Microbial combined microbial inoculant for liquor fermentation and application thereof

Abstract

The application discloses a microbial combined bacterial agent for liquor fermentation and application, and belongs to the technical field of liquor brewing microorganisms. The application provides a microbial combined bacterial agent, which can enhance the generation of characteristic aroma through the synergistic effect of multiple strains, can also inhibit the generation of an undesirable flavor substance, geosmin, and enrich the overall flavor level of the liquor body. The microbial combined bacterial agent can be directly added to the stacking fermentation link, without the need to modify the existing production process, and is suitable for the existing production process of enterprises, and has low application threshold. Compared with a natural fermentation product, the total volatile substance diversity of the sesame-flavor liquor brewed by using the bacterial agent is increased by more than 50%, the content of the core flavor substance, 3-methylthiopropionaldehyde, is increased by 82.8%, the sesame-flavor characteristic is more prominent, the fullness and softness of the liquor body are better, and the overall quality is obviously improved.

Description

Technical Field

[0001] This invention relates to a microbial combination agent for baijiu fermentation and its application, belonging to the field of baijiu brewing microbial technology. Background Technology

[0002] Traditional baijiu production relies heavily on natural inoculation to enrich microorganisms in the environment. Through long-term domestication, a unique brewing microbial system is formed. The stability of flavor and quality depends entirely on the microbial community in the fermentation pits and production environment. Because the abundance of microorganisms in the natural environment fluctuates greatly, it is difficult to stably control the quality and style of baijiu.

[0003] Generally, the amount of bran koji used in the production of sesame-flavored baijiu is much greater than that of daqu (fermented wheat koji). Bran koji includes yeast koji, bacterial koji, and mold koji. Yeast koji mainly includes... Saccharomyces cerevisiae , Pichia kudriavzevii , Wickerhamomyces anomalus , Saccharomycopsis fibuligera , White spp.、 Little Hansen Spp. and other yeasts perform both ethanol fermentation and aroma substance metabolism. Bacterial koji mainly includes Bacillus subtle , Bacillus amyloliquefaciens , Bacillus licheniformis During fermentation, bran koji provides proteases and flavor precursors. Aspergillus is the dominant mold in koji, and its acidic proteases hydrolyze proteins in the raw materials, providing small molecules for the growth and metabolism of other microorganisms, while also providing precursors for the production of volatile flavor compounds. Therefore, bran koji plays a crucial role in shaping the quality and style of sesame-flavored baijiu. However, in actual production, the source of the bran koji microbial community for sesame-flavored baijiu is highly random, and its composition and dynamics are extremely unstable. This natural uncontrollability directly leads to significant differences in the composition ratio of key flavor compounds and the final sensory quality between different batches of baijiu. Therefore, traditional methods struggle to achieve stable and consistent product flavor, and face fundamental bottlenecks and challenges in standardized control, large-scale expansion, and intelligent production transformation.

[0004] In existing technological explorations, some studies have attempted to optimize the fermentation process of baijiu (Chinese liquor) by introducing single-functional strains or constructing simple microbial communities. However, these attempts generally suffer from several key flaws: First, the strain combinations used often lack precise target guidance, failing to specifically screen and match the characteristic flavor substances of sesame-flavored baijiu, resulting in insufficient typicality of the sesame aroma and an indistinct characteristic flavor in the final product. Second, the constructed microbial communities often suffer from an excessive number of strains or severe overlap in functional roles, intensifying the fierce competition for nutrients and space within the community. This causes some introduced functional strains to fail to stably colonize and continuously exert their effects in the fermentation system, resulting in large fluctuations and poor reproducibility in fermentation results. Finally, many studies are limited to idealized laboratory conditions, and the designed microbial agents lack consideration for the adaptability to the complex environment of actual solid-state fermentation (such as material matrix, temperature gradient, water activity, oxygen distribution, etc.). This leads to a significant decline in the metabolic function and colonization capacity of superior strains screened in the laboratory when scaled up to industrial production scenarios, making technology transfer and transformation difficult.

