CN110372769B - Method for extracting steroid product from steroid fermentation liquor - Google Patents
Method for extracting steroid product from steroid fermentation liquor Download PDFInfo
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- CN110372769B CN110372769B CN201811653139.0A CN201811653139A CN110372769B CN 110372769 B CN110372769 B CN 110372769B CN 201811653139 A CN201811653139 A CN 201811653139A CN 110372769 B CN110372769 B CN 110372769B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0003—Androstane derivatives
- C07J1/0011—Androstane derivatives substituted in position 17 by a keto group
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
- C07J5/0007—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa
- C07J5/0015—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa not substituted in position 16
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Steroid Compounds (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention relates to a new method for extracting steroids after fermentation, which comprises the following process steps: heating and inactivating the fermentation liquor; measuring the initial membrane water flux, performing membrane filtration on the fermentation liquor to collect trapped fluid, then performing membrane cleaning, and washing to the initial membrane water flux; extracting the trapped fluid with methanol, layering to obtain methanol extract of steroid product, concentrating to viscous state, adding water, dispersing, and filtering to obtain crude product; heating the crude product with the first solvent, pulping, cooling, filtering to obtain the first extract, dissolving the first extract with the second solvent, decolorizing, and recrystallizing to obtain the final product. The invention has reasonable design, simple process, strong operability and small toxicity of the working environment, not only avoids the phenomenon of difficult separation of emulsification of the traditional process total extraction method, but also provides a novel separation method, avoids the use of toxic extraction organic solvent and solves the problem of wastewater treatment. The method has low cost and high yield, and the final product meets the market quality requirement.
Description
Technical Field
The invention belongs to the technical field of extraction processes after fermentation of steroid series products, and particularly relates to a method for effectively separating a water phase containing a culture medium from an oil phase containing bacteria, oil and steroid products by a membrane separation technology, which solves the problem of difficult oil-water separation in the field of industry and simultaneously facilitates the treatment of wastewater.
Background
Steroids are a group of natural products that are widely found in animals and plants and play an important role in life, such as sterols, vitamin D, bile acids, many sex hormones, adrenocortical hormones, certain carcinogenic hydrocarbons, steroidal saponins, and steroidal alkaloids. The steroid character is represented by the structural characteristics of steroid molecules, the lower part of the steroid character is represented by 'Tian' character which represents A, B, C, D four fused rings, and the upper part of the steroid character is represented by < > which represents three side chains are connected. The general formula shows the general structure of steroid compound, the steroid is mainly composed of A, B, C three six-membered rings and one five-membered ring D, and forms rigid steroid skeleton. Wherein, the R1 and R2 positions are usually methyl, hydroxyl, aldehyde group and the like, and the R3 position is usually a structure of some carbon chains or a substituent containing nitrogen and oxygen.
Due to the difference of side chains and the stereochemical difference of substituents such as double bonds and hydroxyl groups, the structural diversity and specific biological functions of the side chains are determined, and great interest is brought to the research of the chemical compounds. The steroid compound is also an important basic raw material in the pharmaceutical industry, and is synthesized by adopting a semisynthesis method through structural modification to produce various steroid medicines.
4AD, BA, 9 alpha OH-AD and the like are mainly used for synthesizing a plurality of steroid drugs such as sex hormone, progestogen, corticoid and the like, and are the most important upstream intermediates of the steroid drugs in the market at present. The structure is as follows:
the ceramic membrane is a precise ceramic filtering material which is formed by sintering alumina, zirconia and the like at high temperature and has a porous structure, a porous supporting layer, a filtering layer and a micro-filtering membrane layer are asymmetrically distributed, and the inorganic ceramic membrane established on the basis of inorganic material science has some advantages which are incomparable with traditional separation media such as plate frames, centrifuges, diatomite and the like and polymer membranes:
high temperature resistance, organic solvent resistance;
the separation precision is high, and the permeation liquid is clear and transparent, so that the subsequent treatment difficulty is greatly reduced;
the error of the pore size distribution is small, and the diameters of the pores are uniform;
higher filtration accuracy, higher porosity;
the chemical stability is excellent, and the acid resistance, the alkali resistance and the oxidation resistance can be realized;
high mechanical strength and good wear resistance;
easy to clean, can be sterilized on-line with chemicals or at high temperature, and can be backwashed.
