Tinea cystidium and its applications

Abstract

The present invention relates to the field of microbial technology, and discloses a strain of Saccothecium and its application. The present invention for the first time extracts Saccothecium from the rotten wood of Poria cocos cultivation, and discovers that it can be used for the production of β-glucosidase. Through further research, it is also found that the β-glucosidase produced by this strain also has the ability to transform rare ginsenosides CK and Rh1, and can be used for the production of rare ginsenosides and the development of downstream products, thus having broad application prospects and extremely high application value.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a strain of Saccothecium and its application. Background Art

[0002] β-glucosidase can catalyze the hydrolysis reaction of β-glucosidic bonds, converting glycoside compounds in the substrate into corresponding glucose and corresponding ligands. Due to its high efficiency, specificity, and environmental friendliness, it has been widely used in fields such as food processing, medical field, industrial applications, and bioenergy. Especially in the pharmaceutical field, β-glucosidase can be used to transform glycoside compounds in natural products (such as plant extracts, etc.), thereby changing their physiological activities and medicinal values. Especially in plants, many medicinal components exist in the form of glycoside compounds and need to be released by the catalytic action of β-glucosidase.

[0003] For example, as a medicinal plant with high medicinal value and economic value, ginseng is widely used in traditional Chinese medicine and health care fields. Ginsenosides are the main active components of ginseng, and more than 90% of ginseng extracts contain main ginsenosides of PPD (Rb1, Rb2, Rc, and Rd) and PPT (Rg1 and Re) types. In addition to the above main ginsenosides (i.e., ginsenosides with relatively high content and easy extraction in ginseng), ginseng also contains rare ginsenosides (also known as "minor ginsenosides"). For example, rare ginsenoside CK has a low content in nature, but has good curative effects on diseases such as cancer, diabetes, inflammation, allergy, angiogenesis, etc., and also has significant effects in aspects such as anti-aging. Moreover, ginsenoside CK has better neuroprotection and hepatoprotective effects than other minor ginsenosides, making it have broad application prospects in the pharmaceutical and beauty fields. Due to the low content of rare ginsenosides in ginseng and the difficulty in extraction, but having unique biological activities in pharmacology and other aspects, they have extremely high economic value and application value.

[0004] Although β-glucosidase has broad application prospects in the fields of biotechnology and medicine, there are still some problems and challenges in practical applications. The most important challenges currently faced by β-glucosidase include how to find efficient sources of β-glucosidase to meet the needs of industrial production, and how to use β-glucosidase to convert glycoside compounds in natural products into target compounds with medicinal activities (i.e., finding suitable substrates and reaction conditions).

[0005] Therefore, there is an urgent need to develop new methods and related products that can produce β-glucosidase with the required enzyme activity in large quantities and efficiently to meet the production and application needs in this field. Summary of the Invention

[0006] The object of the present invention is to overcome the above problems existing in the prior art, and to provide a Plectosphaerella oligotrophica and its application. The Plectosphaerella oligotrophica strain provided by the present invention has good β-glucosidase production ability, and the β-glucosidase produced by it can also transform ginsenosides, so that the main ginsenosides Rb1, Rg1, etc. are transformed into rare ginsenosides CK and Rh1, laying a foundation for the development and production of derivative products such as drugs, health products, and beauty products.

[0007] To achieve the above object, in the first aspect of the present invention, a Plectosphaerella oligotrophica is provided, and the preservation number of the Plectosphaerella oligotrophica is CGMCC No. 41079.

[0008] In the second aspect of the present invention, a bacterial agent is provided, and the active ingredient of the bacterial agent includes the Plectosphaerella oligotrophica described in the first aspect.

[0009] In the third aspect of the present invention, the use of the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect in the preparation of β-glucosidase is provided.

[0010] In the fourth aspect of the present invention, a method for preparing β-glucosidase is provided, and the method includes contacting the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect with a fermentation substrate, and performing a first fermentation culture.

[0011] In the fifth aspect of the present invention, β-glucosidase prepared by the method described in the fourth aspect is provided.

[0012] In the sixth aspect of the present invention, the use of the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect in the production of rare ginsenosides CK and / or Rh1 is provided.

[0013] In the seventh aspect of the present invention, a method for preparing rare ginsenosides is provided, and the method includes:

[0014] (A) contacting ginsenoside Rb1 with the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect, and performing a second fermentation culture, or contacting ginsenoside Rb1 with the β-glucosidase described in the fifth aspect to obtain rare ginsenoside CK; and / or,

[0015] (B) contacting ginsenoside Rg1 with the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect, and performing a third fermentation culture, or contacting ginsenoside Rg1 with the β-glucosidase described in the fifth aspect to obtain rare ginsenoside Rh1.

[0016] Through the above technical solutions, the present invention can at least achieve the following beneficial effects:

[0017] (1) The present invention for the first time isolated Coniella fibulata from rotten wood of Poria cocos cultivation, and for the first time discovered the ability of strain LB-002 with strong potential for producing β-glucosidase in this strain (through gene annotation, 304 glycoside hydrolase genes were annotated from this strain, among which 11 β-glucosidase genes were annotated. It indicates that strain LB-002 has strong potential for producing β-glucosidase);

[0018] (2) The β-glucosidase produced by Coniella fibulata LB-002 provided by the present invention shows the docking ability with ginsenoside Rb1 and Rg1 molecules in molecular docking, and has the ability to convert them into rare ginsenosides CK and Rh1 respectively, which has never been reported in the current research on Coniella fibulata.

[0019] (3) Through experimental verification, it is confirmed that Coniella fibulata LB-002 provided by the present invention indeed has good ginsenoside conversion ability, and can convert the main ginsenosides Rb1 and Rg1 into rare ginsenosides CK and Rh1, which has a positive impact and broad application prospects in the development and production of derivative products such as health products, drugs, and beauty products. Description of the Drawings

[0020] Figure 1 It is the esculin plate screening map for Coniella fibulata LB-002 to produce β-glucosidase;

[0021] Figure 2A 、 Figure 2B and Figure 2C They are the phylogenetic trees constructed by the neighbor-joining method based on ITS sequence, LSU sequence and β-TUB sequence of Coniella fibulata LB-002 respectively;

[0022] Figure 3 It is the genomic data of Coniella fibulata LB-002, and the annotation result map through the Carbohydrate-Active enZYmes (CAZy) database;

[0023] Figure 4 It is the 3D molecular virtual docking result map of the β-glucosidase protein produced by Coniella fibulata LB-002 with (A) ginsenoside Rb1 and (B) ginsenoside Rg1;

[0024] Figure 5 It is the TLC detection result of the β-glucosidase produced by Coniella fibulata LB-002 to convert ginsenosides;

[0025] Figure 6A and Figure 6B It is the full-scan electrospray mass spectrometry map of the sample solution of Coniella fibulata LB-002;

[0026] Figure 7High performance liquid chromatography (HPLC) chromatograms of the sample solution of Plectosphaerella oligotrophica LB-002, as well as the mixed standard solutions of ginsenoside CK and 20(S)-Rh1.

