Yeast cell wall and use thereof in field of alcohol fermentation
By using the cell wall of Saccharomyces cerevisiae ER18 as an encapsulation carrier for alcoholic fermentation promoters, the problem of yeast nutrient deficiency was solved, encapsulation efficiency and nutritional value were improved, high-concentration alcoholic fermentation was achieved efficiently, and production costs were reduced.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2026-04-02
AI Technical Summary
During high-concentration ethanol fermentation, yeast faces nutritional deficiencies when producing alcohol, leading to decreased yeast activity, reduced budding rate, and increased mortality. This results in high residual sugar levels and incomplete alcohol fermentation. Existing additives such as urea and acidic protease cannot meet the needs of high-density yeast cultivation and high-concentration alcohol fermentation.
The cell wall of Saccharomyces cerevisiae ER18 is used as an encapsulation carrier for alcoholic fermentation promoters. The encapsulation efficiency is improved through preparation methods. It is rich in ergosterol and other sterols and unsaturated fatty acids, providing high-nutritional-value raw materials, replacing urea and reducing the use of acidic proteases.
It improves yeast fermentation performance, increases alcohol yield, meets the requirements for high-concentration alcohol fermentation, complies with food safety regulations, and reduces production costs.
Smart Images
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Abstract
Description
Yeast cell wall and application thereof in the field of alcohol fermentation
[0001] This application claims priority to the Chinese patent application No. 202411335122.6 filed on September 24, 2024, and titled "Yeast cell wall and application thereof in the field of alcohol fermentation", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD
[0002] The present application belongs to the field of food technology, and specifically relates to a yeast cell wall and application thereof in the field of alcohol fermentation. BACKGROUND
[0003] In the process of high-concentration ethanol fermentation, yeast fermentation to produce alcohol usually faces the problem of yeast nutrient deficiency, which leads to the phenomena of decreased yeast activity, reduced budding rate and increased mortality rate in the later stage of alcohol fermentation, thereby resulting in high residual sugar and incomplete alcohol fermentation.
[0004] In actual production, alcohol plants mostly use urea and acid protease to promote alcohol fermentation. However, according to the GB2760-2011 food additive directory, urea cannot be used for edible alcohol production. Moreover, single addition of urea or acid protease cannot meet the requirements of high-density yeast culture and high-concentration alcohol fermentation (alcohol content ≥ 16.5% (v / v)).
[0005] Yeast and nutrient salts are usually added at one time, and excessive nutrition will cause excessive growth of yeast and insufficient nutrition in the later stage. Under a microscope, the yeast with excessive nutrition has a large volume, but there is insufficient nutrition in the later stage of fermentation, which leads to yeast death, showing the attenuation of alcohol production capacity and the slowing down of fermentation speed. Therefore, how to control the growth of yeast, reduce the mortality rate of yeast in the later stage of fermentation, and solve the precise nutritional requirements of yeast are key technical problems for realizing high-concentration ethanol fermentation.
[0006] Yeast cell wall is a by-product of yeast extract production. Yeast cell wall is mainly composed of cytoplasmic membrane, glucan layer and mannose layer. The fat content of yeast cell wall is 15-20%, which is mainly composed of phospholipids constituting the cell membrane, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylserine (PS). Phospholipids are not only an important component of yeast cells, but also an important yeast growth nutrient and regulatory factor, which helps to improve yeast activity and reproductive performance. Meanwhile, ergosterol substances are also contained in the yeast cell wall. In the process of yeast extract production, the contents of yeast cytoplasm, including proteins, polypeptides, amino acids, nucleic acids, liposomes and growth factors, are partially left in the cell membrane cavity, and the nutritional elements and structural proportions meet the growth of yeast and maintain the physiological activity of yeast.
[0007] Meanwhile, the cavity composed of the three-layer structure of the yeast cell wall is a natural nutrient molecule embedding carrier, which makes the nutrient components become the natural adjuvant of the alcohol fermentation yeast nutrition through physical adsorption.
[0008] However, the existing yeast cell wall as the embedding carrier has low embedding efficiency and low nutritional value. Therefore, it is a problem to be solved to provide an embedding carrier with high embedding efficiency and high nutritional value. SUMMARY
[0009] Therefore, the technical problem to be solved by the present application is to provide a yeast cell wall and its application in the field of alcohol fermentation. The yeast cell wall provided by the present application has an embedding efficiency of the alcohol fermentation promoter increased by more than 30%, and is rich in ergosterol and other sterols and various unsaturated fatty acids, has high nutritional value, and can provide nutritional raw materials for alcohol fermentation.
[0010] The present application provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number CCTCC NO: M 2020118.
[0011] Preferably, the yeast cell wall accounts for more than 30wt% of the 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.
