Strain combination for efficiently producing tetramethylpyrazine and guaiacol and co-culture method thereof

Abstract

The invention relates to the field of microbial fermentation, and discloses a strain combination for efficiently producing tetramethylpyrazine and guaiacol and a co-culture method of the strain combination. The combination is prepared by carrying out synergistic fermentation on bacillus velezensis WQ19 with the preservation number of CCTCC (China Center For Type Culture Collection) NO: M20232058 and cronobacter malonate B1292 with the preservation number of CCTCC NO: M2025146 in a mixed substrate of wheat, sorghum and corn. The stable symbiotic micro-ecology is constructed by optimizing the inoculation ratio of the two bacteria at 3: 2, step-by-step temperature control of culturing for 2d at 37 DEG C and culturing for 3d at 45 DEG C, and 15% of sterile water addition amount. Compared with single-bacterium fermentation, the yield of tetramethylpyrazine in co-culture products is increased by 45.52%, and the yield of guaiacol is increased by 72.28%; the solid-state strain is applied to Maotai-flavor liquor small tests, the concentrations of the two substances are increased by 4-6 times and 7-10 times respectively compared with a control group, and a new way is provided for enhancing the flavor and health factors of the liquor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation and liquor brewing, and specifically provides a strain combination for efficient production of tetramethylpyrazine and guaiacol and a co-culturing method thereof. BACKGROUND

[0002] The flavor and health value of liquor are jointly determined by volatile components (such as pyrazines and phenols) and non-volatile components contained therein. Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine) has roasting aroma, nutty aroma and cocoa aroma, and has been confirmed to have physiological activities such as lowering blood lipids and protecting the cardiovascular and cerebrovascular systems. Guaiacol (2-methoxyphenol) can impart fruit aroma, caramel aroma and "grain aroma" to liquor, and also exhibits antioxidant, antitumor and immunomodulatory effects. Both of them are key substances for improving the quality of liquor. However, the natural concentrations of these two active substances in liquor are generally low, which cannot meet the dual demands of consumers for flavor richness and health functions.

[0003] To solve the above problems, the prior art mainly adopts a single-function bacteria strengthening strategy, such as isolating Bacillus subtilis strains from clear-fragrance Daqu to increase the yield of tetramethylpyrazine, or screening Bacillus strains from thick-fragrance pit mud to increase the content of guaiacol. Some technologies apply high-yield single-substance strains to liquor brewing processes to achieve the increase of the content of a single target product. However, the existing technologies still have significant limitations: first, they cannot simultaneously and efficiently increase the contents of tetramethylpyrazine and guaiacol, and single-function bacteria can only specifically strengthen one substance, which cannot meet the dual demands of flavor and health; second, the adaptability of the strains is insufficient, and most of the existing high-yield strains are isolated from non-pao-flavor liquor fermentation systems such as clear-fragrance Daqu and thick-fragrance pit mud, which have poor stability in the special fermentation environment of pao-flavor liquor with high temperature and high acidity, and the actual application effect is limited; third, the process design lacks precision, and most of the existing co-culturing technologies are generalized microbial cooperation, which do not optimize key parameters according to the growth rate and metabolic characteristics of the strains, resulting in an unstable symbiotic system, limited product yield, and inability to meet the needs of industrial production. Therefore, developing a technical solution that can adapt to the pao-flavor liquor fermentation system and simultaneously and efficiently increase the contents of tetramethylpyrazine and guaiacol has become an urgent problem in the industry. SUMMARY

[0004] Therefore, the present application provides a strain combination for efficient production of tetramethylpyrazine and guaiacol and a co-culturing method thereof, which realizes the simultaneous high production of two products by screening special function strains and optimizing the co-culturing process, and meets the needs of pao-flavor liquor brewing.

[0005] The technical scheme of the present application is implemented as follows: the present application provides a strain combination for efficient production of tetramethylpyrazine and guaiacol and a co-culturing method thereof, which adopts Bacillus velezensis WQ19 and Cronobacter malonaticus B1292 to form a functional strain combination, and co-cultures in a mixed substrate composed of wheat, sorghum and corn to prepare solid-state seeds and apply them to the brewing of Maotai-flavor liquor.