The ceramic membrane separation technology is a substance separation technology based on the sieving effect of a porous ceramic medium, and adopts a dynamic cross-flow filtration mode which is completely different from the traditional filtration modes such as 'dead-end filtration' and 'filter cake filtration': under the drive of pressure, the raw material liquid flows at a high speed at a certain flow speed on the surface of the membrane layer on the inner side of the membrane tube, small molecular substances (liquid) penetrate through the microporous membrane along the direction vertical to the raw material liquid, and macromolecular substances (or solid particles) are intercepted by the membrane, so that the purposes of separating, concentrating and purifying the fluid are achieved.
In the extraction of the steroid series oil after fermentation, because a fermentation system mainly comprises thalli, oil, a steroid product, a culture medium, water and the like, how to effectively separate a water phase containing the culture medium from an oil phase containing the thalli, the oil and the steroid product is a key technology of a post-extraction process. With the increasing development of fermentation methods in steroid industries, the market demand of upstream intermediates such as 4AD, BA, 9 alpha OH-AD and the like is larger and larger, and the intermediates are basically obtained by oil fermentation, so how to effectively and quickly separate and purify the intermediate products meeting the market requirements is required by the market and is an important direction for the technical development of the industries.
At present, the oil-water separation process of the products in the market which are produced in a large scale mainly comprises the following steps:
(1) standing and layering: the fermentation liquor is firstly kept stand for layering, most of oil layers are separated, the lower layer is an emulsification system of water layers, thalli and products, the lower layer is settled to obtain a mixture of thalli and products, and the mixture is combined with the former oil layers and then further purified. The method has the defects of long standing time, time consumption of lower-layer sedimentation and incomplete product sedimentation, so that the yield is low, and the final wastewater contains oil and products.
(2) And (3) full extraction: some fermentation liquid can not be layered after standing, so that oil layer is difficult to obtain, and the fermentation liquid is extracted for several times with ethyl acetate or chloroform type water insoluble solvent, and the final extracted liquid is concentrated to recover solvent for further purification. The method has the defects that the extracted wastewater contains organic solvents, a biochemical wastewater system is difficult to treat, and emulsion layers are more, and some of the emulsion layers even cannot be layered at all. Therefore, the process is unstable and the problem of wastewater treatment is serious.
(3) And (3) distillation: some adopt the method of distillation, heat directly and evaporate the water in the fermented solution, the concentrate goes on further purification finally, this method has taken away water, but the distillation energy consumption is high, and it is only water and some volatile substances that take away, other water-soluble nonvolatile substances are still with the oil phase together, have proposed the higher requirement to the further purification craft of the follow-up. Therefore, the method has high energy consumption and cost and complicated subsequent purification process.
Disclosure of Invention
Aiming at the problems in the prior production technology, the invention effectively separates the water phase containing the culture medium and the oil phase containing the bacteria, the oil and the steroid products by a membrane separation technology, has simple operation, avoids the problems of difficult oil-water layering and difficult emulsification separation, has low energy consumption, and can directly treat the generated wastewater in a biochemical system.
The method for extracting the steroid product from the steroid fermentation broth is characterized by comprising the following process steps:
(1) heating the steroid fermentation liquor with the oil content of 2-25% to 50-80 ℃, maintaining for 1-3 h for inactivation, and cooling to room temperature;
(2) recording the water flux before membrane filtration of the feed liquid, performing membrane filtration on the inactivated fermentation liquid, stopping filtration when the filtrate amount is 3/4-9/10 of the fermentation liquid amount, collecting the final trapped liquid, performing membrane cleaning, and washing until the water flux recorded before the feed liquid is filtered;
(3) adding 2-4 times of methanol for extraction according to the amount of trapped fluid (hereinafter referred to as oil phase), stirring for 0.5-3 h at 20-70 ℃, standing for 0.5-3 h, separating out an upper methanol phase, continuously adding 2-4 times of methanol into a lower oil phase according to the same method, extracting for 2-5 times, combining methanol extract, concentrating to be viscous, adding water for dispersion, filtering, and drying to obtain a crude product;
(4) adding 2-8 times of the first solvent according to the amount of the crude product, heating to 50-80 ℃, and pulping for 1-4 hours. Cooling to room temperature, filtering, and oven drying to obtain extract;
(5) adding 2-10 times of the second dissolving according to the amount of the first extract, heating to 50-80 ℃, adding 0.2-2% (w/v relative to the dissolving solution) of activated carbon, decoloring for 1-4 h, filtering to remove the activated carbon, concentrating the filtrate, namely the decoloring solution, 1-3 times, cooling to room temperature, filtering, and drying to obtain the finished product.
