Bacillus amyloliquefaciens and application thereof in inhibition of edwardsiella ictaluri and photobacterium damsonii

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus isothermophilus and application thereof in inhibition of edwardsiella ictaluri and photobacterium damsonii. The invention relates to a strain of bacillus straticola, which is a strain K of bacillus straticola, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 19, 2025, and the preservation number of the strain is CGMCC No.33567. The invention further discloses a preparation method of the bacillus straticola. The strain can be used as a feed additive for preventing and treating edwardsiella ictaluri and photobacterium damsonii in aquaculture, or can be directly prepared into a feed, and the antibacterial effect is remarkable when the bacterial feed is fed in the aquaculture, or the strain can be added into a water body to be used as a water body disinfectant.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus stratosphericus strain and its application in inhibiting Edwardsiella ictaluri and Photobacterium damselae subsp. piscicida. Background Art

[0002] Aquaculture is favored because it can produce a large amount of products in a limited area and overcome the related limitations of capturing wild species. Its output has always been maintained at a high level. It is an important part of agricultural production and plays a crucial role in economic development. More than half of the world's aquatic animal food comes from aquaculture (about 56%). Aquatic products to a certain extent meet the growing demand for nutrients such as protein by people and have become an important source for humans to obtain high-quality animal protein.

[0003] However, in order to meet the growing consumption demand, the intensification level of aquaculture has been continuously improved, and aquatic animal diseases occur frequently, showing a trend of diversification and complexity. Mixed infections, secondary infections, and co-infections are becoming increasingly serious, causing huge economic losses, and the purification of the aquaculture environment faces great pressure and challenges. As one of the most common means at present, antibiotics play an outstanding role in the prevention and control of aquatic diseases. In contrast, problems such as the abuse of antibiotics, the deterioration of the aquaculture environment, the enhancement of pathogenic microorganism drug resistance, and the drug residues in farmed animals emerge in an endless stream. It is urgent to reduce the use of antibiotics and develop disease prevention and control technologies and products that meet the environmental friendliness and sustainable development strategies. The screening of pathogenic microorganism inhibitory strains and the application of their bacterial agents are regarded as an important means to solve the above problems because they have the advantages of convenient use, environmental friendliness, and low price, and are not like antibacterial peptides and proteases that need to be extracted, are prone to loss of activity, and have high storage requirements, and have become a research and application hotspot in this field.

[0004] Pathogenic microorganisms Edwardsiella ictaluri (hereinafter referred to as "EI") and Photobacterium damselae subsp. damselae (hereinafter referred to as "PDD") are two pathogenic microorganisms that pose a greater threat in aquaculture. EI is the most harmful bacterial pathogen in the culture of catfish (such as Pelteobagrus fulvidraco), which can cause red head disease, split head disease, ascites, etc., and the annual infection incidence rate is more than 50%. The disease outbreaks caused by EI are seasonal and usually occur in seasons with water temperatures around 20°C - 30°C. Photobacterium damselae includes two types: subsp. damselae (PDD) and subsp. piscicida. PDD was first isolated from the skin ulcers of diseased fish in the family Pomacentridae in 1981 and was officially named in 1995. Subsequently, it has been isolated from many diseased marine fish such as rainbow trout, sea bass, turbot, half-smooth tongue sole, and Sebastes schlegelii, with a wide range of pathogenicity, and can also infect mammals such as dolphins and humans, being a common pathogenic microorganism in mariculture. In recent years, it has also been found that PDD is pathogenic to Litopenaeus vannamei (also known as white shrimp or Pacific white shrimp, which is the main shrimp variety in China), causing diseases such as drip star disease, resulting in hepatopancreas necrosis and death of shrimp, causing heavy losses to farmers. Currently, the diseases caused by EI and PDD mainly rely on chemical drugs including antibiotics for prevention and treatment, but problems such as antibiotic residues and the generation of drug resistance are very intractable, endangering the health of the people. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Bacillus stratosphericus that is sensitive to common antibiotics and has the ability to significantly inhibit Edwardsiella ictaluri and Photobacterium damselae.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] A strain of Bacillus stratosphericus, which is strain K of Bacillus stratosphericus. This strain was deposited on February 19, 2025, at the China General Microbiological Culture Collection Center (abbreviation: CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101), and its deposit number is: CGMCC No. 33567.

