CN115786133A - Aspergillus thermofuscus strain CLH-22, application thereof, leavening agent and method for preparing 3-oxo-glycyrrhetinic acid - Google Patents

Abstract

The invention discloses an aspergillus caldarius strain CLH-22, application thereof, a leaven and a method for preparing 3-oxo-glycyrrhetinic acid, and belongs to the technical field of microorganisms and biology. The invention provides an Aspergillus thermofuscus (Aspergillus caliidus) strain CLH-22, which has a preservation number of: CGMCC No.40213. The invention also provides application of the strain CLH-22 in preparing 3-oxo-glycyrrhetinic acid, a leavening agent based on the strain and a method for preparing 3-oxo-glycyrrhetinic acid. The strain CLH-22 has the characteristics of strong substrate specificity, high conversion efficiency and no byproduct generation, and the yield of 3-O-GA prepared under the conventional condition is 94.6 percent, which is the highest level at present. The invention fills the production vacancy of domestic 3-O-GA products and provides a simple and effective method for preparing 3-O-GA.

Description

A strain of Aspergillus pyrrogenus strain CLH-22 and its application, fermentation agent and preparation of 3-oxoglycerol oxalic acid method

technical field

The invention belongs to the field of microorganism and biotechnology, and in particular relates to an Aspergillus calidoustus strain CLH-22 and its application, a starter and a method for preparing 3-oxoglycyrrhetinic acid.

Background technique

Licorice (Glycyrrhiza uralensis Fisch.) is an important traditional medicinal plant, mainly distributed in China, Kazakhstan, Kyrgyzstan and Mongolia and other countries. According to historical records, licorice has been used as medicine for four thousand years, which can be described as "old in China, licorice in medicine". Licorice not only has the functions of clearing away heat and detoxifying, invigorating the spleen and replenishing qi, nourishing the lungs and relieving cough, and reconciling various medicines, but also can be used for symptoms such as sore throat, spleen and stomach deficiency, and peptic ulcer; it is widely used clinically to treat respiratory, digestive, etc. , reproductive, cardiovascular and endocrine systems and other diseases.

Glycyrrhizic acid (GL) is one of the important pentacyclic triterpenoids in the traditional plant licorice. and other pharmacological activities. Since the structure of glycyrrhizic acid contains two glucuronic acid groups with strong polarity, that is, the C3 position of glycyrrhizin aglycone is connected by β-1,2 glycosidic bonds and β-1,3 glycosidic bonds, making it difficult to penetrate The cell membrane plays a pharmacological role, and the oral utilization rate is low, so the glycyrrhizic acid molecule is not the best form to exert the drug effect. Glycyrrhetinic acid (Glycyrrhetinic acid, GA), as another important pentacyclic triterpene component in licorice, can be prepared by hydrolyzing two molecules of glucuronic acid groups on the structure of glycyrrhizic acid. In the enzymatic green production process of glycyrrhetinic acid, β-glucuronidase (β-glucuronidase, GUS) is often used to hydrolyze two molecules of glucuronic acid groups on the C3 position of glycyrrhizic acid to realize the conversion to glycyrrhetinic acid. Glycyrrhetinic acid has pharmacological activities similar to glycyrrhizic acid, such as anti-inflammatory, hepatoprotective, antiviral, antitumor, antidiuretic, etc. However, long-term use of this drug may cause hyperaldosteronoid syndrome, and it is easy to cause toxicity to normal cells at high concentrations, which limits its clinical application.

For this reason, using glycyrrhetinic acid as a precursor compound to develop novel glycyrrhetinic acid derivative drugs with low toxicity and high efficacy has become one of the research hotspots. As an important structural modification method, ketolation can usually oxidize hydroxyl to carbonyl to realize group transformation, and has important applications in the modification of natural products, such as pentacyclic triterpenoids 3-oxoursolic acid The activity of ursolic acid on human acute promyelocytic leukemia cells (HL-60) is 3.7 times stronger; in addition, 3-oxooleanolic acid can significantly inhibit the growth of cancer cells in different tissues in vitro, including human oral epidermis Cancer cells (KB), highly metastatic mouse melanoma cells (B16-BL6), human colorectal adenocarcinoma cells (HCT-8), human lung adenocarcinoma cells (A549), human fibrosarcoma cells (HT-1080) , human prostate cancer cells (PC-3M), human kidney cancer cells (Ketr-3) and human liver cancer cells (Bel-7402), etc.

