Bacillus subtilis for producing tetramethylpyrazine and application of bacillus subtilis in white spirit

Abstract

The invention discloses bacillus subtilis for producing tetramethylpyrazine and application of the bacillus subtilis in baijiu, and relates to the field of baijiu brewing and food safety. The preservation number of the bacillus subtilis is CCTCC NO: M20241287, and the bacillus subtilis can be applied to preparation of tetramethylpyrazine and / or fermented products, such as Baijiu, and can remarkably improve the content of tetramethylpyrazine and the content of esters in the Baijiu and reduce the content of methyl mercaptan, so that the quality of the Baijiu is improved, the flavor of the Baijiu is optimized, and the quality of the Baijiu is improved. And a new way is provided for production of tetramethylpyrazine and preparation of high-quality white spirit.

Description

Technical Field

[0001] The invention relates to the field of liquor brewing and food safety, and in particular to a tetramethylpyrazine-producing Bacillus subtilis and application thereof in liquor. Background Art

[0002] Traditional Chinese liquor has a long history of thousands of years. Luzhou-flavor liquor is one of the twelve types of liquor and is widely loved by Chinese people for its unique style. Bacillus is commonly found in Chinese liquor. Related studies have shown that Bacillus interacts with many bacteria, thereby changing the metabolism of individual organisms and ultimately changing the quality of liquor. Many studies have shown that Bacillus plays an important role in liquor, and studying its effect on flavor substances in liquor is of great significance to liquor.

[0003] Tetramethylpyrazine (TTMP), also known as ligustrazine, is a pyrazine substance that exists in cocoa beans, coffee, peanuts and other nuts, soy products and meat. It has the flavor of toast, barbecue and nuts and is one of the important aroma components in liquor. TTMP is produced to varying degrees during the koji making, koji piling and fermentation process of liquor, and penetrates into the liquor through the distillation process. Yu et al. studied the retronasal aroma perception and oral aroma release of liquor, and tetramethylpyrazine was related to the persistence of the retronasal aroma of liquor. Many studies have confirmed that TTMP has anti-inflammatory, liver-protecting and blood sugar-lowering effects, and is also known as a health factor in liquor.

[0004] The current manufacturing technologies of TTMP mainly include three methods: plant extraction, chemical synthesis and biological fermentation. Although the chemical synthesis method has increased the output compared to the direct extraction method, it still faces problems such as increased chemical pollution, rising production costs and difficult to manage the reaction process. At present, the direct extraction of TTMP from plant tissues faces a series of problems, including low TTMP content, limited available ingredients and difficulty in accurately controlling the quality of raw materials. Microbial fermentation is considered to be an environmentally friendly method in the production of TTMP. It has attracted widespread attention for its natural, environmentally friendly, low-pollution and mild reaction characteristics, and has gradually become the research focus in recent years. At present, the production of TTMP using microbial technology still faces a series of problems such as low output, high cost, immature production methods and difficulty in product extraction.

[0005] With the rapid development of the liquor industry, higher requirements are placed on the quality of liquor products. It is forbidden to add non-fermentable substances in the solid-state fermentation process, and it is not feasible to directly add exogenous TTMP. As for commercially available liquor, a certain concentration of TTMP can be detected in Maotai-flavor liquor and sesame-flavor liquor, while it is relatively low in Luzhou-flavor liquor. As one of the main aroma types, Luzhou-flavor liquor should be given more health factors while improving its flavor. Many scholars have studied the source, content and high-yield tetramethylpyrazine strains in Maotai-flavor liquor and sesame-flavor liquor. However, there are limited studies on the high-yield tetramethylpyrazine strains from Luzhou-flavor Daqu and simulated fermentation to evaluate their effects on the flavor of Luzhou-flavor liquor.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a tetramethylpyrazine-producing Bacillus subtilis and application thereof in liquor.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides a Bacillus subtilis, whose taxonomic name is Bacillus subtilis and whose deposit number is CCTCC NO: M20241287.

[0010] In a second aspect, an embodiment of the present invention provides a bacterial agent containing the Bacillus subtilis described in the above embodiment.

