Bacillus subtilis and application thereof

Abstract

The invention discloses a bacillus subtilis strain and application thereof. Belongs to the technical field of probiotic research and development. According to the invention, a strain of bacillus subtilis is identified through traditional isolated culture and morphological, physiological and biochemical and molecular biological methods, and probiotic performance evaluation is carried out on the bacillus subtilis by combining in-vitro and in-vivo tests. An in-vitro test result shows that the strain is resistant to acid (the lowest pH is 2) and salt (the highest NaCl concentration is 10%), does not hemolysis, inhibits pathogenic bacteria and is sensitive to most antibiotics; an in-vivo test result shows that the production performance of animals can be improved, the absorption of small intestines to nutrient substances is promoted, and the diversity of dominant flora in intestinal tracts is increased. According to the invention, not only can the research on bacillus subtilis be expanded, but also a reference basis can be provided for the development and research of probiotic preparations for antagonizing pathogenic bacteria, regulating intestinal microecological balance and the like in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic research and development, and more specifically to a strain of Bacillus subtilis and application thereof. Background Art

[0002] The use of antibiotics and chemical drugs is an important means to inhibit the spread of pathogens and alleviate symptoms. Their widespread use has led to an increase in drug-resistant bacteria, drug residues, and a decline in the quality of the ecological environment, posing an increasingly severe threat and challenge to human health. Probiotics are gradually showing great potential that antibiotics do not have. They not only play a key role in maintaining intestinal health and enhancing the body's immunity, but also become potential substitutes for antibiotics due to their natural and side-effect-free characteristics. They are expected to be widely used in multiple industries such as medical health, food additives, biopesticides and biofertilizers, and environmental governance, and become an important member of disease prevention and control strategies. Therefore, by promoting ecological balance and inhibiting the growth of harmful bacteria, probiotics are expected to provide us with a safer and more effective health protection barrier.

[0003] Bacillus subtilis is a ubiquitous Gram-positive probiotic. As a biocontrol agent, it has attracted widespread attention by inhibiting plant pathogens and promoting plant growth, playing the role of biological pesticides and biological fertilizers in agriculture. Bacillus subtilis colonizes in the intestines, which can effectively prevent pathogens from binding to intestinal mucosal receptors, inhibit the spread of pathogens, and stabilize the intestinal flora. Since Bacillus subtilis produces various volatile fatty acids in physiological metabolism, it provides an acidic environment for the gastrointestinal tract of animals, increases the activity of gastric proteases, hinders the growth of pathogens, and improves the production performance and disease resistance of poultry and livestock. Bacillus subtilis is widely present in soil, plant rhizosphere and leaf sphere, and on the surface and body of animals and plants. It has antibacterial activity and strong stress resistance. It shows good anti-disease and anti-bacterial and stress resistance effects in the fields of crop planting and poultry and livestock breeding. It secretes digestive enzymes, vitamins or antibacterial substances in the animal intestine or environment, regulates intestinal flora, inhibits pathogenic microorganisms, and improves animal growth performance.

[0004] In summary, how to provide a Bacillus subtilis and develop its probiotic function is a scientific problem that technicians in this field need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a Bacillus subtilis strain and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Bacillus subtilis, named HCD03, whose classification name is Bacillus subtilis, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, with the deposit number CGMCC No.31448, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, it is bovine Bacillus subtilis.

[0009] Furthermore, the lowest pH tolerated by the Bacillus subtilis is 2, the highest pH tolerated is 12, the highest salt concentration tolerated is 10%, and the highest temperature tolerated is 95°C.

[0010] The application of the above-mentioned Bacillus subtilis in inhibiting Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae.

[0011] The use of the above-mentioned Bacillus subtilis in the preparation of probiotics.

[0012] Furthermore, the probiotic is used to increase the feed-to-weight ratio, increase the diversity index of intestinal flora and the number of dominant groups.

[0013] A probiotic agent comprises the above-mentioned Bacillus subtilis.

