CN115058347A

CN115058347A - Lepista sordida, preparation method of non-saccharide extract of Lepista sordida and application of non-saccharide extract

Info

Publication number: CN115058347A
Application number: CN202210637638.0A
Authority: CN
Inventors: 徐丽丽; 陆秀华; 郭立忠; 杜伟; 冯阳
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2022-09-16
Anticipated expiration: 2042-06-07
Also published as: CN115058347B

Abstract

The invention discloses a Lepista sordida, a preparation method of a non-saccharide extract thereof and application thereof, wherein the fungus is named as Mongolian L1, and the preservation number is CCTCC M2022511. The preparation method of the non-saccharide extract of Lepista sordida provided by the invention comprises the following steps: s1, inoculating the seed liquid of the Lepista sordida thalli into a liquid culture medium, and performing shake culture for 2-4 days; s2, reducing the culture temperature to 5-15 ℃, and performing shake culture for 24-72 hours; returning the culture temperature to 20-22 deg.C, and continuing culturing at a shaker rotation speed of 150rpm/min to obtain primary fermentation broth; s3, low-temperature refrigeration treatment: storing the fermentation liquor in a low-temperature environment of 1-6 ℃ for low-temperature treatment for 12-96 h; s4, removing mycelium pellets and keeping supernatant; s5, primarily purifying the fermented supernatant to obtain a crude extract; and purifying by HPLC, and separating to obtain refined extract. The preparation method is simple to operate, the solvent types are few, the time consumption is short, the extraction effect is good, the obtained refined extract has the effects of resisting tumors and protecting liver, and the fungus and the extract thereof have good application prospects.

Description

Lepista sordida, preparation method of non-saccharide extract of Lepista sordida and application of non-saccharide extract

Technical Field

The invention relates to the field of medicinal fungus extracts, in particular to Lepista sordida, and preparation and application of a non-saccharide extract thereof.

Background

Lepista sordida belongs to Tricholomataceae, and is a fungus of Lepista. When the pileus is young, the pileus is hemispherical, and then is flat, and when the pileus is wet, the pileus is semitransparent or soaked in water; the edge is curled inwards and has unobvious stripes which are often wavy or petal-shaped; the mushroom flesh is light violet, thin, and the mushroom folds are straight and sometimes slightly bent; the stipe is violet, solid and the base is bent; the basidiospore is oval in width to oval in shape, coarse to have pock marks, and colorless. The strain is widely distributed, and is mainly grown on hilly grassland, vegetable garden, compost and the like or is close to the grassland mushroom ring in summer and autumn.

The flower-face fragrant mill not only has higher nutritive value, but also has higher medicinal value. Ardisia formosana fruiting bodies cultured by artificial substitute materials are measured by 18 elements: the floral face fragrant mill entity is indicated to contain rich essential trace elements such as Se, Zn, Ge, Fe and the like which are necessary for human body to resist oxidation, enhance immunity and resist aging. And the content of Cd, Hg and Pb which are harmful to human bodies in the floral-face spice mill fruiting body is about 20 to 100 times lower than that of 25 wild mushrooms such as European white forest mushroom (Agarcussivoid), Lepistrudua, Lasiosphaera reticulata (Lycoperdonoperculatum) and the like. Through research and comparison, the Se content of the Lepista sordida fruiting body is 40 times of that of rice in Keshan disease areas of New Ministry of Magnolia county of Black Dragon Jiang province of China, and is several times or even dozens of times of that of fruits, vegetables, rice, corns, pork, beef, eggs, milk and the like. Lepista sordida has the effects of enriching selenium, germanium and other anticancer, anti-aging and other trace elements, and harmful heavy metal elements reach the FDA (American) standards of food and drug administration. As a rare edible fungus, Lepista sordida has important medicinal, edible, development and application values.

However, the research on the medicinal value of the floral-face incense mill at present mainly focuses on the research on the components of macromolecular substances such as crude protein and crude polysaccharide, so that the research on the anti-tumor and liver-protecting effects of the bacterium at present is also attributed to the polysaccharide component in hypha; however, the existing research in the field does not develop the value of the small-molecule non-saccharide active ingredients of Lepista sordida, and lacks a method for extracting, preparing and identifying the corresponding non-saccharide active ingredients.

