CN106967775B - Method for biocatalytic preparation of diosgenin and bacterial agent used therefor - Google Patents

Abstract

The invention discloses a method for biocatalytically preparing diosgenin and a bacterial agent used therefor. The method for preparing diosgenin is M1) the method includes culturing Fusarium fungi and Curvus sp The culture medium of Dioscorea genus cultivates Fusarium fungi and Bacillus spp. fungi to obtain diosgenin; M3) the method includes culturing Fusarium fungi with a medium containing Dioscorea scutellariae to obtain diosgenin. The yields of diosgenin from the combined fermentation of Curvus crescentus and Fusarium containing Dioscorea scutellariae were higher than that of the culture medium containing Diospermum scutellariae fermented by C. crescentus alone with Fusarium spp. The sum of the yields of diosgenin in the medium of Dioscorea solani was even higher, and the synergistic effect of Curvus crescentus and Fusarium was produced in the production of diosgenin by fermenting the medium containing Dioscorea solani.

Description

Method for biocatalytic preparation of diosgenin and bacterial agent used therefor

technical field

The invention relates to a method for biocatalytically preparing diosgenin and a fungicide used therefor.

Background technique

Diosgenin (diosgenin), known as diosgenin in industry, is an important active ingredient in Dioscorea L. plants. ". More than 60% of the steroid hormones produced in various countries in the world use diosgenin as the raw material, and the market demand is huge. At present, the method for producing diosgenin is based on the chemical acid hydrolysis method proposed by Rothrock et al. in 1957, which has many problems. How to cure pollution from the source is an urgent problem to be solved in the sustainable development of diosgenin industrialization. The preparation of diosgenin by microbial fermentation or the preparation of diosgenin by biological enzymes is a development direction for the production of diosgenin without acidification.

SUMMARY OF THE INVENTION

A technical problem to be solved by the present invention is how to efficiently prepare diosgenin.

In order to solve the above technical problems, the present invention provides the application of P1) to P3):

P1) the application of Fusarium fungi and Curvus sp. fungi in the preparation of diosgenin or the application of the following C1 or the following C2 in the preparation of diosgenin;

P2) application of Fusarium fungi and Bacillus sp. fungi in the preparation of diosgenin or the application of the following C3 or the following C4 in the preparation of diosgenin;

P3) Application of Fusarium fungus in the preparation of diosgenin.

In order to solve the above technical problems, the present invention provides the method for preparing diosgenin from M1) to M3):

M1) The method comprises culturing Fusarium fungus and Curvus sp. fungus with a medium containing Dioscorea scutellariae to obtain diosgenin;

M2) The method comprises culturing Fusarium fungi and Bacillus sp. fungi with a medium containing Dioscorea scutellariae to obtain diosgenin;

M3) The method includes culturing the Fusarium fungus with a medium containing Dioscorea scutellariae to obtain diosgenin.

In the above method or application, in M1) to M3) and P1) to P3), the fungi of the genus Fusarium can be Fusarium sp. CPCC 400709, Fusarium sp. ) at least one of CPCC 400226 and Fusarium sp. CPCC 400712; the Curvus sp. fungus may be Curvus sp. crescents; the Brachysporium sp. fungus may be Brachysporium sp. Dictyosporium sp. CPCC 400718.

In the above method or application, in M1), M3), P1) and P3), the Fusarium fungus can specifically be in Fusarium sp. CPCC 400709; M2) and P2), the Fusarium sp. The Fusarium fungus can be Fusarium sp. CPCC 400709; M2) and P2), and the Fusarium sp. can also be Fusarium sp. CPCC 400709 and Fusarium sp. In species (Fusarium sp.) CPCC 400226; M2) and P2), the Fusarium sp. fungi can also be Fusarium sp. (Fusarium sp.) CPCC 400709, Fusarium sp. (Fusarium sp.) CPCC 400226 and Fusarium sp. CPCC 400712.

In the above method or application, in M1) and P1), the Curvularialunata 3.4381 may be the curvularialunata.

In the above application, the preparation of diosgenin includes fermentation with a medium containing Dioscorea solani.

In the above method or application, the medium containing Dioscorea scutata can be a solid medium or a liquid medium.

In the above method or application, the Dioscorea scuti can be the rhizome of Dioscorea scutellariae.

In the above method or application, in M1) and P1), the ratio of the number of colony forming units (cfu) of the Fusarium fungus and the Curvus sp. fungus may be 1:(0.5-4), such as 1:2 .

In the above-mentioned application, the above-mentioned P2) may specifically be P21), P22) or P23); the P21), P22) or P23) are the applications of Fusarium fungi and Bacillus sp. fungi in the preparation of diosgenin.

The Fusarium fungus in the P21) may be Fusarium sp. CPCC 400709; in the P21), the Fusarium fungus and the Bacillus sp. The ratio of the number of units (cfu) can be 1:(0.5-4), such as 1:2.

In the P22), the Fusarium fungi can specifically be Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226. In the P22), the colony forming unit (cfu) of Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC 400718 ) number ratio may be 1:(0.5-4):(0.5-4), such as 1:1:1.

In the P23), the Fusarium fungal agent can specifically be Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Fusarium sp. .) CPCC400712. In the P23), Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226, Fusarium sp. CPCC 400712, and Bacillus sp. (Dictyosporium sp.) CPCC 400718 may have a colony forming unit (cfu) number ratio of 1:(0.5-4):(0.5-4):(0.5-4), such as 1:1:1:1.

