A Maotai slow-growing Bacillus and its application in Daqu fermentation

Abstract

The present invention belongs to the technical field of fermentation engineering, and specifically discloses a strain of Moutai slow-growing Bacillus (Lentibacillus maotaii) JN110522, which is stored in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a preservation date of June 29, 2022, and a preservation number of CGMCC No.25214. The strain can simultaneously metabolize and produce high levels of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine, and the output of 2,3,5,6-tetramethylpyrazine can reach 26.2±0.8mg / L, and the content of 2,3,5-trimethylpyrazine can reach 4.1±0.4mg / L. The present invention also discloses that the strain is applied to the compression stage of high-temperature Daqu fermentation process, which can greatly increase the content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in high-temperature Daqu, so that the sauce-flavored flavor of high-temperature Daqu is better prominent, and can be used to prepare high-performance Daqu. The total yield of pyrazine compounds, 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine of the fortified qu fermented by this bacterium is 1 times higher than that of ordinary Maotai slow-growing Bacillus fortified qu, and the application value is very high.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and particularly relates to a Maotai slow-growing Bacillus capable of simultaneously producing 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine and application thereof in Daqu fermentation. Background Art

[0002] 2,3,5,6-tetramethylpyrazine is a heterocyclic nitrogen compound, which is widely found in fermented foods. It has the aroma of baking, cocoa and coffee, and is often used in the blending of flavors and fragrances such as baked foods, cold drinks, dairy products, and cigarettes. 2,3,5-trimethylpyrazine is also a heterocyclic nitrogen compound, which can be used to flavor cream flavors, and can also be used to blend flavors such as coffee, cocoa, chocolate, peanuts, and sesame oil. At present, a large amount of 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethylpyrazine and other pyrazine substances have also been detected in Chinese liquor, especially Maotai-flavor liquor. Among them, 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine are important aroma components in liquor, especially Maotai-flavor liquor, and they are believed to have made an important contribution to the flavor of Maotai-flavor liquor.

[0003] 2,3,5-trimethylpyrazine is derived from chemical synthesis, such as 1,2-propylenediamine and butanediol or methyl oxirane cyclocondensation; while 2,3,5,6-tetramethylpyrazine is derived from three sources: direct extraction from plants, chemical synthesis, and biosynthesis. Among them, the method of direct extraction from plants is less efficient. The content of tetramethylpyrazine in the rhizome of the Chinese medicinal material Chuanxiong is only 0.075%, and the raw materials are difficult to supply on a large scale; the reaction process used in chemical synthesis requires drastic reaction conditions, has high requirements for mechanical equipment, and has the risk of producing by-products; biosynthesis mainly refers to microbial engineering technology, which uses strain screening mutagenesis technology and fermentation engineering technology to produce tetramethylpyrazine. It has the characteristics of green, natural, low cost, and mild conditions. Therefore, it is widely used in the field of winemaking through biosynthesis, especially by improving the fermentation quality of Daqu and increasing the content of pyrazines to improve the flavor and performance of Daqu, thereby improving the flavor of liquor.

[0004] A commonly used method to improve the fermentation quality of Daqu is to enhance Daqu technology, which uses modern microbial technology to isolate and cultivate Daqu functional strains. In the Daqu production process, by combining artificial inoculation of functional bacteria with naturally enriched microorganisms, the ecological occupancy of functional microorganisms can be increased, which is beneficial to their metabolic functions, thereby effectively improving the flavor and performance of Daqu.

