Method for improving ester yield by co-culturing two strains of ester-producing yeast and application thereof

Abstract

The present application belongs to the field of microorganism and fermentation technology, and discloses a method for improving ester content in fermentation by co-culturing two strains of ester-producing yeast, wherein Pichia kudriavzevii PK2 and Wickerhamomyces anomalus W4 are used, and the two strains are fermented simultaneously. The present application applies Pichia kudriavzevii and Wickerhamomyces anomalus to the solid-state brewing process of Maotai-flavor liquor, improves the liquor yield and the ester content of Maotai-flavor liquor, makes the liquor taste softer and the liquor flavor richer, and plays an important role in further improving the liquor quality. Meanwhile, the present application has far-reaching social and economic significance for the intensified utilization of functional brewing microbial resources and the innovation and upgrading of traditional brewing process.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and fermentation technology, and in particular to a method and application of co-culturing two ester-producing yeast strains to improve the ester content of fermentation. Background Technology

[0002] Esters are the largest category in baijiu (Chinese liquor) and play a crucial role in its quality. Their type, content, and proportion are key factors determining the quality of the liquor and have a significant impact on traditional baijiu brewing. Research shows that in traditionally brewed foods, esters mainly originate from microbial fermentation, with smaller amounts arising from chemical reactions during fermentation and introduction from raw materials. Therefore, fully utilizing high-ester-producing microorganisms in fermentation can have a predictable and considerable effect on improving baijiu quality. Previous studies have found that among these ester-producing microorganisms, ester-producing yeasts are the main ester-producing strains in traditionally fermented foods. Obtaining high-ester-producing functional microorganisms from the baijiu brewing system and enhancing their application in the brewing system is of great significance for increasing the ester content in baijiu.

[0003] The raw materials, processes, microorganisms, and environmental conditions during the brewing of baijiu (Chinese liquor) can cause differences in the types and contents of flavor compounds in the liquor. Among these, microorganisms are the most important factor, and yeast is the main microorganism contributing to the flavor of baijiu. Yeast is subjected to various environmental stresses during fermentation, mainly including osmotic pressure, temperature, ethanol, and low pH. These stresses can damage the yeast's cell structure, affecting its growth, metabolism, and physiological functions, leading to lower growth rates and survival rates, ultimately impacting fermentation efficiency. Therefore, yeast strains must possess characteristics such as multi-environment tolerance, broad-spectrum substrate adaptability, and ester production to improve fermentation efficiency, reduce production costs, and improve product quality in industrial production. Among the many aroma-producing yeasts, *Pichia pastoris* and *Wickham's unusual* produce high levels of esters, imparting floral and fruity aromas to baijiu and significantly improving its aroma profile and quality.

[0004] Existing reports include invention patents 201811379487.3 and 201711136361.9, which respectively provide a high-ethanol-producing brewer's yeast and a method for improving the quality of traditional fermented foods by co-culturing it with a high-ester-producing abnormal Wickham yeast. Invention patent 201811379487.3 involves optimizing the co-culture conditions of brewer's yeast YF1914 and abnormal Wickham yeast YF1503 in sorghum enzymatic hydrolysate culture medium, ultimately achieving an ethyl acetate content of 4.30 g / L. Invention patent 201711136361.9 provides a high-ethanol-producing brewer's yeast YF3401 and its optimal ratio for co-fermentation with abnormal Wickham yeast YF3604 to increase ethyl acetate content, as well as its application. These differ from the ester-producing yeasts used in this invention in terms of species and performance, and the optimized co-culture... The culture media used in this invention differ from those used in the previous invention. Simulated solid-state fermentation and in-situ fermentation experiments were employed to apply ester-producing yeast to the seventh round of brewing of Maotai-flavor liquor, making it more practically significant. Furthermore, this invention not only increased the ethyl acetate content in the fermented liquor sample but also significantly improved the content of total esters, ethyl hexanoate, and ethyl lactate. Invention patent 202110120767.8 provides a strain of highly resistant vesicle-coated yeast and its application in co-culturing with Kluyveromyces martensii to produce acetic acid and other organic acids, as well as phenylethyl acetate. This invention uses corn bran solid culture medium, mixing the two yeast strains in a certain proportion for fermentation, and applies it to the rice wine fermentation process. This differs from the strain, fermentation medium, function, and main application environment of this invention. Moreover, the strain of this invention exhibits multi-environmental tolerance, with stronger tolerance and adaptability. Invention patent 201710827683.1 provides a method for producing light-aroma baijiu through multi-strain enhanced fermentation of daqu (a type of starter culture). This method uses brewing yeast, Rhizopus oryzae, Monascus fusca, and Bacillus subtilis in a certain proportion to enhance the daqu, increasing the ethyl acetate / milk ratio in the baijiu. While the strains used in this invention have similar origins to those in the present invention, their functions and application environments differ. Furthermore, the strains in this invention exhibit stronger tolerance. This method not only increases the ester content of the baijiu but also enriches the microbial resource library, demonstrating broad application prospects.

[0005] In summary, current research on using ester-producing yeasts to enhance the fermentation of baijiu (Chinese liquor) has a limited scope in assessing the yeast's tolerance, and most studies involve the application of single-strain fermentation, resulting in the lack of broad applicability and limited functionality of the enhanced strains. Furthermore, current research primarily employs liquid-state fermentation to apply ester-producing yeasts to the brewing process of sauce-flavored baijiu; however, this differs significantly from the actual solid-state fermentation environment in production, limiting the research to laboratory-scale studies and hindering its application in real-world production.

[0006] In contrast, the two ester-producing yeasts in this invention have greater tolerance to multiple environments and stronger ester-producing capacity. Furthermore, the culture conditions for the mixed fermentation of ester-producing yeasts were optimized using sorghum solid culture medium. Through in-situ fermentation experiments, they were applied to the seventh round of brewing of Maotai-flavor liquor to increase the content of esters in the liquor brewing process, thereby improving the flavor of the liquor and making it more mellow and full-bodied, thus enhancing the quality of Maotai-flavor liquor. This not only enriches the strain resource bank but also has higher application value and practical significance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for improving the ester content of fermentation by co-culturing two ester-producing yeast strains.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A method for improving the ester content of fermentation by co-culturing two ester-producing yeast strains, wherein the method uses Pichia kudriavzevii PK2 and Wickerhamomycesanomalus W4, and the two strains ferment simultaneously.