In the above-mentioned method, in the above-mentioned M1), culturing Fusarium fungi and Curvus sp. fungi with the medium containing Dioscorea scutae specifically may be to first insert the Fusarium fungus into the medium containing Dioscorea scutellum to carry out. After culturing, the fungus of the genus Curlyspora is inserted for culturing. For example, the Fusarium fungus is firstly inserted into the medium containing Dioscorea scutellariae, and cultured at 20-30° C. (eg 28° C.) for 15-25 days (eg, 22 days), and then the Curvus sp. fungus is inserted Incubate at 20-30°C (eg 28°C) for 5-10 days (eg 7 days).

The above-mentioned M2) may specifically be M21), M22) or M23).

The M21) is that the method comprises culturing Fusarium fungi and Bacillus sp. fungi with a medium containing Dioscorea scutellariae to obtain diosgenin. In the M21), the Fusarium fungus can be Fusarium sp. CPCC400709; the Brachysporum fungus can be Dictyosporium sp. CPCC 400718. The ratio of the number of colony forming units (cfu) of the Fusarium fungus and the Bacillus sp. fungus may be 1:(0.5-4), such as 1:2. Wherein, culturing Fusarium fungi and Bacillus spp. fungi with a medium containing Dioscorea scutellariae may specifically be inserting the Fusarium fungus into the medium containing Diospermum scutellariae for culturing and then inserting the Fusarium spp. The fungus of the genus Bacillus sp. was cultured. For example, the Fusarium fungus is firstly inserted into the medium containing Dioscorea scutellariae, and cultured at 20-30°C (eg, 28°C) for 15-25 days (eg, 22 days), and then the Fusarium fungus is inserted into the medium Incubate at 20-30°C (eg 28°C) for 5-10 days (eg 7 days).

The M22) is that the method comprises culturing Fusarium fungi and Bacillus sp. fungi with a medium containing Dioscorea scutellariae to obtain diosgenin. In the M22), the Fusarium fungus may specifically be Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226; the Bacillus sp. fungus may be Dictyosporium sp. CPCC 400718. In the M22), the colony forming unit (cfu) of Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC 400718 ) number ratio may be 1:(0.5-4):(0.5-4), such as 1:1:1. Wherein, culturing Fusarium sp. fungi and Bacillus sp. fungi with a medium containing Dioscorea scutellariae may specifically be firstly inserting Fusarium sp. The Bacillus sp. fungus was then inoculated for cultivation, and then inoculated with Fusarium sp. CPCC 400226 for cultivation. For example, Fusarium sp. CPCC 400709 is firstly inserted into the medium containing Dioscorea scutifolia, and cultured at 20-30°C (eg, 28°C) for 15-25 days (eg, 22 days), and then the bricks are added. Dictyosporium sp. CPCC 400718 was cultured at 20-30°C (eg 28°C) for 5-10 days (eg 7 days) and then inserted into Fusarium sp. CPCC 400226 on 20 -30°C (eg 28°C) for 5-10 days (eg 7 days).

The M23) is that the method comprises culturing Fusarium fungi and Bacillus sp. fungi with a medium containing Dioscorea scutellariae to obtain diosgenin. In the M23), the Fusarium fungi can specifically be Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Fusarium sp. ) CPCC400712; the Dictyosporium sp. fungus may be Dictyosporium sp. CPCC 400718. In the M23), Fusarium sp. CPCC 400709, Fusarium sp. CPCC400226, Fusarium sp. CPCC 400712, and Bacillus sp. (Dictyosporium sp.) CPCC 400718 may have a colony forming unit (cfu) number ratio of 1:(0.5-4):(0.5-4):(0.5-4), such as 1:1:1:1. Wherein, the cultivation of Fusarium fungi and Bacillus spp. fungi in the medium containing Dioscorea scutellum may specifically be the first inserting Fusarium sp. Then insert Dictyosporium sp. CPCC 400718 for culture, insert Fusarium sp. CPCC400226 for culture, and insert Fusarium sp. CPCC 400712 to cultivate. For example, Fusarium sp. CPCC 400709 is firstly inserted into the medium containing Dioscorea scutifolia, and cultured at 20-30°C (eg, 28°C) for 15-25 days (eg, 22 days), and then the bricks are added. Dictyosporium sp. CPCC 400718 was cultured at 20-30°C (eg 28°C) for 5-10 days (eg 7 days) and then inserted into Fusarium sp. CPCC 400226 on 20 -30°C (eg 28°C) for 5-10 days (eg 7 days) and then inoculated with Fusarium sp. CPCC 400712 at 20-30°C (eg 28°C) for 7-15 days ( such as 10 days).

In the above-mentioned M3), culturing the Fusarium fungus with the medium containing Dioscorea scutellariae specifically may be to insert the Fusarium fungus into the medium containing Dioscorea scutellariae and cultivate at 20-30° C. (such as 28° C.) for 15 minutes. -25 days (eg 22 days).

In order to solve the above technical problems, the present invention provides a fungicide for preparing diosgenin.

The bacterial agent for preparing diosgenin provided by the present invention is any one of C1 to C4:

The C1 is a complete set of inoculants for preparing diosgenin, which is composed of a Fusarium fungal inoculum and a Curvus sp. , the active ingredient of the Curvus sp. fungal inoculum contains the Curvus sp. fungus;

The C2 is an inoculum for preparing diosgenin containing the Fusarium fungus and the Curvus sp. fungus as active ingredients;

The C3 is a complete set of inoculants for preparing diosgenin, which is composed of a Fusarium fungal inoculant and a Brachysporum fungal inoculum, and the active ingredient of the Fusarium fungal inoculum contains the Fusarium fungus , the active ingredient of the Bacillus sp. fungal inoculum contains the Bacillus sp. fungus;

The C4 is an inoculant for preparing diosgenin containing the Fusarium fungus and the Bacillus sp. fungus as active ingredients.

