Composite microbial agent for reducing higher alcohols and enriching health factors through synergistic interaction

Abstract

The invention provides a compound microbial agent capable of reducing higher alcohols and enriching health factors through synergistic interaction. The compound microbial agent is prepared from saccharomyces cerevisiae JQSC001 (CGMCC NO. 35241), saccharomyces cerevisiae JQSC002 (CGMCC NO. 35242), bacillus subtilis JQBS001 (CGMCC NO. 35239) and aspergillus niger JQAN001 (CGMCC NO. 42130). The invention further provides a preparation method of the compound microbial agent. The compound microorganism bacterium agent is applied to Baijiu brewing, synchronous optimization of four core indexes in the Baijiu fermentation process is achieved, the conversion rate of ethyl alcohol is effectively and synchronously increased, the content of ester substances in Baijiu is increased, synthesis of higher alcohols in the Baijiu is effectively inhibited, the drinking comfort is improved, and the Baijiu brewing quality is improved. The generation of terpene substances (health factors) in the Baijiu is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and specifically relates to a compound microbial agent that synergistically enhances the reduction of higher alcohols and enriches health factors. Background Technology

[0002] Baijiu, a unique distilled spirit in China, owes its distinctive flavor and quality to the metabolic activities of the microbial community during fermentation. Ethanol, as the main component, lays the foundation for its alcohol content; esters impart layers of aroma; and trace components such as terpenes determine both flavor complexity and health benefits. The content of higher alcohols directly affects the drinking experience (excessive consumption can easily lead to a hangover). Therefore, the core technological challenge in baijiu brewing lies in simultaneously achieving efficient ethanol conversion, ester enrichment, precise inhibition of higher alcohols, and enhancement of health-promoting factors (such as terpenes). This "multi-objective synergistic optimization" problem has always been a key direction for technological upgrading in the industry.

[0003] Traditional solid-state fermentation of baijiu relies on the microbial community in the natural environment. Although it can form a unique flavor, it has three inherent defects: First, the microbial community structure is unstable, resulting in significant differences in the quality of different batches of products; second, the utilization rate of raw materials is low, the ethanol conversion rate is insufficient, and the production efficiency is limited; third, the proportion of metabolites is unbalanced, often resulting in problems such as insufficient esters (thin aroma), excessive higher alcohols (discomfort after drinking), or low content of health factors (lack of functionality).

[0004] To address these issues, the industry is gradually shifting towards the artificial screening of microbial agents, but existing technologies still have significant limitations: (1) The focus is on a single function and lacks synergy: Most of the published technical solutions focus on optimizing a single indicator. For example, Chinese patent CN 107988090 A increases the content of esters by combining Saccharomyces cerevisiae, Saccharomyces orientalis, and Aspergillus oryzae, but does not involve the inhibition of higher alcohols and the enhancement of health factors; Chinese patent CN 119320703 A only targets the increase of the yield of β-phenylethanol, a flavor substance, without linking it to ethanol conversion efficiency; Chinese patent CN 118374371 A can increase the content of ethanol and ethyl hexanoate, but does not solve the problems of excessive higher alcohols and insufficient health factors. None of these solutions can achieve the simultaneous optimization of "ethanol conversion, ester synthesis, higher alcohol inhibition, and enrichment of health factors".

[0005] (2) Insufficient rationality of strain combination, functional redundancy or lack: Some compound microbial agents have redundant strain combinations (such as repeated addition of strains with similar functions of the same genus), or lack key functional strains (such as lack of yeast-bacteria-mold combination that can synergistically produce esters and inhibit higher alcohols). For example, although relying solely on the combination of yeast and mold can improve saccharification efficiency, it is difficult to balance the synthesis ratio of higher alcohols and esters due to the lack of metabolic regulation by bacteria.

[0006] (3) Poor process adaptability and limited large-scale application: Existing microbial agents mostly require complex cultivation and compounding processes, and their compatibility with commercial fermentation agents is insufficient, making it difficult to replace traditional yeast or connect with existing processes in actual production, which increases the cost of technological transformation for enterprises.