Glycocholic acid is a newly found biomarker of hepatocellular carcinoma patients, is found in animal bile, can induce apoptosis of liver cancer cells of rats (Wen prescription, 2000, etc.), improves drug resistance of tumor cells to chemotherapy (YL.Lo, 2018), has the efficacy of protecting retinal pigment epithelial cells from oxidative damage (2021), can obviously reduce the content of nitric oxide in serum of inflammatory rats, and has obvious inhibition effect on acute and chronic inflammations (Li Feng, 2006). Glycocholic acid can also improve MDA and SOD activity in liver and brain tissues of mice, and has a certain antioxidation effect on organisms (Wuyunfu, 2013).

At present, a small amount of prior arts disclose methods for chemically synthesizing glycocholic acid, such as patents with publication numbers CN101817860B and IN6800DELNP2011A, but the activity and the application safety are inferior to those of natural glycocholic acid; at present, natural glycocholic acid is mainly extracted from bile of animals such as cattle, sheep and geese, and has the disadvantages of limited source, complex extraction process and high cost. However, no research or report on the extraction of glycocholic acid from bacteria or fungi which are easy to reproduce has been found.

Therefore, the prior art is in need of further improvement.

Disclosure of Invention

Aiming at the problems, the application discovers a fungus, namely Lepista sordida (Menghua L1), which can produce glycocholic acid in a biosynthetic way for the first time, and provides a preparation method for preparing a non-saccharide extract containing glycocholic acid by using the fungus.

The technical scheme of the application is as follows:

in a first aspect, the invention provides a Lepista sordida strain named as flos buddlejae L1, which is preserved for 28 days at 4 months 2022 at the place of China center for type culture Collection (Wuhan university, China) with the preservation number of CCTCC M2022511.

A large number of experiments prove that the non-saccharide extract extracted from Lepista sordida L1 contains glycocholic acid, and has the effect of inhibiting tumors.

In a second aspect, the application also provides an application of the lepista sordida in preparing anti-tumor and liver-protecting medicines. The non-saccharide extract extracted from Lepista sordida L1 has tumor inhibiting effect, and is rich in glycocholic acid as active ingredient, so Lepista sordida L1 can be used for preparing anti-tumor and liver-protecting medicines.

In a third aspect, the invention provides application of the lepista sordida in preparation of the glycine-bile acid extract. The content of the invention shows that the Lepista sordida is rich in glycocholic acid, glycocholic acid substances in the Lepista sordida can be extracted by a separation and purification method, and the purity and the impurities of the obtained glycocholic acid substances are different based on different methods. For example, the small molecule non-saccharide extract obtained by separation and purification by the following preparation method is glycocholic acid substances.

In a fourth aspect, the present invention provides a method for preparing a non-saccharide extract of Lepista sordida, the method comprising the steps of:

s1, inoculating the Lepista sordida thalli into a liquid culture medium, culturing at 20-22 ℃ to obtain a seed solution, inoculating the seed solution into the liquid culture medium, and performing shake culture for 2-4 days. Preferably, the incubation time is 72 h.

And S2, carrying out cooling treatment: the culture temperature is reduced to 5-15 ℃, and shaking culture is carried out for 24-72 h; and then carrying out rewarming treatment: returning the culture temperature to 20-22 deg.C, and continuously culturing for 60-80h at the rotation speed of 150rpm/min to obtain primary fermentation broth.

Experiments prove that no glycocholic acid active ingredient is found in the refined extract prepared by the method without temperature change treatment. Therefore, this step is a critical step, which functions to: reduce the production quantity and speed of Lepista sordida thalli, and provide material basis and time for the production of a secondary metabolite, namely glycocholic acid.

Preferably, in step S2, the conditions of the temperature reduction process are: culturing at 10 deg.C for 48 h. Experiments prove that the yield of glycocholic acid is the highest under the experimental conditions, and the optimal reaction conditions are achieved.

S3, low-temperature refrigeration treatment: and (3) storing the primary fermentation liquor obtained in the last step at a low temperature of 1-6 ℃ for 12-96 h.

In the research and development process, the accidental discovery shows that the output of glycocholic acid in the fermentation liquor is remarkably improved after refrigeration treatment, and the output is increased by about 10-20%. In addition, the experimental result shows that the yield of glycocholic acid in the fermentation broth is gradually increased along with the prolonging of the refrigeration time within a certain time range, but the effect difference between 60 hours of refrigeration and 96 hours of refrigeration is not obvious.

