CN117210370A - Bacillus subtilis for producing bacteriocin and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus subtilis for producing bacteriocin and application thereof. The bacillus subtilis (Bacillus subtilis) T20 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.26358. The strain can efficiently express bacteriocin, has obvious antibacterial performance on main pathogenic bacteria of livestock and poultry such as staphylococcus aureus, escherichia coli, salmonella, clostridium perfringens and aeromonas hydrophila, can endure high temperature of 70 ℃, can be stably stored for a long time, can reduce diarrhea rate after being fed to livestock and poultry such as weaned pigs and broiler chickens, remarkably improves feed conversion efficiency, improves growth performance, can be used as a novel effective antibiotic substitute, and has good application prospect in livestock and poultry cultivation.

Description

A bacteriocin-producing Bacillus subtilis and its application

Technical field

The present invention relates to the technical field of microorganisms, and in particular to a bacteriocin-producing Bacillus subtilis and its application.

Background technique

In recent years, the development of new green feed additives has become a key research and development content in the field of animal husbandry. The development of new feed additives that can replace or partially replace the functions of antibiotics, can effectively inhibit the reproduction of pathogenic bacteria and improve the intestinal health of livestock and poultry is of great importance to the development of animal husbandry. significance.

Bacillus subtilis is a Gram-positive bacterium that is highly resistant to stress and can reproduce in large quantities. Its extensive functions in the field of livestock and poultry breeding have been recognized, but the probiotic functions of Bacillus subtilis are different. The difference is also quite obvious. The breeding of a strain of Bacillus subtilis with excellent performance, especially a new strain with special functions, is the focus of the research and development of Bacillus subtilis preparations.

Bacteriocins are polypeptide small molecule natural products with antibacterial activity produced through ribosomal (RiPP) or non-ribosomal (NSP) synthesis mechanisms during bacterial metabolism. Bacteriocins affect bacterial community dynamics and composition through niche clearance, colonization resistance, and spatial isolation. Compared with antimicrobial peptides, bacteriocins are active in the picomolar (pmol) or nanomolar (nmol) range and have highly effective antibacterial effects. In medicine, bacteriocins are not only used as antibacterial drugs to replace antibiotics, but also as a new type of anti-cancer drug.

In livestock and poultry breeding, the mortality rate of young animals caused by bacterial infection is high and widespread, seriously harming the breeding efficiency. The main pathogenic bacteria include Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella, and Hydrophila Aeromonas, etc. Therefore, there is an urgent need to develop non-antibiotic antibacterial agents against these pathogenic bacteria.

Contents of the invention

The invention provides Bacillus subtilis and its application.

The present invention provides Bacillus subtilis (Bacillus subtilis) T20, which strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (CGMCC for short) on December 29, 2022. Address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), classified as Bacillus subtilis, and deposited as CGMCC No. 26358.

In the present invention, the T20 strain was isolated from a deep soil sample of a primitive forest in Heilongjiang and obtained through ultraviolet mutagenesis and selection. After 16S rRNA sequence analysis and comparison with the NCBI database, it was determined that the strain was Bacillus subtilis (Bacillus subtilis).

The microbiological characteristics of Bacillus subtilis T20 are: Gram-positive bacilli, colony size on LB solid plate is 5 to 8 mm, milky white, opaque colonies, with wrinkles on the surface of the colony, an inward depression in the center, and irregular edges of the colony. Bacillus subtilis T20 is capable of producing bacteriocins. The bacterial liquid and solid fermentation preparations of Bacillus subtilis T20 can effectively inhibit animal pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila, and Bacillus subtilis T20 has better Heat resistance. It has been verified by animal experiments that Bacillus subtilis T20 is highly safe for animals, can reduce animal diarrhea rates, improve animal disease resistance, and can increase animal feed intake, promote animal growth, promote animal weight gain, and improve feed conversion Rate.

The present invention provides a bacterial agent, which contains the above-mentioned Bacillus subtilis T20.

The above-mentioned bacterial agent can be a solid preparation or a liquid preparation.

The above-mentioned microbial agents may also contain carriers or auxiliary materials permitted in the field of microbial preparations, including but not limited to freeze-drying protective agents (such as glycerol), peat soil, talc, attapulgite, diatomaceous earth, etc.

The present invention provides a method for preparing the above-mentioned bacterial agent, which method includes the steps of cultivating the Bacillus subtilis T20 and collecting the culture.

The culture is preferably performed at 20 to 40°C. More preferably, it is carried out at 25 to 40°C.

