A compound Bacillus coagulans bacterial agent, its preparation method and application

Abstract

The present invention discloses a compound Bacillus coagulans bacterium agent and its preparation method and application, belonging to the field of microbial technology. The compound bacterium agent provided by the present invention is obtained by compounding Bacillus coagulans BC77 and Weizmannia coagulans VC77. When the compounding ratio of BC77 and VC77 is 1:2, the mixed strain has excellent antibacterial effect and high mycotoxin degradation ability, the inhibition of pathogenic bacteria reaches more than 84.2%, the degradation rate of aflatoxin reaches more than 86.7%, and the degradation rate of zearalenone reaches more than 92.7%. The compound Bacillus coagulans bacterium agent of the present invention has a positive treatment effect on the abuse of antibiotics and environmental governance.

Description

Technical Field

[0001] The present invention relates to a compounded Bacillus coagulans bacterial agent, a preparation method thereof, and an application thereof, belonging to the field of microbial technology. Background Art

[0002] Inhibiting pathogenic bacteria is an important disease prevention strategy and is crucial for protecting the health of humans, animals, and plants. Currently, the main means of inhibiting pathogenic bacteria is the use of antibiotics. However, the overuse of antibiotics has caused serious problems, leading to multiple adverse consequences, such as the gradual evolution of pathogenic bacteria to form resistance to drugs, the increase in drug resistance, and the increasing difficulty of public health infection control, etc.

[0003] Bacillus coagulans is a common Gram-positive bacterium belonging to the genus Bacillus. This strain is widely distributed in various environments in nature, such as soil, water bodies, and the surfaces of plants. While having excellent antagonistic effects, Bacillus coagulans is also an excellent probiotic. As an edible strain, it can inhibit the growth of pathogenic microorganisms and promote the health of the digestive tract. The excellent probiotic and antagonistic effects of Bacillus coagulans make it one of the important microorganisms in the fields of research and application.

[0004] Therefore, inhibiting pathogenic bacteria by utilizing the antagonistic effects of microorganisms is of great significance for human health, curbing the problem of antibiotic abuse, and reducing environmental pollution. Developing probiotic bacterial agents with antibacterial effects can be widely applied in the environmental field, providing new solutions and research directions for curbing antibiotic abuse, and having broad application prospects and economic value. Summary of the Invention

[0005] In order to address various aspects of existing problems and expand the scope of application of Bacillus coagulans, the present invention provides a compounded Bacillus coagulans bacterial agent. This bacterial agent can efficiently inhibit pathogenic bacteria and simultaneously has the ability to degrade various mycotoxins. The compounded Bacillus coagulans bacterial agent provided by the present invention has a wider scope of application in the environmental field, providing new ideas and methods for solving the problem of antibiotic abuse. The present invention provides a preparation method and an application of a bacterial agent obtained by compounding two strains of Bacillus coagulans, which has more excellent antibacterial effects and mycotoxin degradation effects.

[0006] The first object of the present invention is to provide a microbial bacterial agent obtained by compounding Bacillus coagulans. The bacterial agent contains Bacillus coagulans BC77, which was deposited at the China Center for Type Culture Collection on May 7, 2021, with the deposit number CCTCC NO: M 2021497; and Weissella coagulans VC77, which was deposited at the China Center for Type Culture Collection on June 29, 2023, with the deposit number CCTCC NO: M 20231138.

[0007] The second object of the present invention is to provide a compounded Bacillus coagulans bacterium agent, which contains Bacillus coagulans BC77, preserved in the China Center for Type Culture Collection, Wuhan on May 7, 2021, with the preservation number CCTCC NO: M2021497; and Weissella coagulans VC77, preserved in the China Center for Type Culture Collection, Wuhan on June 29, 2023, with the preservation number CCTCC NO: M 20231138.

[0008] In one embodiment, the viable bacteria ratio of Bacillus coagulans BC77 to Weissella coagulans VC77 in the compounded Bacillus coagulans bacterium agent is 1:2.

