A mutant Bacillus coagulans and its application in the production of ligustrazine

Abstract

The present invention provides a mutagenized Bacillus coagulans and its application in the production of tetramethylpyrazine, belonging to the field of biotechnology. The mutagenized Bacillus coagulans YB-2023 described in the present invention is classified and named as Heyndrickxia coagulans, with the strain number YB-2023; it was deposited at the China Center for Type Culture Collection on March 7, 2024, at the address No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the accession number of the strain is CCTCC NO: M 2024411; the acetoin yield of the mutagenized Bacillus coagulans YB-2023 can reach 12.61 ± 0.34 g / L, and the concentration of tetramethylpyrazine can reach 2.54 ± 0.26 g / L. After the original strain is mutagenized, under normal culture conditions, using glucose and yeast extract as raw materials, while retaining the number of probiotic Bacillus coagulans, it can improve the yield of tetramethylpyrazine, reduce costs, and has significance for the green production process of producing natural tetramethylpyrazine using renewable biomass.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a mutagenized Bacillus coagulans and its application in the production of ligustrazine. Background Art

[0002] Bacillus coagulans is a Gram-positive bacillus with the ability to produce spores. Its spores have strong heat resistance, enabling it to have good viability and the ability to resist 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 homofermentative lactic acid bacterium. Moreover, it is a facultative anaerobe. After entering the intestine, it consumes free oxygen, which is beneficial 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 it produces spore bodies is the key difference between Bacillus coagulans and traditional lactic acid bacteria. Bacillus coagulans belongs to spore-forming probiotics and can form spores under certain conditions, thus having resistance to adverse environments and strong tolerance to acids, heat, and bile salts. It can solve the problems of poor stress resistance, short shelf life, and easy inactivation in the body of other non-spore-forming probiotics. Due to its ability to produce spores, it also has unique biological characteristics such as resistance to gastric acid, resistance to drying, high temperature and high pressure resistance, and easy storage, which 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 buttery fragrance and appears as a white crystalline powder. Acetoin is the third highest content volatile substance in traditional solid-state fermented vinegar in China after acetic acid and furfural, and is an important intermediate affecting the microbial fermentation production of ligustrazine. As an intermediate of the product, acetoin is also a popular food flavor additive. It not only plays an important role in the biological control of agricultural pests, has a good influence on the formation of tobacco leaf aroma, but also has an indirect effect on the modification of drugs. In addition, acetoin can be used as a stabilizer and foaming agent for synthesizing chlorine-containing polymers, and derivatives of acetoin can be used to synthesize novel hydrophilic coatings with antioxidant properties, which can extend the service life of resins.

[0004] Ligustrazine (2,3,5,6 - tetramethylpyrazine) is a compound with cardiovascular and cerebrovascular health care functions. It can promote blood circulation and has the effect of removing stasis and activating blood circulation. It has a protective effect on cerebral ischemia, hypoxia-reperfusion injury and cerebral infarction, can reduce arterial pressure and coronary artery resistance, and its effects are widely used in the treatment of cardiovascular and cerebrovascular diseases such as typical migraine, arteriosclerosis, and ischemic stroke. The traditional production method of ligustrazine mainly relies on chemical synthesis, which has problems of environmental pollution and high cost. In recent years, the microbial fermentation method has received 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. Currently, the main methods for producing ligustrazine are chemical synthesis method and microbial fermentation method. 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, low prices, simple fermentation processes, high production efficiency, high product purity, is suitable for food applications, and has little environmental pollution. In addition, acetoin, as a precursor of ligustrazine, due to its unique property of being easily soluble in water and organic solvents, it is very difficult to separate efficiently. Compared with the separation and extraction of acetoin, the purification of ligustrazine is simpler. Converting acetoin in the fermentation broth into ligustrazine that is easy to separate not only avoids the difficulty of acetoin recovery but also increases the product value.

