A microbial compound inoculant for brewing and its application

Abstract

This invention discloses a microbial compound inoculant for brewing and its application, belonging to the field of microbial materials technology. The inoculant is composed of *Diplostomum cuspidatum* (…). Saccharomycopsis brooch ), Pediococcus acidilactici ( Pediococcus lactic acid ) and Bacillus stearothermia ( Bacillus spizizen This compound microbial agent is obtained through a stepped solid-state fermentation process. Through the synergistic effect of the three strains mentioned above, combined with the stepped solid-state fermentation process, its addition to the brewing process of light-aroma baijiu can produce more aroma and flavor compounds, increase the variety and content of aroma components, and simultaneously reduce the proportion of higher alcohols, resulting in a smoother, sweeter, and cleaner baijiu with a richer flavor. Furthermore, this compound microbial agent exhibits good adaptability to various raw materials and fermentation environments. Its preparation method is simple and cost-effective, making it highly suitable for large-scale baijiu production.

Description

Technical Field

[0001] This invention belongs to the field of microbial materials technology, specifically relating to a microbial compound agent for brewing and its application. Background Technology

[0002] Baijiu, generally speaking, is a distilled spirit with a unique brewing flavor, made primarily from grains and cereals, using various types of yeast as the main saccharifying and fermenting agents, and undergoing artificial fermentation, distillation, storage, and blending. While light-aroma baijiu possesses its own unique characteristics, it also shares commonalities with other distilled spirits.

[0003] Most light-aroma baijiu uses clean cement tanks as the main solid-state fermentation tanks, with grains with high starch content as the main raw material. It is brewed using traditional solid-state distillation methods, going through processes such as low-temperature fermentation, distillation, aging, and blending. In this process, no edible ethanol or non-baijiu edible additives are artificially or directly added. Instead, various aroma and flavor substances are naturally produced during solid-state fermentation, resulting in a complex aroma with ethyl acetate as the main component.

[0004] Based on the different saccharification and fermentation agents used, baijiu can be divided into three categories: daqu (large koji) baijiu, xiaoqu (small koji) baijiu, and bran koji baijiu. Bran koji baijiu is characterized by short fermentation time, high yield, and a clear, crisp body. However, compared to other light-aroma baijiu, bran koji baijiu has fewer aroma compounds, lower content, higher alcohol content, and a bland flavor. Therefore, improving the flavor of bran koji baijiu has become a key research focus. Adding functional bacteria and using combined fermentation methods during alcoholic beverage fermentation can enhance the flavor and improve the quality of the liquor.

[0005] For example, Chinese patent application CN202211494388.6 discloses an isolated strain of *Diospyros koraiensis*, a brewing agent composition, and its application in the production of bran-fermented baijiu. This invention provides an isolated strain of *Diospyros koraiensis*, a brewing agent composition, and its application in the production of bran-fermented baijiu, relating to the fields of microbial engineering and brewing technology. The strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number: M20221117; deposit date: July 15, 2022. This invention uses the bran-fermented koji prepared from the first isolated *Diospyros koraiensis* strain HXJ29, combined with an ester-producing yeast culture, in the brewing of light-aroma bran-fermented baijiu. This increases the content of ethyl acetate and ethyl lactate in the baijiu, enriches the types of sweet substances in the original liquor, increases the mellow sweetness of the original liquor, and also improves the yield.

[0006] While current microbial agents for brewing have shown some effectiveness in enhancing the flavor of wheat bran-based baijiu, several shortcomings remain. Some agents exhibit poor adaptability to different raw materials and fermentation environments, making large-scale application difficult. This results in inconsistent flavor improvement and a limited range of flavor profiles, failing to meet the increasing demand for higher-quality baijiu. Furthermore, self-screened strains are difficult to scale up production, and the high cost of these agents restricts their widespread adoption in large-scale brewing production. Therefore, it is essential to develop a compound microbial agent for brewing that can adapt to various environments, exhibits good synergistic effects among strains, and is cost-effective and easy to promote. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by proposing a compound microbial agent for brewing and its application. The compound agent is prepared by a stepped solid-state fermentation process using *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stenosum*. Through the synergistic effect of these three strains combined with the stepped solid-state fermentation process, more aroma and flavor compounds are produced during the fermentation and brewing of light-aroma baijiu, increasing the variety and content of aroma components while reducing the proportion of higher alcohols, resulting in a smoother, sweeter, and cleaner baijiu with a richer flavor. Furthermore, this compound agent exhibits good adaptability to the fermentation environment, its preparation method is simple, and its cost is controllable, making it highly suitable for large-scale brewing production.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A microbial compound inoculant for brewing, wherein the inoculant is composed of *Saccharomyces cerevisiae* (…). Saccharomycopsis fibuligera ), Pediococcus acidilactici ( Pediococcus acidilactici ) and Bacillus stearothermia ( Bacillus spizizenii It is obtained through step-by-step solid-state fermentation.

[0009] Furthermore, the strain number of *Diplostomum cladosporium* is CGMCC No. 2.5608, and its original deposit date is April 20, 2016; the strain number of *Pediococcus lactis* is CGMCC No. 1.2696, and its original deposit date is November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; the strain number of *Bacillus stearothermiae* is GDMCC NO. 1.136, which was purchased from the Guangdong Provincial Microbiological Culture Collection Center, and its original deposit date is July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0010] Furthermore, the effective viable count of the compound microbial agent is 2-4 × 10⁻⁴. 9 CFU / g.

