A strain of Bacillus tekirae with antagonistic properties against aquatic pathogens and its application

Abstract

This invention relates to the field of microbiology, and particularly to a strain of *Bacillus tekirae* with antagonistic properties against aquatic pathogens and its applications. This invention provides *Bacillus tekirae* with accession number GDMCC No:64960, which possesses antagonistic properties against common aquatic pathogens, effectively antagonizing pathogens such as *Edwardsiella piscinoides*, *Aeromonas hydrophila*, *Aeromonas velutipes*, *Shigella-like*, *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Streptococcus agalactiae*. Furthermore, it can enhance the disease resistance and promote the growth of aquatic animals.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Bacillus tequilensis Bt-CO and its application in promoting growth and controlling bacterial diseases. Background Technology

[0002] In recent years, aquaculture has developed towards a high-density, intensive model. The accompanying various stressful conditions easily put fish in a state of stress, increasing the risk of opportunistic pathogen infection in the water, leading to a proliferation of various fish diseases and even large-scale mortality in aquatic animals. Traditional antibiotic therapy, besides leading to increased drug resistance in pathogens, also has drawbacks such as causing intestinal flora imbalance and the spread of antibiotic resistance genes in aquatic animals. Probiotics can safely and effectively enhance the immunity of aquatic animals, and adding probiotic additives to feed is one of the main strategies for controlling bacterial diseases in aquatic animals.

[0003] Bacillus probiotics are among the safest microbial strains approved for use by the Ministry of Agriculture of my country, exhibiting good efficacy. They possess the following characteristics: acid resistance, adapting to the acidic environment of the digestive tract of aquatic animals; as dominant intestinal bacteria, most Bacillus strains can colonize the intestines, inhibiting the colonization and proliferation of opportunistic pathogens by competing with pathogens for adhesion sites. Bacillus can produce highly resistant dormant spores that are heat-resistant, acid-resistant, and bile-resistant, facilitating their use in feed processing; the bacteria can produce enzymes such as proteases, amylases, and lipases, enhancing the digestive capacity of aquatic animals. Currently, Bacillus subtilis, Bacillus coagulans, and Bacillus licheniformis are commonly used in aquaculture, but with the widespread use of these strains, their effectiveness has gradually declined. There is an urgent need to discover and identify new strains for continuous improvement to meet the diverse needs of aquaculture. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Bacillus tequilensis Bt-CO, which has an antagonistic effect on common pathogens in aquaculture, and can enhance the disease resistance of aquatic animals and promote their growth.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides Bacillus tequilensis, with accession number GDMCCNo:64960.

[0007] The present invention also provides the fermentation broth of the above-mentioned Bacillus tequilensis.

[0008] In some specific embodiments of the present invention, the fermentation culture medium of the above-mentioned fermentation broth includes, by weight, 30 parts corn flour, 50 parts soybean meal flour, 1 part KH2PO4, 1 part K2HPO4, and 2 parts CaCl2.

[0009] In some specific embodiments of the present invention, the preparation method of the above-mentioned fermentation broth includes: inoculating the above-mentioned Bacillus tequilensis seed liquid into the above-mentioned fermentation substrate, and culturing it at 37°C with shaking at a rate of 180 r / min for 60 h.

[0010] In some specific embodiments of the present invention, the seed culture of the above-mentioned fermentation broth is cultured at 37°C and 200 r / min for 16-24 h with shaking.

[0011] The present invention also provides the application of the above-mentioned Bacillus tequilensis or the above-mentioned fermentation broth in the preparation of products for aquaculture;

[0012] The products include formulations or feed.

[0013] In some specific embodiments of the present invention, the above-described product is used to antagonize pathogenic microorganisms;

[0014] The pathogenic microorganisms include at least one of Edwardsiella faecium, Aeromonas hydrophila, Aeromonas versicolor, Shigella-like Orthomonas, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae.

[0015] In some specific embodiments of the present invention, the product described above is used for the prevention and control of diseases;

[0016] The disease is caused by at least one of the following: Edwardsiella faecium, Aeromonas hydrophila, Aeromonas vernix, Shigella-like Orthomonas, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae.

