CN117660199B - A white and yellow brittle mushroom, a bacterial agent, a method for converting liquiritin into liquiritigenin and its application - Google Patents

Abstract

The invention belongs to the technical field of endophyte enzyme-producing biology, and particularly relates to a method for converting white Huang Xiaocui handle mushroom, a microbial inoculum and liquiritin into liquiritigenin and application thereof. The invention provides a white Huang Xiaocui handle mushroom (Candolleomyces candolleanus) KXS-P9 with a preservation number of CGMCC No.40386. The invention uses glycyrrhizin as a substrate and utilizes beta-glucosidase produced by the white Huang Xiaocui handle mushroom KXS-P9 to transform the glycyrrhizin to generate the glycyrrhizin. The results of the examples show that: the white Huang Xiaocui handle mushroom KXS-P9 is converted to generate the glycyrrhizin by taking the glycyrrhizin as a substrate, and the conversion yield of the glycyrrhizin can reach 90% at most. Therefore, the white Huang Xiaocui handle mushroom KXS-P9 can be used for converting liquiritin into liquiritigenin, and provides theoretical basis support for development and utilization of the liquiritigenin and related functional health-care foods, food additives and the like in efficient mass production.

Description

A white and yellow brittle mushroom, a bacterial agent, a method for converting liquiritin into liquiritigenin and its application

Technical Field

The invention belongs to the field of biotechnology of enzyme production by endophytic bacteria, and specifically relates to a white-yellow brittle mushroom, a bacterial agent, and a method and application of converting liquiritigenin into liquiritigenin.

Background technique

As people's health awareness gradually improves, the study of the biological activity of flavonoid aglycone compounds has attracted more and more attention from domestic and foreign researchers. Liquiritin is one of the main flavonoid components in licorice. Liquiritin is a glycoside type formed by the phenolic hydroxyl group of liquiritin and glucose connected by a β-glucoside bond. However, this glycosidic bond form is often not easily absorbed and utilized by the body, and the biological activity is greatly reduced. Liquiritigenin is the aglycone form of liquiritin, which has anti-tumor, anti-inflammatory, antibacterial, antioxidant, liver-protecting, estrogen-like effects, food flavoring and other effects. It is widely used in biomedicine, food field, and cosmetics production. The content of liquiritigenin in Ural licorice is very low, lower than the content of liquiritigenin, and it is difficult to meet the market demand for liquiritigenin.

At present, the production of glycyrrhizin mainly relies on licorice plant extraction and chemical synthesis. However, due to the limited resources of licorice, low yield, slow growth rate, and difficulty in separating flavonoids, the extraction of glycyrrhizin is restricted. Chemical synthesis is also limited by the complex molecular structure of the compound, the use of toxic and expensive chemicals, severe reaction conditions, and the easy generation of isomers. Therefore, the research on the method of preparing glycyrrhizin by microbial transformation is very necessary. There is no report on the preparation of glycyrrhizin by Fragilis alba in the prior art.

Summary of the invention

The purpose of the present invention is to provide a strain of Fragilis leucophylla KXS-P9, which has high β-glucosidase activity and can convert liquiritigenin into liquiritigenin by a microbial transformation method.

The invention provides a Candolleomyces candolleanus KXS-P9, whose preservation number is CGMCC No.40386.

The present invention provides a bacterial agent containing the white yellow brittle mushroom KXS-P9 and/or its metabolites described in the above technical solution.

The present invention provides a method of using the white and yellow brittle mushroom KXS-P9 described in the above technical solution or the bacterial agent described in the above technical solution in one or more of the following 1) to 3);

1) preparing glycyrrhizin;

2) Improve the conversion rate of liquiritigenin to liquiritigenin;

3) Preparation of β-glucosidase.

The present invention provides a method for converting liquiritin into liquiritigenin, comprising the following steps:

The beta-glucosidase enzyme solution prepared by the white yellow brittle mushroom KXS-P9 described in the above technical scheme is converted with liquiritigenin to obtain liquiritigenin.

Preferably, the conversion system further comprises a buffer, and the buffer comprises a phosphate buffer.

Preferably, the preparation method of the β-glucosidase enzyme solution comprises: culturing the seed solution of Pleurotus eryngii KXS-P9 in a culture medium to obtain a culture solution;

Centrifuging the culture solution to obtain a supernatant;

The supernatant is subjected to 60% saturation ammonium sulfate precipitation of β-glucosidase to obtain β-glucosidase enzyme solution;

The culture medium uses water as solvent and comprises: 30-31 g/L wheat bran, 11-12 g/L beef extract, 0.9-1.1 g/L KH ₂ PO ₄ and 0.4-0.6 g/L MgSO ₄ , with a pH value of 7-8.

Preferably, the enzyme activity of the crude β-glucosidase enzyme solution is 50-100 U/mL; the initial addition amount of the β-glucosidase enzyme solution in the transformation system is 15%-20% of the total volume of the transformation system.

Preferably, the initial substrate concentration of liquiritin in the transformation system is 0.1-1.0 mg/mL.

Preferably, the temperature of the conversion is 30° C. to 40° C., the time of the conversion is 6 to 14 hours, and the initial rotation speed of the conversion is 150 to 200 rpm.

Preferably, the initial pH value of the conversion system is 4 to 7.5.

Beneficial effects of the present invention: The present invention provides a white and yellow small crisp mushroom (Candolleomycescandolleanus) KXS-P9, with a preservation number of CGMCC No.40386. The white and yellow small crisp mushroom KXS-P9 can produce β-glucosidase during its growth. β-glucosidase produces an active ingredient in the form of aglycone by hydrolyzing the non-reducing β-D-glucosidic bond at the end of the compound. The present invention uses liquiritin as a substrate and uses the β-glucosidase produced by the white and yellow small crisp mushroom KXS-P9 to convert it into liquiritigenin. The technical scheme of the present invention is an efficient, stable and controllable conversion process. The results of the embodiment show that the white and yellow small crisp mushroom KXS-P9 uses liquiritin as a substrate to convert it into liquiritigenin, and the conversion rate of liquiritigenin can reach up to 90%. It can be seen that the white and yellow small crisp mushroom KXS-P9 of the present invention can be used to convert liquiritin into liquiritigenin, providing a theoretical basis for the efficient large-scale production of liquiritigenin and related functional health foods, food additives, etc.

Biological Deposit Description

Candolleomyces candolleanus KXS-P9 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on February 27, 2023, with the deposit number CGMCC No.40386. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

Fig. 1 is a standard curve diagram of the concentration and peak area of liquiritin;

Fig. 2 is a standard curve diagram of the concentration and peak area of licorice root;

Fig. 3 is a HPLC chart of liquiritigenin and liquiritigenin standards;

FIG4 is an HPLC chart of liquiritin before and after hydrolysis by β-glucosidase obtained in the present invention;

Figure 5 shows the inhibition zones before and after the transformation of liquiritin, where P represents the positive control, streptomycin sulfate; N represents the negative control, sterile distilled water; a represents Escherichia coli; b represents Salmonella; c represents Staphylococcus aureus;

FIG6 is a graph showing the DPPH free radical scavenging rate of liquiritigenin obtained by converting liquiritigenin into β-glucosidase obtained by the present invention and commercial β-glucosidase;

FIG7 is a graph showing the ABTS free radical scavenging rate of liquiritigenin obtained by converting liquiritigenin by β-glucosidase obtained by the present invention and commercial β-glucosidase;

FIG8 is a curve showing the relationship between Fe ²⁺ concentration and absorbance;

FIG9 is a graph showing the total antioxidant capacity of liquiritigenin obtained by converting liquiritigenin into liquiritigenin by β-glucosidase obtained by the present invention and commercial β-glucosidase;

In Figures 6, 7 and 9, CK1 is the β-glucosidase obtained in the present invention before enzymatic hydrolysis of liquiritin; CK2 is the commercial β-glucosidase before enzymatic hydrolysis of liquiritin; conversion group 1 is the β-glucosidase obtained in the present invention after enzymatic hydrolysis of liquiritin; conversion group 2 is the commercial β-glucosidase after enzymatic hydrolysis of liquiritin;

Figure 10 Colony morphology of Pleurotus eryngii KXS-P9 after culturing on PDA medium for 6 days;

Figure 11 Microscopic morphology of mycelium of Pleurotus eryngii KXS-P9;

Fig. 12 Phylogenetic tree of KXS-P9 strain and related fungi based on ITS sequences.

