A synergistic high-alcohol-reducing and health factor-enriching composite microbial agent

Abstract

The present application provides a kind of synergistic high alcohol reduction and enrichment of health factor complex microbial inoculant, the complex microbial inoculant is composed of 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 complex microbial inoculant is applied in liquor brewing, realizes the synchronous optimization of four core indexes in liquor fermentation process: effectively synchronously improves the conversion rate of ethanol, so that the ester content of liquor is improved, effectively inhibits the synthesis of high alcohol in liquor, improves the drinking comfort, strengthens the generation of terpene (health factor) in liquor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a compound microbial inoculum for synergistically reducing higher alcohols and enriching health factors. BACKGROUND

[0002] As a unique distillate liquor category in China, the unique flavor and quality of Baijiu (liquor) are derived from the metabolic activities of microbial communities during the fermentation process. Ethanol, as the main component, lays the foundation for alcohol content. Ester substances impart aroma levels, and trace components such as terpenes determine both the complexity of flavor and health attributes. The content of higher alcohols directly affects the comfort of drinking (excessive consumption can lead to "headiness"). Therefore, the core technical challenge of Baijiu brewing is to simultaneously achieve efficient conversion of ethanol, enrichment of ester substances, precise inhibition of higher alcohols, and strengthening of health factors (such as terpenes). This "multi-objective synergistic optimization" problem has always been the key direction for the industry's technical upgrade.

[0003] Traditional solid-state fermentation of Baijiu relies on microbial communities in the natural environment, which can form unique flavors but has three inherent defects: first, the microbial community structure is unstable, leading to significant quality differences between different batches of products; second, the raw material utilization rate is low, and the ethanol conversion rate is insufficient, limiting production efficiency; third, the proportion of metabolic products is imbalanced, often resulting in insufficient ester substances (thin aroma), excessive higher alcohols (uncomfortable after drinking), or low health factor content (lack of functionality).

[0004] To address the above problems, the industry has gradually shifted towards the direction of artificially selecting microbial inoculums. However, existing technologies still have obvious limitations:

[0005] (1) Single function orientation is obvious, lacking synergy: The disclosed technical solutions mostly focus on optimizing a single indicator. For example, Chinese Patent CN 107988090 A improves the content of ester substances through the combination of Saccharomyces cerevisiae, Issatchenkia orientalis, and Aspergillus oryzae, but does not address the inhibition of higher alcohols and the strengthening of health factors. Chinese Patent CN 119320703 A only aims to increase the yield of β-phenylethanol, a flavor substance, without considering 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. These solutions cannot achieve the simultaneous optimization of "ethanol conversion, ester synthesis, higher alcohol inhibition, and health factor enrichment."

[0006] (2) The strain combination is not reasonable, and there is functional redundancy or lack: some complex microbial agents have redundant strain combinations (such as repeated addition of similar functional strains of the same genus), or lack of key functional strains (such as lack of yeast-bacteria-mold combination that can synergistically produce esters and inhibit higher alcohols). For example, although the combination of only yeast and mold can improve the saccharification efficiency, it is difficult to balance the synthesis ratio of higher alcohols and esters due to the lack of metabolic regulation of bacteria.

[0007] (3) Poor process adaptability, limited for large-scale application: the existing microbial agents require complex culture and compounding processes, and have insufficient compatibility with commercial fermentation agents, which makes it difficult to replace traditional liquor starter or connect with existing processes in actual production, increasing the technical modification cost of enterprises.

[0008] In summary, the existing technology has not broken through the limitation of "single function optimization" and cannot meet the production needs of "high efficiency, high quality and healthy" liquor industry. Therefore, developing a complex microbial agent based on functional complementation, through precise screening of strain combination and proportion regulation, to realize the simultaneous improvement of ethanol conversion rate, ester content, higher alcohol inhibition rate and healthy factor content, has become the key innovative direction to solve the problem of multi-objective synergistic optimization in liquor brewing - which is also the core value of the present application. SUMMARY

[0009] In order to overcome the problems of the prior art, the purpose of the present application is to realize the simultaneous optimization of "ethanol conversion, ester synthesis, higher alcohol inhibition and healthy factor enrichment" in the liquor fermentation process, and a complex microbial agent for synergistically reducing higher alcohols and enriching healthy factors is proposed.

