Bacillus coagulans and application thereof

Abstract

The invention relates to the technical field of screening and application of probiotics, and particularly provides a novel bacillus coagulans strain and application of the novel bacillus coagulans strain. The bacillus coagulans is screened from naturally fermented soybean paste, has good biological safety, strong stress resistance to gastrointestinal tract environment, high yield of exopolysaccharide and protease, strong bacteriostasis and oxidation resistance, can efficiently degrade cholesterol, is beneficial to maintaining intestinal health, and can be widely applied to the fields of food, health care products and the like.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic screening and application, and in particular to a strain of Bacillus coagulans and application thereof. Background Art

[0002] The World Health Organization and the Food and Agriculture Organization of the United Nations define probiotics as live microorganisms that are beneficial to the health of the host when ingested in sufficient quantities. Probiotics have been a hot topic in the field of food and health in recent years. They have the functions of regulating intestinal microbial homeostasis and maintaining mucosal barriers, promoting nutrient absorption and improving the body's immunity.

[0003] Intestinal flora plays an important role in the human body. Probiotics can regulate the composition of intestinal flora and thus affect human health, intervening in the occurrence and development of various diseases. Mao Bingyong et al. found that the use of a composite probiotic preparation can affect the relative abundance of bacteria such as Lactobacillus, Bifidobacterium, Collinsella, Ruminococcus, Dorea and Eubacterium in the human intestinal flora, proving that composite probiotic preparations can increase the abundance of some beneficial bacteria in the intestine and regulate intestinal flora imbalance. In addition to intestinal flora imbalance, diseases lead to intestinal bacterial translocation (BT), that is, the migration of intestinal bacteria or metabolites from the intestinal cavity to extraintestinal sites, which will seriously threaten human health. Probiotics can also play a regulatory and preventive role in bacterial translocation. Minnen et al. found in their study that using a variety of probiotics to modify the intestinal flora can reduce intestinal bacterial translocation during experimental acute pancreatitis and reduce morbidity and mortality.

[0004] Probiotics can promote the body's nutrient absorption and improve nutritional status. Ballini et al. evaluated the effectiveness and safety of preventive probiotics in the pediatric population to improve the nutrient absorption capacity of children, thereby enhancing their overall health and immunity. The results of the study confirmed that the use of probiotics can effectively improve the overall biomarker levels of vitamin, calcium and mineral absorption in the blood of children from baseline to 10 weeks of treatment compared with placebo.

[0005] Currently, the probiotic strains approved by the state for use in food include 38 strains from 17 genera on the "List of Strains That Can Be Used in Food", 14 strains on the "List of Strains That Can Be Used in Infant Food", and 10 strains on the "Regulations on the Declaration and Review of Probiotic Health Foods (Trial)".

[0006] With the continuous development of the food industry, the application scope of probiotics is not limited to the processing of traditional foods. Due to its ability to regulate intestinal flora and enhance immune function, the research on probiotic foods as functional foods and their nutritional and health benefits has gradually attracted attention. At present, probiotic functional foods used in the form of ordinary foods at home and abroad are mainly probiotic fermented dairy products such as cheese, yogurt and milk drinks, as well as some nutritional bars, candies and other probiotic functional foods; probiotic preparations and health products mainly in the form of medicines include powders, hard capsules, soft capsules, granules, oral liquids, microcapsules and tablets.

[0007] In the field of probiotic product development, the exploration of high-quality probiotic germplasm resources is of utmost importance and is also a current research hotspot. Summary of the invention

[0008] The purpose of the present invention is to provide a novel Bacillus coagulans strain and its application. The Bacillus coagulans is screened from naturally fermented soybean paste, has strong antibacterial and antioxidant abilities, can produce high amounts of extracellular polysaccharides, efficiently remove cholesterol, maintain intestinal health, and can be widely used in the fields of food, health products, etc.

[0009] On the one hand, the present invention relates to a strain of Bacillus coagulans, named Bacillus coagulans KDB-18, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on July 1, 2024, and its deposit number is CCTCC NO: M20241408.

[0010] The invention also relates to the application of Bacillus coagulans KDB-18 strain in the production of food, health care products or medicines.

