Saccharomyces cerevisiae strain and use thereof in fermentation production of ethanol

Abstract

Provided are a Saccharomyces cerevisiae strain and use thereof in the fermentation production of ethanol. The strain is a Saccharomyces cerevisiae strain that is resistant to low temperatures, high sugar, ethanol, and low pH. When a sugar solution with a concentration greater than 300 g / L is fermented with the strain at 10 °C to 22 °C, a relatively high fermentation alcohol concentration can be achieved in a shorter period of time, the fermentation is fast, the fermentation period is short, and there is less residual sugar. The present invention effectively solves the problems with the existing alcohol fermentation techniques: when a sugar solution with a high concentration is fermented at a low temperature, the fermentation is slow, the fermentation efficiency is low, and the production costs are high.

Description

A strain of brewer's yeast and its application in the fermentation production of ethanol

[0001] This application claims priority to Chinese Patent Application No. 202411635755.9, filed on November 15, 2024, entitled "A Strain of Saccharomyces cerevisiae and Its Application in Fermentation Production of Ethanol", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of microbial technology, and more particularly to a strain of Saccharomyces cerevisiae and its application in the fermentation of ethanol. Background Technology

[0003] Blended spirits are alcoholic beverages made by blending or further processing fermented wines, distilled spirits, or edible alcohol as a base, adding edible raw and auxiliary materials. They are low in alcohol and offer diverse flavors, making them extremely popular among young consumers. In recent years, the market size has shown a steady growth trend, and the global market value of blended spirits is expected to reach several billion US dollars by 2025. To achieve harmony and stability in flavor, blended spirits often require a clean and aromatic distilled spirit to bring out the characteristics of the added flavorings, achieving a harmonious blend of aromas. Compared to fruit wines, grape wines, beer, and even milk wines, alcohol fermented with sugar water is more suitable as a base spirit for blended spirits because it has a pure flavor, no off-flavors, and is less likely to damage the unique aroma and style of the blended spirit or create unpleasant sensations.

[0004] However, current sugar syrup-based alcohol production technologies require low-temperature fermentation (fermentation temperature <22℃) in Europe and Russia due to climatic conditions. Existing technologies using sugar syrup for low-temperature fermentation typically employ sugar syrup concentrations of 150g / L to 290g / L. When the fermentation temperature is below 22℃, the fermentation time often exceeds 72 hours. Therefore, current technologies generally suffer from low sugar concentrations, long fermentation times, incomplete fermentation in a short period, low alcohol content (generally 14-16%), high residual sugar, low production efficiency, and long production times, leading to significantly increased production costs. Therefore, there is an urgent need to provide yeast strains capable of rapidly fermenting high-concentration sugar syrup at low temperatures to produce high-alcohol content, thereby improving production efficiency. Summary of the Invention

[0005] In view of this, the present invention provides a strain of brewing yeast and its application in the fermentation production of ethanol. This strain is tolerant to low temperatures, high sugar levels, and low pH. It can ferment sugar solutions with a concentration greater than 300 g / L at 10℃-22℃, achieving a higher alcohol content in a shorter time. It has the advantages of fast fermentation speed, short cycle, and low residual sugar, solving the problems of slow fermentation speed, low fermentation efficiency, long fermentation cycle, and high cost inherent in existing low-temperature fermentation techniques for producing alcohol from high-concentration sugar solutions.

[0006] This invention provides a *Saccharomyces cerevisiae* strain MP-8, which was isolated from distiller's grains and deposited at the China Center for Type Culture Collection (CCTCC) on June 14, 2024, with the accession number CCTCC NO: M20241232. Its ITS rDNA sequence is shown in SEQ ID NO: 1.

[0007] Experiments show that strain MP-8 is resistant to low temperature, high sugar, ethanol and low pH. Using this strain to ferment high-concentration sugar solutions to produce alcohol results in fast fermentation speed, short fermentation cycle, high alcohol content and low production cost.