[0011] In a third aspect, an embodiment of the present invention provides a method for preparing the bacterial agent as described in the above embodiment, comprising: inoculating the Bacillus subtilis described in the above embodiment into a culture medium for culturing.

[0012] In a fourth aspect, an embodiment of the present invention provides use of the Bacillus subtilis described in the preceding embodiment, or the bacterial agent described in the preceding embodiment, or the bacterial agent prepared by the preparation method described in the preceding embodiment in the preparation of tetramethylpyrazine.

[0013] In a fifth aspect, an embodiment of the present invention provides a method for preparing tetramethylpyrazine, comprising: inoculating the Bacillus subtilis described in the preceding embodiment or the bacterial agent described in the preceding embodiment or the bacterial agent prepared by the preparation method described in the preceding embodiment into a fermentation medium for fermentation.

[0014] In a sixth aspect, embodiments of the present invention provide use of the Bacillus subtilis described in the preceding embodiments, or the bacterial agent described in the preceding embodiments, or the bacterial agent prepared by the preparation method described in the preceding embodiments in the preparation of fermentation products.

[0015] In a seventh aspect, an embodiment of the present invention provides a method for preparing a fermented product, comprising: adding the Bacillus subtilis described in the aforementioned embodiment or the bacterial agent described in the aforementioned embodiment or the bacterial agent prepared by the preparation method described in the aforementioned embodiment during the fermentation process.

[0016] The present invention has the following beneficial effects:

[0017] The embodiment of the present invention provides a new Bacillus subtilis YR-5, which can be used to prepare tetramethylpyrazine and / or fermented products, such as liquor. It can significantly increase the content of tetramethylpyrazine and ester substances in the liquor and reduce the content of methyl mercaptan, thereby improving the quality of the liquor and optimizing the flavor of the liquor, and provides a new way for the production of tetramethylpyrazine and the preparation of high-quality liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The results of the determination of the production of tetramethylpyrazine of 12 Bacillus strains are shown;

[0020] Figure 2 Heat map of the abundance of volatile flavor compounds in mash (A) and liquor (B) after inoculation with Bacillus; the content of tetramethylpyrazine in mash (C) and liquor (D). DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] On the one hand, an embodiment of the present invention provides a Bacillus subtilis, whose taxonomic name is Bacillus subtilis, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20241287, a deposit date of June 19, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0023] The Bacillus subtilis provided in the embodiment of the present invention has the ability to produce high TTMP, and can be applied to the preparation of fermented products such as liquor, which can significantly increase the content of TTMP and ester substances in the liquor and reduce the content of methyl mercaptan, thereby improving the quality of the liquor and optimizing the flavor of the liquor.

[0024] On the other hand, an embodiment of the present invention provides a bacterial agent, which contains the Bacillus subtilis described in the above embodiment.

[0025] On the other hand, an embodiment of the present invention provides a method for preparing the bacterial agent as described in the above embodiment, comprising: inoculating the Bacillus subtilis described in the above embodiment into a culture medium for cultivation.

[0026] In some embodiments, the culture conditions include: 35-39°C, 36-48h. The temperature can be any one of 35, 36, 37, 37.5, 38, 38.5, 39°C or a range between any two thereof. The time can be any one of 36, 38, 40, 42, 44, 46, 48h or a range between any two thereof. In some embodiments, the culture medium includes a spore-forming medium.

[0027] In some embodiments, the nitrogen source of the spore production medium includes corn flour and ammonium sulfate. Using corn flour and ammonium sulfate as a dual nitrogen source can significantly improve the spore conversion rate, which is better than a single nitrogen source.