Therefore, in step S3, the conditions of the low-temperature refrigeration process are preferably: low-temperature treatment for 60h at 4 ℃.

And S4, removing mycelium pellets in the filtered fermentation liquor after low-temperature refrigeration treatment, and keeping the supernatant. Experiments prove that the active ingredients of the glycine-bile acids are mainly distributed in the supernatant of the fermentation liquor.

S5, primarily purifying the supernatant to obtain a crude extract; and purifying the crude extract solution by preparative HPLC, and separating to obtain a refined extract, namely the micromolecular non-carbohydrate extract.

Mass spectrum detection shows that the main component of the micromolecule non-saccharide extract is glycocholic acid, so that the cholic acid compound is extracted and found from Lepista sordida for the first time, and experiments show that the extract has good anti-tumor and liver-protecting effects and wide application prospects.

Preferably, in step S5, the preliminary purification treatment method includes: carrying out alcohol precipitation on the supernatant, and reserving the obtained supernatant after removing precipitates; dialyzing and reserving 200-500Da supernatant, removing ethanol and water by rotary evaporation to obtain a crude extract, and extracting the crude extract by using ethanol.

Preferably, the HPLC purification is carried out by using a methanol-water elution system, wherein the volume ratio of water to methanol is as follows: 1: (4-10), the flow rate of the extract was 0.2 ml/min. Alternatively, the chromatographic column used is column Hypersil C18 at a column temperature of 30 ℃.

Preferably, the liquid culture medium comprises the following components in percentage by mass: 0.1-0.2% of fulvic acid, 10-20% of wheat straw juice, 1-5% of glucose, 0.1-0.5% of yeast powder and 0.1-0.2% of KH ₂ PO ₄ 0.05 to 0.1 percent of MgSO ₄ And 0.01-0.05% of vitamin B1, wherein the solvent is water.

The fulvic acid (FA for short) has the smallest molecular weight in humic acid, is easy to dissolve in water, and has a pH value of about 5. Experiments prove that the addition of fulvic acid is beneficial to the synthesis of the glycocholic acid component and the improvement of the yield of glycocholic acid so as to meet the extraction requirement. The discovery is realized: the fulvic acid has small molecular weight and high active group content, acts on the metabolism of Lepista sordida by inhibiting or activating enzyme, and plays an important role in the synthesis of a secondary metabolite, namely glycocholic acid.

Further preferably, the liquid culture medium comprises the following components in percentage by mass: 3% of glucose, 0.3% of yeast powder, 0.1% of fulvic acid, 15% of wheat straw juice and MgSO ₄ 0.05％，KH ₂ PO ₄ 0.1 percent, 0.01 percent of vitamin B1 and the solvent is water.

In a fifth aspect, the present application also provides a non-saccharide extract of lepista sordida, which is prepared using the above method.

The invention has the following beneficial effects:

1. the invention discovers a fungus Lepista sordida (Mongolian L1) capable of producing glycocholic acid in a biosynthetic way for the first time, and the fungus Lepista sordida has high edible, medicinal and agricultural application values. Firstly, Lepista sordida L1 is a strain for realizing mass cultivation of mushrooms after domestication and breeding of wild bacteria of a Herenbei grassland in Mongolia, the cultivation cost is low, the yield is high, large-scale artificial cultivation can be realized by efficiently utilizing waste forage, crop straw, animal and poultry manure and the like, outbreak fruiting is shown, mushroom dregs after fruiting can be used for fertilizing soil fertility, and the ecological significance is achieved. And secondly, the Lepista sordida Mongolian L1 has potential application prospects in food processing, medicine processing or health product processing, can be used for preparing glycocholic acid micromolecule non-saccharide extracts, can be widely applied to preparing medicines for resisting tumors, protecting liver, resisting oxidation and the like, and has wide application prospects.

2. The invention provides a preparation method of a small molecule non-saccharide extract in Lepista sordida, which is designed aiming at the properties of the small molecule non-saccharide extract; different from the complicated steps of the existing preparation method of the Lepista sordida extract, the method is simple and easy to operate, the types of the adopted extraction solvents are few, the extraction steps are short in time consumption, and the extraction effect is good.

