A strain of Aspergillus thermophilus CLH-22 and its uses, fermentation agent and method for preparing 3-oxoglycyrrhetinic acid

Abstract

This invention relates to a *Aspergillus calidoustus* strain CLH-22, its uses, a fermentation agent, and a method for preparing 3-oxoglycyrrhetinic acid (3-OGA), belonging to the field of microbiology and biotechnology. This invention provides a *Aspergillus calidoustus* strain CLH-22, with the accession number CGMCC No. 40213. This invention also provides the uses of this strain CLH-22 in the preparation of 3-OGA, as well as a fermentation agent based on this strain and a method for preparing 3-OGA. The strain CLH-22 of this invention exhibits strong substrate specificity, high conversion efficiency, and no byproduct formation. Under conventional conditions, the yield of 3-OGA is 94.6%, which is currently the highest level. This invention fills the production gap of 3-OGA products in China and provides a simple and effective method for preparing 3-OGA.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and biotechnology, specifically relating to a strain of Aspergillus calidoustus CLH-22 and its uses, fermentation agent, and method for preparing 3-oxoglycyrrhetinic acid. Background Technology

[0002] Licorice (Glycyrrhiza uralensis Fisch.) is an important traditional medicinal plant, mainly distributed in China, Kazakhstan, Kyrgyzstan, and Mongolia. Historical records indicate that licorice has been used medicinally for four thousand years, truly embodying the saying "Licorice is ancient in China, and ancient in medicine." Licorice not only possesses the effects of clearing heat and detoxifying, tonifying the spleen and replenishing qi, moistening the lungs and relieving cough, and harmonizing other medicines, but it can also be used for symptoms such as sore throat, spleen and stomach deficiency, and peptic ulcers. Clinically, it is widely used to treat various diseases of the respiratory, digestive, reproductive, cardiovascular, and endocrine systems.

[0003] Glycyrrhizic acid (GL) is an important pentacyclic triterpenoid compound found in the traditional plant licorice, possessing pharmacological activities such as antibacterial, anti-inflammatory, antiviral, antitumor, antioxidant, anti-aging, hepatoprotective, and anti-immune responses. However, due to the presence of two highly polar glucuronic acid groups in its structure—linked at the C3 position by β-1,2 and β-1,3 glycosidic bonds—glycyrrhizic acid does not readily cross cell membranes to exert its pharmacological effects, resulting in low oral bioavailability. Therefore, glycyrrhizic acid is not the optimal form for exerting its therapeutic effects. Glycyrrhetinic acid (GA), another important pentacyclic triterpenoid component of licorice, can be prepared by hydrolyzing two glucuronic acid molecules from glycyrrhizic acid. In the enzymatic green production process of glycyrrhetinic acid, β-glucuronidase (GUS) is commonly used to hydrolyze the two glucuronic acid molecules at the C3 position of glycyrrhizic acid to achieve the conversion to glycyrrhetinic acid. Glycyrrhetinic acid has similar pharmacological activities to glycyrrhizic acid, such as anti-inflammatory, hepatoprotective, antiviral, antitumor, and antidiuretic effects. However, long-term use of this drug may cause hyperaldosteronism, and high concentrations can easily cause toxicity to normal cells, limiting its clinical application.

[0004] Therefore, developing novel glycyrrhetinic acid derivatives with low toxicity and high efficacy, using glycyrrhetinic acid as a precursor compound, has become a research hotspot. Ketolation, as an important structural modification method, typically oxidizes hydroxyl groups to carbonyl groups to achieve group transformation, and has important applications in the modification of natural products. For example, the pentacyclic triterpenoid compound 3-oxoursolic acid is 3.7 times more active than ursolic acid against human acute promyelocytic leukemia cells (HL-60). Furthermore, 3-oxooleanolic acid can significantly inhibit the growth of cancer cells in different tissues in vitro, including human oral epidermal carcinoma 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 renal cancer cells (Ketr-3), and human liver cancer cells (Bel-7402).