[0009] In one embodiment, the compounded bacterium agent contains the freeze-dried bacterium powder of Bacillus coagulans BC77 or Weissella coagulans VC77 obtained by freeze-drying the viable cells of Bacillus coagulans BC77 or Weissella coagulans VC77, immobilized Bacillus coagulans BC77 or Weissella coagulans VC77, the liquid bacterium agent or solid bacterium agent of Bacillus coagulans BC77 or Weissella coagulans VC77, or one or more of the fermentation metabolites, cell lysates, saccharides, lipids, fibers, proteins, and nucleic acid substances prepared from Bacillus coagulans BC77 or Weissella coagulans VC77.

[0010] In one embodiment, the total viable bacteria number of Bacillus coagulans in the compounded Bacillus coagulans bacterium agent is 10 10 ~10 11 CFU / g.

[0011] In one embodiment, the preparation method of the compounded Bacillus coagulans bacterium agent includes the following steps:

[0012] (1) Inoculate both strains of Bacillus coagulans into MRS medium at 2%, and culture at 37°C for 24 hours;

[0013] (2) Centrifuge to collect the bacterial cells, wash the bacterial cells with PBS and then resuspend them in milk;

[0014] (3) Perform vacuum freeze-drying on the resuspended bacterial liquid to obtain the bacterium powder;

[0015] (4) Mix the bacterium powders of Bacillus coagulans BC77 and Weissella coagulans VC77 in a ratio of 1:2 to obtain the compounded Bacillus coagulans bacterium agent.

[0016] The third object of the present invention is to provide the application of the compounded Bacillus coagulans bacterium agent in inhibiting pathogenic bacteria and degrading mycotoxins.

[0017] In one embodiment, the pathogenic bacteria in the application include: Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Streptococcus pneumoniae, Salmonella, Pseudomonas aeruginosa.

[0018] In one embodiment, the mycotoxins in the application include: aflatoxin and zearalenone.

[0019] In one embodiment, the microbial inoculum is used in the environmental field.

[0020] The third object of the present invention is to provide a product for inhibiting pathogenic bacteria and degrading mycotoxins.

[0021] Beneficial effects:

[0022] The present invention provides a preparation method of a compound Bacillus coagulans inoculum. The compounding ratio of Bacillus coagulans BC77 and Weizmannia coagulans VC77 is 1-4:1-4. When the ratio reaches 1:2, its antibacterial effect is the best, and the inhibition of pathogenic bacteria reaches more than 84.2%. At the same time, when the compound inoculum ratio is 1:2, it can efficiently degrade aflatoxin, and its degradation rate reaches more than 86.7%, and the degradation rate of zearalenone reaches more than 92.7%. The various effects of the compound inoculum are far superior to those of a single strain, and the application range is also wider. The compound Bacillus coagulans inoculum provided by the present invention has excellent pathogenic bacteria inhibition ability and mycotoxin degradation ability, and has a positive control effect on antibiotic abuse and environmental treatment.

[0023] Biological material preservation

[0024] Bacillus coagulans BC77, taxonomically named Bacillus coagulans, was deposited at the China Center for Type Culture Collection in Wuhan on May 7, 2021, with the deposit number CCTCC NO: M 2021497;

[0025] Weizmannia coagulans VC77, taxonomically named Weizmannia coagulans, was deposited at the China Center for Type Culture Collection in Wuhan on June 29, 2023, with the deposit number CCTCC NO: M 20231138. Description of the drawings

[0026] Figure 1 It is a colony map of Weizmannia coagulans VC77;

[0027] Figure 2 It is a colony map of Bacillus coagulans BC77. Detailed implementation manners

[0028] The present invention relates to:

[0029] MRS medium / fermentation medium: peptone 10.0 g / L, beef powder 10.0 g / L, yeast powder 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, and agar powder 20.0 g / L.

