Composite fermentation inoculant capable of efficiently producing flavor substances in Baijiu brewing and application thereof

Abstract

The invention discloses a compound fermentation inoculant capable of efficiently producing flavor substances in white spirit brewing and application of the compound fermentation inoculant. The compound fermentation inoculant is prepared from salt-tolerant bacillus G47, lactobacillus plantarum F25-4 and pichia kudriavzevii K44. In the compound fermentation system provided by the invention, the function of the bacillus is not obviously influenced, and the bacillus can stably play a role under the condition of coexistence of multiple microorganisms and has good micro-ecological compatibility. Moreover, after the compound fermentation inoculant is inoculated, experiments prove that the content of the flavor substances is not superposed with the fermentation effect of a single strain, but shows a remarkable synergistic effect, and a feasible technical means is provided for regulation and control of the flavor substances of the white spirit, research on a fermentation mechanism and optimization of related processes.

Description

Technical Field

[0001] This invention relates to the field of baijiu brewing technology, and in particular to a compound fermentation agent that can efficiently produce flavor substances in baijiu brewing and its application. Background Technology

[0002] Baijiu is a traditional Chinese distilled spirit, typically made from starchy plant materials such as sorghum, and produced using a multi-stage solid-state fermentation process. Its brewing process generally includes multiple stages such as steaming, adding yeast, piling, fermentation in pits, distillation, and repeated cycles. The fermentation cycle is relatively long, and environmental factors such as temperature, acidity, moisture, oxygen, and ethanol content continuously change during fermentation, resulting in a complex fermentation system with dynamic variations across multiple factors.

[0003] In the aforementioned fermentation system, a diverse range of microorganisms interact with each other through their metabolic activities, significantly influencing the formation of flavor compounds in baijiu. Among these, phenolic flavor compounds, as a representative class of components in baijiu, play a crucial role in the layers of taste, aftertaste characteristics, and overall flavor profile of the liquor.

[0004] Existing research indicates that phenolic substances in baijiu mainly originate from the release of phenolic precursors from raw materials and starter cultures, as well as the metabolic transformation of these precursors by microorganisms during fermentation. Compared to relying solely on the release of phenolic precursors from raw materials, the transformation of phenolic precursors into phenolic flavor compounds through microbial metabolism offers greater controllability.

[0005] However, the fermentation system of baijiu typically exhibits characteristics such as a gradual increase in acidity, a gradual accumulation of ethanol, and solid-state or near-solid-state fermentation. Under these environmental conditions, some microorganisms struggle to maintain stable metabolic activity, making the transformation process of phenolic precursors susceptible to environmental changes and difficult to regulate stably and reproducibly. Therefore, it remains necessary to provide a technical solution that can adapt to the simulated fermentation environment of baijiu and maintain the transformation behavior of phenolic precursors under these conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a compound fermentation agent that can efficiently produce flavor substances in baijiu brewing and its application. This compound fermentation agent can be used to generate and enrich phenolic flavor substances under the environmental conditions related to baijiu fermentation.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A compound fermentation agent that can efficiently produce flavor substances in the brewing of baijiu is composed of halophilic Bacillus G47, Lactobacillus plantarum F25-4 and Kudria zwibbiac yeast K44. The salt-resistant Bacillus G47, classified as... Bacillus halotolerunsIt is deposited at the China Center for Type Culture Collection (CCTCC) on April 25, 2025, with accession number CCTCC NO: M 2025886; The *Lactobacillus plantarum* F25-4 was classified and named... Lactobacillus plantarum It is deposited at the China Center for Type Culture Collection (CCTCC) on December 9, 2021, with accession number CCTCC NO: M 20211574; The aforementioned *Kudriazwibichthys* K44, classified and named... Pichia kudriavzecii It is deposited at the China Center for Type Culture Collection (CCTCC) on April 25, 2025, with accession number CCTCC NO: M2025887.

[0008] Furthermore, the ratio of the halophilic Bacillus G47, Lactobacillus plantarum F25-4, and Pichia kudrica K44 is 1:1:1.

