Novel bacillus velezensis 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 novel bacillus velezensis strain as well as a separation and screening method and application thereof. According to the invention, an enterobacter strain which is different from spores and produces tetramethylpyrazine is excavated. According to the invention, a bacillus velezensis 17 with high yield of tetramethylpyrazine is separated and screened from yeast for making hard liquor and fermented grains, and the bacillus velezensis 17 can grow and ferment in solid culture media such as wheat, mouldy bran, five grains and the like at the same time and can produce various pyrazines. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 11, 2025, the preservation number is CGMCC No.35896, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, 3 #, 1 # yard, West Beichen Road, Chaoyang District, Beijing, the postcode is 100101, and the classification name is Bacillus velezensis. The strain provided by the invention can adapt to a plurality of common fermented grain substrates, and shows good industrial adaptability and stability, so that the strain has a wide application prospect in white spirit brewing.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a new strain of Bacillus belye and its isolation, screening method and application. Background Technology

[0002] Fermented foods hold an important place in human dietary culture, and their unique flavor and nutritional value are highly favored by consumers. The flavor compounds produced by microorganisms during fermentation are one of the key factors determining product quality. Among them, pyrazine compounds, as important flavor active substances, possess characteristic aromas such as nutty, roasted, and caramel notes, and are widely found in fermented foods, baked goods, and condiments. Pyrazine compounds not only have excellent flavor properties but also certain physiological activities. Studies have shown that some pyrazine compounds possess antioxidant, antibacterial, and neuroprotective activities, which have positive effects on human health.

[0003] Tetramethylpyrazine, a health-promoting factor in baijiu (Chinese liquor), is a key aroma compound in sauce-aroma and sesame-aroma baijiu, exhibiting typical roasted and nutty aromas, and its content is significantly higher than in other aroma types of baijiu. Dimethylpyrazine (including isomers such as 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, and 2,6-dimethylpyrazine) is an important flavor compound in baijiu, possessing a pungent roasted peanut aroma and chocolate and creamy flavors. 2,6-Diethylpyrazine is an important derivative of pyrazine compounds, with significant application value in the food, baijiu, and flavor industries. With the development of the baijiu industry, consumers' demands for baijiu flavor and quality are constantly increasing, making the generation pathways and regulation of pyrazine flavor compounds a research hotspot.

[0004] Traditionally, pyrazine compounds are mainly produced through chemical synthesis, but this method suffers from high costs, environmental pollution, and difficulty in controlling product purity. In recent years, the production of pyrazine compounds using microbial fermentation has become a research hotspot. Microbial fermentation offers advantages such as a wide range of raw material sources, mild production conditions, naturally safe products, and environmental friendliness, aligning with the trend of green production. Currently reported pyrazine-producing strains mainly include lactic acid bacteria, Bacillus, and yeast. Among them, Bacillus, due to its strong tolerance, relatively simple fermentation requirements, and high safety, has become an ideal strain for industrial production. However, existing strains still have shortcomings in terms of pyrazine yield, diversity, and fermentation stability, making it difficult to meet the needs of industrial production. *Bacillus belyssus* (…) Bacillus velezensisAs an important member of the Bacillus genus, *Bacillus belyssiensis* possesses excellent fermentation characteristics and safety. This strain has been widely used in food fermentation, biocontrol, and enzyme production. However, research on pyrazine production by *Bacillus belyssiensis* is relatively limited, and further exploration is needed to optimize pyrazine production and fermentation conditions. Therefore, screening high-pyrazine-producing *Bacillus belyssiensis* strains and studying their fermentation characteristics and flavor compound production patterns is of great significance for developing novel flavor starter cultures, enriching the flavors of fermented foods, and promoting the development of the fermentation industry.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention has discovered a strain of *Bacillus belyss* that differs from spore-forming tetramethylpyrazine-producing strains. This invention isolates and screens a high-tetramethylpyrazine-producing *Bacillus belyss* strain from daqu (a type of starter culture) and fermented mash. Bacillus velezensis This strain (17) can grow and ferment on solid culture media such as wheat, bran koji, and grains, and produces various pyrazines. It was deposited on September 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35896, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China, and is classified as *Bacillus belyesense*. Bacillus velezensis .

