Method for improving quality of fermented grains of Baijiu

Abstract

The invention discloses a method for improving the quality of fermented grains of Baijiu, and belongs to the technical field of Baijiu fermentation regulation. According to the invention, different strains are compounded and screened to obtain the complex microbial inoculant capable of improving the ethyl acetate inhibition and the ethyl hexanoate increase of the candida tropicalis CCTCC NO: M 20222053. On the basis, the performance of the bacillus paralicheniformis CCTCC NO: M 2024869 and the performance of the candida tropicalis CCTCC NO: M 20222053 in different proportions are further detected, and the composite microbial inoculant capable of adjusting the content of ethyl acetate and ethyl hexanoate in the fermented grain fermentation process is prepared.

Description

Technical Field

[0001] This invention relates to a method for improving the quality of baijiu mash, belonging to the field of baijiu fermentation regulation technology. Background Technology

[0002] Baijiu is a traditional fermented liquor made primarily from grains through solid-state or semi-solid-state fermentation, distillation, and aging. Its flavor characteristics are mainly composed of various volatile compounds produced during fermentation, with esters playing a dominant role in its aroma. Ethyl acetate is one of the most abundant and aromatic esters in baijiu, and its concentration and proportion have a crucial impact on the aroma type, taste harmony, and overall quality. Appropriate amounts of ethyl acetate can impart delicate aromas such as fruit and floral notes to baijiu, enhancing its fullness and smoothness, and forming an important foundation for the characteristic "fragrant but not overpowering, smooth but not bland" flavor of baijiu.

[0003] During the fermentation of baijiu (Chinese white liquor), ethyl acetate is mainly produced from acetic acid and ethanol under the action of esterification enzymes. Its production is influenced by various factors, including the composition of fermentation raw materials, microbial community structure, temperature and humidity, and the fermentation cycle. Both excessively high and low ethyl acetate content negatively impact the quality of baijiu. When the ethyl acetate content is too high, while the aroma may be intense, it can also lead to a strong, pungent taste, an unbalanced flavor profile, and a harsh, overpowering character, masking other minor aroma components and resulting in a simplistic and sharp overall flavor. Furthermore, high concentrations of ethyl acetate may cause excessively high volatile acidity, affecting the drinking experience. Conversely, if the ethyl acetate content is too low, the aroma will be weak, the taste bland, and the flavor profile insufficient, making it difficult to showcase the unique complex aroma characteristics of baijiu.

[0004] Currently, the production of ethyl acetate is usually regulated by controlling fermentation process parameters or adjusting the microbial community structure. However, the method of controlling fermentation process parameters is greatly affected by environmental fluctuations, has low control precision, and varies between different fermentation pits, making it difficult to achieve standardized control. On the other hand, adjusting the microbial community structure results in poor microbial community stability, often leading to low fermentation reproducibility and large flavor fluctuations.

[0005] Patent CN116769618A discloses a functional microorganism that inhibits ethyl acetate production. While this strain can significantly reduce ethyl acetate production and increase ethyl hexanoate production, it is pH sensitive and exhibits significant growth inhibition at lower pH conditions, resulting in poor inhibition of ethyl acetate. Patent CN116790421B discloses a *Bacillus desertica* strain that inhibits lactic acid bacteria in fermented mash; patent CN116751693B discloses a lactic acid-resistant *Bacillus baiji* strain; and patent CN120118800A discloses a *Bacillus licheniformis* strain that inhibits lactic acid bacteria. Although numerous strains exist capable of inhibiting lactic acid bacteria or decomposing lactic acid, the metabolic processes of different strains and the complex metabolic interactions between them make it difficult to achieve stable effects.

[0006] Therefore, developing a microbial compound inoculant to achieve precise control of ethyl acetate during the fermentation process of baijiu is of great significance for ensuring stable product quality and consistent flavor. Summary of the Invention

[0007] To address the aforementioned problems, this invention employs a compound microbial agent obtained by combining different strains and screening them to enhance the inhibition of ethyl acetate and the increase of ethyl hexanoate in *Candida tropicalis* CCTCC NO: M 20222053. Based on this, the invention further tested the performance of *Bacillus paralichrysiformis* CCTCC NO: M 2024869 and *Candida tropicalis* CCTCC NO: M20222053 at different ratios, thus preparing a compound microbial agent capable of regulating the content of ethyl acetate and ethyl hexanoate during the fermentation of brewing mash.

[0008] The first objective of this invention is to provide a compound microbial agent containing Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053; wherein the live count ratio of Bacillus paralicheniformis CCTCC NO: M2024869 to Candida tropicalis CCTCC NO: M 20222053 is 3~8:2~7.

[0009] In one embodiment, the viable bacteria count in the compound microbial agent is 3~5×10⁻⁶. 7 CFU / g.

