CN104805169A - Method for producing glycyrrhetinic acid through microbial transformation, and medium thereof - Google Patents
Method for producing glycyrrhetinic acid through microbial transformation, and medium thereof Download PDFInfo
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- CN104805169A CN104805169A CN201410040763.9A CN201410040763A CN104805169A CN 104805169 A CN104805169 A CN 104805169A CN 201410040763 A CN201410040763 A CN 201410040763A CN 104805169 A CN104805169 A CN 104805169A
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Abstract
The invention discloses a method for producing glycyrrhetinic acid through microbial transformation, and a medium thereof. The method comprises the following steps: inoculating an Aspergillus ustus seed liquid into a transformation medium according to an inoculation amount of 3-20V%, and culturing at 25-37DEG C for 48-240h, wherein the transformation medium comprises 1-15g/L of glycyrrhizic acid and 1-10g/L of NaNO3, and the pH value of the medium is 3-7. Glycyrrhizic acid is transformed by Aspergillus ustus in the method for the first time, the final transformation product is single, and the molar transformation rate of glycyrrhetinic acid reaches 96.5%, so the method and the medium are suitable for industrial production of glycyrrhetinic acid.
Description
Technical field
The present invention relates to biological technical field, be specifically related to method and substratum thereof that glycyrrhetinic acid is produced in a kind of microbial transformation.
Background technology
Radix Glycyrrhizae, as a kind of conventional Chinese medicine, is accepted and uses, and its main component is: Potenlini, Liquiritin, flavonoids of Glycyrrhiza, rear curtain are than Tan Su etc.There is removing toxic substances, anti-inflammatory, antibechic, antitumor, antiulcer agent and the effect such as antibacterial.Potenlini (Glycyrrhizic acid, GL) is the main active substances in Radix Glycyrrhizae, and Potenlini and series product thereof have different pharmacologically actives, as liver protecting, suppresses acquired immune deficiency syndrome (AIDS), antiviral and anticancer effect.In addition, Potenlini has higher sweet taste because of it and special local flavor also has purposes widely on food, is often used as foodstuff additive and perfumery base.
On the other hand, Potenlini also by as precursor compound, for the synthesis of other compound.Such as, Potenlini removes two glucal acidic groups, preparing glycyrrhetinic acid (Glycyrrhetinic acid, GA).Glycyrrhetinic acid does not have sweet taste, but its pharmacologically active is stronger than Potenlini, has very strong Hepatocyte protection, antiviral activity and anti-tumor activity.At present, Potenlini preparing glycyrrhetinic acid is utilized to mainly contain two kinds of methods, i.e. chemical process and bioconversion method.Chemical process is by acid, alkali effect, and the glycosyl of Potenlini is removed in hydrolysis, thus preparing glycyrrhetinic acid.But because this method will use a large amount of acid, alkali reagent, environmental pollution is comparatively serious.And strong acid, highly basic also can produce certain destruction to Potenlini structure, thus cause the final yield of glycyrrhetinic acid to decline.Such as, in patent application CN201010192841.9, the yield that applied chemistry method prepares glycyrrhetinic acid is 60% ~ 90%.Therefore, people start trial bioconversion method and produce glycyrrhetinic acid.Just at present about report, by the method for microbial transformation, Potenlini is changed into glycyrrhetinic acid, such as: CN102363796A utilizes flavus (Aspergillus flavus) microbe conversion Potenlini to produce glycyrrhetinic acid.Patent application CN102337319A then adopts preparing glycyrrhetinic acid by converting glycyrrhizic acid with enzymatic.In addition by the endophyte of screening Potenlini, direct preparing glycyrrhetinic acid, such as: CN102643756B, CN102643755B, CN102220247B, CN102660467B but final transformation efficiency is not very high.On the one hand, being because the enzyme amount that can transform Potenlini preparing glycyrrhetinic acid of these microorganism secretions is fewer, on the other hand, is because the vigour of these enzymes is low.Result in the efficiency comparison transforming Potenlini low, bioconversion method commercial application that so far there are no.Therefore, find can the microorganism of effectively hydrolyzing Potenlini just most important.
