CN102363796A - Method for producing glycyrrhetinic acid through microbial fermentation transformation - Google Patents
Method for producing glycyrrhetinic acid through microbial fermentation transformation Download PDFInfo
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Abstract
The invention provides a method for producing glycyrrhetinic acid through microbial fermentation transformation. The method comprises the following steps of: inoculating aspergillus flavus CGMCC No 5144 into a solid potato dextrose agar (PDA) slant culture medium to cultivate; inoculating 1 percent of the cultivated aspergillus flavus CGMCC No 5144 to a culture medium containing fresh seeds from the inclined surface to cultivate; inoculating 30 percent of the strains obtained from the seed culture medium to a fermentation tank containing a fresh fermentation culture medium and performing fermentation culture to obtain fermentation liquid containing the glycyrrhetinic acid; separating a glycyrrhetinic acid coarse product from the fermentation liquid by a membrane separation combined process; and preparing high-purity glycyrrhetinic acid by a purification process. In the glycyrrhetinic acid finally obtained by the method, the fermentation quantity reaches 4.38 g/L, the product purity is more than or equal to 98 percent, the conversion rate is more than or equal to 90 percent, and the technological scale level is reached.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of technology of producing glycyrrhetinic acid with microbial transformation.
Background technology
Radix Glycyrrhizae is China's traditional Chinese medicine material, have the title of " state is old ", and sweet, the property of distinguishing the flavor of is flat.The thoughts of returning home, lung, spleen, stomach.Invigorate the spleen and benefit qi, clearing heat and detoxicating, expelling phlegm for arresting cough, relieving spasm to stop pain, coordinating the actions of various ingredients in a prescription.Be used for weakness of the spleen and the stomach, fatigue and weakness, shortness of breath and palpitation, coughing with a lot of sputum, the anxious pain of gastral cavity abdomen, four limbs contraction, carbuncle sore tumefacting virus.But cushion toxicity, strong.Radix Glycyrrhizae is widely used in fields such as medicine, tobacco, food.Potenlini and glycyrrhetinic acid are the main effective constituent of Radix Glycyrrhizae, Potenlini through the hydrochloric acid in gastric juice hydrolysis or in liver GRD beta-glucuronidase be decomposed to form glycyrrhetinic acid, the pharmacological action of Potenlini comes down to the effectiveness of glycyrrhetinic acid.
The molecular formula of glycyrrhetinic acid (Glycyrrhetinic Acid) is C
30H
46O
4, molecular weight 470.64KD is white crystalline powder, fusing point is 288-297 ℃, structural formula such as Fig. 1.These article are prone to dissolve in pyridine, in ethanol or chloroform, dissolve, and slightly soluble in gasoline or ether, insoluble in water.
In recent years along with the development of modern Chinese herbal medicine, the various countries scholar is on the basis of theory of traditional Chinese medical science, to effective liquorice---and the pharmacological action of glycyrrhetinic acid is furtherd investigate, and is applied to clinical with the new effective constituent of seeking high-efficiency low-toxicity.Discover glycyrrhetinic acid effect such as not only have anti-inflammatory, antiulcer agent, antiviral, reducing blood-fat, antibechic, relieving asthma and eliminate the phlegm; Also can be used for preventing and treating diseases such as viral hepatitis, hyperlipidemia and cancer, is effective interferon inducer and cell immunomodulator.Radix Glycyrrhizae acids medicine and vegeto-alkali, microbiotic, amino acid etc. are composite or process its metallic compound, also can increase stability of drug, improve bioavailability, reduce toxic side effect.Follow the further investigation of glycyrrhetinic acid clinical value, the development and use of glycyrrhetinic acid also more and more receive the concern of domestic and international the world of medicine.
Chemical synthesis is not seen in the preparation of glycyrrhetinic acid.Many at present the extraction through processing with natural glycyrrhizic legume produced, i.e. the plant extract method.The plant extract method is that raw material extracts Potenlini with the Radix Glycyrrhizae, produces glycyrrhetinic acid through acid-hydrolysis method hydrolysis Potenlini again.