[0006] Currently, the existing microbial fermentation method still needs to be improved in terms of the 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 mutagenized Bacillus coagulans and its application in the production of ligustrazine. After the original strain of the present invention is mutagenized, under normal culture conditions, using glucose and yeast extract as raw materials, while retaining the number of probiotic Bacillus coagulans, it can increase the yield of ligustrazine, reduce costs, and has significance for the green production process of producing natural ligustrazine using renewable materials.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a mutagenized Bacillus coagulans YB - 2023, the taxonomic name is Heyndrickxia coagulans, and the strain number is YB - 2023; it was deposited at the China Center for Type Culture Collection on March 7, 2024, at the address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the accession number of the strain is CCTCC NO:M 2024411.

[0010] Preferably, the acetoin production of the mutagenized 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.

[0011] The mutagenized strain Bacillus coagulans of the present invention is obtained by mutagenizing the original strain. The original strain is stored in the form of freeze-dried powder. First, wipe the surface of the ampoule freeze-drying tube with 75% alcohol cotton for disinfection. After uniformly heating the top of the ampoule tube on the outer flame of the alcohol lamp, immediately drop 2-3 drops of sterile water on the heated part to break the tube wall, and knock off the broken part with forceps or other suitable tools. Use a sterile pipette to suck about 0.5 ml of liquid culture medium into the freeze-drying tube to completely dissolve the freeze-dried bacterial powder. Transfer the dissolved bacterial suspension to a test tube containing 4-5 mL of liquid culture medium and mix well. The 1-2 drops of bacterial suspension remaining in the pipette can be transferred to the solid culture medium. After the activated bacteria in the test tube are purified by streaking, pick single colonies and perform static culture on an LB agar slant at 37 °C for 24 h. After the bacteria are stored by freeze-drying, they are in a dormant state. The first-generation bacteria need to be cultured for an appropriate extended time, and the fermentation medium is transferred to 2-3 generations to restore vitality. Take 0.1 mL of the fermentation broth cultured to the exponential growth phase, evenly spread it on a sterile blank petri dish, dry it with sterile air, and perform ion beam implantation after microscopic observation shows no cell overlap. Select nitrogen ion beam implantation, N + The energy is 10~20 keV, and the dose is 20x10 13 ~20x10 13 ions / cm 3 , and the treatment time is 120 s. After the implantation is completed, take out the plate and wash it off with sterile water. Make the strain obtained by screening in the previous step into a cell suspension, adjust the concentration to 10 -6 / ml, take 0.1 ml of the cell suspension and spread it on the sterilized plate. After drying, perform nitrogen ion beam implantation, N + The energy is 10~30 keV, and the dose is 20x10 14 ~20x10 14 ions / cm 3 , and the treatment time is 120 s. After the implantation is completed, wash the petri dish with sterile water, and spread the eluted bacterial liquid on the LB solid medium and incubate it upside down at 37 °C for 24 h. Inoculate the mutagenized bacteria into the seed medium and culture at 37 °C for 10~18 h. The liquid loading volume of the shaking flask is 10% (v / v). Inoculate the bacterial liquid in the seed medium into the fermentation medium at an inoculation amount of 5% and culture at 45 °C for 36 h. Inoculate the bacteria into the LB solid medium and select the strains with good growth conditions. Repeat the re-screening steps, pick single colonies that conform to the colony morphological characteristics of Bacillus coagulans and have good growth, perform shaking flask fermentation culture, measure the number of viable probiotic cells and the number of spores, and finally select a strain with good traits.

[0012] The screened Bacillus coagulans that can retain a large number of probiotic spores is named Bacillus henderickxii, which grows well in LB medium. The observed strain morphology usually presents as round or irregularly round, with a diameter of about 2 - 5 mm, of moderate size, and with a neat or slightly wavy edge. The colony surface is smooth and moist, with a dense and opaque texture, showing milky white or light yellow. As the spores form, the colony surface gradually becomes slightly rough, and the colony shows stress resistance, with the colony color becoming slightly darker, presenting light yellow or light brown, and it is a Gram-positive bacillus.

