A strain of Saccharomyces cerevisiae and its application in producing ergosterol

Abstract

The present invention relates to the field of microbial technology, and more particularly to a strain of Saccharomyces cerevisiae and its application in producing ergosterol. The present invention screens and obtains a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae), and its deposit number is CCTCC M 20241572. The impurities of the ergosterol obtained by fermentation are few and high in purity, wherein the ergosterol content is more than 5% of the yeast dry weight, and the impurity 22,23-dihydroergosterol content is less than 1.5% of the yeast dry weight, which can meet the market demand for high-purity ergosterol products. Compared with traditional strains, the strain of the present invention can produce ergosterol with higher purity in high yield, and subsequent purification process can be simplified. The production process of the present invention is simple, low in cost, and more suitable for industrialized production.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a saccharomyces cerevisiae strain and application thereof in producing ergosterol. Background Art

[0002] Ergosterol, first discovered in ergot, is a sterol derivative based on cyclopentaphenanthrene. It appears as white needle- or flaky crystals at room temperature. It is insoluble in water but readily soluble in organic solvents such as ethanol, petroleum ether, and diethyl ether. Furthermore, ergosterol can be oxidized in air.

[0003] Ergosterol is a crucial component of fungal cell membranes, playing a crucial role in ensuring cell viability, membrane fluidity, membrane-bound enzyme activity, membrane integrity, and substance transport. Ergosterol can combine with fatty acids to form sterol esters, which are stored in lipid droplets. This maintains intracellular sterol balance and regulates the transport efficiency of phospholipases by affecting cell membrane fluidity. Ergosterol can influence nutrient absorption and utilization by regulating the activity of membrane-bound ATPases. Ergosterol can stimulate fungal growth and proliferation and is considered a "fungal hormone."

[0004] Ergosterol is a metabolite of significant economic value. In the pharmaceutical industry, ergosterol is a precursor to vitamin D2 and steroid drugs. It is widely used to prevent rickets, osteoporosis, tetany, and rodent diseases. As a feed additive, it can significantly improve poultry production and hatchability. It is also an intermediate for several new drug products, with potential applications in preventing heart disease, inhibiting tumors, repairing lung disease, regulating blood pressure, and improving the human immune system. It enjoys a broad market both domestically and internationally.

[0005] Current research in China focuses on improving the ergosterol content and extraction process in yeast (or other microorganisms). There are no reports on the structure and content of ergosterol impurities, and the impurity content affects the extraction process and the quality of the final product. Traditional ergosterol-producing strains contain more than 2% of the yeast's dry weight, which makes the subsequent ergosterol extraction process extremely complicated. Therefore, screening a yeast with a low ergosterol impurity content for ergosterol production is of great significance. Summary of the Invention

[0006] In view of this, the present invention provides a strain of Saccharomyces cerevisiae and its use in producing ergosterol. Experiments have shown that the strain can produce high-purity ergosterol and simplifies the purification steps, making the preparation process simpler.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention isolated a yeast strain from baijiuqu (white wine koji), with the strain number AMCC 31780 from Angel Yeast Co., Ltd. It was identified as Saccharomyces cerevisiae by morphology and 26S rDNA, and was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 15, 2004, with the accession number CCTCC M 20241572.

[0009] 22,23-Dihydroergosterol is the most important and difficult impurity to remove during the purification of ergosterol. The lower its content, the higher the purity of the final ergosterol product. Fermentation and extraction of strain CCTCC M20241572 using conventional techniques in the art yields high-purity ergosterol, with intracellular ergosterol content exceeding 5% of the yeast dry weight and the main impurity, 22,23-dihydroergosterol, below 1.5% of the yeast dry weight. This simplifies the production process, shortens the production cycle, reduces costs, and makes it more suitable for industrial production of ergosterol.

[0010] The present invention also provides application of the saccharomyces cerevisiae strain in preparing ergosterol.

[0011] The present invention also provides a method for preparing ergosterol, which comprises: fermenting and culturing the cerevisiae yeast strain CCTCCM20241572 to obtain yeast milk with a dry matter content of 12-20%, and then extracting and purifying to obtain ergosterol.