Detailed ways

The invention provides a Candolleomyces candolleanus KXS-P9, whose preservation number is CGMCC No.40386.

The white and yellow small crispy handle mushroom KXS-P9 of the present invention is an endophytic fungus screened from Ural Glycyrrhiza. The colony morphology of the white and yellow small crispy handle mushroom KXS-P9 of the present invention after culturing on PDA medium for 6 days is shown in Figure 10. The colony is white, the hyphae are dense and thick white, and a small amount of yellow pigment is produced in the later stage. The microscopic morphology of hyphae is shown in Figure 11. The vegetative hyphae are colorless and transparent, thin-walled, septate, and multi-core.

The present invention provides a bacterial agent containing the white yellow brittle mushroom KXS-P9 and/or its metabolites described in the above technical solution.

The present invention provides the use of the white and yellow brittle mushroom KXS-P9 described in the above technical solution or the bacterial agent described in the above technical solution in one or more of the following 1) to 3);

1) preparing glycyrrhizin;

2) Improve the conversion rate of liquiritigenin to liquiritigenin;

3) Preparation of β-glucosidase.

In the embodiment of the present invention, the crude β-glucosidase enzyme solution prepared by Agaricus leucophylla KXS-P9 is used to transform glycyrrhizin, and the conversion rate of glycyrrhizin can reach 81.61%; the commercial β-glucosidase (derived from Aspergillus niger) is used to transform glycyrrhizin, and the conversion rate of glycyrrhizin after the transformation can reach 75.87%. It can be seen that the conversion effect of the β-glucosidase enzyme solution prepared by Agaricus leucophylla KXS-P9 of the present invention is better than that of the commercial β-glucosidase.

The present invention provides a method for converting liquiritin into liquiritigenin, comprising the following steps:

The β-glucosidase enzyme solution prepared from the white and yellow brittle mushroom KXS-P9 described in the above technical scheme is transformed with liquiritigenin to obtain liquiritigenin.

The preparation method of the β-glucosidase enzyme solution of the present invention preferably comprises: culturing the seed liquid of Pleurotus eryngii KXS-P9 in a culture medium to obtain a culture liquid; centrifuging the culture liquid to obtain a supernatant; and precipitating the β-glucosidase in the supernatant with an ammonium sulfate solution with a saturation of 60% to obtain the β-glucosidase enzyme solution.

The present invention has no special restrictions on the preparation of the white and yellow small crispy handle mushroom KXS-P9 seed liquid, and conventional methods can be used. The culture medium of the present invention preferably uses water as a solvent, and the composition includes: 30-31g/L of wheat bran, 11-12g/L of beef extract, _0.9-1.1g /L of _KH2PO4 and _0.4-0.6g /L of MgSO4, and the pH value is 7-8. The wheat bran in the culture medium of the present invention is preferably 30-31g/L, and more preferably 30.6g/L; the beef extract in the culture medium of the present invention is preferably 11-12g/L, and more preferably 11.2g/L; _{the KH2PO4} in the culture medium of the present invention is preferably 0.9-1.1g/L, and more preferably 1.0g/L; the _MgSO4 in the culture medium of the present invention is preferably 0.4-0.6g/L, and more preferably 0.5g/L; the pH value of the culture medium is preferably 7-8, and more preferably 7.23. The inoculation amount of the seed liquid of the white and yellow brittle mushroom KXS-P9 of the present invention is preferably 2% to 3% of the volume of the culture medium, and more preferably 2.6%. The culture temperature of the present invention is preferably 25 to 30°C, and more preferably 30°C; the culture speed is preferably 140 to 160rpm, and more preferably 150rpm; the culture time is preferably 5 to 7d, and more preferably 6d. The speed of the centrifugation of the present invention is preferably 8000rpm, and the time is preferably 10min; the centrifugation is preferably carried out at 4°C. The present invention does not specifically limit the preparation method of the ammonium sulfate solution with a saturation of 60%, and conventional methods can be used. The specific steps of precipitating β-glucosidase with an ammonium sulfate solution with a saturation of 60% of the present invention are not specifically limited, and conventional parameters can be referred to. The enzyme activity of the β-glucosidase solution prepared by the white-yellow brittle mushroom KXS-P9 of the present invention is preferably 50-100 U/mL, further preferably 70-100 U/mL, and more preferably 100 U/mL; the initial addition amount of the β-glucosidase solution of the white-yellow brittle mushroom KXS-P9 in the transformation system of the present invention is preferably 15%-20% of the total volume of the transformation system, and more preferably 18%-20%.

The β-glucosidase enzyme solution prepared by the white and yellow brittle mushroom KXS-P9 of the present invention, liquiritin and phosphate buffer constitute the transformation system. The initial substrate concentration of liquiritin in the transformation system of the present invention is preferably 0.1-1.0 mg/mL, more preferably 0.6-1.0 mg/mL, and more preferably 0.8-1.0 mg/mL. In the present invention, the initial addition amount of the phosphate buffer is preferably 40%-60% of the total volume of the transformation system, and more preferably 50%-60%; the function of the phosphate buffer is to adjust the pH value of the transformation system. The temperature of the transformation of the present invention is preferably 30-40°C, more preferably 35-40°C, and more preferably 40°C; the time of the transformation of the present invention is preferably 6-14h, more preferably 9-13h, and more preferably 12h. The initial pH value of the transformation system of the present invention is preferably 4-7.5, more preferably 6-7.5, and more preferably 7.5.

In the embodiment of the present invention, the β-glucosidase enzyme solution prepared by the white yellow small crispy handle mushroom KXS-P9 is used to transform glycyrrhizin, and the obtained conversion solution has the best inhibitory effect on Staphylococcus aureus, with an inhibition zone of 12.7±0.31mm; followed by the Salmonella inhibition zone of 9.10±0.41mm; the weakest inhibition ability is the Escherichia coli inhibition zone of 6.37±0.62mm. The obtained conversion solution has a strong antibacterial and bactericidal effect on Staphylococcus aureus, with MIC and MBC of 0.058mg/mL and 0.115mg/mL respectively; the minimum inhibitory concentration of the obtained conversion solution for Salmonella is 0.058mg/mL, and the minimum bactericidal concentration for Salmonella is 0.230mg/mL; the MIC and MBC of the obtained conversion solution for Escherichia coli are the highest among the three pathogens, which are 0.115mg/mL and 0.230mg/mL respectively.

