A method for extracting ergosterol using yeast

Abstract

The present invention relates to a method for extracting ergosterol using yeast, which is characterized in that the content of the obtained ergosterol product can reach more than 98%, and the yeast can be recovered during the extraction process for secondary utilization. At the same time, calcium fatty acid can also be co-produced during the production of ergosterol. This method simplifies the washing and concentration processes required during extraction, greatly reduces wastewater generation, and the production process is environmentally friendly and economical.

Description

Technical Field

[0001] The present invention relates to the technical field of fine chemical engineering, and particularly relates to a method for extracting ergosterol. Background Art

[0002] Ergosterol, also known as ergosterol, is an important component of the cell membranes of many microorganisms and plays an important role in ensuring cell viability, cell membrane fluidity, and cell membrane integrity. Ergosterol can enhance the body's ability to resist diseases, has obvious antibacterial and anti-tumor effects, is an important source of fat-soluble vitamin D2, and its metabolite vitamin D2 can be used to promote the absorption of calcium and phosphorus in the human body, prevent the occurrence of rickets and chondropathy, and can also be used as a feed additive in feed to increase the egg production rate and hatching rate of livestock and poultry. Ergosterol is also an important raw material for pharmaceutical and chemical industries and can be used to produce sterol drugs such as "cortisone" and hormone progesterone. This product is insoluble in water and soluble in organic solvents such as ethanol, benzene, ether, and chloroform.

[0003] As an important component of the fungal cell membrane, ergosterol has a relatively high content in yeast and mushrooms. Therefore, in the process of extracting ergosterol, fungi such as yeast or mushrooms are commonly used as raw materials. First, ergosterol in yeast cells is separated through a saponification reaction, and then an ergosterol product is obtained through processes such as extraction, washing, crystallization, and drying. For example, Chen Guoping et al. (patent, patent number: CN114031662A) saponify dry yeast and then perform extraction and washing to obtain a crude product of ergosterol, and then obtain the finished product through recrystallization and filtration. The extraction process of Li Mingyuan et al. (patent, patent number: CN112390842A) is similar, except that the yeast strain is ultrasonically treated before the saponification reaction, and column chromatography is used for purification after saponification extraction, and then the ergosterol finished product is obtained through recrystallization. Li Rongjie et al. (patent, patent number: CN103588843A) utilize the principle that ergosterol is produced as a by-product during the microbial fermentation process to extract ergosterol from the waste mycelium of fermented citric acid. Specifically, the waste mycelium is washed to neutrality and then subjected to a saponification reaction. The filtrate obtained from the reaction is washed until colorless and then saponified and filtered again to obtain a crude product of ergosterol, and the crude product is purified by recrystallization.

[0004] The above several methods for extracting ergosterol obtain the finished product through processes such as extraction and washing purification. The entire process only focuses on the extraction of ergosterol, either the other nutrients in the yeast during the process are not reused, or the yeast after extraction is directly discarded, and a large amount of water is used in the washing process, which is not environmentally friendly and economical. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned prior art, such as the imperfect application of yeast and the large amount of wastewater, the present invention improves and innovates the process of extracting ergosterol from yeast, realizes the secondary utilization of yeast, and reduces the amount of wastewater.

[0006] The method for extracting ergosterol from yeast according to the present invention comprises the following specific steps:

[0007] Step 1: Saponify the yeast raw material in an alkaline solution, and after the reaction, separate the product into a first liquid phase and a first solid phase by solid-liquid separation:

[0008] Step 2: Add a first solvent to the first solid phase obtained in the first step to extract ergosterol, separate the solid and liquid to obtain a second liquid phase and a second solid phase, and dry the second solid phase to obtain defatted yeast;

[0009] Step 3: Concentrate the second liquid phase in the second step under reduced pressure to dryness, dissolve it in a second solvent, then wash with water, separate the liquid, concentrate the organic layer under reduced pressure, add ethanol for crystallization to obtain crude ergosterol, and dissolve and recrystallize the crude product with ethanol to obtain an ergosterol product.

