Saccharomyces cerevisiae strain and application thereof in ergosterol production

Abstract

The invention relates to the technical field of microorganisms, in particular to a saccharomyces cerevisiae strain and application thereof in ergosterol production. A strain of saccharomyces cerevisiae is obtained through screening, the preservation number of the saccharomyces cerevisiae is CCTCC M 20241572, the ergosterol obtained through fermentation of the saccharomyces cerevisiae is few in impurity and high in purity, the content of the ergosterol is 5% or above of the dry weight of the yeast, the content of the impurity 22, 23-dihydroergosterol is 1.5% or below of the dry weight of the yeast, and the requirement of the market for high-purity ergosterol products can be met. Compared with the traditional strain, the strain provided by the invention can produce ergosterol with higher yield and purity, and can simplify the subsequent purification process. The method is simple in production process, low in cost and more suitable for industrial production.

Description

Technical Field

[0001] The 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 was first discovered in ergot and is a type of sterol. Ergosterol is a derivative with cyclopentane polyhydrophenanthrene as the parent nucleus. It is a white needle-shaped or flaky crystal at room temperature, insoluble in water, but easily soluble in organic solvents such as ethanol, petroleum ether, ether, etc. Moreover, ergosterol can be oxidized in the air.

[0003] Ergosterol is an important component of fungal cell membranes and plays an important role in ensuring cell viability, membrane fluidity, membrane-bound enzyme activity, membrane integrity, and material transport. Ergosterol can combine with fatty acids to form sterol esters stored in lipid droplets, maintain the balance of sterols in cells, and regulate the transport efficiency of phospholipase by affecting the fluidity of cell membranes. Ergosterol can affect the absorption and utilization of nutrients by regulating the activity of membrane-bound ATPase. Ergosterol can stimulate the growth and proliferation of fungi and is regarded as a "fungal hormone."

[0004] Ergosterol is a metabolite with important economic value. In the pharmaceutical industry, ergosterol is a precursor of vitamin D2 and steroid drugs. It is widely used to prevent rickets, osteoporosis, tetany, rodent diseases, etc. As a feed additive, it can significantly improve the yield and hatching rate of poultry. It is also an intermediate for some new drugs, which can prevent the occurrence of heart disease, inhibit tumors, repair lung diseases, regulate blood pressure and the human immune system, and has a broad market both at home and abroad.

[0005] The current domestic research direction is mainly to improve 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. The 22,23-dihydroergosterol content of traditional ergosterol production strains exceeds 2% of the dry weight of yeast, which makes the subsequent ergosterol extraction process extremely complicated. Therefore, it is of great significance to screen a yeast with low ergosterol impurity content for the production of ergosterol. Summary of the invention

[0006] In view of this, the present invention provides a strain of Saccharomyces cerevisiae and its application in producing ergosterol. Experiments show 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 isolates a yeast strain from liquor koji, and the strain library number of Angel Yeast Co., Ltd. is AMCC 31780. It is identified as Saccharomyces cerevisiae by morphology and 26S rDNA, and was deposited in China Center for Type Culture Collection on July 15, 24, with the address: Wuhan University, Wuhan, China, and its deposit number is CCTCC M 20241572.

[0009] 22,23-dihydroergosterol is the most important and most difficult impurity to remove during the purification of ergosterol. The lower its content, the higher the purity of the final product ergosterol. The strain CCTCC M20241572 is fermented and extracted according to the conventional process in the field, and the ergosterol obtained is of high purity, with the intracellular ergosterol content reaching more than 5% of the dry weight of the yeast, and the content of the main impurity 22,23-dihydroergosterol is less than 1.5% of the dry weight of the yeast, which simplifies the production process, shortens the production cycle, reduces the cost, and is more suitable for industrial production of ergosterol.

[0010] The 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 saccharomyces cerevisiae 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, 33°C, and the pH value is 4.8-6.0, specifically 4.8, 5.0, 5.2, 5.5, 5.8, 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 components include 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, 0.2-0.8 parts of ammonium dihydrogen phosphate and 100-500 parts of water. After the fermentation is completed, centrifugation is performed 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 units represented by different culture media may be different, such as 1 part in a shake flask culture medium represents 1g, and in a fermentation medium, 1 part represents 1kg. The carbon source is glucose or molasses or a mixture of the two, and the nitrogen source is corn syrup 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 yeast milk containing ergosterol with potassium hydroxide, separating the reaction liquid into solid and liquid, and collecting the first solid phase;

[0018] (2) mixing and reacting the first solid phase and ethanol, separating the reaction liquid 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, and the upper extract is washed with water, separated, and the organic layer is concentrated under reduced pressure; the concentrate is dissolved in 75-80° C. ethanol 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 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.

