Saccharomyces cerevisiae, fermentation inoculant containing saccharomyces cerevisiae, application of saccharomyces cerevisiae and fermentation inoculant and method for producing fruit wine

Abstract

The invention relates to the field of microorganisms and discloses saccharomyces cerevisiae, a fermentation inoculant containing the saccharomyces cerevisiae, application of the saccharomyces cerevisiae and the fermentation inoculant and a method for producing fruit wine. The preservation number of the saccharomyces cerevisiae (Saccharomyces sp.) is CGMCC (China General Microbiological Culture Collection Center) No. 31571. The fermentation inoculant provided by the invention contains the saccharomyces cerevisiae. The invention also provides application of the saccharomyces cerevisiae and the microbial inoculum in production of ethanol and / or wine products. The saccharomyces cerevisiae strain provided by the invention has the properties of high glucose resistance, ethanol resistance, high salt resistance, high acid resistance, sulfur dioxide resistance and the like, has relatively strong ethanol production capacity and ester production capacity, can increase different flavors formed in a wine brewing process, and provides abundant strain resources for improving the flavor of sparkling wine.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and specifically, to a strain of Saccharomyces cerevisiae, a fermentation inoculant containing the strain, their applications, and a method for producing fruit wine. Background Art

[0002] Saccharomyces cerevisiae is a yeast closely related to humans, also known as baker's yeast or budding yeast. Its specific classification is Fungi, Ascomycota, Saccharomycetales, Saccharomycetaceae, Saccharomyces. Saccharomyces cerevisiae has the advantages of a short growth cycle, strong fermentation ability, easy large-scale cultivation, and rich nutritional components such as various proteins, amino acids, vitamins, and bioactive substances, and is widely used in the fields of food, medicine, etc. Yeasts are widely distributed in nature and prefer to grow in environments with a slightly acidic pH and high sugar content, and are most common on the surfaces of fruits, vegetables, nectar, and in orchard soils.

[0003] In recent years, research scholars have isolated excellent wild Saccharomyces cerevisiae strains from different habitats

[0004] , but the viability of wild yeasts is poor, showing varying degrees of instability in environments such as high alcohol, high sugar, high salt, acidic, and high SO2, affecting fermentation performance; different Saccharomyces cerevisiae have different metabolic pathways and enzyme systems, and thus the ethanol and ester production capabilities of the strains also vary, greatly affecting the flavor of brewing; and yeasts will produce trace amounts of H2S during the fermentation of grape juice, bringing unpleasant odors such as the smell of rotten eggs and garlic to the wine, seriously affecting the flavor and sensory quality of the wine. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects of existing wild Saccharomyces cerevisiae, and to provide a strain of Saccharomyces cerevisiae, a fermentation inoculant containing the strain, their applications, and a method for producing fruit wine. This Saccharomyces cerevisiae has strong tolerance to pH, glucose, ethanol, NaCl, and SO2 and excellent fermentation performance.

[0006] To achieve the above purpose, in the first aspect of the present invention, a Saccharomyces cerevisiae (Saccharomyces sp.) is provided, and the preservation number of the Saccharomyces cerevisiae is CGMCC No. 31571.

[0007] In the second aspect of the present invention, a fermentation inoculant is provided, and the fermentation inoculant contains the above-mentioned Saccharomyces cerevisiae.

[0008] Preferably, the fermentation inoculant is at least one of a liquid fermentation inoculant, a semi-liquid fermentation inoculant, and a solid fermentation inoculant.

[0009] Preferably, in the liquid fermentation inoculum, the content of the Saccharomyces cerevisiae in terms of viable cell count is 10 6 CFU / mL or more, more preferably 10 7 -10 8 CFU / mL.

[0010] Preferably, in the semi-liquid fermentation inoculum and the solid fermentation inoculum, the content of the Saccharomyces cerevisiae in terms of viable cell count is 10 8 CFU / mL or more, more preferably 10 9 -10 10 CFU / mL.

[0011] The third aspect of the present invention provides the application of the above-mentioned Saccharomyces cerevisiae and the above-mentioned fermentation inoculum in the production of ethanol and / or the production of alcoholic beverages.

[0012] Preferably, the alcoholic beverage is fruit wine.

[0013] The fourth aspect of the present invention provides a method for producing ethanol, which includes: culturing the Saccharomyces cerevisiae as described above and / or the fermentation inoculum as described above.

[0014] Preferably, the culture medium for the culture contains: yeast extract powder 5-15 g / L, peptone 15-25 g / L, glucose 15-25 g / L.

[0015] Preferably, the culture conditions include: the inoculation amount of the Saccharomyces cerevisiae is not less than 10 5 CFU / mL, the temperature is 20-35 °C, and the rotation speed is 120-180 rpm.

[0016] The fifth aspect of the present invention provides a method for producing fruit wine, which includes: subjecting the Saccharomyces cerevisiae as described above and / or the fermentation inoculum as described above to contact fermentation with fruits.

[0017] Preferably, the process of the contact fermentation includes: juicing and enzymatically hydrolyzing the fruits to obtain fruit juice, and mixing the Saccharomyces cerevisiae and / or the fermentation inoculum with the fruit juice for fermentation.

[0018] Preferably, the inoculation amount of the Saccharomyces cerevisiae in the fruit juice is not less than 10 5 CFU / mL, preferably 1×10 6 -1×10 7 CFU / mL.

[0019] Preferably, the fruits are at least one of grapes, apples, honeydews, pears, kiwifruits, blueberries, and plums.

[0020] Preferably, the fermentation conditions include: temperature of 20 - 35°C and time of 5 - 10 days.