[0006] In some embodiments, the preservation number of the Bacillus velezensis WQ19 is CCTCC NO: M20232058, and the preservation address is China Center of Type Culture Collection, Wuhan, Wuchang, Bayi Road, Lujia Mountain, Wuhan, Hubei Province, China, and the strain was first disclosed in Chinese patent CN120536317A; the preservation number of the Cronobacter malonaticus B1292 is CCTCC NO: M2025146, and the preservation address is China Center of Type Culture Collection, Wuhan University, and the strain was first disclosed in Chinese patent CN120399947A.

[0007] In some embodiments, the co-culturing method of the strain combination includes substrate treatment, seed liquid preparation, inoculation and co-culturing, and product post-treatment steps, specifically: after the wheat, sorghum and corn are mixed and crushed, they are inoculated with the secondary seed liquid after sterilization and cooling, sterile water is added and cultured under the set conditions, and finally the solid-state seeds are obtained after drying and crushing.

[0008] In some embodiments, the secondary seed liquid includes Bacillus velezensis WQ19 secondary seed liquid and Cronobacter malonaticus B1292 secondary seed liquid, and both are prepared under the same conditions: the primary seed liquid is inoculated into a conical flask containing 30 mL of LB liquid medium at an inoculation amount of 0.5%-5% (v / v) from the strain stored in a glycerol tube, and cultured at 37℃ with 180r / min shaking for 24h; the secondary seed liquid is transferred to a conical flask containing 350 mL of LB liquid medium at an inoculation amount of 1%-5% (v / v) from the primary seed liquid, and cultured at 37℃ with 180r / min shaking for 48h.

[0009] In some embodiments, the viable bacterial concentration of the Bacillus velezensis WQ19 secondary seed liquid reaches 8.0×10 5 -1.0×10 7 CFU / mL, and the viable bacterial concentration of the Cronobacter malonaticus B1292 secondary seed liquid reaches 1.0×10 10 -1.0×10 12 CFU / mL.

[0010] In some embodiments, the inoculation ratio of the Bacillus velezensis WQ19 secondary seed liquid to the Cronobacter malonaticus B1292 secondary seed liquid is 3:2 by volume.

[0011] In some embodiments, the addition amount of the Bacillus velezensis WQ19 secondary seed liquid is 15% of the mixed dry weight of wheat, sorghum and corn, and the addition amount of the Cronobacter malonaticus B1292 secondary seed liquid is 10% of the mixed dry weight of wheat, sorghum and corn.

[0012] In some embodiments, the addition amount of the sterile water is 10%-20%, preferably 15%, of the mixed dry weight of wheat, sorghum and corn.

[0013] In some embodiments, the setting condition of the co-culture is to culture at 37℃ for 2d and then at 45℃ for 3d.

[0014] In some embodiments, the drying temperature of the product post-treatment is 50℃, and the addition amount of the solid-state seed is 2%-6% of the weight of the Jiangxiang Baijiu (Chinese liquor) fermented grains.

[0015] The present application has the following beneficial effects over the prior art: The present application screens a functional strain combination specific to Jiangxiang Baijiu koji and optimizes the co-culture process, constructs a stable two-bacterial symbiotic metabolism micro-ecology, successfully realizes the simultaneous and efficient improvement of tetramethylpyrazine and guaiacol, and overcomes the limitation of single functional bacteria that can only specifically strengthen one substance in the prior art. The synergistic improvement effect of the two products is significantly higher than that of the traditional single-bacterial fermentation mode. The selected strains are all derived from the Jiangxiang Baijiu fermentation system and are naturally adapted to the special brewing environment of high temperature and high acidity of Jiangxiang Baijiu, solving the problem of poor adaptability and insufficient stability of existing exogenous strains. The function of Cronobacter malonaticus producing guaiacol is first discovered, breaking through the application boundary of existing functional strains. In terms of process design, through precise matching of inoculation ratio, stepwise temperature control and moisture content and other key parameters, the synergistic adaptation of strain proliferation and product synthesis is realized, avoiding the symbiotic imbalance caused by single parameter optimization. The process stability and controllability are far superior to the existing generalized microbial synergistic technology. In terms of application, the solid-state seed preparation uses the core raw materials of Baijiu (Chinese liquor) brewing, and the product can be directly connected to the existing Jiangxiang Baijiu brewing process without the need to adjust the process parameters, which is convenient to operate and has low application cost. Compared with the existing strengthening technology that needs to change the production process, it has more industrialization promotion value, and provides a new technical path for simultaneously improving the flavor richness and health factors of Baijiu (Chinese liquor) industry. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and other accompanying drawings can be obtained by those skilled in the art without any creative effort.