[0010] The yeast is Saccharomyces cerevisiae, Candida sp., Torulopsis sp., Rhodotorula sp. or Cryptococcus sp., etc.

[0011] The alkaline solution in the first step comprises one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide and sodium methoxide aqueous solutions; the saponification time is 4 to 12 hours; the saponification temperature is 60 to 100 °C. The dosage of calcium chloride is 1 / 5 to 1 / 3 of the weight of the yeast raw material.

[0012] The first solvent comprises any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone and acetonitrile; by mass, the solvent dosage is 10 to 20 times the volume of the yeast raw material, and the ergosterol extraction temperature is maintained between 56 and 120 °C, with reflux for 1 to 3 hours.

[0013] The reduced pressure concentration temperature in the third step is 40 to 80 °C, and the second solvent comprises any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane and chloroform.

[0014] When the crude product is crystallized, by mass, the ethanol usage is 0.5 to 1.5 times that of the yeast raw material, and the crystallization temperature is 0 to 30 °C.

[0015] Preferably, calcium chloride can be added to the first liquid phase obtained in the first step at the same time, and after precipitation, solid-liquid separation is carried out to obtain a third solid phase as a calcium fatty acid product.

[0016] Preferably, by mass, the ratio of yeast to alkali and water in the first step is 1:0.1 to 0.5:5 to 15.

[0017] Preferably, by volume, the amount of the second solvent used in the third step is twice that of the yeast raw material.

[0018] Preferably, by weight, the amount of ethanol used in the recrystallization in the third step is 20 - 50 times the mass of the crude product.

[0019] Preferably, when calcium chloride is added in the form of a solution, the solution concentration is greater than 10%.

[0020] Furthermore, by mass, the ratio of yeast to alkali and water in the first step is 1:0.2 - 0.3:10 - 13.

[0021] Furthermore, the amount of calcium chloride used is 1 / 5 - 1 / 4 of the weight of the yeast raw material.

[0022] Furthermore, when calcium chloride is added in the form of a solution, the solution concentration is greater than 10%.

[0023] The beneficial effects of the present invention are as follows:

[0024] 1. In the saponification process of the present invention, the amount of alkali used is less than that of the prior art. After the saponification is completed, the degreased yeast and the saponification liquid are first separated by solid-liquid separation, and then ergosterol in the degreased yeast is extracted with alcohol. The degreased yeast does not contact toxic reagents. While realizing the extraction of ergosterol, the degreased yeast is recycled and used for the extraction of other extracts, achieving the full utilization of each component of yeast cells.

[0025] 2. After the degreased yeast is further extracted with an organic solvent for ergosterol, only a small amount of water is needed for washing and concentration to obtain a relatively pure crude ergosterol product, and finally a finished product is obtained through refining, and the product content can reach more than 98%. Since the water consumption in the washing process is greatly reduced, the present invention largely solves the environmental protection problem of a large amount of wastewater.

[0026] 3. The saponification liquid separated from the yeast can further co-produce calcium fatty acid with economic value. Specific Embodiments

[0027] The technical solution of the present invention will be further limited below in combination with specific embodiments, but the scope of protection required is not limited only to the description made. The present invention relates to a method for extracting ergosterol from yeast, and the specific steps are as follows:

[0028] First step: Saponify the yeast raw material in an alkali solution, and after the reaction, separate the product by solid-liquid separation to obtain a first liquid phase and a first solid phase;

[0029] Second step: Add a first solvent to the first solid phase obtained in the first step to extract ergosterol, and after solid-liquid separation, obtain a second liquid phase and a second solid phase, and dry the second solid phase to obtain degreased yeast;

[0030] Step 3: Concentrate the second liquid phase in Step 2 under reduced pressure to dryness, dissolve it in a second solvent, then wash with water, separate the layers, concentrate the organic layer under reduced pressure, add ethanol for crystallization to obtain a crude ergosterol product, and dissolve and recrystallize the crude product with ethanol to obtain an ergosterol product.

[0031] The yeast includes, but is not limited to, Saccharomyces cerevisiae, Candida, Torulopsis, Rhodotorula, or Cryptococcus, etc.