In the above-mentioned complete set of inoculants for preparing diosgenin, in the C1, the active ingredient of the Fusarium fungal inoculum may be the Fusarium fungus, and the active ingredient of the Fusarium fungal inoculum may also be: It may contain other biological components or non-biological components, and other active components of the inoculum can be determined by those skilled in the art according to the yield of diosgenin. In the C1, the Fusarium fungus may specifically be Fusarium sp. CPCC 400709. In the C1, the fungus of the genus Curvularia may be Curvularia crescentus; the Curvularia lunata may specifically be Curvularia lunata 3.4381. In the C1, the active ingredient of the fungal inoculum of the genus Curvus sp. may be the fungus of the genus Curvus sp., and the active ingredient of the fungal inoculum of the genus Curvus sp. may also contain other biological components or non-biological components, Other active ingredients of the bacterial agent can be determined by those skilled in the art according to the yield of diosgenin. The Fusarium fungal inoculum and the Curvus sp. fungal inoculum in the C1 are packaged separately. In the C1, the ratio of the number of colony forming units (cfu) of the fungi of the genus Fusarium and the fungus of the genus Curvularia may be 1:(0.5-4), such as 1:2.

In the above-mentioned complete set of microbial inoculants for preparing diosgenin, the C2 may specifically be an microbial inoculum for preparing diosgenin whose active ingredients are the Fusarium fungi and the Curvus sp. fungi; the C2 The active ingredient of C2 may also contain other biological or non-biological ingredients, and the other active ingredients of C2 can be determined by those skilled in the art according to the yield of diosgenin. In the C2, the Fusarium fungus may specifically be Fusarium sp. CPCC 400709. In the C2, the fungus of the genus Curvularia may be Curvularia crescenti; the Curvularia lunata may specifically be Curvularia lunata 3.4381. In the C2, the ratio of the number of colony forming units (cfu) of the fungi of the genus Fusarium and the fungus of the genus Curvularia may be 1:(0.5-4), such as 1:2.

In the above-mentioned complete inoculum for preparing diosgenin, the C3 may specifically be C31, C32 or C33. Said C31, C32 and C33 are all complete sets of inoculants for preparing diosgenin composed of Fusarium fungal inoculants and Bacillus sp. fungal inoculants, and the active ingredient of said Fusarium fungal inoculum contains all the The Fusarium fungus, and the active ingredient of the Bacillus sp. fungus preparation contains the Bacillus sp. fungus. Among the C31, C32 and C33, the fungi of the genus Brachysporium may be Dictyosporium sp. CPCC 400718. In the C31, C32 and C33, the active ingredient of the Brachysporum fungal inoculum may be the Brachysporum fungus, and the active ingredient of the Brachysporum fungal formulation may also contain other biological components or Non-biological components and other active components of the bacterial agent can be determined by those skilled in the art according to the yield of diosgenin.

In the C31, the Fusarium fungus may specifically be Fusarium sp. CPCC 400709. The active ingredient of the Fusarium fungus agent can be the Fusarium fungus, and the active ingredient of the Fusarium fungus agent can also contain other biological components or non-biological components. Those skilled in the art can determine according to the yield of diosgenin. In the C31, each inoculum is packaged independently. In the C31, the ratio of the number of colony forming units (cfu) of the fungi of the genus Fusarium and the fungi of the genus Bracaria may be 1:(0.5-4), such as 1:2.

In the C32, the Fusarium sp. inoculum may specifically be a Fusarium sp. CPCC400709 inoculum and a Fusarium sp. CPCC 400226 inoculum. The active ingredient of the Fusarium sp. CPCC 400709 microbial inoculum can be Fusarium sp. CPCC 400709, and may also contain other biological or abiotic components. Other activities of the microbial inoculum The ingredients can be determined by those skilled in the art according to the yield of diosgenin. The active ingredient of the Fusarium sp. CPCC 400226 inoculant may be Fusarium sp. CPCC 400226, and may also contain other biological or abiotic components. Active ingredients can be determined by those skilled in the art according to the yield of diosgenin. In the C32, each inoculum is packaged independently. In the C32, the colony forming unit (cfu) of Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC400718 The number ratio can be 1:(0.5-4):(0.5-4), eg 1:1:1.

In the C33, the Fusarium sp. CPCC400709 inoculant, the Fusarium sp. CPCC 400226 inoculant, and the Fusarium sp. (Fusarium sp.) CPCC 400712 bacterial agent. The active ingredient of the Fusarium sp. CPCC 400709 microbial inoculum may be Fusarium sp. CPCC 400709, and may also contain other biological or non-biological components. Active ingredients can be determined by those skilled in the art according to the yield of diosgenin. The active ingredient of the Fusarium sp. CPCC 400226 inoculant may be Fusarium sp. CPCC 400226, and may also contain other biological or abiotic components. Active ingredients can be determined by those skilled in the art according to the yield of diosgenin. The active ingredient of the Fusarium sp. CPCC 400712 microbial inoculum may be Fusarium sp. CPCC 400712, and may also contain other biological or non-biological components. Active ingredients can be determined by those skilled in the art according to the yield of diosgenin. In the C33, each inoculum is packaged independently. In the C33, Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226, Fusarium sp. CPCC 400712, and Bacillus sp. (Dictyosporium sp.) CPCC 400718 may have a colony forming unit (cfu) number ratio of 1:(0.5-4):(0.5-4):(0.5-4), such as 1:1:1:1.