[0007] One of the objectives of this invention is to provide a Bacillus belyssus 17, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35896.

[0008] According to a preferred embodiment, the tolerance of Bacillus belye 17 is as follows: minimum tolerant fermentation pH of 3; maximum tolerant salt content of 10%; and maximum tolerant ethanol content of 5%.

[0009] According to a preferred embodiment, the 16S rDNA sequence of Bacillus belyssus 17 is shown in SEQ ID NO: 1.

[0010] One of the objectives of this invention is to provide a composition comprising the above-described Bacillus belye 17.

[0011] One of the objectives of this invention is to provide a method for preparing Bacillus belyssus 17, which includes the following steps: The above-mentioned Bacillus berleis 17 was inoculated into the culture medium and cultured to obtain the bacteria.

[0012] One of the objectives of this invention is to provide the application of the above-mentioned Bacillus berleis 17, the above-mentioned composition, or Bacillus berleis 17 prepared based on the above-mentioned preparation method in fermented grains and pyrazine production.

[0013] One of the objectives of this invention is to provide a method for isolating and screening Bacillus belyssus 17, which includes the following steps: S1 Take 5 g of fermented mash and 5 g of Daqu (a type of starter culture), mix them, add physiological saline to make a suspension, enrich and culture, and then separate and culture. S2 samples were selected and cultured on LB medium with colony morphology characteristics similar to those of Bacillus.

[0014] According to a preferred embodiment, the culture medium used for enrichment culture in S1 comprises: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride and 20 g / L agar.

[0015] According to a preferred embodiment, LB medium (1 L): 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, and 1000 mL distilled water.

[0016] According to a preferred embodiment, the isolation culture medium used for isolation culture in S1 comprises: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride and 20 g / L agar.

[0017] One of the objectives of this invention is to provide a pyrazine-producing bacterial strain, which is prepared from Bacillus belye 17 as described above, the composition described above, or Bacillus belye 17 prepared according to the preparation method described above.

[0018] One of the objectives of this invention is to provide a method for preparing pyrazine-producing bacterial cultures, comprising the following steps: The activated Bacillus berleis 17, the above-described composition, or Bacillus berleis 17 prepared according to the above-described preparation method were inoculated into raw wheat bran and cultured to obtain a prolific bacterial culture.

[0019] One of the objectives of this invention is to provide a type of baijiu (Chinese liquor) prepared using a koji (fermented starter culture) containing Bacillus berleis 17 as described above, the composition described above, or Bacillus berleis 17 prepared according to the preparation method described above.

[0020] According to a preferred embodiment, the fermentation raw materials for baijiu (Chinese liquor) are wheat or five grains. Preferably, the five grains consist of 36% sorghum, 22% rice, 18% glutinous rice, 16% wheat, and 8% corn. More preferably, the five grains, by weight, are 36 parts sorghum, 22 parts rice, 18 parts glutinous rice, 16 parts wheat, and 8 parts corn.

[0021] One of the objectives of this invention is to provide the use of Bacillus berberis 17, as described in this invention, in the production of koji (fermentation starter). The koji is a type of baijiu koji. Wheat is an auxiliary ingredient in the koji.

[0022] One of the objectives of this invention is to provide the use of Bacillus bellis 17, as described herein, in enhancing the flavor of Daqu fermentation. This enhances the flavor of Daqu fermentation and increases the production of tetramethylpyrazine, dimethylpyrazine, and 2,6-diethylpyrazine.

[0023] One of the objectives of this invention is to provide the use of Bacillus berleisi 17 as described herein in improving 3-methylpyrrolo[2,3-b]pyrazine, 2,2'-bipyrazine, vinylpyrazine, pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, 2,6-diethylpyrazine and tetramethylpyrazine in Daqu (a type of Chinese liquor).