3-oxoglycyrrhetinic acid (3-O-GA), also known as 3-ketoglycyrrhetinic acid or 3-oxoglycyrrhetinic acid, is also an important pentacyclic triterpenoid derived from licorice , studies have shown that the carbonylation of the 3-position of glycyrrhetinic acid may be an essential group for neuroprotective activity and plays an important role in neuroprotection. Although 3-oxoglycyrrhetinic acid can be extracted from licorice in a very small amount, it has not been produced in large scale because the planting of licorice is easily affected by the environment and its content is very low. Although the chemical synthesis method can realize the conversion of milligram-level glycyrrhetinic acid to 3-oxoglycyrrhetinic acid, it often has harsh process conditions, consumes a large amount of organic reagents, and easily causes environmental pollution, which greatly limits the production of 3-oxoglycyrrhetinic acid. Batch production of glycyrrhetinic acid. The biocatalytic method has the advantages of mild conditions, environmentally friendly process, low cost, and the ability to prepare a large amount of medicinal precursors, etc., and has become a promising production method for 3-oxoglycyrrhetinic acid. Among them, the conversion form of 3-oxoglycyrrhetinic acid prepared from glycyrrhizic acid by biocatalytic method is shown in Fig. 1 .

At present, there is no domestic report of strains or gene information that can specifically transform glycyrrhetinic acid into 3-oxoglycyrrhetinic acid, and the large-scale production of 3-oxoglycyrrhetinic acid is still in a blank stage. Domestic scholars reported in 2015 the use of the filamentous fungus Cunninghamella blakesleeana for the microbial transformation of glycyrrhetinic acid, through the use of macroporous adsorption resin, silica gel column chromatography and semi-preparative liquid chromatography to extract And separation and purification, obtained 6 conversion products of glycyrrhetinic acid, found that silvery mildew has hydroxylation effect on glycyrrhetinic acid, and can also oxidize the C-3 hydroxyl group to ketone group, but the conversion rate is lower than 1.2%, it is difficult to meet the requirements of large-scale biotransformation of 3-oxoglycyrrhetinic acid.

Foreign researchers reported in 2009 that a strain of Fusarium lini could convert glycyrrhetinic acid into 3-oxoglycyrrhetinic acid, and tests showed that the latter had lipoxygenase resistance (IC ₅₀ =144.2 μM), The lipoxygenase is closely related to the plant's own defense, and participates in the synthesis of antibacterial substances and plant anti-allergic reactions. In addition, the study also provided the NMR identification data of 3-oxoglycyrrhetinic acid, but so far, there has been no report on the transformation efficiency and related gene information of this strain. Therefore, further research on the pharmacological activity of 3-oxoglycyrrhetinic acid is severely limited, and the large-scale production of active components of licorice is hindered.

There is an urgent need in this field to screen a new bacterial strain capable of transforming glycyrrhetinic acid into 3-oxoglycyrrhetinic acid, so as to lay the foundation for the subsequent drug efficacy research and industrial production of 3-oxoglycyrrhetinic acid.

Contents of the invention

Based on the objective problems and needs in this field, the present invention provides an isolated Aspergillus calidoustus strain (Aspergillus calidoustus) CLH-22, which can be used to prepare 3-oxoglycyrrhetinic acid, and has a high preparation rate and fills It fills the vacancy in the domestic industry and lays an important foundation for the industrial production of 3-oxoglycyrrhetinic acid.

Technical scheme of the present invention is as follows:

A Aspergillus calidoustus strain CLH-22, characterized in that its preservation number is CGMCC No.40213.

Use of Aspergillus calidoustus strain CLH-22 with preservation number CGMCC No. 40213 in preparing 3-oxoglycyrrhetinic acid.

The prepared 3-oxoglycyrrhetinic acid has a purity of 93.24%-99.85%.