[0010] In one embodiment, the preparation method of the compound microbial agent is as follows: (1) Bacillus paralichrysogenum CCTCC NO: M 2024869 was activated and inoculated into TB medium and cultured until the viable count reached 10. 8 CFU / mL -1The bacterial cells were collected by centrifugation; the bacterial cells were resuspended in a protectant and freeze-dried to obtain Bacillus paralichrysiformis CCTCC NO: M 2024869 bacterial powder; Candida tropicalis CCTCC NO: M 20222053 was activated and inoculated into molasses medium, and cultured until the viable count reached 10⁻⁶. 8 CFU / mL, centrifuge to collect bacterial cells; resuspend the bacterial cells with a protectant, freeze dry to obtain Candida tropicalis CCTCC NO: M20222053 bacterial powder. (2) Mix Bacillus paralicheniformis CCTCC NO: M 2024869 bacterial powder and Candida tropicalis CCTCC NO: M20222053 bacterial powder to obtain a compound bacterial agent.

[0011] In one embodiment, the culture conditions for Bacillus paralichrysum CCTCC NO: M 2024869 in step (1) are 35~37℃ and 150~200 rpm in shake flask culture.

[0012] In one embodiment, the culture conditions for Candida tropicalis CCTCC NO: M 20222053 in step (1) are 28~30℃ and 150~200 rpm shake flask culture.

[0013] A second objective of this invention is to provide the application of any of the above-mentioned compound microbial agents in the fermentation of baijiu (Chinese liquor).

[0014] In one embodiment, the application is to adjust the content of ethyl acetate and ethyl hexanoate during the fermentation process of baijiu (Chinese liquor).

[0015] The third objective of this invention is to provide a method for adjusting the ester content in fermented mash, characterized in that the compound microbial agent described in any one of claims 1 to 5 is added to the fermented mash for fermentation; The esters mentioned are ethyl acetate and ethyl hexanoate.

[0016] In one embodiment, when the ratio of viable bacteria of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound bacterial agent is 5~7:3~5, the adjustment is to inhibit the formation of ethyl acetate and increase the formation of ethyl hexanoate.

[0017] In one embodiment, when the ratio of viable bacteria of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound bacterial agent is 7~8:2~3, the adjustment is to inhibit the formation of ethyl hexanoate and increase the formation of ethyl acetate.

[0018] Beneficial effects of the present invention This invention obtains a compound bacterial agent that enhances the inhibition of ethyl acetate and the increase of ethyl hexanoate by combining different bacterial strains.

[0019] Specifically: (1) When the ratio of viable counts of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound microbial agent is 5~7:3~5, use 10 5 Adding CFU / g of mash to the mash and fermenting for 4 months significantly inhibited ethyl acetate production and increased ethyl hexanoate production. This effect was superior to using only Candida tropicalis CCTCC NO: M 20222053 (ethyl acetate inhibition increased by more than 10.7%, up to a maximum of 18.7%; ethyl hexanoate increase increased by more than 4.7%, up to a maximum of 28.7%) and Bacillus paralichrysogenum CCTCC NO: M 2024869. (2) When the ratio of viable counts of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound microbial agent is 8:2, at 10 5 Adding CFU / g of mash to the mash significantly inhibited the formation of ethyl hexanoate and increased the formation of ethyl acetate after 4 months of fermentation. Detailed Implementation

[0020] 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.

[0021] Raw materials used in the examples: TB (Terrific Broth) medium: 2% tryptone, 2.4% yeast extract, 0.4% glycerol, 72 mM K2HPO4, 17 mM KH2PO4.

[0022] Molasses culture medium: 80 g·L -1 Magnesium sulfate 0.2 g·L -1 3 g·L ammonium sulfate -1 Potassium dihydrogen phosphate 3 g·L -1 .

[0023] The following patents were disclosed: *Bacillus desertis* CCTCC NO: M 2023422, disclosed on September 22, 2023 in patent CN116790421A; *Candida tropicalis* CCTCC NO: M 20222053, disclosed on September 19, 2023 in patent CN116769618A; *Bacillus baijiensis* CCTCC NO: M 2023729, disclosed on September 15, 2023 in patent CN116751693A; *Bacillus paralicheniformis* CCTCC NO: M 2024869, disclosed on June 10, 2025 in patent CN120118799A; and *Bacillus licheniformis* CCTCC NO: M 2024870, disclosed on June 10, 2025 in patent CN120118800A.

