Composite microbial agent of kodama pichia and d. hansenii and application thereof

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to *Pichia kudrica* (a type of yeast). Pichia kudriavzevii ) and Dell's spore-forming yeast ( Torulaspora delbrueckii This invention relates to a compound microbial agent and its application. Addressing the shortcomings of existing single-strain fermentation methods that result in low ester production efficiency and difficulty in adapting to the fermentation requirements of different baijiu production conditions, this invention combines *Pichia pastoris* MBT-PK97 and *Cyclocarya dauricum* for baijiu brewing. This compound agent can simultaneously increase the levels of the four core esters in baijiu: ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate. It can also significantly reduce the content of isobutanol and isoamyl alcohol, and increase the content of phenylethanol, thereby effectively reducing the spiciness and numbing sensation of the baijiu. Therefore, the compound microbial agent of this invention, when used in baijiu, can significantly improve the quality of baijiu production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a compound inoculum of *Pichia kudrica* and *Cytomyces delleoides* and its application. Background Technology

[0002] Ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate are the four most essential esters in strong-aroma baijiu, and their content and ratio are key indicators for evaluating the quality grade of strong-aroma baijiu. Among them, ethyl hexanoate, as the main aroma component of strong-aroma baijiu, imparts the typical cellar aroma and sweetness to the liquor, and its content directly determines the rate of premium baijiu.

[0003] The formation of esters during the fermentation of baijiu (Chinese white liquor) mainly relies on the metabolic synthesis of microorganisms. These include various microorganisms such as ester-producing yeasts, bacteria, and molds, which catalyze the esterification reaction of acids and alcohols through esterification enzymes, as well as directly synthesizing ester compounds during microbial metabolism. However, traditional strong-aroma baijiu fermentation primarily depends on the naturally enriched microbial communities in the fermentation pit mud and yeast, which suffers from problems such as long fermentation cycles, low ester synthesis efficiency, and poor quality stability. Therefore, some studies have attempted to add functional microorganisms to the fermentation system to enhance the formation of specific esters.

[0004] However, existing technologies still have the following shortcomings: First, most studies focus on adding a single microbial strain to the fermentation system, whose metabolic function is limited, only able to enhance one or two types of esters, making it difficult to achieve synergistic enhancement of the four major esters; second, single strains are easily inhibited by environmental factors (such as temperature, acidity, substrate competition, etc.) in the complex solid-state fermentation system of baijiu, leading to unstable functional expression and difficulty in guaranteeing practical application effects; third, regulating ester production efficiency with a single strain or simple enzyme system is complex to operate and lacks universality, making it difficult to adapt to the fermentation needs under different production conditions. Currently, increasing the content of ethyl hexanoate, reducing the proportion of ethyl lactate, and coordinating the proportions of the four major esters have become urgent technical challenges to be solved in improving the quality of strong-aroma baijiu. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention screens, isolates, and identifies a strain of *Pichia pastoris* that produces high yields of ethyl butyrate during fermentation from strong-aroma daqu (a type of starter culture). Pichia kudriavzevii MBT-PK97 was selected and screened to form a yeast compound agent with the commercially available Dale spore-forming yeast. By utilizing the synergistic effect between strains, it can achieve simultaneous and synergistic enhancement of the four core esters in strong-aroma baijiu: ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate. This provides an efficient and stable solution for improving the flavor and quality of strong-aroma baijiu.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a compound microbial agent containing *Pichia pastoris* (Kudela spp.). Pichia kudriavzevii MBT-PK97 and Dell's spore-forming yeast ( Torulaspora delbrueckii The preservation number of the aforementioned Kudria zwibbichi yeast MBT-PK97 is: CGMCC No. 35720.

[0007] In some embodiments, the mass concentration ratio of the *Pichia pastoris* MBT-PK97 and *Cytomyces delleoides* is 1–3:1–3.

[0008] In some implementations, the Dell spore yeast is a publicly available or commercially available conventional Dell spore yeast.

[0009] In some embodiments, the nucleotide sequence of the 26S rRNA of the *Pichia gondii* MBT-PK97 is shown in SEQ ID NO:1: .

[0010] Secondly, the present invention provides the application of the above-mentioned compound microbial agent in the brewing of baijiu (Chinese liquor), the preparation of mash or daqu (fermentation starter).