[0027] Biological Deposit

[0028] Plectosphaerella oligotrophica LB-002 provided by the present invention has been classified and named as Plectosphaerella oligotrophica, and was deposited at the China General Microbiological Culture Collection Center on January 10, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41079. Detailed Embodiments

[0029] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0030] In the present invention, the numbers for reagents or operations (such as "first liquid medium", "second liquid medium", "first fermentation culture", "second fermentation culture" etc.) are only used for facilitating the distinction of reagents or operations adopted in different steps or scenarios in the description, and have no restrictive effect on the specific reagent components, contents, operation methods, conditions, sequences, etc. involved.

[0031] Plectosphaerella is a type of plant pathogen that may cause plant diseases such as wilting and root rot, and does not have human or animal pathogenicity. Currently, Plectosphaerella is mainly used in research work on the occurrence and prevention of plant diseases. Although certain achievements have been made in the research on Plectosphaerella in aspects such as some mechanisms of plant diseases caused by it or prevention methods, its application potential in production has not been discovered yet.

[0032] However, the inventors of the present invention accidentally isolated a strain of Plectosphaerella during the research and found that it has good β-glucosidase production ability, enabling Plectosphaerella, which was originally only a plant pathogen, to have the potential of "turning waste into treasure". Through further research, the inventors of the present invention also found that this Plectosphaerella can convert the ginsenoside diol group saponins (such as Rb1) and triol group saponins (such as Rg1) with the highest content in ginseng extract into rare ginsenosides CK and Rh1, which has extremely high application value.

[0033] Based on the above findings, in the first aspect of the present invention, a strain of Plectosphaerella oligotrophica is provided, and the preservation number of the Plectosphaerella oligotrophica is CGMCC No. 41079.

[0034] In the second aspect of the present invention, a bacterial agent is provided, and the active ingredient of the bacterial agent includes the Plectosphaerella oligotrophica described in the first aspect.

[0035] In the bacterial agent provided by the present invention, only the Plectosphaerella oligotrophica described in the first aspect can be used as the sole active ingredient, or the Plectosphaerella oligotrophica and other bacteria can be used together as a composite active ingredient.

[0036] According to a preferred embodiment of the present invention, the bacterial agent may further contain excipients. Any excipients commonly used in the preparation of bacterial agents in the art can be applicable to the present invention, such as bacterial agent carriers (such as culture media, etc.), protectants (such as lyophilization protectants, etc.), buffers, preservatives, etc.

[0037] In the present invention, there is no particular limitation on the specific supply dosage form of the bacterial agent. For example, the bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

[0038] According to some preferred embodiments of the present invention, the bacterial agent can be a liquid bacterial agent. Preferably, the liquid bacterial agent can be provided by the culture solution of the Plectosphaerella oligotrophica provided by the present invention.

[0039] According to some preferred embodiments of the present invention, the bacterial agent can be a solid bacterial agent. Preferably, the solid bacterial agent can be obtained by freeze-drying the culture solution (amplified culture solution) of the Plectosphaerella oligotrophica provided by the present invention.

[0040] In the third aspect of the present invention, the use of the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect in the preparation of β-glucosidase is provided.

[0041] In the fourth aspect of the present invention, a method for preparing β-glucosidase is provided, and the method includes contacting the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect with a fermentation substrate and performing a first fermentation culture.

[0042] According to a preferred embodiment of the present invention, the system of the first fermentation culture includes a fermentation substrate and the Plectosphaerella oligotrophica described in the first aspect or the bacterial agent described in the second aspect, and the fermentation substrate is provided by a first liquid medium.

[0043] Preferably, the initial pH value of the first liquid medium is not lower than 5, preferably 5.5 - 6.5. For example, the initial pH value of the first liquid medium can be 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0044] Preferably, the first liquid medium includes cellulose (as the main fermentation substrate), preferably the final concentration of cellulose in the first liquid medium is not lower than 3 g / L, preferably 3 - 15 g / L. For example, the final concentration of cellulose in the first liquid medium can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0045] According to some preferred embodiments of the present invention, wherein the first liquid medium further includes yeast extract and a trace element source. Yeast extract refers to a class of reagents that can be used for microbial culture and are extracted from yeast. Usually, it can be obtained through commercial channels, and the supply forms include yeast extract paste, yeast powder, etc.

[0046] The trace element source refers to a reagent in the first liquid medium that can provide trace elements for the growth of Scytalidium dimidiatum and the production of β - glucosidase. Preferably, the trace element source provides at least one of the elements K, Mg, Na, and Fe for the first liquid medium.

[0047] Any reagent that can provide the above elements and can be used for the culture of Scytalidium dimidiatum is applicable to the present invention. For example, it can be (inorganic or organic) acids, (inorganic or organic) bases, (inorganic or organic) salts, etc. of the above elements.

[0048] According to some preferred embodiments of the present invention, wherein the trace element source can include K 2 HPO 4 、MgSO 4 、NaNO 3 、FeSO 4 and so on, and at least one of their hydrates.

[0049] According to a particularly preferred embodiment of the present invention, wherein the first liquid medium includes:

[0050] K 2 HPO 4 0.01 - 0.3 g / L, MgSO 4 ·7H 2O 0.1 - 1 g / L, NaNO 3 1 - 5 g / L, FeSO 4 ·7H 2 O 0.01 - 0.3 g / L, yeast extract 0.1 - 5 g / L, cellulose 1 - 20 g / L, pH = 5.5 - 6.5.

[0051] According to a preferred embodiment of the present invention, wherein the temperature of the first fermentation culture is 25 - 35 °C.

