CN106967775A - Method for preparing diosgenin by biocatalysis and bacterial agent used therefor - Google Patents

Abstract

The invention discloses a method for preparing diosgenin by biological catalysis and a microbial inoculum used by the method. The method for preparing diosgenin is M1) comprises culturing Fusarium fungus and Curvularia fungus with culture medium containing rhizoma Dioscoreae Zingiberensis to obtain diosgenin; m2) the method comprises culturing fusarium and/or chaga fungi in a medium containing dioscorea zingiberensis to obtain diosgenin; m3) the method comprises culturing fusarium fungus in a medium containing dioscorea zingiberensis to obtain diosgenin. The yield of the diosgenin of the culture medium containing the dioscorea zingiberensis obtained by the combined fermentation of the curvularia lunata and the fusarium is higher than the sum of the yields of the diosgenin of the culture medium containing the dioscorea zingiberensis obtained by the independent fermentation of the curvularia lunata and the culture medium containing the dioscorea zingiberensis obtained by the independent fermentation of the curvularia lunata, and the curvularia lunata and the fusarium produce synergistic effect in the aspect of producing the diosgenin by fermenting the culture medium containing the dioscorea zingiberensis.

Description

Method for preparing diosgenin by biocatalysis and bacterial agent used therefor

technical field

The invention relates to a method for preparing diosgenin by biocatalysis and a bacterial agent used therein.

Background technique

Diosgenin (diosgenin), known as diosgenin in the industry, is an important active ingredient in Dioscorea (Dioscorea L.) plants. As an important pharmaceutical intermediate for the synthesis of steroid hormone drugs, it is called "medicinal gold". ". More than 60% of the steroid hormones produced in the world use diosgenin as raw material, and the market demand is huge. At present, the method of producing diosgenin is based on the chemical acid hydrolysis method proposed by Rothrock et al. in 1957, and there are many problems in this process. How to eradicate pollution from the source is an urgent problem to be solved for 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 of acid-free production of diosgenin.

Contents 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 P1) to P3) application:

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

P2) the application of Fusarium fungi and the genus 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 fungi in the preparation of diosgenin.

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

M1) The method comprises culturing Fusarium fungi and Curvularia fungi with a medium containing Dioscorea scutellaria to obtain diosgenin;

M2) The method comprises cultivating Fusarium fungi and Porphyra fungi with a culture medium containing Dioscorea scutellariae to obtain diosgenin;

M3) The method comprises culturing a fungus of the genus Fusarium with a medium containing Dioscorea scutellaria to obtain diosgenin.

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

In the above method or application, in M1), M3), P1) and P3), the Fusarium fungus can specifically be Fusarium sp. (Fusarium sp.) CPCC 400709; M2) and P2), the said The Fusarium fungus may be Fusarium sp. CPCC 400709; M2) and P2), the Fusarium sp. CPCC 400709 and Fusarium sp. (Fusarium sp.) CPCC 400226; M2) and P2), the Fusarium 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 Curvularia lunata can be Curvularialunata3.4381.

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

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

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

In the above method or application, in M1) and P1), the colony forming unit (cfu) number ratio of the fungus of the genus Fusarium and the fungus of the genus Curvularia can be 1: (0.5-4), such as 1:2 .

Among the above applications, the above P2) can specifically be P21), P22) or P23); said P21), P22) or P23) are all applications of Fusarium fungi and Cortex sp. fungi in the preparation of diosgenin.

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

In the P22), the Fusarium fungi may specifically be Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226. In the P22), the colony forming units (cfu) of Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC 400718 ) ratio can 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 Fusarium 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 Curvularia fungi with a culture medium containing Dioscorea scutellaria can be performed by first inserting the fungi of the genus Fusarium into the medium containing Dioscorea scutellaria After culturing, the fungi of the genus Curvularia were inoculated for culturing. For example, the fungus of the genus Fusarium is first inserted into the medium containing Dioscorea scutellaria, cultured at 20-30°C (such as 28°C) for 15-25 days (such as 22 days), and then the fungus of the genus Curvularia is inserted. Cultivate at 20-30°C (eg 28°C) for 5-10 days (eg 7 days).

The aforementioned M2) may specifically be M21), M22) or M23).

The M21) is that the method comprises cultivating Fusarium fungi and Porphyra fungi with a culture medium containing Dioscorea scutellaria to obtain diosgenin. In the M21), the Fusarium fungus may be Fusarium sp. CPCC400709; the Dictyosporium sp. CPCC 400718 may be the Dictyosporium sp. CPCC400718. The colony-forming unit (cfu) number ratio of the fungus of the genus Fusarium and the fungus of the genus Cortex may be 1:(0.5-4), such as 1:2. Wherein, using the culture medium containing Dioscorea scutellaria to cultivate Fusarium genus fungi and fungi of the genus Trichosporum can specifically be to insert the fungus of the genus Fusarium into the medium containing Dioscorea scutellaria scutellaria for cultivation and then insert the fungus of the genus Fusarium. Cultivation of fungi of the genus Pleurotus spp. For example, the fungus of the genus Fusarium is first inserted into the medium containing Dioscorea scutellaria, cultured at 20-30° C. (such as 28° C.) for 15-25 days (such as 22 days), and then the fungus of the genus Fusarium is inserted. Cultivate at 20-30°C (eg 28°C) for 5-10 days (eg 7 days).