[0010] Furthermore, the aforementioned Pichiakudriavzevii PK2, whose name is PK2, taxonomically named: Pichiakudriavzevii, has the accession number: CGMCC No.29250, accession date: December 6, 2023, and depositary institution: China General Microbiological Culture Collection Center, No.3, No.1 Beichen West Road, Chaoyang District, Beijing;

[0011] The aforementioned abnormal Wickhamomyces W4, with the name W4, taxonomyces anomalus, accession number CGMCC No. 29249, accession date December 6, 2023, depository at: China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0012] Furthermore, the specific steps include the following:

[0013] Using sorghum solid medium as the fermentation ester production medium, with a loading volume of 200g / 500mL, after inoculating with *Bretschneidera sinensis* BD-2 for 36 hours, activated *Pichia pastoris* PK2 and *Wickham's abnormal* W4 were added at a concentration of 10... 6 CFU / mL: 10 8The inoculation ratio was 5% CFU / mL, and the ester production culture was carried out by inoculating the sorghum solid fermentation medium. After inoculation, the medium was incubated at 30℃ for 7 days to complete the fermentation.

[0014] Furthermore, the total ester content obtained by the method reached 1294.62 mg / L.

[0015] Furthermore, the method is a method for improving the quality of baijiu by co-culturing two ester-producing yeast strains, including the following steps:

[0016] Suspensions of *Kudriazwibbi* PK2 and *Wickham's abnormal* W4 were prepared at 10... 6 10 8 The inoculation ratio was mixed, that is, the concentration of Pichia pastoris PK2 was 2×10⁻⁶. 6 CFU / mL, the concentration of abnormal Wickham yeast W4 was 2×10⁻⁶. 8 CFU / mL, inoculation time is B-36, that is, after 36 hours of pile fermentation, the bacterial suspension is inoculated into the mash after six rounds of cooling and mixing with the yeast at an inoculation rate of 5%, and the mash is allowed to ferment naturally in piles and in fermentation pits.

[0017] Furthermore, the total ester content obtained by the method is 11283.83 mg / L.

[0018] The application of the methods described above in food fermentation.

[0019] The application of the methods described above in in-situ fermentation.

[0020] The application of the methods described above in the fermentation of baijiu (Chinese liquor).

[0021] The advantages and positive effects of this invention are as follows:

[0022] 1. This invention applies *Pichia pastoris* and *Wickham's abnormal yeast* to the solid-state brewing process of Maotai-flavor liquor, increasing the liquor yield and ester content, resulting in a smoother taste and richer flavor, which plays a significant role in further improving the quality of the liquor. Simultaneously, the enhanced utilization of functional brewing microbial resources and the innovative upgrading of traditional brewing processes have profound social and economic significance.

[0023] 2. This invention, through research on the co-culture fermentation characteristics of *Pichia kudriavzevii* and *Wickerhamomyces anomalus*, optimized the culture conditions for mixed-strain solid-state fermentation co-culture, thereby increasing the ester content produced during co-culture fermentation. A method for co-culturing ester production was determined, which can be applied in the food fermentation field, such as in the brewing of Maotai-flavor liquor and the development of related functional microbial agents. The practical application effect of the strain co-culture method of this invention is good and can be applied to actual production processes to improve the yield and quality of Maotai-flavor liquor fermentation.

[0024] 3. This invention provides a method for co-culturing two ester-producing yeasts, *Pichia kudriavzevii* and *Wickerhamomyces anomalus*, and applies it to the actual production of Maotai-flavor liquor fermentation. Using this method, the optimized conditions described above are applied to the Maotai-flavor liquor brewing process. The effects of co-culturing these two ester-producing yeasts during the seventh fermentation process on the ester content in the liquor samples were investigated. The practical application effect of the optimized conditions was verified, improving the yield and ester content of the base liquor in the seventh fermentation of Maotai-flavor liquor. This method is beneficial for improving the quality, yield, and overall quality of Baijiu, and has broad industrial application prospects.

[0025] 4. This invention specifically relates to a cultivation method and application of co-culturing two ester-producing yeast strains, namely *Pichia kudriavzevii* PK2 and *Wickerhamomyces anomalus* W4, which were deposited on December 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC), specifically at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number for PK2 is CGMCC No. 29250, and for W4 it is CGMCC No. 29249. These strains originate from the mash of traditional fermented food, soy sauce-flavored baijiu. Both *Pichia kudriavzevii* PK2 and *Wickerhamomyces anomalus* W4 exhibit good tolerance and ester-producing ability. Based on this, the co-culture method of *Pichia pastoris* PK2 and *Wickham's yeast* W4 using sorghum solid-state fermentation medium was optimized, resulting in an ester compound yield of 1294.62 mg / L. Finally, *Pichia pastoris* PK2 and *Wickham's yeast* W4 were applied to the actual production process of Maotai-flavor liquor for in-situ fermentation, ultimately increasing the liquor yield of the seventh fermentation by 2.81 times. GC-MS results showed that the total ester content reached 11283.83 mg / L, with significant increases in all four major ethyl esters. Sensory evaluation showed that the experimental group of liquor had a smoother taste, with prominent "floral," "sweet," and "fruity" aromas. Kudria zweibichi PK2 and Aberrant Wickham yeast W4 have high ester production characteristics, and their co-fermentation can increase the total ester content of Maotai-flavor liquor, playing a role in ester enhancement and aroma enhancement. They can significantly improve the aroma profile and quality of liquor, and harmonize the flavor and taste of liquor. They can be used in the field of food fermentation, such as in brewing and the development of related functional microbial agents.

[0026] 5. The strains used in this invention include *Pichia kudriavzevii* PK2 (CGMCC No. 29250) and *Wickerhamomyces anomalus* W4 (CGMCC No. 29249). These strains are selected from the fermented mash of Maotai-flavor liquor, ensuring a green, natural, safe, and low-cost source.

