A strain of Bacillus licheniformis and its application

Abstract

The present invention discloses a strain of Bacillus licheniformis and its application. It belongs to the technical field of probiotics. A strain of Bacillus licheniformis is separated and identified by traditional identification methods and molecular biological methods, and its probiotic performance and safety in animals are evaluated. At the same time, microbiology is combined with pathology, etc., and it is found that oral administration of the bacteria can reduce organ lesions in mice caused by Salmonella, and can produce antagonistic effects on Salmonella in mice. The present invention can not only expand the research on Bacillus, but also provide a reference basis for the future development of probiotic microecological preparations for promoting digestion and growth in animals, antagonizing pathogens, and regulating intestinal microecological environments.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and more particularly to a strain of Bacillus licheniformis and application thereof. Background Art

[0002] The intestinal microbiota plays a key role in many metabolic, physiological, nutritional and immune processes in animals. Different dietary patterns, obesity, disease and the use of antibiotics can affect the abundance and structure of the intestinal microbiota. In addition, probiotics can also repair inflammation and metabolic disorders caused by intestinal diseases. In recent years, there are two main motivations for the use of probiotics. First, normal people use probiotics and probiotic products to maintain their own health, while enhancing immunity and reducing the risk of disease. Secondly, for specific diseases, the use of probiotics can also be used as an effective treatment, but the probiotic effects produced by different strains may also be very different.

[0003] Probiotics in the genus Bacillus can promote the body's more efficient absorption and utilization of nutrients, while improving the body's resistance to pathogens, maintaining the balance of the intestinal microecological environment, and increasing the colonization of beneficial bacteria in the animal's intestines, thereby ensuring the health of the animal body. Studies have found that dietary supplementation with Bacillus licheniformis can help promote the restoration of ileal microbial community disorders caused by necrotic enteritis to normal steady-state levels. In the application of breeding ruminants such as sheep, adding Bacillus licheniformis to the diet can improve the nitrogen utilization rate of rumen microorganisms in ruminants, change the fermentation pattern, and improve the apparent nutritional digestibility of rumen neutral detergent fiber, acid detergent fiber and organic matter.

[0004] Although some species of Bacillus are currently used as probiotics, finding and introducing new potential probiotic strains that are safe for humans and animals remains one of the most important topics in microbiology and animal husbandry.

[0005] In summary, how to provide a Bacillus licheniformis and study its probiotic properties is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a Bacillus licheniformis strain and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A strain of Bacillus licheniformis, named HCD02, was classified as Bacillus licheniformis and was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 24, 2024, with the deposit number CGMCC No.31449. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, it is bovine Bacillus licheniformis.

[0010] Furthermore, the Bacillus licheniformis tolerates a pH of 2 and a salt concentration of 10%.

[0011] The application of the above-mentioned Bacillus licheniformis in inhibiting Salmonella.

[0012] The use of the above-mentioned Bacillus licheniformis in the preparation of probiotics.

[0013] Furthermore, the probiotics are used to alleviate symptoms of Salmonella infection.

[0014] Furthermore, it is used to increase the abundance of beneficial bacteria in the intestinal flora, alleviate intestinal damage caused by Salmonella infection, and reduce lung and liver lesions caused by Salmonella.

[0015] A probiotic agent comprises the above-mentioned Bacillus licheniformis.

[0016] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0017] A strain of Bacillus licheniformis was isolated and identified by traditional identification methods and molecular biology methods, and its probiotic performance and safety in animals were evaluated. At the same time, microbiology and pathology were combined, and it was found that oral administration of the bacteria could reduce organ lesions in mice caused by Salmonella, and could produce antagonistic effects on Salmonella in mice. The present invention can not only expand the research on Bacillus, but also provide a reference for the future development of probiotic microecological preparations for promoting digestion and growth in animals, antagonizing pathogens, and regulating intestinal microecological environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 It is the technical roadmap of the present invention;

[0020] Figure 2 The colony morphology of strain HCD02 on TSB solid medium in Example 1 of the present invention;

