Novel enterobacter rorrhizae strain as well as separation and screening method and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a new enterobacter rorrhizae strain as well as a separation and screening method and application thereof. In order to enrich microbial resources for producing pyrazine substances through metabolism in the white spirit industry, an enterobacter rorrhizae strain is separated and screened from a mixture of yeast for making hard liquor and fermented grains, and the preservation number is CGMCC No.33262. The strain has good tolerance to pH, salt and alcohol, and can adapt to the growth environment in the white spirit production process; and various pyrazine substances represented by tetramethylpyrazine can be produced. Besides, the strain has strong adaptability to fermented grains, the yield of tetramethylpyrazine is high in a culture medium taking bran, wheat and five grains as the fermented grains, and meanwhile, various flavor substances such as ethyl acetate, ethyl hexanoate, ethyl caprylate, 2, 3-butanediol, guaiacol and 4-vinyl guaiacol can be produced. Therefore, the enterobacter rorrhizae disclosed by the invention can be widely used for brewing white spirit.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and specifically relates to a new strain of Enterobacter roggenkampii, a method for isolating and screening the same, and an application thereof. Background Art

[0002] As a health factor of Chinese liquor, tetramethylpyrazine is a key aroma substance in Maotai-flavor and Sesame-flavor Chinese liquors, with typical baking and nutty aroma characteristics, and its content is significantly higher than that of other flavor types of Chinese liquors. With the development of the Chinese liquor industry, consumers' requirements for the flavor and quality of Chinese liquor are constantly increasing, and the production pathways and regulation of pyrazine flavor substances such as tetramethylpyrazine have become research hotspots.

[0003] Currently, it has been found that Bacillus is the main microorganism for metabolizing and producing pyrazine substances, such as Bacillus subtilis, Bacillus maotaiensis, and Bacillus licheniformis, etc. However, the research on other microorganisms that metabolize and produce pyrazine substances is still in its infancy. For example, Enterobacter roggenkampii, as a member of the Enterobacteriaceae family, is a facultative anaerobe that can grow well in the microaerobic environment during Chinese liquor brewing, with a fast growth rate, strong metabolism, and good utilization ability for various carbohydrate and amino acid substrates.

[0004] However, currently, the research on the production of tetramethylpyrazine by Enterobacter roggenkampii in the Chinese liquor industry is still blank, lacking the screening and application research of high-yield strains. Therefore, screening Enterobacter roggenkampii strains with high yields of tetramethylpyrazine has important theoretical significance and application value for enriching the microbial resources for Chinese liquor brewing and improving the quality of specific flavor types of Chinese liquors. Summary of the Invention

[0005] In order to enrich the microbial resources for metabolizing and producing pyrazine substances in the Chinese liquor industry, the present invention isolates and screens a strain of Enterobacter roggenkampii 32 with high yields of tetramethylpyrazine from the mixture of Daqu and fermented grains. Its production of tetramethylpyrazine in liquid fermentation reaches 1.32 g / L, and it can also grow and ferment in solid media such as wheat, bran koji, and five-grain.

[0006] This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 31, 2024, with the deposit number CGMCC No. 33262. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. The taxonomic name is Enterobacter roggenkampii.

[0007] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0008] In a first aspect, the present invention provides a strain of Enterobacter roggenkampii with a preservation number of CGMCC No. 33262.

[0009] Among them, the tolerance characteristics of the Enterobacter roggenkampii are: pH 3.0 - 12.0, salt tolerance content 0 - 10 V / V%, ethanol tolerance content 1 - 9 V / V%.

[0010] Preferably, the tolerance of the Enterobacter roggenkampii is: the highest tolerated fermentation pH is 12, the highest tolerated salt content is 10 V / V%, and the highest tolerated ethanol content is 9 V / V%.

[0011] Among them, the Enterobacter roggenkampii is circular, with a neat edge, opaque, gray on the front, smooth and bright on the surface, and moist in texture.

