Bacillus licheniformis and its use in the production of acetoin and tetramethylpyrazine

Abstract

The present application relates to the field of microbial fermentation and bioengineering technology, and particularly relates to bacillus licheniformis and application thereof in acetoin and tetramethylpyrazine production. A bacillus licheniformis with high acetoin yield is screened from a large number of strains. The acetoin yield of the strain can reach 98 g / L after 36 h fermentation, which is much higher than that of other natural screening strains, and the liquor yield is increased by 11% and the content of tetramethylpyrazine in the liquor is increased by 57% when the strain is used for liquor brewing, which obviously improves the quality of the liquor.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and bioengineering technology, and in particular to Bacillus licheniformis and its application in the production of acetoin and tetramethylpyrazine. Background Technology

[0002] Acetoin, as an important food flavoring substance and industrial intermediate, has broad application prospects in the food, pharmaceutical, and chemical industries. It naturally exists in many fermented foods and can impart a unique creamy aroma to the products; therefore, it is often used as a flavor enhancer in the food industry. Tetramethylpyrazine (TMP, also known as ligustrazine) is an important flavor active substance and functional component in baijiu (Chinese liquor), and also possesses certain physiological activities, which can add health attributes to the product.

[0003] Microbial fermentation is an important method for producing acetoin, offering advantages such as a wide range of raw material sources, mild reaction conditions, and environmental friendliness, aligning with the trend of green production. Current research reports that *Bacillus licheniformis* can be used for acetoin fermentation, such as Chinese patent CN105969703B, which discloses a *Bacillus licheniformis* strain and its application in acetoin fermentation. However, significant limitations exist: firstly, the acetoin yield of the disclosed strain is low, only 5.6 g / L after 36 hours of fermentation, failing to meet the demands of industrial production for yield and efficiency; secondly, existing *Bacillus licheniformis*-based fermentation systems focus solely on acetoin as a single product, neglecting the synthesis and accumulation of tetramethylpyrazine, thus failing to achieve the synergistic production of these two high-value substances. This makes them particularly unsuitable for scenarios requiring simultaneous consideration of flavor and functional properties, such as baijiu brewing and complex fermented foods. Therefore, developing microbial strains and fermentation technologies that can simultaneously increase acetoin yield and achieve tetramethylpyrazine synthesis has become a crucial issue urgently requiring breakthroughs. Summary of the Invention

[0004] In view of this, the present invention provides Bacillus licheniformis and its use in the production of acetoin and tetramethylpyrazine.

[0005] To address the issues of existing strains being unable to simultaneously produce high yields of acetoin and tetramethylpyrazine, and the high production costs, this invention provides a strain of Bacillus licheniformis with accession number CCTCC NO:M 20251822.

[0006] This invention isolates single colonies from brewing yeast and, using a colorimetric reaction based on acetoin characteristics, screens a large number of strains to obtain a high-acetoin-producing strain, X-11, whose acetoin yield is significantly higher than other strains. Morphological and 16S rRNA gene sequence analysis identifies strain X-11 as a novel strain of the genus *Bacillus*, named *Bacillus licheniformis* X-11. This strain is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20251822.

[0007] The present invention also provides microbial agents, including Bacillus licheniformis with accession number CCTCC NO:M 20251822, and / or fermentation broth, culture, lysate or extract of Bacillus licheniformis with accession number CCTCC NO:M 20251822.

[0008] The formulations of the microbial agents described in this invention include aqueous solutions, emulsions, or powders.

[0009] The present invention also provides a method for preparing the aforementioned microbial agent, comprising the following steps:

[0010] S1: After inoculating and culturing Bacillus licheniformis with accession number CCTCC NO:M 20251822, seed culture was obtained;

[0011] S2: After expanding the fermentation culture of the seed liquid, the bacterial agent is obtained.

