A Bacillus subtilis strain and its applications

Abstract

The present invention discloses a strain of Bacillus subtilis and its application, belonging to the technical field of probiotic R & D. In the present invention, a strain of Bacillus subtilis is identified by traditional isolation and culture, morphological, physiological and biochemical, and molecular biological methods, and its probiotic properties are evaluated by in vitro and in vivo tests. The results of in vitro tests show that the strain can tolerate acid (the lowest pH is 2), tolerate salt (the highest NaCl concentration is 10%), is non-hemolytic, inhibits pathogenic bacteria, and is sensitive to most antibiotics; the results of in vivo tests show that adding the strain can improve the production performance of animals, promote the absorption of nutrients by the small intestine, and increase the diversity of dominant intestinal flora. The present invention can not only expand the research on Bacillus subtilis, but also provide a reference basis for the future development of probiotic preparations for antagonizing pathogenic bacteria and regulating the intestinal microecological balance, etc.

Description

Technical Field

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

[0002] The use of antibiotics and chemical drugs as important means to inhibit the spread of pathogens and relieve symptoms has led to an increase in drug-resistant bacteria, drug residues, and a decline in the quality of the ecological environment due to their extensive application, thus 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, due to their natural and non-toxic side effects, become potential alternatives to antibiotics and are expected to be widely used in multiple industries such as medical health, food additives, biological pesticides, biological fertilizers, and environmental governance, becoming an important part of disease prevention and control strategies. Therefore, probiotics are expected to provide us with a safer and more effective health protection barrier by promoting ecological balance and inhibiting the growth of harmful bacteria.

[0003] Bacillus subtilis is a common Gram-positive probiotic. As a biocontrol agent, it has attracted wide attention in agriculture for its role as a biological pesticide and biological fertilizer by inhibiting plant pathogens and promoting plant growth. Bacillus subtilis colonizes the intestine, can effectively prevent pathogenic bacteria from binding to intestinal mucosal receptors, inhibit the spread of pathogenic bacteria, and stabilize the intestinal flora. Since Bacillus subtilis produces various volatile fatty acids during physiological metabolism, it provides an acidic environment for the animal gastrointestinal tract, can improve the activity of proteases in the stomach, hinder the growth of pathogenic bacteria, and improve the production performance and disease resistance of poultry and livestock. Bacillus subtilis is widely present in soil, the rhizosphere and phyllosphere of plants, and the surfaces and bodies of animals and plants. It has antibacterial activity and strong stress resistance, shows good effects in preventing diseases, inhibiting bacteria, and resisting stress in the fields of crop planting and poultry and livestock breeding, and secretes digestive enzymes, vitamins, or antibacterial substances in the animal intestine or environment to regulate the intestinal flora, inhibit pathogenic microorganisms, and improve animal growth performance.

[0004] In summary, how to provide a Bacillus subtilis strain and develop its probiotic functions is a scientific problem that those skilled in the art urgently need to solve. Summary of the Invention

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

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A strain of Bacillus subtilis, named HCD03, was deposited in the China General Microbiological Culture Collection Center on July 24, 2024. The deposit number is 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 a bovine-derived Bacillus subtilis.

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

[0010] The above-mentioned Bacillus subtilis is used in inhibiting Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae.

[0011] The above-mentioned Bacillus subtilis is used in the preparation of probiotic agents.

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

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

[0014] From the above technical solutions, it can be seen that compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0015] The present invention identified a strain of Bacillus subtilis through traditional isolation and culture, morphological, physiological and biochemical, and molecular biological methods, and evaluated its probiotic properties in vitro and in vivo. The in vitro test results showed that the strain was acid-tolerant (the lowest pH tolerated was 2), salt-tolerant (the highest NaCl concentration was 10%), non-hemolytic, inhibited Staphylococcus aureus (ATCC29213), Streptococcus agalactiae (BNCC185941), and Streptococcus dysgalactiae (BNCC337480), and was sensitive to most antibiotics; the in vivo test results showed that adding the strain could improve the production performance of animals, promote the absorption of nutrients in the small intestine, and increase the diversity of dominant intestinal flora. The present invention can not only expand the research on Bacillus, but also provide a reference basis for the development of probiotic preparations for antagonizing pathogenic bacteria and regulating the intestinal microecological balance in the future. Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

