CN115960156A - Method for extracting ergosterol by using yeast - Google Patents
Method for extracting ergosterol by using yeast Download PDFInfo
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- CN115960156A CN115960156A CN202211723066.4A CN202211723066A CN115960156A CN 115960156 A CN115960156 A CN 115960156A CN 202211723066 A CN202211723066 A CN 202211723066A CN 115960156 A CN115960156 A CN 115960156A
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- yeast
- ergosterol
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- ethanol
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 77
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 title claims abstract description 47
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 title claims abstract description 47
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 title claims abstract description 47
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 title claims abstract description 47
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000000605 extraction Methods 0.000 claims abstract description 19
- 239000011575 calcium Substances 0.000 claims abstract description 16
- 229910052791 calcium Inorganic materials 0.000 claims abstract description 16
- 230000008569 process Effects 0.000 claims abstract description 16
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 15
- 229930195729 fatty acid Natural products 0.000 claims abstract description 15
- 239000000194 fatty acid Substances 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 96
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 74
- 239000007788 liquid Substances 0.000 claims description 45
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000926 separation method Methods 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 30
- 239000007787 solid Substances 0.000 claims description 26
- 238000007127 saponification reaction Methods 0.000 claims description 25
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 22
- 238000001035 drying Methods 0.000 claims description 22
- 239000002904 solvent Substances 0.000 claims description 20
- 238000002425 crystallisation Methods 0.000 claims description 19
- 230000008025 crystallization Effects 0.000 claims description 19
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 18
- 239000001110 calcium chloride Substances 0.000 claims description 18
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 17
- 239000002994 raw material Substances 0.000 claims description 16
- 238000010992 reflux Methods 0.000 claims description 15
- 239000007790 solid phase Substances 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 239000003513 alkali Substances 0.000 claims description 13
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 9
- 239000007791 liquid phase Substances 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- QDRKDTQENPPHOJ-UHFFFAOYSA-N sodium ethoxide Chemical compound [Na+].CC[O-] QDRKDTQENPPHOJ-UHFFFAOYSA-N 0.000 claims description 7
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 6
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- 238000001953 recrystallisation Methods 0.000 claims description 4
- 241001337994 Cryptococcus <scale insect> Species 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 241000223252 Rhodotorula Species 0.000 claims description 3
- -1 calcium fatty acid Chemical class 0.000 claims description 3
- 239000012044 organic layer Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 13
- 150000004665 fatty acids Chemical class 0.000 abstract description 12
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 239000002351 wastewater Substances 0.000 abstract description 4
- 238000003756 stirring Methods 0.000 description 31
- 239000012043 crude product Substances 0.000 description 29
- 239000000243 solution Substances 0.000 description 24
- 238000010438 heat treatment Methods 0.000 description 18
- 239000000047 product Substances 0.000 description 16
- 238000001816 cooling Methods 0.000 description 12
- 239000000284 extract Substances 0.000 description 11
- 239000012071 phase Substances 0.000 description 10
- 239000003814 drug Substances 0.000 description 7
- 238000007670 refining Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 229960002061 ergocalciferol Drugs 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 2
- 235000001892 vitamin D2 Nutrition 0.000 description 2
- 239000011653 vitamin D2 Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 208000005368 osteomalacia Diseases 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 208000007442 rickets Diseases 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for extracting ergosterol by using yeast, which is characterized in that the content of an obtained ergosterol product can reach more than 98%, and the yeast can be recovered in the extraction process for secondary utilization. And simultaneously, fatty acid calcium can be co-produced in the ergosterol production process. The method simplifies the washing and concentrating processes required in the extraction process, greatly reduces the generation of waste water, and has environment-friendly and economic production process.
Description
Technical Field
The invention relates to the technical field of fine chemical engineering, in particular to an ergosterol extraction method.
Background
Ergosterol, also known as ergosterol, is an important component of cell membranes of many microorganisms, and plays an important role in ensuring cell viability, fluidity of cell membranes, integrity of cell membranes, and the like. Ergosterol can enhance the disease resistance of human body, has obvious bacteriostasis and antitumor efficacy, is an important fat-soluble vitamin D2 source, and the vitamin D2 which is a transformation product can be used for promoting the absorption of calcium and phosphorus elements by human body and preventing rickets and osteomalacia, and can also be added into feed as a feed additive to increase the laying rate and the hatching rate of livestock and poultry. Ergosterol is also an important pharmaceutical chemical original family, and can be used for producing sterol drugs such as cortisone and progesterone. The product is insoluble in water, and soluble in organic solvents such as ethanol, benzene, diethyl ether, and chloroform.