[0013] In the present invention, the Bacillus coagulans is a spore-forming bacillus, with an optimal growth temperature of 45 - 55 °C and an optimal pH of 6.6 - 7.0. Its spores have strong heat resistance, can decompose sugars to produce L-lactic acid, is a homofermentative lactic acid bacterium and a facultative anaerobe, effectively enhancing the ability of probiotics to resist adverse external environments and the ability to produce spores.

[0014] In the second aspect, the present invention provides the application of the above-mentioned mutagenized Bacillus spore YB-2023 in the production of tetramethylpyrazine.

[0015] Preferably, the method for the mutagenized Bacillus coagulans strain to produce tetramethylpyrazine is: subjecting Bacillus coagulans to plate culture, slant culture, seed culture, and fermentation culture in sequence.

[0016] Preferably, the medium used for the plate culture contains the following components by mass: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 15 g / L;

[0017] The temperature of the plate culture is 35 - 37 °C, and the time is 24 - 48 h.

[0018] Preferably, the 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;

[0019] The fermentation temperature of the slant culture is 35 - 45 °C, and the fermentation time is 24 - 48 h.

[0020] Preferably, the medium used for the seed culture contains the following components by mass: peptone 10 g / L, edible yeast extract 30 g / L, sodium chloride 1 g / L, glucose 60 g / L;

[0021] The temperature of the seed culture is 35 - 45 °C, and the time is 10 - 20 h.

[0022] Preferably, the culture medium used for the fermentation culture comprises components with the following masses: 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;

[0023] The temperature of the fermentation culture is 45 - 55 °C, and the time is 36 - 46 h.

[0024] Preferably, the number of Bacillus coagulans after the fermentation culture is 8×10 8 ~9×10 8 CFU / mL.

[0025] Beneficial technical effects:

[0026] Compared with the wild strain, the Heyndrickxia coagulans screened by the ion beam engineering technology in the present invention effectively improves the spore-forming ability of the strain, has good genetic stability and also improves the ability to produce tetramethylpyrazine. The production cycle is 36 - 45 hours, the yield is 4.56 g / L, and the number of Bacillus coagulans is 8.91×10 8 CFU / mL. After the original strain is mutagenized, under normal culture conditions, using glucose and yeast extract as raw materials, while retaining the number of probiotic Bacillus coagulans, it improves the yield of tetramethylpyrazine, reduces costs, and has significance for the green production process of producing natural tetramethylpyrazine using renewable materials.

[0027] Biological preservation description:

[0028] The mutagenized Bacillus coagulans YB - 2023 described in the present invention is classified and named as Heyndrickxia coagulans, with the strain number YB - 2023; it was preserved in the China Center for Type Culture Collection on March 7, 2024, at the address No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the accession number of the strain is CCTCC NO: M2024411. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in this invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and modifications can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0034] As used in the present invention, "room temperature" and "normal temperature" are both calculated as 25 ± 2°C unless otherwise specified.

[0035] Unless otherwise specified, the raw materials used in the following examples of the present invention are all commercially available.

[0036] Example 1

[0037] Mutation and low pH screening of strains

[0038] Screening of the starting strain:

[0039] The Bacillus coagulans strain described in the present invention is obtained by mutagenizing the original strain, which is stored in the form of freeze-dried powder. First, wipe the surface of the ampoule freeze-drying tube with 75% alcohol cotton for disinfection. After uniformly heating the top of the ampoule tube on the outer flame of the alcohol lamp, immediately drop 2-3 drops of sterile water on the heated part to break the tube wall, and use forceps or other suitable tools to knock off the broken part. Use a sterile pipette to suck about 0.5 ml of liquid culture medium into the freeze-drying tube to completely dissolve the freeze-dried bacterial powder. Transfer the dissolved bacterial suspension to a test tube containing 4-5 mL of liquid culture medium and mix well. The 1-2 drops of bacterial suspension remaining in the pipette can be transferred to the solid culture medium. The activated bacteria in the test tube are picked with a single colony after being purified by streaking, and statically cultured on an LB agar slant at 37 °C for 24 h. After freeze-drying and preservation, the bacteria are in a dormant state. The first-generation bacteria need to be cultured for an appropriate extended time, and the fermentation medium is transferred to 2-3 generations to restore vitality. Take 0.1 mL of the fermentation broth cultured to the exponential growth phase, evenly spread it on a sterile blank petri dish, dry it with sterile air, and perform ion beam injection under microscopic observation without cell overlap. Select nitrogen ion beam injection, N + The energy is 10~20 keV, and the dose is 20x10 13 ~40x10 13 ions / cm 3 , and the treatment time is 120 s. After the injection is completed, take out the plate and wash it off with sterile water, repeat this process multiple times, and observe and screen out the strains with good growth conditions.