[0024] The beneficial effects of this technical solution are: The *Bacillus belye* 17 strain involved in this invention is derived from baijiu (Chinese liquor) fermentation starter and possesses excellent pyrazine compound synthesis capabilities, particularly capable of producing a variety of pyrazine substances. This strain can generate 10 different pyrazines in wheat culture medium, exhibiting excellent metabolic diversity. When used to prepare bacterial starter, it also produces 10 different pyrazines, indicating its good metabolic stability and adaptability in solid-state fermentation systems.

[0025] This strain exhibits outstanding performance in typical baijiu brewing substrates, particularly in the solid-state fermentation medium of Wuliangye baijiu, where it can generate 12 tetramethylpyrazines, further demonstrating its aroma-producing potential under actual brewing conditions. Furthermore, this strain possesses the ability to efficiently synthesize specific pyrazine compounds, with tetramethylpyrazine yields showing excellent performance across various media: reaching 89.52 mg / kg in wheat bran medium, a high of 714.47 mg / kg in wheat medium, and 156.35 mg / kg in Wuliangye medium.

[0026] In addition to tetramethylpyrazine, this strain can also produce high yields of dimethylpyrazine, with yields of 1.29 mg / kg, 5.33 mg / kg, and 1.74 mg / kg in bran koji, wheat, and grain media, respectively. Simultaneously, this strain also possesses the ability to synthesize 2,6-diethylpyrazine, with yields of 0.045 mg / kg, 0.38 mg / kg, and 0.12 mg / kg in the same three media.

[0027] The strain involved in this invention can adapt to a variety of common fermented grain substrates, exhibiting good industrial adaptability and stability, thus possessing broad application prospects in baijiu (Chinese liquor) brewing. Compared with chemical synthesis methods, this strain produces pyrazine compounds through microbial fermentation, which not only avoids the high cost and environmental problems of chemical synthesis routes, but also has multiple advantages such as safety, greenness, high efficiency, and sustainability, better meeting the development needs of modern food fermentation industry. Attached Figure Description

[0028] Figure 1 This is the preliminary screening result of the polypyrazine-producing strain in Example 1 of the present invention, wherein, Figure 1 a is a streak diagram of bacterial colonies. Figure 1 b is a diagram of bacterial cell morphology; Figure 2 This is a PCR amplification diagram of Bacillus belyssus 17 in Example 1 of the present invention; Figure 3 This is the phylogenetic tree of Bacillus belyssus 17 in Embodiment 1 of the present invention; Figure 4 This is a graph showing the acid tolerance of Bacillus belyssus 17 in Example 2 of this invention; Figure 5 This is a temperature tolerance diagram of Bacillus belyssus 17 in Example 2 of the present invention; Figure 6 This is a graph showing the alcohol tolerance of Bacillus belyssus 17 in Example 2 of this invention. Detailed Implementation

[0029] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0031] Preservation instructions for Bacillus belyssus 17 of this invention: This invention is based on Bacillus belesiensis (B. Bacillus velezensis 17, deposited on September 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 35896, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China, and classified as *Bacillus belyes*. Bacillus velezensis .

[0032] The 16S rDNA sequence of Bacillus belyssus 17 is shown in SEQ ID NO: 1:

[0033] The culture media used in the following examples are as follows: LB medium (1 L): 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, 1000 mL distilled water, sterilized at 121℃ for 15 min.

[0034] Bran koji (200 g): Mix wheat bran and distilled water in a 1:1 ratio and sterilize at 121℃ for 20 min.

[0035] Wheat (100 g): After crushing 100 g of wheat, add 50 mL of distilled water and 0.1% amylase. Heat in a water bath at 100℃ for 2 h, cool down to 60℃, add 0.1% saccharifying enzyme, keep warm at 60℃ for 2 h, and sterilize at 121℃ for 20 min.

[0036] Five Grains (1000 g): Soak 36% sorghum, 22% rice, 18% glutinous rice, 16% wheat, and 8% corn (mass fraction) for 12 hours. Then, mix 20% of the raw material's husk into the grains and steam until fully cooked. After steaming, remove the grain mash, sprinkle 30% of it with 50℃ warm water to replenish moisture, spread it evenly to cool to about 30℃, add 20% of the raw material's koji (a type of starter culture), and mix evenly. Ferment in a sealed container for 14 days.