Ergosterol is used as an important component of a fungal cell membrane, and has high content in yeast and mushroom, so that the ergosterol in yeast cells is usually separated by taking the fungi such as the yeast or the mushroom as a raw material in an ergosterol extraction process through saponification, and then an ergosterol product is obtained through the working procedures of extraction, washing, crystallization, drying and the like. For example, chen Guo apple et al (patent No. CN 114031662A) saponifies dried yeast, extracts and washes to obtain crude ergosterol, and then recrystallizes and filters to obtain the finished product. The extraction process of Limingtie, etc. (patent No. CN 112390842A) is similar, except that before saponification, the yeast strain is firstly treated by ultrasonic treatment, purified by column chromatography after saponification and extraction, and then recrystallized to obtain the finished product of ergosterol. Lirongjie et al (patent No. CN 103588843A) extract ergosterol from waste mycelium of fermented citric acid by using the principle of the production of byproduct ergosterol in the microbial fermentation process, specifically, the waste mycelium is washed to be neutral and then is subjected to saponification reaction, the filtrate obtained by the reaction is washed to be colorless and then is saponified and filtered again to obtain crude product of ergosterol, and the crude product is purified by recrystallization.
The ergosterol extraction methods obtain the finished product through processes of extraction, washing, purification and the like, only concern about the ergosterol extraction in the whole process, or do not recycle other nutrients in the process yeast, or directly discard the extracted yeast, and use a large amount of water in the cleaning process, so that the methods are not environment-friendly and economical.
Disclosure of Invention
Aiming at the defects of insufficient application of yeast and more waste water in the prior art, the invention realizes the secondary utilization of the yeast and reduces the waste water amount by improving and innovating the process for extracting ergosterol from the yeast.
The method for extracting ergosterol by using yeast comprises the following specific steps:
the first step is as follows: carrying out saponification reaction on a yeast raw material in alkali liquor, and carrying out solid-liquid separation on a product after the reaction to obtain a first liquid phase and a first solid phase:
the second step is that: adding a first solvent into the first solid phase obtained in the first step to extract ergosterol, performing solid-liquid separation to obtain a second liquid phase and a second solid phase, and drying the second solid phase to obtain defatted yeast;
the third step: and (3) carrying out pressure concentration on the second liquid in the second step until the second liquid is dry, adding a second solvent for dissolving, then washing with water, separating liquid, carrying out pressure concentration on an organic layer, adding ethanol for crystallization to obtain an ergosterol crude product, and dissolving and recrystallizing the crude product with ethanol to obtain an ergosterol product.
The yeast is Saccharomyces cerevisiae, candida, torulopsis, rhodotorula or Cryptococcus, etc.
In the first step, the alkali liquor 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 60-100 ℃. The dosage of the calcium chloride is 1/5-1/3 of the weight of the yeast raw material.
The first solvent comprises any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone and acetonitrile; the solvent dosage is 10-20 times of the yeast material volume by mass, the ergosterol extraction temperature is kept between 56-120 ℃, and the reflux is carried out for 1-3 hours.
And in the third step, the reduced pressure concentration temperature is 40-80 ℃, 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.
When the crude product is crystallized, the using amount of the ethanol is 0.5 to 1.5 times of that of the yeast raw material by mass, and the crystallization temperature is 0 to 30 ℃.
Preferably, calcium chloride can be added into 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 fatty acid calcium product.
Preferably, the ratio of yeast to alkali and water in the first step is 1.
Preferably, the second solvent used in the third step is twice the amount of the yeast material on a volume basis.
Preferably, the amount of ethanol used in the recrystallization in the third step is 20 to 50 times of the mass of the crude product.
Preferably, the calcium chloride is added as a solution having a concentration greater than 10%
Further, the ratio of yeast to alkali and water in the first step is 1.
Furthermore, the dosage of the calcium chloride is 1/5-1/4 of the weight of the yeast raw material.
Further, when calcium chloride is added in the form of a solution, the concentration of the solution is more than 10%.
