CN116042449A - A kind of Bacillus subtilis and its application in the prevention and treatment of Chinese mitten crab diseases - Google Patents

Abstract

The invention relates to bacillus subtilis, in particular to bacillus subtilis JK-2 which is screened out and has antagonistic effect on pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 can be applied to antagonizing pathogenic bacteria of aquatic animals; the bacillus subtilis JK-2 can obviously improve the activity of lysozyme in serum and liver pancreas of the eriocheir sinensis, can enhance the capability of the eriocheir sinensis organism in eliminating pathogenic bacteria, and can be applied to disease control of the eriocheir sinensis.

Description

A kind of Bacillus subtilis and its application in the prevention and treatment of Chinese mitten crab diseases

technical field

The present invention relates to a kind of Bacillus subtilis and its application in the prevention and treatment of Chinese mitten crab disease, in particular to Bacillus subtilis JK-2 and its microbial agent, and their application in antagonizing aquatic animal pathogenic bacteria. The application in the prevention and treatment of bacterial diseases of Chinese mitten crab belongs to the technical field of aquatic antimicrobial drugs.

Background technique

In recent years, with the rapid development of intensive farming, bacterial diseases have become the most common and most harmful diseases in Chinese mitten crab (Eriocheirsinensis) farming, accounting for the largest proportion of disease outbreaks in each year.

For example, a variety of pathogenic bacteria such as Aeromonas punctatus, Aeromonas hydrophila, Citrobacter freundii, Aeromonas victoria and other pathogens in the aquaculture environment, such as gill rot, enteritis disease, shivering disease, etc. Diseases such as hepatopancreatic necrosis and hepatopancreatic necrosis have caused huge economic losses to Chinese mitten crab farming in my country. In general, many kinds of pathogenic bacteria, fast reproduction speed, strong pathogenicity and high incidence have seriously affected the healthy development of my country's mitten crab aquaculture industry.

In the field of prevention and control of aquatic diseases, hormone drugs, chemical drugs and antibiotic drugs in the past, because of their toxic side effects, drug resistance, especially the damage of drug residues to the aquaculture environment and the threat to the quality of aquatic products, have been It is gradually replaced by highly efficient and zero-residue microbial agents; microbial agents are highly safe, can improve the ecological environment of breeding, and are not easy to produce drug resistance. They can also improve the utilization efficiency of nutrients, enhance the immunity of aquaculture animals, and promote Body metabolism and growth, and also has the advantages of low price and abundant sources.

How to use microbial agents to enhance the resistance of Chinese mitten crabs to bacterial diseases has become an urgent problem to be solved in the aquaculture industry.

At present, enhancing the antioxidant capacity and immunity of Eriocheir sinensis is one of the most effective strategies for the prevention and control of bacterial diseases in Eriocheir sinensis. For example, some excellent strains of the genus Bacillus, such as Bacillus amylase, methylotrophic Bacillus, and Bacillus megaterium, have good antibacterial activity and have good effects in enhancing the body's antioxidant capacity and immunity. , greatly enriching Bacillus as a microbial resource for the development of biological control products for bacterial diseases of Chinese mitten crabs.

Therefore, the field hopes to screen new bacterial strains that antagonize aquatic animal pathogens, and develop effective microbial agents for preventing and treating diseases of mitten crabs.

Contents of the invention

In view of the above-mentioned problems and/or other problems of the related technology, the first aspect of the present invention provides a Bacillus subtilis JK-2, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC No: M20221568.

The second aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the above-mentioned Bacillus subtilis JK-2.

Preferably, the microbial agent comprises the above-mentioned fermentation broth of Bacillus subtilis JK-2 or a bacterial fluid preparation prepared from the fermentation broth or a bacterial powder preparation obtained by drying the fermentation broth.

The third aspect of the present invention provides the application of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent in antagonizing aquatic animal pathogenic bacteria.

Specifically, in this application, pathogenic bacteria in aquatic animals refers to common pathogenic bacteria in freshwater aquaculture environments.

