Bacillus subtilis and application thereof in promoting growth of grass carp and resisting type II grass carp reovirus

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis and application thereof in promoting growth of grass carp and resisting type II grass carp reovirus. The invention provides a bacillus subtilis BS08, and the preservation number of the bacillus subtilis BS08 is CCTCC (China Center for Type Culture Collection) NO: M 2025696. The bacillus subtilis BS08 is separated from the intestinal tract of the grass carp, can be colonized in the intestinal tract of the grass carp, improves the growth performance of the grass carp, can resist II-type grass carp reovirus, does not generate side effects in the use process, has the advantages of safety, harmlessness, low production cost, simple production, good effect and no pollution to the environment, and is suitable for popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis strain and application thereof in promoting grass carp growth and resisting type II grass carp reovirus. Background Art

[0002] Grass carp (Ctenopharyngodon idella), also known as silver carp or white carp, belongs to the Cypriniformes order, Cyprinidae family, and genus Ctenopharyngodon. It is an important freshwater fish species. Due to its primarily plant-based diet, rapid growth, and low aquaculture costs, grass carp aquaculture is often threatened by grass carp reovirus (GCRV), which can cause hemorrhagic disease and severe mortality. GCRV is divided into three genotypes (types I, II, and III), with GCRV-II being the most prevalent. Infection can cause mortality rates as high as 80% in grass carp. Therefore, the development of green and effective products to combat GCRV-II is crucial.

[0003] Currently, the primary approaches to preventing and controlling GCRV-II are drug therapy or vaccines. However, chemical treatments for grass carp reovirus often leave residual disease or develop resistance, while vaccines often suffer from limited protection and a short duration of immunity. Microorganisms, with their widespread availability, ability to survive and colonize in humans and animals, and their lack of residual residues and environmental impact, have become a key research area for GCRV-II prevention and treatment.

[0004] The prior art discloses a strain engineered using Bacillus subtilis WB600 as a vector, which possesses GCRV-II resistance. However, the Bacillus subtilis WB600 disclosed in the prior art is a variant strain derived from Bacillus subtilis BS168, an engineered strain lacking six protease genes. It is not derived from fish and may cause damage to the fish intestines. Its colonization status in the fish intestine is unknown. Summary of the Invention

[0005] In order to solve the problem in the prior art of lacking a fish-derived microbial agent that can colonize in fish, has a growth-promoting effect on grass carp, has an antiviral effect on GCRV-II, and is safe, pollution-free, and free of pesticide residues, the present invention provides a Bacillus subtilis strain and its use in promoting grass carp growth and resisting grass carp reovirus type II, specifically including the following technical solutions:

[0006] The present invention provides a Bacillus subtilis BS08, with a deposit number of CC TCCNO: M 2025696.

[0007] The present invention also provides a bacterial agent, the active ingredient of which includes the Bacillus subtilis BS08 described above.

[0008] Preferably, the concentration of Bacillus subtilis BS08 in the bacterial agent is 5×10 9 ~5×10 10 CFU / g or 5×10 9 ~5×10 10 CFU / mL.

[0009] The present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps:

[0010] The Bacillus subtilis BS08 described above is cultured to obtain a bacterial liquid;

[0011] The bacterial liquid is subjected to solid-liquid separation, and the bacterial bodies are collected and resuspended to obtain a bacterial agent.

[0012] Preferably, the culture medium is a DSM medium, and the components of the DSM medium include: 0.8% broth, 0.1% KCl, 0.025% MgSO4·7H2O, 1mM / L Ca(NO3)2, 10μM / L MnCl2, and the balance water; the percentages in the components are all mass volume percentages; the broth is a nutrient broth medium;

[0013] Preferably, the solid-liquid separation method includes centrifugation, the centrifugal speed is 4000-5000 r / min, and the centrifugal time is 8-12 min.

[0014] Preferably, the resuspending method includes resuspending with PBS.

[0015] The present invention also provides the use of the Bacillus subtilis BS08 or the bacterial agent or the bacterial agent prepared by the preparation method in promoting grass carp growth and / or resisting type II grass carp reovirus.