[0011] The invention also relates to the application of Bacillus coagulans KDB-18 strain in the production of antibacterial agents.

[0012] The present invention also relates to a probiotic preparation, comprising the Bacillus coagulans KDB-18 strain and / or a fermentation product thereof.

[0013] The probiotic preparation further comprises any one or more combinations of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus casei, Lactobacillus fermentum, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Streptococcus thermophilus, and Lactobacillus crispatus.

[0014] The amount of live bacteria of Bacillus coagulans KDB-18 strain in the probiotic preparation is not less than 10 10 CFU / g.

[0015] The present invention also relates to the application of the probiotic preparation in the production of food, health products or medicines.

[0016] The Bacillus coagulans KDB-18 obtained by screening in the present invention is relatively sensitive to common antibiotics such as erythromycin, gentamicin, tetracycline, and ampicillin, and has good biosafety. The strain can produce high amounts of exopolysaccharides and proteases, with the exopolysaccharide yield in the fermentation supernatant reaching 3.64 g / L and the protease activity reaching 358.8 U / mL, achieving unexpected technical effects.

[0017] The Bacillus coagulans KDB-18 has a strong antibacterial ability and is better than commercially available Bacillus coagulans in inhibiting the growth of four pathogenic bacteria, namely Staphylococcus aureus, Escherichia coli, Salmonella and Shigella; among them, the diameters of the inhibition zones for Escherichia coli and Salmonella are as high as 28.32 mm and 31.43 mm, respectively, with significant effects.

[0018] The Bacillus coagulans KDB-18 has a strong antioxidant capacity, and its supernatant has a scavenging rate of 89.35%, 70.19% and 80.56% for DPPH free radicals, hydroxyl free radicals and superoxide anions respectively; it can efficiently remove cholesterol with a removal rate of up to 85.21%, achieving an unexpected technical effect.

[0019] The Bacillus coagulans KDB-18 has high in vitro hypoglycemic activity, and the inhibition rates of its fermentation supernatant on alpha-amylase and glucosidase reach 82.28% and 34.06% respectively, and has potential application prospects for hypoglycemic application.

[0020] The results of the mouse animal experiment showed that the concentrations of pro-inflammatory factors IL-1β, IL-6, TNF-α and the level of C-reactive protein in the serum of mice in the Bacillus coagulans KDB-18 group were significantly lower than those in the Escherichia coli group, indicating that Bacillus coagulans KDB-18 can effectively reduce the expression of pro-inflammatory factors in the serum of mice, reduce the level of C-reactive protein, and effectively enhance the immunity of mice; the contents of three short-chain fatty acids, acetic acid, propionic acid and butyric acid in the cecal contents of mice in the Bacillus coagulans KDB-18 group were increased by 88.6%, 60.2% and 130.3% respectively compared with the Escherichia coli group. It is shown that the Bacillus coagulans KDB-18 provided by the present invention can significantly increase the content of short-chain fatty acids in the intestine of mice, which is beneficial to promote digestion and absorption, improve the body's anti-inflammatory level, maintain intestinal health, and achieve unexpected technical effects.

[0021] The Bacillus coagulans KDB-18 provided by the present invention can be used alone or in combination with probiotics such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus casei, Lactobacillus fermentum, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus crispatus, etc., and is widely used in the fields of food, health care products, medicines, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the colony morphology of Bacillus coagulans. DETAILED DESCRIPTION

[0023] The Bacillus coagulans KDB-18 provided by the present invention is a newly discovered Bacillus coagulans strain, which can produce high amounts of extracellular polysaccharides, effectively inhibit a variety of pathogenic bacteria, and has strong antioxidant and cholesterol degradation capabilities. It can be used as a source of food raw materials, and long-term use will not cause side effects or overdose risks, and has important application value.

[0024] On July 1, 2024, the applicant deposited the Bacillus coagulans KDB-18 in the China Type Culture Collection of Wuhan University, Wuhan, China, with the accession number being CCTCC NO: M20241408.

[0025] The culture medium described in the embodiment of the present invention has the following formula:

[0026] LB liquid culture medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 1000 mL distilled water, pH 7.0-7.4.

