A yeast cell wall and its application in the field of alcohol fermentation

Abstract

The present invention provides a yeast cell wall and its application in the field of alcohol fermentation. The yeast cell wall is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118. The yeast cell wall provided by the present invention, as an embedding carrier of an alcohol fermentation promoter, has an embedding efficiency increased by more than 30%. Moreover, this yeast cell wall is rich in ergosterol and other sterols, as well as various unsaturated fatty acids, and has high nutritional value, which can provide nutrient raw materials for alcohol fermentation, thereby improving the fermentation performance of yeast and increasing the liquor yield of alcohol fermentation.

Description

Technical Field

[0001] The invention belongs to the field of food technology, and particularly relates to a yeast cell wall and application thereof in the field of alcohol fermentation. Background Art

[0002] During the high-concentration ethanol fermentation process, yeast fermentation to produce alcohol usually faces a lack of yeast nutrition, resulting in decreased yeast activity, reduced budding rate, and increased mortality rate in the middle and late stages of alcohol fermentation, which in turn leads to high residual sugar and incomplete alcohol fermentation.

[0003] In practice, alcohol plants often use urea and acid protease to promote alcohol fermentation. However, according to the GB2760-2011 Food Additives Catalogue, urea is not permitted in edible alcohol production. Furthermore, the addition of urea and acid protease alone cannot support high-density yeast cultivation or fermentation of high-concentration alcohol (alcohol content ≥ 16.5% (v / v)).

[0004] Yeast and nutrient salts are typically added all at once, resulting in excessive nutrition, which can lead to rapid yeast growth and subsequent nutrient deficiency. Under a microscope, overnourished yeast appear larger, but nutrient deficiency in the later stages of fermentation can cause yeast death, manifesting as a decrease in alcohol production and a slowdown in fermentation. Therefore, controlling yeast growth, reducing yeast mortality in the later stages of fermentation, and addressing the precise nutritional needs of yeast are key technical challenges in achieving high-concentration ethanol fermentation.

[0005] The yeast cell wall is a byproduct of yeast extract production. It is primarily composed of a cytoplasmic membrane, a glucan layer, and a mannose layer. The yeast cell wall has a fat content of 15-20%, primarily composed of the phospholipids that make up the cell membrane, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylserine (PS). Phospholipids are not only essential components of yeast cells but also serve as important nutrients and regulatory factors for yeast growth, helping to enhance yeast activity and reproductive performance. The yeast cell wall also contains ergosterols. During the yeast extract production process, any remaining contents of the yeast cytoplasm, including proteins, peptides, amino acids, nucleic acids, lipids, and growth factors, remain within the cell membrane cavity. These nutrients and structural proportions provide the necessary nutrients for yeast growth and maintain its physiological activity.

[0006] At the same time, the cavity composed of the three-layer structure of the yeast cell wall is a natural carrier for encapsulating nutrient molecules. By physically adsorbing nutrients, it becomes a natural adjuvant for the nutrition of alcohol fermentation yeast.

[0007] However, the existing yeast cell wall as an embedding carrier has low embedding efficiency and low nutritional value that can be provided. Therefore, providing an embedding carrier with high embedding efficiency and high nutritional value has become a problem to be solved. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a yeast cell wall and its application in the field of alcohol fermentation. The yeast cell wall provided by the present invention, as an embedding carrier for alcohol fermentation promoter, has an embedding efficiency increased by more than 30%, and this yeast cell wall is rich in ergosterol and other sterols, as well as a variety of unsaturated fatty acids, has high nutritional value, and can provide nutritional raw materials for alcohol fermentation.

[0009] The present invention provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M2020118.

[0010] Preferably, the yeast cell wall accounts for more than 30wt% of the cell dry weight of the Saccharomyces cerevisiae, and the yeast cell wall includes ergosterol, and the ergosterol accounts for more than 3wt% of the yeast cell wall.

[0011] The present invention also provides a preparation method of the above yeast cell wall, including the following steps:

[0012] A) Fermenting Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118 to obtain fermented milk;

[0013] B) Autolyzing the fermented milk and then carrying out enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0014] C) Separating and drying the enzymatic hydrolysis product to obtain a yeast cell wall.

[0015] Preferably, the preparation method of the fermented milk includes the following steps:

[0016] (1) Shake flask culture: Inoculating the yeast strain in a shake flask medium for fermentation culture to obtain a shake flask culture solution. The yeast strain is Saccharomyces cerevisiae ER18, preserved in the China Center for Type Culture Collection, CCTCC NO: M 2020118. Among them, the sugar content of the medium for shake flask culture is 5-10%;

[0017] (2) Primary culture: Inoculating the shake flask culture solution obtained in step (1) into a shake flask medium for fermentation culture to obtain a primary fermentation culture solution; the volume of the primary fermentation tank is 0.2~20m 3 ; the sugar content of the medium for primary culture is 5-10%;

[0018] (3) Secondary fermentation: The primary fermentation culture solution obtained in step (2) is inoculated into a secondary culture medium for fermentation to obtain a secondary fermentation culture solution. The volume of the primary fermentation tank is 20 - 60 m 3 ; The sugar content of the secondary fermentation culture medium is 5 - 20%;

[0019] (4) Tertiary fermentation: The secondary fermentation culture solution obtained in step (3) is inoculated into a tertiary culture medium for fermentation to obtain a tertiary fermentation culture solution. The volume of the tertiary fermentation tank is 60 - 150 m 3 ; The sugar content of the tertiary fermentation culture medium is 5 - 20%;

[0020] (5) Commercial fermentation: The tertiary fermentation culture solution obtained in step (3) is inoculated into a commercial culture medium for fermentation to obtain a commercial fermentation culture solution. The volume of the commercial fermentation tank is 150 - 400 m 3 , and the sugar content of the commercial fermentation culture medium is 10 - 30%; Obtain fermented milk mother;

[0021] Preferably, in steps (3) to (5) of the method, the steps can be omitted as needed.

