Complex microbial inoculant for increasing content of isoamyl acetate and isoamyl alcohol in liquor fermentation process

Abstract

The invention discloses a complex microbial inoculant for increasing the content of isoamyl acetate and isoamyl alcohol in a liquor fermentation process, and belongs to the technical field of liquor 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 increasing the content of isoamyl acetate and isoamyl alcohol in the fermentation process of Baijiu is obtained. The mixed bacterial agent prepared by the invention can improve the contents of isoamyl acetate and isoamyl alcohol, and compared with the same amount of pichia kudriavzevii CCTCC NO: M 20252385, the contents of isoamyl acetate and isoamyl alcohol are respectively improved by 158% and 78%.

Description

Technical Field

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

[0002] Baijiu is a distilled spirit made primarily from grains through a process involving fermentation, distillation, and aging using various microorganisms. Its aroma components are complex and diverse, mainly composed of volatile substances such as alcohols, acids, esters, aldehydes, and ketones. Among these, isoamyl acetate and isoamyl alcohol are two important flavor compounds in baijiu, significantly influencing its aroma characteristics and taste.

[0003] Isoamyl acetate, a short-chain fatty acid ethyl ester, is one of the important aroma components in baijiu (Chinese white liquor) that impart fruity and floral aromas. It exhibits distinct banana and pear aromas, and its appropriate presence can enhance the aroma complexity and harmony of baijiu, making the liquor smoother and more rounded. Isoamyl alcohol, a higher alcohol, is one of the main alcohols produced by yeast and some bacteria during baijiu fermentation and has a certain pungent odor. At suitable concentrations, isoamyl alcohol can enhance the fullness and aroma intensity of the liquor.

[0004] Currently, existing technologies for controlling the content of isoamyl acetate and isoamyl alcohol during the fermentation of baijiu include: **Process parameter control method:** This method adjusts fermentation conditions such as temperature, humidity, raw material ratio, ventilation, stack thickness, and fermentation time to alter the redox state and microbial metabolic pathways of the fermentation system, thereby indirectly affecting the formation of isoamyl acetate and isoamyl alcohol. However, this method relies on environmental factors, has low precision, and is significantly affected by differences in fermentation pits and seasonal variations in large-scale production, making it difficult to maintain consistent product flavor. **Microbial control method:** This method involves screening or modifying specific yeast, lactic acid bacteria, or propionic acid bacteria strains to increase the activity of esterases or dehydrogenases, thereby promoting isoamyl acetate synthesis or inhibiting isoamyl alcohol accumulation. While this method can improve the proportion of some flavor components, the complex microbial community structure in the fermentation system and the metabolic interactions between different strains can easily lead to uncontrollable results, poor community stability, and insufficient reproducibility, hindering industrial application. Post-processing flavoring: During the distillation or blending stage, flavor base spirits and ester compounds are artificially blended or added to balance the ratio of isoamyl acetate to isoamyl alcohol, thereby improving the aroma harmony of the finished liquor. Although this method can improve sensory quality in the short term, it cannot regulate the generation of metabolic products from the source of fermentation, and exogenous additions can easily destroy the characteristic flavor of natural fermentation of baijiu, which does not meet the requirements of pure grain brewing for high-end baijiu.

[0005] Therefore, there is an urgent need to develop a technical solution that can effectively regulate the ratio of isoamyl acetate to isoamyl alcohol during the fermentation stage, so as to stabilize and control the aroma components, improve the harmony of the liquor, and thus achieve high-quality and standardized production of baijiu products. Summary of the Invention

[0006] 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 the production of isoamyl acetate and isoamyl alcohol during the fermentation of baijiu.

[0007] The first objective of this invention is to provide a compound microbial agent containing Pichia kudrica (CCTCC NO: M 20252385) and Weizmannii coagulans (CCTCC NO: M 20231139). The viable cell ratio of *Pichia kudrica* CCTCC NO: M 20252385 and *Weizmannii coagulans* CCTCC NO: M20231139 was 1~2:1~2.

[0008] In one embodiment, the viable bacteria count in the compound microbial agent is 7.5~8×10⁻⁶. 7 CFU / g.