Summary of the invention
The technical problem to be solved in the present invention is the defect of the low conversion rate for current microbial transformation Potenlini preparing glycyrrhetinic acid, a kind of microbial transformation is provided to produce the method for glycyrrhetinic acid, the method adopts microbial transformation Potenlini preparing glycyrrhetinic acid, transformation efficiency is high, is applicable to glycyrrhetinic acid suitability for industrialized production.
The present invention solves above-mentioned technical problem by the following technical solutions:
Technical scheme of the present invention is: the method for glycyrrhetinic acid is produced in a kind of microbial transformation, comprise the following steps: by Aspergillus ustus (Aspergillus ustus) seed liquor by volume per-cent 3% ~ 20% inoculum size access bioconversion medium, cultivate 48 hours ~ 240 hours for 25 DEG C ~ 37 DEG C, wherein said bioconversion medium comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, pH3 ~ 7.
Inoculum size described in the present invention is volume percent 3% ~ 20%, more preferably volume percent 8% ~ 15%, it is still further preferred that volume percent 10%.
The temperature that conversion described in the present invention is cultivated is 25 DEG C ~ 37 DEG C, it is still further preferred that 30 DEG C.
The time that conversion described in the present invention is cultivated is 48 hours ~ 240 hours, it is still further preferred that 144 hours.
Preferably shaking culture is cultivated in conversion described in the present invention, and oscillation frequency is preferably 150rpm ~ 240rpm, it is still further preferred that 200rpm.
Bioconversion medium described in the present invention comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, pH3 ~ 7.
Preferably, described bioconversion medium comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, MgSO
47H
2o0.1 ~ 1 grams per liter, KH
2pO
40.1 ~ 5 grams per liter, pH3 ~ 7.
Preferably, also comprise in following component in described bioconversion medium one or both: F
esO
47H
2o0.01 ~ 0.05 grams per liter and C
acl
20.1 ~ 1.5 grams per liter.
In bioconversion medium described in the present invention, the content of Potenlini is 1 ~ 15 grams per liter, preferably 3 ~ 9 grams per liters, it is still further preferred that 5 grams per liters.
In bioconversion medium described in the present invention, NaNO
3content be 1 ~ 10 grams per liter, preferably 3 ~ 8 grams per liters, it is still further preferred that 5 grams per liters.
The pH value of bioconversion medium of the present invention is 3 ~ 7, and preferred pH value is 5.0.
In bioconversion medium described in the present invention,
MgSO
47H
2the content of O is 0.1 ~ 1 grams per liter, preferably 0.3 ~ 0.8 grams per liter, it is still further preferred that 0.5 grams per liter;
KH
2pO
4content be 0.1 ~ 5 grams per liter, preferably 0.5 ~ 2 grams per liter, it is still further preferred that 1 grams per liter;
FeSO
47H
2the content of O is 0.01 ~ 0.05 grams per liter, it is still further preferred that 0.01 grams per liter.
CaCl
2content be 0.1 ~ 1.5 grams per liter, preferably 0.8 ~ 1.3 grams per liter, it is still further preferred that 1.1 grams per liters.
Bioconversion medium the best of the present invention be: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.
The preparation method of described bioconversion medium is that this area is conventional, is mixed by each raw material, is dissolved in the water, regulate pH, autoclaving.Described autoclaving is conventional, preferably, and sterilizing 30 minutes under 121 DEG C of conditions.
Seed liquor described in the present invention is conventional, preferably prepared by the method comprised the following steps and obtain: by Aspergillus ustus (Aspergillus ustus) spore inoculating of slant culture in seed culture medium, 28 ~ 30 DEG C, 200 ~ 240rpm shaking culture, 24 ~ 72h, obtained seed liquor.
Described seed culture medium is preferably:
Substratum 1: glucose 10 grams per liter, maltodextrin 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 2: glucose 10 grams per liter, oatmeal 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 3: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 4: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 5 grams per liter, NaNO
35 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 5:PDA;
Substratum 6: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, NaNO
310 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2.