[0010] The purpose of the present application is achieved as follows:

[0011] The first aspect of the present application provides a complex microbial agent for synergistically reducing higher alcohols and enriching healthy factors, which is composed of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001 and Aspergillus niger JQAN001.

[0012] Saccharomyces cerevisiae JQSC001 is deposited in the China General Microbiological Culture Collection Center, and is classified and named as Saccharomyces cerevisiae Saccharomyces cerevisiae , with the preservation number CGMCC NO. 35241 and the preservation date July 15, 2025;

[0013] Saccharomyces cerevisiae JQSC002 is deposited in the China General Microbiological Culture Collection Center, and is classified and named as Saccharomyces cerevisiae Saccharomyces cerevisiae , with the preservation number CGMCC NO. 35242 and the preservation date July 15, 2025;

[0014] Bacillus subtilis JQBS001, deposited in the China General Microbiological Culture Collection Center, classified as Bacillus subtilis Bacillus subtilis , with the preservation number CGMCC NO. 35239 and the preservation date July 15, 2025;

[0015] Aspergillus niger JQAN001, deposited in the China General Microbiological Culture Collection Center, classified as Aspergillus niger Aspergillus niger , with the preservation number CGMCC NO. 42130 and the preservation date July 15, 2025.

[0016] Further, the composite microbial agent is prepared by mixing the cell suspensions of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001, and the spore suspension of Aspergillus niger JQAN001 in a volume ratio of 2:2:1:1.

[0017] Before mixing, the effective bacterial concentrations of the cell suspensions of Saccharomyces cerevisiae JQSC001 and Saccharomyces cerevisiae JQSC002 are both (6-7) x 10 7 CFU / mL, the effective bacterial concentration of the cell suspension of Bacillus subtilis JQBS001 is (3-4) x 10 7 CFU / mL, and the concentration of the spore suspension of Aspergillus niger JQAN001 is (1-2) x 10 5 CFU / mL.

[0018] In a second aspect, a composite microbial agent for strengthening distiller's yeast is provided, which comprises a commercial Baijiu (Chinese liquor) fermenting agent and the composite microbial agent of the first aspect.

[0019] In a third aspect, a preparation method of the composite microbial agent of the first aspect is provided, which comprises the following steps:

[0020] (1) Preparation of Saccharomyces cerevisiae cell suspension: inoculate the Saccharomyces cerevisiae strain into YPD liquid medium, activate at 30℃ for 24-48 h, draw single colonies on a YPD plate from the activated cell suspension, pick single colonies in YPD liquid medium, and culture at 30℃ for 24-48 h to obtain the Saccharomyces cerevisiae cell suspension;

[0021] (2) Preparation of Bacillus subtilis cell suspension: inoculate the Bacillus subtilis strain into LB liquid medium, activate at 37℃ for 12-20 h, draw single colonies on an LB plate from the activated cell suspension, pick single colonies in LB liquid medium, and culture at 37℃ for 12-16 h to obtain the Bacillus subtilis cell suspension;

[0022] (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.

[0023] (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.

[0024] 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.

[0025] Furthermore, this includes one or more of the following:

[0026] (1) The compound microbial agent or compound microbial agent-enhanced yeast can increase the yield of ethanol;

[0027] (2) The compound microbial agent or compound microbial agent-enhanced yeast can increase the content of esters;

[0028] (3) The compound microbial agent or compound microbial agent-enhanced yeast can reduce the yield of higher alcohols;

[0029] (4) The compound microbial agent or compound microbial agent-enhanced yeast can increase the production of health factors, including terpenes.

