Bacillus coagulans as well as fermentation culture method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus coagulans as well as a fermentation culture method and application thereof. The invention provides the bacillus coagulans with excellent comprehensive performance, and the bacillus coagulans can tolerate the high temperature of 80 DEG C and can grow in an acid environment with the pH value of 1.2. Meanwhile, the strain is sensitive to 18 antibiotics, has no drug resistance and has extremely high biological safety; and the strain has broad-spectrum antibacterial ability, has a remarkable inhibition effect on various pathogenic bacteria, can be widely applied to the field of cultivation, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus coagulans, a fermentation culture method thereof, and an application thereof. Background Art

[0002] Bacillus coagulans belongs to the genus Bacillus, is Gram-positive, motile, and can form endospores. Bacillus coagulans can grow in both aerobic and anaerobic environments, but the amount of lactic acid produced by fermentation under anaerobic conditions is significantly higher than that under aerobic conditions. It not only has the advantages of the production performance of lactic acid bacteria, but also has multiple advantages such as easy germination, ability to colonize, the spores formed having resistance to adverse environments and being able to be stored for a long time. First of all, Bacillus coagulans has the characteristics of lactic acid bacteria-based probiotics, can colonize in the intestine, supplement beneficial flora, adjust the internal environment and the balance of the microflora in the digestive tract, form a biological barrier on the small intestinal wall, prevent and treat digestive disorders and digestive tract infections, and can secrete acidic substances such as lactic acid to regulate the intestinal pH, promote intestinal peristalsis, and improve digestion and absorption functions; secondly, Bacillus coagulans can also form spores, having the characteristics of spore-based probiotics, being able to well tolerate high temperatures, intestinal gastric acid, choline, etc. during the feed processing process, and greatly reducing the inactivation phenomenon during transportation and storage. At the same time, it can produce a variety of digestive enzymes, rich vitamins and unknown growth factors in the digestive tract, etc., which can play a role in assisting digestion and promoting growth. Numerous research results show that Bacillus coagulans can effectively inhibit the growth of harmful bacteria, improve the intestinal microecological environment, promote intestinal development, and enhance intestinal function. It can treat acute and chronic diarrhea, chronic constipation, abdominal distension and indigestion caused by intestinal flora imbalance. Bacillus coagulans also has the effects of promoting the digestion and absorption of animal bodies, improving the immune function of animal bodies, and enhancing the disease resistance of animals, thereby promoting the growth of animals, reducing their mortality rate, culling rate and feed-to-meat ratio. Because the spores of Bacillus coagulans have the characteristics of easy storage and high resurrection rate, it is also an important form of commercialized lactic acid bacteria preparations.

[0003] Bacillus coagulans has a powerful enzyme-producing system. For example, it can produce enzymes such as amylase and protease. When Bacillus coagulans colonizes in the intestine, various enzymes secreted by Bacillus coagulans during its growth and reproduction process can promote the body to absorb nutrients, thereby improving the utilization rate of nutrients. However, the performance of different strains of Bacillus coagulans varies greatly. For example, the enzyme-producing abilities of different strains of Bacillus coagulans are different. Therefore, it is of great significance to screen and obtain a strain of Bacillus coagulans with excellent comprehensive performance.

[0004] In addition, the current fermentation production of Bacillus coagulans has the disadvantages of high contamination rate, low sporulation rate, and low bacterial count, which is not conducive to industrial production and reduces the quality of products containing Bacillus coagulans. Therefore, it is of great value to develop a fermentation culture method suitable for Bacillus coagulans to improve the fermentation level of Bacillus coagulans, especially to improve the sporulation rate of Bacillus coagulans. Summary of the Invention

[0005] To solve the above technical problems, the present invention first provides a Bacillus coagulans with excellent comprehensive performance ( Bacillus coagulans ), which is Bacillus coagulans SDHY-2402, and its preservation number is CGMCC No. 32910.

[0006] This strain was isolated from the soil. This strain was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on December 05, 2024. The address of the depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Taxonomic name: Bacillus coagulans Bacillus coagulans and the preservation number is CGMCC No. 32910.

[0007] Furthermore, the present invention provides a microbial inoculum, which contains the above-mentioned Bacillus coagulans SDHY-2402.

[0008] Preferably, the microbial inoculum is the freeze-dried powder of the fermentation broth of Bacillus coagulans SDHY-2402.

[0009] Preferably, the freeze-dried powder of the fermentation broth also includes a lyoprotectant.

[0010] In the specific implementation process, the lyoprotectant is a conventional lyoprotectant in the art, including but not limited to skim milk powder, dextrin, lactose, sucrose, etc.

