Mutagenic bacillus coagulans and application thereof in production of ligustrazine

Abstract

The invention provides mutagenic bacillus coagulans and application thereof in production of ligustrazine, and belongs to the technical field of biology. The mutagenic bacillus coagulans YB-2023 disclosed by the invention is classified and named as Heynickia coagulans, and the strain number of the mutagenic bacillus coagulans YB-2023 is YB-2023, and the mutagenic bacillus coagulans YB-2023 is named as Heynickia coagulans. The strain is preserved in the China Center for Type Culture Collection on March 7, 2024, the address is No. 299, eight road, Wuchang District, Wuhan City, Hubei Province, and the strain preservation number is CCTCC NO: M 2024411; the acetoin yield of mutagenic bacillus coagulans YB-2023 can reach 12.61 + / -0.34 g / L, and the concentration of ligustrazine can reach 2.54 + / -0.26 g / L. After original strains are mutagenized, glucose and yeast extract are used as raw materials under normal culture conditions, the number of probiotics bacillus coagulans is reserved, the ligustrazine yield is increased, the cost is reduced, and the method is suitable for industrial production. The green production process for producing natural ligustrazine by using renewable biomass has significance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a mutagenized bacillus coagulans strain and application thereof in producing ligustrazine. Background Art

[0002] Bacillus coagulans is a Gram-positive bacillus with the ability to produce spores. Its spores are highly heat-resistant, which gives it good survival and resistance to environmental changes. Bacillus coagulans is rod-shaped, with terminal spores and no flagella. The optimal growth temperature is 45-55°C and the optimal pH is 6.6-7.0. It can decompose sugars to produce L-lactic acid and is a homolactic fermentation bacterium. It is also a facultative anaerobic bacterium. After entering the intestine, it will consume free oxygen, which is conducive to the growth of anaerobic microorganisms such as lactic acid bacteria and bifidobacteria, thereby regulating the balance of the microecological flora in the intestine. Whether spores are produced is the key to distinguishing Bacillus coagulans from traditional lactic acid bacteria. Bacillus coagulans belongs to the Bacillus probiotics. Under certain conditions, it can form spores, thus having resistance to adverse environments. It also has strong tolerance to acid, heat, and bile salts, which can solve the characteristics of other non-spore-producing probiotics, such as poor stress resistance, short shelf life, and easy inactivation in the body. Because it can produce spores and has unique biological characteristics such as resistance to gastric acid, resistance to desiccation, resistance to high temperature and high pressure, and easy storage, it has unique advantages in product processing and storage. Therefore, the research on Bacillus coagulans has become a hot spot in the research and development of probiotic strains.

[0003] Studies have found that Bacillus coagulans can produce acetoin (i.e., 3-hydroxy-2-butanone), the precursor of ligustrazine. Acetoin exists in the form of monomers and dimers at room temperature. The monomer is a colorless or light yellow volatile liquid with a pleasant creamy aroma and a white crystalline powder. Acetoin is the third most abundant volatile substance in traditional solid-state fermented vinegar in my country after acetic acid and furfural, and is an important intermediate product that affects the production of ligustrazine by microbial fermentation. As an intermediate of the product, acetoin is also a popular food flavoring additive. It not only plays an important role in the biological control of agricultural pests, but also has a good effect on the formation of tobacco aroma. It also has an indirect effect on the modification of drugs. In addition, acetoin can also be used to synthesize stabilizers and foaming agents for chlorine-containing polymers. The derivatives of acetoin can be used to synthesize new hydrophilic coatings with antioxidant properties, which can extend the service life of the resin.

[0004] Ligustrazine (2,3,5,6-tetramethylpyrazine) is a compound with cardiovascular and cerebrovascular health care functions. It can promote blood circulation, remove blood stasis and activate blood circulation, protect against cerebral ischemia, hypoxia-reperfusion injury and cerebral infarction, reduce arterial pressure and coronary artery resistance, and is widely used in the treatment of typical migraine, arteriosclerosis, ischemic stroke and other cardiovascular and cerebrovascular diseases. The traditional production method of ligustrazine mainly relies on chemical synthesis, which has problems of environmental pollution and high cost. In recent years, microbial fermentation has attracted attention due to its environmental protection and renewable characteristics.