[0012] In some implementations, the culture medium for expanded fermentation, based on the mass of water, comprises water and the following components in the following mass percentages: glucose 1%–8%, magnesium sulfate heptahydrate 0.02%–0.05%, FM888 1.5%–2.5%, triammonium citrate 0.05%–0.1%, sodium acetate 0.45%–0.65%, dipotassium hydrogen phosphate trihydrate 5%–8%, ferrous sulfate heptahydrate 0.001%–0.003%, zinc sulfate heptahydrate 0.0010%–0.0014%, manganese sulfate monohydrate 0.0001%–0.0004%, with a pH of 6.5–7.0. The above-mentioned mass percentages of components are added based on the mass of water; that is, the percentages of each component represent the mass of each component relative to the mass of water. In some specific embodiments, the culture medium for the expanded fermentation culture includes the following components: 8% glucose, 0.05% magnesium sulfate heptahydrate, 2.5% FM888, 0.1% triammonium citrate, 0.65% sodium acetate, 8% dipotassium hydrogen phosphate trihydrate, 0.003% ferrous sulfate heptahydrate, 0.0014% zinc sulfate heptahydrate, 0.0004% manganese sulfate monohydrate, 100 mL water, and a pH of 6.5-7.0.

[0013] In this invention, the expanded fermentation culture is carried out under specific aeration ratio, tank pressure, stirring, and culture temperature conditions. Specifically, the aeration ratio is 1~1.5 vvm, and can be 1 vvm, 1.1 vvm, 1.2 vvm, 1.3 vvm, 1.4 vvm, or 1.5 vvm. The tank pressure is 0.01~0.05 MPa, and can be 0.01 MPa, 0.02 MPa, 0.03 MPa, 0.04 MPa, or 0.05 MPa. The stirring speed is 180~200 r / min, and can be 180 r / min, 190 r / min, or 200 r / min. The expanded fermentation culture temperature is 34~37℃, and can be 34℃, 35℃, 36℃, or 37℃.

[0014] The present invention also provides a yeast starter, the raw materials of which include: the microbial agent as described above or the microbial agent prepared by the preparation method described above, and a matrix.

[0015] In some embodiments, the substrate includes at least one selected from wheat, wheat bran, corn, rice flour, rice husks, and sorghum. In a specific embodiment of the present invention, the substrate is wheat bran, and the resulting yeast is wheat bran yeast. In some specific embodiments, the wheat bran yeast is prepared from wheat bran and the microbial agent described in the present invention. In a specific embodiment of the present invention, the mass ratio of the wheat bran to the microbial agent is 1:1.

[0016] This invention also provides a method for preparing the yeast starter, comprising mixing the microbial agent and the substrate described in this invention, drying, and stirring. The selection of the substrate is as described above.

[0017] The present invention also provides the use of Bacillus licheniformis as described above, the aforementioned inoculum, the inoculum prepared by the aforementioned method, the aforementioned yeast, or the yeast prepared by the aforementioned method in any of the following:

[0018] (1) Brewing baijiu (Chinese liquor);

[0019] (2) Fermentation to produce acetoin and / or tetramethylpyrazine.

[0020] In the above application, the liquor is a sauce-flavored Daqu liquor.

[0021] Furthermore, the Bacillus licheniformis increases the acetoin content in baijiu (Chinese liquor).

[0022] Furthermore, the Bacillus licheniformis increases the content of methylpyrazine in baijiu (Chinese liquor).

[0023] Experiments show that using Bacillus licheniformis, the inoculant or yeast made from Bacillus licheniformis as described in this invention in the brewing of baijiu can significantly increase the content of flavor substances such as ACT and tetramethylpyrazine in baijiu, effectively improving the taste and flavor of baijiu.

[0024] The present invention also provides a method for preparing sauce-flavored Daqu liquor, comprising: inoculating the Bacillus licheniformis, the microbial agent, the microbial agent prepared by the preparation method, the yeast or the yeast prepared by the preparation method of the present invention into the mash, and fermenting it in a cellar.

[0025] In some implementations, the fermented mash is the mash cooled after the 7th distillation. The temperature of the cooled mash is preferably 32-34°C, specifically 32°C, 33°C, or 34°C.

[0026] This invention provides *Bacillus licheniformis* X-11, with accession number CCTCC NO: M 20251822. This invention also provides inoculants, yeast starters, and their preparation methods and applications made using this strain. Compared with existing *Bacillus licheniformis* strains or other *Bacillus* species producing acetoin and tetramethylpyrazine, this invention has at least the following advantages:

[0027] 1. The high-acetoin-producing strain obtained by screening in this invention has good stability, and the highest acetoin yield can reach 98 g / L acetoin in large-scale culture.