[0040] Re-screening:

[0041] Make the strains obtained in the previous step into cell suspensions, adjust the concentration to 10 -6 / ml, take 0.1 ml of the cell suspension and spread it on the sterilized plate. After drying, perform nitrogen ion beam injection, N + The energy is 10~30 keV, and the dose is 20x10 14 ~40x10 14 ions / cm 3, the processing time is 120 s. After the injection is completed, the petri dish is washed with sterile water, and the eluted bacterial solution is spread on the LB solid medium and cultured upside down at 37 °C for 24 h. The mutagenized strain is inoculated into the seed medium and cultured at 37 °C for 10 - 18 h, and the liquid loading volume of the shaking flask is 10% (v / v). The bacterial solution in the seed medium is inoculated into the fermentation medium at an inoculation amount of 5%, cultured at 45 °C for 36 h, the temperature is raised to 55 °C, 1 g / L calcium carbonate and an appropriate amount of diammonium hydrogen phosphate are added, cultured for 8 h, and the final strain is inoculated into the LB solid medium. Strains with good growth conditions are selected, and the rescreening steps are repeated until the target strain is screened out. The formula of the fermentation medium (components and contents in 1000 ml): yeast extract 30 g, glucose 60 g, sodium acetate 0.5 g, diammonium citrate 0.2 g, magnesium sulfate 0.2 g, manganese sulfate 0.01 g, dipotassium hydrogen phosphate 0.2 g, corn steep liquor 120 g.

[0042] The formula of the seed medium (components and contents in 1000 ml): peptone 10 g, yeast extract 30 g, sodium chloride 1 g, glucose 60 g.

[0043] The screened strain is Gram-positive and negative in the catalase test. The colony morphology usually presents a round or irregular round shape, with a diameter of about 2 - 5 mm, of moderate size, and the edge is neat or slightly wavy. The surface of the colony is smooth and moist, the texture is dense and opaque, showing milky white or light yellow, and as the spores form, the surface of the colony gradually becomes slightly rough. The colony shows stress resistance, and the colony color becomes slightly darker, showing light yellow or light brown.

[0044] Determination of the spore survival rate of the screened strain:

[0045] At 45 °C, the screened mutant strain and the original strain are respectively cultured in the fermentation medium. After 36 h, the growth status is observed. It is found that under each set of conditions, the spore survival rate of the screened strain is higher than that of the original strain, reaching more than 90%, and the growth status of the surviving strains is better than that of the original strain.

[0046] Example 2

[0047] This example illustrates the identification and genetic stability of the strain

[0048] Identification of the strain: On the LB solid medium, the colony is milky white or light yellow, round or irregular round in shape, the surface is smooth and moist, the texture is dense and opaque, and the edge is slightly rough. The suitable growth temperature range of the bacteria is 45 - 55 °C.

[0049] Subculture experiment: The selected mutant bacteria were inoculated into LB solid medium and cultured under anaerobic conditions at 37°C for 24 h, then inoculated into solid medium and cultured for 36 h, and the survival rates of the strains in different generations were recorded.