[0008] A bacterial agent containing the Bacillus stratosphericus described in the present invention. The bacterial agent includes a culture solution containing the Bacillus stratosphericus described in the present invention, the supernatant of this culture solution, etc.

[0009] Preferably, the bacterial agent further contains a pharmaceutically acceptable carrier, and its dosage form is a solution, emulsion, suspension, powder, gel, granule or freeze-dried preparation.

[0010] Use of Bacillus stratosphericus described in the present invention or the bacterial agent described in the present invention in inhibiting Edwardsiella ictaluri.

[0011] Use of Bacillus stratosphericus described in the present invention in preparing a drug, an aquatic feed additive or a water body disinfectant for preventing or treating diseases caused by Edwardsiella ictaluri infection. The aquatic products include at least one of seawater fish, shrimps, crabs, snails or shellfish.

[0012] Use of Bacillus stratosphericus described in the present invention or the bacterial agent described in the present invention in inhibiting Photobacterium damselae subsp. damselae.

[0013] Use of Bacillus stratosphericus described in the present invention in preparing a drug, an aquatic feed additive or a water body disinfectant for preventing or treating diseases caused by Photobacterium damselae infection. The aquatic products include at least one of seawater fish, shrimps, crabs, snails or shellfish.

[0014] A water body disinfectant prepared from Bacillus stratosphericus described in the present invention or the bacterial agent described in the present invention.

[0015] A feed additive, which contains Bacillus stratosphericus described in the present invention or the bacterial agent described in the present invention.

[0016] A fish and shrimp feed, which contains the feed additive described in the present invention.

[0017] The Bacillus stratosphericus strain, the complete nucleotide sequence of its 16S rDNA is as shown in SEQ ID No.1.

[0018] The beneficial effects of the present invention are as follows: The Bacillus stratosphericus K strain provided by the present invention is white, nearly round, slightly convex, wrinkled and easy to pick up on the LB medium ( Figure 2 ), the Gram staining result is positive, and it is sensitive to common antibiotics. The suitable growth pH range of B. stratosphericus K is 4.5 - 9.0; under the conditions of pH 4.5 and 9.0, OD 600 is still around 1.4 and the growth state is good; the optimal range is pH 5.0 - 7.5 (no significant difference, the same lowercase letters). Therefore, for the acid-base environment in general aquaculture water bodies and aquaculture animals, B. stratosphericus K can fully adapt. The optimal growth range of B. stratosphericus K is 25°C to 35°C (no significant difference); under the conditions of 21°C and 37°C, OD 600It is still greater than 1.0 (normal growth); even at 40 °C, it can still grow. This strain can be used as a feed additive for preventing and controlling Edwardsiella ictaluri and Photobacterium damselae subsp. piscicida in aquaculture, or directly made into feed. Feeding the bacterial feed in aquaculture has a significant antibacterial effect, or it can be added to the water body as a water disinfectant. Description of the Drawings

[0019] Figure 1 It is the inhibitory EI photo of strain K (set with 2 replicates);

[0020] Figure 2 It is the colony formed by strain K on LB medium (left: photo of streak plate; right: photo of partial colony);

[0021] Figure 3 It is the phylogenetic tree based on the similarity analysis of the 16S rRNA gene sequence of strain K (the number in parentheses after the bacterial name is the GenBank accession number);

[0022] Figure 4 It is the growth performance of B. stratosphericus K under different pH conditions;

[0023] Figure 5 It is the growth performance of B. stratosphericus K at different temperatures;

[0024] Figure 6 It is the growth performance of B. stratosphericus K under different NaCl concentrations;

[0025] Figure 7 It is the inhibitory performance of B. stratosphericus K against PDD at 2.5% NaCl concentration;

[0026] Figure 8 It is the effect of B. stratosphericus K on the survival rate of the tested Pelteobagrus fulvidraco;

[0027] Figure 9 It is the effect of B. stratosphericus K on the survival rate of the tested Litopenaeus vannamei. Detailed Embodiments

[0028] The following are specific examples to further illustrate the technical solutions of the present invention. It should be understood that the implementation of the present invention is not limited to the following examples, and any formal variation and / or change made to the present invention will fall within the protection scope of the present invention.