[0021] By the above technical solution, the beneficial effects of the present invention are as follows:

[0022] The Saccharomyces cerevisiae strain provided by the present invention or the fermentation inoculant containing this strain exhibits strong fermentation ability, does not produce H2S during the fermentation process, and has stronger ethanol-producing ability and ester-producing ability, which can add different flavors to the wine-making process; this Saccharomyces cerevisiae has strong tolerance to various environmental stresses such as pH, glucose, ethanol, NaCl, and SO2, and can be effectively applied to the actual wine-making production of Xinjiang wineries, increasing the regional characteristics of Xinjiang fruit wines.

[0023] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation section.

[0024] Biological deposit

[0025] The strain provided by the present invention is Saccharomyces sp., and was deposited on August 12, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101, the abbreviation of the depositary institution is CGMCC), with the deposit number of CGMCC No. 31571. Brief description of the drawings

[0026] Figure 1 shows the colony characteristics and cell morphology of strain TY12 on YPD medium in Example 1;

[0027] Figure 2 shows the phylogenetic tree of strain TY12 based on the 26S rDNA D1 / D2 region gene sequence in Example 1;

[0028] Figure 3 shows the determination of the tolerance of strain TY12 to glucose in Example 2;

[0029] Figure 4 shows the determination of the tolerance of strain TY12 to low pH in Example 2;

[0030] Figure 5 shows the determination of the tolerance of strain TY12 to NaCl in Example 2;

[0031] Figure 6 shows the determination of the tolerance of strain TY12 to ethanol in Example 2;

[0032] Figure 7 shows the determination of the tolerance of strain TY12 to SO2 in Example 2. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In a first aspect, the present invention provides a strain of Saccharomyces sp., wherein the deposit number of the Saccharomyces sp. is CGMCC No.31571.

[0036] The brewer's yeast provided by the present invention is isolated from the rhizosphere soil of an orchard in Xinjiang (the surface soil is removed using a sterile shovel and the soil is collected at a distance of about 5 cm from the ground surface). The brewer's yeast can be isolated by a conventional method for isolating new strains in the art, for example, a gradient dilution separation method and an enrichment separation method.

[0037] The gradient dilution separation method can specifically include: mixing the soil sample with sterile water, placing it in a shaker, shaking and incubating it for 25-35 minutes at a temperature of 28-32°C and a rotation speed of 120-160 rpm, and then diluting it into a gradient of 10 -6 , 10 -7 , 10 -8 After diluting the bacterial suspension by 10 times, take 90-110 μL and apply it on the plate containing chloramphenicol, and culture it at 35-40℃ for 3-7 days. After the colonies grow, purify the colonies with different morphologies grown on the plate with the most suitable dilution of the bacterial suspension until a single colony appears. The isolated strains are stored on the slant containing chloramphenicol.

[0038] In the present invention, the culture medium used in the chloramphenicol-containing plate and the chloramphenicol-containing slant contains nutrients required for the growth of Saccharomyces cerevisiae; illustratively, PDA culture medium containing chloramphenicol can be used respectively.

[0039] During the research process, the inventors of the present invention isolated a strain of Saccharomyces sp., and found that this strain has strong fermentation ability, ethanol production ability and ester production ability, and does not produce H2S during the fermentation process, and has strong tolerance to various environmental stresses such as pH, glucose, ethanol, NaCl and SO2. Through further research, it was found that the fruit wine prepared with this strain has good sensory quality and unique flavor.

[0040] This strain was deposited in the China General Microbiological Culture Collection Center on August 12, 2024, with the deposit number CGMCC No. 31571.

[0041] The Saccharomyces sp. provided by the present invention can produce a large amount of viable cells and / or fermentation products of Saccharomyces sp. after cultivation. The present invention has no particular limitation on the cultivation method, as long as the Saccharomyces sp. can be proliferated in large quantities by this cultivation method. For example, viable cells of Saccharomyces sp. can be inoculated into the culture medium at an inoculation amount of not less than 10 5 CFU / mL, and after culturing at a temperature of 20 - 35 °C for not less than 12 h, a culture solution is obtained. Among them, the culture medium can be a culture medium conventionally used in the art. For example, it can be a YPD liquid medium (8 - 12 g / L of yeast extract powder, 15 - 25 g / L of peptone, 15 - 25 g / L of glucose).

[0042] The present invention can further separate the cells of Saccharomyces sp. in the above-mentioned culture solution. There is no particular limitation on the separation method, as long as the cells can be enriched from the culture solution. For example, it can be achieved by centrifugation and / or filtration methods. The conditions of the centrifugation and the filtration can be conventional conditions in the art, which are well known to those skilled in the art and will not be elaborated here.

[0043] In the second aspect, the present invention provides a fermentation inoculant, wherein the fermentation inoculant contains the above-mentioned Saccharomyces sp.

[0044] In the present invention, there is no particular limitation on the specific type of the fermentation inoculant, and it can be any existing inoculant type in the art. According to the preferred embodiment of the present invention, the fermentation inoculant is at least one of a liquid fermentation inoculant, a semi-liquid fermentation inoculant and a solid fermentation inoculant.

[0045] In the present invention, there is no particular limitation on the specific content of Saccharomyces sp. contained in the fermentation inoculant, and specific selection can be made according to specific circumstances. Among them, in different types of fermentation inoculants, the content of Saccharomyces sp. can be the same or different.