[0005] In recent years, relevant researchers have found that microbial metabolism is an important source of 2,3,5,6-tetramethylpyrazine in liquor. There are many studies that use microbial enhancement technology to increase the yield of tetramethylpyrazine in fermented grains, and the strains used involve Bacillus subtilis, Bacillus licheniformis, etc. A large number of microorganisms are involved in the brewing process of Chinese liquor. At present, the functions of a large number of microorganisms have not been verified, and it is not clear which specific microorganism plays an important role in liquor brewing. Therefore, screening new strains that can produce high yields of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine at the same time has important practical significance for the new way to enrich and improve the flavor of liquor in production. At present, there is no record of the discovery of new species of slow-growing Bacillus in the present invention in the prior art, nor is there any application of the slow-growing Bacillus described in the present invention to simultaneously produce tetramethylpyrazine and 2,3,5-trimethylpyrazine. Summary of the invention

[0006] The purpose of the present invention is to provide a new strain of Moutai slow-growing Bacillus, which can metabolize and produce 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine at the same time. The present invention also provides the application of the Moutai slow-growing Bacillus in Daqu fermentation; the present invention also provides a culture medium of the Moutai slow-growing Bacillus and a method for simultaneously metabolizing and producing 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a strain of Lentibacillus maotaii JN110522, which is preserved in the General Microbiology Center of China Culture Collection of Microorganisms (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a preservation date of June 29, 2022 and a preservation number of CGMCC No.25214.

[0009] The Maotai lentiviral Bacillus JN110522 belongs to the genus Lentibacillus, and its 16SrRNA gene sequence is 1468bp in length. The strain with the highest homology is Lentibacillus maotaii (GenBank accession number: SRR17676770), and the sequence identity between the two is 98.03%. The gene sequence is shown in SEQ ID NO: 3.

[0010] The morphological characteristics and physiological and biochemical characteristics of the Maotai slow-growing Bacillus JN110522 are as follows: the colony morphology is milky white, opaque, and has smooth edges; the cell morphology is rod-shaped, with a length and width of approximately 0.3×1.5 μm; it contains spores; it contains flagella; its aerobic characteristics are aerobic, the growth temperature range is 30-45°C, and the salt tolerance range is 0-20g / L.

[0011] The present invention also provides the use of the above-mentioned Moutai lentiviral Bacillus JN110522 in the production of 2,3,5,6-tetramethylpyrazine and / or 2,3,5-trimethylpyrazine, or in the fermentation production of products rich in 2,3,5,6-tetramethylpyrazine and / or 2,3,5-trimethylpyrazine.

[0012] The present invention also provides the use of the Moutai slow-growing Bacillus JN110522 in improving the flavor of Daqu during Daqu fermentation.

[0013] Improving the flavor of Daqu means making the flavor of high-temperature Daqu more prominent, specifically referring to the sauce aroma, baking aroma, floral and fruity aroma, fermentation aroma and exotic aroma.

[0014] Furthermore, the application method is to inoculate the Moutai slow-growing Bacillus JN110522 described in claim 1 during the buckling stage of the Daqu preparation process.

[0015] Furthermore, in the application, the Daqu preparation is high-temperature Daqu preparation.

[0016] Furthermore, in the application, the amount of the inoculated bacterial solution of Moutai slow-growing Bacillus is 1%-8% by weight, and the density of the bacterial solution is 1-10×10 8 CFU / mL.

[0017] Preferably, the amount of the inoculated bacterial solution of Moutai slow-growing Bacillus in the application is 5% by weight, and the density of the bacterial solution is 3×10 8 CFU / mL.

[0018] Furthermore, the improved flavor of Daqu is obtained by increasing the content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in Daqu.

[0019] The present invention also provides Daqu prepared by using the Maotai slow-growing Bacillus JN110522.

[0020] The present invention also provides wine brewed by using the above-mentioned Daqu as a saccharification and fermentation agent.

[0021] The present invention also provides a method for simultaneously producing 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine using the above-mentioned Moutai slow-growing Bacillus JN110522, comprising the following steps: sterilizing the culture medium, inoculating the above-mentioned Moutai slow-growing Bacillus, and culturing with shaking at 35-39° C. under aerobic conditions.

[0022] Furthermore, the method comprises the following steps: sterilizing the culture medium, inoculating the culture medium with Bacillus moutaiensis, and culturing the culture medium on a 37°C constant temperature shaker at a speed of 150 r / min for 120 to 148 hours under aerobic conditions.

