阅读说明：本技术 猴头菇菌株及其培养方法、猴头菌-人参双向固体发酵方法和高效转化稀有人参皂苷的方法 (Hericium erinaceus strain and culture method thereof, hericium erinaceus-ginseng bidirectional solid fermentation method and method for efficiently converting rare ginsenoside ) 是由 王欢 陈长宝 王淑敏 张金亭 唐敏 于 2021-07-13 设计创作，主要内容包括：本发明涉及发酵技术领域,特别涉及猴头菇菌株及其培养方法、猴头菌-人参双向固体发酵方法和高效转化稀有人参皂苷的方法。该猴头菇菌株的保藏编号为CGMCC No.22450。本发明的猴头菇菌株可以高效转化人参中稀有人参皂苷。(The invention relates to the technical field of fermentation, in particular to a hericium erinaceus strain and a culture method thereof, a hericium erinaceus-ginseng bidirectional solid fermentation method and a method for efficiently converting rare ginsenoside. The preservation number of the hericium erinaceus strain is CGMCC No. 22450. The hericium erinaceus strain can efficiently convert rare ginsenoside in ginseng.)

1. A hericium erinaceus strain is characterized in that the preservation number is CGMCC No. 22450.

2. The method for culturing the hericium erinaceus strain according to claim 1, characterized by comprising the steps of:

reviving Hericium erinaceus strain, inoculating to slant culture medium, and slant culturing;

inoculating the hericium erinaceus strain subjected to slant culture into a first-stage liquid culture medium for first-stage seed culture;

inoculating the hericium erinaceus strain subjected to the primary seed culture into a secondary liquid culture medium for secondary seed culture.

3. The culture method according to claim 2, wherein the slant culture medium is PDA solid culture medium; the temperature of slant culture is 24-28 ℃, and the time is 5-20 hours.

4. The culture method according to claim 2, wherein the primary liquid medium is formulated as: 10-30 g of glucose, 5-15 g of peptone and KH₂PO₄ 1～5g，MgSO₄·7H₂0.5-1.5 g of O and 1L of water;

the conditions for the first-stage seed culture are as follows: culturing for 10-20 days at 24-28 ℃ and 150-200 rpm/min in dark condition.

5. The culture method according to any one of claims 2 to 4, wherein the secondary liquid medium has a formulation of: 20-30 g of glucose, 0.1-0.5 g of peptone, 0.1-0.5 g of yeast powder and KH₂PO₄ 2～8g，MgSO₄·7H₂0.5-1.5 g of O and 1L of water;

the temperature of the secondary seed culture is 24-28 ℃, 150-200 rpm/min, and the secondary seed culture is carried out for 5-10 days under a dark condition.

6. A hericium erinaceus-ginseng bidirectional solid fermentation method is characterized by comprising the following steps:

mixing Ginseng radix powder with water, and sterilizing to obtain Ginseng radix matrix;

inoculating the Hericium erinaceus strain of claim 1 to a ginseng medicinal material substrate, and performing fermentation culture.

7. The hericium erinaceus-ginseng bi-directional solid fermentation method according to claim 6, wherein the mass percentage of the water to the ginseng powder is 30-50%;

the inoculation amount of the hericium erinaceus strain is 10-30% (W/V) of a second-level hericium erinaceus strain seed culture solution;

the conditions of the fermentation culture are as follows: the temperature is 23-28 ℃, and the time is 30-50 days.

8. A mycoplasm prepared by the hericium erinaceus-ginseng bidirectional solid fermentation method of claim 6 or 7.

9. A food product comprising the mycoplasm of claim 8 and a dietetically acceptable adjuvant.

10. High-efficiency conversion of rare ginsenoside Rg₃And Rh₁The method is characterized by comprising the following steps:

mixing Ginseng radix powder with water, and sterilizing to obtain Ginseng radix matrix;

inoculating Hericium erinaceus strain of claim 1 to Ginseng radix matrix, fermenting, culturing, and purifying to obtain Rg₃And Rh₁。

Technical Field

The invention relates to the technical field of fermentation, in particular to a hericium erinaceus strain and a culture method thereof, a hericium erinaceus-ginseng bidirectional solid fermentation method and a method for efficiently converting rare ginsenoside.