[0046] Preferably, in the liquid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is 10 6 CFU / mL or more, more preferably 10 7 -10 8 CFU / mL. In the present invention, 10 6 CFU / mL refers to the level of 10 6 CFU / mL, wherein 1×10 6 CFU / mL, 5×10 6 CFU / mL, and 9.99×10 6 CFU / mL, etc. all belong to the level of 10 6 CFU / mL, that is, the level of 10 6 CFU / mL means greater than or equal to 1×10 6 CFU / mL to less than 1×10 7 CFU / mL. That is, in the liquid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is greater than or equal to 1×10 6 CFU / mL, more preferably greater than or equal to 1×10 7 CFU / mL to less than 1×10 9 CFU / mL.

[0047] Preferably, in the semi-liquid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is 10 8 CFU / mL or more, more preferably 10 9 -10 10 CFU / mL. That is, in the semi-liquid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is greater than or equal to 1×10 8 CFU / mL, more preferably greater than or equal to 1×10 9 CFU / mL to less than 1×10 11 CFU / mL.

[0048] Preferably, in the solid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is 10 8 CFU / g or more, more preferably 10 9 -10 10 CFU / g. That is, in the solid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is greater than or equal to 1×10 8 CFU / g, more preferably greater than or equal to 1×10 9 CFU / g to less than 1×10 11 CFU / g.

[0049] The fermentation inoculant provided in the present invention can be prepared by any conventional method in the art. According to a preferred embodiment of the present invention, the fermentation inoculant can be prepared as follows:

[0050] (1) Saccharomyces cerevisiae CGMCC No. 31571 is fermented and cultured (amplified culture) in a fermentation medium to obtain a fermentation broth, which is the liquid inoculant.

[0051] (2) After the fermentation broth obtained in step (1) is concentrated, a semi-liquid yeast inoculant (also called "concentrated fermentation inoculant") is obtained.

[0052] (3) The fermentation broth obtained in step (1) is concentrated, then rinsed with a buffer solution, a protective agent is added, the viable cell concentration is adjusted, and after mixing evenly, it is dried to obtain the solid (powder) inoculant.

[0053] In the present invention, the medium used for fermentation and culture in step (1) is not particularly limited as long as it can be used for the fermentation and culture of Saccharomyces cerevisiae. For example, it can be YPD medium, molasses medium, wort medium, etc.

[0054] In the present invention, any concentration method capable of enriching Saccharomyces cerevisiae in the fermentation broth can be used in step (2). For example, it can be filtration, centrifugation, etc. The conditions of the centrifugation and the filtration can be well-known conditions, which will not be elaborated herein.

[0055] Preferably, in step (2), the concentration multiple of the concentration treatment is 10 - 1000. The "concentration multiple" refers to the multiple relationship between the concentration of Saccharomyces cerevisiae (counted by viable cell number) in the fermentation broth after concentration treatment and the concentration of Saccharomyces cerevisiae (counted by viable cell number) in the fermentation broth before concentration treatment.

[0056] In the present invention, the buffer solution used for rinsing the cells after concentration treatment in step (3) is not particularly limited, and it can be any buffer solution commonly used for cell washing in the art as long as it has no or basically no effect on the activity of Saccharomyces cerevisiae. For example, it can be water, physiological saline, PBS buffer solution, etc.

[0057] In the present invention, the reagent used for adjusting the viable cell concentration in step (3) can be any reagent that has no or basically no effect on the activity of Saccharomyces cerevisiae therein. For example, it can be water, medium, buffer solution, etc. Preferably, the adjusted viable cell concentration makes the content of Saccharomyces cerevisiae in the obtained solid inoculant reach the aforementioned range.

[0058] Preferably, in step (3), the drying method can be selected from at least one of freeze-drying and spray-drying.

[0059] The third aspect of the present invention provides the use of the above-mentioned Saccharomyces cerevisiae and the above-mentioned fermentation inoculum in the production of ethanol and / or the production of alcoholic beverages.

[0060] In the present invention, the Saccharomyces cerevisiae with the preservation number of CGMCC No. 31571 can be used for the production of ethanol through culture fermentation in a culture medium by virtue of its strong fermentation ability and ethanol synthesis ability; or this yeast can also be used for the fermentation of grains or fruits to form alcoholic beverages with unique flavors due to its stronger ethanol-producing ability, ester-producing ability and non-production of H2S.

[0061] In the present invention, preferably, the alcoholic beverage is fruit wine. The fermentation raw material of the fruit wine contains fruits or fruit pulp made from fruits. Any suitable fruit can be used for the fruits, such as grapes, apples, Hami melons, pears, kiwifruits, blueberries, plums, and so on.

[0062] The fourth aspect of the present invention provides a method for producing ethanol, which includes: culturing the Saccharomyces cerevisiae as described above and / or the fermentation inoculum as described above.

[0063] In the present invention, the culture medium used for the culture is not particularly limited as long as it can be used for the fermentation culture of Saccharomyces cerevisiae. Preferably, the culture medium for the culture contains: yeast extract powder 8 - 12 g / L, peptone 15 - 25 g / L, glucose 15 - 25 g / L.

[0064] In the present invention, there is no particular limitation on the specific dosage of the Saccharomyces cerevisiae / fermentation inoculum during the culture process. Preferably, the inoculation amount of the Saccharomyces cerevisiae is not less than 10 5 CFU / mL, more preferably 10 6 -10 7 CFU / mL; that is, in the culture medium for the culture, the inoculation amount of the Saccharomyces cerevisiae in terms of viable cell count is greater than or equal to 1×10 5 CFU / g, more preferably greater than or equal to 1×10 6 CFU / g to less than 1×10 8 CFU / g.

