A method for improving the quality of a fermentation product based on multi-microbial co-fermentation technology

Abstract

The application discloses a method for improving the quality of a fermentation product based on a multi-microorganism co-fermentation technology, and belongs to the field of fermentation engineering and biotechnology. The application screens an abnormal Wickerham yeast Y34 and a Saccharomyces cerevisiae 28-23, and co-fermentation of the two strains helps to improve the alcohol-ester ratio of the fermentation product. When applied in yellow rice wine, compared with the case where the Saccharomyces cerevisiae 28-23 is inoculated alone, the alcohol content is increased by 5.18%, the fusel alcohol content is reduced by 25.63%, the ester content is increased by 57.13%, and the overall aroma quality of the yellow rice wine, especially the fruit aroma and flower aroma characteristics, can be significantly improved, which has important reference significance for improving the quality of fermented alcoholic beverages.

Description

Technical Field

[0001] This invention relates to a method for improving the quality of fermented products based on multi-strain co-fermentation technology, belonging to the fields of fermentation engineering and biotechnology. Background Technology

[0002] Yellow wine, a traditional Chinese fermented beverage, is renowned worldwide for its unique flavor and long history. The brewing process involves complex microbial fermentation, with yeast playing a crucial role and directly influencing the quality and taste of the wine. Previous research on yellow wine has largely focused on traditional brewing yeasts, with relatively little attention paid to the application of non-brewing yeasts. In recent years, with continuous technological advancements and higher demands on the food industry, researchers have increasingly turned their attention to the fermentation process of yellow wine using both non-brewing yeasts and traditional brewing yeasts.

[0003] As the core microorganism in rice wine fermentation, *Saccharomyces cerevisiae* participates in the formation of the vast majority of flavor compounds. Non-*Saccharomyces cerevisiae*, through various interactions, promote the metabolic activities of *Saccharomyces cerevisiae*, thus positively impacting the product's flavor. Lower levels of fusel alcohols and higher levels of esters significantly enhance the drinking comfort and flavor quality of rice wine. As a relatively new microbial resource, non-*Saccharomyces cerevisiae* exhibit significantly different metabolic pathways and characteristics compared to traditional *Saccharomyces cerevisiae*. The metabolites and secreted enzymes of non-*Saccharomyces cerevisiae* promote the degradation of fermentation substrates such as glucose by *Saccharomyces cerevisiae*, producing aromatic compounds such as esters and acids, which positively influence the final flavor of the rice wine product. This difference introduces new possibilities to the rice wine fermentation process, stimulating a pursuit of diversity and innovation.

[0004] In recent years, improving the quality of fermented wines through co-fermentation with both brewing yeast and non-brewing yeast has become a hot research topic. Numerous studies have demonstrated the abundance of non-brewing yeast in the fermentation process of rice wine, but due to tolerance issues with ethanol and other components, these yeasts cannot play a significant positive role. Research indicates that the rational use of non-brewing yeast can effectively improve the quality of alcoholic beverages, creating more diverse styles that are favored by consumers and the brewing industry. Non-brewing yeast has been widely used in both the wine and baijiu industries, and different types may contribute differently to the quality of alcoholic beverages. However, due to the complexity and uniqueness of rice wine production processes, research on non-brewing yeast in rice wine is currently limited. Therefore, this invention addresses the current rice wine production process by establishing a brewing method for rice wine with low fusel oil and high ester production through co-fermentation with both non-brewing yeast and brewing yeast. Different types of non-brewing yeast are evaluated, and superior strains are selected and co-fermented with brewing yeast to optimize the fermentation process. The establishment and application of this method for brewing rice wine with low fusel oil and high ester production through co-fermentation with both non-brewing yeast and brewing yeast is of great significance for improving the drinking comfort, flavor diversity, and quality of modern rice wine. Summary of the Invention

[0005] To address the adverse effects of excessive fusel oil content and the severe homogenization of traditional brewed foods, this invention provides a brewing method for producing low-fuss alcohol and high-ester fermented products through co-fermentation of non-brewing yeast and brewing yeast.

[0006] This invention provides an abnormal Wickhamomyces anomalus Y34 strain isolated from Shaoxing Guyuelongshan wine yeast, classified and named as abnormal Wickhamomyces anomalus Y34, which was deposited at the China Center for Type Culture Collection on May 15, 2024, with accession number CCTCC NO:M 2024923, and deposit address: Wuhan University, Wuhan, China.

[0007] This invention provides a strain of Saccharomyces cerevisiae 28-23, which was deposited at the China Center for Type Culture Collection on May 15, 2024, with accession number CCTCC NO:M 2024924, and the deposit address is Wuhan University, Wuhan, China.

[0008] The present invention also provides a fermentation agent containing the aforementioned brewing yeast 28-23.

[0009] In one embodiment, the fermenting agent further contains the abnormal Wickham yeast Y34.

[0010] In one embodiment, the fermenting agent is obtained by culturing the Saccharomyces cerevisiae 28-23 in a culture medium for a period of time and collecting the cell culture broth as the fermenting agent.

[0011] In one embodiment, the fermenting agent is prepared by culturing the Saccharomyces cerevisiae 28-23 and the Wickham's abnormal yeast Y34 separately in a culture medium for a period of time, and then collecting the cell culture broth and mixing them together as the fermenting agent.

[0012] In one embodiment, the fermenting agent is obtained by co-culturing the Saccharomyces cerevisiae 28-23 and the Wickham's abnormal yeast Y34 in a culture medium for a period of time, and then collecting the cell culture broth as the fermenting agent.

[0013] In one embodiment, the culture medium is a rice saccharification liquid culture medium.

[0014] In one embodiment, the rice saccharification culture medium is prepared by mixing raw rice and water in a certain proportion, adding liquefying enzyme, saccharifying enzyme and raw wheat koji, saccharifying and liquefying at 55-65℃ for 4-5 hours until the sugar content is greater than 13°Brix, and then sterilizing after filtration.

[0015] In one embodiment, the fermentation agent is prepared by:

[0016] (1) Mix raw rice and water in a mass ratio of 1:4, add liquefying enzyme, saccharifying enzyme and raw wheat koji, saccharify and liquefy at 60℃ for 4-5 hours until the sugar content is greater than 13°Brix, filter and sterilize at 115℃ for 20 minutes to obtain rice saccharification liquid culture medium.

[0017] (2) Inoculate the brewing yeast 28-23 into the rice saccharification liquid culture medium prepared in step (1) and culture it in a shaker at 28°C for 36 hours to obtain the brewing yeast 28-23 culture medium.

[0018] In one embodiment, the fermentation agent is prepared by:

[0019] (1) Mix raw rice and water in a mass ratio of 1:4, add liquefying enzyme, saccharifying enzyme and raw wheat koji, saccharify and liquefy at 60℃ for 4-5 hours until the sugar content is greater than 13°Brix, filter and sterilize at 115℃ for 20 minutes to obtain rice saccharification liquid culture medium.

[0020] (2) Inoculate Saccharomyces cerevisiae 28-23 and Wickham yeast Y34 into the rice saccharification liquid culture medium prepared in step (1) and culture them in a shaker at 28°C for 36 hours to obtain Saccharomyces cerevisiae 28-23 culture medium and Wickham yeast Y34 culture medium respectively. Mix the two culture mediums in a certain proportion.

