Pichia kudriavzevii with ethanol tolerance, high yield of ethanol and fatty acid ethyl ester and low yield of isoamyl alcohol and application of pichia kudriavzevii in production of rosa sterilis fruit wine

Abstract

The invention discloses pichia kudriavzevii XJ-YPPKU067-1 with ethanol tolerance, high yield of ethanol and fatty acid ethyl ester and low yield of isoamyl alcohol and application of the pichia kudriavzevii XJ-YPPKU067-1 in production of rosa sterilis fruit wine, and belongs to the technical field of microbial fermentation. The pichia kudriavzevii XJ-YPPKU067-1 disclosed by the invention is separated and screened from Maotai-flavor liquor high-temperature yeast, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31175. The invention further discloses a preparation method of the pichia kudriavzevii XJ-YPPKU067-1. The strain can grow in an environment with ethanol volume percentage concentration of 15%, and has the advantages of high yield of ethanol and a variety of fatty acid ethyl esters and no production of isoamyl alcohol. When the pichia kudriavzevii XJ-YPPKU067-1 is applied to preparation of the fruit wine, the alcohol content of the fruit wine and the content of fatty acid ethyl ester can be improved, the content of harmful isoamyl alcohol is reduced, and then the quality of the fruit wine is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and relates to a Kudriazwiipichii yeast that is resistant to ethanol, produces high levels of ethanol and fatty acid ethyl esters, and produces low levels of isoamyl alcohol, and its application in the production of golden prickly pear fruit wine. Background Technology

[0002] Ethanol plays a crucial role in the brewing industry. The ethanol content in alcoholic beverages determines their alcohol content and strength. It also influences the overall aroma and flavor of the beverage. While not a flavor or aroma compound itself, ethanol is not only a precursor to esters and other aroma compounds in alcoholic beverages but also affects the release of their aromas. In alcohol production, yeast is both the dominant microbial genus and a key producer of ethanol, making significant contributions to the fermentation rate and flavor quality of alcoholic beverages. However, ethanol, a byproduct of fermentation, affects the stability of yeast membranes and metabolic pathways. Its gradual accumulation can hinder yeast growth and metabolism, causing inhibitory and toxic effects. Therefore, the ethanol tolerance of a yeast strain is closely related to its ethanol yield. In light of this, screening for ethanol-tolerant and high-yielding yeast strains and applying them to fruit wine production can shorten the production cycle and reduce production costs, which has profound implications for the fruit wine industry.

[0003] The aroma of fruit wine primarily originates from esters, which impart fruity and floral notes. Most esters are synthesized by yeast or produced through esterification reactions of acids and alcohols in the wine, catalyzed by esterifying enzymes. Among these, ethyl fatty acid esters contribute significantly to the flavor of fruit wine. During anaerobic fermentation, yeast releases large amounts of fatty acids and their related ethyl esters, which are crucial for the aroma and quality of fermented fruit wine. Most ethyl fatty acid esters have low threshold values ​​and primarily contribute fruity or floral aromas, thus enhancing the pleasant and aromatic qualities of the wine. Therefore, increasing the content of ethyl fatty acid esters in fruit wine is a critical issue that urgently needs to be addressed in the fruit wine industry, and it has profound implications for improving the quality of fruit wine.

[0004] Isoamyl alcohol in fruit wine mainly comes from yeast metabolism. It's a double-edged sword in fruit wine; while it imparts sweetness and fruitiness, and in appropriate amounts can improve flavor, making the wine fuller-bodied and more harmonious, excessive isoamyl alcohol can not only cause bitterness and harshness but also potentially damage the drinker's nervous system, causing headaches and a feeling of "headache." Therefore, how to rationally control the isoamyl alcohol content in fruit wine is a crucial issue that urgently needs to be addressed in the fruit wine industry, and it has profound significance for improving the quality of fruit wine. Summary of the Invention

[0005] In some embodiments, the present invention provides a *Pichia kudriavzevii* yeast, named *Pichia kudriavzevii* XJ-YPPKU067-1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31175.

[0006] In some embodiments, the *Kudriazwibichthys* strain does not produce isoamyl alcohol.

[0007] In some implementations, the *Kudriazwibichthys* yeast is derived from Daqu (a type of starter culture).

[0008] In some implementations, the Daqu (a type of starter culture) is derived from high-temperature Daqu.

[0009] In some implementations, the *Kudriazwibichthys* strain is ethanol-resistant.

[0010] In some embodiments, the *Pichia pastoris* species produces high levels of ethanol.

[0011] In some implementations, the *Kudriazwibichthys* strain produces high levels of fatty acid ethyl esters.

[0012] In some embodiments, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl lactate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate.

[0013] In some embodiments, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate.

[0014] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 20% ethanol (v / v).

[0015] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 15% ethanol (v / v).

[0016] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 12% ethanol (v / v).

[0017] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 9% ethanol (v / v).

[0018] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 6% ethanol (v / v).

[0019] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 3% ethanol (v / v).

[0020] In some embodiments, the present invention provides a method for screening Pichia kudrica zwichizosacchari, comprising the following steps:

[0021] (1) After crushing the Daqu of sauce-flavored liquor and shaking it in water, spread it on the lower layer of triphenyltetrazolium chloride (TTC) culture medium and place it in an incubator for incubation;

[0022] (2) Cover the colonies with TTC upper culture medium, place them in an incubator for incubation, and select colonies;

[0023] (3) The selected colonies were inoculated into YPD liquid medium, shaken and cultured, and then the bacterial solution was inoculated into ethanol-resistant medium for culture and the strains that could grow under high concentration of ethanol were screened.

[0024] (4) Take the bacterial culture of the strain that can grow under high concentration of ethanol, inoculate it into high sugar YPD liquid fermentation medium, and after shaking culture, detect the content of ethanol, fatty acid ethyl ester and isoamyl alcohol in the fermentation broth of each strain to obtain the target strain that produces high ethanol and fatty acid ethyl ester and low isoamyl alcohol.

[0025] In some implementations, the temperature of the incubator is 20-30°C.

[0026] In some implementations, the shaking culture is a shaking culture using a shaker.

[0027] In some implementations, the rotation speed of the oscillation culture is 50-250 rpm.

[0028] In some embodiments, the rotation speed of the oscillation culture is 100-180 rpm.

[0029] In some embodiments, the culture temperature for the oscillation culture is 20-30°C.

[0030] In some implementations, in step (1), the incubation time in the incubator is 1-5 days.

[0031] In some implementations, in step (1), the incubator is used for 2-5 days.

[0032] In some implementations, in step (2), the incubation time in the incubator is 1-48 hours.

[0033] In some implementations, in step (2), the incubation time in the incubator is 1-30 hours.