The yield of said preparation of 3-oxoglycyrrhetinic acid was 94.6%.

A starter for preparing 3-oxoglycyrrhetinic acid, including fermentation active ingredients, characterized in that the fermentation active ingredients include: Aspergillus calidoustus strain CLH-22 with preservation number CGMCC No.40213.

The starter for preparing 3-oxoglycyrrhetinic acid also includes auxiliary materials.

A method for preparing 3-oxoglycyrrhetinic acid, characterized in that it comprises: using Aspergillus calidoustus strain CLH-22 with a preservation number of CGMCC No. 40213 to ferment a substrate.

The substrate concentration is 1-4g/L;

Preferably, the substrate is selected from: glycyrrhizic acid or glycyrrhetinic acid.

The method for preparing 3-oxoglycyrrhetinic acid also includes: purifying the fermented product;

Preferably, the purity of the purified product is 93.24%-99.85%;

Preferably, said product refers to 3-oxoglycyrrhetinic acid.

The fermentation refers to: inoculating Aspergillus calidoustus (Aspergillus calidoustus) strain CLH-22 in the culture medium and culturing;

The inoculation refers to: inoculating the strain CLH-22 in the screening medium to obtain a primary seed liquid culture, and/or inoculating the primary seed liquid in the expansion medium to obtain a secondary seed liquid culture, and/or, inoculating the The secondary seed liquid is inoculated in the fermented enzyme production medium and cultured separately;

Preferably, the inoculation amount of the primary seed liquid in the propagation medium is 1%; the inoculum amount of the secondary seed liquid in the fermented enzyme production medium is 2%;

Preferably, the cultivation refers to constant temperature cultivation at 30° C. for 48 hours.

The invention provides an Aspergillus calidoustus strain CLH-22, the preservation number of which is: CGMCC No.40213.

A product for preparing 3-oxoglycyrrhetinic acid, characterized in that the active ingredient of the product includes/is the Aspergillus pylorus CLH-22 described in claim 1.

The product also includes conventional components for preparing 3-oxoglycyrrhetinic acid and/or medium components for cultivating the Aspergillus pylorus CLH-22.

A method for preparing a product for preparing 3-oxoglycyrrhetinic acid, characterized in that, using Aspergillus pylorus CLH-22 according to claim 1 as the active ingredient or one of the active ingredients for preparing the product; and/or

The Aspergillus pyrrosum CLH-22 described in claim 1 is placed in the packaging box marked with the purpose of preparing 3-oxoglycyrrhetinic acid.

A method for preparing 3-oxoglycyrrhetinic acid, characterized in that, in the process of preparing 3-oxoglycyrrhetinic acid, add and/or use Aspergillus pylorum CLH-22 according to claim 1.

The bacterial agent for preparing 3-oxoglycyrrhetinic acid is characterized in that the active ingredient of the bacterial agent comprises the Aspergillus pylorus CLH-22 described in claim 1.

The bacterial agent for preparing 3-oxoglycyrrhetinic acid is characterized in that the active ingredient of the bacterial agent is the Aspergillus pyrimus CLH-22 described in claim 1.

The bacterial agent also includes conventional ingredients for preparing the bacterial agent.

The application of the Aspergillus pylorus CLH-22 in the preparation of 3-oxoglycyrrhetinic acid.

The present invention isolates a strain of fungus from licorice waste residue soil in Tacheng area, Xinjiang. After ITS sequencing, 18SrRNA sequencing, sequence comparison and bacterial morphology identification, it is determined that the strain is Aspergillus calidoustus, which is named as Aspergillus pyrrosum CLH-22.

The present invention obtains for the first time an Aspergillus calidoustus strain CLH-22 capable of fermenting and obtaining 3-oxoglycyrrhetinic acid (3-O-GA), and first verifies that the highest yield of 3-oxoglycyrrhetinic acid is obtained acid. The present invention has verified the effect of the above-mentioned Aspergillus pylorus CLH-22 in preparing 3-oxoglycyrrhetinic acid (3-O-GA), and found that the bacterial strain can convert glycyrrhizic acid or its salt into glycyrrhetinic acid ( GA), and then converted to 3-oxoglycyrrhetinic acid, and the latter has no by-products in the conversion process, and the yield of 3-oxoglycyrrhetinic acid under conventional conditions is 94.6%, which is the highest technical level .