[0030] Information on strain preservation:

[0031] 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;

[0032] 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;

[0033] Bacillus subtilis JQBS001, preserved in the China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Huayuancun, Beijing, China, with a postal code of 100101, and classified as Bacillus subtilis Bacillus subtilis , with a preservation number of CGMCC NO. 35239 and a preservation date of July 15, 2025;

[0034] Aspergillus niger JQAN001, preserved in the China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Huayuancun, Beijing, China, with a postal code of 100101, and classified as Aspergillus niger Aspergillus niger , with a preservation number of CGMCC NO. 42130 and a preservation date of July 15, 2025.

[0035] The advantages and beneficial effects of the present application are:

[0036] 1. Multi-target synergistic optimization is achieved, significantly improving the quality and health attributes of liquor

[0037] The present application realizes the simultaneous optimization of four core indicators in the liquor fermentation process through the specific combination of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001, and Aspergillus niger JQAN001 selected from high-quality liquor starter:

[0038] ① Ethanol conversion rate improvement: efficient utilization of sorghum substrate, ethanol yield increased by more than 15% compared with traditional commercial fermentation agents, significantly improving raw material utilization and production efficiency;

[0039] ② Ester enrichment: high-yield ester yeast and mold synergistic effect, driving esterification enzyme activity, total ester content increased by more than 14%, among which the content of key aroma substances such as ethyl acetate, ethyl lactate, ethyl butyrate, and ethyl hexanoate increased significantly (such as ethyl hexanoate increased by 341%, ethyl lactate increased by 5300%), giving liquor more rich flavor levels;

[0040] ③ Advanced alcohol inhibition: specific inhibition of the synthesis of key advanced alcohols such as isoamyl alcohol, with a content reduced by more than 59.5% compared with the control group, effectively solving the "head" problem after drinking, significantly improving the comfort of drinking;

[0041] ④ Health factor strengthening: promoting the generation of terpene health factors, the number of types increased to 9, the total concentration increased by 28.5% compared with the control group, among which the content of functional ingredients such as linalool (with antibacterial and anticancer activity) and 4-ethyl guaiacol (with hypoglycemic and antioxidant functions) increased significantly, enriching the flavor while enhancing the health attributes of liquor.

[0042] 2. The process is simple, adaptable, and conducive to large-scale production

[0043] The composite microbial inoculant of the application has clear components and simple preparation process: the strains are cultured separately and then mixed according to a specific ratio, without the need for complex purification or modification steps; it has excellent compatibility with commercial liquor fermentation agents (such as Angel fermentation agent), and can be directly compounded into enhanced starter for use, with simple inoculation method (0.4%-0.5% of the raw material quality is added), easy to integrate into the existing liquor brewing process, and reduces the cost of technical transformation of enterprises.

[0044] 3. Improving raw material utilization rate and realizing cost reduction and efficiency increase

[0045] By optimizing the metabolic pathway of microorganisms, the conversion efficiency of raw materials such as sorghum is significantly improved, reducing the waste of substrates in the fermentation process, improving the yield and quality of liquor while reducing the raw material consumption per unit product, providing reliable technical support for liquor enterprises to realize cost reduction and efficiency increase in large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0046] The application will be further described below in conjunction with the drawings and examples.

[0047] Figure 1 The total acid, total ester and alcohol content of the sorghum juice fermentation liquid of the comparative examples and examples are shown;

[0048] Figure 2 The key flavor substance heat map of the sorghum juice of the comparative examples and examples is shown. DETAILED DESCRIPTION

[0049] The examples are given to better illustrate the application, but are not intended to limit the application to the examples only. Therefore, non-essential modifications and adjustments to the embodiments based on the above disclosure can be made by those skilled in the art, and still fall within the scope of the application.