[0005] In conclusion, to overcome the inherent drawbacks of traditional brewing processes and the shortcomings of existing microbial agents, the industry urgently needs to develop a novel, dedicated microbial solution. This solution requires constructing a synthetic microbial community with a streamlined and efficient strain composition, complementary and synergistic metabolic functions, and strong and stable colonization capabilities in a solid-state fermentation environment. Ultimately, this will result in a synthetic microbial combination agent specifically designed for baijiu (Chinese liquor) that can seamlessly integrate with and enhance existing industrial production processes. Summary of the Invention

[0006] [Technical Issues] This invention aims to provide a combined microbial agent for the fermentation of baijiu (Chinese liquor). By screening core strains with well-defined functions and strong complementarity, it solves the problems of unstable microbial communities, uncontrollable flavor, and lack of prominent aroma characteristics in traditional brewing. At the same time, it ensures stable fermentation parameters and a significant improvement in flavor substances, thus meeting the needs of industrialized baijiu production.

[0007] [Technical Solution] This invention provides a microbial combination agent, wherein the microbial combination agent includes Bacillus cereus (… Bacillus vallismortis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Aspergillus oryzae ( ) Aspergillus oryzae ), brewer's yeast ( Saccharomyces cerevisiae ), acid-resistant lactobacillus ( Lactobacillus acetotolerans ), Enterobacter sieboldii ( Weissella paramesenteroides ), Kudria zweipichia yeast ( Pichia kudriavzevii ), abnormal Wickham yeast ( Wickerhamomyces anomalous ).

[0008] In one embodiment of the present invention, the microbial combination agent is composed of Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter strobilus, Pichia pastoris, and Wickham anomala in a ratio of (2-8): (3-10): (1-5): (20-60): (5-15): (1-5): (10-30): (4-16) based on the number of live cells.

[0009] In one embodiment of the present invention, the microbial combination agent is composed of Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter strobilus, Pichia kudrica, and Wickham's yeast in a ratio of 5:6:3:30:10:3:20:8 based on the number of live cells.

[0010] In one embodiment of the present invention, the total number of viable bacterial strains in the microbial combination agent is at least 10. 8 CFU / g.

[0011] In one embodiment of the present invention, the dead Bacillus ( Bacillus vallismortis It was deposited on November 5, 2018, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO. 16704.

[0012] In one embodiment of the present invention, the Bacillus amyloliquefaciens (Bacillus) Bacillus starch liquefier It was deposited at the China Center for Type Culture Collection (CCTCC) on August 12, 2013, with accession number CCTCC M2013368.

[0013] In one embodiment of the present invention, the Aspergillus oryzae ( Aspergillus oryzae It was deposited at the China General Microbiological Culture Collection Center on December 14, 2017, with accession number CGMCCNO.14997.

[0014] In one embodiment of the present invention, the brewing yeast ( Saccharomyces cerevisiae It was deposited at the China General Microbiological Culture Collection Center on May 6, 2016, with accession number CGMCCNO.12417.

[0015] In one embodiment of the present invention, the acid-resistant lactobacillus ( Lactobacillus acetotolerans It was deposited at the China General Microbiological Culture Collection Center on December 9, 2020, with accession number CGMCCNO.21135.

[0016] In one embodiment of the present invention, the Enterobacter sieboldii ( White-bellied paramesenteroid It was deposited at the China General Microbiological Culture Collection Center on April 25, 2021, with accession number CGMCC NO.22238.

[0017] In one embodiment of the present invention, the Kudriazweibichi yeast ( Peach kudriavzevii It was deposited at the China General Microbiological Culture Collection Center on May 6, 2016, with accession number CGMCC NO.12418.

[0018] In one embodiment of the present invention, the abnormal Wickham yeast ( Wickerhamomyces anomalous It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 6, 2016, with accession number CGMCC NO.12416.

[0019] In one embodiment of the present invention, the viable concentration of each microorganism in the microbial composite agent is 10. 8 ~10 10 CFU / g.

[0020] The present invention also provides a method for preparing the aforementioned microbial composite inoculant, as follows: (1) Prepare solid microbial agents of single strains of Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter cytogenes, Pichia kudrica, and Wickham's abnormality, respectively. (2) Mix the solid bacterial agents of each single strain according to the ratio of viable bacteria (2~8): (3~10): (1~5): (20~60): (5~15): (1~5): (10~30): (4~16) to obtain the synthetic microbial combination bacterial agent.

[0021] The present invention also provides the application of the aforementioned microbial combination in the preparation of sesame-flavored baijiu; the baijiu is preferably sesame-flavored baijiu.

