Complex microbial inoculant for increasing flavor substance content in liquor fermentation process

Abstract

The invention discloses a complex microbial inoculant for increasing the content of flavor substances in a white spirit fermentation process, and belongs to the field of white spirit fermentation. According to the invention, pichia kudriavzevii CCTCC NO: M 20252385 is compounded with a plurality of strains of Witzia ciliaris, so that the composite microbial inoculant capable of simultaneously improving a plurality of flavor substances in the fermentation process of white spirit is obtained. Compared with the Pichia kudriavzevii CCTCC NO: M 20252385, the composite microbial inoculant has the advantages that the content of isoamyl acetate is increased by 66% or above, the content of isoamyl alcohol is increased by 44% or above, the content of isobutanol is increased by 31%, and the content of phenethyl alcohol is increased by 18%.

Description

Technical Field

[0001] This invention relates to a compound microbial agent for increasing the content of flavor substances during the fermentation process of baijiu (Chinese liquor), belonging to the field of baijiu fermentation. Background Technology

[0002] Baijiu is a fermented alcoholic beverage made primarily from grains through processes such as steaming, saccharification, fermentation, and distillation. Its flavor compounds mainly originate from the metabolic activities of microorganisms during fermentation, with esters and alcohols being crucial components that contribute to the aroma and flavor profile of baijiu.

[0003] In the baijiu fermentation system, isoamyl acetate, isoamyl alcohol, phenethyl alcohol, and isobutanol are key flavor compounds that influence the aroma type and body style of baijiu. Isoamyl acetate is the main ester aroma component with obvious fruity characteristics, which can enhance the aroma intensity and harmony of baijiu. Isoamyl alcohol is a higher alcohol; when present in appropriate amounts, it can impart a full-bodied feel to the baijiu, but excessive amounts can cause a spicy and pungent sensation. Phenethyl alcohol is an aromatic alcohol compound with floral characteristics, which can improve the aroma balance of baijiu. Isobutanol is a moderately volatile alcohol component that can increase the fullness of the baijiu at a certain concentration. The relative proportions of these flavor compounds have a significant impact on the aroma harmony and taste stability of baijiu.

[0004] The formation of isoamyl acetate, isoamyl alcohol, phenylethyl alcohol, and isobutanol is mainly affected by factors such as the composition of fermentation raw materials, microbial community structure, fermentation conditions, and metabolic pathway regulation. In production practice, if the isoamyl acetate content is too low, the aroma of the baijiu will not be prominent; if its content is too high, it will mask the main aroma characteristics. Similarly, excessive amounts of higher alcohols (such as isoamyl alcohol and isobutanol) will lead to a rough flavor and increased harshness in the baijiu. Therefore, appropriate regulation of these flavor substances is an important step in ensuring the stability of baijiu quality.

[0005] Currently, there are numerous existing technologies for regulating the content of flavor compounds during the fermentation of baijiu. **Process Condition Control Method:** This method alters the balance between esterification and alcohol production metabolism within the fermentation system by adjusting parameters such as fermentation temperature, humidity, pH, raw material ratio, and density. However, this method is highly dependent on the process environment and is significantly affected by climate and raw material differences, resulting in unstable control effects. **Microbial Community Control Method:** This method adjusts the proportion of metabolites by altering the composition of the fermentation microbial community or introducing specific functional strains (such as high-ester-producing yeasts or aroma-producing lactic acid bacteria). However, different strains exhibit substrate competition and metabolic inhibition, leading to poor community stability and insufficient controllability of flavor results. **Exogenous Addition Method:** This method adds aroma compounds such as isoamyl acetate and phenylethanol during distillation or blending to improve product flavor. However, this method struggles to maintain the overall balance of the natural fermentation system and suffers from aroma incoordination and insufficient stability. **Nutritional Factor Regulation and Metabolic Engineering Method:** This method alters metabolic flow and the content of target products by adjusting the concentrations of nitrogen sources, vitamins, and metal ions, or by using genetic engineering to modify fermentation strains. This type of method has limitations such as poor genetic stability of strains, weak process adaptability, and insufficient feasibility for industrialization.