[0027] LB solid medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 2 g agar powder, 1000 mL distilled water, pH 7.0-7.4.

[0028] Sugar production medium: 10g tryptone, 5g yeast extract powder, 10g sodium chloride, 1000mL distilled water, 20g sucrose, pH 7.0-7.4.

[0029] The following examples are provided to better illustrate the present invention, and those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the protection and claims of the present invention are not limited to the examples provided.

[0030] Example 1 Isolation and screening of polysaccharide-producing Bacillus

[0031] 1. Initial screening of Bacillus

[0032] Under sterile conditions, 0.5 g of soybean paste sample collected from naturally fermented Hua Nong Village, Dalian was accurately weighed, the soybean paste was placed in 4.5 mL of sterilized physiological saline cooled to room temperature, vortexed to mix thoroughly, placed in an 80°C water bath for 20 min, and cooled to room temperature to obtain a soybean paste mixture.

[0033] Take 0.5mL of soybean paste mixture into 4.5mL of normal saline, dilute 10 times each time, until it is 10% of the original mixture. -6 , take 10 respectively-4 , 10 -5 、10- 6 100 μL of each diluted sample is added to the center of the LB solid culture medium and spread evenly with a coating rod. Place the plate upside down in a 37°C constant temperature incubator and culture for 20-30 hours. Pick a single colony with the morphology of Bacillus on the plate and streak the plate for culture. Repeat the picking 3-4 times until a single colony and a pure strain are obtained.

[0034] Through morphological observation and Gram staining, the applicant screened and obtained 23 strains of Bacillus, which were named S1, S2, S3, ..., S23.

[0035] 2. Rescreening of mucogenes and sugar-producing Bacillus

[0036] The 23 strains of Bacillus obtained in the initial screening were activated and inoculated on LB plates, cultured at 37°C for 48 hours, and the "sticky" phenotype of the strains was observed, and the length of the filaments formed when the colonies were touched by the inoculation loop was detected. The results showed that the colonies of the three strains of Bacillus, S3, S5 and S18, had the highest viscosity and the longest filaments.

[0037] The polysaccharide content of strains S3, S5 and S18 was further tested using the phenol-sulfuric acid method. The results showed that the extracellular polysaccharide content of the fermentation broth of strain S18 was the highest.

[0038] Example 2 Identification of S18 strain

[0039] 1. Colony morphology identification

[0040] The S18 strain was streaked onto LB solid medium and cultured at 37°C for 24 h. The colony morphology was as follows: Figure 1 As shown. The diameter of a single colony of the S18 strain is 2-3 mm, white and shiny, with a moist, flat surface and neat edges. Observation under a microscope showed that the S18 strain was rod-shaped, arranged singly, in pairs or chains, and the spores were terminal.

[0041] 2. Physiological and biochemical identification

[0042] Refer to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems.

[0043] The results showed that the S18 strain could use D-mannose, amygdalin, glycerol, D-melibiose, sucrose, D-trehalose, D-ribose, D-xylose, maltose, L-rhamnose, D-mannitol, D-raffinose, lactose, inulin, and sodium gluconate as carbon sources, but could not use D-melezitose and L-fucose as carbon sources. The strain had a negative catalase reaction; it fermented glucose to produce acid but not gas.

[0044] 3. 16s rDNA gene sequence identification

[0045] The genomic DNA of the S18 strain was extracted. The genomic DNA was used as a template and PCR amplification was performed using the following primers 27F (AGAGTTTGATCCTGGCTCA) and 1492R (GGTTACCTTGTTACGACTT). The amplified product was sequenced to obtain the 16s rDNA sequence of the S18 strain.

[0046] The 16s rDNA sequence of the S18 strain was compared with the existing sequences in the GenBank database using the BLAST tool in the NCBI database. The results showed that the 16s rDNA sequence of the S18 strain had the highest similarity with Bacillus coagulans. The results of the phylogenetic tree constructed by MEGA7.0 showed that the S18 strain of the present invention had the highest homology with Bacillus coagulans.

[0047] In summary, combined with the colony morphology, physiological and biochemical characteristics and molecular biological identification results of the strain, it can be concluded that the S18 strain screened and obtained in the present invention is a new type of Bacillus coagulans, named Bacillus coagulans KDB-18.