[0021] In the present invention, 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.

[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 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 yeast milk containing ergosterol with potassium hydroxide, collecting the first solid phase after solid-liquid separation of the reaction liquid, wherein the ratio of the 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 separated into solid and liquid, 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, and the upper layer of the 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 brewer's yeast strain CCTCC M20241572 provided by the invention can produce ergosterol with higher purity in high yield, and can directly obtain a high-purity ergosterol product that meets market demand through one crystallization in the extraction and purification step. Compared with traditional strains and processes, the strain provided by the invention can reduce the number of crystallization times, has a simpler production process, lowers cost, 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 The detection results of ergosterol and impurities in yeast milk of strain CCTCC NO: M20241572 of the present invention (5-fold dilution injection, ergosterol retention time 18.4425min; 22,23-dihydroergosterol retention time 21.986min);

[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 result 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 application in producing ergosterol. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article 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 embodiments:

[0038] Example 1 Strain Isolation and Identification

[0039] The present invention isolates and selects a yeast strain from liquor koji, and the strain library number of Angel Yeast Co., Ltd. is AMCC 31780. The specific separation method is: take a koji block weighing 0.2g, and add sterile water to dilute it in accordance with the solid-liquid ratio of 1:10, 1:100, 1:1000, and 1:10000 in sequence, take 50μl of the dilution solution of different concentrations, and 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 rod, and after culturing in a 30°C incubator for 1 to 3 days, pick a milky white, large and thick, smooth, moist, and sticky single colony, and streak culture 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 on the surface, and white or milky white in color.

[0040] The genomic DNA of strain AMCC 31780 was used as a template and PCR amplification was performed using universal primers for strain identification. After the PCR product was qualified, the target band was cut and purified and recovered, and the recovered product was subjected to Sanger sequencing. The measured 26SrDNA gene sequence was submitted to the NCBI database, and the sequence homology analysis was performed using the BLAST tool. Combining the morphological characteristics with the gene sequence analysis results, strain AMCC 31780 can be 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: a ring of Saccharomyces cerevisiae CCTCC NO: M2020118 was inoculated into a 5L shake flask culture medium for activation fermentation culture, wherein the sugar content of the shake flask culture medium was 10%; wherein the shake flask culture medium was composed of: 200g glucose, 50g ammonium sulfate, 10g yeast extract, 2g potassium dihydrogen phosphate and 0.2g magnesium sulfate, and 2L sterile water. The culture time was 28h, the culture temperature was 30°C, and the pH value was 5.2, and the shake flask culture solution was obtained;

[0045] Primary fermentation: The shake flask culture medium is inoculated into a fermentation tank for fermentation culture. The volume of the primary fermentation tank is 5m3. The sugar content of the culture medium is 10%. The composition of the culture medium is: 300kg of carbon source (molasses and glucose mixed in a ratio of 1:1), 75kg of nitrogen source (corn syrup), 6kg of potassium dihydrogen phosphate, 0.3kg of magnesium sulfate, 0.6kg of zinc sulfate, and 3000kg of sterile water. The culture time is 15h, the culture temperature is 32°C, and the pH value is 4.8, and the primary fermentation culture medium is obtained;

[0046] Secondary fermentation: The primary fermentation culture fluid is inoculated into the secondary culture medium for fermentation culture. The volume of the secondary fermentation tank is 60m3; the sugar content of the culture medium is 10%; the composition of the culture medium is: 4000kg of carbon source (molasses and glucose mixed in a ratio of 1:1), 1000kg of nitrogen source (corn syrup), 80kg of potassium dihydrogen phosphate, 4kg of magnesium sulfate, 8kg of zinc sulfate, and 40m3 of sterile water. The culture time is 43h, the culture temperature is 32°C, the pH value is 4.8, the DO is 50%, and the RQ is 1.1, and the secondary fermentation culture fluid is obtained;

[0047] Tertiary fermentation: The secondary fermentation culture fluid 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 (molasses and glucose mixed in a ratio of 1:1), 1250kg of nitrogen source (corn syrup), 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 to add mixed sugar (10%), and according to 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%, and the tertiary fermentation culture fluid is obtained;