[0021] In one embodiment, the fermentation agent is prepared by:

[0022] (1) The brewer's yeast 28-23 was cultured in YPD medium at 26-30℃ for 16-30h;

[0023] (2) The brewing yeast 28-23 cultured in step (1) is cultured in rice saccharification liquid culture medium at 26-30℃ for at least 24h;

[0024] (3) The abnormal Wickham yeast Y34 was cultured in rice saccharification liquid culture medium at 26-30℃ for at least 24h;

[0025] (4) Mix the cell culture medium from steps (2) and (3) at a ratio of 1:(1-1000).

[0026] The present invention also provides a process for preparing alcoholic beverages by co-fermentation of the brewing yeast 28-23 and the abnormal Wickham yeast Y34.

[0027] In one embodiment, the alcoholic beverage includes, but is not limited to, wine, rice wine, sake, rice wine, and fruit wine.

[0028] In one embodiment, the process involves fermenting the brewing yeast 28-23 and the abnormal Wickham yeast Y34 at a ratio of 1:10, with the main fermentation temperature being 20°C.

[0029] In one embodiment, the amount of the brewing yeast 28-23 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL.

[0030] In one embodiment, the quantity of the abnormal Wickham yeast Y34 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL.

[0031] In one embodiment, the method includes:

[0032] (1) Add brewing agent of brewing yeast 28-23 and abnormal Wickham yeast Y34 to the rice-water mixed fermentation system, and add wheat koji and 3%-5% alcohol sweat at a ratio of 12%-15% of the raw rice weight. Complete the material mixing at 25℃-28℃ and let it stand at 20℃ for 5 days for pre-fermentation.

[0033] (2) Lower the temperature of the fermentation tank to 15°C and let it stand for 15 days for post-fermentation.

[0034] (3) The fermented mash obtained in step (2) is pressed by plate and frame pressing and filtered with diatomaceous earth (diatomaceous earth is added at a ratio of 4%-6% and pressure is 0.3-0.5MPa). The filtrate is clarified to obtain clear wine. The clear wine is blended according to the national standard for yellow wine by adding 1‰-3‰ caramel coloring and frying the wine to obtain yellow wine.

[0035] This invention also provides a method for increasing the ester content and / or decreasing the fusel oil content in alcoholic beverages, wherein the method involves co-fermentation of the aforementioned *Wickham's abnormal* yeast Y34 and the aforementioned *Saccharomyces cerevisiae* 28-23; the quantity of *Saccharomyces cerevisiae* 28-23 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL; the quantity of the abnormal Wickham yeast Y34 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL.

[0036] In one embodiment, the fermentation temperature is ≥18°C, specifically 18–36°C, 18–32°C, 18–30°C, 18–25°C, 18–22°C, or 19–21°C.

[0037] In one embodiment, the ratio of the number of cells of the brewing yeast 28-23 to the number of cells of the abnormal Wickham yeast Y34 is 1:1 to 1:10.

[0038] In one embodiment, the ratio of the number of cells of the brewing yeast 28-23 to the number of cells of the abnormal Wickham yeast Y34 is 1:10.

[0039] In one embodiment, the method includes:

[0040] (1) Add a fermentation agent containing the brewing yeast 28-23 and the abnormal Wickham yeast Y34 to the rice-water mixed fermentation system, and add wheat koji and 3%-5% alcohol sweat at a ratio of 12%-15% of the raw rice mass. Complete the material mixing at 25℃-28℃ and pre-ferment at 15-25℃ for 4-5 days.

[0041] (2) Cool down to 14-16℃, let stand and ferment for 14-16 days;

[0042] (3) Filter the fermented mash obtained in step (2) and clarify it to obtain sake.

[0043] In one embodiment, the method further involves adding 1‰-3‰ caramel coloring to sake, followed by simmering the sake to produce yellow wine.

[0044] The present invention also claims protection for the use of the aforementioned abnormal Wickham yeast Y34 and / or the aforementioned brewer's yeast 28-23, alone or in combination, in the preparation of fermented foods.

[0045] In one embodiment, the fermented product is a fermented condiment; the fermented condiment includes, but is not limited to, soy sauce, cooking wine, or vinegar.

[0046] In one embodiment, the rice wine is prepared by adding the brewing yeast 28-23 and abnormal Wickham yeast Y34 as quick brewing starter at an inoculation ratio of 1:10 and a total addition amount of 5%-15% to the cooked or gelatinized raw materials, and then fermenting, pressing, heating, aging, filtering, sterilizing and bottling.

[0047] In one embodiment, the cooking wine is prepared by first fermenting rice wine using the brewing yeast 28-23 and abnormal Wickham yeast Y34 as quick-fermenting starter, and then using the rice wine to prepare the cooking wine.

[0048] In one embodiment, the application is for vinegar brewing, in which the brewing yeast 28-23 and abnormal Wickham yeast Y34 are first used as quick-fermenting starter to ferment rice wine, and then the rice wine is used as raw material for acetic acid fermentation to brew vinegar.

[0049] Beneficial effects:

[0050] (1) This invention screened an excellent brewing yeast strain 28-23, which has the characteristics of high alcohol content, low fusel oil production and high ester production. This strain also has good passage stability, which is beneficial for its application in industrial production.

[0051] (2) This invention screened an excellent non-Saccharomyces anomalus strain—Wickerhamomyces anomalus Y34—which showed the best effect on optimizing the alcohol-ester ratio in the co-fermentation system of fermentation products. When this strain was applied to rice wine, compared with inoculating Saccharomyces cerevisiae 28-23 alone, co-fermentation of Wickerhamomyces anomalus Y34 with Saccharomyces cerevisiae 28-23 resulted in a 14.12% increase in alcohol content, a 14.68% decrease in fusel oil content, and a 20.82% increase in ester content.

[0052] (3) This invention also optimized the co-fermentation process parameters of Saccharomyces cerevisiae 28-23 and Saccharomyces cerevisiae Y34. Saccharomyces cerevisiae 28-23 and Saccharomyces cerevisiae Y34 were inoculated at a ratio of 1:10 (quantity ratio) and fermented at a main fermentation temperature of 20℃. The resulting rice wine had a harmonious balance of alcohols and esters, and a more balanced aroma and taste. The yield of fusel oils was 281.20±5.73mg / L, which was 25.63% lower than that of single-strain fermentation with Saccharomyces cerevisiae 28-23, significantly improving the drinking comfort of rice wine. The yield of esters was 240.02±2.47mg / L, which was 57.13% higher than that of single-strain fermentation with Saccharomyces cerevisiae 28-23. The overall aroma quality of the rice wine, especially the aroma characteristics of fruit and floral aromas, was significantly better than that of single-strain fermentation with Saccharomyces cerevisiae 28-23. The co-fermentation system showed the characteristics of low alcohols and aldehydes and high esters and acids.

[0053] Preservation of biological materials

[0054] Saccharomyces cerevisiae 28-23, classified and named Saccharomyces cerevisiae 28-23, was deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024924, located at Wuhan University, Wuhan, China.