[0011] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (4) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with 6% of Bacillus stearotherm seed liquid. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture in time to ensure oxygen supply and uniform fermentation. (5) Second-stage fermentation: Inoculate the product of the first-stage fermentation with Pediococcus lactis seed liquid at an inoculation amount of 8%, and carry out facultative anaerobic fermentation at 28-30℃ for 36 hours, controlling the oxygen content of the fermentation environment at 5%-10%; (6) Three-stage fermentation: Inoculate the product from the second-stage fermentation with 10% of the *Saccharomyces cerevisiae* seed culture. Perform anaerobic fermentation at 28-30℃ for 48-96 hours, ensuring an anaerobic environment by sealing the fermentation container. The effective viable count should be 3-5 × 10⁻⁵. 9 Fermentation ends when CFU / g is reached; after fermentation, 5% trehalose and 2% skim milk powder by weight of fermented material are added, and the product is then vacuum dried at room temperature until the moisture content is less than 15%, and then pulverized through an 80-mesh sieve to obtain the microbial compound inoculant for brewing.

[0012] Furthermore, the composition of the malt extract liquid culture medium is as follows: 3g yeast extract powder, 10g glucose, 4g peptone, 10g malt extract powder, 1000mL distilled water, dissolved in water and sterilized at 121℃ for 20min.

[0013] An application of a microbial compound agent for brewing, used in the brewing of light-aroma baijiu.

[0014] The microbial agent of this invention is used in the brewing of light-aroma baijiu (Chinese liquor). The specific brewing method is as follows: Soaking the sorghum: After washing the sorghum, add 72℃ water to a level 10cm above the grain surface and soak it. Keep it warm at 60-70℃ for 4-6 hours.

[0015] Initial steaming: After soaking the sorghum, drain the water and steam it at 90-100℃ for 20 minutes.

[0016] Steaming the grain: After the initial steaming, spread the sorghum to cool to about 80℃, add water at 65℃ to a level 10cm above the grain, and soak for 10-30 minutes.

[0017] Re-steaming: After the sorghum has been steamed and drained, it is re-steamed at 95℃ for 20 minutes.

[0018] Spreading the grain and adding koji: After re-steaming, spread the sorghum to cool to between 25-30℃ and add koji. Add the microbial agent of this invention at an addition rate of 1% of the original grain mass.

[0019] Fermentation in the cellar: After adding koji, the sorghum is mixed evenly and put into the fermentation cellar. The cellar opening is sealed and fermented at 25-28℃ for 20 days. During this period, the temperature inside the cellar and the state of the mash are checked regularly.

[0020] Distillation: After fermentation, the mash is distilled, and the distilled liquid is collected. After aging, blending and other processes, it is finally made into light-aroma baijiu.

[0021] Beneficial effects: (1) The present invention screened out three microorganisms with the function of secreting active enzymes: *Saccharomyces cerevisiae*, *Pediococcus lactis* and *Bacillus stearothermiae*, and composed them into core microorganisms. Combined with a step-by-step fermentation process, synergistic enrichment and efficient accumulation of precursor substances were achieved.

[0022] (2) Under the aerobic conditions of the first stage, Bacillus stearothermiae preferentially and rapidly reproduces and secretes abundant hydrolytic enzymes such as amylase and protease, which can efficiently decompose macromolecules (starch and protein) in the bran and rice husk matrix to generate soluble sugars, peptides and amino acids, providing a high-quality nutritional basis for subsequent strains and improving the physical structure of the material.

[0023] (3) In the second stage, the environment is switched to facultative anaerobic and Pediococcus lactis is inoculated. This bacterium uses the products of the first stage to grow rapidly, producing large amounts of organic acids such as lactic acid and acetic acid, which significantly reduces the pH value of the system, effectively inhibits the growth of other bacteria, and purifies the fermentation microenvironment. The accumulated organic acids are also the core flavor precursors for subsequent ester synthesis.

[0024] (4) In the third stage, under strict anaerobic conditions, the yeast can proliferate efficiently and activate its anaerobic metabolic pathway in an optimized environment of rich nutrition, low pH and no interference from other bacteria (although it does not produce a large amount of wine or esters, it lays the enzyme system foundation for subsequent aroma and alcohol production).

[0025] (5) This staged, on-demand oxygen supply "relay" fermentation avoids growth inhibition between strains, allowing each strain to perform its unique function (hydrolysis, acid production, anaerobic metabolic activation) in the optimal environment, forming a powerful synergistic network. The step-by-step process not only enriches live bacteria, but also accumulates functional substances beneficial to subsequent baijiu brewing through the synergistic and targeted accumulation of strains.

