Saccharomyces cerevisiae with high yield of glycerol and its application in wine fermentation in Ningxia region

Abstract

The present application relates to a high-yield glycerol Saccharomyces cerevisiae and its application in Ningxia region grape wine fermentation, belonging to the field of microbial technology. The Saccharomyces cerevisiae ND10 of the present application endows the grape wine from Ningxia region with good flavor and taste. For the local wine-making grape raw materials in Ningxia region, inoculating the Saccharomyces cerevisiae ND10 of the present application can increase the complexity and fullness of the grape wine taste on the basis of not affecting the aroma of the grape wine, so that the taste of the grape wine is round, plump and smooth. Meanwhile, by using the Saccharomyces cerevisiae of the present application to replace the commercial Saccharomyces cerevisiae, it is helpful to improve the defects such as wine quality homogenization caused by using imported commercial Saccharomyces cerevisiae, and better present the characteristics of the grape wine in Ningxia region, so as to help the grape wine industry in Ningxia region form its own local characteristics, promote the development of the whole production area grape wine industry and even the independent innovation development of Chinese grape wine industry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-glycerol-producing brewer's yeast and its application in wine fermentation in Ningxia region. Background Technology

[0002] The fermentation process of wine involves the combined action of multiple microorganisms, characterized by species and quantity diversity, complex interbiotic interactions, and dynamic changes in metabolic products. Among these, *Saccharomyces cevevisiae*, the dominant microorganism, possesses strong colonization and alcoholic fermentation capabilities. Its performance significantly impacts not only the taste and flavor of the wine but also its yield and quality. Furthermore, *Saccharomyces cevevisiae* can tolerate low pH, high sugar and sulfur, low oxygen, and high ethanol concentrations during fermentation, making it well-suited to the harsh conditions of wine fermentation. Compared to natural wine fermentation, when inoculated with *Saccharomyces cevevisiae*, the yeast maintains its dominant position, largely preventing contamination by other microorganisms that could lead to undesirable aroma compounds and fermentation stagnation, thus improving the controllability of the fermentation process. Glycerol is the third most abundant byproduct of alcoholic fermentation in *Saccharomyces cevevisiae*, after ethanol and CO2, and is primarily produced in the early stages of fermentation. Glycerin is a non-volatile substance with viscosity and sweetness. It contributes to a round, full-bodied, and smooth texture, increasing the complexity and fullness of the flavor without affecting the aroma of fruit wine. It is a crucial component that determines the quality of fruit wine. Selecting and breeding high-yield glycerin-producing fermentation strains is an important way to improve the roundness of wine and a key measure to improve the quality of dry and semi-dry wines.

[0003] The Helan Mountain East Foothills wine region is currently my country's most internationally renowned benchmark for premium wines. This region boasts abundant accumulated temperature, suitable sunshine hours, appropriate rainfall ratios, and an arid climate ideal for cultivating a wide variety of wine grapes. The superior ecological environment and geographical location, coupled with strong government support, have fostered a large-scale and brand-driven wine industry. After decades of effort, the Helan Mountain East Foothills has made significant progress in winemaking techniques, approaching international advanced levels. However, research and application of local fermentation microbial resources remain relatively weak. Most wineries generally use commercial yeasts from countries like France and Italy for fermentation, leading to product homogenization, hindering the development of the region's wine quality and style, and failing to fully showcase the unique terroir of the region. Therefore, fully developing and utilizing the local microbial resources of the eastern foothills of the Helan Mountains in Ningxia, and screening for local yeast strains with excellent brewing characteristics, especially those with high glycerol production capacity, can not only increase the roundness and fullness of the wine's taste, but also contribute to the formation of the regional characteristics of Ningxia Hui Autonomous Region wines. This is of great significance for brewing wines with the terroir characteristics of the eastern foothills of the Helan Mountains, and plays a very important role in promoting the international competitiveness of Ningxia wines. In addition, the eastern foothills of the Helan Mountains in Ningxia face problems such as high sugar and low acidity in grape raw materials, resulting in high alcohol content (14.4±1.0% vol) after fermentation, unbalanced body, and insufficient typical flavor characteristics. Therefore, it is urgent to screen for a strain that can produce high glycerol and low alcohol to improve the flavor and quality of wines from the eastern foothills of the Helan Mountains in Ningxia. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a strain that produces high glycerol and low alcohol in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a high-glycerol-producing brewing yeast and its application in wine fermentation in Ningxia. During the screening process, the brewing yeast of this invention exhibited excellent glycerol production capacity, averaging 10.91 g / L, higher than other brewing yeasts screened from the same vineyard and commercially available yeasts. Wines made with this yeast have a smooth and harmonious taste and are of high quality. Therefore, for local Ningxia wine grapes, inoculating with the brewing yeast ND10 of this invention can increase the complexity and fullness of the wine's flavor without affecting its aroma, resulting in a smooth, rich, and creamy taste. Simultaneously, using a local yeast strain instead of commercial brewing yeast helps to mitigate the homogenization of wine quality caused by using imported commercial brewing yeast, better showcasing the characteristics of Ningxia wines, thereby helping the Ningxia wine industry develop its own unique characteristics and promoting the independent and innovative development of the entire wine industry in Ningxia and even the entire Chinese wine industry.

