CN112522134A - Bacillus coagulans and application thereof - Google Patents
Bacillus coagulans and application thereof Download PDFInfo
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- CN112522134A CN112522134A CN202011279981.XA CN202011279981A CN112522134A CN 112522134 A CN112522134 A CN 112522134A CN 202011279981 A CN202011279981 A CN 202011279981A CN 112522134 A CN112522134 A CN 112522134A
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- bacillus coagulans
- hom5301
- fermented
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- bacillus
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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
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to bacillus coagulans and application thereof.
Background
Bacillus coagulans (Bacillus coagulons) is a gram-positive, microaerophilic, lactic acid-producing Bacillus. The bacillus subtilis not only has the characteristics of lactic acid bacteria, but also has the characteristics of strong stress resistance, high temperature resistance, easy storage and the like of the bacillus, and is an excellent choice for being used as probiotics. The species has been listed in the biologics list recommended by the European Union of safety Qualification (QPS) and listed in the International Dairy Federation (IDF) "list of microorganisms with a history of safe use in food" and was identified by the food and drug administration's GRAS (generally recognized as safe substance). In 2016, Bacillus coagulans was also approved by the Ministry of health, China for edible strains.
Clinical studies show that some bacillus coagulans strains have obvious improvement effects on constipation, diarrhea, irritable bowel syndrome or ulcerative colitis and the like. For example, in patent application publication No. CN10056924A, a pharmaceutical bacillus coagulans for the treatment of ulcerative colitis is disclosed. For example, patent application publication No. CN111004732A discloses a bacillus coagulans capable of promoting motilin secretion, which has the effects of up-regulating motilin and promoting gastrointestinal motility. However, the number of Bacillus coagulans strains disclosed in China as having the function of improving specific health conditions of human bodies has remained extremely rare until now. Therefore, it is necessary to find other bacillus coagulans capable of improving specific health conditions of human body.
Disclosure of Invention
In view of the above, the present invention provides a bacillus coagulans and applications thereof. Different from the disclosed bacillus coagulans, the bacillus coagulans can enhance the immunity of organisms, enhance the cellular immune function and the mononuclear-macrophage function, inhibit the proliferation of common pathogenic bacteria, can be used for the production of functional fermented food for enhancing the immunity, can promote the digestion function of the organisms, improve the nutrient availability of the food and improve the mouthfeel.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Bacillus coagulans (Bacillus coagulans) HOM5301, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20383 and the preservation date of 2020, 7 and 16.
The invention provides application of the Bacillus coagulans (Bacillus coagulans) HOM5301 in preparation of a composition for enhancing immunity of organisms.
In some embodiments of the invention, the enhancing immune function includes enhancing cellular immune function and monocyte-macrophage function.
In some embodiments of the invention, the dosage form of the composition comprises any one of a powder, a granule, a solution, or a colloid.
In some embodiments of the invention, the composition comprises a food, nutraceutical, pharmaceutical or feed.
The invention provides a microbial agent which contains the Bacillus coagulans HOM 5301.
In some embodiments of the invention, the microbial agent further comprises an adjuvant.
The invention provides application of the microbial agent in preparation of foods, health-care products, feeds and medicines.
The invention provides a method for preparing the microbial agent, which comprises the following steps:
carrying out amplification culture on the Bacillus coagulans (Bacillus coagulans) HOM5301 in a liquid culture medium, and collecting thalli;
adding a protective agent into the thallus collected after the expanded culture for resuspension, drying and crushing to obtain the active spore microbial inoculum.
The invention also provides a fermentation product, which is obtained by fermenting the Bacillus coagulans (HOM 5301).
In some embodiments of the invention, the fermented product comprises a fermented dairy product, a fermented oat product, or a fermented soy product.
Compared with the prior art, the invention provides a Bacillus coagulans (Bacillus coagulans) HOM5301, the preservation number of which is CGMCC No. 20383. The bacillus coagulans strain has the following effects:
(1) the bacillus coagulans can enhance the immunity of organisms, enhance the cellular immune function and the monocyte-macrophage function and inhibit the proliferation of common pathogenic bacteria.