Preferably, the pathogenic bacteria of aquatic animals are pathogenic bacteria of shrimp and crabs.

Specifically, in this application, pathogenic bacteria of shrimp and crab animals refer to pathogenic bacteria that commonly cause diseases of shrimp and crab animals in the freshwater aquaculture environment, for example, Aeromonas punctatus, Aeromonas hydrophila, Pathogens such as Citrobacter freundii, Aeromonas vietori, and Providencia alcaligenes.

Preferably, the pathogenic bacteria of shrimp and crabs are selected from the group consisting of Aeromonas vernerii, Aeromonas hydrophila, Aeromonas punctatus, Providencia alcaligenes and Citrobacter freundii.

The fourth aspect of the present invention provides a product for antagonizing pathogenic bacteria in aquatic animals, said product comprising the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent.

The fifth aspect of the present invention provides the application of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent in improving the serum lysozyme activity and/or hepatopancreas lysozyme activity of Chinese mitten crab.

The sixth aspect of the present invention provides a feed for Chinese mitten crabs, which comprises feed basic components and the above-mentioned Bacillus subtilis JK-2.

The seventh aspect of the present invention provides the application of the above-mentioned Bacillus subtilis JK-2 or the above-mentioned microbial agent or the above-mentioned Chinese mitten crab feed in the prevention and treatment of Chinese mitten crab bacterial diseases.

The present invention relates to a kind of Bacillus subtilis, in particular to a kind of Bacillus subtilis JK-2 which is screened out and has an antagonistic effect on pathogenic bacteria of aquatic animals; Bacillus subtilis JK-2 of the present invention can be applied to antagonize pathogenic bacteria of aquatic animals bacteria; Bacillus subtilis JK-2 of the present invention can significantly improve the activity of lysozyme in the serum and hepatopancreas of Chinese mitten crab, can enhance the scavenging ability of Chinese mitten crab body to pathogenic bacteria, and can be applied to Chinese mitten crab disease control.

Description of drawings

Fig. 1 is the antagonistic activity of four bacterial strains (JK-1, JK-2, JK-3 and JK-4) and control bacterial strain that primary screening separates to Aeromonas verkisii HXH1;

Fig. 2 is the antagonistic activity of bacterial strain JK-2 to other four known aquatic pathogenic bacteria;

Fig. 3 is the bacterium colony morphology photo that bacterial strain JK-2 forms on nutrient agar plate;

Fig. 4 is the photograph under the 100 times optical microscope of bacterial strain JK-2 after Gram staining;

Fig. 5 is the influence of bacterial strain JK-2 on Chinese mitten crab serum lysozyme content;

Fig. 6 is the influence of bacterial strain JK-2 on Chinese mitten crab hepatopancreas lysozyme content;

Figure 7 is a phylogenetic tree constructed based on the 16S rRNA sequence of bacterial strain JK2.

Detailed ways

The present invention will be further described below through specific embodiments, but the present invention is not limited to these specific embodiments.

The materials and reagents used in the following specific embodiments can be obtained from commercial sources unless otherwise specified. Where no specific technique or condition is indicated, it shall be carried out according to the technique or condition described in the literature in this field or according to the product manual.

One, the following introduces the inventor of the present application from the natural environment to isolate and screen to obtain the new bacterial strain of the prevention and treatment of Chinese mitten crab bacterial disease of the present invention—Bacillus subtilis JK-2 (Bacillus subtilis JK-2) CCTCC No: the operating process of M20221568 .

1) Primary screening

Take 1.0 g of breeding sludge (taken from a breeding pond in Weihai City, Shandong Province) and suspend it in 9 mL of sterile normal saline, shake and mix well, and let it stand in a water bath at 80°C for 10 min, and place it on a nutrient agar plate (the nutrient agar was purchased from Sinopharm Group Chemical Co., Ltd.). Reagent Co., Ltd.) was used to isolate and purify strains by dilution coating plate method and streak plate method.