The invention has the following beneficial effects:
1. the method has the advantages that the alkali consumption in the saponification process is less compared with that in the prior art, after the saponification is finished, the defatted yeast and the saponification liquid are firstly separated into solid and liquid, the ergosterol in the defatted yeast is extracted by using alcohol, the defatted yeast is not contacted with toxic reagents, the defatted yeast is recovered and is used for extracting other extracts while the ergosterol is extracted, and all components of yeast cells are fully utilized.
2. After the ergosterol is extracted by the degreased yeast through an organic solvent, a relatively pure crude product of the ergosterol can be obtained only by washing and concentrating a small amount of water, and finally a finished product is obtained through refining, wherein the content of the product can reach more than 98%. Because the water consumption in the washing process is greatly reduced, the invention solves the environmental protection problem of large waste water amount to a great extent.
3. The saponification liquid separated from the yeast can further co-produce fatty acid calcium with economic value.
Detailed Description
The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description. The invention relates to a method for extracting ergosterol by using yeast, which comprises the following specific steps:
the first step is as follows: carrying out saponification reaction on a yeast raw material in alkali liquor, and carrying out solid-liquid separation on a product after the reaction to obtain a first liquid phase and a first solid phase;
the second step is that: adding a first solvent into the first solid phase obtained in the first step to extract ergosterol, performing solid-liquid separation to obtain a second liquid phase and a second solid phase, and drying the second solid phase to obtain defatted yeast;
the third step: and (3) carrying out pressure concentration on the second liquid in the second step until the second liquid is dry, adding a second solvent for dissolving, then washing with water, separating liquid, carrying out pressure concentration on an organic layer, adding ethanol for crystallization to obtain an ergosterol crude product, and dissolving and recrystallizing the crude product with ethanol to obtain an ergosterol product.
The yeast includes but is not limited to saccharomyces cerevisiae, candida, torulopsis, rhodotorula or cryptococcus, and the like.
In the first step, the alkali liquor 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 60-100 ℃. The dosage of the calcium chloride is 1/5-1/3 of the weight of the yeast raw material.
The first solvent comprises any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone and acetonitrile; according to the mass, the dosage of the solvent is 10 to 20 times of the volume of the yeast raw material, the extraction temperature of the ergosterol is kept between 56 and 120 ℃, and the reflux is carried out for 1 to 3 hours.
And in the third step, the reduced pressure concentration temperature is 40-80 ℃, 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.
When the crude product is crystallized, the using amount of the ethanol is 0.5 to 1.5 times of that of the yeast raw material by mass, and the crystallization temperature is 0 to 30 ℃.
Preferably, calcium chloride can be added into the first liquid phase obtained in the first step at the same time, and after precipitation and solid-liquid separation, a third solid phase can be obtained as a fatty acid calcium product.
Preferably, the ratio of yeast to alkali and water in the first step is 1 by mass; 0.1 to 0.5;5 to 15.
Preferably, the second solvent is used in the third step in an amount twice the amount of the yeast raw material by volume.
Preferably, the amount of ethanol used in the recrystallization in the third step is 20 to 5 times of the mass of the crude product.
Further, the ratio of yeast to alkali and water in the first step is 1.
Furthermore, the dosage of the calcium chloride is 1/5-1/4 of the weight of the yeast raw material.
