Smith yeast for fruit wine fermentation and application of Smith yeast

Abstract

The invention belongs to the field of microorganisms and fermentation industry, and particularly relates to Smith yeast for fruit wine fermentation and application of the Smith yeast. The preservation number of the Smith yeast is CGMCC (China General Microbiological Culture Collection Center) NO.34550, and the Smith yeast is preserved in the China General Microbiological Culture Collection Center on May 15, 2025; the saccharomyces cerevisiae is classified and named as Smith saccharomyces cerevisiae Meyerozyma smithsonii. The Smith yeast Shannon TLT07.79 is separated and screened, and the screening method is simple and efficient; the method has the advantages of high alcoholic strength and good wine flavor; wine fermented by the strain has regional characteristics and good flavor, and can be used for producing high-quality fruit wine.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and fermentation industry, and particularly relates to a Smithsonian yeast strain for fruit wine fermentation and application thereof. Background Art

[0002] As the quality of life continues to improve, people are paying more and more attention to their health. Long-term consumption of high-alcohol wine can damage vital organs such as the heart and liver. Therefore, fruit wines with low alcohol content, rich nutrition and unique flavor are favored by consumers.

[0003] Traditional fruit wine is fermented with brewer's yeast. Compared with commercial brewer's yeast, wild yeast fermentation produces more flavor substances, such as esters and alcohols, during the fermentation process. The types and characteristics of wild yeast in different regions are different, so they can better reflect the local terroir conditions. In addition, wild yeast is a naturally occurring microorganism that is not artificially cultivated or added, and is considered more natural and healthier. However, due to the complexity of the natural living environment of wild yeast, the diversity of its own biological characteristics, and the specific requirements for its functions during fermentation applications, screening biases are easily caused during the screening process; wild strains with special flavor potential are low in the sample, and the target strain may not be screened out; or the characteristics of the strain are changed during the enrichment process, resulting in the screened strain being inconsistent with the original state. Summary of the Invention

[0004] In view of the lack of yeast for fermenting jujube wine in the prior art, the present invention provides a Smithsonian yeast ( Meyerozyma smithsonii When the yeast provided by the present invention is used to ferment jujube wine, the fermentation process is milder, a higher alcohol content can be achieved, and the unique flavor of red dates can be better reflected, so that the jujube wine has more regional characteristics and a richer, more complex and layered aroma.

[0005] Another object of the present invention is to provide the use of the Smithsonian yeast in preparing fruit wine.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides a Smithsonian yeast ( Meyerozyma smithsonii ), the Smithsonian yeast has a deposit number of CGMCC NO.34550 and was deposited in the General Microbiology Center of China Culture Collection Administration on May 15, 2025; it is classified and named Smithsonian yeast Meyerozyma smithsonii .

[0007] Preferably, the gene sequence of the Smithsonian yeast is shown as SEQ ID NO.1.

[0008] The application also provides the application of the above-mentioned Saccharomyces boulardii in the preparation of fruit wine through fermentation.

[0009] The application utilizes Saccharomyces boulardii (Shannan TLT07.79), and the specific fermentation process is as follows: (1) raw material selection: selecting red dates with full grains, no mildew, no metamorphic deterioration, and no insect damage; (2) date juice extraction: washing the red dates, soaking them in water, removing the date pits, adding water to the pitted red dates, beating the mixture to a pulp, adding pectinase, extracting, and squeezing the juice for standby, to obtain red date juice; (3) strain activation: under sterile conditions, a single colony of Saccharomyces boulardii on a solid YPD culture medium is inoculated into a sterilized and cooled liquid YPD culture medium, and oscillation culture is performed, to obtain a Saccharomyces boulardii bacterial solution; (4) fermentation: inoculating Saccharomyces boulardii into the red date juice, and culturing under constant temperature conditions, to obtain fruit wine.

[0010] Preferably, in step (2), the ratio of the red dates to water for beating the pulp is 1:5; the amount of pectinase added in the date pulp mixture is 0.2 g / L; and the extraction is performed at a temperature of 55-60 DEG C for 4-5 hours.