In the embodiments of the present invention, the β-glucosidase enzyme solution prepared by using Pleurotus eryngii KXS-P9 to transform glycyrrhizin has high DPPH free radical scavenging ability, ABTS free radical scavenging activity and total antioxidant capacity, which provides a theoretical basis for the efficient large-scale production of glycyrrhizin and related functional health foods, food additives, etc.

The present invention utilizes the white-yellow brittle mushroom KXS-P9 to transform liquiritin into liquiritigenin by a microbial transformation method with liquiritigenin as a substrate. The technical solution has low production cost and high conversion yield, and is a green and efficient fermentation process for transforming and generating liquiritigenin. The β-glucosidase produced by the white-yellow brittle mushroom KXS-P9 has a good application prospect in the transformation of liquiritigenin into liquiritigenin.

In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

The experimental data results obtained in the embodiments and comparative examples were processed and statistically analyzed using Microsoft Excel 2019 and SPSS 24, and the graphs were drawn using Origin 2021 software.

Unless otherwise specified, all raw and auxiliary materials, reagents and instruments used in the present invention are well known in the art.

Example 1 Isolation, screening and identification of bacterial strains

1. In the autumn of 2021, fresh and healthy Ural red licorice was collected in the Altay region of Xinjiang, placed in a sampling bag and brought back to the laboratory, and stored at 4°C. Take the Ural red licorice plant, rinse it with tap water first, cut it into appropriate small pieces, and disinfect the surface in a sterile operating table: rinse with sterile water 3 times, soak in 75% ethanol for 3 minutes, and rinse with sterile water for 1 minute; soak in a 5% sodium hypochlorite solution for 5 minutes, and rinse with sterile water for 1 minute; rinse with 75% ethanol for 30 seconds, rinse with sterile water for 1 minute, and set aside.

Take 100 μL of the last sterile water washing solution and apply it to NA, TSA and PDA culture media. NA and TSA culture media are cultured at 35℃ for 1 to 5 days, and PDA culture media are cultured at 28℃ for 1 to 5 days. If no colonies are observed to grow, it indicates that the surface of licorice is thoroughly disinfected. The culture was carried out in 3 parallel experiments.

2. Tissue block separation method: After the surface of licorice is disinfected, the two ends of the licorice tissue are removed with a sterilized scalpel, and the remaining part is cut into small pieces of about 1 to 2 cm. Use sterile tweezers to put them into PDA culture medium, with the cut surface close to the culture medium, 3 pieces per dish, and after sealing with sealing film, place them in a constant temperature incubator, and culture the fungi at 28℃ for 1 to 5 days. After waiting for the bacteria to grow, pick the colonies that grow according to the different characteristics of morphology, color, and size, and continuously transfer them to PDA culture medium until a purified strain is obtained. One of the strains is numbered KXS-P9 and stored on a slant.

3. The endophytic fungus strain KXS-P9 was inoculated in a potato glucose water (PDB) medium (composed of: 5.0g potato starch, 20.0g glucose and 1L distilled water, pH 7.0) and cultured at 28°C and 150rpm for 5 days. After that, the culture solution was centrifuged at 4°C and 8000rpm for 5min, and the fungi were collected and quickly ground into powder by adding liquid nitrogen. The DNA of endophytic fungi of licorice was extracted according to the operation steps of the fungal DNA extraction kit. Specifically, PCR amplification was performed using fungal universal primers ITS1 (SEQ ID NO.2: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO.3: 5'-TCCTCCGCTTATTGAATGC-3'). The ITS sequence fragment of the strain was obtained by amplification, and the PCR product of the strain was tested by 1.0% agarose gel electrophoresis. The PCR product with bands was sent to Shanghai Biotech for sequencing. The target sequence fragment with a length of 718bp was obtained by sequencing, as shown in SEQ ID NO.1. The sequencing results of strain KXS-P9 were compared with known model strain sequences in the EzBioCloud database (https://eztaxon-e.ezbiocloud.net) and the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the strain was identified as the genus Pleurotus by online Blast comparison. The sequences of strains of similar species of the genus Pleurotus were downloaded, and a phylogenetic tree was constructed using MEGA7.0 software. The bootstrap value used for tree construction was set to 1000. The phylogenetic tree is shown in Figure 12. The numbers at the branch nodes in Figure 12 represent the credibility. The strain KXS-P9 and the white and yellow brittle mushroom (Candolleomycescandolleanus) (EU520251.1) were clustered into one branch with a similarity of 90%. Combined with morphological and molecular identification, the strain was identified as the white and yellow brittle mushroom (Candolleomyces candolleanus) KXS-P9.

SEQ ID NO.1:

CTTTTCGGTAGGGGTACCTGCGGAAGGATCATTAACGAATATCTATGGCGTTGGTTGTAGCTGGCTCCTAGGAGCATTGTGCACGCCCGTCATTCATATCATCTTTCCACCTGTGAACCATCTGTAGGCCTGGATACCCCTCGCTTTGGCAACAAAGCGGATGCAAGGATTGCTGCGTCGACAAGGCCGGCTCTCTTTGAATTTCCAGGTTCTATGTCTTTTACACACCCCATTTGAATGATTTAGAATGTAGTCAATGGGCTTTCATGCCTATAAAAAACTATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATT GCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATTAAATTCTCAACCTCACCAGTTTTGTAACGAGACAGGTGAAGGCTTGGATGTGGGGGTTTTGCAGGCTGCCTCAGTGCTGGTCTGCTCTCCTGAAATGCATTAGCGAGCTCATATTGAGCTTCCGTCTATTGGTGTGATAATTATCTACGCCGTGGATTGGACTCATGCTTGCTTCTAACCGTCCGCAAGGACAATTTACTTGACCAATTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAAAGGGGGGGAAAGGGAAAAGAA.

Examples 2-3 and Comparative Examples 1-2 used the crude enzyme solution of Agaricus leucophylla KXS-P9 prepared in the same batch, and the conversion time was adjusted.

Example 2 Enzyme production test of Candolleomyces candolleanus KXS-P9

1. Preparation of crude enzyme solution

The white yellow small crispy handle mushroom KXS-P9 was inoculated into the PDB medium for seed culture to obtain seed liquid; the seed liquid was inoculated into the second medium for culture to obtain culture liquid; the inoculation amount of the seed liquid was 2.6% of the volume of the medium, the culture temperature was 30°C, the rotation speed was 150rpm, and the culture time was 6d. The obtained culture liquid was centrifuged at 4°C and 8000g for 10min, and the supernatant was taken. The second culture medium used distilled water as the solvent, and the composition was: 30.6g/L wheat bran, 11.2g/L beef extract, 1.0g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ , and the pH value was 7.23.

The obtained supernatant was precipitated with ammonium sulfate solution to obtain a crude enzyme solution of β-glucosidase (reference: Yan Qing. Isolation, purification and structural analysis of heat-resistant β-glucosidase from Aspergillus niger [D]. Qinhuangdao: Hebei Science and Technology Normal University, 2016.). The results showed that the β-glucosidase activity in the crude enzyme solution precipitated with 60% saturation ammonium sulfate solution was the highest, reaching 167.41U/mL. The enzyme activity was adjusted to 100U/mL with PBS buffer (0.05mol/L, pH 7.5) for standby use.