[0006] The first objective of this invention is to provide a high-glycerol-producing brewer's yeast strain, the brewer's yeast having the accession number CGMCC No. 34040.

[0007] A second objective of this invention is to provide an application of the aforementioned brewing yeast in wine fermentation.

[0008] The third objective of this invention is to provide a method for producing wine, wherein the method involves inoculating the aforementioned wine yeast into a grape juice fermentation system for fermentation and culture, thereby obtaining the wine.

[0009] Furthermore, the grape juice fermentation system is prepared by crushing grapes.

[0010] Furthermore, the fermentation culture temperature is 20-30℃.

[0011] Furthermore, the pH of the fermentation culture is 2.5-8.0.

[0012] Furthermore, the inoculation amount of the brewing yeast is 1%-10%.

[0013] Furthermore, the grape juice fermentation system is sterilized before inoculation with brewer's yeast.

[0014] Furthermore, the grapes are sourced from Ningxia. Because the Ningxia region, particularly the eastern foothills of the Helan Mountains, suffers from high sugar and low acid content in its grape raw materials, resulting in high alcohol content (14.4±1.0% vol) after fermentation, an unbalanced body, and less typical flavor characteristics, the winemaking yeast of this invention, when used to ferment grape raw materials from Ningxia, can increase glycerol production and reduce alcohol content, thereby improving the quality of wines from Ningxia.

[0015] A fourth objective of this invention is to provide a wine produced by the above-described production method.

[0016] The beneficial effects of this invention are:

[0017] The brewing yeast ND10 described in this invention can impart excellent flavor and taste to wines originating from Ningxia. Inoculating local Ningxia grapes with the brewing yeast ND10 of this invention can increase the complexity and fullness of the wine's flavor without affecting its aroma, resulting in a rounder, richer, and smoother taste. Simultaneously, by using a local yeast strain instead of commercial brewing yeast, it helps to overcome the homogenization of wine quality caused by the use of imported commercial brewing yeast, better showcasing the characteristics of Ningxia wines. This helps the Ningxia wine industry develop its own unique local characteristics, promoting the independent and innovative development of the entire wine region and even the Chinese wine industry.

[0018] Preservation of biological materials

[0019] Saccharomyces cerevisiae ND10, which was deposited on April 1, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34040, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Saccharomyces cerevisiae. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0021] Figure 1 It refers to the colony morphology of the strain on WL medium;

[0022] Figure 2 This is a schematic diagram of the glycerol standard curve;

[0023] Figure 3 It refers to the glycerol content in the sample after a small-scale fermentation test of the strain;

[0024] Figure 4 The initial total sugar, residual sugar, glycerol, and alcohol content of the wine obtained from the fermentation test of commercial yeast and ND10 strain are shown. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0026] The culture medium formulations involved in the following examples are as follows:

[0027] YPD solid medium (per 1L): 10g yeast extract, 20g peptone, 20g glucose, 20g agar.

[0028] WL nutrient agar medium (per 1L): 5g casein, 4g yeast extract, 50g glucose, 0.425g KCl, 0.125g CaCl2, 0.55g K2HPO4, 2.5mg FeCl3, 0.125g MgSO4, 22mg bromocresol green, 2.5mg MnSO4, 20g agar.

[0029] Example 1: Isolation, purification and identification of Saccharomyces cerevisiae ND10

[0030] The glycerol-producing brewing yeast of this invention was obtained from the Chardonnay grape variety in the vineyard of Lilan Winery in Yuanlong Village, Yongning County, Yinchuan City, Ningxia Hui Autonomous Region. The screening process is as follows:

[0031] Select whole, ripe grape bunches free of mold and rot, with three replicates per sample. Each grape sample weighs approximately 1 kg and is stored in a sterile bag. Grapes collected on the same day are processed on the same day. Under sterile conditions, the grape samples are destemmed, and whole, ripe grapes free of disease and mold are collected in 500 mL Erlenmeyer flasks. The grapes are crushed in the Erlenmeyer flasks using a sterile spatula. Each flask contains approximately 400 mL of grape must after crushing. Add 0.1042 g / L of potassium metabisulfite, stir well, and seal with sterile sealing film. The grape must is then allowed to ferment naturally at 25°C.