Radix Glycyrrhizae is a medicinal plant, mainly is distributed in the arid and semi-arid lands between the north latitude 30-55 degree, and in recent years owing to Ecological environment worsening, the Radix Glycyrrhizae resource is deficient day by day.In addition, be that raw material is produced glycyrrhetinic acid with the Radix Glycyrrhizae, technology is loaded down with trivial details, product yield is low, cost is higher, has seriously restricted the output of glycyrrhetinic acid.Microbe fermentation method is the glycuronidase that utilizes the mikrobe large scale fermentation to be produced, and the Potenlini in the vitro conversion Radix Glycyrrhizae generates the method for glycyrrhetinic acid.Microbial fermentation production does not receive resource restriction; Have the reaction conditions gentleness, the product specificity is good, technology is simple, cost is low, do not cause advantages such as environmental pollution and aftertreatment be simple; Becoming this area research hot of research and development in recent years, also is the best approach that solves current Radix Glycyrrhizae shortage of resources problem.
At present; The research report of external relevant glycyrrhetinic acid microbial transformation is few; The domestic scientific research institutions such as Sichuan University, Beijing University of Chinese Medicine, Hubei University Of Technology that mainly contain carry out correlative study, but all also are in the experimental study stage, do not form the mass-producing level as yet.
The present invention handles the microbial strains that obtains the high yield glycuronidase through ultraviolet ray and Licl complex mutation; Select the Radix Glycyrrhizae leach cooking liquid for cultivating the fermentation of base band slag; Adopt ultrafiltration and nf membrane combined separation technology; Segmental impurity classification is removed to different molecular weight, has improved conversion rate of products, yield and purity greatly, has reached the industrial scale level.
Summary of the invention
The invention provides a kind of microbial fermentation and transform the method for producing glycyrrhetinic acid, it is characterized in that described mikrobe is flavus Aspergillus flavus, CGMCC No.5144.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, may further comprise the steps:
Step 1, flavus (Aspergillus flavus) CGMCC No 5144 inserted in the solid PDA slant mediums cultivate, from the inclined-plane, be seeded to the fresh seeds substratum then and cultivate by 1% inoculum size;
Step 2, will be in the bacterial classification of cultivating through seed culture medium in vegetative period and be seeded to according to 30% inoculum size and carry out fermentation culture in the fermentor tank that contains fresh fermention medium, fermentation produces the fermented liquid that contains glycyrrhetinic acid;
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, after step 2, also comprises step 3: from fermented liquid, isolate the glycyrrhetinic acid bullion then, and carry out purifying.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the quality percentage composition of seed culture medium is in the step 1: corn saccharification liquid 0.5～1.5%; Wheat bran juice 4.5～5.5%, yeast extract paste 0.5～0.7%, ammonium sulfate 0.05～0.15%; Sal epsom 0.04～0.06%; Potassium primary phosphate 0.09～0.11%, glycyrrhizic acid inclusion compound 0.09～0.11%, all the other are water.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the seed culture medium culture condition is in the step 1: bacterial strain places Erlenmeyer flask to cultivate air flow 0.2m in the shaking table jolting of rotating speed 200rpm
3/ h, incubation time 24 hours.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the quality percentage composition of fermention medium is in the step 2: Potenlini 0.8～1.2%; Ammonium sulfate 0.8～1.0%; Sal epsom 0.03～0.05%, potassium hydrogenphosphate 0.6～0.8%, potassium primary phosphate 0.7～0.9%; All the other are water, initial pH4～5.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the fermentation cylinder for fermentation culture condition is in the step 2: fermention medium 50～70L, 20～40 ℃ of leavening temperatures, air flow 0.3～0.5m
3/ h, stirring velocity is 130～170rmp, fermentation 2～4d.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the separation of glycyrrhetinic acid comprises following steps:
Filter through filter cloth, collect filtrating 1, then gained filtrating 1 is passed through ultra-filtration membrane PES-20, dam particle diameter more than 10nm, the material of molecular weight in 500～500000 scopes collected filtrating 2;
Adopt step gained filtrating 2 in the nf membrane NF-270 filtration, the lower-molecular substance of molecular weight about 200～1000 that dam collected filtrating 3.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, and the purifying of glycyrrhetinic acid comprises following steps:
Earlier said filtrating 3 is carried out separation and purification, obtain the glycyrrhetinic acid bullion through macroporous adsorbent resin H103;
Placed horizontally is in the silicagel column surface after then the bullion of gained glycyrrhetinic acid being processed sample glue; Earlier with pure sherwood oil through post; Use sherwood oil again: the each 20 order wash-out glycyrrhetinic acids of the moving phase of ETHYLE ACETATE=2: 1 (volume ratio); Collect filtrating and carry out spraying drying, substep collection, obtain the pure article of glycyrrhetinic acid at last.