[0037] Example 1 This embodiment relates to the isolation, screening and identification of Bacillus belyssus 17.

[0038] 1. Isolation and screening of Bacillus belyssus 17 S1 initial screening: Take 5 g of soy sauce-flavored daqu and 5 g of soy sauce-flavored mash (the daqu powder sample was taken from a winery in Yibin City, Sichuan Province, and the mash sample was taken from a winery in Luzhou City, Sichuan Province) and add them to a sterile conical flask containing 100 mL of physiological saline. Incubate at 37℃ on a shaker for 12 h and let stand for 30 min.

[0039] Pipette 1 mL of enrichment supernatant into a centrifuge tube containing 9 mL of sterile physiological saline, and perform serial dilutions to obtain 10... -2 10 -3 10 -4 10 -5 10 -6 Diluted bacterial solution.

[0040] Take 10 -3 10 -4 10 -5 100 μL of each of the three dilutions of the bacterial culture was spread on the isolation medium, with three replicates for each dilution. The culture was incubated at 37°C for 24 h.

[0041] Colonies of varying sizes with the same morphological characteristics as Bacillus colonies were selected and isolated by streaking on isolation medium. This operation was repeated until single colonies appeared. Gram staining was performed on strain number 17, and its cell morphology was observed using a 100x oil immersion microscope (e.g., ...). Figure 1 (As shown).

[0042] S2 secondary screening: The strains screened in the initial screening were inoculated into LB liquid medium to prepare seed liquid, and then inoculated into wheat medium at an inoculation rate of 5%. After mixing evenly, the mixture was cultured at 37°C for 14 days, and the flask was shaken every other day to ensure even mixing.

[0043] After fermentation, the flavor compounds produced were determined using GC-MS (internal standard method). Finally, Bacillus bellis 17, which produces multiple pyrazines, was selected and preserved in glycerol.

[0044] 2. Identification of Bacillus belyssus 17 (I) Molecular biological identification DNA was extracted from the strain using the TSINGKE Plant DNA Extraction Kit (General Type).

[0045] PCR amplification was performed using universal primers SEQ ID NO: 2:27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and SEQ ID NO: 3:1492R (5'-TACGGCTACCTTGTTACGAC-3'). The extraction efficiency of the amplified products was detected by 1.5% agarose gel electrophoresis (e.g., ...). Figure 2 (As shown).

[0046] After meeting the requirements, the amplified product was sent to Qingke Biotechnology Co., Ltd. for gene sequencing. The sequencing results are shown in SEQ ID NO: 1.

[0047] Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST) based on the National Center for Biotechnology Information (NCBI) database to construct a phylogenetic tree (e.g., Figure 3 (As shown).

[0048] Based on morphological and physiological-biochemical characteristics and molecular biology, strain 17 was identified as Bacillus belye.

[0049] Example 2 This embodiment relates to the identification of acid resistance, salt resistance, and alcohol resistance of Bacillus belyssus 17.

[0050] 1. Acidity tolerance The activated bacterial culture of strain 17 was inoculated into 100 mL of sterilized fermentation medium at an inoculation rate 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, 12, and 13. After incubation at 37℃ and shaking at 180 r / min for 24 h, the OD value of the bacterial culture at a wavelength of 600 nm was measured to determine the acid resistance of the strain.

[0051] like Figure 4 As shown, the strain tolerates a minimum pH of 3 and a maximum of 10.

[0052] 2. Salt tolerance The activated strain 17 was inoculated at a mass fraction of 1% into MRS liquid medium containing NaCl at concentrations of 0%, 5%, 10%, 15%, 20%, 25%, and 30% (volume). Three replicates were set up for each NaCl concentration. The conical flasks were placed in a shaker 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 salt tolerance of the strain.

[0053] like Figure 5 As shown, strain 17 has a maximum salt tolerance of 10%.