[0007] In summary, existing technologies have not yet broken through the limitations of "single-function optimization" and cannot meet the industry's production demands for "efficient, high-quality, and healthy" baijiu. Therefore, developing a composite microbial agent based on complementary functions, through precise screening of strain combinations and ratio control, to simultaneously improve ethanol conversion rate, ester content, higher alcohol inhibition rate, and health factor content, has become a key innovative direction for solving the multi-objective synergistic optimization problem in baijiu brewing—this is precisely the core value of this invention. Summary of the Invention

[0008] In order to overcome the problems of the prior art, the purpose of this invention is to achieve the simultaneous optimization of "ethanol conversion, ester synthesis, higher alcohol inhibition, and health factor enrichment" in the fermentation process of Baijiu, and proposes a compound microbial agent that synergistically enhances the reduction of higher alcohols and enriches health factors.

[0009] The objective of this invention is achieved as follows: The first aspect of this invention provides a compound microbial agent that synergistically enhances the reduction of higher alcohols and enriches health factors, wherein the compound microbial agent is composed of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001 and Aspergillus niger JQAN001. Saccharomyces cerevisiae JQSC001, deposited at the China General Microbiological Culture Collection Center, is classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35241, and the accession date is July 15, 2025; Saccharomyces cerevisiae JQSC002, deposited at the China General Microbiological Culture Collection Center, is classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35242, and the accession date is July 15, 2025; Bacillus subtilis JQBS001, deposited at the China General Microbiological Culture Collection Center, and classified as Bacillus subtilis. Bacillus subtilis The accession number is CGMCC NO. 35239, and the accession date is July 15, 2025; Aspergillus niger JQAN001, deposited at the China General Microbiological Culture Collection Center, is classified and named Aspergillus niger. Aspergillus nigerThe accession number is CGMCC NO. 42130, and the accession date is July 15, 2025.

[0010] Furthermore, the compound microbial agent is prepared by mixing the bacterial suspensions of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001, and Aspergillus niger JQAN001 spore suspension in a volume ratio of 2:2:1:1. Before mixing, the effective bacterial concentrations of both Saccharomyces cerevisiae JQSC001 and JQSC002 were (6~7)×10⁻⁶. 7 The effective bacterial concentration of Bacillus subtilis JQBS001 bacterial suspension is (3~4) × 10 CFU / mL. 7 The concentration of Aspergillus niger JQAN001 spore suspension was (1~2)×10 CFU / mL. 5 per mL.

[0011] Secondly, a compound microbial agent fortifying yeast is provided, wherein the fortified yeast comprises a commercial baijiu fermentation agent and the compound microbial agent described in the first aspect.

[0012] The third aspect of this invention provides a method for preparing the composite microbial agent described in the first aspect, comprising the following steps: (1) Preparation of Saccharomyces cerevisiae liquid: Saccharomyces cerevisiae inoculated into YPD liquid medium and activated at 30℃ for 24-48 h. Single colonies were streaked on YPD plates after activation. Single colonies were picked and cultured in YPD liquid medium at 30℃ for 24-48 h to obtain Saccharomyces cerevisiae liquid. (2) Preparation of Bacillus subtilis bacterial suspension: Bacillus subtilis strain was inoculated into LB liquid medium and activated at 37°C for 12-20 h. Single colonies were streaked on LB plates after activation. Single colonies were picked and cultured in LB liquid medium at 37°C for 12-16 h to obtain Bacillus subtilis bacterial suspension. (3) Preparation of Aspergillus niger spore suspension: Aspergillus niger hyphae were inoculated onto PDA plates and cultured at 30°C for at least 72 hours. Then, sterile physiological saline was injected into the plates, and the plates were washed with an inoculation stick until the spores were suspended in the liquid to obtain Aspergillus niger spore suspension. (4) The prepared brewer's yeast liquid, Bacillus subtilis liquid and Aspergillus niger spore suspension are mixed in proportion to obtain the composite microbial agent.