[0005] Ligustrazine (i.e. 2,3,5,6-tetramethylpyrazine) has good health care functions for human cardiovascular and cerebrovascular health. At present, the main methods for producing ligustrazine are chemical synthesis and microbial fermentation. The chemical synthesis method is corrosive to the environment and has high requirements for equipment, while the microbial fermentation method uses a wide range of raw materials, which are low in price, has a simple fermentation process, high production efficiency, high product purity, is suitable for food applications, and has little pollution to the environment. In addition, acetoin, as a precursor of ligustrazine, is difficult to separate efficiently due to its unique property of being easily soluble in water and organic solvents. Compared with the separation and extraction of acetoin, the purification of ligustrazine is simpler. Converting acetoin in the fermentation broth into easily separable ligustrazine not only avoids the difficulty of acetoin recovery, but also increases the value of the product.

[0006] At present, the existing microbial fermentation method still needs to be improved in terms of production efficiency and optical purity of ligustrazine. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a mutated Bacillus coagulans and its application in the production of ligustrazine. After the original strain of the present invention is mutated, glucose and yeast extract are used as raw materials under normal culture conditions, while the number of probiotic Bacillus coagulans is retained, the yield of ligustrazine is increased, the cost is reduced, and the green production process of producing natural ligustrazine using renewable materials is meaningful.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides a mutagenized Bacillus coagulans YB-2023, which is classified and named Heyndrickxia coagulans, and the strain number is YB-2023; it was preserved in the China Center for Type Culture Collection on March 7, 2024, with the address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO: M 2024411.

[0009] Preferably, the acetoin production of the induced Bacillus coagulans YB-2023 can reach 12.61±0.34 g / L, and the ligustrazine concentration can reach 2.54±0.26 g / L.

[0010] The mutagenic strain Bacillus coagulans of the present invention is obtained by mutagenesis from the original strain, and the original strain is stored in the form of freeze-dried powder. The surface of the ampoule freeze-dried tube is first wiped with 75% alcohol cotton for disinfection, and the top of the ampoule tube is evenly heated on the outer flame of an alcohol lamp, and 2-3 drops of sterile water are immediately dripped on the heated part to break the tube wall, and the broken part is knocked off with tweezers or other suitable tools. About 0.5 ml of liquid culture medium is sucked into the freeze-dried tube with a sterile pipette to dissolve all the freeze-dried bacterial powder. The dissolved bacterial suspension is transferred to a test tube containing 4-5 mL of liquid culture medium and mixed evenly, and the 1-2 drops of bacterial suspension remaining in the pipette can be transferred to the solid culture medium. After the activated strains in the test tubes were purified by streaking, single bacteria were picked and statically cultured on LB agar slants at 37 °C for 24 h. After freeze-drying, the strains were in a dormant state. The culture time of the first-generation strains needed to be appropriately extended, and the fermentation medium was transferred to the 2-3 generation to restore vitality. 0.1 mL of the fermentation liquid cultured to the exponential growth phase was evenly spread on a sterile blank culture dish, dried with sterile air, and ion beam implanted after microscopic observation without cell overlap. Nitrogen ion beam implantation was selected, and N + The energy is 10~20keV and the dose is 20x10 13 ~20x10 13 ions / cm 3 , the treatment time is 120s. After the injection is completed, take out the plate and wash it with sterile water. Prepare the strains screened in the previous step into a cell suspension and adjust the concentration to 10 -6 / ml, take 0.1ml of cell suspension and spread it on the sterilized plate, blow dry and then perform nitrogen ion beam implantation, N + The energy is 10~30keV and the dose is 20x10 14 ~20x10 14 ions / cm 3 , the treatment time is 120s. After the injection is completed, the plate is washed with sterile water, and the washed bacterial solution is spread on the LB solid medium and inverted at 37℃ for 24h. The induced strain is inoculated into the seed medium and cultured at 37℃ for 10~18h. The volume of the shake flask is 10% (v / v). The bacterial solution in the seed medium is inoculated into the fermentation medium at an inoculation rate of 5%, and cultured at 45℃ for 36h. The strain is inoculated into the LB solid medium and strains with good growth are selected. Repeat the rescreening steps, select single colonies that meet the morphological characteristics of Bacillus coagulans and have good growth for shake flask fermentation culture, determine the number of live probiotic bacteria and the number of spores, and finally select a strain with good properties.