[0052] Preferably, the conditions of the first fermentation culture further include: the dosage of the Scytalidium dimidiatum or the microbial agent is such that in the system of the first fermentation culture, the initial content of Scytalidium dimidiatum is not less than 10 7 CFU / mL, preferably 10 7 -10 9 CFU / mL; the fermentation culture time is not less than 48 h, preferably 50 - 180 h. The initial content of Scytalidium dimidiatum not less than 10 7 CFU / mL means that in the first fermentation culture system, the initial content of Scytalidium dimidiatum is not less than 10 7 CFU / mL level, 1×10 7 CFU / mL, 5×10 7 CFU / mL, 9.9×10 7 CFU / mL, etc. all belong to the 10 7 CFU / mL level. That is, in the present invention, the initial content of Scytalidium dimidiatum in the first fermentation culture system can be in the range of greater than or equal to 1×10 7 CFU / mL to less than 1×10 10 CFU / mL.

[0053] For example, in the first fermentation culture system, the initial content of Scytalidium dimidiatum can be 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 4×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 2×10 9 CFU / mL, 4×10 9 CFU / mL, 6×10 9CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL, 9.5×10 9 CFU / mL, 9.9×10 9 CFU / mL, or it can also be a range formed by any two of the above values, or any intermediate value within that range.

[0054] For example, the time of the first fermentation culture can be 50h, 60h, 70h, 80h, 90h, 100h, 110h, 120h, 130h, 140h, 150h, 160h, 170h, 180h, or it can also be a range formed by any two of the above values, or any intermediate value within that range.

[0055] Preferably, the first fermentation culture is carried out under shaking conditions, and preferably the shaking speed is 100 - 150 rpm.

[0056] According to a preferred embodiment of the present invention, wherein the method further comprises a step of separating β - glucosidase from the first fermentation culture product after the first fermentation culture is completed. Any method that can be used to separate (extract) β - glucosidase in the art can be applied to the present invention.

[0057] The fifth aspect of the present invention provides the β - glucosidase prepared by the method described in the fourth aspect.

[0058] The sixth aspect of the present invention provides the use of the Scytalidium dimidiatum described in the first aspect or the microbial agent described in the second aspect in the production of rare ginsenoside CK and / or Rh1.

[0059] The seventh aspect of the present invention provides a method for preparing rare ginsenosides, the method comprising:

[0060] (A) contacting ginsenoside - Rb1 with the Scytalidium dimidiatum described in the first aspect or the microbial agent described in the second aspect, and performing a second fermentation culture, or contacting ginsenoside - Rb1 with the β - glucosidase described in the fifth aspect to obtain rare ginsenoside CK; and / or,

[0061] (B) contacting ginsenoside - Re with the Scytalidium dimidiatum described in the first aspect or the microbial agent described in the second aspect, and performing a third fermentation culture, or contacting ginsenoside - Re with the β - glucosidase described in the fifth aspect to obtain rare ginsenoside Rh1.

[0062] According to a preferred embodiment of the present invention, wherein in method (A), the system of the second fermentation culture comprises a culture medium, and the Scytalidium dimidiatum described in the first aspect or the microbial agent described in the second aspect, and the culture medium is a second liquid culture medium.

[0063] Preferably, the second liquid medium comprises ginsenoside group A, preferably the final concentration of ginsenoside group A in the second liquid medium is not less than 5 g / L, more preferably 5 - 50 g / L; more preferably the ginsenoside group A comprises ginsenoside Rb1.

[0064] More preferably, the second liquid medium further comprises yeast extract and a trace element source, preferably the trace element source provides at least one element of K, Mg, Na and Fe for the second liquid medium;

[0065] Any reagent that can provide the above elements and can be used for the culture of Sphaerotheca furiicola to convert ginsenoside group A into rare ginsenoside CK can be applicable to the present invention, for example, it can be an (inorganic or organic) acid, (inorganic or organic) base, (inorganic or organic) salt, etc. of the above elements.

[0066] According to some preferred embodiments of the present invention, wherein, the trace element source may comprise K 2 HPO 4 、MgSO 4 、NaNO 3 、FeSO 4 etc., and at least one of their hydrates.

[0067] According to a preferred embodiment of the present invention, wherein, in method (A), the temperature of the second fermentation culture is 25 - 32 °C. For example, it can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, or it can also be a range composed of any two of the above values, or any intermediate value in this range.

[0068] According to a particularly preferred embodiment of the present invention, wherein, the second liquid medium comprises:

[0069] K 2 HPO 4 0.01 - 0.3 g / L, MgSO 4 ·7H 2 O 0.1 - 1 g / L, NaNO 3 1 - 5 g / L, FeSO 4 ·7H 2 O 0.01 - 0.3 g / L, yeast extract 0.1 - 3 g / L, ginsenoside group A 5 - 20 g / L.

[0070] Preferably, the conditions of the second fermentation culture further comprise: the dosage of the Sphaerotheca furiicola or the bacterial agent is such that in the system of the second fermentation culture, the initial content of Sphaerotheca furiicola is not less than 10 7 CFU / mL, preferably 10 7 - 1010 CFU / mL; The fermentation culture time is not less than 150 h, preferably 200 - 400 h. The initial content of Scytalidium dimidiatum is not less than 10 7 CFU / mL means that in the second fermentation culture system, the initial content of Scytalidium dimidiatum is not less than 10 7 CFU / mL level. 1×10 7 CFU / mL, 5×10 7 CFU / mL, 9.9×10 7 CFU / mL, etc. all belong to the 10 7 CFU / mL level. That is to say, in the present invention, the initial content of Scytalidium dimidiatum in the second fermentation culture system can be greater than or equal to 1×10 7 CFU / mL to less than 1×10 11 CFU / mL range.

[0071] For example, in the second fermentation culture system, the initial content of Scytalidium dimidiatum can be 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 4×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 2×10 9 CFU / mL, 4×10 9 CFU / mL, 6×10 9 CFU / mL, 8×10 9 CFU / mL, 1×10 10 CFU / mL, 2×10 10 CFU / mL, 4×10 10 CFU / mL, 6×10 10 CFU / mL, 8×10 10 CFU / mL, 9×10 10 CFU / mL, 9.5×10 10 CFU / mL, 9.9×10 10 CFU / mL, or it can also be the range formed by any two of the above values, or any intermediate value within this range.

[0072] For example, the time of the second fermentation culture can be 200 h, 220 h, 240 h, 260 h, 280 h, 300 h, 320 h, 340 h, 360 h, 380 h, 400 h, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0073] Preferably, the second fermentation culture is carried out under shaking conditions, and preferably the shaking speed is 100 - 150 rpm.