[0011] In some implementations, the liquor is selected from at least one of the following: strong aroma type, sauce aroma type, and light aroma type.

[0012] In some embodiments, the inoculation concentration of the compound microbial agent is 10. 5 ~10 9 CFU / mL or CFU / g.

[0013] Thirdly, the present invention provides the application of the above-mentioned compound microbial agent in simultaneously improving the content of ethyl hexanoate, ethyl acetate, ethyl lactate and ethyl butyrate in fermented foods.

[0014] Fourthly, the present invention provides a method for brewing baijiu (Chinese liquor), which involves adding the above-mentioned compound microbial agent to baijiu raw materials or mash, followed by fermentation.

[0015] In the method described above, the inoculation amount of the compound microbial agent is 10. 5 CFU / g of fermented mash.

[0016] In the method described above, the fermentation temperature is 28–30°C.

[0017] In the method described above, the fermentation time is 30 to 40 days.

[0018] In the method described above, the fermented mash is prepared by adding baijiu daqu (a type of starter culture) to grains.

[0019] Beneficial effects: This invention provides a compound microbial agent containing Pichia kudriezvichiz MBT-PK97 and Pichia delleoides. The Pichia kudriezvichiz MBT-PK97 was isolated, screened and identified from strong-aroma Daqu (a type of Chinese liquor), and its preservation number is CGMCC No. 35720.

[0020] During single-strain fermentation with MBT-PK97, the content of ethyl butyrate in strong-aroma baijiu can be increased, specifically promoting the accumulation of butyric acid and its ester derivatives. However, its effect on increasing ethyl acetate and ethyl lactate is not significant, and is even lower than that of the uninoculated control group, which is not conducive to the synergistic enhancement of the four major esters in strong-aroma baijiu. This invention found that, compared with single-strain fermentation with MBT-PK97 or co-fermentation with other yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Saccharomyces kazakhstan, Saccharomyces baijiu, and Saccharomyces cerevisiae, combining MBT-PK97 with the specific yeast Saccharomyces delle Scovilleae for baijiu fermentation not only overcomes the defects of single-strain fermentation with MBT-PK97, but also significantly activates the esterification metabolic pathway during baijiu brewing, increasing ethyl hexanoate by 40.4%, ethyl acetate by 36.8%, ethyl lactate by 37.7%, and ethyl butyrate by 65.4%. It can also significantly reduce the content of isobutanol and isoamyl alcohol, increase the content of phenylethanol, effectively reduce the spiciness and numbing sensation of the liquor, making it more mellow and smooth, giving the liquor an elegant rose and honey aroma, significantly improving its aroma quality and sophistication, reducing the risk of headaches after drinking, and improving the sensory quality of strong-aroma baijiu.

[0021] Therefore, the compound microbial agent composed of Pichia pastoris MBT-PK97 and Saccharomyces delleoides of this invention has important application value for improving the flavor and quality of baijiu, especially strong-aroma baijiu, by simultaneously and synergistically increasing the levels of the four core esters: ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate.

[0022] Preservation instructions for the strains of this invention: *Pichia pastoris* MBT-PK97, with accession number CGMCC No. 35720, was deposited on August 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification is as follows: Pichia kudriavzevii . Detailed Implementation

[0023] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0024] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0025] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0026] In one embodiment of the present invention, a compound microbial agent is provided, which contains *Pichia kudrica* (a type of yeast). Pichia kudriavzevii MBT-PK97 and Dell's spore-forming yeast ( Torulaspora delbrueckii The preservation number of the aforementioned Kudria zwibbichi yeast MBT-PK97 is: CGMCC No. 35720.

[0027] In some examples, *Pichia pastoris* MBT-PK97 was isolated, screened, and molecularly identified from a strong-aroma type of koji (a type of Chinese starter culture). Its morphological characteristics include the formation of round colonies (2-3 mm in diameter, milky white, smooth and moist with regular edges) after culturing on YPD solid medium at 28–30°C for 48 hours. Under a light microscope, the vegetative cells are elliptical to rod-shaped, reproduce via polygonal budding, and pseudohyphae formation can be observed. Molecular biological identification revealed its 26S rRNA nucleotide sequence as shown in SEQ ID NO:1.