[0027] The *Pichia kudrica* PK2 strain exhibits normal growth and reproduction under various environmental conditions: pH 2.0, ethanol (v / v) 18%, acetic acid 10 g / L, lactic acid 80 g / L, glucose 600 g / L, and temperature 45℃. After fermentation in sorghum liquid fermentation medium, the total ester content reached 843.77 mg / L, demonstrating excellent ester production characteristics. The strain was deposited on December 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 29250.

[0028] The described *Wickhamia lanceolata* W4 strain can grow and reproduce normally under conditions of pH 2.0, ethanol (v / v) 18%, acetic acid 10 g / L, lactic acid 80 g / L, glucose 600 g / L, and temperature 45℃, exhibiting multi-environment tolerance. After fermentation in sorghum liquid fermentation medium, the total ester content reached 796.05 mg / L, demonstrating excellent ester production characteristics. The strain was deposited on December 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 29249 (patent application number: 202410368527.3). Attached Figure Description

[0029] Figure 1 This is a diagram showing the ester production of *Pichia kudrica* PK2 and *Wickhamia leucovora* W4 cultured in sorghum solid medium using different inoculation methods in this invention.

[0030] Figure 2 This is a diagram showing the ester production of *Pichia kudrica* PK2 and *Wickhamia leucovora* W4 cultured in sorghum solid medium at different inoculation ratios in this invention.

[0031] Figure 3 This is a diagram showing the ester production of *Pichia kudrica* PK2 and *Wickhamia leucovora* W4 cultured in sorghum solid medium at different loading volumes in this invention.

[0032] Figure 4 This is a diagram showing the ester production of *Pichia kudrica* PK2 and *Wickhamia leucovora* W4 cultured in sorghum solid medium at different fermentation temperatures in this invention.

[0033] Figure 5This is a graph showing the sensory scores of *Pichia kudrica* PK2 and *Wickham's abnormal* W4 in an in situ fermentation experiment under optimal conditions in this invention.

[0034] Figure 6 The images show the colony morphology (left) and cell morphology (right) of Pichia pastoris PK2 in this invention.

[0035] Figure 7 This is a phylogenetic tree diagram of Pichia kudrica PK2 in this invention;

[0036] Figure 8 The images show the colony morphology (left) and cell morphology (right) of the abnormal Wickham yeast W4 in this invention.

[0037] Figure 9 This is a phylogenetic tree diagram of the abnormal Wickham yeast W4 in this invention. Detailed Implementation

[0038] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0039] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0040] A method for improving the ester content of fermentation by co-culturing two ester-producing yeast strains, wherein the method uses Pichia kudriavzevii PK2 and Wickerhamomycesanomalus W4, and the two strains ferment simultaneously.

[0041] Preferably, the *Pichiakudriavzevii* PK2 is named PK2, classified as *Pichiakudriavzevii*, with accession number CGMCC No. 29250, accession date December 6, 2023, and deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0042] The aforementioned abnormal Wickhamomyces W4, with the name W4, taxonomyces anomalus, accession number CGMCC No. 29249, accession date December 6, 2023, depository at: China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0043] Preferably, the specific steps include the following:

[0044] Using sorghum solid medium as the fermentation ester production medium, with a loading volume of 200g / 500mL, after inoculating with *Bretschneidera sinensis* BD-2 for 36 hours, activated *Pichia pastoris* PK2 and *Wickham's abnormal* W4 were added at a concentration of 10... 6 CFU / mL: 10 8 The inoculation ratio was 5% CFU / mL, and the ester production culture was carried out by inoculating the sorghum solid fermentation medium. After inoculation, the medium was incubated at 30℃ for 7 days to complete the fermentation.

[0045] Preferably, the total ester content obtained by the method reaches 1294.62 mg / L.

[0046] Preferably, the method is a method for improving the quality of baijiu by co-culturing two ester-producing yeast strains, comprising the following steps:

[0047] Suspensions of *Kudriazwibbi* PK2 and *Wickham's abnormal* W4 were prepared at 10... 6 10 8 The inoculation ratio was mixed, that is, the concentration of Pichia pastoris PK2 was 2×10⁻⁶. 6 CFU / mL, the concentration of abnormal Wickham yeast W4 was 2×10⁻⁶. 8 CFU / mL, inoculation time is B-36, that is, after 36 hours of pile fermentation, the bacterial suspension is inoculated into the mash after six rounds of cooling and mixing with the yeast at an inoculation rate of 5%, and the mash is allowed to ferment naturally in piles and in fermentation pits.

[0048] Preferably, the total ester content obtained by the method is 11283.83 mg / L.

[0049] The application of the methods described above in food fermentation.

[0050] The application of the methods described above in in-situ fermentation.

[0051] The application of the methods described above in the fermentation of baijiu (Chinese liquor).

[0052] Specifically, the co-culture method is as follows:

[0053] One loop each of *Kudelazvichi* and *Wickham's Abnormal* yeast was picked from the slant culture and inoculated into the primary culture medium. Activation was performed at 30℃ and 180 rpm for 24 h. 100 μL of the primary culture was then inoculated into the secondary culture medium and activated at 30℃ and 180 rpm for 16 h to obtain seed activation solutions of both yeasts. These solutions were then inoculated into sorghum solid culture medium at appropriate ratios, methods, and loading volumes. Fermentation was carried out using a suitable culture method at a suitable temperature, followed by static incubation at 30℃ for 7 days. The total ester content was then measured. Through a single-factor orthogonal design experiment, the total ester content (calculated as ethyl acetate) of *Kudelazvichi* and *Wickham's Abnormal* yeast co-culture reached 1294.62 mg / L. *Kudelazvichi* and *Wickham's Abnormal* yeast can be used as functional microbial strains in actual production, and the co-culture method has practical significance for improving the quality of baijiu (Chinese liquor).

[0054] The second technical solution provided by this invention is the application of the above-mentioned Pichiakudriavzevii and Wickerhamomyces anomalus, especially in the field of food fermentation, such as its use in the fermentation process of soy sauce-flavored liquor.