In the above-mentioned complete inoculum for preparing diosgenin, the C4 may specifically be C41, C42 or C43. The C41, C42 and C43 are all bacterial agents for preparing diosgenin containing the Fusarium fungus and the Bacillus sp. fungus as active ingredients. The active ingredients of the C41, C42 and C43 can be the Fusarium fungi and the Bacillus spp. fungi, and can also contain other biological or non-biological ingredients. Determined according to the yield of diosgenin. Among the C41, C42 and C43, the fungi of the genus Brachysporium may be Dictyosporium sp. CPCC 400718.

In the C41, the Fusarium fungus may specifically be Fusarium sp. CPCC 400709. In the C41, the ratio of the number of colony forming units (cfu) of the fungi of the genus Fusarium and the fungi of the genus Bracaria may be 1:(0.5-4), such as 1:2.

In the C42, the Fusarium sp. fungi can specifically be Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226. In the C42, the colony forming units (cfu) of Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC 400718 The number ratio can be 1:(0.5-4):(0.5-4), eg 1:1:1.

In the C43, the Fusarium fungi may specifically be Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Fusarium sp. CPCC 400712. In the C43, Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226, Fusarium sp. CPCC 400712 and Bacillus sp. ( Dictyosporium sp.) CPCC 400718 may have a colony forming unit (cfu) number ratio of 1:(0.5-4):(0.5-4):(0.5-4), such as 1:1:1:1.

In addition to the active ingredient, the above-mentioned bacterial agent may also include auxiliary materials, such as water, carbon source and/or nitrogen source, etc. Carbon source is a kind of nutrients for microbial growth, and it is a carbon-containing compound, including fast-acting and delayed-acting carbon sources such as sugars, oils, organic acids and organic acid esters, and small molecular alcohols. Nitrogen sources refer to substances that provide nitrogen elements required for microbial nutrition, including quick-acting and delayed-acting nitrogen sources such as peanut cake flour, soybean cake flour, yeast powder, peptone, ammonia, ammonium salts and nitrates.

In the inoculum, various fungi can exist in the form of cultured living cells and fermentation broths of living cells. The dosage form of the bacterial agent can be various dosage forms, such as liquid, powder or granule.

Experiments have shown that the yield of diosgenin in the combined fermentation of Curvula crescentus and Fusarium containing Diospermum scutellariae is higher than that of C. crescentus alone fermenting the medium containing Diospermum scutellariae alone with Fusarium spp. The sum of the yields of diosgenin from the two fermented medium containing Dioscorea scutellariae was even higher, and the synergy between Curvus crescentus and Fusarium in the production of diosgenin from the fermented medium containing Dioscorea scutellariae effect (Table 1). In terms of the yield of diosgenin, CPCC400709+CPCC 400718 complete set of inoculum treatment increased by 9% compared with Fusarium fungus treatment, CPCC 400709+CPCC400718+CPCC 400226 complete set of inoculant treatment improved than Fusarium fungus treatment Compared with the treatment of Fusarium fungus, the treatment of CPCC 400709+CPCC400718+CPCC 400226+CPCC 400712 increased by 14%.

Description of drawings

Fig. 1 is the chromatogram of the identification of diosgenin by HPLC.

In the figure, (A) is a collection of diosgenin standard product collections, (B) is the HPLC collection of illustrative plates of the fermentation product of Curvus crescentus fungus of 3.3, (C) is the HPLC collection of collections of the fermentation product of Fusarium fungus of 3.4, (D) is the HPLC profile of the sickle+new fermentation product of 3.1, and (E) is the new+ sickle fermentation product of 3.2, wherein the chromatographic peaks of diosgenin are indicated by arrows.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Curvularia lunata 3.4381 in the following examples (Bing Feng, et al. The microbiological transformation of steroidal saponins by Curvularialunata. Tetrahedron 61 (2005) 11758-11763.25 October 2005) The biological material is publicly available from the applicant, the The biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

The Fusarium sp. CPCC 400709 in the following examples has been collected on July 6, 2015 in the China Pharmaceutical Microorganism Culture Collection (ChinaPharmaceutical Culture Collection, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, Abbreviated as CPCC, address: No. 1 Tiantanxili, Beijing, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, zip code: 100050), the public can obtain this strain from CPCC since the collection date.

The Fusarium sp. CPCC 400712 in the following examples has been collected on July 6, 2015 in the China Pharmaceutical Microorganism Culture Collection (ChinaPharmaceutical Culture Collection, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, Abbreviated as CPCC, address: No. 1 Tiantanxili, Beijing, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, zip code: 100050), the public can obtain this strain from CPCC since the collection date.

Dictyosporium sp. CPCC 400718 has been collected on July 6, 2015 in the China Pharmaceutical Microorganism Culture Collection (CPCC for short) at the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, at the address: No. 1 Tiantanxili, Beijing, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, zip code: 100050), the strain can be obtained from CPCC by the public since the collection date.

Aspergillus flavus 3.2792 (A.flavus 3.2792) in the following examples (Hongzhi Huang, et al. Pathways of biotransformation of zingiberen newsaponin from Dioscorea zingiberensis C. H. Wright to diosgenin. Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1-7) The public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

The Fusarium sp. CPCC 400226 in the following examples was collected in the China Pharmaceutical Microorganism Culture Collection (ChinaPharmaceutical Culture Collection, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences on February 1, 2007, Abbreviated as CPCC, address: No. 1 Tiantanxili, Beijing, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, zip code: 100050), the public can obtain this strain from CPCC since the collection date.

Aspergillus niger (Aspergillus niger) CPCC 400524 in the following examples has been collected on July 6, 2015 in the China Pharmaceutical Microorganism Culture Collection (ChinaPharmaceutical Culture Collection, referred to as CPCC, address of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences). : No. 1 Tiantanxili, Beijing, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, zip code: 100050), the strain can be obtained by the public from CPCC since the collection date.