[0014] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0015] The present invention identifies a strain of Bacillus subtilis by traditional separation and culture and morphological, physiological and biochemical and molecular biological methods, and evaluates its probiotic properties in vitro and in vivo. The in vitro test results show that the bacteria are acid-resistant (the lowest pH tolerated is 2) and salt-resistant (the highest NaCl concentration is 10%), non-hemolytic, inhibit Staphylococcus aureus (ATCC29213), Streptococcus agalactiae (BNCC185941) and Streptococcus dysgalactiae (BNCC337480), and are sensitive to most antibiotics; the in vivo test results show that adding the bacteria can improve the production performance of animals, promote the absorption of nutrients by the small intestine, and increase the diversity of the dominant intestinal flora. The present invention can not only expand the research on Bacillus, but also provide a reference basis for the future development of probiotic preparations for antagonizing pathogens and regulating the balance of intestinal microecology. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 It is the technical roadmap of the present invention;

[0018] Figure 2 This is a colony morphology diagram of the isolated and purified strain HCD03 on a solid medium containing TSB and agar in Example 1 of the present invention;

[0019] Figure 3 The figure is the Gram staining microscopic examination result (10×100) of the isolated and purified strain HCD03 in Example 1 of the present invention;

[0020] Figure 4 The agarose gel electrophoresis diagram of the 16S rRNA gene PCR amplification product of the strain HCD03 in Example 1 of the present invention, wherein M: DL2000 Marker; 1: 16S rRNA gene amplification product of the strain HCD03 (fragment size is about 1500bp); 2: negative control;

[0021] Figure 5 The NJ phylogenetic tree of strain HCD03 based on 16S rRNA gene sequence in Example 1 of the present invention;

[0022] Figure 6 This is the hemolysis test result of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0023] Figure 7 is the growth curve of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0024] Figure 8 The optimum pH test result for the growth of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0025] Fig. 9 The salt tolerance test result of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0026] Fig.10 The results of the high temperature resistance test of Bacillus subtilis HCD03 in Example 2 of the present invention are as follows;

[0027] Fig.11The results of the average daily weight gain test of mice in the probiotic group and the control group added with Bacillus subtilis HCD03 in Example 3 of the present invention, wherein ns means that there is no statistically significant difference between the probiotic group and the control group (P>0.05);

[0028] Fig.12 The test results of the feed weight ratio (F / G) of the probiotic group to which Bacillus subtilis HCD03 was added and the control group in Example 3 of the present invention, wherein P<0.05 represents that there is a statistically significant difference between the probiotic group and the control group;

[0029] Fig.13 This is the organ coefficient result of adding Bacillus subtilis HCD03 in Example 3 of the present invention;

[0030] Fig.14 The effect of the probiotic group containing Bacillus subtilis HCD03 in Example 3 of the present invention on the villus length and crypt depth of the mouse intestine (ileum);

[0031] Fig.15 The Shannon index and Simpson index of the fecal flora of mice in the probiotic group to which Bacillus subtilis HCD03 was added in Example 3 of the present invention were analyzed, wherein P<0.05 represented that there was a statistically significant difference between the probiotic group and the control group;

[0032] Fig.16 This is the result of the LEfSe phylogenetic analysis of the dominant flora in mouse feces in the probiotic group to which Bacillus subtilis HCD03 is added in Example 3 of the present invention. Orange represents the significantly dominant flora in the probiotic group to which Bacillus subtilis is added, and green represents the significantly dominant flora in the control group to which Bacillus subtilis is not added. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0035] Example 1

[0036] Isolation, identification and preservation of strains

[0037] (1) Separation and purification

[0038] Use a sterile cotton swab to dip an appropriate amount of cow dung on the solid culture medium, dilute it with physiological saline with a pipette, and spread it evenly with a spreader, and culture it at a constant temperature overnight at 37°C. The next day, use a sterile inoculation loop to pick colonies with consistent morphology and absolute dominance on TSB (with agar) solid culture medium for streak purification. After a single colony appears on the culture medium, perform Gram staining on it, and repeat this process until the staining results reach a consistent bacterial morphology and color. After picking a fresh bacterial lawn of a single bacterium to expand the culture in TSB liquid culture medium, use a pipette to draw 700μL of bacterial solution into a storage tube, add 700μL of 30% glycerol, and finally store it in a -40°C freezer, named HCD03.