[0032] In Step 1, the lye contains one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution; the saponification time is 4 to 12 hours; the saponification temperature is 60 to 100 °C. The dosage of calcium chloride is 1 / 5 to 1 / 3 of the weight of the yeast raw material.

[0033] The first solvent includes any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, and acetonitrile; by mass, the solvent dosage is 10 to 20 times the volume of the yeast raw material, and the ergosterol extraction temperature is maintained between 56 and 120 °C, with reflux for 1 to 3 hours.

[0034] In Step 3, the reduced pressure concentration temperature is 40 to 80 °C, and the second solvent includes any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane, and chloroform.

[0035] When crystallizing the crude product, by mass, the ethanol usage is 0.5 to 1.5 times that of the yeast raw material, and the crystallization temperature is 0 to 30 °C.

[0036] Preferably, calcium chloride can be added to the first liquid phase obtained in Step 1 simultaneously, and after precipitation and solid-liquid separation, the third solid phase is obtained as a calcium fatty acid product.

[0037] Preferably, by mass, the ratio of yeast, alkali, and water in Step 1 is 1; 0.1 to 0.5; 5 to 15.

[0038] Preferably, by volume, the dosage of the second solvent in Step 3 is twice that of the yeast raw material.

[0039] Preferably, by weight, the ethanol usage during recrystallization in Step 3 is 20 to 5 times the mass of the crude product.

[0040] Further, by mass, the ratio of yeast, alkali, and water in Step 1 is 1:0.2 to 0.3:10 to 13.

[0041] Further, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

[0042] Further, when calcium chloride is added in the form of a solution, the solution concentration is greater than 10%.

[0043] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase. The dry yeast is purchased online, and the purchase link is as follows: https: / / fubonshop.angelyeast.com / goods / 77 。

[0044] The sources of ingredients and equipment models used in the preparation process are specifically shown in Tables 1 and 2 below:

[0045] Table 1 Sources of Ingredients

[0046] Drug Name Model / Purity Distributor Sodium ethoxide AR Sinopharm Chemical Reagent Calcium chloride AR Sinopharm Chemical Reagent Potassium hydroxide AR Sinopharm Chemical Reagent Sodium hydroxide AR Sinopharm Chemical Reagent Ethanol Food grade Yichang Tiansheng Trading Co., Ltd. n-Hexane AR Xilong Chemical Co., Ltd. Toluene AR Xilong Chemical Co., Ltd. Ethyl acetate AR Xilong Chemical Co., Ltd. Dried yeast Fubang Feed High-Activity Dried Yeast (Klibiagen) Angel Yeast Co., Ltd. Acetone AR Xilong Chemical Co., Ltd. Isopropanol AR Xilong Chemical Co., Ltd.

[0047] Table 2 Equipment Information

[0048]

[0049] Example 1

[0050] Add 5 kg of water and 0.1 kg of sodium ethoxide to 1 kg of dry yeast, heat to 100 °C with stirring, keep stirring for 5 hours, separate the solid and liquid, add 2 kg of 10% calcium chloride solution dropwise to the solution with stirring, separate the solid and liquid after sufficient stirring, dry the solid at 105 °C to obtain 67.8 g of calcium fatty acid. The solid separated after saponification is added with 15 kg of ethanol, heated to reflux for 2 hours, separate the solid and liquid, dry the solid to obtain 918.5 g of defatted yeast, concentrate the extract to dryness at 50 °C, add 2 L of ethyl acetate to dissolve the residue, add 2 L of water, stir well, let stand for stratification, separate the liquid, concentrate the ethyl acetate phase to dryness, add 0.5 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 15 °C, keep the temperature for crystal precipitation for 2 hours, filter to obtain 11.5 g of crude product, refine the crude product with 460 ml of ethanol according to the crystallization process of the crude product, dry the solid at 80 °C to obtain 5.8 g of crystal, with a purity of 98.6% and a total extraction yield of 52.7%.