[0011] The coagulant Bacillus that can retain a large number of probiotic spores was named as Hendrix coagulans. It grows well in LB medium. The observed strains are usually round or irregularly round, with a diameter of about 2 to 5 mm, a moderate size, and neat or slightly wavy edges. The surface of the colony is smooth and moist, dense and opaque, and appears milky white or light yellow. With the formation of spores, the surface of the colony gradually becomes slightly rough, and the colony shows stress resistance. The colony color becomes slightly darker, showing light yellow or light brown. It is a Gram-positive bacillus.

[0012] The Bacillus coagulans in the present invention is a Bacillus, with an optimum growth temperature of 45-55°C and an optimum pH of 6.6-7.0. Its spores are highly heat-resistant and can decompose sugars to produce L-lactic acid. It is a homolactic acid fermentation bacterium and a facultative anaerobic bacterium, which effectively improves the ability of probiotics to resist adverse external environments and the ability to produce spores.

[0013] In a second aspect, the present invention provides the use of the above-mentioned mutagenized Bacillus YB-2023 in the production of ligustrazine.

[0014] Preferably, the method for inducing mutation of Bacillus coagulans to produce ligustrazine is: subjecting the Bacillus coagulans to plate culture, slant culture, seed culture and fermentation culture in sequence.

[0015] Preferably, the culture medium used for the plate culture contains the following components in terms of mass: 10 g / L peptone, 25 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar; The plate culture temperature is 35-37°C and the time is 24-48h.

[0016] Preferably, the culture medium used for the slant culture contains the following components in terms of mass: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 20 g / L; The fermentation temperature of the slant culture is 35-45°C, and the fermentation time is 24-48 hours.

[0017] Preferably, the culture medium used for seed culture contains the following components in terms of mass: 10 g / L peptone, 30 g / L edible yeast extract, 1 g / L sodium chloride, and 60 g / L glucose; The seed culture temperature is 35-45°C and the time is 10-20 hours.

[0018] Preferably, the culture medium used in the fermentation culture comprises the following components by mass: 30 g / L yeast extract, 60 g / L glucose, 0.5 g / L sodium acetate, 0.2 g / L diammonium citrate, 0.2 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.2 g / L dipotassium hydrogen phosphate, and 120 g / L corn steep liquor; The fermentation culture temperature is 45-55°C and the fermentation time is 36-46h.

[0019] Preferably, the number of Bacillus coagulans after fermentation is 8×10 8 ~9×10 8 CFU / mL.

[0020] Beneficial technical effects: Compared with wild strains, the coagulant Bacillus hendrix obtained by ion beam engineering technology screening in the present invention effectively improves the spore production ability of the strain, has good genetic stability and improved ligustrazine production ability, with a production cycle of 36-45 hours, a yield of 4.56 g / L, and a coagulant Bacillus count of 8.91×10 8 CFU / mL. After the original strain was induced to undergo mutation, glucose and yeast extract were used as raw materials under normal culture conditions. The number of probiotic Bacillus coagulans was retained while increasing the yield of ligustrazine, reducing costs. The green production process of using renewable materials to produce natural ligustrazine is meaningful.

[0021] Description of biological deposit: The mutated Bacillus coagulans YB-2023 described in the present invention is classified and named Heyndrickxia coagulans, and the strain number is YB-2023; it was preserved in the China Center for Type Culture Collection on March 7, 2024, with the address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO: M2024411. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0028] The raw materials used in the following examples of the present invention are commercially available unless otherwise specified.