[0039] (2) Morphological identification

[0040] The isolated and purified strain HCD03 was cultured under appropriate conditions, the phenotypic characteristics of the colonies were observed with the naked eye, and the morphology, color and size were recorded; the isolated strain HCD03 was Gram-stained and then observed under an optical microscope for cell morphological characteristics.

[0041] Depend on Figure 2 It can be seen that the colony morphology of the strain on TSB (with agar) solid medium is round, with irregular edges, rough surface, and grayish white to grayish yellow. Figure 3 It can be seen that the screened strain HCD03 was Gram-stain positive and the microscopic results showed that it was oval or short rod-shaped.

[0042] (3) Bacterial 16S rRNA gene sequencing and identification

[0043] The genomic DNA of HCD03 strain was extracted according to the instructions of TIANamp Bacteria DNA Kit. The genomic DNA of HCD03 strain was used as a template and the universal primers 27F and 1492R of bacterial 16S rRNA gene were used for PCR amplification.

[0044] Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO. 1;

[0045] Downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2.

[0046] The 16S rRNA gene PCR amplification product was detected by 1% agarose gel electrophoresis, and the target DNA band was successfully amplified, with a size of about 1500 bp ( Figure 4 ).

[0047] The length of the sequence obtained by sequencing the 16S rRNA gene of the HCD03 strain was 1494 bp. The sequence was compared by BLAST and the NJ (Neighborjoining) phylogenetic tree was constructed using MEGA X software. It was found that the homology between the HCD03 strain and Bacillus subtilis IAM 12118, DSM 10, NBRC 13719, and JCM 1465 was greater than 99% ( Figure 5 ), indicating that the HCD03 strain is Bacillus subtilis.

[0048]

[0049] (4) Physiological and biochemical identification

[0050] The biochemical tests of the HCD03 isolated strain were carried out with reference to the Common Bacterial Identification Manual and the instructions for the microbiochemical identification tube. The main tests included glycolysis test, urea test, lysine decarboxylase test, hydrogen sulfide test, Simon's citrate test, hydrogen peroxide gas production test, glucose gas production test and MR-VP test. The phenotype of strain HCD03 was further identified based on the test results.

[0051] Table 1 Physiological and biochemical identification results of HCD03 isolated strains

[0052]

[0053] Note: “+” means positive, “-” means negative.

[0054] The results of physiological and biochemical identification showed that the HCD03 isolated strain could ferment glucose, sucrose, mannitol, mannose, fructose, salicin, esculin, and urease, but could not ferment muscarin, lactose, and maltose, and did not form indole. It could hydrolyze starch and reduce nitrate, but could not utilize Simon's citrate and citrate. It produced gas in the hydrogen peroxide test, was positive in the VP test, and was negative in the methyl red reaction, all of which were consistent with the phenotypic characteristics of Bacillus.

[0055] (5) Preservation

[0056] The Bacillus subtilis is named HCD03, and its classification name is Bacillus subtilis. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on July 24, 2024, with the deposit number CGMCC No. 31448, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0057] Example 2

[0058] Evaluation of probiotic properties of strains in vitro

[0059] (1) Hemolysis test

[0060] A single colony of Bacillus subtilis HCD03 was selected, and fresh bacterial moss was picked up with a sterile inoculation loop on the blood agar medium and streaked on the plate. The plate was inverted and placed in a constant temperature incubator (37° C.) for 24 hours to observe whether there was hemolysis.

[0061] The morphology of Bacillus subtilis HCD03 on blood agar medium is as follows Figure 6 As shown, no hemolytic zone appeared, indicating that Bacillus subtilis HCD03 was not hemolytic.

[0062] (2) Growth curve determination

[0063] The Bacillus subtilis HCD03 bacterial solution was inoculated into TSB liquid medium at a ratio of 1%, and cultured at a constant temperature (37°C, 200 rpm). 200 μL of the bacterial solution was taken every 2 hours and the OD was measured using an enzyme marker. 600 Values ​​are used to draw growth curves.