[0050] The survival rates of the strains in different generations are shown in Table 1:

[0051] Table 1

[0052]

[0053] It can be seen from the experimental results that after five consecutive subcultures, the mutant strain has good subculture stability and can be used as a strain for further research and development.

[0054] Example 3

[0055] Cultivation method of Bacillus coagulans

[0056] This example describes the cultivation method of Bacillus coagulans, which includes the following steps:

[0057] 1) Plate culture: Inoculate Bacillus coagulans on plate medium and culture at a temperature of 35 - 45°C for 24 - 48 h;

[0058] 2) Slant culture: Inoculate the Bacillus coagulans cultured on the plate medium in step 1) into slant medium and culture at a temperature of 35 - 45°C for 24 - 48 h;

[0059] 3) Seed culture: Inoculate the slant culture in step 2) into seed medium and culture at a temperature of 35 - 45°C for 10 - 20 h;

[0060] 4) Fermentation culture: Inoculate the seed liquid of Bacillus coagulans cultured in step 3) into fermentation medium at an inoculation amount of 5% (v / v), culture at a temperature of 45°C for 36 h, raise the temperature of the fermentation broth to 55°C, and add 1 g / L calcium carbonate and an appropriate amount of diammonium hydrogen phosphate.

[0061] The plate medium contains the following components by mass: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0062] The slant medium: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0063] The seed medium: peptone 10 g / L, edible yeast extract 30 g / L, sodium chloride 1 g / L, glucose 60 g / L.

[0064] The fermentation medium: 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.

[0065] Example 4

[0066] Production of ligustrazine by strain fermentation

[0067] Select Bacillus coagulans with good growth state and inoculate it into the seed medium. Incubate overnight at 37 °C and 180 r / min to obtain the seed liquid. Inoculate the seed liquid into 500 mL of the fermentation medium at a ratio of 5% (v / v), and shake-flask ferment the strain at 180 rpm for 36 h. Take samples to detect glucose consumption, acetoin production, spore count and viable cell count. Raise the temperature of the fermentation broth to 55 °C, add 1 g / L calcium carbonate and an appropriate amount of diammonium hydrogen phosphate (to make the molar ratio of acetoin to ammonium ion 1:3), and continue to culture for 8 h. At this time, the viable cell count and spore count are basically equal. After the fermentation is completed, determine the contents of acetoin and ligustrazine by gas chromatography (GC).

[0068] The formula of the fermentation medium 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.

[0069] The fermentation temperature is 45 - 55 °C.

[0070] The measurement results show that under the condition of enlarged culture scale, the original strain can ferment and produce 8.4 g / L of acetoin in 36 h, and the optical purity of the obtained product acetoin is 96.3%. The acetoin fermented and produced by the mutagenized strain in 36 h is 14.6 g / L (a 74% increase), and the optical purity of the obtained product L-lactic acid is above 99.8%. The mutant strain is fermented and cultured at pH 8.0 for 46 h, and the ratio of the accumulation number of its bacterial spores to the ligustrazine yield is significantly higher than that of the original strain. The spore accumulation number of the original strain is 6.67×10 8 CFU / mL, while the spore accumulation number of the mutant strain is 8.91×10 8 CFU / mL (a 34% increase). The ligustrazine yield of the original strain is only 1.37 g / L, while the ligustrazine yield of the mutant strain reaches 4.56 g / L, which is 3.33 times that of the original strain.

[0071] Determination of acetoin in the fermentation broth by gas chromatography:

[0072] Instrument: Agilent 7890B gas chromatograph, equipped with a flame ionization detector (FID).

[0073] Chromatographic column: HP-5 capillary column (30 m × 0.32 mm × 0.25 μm).

[0074] Carrier gas: High-purity nitrogen, air flow rate 450 mL / min, hydrogen flow rate 45 mL / min.

[0075] Injector temperature: 280 °C, in split mode, split ratio 30:1.

[0076] Temperature programming: Initial temperature 50 °C, hold for 2 minutes; increase temperature to 250 °C at a rate of 10 °C / min, hold for 8 minutes.