[0011] Furthermore, the present invention also provides a product, which contains the above-mentioned Bacillus coagulans SDHY-2402 or the above-mentioned microbial inoculum; the product is any one selected from bacteriostatic agents, drugs, foods or food additives, feeds or feed additives.

[0012] Furthermore, the present invention also provides the application of the above-mentioned Bacillus coagulans SDHY-2402 or the above-mentioned microbial inoculum in the preparation of a product; the product is any one selected from bacteriostatic agents, drugs, foods or food additives, feeds or feed additives.

[0013] Preferably, the product is used for at least one of the following aspects: (1) Bacteriostasis; (2) Improving the growth performance of livestock and poultry; (3)Improve the intestinal flora of animals; (4)Improve the intestinal digestion and absorption function of animals.

[0014] When Bacillus coagulans is used as a probiotic in feed and clinical applications, the viable cell count and spore rate during the fermentation production of Bacillus coagulans are particularly important. A large amount of lactic acid is produced during the fermentation process of Bacillus coagulans, which reduces the environmental pH value. When the pH value drops to a certain extent, its own growth will be inhibited; moreover, the conditions for spore formation are extremely harsh and are affected by many factors such as nutritional conditions and environmental factors. To achieve high-density fermentation and high spore conversion rate, it is necessary to take into account the appropriate medium components and fermentation process parameters.

[0015] Based on this, in order to improve the fermentation level of Bacillus coagulans, the present invention also provides a fermentation medium for fermenting and culturing the Bacillus coagulans, which comprises the following components in parts by weight: 20-30 parts of yeast extract, 10-15 parts of corn starch, 5-10 parts of glucose, 20-30 parts of peptone, 5-8 parts of beef extract, 6-12 parts of fermented soybean meal, 4-6 parts of sodium acetate, 1-1.5 parts of α-methyl glucoside, 0.1-0.2 parts of potassium sorbate, 2-3 parts of potassium dihydrogen phosphate, 0.5-1.0 part of magnesium sulfate, 0.2-0.3 part of manganese sulfate, 0.2-0.3 part of ferric sulfate, and 0.3-0.5 part of calcium chloride.

[0016] Furthermore, the present invention provides a fermentation culture method for the Bacillus coagulans, comprising: inoculating the Bacillus coagulans described in claim 1 into a fermentation medium (preferably the above fermentation medium), and performing fermentation culture under the conditions of 45°C-47°C, pH 5.5-6.0, and dissolved oxygen not less than 20%.

[0017] In the above fermentation culture method, the spore production rate can reach more than 90%. When the fermentation scale is a 50L fermentation tank, the cell concentration in the fermented broth at the end of fermentation can reach more than 10 billion CFU / mL, and the spore concentration in the fermented broth can reach more than 8 billion CFU / mL, effectively improving the problems of high contamination rate, low spore production rate, and low cell count existing in the traditional fermentation production of Bacillus coagulans, which is beneficial to improving the quality of Bacillus coagulans products.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a strain of Bacillus coagulans with excellent comprehensive performance. This strain can tolerate high temperatures of 80°C and can grow in an acidic environment with a pH of 1.2. At the same time, this strain is sensitive to 18 antibiotics and does not have drug resistance, with extremely high biosafety; moreover, this strain has a broad-spectrum antibacterial ability and has a significant inhibitory effect on a variety of pathogenic bacteria, and can be widely used in the breeding field, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the colony morphology diagram of Bacillus coagulans SDHY-2402.

[0020] Figure 2 This is the microscopic examination diagram of Gram staining of Bacillus coagulans SDHY-2402.

[0021] Figure 3 Growth curve of Bacillus coagulans SDHY-2402.

[0022] Figure 4 Calcium dissolution reaction diagram of Bacillus coagulans SDHY-2402.

[0023] Figure 5 Results of sensitivity test of Bacillus coagulans SDHY-2402 to different antibiotics.

[0024] Figure 6 Inhibitory effect diagram of Bacillus coagulans SDHY-2402 on various livestock and poultry pathogenic bacteria. Detailed implementation mode

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. In the embodiments provided in this specification, for those without specific technical or conditions indicated, the techniques or conditions described in the literature in the field are followed, or the product specifications are followed. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0026] Example 1 Identification of Bacillus coagulans SDHY-2402 strain I. Strain identification The selected pure colony was inoculated into 5 mL of modified bromocresol purple glucose broth medium and cultured in a shaker at 47 °C for 24 h. The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit according to the instructions. Using the 16S rRNA primers of the strain, which include 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID No.1) and 1492R: GGTTACCTTGTTACGACTT (SEQ ID No.2), a 16S rDNA fragment was amplified. The PCR product was sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing, and then BLAST alignment was performed (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). According to the alignment results, the sequence was identified as Bacillus coagulans (Bacillus coagulans strains with homology A > 99% of the 16S rDNA gene sequence as the identification criterion.