[0017] In some specific embodiments of the present invention, the above-described product is used to enhance the disease resistance of aquatic animals;

[0018] The aquatic animals mentioned include tilapia.

[0019] The present invention also provides products for aquaculture, including acceptable excipients or auxiliaries, and:

[0020] (A1) The above-mentioned Bacillus tequilensis; or

[0021] (A2) The above fermentation broth.

[0022] In some specific embodiments of the present invention, the above-mentioned products include formulations or feeds.

[0023] In some specific embodiments of the present invention, the above-mentioned product is a probiotic feed;

[0024] The probiotic feed includes the step of mixing the above-mentioned Bacillus tequilensis with the feed at a weight ratio of 1:2.

[0025] The present invention also provides the use of the above-mentioned Bacillus tequilensis, the above-mentioned fermentation broth, or the above-mentioned product in at least one of the following:

[0026] (B1) Promotes the growth of aquatic animals;

[0027] (B2) Antagonizes common pathogens in aquatic animals;

[0028] (B3) Enhance the disease resistance of aquatic animals.

[0029] The present invention also provides a method for preparing products for aquaculture, including preparation based on the above-mentioned Bacillus tequilensis.

[0030] The present invention also provides a method for aquaculture, including the steps of using the above-mentioned Bacillus equilensis, the above-mentioned fermentation broth, or the above-mentioned product.

[0031] The *Bacillus tekirii* Bt-CO described in this invention has been identified as having multiple functional characteristics, with the main effects including: (1) production of cellulase, amylase, and protease; (2) antagonism against common aquatic pathogens, effectively antagonizing pathogens such as *Edwardsiella piscinoides*, *Aeromonas hydrophila*, *Aeromonas vesiculosus*, *Shigella*-like bacteria, *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Streptococcus agalactiae*; (3) enhancement of disease resistance in aquatic animals; and (4) promotion of aquatic animal growth. This invention can enrich my country's aquatic animal probiotic resource bank and, in the context of the current green development of antibiotic-free feed in aquaculture, provide a scientific basis for accelerating the development of environmentally friendly functional feed additives.

[0032] Biological Preservation Instructions

[0033] Biological material: Bacillus tequilensis Bt-CO, taxonomically named Bacillus tequilensis, was deposited on August 5, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Guangdong Province; accession number: GDMCC No:64960. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0035] Figure 1 The diagram shows the colony morphology and physiological and biochemical information of strain Bt-CO. In section A, the colony morphology, Gram staining results, and hemolytic test results of Bt-CO are shown in sequence. In section B, the results of starch hydrolysis, casein hydrolysis, and sodium cellulose hydrolysis experiments of Bt-CO are shown in sequence.

[0036] Figure 2 Phylogenetic tree of strain Bt-CO;

[0037] Figure 3 The inhibition zone of strain Bt-CO against common aquatic pathogens is shown.

[0038] Figure 4 A statistical chart showing the diameter of the inhibition zone of Bt-CO strain against common aquatic pathogens;

[0039] Figure 5 The fermentation broth of strain Bt-CO shows the inhibition zone against common aquatic pathogens;

[0040] Figure 6 Statistical chart of inhibition zone diameters of Bt-CO fermentation broth against common aquatic pathogens;

[0041] Figure 7 The image shows the antibacterial effect of the fermentation broth of Bt-PI, which is the same species as Bt-CO, isolated in the laboratory, as a control for the antibacterial effect of Bt-CO fermentation broth. Detailed Implementation

[0042] This invention discloses a strain of Bacillus tequilensis (Bt-CO) and its application in promoting growth and controlling bacterial diseases. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0043] This invention provides a strain of Bacillus tequilensis (Bt-CO), a bacterial agent prepared from this bacterium, and applications of the bacterium or its agent. Specifically:

[0044] The first aspect of the present invention is to provide a strain of Bacillus tekirae that has antagonistic effects against common pathogens in aquaculture and can promote the growth of aquatic animals.