[0009] The compound fermentation agent of the present invention can be used to promote the generation of phenolic flavor substances in the brewing of baijiu. The total inoculum amount of halophilic Bacillus G47, Lactobacillus plantarum F25-4 and Pichia pastoris K44 is 3% v / w of the fermentation substrate mass.

[0010] Furthermore, this includes the following steps: (1) Salt-tolerant Bacillus G47 was inoculated into LB liquid medium, Pichia pastoris K44 into YPD liquid medium, and Lactobacillus plantarum F25-4 into MRS liquid medium; all were cultured to the logarithmic growth phase, and the cells were collected by centrifugation, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; (2) Salt-tolerant Bacillus G47, Lactobacillus plantarum F25-4 and Pichia pastoris K44 were inoculated into the fermentation substrate at a ratio of 1:1:1, and the total inoculation amount was 3% v / w of the fermentation substrate mass; (3) The fermentation conditions are: pile culture at 37°C for the first 24 hours, and anaerobic fermentation at 30°C for the next 48-168 hours.

[0011] The method for enriching phenolic flavor compounds in a simulated fermentation system of baijiu using a compound fermentation agent of the present invention includes the following steps: (1) Construct a simulated fermentation system for baijiu (Chinese liquor). The fermentation system is a solid-state fermentation system. During the fermentation process, the pH of the system is controlled at 4.0-6.0, and ethanol is generated and gradually accumulated. (2) Inoculate the simulated fermentation system of Baijiu with Bacillus G47, Lactobacillus plantarum F25-4 and Kudria zwibbiac yeast K44; (3) Cultivate under certain fermentation conditions so that the compound fermentation agent participates in the conversion of phenolic precursor substances from raw materials and / or koji in the fermentation system, thereby obtaining phenolic flavor substances in the fermentation system.

[0012] The phenolic precursor is selected from one or more of ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid.

[0013] The phenolic flavor compounds include one or more of guaiacol, 4-ethylguaiacol, 4-vinylguaiacol, and 4-ethylphenol.

[0014] The fermentation conditions are pH 4.0-6.0 and ethanol volume fraction of 2%-8%.

[0015] The inoculation amount of Bacillus G47, Lactobacillus plantarum F25-4, and Pichia pastoris K44 was 1×10⁻⁶. 5 ~1×10 7 CFU / g fermentation substrate.

[0016] The Bacillus strain provided in this invention originates from a baijiu (Chinese liquor) brewing system. Under simulated solid-state or near-solid-state fermentation conditions, it can participate in the conversion of phenolic precursors in the fermentation system when inoculated alone, thereby generating phenolic flavor compounds. Considering the complex microecological environment where multiple microorganisms coexist in baijiu production, this invention further introduces other microorganisms such as lactic acid bacteria and yeast to form a composite fermentation system. The Bacillus strain can still maintain its ability to convert phenolic precursors and exhibits a significant synergistic effect.

[0017] Compared with the prior art, the outstanding effect of the present invention is as follows: (1) After extensive preliminary experiments, the research team of this invention found a compound fermentation agent composed of Bacillus, lactic acid bacteria and yeast. In this system, the function of Bacillus is not significantly affected and it can play a stable role under the coexistence of multiple microorganisms, showing good microecological compatibility. Moreover, after inoculating the compound fermentation agent of this invention, experiments have shown that the content of flavor substances is not the sum of the fermentation effects of a single strain, but rather shows a significant synergistic effect.

[0018] (2) By using the compound fermentation agent of the present invention, the generation process of phenolic flavor substances is stabilized while maintaining the key environmental characteristics of baijiu fermentation. This provides a practical and feasible technical means for the regulation of baijiu flavor substances, the study of fermentation mechanism and related process optimization.

[0019] (3) Under the conditions of increased acidity, gradual accumulation of ethanol and solid or quasi-solid fermentation during the simulated fermentation process of baijiu, the Bacillus in the compound fermentation agent of the present invention can still maintain its conversion behavior of phenolic precursor substances, and is suitable for the complex fermentation environment involved in baijiu production.

[0020] (4) The Bacillus in the compound fermentation agent of the present invention can participate in the transformation process of phenolic precursor substances even when it exists alone, avoiding dependence on complex microbial combinations or specific microbial community structures, and improving the controllability and repeatability of the technical solution.

