CN112522134B - Bacillus coagulans and application thereof - Google Patents

Abstract

The invention provides bacillus coagulans and application thereof, and belongs to the technical field of microorganisms. The Bacillus coagulans (Bacillus coagulans) HOM5301 disclosed by the invention is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.20383, and the preservation date is 2020, 7 and 16 days. The Bacillus coagulans strain can enhance organism immunity, enhance cellular immunity and monocyte-macrophage function, and inhibit proliferation of common pathogenic bacteria. Meanwhile, the bacillus coagulans strain can be fermented to produce fermented milk, oat milk, soybean milk and other fermented products. Has effects in enhancing immunity, promoting digestion, improving food nutrient availability, and improving taste. Has wide application prospect.

Description

A strain of Bacillus coagulans and its application

technical field

The invention relates to the technical field of microorganisms, in particular to a strain of bacillus coagulans and its application.

Background technique

Bacillus coagulans is a Gram-positive, microaerobic, lactic acid-producing spore. It not only has the characteristics of lactic acid bacteria, but also has the characteristics of strong stress resistance, high temperature resistance, and easy storage of spores. It is an excellent choice for probiotics. The strain has been included in the list of biological agents recommended by the European Union Safety Qualification (QPS), and included in the International Dairy Federation (IDF) "microorganism list with a history of safe use in food", and has passed the US Food and Drug Administration Bureau's GRAS (Generally Recognized as Safe Substances) certification. In 2016, the Chinese Health and Family Planning Commission also approved Bacillus coagulans as an edible strain.

Clinical studies have shown that some strains of Bacillus coagulans can significantly improve constipation, diarrhea, irritable bowel syndrome or ulcerative colitis. For example, in the patent application with publication number CN10056924A, a medicinal Bacillus coagulans for treating ulcerative colitis is disclosed. For example, the patent application document with publication number CN111004732A discloses a Bacillus coagulans capable of promoting the secretion of motilin. The Bacillus coagulans has the effect of up-regulating motilin and promoting gastrointestinal motility. But so far, there are still very few domestically disclosed strains of Bacillus coagulans that can improve the specific health status of the human body. Therefore, it is necessary to find other Bacillus coagulans that can improve specific health conditions in humans.

Contents of the invention

In view of this, the present invention provides a bacillus coagulans and application thereof. Different from the disclosed bacillus coagulans, the bacillus coagulans in the present invention can enhance the immunity of the body, enhance the cellular immune function and monocyte-macrophage function, can inhibit the proliferation of common pathogenic bacteria, and can be used to enhance immunity In the production of functional fermented food, it can promote the digestive function of the body, improve the nutritional utilization of food, and improve the taste.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a strain of Bacillus coagulans (Bacillus coagulans) HOM5301. The Bacillus coagulans (Bacillus coagulans) is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee. The preservation number is CGMCC No.20383, and the preservation date is 2020 July 16.

The present invention provides the application of the above-mentioned Bacillus coagulans (Bacillus coagulans) HOM5301 in the preparation of a composition for enhancing body immunity.

In some embodiments of the present invention, the enhancement of immune function of the body includes enhancement of cellular immune function and monocyte-macrophage function.

In some embodiments of the present invention, the dosage form of the composition includes any one of powder, granule, solution or colloid.

In some embodiments of the present invention, the composition includes food, health product, medicine or feed.

The present invention provides a microbial bacterial agent, which contains the above-mentioned Bacillus coagulans (Bacillus coagulans) HOM5301.

In some embodiments of the present invention, the microbial agent also includes auxiliary materials.

The invention provides the application of the above-mentioned microbial agent in the preparation of food, health products, feed and medicine.

The present invention provides the method for preparing above-mentioned microbial bacterial agent, comprises the following steps:

The above-mentioned Bacillus coagulans (Bacillus coagulans) HOM5301 is expanded and cultivated in a liquid medium, and the bacteria are collected;

Add the protective agent to resuspend the bacteria body collected after the expanded culture, dry and pulverize to obtain the active spore bacteria agent.

The present invention also provides a fermented product, which is obtained by fermenting the above-mentioned Bacillus coagulans (Bacillus coagulans) HOM5301.