[0027] II. Growth characteristics and physiological and biochemical characteristics of Bacillus coagulans SDHY-2402 1. Colony morphology: Take the bacterial solution of Bacillus coagulans SDHY-2402 in the logarithmic growth phase, dilute it to different multiples according to the 10-fold dilution method, coat it on the modified bromocresol purple glucose broth agar plate, culture it at 47 °C for 18 - 36 h, take the plate with the number of colonies between 30 - 200 for observation and photographing, and the colony morphology is as Figure 1 shown. The colony size of this strain on the modified bromocresol purple glucose broth agar plate is 2 - 4 mm, showing a yellow opaque round colony, with a smooth surface and a convex center, and the colony edge is regular.

[0028] 2. Gram staining of Bacillus coagulans SDHY-2402 strain: Take the bacterial solution of Bacillus coagulans SDHY-2402 in the logarithmic growth phase, take 2 - 3 drops and coat them on the glass slide, fix them with the flame of the alcohol lamp, and operate according to the Gram staining kit. After staining, observe under the oil immersion lens of the microscope. The test results are as Figure 2 shown. Bacillus coagulans SDHY-2402 shows blue-purple rod-shaped, indicating that Bacillus coagulans SDHY-2402 is a Gram-positive bacterium.

[0029] 3. Determination of the growth curve of Bacillus coagulans SDHY-2402: The growth curve represents the dynamic changes of the whole process of bacteria growing, reproducing, and finally aging and dying in a new suitable environment. Inoculate Bacillus coagulans SDHY-2402 into the MRS liquid medium at an inoculation amount of 1% (v / v), culture it at 42 °C for 24 hours, use the MRS medium without the bacterial solution as the blank control, and plate count every 2 hours. The experiment is set with three replicates, and the results are averaged, record the data and draw the growth curve. As Figure 3 shown, from 2 - 16 hours, Bacillus coagulans SDHY-2402 is in the logarithmic growth stage with a relatively high reproduction rate. From 16 - 24 hours, the number of Bacillus coagulans SDHY-2402 tends to be stable.

[0030] 4. Physiological and biochemical identification Refer to "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria" for identification.

[0031] The biochemical identification of Bacillus coagulans SDHY-2402 was carried out with reference to the instructions of the HBI biochemical identification strip. The analysis and detection were carried out by two inoculation methods, namely inoculating the bacterial suspension and puncturing of the Bacillus coagulans SDHY-2402 strain, and various biochemical reactions of the strain were detected respectively, including V-P, citrate, gelatin liquefaction, 7% sodium chloride, growth at pH 5.7, nitrate growth, starch hydrolysis, propionate, D-xylose, L-arabinose and D-mannitol. The results are shown in Table 1, and Bacillus coagulans SDHY-2402 conforms to the biochemical characteristics of Bacillus coagulans.

[0032] Table 1 HBI biochemical identification

[0033] The lactic acid production ability of Bacillus coagulans SDHY-2402 was determined by the calcium dissolution reaction. Calcium carbonate generates soluble calcium lactate under the action of lactic acid. The solid MRS medium was sterilized at 121 °C, and calcium carbonate was sterilized separately after adding water. When the temperature of the MRS solid medium dropped to 45 - 55 °C, calcium carbonate was added to the solid medium at a dose of 0.5‰, and after thorough mixing, the plate was quickly poured to avoid calcium carbonate precipitation. The Bacillus coagulans SDHY-2402 strain was streaked and inoculated on the MRS solid medium supplemented with calcium carbonate and cultured at 42 °C for 36 - 48 h, and the size of the calcium dissolution circle was observed. The results are as Figure 4 shown, Bacillus coagulans SDHY-2402 has good lactic acid production ability and conforms to the lactic acid production characteristics of Bacillus coagulans.

[0034] Example 2 Detection of stress resistance of Bacillus coagulans SDHY-2402 1. Heat resistance detection 2 mL of the culture of Bacillus coagulans SDHY-2402 at the end of the growth stage with fully formed spores were taken respectively, centrifuged at 7000 rpm for 10 min at 4 °C, washed three times with PBS, and after resuspension, they were placed in a water bath at 60 °C, 70 °C, 80 °C, and 90 °C for 10 min respectively, and then cooled in an ice-water mixture at 0 °C. The gradient dilution plate coating method was used to determine their survival rate, with 3 replicates / sample. The measurement results are shown in Table 2.

