Process for the preparation of ergosterol and zymosterol by coproduction from yeast

Abstract

The present application relates to the field of yeast extraction, in particular to a method for preparing ergosterol and zymosterol by co-production of yeast. The present application provides a method for preparing ergosterol and zymosterol by co-production of yeast, which utilizes 4-phenyl-3H-1,2,4-triazoline-3,5-dione to selectively react with ergosterol in a mixed solution of sterol, while zymosterol does not participate in the reaction, thereby achieving the purpose of removing ergosterol and purifying zymosterol. The present application realizes the co-production of zymosterol from ergosterol-producing yeast, reduces tail emissions while improving economic value. Compared with the existing process for extracting zymosterol from yeast, the present process does not require multi-step chromatographic separation, greatly simplifying the process, and has high selectivity and high purity of zymosterol.

Description

Technical Field

[0001] This invention relates to the field of yeast extraction, and particularly to a method for preparing ergosterol and yeast sterol from yeast. Background Technology

[0002] Yeast extraction for ergosterol is a mature and currently mainstream commercial ergosterol production method. Yeast sterol is a core intermediate in the biosynthetic pathway of sterols and can be used as a substrate for enzymatic reactions to synthesize sterols such as ergosterol, sterols, and cholesterol. It also has certain drug development potential and can be studied for antifungal targets and anticancer mechanisms.

[0003] During the metabolic process of yeast synthesis of ergosterol, not all accumulated yeast sterols are converted; only a portion is converted into ergosterol and other metabolites. Therefore, yeast producing ergosterol mainly contains two sterol substances: ergosterol and yeast sterol. During yeast production of ergosterol, yeast sterol is removed as an impurity during purification and accumulates in the mother liquor, ultimately being disposed of as waste.

[0004] There are many technical schemes for extracting ergosterol from yeast, but few schemes for preparing yeast sterol, and even fewer schemes for recovering yeast sterol from the mother liquor after ergosterol extraction. The technical scheme used in the article "Sterol synthesis: a simple method for the isolation of zymosterol (5 alpha-cholesta-8, 24-dien-3 beta-ol) from yeast and spectral properties of zymosterol" is as follows: yeast is refluxed in 15% potassium hydroxide ethanol solution for 12 hours, the reaction solution is poured into water, extracted with petroleum ether, washed with water, and the organic phase is collected by column chromatography. The eluent is collected in fractions and subjected to esterification reaction. Yeast sterol ester is obtained by column chromatography again, and finally hydrolyzed to obtain yeast sterol with high purity.

[0005] Since yeast contains both yeast sterol and ergosterol, but their solubility differs significantly, yeast sterol is easily removed as an impurity when extracting ergosterol from yeast. However, if yeast sterol is extracted alone, ergosterol, as an impurity, is difficult to remove. Therefore, the existing technical solution requires several column chromatography steps, as well as esterification and hydrolysis reactions to obtain yeast sterol. The process is lengthy, and the multiple column chromatography steps make the operation cumbersome. In addition, sterols can undergo esterification and hydrolysis reactions, so the selectivity is not high. Summary of the Invention

[0006] In view of this, the present invention provides a method for the co-production of ergosterol and yeast sterol from yeast. This method utilizes the selective reaction of 4-phenyl-3H-1,2,4-triazolline-3,5-dione with ergosterol in a sterol mixture solution, while yeast sterol does not participate in the reaction, thereby achieving the purpose of removing ergosterol and purifying yeast sterol.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing yeast sterols, comprising the following steps:

[0009] Step 1: Obtain the mother liquor of crystalline extract of ergosterol from yeast, concentrate it, and collect the concentrate;

[0010] Step 2: Dissolve the concentrate in ethyl acetate, wash with water, separate the liquids, and collect the organic phase;

[0011] Step 3: React the organic phase with 4-phenyl-3H-1,2,4-triazolline-3,5-dione, concentrate, dissolve in ethanol, perform a first cooling crystallization, collect the filtrate by vacuum filtration, perform a second cooling crystallization, filter, and dry to obtain the yeast sterol; the filtration is vacuum filtration.

[0012] The crystallization in step 3 includes a first crystallization and a second crystallization; the temperature of the first crystallization is 20~25℃; the temperature of the second crystallization is 0~10℃;

[0013] The amount of 4-phenyl-3H-1,2,4-triazolline-3,5-dione used in step 3 is 1.1 to 1.5 equivalents;

[0014] The purity of the 4-phenyl-3H-1,2,4-triazolline-3,5-dione is not less than 94%.