Further, calcium chloride is added as a solution with a concentration of greater than 10%.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The dry yeast is purchased on the Internet, and the purchase links are as follows:https:// fubonshop.angelyeast.com/goods/77。
the sources and equipment types of the ingredients used in the preparation process are specifically shown in the following tables 1 and 2:
TABLE 1 sources of ingredients
| Name of medicine | Type/purity | Dealer |
| Sodium ethoxide | AR | Chemical reagent for national medicine group |
| Calcium chloride | AR | Chemical reagent for national medicine group |
| Potassium hydroxide | AR | Chemical reagent for national medicine group |
| Sodium hydroxide | AR | Chemical reagent for national medicine group |
| Ethanol | Food grade | Yichang City Tiansheng trade Co Ltd |
| N-hexane | AR | Xilong Chemical Co., Ltd. |
| Toluene | AR | Xilong Chemical Co., Ltd. |
| Ethyl acetate | AR | Xilong Chemical Co., Ltd. |
| Dry yeast | Fubang feed high activity dry yeast (Kelibejijian) | Angel fermentStock Limited |
| Acetone (II) | AR | Xilong Chemical Co., Ltd. |
| Isopropyl alcohol | AR | Xilong Chemical Co., Ltd. |
TABLE 2 device information
Example 1
Adding 5kg of water and 0.1kg of sodium ethoxide into 1kg of dry yeast, heating to 100 ℃ under stirring, keeping the temperature and stirring for 5 hours, carrying out solid-liquid separation, adding 2kg of 10% calcium chloride solution into the solution under stirring, fully stirring, carrying out solid-liquid separation, and drying the solid at 105 ℃ to obtain 67.8g of fatty acid calcium. Adding 15kg of ethanol into the solid obtained by separation after saponification, heating and refluxing for 2 hours, carrying out solid-liquid separation, drying the solid to obtain 918.5g of defatted yeast, concentrating the extracting solution at 50 ℃ to dry, adding 2L of ethyl acetate to dissolve the residue, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the ethyl acetate phase to dry, adding 0.5kg of ethanol, heating to reflux and dissolving completely, naturally cooling to room temperature, further cooling to 15 ℃, carrying out heat preservation and crystallization for 2 hours, filtering to obtain 11.5g of crude product, refining the crude product by 460ml of ethanol according to the crude product crystallization process, and drying the solid at 80 ℃ to obtain 5.8g of crystals with the purity of 98.6 percent and the total extraction yield of 52.7 percent.
Example 2
Adding 15kg of water and 0.2kg of potassium hydroxide into 1kg of dry yeast, heating to 80 ℃ under stirring, keeping the temperature and stirring for 8 hours, carrying out solid-liquid separation, adding 200g of calcium chloride into the solution by times under stirring, carrying out solid-liquid separation after full stirring, and drying the solid at 105 ℃ to obtain 68.8g of fatty acid calcium. Adding 10kg of acetone into the solid obtained by separation after saponification, heating and refluxing for 1 hour, performing solid-liquid separation, drying the solid to obtain 915.2g of defatted yeast, concentrating the extracting solution at 40 ℃ until the extract is dry, adding 2L of toluene to dissolve residues, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the toluene phase until the toluene phase is dry, adding 1kg of ethanol, heating until the extract is completely dissolved by refluxing, naturally cooling to room temperature, further cooling to 10 ℃, performing heat preservation and crystallization for 2 hours, filtering to obtain 12.6g of crude product, refining the crude product by 460ml of ethanol according to the crude product crystallization process, and drying the solid at 80 ℃ to obtain 6.3g of crystals, wherein the purity is 98.7%, and the total extraction yield is 57.3%.
Example 3
Adding 15kg of water and 0.5kg of sodium hydroxide into 1kg of dry yeast, heating to 60 ℃ under stirring, keeping the temperature and stirring for 12 hours, carrying out solid-liquid separation, adding a saturated solution containing 200g of calcium chloride into the solution under stirring, carrying out solid-liquid separation after full stirring, and drying the solid at 105 ℃ to obtain 67.1g of fatty acid calcium. Adding 20kg of isopropanol into the solid obtained by separation after saponification, heating and refluxing for 1 hour, carrying out solid-liquid separation, drying the solid to obtain 920.1g of defatted yeast, concentrating the extracting solution at 50 ℃ until the extract is dry, adding 2L of n-hexane to dissolve residues, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the n-hexane phase until the n-hexane phase is dry, adding 1kg of ethanol, heating until the extract is completely dissolved by refluxing, naturally cooling to room temperature, further cooling to 10 ℃, carrying out heat preservation and crystallization for 2 hours, filtering to obtain 10.9g of crude product, refining the crude product by 380ml of ethanol according to the crude product crystallization process, and drying the solid at 80 ℃ to obtain 5.6g of crystals, wherein the purity is 98.2 percent and the total extraction yield is 50.9 percent.
