Bacillus sonorensis and uses thereof

Abstract

The present application belongs to the technical field of biological fermentation, and particularly relates to a Bacillus sonorensis strain and application thereof. In order to enrich microbial resources for producing 4-vinylguaiacol, 4-vinylphenol and other flavor substances, the present application screens and isolates a Bacillus sonorensis strain from high-temperature Daqu, and the preservation number is CCTCC NO: M 2025364. The strain can utilize wheat at high temperature to metabolize 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone and 2,3,5,6-tetramethylpyrazine and other flavor substances. Therefore, the strain has a wide application prospect in the fields of liquor brewing and food fermentation for improving the yield of 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone and other flavor substances, and can also provide new strain resources for studying phenolic acid decarboxylase from bacteria.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to Bacillus sonorensis, which simultaneously produces 4-vinylguaiacol and 4-vinylphenol, and its applications. Background Technology

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

[0003] Both 4-vinylguaiacol and 4-vinylphenol can be used as food flavor additives. Besides chemical synthesis, they can be obtained through microbial fermentation and contribute to the flavor formation of fermented foods. For example, 4-vinylguaiacol and 4-vinylphenol, produced by yeast fermentation during beer brewing, are important aroma compounds that contribute to the typical flavor characteristics of Belgian white beer and German wheat beer. Studies have shown that plant cell walls contain various phenolic acid compounds, including benzoic acid derivatives (gallic acid, protocatechuic acid) and cinnamic acid derivatives (coumaric acid, ferulic acid, caffeic acid, sinapic acid). During fermentation, these compounds undergo non-oxidative decarboxylation reactions catalyzed by phenolic acid decarboxylases to generate the corresponding 4-vinyl derivatives. Ferulic acid and coumaric acid are precursors of 4-vinylguaiacol and 4-vinylphenol, respectively. Research on brewing microorganisms has identified bacteria capable of decarboxylating both ferulic acid and coumaric acid, including yeast, Pediococcus valerate, Enterococcus, and Lactobacillus plantarum, with most being lactic acid bacteria.

[0004] Bacillus sonorensis is a dominant bacterium in the field of winemaking, possessing excellent flavor metabolism capabilities. However, the number of Bacillus sonorensis species discovered so far is limited, and even fewer Bacillus sonorensis species capable of simultaneously producing 4-vinylguaiacol and 4-vinylphenol have been discovered. Summary of the Invention

[0005] To enrich the microbial resources that produce flavor compounds such as 4-vinylguaiacol and 4-vinylphenol, this invention screens and isolates a strain of Sonora desert Bacillus that simultaneously produces multiple flavor compounds such as 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone, and 2,3,5,6-tetramethylpyrazine from high-temperature Daqu (a type of starter culture), and explores its applications.

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

[0007] In a first aspect, the present invention provides a *Bacillus sonorensis*, which was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The accession number is CCTCC NO: M 2025364, and the name is *Bacillus sonorensis* LZLJ2-8.

[0008] The 16S rDNA sequence of the aforementioned Sonora desert spore bacillus is shown in SEQ ID NO:1.

[0009] Among them, the colony characteristics of the aforementioned Sonora desert bacillus are round, with neat edges, yellow in the middle, white at the edges, and opaque.

[0010] Secondly, the present invention provides a method for screening, isolating and identifying the above-mentioned Sonora desert bacillus, which includes the following steps: preparing a sample bacterial suspension by high-temperature Daqu, enriching and culturing it first, and then isolating and culturing it on ISP2 solid medium, screening out strains that produce 4-vinylguaiacol and 4-vinylphenol, and identifying the Sonora desert bacillus by combining morphological, physiological and biochemical characteristics and / or molecular biology.

[0011] The ISP2 solid culture medium consists of: 4.0 g / L yeast extract, 10.0 g / L malt extract, 4.0 g / L glucose, and 15.0 g / L agar.

[0012] The culture temperature is 37℃~55℃, and the culture time is 1~3 days.

[0013] Thirdly, the present invention provides a microbial agent containing the fermentation broth, seed liquid or bacterial cells of the aforementioned Sonora desert Bacillus.

[0014] Fourthly, the present invention provides the application of the above-mentioned Sonora desert Bacillus or microbial agents in the brewing of distilled spirits, fermented wines, or food fermentation.

[0015] The distilled spirit is at least one of the following: baijiu, brandy, whiskey, vodka, rum, gin, tequila, or fruit distilled spirits.

