A Bacillus subtilis and its application in enhancing animal immunity

Abstract

The present invention discloses a Bacillus subtilis strain and its application in improving animal immunity, belonging to the technical field of agricultural microorganisms. The preservation number of the Bacillus subtilis strain is CCTCC NO: M 20232183. It is derived from the permafrost in Russia and has good strain properties. It can significantly tolerate low temperature, high temperature, artificial gastric acid, bile salts, and artificial intestinal juice; has strong inhibitory effects on typical animal intestinal pathogenic bacteria such as Escherichia coli, Salmonella, and pathogenic Staphylococcus aureus, etc.; produces high yields of various enzymes such as protease, xylanase, fructosyltransferase, phytase, amylase, cellulase, and SOD enzyme; can play the function of a vaccine adjuvant, achieve the same immune stimulation effect as traditional adjuvants, enhance animal resistance, reduce the occurrence of diseases, and improve animal welfare.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a Bacillus subtilis strain and its application in improving animal immunity. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] With the advent of the era of total antibiotic ban, the breeding industry is also actively seeking new solutions. New products such as natural plant extracts, beneficial microorganisms, oligosaccharides and polysaccharides, immune active peptides, enzyme preparations, acidifying agents, etc. have set off a "antibiotic substitution" boom. Among them, the development of microecological preparations has attracted wide attention. Microecological preparations are biological products made from one or more normal flora microorganisms, which can improve the balance of microbial flora in the animal intestine, enhance the mucosal immunity of the body, improve the vaccine immunity effect, resist pathogenic microorganism infection, promote nutrient digestion and absorption, improve the feed-to-meat ratio and production performance, save energy and reduce emissions, and effectively improve the health level of livestock and poultry and the economic benefits of the breeding industry.

[0004] The key factor for probiotics to exert their effects lies in the performance of the selected strains. Currently, the strains that can be used for the production of probiotics mainly include Bacillus, Lactobacillus, Saccharomyces cerevisiae, and Bifidobacterium, etc. Li Guangzhi et al. provided a Bacillus subtilis with strong tolerance, broad-spectrum antibacterial properties, and the functions of efficiently degrading starch and cellulose; Wei Kefeng et al. showed through research that Bacillus subtilis can enhance the immune function of mice, expanding the uses of Bacillus; Patent CN109337841A provided a Bacillus subtilis BYS2 with high antibacterial performance, which can withstand high temperature, acid, and bile salts, not only can adapt to the gastrointestinal environment, but also has a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and Clostridium perfringens; Patent CN112239735A provided a Bacillus subtilis with strong abilities to produce protease, amylase, lipase, and cellulase, which can reduce the damage caused by Aeromonas hydrophila to grass carp. However, the current relevant research on the enhancement of vaccine immune effects by strains is not comprehensive enough. Most of the relevant research is to use genetically recombinant strains as vaccine adjuvants. For example, Li Zhihui et al. provided a method of co-incubating surface-displayed FDIBP recombinant Bacillus subtilis spores and Escherichia coli heat-labile enterotoxin (LT) with human IgG (hlgG) as an adjuvant to prepare a vaccine; Hu et al. successfully constructed recombinant Bacillus using the FMDV antigenic site epitope cassette gene and cholera toxin B gene, and orally immunized mice and guinea pigs respectively. The research results showed that the recombinant spores could induce immune responses of antiviral IgA antibodies and serum IgG antibodies, as well as T lymphocyte proliferation and IFN-γ secretion responses in the lung and intestinal lavage fluids of the vaccinated animals; Mou et al. successfully displayed the TGEV spike protein on the surface of Bacillus spores. After orally immunizing Yorkshire piglets, the fecal IgA and serum IgG neutralizing antibody titers increased. There are also some studies that extract the corresponding proteins from strains for immune enhancement against viruses. For example, Wang Xiao'e et al. used ultrasonic disruption and TritonX-100 treatment techniques to extract outer membrane proteins (OMP) from Lactobacillus and Bacillus licheniformis strains, and mixed them with the allantoic fluid of Newcastle disease virus in different ways and contents, and then made immunogens with oil adjuvants. The results showed that the immunogens added with OMP could significantly stimulate immune responses to continuously produce high-titer antibodies and effectively resist the attack of the corresponding virulent strains. However, at present, no strain has been reported to have excellent functions in multiple aspects such as strain tolerance, antibacterial spectrum, enzyme production ability, immune enhancement ability, and directly used as a vaccine adjuvant without recombination. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the object of the present invention is to provide a Bacillus subtilis strain and its application in improving animal immunity. The Bacillus subtilis provided by the present invention can significantly tolerate low temperature, high temperature, artificial gastric acid, bile salts, and artificial intestinal juice; has strong inhibitory effects on typical animal intestinal pathogenic bacteria such as Escherichia coli, Salmonella, and pathogenic Staphylococcus aureus; produces high yields of various enzymes such as amylase, cellulase, and SOD enzyme; can increase intestinal length and improve the digestion and absorption rate of animals; can also play an adjuvant function, achieving the same or better immune stimulation effect as traditional adjuvants, enhancing animal resistance, reducing the occurrence of diseases, and improving animal welfare.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a Bacillus subtilis strain is provided, and the preservation number of this strain is CCTCC NO: M20232183.

[0008] In some embodiments of the present invention, the usage forms of the Bacillus subtilis include one or more of its live bacteria, fermentation broth, and bacterial powder.

[0009] In the second aspect of the present invention, a microbial inoculant is provided, which includes the above-mentioned Bacillus subtilis.