In some embodiments of the present invention, the fermented products include fermented milk products, fermented oat products or fermented soybean products.

Compared with the prior art, the present invention provides a Bacillus coagulans HOM5301 whose preservation number is CGMCC No.20383. The Bacillus coagulans strain has the following effects:

(1) The Bacillus coagulans of the present invention can enhance the body's immunity, enhance cellular immune function and monocyte-macrophage function, and can inhibit the proliferation of common pathogenic bacteria.

(2) The active spore bacterial agent of the present invention has simple production process parameters, is easy to control, and has a short cycle, which ensures a high survival rate of Bacillus coagulans, and the obtained product can be stored for a long time with stable product quality.

(3) The Bacillus coagulans of the present invention can be fermented to produce fermented milk, oat milk, soybean milk and other fermented products. It can enhance the immunity of the human body, promote the digestive function of the body, improve the nutritional utilization of food, and improve the taste.

Description of Biological Preservation:

Biological material: HOM5301, classification name: Bacillus coagulans (Bacillus coagulans), was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on July 16, 2020. The address of the preservation center is: Hospital No. 1, Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences; deposit number is CGMCC No.20383.

Description of drawings

Figure 1 is based on the RAPD cluster analysis diagram of Bacillus coagulans constructed by the UPGMA method;

Fig. 2 is a schematic diagram of test results of cellular immune function and monocyte-macrophage function in different groups.

Detailed ways

The embodiments of the present invention will be further described below in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art will not depart from the spirit and spirit of the present invention Within the scope, some changes and modifications can be made, so the protection scope of the present invention should be defined by the scope of the appended patent application.

The present invention is a novel strain of Bacillus coagulans (Bacillus coagulans) HOM5301. The Bacillus coagulans (Bacillus coagulans) is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee. The preservation number is CGMCC No.20383, and the preservation date is 2020 July 16th. The bacillus coagulans of the invention can enhance body immunity, enhance cellular immune function and mononuclear-macrophage function, and can inhibit the proliferation of common pathogenic bacteria.

The Bacillus coagulans HOM5301 is isolated from dairy products.

The random polymorphic DNA analysis (RAPD) of the isolated Bacillus coagulans HOM5301 shows that HOM5301 is different from some commercial Bacillus coagulans strains and has uniqueness.

The suitable growth temperature of the Bacillus coagulans (Bacillus coagulans) HOM5301 is 37°C-45°C.

The Bacillus coagulans (Bacillus coagulans) HOM5301 has the characteristics of acid resistance and bile salt resistance, and has a relatively high survival rate in the gastrointestinal tract.

The Bacillus coagulans (Bacillus coagulans) HOM5301 has the characteristic of high temperature resistance, and the survival rate is above 50% after being treated at 140°C for 120s.

The Bacillus coagulans (Bacillus coagulans) HOM5301 can produce amylase and has the ability to ferment cereals with high starch content.

The Bacillus coagulans HOM5301 can enhance cellular immune function and monocyte-macrophage function.

The Bacillus coagulans (Bacillus coagulans) HOM5301 has an inhibitory effect on various pathogenic bacteria, and the pathogenic bacteria include Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes and the like.

The Bacillus coagulans (Bacillus coagulans) HOM5301 in the present invention can be applied to the preparation of a composition for enhancing the body's immunity.

Wherein, the dosage form of the composition includes any one of powder, granule, solution or colloid.

The composition includes food, health product, medicine or feed.

The present invention also provides a microbial agent, which contains Bacillus coagulans (Bacillus coagulans) HOM5301.

In some embodiments, the microbial agent also includes auxiliary materials.

The preparation method of described microbial bacterial agent is as follows:

For the isolated Bacillus coagulans (Bacillus coagulans) HOM5301. Carry out amplified culture, collect the bacteria by centrifugation, add appropriate protective agent to resuspend, and then dry to obtain active spore powder.

