Zygosaccharomyces rouxii and its application in fermented food

Abstract

The application discloses a strain of Zygosacharomyces rouxii and application of the strain in fermented food, and belongs to the field of fermentation engineering and biotechnology. The application provides a strain of Zygosacharomyces rouxii MY03, and the preservation number is CCTCC NO: M 20221652. The Zygosacharomyces rouxii MY03 is used for yellow rice wine fermentation, and the alcohol content, fusel and amino acid content of the prepared yellow rice wine have no significant difference compared with a traditional fermentation method, the organic acid content is reduced, the ethyl ester content is increased by 50.82%, the fusel and ester of the produced yellow rice wine are coordinated, the astringency, sourness and alcohol aroma are reduced, the aroma and taste are more balanced, the content of ethyl vinyl guaiacol is increased from 9.16 mu / g to 74.31 mu / g, the herbal aroma is prominent, and the flavor and sensory diversity of the yellow rice wine are increased.

Description

Technical Field

[0001] This invention relates to a strain of *Zygosacchariformis* and its application in fermented foods, belonging to the fields of fermentation engineering and biotechnology. Background Technology

[0002] Traditional rice wine production uses fermentation agents (wheat koji and yeast starter) that provide a rich variety of brewing microorganisms, including bacteria, fungi, and yeast. Modern rice wine production, however, uses pure cultured yeast instead of yeast starter. Compared to traditional methods, traditional rice wine generally has better quality, indicating that microorganisms other than brewer's yeast contribute significantly to the flavor and quality formation of rice wine. Many studies have used high-throughput sequencing (HTS) technology (bacterial 16S rRNA genes, fungal ITS2, and metagenomics) to analyze the microbial diversity of rice wine fermentation agents and their impact on the fermentation process. Based on cultivation methods, a large number of brewing microorganisms besides brewer's yeast have also been screened. How to utilize and leverage the value of these microorganisms in mechanized rice wine production is crucial for improving the quality of rice wine.

[0003] Saccharomyces cerevisiae is the main flavor-producing microorganism in alcoholic beverages, and its fermentation power is a crucial driving force in brewing production. Yeast utilizes sugars in raw materials to produce alcohol, while proteins and fats are converted into organic acids, amino acids, and esters through the action of yeast and other microorganisms such as lactic acid bacteria. The quality of yeast directly affects the production efficiency and flavor of the final product. In mechanized rice wine production, the alcohol metabolism rate is relatively fast during the main fermentation stage. Due to ethanol tolerance issues, many strains that actively contribute to fermentation cannot function effectively. Non-saccharomyces cerevisiae, as important microorganisms in rice wine brewing, can influence the quality of rice wine (ethanol production, volatile flavor compounds, organic acids, amino acids, etc.). Non-saccharomyces cerevisiae can secrete extracellular enzymes such as proteases, pectinases, glucosidases, lipases, and cellulases, which act on relevant substrates in the raw materials to produce alcohols, esters, acids, terpenes, and other substances, thus affecting the quality and flavor of the alcoholic beverage. Numerous studies have demonstrated the abundance of non-saccharomyces cerevisiae during rice wine fermentation, but due to ethanol tolerance issues, they cannot play a significant positive role.

[0004] Studies have shown 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 the wine and beer industries. Different types of non-brewing yeast may make different contributions to the quality of alcoholic beverages. However, due to the complexity and uniqueness of the rice wine production process, there is currently little research on non-brewing yeast in rice wine, and it is basically a blank area. Summary of the Invention

[0005] To address the aforementioned issues, and considering the current lack of superior non-brewing yeasts in the production of traditional fermented foods such as rice wine, cooking wine, and vinegar to enhance product flavor diversity and quality, this invention provides a strain of *Zygosacchariformis* with excellent stress resistance and good fermentation performance, and its application in fermented foods.

[0006] This invention provides a strain of Zygosacharomyces rouxii MY03, classified and named Zygosacharomyces rouxii MY03, which was deposited at the China Center for Type Culture Collection on October 25, 2022, with accession number CCTCC NO:M 20221652, and the deposit address is Wuhan University, Wuhan, China.