[0012] Among them, the 16S rDNA sequence of the Enterobacter roggenkampii 32 is as shown in SEQ ID NO: 1:

[0013] SEQ ID NO: 1: 16S rDNA sequence of Enterobacter roggenkampii 32

[0014]

[0015] In a second aspect, the present invention provides a method for isolating and screening the above-mentioned Enterobacter roggenkampii, comprising the following steps:

[0016] S1. Mix the fermented grains and Daqu, add them to physiological saline to make a suspension, first perform enrichment culture and then separation culture, and initially screen out strains that produce acetoin and show dark red color through the creatine color reaction method;

[0017] S2. Fermentatively culture the initially screened strains, re-screen out the strains with the highest production of tetramethylpyrazine, and identify the above-mentioned Enterobacter roggenkampii by combining morphological, physiological and biochemical characteristics and / or molecular biology.

[0018] Among them, in step S1, the components of the separation medium include: yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0019] Among them, in step S2, the components of the fermentation medium include: tryptone 30 g / L, diammonium hydrogen phosphate 30 g / L, yeast extract powder 10 g / L, glucose 50.0 g / L, pH 7.0.

[0020] Among them, in step S2, the fermentation culture conditions are: inoculum amount 5%, fermentation temperature 37°C, rotation speed 180 r / min, fermentation time 48 h.

[0021] In a third aspect, the present invention provides a microbial inoculant containing the above-mentioned Enterobacter roggenkampii.

[0022] In a fourth aspect, the present invention provides a bacterial koji, which is obtained by activating the above-mentioned Enterobacter roggenkampii and fermenting it in a bran koji medium.

[0023] Among them, the bran koji medium is obtained by mixing bran and water at a mass ratio of 1:1.

[0024] In a fifth aspect, the present invention provides the application of the above-mentioned Enterobacter roggenkampii or microbial inoculant in liquor brewing, koji or fermented grains preparation.

[0025] Among them, in the above application, the fermented grains used include at least one of wheat, bran, and five-grain.

[0026] Among them, the liquor flavor types include at least one of Maotai flavor, strong flavor, light flavor, Feng flavor, rice flavor, Douchi flavor, blended flavor, sesame flavor, special flavor, Laobaigan flavor, Fuyu flavor, and Dong flavor.

[0027] In a sixth aspect, the present invention provides the application of the above-mentioned Enterobacter roggenkampii or microbial inoculant in the production of pyrazine substances.

[0028] Among them, the pyrazine substances include at least one of tetramethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, methylpyrazine or 2,5-dimethylpyrazine.

[0029] Beneficial effects: A strain of Enterobacter roggenkampii 32 was isolated and screened from the mixture of Luzhou-flavor Daqu and Maotai-flavor fermented grains, and its preservation number is CGMCC No. 33262. This strain has good tolerance to pH, salt and alcohol, and can adapt to the growth environment during liquor production; moreover, compared with pyrazine-producing Bacillus or yeast, the strain of the present invention can produce a variety of pyrazine substances, including tetramethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, methylpyrazine and 2,5-dimethylpyrazine. For example, when the strain of the present invention is fermented in liquid, the yield of tetramethylpyrazine is 1.32 g / L. In addition, this strain has strong adaptability to a variety of fermented grains. In the culture media with bran, wheat and five-grain as fermented grains, the yields of tetramethylpyrazine reach 255.88 mg / kg, 40.56 mg / kg and 15.71 mg / kg respectively; at the same time, it can also produce high yields of ethyl esters such as ethyl acetate, ethyl hexanoate and ethyl octanoate, and a variety of flavor substances such as 2,3-butanediol, guaiacol and 4-vinylguaiacol.

[0030] It can be seen that the strain of the present invention not only adapts to the growth environment during liquor production, but also adapts to a variety of fermented grains in liquor brewing, and has good development prospects for liquor brewing. Moreover, compared with the preparation of tetramethylpyrazine by chemical synthesis method, the present invention uses the microbial fermentation method to obtain tetramethylpyrazine, which has the advantages of safety, greenness, high efficiency and sustainability. Description of the Drawings

[0031] Figure 1 is the preliminary screening result of the tetramethylpyrazine-producing strain in Example 1 of the present invention: a: Colony streaking map; b: Creatine test result map;

[0032] Figure 2 is the standard curve graph of tetramethylpyrazine in Example 1 of the present invention;

[0033] Figure 3 is the PCR amplification map of Enterobacter roggenkampii 32 in Example 1 of the present invention;

[0034] Figure 4 is the phylogenetic tree of Enterobacter roggenkampii 32 in Example 1 of the present invention;

[0035] Figure 5 is the acidity tolerance graph of Enterobacter roggenkampii 32 in Example 2 of the present invention;

[0036] Figure 6 is the salinity tolerance graph of Enterobacter roggenkampii 32 in Example 2 of the present invention;

[0037] Figure 7 It is the graph of the alcohol tolerance of Enterobacter roggenkampii 32 in Example 2 of the present invention.