[0028] 2. When the microbial agent of the present invention was applied to the 7th round of production of Maotai-flavor liquor, the content of tetramethylpyrazine in the liquor increased by 57% and the yield increased by 11% compared with the control group.

[0029] Biological Preservation Instructions

[0030] Bacillus licheniformis X-11 was deposited on August 12, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251822. Attached Figure Description

[0031] Figure 1 The result is a phylogenetic tree.

[0032] Figure 2 The yield of acetoin obtained at different cultivation times;

[0033] Figure 3 The content of tetramethylpyrazine in the wine sample obtained from fermentation;

[0034] Figure 4 The yield of the wine sample obtained from fermentation;

[0035] Figure 5 The content of acetoin in the wine sample obtained from fermentation. Detailed Implementation

[0036] This invention provides Bacillus licheniformis and its application in the production of acetoin and tetramethylpyrazine. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0037] The terms “comprising,” “including,” and “having” are used interchangeably in this document to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0038] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0039] This invention relates to Bacillus licheniformis X-11, isolated from the Daqu (fermentation starter) of a Maotai-flavor liquor distillery, with the preservation number CCTCC NO:M 20251822. Experiments show that this strain can significantly increase the yield of acetoin and also significantly increase the content of tetramethylpyrazine, with a short fermentation cycle, making it suitable for industrial production.

[0040] The screening and identification method of Bacillus licheniformis X-11, which produces high levels of acetoin, is as follows:

[0041] 1. Separation and purification

[0042] Daqu (a type of starter culture) was taken from a certain soy sauce-flavored liquor factory, and sterile water was added to prepare a sample suspension. The suspension was heated in an 80°C water bath for 10 min to eliminate non-spore-forming bacteria. The suspension was then transferred to an enrichment medium and cultured for 12 h. The enriched solution was then collected.

[0043] The enriched solution is gradient diluted to a gradient concentration (e.g., 10). -3 ~10 -8Take an appropriate amount of bacterial suspension and drop it onto the center of an isolation medium plate. Spread the bacterial suspension evenly on the medium using a spreader. Incubate upside down in a 37°C incubator for 1-2 days. Based on the morphological characteristics of Bacillus colonies, select colonies with inconsistent morphology and streak them onto the isolation medium plate using a sterile inoculation loop. Incubate at 37°C until single colonies grow. Then, select single colonies and inoculate them onto purification medium for purification.

[0044] Furthermore, the mass percentage of each component in the liquid enrichment culture medium is as follows: glucose 3%, peptone 1%, yeast extract 0.50%, sodium chloride 0.05%, pH 6.0.

[0045] The mass percentages of each component in the separation and purification media are as follows: glucose 3%, peptone 1%, yeast extract 0.50%, sodium chloride 0.05%, agar 2%, pH 6.0.

[0046] 2. Screening of high-acetoin-producing strains

[0047] The purified single colonies were taken and subjected to primary and secondary screening to obtain strain X-11 with high ACT production.

[0048] Initial screening: The creatine colorimetric method was used to screen for strains capable of synthesizing acetoin (ACT). ACT-producing strains can rapidly turn creatine-containing solutions red; the deeper the color, the higher the yield.

[0049] Secondary screening: The strains obtained from the initial screening were inoculated into seed culture medium and cultured on a shaker at 37℃ and 180 r / min for 12–24 h. Then, 5% of the inoculum was added to the fermentation medium and cultured on a shaker at 37℃ and 180 r / min for 72 h. After fermentation, the culture was centrifuged at 12000 r / min for 2 min, and the supernatant was collected. The OD of the fermentation broth was determined colorimetrically. 522 The nm value was used to obtain strain X-11 of this invention, which has a significantly higher ACT yield than other strains.

[0050] The seed culture medium for seed culture contains the following components in terms of mass percentage: glucose 3%, peptone 1%, yeast extract 0.50%, sodium chloride 0.05%, and pH 6.0.

[0051] The fermentation medium used for fermentation culture has the following mass percentages: glucose 8%, peptone 1%, yeast extract 1%, sodium chloride 0.05%, magnesium sulfate heptahydrate 0.03%, calcium chloride dihydrate 0.10%, potassium dihydrogen phosphate 0.03%, ammonium sulfate 0.03%, and pH 6.0.