[0005] 3-O-glycyrrhetic acid (3-O-GA), also known as 3-keto-glycyrrhetic acid or 3-carbonyl-glycyrrhetic acid, is an important pentacyclic triterpenoid compound derived from licorice. Studies have shown that carbonylation at the 3-position of glycyrrhetic acid may serve as an essential group for neuroprotective activity, playing a crucial role in neuroprotection. Although 3-O-glycyrrhetic acid can be extracted in very small quantities from the plant licorice, large-scale production has not been achieved due to the susceptibility of licorice cultivation to environmental factors and its very low content. While chemical synthesis can achieve the conversion of milligram-level glycyrrhetic acid to 3-O-glycyrrhetic acid, it often suffers from stringent process conditions, requires large amounts of organic reagents, and is prone to environmental pollution, significantly limiting the mass production of 3-O-glycyrrhetic acid. In contrast, biocatalysis offers advantages such as mild conditions, environmental friendliness, low cost, and the ability to prepare large quantities of pharmaceutical precursors, making it a highly promising method for the production of 3-O-glycyrrhetic acid. Among them, the conversion form of 3-oxoglycyrrhizic acid to glycyrrhizic acid via biocatalysis is as follows: Figure 1 As shown.

[0006] Currently, no strains or gene information have been reported in China that can specifically convert glycyrrhetinic acid to 3-oxoglycyrrhetinic acid, leaving the field of large-scale production of 3-oxoglycyrrhetinic acid in a blank stage. In 2015, Chinese scholars reported a study on the microbial transformation of glycyrrhetinic acid using the filamentous fungus *Cunninghamella blakesleeana*. Through extraction and purification using macroporous adsorption resin, silica gel column chromatography, and semi-preparative liquid chromatography, six glycyrrhetinic acid transformation products were obtained. It was found that *Cunninghamella blakesleeana* has a hydroxylation effect on glycyrrhetinic acid and can also oxidize the C-3 hydroxyl group to a ketone group, but the conversion rate was less than 1.2%, which is insufficient to meet the requirements for large-scale biotransformation of 3-oxoglycyrrhetinic acid.

[0007] In 2009, foreign researchers reported that a strain of *Fusarium lini* could convert glycyrrhetinic acid to 3-oxoglycyrrhetinic acid, and experiments showed that the latter exhibited lipoxygenase resistance (IC50). 50 =144.2 μM), and lipoxygenase is closely related to plant defense, participating in the synthesis of antibacterial substances and plant anti-allergic responses. In addition, this study also provided NMR identification data for 3-oxoglycyrrhetinic acid, but to date, no transformation efficiency or related gene information for this strain has been reported. Therefore, this severely limits further pharmacological activity research on 3-oxoglycyrrhetinic acid and hinders the large-scale production of glycyrrhiza-based active components.

[0008] There is an urgent need in this field to screen a new strain that can convert glycyrrhetinic acid into 3-oxoglycyrrhetinic acid, so as to lay the foundation for subsequent pharmacodynamic studies and industrial production of 3-oxoglycyrrhetinic acid. Summary of the Invention

[0009] Based on the objective difficulties and needs in this field, this invention provides an isolated Aspergillus calidoustus strain CLH-22, which can be used to prepare 3-oxoglycyrrhetinic acid with high yield, filling a gap in the domestic industry and laying an important foundation for the industrial production of 3-oxoglycyrrhetinic acid.

[0010] The technical solution of the present invention is as follows:

[0011] A strain of Aspergillus calidoustus, CLH-22, is characterized by its preservation number CGMCC No.40213.

[0012] Use of Aspergillus calidoustus strain CLH-22 with accession number CGMCC No.40213 in the preparation of 3-oxoglycyrrhetinic acid.

[0013] The purity of the prepared 3-oxoglycyrrhetinic acid was 93.24%-99.85%.

[0014] The yield of 3-oxoglycyrrhetinic acid was 94.6%.

[0015] A fermentation agent for preparing 3-oxoglycyrrhetinic acid, comprising a fermentation active ingredient, characterized in that the fermentation active ingredient comprises: Aspergillus calidoustus strain CLH-22 with preservation number CGMCC No.40213.