[0048] On July 1, 2024, the applicant deposited the Bacillus coagulans KDB-18 in the China Type Culture Collection of Wuhan University, Wuhan, China, with the accession number being CCTCC NO: M20241408.

[0049] Example 3 Experiment on the tolerance of Bacillus coagulans KDB-18 to antibiotics

[0050] Prepare 2048 μg / mL stock solutions of ampicillin, tetracycline, erythromycin, gentamicin, streptomycin, and clindamycin respectively and store them at -20°C for future use. When used, dilute the stock solutions 2-fold with BSM liquid medium to form working solutions, with a total of 11 gradients of gradient dilution concentrations ranging from 1 to 1024 μg / mL.

[0051] Bacillus coagulans KDB-18 was inoculated into LB liquid culture medium and cultured at 37°C for 24 hours; the fresh bacterial liquid was centrifuged at 12000 rpm for 5 minutes, washed once with sterile saline, and then resuspended with the same volume of saline and diluted 50 times to serve as the inoculum.

[0052] The minimum inhibitory concentration (MIC) of antibiotics against Bacillus coagulans KDB-18 was determined by the microbroth dilution method. The specific results are shown in Table 1.

[0053] Table 1 Antibiotic MIC values ​​of Bacillus coagulans KDB-18 (μg / mL)

[0054]

[0055] It can be seen from the results in Table 1 that the Bacillus coagulans KDB-18 provided by the present invention is relatively sensitive to common antibiotics such as erythromycin, gentamicin, tetracycline, and ampicillin, and has good biosafety.

[0056] Example 4 Application of Bacillus coagulans KDB-18 in the production of exopolysaccharides

[0057] 1. Fermentation broth preparation

[0058] The Bacillus coagulans KDB-18 strain was inoculated into 50 mL LB liquid culture medium, and activated in a constant temperature incubator at 37°C for 24 hours to prepare a first-generation seed solution; the first-generation seed solution was inoculated into 100 mL LB liquid culture medium at a 5% inoculation amount to prepare a second-generation seed solution, and cultured in a constant temperature incubator at 37°C for 24 hours; the second-generation seed solution was inoculated into 1 L sugar-producing culture medium at a 5% inoculation amount, and fermented in a shaker at 37°C at a speed of 160 r / min for 48 hours to obtain a fermentation liquid;

[0059] 2. Extracellular polysaccharide extraction

[0060] The fermentation liquid is centrifuged at 4°C and 12000rpm for 15 minutes to obtain a supernatant. At least 3 volumes of anhydrous ethanol are added to the supernatant and the mixture is allowed to stand. The mixture is centrifuged and the precipitate is collected to obtain the crude extracellular polysaccharide.

[0061] The crude extracellular polysaccharide was washed with 95% ethanol by volume, centrifuged at about 10,000 rpm and 4°C for 15 minutes, the supernatant was discarded, and the precipitate was washed again with 95% ethanol by volume, centrifuged at about 10,000 rpm and 4°C for 15 minutes, the supernatant was discarded, and the final precipitate was dissolved in 85°C distilled water. After removing the protein in the crude polysaccharide using the Sevage method, pure water was used as the dialysate in a dialysis bag with a molecular weight cutoff of 7000 for 48 hours, and the dialysate was replaced every 12 hours. The polysaccharide solution in the dialysis bag was freeze-dried using a freeze dryer. The freeze dryer had a condensation temperature of -40°C, a vacuum degree of 20 Pa, and a freezing time of about 20 hours to obtain dry powdered extracellular polysaccharides.

[0062] The results showed that the extracellular polysaccharide production in the fermentation broth of Bacillus coagulans KDB-18 reached 3.64 g / L.

[0063] Example 5 Analysis of the protease production ability of Bacillus coagulans KDB-18

[0064] The activated Bacillus coagulans KDB-18 was inoculated into LB liquid culture medium, and cultured at 37°C and 160 rpm for 24 hours; centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was collected.

[0065] The protease activity of the obtained fermentation supernatant was determined by referring to Appendix B "Determination of protease activity by Folin method" in the national standard GB / T 23527-2009 "Protease preparations".

