CN115058347B

CN115058347B - Preparation and application of lepista sordida and non-saccharide extract thereof

Info

Publication number: CN115058347B
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: 2023-05-12
Anticipated expiration: 2042-06-07
Also published as: CN115058347A

Abstract

The invention discloses a Lepidium sordidum (Lepidosta) strain, a preparation method of a non-saccharide extract thereof and application thereof, wherein the fungus is named Mongolian L1 and has a preservation number of CCTCC M2022511. The preparation method of the non-sugar extract of the lepista sordida provided by the invention comprises the following steps: s1, inoculating the seed solution of the lepista sordida thalli into a liquid culture medium, and shake culturing for 2-4 d; s2, reducing the culture temperature to 5-15 ℃ and carrying out shake culture for 24-72 h; restoring the culture temperature to 20-22 ℃ for continuous culture, wherein the rotation speed of a shaking table is 150rpm/min, and obtaining a primary fermentation broth; s3, low-temperature refrigeration treatment: the fermentation liquor is stored in the environment of 1-6 ℃ for low-temperature treatment for 12-96 hours; s4, removing mycelium pellets, and reserving supernatant; s5, primarily purifying the fermentation supernatant to obtain a crude extract; purifying by preparative HPLC, and separating to obtain refined extract. The preparation method is simple to operate, the solvent type is few, the time consumption is short, the extraction effect is good, the obtained refined extract has the anti-tumor and liver-protecting effects, and the fungus and the extract thereof have good application prospects.

Description

Preparation and application of lepista sordida and non-saccharide extract thereof

Technical Field

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

Background

Lepidium (Lepidium sordida) belongs to the genus Tricholomataceae. The fungus cover is hemispherical when young, flat after being flattened, and semitransparent or water-immersed when wet; the edge is internally coiled, has no obvious stripes and is often wavy or petaloid; the fungus meat is light violet, thinner, and the fungus folds grow directly and sometimes slightly bent; the pansy is solid, and the basal part is bent more; the basidiomycetes are wide and elliptic to oval, coarse to have pits and colorless. The bacteria are widely distributed, and are clustered or clustered in hillside grasslands, vegetable gardens, compost and the like in summer and autumn, so that grassland mushroom circles are often formed.

The flower face fragrance grinder not only has higher nutritive value, but also has higher medicinal value. Luo Xinyi et al measured 18 elements in fruiting bodies of Lepista sordida cultivated by artificial substitution: the flower face fragrance mill fruiting body is rich in essential microelements such as Se, zn, ge, fe for resisting oxidation, enhancing immunity and resisting aging of human body. The content of Cd, hg and Pb harmful to human body in the flower face fragrance mill fruiting body is about 20-100 times lower than that of 25 wild mushrooms such as European Bai Lin mushrooms (Agarcussilvoid), lepistaudua (Lepistaudua), lasiosphaera seu Calvatia (Lycopendonperlaturtum) and the like. Through research and comparison, the Se content in the lepista sordida fruiting body is 40 times of that in rice in the keshan disease area of New people's society in Magnolia county in Heilongjiang province of China, and is several times or even tens times of that in fruits, vegetables, rice, corn, pork, beef, eggs, milk and the like. Lepista sordida has various trace elements such as selenium, germanium and the like enriched for resisting cancer and aging, and the harmful heavy metal elements reach the FDA (American) food and drug administration standard. As a rare edible fungus, the Lepista sordida has important medical, edible and development application values.

However, the research on the medicinal value of the flower face fragrance mill is mainly focused on the research on macromolecular substance components such as crude protein, crude polysaccharide and the like, so the research on the effects of the fungus such as anti-tumor and liver protection are also often attributed to the polysaccharide components in hyphae; however, the research existing in the art does not develop the value of small-molecule non-saccharide active ingredients of lepista sordida, and lacks methods for extraction preparation and identification of corresponding non-saccharide active ingredients.

Glycocholic acid is a biomarker of a newly discovered hepatocellular carcinoma patient, is found to exist in animal bile, can induce apoptosis of rat liver cancer cells (Huang Wenfang and the like, 2000), 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 inflammation (Li Huifeng, 2006). Glycocholic acid also can improve MDA and SOD activities in liver and brain tissues of mice, and has a certain antioxidation effect on organisms (Wu Yunfu, 2013).