The preparation method of described microbial bacterial agent specifically comprises the following steps:

(1) Strain fermentation

Inoculate Bacillus coagulans (Bacillus coagulans) HOM5301 in sterile TSB liquid medium with an inoculation amount of 0.5-3% (v/v), culture at 37-45°C, 180-250rpm for 16-24 hours, and continuously subculture Twice, to obtain the activated seed culture liquid. The seed culture liquid is inoculated in the fermentation medium with an inoculum amount of 0.5-3% (v/v). 37-45°C, 200-600rpm constant temperature culture. During the fermentation process, sodium hydroxide solution and citric acid solution are automatically added to keep the pH at 5.5-7.0. When the acid supplement is stopped, take the fermentation liquid for microscopic examination, when more than 70-90% of the bacteria have formed spores, that is, the end of the fermentation is reached, and the high-density culture medium of Bacillus coagulans HOM5301 is obtained, and the number of spores can reach 1-4 billion CFU/ More than mL. The composition of the fermentation medium is: glucose 5-20g/L, yeast extract 5-20g/L, manganese sulfate 0.005-0.02g/L, magnesium sulfate 0.1-0.5g/L, dipotassium hydrogen phosphate 1-4g/L, Calcium carbonate 0.5-4g/L, sodium chloride 1-5g/L.

(2) Preparation of bacteria powder

The fermentation liquid of Bacillus coagulans (Bacillus coagulans) HOM5301 was collected by centrifugation. Discard the supernatant, and wash the sludge with 0.9% sterile saline for 1-2 times. Mix the washed bacteria slime with the protective agent. The final concentration of the protective agent is: 50-200g/L skimmed milk powder, 20-60g/L trehalose, 1-5g/L vitamin C, 3-8g/L L-glucose Sodium Amino Acid, followed by vacuum freeze-drying. After the freeze-drying is completed, the bacteria cake is crushed with a fine grinder to obtain the freeze-dried bacteria powder. The number of active spores in the freeze-dried powder is higher than (1-4)×10 ¹¹ CFU/g, and the freeze-dried bacteria powder The spore rate is above 80-90%.

The present invention also provides a fermented product obtained by fermenting with Bacillus coagulans HOM5301.

Specifically, Bacillus coagulans (Bacillus coagulans) HOM5301 can be inoculated into skim milk powder, oat milk or soybean milk, and fermented at 37-45°C to obtain a fermentation product. The fermented product contains a large amount of Bacillus coagulans (Bacillus coagulans) HOM5301, which can enhance the body's immunity, promote digestion, improve the nutrient utilization of food and improve the taste.

In order to further understand the present invention, the Bacillus coagulans provided by the present invention and its application are described in detail below in conjunction with the examples, and the protection scope of the present invention is not limited by the following examples.

Example 1 Isolation and Identification of Bacillus coagulans HOM5301

Weigh 3g of dairy products, shake evenly with 10mL TSB liquid medium to obtain a mixed solution, put in a water bath at 80°C for 10 minutes, absorb 1mL of the mixed solution and dilute the sample by 10-fold dilution method, dilute to 1000 times, and spread the diluted solution on a PCA solid plate Medium, 50 ℃ aerobic culture for 48-72h. Pick a single colony, streak culture, purify 3-4 times until the colony is single, and at the same time perform Gram staining and microscopic examination to observe the shape of the colony. Transfer a single colony to TSB liquid medium for pure culture, keep the seeds in glycerol and number them.

Random polymorphic DNA analysis (RAPD) was performed on the isolated Bacillus coagulans HOM5301. As shown in Figure 1, HOM5301 is different from some commercial Bacillus coagulans strains, with a genotype difference rate of nearly 50%, which is unique.

Embodiment 2 prepares active spore bacteria powder

Cultivation of strains: Bacillus coagulans HOM5301 frozen at -80°C was inoculated in sterile TSB liquid medium with an inoculum of 1% (v/v), cultured at 45°C and 250rpm for 16 hours, and subcultured for two Obtain the seed culture liquid after activation for the first time; The seed culture liquid is inoculated in the fermentation medium with the inoculum size of 2% (v/v), 45 ℃, 400rpm constant temperature cultivation, automatic flow adds sodium hydroxide solution and lemon in the fermentation process The acid solution maintains a constant pH of 6.0. When the acid supplement is stopped, take the fermentation liquid for microscopic examination. When more than 80% of the bacteria have formed spores, the end of the fermentation is reached, and the high-density culture medium of Bacillus coagulans HOM5301 is obtained, and the number of spores can reach More than 2 billion CFU/mL; wherein the formulation of the fermentation medium is shown in Table 1.