The purified single colonies were respectively connected to sterile nutrient broth (purchased from Sinopharm Chemical Reagent Co., Ltd.), shaken and cultivated at 33°C and 180r/min for 24h, and aseptically sucked 0.1mL culture solution evenly. Spread on a nutrient agar plate, incubate at 33°C for 24 hours, wash the bacterial lawn with 0.85% sterile saline to prepare a bacterial suspension with a final concentration of 1.0×10 ⁹ CFU/mL, and store for future use.

In the preliminary screening step, 4 strains (respectively named: JK-1, JK-2, JK-3 and JK-4) were successfully isolated from the culture sludge.

2) Further screening

The pathogenic bacteria "Aeromonas verkirea HXH1" (purchased from the National Aquatic Animal Pathogen Bank) was used as the screening indicator bacteria; 0.1 mL of Aeromonas verkirea HXH1 bacterial suspension was aseptically drawn and spread evenly on the nutrient agar plate, Then aseptically paste a filter paper sheet containing 10 μL of the bacterial suspension (concentration of 1.0×10 ⁹ CFU/mL) of the four strains obtained by the above-mentioned primary screening on the plate, and measure the diameter of the inhibition zone after incubating at 33°C for 24 hours. (H) and the ratio (H/C) of colony diameter (C).

At the same time, with the known Bacillus amyloliquefaciens G1 and Bacillus licheniformis PNB3 (purchased from the National Aquatic Animal Pathogen Bank), as positive control antagonistic strains, the ratio of the diameter of the inhibition zone (H) to the diameter of the colony (C) was determined (H /C).

See Figure 1 for the experimental results. It can be seen from Fig. 1 that the antagonistic activity of the strain JK2 to the pathogenic bacteria Aeromonas virtienii HXH1 is the strongest. Specifically, the H/C ratio of strain JK2 to Aeromonas victorii HXH1 was 2.66, which was 47.78% (P<0.05) and 27.27% (P<0.05) higher than the H/C of strains JK1, JK3, and JK4, respectively. ) and 97.04% (P<0.05); compared with the positive control antagonistic strains (Bacillus amyloliquefaciens G1 and Bacillus licheniformis PNB3), the H/C of Aeromonas victorii HXH1 was 10.83% higher (P<0.05), respectively 22.02% (P<0.05).

The experimental data in this article are expressed as mean ± standard deviation, and SPSS 19.0 software is used for statistical analysis, and P<0.05 indicates that the difference is significant (the same below).

In order to further verify the above antibacterial results, the candidate strains, including strain JK-2, were further tested according to the above-mentioned operations against four known aquatic animal pathogens "Aeromonas hydrophila WS05", "Aeromonas punctatus The ratio of the diameter of the inhibition zone (H) to the diameter of the colony (C) of Monas DD5", "Providenia alcaligenes P3" and "Citrobacter freundii C1" (all purchased from the National Aquatic Animal Pathogen Bank) (H/C).

The experimental results are shown in Figure 2. It can be seen that the strain JK2 is effective against four known aquatic animal pathogens "Aeromonas hydrophila WS05", "Aeromonas punctatus DD5", "Providenia alcaligenes P3 " and "Citrobacter freundii C1" also had good antagonistic activity, with H/C values greater than 1.80.

Therefore, the strain JK-2 was selected as an excellent strain.

Aeromonas vernerii HXH1, Aeromonas hydrophila WS05, Aeromonas punctatus DD5, Alcaligenes Providencia P3, and Citrobacter freundii C1 are common pathogens in shrimp and crabs bacteria. Based on the above results, those skilled in the art can think of applying the screened bacterial strain JK-2 to antagonize aquatic animal pathogens, especially to antagonize shrimp and crab animal pathogens; it is conceivable that the screened bacterial strain JK-2 Products for antagonizing pathogenic bacteria in aquatic animals.

2. The identification process of the strain JK-2 obtained by screening is as follows:

(1) Molecular biological identification

According to the bacterial 16S rRNA sequence, the screened strain JK-2 was identified by molecular biology by phylogenetic analysis.