[0022] Preferably, before autolyzing the fermented milk mother, adjust the concentration of the fermented milk mother to 15 - 20%;

[0023] The temperature of the autolysis is 55 - 60 °C, and the time of the autolysis is 4 - 8 h;

[0024] The enzymes used for enzymatic hydrolysis include papain and bacterial alkaline protease; The dosage ratio of papain to bacterial alkaline protease is 0.5 - 2:1; The amount of the enzyme used for enzymatic hydrolysis is 0.05 - 1.15%;

[0025] The temperature of the enzymatic hydrolysis is 55 - 70 °C, the pH of the enzymatic hydrolysis is 5.5 - 6.0, and the time of the enzymatic hydrolysis is 5 - 20 hours;

[0026] After the enzymatic hydrolysis, sterilize the enzymatic hydrolysis product at 90 - 100 °C.

[0027] The present invention also provides an application of the above yeast cell wall in the preparation of an alcohol fermentation promoter, and the alcohol fermentation promoter includes:

[0028] 29 - 135 parts by mass of an alcohol fermentation promoter matrix;

[0029] 30 - 70 parts by mass of yeast cell wall.

[0030] Preferably, the alcohol fermentation promoter matrix comprises: 10-40 parts of yeast extract, 10-40 parts of ammonium sulfate, 5-30 parts of diammonium hydrogen phosphate, 2-10 parts of magnesium sulfate, 2-10 parts of zinc sulfate, and 2-5 parts of trace elements and vitamins.

[0031] Preferably, a certain amount of alcohol fermentation promoter matrix is coated by the yeast cell wall, wherein the mass ratio of the alcohol fermentation promoter matrix coated by the yeast cell wall to the uncoated alcohol fermentation promoter matrix is 40-95:5-40.

[0032] The present invention also provides a preparation method of the above-mentioned alcohol fermentation promoter, comprising the following steps:

[0033] Dissolve the alcohol fermentation promoter matrix in a solvent to obtain a mixed solution;

[0034] Add yeast cell wall to the mixed solution and stir to obtain a yeast cell wall mixture;

[0035] Dry the yeast cell wall mixture to obtain the alcohol fermentation promoter.

[0036] The present invention also provides an application of the above-mentioned alcohol fermentation promoter in alcohol fermentation, and the addition amount of the alcohol fermentation promoter is 0.2-0.4 kg / ton of alcohol.

[0037] Compared with the prior art, the present invention provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118. The yeast cell wall provided by the present invention, as an embedding carrier of the alcohol fermentation promoter, has an embedding efficiency increased by more than 30%, and this yeast cell wall is rich in ergosterol and other sterols, as well as various unsaturated fatty acids, and has high nutritional value, and can provide nutritional raw materials for alcohol fermentation, thereby improving the fermentation performance of yeast and the liquor yield of alcohol fermentation. Detailed Embodiments

[0038] The present invention provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M2020118.

[0039] The strain of the Saccharomyces cerevisiae is Saccharomyces cerevisiae ER18, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 2020118.

[0040] In the present invention, after the Saccharomyces cerevisiae is used as the starting strain and fermented, the yeast cell wall accounts for more than 30 wt% of the dry cell weight of the Saccharomyces cerevisiae, preferably 30 wt% - 35 wt%, and the ergosterol accounts for more than 3 wt% of the yeast cell wall, preferably 3 wt% - 3.5 wt%.

[0041] The present invention also provides a method for preparing the above yeast cell wall, comprising the following steps:

[0042] A) Fermenting the Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118 to obtain fermented milk of yeast;

[0043] B) Autolyzing the fermented milk of yeast and then performing enzymatic hydrolysis to obtain an enzymatic hydrolysis product;

[0044] C) Separating and drying the enzymatic hydrolysis product to obtain the yeast cell wall.