The M22) is that the method comprises cultivating Fusarium fungi and Porphyra fungi with a medium containing Dioscorea scutellaria to obtain diosgenin. In the M22), the Fusarium fungus can specifically be Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226; the Fusarium sp. can be Dictyosporium sp. CPCC 400718. In the M22), the colony forming units (cfu) of Fusarium sp. CPCC400709, Fusarium sp. CPCC 400226 and Dictyosporium sp. CPCC 400718 ) ratio can be 1:(0.5-4):(0.5-4), such as 1:1:1. Wherein, using the culture medium containing Dioscorea scutellaria to cultivate Fusarium sp. fungi and the fungi of the genus Trichosporum can specifically be to first insert Fusarium sp. Then inoculate the fungus of the genus Fusarium sp. for cultivation, and then inoculate Fusarium sp. CPCC 400226 for cultivation. For example, first inoculate Fusarium sp. CPCC 400709 into the culture medium containing Dioscorea scutellaria, culture at 20-30°C (e.g. 28°C) for 15-25 days (e.g. 22 days) and then inoculate bricks Dictyosporium sp. CPCC 400718 was cultured at 20-30°C (such as 28°C) for 5-10 days (such as 7 days) and then Fusarium sp. CPCC 400226 was inoculated at 20 Cultivate at -30°C (eg 28°C) for 5-10 days (eg 7 days).

The M23) is that the method comprises cultivating Fusarium fungi and Porphyra fungi with a culture medium containing Dioscorea scutellaria to obtain diosgenin. In the M23), the Fusarium fungi may specifically be Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Fusarium sp. ) CPCC400712; the fungus of the genus Dictyosporium may be Dictyosporium sp. CPCC 400718. In the said M23), Fusarium sp. CPCC 400709, Fusarium sp. CPCC400226, Fusarium sp. CPCC 400712 and Fusarium 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, using the medium containing Dioscorea scutellaria to cultivate the Fusarium sp. and the fungus of the genus Fusarium genus can specifically be to insert Fusarium sp. Then insert Dictyosporium sp. CPCC 400718 for culture, then insert Fusarium sp. CPCC400226 for culture, then insert Fusarium sp. CPCC 400712 To cultivate. For example, first inoculate Fusarium sp. CPCC 400709 into the culture medium containing Dioscorea scutellaria, culture at 20-30°C (e.g. 28°C) for 15-25 days (e.g. 22 days) and then inoculate bricks Dictyosporium sp. CPCC 400718 was cultured at 20-30°C (such as 28°C) for 5-10 days (such as 7 days) and then Fusarium sp. CPCC 400226 was inoculated at 20 Cultivate at -30°C (such as 28°C) for 5-10 days (such as 7 days), and then inoculate Fusarium sp. CPCC 400712 at 20-30°C (such as 28°C) for 7-15 days ( such as 10 days).

In the above-mentioned M3), the Fusarium fungus can be cultured with the culture medium containing Dioscorea scutellaria, specifically, the fungus of the genus Fusarium can be inserted into the culture medium containing Dioscorea scutellaria and cultivated at 20-30°C (such as 28°C) for 15 -25 days (like 22 days).

In order to solve the above technical problems, the present invention provides a bacterial agent 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 fungal agents for preparing diosgenin consisting of fungal agents of the genus Fusarium and fungal agents of the genus Curvularia. The active ingredient of the fungal agent of the genus Fusarium contains the fungal agents of the genus Fusarium , the active ingredient of the Curvularia fungal agent contains the Curvularia fungus;

The C2 is an inoculant for preparing diosgenin containing the Fusarium fungus and the Curvularia fungus as active ingredients;

The C3 is a complete set of bacterial agents for the preparation of diosgenin consisting of a Fusarium fungal agent and a Fusarium fungal agent, and the active ingredient of the Fusarium fungal agent contains the Fusarium fungal agent , the active ingredient of the fungus agent of the genus Cortex contains the fungus of the genus Cortex;

The C4 is a fungal agent for preparing diosgenin containing the Fusarium fungus and the Trichosporum fungus as active ingredients.

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

In the above-mentioned complete set of bacterial preparations for preparing diosgenin, the C2 may specifically be a bacterial preparation for preparing diosgenin whose active ingredients are the fungus of the genus Fusarium and the fungus of the genus Curvularia; the C2 The active ingredient of C2 may also contain other biological components or non-biological components, and those skilled in the art can determine the other active ingredients of C2 according to the yield of diosgenin. In the C2, the Fusarium fungus can specifically be Fusarium sp. CPCC 400709. In the C2, the fungus of the genus Curvularia can be Curvularia lunata; specifically, the Curvularia lunata can be Curvularia lunata3.4381. In the C2, the colony forming unit (cfu) ratio of the Fusarium fungus and the Curvularia fungus may be 1:(0.5-4), such as 1:2.