[0012] The present application also provides a preparation method of the above yeast cell wall, comprising the following steps:
[0013] A) fermenting Saccharomyces cerevisiae ER18 with the preservation number CCTCC NO: M 2020118 to obtain yeast milk;
[0014] B) performing enzymolysis on the yeast milk after autolysis to obtain an enzymolysis product;
[0015] C) drying the enzymolysis product after separation to obtain the yeast cell wall.
[0016] Preferably, the preparation method of the yeast milk comprises the following steps:
[0017] (1) shake flask culture: inoculating a yeast strain in a shake flask culture medium for fermentation culture to obtain a shake flask culture solution, wherein the yeast strain is Saccharomyces cerevisiae ER18 preserved in the China Center for Type Culture Collection (CCTCC NO: M 2020118), and the sugar content of the shake flask culture medium is 5-10%;
[0018] (2) primary culture: the shake flask culture solution obtained in step (1) is inoculated into a shake flask culture 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 culture medium of the primary culture is 5-10%;
[0019] (3) secondary fermentation: the primary fermentation culture solution obtained in step (2) is inoculated into a secondary culture medium for fermentation culture to obtain a secondary fermentation culture solution; the volume of the secondary fermentation tank is 20-60m 3 ; the sugar content of the culture medium of the secondary fermentation is 5-20%;
[0020] (4) tertiary fermentation: the secondary fermentation culture solution obtained in step (3) is inoculated into a tertiary culture medium for fermentation culture to obtain a tertiary fermentation culture solution; the volume of the tertiary fermentation tank is 60-150m 3 ; the sugar content of the culture medium of the tertiary fermentation is 5-20%;
[0021] (5) commercial fermentation: the tertiary fermentation culture solution obtained in step (3) is inoculated into a commercial culture medium for fermentation culture to obtain a commercial fermentation culture solution; the volume of the commercial fermentation tank is 150-400m 3 ; the sugar content of the culture medium of the commercial fermentation is 10-30%; a yeast milk is obtained;
[0022] Preferably, steps (3)-(5) in the method can be omitted as needed.
[0023] Preferably, the concentration of the yeast milk is adjusted to 15-20% before autolysis of the yeast milk;
[0024] The temperature of the autolysis is 55-60℃, and the time of the autolysis is 4-8h;
[0025] The enzymes used for the enzymolysis include papain and bacterial alkaline protease; the use amount ratio of the papain and the bacterial alkaline protease is 0.5-2:1; the amount of the enzymes used for the enzymolysis is 0.05-1.15%;
[0026] The temperature of the enzymolysis is 55-70℃, the pH of the enzymolysis is 5.5-6.0, and the time of the enzymolysis is 5-20 hours;
[0027] After the enzymolysis, the enzymolysis product is sterilized at 90-100℃.
[0028] The application further provides a use of the above-mentioned yeast cell wall in preparation of an alcohol fermentation accelerator, and the alcohol fermentation accelerator comprises:
[0029] 29-135 mass parts of an alcohol fermentation accelerator base;
[0030] 30-70 parts by mass of yeast cell walls.
[0031] Preferably, the alcohol fermentation promoter matrix comprises: yeast extract 10-40 parts, ammonium sulfate 10-40 parts, diammonium hydrogen phosphate 5-30 parts, magnesium sulfate 2-10 parts, zinc sulfate 2-10 parts, and trace elements and vitamins 2-5 parts.
[0032] Preferably, the yeast cell wall coats a certain amount of alcohol fermentation promoter matrix, and 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.
[0033] The present application also provides a preparation method of the above-mentioned alcohol fermentation promoter, comprising the following steps:
[0034] dissolving the alcohol fermentation promoter matrix in a solvent to obtain a mixed solution;
[0035] adding yeast cell walls to the mixed solution and mixing to obtain a yeast cell wall mixture;
[0036] drying the yeast cell wall mixture to obtain the alcohol fermentation promoter.
[0037] The present application 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.
[0038] Compared with the prior art, the present application provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number CCTCC NO: M 2020118. The yeast cell wall provided by the present application has an embedding efficiency of more than 30% as an embedding carrier of an alcohol fermentation promoter, and is rich in ergosterol and other sterols and various unsaturated fatty acids, has a high nutritional value, can provide nutritional raw materials for alcohol fermentation, thereby improving the fermentation performance of yeast and the alcohol yield of alcohol fermentation. DETAILED DESCRIPTION
[0039] The present application provides a yeast cell wall, which is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number CCTCC NO: M 2020118.
[0040] The strain of the Saccharomyces cerevisiae is Saccharomyces cerevisiae ER18, which is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number CCTCC NO: M2020118.
[0041] In the present application, the yeast cell wall accounts for more than 30wt% of the dry weight of the Saccharomyces cerevisiae, preferably 30wt%-35wt%, and the ergosterol accounts for more than 3wt% of the yeast cell wall, preferably 3wt%-3.5wt%.