The method for extracting the steroid product from the steroid fermentation liquor is characterized in that in the step (1), the oil content of the steroid fermentation liquor is 2-25%, the inactivation temperature is 50-80 ℃, the inactivation time is 1-3 h, and the steroid product can be 4AD (androstenedione, CAS number 63-05-8), BA (21-hydroxy-20-methylpregna-4-en-3-one, CAS number 60966-36-1), 9 alpha OH-AD (9-hydroxy androstenedione or CAS number 560-62-3) and the like.
The method for extracting the steroid product from the steroid fermentation liquor is characterized in that water flux needs to be recorded before membrane filtration of feed liquid in the step (2), the amount of permeate liquid after membrane filtration is 3/4-9/10 of the amount of fermentation liquor, and the membrane can be a ceramic membrane with the pore diameter of 50nm, a ceramic membrane with the pore diameter of 200nm and the like.
The method for extracting the steroid product from the steroid fermentation liquor is characterized in that the amount of the extracted methanol in the step (3) is 2-4 times of the amount of the membrane interception liquid in the previous step, the extraction temperature is 20-70 ℃, the mixture is stirred for 0.5-3 h, the mixture is kept stand for 0.5-3 h, and the extraction is carried out for 2-5 times.
The method for extracting the steroid product from the steroid fermentation liquor is characterized in that in the step (4), the dosage of the first solvent is 2-8 times of the amount of the crude product, the pulping temperature is 50-80 ℃, and the time is 1-4 hours; the first solvent is selected from one or more of n-hexane, n-heptane, cyclohexane, petroleum ether and toluene.
The method for extracting the steroid product from the steroid fermentation liquor is characterized in that in the step (5), the dosage of the second solvent is 2-10 times of the first precise amount, the dosage of the active carbon is 0.2-2% (relative to w/v of the solution), the decoloring temperature is 50-80 ℃, the decoloring time is 1-4 hours, and the concentration multiple of the filtrate is 1-3 times; the second solvent is selected from one or more of methanol, ethanol, isopropanol, acetone and ethyl acetate.
The invention has reasonable design, simple process, strong operability and small toxicity of the working environment, avoids the phenomenon of difficult separation of emulsification in the traditional process total extraction method, and has low energy consumption and effective separation of water-soluble impurities compared with a distillation method. Moreover, the method is a novel separation method in the industry, avoids the use of toxic extraction organic solvents, and solves the problem of wastewater treatment. The method has low cost and high yield, and the final product also meets the market quality requirement.
Detailed description of the invention
The invention is further illustrated by the following specific examples.
The contents of steroid products in steroid series oil fermentation liquor and post-extraction materials are detected by HPLC, and the yield is calculated by the ratio of the yield of qualified products to the amount of fermentation substrate sterol.
Example 1
35L of 4AD fermentation broth (4.8L of oil, 1.75kg of added amount of substrate sterol) was inactivated at 80 ℃ for 1 hour, and then cooled to room temperature. Recording the initial water flux of a membrane (ceramic membrane: with the aperture of 50nm, the filtration area of 0.12 square meter, which accords with the technical standard of the ocean industry) of 500L/h, carrying out membrane filtration on the inactivated fermentation liquor, stopping filtration after receiving 27L of permeate liquid, collecting about 8L of trapped liquid (namely oil phase), and cleaning the membrane by using a medicament until the water flux is 400-500L/h.
Extracting 8L of trapped solution with 16L of methanol at 60 deg.C for 4 times, mixing the methanol extracts, concentrating to viscous state, dispersing with 7L of water, filtering, and oven drying the filter cake to obtain 1.5kg of crude 4AD product.
1.5kg of 4AD crude product is added with 5.5L of n-hexane, heated to 60 ℃, stirred for 2h, cooled to room temperature, filtered, and the filter cake is dried to obtain 1kg of 4AD primary extract.
Adding 9L methanol into 1kg of 4AD, heating to 60 deg.C for dissolving, adding 30g of activated carbon, decolorizing for 1h, filtering, concentrating the filtrate to 4.5L, cooling to room temperature, filtering, and oven drying the filter cake to obtain 0.88kg of 4AD finished product (purity 99.5%, content 98.7%), yield 50.28%.
Example 2
35LBA fermentation broth (4.8L oil, 1.75kg added substrate sterol) was inactivated at 80 deg.C for 1h, and then cooled to room temperature. Recording the initial water flux of a membrane (ceramic membrane: with the aperture of 50nm, the filtration area of 0.12 square meter, which accords with the technical standard of the ocean industry) of 500L/h, carrying out membrane filtration on the inactivated fermentation liquor, stopping filtration after receiving 27L of permeate liquid, collecting about 8L of trapped liquid (namely oil phase), and cleaning the membrane by using a medicament until the water flux is 400-500L/h.