[0017] Figure 1 Figure for colony morphological characteristics of the B. velezensis WQ19 and C. propinquum B1292 co-cultured in the present application; Figure 2 Figure for colony morphological characteristics of the B. velezensis WQ19 in the present application; Figure 3 Figure for colony morphological characteristics of the C. propinquum B1292 in the present application. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments only constitute some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative effort fall within the scope of protection of the present application.

[0019] Embodiment 1 1. Substrate treatment: mix wheat, sorghum and corn in equal quality ratio, crush, pass through a 40-mesh sieve, take 100 g of mixed dry material per portion, put into a conical flask, sterilize by 121℃ high-pressure steam for 30 min, cool to room temperature for standby.

[0020] 2. Seed liquid preparation: The seed liquid of the strains in the present application: the secondary seed liquid preparation conditions of B. velezensis WQ19 (CCTCC NO: M20232058) and C. propinquum B1292 (CCTCC NO: M2025146) are the same: (1) Primary seed liquid: inoculate the strain stored in a glycerol tube into a conical flask containing 30 mL of LB liquid medium at an inoculation amount of 0.5%-5% (v / v), and culture at 37℃ with 180 r / min shaking for 24 h; (2) Secondary seed liquid: transfer the primary seed liquid into a conical flask containing 350 mL of LB liquid medium at an inoculation amount of 1%-5% (v / v), and culture at 37℃ with 180 r / min shaking for 48 h, so that the viable bacterial concentration of WQ19 reaches 8.0×10 5 -1.0×10 7 CFU / mL, and the viable bacterial concentration of B1292 reaches 1.0×10 10-1.0 x 10 12 CFU / mL.

[0021] Single strain control group seed liquid: seed liquid preparation conditions of WQ19 and B1292 alone are the same as above.

[0022] 3. Inoculation and co-culture: Co-culture experiment group (original Table 1 grouping): inoculate WQ19 and B1292 secondary seed liquid and sterile water into the cooled substrate, set the parameters (inoculum, temperature, and moisture are different) according to C1-C11 in the table below, and place it in a constant temperature incubator for culture;

[0023] WQ19 single strain control group: only inoculate WQ19 secondary seed liquid, and set the parameters according to W1-W11 in the table below for culture;

[0024] B1292 single strain control group (original Table 3 grouping): only inoculate B1292 secondary seed liquid, and set the parameters according to B1-B11 in the table below for culture;

[0025] Defensive comparative example: D1: inoculation ratio (WQ19:B1292) is 1:1, and the rest of the parameters are the same as C7 (15%+10% addition amount, moisture 45%, 37℃ 2d+45℃ 3d); D2: constant temperature 37℃ culture for 5d, the rest of the parameters are the same as C7; D3: sterile water addition amount 10% (substrate moisture 40%), the rest of the parameters are the same as C7; D4: inoculation ratio 1:1 + constant temperature 37℃ culture for 5d, the rest of the parameters are the same as C7.

[0026] 4. Product post-treatment: after the culture is completed, the product is placed in a 50℃ oven and dried to constant weight, crushed through a 60 mesh sieve, and solid seeds (co-culture group) or single strain solid products (single strain control group) are obtained.

[0027] Performance verification steps 1. Sample pretreatment: take 1g of each group product, add 10mL of anhydrous ethanol, ultrasonic extraction for 30min, centrifuge at 8000r / min for 10min, take the supernatant and pass through a 0.22μm organic phase filter membrane.

[0028] 2. Detection method: GC-MS method was used to determine the content of tetramethylpyrazine and guaiacol. The chromatographic column was HP-5MS (30 m x 0.25 mm x 0.25 μm), the injection port temperature was 250℃, the programmed temperature was as follows: the initial temperature was 60℃, maintained for 2 min, then increased to 200℃ at the rate of 10℃ / min, maintained for 5 min; the carrier gas was helium, the flow rate was 1.0 mL / min; the ion source temperature was 230℃, the electron impact energy was 70 eV, and the external standard method was used for quantification.