[0021] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the compound fermentation agent of the present invention, which can efficiently produce flavor substances in the brewing of baijiu (Chinese liquor), and its application.

[0022] Biological Preservation Information Salt-resistant Bacillus G47, classified and named Bacillus halotoleruns It is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on April 25, 2025, with accession number CCTCC NO: M 2025886.

[0023] Lactobacillus plantarum F25-4, classified and named Lactobacillus plantarum The specimen is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on December 9, 2021. The accession number is CCTCC NO: M 20211574.

[0024] Kudria zweipichia korshinskii K44, classified and named Pichia kudriavzecii The specimen is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on April 25, 2025, with accession number CCTCC NO: M2025887. Attached Figure Description

[0025] Figure 1 The colony morphology of halophilic Bacillus G47.

[0026] Figure 2 Microscopic morphology of halophilic Bacillus G47.

[0027] Figure 3 This is the standard curve for 4-VG fluorescence detection.

[0028] Figure 4 This represents the changes in phenolic precursors and corresponding phenolic substances during fermentation. Detailed Implementation

[0029] Example 1: Screening of halophilic Bacillus I. Experimental Materials 1. Sample Source The fermented mash for baijiu is stored in a refrigerator at -20℃.

[0030] 2. Culture medium (1) Isolation and purification medium (NA): 10g peptone, 3g beef extract powder, 5g sodium chloride (NaCl), 20g agar powder, 1L deionized water, adjust pH to 7.2-7.4, sterilize at 121℃ for 20 min.

[0031] (2) Seed culture medium (LB): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1 L deionized water, pH 7.2, sterilized at 121℃ for 20 min.

[0032] (3) Liquid fermentation medium: 30 g glycerol, 20 g yeast extract, 4 g disodium hydrogen phosphate (Na2HPO4), 0.3 g dipotassium hydrogen phosphate (K2HPO4), 1 L deionized water, pH 6-8, sterilized at 121℃ for 20 min.

[0033] (4) Solid fermentation medium: 40.3% wheat bran, 18.3% soybean meal, 2.1% rice husk, 39.3% water, sterilized at 115℃ for 25 min.

[0034] II. Experiment Content 1. Strains screening (1) Initial screening Enrichment culture: Weigh 10 g of fermented mash and add it to a 250 mL shake flask containing 90 mL of sterile physiological saline (0.85% NaCl). Shake for 20 min. Incubate the suspension in an 80℃ water bath for 10 min to inactivate the vegetative cells and obtain a spore suspension.

[0035] Purification culture: After the above suspension has cooled to room temperature, perform a 10-fold serial dilution (10... -1 Up to 10 -6 100 μL of each of the different dilutions were spread onto NA (nutrient agar) plates. The plates were incubated at 30°C for 48 h. After incubation, colonies of different morphologies (rough, dry, irregular edges, and other typical Bacillus characteristics) were streaked onto NA plates for purification (at least three streaking purifications were performed). Finally, single colonies were inoculated onto NA slant tubes and incubated at 30°C for 24 h, then stored at 4°C for later use.

[0036] (2) Secondary screening Liquid fermentation experiment of strain: To verify the ability of each strain to produce phenolic substances, the screened purified strains were subjected to fermentation experiments on fermentation medium. The purified strains were inoculated into 50 mL shake flasks containing 10 mL of LB medium and cultured at 37℃ and 200 rpm for 24 h. The culture medium was then used as seed culture and inoculated at a rate of 2% into 250 mL shake flasks containing 50 mL of liquid fermentation medium, and cultured at 37℃ and 200 rpm for 48 h. 3 mL of the culture medium was transferred to a 10 mL centrifuge tube, centrifuged at 7000 rpm for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm nylon membrane and used for GC-MS to determine the ability of the strains to produce phenolic substances through liquid fermentation.