[0038] Description of the preservation of the strain Enterobacter roggenkampii 32 of the present invention:

[0039] Enterobacter roggenkampii 32 of the present invention was deposited on December 31, 2024 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 33262. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. The taxonomic name is Enterobacter roggenkampii. Detailed implementation manners

[0040] Specific embodiments will be enumerated below to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, the techniques or conditions described in the literature in this field or according to the product specifications shall be followed. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0041] The culture media used in the following embodiments are as follows:

[0042] Isolation medium (1L): yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar 20 g / L.

[0043] LB medium (1L): tryptone 10 g / L, sodium chloride 10 g / L, yeast extract powder 5 g / L, distilled water 1000 mL, sterilized at 121 °C for 15 min.

[0044] Fermentation medium (1L): tryptone 30 g / L, diammonium hydrogen phosphate 30 g / L, yeast extract powder 10 g / L, glucose 50.0 g / L, distilled water 1000 mL, pH 7.0, sterilized at 115 °C for 20 min.

[0045] Creatine (100 mL): creatine 0.01 g / L, 1-naphthol 0.1 g / L, NaOH 0.4 g / L, distilled water 100 mL.

[0046] MRS medium (1 L): Peptone 10 g / L, Beef extract 10 g / L, Yeast extract 5 g / L, Diammonium hydrogen citrate 2 g / L, Sodium acetate anhydrous 5 g / L, Dipotassium hydrogen phosphate 2.62 g / L, Tween 1 mL / L, Magnesium sulfate heptahydrate 0.58 g / L, Manganese sulfate monohydrate 0.25 g / L, Distilled water 1000 mL, Sterilized at 121 °C for 20 min; Glucose 20.0 g / L, Sterilized at 115 °C for 20 min.

[0047] Wheat bran koji (200 g): Wheat bran and distilled water are mixed at a mass ratio of 1:1 and sterilized at 121 °C for 20 min.

[0048] Wheat (100 g): After 100 g of wheat is crushed, 50 mL of distilled water is added, 0.1% amylase is added, and it is heated in a water bath at 100 °C for 2 h. Then it is cooled to 60 °C, 0.1% glucoamylase is added, and it is kept warm at 60 °C for 2 h, and then sterilized at 121 °C for 20 min.

[0049] Five-grain mixture (1000 g): Sorghum 36%, rice 22%, glutinous rice 18%, wheat 16%, and corn 8% are moistened for 12 h according to the mass ratio. Then 20% of the chaff by the mass of the raw materials is mixed into the grains and steamed until cooked through. After steaming, the grain mash is taken out, 30% of warm water at 50 °C is sprinkled to supplement moisture, evenly spread out and cooled to 30 °C, and 20% of the strong-flavor Daqu by the mass of the raw materials is added and mixed evenly. Ferment by sealing for 14 d.

[0050] Example 1 Isolation, Screening and Identification of Enterobacter roggenkampii 32