[0028] The applicant discovered that combining *Pichia kudriazina* MBT-PK97 with *Cyclocarya delle* for the fermentation of strong-aroma baijiu significantly increases the levels of ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate. However, this effect is not achieved by fermenting *Pichia kudriazina* MBT-PK97 or *Cyclocarya delle* alone, or by co-fermenting *Pichia kudriazina* MBT-PK97 with other yeasts such as *Saccharomyces cerevisiae*, *Schizosaccharomyces kazakhstanensis*, *Baier conjugate*, or *V. wickhana*. Therefore, the compound microbial agent of this invention can be used in baijiu brewing or other food fermentation fields to simultaneously increase the levels of ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate in the product.

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. The advantages and features of this invention will become clearer with this description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0030] The culture medium formulations used in the following examples are as follows: YPD liquid medium: 2% peptone, 2% glucose, 1% yeast extract, natural pH.

[0031] YPD solid medium: YPD liquid medium with an additional 2% agar, natural pH.

[0032] Example 1: Screening of Pichia pastoris MBT-PK97 (1) Strain screening Samples were taken from strong-aroma daqu (a type of Chinese liquor), diluted and spread using YPD solid medium, and cultured at 30℃ for 48-72 hours. Single colonies were picked and purified multiple times to obtain a pure culture, which was named MBT-PK97.

[0033] (2) Morphological identification This strain forms round colonies on YPD solid plates, 2-3 mm in diameter, milky white, with smooth, moist surfaces and neat edges. Under an optical microscope, the vegetative cells are elliptical to rod-shaped, reproduce through polygonal budding, and pseudohyphae can be observed. These morphological characteristics are similar to those of the genus *Pichia pastoris* (…). Pichia It matches the characteristics of ).

[0034] (3) Molecular biological identification Genomic DNA was extracted from strain MBT-PK97. The fungal 26S rDNA was amplified and sequenced using the universal primers NL1 and NL4 for fungal 26S rDNA. The D1 / D2 region of the 26S rDNA was amplified and sequenced.

[0035] NL1: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2), NL4: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3).

[0036] The sequencing results were identified by molecular biology of 26S rDNA, as shown in SEQ ID NO:1.

[0037] The obtained sequences were BLAST-aligned with the NCBI database. Pichia kudriavzevii The standard model strain showed the highest sequence homology. Based on comprehensive analysis of cell morphology, physiological and biochemical characteristics, and 26S rDNA gene sequence, strain MBT-PK97 was identified as *Pichia kudrifolia*, and was deposited on August 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is: Pichia kudriavzeviiIts accession number is: CGMCCNo. 35720.

[0038] Example 2: Application of Pichia pastoris MBT-PK97 in the separate fermentation of strong-aroma baijiu The selected *Pichia pastoris* MBT-PK97 strains were inoculated into YPD liquid medium and cultured on a shaker at 170 rpm and 30°C for 12 h. Following a 10... 5 The CFU / g inoculum was inoculated into the mash of strong-aroma baijiu (obtained by fermentation of five grains and 25% rice husks by mass, wherein the five grains consist of 65% sorghum, 10% wheat, 10% glutinous rice, 10% corn, and 5% rice) in 500 mL Erlenmeyer flasks for fermentation experiments over 30 days. After fermentation, the following parameters were determined: weight loss, acidity, reducing sugar content, moisture content, starch content, pH value, ethanol content, lactic acid, acetic acid, propionic acid, butyric acid, aroma compounds, and higher alcohols.

[0039] Table 1: Results of weight loss, acidity, reducing sugar content, moisture, starch content, pH value, ethanol content, acids, aroma compounds and higher alcohol content after single fermentation of Pichia pastoris MBT-PK97.

[0040] As shown in Table 1, MBT-PK97 fermentation alone can produce ethyl acetate, isoamyl alcohol, 2,3-butanediol, phenylethanol and other substances that are beneficial to enhance the flavor of baijiu.