[0007] The present invention also provides a fermentation agent containing the aforementioned *Zygosaccharomyces rouxii* MY03.

[0008] In one embodiment, the fermenting agent contains *Zygosaccharomyces rouxii* MY03 and *Saccharomyces cerevisiae*.

[0009] In one embodiment, the fermenting agent contains *Zygosaccharomyces rouxii* MY03, *Candida tropicalis* CS8, and *Saccharomyces cerevisiae*.

[0010] In one embodiment, the brewing yeast is brewing yeast jiangnan1#, with accession number CCTCC NO:M2021523, which has been disclosed in patent application document CN113621528A.

[0011] In one embodiment, the Candida tropicalis CS8 was deposited on October 25, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20221651, located at Wuhan University, Wuhan, China.

[0012] In one embodiment, the fermentation agent is prepared by:

[0013] (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.

[0014] (2) Inoculate the rice saccharification liquid culture medium prepared in step (1) with Saccharomyces cerevisiae jiangnan1#, Zygosaccharomyces rouxiensis MY03 or Candida tropicalis CS8, and culture in a shaker at 28°C for 36 h to obtain Saccharomyces cerevisiae jiangnan1#, Zygosaccharomyces rouxiensis MY03 or Candida tropicalis CS8 culture medium.

[0015] The present invention also provides a method for co-fermentation of brewing yeast jiangnan1# with non-brewing yeast.

[0016] In one embodiment, the non-Saccharomyces cerevisiae includes Zygomyces rouxii MY03 and / or Candida tropicalis CS8.

[0017] In one embodiment, the method involves fermenting the brewing yeast jiangnan1# and the zygosacchariformis MY03 in a ratio of 1:(1-1000).

[0018] In one embodiment, the method involves fermenting the brewing yeast jiangnan1# and the Lurvii zygosacchari yeast MY03 in a ratio of 1:1, 1:10, or 1:100.

[0019] In one embodiment, the method involves fermenting the brewing yeast jiangnan1# with the yeast MY03 and the Candida tropicalis CS8 in a 1:1:1 ratio.

[0020] In one embodiment, the method involves fermenting the brewing yeast jiangnan1# and the Lurvii zygosacchari yeast MY03 in a ratio of 1:1, 1:10, or 1:100.

[0021] In one embodiment, the quantity of the *Zygosaccharomyces rouxii* MY03 in the fermentation system is ≥1×10⁻⁶. 6 CFU / mL.

[0022] In one embodiment, the quantity of the tropical Candida CS8 in the fermentation system is ≥1×10⁸. 6 CFU / mL.

[0023] In one embodiment, the amount of the brewing yeast jiangnan1# in the fermentation system is ≥1×10⁻⁶. 6 CFU / mL.

[0024] In one embodiment, the method includes:

[0025] (1) Add brewing yeast jiangnan1# and MY03 fermentation agent to the rice-water mixed fermentation system, and add wheat koji at a ratio of 12%-15% of the raw rice mass. Complete the material mixing at 25℃-28℃, and let it stand for 3-5 days for pre-fermentation at 20℃-35℃.

[0026] (2) Lower the temperature of the fermentation tank to 10℃-15℃ and let it stand for 15-20 days for post-fermentation.

[0027] (3) The fermented mash obtained in step (2) is pressed by plate and frame pressing and filtered by 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, and 1‰-3‰ caramel coloring is added to the wine and then it is heated to obtain yellow wine.

[0028] The present invention also provides the application of the said Lurva zygosaccharidus MY03, or the said starter culture, or the said method in the production of fermented condiments.

[0029] In one embodiment, the fermented seasoning includes, but is not limited to, rice wine, cooking wine, or vinegar.

[0030] In one embodiment, the rice wine is prepared by adding the brewing yeast jiangnan1# and non-brewing yeast as quick-brewing starter at an inoculation ratio of 1:1 or 1:10, with a total addition amount of 5%-15%, to the cooked or gelatinized raw materials, and then fermenting, pressing, heating, aging, filtering, sterilizing, and bottling.