Table 1 Fermentation Medium Formula

Preparation of freeze-dried protective agent: use sterile water to mix the protective agent raw materials to prepare a protective agent containing 100g/L skimmed milk powder, 50g/L trehalose, 3g/L vitamin C, and 5g/L L-sodium glutamate;

Preparation of active spore bacteria powder: Centrifuge the cultured Bacillus coagulans HOM5301 fermentation broth at 2-8°C, 6000rpm for 10min, discard the supernatant, collect the sludge, and wash the sludge with 0.9% sterile saline 1 -2 times, mix the washed bacteria slime with the above-mentioned protective agent, so that the bacterial concentration of the mixed bacteria solution reaches more than 10 ¹⁰ CFU/mL, and freeze-dry in a freeze dryer. After the freeze-drying is completed, use a fine grinder to grind The bacteria cake is pulverized to obtain the freeze-dried bacteria powder, the number of live bacteria in the freeze-dried bacteria powder is higher than 1.0×10 ¹¹ CFU/g, and the spore rate of the freeze-dried bacteria powder is more than 80%.

Embodiment 3 resistance to gastrointestinal environment test

Take 1 g of the bacterial powder prepared in Example 2, add it to 9 mL of artificial gastric juice (pH 3.0), mix it and count it, record it as C0, place it in a 37°C incubator for 3 hours and count it, record it as C1. The liquid was centrifuged at 8000rpm for 10min to obtain the sludge, which was transferred to an equal volume of artificial intestinal fluid (pH 6.8), mixed and incubated at 37°C for 3h. MRS medium was used to count viable bacteria on the plate, which was recorded as C2, and the formula for calculating the survival rate was as follows:

Artificial gastric juice 3-hour survival rate (%)=(C1/C0)×100%

Artificial gastric juice 3 hours + artificial intestinal juice 3 hours survival rate (%) = (C2/C0) × 100%

C0=the initial number of viable bacteria, C1=the number of viable bacteria after being treated with gastric juice for 3 hours, C2=the number of viable bacteria after being treated with gastric juice for 3 hours and intestinal juice for 3 hours.

The survival rate of Bacillus coagulans in the simulated gastrointestinal fluid is listed in Table 2, as can be seen from Table 2: after 3 hours of simulated artificial gastric juice treatment, the survival rate of Bacillus coagulans HOM5301 can reach more than 90%. After the liquid was treated with simulated intestinal juice for 3 hours, its survival rate was still as high as 70%, which indicated that Bacillus coagulans had a higher survival rate in gastric juice and intestinal tract.

Table 2 Survival rate of Bacillus coagulans in simulated gastrointestinal fluid

Embodiment 4 high temperature resistance test

Weigh 1 g of the bacterial powder prepared in Example 2 into 9 ml of normal saline, and use the gradient dilution method to sequentially dilute to a suitable gradient, then transfer the bacterial liquid to an MRS solid plate, and count after culturing at 37-45°C. The bacterial powder was treated at 90°C and 140°C for 30s, 60s, 90s, and 120s respectively, counted and recorded the survival rate. The results are shown in Table 3. It can be seen that the survival rate of Bacillus coagulans HOM5301 can still reach more than 50% after being treated at 140°C for 120s, indicating that the active spore powder has strong heat resistance.

Table 3 High temperature resistance of Bacillus coagulans HOM5301

Example 5 Antibiotic Susceptibility Test

The drug susceptibility test was carried out according to the K-B agar method recommended by the National Committee for Clinical Standards (NCCLS), and the drug susceptibility disc was purchased from Beijing Tiantan Pharmaceutical Biotechnology Co., Ltd.