[0034] In some implementations, in step (2), the incubation time in the incubator is 1-12 hours.

[0035] In some implementations, in step (3), the selected colonies are cultured in YPD liquid medium with shaking for 1-5 days.

[0036] In some implementations, in step (3), the selected colonies are cultured in YPD liquid medium with shaking for 1-3 days.

[0037] In some implementations, in step (3), the bacterial culture is cultured in an ethanol-resistant medium for 1-120 hours.

[0038] In some implementations, in step (3), the bacterial culture is cultured in an ethanol-resistant medium for 1-96 hours.

[0039] In some implementations, in step (4), the bacterial culture is cultured in high-sugar YPD liquid fermentation medium for 1-5 days.

[0040] In some implementations, in step (4), the bacterial culture is cultured in high-sugar YPD liquid fermentation medium for 2-5 days.

[0041] In some implementations, the selected colonies are prominent red colonies.

[0042] In some embodiments, the selected colonies are colonies with typical yeast characteristics.

[0043] In some embodiments, the composition of the TTC upper culture medium includes: TTC 0.1-5.0 g / L, glucose 2-30 g / L, agar 5-30 g / L, and water as the solvent.

[0044] In some embodiments, the composition of the TTC upper culture medium includes: TTC 0.5 g / L, glucose 5 g / L, agar 15 g / L, and water as the solvent.

[0045] In some embodiments, the TTC lower layer culture medium comprises: magnesium sulfate 0.2-1.0 g / L, potassium dihydrogen phosphate 0.2-2.0 g / L, yeast extract powder 0.5-30 g / L, peptone 1-30 g / L, glucose 2-30 g / L, agar 5-30 g / L, water as solvent, and pH 4-7.

[0046] In some embodiments, the TTC lower culture medium comprises: 0.4 g / L magnesium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.5 g / L yeast extract, 2 g / L peptone, 10 g / L glucose, 20 g / L agar, with water as the solvent and a pH of 5.5.

[0047] In some embodiments, the YPD liquid culture medium comprises: 0.5-30 g / L yeast extract, 1-30 g / L peptone, 10-200 g / L glucose, and water as the solvent.

[0048] In some embodiments, the YPD liquid culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and water as the solvent.

[0049] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 20%.

[0050] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 15%.

[0051] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 12%.

[0052] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 9%.

[0053] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 6%.

[0054] In some embodiments, the ethanol-tolerant culture medium has an ethanol volume percentage concentration of 0% to 3%.

[0055] In some embodiments, the ethanol-resistant culture medium comprises: 0.5-30 g / L yeast extract, 1-30 g / L peptone, 10-200 g / L glucose, and water as the solvent.

[0056] In some embodiments, the ethanol-resistant culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and water as the solvent.

[0057] In some embodiments, the high-sugar YPD liquid fermentation medium comprises: 0.5-30 g / L yeast extract, 1-30 g / L peptone, 10-200 g / L glucose, and water as the solvent.

[0058] In some embodiments, the high-sugar YPD liquid fermentation medium comprises: 10 g / L yeast extract, 20 g / L peptone, 100 g / L glucose, and water as the solvent.

[0059] In some embodiments, the *Pichiakudriavzevii* yeast is named *Pichiakudriavzevii* XJ-YPPKU067-1 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31175.

[0060] In some embodiments, the present invention provides *Kudriazwibichthys* obtained by the method.

[0061] In some embodiments, the present invention provides a fermentation broth obtained by fermentation of Pichia kudriavzevii, wherein the Pichia kudriavzevii is named Pichia kudriavzevii XJ-YPPKU067-1 and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 31175.

[0062] In some embodiments, the *Kudriazwibichthys* strain does not produce isoamyl alcohol.

[0063] In some implementations, the *Kudriazwibichthys* yeast is derived from Daqu (a type of starter culture).

[0064] In some implementations, the Daqu (a type of starter culture) is derived from high-temperature Daqu.

[0065] In some implementations, the *Kudriazwibichthys* strain is ethanol-resistant.

[0066] In some implementations, the *Kudriazwibichthys* strain produces high levels of ethanol.

[0067] In some implementations, the *Kudriazwibichthys* strain produces high levels of fatty acid ethyl esters.

[0068] In some embodiments, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl lactate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate.

[0069] In some embodiments, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate.

[0070] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 20% ethanol (v / v).

[0071] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 15% ethanol (v / v).

[0072] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 12% ethanol (v / v).

[0073] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 9% ethanol (v / v).

[0074] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 6% ethanol (v / v).

[0075] In some embodiments, the *Kudriazwibichthys* yeast grows well under conditions of 0% to 3% ethanol (v / v).

[0076] In some embodiments, the present invention provides a microbial inoculum containing the aforementioned *Kudriazwibichthys* and / or the aforementioned fermentation broth.

[0077] In some embodiments, the microbial agent is in liquid or solid form.

[0078] In some embodiments, the present invention provides the use of the *Kudriazwibichthys* or the fermentation broth in the preparation of microbial agents.

[0079] In some embodiments, the microbial agent is in liquid or solid form.

[0080] In some embodiments, the present invention provides the use of the *Kudriazwibichthys* yeast, the fermentation broth, or the microbial agent in the preparation of fermentation raw materials, wine, or food.

[0081] In some implementations, the alcoholic beverage includes brewed, distilled, or blended beverages.

[0082] In some implementations, the wine includes fruit wine.

[0083] In some implementations, the wine is golden prickly pear fruit wine.

[0084] In some embodiments, the present invention provides a method for preparing wine, the method comprising using the *Kudriazwich.* yeast, the fermentation broth, or the microbial agent during the winemaking process.

[0085] In some implementations, the alcoholic beverage includes brewed, distilled, or blended beverages.

[0086] In some implementations, the wine includes fruit wine.

[0087] In some implementations, the wine is golden prickly pear fruit wine.

[0088] In some implementations, the method is a method for increasing the types of fatty acid ethyl esters in wine.

[0089] In some embodiments, the method is a method for increasing the total content of fatty acid ethyl esters in wine.

[0090] In some implementations, the method is a method for reducing isoamyl alcohol in wine.

[0091] In some implementations, the method is a method for increasing the ethanol content in wine.

[0092] In some embodiments, the present invention provides a wine prepared by the preparation method described above.

[0093] In some implementations, the alcoholic beverage includes brewed, distilled, or blended beverages.

[0094] In some implementations, the wine includes fruit wine.

[0095] In some implementations, the fruit wine is golden prickly pear fruit wine.