[0052] 3. Observe the morphological characteristics of the strain.

[0053] Straw X-11, which yielded high ACT production, was used to obtain single colonies that were purified and streaked onto the culture medium. The colonies were then incubated at 37°C for 1–2 days. The surface morphology of the colonies was observed. The colonies of this invention were small, round, with a rough surface, light yellow in color, and a diameter of 2–3 mm. They were straight rods, solitary, and produced nearly centrally located elliptical spores.

[0054] 4. 16S rRNA gene sequencing

[0055] The strain was identified as Bacillus licheniformis by amplifying its 16S rRNA gene sequence using universal bacterial primers.

[0056] The method for expanding the culture of Bacillus licheniformis and the fermentation performance test are as follows:

[0057] Step 1: Strain activation: Take the Bacillus licheniformis X-11 as the strain and activate the strain;

[0058] Step 2: Preparation of primary seed culture: The activated Bacillus licheniformis X-11 obtained in Step 1 is cultured to obtain primary seed culture;

[0059] Step 3: Preparation of high-yield acetoin fermentation broth: The primary seed liquid obtained in step 2 is cultured to obtain the fermentation broth of Bacillus licheniformis X-11.

[0060] In some specific embodiments of the present invention, the activation in step 1 is as follows: the Bacillus licheniformis X-11 stored at -80°C is inoculated into an isolation and purification culture medium and cultured at 34~38°C for 12-24 hours to obtain the activated Bacillus licheniformis X-11.

[0061] In some specific embodiments of the present invention, the preparation of primary seed in step 2 is as follows: the activated Bacillus licheniformis X-11 strain is mixed with sterile water and then inoculated into a primary seed culture medium and cultured at 34~38℃ for 12~24h to obtain the primary seed liquid.

[0062] In some specific embodiments of the present invention, the preparation of fermentation broth in step 3 is as follows: the primary seed culture is transferred to the expansion culture fermentation medium at an inoculation amount of 2% to 5% by volume, and cultured at 34 to 38°C, rotation speed of 180 to 220 rpm / min, aeration rate of 0.5 to 0.8 vvm, and a pressure of 0.04 MPa to 0.06 MPa for 1 to 3 days. Samples are taken every 4 hours to detect the acetoin content (the glucose content is controlled at 20 g / L and the pH is 6.5 during the fermentation process).

[0063] The mass percentage of each component in the expanded culture fermentation medium is as follows: glucose 8%, magnesium sulfate heptahydrate 0.05%, FM888 (yeast extract powder from Angel Yeast Co., Ltd.) 2.5%, triammonium citrate 0.1%, sodium acetate 0.65%, dipotassium hydrogen phosphate trihydrate 8%, ferrous sulfate heptahydrate 0.003%, zinc sulfate heptahydrate 0.0014%, and manganese sulfate monohydrate 0.0004%.

[0064] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0065] The present invention will be further illustrated below with reference to the embodiments:

[0066] Example 1: Screening of high-acetoin-producing strains

[0067] 1. Separation and purification

[0068] 1 g of Daqu (a type of starter culture) was weighed from a certain Maotai-flavor liquor factory and placed in a conical flask containing 99 mL of sterile water and sterile glass beads. The Daqu was shaken to disperse it evenly, and a sample suspension was prepared. The suspension was heated in an 80℃ water bath for 10 min to eliminate non-spore-forming bacteria. Then, 1 mL of the suspension was transferred to an enrichment medium and incubated for 12 h.

[0069] Then, the enrichment solution was gradient diluted to 10 in a clean bench. -3 -10 -8 Take 0.2 mL of the bacterial suspension and drop it onto the center of an isolation medium plate. Spread the bacterial suspension evenly on the medium using a spreader and incubate upside down at 37°C for 1–2 days. Based on the morphological characteristics of Bacillus colonies, select colonies with inconsistent morphology and streak them onto the isolation medium plate using a sterile inoculation loop. Incubate at 37°C until single colonies grow, and then select single colonies for continuous purification culture.

[0070] Liquid enrichment medium: 3% glucose, 1% peptone, 0.50% yeast extract, 0.05% sodium chloride, fermented mash extract, pH 6.0, sterilized at 115 ℃ for 20 min.