[0050] The endpoints of the ranges and any values in this disclosure are not limited to the precise values stated. The endpoints of the ranges and values should be construed as being approximate. For ranges, the endpoints are provided as a roughly correct indication of the range. The exact range can depend upon the context and / or operational conditions. For example, the range can be narrower than the range stated. Other ranges can be included in the range stated or substituted for that range. When no range is stated, the range can be understood to be zero to infinity.

[0051] The application will be described in detail below through examples. It should be understood that the following examples are only used to exemplarily further explain and illustrate the content of the application, and are not used to limit the application.

[0052] The culture medium involved in the following examples is as follows:

[0053] The composition of YPD liquid medium is: 2% glucose, 1% yeast extract powder, 2% peptone, pH natural, 121℃ 15 min high pressure steam sterilization;

[0054] The composition of YPD solid medium is: 2% glucose, 1% yeast extract powder, 2% peptone, 2% agar, pH natural, 121℃ 15 min high pressure steam sterilization;

[0055] The composition of PDA liquid medium is: potato extract powder 4g / L, glucose 2g / L, pH natural, 121℃ 15 min high pressure steam sterilization;

[0056] The composition of PDA solid medium is: potato extract powder 4g / L, glucose 20g / L, agar: 15g / L, pH natural, 121℃ 15 min high pressure steam sterilization;

[0057] The composition of LB liquid medium is: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH natural, 121℃ 15 min high pressure steam sterilization;

[0058] The composition of LB solid medium is: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar 15g / L, pH natural, 121℃ 15 min high pressure steam sterilization.

[0059] Bengal red solid medium: peptone 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, Bengal red 0.033g / L, chloramphenicol 0.1g / L, glucose 10g / L, agar 20g / L, pH 6.0, 121℃ 15 min high pressure steam sterilization.

[0060] The following examples and comparative examples involve the following method of making sorghum juice:

[0061] Sorghum was fully ground and passed through a 50 mesh sieve, and distilled water was added at a material to water ratio of 1:6 (w / w) and boiled until thick, then transferred to a 90℃ constant temperature water bath for pasting for 90 min (constant stirring to ensure uniform heating), then transferred to a 60℃ constant temperature water bath for liquefaction and saccharification, 2% of α-liquefying enzyme (enzyme activity > 3700 U / g) was added based on the amount of raw material and incubated for 60 min, then 1% of saccharifying enzyme (enzyme activity > 10,000 U / g) was added and incubated for another 60 min, then filtered, and the filtrate was divided and packaged, 200 mL was placed in a 500 mL triangular flask and sterilized at 121℃ for 20 min for standby.

[0062]

[0063] Comparative example

[0064] The comparative example utilizes the commercial koji in Table 1 to carry out simulated liquid fermentation of sorghum juice, specifically including the following steps:

[0065] Step one: colony count of the commercial koji

[0066] Take 1 g of the commercial liquor starter into 100 ml of sterile normal saline, mix for 30 min, stand for 30 min, take 1 ml of supernatant, dilute with normal saline to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , respectively, take 200 μl of the diluent on YPD plates, LB plates, and Bengal red medium plates, evenly spread the plates with a spreader or sterile glass beads, and place them in 30°C, 37°C, and 30°C incubators for 48 h, 24 h, and 72 h, respectively, until single colonies are completely grown. Count the colonies on the plates with better colony separation, and calculate the total number of colonies in 1 g of the commercial starter.

[0067] Step two: fermentation of the commercial starter with sorghum juice

[0068] According to the dosage indication in the commercial starter, add 0.2 g of Angel starter to the above 200 ml of sorghum juice prepared, set up 3 parallels, and carry out 30°C closed fermentation for 7 d.