[0065] According to the present invention, conditions such as the temperature, rotation speed, and time of the cultivation can be set according to the growth characteristics of the Saccharomyces cerevisiae to meet the proliferation of the Saccharomyces cerevisiae. Preferably, the conditions of the cultivation include: the temperature is 20-35°C, specifically it can be 20°C, 23°C, 26°C, 29°C, 32°C, 35°C, or any value between the above two values; the rotation speed is 120-180 rpm, specifically it can be 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, or any value between the above two values. The inventors have found that under this preferred specific embodiment, the growth rate of the Saccharomyces cerevisiae can be increased, thereby improving the ethanol production capacity.

[0066] The fifth aspect of the present invention provides a method for producing fruit wine, which includes: contacting and fermenting the Saccharomyces cerevisiae and / or the fermentation inoculant as described above with fruits.

[0067] In the present invention, the process of the contact fermentation can be carried out by using any existing method for fruit wine fermentation. Preferably, the process of the contact fermentation includes: juicing and enzymatically hydrolyzing the fruits to obtain fruit juice, and mixing the Saccharomyces cerevisiae and / or the fermentation inoculant with the fruit juice for fermentation.

[0068] In the present invention, generally the fruits are washed, peeled and then juiced (the juice after removing residues). Before the enzymatically hydrolyzing of the juice after removing residues, it can also be subjected to antibacterial treatment. The way of the antibacterial treatment is to add potassium metabisulfite. The process of the enzymatically hydrolyzing includes: mixing the juice after antibacterial treatment with pectinase (the addition concentration is 0.01-0.1 g / L), and carrying out enzymatically hydrolyzing at a temperature of 20-30°C for 5-10 h.

[0069] In the present invention, before fermentation, the fruit juice can also be adjusted in sugar content so that the sugar content of the fruit juice is 20-25°BX.

[0070] According to the present invention, preferably, the inoculation amount of the Saccharomyces cerevisiae is not less than 10 5 CFU / mL, preferably 1×10 6 -1×10 7 CFU / mL. That is, in the fruit juice, the inoculation amount of the Saccharomyces cerevisiae in terms of viable cell count is greater than or equal to 1×10 5 CFU / g, more preferably greater than or equal to 1×10 6 CFU / g to less than 1×10 8 CFU / g.

[0071] According to the present invention, the fruits can be any applicable fruits. Preferably, the fruits are at least one of grapes, apples, honeydews, pears, kiwifruits, blueberries, and plums.

[0072] According to the present invention, preferably, the fermentation conditions include: the temperature is 20 - 35°C, specifically it can be 20°C, 23°C, 26°C, 29°C, 32°C, 35°C, or any value between the above two values; the time is 5 - 10 days, specifically it can be 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any value between the above two values. The inventors found that under this preferred specific embodiment, the growth rate of Saccharomyces cerevisiae can be increased, and the flavor characteristics of Xinjiang fruit wine can be enhanced.

[0073] According to the present invention, Saccharomyces cerevisiae is inoculated into the cultured medium or fruit juice in the form of a seed solution for fermentation culture; specifically, the seed solution of Saccharomyces cerevisiae can be obtained by inoculating Saccharomyces cerevisiae into YPD liquid medium and performing at least one culture.

[0074] The present invention will be described in detail below through examples.

[0075] In the following examples, unless otherwise specified, the experimental materials used are all purchased from conventional biochemical reagent stores.

[0076] Soil sample: In September 2023, rhizosphere soil of an orchard in Xinjiang was collected. At each selected sampling point, sterile gloves were worn and the surface soil was removed using a sterile shovel, and soil was collected at a depth of 5 cm from the ground surface. For each sampling point, the longitude and latitude of the sampling point were located using GPS, and the altitude and temperature of the sampling area were recorded.

[0077] Commercial Saccharomyces cerevisiae CK: Angel Yeast Co., Ltd.

[0078] 2,3,5 - Triphenyltetrazolium chloride (TTC), glucose, anhydrous ethanol, glycerol, potassium metabisulfite, tributyrin, MgSO4, KH2PO4, peptone, yeast extract powder, agar, etc. (all are analytical pure or biochemical reagents) were purchased from Kunming Pinmei Technology Co., Ltd.; PDA medium, WL medium, and Biggy medium were all purchased from Qingdao Hi - tech Industrial Park Haibo Biotechnology Co., Ltd.; primers, 2×Taq enzyme (5U / mL) were purchased from Kunming Qingke Biotechnology Co., Ltd.; Ezup column fungal genomic DNA extraction kit, DNA polymerase, DNA marker were purchased from Sangon Biotech (Shanghai) Co., Ltd.; other reagents are all domestic analytical pure or biochemical reagents.

[0079] The composition of YPD liquid medium is: yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L.

[0080] The components of the YPD solid medium are: yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 20 g / L.

[0081] The components of the TTC upper-layer medium are: TTC 0.5 g / L, glucose 0.5 g / L, agar 15 g / L.

[0082] The components of the TTC lower-layer medium are: MgSO4 0.4 g / L, KH2PO4 1 g / L, yeast extract powder 1.5 g / L, peptone 2 g / L, glucose 10 g / L, agar 20 g / L.

[0083] The components of the ester-producing screening medium are: adding tributyrin 4 g / L to the YPD solid medium.

[0084] The biochemical incubator was purchased from Shanghai Fuma Experimental Equipment Co., Ltd., the stereomicroscope was purchased from Nikon, Japan, the shaking incubator was purchased from the medical equipment factory of Shanghai Boxun Industry Co., Ltd., the autoclave was purchased from HIRAYAMA Co., Japan, and the microplate reader was purchased from BioTek Instruments, Inc., USA.