[0029] Example 1 Mutagenesis of strains and low pH screening Starting strain screening: The strain Bacillus coagulans of the present invention is obtained by induction from the original strain, and the original strain is stored in the form of freeze-dried powder. The surface of the ampoule freeze-dried tube is first wiped with 75% alcohol cotton for disinfection, and the top of the ampoule tube is evenly heated on the outer flame of an alcohol lamp, and 2-3 drops of sterile water are immediately dripped on the heated part to break the tube wall, and the broken part is knocked off with tweezers or other suitable tools. About 0.5 ml of liquid culture medium is sucked into the freeze-dried tube with a sterile pipette to dissolve all the freeze-dried bacterial powder. The dissolved bacterial suspension is transferred to a test tube containing 4-5 mL of liquid culture medium and mixed, and the 1-2 drops of bacterial suspension remaining in the pipette can be transferred to the solid culture medium. After the activated strains in the test tubes were purified by streaking, single bacteria were picked and statically cultured on LB agar slants at 37 °C for 24 h. After freeze-drying, the strains were in a dormant state. The culture time of the first-generation strains needed to be appropriately extended. The fermentation medium was transferred to the 2-3 generation to restore vitality. 0.1 mL of the fermentation liquid cultured to the exponential growth phase was evenly spread on a sterile blank culture dish, dried with sterile air, and ion beam implantation was performed under microscopic observation without cell overlap. Nitrogen ion beam implantation was selected, and N +The energy is 10~20keV and the dose is 20x10 13 ~40x10 13 ions / cm 3 After the injection, the plate was taken out and washed with sterile water. This process was repeated several times to observe and select strains with good growth conditions.

[0030] Rescreening: The strains screened in the previous step were made into cell suspensions and the concentration was adjusted to 10 -6 / ml, take 0.1ml of cell suspension and spread it on the sterilized plate, blow dry and then perform nitrogen ion beam implantation, N + The energy is 10~30keV and the dose is 20x10 14 ~40x10 14 ions / cm 3 , the treatment time is 120s. After the injection is completed, the plate is washed with sterile water, and the washed bacterial solution is spread on the LB solid medium and inverted at 37℃ for 24h. The induced strain is inoculated into the seed medium and cultured at 37℃ for 10~18h. The volume of the shake bottle is 10% (v / v). The bacterial solution in the seed medium is inoculated into the fermentation medium at an inoculation rate of 5%, cultured at 45℃ for 36h, heated to 55℃, added 1 g / L calcium carbonate and appropriate amount of diammonium hydrogen phosphate, cultured for 8h, and the final strain is inoculated into the LB solid medium. The strain with good growth is selected, and the re-screening steps are repeated until the target strain is screened out. The formula of the fermentation medium is (components and contents in 1000ml): 30 g yeast extract, 60 g glucose, 0.5 g sodium acetate, 0.2 g diammonium citrate, 0.2 g magnesium sulfate, 0.01 g manganese sulfate, 0.2 g dipotassium hydrogen phosphate, and 120 g corn steep liquor.

[0031] The formula of seed culture medium (components and contents in 1000 ml): peptone 10 g, yeast extract 30 g, sodium chloride 1 g, glucose 60 g.

[0032] The strains obtained by screening were positive for Gram staining and negative for catalase test. The colony morphology was usually round or irregular, with a diameter of about 2 to 5 mm, a moderate size, and neat or slightly wavy edges. The colony surface was smooth and moist, dense and opaque, and appeared milky white or light yellow. With the formation of spores, the colony surface gradually became slightly rough, and the colony showed stress resistance. The colony color became slightly darker, showing light yellow or light brown.

[0033] Determination of spore survival rate of screening strains: Under a 45°C environment, the screened mutant strains and the original strains were cultured in fermentation medium respectively. The growth conditions were observed after 36 hours. It was found that under each set of conditions, the survival rate of the screened strain spores was higher than that of the original strain, reaching more than 90%, and the growth state of the surviving strains was better than that of the original strains.

[0034] Example 2 This example illustrates the identification and genetic stability of the strain Identification of strains: On LB solid culture medium, the colonies are milky white or light yellow, round or irregular in shape, with a smooth and moist surface, dense and opaque texture, and slightly rough edges. The suitable growth temperature range of the bacteria is 45-55°C.

[0035] Subculture experiment: The mutant bacteria screened were inoculated into LB solid culture medium and cultured at 37°C under anaerobic conditions for 24 h. They were then inoculated into solid culture medium and cultured for 36 h, and the survival rates of strains of different generations were recorded.

[0036] The survival rates of strains of different generations are shown in Table 1: Table 1 The experimental results show that after five consecutive subcultures, the mutant strain has good subculture stability and can be used as a strain for further research and development.