[0016] The fermentation agent for preparing 3-oxoglycyrrhetinic acid also includes: excipients.

[0017] A method for preparing 3-oxoglycyrrhetinic acid, characterized in that it includes fermenting the substrate with Aspergillus calidoustus strain CLH-22, which has the preservation number CGMCCNo.40213.

[0018] The substrate concentration is 1-4 g / L;

[0019] Preferably, the substrate is selected from glycyrrhizic acid or glycyrrhetinic acid.

[0020] The method for preparing 3-oxoglycyrrhetinic acid further includes: purifying the fermentation product;

[0021] Preferably, the purity of the purified product is 93.24%-99.85%;

[0022] Preferably, the product refers to 3-oxoglycyrrhetinic acid.

[0023] The fermentation refers to: inoculating Aspergillus calidoustus strain CLH-22 into a culture medium and culturing it;

[0024] The inoculation refers to: inoculating strain CLH-22 into a screening medium to obtain a primary seed culture, and / or inoculating the primary seed culture into a propagation medium to obtain a secondary seed culture, and / or inoculating the secondary seed culture into a fermentation enzyme production medium for separate culture.

[0025] Preferably, the inoculum amount of the primary seed culture in the propagation medium is 1%; the inoculum amount of the secondary seed culture in the fermentation enzyme production medium is 2%.

[0026] Preferably, the culture refers to a constant temperature culture at 30°C for 48 hours.

[0027] This invention provides a strain of Aspergillus calidoustus, CLH-22, with accession number CGMCC No. 40213.

[0028] The product for preparing 3-oxoglycyrrhetinic acid is characterized in that the active ingredient of the product includes / is Aspergillus thermophilus CLH-22 as described in claim 1.

[0029] The product also includes conventional components for preparing 3-oxoglycyrrhetinic acid and / or culture medium components for culturing Aspergillus thermophilus CLH-22.

[0030] A method for preparing a product containing 3-oxoglycyrrhetinic acid, characterized in that Aspergillus thermophilus CLH-22 as described in claim 1 is used as an active ingredient or one of the active ingredients in the preparation of the product; and / or

[0031] The thermophilic Aspergillus CLH-22 of claim 1 is placed in a packaging box labeled for use in the preparation of 3-oxoglycyrrhetinic acid.

[0032] A method for preparing 3-oxoglycyrrhetinic acid, characterized in that, during the preparation of 3-oxoglycyrrhetinic acid, Aspergillus thermophilus CLH-22 as described in claim 1 is added and / or used.

[0033] The bacterial agent for preparing 3-oxoglycyrrhetinic acid is characterized in that the active ingredient of the bacterial agent includes Aspergillus thermophilus CLH-22 as described in claim 1.

[0034] The bacterial agent for preparing 3-oxoglycyrrhetinic acid is characterized in that the active ingredient of the bacterial agent is Aspergillus thermophilus CLH-22 as described in claim 1.

[0035] The microbial agent also includes conventional components used in its preparation.

[0036] The application of Aspergillus pyrophorus CLH-22 in the preparation of 3-oxoglycyrrhetinic acid.

[0037] This invention isolated a fungus from licorice waste soil in Tacheng region of Xinjiang. Through ITS sequencing, 18S rRNA sequencing, sequence alignment and bacterial morphology identification, the strain was identified as Aspergillus calidoustus and named Aspergillus calidoustus CLH-22.

[0038] This invention provides the first known strain of Aspergillus calidoustus, CLH-22, capable of fermenting to obtain 3-oxoglycyrrhizic acid (3-O-GA), and for the first time, verifies the highest yield of 3-oxoglycyrrhizic acid. The invention validates the effectiveness of the aforementioned Aspergillus calidoustus CLH-22 in preparing 3-oxoglycyrrhizic acid (3-O-GA), finding that this strain can first convert glycyrrhizic acid or its salts to glycyrrhizic acid (GA), and then to 3-oxoglycyrrhizic acid, with no byproducts generated during the latter conversion. Under conventional conditions, the yield of 3-oxoglycyrrhizic acid is 94.6%, representing the highest existing technical level.