[0045] The present invention first prepares the fermented milk of yeast. Among them, the method for preparing the fermented milk of yeast comprises the following steps:

[0046] (1) Shake flask culture: Inoculating the yeast strain in a shake flask medium for fermentation culture to obtain a shake flask culture solution. The yeast strain is Saccharomyces cerevisiae ER18, preserved in the China Center for Type Culture Collection, CCTCC NO: M 2020118. Among them, the sugar content of the medium for shake flask culture is 5 - 10%;

[0047] (2) Primary culture: Inoculating the shake flask culture solution obtained in step (1) into a shake flask medium for fermentation culture to obtain a primary fermentation culture solution; the volume of the primary fermentation tank is 0.2 - 20 m 3 ; the sugar content of the medium for primary culture is 5 - 10%;

[0048] (3) Secondary fermentation: Inoculating the primary fermentation culture solution obtained in step (2) into a secondary medium for fermentation culture to obtain a secondary fermentation culture solution. The volume of the primary fermentation tank is 20 - 60 m 3 ; the sugar content of the medium for secondary fermentation is 5 - 20%;

[0049] (4) Tertiary fermentation: Inoculating the secondary fermentation culture solution obtained in step (3) into a tertiary medium for fermentation culture to obtain a tertiary fermentation culture solution. The volume of the tertiary fermentation tank is 60 - 150 m 3 ; the sugar content of the medium for tertiary fermentation is 5 - 20%;

[0050] (5) Commercial fermentation: Inoculating the tertiary fermentation culture solution obtained in step (3) into a commercial medium for fermentation culture to obtain a commercial fermentation culture solution. The volume of the commercial fermentation tank is 150 - 400 m 3, the sugar content of the culture medium for commodity fermentation is 10-30%; fermented milk is obtained;

[0051] Preferably, in steps (3)-(5) of the method, the steps can be omitted as needed.

[0052] Preferably, in steps (1)-(5), the culture temperature is 28-35°C, preferably 30-33°C; the pH value is 4.5-7.0, preferably 4.8-6.0.

[0053] Among them, the time for shake flask culture is 20-30h, the time for primary fermentation is 8-15h; the time for secondary fermentation is 40-50h, the time for tertiary fermentation is 30-40h; the time for commodity fermentation is 20-30h.

[0054] By weight, the culture medium in steps (1)-(5) comprises the following components: 25-50 parts of carbon source, 2-5 parts of nitrogen source, 0.1-0.6 parts of potassium dihydrogen phosphate, 0.02-0.04 parts of magnesium sulfate, 0.02-0.05 parts of zinc sulfate and 0.2-0.8 parts of ammonium dihydrogen phosphate.

[0055] Among them, the carbon source is selected from one or more of cane molasses, beet molasses, corn hydrolyzate, glucose, sucrose and maltose; the nitrogen source is selected from one or more of corn steep liquor, plant protein, ammonia water, ammonium sulfate and yeast extract.

[0056] Among them, the dissolved oxygen DO for fermentation is 40-80%, preferably, the respiratory quotient RQ is 0.95-1.2.

[0057] After obtaining the fermented milk, the fermented milk is autolyzed and then enzymolyzed to obtain an enzymolysis product.

[0058] Among them, before autolysis, the concentration of the fermented milk is adjusted to 15-20%, which can be 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18, 18.5%, 19%, 19.5%, 20%, or any value between 15-20%. Among them, the concentration of the fermented milk is the percentage of the mass of dry yeast in the fermented milk accounting for the mass of the fermented milk.

[0059] Then, the fermented milk with adjusted concentration is autolyzed. Among them, the temperature of autolysis is 55-60°C, which can be 55, 56, 57, 58, 59, 60, or any value between 55-60°C, and the time of autolysis is 4-8h, which can be 4, 5, 6, 7, 8, or any value between 4-8h.

[0060] Next, the autolyzed yeast milk is enzymatically hydrolyzed. Among them, the enzymes used for the enzymatic hydrolysis include papain and bacterial alkaline protease; the dosage ratio of papain to bacterial alkaline protease is 0.5-2:1, which can be 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, or any value between 0.5-2:1; the amount of the enzyme used for the enzymatic hydrolysis is 0.05-1.15%, which can be 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.15%, or any value between 0.05-1.15%.

[0061] The temperature of the enzymatic hydrolysis is 55-70°C, which can be 55, 60, 65, 70, or any value between 55-70°C. The pH of the enzymatic hydrolysis is 5.5-6.0, which can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, or any value between 5.5-6.0. The time of the enzymatic hydrolysis is 5-20 hours, which can be 5, 10, 12, 15, 18, 20, or any value between 5-20 hours.

[0062] After the enzymatic hydrolysis, the enzymatic hydrolysis product is sterilized at 90-100°C. After the sterilization is completed, the temperature is lowered to 60-65°C, and then separation is carried out. The heavy phase separated is further spray-dried to obtain yeast cell walls.

[0063] The present invention also provides an application of the above yeast cell walls in the preparation of an alcohol fermentation promoter. The alcohol fermentation promoter includes:

[0064] 29-135 parts by mass of an alcohol fermentation promoter matrix;

[0065] 30-70 parts by mass of yeast cell walls.

[0066] The alcohol fermentation promoter provided by the present invention includes 29-135 parts by mass of an alcohol fermentation promoter matrix, which can be 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, or any value between 29-135 parts by mass.

[0067] In the present invention, the alcohol fermentation promoter matrix preferably includes the following raw materials in parts by mass:

[0068] 10-40 parts of yeast extract, 10-40 parts of ammonium sulfate, 5-30 parts of diammonium hydrogen phosphate, 2-10 parts of magnesium sulfate, 2-10 parts of zinc sulfate, and 2-5 parts of trace elements and vitamins.

[0069] The matrix of the alcohol fermentation promoter described in the present invention comprises 10 to 40 parts of yeast extract, which can be 10, 15, 20, 25, 30, 35, 40, or any value between 10 and 40 parts.

