Bacillus licheniformis for inhibiting lactic acid bacteria and application thereof

Abstract

The application discloses a bacillus licheniformis for inhibiting lactic acid bacteria and an application thereof, and belongs to the technical field of microorganisms. The bacillus licheniformis is screened from original strains in Daqu, and can inhibit the growth of lactic acid bacteria without affecting the growth of spores and yeasts in liquor fermentation, so that the effect of reducing lactic acid generation in liquor fermentation is achieved. The bacillus licheniformis LBM14002 screened in the application is derived from Daqu, is safer for liquor fermentation, and can inhibit lactic acid bacteria without destroying the original ecological system of fermented grains.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus licheniformis that inhibits lactic acid bacteria and its application, belonging to the field of microbial technology. Background Technology

[0002] The fermentation process of baijiu involves numerous microorganisms of various types and in large numbers, which produce diverse metabolites through complex metabolic activities. The interaction of these substances gives baijiu its rich and unique flavor. However, if the content of a single component in the liquor is too high, it may lead to an imbalance in flavor and affect the overall taste experience.

[0003] Because baijiu brewing takes place in an open environment, fluctuations in raw material quality and seasonal temperature changes can affect the distribution and metabolic characteristics of the microbial community, thus influencing the final flavor. During fermentation, lactobacilli gain ecological dominance after entering the fermentation pit, causing their metabolite—lactic acid—to accumulate continuously in the mash. As a non-volatile organic acid, lactic acid content increases continuously under the multi-round fermentation process of sauce-aroma baijiu. Furthermore, lactic acid can esterify with ethanol to form ethyl lactate. While ethyl lactate is an important aroma component in various baijiu types, excessively high levels can disrupt the overall harmony of the liquor.

[0004] In recent years, abnormally high lactic acid content in baijiu mash has frequently occurred in baijiu production, a problem that urgently requires a systematic and scientific solution. Currently, measures to reduce lactic acid levels still face challenges such as high costs and difficulty in controlling it at the source. Therefore, exploring more efficient and economical lactic acid reduction strategies has become a key focus for the industry. As a popular traditional alcoholic beverage both domestically and internationally, the market demand for Maotai-flavor baijiu is strong. How to optimize the fermentation process and improve product quality deserves in-depth research.

[0005] Bacillus licheniformis is a microorganism belonging to the genus Bacillus with broad application potential. It has demonstrated unique application value in multiple fields, including plant disease control, feed processing, pharmaceutical development, and environmental pollution control. Bacillus licheniformis can produce a variety of antimicrobial substances, such as antimicrobial proteins and bacteriocins, which have antagonistic effects on a variety of plant pathogens and pathogenic bacteria. The literature "Clinical Application Progress of Live Bacillus licheniformis Preparations; Chinese Journal of Microecology. 2017, 29(09)" discloses the application of Bacillus licheniformis in regulating intestinal flora; patent CN102876614B discloses a strain of Bacillus licheniformis that can inhibit Staphylococcus aureus, Lactobacillus plantarum, and Bacillus subtilis; patent CN117660226A discloses the application of Bacillus licheniformis in inhibiting lactic acid bacteria.

[0006] The aforementioned technology discloses the antibacterial effect of Bacillus licheniformis, and also involves its inhibitory effects on Bacillus subtilis and lactic acid bacteria. However, in the fermentation process of baijiu (Chinese liquor), yeast and Bacillus are important strains. As Bacillus licheniformis is capable of inhibiting microbial growth, the above technology neglects its effect on yeast and Bacillus. If Bacillus licheniformis inhibits the growth of yeast and Bacillus while also inhibiting lactic acid bacteria, it will have a significant impact on baijiu brewing.

[0007] Therefore, screening a strain of Bacillus licheniformis that can inhibit lactic acid bacteria without affecting yeast and Bacillus in the mash is of great practical significance and provides a new approach for more effective biological regulation of lactic acid content. Summary of the Invention

[0008] To address the aforementioned issues, this invention starts with the existing microbial strains in the Daqu (a type of starter culture) and screens strains capable of inhibiting the growth of lactic acid bacteria without affecting the growth of spores and yeast during Baijiu fermentation, thereby reducing lactic acid production during Baijiu fermentation. The Bacillus licheniformis LBM14002 (i.e., strain A8 screened in this invention) is derived from Daqu and is therefore safer for use in Baijiu fermentation, inhibiting lactic acid bacteria without disrupting the original ecosystem of the mash.