[0096] In some embodiments, this invention screened a strain of *Pichia pastoris* XJ-YPPKU067-1 from high-temperature koji (fermentation starter) of soy sauce-flavored baijiu, which is resistant to ethanol, produces high levels of ethanol and fatty acid ethyl esters, and produces low levels of isoamyl alcohol. This strain can grow in an environment with an ethanol content of 15%, and exhibits the ability to produce high levels of ethanol and fatty acid ethyl esters while producing low levels of isoamyl alcohol. During the fermentation experiment of *Pyracantha fortuneana* fruit wine, its advantages of ethanol resistance, high production of ethanol and fatty acid ethyl esters, and low production of isoamyl alcohol were fully demonstrated.

[0097] In some embodiments, this invention can help solve problems such as low alcohol content and poor flavor in fruit wine production caused by yeast's weak ability to produce ethanol and fatty acid ethyl esters and high isoamyl alcohol content. The Kudria zwei Pichia yeast XJ-YPPKU067-1 has significant application value in fruit wine production, helping to increase the alcohol content and flavor substance content of fruit wine, reduce the content of harmful isoamyl alcohol, and thus improve the quality of fruit wine, bringing significant economic benefits to fruit wine enterprises.

[0098] The preservation information of the *Pichiakudriavzevii* strain of the present invention is as follows: The *Pichiakudriavzevii* strain of the present invention is named XJ-YPPKU067-1, and its classification name is *Pichiakudriavzevii*. It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 31175. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC, and the name of the depository is China General Microbiological Culture Collection Center. Attached Figure Description

[0099] Figure 1 The growth curves of the target strain in culture media with different ethanol volume percentages are shown.

[0100] Figure 2 Colony (A) and microscopic morphology (B) of the target strain under suitable conditions.

[0101] Figure 3 Phylogenetic tree of Pichia kudrica XJ-YPPKU067-1.

[0102] The preservation information of the *Pichiakudriavzevii* strain of the present invention is as follows: The *Pichiakudriavzevii* strain of the present invention is named XJ-YPPKU067-1, and its classification name is *Pichiakudriavzevii*. It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 31175. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC, and the name of the depository is China General Microbiological Culture Collection Center. Detailed Implementation

[0103] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0104] The preservation information of the *Pichiakudriavzevii* strain of the present invention is as follows: The *Pichiakudriavzevii* strain of the present invention is named XJ-YPPKU067-1, and its classification name is *Pichiakudriavzevii*. It was deposited on July 4, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 31175. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depository code is CGMCC, and the name of the depository is China General Microbiological Culture Collection Center.

[0105] The technical terms used in this field are explained below:

[0106] High-temperature Daqu: High-temperature Daqu is made from wheat through artificial stacking and natural fermentation. It is usually used as a saccharification and fermentation agent for brewing sauce-flavored Baijiu. It is rich in a variety of microbial systems and various brewing enzyme systems. The fermentation temperature of high-temperature Daqu is high, reaching up to 60-65℃.

[0107] Inoculation: The process of inoculating microorganisms onto a suitable carrier for their growth and reproduction.

[0108] Inoculation volume (%): The ratio of the volume of bacterial culture transferred to the total volume of the culture medium after inoculation.

[0109] Lag phase: When a small amount of bacterial strain is inoculated into a liquid culture medium, it usually does not immediately begin to grow and reproduce. The cell number hardly increases, and may even decrease slightly, with the population growth rate approaching zero. This period is called the lag phase. The length of the lag phase depends on the bacterial strain, its age, the inoculum size, and the culture conditions.

[0110] Volume percentage concentration (V / V): Commonly used for alcoholic beverages, it refers to the volume of solute (in milliliters) in 1 milliliter of solution.

[0111] Sugar content: The number of grams of sucrose dissolved in 100 grams of aqueous solution at 20°C.

[0112] Weight loss (fermentation weight loss): During fermentation, microorganisms carry out metabolic activities and produce gases such as carbon dioxide. These gases escape from the fermentation broth, resulting in a decrease in the mass of the fermentation broth.

[0113] In the embodiments described herein, the materials and reagents used include: glucose, agar, yeast extract powder, peptone, yeast carbon-based basal medium (YCB), yeast nitrogen-based basal medium (YNB), magnesium sulfate, potassium dihydrogen phosphate, triphenyltetrazolium chloride (TTC), sulfuric acid, sucrose, maltose, soluble starch, fructose, galactose, xylose, arabinose, lactose, ammonium chloride, ammonium sulfate, ethanol (chromatographic grade), isoamyl alcohol (chromatographic grade), ethyl acetate (chromatographic grade), and ethyl lactate (chromatographic grade). ), ethyl butyrate (chromatographic grade), ethyl hexanoate (chromatographic grade), ethyl heptanoate (chromatographic grade), ethyl octanoate (chromatographic grade), ethyl nonanoate (chromatographic grade), ethyl decanoate (chromatographic grade), ethyl phenylacetate (chromatographic grade), ethyl isovalerate (chromatographic grade), ethyl laurate (chromatographic grade), ethyl palmitate (chromatographic grade), ethyl 3-octenate (chromatographic grade), ethyl trans-cinnamate (chromatographic grade), ethyl trans-3-hexenoate (chromatographic grade), octanol (chromatographic grade), pectinase, potassium metabisulfite.