[0035] Table 2 Heat resistance detection results

[0036] 2. Tolerance to artificial gastric juice and artificial intestinal juice (artificial gastric juice and artificial intestinal juice were prepared according to the Chinese Pharmacopoeia) Tolerance experiment of artificial gastric juice (pH 1.2): 2 mL of the culture at the end of the growth stage was taken, centrifuged at 7000 rpm for 15 min at 4 °C, washed three times with PBS and then resuspended, and at 10 8Inoculate at 10⁶ CFU / mL into artificial gastric juice, shake well, incubate in a water bath at 37 °C, take samples after 1 h, and perform viable count using the gradient dilution plate coating method, with 3 replicates per sample.

[0037] Tolerance to artificial intestinal juice (pH 6.8): Take 1 mL of the culture at the end of growth, centrifuge at 7000 rpm for 15 min at 4 °C, wash three times with PBS and resuspend, and inoculate at 10⁶ 8 CFU / mL into artificial intestinal juice, take samples after treatment for 2 h, and perform viable count using the gradient dilution plate coating method. The measurement results are shown in Table 3.

[0038] Table 3 Detection results of tolerance to artificial gastric juice and artificial intestinal juice

[0039] It can be seen from the above results that Bacillus coagulans SDHY-2402 can tolerate the environments of artificial gastric juice and artificial intestinal juice.

[0040] 3. Detection of bile salt tolerance Add bile salts to MRS liquid medium at mass fractions of 0.20%, 0.40%, 0.60%, 0.80%, and 1.0% respectively, sterilize at 121 °C for 15 min. Inoculate at 2%, incubate in an incubator at 47 °C, observe the growth of the bacteria, with 3 replicates per sample. "+++" indicates good growth, "++" indicates second best, "+" indicates slight growth, and "-" indicates no growth. The measurement results are shown in Table 4.

[0041] Table 4 Detection results of bile salt tolerance

[0042] 4. Enzyme production characteristics of Bacillus coagulans SDHY-2402 Inoculate the activated Bacillus coagulans SDHY-2402 into MRS liquid medium, culture at 47 °C and 200 r / min for 24 h to prepare a bacterial liquid for standby. Inoculate the bacterial liquid onto screening media containing amylase, protease, and lipase respectively, and culture at 42 °C for 24 h. For the amylase screening medium, add dilute iodine solution to make the starch change color, and then measure the diameter of the enzyme production circle. The measurement results are shown in Table 5.

[0043] Table 5 Evaluation of enzyme production effect

[0044] It can be seen from the results in Table 5 that Bacillus coagulans SDHY-2402 has excellent enzyme production effect.

[0045] Example 3 Sensitivity analysis test of Bacillus coagulans SDHY-2402 to different antibiotics The sensitivity of Bacillus coagulans SDHY-2402 to different antibiotics was determined by antibiotic susceptibility discs. Several common antibiotics were selected as representatives from each category to comprehensively evaluate the drug sensitivity of Bacillus coagulans SDHY-2402. Specifically, 1% of the Bacillus coagulans SDHY-2402 bacterial solution was added to the liquid MRS solid medium and mixed evenly until the MRS medium solidified and dried for 15 min. The tablet was placed in the center of the plate and gently pressed. One tablet was placed on each plate, and the plate was cultured in an incubator at 42 °C for 36 h, and the size of the antibacterial circle was observed. Each tablet was repeated three times. An inhibition zone diameter less than 15 mm was considered resistant, in the range of 16 - 20 mm was considered moderately sensitive, in the range of 21 - 25 mm was considered highly sensitive, and greater than 25 mm was considered extremely sensitive. The test results are as Figure 5 shown. After measuring the diameter of the inhibition zone and analysis, it was shown that Bacillus coagulans SDHY-2402 was sensitive to 18 antibiotics such as penicillin, tetracycline, and erythromycin (Table 6), indicating that Bacillus coagulans SDHY-2402 did not have drug resistance, so it was safe and reliable to be used as a feed probiotic.

[0046] Table 6 Antibiotic Sensitivity Results

[0047] Example 4 Safety Evaluation of Bacillus coagulans SDHY-2402 in Mice In this example, mice were used as experimental animals, and the gavage test method was adopted to evaluate the safety of Bacillus coagulans SDHY-2402. The specific method was as follows: 1. The bacterial solution of Bacillus coagulans SDHY-2402 was freeze-dried to make freeze-dried powder, and the steps were as follows: (1) The vacuum concentration method was used to reduce the volume.