[0023] Furthermore, in the above method, the culture medium contains 10-20 g / L glucose, 2-8 g / L tryptone, 2-7 g / L yeast powder, 0.05-0.15 g / L ferric citrate, 10-20 g / L sodium chloride, 2-7 g / L magnesium chloride, and 0.2-0.7 g / L potassium chloride; the pH value of the culture medium is 7.0-8.0.

[0024] Preferably, in the above method, the culture medium contains 20 g / L glucose, 5 g / L tryptone, 5 g / L yeast powder, 0.1 g / L ferric citrate, 15 g / L sodium chloride, 5 g / L magnesium chloride, 0.5 g / L potassium chloride, and the pH value is 7.5.

[0025] Furthermore, in the above method, the amount of the bacterial solution inoculated with Maotai slow-growing Bacillus is 1%-8% by weight, and the density of the bacterial solution is 1-10×10 8 CFU / mL.

[0026] Preferably, in the above method, the amount of the bacterial solution inoculated with Maotai slow-growing Bacillus is 5% by weight, and the density of the bacterial solution is 3×10 8 CFU / mL.

[0027] The beneficial effects of the present invention are:

[0028] The present invention screens a new strain capable of simultaneously producing 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine from Daqu, and the strain is identified as Lentibacillus maotaii. The 2,3,5,6-tetramethylpyrazine yield of the strain can reach 26.2±0.8 mg / L, and the content of 2,3,5-trimethylpyrazine can reach 4.1±0.4 mg / L. The strain can be applied to fermentation to simultaneously produce 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine; the strain can be applied to the pressing stage of the high-temperature Daqu fermentation process to greatly increase the content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the high-temperature Daqu, so that the sauce-flavored flavor of the high-temperature Daqu is more prominent, and the strain can be used to prepare high-performance Daqu, and the Daqu can also be applied to liquor brewing to improve the flavor of the liquor. The total yield of pyrazine compounds, 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine of the fortified qu fermented by this bacterium was increased by 1 times compared with that of ordinary Maotai slow-growing Bacillus fortified qu, and the application value is very high.

[0029] The Moutai slow-growing Bacillus JN110522 was isolated from the high-temperature Daqu in Moutai Town in July 2022. The Moutai slow-growing Bacillus (Lentibacillus maotaii) JN110522 provided by the present invention is preserved in the General Microbiological Center of China Microorganism Culture Collection Center (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation date is June 29, 2022, and the preservation number is CGMCC No. 25214. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the phylogenetic tree of the Maotai lentiviral Bacillus JN110522 based on the 16S rDNA sequence.

[0031] Figure 2 This is a scanning electron micrograph of the Maotai slow-growing Bacillus JN110522.

[0032] Figure 3 This is the colony morphology of the Maotai slow-growing Bacillus JN110522. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available; the slow-growing Bacillus in the following examples was isolated from the high-temperature Daqu in Maotai Town in June 2022. The Maotai slow-growing Bacillus (Lentibacillus maotaii) JN110522 provided by the present invention is preserved in the General Microbiological Center of the China Microbiological Culture Collection Committee (English name: China General Microbiological Culture Collection Center, referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation date is June 29, 2022, and the preservation number is CGMCC No. 25214.

[0035] Example 1: Screening of Bacillus lentiviridae JN110522

[0036] 1. Experimental Methods

[0037] (1) Isolation and cultivation of slow-growing Bacillus

[0038] In Maotai Town, 10 g of sample was taken from the high temperature Daqu and placed in a 500 mL Erlenmeyer flask mixed with glass beads and 90 mL of sterile water. The sample was shaken at 115 r / min for 30 min at 30 °C. Then 1 mL of the bacterial suspension was diluted 10 times with sterile saline. -3 ~10 -6 The gradient dilution samples were coated on separation medium plates with the composition of the medium as shown in a below. The plates were cultured at 30°C for 7 days. Subsequent steps could be performed after culturing at this temperature for 7-10 days according to actual needs. The abundant colonies on the plates were isolated and purified and then inoculated into slant culture medium with the composition of the medium as shown in a below. The plates were cultured at 30°C for 7 days and stored at 4°C. Meanwhile, the single colonies in the separation / slant culture medium were inoculated into liquid culture medium with the composition of the medium as shown in b below. The plates were cultured at 30°C for 7 days to obtain fermentation broth.