[0114] Among them, the high-temperature Daqu (a type of starter culture) was obtained by pulverizing the finished product after six months of storage at Guizhou Xijiu Co., Ltd.; the golden prickly pear juice was purchased from Guizhou Tianci Guibao Food Co., Ltd.; the glucose was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 63005518; the agar was purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number 9002-18-0; the peptone was purchased from Beijing Aoboxing Biotechnology Co., Ltd., item number 01-001; the yeast extract powder was purchased from OXOID, item number LP0021B; the yeast carbon source basal medium (YCB) was purchased from Shandong Tuopu Bioengineering Co., Ltd., item number M4199B; and the yeast nitrogen source basal medium (YNB) was purchased from Qingdao Gaoke. The following products were purchased from various suppliers: Yuanhaibo Biotechnology Co., Ltd. (item number HB7040-1); Magnesium sulfate (item number 1001-0557) from Tianjin Yongda Chemical Reagent Co., Ltd.; Potassium dihydrogen phosphate (item number 10017618) from Shanghai Hushi Laboratory Equipment Co., Ltd.; TTC (item number BN30765-25g) from Beijing Bairuiji Biotechnology Co., Ltd.; Sulfuric acid (item number BH0065) from Tianjin Jiangtian Chemical Technology Co., Ltd.; Sucrose (item number 10021463) from Shanghai Hushi Laboratory Equipment Co., Ltd.; Maltose (item number 63008534) from Sinopharm Chemical Reagent Co., Ltd.; and Soluble starch (item number [missing information]) from Shanghai Yuanye Biotechnology Co., Ltd. S11007-500g; Fructose was purchased from Tianjin Kemei Chemical Reagent Co., Ltd., item number KMO0008; Galactose was purchased from Sinopharm Chemical Reagent Co., Ltd., item number 63004434; Xylose was purchased from Sinopharm Chemical Reagent Co., Ltd., item number 63012034; Arabinose was purchased from Shanghai Huzhen Industrial Co., Ltd., item number 147-81-9; Lactose was purchased from Beijing Shuangxuan Microbial Culture Medium Products Factory, item number 02-23A; Ammonium chloride was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 10001518; Ammonium sulfate was purchased from Shanghai Hushi Laboratory Equipment Co., Ltd., item number 10002918; Ethanol (chromatographic grade) was purchased from Shanghai Ala The following products were purchased from Aladdin Biochemical Technology Co., Ltd.: E111993-500ml (Catalog No.); Isoamyl alcohol (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No. M116201-5ml); Ethyl acetate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No. E116134-500ml); Ethyl lactate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No. E108228-5ml); Ethyl butyrate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No. E101116-5ml); Ethyl hexanoate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No. E103497-5ml).Ethyl heptaate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E100047-5ml; ethyl octanoate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E101042-5ml; ethyl nonanoate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E156541-100ml; ethyl decanoate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E101444-5ml; ethyl phenylacetate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E103494-5ml; ethyl isovalerate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E108585-1ml; ethyl laurate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E113259-10. 0ml; Ethyl palmitate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number E103506-5ml; Ethyl 3-octenate (chromatographic grade) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., item number E13000-5g; Ethyl trans-cinnamate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number E303690-100g; Ethyl trans-3-hexenoate (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number E117700-5g; 2-octanol (chromatographic grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number O111528-100ml; Pectinase was purchased from DSM (China) Co., Ltd.; Potassium metabisulfite was purchased from Zibo Great Wall Chemical Plant; Angel Yeast SY (hereinafter referred to as "Angel Fruit Wine Yeast SY") was sourced from Angel Yeast Co., Ltd.

[0115] In this embodiment, the instruments and equipment used include: a high-performance liquid chromatograph (HPLC) with a differential refractive index detector (DRPD), a gas chromatograph (GC) with a flame ionization detector (FID), a gas chromatography-mass spectrometry (GC-MS) system, and a growth curve analyzer. Specifically, the HPLC with DDR was purchased from Agilent Technologies, USA, model Agilent 1260 Infinity; the GC with FID was purchased from Agilent Technologies, USA, model Agilent GC8890; the GC-MS system was purchased from Agilent Technologies, USA, model GC 7890-5975MSD; and the growth curve analyzer was purchased from OYGrowth CurvesAb Ltd, Finland, model BIOSCREEN C PRO.

[0116] Example 1: Screening of yeast strains in Daqu (a type of starter culture) that are resistant to ethanol, produce high levels of ethanol and fatty acid ethyl esters, and produce low levels of isoamyl alcohol.

[0117] Weigh 10g of pulverized high-temperature Daqu (a type of starter culture) and place it in 90mL of sterile physiological saline. Shake well for 30min to obtain an extract. Dilute the extract appropriately and spread it on the lower layer of triphenyltetrazolium chloride (TTC) medium. Incubate at 28℃ for 2 days until visible colonies appear. Cover the colonies with the upper layer of TTC medium that has been melted and cooled to 50℃. Incubate at 28℃ for 3 hours. Select strains that are bright red and have typical yeast characteristics.

[0118] Selected colonies were inoculated into YPD liquid medium using an inoculation loop and cultured at 28°C with shaking at 120 rpm for 1 day. The selected bacterial cultures were then transferred at a 2% inoculation rate to ethanol-tolerant media with ethanol concentrations of 0%, 3%, 6%, 9%, 12%, and 15% (v / v), and cultured for 72 hours using a growth curve analyzer preheated to 28°C with rapid shaking.

[0119] Finally, the bacterial culture of the selected strains capable of growing under high-concentration ethanol conditions (i.e., the bacterial culture obtained by shaking and culturing in YPD liquid medium at 28°C and 120 rpm for 1 day) was inoculated at a 2% inoculation rate into high-sugar YPD liquid fermentation medium for liquid fermentation. After culturing at 28°C and 120 rpm for 3 days, the contents of ethanol, fatty acid ethyl esters, and isoamyl alcohol in the fermentation broth of each strain were detected by high-performance liquid chromatography and gas chromatography, respectively. A yeast strain that produces high levels of ethanol and fatty acid ethyl esters but low levels of isoamyl alcohol (hereinafter referred to as the "target strain") was screened. The growth curves of the target strain in media with different ethanol volume percentages were automatically generated by a growth curve measuring instrument, such as... Figure 1 As shown in Table 1, the detection data of the target strain are as follows.

[0120] Depend on Figure 1 It can be seen that the target strain grew well under ethanol volume percentages of 0%, 3%, 6%, 9%, and 12%, and could still grow under ethanol volume percentage of 15%, but with a lag phase. Meanwhile, the cell viability during the stationary phase of the target strain was almost consistent under ethanol volume percentages of 0%, 3%, 6%, and 9% (viability (%) = OD600 value after culture in medium containing different ethanol concentrations / OD600 value after culture in medium without ethanol), but decreased slightly under ethanol volume percentages of 12% and 15%.

[0121] The liquid fermentation culture conditions were as follows: inoculum size of 2%; high-sugar YPD liquid fermentation medium was used; cultured at 28℃, 120 rpm, and on a shaker for 3 days.

[0122] TTC upper culture medium: TTC 0.5g / L, glucose 5g / L, agar 15g / L, dissolved in distilled water, sterilized at 115℃ for 15min.

[0123] TTC lower layer culture medium: magnesium sulfate 0.4 g / L, potassium dihydrogen phosphate 1.0 g / L, yeast extract powder 1.5 g / L, peptone 2 g / L, glucose 10 g / L, agar 20 g / L, dissolved in distilled water, pH adjusted to 5.5, sterilized at 115℃ for 15 min.

[0124] YPD liquid culture medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, dissolved in distilled water and sterilized at 115°C for 15 min.

[0125] Ethanol-tolerant culture media (ethanol volume percentages of 0%, 3%, 6%, 9%, 12%, and 15%): 20 g / L yeast extract, 40 g / L peptone, and 40 g / L glucose were dissolved in distilled water and sterilized at 115°C for 15 min. After sterilization, ethanol and sterilized distilled water were added according to the final ethanol concentration of the prepared culture media to dilute the yeast extract to 10 g / L, the peptone to 20 g / L, and the glucose to 20 g / L, with ethanol volume percentages of 0%, 3%, 6%, 9%, 12%, and 15%, respectively.