[0022] In one embodiment of the present invention, the application is to add the microbial combination agent to a brewing substrate or brewing raw material for fermentation to prepare baijiu (Chinese liquor).

[0023] In one embodiment of the present invention, the amount of the microbial combination inoculant added is 0.1% to 1% of the mass of the brewing substrate.

[0024] The present invention also provides a method for improving the aroma of baijiu, wherein the method comprises adding 0.1% to 1% by weight of a microbial combination agent to the mash.

[0025] In one embodiment of the present invention, the liquor is a sesame-flavored liquor.

[0026] [Beneficial Effects] 1. Concise and complementary strains: The nine core strains selected cover the key functions required for the entire fermentation process. They work synergistically with each other, avoiding microbial redundancy and effectively reducing competition and inhibition between different strains, thereby improving the overall fermentation efficiency and stability.

[0027] 2. Stable and controllable fermentation process: All nine strains can achieve stable colonization in the fermentation system. Their acid production capacity and ethanol yield are comparable to those of traditional complex microbial community processes. They will not disrupt the original dynamic balance of fermentation, ensuring a smooth fermentation process and predictable results.

[0028] 3. Comprehensive optimization of sensory quality: The product boasts a more prominent aroma, significantly improved smoothness and body, enhanced sweetness, and better overall harmony. Professional tastings have shown a 10%–15% increase in sensory scores compared to traditional methods, resulting in a markedly upgraded overall drinking experience.

[0029] 4. Strong industrial adaptability and easy to promote and apply: The preparation process of the microbial agent is simple, the production cost is low, and it is highly compatible with existing conventional production equipment and processes. No large-scale modification is required, so it has good potential for industrialization and market promotion. Detailed Implementation

[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0031] The detection methods involved in the following embodiments: Detection of total volatile substances: Accurately weigh 5 g of fermented mash sample, add 20 mL of ultrapure water, mix thoroughly, sonicate in an ice bath for 30 min, centrifuge at 8,000 × g for 10 min, and collect the supernatant. Place 8 mL of the supernatant, 20 µL of internal standard (108 µg / mL menthol), and 3 g of NaCl into a 20 mL headspace vial. Use a 50 / 30 μm DVB / CAR / PDMS (Supelco, Inc., Bellefonte, PA) extraction head to determine the total volatile compounds using headspace solid-phase microextraction.

[0032] The detection instrument was a gas chromatography-mass spectrometry (GC-MS) system. This instrument was equipped with an automated headspace sampler (Gerstel, Inc., Mülheim, Ruhr, Germany). The detection column was a DB-Wax (30 m × 0.25 mm, 0.25 μm; J&W Scientific, Folsom, USA). The sample was equilibrated for 5 min, adsorbed for 45 min, and desorbed at 250 °C for 5 min.

[0033] The gas chromatographic column oven temperature program was as follows: 50℃ for 2 min, ramped up to 230℃ at 6℃ / min, and held at 230℃ for 20 min, with helium as the carrier gas at a flow rate of 2 mL / min. The volatile matter comparison database was the National Institute of Standards and Technology Mass Spectrometry Library (NIST2014). Volatile substances were semi-quantitatively identified by comparing the peak areas of volatile substances and internal standards.

[0034] Total volatile matter diversity is an indicator that comprehensively characterizes the types and concentrations of total volatile matter. The calculation method is as follows: (1) Normalize the volatile matter content of all fermentation samples to 0-1; (2) Calculate the average value of the normalized values ​​of all volatile matter in each fermentation sample.

[0035] Detection of volatile sulfur compounds (3-methylthiopropionaldehyde): To avoid weighing oxidized samples, 10 g of the fermented mash from the middle of the sample bag was accurately weighed into a sterile 50 mL centrifuge tube treated with argon gas. 20 mL of boiled ultrapure water was added, and the mixture was sonicated in an ice bath for 10 min, then centrifuged at 8,000 × g for 10 min. The supernatant was collected. 8 mL of the supernatant, 50 µL of internal standard (2.2 µg / L menthol), and 3 g of NaCl were placed into a 20 mL headspace vial treated with argon gas. Sulfide content was determined using headspace solid-phase microextraction (HS-SPME) on a triple quadrupole gas chromatography-mass spectrometry (GC-MS) system in full scan mode for qualitative and quantitative analysis of sulfides. The GC column temperature program was as follows: 50 ℃ for 2 min, ramped up to 230 ℃ at 6 ℃ / min, and held at 230 ℃ for 20 min, with helium at a carrier gas flow rate of 2 mL / min. The column used was a DB-Wax.