[0051] 1. Isolation and Screening of Enterobacter roggenkampii 32

[0052] I) Primary screening: Take 5 g of strong-flavor Daqu and 5 g of sauce-flavor fermented grains (the koji powder sample is taken from a distillery in Yibin City, Sichuan Province, and the fermented grains sample is taken from a distillery in Luzhou City, Sichuan Province) and add them to a sterile conical flask containing 100 mL of normal saline, and culture on a shaker at 37 °C for 12 h, then let it stand for 30 min. Pipette 1 mL of the enriched supernatant into a centrifuge tube containing 9 mL of sterile normal saline and perform gradient dilution to obtain dilution bacterial solutions of 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 . Take 10 -3 、10 -4 、10 -5100 μL of the diluted bacterial solutions at three gradients were each spread on the isolation medium, with 3 replicates for each dilution gradient, and cultured in an incubator at 37 °C for 24 h. Colonies with different morphological characteristics in terms of size were selected and streaked on the isolation medium, and this operation was repeated until single colonies appeared. The single colonies on the isolation medium were inoculated into a test tube containing 1 mL of freshly prepared creatine mixture and stirred evenly, and the change in the solution was observed after standing for a few minutes. Strains with the creatine turning red were inoculated into LB medium and cultured on a shaker at 37 °C and 180 rpm / min for 24 h. Through fermentation culture, a creatine color reaction experiment was carried out on the fermentation broth to re-screen the strain with the highest acetoin production, and it was preserved in a glycerol tube ( Figure 1 ).

[0053] II) Re-screening: The strains preliminarily screened were inoculated into LB liquid medium to prepare seed solutions, and then inoculated into the fermentation medium (100 mL / 250 mL) at an inoculation amount of 5%, and parallel tests were also carried out. They were cultured at 37 °C and 180 r / min for 48 h. After fermentation, the content of tetramethylpyrazine in the fermentation broth was determined by HPLC, and the strains with the highest tetramethylpyrazine production were screened out (Table 1).

[0054] Table 1 Screening of strains with high tetramethylpyrazine production

[0055] Strain number Creatine display Tetramethylpyrazine content (g / L) 32 Purple-red 1.32 16 Purple-red 0.64 25 Light red 0.18

[0056] In step II), the method for determining tetramethylpyrazine: Instrument: Shimadzu high-performance liquid chromatograph (LC-2030C 3DPlus), chromatographic column: Agilent HC-C18(2) (4.6 mm × 250 mm, 5 μm), mobile phase: methanol-water (35:65, V / V), flow rate: 1.0 mL / min, column temperature: 40 °C, injection volume: 10 μL, detection wavelength: 278 nm. The tetramethylpyrazine production of the strains was calculated according to the tetramethylpyrazine standard curve ( Figure 2 ).

[0057] 2. Identification of Enterobacter roggenkampii 32

[0058] I) Morphological characteristics:

[0059] The above-mentioned strain numbered 32 was inoculated into LB solid medium, and the colony morphological characteristics were observed after culturing for 24 h. As Figure 1 shown in a, strain 32 was circular, with a neat edge, opaque, gray on the front, without pigment, convex in the middle, smooth and bright on the surface, and moist in texture.

[0060] (II) Molecular biological identification: The DNA of the strain No. 32 in Table 1 was extracted using the TSINGKE Plant DNA Extraction Kit (Universal Type). PCR amplification was performed using the universal primers SEQ ID NO: 2: 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and SEQ ID NO: 3: 1492R (5'-TACGGCTACCTTGTTACGAC-3'). The amplification system was as follows: 45 uL of 1×TSE101 Gold Mix, 2 uL of the universal primers 27F (10P) and 1429R (10P), and 1 uL of the DNA template; the amplification program is shown in Table 2. The amplification product was detected by 1.5% agarose gel to check the extraction effect ( Figure 3 ). After meeting the requirements, the amplified product was sent to Tsingke Biotechnology Co., Ltd. for gene sequencing. The sequencing result is shown as SEQ ID NO: 1. Then, it was submitted to the National Center for Biotechnology Information (NCBI) database for sequence alignment using the Basic Local Alignment Search Tool (BLAST) based on the local alignment algorithm, and a phylogenetic tree was constructed ( Figure 4 ).

[0061] Table 2 Amplification program

[0062]

[0063] Through morphological, physiological and biochemical characteristics, and molecular biological identification, the strain 32 was identified as Enterobacter roggenkampii, and its taxonomic name is Enterobacter roggenkampii. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on December 31, 2024, with the deposit number CGMCC No. 33262.