[0006] Therefore, in order to address the problems of insufficient precision in controlling the content of key flavor substances in baijiu fermentation, strong dependence on environmental conditions, and complex microbial systems, the development of a multifunctional microbial agent has extremely high practical and economic value. Summary of the Invention

[0007] To address the aforementioned issues, this invention combines *Pichia pastoris* CCTCC NO:M 20252385 with multiple strains of *Weizmannia coagulans* to obtain a compound microbial agent capable of simultaneously enhancing various flavor compounds during the fermentation of baijiu.

[0008] The first objective of this invention is to provide a compound microbial agent containing *Pichia gondii* CCTCC NO: M 20252385, *Weizmannii coagulans* CCTCC NO: M 20231138, and *Weizmannii coagulans* CCTCC NO: M 20231139. The viable cell ratio of *Pichia kudrica* CCTCC NO:M 20252385, *Weizmannia coagulans* CCTCC NO:M20231138, and *Weizmannia coagulans* CCTCC NO:M 20231139 was 1~3:1~2:1~2.

[0009] In one embodiment, the viable bacteria count in the compound microbial agent is 5~8×10⁻⁶. 7 CFU / g.

[0010] In one embodiment, the ratio of viable bacteria counts of *Pichia gondii* CCTCC NO: M20252385, *Weizmannii coagulans* CCTCC NO: M 20231138, and *Weizmannii coagulans* CCTCC NO: M 20231139 in the compound microbial agent is 1~3:2:1.

[0011] In one embodiment, the viable count ratio of *Pichia kudrica* CCTCC NO:M 20252385, *Weizmannia coagulans* CCTCC NO:M 20231138, and *Weizmannia coagulans* CCTCC NO:M 20231139 is 1~3:1~2:1.

[0012] Optionally, the viable cell ratio of *Pichia kudrica* CCTCC NO:M 20252385, *Weizmannia coagulans* CCTCC NO:M 20231138, and *Weizmannia coagulans* CCTCC NO:M 20231139 is 2:1:1 or 3:2:1.

[0013] In one embodiment, the compound microbial agent also contains a freeze-drying protectant, which includes skim milk powder, fructooligosaccharides, and monosodium glutamate.

[0014] 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).

[0015] In one embodiment, the application is to increase the content of isoamyl acetate, isoamyl alcohol, isobutanol, and phenylethanol during the fermentation process of baijiu.

[0016] A third objective of this invention is to provide a method for increasing the content of flavor substances during the fermentation of baijiu (Chinese liquor), by adding any of the above-mentioned compound microbial agents during the fermentation process.

[0017] In one embodiment, the flavoring substance is one or more of isoamyl acetate, isoamyl alcohol, isobutanol, and phenylethanol.

[0018] In one embodiment, the mass ratio of the compound microbial agent to the yeast is 5~8:2~5.

[0019] In one embodiment, the mass ratio of the compound microbial agent to the yeast is 7~8:2~3.

[0020] Beneficial effects of the present invention Compared with using only Pichia pastoris CCTCC NO:M 20252385, the compound microbial agent of the present invention increases the content of isoamyl acetate by more than 66%, with a maximum increase of 148%; isoamyl alcohol by more than 44%, with a maximum increase of 81%; isobutanol by 31%, with a maximum increase of 125%; and phenylethanol by 18%, with a maximum increase of 67%.

[0021] Biological Preservation Materials Kudria zwibichi yeast ( Pichia kudriavzevii JGWN-001, taxonomically named: Pichia Kudryavzevii JGWN-001 was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2025, with accession number CCTCC NO:M 20252385. The deposit address is China Center for Type Culture Collection, Wuhan University, Wuhan, China. Detailed Implementation

[0022] 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.

[0023] Raw materials used in the examples: Preparation method of sorghum culture medium: 100 g of sorghum is crushed and mixed with 500 mL of water. 10 U / g of sorghum α-amylase is added and the mixture is cooked at 85~95℃ for 2 h. After cooling to 60℃, 5 U / g of sorghum saccharifying enzyme is added and the mixture is kept warm for 2 h. The mixture is dispensed and sterilized at 115℃ for 20 min to obtain sorghum culture medium.

[0024] Isoamyl acetate, isoamyl alcohol, phenylethanol, isobutanol, and n-propanol are all commercially available.

[0025] The yeast starter is commercially available.