[0008] The present invention also provides a microbial fermentation agent, including at least one of the following: Saccharomyces cerevisiae strain MP-8 or its culture, metabolites, exosomes, and fermentation products.

[0009] This invention does not impose any particular limitation on the form of the microbial fermentation agent; any form commonly used in the art, such as solid preparations, liquid preparations, or semi-solid preparations, is acceptable. In a specific embodiment of this invention, the microbial fermentation agent is a solid agent, namely, the dry yeast of the Saccharomyces cerevisiae strain MP-8.

[0010] The present invention also provides the application of the brewing yeast strain MP-8 or the microbial fermentation agent in the fermentation production of ethanol or in increasing the alcohol content of alcoholic beverages.

[0011] The present invention also provides a method for producing ethanol by fermentation, comprising: adding MP-8 or the microbial fermentation agent to a high sugar solution and fermenting.

[0012] In the method of the present invention, the dry weight of the Saccharomyces cerevisiae strain or the microbial fermentation agent to the mass-volume ratio of the high sugar solution (g / ml) is 0.1% to 2%, preferably 0.5% to 1%, and specifically 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.

[0013] In the method described in this invention, the fermentation is a high-sugar fermentation; the sugar concentration of the high sugar is less than 400 g / L, including but not limited to this concentration. In some specific embodiments of this invention, the sugar concentration of the high sugar is 300–360 g / L, specifically 300 g / L, 310 g / L, 320 g / L, 330 g / L, 340 g / L, 350 g / L, or 360 g / L.

[0014] Furthermore, the fermentation is a low-temperature fermentation, and the low temperature is 10-38℃, preferably 10-22℃, specifically 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, and 22℃.

[0015] Furthermore, the fermentation time is ≤120h, preferably 48h-120h, specifically 48h, 60h, 72h, 84h, 96h, 108h, or 120h.

[0016] In the method described in this invention, the sugar solution also includes urea and a fermentation promoter.

[0017] In some embodiments, the concentration of urea in the sugar solution is 1 g / L-5 g / L, specifically 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L (i.e., 2.5‰), 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, and 5 g / L. In this invention, the concentration of the fermentation promoter is 0.2-0.8 g / L, specifically 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, and 0.8 g / L. This invention does not impose any special restrictions on the specific type of fermentation promoter; any commonly used fermentation promoter for ethanol production in the art is acceptable. In a specific embodiment of this invention, the fermentation promoter is NutrienFast, a nutritional product of Angel Yeast.

[0018] The brewing yeast strain MP-8 provided by this invention can ferment sugar solutions with a concentration greater than 300g / L at 10℃-22℃, achieving a higher alcohol content in a shorter time, with faster fermentation speed, shorter cycle, and lower residual sugar. This solves the problems of slow fermentation speed, low fermentation efficiency, and high production cost in existing alcohol fermentation technologies when fermenting high-concentration sugar water at low temperatures. It is suitable for industrial fermentation production of various alcoholic products.

[0019] Biological Preservation Instructions

[0020] Saccharomyces cerevisiae MP-8 was deposited on June 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO:M 20241232. Attached Figure Description

[0021] Figure 1 shows the process flow diagram for the preparation of dry yeast;

[0022] Figure 2 shows the process flow diagram of the fermentation production of alcohol according to the present invention. Detailed Implementation

[0023] This invention provides a strain of brewing yeast and its application in the fermentation production of ethanol. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0024] The Saccharomyces cerevisiae strain MP-8 provided by this invention was deposited at the China Center for Type Culture Collection on June 14, 2024, with the accession number CCTCC NO: M20241232. Its ITS rDNA sequence is shown in SEQ ID NO: 1.

[0025] The sequence of SEQ ID NO: 1 is as follows:

[0026] Another object of the present invention is to provide the application of the above-mentioned strain in the fermentation production of ethanol (or alcohol).