[0031] In one embodiment, the cooking wine is prepared by first fermenting rice wine using the brewing yeast jiangnan1# and non-brewing yeast as quick-fermenting starter, and then using the rice wine to prepare the cooking wine.

[0032] In one embodiment, the application is used for vinegar brewing. First, the brewing yeast jiangnan1# and non-brewing yeast are used as quick-fermenting starter to ferment rice wine, and then the rice wine is used as a raw material for acetic acid fermentation to brew vinegar.

[0033] In one embodiment, the non-Saccharomyces cerevisiae includes Zygomyces rouxii MY03 and / or Candida tropicalis CS8.

[0034] Beneficial effects:

[0035] (1) The excellent Zygosacharomyces rouxii MY03 strain obtained by this invention has good tolerance. When applied to rice wine, there is no significant difference in alcohol content, fusel oil and amino acid content, but the organic acid content is reduced, while the content of ethyl esters is increased by 50.82%.

[0036] (2) The use of non-brewing yeast, *Saccharomyces rouxii* MY03, produces rice wine with a more balanced aroma and taste, and the content of p-vinylguaiacol in the produced rice wine, cooking wine and vinegar increases significantly (P<0.01), from 9.16 μg / L to 74.31 μg / L, with a prominent herbal aroma.

[0037] (3) Simultaneous inoculation with two non-brewing yeasts and brewing yeast jiangnan1# can co-ferment rice wine. The resulting rice wine has different sensory characteristics and a better overall taste (the sourness, astringency and bitterness are reduced, and the sweetness and umami are enhanced), which is closer to the sensory style characteristics of traditional rice wine.

[0038] (4) The present invention also allows the use of Lubricated yeast MY03 in fermented foods such as vinegar and soy sauce, which is of great significance for the flavor diversity and quality improvement of alcoholic beverages such as rice wine and vinegar and soy sauce.

[0039] Preservation of biological materials

[0040] Zygosacharomyces rouxii MY03, taxonomically named Zygosacharomyces rouxii MY03, was deposited on October 25, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221652, located at Wuhan University, Wuhan, China.

[0041] Candida tropicalis CS8, classified as Candida tropicalis CS8, was deposited on October 25, 2022, at the China Center for Type Culture Collection (CCTCCNO: M20221651), Wuhan University, Wuhan, China. Attached Figure Description

[0042] Figure 1 This is a colony morphology diagram of Zygosacharomyces rouxii MY03, the present invention.

[0043] Figure 2 Sensory quality of co-fermentation of brewer's yeast jiangnan1# and Zygosacharomyces rouxii MY03.

[0044] Figure 3 The amino acid content of *Saccharomyces cerevisiae* jiangnan1# and *Zygosacharomyces rouxii* MY03 co-fermented.

[0045] Figure 4 The amino acid content was determined by co-fermentation of *Saccharomyces cerevisiae* jiangnan1# with *Zygosacharomyces rouxii* MY03 and *Candida tropicalis* CS8.

[0046] Figure 5 Sensory qualities of *Saccharomyces cerevisiae* jiangnan1#, *Zygosacharomyces rouxii* MY03, and *Candida tropicalis* CS8 were evaluated. Detailed Implementation

[0047] 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 acids and amino acid content were determined using high-performance liquid chromatography (HPLC), and volatile flavor substances 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.

[0048] 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.

[0049] YPD medium: 10g yeast extract, 20g peptone, 20g glucose, add water to 1000mL, 2% agar, autoclave at 121℃ for 20min, cool and set aside.

[0050] 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 add saccharifying enzyme (150U / g-300U / g) and liquefying enzyme (200U / g-400U / g) based on the rice raw material, and add raw wheat koji (10% of the rice weight). Saccharify at 55℃-65℃ for 4-5 hours until the sugar content is above 13 Brix. Dispense into containers and autoclave at 121℃ for 15-20 minutes. Cool and set aside for later use.