(2) The production process of the active spore microbial inoculum has simple parameters, easy control and short period, ensures the high survival rate of the bacillus coagulans, and the obtained product can be stored for a long time and has stable product quality.
(3) The bacillus coagulans 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.
Biological preservation description:
biological material: HOM5301, category name: bacillus coagulans (Bacillus coagulans) is preserved in China general microbiological culture Collection center (CGMCC) in 7 months and 16 days of 2020, and the preservation center addresses are as follows: the institute of microbiology, national academy of sciences No. 3, Xilu 1, Beijing, Chaoyang, Beijing; the preservation number is CGMCC No. 20383.
Drawings
FIG. 1 shows RAPD cluster analysis chart of Bacillus coagulans constructed based on UPGMA method;
FIG. 2 is a graph showing the results of tests of cellular immune function and monocyte-macrophage function in different groups.
Detailed Description
The following description of the present invention is provided in connection with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention, and it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention, and it is intended to cover all modifications and equivalents as may fall within the true spirit and scope of the invention.
The invention relates to a novel Bacillus coagulans (Bacillus coagulans) HOM5301, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.20383 and the preservation date of 2020, 7 and 16. The bacillus coagulans can enhance the immunity of organisms, enhance the cellular immune function and the monocyte-macrophage function and inhibit the proliferation of common pathogenic bacteria.
The Bacillus coagulans HOM5301 is isolated from a dairy product.
Random polymorphic DNA analysis (RAPD) is carried out on the separated Bacillus coagulans HOM5301, and the result shows that the HOM5301 is different from part of commercial Bacillus coagulans strains and has uniqueness.
The growth temperature of the Bacillus coagulans HOM5301 is 37-45 ℃.
The Bacillus coagulans (Bacillus coagulans) HOM5301 has the characteristics of acid resistance and cholate resistance, and has a 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 more than 50% after the Bacillus coagulans (Bacillus coagulans) HOM5301 is processed at 140 ℃ for 120 s.
The Bacillus coagulans HOM5301 can produce amylase and has the capability of fermenting grain substances with high starch content.
The Bacillus coagulans (Bacillus coagulans) HOM5301 can enhance the functions of cellular immunity and mononuclear-macrophage.
The Bacillus coagulans (Bacillus coagulans) HOM5301 has an inhibiting effect on various pathogenic bacteria, wherein the pathogenic bacteria comprise escherichia coli, salmonella, staphylococcus aureus, pseudomonas aeruginosa, listeria monocytogenes and the like.
The Bacillus coagulans (Bacillus coagulans) HOM5301 can be applied to preparation of a composition for enhancing the immunity of organisms.
Wherein the dosage form of the composition comprises any one of powder, granule, solution or colloid.
The composition comprises food, health product, medicinal product or feed.
The invention also provides a microbial agent which contains the Bacillus coagulans HOM 5301.
In some embodiments, the microbial agent further comprises an adjuvant.
The preparation method of the microbial agent is as follows:
the isolated Bacillus coagulans HOM5301 was treated. Carrying out amplification culture, centrifugally collecting thalli, adding a proper protective agent for heavy suspension, and drying to obtain active spore bacterium powder.
The preparation method of the microbial agent specifically comprises the following steps:
(1) fermentation of bacterial species
Inoculating Bacillus coagulans (Bacillus coagulans) HOM5301 into a sterile TSB liquid culture medium in an inoculation amount of 0.5-3% (v/v), culturing at 37-45 ℃ and 180-250rpm for 16-24 hours, and continuously subculturing twice to obtain an activated seed culture liquid. The seed culture was inoculated into the fermentation medium at an inoculum size of 0.5-3% (v/v). Culturing at 37-45 ℃ and 200-600 rpm. Automatically feeding sodium hydroxide solution and citric acid solution during fermentation to maintain pH at 5.5-7.0. When acid supplementation is stopped, taking fermentation liquor for microscopic examination, and obtaining a high-density culture solution of the bacillus coagulans HOM5301 when more than 70-90% of thalli form spores, wherein the number of the spores can reach more than 10-40 hundred million CFU/mL. The fermentation medium comprises the following components: 5-20g/L of glucose, 5-20g/L of yeast extract, 0.005-0.02g/L of manganese sulfate, 0.1-0.5g/L of magnesium sulfate, 1-4g/L of dipotassium phosphate, 0.5-4g/L of calcium carbonate and 1-5g/L of sodium chloride.