[0064] Depend on Figure 7 It can be seen that Bacillus subtilis HCD03 grew slowly from 0 to 2 hours and was in the lag phase; it grew rapidly after 2 hours, and the OD 600 The value increased rapidly and entered the logarithmic growth phase; after 6 hours, the growth slowed down and gradually entered the stable phase; after 24 hours, the growth of Bacillus subtilis HCD03 decreased and entered the decline phase.

[0065] (3) Drug sensitivity test

[0066] The drug sensitivity test adopts the Kirly-Baue method, and β-lactams, quinolones, macrolides, tetracyclines, lincosamides, aminoglycosides, chloramphenicol, etc. are selected for drug resistance and drug sensitivity tests. 70 μL of Bacillus subtilis HCD03 culture fluid is evenly spread on TSB solid medium until the bacterial solution is observed to be fully absorbed, and then the drug sensitivity sheet is pasted with tweezers. Three parallel drug sensitivity sheets for each drug are set up, and the diameter of the inhibition zone is measured with a vernier caliper after inverted culture in a 37°C constant temperature incubator for one day (24h). The test results obtained are compared with the "Standard for Determining the Diameter of the Inhibition Zone of the Paper Method Drug Sensitivity Test", and the results are evaluated as sensitive (S), intermediate (I), and resistant (R).

[0067] Table 2 Bacillus subtilis HCD03 drug sensitivity test results

[0068]

[0069]

[0070] As shown in Table 2, Bacillus subtilis HCD03 was resistant to azithromycin; it was intermediate to penicillin G, cefotaxime, amoxicillin, chloramphenicol, erythromycin, midecamycin, doxycycline, streptomycin, kanamycin, neomycin, and gentamicin; and it was sensitive to cefradine, ceftriaxone, cephalexin, quinolones, clindamycin, and spectinomycin.

[0071] (4) Antibacterial test

[0072] The antibacterial test used the Oxford cup agar diffusion method. First, the activated Bacillus subtilis HCD03 bacterial solution was inoculated into the corresponding liquid culture medium at a ratio of 1%, and then cultured in a constant temperature incubator for 24 hours. The next day, centrifuged for 10 minutes to prepare the cell-free supernatant. The five indicator bacteria selected in the experiment were Staphylococcus aureus ATCC29213, Streptococcus agalactiae BNCC185941, Streptococcus dysgalactiae BNCC337480, Salmonella CVCC2184 and Escherichia coli ATCC25922; First, the indicator bacteria were cultured and the OD of the indicator bacteria was adjusted. 600 is 0.5; 70 μL of indicator bacteria were spread on TSB agar solid culture medium respectively; an Oxford cup was placed on each culture medium, 200 μL of supernatant of Bacillus subtilis HCD03 was added to the Oxford cup, and finally it was placed in a constant temperature incubator for 24 hours, and the diameter of the inhibition zone was measured.

[0073] The diameter of the inhibition zone of Bacillus subtilis HCD03 against Staphylococcus aureus is about 15.0 mm, the diameter of the inhibition zone against Streptococcus agalactiae is about 20.0 mm, and the diameter of the inhibition zone against Streptococcus dysgalactiae is about 12.0 mm. It has no inhibitory ability against other reference bacteria.

[0074] (5) Acid resistance test

[0075] The overnight cultured Bacillus subtilis HCD03 was inoculated into TSB liquid culture medium with different pH values ​​(i.e., 2, 3, 4) at a 1% inoculation amount, and cultured at constant temperature (37° C., 200 rpm) for 24 hours to observe whether the culture medium became turbid. The survival rate of the strain was calculated according to the following formula.

[0076] Survival rate = OD of treated culture medium 600 Value / OD of untreated culture medium 600 Value × 100%

[0077] (OD of untreated medium 600 value = 0.350)

[0078] Table 3 Bacillus subtilis HCD03 acid resistance test results

[0079]

[0080] The results are shown in Table 3. Under the strong acidic environment with a pH value of 2 to 4, the survival rate of Bacillus subtilis HCD03 was 16.94 to 17.89%, indicating that Bacillus subtilis HCD03 has acid resistance.