[0016] Preferably, the application method comprises: mixing the Bacillus subtilis BS08 or bacterial agent with a basic feed and then feeding the mixture to grass carp.

[0017] The present invention also provides a feed for promoting grass carp growth and / or resisting type II grass carp reovirus, the feed comprising active ingredients and basic feed;

[0018] The active ingredient includes the Bacillus subtilis BS08 or the bacterial agent as described above or the bacterial agent prepared by the preparation method.

[0019] The beneficial effects of the present invention are:

[0020] The present invention provides a Bacillus subtilis BS08, with a deposit number of CCTCCNO: M 2025696. The Bacillus subtilis BS08 of the present invention can colonize the intestinal tract of grass carp, improve the growth performance and survival rate of grass carp after infection with GCRV-II, and resist the grass carp GCRV-II virus. Furthermore, the Bacillus subtilis BS08 does not produce side effects during use and is safe and harmless. The Bacillus subtilis BS08 of the present invention has the advantages of a simple production method, low production cost, good application effect, and no pollution to the environment, making it suitable for widespread use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 are briefly introduced below.

[0022] Figure 1 This is a schematic diagram of the colonization effect of Bacillus subtilis BS08 in the intestine of grass carp in Example 5 of the present invention;

[0023] Figure 2 The survival rates of grass carp in the experimental group, positive control group, and negative control group in Example 6 of the present invention are as follows;

[0024] Figure 3 This is a schematic diagram of the effect of Bacillus subtilis BS08 on the growth of grass carp in Example 6 of the present invention.

[0025] Biological Deposit Description

[0026] Bacillus subtilis BS08, classification name: Bacillus subtilis, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan City, Hubei Province, China, collection number: CCTCC NO: M 2025696, preservation date: April 3, 2025. DETAILED DESCRIPTION

[0027] The present invention provides a Bacillus subtilis BS08, with a deposit number of CC TCCNO: M 2025696.

[0028] The colony surface of the Bacillus subtilis BS08 of the present invention is wrinkled, has irregular edges, and is milky white.

[0029] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent includes the above-mentioned Bacillus subtilis BS08. As an embodiment, the concentration of Bacillus subtilis BS08 in the microbial agent of the present invention is 5×10 9 ~5×10 10 CFU / g or 5×10 9 ~5×1010 CFU / mL; the concentration of Bacillus subtilis BS08 in the bacterial agent can be any value within the above range. As an optional embodiment, the concentration of Bacillus subtilis BS08 in the bacterial agent of the present invention can be 5×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 5×10 9 CFU / mL, 1×10 10 CFU / mL or 5×10 10 As an embodiment, the formulation of the bacterial agent of the present invention can be a conventional formulation of bacterial agents in the art, such as a liquid, powder, granule, suspension or freeze-dried powder.

[0030] The present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps:

[0031] The Bacillus subtilis BS08 described above is cultured to obtain a bacterial solution; the bacterial solution is subjected to solid-liquid separation, and the bacterial bodies are collected and resuspended to obtain a bacterial agent.

[0032] The present invention cultivates Bacillus subtilis BS08 to obtain a bacterial liquid. In one embodiment, the culture medium used in the culture process is a DSM culture medium, comprising: 0.8% broth, 0.1% KCl, 0.025% MgSO4·7H2O, 1 mM / L Ca(NO3)2, 10 μM / L MnCl2, and the remainder water, with the percentages of the components being by mass and volume. In one embodiment, the broth described in the DSM culture medium components is a nutrient broth culture medium, which is used as one of the components of the DSM culture medium in the present invention. In one embodiment, the culture temperature is 36-38°C; in an optional embodiment, the culture temperature can be 36°C, 37°C, or 38°C. As an embodiment, the culture time can be any value between 60 and 84 hours; as an optional embodiment, the culture time can be 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours or 84 hours.