The present invention provides an antibacterial agent, which contains the above-described Bacillus subtilis T20 or is prepared from the above-described Bacillus subtilis T20.

The above-mentioned antibacterial agents can effectively inhibit animal pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila.

The present invention provides a feed, feed additive or medicine, which contains the above-mentioned Bacillus subtilis T20 or is prepared from the above-mentioned Bacillus subtilis T20.

The above-mentioned feeds, feed additives or drugs have one or more of the following functions: reducing animal diarrhea rates, improving animal disease resistance, increasing animal feed intake, promoting animal growth, promoting animal weight gain, and improving feed conversion rate.

Preferably, in the feed additive, the viable bacterial count of Bacillus subtilis T20 is 5.0×10 ⁹ to 9.9×10 ⁹ CFU/g.

Preferably, the viable bacterial count of Bacillus subtilis T20 in the feed is 5.0×10 ⁷ to 5.0×10 ⁹ CFU/kg.

Based on the characteristics and functions of Bacillus subtilis T20, the present invention provides the following uses of this strain:

Bacillus subtilis T20 can effectively inhibit animal pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila.

The present invention provides the use of the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent in preparing products for inhibiting animal pathogenic bacteria.

Preferably, in the above applications, the animal pathogenic bacteria include enteric pathogenic bacteria. Specifically, the animal pathogenic bacteria include one or more selected from the group consisting of Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila.

The present invention provides the use of the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent in bacteriostasis for non-disease treatment purposes.

The above-mentioned bacteriostatic bacteria for non-disease treatment purposes include one or more selected from the group consisting of Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila.

The above-mentioned bacteriostasis for non-disease treatment purposes includes bacteriostasis in the external environment.

The present invention provides the use of the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent in the preparation of bacteriocins.

The present invention provides the use of the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent in preparing products for reducing animal diarrhea rates and/or improving animal disease resistance.

The above-mentioned products include feed, feed additives or pharmaceuticals.

The present invention provides the use of the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent in the preparation of feed, feed additives or medicines.

The present invention provides the above-mentioned Bacillus subtilis T20 or the bacterial agent or the antibacterial agent or the feed, feed additive or medicine for improving animal feed intake, promoting animal growth, promoting animal weight gain and/or improving feed Application in conversion rate.

The present invention has verified through animal experiments that Bacillus subtilis T20 has the effects of relieving diarrhea in weaned piglets, promoting piglet growth, increasing piglet feed intake, increasing piglet average daily weight gain, and improving feed conversion efficiency. The present invention has verified through animal experiments that Bacillus subtilis T20 can promote the growth of broiler chickens, increase the feed intake of broiler chickens, increase the average daily weight gain of broiler chickens, and improve the feed conversion efficiency in broiler chicken breeding. The present invention has verified through animal experiments that Bacillus subtilis T20 can improve the milk production and feed utilization of Holstein cows in dairy farming.

The above animal experiments are only examples. In the above applications, the animals include but are not limited to pigs, chickens, cows, etc.

The invention provides a liquid submerged fermentation-solid aerobic fermentation method of Bacillus subtilis T20, which includes the following steps:

(1) Bacteria recovery and activation

Thaw the cryopreservation tube of Bacillus subtilis T20 and streak inoculate it on the solid LB medium plate. Cultivate it at 37°C for 12 to 24 hours until a single colony grows. Pick the single colony and streak it on the LB solid plate again. Purify and obtain the second Plates with single colonies were used as seed plates.

(2) Preparation of first-level seed liquid

Select a single colony from the seed plate and inoculate it into an LB liquid shake flask, and culture it on a 37°C shaker at 200 r/min for 14 to 18 hours as the first-level seed liquid, with an OD ₆₀₀ of 1.5 to 2.0.

(3) Preparation of secondary seed liquid

Inoculate the first-level seed liquid into the LB liquid seed tank at a ratio of 0.5% to 5%, and culture it at 20-40°C for 10-20 hours. The dissolved oxygen control range is 5%-30%, and the liquid volume is 15-35L. As a second-level seed liquid, Grade seed liquid.

(4) Three-level liquid submerged fermentation

Inoculate the secondary seed liquid into the liquid deep fermentation tank at a proportion of 0.5% to 5%, and culture it at 20 to 40°C for 10 to 20 hours. The dissolved oxygen control range is 5% to 30%. The liquid volume is 500 to 2000L. The fermentation time Control it to 10~18h.