[0010] In the third aspect of the present invention, the above-mentioned Bacillus subtilis or the above-mentioned microbial inoculant is provided for use in preparing any one of the following products:

[0011] (1) For the production of xylanase, fructosyltransferase, protease, amylase, cellulase, phytase, and SOD enzyme;

[0012] (2) For inhibiting livestock and poultry intestinal pathogenic bacteria;

[0013] (3) For increasing the intestinal length of livestock and poultry and improving the digestion and absorption rate;

[0014] (4) For improving the immune ability of livestock and poultry;

[0015] (5) Used as a vaccine adjuvant for livestock and poultry.

[0016] In some embodiments of the present invention, the improvement of the immune ability of livestock and poultry includes increasing the average daily gain, average daily feed intake, and feed-to-weight ratio of livestock and poultry, reducing the diarrhea rate of livestock and poultry, increasing the level of immunoglobulin in the body of livestock and poultry, and improving immunity.

[0017] In some embodiments of the present invention, the livestock and poultry intestinal pathogenic bacteria include, but are not limited to, Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, and Aeromonas hydrophila.

[0018] In some embodiments of the present invention, the vaccine adjuvant can be used in combination with various vaccines to enhance the immune response, reduce the amount of antigen used, efficiently activate the immune system, and prolong the titer level.

[0019] The fourth aspect of the present invention provides a product, including any one of the following:

[0020] (1) The above-mentioned Bacillus subtilis;

[0021] (2) The above-mentioned microbial inoculum.

[0022] The fifth aspect of the present invention provides a method for improving the immunity of livestock and poultry, which is to feed the above-mentioned Bacillus subtilis or the above-mentioned microbial inoculum to livestock and poultry.

[0023] In some embodiments of the present invention, the above-mentioned Bacillus subtilis is fed to livestock and poultry, and the use form of the Bacillus subtilis is the strain spray-dried powder, and the viable count in the strain spray-dried powder ≥ 1.0×10 11 CFU / g.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention reports for the first time a strain of Bacillus subtilis, which is derived from the sampling of loam in the permafrost of Russia and has good strain performance. Specifically, it can significantly tolerate low temperature, high temperature, artificial gastric acid, bile salts, and artificial intestinal juice; has strong inhibitory effects on typical animal intestinal pathogenic bacteria such as Escherichia coli, Salmonella, and pathogenic Staphylococcus aureus; produces high yields of various enzymes such as protease, xylanase, fructosyltransferase, phytase, amylase, cellulase, and superoxide dismutase (SOD); can play the function of an immune adjuvant, achieving the same or better immune stimulation effect as traditional adjuvants, enhancing animal resistance, reducing the occurrence of diseases, and improving animal welfare. The Bacillus subtilis of the present invention has been shown to significantly improve the immune level of piglets through animal immune level tests and immune adjuvant effect tests, and can also achieve an immune stimulation effect equivalent to that of traditional adjuvants, having good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0027] Figure 1 It is a colony morphology diagram of Bacillus subtilis NBL-BQ218 in an embodiment of the present invention;

[0028] Figure 2 It is the microscopic examination morphology of Bacillus subtilis NBL-BQ218 in an embodiment of the present invention;

[0029] Figure 3 It is the phylogenetic tree analysis of Bacillus subtilis NBL-BQ218 in the embodiments of the present invention;

[0030] Figure 4 It is the hemolytic test result of Bacillus subtilis NBL-BQ218 in the embodiments of the present invention. Specific Embodiments

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] Example 1: Isolation of Strains

[0033] 1.1 Test Materials

[0034] Samples: Sampling of loam in permafrost was carried out in the Kungara region of the Sakha (Yakutia) Republic. Five samples were taken from each of two different depths of 0-10 cm and 10-20 cm in each test area, and a total of samples from 3 test areas were taken. Each sample was 100 g, stored at 4 °C, and transported back to the laboratory.

[0035] NA medium and NB medium were purchased from Qingdao Haibo Biotechnology.

[0036] Normal saline: 0.9 g of NaCl, 100 mL of distilled water, sterilized at 121 °C for 20 min.

[0037] 1.2 Instruments and Equipment

[0038] Laminar flow hood, constant temperature shaker, oscillator, electrothermal constant temperature incubator, autoclave, electron microscope

[0039] 1.3 Screening of Strains

[0040] Isolation: Take 10 g and place it in a conical flask containing 90 mL of 0.9% normal saline, and shake it in a constant temperature shaker at 30 °C and 180 r / min for 30 min. Pipette 1 mL of the enriched culture solution into the normal saline and dilute it to 10 6 、10 7 、10 8 Concentration gradients for NA plate coating, and incubate at 37 °C for 1-2 d.

[0041] Purification: Pick colonies with different morphologies for purification until a purified colony with a single morphology is obtained. Culture the single colony on a slant preservation medium for 1-2 d to form spores and then store it at 4 °C for standby.

[0042] Example 2: Identification of Strains

[0043] 2.1 Morphological Characteristics of Strains

[0044] A single colony was streaked onto an NA plate, and the plate was inverted and placed in an incubator at 30 °C for 24 h to form irregular milky white colonies, which were rough, dull, semi-transparent to opaque, flat, with wavy edges, and viscous in texture. See Figure 1 . The cell morphology was rod-shaped, arranged in chains, clusters and singly, without a capsule, and endospores were formed. The spores were oval, located in the center, and Gram-positive. See Figure 2 .

[0045] 2.2 Molecular identification:

[0046] The strain was sent to Sangon Biotech (Shanghai) Co., Ltd. for whole-genome sequencing. The 16srRNA sequence (SEQ ID NO.1) predicted by gene was compared with the 16s database of NCBI using Blast, and the parameter identify>95 was set. Then, the top 30 16srRNA sequences with the highest identify (take all if less than 30) were selected, and after multiple sequence alignment using the muscle software, a phylogenetic tree was constructed using the FastTree software, which had the highest homology with Bacillus subtilis strain. This strain was named NBL-BQ218. Bacillus subtilis strain NBL-BQ218 was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC) on November 10, 2023. The deposit address is the China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072 and a deposit number of CCTCC NO: M20232183.