[0009] The first objective of this invention is to provide a strain of Bacillus licheniformis LBM14002, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2024870 and deposit date of May 8, 2024.

[0010] The Bacillus licheniformis LBM14002 strain of the present invention has the following effects:

[0011] (1) The metabolites of Bacillus licheniformis LBM14002 of the present invention can inhibit the growth of a variety of lactic acid bacteria, with a half-inhibitory concentration of 500 μL·mL. -1 ;

[0012] (2) At half-inhibitory concentration, the metabolites of Bacillus licheniformis LBM14002 can delay the time when acid-fast Lactobacillus enters the logarithmic growth phase by 7 hours and reduce the number of acid-fast Lactobacillus in the logarithmic growth phase by 12.67%.

[0013] (3) The Bacillus licheniformis LBM14002 strain of the present invention does not inhibit yeast and Bacillus in the fermentation of Baijiu; and its inhibition of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus fructose is increased by 18.9%, 16.4% and 21.5% respectively compared with the existing strain MX8.

[0014] (4) The Bacillus licheniformis LBM14002 of the present invention was screened from the Daqu for Baijiu fermentation, making it safer for Baijiu fermentation.

[0015] A second objective of this invention is to provide a microbial agent containing metabolites of Bacillus licheniformis LBM14002 or Bacillus licheniformis LBM14002.

[0016] In one embodiment of the present invention, the microbial agent is a liquid agent or a solid agent.

[0017] In one embodiment of the present invention, the viable count of Bacillus licheniformis LBM14002 per gram or milliliter of microbial agent is 10. 5 ~10 8 CFU.

[0018] A third objective of this invention is to provide the application of Bacillus licheniformis LBM14002 or any of the above-mentioned microbial agents in the food industry.

[0019] In one embodiment of the present invention, the food field includes brewing and koji making.

[0020] A fourth objective of this invention is to provide a method for inhibiting the growth of lactic acid bacteria during the fermentation of baijiu (Chinese liquor), by adding the aforementioned Bacillus licheniformis A8 or any of the aforementioned microbial agents to the mash or yeast.

[0021] In one embodiment of the present invention, the lactic acid bacteria include: acid-resistant lactobacillus, fermentative lactobacillus, plant lactobacillus, and fructose-eating lactobacillus.

[0022] A fifth object of the present invention is to provide a composition for brewing, said composition containing the above-described Bacillus licheniformis A8 or using any of the above-described microbial agents.

[0023] In one embodiment of the present invention, the composition further includes: lactose, chitosan, sucrose, glucose, glycerol, whey protein, and peptone.

[0024] Beneficial effects

[0025] This invention starts with the original strains in Daqu (a type of starter culture) and screens out Bacillus licheniformis LBM14002, which can inhibit the growth of lactic acid bacteria without affecting the growth of spores and yeasts in Baijiu fermentation, thus reducing the production of lactic acid in Baijiu fermentation.

[0026] Specifically:

[0027] (1) The metabolites of Bacillus licheniformis LBM14002 of the present invention can inhibit the growth of a variety of lactic acid bacteria, with a half-inhibitory concentration of 500 μL·mL. -1 Compared with the existing Bacillus licheniformis strain MX8 (disclosed in patent CN117660226A), the efficacy against Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus fructosolicum was improved by 18.9%, 16.4%, and 21.5%, respectively.

[0028] (2) At half-inhibitory concentration, the metabolites of LBM14002 can delay the time when acid-fast Lactobacillus enters the logarithmic growth phase by 7 hours and reduce the number of acid-fast Lactobacillus in the logarithmic growth phase by 12.67%.

[0029] (3) The Bacillus licheniformis LBM14002 of the present invention will not inhibit yeast and Bacillus in the fermentation of Baijiu;

[0030] (4) The Bacillus licheniformis LBM14002 of the present invention is screened from the Daqu of Baijiu fermentation, making it safer to use in Baijiu fermentation.

[0031] biomaterials

[0032] Bacillus licheniformis LBM14002, classified as Bacillus licheniformis LBM14002, was deposited on May 8, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2024870, located at Wuhan University, Wuhan, China.

[0033] Bacillus sonorensis LBM13002 was disclosed in patent CN116790421A on September 22, 2023. Attached Figure Description

[0034] Figure 1 The inhibition zones A31, A42, A12, A27, and A8 were obtained through screening in Example 1.