[0055] Wickerhamomyces anomalus Y34, classified as Wickerhamomyces anomalus Y34, was deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2024923, located at Wuhan University, Wuhan, China. Attached Figure Description

[0056] Figure 1 The present invention relates to the co-fermentation of different non-Saccharomyces yeasts (NSYs) with Saccharomyces cerevisiae 28-23 to produce fusel alcohols.

[0057] Figure 2 The present invention relates to the co-fermentation of different non-Saccharomyces cerevisiae (NSYs) with Saccharomyces cerevisiae 28-23 to produce esters.

[0058] Figure 3 The yields of fusel alcohols and esters in the co-fermentation groups included in the secondary screening of different non-Saccharomyces cerevisiae (NSYs) and Saccharomyces cerevisiae (S. cerevisiae) 28-23 according to the present invention.

[0059] Figure 4 The OAV values ​​of ester production in rice wine produced by co-fermentation of Wickerhamomyces anomalus Y34 and Saccharomyces cerevisiae 28-23 in different addition ratios are given in this invention.

[0060] Figure 5 Cluster analysis of volatile flavor compounds generated by co-fermenting rice wine with Wickerhamomyces anomalus Y34 and Saccharomyces cerevisiae 28-23 at different addition ratios, for the present invention.

[0061] Figure 6 The invention relates to the yield of fusel oils and the OAV value of esters in rice wine co-fermented with Wickerhamomyces anomalus Y34 and Saccharomyces cerevisiae 28-23 at different primary fermentation temperatures.

[0062] Figure 7 The present invention relates to the differences in two key aroma compounds between rice wine co-fermented with Wickerhamomyces anomalus Y34 and Saccharomyces cerevisiae 28-23 and rice wine fermented by Saccharomyces cerevisiae 28-23 alone. Detailed Implementation

[0063] The specific embodiments of the present invention are described below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0064] Technical terms:

[0065] As used in this invention, the term "strain" refers to a specific species of microorganism that shares common characteristics. Unless otherwise indicated, the terms "strain" and "cell" are used interchangeably in this invention.

[0066] The term "plate" as used in this invention refers to plate culture medium, which is the most commonly used form of solid culture medium for obtaining pure cultures of microorganisms. It is a solid surface of culture medium formed in a sterile petri dish after the solidified culture medium has cooled and solidified. It is often simply referred to as a culture plate or plate.

[0067] As used in this invention, the term "culture medium" refers to a culture medium that includes the chemical elements required for the growth of the microorganisms, along with at least one carbon source and one nitrogen source.

[0068] The term "culture" as used in this invention refers to the continuous cultivation of the microorganisms over a period of time until the desired goal is achieved.

[0069] The term "fusel" as used in this invention refers to n-propanol, isobutanol, isoamyl alcohol, and phenylethanol.

[0070] The term "ester" used in this invention refers to ethyl acetate, ethyl isobutyrate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl 2-hydroxy-4-methylvalerate, ethyl benzoate, ethyl 2-phenylethyl acetate, ethyl phenylacetate, ethyl 3-phenylpropionate, diethyl succinate, ethyl butyrate, etc.

[0071] The term “OAV” used in this invention refers to the aroma activity value, which is the “sensory intensity” of a certain aroma substance to a human. Volatile components with an OAV greater than 1 are aroma active components that contribute to flavor. The larger the OAV, the greater the contribution of the component to the flavor of the food. OAV can be used to more reliably evaluate the characteristic aroma substances in fermented products.

[0072] The term "fermentation broth" as used in this invention refers to a liquid culture medium inoculated with microbial strains. After a period of cultivation, the microorganisms utilize the nutrients in the culture medium to synthesize cell bodies and secrete products. This liquid, after being metabolized by the microorganisms, is called fermentation broth.

[0073] Materials and reagents involved in the following embodiments:

[0074] "Wheat koji" is provided by Gu Yue Long Shan. It is made from wheat through a process of pulverizing, adding water, mixing, treading, cutting, and arranging the wheat to form a saccharifying agent rich in beneficial microorganisms. It also provides amylase, protease, etc.

[0075] "Jiuhan" is provided by Guyuelongshan. It is a distilled spirit obtained by fermenting the lees after filtering rice wine and then distilling it.

[0076] The liquefying enzyme was obtained from Beijing Solarbio Science & Technology Co., Ltd., product number A8750, with an enzyme activity of 4.0 × 10⁻⁶. 4 U / g.

[0077] The "saccharifying enzyme" was obtained from Yuanye Biotechnology Co., Ltd., product number S10018, with an enzyme activity of 1.0 × 10⁻⁶.5 U / mL.

[0078] The culture media involved in the following examples are as follows:

[0079] YPD medium: 10g yeast extract, 20g peptone, 20g glucose, add water to 1000mL, 2% agar, autoclave at 121℃ for 20min, cool and set aside.

[0080] Rice saccharification culture medium: Take an appropriate amount of high-quality rice raw material, soak the rice in a 60℃ water bath for 30 minutes, and cook it under normal pressure for 20 minutes. Then, based on the rice raw material, add 150U / g-300U / g 1‰ saccharifying enzyme and 200U / g-400U / g 2‰ liquefying enzyme, and add 10% of the rice weight of raw wheat koji. Saccharify at 55℃-65℃ for 4-5 hours until the sugar content is above 13 Brix. Dispense into containers, autoclave at 121℃ for 15-20 minutes, and cool for later use.

[0081] The detection methods involved in the following embodiments are as follows:

[0082] The physicochemical indicators of rice wine were determined as follows: alcohol content, amino acid nitrogen, and total acid were determined according to GB / T 13662-2018 Rice Wine. Organic acid and amino acid content were determined using high-performance liquid chromatography (HPLC), while ethyl carbamate and volatile flavor compounds such as higher alcohols and esters were detected using gas chromatography-mass spectrometry (GC-MS). Reducing sugar content was determined using the DNS method. Microbial concentration was determined spectrophotometrically. The higher alcohols (also known as fusel alcohols) in rice wine mainly include four types: n-propanol, isobutanol, isoamyl alcohol, and 2-phenylethanol. Dispersion liquid-liquid microextraction (DLLME) was used, followed by GC-MS detection, with 4-methyl-2-pentanol as an internal standard, and an external standard curve was established for quantitative determination of fusel alcohol content. The strains used in establishing the method were obtained from the Traditional Fermentation Food Research Center of Jiangnan University. The strain information is shown in Table 1 and is publicly available on the website of the Traditional Fermentation Food Research Center of Jiangnan University: https: / / rctff.jiangnan.edu.cn / info / 1087 / 1663.htm.

[0083] Table 1. Strain Information

[0084]

[0085]

[0086] Example 1: Screening of brewing yeast

[0087] Saccharomyces jiangnan1# was isolated from Shaoxing handmade rice wine fermentation mash samples using natural selection. Then, following the method described in the paper "Selection of high-yield β-phenylethanol rice wine yeast", it was purified by UV mutagenesis, ARTP, and sporulation purification to obtain Saccharomyces 28-23. This strain showed good genetic stability, and after five generations, there were no significant differences in any of the indicators (P>0.05).