[0047] 16sRNA gene sequence of strain NBL-BQ218: (SEQ ID NO.1)

[0048] AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCT

[0049] 2.3 Physiological and biochemical identification of strains

[0050] The optimal growth temperature is 37 °C. Catalase test is positive; gelatin liquefaction test is positive.

[0051] Halotolerance: Grows in 4% NaCl.

[0052] It can hydrolyze starch and decompose casein. It can ferment glucose, sucrose, maltose, trehalose, mannose, mannitol, fructose, sorbitol, raffinose, cellobiose to produce acid, but does not form gas, does not ferment arabinose, xylose, galactose, lactose, inulin, rhamnose, and does not produce indole or hydrogen sulfide. It grows in the presence of up to 4% petroleum.

[0053] Example 3: Strain tolerance

[0054] 3.1 Bile salt tolerance test

[0055] Add 0.3% porcine bile salt to the liquid medium (3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 1 L of distilled water, pH 7.2), and sterilize it at 121 °C under high pressure for 25 min. Inoculate the strain NBL-BQ218 in Example 2 into the above medium, and culture it at 37 °C on a shaker (180 rpm). Take samples at 0, 4, 8, and 24 h for plate count detection, and calculate the survival rate.

[0056] Survival rate of the strain (%) = N t / N0 × 100%

[0057] where N t represents the number of viable bacteria at different sampling times; N0 represents the number of viable bacteria at 0 h.

[0058] The test results are shown in Table 1.

[0059] Table 1 Tolerance of strain NBL-BQ218 to bile salt

[0060] Time (h) 0 4 8 24 Survival rate (%) 100 92 75 56

[0061] As can be seen from Table 1, after 24 h of treatment under the action of 0.3% bile salt, the strain NBL-BQ218 can still maintain a viable bacteria count of more than 10 8 CFU / mL, with a survival rate of about 56%.

[0062] 3.2 Gastric acid tolerance test

[0063] Preparation of artificial gastric juice: 1% pepsin (Amresco), 0.85% sodium chloride solution, adjust the pH to 2.0 with hydrochloric acid, and then filter it with a bacterial filter. Inoculate the strain NBL-BQ218 in Example 2 into the above artificial gastric juice, and culture it at 37 °C on a shaker (180 rpm). Take samples at 0, 4, 8, and 24 h for plate count detection, and calculate the survival rate.

[0064] Survival rate of the strain (%) = N t / N0 × 100%

[0065] where N t represents the number of viable bacteria at different sampling times; N0 represents the number of viable bacteria at 0 h.

[0066] The test results are shown in Table 2.

[0067] Table 2 Tolerance of strain NBL-BQ218 to gastric acid

[0068] Time (h) 0 4 8 24 Survival rate (%) 100 71 49 35

[0069] As can be seen from Table 2, with the extension of the treatment time in artificial gastric juice, the viable count of strain NBL-BQ218 gradually decreased. After 24 hours of treatment, there was still a survival rate of about 35%, and the viable count remained above 10 8 CFU / mL, indicating that this bacterium can tolerate the gastric acid environment.

[0070] 3.3 Intestinal juice tolerance test

[0071] Preparation of artificial intestinal juice: Dissolve 6.8 g of Na2HPO4 in 500 mL of distilled water, adjust the pH to 6.8 with 0.4% NaOH solution, make up the volume to 1 L, autoclave at 121 °C for 25 min and then cool. Subsequently, add 10 g of trypsin (Amresco). Inoculate strain NBL-BQ218 in Example 2 into the above artificial intestinal juice, culture at 37 °C on a shaker (180 rpm), and take samples at 0, 4, 8, and 24 h for plate count detection to calculate the survival rate.

[0072] Survival rate of strain (%) = N t / N0 × 100%

[0073] where N t represents the viable count at different sampling times; N0 represents the viable count at 0 h.

[0074] The test results are shown in Table 3.

[0075] Table 3 Tolerance of NBL-BQ218 to intestinal juice

[0076] Time (h) 0 4 8 24 Survival rate (%) 100 95 89 79

[0077] As can be seen from Table 3, after 24 hours of treatment with artificial intestinal juice, the viable count of Bacillus remained above 10 8 CFU / mL, and the survival rate remained above 70%, indicating that strain NBL-BQ218 can tolerate artificial intestinal juice and can colonize and play a role in the intestine.

[0078] 3.4 High temperature tolerance test

[0079] Take 5 mL of the culture solution obtained by culturing strain NBL-BQ218 in Example 2 for 24 h and place it in a test tube, and place it in an incubator at 60 °C for treatment. Samples were taken before heat treatment, 40 min and 60 min after heat treatment of the strain for counting, and the survival rate was calculated.

[0080] The results showed that the survival rate of strain NBL-BQ218 was 85.21% after heat treatment at 60 °C for 40 min; and the survival rate was 62.76% after heat treatment at 60 °C for 60 min. This indicates that strain NBL-BQ218 has good high temperature tolerance.

[0081] 3.5 Low-temperature Tolerance Test

[0082] The strain NBL-BQ218 in Example 2 was cultured overnight and inoculated into 100 mL of liquid medium at an appropriate inoculum size (the initial viable cell count was 1×10 6 CFU / mL), and the viable cell count was measured after culturing on a shaker (180 rpm) at 4°C, 10°C, 25°C, and 37°C for 18 h respectively.