[0032] Samples were taken under aseptic conditions on days 0, 2, 4, 6, 10, and 13 of natural fermentation. The samples were serially diluted with sterile water to three appropriate concentrations (100 μL each), and spread onto sterilized WL nutrient agar plates. After incubation at 28°C for 72 hours, plates with 30-300 colonies were selected. Based on the WL nutrient agar classification method for yeast, colonies with different characteristics were classified, numbered, and their characteristics and numbers were recorded and photographed for archiving. From each yeast group with different colony morphologies and emergence times, 2-3 strains were selected and preserved in YPD slant agar at 4°C. After initial yeast screening, the strains were purified to prevent impurity. Single colonies were picked and purified on WL nutrient agar plates using the streak plate method. The plates were incubated upside down at 28°C for 72 hours. The characteristics of the colonies after each purification were recorded and photographed for archiving. Each strain was purified 2-3 times. The purified yeast was stored in 50% glycerol cryovials at -80°C for molecular identification.

[0033] The yeast to be tested was pre-activated and inoculated into YPD liquid medium, and cultured at 28℃ with constant temperature shaking at 160 rpm for 12-16 h until the logarithmic growth phase. After treatment with the M5 high-speed mix (catalog number MF848-10) from Beijing Polymer Biotechnology Co., Ltd., PCR amplification was performed. Primers for the 26S rDNA D1 / D2 region amplification were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are as follows: forward primer NL1: 5'-GCATATCAATAAGCGGAGGAAAAG-3'; reverse primer NL4: 5'-GGTCCGTGTTTCAAGACGG-3'. The PCR reaction system consisted of 20 μL: 2 μL template DNA, 1.5 μL of 10 μM NL1 primer, 1.5 μL of 10 μM NL4 primer, 10 μL of 2x M5 Hiper high-AT high-speed mix, and 5 μL of double-distilled water. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 5 min. 3 μL of amplification product was detected by 1% agarose gel electrophoresis.

[0034] PCR product sequencing was performed by Shanghai Sangon Biotech Co., Ltd.

[0035] The target sequence of the test strain was searched using the BLAST tool on the website of the National Center for Biotechnology Information (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Similarity sequences were searched, and the species position of strain ND10 was preliminarily determined based on the similarity of homologous sequences. Simultaneously, the sequencing chromatogram was corrected. The similarity between ND10 and corresponding sequences of known yeasts was compared; the similarity exceeded 99%. The species position of the test strain was finally identified by the colony morphology of ND10 on WL nutrient agar. The identified strain ND10 was *Saccharomyces cerevisiae*.

[0036] Example 2: Glycerol production capacity test of Saccharomyces cerevisiae ND10

[0037] Remove the strain frozen in glycerol tubes, thaw it, and use an inoculation loop to streak a small amount of bacterial solution onto WL nutrient agar medium in four zones. Incubate the streaked plates at 28°C for 48 hours, and purify twice. Add commercial dry yeast powder from the winery to 5% glucose water at a ratio of 1:10 (w / v), and incubate at 35°C for 20-25 minutes until dense bubbles appear, indicating yeast activation. Use an inoculation loop to take one loopful of bacterial solution and purify it using the four-zone streak method on WL nutrient agar medium, incubating at 28°C for 48 hours, and purifying twice.

[0038] After purifying the yeast twice on WL nutrient agar, single colonies from the WL plates were picked using an inoculation loop and inoculated into liquid YPD medium for 12-16 hours. Then, 1% of the activated bacterial culture was inoculated into 10 ml of YPD liquid medium for further culture. The absorbance of the pre-cultured bacterial solution at 600 nm was measured using a UV spectrophotometer, and the OD value of the bacterial solution was adjusted with 0.85% physiological saline. 600 Value up to 1.

[0039] Thaw the Cabernet Sauvignon grape juice from Ningxia region that was frozen at -20℃, sterilize it at 70℃ for 30 min, and add it in equal batches to 50mL centrifuge tubes. Inoculate the bacterial solution obtained in (2) with adjusted concentration into the centrifuge tubes. The inoculation amount is 1%. Each bacterial solution is inoculated into 3 centrifuge tubes as a parallel experiment. Record the single colony number corresponding to each centrifuge tube and start the fermentation process. During the fermentation, the ambient temperature is set to 25℃. Monitor the change of Brix value every day. When Brix does not decrease for 3 consecutive days, the alcoholic fermentation is considered to be over.