[0029] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0030] The reagents used in the following examples can be purchased from a conventional biochemical reagent store unless otherwise specified.

[0031] Edwardsiella ictaluri (hereinafter referred to as "EI"); Photobacterium damselae subsp. damselae (hereinafter referred to as "PDD").

[0032] Example 1 Screening of Strains

[0033] On February 16, 2024, three sites were randomly selected for isolation matrix sampling at the edge of the pond on the Donghu Campus of Zhejiang A&F University, Lin'an District, Hangzhou City, Zhejiang Province. The "soil shaking method" was used to collect the rhizosphere soil of plants near the sites, or the mud attached to the plant roots. Two to three isolation matrix samples were collected at each site and placed in a pre-sterilized petri dish, and then quickly taken back to the laboratory and mixed evenly with a sterile glass rod to make one site sample (a total of 3). With an inoculation amount of 10% (w / v), the mixed samples of the three sites were respectively inoculated into Erlenmeyer flasks pre-filled with sterile water, and several small glass beads were placed in the Erlenmeyer flasks. The Erlenmeyer flasks were placed on a shaker and shaken at 150 rpm for 1 hour. Take 100 μL for plate dilution coating. To avoid clogging of the pipette tips, the front ends of the pipette tips were appropriately trimmed with scissors before sterilization. The medium used was nutrient agar medium (each Erlenmeyer flask contained 200 mL of the medium). When the medium was in a liquid state and not too hot to touch, 1 mL of the pre-cultured EI and PDD bacterial solutions with an OD 600 of 1.0 (one pathogenic bacterium per bottle, a total of 6 bottles) were respectively added to each Erlenmeyer flask, mixed evenly and then poured onto plates. The coated plates were placed in an incubator and cultured at 28 °C for 3 days. Colonies with transparent or semi-transparent antibacterial zones on each plate were selected and purified by streaking. The purified strains were numbered in lowercase alphabetical order, a total of 7 strains.

[0034] The 7 strains were respectively spot-inoculated onto the above-mentioned medium containing EI or PDD bacterial solutions and cultured at 28 °C for 2 days. It was found that only 1 strain (numbered K) could form antibacterial zones on both pathogenic bacterium media. By the filter paper method measurement (diameter 6 mm, 5 μL of bacteria inoculated per piece), the antibacterial zone diameter of strain K against EI was 30 ± 3.1 mm ( Figure 1) The diameter of the inhibition zone against PDD was 24 ± 2.2 mm. The bacterial strain was preserved at -20 °C using a glycerol-bacterial suspension mixture with a final concentration of 30% (v / v). The K strain of the present invention is the first microorganism that simultaneously has the ability to significantly inhibit EI and PDD.

[0035] The K strain was white, nearly round, slightly convex, wrinkled, and easy to pick up on the LB medium Figure 2 ), and the result of Gram staining was positive. Combining physiological and biochemical tests and 16S rRNA gene sequence similarity analysis Figure 3 ), its taxonomic status was identified as Bacillus stratosphericus, named Bacillus stratosphericus K.

[0036] After that, the inhibitory ability of the strain against Citrobacter freundii, Klebsiella pneumoniae, and Streptococcus iniae was tested (the method was the same as before). It was found that B. stratosphericus K had an inhibitory ability against Citrobacter freundii and could form an inhibition zone, but the effect was not as obvious as its inhibition of EI and PDD.