Extracting 8L of trapped solution with 16L of methanol at 60 deg.C for 4 times, mixing the methanol extracts, concentrating to viscous state, dispersing with 7L of water, filtering, and oven drying the filter cake to obtain 1.3kg BA crude product.
Adding 5.5L n-heptane into 1.3kg of BA crude product, heating to 60 ℃, stirring for 2h, cooling to room temperature, filtering, and drying a filter cake to obtain 0.9kg of BA essence.
Adding 9L methanol into 0.9kg BA, heating to 60 deg.C to dissolve, adding 30g active carbon, decolorizing for 1h, filtering, concentrating the filtrate to 4.5L, cooling to room temperature, filtering, and oven drying the filter cake to obtain 0.8kg BA product (purity 99.5%, content 98.9%), yield 45.71%.
Example 3
35L of 9 alpha OH-AD fermentation broth (4.8L of oil, 1.75kg of substrate sterol added) was inactivated at 80 ℃ for 1h and then cooled to room temperature. Recording the initial water flux of a membrane (ceramic membrane: with the aperture of 50nm, the filtration area of 0.12 square meter, which accords with the technical standard of the ocean industry) of 500L/h, carrying out membrane filtration on the inactivated fermentation liquor, stopping filtration after receiving 27L of permeate liquid, collecting about 8L of trapped liquid (namely oil phase), and cleaning the membrane by using a medicament until the water flux is 400-500L/h.
Extracting 8L of trapped solution with 16L of methanol at 60 ℃ for 4 times, combining methanol extract liquid, concentrating to be viscous, adding 7L of water for dispersing, filtering, and drying filter cake to obtain 1.2kg of 9 alpha OH-AD crude product.
1.3kg of 9 alpha OH-AD crude product is added with 7L of toluene, heated to 60 ℃, stirred for 2h, cooled to room temperature, filtered, and the filter cake is dried to obtain 1kg of 9 alpha OH-AD primary extract.
1kg of 9 alpha OH-AD I is added with 9L of ethanol and heated to 60 ℃ for dissolution, 30g of activated carbon is added for decoloration for 1h, filtration is carried out, the filtrate is concentrated to 4.5L, the temperature is reduced to room temperature, filtration and filter cake drying are carried out, thus obtaining 0.85kg of 9 alpha OH-AD finished product (with the purity of 99.5 percent and the content of 98 percent) and the yield of 48.57 percent.
Claims (1)
1. A method for extracting steroid products from steroid fermentation liquor is characterized by comprising the following process steps:
(1) heating the steroid fermentation liquor with the oil content of 2-25% to 50-80 ℃, maintaining for 1-3 h for inactivation, and cooling to room temperature;
(2) recording the water flux before membrane filtration of the feed liquid, performing membrane filtration on the inactivated fermentation liquid, stopping filtration when the filtrate amount is 3/4-9/10 of the fermentation liquid amount, collecting the final trapped liquid, performing membrane cleaning, and washing until the water flux recorded before the feed liquid is filtered;
(3) adding 2-4 times of methanol for extraction according to the amount of trapped liquid, namely an oil phase, stirring for 0.5-3 h at 20-70 ℃, standing for 0.5-3 h, separating out an upper methanol phase, continuously adding 2-4 times of methanol into a lower oil phase according to the same method, extracting for 2-5 times, combining methanol extract liquor, concentrating to be viscous, adding water for dispersion, filtering and drying to obtain a crude product;
(4) adding 2-8 times of a first solvent according to the amount of the crude product, heating to 50-80 ℃, pulping for 1-4 h, cooling to room temperature, filtering, and drying to obtain a first extract; the first solvent is selected from one or more of n-hexane, n-heptane, cyclohexane, petroleum ether and toluene;
(5) adding 2-10 times of a second solvent according to the amount of the first solvent, heating to 50-80 ℃, adding 0.2-2% of activated carbon relative to w/v of the solution, decoloring for 1-4 h, filtering to remove the activated carbon, concentrating the filtrate, namely the decolored solution, 1-3 times, cooling to room temperature, filtering, and drying to obtain a finished product; the second solvent is selected from one or more of methanol, ethanol, isopropanol, acetone and ethyl acetate;
wherein the steroid product is androstenedione, 21-hydroxy-20-methylpregna-4-en-3-one or 9-hydroxyandrostenedione;
the membrane is a ceramic membrane with the pore diameter of 50 nm.
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