[0013] The fourth aspect provides the application of the compound microbial agent described in the first aspect, the compound microbial agent fortified yeast described in the second aspect, or the compound microbial agent prepared by the method described in the third aspect in the brewing of baijiu.

[0014] Furthermore, including one or more of the following: (1) The compound microbial agent or compound microbial agent-enhanced yeast can increase the yield of ethanol; (2) The compound microbial agent or compound microbial agent-enhanced yeast can increase the content of esters; (3) The compound microbial agent or compound microbial agent-enhanced yeast can reduce the yield of higher alcohols; (4) The compound microbial agent or compound microbial agent-enhanced yeast can increase the production of health factors, including terpenes.

[0015] Information on strain preservation: Saccharomyces cerevisiae JQSC001, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35241, and the accession date is July 15, 2025; Saccharomyces cerevisiae JQSC002, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35242, and the accession date is July 15, 2025; Bacillus subtilis JQBS001, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; classified as Bacillus subtilis. Bacillus subtilis The accession number is CGMCC NO. 35239, and the accession date is July 15, 2025; Aspergillus niger JQAN001, deposited at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; classified and named as Aspergillus niger. Aspergillus niger The accession number is CGMCC NO. 42130, and the accession date is July 15, 2025.

[0016] The advantages and beneficial effects of this invention are: 1. Achieve multi-objective synergistic optimization to significantly improve the quality and health attributes of baijiu. This invention achieves simultaneous optimization of four core indicators during the fermentation process of baijiu (Chinese liquor) through a specific combination of brewing yeast JQSC001, brewing yeast JQSC002, Bacillus subtilis JQBS001, and Aspergillus niger JQAN001, selected from high-quality brewing yeasts: ① Improved ethanol conversion rate: By efficiently utilizing sorghum substrate, ethanol production is increased by more than 15% compared to traditional commercial fermentation agents, significantly improving raw material utilization and production efficiency; ②Ester enrichment: The synergistic effect of high ester-producing yeasts and molds drives esterase activity, increasing the total ester content by more than 14%. Among them, the content of key aroma substances such as ethyl acetate, ethyl lactate, ethyl butyrate, and ethyl hexanoate is significantly increased (e.g., ethyl hexanoate increased by 341% compared with the control group, and ethyl lactate increased by 5300%), giving the liquor a richer flavor profile. ③ Deep inhibition of higher alcohols: Specifically inhibits the synthesis of key higher alcohols such as isoamyl alcohol, reducing their content by more than 59.5% compared with the control group, effectively solving the problem of "headache" after drinking and significantly improving the comfort of drinking; ④ Enhancement of health factors: Promotes the generation of terpenoid health factors, increasing the number of types to 9, with the total concentration increasing by 28.5% compared to the control group. Among them, the content of functional components such as damascene (with antibacterial and anticancer activities) and 4-ethylguaiacol (with hypoglycemic and antioxidant functions) is significantly increased, which enhances the health attributes of baijiu while enriching the flavor.

[0017] 2. The process is simple, highly adaptable, and conducive to large-scale production. The composite microbial agent of this invention has clearly defined components and a simple preparation process: each strain can be cultured separately and then mixed in a specific ratio without the need for complex purification or modification steps; it has excellent compatibility with commercial baijiu fermentation agents (such as Angel fermentation agents) and can be directly compounded into fortified koji for use; the inoculation method is simple (add at 0.4%-0.5% of the raw material mass), and it is easy to integrate into existing baijiu brewing processes, reducing the cost of technological transformation for enterprises.