[0039] The preservation information of Aspergillus thermophilus CLH-22 of the present invention is as follows:

[0040] Preservation name of strain: CLH-22

[0041] Collection Number: CGMCC No. 40213

[0042] Classification and nomenclature: Aspergillus thermophilus

[0043] Latin name: Aspergillus calidoustus

[0044] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0045] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0046] Date of preservation: June 16, 2022. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the conversion of glycyrrhizic acid monoammonium salt to 3-oxoglycyrrhetinic acid by Aspergillus calidoustus CLH-22.

[0048] Figure 2 The growth morphology of Aspergillus thermophilus CLH-22 in Experiment Example 1 is shown on a PDA plate, where A is the front side of the plate and B is the back side of the plate.

[0049] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the conversion of glycyrrhizic acid monoammonium salt to 3-oxoglycyrrhetinic acid by Aspergillus pyrolysis CLH-22 in Experiment Example 2. In the figure, A: glycyrrhizic acid (before conversion); B: glycyrrhetinic acid standard; C: after conversion.

[0050] Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of purified 3-oxoglycyrrhetinic acid obtained by semi-preparative HPLC purification in Experiment Example 3.

[0051] Figure 5 This is a liquid chromatography-mass spectrometry (LC-MS) chromatogram of the conversion of glycyrrhizic acid monoammonium salt to 3-oxoglycyrrhizic acid by Aspergillus pyrolysis CLH-22 in Experiment Example 3. In the figure, A is the mass spectrum of glycyrrhizic acid and B is the mass spectrum of 3-oxoglycyrrhizic acid.

[0052] Figure 6 The 700 M NMR of 3-oxoglycyrrhetinic acid obtained after semi-preparative liquid phase purification in Experimental Example 3 is shown. 1 H-spectral analysis diagram.

[0053] Figure 7 The 700 M NMR of 3-oxoglycyrrhetinic acid obtained after semi-preparative liquid phase purification in Experimental Example 3 is shown. 13 C-spectral analysis diagram. Detailed Implementation

[0054] The present invention will be further illustrated below through specific embodiments and experimental examples. It should be understood that these embodiments and experimental examples are for explanation and illustration only, and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the reagents or materials used are all commercially available.

[0055] The glycyrrhizic acid monoammonium salt used in Experiment Example 2 had a purity of 74.3% and was purchased from Kunshen Plant Extract Co., Ltd. in Tumushuke City.

[0056] The following are the culture media used in the experimental examples:

[0057] Screening medium: 2 g / L glycyrrhizic acid monoammonium salt, 3 g / L NH4Cl, 3 g / L NaNO3, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L KCl, 0.01 g / L FeSO4·7H2O, natural pH, sterilized at 121℃ for 15 min; if preparing solid medium, i.e. screening plates, add 20 g / L agar powder to the above components.

[0058] Propagation medium: 2 g / L glycyrrhizic acid monoammonium salt, 1 g / L glucose monohydrate, 3 g / L NH4Cl, 3 g / L NaNO3, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L KCl, 0.01 g / L FeSO4·7H2O, natural pH, sterilized at 115℃ for 15 min.

[0059] Fermentation enzyme production medium: 4 g / L glycyrrhizic acid monoammonium salt, 3 g / L NH4Cl, 3 g / L NaNO3, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L KCl, 0.01 g / L FeSO4·7H2O, pH 5.5, sterilized at 121℃ for 15 min.

[0060] PDA medium: Potato glucose agar medium. After peeling the potatoes, cut them into small pieces. Weigh 200g of potato pieces, add 900mL of purified water, heat to boiling for 30min, filter with gauze, discard the filter residue, add 20g of glucose and 20g of agar powder to the filtrate, dissolve, add water to 1L, maintain natural pH, and sterilize at 115℃ for 20min.

[0061] GA reaction buffer: Weigh 1g of 99% glycyrrhetinic acid and 4.1g of sodium acetate, add them to 950mL of deionized water, adjust the pH to 5.5 with acetic acid, and then bring the volume to 1L with deionized water to obtain 1g / L GA reaction buffer.