[0042] The present application also provides a preparation method of the yeast cell wall, comprising the following steps:
[0043] A) fermenting the Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118 to obtain a yeast milk;
[0044] B) performing enzymolysis on the yeast milk after autolysis to obtain an enzymolysis product;
[0045] C) drying the enzymolysis product after separation to obtain the yeast cell wall.
[0046] The present application first prepares a yeast milk, wherein the preparation method of the yeast milk comprises the following steps:
[0047] (1) shake flask culture: inoculating a yeast strain in a shake flask culture medium to perform fermentation culture to obtain a shake flask culture solution, wherein the yeast strain is Saccharomyces cerevisiae ER18 preserved in the China Center for Type Culture Collection (CCTCC NO: M 2020118), and the sugar content of the shake flask culture medium is 5-10%;
[0048] (2) primary culture: inoculating the shake flask culture solution obtained in step (1) in a shake flask culture medium to perform 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 primary culture medium is 5-10%;
[0049] (3) secondary fermentation: inoculating the primary fermentation culture solution obtained in step (2) in a secondary culture medium to perform fermentation culture to obtain a secondary fermentation culture solution; the volume of the secondary fermentation tank is 20-60m 3 ; the sugar content of the secondary culture medium is 5-20%;
[0050] (4) tertiary fermentation: inoculating the secondary fermentation culture solution obtained in step (3) in a tertiary culture medium to perform fermentation culture to obtain a tertiary fermentation culture solution; the volume of the tertiary fermentation tank is 60-150m 3 ; the sugar content of the tertiary culture medium is 5-20%;
[0051] (5) commercial fermentation: inoculating the tertiary fermentation culture solution obtained in step (3) in a commercial culture medium to perform fermentation culture to obtain a commercial fermentation culture solution; the volume of the commercial fermentation tank is 150-400m 3The sugar content of the medium for commercial fermentation is 10-30%; and a yeast milk is obtained;
[0052] Preferably, steps (3)-(5) in the method can be omitted as needed.
[0053] Preferably, the culture temperature in steps (1)-(5) is 28-35℃, preferably 30-33℃; and the pH value is 4.5-7.0, preferably 4.8-6.0.
[0054] 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, and the time for commercial fermentation is 20-30h.
[0055] The medium in steps (1)-(5) contains the following components by weight: carbon source 25-50 parts, nitrogen source 2-5 parts, potassium dihydrogen phosphate 0.1-0.6 parts, magnesium sulfate 0.02-0.04 parts, zinc sulfate 0.02-0.05 parts, and ammonium dihydrogen phosphate 0.2-0.8 parts.
[0056] The carbon source is selected from one or more of the following: sugarcane molasses, beet molasses, corn hydrolysis sugar, glucose, sucrose, and maltose; and the nitrogen source is selected from one or more of the following: corn steep liquor, plant protein, ammonia water, ammonium sulfate, and yeast extract.
[0057] The dissolved oxygen DO during fermentation is 40-80%, and preferably the respiratory quotient RQ is 0.95-1.2.
[0058] After obtaining the yeast milk, the yeast milk is subjected to autolysis and then enzymatic hydrolysis to obtain an enzymatic hydrolysis product.
[0059] Before autolysis, the concentration of the yeast 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% and 20%. The concentration of the yeast milk is the percentage of the mass of dry yeast in the yeast milk to the mass of the yeast milk.
[0060] Then, the yeast milk with adjusted concentration is subjected to autolysis, wherein the temperature for autolysis is 55-60℃, which can be 55, 56, 57, 58, 59, 60, or any value between 55 and 60℃, and the time for autolysis is 4-8h, which can be 4, 5, 6, 7, 8, or any value between 4 and 8h.
[0061] Then, the yeast milk after autolysis is subjected to enzymolysis, wherein the enzymes used in the enzymolysis include papain and bacterial alkaline protease; the use amount ratio of the papain and the 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 and 2:1; the amount of the enzymes used in the enzymolysis 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 and 1.15%;
[0062] The temperature of the enzymolysis is 55-70℃, which can be 55, 60, 65, 70, or any value between 55 and 70℃; the pH of the enzymolysis 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 and 6.0; and the time of the enzymolysis is 5-20 hours, which can be 5, 10, 12, 15, 18, 20, or any value between 5 and 20 hours.
[0063] After the enzymolysis, the enzymolysis product is sterilized at 90-100℃. After the sterilization is completed, the temperature is lowered to 60-65℃, and then the product is separated, and the heavy phase is further subjected to spray drying to obtain the yeast cell wall.
[0064] The application further provides a use of the yeast cell wall in the preparation of an alcohol fermentation accelerator.
[0065] 29-135 parts by mass of an alcohol fermentation accelerator base;
[0066] 30-70 parts by mass of the yeast cell wall.
[0067] The alcohol fermentation accelerator provided by the application includes 29-135 parts by mass of an alcohol fermentation accelerator base, which can be 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, or any value between 29 and 135 parts by mass.