Example 1. Preparation of diosgenin by cultivating Fusarium fungi and Curvus sp. or Fusarium spp

1. Preparation of the culture medium used in this example

Potato dextrose agar (PDA) medium: peel the potatoes, cut 200g into small pieces, add water and boil for 30min, filter with 4 layers of gauze, add 20g glucose, 17g agar, dilute to 1000mL with distilled water, boil and mix well, sterilize at 121°C The bacteria were incubated for 20 minutes to obtain PDA medium.

Seed medium: glucose 2g, sucrose 1g, soybean powder 0.2g, peptone 1g, dipotassium hydrogen phosphate 0.03g, polyethylene glycol 0.25g, sodium nitrate 0.3g, ammonium sulfate 0.3g, dilute to 100mL with water, pH 6.0 . Sterilize at 121°C for 20 minutes to obtain seed medium.

Solid state fermentation medium (medium containing Dioscorea scuti): 1 g glucose, 1 g yeast extract, 20 g rice husk, 10 g bran, 1 g K ₂ HPO ₄ , 0.5 g MgSO ₄ ·7H ₂ O, 5 g dried scutellum leaves Dioscorea rhizome powder was soaked in 21 mL of deionized water, adjusted to pH 6.0, and sterilized at 121° C. for 20 minutes to obtain a solid-state fermentation medium.

2. Preparation of diosgenin-producing bacterial agent

2.1. Preparation of Curvus crescentus fungicide

Curvularia lunata 3.4381 was cultured on the slant of PDA medium at 28°C for 5 days, and then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and the crescent moon was obtained by shaking at 28°C for 2 days. Fermentation broth of Curvularia lunata 3.4381. The fermented liquid of Curvularia lunata 3.4381 is a curvularia fungus agent.

2.2. Preparation of Fusarium fungal agent

Fusarium sp. CPCC 400709 was cultured on the slant of PDA medium at 28°C for 5 days, and then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and shaken at 28°C for 2 days. Fusarium sp. CPCC 400709 fermentation broth was obtained. The Fusarium sp. CPCC 400709 fermentation broth is a Fusarium sp. fungal agent.

2.3 Preparation of a complete set of inoculants producing diosgenin

After separately packaging the 2.1 curvularia fungus agent and the 2.2 Fusarium fungal agent, according to the colony forming unit of Curvularia lunata 3.4381 and Fusarium sp. CPCC 400709 ( cfu) in a ratio of 2:1 and packed in a large package to obtain a diosgenin-producing microbial inoculum, which is named Curvus crescentus + Fusarium fungal microbial inoculum.

2.4 Preparation of Aspergillus flavus agent

The Aspergillus flavus 3.2792 was cultured on the slant of the PDA medium at 28°C for 5 days, then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and the Aspergillus flavus 3.2792 was fermented by shaking at 28°C for 2 days. liquid. The Aspergillus flavus 3.2792 fermentation broth is the Aspergillus flavus agent.

2.5 Preparation of Aspergillus flavus + Fusarium fungus set

After separately packaging the Aspergillus flavus agent of 2.4 and the Fusarium fungus agent of 2.2, the ratio of the number of colony forming units (cfu) according to Aspergillus flavus 3.2792 and Fusarium sp. CPCC 400709 is: The ratio of 2:1 is packed in a large package to obtain a complete set of Aspergillus flavus + Fusarium fungus.

2.6 Preparation of Aspergillus niger

Aspergillus niger (Aspergillus niger) CPCC 400524 was cultivated at 28 ° C on the slant of PDA medium for 5 days, and then the mycelium block was picked and inoculated in a 500 mL conical flask equipped with 100 mL of seed medium, and the culture was shaken at 28 ° C for 2 days to obtain Aspergillus niger ( Aspergillus niger) CPCC 400524 fermentation broth. The Aspergillus niger CPCC 400524 fermentation broth is an Aspergillus niger agent.

2.7 Preparation of Aspergillus niger + Fusarium fungus complete set

After separately packaging the Aspergillus niger agent of 2.6 and the Fusarium fungus agent of 2.2, according to the colony forming unit (cfu) of Aspergillus niger CPCC 400524 and Fusarium sp. CPCC 400709 The number ratio is 2:1 in a large package to obtain a complete set of Aspergillus niger + Fusarium fungus.

2.8 Preparation of CPCC 400709+CPCC 400718 Complete Inoculum

Dictyosporium sp. CPCC 400718 was cultured at 28°C on the slant of the PDA medium for 5 days, and then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and the culture was shaken at 28°C for 2 days. The fermentation broth of Dictyosporium sp. CPCC 400718 was obtained. The Dictyosporium sp. CPCC 400718 fermentation broth is the CPCC 400718 bacterial agent.

After the CPCC 400718 inoculum and the Fusarium fungal inoculum of 2.2 were individually packaged, the colony-forming units of Dictyosporium sp. CPCC 400718 and Fusarium sp. CPCC 400709 (cfu) The ratio of number ratio is 2:1 and packed in a large package to obtain a complete set of inoculants of CPCC 400709+CPCC 400718.

2.9 Preparation of CPCC 400709+CPCC 400718+CPCC 400226 Complete Set of Inoculants

Fusarium sp. CPCC 400226 was cultured on the slant of PDA medium at 28°C for 5 days, and then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and the culture was shaken at 28°C for 2 days. Fusarium sp. CPCC 400226 fermentation broth was obtained. The Fusarium sp. CPCC 400226 fermentation broth is the CPCC 400226 inoculum.