[0055] The specific method for the application is as follows:

[0056] The seed cultures of *Pichia pastoris* and *Wickham's abnormal* were inoculated into the seventh-round fermentation mash of a sauce-flavored baijiu under the determined inoculation conditions described above, and an in-situ fermentation experiment was conducted. The mash from the fermentation pits was then distilled in small batches, and the alcohol yield was calculated. Sensory analysis was performed on the distilled samples, and the content of volatile substances was determined. After in-situ fermentation, the alcohol yield in the experimental group increased by 2.81 times, and the total ester content of the experimental group reached 11283.83 mg / L, which was 2.26 times higher than that of the control group. The results of volatile flavor compound analysis showed that the experimental group had higher content and types of esters than the control group, with ethyl hexanoate content increasing by 1.95 times, ethyl acetate content increasing by 3.1 times, and ethyl lactate content increasing by 2.25 times. Sensory analysis results showed that the experimental group had prominent fruity and sweet aromas, higher smoothness than the control group, and a higher overall score. The total ester content was determined using the saponification reflux method. The specific content and types of esters in the in-situ fermentation experiment were detected using headspace solid-phase microextraction gas chromatography-mass spectrometry.

[0057] Specifically, the relevant preparation and testing methods are as follows:

[0058] Example 1: A method for improving ester production capacity through solid-state co-culture fermentation of mixed *Pichia pastoris* PK2 and *Wickham's abnormal* W4.

[0059] Preparation of sorghum solid culture medium: Sorghum was crushed into half-grain form and soaked in water for 18 hours at a material-to-water ratio of 1:1. The water was a mixture of 70% hot water and 30% cold water by mass. After soaking, the mixture was dispensed into Erlenmeyer flasks, sealed with gauze and kraft paper, and sterilized at 115℃ for 20 minutes. After cooling to room temperature, 1×10⁻⁶ sorghum grains were inoculated into the culture medium. 6 CFU / mL of *Saccharomyces boulardii* BD-2 was used as the original culture medium for simulating solid-state fermentation. *Saccharomyces boulardii* BD-2 is a strain of brewing yeast previously screened from soy sauce-flavored liquor mash (patent name: A strain of *Saccharomyces boulardii* and its application, Chinese patent application number: CN202211096575.9).

[0060] The total ester content was determined using the saponification reflux method, as detailed below:

[0061] (1) Sample preparation: Weigh 100g of fermented mash into a 1000mL round-bottom flask, add 200mL of water along the flask wall to ensure that any remaining mash at the flask mouth and wall is flushed down. Connect the reflux condenser and use a 100mL volumetric flask as the receiver. Turn on the cooling water and slowly heat the distillation. When the distillate is collected to near the mark, remove the volumetric flask and seal it. Cool to room temperature, add water to make up to the mark, shake well, and obtain the sample to be tested. Let it stand for later use.

[0062] (2) Determination of total ester content: Pipette 50 mL of the sample into a 250 mL reflux flask, add 2 drops of phenolphthalein indicator, and titrate with 0.1 mol / L sodium hydroxide standard titrant until a faint red color persists for 30 seconds. Record the volume of sodium hydroxide standard titrant consumed. Accurately add 25 mL of sodium hydroxide standard titrant, shake well, attach a condenser, reflux in a boiling water bath for 30 min, remove, and cool. Titrate with 0.1 mol / L sulfuric acid standard titrant until the red color just completely disappears as the endpoint. Record the volume of sulfuric acid standard titrant consumed, V1. Simultaneously, pipette 50 mL of ethanol solution (50%, volume fraction) and perform a blank test using the same method as above, recording the volume of sulfuric acid standard titrant consumed, V0.

[0063] (3) Data analysis: The total ester content in the sample is calculated using the following formula.

[0064]

[0065] In the formula

[0066] X1—Total ester content in the sample, expressed as mass concentration (calculated as ethyl acetate), in grams per liter (g / L);

[0067] c1—The actual molar concentration of the sulfuric acid standard titration solution, in moles per liter (mol / L);

[0068] V0—The volume of sulfuric acid standard titration solution consumed by the blank test sample, in milliliters (mL);

[0069] V1 — The volume of sulfuric acid standard titration solution consumed by the sample, in milliliters (mL);

[0070] 88 — Molar mass of ethyl acetate, in grams per mole (g / mol) [M(CH3COOC2H5)=88];

[0071] 50.0 — The volume of sample taken, in milliliters (mL).

[0072] The calculation result is expressed to two decimal places.

[0073] The method for improving ester production capacity through solid-state co-culture fermentation of mixed *Pichia pastoris* PK2 and *Wickham's abnormal* W4, which relates to this invention, is as follows:

[0074] Method 1: Culture methods with different inoculation methods under solid-state fermentation culture conditions

[0075] A method for enhancing ester production capacity through mixed symbiotic fermentation of *Pichia kudriezii* PK2 and *Wickham's abnormal* W4, wherein the method uses sorghum solid medium as the fermentation medium for ester production, and the activated *Pichia kudriezii* PK2 and *Wickham's abnormal* W4 are mixed at a concentration of 10... 6 CFU / mL: 10 6 The inoculation ratio was 5% (CFU / mL). Different inoculation methods were used (PK2 and W4 fermented separately as PK2 and W4, mixed fermentation as PW, simultaneous inoculation with Saccharomyces boulardii BD-2 as B-0, inoculation with Saccharomyces boulardii BD-2 first for 24 hours as B-24, inoculation with Saccharomyces boulardii BD-36 as B-36, and inoculation with Saccharomyces boulardii BD-48 as B-48) to inoculate into sorghum solid-state fermentation medium for ester production culture. The loading volume was 250g / 500mL. After inoculation, the medium was statically cultured at 30℃ for 7 days. After fermentation, the mash was taken for distillation, and the total ester content in the liquor sample was determined.

[0076] Under the above culture conditions, the specific details of the ester production by the mixed co-culture fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 are as follows: Figure 1As can be seen, under the same inoculation time, the ester content of mixed-culture co-fermentation (PW) in each group was significantly higher than that of the two yeast strains fermenting alone. This indicates that different inoculation methods have a certain impact on the ester content of baijiu fermented by ester-producing yeasts, proving that mixed-culture fermentation is more conducive to ester formation. Further comparison of the ester content of mixed-culture fermentation at different inoculation times showed that the lowest ester content was obtained when *Saccharomyces boulardii* BD-2 was inoculated 24 hours prior. However, the highest total ester content (813.64 mg / L) was obtained when *Saccharomyces boulardii* BD-2 was inoculated 36 hours prior. Therefore, the optimal inoculation method is to first inoculate *Saccharomyces boulardii* BD-2 for 36 hours, followed by *Pichia pastoris* PK2 and *Wickham's abnormal* W4 for mixed-culture co-fermentation. The ester content decreased again after *Saccharomyces boulardii* BD-2 was inoculated 48 hours prior. Therefore, three conditions with relatively high total ester content (B-0, B-36, and B-48) were ultimately selected as the three levels for subsequent orthogonal experiment optimization.