[0066] The test results show that the protease activity in the fermentation supernatant of Bacillus coagulans KDB-18 provided by the present invention reaches 358.8 U / mL, achieving an unexpected technical effect.

[0067] Example 6 Analysis of antibacterial ability of Bacillus coagulans KDB-18

[0068] Staphylococcus aureus, Escherichia coli, Shigella, and Salmonella were inoculated into LB liquid culture medium at a volume ratio of 1%, and then cultured at 37°C for 12 h. The suspension of pathogenic bacteria was adjusted to 10% with sterile saline. 6 -10 7 CFU / mL.

[0069] The activated Bacillus coagulans KDB-18 was inoculated into LB liquid medium at a volume ratio of 1% and cultured at 37°C for 24 hours. 100 μL of pathogenic bacteria suspension was evenly spread on LB solid medium, a sterile Oxford cup was vertically placed in the solid medium and punched, then taken out, 200 μL of Bacillus coagulans KDB-18 fermentation liquid was added to the hole, and a commercially available Bacillus coagulans was used as a control, cultured at 37°C for 24 hours, and the diameter of the inhibition zone was measured. The experiment was repeated three times and the average value was taken.

[0070] See Table 2 for specific results.

[0071] Table 2 Bacillus coagulans KDB-18 antibacterial results

[0072]

[0073] It can be seen from the data in Table 2 that, compared with commercially available Bacillus coagulans, the Bacillus coagulans KDB-18 provided by the present invention has a stronger inhibitory effect on four pathogenic bacteria, namely Staphylococcus aureus, Escherichia coli, Salmonella and Shigella, and the diameters of the inhibition zones for Escherichia coli and Salmonella are as high as 28.32 and 31.43 mm, respectively, achieving unexpected technical effects.

[0074] Example 7 Determination of Antioxidant Capacity of Bacillus coagulans KDB-18

[0075] 1. Scavenging rate of DPPH free radicals

[0076] 2 mL of Bacillus coagulans KDB-18 fermentation supernatant was mixed evenly with anhydrous ethanol solution containing 0.2 mmol / L DPPH, vortexed for 1 min, allowed to stand at room temperature in the dark for 30 min, centrifuged at 6000 r / min for 10 min, and the supernatant was taken to measure the absorbance at a wavelength of 517 nm. An equal amount of anhydrous ethanol was used to replace DPPH in the blank group, and equal amounts of distilled water and ethanol were used as blank zero adjustment, and an equal volume of blank solvent was used to replace the sample solution as the control group.

[0077] The calculation formula is as follows:

[0078] Clearance % = [1-(A 样品 -A 空白 ) / A 对照 ]×100%.

[0079] Where: A i A is the absorbance value of the supernatant; j is the absorbance value of the blank group; A c is the absorbance value of the control group.

[0080] (2) Hydroxyl free radical scavenging rate:

[0081] The operation was carried out according to the instructions of the hydroxyl free radical determination kit (Fenton colorimetric method), the absorbance was measured at 550 nm and the scavenging ability of the fermentation supernatant of the strain on hydroxyl free radicals was calculated.

[0082] (3) Superoxide anion clearance rate:

[0083] The superoxide anion scavenging ability of the strain was determined by the pyrogallol auto-oxidation method. 1 mL of 150 mmol / L Tris-HCl (pH 8.2), 1.2 mmol / L pyrogallol and 3 mmol / L diethylenetriaminepentaacetic acid were added to the sample, kept at 25°C for 10 min, and the absorbance was measured at 325 nm.

[0084] The calculation formula is as follows:

[0085] Superoxide anion clearance rate (%) = [1-(A b -A c ) / (A d -A e )]×100%.

[0086] Where: A b is the absorbance value without sample and pyrogallol; A c A is the absorbance value without sample and containing pyrogallol; d A is the absorbance value of the sample without pyrogallol; e is the absorbance value of the sample and pyrogallol.

[0087] The results showed that Bacillus coagulans KDB-18 had a strong antioxidant function, and its supernatant had a scavenging rate of 89.35%, 70.19% and 80.56% for DPPH radicals, hydroxyl radicals and superoxide anions, respectively, achieving unexpected technical results.