[0045] A second aspect of the present invention is to provide a fermentation broth of the Bacillus tekirae strain as described in the first aspect of the present invention.

[0046] A third aspect of the present invention is to provide a formulation containing the Bacillus tekiria strain described in the first aspect of the present invention or the fermentation broth described in the second aspect of the present invention.

[0047] A fourth aspect of the present invention is to provide the use of the *Bacillus tekirii* strain as described in the first aspect of the present invention or the fermentation broth as described in the second aspect of the present invention in antagonizing *Edwardsiella pisciformis*, *Aeromonas hydrophila*, *Aeromonas versicolor*, *Shigella*-like *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Streptococcus agalactiae*.

[0048] The fifth aspect of the present invention is to provide the use of the Bacillus tekirii strain as described in the first aspect of the present invention or the fermentation broth as described in the second aspect of the present invention in the prevention and control of diseases caused by Edwardsiella tekirii.

[0049] The sixth aspect of the present invention is to provide new uses of the Bacillus tekirii strain as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the preparation as described in the third aspect of the present invention, such as the use in any of A1 to A4: (A1) promoting the growth of aquatic animals; (A2) antagonizing common pathogens of aquatic animals; (A3) enhancing the disease resistance of aquatic animals; (A4) serving as a probiotic for aquatic animals.

[0050] The Bacillus tequilensis Bt-CO strain described in this invention was tested for antibiotic susceptibility and blood agar plate culture. It was sensitive to multiple antibiotics and showed negative hemolytic activity, indicating that it has low environmental risk and is harmless to humans and animals.

[0051] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0052] The present invention will be further illustrated below with reference to the embodiments.

[0053] Example 1: Bt-CO separation and identification

[0054] (a) Isolation and screening of strains

[0055] Coral samples were collected from Huaguang Reef in the Xisha Islands of China. Beef extract peptone medium was used, and microorganisms were isolated and purified by grinding and multi-concentration gradient dilution plating. Single colonies with clear morphology were picked from the medium and streaked on LB medium for 3 days, and then preserved in glycerol tubes at -20℃.

[0056] The beef extract peptone culture medium was prepared according to the following components in the indicated weight-volume ratios: 3 g / L beef extract, 10 g / L peptone, 5.0 g / L sodium chloride, and 20 g / L agar. It was autoclaved at 121°C for 20 min.

[0057] The LB solid culture medium was prepared according to the following components in the indicated weight-volume ratios: 10.0 g / L tryptone, 5.0 g / L yeast extract, 5.0 g / L sodium chloride, and 20 g / L agar. It was autoclaved at 121°C for 20 min.

[0058] (II) Identification of Bt-CO

[0059] 1. Morphological characteristics

[0060] Morphological observation of Bt-CO was performed using streak plating, including shape, color, surface, and viscosity. Results showed that Bt-CO colonies were opaque with irregular edges, initially slightly whitish in color, and initially at high humidity, with the colony surface becoming drier in the later stages of cultivation. Figure 1 ).

[0061] 2. Physiological and biochemical characteristics

[0062] Gram staining: The bacterial cells were cultured on LB medium to the logarithmic growth phase, stained according to the kit instructions (Solepro), and observed under a light microscope. The results showed that strain Bt-CO was Gram-positive. Figure 1 ).

[0063] MR test: The strain was inoculated into glucose phosphate peptone water medium and cultured at 37°C at 180 rpm for 48 h. Then, 5 mL of fermentation broth was added to a clean test tube, along with 1-2 drops of methyl red reagent. The mixture was thoroughly shaken and the color change was observed. The results showed that strain Bt-CO was negative for MR.

[0064] VP test: The strain was inoculated into glucose phosphate peptone water medium and cultured at 37℃ and 180 rpm for 48 h. 5 mL of fermentation broth was placed in a clean test tube, and the VP test reagent was added. The mixture was thoroughly shaken and observed for color change; a positive reaction was indicated by a red color. The results show that strain Bt-CO is VP positive.