[0033] After obtaining the bacterial liquid, the present invention performs solid-liquid separation on the bacterial liquid, collects the bacterial bodies and resuspends them to obtain a bacterial agent. As an embodiment, the solid-liquid separation method of the present invention includes centrifugation. As an embodiment, the centrifugation time can be any value between 8 and 12 minutes; as an optional embodiment, the centrifugation time can be 8 minutes, 10 minutes or 12 minutes. As an embodiment, the centrifugation speed can be any value between 4000 and 5000 r / min; as an optional embodiment, the centrifugation speed can be 4000 r / min, 4500 r / min or 5000 r / min. As an embodiment, the resuspending method includes resuspending with PBS. As an embodiment, after resuspending with PBS, the concentration of Bacillus subtilis BS08 in the bacterial agent can be 5×10 9 CFU / mL~5×10 10 CFU / mL; As an optional embodiment, after resuspending with PBS, the concentration of Bacillus subtilis BS08 in the bacterial agent can be 5×10 9 CFU / mL, 1×10 10 CFU / mL or 5×10 10 CFU / mL.

[0034] The present invention also provides the use of the Bacillus subtilis BS08 or the bacterial agent or the bacterial agent prepared by the preparation method in promoting grass carp growth and / or resisting type II grass carp reovirus.

[0035] As an embodiment, the application method of the present invention comprises: mixing the Bacillus subtilis BS08 or the bacterial agent with the basic feed and then feeding it to grass carp. As an embodiment, the ratio of the Bacillus subtilis to the basic feed is 1×10 10 CFU: 1g.

[0036] The present invention also provides a feed for promoting grass carp growth and / or resisting type II grass carp reovirus, comprising an active ingredient and a base feed; the active ingredient comprises the above-mentioned Bacillus subtilis BS08 or the bacterial agent or the bacterial agent prepared by the preparation method. As an embodiment, the ratio of Bacillus subtilis BS08 to the base feed in the feed can be 5×10 9 CFU~5×10 10 CFU: any point value in 1g. As an embodiment, the basic feed is a conventional commercial grass carp feed.

[0037] To further illustrate the present invention, a strain of Bacillus subtilis provided by the present invention and its application in promoting grass carp growth and resisting type II grass carp reovirus are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0038] Example 1 Isolation and Identification of Bacillus

[0039] In this example, grass carp purchased from various farmers' markets and farms in Wuhan were anesthetized with a fish tranquilizer (MS-222), cleaned with 75% alcohol, and their intestines removed. The intestinal wall was cleaned of blood and contents with PBS buffer, and the intestines were minced and homogenized.

[0040] The homogenate was heated in an 80°C water bath for 20 min, spread on a TSA culture plate, and cultured in a 37°C incubator for 12 h. After colonies grew, single colonies with wrinkled surfaces, irregular edges, and milky white color were selected, streaked onto a new TSA culture plate, and purified in a 37°C incubator. The colonies were named BS08.

[0041] The 16S rDNA of the purified strain BS08 was amplified and sequenced using the upstream and downstream primers shown in SEQ ID NO: 1 and SEQ ID NO: 2, and homology comparison was performed in the NCBI database.

[0042] Upstream primer 27F (SEQ ID NO: 1): 5′-AGAGTTTGATCCTGGCTCA-3′;

[0043] Downstream primer 1492R (SEQ ID NO: 2): 5′-GGTTACCTTGTTACGACTT-3′.

[0044] Based on the results of homology comparison, the isolated and purified strain BS08 was determined to be Bacillus subtilis and was biologically preserved.

[0045] Example 2 Hemolysis experiment of Bacillus

[0046] Some bacteria can produce hemolysin during their growth process, causing rupture and lysis of red blood cells, resulting in low biosafety. Therefore, this example tested the biosafety of Bacillus subtilis BS08 through a hemolysis experiment.

[0047] Single colonies of Bacillus subtilis BS08 were selected and spotted on Columbia blood agar plates, which were then incubated at 37°C for 20 hours. The results showed that no clear transparent areas appeared around the Bacillus subtilis BS08 colonies, indicating no hemoglobin degradation.

[0048] In conclusion, Bacillus subtilis BS08 did not cause hemolysis during the experiment and is safe for fish in clinical applications.