[0026] The following patents were disclosed on December 22, 2023: *Weizmannii coagulans* VC77CCTCC NO:M 20231138; *Weizmannii coagulans* VC99 CCTCC NO:M 20231139; and *Bacillus coagulans* BC77 CCTCC NO:M 2021497.

[0027] Test method: The content of isoamyl acetate, isoamyl alcohol, phenylethanol, isobutanol, and n-propanol was determined by gas chromatography.

[0028] Distillation method: Take the fermented sorghum culture medium and distill it at 95℃ for 30 min to obtain the distillation product.

[0029] Example 1: Preparation of compound bacterial agent The following steps were taken to prepare bacterial inoculum: *Pichia pastoris* CCTCC NO: M 20252385, *Weizmannii coagulans* VC77 CCTCC NO: M 20231138, *Weizmannii coagulans* VC99 CCTCC NO: M 20231139, and *Bacillus coagulans* BC77 CCTCC NO: M2021497. After activation, *Weizmannii coagulans* VC77 CCTCC NO: M 20231138, *Weizmannii coagulans* VC99 CCTCC NO: M20231139, and *Bacillus coagulans* BC77 CCTCC NO: M 2021497 were inoculated into MRS 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 in a protective solution (10 g / 100 mL skim milk powder, 10 g / 100 mL fructooligosaccharides, 5 g / 100 mL monosodium glutamate), and lyophilized to obtain *Weizmannii coagulans* VC77, *Weizmannii coagulans* VC99, and *Bacillus coagulans* BC77 bacterial powders, respectively. After activation, Pichia pastoris CCTCC NO:M 20252385 was inoculated into MRS medium at a 1% v / v inoculum and cultured at 30℃ and 300 r / min until the viable count reached 10⁻⁶. 8 CFU / mL, centrifuge to collect bacterial cells; resuspend the bacterial cells in a protectant solution (5 g / 100 mL skim milk powder, 8 g / 100 mL sucrose, 5 g / 100 mL monosodium glutamate), freeze-dry to obtain Pichia kudrica zwiecchii CCTCC NO:M 20252385 bacterial powder.

[0030] The live bacteria powder of *Pichia kudriezvichirosa* CCTCC NO:M 20252385 was mixed with *Weizmannii coagulans* VC77, *Weizmannii coagulans* VC99, and *Bacillus coagulans* BC77 at a live bacteria ratio of 1:1 to obtain mixed bacterial agents, which were named compound bacterial agent 1 (*Pichia kudriezvichirosa* + VC77), compound bacterial agent 2 (*Pichia kudriezvichirosa* + VC99), and compound bacterial agent 3 (*Pichia kudriezvichirosa* + BC77), respectively.

[0031] The viable bacteria count in the compound microbial agent was measured to be 7.5~8×10⁻⁶. 7 CFU / g.

[0032] Example 2: Preparation of compound bacterial agent The compound microbial agent prepared in Example 1 was used to test its effect on flavor compounds, including the following steps: The compound microbial agent and the yeast were mixed at a mass ratio of 8:2 and added to the sorghum culture medium at a dosage of 10% w / w. Fermentation was carried out for 72 h. The fermented sorghum culture medium was collected and distilled, and the content of flavor substances in the distillate was detected by gas chromatography. An equal amount of *Pichia pastoris* CCTCC NO:M 20252385, *Weizmannii coagulans* VC77, *Weizmannii coagulans* VC99, and *Bacillus coagulans* BC77 were inoculated as a control. The results are shown in Table 1.

[0033] Table 1. Results of Flavor Substance Detection

[0034] It is evident that different strains can produce different effects when combined; the combination of *Weizmannia coagulans* VC99 and *Pichia kudrica* can simultaneously increase the content of isoamyl acetate and isoamyl alcohol; while the use of *Weizmannia coagulans* VC77 or *Bacillus coagulans* BC77 cannot increase the content of isoamyl acetate and isoamyl alcohol. The combination of Weizmann's coagulans VC77 and Pichia kudrica can simultaneously increase the content of isobutanol and phenylethanol; while the use of Weizmann's coagulans VC99 or Bacillus coagulans BC77 cannot increase the content of isobutanol and phenylethanol.