[0077] Detector temperature: 300 °C.

[0078] Determination of tetramethylpyrazine in fermentation broth by gas chromatography:

[0079] Instrument: Agilent 7890B gas chromatograph, equipped with a flame ionization detector (FID).

[0080] Chromatographic column: HP-5 capillary column (30 m × 0.32 mm × 0.25 μm).

[0081] Carrier gas: High-purity nitrogen, air flow rate 450 mL / min, hydrogen flow rate 45 mL / min.

[0082] Injector temperature: 280 °C, in split mode, split ratio 30:1.

[0083] Temperature programming: Initial temperature 50 °C, hold for 2 minutes; increase temperature to 250 °C at a rate of 10 °C / min, hold for 8 minutes.

[0084] Detector temperature: 300 °C.

[0085] Prepare acetoin standard solutions with different concentrations (1 - 25 g / L) and tetramethylpyrazine standard solutions (0.01 - 10 g / L). After sample treatment, the peak area ratios obtained by GC detection and analysis are combined with the standard curve for quantitative analysis. The retention time of acetoin is about 4.6 min, and the retention time of tetramethylpyrazine is about 13 min. The ratios of the peak areas of acetoin and tetramethylpyrazine to the peak area of n-hexanol are obtained, and the yields of acetoin and tetramethylpyrazine are calculated based on the peak areas.

[0086] Industrial applicability

[0087] The above research shows that the strain of the present invention is significantly superior to the original strain without mutagenesis in terms of the number of probiotic spores accumulated, the production rate of tetramethylpyrazine, and the optical purity (the number of spores increased by 34%, the production rate increased by 3.33 times, and the optical purity increased to 99%). The present invention provides a new strain with low fermentation cost, fast production rate of tetramethylpyrazine, and high optical purity of the product. This strain can increase the yield of tetramethylpyrazine while retaining the number of probiotic Bacillus coagulans, reduce costs, and achieve the purpose of reducing environmental pollution through the green production process of producing natural tetramethylpyrazine using renewable biomass, and is of great significance for the industrial microbial fermentation production of tetramethylpyrazine.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A mutagenized Bacillus coagulans YB-2023, characterized in that, Its taxonomic name is Bacillus coagulans ( Heyndrickxia coagulans ), and the strain number is YB-2023. It was deposited at the China Center for Type Culture Collection on March 14, 2024. The address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The accession number of the strain deposit is CCTCC NO: M2024411.

2. Use of the mutagenized Bacillus coagulans YB-2023 as claimed in claim 1 in the production of tetramethylpyrazine.

3. The application according to claim 2, characterized in that The method for producing tetramethylpyrazine by the mutagenized Bacillus coagulans strain is as follows: The Bacillus coagulans is successively subjected to plate culture, slant culture, seed culture, and fermentation culture.

4. The application according to claim 3, characterized in that, The medium used for the plate culture contains the following components by mass: peptone 10 g / L, yeast extract 25 g / L, sodium chloride 10 g / L, agar 15 g / L; The temperature for the plate culture is 35 - 37 °C, and the time is 24 - 48 h.

5. The application according to claim 3, characterized in that, The 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 for the slant culture is 35 - 45 °C, and the fermentation time is 24 - 48 h.

6. The application according to claim 3, wherein The medium used for the seed culture contains the following components by mass: peptone 10 g / L, edible yeast extract 30 g / L, sodium chloride 1 g / L, glucose 60 g / L; The temperature for the seed culture is 35 - 45 °C, and the time is 10 - 20 h.

7. The application according to claim 3, characterized in that, The medium used for the fermentation culture contains the following components by mass: 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; The temperature for the fermentation culture is 45 - 55 °C, and the time is 36 - 46 h.

8. The application according to claim 3, characterized in that, The inoculation ratio of the seed liquid in the fermentation culture is 5% v / v.

9. The application according to claim 3, wherein The number of Bacillus coagulans after fermentation culture is 8×10 8 ~9×10 8 CFU / mL.

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