The preservation information of Aspergillus pylorus CLH-22 of the present invention is as follows:

Name of strain deposit: CLH-22

Deposit number: CGMCC No.40213

Taxonomic name: Aspergillus pyrrosum

Latin name: Aspergillus calidoustus

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: June 16, 2022.

Description of drawings

Fig. 1 is a schematic diagram of the conversion of Aspergillus calidoustus CLH-22 into 3-oxoglycyrrhetinic acid by converting monoammonium glycyrrhizinate.

Fig. 2 is the growth form of Aspergillus pycnogenus CLH-22 in Experimental Example 1 on the PDA plate, wherein, A is the front side of the plate, and B is the reverse side of the plate.

Fig. 3 is the high-efficiency liquid chromatographic analysis figure of the conversion of glycyrrhizic acid monoammonium salt into 3-oxoglycyrrhetinic acid by Aspergillus pyrorrhizinum CLH-22 of experimental example 2, wherein, A: glycyrrhizic acid (before conversion); B: glycyrrhizinic acid Acid standard; C: after conversion.

Fig. 4 is an HPLC analysis chart of purified 3-oxoglycyrrhetinic acid obtained by semi-preparative liquid phase purification in Experimental Example 3.

Fig. 5 is the liquid chromatography-mass spectrometry analysis figure that the Aspergillus pylorus CLH-22 of experimental example 3 converts glycyrrhetinic acid monoammonium salt to generate 3-oxoglycyrrhetinic acid, wherein, A is the mass spectrum of glycyrrhetinic acid, B It is the mass spectrum of 3-oxoglycyrrhetinic acid.

Fig. 6 is a 700M nuclear magnetic resonance ¹ H spectrum analysis diagram of 3-oxoglycyrrhetinic acid obtained after semi-preparative liquid phase purification in Experimental Example 3.

Fig. 7 is a 700M nuclear magnetic resonance ¹³ C spectrum analysis chart of 3-oxoglycyrrhetinic acid obtained after semi-preparative liquid phase purification in Experimental Example 3.

Detailed ways

The present invention will be further described below through specific examples and experimental examples. It should be understood that these examples and experimental examples are only used as explanations and descriptions of the present invention, rather than limiting the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the reagents or materials used can be obtained from commercial sources.

The purity of monoammonium glycyrrhizinate used in Experimental Example 2 was 74.3%, and they were all purchased from Tumushuke Kunshen Plant Extraction Co., Ltd.

The following are the media used in the experimental examples:

Screening medium: monoammonium glycyrrhizinate 2g/L, NH ₄ Cl 3g/L, NaNO ₃ 3g/L, K ₂ HPO ₄ 3g/L, MgSO ₄ 7H ₂ O 0.5g/L, KCl 0.5g/L , FeSO ₄ ·7H ₂ O 0.01g/L, natural pH, sterilized at 121°C for 15 minutes; if a solid medium is configured, that is, a screening plate, add 20g/L agar powder on the basis of the above ingredients.

Proliferation medium: monoammonium glycyrrhizinate 2g/L, glucose monohydrate 1g/L, NH ₄ Cl 3g/L, NaNO ₃ 3g/L, K ₂ HPO ₄ 3g/L, MgSO ₄ 7H ₂ O 0.5g /L, KCl 0.5g/L, FeSO ₄ ·7H ₂ O 0.01g/L, natural pH, sterilized at 115°C for 15min.

Fermentation enzyme production medium: monoammonium glycyrrhizinate 4g/L, NH ₄ Cl 3g/L, NaNO ₃ 3g/L, K ₂ HPO ₄ 3g/L, MgSO ₄ 7H ₂ O 0.5g/L, KCl 0.5g /L, FeSO ₄ ·7H ₂ O 0.01g/L, pH5.5, sterilized at 121°C for 15min.