[0062] The yield of 3-oxoglycyrrhetinic acid (3-O-GA) in this invention is defined as follows:

[0063]

[0064] Group 1 Examples, the strain CLH-22 of the present invention

[0065] This set of embodiments provides a strain of Aspergillus calidoustus CLH-22, characterized in that its preservation number is CGMCC No.40213.

[0066] Any use, sale, offer for sale, production, preparation, cultivation, propagation, fermentation, or preservation of Aspergillus calidoustus strain CLH-22 with CGMCC No. 40213 falls within the protection scope of this invention.

[0067] Based on the teachings and inspiration of this invention, and for actual production needs, those skilled in the art can select appropriate excipients and formulate various dosage forms, such as powders, tablets, and liquids, from the Aspergillus calidoustus strain CLH-22 (accession number CGMCC No. 40213) that meet the requirements of various production processes, in combination with commonly used techniques in the field of microbial processes.

[0068] Group 2 Examples, Uses of the Invention Straw C1H-22

[0069] This set of examples provides the use of Aspergillus calidoustus strain CLH-22 (accession number CGMCC No. 40213) in the preparation of 3-oxoglycyrrhetinic acid.

[0070] In a specific embodiment, the purity of the prepared 3-oxoglycyrrhetinic acid was 93.24%-99.85%.

[0071] In other embodiments, the yield of 3-oxoglycyrrhetinic acid was 94.6%.

[0072] Group 3 Examples, Fermentation Agent of the Present Invention

[0073] This set of embodiments provides a starter culture for preparing 3-oxoglycyrrhetinic acid. All embodiments in this set share the following common feature: the starter culture includes a fermentation-active ingredient, which includes: Aspergillus calidoustus strain CLH-22 with accession number CGMCCNo.40213.

[0074] In further detail, the fermentation agent for preparing 3-oxoglycyrrhetinic acid also includes: excipients.

[0075] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0076] According to the present invention, based on different needs in actual production applications, and combined with conventional technical means in the fields of microbial preparation, production, and processing (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare the Aspergillus calidoustus strain CLH-22 with accession number CGMCC No.40213 into different dosage forms, such as powders, tablets, liquids, etc.

[0077] Group 4 Examples, the method for preparing 3-oxoglycyrrhetinic acid according to the present invention

[0078] This set of embodiments provides a method for preparing 3-oxoglycyrrhetinic acid. All embodiments in this set share the following common feature: the method includes fermenting the substrate using Aspergillus calidoustus strain CLH-22 with accession number CGMCC No. 40213.

[0079] In a specific embodiment, the substrate concentration is 1-4 g / L;

[0080] Preferably, the substrate is selected from glycyrrhizic acid or glycyrrhetinic acid.

[0081] In a further embodiment, the method for preparing 3-oxoglycyrrhetinic acid further includes: purifying the fermentation product;

[0082] Preferably, the purity of the purified product is 93.24%-99.85%;

[0083] Preferably, the product refers to 3-oxoglycyrrhetinic acid.

[0084] In some embodiments, the fermentation refers to: inoculating Aspergillus calidoustus strain CLH-22 into a culture medium and culturing it;

[0085] The inoculation refers to: inoculating strain CLH-22 into a screening medium to obtain a primary seed culture, and / or inoculating the primary seed culture into a propagation medium to obtain a secondary seed culture, and / or inoculating the secondary seed culture into a fermentation enzyme production medium for separate culture.

[0086] Preferably, the inoculum amount of the primary seed culture in the propagation medium is 1%; the inoculum amount of the secondary seed culture in the fermentation enzyme production medium is 2%.

[0087] Preferably, the culture refers to a constant temperature culture at 30°C for 48 hours.