[0077] Meanwhile, it can also be seen that the simultaneous fermentation of Kudriazvipichia PK2 and Wickham's abnormal yeast W4 in the method of the present invention can synergistically increase the total ester content in the prepared wine sample, indicating that Kudriazvipichia PK2 and Wickham's abnormal yeast W4 have a synergistic effect in the method of the present invention.

[0078] Method 2: Culture methods with different inoculation ratios under solid-state fermentation conditions

[0079] A method for enhancing ester production capacity through mixed symbiotic fermentation of *Pichia kudrieziva* PK2 and *Wickham's abnormal yeast* W4, wherein the method uses sorghum solid medium as the fermentation medium for ester production, and the activated *Pichia kudrieziva* PK2 and *Wickham's abnormal yeast* W4 are cultured at different bacterial concentration ratios (PK2:W4 = 10:10). 5 CFU / mL: 10 5 CFU / mL, 10 6 CFU / mL: 10 6 CFU / mL, 10 6 CFU / mL: 10 7 CFU / mL, 10 6 CFU / mL: 10 8 The total ester content (CFU / mL) was inoculated into the sorghum solid-state fermentation medium at a 5% inoculation rate, simultaneously with the yeast strain BD-2, for ester production culture. The loading volume was 250g / 500mL. After inoculation, the medium was statically cultured at 30℃ for 7 days. After fermentation, the mash was distilled and the total ester content in the liquor sample was determined.

[0080] Under the above culture conditions, the specific details of the ester production by the mixed co-culture fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 are as follows: Figure 2 It can be seen that different inoculation ratios result in different ester production during fermentation. In mixed-culture fermentation, the ester production initially increases and then decreases with increasing bacterial concentration. When the bacterial concentration ratio of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 is 10... 6 CFU / mL: 10 7 At a concentration of CFU / mL, the ester content reached its highest level, 776.42 mg / L. Therefore, *Pichia pastoris* PK2 and *Wickham's abnormal* W4 were preferred at a concentration of 10... 6 CFU / mL: 10 7 Inoculating the sorghum solid-state fermentation medium with a CFU / mL ratio for mixed-culture co-fermentation yielded the highest total ester content in the liquor sample, which was determined to be the optimal inoculation ratio. The next highest total ester content was achieved with a bacterial concentration ratio of 10:1. 6 CFU / mL: 10 8 CFU / mL and 10 6 CFU / mL: 10 6 CFU / mL CFU / mL. Therefore, the three conditions with relatively high total ester content were ultimately selected (bacterial concentration ratio 10). 6 10 6 10 6 10 7 10 6 10 8 (CFU / mL) was used as the three levels for subsequent orthogonal experiment optimization.

[0081] At the same time, it can also be seen that in the method of the present invention, *Pichia pastoris* PK2 and *Wickham's abnormal yeast* W4 are used at a concentration of 10... 6 CFU / mL: 10 7 Fermentation at a CFU / mL ratio can synergistically increase the total ester content in the prepared wine sample. This demonstrates that in the method of this invention, *Pichia pastoris* PK2 and *Wickham's abnormal* W4, at a concentration of 10... 6 CFU / mL: 10 7 Fermentation is carried out at a CFU / mL ratio, and the two have a synergistic effect.

[0082] Method 3: Cultivation methods with different loading volumes under solid-state fermentation conditions

[0083] A method for enhancing ester production capacity through mixed symbiotic fermentation of *Pichia kudriezii* PK2 and *Wickham's abnormal* W4, wherein the method uses sorghum solid medium as the fermentation medium for ester production, and the activated *Pichia kudriezii* PK2 and *Wickham's abnormal* W4 are mixed at a concentration of 10... 6 CFU / mL: 10 6 The inoculation ratio of CFU / mL was 5%, with different filling amounts (150g / 500mL, 200g / 500mL, 250g / 500mL, 300g / 500mL), and was simultaneously inoculated into the sorghum solid-state fermentation medium with Saccharomyces boulardii BD-2 for ester production culture. After inoculation, the medium was statically cultured at 30℃ for 7 days. After fermentation, the mash was taken for distillation, and the total ester content in the liquor sample was determined.

[0084] Under the above culture conditions, the specific details of the ester production by the mixed co-culture fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 are as follows: Figure 3 It can be seen that different filling volumes result in different ester production during fermentation. The ester content of mixed-culture fermentation first increases and then decreases with increasing filling volume. The lowest ester content (367.32 mg / L) is achieved at a filling volume of 300 g / 500 mL, while the highest (784.82 mg / L) is achieved at a filling volume of 200 g / 500 mL. Therefore, the optimal filling volume is 200 g / 500 mL for mixed-culture co-fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4, as this yields the highest total ester content in the wine sample. The next highest total ester contents are found at filling volumes of 150 g / 500 mL and 250 g / 500 mL. Therefore, 150 g / 500 mL, 200 g / 500 mL, and 250 g / 500 mL were subsequently selected as the three filling volume levels for the orthogonal experiment.

[0085] Method 4: Cultivation methods at different fermentation temperatures under solid-state fermentation conditions.

[0086] A method for enhancing ester production capacity through mixed symbiotic fermentation of *Pichia kudriezii* PK2 and *Wickham's abnormal* W4, wherein the method uses sorghum solid medium as the fermentation medium for ester production, and the activated *Pichia kudriezii* PK2 and *Wickham's abnormal* W4 are mixed at a concentration of 10... 6 CFU / mL: 10 6The inoculation ratio was 5% (CFU / mL), and the inoculation was carried out simultaneously with Saccharomyces boulardii BD-2 in sorghum solid-state fermentation medium for ester production culture. The loading volume was 250g / 500mL. After inoculation, the mash was statically cultured for 7 days at different fermentation temperatures (20℃, 25℃, 30℃, 35℃). After fermentation, the mash was taken for distillation, and the total ester content in the liquor sample was determined.