[0036] Example 1: Preparation of Microbial Combination Inoculants Table 1. Strains Information Table

[0037] 1. Preparation of culture medium Spore culture medium (g / L): potato flour 200, glucose 20, agar 15-20, pH natural, sterilized; Basic liquid culture medium 1 (g / L): yeast extract 10, peptone 20, glucose 20, pH natural, sterilized; Basic liquid culture medium 2 (g / L): beef extract 10, glucose 10, NaCl 5, pH 7.0, 1×10 5 Sterilize for 20 minutes using Pa. Basic liquid culture medium 3 (g / L): beef extract 5, peptone 10, yeast powder 4, MgSO4·7H2O 0.2, NaCl 5, Tween-80 1, pH 7.0, sterilized.

[0038] The liquid inoculum culture medium is prepared by crushing sorghum raw material samples, mixing them with water, adding 30-50 U / g of high-temperature α-amylase, cooking for 1-3 hours, cooling, adding 10-50 U / g of saccharifying enzyme, letting stand for 2-10 hours, filtering, centrifuging the filtrate, adjusting the sugar content to 6-12°Bx, adjusting the pH to 4-6, and sterilizing. The solid culture medium is prepared by mixing raw wheat bran with water and then steaming at 80-100℃ for 30 minutes.

[0039] 2. Preparation of solid microbial inoculants Preparation of solid inoculants of *Saccharomyces cerevisiae* CGMCC No. 12417, *Pichia kudriezvichi* CGMCC No. 12418, and *Wickham's Aberrant* CGMCC No. 12416: One loopful of cells was inoculated into basal liquid medium 1 and activated at 30℃ for 24 h to prepare a culture solution. The culture solution was then transferred to liquid inoculant medium and cultured at 30℃ for 20 h to prepare a seed culture. The prepared seed culture was then inoculated into solid medium and cultured at 30℃ for 20 h to obtain solid inoculants of *Saccharomyces cerevisiae* CGMCC No. 12417, *Pichia kudriezvichi* CGMCC No. 12418, and *Wickham's Aberrant* CGMCC No. 12416, respectively. Preparation of Aspergillus oryzae CGMCC NO.14997 solid inoculum: One loop of Aspergillus oryzae mycelium was inoculated into a spore medium and cultured at 30℃ for 48 h. After spores were washed away with buffer to obtain spore solution, the spore solution was inoculated into a solid medium and cultured at 30℃ for 24 h to obtain Aspergillus oryzae CGMCC NO.14997 solid inoculum. Preparation of solid bacterial agents of *Bacillus oryzae* CGMCC NO. 16704 and *Bacillus amyloliquefaciens* CCTCC M2013368: One loopful of bacterial cells was inoculated into basic liquid culture medium 2 and cultured at 37℃ for 16 h to prepare a culture solution. The culture solution was then transferred to liquid bacterial agent culture medium and cultured at 37℃ for 14 h to prepare a seed culture. The seed culture was inoculated into solid culture medium and cultured at 37℃ for 20 h to prepare solid bacterial agents of *Bacillus oryzae* CGMCC NO. 16704 and *Bacillus amyloliquefaciens* CCTCC M2013368, respectively. Preparation of solid bacterial agents of *Lactobacillus acid-fast bacillus* CGMCC No. 21135 and *Westernella enterica* CGMCC No. 22238: One loopful of bacterial cells was inoculated into basal liquid medium 3 and cultured at 37℃ for 36 h to prepare a culture solution. The culture solution was then transferred to liquid bacterial agent medium and cultured at 37℃ for 72 h to prepare a seed culture. The seed culture was then inoculated into solid medium and cultured at 37℃ for 48 h to prepare solid bacterial agents of *Lactobacillus acid-fast bacillus* CGMCC No. 21135 and *Westernella enterica* CGMCC No. 22238, respectively.