[0070] The matrix of the alcohol fermentation promoter described in the present invention further comprises 10 to 40 parts of ammonium sulfate, which can be 10, 15, 20, 25, 30, 35, 40, or any value between 10 and 40 parts.

[0071] The matrix of the alcohol fermentation promoter described in the present invention further comprises 5 to 30 parts of diammonium hydrogen phosphate, which can be 5, 10, 15, 20, 25, 30, or any value between 5 and 30 parts.

[0072] The matrix of the alcohol fermentation promoter described in the present invention further comprises 2 to 10 parts of magnesium sulfate, which can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 2 and 10 parts.

[0073] The matrix of the alcohol fermentation promoter described in the present invention further comprises 2 to 10 parts of zinc sulfate, which can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 2 and 10 parts.

[0074] The matrix of the alcohol fermentation promoter described in the present invention further comprises 2 to 5 parts of trace elements and vitamins, which can be 2, 2.5, 3, 3.5, 4, 4.5, 5, or any value between 2 and 5 parts.

[0075] The alcohol fermentation promoter provided by the present invention further comprises 30 to 70 parts by mass of yeast cell wall, which can be 30, 35, 40, 45, 50, 55, 60, 65, 70, or any value between 30 and 70 parts by mass. The yeast cell wall is selected from the yeast cell walls described above.

[0076] The yeast cell wall encapsulates a certain amount of the matrix of the alcohol fermentation promoter. Among them, the mass ratio of the matrix of the alcohol fermentation promoter encapsulated by the yeast cell wall to the unencapsulated matrix of the alcohol fermentation promoter is 40 to 95:5 to 40, which can be 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 50:40, 40:40, or any value between 40 and 95:5 and 40.

[0077] The present invention uses the above-mentioned yeast cell wall as an adsorption carrier, which has a higher embedding rate; at the same time, because the cell wall provided by the present invention has a high fat content, is rich in ergosterol and other sterols, and a variety of unsaturated fatty acids, the alcohol fermentation promoter provided by the present invention has better nutritional effects.

[0078] The alcohol fermentation promoter provided by the present invention can effectively solve the problems of excessive nutrition in the early stage and insufficient nutrition in the later stage of yeast fermentation throughout the whole process of yeast fermentation due to the high embedding rate of yeast cell walls and the various yeast nutrients (phospholipids, sterols and intracellular yeast nutrients) rich in the embedding carrier, thereby improving the yeast fermentation performance, increasing the liquor yield of alcohol fermentation, and having a relatively good market nutrition prospect.

[0079] The present invention also provides a preparation method of the above alcohol fermentation promoter, comprising the following steps:

[0080] Dissolve the alcohol fermentation promoter matrix in a solvent to obtain a mixed solution;

[0081] Add yeast cell walls to the mixed solution and stir to obtain a yeast cell wall mixture;

[0082] Dry the yeast cell wall mixture to obtain the alcohol fermentation promoter.

[0083] In the present invention, the alcohol fermentation promoter matrix is dissolved in a solvent to obtain a mixed solution. Among them, the solvent is selected from food-grade organic solvents, and the food-grade organic solvent is ethanol. The concentration of the food-grade organic solvent is 10-95% (v / v), which can be 10%, 20%, 30%, 40%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or any value between 10-95% (v / v). Preferably, it is 55%-95% (v / v). Yeast cell walls are prone to stick together in water, resulting in a decrease in specific surface area, a reduction in the efficiency of nutrients entering the cell walls, and a decrease in the embedding efficiency. However, yeast cell walls are not easily stuck together in a high-concentration alcohol solution, the yeast has a larger specific surface area, and nutrients are more likely to enter the interior of the cell walls, improving the embedding efficiency. A better embedding effect can make the nutrient release more stable, stably provide nutrients for yeast, and thus enhance the yeast alcohol fermentation ability.

[0084] Then, add yeast cell walls to the mixed solution and stir to obtain a yeast cell wall mixture.

[0085] Specifically, slowly add the dry yeast cell wall powder to the mixed solution, stir while adding until it is evenly mixed. Through physical adsorption, the yeast cell wall can embed the alcohol fermentation promoter matrix. The yeast cell wall serves as an embedding carrier for the alcohol fermentation promoter, increasing the embedding efficiency by more than 30%.

[0086] Then, the yeast cell wall mixture is dried. In the present invention, the drying is preferably high-temperature vacuum drying. The drying temperature is 60-100 °C, which can be 60, 65, 70, 75, 80, 85, 90, 95, 100, or any value between 60-100 °C, and the time is 2-6 h, which can be 2, 3, 4, 5, 6, or any value between 2-6 h. The organic solvent recovered during the drying process can be recycled.

[0087] Finally, the dried alcohol fermentation promoter is cooled and packaged.

[0088] The alcohol fermentation promoter provided by the present invention can completely replace the use of urea in alcohol fermentation and meet the needs of alcohol factories to comply with food supervision regulations. The present invention can reduce the usage amount of acidic protease in alcohol factories by more than 30% and reduce the usage cost of alcohol factories.