After the CPCC 400226 inoculum, the CPCC 400718 inoculum in 2.8, and the Fusarium fungus in 2.2 were packaged separately, according to the Fusarium sp. CPCC 400226, Dictyosporium sp. ) CPCC 400718 and Fusarium sp. CPCC 400709 in a ratio of 1:1:1 in the number of colony forming units (cfu) are packed in a large package to obtain a complete set of CPCC 400709+CPCC 400718+CPCC 400226 Bacterial agent.

2.10 Preparation of CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 Complete Set of Inoculants

Fusarium sp. CPCC 400712 was cultured on the slant of PDA medium at 28°C for 5 days, then the mycelium block was picked and inoculated into a 500mL conical flask containing 100mL of seed medium, and cultured with shaking at 28°C for 2 days Fusarium sp. CPCC 400712 fermentation broth was obtained. The Fusarium sp. CPCC 400712 fermentation broth is the CPCC 400712 inoculum.

After the CPCC 400712 inoculum, the CPCC 400226 inoculum of 2.9, the CPCC 400718 inoculum of 2.8, and the Fusarium fungus inoculum of 2.2 were individually packaged, the inoculum of Fusarium sp. CPCC 400712, Fusarium sp. The colony forming unit (cfu) number ratio of Fusarium sp. CPCC 400226, Dictyosporium sp. CPCC 400718 and Fusarium sp. CPCC 400709 was 1:1 : 1:1 ratio is packed in a large package to obtain a complete set of inoculants of CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712.

3. Preparation of Diosgenin

The inventor of the present invention found in the course of the experiment that inoculating two fungi at the same time would cause the phenomenon that neither of the two fungi could grow normally. Therefore, this experiment adopts the combined sequential fermentation, that is, first inoculate a fungus for fermentation, ferment for a certain period of time, and then inoculate the second fungus for fermentation. This method is more complicated in operation than the combined simultaneous fermentation, but it can make each bacteria get good proliferation, thereby improving the yield of diosgenin. The inventors of the present invention found in the experimental process that the yield of diosgenin was the highest when a fungus was fermented alone for 22 days, so in the following treatments, the time for a single fungus fermentation was set to 22 days.

There are 13 treatments in the experiment, as follows:

3.1 Curvula crescentus + Fusarium fungus complete set of fusarium + new treatment

Diosgenin is prepared with the complete inoculum of 2.3 producing diosgenin, and the concrete method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, on the 23rd day, the 2.1 Curvularia lunata mold agent was added, and 2×10 ⁷ cfu Curvularia lunata 3.4381 was added to each gram of solid-state fermentation medium, Mix well, continue to culture at 28°C for 7 days, collect all the substances in the culture vessel, and obtain sickle+new fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.2 Curvula crescentus + Fusarium fungus complete set of new + fusarium treatment

Diosgenin is prepared with the complete inoculum of 2.3 producing diosgenin, and the concrete method is as follows:

Add 2.1 of Curvularia lunata mold agent into a 500mL conical flask containing 50g solid-state fermentation medium, and each gram of solid-state fermentation medium is inserted with 2×10 ⁷ cfu Curvularia lunata 3.4381, and mix well. , cultured at 28°C for 22 days, on the 23rd day, Fusarium sp. CPCC 400709 was added to each gram of solid-state fermentation medium with 1×10 ⁷ cfu of Fusarium sp. Mix well, continue to culture at 28°C for 7 days, collect all the substances in the culture vessel, and obtain the new + sickle fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.3 Curvus crescentus fungicide treatment

The diosgenin was prepared with the 2.1 Curvus crescentus fungicide, and the specific method was as follows:

Add 2.1 Curvularia lunata fungicide into a 500mL conical flask containing 50g solid-state fermentation medium, and 3×10 ⁷ cfu Curvularia lunata 3.4381 per gram of solid-state fermentation medium, and mix well. , cultivated at 28°C for 22 days, collected all the substances in the culture vessel, and obtained the fermentation product of Curvus crescentus fungus agent. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.4 Fusarium fungicide treatment

Diosgenin is prepared with Fusarium fungal agent, and the concrete method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 3×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed evenly, cultured at 28°C for 22 days, collected all the substances in the culture container, and obtained the fermentation product of Fusarium fungal agent. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.5 Aspergillus flavus agent treatment

Prepare diosgenin with the Aspergillus flavus agent of 2.4, the concrete method is as follows:

The Aspergillus flavus agent of 2.4 was inserted into a 500mL conical flask containing 50g of solid-state fermentation medium, and 3×10 ⁷ cfu of Aspergillus flavus 3.2792 was inserted into each gram of solid-state fermentation medium, mixed well, and cultured at 28°C for 22 On the next day, collect all the substances in the culture vessel to obtain the fermentation product of Aspergillus flavus agent. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.6 Aspergillus flavus + Fusarium fungus complete set of inoculum yellow + fusarium treatment

Prepare diosgenin with 2.5 Aspergillus flavus+Fusarium fungus complete set, the concrete method is as follows:

The Aspergillus flavus agent of 2.4 was inserted into a 500mL conical flask containing 50g of solid-state fermentation medium, and 2×10 ⁷ cfu of Aspergillus flavus 3.2792 was inserted into each gram of solid-state fermentation medium, mixed well, and cultured at 28°C for 22 On the 23rd day, Fusarium sp. CPCC ⁴⁰⁰⁷⁰⁹ was added to each gram of solid-state fermentation medium, and the Fusarium sp. Cultivated at °C for 7 days, and collected all the substances in the culture vessel to obtain a yellow + sickle fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.7 Aspergillus flavus + Fusarium fungus complete set of fusarium + yellow treatment