(2) Preparation of fungal powder
And (3) centrifugally collecting fermentation liquor of Bacillus coagulans HOM 5301. Discarding the supernatant, and washing the bacterial sludge with 0.9% sterile normal saline for 1-2 times. Mixing the washed bacterial sludge with a protective agent, wherein the final concentration of the protective agent is as follows: 50-200g/L of skimmed milk powder, 20-60g/L of trehalose, 1-5g/L of vitamin C and 3-8g/L L of sodium glutamate, and then carrying out vacuum freeze drying. After freeze-drying, pulverizing the fungus cake with a fine grinder to obtain freeze-dried fungus powder, wherein the number of active spore fungi in the freeze-dried powder is higher than (1-4) x 1011CFU/g, the spore rate of the freeze-dried fungus powder is more than 80-90%.
The invention also provides a fermentation product, which is obtained by fermenting Bacillus coagulans (Bacillus coagulans) HOM 5301.
Specifically, Bacillus coagulans (Bacillus coagulans) HOM5301 can be inoculated into skimmed milk powder, oat milk or soybean milk, and fermented at 37-45 ℃ to obtain a fermentation product. The fermentation product contains a large amount of Bacillus coagulans (Bacillus coagulans) HOM5301, and can enhance the immunity of organisms, promote digestion, improve the nutrient availability of food and improve the mouthfeel.
For further understanding of the present invention, the following examples are given to illustrate the bacillus coagulans and its use, and the scope of the present invention is not limited by the following examples.
Example 1 isolation and identification of Bacillus coagulans HOM5301
Weighing 3g of dairy product, shaking the dairy product uniformly by using 10mL of TSB liquid culture medium to obtain a mixed solution, carrying out water bath at 80 ℃ for 10min, sucking 1mL of the mixed solution, diluting a sample by using a 10-time dilution method, diluting the sample to 1000 times, coating the diluted solution on a PCA solid plate, and carrying out aerobic culture at 50 ℃ for 48-72 h. Selecting single colony, streaking, purifying for 3-4 times until the colony is single, and simultaneously performing gram staining and microscopic examination to observe colony morphology. Transferring the single colony to a TSB liquid culture medium for pure culture, preserving the strain by glycerol and numbering.
Random polymorphic DNA analysis (RAPD) was performed on the isolated Bacillus coagulans HOM5301, and as shown in FIG. 1, HOM5301 has a genotype difference rate of nearly 50% and uniqueness, unlike some commercial Bacillus coagulans strains.
EXAMPLE 2 preparation of active spore bacteria powder
Culturing strains: inoculating bacillus coagulans HOM5301 which is frozen and preserved at the temperature of minus 80 ℃ into a sterile TSB liquid culture medium in an inoculation amount of 1% (v/v), culturing for 16 hours at the temperature of 45 ℃ and at the speed of 250rpm, and carrying out subculture twice to obtain an activated seed culture liquid; inoculating a seed culture solution into a fermentation culture medium in an inoculation amount of 2% (v/v), culturing at a constant temperature of 45 ℃ and 400rpm, automatically feeding a sodium hydroxide solution and a citric acid solution in a flow manner in the fermentation process to keep constant pH of 6.0, taking a fermentation liquid for microscopic examination when acid supplementation is stopped, and obtaining a bacillus coagulans HOM5301 high-density culture solution when more than 80% of thalli form spores, namely reaching the end point of fermentation, wherein the number of the spores can reach more than 20 hundred million CFU/mL; the formulation of the fermentation medium is shown in Table 1.