[0037] Strains of wheat bran solid-state fermentation experiment: The purified strain was inoculated into a 50 mL shake flask containing 10 mL of fermentation medium and cultured at 37°C and 200 rpm for 24 h. The culture medium was then used as seed culture and inoculated at a rate of 2% into a 250 mL shake flask containing 50 g of wheat bran culture medium, and cultured at 37°C for 48 h. After culturing, approximately 20 g of the solid culture was weighed, and 120 mL of 12% ethanol was added. The mixture was sonicated for 20 min and then allowed to stand overnight. 10 mL of the supernatant was centrifuged at 7000 rpm for 10 min, filtered through a 0.22 μm nylon membrane, and then used for GC-MS to determine the strain's ability to produce phenolic substances through solid-state fermentation of wheat bran.

[0038] Based on the comprehensive testing of the above liquid and solid fermentation performance, the superior strain G47, which has both high phenol production capacity and excellent fermentation characteristics, was selected.

[0039] 2. Strain identification (1) Morphological observation The selected strain G47 was streaked onto NA solid medium, and the characteristics of single colonies, such as color, transparency, smoothness, moisture, and whether the edges were neat, were observed. Single colonies were also selected for morphological observation under a microscope.

[0040] The results are as follows Figure 1 As shown. The colonies of this bacterium are milky white and opaque, mostly round to irregularly round, scattered along the direction of the streaks, with irregular, slightly wavy edges, a rough and somewhat dry surface, and a powdery or cheese-like texture. The degree of elevation ranges from slightly raised to convex. Under a microscope, they can be observed to be (…). Figure 2The bacterial cells are primarily rod-shaped single-celled structures, with relatively uniform cell size, mostly scattered or arranged in short chains. The cells are uniformly stained, with clear structures, and no obvious contamination by other bacteria was observed. Its typical rod-shaped morphology is consistent with the morphological characteristics of Bacillus bacteria.

[0041] (2) DNA extraction The selected salt-tolerant strain G47 was inoculated into LB liquid medium and cultured at 37℃ (or 30℃) and 200 rpm for 12–24 h. One mL of the bacterial culture was centrifuged at 4℃ for 1 min, the supernatant was discarded, and the bacterial cells were collected. Total bacterial genomic DNA was extracted using a rapid bacterial genomic DNA extraction kit according to the manufacturer's instructions.

[0042] (3) PCR amplification The 16S rDNA gene of strain G47 was amplified by PCR using universal bacterial PCR primers 27F and 1492R (as shown in SEQ ID NO: 2-3). The integrity of the amplified bands was detected by 1%–2% agarose gel electrophoresis.

[0043] Table 1 Primer Sequences

[0044] (4) Sequencing identification The sequencing and identification were commissioned to General Biotech (Anhui) Co., Ltd. The sequencing results are shown in SEQ ID NO: 1.

[0045] Sequence alignment on the NCBI platform revealed that strain G47 was similar to... Bacillus halotolerans The strain showed the highest homology, therefore it was named halophilic Bacillus G47. Bacillus halotoleruns G47).

[0046] Example 2: Application of halophilic Bacillus G47 in a simulated fermentation system for Baijiu (Chinese liquor) A simulated fermentation system for baijiu (Chinese liquor) was constructed using sorghum as the main raw material. The sorghum, after appropriate crushing and gelatinization, served as the fermentation substrate. The system's moisture content was adjusted to 38-42%, and the sorghum was sterilized before use. In the fermentation system, exogenous enzymes assisted in the starch hydrolysis process.

[0047] The above-mentioned simulated fermentation system for baijiu was inoculated with halophilic Bacillus G47 at an inoculation rate of 1×10⁻⁶. 6 CFU / g fermentation substrate.

[0048] Fermentation was carried out at 28-30℃, with an acidic environment and gradual accumulation of ethanol during the fermentation process, lasting 5-7 days. After fermentation, the phenolic substances in the fermentation products were analyzed, and the results are shown in Table 2.

[0049] Table 2. Phenolic substances of Bacillus halophilus G47 in a simulated Baijiu fermentation system

[0050] The test results showed that, compared with the control system without Bacillus inoculation, the simulated fermentation system of Baijiu inoculated with Bacillus inoculated with Bacillus could promote the generation of phenolic flavor substances, indicating that Bacillus can participate in the conversion process of phenolic precursor substances under this fermentation environment.