[0069] Example 1

[0070] This example utilizes the enhanced koji A in Table 1 to carry out simulated liquid fermentation of sorghum juice. Example 1 aims to verify the performance of the complex microbial inoculant when the number of strains is 1:1:1 of Saccharomyces cerevisiae, bacteria, and molds, and the Saccharomyces cerevisiae JQSC002 only contains a single Saccharomyces cerevisiae, specifically including the following steps:

[0071] Step one: preparation of seed liquid of each strain

[0072] (1) Preparation method of Saccharomyces cerevisiae liquid: inoculate the Saccharomyces cerevisiae strain into YPD liquid medium and activate at 30°C for 48 h, draw a single colony on a YPD plate with the activated liquid, pick a single colony into YPD liquid medium and culture at 30°C for 24 h to obtain the Saccharomyces cerevisiae liquid, detect the cell concentration OD 600 , dilute the liquid to a final concentration OD 600 =2 with sterile normal saline, and obtain the Saccharomyces cerevisiae seed liquid for standby.

[0073] (2) Preparation method of Bacillus subtilis liquid (bacteria): inoculate Bacillus subtilis strain into LB liquid medium, activate at 37°C for 12-20 h, draw single colony on LB plate, pick single colony into LB liquid medium, and culture at 37°C for 16 h to obtain Bacillus subtilis liquid, and detect the bacterial concentration OD 600 Dilute the bacterial liquid with sterile normal saline to a final concentration OD 600 =0.5 to obtain Bacillus subtilis seed liquid for standby.

[0074] (3) Preparation method of Aspergillus niger spore suspension: inoculate Aspergillus niger mycelium into PDA plate, culture at 30°C for at least 72 h, inject sterile normal saline into the plate, and wash the plate with an inoculation stick until the spores are suspended in the liquid. Obtain Aspergillus niger spore suspension, and detect the spore liquid concentration OD 630 Dilute the spore liquid with sterile normal saline to a final concentration OD 630 =0.17 to obtain Aspergillus niger spore suspension for standby.

[0075] Step two: preparation of enhanced wine starter A:

[0076] Mix the Saccharomyces cerevisiae JQSC001 seed liquid, Bacillus subtilis JQBS001 seed liquid, and Aspergillus niger JQAN002 spore suspension according to the volume ratio of 4:1:1 to obtain a composite microbial inoculant A; when used, mix the commercial fermentation agent with the composite microbial inoculant A according to the ratio of 1:1 of the number of colonies, i.e. the mass M (g) / volume V (ml) is 1:7.5, to obtain the enhanced wine starter A.

[0077] Step three: fermentation of highland millet juice with enhanced wine starter A:

[0078] According to the amount of commercial fermentation agent added in the comparative example, mix 0.1 g of Angel fermentation agent with 0.75 ml of composite microbial inoculant A, and add it to the above prepared 200 ml of highland millet juice. Set three parallels, and ferment at 30°C for 7 days.

[0079] Example 2

[0080] This example uses the enhanced wine starter B in Table 1 for highland millet juice simulation liquid fermentation. Example 2 aims to verify the performance of the composite microbial inoculant when the number of strains is yeast:bacteria:mold=1:1:1, i.e. lacking Saccharomyces cerevisiae JQSC002, containing only a single Saccharomyces cerevisiae, and changing the mold strain and yeast strain combination. The specific steps are carried out according to the steps in Example 1, with the difference being:

[0081] Replace the enhanced wine starter A in Example 1 with the enhanced wine starter B for highland millet juice simulation liquid fermentation.

[0082] Preparation of the fortified starter B: the seed liquid of Saccharomyces cerevisiae JQSC002, the seed liquid of Bacillus subtilis JQBS001, and the spore suspension of Aspergillus niger JQAN002 were mixed in a volume ratio of 4:1:1 to obtain the compound microbial inoculant B; when used, the commercial starter was mixed with the compound microbial inoculant B in a strain number ratio of 1:1, i.e. a mass M (g) / volume V (ml) ratio of 1:7.5, to obtain the fortified starter B.