[0087] Under the above culture conditions, the specific details of the ester production by the mixed co-culture fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 are as follows: Figure 4 It can be seen that fermentation temperature has a significant impact on the ester production of the strains. The ester content of mixed-strain fermentation first increases and then decreases with increasing fermentation temperature. The highest ester content (502.46 mg / L) was observed at 30℃. Therefore, *Pichia pastoris* PK2 and *Wickham's abnormal* W4 were selected for mixed-strain co-fermentation at 30℃, as this temperature resulted in the highest total ester content in the wine sample, which was determined as the optimal fermentation temperature. Secondly, fermentation temperatures of 25℃ and 35℃ also showed relatively high total ester content. Therefore, three conditions with relatively high total ester content (25℃, 30℃, and 35℃) were selected as the three temperature levels in the orthogonal experiment.

[0088] Method 5: In a mixed-culture solid-state simulated fermentation system, orthogonal experiments were used to optimize the cultivation method to improve ester production capacity.

[0089] A method for enhancing ester production capacity through mixed symbiotic fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 is disclosed. The method uses sorghum solid medium as the fermentation medium for ester production. Activated *Pichia pastoris* PK2 and *Wickham's abnormal* W4 are fermented under different conditions and at different levels. Based on the above experiments, a four-factor, three-level orthogonal experiment was conducted with ester production content as the indicator. The specific fermentation conditions and factor levels are shown in Table 1.

[0090] Table 1 Orthogonal Experimental Design Table

[0091]

[0092] According to the orthogonal experimental results, the group with the highest total ester content was A1B2C3D2, reaching 1149.28 mg / L. According to the range analysis, the optimal combination for ester production was A3B2C2D2; however, this combination was not included in the orthogonal experimental groups. Therefore, further verification of the ester production capacity of the two groups was conducted. Based on the results, the optimal combination for ester production capacity of *Pichia pastoris* PK2 and *Wickhamia lanceolata* W4 was A3B2C2D2, i.e., an inoculum concentration ratio of 10:1. 6 CFU / mL: 108 The total ester content reached 1294.62 mg / L, with a CFU / mL (PK2:W4) concentration, a loading volume of 200 g / 500 mL, an inoculation time of 36 h, a fermentation temperature of 30 °C.

[0093] Example 2: Application of Pichia pastoris PK2 and Wickham's abnormal yeast W4 in in situ fermentation

[0094] The suspensions of *Kudela sibirica* PK2 and *Wickham's abnormal* W4 were prepared at a bacterial concentration of 10. 6 CFU / mL: 10 8 The inoculation ratio was mixed at a rate of CFU / mL, i.e., the concentration of Pichia pastoris PK2 was 2 × 10⁻⁶. 6 CFU / mL, the concentration of abnormal Wickham yeast W4 was 2×10⁻⁶. 8 At an inoculation time of B-36 (36 hours of stacked fermentation), a 5% inoculation rate of the bacterial suspension was added to 50 kg of fermented mash after six rounds of cooling and mixing with the starter culture. The mixture was then evenly distributed into two pure cotton gauze bags and placed in different locations as the experimental group. An equal volume of physiological saline was added to the same 50 kg of fermented mash after six rounds of cooling and mixing with the starter culture, and this mixture was also evenly distributed into two pure cotton gauze bags as the control group. Samples of stacked fermented mash and fermented mash from different locations during the actual fermentation process were mixed and used as the blank group. After the stacked fermentation and fermentation in the fermentation pits were completed, the total ester content of the liquor samples was measured, the alcohol yield was calculated, and the types and contents of volatile substances in the liquor samples were analyzed.

[0095] The total ester content in the sample was determined by the saponification reflux method, and the specific method was carried out according to the method described in Example 1 above.

[0096] After fermentation, the mash is distilled in small pots, and the specific method is as follows:

[0097] (1) Sample preparation: Weigh 5 kg of mash, add about 20% of the mash mass of rice husks, mix well and pour into a small steamer, install the gas delivery pipe, add water and seal, insert the water pump, connect the condenser reflux device, and record the alcohol content of the distilled liquor.

[0098] (2) Method for calculating alcohol yield:

[0099] The analysis of flavor compounds in the wine samples was performed using gas chromatography-mass spectrometry (GC-MS), and the specific method is as follows:

[0100] (1) Sample pretreatment: Headspace solid-phase microextraction (SPME) was used. 8 mL of wine sample was diluted to 10% (v / v) and poured into a headspace vial. 3 g NaCl and 10 μL of internal standard (876 mg / L n-amyl acetate and 890 mg / L menthol) were added. A magnetic stirrer was placed in the vial, and the vial was sealed. The sample was placed in a constant temperature (60℃) water bath for 10 min to equilibrate. The aged 50 / 30 μm DVB / CAR / PDMS extraction head was inserted into the headspace vial for adsorption for 60 min, with shaking at 400 rpm. The vial was then transferred to a high-temperature vaporization chamber for desorption for 5 min, followed by gas chromatography-mass spectrometry (GC-MS) analysis.

[0101] (2) Gas chromatography conditions: The capillary column was DB-WAX with a specification of (60m×0.25mm×0.50μm); the carrier gas was high-purity He (purity>99.999%), the carrier gas flow rate was 1mL / min, the column temperature program was 40℃, held for 3min, increased to 60℃ at a rate of 2℃ / min, held for 2min, increased to 100℃ at a rate of 3℃ / min, increased to 230℃ at a rate of 4℃ / min, and held for 12min, the total run time was 72.833min, the injection mode was splitless injection, the injection port temperature was 250℃, and the auxiliary heating zone temperature was 280℃.

[0102] (3) Mass spectrometry conditions: quadrupole temperature 150℃, ion source temperature 230℃, solvent delay 5min, acquisition mode full scan, acquisition range 35~500amu.