[0027] This invention also provides a method for producing ethanol by fermentation using the strain MP-8 of this invention, comprising: adding MP-8 or the microbial fermentation agent to a high-sugar solution and fermenting. The method specifically includes the following steps:

[0028] Step 1: Prepare a high-sugar-concentration sugar solution by adding 2.5‰ (w / v) of urea and a fermentation promoter;

[0029] Step 2: After adding a certain amount of dry yeast made from strain MP-8, stir thoroughly to completely dissolve the dry yeast.

[0030] Step 3: Place in an incubator at a certain temperature for cultivation, stir two to three times a day, measure specific gravity and sugar content every 24 hours, and measure fermentation alcohol content after 72 hours of fermentation.

[0031] In the first step, the sugar solution is selected from sucrose or glucose, and its fermentation sugar concentration is less than 400 g / L, preferably 320 g / L-360 g / L.

[0032] In the second step, the amount of yeast added is 0.1%-2% based on the mass-volume content of dry weight and fermentation liquid volume, preferably 0.5%-1%.

[0033] In the third step, the fermentation temperature is 10℃-38℃, preferably 15-22℃, and the fermentation time is within 120 hours, preferably 72 hours.

[0034] This invention does not impose any particular limitation on the method for producing dry yeast from strain MP-8; any method commonly used in the art is acceptable. In this embodiment of the invention, the dry yeast is produced according to the process shown in Figure 1.

[0035] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0036] The present invention will be further illustrated below with reference to the embodiments:

[0037] Example 1

[0038] Multiple yeast strains were isolated from soil samples of natural wine cellars in Yibin, Sichuan Province. These strains underwent initial screening for ethanol tolerance and acclimatization to high sugar content. Finally, a yeast strain capable of tolerating 30% sucrose and 10%-15% ethanol was selected. Using this strain, a study was conducted on low-temperature fermentation of high-concentration sugar water to produce alcohol. The results showed that this strain exhibited rapid fermentation and strong alcohol production capacity. Molecular biological identification confirmed it as a *Saccharomyces cerevisiae* strain, named MP-8.

[0039] The specific isolation and purification method is as follows: Take 0.5g of koji block and dilute it with sterile water at solid-liquid ratios of 1:100, 1:1000, and 1:10000. Take 50μL of each dilution and spread it on a solid culture medium (8% sucrose, 2% yeast extract, 0.1% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 2% agar powder, and 50μg / ml ampicillin). After incubating at 30℃ for 1-3 days, pick several large, thick, smooth, moist, and viscous single colonies and streak them on the solid culture medium. Repeat twice to obtain several pure yeast strains. After initial screening for ethanol tolerance and acclimatization to high sugar tolerance, a strain with strong tolerance was selected. Studies on low-temperature fermentation of high-concentration sugar water to produce alcohol showed that its fermentation performance was outstanding. Molecular biological identification confirmed it as a Saccharomyces cerevisiae strain, named MP-8.

[0040] The invention utilizes strain MP-8 and existing strains for low-temperature fermentation of a high-sugar solution to produce ethanol. The fermentation method is shown in the process flow diagram in Figure 1, and specifically includes the following steps:

[0041] Step 1: Prepare high sugar concentration sugar solutions (300g / L, 340g / L, 350g / L, 360g / L), add 2.5‰ (w / v) urea and fermentation promoter (Angel Nutrition product NutrienFast, product batch numbers 240523061-1, 240523061-2, 240523061-3 or 240523061-4) to make the urea concentration 2.5g / L and the fermentation promoter concentration 0.4g / L;

[0042] Step 2: Add 0.8%-1% of the dry yeast prepared by strain (the strain of this invention and comparative strain 1-2, add 0.1-2g of dry yeast per 100ml of sugar solution) to the prepared sugar solution, stir thoroughly until the dry yeast is completely dissolved;

[0043] Step 3: Place the mixture in a static incubator (purchased from Shanghai Yuejin Medical Equipment Co., Ltd.) at a specific temperature of 10℃. Stir two to three times a day during the static incubation period, and measure the specific gravity and sugar content every 24 hours. After 72 hours of fermentation, measure the fermentation alcohol content, residual sugar, and specific gravity of the fermentation liquid. Comparative strain 1 was a high-alcohol yeast with preservation number CCTCC NO:2023130; comparative strain 2 was Angel Yeast from the article "The Influence of NutrienFast Addition Period on Wine Fermentation".