[0018] 3. Improve raw material utilization rate to reduce costs and increase efficiency. By optimizing microbial metabolic pathways, this invention significantly improves the conversion efficiency of raw materials such as sorghum, reduces substrate waste during fermentation, and reduces raw material consumption per unit of product while increasing the yield and quality of baijiu. This provides reliable technical support for baijiu enterprises to achieve cost reduction and efficiency improvement in large-scale production. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 The results of total acid, total esters, and alcohol content of the sorghum juice fermentation broth in the comparative and example samples are shown. Figure 2 Heat maps of key flavor compounds in sorghum juice for comparative and example cases. Detailed Implementation

[0021] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0024] The culture media involved in the following examples are as follows: The YPD liquid culture medium consists of 2% glucose, 1% yeast extract, and 2% peptone, with a natural pH, and is autoclaved at 121°C for 15 minutes. The YPD solid culture medium consists of 2% glucose, 1% yeast extract, 2% peptone, and 2% agar, with a natural pH, and is autoclaved at 121°C for 15 minutes. The composition of PDA liquid culture medium is: 4 g / L potato extract powder, 2 g / L glucose, natural pH, autoclaved at 121℃ for 15 min. The components of PDA solid medium are: potato extract powder 4g / L, glucose 20g / L, agar 15g / L, natural pH, autoclaved at 121℃ for 15 min. The composition of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH, autoclaved at 121℃ for 15 min; The components of LB solid medium are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, natural pH, and autoclaved at 121°C for 15 min.

[0025] Bengal Red Solid Medium: Peptone 5 g / L, Potassium dihydrogen phosphate 1 g / L, Magnesium sulfate 0.5 g / L, Bengal Red 0.033 g / L, Chloramphenicol 0.1 g / L, Glucose 10 g / L, Agar 20 g / L, pH 6.0, Autoclaved at 121℃ for 15 min.

[0026] The following comparative examples and embodiments involve the preparation methods of sorghum juice: Thoroughly crush the sorghum, pass it through a 50-mesh sieve, add distilled water at a material-to-water ratio of 1:6 (w / w), and boil until viscous. Then, heat in a 90℃ constant temperature water bath for 90 min to gelatinize (stirring constantly to ensure even heating). Next, transfer to a 60℃ constant temperature water bath for liquefaction and saccharification. Add α-liquefying enzyme (enzyme activity > 3700 U / g) at 2% of the raw material amount and heat for liquefaction for 60 min. Then, add 1% saccharifying enzyme (enzyme activity > 10,000 U / g) and continue heating for 60 min. Filter the solution, and dispense 200 mL into 500 mL Erlenmeyer flasks. Sterilize at 121℃ for 20 min for later use.

[0027]

[0028] Comparative Example This comparative example uses commercial yeast starter listed in Table 1 to simulate liquid fermentation of sorghum juice, specifically including the following steps: Step 1: Colony counting of commercial brewing yeast: Weigh 1g of commercial baijiu fermentation agent into 100ml of sterile physiological saline, mix well for 30min, let stand for 30min, take 1ml of supernatant, and serially dilute with physiological saline to a concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 Take 200 μl of the diluted solution and spread it evenly on YPD plates, LB plates, and Bengal Red agar plates. Incubate the plates at 30℃, 37℃, and 30℃ for 48 h, 24 h, and 72 h, respectively, until single colonies have grown completely. Count the colonies on the plates with good colony isolation and calculate the total number of colonies in 1 g of commercial starter.

[0029] Step Two: Fermentation of sorghum juice using commercial fermentation agent: Add 0.2g of Angel Yeast starter to 200ml of sorghum juice prepared above, following the dosage instructions on the commercial starter. Set up 3 parallel fermentations and ferment at 30℃ in a sealed environment for 7 days.

[0030] Example 1 This embodiment utilizes the fortified yeast A from Table 1 to simulate liquid fermentation of sorghum juice. Example 1 aims to verify the performance of the compound microbial agent when it contains only a single brewing yeast (JQSC002) with a yeast:bacterial:mold ratio of 1:1:1. The specific steps include: Step 1: Preparation of seed culture for each strain (1) Preparation method of Saccharomyces cerevisiae liquid: Saccharomyces cerevisiae inoculated into YPD liquid medium and activated at 30℃ for 48h. Single colonies were streaked on YPD plates after activation. Single colonies were picked and cultured in YPD liquid medium at 30℃ for 24h to obtain Saccharomyces cerevisiae liquid. The cell concentration OD was then measured. 600 The bacterial culture was diluted to a final concentration OD using sterile physiological saline. 600 =2, obtain brewer's yeast seed liquid, for later use.

