehead after being infected by nocardia seriolae, improves the growth performance, reduces the blood sugar and blood fat content in serum, and simultaneously has the characteristics of reducing the use amount and the residual amount of antibiotics in the breeding process of the hybrid snakehead, reducing the production cost, improving the production performance and the like.

Description

Bacillus subtilis for preventing and treating nocardia and regulating blood sugar and blood fat and application thereof

Technical Field

The invention relates to bacillus subtilis, in particular to bacillus subtilis for preventing and treating nocardia and regulating blood sugar and blood fat. The invention also relates to application of the bacillus subtilis.

Background

The hybrid snakehead (Hybrid snakehead) is obtained by taking snakehead (Channa argus) and snakehead (Channa maculata) as parents and performing artificial hybridization. The hybridization characteristics have remarkable advantages, such as high growth speed, easy domestication of artificial feed, low temperature and low oxygen resistance, strong disease resistance, large body size, good edible taste and the like. In recent years, the cultivation scale of hybrid snakeheads is gradually enlarged, and the snakeheads are widely cultivated in the whole country, and the yield is over 50 ten thousand tons. Especially in the bead triangle area, the parent is replaced to become the main breeding variety in snakehead fish, the per mu yield can reach 5000Kg, and the per mu yield in some areas breaks through 15000Kg. However, with the rapid development of the hybrid snakehead breeding industry, new breeding modes are continuously developed, the degree of intensification is higher and higher, the problem of breeding diseases is serious, and the problem of diseases becomes one of the reasons for restricting the continuous and stable development of the hybrid snakehead breeding industry. Meanwhile, high-strength feed feeding is easy to cause glycolipid metabolic disturbance of the hybrid snakehead organisms, so that disease outbreaks in the cultivation process are further aggravated, and great economic losses are caused for the farmers.

For the above cases, antibiotics are generally used for disease control, but the use of antibiotics has potential risks to consumers and the environment because they have a tendency to bioaccumulate and remain in fish bodies, which not only causes degradation of the quality of the hybrid snakeheads, but also damages the health of consumers. In addition, there is a major problem that excessive use of antibiotics in the cultivation process may cause the generation of antibiotic resistant strains, and destroy and interfere with the ecological balance of microorganisms in the cultivation environment, increasing the difficulty in disease control of aquatic animals.

Probiotics are considered to be an ideal antibiotic replacement. In the aquaculture industry, probiotics can obviously improve the production performance of the aquaculture industry, enhance the resistance to diseases, reduce the occurrence of diseases, reduce the use amount of chemical medicines such as antibiotics and the like, improve the quality of water products and have better social and economic benefits.

However, most probiotics have strong host specificity, i.e. probiotics obtained from non-homologous organisms or in culture environments have unsatisfactory application effects in the culture production process of other animals, and have good stability and high efficiency. Therefore, the unique strain which has a good probiotic effect on the hybrid snakeheads and is suitable for the cultivation of the hybrid snakeheads is particularly important to be selected from a plurality of probiotics.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention separates and screens out a bacillus subtilis (Bacillus subtilis) from healthy intestinal tracts of hybrid snakeheads, and the bacillus subtilis is named as: HS-B2. Experiments prove that the strain has the activities of producing protease, amylase and cellulase in vitro, has an inhibiting effect on common pathogenic bacteria of aeromonas sulburi and nocardia seriolae in the process of breeding hybrid snakeheads, and passes safety and tolerance tests. In the culture test, the strain effectively reduces the death rate of the hybrid snakehead infected with nocardia sericata, improves the growth performance, reduces the content of blood sugar and blood fat in serum, and has the characteristics of reducing the use amount and the residual amount of antibiotics in the culture process of the hybrid snakehead, reducing the production cost, improving the production performance and the like.

The first object of the present invention is to provide a strain of Bacillus subtilis (Bacillus subtilis), designated as: HS-B2. The strain is separated and screened from healthy intestinal tracts of hybrid snakeheads, has the activities of producing protease, amylase and cellulase in vitro, and has an inhibiting effect on common pathogenic bacteria of aeromonas schubertii and nocardia seriolae in the process of culturing the hybrid snakeheads.