[0083] The test results are shown in Table 4. As can be seen from Table 4, when the strain NBL-BQ218 was cultured at 4°C, the viable cell count could be maintained above 10 7 CFU / mL.

[0084] Table 4 Low-temperature Tolerance of Strain NBL-BQ218

[0085]

[0086] Example 4: Determination of Enzyme Activity of the Strain

[0087] The strain NBL-BQ218 in Example 2 was inoculated into a liquid medium (3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 1 L of distilled water, pH 7.2) at an inoculum size of 3%, and cultured at 37°C on a shaker (180 rpm). The amylase (refer to GB 8275-2009) and phytase (GOST 31487-2012) were measured on the 1st day of culture, and the xylanase activity (GOST R 55302-2012) and fructosyltransferase (the amount of enzyme that catalyzes the formation of 1 μM glucose per minute at 55°C and pH 5.5), protease activity (including alkaline protease, acid protease and neutral protease, refer to SB / T10317-1999), cellulase activity (CMC saccharification method), and SOD enzyme activity (Nanjing Jiancheng Bioengineering Institute) were measured on the 2nd day of culture.

[0088] The test results are shown in Table 5.

[0089] Table 5 Enzyme Production Characteristics of Strain NBL-BQ218

[0090]

[0091] Example 5: Determination of the Safety of the Strain

[0092] 1. Hemolysis Test

[0093] The strain NBL-BQ218 in Example 2 was inoculated onto a nutrient agar solid medium containing blood cells (0.8 mL per 10 mL of agar) by streaking, and cultured at 37°C for 1 d, and the presence or absence of a zone phenomenon was observed.

[0094] The results showed that ( Figure 4 ), there was no zone of inhibition around the inoculated strain, and the strain NBL-BQ218 had no hemolytic activity.

[0095] 2. Mouse safety test

[0096] The strain NBL-BQ218 in Example 2 was fermented and spray-dried to form a bacterial powder with a viable count of 100 billion CFU / g. Forty healthy Kunming white mice, 20 males and 20 females, were randomly divided into two groups of 10 according to body weight. The experiment was divided into an experimental group (each mouse was gavaged with 5000 mg / kg of the bacterial powder, administered in two doses within 24 h) and a control group (each mouse was gavaged with an equal volume of normal saline). Before the experiment, the mice were fasted overnight for 16 h without water restriction. After administration, the toxic manifestations and deaths of the animals were immediately observed, and then at least once a day for 14 consecutive days. During the observation period, the body weight and death of the animals were recorded, and the dead animals were autopsied to observe whether the tissues and organs were abnormal.

[0097] The test results are shown in Tables 6 and 7.

[0098] Table 6 Effect of strain NBL-BQ218 on the body weight of mice

[0099]

[0100] Table 7 Effect of strain NBL-BQ218 on poisoning and death of mice

[0101]

[0102]

[0103] As can be seen from Tables 6 and 7, no poisoning symptoms or deaths were observed in the mice during the 14-day observation period after gavage with the bacterial powder, and the animals of each sex showed an increasing trend. After the observation period, the animals were dissected, and no abnormal symptoms were found in the organs and tissues, indicating that the LD 50 of strain NBL-BQ218 > 5000 mg / kg, belonging to the practically non-toxic class.

[0104] Example 6: Bacteriostatic effect test

[0105] The strain NBL-BQ218 in Example 2 was inoculated into 50 mL of NB medium (Qingdao Haibo), cultured at 37 °C on a shaker (180 rpm) for 24 h. After that, it was centrifuged at 5000 rpm for 20 min, and the supernatant was used for the bacteriostatic test.

[0106] Common animal pathogenic bacteria Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, and Aeromonas hydrophila were used as indicator bacteria and were respectively mixed evenly with tryptone soya agar (TSA, Qingdao Haibo) medium at 45°C and poured into petri dishes. After the medium cooled, holes with a diameter of 0.7 cm were punched in the medium using a hole puncher, and 80 μL of the supernatant was added to each hole until it did not overflow. The plates were incubated at 37°C for 24 h, and the size of each inhibition zone was observed and measured.

[0107] The test results are shown in Table 8.

[0108] Table 8 Inhibitory effects of strain NBL-BQ218 on common animal pathogenic bacteria

[0109]

[0110] As can be seen from Table 8, strain NBL-BQ218 has good inhibitory effects on common animal pathogenic bacteria such as Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, and Aeromonas hydrophila.

[0111] Example 7: Determination of the immune level of strain in piglets

[0112] The strain NBL-BQ218 in Example 2 was fermented and spray-dried to form a bacterial powder with a viable count of 100 billion CFU / g.

[0113] Sixty weaned piglets aged (30 ± 2) days with an average body weight of (8.15 ± 0.05) kg were selected and randomly divided into 2 groups: a blank control group and an experimental treatment group. Each group had 5 replicates (half male and half female), with 6 piglets in each replicate, and each replicate was in 1 pen. The blank control group was fed a basal diet; the experimental treatment group was supplemented with NBL-BQ218 bacterial powder in each ton of the basal diet, so that the content of probiotics in the diet was 10 9 CFU / kg. The pre-trial period was 3 d, and the trial period was 28 d. All piglets were raised in a closed pig house with a cement floor, free access to food and water, and deworming, disinfection, and immunization were carried out according to the conventional pig farm feeding management procedures. The basal diet was formulated according to the NRC (2012) standard. The components and contents of the basal diet are shown in Table 9.