First inoculate Bacillus coagulans HOM5301 on a TSA agar plate, culture at 45°C for 18-24 hours, then pick pure colonies, place them in sterile saline, and make a bacterial suspension with a 0.5 McFarland turbidity standard. Then mix the bacterial suspension with TSB agar medium evenly, pour it into the plate, and place the drug-sensitive paper on the plate after the plate solidifies. Place the plate in a 45°C incubator and incubate for 24-48 hours, take it out, measure and record the diameter of the bacterial circle with a ruler, and finally interpret it according to the CLSI criteria. The results are shown in Table 4. The results showed that HOM5301 was sensitive to all nine antibiotics.

Table 4 Bacillus coagulans HOM5301 to 9 kinds of antibiotic susceptibility assay results

S (susceptible) means sensitive; I (intermediate) means medium; R (resistance) means resistance

Embodiment 6 inhibits common pathogenic bacteria ability test

The indicator bacteria (Escherichia coli ATCC8739; Staphylococcus aureus ATCC6538; Salmonella typhimurium ATCC14028; Pseudomonas aeruginosa ATCC9027; Listeria monocytogenes ATCC19111) were inoculated in TSB medium with 1% (v/v) inoculum Medium, 37°C, cultured for 24h for later use. Then Bacillus coagulans HOM5301 was respectively inoculated into the sterilized MRS medium at an inoculation amount of 1% (v/v), shaken and cultured at 37-45° C. for 24 hours, and activated twice to obtain a fermentation broth. 8000rpm, centrifuge for 10min, take the supernatant, pass through a 0.22μm filter membrane, freeze-dry, dissolve the freeze-dried powder, and obtain a ten-fold concentrated fermentation supernatant, which is dissolved in a phosphate buffer (pH=7.0) with a concentration of 50mmol/L Prepare catalase as a mother liquor, add it to the supernatant at a final concentration of 1 mg/mL, take it out after warming at 37°C for 2 hours, then boil it in a water bath at 100°C for 5 minutes to inactivate the enzyme, and then adjust to pH 5.0 to conduct the antibacterial test . Then, add the indicator bacteria into the TSA medium, shake evenly, pour into the pre-prepared TSA agar plate, and let it stand to solidify. Finally, place the Oxford cup on a solid nutrient agar plate with sterile tweezers, add 0.2 mL of the supernatant of the Bacillus coagulans HOM5301 fermentation broth to be tested into the wells, place it in a refrigerator at 4°C for 4 hours, and place it at 37°C Cultivate in the incubator for 12-18 hours, observe and measure the diameter of the inhibition zone. MRS ten-fold concentrated solution was used as a negative control, and three parallels were performed for each sample. It can be seen from Table 5 that Bacillus coagulans HOM5301 has inhibitory effect on the five pathogenic bacteria, and it is speculated that Bacillus coagulans HOM5301 may produce antibacterial substances other than organic acids and hydrogen peroxide.

The inhibitory effect of table 5 Bacillus coagulans HOM5301 on pathogenic bacteria

Note: "-" has no antibacterial activity; "+" has antibacterial activity.

Embodiment 7 produces amylase activity test

Amylase producing medium formula: peptone 10g, yeast powder 5g, soluble starch 5g, potassium dihydrogen phosphate 2g, magnesium sulfate heptahydrate 0.5g, calcium chloride dihydrate 0.2g, water 1L, pH 7.0. Sterilized at 121°C for 20 minutes before use.

Transfer Bacillus coagulans HOM5301 to TSB liquid medium, activate it twice at 45°C, transfer to amylase-producing medium, shake and culture at 45°C for 24h-48h, centrifuge at 8000rpm for 10min, and take the supernatant to pass through 0.22μm Sterilize through a membrane and set aside. According to the operation of the amylase kit (Nanjing Jiancheng C016-1-1), the starch hydrolysis rate and amylase activity of the bacterial strain were measured, and the results are shown in Table 6. It can be seen from the data in Table 6 that Bacillus coagulans HOM5301 is rich in amylase, which can partially hydrolyze starch, the hydrolysis rate can reach 12.3±0.6%, and the amylase activity is 476.4±5.5U/dL. This shows that the strain has the ability to ferment high starch content cereals.