[0064] Example 2 Acid tolerance, salt tolerance, and alcohol tolerance of Enterobacter roggenkampii 32

[0065] 1. Acid tolerance

[0066] The activated strain 32 bacterial solution was inoculated into 100 mL of sterilized fermentation medium at an inoculation amount of 1%. The pH of the medium (adjusted with 1 mol / L glacial acetic acid solution) was 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. After shaking culture at 37°C and 180 r / min for 24 h, the OD value of the bacterial solution at a wavelength of 600 nm was measured to determine the acid tolerance of the strain.

[0067] As Figure 5 shown, the minimum tolerable pH of this strain is 3 and the maximum is 12.

[0068] 2. Salt Tolerance

[0069] The activated strain 32 bacterial liquid was inoculated into MRS liquid medium with NaCl volume concentrations of 0%, 5%, 10%, 15%, 20%, 25%, and 30% at an inoculation amount of 1%. Three parallels were set for each NaCl concentration. The conical flasks were placed in a shaker at 37°C and 180 r / min for 24 h, and the OD value of the bacterial liquid at a wavelength of 600 nm was measured to determine the salt tolerance of the strain.

[0070] As Figure 6 shown, the highest salt tolerance of strain 32 is 10%.

[0071] 3. Alcohol Tolerance

[0072] 99 mL, 97 mL, 95 mL, 93 mL, 91 mL, 89 mL, 87 mL, and 85 mL of fermentation medium were respectively placed in 250 mL conical flasks, autoclaved at 121°C for 20 minutes, and then cooled to room temperature. Subsequently, 1 mL, 3 mL, 5 mL, 7 mL, 9 mL, 11 mL, 13 mL, and 15 mL of absolute ethanol were respectively added to each conical flask under sterile operation conditions, so that the alcohol content (V / V) in the final medium was 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15% respectively. The strain was inoculated, and after shaking culture at 37°C and 180 r / min for 24 h, the OD value of the bacterial liquid at a wavelength of 600 nm was measured to determine the ethanol tolerance of the strain.

[0073] As Figure 7 shown, the highest alcohol tolerance of strain 32 is 9%.

[0074] Example 3 Determination of the Flavor of Liquid Fermentation of Enterobacter roggenkampii 32

[0075] Preparation of bacterial seed liquid: A single colony of strain 32 was picked and inoculated into 100 mL of LB liquid medium, and cultured in a shaker at 37°C and 180 rpm / min for 24 h to prepare a triangular flask seed liquid.

[0076] The newly prepared bacterial seed liquid was inoculated into the fermentation medium at an inoculation amount of 5%, and liquid fermentation was carried out under fermentation conditions (fermentation temperature 37°C, rotation speed 180 r / min, fermentation time 48 h). After centrifugation of the fermentation broth (8000 rpm, 10 min), it was filtered through a 0.22 μm filter membrane, and the produced tetramethylpyrazine was determined by HPLC. As shown in Table 1, the yield of tetramethylpyrazine of strain 32 during fermentation was 1.32 g / L, much higher than that of similar strains, and it could also produce flavor substances such as acetoin, trimethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, and 2,3-butanediol.

[0077] Preparation of Bacillus subtilis koji producing tetramethylpyrazine by Enterobacter roggenkampii in Example 4

[0078] Cultivation of Bacillus subtilis koji: The newly prepared bacterial seed liquid was inoculated into the koji medium at an inoculation amount of 5%, and after mixing evenly, it was cultured at 37 °C for 72 h, and the koji was turned over every 6 - 8 h. After the cultivation was completed, it was dried at 50 °C for standby. The flavor substances produced were determined by GC-MS method (internal standard method). As can be seen from Table 3, strain 32 also produced flavor substances such as ethyl acetate, ethyl hexanoate, trimethylpyrazine, 2,3-dimethylpyrazine, (2R,3R)-2,3-butanediol, 2,3-butanedione, guaiacol, 4-vinylguaiacol, etc. during the fermentation process.

[0079] GC-MS method: Take 2 g of the product and place it in a 10 mL headspace sample bottle, add 20 μL of 2-acetylpyridine as the internal standard, and tighten the sample bottle; after the extraction head is aged at 210 °C for 30 min, insert it into the sample bottle, equilibrate at 50 °C for 5 min, and then insert a 50 μm / 30 μm VB / CAR on PDMS extraction head, extract for 50 min, and desorb for 4 min.