[0051] Example 3: Application of halophilic Bacillus G47 in the preparation of fortified Daqu (a type of starter culture) 1. Experimental Materials Experimental strain: Salt-tolerant Bacillus G47, which produces phenolic acid decarboxylase (PAD) activity.

[0052] Raw materials for making koji: high-quality wheat, with 30%-40% of the wheat passing through a 20-mesh sieve and the remainder passing through an 8-mesh sieve.

[0053] 2. Preparation method of enhanced Daqu (a type of starter culture) The specific steps for preparing enhanced Daqu (a type of starter culture) using bio-fortification technology are as follows: Preparation of bacterial suspension: Bacillus G47 was activated and cultured on a large scale. The bacterial cells were collected by centrifugation and resuspended in sterile water to prepare a high-concentration bacterial suspension (1×10⁻⁶). 8 (CFU / mL) for later use.

[0054] Mixing and Inoculation: Take the crushed wheat raw material and add an appropriate amount of water (approximately 37%-40% of the raw material mass) and stir to moisten. During the mixing process, evenly spray the prepared G47 bacterial suspension into the mixing water, controlling the inoculation amount to 1×10⁻⁶. 6 CFU / g of koji blank (based on the wet weight of the koji blank).

[0055] Treading and shaping: The inoculated raw materials are put into a mold and shaped by manual or mechanical treading to form koji blanks.

[0056] Fermentation: The koji blanks are transferred to the fermentation room and managed according to the traditional medium-high temperature koji cultivation process. The stages of initial fermentation, low-temperature growth, and high-temperature conversion are controlled, with the highest product temperature controlled at 60℃±2℃, and the cultivation cycle is approximately 30 days.

[0057] Finished product storage: After the koji is cultivated, it is aged and stored for 3 months to obtain "G47 reinforced koji".

[0058] Preparation of the control group: Except for the absence of Bacillus G47 bacterial culture (which was replaced with an equal amount of sterile water), the raw materials, processes and culture conditions were exactly the same, and "ordinary control koji" was prepared.

[0059] 3. Methods for determining PAD enzyme activity (1) Preparation of crude enzyme solution: Take the sample to be tested Sample preparation: Weigh 1.0 g of enhanced Daqu (fermented koji), add 9.0 mL of 50 mM phosphate buffer (PBS, pH 6.0), and extract by shaking at 4°C for 30 min. After extraction, centrifuge at 12000 rpm for 10 min at 4°C, and collect the supernatant as crude enzyme solution.

[0060] (2) Reaction system: Buffer: 50 mM phosphate buffer (pH 6.0); Final substrate concentration: 2.0 mM ferulic acid; Total reaction volume: 1 mL (500 μL substrate buffer + 500 μL crude enzyme solution); Reaction temperature: 37℃; Reaction time: 5-10 min; Blank / control: Heat inactivation control: Crude enzyme solution was treated at 100℃ for 10 min and then added to the reaction system.

[0061] (3) Termination of reaction and sample preparation The reaction was terminated by adding 1 mL of methanol, followed by centrifugation at 12000 rpm for 5 min, and the supernatant was collected for analysis.

[0062] (4) Fluorescence detection Take the supernatant and measure the fluorescence intensity of Ex 258 / Em 345 in fluorescence mode. Use 4-VG standard to make a standard curve and convert the fluorescence intensity to 4-VG concentration according to the standard curve.

[0063] (5) Definition of enzyme activity 1 U can be defined as: the amount of enzyme that generates 1 μmol of 4-VG per minute under the conditions specified above. This is then converted to U / g sample based on the enzyme volume and reaction time.

[0064] (6) 4-VG standard curve Standard solutions with gradient concentrations were prepared using 4-VG. The fluorescence intensity of Ex 258 / Em 345 was measured in fluorescence mode. Standard curves were plotted with 4-VG concentration and Ex 258 / Em 345 fluorescence intensity as the x and y axes, respectively. The results are shown below. Figure 3 As shown, there is a good linear correlation between fluorescence intensity and concentration (R0). 2 > 0.999).