[0114] Table 9 Composition and nutrient components of the basal diet (air-dried basis)

[0115]

[0116]

[0117] Note: ① The premix (without antibiotics) provides per kilogram of diet: iron: 110 mg, zinc: 100 mg, copper: 20 mg, manganese: 15 mg, selenium: 0.3 mg, iodine: 0.4 mg, vitamin A: 6000 IU, vitamin D3: 400 IU, vitamin E: 50 IU, vitamin K3: 2 mg, vitamin B1: 3.5 mg, vitamin B2: 5.5 mg, vitamin B6: 3.5 mg, vitamin B 12 : 25.0 μg, biotin: 0.05 mg, folic acid: 0.3 mg, D-pantothenic acid: 20 mg, niacin: 20 mg, choline chloride: 500 mg. ② The calculated value of digestible energy in the nutrient components, and the rest (contents such as crude protein, calcium, total phosphorus, available phosphorus, etc.) are measured values.

[0118] Determination indexes:

[0119] Growth performance: The feed intake was recorded in units of pens throughout the experiment. On the 1st and 28th days, each piglet was weighed on an empty stomach, and the average daily gain (ADG), average daily feed intake (ADFI), and feed-to-gain ratio (F / G) from 1 to 28 days were calculated. Specific calculation methods: ADG = total weight gain of the pen / (number of piglets in the pen × number of experimental days), ADFI = total feed intake of the pen / (number of piglets in the pen × number of experimental days), F / G = ADFI / ADG. The test results are shown in Table 10.

[0120] Diarrhea rate: The diarrhea situation of piglets was observed and recorded every day during the experimental period. Each day of diarrhea for each piglet was recorded as 1 diarrhea episode. After the experiment, the diarrhea rate (DR) of each group was calculated. DR = [total number of diarrhea episodes / (total number of experimental piglets × total number of experimental days)] × 100%. The test results are shown in Table 10.

[0121] Intestinal mucosal sIgA: On the 0th and 28th days, 1 pig was randomly selected from each replicate of each treatment, and 20 cm of the middle segment of the jejunum and ileum was taken. The intestinal tract was cut along the longitudinal axis, and the outer wall and contents were rinsed with ice-cold PBS solution. The intestinal mucus was gently scraped onto a 10 mL centrifuge tube with a glass slide, 2 times the volume of 0.9% physiological saline was added, and it was fully shaken and mixed at low temperature for 30 min. All the supernatant obtained after centrifugation at 12000 g and 4 °C for 20 min was stored at -20 °C for later use. The content of sIgA in the intestinal mucosa was detected using a porcine secretory immunoglobulin A (sIgA) detection kit. The test results are shown in Table 11.

[0122] Table 10 Effects of strain NBL-BQ218 on the growth performance and diarrhea rate of weaned piglets

[0123]

[0124] Note: "*" indicates significant difference from the control group (P < 0.05), the same as the following table.

[0125] As can be seen from Table 10, there were no significant differences in the initial body weights among groups (P>0.05). Compared with the blank control, the final body weights and average daily gains (ADG) of the experimental treatment groups were significantly increased (P<0.05); there were no significant differences in the average daily feed intakes (ADFI) (P>0.05), and the feed-to-gain ratios (F / G) and diarrhea rates were significantly decreased (P<0.05).

[0126] Table 11 Effects of Strain NBL-BQ218 on the Intestinal Mucosal sIgA Levels of Weaned Piglets

[0127]

[0128]

[0129] As can be seen from Table 11, at 28 d, the jejunal sIgA content in the experimental treatment group was 7.92% higher than that in the blank control group, but the difference was not significant (P>0.05); the sIgA content in the ileal contents was significantly higher than that in the blank control group, with a 18.31% increase (P<0.05).

[0130] Example 8: Effects of the Strain on the Growth and Immunity of Broiler Chickens

[0131] 1. Experimental Method

[0132] 1.1 Experimental Grouping

[0133] Healthy 1-day-old broiler chickens were selected and divided into 3 treatment groups according to the principle of no difference in body weight. Each treatment had 3 replicates, and each replicate had 6 chickens. There were a total of 54 chickens, and the pre-feeding period was 7 days.

[0134] The control group (CK) was fed a basal diet;

[0135] The low-dose group was added with 5×10 5 CFU / g of strain NBL-BQ218;

[0136] The high-dose group was added with 1×10 6 CFU / g of strain NBL-BQ218.

[0137] Commercial broiler feed was used as the basal diet, and the chickens had free access to feed and water. The probiotics were added by mixing with the feed. The body weights and feed intakes were recorded weekly. Immunization program: Newcastle disease at 7 days, infectious bursal disease at 14 d.

[0138] 1.2 Sample Collection

[0139] Commercial broiler feed was used as the basal diet, and the chickens had free access to feed and water. The daily feed addition amount was recorded, the water was changed, the experimental feed was mixed every 2 - 3 days, and the feces were cleared. The probiotics were added by mixing with the feed. The body weights and feed intakes were recorded weekly.

[0140] 1.3 Index Detection

[0141] 1.3.1 Growth performance (statistical weekly)

[0142] The experimental broilers are weighed and fed weekly, and the average daily gain (ADG) and feed-to-gain ratio (F:G) of broilers at each stage are statistically analyzed.

[0143] 1.3.2 Slaughter performance

[0144] Dressed weight: The weight after bleeding, removing feathers, the cuticle of feet, toe shells and beak shells is the dressed weight.