[0037] Example 3 Cultivation method of Bacillus coagulans This example illustrates a method for culturing Bacillus coagulans, comprising the following steps: 1) Plate culture: Inoculate Bacillus coagulans onto a plate culture medium at a temperature of 35-45°C for 24-48 hours; 2) Slant culture: inoculate the Bacillus coagulans cultured on the plate medium in step 1) into the slant medium for culture at a temperature of 35-45°C for 24-48 hours; 3) Seed culture: inoculate the slant culture in step 2) into the seed culture medium for culture at a temperature of 35-45°C for 10-20 hours; 4) Fermentation culture: Inoculate the seed solution of Bacillus coagulans cultured in step 3) into the fermentation medium at an inoculum rate of 5% (v / v), the culture temperature is 45°C, the culture time is 36h, the fermentation liquid temperature is raised to 55°C, and 1 g / L calcium carbonate and an appropriate amount of diammonium hydrogen phosphate are added.

[0038] The plate culture medium comprises the following components by mass: 10 g / L peptone, 25 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar.

[0039] The slant culture medium includes: 10 g / L peptone, 25 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar. The seed culture medium comprises: 10 g / L peptone, 30 g / L edible yeast extract, 1 g / L sodium chloride, and 60 g / L glucose.

[0040] The fermentation medium comprises: yeast extract: 30 g / L, glucose: 60 g / L, sodium acetate: 0.5 g / L, diammonium citrate: 0.2 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.01 g / L, dipotassium hydrogen phosphate: 0.2 g / L, and corn steep liquor: 120 g / L.

[0041] Example 4 Production of Ligustrazine by Strain Fermentation Bacillus coagulans with good growth status were selected and inoculated into the seed culture medium, and the seed solution was obtained by overnight culture at 37 ℃ and 180 r / min. The seed solution was inoculated at a ratio of 5% (v / v) in 500mL fermentation medium, and the strain was fermented in a shaking table at 180rpm for 36h. The glucose consumption, acetoin production, spore count and viable bacteria count were sampled and tested. The fermentation liquid temperature was raised to 55℃, 1 g / L calcium carbonate and an appropriate amount of diammonium hydrogen phosphate (so that the molar ratio of acetoin to ammonium ion was 1:3) were added, and the culture was continued for 8 hours. At this time, the number of viable bacteria and the number of spores were basically equal. After the fermentation, the content of acetoin and ligustrazine was determined by gas chromatography (GC).

[0042] The fermentation medium formula is: yeast extract: 30 g / L, glucose: 60 g / L, sodium acetate: 0.5 g / L, diammonium citrate: 0.2 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.01 g / L, dipotassium hydrogen phosphate: 0.2 g / L, corn steep liquor: 120 g / L.

[0043] The fermentation temperature is 45-55°C.

[0044] The results of the test showed that, under the condition of amplified culture scale, the original strain could produce 8.4 g / L of acetoin in 36 h, and the optical purity of the obtained product was 96.3%. The acetoin produced by the induced strain in 36 h was 14.6 g / L (an increase of 74%), and the optical purity of the obtained product L-lactic acid was above 99.8%. The mutant strain was fermented and cultured at pH 8.0 for 46 h, and the number of bacterial spore accumulation and the yield of ligustrazine were significantly higher than those of the original strain. The number of spore accumulation of the original strain was 6.67×10 8 CFU / mL, while the spore accumulation number of the mutant strain was 8.91×10 8CFU / mL (increase of 34%), the ligustrazine production of the original strain was only 1.37g / L, while the ligustrazine production of the mutant strain reached 4.56g / L, which was 3.33 times that of the original strain.

[0045] Determination of acetoin in fermentation broth by gas chromatography: Instrument: Agilent 7890B gas chromatograph equipped with flame ionization detector (FID).

[0046] Chromatographic column: HP-5 capillary column (30 m × 0.32 mm × 0.25 μm).

[0047] Carrier gas: high-purity nitrogen, air flow rate 450 mL / min, hydrogen flow rate 45 mL / min.

[0048] The injection port temperature was 280 °C, the split mode was adopted, and the split ratio was 30:1.

[0049] Heating program: initial temperature 50°C, hold for 2 minutes; increase the temperature to 250°C at a rate of 10°C / min, hold for 8 minutes.