In the above-mentioned complete set of microbial agents for preparing diosgenin, the C3 can specifically be C31, C32 or C33. The C31, C32 and C33 are all sets of bacterial agents for the preparation of diosgenin composed of Fusarium fungal inoculum and Trichosporum fungal inoculum, and the active ingredients of the Fusarium fungal inoculum contain the The Fusarium fungus, the active ingredient of the Fusarium fungus inoculant contains the Fusarium fungus. In the C31, C32 and C33, the fungus of the genus Dictyosporium can be Dictyosporium sp. CPCC 400718. In the C31, C32 and C33, the active ingredient of the fungal agent of the genus Cortella can be the fungus of the genus Cortella, and the active ingredient of the fungal agent of the genus Cortex can also contain other biological components or Non-biological components, 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 can specifically be Fusarium sp. CPCC 400709. The active ingredient of the Fusarium fungal inoculum can be the Fusarium fungus, and the active ingredient of the Fusarium fungal inoculum can also contain other biological components or non-biological components, and the other active ingredients of the Fusarium inoculum are Those skilled in the art can determine according to the yield of diosgenin. In the C31, each bacterial agent is packaged independently. In the C31, the ratio of the number of colony-forming units (cfu) between the fungus of the genus Fusarium and the fungus of the genus Porphyria may be 1:(0.5-4), such as 1:2.

In the C32, the Fusarium fungal inoculum can specifically be Fusarium sp. CPCC400709 inoculum and Fusarium sp. CPCC 400226 inoculum. The active ingredient of the Fusarium sp. (Fusarium sp.) CPCC 400709 bacterial agent can be Fusarium sp. (Fusarium sp.) CPCC 400709, and can also contain other biological components or non-biological components. Other active ingredients of the bacterial agent Components can be determined by those skilled in the art according to the yield of diosgenin. The active ingredient of the Fusarium sp. (Fusarium sp.) CPCC 400226 bacterial agent can be Fusarium sp. (Fusarium sp.) CPCC 400226, and can also contain other biological components or non-biological components. Active ingredients can be determined by those skilled in the art according to the yield of diosgenin. In the C32, each bacterial agent is packaged independently. In the C32, the colony forming units (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), such as 1:1:1.

In the C33, the Fusarium fungal inoculum can specifically be Fusarium sp. CPCC400709 inoculum, Fusarium sp. CPCC 400226 inoculum and Fusarium sp. (Fusarium sp.) CPCC 400712 bacterial agent. The active ingredient of the Fusarium sp. (Fusarium sp.) CPCC 400709 bacterial agent can be Fusarium sp. (Fusarium sp.) CPCC 400709, and can also contain other biological components 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. (Fusarium sp.) CPCC 400226 bacterial agent can be Fusarium sp. (Fusarium sp.) CPCC 400226, and can also contain other biological components 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. (Fusarium sp.) CPCC 400712 bacterial agent can be Fusarium sp. (Fusarium sp.) CPCC 400712, and can also contain other biological components 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 bacterial agent is packaged independently. Among the C33, Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226, Fusarium sp. CPCC 400712 and Fusarium 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 set of microbial agents for preparing diosgenin, the C4 can specifically be C41, C42 or C43. The C41, C42 and C43 are all bacterial agents for preparing diosgenin, which contain the Fusarium fungus and the Porphyra fungus as active ingredients. The active ingredients of the C41, C42 and C43 can be the Fusarium genus fungus and the Porphyra genus fungus, and can also contain other biological components or non-biological components. Determined according to the yield of diosgenin. In the C41, C42 and C43, the fungus of the genus Dictyosporium can be Dictyosporium sp. (Dictyosporium sp.) CPCC 400718.

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

In the C42, the Fusarium fungi may 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), such as 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. Among the C43, Fusarium sp. CPCC 400709, Fusarium sp. CPCC400226, Fusarium sp. CPCC 400712 and Fusarium sp. ( The colony forming unit (cfu) number ratio of Dictyosporium sp.) CPCC 400718 can be 1:(0.5-4):(0.5-4):(0.5-4), such as 1:1:1:1.

In addition to the active ingredient, the above bacterial agent may also include auxiliary materials, such as water, carbon source and/or nitrogen source and the like. Carbon source is a type of nutrient for microbial growth, and it is a carbon-containing compound, including sugars, oils, organic acids and organic acid esters, and small molecule alcohols. Nitrogen sources refer to substances that provide nitrogen elements required for microbial nutrition, including peanut cake powder, soybean cake powder, yeast powder, peptone, ammonia water, ammonium salt and nitrate and other quick-acting and late-acting nitrogen sources.

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

Experiments have shown that the yield of diosgenin in the joint fermentation of Curvularia crescens and the fungus of the genus Fusarium containing Dioscorea shield is higher than that of the culture medium containing Dioscorea shield leaf fermented by Curvularia crescens alone and the fungus of the genus Fusarium alone. The sum of the yields of diosgenin from the fermentation of the medium containing Dioscorea scutellaria was even higher, and Curvularia lunata and Fusarium fungi produced synergy in the production of diosgenin in the fermentation of the medium containing Dioscorea scutellaria role (Table 1). In terms of yield of diosgenin, CPCC400709+CPCC 400718 complete set of inoculum treatment was 9% higher than Fusarium fungal inoculum treatment, and CPCC 400709+CPCC400718+CPCC 400226 complete set of inoculum treatment was higher than Fusarium fungal inoculum treatment 16%, CPCC 400709+CPCC400718+CPCC 400226+CPCC 400712 complete set of inoculum treatment increased by 14% compared with Fusarium fungus inoculum treatment.