[0081] (6) Optimal growth pH range test

[0082] The overnight cultured Bacillus subtilis HCD03 was inoculated into TSB liquid medium with different pH values ​​(i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) at a 1% inoculum, and the OD was measured after culturing at constant temperature and shaking (37°C, 200 rpm) for 24 h. 600 value.

[0083] like Figure 8 As shown, the optimal pH range for the growth of Bacillus subtilis HCD03 is 5.0-9.0, and its proliferation ability is significantly inhibited in an acidic environment of pH 2 to 4 and an alkaline environment of pH 10 to 12.

[0084] (7) Salt resistance test

[0085] TSB liquid culture medium containing NaCl was prepared respectively, and the concentration gradient was set to 2%, 4%, 6%, 8% and 10%, and high pressure steam sterilization (121°C, 15min) was performed after preparation. Bacillus subtilis HCD03 was inoculated into the corresponding culture medium at an inoculum rate of 1%, and the OD of the bacterial solution was measured after constant temperature culture (37°C, 200rpm) for 24h. 600 value.

[0086] like Fig. 9 As shown, with the increase of NaCl concentration gradient, the turbidity of the bacterial culture of Bacillus subtilis HCD03 showed an overall downward trend, and turbidity was produced in the liquid culture medium containing NaCl, indicating that it was able to grow and reproduce, indicating that Bacillus subtilis HCD03 has a certain salt tolerance.

[0087] (8) High temperature resistance test

[0088] 50mL of overnight cultured Bacillus subtilis HCD03 bacterial solution was divided into 5 clean test tubes, each tube containing 10mL of bacterial solution, and then the bacterial solution of each 10mL test tube was further divided into 4 test tubes, each tube 2.5mL (each 4 tubes were designed as a group, a total of 5 groups of test tubes). Take any 3 test tubes (three replicates) from each group of test tubes, and place them in a constant temperature water bath at 80, 85, 90, 95, and 100℃ for 30min (treatment group), and keep 1 test tube in each group at room temperature as a control group. After the water bath, use a pipette to draw 20μL of bacterial solution and spread it on TSB nutrient agar medium, and add 2dNaCl (to adjust the osmotic pressure of the medium) at the same time. After the coating is completed, the TSB nutrient agar medium is placed in a constant temperature incubator at 37℃ for 24h. The above experiment was repeated three times, and the survival rates of Bacillus subtilis HCD03 at 80, 85, 90, 95, and 100°C were calculated.

[0089] Survival rate / % = (number of viable bacteria in the treatment group ÷ number of viable bacteria in the control group) × 100%

[0090] like Fig.10 As shown, the survival rate of Bacillus subtilis HCD03 decreased with increasing temperature. The survival rate was 16% at 90°C and was only 2% when the temperature increased to 100°C.

[0091] (9) Adhesion performance measurement

[0092] ①Self-cohesion test

[0093] After incubating Bacillus subtilis HCD03 in TSB medium at 37°C for 24 h, the cells were collected by centrifugation at 4°C and 8000 r / min for 10 min, and then washed twice with sterilized PBS (pH 7.4) and centrifuged under the same conditions as above. Finally, the cells were resuspended in PBS to an initial OD of 1.0 600 The value was 0.5±0.02. The bacterial suspension was placed at 37°C for 24 hours, and the supernatant was taken at 0, 2, 4, and 24 hours respectively. Sterile PBS was used as a blank control, and the absorbance was detected at a wavelength of 600 nm. The self-aggregation ability of Bacillus subtilis HCD03 was calculated according to the following formula.

[0094] Self-cohesion = (A0-A t ) / A0×100%

[0095] Where: A0 is OD at 0h 600 , A t OD when th 600

[0096] The self-aggregation classification levels of bacteria are: low (below 35%), medium (35%-50%) and high (above 50%).

[0097] Table 4 Bacillus subtilis HCD03 self-aggregation test results

[0098]

[0099] As shown in Table 4, the self-aggregation ability of Bacillus subtilis HCD03 in 2 h was 60%, which belongs to the high self-aggregation range, indicating that Bacillus subtilis HCD03 has self-aggregation ability.