[0011] Preferably, in step (3), the strain activation conditions are as follows: one single colony of Saccharomyces boulardii is picked from a solid YPD culture medium and inoculated into a liquid YPD culture medium, and oscillation culture is performed at 28-30 DEG C and 180 rpm for 24-48 hours.

[0012] Preferably, in step (4), the inoculation amount of Saccharomyces boulardii is 1-5%.

[0013] Preferably, in step (4), the culturing is performed at a temperature of 28-30 DEG C for 10-12 days.

[0014] The wild yeast strain screened from the date fermentation liquor is more conducive to the fermentation of date wine. This provides a theoretical basis and data support for the further development and utilization of wild Saccharomyces cerevisiae for fruit wine, and has important academic value and practical significance.

[0015] The application has the following beneficial effects: (1) The application separates and screens a strain of Saccharomyces boulardii, Shannan TLT07.79, and the screening method is simple and efficient; (2) The Saccharomyces boulardii screened in the application has the advantages of high alcohol yield and good wine brewing flavor; and the fermented wine has regional characteristics, good flavor, and can produce high-quality fruit wine.

[0016] Preservation information Preservation time: May 15, 2025; Collection Number: CGMCC No.34550; Collection Number: CGMCC No.34550; Address of Collection Unit: No.3, Yuanmingyuan West Road, Beijing, China; Post Code: 100101; Classification Name: Saccharomyces boulardii Meyerozyma smithsonii . BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is a cell morphology chart of Saccharomyces boulardii under microscope; Figure 2 Figure 2 is a colony morphology chart of Saccharomyces boulardii; Figure 3 Figure 3 is a phylogenetic tree chart of Saccharomyces boulardii; Figure 4 Figure 4 is an ethanol tolerance experiment chart of Saccharomyces boulardii and commercial Angel yeast. DETAILED DESCRIPTION

[0018] The present application will be described in detail below in combination with specific drawings and examples. The following examples are only the preferred embodiments of the present application, and it should be noted that the following description is only for the purpose of explaining the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification made according to the technical essence of the present application to the embodiments are within the scope of the technical solutions of the present application.

[0019] In the following examples, the materials, reagents and the like used are obtained from commercial channels unless otherwise specified.

[0020] The present application utilizes Saccharomyces boulardii to brew fruit wine, which specifically comprises the following steps: (1) Raw material selection: select red dates with full grains, no mildew, no metamorphic deterioration and no insect pests.

[0021] (2) Date juice extraction: beat the pulp according to the ratio of 1:5 to obtain date pulp, add pectinase according to the ratio of 0.2 g / L, extract at 55°C for 4 hours, and filter for use.

[0022] (3) Strain activation: under sterile conditions, pick a single colony of Saccharomyces boulardii on solid YPD medium, inoculate into sterilized and cooled liquid YPD medium (20 g / L of anhydrous glucose, 20 g / L of protein peptone, 10 g / L of yeast extract powder, sterilized at 115°C for 20 minutes), and cultivate overnight at 28°C with 180 rpm oscillation.

[0023] (4) Fermentation: inoculate 4.5% of the Smith yeast (Shannan TLT07.79), 28℃ in incubator for 10-12 days.

[0024] Example 1 Screening of Smith yeast Take Jinsixiaozao in Leling, Shandong, which is not moldy, no disease, add sterile water and seal for natural fermentation. When the fermentation liquid produces a large amount of bubbles, dilute the fermentation liquid 10 times with sterile water (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ) gradient, each gradient 2 parallel, take 100 μL diluent with sterile glass beads on solid YPD medium, 28℃ constant temperature culture 2-3 days, pick up the single colony with typical yeast characteristics of white, smooth, easy to pick up, in YPD medium, 28℃, 180 rpm vibration culture 36h, pure strain with volume fraction of 30% glycerol at-80℃ preservation, named as Shannan TLT07.79.

[0025] Solid YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, agar 17.5 g / L, 115℃ sterilization 20 minutes.