[0088] Example 2: Study on the fermentation characteristics of brewer's yeast

[0089] The brewing yeast 28-23 was fermented according to the method of Example 1 in patent application CN113621528B, the difference being that 3%-5% of rice wine was added to the raw material ratio of traditional rice wine fermentation; the fermentation results were tested. The results showed that the brewing yeast 28-23 had good fermentation degree, high alcohol content, and low fusel oil and high ester production. The alcohol yield of strain 28-23 was higher than 20% vol, and the fusel oil yield was 378.16 ± 28.76 mg / L, which was 21.41% lower than the control strain S. cerevisiae jiangnan1# (disclosed in patent publication number CN113621528B). Among them, the yield of isobutanol was reduced by 17.07%, isoamyl alcohol by 24.52%, and phenylethanol by 24.22%, showing stable fusel oil reduction performance. The ester content was 152.86 ± 19.73 mg / L, which was 35.57% higher than the control strain. Among them, the content of acetate increased by 44.07%, ethyl isobutyrate by 119.04%, diethyl succinate by 17.45%, ethyl butyrate by 27.03%, and nonanolide by 64.90%, showing stable ester enhancement performance, laying the foundation for the industrial production of strain 28-23.

[0090] Example 3 Screening of non-Saccharomyces cerevisiae

[0091] The *S. cerevisiae* 28-23 strains selected in Example 1 were co-fermented with 52 strains of non-saccharomyces yeasts (NSYs) with potential ester-producing capabilities from the strain bank of the Traditional Brewing Food Research Center of Jiangnan University at a ratio of 1:10 (volume ratio, determined through preliminary experiments). Only *S. cerevisiae* 28-23 was inoculated as a control group. After fermentation, the physicochemical indicators, fusel oils, and volatile flavor compounds were measured to determine the optimal co-fermentation combination.

[0092] The specific steps are as follows:

[0093] (1) Liquid culture of yeast strains: Take a loop of yeast preserved in glycerol tube with a 10 μL inoculation loop, streak it in zone 3 on a YPD plate to obtain single colonies, pick the single colonies and culture them in 100 mL of YPD medium at 37℃ for 24 h to obtain yeast liquid.

[0094] (2) Soaking rice: Weigh out the glutinous rice as needed, add tap water, mix the rice and water well, and make sure the water level is more than 7cm above the liquid surface to allow the rice to fully absorb water. Soak the rice in a constant temperature water bath at 60℃ for 20 minutes.

[0095] (3) Steaming rice: Boil the water in the steamer, soak the rice and then drain the soaking water. Spread the wet rice evenly on two layers of gauze and steam for 20 minutes, spraying it with 80℃ hot water during the process.

[0096] (4) Cooling the rice: Spread out the steamed rice and let it cool until the temperature is below 60°C.

[0097] (5) Rice saccharification liquid culture medium: Mix raw rice and water (1:4 mass ratio), and add 2‰ (by mass of raw rice) of liquefying enzyme (enzyme activity 4.0 × 10⁻⁶). 4 U / mL), 1‰ of saccharifying enzyme (enzyme activity 1.0×10⁻⁶). 5 Mix 10% raw wheat koji (U / mL) with 10% raw wheat koji, and saccharify and liquefy at 60℃ for 4-5 hours. Measure the sugar content using a refractometer until the sugar content is greater than 13°Brix. After saccharification, filter the mixture through filter bags, dispense it, sterilize it at 115℃ for 20 minutes, and cool it for later use.

[0098] (6) Preparation of yeast culture medium: The yeast cultured in liquid culture in step (1) was inoculated into the rice saccharification liquid culture medium prepared in step (5) at a ratio of 1‰ (volume ratio), and cultured in a shaker at 28℃ for 36h to obtain yeast culture medium. Then, it was transferred into rice saccharification liquid culture medium at a ratio of 5%, and then serially diluted 10% at equal proportions. -1 -10 -7 Take 100 μL of 10 -5 10 -6 and 10 -7 The yeast culture medium was spread on YPD plates and cultured at 28°C for 36 hours. Plate counting was performed to determine the number of yeasts in the Erlenmeyer flask yeast culture medium. At the same time, 1.5 mL of the yeast cultured in step (1) was stored in an EP tube at low temperature (4°C) as seed culture for subsequent inoculation.

[0099] (7) Preparation of starter culture: Take the yeast culture obtained in step (6) and inoculate it into rice saccharification liquid culture medium at a ratio of 1‰. Culture in a shaker at 28℃ for 36h to obtain a pure yeast starter culture containing only non-Saccharomyces cerevisiae or Saccharomyces cerevisiae. Then, transfer it into rice saccharification liquid culture medium at a ratio of 5%. After testing, the concentration of Saccharomyces cerevisiae reached 1×10⁻⁶. 8CFU / mL, non-Saccharomyces cerevisiae concentration is 1×10 7 CFU / mL ~ 1×10 8 CFU / mL. The concentration of *Saccharomyces cerevisiae* 28-23 starter culture (1×10⁻⁶ cells / mL) was determined based on the dilution plate count results. 8 CFU / mL) and non-Saccharomyces cerevisiae starter (crystal concentration of 1×10⁻⁶) 8 The inoculation ratio (CFU / mL) was 1:10 (5mL+45mL) into a 1.5L fermentation system, with 28-23 (50mL) single-cell inoculation as a control.

[0100] (8) Co-fermentation of non-brewing yeast and brewing yeast shall be carried out in the following manner:

[0101] a) Preparation of fermented rice: Add water to the raw rice of the production quantity to a level 10cm above the liquid surface and soak the rice for 3-5 days. The acidity of the rice slurry reaches 4.5g / L or higher. Drain the water to obtain wet rice. Steam the wet rice in a rice steamer at 121℃ for 20-30 minutes until the rice is cooked but not mushy, with no white core in the rice grains, and the rice has a sour taste and aroma. The yield is 140%-160%.

[0102] b) Follow the traditional raw material ratio for rice wine fermentation, including the following steps:

[0103] S1. Add 750g of raw rice (500g of raw rice) and 625.25ml of water to a sterilized fermentation container. Add 50mL of the fermentation agent prepared in step (7) of this embodiment to the rice-water mixed fermentation system (1.5L). Add 12%-15% of wheat koji and 3%-5% of alcohol sweat according to the weight of the raw rice. Complete the material mixing at 25℃-28℃ and let it stand at 28℃ for pre-fermentation for 3-5 days.

[0104] S2. Lower the temperature of the fermentation tank to 15°C and let it stand for 15-20 days for post-fermentation.

[0105] S3. The fermentation mash obtained in S2 is processed through a plate and frame press (4 feeds, mash pressure of 0.2-0.6 MPa, filtration area of ​​100 m²). 2 The process involves filtering with a filter plate diameter of 1m and using diatomaceous earth (diatomaceous earth added at a ratio of 4%-6%, pressure 0.3-0.5MPa). The resulting filtrate is clarified to obtain rice wine. The rice wine is then blended according to the national standard for rice wine by adding 1‰-3‰ caramel coloring and simmering the wine to obtain rice wine.