Specifically, the strain JK-2 obtained by screening was inoculated into 100 mL of sterile nutrient broth, cultured at 33 °C and 180 r/min for 24 h, centrifuged at 4 °C and 12000 r/min for 1 min, the supernatant was discarded, and then Genomic DNA of strain JK-2 was extracted using Ezup column genomic DNA extraction kit.

Then the obtained genomic DNA was used as a template, and its 16S rRNA was amplified by PCR. Among them, the forward primer is 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, the reverse primer is 1492R: 5′-GGTTACCTTGTTACGACTT-3′; the PCR amplification system (20 μL) is Premix Taq 10 μL, upstream and downstream primers 1 μL, template 1 μL , ddH ₂ O 7 μL; PCR amplification conditions were: 94°C for 3 minutes; 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, a total of 35 cycles. The amplified 16S rRNA gene was sequenced by Shanghai Maipu Biotechnology Co., Ltd.

The DNA sequence corresponding to the 16S rRNA of the measured strain JK-2 is as follows (SEQ ID NO.1):

ACTTTTTGTTCACTTCGGGCGGCTGGCTCCTAAAGGTTACTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACC TCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCA CCACCTGTCACTCTGCCCCCGAAGGGGGACGTCCTATTCTCTAGGATTGTCAGAGGATGTCCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACAC TTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCA GACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGT CCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCACGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACTTTTATGTTTGAACCATGCGGTTCAAAACAAGCATCCGGTATTAGCC CCGGTTTCCCGGAGTTATTCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTGCATTATAGCAGGCCCCCCCGCCC.

Using BLAST of NCBI to compare the homology of the 16S rRNA sequence of strain JK-2, from the comparison results, the 16S rRNA sequence of strain JK-2 is naturally clustered with the 16S rRNA sequence of Bacillus subtilis in the GenBank database, homologous The resistance is more than 99%.

Then use the MEGA 5.0 software to construct a phylogenetic tree using the Neighbor-joining method (Neighbor-joining, repeatability F1000), which further proves that the strain JK-2 has the closest genetic relationship with Bacillus subtilis A65.1 (GenBank accession number: ON366398); for details, see Fig. 7.

(2) Morphological identification

See Figure 3 for the colony morphology formed by the strain JK-2 on the nutrient agar plate. The colony is characterized by white, irregular edges, smooth and easy to pick.

Strain JK-2 is a Gram-positive bacterium. After Gram-staining, its photo under a 100-fold optical microscope is shown in Figure 4. It can be seen that its shape is rod-shaped. It is similar in shape to the common Bacillus subtilis.

(3) Physiological and biochemical identification

The physiological and biochemical test traits were selected by referring to the relevant content of the corresponding genus and species identification in the "Common Bacteria System Identification Manual" (first edition) edited by Dong Xiuzhu et al., and the physiological and biochemical identification of strain JK-2 was carried out using bacterial trace biochemical identification tubes.

The identification results are shown in Table 1 below:

It can be seen from Table 1 above that the physiological and biochemical characteristics of strain JK-2 are completely consistent with Bacillus subtilis B25 (Document: Lin Baoying, Partial characteristic analysis and isolation and purification of antifungal active substances of Bacillus subtilis B25, Hainan University, 2013).

In summary, combined with the results of 16S rRNA molecular identification, morphological observation, and physiological and biochemical identification, it can be determined that the strain JK-2 obtained by the screening of the present invention is Bacillus subtilis.

3. About the preservation of strains

The bacterial strain JK-2 of the present invention belongs to Bacillus subtilis and has been preserved in the China Type Culture Collection Center. The address of the preservation unit is: Wuhan, China. The preservation date is October 14, 2022, and its preservation number is CCTCC No. : M 20221568.

Four, the fermentation culture method of bacterial strain JK-2 of the present invention

Inoculate a single colony of the strain JK-2 of the present invention into 20mL of nutrient broth or other Bacillus fermentation medium, and culture it on a shaking table at 33°C and 180r/min for 24 hours, so that the concentration of the bacterial liquid is greater than 10 ⁸ CFU/mL Then transfer it to 200mL nutrient broth or other bacillus fermentation medium with 10% inoculum size, and cultivate it for 24 hours at 33°C and 180r/min shaker shaker, and the fermented liquid obtained is bacterial strain JK-2 of the present invention of fermentation broth. After testing, the bacterial concentration of the fermentation broth is about 10 ⁹ -10 ¹⁰ CFU/mL.