Described PDA is the substratum of this area routine, can be purchased and obtain, and also can prepare voluntarily according to existing formula.The formula of PDA liquid nutrient medium is: potato 200g, glucose 20g, water 1000ml, pH nature.Concrete compound method is as follows: peeling potatoes is cut into small pieces and weighs 200g, and boiling water boils 30min, and 4 layers of filtered through gauze, add glucose 20g, and adding water after dissolving is settled to 1000ml, and pH nature, 121 DEG C of sterilizing 30min, to obtain final product.As this area is conventional, in liquid medium within, add appropriate agar, namely obtain solid or semisolid medium.
What described seed culture medium was better is substratum 2,3, and best is substratum 5.
The temperature of described seed culture is preferably 28 ~ 30 DEG C, and best is 28 DEG C,
The time of described seed culture is 24 ~ 72 hours, is preferably 36 ~ 60 hours, and best is 48 hours.
The oscillation frequency of described seed culture is preferably 200 ~ 240rpm, and that best is 240rpm.
In the present invention, described slant culture is conventional, and generally on slant medium 28 DEG C, quiescent culture 3 ~ 7 days, namely reaches spore and enrich period, namely can be used for inoculating seed culture medium.Slant medium is conventional, is generally the agar adding mass volume ratio 1.8% on the basis of PDA liquid nutrient medium.
In the present invention, after conversion cultivation terminates, namely containing glycyrrhetinic acid in nutrient solution.Glycyrrhetinic acid can be obtained from nutrient solution.
Aspergillus ustus (Aspergillus ustus) described in the present invention refers to any bacterial strain belonging to this genus Aspergillus (Aspergillus) ustus kind, such as can be selected from one or more in strains A spergillus ustusCGMCC3.1432, Aspergillus ustus CGMCC3.3937, Aspergillus ustus CGMCC3.5298.
In the present invention, unless otherwise noted, described per-cent is mass percent.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: the present invention utilizes Aspergillus ustus (Aspergillus ustus) to transform Potenlini innovatively first, obtains glycyrrhetinic acid.The method comprises three aspects:
1, Aspergillus ustus is utilized to transform Potenlini first;
2, final converted product is single, and the molar yield of glycyrrhetinic acid, up to 96.5%, is applicable to the suitability for industrialized production of glycyrrhetinic acid;
3, strain culturing technique is simple, reaction conditions is gentle, substratum is simple, and cost is low.For the suitability for industrialized production of glycyrrhetinic acid provides foundation.
Accompanying drawing explanation
Fig. 1 utilizes microorganism to be glycyrrhetinic acid by Potenlini substrate conversion.1, Potenlini; 2, glycyrrhetinic acid.
Fig. 2 is glycyrrhetinic acid mark product.No. 2 peak representatives: glycyrrhetinic acid.
Fig. 3 is the collection of illustrative plates of strains A spergillus ustus CGMCC3.1432 converted product.No. 1 peak representative: Potenlini; No. 2 peak representatives: glycyrrhetinic acid.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Being calculated as follows of mole production rate in embodiment:
The molar mass 470.68g/mol of Potenlini; The molar mass 822.93g/mol of glycyrrhetinic acid.
Embodiment raw material used is as follows:
Aspergillus ustus (Aspergillus ustus) CGMCC3.1432, Aspergillus ustus CGMCC3.3937, Aspergillus ustus3.5298 are purchased from China General Microbiological culture presevation administrative center.Aspergillus ustus (Aspergillus ustus) CGMCC3.1432, Aspergillus ustus CGMCC3.3937, Aspergillus ustus CGMCC3.5298 are respectively used to embodiment 1 ~ 36,37,38.
Slant medium: for adding the agar of mass volume ratio 1.8% on the basis of PDA liquid nutrient medium.
Embodiment 1
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: with the slant culture of gained, be inoculated in seed culture medium, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.This seed liquor is used for embodiment 1 ~ 27.
Wherein, seed culture medium is: PDA liquid nutrient medium.
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 25 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 82.8%.
Embodiment 2
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 96.5%.
Embodiment 3
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 37 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 69.8%.