(5)Solid state fermentation

Sterilize the solid fermentation material at 110°C to 121°C for 10 to 20 minutes and then cool it for later use. Inoculate the deep fermentation liquid at a ratio of 10% to 50% and mix it evenly in the solid fermentation medium. Control the stirring speed and fermentation temperature of the solid fermentation to maintain The temperature is 25-40°C and fermented for 12-36 hours to prepare a solid fermentation agent. After drying and crushing the solid fermentation agent, the number of viable bacteria in the solid product can reach 5.0-9.9×10 ⁹ CFU/g.

Bacillus subtilis T20 can efficiently express bacteriocins through liquid submerged fermentation-solid aerobic fermentation.

Preferably, in the above method, the liquid submerged fermentation medium contains the following components by weight: 45-55 parts of corn steep liquor dry powder, 70-80 parts of defatted soybean meal powder, 35-45 parts of yeast extract powder, sodium chloride 5-7 parts, ferrous chloride 1-3 parts, dipotassium hydrogen phosphate 5-7 parts, magnesium sulfate heptahydrate 8-12 parts, defoaming agent 1-30 parts.

Preferably, in the above method, the solid aerobic fermentation medium contains the following components by weight: 0.4-0.6 parts of corn flour, 0.4-0.6 parts of soybean meal powder, 1-3 parts of defatted rice bran, and 2-3 parts of corn cob powder. 4 parts.

The beneficial effects of the present invention at least include: the present invention provides Bacillus subtilis T20, which strain can efficiently express bacteriocins and is effective against Staphylococcus aureus, Escherichia coli, Salmonella, Clostridium perfringens, and Aeromonas hydrophila, the main pathogenic bacteria of livestock and poultry. The bacterium has obvious antibacterial properties, can withstand high temperatures of 70°C, and can be stored stably for a long time. After feeding weaned piglets, broilers and other livestock and poultry, it can reduce diarrhea rates, significantly improve feed conversion efficiency, and improve growth performance. It can be used as a New and effective antibiotic alternatives have good application prospects in livestock and poultry breeding.

Description of the drawings

In order to explain the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are of the present invention. For some embodiments of the invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 is a colony morphology diagram of Bacillus subtilis T20 in Example 1 of the present invention.

Figure 2 is a Gram staining diagram of Bacillus subtilis T20 in Example 1 of the present invention.

Figure 3 is the growth curve of Bacillus subtilis T20 in LB liquid culture medium in Example 1 of the present invention.

Figure 4 is a morphological diagram of the product after processing the solid fermentation product of Bacillus subtilis T20 in Example 2 of the present invention.

Figure 5 shows the heat resistance test results of Bacillus subtilis T20 in Example 3 of the present invention.

Figure 6 is the test result of the antibacterial performance of Bacillus subtilis T20 against Staphylococcus aureus 25923 in Example 3 of the present invention.

Figure 7 is the test result of the antibacterial performance of Bacillus subtilis T20 against Clostridium perfringens 2027 in Example 3 of the present invention.

Figure 8 is the test result of the antibacterial performance of Bacillus subtilis T20 against Escherichia coli K88 in Example 3 of the present invention.

Figure 9 is the test result of the antibacterial performance of Bacillus subtilis T20 against Salmonella 1791 in Example 3 of the present invention.

Figure 10 shows the drug sensitivity test results of Bacillus subtilis T20 in Example 3 of the present invention. The upper figure from left to right shows penicillin (P), chloramphenicol (C), erythromycin (E), and kanamycin. K), the picture below from left to right is doxycycline (DOX), ciprofloxacin (CIP), norfloxacin (NOR), and cefradine (RAD).

Figure 11 shows the results of morphological observation of mouse tissues after the mouse safety evaluation of Bacillus subtilis T20 in Example 4 of the present invention. The upper picture from left to right is the liver, spleen and kidney respectively, and the lower picture is from From left to right are the jejunum, ileum and colon

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Example 1 Obtaining Bacillus subtilis T20

The strain provided by the invention is isolated from a deep soil sample of a primitive forest in Heilongjiang and obtained through ultraviolet mutagenesis and breeding. The strain is named T20. The 16S rRNA sequence sequencing results of the strain were compared in the NCBI database, and the results showed that the strain was Bacillus subtilis. Bacillus subtilis (Bacillus subtilis) T20 has been deposited in the General Microbiology Center of the Chinese Committee for the Collection of Microbial Cultures (CGMCC for short) on December 29, 2022. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Microbiology Research Institute, Postal Code 100101), the classification name is Bacillus subtilis, and the deposit number is CGMCC No. 26358.