[0016] Preferably, the liquor includes at least one of the following: strong aroma type, sauce aroma type, light aroma type, or mixed aroma type.

[0017] The fermented wine is at least one of rice wine, beer, wine, fruit wine, sake, or milk wine.

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

[0019] Fifthly, the present invention provides the application of the above-mentioned Sonora desert Bacillus or microbial agents in the production of flavor substances; the flavor substances include at least one of the following: benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrax alcohol, gamma-caprolactone, 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-methylacetaniline.

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

[0021] In the aforementioned application, the flavor substance is obtained by fermenting and culturing the above-mentioned Sonora desert Bacillus or microbial inoculants.

[0022] Preferably, the inoculation amount of the Sonora desert bacillus or microbial agent is 5-10 (v / v)%.

[0023] Preferably, the fermentation temperature is 37℃~63℃.

[0024] Preferably, the fermentation time is 1 to 3 days.

[0025] In a sixth aspect, the present invention provides a method for producing flavor substances, comprising the following steps: activating the above-mentioned Sonora desert Bacillus or microbial inoculant, inoculating it into a wheat solid culture medium, and performing gradient temperature culture to produce a variety of flavor substances;

[0026] The flavoring substances include at least one of the following: benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrax alcohol, gamma-caprolactone, 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-methylacetaniline.

[0027] Furthermore, the inoculation dose is 5–10 (v / v)%.

[0028] Furthermore, the gradient culture temperature is 37℃-48℃-52℃-55℃-59℃-63℃-63℃, and each gradient is cultured for 1 to 3 days.

[0029] Beneficial Effects: This invention screened, isolated, and identified a strain of *Bacillus sonorensis* from high-temperature koji (fermentation starter culture), with the preservation number CCTCC NO: M 2025364. The *Bacillus sonorensis* provided by this invention can utilize wheat at high temperatures to metabolize and produce various flavor compounds, including 4-vinylguaiacol, 4-vinylphenol, 3-hydroxy-2-butanone, and 2,3,5,6-tetramethylpyrazine. Therefore, it has broad application prospects in the fields of baijiu brewing and food fermentation for increasing the yield of flavor compounds such as 4-vinylguaiacol, 4-vinylphenol, and 3-hydroxy-2-butanone, and can also provide new strain resources for the study of bacterial phenolic acid decarboxylases. Attached Figure Description

[0030] Figure 1 Example 1: Colony morphology of Sonora desert bacillus LZLJ2-8;

[0031] Figure 2 This is a GC-MS fragment mass spectrum of 4-vinylguaiacol produced by fermentation in Example 1;

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

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

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

[0035] Figure 6 This is a GC-MS image of volatile substances in the wheat solid-state fermentation sample from Example 3.

[0036] Instructions for strain preservation:

[0037] The *Bacillus sonorensis* provided in this invention was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The accession number is CCTCC NO: M 2025364, and the name is *Bacillus sonorensis* LZLJ2-8. Detailed Implementation

[0038] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0039] In one embodiment of the present invention, a strain of *Bacillus sonorensis* was screened, isolated, and identified from high-temperature koji (a type of Chinese liquor), with accession number CCTCC NO: M 2025364. It was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. It was named *Bacillus sonorensis* LZLJ2-8.

[0040] Molecular biological identification confirmed that the 16S rDNA sequence of the aforementioned Sonora desert bacillus is shown in SEQ ID NO:1:

[0041] SEQ ID NO:1:

[0042]

[0043] Among them, the colony characteristics of the aforementioned Sonora desert spore bacillus on ISP2 solid medium are round, with neat edges, yellow in the middle, white at the edges, and opaque.

[0044] The ISP2 solid culture medium consists of: 4.0g yeast extract, 10.0g malt extract, 4.0g glucose, and 15.0g agar, with water added to 1000mL, pH adjusted to 7.0, and autoclaved at 121℃ for 15 minutes.

[0045] In some embodiments of the present invention, a microbial agent is prepared, which contains the fermentation broth, seed liquid or cell of the above-mentioned Sonora desert Bacillus.

[0046] In one embodiment of the present invention, the above-mentioned Sonora desert Bacillus or microbial inoculant is inoculated into a wheat solid culture medium to simulate high-temperature fermentation. It can metabolize and produce a variety of flavor substances, including benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrax alcohol, gamma-caprolactone, 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-methylacetaniline.