[0029] 3. Validation of detection methodology: Standard curve: the concentration range of tetramethylpyrazine standard was 0.1-100 μg / mL, the linear regression equation was y=12543x+215.6, the correlation coefficient R 2 =0.9996; the concentration range of guaiacol standard was 0.05-50 μg / mL, the linear regression equation was y=9876x+189.3, the correlation coefficient R 2 =0.9993; Spiked recovery rate: the solid seed sample with known content was added with low, medium and high concentration standards, the recovery rate was 88.5%-103.2%, and the RSD was ≤2.8% (n=3); Limit of detection (LOD) and limit of quantification (LOQ): the LOD of tetramethylpyrazine was 0.02 μg / mL, the LOQ was 0.06 μg / mL; the LOD of guaiacol was 0.01 μg / mL, the LOQ was 0.03 μg / mL.

[0030] Validation results Table 1 Co-culture experimental group

[0031] Table 2 WQ19 single bacteria control group

[0032] Table 3 B1292 single bacteria control group

[0033] Table 4 Comparative product data

[0034] Note: The calculation method of the comprehensive improvement range of double products is: (the yield of tetramethylpyrazine in the co-culture group + the yield of guaiacol in the co-culture group) - (the yield of WQ19 single bacteria + the yield of B1292 single bacteria under the same process parameters) ÷ (the yield of WQ19 single bacteria + the yield of B1292 single bacteria under the same process parameters) × 100%; wherein, the C7 group corresponds to the superposition value of W7 single bacteria (463521.5 μg / kg) + B7 single bacteria (101297.6 μg / kg), and the D1-D4 group corresponds to the yield of single bacteria under the same inoculation ratio / temperature condition (for example, the D1 group corresponds to the yield of WQ19 single bacteria + the yield of B1292 single bacteria when the inoculation ratio is 1:1).

[0035] Example 2 Preparation step 1. Substrate treatment, seed liquid preparation (strains of the present application): same as steps 1-2 of Example 1.

[0036] 2. Seed liquid preparation of control strains: Bacillus subtilis q94, Bacillus licheniformis, Bacillus cavenae Y2: cultured according to the seed liquid preparation conditions of Example 1, to ensure that the viable bacterial concentration is consistent with that of the secondary seed liquid of the strains of the present application; Common Cronobacter (standard strain ATCC27506): cultured according to the seed liquid preparation conditions of Example 1, and the viable bacterial concentration reached 5.0×10 11 CFU / mL.

[0037] 3. Inoculation and co-culture: all groups used the optimal process parameters of C7 in Example 1 (inoculation ratio 3:2, moisture 45%, 37°C for 2 days + 45°C for 3 days), only the strain combination was changed: Experimental group S1: WQ19 + B1292 (combination of the present application); Comparative example S2: Bacillus subtilis q94 + Bacillus cavenae Y2; Comparative example S3: Bacillus licheniformis + Common Cronobacter (ATCC27506); Comparative example S4: WQ19 + Bacillus cavenae Y2; Comparative example S5: Bacillus subtilis q94 + B1292; Comparative example W7: only WQ19 single bacteria; Comparative example B7: only B1292 single bacteria.

[0038] 4. Product post-treatment: same as step 4 of Example 1.

[0039] Performance verification step Same as the performance verification step of Example 1.

[0040] Verification results

[0041] Example 3 Preparation step 1. Substrate treatment: same as step 1 of Example 1.

[0042] 2. Preparation of seed liquid of strains from different sources: Experimental group Y1: WQ19 (separated from high-temperature Daqu of Maotai-flavor type) + B1292 (separated from wine Daqu of Maotai-flavor type) of the application, the seed liquid was prepared according to Example 1. Comparative example Y2: Bacillus velezensis standard strain (CCTCC AB93061) + Cronobacter malonaticus standard strain (ATCC 27506), the seed liquid was prepared according to Example 1. Comparative example Y3: Bacillus subtilis q94 separated from Daqu of Qingxiang type + Bacillus sp. Y2 separated from pit mud of Nongxiang type, the seed liquid was prepared according to Example 1.

[0043] 3. Inoculation and co-culture: all groups used the optimal process parameters of C7 in Example 1, and the fermentation environment of Maotai-flavor liquor was simulated (sterile air containing 10% vol ethanol was introduced during the culture process, and the pH of the culture medium was adjusted to 3.5).

