CN106636290A - Method for producing glycyrrhetinic acid monoglucuronide by fermentation, and application - Google Patents
Method for producing glycyrrhetinic acid monoglucuronide by fermentation, and application Download PDFInfo
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Abstract
The invention discloses a method for producing glycyrrhetinic acid monoglucuronide by fermentation, and application, and belongs to the technical field of bioengineering. According to the invention, glycyrrhizinic acid is taken as a carbon source, a talaromyces fungus is introduced to produce beta-glucuronidase, and glycyrrhetinic acid monoglucuronide can be prepared in large scale on the premise of being friendly to the environment. The method is simple in process and low in cost, the GAMG yield reaches 4.05g/L, the productivity is 88.00%, the purity is 80% or higher than 80% after purification by macroporous resin, the product purity is high, no structural alienation occurs, and the method has the advantages of being short in production cycle, easy to operate, low in energy consumption and the like, thereby being suitable for characteristics of later industrial production.
Description
Technical field
The present invention relates to a kind of methods and applications of fermenting and producing Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide, belong to bioengineering skill
Art field.
Background technology
Radix Glycyrrhizae is pulse family glycyrrhiza genus, is traditional Chinese herbal medicine, and its medicinal part is dry root and rhizome.Triterpenes
Compound glycyrrhizic acid is the main pharmacodynamics composition in Radix Glycyrrhizae, is the research emphasis in current Radix Glycyrrhizae direction, Chinese scholars to its
Further investigation shows, glycyrrhizic acid (Glycyrrhizin, GL) has certain anti-inflammatory, liver protection, antiviral, antiallergy, lipid-loweringing, anti-
The effect of the aspect such as atherosclerotic, antitumor, removing toxic substances.
GAMG (Glycyrrhetinic acid monoglucuronide, GAMG), by one
The enoxolone of molecule glucose aldehydic acid and a molecule is formed by connecting by glycosidic bond.Its chemical formula is C36H54O10, molecular weight is
648.8g mol-1, according to the difference of the plane of its H, including two kinds of isomers of 18 β-GAMG and 18 α-GAMG.GAMG containing only
There is a glucuronic acid base, its polarity can smoothly enter cell between glycyrrhizic acid and enoxolone, and lipophilicity is not
Too strong, the solubility and transmembrane transport ability in body is stronger than glycyrrhizic acid and enoxolone.Thus have more preferable biological utilisation
Degree, also can be than glycyrrhizic acid and the more preferable performance drug effect of enoxolone.Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide (GAMG) is used as glycyrrhizic acid
Derivative, both have similar biologically active, its anti-hepatitis, antiallergy, anticancer, anti-virus aspect it is evident in efficacy
Higher than GL.Therefore, developing GAMG as the efficient new drug in terms of anti-hepatitis class, antiallergy, anticancer there is wide exploitation to answer
Use prospect.The LD of GAMG50The LD of=5000mg/kg, GL50=805mg/kg, therefore it is more safer than glycyrrhizic acid, GAMG conducts
A kind of new natural sweetener, sugariness is approximately 5 times of glycyrrhizic acid, having a extensive future in terms of food additives.Changing
Cosmetic aspect, the property of GAMG is relatively stable, not only high temperature resistant, acidproof and pressure, and has preferable emulsibility and foaming characteristic.
In existing technology, preparing GAMG includes chemical method and microbial method.Glycyrrhizic acid is aglycon enoxolone difference
By β -1,2 glycosidic bonds and β -1, what 3 glycosidic bonds and two molecule glucose aldehydic acid were formed by connecting, the bond energy of the two glycosidic bonds
It is similar, selectivity of the chemical hydrolysis to the two glycosidic bonds is very low, is prepared using high energy consumption and the chemical method of high pollution
During product GAMG, substantial amounts of accessory substance enoxolone can be also generated.Therefore, the research of chemical method preparation production GAMG is more stranded
It is difficult and rare.Microorganism prepares Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide, its essence is beta-glucuronic acid of the microorganism by generation
Glycyrrhizic acid is substrate by glycosides enzyme, and β -1 between two glycosyls of selective fracture, 2 glycosidic bonds are sweet so as to list glucuronic acid is obtained
Careless hypo acid.There is purpose product and yield poorly in microbe fermentation method, the defect more than accessory substance, prevent fermentation from meeting the industry of the country
Change Production requirement.
The content of the invention
It is an object of the invention to provide a kind of method of Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide, is by basket fungi
(Talaromyces sp) is seeded to using glycyrrhizic acid as carbon source and derivant, sodium nitrate by volume with 5～10% inoculum concentration
In the fermentation medium of nitrogen source, to control initial pH 4.5～6.5, ferment 120-144h in 25～37 DEG C.
In one embodiment of the invention, the sodium nitrate concentration is 2-4g/L.
In one embodiment of the invention, the sodium nitrate concentration is 3g/L.