[0080] Gas chromatography conditions: Capillary chromatographic column DB-WAX, with specifications of (30 m × 320 μm × 0.25 μm); inlet temperature 250 °C, splitless mode; programmed temperature rise: hold at 40 °C for 3 min, rise to 180 °C at a rate of 5 °C / min, and finally rise to 230 °C at a rate of 2.5 °C / min and hold for 10 min, for a total of 50 min; carrier gas: high-purity helium, with a flow rate of 1 mL / min.

[0081] Mass spectrometry conditions: Electron impact ionization (EI) ion source, electron energy 70 eV, emission current 200 A, voltage 350 V, ion source temperature 200%, interface temperature 250 °C, scanning mass range 33 - 450 u, scanning range 20 - 500 u.

[0082] Table 3 Determination results of flavor substances in the koji fermentation of strain 32

[0083]

[0084]

[0085] Example 5 Determination of the flavor of wheat fermented by Enterobacter roggenkampii 32

[0086] The newly prepared Enterobacter roggenkampii 32 bacterial seed solution was inoculated into the wheat medium at an inoculation amount of 5%, and after mixing evenly, it was cultured at 37 °C for 14 days, and the flask was shaken every other day to make it mix evenly. After the fermentation was completed, the flavor substances produced were determined by GC-MS method (internal standard method). As can be seen from Table 4, the strain of the present invention can produce tetramethylpyrazine in the wheat solid fermentation medium, and the yield is 40.56 mg / kg. It can not only produce tetramethylpyrazine, but also produce flavor substances such as trimethylpyrazine, guaiacol, 4-vinylguaiacol, 2,5-dimethylpyrazine, etc.

[0087] Table 4 Determination results of flavor substances in wheat fermentation by strain 32

[0088]

[0089]

[0090] Example 6 Determination of flavor of Enterobacter roggenkampii 32 in five-grain fermentation

[0091] The newly prepared Enterobacter roggenkampii 32 bacterial seed solution was inoculated into the five-grain medium at an inoculation amount of 5%, and after mixing evenly, it was cultured at 37 °C for 14 days, and the flask was shaken every other day to make it mix evenly. After the fermentation was completed, the flavor substances produced were determined by GC-MS method (internal standard method). As can be seen from Table 5, the strain of the present invention can produce tetramethylpyrazine in the five-grain solid fermentation medium, and the yield is 15.71 mg / kg. It can not only produce tetramethylpyrazine, but also produce flavor substances such as ethyl acetate, ethyl hexanoate, ethyl octanoate, guaiacol, 4-vinylguaiacol, trimethylpyrazine, ethyl lactate, etc.

[0092] Table 5 Determination results of flavor substances in five-grain fermentation by strain 32

[0093]

[0094]

Claims

1. Enterobacter roggenkampii, characterized in that: The deposit number is CGMCC No.33262.

2. The method for separating and screening Enterobacter logigensis according to claim 1, characterized in that: The following steps are involved: S1. Mix the fermented grains and Daqu and add them to physiological saline to prepare a suspension, first perform enrichment culture and then separate culture, and preliminarily screen out strains that produce acetoin and are dark red by creatine colorimetric method; S2. Ferment and culture the strains after the initial screening, re-screen the strains with the highest tetramethylpyrazine production, and identify the said Enterobacter logenii by combining morphological, physiological and biochemical characteristics and / or molecular biology.

3. A microbial agent, characterized in that: Contains the Enterobacter logenii according to claim 1.

4. A bacterial song, characterized in that: The method is obtained by activating the Enterobacter logenii described in claim 1 and fermenting it in a bran culture medium.

5. Use of the Enterobacter logenii described in claim 1 or the microbial agent described in claim 3 in liquor brewing, koji or fermented grains preparation.

6. The use according to claim 5, characterized in that: The fermented grains used include at least one of wheat, bran or five grains.

7. Use of the Enterobacter logenii described in claim 1 or the microbial agent described in claim 3 in producing pyrazines.

8. The use according to claim 7, characterized in that: The pyrazine substance includes at least one of tetramethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, methylpyrazine or 2,5-dimethylpyrazine.

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