Bacillus sorola and application thereof

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a bacillus sorola and application thereof. In order to enrich microbial resources for producing flavor substances such as 4-vinyl guaiacol, 4-vinyl phenol and the like, a strain of bacillus sonorrensis is screened and separated from high-temperature yeast for making hard liquor, and the preservation number of the bacillus sonorrensis is CCTCC (China Center for Type Culture Collection) NO: M 2025364. The strain can produce a plurality of flavor substances such as 4-vinyl guaiacol, 4-vinylphenol, 3-hydroxy-2-butanone, 2, 3, 5, 6-tetramethylpyrazine and the like by metabolism of wheat at a high temperature. Therefore, the strain has a wide application prospect in improving the yield of flavor substances such as 4-vinyl guaiacol, 4-vinylphenol and 3-hydroxy-2-butanone in the fields of white spirit brewing and food fermentation, and a new strain resource can be provided for researching phenolic acid decarboxylase derived from bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and specifically relates to Bacillus sonorensis capable of simultaneously producing 4-vinylguaiacol and 4-vinylphenol, and its application. Background Art

[0002] 4-Vinylguaiacol, also known as 2-methoxy-4-vinylphenol and p-vinylguaiacol, has a strong spice, clove, and fermented fragrance, and is an important aroma substance in foods such as liquor, beer, wine, soy sauce, tea, and cheese. 4-Vinylphenol, also known as p-hydroxystyrene, has a medicinal smell and a meaty fragrance, and is widely present in foods such as black tea, coffee, and wine.

[0003] Both 4-vinylguaiacol and 4-vinylphenol can be used as food flavor additives. In addition to chemical synthesis, they can be obtained through microbial fermentation and act on the formation of the flavor of fermented foods. For example, 4-vinylguaiacol and 4-vinylphenol produced by yeast fermentation during beer brewing are important flavor substances that form the typical characteristic flavors of Belgian white beer and German wheat beer. Research shows that there are various phenolic acid compounds in plant cell walls, including benzoic acid derivatives (gallic acid, protocatechuic acid) and cinnamic acid derivatives (p-coumaric acid, ferulic acid, caffeic acid, sinapic acid). During the fermentation process, non-oxidative decarboxylation reactions occur under the catalysis of phenolic acid decarboxylase to generate corresponding 4-vinyl derivatives. Among them, ferulic acid and p-coumaric acid are the precursor substances of 4-vinylguaiacol and 4-vinylphenol, respectively. In the research on brewing microorganisms, bacteria such as yeast, Pediococcus pentosaceus, Enterococcus, Lactobacillus plantarum, etc. that have the ability to decarboxylate both ferulic acid and p-coumaric acid have been found, and most of the bacteria are lactic acid bacteria.

[0004] Bacillus sonorensis is a dominant bacterium in the field of liquor brewing, and it has good flavor metabolism ability. However, the currently discovered species of Bacillus sonorensis are limited, and it is even rarer to discover Bacillus sonorensis that can simultaneously produce 4-vinylguaiacol and 4-vinylphenol. Summary of the Invention

[0005] In order to enrich the microbial resources that produce flavor substances such as 4-vinylguaiacol and 4-vinylphenol, the present invention screens and isolates a strain of Bacillus sonorensis from high-temperature Daqu that can simultaneously produce various flavor substances such as 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone, and 2,3,5,6-tetramethylpyrazine, and its application.

[0006] To achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In the first aspect, the present invention provides a Bacillus sonorensis, which was deposited at the China Center for Type Culture Collection (CCTCC) on March 4, 2025. The address of the deposit center is the Culture Collection Center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072. The deposit number is CCTCC NO: M 2025364, named Bacillus sonorensis LZLJ2-8.

[0008] Among them, the 16S rDNA sequence of the above-mentioned Bacillus sonorensis is shown in SEQ ID NO: 1.

[0009] Among them, the colony characteristics of the above-mentioned Bacillus sonorensis are round, with a neat edge, yellow in the middle, white at the edge, and opaque.