[0048] (2) The freeze-drying protectant sucrose was added to prevent protein denaturation.

[0049] (3) Pre-freezing was carried out at -40 °C to -80 °C to ensure complete freezing.

[0050] (4) Under vacuum conditions, the temperature was slowly raised to -20 °C to -30 °C to sublime and remove ice crystals.

[0051] (5) The temperature was further raised to 20 °C to 30 °C to remove residual moisture.

[0052] (6) The freeze-dried block was crushed into a uniform powder.

[0053] (7) Sealed and packaged, and stored in a dry and cool place.

[0054] Determined by plate counting, the number of bacteria of Bacillus coagulans SDHY-2402 was 1×10 9CFU / g.

[0055] 2. Select 72 mice at about 8 weeks old and randomly divide them into 4 groups (Group A is the control group, intragastrically administered with sterile normal saline; Group B is the high-dose group, intragastrically administered with bacterial solution at a dose of 1×10 9 CFU / mouse; Group C is the medium-dose group, intragastrically administered with bacterial solution at a dose of 1×10 8 CFU / mouse; Group D is the low-dose group, intragastrically administered with bacterial solution at a dose of 1×10 7 CFU / mouse). Each group has 3 replicates, and each replicate has 6 mice.

[0056] 3. Administer intragastric gavage once at 3 pm every day for 21 consecutive days.

[0057] The temperature and humidity of the experimental mouse house are kept constant, with natural light. The mice are allowed to freely eat and drink water, and the mouse cages are cleaned once every 7 days. During the experiment, the status, survival situation, and any clinical abnormal symptoms of the mice are observed and recorded every day.

[0058] Detection indexes: (1) On the day of the end of the experiment, collect blood samples from the experimental mice by cardiac puncture. After static centrifugation, obtain serum for detecting blood biochemical indexes such as albumin, total protein, high-density lipoprotein, low-density lipoprotein, triglyceride, cholesterol, urea, and tumor necrosis factor in the serum.

[0059] (2) Take the intact heart, liver, spleen, and kidneys (bilateral) and weigh them wet. Calculate the heart index = (wet weight of the heart / body weight) × 100%, liver index = (wet weight of the liver / body weight) × 100%, spleen index = (wet weight of the spleen / body weight) × 100%, and kidney index = (wet weight of the kidney / body weight) × 100% respectively.

[0060] The survival situations of mice in different treatment groups are shown in Table 7.

[0061] Table 7 Survival situations of mice in different treatment groups

[0062] As can be seen from Table 7, after intragastrically administering Bacillus coagulans SDHY-2402 to mice for 21 days, all the mice in each treatment group survived, indicating that the above-mentioned Bacillus coagulans SDHY-2402 is safe for animals.

[0063] The organ coefficients of mice in different treatment groups are shown in Table 8.

[0064] Table 8 Organ coefficients of mice in different treatment groups

[0065] As can be seen from Table 8, there were no significant changes in the organ indices of the mice in the treatment group compared with those in the control group, indicating that Bacillus coagulans SDHY-2402 did not cause abnormalities in the organs of the mice.

[0066] In addition, biochemical analyzers were used to detect albumin, total protein, high-density lipoprotein, low-density lipoprotein, triglyceride, cholesterol, urea, tumor necrosis factor, etc. in the serum of the mice, and the results all showed normal, indicating that the preparation containing Bacillus coagulans SDHY-2402 had no impact on the physiological indices of the mice.

[0067] Example 5 In vitro antibacterial test Take the Bacillus coagulans SDHY-2402 bacterial liquid in the logarithmic growth phase, inoculate it into the MRS medium at a ratio of 1%, culture it at 42-47 °C for 24-36 h, centrifuge the cultured bacterial liquid at 7000 rpm for 15 min to separate the bacteria and the supernatant. The supernatant obtained from the first centrifugation is reserved for later use. The bacteria are resuspended with an equal volume of physiological saline, centrifuged at 7000 rpm for 15 min, and then the supernatant is discarded to obtain the bacteria. The bacteria are washed 3 times with physiological saline to obtain the bacteria.

[0068] Take Clostridium perfringens CVCC2027 preserved in a cryopreservation tube and inoculate it into the sterilized RCM liquid medium. Seal the surface with sterile liquid paraffin, and culture it statically at 47 °C for 12 h. Dilute the bacterial liquid with physiological saline to make the OD 600 between 0.04 and 0.06, inoculate it at a ratio of 1% into the solid RCM medium at 45-55 °C, mix well, pour 20 mL per plate onto the plain agar plate with Oxford cups placed on it, wait for it to cool and solidify, and then take out the Oxford cups. Add 200 μL of the test samples with different concentrations into the wells, put them into an anaerobic bag, add an anaerobic pack and seal it, and culture it at 37 °C for 16 h. The results are as Figure 6 shown.