[0088] Example 8 In vitro cholesterol degradation test with Bacillus coagulans KDB-18

[0089] Accurately weigh 1 g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100 mL. Filter and sterilize it with a 0.22 μm microporous filter membrane under sterile conditions.

[0090] Add the above cholesterol solution to the LB liquid medium to make the final cholesterol concentration 0.1%. Inoculate fresh bacterial solution at an inoculum volume of 0.1%, culture at 37°C for 48 hours, then take 0.2 ml of bacterial solution, add 1.8 ml of anhydrous ethanol, mix, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, take the supernatant, and detect the cholesterol content in the supernatant according to GB / T5009.128-2016 "Determination of Cholesterol in Foods", and calculate the degradation rate of cholesterol by Bacillus coagulans KDB-18. At the same time, a commercially available Bacillus coagulans strain was used as a control strain. The specific results are shown in Table 3.

[0091] Cholesterol removal rate (%) = (initial content - final content) / initial content × 100%.

[0092] Table 3 Effect of Bacillus coagulans KDB-18 on cholesterol removal

[0093] strain Cholesterol removal rate Bacillus coagulans KDB-18 85.21％ Bacillus coagulans 29.75％

[0094] From the data in Table 3, it can be seen that the Bacillus coagulans KDB-18 provided by the present invention can efficiently remove cholesterol, with a removal rate of up to 85.21%, which is significantly higher than that of commercially available Bacillus coagulans, and achieves unexpected technical effects.

[0095] Example 9 Evaluation of in vitro hypoglycemic activity of Bacillus coagulans KDB-18

[0096] In this example, α-amylase and glucosidase inhibition experiments were used to evaluate the in vitro hypoglycemic activity of Bacillus coagulans KDB-18. The inhibition rates of α-amylase and glucosidase of the fermentation supernatant of Bacillus coagulans KDB-18 were determined according to the instructions of the kit.

[0097] The results showed that the inhibition rates of Bacillus coagulans KDB-18 on α-amylase and glucosidase were 82.28% and 34.06%, respectively, indicating that the Bacillus coagulans KDB-18 provided by the present invention has high in vitro hypoglycemic activity, has potential application prospects in hypoglycemic treatment, and has achieved unexpected technical effects.

[0098] Example 10 Effect of Bacillus coagulans KDB-18 on intestinal health of mice

[0099] The experimental animals were Kunming male mice, 5 weeks old, weighing (22±3) g.

[0100] The mice used in this example were acclimatized to the environment for one week before the experiment. During this period, the temperature was 23±2° C., the humidity was 50%±10%, the light-dark cycle was 12 h, and drinking water and food were provided normally.

[0101] The experimental mice were randomly divided into 3 groups, namely normal control group, Escherichia coli group and Bacillus coagulans KDB-18 group, with 10 mice in each group. The mice were fasted for 24 hours before the experiment. The treatment methods of mice in each group are as follows:

[0102] The mice in the E. coli group and the Bacillus coagulans KDB-18 group were gavaged with 400 μL of an equal proportion of E. coli O157 and E. coli H7 suspension (10 9 After the inflammation model was successfully established, the mice in the E. coli group were gavaged with 400 μL of normal saline daily, and the mice in the Bacillus coagulans KDB-18 group were gavaged with 400 μL of Bacillus coagulans KDB-18 suspension daily (10 8 CFU / mL) for 5 consecutive days. The mice in the normal control group were gavaged with 400 μL of normal saline every day.

[0103] During the experiment, mice were free to eat and drink water, and their body weight and food intake were measured every day.

[0104] After the experiment, the mice were fasted for 12 h and then killed by anesthesia.

[0105] Blood was collected from the eyeballs of all mice and centrifuged at 4°C and 3500 rpm for 15 minutes to obtain serum. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the expression of biochemical indicators and inflammatory factors in the serum of mice in each group.