The bacillus subtilis (Bacillus subtilis) strain HS-B2 provided by the invention is delivered to the Guangdong province microorganism strain collection for preservation in 2023, 7 and 19 days, and is received with a strain preservation registration number of GDMCC No:63663.

The second object of the present invention is a microecological preparation. Specifically, the active ingredient of the microecological preparation is bacillus subtilis HS-B2 or fermentation liquor or bacterial suspension or fermentation product or fermentation degerming supernatant thereof.

The third object of the invention relates to the application of the bacillus subtilis strain HS-B2 and the microecological preparation. In particular to application of the bacillus subtilis HS-B2 in the culture of hybrid snakeheads. Relates to the application of the microecological preparation in the cultivation of hybrid snakeheads.

Specifically, the bacillus subtilis strain HS-B2 or the microecological preparation thereof is fed weekly in the process of breeding the hybrid snakehead.

According to the invention, the bacillus subtilis HS-B2 and the microecological preparation are used in the process of breeding the hybrid snakeheads, and the feeding test for 8 weeks shows that the bacillus subtilis HS-B2 has the functions of improving the growth performance of the hybrid snakeheads, reducing the blood sugar and triglyceride level in serum and improving the resistance of the hybrid snakeheads to nocardia seriolae infection.

Drawings

FIG. 1 shows colony morphology and gram staining of Bacillus subtilis strain HS-B2.

FIG. 2 is a 16S rRNA phylogenetic tree of B.subtilis strain HS-B2.

FIG. 3 is a hemolytic assay of Bacillus subtilis strain HS-B2;

a: the front of the hemolysis test; reverse side of hemolysis test; c: hemolysis test micro distance.

FIG. 4 is a test of extracellular hydrolase activity of Bacillus subtilis strain HS-B2;

A: qualitative protease culture medium; b, amylase qualitative culture medium; c: cellulase qualitative culture medium.

FIG. 5 is a graph showing the results of a drug susceptibility test of Bacillus subtilis HS-B2;

1: enrofloxacin; 2: ciprofloxacin; 3: compound neonolamine; 4: neomycin; 5: chloramphenicol; 6: erythromycin; 7: gentamicin; 8: amoxicillin; 9: kanamycin.

FIG. 6 is a B.subtilis strain HS-B2 pathogen antagonism assay;

A: aeromonas sulbert; nocardia Seriola.

FIG. 7 is a plot of mortality from nocardia seriolae infection after feeding a control or probiotic-containing feed;

". Times." indicates significant differences between the two groups (P < 0.05).

Detailed Description

In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings.

1. Material

Experimental animals: healthy hybrid snakehead provided by Guangdong certain fine variety field

Test strain: the strain is obtained by separating from the intestinal tract of healthy hybrid snakehead as a test strain.

Pathogen-indicative strains: aeromonas sulbactam WL-2 and Nocardia seriolae NK-3.

2. Main reagent and instrument

LB medium, BHI medium and blood agar medium, purchased from Guangdong Crypto microorganism technologies Co., ltd; gram staining kit, purchased from beijing solibao technologies limited; antibiotic drug sensitive paper purchased from Hangzhou microbial agents, inc.; PCR instrument, available from Hangzhou cypress constant technology Co., ltd; biochemical incubator, purchased from tai macro medical instruments limited; a high-speed bench-top refrigerated centrifuge was purchased from Ai Ben Germany. pH analyzer, available from WTW, germany. Primers used in the experiments were supplied by the gene technologies limited company, san francisco, guangzhou (table 1).