[0068] In the application, the alcohol fermentation accelerator base preferably includes the following raw materials in parts by mass:
[0069] Yeast extract 10-40 parts, ammonium sulfate 10-40 parts, diammonium hydrogen phosphate 5-30 parts, magnesium sulfate 2-10 parts, zinc sulfate 2-10 parts, and trace elements and vitamins 2-5 parts.
[0070] The alcohol fermentation accelerator substrate of the present application comprises 10-40 parts of yeast extract, which can be 10, 15, 20, 25, 30, 35, 40, or any value between 10-40 parts.
[0071] The alcohol fermentation accelerator substrate of the present application further comprises 10-40 parts of ammonium sulfate, which can be 10, 15, 20, 25, 30, 35, 40, or any value between 10-40 parts.
[0072] The alcohol fermentation accelerator substrate of the present application further comprises 5-30 parts of diammonium hydrogen phosphate, which can be 5, 10, 15, 20, 25, 30, or any value between 5-30 parts.
[0073] The alcohol fermentation accelerator substrate of the present application further comprises 2-10 parts of magnesium sulfate, which can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 2-10 parts.
[0074] The alcohol fermentation accelerator substrate of the present application further comprises 2-10 parts of zinc sulfate, which can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 2-10 parts.
[0075] The alcohol fermentation accelerator substrate of the present application further comprises 2-5 parts of trace elements and vitamins, which can be 2, 2.5, 3, 3.5, 4, 4.5, 5, or any value between 2-5 parts.
[0076] The alcohol fermentation accelerator provided by the present application further comprises 30-70 parts by mass of yeast cell wall, which can be 30, 35, 40, 45, 50, 55, 60, 65, 70, or any value between 30-70 parts by mass. The yeast cell wall is selected from the yeast cell wall described above.
[0077] The yeast cell wall coats a certain amount of alcohol fermentation accelerator substrate, wherein the mass ratio of the alcohol fermentation accelerator substrate coated by the yeast cell wall to the alcohol fermentation accelerator substrate without coating is 40-95:5-40, 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 40-95:5-40.
[0078] The alcohol fermentation accelerator provided by the present application has higher embedding rate with the above-mentioned yeast cell wall as the adsorption carrier. Meanwhile, the cell wall provided by the present application has high fat content, rich in ergosterol and other sterols, and various unsaturated fatty acids, so that the alcohol fermentation accelerator provided by the present application has better nutritional effect.
[0079] The alcohol fermentation accelerator provided by the application can effectively solve the problems of excessive nutrition in the early stage and insufficient nutrition in the later stage of the whole yeast fermentation process, thereby improving the yeast fermentation performance and the alcohol yield of alcohol fermentation.
[0080] The application further provides a preparation method of the alcohol fermentation accelerator.
[0081] The alcohol fermentation accelerator matrix is dissolved in a solvent to obtain a mixed solution.
[0082] Yeast cell walls are added to the mixed solution and mixed to obtain a yeast cell wall mixture.
[0083] The yeast cell wall mixture is dried to obtain the alcohol fermentation accelerator.
[0084] The alcohol fermentation accelerator matrix is dissolved in a solvent to obtain a mixed solution, wherein the solvent is selected from food-grade organic solvents, and the food-grade organic solvent is ethanol. The solubility 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 easy to stick together in water, which reduces the specific surface area and reduces the efficiency of nutrients entering the cell wall and the embedding efficiency. Yeast cell walls are not easy to stick together in a high-concentration alcohol solution, the specific surface area of yeast is larger, nutrients are more easily absorbed into the cell wall, and the embedding efficiency is improved. Better embedding effect can make the release of nutrients more stable, provide stable nutrition for yeast, and thus improve the alcohol fermentation capacity of yeast.
[0085] Then, yeast cell walls are added to the mixed solution and mixed to obtain a yeast cell wall mixture.
[0086] Specifically, dry yeast cell wall powder is slowly added to the mixed solution and stirred until evenly mixed. Through physical adsorption, the yeast cell wall can embed the alcohol fermentation accelerator matrix, and the yeast cell wall serves as an embedding carrier of the alcohol fermentation accelerator, which improves the embedding efficiency by more than 30%.
[0087] Then, the yeast cell wall mixture is dried, in the present application, the drying is preferably high-temperature vacuum drying, the temperature of the drying is 60-100℃, which can be 60, 65, 70, 75, 80, 85, 90, 95, 100, or any value between 60-100℃, and the time is 2-6h, which can be 2, 3, 4, 5, 6, or any value between 2-6h. The organic solvent recovered during the drying process can be recycled.
[0088] Finally, the dried alcohol fermentation accelerator is cooled and packaged.
[0089] The alcohol fermentation accelerator provided by the present application can completely replace the use of urea in alcohol fermentation, meeting the needs of alcohol plants to comply with food regulatory regulations. The present application can reduce the use of acid protease in alcohol plants by more than 30%, reducing the use cost of alcohol plants.