Description of drawings

Fig. 1 is a spectrum of identification of diosgenin by HPLC.

Among the figure, (A) is the diosgenin standard product collection of illustrative plates, (B) is the HPLC collection of illustrative plates of the fermentation product of Curvularia lunata fungi of 3.3, (C) is the HPLC collection of collections of the fermentation product of the Fusarium genus fungus of 3.4, (D) is the HPLC spectrum of the 3.1 sickle + new fermentation product, (E) is the 3.2 new + sickle fermentation product, wherein the chromatographic peak of diosgenin is shown by the arrow.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Curvularia lunata 3.4381 (Bing Feng, etal. Themicrobiological transformation of steroidal saponins by Curvularialunata. Tetrahedron 61 (2005) 11758-11763.25 October 2005) in the following examples The public can obtain this biological material from the applicant, the Biological materials are only used for repeating related experiments of the present invention, and cannot be used for other purposes.

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

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

Dictyosporium sp. CPCC 400718 was collected on July 6, 2015 at the China Pharmaceutical Culture Collection (CPCC) of the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences, address: Beijing Tiantan Xili No. 1, Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences, postal code: 100050), since the collection date, the public can obtain the strain from CPCC.

Aspergillus flavus 3.2792(A.flavus 3.2792) in the following examples The public can obtain the biological material from the applicant, and the biological material is only used for repeating related experiments of the present invention, and cannot be used for other purposes.

The Fusarium sp. (Fusarium sp.) CPCC 400226 in the following examples has been stored in the China Pharmaceutical Culture Collection (China Pharmaceutical Culture Collection, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences) on February 1, 2007. Abbreviated as CPCC, address: No. 1 Xili, Tiantan, Beijing, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, postal code: 100050), and the public can obtain the strain from CPCC from the collection date.

Aspergillus niger (Aspergillus niger) CPCC 400524 in the following examples has been collected on July 6, 2015 in the China Pharmaceutical Microbiology Culture Collection Center (China Pharmaceutical Culture Collection, CPCC, address : Beijing Tiantan Xili No. 1, Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, postal code: 100050), the public can obtain this bacterial strain from CPCC since the collection date.

Embodiment 1, prepare diosgenin by culturing Fusarium fungi and Curvularia or Fusarium fungi with a medium containing Dioscorea scutellaria

1. The preparation of the culture medium used in this embodiment

Potato Dextrose Agar (PDA) Medium: Peel potatoes, cut 200g into small pieces, add water to boil for 30min, filter through 4 layers of gauze, add 20g of glucose, 17g of agar, distilled water to 1000mL, boil and mix well, sterilize at 121℃ Bacteria for 20 minutes to obtain PDA medium.

Seed medium: glucose 2g, sucrose 1g, soybean flour 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 a seed culture medium.

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

2. Preparation of bacterium agent producing diosgenin

2.1. Preparation of Curvularia crescens fungicide

Cultivate Curvularia lunata 3.4381 on a slope of PDA medium at 28°C for 5 days, then pick mycelia pieces and inoculate them in a 500mL Erlenmeyer flask containing 100mL of seed medium, and cultivate them with shaking at 28°C for 2 days to obtain a crescent Spore mold Curvularia lunata 3.4381 fermentation broth. The fermentation broth of Curvularia lunata 3.4381 is the Curvularia lunata fungal agent.

2.2. Preparation of Fusarium fungal agent

Fusarium sp. CPCC 400709 was cultured on the slope of PDA medium at 28°C for 5 days, and then the hyphae pieces were picked and inoculated in a 500mL Erlenmeyer flask containing 100mL seed medium, and cultured with shaking at 28°C for 2 days The fermentation liquid of Fusarium sp. CPCC 400709 was obtained. The Fusarium sp. CPCC 400709 fermentation liquid is the Fusarium sp. fungal agent.

2.3 Preparation of a complete set of bacterial agents producing diosgenin

After the Curvularia lunata fungal agent of 2.1 and the Fusarium fungal agent of 2.2 are individually packaged respectively, according to the colony forming unit of Curvularia lunata 3.4381 and Fusarium sp. cfu) with a ratio of 2:1 in a large package to obtain a complete set of diosgenin-producing inoculum, which is named as Curvularia lunata + Fusarium fungus complete set of inoculum.

2.4 Preparation of Aspergillus flavus agent

Cultivate Aspergillus flavus 3.2792 on the slope of PDA medium at 28°C for 5 days, then pick mycelia pieces and inoculate them in a 500mL Erlenmeyer flask containing 100mL of seed medium, and culture them with shaking at 28°C for 2 days to obtain Aspergillus flavus 3.2792 fermentation liquid. The fermented liquid of Aspergillus flavus 3.2792 is the aspergillus flavus agent.

2.5 Preparation of Aspergillus flavus + Fusarium fungus complete set

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

2.6 Preparation of Aspergillus niger inoculant

Aspergillus niger (Aspergillus niger) CPCC 400524 was cultured on a PDA medium slope at 28°C for 5 days, then the hyphae pieces were picked and inoculated in a 500mL Erlenmeyer flask containing 100mL seed medium, and cultured with shaking at 28°C for 2 days to obtain Aspergillus niger ( Aspergillus niger) CPCC 400524 fermentation broth. The Aspergillus niger (Aspergillus niger) CPCC 400524 fermentation broth is the Aspergillus niger inoculant.