[0012] In the method for preparing ergosterol provided by the present invention, the fermentation culture temperature is 30-33°C, specifically 30°C, 31°C, 32°C, or 33°C, and the pH value is 4.8-6.0, specifically 4.8, 5.0, 5.2, 5.5, 5.8, or 6.0.

[0013] In the present invention, the fermentation culture includes primary fermentation, secondary fermentation, tertiary fermentation and commercial fermentation, wherein the time of primary fermentation is 8 to 15 hours, the time of secondary fermentation is 40 to 50 hours, the time of tertiary fermentation is 30 to 40 hours, and the time of commercial fermentation is 20 to 30 hours.

[0014] In the present invention, the fermentation medium comprises 25-50 parts of a carbon source, 2-5 parts of a 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, 0.2-0.8 parts of ammonium dihydrogen phosphate, and 100-500 parts of water. After fermentation, the fermentation medium is centrifuged to obtain yeast milk.

[0015] In the present invention, the mass unit "part" used for each component involved in the culture medium is mass part. In the same culture medium, "1 part" represents the same mass, and the mass unit represented may be different between different culture media. For example, 1 part in a shake flask culture medium represents 1 g, while in a fermentation culture medium, 1 part represents 1 kg. The carbon source is glucose or molasses, or a mixture of the two, and the nitrogen source is corn steep liquor or ammonia water, or a mixture of the two.

[0016] In the method for preparing ergosterol provided by the present invention, the extraction and purification comprises the following steps:

[0017] (1) reacting the ergosterol-containing yeast milk with potassium hydroxide, subjecting the reaction solution to solid-liquid separation, and collecting a first solid phase;

[0018] (2) mixing and reacting the first solid phase and ethanol, separating the reaction solution into solid and liquid, and collecting the second liquid phase;

[0019] (3) After the second liquid phase is concentrated to dryness under reduced pressure, equal volumes of toluene and water are added for extraction, the upper extract is washed with water, separated, and the organic layer is concentrated under reduced pressure; the concentrate is dissolved in ethanol at 75-80°C and then cooled to crystallize at a crystallization temperature of 5-30°C, and the crystals are collected to obtain ergosterol.

[0020] In the present invention, in step (1), the ratio of yeast milk to potassium hydroxide is 1:0.1-0.5, wherein the yeast milk is calculated on a dry basis, the reaction temperature is 60-100° C., and the reaction time is 4-12 hours.

[0021] In the present invention, in step (2), the amount of ethanol used is 10 to 20 times the dry weight of the yeast milk, the reaction temperature is maintained between 56 and 120° C., and the reaction time is 1 to 3 hours.

[0022] In the present invention, in the step (3), the amount of toluene used is 1.5 to 3.0 times the dry weight of the yeast milk.

[0023] In some specific embodiments of the present invention, the method for preparing ergosterol specifically comprises the following steps:

[0024] (1) reacting the ergosterol-containing yeast milk with potassium hydroxide, subjecting the reaction solution to solid-liquid separation, and collecting a first solid phase. The ratio of yeast milk to potassium hydroxide is 1:0.1-0.5, wherein the yeast milk is calculated on a dry weight basis, the reaction temperature is 60-100° C., and the reaction time is 4-12 hours.

[0025] (2) The first solid phase and ethanol are mixed and reacted, and the reaction liquid is subjected to solid-liquid separation, and the second liquid phase is collected. The amount of ethanol used is 10 to 20 times the dry weight of the yeast milk, the reaction temperature is maintained between 56 and 120° C., and the reaction time is 1 to 3 hours.

[0026] (3) After the second liquid phase is concentrated to dryness under reduced pressure, equal volumes of toluene and water are added for extraction, wherein the amount of toluene is 1.5 to 3 times the dry weight of the yeast milk, the upper extract is washed with water, separated, and the organic layer is concentrated under reduced pressure; the concentrated product is dissolved in ethanol at 75 to 80°C and then cooled to crystallize at a crystallization temperature of 5 to 30°C, and the crystals are collected to obtain ergosterol.

[0027] The cerevisiae yeast strain CCTCC M20241572 provided by the present invention can produce high-purity ergosterol. A high-purity ergosterol product that meets market demand can be directly obtained through a single crystallization in the extraction and purification step. Compared with traditional strains and processes, the strain of the present invention can reduce the number of crystallizations, has a simpler production process, lowers costs, and is more suitable for industrial production.