[0051] Example 1: Isolation and screening of Zygomycosis MY03

[0052] (1) Sample preparation and strain isolation

[0053] Juice from fresh kiwifruit was collected and serially diluted with sterile water (10⁻⁶). -1 -10 -5 Take 100 μL of the diluted sample and spread it on a YPD plate. Incubate at 28°C upside down for 48 h. Select dilutions with single colonies and choose strains whose colony morphology, color, and appearance conform to the physiological morphology of yeast. Perform multiple streaking tests to purify the strains. Finally, number and preserve the obtained pure strains.

[0054] (2) Strain identification

[0055] Based on the morphological characteristics of the strain, ITS sequencing and comparison analysis with the NCBI database showed that the homology between the strain and Candida tropicalis was greater than 99%, thus identifying Zygomyces rouxii. It was deposited at the China Center for Type Culture Collection on October 25, 2022, with accession number CCTCC NO:M 20221652, and the deposit address is Wuhan University, Wuhan, China.

[0056] Based on the morphological characteristics of the strain, ITS sequencing and comparison analysis with the NCBI database showed that the homology between the strain and *Candida tropicalis* was greater than 99%, thus confirming the identification of *Candida tropicalis*. The strain was deposited on October 28, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20221651, located at Wuhan University, Wuhan, China.

[0057] Example 2: Preparation of fermentation agent for a co-fermentation system of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae

[0058] (1) Liquid culture of yeast strains: Take a loop of glycerol tube containing Saccharomyces cerevisiae jiangnan1# or Zygosaccharomyces rouxii MY03 or Candida tropicalis CS8 strains using a 10 μL inoculation loop, streak in zone 3 on a YPD plate to obtain single colonies, pick single colonies and culture them in 100 mL of YPD medium at 37 °C for 24 h to obtain Saccharomyces cerevisiae jiangnan1# liquid culture, or Zygosaccharomyces rouxii MY03 liquid culture, or Candida tropicalis CS8 liquid culture.

[0059] (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.

[0060] (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.

[0061] (4) Cooling the rice: Spread out the steamed rice and let it cool until the temperature is below 60°C.

[0062] (5) Rice saccharification liquid culture medium: Mix raw rice and water (1:4 mass ratio), add liquefying enzyme (2‰ of the mass of cooked rice), saccharifying enzyme (1‰) and raw wheat koji (10%), control the temperature at 60℃ in a water bath, saccharify and liquefy for about 4 hours, measure the sugar content with a refractometer until the sugar content is greater than 13°Brix, filter through filter bags after saccharification, dispense, sterilize at 115℃ for 20 minutes, cool and use for later use.

[0063] (6) Erlenmeyer flask yeast culture medium: The yeast cultured in liquid culture in (1) was inoculated into rice saccharification liquid culture medium at a ratio of 1‰, and cultured in a shaker at 28℃ for 36h to obtain the culture medium of Saccharomyces cerevisiae jiangnan1#, and then serially diluted 10% at equal ratios. -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℃ for 36h. The number of yeasts in the Erlenmeyer flask yeast culture medium was determined by plate counting. At the same time, 1.5mL of the yeast cultured in step (1) was stored in EP tubes at low temperature (4℃) as seed culture for subsequent inoculation.

[0064] (7) Preparation of fermentation agent: Take the culture medium of Saccharitomyces cerevisiae jiangnan1# 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 fermentation agent containing only Saccharitomyces cerevisiae jiangnan1#.

[0065] Take the MY03 culture of Zygosacchariformis or the CS8 culture of Candida tropicalis obtained in step (1), and inoculate them into rice saccharification liquid culture medium at a ratio of 1‰. Culture them in a shaker at 28℃ for 36h to obtain a pure yeast starter containing only the MY03 culture of Zygosacchariformis or the CS8 culture of Candida tropicalis.

[0066] The concentration of S. cerevisiae jiangnan1# was measured to be 1×10⁻⁶. 8 CFU / mL, the concentration of *Zygosaccharomyces rouxii* MY03 or *Candida tropicalis* CS8 is 1×10⁻⁶. 7 CFU / mL ~ 1×10 8 CFU / mL.