[0015] In some specific embodiments of the present invention, the mass ratio of ergosterol in the crystallization mother liquor to 4-phenyl-3H-1,2,4-triazolline-3,5-dione is 2.4:(1.16~1.59).

[0016] In some specific embodiments of the present invention, the mass ratio of ergosterol in the crystallization mother liquor to the 4-phenyl-3H-1,2,4-triazolline-3,5-dione is 2.4:1.16, 2.4:1.38, or 2.4:1.59.

[0017] In some specific embodiments of the present invention, the first concentration in step 1 is a vacuum concentration at 50~60°C.

[0018] In some specific embodiments of the present invention, the temperature of the reaction in step 3 is 25℃±5℃, and the reaction time is 2h.

[0019] In some specific embodiments of the present invention, the concentration in step 3 is a vacuum concentration at 40~50°C.

[0020] In some specific embodiments of the present invention, the dissolution temperature in step 3 is 70~80°C, preferably 78°C.

[0021] In some specific embodiments of the present invention, the temperature of the first cooling crystallization in step 3 is 20~25°C, preferably 20°C.

[0022] In some specific embodiments of the present invention, the temperature of the second cooling crystallization in step 3 is 0~10℃, preferably 0℃.

[0023] In some specific embodiments of the present invention, the method for preparing the crystallization mother liquor after yeast extraction of ergosterol in step 1 includes:

[0024] Step 1-1: Take yeast and saponify it in an alkaline substance and water. After solid-liquid separation, collect the first solid phase.

[0025] Steps 1-2: Add the first solvent to the first solid phase to extract ergosterol, and obtain the second liquid phase and the second solid phase by solid-liquid separation. Dry the second solid phase to obtain defatted yeast.

[0026] Steps 1-3: Concentrate the second liquid phase to dryness under reduced pressure, add the second solvent to dissolve, wash with water, separate the liquid, collect the organic layer and concentrate under reduced pressure, add ethanol to crystallize and obtain ergosterol, collect the crystallization mother liquor, which is the crystallization mother liquor after yeast extraction of ergosterol;

[0027] In steps 1-2, the first solvent includes any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, and acetonitrile;

[0028] In steps 1-3, the second solvent includes any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane, and chloroform.

[0029] In some specific embodiments of the present invention, in step 1-1, the alkaline substance comprises one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution;

[0030] In step 1-1, the mass ratio of the yeast, the alkaline substance, and water is 1:(0.1-0.5):(5-15).

[0031] In step 1-1, the saponification reaction takes 4 to 12 hours and the saponification reaction takes 60 to 100°C.

[0032] In some specific embodiments of the present invention, in step 1-1, the temperature of the saponification reaction is 60~80°C.

[0033] In some specific embodiments of the present invention, in steps 1-2, the mass-volume ratio of the yeast to the first solvent is 1:(10-20) in kg / L.

[0034] In steps 1-2, the extraction temperature is 56~120℃, and the mixture is refluxed for 1~3 hours.

[0035] In some specific embodiments of the present invention, in steps 1-2, the ratio of the yeast, the alkaline substance and water by mass is 1:(0.2-0.3):(10-13).

[0036] In some specific embodiments of the present invention, in steps 1-3, the amount of the second solvent is twice the volume of the yeast;

[0037] In steps 1-3, the vacuum concentration temperature is 40-80℃;

[0038] In steps 1-3, the mass ratio of ethanol to yeast used in the crystallization is (0.5-1.5), and the crystallization temperature is 0-30℃.

[0039] In some specific embodiments of the present invention, the yeast includes one or more of Saccharomyces cerevisiae, Candida albicans, Gastrodia elata, Rhodotorula rubra, or Cryptococcus pluvialis.

[0040] Secondly, the present invention also provides a method for preparing ergosterol and yeast sterol by co-production of yeast, comprising the following steps:

[0041] Ergosterol is extracted from yeast;

[0042] Collect the mother liquor from the yeast extract of ergosterol, and obtain yeast sterol according to the above-described method for preparing yeast sterol.

[0043] In some specific embodiments of the present invention, the method for extracting ergosterol from yeast includes:

[0044] Step 1-1: Take yeast and saponify it in an alkaline substance and water. After solid-liquid separation, collect the first solid phase.