[0041] Example 3: Application of Pichia pastoris MBT-PK97 in the intensified fermentation of strong-aroma baijiu To further verify the actual effect of Pichia kudriezvich on the production of strong-aroma baijiu, the Pichia kudriezvich MBT-PK97 activated in YPD liquid medium in Example 2 was inoculated at the same amount (10 5 CFU / g of fermented mash was inoculated into actual strong-aroma baijiu mash (i.e., obtained by adding 30% by weight of Wuliangye strong-aroma baijiu Daqu for fermentation to the mash described in Example 2) to simulate the actual brewing of strong-aroma baijiu. A control group (CK) (i.e., the enhanced fermentation experimental group without any added microbial agents) was also set up to verify the actual effect of Pichia pastoris MBT-PK97 on strong-aroma baijiu. Fermentation experiments were conducted in 500 mL Erlenmeyer flasks for 30 days. After fermentation, the following parameters were measured using the same methods: weight loss, acidity, reducing sugar content, moisture content, pH value, ethanol content, lactic acid, acetic acid, propionic acid, butyric acid, aroma compounds, and higher alcohol content.

[0042] Table 2: Results of weight loss, acidity, reducing sugar content, moisture content, pH value, ethanol content, lactic acid, acetic acid, propionic acid, butyric acid, aroma compounds and higher alcohol content after enhanced fermentation with Pichia pastoris MBT-PK97

[0043] Table 2 shows that when Pichia pastoris MBT-PK97 was used for single-strain fermentation of strong-aroma baijiu, it significantly increased the content of ethyl butyrate, but the content of ethyl acetate and ethyl lactate was lower than that of the control group. This is not conducive to the synergistic enhancement of the four major esters.

[0044] Example 4: Application of Pichia pastoris MBT-PK97 combined with different yeast strains in the intensified fermentation of strong-aroma baijiu To further verify the actual synergistic effect of Pichia pastoris MBT-PK97 combined with other existing yeast strains (commercially available), numbered AY12 (Saccharomyces cerevisiae), SP65 (Schizosaccharomyces cerevisiae), T4 (Cytomyces delleoides), K4 (Kazakhstan yeast), BMO (Baier conjugate yeast), and W3 (V. ve. hansenii), the following treatment groups were set up: the control group was inoculated with only MBT-PK97; the experimental groups were inoculated with MBT-PK97 plus an equal amount of the other yeast strains mentioned above, specifically including experimental group 1 (MBT-PK97 + AY12), experimental group 2 (MBT-PK97 + SP65), experimental group 3 (MBT-PK97 + T4), experimental group 4 (MBT-PK97 + K4), experimental group 5 (MBT-PK97 + BMO), and experimental group 6 (MBT-PK97 + W3).

[0045] Each of the above-mentioned strains was inoculated into YPD liquid medium and cultured on a shaker at 170 rpm and 30℃ for 12 h. After culture, the control group was treated with 10... 5 The inoculation amount of cfu / g fermented mash was only MBT-PK97; each experimental group was inoculated with a total inoculation amount of 10 5CFU / g of fermented mash and compounded yeast strains were mixed in equal amounts at a 1:1 concentration ratio and inoculated into the mash of strong-aroma baijiu (raw materials: 65% sorghum, 10% wheat, 10% glutinous rice, 10% corn, 5% rice, with an additional 25% rice husk by weight of raw materials, and 30% Daqu prepared according to conventional processes). Simultaneously, a standard amount of medium-high temperature strong-aroma baijiu Daqu was added to simulate the actual brewing process. Fermentation experiments were conducted in 500 mL Erlenmeyer flasks for 30 days. After fermentation, acidity, reducing sugar content, ethanol content, aroma compounds, acid content, and higher alcohol content were measured. Comparative analysis was used to verify whether the combination of MBT-PK97 with the aforementioned different yeast strains (AY12, SP65, T4, K4, BMO, W3) had a synergistic effect in enhancing flavor.

[0046] Table 3. Acidity, reducing sugar content, ethanol content, aroma compounds, acid and higher alcohol content after enhanced fermentation of Pichia pastoris MBT-PK97 combined with different yeast strains.