[0026] (6) The large amount of hydrolytic enzymes (amylase, protease) produced by Bacillus stearothermiae in the first stage are retained in the inoculum, providing a strong impetus for grain saccharification in the early stage of brewing. The lactic acid and acetic acid produced by Pediococcus lactis in the second stage serve as precursors of core acids and are effectively enriched in the solid matrix, becoming direct substrates for the yeast to synthesize the main aroma (ethyl acetate) and mellow sweetness (ethyl lactate) in the subsequent brewing stages. Although Diplostomum tumefaciens does not synthesize a large amount of esters in the third stage, its intracellular ester synthesis enzyme system is effectively induced and preserved, preparing it for subsequent efficient catalytic esterification reactions.

[0027] (7) During the subsequent fermentation process, the three strains in this compound microbial agent can further exert a synergistic effect. The hydrolytic enzymes produced by Bacillus stearothermiae in the early stage continue to deeply decompose the starch and protein in raw materials such as sorghum, providing more nutrients such as sugars and amino acids for fermentation, and promoting the growth and metabolism of microorganisms. The organic acids accumulated by Pediococcus lactis in the early stage provide good conditions for ester synthesis, and can better cooperate with Diplostomum tumefaciens at this stage. Diplostomum tumefaciens utilizes the existing nutrients and the high-quality environment of low pH and no interference from other microorganisms to further activate its anaerobic metabolic pathway, proliferate efficiently, and provide more enzyme support for aroma and alcohol production reactions.

[0028] (8) Through this synergistic effect, more aroma and flavor substances, such as various esters and alcohols, can be produced during the fermentation process, making the aroma of the mash more intense and rich, and the proportion of higher alcohols can be effectively controlled, thereby improving the quality and flavor of the final light aroma type baijiu, and making the liquor more mellow, sweet and refreshing.

[0029] (9) By adding the microbial agent of the present invention during the brewing process of light aroma baijiu, the flavor of bran koji baijiu can be effectively improved, solving the problems of low aroma components, low content, high content of higher alcohols and bland flavor in existing bran koji light aroma baijiu, thus improving the quality and taste of bran koji baijiu. Attached Figure Description

[0030] Figure 1 This is a graph showing the pH tolerance results of the *Saccharomyces cerevisiae* CGMCC No. 2.5608 of this invention; Figure 2 This is a graph showing the alcohol tolerance results of the *Saccharomyces cerevisiae* CGMCC No. 2.5608 of this invention; Figure 3 This is a graph showing the pH tolerance results of *Pediococcus lactis* CGMCC No. 1.2696, as described in this invention. Figure 4 This is a graph showing the alcohol tolerance results of *Pediococcus lactis* CGMCC No. 1.2696, as described in this invention. Figure 5 This is a graph showing the pH tolerance results of Bacillus stearothermiae GDMCC NO.1.136 in this invention; Figure 6 This is a graph showing the alcohol tolerance results of Bacillus stearothermia GDMCC NO.1.136 in this invention. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0032] Example 1 A microbial compound agent for brewing, wherein the agent is obtained by stepwise solid-state fermentation of *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stearothermiae*.

[0033] The strain of *Diplostomum cladosporium* is CGMCC No. 2.5608, with an original deposit date of April 20, 2016; the strain of *Pediococcus lactis* is CGMCC No. 1.2696, with an original deposit date of November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; the strain of *Bacillus stearothermiae* is GDMCC NO. 1.136, purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0034] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (4) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with 6% of Bacillus stearotherm seed liquid. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture in time to ensure oxygen supply and uniform fermentation. (5) Second-stage fermentation: Inoculate the product of the first-stage fermentation with Pediococcus lactis seed liquid at an inoculation amount of 8%, and carry out facultative anaerobic fermentation at 28-30℃ for 36 hours, controlling the oxygen content of the fermentation environment at 5%-10%; (6) Three-stage fermentation: Inoculate the product from the second-stage fermentation with *Streptomyces hygroscopicus* seed culture at an inoculation rate of 10%, and anaerobic ferment at 28-30℃ for 48-96 hours. Ensure an anaerobic environment by sealing the fermentation container. The effective viable count should be 3 × 10⁻⁶. 9 Fermentation ends at CFU / g; after fermentation, add 5% trehalose and 2% skim milk powder by weight of the fermented product, then vacuum dry the product at room temperature until the moisture content is below 15%, pulverize and pass through an 80-mesh sieve to obtain the microbial compound agent for brewing, wherein the effective viable count of the compound agent is 2-3 × 10⁻⁶. 9 CFU / g.

[0035] The composition of the malt extract liquid culture medium is as follows: 3g yeast extract powder, 10g glucose, 4g peptone, 10g malt extract powder, 1000mL distilled water, dissolved in water and sterilized at 121℃ for 20min.

[0036] Strain tolerance test: Seed cultures of *Streptomyces hygroscopicus*, *Pediococcus lactis*, and *Bacillus steudensis* were prepared according to the method described in this embodiment. The seed cultures were inoculated at 5% (v / v) into LB liquid medium with different pH values ​​(2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5) and different ethanol volume fractions (3%, 6%, 9%, 12%, 15%, 18%). After incubation at 30℃ and 160 r / min for 24 h, the absorbance of the culture was measured at a wavelength of 600 nm. The test results are as follows: Figure 1-5 As shown, The results showed that the three strains screened in this invention exhibited good tolerance to different pH and ethanol environments. This means they can stably survive and function in complex and variable brewing environments, further ensuring the smooth progress of the brewing process and the stability of the liquor quality. At the same time, this good tolerance also reflects the excellent characteristics of the strains selected in this invention, providing a strong microbial foundation for achieving efficient and high-quality light-aroma baijiu brewing.