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

[0018] Figure 2 It is the colony morphology diagram of the isolated and purified strain HCD03 on the solid medium with agar added to TSB in Example 1 of the present invention;

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

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

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

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

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

[0024] Figure 8 It is the test result of the optimal growth pH of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0025] Figure 9 It is the salt tolerance test result of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0026] Figure 10 It is the high temperature tolerance test result of Bacillus subtilis HCD03 in Example 2 of the present invention;

[0027] Figure 11This is the test result of the average daily weight gain of the probiotic group supplemented with Bacillus subtilis HCD03 and the control group of mice in Example 3 of the present invention. Among them, ns represents no significant difference between the probiotic group and the control group statistically (P>0.05);

[0028] Figure 12 This is the test result of the feed to gain ratio (F / G) of the probiotic group supplemented with Bacillus subtilis HCD03 and the control group in Example 3 of the present invention. Among them, P<0.05 represents a significant difference between the probiotic group and the control group statistically;

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

[0030] Figure 14 This is the effect of the probiotic group supplemented with Bacillus subtilis HCD03 on the villus length and crypt depth of the small intestine (ileum) of mice in Example 3 of the present invention;

[0031] Figure 15 This is the analysis of the Shannon index and Simpson index of the fecal flora of the probiotic group supplemented with Bacillus subtilis HCD03 in Example 3 of the present invention. Among them, P<0.05 represents a significant difference between the probiotic group and the control group statistically;

[0032] Figure 16 This is the analysis result of the LEfSe phylogenetic tree diagram of the dominant fecal flora of the probiotic group supplemented with Bacillus subtilis HCD03 in Example 3 of the present invention. Orange represents the taxa with significant advantages in the probiotic group supplemented with Bacillus subtilis, and green represents the taxa with significant advantages in the control group without adding Bacillus subtilis. Detailed implementation mode

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] The medicaments required for the present invention are conventional experimental medicaments, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0035] Example 1

[0036] Isolation, identification, and preservation of strains

[0037] (1) Isolation and purification

[0038] Dip a sterile cotton swab into an appropriate amount of cow manure and smear it on a solid medium. Use a pipette to add physiological saline for dilution, and then spread it evenly with a spreader. Incubate overnight at a constant temperature of 37°C. The next day, use a sterile inoculation loop to pick colonies with consistent morphology and an absolute predominance in number onto a TSB (agar added) solid medium for streak purification. After single colonies appear on the medium, perform Gram staining on them, and repeat this process until the staining results show consistent cell morphology and color. Pick a fresh bacterial lawn of a single bacterium into a TSB liquid medium for subculture. Then, use a pipette to aspirate 700 μL of the bacterial liquid into a preservation tube, add 700 μL of 30% glycerol, and finally store it frozen in a -40°C freezer, named HCD03.

[0039] (2) Morphological identification

[0040] Cultivate the isolated and purified strain HCD03 under suitable conditions, visually observe the phenotypic characteristics of the colonies and record their morphology, color, and size; after performing Gram staining on the isolate HCD03, observe the cell morphological characteristics under an optical microscope.

[0041] It can be seen from Figure 2 that the colony morphology of the strain on the TSB (agar added) solid medium is round, with an irregular edge, a rough surface, and a color ranging from off-white to grayish-yellow. It can be seen from Figure 3 that the strain HCD03 obtained by screening is Gram-positive, and the microscopic examination result shows an oval or short rod shape.

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

[0043] Extract the genomic DNA of strain HCD03 according to the instructions of the bacterial genomic DNA extraction kit (TIANamp Bacteria DNA Kit). Using the genomic DNA of strain HCD03 as a template, select the universal primers 27F and 1492R for bacterial 16S rRNA gene for PCR amplification.

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

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

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

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

[0048]

[0049] (4) Physiological and biochemical identification

[0050] The biochemical tests of the isolated strain HCD03 were carried out with reference to the common bacterial identification manual and the instruction manual of the micro biochemical identification tube. The main tests included glycolysis test, urea test, lysine decarboxylase test, hydrogen sulfide test, Simmons citrate test, hydrogen peroxide gas production test, glucose gas production test, and MR-VP test, etc. According to the test results, the phenotype of the strain HCD03 was further identified.