[0037] Example 2 Strain Characterization Test

[0038] 1. Growth performance of B. stratosphericus K under different pH conditions

[0039] Single colonies of B. stratosphericus K were selected on the LB plate and inoculated into a test tube containing 10 mL of LB liquid medium. The culture was shaken overnight at 28 °C and 150 rpm. Referring to the aseptic operation specification, the OD of the bacterial suspension was adjusted 600 to 0.6, and then inoculated into test tubes of LB liquid medium with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.4, 6.8, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 at an inoculation amount of 1% (v / v). After shaking culture at 30 °C and 150 rpm for 24 hours, the absorbance value was recorded. Each treatment was set with 3 replicates. Microsoft Excel 2019 was used for data sorting and statistical analysis, and DPS 2005 was used for significant analysis and multiple comparisons of the data. The significance level was set at P = 0.05 (the same below). The results are shown in Figure 4 .

[0040] pH is crucial for the growth and function of microorganisms. The wider the suitable pH range, the better the characteristics. Inhibitory strains are different from water purification microorganisms in aquaculture applications. The latter only need to adapt to the water environment. The actual environments where inhibitory strains are located can be divided into two categories: aquaculture water and inside aquaculture animals. Taking Pelteobagrus fulvidraco as an example, Pelteobagrus fulvidraco can adapt to water with a pH of 6.0 - 9.0. However, to ensure aquaculture benefits, farmers will try to control the pH within 7.0 - 8.5. The stomach of Pelteobagrus fulvidraco is acidic (about 4.5), and the intestinal pH is relatively high (6.5 - 8.0). Another example is Litopenaeus vannamei (also known as the white - leg shrimp), which can adapt to water with a pH of 6.5 - 9.0, but the suitable range is 7.0 - 8.5. The stomach pH of Litopenaeus vannamei is 5.1 - 5.3, the hepatopancreas pH is 5.9 - 6.1, and the intestinal pH is slightly higher (6.7 - 7.0). As Figure 4 shown, the suitable growth pH range of B.stratosphericus K is 4.5 - 9.0; at pH 4.5 and 9.0, the OD 600 is still around 1.4, and the growth state is good; the optimal range is pH 5.0 - 7.5 (no significant difference, the same lowercase letters). Therefore, for the acid - base environments in general aquaculture water and inside aquaculture animals, B.stratosphericus K can fully adapt.

[0041] 2. Growth performance of B.stratosphericus K at different temperatures

[0042] To study the effect of temperature on the growth of B.stratosphericus K, seven treatments of 21℃, 25℃, 28℃, 30℃, 35℃, 37℃ and 40℃ were set. The results are as Figure 5 shown. The optimal growth range of B.stratosphericus K is from 25℃ to 35℃ (no significant difference); at 21℃ and 37℃, the OD 600 is still greater than 1.0 (normal growth); even at 40℃, it can still grow.

[0043] The suitable water temperatures for the culture of warm - water and hot - water fish are 20℃ to 34℃, and the suitable water temperatures for the culture of shrimps (such as Litopenaeus vannamei) are 22℃ to 30℃. As can be seen from the above, B.stratosphericus K is in the optimal or normal growth state at these aquaculture water temperatures, and its functional role is not affected.

[0044] 3. Growth performance of B.stratosphericus K at different salt concentrations

[0045] To study the effect of salt concentration on the growth of B. stratosphericus K, the mass concentrations of NaCl were set at 0, 0.5%, 1.2%, 1.5%, 2.5%, 3.5%, 5.0%, 10.0% and 20.0%. The results are as Figure 6 shown. The optimal growth range of B. stratosphericus K was from 0 to 2.5% (no significant difference, the maximum OD 600 was read at 1.2%); under the condition of 3.5%, OD 600 was greater than 1.4. According to the differences in the tolerance and requirement of microorganisms for salt during growth, Kushner classified them into: non-halophilic bacteria (the optimal growth NaCl concentration is less than 1.17%), slightly halophilic bacteria (1.17% to 2.93%), moderately halophilic bacteria (2.93% to 14.63%), and extremely halophilic bacteria (14.63% to 30.4%) (Kushner D J. 1978. Life in high salt and solute concentrations: Halophilic bacteria / / In: Kushner DJ (ed). Microbial life in extreme environments[M]. London: Academic. 317 - 368). Accordingly, B. stratosphericus K was classified as slightly halophilic bacteria.