[0028] Biological Deposit Description

[0029] Saccharomyces cerevisiae AMCC 31780 (D1) was deposited in the China Center for Type Culture Collection on July 15, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20241572. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The colony and bacterial morphology of the strain CCTCC NO: M 20241572 of the present invention;

[0031] Figure 2 The test results of ergosterol and impurities in yeast milk of traditional strain CCTCC NO: M2020118 (ergosterol retention time 18.473min; 22,23-dihydroergosterol retention time 22.002min);

[0032] Figure 3 Detection results of ergosterol and impurities in yeast milk of strain CCTCC NO: M20241572 of the present invention (sample diluted 5 times, ergosterol retention time 18.4425 min; 22,23-dihydroergosterol retention time 21.986 min);

[0033] Figure 4 Test results of ergosterol products prepared by traditional strain CCTCC NO: M2020118 (ergosterol retention time 18.129 min; 22,23-dihydroergosterol retention time 21.641 min);

[0034] Figure 5The detection results of the ergosterol product prepared by the strain CCTCC NO: M20241572 of the present invention (ergosterol retention time 18.367 min; 22,23-dihydroergosterol retention time 21.869 min). DETAILED DESCRIPTION

[0035] The present invention provides a strain of Saccharomyces cerevisiae and its use in producing ergosterol. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0037] The present invention will be further described below in conjunction with the embodiments:

[0038] Example 1 Strain isolation and identification

[0039] The present invention isolates and screens a yeast strain from baijiuqu, and the strain library number of Angel Yeast Co., Ltd. is AMCC 31780. The specific isolation method is as follows: take 0.2g of koji block, dilute it with sterile water in the order of solid-liquid ratio of 1:10, 1:100, 1:1000, and 1:10000, take 50μl of the dilutions of different concentrations, spread it on YPD solid culture medium (5% sucrose, 2% yeast extract, 0.1% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 1.5% agar powder, and add 50μg / ml ampicillin) with a spreading stick, culture it in a 30°C incubator for 1-3 days, pick out a milky white, large and thick, smooth, moist and sticky single colony, streak culture it on YPD solid culture medium, repeat twice, and obtain a pure strain of yeast. Figure 1 It can be seen that the colonies are round, relatively regular in shape, smooth in surface, and white or milky white in color.

[0040] PCR amplification was performed using genomic DNA from strain AMCC 31780 as a template using universal primers for strain identification. After the PCR product qualified, the target band was excised and purified, and the recovered product was subjected to Sanger sequencing. The determined 26S rDNA gene sequence was submitted to the NCBI database for sequence homology analysis using the BLAST tool. Combining morphological characteristics with the gene sequence analysis results, strain AMCC 31780 was identified as Saccharomyces cerevisiae.

[0041] Example 2 Preparation of ergosterol

[0042] 2.1 Preparation of ergosterol from traditional strains

[0043] The traditional brewer's yeast CCTCC NO: M2020118 in patent CN113980825B was used to prepare ergosterol, wherein the fermentation culture was carried out according to Example 5 in patent CN113980825B.

[0044] Seed activation culture: One ring of Saccharomyces cerevisiae CCTCC No. M2020118 was inoculated into a 5 L shake flask medium containing 10% sugar and containing 200 g of glucose, 50 g of ammonium sulfate, 10 g of yeast extract, 2 g of potassium dihydrogen phosphate, 0.2 g of magnesium sulfate, and 2 L of sterile water for 28 hours at 30°C and a pH of 5.2 to obtain a shake flask culture solution.

[0045] Primary fermentation: The shake flask culture was inoculated into a 5m³ fermentor for fermentation. The culture medium had a sugar content of 10% and consisted of: 300 kg of a carbon source (a 1:1 mixture of molasses and glucose), 75 kg of a nitrogen source (corn steep liquor), 6 kg of potassium dihydrogen phosphate, 0.3 kg of magnesium sulfate, 0.6 kg of zinc sulfate, and 3000 kg of sterile water. The culture was maintained for 15 hours at 32°C and a pH of 4.8 to obtain the primary fermentation culture medium.