2.7 Preparation of Aspergillus niger + Fusarium fungus complete set

After the Aspergillus niger inoculant of 2.6 and the Fusarium fungal inoculum of 2.2 are packaged separately, the colony forming units (cfu) according to Aspergillus niger (Aspergillus niger) CPCC 400524 and Fusarium sp. (Fusarium sp.) CPCC 400709 The number ratio is 2:1 and packed in a large package to obtain a complete set of Aspergillus niger + Fusarium fungi.

2.8 Preparation of CPCC 400709+CPCC 400718 set of bacterial agents

Dictyosporium sp. CPCC 400718 was cultured on the slope of PDA medium at 28°C for 5 days, and then the mycelium pieces were picked and inoculated in a 500mL Erlenmeyer flask containing 100mL seed medium, and cultured with shaking at 28°C for 2 The fermentation broth of Dictyosporium sp. CPCC 400718 was obtained on the following day. The fermentation broth of Dictyosporium sp. CPCC 400718 is the CPCC 400718 bacterial agent.

After CPCC 400718 inoculum and 2.2 Fusarium fungal inoculum are packaged separately, the colony forming units of Dictyosporium sp. CPCC 400718 and Fusarium sp. CPCC 400709 (cfu) number ratio is 2:1 and packed in a large package to get CPCC 400709+CPCC 400718 complete bacterial agent.

2.9 Preparation of CPCC 400709+CPCC 400718+CPCC 400226 complete set of bacterial agents

Fusarium sp. CPCC 400226 was cultured on the slope of PDA medium at 28°C for 5 days, and then the hyphae pieces were picked and inoculated in a 500mL Erlenmeyer flask containing 100mL seed medium, and cultured with shaking at 28°C for 2 days The fermentation broth of Fusarium sp. CPCC 400226 was obtained. The fermentation broth of Fusarium sp. CPCC 400226 is the CPCC 400226 bacterial agent.

After this CPCC 400226 inoculum, CPCC 400718 inoculum of 2.8 and Fusarium sp. ) CPCC 400718 and Fusarium sp. (Fusarium sp.) CPCC 400709 have a colony-forming unit (cfu) number ratio of 1:1:1 and put them in a large package to obtain a complete set of CPCC 400709+CPCC 400718+CPCC 400226 Bacteria.

2.10 Preparation of CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 complete set of bacterial agents

Fusarium sp. CPCC 400712 was cultured on the slope of PDA medium at 28°C for 5 days, and then the hyphae pieces were picked and inoculated in a 500mL Erlenmeyer flask containing 100mL seed medium, and cultured with shaking at 28°C for 2 days The fermentation broth of Fusarium sp. CPCC 400712 was obtained. The fermentation broth of Fusarium sp. CPCC 400712 is the CPCC 400712 microbial agent.

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, according to Fusarium sp. (Fusarium sp.) CPCC 400712, Fusarium sp. The colony forming unit (cfu) ratio of Fusarium sp. CPCC 400226, Dictyosporium sp. CPCC 400718 and Fusarium sp. CPCC 400709 is 1:1 : The ratio of 1:1 is packed in a large package, and you can get CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 complete set of bacterial agents.

3. Preparation of Diosgenin

The inventors of the present invention found during the experiment that two kinds of fungi could not grow normally when inoculated with two kinds of fungi at the same time. Therefore, this experiment adopts joint sequential fermentation, that is, inoculate one fungus for fermentation first, and then insert the second fungus for fermentation after a certain period of time. This method is more complex in operation than combined simultaneous fermentation, but it can make each bacteria proliferate well, thereby increasing the yield of diosgenin. The inventors of the present invention found during the experiment 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 fungus to be fermented alone was set to 22 days.

The experiment set up 13 treatments, as follows:

3.1 Curvularia crescentus + Fusarium fungus complete set of fungus Fusarium + new treatment

Prepare diosgenin with the complete set of bacterial agents producing diosgenin in 2.3, the specific method is as follows:

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

3.2 Curvularia crescentus + Fusarium fungus complete set of inoculum new + Fusarium treatment

Prepare diosgenin with the complete set of bacterial agents producing diosgenin in 2.3, the specific method is as follows:

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

3.3 Curvularia crescentus fungicide treatment

Prepare diosgenin with the Curvularia lunata mold agent of 2.1, and the specific method is as follows:

Put the 2.1 Curvularia lunata fungal agent into a 500mL Erlenmeyer flask with 50g of solid-state fermentation medium, add 3×10 ⁷ cfu of 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 container, and obtained the fermentation product of Curvularia lunata fungal agent. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

3.4 Fusarium fungal agent treatment

Prepare diosgenin with Fusarium fungal agent, the specific method is as follows:

Put the Fusarium fungal agent of 2.2 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, and insert 3×10 ⁷ cfu of Fusarium sp. CPCC into each gram of solid-state fermentation medium 400709, mix well, culture at 28°C for 22 days, collect all the substances in the culture container, and obtain the fermentation product of Fusarium fungi. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

3.5 Aflatoxin treatment

Prepare diosgenin with the aspergillus flavus agent of 2.4, the specific method is as follows:

Put the Aspergillus flavus agent of 2.4 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, insert 3×10 ⁷ cfu of Aspergillus flavus 3.2792 into each gram of solid-state fermentation medium, mix well, and culture at 28°C for 22 day, collect all the substances in the culture container to obtain the fermentation product of Aspergillus aflatoxin. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

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

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

Put the Aspergillus flavus agent of 2.4 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, insert 2×10 ⁷ cfu of Aspergillus flavus 3.2792 into each gram of solid-state fermentation medium, mix well, and culture at 28°C for 22 On the 23rd day, add 2.2 Fusarium fungal inoculum, add 1×10 ⁷ cfu Fusarium sp. CPCC 400709 per gram of solid-state fermentation medium, mix well, and continue for 28 Cultivate for 7 days, collect all the substances in the culture container, and obtain the yellow + sickle fermentation product. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

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

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

Put the Fusarium fungal agent of 2.2 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, and insert 1×10 ⁷ cfu of Fusarium sp. CPCC per gram of solid-state fermentation medium 400709, mix well, culture at 28°C for 22 days, add 2.4 aflatoxin agent on the 23rd day, insert 2×10 ⁷ cfu Aspergillus flavus 3.2792 per gram of solid-state fermentation medium, mix well, continue for 28 Cultivate at ℃ for 7 days, collect all the substances in the culture vessel, and obtain the sickle+yellow fermentation product. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

3.8 Aspergillus niger agent treatment

Prepare diosgenin with the Aspergillus niger inoculant of 2.6, the specific method is as follows:

Put the Aspergillus niger agent of 2.6 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, insert 3×10 ⁷ cfu of Aspergillus niger (Aspergillus niger) CPCC 400524 into each gram of solid-state fermentation medium, mix well, 28 Cultivate for 22 days, collect all the substances in the culture container, and obtain the fermentation product of Aspergillus niger agent. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

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

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

Put the Aspergillus niger agent of 2.6 into a 500mL Erlenmeyer flask equipped with 50g of solid-state fermentation medium, insert 2×10 ⁷ cfu of Aspergillus niger (Aspergillus niger) CPCC 400524 per gram of solid-state fermentation medium, mix well, 28 Cultivate at ℃ for 22 days, then inoculate 2.2 Fusarium fungal inoculum on the 23rd day, inoculate 1×10 ⁷ cfu Fusarium sp. CPCC 400709 per gram of solid-state fermentation medium, mix well , continue to cultivate at 28°C for 7 days, collect all the substances in the culture container, and obtain the black + sickle fermentation product. The experiment was repeated three times, and 10 Erlenmeyer flasks were set for each repetition.

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

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

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

3.11 CPCC 400709+CPCC 400718 complete set of microbial treatment

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

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

3.12 CPCC 400709+CPCC 400718+CPCC 400226 complete set of microbial treatment

Use 2.9 CPCC 400709+CPCC 400718+CPCC 400226 complete set of bacterial agents to prepare diosgenin, the specific method is as follows:

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

3.13 CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 complete set of microbial treatment

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

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

4. The yield of diosgenin

The Fusarium + new fermentation product of 3.1, the new + sickle fermentation product of 3.2, the fermentation product of Curvularia lunata fungi of 3.3, the fermentation product of Fusarium fungus of 3.4, the fermentation product of Aspergillus flavus of 3.5, and the fermentation product of 3.6 Yellow + sickle fermentation product, 3.7 sickle + yellow fermentation product, 3.8 Aspergillus niger fermentation product, 3.9 black + sickle fermentation product, 3.10 sickle + black fermentation product, 3.11 CPCC 400709+CPCC 400718 complete set of microbial fermentation Product, 3.12 CPCC 400709+CPCC 400718+CPCC 400226 complete set of bacterial agent fermentation product and 3.13 CPCC 400709+CPCC 400718+CPCC 400226+CPCC 400712 complete set of bacterial agent fermentation product were dried and ground into coarse powder, 10 times the volume of acetic acid Ethyl ester was refluxed for 1 h, and the extraction was repeated twice, and the obtained extracts were combined and evaporated to dryness, dissolved in methanol, passed through a 0.22 μm filter membrane, and analyzed by HPLC to calculate the yield of diosgenin: the mass of diosgenin (mg)/ The quality of the medicinal material of Dioscorea scutellariae (mg)×100%. Take the diosgenin standard substance, dissolve it in methanol to a constant volume, and perform HPLC to prepare a standard curve. HPLC chromatographic conditions are as follows: Agilent 1100, equipped with ELSD detector and Chemstation chromatographic workstation; XDB-C18 chromatographic column (250 * 4.6mm, 5 μm); 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 all 13 treatments had the production of diosgenin (Fig. 1 and table 1):

1. The total yield of diosgenin in Curvularia lunata + Fusarium fungal agent set Fusarium + new treatment is higher than the sum of Curvularia lunata fungal agent treatment and Fusarium fungal agent treatment, indicating that in Curvularia lunata + Fusarium fungus complete inoculum Fusarium + new treatment, Curvularia lunata Curvularia lunata 3.4381 and Fusarium sp. (Fusarium sp.) CPCC400709 fermented yams containing yams There was a synergistic effect on saponin; and Curvularia crescentus + Fusarium fungus complete set of inoculum new + Fusarium treatment than Fusarium fungus inoculum treatment of diosgenin yield is even lower, indicating that in crescent crescent Diosgenin was produced by fermenting the culture medium containing Dioscorea scutellaris by Curvularia lunata 3.4381 and Fusarium sp. CPCC 400709 in the new + Fusarium sp. did not produce synergies.