The following is a brief description of the general process of obtaining Bacillus subtilis T20 and the identification process:

1. Isolation and mutagenesis screening of Bacillus subtilis T20

Take 2g of soil sample, add 18mL of sterile distilled water, add sterilized glass beads, shake and mix to obtain a 1:10 dilution of soil, and perform gradient dilution of the dilution according to 10 ^-1 , 10-2, ^{10 -3} , and 10 ^-4 , select dilutions of different concentrations and apply them on LB solid medium in an amount of 0.1 mL, and incubate at 37°C for 48 to 72 hours. When colonies with a suitable density and concentration grow, pick a single colony and streak it on an LB plate for purification again.

After preparing the bacterial liquid from the isolated strain with excellent performance, dilute the bacterial liquid concentration to 1.0-9.9×10 ⁷ CFU/mL, put it into a 10cm dish according to the dosage of 10-20mL, and mutagenize it with a 15W ultraviolet lamp for 60-120s to kill it. The rate is controlled at 60% to 80%. The mutated bacterial solution is spread on an LB solid plate and cultured in the dark for 24 to 48 hours.

Bacillus subtilis T20 was obtained through mutagenesis screening.

2. Observation of colony morphology

During the logarithmic growth phase, take Bacillus subtilis T20 bacterial liquid, dilute it to different multiples according to the 10-fold dilution method and spread it on LB solid plates. Incubate at 37°C for 24 to 48 hours. Select plates with appropriate density to observe colony morphology and take photos to record them. .

The results are shown in Figure 1. The colony size of the T20 strain on the LB solid plate was 5 to 8 mm, milky white, and opaque. The surface of the colony was wrinkled, the center was sunken inward, and the edges of the colony were irregular.

3. Gram stain detection

During the logarithmic growth phase, take 1 to 2 drops of Bacillus subtilis T20 bacterial solution and apply it on a glass slide, fix it with a flame, and stain it according to the Gram staining kit method. After completion, observe it under a microscope. Gram-positive bacteria will appear. Blue-purple, Gram-negative bacteria appear red.

The results are shown in Figure 2. Bacillus subtilis T20 is a Gram-positive bacterium.

4. Bacterial 16S rRNA sequence homology analysis

Total bacterial DNA was extracted using a bacterial genomic DNA extraction kit. The extracted samples were sent to Shanghai Sangon Bioengineering Co., Ltd. for Sanger sequencing. The measurement results (shown in SEQ ID NO. 1) were subjected to BLAST homology comparison in the NCBI database, and the species category was determined to be Bacillus subtilis (Bacillus subtilis), with a similarity of 99.86%.

The growth curve of Bacillus subtilis T20 in LB liquid medium is shown in Figure 3.

Example 2 Solid-state fermentation of Bacillus subtilis T20

The solid-state fermentation method of Bacillus subtilis T20 includes the following steps:

(1) Bacteria recovery and activation

Thaw the cryopreservation tube of Bacillus subtilis T20 and streak inoculate it on the solid LB medium plate. Cultivate it at 37°C for 12 to 24 hours until a single colony grows. Pick the single colony and streak it on the LB solid plate again. Purify and obtain the second Use single colony plates as seed plates.

(2) Preparation of first-level seed liquid

Select a single colony from the seed plate and inoculate it into an LB liquid shake flask, and culture it on a 37°C shaker at 200 r/min for 14 to 18 hours as the first-level seed liquid. The OD ₆₀₀ range is 1.5 to 2.0.

(3) Preparation of secondary seed liquid

Inoculate the first-level seed liquid into the LB liquid seed tank at a ratio of 0.5% to 5%, and culture it at 20-40°C for 10-20 hours. The dissolved oxygen control range is 5%-30%, and the liquid volume is 15-35L. As a second-level seed liquid, Grade seed liquid.

(4) Three-level liquid submerged fermentation

Inoculate the secondary seed liquid into the liquid deep fermentation tank at a proportion of 0.5% to 5%, and culture it at 20 to 40°C for 10 to 20 hours. The dissolved oxygen control range is 5% to 30%. The liquid volume is 500 to 2000L. The fermentation time Control it to 10~18h.