[0085] Example 1

[0086] 1.1 Isolation and screening of yeast

[0087] The gradient dilution separation method and enrichment separation method were used to screen the strains: Weigh 5 g of soil, add 45 mL of sterile water, place it in a shaker and incubate it at a temperature of 30 °C and a rotation speed of 150 rpm for 30 min, and then dilute it into gradient dilutions of 10 -6 、10 -7 、10 -8 times of diluted bacterial suspensions. Then, take 100 μL respectively and coat them on PDA plates containing chloramphenicol (content 0.1 g / L), and culture them at 37 °C for 3 - 7 days. After the colonies grow, purify the colonies with different morphologies growing on the plates of the most suitable dilution multiple of the coated bacterial liquid until single colonies appear;

[0088] The growth performance, fermentation characteristics and other aspects of the screened single colonies were studied. After multiple rounds of research and demonstration, a separated strain TY12 with good performance in all aspects was finally screened from the numerous separated strains.

[0089] The separated strain TY12 was stored on the slant of PDA medium containing chloramphenicol (content 0.1 g / L).

[0090] 1.2 Morphological observation of the separated strain

[0091] The isolated strain TY12 was inoculated onto YPD solid medium for purification and cultured upside down at 28°C for 3 - 5 days. Observe and record the growth characteristics of the colonies, such as color, morphology, smoothness, wetness, and transparency. The colony characteristics and cell morphology of strain TY12 on YPD solid medium are shown in Figure 1 ; as Figure 1 shown, the colonies of strain TY12 are all spherical, opaque and easy to pick up, with a dry surface; the edges of the colonies are slightly irregular and milky white.

[0092] 1.3 Molecular biological identification of the isolated strain

[0093] The isolated strain TY12 was subjected to molecular biological identification. The genomic DNA was extracted using a DNA extraction kit, and the 26S rDNA D1 / D2 region gene sequence of the isolated strain was PCR amplified using primers NL1 (5'-GCATCAATAAGCGGAG - GAAAAG-3') and NL4 (5'-GGTCCGTTTCAAGACGG-3');

[0094] PCR amplification system (25 μL): 1 μL of DNA template, 0.5 μL each of NL1 and NL4, 11 μL of Taq PCR Master Mix, and 12 μL of double-distilled water (dd H2O);

[0095] PCR amplification program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 40 s, extension at 72°C for 1 min, 30 cycles, and further extension at 72°C for 10 min.

[0096] After the PCR amplification products were qualified by 1% agarose gel electrophoresis, they were entrusted to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing.

[0097] The measurement results were submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) in the United States for Basic Local Alignment Search Tool (BLAST) alignment. The 26S rDNA D1 / D2 region gene sequences of the reference strains with higher homology were selected, and the neighbor-joining (NJ) method in MEGA 11 software was used to construct a phylogenetic tree to analyze the taxonomic status of the strain.

[0098] Based on the 26S rDNA D1 / D2 region gene sequence, a phylogenetic tree of strain TY12 was constructed, and the results are shown in Figure 2 . From Figure 2It can be seen that strain TY12 and Saccharomyces cerevisiae ATCC 18824 cluster in the same branch; combined with the morphological observation results, strain TY12 was finally identified as Saccharomyces cerevisiae. Strain TY12 was proposed to be classified and named as Saccharomyces sp., and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 12, 2024, with the deposit number CGMCC No. 31571.

[0099] Example 2 Study on the Fermentation Characteristics of Yeast

[0100] 2.1 Gas Production Experiment of Yeast

[0101] The strain TY12 isolated and purified in Example 1 was inoculated into a test tube containing 5 mL of YPD liquid medium and cultured at 28 °C and 150 rpm for 24 h to obtain a seed solution for standby.

[0102] Using YPD liquid medium as the growth medium, 10 mL was transferred into a test tube and an inverted clean Durham tube was placed, stoppered, sterilized at 121 °C for 15 min, and cooled to room temperature; the seed solution of strain TY12 was inoculated into the sterilized YPD liquid medium test tube at an inoculation amount of 2% (v / v) and cultured statically at 28 °C for 24 h, with each treatment repeated 3 times. The gas production in the Durham tube was observed every 2 h, and the gas production was used as an index, with the commercial brewing yeast CK as a control; the results are shown in Table 1.

[0103] Table 1 Results of Gas Production Experiment

[0104] Strain TY12 CK 14h ++ ++ 16h +++ +++ 18h ++++ ++++ 20h ++++ ++++ 22h ++++ ++++

[0105] Note: In Table 1, "-" indicates no gas production; "+" indicates that the gas fills 1 / 4 of the Durham tube; "++" indicates that the gas fills 1 / 2 of the Durham tube; "+++" indicates that the gas fills 3 / 4 of the Durham tube; "++++" indicates that the gas completely fills the Durham tube.

[0106] Yeast produces CO2 gas during fermentation, and the time when the gas fills the Durham tube can directly reflect the fermentation ability of the strain. A fast CO2 gas production rate indicates that the yeast has strong fermentation ability and a fast fermentation start speed. As can be seen from Table 1, the fermentation speed of strain TY12 is similar to that of commercial yeast CK, and the gas produced can completely fill the Durham tube in 18 h. It can be seen that the isolated strain TY12 is a yeast with a fast fermentation start, a large gas production, and excellent aroma.

[0107] 2.2 Study on the Physiological Characteristics of the Strain

[0108] (1) H2S production detection: The strain TY12 isolated and purified in Example 1 was streaked on YPD solid medium and cultured at 28 °C for 2-3 days for activation. After that, a sterilized toothpick was used to spot-inoculate it onto Biggy medium. There were three parallels for each bacterial spot, and four bacteria were inoculated on one plate. It was cultured at 28 °C for 4 days. The ability of the strain to produce H2S was judged by observing the depth of the brown color appearing in the center of the colony. The commercial Saccharomyces cerevisiae CK was used as a control. The results are shown in Table 2.