2. Conversion of Liquorice glycosides

A total of 5 mL of the basic conversion reaction system: 1 mL of 4 mg/mL liquiritin solution, 1 mL of crude enzyme solution obtained in step 1, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5) were placed in a test tube for conversion. The temperature during the conversion was set to 40°C, the rotation speed was 150 rpm, the starting pH value of the conversion was 7.5, and the conversion time was 6 h. After the conversion was completed, it was immediately taken out and the reaction was stopped at 4°C. The resulting conversion reaction system was filtered through a 0.22 μm microporous membrane, and the filtered product was used to detect the content of liquiritin and liquiritigenin by HPLC, and the molar conversion yield of the product liquiritigenin was calculated. Three parallel experiments were set for the conversion of liquiritin.

Example 3

The crude enzyme solution obtained in step 1 of Example 2 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 2, except that the transformation time was 18 h, and three parallel experiments were set.

Comparative Example 1

The crude enzyme solution obtained in step 1 of Example 2 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 2, except that the transformation time was 24 h, and three parallel experiments were set.

Comparative Example 2

The crude enzyme solution obtained in step 1 of Example 2 was used to transform liquiritin. The transformation of liquiritin was the same as in Example 2, except that the transformation time was 30 h, and three parallel experiments were set.

In Examples 2 to 3 and Comparative Examples 1 to 2, the contents of liquiritigenin and liquiritigenin were detected by HPLC, and the molar conversion rate of the product liquiritigenin was calculated as follows:

Molar conversion rate = (amount of substance of liquiritigenin/amount of substance of liquiritigenin) × 100% = ( _{Mliquiritigenin Cliquiritigenin} / _{Mliquiritigenin Cliquiritigenin} ) × 100%. In the formula, _{Cliquiritigenin} refers to the concentration of liquiritigenin generated after the conversion of liquiritigenin, μg/mL; _{Cliquiritigenin} refers to the initial concentration when the substrate liquiritigenin is added, μg/mL.

The specific method of HPLC detection is:

(1) Instrumental method

Chromatographic column: Elite C18 (4.6mm*150mm*5μm); parameter settings: flow rate 1.0mL/min, wavelength 237nm, detection time 40min, column temperature 25℃, sample volume 10μL.

(2) Content determination

The content of liquiritin and liquiritigenin was determined according to the steps of high performance liquid chromatography in the 2020 version of the Chinese Pharmacopoeia. Mobile phase A: acetonitrile; mobile phase B: 0.05% phosphoric acid water; mobile phase gradient is shown in Table 1 below:

Table 1 HPLC mobile phase gradient conditions

(3) Drawing of standard curve

Prepare the standard stock solutions of liquiritin and liquiritigenin respectively, specifically: accurately weigh 10 mg of liquiritin and liquiritigenin standard respectively, dissolve with methanol and dilute to 10 mL volumetric flask to obtain 1 mg/mL standard stock solution, draw 1 mL from the standard stock solution respectively, dilute with methanol to the mass concentration of 1, 5, 10, 20, 50, 100, 400 μg/mL to be tested, take the peak area (y) as the ordinate and the sample concentration (x) as the abscissa, perform regression analysis on the relationship between the concentration of each sample and the peak area, and draw the standard curves as shown in Figures 1 and 2. The standard curve of liquiritin: y = 0.2888x + 0.0929, R ² = 0.9999; the standard curve of liquiritigenin: y = 0.6110x + 0.6450, R ² = 0.9998. According to the chromatographic conditions in Table 1, the standard solutions of liquiritin and liquiritigenin are prepared into a mixed product and analyzed, and the HPLC chart of the mixed product is measured, as shown in Figure 3. As shown in Figure 3, under certain chromatographic conditions, liquiritigenin and liquiritigenin were well separated, with retention times of 8.757 min and 22.280 min, respectively, and the determination method was feasible.

Examples 2 to 3 and Comparative Examples 1 to 2 used the above HPLC method to detect the content of liquiritigenin and liquiritigenin, and calculated the conversion rate of liquiritigenin, and the results are shown in Table 2. According to Table 2, when the conversion time is 6 to 18 hours, the conversion rate of liquiritigenin increases with the extension of the reaction time of the enzyme and the substrate; as the conversion time is extended until 30 hours, the conversion rate of liquiritigenin increases slowly, and the enzyme reacts with the liquiritigenin substrate almost completely, so the optimal conversion time under this condition is 6 to 18 hours.

Table 2 Conversion rate of glycyrrhizin in Examples 2-3 and Comparative Examples 1-2

Example 4 and Comparative Examples 3 to 5 used the crude enzyme solution of Pleurotus eryngii KXS-P9 prepared in the same batch, and the conversion temperature was adjusted.

Example 4

1. Preparation of crude enzyme solution. A crude enzyme solution was prepared according to Example 2. The enzyme activity of the crude enzyme solution was adjusted to 100 U/mL.

2. Conversion of Liquorice glycosides

The basic conversion reaction system is 5 mL in total: 1 mL of 4 mg/mL liquiritin solution, 1 mL of crude enzyme solution, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5), which are placed in a test tube for conversion. The concentration of liquiritin in the reaction system is 0.8 mg/mL. The temperature of the conversion is 30°C, the speed of the conversion is 150 rpm, the time of the conversion is 6 h, and the starting pH value of the conversion is 7.5. After the conversion is completed, the sample is immediately taken and placed at 4°C to stop the reaction. The sample is filtered through a 0.22 μm microporous membrane, and the content of liquiritin and liquiritigenin is detected by HPLC, and the molar conversion yield of the product liquiritigenin is calculated. Three parallel experiments were set up for the conversion of liquiritin.

Comparative Example 3

The crude enzyme solution obtained in step 1 of Example 4 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 4, except that the transformation temperature was 50° C., and three parallel experiments were set.

Comparative Example 4

The crude enzyme solution obtained in step 1 of Example 4 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 4, except that the transformation temperature was 60° C., and three parallel experiments were set.

Comparative Example 5

The crude enzyme solution obtained in step 1 of Example 4 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 4, except that the transformation temperature was 70° C., and three parallel experiments were set.

The concentration of glycyrrhizin was analyzed by the HPLC method for Example 4 and Comparative Examples 3 to 5, and the conversion yield was calculated. The results are shown in Table 3. According to the results of Table 3 and Example 2, when the temperature is between 30 and 40°C, the conversion yield of glycyrrhizin increases continuously with the increase of temperature; but when the temperature exceeds 40°C, the enzyme is inactivated due to the continued increase in temperature, and the conversion yield decreases rapidly, so the conversion temperature is best at 40°C.

Table 3 Liquoricerin conversion rate of Example 4 and Comparative Examples 3 to 5

Examples 5 to 7 and Comparative Examples 6 to 7 used the crude enzyme solution of Agaricus leucophylla KXS-P9 prepared in the same batch, and the conversion temperature was adjusted.

Example 5

1. Preparation of crude enzyme solution.

The white and yellow small crispy handle mushroom KXS-P9 was inoculated into PDB medium for seed culture to obtain seed liquid; the seed liquid was inoculated into the second medium for culture to obtain culture liquid; the inoculation amount of the seed liquid was 2.6% of the volume of the medium, the culture temperature was 30℃, the rotation speed was 150rpm, and the culture time was 6d. The obtained culture liquid was centrifuged at 4℃ and 8000g for 10min, and the supernatant was taken. The second culture medium used distilled water as solvent, and the composition was: 30.6g/L wheat bran, 11.2g/L beef extract, 1.0g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ , and the pH value was 7.23. The obtained supernatant was precipitated with ammonium sulfate solution to obtain β-glucosidase (reference: Yan Qing. Isolation, purification and structural analysis of heat-resistant β-glucosidase of Aspergillus niger [D]. Qinhuangdao: Hebei Science and Technology Normal University, 2016.).