[0069] Respectively pick Escherichia coli K99, Escherichia coli K88, Escherichia coli CVCC519, pathogenic Escherichia coli, Proteus CVCC1791, Aeromonas hydrophila bio-52500, Salmonella typhimurium SL1344, Pseudomonas aeruginosa, Staphylococcus epidermidis ATCC49134, Vibrio parahaemolyticus ATCC17802, Bordetella, Bacillus cereus and inoculate them into the sterilized LB liquid medium. After culturing at 37 °C for 12-14 h, dilute them with physiological saline to 1×10 8 CFU / mL, inoculate them at a ratio of 1% into the solid LB medium at 45-55 °C, mix well, pour 25 mL per plate onto the plain agar plate with Oxford cups placed on it, wait for it to cool and solidify, and then take out the Oxford cups. Add 200 μL of the test samples with different concentrations into the wells, let them stand at 4 °C for 2-4 h and then culture them at 37 °C for 16 h. The results are as Figure 6 shown.

[0070] The diameters of the antibacterial zones were measured, and the results are shown in Table 9. The in vitro antibacterial test showed that Bacillus coagulans SDHY-2402 had strong inhibitory effects on 13 kinds of pathogenic bacteria of livestock and poultry, including Escherichia coli K99, Escherichia coli K88, Escherichia coli CVCC519, pathogenic Escherichia coli, Proteus CVCC1791, Aeromonas hydrophila bio-52500, Salmonella typhimurium SL1344, Pseudomonas aeruginosa, Clostridium perfringens CVCC2027, Staphylococcus epidermidis ATCC49134, Vibrio parahaemolyticus ATCC17802, Bordetella, and Bacillus cereus. It can replace antibiotics in the intestine to inhibit the growth of harmful bacteria and has a good effect on adjusting the intestinal microecological environment.

[0071] Table 9 Statistical results of the diameters of antibacterial zones

[0072] Example 6 Fermenter test In this example, taking a 50L fermenter as an example, a fermentation and culture method for Bacillus coagulans is provided, including the following steps: (1) Inoculate the rejuvenated single colony of Bacillus coagulans SDHY-2402 into a shake flask and culture it by shaking for 24 h. The formula of the seed liquid medium: 30 g / L yeast extract powder, 30 g / L peptone, 10 g / L glucose, 8 g / L beef extract, 6.0 g / L sodium acetate, 3.0 g / L dipotassium hydrogen phosphate, 1.0 g / L magnesium sulfate, 0.5 g / L calcium carbonate, and 0.2 g / L manganese sulfate. Adjust the pH value of the medium to 6.2 - 6.5 and sterilize it at 115 °C for 20 min. The liquid loading volume in a 2000 mL shake flask is 500 mL, and the culture conditions: the rotation speed of the shake flask is 200 r / min, and the temperature is 42 °C.

[0073] (2) Inoculate the seed liquid cultured in the shake flask into the fermenter, with an inoculation amount of 2% - 3%, a fermenter volume of 50 L, and a liquid loading volume of 60%. The culture conditions: temperature 47 °C, initial rotation speed 150 rpm, maximum rotation speed 500 rpm, initial ventilation volume 15 L / min, maximum ventilation volume 65 L / min, tank pressure 0.04 Mpa, dissolved oxygen maintained at 20%, pH controlled at 5.5 - 6.0, culture time 24 - 26 h, and the composition of the fermentation medium: 25 g / L yeast extract powder, 10 g / L corn starch, 5 g / L glucose, 25 g / L peptone, 5 g / L beef extract, 10 g / L fermented soybean meal, 6 g / L sodium acetate, 1.0 g / L α-methyl glucoside, 0.1 g / L potassium sorbate, 0.3 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 0.3 g / L manganese sulfate, 0.3 g / L ferric sulfate, and 0.5 g / L calcium chloride.

[0074] (3) The fermentation broth was separated by a tubular centrifuge, and the bacterial sludge was collected. The Bacillus coagulans SDHY-2402 bacterial powder was obtained by spray drying. The inlet air temperature of the spray drying tower was 200 °C, and the outlet air temperature was 90 °C.

[0075] After detection, the cell concentration in the fermentation broth before discharging was 11 billion CFU / mL, and the sporulation rate could reach 90.7%; the number of spores in the bacterial powder was 8×10 11 CFU / g.