[0044] The sources of the above raw materials and the models of the equipment are shown in Tables 1 and 2. The residual sugar content and specific gravity were measured using a hydrometer. The method for determining the fermentation alcohol content is as follows, and the results are shown in Tables 3 to 6.

[0045] Methods for determining the alcohol content of fermented food:

[0046] (1) Distillation

[0047] After fermentation, shake well. Use a 100mL graduated cylinder to take 100mL of fermentation mash and pour it into a 1000mL distillation flask. Add 100-150mL of tap water to the distillation flask, add 2 drops of defoaming agent, and proceed with distillation. Collect the distillate in a 100mL volumetric flask (with an external cold water bath to control the temperature of the distillate below 25℃). When the distillate reaches approximately 95mL, stop distillation, remove the flask, and bring the volume to 100mL. Shake well.

[0048] (2) Measuring alcohol content

[0049] Pour the distillate to a clean, dry 100mL graduated cylinder and let it stand for several minutes until the bubbles disappear. Then, place the cylinder into a clean, dry precision alcohol meter and gently press it down. After standing, observe the reading at the point tangent to the meniscus while simultaneously recording the temperature using a thermometer. Based on the measured temperature and alcohol meter reading, convert the alcohol concentration to 20℃ using the "Alcohol Meter Temperature-Concentration Conversion Table" shown in Appendix E of the national standard GB / T 20886.1-2021bz (Quality Requirements for Yeast for Food Processing), retaining one decimal place. Repeat the test once more and use the higher of the two readings.

[0050] Table 1 Source of Ingredients

[0051] Table 2 Equipment Information

[0052] Table 3. Sugar content, specific gravity, and fermentation alcohol content under the conditions of initial sugar concentration of 300 g / L and culture temperature of 10℃.

[0053] Table 4. Sugar content, specific gravity, and fermentation alcohol content under the conditions of initial sugar concentration of 340 g / L and culture temperature of 17℃.

[0054] Table 5. Sugar content, specific gravity, and fermentation alcohol content under the conditions of initial sugar concentration of 350 g / L and culture temperature of 20℃.

[0055] Table 6. Sugar content, specific gravity, and fermentation alcohol content under the conditions of initial sugar concentration of 360 g / L and culture temperature of 21℃.

[0056] The results showed that, compared with control strains 1-2, with an initial sugar concentration in the range of 300g / L-360g / L and a fermentation temperature in the range of 10℃-22℃, the strain of the present invention achieved a higher alcohol content and lower residual sugar and specific gravity at the same fermentation time of 72 hours (i.e., three days). This indicates that the strain of the present invention has the advantages of shorter fermentation time and higher efficiency compared with control strains 1-2.

[0057] Example 2

[0058] The difference between this fermentation method and Example 1 is that the culture temperature was uniformly set at 20℃, the fermentation completion time for each strain was recorded, and other fermentation conditions and measurement methods were the same as in Example 1. The alcohol content and residual sugar after fermentation were also measured. The results are shown in Tables 7-9.

[0059] Table 7. Fermentation end time, alcohol content, residual sugar, and specific gravity of different strains under the conditions of initial sugar concentration of 340 g / L and culture temperature of 20 °C.

[0060] The results showed that the fermentation of the strain of the present invention was completed after 72 hours of fermentation under the conditions of initial sugar concentration of 340 g / L and culture temperature of 20 °C, while the fermentation of control strains 1 and 2 took 96 hours to approach the end of fermentation.

[0061] Table 8. Fermentation results of different strains under the conditions of initial sugar concentration of 350 g / L and culture temperature of 20 °C.