Preferably, described microbial fermentation transforms in the method for producing glycyrrhetinic acid, flavus Aspergillus flavus, and CGMCC No.5144 obtains for the Aspergillus flavus of handling glucuronic acid through ultraviolet ray and Licl complex mutation.
The invention provides a kind of microbial transformation and produce the method for glycyrrhetinic acid, may further comprise the steps:
1) in solid PDA slant medium, flavus (Aspergillus flavus) CGMCC No 5144 is cultivated earlier; Getting a ring from the inclined-plane with the inoculation shovel then is seeded to the Erlenmeyer flask that contains seed culture medium; Inoculum size is 1%; Culture condition is: the pH nature, air flow 0.2m is cultivated in jolting in the shaking table of rotating speed 200rpm
3/ h, incubation time 24 hours.
2) cultured bacterial strain in the seed bottle is transferred in the fermentor tank of 100L according to 30% inoculum size, dress 60L is through the fermention medium of sterilization in the fermentor tank, and the adjustment leavening temperature is 30 ℃, air flow 0.4m
3/ h, stirring velocity is 150rmp, fermentation 3d obtains containing the fermented liquid of glycyrrhetinic acid.
3) fermented liquid that above-mentioned gained is contained glycyrrhetinic acid is earlier removed macromolecular substance through filter cloth, collects filtrating 1, then with fermented liquid 1 through ultra-filtration membrane PES-20; Dam particle diameter more than 10nm; The material of molecular weight in 500～500000 scopes collected filtrating 2, adopts nanofiltration NF-270 2 to carry out thickening filtration to filtrating again; The lower-molecular substance of molecular weight about 200～1000 that dam collected filtrating 3.
4) will filtrate and 3 separate with purifying through macroporous adsorbent resin H103 and to obtain crude extract, and measure crude extract 200ml and be concentrated to certain volume and put into china bowl, add 80～100 purpose silica gel 5g; Stir fast; Put to heat on 60 ℃ of water-baths and do not stop to stir, treat promptly to make sample glue after solvent evaporates completely, in silicagel column, add 300～400 purpose silica gel 100g (separation gel); Keep upper surfaces level; Reinstall dry good sample glue, earlier with pure sherwood oil through post, use sherwood oil again: the each 20 order wash-out glycyrrhetinic acids of the moving phase of ETHYLE ACETATE=2: 1 (volume ratio) then; Collect filtrating and carry out spraying drying, substep collection, obtain the pure article of glycyrrhetinic acid at last.
Microbial fermentation provided by the invention transforms the method for producing glycyrrhetinic acid and has following outstanding feature:
(1) adopt ultraviolet ray and Licl complex mutation to handle the screening superior strain.The obtained strains heritability is stable, and secreted glycuronidase ability orientation is the β of enzymolysis Potenlini-glucose glycoside key efficiently, has improved the transformation efficiency of Potenlini greatly;
(2) selecting the Radix Glycyrrhizae leach cooking liquid is substratum; Be not merely growth abundant carbon source, nitrogenous source is provided; And solved raw material Potenlini in the chemical acid hydrolyzation and must pass through complicated process for refining problem, improved raw material availability, reduced discharging of waste liquid and environmental pollution;
(3) adopt ultrafiltration and nf membrane combined separation technology, segmental impurity classification is removed to different molecular weight, has improved product yield and purity, has reduced cost.
The present invention makes the amount of fermentation of glycyrrhetinic acid reach 4.38g/L through optimizing the separation purifying technique of fermentation culture and target product, and conversion rate of products >=90% greatly reduces the industrial production cost of glycyrrhetinic acid.
Description of drawings
The process flow sheet of glycyrrhetinic acid is produced in Fig. 1 microbial transformation provided by the invention.
Embodiment
Through embodiment the present invention is further specified below, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Flavus Aspergillus flavus; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, 100101; Preserving number CGMCC No.5144, preservation date: on 08 16th, 2011.
Embodiment 1:
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), the pH nature, and inoculum size is 1%, and rotating speed is 200rpm, and air flow is 0.2m
3/ h, incubation time are 24 hours.
Above cultivation bacterial strain is transferred (interior dress 60L is through the fermention medium of sterilization) in the fermentor tank of 100L according to 30% inoculum size, and the adjustment leavening temperature is 10 ℃, air flow 0.4m
3/ h, stirring velocity 150rmp, fermentation 3d.