[0065] Catalase detection: A small amount of bacterial cells was scraped and placed on a clean glass slide, and then a small amount of 3% H2O2 solution was applied to its surface. Numerous bubbles appeared. The results indicate that strain Bt-CO can produce catalase.

[0066] Gelatin liquefaction: The strain was inoculated into gelatin medium by stab and cultured for 7 days, with liquefaction observed daily. If the room temperature was found to be too high during the experiment, the medium might dissolve itself; therefore, it was frozen in a refrigerator for 30 minutes before being removed and the liquefaction observed. The results showed that strain Bt-CO can hydrolyze gelatin.

[0067] Protease detection: The strain was inoculated onto protease detection medium and incubated at 37°C for 48 h. The plate phenomenon was observed, and a clear zone appeared. Results are shown below. Figure 1 This indicates that strain Bt-CO can produce proteases.

[0068] Cellulase detection: The strain was inoculated onto the test medium and cultured at 37°C for 48 hours. Then, the entire medium was covered with 0.5% Congo red solution and kept covered for 20 minutes. The Congo red solution was then discarded, and 5% NaCl solution was added, maintaining the coverage for another 1 hour. After 1 hour, the solution was discarded, and a clear zone appeared. Results are shown below. Figure 1 This indicates that strain Bt-CO can produce cellulase.

[0069] Amylase detection: The strain was inoculated into amylase detection plates and incubated at 37°C for 3–4 days. Lugol's iodine solution was then applied to the plates; a clear zone appeared. Results are shown below. Figure 1 This indicates that strain Bt-CO can produce amylase.

[0070] Table 1 summarizes the results above.

[0071] Table 1: Morphological and physiological-biochemical characteristics of the strains

[0072]

[0073] Note: Positive is "+"; negative is "-".

[0074] 3. Molecular biological identification

[0075] Using bacterial DNA as a template, 16S rRNA fragments were amplified and sequenced using universal primers: 27F: 5'-GAGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1) and 1492R: 5'-ACGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). During the pre-denaturation phase, the temperature was adjusted to 95°C for 5 minutes; during the cycling phase, the temperature was adjusted to 95°C for 1 minute; then 60°C for 45 seconds; and 72°C for 60 seconds. During the extension phase, the temperature was adjusted to 72°C for 10 minutes; finally, the temperature was lowered to 4°C. After sequencing was completed, the BLAST search program of NCBI (https: / / www.ncbi.nlm.nih.gov / ) was used to compare the sequences in GenBank with the obtained 16S rRNA gene sequences. Strains with high 16S rRNA sequence similarity were selected, and phylogenetic trees were constructed using the neighbor-joining method in MEGA 11 software to better understand and describe the phylogenetic process. The phylogenetic trees of Bt-CO and related strains are shown below. Figure 2 .

[0076] (III) Detection of common aquatic pathogens antagonized by Bt-CO

[0077] The purified strains were inoculated into LB liquid medium and cultured on a shaker (37℃, 180 r / min) for 1 day. Eight pathogenic bacteria (Edwardsiella faecium, Aeromonas hydrophila, Aeromonas verrucosa, Shigella-like bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae) were cultured to OD500. 600 When the concentration is approximately 0.5, mix with LB liquid medium and pour onto a plate. Spot 3 μL of the cultured Bt-CO bacterial suspension onto the pathogen plate to detect the antibacterial activity of the target strain. Incubate at 28℃ for 24–72 h and observe the size of the inhibition zone. If the strain can inhibit the indicator bacteria, a clear zone will appear on the plate; otherwise, it will not. Results are as follows: Figure 3 As shown, the diameter of the inhibition zone is statistically analyzed as follows: Figure 4 As shown.

[0078] (iv) Safety testing of Bt-CO

[0079] Hemolytic activity: Bt-CO strain was inoculated into blood agar medium and incubated at 37°C for 3 days. The presence or absence of a hemolytic zone was observed. The appearance of a hemolytic zone indicates that the strain has hemolytic activity and poses a potential threat to humans and animals; therefore, the strain should not be used in microbial fertilizers. The absence of a hemolytic zone indicates that the strain has no hemolytic activity and is a safe strain that can be used as a microbial fertilizer. Strain Bt-CO did not show a hemolytic zone, as shown in the results below. Figure 1 As shown.