Example 4
Adding 10kg of water and 0.3kg of potassium hydroxide into 1kg of dry yeast, heating to 100 ℃ under stirring, keeping the temperature and stirring for 8 hours, carrying out solid-liquid separation, adding 1kg of 20% calcium chloride solution into the solution under stirring, stirring the solution to flow downwards, carrying out solid-liquid separation after full stirring, and drying the solid at 105 ℃ to obtain 71.8g of fatty acid calcium. Adding 15kg of ethanol into the solid obtained by separation after saponification, heating and refluxing for 2 hours, carrying out solid-liquid separation, drying the solid to obtain 910.2g of defatted yeast, concentrating the extracting solution at 50 ℃ until the extract is dry, adding 2L of toluene to dissolve residues, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the toluene phase until the toluene phase is dry, adding 0.5kg of ethanol, heating until the reflux and dissolution are complete, naturally cooling to room temperature, further cooling to 5 ℃, carrying out heat preservation and crystallization for 2 hours, filtering to obtain 13.4g of crude product, refining the crude product by using 600ml of ethanol according to the crude product crystallization process, and drying the solid at 80 ℃ to obtain 6.9g of crystals, wherein the purity is 98.7 percent and the total extraction yield is 62.7 percent.
Comparative example 1
Adding 5kg of water and 0.1kg of sodium ethoxide into 1kg of dry yeast, heating to 100 ℃ under stirring, keeping the temperature and stirring for 5 hours, carrying out solid-liquid separation, adding 1.5kg of 10% calcium chloride solution into the solution under stirring, fully stirring, carrying out solid-liquid separation, and drying the solid at 105 ℃ to obtain 50.8g of fatty acid calcium. Adding 15kg of ethanol into the solid obtained by separation after saponification, heating and refluxing for 2 hours, carrying out solid-liquid separation, drying the solid to obtain 918.5g of defatted yeast, concentrating the extracting solution at 50 ℃ until the extract is dry, adding 2L of ethyl acetate to dissolve residues, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the ethyl acetate phase until the extract is dry, adding 0.5kg of ethanol, heating until the extract is completely dissolved by refluxing, naturally cooling to room temperature, further cooling to 15 ℃, carrying out heat preservation and crystallization for 2 hours, filtering to obtain 11.5g of crude product, refining the crude product by 460ml of ethanol according to the crude product crystallization process, drying the solid at 80 ℃ to obtain 5.8g of crystals, wherein the purity is 98.6%, and the total extraction yield is 52.7%.
Comparative example 2
Adding 3kg of water and 0.08kg of sodium ethoxide into 1kg of dry yeast, heating to 55 ℃ under stirring, keeping the temperature and stirring for 4 hours, carrying out solid-liquid separation, adding 2kg of 10% calcium chloride solution into the solution under stirring, fully stirring, carrying out solid-liquid separation, and drying the solid at 105 ℃ to obtain 43.3g of fatty acid calcium. Adding 15kg of ethanol into the solid obtained by separation after saponification, heating and refluxing for 2 hours, carrying out solid-liquid separation, drying the solid to obtain 932.8g of defatted yeast, concentrating the extracting solution at 50 ℃ until the extracting solution is dry, adding 2L of ethyl acetate to dissolve residues, adding 2L of water, stirring uniformly, standing for layering, separating liquid, concentrating the ethyl acetate phase until the ethyl acetate phase is dry, adding 0.5kg of ethanol, heating until the reflux and dissolution are complete, naturally cooling to room temperature, further cooling to 15 ℃, carrying out heat preservation and crystallization for 2 hours, filtering to obtain 11.5g of crude product, refining the crude product by 460ml of ethanol according to the crystallization process of the crude product, and drying the solid at 80 ℃ to obtain 3.4g of crystals, wherein the purity is 96.6 percent, and the total extraction yield is 30.9 percent.
As can be seen from the above embodiments, in examples 1 to 4, the yeast: alkali: the ratios of water are 1;0.1 to 0.5; 5-15, and the final ergosterol obtained is only 3.4g, much less than 5.8g, 6.3g, 5.6g and 6.9g obtained in examples 1-4, where the ratio of yeast to base and water is within the range described in the present invention. Also, in this case, although the ratio of calcium chloride to dry yeast was 1/5 in both the examples and comparative examples, the solution described in comparative example 2 yielded only 43.3g of calcium fatty acid, which was significantly less than the 67.8g, 68.8g, 67.1g and 71.8g obtained in the example solution. Compared with the comparative example 1, the proportion of yeast, alkali and water in the saponification reaction process is the same, the addition amount of calcium chloride in the example 2 is 1/5 of the mass of the yeast, the addition amount of calcium chloride in the comparative example 1 is 15% and is less than 1/5 of the mass of the yeast, and the obtained fatty acid calcium is only 50.8g and is obviously less than that in the example 2. In all examples, the amount of water used in the washing process after the second solvent has dissolved ergosterol is comparable to the amount of solvent, only twice the weight of the dry yeast.