The growth rate of the bacterial strain JK-2 of the present invention is fast, and the operating conditions for fermentation and cultivation are relatively simple. Rapid and high-density fermentation is carried out in an aerobic and aseptic fermentation medium environment, which saves energy and reduces the pollution of miscellaneous bacteria. The obtained fermented product Quality controllable. In conclusion, the bacterial strain JK-2 of the present invention is an excellent bacterial strain suitable for industrialized fermentation production.

Five, the safety test of Bacillus subtilis JK-2 of the present invention to Chinese mitten crab

The test set up 1 control group and 5 experimental groups, and each group had 3 parallel groups. 10 healthy Chinese mitten crabs were placed in plastic aquariums (76cm×45cm×55cm) filled with 60L aerated tap water, and the water temperature was controlled. at 28°C. The Chinese mitten crabs in the test group were injected with 0.2 mL of Bacillus subtilis at the base of the third leg with a final concentration of 1.0×10 ⁵ , 1.0×10 ⁶ , 1.0×10 ⁷ , 1.0×10 ⁸ , and 1.0×10 ⁹ CFU/mL. JK-2 bacterial suspension (bacterial suspension formed in sterile normal saline); the control group was injected with the same amount of sterile normal saline. The test period is 7d. The number of dead Chinese mitten crabs was recorded every day during the test, and the median lethal concentration (LC ₅₀ ) of Bacillus subtilis JK-2 to Chinese mitten crabs was calculated by Bliss method.

The safety test results of Bacillus subtilis JK-2 on Chinese mitten crabs showed that when the strain JK2 with a concentration of 1.0×10 ⁵ ~1.0×10 ⁹ CFU/mL was injected into healthy Chinese mitten crabs, the vitality of Chinese mitten crabs was normal, and no Weakness of appendages, fighting and death occurred, and no death or abnormality occurred in the control group, indicating that the LC ₅₀ of Bacillus subtilis JK-2 against Chinese mitten crabs was >1.0×10 ⁹ CFU/mL.

Six, the application effect of Bacillus subtilis JK-2 of the present invention to the prevention and treatment of Chinese mitten crab bacterial disease

Choose healthy Chinese mitten crabs with no disease, strong constitution, and uniform specifications and put them in the aquarium. Put tiles in the box as concealed objects, and domesticate the Chinese mitten crabs with basic feed. Start the test after stabilization.

The experiment consisted of 3 test groups and 1 control group, and each group had 3 parallel aquariums with 40 animals in each aquarium.

Test group: add Bacillus subtilis JK-2 of the present invention in the feedstuff fed; Bacillus JK-2 bacterial suspension (bacterial suspension formed in sterile water), so that the final content of Bacillus subtilis JK-2 in the feed is 1.0×10 ⁵ , 1.0×10 ⁶ , 1.0×10 ⁷ CFU /g feed, as the feed for the three test groups.

Control group: fed with basal feed (no microbial bacteria added).

Feed once a day at 8:00 and 18:00, and the amount of feeding is 3% of the crab body weight. The test period is 20d. During the test, keep the water temperature at 28°C, pH ≤ 8.0, ammonia nitrogen ≤ 0.20mg/L, nitrite ≤ 0.01mg/L, dissolved oxygen ≥ 5.5mg/L, change the water once a day, and the water change volume is 1/3 of the water capacity , using a 40W fluorescent lamp as the light source, and the duration of light and darkness are 12 hours respectively.

After the test, 5 crabs were randomly selected from each group to collect their hemolymph and hepatopancreas, and the serum and hepatopancreas were determined according to the instructions of the kit (LZM test box from Nanjing Jiancheng Bioengineering Institute). The content of lysozyme, the results are shown in Figure 5 and Figure 6.