In one embodiment of the invention, control Radix Glycyrrhizae acid concentration in sweat to be induced for 1-8g/L.
In one embodiment of the invention, the Radix Glycyrrhizae acid concentration is 5g/L.
In one embodiment of the invention, the inoculation is inoculation seed liquor, and the seed liquor is by basket fungi
In being seeded to seed culture medium, in 25～40 DEG C 36-48h is cultivated.
In one embodiment of the invention, the seed culture medium contains per L：Peptone 5g, glucose 10g, phosphorus
Acid dihydride potassium 1g, magnesium sulfate 0.4g.
In one embodiment of the invention, the fermentation medium contains per L：Glycyrrhizic acid 1-8g, sodium nitrate 2-4g,
Potassium dihydrogen phosphate 1-3g, magnesium sulfate 0.5-2g, ferrous sulfate 0.01-0.05g.
In one embodiment of the invention, the fermentation medium contains per L：Glycyrrhizic acid 5g, sodium nitrate 3g, phosphoric acid
Potassium dihydrogen 2g, magnesium sulfate 1g, ferrous sulfate 0.01g.
Beneficial effect：The invention provides with glycyrrhizic acid as carbon source, inducing basket fungi (Talaromyces sp) production
The method of GAMG, can be on the premise of environmentally friendly, and a large amount of preparation has widely studied valency
The product GAMG of value, meanwhile, the microbial fermentation processes are that the large-scale production for preparing this triterpene compound has been established newly
Basis.Method of the present invention process is simple, low cost, GAMG yield reaches 4.05g/L, and yield is 88.00%, Jing macropore trees
After purification, its purity reaches more than 80% to fat, and product purity is high, not recurring structure alienation, and with it is with short production cycle, be easy to behaviour
Make, low power consumption and other advantages, the characteristics of be suitable for the industrialized production in later stage.
Description of the drawings
Fig. 1 is the liquid chromatogram that fermentation after fermentation liquid is carried out according to the method for the present invention；
Fig. 2 is the zymotic fluid liquid chromatogram after being fermented according to the method for reference examples.
Specific embodiment
GAMG assay methods：Assay method referring to《The research that Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide is isolated and purified》, author wears
Swallow, is disclosed in 2006.
Embodiment 1
Seed culture medium contains per L：Peptone 5g, glucose 10g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.4g.
Fermentation medium contains per L：Glycyrrhizic acid 5g, sodium nitrate 3g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, ferrous sulfate
0.01g。
During basket fungi (Talaromyces sp) is seeded into seed culture medium, in 25～40 DEG C 36-48h is cultivated.Press
Volume is forwarded in the fermentation medium with sodium nitrate as nitrogen source with 5～10% inoculum concentration, controls initial pH 4.5～6.5,
Ferment 120-144h in 25～37 DEG C, and final concentration 5g/L glycyrrhizic acids are controlled in sweat as carbon source and derivant.
Yield, purity, the conversion ratio of purpose product in detection fermentation ends after fermentation liquid, as a result as shown in figure 1, when retaining
Between reach 4.05g/L for 16.91 product GAMG yield, yield is 88.00%, and without reserve the time be 24.40 Radix Glycyrrhizae time
This accessory substance of acid is generated, and Jing after macroporous resin purification, its purity reaches more than 80%.
Reference examples 1
Embodiment is to induce without glycyrrhizic acid in sweat with embodiment 1, difference.Fermentation results are carried out
Determine, as a result show, GAMG yield is close to 0.
Reference examples 2
Embodiment is that the glycyrrhizic acid of 5g/L is comprised only in fermentation medium with embodiment 1, difference, is added without other
Nutriment.Fermentation results are measured, are as a result shown, be not detected by GAMG, the bacterial strain does not almost utilize glycyrrhizic acid.
Reference examples 3
Embodiment is with embodiment 1, difference, in fermentation medium respectively the glucose containing 5g/L and glycyrrhizic acid,
Or the sucrose containing 5g/L and glycyrrhizic acid respectively, its yield may be substantially close to 0.
Reference examples 4
Embodiment is that Radix Glycyrrhizae acid concentration is 1g/L, and the yield of its GAMG is 42.55% (Fig. 2 with embodiment 1, difference
It is shown).
Reference examples 5
Embodiment is that nitrogen source is replaced with respectively beef extract, urea, the dusty yeast of 3g/L with embodiment 1, difference,
Fermentation results are measured, are as a result shown, GAMG yields are respectively 25.87%, 24.60%, 40.28%.
Reference examples 6
Embodiment is that initial pH difference 3.5 and 4.0 is measured, as a result to fermentation results with embodiment 1, difference
Show, GAMG yields are respectively 23.20%, 34.70%.
Reference examples 7
Embodiment is with embodiment 1, difference, temperature be respectively 20 DEG C, 24 DEG C when, fermentation results are measured,
As a result show, GAMG yields are respectively 11.50%, 14.86%.