[0089] The present invention also provides an application of the above-mentioned alcohol fermentation promoter in alcohol fermentation. The addition amount of the alcohol fermentation promoter is 0.2-0.4 kg / ton of alcohol, which can be 0.2, 0.25, 0.3, 0.35, 0.4, or any value between 0.2-0.4 kg / ton of alcohol.

[0090] In the present invention, the alcohol fermentation promoter is applied to the alcohol fermentation production using starch, sugar, and biomass hydrolysate as carbon source raw materials.

[0091] The present invention entirely uses food-grade raw materials and a clean, safe, and hygienic processing technology, making the fermentation promoter provided by the present invention applicable to food-grade alcohol and industrial alcohol, meeting the requirements of national regulations.

[0092] While the yeast cell wall provided by the present invention entraps the alcohol fermentation promoter matrix as an embedding material, it can also serve as a nutritional raw material, providing the phospholipids, sterols, and intracellular nutrients required for yeast growth and maintaining activity, and delaying yeast activity.

[0093] To further understand the present invention, the following examples are used to illustrate the yeast cell wall provided by the present invention and its application in the field of alcohol fermentation. The protection scope of the present invention is not limited by the following examples.

[0094] Preparation of yeast milk:

[0095] Using Saccharomyces cerevisiae ER18 (preserved in the China Center for Type Culture Collection (CCTCC), preservation number CCTCC NO: M 2020118) as the starting strain, a method for fermenting Saccharomyces cerevisiae ER18 strain to produce yeast milk rich in ergosterol includes the following steps:

[0096] (1) Seed activation culture: Inoculate one loop of Saccharomyces cerevisiae ER18 strain into a 5L shake flask medium for activation fermentation culture. Among them, the sugar content of the shake flask culture medium is 6%; the composition of the shake flask medium is: carbon source (glucose) 20g, nitrogen source (ammonium sulfate) 8g, yeast extract 4g, potassium dihydrogen phosphate 2g, magnesium sulfate 0.2g, and sterile water 2L. The culture time is 24h, the culture temperature is 30°C, and the pH value is 5.4 to obtain the shake flask culture solution;

[0097] (2) Primary culture: Inoculate the shake flask culture solution obtained in step (1) into a fermenter for fermentation culture. The volume of the primary fermenter is 0.5m 3 ; the sugar content of the medium is 10%; the composition of the medium is: carbon source (glucose) 50kg, nitrogen source (corn steep liquor) 12.5kg, potassium dihydrogen phosphate 2.5kg, magnesium sulfate 0.1kg, zinc sulfate 0.1kg, and sterile water 450kg. The culture time is 15h, the culture temperature is 32°C, and the pH value is 5.0 to obtain the primary fermentation culture solution;

[0098] (3) Secondary fermentation: Inoculate the primary fermentation culture solution obtained in step (2) into the secondary medium for fermentation culture. The volume of the secondary fermenter is 50m 3 ; the sugar content of the medium is 10%: the composition of the medium is: carbon source (cane molasses) 3000kg, nitrogen source (corn steep liquor) 750kg, potassium dihydrogen phosphate 60kg, magnesium sulfate 3kg, zinc sulfate 6kg, and sterile water 30000kg. The culture time is 40h, the culture temperature is 30°C, the pH value is 5.2, the DO is 40 - 45%, and the RQ is 0.95 to obtain the secondary fermentation culture solution:

[0099] (4) Commercial fermentation: Inoculate the secondary fermentation culture solution obtained in step (3) into the commercial medium for fermentation culture. The volume of the commercial fermenter is 150m 3 , the sugar content of the medium is 10%; the composition of the medium is: carbon source (cane molasses) 5000kg, nitrogen source (ammonia water) 1250kg, potassium dihydrogen phosphate 100kg, ammonium dihydrogen phosphate 100kg, magnesium sulfate 5kg, zinc sulfate 10kg, and sterile water 50m 3 . When the RQ drops to 1.1, start to feed glucose (10%), control it at 0.9 ± 0.05 according to the RQ value, gradually increase the glucose feeding rate from 800 - 1600L / h, and the ammonia water feeding rate is 200L / h, so that the pH is kept constant at 5.2 - 5.3. The culture time is 20h, the culture temperature is 30°C, and the DO is controlled at 40% - 50% to obtain the yeast milk rich in ergosterol.

[0100] Select the yeast milk above for the preparation of yeast cell walls in Examples 1 - 3

[0101] Example 1:

[0102] Adjust the concentration of yeast milk to 15%, heat it to 55 °C for autolysis for 4 h, then heat it to 70 °C, adjust the pH to 5.5, add 0.05% papain and the ratio of bacterial alkaline protease is 0.5:1. After enzymatic hydrolysis, heat it to 90 °C for sterilization, cool it to 60 °C for separation, and further spray-dry the separated heavy phase to obtain the yeast cell wall of the present invention.

[0103] Example 2:

[0104] Adjust the concentration of yeast milk to 20%, heat it to 58 °C for autolysis for 6 h, then heat it to 70 °C, adjust the pH to 6.0, add 0.05% papain and the ratio of bacterial alkaline protease is 1:1. After enzymatic hydrolysis, heat it to 95 °C for sterilization, cool it to 60 °C for separation, and further spray-dry the separated heavy phase to obtain the yeast cell wall of the present invention.