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

[0052]

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

[0054] The physiological and biochemical identification results showed that the isolated strain HCD03 could ferment glucose, sucrose, mannitol, mannose, fructose, salicin, esculin, and urease, could not ferment trehalose, lactose, maltose, did not form indole, could hydrolyze starch, reduce nitrate, could not utilize Simmons citrate and citrate, 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 in line with the phenotypic characteristics of Bacillus.

[0055] (5) Preservation

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

[0057] Example 2

[0058] Evaluation of the in vitro probiotic performance of the strain

[0059] (1) Hemolysis test

[0060] A single colony of Bacillus subtilis HCD03 was selected. A fresh bacterial lawn was picked with a sterile inoculation loop on the blood agar medium and streaked on the plate. After being inverted and cultured in a constant temperature incubator (37°C) for 24 h, the hemolysis phenomenon was observed.

[0061] The morphology of Bacillus subtilis HCD03 on the blood agar medium was as Figure 6 shown, and no hemolysis zone appeared, indicating that Bacillus subtilis HCD03 had no hemolytic property.

[0062] (2) Growth curve determination

[0063] The Bacillus subtilis HCD03 bacterial solution was inoculated into the TSB liquid medium at a ratio of 1%, and cultured with constant temperature oscillation (37 °C, 200 rpm). Every 2 h, 200 μL of the bacterial solution was taken, and the OD 600 value was measured with a microplate reader to plot the growth curve.

[0064] It can be seen from Figure 7 that the growth rate of Bacillus subtilis HCD03 was slow in the range of 0 - 2 h, and it was in the lag phase; after 2 h, the growth was rapid, and the OD 600 value increased rapidly and entered the logarithmic growth phase; after 6 h, the growth slowed down and gradually entered the stationary phase; after 24 h, the growth amount of Bacillus subtilis HCD03 decreased and entered the decline phase.

[0065] (3) Drug sensitivity test

[0066] The Kirby - Bauer disk diffusion method was used for the drug sensitivity test. β - lactams, quinolones, macrolides, tetracyclines, lincosamides, aminoglycosides, chloramphenicols, etc. were selected for the drug resistance and drug sensitivity tests. 70 μL of the Bacillus subtilis HCD03 culture solution was pipetted onto the TSB solid medium and spread evenly until it was observed that the bacterial solution had been fully absorbed, and then the drug sensitivity disks were pasted with forceps. Three parallels were set for each drug's sensitivity disk. After culturing in an inverted position in a 37 °C constant temperature incubator for one day (24 h), the diameter of the inhibition zone was measured with a vernier caliper. The test results obtained were compared with the "Judgment Standard for the Diameter of the Inhibition Zone in Disk Diffusion Antibiotic Susceptibility Test", and the results were evaluated as sensitive (S), intermediate (I), and resistant (R).

[0067] Table 2 Results of the drug sensitivity test of Bacillus subtilis HCD03

[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, gentamicin; it was sensitive to cefradine, ceftriaxone, cefalexin, quinolones, clindamycin, spectinomycin.

[0071] (4) Bacteriostatic test

[0072] The antibacterial test was carried out by the Oxford cup agar diffusion method. First, the activated Bacillus subtilis HCD03 bacterial liquid was inoculated into the corresponding liquid medium at a ratio of 1%, and then cultured in a constant temperature incubator for 24 h. The next day, it was centrifuged for 10 min to prepare a cell-free supernatant. The 5 indicator bacteria selected for the experiment were Staphylococcus aureus ATCC29213, Streptococcus agalactiae BNCC185941, Streptococcus dysgalactiae subsp. dysgalactiae BNCC337480, Salmonella CVCC2184, and Escherichia coli ATCC25922. First, the indicator bacteria were cultured and the OD 600 was adjusted to 0.5. 70 μL of each indicator bacterium was respectively spread on the TSB agar solid medium. The Oxford cup was placed on each medium, and 200 μL of the supernatant of the cultured Bacillus subtilis HCD03 was added to the Oxford cup. Finally, it was placed in a constant temperature incubator and cultured for 24 h, and the diameter of the antibacterial circle was measured.

[0073] The diameter of the antibacterial circle of Bacillus subtilis HCD03 against Staphylococcus aureus was about 15.0 mm, the diameter of the antibacterial circle against Streptococcus agalactiae was about 20.0 mm, the diameter of the antibacterial circle against Streptococcus dysgalactiae subsp. dysgalactiae was about 12.0 mm, and it had no inhibitory ability against other reference bacteria.