[0039] a. The compositions of separation and slant culture media are: 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.008 g / L disodium hydrogen phosphate, and 17 g / L agar.

[0040] b. Liquid culture medium: peptone 5.0 g / L, yeast powder 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, disodium hydrogen phosphate 0.008 g / L.

[0041] (2) Gas chromatography-mass spectrometry was used to screen strains that simultaneously produced 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the fermentation broth.

[0042] According to the following gas chromatography and mass spectrometry analysis conditions, the corresponding settings were made, and then the GC-MS was used to determine the content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the fermentation broth according to the following method. The fermentation broth was first pretreated and then subjected to GC-MS analysis, specifically:

[0043] 1) Preprocessing

[0044] Pretreatment: Pipette 6 mL of fermentation broth into a 20 mL headspace solid phase microextraction bottle, add 2.5 g of NaCl, shake evenly, add 10 μl of tert-amyl alcohol (8.05 g / L) as an internal standard, cover the bottle cap, and record the label number.

[0045] 2) GC-MS analysis

[0046] A gas chromatograph-mass spectrometer (model US1845Q002) from Agilent, USA, was used for injection; the extraction head of the coupling instrument was a 50 / 30μm DVB / CAR on PDMS extraction head, the gas chromatography analysis conditions were as shown in ① in this embodiment, and the mass spectrometry analysis conditions were as shown in ② in this embodiment. The injection method was automatic injection, the adsorption time was 40min, the stirring speed was 500r / min, the analysis temperature was 250°C, and the analysis time was 5min. The contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine were determined, and the fermentation broth containing both 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine was screened, and the strain corresponding to the fermentation broth was the strain that simultaneously metabolized and produced 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine.

[0047] ① Gas chromatography analysis conditions are as follows: chromatographic column: DB-Wax capillary column, column length 30m, inner diameter 0.32mm, liquid film thickness 0.25μm; carrier gas: helium He; flow rate: 1.0mL / min, split ratio 2:1; column temperature: the injection port temperature is maintained at 250℃, the initial gas chromatography column temperature is maintained at 40℃ for 3min, then increased to 120℃ at 5℃ / min, and then increased to 250℃ at 10℃ / min and maintained for 5min.

[0048] ②Mass spectrometry analysis conditions are: ion source temperature 250°C; interface temperature 280°C; ionization mode: EI+; electron energy: 70ev; scanning mass range: 33-350amu.

[0049] 2. Experimental results

[0050] The samples were analyzed according to the above GC-MS conditions to obtain the total ion current of the fermentation broth. Each chromatographic peak was analyzed by fragmentation and qualitative analysis by consulting the NIST2.0 mass spectrum library to determine the chemical structure of the product. A strain that simultaneously produced 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine was obtained. The screening was considered successful and the strain was recorded as JN110522.

[0051] Example 2: Molecular identification of Bacillus lentiviridae JN110522

[0052] 1. Experimental Methods

[0053] For the strain JN110522 obtained by purification and screening in Example 1, fresh bacterial liquid in the exponential growth phase was taken, and the bacterial bodies were collected by centrifugation, and genomic DNA was extracted using a bacterial genome extraction kit. The following bacterial universal primers P0 and P6 in this example were used to amplify PCR under ① reaction system and ② reaction program ③, and the PCR product was separated and tested by 1.0% agarose gel electrophoresis, the voltage was about 11V / cm, and the electrophoresis time was 20min. The purification of the amplified product was obtained, and the purification of the amplified product was carried out according to the instructions of the gel recovery PCR product purification kit of Shanghai Shenggong Biotechnology Co., Ltd., and the full-length sequence sequencing of the 16S rRNA gene was completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0054] ①Universal primer:

[0055] P0: 5'-GAG AGT TTG ATC CTG GCT CAG-3', sequence see SEQ ID NO: 1

[0056] P6: 5'-CTA CGG CTA CCT TGT TAC GA-3', sequence as shown in SEQ ID NO: 2

[0057] ②Reaction system (50μL)

[0058] 10×buffer(Mg2 + )5.0μL

[0059] dNTPs (2.5 mM) 4.0 μL

[0060] P0(12.5μM)2.0μL

[0061] P6 (12.5 μM) 2.0 μL

[0062] ExTaq DNA polymerase (5U / uL) 1.0μL

[0063] Template DNA 2.0μL

[0064] dH 2 O 34 μL

[0065] ③ Reaction procedure (according to the following sequence, from pre-deformation to final extension, PCR products are obtained)

[0066]

[0067] 2. Experimental results

[0068] The full length of the 16S rRNA gene sequence of strain JN110522 is 1468 bp, and the sequence is shown in SEQ ID NO: 3. The species was determined by BLAST comparison in GenBank. The phylogenetic tree based on the 16S rRNA gene sequence is shown in Figure 1 As shown, the strain with the highest homology is Lentibacillus maotaii (GenBank accession number: SRR17676770); further analysis of the sequence consistency of the two showed that the sequence consistency of the two was 98.03%, with a difference of 17 bases.

[0069] The strain was identified as Lentibacillus maotaii JN110522 and was deposited in the General Microbiology Center of China Culture Collection Administration on June 29, 2022, with the deposit number CGMCC No.25214.

[0070] Example 3: Physiological and biochemical characteristics of Maotai slow-growing Bacillus

[0071] 1. Experimental Methods

[0072] The strain was inoculated into the separation medium and cultured at 30°C for 4 days. The cell morphology of the strain, including the size, spores and flagella, was observed by transmission electron microscopy and scanning electron microscopy. The strain was inoculated into the liquid culture medium and the optical density (OD) of the culture medium at 600 nm was measured. 600 ) to detect the aerobic characteristics, suitable growth temperature range and salt tolerance of the strain and other biochemical characteristics. The above method is mainly based on the "Common Bacteria System Identification Manual" (Dong Xiuzhu, Cai Miaoying. 2001. Common Bacteria System Identification Manual. Beijing: Science Press).

[0073] 2. Experimental results

[0074] Cell morphology and physiological and biochemical characteristics of Bacillus moutaiensis JN110522 Figure 2-Figure 3 As shown in Table 1, the morphological and physiological and biochemical characteristics of the Moutai slow-growing Bacillus are as follows: the colony morphology is milky white, opaque, and has smooth edges ( Figure 3 ); The cell morphology is rod-shaped, with a length and width of approximately 0.3×1.5μm ( Figure 2 ); contains spores; contains flagella; aerobic characteristics are aerobic, the growth temperature range is 30-45℃, and the salt tolerance range is 0-20g / L.

[0075] Table 1 Morphological characteristics and physiological and biochemical properties of Maotai lentiviral Bacillus JN110522

[0076] Bacteria JN110522 Experimental Project characteristic Colony morphology Milky white, opaque, smooth edges Cell morphology (size, μm) Rod-shaped (0.3×1.5) Spore have flagellum have Aerobic characteristics Aerobic Growth temperature range (℃) 30-45 Salt tolerance range (g / L) 0-20

[0077] Example 4 Method for simultaneously producing high content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine

[0078] 1. Experimental Methods

[0079] (1) Fermentation of Maotai slow-growing Bacillus JN110522

[0080] 100 mL of liquid culture medium was added to a 500 mL conical flask. The contents of the components in the culture medium were as follows: glucose 20 g / L, tryptone 5 g / L, yeast powder 5 g / L, ferric citrate 0.1 g / L, sodium chloride 15 g / L, magnesium chloride 5 g / L, potassium chloride 0.5 g / L, and the pH value was 7.5. After the culture medium was sterilized, the above-mentioned Maotai slow-growing Bacillus JN110522 was inoculated. The inoculation amount of the culture medium was 5%, and the concentration of the culture medium was 3 × 10 8 , cultured on a 37℃ constant temperature shaker at 150r / min for 130h as the experimental group.