[0071] Isolation and purification culture medium: glucose 3%, peptone 1%, yeast extract 0.50%, sodium chloride 0.05%, agar 2%, pH 6.0, sterilized at 115℃ for 20 min.

[0072] 2. Screening of high-yield acetoin strains

[0073] Initial screening: The creatine colorimetric method was used to screen strains capable of synthesizing acetoin (ACT). The method was as follows: 1 g of 1-naphthol, 0.1 g of creatine, and 4 g of NaOH were mixed to prepare a 100 mL creatine solution. A loopful of Bacillus strains screened from Daqu was gently scraped into a test tube containing 1 mL of the solution. Strains capable of producing ACT would quickly turn the solution red, and the deeper the color, the higher the yield.

[0074] Secondary screening: The strains obtained from the initial screening were inoculated into 50 mL of seed culture medium and cultured on a shaker at 37℃ and 180 r / min for 12–24 h. Then, 5% of the inoculum was added to 50 mL of fermentation medium and cultured on a shaker at 37℃ and 180 r / min for 72 h. After fermentation, the culture was centrifuged at 12000 r / min for 2 min, and the supernatant was collected. The OD of the fermentation broth was determined colorimetrically. 522 The nm value was used to obtain strain X-11 with high ACT yield and control strain X-12 with low ACT yield. 40% glycerol was mixed with the bacterial solution at a 1:1 ratio and stored at -80℃.

[0075] Seed culture medium: 3% glucose, 1% peptone, 0.50% yeast extract, 0.05% sodium chloride, pH 6.0, sterilized at 115℃ for 20 min.

[0076] Fermentation medium: 8% glucose, 1% peptone, 1% yeast extract, 0.05% sodium chloride, 0.03% magnesium sulfate heptahydrate, 0.10% calcium chloride dihydrate, 0.03% potassium dihydrogen phosphate, 0.03% ammonium sulfate, pH 6.0, sterilized at 115℃ for 20 min.

[0077] 3. Observe the morphological characteristics of the strain.

[0078] Straw X-11, which yielded high ACT production, was used to obtain single colonies that were purified and streaked onto the culture medium. The colonies were then incubated at 37°C for 1–2 days. The surface morphology of the colonies was observed. The colonies of this invention were small, round, with a rough surface, light yellow in color, and a diameter of 2–3 mm. They were straight rods, solitary, and produced nearly centrally located elliptical spores.

[0079] 4. 16S rRNA gene sequencing

[0080] 16S rDNA Sequencing and Analysis

[0081] (1) DNA extraction

[0082] Purified single colonies of different morphologies were inoculated into fermentation broth and incubated at 37°C for 24 h. 2 mL of culture was then transferred to a sterile centrifuge tube and centrifuged at 13000 rpm for 10 min. The supernatant was discarded, and the bacterial pellet was collected (repeated 2-3 times). Genomic DNA was extracted from the bacterial pellet using a rapid bacterial genomic DNA extraction kit following the prescribed procedure.

[0083] (2) PCR amplification and construction of phylogenetic tree

[0084] The DNA solution obtained in step (1) was used as an amplification template, and PCR amplification was performed using the bacterial universal primer 27F-1492R;

[0085] The PCR amplification system is: 5×Buffer (containing Mg) 2+ 10 μL, 200 μmol / L dNTPs 1 μL, forward and reverse primers 1 μL each, Taq DNA polymerase 1 μL, template DNA 3 μL, add sterile water to make up to 50 μL;

[0086] The amplification conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min; 55℃ annealing for 1 min; 72℃ extension for 1.5 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min.

[0087] The amplification products were detected by 1% agarose gel electrophoresis, and the PCR products were sequenced at BGI Genomics Co., Ltd. After cutting the sequenced sequences using BioEdit, reliable sequence fragments were uploaded to the NCBI nucleic acid alignment website for comparison. The sequences with the highest homology were selected, and a phylogenetic tree was constructed using MEGA 5.0 software. The phylogenetic tree based on 16S rDNA is shown below. Figure 1 As shown.

[0088] The strain was identified as Bacillus licheniformis by 16S rDNA and named Bacillus licheniformis X-11.