The results showed that the β-glucosidase activity in the crude enzyme solution obtained by precipitation with 60% saturation ammonium sulfate solution was the highest, reaching 167.41 U/mL. The enzyme activity was adjusted to 100 U/mL with PBS buffer (0.05 mol/L, pH 7.5) for use.

2. Conversion of Liquorice glycosides

The basic conversion reaction system is 5 mL in total: 1 mL of 0.5 mg/mL liquiritin solution, 1 mL of crude enzyme solution, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5). The above liquids are placed in a test tube for conversion. The concentration of liquiritin in the basic conversion reaction system is 0.1 mg/mL. The temperature of the conversion is 40°C, the speed of the conversion is 150 rpm, the time of the conversion is 6 h, and the pH value of the conversion is 7.5. After the conversion is completed, a sample is taken immediately, and the reaction is stopped at 4°C. The sample is filtered through a 0.22 μm microporous membrane, and the content of liquiritin and liquiritigenin is detected by HPLC, and the molar conversion yield of the product liquiritigenin is calculated. Three parallel experiments were set for the conversion of liquiritin.

Example 6

The crude enzyme solution obtained in step 1 of Example 5 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 5, except that the concentration of liquiritin in the basic transformation reaction system was 0.4 mg/mL. Three parallel experiments were set up.

Example 7

The crude enzyme solution obtained in step 1 of Example 5 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 5, except that the concentration of liquiritin in the basic transformation reaction system was 0.8 mg/mL. Three parallel experiments were set up.

Comparative Example 6

The crude enzyme solution obtained in step 1 of Example 5 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 5, except that the concentration of liquiritin in the basic transformation reaction system was 1.2 mg/mL. Three parallel experiments were set up.

Comparative Example 7

The crude enzyme solution obtained in step 1 of Example 5 was used to transform liquiritin. The transformation of liquiritin was the same as that of Example 5, except that the concentration of liquiritin in the basic transformation reaction system was 1.6 mg/mL. Three parallel experiments were set up.

The concentration of glycyrrhizin was analyzed by the above HPLC method for Examples 5 to 7 and Comparative Examples 6 to 7, and the conversion yield was calculated, as shown in Table 4. According to Table 4, when the substrate glycyrrhizin was in the concentration range of 0.1 to 0.8 mg/mL, the glycyrrhizin conversion yield did not decrease significantly with the increase of the substrate concentration; with the increase of the substrate concentration of 0.1 to 0.8 mg/mL, the enzyme in the reaction system was almost saturated, and when the substrate concentration was too high to 1.2 to 1.6 mg/mL, the glycyrrhizin conversion yield decreased accordingly, so the glycyrrhizin concentration of 0.8 mg/mL was selected as the optimal substrate concentration for conversion.

Table 4 Conversion rate of glycyrrhizin in Examples 5 to 7 and Comparative Examples 6 to 7

Examples 8 to 11 and Comparative Example 8 used crude enzyme solution of Agaricus leucophylla KXS-P9 prepared in the same batch, and the initial pH of the basic conversion reaction system was adjusted.

Example 8

1. Preparation of crude enzyme solution. Crude enzyme solution was prepared according to the method of Example 2. The enzyme activity of the crude enzyme solution was 100 U/mL.

2. Conversion of Liquorice glycosides

The basic conversion reaction system has a total of 5mL, 1mL of 4mg/mL liquiritin solution, 1mL of crude enzyme solution, and 3mL of PBS buffer (0.05mol/L, pH 7.5). The above liquids are placed in a test tube for conversion. The concentration of liquiritin in the basic conversion reaction system is 0.8mg/mL, the initial pH value of the basic conversion reaction system is adjusted to 4.5, and the conversion is carried out at 40°C for 6h, and the conversion speed is 150rpm. After the conversion is completed, a sample is taken immediately, and the reaction is stopped at 4°C, filtered through a 0.22μm microporous membrane, and the content of liquiritin and liquiritigenin is detected by HPLC, and the molar conversion yield of the product liquiritigenin is calculated. Three parallel experiments are set.

Example 9

The crude enzyme solution obtained in step 1 of Example 8 was used to transform liquiritin. The transformation of liquiritin was the same as in Example 8, except that the initial pH value of the basic transformation reaction system was adjusted to 5.5. Three parallel experiments were set.

Example 10

The crude enzyme solution obtained in step 1 of Example 8 was used to transform liquiritin. The transformation of liquiritin was the same as in Example 8, except that the initial pH value of the basic transformation reaction system was adjusted to 6.5. Three parallel experiments were set.

Embodiment 11

The crude enzyme solution obtained in step 1 of Example 8 was used to transform liquiritin. The transformation of liquiritin was the same as in Example 8, except that the initial pH value of the basic transformation reaction system was adjusted to 7.5. Three parallel experiments were set.

Comparative Example 8

The crude enzyme solution obtained in step 1 of Example 8 was used to transform liquiritin. The transformation of liquiritin was the same as in Example 8, except that the initial pH value of the basic transformation reaction system was adjusted to 8.5. Three parallel experiments were set.

The conversion yields of glycyrrhizin in Examples 8 to 11 and Comparative Example 8 are shown in Table 5. According to Table 5, when the pH value of the basic conversion reaction system is in the range of 4.5 to 6.5, the conversion yield of glycyrrhizin increases slowly; as the pH value increases, when the pH value is between 6.5 and 7.5, the conversion yield of glycyrrhizin rises rapidly, reaches the highest when the pH value is 7.5, and then decreases rapidly. Therefore, the optimum initial pH value for the conversion of glycyrrhizin by the β-glucosidase obtained in the present invention is about 7.5. The research results in the prior art show that the optimum reaction pH value of β-glucosidase is between 3.0 and 7.0, most of which are between 3.5 and 5.5, and a few are alkaline.

Table 5 Liquoricerin conversion rate of Examples 10 to 13 and Comparative Example 8

Example 12

The basic conversion reaction system is 5 mL in total: 1 mL of liquiritin solution, 1 mL of crude enzyme solution, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5). The above liquids are placed in a test tube for conversion. The crude enzyme solution is prepared according to the method for preparing the crude enzyme solution in Example 2, and the enzyme activity of the crude enzyme solution is adjusted to 100 U/mL.

Four factors, namely, conversion time (A), conversion temperature (B), liquiritin substrate concentration (C), and initial pH value of the basic conversion reaction system (D), were selected for the L9 (3 ⁴ ) orthogonal experiment. The experimental design and experimental results are shown in Table 6.

Table 6 Orthogonal test results

Through the intuitive analysis of the k ₁ , k ₂ , k ₃ values and range R of each factor in Table 6, the results show that the order of influence of each factor on the molar conversion yield of liquiritin is: B (temperature) > D (pH) > A (time) > C (substrate concentration). The variance analysis uses the liquiritin substrate concentration C with the smallest range as the error term, and the results of the variance analysis are shown in Table 7. According to Table 7, the conversion temperature has a significant effect on the conversion yield of liquiritin to liquiritigenin, and time, liquiritin substrate concentration, and pH have no significant effect on the conversion yield of liquiritin.

Table 7 Analysis of variance of orthogonal test results

The optimal conversion conditions for liquiritin to liquiritigenin were determined by the values of k ₁ , k ₂ , and k ₃ to be A ₂ B ₁ C ₂ D ₂ , ie, a conversion time of 12 h, a temperature of 37° C., a liquiritin substrate concentration of 0.8 mg/mL, and a pH value of 7.5.