[0106] The physicochemical indicators at the end of fermentation are shown in Table 2. The results showed that the alcohol content of all NSYs co-fermentation groups was greater than 15.00% vol, but some groups had excessively high total acid content (>8.00 mg / L), resulting in spoilage. Based on these results, NSYs co-fermentation groups with a total acid content exceeding 8.00 g / L were not further analyzed. Further research was conducted on the remaining 46 groups whose alcohol content, total acid content, and amino acid nitrogen all met the requirements for rice wine fermentation, and the positive impact of NSYs on the quality of rice wine was evaluated.

[0107] Table 2 Physicochemical indices of different NSYs co-fermented with S. cerevisiae

[0108]

[0109]

[0110]

[0111] Note: Total acid content is calculated as lactic acid.

[0112] according to Figure 1 The co-fermentation group was selected where the fusel oil content did not increase significantly compared to the control (S. cerevisiae 28-23 single-strain fermentation); further determination of ester content yielded the following results: Figure 2 As shown, the acetate content of the seven co-fermentation combinations 28-23+Y4(ZY), 28-23+Y5(ZY), 28-23+Y15, 28-23+Y17, 28-23+Y21, 28-23+Y34, and 28-23+Y36 was higher than that of the control (S. cerevisiae). (Single-strain fermentation of 28-23); the ethyl butyrate content of 28-23+Y4(ZY) was higher than that of the control; the content of nonanolide in the following 10 co-fermentation combinations was higher than that of the control: 28-23+SY2107, 28-23+Y4(ZY), 28-23+Y5(ZY), 28-23+Y5(YC), 28-23+Y15, 28-23+Y17, 28-23+Y21, 28-23+Y34, 28-23+Y36, and 28-23+Y44.

[0113] Based on fermentation indicators such as fusel oils and esters, NSYs strains were re-screened, and a total of 10 different NSYs strains were identified, namely: *Saccharomyces fibuligera* SY2107, *P. kudriavzevii* Y4(ZY), *P. kudriavzevii* Y5(ZY), *W. anomalus* Y5(YC), *W. anomalus* Y15, *W. anomalus* Y17, *W. anomalus* Y21, *W. anomalus* Y34, *W. anomalus* Y36, and *W. anomalus* Y44.

[0114] The physicochemical properties of the rice wine obtained by co-fermentation of the re-screened strains with S. cerevisiae 28-23 are shown in Table 3: the alcohol content, acidity, and amino acid nitrogen content all meet the national standards for rice wine. Figure 3 It can be seen that the co-fermentation group with added W. anomalus Y34 had the lowest fusel oil production and the highest ester production, and the co-fermentation group of S. cerevisiae 28-23+W. anomalus Y34 was finally determined.

[0115] Table 3 Physicochemical indicators of different NSYs co-fermented with S. cerevisiae rice wine

[0116]

[0117]

[0118] Note: Total acid content is expressed as lactic acid; the experimental group samples were inoculated with NSYs, and only inoculated with mutant strains 28-23 as the control group; different letters in the same column of data indicate significant differences (P < 0.05).

[0119] Example 4: Optimization of co-fermentation process of abnormal Wickham yeast Y34 and Saccharomyces cerevisiae 28-23

[0120] 1. Optimization of vaccination ratio

[0121] The preparation method of the rice wine fermentation agent and the rice wine fermentation are the same as in Example 3, the difference being that W. anomalus Y34 (with a concentration of 1×10⁻⁶) is used. 8 CFU / mL) and S. cerevisiae 28-23 fermentation spawn (1×10⁻⁶ CFU / mL) 8The rice wine was inoculated with CFU / mL at ratios of 1:1, 1:10, 1:100, and 1:1000, respectively, and fermented in small-scale systems. The specific steps are as follows:

[0122] a) Preparation of fermented rice: Add water to the raw rice of the production quantity to a level 10cm above the liquid surface and soak the rice for 3-5 days. The acidity of the rice slurry reaches 4.5g / L or higher. Drain the water to obtain wet rice. Steam the wet rice in a rice steamer at 121℃ for 20-30 minutes until the rice is cooked but not mushy, with no white core in the rice grains, and the rice has a sour taste and aroma. The yield is 140%-160%.

[0123] b) Follow the traditional raw material ratio for rice wine fermentation, including the following steps:

[0124] S1. Add the raw rice and water to the sterilized fermentation container. Add 50mL of brewing yeast 28-23 and abnormal Wickham yeast Y34 (volume ratio of bacteria to liquid is 1:1, 1:10, 1:100, 1:1000) to the 1.5L rice-water mixed fermentation system. Add wheat koji (12%-15% of the weight of raw rice) and alcohol sweat (3%-5%). Complete the material mixing at 25℃-28℃ and let it stand at 28℃ for pre-fermentation for 3-5 days.

[0125] S2. Lower the temperature of the fermentation tank to 10℃-15℃ and let it stand for 15-20 days for post-fermentation.

[0126] S3. The fermentation mash obtained in S2 is processed through a plate and frame press (4 feeds, mash pressure of 0.2-0.6 MPa, filtration area of ​​100 m²). 2 The process involves filtering with a filter plate diameter of 1m and using diatomaceous earth (diatomaceous earth added at a ratio of 4%-6%, pressure 0.3-0.5MPa). The resulting filtrate is clarified to obtain rice wine. The rice wine is then blended according to the national standard for rice wine by adding 1‰-3‰ caramel coloring and simmering the wine to obtain rice wine.

[0127] Based on the results of the fermentation completion indicators (Table 4), it can be seen that when W. anomalus Y34 was inoculated as a single strain, the acidity was >8 g / L, and the system became rancid. When the proportion of NSYs was too high (1:100, 1:1000), the number of dominant strains S. cerevisiae was small, the alcohol content increased slowly, leading to the large-scale growth and reproduction of miscellaneous bacteria and acid production. This resulted in a system with low alcohol content and high total acidity, making fermentation prone to stagnation. However, when the inoculation ratio was 1:1 and 1:10, it was clearly shown that the co-fermentation system could not only ferment normally, but the obtained physicochemical indicators all met the requirements for rice wine fermentation. Moreover, the yield of fusel oil per unit alcohol content was significantly lower than that of S. cerevisiae 28-23 single-strain fermentation. Figure 4It can be seen that the ester content of the co-fermentation system increases with the increase of W. anomalus Y34 addition. Combining the physicochemical indicators of the rice wine and the formation of fusel oils, it was finally determined that a co-fermentation inoculation ratio of S. cerevisiae 28-23 to W. anomalus Y34 of 1:10 achieves the optimal alcohol-ester ratio, with lower fusel oil content and higher ester content at this ratio; and... Figure 5 As shown, the rice wine fermented at this inoculation ratio exhibits higher ester content and lower alcohol and aldehyde content compared to the control, which is beneficial for improving the flavor of the rice wine.

[0128] Table 4. Indicators of co-fermentation of 28-23 and Y34 at different inoculation ratios

[0129]

[0130] Note: Units for fusel oil content: (mg / L / %vol); different letters in the same column indicate significant differences (P<0.05).