[0021] Figure 3 The Gram staining microscopic examination results of strain HCD02 in Example 1 of the present invention (10×100);

[0022] Figure 4 is the NJ phylogenetic tree of strain HCD02 in Example 1 of the present invention;

[0023] Figure 5 The results of functional gene detection of strain HCD02 in Example 1 of the present invention are shown in the figure on the left, where 1 represents racE; 2 represents gyrA; 3 represents gyrB; 4 represents negative control; and M represents 500 bp Marker; in the figure on the right, 1 represents negative control; 2 represents pelA; 3 represents AST; and M represents 2000 bp Marker;

[0024] Figure 6 This is the hemolysis test result of Bacillus licheniformis HCD02 in Example 2 of the present invention;

[0025] Figure 7 is the growth curve of Bacillus licheniformis HCD02 in Example 2 of the present invention;

[0026] Figure 8 The weight gain test results of the Bacillus licheniformis group in Example 3 of the present invention;

[0027] Fig. 9 The test results of the feed-to-weight ratio (F / G) in Example 3 of the present invention, wherein the same and different lowercase letters represent no significant difference (P>0.05) and significant difference (P<0.05), respectively;

[0028] Fig.10 The organ coefficient result in Example 3 of the present invention;

[0029] Fig.11 This is a diagram of the autopsy of mice in Example 3 of the present invention, wherein a is the control group; b is the pathogenic bacteria group; and c is the protection group;

[0030] Fig.12 The effect of Bacillus licheniformis HCD02 antagonizing Salmonella on the intestinal morphology of mice in Example 3 of the present invention;

[0031] Fig.13 The effect of Bacillus licheniformis HCD02 in Example 3 of the present invention on the antagonism of Salmonella on the liver and lung tissues of mice;

[0032] Fig.14 The OTU annotation Venn diagram in Example 3 of the present invention, wherein CK is the control group; Blic1 is the group that was gavaged with Bacillus licheniformis for 7 consecutive days; Blic2 is the group that was gavaged with Bacillus licheniformis for 14 consecutive days; BlicP1 is the group that was gavaged with Bacillus licheniformis for 7 consecutive days and then attacked with Salmonella, i.e., the 7th day of antagonism; BlicP2 is the second stage of Bacillus licheniformis antagonism against Salmonella, i.e., the 14th day of antagonism;

[0033] Fig.15 The Chao index and Shannon index analysis of fecal flora in Example 3 of the present invention;

[0034] Fig.16 is the rarefaction curve of each group of samples in Example 3 of the present invention;

[0035] Fig.17 : is the grade abundance curve of each group of samples in Example 3 of the present invention, wherein the control group CKAn is not administered with the test bacteria; the probiotic group Blic1: the test probiotic (Bacillus licheniformis) was administered for 7 consecutive days; the probiotic group Blic2: the test probiotic was administered for 14 consecutive days; the antagonistic group BlicP1: the test probiotic was administered for 7 consecutive days and then attacked with Salmonella, i.e., the 7th day of antagonism (Bacillus licheniformis antagonistic Salmonella group); the antagonistic group BlicP2: the second stage after the attack with Salmonella, i.e., the 14th day of antagonism;

[0036] Fig.18 The results of the phylogenetic analysis of the mouse fecal flora based on LEfSe in Example 3 of the present invention, wherein (a) is a comparison of the dominant bacterial groups in mouse feces between the probiotic Blic group and the control CKAn group; (b) is a comparison of the dominant groups between the probiotic antagonistic Salmonella group BlicP and the control CKAn group;

[0037] Fig.19 The PCoA analysis result of the species similarity of the fecal flora of mice in Example 3 of the present invention is shown, wherein CKAn is the control group; Blic is the probiotic group (the group administered with Bacillus licheniformis); and BlicP is the antagonistic group (the group antagonizing Salmonella with Bacillus licheniformis);

[0038] Fig. 20 This is a PCA diagram of the characteristic genera / differential genera in the feces of mice in the control CKAn group, the probiotic Blic group, and the antagonistic BlicP group in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] 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.