[0010] In the second aspect, the present invention provides a method for screening, isolating, and identifying the above-mentioned Bacillus sonorensis, which includes the following steps: making a sample bacterial suspension from high-temperature Daqu, first enriching and culturing, then isolating and culturing on an ISP2 solid medium, screening strains that produce 4-vinylguaiacol and 4-vinylphenol, and identifying the above-mentioned Bacillus sonorensis by combining morphological, physiological and biochemical characteristics and / or molecular biology.

[0011] Among them, the composition of the ISP2 solid medium includes: yeast extract 4.0 g / L, malt extract 10.0 g / L, glucose 4.0 g / L, and agar 15.0 g / L.

[0012] Among them, the culture temperature is 37°C to 55°C, and the culture time is 1 to 3 days.

[0013] In the third aspect, the present invention provides a microbial inoculant, which contains the fermentation broth, seed liquid, or bacterial cells of the above-mentioned Bacillus sonorensis.

[0014] In the fourth aspect, the present invention provides the application of the above-mentioned Bacillus sonorensis or microbial inoculant in the brewing of distilled spirits, fermented spirits, or food fermentation.

[0015] Among them, the distilled spirits are at least one of white liquor, brandy, whisky, vodka, rum, gin, tequila, or fruit distilled spirits.

[0016] Preferably, the white liquor includes at least one of strong aroma type, sauce aroma type, light aroma type, or blended aroma type.

[0017] Among them, the fermented wine is at least one of yellow rice wine, beer, wine, fruit wine, sake or milk wine.

[0018] Among them, the food includes at least one of soy sauce, tea, cheese or coffee.

[0019] In a fifth aspect, the present invention provides the application of the above-mentioned Bacillus sonorensis or microbial inoculum in producing flavor substances; the flavor substances include: at least one of benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styralyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline or 4-methylacetanilide.

[0020] Preferably, the flavor substances are 4-vinylguaiacol and 4-vinylphenol.

[0021] Among them, in the above application, the flavor substances are obtained by fermenting and culturing the above-mentioned Bacillus sonorensis or microbial inoculum.

[0022] Preferably, the inoculation amount of the Bacillus sonorensis or microbial inoculum is 5-10 (v / v)%.

[0023] Preferably, the fermentation temperature is 37°C to 63°C.

[0024] Preferably, the fermentation time is 1-3 d.

[0025] In a sixth aspect, the present invention provides a method for producing flavor substances, including the following steps: After activating the above-mentioned Bacillus sonorensis or microbial inoculum, inoculating it into a wheat solid medium and performing gradient temperature culture, various flavor substances can be produced;

[0026] Among them, the flavor substances include at least one of benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrallyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline or 4-methylacetanilide.

[0027] Further, the inoculum amount is 5-10 (v / v)%.

[0028] Further, the gradient culture temperatures are 37°C - 48°C - 52°C - 55°C - 59°C - 63°C - 63°C, and each gradient is cultured for 1-3 d.

[0029] Beneficial effects: A strain of Bacillus sonorensis was screened, isolated and identified from high-temperature Daqu, and the preservation number is CCTCC NO: M 2025364. The Bacillus sonorensis provided by the present invention can utilize wheat at high temperature to metabolize and produce various flavor substances such as 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone and 2,3,5,6-tetramethylpyrazine. Therefore, it has a wide application prospect in improving the yields of flavor substances such as 4-vinylguaiacol, 4-vinylphenol and 3-hydroxy-2-butanone in the field of liquor brewing and food fermentation, and can also provide new strain resources for studying phenolic acid decarboxylase derived from bacteria. Description of the Drawings

[0030] Figure 1 It is the colony morphology diagram of Bacillus sonorensis LZLJ2-8 in Example 1;

[0031] Figure 2 It is the GC-MS molecular fragment mass spectrum diagram of 4-vinylguaiacol produced by fermentation in Example 1;

[0032] Figure 3 It is the GC-MS molecular fragment mass spectrum diagram of the 4-vinylguaiacol standard product in Example 1;

[0033] Figure 4 It is the GC-MS molecular fragment mass spectrum diagram of 4-vinylphenol produced by fermentation in Example 1;

[0034] Figure 5 It is the GC-MS molecular fragment mass spectrum diagram of the 4-vinylphenol standard product in Example 1;

[0035] Figure 6 GC-MS chromatogram of volatile substances in the wheat solid-state fermentation sample of Example 3.