[0074] (5) Acid tolerance experiment

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

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

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

[0078] Table 3 Results of the acid tolerance test of Bacillus subtilis HCD03

[0079]

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

[0081] (6) Optimal growth pH range test

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

[0083] As Figure 8 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 the acidic environment with pH values of 2 to 4 and the alkaline environment with pH values of 10 to 12.

[0084] (7) Salt tolerance test

[0085] Prepare TSB liquid media containing NaCl respectively, with the concentration gradients set as 2%, 4%, 6%, 8%, and 10%. After preparation, perform autoclaving (121 °C, 15 min). Inoculate Bacillus subtilis HCD03 into the corresponding media at an inoculation amount of 1% respectively, and measure the OD of the bacterial solution after culturing at a constant temperature (37 °C, 200 rpm) for 24 h. 600 value.

[0086] As Figure 9 shown, as the NaCl concentration gradient increases, the turbidity of the bacterial solution of Bacillus subtilis HCD03 generally shows a downward trend, and turbidity occurs in all the liquid media containing NaCl, indicating that it can grow and reproduce, suggesting that Bacillus subtilis HCD03 has a certain salt tolerance ability.

[0087] (8) High temperature tolerance test

[0088] Dispense 50 mL of the overnight-cultured bacterial solution of Bacillus subtilis HCD03 into 5 clean test tubes, with 10 mL of the bacterial solution in each tube. Then further dispense the 10 mL bacterial solution in each test tube into 4 test tubes, with 2.5 mL in each tube (each group of 4 test tubes is designed as a set, and there are 5 sets of test tubes in total). Take any 3 test tubes from each set of test tubes (three replicates), and place them in a water bath at 80, 85, 90, 95, and 100 °C respectively for 30 min of constant temperature water bath (treatment group), and keep 1 test tube in each set at room temperature as the control group. After the water bath, use a pipette to aspirate 20 μL of the bacterial solution and spread it on the TSB nutrient agar medium, and at the same time add 2 drops of NaCl (to adjust the osmotic pressure of the medium). After spreading, place the TSB nutrient agar medium in an incubator at 37 °C for 24 h of constant temperature culture. Repeat the above experiment 3 times, and calculate the survival rates of Bacillus subtilis HCD03 at 80, 85, 90, 95, and 100 °C respectively.

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

[0090] like Figure 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 represent the OD of the aqueous phase before and after mixing with xylene 600 value

[0104] Hydrophobicity of 40% or more is judged as good.

[0105] Table 5 Results of the surface hydrophobicity test of Bacillus subtilis HCD03

[0106]

[0107] As shown in Table 5, the hydrophobicity of Bacillus subtilis HCD03 can reach 78.52%, belonging to highly hydrophobic strains.

[0108] Example 3

[0109] Evaluation of probiotic performance in vivo of strains

[0110] Test animals: SPF-grade Kunming mice aged 4 - 6 weeks, all male, 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 feed intake test

[0113] Select 20 male mice, randomly divide them into 2 groups with 10 mice in each group, and conduct the test after one week of adaptive breeding. Use a mouse gavage needle to administer once a day. The mice in the probiotic group (added with Bacillus subtilis) were gavaged with the bacterial solution of Bacillus subtilis HCD03, and the control group (without added Bacillus subtilis) was gavaged with the same volume of sterile normal saline. The gavage volume was 0.2 mL / mouse, and the test period was 7 days.

[0114] During the test, observe the mental state, appetite, hair color, feces condition, etc. of the mice. Weigh each mouse once a day within 7 days, and at the same time weigh the feed intake of the mice every day, and calculate the average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F / G) of the mice.

[0115]

[0116] Table 6 Effects of adding or not adding Bacillus subtilis HCD03 on mouse weight gain

[0117]

[0118] In the mouse weight gain experiment, within 7 days of gavage for each group, the mice did not show any adverse reactions, had good mental state, normal hair color and fecal color, and no diarrhea. The measured experimental data were statistically sorted using Excel software, and the results were expressed as "mean ± standard deviation". The average daily weight gain results of the mice in each group were subjected to a T-test using Graphpad Prism Version 9.5.1 software. At the same stage, the same lowercase letters indicate no significant difference (P>0.05), and different lowercase letters indicate a significant difference (P<0.05). According to Table 6 and Figure 11 It can be seen that the weights of the mice in the probiotic group and the control group increased within 7 days of gavage. The average daily weight gain of the probiotic group was slightly higher than (P>0.05) that of the control group, indicating that the probiotic has better growth-promoting performance.