[0109] Using bismuth in Biggy medium as an indicator, it reacts with hydrogen sulfide produced by yeast to form bismuth sulfide precipitate. According to the number of aggregates of bismuth precipitate, the depth of the colony color is determined; the deeper the color, the more hydrogen sulfide is produced. That is, the unproduced hydrogen sulfide is white, indicated by "-" in Table 2, the low-produced hydrogen sulfide is light brown, indicated by "+" in Table 2, and the high-produced hydrogen sulfide is dark brown, indicated by "++" in Table 2.

[0110] (2) Ethanol production ability detection: The strain TY12 isolated and purified in Example 1 was streaked on the lower layer of TTC solid medium and statically cultured at 28 °C for 2 days. The commercial Saccharomyces cerevisiae CK was used as a control, and each treatment was repeated 3 times. Then, the upper layer of TTC medium was poured. After cooling and solidifying, it was statically cultured at 28 °C for 5 h. The color development of each strain on the TTC medium was observed and recorded. The results are shown in Table 2. The strength of the respiratory enzyme activity of the yeast strain determines the colony color. The colony showing pink is defined as low ethanol production, indicated by "-" in Table 2; if it is red, it is medium ethanol production, indicated by "+" in Table 2; if it is dark red, it is high ethanol production, indicated by "++" in Table 2.

[0111] (3) Ester production ability detection: The strain TY12 isolated and purified in Example 1 was streaked in a Z shape on the ester production medium and cultured at 28 °C for 3 days. The colony color of each yeast on the ester production medium was observed and recorded, and each treatment was repeated 3 times. The commercial Saccharomyces cerevisiae CK was used as a control. The results are shown in Table 2. According to the colony color, the colonies of low-ester-producing yeast strains are milky white, indicated by "-" in Table 2; the colonies of medium-ester-producing yeast are light yellow, indicated by "+" in Table 2; the colonies of high-ester-producing yeast are bright yellow, indicated by "++" in Table 2.

[0112] The physiological characteristics of the isolated strain TY12 are shown in Table 2. Through the preliminary determination of H2S production ability, it was found that neither the strain TY12 nor the commercial Saccharomyces cerevisiae CK produced H2S; through the preliminary determination of ethanol production ability, it was found that compared with the commercial Saccharomyces cerevisiae CK, the strain TY12 had a stronger ethanol production ability; through the preliminary determination of ester production ability, it was found that the strain TY12 had a stronger ester production ability, while the ester production ability of the commercial Saccharomyces cerevisiae CK was weaker.

[0113] Table 2 Research Results of Physiological Characteristics

[0114] Strain TY12 CK <![CDATA[Producing H2S]]> - - Ethanol production ++ + Ester production ++ -

[0115] 2.3 Strain Tolerance Study

[0116] The strain TY12 isolated and purified in Example 1 was subjected to tolerance screening, namely alcohol tolerance, sulfur dioxide tolerance, high sugar tolerance, low pH tolerance and temperature tolerance tests.

[0117] High sugar experiment: The seed liquid of strain TY12 was inoculated into YPD liquid medium supplemented with different concentrations of glucose (150, 400, 550 g / L) at an inoculation amount of 2% (v / v). There were 3 parallels for each treatment. After culturing in a shaker at 28°C and 150 rpm for 24 h, OD was measured 600 , and the results are shown in Figure 3 .

[0118] As Figure 3 shown, the growth of strain TY12 was inhibited as the glucose concentration increased. When the glucose concentration was 15%, strain TY12 could grow vigorously. When the glucose concentration was increased to 40%, strain TY12 began to be inhibited but still could grow vigorously; when the glucose concentration reached 55%, the growth of strain TY12 was inhibited but it still survived, and its growth activity was significantly higher than that of commercial brewing yeast CK. It can be seen that strain TY12 has strong high sugar tolerance ability.

[0119] pH experiment: The seed liquid of strain TY12 was inoculated into YPD liquid medium supplemented with different pH values (2, 2.5, 3) at an inoculation amount of 2% (v / v). There were 3 parallels for each treatment. After culturing in a shaker at 28°C and 150 rpm for 24 h, OD was measured 600 , and the results are shown in Figure 4 .

[0120] As Figure 4 shown, the pH of the medium had a more obvious effect on the growth of yeast. When the pH range was 2.0, the growth of strain TY12 was significantly inhibited, but it still survived; when the pH range was 2.5 and 3.0, strain TY12 showed good growth characteristics, and its growth was significantly better than that of commercial brewing yeast CK.

[0121] Salt tolerance experiment: The seed liquid of strain TY12 was inoculated into YPD liquid medium supplemented with different concentrations of NaCl (40, 60, 80 g / L) at an inoculation amount of 2% (v / v). There were 3 parallels for each treatment. After culturing in a shaker at 28°C and 150 rpm for 24 h, OD was measured 600 , and the results are shown in Figure 5 .

[0122] As shown Figure 5 in the figure, the growth of strain TY12 was inhibited to a certain extent with the increase of sodium chloride concentration. However, when the NaCl concentration was increased to 60 g / L and 80 g / L, strain TY12 maintained good growth characteristics. It can be seen that strain TY12 has the ability to tolerate high salt stress.