[0037] Example 2 A microbial compound agent for brewing, wherein the agent is obtained by stepwise solid-state fermentation of *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stearothermiae*.

[0038] The strain of *Diplostomum cladosporium* is CGMCC No. 2.5608, with an original deposit date of April 20, 2016; the strain of *Pediococcus lactis* is CGMCC No. 1.2696, with an original deposit date of November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; the strain of *Bacillus stearothermiae* is GDMCC NO. 1.136, purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0039] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (4) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with 6% of Bacillus stearotherm seed liquid. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture in time to ensure oxygen supply and uniform fermentation. (5) Second-stage fermentation: Inoculate the product of the first-stage fermentation with Pediococcus lactis seed liquid at an inoculation amount of 8%, and carry out facultative anaerobic fermentation at 28-30℃ for 36 hours, controlling the oxygen content of the fermentation environment at 5%-10%; (6) Three-stage fermentation: Inoculate the product from the second-stage fermentation with *Streptomyces hygroscopicus* seed culture at an inoculation rate of 10%, and anaerobic ferment at 28-30℃ for 48-96 hours. Ensure an anaerobic environment by sealing the fermentation container. The effective viable count should be 5 × 10⁻⁶. 9 Fermentation was terminated when CFU / g was reached. After fermentation, 5% trehalose and 2% skim milk powder (by weight of fermented product) were added, and the product was then vacuum-dried at room temperature until the moisture content was below 15%. The product was then pulverized and passed through an 80-mesh sieve to obtain a microbial compound agent for brewing. The effective viable count of the compound agent was 3-4 × 10⁻⁴ CFU / g. 9 CFU / g.

[0040] The composition of the malt extract liquid culture medium is as follows: 3g yeast extract powder, 10g glucose, 4g peptone, 10g malt extract powder, 1000mL distilled water, dissolved in water and sterilized at 121℃ for 20min.

[0041] Example 3 An application of a microbial compound agent for brewing, used in the brewing of light-aroma baijiu.

[0042] The microbial agent of this invention is used in the brewing of light-aroma baijiu. The specific brewing method is as follows: Soaking the sorghum: After washing the sorghum, add 72℃ water to a level 10cm above the grain surface and soak it. Keep it warm at 60-70℃ for 4-6 hours.

[0043] Initial steaming: After soaking the sorghum, drain the water and steam it at 90-100 ℃ for 20 minutes.

[0044] Steaming the grain: After the initial steaming, spread the sorghum to cool to about 80℃, add water at 65℃ to a level 10cm above the grain, and soak for 30 minutes to steam the grain.

[0045] Re-steaming: After the sorghum has been steamed and drained, it is re-steamed at 95℃ for 20 minutes.

[0046] Spreading the grain and adding koji: After re-steaming, spread the sorghum to cool to between 25-30℃ and add koji. Add the microbial agent of this invention at an addition rate of 1% of the original grain mass.

[0047] Fermentation in the cellar: After adding koji, the sorghum is mixed evenly and put into the fermentation cellar. The cellar opening is sealed and fermented at 25-28℃ for 20 days. During this period, the temperature inside the cellar and the state of the mash are checked regularly.

[0048] Distillation: After fermentation, the mash is distilled, and the distilled liquid is collected. After aging, blending and other processes, it is finally made into light-aroma baijiu.

[0049] Comparative Example 1 In this comparative example, the treatment only used a compound microbial agent composed of *Diplostomum cladosporium* and *Pediococcus lactis*; all other raw materials and process steps were the same as in Example 1. That is: A microbial compound inoculant for brewing, wherein the inoculant is obtained by stepwise solid-state fermentation of *Diplostomum spp.* and *Pediococcus lactis*.

[0050] The strain of *Cyclocarya paliurus* is numbered CGMCC No. 2.5608, and its original deposit date was April 20, 2016; the strain of *Pediococcus lactis* is numbered CGMCC No. 1.2696, and its original deposit date was November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; both strains are commercially available and do not require biological preservation.

[0051] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) First-stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with Pediococcus lactis seed liquid at an inoculation rate of 8%. Perform facultative anaerobic fermentation at 28-30℃ for 36 hours, and control the oxygen content of the fermentation environment at 5%-10%. (4) Second-stage fermentation: Inoculate the first-stage fermentation product with *Streptomyces chrysogenum* seed culture at an inoculation rate of 10%, and anaerobic ferment at 28-30℃ for 48-96 hours. Seal the fermentation container to ensure an anaerobic environment, and detect the effective viable count as 3×10⁶. 9 Fermentation ends at CFU / g; after fermentation, add 5% trehalose and 2% skim milk powder by weight of the fermented product, then vacuum dry the product at room temperature until the moisture content is below 15%, pulverize and pass through an 80-mesh sieve to obtain the microbial compound agent for brewing, wherein the effective viable count of the compound agent is 2-3 × 10⁻⁶. 9 CFU / g.