[0083] Example 3

[0084] This example uses the fortified starter C in Table 1 for the simulation of liquid fermentation of sorghum juice. Example 3 aims to verify the performance of the compound microbial inoculant when the bacterial strain combination is 2 strains of Bacillus amyloliquefaciens and the strain number is yeast:bacteria:mold = 1:2:1, specifically including the following steps:

[0085] Step one: preparation of the seed liquid of each strain

[0086] The seed liquid of each strain was prepared according to Step one in Example 1.

[0087] Step two: preparation of the fortified starter C

[0088] The seed liquid of Saccharomyces cerevisiae JQSC001, the seed liquid of Bacillus amyloliquefaciens JQBA001, the seed liquid of Bacillus amyloliquefaciens JQBA002, and the spore suspension of Aspergillus niger JQAN002 were mixed in a volume ratio of 4:0.5:0.5:1 to obtain the compound microbial inoculant C; when used, the commercial starter was mixed with the compound microbial inoculant C in a strain number ratio of 1:1, i.e. a mass M (g) / volume V (ml) ratio of 1:7.5, to obtain the fortified starter C.

[0089] Step three: simulation of liquid fermentation of sorghum juice using the fortified starter C

[0090] According to the amount of the commercial starter added in the comparative example, 0.1 g of Angel starter was mixed with 0.75 ml of the compound microbial inoculant C and added to the above prepared 200 ml of sorghum juice, and 3 parallel tests were set up for 7 days of closed fermentation at 30°C.

[0091] Example 4

[0092] This example uses the fortified starter D in Table 1 for the simulation of liquid fermentation of sorghum juice. Example 4 aims to verify the effect of changing the Bacillus species so that the bacterial strain combination is 2 strains of Bacillus subtilis and the strain number is yeast:bacteria:mold = 1:2:1 on the function of the microbial inoculant, and the specific steps are performed according to the steps in Example 3, with the difference being that:

[0093] Adjust the two strains of Bacillus amyloliquefaciens in Example 3 to two strains of Bacillus subtilis, and adjust the fermentation agent in Example 3 to the fortified starter D to perform the simulation liquid fermentation of the sorghum juice.

[0094] Example 5

[0095] This example uses the fortified starter E in Table 1 to perform the simulation liquid fermentation of the sorghum juice, and Example 5 aims to verify the influence of changing the Bacillus species and making the bacterial strain combination Bacillus subtilis + Bacillus amyloliquefaciens, and making the strain number be yeast:bacteria:mold = 1:2:1 on the function of the microbial agent. The specific steps are performed according to the steps in Example 3, and the difference lies in that:

[0096] Adjust the Bacillus amyloliquefaciens JQBA001 and Bacillus amyloliquefaciens JQBA002 in Example 3 to Bacillus amyloliquefaciens JQBA003 and Bacillus subtilis JQBS001, and adjust the fermentation agent in Example 3 to the fortified starter E to perform the simulation liquid fermentation of the sorghum juice.

[0097] Example 6

[0098] This example uses the fortified starter F in Table 1 to perform the simulation liquid fermentation of the sorghum juice, and Example 6 aims to verify the influence of increasing the Saccharomyces cerevisiae species and making the strain number be yeast:bacteria:mold = 2:1:1 on the function of the microbial agent. The specific steps include the following steps:

[0099] Step one: preparation of seed liquid of each strain:

[0100] Prepare the seed liquid of each strain according to Step one in Example 1.

[0101] Step two: preparation of the fortified starter F:

[0102] Mix the seed liquid of Saccharomyces cerevisiae JQSC001, the seed liquid of Saccharomyces cerevisiae JQSC002, the seed liquid of Bacillus subtilis JQBS001, and the spore suspension of Aspergillus niger JQAN001 according to the volume ratio of 2:2:1:1 to obtain the compound microbial agent F. When used, mix the commercial fermentation agent with the compound microbial agent F according to the ratio of 1:1 of the number of colonies, that is, the mass M (g) to the volume V (ml) is 1:7.5, to obtain the fortified starter F.