[0047] The Sonora desert bacillus or microbial agents provided by this invention can metabolize and produce a variety of flavor substances, including but not limited to benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styraxol, gamma-caprolactone, 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-methylacetaniline. Therefore, they have wide applications in distilled spirits, fermented wine brewing, and food fermentation.

[0048] More specifically, the distilled spirit is at least one of baijiu, brandy, whiskey, vodka, rum, gin, tequila, or fruit-based distilled spirits. Preferably, the baijiu includes at least one of strong-aroma, sauce-aroma, light-aroma, or mixed-aroma types.

[0049] The fermented wine is at least one of 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] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0052] The culture medium formulations involved in the examples are as follows:

[0053] ISP2 solid culture medium: 4.0g yeast extract, 10.0g malt extract, 4.0g glucose, 15.0g agar, add water to 1000mL, adjust pH to 7.0, and autoclave at 121℃ for 15 minutes.

[0054] TSB liquid culture medium: 17.0g tryptone, 5.0g sodium chloride, 3.0g soybean papain hydrolysate, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, add water to 1000mL, adjust pH to 7.2, autoclave at 121℃ for 15 minutes.

[0055] Fermentation medium: 17.0g tryptone, 5.0g sodium chloride, 3.0g soybean papain hydrolysate, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, 2mg ferulic acid, 2mg coumaric acid, add water to 1000mL, adjust pH to 7.0, and autoclave at 121℃ for 15 minutes.

[0056] Wheat solid culture medium: Take crushed wheat, add water at a ratio of 1:0.3 and mix evenly. Moisten for 1 hour, then dispense into 100mL Erlenmeyer flasks, with about 40g of material per flask. Sterilize at 121℃ for 30 minutes. After cooling, add 16mL of sterile water to each flask and stir evenly.

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

[0058] Take the sample and add it to the headspace vial. Add 2g NaCl, 5mL pure water, and 5μL internal standard (0.264mg / mL 2-octanol). After equilibration at 55℃ for 5min, extract the sample at 55℃ for 45min using a 50 / 30μm DVB / CAR / PDMS extraction head. After extraction, desorb the sample at 250℃ for 5min at the GC inlet.

[0059] GC-MS detection chromatographic conditions

[0060] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: initial temperature 40℃, hold for 1 min, increase to 75℃ at 2.5℃ / min, increase to 162℃ at 3℃ / min, increase to 230℃ at 6℃ / min, hold for 5 min; carrier gas is high-purity helium (1.0mL / min); injection port temperature 250℃, 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 0.264 mg / mL 2-octanol as an internal standard, the compound search results were matched with the NIST standard spectral library, and the similarity was more than 80%, confirming it as the target compound.

[0063] Example 1: Screening of strains

[0064] Weigh 10g of high-temperature Daqu (a type of starter culture) and add it to a 250mL Erlenmeyer flask containing 90mL of sterile water (or physiological saline) with glass bulbs. Shake on a shaker at room temperature and 150rpm for 30min to prepare a bacterial suspension for later use. Transfer 1mL of the Daqu bacterial suspension supernatant and 9mL of sterile water (or physiological saline) to a 15mL centrifuge tube to prepare 10... -2 The diluted solution was serially diluted using this method to prepare 10 solutions. -2 10 -3 10 -4 10 -5 10 -6 The dilution was prepared by spreading 100 μL of each dilution onto ISP2 solid medium and incubating at 55°C for 1–3 days. Vigorous single colonies were then streaked multiple times until all single colonies were purified. The purified strain was inoculated into fermentation medium and incubated at 37°C with a shaking speed of 120 rpm for 2–3 days. 0.2 mL of the bacterial solution was then analyzed using HS-SPME / GC-MS to detect volatile metabolites in the sample. A strain producing 4-vinylguaiacol (VV) was screened. Figures 2-3 ) and 4-vinylphenol ( Figures 4-5 The strain with the ability to [analyze] was designated LZLJ2-8. This strain was preserved at -80°C in 30% glycerol for further analysis.