[0088] Experiment 1: Isolation, purification, and screening of bacterial strains

[0089] Soil samples were collected from licorice waste piles and nearby plant root soil in Xinjiang. 10g of soil sample was weighed and placed in 30mL of sterile water, shaken thoroughly for 20 minutes to ensure complete dispersion, and then allowed to stand for 10 minutes. Under sterile conditions, 1mL of the supernatant was collected and serially diluted with sterile water to prepare 10 mL of the supernatant. -1 Up to 10 -6 Prepare dilutions at multiple concentrations. Take 500 μL of each dilution and spread it evenly on a screening plate. Incubate upright for 30 min, then invert the plate and incubate at 30°C. Observe the growth of bacteria on the screening plate every 12 h. After 2-7 days, further streak the newly grown colonies to obtain pure cultures.

[0090] Pure strains were picked from screening plates and transferred to screening medium, and cultured in a constant temperature shaking incubator at 30℃ and 200rpm for 3-5 days. 100μL of the fermentation broth was added to 900μL of methanol, mixed thoroughly, filtered through a 0.22μm organic filter membrane, and analyzed by high-performance liquid chromatography (HPLC). Strains with the functions of hydrolyzing glycyrrhizic acid and oxidizing glycyrrhetinic acid were preliminarily screened, and one fungus was obtained and named CLH-22.

[0091] The strain CLH-22 was cultured on PDA plates, and its growth morphology in the nutrient-rich medium was observed. The results are as follows: Figure 2 As shown.

[0092] The ITS and 18S rRNA sequences of strain CLH-22 were sequenced, and their nucleotide sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. BLAST alignment of these sequences revealed that the ITS sequence of CLH-22 was 584 bp in length, showing 96%-100% homology with *Aspergillus*, and the 18S rRNA sequence was 1683 bp in length, showing 98%-100% homology with *Aspergillus*. Based on colony morphology analysis, it was identified as *Aspergillus calidoustus* and named CLH-22. Strain CLH-22 was deposited at a microbial culture center; the preservation information is as follows:

[0093] Preservation name of strain: CLH-22

[0094] Collection Number: CGMCC No. 40213

[0095] Classification and nomenclature: Aspergillus thermophilus

[0096] Latin name: Aspergillus calidoustus

[0097] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0098] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0099] Date of preservation: June 16, 2022.

[0100] Experimental Example 2: Enzyme Production Culture and Substrate Transformation of Aspergillus thermophilus CLH-22

[0101] Aspergillus thermophilus CLH-22 was inoculated into selection medium as primary seed culture and cultured in a 30℃ constant temperature shaking incubator for 48 h. At a volume ratio of 1%, the primary seed culture was transferred to propagation medium as secondary seed culture and cultured in a 30℃ constant temperature shaking incubator for 48 h. At a volume ratio of 2%, the secondary seed culture was transferred to fermentation enzyme production medium and cultured in a 30℃ constant temperature shaking incubator. Samples were taken every 12 h, and the consumption of glycyrrhizic acid monoammonium salt and the production of glycyrrhetinic acid and 3-oxoglycyrrhetinic acid were determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 3As shown in the figure. The high-performance liquid chromatography (HPLC) detection method used a mobile phase of methanol:0.6% acetic acid = 84:16, a flow rate of 0.8 mL / min, a Kromasil 100-3.5-C18 column, and a detection wavelength of 254 nm. The peak elution times were 3.175 min for the substrate glycyrrhizic acid, 9.414 min for the glycyrrhetinic acid standard, and 9.703 min for the final product 3-oxoglycyrrhetinic acid. The results indicate that glycyrrhetinic acid and 3-oxoglycyrrhetinic acid were detected simultaneously after 48 h of fermentation, but the accumulation of glycyrrhetinic acid during the conversion process was very low. After 84 h, the conversion of glycyrrhizic acid monoammonium salt was complete, and there was no significant increase in 3-oxoglycyrrhetinic acid. The calculated yield of 3-oxoglycyrrhetinic acid was 94.6%.

[0102] Based on the above test results, the ability of Aspergillus thermophilus CLH-22 to directly convert glycyrrhetinic acid to 3-oxoglycyrrhetinic acid was verified in vitro.