[0103] Step three: simulation liquid fermentation of the fortified starter F:

[0104] According to the addition amount of the commercial fermentation agent in the comparative example, mix 0.1 g of Angel fermentation agent with 0.75 ml of compound microbial agent F, and add it to the above prepared 200 ml of sorghum juice. Set three parallels, and perform closed fermentation at 30°C for 7 days.

[0105] Test Example 1:

[0106] 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:

[0107] 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.

[0108] Figure 1 The results are for the detection of physicochemical indicators.

[0109] 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).

[0110] Test Example 2:

[0111] The volatile flavor substances in the fermentation liquor obtained in the above comparative examples and each example were compared, and the headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used. The specific method was as follows:

[0112] The headspace SPME condition was as follows: 10 mL of the sample was taken into a 20 mL headspace bottle, 1.5 g of NaCl and 5 μl of an internal standard (2-octanol, 1 mg / mL) were added, and solid-phase microextraction (SPME) was performed. The sample was equilibrated at 50℃ for 15 min, and then the extraction fiber was inserted for adsorption for 30 min. After adsorption, pyrolysis was performed at the gas chromatography inlet at 250℃ for 3 min for the next analysis.

[0113] The GC condition was as follows: a DB-WAX chromatographic column (60 m x 0.25 mm, 0.25 μm) was used, He was used as the carrier gas, the flow rate was set to 1 mL / min. The column temperature program was as follows: 40℃ was kept for 3 min, then increased to 160℃ at a rate of 4℃ / min, then increased to 230℃ at a rate of 7℃ / min, and kept at this temperature for 8 min. The connecting rod temperature was set to 230℃. The MS condition was as follows: the full scan range was 33-450 amu, and the scanning was 1 time per second. EI was used as the ionization source, the ion source temperature was 230℃, the electron energy was 70 eV, the filament flow was 0.2 mA, and the detector voltage was 350 V. +

[0114] The volatile flavor substances in the fermentation liquor obtained in the above comparative examples and each example were analyzed by using the Qualitative Analysis software.

[0115] The results are shown in Table 1. Figure 2 As shown in Table 1, the contents of the key ester substances in the liquor in Example 6, such as ethyl acetate, ethyl butyrate, ethyl hexanoate and ethyl lactate, were significantly improved compared with the comparative examples, which were increased by 20%, 23%, 341% and 5300% respectively compared with the comparative examples. At the same time, the key higher alcohols such as isoamyl alcohol were deeply inhibited, which were reduced by 59.5% compared with the control group. It is proved that the compound microbial inoculant can improve the synthesis of ester substances, which verifies the above conclusion; at the same time, it can significantly reduce the synthesis of higher alcohols, and improve the drinking comfort.

[0116] The terpene substances in the fermentation liquor obtained in the above comparative examples and each example were analyzed by using the Qualitative Analysis software.

[0117] ​The results are shown in Table 2, 9 kinds of terpenes are detected in the fermentation liquor obtained in Example 6, the kinds of terpenes are improved compared with the comparative examples; among them, the content of 1-octene-3-ol, linalool, beta-ionone, damascone, 4-ethyl-guaiacol, geraniol is obviously higher than that in the comparative examples, the total concentration reaches 15.51 mg / L, which is increased by 28.5% compared with the comparative examples. It shows that the compound microbial inoculant of the present application can strengthen the generation of terpenes, not only make the flavor substances more full, but also improve the health factors.