Table 6 Determination of starch hydrolysis rate and amylase activity of Bacillus coagulans HOM5301

Example 8 In vitro cytokine secretion test

Bacillus coagulans HOM5301 was inoculated in TSB medium, and after two generations of continuous activation, it was used for counting viable bacteria and cell experiments. After centrifuging at 8000rpm for 10min, after collecting the bacteria, use DMEM complete medium without antibiotics to adjust to the working concentration (about 5.0×10 ⁵ CFU/mL); mouse macrophage RAW264.7 (about 5×10 ⁵ cell/mL) into a 24-well culture plate, 1 mL per well; after 2 hours of attachment, discard the medium, and add 1 mL of bacteria-containing medium to each well; set a blank control group, add 1 mL of DMEM medium; collect after 24 hours of co-cultivation The cell supernatant was assayed for the contents of TNF-α and IL-6 in the cell supernatant by enzyme-linked immunosorbent assay (ELISA) according to the instructions of the kit, and the results are shown in Table 7. It can be seen from Table 7 that Bacillus coagulans HOM5301 can significantly increase the secretion of TNF-α and IL-6 from RAW264.7 cells. It can be seen that Bacillus coagulans HOM5301 has potential immune regulation ability.

Table 7 HOM5301 promotes mouse macrophages to secrete cytokines

Note: **Compared with HOM5301 control group, P<0.01

Embodiment 9 Immunity-enhancing animal test

96 18g-20g SPF grade KM female mice bred by Beijing Huafukang Biotechnology Co., Ltd. were selected. After the animals were adapted to feeding and observed, they were randomly divided into four batches, with 2 groups of 12 in each group. The experimental group was fed with active bacterial powder of Bacillus coagulans HOM5301, each mouse was fed with 10 ⁹ CFU per day for 30 consecutive days. The control group was administered with normal saline at the same time. The first batch of experiments was carried out with carbon clearance experiments; the second batch of experiments was carried out with organ/body weight ratio determination and delayed type hypersensitivity experiments; Cell transformation experiments. For specific experimental methods, refer to "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition), and the data of the test results were statistically analyzed using SPSS software. For specific experimental test results, please refer to FIG. 2 and Tables 8 to 11.

As can be seen from the results in Table 8, compared with the control group, there was no significant difference in the body weight of the mice in the HOM5301 group before and after the experiment (P>0.05), which indicated that the active Bacillus coagulans HOM5301 powder had no effect on the body weight of the mice.

As can be seen from the results in Table 9, compared with the control group, there was no significant difference in the spleen/body and thymus/body of the mice in the HOM5301 group (P>0.05). No effect.

From the results in Table 10, it can be seen that the active bacterial powder of Bacillus coagulans HOM5301 can significantly improve the degree of thickening of mouse paws induced by sheep red blood cells (SRBC) (P<0.05) and the proliferation ability of mouse spleen lymphocytes induced by ConA (P<0.05 ), which shows that the active bacterial powder of Bacillus coagulans HOM5301 can significantly enhance the cellular immune function of mice.

From the results in Table 11, it can be seen that the active bacterial powder of Bacillus coagulans HOM5301 can significantly improve the carbon clearance ability of mice (P<0.01), the phagocytosis rate of chicken red blood cells by mouse macrophages (P<0.01) and the phagocytosis index (P<0.01) , which shows that the active bacterial powder of Bacillus coagulans HOM5301 can significantly enhance the function of mouse monocyte-macrophage.

In summary, compared with the control group, the active bacterial powder of Bacillus coagulans HOM5301 can significantly enhance the cellular immune function and monocyte-macrophage function of mice, but has no effect on the body weight, thymus and spleen weight of mice. In summary, the active bacterial powder of Bacillus coagulans HOM5301 has the function of enhancing immunity.

Table 8 Effect of HOM5301 bacterial powder on body weight of mice

Table 9 Effect of HOM5301 active bacterial powder on the visceral/body ratio of mice

Table 10 Effect of HOM5301 bacterial powder on sheep red blood cell (SRBC)-induced mouse DTH and effect on ConA-induced proliferation of mouse spleen lymphocytes

Note: *P<0.05 compared with the control group.

Table 11 Effect of HOM5301 bacterial powder on carbon clearance ability and macrophage phagocytosis of chicken red blood cells

Note: **P<0.01 compared with the control group.