[0040] The viable count of each solid microbial agent was tested. For Bacillus-based solid microbial agents (Bacillus glutenosa CGMCC NO. 16704 and Bacillus amyloliquefaciens CCTCC M2013368), the viable count was not less than 1.0 × 10⁻⁶. 8 The CFU / g count of yeasts (Saccharomyces cerevisiae CGMCC No. 12417, Pichia kudrica CGMCC No. 12418, and Wickham's abnormal CGMCC No. 12416) also reached 1.0 × 10⁻⁶. 8 CFU / g or higher. Furthermore, the number of Aspergillus oryzae CGMCC NO.14997 spores in the product is guaranteed to be no less than 1.0 × 10⁻⁶ per gram. 8 The total viable counts of *Lactobacillus acid-fast bacillus* CGMCC No. 21135 and *Westernella enteritidis* CGMCC No. 22238 were both not less than 1.0 × 10⁻⁶. 8 CFU / g. Furthermore, the prepared solid microbial agent was strictly controlled to have a contamination rate of less than 1.0%, while the moisture content was controlled to be below 10.0%.

[0041] Example 2: Application of Microbial Combination Agents in the Preparation of Sesame-Flavored Baijiu 1. Preparation of Microbial Combination Inoculants Solid inoculum of *Bacillus oryzae* (CGMCC NO. 16704), *Bacillus amyloliquefaciens* (CCTCC M2013368), *Aspergillus oryzae* (CGMCC NO. 14997), *Saccharomyces cerevisiae* (CGMCC No. 12417), *Lactobacillus acid-fast bacillus* (CGMCC No. 21135), *Westernella enteroides* (CGMCC No. 22238), *Pichia pastoris* (CGMCC No. 12418), and *Wickham's aberrant* (CGMCC No. 12416) was mixed in a ratio of 5:6:3:30:10:3:20:8, resulting in a total viable count of 1.8 × 10⁻⁶ cells. 10 CFU / g.

[0042] 2. Application of combined microbial inoculants The microbial inoculum prepared in step 1 was added to the fermented mash used in the production of sesame-flavored baijiu at a rate of 0.5% of the mash weight. After fermentation for 40 hours, the mash was transferred to a fermentation tank for 30 days. Three independent fermentation batches were conducted in both the experimental and control groups. The total volatile matter diversity, 3-methylthiopropionaldehyde, and sensory evaluations were performed on the baijiu.

[0043] Table 2 Comparison of Detection Indicators

[0044] Note: The experimental group was the fermentation group with the addition of the microbial combination agent of this invention, and the control group was the traditional natural fermentation group without the addition of this agent. ns indicates no significant difference.

[0045] As can be seen from the test results in Table 2, after the microbial combination agent of the present invention was applied to the fermentation of sesame-flavored baijiu, the diversity of total volatile substances increased by 51.2%, and the characteristic flavor substance 3-methylthiopropionaldehyde increased by 82.8%, with both improvement effects reaching a highly significant level. At the same time, the acidity and ethanol yield during the fermentation process were not significantly different from the control group, proving that the combination agent does not disrupt the acid-base balance and alcohol production capacity of the original fermentation system, and fully meets the fermentation requirements of traditional sesame-flavored baijiu.

[0046] Next, the base spirit obtained from distillation underwent professional sensory evaluation. The sensory evaluation team consisted of 15 members, including 7 men and 8 women, aged between 30 and 55, including 2 national-level judges, and the remaining judges were all engaged in baijiu flavor research. This evaluation was based on sesame-flavored baijiu. In sensory analysis, eight sensory terms commonly used in sensory analysis were selected: "aroma intensity", "acidity", "sweetness", "spiciness", "fullness", "sesame aroma", "soy sauce aroma" and "smoothness". Under the temperature conditions of 20-25℃, a six-point structured scale ("none", "very weak", "weak", "medium", "strong", "very strong") was used to score the intensity of the eight sensory terms of sesame-flavored base liquor from 0 to 5.

[0047] Table 3 Sensory Evaluation

[0048] The results are shown in Table 3. The judges generally described the experimental group's base liquor as having "a prominent sesame aroma with a pleasant sweetness, and an elegant and lasting fragrance," while the control group's aroma was described as "a weak sesame aroma, a prominent acidity, and insufficient aroma complexity." Overall, the addition of this combination of microbial agents clearly improved the flavor and quality of the base liquor.