The transformation experiment was repeated under the optimal transformation conditions: the basic transformation reaction system was 5 mL in total: 1 mL of liquiritin solution, 1 mL of crude enzyme solution, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5). According to the optimal parameter conditions: the transformation time was 12 h, the temperature was 37 ° C, the liquiritin substrate concentration was 0.8 mg/mL, and the initial pH value was 7.5. The HPLC graph of liquiritin before and after hydrolysis by β-glucosidase is shown in Figure 4. According to Figure 4, the active substance before transformation was mainly liquiritin, and the active substance after conversion by crude enzyme solution was mainly liquiritigenin. It can be calculated that the conversion rate of liquiritigenin after liquiritin was converted by β-glucosidase reached 93.09%.

Example 13

Crude β-glucosidase solution was prepared using Agaricus pyogenes KXS-P9, and the conversion rates of liquiritin to glycyrrhizin using crude β-glucosidase solution prepared from Agaricus pyogenes KXS-P9 and commercial β-glucosidase were compared.

The crude enzyme solution of β-glucosidase was prepared from Agaricus leucophylla KXS-P9 according to the method of Example 2, which was recorded as the crude enzyme solution of β-glucosidase (A).

Treatment 1: Basic conversion reaction system: 1 mL of crude enzyme solution of β-glucosidase (A) (the enzyme activity of the crude enzyme solution is 50 U/mL), 1 mL of substrate liquiritin solution and 3 mL of PBS buffer (0.05 mol/L, pH 7.5) were added to form a basic conversion reaction system; the concentration of liquiritin in the basic conversion reaction system was 0.8 mg/mL, and the initial pH value was 7.5. The conversion was carried out at 40°C for 6 h, and the conversion speed was 150 rpm. Three parallel experiments were set up.

Treatment 2: Same as treatment 1, except that the crude enzyme solution of β-glucosidase (A) with an enzyme activity of 75 U/mL was added. Three parallel experiments were set up.

Treatment 3: Same as treatment 1, except that the crude enzyme solution of β-glucosidase (A) with an enzyme activity of 125 U/mL was added. Three parallel experiments were set up.

Treatment 4: Same as treatment 1, except that the crude enzyme solution of β-glucosidase (A) with an enzyme activity of 150 U/mL was added. Three parallel experiments were set up.

Control 1: Basic conversion reaction system: 1 mL of commercial β-glucosidase (B) enzyme solution (enzyme activity of the enzyme solution is 50 U/mL), add 1 mL of substrate liquiritin solution and PBS buffer (0.05 mol/L, pH 7.5). The concentration of liquiritin in the basic conversion reaction system is 0.8 mg/mL, and the initial pH value is 7.5. Conversion is carried out at 40°C for 6 hours, and the conversion speed is 150 rpm. Set up 3 parallel experiments. The commercial β-glucosidase (B) enzyme solution is prepared using PBS buffer (0.05 mol/L, pH 7.5), the same below.

Control 2: Same as control 1, except that the commercial β-glucosidase (B) enzyme solution with an enzyme activity of 75 U/mL was added; three parallel experiments were set up.

Control 3: Same as control 1, except that the commercial β-glucosidase (B) enzyme solution with an enzyme activity of 125 U/mL was added; three parallel experiments were set up.

Control 4: Same as control 1, except that commercial β-glucosidase (B) enzyme solution with an enzyme activity of 150 U/mL was added, and three parallel experiments were set up.

The concentration of glycyrrhizin in treatments 1 to 4 and controls 1 to 4 was analyzed by the above HPLC method, and the conversion rate of glycyrrhizin to glycyrrhizin was calculated. The results are shown in Table 8. According to Table 8, within the range of enzyme activity of 50 to 75 U/mL, with the increase of the dosage of β-glucosidase (A) crude enzyme solution and commercial β-glucosidase (B), the effects of both on the conversion rate of glycyrrhizin have the same trend, that is, they both increase with the increase of enzyme dosage. As the enzyme dosage continues to increase, the effect of the two enzymes on the increase of glycyrrhizin conversion rate is not obvious. Overall, under the same enzyme dosage conditions, the effect of β-glucosidase (A) crude enzyme solution on the conversion of glycyrrhizin to glycyrrhizin is better than that of commercial β-glucosidase (B), and the highest conversion rates of glycyrrhizin after conversion are 81.28% and 75.87%, respectively. Therefore, the β-glucosidase produced by the white yellow small crispy handle mushroom KXS-P9 has a good application prospect in the preparation of glycyrrhizin from glycyrrhizin.

Table 8 Conversion rate of glycyrrhizin in treatments 1 to 4 and controls 1 to 4 (%)

Embodiment 14

1. Method for converting liquiritin into liquiritigenin: A total of 5 mL of the basic conversion reaction system: 1 mL of 4 mg/mL liquiritin solution, 1 mL of crude enzyme solution with an enzyme activity of 100 U/mL, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5), the above liquids are placed in a test tube for conversion. The initial pH value of the basic conversion reaction system is 7.5, the conversion temperature is 37°C, the conversion time is 12 h, the liquiritin substrate concentration in the basic conversion reaction system is 0.8 mg/mL, and a mixed solution is obtained after the conversion (the mixed solution is the converted solution).

Solution before transformation: concentration of 4 mg/mL liquiritin solution.

2. Determination of inhibition zone

Using Escherichia coli, Salmonella and Staphylococcus aureus as indicator bacteria for antibacterial activity, the punching method was used to evaluate the antibacterial activity of glycyrrhizin before and after transformation (reference: Liu Wenjie, Li Longqiang, Tang Jiahui, et al. Study on the diversity and antibacterial activity of endophytic fungi from licorice [J]. Natural Product Research and Development, 2021, 33(02): 256-267.).

(1) Escherichia coli. The pathogenic bacteria Escherichia coli was inoculated into nutrient broth medium and cultured at 35°C for 12 h. The suspension was diluted with sterile water to a total bacterial count of 10 ⁶ CFU/mL.

The solution before the conversion of liquiritin after membrane filtration was recorded as solution 1; the solution after the conversion of liquiritin after membrane filtration was recorded as solution 2; and the streptomycin sulfate solution with a mass concentration of 0.5 mg/mL was recorded as solution 3.

Take 100 μL of the above Escherichia coli suspension and evenly spread it on a NA medium plate, and use an 8 mm puncher to punch 4 holes on the medium, wherein 100 μL of solution 1 was added to the first well; 100 μL of solution 2 was added to the second well; 100 μL of sterile distilled water was added to the third well (negative control); and 100 μL of solution 3 was added to the fourth well (as a positive control for antibacterial activity determination).

After addition, the NA medium plates were cultured at 35°C for 24 h, and the diameters of the growth inhibition zones of the three pathogens were measured using the cross method. Three parallel experiments were set up and the average value was taken.

(2) Salmonella. The method for determining the inhibitory activity of glycyrrhizin against Salmonella before and after transformation is the same as that for Escherichia coli.

(3) Staphylococcus aureus. The method for determining the inhibitory activity of glycyrrhizin on Staphylococcus aureus before and after transformation is the same as that for Escherichia coli.

The antibacterial effect of liquiritin before and after β-glucosidase hydrolysis was studied, and the results are shown in Table 9 and Figure 5. According to Table 9 and Figure 5, liquiritin had no antibacterial activity against the three pathogens before transformation, while the transformation solution containing liquiritin after transformation showed an inhibitory effect on the three pathogens. Among them, the transformation solution had the best inhibitory effect on Staphylococcus aureus, with an inhibition zone of 12.7±0.31mm, followed by Salmonella 9.10±0.41mm, and the weakest inhibitory ability was Escherichia coli 6.37±0.62mm.