[0051] Example 2

[0052] Add 15 kg of water and 0.2 kg of potassium hydroxide to 1 kg of dry yeast. While stirring, heat up to 80 °C and keep stirring for 8 hours. Then perform solid-liquid separation. Add 200 g of calcium chloride to the solution in portions while stirring. After sufficient stirring, perform solid-liquid separation again. Dry the solid at 105 °C to obtain 68.8 g of calcium fatty acid. Add 10 kg of acetone to the solid separated after saponification, heat to reflux for 1 hour, perform solid-liquid separation, and obtain 915.2 g of defatted yeast after drying the solid. Concentrate the extract to dryness at 40 °C, add 2 L of toluene to dissolve the residue, add 2 L of water, stir well, let it stand for layering, separate the liquid. Concentrate the toluene phase to dryness, add 1 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 10 °C. Keep the temperature for crystal precipitation for 2 hours, filter to obtain 12.6 g of crude product. Refine the crude product with 460 ml of ethanol according to the crystallization process of the crude product. Dry the solid at 80 °C to obtain 6.3 g of crystal with a purity of 98.7% and a total extraction yield of 57.3%.

[0053] Example 3

[0054] Add 15 kg of water and 0.5 kg of sodium hydroxide to 1 kg of dry yeast. While stirring, heat up to 60 °C and keep stirring for 12 hours. Then perform solid-liquid separation. Add a saturated solution containing 200 g of calcium chloride to the solution while stirring. After sufficient stirring, perform solid-liquid separation again. Dry the solid at 105 °C to obtain 67.1 g of calcium fatty acid. Add 20 kg of isopropanol to the solid separated after saponification, heat to reflux for 1 hour, perform solid-liquid separation, and obtain 920.1 g of defatted yeast after drying the solid. Concentrate the extract to dryness at 50 °C, add 2 L of n-hexane to dissolve the residue, add 2 L of water, stir well, let it stand for layering, separate the liquid. Concentrate the n-hexane phase to dryness, add 1 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 10 °C. Keep the temperature for crystal precipitation for 2 hours, filter to obtain 10.9 g of crude product. Refine the crude product with 380 ml of ethanol according to the crystallization process of the crude product. Dry the solid at 80 °C to obtain 5.6 g of crystal with a purity of 98.2% and a total extraction yield of 50.9%.

[0055] Example 4

[0056] Add 10 kg of water and 0.3 kg of potassium hydroxide to 1 kg of dry yeast. Heat the mixture to 100 °C with stirring and keep stirring for 8 hours. Then perform solid-liquid separation. While stirring, add 1 kg of 20% calcium chloride solution to the solution. After sufficient stirring, perform solid-liquid separation again. Dry the solid at 105 °C to obtain 71.8 g of calcium fatty acid. Add 15 kg of ethanol to the solid separated after saponification, heat to reflux for 2 hours, perform solid-liquid separation, and dry the solid to obtain 910.2 g of defatted yeast. Concentrate the extract to dryness at 50 °C, add 2 L of toluene to dissolve the residue, add 2 L of water, stir well, let it stand for layering, separate the liquid, concentrate the toluene phase to dryness, add 0.5 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 5 °C. Keep the temperature for crystal precipitation for 2 hours, filter to obtain 13.4 g of crude product. Refine the crude product with 600 ml of ethanol according to the crystallization process of the crude product, dry the solid at 80 °C to obtain 6.9 g of crystal with a purity of 98.7% and a total extraction yield of 62.7%.

[0057] Comparative Example 1

[0058] Add 5 kg of water and 0.1 kg of sodium ethoxide to 1 kg of dry yeast. Heat the mixture to 100 °C with stirring and keep stirring for 5 hours. Then perform solid-liquid separation. While stirring, add 1.5 kg of 10% calcium chloride solution to the solution. After sufficient stirring, perform solid-liquid separation again. Dry the solid at 105 °C to obtain 50.8 g of calcium fatty acid. Add 15 kg of ethanol to the solid separated after saponification, heat to reflux for 2 hours, perform solid-liquid separation, and dry the solid to obtain 918.5 g of defatted yeast. Concentrate the extract to dryness at 50 °C, add 2 L of ethyl acetate to dissolve the residue, add 2 L of water, stir well, let it stand for layering, separate the liquid, concentrate the ethyl acetate phase to dryness, add 0.5 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 15 °C. Keep the temperature for crystal precipitation for 2 hours, filter to obtain 11.5 g of crude product. Refine the crude product with 460 ml of ethanol according to the crystallization process of the crude product, dry the solid at 80 °C to obtain 5.8 g of crystal with a purity of 98.6% and a total extraction yield of 52.7%.