Investigate the output of glycyrrhetinic acid.Detect glycyrrhetinic acid with sulfuric acid Vanillin method:
(1) reference substance solution preparation
Precision takes by weighing 100 ℃ of glycyrrhetinic acid standard substance 20mg that are dried to constant weight, puts in the 20ml measuring bottle, adds absolute ethyl alcohol to scale, and obtaining glycyrrhetinic acid reference liquid content is 1mg/mL.
(2) preparation of typical curve
Precision is measured standardized solution (1mg/mL) 0.1,1.5,2.5; Add 0.5ml ETHYLE ACETATE respectively in test tube 4.5ml volatilize, add 95% ethanol 3ml more successively, 1% Vanillin aqueous solution 0.2ml; Be placed in the frozen water, add 80% sulfuric acid 2ml, mix the test tube jam-pack; Place immediately in 50 ℃ of water-baths and be incubated 10min, put again, be diluted to scale 20ml to room temperature.Get 5ml and on ultraviolet spectrophotometer, scan absorption spectrum, make blank reference with 0.0mg/ml concentration at the 500-600nm wave band.Glycyrrhetinic acid reaction solution maximum absorption band is surveyed the O.D value as the sample determination absorption peak, and its result is the drawing standard curve.
(3) sample is prepared
Radix Glycyrrhizae liquid fermentation production 2.5ml adds the 5ml ethyl acetate extraction successively.Draw ETHYLE ACETATE supernatant 1ml, the 3ml ethanolic soln, 0.2ml1% Vanillin and 2ml80% sulphuric acid soln are diluted to scale 20ml in 50 ℃ of colour developing 10min, measure O.D.
Embodiment 2
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), the pH nature, and inoculum size is 1%, and rotating speed is 200rpm, and air flow is 0.2m
3/ h, incubation time are 24 hours.
Above cultivation bacterial strain is transferred (interior dress 60L is through the fermention medium of sterilization) in the fermentor tank of 100L according to 30% inoculum size, and the adjustment leavening temperature is 20 ℃, air flow 0.4m
3/ h, stirring velocity 150rmp, fermentation 3d, strain fermentation concentration reaches 3.1g/L.
The glycyrrhetinic acid detection method is the same.
Embodiment 3
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), the pH nature, and inoculum size is 1%, and rotating speed is 200rpm, and air flow is 0.2m
3/ h, incubation time are 24 hours.
Above cultivation bacterial strain is transferred (interior dress 60L is through the fermention medium of sterilization) in the fermentor tank of 100L according to 30% inoculum size, and the adjustment leavening temperature is 30 ℃, air flow 0.4m
3/ h, stirring velocity 150rmp, fermentation 3d, strain fermentation concentration reaches 4.2g/L.
The glycyrrhetinic acid detection method is the same.
Embodiment 4
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), the pH nature, and inoculum size is 1%, and rotating speed is 200rpm, and air flow is 0.2m
3/ h, incubation time are 24 hours.
Above cultivation bacterial strain is transferred (interior dress 60L is through the fermention medium of sterilization) in the fermentor tank of 100L according to 30% inoculum size, and the adjustment leavening temperature is 40 ℃, air flow 0.4m
3/ h, stirring velocity 150rmp, fermentation 3d, strain fermentation concentration reaches 3.3g/L.
The glycyrrhetinic acid detection method is the same.
Embodiment 5
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), and the percentage composition of seed culture medium quality is: corn saccharification liquid 0.5%, wheat bran juice 4.5%; Yeast extract paste 0.5%, ammonium sulfate 0.05%, sal epsom 0.04%; Potassium primary phosphate 0.09%, glycyrrhizic acid inclusion compound 0.09%, all the other are water pH nature; Inoculum size is 1%, and rotating speed is 180rpm, and air flow is 0.15m
3/ h, incubation time are 20 hours.
Above cultivation bacterial strain is transferred in the fermentor tank of 100L according to 30% inoculum size, include the 50L fermention medium, the quality percentage composition of fermention medium: be Potenlini 0.8%; Ammonium sulfate 0.8%, sal epsom 0.03%, potassium hydrogenphosphate 0.6%; Potassium primary phosphate 0.7%, all the other are water, initial pH4; The adjustment leavening temperature is 30 ℃, air flow 0.3m
3/ h, stirring velocity 130rmp, fermentation 2d obtains containing the fermented liquid of glycyrrhetinic acid.