TABLE 1 fermentation Medium formulation
Preparation of the freeze-drying protective agent: sterile water is mixed with a protective agent raw material to prepare a protective agent containing 100g/L of skimmed milk powder, 50g/L of trehalose, 3g/L of vitamin C and 5g/L L-sodium glutamate;
preparing active spore bacterium powder: centrifuging the cultured Bacillus coagulans HOM5301 zymocyte liquid at 2-8 deg.C and 6000rpm for 10min, discarding supernatant, collecting bacterial sludge, washing bacterial sludge with 0.9% sterile normal saline for 1-2 times, mixing the washed bacterial sludge with the above protective agent to make the bacterial concentration of the mixed bacterial liquid reach 1010Freeze drying in a freeze dryer until the freeze drying is finished, pulverizing the fungus cake with a fine grinder to obtain freeze-dried fungus powder with viable count higher than 1.0 × 1011CFU/g, the spore rate of the freeze-dried fungus powder is more than 80%.
Example 3 gastrointestinal environmental resistance test
1g of the bacterial powder prepared in example 2 was added into 9mL of artificial gastric juice (pH 3.0), mixed uniformly and counted as C0, and the mixture was cultured in an incubator at 37 ℃ for 3 hours and counted as C1. Centrifuging the liquid at 8000rpm for 10min to obtain bacterial sludge, transferring into artificial intestinal juice (pH 6.8) of the same volume, mixing, and culturing at 37 deg.C for 3 hr. The viable count of the plate in MRS culture medium is marked as C2, and the survival rate is calculated according to the following formula:
the 3-hour survival rate (%) of the artificial gastric juice is (C1/C0). times.100%
3-hour artificial gastric juice + 3-hour artificial intestinal juice survival rate (%) (C2/C0) × 100%
C0-initial viable count, C1-viable count after 3 hours of gastric juice treatment, C2-viable count after 3 hours of gastric juice treatment and 3 hours of intestinal juice treatment.
The survival rates of bacillus coagulans in simulated gastrointestinal fluids are listed in table 2, and it can be seen from table 2 that: after 3 hours of simulated artificial gastric juice treatment, the survival rate of the bacillus coagulans HOM5301 can reach more than 90%, and after 3 hours of simulated intestinal juice treatment, the survival rate of the bacillus coagulans HOM5301 can still reach more than 70%, which indicates that the bacillus coagulans has higher survival rates in gastric juice and intestinal tracts.
TABLE 2 survival rate of Bacillus coagulans in simulated gastrointestinal fluids
Example 4 high temperature test
Weighing 1g of the bacterial powder prepared in the example 2 into 9ml of physiological saline, sequentially diluting to a proper gradient by using a gradient dilution method, transferring the bacterial liquid into an MRS solid plate, culturing at 37-45 ℃, and counting. The powder was treated at 90 ℃ and 140 ℃ for 30s, 60s, 90s, 120s, respectively, counted and the survival rate was recorded. The results are shown in Table 3, and it can be seen that the survival rate of Bacillus coagulans HOM5301 can still reach more than 50% after being treated at 140 ℃ for 120s, which indicates that the active spore bacteria powder has strong heat resistance.
TABLE 3 high temperature resistance of Bacillus coagulans HOM5301
Example 5 antibiotic susceptibility testing
The drug sensitivity test was performed according to the K-B agar method recommended by the American clinical standards Committee (NCCLS), and the drug sensitivity paper was purchased from Beijing Temple pharmaceutical Biotech, Inc.
Firstly, inoculating bacillus coagulans HOM5301 to a TSA agar plate, culturing for 18-24 hours at 45 ℃, then picking out pure colonies, placing the pure colonies into sterile physiological saline, and preparing into bacterial suspension with 0.5 McLeod turbidity standard. And uniformly mixing the bacterial suspension and the TSB agar culture medium, pouring the mixture into a flat plate, and placing a drug sensitive paper sheet on the flat plate after the flat plate is solidified. And (3) placing the flat plate in an incubator at 45 ℃ for culturing for 24-48 h, taking out, measuring and recording the diameter of a bacteria ring by using a ruler, and finally, judging and reading according to CLSI (CLSI standard), wherein the result is shown in table 4. The results indicate that HOM5301 is sensitive to all nine antibiotics.