TABLE 1PCR amplification primer details

3. Preparation of culture medium

LB medium was used for isolated culture of the strain, BHI medium was used for culture of pathogenic indicator bacteria, and all were prepared according to the commercial instructions. Qualitative culture medium for producing amylase: 10g of tryptone, 10g of yeast extract, 5g of NaCl, 20g of soluble starch, 15g of agar and 1000mL of distilled water; adjusting the pH to 7.2-7.4, sterilizing for 15min at 121 ℃. Qualitative culture medium for producing cellulase: 10g of tryptone, 10g of yeast extract, 5g of NaCl, 10g of sodium carboxymethylcellulose (CMC-Na), 15g of agar, 1000mL of distilled water, pH 7.0 and sterilization at 121 ℃ for 15min. Qualitative culture medium for producing lipase: 10g of tryptone, 10g of yeast extract, 5g of NaCl, 10g of tributyrin, 1000mL of distilled water and sterilization at 121 ℃ for 15min. Qualitative protease production medium: 10g of tryptone, 10g of yeast extract, 5g of NaCl, 20g of skim milk powder, 15g of agar, 1000mL of distilled water, pH of 7.2-7.4 and sterilization at 121 ℃ for 15min.

0.03G of pig bile salt is added into 100mL of LB culture medium, after complete dissolution and uniform mixing, 0.4% (w/v) NaOH solution and HCl solution are used for adjusting the pH value of the bile salt culture medium to be=6.8, and a 0.22 mu m degerming filter is used for filtering the culture medium, thus obtaining the bile salt culture medium. Taking 1.64mL of dilute hydrochloric acid, adding 80mL of distilled water and 1.0g of pepsin, shaking uniformly, adding into a 100mL volumetric flask, fixing the volume by distilled water, sterilizing by a 0.22 mu m filter, and mixing with LB liquid culture medium 1:1 with pH=3 to obtain the gastric juice culture medium. KH ₂PO₄ 1.36.36 g was added to 100mL of LB liquid medium, and after complete dissolution, 1.0g of trypsin was added, and the mixture was thoroughly mixed, and the pH value was adjusted to 6.8,0.22 μm, and the mixture was filtered and sterilized to obtain an intestinal juice medium.

4. Acquisition of strains

Collecting intestinal tracts of healthy hybrid snakeheads under the aseptic condition, cleaning the content, shearing and washing the intestinal tracts with aseptic normal saline, homogenizing the obtained intestinal tissues, centrifuging, taking supernatant to dilute the supernatant to obtain tissue suspensions with different concentration gradients, coating the tissue suspensions on an LB solid medium for culture, and purifying and culturing single colony to obtain isolated strains; screening the separated strain with enzyme-producing activity and pathogenic bacteria antagonism to obtain the strain with the strongest comprehensive probiotics effect, and further screening and identifying to obtain the strain with the number of HS-B2.

5. Morphological observation

According to the handbook of the identification of common bacterial systems, the strain HS-B2 obtained by screening was streaked onto LB solid plates, and after culturing at 37℃for 12 hours, the colony morphology was observed. Gram staining is carried out on the strain HS-B2, single colony is transferred into LB liquid medium, the culture is carried out for 6 hours at 37 ℃ and 180rpm, a small amount of bacterial liquid is sucked on a glass slide by a sterile suction tube, gram staining is carried out according to a conventional method, and the bacterial morphology is observed.

As a result, as shown in FIG. 1, the colony surface of the HS-B2 strain on the LB medium was rough and opaque, and was stained white, rough and irregular in edge. After gram-dyeing and color-testing, the bacterial cells are found to be dyed into purple, the form is in a short rod shape, most of the bacterial cells are arranged in pairs or chains, and few bacterial cells are arranged singly, so that the bacterial cells are gram-positive bacteria.

6. Molecular biological identification

Genomic DNA of strain HS-B2 was extracted. The target gene amplification was performed using the 16S rRNA gene universal primer. The PCR reaction conditions of the 16SrRNA gene are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 30 cycles; and finally, fully extending for 10min at 72 ℃. After 5. Mu.L of the amplified product was electrophoresed on a 1% agarose gel, the amplified product was observed, and the positive PCR product was subjected to sequencing analysis. The measured gene sequences were aligned in the GenBank database for Nucleotide BLAST sequences, and a phylogenetic tree was constructed using MEGA7.0 in-software adjacency (NJ) to further identify the strain as bacillus subtilis (fig. 2).