[0040] 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.

[0041] Example 1

[0042] Isolation, identification and preservation of strains

[0043] (1) Isolation and purification of bacterial strains

[0044] Fecal samples: The present invention selects the grassland of Simmental dairy cows in Siziwang Banner, Ulanqab City, Inner Mongolia Autonomous Region (41°10′~43°22′N, 110°20′~113°00′E) to collect dairy cow fecal samples, and the cow fecal samples are placed in sterile bags and stored at low temperature for subsequent separation and screening of potential probiotics.

[0045] Use a sterile cotton swab to dip an appropriate amount of cow dung on the solid culture medium, add physiological saline with a pipette to dilute, and apply 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 solid culture medium for streaking purification. After a single colony appears on the culture medium, perform Gram staining on it, and repeat this process until the staining results are consistent in bacterial morphology and color. Pick a fresh bacterial lawn of a single bacterium and expand it in TSB liquid culture medium. Use a pipette to draw 1mL of bacterial solution into a storage tube, add 1mL of 30% glycerol, and finally freeze it in a -40°C freezer for storage, named HCD02.

[0046] (2) Morphological identification

[0047] The isolated and purified strains were 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 strains were Gram-stained and then observed under an optical microscope for cell morphological characteristics.

[0048] Depend on Figure 2 It can be seen that strain HCD02 appears as flat, irregularly edged, and rough, wrinkled milky white colonies on TSB solid medium. After long-term culture, the edges appear hair-like. Gram staining observation shows that Figure 3 As shown, strain HCD02 is a long rod-shaped Gram-positive bacterium arranged in single / paired / chain forms.

[0049] (3) Bacterial 16S rRNA gene sequencing and identification

[0050] The gene was extracted according to the instructions of the TIANamp Bacteria DNA Kit, and the extracted isolated bacterial genome was used as a template to perform PCR amplification using universal primers for bacterial 16S rRNA gene. The upstream and downstream primers were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1 and 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2.

[0051] The amplified products were sequenced, and after sequencing, BLAST homology comparison was performed, and the NJ (Neighborjioning) phylogenetic tree was constructed using MEGA X software.

[0052] The sequencing results of the amplified 16S rRNA gene of strain HCD02 are as follows, and the sequence length is 1313 bp.

[0053]

[0054] The 16S rRNA gene sequence of strain HCD02 was compared by BLAST sequence comparison, and the NJ (Neighbor-Joining) phylogenetic tree was constructed using MEGA X software. Figure 4 As shown, the homology between strain HCD02 and Bacillus licheniformis SMIA-2 is 100%, and the homology between strain HCD02 and strains such as Bacillus licheniformis RSP-09, A-2-11B-AP, and NB STEM 4 is greater than 99%, indicating that strain HCD02 is Bacillus licheniformis.

[0055] (4) PCR detection of functional genes

[0056] The primer sequences involved are shown in Table 1. The primers used were all synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0057] Table 1 Primer sequences for PCR amplification of functional genes

[0058]

[0059]

[0060] Results Figure 5 Obvious bands appeared in the gyrA gene and the gyrB gene. The electrophoresis result of the amplified product at the gyrA gene was about 300-400 bp, which was the same as the expected fragment size (346 bp); the band size at the gyrB gene was about 500 bp, which was consistent with the expected result (507 bp); no bright bands appeared at the racE, pelA and AST genes. The above results indicate that strain HCD02 contains gyrA and gyrB genes, but does not contain racE, pelA and AST genes.

[0061] (5) Biochemical identification

[0062] The biochemical tests of isolated strains were carried out with reference to the Common Bacteria Identification Manual and the instructions for microbiochemical identification tubes. 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 strains were further identified based on the test results.

[0063] Table 2 Biochemical identification results of isolated bacteria

[0064]

[0065] Note: “+” means positive, “-” means negative.