Note: In Figure 5 and Figure 6, T0 group represents the control group fed with basal feed (without adding microbial bacteria); ^T1 group represents the control group fed with Bacillus subtilis JK- 2; T2 group means the experimental group 2 fed with the feed of Bacillus subtilis JK-2 with a final content of 1.0×10 ⁶ CFU/g feed; T3 group means fed with a final content of Test group 3 fed with 1.0×10 ⁷ CFU/g of Bacillus subtilis JK-2.

From the experimental results in Figure 5, it can be seen that in group T1-3 (using the feed with the final content of strain JK2 being 1.0×10 ⁵ to 1.0×10 ⁷ CFU/g, the Chinese mitten crab was continuously fed for 20 days), the Chinese mitten crab The activity of serum lysozyme was significantly enhanced, specifically, it was increased by 5.91%-10.97% (P<0.05) compared with the results of T0 group (fed with basal feed).

From the experimental results in Figure 6, it can be seen that in group T1-3 (feeding mitten crabs with strain JK2 whose final content was 1.0×10 ⁵ ~1.0×10 ⁷ CFU/g continuously for 20 days), Chinese mitten crabs Hepatopancreatic lysozyme activity was significantly enhanced, specifically, compared with T0 group (fed with basal feed), the results were increased by 71.77% to 132.26% (P<0.05); and the hepatopancreatic lysozyme activity increased with the content of strain JK2 increased significantly.

This shows that adding Bacillus subtilis JK-2 of the present invention in the basal feed can promote the serum of Chinese mitten crab and the activity of lysozyme in the hepatopancreas, especially significantly improve the activity of lysozyme in the hepatopancreas, which can enhance The scavenging ability of Eriocheir sinensis against pathogenic bacteria.

Based on the above results, those skilled in the art can think of applying Bacillus subtilis JK-2 of the present invention to improve the serum lysozyme activity and/or hepatopancreas lysozyme activity of Chinese mitten crab, and to improve the body's resistance to The scavenging ability of pathogenic bacteria is applied to the prevention and treatment of bacterial diseases of Chinese mitten crab.

Based on the above results, those skilled in the art can think of adding the Bacillus subtilis JK-2 of the present invention to the feed of Chinese mitten crabs. In view of the LC ₅₀ of Bacillus subtilis JK-2 on Eriocheir sinensis >1.0×10 ⁹ CFU/mL, and the increase of serum lysozyme and hepatopancreatic lysozyme activity with the content of strain JK2 (based on the results in Figure 5 and Figure 6 Reasonable speculation), therefore, the concentration of Bacillus subtilis JK-2 in the feed of mitten crabs can be 1.0×10 ⁵ ~1.0×10 ⁹ CFU/g feed, for example, 1.0×10 ⁵ , 1.0×10 ⁶ , 1.0× 10 ⁷ , 1.0×10 ⁸ or 1.0×10 ⁹ CFU/g feed.

In order to further verify that "Bacillus subtilis JK-2 of the present invention can strengthen the scavenging ability of the Chinese mitten crab body to pathogenic bacteria", 10 crabs were randomly selected from each group to carry out the artificial infection test, respectively for the control group and the test group. Chinese mitten crabs were fed with the basal feed containing Aeromonas victorii HXH1 at a concentration of 1.0×10 ⁷ CFU/g feed. The bacteria feed was fed once a day at 8:00 and 18:00, and the feeding amount was 3% of the crab body mass. After 7 days of continuous feeding, the death of Chinese mitten crabs in each group was observed and recorded, and the mortality rate was calculated. (%); and calculate the disease resistance protection rate (%) according to the following formula (1).

The statistical results are shown in Table 2 below:

As can be seen from the results in Table 2, the ability of the Chinese mitten crab antagonizing pathogenic bacteria (Aeromonas victoria HXH1) infection of the test group (feeding the feed containing Bacillus subtilis JK-2 of the present invention) is significant enhanced. The mortality rate of Chinese mitten crabs in the T1-T3 test group was reduced by 43.34%-66.67% compared with the mortality rate of the control group T0 group (P<0.05).