[0049] Example 3 Enzyme production performance of Bacillus subtilis BS08

[0050] The enzyme production capacity of Bacillus subtilis BS08 was tested by the spot inoculation method. The test steps are as follows:

[0051] Single colonies of Bacillus subtilis BS08 were picked with a sterilized toothpick and spotted into protease-producing medium, amylase-producing medium, and cellulase-producing medium, respectively. The above culture media were cultured at 28° C. for 2 days.

[0052] The protease-producing culture medium is MH broth culture medium (Mueller-Hinton Broth), and the raw materials of the culture medium are as follows: 6g / L beef extract powder, 1.5g / L soluble starch, 17.5g / L acid hydrolyzed casein and 17g / L agar.

[0053] The raw material composition of the amylase production culture medium is: 5g / L beef extract, 10g / L peptone, 5g / L NaCl, 2g / L soluble starch and 15g / L agar powder.

[0054] The mass percentages of the cellulase-producing culture medium are: 10% carboxymethyl cellulose, 4% KH2PO4, 4% Na2HPO4, 2% tryptone, 0.2% MgSO4·7H2O, 0.001% CaCl2, 0.001% FeSO4·7H2O, 15% agar powder and the balance water.

[0055] After the culture was completed, the presence of hydrolysis zones on each plate was observed and their diameters were measured. The enzyme production capacity of the strain was determined by the ratio of the hydrolysis zone diameter (Dh) to the colony diameter (Dc) (Dh / Dc). The results are shown in Table 1.

[0056] Table 1 Enzyme production capacity of Bacillus subtilis BS08

[0057]

[0058] Note: Colony diameter, hydrolysis zone diameter and enzyme production capacity are expressed as mean ± standard deviation (mean ± SD, n = 3).

[0059] As can be seen from Table 1, the Bacillus subtilis BS08 screened out in the present invention has the ability to produce protease, amylase and cellulase.

[0060] Example 4 Drug sensitivity test of Bacillus subtilis BS08

[0061] When probiotics colonize the body, there's a potential for transferring antibiotic resistance genes to pathogens within the intestinal environment. Therefore, this example uses the drug sensitivity of Bacillus subtilis BS08 as a safety assessment measure for probiotics. This example tested the sensitivity of Bacillus subtilis BS08 to 22 antimicrobial drugs using the following method:

[0062] A single colony of Bacillus subtilis BS08 was selected and inoculated into DSM medium and cultured at 37°C for 72 h to obtain a bacterial solution. The concentration of the Bacillus subtilis BS08 bacterial solution was adjusted to 1×10 8 CFU / mL, a Bacillus subtilis BS08 bacterial suspension was evenly spread on the surface of MH solid culture medium. Sterile tweezers were then used to hold antibiotic-sensitive paper strips with different antibiotic dosages as shown in Table 2 and affixed to the surface of the culture medium. The culture medium was then incubated in an inverted manner at 37°C for 24 hours. The diameter of the inhibition zone was measured to determine the sensitivity of Bacillus subtilis to the antibiotics. The results are shown in Table 2.

[0063] Table 2 Drug sensitivity test results of Bacillus subtilis BS08

[0064]

[0065]

[0066] Note: In Table 2, R indicates resistance; I indicates intermediate sensitivity; and S indicates highly sensitive.

[0067] From the results in Table 2, it can be seen that Bacillus subtilis BS08 is highly sensitive to 19 common antibiotics and moderately sensitive to 3 antibiotics, and can be safely used in production.

[0068] Example 5 Colonization experiment of Bacillus subtilis BS08 in the intestinal tract of grass carp

[0069] A single colony of Bacillus subtilis BS08 was selected and inoculated into DSM medium and cultured at 37°C for 72 h to obtain a bacterial solution containing Bacillus subtilis BS08 spores. The bacterial solution concentration was adjusted to 10 10 After quantifying CFU / mL, the supernatant was discarded after centrifugation and the bacterial suspension was resuspended in PBS to obtain a bacterial suspension.