[0103] Take 4 mL of fermentation broth, centrifuge at 12000 rpm for 10 min, discard the supernatant, collect the bacterial precipitate in the same centrifuge tube, wash the bacterial cells twice with sterile water, centrifuge at 12000 rpm for 5 min, and discard the supernatant. Add an appropriate amount of mixed Glass bead and Zarconia / Silica bead disruptors, and add 1 mL of 1 g / L GA reaction buffer (GA purity 99%) to the centrifuge tube. Disrupt the bacterial cells using a bead disruptor. After disruption, place the centrifuge tube in a constant temperature shaking incubator at 30℃ and 200 rpm for 24 h. High-performance liquid chromatography (HPLC) analysis showed that the crude enzyme solution of *Aspergillus pyrophorus* CLH-22 could directly convert GA to 3-oxoglycyrrhetinic acid in vitro without any byproducts, consistent with the previous results. Combining enzyme production culture and in vitro verification, it was shown that *Aspergillus thermophilus* CLH-22 contains not only a β-glucuronidase capable of hydrolyzing glycine (GL) to produce glycyrrhizic acid (GA), but also a C3-hydroxy oxidase capable of converting GA to 3-oxoglycyrrhizic acid. The synergistic effect of these two enzymes enables a highly efficient conversion from GL to 3-oxoglycyrrhizic acid.

[0104] Experimental Example 3: Preparation and Identification of Pure 3-O-Glycyrrhetinic Acid

[0105] Following the fermentation method described in Example 2, 1 L of the converted fermentation medium was centrifuged at 12000 rpm for 15 min, and the supernatant was discarded. 100 mL of ethanol was added to the precipitate, and the mixture was stirred in a 50°C water bath for 20 min until fully dissolved. The mixture was then filtered while hot, retaining the filtrate. 100 mL of pre-cooled purified water was added to the filtrate, and the mixture was stirred for 10 min, then allowed to stand at room temperature for 1 h. At this point, a significant amount of solid precipitated. The filtrate was centrifuged at 8000 rpm for 10 min, and the filter cake was collected and dried using a vacuum rotary evaporator for 2 h to evaporate residual ethanol, obtaining a first-stage purified product. The first-stage purified product was further frozen at -80°C and then freeze-dried under vacuum to obtain the crude product. The purity of the crude product was determined using high-performance liquid chromatography (HPLC). The calculated purity of 3-oxoglycyrrhetinic acid in the crude product was 93.24%.

[0106] The product was further purified using semi-preparative liquid chromatography, as follows:

[0107] 100 mg of the crude product obtained above was added to 21.5 mL of methanol and 3.5 mL of 0.6% acetic acid. The mixture was stirred for 30 min to ensure complete dissolution. Further purification was then performed using semi-preparative liquid chromatography (HPLC) to obtain a purified sample solution. The mobile phase ratio was methanol:0.6% acetic acid = 84:16, the flow rate was 5 mL / min, and the detection wavelength was 254 nm. The sample solution purified by semi-preparative HPLC was dried using a vacuum rotary evaporator for 2 h to evaporate methanol and acetic acid, obtaining a secondary purified product. This secondary purified product was further frozen at -80℃ and then freeze-dried under vacuum to obtain the purified product. The purity of the purified product was determined using high-performance liquid chromatography (HPLC), and the results are as follows: Figure 4 As shown. Calculations show that the purity of 3-oxoglycyrrhetinic acid after secondary purification is 99.85%.

[0108] The high-purity 3-oxoglycyrrhetinic acid obtained after secondary purification was analyzed by liquid chromatography-mass spectrometry, and the results are as follows: Figure 5 As shown. Figure 5 A-mass spectrometry results showed that glycyrrhetinic acid was measured at (MH). - The value is 469.3323. Figure 5 B-mass spectrometry results showed that the predicted 3-oxoglycyrrhetinic acid (MH) - The measured concentration (MH) of the tested compound was 467.3167. - The molecular weight was 467.3168, meaning the molecular weight of the detected compound matched the predicted molecular weight of 3-oxoglycyrrhetinic acid, confirming that the molecular weight of the secondary purified product was consistent with that of 3-oxoglycyrrhetinic acid.