PDA medium: Potato dextrose agar medium. After peeling the potatoes, cut them into small pieces, weigh 200g of potato pieces, add 900mL of purified water, heat and boil for 30min, filter with gauze, discard the filter residue, and add to the filtrate Add 20g of glucose and 20g of agar powder, dissolve and replenish water to 1L, with natural pH, and sterilize at 115°C for 20min.

GA reaction buffer: Weigh 1g of glycyrrhetinic acid with a purity of 99%, 4.1g of sodium acetate, add 950mL of deionized water, adjust the pH to 5.5 with acetic acid, and then dilute to 1L with deionized water to obtain 1g/L GA reaction buffer.

Among the present invention, the productive rate of 3-oxoglycyrrhetinic acid (3-O-GA) is defined as:

Example 1, bacterial strain CLH-22 of the present invention

The embodiment of this group provides an Aspergillus calidoustus strain CLH-22, which is characterized in that its preservation number is CGMCC No.40213.

Any act of utilizing, using, selling, promising to sell, producing, preparing, cultivating, propagating and fermenting Aspergillus calidoustus strain CLH-22 with the preservation number CGMCC No. 40213 falls within the protection scope of the present invention.

According to the teachings and inspirations of the present invention, those skilled in the art select suitable adjuvants for deployment in combination with common technical means in the field of microbiological technology for actual production needs, and prepare Aspergillus calidoustus (Aspergillus calidoustus) with the preservation number of the present invention as CGMCC No.40213 Strain CLH-22 can be made into various dosage form products that meet the production requirements of various processes, for example, powder, tablet, and liquid.

The 2nd group embodiment, the purposes of bacterial strain CLH-22 of the present invention

This group of examples provides the use of Aspergillus calidoustus strain CLH-22 with the deposit number CGMCC No. 40213 in preparing 3-oxoglycyrrhetinic acid.

In a specific embodiment, the prepared 3-oxoglycyrrhetinic acid has a purity of 93.24%-99.85%.

In other embodiments, the yield of preparing 3-oxoglycyrrhetinic acid is 94.6%.

The 3rd group of embodiment, leavening agent of the present invention

The examples of this group provide a starter for preparing 3-oxoglycyrrhetinic acid. All the examples in this group have the following common features: the starter contains fermentation active ingredients, and the fermentation active ingredients include: Aspergillus calidoustus strain CLH-22 with a preservation number of CGMCC No. 40213.

In a further fact, the starter for preparing 3-oxoglycyrrhetinic acid also includes: auxiliary materials.

In a more specific embodiment, the auxiliary materials are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers , Surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, etc.

According to the content of the present invention, due to the different needs in actual production and application, combined with the conventional technical means in the field of bacterial agent preparation, production, and processing technology (for example, "Encyclopedia of Preparation Technology", "Pharmaceutical Preparation Technology", etc.), this Those skilled in the art can select and formulate the above-mentioned excipients, and make the Aspergillus calidoustus strain CLH-22 with the preservation number CGMCC No. 40213 into different dosage forms, such as powder, tablet, liquid and so on.

The 4th group embodiment, the method for preparing 3-oxoglycyrrhetinic acid of the present invention

This group of examples provides a method for preparing 3-oxoglycyrrhetinic acid. All the examples in this group have the following common features: the method includes: using Aspergillus calidoustus strain CLH-22 with the preservation number CGMCC No. 40213 to ferment the substrate.

In a specific embodiment, the substrate concentration is 1-4g/L;

Preferably, the substrate is selected from: glycyrrhizic acid or glycyrrhetinic acid.

In a further embodiment, the method for preparing 3-oxoglycyrrhetinic acid further includes: purifying the fermented product;

Preferably, the purity of the purified product is 93.24%-99.85%;

Preferably, said product refers to 3-oxoglycyrrhetinic acid.