7. Biological safety evaluation

After the HS-B2 strain is cultured to the logarithmic growth phase, the strain is diluted to 1X 10 ⁴ CFU/mL, a sterile inoculating loop is used for inoculating the bacterial liquid onto a defatted sheep blood agar plate, and the defatted sheep blood agar plate is placed in a 28 ℃ incubator for culturing for 24 hours, and whether hemolysis loops exist around the colony or not is observed. The results showed that the HS-B2 strain had no hemolytic ring around the colony, i.e., the strain was not hemolytic, and had high biosafety (FIG. 3).

Example 1 extracellular hydrolase Activity assay

The activity of extracellular hydrolases such as amylase, cellulase and protease of the strain HS-B2 was examined.

Sterilized oxford cups (diameter 6 mm) were placed on qualitative medium, 200. Mu.L of overnight culture broth was added to each oxford cup well, 3 replicates were set, and incubated at 30℃for 20h. Adding a proper amount of 1% Congo red dye solution into a cellulase qualitative culture medium, covering the whole plate, pouring out the Congo red dye solution after dyeing for 30min, adding 1mol/mL NaCl solution for decoloring, pouring out the NaCl solution after standing for 30min, and recording the diameter of a transparent hydrolysis ring. Adding a proper amount of iodine solution into an amylase qualitative culture medium, uniformly coating a cover plate, standing for 10min, and recording the condition of color development rings on a blue-violet plate dyed by the iodine solution. The diameter of the transparent circle on the protease qualitative medium was measured and recorded.

As shown in FIG. 4, the strain HS-B2 can form hydrolysis circles in the corresponding qualitative culture medium, which indicates that the strain HS-B2 has certain hydrolysis capability on starch, cellulose and protein and has the capability of secreting extracellular amylase, cellulase and protease.

EXAMPLE 2 drug sensitivity assay

The sensitivity of the strain HS-B2 to the antibacterial agent was examined by the sheet agar diffusion method. Inoculating the strain to be tested into BHI liquid culture medium, culturing overnight at 30deg.C with 200rpm, regulating bacteria concentration to 0.5 McAb turbidity with 0.75% sterile physiological saline, inoculating 200 μl into MH agar culture medium by coating method, uniformly attaching drug sensitive paper sheet onto the surface of culture medium with sterile forceps after drying, inverting the plate, culturing at 30deg.C for 20h, and measuring diameter of antibacterial zone. And judging the drug sensitivity characteristics of the strain by referring to the drug sensitive paper sheet instruction. As shown in Table 2 and FIG. 5, the strain HS-B2 is sensitive to 9 antibacterial drugs such as enrofloxacin, which indicates that the strain HS-B2 may not contain the drug resistance genes of the drugs.

TABLE 2 results of drug sensitivity test on strain HS-B2

R: resistance; i: intermediate is moderately sensitive; s: susceptible sensitivity

EXAMPLE 3 pathogen antagonism assay

The antagonistic activity of the strain HS-B2 on common aquatic pathogenic bacteria, aeromonas sulbernati and Nocardia seriolae in the process of breeding the hybrid snakehead is detected by adopting an oxford cup method. The concentration of the fresh overnight cultured indicator bacteria liquid is adjusted to be 1 multiplied by 10 ⁷ CFU/mL, 200 mu L of the indicator bacteria liquid is respectively and evenly coated on an MH agar plate, sterilized oxford cups (with the diameter of 6 mm) are placed on the plate (3 repeats), and 200 mu L of HS-B2 bacteria liquid which is cultured until OD ₆₀₀ =1 is added into each oxford cup. Placing the mixture in a constant temperature incubator at 30 ℃ for culturing for 24 hours, and observing and measuring the diameter of the inhibition zone.