Table 9 Antibacterial effect of liquiritin before and after transformation

Note: “+” indicates bacterial growth, and “-” indicates sterile growth.

3. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The minimum inhibitory concentration of the sample was determined by serial dilution. The specific steps are as follows:

(1) The sample is the mixed solution obtained in step 1, and the content of glycyrrhizin in the mixed solution is 0.46 mg/mL.

(2) Add 100 μL of NB nutrient broth medium to wells 1 to 8 of a 96-well cell culture plate, take 100 μL of the sample and add it to well 1, mix well, then pipette 100 μL and add it to well 2, and so on, and dilute it continuously to well 8, which is equivalent to the sample in well 1 being diluted 2 times, the sample in well 2 being diluted 4 times, the sample in well 3 being diluted 8 times, the sample in well 4 being diluted 16 times, the sample in well 5 being diluted 32 times, the sample in well 6 being diluted 64 times, the sample in well 7 being diluted 128 times, and the sample in well 8 being diluted 256 times. The above dilution multiples are set for 9 parallel experiments, with a total of 72 wells.

(3) Three pathogens (Escherichia coli, Salmonella, and Staphylococcus aureus) were inoculated into NB medium and cultured at 35°C for 12 h. The suspensions were diluted with sterilized NB medium to a concentration of 10 ⁶ CFU/mL for later use.

(4) 100 μL of the pathogen suspension in step (3) was respectively aspirated and added to the wells of the cell culture plate in step (2) to mix evenly. Specifically, the Escherichia coli pathogen solution was added to wells 1 to 24, with 8 wells as a group and 3 parallel experiments set up for a total of 24 wells; the Salmonella pathogen solution was added to wells 25 to 48, with 8 wells as a group and 3 parallel experiments set up for a total of 24 wells; the Staphylococcus aureus pathogen solution was added to wells 49 to 72, with 8 wells as a group and 3 parallel experiments set up for a total of 24 wells; and then the cells were cultured at 30° C. for 24 h.

(5) Using sterile NB medium as negative control, set up three parallel experiments;

(6) Three pathogenic bacterial suspensions were used as positive controls, and three parallel experiments were set up for each.

After 24 hours, observe the culture fluid in the wells of the above cell culture plate. The clear culture fluid represents the sample with antibacterial activity, and the lowest concentration of the sample added is the minimum inhibitory concentration (MIC). Take 100 μL of the culture fluid in the wells of the cell culture plate where no pathogenic bacteria grow, spread it on the NA culture dish, and place it in a constant temperature incubator at 35°C for 24 hours. The lowest sample concentration at which no pathogenic bacteria grow on the plate is the minimum bactericidal concentration (MBC).

The results are shown in Table 10, and the data in Table 10 are the average values of three parallel experiments. The mixed solution after transformation has strong antibacterial and bactericidal effects on Staphylococcus aureus, with MIC and MBC of 0.058 mg/mL and 0.115 mg/mL respectively; the minimum inhibitory concentration of Salmonella is 0.058 mg/mL, and the minimum bactericidal concentration is 0.230 mg/mL; the MIC and MBC of Escherichia coli are the highest among the three pathogens, 0.115 mg/mL and 0.230 mg/mL respectively, indicating that the antibacterial activity against Escherichia coli is slightly poor. Combined with the inhibition zone of pathogenic bacteria, the inhibitory effect of the transformation solution on the three pathogenic bacteria is in the order of Staphylococcus aureus, Salmonella, and Escherichia coli.

Table 10 MIC and MBC of the mixture after liquiritin conversion

Embodiment 15

1. Transformation Group 1: A method for converting liquiritin into glycyrrhizin using β-glucosidase (denoted as β-glucosidase A) from Agaricus leucophylla KXS-P9:

Preparation of crude enzyme solution of β-glucosidase (β-glucosidase A) from Agaricus leucophylla KXS-P9: The preparation method is the same as that in Example 2, and the enzyme activity is adjusted to 100 U/mL with PBS buffer (0.05 mol/L, pH 7.5) as the conversion enzyme solution.

Transformation group 1 was divided into 3 treatments. Treatment group 1: 5 mL of basic transformation reaction system: 1 mL of 4 mg/mL liquiritin solution, 1 mL of crude enzyme solution with an enzyme activity of 100 U/mL, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5). The above liquids were placed in a test tube for transformation. The initial pH value of the basic transformation reaction system was 7.5, the transformation temperature was 37 ° C, the transformation time was 12 h, the liquiritin substrate concentration in the transformation reaction system was 0.8 mg/mL, and a mixed solution 1 was obtained after the transformation (the mixed solution was the solution after transformation). Three parallel experiments were set up.

Treatment group 2: Same as treatment group 1, the only difference is that the concentration of liquiritin substrate in the basic conversion reaction system is 0.4 mg/mL; 3 parallel experiments are set.

Treatment group 3: Same as treatment group 1, the only difference is that the concentration of liquiritin substrate in the basic conversion reaction system is 0.6 mg/mL; 3 parallel experiments are set.

2. CK1: The basic conversion reaction system solutions of treatment group 1, treatment group 2 and treatment group 3 of conversion group 1 were mixed and measured immediately.

3. Transformation group 2: 5 mL of the basic transformation reaction system of commercial β-glucosidase (denoted as β-glucosidase B), β-glucosidase B is derived from Aspergillus niger and produced by Shanghai Yuanye Biotechnology Co., Ltd.: Transformation group 2 is divided into 3 treatments, specifically:

Treatment group 4: 1 mL of 4 mg/mL liquiritin solution, 1 mL of β-glucosidase B enzyme solution with an enzyme activity of 100 U/mL, and 3 mL of PBS buffer (0.05 mol/L, pH 7.5), the above liquids were placed in a test tube for conversion. The initial pH value of the basic conversion reaction system was 7.5, the conversion temperature was 37°C, the conversion time was 12 h, the liquiritin substrate concentration in the conversion reaction system was 0.8 mg/mL, and after the conversion, a mixed solution 2 was obtained (the mixed solution was the converted solution). Three parallel experiments were set up.

Treatment group 5: Same as treatment group 4, the only difference is that the concentration of liquiritin substrate in the basic conversion reaction system is 0.4 mg/mL; 3 parallel experiments are set.

Treatment group 6: Same as treatment group 4, the only difference is that the concentration of liquiritin substrate in the basic conversion reaction system is 0.6 mg/mL; 3 parallel experiments are set.

4. CK2: The basic conversion reaction system solutions of treatment groups 4, 5 and 6 of conversion group 2 were mixed and measured immediately.

The mixed solutions obtained after the transformation of each treatment group of CK1, CK2, transformation group 1 and transformation group 2 were immediately subjected to the following assays.

1. Determination of DPPH free radical scavenging activity

Sample DPPH clearance rate = [A0-(A1-A2)]/A0*100%

A0 = absorbance at 517 nm of the blank group, which is a mixture of sample solution and methanol solution; A1 = absorbance at 517 nm of the sample measurement group, which is a mixture of sample solution and DPPH solution; A2 = absorbance at 517 nm of the sample control group, which is a mixture of methanol solution and DPPH solution.