[0066] As shown in Table 2, strain HCD02 ferments glucose to produce acid but not gas; can utilize most types of sugars, such as muscarinic acid, sucrose, lactose, etc.; in the sugar alcohol test, can utilize mannitol, but does not show any utilization results for sorbitol; does not ferment urea, does not decompose urease; produces lysine decarboxylase; does not produce hydrogen sulfide, but produces Simon's citrate; can decompose salicin and esculin; can decompose hydrogen peroxide and produce gas; MR-VP test is negative. The biochemical test results of strain HCD02 are basically consistent with the biochemical characteristics of Bacillus.

[0067] (6) Preservation

[0068] The Bacillus licheniformis is named HCD02, and its classification name is Bacillus licheniformis. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 24, 2024, with the deposit number CGMCC No.31449, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0069] Example 2

[0070] Evaluation of probiotic properties of strains in vitro

[0071] (1) Hemolysis test

[0072] A single colony of Bacillus licheniformis HCD02 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 placed upside down in a constant temperature incubator (37° C.) and cultured for one day (24 h) to observe whether there was hemolysis.

[0073] The morphology of Bacillus licheniformis HCD02 on blood agar medium is as follows Figure 6 As shown, no hemolytic zone appeared, indicating that Bacillus licheniformis HCD02 was not hemolytic.

[0074] (2) Drug sensitivity test

[0075] The drug sensitivity test uses the Kirly-Baue method, and selects antibacterial drugs such as β-lactams, chloramphenicol, quinolones, sulfonamides, and tetracyclines. 200 μL of bacterial culture fluid is evenly spread on the solid culture medium and dried at room temperature for 3 to 5 minutes until the bacterial fluid is observed to be fully absorbed and then the drug sensitivity sheet is pasted with tweezers. Three parallel drug sensitivity sheets are set for each drug, 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 (24 hours). The test results obtained are compared with the "Standards for Determining the Diameter of the Inhibition Zone of the Paper Disk Method Drug Sensitivity Test", and the results are divided into sensitive (S), intermediate (I), and resistant (R).

[0076] Table 3 Drug sensitivity test results of Bacillus licheniformis HCD02

[0077]

[0078]

[0079] Note: “R” stands for resistant; “I” stands for intermediate; “S” stands for sensitive.

[0080] The resistance and sensitivity of Bacillus licheniformis HCD02 to common antibiotics are shown in Table 3. The results showed that Bacillus licheniformis HCD02 was sensitive to cefradine, cephalexin, norfloxacin, ofloxacin, enrofloxacin, doxycycline, spectinomycin, streptomycin, neomycin and gentamicin; intermediate to amoxicillin; resistant to penicillin G, cefotaxime, ceftriaxone, chloramphenicol, co-trimoxazole, erythromycin, azithromycin, clindamycin and kanamycin.

[0081] (3) Antibacterial test

[0082] The antibacterial test was carried out by the Oxford cup double-layer plate diffusion method. Prepare solid agar medium, take it out after high-temperature sterilization, control the amount of each culture dish to be about 10mL, and make the lower plate after cooling and solidification, and place the Oxford cup at a certain interval. Repeat the above steps to prepare the upper plate and pour it on the lower plate, wait for it to solidify and pull out the Oxford cup. Select Salmonella (Salmonella) CVCC2184, Streptococcus dysgalactiaeequisimilis (Streptococcus dysgalactiaeequisimilis) BNCC337480 and Staphylococcus aureus (Staphylococcus aureus) ATCC29213 as pathogenic indicator bacteria, take 50μL of bacterial solution from each and evenly spread it on the culture medium; add 200μL of the culture solution of the isolated strain to the small hole, place it in a 37℃ constant temperature box for one day (24h), observe whether the antibacterial circle appears and measure the diameter of the antibacterial circle.

[0083] Table 4 Antibacterial test results of isolated bacteria

[0084]

[0085] The in vitro antibacterial test of the isolated bacteria was carried out by the Oxford cup double-layer plate diffusion method. The results are shown in Table 4. Bacillus licheniformis HCD02 had no inhibitory effect on Staphylococcus aureus, had an inhibitory effect on Salmonella, and had no inhibitory effect on Streptococcus dysgalactiae.