[0047] Table 3 shows that, compared with the control group (MBT-PK97), the addition of Pichia pastoris MBT-PK97 and the addition of T4 strain for enhanced fermentation resulted in significant differences in acidity, reducing sugar content, and ethanol content. Acidity increased from 0.35±0.07 mmol / 10g to 0.75±0.07 mmol / 10g, reducing sugar content increased from 2.28±0.21% to 2.92±0.21%, and ethanol content decreased from 26.47±2.13 g / Kg to 22.02±2.03 g / Kg. Excessive acidity leads to a sharp, pungent taste and unbalanced flavor, while excessively low acidity results in a thin, bland taste and lack of aroma. A moderate increase in acidity enhances the body, makes the aroma richer and fuller, and improves the overall harmony of the flavor. Excessive reducing sugar content makes the wine taste cloyingly sweet and dull, while insufficient reducing sugar content results in a bland body and a lack of sweetness. A moderate increase in reducing sugar content imparts a sweet and mellow taste, enhancing its smoothness and sweet aftertaste. Adding the PK97+T4 combination keeps acidity and reducing sugar content within an appropriate range, increasing the wine's body, making the aroma more rich and full-bodied, enhancing its smoothness and sweet aftertaste, and creating a more harmonious flavor without negatively impacting the wine's quality.

[0048] Regarding acid components, the combinations exhibited different patterns of change compared to the control group. In the MBT-PK97+T4 combination, the acetic acid content decreased from 409.5 mg / L to 384.6 mg / L, butyric acid from 11.9 mg / L to 6.4 mg / L, isovaleric acid from 9.0 mg / L to 15.7 mg / L, and caprylic acid from 200.7 mg / L to a significant decrease to 14.2 mg / L. The moderate decrease in acetic acid, the significant drop in butyric acid, and the slight increase in isovaleric acid collectively constitute a rebalancing of the acid composition. Although the content of some acids, such as acetic acid and butyric acid, decreased, the total acidity (acidity value) actually increased, possibly due to the contribution of other untested organic acids. This resulted in a more robust overall acidity structure, contributing to a mellow and sweet taste while avoiding the sharpness that can result from excessive levels of a single acid. The significant decrease in caprylic acid helps reduce potential rancidity, resulting in a purer aroma.

[0049] Regarding ester components, compared with the control group, the ethyl lactate content of the MBT-PK97+T4 combination increased from 1331.0 mg / L to 1832.9 mg / L, the ethyl acetate content significantly increased from 1452.8 mg / L to 1987.6 mg / L, the ethyl butyrate content increased from 814.3 mg / L to 1347.3 mg / L, and the ethyl hexanoate content increased from 1198.2 mg / L to 1681.9 mg / L. In addition, the concentrations of isoamyl acetate increased from 103.8 mg / L to 150.5 mg / L, ethyl valerate from 5.1 mg / L to 10.0 mg / L, ethyl heptaate from 10.1 mg / L to 16.6 mg / L, ethyl octanoate from 90.5 mg / L to 158.6 mg / L, ethyl nonanoate from 50.3 mg / L to 55.3 mg / L, ethyl decanoate from 110.5 mg / L to 183.3 mg / L, diethyl succinate from 49.9 mg / L to 46.8 mg / L, ethyl benzoate from 19.1 mg / L to 17.4 mg / L, ethyl phenylacetate from 72.1 mg / L to 81.7 mg / L, and ethyl phenylpropionate from 24.1 mg / L to 15.9 mg / L. Overall, most esters showed an upward trend.