[0031] (2) Preparation method of Bacillus subtilis bacterial suspension (bacteria): Bacillus subtilis strain was inoculated into LB liquid medium and activated at 37℃ for 12-20 h. Single colonies were streaked on LB plates after activation. Single colonies were picked and cultured in LB liquid medium at 37℃ for 16 h to obtain Bacillus subtilis bacterial suspension. The bacterial concentration OD was then measured. 600 The bacterial culture was diluted to a final concentration OD using sterile physiological saline. 600 =0.5, obtain Bacillus subtilis seed liquid for later use.

[0032] (3) Preparation method of Aspergillus niger spore suspension: Aspergillus niger mycelia were inoculated onto PDA plates and cultured at 30°C for at least 72 h. Sterile physiological saline was then injected into the plates, and the plates were washed with an inoculation rod until the spores were suspended in the liquid. The Aspergillus niger spore suspension was obtained, and its spore concentration (OD) was measured. 630 The spore solution was diluted to a final concentration OD using sterile physiological saline. 630 =0.17, obtain Aspergillus niger spore suspension for later use.

[0033] Step 2: Preparation of fortified yeast A: A compound microbial agent A is prepared by mixing brewing yeast JQSC001 seed liquid, Bacillus subtilis JQBS001 seed liquid, and Aspergillus niger JQAN002 spore suspension at a volume ratio of 4:1:1. When using, the commercial fermentation agent is mixed with the compound microbial agent A at a colony count ratio of 1:1, i.e., the mass M (g) / volume V (ml) is 1:7.5, to obtain fortified yeast A.

[0034] Step 3: Fortify the fermentation of sorghum juice with yeast A: Referring to the amount of commercial starter culture added in the comparative example, 0.1g of Angel starter culture and 0.75ml of compound microbial agent A were mixed and added to 200ml of sorghum juice prepared above. Three parallel fermentations were set up and fermented in a sealed environment at 30℃ for 7 days.

[0035] Example 2 This embodiment uses the fortified yeast B in Table 1 to simulate liquid fermentation of sorghum juice. Example 2 aims to verify the performance of the compound microbial agent when only a single brewing yeast (JQSC002) is present and the combination of mold and yeast strains is changed to a yeast:bacterial:mold ratio of 1:1:1. The specific steps are the same as in Example 1, with the difference being: In Example 1, fortified yeast A was replaced with fortified yeast B to simulate liquid fermentation of sorghum juice.

[0036] Preparation of fortified yeast starter B: The seed liquid of brewing yeast JQSC002, the seed liquid of Bacillus subtilis JQBS001, and the spore suspension of Aspergillus niger JQAN002 were mixed at a volume ratio of 4:1:1 to obtain compound microbial agent B; when using, the commercial fermentation agent and compound microbial agent B were mixed at a colony count ratio of 1:1, that is, the mass M (g) / volume V (ml) was 1:7.5 to obtain fortified yeast starter B.

[0037] Example 3 This embodiment uses the fortified yeast C from Table 1 to simulate liquid fermentation of sorghum juice. Example 3 aims to verify the performance of the compound microbial agent when the number of Bacillus species is increased to two strains of Bacillus amyloliquefaciens, and the strain ratio is yeast:bacteria:mold = 1:2:1. The specific steps include: Step 1: Preparation of seed culture for each strain: Seed solutions for each strain were prepared according to step one in Example 1.

[0038] Step 2: Preparation of fortified yeast C: A compound microbial agent C was prepared by mixing the seed liquid of brewing yeast JQSC001, the seed liquid of Bacillus amyloliquefaciens JQBA001, the seed liquid of Bacillus amyloliquefaciens JQBA002, and the spore suspension of Aspergillus niger JQAN002 at a volume ratio of 4:0.5:0.5:1. When using the agent, the commercial fermentation agent was mixed with the compound microbial agent C at a colony count ratio of 1:1, i.e., the mass M (g) / volume V (ml) ratio was 1:7.5, to obtain the fortified yeast C.

