CN103992953A - Dongxiang wild rice endophytic fungus for converting glycyrrhizic acid to generate glycyrrhetinic acid glycoside - Google Patents

Abstract

The Dongxiang wild rice endophytic fungus for transforming glycyrrhizic acid to generate glycyrrhetinic acid is identified and named as Aspergillus flavus DX-SEL 2. Has been preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2013686. The strain can induce the production of beta-glucuronidase in a conversion culture medium with glycyrrhizic acid as an inducer, and then hydrolyze to remove a glucuronide group at the tail end of a glycyrrhizic acid molecule to generate the glycyrrhetinic acid glycoside (secreted in a fermentation liquid). Further separating and purifying by organic solvent extraction, normal phase silica gel column chromatography, reverse phase column chromatography, etc. to obtain glycyrrhetinic glycoside. The invention adopts the microorganism catalytic conversion, and has the advantages of environmental protection, no pollution, simple conversion step, single product, high conversion rate and the like.

Description

An endophytic fungus from Dongxiang wild rice that transforms glycyrrhizic acid to glycyrrhetin

technical field

The invention relates to the technical field of biotransformation, in particular to an endophytic fungus of Dongxiang wild rice that transforms glycyrrhizic acid into glycyrrhetin.

Background technique

Glycyrrhizic acid (GL) is one of the main active ingredients in licorice. The structure of glycyrrhizic acid is shown in Figure 3, which is composed of two glucuronic acids connected by pentacyclic triterpene saponins through glycosidic bonds. Glycyrrhizic acid is hydrolyzed by β-glucuronidase to remove a glucuronic acid group at its terminal to generate glycyrrhetin (Figure 3).

Glycyrrhetin is also known as monoglucuronyl glycyrrhetinic acid (GAMG). The application value of glycyrrhizin (GAMG) is far greater than that of glycyrrhizic acid. The sweetness of glycyrrhizin is 5 times that of glycyrrhizic acid and 941 times that of sucrose. It is a new type of sweetener with high sweetness and low calories, which can effectively reduce obesity and diabetes caused by the intake of high-calorie sweeteners. , hyperlipidemia, dental caries and other diseases; Glycyrrhetin is stable in nature, resistant to high temperature, acid and high pressure, and has a wider range of applications in the food field; as a drug, Glycyrrhetin and Glycyrrhizic acid have the same (or stronger) pharmacological activity , such as anti-inflammation, anti-virus, anti-tumor, anti-digestive ulcer, anti-allergic, liver protection, lowering blood lipids, etc., but glycyrrhizin has medium polarity, better solubility in the body and easier transmembrane transport. The bioavailability of glycyrrhizic acid is better; the most important thing is that glycyrrhizin is safer than glycyrrhizic acid, the LD50 of glycyrrhizin is 5000mg/kg, and the LD50 of glycyrrhizic acid is 805mg/kg. Therefore, the production and development of Glycyrrhizin has very important application value and practical significance.

Kanaoka M used chemical synthesis to synthesize glycyrrhizin (M Kanaoka, Chem pharm Bull (Tokyo), 1986, 34(12):4978-4983.), but the synthetic route was long and the yield was low. The traditional chemical hydrolysis method removes the glucuronic acid group of glycyrrhizic acid to generate glycyrrhetin, but this method has low selectivity for the two glycosidic bonds of glycyrrhizic acid, and cannot be directional hydrolyzed to generate glycyrrhetin. There are many by-products and the yield is relatively low. Low, at the same time there are high energy consumption, high pollution shortcomings. In addition, according to the legislation of the European Union and the United States, products obtained by chemical methods are not natural substances, and their application in the food field is restricted, while substances obtained by enzymatic or microbial methods (ie, biotransformation methods) are considered natural substances. Biotransformation refers to the biochemical reaction process of using microorganisms, animals, plants and culture systems or the enzymes produced to modify the structure of exogenous compounds. Its essence is to use the enzymes produced by biological systems to perform enzymatic reactions on exogenous compounds. The conversion of glycyrrhizic acid to glycyrrhetin by biotransformation has also been reported. According to previous reports, intestinal bacteria and animal tissues contain β-glucuronidase, which can convert glycyrrhizic acid into glycyrrhetin, but its ubiquitous transformation has low specificity (not only producing glycyrrhizin, but also producing A large number of by-products), the disadvantages of low conversion efficiency. Extracting β-glucuronidase from animal tissues is costly and complicated to prepare.