[0040] After the alcoholic fermentation was completed, the bacterial culture was centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm polyethersulfone (PES) filter membrane. Glycerol was qualitatively identified by high performance liquid chromatography based on the elution time of glycerol standards (see Table 1). Glycerol was quantified by plotting a standard curve of the standards (see Table 2), thereby evaluating the glycerol production capacity of different strains.

[0041] Table 1. Setup of the glycerol standard sample group

[0042] Sample number 1 2 3 4 5 6 Glycerin (Gly) g / L 15 9 3 1.5 0.75 0.15

[0043] Table 2 Glycerin Standard Curve

[0044] compound Peak time Standard curve <![CDATA[R 2 ]]> glycerin 16.04 y = 2441.8x - 30.417 0.9999

[0045] Using grape juice without added yeast as a blank control, the following were tested as experimental controls: different genotypes of Saccharomyces cerevisiae strains C15, D18, E22, E26, F11, and F13 from the same batch of naturally fermented wine samples, as well as a previously screened Saccharomyces cerevisiae strain NW9 (preservation number CGMCC No. 28504) with good fermentation performance, and commercial dry yeast powder (SY) used by the winery. The results of glycerol production of each strain are shown in Table 3.

[0046] Table 3 shows the basic glycerol yield of the wine samples after small-scale fermentation experiments with strain 3.

[0047]

[0048] Centrifuge tube fermentation experiments yielded three strains with high glycerol production (>8 g / L), of which only one strain, *Saccharomyces cerevisiae* ND10, produced glycerol >10 g / L. In conclusion, the selected wild *Saccharomyces cerevisiae* strain ND10 demonstrated excellent glycerol production during grape juice fermentation in Ningxia, making it a high-quality strain capable of enhancing the quality characteristics of local wines and possessing commercial application potential.

[0049] Example 3: Comparative Fermentation Experiment of Commercial Saccharomyces cerevisiae and ND10

[0050] The winery used commercial yeast to ferment Cabernet Sauvignon grape must in a large fermentation tank (2 tons) and obtained the basic physicochemical properties shown in Table 4.

[0051] Table 4 Basic Physicochemical Indicators of Large-Scale Commercial Yeast Fermentation

[0052]

[0053] Because the grapes grown on the eastern foothills of the Helan Mountains in Ningxia have high sugar and low acid content, resulting in a high alcohol content (14.4±1.0% vol) after fermentation, an unbalanced body, and a lack of typical flavor characteristics, the grape juice fermentation system was expanded to 100 mL according to the experimental method in Example 2, and fermentation experiments were conducted on commercial yeast and ND10 strain. Figure 4 As shown, after fermentation, there was no significant difference in residual sugar content, but the glycerol yield was 3.53 ± 0.25 g / L higher than that of commercial yeast, and the alcohol content was 8.6% vol, which was 3.7% vol lower than that of Saccharomyces cerevisiae. In conclusion, the selected wild Saccharomyces cerevisiae strain ND10 exhibits excellent high glycerol and low alcohol production capabilities during grape juice fermentation in Ningxia, making it a high-quality Saccharomyces cerevisiae strain capable of bringing out the unique characteristics of local wines and possessing the potential for commercial application.

[0054] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A high-glycerol-producing brewing yeast, characterized in that, The brewing yeast ( Saccharomyces cerevisiae The accession number of the object is CGMCC No. 34040.

2. The application of the brewing yeast as described in claim 1 in wine fermentation.

3. A method for producing wine, characterized in that, The production method is as follows: the wine yeast described in claim 1 is inoculated into a grape juice fermentation system for fermentation and culture to obtain the wine.

4. The production method according to claim 3, characterized in that, The grape juice fermentation system is prepared by crushing grapes.

5. The production method according to claim 3, characterized in that, The fermentation culture temperature is 20-30℃.

6. The production method according to claim 3, characterized in that, The pH of the fermentation culture is 2.5-8.

0.

7. The production method according to claim 3, characterized in that, The inoculation amount of the brewing yeast is 1%-10%.

8. The production method according to claim 3, characterized in that, The grape juice fermentation system is sterilized before inoculation with brewer's yeast.

9. The production method according to claim 4, characterized in that, The grapes mentioned are from Ningxia.

10. Wine produced by the production method according to any one of claims 3-9.

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