[0118]

[0119] Finally, it should be noted that the above is only to illustrate the technical solutions of the present application rather than limiting, although the present application is described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalent, without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A synergistic high-alcohol-reducing and health factor-enriching composite microbial inoculant, characterized in that, The complex microbial agent is composed of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001 and Aspergillus niger JQAN001; The Saccharomyces cerevisiae JQSC001 is deposited in the China General Microbiological Culture Collection Center, classified and named as Saccharomyces cerevisiae, has a preservation number of CGMCC NO. 35241, and was deposited on July 15, 2025; The Saccharomyces cerevisiae JQSC002 is deposited in the China General Microbiological Culture Collection Center, classified and named as Saccharomyces cerevisiae, has a preservation number of CGMCC NO. 35242, and was deposited on July 15, 2025; The Bacillus subtilis JQBS001 is deposited in the China General Microbiological Culture Collection Center, classified and named as Bacillus subtilis, has a preservation number of CGMCC NO. 35239, and was deposited on July 15, 2025; The Aspergillus niger JQAN001 is deposited in the China General Microbiological Culture Collection Center, classified and named as Aspergillus niger, has a preservation number of CGMCC NO. 42130, and was deposited on July 15, 2025; The complex microbial agent is prepared by mixing the bacterial liquid of Saccharomyces cerevisiae JQSC001, Saccharomyces cerevisiae JQSC002, Bacillus subtilis JQBS001 and the spore suspension of Aspergillus niger JQAN001 at a volume ratio of 2:2:1:1; The effective bacteria concentration of Saccharomyces cerevisiae JQSC001 and Saccharomyces cerevisiae JQSC002 liquid before mixing was (6-7)× CFU / mL, the effective bacteria concentration of Bacillus subtilis JQBS001 liquid was (3-4)× CFU / mL, and the concentration of Aspergillus niger JQAN001 spore suspension was (1-2)× CFU / mL.

2. A composite microbial inoculant fortified distiller's yeast, characterized in that, The fortified distiller's yeast comprises a commercial liquor fermentation agent and the complex microbial agent of claim 1.

3. A method for preparing the composite microbial agent as described in claim 1, characterized in that, The preparation method comprises the following steps: (1) Preparation of Saccharomyces cerevisiae bacterial liquid: inoculate the Saccharomyces cerevisiae strain in YPD liquid medium, activate at 30℃ for 24-48 h, draw single colonies on a YPD plate, pick single colonies in YPD liquid medium and culture at 30℃ for 24-48 h to obtain the Saccharomyces cerevisiae bacterial liquid; (2) Preparation of Bacillus subtilis bacterial liquid: inoculate the Bacillus subtilis strain in LB liquid medium, activate at 37℃ for 12-20 h, draw single colonies on an LB plate, pick single colonies in LB liquid medium and culture at 37℃ for 12-16 h to obtain the Bacillus subtilis bacterial liquid; (3) Preparation of Aspergillus niger spore suspension: inoculate the Aspergillus niger mycelium on a PDA plate, culture at 30℃ for at least 72 h, inject sterile normal saline into the plate, and wash the plate with an inoculation stick until the spores are suspended in the liquid to obtain the Aspergillus niger spore suspension; (4) Mix the prepared Saccharomyces cerevisiae bacterial liquid, Bacillus subtilis bacterial liquid and Aspergillus niger spore suspension in proportion to obtain the complex microbial agent.

4. The application of the composite microbial inoculant of claim 1, the composite microbial inoculant fortified distiller's yeast of claim 2 or the composite microbial inoculant prepared by the method of claim 3 in the production of Baijiu.

5. Use according to claim 4, characterized in that, The application comprises one or more of the following: (1) The composite microbial inoculant or the composite microbial inoculant fortified distiller's yeast increases the yield of ethanol; (2) The composite microbial inoculant or the composite microbial inoculant fortified distiller's yeast increases the content of ester substances; the ester substances are ethyl acetate, ethyl lactate, ethyl butyrate and ethyl hexanoate; (3) The composite microbial inoculant or the composite microbial inoculant fortified distiller's yeast reduces the yield of higher alcohols; the higher alcohols are isoamyl alcohol; (4) The composite microbial inoculant or the composite microbial inoculant fortified distiller's yeast increases the yield of 1-octen-3-ol, linalool, β-ionone, damascone and 4-ethyl-guaiacol.

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