Elgibberella thermophilus and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to bacillus Elgibbereli and application thereof. In order to develop a microbial resource which can produce more 2, 4, 5-trimethyl oxazole and is high in yield, the invention provides a new strain of the bacillus erzumurae, and the preservation number of the new strain is CCTCC (China Center for Type Culture Collection) NO: M 2025368. According to the present invention, the Elgibler thermophilic bacillus can produce 2, 4, 5-trimethyloxazole by using bran and wheat, and can be used in leavening agents such as mouldy bran, medium-high temperature yeast, high temperature yeast and the like. And the strain can also produce other flavor substances such as isovaleric acid, 3-hydroxy-2-butanone, 2, 3, 5, 6-tetramethylpyrazine and the like through metabolism, so that an excellent functional strain is provided for strengthening a fermented food process and improving the quality.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to Erzurum thermophilic bacillus and applications thereof. Background Art

[0002] 2,4,5-Trimethyloxazole can be used as a food flavoring additive in chocolate, Dutch cocoa, coffee, malt liquor, toast, and various meat-flavored flavors. It also occurs naturally in some fermented foods, such as cooked beef, cocoa, cooked pork, French fries, autolyzed yeast, boiled eggs, and fried chicken. Studies have shown that 2,4,5-trimethyloxazole is the key characteristic aroma compound in Zhenjiang vinegar, the most abundant oxazole compound, and its concentration increases with aging (Sun Zongbao, "Determination of the Characteristic Aroma Components of Zhenjiang Vinegar by HS-SPME / GC-MS / GC-O," Journal of Jiangsu University (Natural Science Edition) 002 (2010): 031). Furthermore, 2,4,5-trimethyloxazole in fermented foods is derived from microbial metabolism. For example, Zhang Xiaolong et al. (Zhang Xiaolong et al., "Relationship between Volatile Components and Microbial Metabolism in Sauce-Flavored Daqu." China Brewing 39.12(2020):7) screened 11 strains of sauce-flavor-producing bacteria from sauce-flavored Daqu. These strains, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis, were all capable of producing 2,4,5-trimethyloxazole using solid-state culture medium, but at relatively low levels, ranging from 0.074% to 1.764%. Therefore, developing more high-yield microbial resources for 2,4,5-trimethyloxazole has broader application prospects in the food and fermentation fields. Summary of the Invention

[0003] In order to develop microbial resources that are more productive and have high yield of 2,4,5-trimethyloxazole, the present invention provides a Calidifontibacillus erzurumensis and applications thereof.

[0004] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0005] In a first aspect, the present invention provides a new strain of Thermophilic Bacillus erzurum (Calidifontibacilluserzurumensis), which was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072. The deposit number is CCTCCNO: M 2025368, and it is named Thermophilic Bacillus erzurum LZLJ2-12 (Calidifontibacilluserzurumensis LZLJ2-12).

[0006] The 16S rDNA sequence of the aforementioned Thermobacillus Erzurum is shown in SEQ ID NO: 1.

[0007] Among them, the biological characteristics of the above-mentioned Erzurum thermophilic Bacillus are: on the DSMZ 1 solid culture medium, the colonies are round, neat, wrinkled, milky white, and opaque.

[0008] Among them, the above-mentioned Erzurum thermophilic Bacillus produces 2,4,5-trimethyloxazole.

[0009] In a second aspect, the present invention provides a method for isolating, screening and identifying the above-mentioned Thermophilic Bacillus of Erzurum, comprising the following steps:

[0010] The high-temperature Daqu is prepared into a sample bacterial suspension, which is first enriched and cultured and then isolated and cultured on a DSMZ 1 solid culture medium. After purification, the suspension is fermented to screen out a strain producing 2,4,5-trimethyloxazole, and the Erzurum thermophilic Bacillus is identified by combining morphological, physiological and biochemical characteristics and / or molecular biology.

[0011] The DSMZ 1 solid culture medium comprises the following components: 5.0 g of peptone, 3.0 g of meat extract powder, 15.0 g of agar, water is added to 1000 mL, and the pH is adjusted to 7.0.

[0012] The fermentation medium is bran filtered after cooking, glucose is added to a final concentration of 5 to 10 g / L, and the pH is adjusted to 6.0 to 6.4.