[0070] Add conventional grass carp feed to the bacterial suspension to prepare 10 10 CFU / g of bacteria-carrying feed, the main ingredients of the conventional grass carp feed are: fish meal, soybean cake, vegetable cake, barley flour, wheat flour, minerals and vitamins.

[0071] The control group feed was prepared by adding PBS solution in the same amount as the bacterial suspension to conventional grass carp feed.

[0072] Thirty grass carps with an average weight of 10.50±1.40 g were prepared and divided into two groups for the Bacillus subtilis BS08 colonization experiment, one of which was the experimental group and the other was the control group.

[0073] The experimental group of grass carp was fed once with a bacterial feed at a feeding amount of 2% of the grass carp's body weight. The control group of grass carp was fed once with an equal amount of the control group feed. 3 fish were taken from each group 1 hour, 6 hours, 24 hours, 48 hours, and 72 hours after feeding the feed. The grass carp intestines were removed and homogenized according to the method described in Example 1. The homogenate was treated at 80°C for 20 minutes, and after gradient dilution, it was spread on a TSA culture plate. After incubation at 37°C for 12 hours, the number of colonies was counted. The results are as follows: Figure 1 shown.

[0074] Depend on Figure 1 It can be seen that after feeding the contaminated feed for 72 hours, the number of Bacillus subtilis BS08 in the intestine of grass carp was higher than that in the control group, which shows that Bacillus subtilis BS08 can colonize in the intestine of grass carp for 72 hours; It should be noted that Figure 1 The value of the PBS group is not 0, but it is only in the single digit. Figure 1 In summary, there were significant differences between the results of the BS08 and PBS groups.

[0075] Example 6 Effect of Bacillus subtilis BS08 on grass carp resistance to GCRV-Ⅱ and its growth performance

[0076] 90 grass carps were prepared, with an average weight of 10.50±1.40g. The grass carps were temporarily kept in the laboratory for 14 days to adapt to the laboratory environment, and then randomly divided into 3 groups, with 30 fish in each group. The three groups were BS08 group, positive control group and negative control group. The BS08 group was fed with feed supplemented with Bacillus subtilis BS08 spores once every 3 days, with a Bacillus subtilis spore concentration of 10 10 CFU / g, and fed the same conventional grass carp feed as described in Example 5 at other times, the feed feeding amount was 2% of the fish body weight, and the fish were raised for 30 days. After 30 days, 100μL GCRV-Ⅱ was injected into the grass carp. The positive control group was fed with the same amount of conventional feed as the BS08 group every day. After feeding for 30 days, the grass carp was injected with the same amount of GCRV-Ⅱ as the BS08 group. The negative control group was fed with the same amount of conventional feed as the BS08 group every day. After feeding for 30 days, the grass carp was injected with the same amount of PBS solution as the GCRV-Ⅱ of the BS08 group. After the injection of GCRV-Ⅱ / PBS solution, the survival of the grass carp in the positive control group, the negative control group and the BS08 group was observed and counted respectively until the mortality rate of the grass carp stabilized after 14 days. The survival rate of the grass carp within 14 days after the injection was counted, and the results are as follows Figure 2 and as shown in Table 3.

[0077] Table 3 Survival rate of grass carp in different groups (%)

[0078] Number of days Positive control group Negative control group BS08 Group 1d 100 100 100 2d 100 100 100 3d 96.7 100 90 4d 86.7 100 86.7 5d 73.3 100 86.7 6d 73.3 100 86.7 7d 70 100 83.3 8d 70 100 83.3 9d 70 100 80 10d 66.7 100 80 11d 63.3 100 80 12d 60 100 80 13d 60 100 80 14d 60 100 80

[0079] From Table 3 and Figure 2 It can be seen that the survival rate of grass carp in the BS08 group fed with the contaminated feed containing Bacillus subtilis BS08 was significantly increased compared with the survival rate of grass carp in the positive control group. The protection rate of Bacillus subtilis BS08 in the BS08 group against grass carp infected with GCRV-Ⅱ was 50%.