Background

Ginseng (panaxginng c.a.mey) is a perennial root negative herbaceous dicotyledon, is a traditional and valuable Chinese medicinal material in China, is mainly produced in the northeast of China, and is praised as 'king of all grass'. According to records in Chinese pharmacopoeia, ginseng has multiple medicinal effects of reinforcing primordial qi, recovering pulse, relieving depletion, tranquilizing and the like. The research shows that the pharmacological activity of ginseng is mainlyIs dependent on ginsengenin and rare ginsenosides Rd and Rg₁、 Rg₃、Rh₁、Rh₂And the like. However, the content of the natural saponins is very low, the extraction and separation are complex, the yield is very low, and the industrial production cannot be carried out, so that the conversion of the ginsenoside into rare saponin has profound significance.

The ginseng solid fermentation technology takes ginseng medicinal materials or dregs of a decoction as a medicinal substrate, hydrolyzes cellulose substances in the medicinal substrate through an abundant enzyme system of microorganisms, and provides energy for the growth and metabolism of the microorganisms. The ginsenoside is structurally modified through the physiological metabolism and biochemical reaction process of microbes to produce new bioactive components and new medicinal effect. By the microbial conversion, the sugar chain structure of the saponin can be changed rapidly and effectively, so that the prototype ginsenoside is converted into the rare ginsenoside, and the biological activity of the ginsenoside is improved. For example, Lactobacillus plantarum KCCM 11613P can convert ginsenoside Rb₂、Rb₃Converted into ginsenoside Rd, Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 can be used for treating ginsenoside Rb₁Rc and Rb₂And (3) carrying out conversion to obtain the specific deglycosylated ginsenoside Rd as a final metabolite. Hasegawa et al can separate and screen bacterial strain Prevotella oris from intestinal anaerobic microorganisms to obtain various ginsenosides (such as Rb)₁Rc, Rd, etc.) into the rare ginsenoside component. Qin et al expressed the diol-type ginsenoside Rb in Paecilomyces Bainier sp.229 by beta-glucosidase₁Converted into the rare ginsenoside CK. Choi from the viewpoint of protein engineering converts ginsenoside Rc into rare ginsenoside CK by hydrolyzing specific glycosides bound to ginsenoside with specific enzymes in microorganisms.

The ginseng medicinal material after the microbial solid fermentation has the advantages that the fermentation product is more beneficial to human body digestion and absorption, the original toxicity of the medicinal material is reduced, and the medicinal value of the medicinal material is greatly improved. For example, rare ginsenoside Rg obtained by conversion of prototype ginsenoside₂、Rg₃Has more effective antianxiety, antiinflammatory, and anticancer activities, and is rich in rare saponins CK and F₁₂Has antitumor, antimutagenic, antiinflammatory, hepatoprotective, and asthma effectsShows good pharmacodynamic activity and has higher pharmacological activity compared with the prototype ginsenoside. The Wanghoufeng etc. in the research of chemical composition change in the process of bidirectional solid fermentation of ginseng, it is found that a large amount of rare ginsenoside is detected in the ginseng medicinal mycoplasm, and the ginsenoside Rg is guessed₁Re and Rb can be converted into rare ginsenoside Rg₃Rh, etc. Among them, the biological conversion technology of bidirectional fermentation can greatly improve the conversion efficiency without producing harmful by-products, compared with the chemical conversion method. For example, ginsenoside Rd is converted into a large amount of rare ginsenoside through deglycosylation.