[0081] The control group was distinguished by a liquid culture medium without bacteria, which was inoculated in equal amounts into the same culture medium as above, and the culture conditions remained consistent.

[0082] (2) Determination of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in fermentation broth

[0083] The contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the fermentation broth of the control group and the experimental group were determined by referring to part (2) of Example 1, and the gas chromatography-mass spectrometry method was used to screen the fermentation broth that simultaneously produced 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine.

[0084] 2. Experimental results

[0085] Gas chromatography-mass spectrometry was used to determine the contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the fermentation broth. The results are shown in Table 2. The yield of 2,3,5,6-tetramethylpyrazine in the fermentation broth of the experimental group can reach 26.2±0.8 mg / L, and the content of 2,3,5-trimethylpyrazine can reach 4.1±0.4 mg / L. Compared with the control group, the total amount of pyrazines and the contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in the fermentation broth of this bacteria were significantly increased.

[0086] Table 2 Content of pyrazine compounds in fermentation broth

[0087]

[0088] Example 5: Using Moutai slow-growing Bacillus JN110522 to strengthen the fermentation process of high-temperature Daqu

[0089] 1. Experimental Methods

[0090] (1) Preparation of ordinary high-temperature Daqu and fortified Daqu.

[0091] Ordinary high-temperature Daqu is Daqu produced by fermentation according to the traditional high-temperature Daqu process. The high-abundance members of the bacterial community mainly include: Kroppenstedtella, Bacillus, Marine Bacillus, Cladosporium, Saccharopolyspora, Scopulibacillus, Staphylococcus, and Pediococcus.

[0092] The fortified koji is: during the koji pressing and embryo making stage of high temperature koji production, add Moutai slow-growing Bacillus JN110522. The screened Moutai slow-growing Bacillus JN110522 is activated with fermentation medium and cultured for 5 days until 3×10 8 CFU / mL, after the seed liquid is expanded, the bacterial liquid is directly inoculated into the raw koji and fermented to make the fortified koji.

[0093] (2) Ordinary high-temperature Daqu in the koji room was used as the control group, and the fortified koji was used as the fortified group. Fortified group 1 was fortified koji fermented with the above-mentioned Moutai slow-growing Bacillus JN110522, and fortified group 2 was fortified koji fermented with the same concentration of ordinary Moutai slow-growing Bacillus (commercially available) as fortified group 1. The fermented samples were subjected to sensory analysis. The sensory analysis method was as follows: referring to the sensory evaluation method of Daqu aroma characteristics (Shen Shiming, et al. Preliminary construction of the flavor wheel of high-temperature Daqu with sauce aroma and analysis of its aroma characteristics. [J]. Brewing Technology, 2022: 10.), a 5-point system was used to describe the sensory intensity of the samples. The larger the value, the stronger the intensity. The aroma intensities corresponding to scores of 1, 2, 3, 4, 5, and 6 were none, weak, relatively weak, medium, relatively strong, and strong, respectively. The aroma was divided into five types: sauce aroma, baking aroma, floral and fruity aroma, fermentation aroma, and foreign aroma.

[0094] (3) The contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in Daqu were determined by gas chromatography-mass spectrometry in Example 1. Specifically, the sample was analyzed according to the GC-MS conditions in Example 1 to obtain a total ion current diagram in the fermentation broth. Each chromatographic peak in the total ion current diagram was subjected to fragment analysis and qualitative analysis by consulting the NIST2.0 mass spectrum library. After the compound was determined according to the analysis, the relative contents of pyrazine compounds, 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine of each component were obtained by analysis using the corrected peak area normalization method.