[0048] Commercial fermentation: The tertiary fermentation culture fluid is inoculated into the commercial culture medium for fermentation culture. The volume of the commercial fermentation tank is 400m3, and the sugar content of the culture medium is 5%; the composition of the culture medium is: carbon source (molasses and glucose mixed in a ratio of 1:1) 7500kg, nitrogen source (ammonia water) 3750kg, potassium dihydrogen phosphate 300kg, ammonium dihydrogen phosphate 300kg, magnesium sulfate 15kg, zinc sulfate 30kg, sterile water 150m3. When RQ drops to 1.05, start to add glucose (10%), and according to the RQ value controlled at 1.05±0.05, gradually increase the glucose flow rate from 800 to 1600L / h, and the ammonia flow rate 200L / h, so that the pH fluctuates constantly at 5.2, the culture time is 26h, the culture temperature is 30℃, and the DO is controlled at 55%. After the fermentation, a disc centrifuge was used to separate the yeast milk 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] Extraction and purification of ergosterol: add 4.68 tons of potassium hydroxide to the yeast milk, heat to 100°C under stirring, keep warm and stir for 8 hours, separate solid and liquid, collect solids, and obtain the first solid phase; add 234 tons of ethanol to the first solid phase, heat to about 80 degrees and react for 2 hours, separate solid and liquid, collect liquid phase, and obtain the second liquid phase. The second liquid phase is concentrated to dryness at 50°C, 31 tons of toluene and 31 tons of water are added to dissolve the residue, stir well and stand for stratification, separate liquids, concentrate the toluene phase to dryness, add 7.8 tons of ethanol and heat to about 78 degrees to dissolve completely, cool naturally to room temperature and then further cool to 5°C, keep warm and crystallize for 2 hours, that is, primary crystallization, filter to obtain crude ergosterol, refer to the above crystallization steps for secondary crystallization, and obtain 321.9 kg of ergosterol final product.

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

[0051] The saccharomyces cerevisiae CCTCC NO: M20241572 of the present invention is used to prepare ergosterol, and the process is referred to process 2.1. The experiment obtains 98 tons of yeast milk, with a dry matter content of 16.3%, and 16.0 tons of dry yeast. After the yeast milk is extracted and purified, 524.1 kg of ergosterol final product is obtained by one crystallization.

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

[0053] The ergosterol and impurity 22,23-dihydroergosterol in the yeast milk in 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 in 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 detection

[0059]

[0060] From the results in Table 1 and Table 2, it can be seen that when ergosterol is prepared by using yeast milk prepared by the strain CCTCC NO: M 20241572 of the present invention, ergosterol can be obtained by one 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] The yeast milk prepared by the traditional strain CCTCC NO: M2020118 had an ergosterol content of 96.8% and a 22,23-dihydroergosterol content of 2.0% after secondary crystallization. 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, and can simplify the purification process, directly obtain a final product with higher purity that meets market demand, with lower production cost, and is 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 M20241572.

2. The Saccharomyces cerevisiae strain according to claim 1, characterized in that The intracellular ergosterol content is more than 5% of the dry weight of the yeast, and the impurity 22,23-dihydroergosterol content is less than 1.5% of the dry weight of the yeast.

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

4. A method for preparing ergosterol, characterized in that: include: Fermenting and culturing the brewer's yeast strain according to claim 1 or 2, and obtaining yeast milk by centrifugation; The yeast milk is extracted and purified to obtain ergosterol.

5. The method according to claim 4, 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 8-15h, the secondary fermentation time is 40-50h, the tertiary fermentation time is 30-40h, and the commercial fermentation time is 20-30h.

6. The method according to claim 4, characterized in that The fermentation medium components include: 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, 0.2-0.8 parts of ammonium dihydrogen phosphate and 100-500 parts of water.

7. The method according to claim 4, characterized in that The extraction and purification comprises the following steps: (1) reacting the yeast milk containing ergosterol with potassium hydroxide, separating the reaction liquid into solid and liquid, and collecting the first solid phase; (2) mixing and reacting the first solid phase and ethanol, separating the reaction liquid 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, and the upper extract is washed with water, separated, and the organic layer is concentrated under reduced pressure; the concentrate is dissolved in 75-80° C. ethanol and then cooled to crystallize at a crystallization temperature of 5-30° C., and the crystals are collected to obtain ergosterol.

8. The method according to claim 7, 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.

9. The method according to claim 7, 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.

10. The method according to claim 7, 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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