[0067] Example 3: Sequential inoculation and co-fermentation process of *Saccharomyces rouxii* MY03 and *Saccharomyces cerevisiae*

[0068] Sequential inoculation and co-fermentation process of *Zygosacchariformis* MY03 and *Sacchariformis jiangnan1#*: The concentration of *Sacchariformis jiangnan1#* starter culture was determined based on the dilution plate count results (1×10⁻⁶ cells / year). 8 CFU / mL) and the fermentation agent of *Zygosaccharomyces rouxii* MY03 (concentration of 1×10⁻⁶ CFU / mL) 8 The inoculation ratios (CFU / mL) were 1:1 (25mL+25mL), 1:10 (5mL+45mL), and 1:100 (0.5mL+50mL) to a 1.5L fermentation system, with jiangnan1# (50mL) single inoculation and non-Saccharomyces cerevisiae (50mL) single inoculation as controls.

[0069] To explore the potential application value of non-Sacchariform yeasts in rice wine brewing, yeast saccharification broth culture media (secondary seed culture) prepared from different yeast strains were used as fermentation agents to evaluate non-Sacchariform yeasts, following the method in Example 2. First, the sequential inoculation method was investigated. Sacchariform yeast jiangnan1# and Zygomyces rouxii MY03 were inoculated at ratios of 1:1, 1:10, and 1:100 (volume ratios). Zygomyces rouxii MY03 was inoculated first for fermentation, followed by S. cerevisiae jiangnan1# 24 hours later.

[0070] The preparation method of the rice wine fermentation agent is the same as in Example 2, and the rice wine fermentation is carried out in the following manner:

[0071] 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%.

[0072] b) The raw materials are added and fermented according to the traditional proportions for rice wine fermentation. The specific steps are as follows:

[0073] S1. Add the raw rice and water to the sterilized fermentation container. Add 50 mL of the prepared starter culture to the rice-water mixed fermentation system (1.5 L). Inoculate with Saccharomyces cerevisiae jiangnan1# and Saccharomyces rouxii MY03 in a ratio of 1:1, 1:10 and 1:100 respectively. First, inoculate with Saccharomyces rouxii MY03 for fermentation. Then, inoculate with S. cerevisiae jiangnan1# 24 h after inoculation. Add wheat koji in a ratio of 12%-15% of the raw rice mass. Complete the material mixing at 25℃-28℃. Let it stand at 28℃ for pre-fermentation for 3-5 days.

[0074] S2. Lower the temperature of the fermentation tank to 15℃ and let it stand for 15-20 days for post-fermentation.

[0075] S3. The fermented mash obtained in S2 is processed through plate and frame pressing (4 feedings, mash pressure of 0.2-0.6 MPa, filtration area of ​​100 m2, filter plate diameter of 1 m) and diatomaceous earth filtration (diatomaceous earth addition ratio of 4%-6%, pressure of 0.3-0.5 MPa). The filtrate is clarified to obtain clear wine. The clear wine is blended according to the national standard for yellow wine, with the addition of 1‰-3‰ caramel coloring and decoction to obtain yellow wine.

[0076] The results showed that inoculating *Zygosacchariformis* MY03 first, followed by *Saccharitomyces cerevisiae* jiangnan1#, resulted in an alcohol content <14% vol and a total acid content >8.0 mg / L, leading to rancidity. However, starch saccharification and liquefaction (residual sugar >50.0 g / L) were normal. The reason for incomplete fermentation is that the fermentation process of rice wine is spontaneous, involving a variety of microorganisms, including pure culture microorganisms, environmental microorganisms, and microorganisms from the koji (fermentation starter). In the early stage of fermentation (the first 24 hours), the yeast produced relatively little ethanol, which was insufficient to inhibit the growth of other microbial communities. Meanwhile, the acid-producing bacteria produced excessively high acid concentrations, which may have led to rancidity when inoculating *Zygosacchariformis* MY03 first, followed by *S. cerevisiae* jiangnan1#.