[0086] (4) Growth curve

[0087] The isolated bacteria were inoculated into the corresponding liquid culture medium and cultured at a constant temperature with shaking (37°C, 200 rpm). The bacterial solution was collected every 2 hours (i.e., after 2h, 4h, 6h, 8h, 10h, 12h, 14h, and 16h), and the absorbance value (OD600) at a wavelength of 600nm was measured using an enzyme marker to draw a growth curve.

[0088] Depend on Figure 7 It can be seen that Bacillus licheniformis HCD02 entered the logarithmic growth phase after 4 hours, the growth rate decreased slightly after 8 hours, but it still showed an overall growth trend. It entered the stable phase after 10 hours, and no decline occurred within 16 hours of culture.

[0089] (5) Acid resistance test

[0090] The isolated bacteria cultured overnight were inoculated into liquid culture media with different pH values ​​(i.e., 2, 3, and 4) at an inoculum size of 1%, and cultured at a constant temperature with shaking (37° C., 200 rpm) for 24 hours to observe whether the culture media became turbid.

[0091] Table 5 Acid resistance test results of isolated bacteria

[0092]

[0093] Note: “+” indicates turbidity.

[0094] The results of the acid resistance test of the strain are shown in Table 5. After overnight culture in liquid culture medium with pH values ​​of 2, 3, and 4, turbidity can be produced, indicating that the strain has a certain degree of acid resistance.

[0095] (6) Salt resistance test

[0096] 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. The isolated bacteria were inoculated into the corresponding culture medium at an inoculum volume of 1%, and the OD600 value of the bacterial solution was measured with an ELISA instrument after constant temperature culture (37°C, 200rpm) for 24h.

[0097] Table 6 Results of isolated bacterial salt tolerance test

[0098]

[0099] Note: “+” indicates turbidity.

[0100] According to the results in Table 6, the turbidity of the bacterial solution of Bacillus licheniformis HCD02 showed an overall downward trend with the increase of the NaCl concentration gradient, but turbidity was produced after 24 hours of cultivation in the liquid culture medium with five NaCl contents, indicating that it was able to grow and reproduce, indicating that Bacillus licheniformis HCD02 has a certain salt tolerance.

[0101] (7) Coagulation test

[0102] The isolated bacteria were inoculated into liquid culture medium at an inoculum size of 1%, cultured overnight with constant temperature shaking (37°C, 200 rpm), and then mixed solutions were prepared according to the bacterial liquid / skim milk ratios of 1:5, 1:10, 1:15, 1:20, and 1:25, and placed in a 37°C constant temperature incubator. After 24 hours, whether coagulation occurred was observed and the pH value of the mixed solution was measured.

[0103] Table 7 Separation of bacterial coagulation test results and pH value determination results

[0104]

[0105]

[0106] Note: “-” indicates no coagulation occurs.

[0107] The results of the coagulation test and the measured pH value are shown in Table 7. When the ratio of Bacillus licheniformis HCD02 bacterial solution to skim milk was 1:10, the pH value reached the lowest, but coagulation still did not occur, indicating that Bacillus licheniformis HCD02 did not have the ability to coagulate milk.

[0108] Example 3

[0109] Evaluation of the probiotic and safety performance of the strain 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 the isolated bacteria into TSB liquid culture 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. In the same way, Salmonella was inoculated into TSB medium and cultured overnight with constant temperature shaking. The concentration of the bacterial solution was adjusted to 1×10 7 CFU / mL, for future use.