2. In terms of the yield of diosgenin, whether it is Aspergillus flavus + Fusarium fungus complete set of inoculant yellow + Fusarium treatment or Aspergillus flavus + Fusarium fungal set of inoculum Fusarium + yellow treatment is higher than Fusarium Treatment and Aspergillus flavus treatment were all low, indicating that Aspergillus flavus 3.279 and Fusarium sp. CPCC 400709 had an antagonistic effect on the production of diosgenin in the fermentation medium containing Dioscorea scutellaria.

3. In terms of the yield of diosgenin, whether it is Aspergillus niger + Fusarium fungus complete set of inoculant black + Fusarium treatment or Aspergillus niger + Fusarium fungus complete set of inoculant Fusarium + black treatment is higher than Fusarium fungal inoculant The treatment was low, indicating that Aspergillus niger (Aspergillus niger) CPCC 400524 and Fusarium sp. (Fusarium sp.) CPCC 400709 had an antagonistic effect on the production of diosgenin in the fermentation medium containing Dioscorea scutellaria.

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

Table 1, the output and yield of each treatment diosgenin

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

Claims (10)

1. The method for preparing diosgenin is any one of M1) to M3): M1) The method comprises culturing Fusarium fungi and Curvularia fungi with a medium containing Dioscorea scutellaria to obtain diosgenin; M2) The method comprises cultivating Fusarium fungi and Porphyra fungi with a culture medium containing Dioscorea scutellariae to obtain diosgenin; M3) The method comprises culturing a fungus of the genus Fusarium with a medium containing Dioscorea scutellaria to obtain diosgenin. 2. The method according to claim 1, characterized in that: in the M1) to M3), the Fusarium fungus is Fusarium sp. (Fusarium sp.) CPCC 400709, Fusarium sp. ( At least one of Fusarium sp.) CPCC 400226 and Fusarium sp. (Fusarium sp.) CPCC 400712; the fungus of the genus Curvularia is Curvularia lunata; Genus species (Dictyosporium sp.) CPCC 400718. 3. The method according to claim 1 or 2, characterized in that: in the M1) and M3), the Fusarium fungus is Fusarium sp. (Fusarium sp.) CPCC 400709; Said M2) is M21), M22) or M23); Said M21) is that said method comprises cultivating Fusarium fungus and Trichosporum fungus with a culture medium containing Dioscorea scutellaria to obtain diosgenin; in said M21), said Fusarium fungus is Fusarium Bacterial species (Fusarium sp.) CPCC400709; the fungus of the genus Dictyosporium is Dictyosporium sp. (Dictyosporium sp.) CPCC 400718; Said M22) is that said method comprises cultivating Fusarium fungi and Trichosporum fungi with a medium containing Dioscorea scutellaria to obtain diosgenin; in said M22), said Fusarium fungus is Fusarium Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226; the Dictyosporium sp. CPCC 400718; Said M23) is that said method comprises culturing Fusarium fungi and Trichosporum fungi with a culture medium containing Dioscorea scutellaria to obtain diosgenin; in said M23), said Fusarium fungi are Fusarium fungi Bacterial classification (Fusarium sp.) CPCC400709, Fusarium sp. (Fusarium sp.) CPCC 400226 and Fusarium sp. (Fusarium sp.) CPCC400712; The fungus of the genus Dictyosporium is Dictyosporium sp.) CPCC 400718. 4. The method according to claim 3, characterized in that: in the M1), cultivating Fusarium fungi and Curvularia fungi with the culture medium containing Dioscorea scutellaria is in the medium containing Dioscorea scutellaria Inoculating the Fusarium fungus for cultivation first, and then inserting the Curvularia fungus for cultivation; In said M21), using the culture medium containing Dioscorea scutellaria to cultivate Fusarium fungi and the fungi of the genus Porphyria is to first insert the fungi of the genus Fusarium into the medium containing Dioscorea scutellaria for cultivation and then insert The fungus of the genus Pleurotus spp. is cultivated; In the M22), using the culture medium containing Dioscorea scutellaria to cultivate Fusarium sp. fungi and the genus Fusarium is to insert Fusarium sp. (Fusarium sp.) CPCC 400709 into the medium containing Dioscorea scutellaria After culturing, insert Dictyosporium sp. CPCC 400718 for cultivation, then insert Fusarium sp. CPCC 400226 for cultivation; In the M23), using the culture medium containing Dioscorea scutellaria to cultivate Fusarium sp. fungi and the genus Fusarium sp. is to insert Fusarium sp. (Fusarium sp.) CPCC 400709 into the medium containing Dioscorea scutellaria After culture, insert Dictyosporium sp. CPCC 400718 for culture, then insert Fusarium sp. CPCC400226 for culture, then insert Fusarium sp. CPCC 400712 was used for cultivation. 