[0045] Steps 1-2: Add the first solvent to the first solid phase to extract ergosterol, and obtain the second liquid phase and the second solid phase by solid-liquid separation. Dry the second solid phase to obtain defatted yeast.

[0046] Steps 1-3: Concentrate the second liquid phase to dryness under reduced pressure, add the second solvent to dissolve, wash with water, separate the liquid and liquid, collect the organic layer, concentrate under reduced pressure, add ethanol to crystallize and obtain ergosterol;

[0047] In steps 1-2, the first solvent includes any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, and acetonitrile;

[0048] In steps 1-3, the second solvent includes any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane, and chloroform.

[0049] In some specific embodiments of the present invention, in step 1-1, the alkaline substance comprises one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution;

[0050] In step 1-1, the mass ratio of the yeast, the alkaline substance, and water is 1:(0.1-0.5):(5-15).

[0051] In step 1-1, the saponification reaction takes 4 to 12 hours and the saponification reaction temperature is 60 to 100°C.

[0052] In step 1-1, the temperature of the saponification reaction is 60~80℃;

[0053] In steps 1-2, the mass-volume ratio of the yeast to the first solvent is 1:(10-20) in kg / L.

[0054] In steps 1-2, the extraction temperature is 56~120℃, and the mixture is refluxed for 1~3 hours;

[0055] In steps 1-2, the ratio of yeast, alkaline substance, and water by mass is 1:(0.2-0.3):(10-13).

[0056] In steps 1-3, the amount of the second solvent used is twice the volume of the yeast;

[0057] In steps 1-3, the vacuum concentration temperature is 40-80℃;

[0058] In steps 1-3, the mass ratio of ethanol to yeast used in the crystallization is (0.5-1.5), and the crystallization temperature is 0-30℃.

[0059] In some specific embodiments of the present invention, the yeast includes one or more of Saccharomyces cerevisiae, Candida albicans, Gastrodia elata, Rhodotorula rubra, or Cryptococcus pluvialis.

[0060] This invention provides a method for the co-production of ergosterol and yeast sterol from yeast. The method utilizes 4-phenyl-3H-1,2,4-triazolline-3,5-dione to selectively react with ergosterol in a sterol mixture solution, while yeast sterol does not participate in the reaction, thereby achieving the purpose of removing ergosterol and purifying yeast sterol.

[0061] The beneficial effects of the present invention include, but are not limited to:

[0062] (1) The co-production of yeast sterols from yeast that produces ergosterol has been achieved, reducing waste emissions while increasing economic value.

[0063] (2) Compared with the existing process of extracting yeast sterols from yeast, this process does not require multi-step chromatographic separation, greatly simplifies the process, and has high selectivity and high purity of yeast sterols. Attached Figure Description

[0064] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0065] Figure 1 This shows the ratio of yeast sterols and ergosterols in existing yeast extracts;

[0066] Figure 2 This shows the ratio of ergosterol and yeast sterol in the existing ergosterol crystallization mother liquor;

[0067] Figure 3 This invention presents a process flow diagram;

[0068] Figure 4 This is a schematic diagram of the invention;

[0069] Figure 5 Example 1 shows the HPLC chromatogram of yeast sterols;

[0070] Figure 6 Example 2 shows the HPLC chromatogram of yeast sterols;

[0071] Figure 7 Example 3 shows the HPLC chromatogram of yeast sterols;

[0072] Figure 8 Example 4 shows the HPLC chromatogram of yeast sterols;

[0073] Figure 9 The HPLC chromatogram of yeast sterols in Comparative Example 1 is shown.

[0074] Figure 10 The HPLC chromatogram of yeast sterols in Comparative Example 2 is shown.

[0075] Figure 11 The HPLC chromatogram of yeast sterols in Comparative Example 3 is shown.

[0076] Figure 12 The HPLC overlay chromatograms of each sample and the yeast sterol standard are shown. Detailed Implementation

[0077] This invention discloses a method for the co-production of ergosterol and yeast sterol from yeast. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0078] Technical solution flow of this invention:

[0079] (1) Referring to our authorized patent CN115960156B "A method for extracting ergosterol using yeast", the ergosterol extraction method is as follows: after the product is separated by crystallization, a crystallization mother liquor is obtained. The mother liquor is concentrated and the solvent is recovered. The concentrated liquid is dissolved in ethyl acetate, washed with water, and separated to obtain an ethyl acetate solution containing yeast sterol and ergosterol.