[0046] Secondary fermentation: The primary fermentation broth was inoculated into the secondary culture medium for fermentation in a 60 m³ fermentation tank. The culture medium had a sugar content of 10% and consisted of: 4000 kg of a carbon source (a 1:1 mixture of molasses and glucose), 1000 kg of a nitrogen source (corn steep liquor), 80 kg of potassium dihydrogen phosphate, 4 kg of magnesium sulfate, 8 kg of zinc sulfate, and 40 m³ of sterile water. The culture was incubated for 43 hours at a temperature of 32°C, a pH of 4.8, a DO of 50%, and an RQ of 1.1 to obtain the secondary fermentation broth.

[0047] Tertiary fermentation: The secondary fermentation broth is inoculated into the tertiary culture medium for fermentation culture. The volume of the tertiary fermentation tank is 150m3; the sugar content of the culture medium is 5%; the composition of the culture medium is: 2500kg of carbon source (a mixture of molasses and glucose in a ratio of 1:1), 1250kg of nitrogen source (corn steep liquor), 100kg of potassium dihydrogen phosphate, 5kg of magnesium sulfate, 10kg of zinc sulfate, and 50m3 of sterile water. When the RQ drops to 0.85, start feeding mixed sugar (10%). Based on the RQ value controlled at 0.9±0.05, gradually increase the glucose flow rate from 200 to 800L / h, and accelerate the ammonia flow by 50L / h, so that the pH fluctuates constantly at 5.0. The culture time is 38h, the culture temperature is 30℃, and the DO is 40%, to obtain the tertiary fermentation culture medium.

[0048] Commercial fermentation: The tertiary fermentation broth was inoculated into commercial culture medium for fermentation in a 400 m³ commercial fermentor tank with a 5% sugar content. The culture medium consisted of 7500 kg of carbon source (a 1:1 mixture of molasses and glucose), 3750 kg of nitrogen source (ammonia), 300 kg of potassium dihydrogen phosphate, 300 kg of ammonium dihydrogen phosphate, 15 kg of magnesium sulfate, 30 kg of zinc sulfate, and 150 m³ of sterile water. After the RQ dropped to 1.05, glucose (10%) was fed. While maintaining the RQ at 1.05 ± 0.05, the glucose feed rate was gradually increased from 800 to 1600 L / h and the ammonia feed rate at 200 L / h to maintain a constant pH of 5.2. The culture duration was 26 hours, the temperature was 30°C, and the DO was controlled at 55%. After the fermentation, the yeast was separated by a disc centrifuge at a speed of 5000 rpm and washed with process water to obtain 96 tons of ergosterol-rich yeast milk with a dry matter content of 15.6%, equivalent to 15.6 tons of dry yeast.

[0049] Ergosterol extraction and purification: 4.68 tons of potassium hydroxide were added to the yeast milk, heated to 100°C with stirring, and stirred for 8 hours. The mixture was separated into solids and liquids, and the solids were collected to obtain a first solid phase. 234 tons of ethanol were added to the first solid phase, and the mixture was heated to approximately 80°C for 2 hours. The solids and liquids were separated, and the liquid phase was collected to obtain a second liquid phase. The second liquid phase was concentrated to dryness at 50°C, and 31 tons of toluene and 31 tons of water were added to dissolve the residue. The mixture was stirred well, allowed to stand, and separated. The toluene phase was concentrated to dryness, and 7.8 tons of ethanol was added. The mixture was heated to approximately 78°C for complete dissolution. The mixture was cooled naturally to room temperature and then further cooled to 5°C. The mixture was kept warm for 2 hours for crystallization, i.e., primary crystallization. Filtering was performed to obtain crude ergosterol. Secondary crystallization was performed according to the above crystallization steps to obtain 321.9 kg of the final ergosterol product.

[0050] 2.2 Preparation of ergosterol by the strain of the present invention

[0051] Ergosterol was prepared using the present invention's Saccharomyces cerevisiae CCTCC NO: M20241572, following the process described in 2.1. The resulting yeast milk produced 98 tons of yeast, with a dry matter content of 16.3%, equivalent to 16.0 tons of dry yeast. After extraction and purification, the yeast milk was subjected to primary crystallization to yield 524.1 kg of the final ergosterol product.