Embodiment 4
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 150rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 86.2%.
Embodiment 5
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 240rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 70.4%.
Embodiment 6
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 48h, mole production rate of glycyrrhetinic acid is 50.5%.
Embodiment 7
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 240h, mole production rate of glycyrrhetinic acid is 94.7%.
Embodiment 8
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 1 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 91.5%.
Embodiment 9
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 15 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 90.5%.
Embodiment 10
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.1 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 83.9%.
Embodiment 11
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o1 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 88.1%.
Embodiment 12
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
31 grams per liter, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 87.3%.
Embodiment 13
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
310 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 89.5%.
Embodiment 14
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.05 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 81.4%.
Embodiment 15
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
40.1 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 83.1%.
Embodiment 16
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
45 grams per liters, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 86.2%.
Embodiment 17
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, CaCl
20.1 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 86.1%.
Embodiment 18
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, CaCl
21.1 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 90.8%.
Embodiment 19
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, CaCl
21.5 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 84.9%.
Embodiment 20
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH3.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 18.3%.
Embodiment 21
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH7.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 83.6%.
Embodiment 22
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 3% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 82.3%.
Embodiment 23
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 20% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 83.8%.
Embodiment 24
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, ZnSO
47H
2o2.87 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 9.4%.
Embodiment 25
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, MnSO
4h
2o1.69 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 10.7%.
Embodiment 26
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, NaNO
35 grams per liters, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 85.1%.
Embodiment 27
Seed liquor is seeded in the Erlenmeyer flask containing bioconversion medium with the inoculum size of 10% (v/v), the composition of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, KH
2pO
41 grams per liter, pH5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 87.8%.
Embodiment 28
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, the composition of seed culture medium: glucose 10 grams per liter, maltodextrin 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4, 10mmol CaCl
2, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.
By seed liquor with 10% inoculum size be seeded in the Erlenmeyer flask containing bioconversion medium, consisting of of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, regulates pH to 5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 84.0%.
Embodiment 29
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, the composition of seed culture medium: glucose 10 grams per liter, oatmeal 10 grams per liter, NaNO
310 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4, 10mmol CaCl
2, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.
By seed liquor with 10% inoculum size be seeded in the Erlenmeyer flask containing bioconversion medium, consisting of of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, regulates pH to 5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 87.9%.
Embodiment 30
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, the composition of seed culture medium: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4, 10mmol CaCl
2, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.
By seed liquor with 10% inoculum size be seeded in the Erlenmeyer flask containing bioconversion medium, consisting of of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, regulates pH to 5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 87.0%.
Embodiment 31
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, the composition of seed culture medium: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 5 grams per liter, NaNO
35 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4, 10mmol CaCl
2, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.
By seed liquor with 10% inoculum size be seeded in the Erlenmeyer flask containing bioconversion medium, consisting of of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, regulates pH to 5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 86.3%.
Embodiment 32
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, the composition of seed culture medium: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, NaNO
310 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4, 10mmol CaCl
2, 28 DEG C, 240rpm shaking culture 48h, obtains seed liquor.
By seed liquor with 10% inoculum size be seeded in the Erlenmeyer flask containing bioconversion medium, consisting of of bioconversion medium: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, regulates pH to 5.0.Automatic control leavening temperature 30 DEG C, 200rpm shaking culture 144h, mole production rate of glycyrrhetinic acid is 86.5%.
Embodiment 33
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium, 28 DEG C, and 240rpm shaking culture 48h, obtains seed liquor.Detect the pmv value (thalline weight in wet base value) of the thalline of gained seed liquor.