[0126] High-sugar YPD liquid fermentation medium: 10 g / L yeast extract, 20 g / L peptone, and 100 g / L glucose were dissolved in distilled water and sterilized at 115°C for 15 min.

[0127] Pretreatment for high performance liquid chromatography detection of ethanol in fermentation broth: Pipette the fermentation broth into a centrifuge tube, centrifuge at 12000 rpm for 10 min, and then filter the centrifuged liquid through a 0.22 μm oil-based filter membrane.

[0128] High-performance liquid chromatography (HPLC) detection conditions for ethanol in fermentation broth: Detector: differential refractive index detector; Column: Bio-Rad Aminex HPX-87H (300 mm × 7.8 mm); Mobile phase: 5 mmol / L dilute sulfuric acid; Flow rate: 0.6 mL / min; Detector temperature: 45 °C; Column temperature: 65 °C; Injection volume: 10 μL.

[0129] Ethanol was qualitatively determined using retention time, standards, parent ion, and secondary fragment ions, while ethanol was quantitatively determined using the external standard method.

[0130] Pretreatment of fermentation broth for ethyl fatty acid ester and isoamyl alcohol gas chromatography detection: The fermentation broth was aspirated into a centrifuge tube, centrifuged at 12000 rpm for 10 min, and then filtered through a 0.22 μm oil-based filter membrane to obtain the sample to be tested.

[0131] Gas chromatographic detection conditions for fatty acid ethyl esters and isoamyl alcohol in fermentation broth: detector: flame ionization detector; column: DB-WAX column (30m×0.2mm×0.25μm); injection port temperature: 240℃; injection volume: 1μL; split ratio: 30:1; carrier gas: nitrogen (purity ≥99.999%); column flow rate: 1mL / min; temperature program: initial 30℃, hold for 3min, increase to 90℃ at 3℃ / min, do not hold, then increase to 180℃ at 5℃ / min, hold for 25min, flame ionization detector temperature: 250℃.

[0132] The qualitative analysis of fatty acid ethyl esters and isoamyl alcohol was performed using retention time, standards, parent ions, and secondary fragment ions. The quantitative analysis of fatty acid ethyl esters and isoamyl alcohol was performed using the internal standard method.

[0133] Table 1. Contents of ethanol, ethyl fatty acid esters, and isoamyl alcohol in the fermentation broth of the target strain after liquid fermentation.

[0134] project Target strain (mean ± standard deviation) Ethanol (g / L) 40.95±1.03 Ethyl acetate (mg / L) 1249.49±57.23 Ethyl lactate (mg / L) 0.00±0.00 Ethyl butyrate (mg / L) 274.81±13.91 Ethyl hexanoate (mg / L) 1442.33±72.12 Ethyl heptaate (mg / L) 424.50±18.72 Ethyl octanoate (mg / L) 40.56±1.07 Ethyl nonanoate (mg / L) 56.62±1.70 Ethyl decanoate (mg / L) 28.79±1.14 Ethyl phenylacetate (mg / L) 109.64±5.47 Ethyl isovalerate (mg / L) 18.52±0.14 Ethyl lauryl acid (mg / L) 35.74±1.79 Ethyl palmitate (mg / L) 1346.68±67.87 Ethyl 3-octenate (mg / L) 23.95±0.59 Ethyl trans-cinnamate (mg / L) 6.35±0.12 Ethyl trans-3-hexenoate (mg / L) 9.27±0.39 Isoamyl alcohol (mg / L) 0.00±0.00

[0135] Example 2: Species identification of the target strain

[0136] 2.1 Strain morphology

[0137] The colony morphology and microscopic morphology of the target strain described in Example 1 are as follows: Figure 2 A, Figure 2 As shown in B. From Figure 2 In sample A, the colonies appear as milky-white raised bumps with a dry, opaque surface. The surface and edges exhibit hyphae-like structures. Figure 2 B shows that the target strain exhibits an oval or elliptical cell morphology.

[0138] 2.2 Molecular biological identification of the strain

[0139] 2.2.1 DNA extraction from strains

[0140] Use EZNA TM The Mag-Bind Soil DNAKit was used to extract DNA from the target strain described in Example 1.

[0141] 2.2.2 PCR amplification

[0142] The extracted DNA sample was used as a template for PCR amplification. The universal primers selected for amplifying the 26S rDNA nucleotide sequence of the target strain were: NL1 (SEQ ID NO:1): 5'-GCATATCAATAAGCGGAGGAAAAG-3', NL4 (SEQ ID NO:2): 5'-GGTCCGTGTTTCAAGACGG-3'.

[0143] DNA polymerase used was 2×SanTaq PCR Mix premix (containing blue dye) from Sangon Biotech (Shanghai) Co., Ltd. Amplification system (50 μL): 25 μL 2×SanTaq PCR Mix premix, 2 μL NL1 (10 μmol / L), 2 μL NL4 (10 μmol / L), 2 μL DNA template, and ddH2O to a final volume of 50 μL.

[0144] The amplification program for the target fragment was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 1 min, repeated for 35 cycles; and extension at 72℃ for 10 min.

[0145] 2.2.3 Identification of 26S rDNA of the strain

[0146] The amplified target fragment of approximately 600 bp from the target strain was sent to Shanghai Sangon Biotech for sequencing. The sequencing results (i.e., the 26S rDNA sequence of the strain, as shown in SEQ ID NO:3) were then compared with existing data in the NCBI database to perform sequence alignment analysis and construct a phylogenetic tree. Figure 3 As shown, the 26S rDNA sequence of the target strain screened in this invention has a high homology of 99.83% with Pichia kudriavzevii registered with NCBI. Therefore, the strain is identified as Pichia kudriavzevii and named Pichia kudriavzevii XJ-YPPKU067-1. It was deposited at the China General Microbiological Culture Collection Center on July 4, 2024, with accession number CGMCC No. 31175.

[0147] The 26S rDNA nucleotide sequence of the *Pichia pastoris* XJ-YPPKU067-1 is as follows (SEQ ID NO:3):

[0148] Example 3: Physiological and biochemical identification of Pichia gondii XJ-YPPKU067-1

[0149] According to the "Manual of Characteristics and Identification of Yeasts" (Barnett et al. Manual of Characteristics and Identification of Yeasts [M]. Qingdao Ocean University Press, 1991.), the physiological and biochemical experiments of yeasts mainly include: nitrogen source assimilation, carbon source assimilation, and sugar fermentation experiments.