[0026] Liquid YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, 115℃ sterilization 20 minutes.

[0027] Example 2 Morphological observation and physiological and biochemical characteristics identification of strain Shannan TLT07.78 Under the microscope, observe the colony characteristics of Shannan TLT07.79, such as colony morphology, size, color, texture, transparency, edge flatness, and record the cell shape, size, etc., the results are shown in Figure 1 and Figure 2 The physiological and biochemical characteristics identification test of strain TLT07.79 was carried out at 28℃.

[0028] The biological characteristics of strain Shannan TLT07.79 are: usually oval, round or nearly round, single cell size is (2.0 μm ~ 5.0 μm) x (3.0 μm ~ 10.0 μm). The colony is round, with a raised middle, white, opaque, dry surface, easy to pick up, and the edge is neat when cultured at 28℃.

[0029] The physiological and biochemical characteristics of the strain Shannontlt07.79 are as follows: facultative anaerobic, the optimum growth temperature is 25-28℃, and a relatively obvious wine taste is produced in the fermentation process.

[0030] The strain Shannontlt07.79 screened in Example 1 is entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing result is shown in SEQ ID NO. 1; the obtained 16S rRNA sequence is subjected to BLAST analysis with the existing sequences in the NCBI database, and the strains with similar homology are selected, a phylogenetic tree is constructed by using MEGA 11.0 software, and the construction method is Neighbor joining, and it is found that the similarity of the strain Shannontlt07.79 screened above and Meyerozyma smithsoniiATCC MYA-4323 is 99%, and the evolutionary distance is relatively close, and combined with the physiological and biochemical characteristics of the strain, it is identified as the Saccharomyces boulardii (Meyerozyma smithsonii) .

[0031] SEQ ID NO. 1: >.

[0032] Example 3 Analysis of ethanol tolerance of strain Shannong TLT07.79 Ethanol tolerance test of Shannong TLT07.79 strain: 2% inoculum of Shannong TLT07.79 was added to 60 mL of liquid YPD medium with different ethanol contents (ethanol content was 6%, 9%, 12%, 15%, and 18%) in a 100 mL conical flask. The culture was kept in an incubator at 28°C for 48 hours. Two replicates were set up, and commercial Angel Yeast was used as a control. The growth of the strain was measured at OD 600nm value.

[0033] OD 600nm Detection of the value: Take 3 mL of the bacterial solution and measure it using a UV spectrophotometer at a wavelength of 600 nm. If the fermentation solution is too turbid (high bacterial density), dilute the solution 10-fold with sterile water before measurement.

[0034] The OD values ​​of Angel Yeast and Shannong TLT07.79 increased with the increase of ethanol concentration.600 The OD values of the S. cerevisiae Angelus and the S. cerevisiae S. TLT07.79 showed a downward trend, and the OD values of the S. cerevisiae Angelus were relatively stable, although they also showed a downward trend, but the downward trend was relatively small. The OD value of the S. cerevisiae Angelus was close to 0.5 when the ethanol concentration was 18%, and the OD value of the S. cerevisiae S. TLT07.79 was also close to 0.5 when the ethanol concentration was 18%. 600 The OD values of the S. cerevisiae Angelus and the S. cerevisiae S. TLT07.79 showed a downward trend, and the OD values of the S. cerevisiae Angelus were relatively stable, although they also showed a downward trend, but the downward trend was relatively small. The OD value of the S. cerevisiae Angelus was close to 0.5 when the ethanol concentration was 18%, and the OD value of the S. cerevisiae S. TLT07.79 was also close to 0.5 when the ethanol concentration was 18%. 600 The OD values of the S. cerevisiae Angelus and the S. cerevisiae S. TLT07.79 showed a downward trend, and the OD values of the S. cerevisiae Angelus were relatively stable, although they also showed a downward trend, but the downward trend was relatively small. The OD value of the S. cerevisiae Angelus was close to 0.5 when the ethanol concentration was 18%, and the OD value of the S. cerevisiae S. TLT07.79 was also close to 0.5 when the ethanol concentration was 18%. 600 The OD values of the S. cerevisiae Angelus and the S. cerevisiae S. TLT07.79 showed a downward trend, and the OD values of the S. cerevisiae Angelus were relatively stable, although they also showed a downward trend, but the downward trend was relatively small. The OD value of the S. cerevisiae Angelus was close to 0.5 when the ethanol concentration was 18%, and the OD value of the S. cerevisiae S. TLT07.79 was also close to 0.5 when the ethanol concentration was 18%. Figure 4 The OD values of the S. cerevisiae Angelus and the S. cerevisiae S. TLT07.79 showed a downward trend, and the OD values of the S. cerevisiae Angelus were relatively stable, although they also showed a downward trend, but the downward trend was relatively small. The OD value of the S. cerevisiae Angelus was close to 0.5 when the ethanol concentration was 18%, and the OD value of the S. cerevisiae S. TLT07.79 was also close to 0.5 when the ethanol concentration was 18%.