[0131] 2. Optimization of primary fermentation temperature

[0132] The preparation method of the rice wine fermentation agent and the rice wine fermentation are the same as in Example 3, the difference being that W. anomalus Y34 (with a concentration of 1×10⁻⁶) is used. 8 CFU / mL) and S. cerevisiae 28-23 fermentation spawn (1×10⁻⁶ CFU / mL) 8 The rice wine was co-inoculated with CFU / mL at a ratio of 1:10, and the main fermentation temperatures were set at 20℃, 24℃, 28℃, 32℃, and 36℃ respectively for small-scale rice wine fermentation. The specific steps are as follows:

[0133] a) Preparation of fermented rice: Add water to the raw rice of the production quantity to a level 10cm above the liquid surface and soak the rice for 3-5 days. The acidity of the rice slurry reaches 4.5g / L or higher. Drain the water to obtain wet rice. Steam the wet rice in a rice steamer at 121℃ for 20-30 minutes until the rice is cooked but not mushy, with no white core in the rice grains, and the rice has a sour taste and aroma. The yield is 140%-160%.

[0134] b) Follow the traditional raw material ratio for rice wine fermentation, including the following steps:

[0135] S1. Add the raw rice and water to the sterilized fermentation container. Add 50mL of brewing yeast 28-23 and abnormal Wickham yeast Y34 (1:10 volume ratio) to the 1.5L rice-water mixed fermentation system. Add 12%-15% of wheat koji and 3%-5% of alcohol by weight of raw rice. Complete the material mixing at 25℃-28℃. Allow the mixture to stand at 20℃, 24℃, 28℃, 32℃ and 36℃ for pre-fermentation for 3-5 days.

[0136] S2. Lower the temperature of the fermentation tank to 10℃-15℃ and let it stand for 15-20 days for post-fermentation.

[0137] S3. The fermentation mash obtained in S2 is processed through a plate and frame press (4 feeds, mash pressure of 0.2-0.6 MPa, filtration area of ​​100 m²). 2 The process involves filtering with a filter plate diameter of 1m and using diatomaceous earth (diatomaceous earth added at a ratio of 4%-6%, pressure 0.3-0.5MPa). The resulting filtrate is clarified to obtain rice wine. The rice wine is then blended according to the national standard for rice wine by adding 1‰-3‰ caramel coloring and simmering the wine to obtain rice wine.

[0138] The physicochemical properties of rice wine co-fermented with S. cerevisiae 28-23 and W. anomalus Y34 at different primary fermentation temperatures are shown in Table 5. It can be found that when the primary fermentation temperature is 36℃, the alcohol content of the co-fermentation system is less than 15% vol. The higher temperature inhibits the normal growth and fermentation activity of the yeast, resulting in the inability to complete the fermentation of rice wine. The fermentation properties at other temperatures all meet the national standards for rice wine.

[0139] Table 5 Physicochemical properties of co-fermentation at different primary fermentation temperatures

[0140]

[0141] Note: Different letters in the same column of data indicate significant differences (P < 0.05).

[0142] The effect of primary fermentation temperature on the formation of fusel oils and esters in the co-fermentation system is as follows: Figure 6 As shown, the alcohol-ester ratio was lowest at a fermentation temperature of 20℃. With increasing fermentation temperature, the alcohol-ester ratio first increased and then decreased, indicating that yeast's ability to produce fusel oils at high temperatures was significantly higher than at low temperatures. Higher fermentation temperatures were therefore unfavorable for the intracellular metabolism of esters in yeast. Thus, the optimal primary fermentation temperature for the co-fermentation of *S. cerevisiae* 28-23 and *W. anomalus* Y34 was determined to be 20℃, providing some data support for the low-temperature fermentation of rice wine.

[0143] Example 5: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in traditional rice wine production.

[0144] Fermentation process using mixed inoculation of Saccharomyces cerevisiae 28-23 and Saccharomyces aberrantus strain Y34: The concentration of S. cerevisiae 28-23 (1×10⁻⁶ cells / year) was determined based on dilution plate counts. 8 CFU / mL) and W. anomalus Y34 (bacterial concentration of 1×10⁻⁶) 8 Inoculate with CFU / mL at a ratio of 1:10; use S. cerevisiae 28-23 as a control.

[0145] Rice saccharification liquid culture medium was prepared according to the method of Example 3. The fermentation conditions for rice wine were the same as in Example 4, and the main fermentation temperature was controlled at 20°C.

[0146] The main indicators of the fermented rice wine under these conditions are shown in Table 6. It can be observed that the basic physicochemical indicators of the fermentation system meet the national standards for rice wine. The fusel oil yield is 281.20±5.73 mg / L, which is 25.63% lower than that of single-strain fermentation with *S. cerevisiae* 28-23 (among which, n-propanol content decreased by 47.28%, isobutanol content decreased by 6.83%, and isoamyl alcohol content decreased by 40.39%), effectively improving the drinking comfort of the rice wine. The ester yield is 240.02±2.47 mg / L, which is 57.13% higher than that of single-strain fermentation with *S. cerevisiae* 28-23 (among which, acetate content increased by 57.01%, ethyl lactate by 67.29%, nonyl lactone by 27.83%, and ethyl propionate by 35.33%), effectively enriching the flavor diversity of the rice wine. Figure 7 As shown, there are significant differences in flavor compounds between the co-fermentation system and the single-strain fermentation system. The content of key aroma compounds in rice wine, such as butyric acid (cheese flavor), hexanoic acid (cheese flavor), ethyl hexanoate (apple aroma, fruit aroma), ethyl caprylate (fruit aroma), ethyl lactate (fruit aroma), and isoamyl acetate (banana aroma, fruit aroma), is significantly higher in the co-fermentation system than in the single-strain fermentation system. The overall aroma quality of rice wine products obtained by co-fermentation of S. cerevisiae 28-23 and W. anomalus Y34, especially the aroma characteristics of fruit and floral aromas, is significantly better than that obtained by single-strain fermentation of S. cerevisiae 28-23.

[0147] Table 6. Main indicators of co-fermentation under optimal process conditions.

[0148]

[0149] Note: Different letters in the same row of data indicate significant differences (P < 0.05).

[0150] Example 6: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in millet rice wine

[0151] The raw material ratio selected for millet rice wine fermentation in this embodiment is as follows:

[0152] Millet (standard): 500g; Water: 500g (mL) - 625g (mL); Raw wheat koji: 50g - 75g; Cooked wheat koji: 9g; Brewing yeast 28-23 and abnormal Wickham yeast Y34 starter culture: 50mL; When using Angel brewing high-activity dry yeast and Angel brewing koji as activating starter culture, replace the raw wheat koji, cooked wheat koji and brewing yeast 28-23 and abnormal Wickham yeast Y34 starter culture with the activating starter culture.

[0153] Experimental group: The starter culture was prepared by using low fusel oil and high ester production brewing yeast 28-23 and abnormal Wickham yeast Y34, following the steps (1)-(7) of Example 3.

[0154] Control group 1: The quick-brewing yeast culture medium was prepared by using the superior brewing yeast 28-23 as the pure yeast according to the steps (1)-(6) of Example 3.