[0103] (4) Preparation and establishment methods of standard solutions:

[0104] Preparation of standard solutions: Weigh or transfer appropriate amounts of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate and other ester compounds as standards, dissolve them in chromatographic grade anhydrous ethanol and dilute to 10 mL in a volumetric flask to prepare stock solutions of each standard. Store at 4°C for later use.

[0105] Establishment of the standard curve: Using a 10% (v / v) ethanol-water solution as the solvent, add an appropriate concentration of the compound standard, and sequentially dilute to six concentration gradients. Extraction is performed using headspace solid-phase microextraction, followed by GC-MS analysis. The linear standard curve of the compound is calculated by plotting the ratio of the known compound concentration to the internal standard concentration on the x-axis and the ratio of the known compound quantitative ion peak area to the internal standard quantitative ion peak area on the y-axis.

[0106] (5) Data Analysis

[0107] Qualitative analysis: Mass spectra of detected volatile substances were used to search for unknown compounds using GC-MS data analysis software. The results were compared with the NIST11 spectral library, and only those with a match score greater than 800 (maximum value 1000) were reported.

[0108] Quantitative analysis: External standard method and internal standard method were used to quantitatively analyze volatile substances in fermented wine samples.

[0109] Sensory evaluation was conducted in accordance with GB / T 10345-2022 "Analytical Methods for Baijiu" and relevant literature. The specific methods are as follows:

[0110] A 10-member sensory evaluation team was established for baijiu (Chinese liquor). All sensory evaluators (aged 20-50) were in good health and had received training. The sensory characteristics of the baijiu samples were described using the "baijiu flavor wheel" sensory terminology. The aroma attributes (fruity, grainy, caramel, sweet) and taste attributes (persistence, smoothness, spiciness, bitterness, sourness) of the baijiu samples were determined, and the intensity of perception was described on a 5-point scale, with "0" representing no sensation and "5" representing the strongest. Each sensory evaluator evaluated each sample three times. The evaluation result for a single sample was the average of the three evaluation results from each sensory evaluator, and a sensory profile diagram was drawn.

[0111] Under the above culture conditions, the volatile substances in the wine samples were compared between the experimental group (wine mash supplemented with *Pichia pastoris* PK2 suspension + *Wickham's abnormal* W4 suspension), the control group (wine mash supplemented with the same volume of physiological saline as the experimental group), and the blank group (wine mash from actual brewing production without any samples added). The results of some ester compound analyses are shown in Table 2. According to the results, the experimental group samples showed a richer variety and quantity of volatile compounds. Among them, the difference in the variety and quantity of esters was the most significant, with 81 ester compounds detected in the experimental group and 60 ester compounds detected in the blank group. Compared to the control group, the experimental group showed a 2.26-fold increase in total ester content (11283.83 mg / L), with ethyl hexanoate at 2395.64 mg / L (1.95-fold increase), ethyl acetate at 1297.33 mg / L (3.1-fold increase), and ethyl lactate at 4659.97 mg / L (2.25-fold increase). Furthermore, the alcohol yield in the experimental group was 2.81 times higher than in the control group. Sensory evaluation results showed that, compared to the control group, the experimental group had higher scores in fruit, sweet, and caramel aromas, weaker irritation and spiciness, and a smoother, more persistent taste. See the detailed sensory evaluation chart below. Figure 5According to the present invention, the symbiotic fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 significantly increases the content of ester compounds in baijiu fermentation, and can be used as functional microorganisms in the baijiu brewing process to play a role in ester enhancement and aroma enhancement. Based on existing technology, in studies using ester-producing yeasts to produce baijiu from leftover grains, the alcohol yield increased from 6.57% to 12.90%, increasing the content of esters, alcohols, etc. in the baijiu; in studies using *Wickham's abnormal* to simulate the fermentation of special-aroma baijiu, the content of ethyl esters was effectively increased, with ethyl butyrate, ethyl lactate, and ethyl hexanoate increasing by 0.54 mg / L, 3.27 mg / L, and 0.58 mg / L, respectively. The co-fermentation of *Pichia pastoris* PK2 and *Wickham's abnormal* W4 provided in this invention not only increases the alcohol yield but also significantly improves the total ester content, ethyl acetate, ethyl hexanoate, and ethyl lactate content in baijiu. Sensory evaluation reveals a more intense fruity aroma, a smoother taste, and higher acceptability. Therefore, the culture conditions for the two ester-producing yeasts disclosed in this invention can be applied to traditional food fermentation processes, significantly improving the flavor and quality of baijiu, and have practical application significance.

[0112] Table 2 Information on some ester compounds in the in-situ fermentation experiment.

[0113]

[0114]

[0115] Note: "-" indicates not detected.

[0116] Example 3 Screening and Identification of Pichia kudrica PK2

[0117] I. Screening and isolation of Pichia pastoris PK2:

[0118] Pichia kudriavzevii, whose name is PK2 and taxonomic name is Pichia kudriavzevii, has the accession number CGMCC No.29250, the deposit date is December 6, 2023, and the depositary institution is the China General Microbiological Culture Collection Center, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0119] YPD solid culture medium composition: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar 20 g / L, the remainder being water. Preparation method: Dissolve all the above components in 1 L of distilled water, then dispense into 100 mL Erlenmeyer flasks and sterilize at 115 °C for 20 min. To eliminate bacterial interference during the screening process, add 60 μg / mL of chloramphenicol.

[0120] The composition of YPD liquid culture medium is the same as that of YPD solid culture medium, except that no agar is added.

[0121] Take 10g of soy sauce-flavored baijiu mash into a 250mL sterile Erlenmeyer flask, add 90mL of sterile physiological saline, seal the flask mouth with a breathable sealing film, and activate at 30℃ for 30min; take out the bacterial suspension and dilute it with sterile water to obtain a diluted solution, spread it on YPD medium, and incubate at 30℃ for 1-2 days. Isolate and streak-purify strains with typical yeast colony characteristics (purify 2-3 times depending on the isolation effect) until a single strain is obtained.

[0122] Among them, the colony morphology of strain PK2 is milky white, opaque, wrinkled, dull, with velvety edges and a raised center; it is moist and easily picked up. Under a light microscope, the cell morphology is elliptical, and the reproductive mode is budding. Colony morphology and light microscope morphology are as follows: Figure 6 .