[0024] Example 1: Preparation of compound microbial agent The following are the steps for preparing inoculum agents: *Bacillus desertica* (CCTCC NO: M 2023422), *Saccharomyces cerevisiae* (CCTCC NO: M2023729), *Candida tropicalis* (CCTCC NO: M 20222053), *Bacillus paralichrysum* (CCTCC NO: M 2024869), and *Bacillus lichrysum* (CCTCC NO: M 2024870). After activation, *Bacillus paralicheniformis* CCTCC NO: M 2024869, *Bacillus licheniformis* CCTCC NO: M 2024870, and *Bacillus sonosus* CCTCC NO: M 2023422 were inoculated into TB medium at a 1% v / v inoculum and cultured at 37°C and 200 r / min until the viable count reached 10⁻⁶. 8 CFU / mL, the bacterial cells were collected by centrifugation; the bacterial cells were resuspended with a protectant and lyophilized to obtain Bacillus paralicheniformis CCTCC NO: M 2024869 bacterial powder, Bacillus licheniformis CCTCC NO: M 2024870 bacterial powder, and Bacillus sonosus desertis CCTCC NO: M 2023422 bacterial powder, respectively.

[0025] Bayer Zygosacchariformis (CCTCC NO: M 2023729) and Candida tropicalis (CCTCC NO: M 20222053) were activated and inoculated into molasses medium, and cultured at 30°C and 200 r / min until the viable count reached 10⁻⁶. 8 CFU / mL, the bacterial cells were collected by centrifugation; the bacterial cells were resuspended with a protectant and lyophilized to obtain Bayer Zygomyces zygomyces CCTCC NO: M 2023729 and Candida tropicalis CCTCC NO: M 20222053 bacterial powders, respectively.

[0026] Bayer zygosacchariformis CCTCC NO: M 2023729, Bacillus paralicheniformis CCTCC NO: M 2024869, Bacillus licheniformis CCTCC NO: M 2024870, and Bacillus desertis CCTCC NO: M 2023422 were mixed with Candida tropicalis CCTCC NO: M 20222053 at a 1:1 ratio of live bacteria count to obtain mixed bacterial agents, which were named compound bacterial agent 1 (Bayer zygosacchariformis-Candida tropicalis), compound bacterial agent 2 (Bacillus paralicheniformis-Candida tropicalis), compound bacterial agent 3 (Bacillus licheniformis-Candida tropicalis), and compound bacterial agent 4 (Bacillus desertis-Candida tropicalis).

[0027] The viable bacteria count in the compound microbial agent was determined by plate counting, and was found to be 3-5 × 10⁻⁶. 7 CFU / g.

[0028] Example 2: Application of compound microbial agents in regulating esters in wine mash 1. Effects of different compound microbial agents on the content of ethyl acetate and ethyl hexanoate The compound bacterial agents 1-4 prepared in Example 1 were used to test their effect on inhibiting ethyl acetate formation. The steps are as follows: The same batch of fermented mash was divided into 12 equal portions, and 4 of these portions were taken and divided into 10 portions each. 5 CFU / g fermented mash was inoculated with compound microbial agent 1-4 and fermented for 4 months; 6 portions were taken, one without inoculation with compound microbial agent and the other with only inoculation with 10 CFU / g compound microbial agent. 5 CFU / g Candida tropicalis CCTCC NO: M20222053 powder, inoculated only at 10 5 CFU / g Bayer Zygomycin CCTCC NO: M 2023729 bacterial powder, inoculated only at 10 5 CFU / g Bacillus paralichrysogenus CCTCC NO: M 2024869 bacterial powder, inoculated only at 10 5 CFU / g Bacillus licheniformis CCTCC NO: M2024870 bacterial powder and inoculation with only 10 5 CFU / g Sonos Desert Bacillus CCTCC NO: M 2023422 fermented mash was used as a control.

[0029] The contents of ethyl acetate and ethyl hexanoate in the mash were determined by gas chromatography, and the results are shown in Table 1.

[0030] Table 1. Ethyl acetate and ethyl hexanoate content under different compound bacterial agents

[0031] As shown in Table 1, among Bacillus paralichrysum CCTCC NO: M 2024869, Bacillus lichrysum CCTCC NO: M2024870, Bacillus sonosus desert CCTCC NO: M 2023422, and Bayer zygosaccharomyces CCTCC NO: M 2023729, only Bacillus paralichrysum CCTCC NO: M 2024869 showed a more significant inhibition of ethyl acetate and an increase in ethyl hexanoate formation when combined with Candida tropicalis CCTCC NO: M 20222053. Although the combination of Bayer's zygosacchariformis CCTCC NO: M 2023729 and Candida tropicalis CCTCC NO: M 20222053 has a certain ability to inhibit ethyl acetate, it cannot increase the content of ethyl hexanoate. Neither Bacillus licheniformis CCTCC NO: M 2024870 nor Bacillus sonosus CCTCC NO: M 2023422 showed any ability to alter ethyl acetate or ethyl hexanoate.