[0035] Table 1 Example 4 Application of the S. cerevisiae S. TLT07.79 in the Fermentation of Fruit Wine (1) Strain selection: S. cerevisiae S. TLT07.79; (2) Strain activation: inoculate the S. cerevisiae S. TLT07.79 on the strain activation solid culture medium, and incubate at 28°C for 24-48 h.

[0036] (3) Seed culture: inoculate the colonies in step (2) into 100 mL of liquid seed culture medium, and incubate at 28°C, 180 rpm for 36 h.

[0037] (4) Fermentation culture: inoculate 4.5% of the S. cerevisiae S. TLT07.79 into a conical flask (500 mL) containing 400 mL of jujube juice; (5) Ethanol production ability test: after 7 days of culture, the alcohol content of the jujube wine was measured every day, and the results showed that the alcohol content reached 11° on the 9th day and reached 12° on the 12th day, as shown in Table 2.

[0038] Table 2

Claims

1. A strain of Smithsonian yeast ( Meyerozyma smithsonii ), characterized in that The Smithsonian yeast is deposited with CGMCC NO.34550 on May 15, 2025 at the General Microbiology Center of China Culture Collection Administration; and is classified as Smithsonian yeast. Meyerozyma smithsonii .

2. The Smithsonian yeast according to claim 1, characterized in that The gene sequence of the Smithsonian yeast is shown in SEQ ID NO.

1.

3. Use of the Smithsonian yeast according to claim 1 or 2 in fermenting and preparing fruit wine.

4. The use according to claim 3, characterized in that The specific fermentation process is: (1) Raw material screening: Select red dates with full grains, no mold, no deterioration, and no insect pests; (2) Jujube juice extraction: clean the red dates and soak them in water; remove the pits, add water to the pitted red dates, beat them, add pectinase, extract, and squeeze the juice for later use to obtain red date juice; (3) Activation of the strain: Under sterile conditions, a single colony of Smithsonian yeast on solid YPD medium was picked and inoculated into sterilized and cooled liquid YPD medium, and cultured with shaking to obtain Smithsonian yeast culture liquid; (4) Fermentation: Inoculate the red date juice with Smithsonian yeast and culture it under constant temperature to obtain fruit wine.

5. The use according to claim 4, characterized in that In step (2), the material-liquid ratio of the red dates and water for pulping is 1:5; the amount of pectinase added to the date pulp mixture is 0.2 g / L; and the extraction is performed at a temperature of 55-60° C. for 4-5 hours.

6. The use according to any one of claims 3 to 5, characterized in that: In step (3), the conditions for strain activation are to pick a single colony of Smithsonian yeast from solid YPD medium and transfer it to liquid YPD medium, and culture it at 28-30°C and 180 rpm for 24-48 hours.

7. The use according to any one of claims 3 to 6, characterized in that: In step (4), the inoculation amount of the Smithsonian yeast is 1-5%.

8. The use according to claim 7, characterized in that In step (4), the culture is carried out at a temperature of 28-30°C for 10-12 days.

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