[0089] Example 2: Strain expansion culture and fermentation performance test

[0090] The methods for scaling up the culture of Bacillus licheniformis X-11 and the fermentation performance tests were conducted, including:

[0091] Step 1: Strain activation: The Bacillus licheniformis X-11 stored at -80℃ is inoculated into the isolation and purification medium and cultured at 34~38℃ for 12~24 hours to obtain the activated Bacillus licheniformis X-11.

[0092] Step 2: Preparation of primary seed culture: Take the activated Bacillus licheniformis X-11 strain, inoculate it into primary seed culture medium, and culture it at 34~38℃ for 12~24h to obtain the primary seed culture.

[0093] Step 3: Preparation of high-yield acetoin fermentation broth: The primary seed culture was transferred to a 100L fermenter containing a large-scale fermentation medium at an inoculation rate of 2%–5% by volume. The fermentation was carried out at 34–38℃, a rotation speed of 180–220 rpm / min, an aeration rate of 0.5–0.8 vvm, and an aeration rate of 0.04 MPa–0.06 MPa for 1–3 days. Samples were taken every 4 hours to detect the acetoin content (the fermentation process was controlled at glucose 20 g / L and pH 6.5). The large-scale fermentation medium consisted of water and the following components by mass percentage: glucose 8%, magnesium sulfate heptahydrate 0.05%, FM888 2.5%, triammonium citrate 0.1%, sodium acetate 0.65%, dipotassium hydrogen phosphate trihydrate 8%, ferrous sulfate heptahydrate 0.003%, zinc sulfate heptahydrate 0.0014%, and manganese sulfate monohydrate 0.0004%.

[0094] The control strain X-12 was cultured on a large scale and its fermentation performance was tested according to the above method. The yield of acetoin was determined, and the results are shown in Table 1. The acetoin yield of strain X-11 of this invention is shown in Table 1. Figure 2 .

[0095] Table 1. Yield of acetoin during fermentation.

[0096]

[0097] As shown in Table 1, under the same fermentation conditions, after 36 hours of cultivation, strain X-11 produced 98 g / L of acetoin, while the control strain X-12 produced only 33 g / L, far lower than the strain of this invention. This fully demonstrates that strain X-11 of this invention can utilize the nutrients in the culture medium more efficiently, synthesize and accumulate a large amount of acetoin in a short time, and its performance in fermenting acetoin production is significantly better than that of the control strain X-12, possessing the potential for industrial-scale production of acetoin.

[0098] Example 3: Application of Bacillus licheniformis X-11 in the production of Maotai-flavor liquor

[0099] Step 1: Cultivation of Acetoin Bacteria in Broth

[0100] Scale-up of high-yield acetoin strain: Single colonies of the activated strain were picked from plates and inoculated into fermentation medium. The cultures were then shake-cultured at 34–38°C and 180–200 rpm for 12–48 h, yielding a viable count of 1.8 × 10⁻⁶ cells / ml. 8The primary seed culture was prepared at CFU / mL. A fermenter was then selected as the seed tank, into which 50% of the expansion fermentation medium was added, along with 0.3% (v / v) of polyether defoamer. The pH was adjusted to 6.5 with sodium hydroxide. The mixture was then sterilized at 121°C for 20 min, cooled to 37°C, and then inoculated into the expansion fermentation medium in the seed tank at an inoculation rate of 2%–5% (the expansion fermentation medium comprises the following components by mass fraction: glucose 8%, magnesium sulfate heptahydrate 0.05%, FM...). 8882.5%, triammonium citrate 0.1%, sodium acetate 0.65%, dipotassium hydrogen phosphate trihydrate 8%, ferrous sulfate heptahydrate 0.003%, zinc sulfate heptahydrate 0.0014%, manganese sulfate monohydrate 0.0004%, water 100mL, pH 6.5~7.0), cultured under the conditions of an aeration ratio of 1.5 vvm, tank pressure of 0.05 MPa, stirring speed of 1801~200 r / min, and 37℃, with samples taken every 4 hours for testing. When the number of cells in the fermentation broth reached 16 × 10⁻⁶, the fermentation was considered complete. 8 Once the concentration of CFU / mL is reached, the mixture can be transferred to a fermentation tank to obtain the fermentation broth for later use.

[0101] Step 2: Making bran koji

[0102] Mix the acetoin fermentation broth obtained in step 1 with wheat bran in a 1:1 ratio (by mass), then place it in an oven to dry, stirring regularly until it is completely dry (moisture content < 10%).