[0150] The detection and analysis of physiological and biochemical indicators were based on the above-mentioned "Manual of Characteristics and Identification of Yeasts", the literature published by Lin Liangcai et al. (Lin Liangcai, Bai Ru, Gao Ying, et al. Screening and application of highly resistant Pichia pastoris [J]. Food and Fermentation Industries, 2023, 49(03):60-67.DOI:10.13995 / j.cnki.11-1802 / ts.031854.), and the literature published by Luo Ling et al. (Luo Ling, He Jing, Xu Liping, [References: Tao Sen et al. Isolation, Identification and Application of Yeasts in Kiwi Fruit Juice [J]. China Brewing, 2023, 42(08): 103-110.; Tao Sen et al. Isolation, Identification and Physiological and Biochemical Characteristics of Salt-Tolerant Yeasts in Salted Fish [J]. Food Industry Technology, 2019, 40(02): 136-141+147. DOI: 10.13386 / j.issn1002-0306.2019.02.02]

[0151] 4.) The nitrogen source assimilation experiment was conducted using yeast carbon source basal medium (YCB), while the carbon source assimilation experiment and sugar fermentation experiment were conducted using yeast nitrogen source basal medium (YNB). The physiological and biochemical identification results of Pichia kudriavzevii XJ-YPPKU067-1 are shown in Table 2, which are consistent with the characteristics of Pichia kudriavzevii.

[0152] Table 2. Physiological and biochemical identification results of Pichia gondii XJ-YPPKU067-1

[0153] Sugar fermentation result Carbon source assimilation result Nitrogen source assimilation result glucose + fructose + ammonium chloride - sucrose + Galactose + ammonium sulfate - maltose - Xylose - yeast extract powder + Soluble starch - Arabic sugar - peptone + lactose -

[0154] Note: "+" indicates that fermentation or growth is possible, and "-" indicates that fermentation or growth is not possible.

[0155] Example 4: Application of Pichia kudrica xJ-YPPKU067-1 in the fermentation of golden prickly pear wine

[0156] The sugar content of the golden prickly pear juice was measured using a handheld saccharimeter. Based on the measured results, sucrose was added to adjust the sugar content to 22°Bx. The adjusted juice was then dispensed into three fruit wine fermentation tanks equipped with one-way exhaust valves, with 300 mL of juice dispensed into each tank. Additionally, 0.45 mL of pectinase and 30 mg of potassium metabisulfite were added to each tank. During the fermentation process, *Pichia pastoris* XJ-YPPKU067-1 was inoculated, with the total inoculum size approximately controlled at 9.60 × 10⁻⁶. 9CFU. After inoculation with the strain, the fermenters were placed in an incubator at 28°C for fermentation. Each fermenter was weighed daily to monitor the weight loss during the fermentation process. Fermentation was stopped when the daily weight loss of each fermenter was less than 0.2 g / 300 mL.

[0157] The results of detecting the contents of ethanol, fatty acid ethyl esters, and isoamyl alcohol in the fermented prickly pear wine by *Pichia kudriazina* XJ-YPPKU067-1 are shown in Table 3. The isoamyl alcohol content in the fermented prickly pear wine by *Pichia kudriazina* XJ-YPPKU067-1 was 0, consistent with the results of the liquid fermentation experiment in Example 1.

[0158] Pretreatment for high performance liquid chromatography detection of ethanol in golden prickly pear fruit wine: Pipette golden prickly pear fruit wine into a centrifuge tube, centrifuge at 12000 rpm for 10 min, and then filter the centrifuged liquid using a 0.22 μm oil-based filter membrane.

[0159] The ethanol detection conditions were the same as in Example 1.

[0160] The qualitative analysis of ethanol in golden prickly pear fruit wine was performed using retention time, standards, parent ions, and secondary fragment ions. The quantitative analysis of ethanol was performed using the external standard method.

[0161] Pretreatment for the detection of fatty acid ethyl esters and isoamyl alcohol in prickly pear fruit wine using gas chromatography-mass spectrometry (GC-MS / MS) (Agilent Technologies GC7890-5975MSD, USA): Pipette prickly pear fruit wine into a centrifuge tube and centrifuge at 12000 rpm for 10 min. Transfer 6 mL of the centrifuged liquid to a headspace vial and add 3 g NaCl and 20 μL of 29.9899 mg / L octanol internal standard solution (prepared with 53% ethanol by volume). Use an SPME fiber extraction head (Supelco, USA, 50 μm / 30 μm CAR / DVB / PDMS) for the extraction of flavor compounds from prickly pear fruit wine. The sample extraction oscillation speed is 250 rpm, the sample extraction temperature is 50℃, the sample extraction time is 30 min, and the sample desorption time is 5 min.

[0162] Gas chromatography-mass spectrometry (GC-MS) conditions for detecting fatty acid ethyl esters and isoamyl alcohol in prickly pear fruit wine: GC conditions: DB-FFAP capillary column (60m×0.25mm, 0.25μm), high-purity helium carrier gas, flow rate 1.0mL / min, splitless injection, temperature program: initial temperature 50℃, hold for 0min, increase to 150℃ at 5℃ / min, hold for 5min, increase to 230℃ at 10℃ / min, hold for 5min; Mass spectrometry conditions: EI ion source, ion source temperature 230℃, quadrupole temperature 150℃, mass scan range 30~350u.

[0163] The qualitative analysis of fatty acid ethyl esters and isoamyl alcohol in golden prickly pear fruit wine was performed using retention time, standards, parent ions, and secondary fragment ions. The quantitative analysis of fatty acid ethyl esters and isoamyl alcohol was performed using the internal standard method.

[0164] Table 3. Contents of ethanol, fatty acid ethyl esters, and isoamyl alcohol in fermented prickly pear wine using *Pichia pastoris* XJ-YPPKU067-1.

[0165]

[0166]

[0167] Comparative Example: Angel Yeast SY applied to the fermentation of golden prickly pear wine

[0168] The sugar content of the golden prickly pear juice (same as in Example 4) was measured using a handheld saccharimeter. Based on the measured results, sucrose was added to adjust the sugar content to 22°Bx. The adjusted golden prickly pear juice was then dispensed into three fruit wine fermentation tanks equipped with one-way exhaust valves, with 300 mL dispensed into each tank. Additionally, 0.45 mL of pectinase and 30 mg of potassium metabisulfite were added to each fermentation tank. During the fermentation process, Angel Yeast SY was inoculated, with the total inoculum size of Angel Yeast SY approximately controlled at 9.60 × 10⁻⁶. 9 CFU. After inoculation with the strain, the fermenters were placed in an incubator at 28°C for fermentation. Each fermenter was weighed daily to monitor the weight loss during the fermentation process. Fermentation was stopped when the daily weight loss of each fermenter was less than 0.2 g / 300 mL.

[0169] The results of the detection of ethanol, fatty acid ethyl esters and isoamyl alcohol content in Angel Yeast SY fermented golden prickly pear fruit wine are shown in Table 4.

[0170] The pretreatment for ethanol detection in golden prickly pear fruit wine by high performance liquid chromatography is the same as in Example 4.