[0155] Control group 2: 1g of Angel Yeast high-activity dry yeast and 2.5g of Angel Yeast brewing koji were activated in advance with 30mL of sugar water containing 20mg / mL glucose at 30℃-35℃ for 20-30 minutes as the activating starter. The yeast count in the activating starter was >10. 8 CFU / mL.

[0156] Millet rice wine brewing process:

[0157] a) The preparation method of fermented raw rice is as follows: Soak millet raw rice in water at room temperature for 1-3 days with 2-3 times the weight of raw millet rice until the acidity is greater than 3mg / L. Drain the water to obtain wet rice. Steam the wet rice in a rice steamer at 121℃ for 30-50 minutes until the rice is cooked but not mushy, and there is no white core in the rice grains. The yield is 140%-160%.

[0158] b) According to the raw material ratio for millet rice wine fermentation, the experimental group and the control group were cooled to 25℃-28℃ for pre-fermentation. Pre-fermentation: fermentation temperature is 20℃, fermentation for 3-7 days to enter post-fermentation. Post-fermentation: 15℃, post-fermentation for 15-20 days to obtain fermented mash.

[0159] c) In factory production, the fermented mash is pressed using a plate and frame press and filtered through diatomaceous earth. The resulting filtrate is clarified to obtain sake. The sake is then blended with distilled spirits to produce rice wine. After aging, ingredients such as goji berries, red dates, honey, astragalus, ginseng, and preserved plums can be added for further blending. This embodiment uses a small-scale laboratory system to simulate fermentation, and filtration is performed using gauze filtration and centrifugation.

[0160] After fermentation, the final millet fermentation mash obtained from the control and experimental groups was tested, and the results are shown in Table 6.

[0161] Table 6. Main indicators of millet rice wine fermentation

[0162]

[0163] The results showed that the final alcohol content was 12% (v / v)-18% (v / v). The yield of fusel oil in the rice wine co-fermented by strain Y34 and 28-23 was less than 300 mg / L. The fusel oil content per unit alcohol content was more than 10% lower than that in control group 1 and control group 2. Other indicators met the national standards for rice wine. The fermented millet rice wine was clear, bright in color, and refreshing in taste. The final millet rice wine co-fermented by strain Y34 and 28-23 had fruity and ester aromas, which can meet consumers' demand for alcoholic beverages with low fusel oil, high ester content, and nutritional health benefits.

[0164] Example 7: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in red yeast rice wine.

[0165] The red yeast rice wine production application is carried out according to the method in Example 4, the difference being that red yeast rice is used instead of wheat yeast rice in step S3, and after fermentation for 15-20 days in step S5, the fermented mash is pressed in a plate and frame and filtered through diatomaceous earth. The filtrate is clarified to obtain clear wine, which is then boiled to obtain red yeast rice wine (without adding caramel coloring).

[0166] Low-fuss alcohol and high-ester brewing yeast 28-23 and abnormal Wickham yeast Y34 were used as experimental strains to prepare the starter culture according to the method in Example 3. Angel brewing high-activity dry yeast and Angel brewing koji were used to replace red yeast rice and starter culture, respectively, as control groups. Red yeast rice wine was fermented according to the raw material ratio. After fermentation, the physicochemical indicators such as alcohol content and flavor indicators such as fusel alcohol were measured. The results are shown in Table 7.

[0167] Table 7: Main Fermentation Indicators of Red Yeast Rice Wine

[0168]

[0169] The results showed that after fermentation, the alcohol content of the red yeast rice wine obtained by the control group and the experimental group was 16% (v / v)-19% (v / v). The red yeast rice wine was brown or dark brown in color, with a refreshing taste and no off-flavors. The wine was well-balanced. The red yeast rice wine obtained by co-fermentation of brewing yeast 28-23 and abnormal Wickham yeast Y34 had a rich fruity and ester aroma.

[0170] Compared with the control group, the fusel oil content in the rice wine obtained by co-fermenting Saccharomyces cerevisiae 28-23 and Saccharomyces aberrantis Y34 was 16.5 mg / L, which is more than 20% lower than the unit alcohol content. The fusel oil reduction effect was significant, and other indicators met the national standards for rice wine.

[0171] Example 8: Application of Wickham Saccharomyces Y34, a strain with abnormally low fusel alcohol and high ester production, in rice wine.

[0172] Using rice as the main raw material, wash it with clean water and soak it at room temperature for about 4 hours until it is fully hydrated. The rice should be easily crushed by hand after soaking and have no hard core. Drain the soaked rice and steam it under normal pressure for about 30 minutes until there is no white core in the rice. After the rice is cooked, cool it with cold boiled water to about 25℃-28℃. Add 0.4%-0.8% of sweet wine yeast by weight of rice and saccharify at 28℃±2℃ for 36-42 hours. After saccharification, inoculate with 5%-10% of the abnormal Wickham yeast Y34 and brewer's yeast 28-23 prepared according to Example 3, stir well, and add drinking water at 1-1.5 times the weight of the raw rice. Ferment at 26℃-34℃ for 3-5 days, then lower the temperature to 10℃-15℃ and ferment for 10-15 days. After fermentation, press and filter to obtain rice wine. The fermented rice wine has a high ester content and is rich in rice and fruit aromas; it has a low content of higher alcohols, with fusel alcohol content of less than 18 mg / L per unit alcohol content, making it less likely to cause intoxication after drinking.

[0173] Example 9: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in beer.

[0174] Using barley malt (15kg-20kg), hops (50g-70g), and water (100L-120L) as the main raw materials, the beer is prepared using a multi-step saccharification process. A low-fuss alcohol, high-ester fermentation agent (non-brewing yeast Y34) and brewing yeast 28-23, prepared according to the method in Example 3, are added to the wort at a rate of 0.2%-1%. The beer undergoes malt crushing, saccharification, filtration, wort boiling, and fermentation. Top-fermenting yeast is used, with pre-fermentation at 18℃-23℃ for 2-3 days at a fermentation pressure of 0.15 MPa. During pre-fermentation, the sugar content is adjusted daily until it drops below 5 Brix, at which point the beer is cooled to 5℃ for storage. The resulting beer has an alcohol content of 3%-5% (v / v), total sugar (as glucose) <50g / L, a prominent but not overpowering aroma with malt and fruit notes, fusel alcohol content below 100mg / L, and other physicochemical indicators are normal.

[0175] Example 10: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in cooking wine.

[0176] First, fermentation is carried out according to the method of Example 3 to obtain rice wine. Take a portion of the rice wine sample and add 10% edible salt. Edible water, spices and caramel color can be added according to product needs. After sterilization, the rice wine co-fermented by non-brewing yeast Y34 and brewing yeast 28-23 is used as the main raw material to prepare cooking wine. The alcohol content is 10% (v / v)-15% (v / v), the amino nitrogen content is higher than 0.5g / L, the brewed cooking wine has a high ester content, is rich in amino acids, has a good flavor, and the aroma and taste components are more coordinated. The product meets the requirements of SB / T10416-2007 seasoning wine.

[0177] Example 11: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in vinegar.

[0178] Yellow rice wine was prepared according to the method in Example 3, and the prepared yellow rice wine was used as raw material for acetic acid fermentation.