[0013] The fermentation culture temperature is 40-50° C., and the culture time is 1-3 days.

[0014] In a third aspect, the present invention provides a microbial agent comprising a fermentation liquid, a seed liquid or a bacterial cell of the aforementioned Thermophilic Bacillus Erzurum.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned Erzurum Thermophilic Bacillus or microbial agent in the brewing of liquor, malt liquor, food fermentation, and the preparation of bran koji or daqu.

[0016] The liquor comprises at least one of strong-flavor, sauce-flavor, light-flavor or mixed-flavor types.

[0017] Wherein, the food comprises at least one of soy sauce, fermented black beans, fermented bean curd, pickles or balsamic vinegar.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned Erzurum Thermophilic Bacillus or microbial agent in producing at least one of the following flavor substances, wherein the flavor substances include: 2,4,5-trimethyloxazole, 2-ethylhexanol, phenylethyl alcohol, gamma-nonalactone, isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, butyric acid, nonanal, benzaldehyde, α-ethylidene-phenylacetaldehyde, acetophenone, 2-nonanone, 3-hydroxy-2-butanone, 2-undecanone, phenol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine or 2-acetyl-3-5-dimethylpyrazine.

[0019] Preferably, the flavoring substance is 2,4,5-trimethyloxazole.

[0020] The flavor substance is obtained by fermenting the Erzurum thermophilic bacillus or microbial agent with wheat as raw material.

[0021] Furthermore, the inoculation amount of the Erzurum Thermophilic Bacillus or microbial agent is 5 to 10 (v / v)%.

[0022] Furthermore, the fermentation temperature is 37-55°C.

[0023] Furthermore, the fermentation time is 1 to 12 days.

[0024] In a sixth aspect, the present invention provides a method for producing flavor substances, comprising the following steps: activating the above-mentioned Erzurum Thermophilic Bacillus or microbial agent, inoculating it into a wheat solid culture medium, and conducting gradient temperature culture to produce a variety of flavor substances; the flavor substances include: at least one of 2,4,5-trimethyloxazole, 2-ethylhexanol, phenylethyl alcohol, gamma-nonalactone, isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, butyric acid, nonanal, benzaldehyde, α-ethylidene-phenylacetaldehyde, acetophenone, 2-nonanone, 3-hydroxy-2-butanone, 2-undecanone, phenol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine or 2-acetyl-3-5-dimethylpyrazine.

[0025] Wherein, the inoculation amount of the Erzurum thermophilic bacillus or microbial agent is 5-10 (v / v)%.

[0026] The gradient culture temperature is 37°C-48°C-52°C-55°C, and each gradient culture is 1 to 3 days.

[0027] Beneficial Effects: The present invention screened, isolated, and identified a strain of Thermophilic Bacillus erzurumensis from high-temperature Daqu (CCTCC No. M 2025368). This strain can utilize bran and wheat to produce 2,4,5-trimethyloxazole and can be used in fermentation cultures such as bran koji, medium- and high-temperature Daqu, and high-temperature Daqu. It can also metabolize and produce various other flavor compounds, including isovaleric acid, 3-hydroxy-2-butanone, and 2,3,5,6-tetramethylpyrazine, providing an excellent functional strain for enhancing fermented food processing and improving quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the phylogenetic tree of the strain Thermophilic Bacillus erzurum LZLJ2-12 from Example 2;

[0029] Figure 2 This is a colony morphology diagram of the strain Thermophilic Bacillus Erzurum LZLJ2-12 from Example 1;

[0030] Figure 3 This is a GC-MS molecular fragmentation mass spectrum of 2,4,5-trimethyloxazole produced by the strain Thermobacillus Erzurum LZLJ2-12 in Example 1;

[0031] Figure 4 This is the GC-MS molecular fragmentation mass spectrum of the 2,4,5-trimethyloxazole standard in Example 1;

[0032] Figure 5 This is the GC-MS graph of volatile substances in the wheat solid-state fermentation sample in Example 3.

[0033] Strain preservation instructions:

[0034] The novel strain of Thermophilic Bacillus erzurumensis provided herein was deposited on March 4, 2025, with the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072. The deposit number is CCTCC NO: M 2025368, and the strain is named Thermophilic Bacillus erzurumensis LZLJ2-12. DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0036] In one embodiment of the present invention, a strain of Thermophilic Bacillus erzurumensis (Calidifontibacillus erzurumensis) was screened, isolated, and identified from high-temperature Daqu. The strain has a CCTCC NO: M 2025368 and was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University Collection Center, 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. The strain was named Thermophilic Bacillus erzurumensis LZLJ2-12.