[0030] BHI medium: brain extract 8 g / L, heart extract 10 g / L, meat extract 10 g / L, glucose 2 g / L, yeast extract 5 g / L, sodium chloride 5 g / L.

[0031] TSB medium: peptone 17 g / L, soybean peptone 3 g / L, pancreatic digest of casein 5 g / L, soybean casein digest 5 g / L, sodium chloride 5 g / L, glucose 2 g / L.

[0032] Sheep blood medium: peptone 10 g / L, beef extract 3 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, glucose 2 g / L, sheep blood 50 mL / L.

[0033] LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0034] NA medium: peptone 5 g / L, beef powder 3 g / L, table salt 5 g / L, agar 15 g / L.

[0035] Strains: Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Actinobacillus actinomycetemcomitans (ATCC 700685), Streptococcus mutans (ATCC 25175), Staphylococcus aureus subsp (ATCC 6538P), Escherichia coli (ATCC 13706), Listeria monocytogenes (ATCC 23074), Shigella Sp. (ATCC 12038), Pneumococcal Polysaccharide Powder Type 1 (ATCC 15-X), Salmonella Enterica Subsp. Enterica (ATCC 29629), Pseudomonas Aeruginosa (ATCC 10145).

[0036] Weizmannia coagulans VC77 used in the present invention was screened from pickled kimchi, and it has inhibitory effects on various common pathogenic bacteria and has the following characteristics: (1) It can inhibit a variety of potential oral pathogenic bacteria; (2) Low acid production; (3) It does not produce volatile sulfides such as hydrogen sulfide, methanethiol, dimethyl disulfide, and dimethyl trisulfide.

[0037] Example 1: Preparation of bacterial powder from strains

[0038] This preparation example provides a bacterial powder of Bacillus coagulans BC77 and Weizmannia coagulans VC77, which is prepared by the following method: Bacillus coagulans BC77 and Weizmannia coagulans VC77 are respectively inoculated into MRS liquid medium and cultured at 37 °C for 12 h for activation, and the activation is carried out continuously for 2 times to obtain an activated solution; the activated solution is inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37 °C for 24 h to obtain a bacterial solution; the bacterial solution is centrifuged at 5000 rpm for 10 min to obtain the cells of Bacillus coagulans BC77 and Weizmannia coagulans VC77 respectively; BC77 and VC77 are resuspended in an aqueous solution of skimmed milk powder with a mass concentration of 10% at a ratio of 1:2 to a concentration of 1×10 11 CFU / mL to obtain a bacterial suspension; the bacterial suspension is cultured at 37 °C for 1 h and then freeze-dried to obtain a compound bacterial agent powder.

[0039] Example 2: Inhibitory effect of compounded Bacillus coagulans on pathogenic bacteria

[0040] Grouping: Inoculate Bacillus coagulans into MRS medium and culture at 37 °C for 24 h.

[0041] Preparation of metabolite of compounded strain: Take 1 mL of the cultured bacterial liquid (the viable count is 10 9 CFU / mL), centrifuge at 4000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm filter membrane, and collect the supernatant as the metabolite of the strain.

[0042] Preparation of lysate of compounded strain: Take 1 mL of the cultured bacterial liquid (the viable count is 10 9 CFU / mL), centrifuge at 4000 rpm for 10 min, add PBS to wash the bacterial cells to remove the residual medium, resuspend the collected bacterial cells with 1 mL of PBS, and perform homogenization and crushing to obtain the lysate of the strain.

[0043] Preparation of bacterial suspension of compounded strain: Take 1 mL of the cultured bacterial liquid (the viable count is 10 9 CFU / mL), centrifuge at 4000 rpm for 10 min, discard the supernatant, add PBS to wash the bacterial cells to remove the residual medium, centrifuge again at 4000 rpm for 10 min, discard the supernatant, and then add 1 mL of PBS to resuspend to obtain the bacterial suspension of the strain.