The main component of the non-saccharide extract obtained by the method is glycocholic acid, which is a cholic acid compound extracted from Lepista sordida for the first time and is found to have good anti-tumor and liver-protecting effects; the glycocholic acid compound is mainly obtained by a chemical synthesis mode, which is the extraction of glycocholic acid substances from fungi for the first time, so that the glycocholic acid compound not only has better application prospect, but also fully develops the application value of Lepista sordida and improves the utilization rate of the medicinal fungi.

Drawings

FIG. 1 shows the spore imprints and basidiospore electron microscopic morphology of the fruit body of Mongolian flower L1;

FIG. 2 shows the hypha growth state and domesticated cultivar entity morphology of the strain flos Buddlejae L1;

FIG. 3 is a laboratory pilot test and a greenhouse pilot test fruiting test of a strain Mongolian flowers L1;

FIG. 4 is HPLC chromatogram of anion of Lepista sordida;

FIG. 5 is an anion mass spectrum of Lepista sordida extract;

FIG. 6 is a HPLC chromatogram of Liriopsis sordida extract HPLC using cation;

FIG. 7 is a cation mass spectrum of Lepista sordida extract;

FIG. 8 shows the result of the chromatographic peak area integration ratio in example 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example 1 domestication and identification of Lepistasdora sordida (Lepistasoda) Strain, Mongolian L1

1. Mongolian L1 strain separation and domestication cultivation

(1) The experimental method comprises the following steps: the present inventors have collected wild mushroom sporocarp from inner Mongolia grassland, performed tissue separation and purification in an aseptic environment, optimized culture conditions, and cultivated mushrooms. And then the fruiting bodies after the fruiting are screened out are subjected to tissue separation and purification again, strains are prepared and cultivated for fruiting, and after laboratory bench tests and greenhouse pilot tests are carried out in sequence, cultivated strains suitable for large-area popularization are screened out and are named as Mongolian flower L1.

(2) Experimental results and analysis: the colony of the Mongolian L1 has regular edges, and the hypha grows divergently, is dense and strong, and is light purple at the later stage of growth, mainly stolon hypha, and has a small amount of aerial hypha (see figure 2).

2. Identification

(1) The experimental method comprises the following steps: method for identifying binding molecules by morphological identification

(2) Experimental results and analysis:

A. morphological identification:

the fungus fruiting body is medium-sized, the pileus is flat and semi-spherical to flat, sometimes the middle part is slightly concave, the pileus is thin, when the pileus is wet, the pileus is soaked in water, the pileus is white to light purple, and the old edge is wavy or petaloid. The mushroom flesh is light purple, and the mushroom fold is light purple, slightly diluted, and directly grown, and sometimes slightly extended, unequal in length. The stipe is colored with pileus and is bent and solid when being close to the base. The spore print is light yellow, the spore is oval under an electron microscope, the length is about 4.5-5 um, the width is about 2.6-3 um, and the surface is uneven. The lepista sordida is identified as lepista sordida according to the records of aralan (2009) 'Chinese mushroom fungus' and the like on lepista sordida.

B. And (3) identifying the strain molecules:

extracting hypha DNA of the Mongolian L1 by using an EZNA Fungal DNA Kit, carrying out PCR amplification on the ITS zone of rDNA of a sample by using a T5 Direct PCR Kit (Plant) Kit, and sending an amplification product to Shanghai bio-corporation for sequencing. The sequence obtained by sequencing is shown as SEQ ID NO:1 in a sequence table, and the gene sequence is subjected to homology comparison with a sequence which is sequenced successfully in GenBank through NCBI online Blast, and the result shows that the ITS sequence of the fungus has the highest homology with Lepistasoda sordida (Lepistasoda) and reaches 99 percent.

Therefore, the fungus was identified as a new Lepistasoda (Lepistasoda) by combining the above morphological identification and molecular identification. Subsequently, the lepistanida strain, namely the Mongolian flower L1, is preserved for 2022 years, 4 months and 28 days at the preservation place of China center for type culture Collection (Wuhan, Wuhan university, China) with the preservation number of CCTCC M2022511.

Example 2 preparation of non-saccharide extract of Lepista sordida- -Mongolian L1

1. The experimental method comprises the following steps:

(1) culture and fermentation of Lepista sordida

The formula of the liquid culture medium is as follows: 3% of glucose, 0.3% of yeast powder, 0.1% of fulvic acid, 15% of wheat straw juice and MgSO ₄ 0.05％，KH ₂ PO ₄ 0.1 percent, vitamin B1, 0.01 percent and the balance of water. The prepared liquid medium was autoclaved.