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

[0113] Ten male mice were randomly divided into two groups, 5 mice in each group, and the experiment was conducted after one week of adaptive breeding. The mice in the experimental group (Bacillus licheniformis group) were gavaged with Bacillus licheniformis HCD02 bacterial solution once a day, and the control group was gavaged with the same volume of sterile saline, with the gavage volume of 0.2 mL / mouse, and the experimental period was 14 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 14 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 8 Mouse weight gain test results

[0117]

[0118] In the mouse weight gain test, within 14 days of gavage in each group, the mice had no adverse reactions, their mental state was normal, their fur and feces were normal, and there was no diarrhea. The measured and recorded test data were statistically organized using Excel software, and the results were expressed as "mean ± standard deviation". According to Table 8, all groups showed a trend of weight growth within 14 days of gavage, and the average daily weight gain of each group of mice within 7 days (half cycle) was greater than that within 14 days (one cycle), which shows that the weight gain of mice was faster in a short period, and then the growth condition gradually stabilized.

[0119] The average daily weight gain of mice in each group was tested by T test using Graphpad Prism Version 9.5.1 software. In the same stage, "ns" indicates no significant difference (P>0.05), "*" indicates significant difference (P<0.05); the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05). Figure 8 As shown, although oral administration of Bacillus licheniformis within half a cycle had a weight gain effect on mice, there was no significant difference compared with the control group (P>0.05); from the perspective of the entire experimental cycle, Bacillus licheniformis could promote the growth of mice, but there was no significant difference between them and mice that were not oral administered with the bacterial solution (P>0.05).

[0120] Table 9 Mouse food intake test results

[0121]

[0122] The results of the average daily feed intake of mice are shown in Table 9, and the feed-to-weight ratio results are shown in Fig. 9 Compared with the control group, the feed-to-weight ratio of the group administered with Bacillus licheniformis was higher than that of the control group in both the half-cycle and the whole cycle, indicating that no obvious growth-promoting effect was found during this period.

[0123] (2) Mouse organ coefficient determination test

[0124] Ten male mice were randomly divided into two groups and gavage once a day. One group was gavaged with Bacillus licheniformis solution, and the remaining group was gavage with sterile saline as a blank control. The mice were autopsied on the 7th day, and the hearts, livers, kidneys, and lungs were removed. At the same time, the tissues were observed for visible lesions, and the organ coefficients were measured.

[0125]

[0126] 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.10 As shown, there was no significant difference in the organ coefficient of mice administered with Bacillus licheniformis HCD02 compared with the control group (P>0.05), indicating that the bacteria did not cause edema or hyperplasia, or degenerative changes such as atrophy, in the immune organs of mice.

[0127] (3) Antagonism test in mice

[0128] Fifteen mice were randomly divided into three groups, with five mice in each group. The protective group was the Bacillus licheniformis group (i.e., the antagonistic Salmonella group). The mice in the protective group were gavaged with 0.2 mL of Bacillus licheniformis solution daily; the pathogenic bacteria group and the control group were gavaged with the same volume of sterile saline daily. After 7 consecutive days of gavage, according to the results of the preliminary test, the mice were gavaged with Salmonella, and the control group was not treated. After 96 hours, the mice were randomly dissected to observe whether the organs had lesions. The lungs, livers, small intestines, and colons of the mice were collected, fixed with a universal tissue fixative, and sent to Wuhan Seville Biological Company for HE staining and preparation of pathological tissue sections.

[0129] After being challenged with Salmonella, the mice became listless and showed symptoms such as shortness of breath and diarrhea. There was no death in the control group and the protection group, and their mental state and defecation were normal. Fig.11 As shown, there were no abnormalities in the liver and intestines of the mice in the control group; the mice in the pathogenic bacteria group had visible lesions such as bleeding and bloating in the intestines; after Bacillus licheniformis antagonized Salmonella, the symptoms of tissue lesions in the mice were alleviated.

[0130] After in vivo antagonism between Bacillus licheniformis and Salmonella, the intestinal morphology of mice was Fig.12As shown. Compared with the control group, the mice in the pathogenic bacteria group had shorter intestinal villi and a large number of intestinal villus epithelial cells fell off; the mucosal layer of the colon tissue produced wrinkles and focal erosions, uneven thickness, necrosis and shedding of mucosal epithelial cells, a decrease in the number of intestinal glands, and a very small amount of connective tissue hyperplasia, accompanied by a very small amount of granulocyte infiltration. In the mice in the protection group, the small intestinal muscle layer structure and muscle cells were neatly arranged, and there was no significant change in the length of the intestinal villi compared with the control group. Compared with the pathogenic bacteria group, the villi became longer, the shedding of epithelial cells was significantly reduced, and the intestinal morphology was improved; there was no focal erosion in the colon tissue, and there was no significant change in the goblet cells. Therefore, Bacillus licheniformis HCD02 can alleviate the intestinal damage caused by Salmonella infection.