The inventors isolated and screened an endophytic fungus, Aspergillus flavus DX-SEL2, which can directional transform glycyrrhizic acid into glycyrrhetin from the leaf tissue of the gramineous plant Dongxiang wild rice. The number is CCTCCNO: M 2013686. Under the induction of glycyrrhizic acid (or ammonium glycyrrhizic acid), the bacterium can directional transform glycyrrhizic acid to generate glycyrrhetinin, and the conversion rate is high, the conversion step is simple, and it is environmentally friendly and pollution-free.

In addition, the present invention discovers for the first time that endophytic fungi transform glycyrrhizic acid into glycyrrhetin, and it is an endophytic fungus of the gramineous plant Dongxiang wild rice that has nothing to do with licorice, which provides a new research idea for searching for related efficient transformation strains .

Contents of the invention

The purpose of the present invention is to provide a Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 to convert glycyrrhizic acid into glycyrrhetin, which overcomes the poor catalytic orientation, environmental pollution, high energy consumption and low conversion rate in the prior art. insufficient.

The Dongxiang wild rice endophytic fungus that transforms glycyrrhizic acid into glycyrrhetin is classified as Aspergillus flavus DX-SEL2, and is obtained from the leaf tissue of Dongxiang wild rice plant by using endophytic fungus separation and purification technology. Preserved in China Center for Type Culture Collection, the date of deposit is December 23, 2013, and the deposit number is CCTCC NO: M 2013686.

The morphological characteristics of the Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 of the present invention are as follows: on the PDA medium, cultivated at 28 ± 2°C for 2 days, the diameter of the colony is 30-32mm, and the diameter of the colony is 61-63mm after 3 days. The sky covers the entire Petri dish; the texture is velvety, thicker, and the center is flocculent, the front of the colony is olive green, and the edge is slightly white; the back is light yellow. Conidiophores are colorless or light brown, with a diameter of 10-15 μm; conidia are nearly spherical, with a diameter of 50-65 μm, and conidia are spherical to subspherical, with a diameter of 2.5-4.5 μm.

The accession number of the Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 gene of the present invention is KC871017.

The Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 of the present invention converts glycyrrhizic acid to generate glycyrrhetin, which comprises the following steps:

Step 1. Inoculate Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 into PDA slant medium, cultivate and activate for 72-84 hours, and make spore suspension, then inoculate into seed medium, 28±1°C, 150 ~300r/min, shaker culture to logarithmic growth phase.

Step 2: Inoculate the seed liquid in the logarithmic growth phase into the transformation medium containing glycyrrhizic acid according to the inoculum amount of 10%-30%, and transform to produce glycyrrhizin until the content is basically constant.

Step 3, separating and purifying glycyrrhizin from the fermentation broth.

Preferably, the Aspergillus flavus DX-SEL2 converts glycyrrhizic acid to generate glycyrrhizin directionally, and the composition of the seed medium in step 1 is: 10-40 g of glucose per liter of medium, glycyrrhizic acid (or ammonium glycyrrhizinate ) 0.1~1g, KH ₂ PO ₄ 1.0~3.0g, NH ₄ NO ₃ 2.0~5.0g, NaCl 0.3~0.8 g, yeast powder 0.05~0.3 g, MgSO ₄ 0.1~0.5g, CaCl ₂ 0.01~0.5g, FeSO ₄ 0.014g, ZnSO ₄ 7H ₂ O 2.9mg, MnCl ₂ 4H ₂ O 2.0mg, CuSO ₄ 5H ₂ O 0.25mg, CoCl ₂ 6H ₂ O 0.24mg, Na ₂ MoO ₄ 2H ₂ O 0.24 mg, H ₃ BO ₃ 0.03mg, the rest is pure water, adjust the pH to 5.0-7.0.

Preferably, the Aspergillus flavus DX-SEL2 transforms glycyrrhizic acid to generate glycyrrhetin in a directed manner, and the composition of the transformation medium in step 2 is: 2-30 g of glycyrrhizic acid (or ammonium glycyrrhizinate) per liter of medium, KH ₂ PO ₄ 1.0～3.0g, NH ₄ NO ₃ 2.0～4.0g, NaCl 0.3～0.8g, yeast powder 0.05～0.1g, MgSO ₄ 0.1～0.5g, CaCl ₂ 0.01～0.5g, FeSO ₄ 0.014g, ZnSO ₄ 7H ₂ O 2.9mg, MnCl ₂ 4H ₂ O 2.0mg, CuSO ₄ 5H ₂ O 0.25mg, CoCl ₂ 6H ₂ O 0.24mg, Na ₂ MoO ₄ 2H ₂ O 0.24mg, H ₃ BO ₃ 0.03mg, the rest is pure water, adjust the pH to 3.5-9.0. Among them, glycyrrhizic acid (or ammonium glycyrrhizinate) is an inducer of β-glucuronidase produced by bacteria.