[0028] In some embodiments, the spore production medium includes the following components by weight: 90-100 parts of bran powder, 4-6 parts of glucose, 2-3 parts of corn flour, 1.5-2 parts of ammonium sulfate, 1.5-2 parts of dipotassium hydrogen phosphate, 1-5 parts of sodium chloride, 0.3-0.5 parts of magnesium sulfate, 0.008-0.016 parts of zinc acetate, 0.2-0.3 parts of manganese sulfate, and 800-1200 parts of water. Specifically, the weight of bran powder can be any one of 90, 92, 94, 96, 98, and 100 parts, or a range between any two. The weight of glucose can be any one of 4, 5, and 6 parts, or a range between any two. The weight of corn flour can be any one of 2, 2.5, and 3 parts, or a range between any two. The weight of ammonium sulfate can be any one of 1.5, 1.6, 1.7, 1.8, 1.9, and 2 parts, or a range between any two. The weight portion of dipotassium hydrogen phosphate can be any one of 1.5, 1.6, 1.7, 1.8, 1.9, 2 parts or a range between any two. The weight portion of sodium chloride can be any one of 1, 2, 3, 4, 5 parts or a range between any two. The weight portion of magnesium sulfate can be any one of 0.3, 0.4, 0.5 parts or a range between any two. The weight portion of zinc acetate can be any one of 0.008, 0.009, 0.01, 0.012, 0.014, 0.016 parts or a range between any two. The weight portion of manganese sulfate can be any one of 0.2, 0.25, 0.3 parts or a range between any two. The weight portion of water can be any one of 800, 900, 1000, 1100, 1200 parts or a range between any two. Water can be distilled water or sterile water. The pH of the culture medium may be 6 to 8, specifically any one of 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, or a range between any two of them.

[0029] In some embodiments, before inoculating Bacillus subtilis into the spore-forming medium, the preparation method further comprises: first inoculating Bacillus subtilis into the seed medium to obtain a seed solution. The seed solution is used to inoculate the spore-forming medium.

[0030] In some embodiments, the inoculation amount of Bacillus subtilis in the seed culture medium is 1% to 10% (volume percentage), specifically any one of 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10% or a range between any two of them.

[0031] In some embodiments, the inoculation amount of the seed solution in the spore production medium is 8% to 12% (volume percentage), specifically any one of 8%, 9%, 10%, 11%, 12% or a range between any two of them.

[0032] In some embodiments, the number of viable bacteria in the bacterial agent is 10 8 ~10 9 cfu / mL.

[0033] On the other hand, an embodiment of the present invention provides use of the Bacillus subtilis described in the preceding embodiment, or the bacterial agent described in the preceding embodiment, or the bacterial agent prepared by the preparation method described in any preceding embodiment in the preparation of tetramethylpyrazine.

[0034] On the other hand, an embodiment of the present invention provides a method for preparing tetramethylpyrazine, which comprises: inoculating the Bacillus subtilis described in the above embodiment or the bacterial agent described in the above embodiment or the bacterial agent prepared by the preparation method described in any of the above embodiments into a fermentation medium for fermentation.

[0035] In some embodiments, the fermentation comprises: inoculating the Bacillus subtilis or the bacterial agent into a first fermentation medium for a first stage fermentation to obtain a first fermentation liquid; inoculating the first fermentation liquid into a second fermentation medium for a second stage fermentation to obtain a second fermentation liquid;

[0036] In some embodiments, the conditions of the first stage fermentation include: 30-45°C, 15-18h. The temperature can be any one of 30, 32, 34, 36, 37, 38, 40, 42, 44, 45°C or a range between any two thereof. The time can be any one of 15, 16, 17, 18h or a range between any two thereof.

[0037] In some embodiments, the first fermentation medium comprises broth.

[0038] In some embodiments, the components of the first fermentation medium and the final concentrations of the components thereof include: 1-10 g / L yeast extract, 5-15 g / L peptone and 1-10 g / L NaCl. Specifically, the final concentration of yeast extract can be any one of 1, 2, 4, 5, 6, 8, 10 g / L or a range between any two thereof. The final concentration of peptone can be any one of 5, 6, 8, 10, 12, 14, 15 g / L or a range between any two thereof. The final concentration of NaCl can be any one of 1, 2, 4, 5, 6, 8, 10 g / L or a range between any two thereof.

[0039] In some embodiments, the second fermentation broth contains glucose.