[0123] Ethanol tolerance: The seed liquid of strain TY12 was inoculated into YPD liquid medium supplemented with different concentrations of ethanol (3%, 6%, 9%) (v / v) at an inoculation amount of 2% (v / v). There were 3 parallels for each treatment. After culturing in a shaker at 28 °C and 150 rpm for 24 h, OD was measured 600 , and the results are shown Figure 6 .

[0124] As shown Figure 6 in the figure, with the increase of ethanol concentration, the growth of strain TY12 was gradually inhibited. When the ethanol concentration was 3%, the growth activity of commercial brewing yeast CK was slightly higher than that of strain TY12. However, with the increase of ethanol concentration, strain TY12 showed good ethanol adaptation ability, and the growth activity of strain TY12 was higher than that of commercial brewing yeast CK.

[0125] SO2 tolerance: The seed liquid of strain TY12 was inoculated into YPD liquid medium supplemented with different concentrations of SO2 (300, 400, 500 mg / L) at an inoculation amount of 2% (v / v). There were 3 parallels for each treatment. After culturing in a shaker at 28 °C and 150 rpm for 24 h, OD was measured 600 , and the results are shown Figure 7 .

[0126] As shown Figure 7 in the figure, the growth of strain TY12 was relatively stable at different sulfur dioxide concentrations. When the sulfur dioxide concentration increased from 300 mg / L to 500 mg / L, the growth inhibition of strain TY12 was small, and its growth stability at different sulfur dioxide concentrations was significantly better than that of commercial brewing yeast CK. During the process of fruit wine brewing, in order to prevent harmful bacteria from adhering to the fruit peel, sulfurous acid solution is generally added, which can play roles such as inhibiting the growth of harmful bacteria and antioxidant. It has been verified that strain TY12 has the ability to tolerate sulfur dioxide and is especially suitable for fruit wine fermentation.

[0127] In summary, the strain TY12 isolated in Example 1 has a fast fermentation start speed, the characteristics of not producing H2S gas, high yield of esters and ethanol, and can tolerate a variety of fermentation stress conditions, showing good application prospects, laying a theoretical basis for the further development and utilization of the strain.

[0128] Example 3 Peach wine fermentation

[0129] Fruit juice

[0130] Preparation of bacterial suspension: The strains TY12 isolated in Example 1, non-Saccharomyces cerevisiae N14 (Naganishia diffluens, self-screened, preserved and provided by the Institute of Microbiology Application, Xinjiang Academy of Agricultural Sciences, isolated from the same soil sample as strain TY12), and Angel yeast RV171 were respectively cultured in YPD liquid medium at 28 °C and 180 rpm for 24 h, and then inoculated into fresh YPD liquid medium at an inoculation amount of 0.03% (v / v), and statically cultured at 28 °C and 180 rpm for 24 h, centrifuged, and resuspended with sterile normal saline to form a bacterial suspension with a cell number of 1×10 8 CFU / mL for standby.

[0131] Fermentation method: The bacterial suspension of the activated yeast was set in the following 4 groups (3 parallels in each group, a total of 12 fermentation samples), inoculated into the boiled peach juice at an inoculation amount of 0.03% (v / v), and fermented at 22 - 26 °C for 10 d. Physical and chemical indexes were measured by sampling every day; each tank was 1200 mL, a total of 14.4 L of peach juice was required, and 0.36 mL of the bacterial suspension of the corresponding yeast was inoculated into each tank;

[0132] (1) Only add Angel yeast RV171 for single-strain fermentation;

[0133] (2) Only add Saccharomyces cerevisiae TY12 for single-strain fermentation;

[0134] (3) Strains N14 and Angel yeast RV171 were sequentially mixed and fermented in a ratio of 1:1;

[0135] (4) Strains N14 and Saccharomyces cerevisiae TY12 were sequentially mixed and fermented in a ratio of 1:1.

[0136] The specific fermentation operation is as follows. Taking groups (3) and (4) as examples, the operations of the other groups are the same as theirs, only the inoculation is a single strain:

[0137] 1. Adjust the sugar degree: First, take out the pre-prepared peach juice, thaw it and heat it to boiling, and add granulated sugar to make the sugar degree reach 20 Brix;

[0138] 2. Add potassium metabisulfite: Add 5 mL of potassium metabisulfite solution with a concentration of 24 mg / mL (7.2 g dissolved in 300 mL of sterile water) to make the final concentration of potassium metabisulfite in the peach juice 100 mg / L, and let it stand for 2 hours;

[0139] 3. Enzymolysis: Add 2 mL of pectinase solution with a concentration of 36 mg / mL (1.26 g dissolved in 35 mL of sterile water) to make the final concentration of pectinase in the peach juice 60 mg / L, and enzymolyze at room temperature for 8 h;

[0140] 4. Inoculation of ester-producing yeast (optional): Add the ester-producing yeast (N14) bacterial suspension at an inoculation amount of 0.03% (i.e., 0.36 mL / tank) and ferment for 24 / 36 h;

[0141] 5. Inoculation of Saccharomyces cerevisiae: Inoculate the Saccharomyces cerevisiae TY12 or Angel yeast RV171 bacterial suspension respectively at an inoculation amount of 0.03% (i.e., 0.36 mL / tank);

[0142] 6. Fermentation: Static fermentation at 20 - 26 °C for 10 days (sampling once a day). After the residual sugar no longer decreases / stabilizes, add 100 mg / L potassium metabisulfite solution to stop fermentation;

[0143] 7. Solid-liquid separation: After stopping fermentation, filter the fermentation broth with a nylon mesh / gauze to obtain the original peach wine;

[0144] 8. Clarification and filtration: Centrifuge the original peach wine at 8000 rpm for 5 min to obtain the finished product.