[0080] Antibiotic susceptibility: Bt-CO strain was inoculated into liquid LB and cultured overnight at 37°C. 100 μL of the culture was spread on a plate. After spreading, different types of drug susceptibility plates (6 mm in diameter) were inoculated onto the plate and cultured at 37°C for 2 days. The presence of a clear zone was then observed.

[0081] Table 2: Antibiotic Susceptibility

[0082]

[0083] Example 2: Detection of antagonistic effects of Bt-CO fermentation broth on common aquatic pathogens

[0084] (I) Preparation of Bt-CO fermentation broth

[0085] The Bt-CO strain was inoculated into LB liquid medium and cultured at 37°C with shaking at 200 r / min for 16–24 h to obtain the seed culture. The fermentation medium was sterilized at 121°C for 20 min, cooled, and then 1% Bt-CO seed culture was added. The culture was then carried out at 37°C with shaking at 180 r / min for 60 h.

[0086] Fermentation medium: 30 g corn flour, 50 g soybean meal, 1 g KH2PO4, 1 g K2HPO4, 2 g CaCl2, 1000 mL distilled water.

[0087] (II) Experimental Methods

[0088] The purified strains were inoculated into LB liquid medium and cultured on a shaker (37℃, 180 r / min) for 1 day. Eight pathogenic bacteria (Edwardsiella faecium, Aeromonas hydrophila, Aeromonas verrucosa, Shigella-like bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae) were cultured to OD500. 600 When the concentration is approximately 0.5, mix with LB liquid medium and pour onto a plate. Use a 1 mL sterile blue pipette tip to make a well in the center of the plate. Centrifuge the fermentation medium, collect the supernatant, filter through a 0.22 μm filter membrane, and add 50 μL to the wells of the pathogen plate. Test the antibacterial activity of the Bt-CO sterile fermentation broth. Incubate at 28℃ for 24–48 h and observe the size of the inhibition zone. If the sterile fermentation broth can inhibit the indicator bacteria, a clear zone will appear on the plate; otherwise, it will not. Results are as follows: Figure 5 As shown, the diameter of the inhibition zone is as follows: Figure 6 As shown.

[0089] Example 3: Bt-CO promotes weight gain in tilapia

[0090] Preparation of Bt-CO probiotic feed: The preparation of Bt-CO fermentation liquid is the same as in Example 2. After filtration, the fermentation liquid is mixed with commercially available Tongwei tilapia feed at a weight ratio of 1:2. After drying, probiotic feed with added Bacillus teslaeri Bt-CO is obtained.

[0091] Preparation of Bt-PI bacterial feed: The preparation method of Bt-PI fermentation broth is the same as that of Bt-CO fermentation broth in Example 2. After filtration, the fermentation broth is mixed with commercially available Tongwei tilapia feed at a weight ratio of 1:2. After drying, bacterial feed containing Bacillus teslaeri Bt-PI is obtained.

[0092] Preparation of control group feed: After filtering the fermentation medium, it was mixed with commercially available Tongwei tilapia feed at a weight ratio of 1:2, and then dried to obtain the control group feed.

[0093] Tilapia fry were sourced from the Lingshan Tilapia Farm in Hainan Province, with an initial weight of approximately 6.6 g. Healthy and active tilapia fry of uniform size were selected for rearing. Three control groups, three Bt-CO treatment groups, and three Bt-PI treatment groups were established. The Bt-CO treatment groups were fed a diet supplemented with Bacillus teslaeri Bt-CO probiotics, the Bt-PI treatment groups were fed a diet supplemented with Bacillus teslaeri Bt-PI bacteria, and the control groups were fed commercial tilapia feed without added bacteria. Tilapia were fed a 30-minute satiety period, twice daily (morning and evening). After four weeks of feeding, the weight of each fish in each group was measured and recorded, and the weight gain rate was calculated. The results are shown in Table 3.