[0131] like Fig.13 As shown in the figure, after Salmonella challenged mice, compared with the control group, the lung tissue of the mice showed granulocyte infiltration of the alveolar wall, mild thickening of the focal alveolar wall, widening of the alveolar septum, alveolar hemorrhage, irregular arrangement of the bronchiolar epithelium, and a small amount of eosinophilic substances in the cavity. After oral administration of Bacillus licheniformis HCD02 to antagonize Salmonella, no obvious thickening of the alveolar wall was observed, and only a small amount of granulocyte infiltration was observed. In the liver tissue of mice challenged with Salmonella, it can be seen that the boundaries of the liver lobules were not obvious, and symptoms such as hepatocyte hydropic degeneration and venous congestion appeared. Although the liver of the mice in the protection group was damaged, the symptoms were alleviated compared with the pathogenic bacteria group. Therefore, oral administration of Bacillus licheniformis HCD02 for in vivo Salmonella antagonism test can reduce the lung and liver lesions caused by Salmonella.

[0132] (4) High-throughput sequencing of 16S rRNA gene of mouse fecal bacterial community

[0133] The above-mentioned fresh mouse fecal samples were collected on the 7th and 14th days, respectively, and the fecal samples were packaged in sterile cryopreservation tubes, with a sample volume of approximately 0.5 to 2.0 g in each tube. 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.

[0134] Cluster analysis was performed on the species sequences of each sample, and bioinformatics statistical analysis was performed at a 97% similarity level. The distribution of the number of OTUs between different groups was compared based on the Venn diagram. The clustering and species annotation results are shown in Fig.14 As shown. The number of OTUs in the Blic1, BlicP1, Blic2, and BlicP2 groups were 171, 167, 111, and 138, respectively. The number of OTUs after oral administration of Bacillus licheniformis and antagonism of Salmonella was different from that of the corresponding control group.

[0135] like Fig.15 As shown, in the first week, the species diversity and species abundance of the Bacillus licheniformis group were improved compared with the control group.

[0136] The rarefaction curve can reflect the impact of changes in sequencing depth on the diversity of each group of samples. Fig.16 With the increase of sampling depth, the rarefaction curve of each group of samples gradually reaches a flat state, indicating that the sequencing results can reflect the diversity contained in the current samples and the Alpha diversity has reached saturation.

[0137] The rank abundance curve can reflect the uniformity of community composition. Fig.17 The grade abundance curve shown has gradually flattened, indicating that the abundance differences between ASV / OTUs in the sample communities detected in this experiment are gradually decreasing, the number of new species is no longer increasing with continuous sampling, the sequencing depth is sufficient, and the amount of sequencing data is reasonable.

[0138] Table 10 Relative abundance of mouse fecal flora at the phylum level (%)

[0139]

[0140] Table 11 Relative abundance of mouse fecal flora at the genus level (%)

[0141]

[0142]

[0143] The structure of the fecal microbial community of mice in different groups was analyzed at the phylum and genus levels, and the composition abundance is shown in Tables 10 and 11. There are two main phylum levels: one is the Bacteroidetes, which is one of the absolute dominant flora in mouse feces, among which the genus Bacteroides has a higher abundance at the genus level. The relative abundance of Bacteroidetes in the Blic1 group (70.643%) was higher than that in the control CKAn1 group (66.682%); the relative abundance of Bacteroides in the Blic1 group (4.228%) was higher than that in the control group CKAn1 (1.938%); the second is the Firmicutes, which mainly includes the genus Lactobacillus; the relative abundance of Lactobacillus in the fecal samples collected on the 7th day was higher in each group than in the control group CKAn1 (1.719%). The results showed that oral administration of Bacillus licheniformis HCD02 could increase the abundance of beneficial bacteria in the intestines of mice, and after antagonizing Salmonella, the bacterial flora gradually restored balance compared with the control group.