[0040] In some embodiments, the components of the second fermentation medium and the final concentrations of their components include: 5-15 g / L yeast extract, 10-30 g / L peptone, 1-5 g / L K2HPO4, 1-10 g / L NaCl, 150-200 g / L glucose and 20-40 g / L (NH4)2HPO4. Specifically, the final concentration of yeast extract can be any one of 5, 6, 8, 10, 12, 14, 15 g / L or a range between any two. The final concentration of peptone can be any one of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 g / L or a range between any two. The final concentration of K2HPO4 can be any one of 1, 2, 3, 4, 5 g / L or a range between any two. The final concentration of NaCl can be any one of 1, 2, 4, 6, 8, 10 g / L or a range between any two. The final concentration of glucose can be any one of 150, 160, 170, 180, 190, 200 g / L or a range between any two thereof. The final concentration of (NH4)2HPO4 can be any one of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 g / L or a range between any two thereof.

[0041] In some embodiments, the conditions of the second stage fermentation include: 30-45°C, 20-30h; the temperature can be any one of 30, 32, 34, 36, 38, 40, 42, 44, 45°C or a range between any two thereof. The time can be any one of 20, 22, 24, 26, 28, 30h or a range between any two thereof.

[0042] In some embodiments, the preparation method further comprises extracting tetramethylpyrazine from the second fermentation broth.

[0043] On the other hand, an embodiment of the present invention provides use of the Bacillus subtilis described in the preceding embodiment, or the bacterial agent described in the preceding embodiment, or the bacterial agent prepared by the preparation method described in any preceding embodiment in the preparation of a fermentation product.

[0044] In some embodiments, the fermented product includes liquor.

[0045] In some embodiments, the liquor comprises Luzhou-flavor liquor.

[0046] In addition, an embodiment of the present invention further provides a method for preparing a fermented product, which comprises: adding the Bacillus subtilis described in the above embodiment or the bacterial agent described in the above embodiment or the bacterial agent prepared by the preparation method described in any of the above embodiments during the fermentation process.

[0047] In some embodiments, the fermented product includes liquor;

[0048] In some embodiments, the liquor includes Luzhou-flavor liquor;

[0049] In some embodiments, the fermentation process of the liquor includes adding the Bacillus subtilis or the bacterial agent to the fermentation material for fermentation.

[0050] In some embodiments, during the fermentation process, the inoculation amount of the Bacillus subtilis or the bacterial agent added to the fermentation material can be 1% to 10% (mass volume percentage), specifically any one of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of them.

[0051] In some embodiments, the fermented material includes any one or more of koji and mash.

[0052] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0053] Example 1

[0054] 1. Isolation and purification of Bacillus from Luzhou-flavor Daqu

[0055] Eighteen strains of bacteria were isolated and purified from Luzhou-flavor Daqu, and identified by morphological observation and molecular biology. The 18 strains of Bacillus were identified as 6 species with a species homology higher than 99%, namely Bacillus amyloliquefaciens (5 strains), Bacillus velezensis (6 strains), Bacillus subtilis (4 strains), Bacillus vallismortis (1 strain), Bacillus mojavensis (1 strain) and Bacillus tequilensis (1 strain).

[0056] The bacteria were cultured in broth medium (5 g yeast extract, 10 g peptone, 5 g NaCl, and 1000 mL distilled water).

[0057] 2. Cultivation of strains

[0058] The purified strain was inoculated into nutrient broth medium (5 g yeast extract, 10 g peptone, 5 g NaCl, 1000 mL distilled water), cultured at 37°C and 180 r / min on a shaking table for 15-18 h, and then inoculated into 100 mL fermentation medium (10 g yeast extract, 20 g peptone, 3 g K2HPO4, 5 g NaCl, 100 g glucose, 30 g (NH4)2HPO4, 1000 mL distilled water) at an inoculum rate of 3%, cultured at 37°C and shaken for 24 h to obtain a fermentation broth.

[0059] 3. Determination of the ability to produce tetramethylpyrazine

[0060] Take 20 mL of fermentation liquid in a separatory funnel, extract with 5 mL of n-hexane, shake gently to obtain the extract, and determine the TTMP content using HS-SPME-GC-MS. Figure 1 shown.