[0145] Dressing percentage (%) = dressed weight / live weight × 100%

[0146] Eviscerated weight: The weight after removing the trachea, esophagus, crop, glandular stomach, muscular stomach, abdominal fat, intestines, spleen, pancreas, gallbladder, reproductive organs, head and feet from the carcass. Eviscerated percentage (%) = eviscerated weight / live weight × 100%

[0147] Breast muscle percentage: Cut the skin along the sternal ridge and peel it towards the back, cut off the muscle attached to the side of the sternal ridge and the tendon of the scapular part with a knife, then the whole peeled breast muscle can be peeled off and weighed. Breast muscle percentage = bilateral breast muscle / live weight × 100%

[0148] 1.3.3 Immunity determination

[0149] (1) Immune organ index

[0150] After the experimental chickens are slaughtered, the thymus, spleen and bursa of Fabricius are accurately removed, the excess tissue is removed and weighed fresh, and the immune organ index is calculated according to the weight of each immune organ and the live weight before slaughter. The calculation formula is as follows:

[0151] Immune organ index (g / kg) = immune organ weight (g) / live weight before slaughter (kg)

[0152] (2) Serum immune factors (ELISA detection kit)

[0153] Immune factors: IgG, IgA, IgM

[0154] 1.3.4 Serum biochemical indexes

[0155] After the feeding experiment is completed, about 5 mL of venous blood under the wing is collected, left standing at 37 °C for 30 min, centrifuged at 3000 r / min and 4 °C for 15 min to separate the serum, and the contents or activities of serum albumin (ALB), total cholesterol (TC), glucose (GLU), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea (UREA), total protein (TP) and creatinine (CREA) are detected using an automatic biochemical analyzer.

[0156] 2. Test Results

[0157] 2.1 Effects of Different Test Strains on Growth Performance of Broiler Chickens

[0158] Table 12 Growth Performance of Test Broiler Chickens during the Whole Period

[0159]

[0160] Note: Different superscript letters in the same column indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0161] As can be seen from Table 12, in the low-dose group (5×10 5 CFU / g) with the addition of NBL-BQ218, the feed-to-meat ratio was the lowest, significantly lower than that of the control group, the average daily gain was the highest, and the growth performance was the best.

[0162] 2.2 Effects of Different Test Strains on Slaughter Performance of Broiler Chickens

[0163] Table 13 Slaughter Performance of Test Broiler Chickens

[0164]

[0165] Note: Different superscript letters in the same column indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0166] As can be seen from Table 13, with the addition of strain NBL-BQ218, compared with the control group, although the slaughter rate was slightly lower, the eviscerated yield and breast muscle rate were both higher than those of the control group. The eviscerated yield of the high-dose group of NBL-BQ218 was significantly higher than that of the control group by 6.37%.

[0167] 2.3 Effects of Different Test Strains on Immune Organ Index of Broiler Chickens

[0168] Table 14 Immune Organ Index of Test Broiler Chickens

[0169]

[0170] Note: Different superscript letters in the same column indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0171] As can be seen from Table 14, the thymus and spleen indices were both higher with the addition of NBL-BQ218. Among them, the spleen index of the low-dose group was significantly higher than that of the control group, and the thymus index of the high-dose group was the highest.

[0172] 2.4 Effects of Different Test Strains on Relative Intestinal Length of Broiler Chickens

[0173] Table 15 Relative Intestinal Length of Test Broiler Chickens

[0174]

[0175] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), and the same superscript letters or no letters indicate no significant differences (P > 0.05).

[0176] After adding strain NBL-BQ218, the relative length of the duodenum was higher than that of the control group in all cases; especially in the low-dose group, the relative lengths of the duodenum, jejunum, and ileum were the highest.

[0177] 2.5 Effects of different test strains on immune factors in broiler serum

[0178] Table 16 Levels of immune factors in the serum of experimental broilers

[0179]

[0180] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), and the same superscript letters or no letters indicate no significant differences (P > 0.05).

[0181] As can be seen from Table 16, generally, adding strain NBL-BQ218 can increase the levels of IgG, IgA, and IgM.

[0182] 2.7 Effects of different test strains on blood glucose and blood lipids in broilers

[0183] Table 17 Effects of different test strains on blood glucose and blood lipids in experimental broilers

[0184]

[0185] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), and the same superscript letters or no letters indicate no significant differences (P > 0.05).

[0186] As can be seen from Table 17, compared with the control group, the blood glucose content in each test group increased, and the total cholesterol (TC) content decreased, but there were no significant differences.

[0187] With the increase of the addition amount, the triglyceride (TG) content in the serum decreased. The TG content in the high-dose group of NBL-BQ218 was significantly lower than that of the control group, indicating a certain ability to reduce blood lipids.

[0188] 2.8 Effects of different test strains on serum protein content in broilers

[0189] Table 18 Effects of different test strains on serum protein content in experimental broilers

[0190]

[0191] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0192] Total protein includes albumin and globulin. There was no significant difference in serum protein levels between the experimental group and the control group, indicating that the test strains had no adverse effects on the protein metabolism of broilers.

[0193] 2.9 Effects of different test strains on the liver and kidney functions of broilers

[0194] Table 19 Liver function indexes of experimental broilers

[0195]

[0196] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0197] Higher levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in liver function indicate liver abnormalities, while a decrease has no pathological significance. As shown in Table 19, there were no significant differences between each group of NBL - BQ218 and the control group.

[0198] Table 20 Kidney function indexes of experimental broilers

[0199]

[0200]

[0201] Note: Different superscript letters in the same column of data indicate significant differences (P < 0.05), while the same superscript letters or no superscript letters indicate no significant differences (P > 0.05).

[0202] CREA is a metabolite of muscle metabolism in the body. CREA in the blood is mainly filtered by the kidneys and excreted through urine. The level of CREA represents kidney function. UREA can reflect the digestion and absorption of protein by animals and the utilization of nitrogen. An increase in creatinine (CREA) and blood urea nitrogen (UREA) in kidney function indicates problems with the kidney's detoxification function and kidney function impairment, while a decrease has no pathological significance. The results showed that there were no significant differences in CREA and UREA between each experimental group and the control group.