[0044] Oxford cup inhibition experiment to preliminarily screen the compounding ratio:

[0045] 1. Prepare the medium required for pathogenic bacteria: Select an appropriate medium according to the experimental needs, such as agar medium, glucose agar medium, etc., and prepare the medium according to the preparation method.

[0046] 2. Preparation of pathogenic strains: Culture the pathogenic bacteria and perform pre-culture. Liquid medium can be used for pre-culture, and the bacteria are incubated under appropriate conditions until the logarithmic growth phase to obtain a higher colony concentration.

[0047] 3. Prepare Oxford cups: Prepare a set of Oxford cups, which are round transparent plastic cups with small holes at the bottom for containing the test substances. Ensure that the surface of the Oxford cups is clean and sterile.

[0048] 4. Uniformly coat the bacterial liquid: Uniformly coat the pre-cultured pathogenic bacteria strain on the corresponding culture medium plate.

[0049] 5. Place the Oxford cups: Gently press the prepared Oxford cups on the agar plate so that the small holes at the bottom are aligned with the bacterial plaque, ensuring that the Oxford cups are stable and closely fit with the agar medium.

[0050] 6. Adding the substance to be tested: Add 200 μL of the resuspended bacterial solution of the BC77 or VC77 mixed bacteria, metabolite, or bacterial strain lysate to the Oxford cup, ensuring that the bottom of the Oxford cup is completely wetted by the substance to be tested, and pay attention to avoiding overflow or contamination.

[0051] 7. Incubation and culture: Place the culture medium plate together with the petri dish containing the Oxford cup in an incubator and incubate at an appropriate temperature and time according to the requirements of the studied bacterial strain.

[0052] 8. Result observation: After the culture is completed, observe the growth of the bacterial plaque. Note whether an antibacterial zone is formed around the Oxford cup. Measure the diameter or area of the antibacterial zone to evaluate the inhibitory effect of the substance to be tested on the bacterial strain.

[0053] The antibacterial effects of the metabolite of the compounded bacterial strain are as follows (the total amount of the metabolite remains the same among the experimental groups with different compounding ratios (volume ratio)), and the antibacterial results are as follows:

[0054] Table 1 Antibacterial effects of the compounded bacterial strain with different ratios (diameter cm)

[0055]

[0056] The results show that the effects are average when the metabolite ratios of BC77 and VC77 are 1:4 and 4:1. The remaining ratios are used to continue compounding the bacterial strain lysate (the total amount of the lysate remains the same among the experimental groups with different compounding ratios (volume ratio)), and the antibacterial results are as follows:

[0057] Table 2 Antibacterial effects of the compounded bacterial strain lysate with different ratios (diameter cm)

[0058]

[0059]

[0060] It can be seen from the results that both the metabolite of the compounded bacterial strain and the lysate of the compounded bacterial strain have good antibacterial effects, and the antibacterial effect is the best when the ratio of BC77 to VC77 is 1:2 (volume ratio).

[0061] On this basis, the inhibition rates of BC77, VC77 and the mixed bacteria against pathogenic bacteria were measured in detail. The pathogenic bacteria were evenly spread on the agar medium. A series of small holes were made on the agar medium using a sterilized plastic rod or borer. These small holes would be used to place single strains of bacteria (Bacillus coagulans BC77, Weizmannia coagulans VC77) and the mixed bacteria mixed in a 1:2 ratio. The inhibitory strains were inoculated into the made small holes to ensure that each small hole had enough inhibitory strains. After culturing for 24 hours, the pathogenic bacteria would grow into colonies in the areas not inhibited, while there might be no or only small colonies in the inhibited areas. The diameter of the inhibition zone was measured to evaluate the inhibitory effect. By measuring the diameter of the inhibition zone and comparing it with the area of the uninhibited pathogenic bacteria, the inhibition rate could be calculated. Usually, the inhibition rate is expressed as a percentage, and the formula is: Inhibition rate (%) = [1 - (diameter of the inhibition zone / diameter of the pathogenic bacteria zone)] × 100%.