According to the scheme for extracting ergosterol by using yeast, the consumption of alkali and water is low, the obtained ergosterol product is high in purity, the yeast is recovered, and the fatty acid calcium is co-produced, so that the yeast raw material is utilized as much as possible, the consumption of medicines and water is saved, the production scale is enlarged, and the generated economic and environmental benefits are not insignificant.
The above-mentioned embodiments are only some embodiments of the technical solutions of the present invention, and therefore should not be understood as limiting the present invention in any way, and all modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention are required to be included in the protection scope of the present invention.
Claims (8)
1. A method for extracting ergosterol by using yeast specifically comprises the following steps:
the first step is as follows: the yeast raw material is subjected to saponification reaction in alkali liquor, and after the saponification reaction, the product is subjected to solid-liquid separation to obtain a first liquid phase and a first solid phase;
the second step: adding a first solvent into the first solid phase obtained in the first step to extract ergosterol, performing solid-liquid separation to obtain a second liquid phase and a second solid phase, and drying the second solid phase to obtain defatted yeast;
the third step: and (3) carrying out vacuum concentration on the second liquid in the second step until the second liquid is dry, adding a second solvent for dissolving, then washing with water, separating liquid, carrying out vacuum concentration on an organic layer, and adding ethanol for crystallization to obtain ergosterol.
2. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae, candida, torulopsis, rhodotorula, or Cryptococcus; preferably, the ergosterol obtained is dissolved in ethanol and recrystallized to obtain the ergosterol product.
3. The process according to any one of claims 1 or 2, characterized in that the lye in the first step comprises one or more of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium ethoxide and sodium methoxide in water; preferably, the ratio of yeast to alkaline substance and water is 1.1 to 0.5, preferably 1;
more preferably the saponification time is 4 to 12 hours; and/or the saponification temperature is 60 to 100 ℃, preferably 60 to 80 ℃.
4. A process according to any one of claims 1-3, characterized in that calcium chloride is added to the first liquid phase obtained in the first step to precipitate calcium fatty acid, and after solid-liquid separation a third solid phase is obtained as calcium fatty acid product.
5. The method according to claim 4, wherein the amount of calcium chloride is 1/5 to 1/3, preferably 1/5 to 1/4; the calcium chloride can be added in the form of solid or solution, and the concentration of the solution is more than 10%.
6. The method according to any one of claims 1 to 5, wherein the first solvent in the second step comprises any one or more of ethanol, n-propanol, isopropanol, n-butanol, acetone, acetonitrile; preferably, the amount of the solvent is 10 to 20 times the volume of the yeast raw material by mass; preferably, the ergosterol extraction temperature is maintained between 56-120 deg.C, and reflux is carried out for 1-3 hours.
7. The method according to any one of claims 1 to 6, wherein the reduced pressure concentration temperature is 40 to 80 ℃, and/or the second solvent is any one or more of n-hexane, toluene, ethyl acetate, petroleum ether, cyclohexane, methyl tert-butyl ether, dichloromethane and chloroform, and preferably the amount of the solvent is twice the volume of the yeast raw material.
8. The method according to any one of claims 1 to 7, wherein ethanol is used in an amount of 0.5 to 1.5 times by mass of the yeast raw material during the crystallization, preferably at a temperature of 0 to 30 ℃; more preferably, the amount of ethanol used in recrystallization is 20 to 50 times the mass of the crude ergosterol product.
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| CN116970018A (en) * | 2023-07-27 | 2023-10-31 | 淮南市健坤制药股份有限公司 | Ergosterol preparation and extraction method |
| CN118085001A (en) * | 2024-01-19 | 2024-05-28 | 成都亚中生物制药有限责任公司 | Method for extracting ergosterol from yeast cell wall and application thereof |
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| CN118085001A (en) * | 2024-01-19 | 2024-05-28 | 成都亚中生物制药有限责任公司 | Method for extracting ergosterol from yeast cell wall and application thereof |
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