Hericium erinaceus (Hericium erinaceus) belonging to the order of the Agaricales (Russules) of the Basidiomycota (Basidiomycota) Hymenomycetes (Agaricaomycetes) and Hericium (Hericium), fruiting bodies are annual, petiolless or short-lived lateral petioles, fresh fleshy, later-stage soft leathery, odorless and tasteless, and dried cheese or cork suppository has slight rancid smell. The growth process of the plant cell is accompanied with the generation of secondary metabolites such as steroid compounds, alkaloid compounds, aromatic compounds, terpenoid compounds, fatty acid and lipid thereof, and the like, and the plant cell has certain effects of oxidation resistance, antibiosis, aging resistance, tumor resistance, neuroprotection, inflammation resistance and the like. The potential medicinal value of these secondary metabolites of Hericium erinaceus has also received a great deal of attention.

Many microorganisms in nature can grow on solid substrates, particularly filamentous fungi, which can grow in the absence of water in adherence to solid pharmaceutical substrates. Hericium erinaceus, as a precious large-scale edible and medicinal fungus in nature, can be subjected to bidirectional fermentation with solid substrates such as medicinal materials or herb residues. In the solid fermentation process, a plurality of secondary metabolites generated by the metabolism of the fungi can modify the material basis of the solid matrix, thereby improving the content of active substances in the matrix. For example, Liuming and the like are subjected to bidirectional fermentation by utilizing hericium erinaceus and a wheat solid culture medium, and the result shows that compared with a control group, the hericium erinaceus fermentation group obviously improves the content of protein in a wheat matrix. After the hericium erinaceus and the soybean solid culture medium are fermented, the contents of total flavone and total triterpene are reduced, and the contents of total polyphenol and anthocyanin are increased. The Hericium erinaceus and the corn protein powder are fermented, and the antioxidant activity of the fermented product is obviously improved.

The hericium erinaceus solid fermentation process simulates living conditions of many higher filamentous fungi, and in the process, the hericium erinaceus can enhance the pharmacological activity of a fermentation product by changing the component structure of active substances. Compared with fungal spores produced by liquid fermentation, the biological enzyme, fungal spores and metabolites under the culture condition of solid fermentation have stronger environmental adaptability. The previous research shows that the spores produced by the hericium erinaceus through solid fermentation have higher stability, stronger drying resistance and higher germination rate in a longer time after freeze-drying.

Saponins such as Rb with high content in Ginseng radix₁，Rb₂Rc, Rd, Re and Rg₁And the like. Rare ginsenoside such as F₁、F₂、Rg₃、Rh₁、Rh₂CK, CY and CMc, etc., are contained in natural ginseng in low content, but the pharmacological activity is often better. Since rare ginsenosides have the same parent nucleus structure as high-content saponins except for the number of sugar residues, rare ginsenosides can be prepared by hydrolyzing the sugar residues in high-content saponins. Common methods include acid hydrolysis, high temperature and pressure, microbial transformation, and enzymatic transformation. The development and application of the chemical methods such as acid hydrolysis, high temperature and high pressure are limited due to the defects of severe reaction conditions, low conversion efficiency, more byproducts, difficult separation and purification and the like. The biotransformation method has become the most potential method due to mild reaction conditions, high transformation efficiency and good specificity, and has achieved certain achievements in the field at home and abroad. Bioconversion generally includes microbial conversion and enzymatic conversion.

The microorganisms mainly used for ginsenoside conversion at present are as follows:

1. human intestinal bacteria, including the genera polymorphous, bifidobacterium, clostridium, lactobacillus, etc., have received much attention from researchers in degrading ginsenosides because they can metabolize the oral ginsenosides and can be safely used in foods. The microorganisms can grow in an anaerobic environment using ginsenoside as a carbon source. However, the transformation of ginsenoside by using intestinal bacteria also has the problems of high culture medium cost, low yield and the like.

2. The microorganism isolated from the soil for ginseng planting includes Curvularia lunata, Aspergillus oryzae, Absidia lanchoe, Fusarium saccharum, Acremonium erectum, Paecilomyces, etc., and the bacteria include Bacillus megaterium, Sphingomonas echinocandii, Aureobasidium, Microsporum, etc., and these fungi and bacteria are used for transforming ginsenoside. Compared with intestinal bacteria, soil microorganisms can grow rapidly in a common culture medium, and the method has obvious economic advantages in the conversion of ginsenoside. However, soil microorganisms must be identified as safe to be used in the food industry. Although non-pathogenic microorganisms can be applied to the food industry, the microbial transformation method still has the disadvantages of poor selectivity, low yield and the like, and enzymes secreted by the microorganisms and involved in degradation are difficult to determine.