[0039] Step 3: Strengthen the yeast C sorghum juice to simulate liquid fermentation: Referring to the amount of commercial starter culture added in the comparative example, 0.1g of Angel starter culture and 0.75ml of compound microbial agent C were mixed and added to 200ml of sorghum juice prepared above. Three parallel fermentations were set up and the mixture was sealed at 30℃ for 7 days.

[0040] Example 4 This embodiment uses the fortified yeast D from Table 1 to simulate liquid fermentation of sorghum juice. Example 4 aims to verify the effect of changing the type of Bacillus to two strains of Bacillus subtilis, with the strain ratio of yeast:bacteria:mold = 1:2:1, on the function of the inoculant. The specific steps are the same as those in Example 3, with the difference being: In Example 3, the two strains of Bacillus amyloliquefaciens were replaced with two strains of Bacillus subtilis, and the fermenting agent in Example 3 was changed to fortified yeast D to simulate liquid fermentation of sorghum juice.

[0041] Example 5 This embodiment uses the fortified yeast E in Table 1 to simulate liquid fermentation of sorghum juice. Example 5 aims to verify the effect of changing the type of Bacillus to Bacillus subtilis + Bacillus amyloliquefaciens, with a yeast:bacterial:mold ratio of 1:2:1, on the function of the inoculant. The specific steps are the same as those in Example 3, with the difference being: In Example 3, Bacillus amyloliquefaciens JQBA001 and Bacillus amyloliquefaciens JQBA002 were replaced with Bacillus amyloliquefaciens JQBA003 and Bacillus subtilis JQBS001. The fermenting agent in Example 3 was also adjusted to be fortified yeast E for simulated liquid fermentation of sorghum juice.

[0042] Example 6 This embodiment uses the fortified yeast F from Table 1 to simulate liquid fermentation of sorghum juice. Example 6 aims to verify the effect of increasing the types of brewing yeast, so that the strain ratio is yeast:bacteria:mold = 2:1:1, on the function of the inoculant. Specifically, it includes the following steps: Step 1: Preparation of seed culture for each strain: Seed solutions for each strain were prepared according to step one in Example 1.

[0043] Step 2: Preparation of fortified yeast F: The seed liquids of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001, and Aspergillus niger JQAN001 spore suspension were mixed in a volume ratio of 2:2:1:1 to obtain compound microbial agent F. When using, the commercial fermentation agent was mixed with compound microbial agent F at a colony count ratio of 1:1, that is, the mass M (g) / volume V (ml) was 1:7.5 to obtain fortified yeast F.

[0044] Step 3: Strengthen the yeast starter F and simulate liquid fermentation of sorghum juice: Referring to the amount of commercial starter culture added in the comparative example, 0.1g of Angel starter culture and 0.75ml of compound microbial agent F were mixed and added to 200ml of sorghum juice prepared above. Three parallel fermentations were set up and the mixture was sealed at 30℃ for 7 days.

[0045] Test Example 1: This test example compares the physicochemical properties of the fermentation broth obtained from the above comparative examples and various embodiments, including alcohol content, total acid, and total esters. The specific method is as follows: The fermentation broth was distilled, and the volume of the distillate was accurately measured using a graduated cylinder. Distillation was stopped when the volume of the distillate reached 100 ml. The alcohol content, total acid, and total esters of the distillate were tested according to the methods specified in national standards GB 5009.225-2016, GB / T 10345-2007, and GB / T 10345-2007, respectively.

[0046] Figure 1 The results are for the detection of physicochemical indicators.