[0105] Example 3:

[0106] Adjust the concentration of yeast milk to 20%, heat it to 60 °C for autolysis for 8 h, then heat it to 70 °C, adjust the pH to 6.0, add 0.05% papain and the ratio of bacterial alkaline protease is 2:1. After enzymatic hydrolysis, heat it to 100 °C for sterilization, cool it to 65 °C for separation, and further spray-dry the separated heavy phase to obtain the yeast cell wall of the present invention.

[0107] Example 4 Fat content and sterol content of cell walls from different strain sources

[0108] Select three strains of yeast, namely Saccharomyces cerevisiae with high sugar tolerance, Saccharomyces cerevisiae with low sugar tolerance, and Saccharomyces cerevisiae ER18, for yeast fermentation to obtain yeast milk.

[0109] Among them, both Saccharomyces cerevisiae with high sugar tolerance and Saccharomyces cerevisiae with low sugar tolerance are purchased from Angel Yeast Co., Ltd. The preparation methods of their yeast milk are the same as that of the yeast milk of Saccharomyces cerevisiae ER18 above, and only the yeast strains need to be replaced.

[0110] Prepare cell walls from the three kinds of yeast milk obtained above according to the preparation process of Example 1. Respectively obtain the cell walls of Saccharomyces cerevisiae with high sugar tolerance, Saccharomyces cerevisiae with low sugar tolerance, and Saccharomyces cerevisiae ER18, and detect the fat and sterol contents respectively.

[0111] Table 1 Fat and sterol contents of yeast cell walls

[0112]

[0113] The cell wall of Saccharomyces cerevisiae ER18 has a sterol content more than 50% higher and a fat content more than 100% higher compared to the cell walls of high-sugar-tolerant Saccharomyces cerevisiae and low-sugar Saccharomyces cerevisiae.

[0114] Example 5 Embedding Efficiency of Different Cell Walls

[0115] Dissolve 20 parts of ammonium sulfate, 30 parts of diammonium hydrogen phosphate, 3 parts of magnesium sulfate, and 2 parts of zinc sulfate in 95% (v / v) food-grade ethanol. Slowly add and stir 45 parts of the dry powder of the 3 kinds of yeast cell walls obtained in Example 4 into the above ethanol solution until the ethanol solvent is completely absorbed, forming 3 kinds of yeast cell wall mixtures. Centrifuge the above yeast cell wall mixtures at a centrifuge speed of 12,000 rpm for 10 min. Take the supernatant to measure the contents of ammonium sulfate, diammonium hydrogen phosphate, magnesium sulfate, and zinc sulfate, and calculate the embedding efficiency. The formula for calculating the embedding efficiency is as follows:

[0116]

[0117] M: Total content of nutrients (ammonium sulfate / diammonium hydrogen phosphate / magnesium sulfate / zinc sulfate);

[0118] m: Content of nutrients in the centrifuged supernatant (ammonium sulfate / diammonium hydrogen phosphate / magnesium sulfate / zinc sulfate).

[0119] Detection method for ammonium sulfate: Appendix A.4 of GB 29206-2012;

[0120] Detection method for diammonium hydrogen phosphate: Appendix A.3 of GB 1886.331-2021;

[0121] Detection method for magnesium sulfate: Appendix A.4 of GB 29207-2012;

[0122] Detection method for zinc sulfate: Appendix A.4 of GB 29207-2012.

[0123] Table 2 Embedding Efficiency of Yeast Cell Wall for Nutrients and Inorganic Salts

[0124]

[0125] Compared with the cell walls of low-sugar Saccharomyces cerevisiae and high-sugar-tolerant Saccharomyces cerevisiae, the cell wall of Saccharomyces cerevisiae ER18 has an embedding efficiency of more than 30% higher for ammonium sulfate, diammonium hydrogen phosphate, magnesium sulfate, and zinc sulfate, and can effectively embed nutrients and inorganic salts and control the release of nutrients and inorganic salts.

[0126] Example 6

[0127] Dissolve 20 parts of yeast extract, 20 parts of ammonium sulfate, 15 parts of diammonium hydrogen phosphate, 5 parts of magnesium sulfate, 5 parts of zinc sulfate, 1.5 parts of vitamin B1, 1.0 part of calcium pantothenate, 1.0 part of nicotinic acid, and 1.5 parts of inositol in 95% (v / v) food-grade ethanol. Slowly add 30 parts of the dry yeast cell wall powder from Example 1 to the above ethanol solution while stirring until the ethanol solvent is completely absorbed to form a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0128] Example 7

[0129] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 15 parts of diammonium hydrogen phosphate, 11 parts of magnesium sulfate, 12 parts of zinc sulfate, 0.6 part of vitamin B1, 0.4 part of calcium pantothenate, 0.4 part of nicotinic acid, and 0.6 part of inositol in 95% (v / v) food-grade ethanol. Slowly add 40 parts of the dry yeast cell wall powder from Example 1 to the above ethanol solution while stirring until the ethanol solvent is completely absorbed to form a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0130] Example 8