[0035] Figure 2 The concentrations of A8, A27, and LBM13002 in Example 2 are equal to or equal to the concentrations of LBM13002.

[0036] Figure 3 The OD of A8 delayed logarithmic growth phase in Example 3 of Lactobacillus acidophilus 600 Test results.

[0037] Figure 4 The inhibitory effects of A8 and A27 on other lactic acid bacteria in the mash in Example 4 are shown; where 1 is sterile water; 2 is A8 fermentation broth; and 3 is A27 fermentation broth.

[0038] Figure 5 The inhibitory effects of A8, A27, and LBM13002 on Bacillus spores in Daqu (a type of starter culture) in Example 4 are shown in Figure 4. 1 represents sterile water; 2 represents A8 fermentation broth; 3 represents A27 fermentation broth; and 4 represents LBM13002 fermentation broth.

[0039] Figure 6 The inhibitory effects of A8, A27, and LBM13002 on yeast in Daqu (a type of starter culture) in Example 4 are shown in Figure 4. 2 represents the fermentation broth of A8, A27, and LBM13002. Detailed Implementation

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] Raw materials used in the examples:

[0042] TB solid medium: peptone 12 g·L -1 Yeast extract 24g·L -1 Anhydrous dipotassium hydrogen phosphate, 12.54 g·L -1 Anhydrous potassium dihydrogen phosphate 2.31 g·L -1 4 mL·L glycerol -1 .

[0043] MRS medium was purchased from Oxoid.

[0044] Acid-fast Lactobacillus (NS), Lactobacillus fermentum (JSA), Lactobacillus plantarum (JD19), Pediococcus pentosaceus (B24), Lactobacillus fructose (LP), Bacillus daquica, Bacillus sonos, Bacillus subtilis, Bacillus amyloliquefaciens, Bayer conjugated yeast, Pichia pastoris, and Saccharomyces cerevisiae were all preserved in the laboratory of Jiangnan University.

[0045] Example 1: Screening for Bacillus licheniformis A8

[0046] The method of room temperature dilution plate coating was used to isolate and screen strains that inhibit lactic acid bacteria in the samples.

[0047] All samples of Daqu (a type of starter culture) were collected from a sauce-flavored liquor factory in Guizhou Province. Three starter culture rooms were selected, and three samples of black, white, and yellow Daqu were taken respectively. The Daqu was ground into powder and mixed evenly. Each sample was 200g and stored in a self-sealing bag at 4℃ for subsequent microbial screening.

[0048] Take 10g each of the yellow, white, and black high-temperature Daqu (fermented starter culture) and add them to 90mL of sterile PBS buffer (pH 7.2–7.4). Mix on a shaker at 37℃ and 200rpm for 30 minutes to obtain bacterial suspensions. Dilute the bacterial suspensions 10... -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Six gradients were evenly spread on LB solid medium supplemented with nystatin (1‰ v / v) to inhibit fungal growth, and single colonies were obtained.

[0049] A single colony was inoculated into a test tube and cultured on a shaker at 37°C and 200 rpm for 24 hours. 1 mL of the bacterial culture was then centrifuged to obtain the inhibition supernatant, which was used for the inhibition zone experiment. Acid-fast Lactobacillus was cultured to the logarithmic growth phase (1×10⁻⁶). 8 CFU·mL -1 Inoculate 1‰ v / v into solid MRS medium cooled to a temperature that is not too hot to touch. Place the plate at 4℃ and let it solidify for 20 min. Then punch holes and add 50 μL of the antibacterial supernatant to each well. Incubate at 37℃ for 12 h. If an inhibition zone appears, it is an antibacterial microorganism.

[0050] The results of the inhibition zone are as follows Figure 1 As shown, the selected bacterial strains were named A42, A31, A12, A8, and A27. DNA from the inhibiting strains was extracted using the PlantGenomic DNA Extraction Kit. Universal bacterial primers 27F and 1492R were used for amplification. PCR reaction conditions were: 94℃ pre-denaturation for 6 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, 30 cycles, followed by a final extension at 72℃ for 10 min. The concentration of the PCR product was measured, and the reliability was verified by nucleic acid gel electrophoresis. The qualified DNA samples were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results were compared using BLAST in the National Center for Biotechnology Information (NCBI) database.

[0051] The comparison results showed that A42, A31, A12, and A8 are Bacillus licheniformis; A27 is Bacillus paralicheniformis. Strain A8 was deposited at the China Center for Type Culture Collection on May 8, 2024, with accession number CCTCC NO: M2024870, deposit address: Wuhan University, Wuhan, China, and classified as Bacillus licheniformis LBM14002.