[0109] The structure was identified using a Bruker Ascend 700M nuclear magnetic resonance spectrometer, as follows:

[0110] 15 mg of high-purity 3-oxoglycyrrhetinic acid obtained from secondary purification was dissolved in 1 mL of deuterated chloroform, and subjected to Bruker Ascend 700M nuclear magnetic resonance. 1 H spectrum and 13 C-spectral analysis 1 The H spectrum results are as follows Figure 6 As shown, 13 The C-spectrum results are as follows Figure 7 As shown. The predicted chemical shift of glycyrrhetinic acid in the 1H NMR spectrum shows a 3-OH formation chemical shift of 4.77 ppm, while in this invention... 1 The 1H NMR spectroscopy results showed that no 3-OH formation was detected at the 3-position of the predicted compound. Predicted 3C NMR chemical shifts for glycyrrhetinic acid and 3-oxoglycyrrhetinic acid showed 3C formation chemical shifts of 78.6 ppm and 217.0 ppm, respectively. However, in this invention… 13 The C-ray spectroscopy results showed that only a characteristic chemical shift of 217.309 ppm was present, while the characteristic chemical shift at position 3 for glycyrrhetinic acid formation was absent. Therefore, based on the combined analysis of high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, the compound purified in this invention was determined to be 3-oxoglycyrrhetinic acid.

Claims

1. A strain of Aspergillus thermophilus ( Aspergillus calidoustus strain CLH-22, characterized in that, Its accession number is CGMCC No.40213.

2. Aspergillus pyrophorus with accession number CGMCC No. 40213 ( Aspergillus calidoustus Use of strain CLH-22 in the preparation of 3-oxoglycyrrhetinic acid.

3. The Aspergillus pyrophorus with accession number CGMCC No. 40213 as described in claim 2 ( Aspergillus calidoustus The use of strain CLH-22 in the preparation of 3-oxoglycyrrhetinic acid, characterized in that, The purity of the prepared 3-oxoglycyrrhetinic acid was 93.24%-99.85%.

4. The Aspergillus thermophilus with accession number CGMCC No. 40213 as described in claim 2 or 3. Aspergillus calidoustus The use of strain CLH-22 in the preparation of 3-oxoglycyrrhetinic acid, characterized in that, The yield of 3-oxoglycyrrhetinic acid was 94.6%.

5. A fermentation agent for preparing 3-oxoglycyrrhetinic acid, comprising a fermentation-active ingredient, characterized in that, The fermentation active ingredient includes: *Aspergillus thermophilus* with accession number CGMCC No. 40213. Aspergillus calidoustus ) strain CLH-22.

6. The fermentation agent for preparing 3-oxoglycyrrhetinic acid according to claim 5, characterized in that, Also includes: Auxiliary materials.

7. A method for preparing 3-oxoglycyrrhetinic acid, characterized in that, include: The sample was obtained from Aspergillus thermophilus with accession number CGMCC No. 40213. Aspergillus calidoustus The strain CLH-22 was used to ferment the substrate; the substrate concentration was 1-4 g / L; the substrate was selected from glycyrrhizic acid or glycyrrhetinic acid.

8. The method for preparing 3-oxoglycyrrhetinic acid according to claim 7, characterized in that, It also includes: purifying the fermentation products; And / or, the purity of the purified product is 93.24%-99.85%; And / or, the product refers to 3-oxoglycyrrhetinic acid.

9. A method for preparing 3-oxoglycyrrhetinic acid according to claim 7 or 8, characterized in that, The fermentation refers to: processing Aspergillus pyroximate (…). Aspergillus calidoustus Strain strain CLH-22 was inoculated into the culture medium and cultured. The inoculation refers to: inoculating strain CLH-22 into a screening medium to obtain a primary seed culture, and / or inoculating the primary seed culture into a propagation medium to obtain a secondary seed culture, and / or inoculating the secondary seed culture into a fermentation enzyme production medium for separate culture. And / or, the inoculum size of the primary seed culture in the propagation medium is 1%; The inoculum size of the secondary seed culture in the enzyme-producing fermentation medium was 2%. And / or, the culture refers to: constant temperature culture at 30℃ for 48 hours.

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