[0074] According to a preferred embodiment of the present invention, in method (A), the method further includes a step of separating ginsenoside CK from the product of the second fermentation culture. Any method in the art that can separate ginsenoside CK from the fermentation broth system can be applied to the present invention.

[0075] According to a preferred embodiment of the present invention, in method (A), the conditions for contacting ginsenoside - diol - type saponins with β - glucosidase include: temperature 25 - 35°C, time 10 - 40 h.

[0076] For example, the temperature for contacting ginsenoside - diol - type saponins with β - glucosidase can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0077] For example, the time for contacting ginsenoside - diol - type saponins with β - glucosidase can be 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0078] According to a preferred embodiment of the present invention, in method (B), the system for the third fermentation culture includes a culture medium, and the Xylohypha sp. or the microbial agent, and the culture medium is a third liquid culture medium.

[0079] Preferably, the third liquid culture medium includes ginsenoside - triol - type saponins. Preferably, the final concentration of ginsenoside - triol - type saponins in the third liquid culture medium is not less than 5 g / L, more preferably 5 - 50 g / L; more preferably, the ginsenoside - triol - type saponins include ginsenoside Rg1.

[0080] More preferably, the third liquid culture medium further includes yeast extract and a trace element source. Preferably, the trace element source provides at least one of the elements K, Mg, Na, and Fe for the third liquid culture medium.

[0081] Any reagent that can provide the above elements and can be used for culturing Tolypocladium inflatum to convert ginsenoside group A into rare ginsenoside CK can be applicable to the present invention. For example, it can be an (inorganic or organic) acid, (inorganic or organic) base, (inorganic or organic) salt, etc. of the above elements.

[0082] According to some preferred embodiments of the present invention, the trace element source may include K 2 HPO 4 , MgSO 4 , NaNO 3 , FeSO 4 , etc., and at least one of their hydrates.

[0083] According to a preferred embodiment of the present invention, in method (B), the temperature of the third fermentation culture is 25-32 °C. For example, it can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, or it can also be a range composed of any two of the above values, or any intermediate value within this range.

[0084] According to a particularly preferred embodiment of the present invention, the third liquid medium includes:

[0085] K 2 HPO 4 0.01-0.3 g / L, MgSO 4 ·7H 2 O 0.1-1 g / L, NaNO 3 1-5 g / L, FeSO 4 ·7H 2 O 0.01-0.3 g / L, yeast extract 0.1-3 g / L, ginsenoside group C 5-20 g / L.

[0086] Preferably, the conditions for the third fermentation culture further include: the dosage of the Tolypocladium inflatum or the bacterial agent is such that in the system of the third fermentation culture, the initial content of Tolypocladium inflatum is not less than 10 7 CFU / mL, preferably 10 7 -10 10 CFU / mL; the fermentation culture time is not less than 150 h, preferably 200-400 h. The initial content of Tolypocladium inflatum not less than 10 7 CFU / mL means that in the system of the third fermentation culture, the initial content of Tolypocladium inflatum is not less than 10 7 CFU / mL level. 1×10 7 CFU / mL, 5×10 7 CFU / mL, 9.9×10 7 CFU / mL, etc. all belong to 107 CFU / mL level. That is, in the present invention, the initial content of Scytalidium dimidiatum in the third fermentation culture system can be greater than or equal to 1×10 7 CFU / mL to less than 1×10 11 CFU / mL.

[0087] For example, in the third fermentation culture system, the initial content of Scytalidium dimidiatum can be 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 4×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 2×10 9 CFU / mL, 4×10 9 CFU / mL, 6×10 9 CFU / mL, 8×10 9 CFU / mL, 1×10 10 CFU / mL, 2×10 10 CFU / mL, 4×10 10 CFU / mL, 6×10 10 CFU / mL, 8×10 10 CFU / mL, 9×10 10 CFU / mL, 9.5×10 10 CFU / mL, 9.9×10 10 CFU / mL, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0088] For example, the time of the third fermentation culture can be 200h, 220h, 240h, 260h, 280h, 300h, 320h, 340h, 360h, 380h, 400h, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0089] Preferably, the third fermentation culture is carried out under shaking conditions, and preferably the shaking speed is 100 - 150 rpm.

[0090] According to a preferred embodiment of the present invention, in method (B), the method further includes a step of separating ginsenoside Rh1 from the third fermentation culture product after the end of the third fermentation culture. Any method in the art that can separate ginsenoside Rh1 from the fermentation broth system can be applied to the present invention.

[0091] According to a preferred embodiment of the present invention, in method (B), the conditions for contacting the ginsenoside triol group with β-glucosidase include: temperature 25 - 35°C, time 10 - 40 h.

[0092] For example, the temperature for contacting the ginsenoside triol group with β-glucosidase can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0093] For example, the time for contacting the ginsenoside triol group with β-glucosidase can be 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0094] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not used to limit the present invention.

[0095] In the following examples, unless otherwise specified, the reagents or materials used are all commercially available products purchased from regular biological or chemical reagent / material suppliers, and the reagents are all of analytical grade.

[0096] The formula / source of the (liquid) culture medium used in the following examples is as follows:

[0097] Ginsenoside diol group (mainly composed of Rb1) and ginsenoside triol group (mainly composed of Rg1): Purchased from Shanghai Standard Technology Service Co., Ltd.

[0098] Rose Bengal medium: Purchased from Haibo Biotechnology Co., Ltd. (product number HB0237 - 2).

[0099] Esculin medium: Esculin 1 g, ferric citrate 0.5 g, NaCl 2 g, MgSO 4 ·7H 2 O 0.5 g, KH 2 PO 4 1 g, agar 20 g, made up to 1 L with deionized water, natural pH.

[0100] Fermentation medium: K 2 HPO 4 0.1 g, MgSO 4 7H 2 O 0.5g, NaNO 3 3g, FeSO 4 7H 2 O 0.1g, yeast extract 1g, cellulose powder 5g, deionized water to 1L, pH adjusted to 6.

[0101] Potato glucose liquid culture medium: 5g potato powder, 15g glucose, dilute to 1L with deionized water, natural pH.

[0102] After the above culture media are prepared, sterilize them at 121℃ for 20min, cool to room temperature before use or store in a refrigerator. If solid culture medium plates are used, add 20g / L agar powder before sterilization on the basis of preparing liquid culture medium. After sterilization, pour the plates after cooling until they are not hot (about 50℃). After cooling and solidification, the culture plates of the corresponding culture medium are obtained.