Preferably, said Aspergillus flavus DX-SEL2 converts glycyrrhizic acid to produce glycyrrhetin in a directed manner, and the culture conditions in step 2 are: 28°C-60°C, rotation speed 150-300r/min.

Preferably, said Aspergillus flavus DX-SEL2 converts glycyrrhizic acid to generate glycyrrhetin in a directed manner, and the separation and purification of glycyrrhetin in step 3 comprises the following steps:

Buchner funnel suction filtration or six-layer gauze filtration, collect the filtrate, and extract 2 to 4 times with equal volume of chloroform (to extract impurities with low polarity, but not to extract glycyrrhetin). The aqueous phase was extracted 3-5 times with equal volume of ethyl acetate, and the obtained ethyl acetate phase was concentrated under reduced pressure to obtain crude glycyrrhetin. Then use methods such as normal phase silica gel column chromatography or reverse phase column chromatography to further purify glycyrrhetin.

The beneficial effects of the Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 converting glycyrrhizic acid to produce glycyrrhetin are mainly reflected in the following aspects: the enzyme produced by microorganisms is used as a catalyst, the reaction efficiency is high, the specificity is strong, and the by-product Less, simple steps, green and pollution-free.

Description of drawings

Fig. 1 is the bacterium colony form of endophytic fungus Aspergillus flavus DX-SEL2 of Dongxiang wild rice of the present invention;

Fig. 2 is the sporangia and spore morphology under the scanning electron microscope of the endophytic fungus Aspergillus flavus DX-SEL2 of Dongxiang wild rice of the present invention

Figure 3 Glycyrrhizic acid is hydrolyzed by β-glucuronidase to produce glycyrrhetin

Fig. 4 is the HPLC detection of the glycyrrhizic acid product transformed by Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 of the present invention;

Figure 5 LC-MS detection of glycyrrhizic acid products converted by the endophytic fungus Aspergillus flavus DX-SEL2.

Detailed ways

The present invention is described in detail below in conjunction with example.

Embodiment 1: Isolation of the Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 of the present invention

(1) Collect complete Dongxiang wild rice and process it immediately after returning to the laboratory.

(2) Roots, stems and leaves are preliminarily sterilized by soaking in 75% ethanol for 5 minutes, then soaking in 0.1% mercuric acid for 8 minutes, and rinsing with sterile water to sterilize the surface of the tissues.

(3) Cut off the two ends of the above-mentioned treated stem with sterilized scissors, then cut the stem with two ends longitudinally, divide it into two, and cut it into small pieces of 0.1×0.5 cm. The edge of the leaf is cut off with sterilized scissors, and the edge-cut blade is also cut into small pieces of about 0.2×0.5 cm. They were respectively placed on PDA medium supplemented with 60 μg/L streptomycin and 0.5 g/L potassium dichromate, and cultured in an incubator at 28°C. In order to check the surface sterilization effect, set up a control to test the sterilization effect. Control I: The roots, stems and leaves that have been sterilized are not cut off at both ends and edges, and then their surfaces are taken out after contacting the solid plate, and the petri dish is placed in an incubator for cultivation; Control II: the last washing Sterile water was added to the medium. Each treatment was repeated 3 times.

(4) Colonies were found around the inoculated tissue, and the front end of the hyphae was picked up with an inoculation needle, and inoculated on another medium. Observe once a day, if colonies grow, pick them out immediately.

(5) After the selected fungus forms colonies, use an inoculation needle to pick the front end of the mycelia, and then inoculate it on another medium, repeat the purification 4 times in this way, and store the bacteria on the slant medium at 4°C

Example 2: The present invention screened out Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2, which was directed to transform glycyrrhizic acid into glycyrrhetin.

(1) Plate primary screening: The endophytic fungi isolated above from Dongxiang wild rice were activated and inoculated in a solid screening medium with glycyrrhizic acid as the sole carbon source. In the control group, glucose was used instead of glycyrrhizic acid as the sole carbon source. At 28°C, cultivate for 7 days, and screen out the strains that can grow well.