The measuring method of pmv value: get 10ml seed liquor, the centrifugal 10min of 3000rpm, surveys the volume V(ml of supernatant liquor),
Wherein said seed culture medium is as follows respectively:
Substratum 1: glucose 10 grams per liter, maltodextrin 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 2: glucose 10 grams per liter, oatmeal 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 3: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 4: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 5 grams per liter, NaNO
35 grams per liters, 10mmol MgSO47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2;
Substratum 5:PDA;
Substratum 6: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, NaNO
310 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2.The thalline pmv value of gained seed liquor is as follows respectively:
| Substratum | 1 | 2 | 3 | 4 | 5 | 6 |
| The pmv value (%) of thalline | 7 | 11 | 10 | 7 | 15 | 9 |
Embodiment 34
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, be inoculated in seed culture medium (seed culture medium is PDA), in different temperature, 240rpm shaking culture 48h, obtains seed liquor.The thalline pmv value of gained seed liquor is as follows respectively:
| Temperature DEG C | 27 | 28 | 29 | 30 | 31 |
| The pmv value (%) of thalline | 13 | 15 | 13 | 12 | 12 |
Embodiment 35
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, be inoculated in seed culture medium (seed culture medium is PDA), at 28 DEG C, 240rpm shaking culture certain hour, obtains seed liquor.Thalline pmv value in gained seed liquor is as follows respectively:
| Time (h) | 12 | 24 | 36 | 48 | 60 | 72 |
| The pmv value (%) of thalline | 1 | 4 | 10 | 15 | 16 | 17 |
Embodiment 36
Slant culture: by inoculation slant medium, 28 DEG C, quiescent culture 3 ~ 7d.
Seed culture: by the slant culture of gained, is inoculated in seed culture medium (seed culture medium is PDA), at 28 DEG C, cultivates 48h, obtain seed liquor at different oscillation frequency.Thalline pmv value in gained seed liquor is as follows respectively:
| Oscillation frequency (rpm) | 180 | 200 | 220 | 240 | 260 |
| The pmv value (%) of thalline | 10 | 11 | 12 | 15 | 9 |
Embodiment 37
Adopt Aspergillus ustus (Aspergillus ustus) CGMCC3.3937, as embodiment 1 carries out slant culture and seed culture, seed culture fluid adopts the condition with embodiment 2 to carry out conversion and cultivates.Mole production rate of glycyrrhetinic acid is 87.5%.
Embodiment 38
Adopt Aspergillus ustus (Aspergillus ustus) CGMCC3.5298, as embodiment 1 carries out slant culture and seed culture, seed culture fluid adopts the condition with embodiment 2 to carry out conversion and cultivates.Mole production rate of glycyrrhetinic acid is 89.3%.
Comparative example 1
Flavus (Aspergillusflavus) CGMCC NO.5144 in patent CN102363796A is replaced the Aspergillus ustus CGMCC3.1432 in the present invention, as embodiment 1 carries out slant culture and seed culture, seed culture fluid adopts the condition with embodiment 2 to carry out conversion and cultivates.Mole production rate of glycyrrhetinic acid is 69.8%.
Effect example 1
In above embodiment, adopt UPLC to detect glycyrrhetinic acid, method is as follows:
Reference substance solution is prepared: accurately take the quantitative glycyrrhetinic acid (HPLC purity >=95%) being dried to constant weight respectively, be mixed with the ethanolic soln of 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 2.5mg/ml, UPLC measures peak area corresponding to different concns reference substance, drawing standard curve.
The preparation of sample: Potenlini liquid fermentation production, adds the acetone of 2 times of volumes, vibration, and leave standstill soaked overnight, get the acetone soak solution of 1ml, 14000rpm high speed centrifugation 3min, supernatant liquor aperture is get final product test sample after organic membrane filtration of 0.22 μm.
UPLC testing conditions:
Chromatographic column: ultimate XB ~ C18(2.1 × 100mm, 1.8 μm);
Gradient elution:
posttime:5min；
Flow velocity 0.22ml/min, determined wavelength 254nm, sample size 2 μ L, column temperature 35 DEG C.
Wherein, Fig. 2 is shown in by the UPLC collection of illustrative plates of glycyrrhetinic acid mark product.
Wherein, Fig. 3 is shown in by the collection of illustrative plates of strains A spergillus ustus CGMCC3.1432 converted product.
The glycyrrhetinic acid of UPLC purifying is carried out Mass Spectrometric Identification, and mass-spectrometric data is as follows:
The mass-spectrometric data of glycyrrhetinic acid:
ESI ~ TOF ~ MS(negative) m/z:469.33 [M ~ H] ~ (C
30h
45o
4; Calculated value: 469.33).