[0046] For fresh water, brackish water, and even most of the salt concentrations in seawater aquaculture, B. stratosphericus K can grow normally with excellent traits. Taking Litopenaeus vannamei aquaculture as an example, its optimal salt concentration is 1.5% to 2%. Within this range, it can exhibit the best growth performance, survival rate, and a relatively low feed coefficient, and this range is within the optimal growth salt concentration range of B. stratosphericus K.

[0047] 4. Test on the ability of B. stratosphericus K to inhibit pathogenic bacteria at 2.5% NaCl concentration

[0048] 5 μL of the B. stratosphericus K bacterial solution with an OD 600 of 0.6 was added to the filter paper in the center of the LB plate mixed with PDD, and cultured at 28 °C for 2 days. The results are as Figure 7 shown. An inhibition zone with a diameter of 18 ± 0.3 mm was formed, and the inhibitory effect was obvious. EI could not grow at 2.5% NaCl concentration, so it was not measured.

[0049] 5. Antibiotic sensitivity test of B. stratosphericus K

[0050] The sensitivity of B. stratosphericus K to various antibiotics was tested by the disk diffusion method. The antibiotic disks for testing were purchased from Hangzhou Microbial Reagent Co., Ltd. The specific method is as follows: Pick a single colony of B. stratosphericus K and culture it by shaking in LB liquid medium; adjust the OD of the bacterial liquid 600 to 0.6, mix it with LB medium in an amount of 1% (v / v), and pour the plate; after the medium solidifies, use sterile forceps to place 13 antibiotic disks on the center of the medium surface respectively, and gently press to ensure contact. Three replicates were set for each antibiotic; after culturing for 48 hours, measure the diameter of the inhibition zone, and complete the sensitivity judgment according to the standards provided by the manufacturer. A total of 13 antibiotics in 8 categories were tested, and the sites of action included cell wall, cell membrane and ribosome. The results are shown in Table 1. B. stratosphericus K was sensitive to all the tested antibiotics.

[0051] Table 1. Results of antibiotic sensitivity test of B. stratosphericus K

[0052]

[0053] Through the above tests, it can be clarified that B. stratosphericus K does not belong to the category of "super bacteria" and has high safety.

[0054] Effect of adding B. stratosphericus K on yellow catfish infected with EI

[0055] Two hundred yellow catfish with a size of 7 - 11 cm were purchased from fish farmers and placed in a glass tank for 15 days to adapt to the environment: the water temperature was controlled at about 23 °C with a heating rod, pH 7.2, normal aeration, and fed with the feed provided by the fish farmers according to their suggestions (once in the morning and once in the evening), and the feces and residual feed were cleaned regularly. Then, 180 yellow catfish with similar size and normal vitality were randomly divided into control group 1, control group 2 and treatment group, with 3 replicates set for each group (20 tails in each replicate).

[0056] Control group 1 and control group 2 used the feed provided by the fish farmers,

[0057] The feed for the treatment group was prepared as follows: Centrifuge the bacterial liquid of B. stratosphericus K cultured in LB medium, collect the bacterial cells, and resuspend them with sterile water; repeat the centrifugation - resuspension step 3 times, then mix it with the feed. Each kilogram of feed contained about 1×10 8 B. stratosphericus K; The feed was prepared once every 3 days and stored in a cool place when not in use.

[0058] Cultivation processes of Control Group 1, Control Group 2, and the treatment group: Feed the original feed or the feed added with B. stratosphericus K (hereinafter referred to as "bacterial feed") once in the morning and once in the evening for 30 days. After that, each fish in Control Group 2 and the treatment group was intraperitoneally injected with 0.15 ml of EI bacterial solution (without culture medium components) at a concentration of 1×10 7 CFU / ml, and Control Group 1 was injected with an equal amount of sterilized normal saline. Observe for 15 days. During this period, the feed feeding was the same as before. Record the status of Pelteobagrus fulvidraco. Remove the dead fish in time and weigh each fish on the 15th day.