[0077] Example 4: Determination of inoculation method and ratio for the co-fermentation system of *Zygosacchariformis MY03* and *Sacchariformis sacchariformis*.

[0078] The preparation method of the rice wine fermentation agent is the same as in Example 2. The rice wine fermentation is carried out according to Example 3, except that the fermentation agent is inoculated simultaneously: S. cerevisiae jiangnan1# fermentation agent and zygosacchariformis MY03 fermentation agent are co-inoculated at addition ratios of 1:1, 1:10 and 1:100 respectively to carry out small-scale rice wine fermentation. The specific steps are as follows:

[0079] 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%.

[0080] b) The raw materials are added and fermented according to the traditional proportions for rice wine fermentation. The specific steps are as follows:

[0081] S1. Add the raw rice and water to the sterilized fermentation container. Add 50mL of brewing yeast jiangnan1# and Lubricated yeast MY03 (volume ratio of bacteria to liquid is 1:1, 1:10 or 1:10) to the 1.5L rice-water mixed fermentation system. Add wheat koji at a ratio of 12%-15% of the raw rice weight. Complete the material mixing at 25℃-28℃ and let it stand for pre-fermentation for 3-5 days at 20℃-35℃.

[0082] S2. Lower the temperature of the fermentation tank to 10℃-15℃ and let it stand for 15-20 days for post-fermentation.

[0083] 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.

[0084] The results showed that most of the co-fermentation groups of *Saccharomyces cerevisiae* jiangnan1# and *Zygosaccharomyces rouxii* MY03 at inoculation ratios of 1:1 and 1:10 were able to complete fermentation. Different mixed fermentation ratios had different effects on the fermentation of rice wine. Although fermentation with *Zygosaccharomyces rouxii* MY03 alone could produce more flavor compounds, it usually resulted in spoilage and could not complete the fermentation of rice wine. When *S. cerevisiae* was inoculated with non-saccharomyces cerevisiae at an inoculation ratio of 1:100, the small amount of *S. cerevisiae* led to a slow increase in alcohol content in the early stage of primary fermentation, which could not inhibit the growth of other microorganisms. After fermentation, the alcohol content was low and the total acid was high, which posed a risk of fermentation stagnation or termination. When the addition ratio of *S. cerevisiae* to non-saccharomyces cerevisiae* was 1:1 and 1:10, most non-saccharomyces cerevisiae were able to ferment normally, and the physicochemical indicators met the national standards for rice wine.

[0085] Simultaneous inoculation of *Saccharomyces cerevisiae* jiangnan1# and *Zygosacchariformis* MY03 at a 1:1 ratio met the fermentation requirements of rice wine, with no significant differences in physicochemical indicators. The total amounts of the four main fusel oils showed no significant difference. Volatile substances showed a decrease in acetate and an increase in ethyl acetate content, with a significant increase in p-vinylguaiacol content, from 9.16 μg / L to 74.31 μg / L. Compared with non-*Saccharomyces cerevisiae* MY03, there was no significant difference in the total amino acid content between *S. cerevisiae* jiangnan1# and the control, but the contents of lactic acid and acetic acid were significantly lower (P<0.05), while the contents of oxalic acid, tartaric acid, and citric acid were higher (P<0.05). Sensory results showed that the overall taste and aroma were lower than the control (P<0.01), astringency, sourness and mellowness were significantly reduced (P<0.05), smoky aroma, caramel aroma, fruity aroma and honey aroma were significantly reduced (P<0.05), and herbal aroma was significantly increased (P<0.05). The produced rice wine had a harmonious balance of alcohols and esters, and a more balanced aroma and taste.

[0086] Table 1. Physicochemical indicators of mixed fermentation of non-Saccharomyces cerevisiae MY03 and S. cerevisiae jiangnan1#

[0087]

[0088] Note: a The total acid content, expressed as lactic acid, is represented by the mean ± standard deviation of at least three independent tests.

[0089] Table 2. Volatile Matter Content in Mixed Fermentation of Non-Saccharomyces cerevisiae MY03 and S. cerevisiae jiangnan1#

[0090]

[0091] Table 3 shows the organic acids in rice wine co-fermented with MY03 and jiangnan1# and rice wine fermented separately with jiangnan1#.