[0131] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 20 parts of diammonium hydrogen phosphate, 2 parts of magnesium sulfate, 2 parts of zinc sulfate, 0.3 part of vitamin B1, 0.2 part of calcium pantothenate, 0.2 part of nicotinic acid, and 0.3 part of inositol in 95% (v / v) food-grade ethanol. Slowly add 45 parts of the dry yeast cell wall powder from Example 1 to the above ethanol solution while stirring until the ethanol solvent is completely absorbed to form a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0132] Comparative Example 1

[0133] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 20 parts of diammonium hydrogen phosphate, 2 parts of magnesium sulfate, 2 parts of zinc sulfate, 0.3 part of vitamin B1, 0.2 part of calcium pantothenate, 0.2 part of nicotinic acid, and 0.3 part of inositol in 95% (v / v) food-grade ethanol. Slowly add 45 parts of the dry low-sugar brewing yeast cell wall powder to the above ethanol solution while stirring until the ethanol solvent is completely absorbed to form a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0134] Comparative Example 2

[0135] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 20 parts of diammonium hydrogen phosphate, 2 parts of magnesium sulfate, 2 parts of zinc sulfate, 0.3 part of vitamin B1, 0.2 part of calcium pantothenate, 0.2 part of nicotinic acid, and 0.3 part of inositol in 95% (v / v) food-grade ethanol. Slowly add 45 parts of dry high-sugar brewing yeast cell wall powder to the above ethanol solution while stirring until the ethanol solvent is completely absorbed, forming a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0136] Comparative Example 3

[0137] Mix 20 parts of yeast extract, 20 parts of ammonium sulfate, 50 parts of diammonium hydrogen phosphate, 4 parts of magnesium sulfate, 4 parts of zinc sulfate, 0.6 part of vitamin B1, 0.4 part of calcium pantothenate, 0.4 part of nicotinic acid, and 0.6 part of inositol to form an alcohol fermentation promoter.

[0138] Comparative Example 4

[0139] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 20 parts of diammonium hydrogen phosphate, 2 parts of magnesium sulfate, 2 parts of zinc sulfate, 0.3 part of vitamin B1, 0.2 part of calcium pantothenate, 0.2 part of nicotinic acid, and 0.3 part of inositol in deionized water. Slowly add 45 parts of the yeast cell wall dry powder in Example 1 to the above aqueous solution while stirring until the water solvent is completely absorbed, forming a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0140] Comparative Example 5

[0141] Dissolve 20 parts of yeast extract, 10 parts of ammonium sulfate, 20 parts of diammonium hydrogen phosphate, 2 parts of magnesium sulfate, 2 parts of zinc sulfate, 0.3 part of vitamin B1, 0.2 part of calcium pantothenate, 0.2 part of nicotinic acid, and 0.3 part of inositol in 50% (v / v) food-grade ethanol. Slowly add 45 parts of the yeast cell wall dry powder in Example 1 to the above alcohol solution while stirring until the alcohol solvent is completely absorbed, forming a yeast cell wall mixture. Vacuum dry and cool the above yeast cell wall mixture to form an alcohol fermentation promoter.

[0142] Experimental Example 1

[0143] Weigh 100 g of corn flour separately, transfer it into a clean and dry sample cup of the color tester, add 0.2 ml of 1.5% dilute sulfuric acid, add 9.0 u / g of amylase based on the corn flour, add 181 - 182 g of deionized water, stir well, put it into the color tester, with the liquefaction temperature at 101°C and maintain for 75 minutes; cool down to 45°C to end. During this period, the heating and cooling rate is 3°C per minute. Rapidly cool it to 30 - 33°C with a cooling water bath. Then pour the cooled corn mash into a 500 mL Erlenmeyer flask and add water to make the material-water ratio 1:1.9. Add 150 u / g of glucoamylase based on the corn flour to each Erlenmeyer flask and keep it warm for half an hour. Then add 0.08 g of yeast to each Erlenmeyer flask, and add 0.013 g of the alcohol fermentation promoter in Examples 6 - 8 and Comparative Examples 1 - 5 respectively, and then place it in a shaker for fermentation. Set the fermentation temperature at 33°C and the rotation speed at 170 rpm. Measure the reducing sugar concentration, yeast cell count, and alcohol degree every 8 h.

[0144] The experimental results are as follows:

[0145] Table 3 Application effects of alcohol fermentation promoters

[0146]

[0147] (1) The fermentation process of Example 8 of the present invention is the most stable, still maintaining a strong fermentation ability in the middle and late stages of fermentation. The viable cell count of yeast in the late stage of fermentation is significantly higher than that of the comparative examples. After 72 h of fermentation, the residual reducing sugar is the lowest and the alcohol degree is the highest. Among them, the alcohol degree is more than 5% higher than that of the comparative examples. Compared with Comparative Examples 1 - 5, the fermentation processes of Examples 6 - 7 are more stable, the reducing sugar at the end of fermentation is lower, and the alcohol degree is higher. In Comparative Example 3, yeast cell wall was not added, and the nutrient release was the fastest. The yeast cell count was relatively high in the early stage of alcohol fermentation, the alcohol fermentation speed was fast, the reducing sugar was low, and the alcohol degree was high. However, in the middle and late stages of alcohol fermentation, the yeast fermentation ability decreased relatively fast, the reducing sugar at the end of fermentation was the highest, and the alcohol degree was the lowest.