[0052] A8 and A27, which have larger inhibition zones, were selected for subsequent experiments.

[0053] Example 2: Antibacterial supernatant IC 50 Detection

[0054] The half-maximal inhibitory concentration (IC50) of the antibacterial supernatant was determined using the micro-dilution method. 50 The strain Bacillus sonorensis LBM13002 was used as a control. The strain Bacillus sonorensis LBM13002 was disclosed in patent CN116790421A on September 22, 2023.

[0055] Strains Bacillus. Paralicheniformis A27 (hereinafter referred to as A27), Bacillus. Licheniformis A8 (i.e., Bacillus licheniformis LBM14002; hereinafter referred to as A8), and Bacillus. sonorensis LBM13002 (hereinafter referred to as LBM13002) were inoculated into TB medium at 200 r / min and 37℃, and cultured until OD. 600 1×10 10 CFU·mL -1 The bacterial culture was centrifuged at 8000 rpm for 15 min at 4°C, and then further sterilized by passing through a 0.22 μm filter membrane to obtain the antibacterial supernatant. The antibacterial supernatant was diluted with sterile MRS liquid medium using the two-fold dilution method, and each supernatant was diluted to 500 μL / mL. -1 250 μL·mL -1 125 μL·mL -1 and 62.5 μL·mL -1 Four concentration gradients were used, with MRS liquid medium without added antibacterial supernatant serving as the control group.

[0056] After the acid-fast Lactobacillus cultured to the logarithmic growth phase, it was inoculated with 1% v / v (inoculation amount of 1×10⁻⁶). 6 CFU·mL -1), OD measured 12 hours later 600 Plotting concentration on the x-axis, OD 600 The value is on the ordinate, compared with the final OD of the control group. 600 Compared to the control group, OD 600 The corresponding concentration is IC50. 50 .

[0057] IC 50 The results are as follows Figure 2 As shown, the results indicate that when the concentration of the supernatant of A8 is 500 μL·mL -1 At that time, the OD of acid-fast lactobacilli 600 The value was 0.757; when the concentration of the supernatant of A27 and LBM13002 was 500 μL·mL -1 and 250 μL·mL -1 At this time, acid-resistant Lactobacillus was completely inhibited and almost stopped growing. Calculations showed that the half-maximal inhibitory concentrations (WMCs) for A8, A27, and LBM13002 were 500 μL·mL⁻¹. -1 100 μL·mL -1 330 μL·mL -1 .

[0058] Example 3: Bacillus licheniformis A8 delays the growth of lactic acid bacteria

[0059] Based on the results of Example 2, respectively in IC 50 1 / 2 IC 50 Acid-fast Lactobacillus was cultured in A8 antibacterial supernatant at a concentration of 100 mg / L, with the control group being the fermentation broth without antibacterial addition. The time required for the acid-fast Lactobacillus to grow to the logarithmic growth phase and the peak OD of the acid-fast Lactobacillus were measured. 600 Acid-fast Lactobacillus was inoculated with 1% v / v (seed culture concentration of 1×10⁻⁶). 6 CFU·mL -1 ) containing 500 μL·mL -1 250 μL·mL -1 The A8 supernatant was cultured in MRS medium for 24 hours, and OD was measured every 2.5 hours. 600 .

[0060] The results are as follows Figure 3 As shown, the results indicated that at the half-inhibition concentration, the A8 supernatant delayed the logarithmic growth phase of *Lactobacillus acid-fast bacilli* by 9 hours; at the half-inhibition concentration, the A8 supernatant delayed the logarithmic growth phase of *Lactobacillus acid-fast bacilli* by 7 hours. After *Lactobacillus acid-fast bacilli* reached the stationary phase, the OD of *Lactobacillus acid-fast bacilli* in the A8 supernatant at the half-inhibition concentration... 600 It decreased by 12.67%.

[0061] Example 4: Detection of the inhibitory effect of Bacillus licheniformis A8 on other lactic acid bacteria, yeasts, and Bacillus.

[0062] 1. Inhibits other lactic acid bacteria

[0063] The inhibitory effects of A8 and A27 on *Lactobacillus fermentum*, *Lactobacillus plantarum*, *Pediococcus pentosus*, and *Lactobacillus fructose* screened from baijiu mash were tested according to the method in Example 1.