[0047] Depend on Figure 1 It can be seen that the fortified yeast F obtained by combining commercial fermentation agent and compound microbial agent F in Example 6 significantly improved the substrate utilization rate compared with the comparative example. The ethanol yield of the comparative example was 66.7 g / L, while the ethanol yield of Example 6 was 76.6 g / L, representing an increase of 15% in ethanol yield. p <0.05), and improved the synthesis efficiency of esters. The total ester content in the comparative example was 0.21 g / L, and the total ester content in Example 6 was 0.24 g / L, representing a 14.2% increase in total ester content compared to the control group. p<0.05). This demonstrates that the compound microbial agent of the present invention can effectively enhance ethanol conversion and ester synthesis. In Examples 1 and 2, which only contained a single type of *Saccharomyces cerevisiae*, the total ester content was 0.19 g / L and 0.17 g / L, respectively, which was 9.5% and 19% lower than the comparative example, and 20.8% and 29% lower than Example 6; the ethanol yield was 61.6 g / L and 56.2 g / L, respectively, which was 7.5% and 15.6% lower than the comparative example, and 19.5% and 26.5% lower than Example 6. This illustrates the important role of the synergistic effect of *Saccharomyces cerevisiae* JQSC001 and JQSC002 in improving the ester and ethanol production capacity of the compound microbial agent. Examples 3-5, due to the use of only a single *Saccharomyces cerevisiae* strain and an increased number of *Bacillus* strains, showed total ester contents of 0.16 g / L, 0.11 g / L, and 0.12 g / L, respectively, which were 23.8%, 47.6%, and 42.8% lower than the comparative example, and 33.3%, 54.2%, and 50% lower than Example 6. Ethanol yields were 64.8 g / L, 63.8 g / L, and 66.4 g / L, respectively, which were 2.8%, 4.3%, and 0.4% lower than the comparative example, and 15.4%, 16.7%, and 13.3% lower than Example 6. Increasing the number of *Bacillus* strains narrowed the gap in ethanol production capacity compared to the comparative example and Example 6, but it could not surpass them. The difference in ester production capacity became more significant, indicating that simply increasing the number of *Bacillus* strains was insufficient to improve the ethanol and ester production capacity of the compound microbial agent. The above comparisons verified the synergistic effect of Example 6 (the core microbial strain combination of this invention).

[0048] Test Example 2: This test example compares the volatile flavor compounds in the fermentation broths obtained from the above comparative examples and various embodiments. Headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used. The specific method is as follows: Headspace SPME conditions: 10 mL of sample was placed in a 20 mL headspace vial, and 1.5 g NaCl and 5 μl of internal standard (2-octanol, 1 mg / mL) were added for solid-microextraction (SPME). After equilibration at 50 °C for 15 min, the sample was then inserted into the extraction fiber for adsorption for 30 min. Immediately after adsorption, the sample was thermally desorbed at 250 °C for 3 min at the gas chromatograph injection port in preparation for further analysis.

[0049] GC conditions: DB-WAX column (60m × 0.25 mm, 0.25μm), He as carrier gas, flow rate set to 1 mL / min. Column temperature program: 40℃ for 3 min, ramped to 160℃ at 4℃ / min, ramped to 230℃ at 7℃ / min, held at this temperature for 8 min. Connector temperature set to 230℃. MS conditions: Full scan range 33–450 amu, scan once per second. EI +The ion source has an ionization temperature of 230℃, an electron energy of 70eV, a filament flow rate of 0.2mA, and a detector voltage of 350V.

[0050] The volatile flavor compounds in the fermentation broths obtained from the comparative examples and each embodiment were analyzed using Qualitative Analysis software.

[0051] The results are as follows Figure 2 As shown, the content of key esters in the liquor of Example 6, such as ethyl acetate, ethyl butyrate, ethyl hexanoate, and ethyl lactate, was significantly increased compared to the comparative example, by 20%, 23%, 341%, and 5300%, respectively. Simultaneously, key higher alcohols such as isoamyl alcohol were deeply inhibited, decreasing by 59.5% compared to the control group. This demonstrates that the composite microbial agent of the present invention can enhance the synthesis of esters, confirming the above conclusions; at the same time, it can significantly reduce the synthesis of higher alcohols, improving the drinking experience.

[0052] The terpenoids in the fermentation broths obtained from the comparative examples and each embodiment were analyzed using Qualitative Analysis software.