[0009] In one embodiment, the ratio of viable bacteria counts of *Pichia gondii* CCTCC NO: M20252385 and *Weizmannii coagulans* CCTCC NO: M 20231139 in the compound microbial agent is 1:1.

[0010] In one embodiment, the compound microbial agent also contains a freeze-drying protectant, which includes skim milk powder, fructooligosaccharides, and monosodium glutamate.

[0011] 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).

[0012] In one embodiment, the application is to increase the content of isoamyl acetate and isoamyl alcohol during the fermentation of baijiu (Chinese liquor).

[0013] The third objective of this invention is to provide a method for increasing the content of flavor substances during the fermentation of baijiu by adding any of the above-mentioned compound microbial agents during the fermentation process.

[0014] In one embodiment, the flavoring substance is one or more of isoamyl acetate and isoamyl alcohol.

[0015] In one embodiment, the mass ratio of the compound microbial agent to the yeast is 5~8:2~5.

[0016] In one embodiment, the mass ratio of the compound microbial agent to the yeast is 7~8:2~3.

[0017] Beneficial effects of the present invention This invention mixes Pichia kudrizia citrate CCTCC NO:M 20252385 with Weizmannia coagulans CCTCC NO:M 20231139 at a viable cell ratio of 1:1. The resulting mixed inoculum increases the content of isoamyl acetate and isoamyl alcohol. Compared with using only an equal amount of Pichia kudrizia citrate CCTCC NO:M 20252385, the content of isoamyl acetate and isoamyl alcohol increases by 158% and 78%, respectively.

[0018] 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

[0019] 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.

[0020] 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.

[0021] The content of isoamyl acetate and isoamyl alcohol was determined by gas chromatography.

[0022] The yeast starter is commercially available.

[0023] The following patents were disclosed on December 22, 2023: *Weizmannii coagulans* VC77 CCTCC NO: M 20231138, *Weizmannii coagulans* VC99 CCTCC NO: M 20231139 ...7 CCTCC NO: M 20231138, *Weizmannii coagulans* VC97 CCTCC NO: M 20231138, *Weizmannii coagulans* VC97 CCTCC NO: M 20231138, *Weizmannii coagulans* VC97 CCTCC NO: M 20231138, *Weizmannii coagulans* VC97 CCTCC NO: M 20231138, *Weizmannii coagulans* VC97 CCTCC NO: M 20231138, *Weizmannii coagulans* VC9

[0024] Test method: The content of isoamyl acetate, isoamyl alcohol, and n-propanol was determined by gas chromatography.

[0025] Distillation method: Take the fermented sorghum culture medium and distill it at 95℃ for 30 min to obtain the distillation product.

[0026] 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.

[0027] 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.

[0028] The viable bacteria count in the compound microbial agent was measured to be 7.5~8×10⁻⁶. 7 CFU / g.

[0029] 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 5% w / w. Fermentation was carried out for 72 h. The fermentation broth was collected, and the content of flavor substances 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.

[0030] Table 1. Results of Flavor Substance Detection

[0031] It is evident that only when *Weizmannia coagulans* VC99 and *Pichia kudrica* CCTCC NO:M 20252385 are combined can the content of isoamyl acetate and isoamyl alcohol be increased simultaneously. Using *Weizmannia coagulans* VC77 and *Pichia kudrica* CCTCC NO:M 20252385 could not increase the content of isoamyl acetate and isoamyl alcohol; while using *Pichia kudrica* CCTCC NO:M 20252385 and *Weizmannia coagulans* BC77 could increase the content of n-propanol, but the content of isobutanol and phenylethanol was low.

[0032] 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 and *Weizmannii coagulans* CCTCC NO: M 20231139. The viable cell ratio of *Pichia kudrica* CCTCC NO: M 20252385 and *Weizmannii coagulans* CCTCC NO: M20231139 was 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 cell ratio of Pichia pastoris CCTCC NO:M 20252385 and Weizmannii coagulans CCTCC NO:M 20231139 in the compound microbial agent is 1:

1.

4. The compound microbial agent according to claim 3, characterized in that, The compound microbial agent also contains a freeze-drying protectant, which includes skim milk powder, fructooligosaccharides, and monosodium glutamate.

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 and isoamyl alcohol 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 and isoamyl alcohol.

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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