TABLE 4 determination of the sensitivity of Bacillus coagulans HOM5301 to 9 antibiotics
S (susceptable) indicates sensitivity; i (intermediate) means medium; r (resistance) represents drug resistance
Example 6 ability to repress common pathogenic bacteria test
The indicator bacteria (Escherichia coli ATCC 8739; Staphylococcus aureus ATCC 6538; Salmonella typhimurium ATCC 14028; Pseudomonas aeruginosa ATCC 9027; Listeria monocytogenes ATCC19111) were inoculated in the TSB medium at an inoculation amount of 1% (v/v) and cultured at 37 ℃ for 24 hours for use. Then respectively inoculating the bacillus coagulans HOM5301 into a sterilized MRS culture medium in an inoculation amount of 1% (v/v), carrying out shake culture at 37-45 ℃ for 24 hours, and activating twice to obtain fermentation liquor. Centrifuging at 8000rpm for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, freeze drying, dissolving lyophilized powder to obtain ten times concentrated fermentation supernatant, dissolving catalase in 50mmol/L phosphate buffer (pH 7.0) to obtain mother liquor, adding into the supernatant at a final concentration of 1mg/mL, taking out after warm bath at 37 deg.C for 2 hr, boiling in water bath box at 100 deg.C for 5min to inactivate enzyme, adjusting pH to 5.0, and performing antibacterial test. Then, the indicator bacterium was added to the TSA medium, shaken well, poured into a prepared TSA agar plate, and allowed to stand for coagulation. And finally, placing the Oxford cup on a solid nutrient agar plate by using sterile forceps, respectively adding 0.2mL of the supernatant of the fermentation liquor of the bacillus coagulans HOM5301 to be detected into the holes, placing the holes in a refrigerator at 4 ℃ for diffusion for 4 hours, then culturing the holes in an incubator at 37 ℃ for 12 to 18 hours, and observing and determining the diameter of a bacteriostatic circle. Ten fold MRS concentrate was used as negative control, and 3 replicates were run for each sample. As can be seen from table 5, bacillus coagulans HOM5301 has an inhibitory effect on 5 pathogenic bacteria, and it is presumed that bacillus coagulans HOM5301 may produce bacteriostatic substances other than organic acids and hydrogen peroxide.
TABLE 5 inhibitory Effect of Bacillus coagulans HOM5301 on pathogenic bacteria
Note: "-" no bacteriostatic activity; "+" has bacteriostatic activity.
Example 7 Amylase Activity assay
The formula of the culture medium for producing the amylase comprises the following components: 10g of peptone, 5g of yeast powder, 5g of soluble starch, 2g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.2g of calcium chloride dihydrate and 1L of water, wherein the pH value is 7.0. Sterilizing at 121 deg.C for 20 min.
Transferring the bacillus coagulans HOM5301 into a TSB liquid culture medium, activating twice at 45 ℃, transferring to an amylase production culture medium, carrying out shake culture at 45 ℃ for 24-48 h, centrifuging at 8000rpm for 10min, taking supernatant, and carrying out membrane filtration sterilization with a thickness of 0.22 mu m for later use. The starch hydrolysis rate and amylase activity of the strain were determined according to the procedure of the amylase kit (Nanjing Jian C016-1-1), and the results are shown in Table 6. As can be seen from the data in Table 6, Bacillus coagulans HOM5301 is rich in amylase, can partially hydrolyze starch, and has a hydrolysis rate of 12.3 +/-0.6% and an amylase activity of 476.4 +/-5.5U/dL. This demonstrates the ability of the strain to ferment high starch content grain-like materials.
TABLE 6 determination of starch hydrolysis and amylase activity in Bacillus coagulans HOM5301
Example 8 in vitro cytokine secretion assay
The bacillus coagulans HOM5301 is inoculated in a TSB culture medium and used for viable count and cell experiments after two successive generations of activation. After centrifugation at 8000rpm for 10min to collect the cells, the cells were adjusted to working concentration (about 5.0X 10) using DMEM complete medium without antibiotic5CFU/mL); mouse macrophage RAW264.7 (about 5X 10)5cell/mL) was added to a 24-well plate, 1mL per well; after the wall is attached for 2 hours, removing the culture medium, and adding 1mL of a bacterium-containing culture medium into each hole; setting a blank control group, and adding 1mL of DMEM medium; collecting cell supernatant after 24h of co-culture, and adopting enzyme-linked immunosorbent assayThe contents of TNF-. alpha.and IL-6 in the cell supernatants were determined by immunoadsorption (ELISA) according to the kit instructions and the results are shown in Table 7. Table 7 shows that Bacillus coagulans HOM5301 can remarkably improve TNF-alpha and IL-6 secretion of RAW264.7 cells. As can be seen, Bacillus coagulans HOM5301 has potential immunomodulatory capacity.