[0061] The results show that among the 18 strains of Bacillus screened, 12 strains have the ability to produce TTMP, indicating that there are abundant Bacillus groups producing pyrazine compounds in Daqu. The names and taxonomic names of the 12 strains are shown in Table 1. Among them, Bacillus subtilis YR-5 has the best TTMP production capacity, reaching 761.32 mg / L, followed by Bacillus velezensis YR-3 and Bacillus velezensis YR-12, with production reaching 553.25 mg / L and 341.19 mg / L respectively.

[0062] Table 1 Names and taxonomic names of the 12 bacterial strains

[0063]

[0064]

[0065] The 16S rRNA sequence of YR-5 is shown in SEQ ID NO:1.

[0066] Liu et al. (Liu, Y., Li, M., Hong, X., Li, H., Huang, R., Han, S., Hou, J., & Pan, C. (2023). Screening and identification of high yield tetramethylpyrazine strains in Nongxiangxing liquor Daqu and study on the mechanism of tetramethylpyrazine production. Journal of the Science of Food and Agriculture, 103(14), 6849-6860. https: / / doi.org / 10.1002 / jsfa.12773) screened out a strain with high yield of tetramethylpyrazine (TTMP) from Luzhou-flavor liquor Daqu, with a yield of 29.83 μg / mL. The TTMP produced by Bacillus subtilis YR-5 was 25.5 times higher than that produced by the strain screened by Liu et al. It can be seen that the Bacillus subtilis YR-5 screened in the present invention can produce TTMP761.32 mg / L, indicating that it has great potential for producing tetramethylpyrazine, and provides a good strain for the optimization and practical application of high TTMP yield strains of Daqu Luzhou-flavor liquor.

[0067] 4. Seed solution preparation

[0068] The purified strain YR-5 was inoculated into 45 mL of seed culture medium (10 g peptone, 5 g yeast extract, 5 g NaCl, 1000 mL distilled water) and cultured at 200 r / min and 37°C for 12 h to prepare the corresponding seed solution. The seed solution was then centrifuged, the supernatant was discarded, and the bacterial precipitate was washed twice with sterile saline. The OD value of the bacterial solution was calculated. 600 The value was diluted to between 0.2-0.8, and the absorbance was measured by UV spectrophotometer. Finally, the measured absorbance was multiplied by the dilution factor to ensure the accuracy of the data.

[0069] YR-5 (Bacillus subtilis YR-5). The strain was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20241287, the deposit date was 2024.06.19, and the deposit location was Wuhan University.

[0070] The culture medium was nutrient broth, and the culture time was 24 hours at a constant temperature of 37℃. The bacteria were rod-shaped under microscopic examination, with a diameter of about 0.7-0.8μm*(1.5-3)μm. The colony morphology showed the typical morphology of Bacillus, which was white and opaque, irregular in shape, with a central bulge, a rough and wrinkled surface, irregular edges, and moist and sticky. After Gram staining, it was found to be Gram-positive bacteria.

[0071] Physiological and biochemical characteristics: The results are shown in Table 2.

[0072] Table 2 Physiological and biochemical characteristics of YR-5

[0073] Characteristics result Characteristics result Hydrolyzed starch + D-Fructose - lactose - sucrose - VP Experiment + Indole experiment - 10% NaCl + 14% NaCl - Citrate Utilization - Anaerobic growth -

[0074] 5. Effect of simulated fermentation on liquor flavor

[0075] Strains YR-12, YR-5 and YR-3 were obtained, and the bacterial concentration was adjusted (the value measured by ultraviolet spectrophotometer was 0.61 at a wavelength of 600nm), and the strains were inoculated into solid-state simulated fermentation medium with equal concentration and volume (5% inoculation amount) for solid-state simulated fermentation. The blank control was made with the strains not inoculated, and three groups were made in parallel for each. After the jar was sealed with liquid, the fermentation temperature was controlled to be 30°C, and the fermentation time was 35d. The lees after 35 days of fermentation in the jar were placed in a distiller for heating and distillation, and 30mL of the head of the wine was removed. The distillation was stopped when the alcohol content in the bottle was 52%vol, and the flavor compounds of the obtained wine sample were detected. Among them, the composition of the simulated solid-state fermentation medium is: 2.7kg of broken sorghum, 200g of rice husk, and 580g of Daqu powder.