[0095] 2. Experimental results

[0096] (1) Sensory evaluation of ordinary high-temperature Daqu and enhanced Daqu fermented with Maotai slow-growing Bacillus

[0097] The aroma and flavor characteristics of the solid-state fermented koji samples are shown in Table 3. As shown in Table 3, the sensory score of the Daqu fermented with Moutai slow-growing Bacillus JN110522 was significantly higher than that of the control group, and the sauce aroma, baking aroma, and floral and fruity aroma of the high-temperature Daqu in the enhanced group were significantly improved compared with the aroma and flavor of the high-temperature Daqu in the control group. The enhanced koji fermented with Moutai slow-growing Bacillus JN110522 had a prominent sauce aroma, and the sauce aroma and baking aroma were better than those of the enhanced group 2.

[0098] Table 3 Sensory scores of ordinary Daqu and enhanced Daqu

[0099] Finished Song Sauce Baking aroma Floral and fruity Fermented aroma Odor Control group 3 3 3 4 1 Reinforcement Group 1 6 5 5 5 1 Enhanced Group 2 4 4 7 5 1

[0100] (2) Content of pyrazine compounds in ordinary Daqu and fortified Daqu

[0101] The contents of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in high-temperature Daqu were determined by gas chromatography-mass spectrometry. The results are shown in Table 4. Compared with the control group, the content of 2,3,5,6-tetramethylpyrazine in the high-temperature Daqu of the enhanced group increased from 0.07 g / 100 g to 0.22 g / 100 g, an increase of 2.1 times; 2,3,5-trimethylpyrazine increased from 0.01 g / 100 g to 0.04 g / 100 g. 100g, increased by 3 times; the total amount of pyrazine compounds increased from 0.14g / 100g to 0.46g / 100g, increased by 2.3 times. The total yield of pyrazine compounds, 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine of the enhanced qu fermented by Moutai slow-growing Bacillus JN110522 were compared with those of ordinary Moutai slow-growing Bacillus enhanced daqu, and the yield of each pyrazine compound increased by 1 times.

[0102] Table 4 Content of enhanced pyrazine compounds in fermentation with Maotai slow-growing Bacillus

[0103]

[0104] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lentibacillus maotaii JN110522, characterized in that: The Moutai slow-growing Bacillus is preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a preservation date of June 29, 2022 and a preservation number of CGMCC No.25214.

2. Use of the Moutai slow-growing Bacillus JN110522 described in claim 1 in the production of 2,3,5,6-tetramethylpyrazine and / or 2,3,5-trimethylpyrazine, or in the fermentation production of products rich in 2,3,5,6-tetramethylpyrazine and / or 2,3,5-trimethylpyrazine.

3. Use of the Maotai slow-growing Bacillus JN110522 described in claim 1 in improving the flavor of Daqu during Daqu fermentation.

4. The use according to claim 3, characterized in that: The application method is to inoculate the Moutai slow-growing Bacillus JN110522 described in claim 1 during the buckling stage of the Daqu preparation process.

5. The application according to claim 3, characterized in that: The improved flavor of Daqu is obtained by increasing the content of 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine in Daqu.

6. The Daqu prepared by using the Maotai slow-growing Bacillus JN110522 described in claim 1.

7. The wine brewed using the Daqu as a saccharifying and fermenting agent according to claim 6.

8. A method for simultaneously producing 2,3,5,6-tetramethylpyrazine and 2,3,5-trimethylpyrazine using the Maotai lentiviral Bacillus JN110522 of claim 1, characterized in that: The method comprises the following steps: sterilizing the culture medium, inoculating the Maotai slow-growing Bacillus according to claim 1, and culturing with shaking at 35-39° C. under aerobic conditions.

9. The method according to claim 8, characterized in that The culture medium contains 10-20 g / L glucose, 2-8 g / L tryptone, 2-7 g / L yeast powder, 0.05-0.15 g / L ferric citrate, 10-20 g / L sodium chloride, 2-7 g / L magnesium chloride, and 0.2-0.7 g / L potassium chloride; the pH value of the culture medium is 7.0-8.

0.

10. The method according to claim 8, characterized in that The amount of the bacterial solution inoculated with Maotai slow-growing Bacillus is 1%-8% by weight, and the density of the bacterial solution is 1-10×10 8 CFU / mL.

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