And the traditional Chinese medicines of red yeast rice, phellinus igniarius, lactobacillus plantarum, saccharomycetes and the like are used for fermentation and transformation, at present, the bacteria, the saccharomycetes and the mould are mainly used for biotransformation of ginsenoside, and the research of using hericium erinaceus strain and ginseng for co-fermentation transformation of rare ginsenoside by using a two-way solid fermentation technology is not available.

Disclosure of Invention

In view of the above, the invention provides a hericium erinaceus strain and a culture method thereof, a hericium erinaceus-ginseng bidirectional solid fermentation method and a method for efficiently converting rare ginsenoside. The Hericium erinaceus strain can efficiently convert rare ginsenoside in Ginseng radix.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a hericium erinaceus strain, the preservation number of which is CGMCC No. 22450.

The invention also provides a culture method of the hericium erinaceus strain, which comprises the following steps:

reviving Hericium erinaceus strain, inoculating to slant culture medium, and slant culturing;

inoculating the hericium erinaceus strain subjected to slant culture into a first-stage liquid culture medium for first-stage seed culture;

inoculating the hericium erinaceus strain subjected to the primary seed culture into a secondary liquid culture medium for secondary seed culture.

Preferably, the slant culture medium is PDA solid culture medium.

Preferably, the temperature of the slant culture is 24-28 ℃ and the time is 5-20 hours.

In the specific embodiment provided by the present invention, the temperature of the slant culture is 26 ℃.

Preferably, the formula of the primary liquid culture medium is as follows: 10-30 g of glucose, 5-15 g of peptone and KH₂PO₄ 1～5g，MgSO₄·7H₂0.5-1.5 g of O and 1L of water;

preferably, the conditions for the primary seed culture are: culturing for 10-20 days at 24-28 ℃ and 150-200 rpm/min in dark condition.

In the specific embodiment provided by the invention, the formula of the primary liquid culture medium is as follows: glucose 20g, peptone 10g, KH₂PO₄ 2g，MgSO₄·7H₂O1 g and water 1L.

In the specific embodiment provided by the invention, the conditions for primary seed culture are as follows: 26 ℃, 160rpm/min, dark conditions, cultured for 15 days.

Preferably, the formula of the secondary liquid culture medium is as follows: 20-30 g of glucose, 0.1-0.5 g of peptone, 0.1-0.5 g of yeast powder and KH₂PO₄ 2～8g，MgSO₄·7H₂0.5-1.5 g of O and 1L of water;

preferably, the temperature of the secondary seed culture is 24-28 ℃, 150-200 rpm/min, and the secondary seed culture is carried out for 5-10 days under dark conditions.

Preferably, the inoculation amount of the hericium erinaceus strain after the first-stage seed culture is 1 (10-30) (V/V).

In the specific embodiment provided by the invention, the inoculation amount of the hericium erinaceus strain after the first-stage seed culture is 1:20 (V/V).

In the specific embodiment provided by the invention, the formula of the secondary liquid culture medium is as follows: 22g of glucose, 0.2g of peptone, 0.2g of yeast powder and KH₂PO₄ 5g，MgSO₄·7H₂O1 g, water 1L;

in the specific embodiment provided by the invention, the conditions for secondary seed culture are as follows: 26 ℃, 160rpm/min, dark conditions, cultured for 7 days.

The invention also provides a hericium erinaceus-ginseng bidirectional solid fermentation method, which comprises the following steps:

mixing Ginseng radix powder with water, and sterilizing to obtain Ginseng radix matrix;

inoculating Hericium erinaceus strain to Ginseng radix matrix, and fermenting and culturing.

Preferably, the mass percentage of the water and the ginseng powder is 30-50%.

In the specific embodiment provided by the invention, the mass percentage of the water and the ginseng powder is 40%.