[0036] Description of strain preservation:

[0037] The Bacillus sonorensis provided by the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on March 4, 2025. The address is the preservation center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the postal code 430072. The deposit number is CCTCC NO: M 2025364, named Bacillus sonorensis LZLJ2-8. Detailed implementation manners

[0038] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer, the present application will be further described in detail below in conjunction with the implementation manners. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0039] In one implementation manner of the present invention, a strain of Bacillus sonorensis was screened, isolated and identified from high-temperature Daqu, and the deposit number is CCTCC NO: M 2025364. It was deposited at the China Center for Type Culture Collection (CCTCC) on March 4, 2025. The address is the preservation center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the postal code 430072. It is named Bacillus sonorensis LZLJ2-8.

[0040] Through molecular biological identification, the 16S rDNA sequence of the above-mentioned Bacillus sonorensis is shown as SEQ ID NO:1:

[0041] SEQ ID NO:1:

[0042]

[0043] Among them, the colony characteristics of the above-mentioned Bacillus sonorensis on ISP2 solid medium are round, with neat edges, yellow in the middle, white at the edges, and opaque.

[0044] The components of the ISP2 solid medium include: 4.0 g of yeast extract, 10.0 g of malt extract, 4.0 g of glucose, 15.0 g of agar, add water to 1000 mL, adjust the pH to 7.0, and sterilize by high-pressure steam at 121 °C for 15 minutes.

[0045] In some embodiments of the present invention, a microbial inoculant is prepared, which contains the fermentation broth, seed liquor or thallus of the above-mentioned Bacillus sonorensis.

[0046] In one embodiment of the present invention, the above-mentioned Bacillus sonorensis or microbial inoculant is inoculated into a wheat solid medium to simulate the fermentation of high-temperature Daqu, and it can metabolize and produce a variety of flavor substances, including benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styralyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline and 4-methylacetanilide.

[0047] The above-mentioned Bacillus sonorensis or microbial inoculant provided by the present invention can metabolize and produce a variety of flavor substances, including but not limited to benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styralyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline and 4-methylacetanilide. Therefore, it has a wide range of applications in the brewing of distilled spirits, fermented spirits or food fermentation.

[0048] More specifically, the distilled spirit is at least one of white liquor, brandy, whisky, vodka, rum, gin, tequila or fruit distilled spirit. Preferably, the white liquor includes at least one of strong aroma type, sauce aroma type, light aroma type or mixed aroma type.

[0049] The fermented wine is at least one of yellow rice wine, beer, grape wine, fruit wine, sake or milk wine.

[0050] The food includes at least one of soy sauce, tea, cheese or coffee.

[0051] Specific embodiments will be listed 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, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] The medium formula involved in the embodiments:

[0053] ISP2 solid medium: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, agar 15.0 g, add water to 1000 mL, adjust the pH to 7.0, and sterilize by high-pressure steam at 121 °C for 15 minutes.

[0054] TSB liquid medium: tryptone 17.0 g, sodium chloride 5.0 g, soy peptone 3.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g, add water to 1000 mL, adjust the pH to 7.2, and sterilize by high-pressure steam at 121 °C for 15 minutes.

[0055] Fermentation medium: tryptone 17.0 g, sodium chloride 5.0 g, soy peptone 3.0 g, dipotassium hydrogen phosphate 2.5 g, glucose 2.5 g, ferulic acid 2 mg, coumaric acid 2 mg, add water to 1000 mL, adjust the pH to 7.0, and sterilize by high-pressure steam at 121 °C for 15 minutes.

[0056] Wheat solid medium: Take the crushed wheat, add water and mix evenly according to the material-water ratio of 1:0.3, moisten for 1 h, dispense into 100 mL Erlenmeyer flasks, about 40 g of material in each flask, sterilize at 121 °C for 30 min, and add 16 mL of sterile water to each flask after cooling, and stir evenly.

[0057] The specific method for determining volatile products by headspace solid-phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) is as follows:

[0058] Take the sample and add it to a headspace vial. Add 2 g of NaCl, 5 mL of pure water, and 5 μL of internal standard (2-octanol at 0.264 mg / mL). After equilibrating the prepared sample at 55 °C for 5 min, extract it with a 50 / 30 μm DVB / CAR / PDMS extraction head at 55 °C for 45 min. After extraction, desorb it at 250 °C in the GC injection port for 5 min.