[0037] Molecular biological identification showed that the 16S rDNA sequence of the Erzurum thermophilic Bacillus was as shown in SEQ ID NO: 1.

[0038] SEQ ID NO: 1: 16S rDNA sequence of Thermobacillus erzurum

[0039]

[0040] Among them, the biological characteristics of the above-mentioned Erzurum thermophilic Bacillus are: on the DSMZ 1 solid culture medium, the colonies are round, neat, wrinkled, milky white, and opaque.

[0041] In some embodiments of the present invention, a microbial agent is provided, which contains the fermentation liquid, seed liquid or bacterial cells of the above-mentioned Thermophilic Bacillus Erzurum.

[0042] In some embodiments of the present invention, the above-mentioned Erzurum Thermophilic Bacillus or microbial agent is activated and inoculated into a wheat solid culture medium, and cultured at a gradient temperature to produce a variety of flavor substances; the flavor substances include: at least one of 2,4,5-trimethyloxazole, 2-ethylhexanol, phenylethyl alcohol, gamma-nonalactone, isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, butyric acid, nonanal, benzaldehyde, α-ethylidene-phenylacetaldehyde, acetophenone, 2-nonanone, 3-hydroxy-2-butanone, 2-undecanone, phenol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine or 2-acetyl-3-5-dimethylpyrazine.

[0043] In some preferred embodiments of the present invention, the inoculation amount of the Erzurum Thermophilic Bacillus or microbial agent is 5-10 (v / v)%.

[0044] In some preferred embodiments of the present invention, the gradient culture temperature is 37° C.-48° C.-52° C.-55° C., and each gradient culture is 1 to 3 days.

[0045] Since the Erzurum thermophilic bacillus or microbial agent of the present invention can produce the above-mentioned multiple flavor substances, it can be used in the brewing of white wine, malt wine, food fermentation, and the preparation of bran koji or daqu.

[0046] More specifically, the liquor includes at least one of strong-flavor, sauce-flavor, light-flavor or mixed-flavor types.

[0047] More specifically, the food comprises at least one of soy sauce, fermented black beans, fermented bean curd, kimchi or balsamic vinegar.

[0048] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0049] The culture medium formula involved in the embodiment is:

[0050] DSMZ 1 solid medium: 5.0 g of peptone, 3.0 g of meat extract powder, 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.

[0051] Fermentation medium: 200 g bran, add 800 mL of water, cook at 100°C for 30 min, filter through 3 layers of gauze, add glucose to a final concentration of 5 g / L, adjust the pH to 6.2, and sterilize at 121°C for 30 min.

[0052] TSB liquid medium: 17.0 g of trypticase, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolysate, 2.5 g of dipotassium hydrogen phosphate, and 2.5 g of glucose. Add water to 1000 mL, adjust the pH to 7.2, and sterilize by high-pressure steam at 121°C for 15 min.

[0053] Wheat solid culture medium: Take the crushed wheat, add water at a material-water ratio of 1:0.3 and mix evenly, moisten for 1 hour, and divide into 100mL Erlenmeyer flasks, with about 40g of material in each bottle, sterilize at 121℃ for 30min, add 16mL of sterile water to each bottle after cooling, and stir evenly.

[0054] The specific method for determining volatile products using headspace solid phase microextraction / gas chromatography-mass spectrometry (HS-SPME / GC-MS) is as follows:

[0055] The sample was placed in a headspace vial, and 2 g of NaCl, 5 mL of pure water, and 5 μL of internal standard (0.264 mg / mL 2-octanol) were added. The prepared sample was kept equilibrated at 55°C for 5 minutes, and then extracted with a 50 / 30 μm DVB / CAR / PDMS extraction head at 55°C for 45 minutes. After the extraction, it was desorbed at the GC injection port at 250°C for 5 minutes.