TABLE 7 HOM5301 cytokine secretion by mouse macrophages
Note: comparison of P <0.01 with control group of HOM5301
Example 9 Immunity-enhancing animal test
96 female mice of 18g-20g SPF KM bred by Beijing Huafukang biotech GmbH are selected, and are randomly divided into four batches of 2 groups of 12 mice each after being subjected to animal adaptive feeding observation. Experimental group of active bacterium powder of Bacillus coagulans HOM5301 for intragastric administration 10/day for each mouse9CFU, continuous gavage for 30 days. The control group was also perfused with gastric saline. Carrying out carbon clearance experiments in a batch of experiments; the experiment two batches are used for measuring the ratio of viscera to body weight and carrying out delayed allergy experiments; experiment three batches are carried out for mouse abdominal cavity macrophage phagocytosis chicken erythrocyte experiment; experiment four batches of experiments were performed for ConA-induced mouse lymphocyte transformation. The specific experimental method refers to 'health food inspection and evaluation technical specification' (2003 edition), and the test result data is statistically analyzed by SPSS software. Specific experimental test results can be seen in fig. 2 and tables 8 to 11.
As can be seen from the results in Table 8, there was no significant difference in the weight average of the mice in HOM5301 group before and after the experiment (P >0.05) compared with the control group, which indicates that the powder of Bacillus coagulans HOM5301 active bacteria has no effect on the body weight of the mice.
From the results in Table 9, it was found that there was no significant difference in spleen/body and thymus/body in the HOM5301 group mice compared to the control group (P >0.05), indicating that the active powder of Bacillus coagulans HOM5301 had no effect on the weight of spleen and thymus in the mice.
The results in table 10 show that the bacillus coagulans HOM5301 active bacterium powder can significantly improve the mouse plantar thickening degree (P <0.05) induced by Sheep Red Blood Cells (SRBC) and the mouse spleen lymphocyte proliferation capacity (P <0.05) induced by ConA, which indicates that the bacillus coagulans HOM5301 active bacterium powder has the function of significantly enhancing the mouse cellular immune function.
The results in table 11 show that the bacillus coagulans HOM5301 active bacterium powder can significantly improve the mouse carbon clearance capacity (P <0.01), the phagocytosis rate (P <0.01) of chicken erythrocytes by mouse macrophages and the phagocytosis index (P <0.01), which indicates that the bacillus coagulans HOM5301 active bacterium powder can significantly enhance the mouse mononuclear-macrophage function.
In conclusion, compared with a control group, the bacillus coagulans HOM5301 active bacterium powder can obviously enhance the cell immune function and the monocyte-macrophage function of a mouse, and has no influence on the weight of the mouse, the thymus and the spleen. In conclusion, the active bacillus coagulans HOM5301 powder has the function of enhancing immunity.
TABLE 8 Effect of HOM5301 fungal powder on mouse body weight
TABLE 9 Effect of HOM5301 active bacterial powder on mouse organ/body ratio
TABLE 10 influence of HOM5301 bacterial powder on Sheep Red Blood Cell (SRBC) induced mouse DTH and ConA induced mouse splenic lymphocyte proliferation
Note: p <0.05 compared to control group.
TABLE 11 influence of HOM5301 bacterial powder on carbon clearance and phagocytosis of chicken erythrocytes by macrophages
Note: p <0.01 compared to control group.