[0035] Example 3: Preparation of compound bacterial agent In Example 2, both *Weizmannii coagulans* VC77 and VC99, when mixed with *Pichia kudrica* CCTCC NO: M20252385, showed an increase in the content of certain flavor compounds. Therefore, different ratios of the three strains were blended (*Pichia kudrica* CCTCC NO: M 20252385: *Weizmannii coagulans* VC77: *Weizmannii coagulans* VC99) to screen for formulations that could simultaneously increase the content of flavor compounds. The results are shown in Table 2.

[0036] Table 2 Effects of different proportions on flavor compounds

[0037] The results showed that when Pichia kudrica citrate CCTCC NO:M 20252385, Weizmannia coagulans VC77 and VC99 were mixed in equal proportions, the contents of isoamyl acetate and isoamyl alcohol increased, but the contents of isobutanol and phenylethanol did not change significantly. Increasing the proportion of Pichia kudriezvich CCTCC NO:M 20252385 (2:1:1) improved the content of each flavor compound, but it was still lower than the result of the combination of the two strains. On this basis, reducing the proportion of Pichia kudriezvich CCTCC NO:M 20252385 and increasing the proportion of VC77 or VC99 (2:1:2, 2:2:1) made the content of some flavor compounds closer to the result of the combination of the two strains, but it could not achieve a simultaneous increase in all four flavor compounds. After further increasing the proportion of Pichia kudriezvich CCTCC NO:M 20252385 (3:1:1), the content of each flavor substance decreased compared to 2:1:1. On this basis, reducing the proportion of Pichia kudriezvich CCTCC NO:M20252385 and VC99, and only increasing the proportion of VC77, the results improved. The content of isoamyl acetate and isoamyl alcohol was close to that of compound inoculum 2 (Pichia kudriezvich CCTCC NO:M 20252385 and VC99 in a 1:1 ratio), and the content of isobutanol and phenylethanol was slightly better than that of compound inoculum 1 (Pichia kudriezvich CCTCC NO:M20252385 and VC77 in a 1:1 ratio), which can simultaneously increase the content of isoamyl acetate, isoamyl alcohol, isobutanol and phenylethanol.

[0038] 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 *Pichia kudrica* CCTCC NO: M20252385, *Weizmannia coagulans* CCTCC NO: M 20231138, and *Weizmannia coagulans* CCTCC NO: M 20231139. The viable cell ratio of *Pichia kudrica* CCTCC NO:M 20252385, *Weizmannia coagulans* CCTCC NO:M20231138, and *Weizmannia coagulans* CCTCC NO:M 20231139 was 1~3:1~2:1~2.

2. The compound microbial agent according to claim 1, characterized in that, The viable count in the compound microbial agent is 7.5~8×10⁻⁶. 7 CFU / g.

3. The compound microbial agent according to claim 1, characterized in that, The viable count ratio of Pichia pastoris CCTCC NO:M 20252385, Weizmann's coagulans CCTCC NO:M 20231138, and Weizmann's coagulans CCTCC NO:M 20231139 in the compound microbial agent is 1~3:1~2:

1.

4. The compound microbial agent according to claim 3, characterized in that, The viable cell ratio of Pichia kudrica (CCTCC NO: M20252385), Weizmann's coagulans (CCTCC NO: M 20231138), and Weizmann's coagulans (CCTCC NO: M 20231139) was 2:1:1 or 3:2:

1.

5. The application of the compound microbial agent according to any one of claims 1 to 4 in the fermentation of baijiu.

6. The application according to claim 5, characterized in that, The application is to increase the content of isoamyl acetate, isoamyl alcohol, isobutanol, and phenylethanol during the fermentation process of baijiu.

7. A method for increasing the content of flavor substances during the fermentation of baijiu (Chinese liquor), characterized in that, The compound microbial agent described in any one of claims 1 to 4 is added during the fermentation process of baijiu.

8. The method according to claim 7, characterized in that, The flavoring substance is one or more of isoamyl acetate, isoamyl alcohol, isobutanol, and phenylethanol.

9. The method according to claim 7, characterized in that, The mass ratio of compound microbial agent to yeast is 5~8:2~5.

10. The method according to claim 9, characterized in that, The mass ratio of compound microbial agent to yeast is 7~8:2~3.

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