The fermented liquid that above-mentioned gained is contained glycyrrhetinic acid is removed macromolecular substance through filter cloth earlier, collects filtrating 1, then fermented liquid 1 is passed through ultra-filtration membrane PES-20; Dam particle diameter more than 10nm; The material of molecular weight in 500～500000 scopes collected filtrating 2, adopts nanofiltration NF-270 2 to carry out thickening filtration to filtrating again; The lower-molecular substance of molecular weight about 200～1000 that dam collected filtrating 3.
To filtrate and 3 separate with purifying through macroporous adsorbent resin H103 and to obtain crude extract, and measure crude extract 200ml and be concentrated to certain volume and put into china bowl, add 80～100 purpose silica gel 5g; Stir fast; Put to heat on 60 ℃ of water-baths and do not stop to stir, treat promptly to make sample glue after solvent evaporates completely, in silicagel column, add 300～400 purpose silica gel 100g (separation gel); Keep upper surfaces level; Reinstall dry good sample glue, earlier with pure sherwood oil through post, use sherwood oil again: the each 20 order wash-out glycyrrhetinic acids of the moving phase of ETHYLE ACETATE=2: 1 (volume ratio) then; Collect filtrating and carry out spraying drying, substep collection, obtain the pure article of glycyrrhetinic acid at last.
Obtain glycyrrhetinic acid product purity >=98% at last, transformation efficiency >=90%.
Embodiment 6
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), and the percentage composition of seed culture medium quality is: corn saccharification liquid 1.0%, wheat bran juice 5.0%; Yeast extract paste 0.6%, ammonium sulfate 0.1%, sal epsom 0.05%; Potassium primary phosphate 0.1%, glycyrrhizic acid inclusion compound 0.1%, all the other are water pH nature; Inoculum size is 1%, and rotating speed is 180rpm, and air flow is 0.15m
3/ h, incubation time are 20 hours.
Above cultivation bacterial strain is transferred in the fermentor tank of 100L according to 30% inoculum size, include the 60L fermention medium, the quality percentage composition of fermention medium: be Potenlini 1.0%; Ammonium sulfate 0.9%, sal epsom 0.04%, potassium hydrogenphosphate 0.7%; Potassium primary phosphate 0.8%, all the other are water, initial pH4.5; The adjustment leavening temperature is 32 ℃, air flow 0.4m
3/ h, stirring velocity 150rmp, fermentation 3d obtains containing the fermented liquid of glycyrrhetinic acid.
The separation of glycyrrhetinic acid and purification step obtain glycyrrhetinic acid product purity >=98% at last with embodiment 5, transformation efficiency >=90%.
Embodiment 7
CGMCC No 5144 is seeded in the Erlenmeyer flask that contains seed culture medium with flavus (Aspergillus flavus), and the percentage composition of seed culture medium quality is: corn saccharification liquid 1.5%, wheat bran juice 5.5%; Yeast extract paste 0.7%, ammonium sulfate 0.15%, sal epsom 0.06%; Potassium primary phosphate 0.11%, glycyrrhizic acid inclusion compound 0.11%, all the other are water pH nature; Inoculum size is 1%, and rotating speed is 180rpm, and air flow is 0.15m
3/ h, incubation time are 20 hours.
Above cultivation bacterial strain is transferred in the fermentor tank of 100L according to 30% inoculum size, include the 70L fermention medium, the quality percentage composition of fermention medium is: Potenlini 1.2%; Ammonium sulfate 1.0%, sal epsom 0.05%, potassium hydrogenphosphate 0.8%; Potassium primary phosphate 0.9%, all the other are water, initial pH5; The adjustment leavening temperature is 30 ℃, air flow 0.5m
3/ h, stirring velocity 170rmp, fermentation 4d obtains containing the fermented liquid of glycyrrhetinic acid.
The separation of glycyrrhetinic acid and purification step obtain glycyrrhetinic acid product purity >=98% at last with embodiment 5, transformation efficiency >=90%.
Embodiment 8
To from the soil on licorice planting ground, gather the Aspergillus flavus Aspergillus flavus that target sample process primary dcreening operation, ultraviolet mutagenesis and the mutagenesis of Licl composite multiple obtain the high yield glucuronic acid, CGMCC No.5144.