Example 10 fermented milk test
Weighing 12g of skimmed milk powder, 2g of glucose and 2g of desalted whey powder, adding double distilled water to 100mL, stirring uniformly, homogenizing by a high-pressure homogenizer, sterilizing at 95-100 ℃ for 5-10min, and cooling to 37 ℃ for later use;
inoculating the seed liquid of the bacillus coagulans HOM5301 into a TSB liquid culture medium, carrying out shaking culture at 37-45 ℃ for 24h, carrying out passage for 2 times according to the method to obtain high-activity bacterial liquid, and preserving the high-activity bacterial liquid in a refrigerator at 4 ℃ for later use;
the bacterial solution was eluted 2 times with 0.9% physiological saline at 1X 107Inoculating the CFU/mL inoculation amount to prepared milk, uniformly mixing, standing and fermenting at the constant temperature of 37-45 ℃ for 16h, and filling into a packaging container to obtain a fermented milk product;
the fermented milk samples were sampled, and counted by plate counting after gradient dilution. The content of active bacillus coagulans HOM5301 in the fermented milk is higher than 5.0 x 108CFU/mL, pH up to 5.00, and good taste.
Example 11 fermented oat milk test
Weighing 120-150g of enzymolysis oat powder, adding double distilled water to 1000mL, homogenizing by a high-pressure homogenizer, sterilizing at 95-100 ℃ for 5-10min, and cooling to 37 ℃ for later use;
inoculating the seed liquid of the bacillus coagulans HOM5301 into a TSB liquid culture medium, carrying out shaking culture at 37-45 ℃ for 24h, carrying out passage for 2 times according to the method to obtain high-activity bacterial liquid, and preserving the high-activity bacterial liquid in a refrigerator at 4 ℃ for later use;
the bacterial solution was eluted 2 times with 0.9% physiological saline at 1X 107Inoculating the CFU/mL inoculum size into the prepared oat pulp, uniformly mixing, standing and fermenting at the constant temperature of 37-45 ℃ for 16h, and filling into a packaging container to obtain fermented oat milk;
the fermented milk was diluted in a gradient and counted by plate counting method. The viable count of the active bacillus coagulans HOM5301 in the fermented milk is higher than 1 × 108CFU/mL, pH up to 4.50, and good taste.
Example 12 fermented soymilk test
Weighing 120-150g of soybeans, soaking overnight, adding 20g of food-grade yeast powder, adding double distilled water to 1000mL, performing wall breaking to obtain soybean milk, sterilizing at 95-100 ℃ for 5-10min, and cooling to 37 ℃ for later use;
inoculating the seed liquid of the bacillus coagulans HOM5301 into a TSB liquid culture medium, carrying out shaking culture at 37-45 ℃ for 24h, carrying out passage for 2 times according to the method to obtain high-activity bacterial liquid, and preserving the high-activity bacterial liquid in a refrigerator at 4 ℃ for later use;
the bacterial solution was eluted 2 times with 0.9% physiological saline at 1X 107Inoculating the CFU/mL inoculation amount into the soybean milk, uniformly mixing, standing and fermenting at the constant temperature of 37-45 ℃ for 16h, and filling into a packaging container to obtain fermented soybean milk;
the fermented milk was diluted in a gradient and counted by plate counting method. The content of active bacillus coagulans HOM5301 in the fermented milk is higher than 8 x 108CFU/mL, pH up to 5.00, and mellow taste.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The Bacillus coagulans strain is characterized in that the Bacillus coagulans strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No.20383, and the preservation date is 2020, 7 and 16 days.
2. Use of Bacillus coagulans (Bacillus coaguluns) according to claim 1 for the preparation of a composition for enhancing immunity in the body.
3. The use according to claim 2, wherein the enhancement of immune function comprises enhancement of cellular immune function and monocyte-macrophage function.
4. The use according to claim 2, wherein the composition is in a dosage form comprising any one of a powder, a granule, a solution, or a colloid.
5. The use as claimed in claim 2, wherein the composition comprises a food, nutraceutical, pharmaceutical or feed product.
6. A microbial agent, characterized in that it contains the Bacillus coagulans (Bacillus coagulans) of claim 1.
7. The microbial inoculant according to claim 6, comprising adjuvants.
8. Use of the microbial inoculant of claim 6 or 7 in the preparation of a food, health product, feed, pharmaceutical product.