[0145] During the fermentation process, 50 mL of samples are taken every day. After centrifuging at 8000 rpm for 5 min, the physical and chemical indexes are measured, including the total acid content (calculated as tartaric acid), soluble solids, total phenol content, reducing sugar content, and the final alcohol content. The results are shown in Tables 3 to 5.

[0146] Among them, the detection method of the total acid content refers to "GB 12456—2021 Determination of Total Acid in Foods", the detection method of soluble solids refers to "GB / T 12143-2008 General Analytical Methods for Beverages", and the detection method of alcohol content refers to "GB5009.225-2016 National Food Safety Standard Determination of Ethanol Concentration in Wine"; the detection of total phenol content adopts the Folin-Ciocalteu colorimetric method. The specific process is as follows: Weigh 3 g of anhydrous sodium carbonate and make up the volume to 50 mL with water to obtain a sodium carbonate solution; accurately pipette 0.5 mL of the wine sample diluted by a certain multiple, add 1.0 mL of Folin-Ciocalteu reagent and mix well. After standing for 5 min, pipette and add 2.0 mL of 10% sodium carbonate solution, make up the volume to 10 mL, mix well thoroughly with a vortex shaker, heat in a water bath at 70 °C for 10 min and cool to room temperature, and measure the absorbance at a wavelength of 765 nm. Each group of samples is repeated three times; the measured results are substituted into the regression equation y = 0.059x + 0.0783 to calculate the total phenol content of the sample.

[0147] The detection method for the content of reducing sugar is as follows: Take 0.5 mL of wine sample and make up the volume to 100 mL as the sample. Then take 0.5 mL of the sample into a 25-mL test tube, add 2 mL of 3,5-dinitrosalicylic acid (DNS) reagent, mix well, heat in a boiling water bath for 5 min, and then quickly cool with running water. Add distilled water to make up the volume to 10 mL, mix well, and measure the OD value at 540 nm; Use water instead of the sample and perform the same operation as the blank; Perform the same operation with glucose standard solutions of different concentrations (0.2 - 1.0 mg / mL, as x), and measure the OD value at 540 nm (as y), draw a standard curve, and the linear regression equation is y = 1.1414x - 0.0120, R 540 = 0.9994; Calculate the content of reducing sugar in the wine sample according to the standard curve. 2

[0148] Table 3

[0149]

[0150] Table 4

[0151]

[0152] Table 5

[0153] Group Alcohol content (% vol) Group (1) 10.83 Group (2) 11.87 Group (3) 11.03 Group (4) 11.73

[0154] By comparing group (1) with group (2), and group (3) with group (4), the Saccharomyces cerevisiae strain provided by the present invention can significantly increase the total acid content and total phenol content in peach wine, and has a high alcohol content. It can be seen that the Saccharomyces cerevisiae strain provided by the present invention has stronger ethanol-producing ability and ester-producing ability, adding different flavors to the wine-making.

[0155] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.​

Claims

1. A Saccharomyces sp., characterized in that, The preservation number of the Saccharomyces cerevisiae is CGMCC No. 31571.

2. A fermentation inoculant, characterized in that, The fermentation inoculant contains the Saccharomyces cerevisiae described in claim 1.

3. The fermenting inoculant according to claim 2, wherein, The fermentation inoculant is at least one of a liquid fermentation inoculant, a semi-liquid fermentation inoculant, and a solid fermentation inoculant.

4. The fermenting inoculant according to claim 3, characterized in that, In the liquid fermentation inoculum, the content of Saccharomyces cerevisiae in terms of viable cell count is 10 6 CFU / mL or more, more preferably 10 7 -10 8 CFU / mL; Preferably, in the semi-liquid fermentation inoculant and the solid fermentation inoculant, the content of Saccharomyces cerevisiae in terms of viable cell count is 10 8 CFU / mL or more, more preferably 10 9 -10 10 CFU / mL.

5. Use of the Saccharomyces cerevisiae described in claim 1 and the fermentation inoculant described in any one of claims 2 to 4 in the production of ethanol and / or the production of alcoholic beverages.

6. The application according to claim 5, characterized in that, The alcoholic beverage is a fruit wine.

7. A method for producing ethanol, characterized in that, The method includes: culturing the Saccharomyces cerevisiae described in claim 1 and / or the fermentation inoculant described in any one of claims 2 to 4.

8. The method according to claim 7, wherein The culture medium for the culturing contains: yeast extract powder 5 - 15 g / L, peptone 15 - 25 g / L, glucose 15 - 25 g / L; Preferably, the culture conditions include: the inoculation amount of the Saccharomyces cerevisiae is not less than 10 5 CFU / mL, the temperature is 20-35 °C, and the rotation speed is 120-180 rpm.

9. A method for producing fruit wine, characterized in that, The method includes: subjecting the Saccharomyces cerevisiae described in claim 1 and / or the fermentation inoculant described in any one of claims 2 to 4 to contact fermentation with fruits.

10. The method according to claim 9, wherein The process of the contact fermentation includes: juicing and enzymatically hydrolyzing the fruits to obtain fruit juice, and mixing the Saccharomyces cerevisiae and / or the fermentation inoculant with the fruit juice for fermentation; Preferably, the inoculation amount of the Saccharomyces cerevisiae in the fruit juice is not less than 10 5 CFU / mL, preferably 10 6 -10 7 CFU / mL; Preferably, the fruits are at least one of grapes, apples, honeydews, pears, kiwifruits, blueberries, and plums; Preferably, the conditions for the fermentation include: a temperature of 20 - 35 °C and a time of 5 - 10 days.

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