[0100] ②Surface hydrophobicity test

[0101] The same treatment as in the autoaggregation test was used to make the initial OD of the bacterial suspension 600 The value was 0.5±0.02. Take an appropriate amount of bacterial suspension, add an equal volume of xylene, vortex for 2 minutes, place at room temperature at 25℃ for 30 minutes, separate into two phases, use sterile PBS as the blank group, and detect the absorbance of the lower aqueous phase at a wavelength of 600nm.

[0102] Surface hydrophobicity = (A0-A t) / A0×100%

[0103] Where: A0 and A t Indicates the OD of the water phase before and after mixing with xylene 600 value

[0104] A hydrophobicity of 40% or more was judged to be good.

[0105] Table 5 Surface hydrophobicity test results of Bacillus subtilis HCD03

[0106]

[0107] As shown in Table 5, the hydrophobicity of Bacillus subtilis HCD03 can reach 78.52%, which is a highly hydrophobic strain.

[0108] Example 3

[0109] Evaluation of probiotic properties of strains in vivo

[0110] Experimental animals: 4-6 week old SPF grade Kunming mice were selected. All the experimental mice were male and purchased from Inner Mongolia Medical University.

[0111] Preparation of bacterial solution: Inoculate Bacillus subtilis HCD03 into TSB liquid medium at a ratio of 1%, incubate overnight at 37°C and 200 rpm, and adjust the bacterial solution concentration to 1×10 8 CFU / mL.

[0112] (1) Mouse weight gain and food intake test

[0113] Twenty male mice were selected and randomly divided into two groups, 10 mice in each group, and the experiment was conducted after one week of adaptive breeding. The mice were gavaged once a day using a mouse gavage needle. The probiotic group (added with Bacillus subtilis) was gavaged with Bacillus subtilis HCD03 bacterial solution, and the control group (without Bacillus subtilis) was gavaged with the same volume of sterile saline, with the gavage volume of 0.2 mL / mouse, and the experimental period was 7 days.

[0114] During the experiment, the mice's mental state, appetite, coat color, feces, etc. were observed. Each mouse was weighed once a day for 7 days, and the feed intake of the mice was weighed daily to calculate the average daily weight gain (ADG), average daily feed intake (ADFI) and feed-to-weight ratio (F / G).

[0115]

[0116] Table 6 Effect of adding or not adding Bacillus subtilis HCD03 on weight gain of mice

[0117]

[0118] In the mouse weight gain test, within 7 days of oral gavage, the mice in each group had no adverse reactions, were in good mental state, had normal fur and feces color, and had no diarrhea. The measured and recorded test data were statistically organized using Excel software, and the results were expressed as "mean ± standard deviation". GraphpadPrism Version 9.5.1 software was used to perform a T test on the average daily weight gain results of each group of mice. At the same stage, the same lowercase letters indicate no significant difference (P>0.05), and different lowercase letters indicate significant difference (P<0.05). According to Table 6 and Fig.11 It can be seen that the body weight of mice in the probiotic group and the control group increased within 7 days of gavage, and the average daily weight gain of mice in the probiotic group was slightly higher (P>0.05) than that of mice in the control group, which shows that probiotics have better growth-promoting performance.

[0119] Table 7 Effect of adding Bacillus subtilis HCD03 on the food intake of mice

[0120]

[0121] The average daily feed intake of 10 mice is shown in Table 7, and the feed-to-weight ratio is shown in Fig.12 Within 7 days, the feed-to-weight ratio of the probiotic group administered with Bacillus subtilis HCD03 was significantly higher (P<0.05) than that of the control group, indicating that the addition of probiotics can promote the absorption and conversion of nutrients in mice and improve the growth performance of mice.

[0122] (2) Mouse organ coefficient determination test

[0123] Twenty male mice were randomly divided into two groups and orally administered once a day. The probiotic group was orally administered with Bacillus subtilis HCD03 solution, and the control group was orally administered with sterile saline. The mice were dissected on the 7th day, and the heart, liver, kidney, lung, and spleen were removed. At the same time, the tissues were observed for visible lesions, and the organ coefficients were measured.