9. A fermented product obtained by fermentation using Bacillus coagulans as described in claim 1.
10. The fermented product according to claim 9, wherein the fermented product comprises a fermented dairy product, a fermented oat product or a fermented soy product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN112852679A (en) * | 2021-03-17 | 2021-05-28 | 武汉微康益生菌研究院有限公司 | Probiotic bacillus coagulans and application thereof |
| CN114145428A (en) * | 2021-12-16 | 2022-03-08 | 广东广益科技实业有限公司 | Fermented soybean milk steamed cake and preparation method thereof |
| CN114231464A (en) * | 2021-12-31 | 2022-03-25 | 上海新溢生物科技工程研究中心(有限合伙) | Bacillus coagulans and application thereof |
| CN114391648A (en) * | 2021-11-15 | 2022-04-26 | 济南瑞隆安生物技术有限公司 | Preparation method of probiotic fermented oat composition with effects of reducing blood sugar and blood fat |
| CN114908023A (en) * | 2022-06-17 | 2022-08-16 | 江南大学 | Bacillus coagulans for improving relative abundance of intestinal actinomycete phylum and inhibiting expression level of proinflammatory factors |
| CN115074299A (en) * | 2022-07-29 | 2022-09-20 | 江南大学 | Bacillus coagulans capable of stably producing odor substances of strong-smelling preserved bean curd |
| CN115651860A (en) * | 2022-08-18 | 2023-01-31 | 北京农学院 | Bacillus coagulans BC-HYC strain and application thereof |
| CN116731937A (en) * | 2023-08-15 | 2023-09-12 | 天津创源生物技术有限公司 | Preparation method and application of Wittman coagulans IOB502 zymocyte powder |
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| CN112852679A (en) * | 2021-03-17 | 2021-05-28 | 武汉微康益生菌研究院有限公司 | Probiotic bacillus coagulans and application thereof |
| CN112852679B (en) * | 2021-03-17 | 2023-01-17 | 武汉微康益生菌研究院有限公司 | Probiotic bacillus coagulans and application thereof |
| CN114391648A (en) * | 2021-11-15 | 2022-04-26 | 济南瑞隆安生物技术有限公司 | Preparation method of probiotic fermented oat composition with effects of reducing blood sugar and blood fat |
| CN114145428A (en) * | 2021-12-16 | 2022-03-08 | 广东广益科技实业有限公司 | Fermented soybean milk steamed cake and preparation method thereof |
| CN114145428B (en) * | 2021-12-16 | 2024-01-05 | 广东广益科技实业有限公司 | Fermented soybean steamed cake and preparation method thereof |
| CN114231464A (en) * | 2021-12-31 | 2022-03-25 | 上海新溢生物科技工程研究中心(有限合伙) | Bacillus coagulans and application thereof |
| CN114231464B (en) * | 2021-12-31 | 2024-05-10 | 上海新溢生物科技工程研究中心(有限合伙) | Bacillus coagulans and application thereof |
| CN114908023A (en) * | 2022-06-17 | 2022-08-16 | 江南大学 | Bacillus coagulans for improving relative abundance of intestinal actinomycete phylum and inhibiting expression level of proinflammatory factors |
| CN114908023B (en) * | 2022-06-17 | 2023-08-25 | 江南大学 | Bacillus coagulans for improving relative abundance of actinomycota in intestinal tract and inhibiting expression quantity of proinflammatory factors |
| CN115074299A (en) * | 2022-07-29 | 2022-09-20 | 江南大学 | Bacillus coagulans capable of stably producing odor substances of strong-smelling preserved bean curd |
| CN115074299B (en) * | 2022-07-29 | 2023-10-27 | 江南大学 | Bacillus coagulans capable of stably producing stinky tofu smell substances |
| CN115651860A (en) * | 2022-08-18 | 2023-01-31 | 北京农学院 | Bacillus coagulans BC-HYC strain and application thereof |
| CN116731937A (en) * | 2023-08-15 | 2023-09-12 | 天津创源生物技术有限公司 | Preparation method and application of Wittman coagulans IOB502 zymocyte powder |
| CN116731937B (en) * | 2023-08-15 | 2023-11-10 | 天津创源生物技术有限公司 | Preparation method and application of Wittman coagulans IOB502 zymocyte powder |
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