Preferably, the fineness of the ginseng powder is 10 to 20 mesh.

Preferably, the sterilization is autoclaving and/or uv sterilization.

Preferably, the inoculation amount of the hericium erinaceus strain is 10-30% (W/V) of the second-level hericium erinaceus seed culture solution.

In the specific embodiment provided by the invention, the inoculation amount of the hericium erinaceus strain is 20% (W/V) of the secondary hericium erinaceus strain seed culture solution.

Preferably, the conditions of the fermentation culture are: the temperature is 23-28 ℃, and the time is 30-50 days.

After solid fermentation, the co-fermentation product (ginseng medicinal mycoplasm) can be made into products (medicines, health products, functional foods and foods) with similar effects of ginseng and hericium erinaceus, such as tablets, granules, effervescent tablets, pills, oral liquids and the like.

The invention also provides the mycoplasm prepared by the hericium erinaceus-ginseng bidirectional solid fermentation method.

The invention also provides a food which comprises the mycoplasm and auxiliary materials acceptable in food science.

The invention also provides a method for efficiently converting rare ginsenoside Rg₃And Rh₁In the method of (a) to (b),the method comprises the following steps:

mixing Ginseng radix powder with water, and sterilizing to obtain Ginseng radix matrix;

inoculating Hericium erinaceus strain to Ginseng radix matrix, fermenting, culturing, and purifying to obtain Rg₃And Rh₁。

The invention provides a hericium erinaceus strain and a culture method thereof, a hericium erinaceus-ginseng bidirectional solid fermentation method and a method for efficiently converting rare ginsenoside. The preservation number of the hericium erinaceus strain is CGMCC No. 22450. The invention has the following advantages:

1. hericium erinaceus is a traditional edible and medicinal fungus in China, has the functions of benefiting five internal organs, invigorating spleen and benefiting stomach, and has good safety and efficacy when being used as food and medicine.

2. According to the invention, hericium erinaceus and 4-year-old ginseng are utilized for bidirectional solid fermentation for the first time, so that the contents of rare ginsenoside Rg3 and Rh1 are increased, and the research is not reported yet.

3. The process can efficiently convert rare ginsenoside Rg₃And Rh₁。

Biological preservation Instructions

CCUCM-HE001, named after classification: hericium erinaceus (Hericium erinaceus) is preserved in China general microbiological culture Collection center (CGMCC) at 10.6.1.1.Navy, the address of the CGMCC is No. 3 of the national institute of microbiology, national institute of sciences, North Chen Lu No. 1, Beijing, and the preservation number is CGMCC No. 22450.

Drawings

FIG. 1 strain CCUCM-HE-001 colony morphology;

FIG. 2 strain CCUCM-HE-001 hyphal morphology;

FIG. 3 ITS phylogenetic Tree of strain CCUCM-HE 001;

FIG. 4 shows the comparison of the content of diol-type and oleanolic acid-type ginsenosides; note: represents a very significant difference compared to day 0 (P < 0.001); the # indicates that the difference is very significant (P is less than 0.001) when the fermentation is carried out for 30 days compared with 40 days;

FIG. 5 comparison of the content of triol-type ginsenosides; note: represents a very significant difference compared to day 0 (P < 0.001); represents significant difference compared to day 0 (P < 0.005); # # indicates that the difference is very significant (P < 0.001) when 30 days of fermentation are compared with 40 days.

Detailed Description

The invention discloses a hericium erinaceus strain, a culture method thereof and a hericium erinaceus-ginseng two-way solid fermentation method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The culture medium, reagents and the like used in the present invention are commercially available.

The invention is further illustrated by the following examples:

example 1 identification of Hericium erinaceum species

1. Hericium erinaceus strain culture and strain morphology identification

Inoculating the strain CCUCM-HE001 on a PDA plate medium, culturing at a constant temperature of 28 ℃ for about 10 days, observing the growth condition of a colony, and performing microsoform classification and identification by using a solid medium slide method according to the characteristics of fungus colony color, hypha growth condition, spore morphology and the like. The strain is cultured by using PDA culture medium, the colony is circular, the edge is irregular, the hypha is white villous, the hypha radially diffuses from the inoculation point to the periphery, the early growth of the hypha is slow, the hypha in the later period is more, the pigment can be produced, and the culture medium is changed into brown yellow (figure 1). Hyphae were thick, there was no gap, and spores were round (FIG. 2).