[0171] The ethanol detection conditions for golden prickly pear fruit wine using high-performance liquid chromatography were the same as in Example 1.

[0172] The qualitative analysis of ethanol in golden prickly pear fruit wine was performed using retention time, standards, parent ions, and secondary fragment ions. The quantitative analysis of ethanol was performed using the external standard method.

[0173] The pretreatment of fatty acid ethyl esters and isoamyl alcohol in golden prickly pear fruit wine for detection using gas chromatography-mass spectrometry (Agilent Technologies GC7890-5975MSD, USA) was the same as in Example 4.

[0174] The detection conditions for fatty acid ethyl esters and isoamyl alcohol in the prickly pear fruit wine were the same as in Example 4.

[0175] The qualitative analysis of fatty acid ethyl esters and isoamyl alcohol in golden prickly pear fruit wine was performed using retention time, standards, parent ions, and secondary fragment ions. The quantitative analysis of fatty acid ethyl esters and isoamyl alcohol was performed using the internal standard method.

[0176] Calculations showed that in the fermentation experiment of golden prickly pear fruit wine, the ethanol yield of the experimental group with added Kudria zweipichia yeast XJ-YPPKU067-1 was 0.7% higher than that of the control group with added Angel yeast SY. The content of fatty acid ethyl esters in the experimental group was generally higher than that in the control group: ethyl acetate, ethyl hexanoate, and ethyl isovalerate increased from none to present; ethyl butyrate increased by about 87.2%; ethyl heptanoate increased by about 52.2%; ethyl octanoate increased by about 49.0%; ethyl nonanoate increased by about 67.1%; ethyl decanoate increased by about 102.8%; ethyl laurate increased by about 733.5%; ethyl palmitate increased by about 38.5%; ethyl 3-octenoate increased by about 83.3%; ethyl trans-cinnamate increased by about 4.0%; ethyl trans-3-hexenoate increased by about 71.7%; and ethyl phenylacetate decreased slightly by 32.3%. The content of isoamyl alcohol decreased particularly significantly compared with the control group.

[0177] Meanwhile, tasters examined and analyzed the sensory characteristics of the golden prickly pear wine prepared by different strains, including color, aroma, and taste. The results are shown in Table 5 below. Sensory analysis revealed that the experimental group wine with added *Pichia pastoris* XJ-YPPKU067-1 bacterial solution exhibited a reduced original sourness and astringency compared to the control group wine with added *Angel Wine Yeast SY*. It also possessed a slight jasmine aroma. This confirms that fatty acid ethyl esters have floral or fruity aroma characteristics, which can impart a richer floral and fruity aroma to the wine, thus improving its sensory quality.

[0178] Table 4. Content of ethanol, ethyl fatty acid esters, and isoamyl alcohol in golden prickly pear fruit wine fermented with Angel Yeast SY

[0179]

[0180] Table 5. Sensory analysis results

[0181] Golden prickly pear fruit wine (inoculated with XJ-YPPKU067-1) Golden prickly pear fruit wine (inoculated with Angel Yeast SY) Color Golden color Golden color aroma Aroma of golden prickly pear with a hint of jasmine. Golden prickly pear fruit fragrance taste Mildly sour and astringent It has a strong sour and astringent taste.

[0182] The above embodiments are only for further elaboration and explanation of the technical solutions of the present invention, so that those skilled in the art can more accurately understand the inventive concept and operation scheme of the present invention, and are not intended to further limit the present invention. Any non-prominent substantive features and non-significant improvements made by those skilled in the art on this basis shall fall within the protection scope of the present invention.

Claims

1. A Pichia coudriavzwiis strain, characterized in that, The Pichia kudriavzevii is named Pichia kudriavzevii XJ-YPPKU067-1, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 31175.

2. The K. codiierei of claim 1, wherein, The Pichia kudriavzevii does not produce isoamyl alcohol; Preferably, the Pichia kudriavzevii is derived from Daqu; Preferably, the Daqu is derived from high-temperature Daqu; Preferably, the Pichia kudriavzevii is ethanol-tolerant; Preferably, the Pichia kudriavzevii is high in ethanol production; Preferably, the Pichia kudriavzevii is high in fatty acid ethyl ester production; Preferably, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenoate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate; Preferably, the fatty acid ethyl ester includes one or more of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenoate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate; Preferably, the Pichia kudriavzevii grows well under the condition of 0% to 20% ethanol volume percentage; Preferably, the Pichia kudriavzevii grows well under the condition of 0% to 15% ethanol volume percentage; Preferably, the Pichia kudriavzevii grows well under the condition of 0% to 12% ethanol volume percentage; Preferably, the Pichia kudriavzevii grows well under the condition of 0% to 9% ethanol volume percentage; Preferably, the Pichia kudriavzevii grows well under the condition of 0% to 6% ethanol volume percentage; preferably, the Pichia kudriavzevii grows well under the condition of 0% to 3% ethanol volume percentage.

3. A method of screening Pichia coudriavzveii for a library, characterized by, The method comprises the following steps: (1) crushing Daqu for Jiang-flavor liquor and placing it in water, shaking well, and then coating it on a lower layer medium of triphenyltetrazolium chloride (TTC), and placing it in an incubator for culture; (2) covering the colonies with a TTC upper layer medium, placing it in an incubator for culture, and selecting colonies; (3) inoculating the selected colonies in YPD liquid medium, performing shaking culture, taking the bacterial liquid to inoculate in ethanol-tolerant medium for culture, and screening strains capable of growing under the condition of high-concentration ethanol; (4) taking the bacterial liquid of the strains capable of growing under the condition of high-concentration ethanol, inoculating in high-sugar YPD liquid fermentation medium, performing shaking culture, and then detecting the contents of ethanol, fatty acid ethyl ester, and isoamyl alcohol in the fermentation liquid of each strain, to obtain a target strain high in ethanol and fatty acid ethyl ester production and low in isoamyl alcohol production; Preferably, the temperature of the incubator is 20-30℃; Preferably, the shaking culture is shaking culture using a shaking table. Preferably, the rotation speed of the oscillation culture is 50-250 rpm; Preferably, the rotation speed of the oscillation culture is 100-180 rpm; Preferably, the culture temperature for the oscillation culture is 20-30℃; Preferably, in step (1), the incubation time in the incubator is 1-5 days; Preferably, in step (1), the incubation time in the incubator is 2-5 days; Preferably, in step (2), the incubation time in the incubator is 1-48 hours; Preferably, in step (2), the incubation time in the incubator is 1-30 hours; Preferably, in step (2), the incubation time in the incubator is 1-12 hours; Preferably, in step (3), the selected colonies are cultured in YPD liquid medium with shaking for 1-5 days; Preferably, in step (3), the selected colonies are cultured in YPD liquid medium with shaking for 1-3 days; Preferably, in step (3), the bacterial culture is cultured in an ethanol-resistant medium for 1-120 hours. Preferably, in step (3), the bacterial culture is cultured in an ethanol-resistant medium for 1-96 hours; Preferably, in step (4), the bacterial culture is cultured in high-sugar YPD liquid fermentation medium for 1-5 days; Preferably, in step (4), the bacterial culture is cultured in high-sugar YPD liquid fermentation medium for 2-5 days; Preferably, the selected colonies are prominent red colonies; Preferably, the selected colonies are colonies with typical yeast characteristics; Preferably, the composition of the TTC upper culture medium includes: TTC 0.1-5.0 g / L, glucose 2-30 g / L, agar 5-30 g / L, and water as the solvent; Preferably, the composition of the TTC upper culture medium includes: TTC 0.5 g / L, glucose 5 g / L, agar 15 g / L, and water as the solvent; Preferably, the composition of the lower TTC culture medium includes: magnesium sulfate 0.2-1.0 g / L, potassium dihydrogen phosphate 0.2-2.0 g / L, yeast extract powder 0.5-30 g / L, peptone 1-30 g / L, glucose 2-30 g / L, agar 5-30 g / L, water as solvent, and pH 4-7. Preferably, the composition of the lower TTC culture medium includes: 0.4 g / L magnesium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.5 g / L yeast extract, 2 g / L peptone, 10 g / L glucose, 20 g / L agar, with water as the solvent and a pH of 5.