[0080] The grass carp in the experimental and negative control groups were weighed at the beginning of feeding and after 30 days of feeding, and the weight gain rate (PWG), specific growth rate (SGR) and feed conversion rate (FCR) of grass carp were calculated according to the following formulas. The results are shown in the figure: Figure 3 and as shown in Table 4.

[0081] Body weight gain rate (%) = 100 × [(final body weight - initial body weight) / initial body weight].

[0082] Specific growth rate (% / day) = 100 × [(ln final body weight - ln initial body weight) / number of days of feeding].

[0083] Feed conversion rate = amount of feed fed (g) / fish weight gain (g).

[0084] Table 4 Effects of Bacillus subtilis BS08 on grass carp growth

[0085] control BS08 Group PWG (%) <![CDATA[24.73±1.03 a ]]> <![CDATA[40.79±1.84 b ]]> SGR (d / %) <![CDATA[0.74±0.03 a ]]> <![CDATA[1.14±0.04 b ]]> FCR <![CDATA[0.81±0.02 a ]]> <![CDATA[0.49±0.02 b ]]>

[0086] Note: PWG, SGR and FCR are expressed as mean ± standard deviation (mean ± SD, n = 30). Different letters indicate significant differences in data quality inspection among peers (P < 0.01).

[0087] As can be seen from Table 4, the PWG and SGR of grass carp were significantly increased and the FCR was significantly reduced after feeding the contaminated feed for 30 days, which shows that the Bacillus subtilis BS08 of the present invention plays a role in promoting the growth of grass carp.

[0088] In summary, the present invention provides a strain of Bacillus subtilis BS08. The Bacillus subtilis BS08 of the present invention is isolated from the intestine of grass carp and can colonize in the intestine of grass carp, thereby promoting the growth of grass carp and resisting type II grass carp reovirus. The Bacillus subtilis BS08 of the present invention has low production cost, simple production method, no pollution to the environment, and high biosafety, providing a new application method for improving the efficiency of grass carp farming.

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments like this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis BS08, deposited with CCTCC NO: M2025696.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Bacillus subtilis BS08 according to claim 1.

3. The bacterial agent according to claim 2, wherein The concentration of Bacillus subtilis BS08 in the bacterial agent is 5×10 9 ~5×10 10 CFU / g or 5×10 9 ~5×10 10 CFU / mL.

4. The method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The steps include: Cultivating the Bacillus subtilis BS08 according to claim 1 to obtain a bacterial liquid; The bacterial liquid is subjected to solid-liquid separation, and the bacterial bodies are collected and resuspended to obtain a bacterial agent.

5. The preparation method according to claim 4, wherein The culture medium is DSM medium, and the components of the DSM medium include: 0.8% broth, 0.1% KCl, 0.025% MgSO4·7H2O, 1mM / L Ca(NO3)2, 10μM / L MnCl2 and the balance water; the percentages in the components are all weight volume percentages; the broth is a nutrient broth medium; The culture temperature is 36-38° C., and the culture time is 60-84 hours.

6. The preparation method according to claim 4, wherein The solid-liquid separation method includes centrifugation, the centrifugal speed is 4000-5000 r / min, and the centrifugal time is 8-12 minutes.

7. The preparation method according to claim 4, wherein The resuspending method includes resuspending with PBS.

8. Use of the Bacillus subtilis BS08 according to claim 1, the bacterial agent according to claim 2 or 3, or the bacterial agent prepared by the preparation method according to any one of claims 4 to 7 in promoting grass carp growth and / or resisting type II grass carp reovirus.

9. The use according to claim 8, characterized in that The application method comprises: mixing the Bacillus subtilis BS08 or the bacterial agent with a basic feed and then feeding the mixture to grass carp.

10. A feed for promoting grass carp growth and / or resisting type II grass carp reovirus, characterized in that: The feed comprises active ingredients and basic feed; The active ingredient includes the Bacillus subtilis BS08 according to claim 1, the bacterial agent according to claim 2 or 3, or the bacterial agent prepared by the preparation method of any one of claims 4 to 7.

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