[0094] Table 3: Weight gain of tilapia after feeding with Bacillus tekirae Bt-CO and Bt-PI

[0095]

[0096] The results showed that feeding tilapia with probiotic feed supplemented with Bacillus tektii Bt-CO could significantly increase the body weight of tilapia, with an average weight gain of 8.72 g and a weight gain rate of 130.1%. Compared with the control group, the weight gain rate was increased by 66.4%, and compared with the Bt-PI treatment group, the weight gain rate was increased by 50.5%.

[0097] Example 4: Bt-CO control of diseases caused by Edwardsiella tarda (fish killer)

[0098] Feeding, intraperitoneal injection challenge, and disease control: Healthy tilapia (7±1.5 g), 20 fish per group. Three groups were fed a diet supplemented with Bacillus teslaeri Bt-CO, three groups were fed a diet supplemented with Bacillus teslaeri Bt-PI, and three groups were fed the control diet. The diet preparation was the same as in Example 3. The fish were fed for 4 weeks, with a 30-minute feeding period, twice a day (morning and evening). After a one-day fast, the fish were challenged with intraperitoneal injection of Edwardsiella tarda. 10 7 Edwardsiella tectoris was resuspended in PBS at a concentration of CFU / mL and injected into each tilapia at a rate of 50 μL. The incidence and mortality of tilapia infected with Edwardsiella tectoris were observed and recorded for 7 consecutive days. Two batches of experiments were conducted. The results are shown in Table 4.

[0099] Table 4: Mortality rates of fish after intraperitoneal injection of Edwardsiella tarda (a deadly bacterium)

[0100]

[0101] On day 7, the mortality rate of tilapia in the control group was 78.5%, while the mortality rate in the Bt-CO probiotic group was 26.7%, with a protection rate of 66.0%. The mortality rate in the Bt-PI group was 73.3%, with a protection rate of 6.6%. The results indicate that Bt-CO has an excellent effect on the prevention and control of diseases caused by Edwardsiella tarda.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacillus tequilensis, characterized by, The accession number is GDMCC No:64960.

2. The fermentation broth of Bacillus tequilensis as described in claim 1.

3. The fermentation broth as described in claim 2, characterized in that, The fermentation medium consists of 30 parts corn flour, 50 parts soybean meal, 1 part KH2PO4, 1 part K2HPO4, and 2 parts CaCl2 by weight.

4. The application of Bacillus tequilensis as described in claim 1 or the fermentation broth as described in claim 2 or 3 in the preparation of products for aquaculture; The products include formulations or feed.

5. The application as described in claim 4, characterized in that, The product is used for at least one of the following: (A1) Antagonistic to pathogenic microorganisms; (A2) Disease prevention and control; (A3) Enhance the disease resistance of aquatic animals; The pathogenic microorganisms include at least one of Edwardsiella pisciformis, Aeromonas hydrophila, Aeromonas versicolor, Shigella-like Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae. The disease is caused by at least one of Edwardsiella faecium, Aeromonas hydrophila, Aeromonas versicolor, Shigella-like Orthomonas, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus agalactiae. The aquatic animals mentioned include tilapia.

6. Products for aquaculture, including formulations or feeds, characterized in that, Including acceptable excipients or adjuvants, and: (B1) Bacillus tequilensis as described in claim 1; or (B2) The fermentation broth as described in claim 2 or 3.

7. The product as described in claim 6, characterized in that, It is a probiotic feed; The probiotic feed includes the step of mixing Bacillus tequilensis as described in claim 1 with the feed at a weight ratio of 1:

2.

8. A method for preparing a product for aquaculture, characterized in that, Prepared based on Bacillus tequilensis as described in claim 1.

9. The use of Bacillus tequilensis as described in claim 1, the fermentation broth as described in claim 2 or 3, or the product as described in claim 6 or 7 in promoting the growth of aquatic animals.

10. Aquaculture methods for non-therapeutic purposes, characterized in that, The steps include using Bacillus tequilensis as described in claim 1, the fermentation broth as described in claim 2 or 3, or the product as described in claim 6 or 7.

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