[0052] 2.3 Detection of ergosterol and 22,23-dihydroergosterol

[0053] The ergosterol and impurity 22,23-dihydroergosterol in the yeast milks of Examples 2.1 and 2.2 were determined by liquid chromatography. The results are shown in Table 1.

[0054] The ergosterol and impurity 22,23-dihydroergosterol in the ergosterol final products of Examples 2.1 and 2.2 were determined by liquid chromatography, and the ergosterol yield was calculated. The results are shown in Table 2.

[0055] Ergosterol yield % = ergosterol product mass T / (yeast dry weight T*ergosterol content in yeast %)*100%.

[0056] Table 1 Yeast milk detection

[0057]

[0058] Table 2 Ergosterol product testing

[0059]

[0060] As shown in Tables 1 and 2, ergosterol can be prepared by using yeast milk prepared by the strain CCTCC NO: M 20241572 of the present invention. ergosterol can be obtained by a single crystallization without the need for secondary crystallization. The content of the obtained ergosterol product is 98.7%, the content of 22,23-dihydroergosterol is 0.9%, and the final yield of the ergosterol product is 63.0%.

[0061] Yeast milk prepared with the traditional strain CCTCC NO: M2020118, after secondary crystallization, obtained an ergosterol content of 96.8% and a 22,23-dihydroergosterol content of 2.0%. The secondary crystallization also resulted in a final yield of ergosterol product of 54.3%.

[0062] The above results show that the strain of the present invention can produce ergosterol with higher purity in high yield, and can simplify the purification process, directly obtaining a final product with higher purity that meets market demand, with lower production cost and more suitable for industrial production.

[0063] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Saccharomyces cerevisiae strain, characterized in that Its deposit number is CCTCC NO:M 20241572.

2. Use of the Saccharomyces cerevisiae strain according to claim 1 in the preparation of ergosterol.

3. A method for preparing ergosterol, characterized in that: include: Fermenting and culturing the Saccharomyces cerevisiae strain according to claim 1, and obtaining yeast milk by centrifugation; The yeast milk is extracted and purified to obtain ergosterol.

4. The method according to claim 3, characterized in that The fermentation culture temperature is 30-33° C., and the pH value is 4.8-6.0; the fermentation culture includes primary fermentation, secondary fermentation, tertiary fermentation and commercial fermentation; wherein the primary fermentation time is 8-15 hours, the secondary fermentation time is 40-50 hours, the tertiary fermentation time is 30-40 hours, and the commercial fermentation time is 20-30 hours.

5. The method according to claim 3, characterized in that The culture medium components for the fermentation culture include: 25-50 parts of a carbon source, 2-5 parts of a 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, 0.2-0.8 parts of ammonium dihydrogen phosphate and 100-500 parts of water.

6. The method according to claim 3, characterized in that The extraction and purification comprises the following steps: (1) reacting the yeast milk with potassium hydroxide, subjecting the reaction solution to solid-liquid separation, and collecting a first solid phase; (2) mixing and reacting the first solid phase and ethanol, separating the reaction solution into solid and liquid, and collecting the second liquid phase; (3) After the second liquid phase is concentrated to dryness under reduced pressure, equal volumes of toluene and water are added for extraction, the upper extract is washed with water, separated, and the organic layer is concentrated under reduced pressure; the concentrate is dissolved in ethanol at 75-80°C and then cooled to crystallize at a crystallization temperature of 5-30°C, and the crystals are collected to obtain ergosterol.

7. The method according to claim 6, characterized in that In the step (1), the ratio of yeast milk to potassium hydroxide is 1:0.1-0.5, wherein the yeast milk is calculated on a dry basis, the reaction temperature is 60-100° C., and the reaction time is 4-12 hours.

8. The method according to claim 6, characterized in that In the step (2), the amount of ethanol used is 10 to 20 times the dry weight of the yeast milk, the reaction temperature is maintained between 56 and 120° C., and the reaction time is 1 to 3 hours.

9. The method according to claim 6, characterized in that In the step (3), the amount of toluene used is 1.5 to 3.0 times the dry weight of the yeast milk.

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