2 molecular biological identification

The results of the sequencing and the phylogenetic tree are shown below by performing homology analysis on the sequences and constructing the phylogenetic tree. The ITS zone sequence length of the genome DNA of the strain CCUCM-HE001 is 606bp, and the sequence similarity rate of the strain CCUCM-HE-001 and Hericium erinaceus (Hericium erinaceum) can be over 99 percent by using the online comparison result of a GenBank database BLAST in NCBI. And (3) selecting a sequence with greater similarity to the strain to construct a phylogenetic tree, and further determining the species relationship of the strain. Taking Mycoleptodonoides aitchisonii (Mycoleptodonoides aitchisonii) as an exogenous strain, pairwise comparing strain sequences of a developmental tree according to a Neighbour-Joining method, calculating the evolution or genetic relationship among the sequences, and constructing a phylogenetic tree as shown in figure 3. The research result of the phylogenetic tree constructed by ITS sequence comparison analysis shows that the strain CCUCM-HE001 has higher homology relation with Hericium erinaceus (Hericium erinaceum) and closer genetic relation, which indicates that the strain CCUCM-HE001 belongs to Hericium erinaceus.

Example 2 two-way solid fermentation technique for Hericium erinaceus-ginseng

1. Slant culture of Hericium erinaceus

Recovering strain of Hericium erinaceus stored in a refrigerator at 4 deg.C, transferring into newly prepared PDA solid culture medium under aseptic condition, culturing in a constant temperature incubator at 26 deg.C in dark for 7 days, subculturing again, repeating the above steps, and observing growth of Hericium erinaceus mycelium to cover the whole culture medium surface to obtain liquid culture medium.

2. Preparation of first-grade and second-grade hericium erinaceus liquid strains

Inoculating appropriate amount of the solid culture of Hericium Erinaceus into first-stage liquid culture medium (glucose 20g, peptone 10g, KH)₂PO₄ 2g，MgSO₄·7H₂O1 g, distilled water 1L), culturing at 26 deg.C under dark condition of 160rpm/min for 15 days, transferring the primary liquid strain to Hericium Erinaceus secondary liquid culture medium (glucose 22g, peptone 0.2g, yeast powder 0.2g, KH) at a ratio of 1:20(V/V)₂PO₄ 5 g，MgSO₄·7H₂O1 g, 1L of distilled water) under the above conditions for 7 days. When the second flask was observed to be filled with uniformly shaped yellowish pellets, the fermentation was terminated.

Example 3 preparation of Hericium erinaceum-Ginseng bidirectional solid fermentation mycoplasm

In the reference literature (Chentianli, compound ginseng and grass mycoplasm particle preparation process and research on antitumor activity thereof [ D ] Changchun Chinese medicine university, 2019; Tanzwhen & North cordyceps solid fermentation conditions influence on the content of main active ingredients [ D ] Hunan agriculture university, 2013.), the ginseng and hericium erinaceus are subjected to bidirectional solid fermentation. Pulverizing Ginseng radix, and sieving with 16 mesh pharmacopeia sieve to obtain Ginseng radix powder with uniform particle size. Weighing 30g of the ginseng powder, placing the ginseng powder into a 200mL culture bottle, adding 40% of distilled water as an initial water adding amount, uniformly mixing the water and the ginseng powder, filling the mixture into the culture bottle, and carrying out autoclaving at 121 ℃ for two times, wherein each time is 30 minutes. Transferring the sterilized ginseng medicinal material matrix to aseptic condition, cooling, shaking the matrix to loosen, and sterilizing under ultraviolet lamp for 30 min. A20% (W/V) second-level hericium erinaceus seed culture solution is inoculated into a ginseng solid culture medium to serve as an experimental group, and a ginseng solid culture medium which is not inoculated with the second-level hericium erinaceus seed culture solution is used as a control group. When the whole culture bottle is full of hericium erinaceus mycelium and an obvious bud structure is formed, the fermentation end point is determined (30 days), and then the fermentation is continued for 10 days. Taking out 3 bottles of the experimental group and sample group samples every 3 days, drying at 40 ℃, crushing, sieving by a No. 3 sieve, and storing in a dark sealed manner.