[0050] Most notably, a horizontal comparison between the MBT-PK97+T4 combination and other combinations containing AY12, SP65, K4, BMO, and W3 revealed that the MBT-PK97+T4 combination had the highest content of ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate among all combinations. The ethyl acetate content reached 1987.6 mg / L, significantly higher than AY12 (1847.5 mg / L), SP65 (1745.6 mg / L), K4 (1837.8 mg / L), BMO (1712.6 mg / L), and W3 (1840.8 mg / L); the ethyl lactate content reached 1832.9 mg / L, significantly higher than AY12 (1432.6 mg / L), SP65 (1011.4 mg / L), K4 (1606.8 mg / L), BMO (1160.2 mg / L), and W3 (1196.9 mg / L); and the ethyl butyrate content reached 1347.3 mg / L, far higher than AY12 (516.2 mg / L), SP65 (1000.1 mg / L), K4 (904.6 mg / L), and BMO (1028.5 mg / L). The concentrations of ethyl hexanoate and BMO were 1174.4 mg / L and 912.1 mg / L, respectively. The ethyl hexanoate content reached 1681.9 mg / L, significantly higher than AY12 (1174.4 mg / L), SP65 (1164.4 mg / L), BMO (1176.8 mg / L), and W3 (1184.4 mg / L), while slightly higher than K4 (1617.1 mg / L). Compared with the control group (PK97), the MBT-PK97+T4 combination showed increases of 40.4% (ethyl hexanoate), 36.8% (ethyl acetate), 37.7% (ethyl lactate), and 65.4% (ethyl butyrate), respectively. These results indicate that, under the same conditions of combining different strains with MBT-PK97 as the base, the synergistic effect between T4 (Cytomyces delleoides) and MBT-PK97 (Pichia kudriezweig) is the most prominent, exhibiting the best promoting effect on the synthesis of core esters in strong-aroma baijiu. The increases in ethyl lactate and ethyl acetate corroborate the aforementioned changes in acid composition. Acetic acid, as a precursor to esterification, provides a more abundant material basis for the esterification reaction, thereby shifting the reaction equilibrium to the right. The significant increase in ethyl butyrate and ethyl hexanoate directly reflects the enhancement of butyric acid and hexanoic acid-related metabolic pathways. Even with a decrease in the content of free butyric acid, the surge in esterification products indicates a substantial improvement in esterification efficiency.

[0051] Regarding higher alcohols, compared to the control group, the MBT-PK97+T4 combination showed a significant decrease in isobutanol from 33.9 mg / L to 5.6 mg / L, isoamyl alcohol from 163.3 mg / L to 141.9 mg / L, phenethyl alcohol from 235.1 mg / L to 256.8 mg / L, and 2,3-butanediol from 24.9 mg / L to 10.6 mg / L. The marked reduction in isobutanol and isoamyl alcohol effectively alleviates the spiciness and numbing sensation of the spirit, making it more mellow and smooth, and reducing the masking of the main aromas, allowing the cellar and grain aromas to stand out more, thus reducing the risk of headaches after drinking. The appropriate increase in phenethyl alcohol imparts elegant rose and honey aromas to the spirit, significantly enhancing its aroma quality and sophistication.

[0052] The MBT-PK97+T4 combination significantly increased the content of four key esters—ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate—reaching the highest levels among all combinations. The core mechanism lies in the synergistic introduction of MBT-PK97 yeast and the T4 strain. Instead of directly producing large quantities of these esters, they act as an "ecological engine" and "strategic logistics manager," completely reshaping the microbial ecosystem within the fermenting mash. This forms a highly efficient symbiotic alliance with the indigenous microbial community, greatly enhancing the two key biochemical pathways of "acid production" and "esterification." During the early stages of fermentation, MBT-PK97 and T4 undergo vigorous respiration and sugar metabolism, producing large amounts of hydrogen and organic acids (such as acetic acid), providing electron donors and carbon sources for the strictly anaerobic acid-producing bacteria, stimulating their activity. Simultaneously, their rapid growth consumes residual oxygen, creating a highly reducing environment for the anaerobic bacteria, maximizing their metabolic capacity. Butyric acid and hexanoic acid, as direct precursors of ethyl butyrate and ethyl hexanoate, may be rapidly consumed in their free forms in the mash due to esterification. However, the esterification reaction follows the principle of chemical equilibrium: acid + ethanol + ester + water. The high concentration of precursor acid and sufficient ethanol significantly shift the reaction equilibrium to the right (Le Chatelier's principle), generating far greater amounts of ethyl butyrate and ethyl hexanoate than usual. This is corroborated by the aforementioned moderate reduction in ethanol content. Simultaneously, the appropriate decrease in acetic acid content (consumed by esterification) leads to a significant increase in ethyl acetate, further validating the improved esterification efficiency.