[0092]

[0093]

[0094] Note: Values ​​are the mean ± standard deviation of at least three independent tests, * indicates a significant difference (P<0.05).

[0095] Example 5: Application of co-fermentation of *Zygosaccharomyces rouxii* MY03, *Candida tropicalis* CS8, and *Saccharomyces cerevisiae* jiangnan1# in the production of Shaoxing rice wine.

[0096] The preparation method of the rice wine fermentation agent is the same as in Example 2. The rice wine fermentation is carried out according to Example 4. The difference is that the fermentation agents of S. cerevisiae jiangnan1#, Zygomycosis rouxii MY03 and Candida tropicalis CS8 are inoculated in a ratio of 1:1:1.

[0097] The results showed that the co-fermentation method could meet the requirements of rice wine fermentation, with no significant differences in physicochemical indicators. The total amounts of the four main fusel oils were also not significantly different. Volatile substances showed a decrease in acetate and an increase in ethyl acetate content, with p-vinylguaiacol content significantly increasing to 91.62 μg / L. Compared with non-Sacchariform yeast MY03, the total amino acid content of S. cerevisiae jiangnan1# inoculated with this method was not significantly different, but the contents of lactic acid and acetic acid were significantly lower than the control (P<0.05), while the contents of oxalic acid, tartaric acid, malic acid, and citric acid were higher than the control (P<0.05). Sensory results showed that, compared with the control, the overall taste intensity increased (P<0.05), which was associated with a significant decrease in astringency, sourness, and acidity (P<0.01) and a significant increase in sweetness (P<0.05). The overall aroma intensity decreased (P<0.05), which was associated with a significant decrease in mellow aroma, caramel aroma, fruit aroma, and honey aroma (P<0.05) and a significant increase in yeast aroma and herbal aroma (P<0.05). The produced rice wine had a harmonious balance of alcohols and esters, and its sensory style was closer to traditional rice wine than that of Jiangnan No. 1# new wine.

[0098] Table 4 Physicochemical indicators of mixed fermentation of non-Saccharomyces cerevisiae MY03, CS8 and S. cerevisiae jiangnan1#

[0099]

[0100] Note: a The total acid content, expressed as lactic acid, is represented by the mean ± standard deviation of at least three independent tests.

[0101] Table 5. Volatile Matter Content in Mixed Fermentation of Non-Saccharomyces cerevisiae MY03, CS8, and S. cerevisiae jiangnan1#

[0102]

[0103] Table 6. Organic acids in rice wines co-fermented with MY03, CS8, and jiangnan1#, and rice wines fermented solely by jiangnan1#.

[0104]

[0105]

[0106] Note: Values ​​are the mean ± standard deviation of at least three independent tests, * indicates a significant difference (P<0.05).

[0107] Example 6: Application of co-fermentation of non-brewing yeast and brewing yeast jiangnan1# in cooking wine

[0108] First, fermentation is carried out according to the method of Example 4 or 5 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 fermented by non-brewing yeast MY03 alone or co-fermented with brewing yeast jiangnan1# 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, significantly increases the content of vinyl guaiacol, and has a more harmonious aroma and taste components. The product meets the requirements of SB / T 10416-2007 for seasoning wine.

[0109] Example 7: Application of co-fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae jiangnan1# in vinegar.

[0110] The rice wine obtained by referring to the method in Example 4 or 5 was used as raw material for acetic acid fermentation.

[0111] The vinegar brewing process employs solid-state fermentation: Yellow rice wine, wheat bran, and rice husks are mixed in a mass ratio of 10:4:1. 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 an acetic acid content of 50-80 g / L and a significantly increased content of vinyl guaiacol. It has a mellow, clean taste and a unique flavor.