[0054] 3. Alcohol tolerance Fermentation medium was placed in 250 mL Erlenmeyer flasks at concentrations of 99 mL, 97 mL, 95 mL, 93 mL, 91 mL, 89 mL, 87 mL, and 85 mL, respectively. The flasks were then autoclaved at 121 °C for 20 minutes and cooled to room temperature.

[0055] Subsequently, 1 mL, 3 mL, 5 mL, 7 mL, 9 mL, 11 mL, 13 mL, and 15 mL of anhydrous ethanol were added to each conical flask under aseptic conditions, so that the final alcohol content (V / V) in the culture medium was 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15%, respectively.

[0056] After inoculating the bacterial strain and culturing it at 37℃ 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 ethanol tolerance of the strain.

[0057] like Figure 6 As shown, strain 17 has a maximum alcohol tolerance of 5%.

[0058] Example 3 This embodiment relates to the preparation of Bacillus berberis bacterial koji that produces tetramethylpyrazine.

[0059] Cultivation of bacterial koji: The freshly prepared bacterial seed solution was inoculated into the koji culture medium at an inoculation rate of 5%. After mixing evenly, it was incubated at 37℃ for 72 h. The koji was turned over every 6-8 h. After the incubation was completed, it was dried at 50℃ for later use. The flavor substances produced were determined by GC-MS (internal standard method).

[0060] Table 1 shows that strain 17 produces 10 pyrazines during fermentation, including 3-methylpyrrolo[2,3-b]pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, pyrazine, trimethylpyrazine, 2,6-diethylpyrazine, tetramethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, and 2,3-dimethylpyrazine. This strain can produce high yields of tetramethylpyrazine (89.52 mg / kg on bran koji medium), dimethylpyrazine (1.29 mg / kg on bran koji medium), and 2,6-diethylpyrazine (0.045 mg / kg on bran koji medium). Strain 17 not only produces abundant pyrazines but also flavor compounds such as guaiacol and ethyl hexanoate.

[0061] GC-MS method: Take 4 g of product and place it in a 10 mL headspace vial. Add 20 μL of 2-acetylpyridine as an internal standard and tighten the vial. After aging the extraction head at 210℃ for 30 min, insert it into the vial and equilibrate at 50℃ for 5 min. Then insert a 50 μm / 30 μm VB / CAR on PDMS extraction head and extract for 50 min, followed by desorption for 4 min.

[0062] Gas chromatography conditions: DB-WAX capillary column (30 m × 320 μm × 0.25 μm); injection port temperature 250℃, splitless mode; temperature program: 40℃ for 3 min, 5℃ / min to 180℃, and finally 230℃ at 2.5℃ / min for 10 min, for a total of 50 min; carrier gas: high-purity helium, flow rate 1 mL / min.

[0063] 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.

[0064] The strain of this invention can produce high yields of tetramethylpyrazine, with yields of 89.52 mg / kg on bran culture medium, 714.47 mg / kg on wheat culture medium, and 156.35 mg / kg on grain culture medium.

[0065] The strain of this invention can produce high yields of dimethylpyrazine, with yields of 1.29 mg / kg on bran culture medium, 5.33 mg / kg on wheat culture medium, and 1.74 mg / kg on grain culture medium.

[0066] The strain of this invention can produce high yields of 2,6-diethylpyrazine, with yields of 0.045 mg / kg on bran culture medium, 0.38 mg / kg on wheat culture medium, and 0.12 mg / kg on grain culture medium.

[0067] Table 1

[0068] Example 4 This embodiment relates to the determination of the fermentation flavor of wheat by Bacillus vesiculosus 17.

[0069] The newly prepared Bacillus belye 17 bacterial seed culture was inoculated into wheat culture medium at an inoculation rate of 5%. After being mixed evenly, the culture was incubated at 37°C for 14 days, and the flask was shaken every other day to ensure even mixing.