[0103] Example 1

[0104] This example is used to illustrate the separation, screening, identification and preservation of the tinea cysticercus LB-002 provided by the present invention.

[0105] 1. Strain Isolation and Purification

[0106] The strains were isolated by three-point inoculation method using Bengal red culture plates, and then purified by streak method (the culture temperature during the separation and purification process was 28°C, the culture time was 5-7 days, and the growth of the colonies on the plates was observed every day during the culture period).

[0107] Using the above method, a fungus was obtained from a sample of rotten wood grown under the cultivation of Poria cocos in Shuangbai County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, and was named LB-002.

[0108] 2. Screening of β-glucosidase-producing strains

[0109] β-glucosidase can hydrolyze aesculin into glucose and aesculin, which can react with Fe in the culture medium. 3+ According to the property of generating black compounds, the strain LB-002 isolated and purified in Experiment 1 and other strains isolated and purified in the same batch were inoculated on aesculin solid culture plate by the spot-grafting method and cultured at 28°C for 7 days.

[0110] After the culture was completed, the color of the culture medium around strain LB-002 turned brown-black ( Figure 1 ), indicating that the strain has the ability to produce β-glucosidase.

[0111] 3. Strain Identification

[0112] The ribosomal internal transcribed spacer (ITS) sequencing, ribosomal large subunit (LSU) sequencing, and β-Tubulin (β-TUB) gene sequencing were performed on the strain LB-002 isolated in Experiment 1 for strain identification. The above-mentioned sequencing was commissioned to Beijing Qingke Biotechnology (Kunming) Co., Ltd. Among them, the amplification primers used for the ITS sequence were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO:1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:2), the amplification primers used for the LSU sequence were NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3', SEQ ID NO:3) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3', SEQ ID NO:4), and the amplification primers used for the β-TUB sequence were T1 (5'-AACATGCGTGAGATTGTAAGT-3', SEQ ID NO:5) and BT2b (5'-ACCCTCAGTGTAGTGACCCTTGGC-3', SEQ ID NO:6).

[0113] The sequencing results are shown in Table 1 in detail. By comparing the sequencing results with the sequences in the NCBI database, the results showed that the consistency of the amplified fragment of the ITS gene of strain LB-002 with Plectosphaerella oligotrophica CGMCC 3.15078 was 99.42%, the consistency of the amplified fragment of the LSU gene with Plectosphaerella oligotrophica CGMCC 3.15078 was 100%, and the consistency of the amplified fragment of the β-TUB gene with Plectosphaerella oligotrophica LC1990 was 98.28%.

[0114] Table 1

[0115]

[0116] The neighbor-joining method was used to construct a phylogenetic tree (the results are shown in Figure 2A 、 Figure 2B and Figure 2C ), and the results showed that strain LB-002 and the standard control strain of the genus Plectosphaerella clustered in the same branch.

[0117] In summary, it is determined that the strain LB002 belongs to the microorganism of the genus Plectosphaerella taxonomically. The isolation of Plectosphaerella from the rotten wood of Poria cocos cultivation has never been reported in the relevant research in this field.

[0118] 4. Strain preservation

[0119] On January 10, 2024, the above-mentioned screened LB-002 was deposited with the China General Microbiological Culture Collection Center at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number of CGMCC No. 41079.

[0120] Example 2

[0121] This example is used to illustrate the whole-genome sequencing and genomic analysis results of the Plectosphaerella LB-002 provided by the present invention.

[0122] 1. Genome sequencing

[0123] The strain LB-002 was inoculated into a potato dextrose liquid medium and cultured at 30 °C at 150 rpm for 5 days. Then the cells were collected by centrifugation, and the genomic sequencing of the strain was entrusted to Shanghai Majorbio Bio-pharm Technology Co., Ltd. using the Illumina second-generation sequencing platform.

[0124] 2. Gene prediction

[0125] The short sequence assembly software SOAPdenovo2 (http: / / soap.genomics.org.cn / ) was used to perform de novo assembly on the sequencing data. The Maker2 software was used for gene prediction of fungi, and the Barrnap 0.4.2 and tRNAscan-SE v1.3.1 software were used to predict the rRNA and tRNA contained in the genome.

[0126] The total length of the whole genome of the strain LB-002 is 53,325,194 bp, the G+C (%) content is 55.01%, 309 scaffolds were assembled, 15,156 coding genes were predicted, the total length of the genes is 30,560,308 bp, accounting for 57.31% of the genome, and the average length is 2,016.38 bp. 564 tRNAs and 74 rRNAs (73 5S rRNAs and 1 18S rRNA) were predicted.

[0127] 3. Gene function annotation

[0128] The predicted coding genes were compared and functionally annotated with the databases COG, GO, KEGG, and the Carbohydrate-Active Enzyme database (CAZy). The results are as follows:

[0129] The coding genes of the genome of strain LB-002 were analyzed by alignment in the GOG database. A total of 8,419 coding genes were annotated. When analyzed by alignment in the GO database, a total of 9,282 coding genes were annotated. When analyzed by alignment in the KEGG database, a total of 6,599 coding genes were annotated to 6 major metabolic pathways including metabolism, cellular processes, genetic information processing, environmental information processing, and organismal systems. Among them, there were 5,188 genes in metabolism, and 721 and 565 genes were annotated to carbohydrate metabolism and amino acid metabolism respectively.

[0130] The genomic data of strain LB-002 was analyzed by alignment in the CAZy database, and 712 genes were annotated. Among them, the glycoside hydrolase (GH) genes were the most (a total of 304 glycoside hydrolase GH genes were annotated. The results are as Figure 3 shown). This result shows the diversity and adaptability of strain LB-002 in the utilization of carbohydrates. The 304 glycoside hydrolase genes annotated in strain LB-002 belong to 69 families and 4 subfamilies, and 11 of them are β-glucosidase genes. This annotation result indicates that strain LB-002 has strong potential for producing β-glucosidase.

[0131] Example 3

[0132] This example is used to illustrate the β-glucosidase production ability of the dermatophyte Saccothecium sp. LB-002 provided by the present invention.