Pathogenic bacteria antagonism experiments showed that the strains HS-B2 Aeromonas sulbactam and nocardia seriolae have an inhibitory effect (FIG. 6). In the antibacterial test of the Nocardia seriolae of HS-B2, the Nocardia seriolae does not grow on a culture medium, which indicates that the bacillus subtilis HS-B2 has stronger antibacterial activity on the Nocardia seriolae. Nocardia seriolae is unable to grow due to nutritional competition.

EXAMPLE 4 Strain tolerance test

And (3) picking the purified single bacterial colony, inoculating the single bacterial colony into a liquid culture medium for expansion culture, respectively sucking 500 mu L of bacterial liquid subjected to expansion culture into 5mL of basic liquid culture medium, bile salt, intestinal and gastric juice culture medium by using a pipetting gun in an ultra clean bench for 16-18 hours, starting timing after 5 minutes, taking the cultured bacterial liquid for concentration gradient dilution to 10 ^-5 hours, taking 100 mu L of diluted bacterial liquid, coating the bacterial liquid on a solid culture medium, and culturing for 24-48 hours, and then counting bacterial colonies.

The tolerance test result shows that the strain HS-B2 has better tolerance and can survive after being cultured for 2 hours in the culture environment of gastric juice, bile salt and intestinal juice, and the survival rate of the strain HS-B2 in the gastric juice is 59.75+/-7.28%; the survival rate in intestinal juice is 87.42 +/-2.35%; the survival rate in bile salts is 65.41 +/-3.21 percent.

Example 5 Strain feeding test

Taking 540 hybrid snakeheads (17.36+/-0.25 g) with good health status, carrying out temporary culture 14d and bacteria separation detection and microscopic examination, and randomly dividing the snakeheads into 3 groups (three groups are parallel and 60 groups are parallel): strain HS-B2 group, bacillus belicus group (HS-B4) and PBS control group. About 5% of feed is fed daily according to the body weight of the hybrid snakehead, 2 times daily (8:00 and 17:00), 8 weeks total, and the water temperature is 28+/-2 ℃. Each barrel is continuously filled with air and oxygen, and 33% of the culture water is replaced after the sewage is sucked every day.

Preparing probiotics feed: the HS-B2 strain and Bacillus belicus (HS-B4) were grown in an enlarged manner, washed 3 times with PBS, resuspended in PBS, and OD ₆₀₀ measured using a UV spectrophotometer to adjust to the desired concentration of 1X 10 ⁸ CFU/mL.

The nutritional composition of the test compound feed is detailed in table 3. The feed was purchased from Fenghua feed industries, inc. in Shunde district of Buddha. The probiotic bacteria liquid was coated onto the feed pellets using a spray can with sterile Phosphate Buffer (PBS) (ph=7.4) as carrier. An equal amount of sterile Phosphate Buffer (PBS) (ph=7.4) was added to the control feed. After the wet feed is uniformly sprayed, the feed is naturally air-dried in the air, and then the feed is put into a sterile sealed container and is stored in a refrigerator at 4 ℃. Probiotic feed formulation was performed once a week.

Table 3 test of the nutritional composition of the Compound feed (in% of dry matter)

The raw materials comprise: fish meal, soybean meal, rapeseed meal, peanut meal, wheat and processed products thereof, minerals, mineral elements and complex (chelate) compounds thereof, vitamins, amino acids, amino acid salts and analogues thereof, enzyme preparations, preservatives and the like.

After feeding, after 1d of feeding, anesthesia is carried out, body length is measured, body weight, liver weight and viscera mass are weighed, and serum and immune related tissue organs are collected. The growth index calculation method comprises the following steps:

Weight Gain Rate (WGR) = (final body weight-initial body weight)/initial body weight×100%

Specific Growth Rate (SGR) = (ln end average weight-ln initial average weight)/test period×100%

Feed Conversion Ratio (FCR) =feed amount fed/(final body weight-initial body weight) ×100%

Liver volume ratio (HSI) =liver weight/end body weight×100%

Visceral volume ratio (VSI) =visceral weight/terminal weight x 100%

Full length (CF) = (end body weight/end body length ³) ×100%

Survival (SR) =last/first mantissa×100%

Serum immunization indicators include: blood Glucose (GLU), serum total Cholesterol (CHO), glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP), triglycerides (TG), total Protein (TP), albumin (ALB), lactate Dehydrogenase (LDH), and Glutathione Reductase (GR). Serum samples stored at-80℃were sent to Guangzhou New sea Hospital and tested using Hitachi full-automatic Biochemical Analyzer (HITACHI 7180, japan).