[0124]

[0125] During the mouse organ coefficient test, it was found that the organ color and size of each group of mice were normal, and there were no visible lesions. The organ coefficient results of each group of mice are as follows Fig.13 As shown, there was no significant difference in the organ coefficient of mice in the group that was orally administered with Bacillus subtilis HCD01 probiotics compared with the control group (P>0.05), indicating that the bacteria did not cause edema or hyperplasia or other pathological changes in the immune organs of mice, nor did it cause degenerative changes such as atrophy.

[0126] (3) HE staining test of mouse ileum tissue sections

[0127] Twenty male mice were randomly divided into two groups and orally administered once a day. The probiotic group was orally administered with Bacillus subtilis HCD03 solution, and the control group was orally administered with sterile saline. The mice were autopsied on the 7th day, and the ileum tissues of the mice were cut for paraffin sectioning and HE staining. The changes in villus length and crypt depth were observed, and the ratio of villus length to crypt depth was calculated simultaneously.

[0128] Mouse ileum tissue sections and HE staining results are shown in Fig.14 As shown, the ratio of villus length / crypt depth of the ileum in the probiotic group and the control group were 0.023±0.004 and 0.018±0.002, respectively. Statistical analysis showed that the ratio of villus length / crypt depth of the ileum in the probiotic group was significantly (P<0.05) greater than that in the control group, indicating that the addition of probiotics can promote the absorption of nutrients in the ileum.

[0129] (4) High-throughput sequencing of 16S rRNA gene of mouse fecal bacterial community

[0130] On the 7th day, fresh mouse fecal samples were collected from the probiotic group and the control group and packaged into sterile cryopreservation tubes, with each tube containing approximately 0.5 to 2.0 g of sample. After packaging, they were immediately transported on dry ice and sent to Nanjing Paisonno Gene Technology Co., Ltd. for sequencing. The sequencing results were analyzed using the Paisonno Gene Cloud Platform.

[0131] Cluster analysis was performed on the sequences of each sample species, and bioinformatics statistical analysis was performed at a 97% similarity level. Fig.15 As shown in the figure, the Shannon index and Simpson index of the probiotic group supplemented with Bacillus subtilis HCD03 bacterial solution were significantly (P<0.05) higher than those of the control group, indicating that the addition of probiotics can significantly increase the diversity of intestinal flora and improve the homeostasis of intestinal flora.

[0132] The LDA threshold was set to 2, and the comparison strategy was selected as one-against-all to draw the LEfSe-based phylogenetic map of mouse fecal flora. Fig.16The results showed that there were significant differences in the relative abundance of the dominant phylum (p), class (c), order (o), family (f), and genus (g) of the intestinal flora between the probiotic group and the control group (P<0.05). The number of dominant groups (35 in total) in the probiotic group with Bacillus subtilis (orange) was significantly higher than that in the control group without Bacillus subtilis (10 in total). Among them, the probiotic group with Bacillus subtilis had 1 dominant phylum and 1 dominant class, 4 dominant orders, 9 dominant families, and 20 dominant genera; the control group without Bacillus subtilis had 1 dominant phylum, 1 dominant class, 2 dominant orders, 3 dominant families, and 3 dominant genera; this showed that the addition of probiotics could significantly improve the diversity of the intestinal flora of the tested mice. The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0133] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus subtilis strain, characterized in that: Named HCD03, its classification name is Bacillus subtilis, and it was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 24, 2024, with the deposit number CGMCC No.31448. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Bacillus subtilis according to claim 1, characterized in that It is Bacillus subtilis from bovine origin.

3. The Bacillus subtilis according to claim 1, characterized in that The lowest pH tolerated by the Bacillus subtilis is 2, the highest pH tolerated is 12, the highest salt concentration tolerated is 10%, and the highest temperature tolerated is 95°C.

4. Use of the Bacillus subtilis according to claim 1 in inhibiting Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae.

5. Use of the Bacillus subtilis according to claim 1 in the preparation of a probiotic.

6. The use according to claim 5, characterized in that The probiotic is used to increase the feed-to-weight ratio, and increase the diversity index of intestinal flora and the number of dominant groups.

7. A probiotic, characterized in that: The invention comprises the Bacillus subtilis described in claim 1.

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