[0059] Comparative Example 2

[0060] Add 3 kg of water and 0.08 kg of sodium ethoxide to 1 kg of dry yeast. Heat the mixture to 55 °C with stirring and keep stirring for 4 hours. Then, perform solid-liquid separation. While stirring, add 2 kg of 10% calcium chloride solution to the solution. After sufficient stirring, perform solid-liquid separation again. Dry the solid at 105 °C to obtain 43.3 g of calcium fatty acid. Add 15 kg of ethanol to the solid separated after saponification, heat to reflux for 2 hours, perform solid-liquid separation, and dry the solid to obtain 932.8 g of defatted yeast. Concentrate the extract to dryness at 50 °C, add 2 L of ethyl acetate to dissolve the residue, add 2 L of water, stir well, let it stand for layering, separate the liquid, concentrate the ethyl acetate phase to dryness, add 0.5 kg of ethanol, heat to reflux until completely dissolved, naturally cool to room temperature, and then further cool to 15 °C. Keep the temperature for crystal precipitation for 2 hours, filter to obtain 11.5 g of crude product. Refine the crude product with 460 ml of ethanol according to the crystallization process of the crude product, dry the solid at 80 °C to obtain 3.4 g of crystal with a purity of 96.6% and a total extraction yield of 30.9%.

[0061] As can be seen from the above specific implementation examples, in Examples 1-4, the ratios of yeast: alkali: water in the saponification reaction process are 1:0.1:5, 1:0.2:15, 1:0.5:15, and 1:0.3:10 respectively. In Comparative Example 2, the ratio of yeast, alkali, and water is 1:0.08:3, and the ratios of water and alkali are both less than 1; 0.1-0.5; 5-15 disclosed in the present invention. Finally, only 3.4 g of ergosterol is obtained, which is much less than the 5.8 g, 6.3 g, 5.6 g, and 6.9 g obtained in Examples 1-4 where the ratios of yeast, alkali, and water are within the range described in the present invention. Moreover, in this case, although the ratio of calcium chloride to dry yeast in both the examples and the comparative examples is 1 / 5, only 43.3 g of calcium fatty acid is obtained in the scheme of Comparative Example 2, which is significantly less than the 67.8 g, 68.8 g, 67.1 g, and 71.8 g obtained in the example schemes. Comparing Example 2 with Comparative Example 1, the ratios of yeast, alkali, and water in the saponification reaction process are the same for both. In Example 2, the addition amount of calcium chloride is 1 / 5 of the yeast mass described in the present invention, while in Comparative Example 1, the addition amount of calcium chloride is 15% of the yeast mass, which is less than 1 / 5, and only 50.8 g of calcium fatty acid is obtained, which is significantly less than that in Example 2. In all examples, the amount of water used in the cleaning process after dissolving ergosterol with the second solvent is equivalent to the amount of the solvent, which is only twice the weight of the dry yeast.

[0062] The scheme for extracting ergosterol from yeast proposed by the present invention uses less amounts of alkali and water, obtains an ergosterol product with a higher purity, recovers the yeast at the same time, and co-produces calcium fatty acid, thus making the most of the yeast raw material as much as possible, and saving the usage of drugs and water. When scaled up to the production scale, the economic and environmental benefits generated cannot be underestimated.

[0063] The above embodiments are only some implementation manners of the technical solution of the present invention, and thus should not be construed as imposing any form of limitation on the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall all be included within the protection scope of the present invention.