(5)Solid state fermentation

Sterilize the solid fermentation material at 110°C to 121°C for 10 to 20 minutes and then cool it for later use. Inoculate the deep fermentation liquid at a ratio of 10% to 50% and mix it evenly in the solid fermentation medium. Control the stirring speed and fermentation temperature of the solid fermentation to maintain The temperature is 25-40°C and fermented for 12-36 hours to obtain a solid fermentation agent. The solid fermentation agent is dried and crushed to obtain a solid fermentation product, in which the number of viable bacteria can reach 5.0-9.9×10 ⁹ CFU/g. The product appearance is shown in Figure 4.

In the above method, the medium for liquid submerged fermentation contains the following components: 50 parts of dry corn steep powder, 75 parts of defatted soybean meal powder, 40 parts of yeast extract powder, 6 parts of sodium chloride, 2 parts of ferrous chloride, and dipotassium hydrogen phosphate 6 parts, 10 parts magnesium sulfate heptahydrate, 20 parts defoaming agent.

The solid fermentation medium contains the following components: 0.5 parts of corn flour, 0.5 parts of soybean meal powder, 2 parts of defatted rice bran, and 3 parts of corn cob powder.

Example 3 Performance Testing of Bacillus subtilis T20

1. Detection of heat resistance of Bacillus subtilis T20

The solid-state fermentation product of Bacillus subtilis T20 was treated at 70°C, 80°C, 90°C and 100°C for 3, 5, 10 and 15 minutes respectively. Three replicate samples were designed for each treatment. After the treatment, the samples were compared with those stored at room temperature. The number of viable bacteria was used to evaluate the heat resistance of Bacillus subtilis T20. The results show (Figure 5) that the initial viable bacterial count of Bacillus subtilis T20 is 9.4×10 ⁹ CFU/g. After being treated at 70°C for 15 minutes, the viable bacterial count can still be maintained at 2.8×10 ⁹ CFU/g, which is consistent with the final product. Minimum requirements for viable bacterial count.

2. Determination of antibacterial properties of Bacillus subtilis T20

After solid-state fermentation, Bacillus subtilis T20 was diluted with sterile physiological saline at different times, and the effects of different concentrations of solid-state fermentation product liquids on Staphylococcus aureus, Clostridium perfringens, and For the inhibitory effect of Escherichia coli, Salmonella and Aeromonas hydrophila, the sample volume is 100-200 μL, let stand at 4°C for 2-4 hours, and then incubate at 37°C for 18-24 hours. The antibacterial performance is tested. The results are shown in Table 1 and the figure. 6. Figure 7, Figure 8, Figure 9. The results show that Bacillus subtilis T20 has significant inhibitory effect on the main pathogenic bacteria of livestock and poultry: Staphylococcus aureus, Clostridium perfringens, Escherichia coli, Salmonella and Aeromonas hydrophila. effect.

Table 1

Pathogen type Solid fermentation product liquid dilution ratio Inhibition zone diameter/mm E. coli K88 8000 times 14 Clostridium perfringens CVCC2027 4000 times 11 Staphylococcus aureus ATCC25923 8000 times 13 Salmonella ATCC14028 2000 times 13 Aeromonas hydrophila ATCC49140 2000 times 14

3. Drug susceptibility testing of Bacillus subtilis T20

Take the Bacillus subtilis T20 bacterial liquid in the logarithmic growth phase and inoculate it into solid LB medium at 45-50°C at a concentration of 1% to 50%. Prepare an LB plate containing bacteria. Place different drug-sensitive paper sheets in the center of the petri dish and culture for 24 ~48h, detect the size of the inhibition zone, take photos and record. The results are shown in Figure 10 and Table 2. The results show that Bacillus subtilis T20 is extremely sensitive (S) to different types of antibiotics, has no potential risk of drug resistance, and has reliable safety as a probiotic.

Table 2 Bacillus subtilis T20 drug susceptibility test results

antibiotic Inhibition zone/mm sensitivity Penicillin(P) 28.5 S Chloramphenicol(C) 29 S Erythromycin(E) 34.5 S Kanamycin (K) 30.5 S Doxycycline (DOX) 30 S Ciprofloxacin (CIP) 42.5 S Norfloxacin (NOR) 35.5 S Cefradine (RAD) 50 S

Example 4 Safety evaluation of Bacillus subtilis inoculant T20 in mice

In the experiment, 18 mice aged 4 to 5 weeks were selected and randomly divided into 3 groups (CON: control group, each mouse was gavaged with 100 μL sterile physiological saline every day; LOW: low-dose group, each mouse was gavaged with 100 μL 1.5×10 ⁷ CFU/mL Bacillus subtilis T20 bacterial agent; HIGH: high-dose group, each animal was gavaged with 100 μL 1.5×10 ⁷ CFU/mL Bacillus subtilis T20 bacterial agent every day, 3 replicates per group, 2 animals per replicate, Feed by gavage for 28 days.