[0052] Comparative Example 2 In this comparative example, the treatment only used a compound microbial agent composed of *Bacillus spp.* and *Bacillus stearothermiae*; all other raw materials and process steps were the same as in Example 1. That is: A microbial compound inoculant for brewing, wherein the inoculant is obtained by stepwise solid-state fermentation of *Bacillus subtilis* and *Bacillus stearothermiae*.

[0053] The strain of *Cyclocarya spp.* is CGMCC No. 2.5608, with an original deposit date of April 20, 2016; the strain of *Bacillus stearothermiae* is GDMCC NO. 1.136, purchased from the Guangdong Provincial Microbial Culture Collection Center, with an original deposit date of July 1, 2003. Both strains are commercially available and do not require biological preservation.

[0054] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (3) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with 6% of Bacillus stearotherm seed liquid. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture in time to ensure oxygen supply and uniform fermentation. (4) Second-stage fermentation: Inoculate the first-stage fermentation product with *Streptomyces chrysogenum* seed culture at an inoculation rate of 10%, and anaerobic ferment at 28-30℃ for 48-96 hours. Seal the fermentation container to ensure an anaerobic environment, and detect the effective viable count as 3×10⁶. 9 Fermentation ends at CFU / g; after fermentation, add 5% trehalose and 2% skim milk powder by weight of the fermented product, then vacuum dry the product at room temperature until the moisture content is below 15%, pulverize and pass through an 80-mesh sieve to obtain the microbial compound agent for brewing, wherein the effective viable count of the compound agent is 2-3 × 10⁻⁶. 9 CFU / g.

[0055] Comparative Example 3 In this comparative example, the treatment only used a compound microbial agent composed of *Pediococcus lactis* and *Bacillus steudensis*; all other raw materials and process steps were the same as in Example 1. That is: A microbial compound inoculant for brewing, wherein the inoculant is obtained by step solid-state fermentation of Pediococcus lactis and Bacillus stearothermia.

[0056] The *Pediococcus lactis* strain numbered CGMCC No. 1.2696, with an original deposit date of November 23, 2000; the *Bacillus stearothermiae* strain numbered GDMCC NO. 1.136, purchased from the Guangdong Provincial Microbial Culture Collection Center, with an original deposit date of July 1, 2003. Both strains are commercially available and do not require biological preservation.

[0057] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (2) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (3) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with 6% of Bacillus stearotherm seed liquid. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture in time to ensure oxygen supply and uniform fermentation. (4) Second-stage fermentation: Inoculate the product from the first-stage fermentation with *Pediococcus lactis* seed culture at an inoculation rate of 8%, and perform facultative anaerobic fermentation at 28-30℃ for no less than 36 hours, controlling the oxygen content of the fermentation environment at 5%-10%; the effective viable count is 3×10⁻⁶. 9 Fermentation ends at CFU / g; after fermentation, add 5% trehalose and 2% skim milk powder by weight of the fermented product, then vacuum dry the product at room temperature until the moisture content is below 15%, pulverize and pass through an 80-mesh sieve to obtain the microbial compound agent for brewing, wherein the effective viable count of the compound agent is 2-3 × 10⁻⁶. 9 CFU / g.

[0058] Comparative Example 4 In this comparative example, except for changing the strain type of *Streptomyces cladosporium* (i.e., using other commercially available strains), the raw materials and process steps are the same as in Example 1. That is: A microbial compound agent for brewing, wherein the agent is obtained by stepwise solid-state fermentation of *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stearothermiae*.

[0059] The *Diplostomum tumefaciens* strain number B94979 was purchased from Mingzhou Biotechnology, located at 3rd Floor, Building E4, Zhizao Port, No. 33, Lane 866, Jinghui Road, High-tech Zone, Ningbo City, Zhejiang Province; the *Pediococcus lactis* strain number CGMCC No. 1.2696, with an original deposit date of November 23, 2000, was purchased from the China General Microbiological Culture Collection Center; the *Bacillus stearothermii* strain number GDMCC NO. 1.136 was purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0060] Comparative Example 5 In this comparative example, except for changing the strain of *Pediococcus lactis* (i.e., using other commercially available strains), the raw materials and process steps are the same as in Example 1. That is: A microbial compound agent for brewing, wherein the agent is obtained by stepwise solid-state fermentation of *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stearothermiae*.

[0061] The *Cyclocarya paliurus* strain numbered CGMCC No. 2.5608, with an original deposit date of April 20, 2016, was purchased from the China General Microbiological Culture Collection Center; the *Pediococcus lactis* strain numbered HZB224879 was purchased from Gray Algae Biotechnology, Room 302, Building 4, No. 33, Shidi Street, Qingshanhu Subdistrict, Lin'an District, Hangzhou City, Zhejiang Province; and the *Bacillus stearothermii* strain numbered GDMCC NO. 1.136 was purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0062] Comparative Example 6 In this comparative example, except for changing the strain type of Bacillus stearothermiae (i.e., using other commercially available strains), the raw materials and process steps are the same as in Example 1. That is: A microbial compound agent for brewing, wherein the agent is obtained by stepwise solid-state fermentation of *Diplostomum spp.*, *Pediococcus lactis*, and *Bacillus stearothermiae*.