[0059] During the observation period, before the death of Pelteobagrus fulvidraco in Control Group 2 and the treatment group, the swimming speed became slower, the feeding condition became worse, and the abdomen became swollen. The results of the effects of Control Group 1, Control Group 2, and the treatment group on the survival rate of the tested Pelteobagrus fulvidraco are shown in Figure 8 . At the end of the experiment, there was no significant difference in the weight of Pelteobagrus fulvidraco in each group. The survival rates were Control Group 1 (96.67±2.89)%, Control Group 2 (28.33±2.89)%, and the treatment group (71.67±7.64)%. There was a significant difference between Control Group 2 and the treatment group, and the antibacterial effect of feeding the bacterial feed was significant.

[0060] Effect of adding B. stratosphericus K on Litopenaeus vannamei infected with PDD in the application example

[0061] Select 180 healthy Litopenaeus vannamei with the same specifications and cultured in the laboratory for 10 days, and randomly place them into 9 plastic buckets numbered Control Group 1, Control Group 2, and the treatment group (3 replicates for each treatment), with 20 tails in each bucket. Subsequently, Control Group 2 (added with PDD) and the treatment group (added with PDD and B. stratosphericus K) were added with PDD and B. stratosphericus K without culture medium components at a final concentration of 1×10 4 CFU / ml on the first day and the third day of the bacteria addition experiment (2 times for each group). During the experiment, the water temperature was about 26°C, pH was 8.2, and the salt concentration was 1.8%. Oxygen was continuously supplied. Feed once in the morning and once in the evening according to the suggestions of farmers, clean the residual bait and feces irregularly, and remove the dead shrimp in time. Observe the disease occurrence of shrimp in each group within 7 days and record the number of dead shrimp. During this period, the vitality of diseased shrimp weakened, they lay on the bottom of the bucket, gradually stopped eating, and the edge of the hepatopancreas became blurred and atrophied.

[0062] The results of the effects of Control Group 1, Control Group 2, and the treatment group on the survival rate of the tested Litopenaeus vannamei are shown in Figure 9 . At the end of the experiment, as Figure 9As shown, the survival rates of Litopenaeus vannamei were (98.33±2.89)%, (65.00±8.66)% in control group 1 and control group 2 respectively, and (98.33±2.89)% in the treatment group. There were significant differences between control group 2 and the other two groups, which confirmed that B. stratosphericus K had a significant inhibitory effect on PDD and was beneficial to the survival of Litopenaeus vannamei.

[0063] In summary, the B. stratosphericus strain K described in the present invention can be used as a feed additive for preventing and controlling Edwardsiella ictaluri and Photobacterium damselae subsp. piscicida in aquaculture, or directly made into feed. The antibacterial effect of feeding bacterial feed in aquaculture is significant, or it can be added to the water body as a water disinfectant.

[0064] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0065] The above has introduced in detail a strain of Bacillus stratosphericus provided by the present invention and its application in inhibiting Edwardsiella ictaluri and Photobacterium damselae subsp. piscicida. Specific examples are used in this article to elaborate on the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A strain of Bacillus stratosphericus, which is Bacillus stratosphericus strain K, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 19, 2025, and its deposit number is: CGMCC No.33567.

2. A bacterial agent containing the stratosphere bacillus according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent also contains a pharmaceutically acceptable carrier, and its dosage form is a solution, emulsion, suspension, powder, gel, granule or lyophilized agent.

4. Use of the stratospheric bacillus according to claim 1 or the bacterial agent according to claim 2 in inhibiting Edwardsiella ictaluri.

5. Use of the stratospheric bacillus according to claim 1 or the bacterial agent according to claim 2 in inhibiting Photobacterium damselae subsp. damselae.

6. A water disinfectant prepared from the stratospheric bacillus according to claim 1 or the bacterial agent according to claim 2.

7. A feed additive, characterized in that: The feed additive comprises the stratosphere bacillus according to claim 1 or the bacterial agent according to claim 2.

8. A fish and shrimp feed, characterized in that: The fish and shrimp feed comprises the feed additive according to claim 8.

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