In some embodiments, the fermentation refers to: inoculating Aspergillus calidoustus (Aspergillus calidoustus) strain CLH-22 in the medium for cultivation;

The inoculation refers to: inoculating the strain CLH-22 in the screening medium to obtain a primary seed liquid culture, and/or inoculating the primary seed liquid in the expansion medium to obtain a secondary seed liquid culture, and/or, inoculating the The secondary seed liquid is inoculated in the fermented enzyme production medium and cultured separately;

Preferably, the inoculation amount of the primary seed liquid in the propagation medium is 1%; the inoculum amount of the secondary seed liquid in the fermented enzyme production medium is 2%;

Preferably, the cultivation refers to constant temperature cultivation at 30° C. for 48 hours.

Experimental Example 1, Isolation, Purification and Screening of Bacterial Strains

Soil samples were collected from the licorice waste dump in Xinjiang and the root soil of nearby plants. Weigh 10g of the soil sample, put it into 30mL of sterile water, shake it fully for 20min to fully disperse the sample, and let it stand for 10min. Under a sterile environment, draw 1mL of the supernatant, and then perform gradient dilution with sterile water to prepare dilutions of 10 ^-1 to 10 ^-6 times concentration respectively. Take 500 μL of the dilutions of each concentration, spread evenly on the screening plate, put it upright for 30 minutes, and then place it upside down in a constant temperature incubator at 30°C for cultivation. Observe the growth of the bacteria on the screening plate every 12 hours. After 2-7 days, the newly grown colonies were further streaked and separated to obtain pure strains.

The pure strains were picked from the screening plate to the screening medium, and placed in a constant temperature shaking incubator at 30° C. at 200 rpm for 3-5 days. Take 100 μL of the fermentation culture solution, add it to 900 μL methanol, mix well, filter through a 0.22 μm organic filter membrane, and analyze it by high-performance liquid chromatography. A fungus was obtained and named CLH-22.

The strain CLH-22 was cultured on a PDA plate, and its growth form in the nutrient-rich medium was observed. The results are shown in Figure 2.

The ITS and 18S rRNA sequences of strain CLH-22 were sequenced, and the nucleotide sequences thereof are shown in SEQ ID No.1 and SEQ ID No.2, respectively. The above-mentioned sequence is carried out BLAST sequence comparison, the ITS sequence length of CLH-22 bacterium is 584bp, has 96%-100% homology with Aspergillus (Aspergillus), 18S rRNA sequence length is 1683bp, has 98%-100% homology with Aspergillus % homology. Combined with its colony morphology analysis, it was identified as Aspergillus calidoustus and named CLH-22. Send the CLH-22 strain to the strain preservation center for preservation. The preservation information of the strain is as follows:

Name of strain deposit: CLH-22

Deposit number: CGMCC No.40213

Taxonomic name: Aspergillus pyrrosum

Latin name: Aspergillus calidoustus

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: June 16, 2022.

Experimental example 2, enzyme production culture and substrate transformation of Aspergillus pylorus CLH-22

Aspergillus pylorus CLH-22 was inoculated in the screening medium as the primary seed liquid, and cultured in a constant temperature shaking incubator at 30°C for 48 hours. The inoculum amount of 1% by volume was transferred to the multiplication medium as the secondary seed liquid, and cultured in a constant temperature shaking incubator at 30°C for 48 hours. With an inoculum volume of 2% by volume, transfer the secondary seed liquid to the fermented enzyme production medium, cultivate it in a constant temperature shaking incubator at 30°C, and take samples every 12 hours, and use high performance liquid chromatography to detect monoammonium glycyrrhizinate The results of salt consumption, glycyrrhetinic acid and 3-oxoglycyrrhetinic acid production are shown in Figure 3. Wherein, the HPLC detection method: the mobile phase is methanol:0.6% acetic acid=84:16, the flow rate is 0.8mL/min, the chromatographic column is Kromasil100-3.5-C18, and the detection wavelength is 254nm. Among them, the peak elution time of the substrate glycyrrhetinic acid was 3.175 min, the peak elution time of the standard glycyrrhetinic acid was 9.414 min, and the peak elution time of the final product 3-oxoglycyrrhetinic acid was 9.703 min. The results showed that the production of glycyrrhetinic acid and 3-oxoglycyrrhetinic acid could be detected at the same time after 48 hours of fermentation, but the accumulation of glycyrrhetinic acid was very low during the conversion process; Glycyrrhetinic acid did not increase significantly. After calculation, the yield of product 3-oxoglycyrrhetinic acid is 94.6%.