[0063] The strain of *Cyclocarya pallida* is CGMCC No. 2.5608, with an original deposit date of April 20, 2016; the strain of *Pediococcus lactis* is CGMCC No. 1.2696, with an original deposit date of November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; the strain of *Bacillus stearothermiae* is GDMCC NO. 1.2250, purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of September 9, 2020. All three strains are commercially available and do not require biological preservation.

[0064] Comparative Example 7 This comparative example differs from Example 1 only in that it uses mixed fermentation, except for the altered step-fermentation process. All other raw materials and process steps remain the same. A microbial compound inoculant for brewing, wherein the inoculant is composed of *Saccharomyces cerevisiae* (…). Saccharomycopsis fibuligera ), Pediococcus acidilactici ( Pediococcus acidilactici ) and Bacillus stearothermia ( Bacillus spizizenii It is obtained through step-by-step solid-state fermentation.

[0065] The strain of *Diplostomum cladosporium* is CGMCC No. 2.5608, with an original deposit date of April 20, 2016; the strain of *Pediococcus lactis* is CGMCC No. 1.2696, with an original deposit date of November 23, 2000. Both strains were purchased from the China General Microbiological Culture Collection Center; the strain of *Bacillus stearothermiae* is GDMCC NO. 1.136, purchased from the Guangdong Provincial Microbiological Culture Collection Center, with an original deposit date of July 1, 2003. All three strains are commercially available and do not require biological preservation.

[0066] A method for preparing a microbial compound inoculant for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum*. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (4) Mixed fermentation: Wheat bran and rice husks were mixed evenly at a mass ratio of 7:3 and the moisture content was adjusted to 50%. The mixture was sterilized at 121℃ for 30 minutes to obtain a mixed material. Bacillus stearothermii seed liquid was inoculated at 6%, Pediococcus lactis seed liquid at 8%, and Diplostomum tumefaciens seed liquid at 10% simultaneously into the mixed material. Aerobic fermentation was carried out at 28-30℃ for 48 hours, facultative anaerobic fermentation was carried out at 28-30℃ for 36 hours, and finally anaerobic fermentation was carried out at 28-30℃ for 48-96 hours. The effective viable bacteria count was detected to be 3×10⁻⁶. 9 After fermentation at CFU / g, 5% trehalose and 2% skim milk powder (by weight of fermented product) are added. The product is then vacuum-dried at room temperature until the moisture content is below 15%, pulverized, and passed through an 80-mesh sieve to obtain a microbial compound agent for brewing. The effective viable count of the compound agent is 2-3 × 10⁻⁶. 9 CFU / g.

[0067] Performance testing Determination of esterification power, liquefaction power and saccharification power of different bacterial agents: The microbial agent powder was prepared according to the methods of Examples 1-2 and Comparative Examples 1-7. Each group of experiments was repeated three times, and the average value of the results was taken.

[0068] (1) Determination of esterification power: Preparation of esterification solution: Accurately weigh 5.0 g of the microbial agent to be tested, and place it into a 250.0 mL Erlenmeyer flask containing 0.75 mL of hexanoic acid, 12.5 mL of anhydrous ethanol, and 37.5 mL of distilled water. Use a solution without added koji powder as a blank control. Treat the solution in a constant temperature water bath at 30℃ with shaking at 100 r / min for 10 min, and then esterify at 30℃ for 100 h.

[0069] Transfer the esterified solution to a 250.0 mL distillation flask, repeatedly wash the conical flask with 50 mL of 30% ethanol, pour the washing solution into the distillation flask, shake well, heat slowly to distill, and collect 50.0 mL of distillate into a 100.0 mL conical flask for esterification power determination.

[0070] Esterification power determination: Add 2 drops of 1% phenolphthalein indicator to a stoppered conical flask, add sodium hydroxide standard solution to neutralize until the distillate turns light pink, record the volume of sodium hydroxide standard solution consumed, then accurately add 25.0 mL of sodium hydroxide standard solution, mix well, reflux in a boiling water bath for 30 min, cool to room temperature, add sulfuric acid standard solution until the light pink color just disappears, record the volume of sulfuric acid standard solution consumed.

[0071] The esterification power is calculated using the formula: A = (C1×25-C2×V1) / 50 ×142; A = A1 - A0; A: esterification power of the sample, C1: standard concentration of sodium hydroxide, C2: standard concentration of sulfuric acid solution, V1: volume of sulfuric acid consumed.

[0072] (2) Determination of saccharification power: Preparation of saccharification solution: Accurately weigh 5.0 g of the microbial inoculant powder to be tested and place it in a 100 mL volumetric flask containing 10 mL of acetate-sodium acetate buffer solution. Shake well and add distilled water to bring the volume to 100 mL. Place the volumetric flask in a constant temperature water bath at 35℃ for 1 h, filter, and collect the filtrate for saccharification power determination.

[0073] Saccharification power determination: Pipette 10.0 mL of extract, 50.0 mL of soluble starch, and 2 mL of 20% sodium hydroxide solution into an Erlenmeyer flask, shake well, and add 10.0 mL as a blank control to a 150.0 mL Erlenmeyer flask containing 10 mL each of Fehling's reagent A and B. Add 2 drops of methylene blue indicator, heat to a gentle boil on an electric furnace and maintain for 2 min, and add glucose standard solution until the blue color of the solution completely disappears and turns pale yellow. Record the volume of glucose standard solution. This titration operation must be completed within 1 min.