Primary dcreening operation is that extracting liquorice ground soil sample adds sterile distilled water, after shaking up, leaving standstill, gets supernatant 1ml gradient dilution, respectively draws 0.1ml then on the primary dcreening operation substratum, cultivating 3d for 30 ℃ by conventional coating process; The quality percentage composition that primary dcreening operation is dull and stereotyped: 90% glycyrrhizic acid inclusion compound 1.0g, sal epsom 0.05g, SODIUMNITRATE 0.5g, ferrous sulfate 0.001g; Potassium primary phosphate 0.1g, agar 1.8g, all the other are water; PH5 picks out the single bacterium colony that grows fine, and preservation is that the primary dcreening operation bacterial classification is for use.The access of primary dcreening operation bacterial classification is sieved in the substratum again, sieve the quality percentage composition of substratum again: 10% Radix Glycyrrhizae leach cooking liquid (w/v), pH nature, 30 ℃, fermentation culture 3d in the shaking table of 150rpm.Fermented liquid is made product TLC and is detected, and the bacterial strain that on thin layer chromatography board, has the glycyrrhetinic acid spot to occur is aimed strain.Get the bacterium colony of aimed strain and on the test tube slant substratum, cultivate, cultivate 1～2d and can obtain slant strains.
Ultraviolet mutagenesis is that slant strains is scraped spore in the sterilized water triangular flask of 50ml gently with transfering loop, firmly shakes the 5min homodisperse, gets the plate that 5ml places Ф 10cm, the 30W uv lamp, and apart from 30cm, irradiation 20s.
Licl mutagenesis is that lucifuge is cultivated 5～7d, mutagenesis substratum: glucose 4% for 25 ℃ with the bacterium liquid dilution coating mutagenesis culture medium flat plate through ultraviolet mutagenesis; Ammonium sulfate 0.4%, potassium primary phosphate 0.15%, sal epsom 0.05%; Yeast extract paste 0.01%, sodium-acetate 0.2%, zinc acetate 6 μ g/ml; Copper sulfate 0.5 μ g/ml, lithium chloride 0.5%, agar 2.0%.
It is emphasized that at last: above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although the present invention is specified with reference to preferred embodiment; Those of ordinary skill in the art is to be understood that; Can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the aim and the scope of present technique scheme, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (10)
1. a microbial fermentation transforms the method for producing glycyrrhetinic acid, it is characterized in that described mikrobe is flavus Aspergillus flavus, CGMCC No.5144.
2. microbial fermentation as claimed in claim 1 transforms the method for producing glycyrrhetinic acid, it is characterized in that, may further comprise the steps:
Step 1, with flavus Aspergillus flavus, CGMCC No.5144 inserts in the solid PDA slant medium and cultivates, and from the inclined-plane, is seeded to the fresh seeds substratum by 1% inoculum size then and cultivates;
Step 2, will be in the bacterial classification of cultivating through seed culture medium in vegetative period and be seeded to according to 30% inoculum size and carry out fermentation culture in the fermentor tank that contains fresh fermention medium, fermentation produces the fermented liquid that contains glycyrrhetinic acid.
3. microbial fermentation as claimed in claim 1 transforms the method for producing glycyrrhetinic acid, it is characterized in that, after step 2, also comprises step 3: from fermented liquid, isolate the glycyrrhetinic acid bullion then, and carry out purifying.
4. microbial fermentation as claimed in claim 2 transforms the method for producing glycyrrhetinic acid, and wherein, the quality percentage composition of seed culture medium is in the step 1: corn saccharification liquid 0.5～1.5%; Wheat bran juice 4.5～5.5%, yeast extract paste 0.5～0.7%, ammonium sulfate 0.05～0.15%; Sal epsom 0.04～0.06%; Potassium primary phosphate 0.09～0.11%, glycyrrhizic acid inclusion compound 0.09～0.11%, all the other are water.
5. microbial fermentation as claimed in claim 4 transforms the method for producing glycyrrhetinic acid, and wherein, the seed culture medium culture condition is in the step 1: bacterial strain is placed into jolting cultivation on the shaking table of rotating speed 200rpm in the Erlenmeyer flask, air flow 0.2m
3/ h, incubation time 24 hours.
6. microbial fermentation as claimed in claim 2 transforms the method for producing glycyrrhetinic acid, wherein, and the quality percentage composition of fermention medium in the step 2: be Potenlini 0.8～1.2%; Ammonium sulfate 0.8～1.0%; Sal epsom 0.03～0.05%, potassium hydrogenphosphate 0.6～0.8%, potassium primary phosphate 0.7～0.9%; All the other are water, initial pH4～5.