[0062] Table 3 Inhibitory rates of BC77, VC77, and the 1:2 mixed bacteria

[0063] Inhibition rate BC77 VC77 1:2 mixed bacteria Fusobacterium nucleatum 37.4％ 67.4％ 88.7％ Porphyromonas gingivalis 48.9％ 52.8％ 91.5％ Actinobacillus actinomycetemcomitans 42.5％ 65.1％ 85.9％ Streptococcus mutans 30.2％ 54.3％ 84.2％ Staphylococcus aureus 31.6％ 62.7％ 94.1％ Escherichia coli 49.3％ 51.9％ 84.8％ Listeria monocytogenes 44.1％ 66.2％ 86.3％ Shigella 33.8％ 68.0％ 92.3％ Streptococcus pneumoniae 46.5％ 53.7％ 90.5％ Salmonella 39.7％ 57.5％ 93.7％ Pseudomonas aeruginosa 35.0％ 61.4％ 87.6％

[0064] Example 4: Mycotoxin degradation experiment

[0065] Grouping: Strains BC77 and VC77 were inoculated into MRS medium at an inoculation amount of 2% (v / v) and cultured at 37°C for 24 hours to prepare the bacterial fermentation broth.

[0066] (1) Application for degrading aflatoxin:

[0067] The peanut meal sample containing aflatoxin was crushed and passed through a 40-mesh sieve, then mixed evenly. Sampling was carried out according to the method of GB / T 14699.1-2005, and 5.00 g was weighed and placed in a glass container. It was sealed with four layers of gauze and newspaper and sterilized in a high-temperature and high-pressure autoclave at 121 °C for 20 min. The sterilized peanut meal was transferred to a fermentation dish, and 1 mL of the fermentation culture solution of Bacillus coagulans BC77, the fermentation culture solution of Weissella confusa VC77, and the compound culture solution (mixed with different volume ratios of BC77 and VC77 culture solutions) were added to the above-treated sample. Then, an appropriate amount of sterile distilled water was added. After stirring and mixing evenly, it was subjected to constant-temperature solid-state fermentation at 38 °C. After 48 h of fermentation, the sample after removing four aflatoxins by solid-state fermentation was obtained. The fermented samples were collected, dried, and each portion of peanut meal was introduced into a centrifuge tube. Acetonitrile-aqueous solution (70%, 30%) was added for extraction, and the fermentation dish was rinsed and introduced into the centrifuge tube together to avoid the influence of uneven distribution of toxins in peanut meal on the experimental results; after mixing evenly, it was ultrasonically treated for 10 min, shaken for 30 min, centrifuged at 8000 r / min for 10 min, and the supernatant after filtration was passed through a solid-phase purification column. After collecting the liquid, it was frozen and concentrated, redissolved with methanol-aqueous solution (50%, 50%) and filtered through a membrane, and then detected by LC-MS / MS. Chromatographic conditions: Chromatographic column: BEH C18 column (2.1 mm × 100 mm, 1.7 μm), mobile phase A was acetonitrile, B was ammonium formate solution, column temperature: 45 °C, flow rate: 0.3 mL / min, injection volume: 5 μL; The above method was used to detect the residual amounts of the four toxins in the sample after removing aflatoxin, and the removal rates of the corresponding four aflatoxins were calculated.

[0068]

[0069] The results showed that the above method was used to detect the residual amounts of the four toxins in the sample after removing aflatoxin, and the removal rates of the corresponding four aflatoxins were calculated. The results showed that compared with the sample without adding the strain fermentation broth as a control, the best effect was obtained when the compound ratio of aflatoxin AFTB1, AFTB2, AFTG1, and AFTG2 in BC77 and VC77 was 1:2, and the removal rates were 86.7%, 91.2%, 88.4%, and 89.9% respectively.