[0203] Example 9: Test on the immune enhancement effect of strains

[0204] The strain NBL - BQ218 in Example 2 was fermented and spray - dried to form a bacterial powder with a viable count of 100 billion CFU / g.

[0205] Sixty 1-day-old Sanhuang chickens were randomly divided into 2 groups, with 30 chickens in each group. The control group was fed a basal diet, and the experimental group was supplemented with 10 6 CFU / g of bacterial powder. From 1 to 3 weeks of age: crude protein 20.79%, metabolic energy 12.26 MJ / kg, calcium 0.94%, phosphorus 0.64%, available phosphorus 0.48%, lysine 1.10%, methionine 0.44%, crude fiber 4.66%; at 4 weeks of age: crude protein 18.30%, metabolic energy 12.43 MJ / kg, calcium 0.88%, phosphorus 0.63%, available phosphorus 0.46%, lysine 0.95%, methionine 0.35%, crude fiber 5.0%.

[0206] The Sanhuang chickens were fed in cages, and the temperature was controlled at about 30 °C with incandescent lamps. At 7 days of age, the chickens were vaccinated for the first time by intranasal drip with Newcastle disease vaccine strain La Sota, and the second vaccination was carried out at 21 days of age.

[0207] When the chicks were fed to 28 days of age, 5 chickens were randomly selected from each group, bled to death, 5 - 10 mL of blood was collected, the thymus, spleen, and bursa of Fabricius were quickly removed, connective tissue was removed, the blood was blotted dry, and weighed. The blood was separated to obtain serum, and the hemagglutination inhibition titer against Newcastle disease was measured; the organ coefficients were calculated for the thymus, spleen, and bursa of Fabricius (organ coefficient = wet weight of organ: body weight).

[0208] The test results are shown in Tables 21 and 22.

[0209] Table 21 Effects of the strain on the immune organs of broilers

[0210]

[0211]

[0212] As can be seen from Table 21, the immune organ indices of the experimental group were all higher than those of the control group. Among them, the thymus index increased by 10.3%, the spleen index increased by 18.2%, and the bursa of Fabricius index increased by 11.2%.

[0213] Table 22 Effects of the strain on the hemagglutination inhibition titer of broilers against Newcastle disease

[0214] Group X±SE Xtest - Xcontrol Treatment group 4.12±0.55 1.17 Control group 2.95±0.43 /

[0215] As can be seen from Table 22, the hemagglutination inhibition titer of the experimental group against Newcastle disease was higher than that of the control group, and the speed of producing specific antibodies was faster than that of the control group.

[0216] Example 10: NBL-BQ218 enhances the antibody titer against Newcastle disease

[0217] 1. Experimental animals: 60 7-day-old laying hens

[0218] 2. Materials and Reagents: 96-well V-shaped microtiter plates, micro-oscillators, 1% red blood cell suspension, normal saline, micropipettes, syringes, anticoagulants, centrifuges, 37°C incubators, sera of chickens to be tested, formalin, SPF chicken embryos, etc.; Newcastle disease LaSota strain live attenuated vaccine; Newcastle disease hemagglutination antigen, etc.

[0219] 3. Antigen Preparation: Inoculate the Newcastle disease LaSota strain into 9 - 11-day-old chicken embryos, harvest allantoic fluid, and after detecting that the hemagglutination titer (HA) ND ≥ 1∶640 and the virus content in the allantoic cavity injection infection dose ≥ 10 6 EID 50 / 0.1 mL, it can be used for the test.

[0220] 4. Antigen Inactivation: Add formalin with a concentration of 0.12% to the harvested allantoic fluid, mix evenly and incubate at 37°C for 48 h.

[0221] 5. Preparation of Inactivated Bacillus subtilis Bacterial Solution: Inoculate the strain NBL-BQ218 in Example 2 into LB medium, shake culture at 37°C for 12 h. After culturing for 3 generations, calculate the bacterial solution concentration by colony plate counting method. Then inactivate it by autoclaving for 40 min and resuspend it with PBS.

[0222] 6. Test Grouping and Immunization Treatment: Randomly divide 60 7-day-old laying hens (the average maternal antibody of Newcastle disease is 4.1 log2) into 4 groups, with 15 hens in each group, namely: control group, virus group, virus + adjuvant group, virus + inactivated bacteria group. Among them, each hen in the control group is inoculated with 0.2 mL PBS; each hen in the virus group is inoculated with a mixture of 0.1 mL inactivated allantoic fluid (10 6 EID 50 ) and 0.1 mL PBS; each hen in the virus + adjuvant group is inoculated with a mixture of 0.1 mL inactivated allantoic fluid (10 6 EID 50 ) and 0.1 mL Freund's adjuvant; each hen in the virus + inactivated bacteria group is inoculated with a mixture of 0.1 mL inactivated allantoic fluid (10 6 EID 50 ) and 0.1 mL inactivated bacterial solution (10 9 CFU / mL). Each group is immunized by intramuscular injection. The first immunization is carried out at 7 days old for each group, and the second immunization is carried out at 21 days old.

[0223] 7. Antibody Titer Detection: Blood is collected from the wing vein of each group at 7 days (first immunization), 21 days (second immunization), 28 days, 35 days, and 42 days respectively, and 5 hens are randomly selected from each group. Antibody detection is carried out according to the method of micro-hemagglutination inhibition test (HI) for Newcastle disease virus.