The body weight of a small test was recorded every 7 days. The results are shown in Table 3. After the mice were sacrificed by cervical dislocation, the complete heart, liver, spleen, and kidney were dissected and removed. The samples were placed in 4% paraformaldehyde and used to fix the samples. Different representatives were collected. The intestinal segments were placed in 4% paraformaldehyde and used to fix the samples. They were sectioned by HE staining and used to observe the tissue morphology. The tissue morphology results are shown in Figure 11. The results showed that there was no obvious damage to the main organs of the mice. Bacillus strain T20 has no safety risk to mice.

Table 3 Mouse safety evaluation of Bacillus subtilis T20

Example 5 Application of Bacillus subtilis T20 in weaned piglets

The experiment selected 28-day-old Durian × Long × Da three-way cross weaned piglets and divided them into 3 groups, with 6 replicates in each group and 7 pigs in each replicate, totaling 126 pigs. Group A: fed control basal diet; Group B: fed Bacillus subtilis T20 (according to 500g/t dosage, effective viable bacterial count is 1.5~5.0×10 ⁹ CFU/kg); Group C: third-party product control Group, the product is an antimicrobial peptide product collected from the market for weaned piglets, and the dosage is 500g/t. The experiment was fed for 28 days, and feed intake and daily weight gain were recorded every 14 days. During the experiment, the diarrhea status of the piglets was observed, and the diarrhea rate was recorded and calculated. The results are shown in Table 4. The results show that Bacillus subtilis T20 can effectively increase piglet feed intake, increase average daily weight gain, improve feed conversion efficiency, and significantly reduce diarrhea rate.

Table 4 Effect of Bacillus subtilis T20 on the growth performance of weaned piglets

Group Average daily feed intake (g/d) Average daily weight gain (g/d) Feed weight gain ratio Diarrhea rate Group A 694.2 321.1 2.17 17.7 Group B 725.3 360.3 2.02 13.7 Group C 693.13 347.7 2.02 10.8

Example 6 Application of Bacillus subtilis T20 in broilers

The experiment selected 96 1-day-old AA broiler chickens and divided them into 2 groups, with 8 replicates in each group and 6 chickens in each replicate. The control group was fed a basic diet, and the T20 group was supplemented with 500g/t product (the number of effective viable bacteria was 1.5～5.0×10 ⁹ CFU/kg), fed for 42 days, weighed once every 21 days during the feeding period, and changed to post-feeding after 21 days of age. During the test period, the test operations were carried out according to the daily management of the chicken house. The results are shown in Table 5. The results show that Bacillus subtilis T20 can effectively increase the feed intake and average daily weight gain of AA broilers.

Table 5 Effect of Bacillus subtilis inoculant T20 on the growth performance of AA broilers

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The bacillus subtilis (Bacillus subtilis) T20 is characterized by being preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 26358.

2. A microbial agent comprising bacillus subtilis (Bacillus subtilis) T20 according to claim 1.

3. An antibacterial agent comprising the bacillus subtilis (Bacillus subtilis) T20 of claim 1 or prepared from the bacillus subtilis (Bacillus subtilis) T20 of claim 1.

4. A feed, feed additive or medicament comprising the bacillus subtilis (Bacillus subtilis) T20 of claim 1 or prepared from the bacillus subtilis (Bacillus subtilis) T20 of claim 1.

5. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or a microbial agent according to claim 2 or an antimicrobial agent according to claim 3 for the preparation of a product for inhibiting animal pathogenic bacteria.

6. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or a microbial agent according to claim 2 or an antimicrobial agent according to claim 3 for bacteriostasis for non-disease treatment purposes.

7. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or a microbial agent according to claim 2 or an antimicrobial agent according to claim 3 in bacteriocin preparation.

8. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or a microbial agent according to claim 2 or an antimicrobial agent according to claim 3 for the preparation of a product for reducing diarrhea rate and/or improving disease resistance in an animal.

9. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or a microbial agent according to claim 2 or an antimicrobial agent according to claim 3 for the preparation of a feed, feed additive or medicament.

10. Use of bacillus subtilis (Bacillus subtilis) T20 according to claim 1 or the microbial agent according to claim 2 or the antimicrobial agent according to claim 3 or the feed, feed additive or medicament according to claim 4 for increasing feed intake, promoting animal growth, promoting animal weight gain and/or increasing feed conversion rate in animals.