[0062] As shown in Table 8, the strain of this invention fermented completely after 72 hours of cultivation under the conditions of an initial sugar concentration of 350 g / L and a culture temperature of 20°C, while the control strains 1 and 2 fermented to near the fermentation endpoint after 120 hours. The fermentation endpoint was determined based on both specific gravity and residual sugar; generally, a specific gravity below 1.0 and residual sugar close to 0 were considered the fermentation endpoint.

[0063] Table 9. Fermentation results of different strains under the conditions of initial sugar concentration of 360 g / L and culture temperature of 20 °C.

[0064] As shown in Table 9, the fermentation of the strain of the present invention ended after 72 hours of culture, while the fermentation of comparative strains 1 and 2 was not complete even after 120 hours.

[0065] The results above show that, under the same fermentation conditions, compared with control strains 1-2, the strain of this invention has a higher alcohol content, shorter fermentation time, and lower residual sugar. This indicates that the strain of this invention has significant advantages in low-temperature tolerance, sugar tolerance, and ethanol tolerance, and is more suitable for low-temperature fermentation of sugar water to produce alcohol.

[0066] Example 3

[0067] The difference between this embodiment and Example 1 in terms of fermentation method is as follows: the initial sugar concentration is 250 g / L, the culture temperature is 28℃, and the pH is 3.5. Other fermentation conditions and measurement methods are the same as in Example 1. The fermentation end time of the two strains was recorded, and the residual sugar and alcohol content after fermentation were measured. The results are shown in the table below.

[0068] Table 10

[0069] The results above show that, under low pH conditions, this strain has a higher alcohol content and a faster fermentation rate compared to control strain 1, indicating that this strain has a significant advantage in tolerance to low pH.

[0070] Example 4

[0071] Using the Saccharomyces cerevisiae with accession number CCTCC M20231997 in patent CN117603826 as control strain 3, the present invention strain MP-8 and control strain 3 were fermented and cultured for 5 to 10 days according to the fermentation method in Example 8 of patent CN117603826. The alcohol content of simulated grape juice with different sugar contents was measured after 5 days of fermentation by the present invention strain. The measurement method is the same as in Example 1 of the present invention. The results are shown in Table 10.

[0072] Table 11

[0073] The results showed that, compared with control strain 3, strain MP-8 of the present invention produced higher alcohol content, shorter fermentation time, and lower cost, resulting in greater economic benefits.

[0074] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Saccharomyces cerevisiae strain MP-8, characterized in that, Its accession number is CCTCCNO: M20241232.

2. A microbial fermentation inoculant characterized by, It includes at least one of the following: the Saccharomyces cerevisiae strain or its culture, metabolites, exosomes, and fermentation products as described in claim 1.

3. The application of the brewing yeast strain MP-8 according to claim 1 or the microbial fermentation agent according to claim 2 in the fermentation production of ethanol or in increasing the alcohol content of alcoholic beverages.

4. Process for the fermentative production of ethanol, characterized in that, The *Saccharomyces cerevisiae* strain of claim 1 or the microbial fermentation agent of claim 2 is inoculated into a high-sugar solution for fermentation.

5. The method of claim 4, wherein, The dry weight to high sugar solution mass-volume ratio (g / ml) of the Saccharomyces cerevisiae strain or the microbial fermentation agent is 0.1% to 2%, preferably 0.5% to 1%.

6. The method according to claim 4 or 5, characterized in that, The fermentation is a high-sugar fermentation; the sugar concentration of the high sugar is <400g / L; preferably, the sugar concentration of the high sugar is 300-360g / L.

7. The method according to any one of claims 4 to 6, characterized in that, The fermentation is a low-temperature fermentation, and the low temperature is 10-38℃.

8. The method of claim 4, wherein, The fermentation time is 48 to 120 hours, preferably 72 hours.

9. The method according to any one of claims 4 to 8, characterized in that, The sugar solution also includes urea and a fermentation promoter.

10. The method of claim 9, wherein, In the sugar solution, the concentration of urea is 1 g / L to 5 g / L, and the concentration of fermentation promoter is 0.2 g / L to 0.8 g / L.

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