[0224] 8. Test Results:

[0225] Average Newcastle disease antibody titer (log2) of each group and each period in Table 23

[0226] Grouping 7d 21d 28d 35d 42d Control group <![CDATA[3.6±0.548 a > <![CDATA[2.8±0.447 c > <![CDATA[2.2±0.447 c > <![CDATA[0.6±0.548 c > <![CDATA[0 c > Virus group <![CDATA[4.0±0 a > <![CDATA[4.4±0.548 b > <![CDATA[5.4±0.548 b > <![CDATA[7.0±0 b > <![CDATA[7.8±0.447 b > Virus + adjuvant group <![CDATA[3.6±0.548 a > <![CDATA[5.2±0.447 a > <![CDATA[6.2±0.447 a > <![CDATA[7.4±0.548 b > <![CDATA[8.4±0.548 ab > Virus + inactivated bacteria group <![CDATA[3.8±0.447 a > <![CDATA[5.4±0.548 a > <![CDATA[6.4±0.548 a > <![CDATA[8.2±0.447 a > <![CDATA[8.8±0.837 a >

[0227] From the detection results of the average antibody titer, it can be seen that the antibody level in the control group gradually decreased and dropped to 0 at 42 d; since 21 d, the antibody titers in the virus + adjuvant group and the virus + inactivated bacteria group were significantly higher than those in the pure virus group, indicating that both Freund's adjuvant and inactivated bacteria solution could play an immune-enhancing role. At 35 d, the antibody level in the virus + inactivated bacteria group was significantly higher than that in the virus + adjuvant group, suggesting that inactivated bacteria could mediate immune response faster than Freund's adjuvant, enhance the antibody level, and play a better protective role.

[0228] Example 11: Study on enhancing the titer of avian influenza virus by NBL-BQ218

[0229] 1. Experimental animals: 30 14-day-old AA broilers

[0230] 2. Materials and reagents: 96-well V-shaped microtiter plates, 1% erythrocyte suspension, physiological saline, micropipettes, syringes, centrifuges, 37 °C incubators, formalin, SPF chicken embryos, etc.; avian influenza H9 subtype virus; H9 hemagglutination antigen, etc.

[0231] 3. Antigen preparation: Dilute the avian influenza H9 virus 1000-fold, inoculate 9- to 11-day-old chicken embryos, harvest the allantoic fluid, and after detecting that the virus content in the allantoic cavity injection infection dose ≥ 10 6 EID 50 / 0.1 mL, it can be used for the experiment.

[0232] 4. Antigen inactivation: Add formalin with a concentration of 0.12% to the harvested allantoic fluid, mix evenly, and incubate at 37 °C for 48 h.

[0233] 5. Preparation of inactivated Bacillus subtilis bacterium solution: Inoculate Bacillus subtilis in LB medium, shake culture at 37 °C for 12 h, calculate the bacterium solution concentration by colony plate counting method after culturing for 3 generations. Then inactivate by autoclaving for 40 min and resuspend with PBS.

[0234] 6. Experimental grouping and immunization treatment: Randomly divide 30 14-day-old AA broilers into 3 groups, with 10 in each group, namely: control group, virus group, virus + inactivated bacteria group. Among them, each chicken in the control group was inoculated with 0.2 mL of PBS; each chicken in the virus group was inoculated with a mixture of 0.1 mL of inactivated antigen (10 6 EID 50 ) and 0.1 mL of PBS; each chicken in the virus + inactivated bacteria group was inoculated with a mixture of 0.1 mL of inactivated antigen (10 6 EID 50 ) and 0.1 mL of inactivated bacterium solution (109 A mixed solution of (CFU / mL). Each group was immunized by intramuscular injection. The first immunization was carried out at 14 days old in each group, and the second immunization was carried out at 28 days old.

[0235] 7. Antibody titer detection Blood was collected from the wing vein of each group at 14 days after the first immunization, 28 days after the second immunization, 35 days, and 42 days. 5 were randomly selected from each group, and antibody detection was carried out with reference to the hemagglutination inhibition test (HI) method.

[0236] 8. Test results

[0237] Table 24 Average H9 antibody titers (log2) of each group at each stage

[0238]

[0239]

[0240] The results showed that the antibody titers of the virus + inactivated bacteria group were higher than those of the pure virus group, and the differences were significant at 35 days and 42 days, indicating that the inactivated bacteria solution could indeed play an immune-enhancing role.

[0241] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis ), which was named Bacillus subtilis NBL-BQ218, and its deposit number is CCTCC NO: M 20232183.

2. A microbial agent, characterized in that: The invention comprises the Bacillus subtilis described in claim 1.

3. Use of the Bacillus subtilis according to claim 1 or the microbial agent according to claim 2 in the preparation of any of the following products: (1) Products used for the production of xylanase, fructosyltransferase, protease, amylase, cellulase, phytase and SOD; (2) Products used to inhibit pathogenic bacteria in the intestines of livestock and poultry; (3) Products used to increase the intestinal length of livestock and poultry and improve the digestion and absorption rate of feed; (4) Products used to improve the immunity of livestock and poultry; (5) Products used as vaccine adjuvants for livestock and poultry; in, The livestock and poultry intestinal pathogens are Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens and Aeromonas hydrophila.

4. The use according to claim 3, characterized in that The improving the immunity of livestock and poultry includes increasing the average daily weight gain, average daily feed intake and feed-to-weight ratio of livestock and poultry, reducing the diarrhea rate of livestock and poultry, increasing the level of immunoglobulin in livestock and poultry, and improving immunity.

5. The use according to claim 3, characterized in that The vaccine adjuvant can be used in combination with a vaccine to enhance the immune response, reduce the amount of antigen used, efficiently activate the immune system and prolong the potency level.

6. A product, characterized in that Include any of the following: (1) The Bacillus subtilis according to claim 1; (2) The microbial agent according to claim 2.

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