[0056] GC-MS detection chromatographic conditions

[0057] Gas chromatography conditions: HP-INNOWAX column (60 m × 0.25 mm × 0.25 μm); temperature program: starting temperature 40°C, hold for 1 min, increase to 75°C at 2.5°C / min, increase to 162°C at 3°C / min, increase to 230°C at 6°C / min, hold for 5 min, carrier gas is high-purity helium (1.0 mL / min); inlet temperature 250°C, no split.

[0058] 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.

[0059] Using 0.264 mg / mL 2-octanol as the internal standard, the compound search results were matched with the NIST standard spectral library, and the target compound was confirmed with a similarity of more than 80%.

[0060] Example 1: Screening of strains

[0061] Weigh 10g of high-temperature Daqu and add 90mL of sterile water with glass balls into a 250mL conical flask. Shake on a shaker at room temperature and 150r / min for 30min to prepare a sample bacterial suspension. Pipette 1mL of Daqu suspension supernatant and 9mL of sterile water (or saline) into a 15mL centrifuge tube to prepare 10 -2 The dilution was diluted in this way to prepare 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilution; 100 μL of each dilution was spread on DSMZ 1 solid culture medium and cultured in an incubator at 45°C for 1 to 3 days; then, a single colony with vigorous growth was picked and streaked multiple times until all single colonies were purified. A total of 20 strains were purified, and the 20 purified strains were inoculated into the fermentation medium respectively. The culture temperature was 45°C and the shaker speed was 120 rpm. After culturing for 2 to 3 days, 0.2 mL of the bacterial solution was taken and the volatile metabolites in the sample were detected by HS-SPME / GC-MS method. The results are shown in Table 1. A strain with high 2,4,5-trimethyloxazole production was screened and obtained. The strain number was 12. The 2,4,5-trimethyloxazole content in its bran extract fermentation sample was 4.728 μg / mL. The GC-MS molecular fragment mass spectrum of the 2,4,5-trimethyloxazole produced was shown in Figure 1. Figure 3 As shown, the colonies are round, neat, wrinkled, milky white, and opaque. The strain was stored at -80°C with 30% glycerol for further analysis.

[0062] Table 1 2,4,5-trimethyloxazole content in strain fermentation samples (μg / mL)

[0063] strain number 2,4,5-Trimethyloxazole content strain number 2,4,5-Trimethyloxazole content 1 -- 11 1.016 2 0.031 12 4.728 3 0.042 13 0.616 4 0.036 14 -- 5 0.752 15 -- 6 0.290 16 0.234 7 -- 17 0.282 8 1.756 18 0.058 9 -- 19 -- 10 0.166 20 --

[0064] Note: “--” not detected

[0065] Example 2: Molecular identification of strains

[0066] After amplification of the target strain numbered 12, fresh bacterial culture was collected during the logarithmic growth phase and centrifuged. Genomic DNA was extracted using the Ezup Column-Based Bacterial Genomic DNA Extraction Kit from Shanghai Sangon Biotechnology Co., Ltd. The DNA sequence encoding the 16S rRNA 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] 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 analyzed by 1.0% agarose gel electrophoresis at approximately 11 V / cm for 20 min. PCR products were purified according to the instructions of the Shanghai Sangon Biotechnology Co., Ltd. mini-gel recovery PCR product purification kit. Sequencing was performed by Shanghai Sangon Biotechnology Co., Ltd.

[0074] The DNA sequence encoding 16S rRNA was obtained by sequencing, and the DNA sequence encoding 16S rRNA of the strain with high similarity was downloaded from the GemBank nucleic acid sequence database to construct a phylogenetic tree. Figure 1 As shown, combined with colony morphology, such as Figure 2 As shown, on DSMZ 1 solid medium, the colonies are round, neat, wrinkled, milky white, and opaque. The strain was identified as Thermophilic Bacillus erzurumensis (Calidifontibacillus erzurumensis) and named Thermophilic Bacillus erzurumensis LZLJ2-12. It was deposited with the China Center for Type Culture Collection (CCTCC) on March 4, 2025, at the Collection Center of Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei 430072, China. The deposit number is CCTCC NO: M 2025368.