Inoculating mycelia of Lepista sordida to 100ml of liquid culture medium, inoculating Lepista sordida thalli to the liquid culture medium, performing shake culture at 20-22 ℃ for 60h to obtain a seed solution, inoculating the seed solution to 200ml of the liquid culture medium according to the inoculum size of 10%, performing shake culture for 72h, reducing the culture temperature to 10 ℃, performing shake culture for 48h, recovering the culture temperature to 20 ℃, and continuing to perform culture for 72h, wherein the rotation speed of a shaking table is 150rpm/min to obtain fermentation liquor.

(2) Temperature-changing culture and low-temperature treatment of Lepista sordida.

After culturing for 72h, reducing the culture temperature to 10 ℃, shaking for 48h, then returning the culture temperature to 20 ℃, continuing culturing for 72h, and storing the culture solution in a 4 ℃ environment for low-temperature treatment for 60h, wherein the step is beneficial to the target product of the lepista sordida induced expression in adverse circumstances.

(3) Preparation of crude extract

The cryogenically treated broth is filtered to remove the mycelial pellets and leave only the liquid portion of the broth (i.e., the supernatant). Then carrying out alcohol precipitation on the liquid part, and removing the precipitate to leave supernatant.

Dialyzing the supernatant, and reserving the supernatant of 200-500Da, and performing rotary evaporation to remove ethanol and water to obtain a crude extract.

(4) Extraction of active ingredients

Extracting the crude extract with ethanol as solvent, soaking for 12 hr, filtering with filter paper, and removing impurities to obtain primary extractive solution.

(5) Dialysis treatment of primary extract

Taking 2 sections of dialysis bags, intercepting the primary extracting solution with the molecular weight of 200-. After the dialysis treatment, macromolecular substance molecules in the primary extracting solution are removed to obtain a secondary extracting solution.

(6) Purification treatment

The secondary extract was purified by HPLC using a Hypersil C18 column (specification 2.1X 100mm,5 μm; Thermo Fisher Scientific, Waltham, USA) at a column temperature of 30 ℃ in a two-dimensional nano-liquid system at a flow rate of 0.2 ml/min.

In the elution process, the eluent A adopts water, the eluent B adopts a methanol elution system, and the volume ratio of the two eluents is as follows: 1, (4-10) under the elution condition, carrying out chromatographic retention for 15-16min, and collecting a purified product, namely the lepista sordida ethanol refined extract. And identifying the structure of the purified small-molecule non-saccharide extract by adopting liquid chromatography-mass spectrometry.

(7) Mass spectrometric analysis

Carrying out mass spectrometry on the ethanol refined extract, carrying out electrospray mass spectrometry on the ethanol refined extract, and carrying out positive ion and negative ion full scanning in the nuclear-mass ratio range of 50-1500m/z, wherein nitrogen is used as atomizing gas and the flow rate is 6L/min; the temperature is 180 ℃; the pressure was 1.0Bar and the data acquisition and processing used LC/MS data analysis software supplied with the instrument (version 4.1). The nuclear to proton ratio data corresponding to a particular elemental composition is calculated using formula prediction software provided with the instrument. The error between the measured nuclear-to-cytoplasmic ratio and the standard nuclear-to-cytoplasmic ratio of the substance is required to be no more than 5 ppm.

2. Results and analysis of the experiments

(1) And identifying the purified small molecule non-saccharide extract, wherein the anion chromatogram is shown in figure 4, and the cation chromatogram is shown in figure 6.

(2) The anion mass spectrum result is shown in figure 5, the analysis of the mass spectrum shows that the maximum peak is the molecular ion peak, the M/Z value of the anion mass spectrum is 464.3052, and the molecular formula of the main active ingredient is C in the anion mass spectrum determination ₂₇ H ₃₉ N ₅ O ₂ (anion C) ₂₇ H ₃₈ N ₅ O ₂ Adding H); because the compound has amino group, cation mass spectrometry also determines that the cation of the substance has M/Z value of 466.3188 (see figure)7) The cationic ion mass spectrum shows that the molecular formula is C ₂₇ H ₃₉ N ₅ O ₂ (anion C) ₂₇ H ₄₀ N ₅ O ₂ Minus H), in combination with data from analysis of anion and cation mass spectra, the final determination: the compound is glycocholic acid substance.