Claims (10)
1. the method for a microbial transformation production glycyrrhetinic acid, it is characterized in that, comprise the following steps: by Aspergillus ustus (Aspergillus ustus) seed liquor by volume per-cent 3% ~ 20% inoculum size access bioconversion medium, cultivate 48 hours ~ 240 hours for 25 DEG C ~ 37 DEG C, wherein said bioconversion medium comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, pH3 ~ 7.
2. the method for claim 1, it is characterized in that, described seed liquor is prepared by the method comprised the following steps and obtains: by Aspergillus ustus (Aspergillus ustus) spore inoculating of slant culture in seed culture medium, 28 ~ 30 DEG C, 200 ~ 240rpm shaking culture 24 ~ 72 hours, obtained seed liquor.
3. method as claimed in claim 2, it is characterized in that, described seed culture medium is PDA; Or comprise following component: glucose 10 grams per liter, oatmeal 10 grams per liter, NaNO
310 grams per liters, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2; Or comprise following component: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2.
4. the method for claim 1, is characterized in that, described bioconversion medium comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, MgSO
47H
2o0.1 ~ 1 grams per liter, KH
2pO
40.1 ~ 5 grams per liter, pH3 ~ 7.
5. the method as described in claim 1 or 4, is characterized in that, also comprises one or both in following component: FeSO in described bioconversion medium
47H
2o0.01 ~ 0.05 grams per liter and CaCl
20.1 ~ 1.5 grams per liter.
6. produce a bioconversion medium for glycyrrhetinic acid for microbial transformation, it is characterized in that, comprise following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, pH3 ~ 7.
7. bioconversion medium as claimed in claim 6, it is characterized in that, described bioconversion medium comprises following component: Potenlini 1 ~ 15 grams per liter, NaNO
31 ~ 10 grams per liter, MgSO
47H
2o0.1 ~ 1 grams per liter, KH
2pO
40.1 ~ 5 grams per liter, pH3 ~ 7.
8. bioconversion medium as claimed in claim 6, is characterized in that, also comprises one or both in following component: FeSO in described bioconversion medium
47H
2o0.01 ~ 0.05 grams per liter and CaCl
20.1 ~ 1.5 grams per liter.
9. bioconversion medium as claimed in claim 6, it is characterized in that, described bioconversion medium is: Potenlini 5 grams per liter, MgSO
47H
2o0.5 grams per liter, NaNO
35 grams per liters, FeSO
47H
2o0.01 grams per liter, KH
2pO
41 grams per liter, pH5.0.
10. produce a seed culture medium for glycyrrhetinic acid for microbial transformation, it is characterized in that, described seed culture medium comprises following component: glucose 10 grams per liter, oatmeal 10 grams per liter, NaNO
310 grams per liters, 10mmol MgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2; Or comprise following component: glucose 10 grams per liter, oatmeal 10 grams per liter, fish meal protein peptone 10 grams per liter, 10mmolMgSO
47H
2o, 10mmol KH
2pO
4with 10mmol CaCl
2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101603067A (en) * | 2009-07-23 | 2009-12-16 | 北京理工大学 | A kind of method of oriented biosynthesis of GAMG |
| CN102363796A (en) * | 2011-11-10 | 2012-02-29 | 中科医药行业生产力促进中心有限公司 | Method for producing glycyrrhetinic acid through microbial fermentation transformation |
| CN102643756A (en) * | 2012-04-26 | 2012-08-22 | 黑龙江大学 | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603067A (en) * | 2009-07-23 | 2009-12-16 | 北京理工大学 | A kind of method of oriented biosynthesis of GAMG |
| CN102363796A (en) * | 2011-11-10 | 2012-02-29 | 中科医药行业生产力促进中心有限公司 | Method for producing glycyrrhetinic acid through microbial fermentation transformation |
| CN102643756A (en) * | 2012-04-26 | 2012-08-22 | 黑龙江大学 | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice |
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