The results are shown in Table 11 and Figure 6, which are the results of DPPH radical scavenging activity determination of CK1, CK2, conversion group 1 and conversion group 2 at different liquiritin substrate concentrations. The two β-glucosidases converted three concentrations of substrate liquiritin. In the range of 0.4-0.8 mg/mL of liquiritin concentration, the DPPH radical scavenging rate showed an increasing trend with the increase of substrate liquiritin concentration. The antioxidant activity was highest when the liquiritin concentration was 0.8 mg/mL. Under this condition, the DPPH radical scavenging rate of conversion group 1 was significantly higher than that of conversion group 2, with the scavenging rates of 52.90% and 46.64%, respectively, indicating that the ability of β-glucosidase produced by Pleurotus eryngii KXS-P9 to convert liquiritin was stronger than that of commercial β-glucosidase. The above two enzymes were used to convert liquiritin to liquiritigenin, and the results showed that the DPPH radical scavenging rate after β-glucosidase hydrolysis was significantly higher than that before conversion (P<0.05), indicating that the DPPH radical scavenging ability of liquiritigenin was stronger than that of liquiritin. The data in Figure 6 are the average values of parallel experiments.

Table 11 Determination of DPPH radical scavenging activity at different liquiritin substrate concentrations

2. Determination of ABTS free radical scavenging activity

The antioxidant activity of the sample solutions of CK1, CK2, transformation group 1 and transformation group 2 was determined using an antioxidant capacity detection kit (ABTS microplate method). Free radical scavenging rate of the sample = (A blank - A determination) / A blank * 100%, where A blank represents the absorbance value of the working solution, and A determination represents the absorbance value after the reaction of the working solution and the sample solution.

The results of ABTS free radical scavenging activity determination of CK1, CK2, conversion group 1 and conversion group 2 at different liquiritin substrate concentrations are shown in Figure 7 and Table 12. In the concentration range of liquiritin concentration of 0.4-0.8 mg/mL, the ABTS free radical scavenging rate of the mixed solution showed an upward trend with the increase of substrate concentration. The scavenging rate of ABTS free radicals was the highest when the liquiritin concentration was 0.8 mg/mL, and the scavenging rate was conversion group 1 (81.52%) > conversion group 2 (69.10%) > CK1 (62.75%) > CK2 (60.72%). The ABTS free radical scavenging rate of conversion group 1 at three substrate concentrations was significantly stronger than that of conversion group 2, and was significantly higher than that of the control group (P<0.05). This indicates that the content of liquiritigenin in conversion group 1 is higher, which is consistent with the above experimental results. The β-glucosidase produced by the white yellow small crispy handle mushroom KXS-P9 has great application potential in converting liquiritigenin to liquiritigenin. The data in Figure 7 and Table 12 are the average values of parallel experiments.

Table 12 ABTS free radical scavenging rate determination results at different liquiritin substrate concentrations

3. Determination of total antioxidant capacity (FRAP method)

The total antioxidant activity of the sample solutions of CK1, CK2 conversion group 1 and conversion group 2 was determined using a total antioxidant capacity detection kit (FRAP microplate method). The kit uses the concentration of Fe ²⁺ to represent the total antioxidant capacity. The standard curve of the relationship between the concentration of Fe ²⁺ and the absorbance, y=1.1928x+0.0886 (R ² =0.9979), was prepared using the reagents in the kit. The standard curve is shown in FIG8 . The total antioxidant capacity of the four groups of samples was determined using the standard curve.

The results of total antioxidant capacity determination of CK1, CK2, conversion group 1 and conversion group 2 under different liquiritin substrate concentrations are shown in Figure 9 and Table 13. In the concentration range of liquiritin concentration of 0.4-0.8 mg/mL, with the increase of substrate concentration, the total antioxidant capacity of the conversion liquid of CK1, CK2, conversion group 1 and conversion group 2 increased to varying degrees. The total antioxidant capacity was the best when the liquiritin concentration was 0.8 mg/mL, and the order of strength was conversion group 1 (0.141 mmol/L) > conversion group 2 (0.105 mmol/L) > CK1 (0.094 mmol/L) > CK2 (0.086 mmol/L). The ABTS free radical scavenging rate of conversion group 1 was significantly stronger than that of conversion group 2 under the three substrate concentrations (P<0.05), but there was no significant difference in the total antioxidant capacity of the control group CK1 and CK2. The reason is that liquiritin is converted into liquiritigenin after enzymatic hydrolysis, which has stronger antioxidant activity and reduces Fe ³⁺ to Fe ²⁺ , which is manifested as an increase in total antioxidant capacity. Similarly, the total antioxidant capacity of commercial β-glucosidase is significantly lower than that of β-glucosidase produced by Pleurotus eryngii KXS-P9 (P<0.05). The data in Figure 9 are the average values of parallel experiments.

Table 13 Determination results of total antioxidant capacity at different liquiritin substrate concentrations (mmol/L)

In summary, the present invention uses the white and yellow brittle mushroom KXS-P9 to transform liquiritin into liquiritigenin through a microbial transformation method with liquiritigenin as a substrate. The technical solution has low production cost and high conversion rate, and is a green and efficient fermentation process for transforming liquiritigenin. The β-glucosidase produced by the white and yellow brittle mushroom KXS-P9 has a good application prospect in the transformation of liquiritigenin into liquiritigenin.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (10)

1. A strain of white Huang Xiaocui handle mushroom (Candolleomyces candolleanus) KXS-P9 with a preservation number of CGMCC No.40386.

2. A microbial agent comprising the Bai Huangxiao panus crispatus KXS-P9 and/or a metabolite thereof of claim 1.

3. Use of Bai Huangxiao, agrocybe cylindracea KXS-P9 of claim 1 or the microbial inoculum of claim 2 in one or more of the following 1) to 3);

1) Preparing glycyrrhizin;

2) The conversion yield of the liquiritin to the liquiritin is improved;

3) Preparing beta-glucosidase.

4. A method for converting liquiritin to liquiritin, comprising the steps of:

The beta-glucosidase enzyme solution prepared by using Bai Huangxiao XUANGGU KXS-P9 in claim 1 is converted with liquiritin to obtain liquiritin.

5. The method of claim 4, wherein the conversion system further comprises a buffer comprising a phosphate buffer.

6. The method according to claim 5, wherein the preparation method of the beta-glucosidase enzyme solution comprises the following steps: culturing the seed solution of the Huang Xiaocui handle mushroom KXS-P9 in a culture medium to obtain a culture solution;

centrifuging the culture solution to obtain a supernatant;

Precipitating beta-glucosidase by adopting an ammonium sulfate solution with the saturation degree of 60% to obtain beta-glucosidase enzyme solution;

The culture medium takes water as a solvent, and comprises the following components: 30-31 g/L of wheat bran, 11-12 g/L, KH ₂PO₄ 0.9.9-1.1 g/L of beef extract and 0.4-0.6 g/L of MgSO ₄, and the pH value is 7-8.

7. The method according to claim 6, wherein the enzyme activity of the beta-glucosidase enzyme solution is 50-100U/mL; the initial addition amount of the beta-glucosidase enzyme liquid in the conversion system is 15% -20% of the total volume of the conversion system.

8. The method according to claim 4, wherein the initial substrate concentration of glycyrrhizin in the transformation system is 0.1-1.0 mg/mL.

9. The method according to claim 4, wherein the temperature of the conversion is 30-40 ℃, the time of the conversion is 6-14 hours, and the initial rotation speed of the conversion is 150-200 rpm.

10. The method according to claim 4, wherein the initial pH of the conversion system is 4 to 7.5.