[0123] II. Identification of Pichia pastoris PK2

[0124] Pure PK2 strains were inoculated into YPD liquid medium and activated by incubation at 30°C for 24 hours. An appropriate amount of the activated bacterial culture was collected by centrifugation. Genomic DNA was extracted from the pure cultured target strain using a kit. The conserved region of its 26S rDNA gene was amplified using upstream primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3' and downstream primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3', and then sent to a sequencing company for sequencing analysis.

[0125] The PCR reaction conditions were: denaturation at 95°C for 15 seconds; annealing at 55°C for 15 seconds; extension at 72°C for 15 seconds; 30 cycles, with an extension at 72°C for 10 minutes.

[0126] Sequence alignment was performed in the GenBank database using NCBI's BLAST function. See the phylogenetic tree below. Figure 7 Analysis determined that strain PK2 was classified as *Pichia kudriavzevii*, and therefore named it *Pichia kudriavzevii*. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 29250.

[0127] The 26S rDNA gene sequence of PK2 is as follows (SEQ ID NO.1):

[0128] .

[0129] Example 4: Screening and Identification of Abnormal Wickham Saccharomyces W4

[0130] I. Screening and isolation of abnormal Wickham yeast W4:

[0131] The species is *Wickerhamomyces anomalus*, with the name W4 and taxonomic name W4. Its accession number is CGMCC No. 29249, the accession date is December 6, 2023, and the depository is China General Microbiological Culture Collection Center, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0132] YPD solid culture medium composition: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar 20 g / L, the remainder being water. Preparation method: Dissolve all the above components in 1 L of distilled water, then dispense into 100 mL Erlenmeyer flasks and sterilize at 115 °C for 20 min. To eliminate bacterial interference during the screening process, add 60 μg / mL of chloramphenicol.

[0133] The composition of YPD liquid culture medium is the same as that of YPD solid culture medium, except that no agar is added.

[0134] Take 10g of soy sauce-flavored baijiu mash into a 250mL sterile Erlenmeyer flask, add 90mL of sterile physiological saline, seal the flask mouth with a breathable sealing film, and activate at 30℃ for 30min; take out the bacterial suspension and dilute it with sterile water to obtain a diluted solution, spread it on YPD medium, and incubate at 30℃ for 1-2 days. Isolate and streak-purify strains with typical yeast colony characteristics (purify 2-3 times depending on the isolation effect) until a single strain is obtained.

[0135] Among them, strain W4 colonies on solid YPD medium are cream-colored, smooth, glossy, opaque, with neat edges, a raised center, and are moist and easily picked up; under a light microscope, the cell morphology is observed to be oval, exhibiting budding reproduction. See details... Figure 8 .

[0136] II. Identification of Abnormal Wickham Saccharomyces W4

[0137] Pure strain W4 was inoculated into YPD liquid medium and activated by incubation at 30°C for 24 hours. An appropriate amount of the activated bacterial solution was collected by centrifugation. Genomic DNA of the pure cultured target strain was extracted using a kit. The conserved region of its 26S rDNA gene was amplified using the upstream primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3' and the downstream primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3', and then sent to a sequencing company for sequencing analysis.

[0138] The PCR reaction conditions were: denaturation at 95°C for 15 seconds; annealing at 55°C for 15 seconds; extension at 72°C for 15 seconds; 30 cycles, with an extension at 72°C for 10 minutes.

[0139] Sequence alignment was performed in the GenBank database using NCBI's BLAST function. See the phylogenetic tree below. Figure 9Analysis determined that strain W4 was classified as Wickerhamomyces anomalus, and therefore named it Wickerhamomyces anomalus. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29249.

[0140] The 26S rDNA gene sequence of W4 is as follows (SEQ ID NO.2):

[0141] .

[0142] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for improving the ester content during fermentation by co-culturing two ester-producing yeast strains, characterized in that: The method uses Kudria zweibichi yeast. Pichia kudriavzevii PK2, Abnormal Wickham Yeast ( Wickerhamomyces anomalus W4, and both strains fermented simultaneously; The aforementioned *Pichia kudrica* PK2, with accession number CGMCC No. 29250, is deposited at the China General Microbiological Culture Collection Center (CGMCC). The abnormal Wickham yeast W4, whose name is W4, is classified as follows: Wickerhamomyces anomalus The accession number is CGMCC No. 29249, and the depository is the China General Microbiological Culture Collection Center.

2. The method according to claim 1, characterized in that: Specifically, the steps include the following: Using sorghum solid medium as the fermentation ester production medium, with a loading volume of 200 g / 500 mL, after inoculating with *Bretschneidera sinensis* BD-2 for 36 h, activated *Kudelazvichia PK2* and *Wickhamia lanceolata* W4 were added at a concentration of 10... 6 CFU / mL: 10 8 The inoculation ratio was 5% CFU / mL, and the ester production culture was carried out by inoculating the sorghum solid-state fermentation medium. After inoculation, the medium was incubated at 30 °C for 7 days to complete the fermentation.

3. The method according to claim 2, characterized in that: The total ester content obtained by the method reached 1294.62 mg / L.

4. The method according to claim 1, characterized in that: The method described is a method for improving the quality of baijiu by co-culturing two ester-producing yeast strains, including the following steps: Suspensions of *Kudriazwibbi* PK2 and *Wickham's abnormal* W4 were prepared at 10... 6 10 8 The inoculation ratio was mixed, that is, the concentration of Pichia pastoris PK2 was 2×10⁻⁶. 6 CFU / mL, the concentration of abnormal Wickham yeast W4 was 2×10⁻⁶. 8 After fermentation for 36 hours, the bacterial suspension was inoculated into the fermented mash after six rounds of cooling and mixing with the starter culture at a rate of 5% (CFU / mL). The fermentation of the mash was allowed to continue naturally until the fermentation in the fermentation pits was completed.

5. The method according to claim 4, characterized in that: The total ester content obtained by the method was 11283.83 mg / L.

6. The application of the method as described in any one of claims 1 to 5 in the field of food fermentation.

7. The application of the method as described in any one of claims 1 to 5 in in situ fermentation.

8. The application of the method as described in any one of claims 1 to 5 in the fermentation of baijiu (Chinese liquor).

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