7. microbial fermentation as claimed in claim 6 transforms the method for producing glycyrrhetinic acid, and wherein, the fermentation cylinder for fermentation culture condition is in the step 2: fermention medium 50～70L, 20～40 ℃ of leavening temperatures, air flow 0.3～0.5m
3/ h, stirring velocity is 130～170rmp, fermentation 2～4d.
8. microbial fermentation as claimed in claim 3 transforms the method for producing glycyrrhetinic acid, and wherein, the separation of glycyrrhetinic acid comprises following steps:
Filter through filter cloth, collect filtrating 1, then gained filtrating 1 is passed through ultra-filtration membrane PES-20, dam particle diameter more than 10nm, the material of molecular weight in 500～500000 scopes collected filtrating 2;
Adopt step gained filtrating 2 in the nf membrane NF-270 filtration, the lower-molecular substance of molecular weight about 200～1000 that dam collected filtrating 3.
9. microbial fermentation as claimed in claim 3 transforms the method for producing glycyrrhetinic acid, and wherein, the purifying of glycyrrhetinic acid comprises following steps:
Earlier said filtrating 3 is carried out separation and purification, obtain the glycyrrhetinic acid bullion through macroporous adsorbent resin H103;
Placed horizontally is in the silicagel column surface after then the bullion of gained glycyrrhetinic acid being processed sample glue; Earlier with pure sherwood oil through post; Use sherwood oil again: the each 20 order wash-out glycyrrhetinic acids of the moving phase of ETHYLE ACETATE=2: 1 (volume ratio); Collect filtrating and carry out spraying drying, substep collection, obtain the pure article of glycyrrhetinic acid at last.
10. microbial fermentation as claimed in claim 1 transforms the method for producing glycyrrhetinic acid, wherein, flavus Aspergillus flavus, CGMCC No.5144 obtains for the Aspergillus flavus of handling glucuronic acid through ultraviolet ray and Licl complex mutation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660467A (en) * | 2012-05-29 | 2012-09-12 | 黑龙江大学 | Liquorice endophytic fungus for producing glycyrrhetinic acids |
| CN103992953A (en) * | 2014-02-20 | 2014-08-20 | 江西师范大学 | Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide |
| CN104357525A (en) * | 2014-11-04 | 2015-02-18 | 黑龙江大学 | Method for producing glycyrrhetinic acid by using microbial fermentation |
| CN104805169A (en) * | 2014-01-27 | 2015-07-29 | 上海医药工业研究院 | Method for producing glycyrrhetinic acid through microbial transformation, and medium thereof |
| CN107058443A (en) * | 2017-06-07 | 2017-08-18 | 江苏天晟药业股份有限公司 | A kind of preparation method of enoxolone |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660467A (en) * | 2012-05-29 | 2012-09-12 | 黑龙江大学 | Liquorice endophytic fungus for producing glycyrrhetinic acids |
| CN102660467B (en) * | 2012-05-29 | 2013-04-24 | 黑龙江大学 | Liquorice endophytic fungus for producing glycyrrhetinic acids |
| CN104805169A (en) * | 2014-01-27 | 2015-07-29 | 上海医药工业研究院 | Method for producing glycyrrhetinic acid through microbial transformation, and medium thereof |
| CN104805169B (en) * | 2014-01-27 | 2018-04-27 | 上海医药工业研究院 | A kind of method and its culture medium of microorganism conversion production enoxolone |
| CN103992953A (en) * | 2014-02-20 | 2014-08-20 | 江西师范大学 | Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide |
| CN103992953B (en) * | 2014-02-20 | 2016-10-05 | 江西师范大学 | One strain conversion glycyrrhizic acid generates the Dongxiang Wild Rice endogenetic fungus of liquorice enoxolone |
| CN104357525A (en) * | 2014-11-04 | 2015-02-18 | 黑龙江大学 | Method for producing glycyrrhetinic acid by using microbial fermentation |
| CN104357525B (en) * | 2014-11-04 | 2017-06-06 | 黑龙江大学 | A kind of method that utilization microbial fermentation produces enoxolone |
| CN107058443A (en) * | 2017-06-07 | 2017-08-18 | 江苏天晟药业股份有限公司 | A kind of preparation method of enoxolone |
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|---|---|
| CN102363796B (en) | 2013-11-20 |
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