Prepare diosgenin with 2.5 Aspergillus flavus+Fusarium fungus complete set, the concrete method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, on the 23rd day, the Aspergillus flavus agent of 2.4 was added, and 2 × 10 ⁷ cfu Aspergillus flavus 3.2792 was added to each gram of solid-state fermentation medium, mixed well, and continued for 28 Cultivated at °C for 7 days, and collected all the substances in the culture vessel to obtain a sickle+yellow fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.8 Aspergillus niger treatment

Prepare diosgenin with the Aspergillus niger agent of 2.6, the concrete method is as follows:

The Aspergillus niger agent of 2.6 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 3 × 10 ⁷ cfu of Aspergillus niger CPCC 400524 was inserted into each gram of solid-state fermentation medium, and the mixture was uniformly mixed for 28 Cultivated at ℃ for 22 days, and collected all the substances in the culture vessel to obtain the fermentation product of Aspergillus niger. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.9 Aspergillus niger + Fusarium fungus complete set of inoculum black + fusarium treatment

Prepare diosgenin with 2.7 Aspergillus niger+Fusarium fungus complete set, the concrete method is as follows:

The Aspergillus niger agent of 2.6 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 2 × 10 ⁷ cfu of Aspergillus niger CPCC 400524 was inserted into each gram of solid-state fermentation medium, mixed well, and 28 Cultivated at ℃ for 22 days, on the 23rd day, the Fusarium fungus agent of 2.2 was added, and 1×10 ⁷ cfu Fusarium sp. CPCC 400709 was added to each gram of solid-state fermentation medium, and mixed well. , continue to cultivate at 28°C for 7 days, collect all the substances in the culture vessel, and obtain the black + sickle fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.10 Aspergillus niger + Fusarium fungus complete set of fusarium + black treatment

Prepare diosgenin with 2.7 Aspergillus niger+Fusarium fungus complete set, the concrete method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, on the 23rd day, the Aspergillus niger agent of 2.6 was added, and 2×10 ⁷ cfu Aspergillus niger CPCC 400524 was added to each gram of solid fermentation medium, and mixed well , continue to cultivate at 28°C for 7 days, collect all the substances in the culture vessel, and obtain the sickle + black fermentation product. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.11 CPCC 400709+CPCC 400718 Complete Bacteria Treatment

Prepare diosgenin with the CPCC 400709+CPCC 400718 complete inoculum of 2.8, the specific method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, and then inoculated with 2.8 of CPCC 400718 bacterial agent on the 23rd day, each gram of solid fermentation medium was inoculated with 2×10 ⁷ cfu Dictyosporium sp. CPCC 400718, mix well, continue to culture at 28° C. for 7 days, collect all the substances in the culture vessel, and obtain a complete set of inoculum fermentation products of CPCC 400709+CPCC 400718. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.12 CPCC 400709+CPCC 400718+CPCC 400226 Complete Bacteria Treatment

Prepare diosgenin with the CPCC 400709+CPCC 400718+CPCC 400226 complete inoculum of 2.9, the concrete method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, and then added 2.8 of CPCC 400718 inoculum on the 23rd day, and added 1×10 ⁷ cfu Dictyosporium sp. per gram of solid-state fermentation medium. CPCC 400718, mix well, continue to cultivate at 28°C for 7 days, and then add 2.9 of CPCC 400226 inoculum on the 30th day, and insert 1×10 ⁷ cfu Fusarium sp. per gram of solid-state fermentation medium. ) CPCC 400226, mix evenly, continue to culture at 28° C. for 7 days, collect all substances in the culture vessel, and obtain a complete set of inoculum fermentation products of CPCC 400709+CPCC 400718+CPCC 400226. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

3.13 CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 Complete Bacteria Treatment

Prepare diosgenin with the complete set of inoculum of CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 of 2.10, the specific method is as follows:

The Fusarium sp. fungal agent of 2.2 was inserted into a 500 mL conical flask containing 50 g of solid-state fermentation medium, and 1×10 ⁷ cfu of Fusarium sp. CPCC was inserted into each gram of solid-state fermentation medium. 400709, mixed well, cultured at 28°C for 22 days, and then added 2.8 of CPCC 400718 inoculum on the 23rd day, and added 1×10 ⁷ cfu Dictyosporium sp. per gram of solid-state fermentation medium. CPCC 400718, mix well, continue to cultivate at 28°C for 7 days, and then add 2.9 of CPCC 400226 inoculum on the 30th day, and insert 1×10 ⁷ cfu Fusarium sp. per gram of solid-state fermentation medium. ) CPCC 400226, mix well, continue to cultivate at 28°C for 7 days, and on the 37th day, add CPCC 400712 inoculum of 2.10, and insert 1×10 ⁷ cfu Fusarium sp. per gram of solid-state fermentation medium. .) CPCC 400712, mix well, continue to culture at 28° C. for 10 days, collect all the substances in the culture vessel, and obtain a complete set of inoculum fermentation products of CPCC 400709+CPCC 400718+CPCC 400226+CPCC400712. The experiment was repeated three times, with 10 Erlenmeyer flasks set for each repetition.