[0144] 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.18(a) The relative abundance of intestinal flora in the CKAn control group and the Blic probiotic group was significantly different at the phylum (p), class (c), order (o), family (f), and genus (g) levels (P<0.05). 19 dominant groups and 7 dominant groups (e.g., important probiotics in the intestine - Enterococcaceae and Enterococcus and Akkermansia) were screened out in the CKAn control group (green fan ring) and the Blic probiotic group (yellow fan ring), respectively.

[0145] Depend on Fig.18 (b) It can be seen that there were significant differences in the relative abundance of intestinal flora at the phylum (p), class (c), order (o), family (f) and genus (g) levels between the control group CKAn and the antagonistic Salmonella group BlicP (P<0.05); 16 dominant groups and 9 dominant groups were screened out in the control group CKAn (green fan ring) and the antagonistic Salmonella group BlicP (yellow fan ring), respectively.

[0146] Fig.19 The bray_curtis distance algorithm was used for principal coordinate analysis (PCoA) to characterize the level of β diversity. The distance of the control group CKAn sample projected on the first axis (PCo1=24.4%) was in the middle, and the distance projected on the second axis (PCo2=15.9%) was the largest, with a high overall dispersion, indicating that the β diversity of the control group CKAn was high; the distance of the probiotic group Blic sample projected on the first axis (PCo1=24.4%) was the largest, and the distance on the second axis (PCo2=15.9%) was the smallest, with a high overall dispersion, indicating that the β diversity of the probiotic group Blic was high; the distance of the antagonistic Salmonella group BlicP projected on the first axis (PCo1=24.4%) was the smallest, and the distance on the second axis (PCo2=15.9%) was in the middle, with a medium overall dispersion, indicating that the β diversity of the antagonistic Salmonella group BlicP was medium.

[0147] Fig. 20Principal coordinate analysis (PCA) was used to screen the iconic / differential genus. The explanatory power of the first axis (PCA1) was as high as 84.4%, and the explanatory power of the second axis (PCA2) was 10.1%, indicating that the first axis and the second axis played a major and minor role, respectively. The distance of the control group CKAn samples on the first axis (PCA1 = 84.4%) was the smallest, and the degree of aggregation was high, indicating that the control group CKAn had the least iconic / differential genus; the distance of the probiotic group Blic and the antagonistic Salmonella group BlicP samples on the first axis (PCA1 = 84.4%) was large, and the degree of dispersion was high, indicating that the probiotic group Blic and the antagonistic Salmonella group BlicP had more iconic / differential genus; in addition, the distance of the probiotic group Blic projection on the second axis (PCo2 = 15.9%) was much greater than the distance of the antagonistic Salmonella group BlicP on the second axis (PCo2 = 15.9%). In summary, the number of characteristic / differential genera in the probiotic group Blic>the antagonistic Salmonella group BlicP>the control group CKAn.

[0148] 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.

[0149] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 strain of Bacillus licheniformis, characterized in that: Named HCD02, its classification name is Bacillus licheniformis ( Bacillus licheniformis , deposited in the General Microbiology Center of China Microorganism Culture Collection on July 24, 2024, with the deposit number CGMCC No.31449, 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 licheniformis according to claim 1 in the preparation of probiotics.

3. The use according to claim 2, characterized in that The probiotics are used to alleviate the symptoms of Salmonella infection.

4. The use according to claim 3, characterized in that Used to increase the abundance of beneficial bacteria in the intestinal flora, alleviate intestinal damage caused by Salmonella infection, and reduce lung and liver lesions caused by Salmonella.

5. A probiotic, characterized in that: The invention comprises the Bacillus licheniformis according to claim 1.

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