(3) In the control group, the fine extract of lepista sordida extracted from the control group was examined without adding 0.1% fulvic acid and subjecting to a temperature-changing treatment, and it was found that: the glycocholic acid compound was not found in the concentrate of the control group, which indicates that: the Lepista sordida, Mongolian L1, which was not subjected to temperature swing treatment and 0.1% fulvic acid addition treatment, failed to express sufficient quantities of extractable glycocholic acid.

Example 3 in vitro tumor inhibition assay of Lepista sordida Small molecule non-saccharide extract

1. Culturing of tumor cells:

human hepatoma cell line SMMC (purchased from Weifang medical college) is cultured in RPMI1640 culture medium containing 10% fetal calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin at 37 ℃ and 5% CO ₂ Culturing in an incubator under the conditions of (1) to obtain the human liver tumor cells after the cells grow in logarithmic phase.

2. Sample set setting:

experimental and control groups were set up separately as follows, with at least 3 replicates per group:

experimental group 1: preparing the Lepista sordida ethanol refined extract according to the complete experimental steps, and taking the Lepista sordida ethanol refined extract as a sample; experimental group 2: basically the same as the method of the experimental group 1, only the steps of temperature change treatment and 0.1% humic acid addition treatment are omitted, and the extract is collected as a sample; positive control group: chemically synthesized glycocholic acid was used as a sample.

And respectively adding the samples of each experimental group into sterile distilled water for dissolving, and preparing the prepared solution for later use.

3. Determination of tumor-inhibiting Activity

(1) Detection of apoptosis: the experimental group 1, the experimental group 2 and the positive control group are used for detecting by a flow cytometer after staining after a sample with 350 mu mol/L dose acts on the liver cancer SMMC cells for 72 hours.

(2) Calculation method for tumor inhibition: analysis line t test analysis was performed with SPSS software, P <0.05 was statistically significant.

(3) Apoptosis was detected using the annexin V-FITC marker.

4. Results and analysis of the experiments

(1) As can be seen from the results in table 1, the apoptosis rates of the experimental group 1 and the positive control group are equivalent, and the apoptosis rate of the experimental group 1 is higher than that of the positive control group; the results show that the main component of the non-saccharide extract prepared by the method is the biosynthetic glycocholic acid, which has higher tumor inhibition activity than the chemically synthesized glycocholic acid, and the molecular structure of the biosynthetic glycocholic acid is higher than the biological activity of the chemically synthesized glycocholic acid, so that the temperature-variable fermentation production of glycocholic acid by lepista sordida has good application prospect, and a new idea is brought to prevention and treatment of liver cancer.

(2) Compared with the experimental group 2, the apoptosis rate of the experimental group 1 is obviously increased, and the results are obviously different, which shows that: the refined extract of the tumor inhibiting active ingredients prepared by the method exists under the conditions of temperature-variable culture and humic acid addition, so that the temperature-variable culture and humic acid addition are important experimental steps.

TABLE 1 apoptosis rate of liver cancer SMMC cells for 72h

Note: n is 3, P <0.05 to control

Example 4 optimization experiment of temperature-variable treatment conditions for the preparation method

1. The experimental method comprises the following steps:

(1) 12 experimental groups are set and respectively placed at 5 ℃, 10 ℃ and 15 ℃ for temperature change treatment for 24h, 48h, 72h and 96h, and other steps are the same as the other steps of the example 2. The high and low yield of glycocholic acid is shown by a chromatographic peak area integral ratio method.

2. Results and analysis of the experiments

From the experimental results of fig. 8, it was shown that the temperature-swing treatment at 10 ℃ for 48 hours was the most suitable, and the yield of glycocholic acid in the extract prepared under such conditions was the highest, so that the temperature-swing treatment condition of example 2 was selected as 10 ℃ for 48 hours.

EXAMPLE 5 optimization of the culture Medium

1. Experiment setting:

carbon source, nitrogen source, fulvic acid and Mg for the screened media according to the settings of Table 2 ²⁺ Orthogonal experiments were performed. Let 9 treatments, 5 replicates each. The water is distilled water containing 15% of wheat straw juice, and 0.1% of KH ₂ PO ₄ 0.01% of vitamin B1.