[0032] Example 3: Application of different live bacteria ratios in regulating esters in wine mash Take the best-performing compound microbial agent 2 from Example 2 (i.e., the compound microbial agent of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053), and change the live bacteria ratio of Bacillus paralicheniformis CCTCC NO: M2024869 and Candida tropicalis CCTCC NO: M 20222053 to 3:7 and 8:2, and use the remaining 3 portions of the same batch of mash (the total inoculation amount is still 10) as in Example 2. 5 The effect of CFU / g on ester formation is shown in Table 2.

[0033] Table 2. Content of ethyl acetate and ethyl hexanoate at different viable bacteria count ratios.

[0034] The results showed that when the live bacteria ratio of Bacillus paralichrysogenum CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M20222053 was 3:7, the compound bacterial agent had a stronger inhibitory effect on ethyl acetate and a stronger ability to generate ethyl hexanoate; however, when the ratio was 8:2, the bacterial agent inhibited ethyl hexanoate and promoted the formation of ethyl acetate. It is evident that *Bacillus paralichrysogenus* CCTCC NO: M 2024869 and *Candida tropicalis* CCTCC NO: M 20222053 exhibit a synergistic effect. When the viable cell count of *Bacillus paralichrysogenus* CCTCC NO: M 2024869 is lower than or comparable to that of *Candida tropicalis* CCTCC NO: M 20222053, it enhances the inhibition of ethyl hexanoate enhancement by *Candida tropicalis* CCTCC NO: M 20222053. Conversely, when the viable cell count of *Bacillus paralichrysogenus* CCTCC NO: M 2024869 is significantly higher than that of *Candida tropicalis* CCTCC NO: M 20222053, it inhibits the enhancement of ethyl hexanoate enhancement by ethyl hexanoate. Therefore, the composite bacterial agent prepared in this application possesses a stronger ability to regulate the levels of ethyl acetate and ethyl hexanoate.

[0035] 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 compound microbial agent, characterized in that, The bacterial agent contains Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053; wherein the live count ratio of Bacillus paralicheniformis CCTCC NO: M 2024869 to Candida tropicalis CCTCC NO: M 20222053 is 3~8:2~7.

2. The compound microbial agent according to claim 1, characterized in that, The viable bacteria count in the compound microbial agent is 3~5×10⁻⁶. 7 CFU / g.

3. The compound microbial agent according to claim 1, characterized in that, The preparation method of the compound microbial agent is as follows: (1) Bacillus paralichrysogenum CCTCC NO: M 2024869 was activated and inoculated into TB medium and cultured until the viable count reached 10. 8 CFU / mL -1 The bacterial cells were collected by centrifugation; the bacterial cells were resuspended in a protective agent and freeze-dried to obtain Bacillus paralichrysiformis CCTCC NO: M 2024869 bacterial powder; Candida tropicalis CCTCC NO: M 20222053 was activated and inoculated into molasses medium, and cultured until the viable count reached 10⁻⁶. 8 CFU / mL, centrifuge to collect bacterial cells; resuspend the bacterial cells with a protectant, freeze dry to obtain Candida tropicalis CCTCC NO: M20222053 bacterial powder. (2) Mix Bacillus paralicheniformis CCTCC NO: M 2024869 bacterial powder and Candida tropicalis CCTCC NO: M 20222053 bacterial powder to obtain a compound bacterial agent.

4. The compound microbial agent according to claim 3, characterized in that, In step (1), the culture conditions for Bacillus paralichrysiformis CCTCCNO: M 2024869 are 35~37℃ and 150~200 rpm in shake flask culture.

5. The compound microbial agent according to claim 3, characterized in that, In step (1), the culture conditions for Candida tropicalis CCTCC NO: M20222053 are 28~30℃ and 150~200 rpm in shake flask culture.

6. The application of the compound microbial agent according to any one of claims 1 to 5 in the fermentation of baijiu.

7. The application according to claim 6, characterized in that, The application is to adjust the content of ethyl acetate and ethyl hexanoate during the fermentation process of baijiu (Chinese liquor).

8. A method for adjusting the ester content in fermented mash, characterized in that, Add the compound microbial agent according to any one of claims 1 to 5 to the mash for fermentation; The esters mentioned are ethyl acetate and ethyl hexanoate.

9. The method according to claim 8, characterized in that, When the ratio of viable bacteria of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound bacterial agent is 5~7:3~5, the adjustment is to inhibit the formation of ethyl acetate and increase the formation of ethyl hexanoate.

10. The method according to claim 8, characterized in that, When the ratio of viable bacteria of Bacillus paralicheniformis CCTCC NO: M 2024869 and Candida tropicalis CCTCC NO: M 20222053 in the compound bacterial agent is 7~8:2~3, the adjustment is to inhibit the formation of ethyl hexanoate and increase the formation of ethyl acetate.

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