This shows that feeding the basal feed with the Bacillus subtilis JK-2 of the present invention can significantly improve the anti-infection ability of Chinese mitten crabs to pathogenic bacteria (Aeromonas victoria HXH1).

In summary, based on the bacteriostasis test results of the above-mentioned Bacillus subtilis JK-2 of the present invention (antagonistic pathogenic bacteria Aeromonas victorii HXH1 of Fig. 1 and Fig. 2, Aeromonas hydrophila WS05, Aeromonas dots Bacillus DD5, Alcaligenes Providencia P3 and Citrobacter freundii C1), physiological and biochemical identification results, and the safety test and control of Chinese mitten crab in the fifth and sixth parts As a result of the application experiment on bacterial diseases, those skilled in the art can use the Bacillus subtilis JK-2 of the present invention as a biocontrol agent to antagonize aquatic animal pathogens, especially shrimp and crab pathogens. It can be added to the feed of Chinese mitten crabs as a biocontrol agent to enhance the anti-infection ability of the Chinese mitten crab body (the ability to scavenge pathogenic bacteria), and be applied to the prevention and treatment of Chinese mitten crab bacterial diseases.

Those skilled in the art can ferment and produce Bacillus subtilis JK-2 of the present invention by conventional Bacillus subtilis fermentation method (such as the fourth part above), and can obtain Bacillus subtilis JK-2 fermentation broth or The bacterial liquid preparation obtained by the fermented bacterial liquid or the bacterial powder preparation obtained by drying the fermented bacterial liquid.

It should be understood that although this description is described according to implementation modes, not each implementation mode only contains an independent technical solution, and this description in the description is only for clarity, and those skilled in the art should take the description as a whole, and each The technical solutions in the embodiments can also be properly combined to form other embodiments that can be understood by those skilled in the art.

The series of detailed descriptions listed above are only specific descriptions for feasible implementations of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent implementation or implementation that does not depart from the technical spirit of the present invention All changes should be included within the protection scope of the present invention.

Claims (10)

1. A Bacillus subtilis JK-2, the Bacillus subtilis JK-2 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC No: M 20221568. 2. A microbial inoculum, characterized in that: said microbial inoculum comprises Bacillus subtilis JK-2 as claimed in claim 1. 3. The microbial inoculum agent according to claim 2, characterized in that: the microbial inoculum agent comprises the fermented bacterial liquid of Bacillus subtilis JK-2 as claimed in claim 1 or the bacterium obtained by the fermented bacterial liquid. Liquid preparation or bacterial powder preparation obtained by drying the fermented bacterial liquid. 4. The application of Bacillus subtilis JK-2 as claimed in claim 1 or the microbial agent as claimed in claim 2 in antagonizing aquatic animal pathogenic bacteria. 5. The application according to claim 4, characterized in that: the aquatic animal pathogenic bacteria are shrimp and crab animal pathogenic bacteria. 6. Application as claimed in claim 5, characterized in that: said shrimp and crab animal pathogenic bacteria are selected from Aeromonas victorii, Aeromonas hydrophila, Aeromonas punctatus, Alcaligenes Provitamin Dendrobium and Citrobacter freundii. 7. A product for antagonizing pathogenic bacteria in aquatic animals, characterized in that the product comprises Bacillus subtilis JK-2 as claimed in claim 1 or the microbial agent as claimed in claim 2. 8. the application of Bacillus subtilis JK-2 as claimed in claim 1 or the microbial agent as claimed in claim 2 in improving the serum lysozyme activity and/or hepatopancreas lysozyme activity of Chinese mitten crab. A Chinese mitten crab feed, characterized in that: the Chinese mitten crab feed comprises feed basic components and Bacillus subtilis JK-2 as claimed in claim 1. 10. the effect of Bacillus subtilis JK-2 as claimed in claim 1 or microbial agent as claimed in claim 2 or Chinese mitten crab feed as claimed in claim 9 in the prevention and treatment of Chinese mitten crab bacterial disease application.

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