The screening medium is; each liter medium contains 3.0 g of glycyrrhizic acid, 2.2 g of KH ₂ PO ₄ , 3.0 g of NH ₄ NO ₃ , 0.5 g of NaCl, 0.05 g of yeast powder, 0.12 g of MgSO ₄ , and 0.014 g of CaCl ₂ , FeSO ₄ 0.014g, ZnSO ₄ 7H ₂ O 2.9mg, MnCl ₂ 4H ₂ O 2.0mg, CuSO ₄ 5H ₂ O 0.25mg, CoCl ₂ 6H ₂ O 0.24mg, Na ₂ MoO ₄ 2H ₂ O 0.24mg, H ₃ BO ₃ 0.03mg, agar powder 20g (not added to liquid medium), the rest is pure water, pH6.0.

(2) Shaking flask re-screening: inoculate the strains screened out on the above plate into the seed medium (replacing the 3.0g/L glycyrrhizic acid in the screening medium with 20g/L glucose) and culture at 28°C and 150 rpm for 2d , and then transferred to the liquid screening medium with glycyrrhizic acid as the only carbon source according to the inoculation amount of 20%, and two controls were set up: one was that the bacterial cells were not inserted into the liquid screening medium to detect whether glycyrrhizic acid was Decomposed in the medium), the second is to transfer the strain to the selection medium with glucose instead of glycyrrhizic acid as the only carbon source. 28 ℃, 150 rpm shaker culture for 4 days, the fermentation broth was collected, and the transformed products were detected by TLC, HPLC and LC-Ms.

The experimental results show that the Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 can directional transform into glycyrrhizin (as shown in Figure 4 and Figure 5), and the conversion rate of glycyrrhizic acid ((initial GL molar concentration to transformed GL molar concentration)/ The initial GL molar concentration×100%) was 72.4%, and the yield of glycyrrhetin (GAMG molar concentration after conversion/initial GL molar concentration×100%) was 67.3%.

Example 3: Endophytic fungus Aspergillus flavus DX-SEL2 converts glycyrrhizic acid to produce glycyrrhetin

The activated Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 was inserted into the seed medium for 3 days at 28°C and 250 rpm, and then transferred to the transformation medium at 35°C with 250 rpm shaking. Bed culture 7d. The bacteria were removed by centrifugation, the fermentation broth was collected, and the transformation product was detected by HPLC.

The composition of the seed medium is as follows: every liter of medium contains 25g of glucose, 0.2g of glycyrrhizic acid (or ammonium glycyrrhizinate), 2.0g of KH ₂ PO ₄ , 3g of NH ₄ NO ₃ , 0.5g of NaCl, and 0.1 g of yeast powder. g, MgSO ₄ 0.12g, CaCl ₂ 0.2g, FeSO ₄ 0.014g, ZnSO ₄ 7H ₂ O 2.9mg, MnCl ₂ 4H ₂ O 2.0mg, CuSO ₄ 5H ₂ O 0.25mg, CoCl ₂ 6H ₂ O 0.24mg, Na ₂ MoO ₄ ·2H ₂ O 0.24mg, H ₃ BO ₃ 0.03mg, the rest is pure water, adjust the pH to 6.0.

The composition of the transformation medium is as follows: each liter of medium contains 5 g of monoammonium glycyrrhizinate, 2.2 g of KH ₂ PO ₄ , 3 g of NH ₄ NO ₃ , 0.5 g of NaCl, 0.1 g of yeast powder, 0.12 g of MgSO ₄ , and CaCl ₂ 0.4g, FeSO ₄ 0.014g, ZnSO ₄ 7H ₂ O 2.9mg, MnCl ₂ 4H ₂ O 2.0mg, CuSO ₄ 5H ₂ O 0.25mg, CoCl ₂ 6H ₂ O 0.24mg, Na ₂ MoO ₄ 2H ₂ O 0.24mg, H ₃ BO ₃ 0.03mg, the rest is pure water, adjust the pH to 7.0.

After the transformation, the filtrate was obtained by suction filtration to remove the bacteria and the transformation product was detected. The detection results showed that the conversion rate of glycyrrhizic acid was 90.5%, and the yield of glycyrrhizin was 85.3%.

The resulting filtrate was extracted three times with equal volumes of chloroform to remove less polar impurities without extracting glycyrrhetin. The aqueous phase was further extracted with equal volume of ethyl acetate for 3 to 5 times, and the obtained ethyl acetate phase was concentrated under reduced pressure to obtain crude glycyrrhetin with a detection purity of 84.8%.