[0112] Example 8: Application of non-brewing yeast MY03 in high-salt, dilute-state soy sauce fermentation

[0113] Soak soybeans in water at a 1:2 ratio until fully cooked. Drain the soybeans, steam them until cooked, and after cooling, mix them with flour at a 5:1 mass ratio. Simultaneously, add 0.05‰-0.1‰ of Aspergillus oryzae spore powder (10g) of the total raw material weight. 8The koji (CFU / g) was cultured at a constant temperature of 30℃ and a relative humidity of 70%-90% in a biochemical incubator. The koji was turned frequently, and after 48 hours, soy sauce koji was obtained, which can be used for soy sauce fermentation. The soy sauce koji was mixed with a 250g / L saline solution at a mass ratio of 1:2, and simultaneously, 10g of the *Gnaphalium rayense* selected in Example 1 of this invention was added. 6 -10 8 Fermentation was carried out at 30℃ for 60-90 days using a starter culture of (CFU / g) alone or in combination with *Saccharomyces cerevisiae* jiangnan1#. After fermentation, the resulting soy sauce mash was mixed with brine at a 1:1 mass ratio and soaked for 48 hours. The extract was then extracted, and the salt concentration was adjusted with brine as needed to obtain low-salt, diluted soy sauce. Flavor component analysis showed that the soy sauce fermented with *Zygosaccharomyces rouxii* MY03 had a significantly higher relative content of esters than naturally fermented soy sauce. Furthermore, the soy sauce fermented with both *Zygosaccharomyces rouxii* MY03 and *Saccharomyces cerevisiae* jiangnan1# had significantly higher relative contents of esters, organic acids, amino acids, and p-vinylguaiacol than naturally fermented soy sauce.

[0114] Comparative Example 1:

[0115] The specific implementation method is the same as in Example 4, except that the MY03 strain of *Zygosacchariformis* was replaced with another strain of *Zygosacchariformis*, MY04. The results showed that the rice wine prepared by co-fermentation became rancid and could not ferment normally.

[0116] Table 7 Comparison of physicochemical properties of different *Zygosacchariformis* co-fermented with jiangnan1# yeast

[0117]

[0118] 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. Luvian zygosacchari ( Zygosacharomyces rouxii MY03 was deposited at the China Center for Type Culture Collection on October 25, 2022, with accession number CCTCC NO:M 20221652.

2. A fermenting agent, characterized in that, The fermentation agent contains the *Zygosaccharomyces rouxii* MY03 as described in claim 1 and *Saccharomyces cerevisiae* jiangnan1#; the preservation number of *Saccharomyces cerevisiae* jiangnan1# is CCTCCNO:M 2021523.

3. The fermenting agent according to claim 2, characterized in that, The fermentation agent contains *Zygosaccharomyces rouxii* MY03 and *Candida tropicalis* (…). Candida tropicalis ) CS8 and Saccharomyces cerevisiae jiangnan1#; the aforementioned Candida tropicalis CS8 was deposited at the China Center for Type Culture Collection on October 25, 2022, with accession number CCTCC NO:M20221651.

4. The fermenting agent according to claim 2 or 3, characterized in that, The cell concentration in the fermentation agent is ≥1×10⁻⁶. 7 CFU / mL.

5. A method for co-fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae, characterized in that, The *Zygosacchariformis* MY03 of claim 1 was co-fermented with *Sacchariformis jiangnan1#*; the preservation number of *Sacchariformis jiangnan1#* is CCTCCNO:M 2021523.

6. The method according to claim 5, characterized in that, The brewing yeast jiangnan1# and the zygosacchariformis MY03 were fermented in a ratio of 1:(1~1000).

7. A method for co-fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae, characterized in that, The brewing yeast jiangnan1# was fermented with the Zygosaccharomyces rouxii MY03 and Candida tropicalis CS8 described in claim 1 in a ratio of 1:1:1; the Candida tropicalis CS8 was deposited at the China Center for Type Culture Collection on October 25, 2022, with accession number CCTCC NO:M20221651; the brewing yeast jiangnan1# has accession number CCTCC NO:M 2021523.

8. The application of the yeast MY03 of claim 1, or the starter culture of any one of claims 2 to 4, or the method of any one of claims 5 to 7 in the production of fermented condiments.

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