[0106] Colon tissues were collected from all mice, and feces in the cecal contents were collected. The feces and ether were mixed in a 1:1 ratio, vortexed for 2 minutes, ultrasonicated for 3 minutes to extract short-chain fatty acids, centrifuged for 10 minutes to remove the precipitate, and the supernatant was passed through a 0.22-micron organic filter membrane, wrapped with a sealing film and stored in a -20°C refrigerator for determination. A short-chain fatty acid standard curve was prepared using a gas chromatograph to determine the content of short-chain fatty acids in the cecal contents of mice in different groups.

[0107] 1. Effect of Bacillus coagulans KDB-18 on the body weight of mice

[0108] Compared with the normal control group, the body weight of mice in the Escherichia coli group was significantly reduced, and the body weight of mice showed a significant decline on the 4th day, while the body weight of mice in the Bacillus coagulans KDB-18 group was significantly higher than that in the Escherichia coli group.

[0109] 2. Regulatory effect of Bacillus coagulans KDB-18 on immune response in mice

[0110] Compared with the normal control group, the concentrations of pro-inflammatory factors IL-6, TNF-α, IL-1β, and C-reactive protein in the serum of mice in the E. coli group were significantly increased; while the concentrations of IL-1β, IL-6, and TNF-α in the serum of mice in the Bacillus coagulans KDB-18 group were significantly lower than those in the E. coli group. This shows that the Bacillus coagulans KDB-18 provided by the present invention can effectively reduce the expression of pro-inflammatory factors in the serum of mice, reduce the level of C-reactive protein, and effectively enhance the body immunity of mice.

[0111] 3. Effects of Bacillus coagulans KDB-18 on the level of short-chain fatty acids (SCFAs) in the cecum of mice

[0112] Compared with the normal control group, the contents of acetic acid, propionic acid and butyric acid in the cecal contents of the Escherichia coli group mice were significantly reduced, while the contents of three short-chain fatty acids, acetic acid, propionic acid and butyric acid, in the cecal contents of the Bacillus coagulans KDB-18 group were increased by 88.6%, 60.2% and 130.3% respectively compared with the Escherichia coli group. It is thus shown that the Bacillus coagulans KDB-18 provided by the present invention can significantly increase the content of short-chain fatty acids in the intestine of mice, which is beneficial to promote digestion and absorption, improve the body's anti-inflammatory level, maintain intestinal health, and achieve unexpected technical effects.

[0113] In summary, the coagulant bacillus KDB-18 obtained by the present invention is more sensitive to common antibiotics, has good biosafety, has strong resistance to the gastrointestinal environment, can produce high-quality extracellular polysaccharides and proteases, has strong antibacterial and antioxidant abilities, can efficiently degrade cholesterol, and reduce blood sugar. Animal experiment results show that the strain can effectively reduce the expression of proinflammatory factors in mouse serum, significantly improve the content of short-chain fatty acids in the intestine, thereby helping to promote digestion and absorption, improve the body's anti-inflammatory level and immunity, and maintain intestinal health. Coagulant bacillus KDB-18 can be alone, or it can be combined with probiotics such as lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus paracasei, bifidobacterium lactis, lactobacillus casei, fermented lactobacillus, lactococcus lactis, bulgaricus, lactobacillus gasseri, thermophilic streptococcus, and lactobacillus crispatus, and is widely used in the fields of food, health products, and medicines, and has broad application prospects.

Claims

1. A strain of Bacillus coagulans ( Bacillus coagulans ), characterized in that, The deposit number of the Bacillus coagulans is CCTCC NO: M20241408.

2. Use of the Bacillus coagulans according to claim 1 in the production of food, health products or medicines.

3. Use of the Bacillus coagulans according to claim 1 in the production of antibacterial agents.

4. A probiotic preparation, characterized in that The probiotic preparation comprises the Bacillus coagulans and / or its fermentation product according to claim 1.

5. The probiotic preparation according to claim 4, characterized in that The probiotic preparation further comprises any one or more combinations of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus casei, Lactobacillus fermentum, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Streptococcus thermophilus, and Lactobacillus crispatus.

6. The probiotic preparation according to claim 4 or 5, characterized in that: The amount of live bacteria of Bacillus coagulans in the probiotic preparation is not less than 10 10 CFU / g.

7. Use of the probiotic preparation according to any one of claims 4 to 6 in the production of food, health products or medicines.

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