[0064] The results are as follows Figure 4 As shown, the results indicate that A27 can inhibit the growth of all lactobacilli, with the most significant inhibition against fermenting lactic acid bacteria; A8 has antibacterial effects against fermenting lactobacilli, plant lactobacilli, and fructose lactobacilli, but E does not show antibacterial activity against Pediococcus saccharidosis.

[0065] 2. Does not inhibit yeast and Bacillus.

[0066] Following the method in Example 1, the inhibitory effects of A8 and A27 on the Bacillus and yeast strains screened in the Daqu (fermentation starter) of Maotai-flavor liquor were tested. The strains were Bacillus rockella, Bacillus sonos, Bacillus subtilis, Bacillus amyloliquefaciens, Bayer conjugate yeast, Pichia pastoris, and Saccharomyces cerevisiae.

[0067] The results are as follows Figure 5 , Figure 6 As shown, the results indicate that A8 and A27 have no inhibitory effect on the Bacillus and yeasts screened in Baijiu, meaning that Bacillus. Licheniformis A8 and Bacillus. Paralicheniformis A27 do not affect the growth of Bacillus and yeasts during the Baijiu brewing process.

[0068] Example 5: Preparation of Bacillus licheniformis A8 inoculum

[0069] Bacillus licheniformis A8 was inoculated at 1% v / v in TB medium and cultured on a shaker at 37°C and 200 rpm for 24 h. The bacterial cells and supernatant were collected by centrifugation. The bacterial cells were resuspended in a protectant with the following formulation (g / L): 20% milk powder, 10% trehalose, 2% glycerol (v / v), and 5% xylooligosaccharides. The resulting Bacillus licheniformis A8 bacterial agent was lyophilized, and the viable count of Bacillus licheniformis A8 in the agent was 10⁻⁶. 5 ~10 8 CFU / g.

[0070] Optionally, the supernatant is freeze-dried to obtain a bacterial metabolite powder, and the bacterial metabolite powder is mixed with Bacillus licheniformis A8 bacterial agent to obtain a bacterial agent containing Bacillus licheniformis A8 and its metabolites.

[0071] Example 6: Comparison of antibacterial effects of other Bacillus licheniformis strains

[0072] 1. Detection of spore and yeast inhibition effects

[0073] Bacillus licheniformis A42, A31, and A12 obtained from screening in Example 1 were used. Following the method in Example 4, the inhibitory effects of Bacillus licheniformis A42, A31, and A12 on Bacillus and yeast were tested.

[0074] The results showed that Bacillus licheniformis A42, A31, and A12 all inhibited the growth of one or more strains of Bacillus or yeast.

[0075] 2. Lactic acid bacteria inhibition effect test

[0076] Bacillus licheniformis A8 and MX8 were taken, and their lactic acid bacteria inhibitory effects were compared according to the method in Example 4.

[0077] The results showed that Bacillus licheniformis A8 had a better inhibitory effect than MX8, and its inhibitory effect on Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus fructosamine was significantly higher than that on MX8. 抑菌圈直径 / MX8 抑菌圈直径 -1) 18.9%, 16.4%, 21.5%.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bacillus licheniformis ( Bacillus licheniformis LBM14002, characterized in that, The Bacillus licheniformis LBM14002 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024870 and deposit date of May 8, 2024.

2. A microbial inoculant, characterized in that, The microbial agent contains Bacillus licheniformis LBM14002 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, Microbial inoculants can be liquid or solid.

4. The microbial agent according to claim 2, characterized in that, The viable count of Bacillus licheniformis LBM14002 per gram or milliliter of microbial agent is 10. 5 ~10 8 CFU.

5. The application of Bacillus licheniformis LBM14002 as described in claim 1 or any of the microbial agents described in claims 2 to 3 in brewing.

6. A method for inhibiting the growth of lactic acid bacteria during the fermentation of baijiu (Chinese liquor), characterized in that, The Bacillus licheniformis LBM14002 of claim 1 or any of the microbial agents of claims 2 to 4 are added to the fermentation mash, wherein the lactic acid bacteria are Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus fructose.

7. A composition for brewing, characterized in that, The composition contains Bacillus licheniformis LBM14002 as described in claim 1 or any of the microbial agents described in claims 2 to 4.

8. The composition according to claim 7, characterized in that, The composition also includes lactose, chitosan, sucrose, glucose, glycerol, whey protein, and peptone.

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