[0074] Pipette 10.0 mL of Daqu extract and 50.0 mL of soluble starch into an Erlenmeyer flask, shake well, and place in a 35℃ constant temperature water bath for saccharification for 1 h. Add 2 mL of 20% sodium hydroxide solution and shake well. Pipette 10.0 mL of the saccharified solution into a 150.0 mL Erlenmeyer flask containing 10 mL each of Fehling's reagent A and B. Add 2 drops of methylene blue indicator, place in an electric furnace, heat to a gentle boil, and maintain for 2 min. Add glucose standard solution dropwise until the blue color of the solution completely disappears and turns pale yellow. Record the volume of glucose standard solution. This titration operation must be completed within 1 min.

[0075] The saccharifying power is calculated using the formula: X = (V1-V2)×2.5×30 / (0.25×5) V1: The volume of glucose standard solution consumed during blank titration; V2: The volume of glucose standard solution consumed during sample titration; (3) Liquefaction power determination Preparation of liquefaction solution: Accurately weigh 5.0 g of the microbial inoculant powder to be tested, place it in a 100 mL volumetric flask containing 10 mL of acetic acid-sodium acetate buffer solution (pH 4.6), shake well, and add distilled water to bring the volume to 100 mL. Place the volumetric flask in a constant temperature water bath at 35℃ for 1 h, filter, and collect the solution for determining liquefaction power.

[0076] Liquefaction power determination: Pipette 20 mL of soluble starch and 5 mL of pH 4.6 acetate-sodium acetate buffer solution into a test tube, shake well, and place in a 35℃ water bath to preheat the solution to 35℃. Add 5% koji powder extract and shake well. Pipette 1 mL of the reaction solution into the cavity of a white porcelain drop plate containing 3 mL of dilute iodine solution, and start timing immediately. Compare with the standard colorimetric solution until the iodine solution no longer turns blue, at which point the reaction is terminated. Record the liquefaction time t.

[0077] The liquefaction force is calculated using the formula: X = 20 × 0.02 × 60 × V / (10 × 10 × t). V: Volume of enzyme solution; t: Reaction time Table 1. Results of activity assays for different microbial agents The data in Table 1 shows that the microbial agents prepared in Examples 1-2 are superior to those prepared in Comparative Examples 1-7 in terms of esterification power, liquefaction power, and saccharification power. In terms of esterification power, Example 1 has 158.9 U / g, Example 2 has 166.7 U / g, while the highest among the comparative examples is only 105.6 U / g. In terms of liquefaction power, Example 1 has 2.2 U / g, and Example 2 reaches 2.5 U / g, significantly higher than the highest 1.2 U / g among the comparative examples. In terms of saccharification power, Example 1 has 112.3 U / g, and Example 2 has 121.5 U / g, also significantly higher than the values ​​in the comparative examples. This indicates that the microbial compound agent for brewing prepared using the specific raw material strains and stepped solid-state fermentation process of this invention has significant performance advantages, providing more efficient microbial support for the brewing process and helping to improve the quality and efficiency of brewing. Comparative Examples 1-3, which altered the composition of the microbial inoculant, and Comparative Examples 3-6, which changed the strain types, showed decreased fermentation efficiency, resulting in inoculants with lower esterification, liquefaction, and saccharification capabilities compared to the examples. Comparative Example 7, although using the same raw material strains, also exhibited significantly lower inoculant performance than the examples after switching to a mixed fermentation process. This demonstrates that the selection of raw material strains and the design of the fermentation process play a crucial role in the performance of microbial compound inoculants for brewing. Only by rationally combining raw material strains and employing appropriate fermentation processes can high-performance compound inoculants be prepared, thereby providing sufficient biological basis and material accumulation for subsequent brewing processes.

[0078] Baijiu brewing: Baijiu was brewed using the microbial agents of Examples 1-2 and Comparative Examples 1-7, according to the method described in Example 3 of this invention. Each group of experiments was repeated three times, and the average value of the results was taken. The existing commercial light-aroma baijiu bran koji (produced and provided by the brewing workshop of Feixian Wenhe Winery) was used as a control. Flavor component detection: Gas chromatography was used to quantitatively detect the flavor components in baijiu. Sample pretreatment, injection analysis and chromatographic conditions were in accordance with the conditions in the literature (Sun Xizhen, Du Jiawei, Huang Pan, et al. Sensory characterization and flavor component analysis of light-aroma baijiu brewed by modern and traditional processes [J]. Food Science, 2021, 42(6): 282-290).

[0079] Calculation of alcohol yield: The weight of the freshly distilled liquor is converted into the weight of 65% vol liquor. The ratio of the weight of liquor to the amount of grain used is the alcohol yield.

[0080] Sensory evaluation of the liquor samples: The evaluation was conducted in accordance with GB / T 33404-2016 and GB / T 10781.2-2022 Guidelines for Sensory Evaluation of Baijiu.