[0065] 4. Enhanced assay of Daqu enzyme activity "G47 Enhanced Daqu" and "Ordinary Control Daqu" that had reached the end of their storage period were taken separately, ground and pulverized, and crude enzyme solutions were extracted according to the above method. The activity of phenolic acid decarboxylase (PAD) was then measured, and the results are shown in Table 3. The test results showed that the phenolic acid decarboxylase activity in the enhanced Daqu was 3.2-3.4 times that of the ordinary Daqu.

[0066] Table 3. Phenolic acid decarboxylase activity in enhanced Daqu and traditional Daqu

[0067] Example 4 Preparation of Compound Fermentation Agent 1. Experimental strain Phenolic Bacillus G47: Derived from the brewing system related to Baijiu (Chinese liquor), it has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025886.

[0068] Lactobacillus plantarum F25-4: Derived from the rice wine production system, it has been deposited at the China Center for Type Culture Collection, with biological accession number CCTCC NO: M 20211574.

[0069] Kudria zweipichia K44: Derived from Baijiu mash, it has been deposited at the China Center for Type Culture Collection, with biological accession number CCTCC NO: M 2025887.

[0070] 2. Simulated solid-state fermentation medium Guizhou red sorghum was selected as the raw material. The sorghum grains were partially crushed, so that each grain was broken into 4-6 pieces, and then mixed with the uncrushed whole sorghum grains at a mass ratio of 1:4. An appropriate amount of water was added to the mixture to adjust its moisture content to about 40%.

[0071] The mixture was cooked at 115°C for 15 min, cooled, and then liquefied by adding 0.3% (w / w) α-amylase. After liquefaction, 0.3% (w / w) saccharifying enzyme was added to carry out saccharification until the soluble sugar content was basically stable. The saccharified substrate was then sterilized at 121°C for 20 min and cooled to below 28°C for later use as a sorghum-based culture medium.

[0072] 3. Seed liquid preparation G47 bacteria were inoculated onto LB broth, K44 bacteria onto YPD broth, and F25-4 bacteria onto MRS broth. After incubation to the logarithmic growth phase, the cells were collected by centrifugation, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL.

[0073] 4. Experimental grouping and inoculation Four parallel experimental groups were set up, with the total inoculum amount uniformly controlled at 3% (v / w) of the fermentation substrate mass. The specific design is as follows: Control group A: Inoculated with 3% G47 alone; Control group B: Inoculated with 3% K44 alone; Control group C: Inoculated alone with 3% F25-4; Experimental group D (synergistic group): mixed inoculation of 1% G47 bacteria + 1% K44 bacteria + 1% F25-4 bacteria (ratio 1:1:1).

[0074] 5. Fermentation conditions First 24 hours: Stack culture at 37℃ (microaerophilic, conducive to Bacillus growth and enzyme production); The following 48-168 hours: Anaerobic fermentation at 30℃ (which is beneficial for the metabolism of yeast and lactic acid bacteria to produce alcohol and acid).

[0075] 6. Flavor compound detection and result analysis The fermentation samples after fermentation were analyzed for volatile flavor compounds using GC-MS, as shown in Table 4. The results indicate that the flavor compound content of the three-strain mixed inoculation (experimental group D) is not a simple additive effect of single strains, but rather exhibits a significant synergistic effect.

[0076] Table 4. Content of key volatile flavor compounds (μg / kg) in simulated Baijiu fermentation samples from different treatment groups.

[0077] Example 5: Application of compound fermentation inoculum under phenolic precursor enhancement conditions Based on Example 4, a phenolic precursor substance, selected from ferulic acid, was added to the simulated fermentation system of Baijiu before fermentation. Subsequently, Bacillus phenologenus G47, Lactobacillus plantarum F25-4, and yeast K44 were inoculated to construct a composite microbial fermentation system, which was then cultured under the same fermentation conditions.