[0133] 1. Fermentation for enzyme production and preparation of crude enzyme solution

[0134] An appropriate amount of mycelium was scraped from the slant medium with an inoculation loop into a 250 mL conical flask containing 50 mL of liquid fermentation medium (the initial content of Saccothecium sp. in the medium after inoculation was about 1.5×10 7 CFU / mL), and cultured with shaking at 30 °C and 150 rpm for 5 d. After the culture was completed, 2 mL of the fermentation broth was centrifuged at 12,000 rpm for 15 min, and the obtained supernatant was the crude enzyme solution.

[0135] 2. Determination of the enzyme activity of β-glucosidase in the crude enzyme solution

[0136] 2.1. Preparation of the p-nitrophenol (pNP) standard curve

[0137] Weigh 139 mg of pNP, dissolve it in distilled water and make up the volume to 100 mL to obtain a stock solution of 0.01 mmol / mL. Dilute it with distilled water to prepare gradient dilution standard solutions of pNP with final concentrations of 0 μmol / mL (i.e., pure water), 0.2 μmol / mL, 0.4 μmol / mL, 0.6 μmol / mL, 0.8 μmol / mL, 1 μmol / mL, 1.2 μmol / mL, and 1.4 μmol / mL.

[0138] Take 200 μL of each of the above standard solutions and mix them with 2 mL of citric acid - disodium hydrogen phosphate buffer. Then add 2 mL of 1 mol / L Na 2 CO 3 solution to each mixture. Mix well and measure the absorbance at 410 nm using a microplate reader. Plot a standard curve based on the measured values.

[0139] The standard curve obtained by the above method is y = 0.4156x + 0.0734, where R 2 = 0.9992. Here, x is the pNP concentration (μmol / mL) and y is the absorbance value corresponding to this concentration.

[0140] 2.2. Determination of enzyme activity

[0141] Enzyme activity unit (U): Under the condition of 50 °C, the amount of enzyme required to hydrolyze pNPG to generate 1 μmol of pNP per minute is defined as one enzyme activity unit.

[0142] Take 2 mL of citric acid - disodium hydrogen phosphate buffer and 0.5 mL of 5 mmol / L pNPG solution and add them to a test tube. After preheating in a water bath, add 0.5 mL of crude enzyme solution and incubate in a 50 °C water bath for 30 min. Then add 2 mL of 1 mol / L Na 2 CO 3 solution to terminate the reaction. Measure the OD 410 of the reaction solution (using the crude enzyme solution inactivated by boiling for 10 min as a blank control, repeating 3 times), and then calculate the enzyme activity of β - glucosidase in the crude enzyme solution according to the standard curve drawn in step 2.1.

[0143] The results show that the enzyme activity of β - glucosidase produced by strain LB - 002 is 10.1 ± 0.8 mU / mL.

[0144] Example 4

[0145] This example is used to illustrate the results of virtual binding analysis of β - glucosidase produced by Trichophyton concentricum LB - 002 provided by the present invention.

[0146] 1. Prediction of the tertiary structure of β - glucosidase protein produced by strain LB - 002

[0147] Use the web interface provided by AlphaFold Colab (https: / / colab.research.google.com / github / sokr ypton / ColabFold / blob / main / AlphaFold2.ipynb) to call AlphaFold2 to predict the protein structure, and use the pLDDT (protein local distance difference test) and pTM (predicted TM score) information output by AlphaFold2 to measure the accuracy and reliability of the predicted protein structure.

[0148] Predict the tertiary structures of the proteins encoded by 11 β-glucosidase genes obtained from the genomic analysis of strain LB-002 in Example 2, and the structure prediction scores are shown in Table 2.

[0149] Table 2

[0150]

[0151] It can be seen from the data in Table 2 that the predicted pLDDTs of the β-glucosidase proteins encoded by the three enzyme genes gene13502, gene04198, and gene11193 are all higher than 80, indicating that the predicted local protein structure has good quality, but the pTM is relatively low. The predicted pLDDTs of the remaining 8 enzyme proteins are all higher than 90, indicating that the predicted local protein structure is very accurate, but the pTMs of the β-glucosidase proteins encoded by gene10548 and gene04310 are relatively low. In addition, the predicted pLDDTs and pTMs of the other 6 enzyme proteins are higher than 90 and 0.9 respectively, indicating that they have good structural stability and accuracy.

[0152] 2. Molecular docking of β-glucosidase encoded by strain LB-002 gene with ginsenoside Rb1 and Rg1 molecules

[0153] Select the enzyme proteins with predicted pLDDT scores higher than 90 and pTM scores higher than 0.9 as receptors, and the 3D structures of ginsenoside Rb1 and Rg1 molecules as ligands respectively, and use AutoDock Vina software to conduct molecular docking studies.

[0154] The docking results of the β-glucosidase proteins encoded by 6 genes with ginsenoside Rb1 and Rg1 molecules are shown in detail in Table 3. The 3D docking schematic diagram is as Figure 4 shown.

[0155] Table 3

[0156]

[0157] From the above results, it can be seen that the docking binding energy between the β-glucosidase protein encoded by the β-glucosidase gene of strain LB-002 and ginsenoside Rb1 and Rg1 molecules is lower than -7kcal / mol; among them, the lowest binding energy between the β-glucosidase protein encoded by the gene07544 gene and ginsenoside Rb1 and Rg1 molecules is -9.3kcal / mol.

[0158] The virtual docking results showed that the β-glucosidase protein of strain LB-002 could effectively bind to ginsenosides Rb1 and Rg1, and had the potential to transform the corresponding ginsenosides.

[0159] Through literature comparison, it can be seen that the minimum binding energy of the β-glucosidase encoded by the β-glucosidase gene of the strain LB-002 and the ginsenoside Rb1 molecule docking is generally lower than the binding energy values ​​of the reported β-glucosidase, indicating that the β-glucosidase encoded by the β-glucosidase gene of the tinea cysticercus LB-002 and the ginsenoside Rb1 have higher affinity potential and catalytic conversion potential; in addition, there has been no report on the molecular docking of the β-glucosidase of the tinea cysticercus and the ginsenoside Rg1 in the field.

[0160] Example 5

[0161] This example is used to illustrate the ability of the β-glucosidase produced by the tinea cysticeris LB-002 provided by the present invention to transform ginsenosides.