The growth performance and survival of the hybridized snakes fed with the control or probiotic-containing feed for 8 weeks are shown in Table 4. The weight gain rate of the hybrid snakehead fed with the probiotic feed containing HS-B2 is 212.91 +/-15.13 percent, which is obviously higher than Yu Siwei bacillus belicus (160.96 +/-4.60 percent) and the control group (182.83 +/-1.56 percent) (P < 0.05); specific growth rate of hybrid snakehead fed with the probiotic feed containing HS-B2 is (2.04+/-0.09%), which is obviously higher than Yu Siwei bacillus belius (HS-B4) (1.71+/-0.03%) and that of the control group (1.86+/-0.01%); the fullness of the hybrid snakehead fed with the probiotic feed containing HS-B2 is (1.53 plus or minus 0.20 percent) which is obviously higher than that of the control group (1.31 plus or minus 0.26 percent); the bait coefficient (1.12.+ -. 0.03%) was significantly lower (P < 0.05) in the HS-B2 group and the survival rate (98.89.+ -. 0.96%) was significantly higher (P < 0.05) in the HS-B2 group compared to the control group and Bacillus bailii (HS-B4).

TABLE 4 growth performance and survival rate of hybrid snakeheads fed control or probiotic-containing feed for 8 weeks

Note that: data are expressed as mean ± standard deviation (n=3) the representations with different superscripts in the same row have significant differences (P < 0.05); IBW: initial weight; FBW: weight of the powder; WGR: weight gain rate; SGR: a specific growth rate; FCR: feed conversion rate; HIS: liver mass ratio; VSI: a dirty body ratio; CF: full of fertilizer; SR: survival rate.

Serum biochemical results are shown in table 5. The average blood sugar in the serum of the control group and the HS-B4 group is 3.99+/-1.13 g/L and 2.61+/-1.19 g/L, and the average blood sugar in the serum of the hybrid snakehead added with the strain HS-B2 is 2.11+/-0.38 g/L, which is obviously lower than that of the control group and the HS-B4 group; the triglyceride content in the serum fed with the hybridized snakehead containing the strain HS-B2 is 0.70+/-0.08 mmol/L, which is obviously lower than 1.29+/-0.54 mmol/L and 2.25+/-1.05 mmol/L (P < 0.05) of the control group and the HS-B4 group; the alkaline phosphatase content in serum of the hybridized snakehead fed with the strain HS-B2 is 6.33+/-1.15U/L, which is obviously lower than 12.67+/-2.30U/L and 11.67+/-2.88U/L (P < 0.05) of the control group and the HS-B4 group.

TABLE 5 Biochemical index of serum of hybrid snakeheads fed with control or probiotic-containing feed

Note that: data are expressed as mean ± standard deviation (n=3) the representations with different superscripts in the same row have significant differences (P < 0.05). TP: total protein; ALB: albumin; GLU: blood sugar; CHO: cholesterol; TG: triglycerides; ALP: alkaline phosphatase; AST: glutamic-oxaloacetic transaminase; ALT: glutamic-pyruvic transaminase; GR: glutathione; LDH: lactate dehydrogenase.

Carbohydrates are used as one of necessary metabolic energy supply substrates of brain, gill tissues, red blood cells and the like of fish, and are closely related to normal physiological functions and viability of fish bodies. But simultaneously, the carnivorous fishes mainly use protein and fat as energy sources, the capability of utilizing carbohydrate is poor, and the excessive high level of carbohydrate in feed can lead to the continuous high level of blood sugar and even inhibit the growth of fish bodies.