[0119] Table 7 Effects of the addition or not of Bacillus subtilis HCD03 on the feed intake of mice

[0120]

[0121] The average daily feed intake results of 10 mice are shown in Table 7, and the feed-to-weight ratio results are shown in Figure 12 . Within 7 days, the feed-to-weight ratio of the probiotic group administered with Bacillus subtilis HCD03 was significantly (P<0.05) higher 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 experiment

[0123] Twenty male mice were randomly divided into 2 groups and gavaged once a day. The probiotic group was gavaged with Bacillus subtilis HCD03 bacterial solution, and the control group was gavaged with sterile normal saline. On the 7th day, the mice were dissected, and the hearts, livers, kidneys, lungs, and spleens of the mice were removed. At the same time, whether there were gross lesions in each tissue was observed, and the organ coefficients were measured.

[0124]

[0125] During the mouse organ coefficient determination experiment, it was found that the colors, sizes, etc. of the organs of the mice in each group were normal, and there were no gross lesions. The organ coefficient results of the mice taken from each group are as Figure 13 shown. There was no significant difference (P>0.05) in the organ coefficients of the mice in the probiotic group administered with Bacillus subtilis HCD01 compared with the control group, indicating that the bacteria did not cause lesions such as edema or hyperplasia in the immune organs of the mice, nor degenerative changes such as atrophy.

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

[0127] Twenty male mice were randomly divided into two groups and gavaged once a day. The probiotic group was gavaged with Bacillus subtilis HCD03 bacterial solution, and the control group was gavaged with sterile saline. On the 7th day, the mice were dissected, and the ileum tissues of the mice were collected for paraffin section and HE staining to observe the changes in villus length and crypt depth, and the ratio of villus length to crypt depth was calculated synchronously.

[0128] The results of the ileum tissue sections and HE staining of the mice are as Figure 14 shown. The ratios of villus length / crypt depth in the ileum of 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 in the ileum of the probiotic group was significantly (P<0.05) greater than that of the control group, indicating that the addition of probiotics could promote the absorption of nutrients in the ileum.

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

[0130] On the 7th day, fresh fecal samples of the mice in the probiotic group and the control group were collected, and the fecal samples were aliquoted into sterile cryotubes, with about 0.5 - 2.0 g of each tube sample. After aliquoting, they were immediately transported by dry ice and sent to Nanjing Personal Biotechnology Co., Ltd. for sequencing, and the sequencing results were analyzed using the Personal Biotechnology Cloud Platform.

[0131] Cluster analysis was performed on the species sequences of each sample, and bioinformatics statistical analysis was performed at a 97% similarity level. As Figure 15 shown, both the Shannon index and the 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 could significantly increase the diversity of the intestinal flora and improve the intestinal flora homeostasis.

[0132] The LDA threshold was set to 2, the comparison strategy was selected as one-against-all, and a phylogenetic tree of the mouse fecal flora based on LEfSe was drawn. Figure 16It was shown that there were significant differences in the relative abundances at the phylum (p), class (c), order (o), family (f), and genus (g) levels of the intestinal flora between the probiotic group and the control group of mice (P<0.05). The probiotic group supplemented with Bacillus subtilis (orange) had significantly more dominant taxa (a total of 35) at the phylum (p_phylum), class (c_class), order (o_order), family (f_family), and genus (g_genus) levels than the control group without Bacillus subtilis supplementation (a total of 10). Among them, the probiotic group supplemented with Bacillus subtilis had 1 dominant phylum, 1 dominant class, 4 dominant orders, 9 dominant families, and 20 dominant genera; the control group without Bacillus subtilis supplementation had 1 dominant phylum, 1 dominant class, 2 dominant orders, 3 dominant families, and 3 dominant genera; indicating that adding probiotics can significantly increase the diversity of the intestinal flora of the test mice. Each example in this specification is described in a progressive manner, and the key point of each example is to illustrate the differences from other examples. For the same or similar parts between the examples, reference can be made to each other.

[0133] The above description of the disclosed examples enables those skilled in the art to implement or use the present invention. Various modifications to these examples will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other examples without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these examples shown herein, but will conform 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 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.

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

3. The use according to claim 2, 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.

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

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