Claims (8)

1. one strain transforms the Dongxiang Wild Rice endogenetic fungus of Potenlini generation Starrhizin, it is characterized in that: described endogenetic fungus is from the leaf tissue of Dongxiang Wild Rice plant living body, to adopt endogenetic fungus separating and purifying technology to separate to obtain, through microbial taxonomy qualification called after aspergillus flavusdX-SEL2, has been preserved in Chinese Typical Representative culture collection center, and preservation date is on December 23rd, 2013, and preserving number is CCTCCNO:M 2013686, and this bacterium can transform Potenlini and generate Starrhizin.

2. as claimed in claim 1 aspergillus flavusdX-SEL2 bacterial strain, is characterized in that: on PDA substratum, cultivates 2 days for 28 ± 1 DEG C, and colony diameter 30～32mm, 3 days colony diameters are 61～63mm, within 4 days, cover with whole culture dish; Quality velvet shape, thicker, central authorities are existing cotton-shaped, and bacterium colony front is olive-green, and edge is white slightly; Reverse side is faint yellow, the colourless or light brown of conidiophore, and diameter is 10～15 μ m; Conidium top capsule is close to spherical, and diameter is 50～65 μ m, and conidium is spherical to subsphaeroidal, and diameter is 2.5～4.5 μ m.

3. as claimed in claim 1 aspergillus flavusdX-SEL2 bacterial strain, is characterized in that: its gene accession number is KC871017.

4. endogenetic fungus as claimed in claim 1 aspergillus flavusdX-SEL2 transforms the method for Potenlini generation Starrhizin, it is characterized in that comprising the following steps:

Step 1, by endogenetic fungus aspergillus flavusdX-SEL2, is inoculated in PDA slant medium and cultivates and activate 72～84 hours, and make spore suspension, is then seeded in seed culture medium, and 25～35 DEG C, 150～300r/min, shaking table is cultured to logarithmic phase;

Step 2, inoculum size according to 10%～40% are seeded to the seed liquor of above-mentioned logarithmic phase in the conversion substratum containing Potenlini, transform and produce Starrhizin to content substantially constant;

Step 3, from fermented liquid separation and purification Starrhizin.

5. endogenetic fungus as claimed in claim 4 transforms Potenlini and generates the method for Starrhizin, it is characterized in that the consisting of of seed culture medium in step 1: in every liter of substratum containing glucose 10～40g, Potenlini (or ammonium glycyrrhizunate) 0.1～1g, KH ₂pO ₄1.0～3.0g, NH ₄nO ₃2.0～5.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.3 g, MgSO ₄0.1～0.5g, CaCl ₂0.01～0.5g, FeSO ₄0.014g, ZnSO ₄7H ₂o 2.9mg, MnCl ₂4H ₂o 2.0mg, CuSO ₄5H ₂o 0.25mg, CoCl ₂6H ₂o 0.24mg, Na ₂moO ₄2H ₂o 0.24mg, H ₃bO ₃0.03mg, all the other are pure water, adjusting pH is 5.0～7.0.

6. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, transforms substratum and consist of in step 2: in every liter of substratum, contain Potenlini (or ammonium glycyrrhizunate) 2～30g, KH ₂pO ₄1.0～3.0g, NH ₄nO ₃2.0～4.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.1g, MgSO ₄0.1～0.5g, CaCl ₂0.01～0.5g, FeSO ₄0.014g, ZnSO ₄7H ₂o 2.9mg, MnCl ₂4H ₂o 2.0mg, CuSO ₄5H ₂o 0.25mg, CoCl ₂6H ₂o 0.24mg, Na ₂moO ₄2H ₂o 0.24mg, H ₃bO ₃0.03mg, all the other are pure water, adjusting pH is 3.5～9.0; Wherein Potenlini (or ammonium glycyrrhizunate) is the inductor that thalline produces beta-glucuronidase enzyme.

7. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, in step 2, culture condition is: 28 DEG C～60 DEG C, and rotating speed 150～300r/min.

8. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, in step 3, the separation and purification of Starrhizin comprises following steps: Büchner funnel suction filtration or six layers of filtered through gauze, collect filtrate, with chloroform equal-volume extraction 2～4 times, water continues with ethyl acetate equal-volume extraction 3～5 times, obtain ethyl acetate phase concentrating under reduced pressure and be Starrhizin crude product, and then be further purified Starrhizin by methods such as purification on normal-phase silica gel column chromatography or reversed phase column chromatographies.