[0065] Example 2 Molecular identification of strains

[0066] After amplification of the target strain LZLJ2-8, fresh bacterial culture in the logarithmic growth phase was collected by centrifugation, and genomic DNA was extracted using the Ezup column-based bacterial genomic DNA extraction kit from Shanghai Sangon Biotech Co., Ltd. The full-length 16S rDNA sequence was amplified using 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] ①Reaction system (25 μL)

[0070]

[0071] ②Reaction Procedure

[0072]

[0073] Following the above procedure, 30 cycles of denaturation-annealing-extension PCR were performed. PCR products were separated and tested by 1.0% agarose gel electrophoresis at 11 V / cm for 20 min. PCR product purification was performed according to the instructions of the small-volume gel recovery PCR product purification kit from Shanghai Sangon Biotech Co., Ltd., and sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0074] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST algorithm, combined with colony morphology, such as... Figure 1 As shown, the colonies on ISP2 solid medium are characterized by being round, with neat edges, a yellow center, a white edge, and opaque. The strain was identified as *Bacillus sonorensis* and named *Bacillus sonorensis* LZLJ2-8. It was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The accession number is CCTCCNO: M 2025364.

[0075] Example 3: Wheat solid-state culture medium fermentation experiment

[0076] 10% of the glycerol culture tubes of *Bacillus desertica* LZLJ2-8 were inoculated into 1 mL of TSB liquid culture medium and cultured at 37℃ and 120 rpm for 1 day to activate the strain. The bacterial culture was then transferred to 5 mL of TSB liquid culture medium and cultured at 37℃ and 120 rpm for 1 day to prepare seed culture. 5% of the seed culture was inoculated into wheat solid culture medium, and a gradient temperature program simulating the high-temperature fermentation process was used for culture: 37℃-48℃-52℃-55℃-59℃-63℃-63℃, with each gradient cultured for 1 day. A blank control group was also included under the same culture conditions. After the experiment, phenolic substances in the samples were detected using HS-SPME / GC-MS, with the culture medium without bacterial fermentation serving as the blank control group. The content of phenolic substances was calculated.

[0077] Experimental results are as follows Figure 6 As shown in Table 1, the results of flavor metabolite analysis indicate that *Bacillus desertica* LZLJ2-8 produced 4-vinylguaiacol and 4-vinylphenol via wheat fermentation, with contents of 8.112 μg / g and 0.276 μg / g, respectively. It was also able to metabolize various other flavor compounds, including alcohols such as benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, and styraxol; esters such as gamma-caprolactone and methyl phenylacetate; acids such as 2-ethylbutyric acid and isovaleric acid; and aldehydes such as benzaldehyde. Ketones such as 3-hydroxy-2-butanone, 2-hexanone, acetophenone, and phenylacetone; phenols such as phenol, 2,4-di-tert-butylphenol, and guaiacol; pyrazines such as 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; and anilines such as 2-methyl-4-methoxyaniline and 4-methylacetaniline.

[0078] Table 1. Volatile substance content (μg / g) of Sonora desert Bacillus LZLJ2-8 solid-state fermentation samples.

[0079]

[0080]

Claims

1. Sonora desert bacillus ( Bacillus sonorensis ), characterized in that: The accession number is CCTCC NO: M2025364.

2. A microbial inoculant, characterized in that: Fermentation broth, seed culture, or cells containing the Sonora desert bacillus as described in claim 1.

3. The application of Sonora desert bacillus as described in claim 1 or the microbial agent as described in claim 2 in the brewing of distilled spirits, fermented wines, or food fermentation.

4. The application according to claim 3, characterized in that: The distilled spirit is at least one of baijiu, brandy, whiskey, vodka, rum, gin, tequila, or fruit distilled spirits; the fermented wine is at least one of huangjiu, beer, wine, fruit wine, sake, or milk wine.

5. The application according to claim 4, characterized in that: The baijiu includes at least one of the following: strong aroma, sauce aroma, light aroma, or mixed aroma.

6. The application of *Bacillus desertica* as described in claim 1 or the microbial agent as described in claim 2 in the production of flavor substances, characterized in that: The flavoring substances include at least one of the following: benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrax alcohol, gamma-caprolactone, 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-methylacetaniline.

7. The use of Sonora desert bacillus as described in claim 1 or the microbial agent as described in claim 2 in the production of 4-vinylguaiacol and 4-vinylphenol.

8. A method for producing flavor substances, characterized in that: Includes the following steps: After activating the Sonora desert bacillus as described in claim 1 or the microbial agent as described in claim 2, it is inoculated into wheat solid culture medium and cultured at gradient temperature to produce a variety of flavor substances. The flavoring substances include at least one of the following: benzyl alcohol, phenethyl alcohol, 2-ethylhexanol, styrax alcohol, gamma-caprolactone, 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-methylacetaniline.

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

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

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