[0148] (2) The alcohol fermentation promoter in Example 8 effectively adsorbs and entraps nutrients and trace elements, making the nutrient release more uniform and lasting, solving the problem of excessive nutrients in the early stage and insufficient nutrients in the late stage caused by using conventional inorganic nutrients, reducing the mortality rate of alcohol yeast in the late stage of fermentation, enabling it to still maintain good alcohol fermentation ability in the middle and late stages of alcohol fermentation, fermenting more thoroughly, having a higher fermentation alcohol degree, improving the utilization rate of fermentation raw materials, and increasing the economic benefits of alcohol production enterprises.

[0149] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An alcohol fermentation promoter, characterized in that, The alcohol fermentation promoter includes: 29 - 135 parts by mass of an alcohol fermentation promoter matrix; 30 - 70 parts by mass of yeast cell wall; the yeast cell wall is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118; The alcohol fermentation promoter matrix includes: 10 - 40 parts of yeast extract, 10 - 40 parts of ammonium sulfate, 5 - 30 parts of diammonium hydrogen phosphate, 2 - 10 parts of magnesium sulfate, 2 - 10 parts of zinc sulfate, and 2 - 5 parts of trace elements and vitamins; The yeast cell wall coats a certain amount of the alcohol fermentation promoter matrix, wherein the mass ratio of the alcohol fermentation promoter matrix coated by the yeast cell wall to the uncoated alcohol fermentation promoter matrix is 40 - 95:5 - 40; The preparation method of the alcohol fermentation promoter includes the following steps: Dissolve the alcohol fermentation promoter matrix in a solvent to obtain a mixed solution, and the solvent is selected from ethanol solutions with a volume fraction of 55% - 95%; Add the yeast cell wall to the mixed solution and stir to obtain a yeast cell wall mixture; Dry the yeast cell wall mixture to obtain the alcohol fermentation promoter.

2. The alcohol fermentation promoter according to claim 1, wherein The yeast cell wall accounts for more than 30 wt% of the cell dry weight of Saccharomyces cerevisiae, and the yeast cell wall includes ergosterol, and the ergosterol accounts for more than 3 wt% of the yeast cell wall.

3. The alcohol fermentation promoter according to claim 1, characterized in that, The preparation method of the yeast cell wall includes the following steps: A) Ferment Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118 to obtain fermented milk; B) Autolyze the fermented milk and then carry out enzymatic hydrolysis to obtain an enzymatic hydrolysis product; C) Separate and dry the enzymatic hydrolysis product to obtain the yeast cell wall.

4. The alcohol fermentation promoter according to claim 3, characterized in that, The preparation method of the fermented milk includes the following steps: (1) Shake flask culture: Inoculate the yeast strain into a shake flask medium for fermentation culture to obtain a shake flask culture solution. The yeast strain is Saccharomyces cerevisiae ER18, preserved in the China Center for Type Culture Collection, CCTCC NO: M 2020118. Among them, the sugar content of the medium for shake flask culture is 5 - 10%; (2) Primary culture: Inoculate the shake flask culture solution obtained in step (1) into a shake flask medium for fermentation culture to obtain a primary fermentation culture solution; the volume of the primary fermentation tank is 0.2 - 20 m 3 ; the sugar content of the medium for primary culture is 5 - 10%; (3) Secondary fermentation: inoculate the primary fermentation culture solution obtained in step (2) into the secondary medium for fermentation culture to obtain the secondary fermentation culture solution. The volume of the primary fermentation tank is 20 - 60 m 3 ; the sugar content of the medium for secondary fermentation is 5 - 20%; (4) Tertiary fermentation: inoculate the secondary fermentation culture solution obtained in step (3) into a tertiary culture medium for fermentation culture to obtain a tertiary fermentation culture solution, and the volume of the tertiary fermentation tank is 60 - 150 m 3 ; the sugar content of the culture medium for tertiary fermentation is 5 - 20%; (5) Commercial fermentation: Inoculate the tertiary fermentation culture solution obtained in step (3) into a commercial culture medium for fermentation culture to obtain a commercial fermentation culture solution. The volume of the commercial fermentation tank is 150 - 400 m 3 , and the sugar content of the commercial fermentation culture medium is 10 - 30%; obtain fermented milk; One or more of the steps (3) - (5) in the method can be omitted as needed.

5. The alcohol fermentation promoter according to claim 3, characterized in that, Before autolyzing the fermented milk, adjust the concentration of the fermented milk to 15 - 20%; The temperature of the autolysis is 55 - 60 °C, and the time of the autolysis is 4 - 8 h; The enzymes used for the enzymatic hydrolysis include papain and bacterial alkaline protease; the dosage ratio of papain and bacterial alkaline protease is 0.5 - 2:1; the amount of the enzymes used for the enzymatic hydrolysis is 0.05 - 1.15%; The temperature of the enzymatic hydrolysis is 55 - 70 °C, the pH of the enzymatic hydrolysis is 5.5 - 6.0, and the time of the enzymatic hydrolysis is 5 - 20 hours; After the enzymatic hydrolysis, sterilize the enzymatic hydrolysis product at 90 - 100 °C.

6. The alcohol fermentation promoter according to claim 1, characterized in that, The addition amount of the alcohol fermentation promoter is 0.2 - 0.4 kg / ton of alcohol.

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