5. Preferably, the YPD liquid culture medium comprises: 0.5-30 g / L yeast extract, 1-30 g / L peptone, 10-200 g / L glucose, and water as the solvent; Preferably, the YPD liquid culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and water as the solvent; Preferably, the ethanol volume percentage concentration of the ethanol-resistant culture medium is 0% to 20%; Preferably, the ethanol volume percentage concentration of the ethanol-resistant culture medium is 0% to 15%; Preferably, the ethanol-resistant medium has an ethanol volume percentage concentration of 0% to 12%; Preferably, the ethanol-resistant medium has an ethanol volume percentage concentration of 0% to 9%; Preferably, the ethanol-resistant medium has an ethanol volume percentage concentration of 0% to 6%; Preferably, the ethanol-resistant medium has an ethanol volume percentage concentration of 0% to 3%; Preferably, the composition of the ethanol-resistant medium comprises: yeast extract powder 0.5-30 g / L, peptone 1-30 g / L, glucose 10-200 g / L, and water as the solvent; Preferably, the composition of the ethanol-resistant medium comprises: yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, and water as the solvent; Preferably, the composition of the high-sugar YPD liquid fermentation medium comprises: yeast extract powder 0.5-30 g / L, peptone 1-30 g / L, glucose 10-200 g / L, and water as the solvent; Preferably, the composition of the high-sugar YPD liquid fermentation medium comprises: yeast extract powder 10 g / L, peptone 20 g / L, glucose 100 g / L, and water as the solvent; Preferably, the Pichia kudriavzevii XJ-YPPKU067-1 is deposited with the China General Microbiological Culture Collection Center, and has a deposit number of CGMCC No. 31175.

4. The Pichia kudriavzevii obtained by the method of claim 3.

5. A fermentation broth obtained by fermentation of Pichia kudriavzevii, characterized in that, Preferably, the Pichia kudriavzevii XJ-YPPKU067-1 is deposited with the China General Microbiological Culture Collection Center, and has a deposit number of CGMCC No. 31175; Preferably, the Pichia kudriavzevii does not produce isoamyl alcohol; Preferably, the Pichia kudriavzevii is derived from Daqu; Preferably, the Daqu is derived from high-temperature Daqu; Preferably, the Pichia kudriavzevii is ethanol-resistant; Preferably, the Pichia kudriavzevii produces a large amount of ethanol; Preferably, the Pichia kudriavzevii produces a large amount of fatty acid ethyl ester; Preferably, the fatty acid ethyl ester comprises one or more of ethyl acetate, ethyl lactate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenoate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate; Preferably, the fatty acid ethyl ester comprises one or more of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl phenylacetate, ethyl isovalerate, ethyl laurate, ethyl palmitate, ethyl 3-octenoate, ethyl trans-cinnamate, and ethyl trans-3-hexenoate; Preferably, the Pichia kudriavzevii grows well under the condition of an ethanol volume percentage concentration of 0% to 20%. Preferably, the Pichia kudriavzevii grows well at an ethanol concentration of 0% to 15% (v / v); Preferably, the Pichia kudriavzevii grows well at an ethanol concentration of 0% to 12% (v / v); Preferably, the Pichia kudriavzevii grows well at an ethanol concentration of 0% to 9% (v / v); Preferably, the Pichia kudriavzevii grows well at an ethanol concentration of 0% to 6% (v / v); Preferably, the Pichia kudriavzevii grows well at an ethanol concentration of 0% to 3% (v / v).

6. A microbial inoculant, characterized in that, The Pichia kudriavzevii of any one of claims 1-2 and / or the fermentation broth of claim 5; Preferably, the microbial inoculum is in liquid or solid form.

7. Use of the Pichia kudriavzevii of any one of claims 1-2 or the fermentation broth of claim 5 in the preparation of a microbial inoculum; Preferably, the microbial inoculum is in liquid or solid form.

8. Use of the Pichia kudriavzevii of any one of claims 1-2 or the fermentation broth of claim 5 or the microbial inoculum of claim 6 in the preparation of a fermentation feedstock, a wine or a foodstuff; Preferably, the wine includes a brewed wine, a distilled wine or a compounded wine; Preferably, the wine includes a fruit wine; Preferably, the wine is a Rosa roxburghii fruit wine.

9. A method of making a wine, characterized in that, The method includes using the Pichia kudriavzevii of any one of claims 1-2 or the fermentation broth of claim 5 or the microbial inoculum of claim 6 in the preparation of a wine; Preferably, the wine includes a brewed wine, a distilled wine or a compounded wine; Preferably, the wine includes a fruit wine; Preferably, the wine is a Rosa roxburghii fruit wine; Preferably, the method is a method for increasing the number of fatty acid ethyl esters in a wine; Preferably, the method is a method for increasing the total content of fatty acid ethyl esters in a wine; Preferably, the method is a method for reducing the content of isoamyl alcohol in a wine; Preferably, the method is a method for increasing the content of ethanol in a wine.

10. A wine prepared by the method of claim 9; Preferably, the wine includes a brewed wine, a distilled wine or a compounded wine; Preferably, the wine includes a fruit wine; Preferably, the fruit wine is a Rosa roxburghii fruit wine.

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