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

[0076] A glycerol stock of Thermobacillus erzurum LZLJ2-12 was inoculated with 1 mL of TSB liquid culture medium at 10% of its original volume and cultured at 37°C with a shaker at 120 rpm for 1 day to activate the strain. A 10% inoculum of the bacterial suspension was transferred to 5 mL of TSB liquid culture medium and cultured at 37°C with a shaker at 120 rpm for 1 day to prepare a seed solution. A 5% inoculum of the seed solution was then inoculated into wheat solid culture medium and cultured at a gradient temperature from 37°C to 48°C, then to 52°C, and finally to 55°C for 2 days. A blank control group was also established under the same culture conditions. After the experiment, volatile compounds in the samples were analyzed using HS-SPME / GC-MS. The content of volatile compounds in the culture medium without the added bacteria was calculated using the blank control.

[0077] By the above method, the GC-MS detection spectrum is as follows Figure 5 The analysis results of flavor metabolites are shown in Table 2. The content of 2,4,5-trimethyloxazole produced by the fermentation of strain Erzurum Thermobacillus LZLJ2-12 was 25.086 μg / g, accounting for 6.07% of the total content. In addition, Erzurum Thermophilic Bacillus LZLJ2-12 can metabolize and produce a variety of other volatile flavor substances, including 2-ethylhexanol and phenylethanol as alcohols; gamma-nonanolide as lactones; isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, and butyric acid as acids; nonanal, benzaldehyde, and α-ethylidene-phenylacetaldehyde as aldehydes; acetophenone, 2-nonanone, 3-hydroxy-2-butanone, and 2-undecanone as ketones; phenol as phenols; and 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, and 2-acetyl-3-5-dimethylpyrazine as pyrazines.

[0078] Table 2 Volatile substance content of solid-state fermentation samples of strain LZLJ2-12 (μg / g)

[0079]

[0080]

Claims

1. A new strain of Calidifontibacillus erzurumensis, characterized by: The deposit number is CCTCC NO: M 2025368.

2. A microbial agent, characterized in that: A fermentation liquid, seed liquid or bacterial cell containing the Erzurum thermophilic Bacillus according to claim 1.

3. Use of the Erzurum thermophilic bacillus according to claim 1 or the microbial agent according to claim 2 in the brewing of liquor, malt liquor, food fermentation, and the preparation of bran koji or daqu.

4. The use according to claim 3, characterized in that: The liquor includes at least one of strong-flavor, sauce-flavor, light-flavor or mixed-flavor types; the food includes at least one of soy sauce, fermented black beans, fermented bean curd, pickles or aromatic vinegar.

5. Use of the Erzurum Thermobacillus according to claim 1 or the microbial agent according to claim 2 for producing flavor substances, characterized in that: The flavoring substances include at least one of the following: 2,4,5-trimethyloxazole, 2-ethylhexanol, phenylethyl alcohol, gamma-nonalactone, isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, butyric acid, nonanal, benzaldehyde, α-ethylidene-phenylacetaldehyde, acetophenone, 2-nonanone, 3-hydroxy-2-butanone, 2-undecanone, phenol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine or 2-acetyl-3-5-dimethylpyrazine.

6. The use according to claim 5, characterized in that: The flavor substance is obtained by fermenting Erzurum thermophilic bacillus or microbial inoculant with wheat as raw material.

7. The use according to claim 6, characterized in that: Meet at least one of the following: The inoculation amount of the Erzurum thermophilic bacillus or microbial agent is 5-10%; The fermentation temperature is 37-55°C; The fermentation time is 1 to 12 days.

8. A method for producing flavor substances, characterized in that: The following steps are involved: After the Erzurum thermophilic bacillus according to claim 1 or the microbial agent according to claim 2 is activated, it is inoculated into a wheat solid culture medium and cultured at a gradient temperature to produce a variety of flavor substances; the flavor substances include: at least one of 2,4,5-trimethyloxazole, 2-ethylhexanol, phenylethyl alcohol, gamma-nonalactone, isobutyric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, octanoic acid, butyric acid, nonanal, benzaldehyde, α-ethylidene-phenylacetaldehyde, acetophenone, 2-nonanone, 3-hydroxy-2-butanone, 2-undecanone, phenol, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine or 2-acetyl-3-5-dimethylpyrazine.

9. The method for producing flavor substances according to claim 8, wherein: The inoculation amount of the Erzurum thermophilic bacillus or microbial agent is 5-10%.

10. The method for producing flavor substances according to claim 8, wherein: The gradient culture temperature is 37° C.-48° C.-52° C.-55° C., and each gradient culture is 1 to 3 days.

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