[0049] The microbial combination agent of the present invention is simple to apply. It only needs to be added to the mash during the stacking process. It does not require changing the original production process of sesame-flavored baijiu. It is compatible with existing traditional production equipment and processes and will not add extra production steps or costs. It is suitable for promotion and application in the industry. It can help production enterprises to steadily improve the product quality of sesame-flavored baijiu, strengthen the product's style characteristics, and solve the common problems of unstable flavor substance content and lack of prominent sesame flavor in traditional natural fermentation.

[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A microbial composite inoculant, characterized in that, The microbial combination agent includes Bacillus cereus (Bacillus subtilis) Bacillus vallismortis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Aspergillus oryzae ( ) Aspergillus oryzae ), brewer's yeast ( Saccharomyces cerevisiae ), acid-resistant lactobacillus ( Lactobacillus acetotolerans ), Enterobacter sieboldii ( Weissella paramesenteroides ), Kudria zweipichia yeast ( Pichia kudriavzevii ), abnormal Wickham yeast ( Wickerhamomyces anomalus ).

2. The microbial composite inoculant according to claim 1, characterized in that, In the microbial combination agent, Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter cylindrica, Pichia pastoris, and Wickham anomala are mixed in the following proportions according to the number of live cells: (2~8): (3~10): (1~5): (20~60): (5~15): (1~5): (10~30): (4~16).

3. The microbial composite inoculant according to claim 2, characterized in that, In the microbial combination inoculant, Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter strobilus, Pichia kudrica, and Wickham's abnormal yeast are mixed in a ratio of 5:6:3:30:10:3:20:8 based on the number of live cells.

4. The microbial composite inoculant according to claim 1, characterized in that, The total viable bacterial strains in the microbial combination agent are at least 10. 8 CFU / g.

5. The microbial composite inoculant according to any one of claims 1 to 4, characterized in that, The dead Bacillus ( Bacillus vallismortis It was deposited on November 5, 2018 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 16704; The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens It was deposited at the China Center for Type Culture Collection (CCTCC) on August 12, 2013, with accession number CCTCC M2013368; Aspergillus oryzae ( Aspergillus oryzae It was deposited at the China General Microbiological Culture Collection Center on December 14, 2017, with accession number CGMCC NO.14997; The brewing yeast ( Saccharomyces cerevisiae It was deposited at the China General Microbiological Culture Collection Center on May 6, 2016, with accession number CGMCC NO.12417; The acid-resistant lactobacillus ( Lactobacillus acetotolerans It was deposited at the China General Microbiological Culture Collection Center on December 9, 2020, with accession number CGMCC NO.21135; The enteromorpha Weissella ( Weissella paramesenteroides It was deposited at the China General Microbiological Culture Collection Center on April 25, 2021, with accession number CGMCC NO.22238; The Kudriazwibichi yeast ( Pichia kudriavzevii It was deposited at the China General Microbiological Culture Collection Center on May 6, 2016, with accession number CGMCC NO.12418; The abnormal Wickham yeast ( Wickerhamomyces anomalus It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 6, 2016, with accession number CGMCC NO.12416.

6. The microbial composite inoculant according to claim 1, characterized in that, In the aforementioned microbial composite inoculant, the viable cell concentration of each microorganism is 10. 8 ~10 10 CFU / g.

7. A method for preparing the microbial composite inoculant according to any one of claims 1 to 6, characterized in that, Specifically as follows: (1) Prepare solid microbial agents of single strains of Bacillus cereus, Bacillus amyloliquefaciens, Aspergillus oryzae, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterobacter cytogenes, Pichia kudrica, and Wickham's abnormality, respectively. (2) Mix the solid bacterial agents of each single strain according to the ratio of viable bacteria (2~8): (3~10): (1~5): (20~60): (5~15): (1~5): (10~30): (4~16) to obtain the synthetic microbial combination bacterial agent.

8. The application of the microbial combination agent according to any one of claims 1 to 6 in the preparation of baijiu; preferably sesame-flavored baijiu.

9. The application according to claim 8, characterized in that, The application involves adding any of the microbial combination agents described in claims 1 to 6 to a brewing substrate or brewing raw materials for fermentation to prepare baijiu (Chinese liquor). Preferably, the amount of the microbial combination inoculant added is 0.1% to 1% of the mass of the brewing substrate.

10. A method for enhancing the aroma of baijiu (Chinese liquor), characterized in that, The method involves adding 0.1% to 1% of the microbial combination agent described in any one of claims 1 to 6 to the fermented mash.

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