Example 4 UPLC-QQQ-MS/MS technique for analyzing and monitoring content changes of 13 ginsenosides in Hericium erinaceus-ginseng mycoplasm

1. Preparation of control solutions

Precisely weighing ginsenoside reference substance Rb₁、Rb₂、Rc、Rd、Re、Rf、Rg₁、Rg₂、Rg₃、 F₂、F₃、Rh₁And adding a proper amount of Ro reference substance into chromatographic methanol for dissolving to obtain a mixed reference substance solution, shaking uniformly, and filtering with a 0.22-micron organic filter membrane for later use. The mixed control solution is stored in a refrigerator at 4 ℃ for later use.

2. Preparation of test solution

According to the method in Chinese pharmacopoeia, 1.0g of mycoplasm is precisely weighed, the mycoplasm is wrapped by filter paper, the wrapped mycoplasm is placed in a Soxhlet extractor, chloroform is added, heating reflux is carried out for 3 hours, chloroform liquid is discarded, a herb residue bag is taken out, a solvent is volatilized, the filter paper bag is transferred to a 100mL conical flask with a plug, 50mL of water saturated n-butanol is added, a sealing plug is kept still for overnight, ultrasonic treatment (power is 250W and frequency is 50kHz) is carried out for 30 minutes, filtration is carried out, primary filtrate is discarded, 25mL of secondary filtrate is precisely taken, the secondary filtrate is placed in an evaporating dish and evaporated to dryness, residues are dissolved by methanol and transferred to a 5mL volumetric flask, the methanol is added to be diluted to a scale, the mixture is shaken uniformly, the mixture passes through a 0.22 mu m organic filter membrane, and the mixture is stored in a 4 ℃ refrigerator for standby.

3. Liquid chromatography separation conditions

Thermo C₁₈Column (50mm × 3mm, 1.7 μm), mobile phase 0.1% formic acid (A) -acetonitrile (B), gradient elution: 0-5 min, 19% B; 5-29 min, 19% -25% of B; 29-72 min, 25% -40% of B; 72-77 min, 40% -90% B; 77-80 min, 90% B; 80-83 min, 90-19% B; 83-88 min, 19% B; the flow rate is 0.2mL/min, the column temperature is 35 ℃, the sample chamber temperature is 4 ℃, and the sample injection amount is 5 mu L.

4. Mass spectrometric detection conditions

Carrying out ionization in an electrospray mode, carrying out negative ion mode, and carrying out electrospray ion source (ESI) full-scan detection; the mass scanning range m/z is 100-1500; spraying voltage: 2500V; sheath gas pressure: 35 arb; auxiliary gas pressure 10 arb; the temperature of the transmission capillary is 350 ℃; temperature of the mist sprayer: at 300 ℃.

Test example 1 determination of content of Hericium erinaceum-ginseng mycoplasm saponin

The content of ginsenoside was measured by the method of example 4 on the fermentation biomass of example 3 at different fermentation stages, the peak area was recorded and the content of 13 saponins was calculated according to the corresponding linear regression equation.

The saponin content in the mycoplasm at different fermentation times was measured, and the results are shown in fig. 4 and 5. The results show that the content of each ginsenoside is increased and decreased to different degrees after the ginseng medicinal materials are subjected to bidirectional fermentation. Compared with 0 day, when the mycoplasm is fermented to 30 days, the rare saponin Rg₃41.07% of oleanolic acid type saponin Ro content, 64.18% of rare saponin Rh₁The increase is 63.57%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.