According to the above test results, the ability of Aspergillus pylorus CLH-22 to directly convert glycyrrhetinic acid into 3-oxoglycyrrhetinic acid was verified in vitro.

Take 4mL of fermentation broth, centrifuge at 12000rpm for 10min, discard the supernatant, collect the precipitated bacteria in the same centrifuge tube, wash the bacteria twice with sterile water, centrifuge at 12000rpm for 5min, discard the supernatant. Add an appropriate amount of mixed Glass bead broken beads and Zarconia/Silica broken beads, and add 1 mL of 1 g/L GA reaction buffer (GA purity 99%) to the centrifuge tube. A bead-beating instrument was used to crush the bacteria. After the bacteria were crushed, the centrifuge tube was placed in a constant temperature shaking incubator at 30° C. and 200 rpm to react for 24 hours. The results of high-performance liquid chromatography showed that the crude enzyme solution of Aspergillus pyromyces CLH-22 could directly convert GA into 3-oxoglycyrrhetinic acid in vitro without any by-products, which was consistent with the above results. Combined with enzyme production culture and in vitro verification, it was shown that there is not only β-glucuronidase that can hydrolyze GL to produce GA in Aspergillus pyrrosum CLH-22, but also the C3 position that can convert GA into 3-oxoglycyrrhetinic acid hydroxyl oxidase. Under the synergistic action of the two enzymes, the efficient conversion from GL to 3-oxoglycyrrhetinic acid can be realized.

Pure product preparation and identification method of experimental example 3, 3-oxoglycyrrhetinic acid

According to the fermentation method described in Experimental Example 2, 1 L of the fermentation medium after conversion was centrifuged at 12000 rpm for 15 min, and the supernatant was discarded. Add 100mL of ethanol to the precipitated part, stir in a water bath at 50°C for 20min, fully dissolve, filter while hot, and keep the filtrate part. Add 100 mL of pre-cooled purified water to the filtrate, stir for 10 minutes, and then let it stand at room temperature for 1 hour. At this time, it can be observed that more solids are precipitated. Centrifuge at 8000rpm for 10min, collect the filter cake part, and dry it with a vacuum rotary evaporator for 2h to volatilize the residual ethanol to obtain a primary purified product. Further, the primary purified product was frozen at -80°C, and then subjected to vacuum freeze-drying to obtain a crude product. The purity of the crude product was checked by high performance liquid chromatography. After calculation, the purity of 3-oxoglycyrrhetinic acid in the crude product is 93.24%.

The product was further purified by semi-preparative liquid phase as follows:

Take 100 mg of the crude product obtained above, add 21.5 mL of methanol and 3.5 mL of acetic acid with a concentration of 0.6%, stir for 30 min, and fully dissolve, then use semi-preparative liquid chromatography for further purification to obtain a purified sample liquid. Wherein, the mobile phase ratio is methanol:0.6% acetic acid=84:16, the flow rate is 5mL/min, and the detection wavelength is 254nm. The sample liquid purified by semi-preparative liquid chromatography was dried with a vacuum rotary evaporator for 2 hours to volatilize methanol and acetic acid to obtain a secondary purified product. Further, the secondary purified product was frozen at -80°C, and then vacuum freeze-dried to obtain the purified product. The purity of the purified product was detected by high performance liquid chromatography, and the results are shown in Figure 4. After calculation, the purity of the secondary purified 3-oxoglycyrrhetinic acid is 99.85%.

The high-purity 3-oxoglycyrrhetinic acid obtained from the secondary purification was analyzed by liquid chromatography-mass spectrometry, and the results are shown in Figure 5. The mass spectrometry results in Figure 5A show that the measured (MH) ^- of glycyrrhetinic acid is 469.3323. Figure 5B mass spectrometry results show that the predicted (MH ⁾ of 3-oxoglycyrrhetinic acid is 467.3167, and the measured (MH) of the detected compound ^is 467.3168, that is, the molecular weight of the detected compound and the predicted molecular weight of 3-oxoglycyrrhetinic acid Consistent, confirm that the molecular weight of the secondary purification product is consistent with 3-oxoglycyrrhetinic acid.