[0162] In order to verify whether the β-glucosidase produced by strain LB-002 has the actual ability to transform ginsenosides Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1, the enzyme solution after fermentation of the strain was used to transform ginsenosides Rb1 and Rg1, and the transformation products were preliminarily detected by thin layer chromatography (TLC), and then further analyzed by mass spectrometry (MS) and high performance liquid chromatography (HPLC). The specific methods are as follows:

[0163] 1. Inoculation of strains

[0164] Under sterile conditions, use a 0.7 cm diameter puncher to take 3 bacterial cakes and inoculate them into 50 mL of fermentation medium after cooling by burning with the outer flame of an alcohol lamp. The inoculated medium is placed in a constant temperature shaker at 28°C and 150 rpm for 7 days, and the growth status of the microorganisms during the fermentation process is monitored regularly.

[0165] 2. Preparation of crude enzyme solution

[0166] After the culture was completed, the fermentation broth was centrifuged at 12,000 rpm for 15 min, and the supernatant was filtered using a sterile filter (d=0.22 μm) to remove the bacteria and other impurities, and then used for ginsenoside substrate conversion.

[0167] 3. Ginsenoside transformation and sample preparation

[0168] Take 20 mL of the above enzyme solution and mix it with 2 g of ginsenosides (1 g each of diol-group ginsenosides and triol-group ginsenosides), place it in a constant-temperature shaker at 30 °C and 150 rpm for reaction for 24 h, with no addition of enzyme solution as the blank control.

[0169] After the reaction is completed, filter the reaction solution. Add 20 mL of 95% ethanol to the filter residue, soak for 24 h and then filter again. Collect the filtrate, and then make the volume of the filtrate constant to 20 mL again. Take out 10 mL of the filtrate and concentrate it by rotary evaporation, and precisely pipette 1 mL of methanol to dissolve it.

[0170] 4. Detection of transformation products by thin-layer chromatography

[0171] Spot the sample on a G60 silica gel plate (5 cm. For detection of transformation), dry it with a hair dryer and then place it in the developing agent to allow upward development. The developing agent is chloroform:methanol:water = 87:13:1 (v / v / v). After development is completed, dry it with a hair dryer and place it in an iodine tank to make the ginsenosides develop color. Compare the unknown ginsenoside products (i.e., the ginsenoside compounds contained in the products obtained from the treatment in step 1.3) with the known-structure ginsenoside standards (purchased from Shanghai Standard Technology Service Co., Ltd.), and preliminarily identify the ginsenoside products by the retention factor (Rf).

[0172] By TLC detection, compare the unknown ginsenoside products with the known-structure ginsenoside standards. The results are as Figure 5 shown. The S1 band and the S2 band correspond to the ginsenoside CK and 20(S)-Rh1 standards respectively. It can be seen from this that the strain LB-002 can transform ginsenoside Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1 respectively.

[0173] 5. Analysis of transformation products by mass spectrometry

[0174] Mass spectrometry conditions: Agilent HPLC1100 MSD / TOF, electrospray ionization (ESI) ion source, positive or negative ion mode, mobile phase methanol / water (containing 0.1% formic acid, 80:20, v / v), flow rate 0.2 mL / min, ion source temperature 325 °C, drying gas flow rate 7 L / min, fragmentation voltage 150 V.

[0175] Scrape the bands at positions S1, S2 and S3 in Figure 5 , dissolve them with 1 mL of methanol, then filter to remove the silica gel with a filter (d = 0.45 μm), collect the filtrate and perform on-machine testing.

[0176] The obtained mass spectrometry diagrams are as Figure 6A and Figure 6BAs shown, it can be seen from the figure that ginsenoside CK and 20(S)-Rh1 are detected in the form of [M+Na] + ions, and [M+Na:645.43] of band S1 + , indicating that its molecular formula is C 36 H 62 O 8 , with a molecular weight of 622.43; [M+Na:661.43] of band S2 + , indicating that its molecular formula is C 36 H 62 O 9 , with a molecular weight of 638.43. Since the molecular formulas and molecular weights of bands S1 and S2 match those of ginsenoside CK and 20(S)-Rh1, the compounds in these two bands are respectively identified as ginsenoside CK and 20(S)-Rh1.

[0177] 6. Determination of the concentration of transformation products by high performance liquid chromatography

[0178] Chromatographic conditions: Agilent HPLC1100, C18 chromatographic column, 4.6, ent HP; flow rate 1 mL / min; column temperature 30 m; wavelength 203 nm; mobile phase acetonitrile / water; injection volume 20 μL.

[0179] Preparation of the mixed standard solution of ginsenoside CK and 20(S)-Rh1 reference substances: Weigh accurately 0.50 mg of ginsenoside CK and 20(S)-Rh1 reference substances respectively into the same sample bottle, and accurately pipette 1 mL of methanol with a pipette gun to dissolve and shake well to obtain a mixed standard solution of reference substances with a concentration of 0.50 mg / mL.

[0180] Determination: Filter the concentrated filtrate dissolved in methanol in step 1.3 with a 0.45 μm microporous filter membrane to obtain the sample solution. Under the above chromatographic conditions, measure the peak areas of ginsenoside CK and Rh1, and calculate the concentrations (mg / mL) of ginsenoside CK and 20(S)-Rh1 in the sample through the single-point quantitative formula.

[0181] The chromatograms of the reference substances and the sample obtained by HPLC test are as Figure 7 shown. The peak areas of ginsenoside CK and 20(S)-Rh1 in the reference substances are 4983.68213 and 4338.94727 (mAU*s) respectively. Comparing the chromatogram and retention time of the reference substances, the peak areas of the transformation products CK and 20(S)-Rh1 chromatographic peaks are 61344.1 and 48063.6 (mAU*s) respectively. It is calculated that the concentrations of ginsenoside CK and 20(S)-Rh1 are 6.15 mg / mL and 5.54 mg / mL respectively.

[0182] The above experimental results verified that the β-glucosidase produced by Plectosphaerella sp. LB-002 could convert ginsenoside Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1, respectively.

[0183] In addition, after directly adding diol-type ginsenosides and triol-type ginsenosides into the fermentation medium, Plectosphaerella sp. LB-002 was cultured (the components of the medium were as follows: K 2 HPO 4 0.1 g, MgSO 4 ·7H 2 O 0.5 g, NaNO 3 3 g, FeSO 4 ·7H 2 O 0.1 g, yeast extract 1 g, diol-type ginsenosides and triol-type ginsenosides (including 10 g of Rb1 and 10 g of Rg1 each), made up to 1 L with deionized water, natural pH; the culture temperature was 28 °C, and it was cultured in a constant temperature shaker at 150 rpm for 2 weeks), and mass spectrometry detection was performed on the obtained culture solution. It was found that the diol-type ginsenosides and triol-type ginsenosides originally added in the medium were converted into rare ginsenosides CK and 20(S)-Rh1 (the mass spectrometry patterns were similar to Figure 6A and 6B ). This was the first discovery in this field that strains of the genus Plectosphaerella had the ability to convert ginsenoside Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1, respectively.