TABLE 2 Lepista sordida liquid fermentation orthogonal factors level table L9 (3) ⁴ )

2. Results and analysis of the experiments

From the results in table 3, it can be seen that the optimal formulation of the broth according to range analysis is A3B3C1D3, and therefore suitable formulations for hyphal fermentation are: 3% of glucose, 0.3% of yeast powder, 0.1% of fulvic acid, 15% of wheat straw juice and MgSO ₄ 0.05％，KH ₂ PO ₄ 0.1%, vitamin B1, 0.01%.

TABLE 3 Effect of Medium composition on liquid fermentation of Mongolian flos L1

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Sequence listing

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<120> Lepista sordida, preparation method and application of non-saccharide extract thereof

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gattggcttg gatgtggagg ttattgcggg cttctctaga agtcggctcc tcttaaatgc 480

attagcggaa cctttgtgga ccagcttttg gtgtgataat tatctacgcc atggttgtga 540

agcagcttta acatggggtt cagcttctaa cagtccattg acttggacaa atttatgaca 600

tttttgacct caaatcaggt aggactaccc gctgaactta agcatatcaa taaagcggag 660

ggaatttttc atttcgaata a 681

Claims

1. A Lepista sordida (Lepista sordida) is named as Menghua L1 and has a preservation number of CCTCC M2022511.

2. Use of Lepista sordida according to claim 1 for the preparation of glycocholic acid extract.

3. A method for preparing a non-saccharide extract of Lepista sordida, comprising the steps of:

s1, inoculating the Lepista sordida thalli of claim 1 into a liquid culture medium, culturing at 20-22 ℃ to obtain a seed solution, inoculating the seed solution into the liquid culture medium, and performing shake culture for 2-4 days;

and S2, carrying out cooling treatment: the culture temperature is reduced to 5-15 ℃, and shaking culture is carried out for 24-72 h; and then carrying out rewarming treatment: returning the culture temperature to 20-22 deg.C, and continuing culturing for 60-80h at a shaker rotation speed of 150rpm/min to obtain primary fermentation broth;

s3, low-temperature refrigeration treatment: storing the primary fermentation liquor obtained in the last step at a low temperature of 1-6 ℃ for 12-96 h;

s4, removing mycelium pellets in the filtered fermentation liquor after low-temperature refrigeration treatment, and reserving supernatant;

s5, primarily purifying the supernatant to obtain a crude extract; and purifying the crude extract solution by preparative HPLC, and separating to obtain a refined extract, namely the glycocholic acid extract.

4. The method for preparing the non-saccharide extract of Lepista sordida according to claim 3, wherein the cooling process is performed under the following conditions in step S2: culturing at 10 deg.C for 48 h.

5. The method for preparing the non-saccharide extract of Lepista sordida according to claim 3, wherein the conditions of the low-temperature refrigeration treatment in step S3 are as follows: low-temperature treatment for 60h at 4 ℃.

6. The preparation method of the non-saccharide extract of Lepista sordida according to claim 3, characterized in that the liquid culture medium comprises the following components by mass percent: 0.1-0.2% of fulvic acid and 10%20 percent of wheat straw juice, 1 to 5 percent of glucose, 0.1 to 0.5 percent of yeast powder and 0.1 to 0.2 percent of KH ₂ PO ₄ 0.05 to 0.1 percent of MgSO ₄ And 0.01-0.05% of vitamin B1, wherein the solvent is water.

7. The method for preparing the non-saccharide extract of Lepista sordida according to claim 3, wherein the preliminary purification treatment in step S5 is: precipitating the supernatant with ethanol, and removing precipitate and retaining the supernatant; dialyzing and reserving 200-500Da supernatant, removing ethanol and water by rotary evaporation to obtain a crude extract, and extracting the crude extract by using ethanol.

8. The method for preparing the non-saccharide extract of Lepista sordida according to claim 3, wherein HPLC purification is performed using a methanol-water elution system, and the volume ratio of water to methanol is 1: (4-10), the flow rate of the extract was 0.2 ml/min.

9. A non-saccharide extract of Lepista sordida, which is prepared by the method according to any one of claims 3 to 8.

10. Use of Lepista sordida according to claim 1 for the preparation of anti-tumor and liver-protecting medicaments.