[0076] It should be noted that the fermentation and culture method provided in this example is applicable to the shake flask fermentation stage, small-scale test stage, pilot-scale test stage, large-scale trial production stage, and industrial production stage. The fermentation scale includes but is not limited to 50L fermenters, 100L fermenters, 200L fermenters, 500L fermenters, 3-ton fermenters, 5-ton fermenters, etc. Those skilled in the art can prepare the seed liquid step by step according to the fermentation scale and relying on the professional knowledge they have mastered.

[0077] Comparative Example 1 This comparative example provides a fermentation and culture method of Bacillus coagulans, in which the fermentation scale is a 50L fermenter. Compared with the fermentation process of Example 6, the difference in the fermentation process of this comparative example is only that: the fermentation medium is different.

[0078] The fermentation medium of this comparative example is as follows: 20 g / L yeast extract powder, 20 g / L peptone, 2 g / L beef extract, 5 g / L fermented soybean meal, 3 g / L sodium acetate, 0.5 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.1 g / L manganese sulfate, and 0.1 g / L ferric sulfate.

[0079] In this comparative example, the fermentation broth before discharging was diluted and spread on an MRS medium plate. Three parallel samples were set for each dilution. After static culture at 45 °C for 24 h, counting was carried out. It can be known from the counting that the cell concentration in the fermentation broth before discharging was 5.6 billion CFU / mL, the spore concentration was 3 billion CFU / mL, and the number of spores in the bacterial powder was 2.2×10 11 CFU / g.

[0080] Comparative Example 2 This comparative example provides a fermentation and culture method of Bacillus coagulans, in which the fermentation scale is a 50L fermenter. Compared with the fermentation process of Example 6, the difference in the fermentation process of this comparative example is only that: the fermentation medium is different.

[0081] The fermentation medium of this comparative example is as follows: 25 g / L yeast extract powder, 25 g / L peptone, 5 g / L beef extract, 8 g / L fermented soybean meal, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.2 g / L manganese sulfate, and 0.2 g / L ferric sulfate.

[0082] In this comparative example, the fermented broth taken out of the fermenter was diluted and spread on MRS medium plates. Three parallel samples were set for each dilution. After static cultivation at 45 °C for 24 h, the count was performed. It was found through counting that the cell concentration in the fermented broth taken out of the fermenter was 7.2 billion CFU / mL, the spore concentration was 4.5 billion CFU / mL, and the number of spores in the bacterial powder was 3.4×10 11 CFU / g.

[0083] Comparative Example 3 This comparative example provides a fermentation process for Bacillus coagulans, and the fermentation scale is a 50 L fermenter. Compared with the fermentation process of Example 6, the difference in the fermentation process of this comparative example lies in: the fermentation media are different.

[0084] The fermentation medium of this comparative example is: 10 g / L glucose, 20 g / L yeast extract powder, 20 g / L peptone, 2 g / L beef extract, 5 g / L fermented soybean meal, 3 g / L sodium acetate, 0.5 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.1 g / L manganese sulfate, and 0.1 g / L ferric sulfate.

[0085] In this comparative example, the fermented broth taken out of the fermenter was diluted and spread on MRS medium plates. Three parallel samples were set for each dilution. After static cultivation at 45 °C for 24 h, the count was performed. It was found through counting that the cell concentration in the fermented broth taken out of the fermenter was 8.7 billion CFU / mL, the spore concentration was 1.5 billion CFU / mL, and the number of spores in the bacterial powder was 1.1×10 11 CFU / g.

[0086] Application of Bacillus coagulans SDHY - 2402 in broiler feeding and its regulation effect on intestinal flora I. Effect of Bacillus coagulans SDHY - 2402 on the growth performance of AA broilers The bacterial powder prepared by fermenting Bacillus coagulans SDHY-2402 according to Example 6 was added to the basal diet of AA broilers at a weight ratio of 0.5‰. A total of 192 one-day-old AA broilers were selected for the experiment and divided into two treatment groups, with six replicates in each treatment group and 16 chickens in each replicate. The blank control group was fed the basal diet, and the treatment group was fed the basal diet + 0.5‰ of the fermented bacterial powder. The experiment was divided into the early stage and the late stage. The early stage was from 1 to 21 days, and the late stage was from 22 to 42 days. During the experiment, the temperature and humidity of the chicken house were observed and adjusted daily. The body weights of the broilers were weighed on days 1, 21, and 42, and the feed intakes of the broilers were also weighed. After the experiment, the average daily gain, average daily feed intake, and feed-to-weight ratio were calculated for each replicate. The experimental results showed that adding 0.5‰ of Bacillus coagulans SDHY-2402 bacterial powder to the diet could significantly increase the average daily gain of broilers, improve the feed intake, and enhance the feed conversion efficiency. Among them, the average daily gain increased by 12.31%, the average daily feed intake increased by 9.52%, and the feed-to-weight ratio decreased by 2.04% (Table 10).