[0044] 4. Post-treatment of product: same as step 4 of Example 1.

[0045] Performance verification step 1. Detection of survival rate of strains: after the culture was completed, 1 g of substrate sample was taken, 9 mL of sterile normal saline was added, and gradient dilution was performed to 10 -6 ~10 -8 times, 0.1 mL of the diluent was taken and spread on LB plates, 3 parallel plates were set for each dilution, and the number of colonies was counted after 24 h of culture at 37℃; the number of viable bacteria before inoculation was determined by gradient dilution and spreading of the seed liquid before inoculation, and the survival rate = (number of viable bacteria after culture ÷ number of viable bacteria before inoculation) × 100%, with the result being rounded to one decimal place; 2. Detection of product content: same as the performance verification step of Example 1.

[0046] Verification result

[0047] Example 4 Preparation step 1. Preparation of solid seed: the solid seed of the application was prepared according to the optimal scheme of C7 in Example 1, and the solid seed of WQ19 only (used in group A8), the solid seed of B1292 only (used in group A9), and the "Bacillus licheniformis enhanced Daqu" disclosed by Ge Xiangyang (used in group A7) were also prepared.

[0048] 2. Small-scale brewing of Maotai-flavor liquor: Experimental groups A2, A4, A6: add 2%, 4%, 6% of the solid starter of the application to the heavy sediment, respectively, by weight; Control group A0: no addition of solid starter (blank group); Comparative example A7: add 6% of "Bacillus licheniformis enhanced Daqu" (consistent with the A6 addition amount); Comparative example A8: add 6% of WQ19 solid starter only; Comparative example A9: add 6% of B1292 solid starter only; Comparative example A10: add 6% of inactivated solid starter (500°C baking for 30 min, verified by plate coating that there is no live bacteria), and the rest of the process is the same as group A6.

[0049] 3. Brewing process: all groups use the traditional process of Jiangxiang Baijiu (Luzhou) to pile up in the airing room until the product temperature reaches 45°C, then enter the cellar, and after 30 days of fermentation, distill to obtain the raw liquor.

[0050] Performance verification steps 1. Detection of target products in raw liquor: take 10 mL of raw liquor from each group, directly pass through a 0.22 μm organic phase filter membrane, and use the GC-MS method of Example 1 to determine the concentrations of tetramethylpyrazine and guaiacol; 2. Sensory evaluation: a panel of 10 professional liquor tasters is formed, and blind evaluation is conducted according to the sensory evaluation standards of Jiangxiang Baijiu (aroma, taste, style), with a full score of 100 points, and the average value is taken.

[0051] 3. Product improvement rate: Tetramethylpyrazine improvement multiple = (experimental group tetramethylpyrazine concentration - A0 group tetramethylpyrazine concentration) ÷ A0 group tetramethylpyrazine concentration Guaiacol improvement multiple = (experimental group guaiacol concentration - A0 group guaiacol concentration) ÷ A0 group guaiacol concentration Verification results

[0052] Example 5 Preparation steps 1. Continuous batch solid starter preparation (P1-P3): according to the optimal scheme of C7 in Example 1, continuously prepare 3 batches of solid starter, each with a feed amount of 10 kg (laboratory scale-up), record the live bacteria concentration of each batch of seed liquid, substrate temperature change during culture, and product drying time.

[0053] 2. Pilot scale-up preparation (P4): A pilot test was carried out in a 100 L fermenter, and the substrate treatment, seed preparation, and process parameters were consistent with the small test, and the feeding amount was 50 kg; the temperature control of the fermenter: the temperature difference between the upper and lower layers was maintained ≤2℃ by the tank wall segmented heating device, and was monitored and recorded in real time; the moisture content was detected every 2 h, and the moisture content was maintained at 45%±1% by automatic addition of sterile water; the aeration amount was 0.5 vvm, and the stirring speed was 50 r / min (according to the power amplification principle, the stirring intensity was adjusted by 1:50 ratio according to the small test).

[0054] 3. Application of Jiang-flavor liquor pilot test: The solid-state seed prepared by the pilot test was added at 6% based on the weight of the fermented grains, and a 500 kg fermented grains was used for the pilot test of Jiang-flavor liquor, and the process was the same as that of Example 4.