Embodiment 10 fermented milk test

Weigh 12g of skimmed milk powder, 2g of glucose and 2g of desalted whey powder, make up to 100mL with double distilled water, stir evenly, homogenize with a high-pressure homogenizer, sterilize at 95°C-100°C for 5min-10min, cool to 37°C for use;

Inoculate the seed solution of Bacillus coagulans HOM5301 into TSB liquid medium, culture it with shaking at 37°C-45°C for 24 hours, pass this method twice to obtain a highly active bacterial solution, and store it in a refrigerator at 4°C for later use;

The bacterial solution was eluted twice with 0.9% normal saline, inoculated into the prepared milk with an inoculation amount of 1×10 ⁷ CFU/mL, mixed evenly, left to ferment at a constant temperature of 37-45°C for 16 hours, and filled into a packaging container , to obtain fermented milk products;

The above-mentioned fermented milk samples were sampled and counted by plate counting method after gradient dilution. The content of active Bacillus coagulans HOM5301 in the fermented milk is higher than 5.0×10 ⁸ CFU/mL, the pH can reach 5.00, and the taste is good.

Example 11 Fermented oat milk test

Weigh 120-150g of enzymatic oat flour, make up to 1000mL with double distilled water, homogenize with a high-pressure homogenizer, sterilize at 95-100°C for 5-10min, cool to 37°C for use;

Inoculate the seed solution of Bacillus coagulans HOM5301 into TSB liquid medium, culture it with shaking at 37°C-45°C for 24 hours, pass this method twice to obtain a highly active bacterial solution, and store it in a refrigerator at 4°C for later use;

The bacterial solution was eluted twice with 0.9% physiological saline, inoculated into the prepared oat slurry with an inoculation amount of 1×10 ⁷ CFU/mL, mixed evenly, left to ferment at a constant temperature of 37-45°C for 16 hours, and filled into a packaging container , to obtain fermented oat milk;

The above-mentioned fermented milk was counted by plate counting method after gradient dilution. The number of live bacteria of the active Bacillus coagulans HOM5301 in the fermented milk is higher than 1×10 ⁸ CFU/mL, the pH can reach 4.50, and the taste is rich.

Embodiment 12 fermented soybean milk test

Weigh 120-150g of soybeans, soak them overnight, add 20g of food-grade yeast powder, make up to 1000mL with double distilled water, put them into a wall breaker to make soybean milk, sterilize at 95-100°C for 5-10min, cool to 37°C for use;

Inoculate the seed solution of Bacillus coagulans HOM5301 into TSB liquid medium, culture it with shaking at 37°C-45°C for 24 hours, pass this method twice to obtain a highly active bacterial solution, and store it in a refrigerator at 4°C for later use;

The bacterial solution was eluted twice with 0.9% physiological saline, inoculated into soybean milk with an inoculation amount of 1×10 ⁷ CFU/mL, mixed evenly, left to ferment at a constant temperature of 37-45°C for 16 hours, filled into a packaging container, and obtained fermentation soy milk;

The above-mentioned fermented milk was counted by plate counting method after gradient dilution. The content of active Bacillus coagulans HOM5301 in the fermented milk is higher than 8×10 ⁸ CFU/mL, the pH can reach 5.00, and the taste is mellow.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. A strain of Bacillus coagulans (Bacillus coagulans), characterized in that, the Bacillus coagulans (Bacillus coagulans) is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCC No.20383, and the preservation date is 2020 July 16th. 2. the application of bacillus coagulans (Bacillus coagulans) described in claim 1 in the composition of preparation enhancing body immunity. 3. The application according to claim 2, characterized in that said enhancing the body's immune function includes enhancing cellular immune function. 4. The application according to claim 2, wherein the dosage form of the composition comprises any one of powder, granule, solution or colloid. 5. The application according to claim 2, characterized in that the composition comprises food, health product, medicine or feed. 6. A microbial inoculum, characterized in that, the microbial inoculum contains Bacillus coagulans (Bacillus coagulans) according to claim 1. 7. The microbial inoculant according to claim 6, characterized in that, it comprises auxiliary materials. 8. the application of the microbial bacterial agent described in claim 6 or 7 in the preparation of food, health products, feed, medicine.

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