[0076] The volatile flavor substances were evaluated and the results were as follows Figure 2 shown.

[0077] A total of 75 flavor compounds were detected in the mash. After adding YR-5, TTMP increased from 344.16 mg / L in the blank group to 902.84 mg / L, an increase of 162.37%.

[0078] A total of 122 flavor substances were detected in the liquor. After adding YR-5, the tetramethylpyrazine in the liquor increased from 192.65 mg / L in the blank group to 920.47 mg / L, an increase of 3.78 times.

[0079] Esters can produce the main flavor characteristics of liquor, including flavor, aroma, grade and style. After inoculation with YR-5, the content of ester substances such as ethyl 3-methylbutyrate, ethyl decanoate and ethyl palmitate with fruity and honey aroma increased, which has potential application value in improving the quality of liquor.

[0080] Example 2

[0081] Verify whether the ability of Bacillus subtilis YR-5 to produce tetramethylpyrazine in actual production can reach the level shown in simulated fermentation.

[0082] 1. Preparation of Bacillus subtilis liquid

[0083] The seed liquid of Bacillus subtilis YR-5 was transferred to a container filled with spore-forming culture medium at an inoculation rate of 10% by mass volume to obtain a bacterial liquid.

[0084] The spore production medium includes the following raw materials in parts by weight: 100 parts of bran powder, 5 parts of glucose, 3 parts of corn flour, 2 parts of ammonium sulfate, 2 parts of dipotassium hydrogen phosphate, 3 parts of sodium chloride, 0.5 parts of magnesium sulfate, 0.016 parts of zinc acetate, 0.3 parts of manganese sulfate, 1000 parts of distilled water, H (humidity) natural, pH 7.

[0085] The culture conditions were 37°C, 150 r / min for 48 h, and the number of viable bacteria was detected at 10 9 CFU / mL.

[0086] 3. Entering the cellar and sealing the cellar

[0087] Before fermentation, add 5% bacterial solution (10 8 -10 9 cfu / mL), mixed evenly with the koji medicine, and fermented together in the cellar.

[0088] The volatile flavor compounds of the liquor obtained after fermentation were analyzed, and the results are shown in Table 3.

[0089] Table 3 Flavor of liquor produced and fermented in practice

[0090]

[0091]

[0092] Sulfur organic compounds in liquor usually produce unpleasant odors, such as methyl mercaptan, which tastes like rubber and gasoline in liquor. After adding YR-5, the methyl mercaptan content in liquor decreased by about 33%, which is beneficial to the flavor of liquor. Ethyl 3-methylbutyrate has a fruity and sweet taste. In the liquor inoculated with YR-5, ethyl 3-methylbutyrate increased from ND to 349.09±25.63μg / mL. Ethyl linoleate has the effect of lowering cholesterol and blood lipid levels. In the liquor inoculated with YR-5, ethyl linoleate increased significantly.

[0093] After inoculation of YR-5, the content of ester substances such as fruity and honey aroma increased. This shows that Bacillus YR-5 can increase the content of ester substances in the fermentation system and enhance the aroma of liquor, and has potential application value in improving the quality of liquor.

[0094] The tetramethylpyrazine content of 4.92 mg / L produced in this example is 1.49 times that of the uninoculated strain. Wang Guoming et al. analyzed the pyrazine substances in "Shanzhuang Laojiu" and found that the tetramethylpyrazine content was only 100.49 μg / L. Zhang Wenqing et al. showed that Xuanjiu sesame-flavored liquor contains a high level of pyrazine health function factors, among which the content of tetramethylpyrazine reaches 1.20 mg / L. In addition, Yan et al. selected 27 commercial liquor samples of 4 aroma types for testing. The concentration of tetramethylpyrazine in sauce-flavor liquor (474-1861 μg / L) was significantly higher than that in sesame-flavor liquor (13-157 μg / L), strong-flavor liquor (18-95 μg / L) and light-flavor liquor (76-201 μg / L), and its content was lower than the tetramethylpyrazine content produced by actual fermentation of YR-5, indicating that the Bacillus subtilis YR-5 screened in the present invention has great potential for producing tetramethylpyrazine, and new strains with higher TTMP production can be bred subsequently through mutagenesis breeding, genetic engineering breeding, etc.