[0053] The addition of Pichia pastoris MBT-PK97 and the combination with Saccharomyces delleoides strain to enhance fermentation resulted in a significant increase in the levels of four major esters—ethyl hexanoate, ethyl acetate, ethyl lactate, and ethyl butyrate—to the highest level among all combinations, representing the ultimate enhancement of flavor quality. Ethyl hexanoate, as the "main aroma" and "soul of cellar aroma" in strong-aroma baijiu, saw its content increase by 40.4% to an absolute value of 1681.9 mg / L (higher than K4's 1617.1 mg / L and other combinations), meaning the cellar aroma of the baijiu became extremely rich, pure, elegant, and penetrating; ethyl acetate increased by 36.8% to an absolute value of 1987.6 mg / L (higher than AY12's 1847.5 mg / L, etc.), giving the baijiu a fresher and more pleasant fruity and sweet aroma, making the aroma layers richer and brighter; ethyl lactate increased by 37.7% to an absolute value of 1832.9 mg / L (higher than K4's 1606.8 mg / L and AY12's 1432.6 mg / L, etc.), giving the baijiu a more mellow and full-bodied sweetness, significantly improving its smoothness; ethyl butyrate increased by 65.4% to an absolute value of 1347.3 mg / L. The concentration of mg / L (significantly higher than AY12's 516.2 mg / L and SP65's 1000.1 mg / L) further enhances the intensity and complexity of the cellar and aged aromas. The synergistic effect of these four elements perfectly blends the cellar, fruit, sweet, and aged aromas, achieving a top-tier level of aroma complexity and layering. This perfectly embodies the essence of "millennial cellars and ten-thousand-year lees," resulting in an explosively sweet and smooth taste, a silky texture, and a long, lingering finish. The fullness, harmony, and richness of the flavor achieve a qualitative leap. The abundant free acids accumulated in the wine serve as a valuable precursor pool, continuously undergoing esterification reactions with ethanol during the long aging process, constantly generating new esters, making the aroma increasingly rich and complex over time.

[0054] In summary, the combination of Pichia pastoris MBT-PK97 and Saccharomyces delleoides provided by this invention significantly activates the esterification metabolic pathway during baijiu brewing, increasing ethyl hexanoate by 40.4%, ethyl acetate by 36.8%, ethyl lactate by 37.7%, and ethyl butyrate by 65.4%. The content of these four esters is the highest among all combination blends, with a simultaneous and significant increase in the four core esters of strong-aroma baijiu. This mechanism ultimately endows the baijiu with a rich and pure cellar aroma, a mellow and sweet taste, and a revolutionary controllability and stability in flavor production. Furthermore, the addition of the MBT-PK97+T4 combination significantly reduces the content of isobutanol and isoamyl alcohol while increasing the content of phenylethanol, effectively reducing the spiciness and numbing sensation of the baijiu, making it more mellow and smooth, and imparting an elegant rose and honey aroma. This significantly enhances its aroma quality and sophistication, reduces the risk of headaches after drinking, and improves the sensory quality of strong-aroma baijiu.

[0055] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements to the present invention without departing from the inventive concept, and all such improvements fall within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, Contains Kudria zweipichia yeast ( Pichia kudriavzevii MBT-PK97 and Dell's spore-forming yeast ( Torulaspora delbrueckii The preservation number of the aforementioned Kudria zwibbichi yeast MBT-PK97 is: CGMCC No. 35720.

2. The compound microbial agent according to claim 1, characterized in that, The mass concentration ratio of *Pichia pastoris* MBT-PK97 to *Cytomyces delleoides* was 1–3:1–3.

3. The compound microbial agent according to claim 1, characterized in that, The nucleotide sequence of the 26S rRNA of *Pichia kudrica* MBT-PK97 is shown in SEQ ID NO:

1.

4. The application of the compound microbial agent according to claim 1 or 2 in the brewing of baijiu, the preparation of mash or daqu.

5. The application according to claim 4, characterized in that, The baijiu is selected from at least one of the following: strong aroma, sauce aroma, and light aroma.

6. The application according to claim 4, characterized in that, The inoculation concentration of the compound microbial agent is 10. 5 ~10 9 CFU / mL or CFU / g.

7. The compound microbial agent according to claim 1 or 2 is used to simultaneously improve the levels of ethyl hexanoate, ethyl acetate, ethyl lactate and ethyl butyrate in fermented foods.

8. A method for brewing baijiu (Chinese liquor), characterized in that, It involves adding the compound microbial agent described in claim 1 or 2 to the raw materials or mash of baijiu, followed by fermentation.

9. The method according to claim 8, characterized in that, The inoculum size of the compound microbial agent is 10. 5 CFU / g.

10. The method according to claim 8, wherein the fermentation temperature is 28–30°C.

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