[0053] The results are shown in Table 2. Nine terpenoids were detected in the fermentation broth obtained in Example 6, which is an increase in the number of terpenoids compared to the comparative example. Among them, the contents of 1-octen-3-ol, linalool, β-ionone, damascene, 4-ethyl-guaiacol, and geraniol were significantly higher than those in the comparative example, with a total concentration of 15.51 mg / L, which is 28.5% higher than that in the comparative example. This indicates that the compound microbial agent of the present invention can enhance the generation of terpenoids, not only making the flavor more abundant, but also improving health factors.

[0054]

[0055] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A compound microbial agent that synergistically reduces higher alcohols and enriches health factors, characterized in that, The compound microbial agent is composed of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001 and Aspergillus niger JQAN001; Saccharomyces cerevisiae JQSC001, deposited at the China General Microbiological Culture Collection Center, is classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35241, and the accession date is July 15, 2025; Saccharomyces cerevisiae JQSC002, deposited at the China General Microbiological Culture Collection Center, is classified and named Saccharomyces cerevisiae. Saccharomyces cerevisiae The accession number is CGMCC NO. 35242, and the accession date is July 15, 2025; Bacillus subtilis JQBS001, deposited at the China General Microbiological Culture Collection Center, and classified as Bacillus subtilis. Bacillus subtilis The accession number is CGMCC NO. 35239, and the accession date is July 15, 2025; Aspergillus niger JQAN001, deposited at the China General Microbiological Culture Collection Center, is classified and named Aspergillus niger. Aspergillus niger The accession number is CGMCC NO. 42130, and the accession date is July 15, 2025.

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent is prepared by mixing the bacterial suspensions of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001, and Aspergillus niger JQAN001 spore suspension in a volume ratio of 2:2:1:

1. Before mixing, the effective bacterial concentrations of both Saccharomyces cerevisiae JQSC001 and JQSC002 were (6~7)×10⁻⁶. 7 The effective bacterial concentration of Bacillus subtilis JQBS001 bacterial suspension is (3~4) × 10 CFU / mL. 7 The concentration of Aspergillus niger JQAN001 spore suspension was (1~2)×10 CFU / mL. 5 per mL.

3. A compound microbial inoculant fortified brewing yeast, characterized in that, The fortified yeast comprises commercial baijiu fermentation agent and the compound microbial agent as described in claim 1 or 2.

4. A method for preparing the composite microbial agent as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Preparation of brewing yeast culture: Inoculate brewing yeast strain into YPD liquid medium and activate at 30℃ for 24-48h. After activation, single colonies are drawn on YPD plates. Single colonies are picked and cultured in YPD liquid medium at 30℃ for 24-48h to obtain brewing yeast culture. (2) Preparation of Bacillus subtilis bacterial suspension: Bacillus subtilis strain was inoculated into LB liquid medium and activated at 37°C for 12-20 h. Single colonies were streaked on LB plates after activation. Single colonies were picked and cultured in LB liquid medium at 37°C for 12-16 h to obtain Bacillus subtilis bacterial suspension. (3) Preparation of Aspergillus niger spore suspension: Aspergillus niger mycelium was inoculated onto a PDA plate and cultured at 30°C for at least 72 h. Then, sterile physiological saline was injected into the plate and the plate was washed with an inoculation stick until the spores were suspended in the liquid to obtain Aspergillus niger spore suspension. (4) The prepared brewer's yeast liquid, Bacillus subtilis liquid and Aspergillus niger spore suspension are mixed in proportion to obtain the composite microbial agent.

5. The application of the compound microbial agent according to claim 1 or 2, the compound microbial agent for strengthening the yeast according to claim 3, or the compound microbial agent prepared by the method according to claim 4 in the brewing of baijiu.

6. The application according to claim 5, characterized in that, Includes one or more of the following: (1) The compound microbial agent or compound microbial agent-enhanced yeast can increase the yield of ethanol; (2) The compound microbial agent or compound microbial agent-enhanced yeast can increase the content of esters; (3) The compound microbial agent or compound microbial agent-enhanced yeast can reduce the yield of higher alcohols; (4) The compound microbial agent or compound microbial agent-enhanced yeast can increase the production of health factors, including terpenes.

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