[0059] GC-MS detection chromatographic conditions

[0060] Gas chromatography conditions: HP-INNOWAX chromatographic column (60 m × 0.25 mm × 0.25 μm); temperature programming: initial temperature 40 °C, hold for 1 min, rise to 75 °C at 2.5 °C / min, rise to 162 °C at 3 °C / min, rise to 230 °C at 6 °C / min, hold for 5 min, carrier gas is high-purity helium (1.0 mL / min); injection port temperature 250 °C, splitless.

[0061] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40 - 450 m / z.

[0062] Using 2-octanol at 0.264 mg / mL as the internal standard, the compound retrieval results are matched with the NIST standard spectral library, and compounds with a similarity of over 80% are confirmed as the target compounds.

[0063] Screening of the strain in Example 1

[0064] Weigh 10 g of high-temperature Daqu and add it to a 250 mL conical flask containing 90 mL of sterile water with glass beads (saline can be used). Shake it on a shaker at room temperature with a rotation speed of 150 r / min for 30 min to prepare a sample bacterial suspension for standby. Pipette 1 mL of the supernatant of the Daqu bacterial suspension and 9 mL of sterile water (or saline) into a 15 mL centrifuge tube to prepare a dilution of 10 -2 . Dilute it in gradient in this way to obtain dilutions of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 respectively. Take 100 μL of each dilution and spread it on an ISP2 solid medium, and culture it in an incubator at 55 °C for 1 - 3 d; then pick out the vigorously growing single colonies and streak them multiple times until all single colonies are purified. Inoculate the purified strains into a fermentation medium, culture at a temperature of 37 °C and a shaker rotation speed of 120 rpm. After culturing for 2 - 3 days, pipette 0.2 mL of the bacterial liquid and use the HS-SPME / GC-MS method to detect the volatile metabolites in the sample, and screen out a strain that can produce 4-vinylguaiacol ( Figures 2 to 3 ) and 4-vinylphenol (Figures 4 to 5 ) A strain with the ability, numbered LZLJ2-8. This strain was preserved at -80 °C with 30% glycerol for further analysis.

[0065] Example 2 Molecular Identification of the Strain

[0066] After the target strain LZLJ2-8 was amplified and cultured, fresh bacterial liquid in the logarithmic growth phase was taken, the cells were collected by centrifugation, and genomic DNA was extracted using the Ezup Column Bacterial Genomic DNA Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd. The full-length 16S rDNA sequence was amplified using the universal bacterial primers 27F / 1492R as follows:

[0067] SEQ ID NO:2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0068] SEQ ID NO:3: 1492R (5′-GGTTACCTTGTTACGACTT-3′)

[0069] ① Reaction System (25 μL)

[0070]

[0071] ② Reaction Program

[0072]

[0073] According to the above process, a PCR reaction of 30 cycles of "denaturation-annealing-extension" was carried out. The PCR products were separated and examined by 1.0% agarose gel electrophoresis at a voltage of 11 V / cm for 20 min. The purification of the PCR products was carried out according to the instructions of the Small Volume Gel Recovery PCR Product Purification Kit from Sangon Biotech (Shanghai) Co., Ltd., and the sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0074] The gene sequence of the 16S rDNA fragment obtained by sequencing was aligned by BLAST of NCBI, combined with the colony morphology. As Figure 1 shown, the colony characteristics on ISP2 solid medium were round, with neat edges, yellow in the middle, white at the edges, and opaque. The species information of the strain was determined, and it was identified as Bacillus sonorensis, and named Bacillus sonorensis LZLJ2-8. It was deposited in the China Center for Type Culture Collection (CCTCC) on March 4, 2025, at the Preservation Center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072. The deposit number is CCTCC NO: M 2025364.