The snakehead fish used as a carnivorous animal rarely has carbohydrate intake in a natural environment, and the problem of hyperglycemia does not exist naturally. However, under the cultivation condition, starch is necessarily present in the production process of the puffed feed, which creates an objective condition for hyperglycemia production of the hybrid snakeheads. This is because the hybrid snakehead, which is a carnivorous fish, has a small number of insulin receptors and a low affinity as compared with the omnivorous fish. When ingested with carbohydrates, the hybrid snakeheads have weaker glycolytic and gluconeogenic effects. Therefore, the speed of decomposing carbohydrates by the hybridized snakeheads is slower, the hyperglycemia phenomenon lasts longer, and the hyperglycemia can lead to accumulation of liver glycogen, thereby causing hepatomegaly, slow growth and outbreak of diseases.

In addition, in order to promote the rapid growth of farmed fish, farmers often overfeed feed, use various growth-promoting antibiotics, hormones and the like, which cause the liver and digestive system of the fish to be too heavy in burden, cause intestinal tract and liver lesions, and cause hepatobiliary syndrome to cause large-area death.

The result shows that the strain HS-B2 can obviously reduce the blood sugar and the triglyceride in the serum of the hybrid snakehead when being fed to improve the growth performance and the survival rate of the hybrid snakehead, and is probably a new way for regulating the blood sugar and the blood fat of fish. Alkaline phosphatase is mainly present in liver tissue, and the increase of alkaline phosphatase content in serum represents that the organism has liver and gall diseases, and under the condition of feeding of strain HS-B2, the content of alkaline phosphatase in serum is reduced, and the method is probably a new way for regulating and controlling liver and gall syndrome of fish. The strain HS-B2 provides new microorganism resources for improving the utilization rate of the feed of the hybrid snakehead, improving the growth index, reducing the production cost and promoting the high-quality healthy cultivation.

EXAMPLE 6 pathogen challenge test

And (3) recovering Nocardia seriolae (NK-3), then scraping and inoculating the bacterial colony into a BHI liquid culture medium for expansion culture, and culturing for 56 hours at 120r/min in a shaking table at 30 ℃. The bacterial solution was then centrifuged at 4℃at 5000rpm for 15min, then ground using a glass grinder, washed 3 times with sterile PBS and resuspended in PBS. OD ₆₀₀ was measured by a spectrophotometer, and the bacterial liquid concentration was adjusted to 3X 10 ⁷ CFU/mL. After the cultivation experiment is finished, 15 fish are selected for temporary cultivation for 2 days in each group, and then the toxicity attack experiment is started. 200 mu L of bacterial liquid is injected into each fish abdominal cavity. The health status of the fish was observed and the mortality of the fish was recorded daily for 14 days.

The cumulative mortality after toxicity challenge of Norcardia quince by intraperitoneal injection was 97.78% + -3.86%, 64.44% + -10.19% and 82.22+ -7.73% (FIG. 7), respectively, showing that the survival rate of the strain HS-B2 fed with the mixture was significantly higher than that of the control group (P < 0.05), and the relative protection rate was calculated to indicate that RPS=33.34% after feeding the strain HS-B2. The calculation method of RPS is rps= [1- (experimental group cumulative mortality/control group cumulative mortality) ]x100%. From this, it was shown that feeding strain HS-B2 significantly improved the resistance of the hybrid snakehead to North Carbarbus quinquefolium infection.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (4)

1. A bacillus subtilis (Bacillus subtilis) strain HS-B2, having a deposit accession number GDMCC No:63663.

2. A microecological preparation, characterized in that the active ingredient is the bacillus subtilis strain HS-B2 or the fermentation liquid or the bacterial suspension thereof according to claim 1.

3. Use of the bacillus subtilis strain HS-B2 according to claim 1 in the cultivation of hybrid snakeheads.

4. Use of the microecological formulation of claim 2 in the cultivation of hybrid snakeheads.