[0090] The present application also provides an application of the above-mentioned alcohol fermentation accelerator in alcohol fermentation, and the addition amount of the alcohol fermentation accelerator is 0.2-0.4kg / ton of alcohol, which can be 0.2, 0.25, 0.3, 0.35, 0.4, or any value between 0.2-0.4kg / ton of alcohol.
[0091] In the present application, the alcohol fermentation accelerator is applied to the production of alcohol fermentation with starch, sugar and biomass hydrolysate as carbon source raw materials.
[0092] The present application uses food-grade raw materials and clean, safe and sanitary processing technology, so that the fermentation accelerator provided by the present application can be used for food-grade alcohol and industrial alcohol, which meets the requirements of national regulations.
[0093] The yeast cell wall provided by the present application can be used as an embedding material to embed the alcohol fermentation accelerator matrix, and also can be used as a nutrient raw material to provide phospholipids, sterols and yeast intracellular nutrients required for yeast growth and maintenance of activity, and delay yeast activity.
[0094] In order to further understand the present application, the yeast cell wall provided by the present application and its application in the field of alcohol fermentation are described below in combination with examples, and the protection scope of the present application is not limited by the following examples.
[0095] Preparation of yeast milk:
[0096] The method for producing yeast milk rich in ergosterol by using Saccharomyces cerevisiae ER18 (preserved in China Center for Type Culture Collection (CCTCC), preservation number CCTCC NO: M 2020118) as a starting strain, includes the following steps:
[0097] (1) Seed activation culture: a ring of Saccharomyces cerevisiae ER18 strain was inoculated in 5L flask culture medium for activation fermentation culture, wherein the sugar content of the flask culture medium was 6%; wherein the composition of the flask culture medium was: carbon source (glucose) 20g, nitrogen source (ammonium sulfate) 8g, yeast extract 4g, potassium dihydrogen phosphate 2g and magnesium sulfate 0.2g, and sterile water 2L. The culture time was 24h, the culture temperature was 30℃, the pH value was 5.4, and the flask culture liquid was obtained;
[0098] (2) Primary culture: the flask culture liquid obtained in step (1) was inoculated into a fermentation tank for fermentation culture, and the volume of the primary fermentation tank was 0.5m 3 ; the sugar content of the culture medium was 10%; wherein the composition of the culture medium was: carbon source (glucose) 50kg, nitrogen source (corn syrup) 12.5kg, potassium dihydrogen phosphate 2.5kg, magnesium sulfate 0.1kg, zinc sulfate 0.1kg, and sterile water 450kg. The culture time was 15h, the culture temperature was 32℃, the pH value was 5.0, and the primary fermentation culture liquid was obtained;
[0099] (3) Secondary fermentation: the primary fermentation culture liquid obtained in step (2) was inoculated into a secondary culture medium for fermentation culture, and the volume of the secondary fermentation tank was 50m 3 ; the sugar content of the culture medium was 10%; wherein the composition of the culture medium was: carbon source (cane molasses) 3000kg, nitrogen source (corn syrup) 750kg, potassium dihydrogen phosphate 60kg, magnesium sulfate 3kg, zinc sulfate 6kg, and sterile water 30000kg. The culture time was 40h, the culture temperature was 30℃, the pH value was 5.2, and the DO was 40-45%, and the RQ was 0.95, and the secondary fermentation culture liquid was obtained:
[0100] (4) Commercial fermentation: the secondary fermentation culture liquid obtained in step (3) was inoculated into a commercial culture medium for fermentation culture, and the volume of the commercial fermentation tank was 150m 3 , and the sugar content of the culture medium was 10%; wherein the composition of the culture medium was: 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 reached 1.1, the glucose (10%) was started to be added, and the glucose addition rate was gradually increased from 800-1600L / h according to the RQ value control at 0.9±0.05, and the ammonia water addition rate was 200L / h, so that the pH was constant at 5.2-5.3 fluctuation. The culture time was 20h, the culture temperature was 30℃, the DO was controlled at 40%-50%, and the ergosterol-rich yeast milk was obtained.
[0101] The yeast milk in the above was selected for the preparation of yeast cell wall in Examples 1-3
[0102] Example 1:
[0103] Adjust the concentration of yeast milk to 15%, and then raise the temperature to 55°C for autolysis for 4h. Then raise the temperature to 70°C, adjust the pH to 5.5, and add 0.05% papain and bacterial alkaline protease at a ratio of 0.5:1. After enzymolysis, raise the temperature to 90°C for sterilization, reduce the temperature to 60°C for separation, and further spray dry the heavy phase to obtain the yeast cell wall of the present application.