[0070] After fermentation, the flavor compounds produced were determined by GC-MS (internal standard method). Table 2 shows that the strain of this invention, when used in solid-state fermentation medium for wheat, can produce 10 pyrazines, including methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, 2,6-diethylpyrazine, 2,5-dimethyl-3-isopropylpyrazine, tetramethylpyrazine, 3,5-diethyl-2-methylpyrazine, and (E)-2,3-dimethyl-5-(1-propenyl)pyrazine. Among these, the yield of tetramethylpyrazine is as high as 714.47 mg / kg, the yield of dimethylpyrazine is as high as 5.33 mg / kg, and the yield of 2,6-diethylpyrazine is as high as 0.38 mg / kg. This strain of the present invention not only produces a large amount of pyrazines but also produces flavor compounds such as guaiacol and ethyl hexanoate. Table 2 shows the results of flavor compound determination in wheat fermentation using Bacillus belyssus 17.

[0071] Table 2

[0072] Example 5 This embodiment relates to the determination of the fermentation flavor of Wuliangye by Bacillus vesiculosus 17.

[0073] The newly prepared Bacillus belye 17 bacterial seed culture was inoculated into Wuliang culture medium at an inoculation rate of 5%. After mixing evenly, it was cultured at 37°C for 14 days, and the flask was shaken every other day to ensure even mixing.

[0074] After fermentation, the flavor compounds produced were determined by GC-MS (internal standard method). Table 3 shows that the strain of this invention, when used in the solid-state fermentation medium of Wuliangye grains, can produce 12 pyrazines, including 3-methylpyrrolo[2,3-b]pyrazine, 2,2'-bipyrazine, vinylpyrazine, pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6-diethylpyrazine, and tetramethylpyrazine. Among them, the yield of tetramethylpyrazine is as high as 156.35 mg / kg, the yield of dimethylpyrazine is as high as 1.74 mg / kg, and the yield of 2,6-diethylpyrazine is as high as 0.12 mg / kg. In addition to producing polypyrazines, it can also produce flavor compounds such as acetoin, 2,3-butanediol, phenylethyl alcohol, guaiacol, ethyl hexanoate, and hexanoic acid.

[0075] Table 3

[0076] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A type of Bacillus belye 17, characterized in that, The Bacillus berleis 17 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35896.

2. The method for preparing Bacillus belyssus 17 according to claim 1, characterized in that, Includes the following steps: The Bacillus berleis 17 of claim 1 was inoculated into a culture medium and cultured to obtain the bacterium.

3. A composition, characterized in that, It contains Bacillus berleis 17 as described in claim 1.

4. The use of Bacillus berleis 17 as described in claim 1, Bacillus berleis 17 prepared according to the preparation method of claim 2, or the composition of claim 3 in fermented grains and pyrazine production.

5. A method for isolating and screening Bacillus belye 17, characterized in that, Includes the following steps: S1 Take the fermented mash and Daqu (a type of starter culture), mix them, add physiological saline to make a suspension, enrich and culture, and then separate and culture. S2 samples were selected and cultured on LB medium with colony morphology characteristics similar to those of Bacillus.

6. The method for isolating and screening Bacillus belye 17 according to claim 5, characterized in that, The isolation medium used for isolation culture in S1 consisted of: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 20 g / L agar.

7. A pyrazine-producing bacterial strain, characterized in that, The pyrazine-producing bacteria are prepared from Bacillus berleis 17 as described in claim 1, Bacillus berleis 17 prepared according to the preparation method described in claim 2, or the composition described in claim 3.

8. A method for preparing a pyrazine-producing bacterial koji, characterized in that, Includes the following steps: The activated Bacillus berleis 17 of claim 1, Bacillus berleis 17 prepared according to the preparation method of claim 2, or the composition of claim 3 are inoculated into raw wheat bran and cultured to obtain the prolific bacterial scion.

9. A type of baijiu (Chinese liquor), characterized in that, The baijiu is prepared using daqu (a type of starter culture) prepared by mixing Bacillus vesicularis 17 as described in claim 1, Bacillus vesicularis 17 prepared according to the preparation method described in claim 2, or the composition described in claim 3.

10. The liquor according to claim 9, characterized in that, The fermentation raw materials for the liquor are wheat or five grains.

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