[0081] Table 1. Quality and Evaluation of Wines from Examples and Comparative Samples The baijiu brewed in Examples 1-2 showed advantages over those brewed with Comparative Examples 1-7 and conventional bran koji in terms of ethyl acetate and ethyl lactate content, as well as the ratio of ethyl acetate to ethyl lactate. Furthermore, Examples 1-2 had lower total higher alcohol content and higher yield. Among all tested samples, Example 2 achieved the best results. Comparative Examples 1-7, by altering or reducing the strains, exhibited significantly reduced fermentation efficiency with their prepared microbial agents. This is likely due to weakened synergistic effects among the three strains, disrupting the balance and leading to a weaker fermentation effect. Comparative Example 7, using a one-step fermentation method, had the lowest ethyl acetate and ethyl lactate content, the highest total higher alcohol content, and a lower yield. This indicates that multi-step fermentation, compared to one-step fermentation, is more conducive to synergistic effects between microorganisms and the accumulation of precursor substances, thereby improving the quality and yield of the baijiu. In summary, the specific strain combination and multi-step fermentation process adopted in this invention have significant effects on improving the performance of microbial agents and the quality of baijiu brewing, providing a more effective technical solution for the baijiu brewing industry.

[0082] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A microbial compound inoculant for brewing, characterized in that, The inoculum is composed of *Saccharomyces cerevisiae* (…). Saccharomycopsis fibuligera ), Pediococcus acidilactici ( Pediococcus acidilactici ) and Bacillus stearothermia ( Bacillus spizizenii The compound microbial agent was obtained through a stepped solid-state fermentation process. The strain number of *Diplostomum tumefaciens* was CGMCC No. 2.5608, and the strain number of *Pediococcus lactis* was CGMCC No. 1.2696, both purchased from the China General Microbiological Culture Collection Center. The strain number of *Bacillus stearothermiae* was GDMCC No. 1.136, purchased from the Guangdong Provincial Microbiological Culture Collection Center. The effective viable count of the compound microbial agent was 2-4 × 10⁻⁴. 9 CFU / g; The preparation method of the microbial compound agent for brewing includes the following preparation steps: (1) Preparation of Seed Culture of *Cytomyces clavatum*: Pick one loop of *Cytomyces clavatum* and inoculate it into a test tube containing 10 mL of malt extract liquid culture medium. Shake at 28-30℃ and 120 r / min for 24 h. After activation culture, inoculate it into 100 mL of malt extract liquid culture medium at an inoculation rate of 10% V / V. Shake culture at 28-30℃ and 120 r / min for 24 h to obtain the seed culture of *Cytomyces clavatum* for later use. (2) Preparation of Pediococcus lactis seed culture: Pick 1 loop of Pediococcus lactis and inoculate it into a test tube containing 10 mL of MRS liquid medium. Shake culture at 28-30℃ and 120 r / min for 18 h. After activation culture, inoculate it into 100 mL of MRS liquid medium at an inoculation rate of 8% V / V. Shake culture at 28-30℃ and 120 r / min for 20 h to obtain Pediococcus lactis seed culture for later use. (3) Preparation of Bacillus stearothermia seed culture: Pick 1 loop of Bacillus stearothermia and inoculate it into a test tube containing 10 mL of LB liquid medium. Shake culture at 28-30℃ and 150 r / min for 16 h. After activation culture, inoculate it into 100 mL of LB liquid medium at an inoculation rate of 12% V / V. Shake culture at 28-30℃ and 150 r / min for 18 h to obtain Bacillus stearothermia seed culture for later use. (4) First stage fermentation: Mix wheat bran and rice husks evenly at a mass ratio of 7:3 and adjust the moisture content to 50%. Sterilize at 121℃ for 30 minutes to obtain a mixture. Inoculate the mixture with Bacillus stearothermia seed liquid at a 6% inoculation rate. Ferment aerobicly at 28-30℃ for 48 hours. Turn the mixture over in time during the period to ensure oxygen supply and uniform fermentation. (5) Second-stage fermentation: Inoculate the product of the first-stage fermentation with Pediococcus lactis seed liquid at an inoculation amount of 8%, and carry out facultative anaerobic fermentation at 28-30℃ for 36 hours, controlling the oxygen content of the fermentation environment at 5%-10%; (6) Three-stage fermentation: Inoculate the product from the second-stage fermentation with 10% of the *Saccharomyces cerevisiae* seed culture. Perform anaerobic fermentation at 28-30℃ for 48-96 hours, ensuring an anaerobic environment by sealing the fermentation container. The effective viable count should be 3-5 × 10⁻⁵. 9 Fermentation ends when CFU / g is reached; after fermentation, 5% trehalose and 2% skim milk powder by weight of fermented material are added, and the product is then vacuum dried at room temperature until the moisture content is less than 15%, and then pulverized through an 80-mesh sieve to obtain the microbial compound inoculant for brewing.

2. The microbial compound inoculant for brewing according to claim 1, characterized in that, The composition of the malt extract liquid culture medium is as follows: 3g yeast extract powder, 10g glucose, 4g peptone, 10g malt extract powder, 1000mL distilled water, dissolved in water and sterilized at 121℃ for 20min.

3. The application of the microbial compound inoculant for brewing as described in claim 1, characterized in that, Used in the brewing of light-aroma baijiu.

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