[0103] Step 3, Application of bran koji

[0104] In the conventional fermentation process of Maotai-flavor liquor, the mash after the 7th distillation is first cooled (to 32-34℃), then 2-5% of microbial powder and 10-12% of Daqu (a type of starter culture) are added by weight. After mixing, the mash is piled up (for 2-3 days) and then fermented in a pit. The experimental group had microbial powder added, while the control group did not. The microbial powder was the bran starter culture obtained in step 2. After fermentation, the mash is distilled, and the liquor samples are analyzed. The results are shown below. Figures 3-5 .

[0105] Depend on Figures 3-5 It was found that the acetoin content in the experimental group and the control group was 25.6 mg / L and 12.8 mg / L, respectively, with the acetoin content in the experimental group being twice that of the control group. The tetramethylpyrazine content in the experimental group and the control group was 13.5 mg / L and 8.6 mg / L, respectively, with the tetramethylpyrazine content in the experimental group increasing by 57% compared to the control group. Furthermore, the yield of the experimental group (60% vol) was 11% higher than that of the control group.

[0106] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. Bacillus licheniformis, characterized by, Its accession number is CCTCC NO:M20251822.

2. A microbial agent, characterized in that, Including Bacillus licheniformis with accession number CCTCC NO:M 20251822, and / or fermentation broth of Bacillus licheniformis with accession number CCTCC NO:M 20251822.

3. The method for preparing the microbial agent according to claim 2, characterized in that, Includes the following steps: S1: After inoculating and culturing Bacillus licheniformis with accession number CCTCC NO:M 20251822, seed culture was obtained; S2: The seed liquid is subjected to large-scale fermentation culture to obtain the microbial agent.

4. The preparation method according to claim 3, characterized in that, Based on the quality of water, the culture medium for the expanded fermentation culture includes water and the following components in the following mass percentages: glucose 1%~8%, magnesium sulfate heptahydrate 0.02%~0.05%, FM888 1.5%~2.5%, triammonium citrate 0.05%~0.1%, sodium acetate 0.45%~0.65%, dipotassium hydrogen phosphate trihydrate 5%~8%, ferrous sulfate heptahydrate 0.001%~0.003%, zinc sulfate heptahydrate 0.0010%~0.0014%, manganese sulfate monohydrate 0.0001%~0.0004%, and pH 6.5~7.

0.

5. The preparation method according to claim 3, characterized in that, The conditions for the expanded fermentation culture include: an aeration ratio of 1.5 vvm, a tank pressure of 0.05 MPa, a stirring speed of 180-200 r / min, and a culture temperature of 34-37℃.

6. The yeast starter, characterized in that, Its raw materials include: the microbial agent as described in claim 2 and the substrate.

7. The yeast starter according to claim 6, characterized in that, The substrate includes at least one of wheat, bran, corn, sorghum, rice flour, and rice husk.

8. The yeast starter according to claim 6, characterized in that, The yeast starter is a bran yeast starter, which is prepared from wheat bran and the microbial agent described in claim 2 at a mass ratio of 1:

1.

9. The method for preparing yeast according to any one of claims 6 to 8, characterized in that, Mix the bacterial agent and substrate as described in claim 2, dry them, and stir.

10. The use of Bacillus licheniformis according to claim 1, the inoculum according to claim 2, or the yeast according to any one of claims 6-8 in any of the following: (1) Brewing baijiu (Chinese liquor); (2) Fermentation to produce acetoin and / or tetramethylpyrazine.

11. The application according to claim 10, characterized in that, The liquor in question is a sauce-flavored type of Daqu liquor.

12. The application according to claim 10, characterized in that, The Bacillus licheniformis increases the content of acetoin and / or tetramethylpyrazine in baijiu.

13. A method for preparing soy sauce-flavored Daqu liquor, characterized in that, include: The Bacillus licheniformis of claim 1, the inoculum of claim 2, or the yeast of any one of claims 6 to 8 are inoculated into the mash and then fermented in a cellar.

14. The preparation method according to claim 13, characterized in that, The fermented mash referred to is the mash that has been cooled after the 7th round of distillation.

15. The preparation method according to claim 14, characterized in that, The temperature of the cooled fermented mash is 32~34℃.

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