Claims

1. A method for extracting ergosterol using yeast, which specifically includes the following steps: The first step: The yeast raw material undergoes a saponification reaction in an alkali solution. After the reaction, the product is separated into a first liquid phase and a first solid phase by solid-liquid separation; The yeast is Saccharomyces cerevisiae, Candida sp., Torulopsis sp., Rhodotorula sp. or Cryptococcus sp.; In the first step, by mass, the ratio of the yeast raw material to the alkali substance and water is 1:0.2 - 0.3:10 - 15; The saponification time is 4 - 12 hours; and / or the saponification temperature is 80 - 100 °C; The second step: Add a first solvent to the first solid phase obtained in the first step to extract ergosterol. After solid-liquid separation, a second liquid phase and a second solid phase are obtained. The second solid phase is dried to obtain defatted yeast; The first solvent includes any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, and acetonitrile; by mass, the solvent dosage is 10 - 20 times the volume of the yeast raw material; Add calcium chloride to the first liquid phase obtained in the first step to precipitate calcium fatty acid. After solid-liquid separation, the third solid phase is obtained as the calcium fatty acid product; the dosage of calcium chloride is 1 / 5 - 1 / 3 of the weight of the yeast raw material; The third step: Concentrate the second liquid phase in the second step to dryness under reduced pressure, add a second solvent to dissolve it, then wash with water and separate the liquid. The organic layer is concentrated under reduced pressure, and ethanol is added for crystallization to obtain ergosterol; The second solvent is any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane, and chloroform; the solvent dosage is twice the volume of the yeast raw material; The usage amount of ethanol is 0.5 - 1.5 times that of the yeast raw material.

2. According to the method described in claim 1, the ergosterol obtained in the third step is redissolved in ethanol and recrystallized to obtain an ergosterol product.

3. According to the method described in claim 1, characterized in that in the first step, the alkali solution includes one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution.

4. According to the method described in claim 2, characterized in that in the first step, the alkali solution includes one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution.

5. According to the method described in claim 1, characterized in that in the first step, the saponification temperature is 60 - 80 °C.

6. According to the method described in claim 2, characterized in that in the first step, the saponification temperature is 60 - 80 °C.

7. According to the method described in claim 3, characterized in that in the first step, the saponification temperature is 60 - 80 °C.

8. According to the method described in claim 4, characterized in that in the first step, the saponification temperature is 60 - 80 °C.

9. According to the method described in claim 1, characterized in that calcium chloride can be added in solid or solution form, and the solution concentration is greater than 10%.

10. According to the method described in claim 2, characterized in that calcium chloride can be added in solid or solution form, and the solution concentration is greater than 10%.

11. According to the method described in claim 3, characterized in that calcium chloride can be added in solid or solution form, and the solution concentration is greater than 10%.

12. According to the method described in claim 5, characterized in that Calcium chloride can be added in solid or solution form, and the solution concentration is greater than 10%.

13. According to the method described in claim 1, characterized in that, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

14. According to the method described in claim 2, characterized in that, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

15. According to the method described in claim 3, characterized in that, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

16. According to the method described in claim 5, characterized in that, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

17. According to the method described in claim 9, characterized in that, the dosage of calcium chloride is 1 / 5 to 1 / 4 of the weight of the yeast raw material.

18. According to the method described in claim 1, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

19. According to the method described in claim 2, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

20. According to the method described in claim 3, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

21. According to the method described in claim 5, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

22. According to the method described in claim 9, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

23. According to the method described in claim 13, characterized in that, in the second step, the extraction temperature of ergosterol is maintained between 56 - 120 °C, and reflux for 1 - 3 hours.

24. According to the method described in any one of claims 1 - 23, characterized in that, in the third step, the reduced pressure concentration temperature is 40 - 80 °C.

25. According to the method described in any one of claims 1 - 23, characterized in that, in the third step, during the crystallization process, the temperature is 0 - 30 °C.

26. According to the method described in claim 24, characterized in that, in the third step, during the crystallization process, the temperature is 0 - 30 °C.

27. According to the method described in claim 2, 4, 6, 10, 14 or 19, characterized in that, by mass, the ethanol dosage during recrystallization is 20 - 50 times the mass of the crude ergosterol.

Images