[0055] Performance verification steps 1. Solid-state seed stability detection: The contents of tetramethylpyrazine and guaiacol in 3 batches of laboratory scale-up solid-state seeds and pilot solid-state seeds were detected, and the relative standard deviation (RSD) was calculated; 2. Raw liquor effect verification: The concentration of target product in the pilot test raw liquor and the sensory score were detected, and compared with the small test A6 group.

[0056] Verification results

[0057] The experimental results of Examples 1-5 show that, by combining Bacillus velezensis WQ19 and the exclusive strain of Cronobacter B1292, using the optimization process of 3:2 inoculation ratio, 37℃ culture for 2d+45℃ culture for 3d stepwise temperature control, and 15% sterile water addition amount, a stable symbiotic metabolic microecology is successfully constructed. According to the data of the complete co-culture group (original table 1, 4) and the single bacteria control group (original table 2, 3, 5, 6) in Example 1, the co-culture product is significantly improved compared with the double product superposition value of single bacteria fermentation, the yield of tetramethylpyrazine is increased by 45.52%, and the yield of guaiacol is increased by 72.28%, which is much higher than the single parameter optimization group and the conventional strain combination. After the solid-state seed is applied to the brewing of Jiang-flavor liquor, the concentrations of the two products are significantly improved compared with the blank group and the existing technology strengthening scheme, and the process stability is verified by continuous batch and pilot test, which fully proves the innovativeness and practicability of the scheme of the present application.

[0058] The above only describes the preferred embodiments of the present application and should not be used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A strain combination for efficient production of tetramethylpyrazine and guaiacol and its co-culture method, characterized in that, Includes the following steps: (1) Selected functional strains: Bacillus velezensis WQ19 with accession number CCTCC NO: M20232058 and Cronobactermalonaticus B1292 with accession number CCTCC NO: M2025146; (2) Substrate treatment: Wheat, sorghum and corn are mixed and crushed, sterilized and cooled to room temperature; (3) Co-culture: Inoculate the two strains of bacteria into the cooled substrate with secondary seed culture, add sterile water, and culture under the set conditions; (4) Product preparation: The cultured product is dried and pulverized to obtain solid seed.

2. The method according to claim 1, characterized in that, The secondary seed culture mentioned in step (3) includes Bacillus belyssus WQ19 secondary seed culture and Cronobacter malonic acid B1292 secondary seed culture, and the preparation conditions for both are the same, specifically: (1) Preparation of primary seed culture: The strain preserved in glycerol tubes was inoculated into a conical flask containing 30 mL of LB liquid medium at an inoculation rate of 0.5%-5% (v / v) and cultured at 37℃ and 180 r / min for 24 h with shaking. (2) Preparation of secondary seed culture: The primary seed culture was transferred to an Erlenmeyer flask containing 350 mL LB liquid medium at an inoculation rate of 1%-5% (v / v) and cultured at 37℃ and 180 r / min for 48 h.

3. The method according to claim 2, characterized in that, The inoculation ratio of the Bacillus belyssus WQ19 secondary seed culture to the Cronobacter malonic acid B1292 secondary seed culture is 3:2 by volume.

4. The method according to claim 2, characterized in that, The amount of the Bacillus vesiculosus WQ19 secondary seed liquid added is 15% of the dry weight of the mixed wheat, sorghum and corn, by mass.

5. The method according to claim 4, characterized in that, The amount of the secondary seed culture of Cronobacter malonic acid B1292 added is 10% of the dry weight of the mixture of wheat, sorghum and corn, by mass.

6. The method according to claim 1, characterized in that, The amount of sterile water added is 10%-20% of the dry weight of the mixture of wheat, sorghum and corn, by mass.

7. The method according to claim 6, characterized in that, The amount of sterile water added is 15% of the dry weight of the mixture of wheat, sorghum, and corn.

8. The method according to claim 1, characterized in that, The culture conditions set in step (3) are: first culture at 37℃ for 2 days, and then culture at 45℃ for 3 days.

9. The method according to claim 1, characterized in that, The drying temperature in step (4) is 50°C.

10. The method according to any one of claims 1-9, characterized in that, The solid-state seed is used in the brewing of Maotai-flavor liquor, and the amount of solid-state seed added is 2%-6% of the weight of the Maotai-flavor liquor mash by mass.

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