4. Yield of diosgenin

The fusarium + new fermentation product of 3.1, the new + fusarium fermentation product of 3.2, the fermentation product of Curvus crescentus fungus agent of 3.3, the fermentation product of Fusarium fungus agent of 3.4, the fermentation product of Aspergillus flavus agent of 3.5, the fermentation product of 3.6 Yellow+Fusarium fermentation product, 3.7 sickle+yellow fermentation product, 3.8 Aspergillus niger fermentation product, 3.9 black+Fusarium fermentation product, 3.10 sickle+black fermentation product, 3.11 CPCC 400709+CPCC 400718 complete inoculum fermentation Product, 3.12 CPCC 400709+CPCC 400718+CPCC 400226 complete inoculum fermentation product and 3.13 CPCC 400709+CPCC 400718+CPCC 400226+CPCC400712 complete inoculum fermentation product were dried and ground into coarse powder, 10 times the volume of acetic acid Ethyl ester reflux extraction for 1 hour, and then repeated the extraction twice, the obtained extracts were combined and evaporated to dryness, dissolved in methanol, and passed through a 0.22 μm filter membrane for HPLC analysis, and the yield of diosgenin was calculated: diosgenin mass (mg)/ The quality of the medicinal material of Dioscorea Shields (mg)×100%. Take the standard product of diosgenin, dissolve it with methanol to constant volume, and perform HPLC to make a standard curve. The HPLC chromatographic conditions were as follows: Agilent 1100 equipped with ELSD detector and Chemstation chromatography workstation; XDB-C18 column (250×4.6 mm, 5 μm); 90% acetonitrile-water solution (acetonitrile and water in a volume ratio of 9:1) Mix the obtained liquid) for elution, the flow rate is 1 mL/min; the column temperature is 30 °C; the injection volume is 10 μL.

The results showed that diosgenin was produced in all 13 treatments (Figure 1 and Table 1):

1. The total yield of diosgenin of Curvus crescentus + Fusarium fungal inoculant Fusarium + new treatment is higher than the sum of the yields of Curvus crescenti and Fusarium fungal inoculants, indicating that in Curvularia lunata 3.4381 and Fusarium sp. CPCC400709 were fermented in the medium containing Dioscorea scutellum to produce Dioscorea A synergistic effect was produced in terms of sapogenin; while the curlyspora crescentus + Fusarium fungus complete set of inoculants new + fusarium treatment had a lower yield of diosgenin than the Fusarium fungal inoculant treatment, indicating that in crescent crescent In the new + Fusarium treatment, Curvularia lunata 3.4381 and Fusarium sp. CPCC 400709 produced diosgenin in the fermentation medium containing Dioscorea spp. There is no synergy.

2. In terms of the yield of diosgenin, whether it is Aspergillus flavus + Fusarium fungus complete set of inoculum yellow + fusarium treatment or Aspergillus flavus + Fusarium fungus complete set of inoculum + yellow treatment is better than Fusarium fungus inoculum Both the treatment and the A. flavus agent treatment were low, indicating that Aspergillus flavus 3.279 and Fusarium sp. CPCC 400709 had antagonistic effects on the production of diosgenin in the fermentation medium containing Dioscorea solani.

3. In terms of the yield of diosgenin, whether it is Aspergillus niger + Fusarium fungus complete set of inoculum black + fusarium treatment or Aspergillus niger + Fusarium fungus complete set of inoculum + black treatment is better than Fusarium fungus inoculum The treatment was low, indicating that Aspergillus niger CPCC 400524 and Fusarium sp. CPCC 400709 had antagonistic effects on the production of diosgenin by fermenting the medium containing Dioscorea scutellariae.

4. In terms of the yield of diosgenin, the treatment of CPCC 400709+CPCC 400718 complete set of inoculum is 9% higher than that of Fusarium fungus, and the treatment of CPCC 400709+CPCC 400718+CPCC 400226 complete set of inoculum is higher than that of Fusarium fungus. The inoculum treatment increased by 16%, and the CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 complete inoculum treatment increased by 14% compared with the Fusarium fungus treatment.

Table 1, the output and yield of diosgenin of each treatment

Note: The yield (mg) of sapogenin per gram of Dioscorea officinalis rhizome powder is based on dry weight.

Claims (6)

1. a method for preparing diosgenin, characterized in that: the method comprises culturing Fusarium fungus and Curvus sp. fungus with a medium containing Diospermum scutellum to obtain diosgenin; the Fusarium fungus is Fusarium Fusarium sp. CPCC 400709; Said Curvularia sp. fungus is Curvularia lunata 3.4381; Said cultivation of Fusarium sp. fungi and Curvularia sp. The fungus is firstly inoculated with the Fusarium fungus into the medium containing Dioscorea scutellariae for cultivation, and then inoculated with the Curvus sp. fungus for cultivation. 2. the microbial inoculum for preparing diosgenin, is characterized in that: described microbial inoculum is the complete set of microbial inoculum for preparing diosgenin composed of Fusarium fungal microbial inoculum and Curvus sp. The active ingredient of the Fusarium sp. fungicide contains Fusarium sp. CPCC 400709, and the active ingredient of the Curvuria sp. fungus contains the Curvus sp. fungus; the Curvus sp. fungus It is Curvularia lunata 3.4381; the Fusarium fungal inoculum and the Curvularia fungal inoculum are individually packaged. 3. the microbial inoculum for preparing diosgenin, is characterized in that: described microbial inoculum is that active ingredient contains Fusarium sp. (Fusarium sp.) CPCC 400709 and Curvus sp. Bacterial agent; the fungus of the genus Curvularia is Curvularia lunata 3.4381; the fungus of the genus Fusarium sp. CPCC400709 and the fungus of the genus Curvularia are individually packaged. 4. The application of Fusarium fungus and Curvus sp. fungus in the preparation of diosgenin, the Fusarium sp. fungus is Fusarium sp. CPCC 400709, and the Curvus sp. fungus is a new Curvularia lunata 3.4381. 5. the application of the microbial inoculum of claim 2 in the preparation of diosgenin. 6. the application of the microbial inoculum of claim 3 in the preparation of diosgenin.

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