[0087] Table 10 Effects of Bacillus coagulans SDHY-2402 on the growth performance of AA broilers

[0088] II. Regulatory effect of Bacillus coagulans SDHY-2402 on the intestinal flora of AA broilers After the above feeding experiment, jejunum, ileum, and tissue samples of AA broilers were collected and fixed in 4% paraformaldehyde. After making sections by HE staining method, they were used to observe the tissue morphology. The results showed that feeding 0.5‰ of the fermented bacterial powder had no obvious damage to the small intestine of AA broilers. The intestinal villus height was measured using ImageJ software. The experimental results showed that Bacillus coagulans SDHY-2402 could significantly increase the ileal villus height and improve the intestinal digestion and absorption function of AA broilers.

[0089] The ileal chyme of AA broilers was collected for analysis of the intestinal microbial composition. The chyme samples were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for microbiome detection and analysis. The 16S rDNA sequencing method was used to analyze the ileal chyme samples. The results showed that the abundance-based coverage estimator index (ACE) and Chao1 index values of the treatment group supplemented with Bacillus coagulans SDHY-2402 were higher than those of the control group. Using NMDS to analyze β-diversity, the results showed that there was no obvious difference between the treatment group supplemented with Bacillus coagulans SDHY-2402 and the control group.

[0090] From the analysis at the microbial genus level, after adding Bacillus coagulans SDHY-2402, the ileal flora changed significantly, and the dominant flora changed from Romboutsia Rothia Lactobacillus, in addition, the addition of strain SDHY-2402 reduced Romboutsia , Turicibacter Turicibacter , Candida albicans Candidatus_Arthromitus and the abundance of Rothia mucilaginosa, and the abundances of Lactobacillus Lactobacillus , Streptococcus Streptococcus , Peptostreptococcus Peptostreptococcaceae , Bacillus Bacillus and Eubacterium lentum Blautia increased, which are related to probiotics.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus coagulans ( Bacillus coagulans ), characterized in that It is Bacillus coagulans SDHY-2402, and its preservation number is CGMCC No. 32910.

2. A microbial inoculant, characterized in that, It contains Bacillus coagulans SDHY-2402 described in claim 1.

3. The microbial inoculant according to claim 2, characterized in that, The microbial inoculum is the freeze-dried powder of the fermentation broth of Bacillus coagulans SDHY-2402.

4. The microbial inoculum according to claim 3, characterized in that, The freeze-dried powder of the fermentation broth also includes a freeze-drying protectant.

5. A product, characterized in that, The product contains Bacillus coagulans SDHY-2402 described in claim 1 or the microbial inoculum described in any one of claims 2 to 4; the product is any one selected from bacteriostatic agents, drugs, foods or food additives, feeds or feed additives.

6. Use of Bacillus coagulans SDHY-2402 described in claim 1 or the microbial inoculum described in any one of claims 2 to 4 in the preparation of a product; the product is any one selected from bacteriostatic agents, drugs, foods or food additives, feeds or feed additives.

7. The application according to claim 6, wherein The product is used in at least one of the following aspects: (1) Bacteriostasis; (2) Improving the growth performance of livestock and poultry; (3) Improving the animal intestinal flora; (4) Improving the animal intestinal digestion and absorption function.

8. The fermentation medium for fermenting and culturing Bacillus coagulans described in claim 1, characterized in that, It includes the following components in parts by weight: 20-30 parts of yeast extract, 10-15 parts of corn starch, 5-10 parts of glucose, 20-30 parts of peptone, 5-8 parts of beef extract, 6-12 parts of fermented soybean meal, 4-6 parts of sodium acetate, 1-1.5 parts of α-methylglucoside, 0.1-0.2 parts of potassium sorbate, 2-3 parts of potassium dihydrogen phosphate, 0.5-1.0 part of magnesium sulfate, 0.2-0.3 part of manganese sulfate, 0.2-0.3 part of ferric sulfate, and 0.3-0.5 part of calcium chloride.

9. The fermentation and culture method of Bacillus coagulans according to claim 1, characterized in that, It includes: Inoculate Bacillus coagulans described in claim 1 into a fermentation medium, and carry out fermentation culture under the conditions of 45°C - 47°C, pH 5.5 - 6.0 and dissolved oxygen not less than 20%.

10. The fermentation culture method according to claim 9, characterized in that, The fermentation medium is the fermentation medium described in claim 8.

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