[0104] Example 2:
[0105] Adjust the concentration of yeast milk to 20%, and then raise the temperature to 58°C for autolysis for 6h. Then raise the temperature to 70°C, adjust the pH to 6.0, and add 0.05% papain and bacterial alkaline protease at a ratio of 1:1. After enzymolysis, raise the temperature to 95°C for sterilization, reduce the temperature to 60°C for separation, and further spray dry the heavy phase to obtain the yeast cell wall of the present application.
[0106] Example 3:
[0107] Adjust the concentration of yeast milk to 20%, and then raise the temperature to 60°C for autolysis for 8h. Then raise the temperature to 70°C, adjust the pH to 6.0, and add 0.05% papain and bacterial alkaline protease at a ratio of 2:1. After enzymolysis, raise the temperature to 100°C for sterilization, reduce the temperature to 65°C for separation, and further spray dry the heavy phase to obtain the yeast cell wall of the present application.
[0108] Example 4 Fatty content and sterol content of cell walls from different strains
[0109] Select high-sugar-resistant S. cerevisiae, low-sugar S. cerevisiae and S. cerevisiae ER18 for yeast fermentation to obtain yeast milk.
[0110] Among them, the high-sugar-resistant S. cerevisiae and the low-sugar S. cerevisiae are purchased from Angel Yeast Co., Ltd. The preparation methods of the yeast milk of the two are the same as the preparation method of the yeast milk of S. cerevisiae ER18 described above, and only the yeast strain is replaced.
[0111] Prepare the three kinds of yeast milk to prepare cell walls according to the preparation process of Example 1. High-sugar-resistant S. cerevisiae cell wall, low-sugar S. cerevisiae cell wall and S. cerevisiae ER18 cell wall are obtained, and the fatty and sterol contents are detected respectively.
[0112] Table 1 Fatty and sterol contents of yeast cell walls
[0113] The sterol content of S. cerevisiae ER18 cell wall is more than 50% higher than that of high-sugar-resistant S. cerevisiae cell wall and low-sugar S. cerevisiae cell wall, and the fatty content is more than 100% higher.
[0114] Embedding efficiency of different cell walls
[0115] Ammonium sulfate 20 parts, diammonium hydrogen phosphate 30 parts, magnesium sulfate 3 parts, zinc sulfate 2 parts were dissolved in 95% (v / v) food grade ethanol. 45 parts of each of the three dry yeast cell wall powders obtained in Example 4 were slowly added to the above ethanol solution while stirring until the ethanol solvent was completely absorbed to form three yeast cell wall mixtures. The yeast cell wall mixtures were centrifuged at 12000 rpm for 10 min, and the supernatant was taken to determine the content of ammonium sulfate, diammonium hydrogen phosphate, magnesium sulfate and zinc sulfate, and the embedding efficiency was calculated. The embedding efficiency calculation formula is as follows:
[0116] M: total content of nutrient salts (ammonium sulfate / diammonium hydrogen phosphate / magnesium sulfate / zinc sulfate);
[0117] m: content of nutrient salts (ammonium sulfate / diammonium hydrogen phosphate / magnesium sulfate / zinc sulfate) in the centrifugal supernatant.
[0118] Ammonium sulfate detection method: GB 29206-2012 Appendix A.4;
[0119] Diammonium hydrogen phosphate detection method: GB 1886.331-2021 Appendix A.3;
[0120] Magnesium sulfate detection method: GB 29207-2012 Appendix A.4;
[0121] Zinc sulfate detection method: GB 29207-2012 Appendix A.4.
[0122] Table 2 Embedding efficiency of yeast cell walls on nutrients and inorganic salts
[0123] Compared with low-sugar S. cerevisiae cell walls and high-sugar S. cerevisiae cell walls, S. cerevisiae ER18 cell walls have an embedding efficiency of more than 30% on ammonium sulfate, diammonium hydrogen phosphate, magnesium sulfate and zinc sulfate, which can effectively embed nutrients and inorganic salts and control the release of nutrients and inorganic salts.