[0184] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. One plant Plectosphaerella oligotrophica , characterized in that, Said Plectosphaerella oligotrophica The deposit number is CGMCC No.41079.

2. A bacterial agent, characterized in that The active ingredients of the bacterial agent include the Plectosphaerella oligotrophica .

3. The method according to claim 1 Plectosphaerella oligotrophica Or use of the bacterial agent according to claim 2 in the preparation of β-glucosidase.

4. A method for preparing β-glucosidase, characterized in that: The method comprises the step of Plectosphaerella oligotrophica Or the bacterial agent according to claim 2 is contacted with a fermentation substrate and a first fermentation culture is performed.

5. The method according to claim 4, wherein: The first fermentation culture system comprises a fermentation substrate and the Plectosphaerella oligotrophica or the bacterial agent according to claim 2, wherein the fermentation substrate is provided by the first liquid culture medium; And / or, the temperature of the first fermentation culture is 25-35°C; And / or, the method further comprises the step of separating β-glucosidase from the first fermentation culture product after the first fermentation culture is completed.

6. The method according to claim 5, wherein: The initial pH value of the first liquid culture medium is not less than 5; and / or, the first liquid culture medium comprises cellulose; And / or, the first fermentation culture conditions further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the first fermentation culture system, Plectosphaerella oligotrophica The initial content is not less than 10 7 CFU / mL; fermentation culture time is not less than 48h; And / or, the first fermentation culture is carried out under shaking conditions.

7. The method according to claim 6, wherein: The initial pH value of the first liquid culture medium is 5.5-6.5; and / or, the final concentration of cellulose in the first liquid culture medium is not less than 3 g / L; And / or, the first fermentation culture conditions further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the first fermentation culture system, Plectosphaerella oligotrophica The initial content is 10 7 -10 9 CFU / mL; fermentation culture time is 50-180h; And / or, the shaking speed is 100-150 rpm.

8. The method according to claim 7, wherein: The final concentration of cellulose in the first liquid culture medium is 3-15 g / L.

9. The method according to any one of claims 5 to 8, wherein: The first liquid culture medium also includes yeast extract and a trace element source.

10. The method according to claim 9, wherein: The trace element source provides at least one element of K, Mg, Na and Fe to the first liquid culture medium.

11. The method according to claim 1 Plectosphaerella oligotrophica Or use of the bacterial agent according to claim 2 in the production of rare ginsenoside CK and / or Rh1.

12. A method for preparing rare ginsenosides, characterized in that: The method comprises: (A) Panaxadiol saponins and the Plectosphaerella oligotrophica or the bacterial agent according to claim 2, and then subjected to a second fermentation culture to obtain rare ginsenoside CK; and / or, (B) Panaxatriol saponins and the Plectosphaerella oligotrophica Or the bacterial agent according to claim 2, and carry out a third fermentation culture to obtain the rare ginsenoside Rh1.

13. The method according to claim 12, wherein: In method (A), the second fermentation culture system includes a culture medium and the Plectosphaerella oligotrophica or the bacterial agent, wherein the culture medium is a second liquid culture medium; And / or, in mode (A), the temperature of the second fermentation culture is 25-32°C; And / or, in aspect (A), the method further comprises the step of isolating ginsenoside CK from the second fermentation product after the second fermentation is completed.

14. The method according to claim 13, wherein: The second liquid culture medium includes panaxadiol saponins; And / or, the second fermentation culture conditions further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the second fermentation culture system, Plectosphaerella oligotrophica The initial content is not less than 10 7 CFU / mL; fermentation culture time is not less than 150h; And / or, the second fermentation culture is carried out under shaking conditions.

15. The method according to claim 14, wherein: The final concentration of ginsenosides in the second liquid culture medium is not less than 5 g / L; And / or, the second fermentation culture conditions further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the second fermentation culture system, Plectosphaerella oligotrophica The initial content is 10 7 -10 10 CFU / mL; fermentation culture time is 200-400h; And / or, the shaking speed is 100-150 rpm.

16. The method according to claim 15, wherein: The final concentration of panaxadiol saponins in the second liquid culture medium is 5-50 g / L.

17. The method according to claim 16, wherein: The ginsenosides of the ginsenoside group include ginsenoside Rb1.

18. The method according to any one of claims 13 to 16, wherein: The second liquid culture medium further comprises yeast extract and a trace element source.

19. The method according to claim 18, wherein: The trace element source provides at least one element of K, Mg, Na and Fe to the second liquid culture medium.

20. The method according to claim 12, wherein: In mode (B), the third fermentation culture system includes a culture medium and the Plectosphaerella oligotrophica or the bacterial agent, wherein the culture medium is a third liquid culture medium; And / or, the temperature of the third fermentation culture is 25-32°C; And / or, the method further comprises the step of separating ginsenoside Rh1 from the third fermentation culture product after the third fermentation culture is completed.

21. The method according to claim 20, wherein: The third liquid culture medium includes panaxatriol saponins; And / or, the conditions of the third fermentation culture further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the third fermentation culture system, Plectosphaerella oligotrophica The initial content is not less than 10 7 CFU / mL; fermentation culture time is not less than 150h; And / or, the third fermentation culture is carried out under shaking conditions.

22. The method according to claim 21, wherein: The final concentration of panaxatriol saponins in the third liquid culture medium is not less than 5 g / L; And / or, the conditions of the third fermentation culture further include: Plectosphaerella oligotrophica Or the amount of the bacterial agent is such that in the third fermentation culture system, Plectosphaerella oligotrophica The initial content is 10 7 -10 10 CFU / mL; fermentation culture time is 200-400h; And / or, the shaking speed is 100-150 rpm.

23. The method according to claim 22, wherein: The final concentration of panaxatriol saponins in the third liquid culture medium is 5-50 g / L.

24. The method according to claim 23, wherein: The ginsenosides of the ginsenoside group include ginsenoside Rg1.

25. The method according to any one of claims 20 to 24, wherein: The third liquid culture medium also includes yeast extract and a trace element source.

26. The method according to claim 25, wherein: The trace element source provides at least one element of K, Mg, Na and Fe to the third liquid culture medium.

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