[0179] The vinegar brewing process employs solid-state fermentation: Yellow rice wine, wheat bran, and rice husks are mixed in a 10:4:1 mass ratio. After inoculating the mixture with 3%-8% vinegar mash, the mash is turned, maintaining a fermentation temperature of 35℃-40℃. For the first two days, the surface of the mash is turned. From day 2-8, the mash is turned from top to bottom. From day 8-12, the mash is turned from bottom to top to cool it down. After fermentation, the raw vinegar is obtained by leaching, sterilized, and then aged in open-air tanks. Vinegar from different years requires high-temperature sterilization at 85℃ for 30 minutes before hot-pipe bottling. The resulting vinegar has normal physicochemical properties after fermentation, with acetic acid content of 50-80 g / L and low ethyl carbamate content, improving safety.

[0180] Example 12: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in fruit wine.

[0181] Using green plums as raw material, the plums are washed, pitted, drained, and crushed. Then, a 2-3 times volume of fructose syrup solution is added to obtain 100L of the mixture, which is then packaged into 150L fermenters. 20mg / L-40mg / L pectinase (enzyme activity 20000U / g) and 40mg / L-80mg / L potassium metabisulfite are added, and each is inoculated at a rate of 1% (v / v)-5% (v / v) with 1x10 [units of fertilizer]. 7A fermentation culture of *Saccharomyces cerevisiae* Y34 and *Saccharomyces cerevisiae* 28-23 at a concentration of CFU / mL was carried out for primary fermentation at 20℃-25℃ and pH 2.5-3.5 for 10-15 days, followed by secondary fermentation at 5℃-8℃ for 5-10 days to obtain fermented plum wine. Each group had two parallel fermentation tanks. The fermented plum wine had an alcohol content of 11% (v / v)-13% (v / v) and a total sugar content (as glucose) of <40g / L. Sensory evaluation results showed that the plum wine brewed with the added *Saccharomyces cerevisiae* Y34 and *Saccharomyces cerevisiae* 28-23 fermentation agents had a harmonious body, a smooth taste, and better fruit wine quality. Other indicators of the fermented plum wine met the requirements of GB 2758-2012 and QB / T 5476-2020 General Technical Requirements for Fruit Wine.

[0182] Example 13 Application of Wickham's abnormal yeast strain Y34 with low fusel alcohol production and high ester production in fruit vinegar

[0183] Using the fruit wine obtained in Example 12 as raw material, acetic acid fermentation was carried out. The fruit vinegar was brewed using a liquid fermentation process, inoculated with 1%-3% acetic acid bacteria, and fermented at 34℃ for 16 days. After leaching, fining, clarification, degassing, and sterilization, the finished fruit vinegar was obtained. The resulting fruit vinegar had an acetic acid content of 3%-8% (g / 100mL), and other physicochemical indicators were normal, meeting the national standard "Apple Cider Vinegar Beverage" GB / T 30884-2014. It had a rich fruit vinegar flavor, a smooth and refreshing taste, and a unique flavor.

[0184] Example 14: Application of the abnormally low fusel oil and high ester production strain Y34 of Wickham's yeast in fermented nutritional ice cream.

[0185] The main ingredients are drinking water, pressed fruit juice, sugar, and honey, mixed in a ratio of (2-4):(0.05-0.2):(0.05-0.2):(0.01-0.05) to achieve a sugar concentration of 10 Brix-20 Brix. The pH is adjusted to 4-5 with food-grade lactic acid. The culture is then inoculated with 5%-10% malt extract to achieve a bacterial concentration of 10 Brix. 6 -10 8Atypical Wickham Y34 and Saccharomyces cerevisiae 28-23 starter cultures (CFU / mL) were cultured in a constant temperature incubator at 30℃±2℃ for 24-48 hours with shaking. After fermentation, the mixture was centrifuged, and the supernatant or the rice wine obtained in Example 3 was used to replace drinking water. 30%-50% whole milk powder, 1%-5% thickener, 1%-5% emulsifier, 1%-5% leavening agent, and 5%-10% non-dairy creamer were added to the mixture until the total dry matter content was 20%-30%. The mixture was then pasteurized, homogenized, and then aged at 4℃ for about 1 hour. After freezing and stirring, the mixture was dispensed into molds, frozen, and unmolded to obtain a nutritious fermented ice cream. The resulting ice cream has a light, sweet, mellow aroma with floral and fruity notes. While providing a cooling and refreshing effect, it has a smooth taste and reduces the initial irritation and sweetness of the ice cream.

[0186] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A fermenting agent, characterized in that, Contains abnormal Wickham yeast ( Wickerhamomyces anomalus Y34 and brewer's yeast ( Saccharomyces cerevisiae )28-23; The abnormal Wickham yeast Y34 was deposited at the China Center for Type Culture Collection on May 15, 2024, with accession number CCTCC NO: M 2024923; The brewing yeast 28-23 was deposited at the China Center for Type Culture Collection on May 15, 2024, with accession number CCTCC NO: M 2024924.

2. The method for preparing the fermenting agent according to claim 1, characterized in that, The *Saccharomyces cerevisiae* 28-23 and *Saccharomyces cerevisiae* Y34 were cultured separately in a culture medium for a period of time, and the cell culture broth was collected and mixed to serve as a starter culture; or the *Saccharomyces cerevisiae* 28-23 and *Saccharomyces cerevisiae* Y34 were co-cultured in a culture medium for a period of time, and the cell culture broth was collected to serve as a starter culture.

3. The method according to claim 2, characterized in that, The culture medium is rice saccharification liquid culture medium.

4. The application of the fermenting agent according to claim 1 in the preparation of alcoholic beverages, characterized in that, The alcoholic beverages mentioned are rice wine, sake, rice wine, or fruit wine.

5. The application according to claim 4, characterized in that, The quantity of the brewing yeast 28-23 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL; the quantity of the abnormal Wickham yeast Y34 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL.

6. A method for increasing the ester content and / or decreasing the fusel oil content in alcoholic beverages, characterized in that, Co-fermentation is carried out using the fermentation agent according to claim 1; the esters include acetate, ethyl lactate, ethyl propionate, ethyl hexanoate, ethyl octanoate, and isoamyl acetate.

7. The method according to claim 6, characterized in that, The quantity of the brewing yeast 28-23 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL; the quantity of the abnormal Wickham yeast Y34 in the fermentation system is ≥1×10⁻⁶. 7 CFU / mL.

8. The method according to claim 6, characterized in that, The method includes: (1) Add a fermentation agent containing the brewing yeast 28-23 and the abnormal Wickham yeast Y34 to the rice-water mixed fermentation system, and add wheat koji and 3%-5% alcohol sweat at a ratio of 12%-15% of the raw rice mass. Complete the material mixing at 25℃-28℃ and pre-ferment at 15~25℃ for 4~5 days. (2) Cool down to 14~16℃, let stand and ferment for 14~16 days; (3) Filter the fermented mash obtained in step (2) and clarify it to obtain an alcoholic beverage.

9. The method according to claim 8, characterized in that, Yellow wine beverages can also be made by adding 1‰-3‰ caramel coloring to alcoholic beverages and then frying them.

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