5. The bacterial agent for preparing diosgenin, characterized in that: the bacterial agent is any one of C1 to C4: The C1 is a complete set of bacterial agents for preparing diosgenin consisting of Fusarium fungal agents and Curvularia fungal agents, and the active ingredient of the Fusarium fungal agent contains the Any one of the fungi of the genus Fusarium, the active ingredient of the fungal agent of the genus Curvularia contains the fungus of the genus Curvularia according to any one of claims 1 to 3; The C2 is an active ingredient containing the fungus of the genus Fusarium according to any one of claims 1 to 3 and the fungus of the genus Curvularia according to any one of claims 1 to 3, and is used for the preparation of diosgenin; The C3 is a complete set of bacterial agents for preparing diosgenin consisting of Fusarium fungal agents and Trichosporum fungal agents, and the active ingredient of the Fusarium fungal agent contains the Any one of the Fusarium fungi, the active ingredient of the Fusarium fungus inoculant contains the Fusarium fungus described in any one of claims 1 to 3; The C4 is a fungal agent for preparing diosgenin, which contains the Fusarium fungus described in any one of claims 1 to 3 and the fungus of the genus Tricharia according to any one of claims 1 to 3 as active ingredients. 6. The bacterium agent for preparing diosgenin according to claim 5, characterized in that: in the C1 and the C2, the Fusarium fungus is Fusarium sp. (Fusarium sp.) CPCC 400709; the fungus of the genus Curvularia is Curvularia lunata; The C3 is C31, C32, or C33; the C31, C32, and C33 are all sets of bacterial agents for the preparation of diosgenin composed of Fusarium fungal agents and Fusarium fungal agents, and the Fusarium The active ingredient of the genus fungal agent contains the Fusarium fungus described in any one of claims 1 to 3, and the active ingredient of the fungal agent of the genus Porphyria contains the brick described in any one of claims 1 to 3. Dictyosporium fungus; in the C31, C32 and C33, the Dictyosporium fungus is Dictyosporium sp. (Dictyosporium sp.) CPCC 400718; In the C31, the Fusarium fungus is Fusarium sp. CPCC 400709; In the C32, the Fusarium fungal inoculum is Fusarium sp. CPCC 400709 inoculum and Fusarium sp. CPCC 400226 inoculum; the Fusarium sp. The active ingredient of Fusarium sp. CPCC 400709 bacterial agent contains Fusarium sp. CPCC 400709; the active ingredient of Fusarium sp. CPCC 400226 bacterial agent contains Fusarium sp. Species (Fusarium sp.) CPCC 400226; In the C33, the Fusarium fungal inoculum is Fusarium sp. (Fusarium sp.) CPCC 400709 inoculum, Fusarium sp. (Fusarium sp.) CPCC 400226 inoculum and Fusarium sp. ( Fusarium sp.) CPCC400712 microbial agent; the active ingredient of the Fusarium sp. (Fusarium sp.) CPCC 400709 bacterial agent contains Fusarium sp. (Fusarium sp.) CPCC 400709; sp.) The active ingredient of CPCC 400226 inoculum contains Fusarium sp. CPCC 400226; the active ingredient of the Fusarium sp. CPCC400712 inoculum contains Fusarium sp. sp.) CPCC400712; The C4 is C41, C42 or C43; the C41, C42 and C43 are all active ingredients containing the Fusarium fungus described in any one of claims 1 to 3 and the brick described in any one of claims 1 to 3 A bacterial agent of Dictyosporium fungus for preparing diosgenin; in the C41, C42 and C43, the Dictyosporium fungus is Dictyosporium sp. (Dictyosporium sp.) CPCC400718; In the C41, the Fusarium fungus is Fusarium sp. CPCC 400709; In the C42, the Fusarium fungi are Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226; In the C43, the Fusarium fungi are Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Fusarium sp. CPCC 400712 . 7. Any application in P1) to P3): P1) The application of Fusarium fungi and Curvularia fungi in the preparation of diosgenin or the application of C1 or C2 in claim 5 or 6 in the preparation of diosgenin; P2) the application of Fusarium fungus and the fungus of the genus Porphyra in the preparation of diosgenin or the application of C3 or C4 in claim 5 or 6 in the preparation of diosgenin; P3) Application of Fusarium fungi in the preparation of diosgenin. 8. The application according to claim 7, characterized in that: in the P1) to P3), the Fusarium fungus is Fusarium sp. (Fusarium sp.) CPCC 400709, Fusarium sp. ( At least one of Fusarium sp.) CPCC400226 and Fusarium sp. (Fusarium sp.) CPCC 400712; the fungus of the genus Curvularia is Curvularia lunata; Dictyosporium sp. CPCC 400718. 9. The application according to claim 7 or 8, characterized in that: said P2) is P21), P22) or P23); said P21), P22) or P23) are Fusarium fungi and brick lattice Application of fungi of the genus Sporus in the preparation of diosgenin; The Fusarium fungus in P21) is Fusarium sp. CPCC 400709; In the P22), the Fusarium fungi are Fusarium sp. CPCC 400709 and Fusarium sp. CPCC 400226; In the P23), the Fusarium fungal agent is Fusarium sp. CPCC 400709, Fusarium sp. CPCC 400226 and Fusarium sp. ) CPCC 400712. 10. The method according to any one of claims 1-4, the bacterium agent for preparing diosgenin according to claim 5 or 6, or the application according to any one of claims 7-9, characterized in that : said Curvularialunata is Curvularialunata 3.4381.