[0080] (2) Add an ethyl acetate solution of 4-phenyl-3H-1,2,4-triazolline-3,5-dione to the solution. 4-phenyl-3H-1,2,4-triazolline-3,5-dione reacts with ergosterol, while yeast sterol does not participate in the reaction.

[0081] (3) Concentrate to remove ethyl acetate, dissolve the residue with ethanol at elevated temperature, and then cool to 20-25°C to precipitate the solid after the ergosterol reaction.

[0082] (4) Filter to remove solids, and further cool the solution to 0-10℃ to crystallize. After filtration and drying, yeast sterol is obtained.

[0083] This invention uses the amount of 4-phenyl-3H-1,2,4-triazoline-3,5-dione and the crystallization temperature of yeast sterols as variables. The former mainly affects the purity of yeast sterols, while the latter mainly affects the yield. The amount of 4-phenyl-3H-1,2,4-triazoline-3,5-dione is preferably 1.1-1.5 equivalents (purity above 94%). Too much will result in waste, while too little will lead to incomplete ergosterol reaction and reduce the purity of the yeast sterol sample. The crystallization temperature is preferably below 0-10℃, where the yield does not change significantly. Too low a temperature wastes energy, while too high a temperature will lead to a decrease in yield.

[0084] The raw materials and reagents used in the method for preparing ergosterol and yeast sterol by co-production of yeast provided by the present invention are all commercially available.

[0085] The present invention will be further illustrated below with reference to the embodiments:

[0086] Example 1:

[0087] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60 °C to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.16 g (1.1 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50 °C to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 25 °C to crystallize, filter, and further cool the filtrate to 0 °C to crystallize. Filter and dry to obtain 5.62 g of yeast sterol with a purity of 94.5%. Figure 5 As shown.

[0088] Example 2:

[0089] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.38 g (1.3 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 20℃ to crystallize, filter, and further cool the filtrate to 0℃ to crystallize. Filter and dry to obtain 5.59 g of yeast sterol with a purity of 94.8%. Figure 6 As shown.

[0090] Example 3:

[0091] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.59 g (1.5 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 23℃ to crystallize, filter, and further cool the filtrate to 5℃ to crystallize. Filter and dry to obtain 5.53 g of yeast sterol with a purity of 95.2%. Figure 7 As shown.

[0092] Example 4:

[0093] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.38 g (1.3 eq) 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 25℃ to crystallize, filter, and further cool the filtrate to 10℃ to crystallize. Filter and dry to obtain 5.49 g of yeast sterol with a purity of 95.0%. Figure 8 As shown.

[0094] Comparative Example 1:

[0095] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.06 g (1.0 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 20℃ to crystallize, filter, and further cool the filtrate to 5℃ to crystallize. Filter and dry to obtain 5.73 g of yeast sterol with a purity of 92.6%. Figure 9 As shown.

[0096] Comparative Example 2:

[0097] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.69 g (1.6 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 20℃ to crystallize, filter, and further cool the filtrate to 15℃ to crystallize. Filter and dry to obtain 4.64 g of yeast sterol with a purity of 95.2%. Figure 10 As shown.

[0098] Comparative Example 3:

[0099] Take 2 L of ergosterol crystallization mother liquor (containing 2.4 g of ergosterol and 6.2 g of yeast sterol), concentrate it under reduced pressure at 50-60℃ to a solid, add 1 L of ethyl acetate to dissolve it, then add 2 L of water and stir for 10 min. After standing and separating, take the organic phase, add 50 g of anhydrous magnesium sulfate to dry it, and add 20 ml of ethyl acetate solution containing 1.38 g (1.3 eq) of 4-phenyl-3H-1,2,4-triazolline-3,5-dione dropwise with stirring at room temperature. React for 2 h, concentrate under reduced pressure at 40-50℃ to an oily substance, add 100 ml of ethanol and heat to reflux to dissolve it, cool to 20℃ to crystallize, filter, and further cool the filtrate to 15℃ to crystallize. Filter and dry to obtain 4.73 g of yeast sterol with a purity of 94.8%. Figure 11 As shown.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing yeast sterols, characterized in that, Includes the following steps: Step 1: Obtain the mother liquor of crystalline extract of ergosterol from yeast, concentrate it, and collect the concentrate; Step 2: Dissolve the concentrate in ethyl acetate, wash with water, separate the liquids, and collect the organic phase; Step 3: React the organic phase with 4-phenyl-3H-1,2,4-triazolline-3,5-dione, concentrate, dissolve in ethanol, crystallize under first cooling, collect the filtrate by suction filtration, crystallize under second cooling, filter, and dry to obtain the yeast sterol; The temperature for the first cooling crystallization is 20~25℃; the temperature for the second cooling crystallization is 0~10℃; The amount of 4-phenyl-3H-1,2,4-triazolline-3,5-dione used in step 3 is 1.1 to 1.5 equivalents of ergosterol. The purity of the 4-phenyl-3H-1,2,4-triazolline-3,5-dione is not less than 94%. The method for preparing the crystallization mother liquor after yeast extraction of ergosterol in step 1 includes: Step 1-1: Take yeast and saponify it in an alkaline substance and water. After solid-liquid separation, collect the first solid phase. Steps 1-2: Add the first solvent to the first solid phase to extract ergosterol, and obtain the second liquid phase and the second solid phase by solid-liquid separation. Dry the second solid phase to obtain defatted yeast. Steps 1-3: Concentrate the second liquid phase to dryness under reduced pressure, add the second solvent to dissolve, wash with water, separate the liquid, collect the organic layer and concentrate under reduced pressure, add ethanol to crystallize and obtain ergosterol, collect the crystallization mother liquor, which is the crystallization mother liquor after yeast extraction of ergosterol; In steps 1-2, the first solvent is any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, and acetonitrile; In steps 1-3, the second solvent is any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane, and chloroform.

2. The preparation method according to claim 1, characterized in that, The mass ratio of ergosterol to 4-phenyl-3H-1,2,4-triazololine-3,5-dione in the mother liquor of crystallization is 2.4:(1.16~1.59).

3. The preparation method according to claim 1, characterized in that, The mass ratio of ergosterol to 4-phenyl-3H-1,2,4-triazolline-3,5-dione in the crystallization mother liquor is 2.4:1.16, 2.4:1.38, or 2.4:1.

59.

4. The preparation method according to claim 1, characterized in that, The concentration described in step 1 is a vacuum concentration at 50~60℃.

5. The preparation method according to claim 1, characterized in that, The reaction temperature in step 3 is 25℃±5℃, and the reaction time is 2h.

6. The preparation method according to claim 1, characterized in that, The concentration described in step 3 is a vacuum concentration at 40~50℃.

7. The preparation method according to claim 1, characterized in that, The dissolution temperature in step 3 is 70~80℃.

8. The preparation method according to claim 1, characterized in that, In step 1-1, the alkaline substance is one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide, and sodium methoxide aqueous solution; In step 1-1, the mass ratio of the yeast, the alkaline substance, and water is 1:(0.1-0.5):(5-15). In step 1-1, the saponification reaction takes 4 to 12 hours and the saponification reaction temperature is 60 to 100°C.

9. The preparation method according to claim 1, characterized in that, In step 1-1, the temperature of the saponification reaction is 60~80℃.

10. The preparation method according to claim 1, characterized in that, In steps 1-2, the mass-volume ratio of the yeast to the first solvent is 1:(10-20) in kg / L. In steps 1-2, the extraction temperature is 56~120℃, and the mixture is refluxed for 1~3 hours.

11. The preparation method according to claim 1, characterized in that, In step 1-1, the ratio of yeast, alkaline substance and water by mass is 1:(0.2-0.3):(10-13).

12. The preparation method according to claim 1, characterized in that, In steps 1-3, the amount of the second solvent used is twice the volume of the yeast; In steps 1-3, the vacuum concentration temperature is 40-80℃; In steps 1-3, the mass ratio of ethanol to yeast used in the crystallization is 0.5 to 1.5; the crystallization temperature is 0 to 30°C.

13. The preparation method according to claim 1, characterized in that, The yeast is one or more of the following: Saccharomyces cerevisiae, Candida albicans, Gastrodia elata, Rhodotorula rubra, or Cryptococcus pluvialis.

14. A method for the co-production of ergosterol and yeast sterol from yeast, characterized in that, Includes the following steps: Ergosterol is extracted from yeast; Collect the mother liquor from the yeast extract of ergosterol and obtain yeast sterol by the preparation method according to any one of claims 1 to 13.

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