[0124] Example 6
[0125] Yeast extract 20 parts, ammonium sulfate 20 parts, diammonium phosphate 15 parts, magnesium sulfate 5 parts, zinc sulfate 5 parts, vitamin B1 1.5 parts, calcium pantothenate 1.0 part, nicotinic acid 1.0 part, inositol 1.5 parts were dissolved in 95% (v / v) food grade ethanol. While slowly adding 30 parts of the dry yeast cell wall powder of Example 1 to the above ethanol solution with stirring, the ethanol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0126] Example 7
[0127] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 15 parts, magnesium sulfate 11 parts, zinc sulfate 12 parts, vitamin B1 0.6 parts, calcium pantothenate 0.4 part, nicotinic acid 0.4 part, inositol 0.6 parts were dissolved in 95% (v / v) food grade ethanol. While slowly adding 40 parts of the dry yeast cell wall powder of Example 1 to the above ethanol solution with stirring, the ethanol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0128] Example 8
[0129] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 20 parts, magnesium sulfate 2 parts, zinc sulfate 2 parts, vitamin B1 0.3 parts, calcium pantothenate 0.2 part, nicotinic acid 0.2 part, inositol 0.3 parts were dissolved in 95% (v / v) food grade ethanol. While slowly adding 45 parts of the dry yeast cell wall powder of Example 1 to the above ethanol solution with stirring, the ethanol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0130] Comparative Example 1
[0131] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 20 parts, magnesium sulfate 2 parts, zinc sulfate 2 parts, vitamin B1 0.3 parts, calcium pantothenate 0.2 part, nicotinic acid 0.2 part, inositol 0.3 parts were dissolved in 95% (v / v) food grade ethanol. While slowly adding 45 parts of the dry yeast cell wall powder of Example 1 to the above ethanol solution with stirring, the ethanol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0132] Comparative Example 2
[0133] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 20 parts, magnesium sulfate 2 parts, zinc sulfate 2 parts, vitamin Bl 0.3 part, calcium pantothenate 0.2 part, nicotinic acid 0.2 part, and inositol 0.3 part were dissolved in 95% (v / v) food grade ethanol. While slowly adding 45 parts of dry powder of high sugar tolerant Saccharomyces cerevisiae cell wall to the above ethanol solution with stirring, the ethanol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0134] Comparative Example 3
[0135] Yeast extract 20 parts, ammonium sulfate 20 parts, diammonium phosphate 50 parts, magnesium sulfate 4 parts, zinc sulfate 4 parts, vitamin Bl 0.6 part, calcium pantothenate 0.4 part, nicotinic acid 0.4 part, and inositol 0.6 part were mixed to form an alcohol fermentation accelerator.
[0136] Comparative Example 4
[0137] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 20 parts, magnesium sulfate 2 parts, zinc sulfate 2 parts, vitamin Bl 0.3 part, calcium pantothenate 0.2 part, nicotinic acid 0.2 part, and inositol 0.3 part were dissolved in deionized water. While slowly adding 45 parts of dry powder of yeast cell wall in Example 1 to the above aqueous solution with stirring, the water solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0138] Comparative Example 5
[0139] Yeast extract 20 parts, ammonium sulfate 10 parts, diammonium phosphate 20 parts, magnesium sulfate 2 parts, zinc sulfate 2 parts, vitamin Bl 0.3 part, calcium pantothenate 0.2 part, nicotinic acid 0.2 part, and inositol 0.3 part were dissolved in 50% (v / v) food grade ethanol. While slowly adding 45 parts of dry powder of yeast cell wall in Example 1 to the above alcoholic solution with stirring, the alcohol solvent was completely absorbed to form a yeast cell wall mixture. The yeast cell wall mixture was vacuum dried and cooled to form an alcohol fermentation accelerator.
[0140] Experimental Example 1
[0141] Take 100 g of corn flour, respectively, into a clean and dry sample cup of colorimeter, add 1.5% dilute sulfuric acid 0.2 ml, add amylase 9.0 u / g corn flour, add deionized water 181-182 g, stir well, put into the colorimeter, the liquefaction temperature is 101℃, maintain 75 minutes; the temperature is reduced to 45℃ during the period. The temperature rising and falling speed is 3℃ per minute. Cool to 30-33℃ with a cooling water bath. Then pour the cooled corn mash into a 500 mL triangular flask and add water to a material-water ratio of 1:1.9, add saccharifying enzyme 150 u / g corn flour to each triangular flask, respectively, and incubate for half an hour. Then add yeast 0.08 g to each triangular flask, and add the alcohol fermentation accelerators in Examples 6-8 and Comparative Examples 1-5, respectively, 0.013 g, and then place in a shaking bed for fermentation, with the fermentation temperature set at 33℃ and the rotation speed at 170 rpm. The reducing sugar concentration, yeast cell number and alcohol degree are measured every 8 hours.
[0142] The experimental results are as follows:
[0143] Table 3 Effect of alcohol fermentation accelerator application
[0144] (1) The fermentation process of Example 8 of the present application is the most stable, and still maintains strong fermentation capacity in the later stage of fermentation. The number of viable yeast cells in the later stage of fermentation is significantly higher than that of the comparative examples, and the residual reducing sugar is the lowest and the alcohol degree is the highest after 72 hours of fermentation, wherein the alcohol degree is more than 5% higher than that of the comparative examples. Examples 6-7 have a more stable fermentation process than Comparative Examples 1-5, lower residual reducing sugar and higher alcohol degree at the end of fermentation.
[0145] (2) The alcohol fermentation accelerator in Example 8 effectively adsorbs and embeds nutrients and trace elements, making the release of nutrients more uniform and lasting, solving the problem of excessive nutrients in the early stage of fermentation and insufficient nutrients in the later stage of fermentation caused by the use of conventional inorganic nutrients, reducing the mortality rate of alcohol yeast in the later stage of fermentation, enabling it to maintain good alcohol fermentation capacity in the middle and later stages of fermentation, making the fermentation more complete, the alcohol degree higher, the utilization rate of fermentation raw materials improved, and the economic benefits of alcohol production enterprises improved.