[0050] Detector temperature: 300°C.

[0051] Determination of ligustrazine in fermentation broth by gas chromatography: Instrument: Agilent 7890B gas chromatograph equipped with flame ionization detector (FID).

[0052] Chromatographic column: HP-5 capillary column (30 m × 0.32 mm × 0.25 μm).

[0053] Carrier gas: high-purity nitrogen, air flow rate 450 mL / min, hydrogen flow rate 45 mL / min.

[0054] The injection port temperature was 280 °C, the split mode was adopted, and the split ratio was 30:1.

[0055] Heating program: initial temperature 50°C, hold for 2 minutes; increase the temperature to 250°C at a rate of 10°C / min, hold for 8 minutes.

[0056] Detector temperature: 300°C.

[0057] Acetoin standard solutions (1-25 g / L) and ligustrazine standard solutions (0.01-10 g / L) of different concentrations were prepared. After sample treatment, the peak area ratio obtained by GC detection analysis was combined with the standard curve for quantitative analysis. The retention time of acetoin was about 4.6 min, and the retention time of ligustrazine was about 13 min. The ratios of the peak areas of acetoin and ligustrazine to the peak area of ​​n-hexanol were obtained, and the yields of acetoin and ligustrazine were calculated based on the peak areas.

[0058] Industrial Applicability The above studies show that the strain of the present invention is significantly superior to the original strain without mutation in terms of probiotic spore accumulation number, ligustrazine production speed and optical purity (the number of spores increased by 34%, the production speed increased by 3.33 times, and the optical purity increased to 99%). The present invention provides a novel strain with low fermentation cost, fast ligustrazine production speed and high product optical purity. This strain can increase the yield of ligustrazine while retaining the number of probiotic Bacillus coagulans, reduce costs, and use renewable biomass to produce natural ligustrazine in a green production process to achieve the purpose of reducing environmental pollution, and is of great significance for the industrial microbial fermentation production of ligustrazine.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutagenic Bacillus coagulans YB-2023, characterized in that: Its taxonomic name is Hyndrickella coagulans ( Heyndrickxia coagulans ), strain numbered YB-2023; it was deposited in the China Center for Type Culture Collection on March 7, 2024, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO:M2024411.

2. The mutated Bacillus coagulans YB-2023 according to claim 1, characterized in that: The acetoin production of the induced Bacillus coagulans YB-2023 could reach 12.61±0.34 g / L, and the ligustrazine concentration could reach 2.54±0.26 g / L.

3. Use of the mutagenic Bacillus YB-2023 according to claim 1 in the production of ligustrazine.

4. The use according to claim 3, characterized in that: The method for inducing mutation of Bacillus coagulans to produce ligustrazine is as follows: subjecting the Bacillus coagulans to plate culture, slant culture, seed culture and fermentation culture in sequence.

5. The use according to claim 4, characterized in that: The culture medium used for the plate culture contains the following components by mass: 10 g / L peptone, 25 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar; The plate culture temperature is 35-37°C and the time is 24-48h.

6. The use according to claim 4, characterized in that: The culture medium used for the slant culture contains the following components by mass: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 20 g / L; The fermentation temperature of the slant culture is 35-45°C, and the fermentation time is 24-48 hours.

7. The use according to claim 4, characterized in that: The culture medium used for seed culture contains the following components by mass: 10 g / L peptone, 30 g / L edible yeast extract, 1 g / L sodium chloride, and 60 g / L glucose; The seed culture temperature is 35-45°C and the time is 10-20 hours.

8. The use according to claim 4, characterized in that: The culture medium used in the fermentation culture contains the following components by mass: 30 g / L yeast extract, 60 g / L glucose, 0.5 g / L sodium acetate, 0.2 g / L diammonium citrate, 0.2 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.2 g / L dipotassium hydrogen phosphate, and 120 g / L corn steep liquor; The fermentation culture temperature is 45-55°C and the fermentation time is 36-46h.

9. The use according to claim 4, characterized in that: The inoculation ratio of the seed solution in the fermentation culture is 5% v / v.

10. The use according to claim 4, characterized in that: The number of Bacillus coagulans after fermentation culture is 8×10 8 ~9×10 8 CFU / mL.

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