[0095] The sequence shown in SEQ ID NO: 1 is as follows (5'-3'):

[0096]

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis, characterized in that Its taxonomic name is Bacillus subtilis and its deposit number is CCTCC NO:M20241287.

2. A bacterial agent, characterized in that It contains the Bacillus subtilis according to claim 1.

3. The method for preparing the bacterial agent according to claim 2, characterized in that: It includes: inoculating the Bacillus subtilis according to claim 1 into a culture medium for cultivation; Optionally, the culture conditions include: 37-39° C., 36-48 h; Optionally, the culture medium comprises a spore-forming medium; Optionally, the spore-forming culture medium includes the following components in parts by weight: 90 to 100 parts of bran powder, 4 to 6 parts of glucose, 2 to 3 parts of corn flour, 1.5 to 2 parts of ammonium sulfate, 1.5 to 2 parts of dipotassium hydrogen phosphate, 1 to 5 parts of sodium chloride, 0.3 to 0.5 parts of magnesium sulfate, 0.008 to 0.016 parts of zinc acetate, 0.2 to 0.3 parts of manganese sulfate, and 800 to 1200 parts of water.

4. Use of the Bacillus subtilis according to claim 1, the bacterial agent according to claim 2, or the bacterial agent prepared by the preparation method according to claim 3 in the preparation of tetramethylpyrazine.

5. A method for preparing tetramethylpyrazine, characterized in that: It includes: The fermentation is carried out using the Bacillus subtilis described in claim 1, the bacterial agent described in claim 2, or the bacterial agent prepared by the preparation method described in claim 3.

6. The preparation method according to claim 5, characterized in that: The fermentation comprises: inoculating the Bacillus subtilis or the bacterial agent into a first fermentation medium for a first stage fermentation to obtain a first fermentation liquid; inoculating the first fermentation liquid into a second fermentation medium for a second stage fermentation to obtain a second fermentation liquid; Optionally, the conditions of the first stage fermentation include: 30-45°C, 15-18h; Optionally, the first fermentation medium comprises broth medium; Optionally, the components of the first fermentation medium and the final concentrations of the components thereof include: 1-10 g / L yeast extract, 5-15 g / L peptone and 1-10 g / L NaCl; Optionally, the second fermentation broth contains glucose; Optionally, the components of the second fermentation medium and the final concentrations of the components thereof include: 5-15 g / L yeast extract, 10-30 g / L peptone, 1-5 g / L K2HPO4, 1-10 g / L NaCl, 150-200 g / L glucose and 20-40 g / L (NH4)2HPO4; Optionally, the conditions of the second stage fermentation include: 30-45° C., 20-30 h.

7. Use of the Bacillus subtilis according to claim 1, the bacterial agent according to claim 2, or the bacterial agent prepared by the preparation method according to claim 3 in the preparation of fermentation products.

8. The use according to claim 7, characterized in that: The fermented products include liquor; Optionally, the liquor includes Luzhou-flavor liquor.

9. A method for preparing a fermented product, characterized in that: It includes: During the fermentation process, the Bacillus subtilis according to claim 1 or the bacterial agent according to claim 2 or the bacterial agent prepared by the preparation method according to claim 3 is added.

10. The preparation method according to claim 9, characterized in that: The fermented products include liquor; Optionally, the liquor includes Luzhou-flavor liquor; Optionally, the fermentation process of the liquor includes adding the Bacillus subtilis or the bacterial agent to a fermentation material for fermentation; Optionally, the fermented material includes any one or more of distiller's yeast and mash.

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