[0146] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.
Claims
1. A yeast cell wall, characterized in that, The yeast cell wall is the yeast cell wall of Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118.
2. The yeast cell wall according to claim 1, characterized in that, The yeast cell wall accounts for more than 30 wt% of the dry weight of the cells of the Saccharomyces cerevisiae, and the yeast cell wall contains ergosterol, which accounts for more than 3 wt% of the yeast cell wall.
3. A process for the preparation of yeast cell walls as claimed in claim 1 or 2, characterized in that, The method comprises the following steps: A) fermenting Saccharomyces cerevisiae ER18 with the preservation number of CCTCC NO: M 2020118 to obtain a yeast milk; B) performing enzymolysis on the yeast milk after autolysis to obtain an enzymolysis product; C) drying the enzymolysis product after separation to obtain a yeast cell wall.
4. The production method according to claim 3, characterized by, The preparation method of the yeast milk comprises the following steps: (1) shake flask culture: inoculating a yeast strain in a shake flask culture medium to perform fermentation culture, to obtain a shake flask culture solution, wherein the yeast strain is Saccharomyces cerevisiae ER18, which is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M 2020118, and the sugar content of the shake flask culture medium is 5-10%; (2) primary culture: the shake flask culture solution obtained in step (1) is inoculated into a shake flask culture 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 culture medium of the primary culture is 5-10%; (3) secondary fermentation: inoculating the primary fermentation broth obtained in step (2) into a secondary culture medium to perform fermentation culture to obtain a secondary fermentation broth, the volume of the primary fermentation tank is 20-60 m 3 ; the sugar content of the secondary fermentation culture medium is 5-20%; (4) Third-stage fermentation: inoculating the secondary fermentation broth obtained in step (3) into a third-stage culture medium to perform fermentation culture to obtain a third-stage fermentation broth, the volume of the third-stage fermentation tank is 60-150m 3 ; the sugar content of the third-stage fermentation culture medium is 5-20%; (5) Commercial fermentation: inoculating the third-stage fermentation broth obtained in step (3) into a commercial culture medium to perform fermentation culture, obtaining a commercial fermentation broth, the volume of the commercial fermentation tank is 150-400m 3 , the sugar content of the culture medium of the commercial fermentation is 10-30%; obtaining a yeast milk; (6) Concentration: concentrating the commercial fermentation broth obtained in step (5) to obtain a concentrated fermentation broth, the concentration ratio is 1:2-1:4, the concentration temperature is 60-80°C, the concentration time is 1-3h, the concentration pressure is 0.1-0.3MPa, the concentration pH is 4.0-5.0, the concentration degree of the concentrated fermentation broth is 20-30% One or more steps in steps (3)-(5) in the method can be omitted as needed.
5. The preparation method according to claim 3, characterized in that, Before the autolysis of the yeast milk, the concentration of the yeast milk is adjusted to 15-20%; The temperature of the autolysis is 55-60℃, and the autolysis time is 4-8h; The enzymes used for the enzymolysis include papain and bacterial alkaline protease, and the use amount ratio of the papain to the bacterial alkaline protease is 0.5-2:1, and the amount of the enzymes used for the enzymolysis is 0.05-1.15%; The temperature of the enzymolysis is 55-70℃, the pH of the enzymolysis is 5.5-6.0, and the time of the enzymolysis is 5-20 hours; After the enzymolysis, the enzymolysis product is sterilized at 90-100℃.
6. Use of a yeast cell wall as claimed in claim 1 or 2 for the preparation of an alcohol fermentation accelerator, characterized in that, The alcohol fermentation accelerator comprises: 29-135 parts by mass of an alcohol fermentation accelerator base; 30-70 parts by mass of a yeast cell wall.
7. Use according to claim 6, characterized in that, The alcohol fermentation accelerator base 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.
8. Use according to claim 6, characterized in that, The yeast cell wall coats a certain amount of the alcohol fermentation accelerator base, and the mass ratio of the alcohol fermentation accelerator base coated by the yeast cell wall to the alcohol fermentation accelerator base not coated is 40-95:5-40.
9. The use according to claim 6, wherein the preparation method of the alcohol fermentation accelerator comprises the following steps: dissolving the alcohol fermentation accelerator base in a solvent to obtain a mixed solution; adding the yeast cell wall into the mixed solution and mixing to obtain a yeast cell wall mixture; drying the yeast cell wall mixture to obtain the alcohol fermentation accelerator.
10. Use of an alcohol fermentation accelerator in the use according to claim 6 in alcohol fermentation, characterized in that, The addition amount of the alcohol fermentation accelerator is 0.2-0.4 kg / ton of alcohol.