Reference examples 8
Embodiment is that basket fungi is replaced with into aspergillus niger with embodiment 1, difference, and fermentation results are measured,
As a result show, GAMG yields are 51%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
Claims (9)
1. a kind of method for producing Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide, it is characterised in that by basket fungi (Talaromyces
Sp) it is seeded to using glycyrrhizic acid as carbon source, in the fermentation medium with sodium nitrate as nitrogen source, controls initial pH 4.5～6.5, in
25～37 DEG C of fermentation 120-144h.
2. method according to claim 1, it is characterised in that the inoculation is with fermentation medium volume 5～10%
Ratio is inoculated with.
3. method according to claim 1, it is characterised in that the sodium nitrate concentration is 2-4g/L.
4. method according to claim 1, it is characterised in that Radix Glycyrrhizae acid concentration is controlled in sweat to be carried out for 1-8g/L
Induction.
5. method according to claim 1, it is characterised in that the fermentation medium contains per L：Glycyrrhizic acid 1-8g, nitre
Sour sodium 2-4g, potassium dihydrogen phosphate 1-3g, magnesium sulfate 0.5-2g, ferrous sulfate 0.01-0.05g.
6. method according to claim 5, it is characterised in that the fermentation medium contains per L：Glycyrrhizic acid 5g, nitric acid
Sodium 3g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, ferrous sulfate 0.01g.
7. method according to claim 1, it is characterised in that the inoculation is inoculation seed liquor, the seed liquor be by
The inoculation of the basket fungi cultivates 36-48h into seed culture medium in 25～40 DEG C.
8. method according to claim 6, it is characterised in that the seed culture medium contains per L：Peptone 5g, grape
Sugared 10g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.4g.
9. the arbitrary methods described of claim 1-8 is prepared in food, biology, field of medicaments and contains GAMG
Product in application.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047713A (en) * | 2016-04-19 | 2016-10-26 | 北京理工大学 | Talaromyces pinophilum strain Li-93 and application 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 |
| CN108576764A (en) * | 2018-03-20 | 2018-09-28 | 亿利耐雀生物科技有限公司 | A kind of Antialcoholic liver-protecting compound sweetener and preparation method thereof |
| CN112544966A (en) * | 2019-09-25 | 2021-03-26 | 天津科技大学 | Mouth-clearing buccal tablet with anti-inflammatory effect and preparation method thereof |
| CN112608800A (en) * | 2020-12-17 | 2021-04-06 | 江苏耐雀生物工程技术有限公司 | Handmade soap with anti-allergy function and preparation method thereof |
| CN115044520A (en) * | 2022-08-12 | 2022-09-13 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
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| CN102367463A (en) * | 2011-11-03 | 2012-03-07 | 北京理工大学 | Method for producing glycyrrhetinic acid monoglucuronide through intermittent feed supplement fermentation |
| CN103509843A (en) * | 2012-06-29 | 2014-01-15 | 江苏天晟药业有限公司 | Method for high-yield preparation of glycyrrhetinic acid monoglucuronide |
| CN103788167A (en) * | 2012-10-31 | 2014-05-14 | 江苏汉邦科技有限公司 | Preparation method for glycyrrhetinic acid monoglucuronide (GAMG) |
| CN105669821A (en) * | 2016-01-08 | 2016-06-15 | 中国农业大学 | Preparation method of glycyrrhetinic acid monoglucuronide |
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| CN102367463A (en) * | 2011-11-03 | 2012-03-07 | 北京理工大学 | Method for producing glycyrrhetinic acid monoglucuronide through intermittent feed supplement fermentation |
| CN103509843A (en) * | 2012-06-29 | 2014-01-15 | 江苏天晟药业有限公司 | Method for high-yield preparation of glycyrrhetinic acid monoglucuronide |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047713A (en) * | 2016-04-19 | 2016-10-26 | 北京理工大学 | Talaromyces pinophilum strain Li-93 and application thereof |
| CN106047713B (en) * | 2016-04-19 | 2019-07-02 | 北京理工大学 | The basket bacteria strain Li-93 of one plant of thermophilic pine and its application |
| 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 |
| CN108576764A (en) * | 2018-03-20 | 2018-09-28 | 亿利耐雀生物科技有限公司 | A kind of Antialcoholic liver-protecting compound sweetener and preparation method thereof |
| CN112544966A (en) * | 2019-09-25 | 2021-03-26 | 天津科技大学 | Mouth-clearing buccal tablet with anti-inflammatory effect and preparation method thereof |
| CN112608800A (en) * | 2020-12-17 | 2021-04-06 | 江苏耐雀生物工程技术有限公司 | Handmade soap with anti-allergy function and preparation method thereof |
| CN115044520A (en) * | 2022-08-12 | 2022-09-13 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
| CN115044520B (en) * | 2022-08-12 | 2022-11-15 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
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