[0078] During fermentation, changes in phenolic precursors and corresponding phenolic substances are monitored (e.g., Figure 4 (As shown). The results indicate that even with the addition of phenolic precursors, the conversion of phenolic precursors into phenolic flavor compounds can still be observed in the composite microbial system, further verifying the applicability of the Bacillus strain in a simulated baijiu fermentation environment.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound fermentation agent that can efficiently produce flavor substances in the brewing of baijiu (Chinese liquor), characterized in that: It is composed of halophilic Bacillus G47, Lactobacillus plantarum F25-4 and Pichia kudricazvichid K44; The salt-resistant Bacillus G47, classified as... Bacillus halotoleruns It is deposited at the China Center for Type Culture Collection (CCTCC) on April 25, 2025, with accession number CCTCC NO: M 2025886; The *Lactobacillus plantarum* F25-4 was classified and named... Lactobacillus plantarum It is deposited at the China Center for Type Culture Collection (CCTCC) on December 9, 2021, with accession number CCTCC NO: M 20211574; The aforementioned *Kudriazwibichthys* K44, classified and named... Pichia kudriavzecii It is deposited at the China Center for Type Culture Collection (CCTCC) on April 25, 2025, with accession number CCTCC NO: M 2025887.

2. The compound fermentation agent according to claim 1, characterized in that: The ratio of the salt-tolerant Bacillus G47, Lactobacillus plantarum F25-4, and Pichia kudricazvichid K44 is 1:1:

1.

3. The application of the compound fermentation agent according to claim 1 or 2 in promoting the generation of phenolic flavor substances in the brewing of baijiu.

4. The application of the compound fermentation agent according to claim 3 in the brewing of Baijiu (Chinese liquor), characterized in that: The total inoculum size of halophilic Bacillus G47, Lactobacillus plantarum F25-4, and Pichia kudrica K44 was 3% v / w of the fermentation substrate mass.

5. The application of the compound fermentation agent according to claim 4 in Baijiu brewing, characterized in that, Includes the following steps: (1) Salt-tolerant Bacillus G47 was inoculated into LB liquid medium, Pichia pastoris K44 into YPD liquid medium, and Lactobacillus plantarum F25-4 into MRS liquid medium; all were cultured to the logarithmic growth phase, and the cells were collected by centrifugation, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; (2) Salt-tolerant Bacillus G47, Lactobacillus plantarum F25-4 and Pichia pastoris K44 were inoculated into the fermentation substrate at a ratio of 1:1:1, and the total inoculation amount was 3% v / w of the fermentation substrate mass; (3) The fermentation conditions are: pile culture at 37°C for the first 24 hours, and anaerobic fermentation at 30°C for the next 48-168 hours.

6. The method for enriching phenolic flavor substances in a simulated fermentation system of baijiu using the compound fermentation agent according to claim 1 or 2, characterized in that, Includes the following steps: (1) Construct a simulated fermentation system for baijiu (Chinese liquor). The fermentation system is a solid-state fermentation system. During the fermentation process, the pH of the system is controlled at 4.0-6.0, and ethanol is generated and gradually accumulated. (2) Inoculate the simulated fermentation system of Baijiu with Bacillus G47, Lactobacillus plantarum F25-4 and Kudria zwibbiac yeast K44; (3) Cultivate under certain fermentation conditions so that the compound fermentation agent participates in the conversion of phenolic precursor substances from raw materials and / or koji materials in the fermentation system, thereby obtaining phenolic flavor substances in the fermentation system.

7. The method for enriching phenolic flavor substances in a simulated fermentation system of baijiu using the compound fermentation agent according to claim 6, characterized in that: The phenolic precursor is selected from one or more of ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid.

8. The method for enriching phenolic flavor substances in a simulated fermentation system of baijiu using the compound fermentation agent according to claim 6, characterized in that: The phenolic flavor compounds include one or more of guaiacol, 4-ethylguaiacol, 4-vinylguaiacol, and 4-ethylphenol.

9. The method for enriching phenolic flavor substances in a simulated fermentation system of baijiu using the compound fermentation agent according to claim 6, characterized in that: Fermentation conditions are pH 4.0-6.0 and ethanol volume fraction of 2%-8%.

10. The method for enriching phenolic flavor substances in a simulated fermentation system of baijiu using the compound fermentation agent according to claim 6, characterized in that: The inoculation amounts of Bacillus G47, Lactobacillus plantarum F25-4, and Pichia pastoris K44 were all 1×10⁻⁶. 5 ~1×10 7 CFU / g fermentation substrate.

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