[0070] (2) Application of degrading zearalenone

[0071] Take a number of moldy feeds, crush and mix them evenly, and mix them with mold-free feeds to adjust the concentration of zearalenone (ZEA) to 60 mg / kg and the pH to 7.4 - 7.6; centrifuge the activated Bacillus coagulans BC77, Weissella confusa VC77, and compound bacterium agent, discard the supernatant, take the live bacteria, and adjust their concentration to 10 12CFU / mL; Take 100 g of moldy feed with a zearalenone concentration of 60 mg / kg, evenly spray the corresponding groups on the feed according to a ratio of 5% v / v, and then place it in an incubator at 37°C for static culture. Take a feed sample every 1 h to measure the remaining zearalenone concentration until the degradation concentration is stable three times (i.e., the measurement error < 0.02 each time). Take the average of the three measurements to evaluate the ability of Bacillus coagulans to degrade zearalenone under normal conditions.

[0072] The calculation method of the zearalenone degradation rate is (mycotoxin content 60 mg / kg - mycotoxin residue) / mycotoxin content 60 mg / kg × 100%

[0073]

[0074]

[0075] The results show that the above method was used to detect the zearalenone residue and calculate the corresponding degradation rate of zearalenone. The results show that the degradation rates of zearalenone by Bacillus coagulans BC77, Weizmannia coagulans VC77, and the compound microbial agent with a ratio of 1:2 are 53.6%, 75.1%, and 92.7% respectively.

[0076] It should be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and its inventive concept, and all such changes or substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A composite Bacillus coagulans inoculant, characterized in that: The bacterial agent comprises Bacillus coagulans ( Bacillus coagulans ) BC77, deposited in China Center for Type Culture Collection on May 7, 2021, with the deposit number CCTCC NO: M2021497; Weizmannella coagulans ( Weizmannia coagulans ) VC77, deposited in China Center for Type Culture Collection on June 29, 2023, with the deposit number CCTCC NO: M 20231138; The total number of viable bacteria in the bacterial agent is 10 10 ~10 11 CFU / g; the ratio of live bacteria of Bacillus coagulans BC77 to Weizmannella coagulans VC77 in the bacterial agent is 1: (2~3).

2. The composite Bacillus coagulans agent according to claim 1, characterized in that: In the bacterial agent, the ratio of live bacteria of Bacillus coagulans BC77 to live bacteria of Weizmannella coagulans VC77 is 1:

2.

3. The composite Bacillus coagulans agent according to claim 1, characterized in that: The bacterial agent uses Bacillus coagulans BC77 and Weizmannella coagulans VC77 as main microorganisms.

4. The composite Bacillus coagulans agent according to claim 1, characterized in that: The bacterial agent is a liquid bacterial agent or a solid bacterial agent.

5. The composite Bacillus coagulans agent according to claim 4, characterized in that: Solid bacterial agent is freeze-dried bacterial powder.

6. Use of the composite Bacillus coagulans agent according to claim 1 in the preparation of a microbial preparation for inhibiting pathogens or degrading toxins, characterized in that: The pathogen is Fusobacterium nucleatum polymorpha subspecies ( Fusobacterium nucleatum subsp. polymorphum gingivalis ( Porphyromonas gingivalis ), Actinobacillus actinomycetemcomitans ( Actinobacillus actinomycetemcomitans )、Streptococcus mutans( Streptococcus mutansClarke ), Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus ), Escherichia coli ( Escherichia coli )、Listeria monocytogenes( Listeria monocytogenes ), Salmonella enterica subsp. Salmonella enterica subsp. enterica )、Pseudomonas aeruginosa( Pseudomonas Aeruginosa ); The toxins are aflatoxins and zearalenone; among them, the aflatoxins are AFTB1, AFTB2, AFTG1, and AFTG2.

7. The use according to claim 6, characterized in that: The microbial agent is used in the environmental field.

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