Bruker Ascend 700M nuclear magnetic resonance spectrometer was used for structural identification, the method is as follows:

Take 15 mg of the high-purity 3-oxoglycyrrhetinic acid obtained from secondary purification and dissolve it in 1 mL of deuterated chloroform, and perform Bruker Ascend 700M nuclear magnetic resonance ¹ H spectrum and ¹³ C spectrum analysis respectively. ^{The 1} H spectrum results are shown in Figure 6, The results of ¹³ C spectrum are shown in Fig. 7 . The hydrogen spectrum chemical shift of glycyrrhetinic acid is predicted, and the chemical shift of 3-OH formation is 4.77ppm, while the results of ¹ H spectrum in the present invention show that no chemical shift of 3-OH formation is detected at the 3-position of the predicted compound. The carbon spectrum chemical shifts of glycyrrhetinic acid and 3-oxoglycyrrhetinic acid are predicted respectively, and the chemical shifts formed by 3C are 78.6ppm and 217.0ppm respectively, while the results of ¹³ C spectrum in the present invention show that there is only a characteristic of 217.309ppm chemical shift without the characteristic 3-position chemical shift for glycyrrhetinic acid formation. Therefore, comprehensive high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance analysis, it is determined that the compound purified by the present invention is 3-oxoglycyrrhetinic acid.

Claims (10)

1. Aspergillus caldolatus strain CLH-22 is characterized in that the preservation number is CGMCC No.40213.

2. Application of Aspergillus caldolatus strain CLH-22 with preservation number of CGMCC No.40213 in preparation of 3-oxo-glycyrrhetinic acid.

3. The use of the Aspergillus thermofuscus (Aspergillus caliidus) strain CLH-22 with the collection number of CGMCC No.40213 for the preparation of 3-oxoglycyrrhetinic acid according to claim 2, wherein the purity of the prepared 3-oxoglycyrrhetinic acid is 93.24% to 99.85%.

4. Use of the Aspergillus thermofuscus (Aspergillus caliidus) strain CLH-22 with collection number CGMCC No.40213 for the preparation of 3-oxoglycyrrhetinic acid according to claim 2 or 3, characterized in that the yield of the preparation of 3-oxoglycyrrhetinic acid is 94.6%.

5. A leaven for preparing 3-oxo glycyrrhetinic acid comprises fermentation active ingredients, and is characterized in that the fermentation active ingredients comprise: aspergillus calidoticus (Aspergillus caliidus) strain CLH-22 with the preservation number of CGMCC No.40213.

6. The starter for producing 3-oxoglycyrrhetinic acid according to claim 5, further comprising: and (5) auxiliary materials.

7. A method for preparing 3-oxoglycyrrhetinic acid, comprising: fermenting the substrate by adopting an Aspergillus thermofuscus (Aspergillus caliidus) strain CLH-22 with the preservation number of CGMCC No.40213.

8. The method of claim 7, wherein the substrate concentration is 1-4g/L;

and/or, the substrate is selected from: glycyrrhizic acid or glycyrrhetinic acid.

9. The method of claim 7, further comprising: purifying the fermented product;

and/or the purity of the purified product is 93.24% -99.85%;

and/or, the product refers to 3-oxo glycyrrhetinic acid.

10. The method of claim 7 or 9, wherein the fermentation is: inoculating Aspergillus thermofuscus (Aspergillus caliidus) strain CLH-22 into culture medium for culture;

the inoculation is as follows: inoculating the strain CLH-22 to a screening culture medium to obtain a first-stage seed liquid for culture, and/or inoculating the first-stage seed liquid to a propagation culture medium to obtain a second-stage seed liquid for culture, and/or inoculating the second-stage seed liquid to a fermentation enzyme production culture medium for respective culture;

and/or the inoculation amount of the first-class seed liquid in the propagation culture medium is 1 percent; the inoculation amount of the secondary seed liquid in the fermentation enzyme production culture medium is 2 percent;

and/or, the culturing refers to: culturing at 30 deg.C for 48h.

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