[0075] Example 3 Wheat Solid Medium Fermentation Experiment

[0076] Take the glycerol preservation tube of Bacillus sonorensis LZLJ2-8 and inoculate it into 1 mL of TSB liquid medium at an inoculation amount of 10% respectively, and culture it on a shaker at 37 °C and 120 r / min for 1 day to activate the strain; transfer the bacterial liquid to 5 mL of TSB liquid medium at an inoculation amount of 10% and culture it on a shaker at 37 °C and 120 r / min for 1 day to prepare the seed liquid; inoculate the seed liquid into the wheat solid medium at an inoculation amount of 5%, simulate the temperature-rising program of high-temperature Daqu, and perform gradient temperature culture. The culture temperatures are 37 °C - 48 °C - 52 °C - 55 °C - 59 °C - 63 °C - 63 °C, and each gradient is cultured for 1 day, and a blank control group experiment is set under the same culture conditions. After the experiment, HS-SPME / GC-MS method is used to detect the phenolic substances in the samples, and the culture solution fermented without adding bacteria is used as the blank control group to calculate the content.

[0077] The experimental results are as Figure 6 shown. The analysis results of flavor metabolites are shown in Table 1. Bacillus sonorensis LZLJ2-8 uses wheat to ferment to produce 4-vinylguaiacol and 4-vinylphenol, and the contents are 8.112 μg / g and 0.276 μg / g respectively, and it can metabolize many other flavor substances, including benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, and styrallyl alcohol of alcohols; gamma-hexalactone and methyl phenylacetate of esters; 2-ethylbutyric acid and isovaleric acid of acids; benzaldehyde of aldehydes; 3-hydroxy-2-butanone, 2-hexanone, acetophenone, and phenylacetone of ketones; phenol, 2,4-di-tert-butylphenol, and guaiacol of phenols; 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, and 2,5-dimethylpyrazine of pyrazines; 2-methyl-4-methoxyaniline and 4-methylacetanilide of anilines.

[0078] Table 1 Content of volatile substances in the solid fermentation sample of Bacillus sonorensis LZLJ2-8 (μg / g)

[0079]

[0080]

Claims

1. Bacillus sonorensis, characterized in that: The preservation number is CCTCC NO: M2025364.

2. A microbial inoculant, characterized in that: Containing the fermentation broth, seed liquor or bacterial cells of Bacillus sonorensis described in claim 1.

3. Use of the Bacillus sonorensis described in claim 1 or the microbial inoculum described in claim 2 in the brewing of distilled spirits, fermented spirits or food fermentation.

4. The application according to claim 3, wherein: The distilled spirits are at least one of Chinese liquor, brandy, whisky, vodka, rum, gin, tequila or fruit distilled spirits; the fermented spirits are at least one of yellow rice wine, beer, wine, fruit wine, sake or milk wine.

5. The application according to claim 4, wherein: The Chinese liquor includes at least one of strong aroma type, Maotai aroma type, light aroma type or blended aroma type.

6. Use of Bacillus sonorensis according to claim 1 or the microbial inoculum according to claim 2 in producing flavor substances, characterized in that: The flavor substances include: at least one of benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrallyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline or 4-methylacetanilide.

7. Use of the Bacillus sonorensis described in claim 1 or the microbial inoculum described in claim 2 in the production of 4-vinylguaiacol and 4-vinylphenol.

8. A method for producing flavor substances, characterized in that: Comprising the following steps: After activating the Bacillus sonorensis described in claim 1 or the microbial inoculum described in claim 2, inoculating it into a wheat solid medium and performing gradient temperature cultivation to produce a variety of flavor substances; Wherein, the flavor substances include: at least one of benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrallyl alcohol, gamma-hexalactone, methyl phenylacetate, 2-ethylbutyric acid, isovaleric acid, benzaldehyde, 3-hydroxy-2-butanone, 2-hexanone, acetophenone, phenylacetone, phenol, 4-vinylguaiacol, 2,4-di-tert-butylphenol, guaiacol, 4-vinylphenol, 2,3,5-trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, 2,3,5,6-tetramethylpyrazine, 5-(sec-butyl)-2,3-dimethylpyrazine, 2,3,5-trimethyl-6-propylpyrazine, 2,5-dimethylpyrazine, 2-methyl-4-methoxyaniline or 4-methylacetanilide.

9. The method for producing flavor substances according to claim 8, characterized in that: The inoculation amount is 5-10%.

10. The method for producing flavor substances according to claim 8, characterized in that: The gradient cultivation temperature is 37°C - 48°C - 52°C - 55°C - 59°C - 63°C - 63°C, and each gradient is cultured for 1-3 days.

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