At present, a small amount of prior art discloses a method for chemically synthesizing glycocholic acid, such as a patent with publication number of CN101817860B, IN6800DELNP2011A, but the activity and application safety of the glycocholic acid are inferior to those of natural glycocholic acid; at present, natural glycocholic acid is mainly extracted from bile of animals such as cattle, sheep, goose and the like, so that the source is limited, the extraction process is complex, and the cost is high. However, no studies and reports have been found on the extraction of glycocholic acid from bacteria or fungi which are easy to reproduce.

Accordingly, there is a need for further improvements in the art.

Disclosure of Invention

Aiming at the problems, the application discovers a fungus-Lepista sordida L1 capable of producing 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, which is simple to operate and lower in cost than the prior art, and the prepared small molecular non-saccharide extract has good anti-tumor and liver protection effects and good application prospect.

The technical scheme of the application is as follows:

in a first aspect, the present invention provides a Lepidium sordida (Lepidium sordida), designated Mongolian L1, deposited for a period of 2022, 4 months and 28 days, at China center for type culture Collection (university of Chinese Wuhan), with a accession number CCTCC M2022511.

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

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

In a third aspect, the invention provides an application of the lepista sordida in preparing glycocholic acid extracts. The content of the invention shows that the lepista sordida is rich in glycocholic acid, and the glycocholic acid substances in the lepista sordida can be extracted by a separation and purification method, and the purity and the impurity 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 substance.

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 seed liquid, inoculating the seed liquid into the liquid culture medium, and shake culturing for 2-4 d. Preferably, the incubation time is 72 hours.

S2, performing cooling treatment: reducing the culture temperature to 5-15 ℃ for shake culture for 24-72 h; and then carrying out rewarming treatment: and (3) returning the culture temperature to 20-22 ℃ for continuous culture for 60-80 hours, wherein the rotation speed of a shaking table is 150rpm/min, and obtaining the primary fermentation liquor.

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 key step, and the function of this step is: the production quantity and speed of the lepista sordida thalli are reduced, and a material basis and time are provided for the production of secondary metabolite glycocholic acid.

Preferably, in the step S2, the conditions for the cooling process are as follows: culturing at 10 ℃ for 48 hours. Experiments prove that under the experimental conditions, the yield of glycocholic acid is the highest, and the optimal reaction conditions are adopted.

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

In the research and development process, the yield of glycocholic acid in fermentation liquor is obviously improved by about 10-20% after refrigeration treatment. Moreover, the experimental results show that the yield of glycocholic acid in the fermentation broth gradually increases with the extension of the refrigeration time within a certain time range, but the difference of the refrigeration effect between 60h and 96h is not obvious.

Therefore, in the above step S3, preferably, the conditions of the low-temperature refrigeration process are: and (5) carrying out low-temperature treatment for 60h at the temperature of 4 ℃.

S4, removing mycelium pellets in the fermentation liquor after the low-temperature refrigeration treatment, and reserving supernatant. Experiments prove that the glycocholic acid active ingredients are mainly distributed in the supernatant of the fermentation broth.

S5, performing preliminary purification treatment on the supernatant to obtain a crude extract; and purifying the crude extract solution by preparative HPLC, and separating to obtain a refined extract, namely a small molecule non-saccharide extract.

The main component of the small-molecule non-saccharide extract is glycocholic acid, which is found by extracting cholic acid compounds from lepista sordida for the first time, and experiments show that the extract has good anti-tumor and liver-protecting effects and has wide application prospect.

Preferably, in the step S5, the preliminary purification treatment method is as follows: precipitating the supernatant with ethanol, removing precipitate, and retaining the supernatant; dialyzing and collecting 200-500Da supernatant, removing ethanol and water by rotary evaporation to obtain crude extract, and extracting with ethanol.

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

Preferably, the liquid culture medium comprises the following components in percentage by mass: 0.1 to 0.2 percent of fulvic acid and 10 percent ofAbout 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, and 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 glycocholic acid components and the improvement of the yield of glycocholic acid so as to meet the extraction requirement. The realization is found: the fulvic acid has small molecular weight and high active group content, acts on the metabolism of the lepista sordida by inhibiting or activating enzyme, and plays an important role in the synthesis of the secondary metabolite glycocholic acid.

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

In a fifth aspect, the present application also provides a non-saccharide extract of lepista sordida, prepared by 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 has high edible, medicinal and agricultural application values. Firstly, the lepidoptera murraya L1 is a strain for realizing mass cultivation and fruiting after domestication and breeding of wild mushrooms in the inner Mongolian Hulen's grassland in China, has low cultivation cost and high yield, can efficiently utilize waste forage, crop straws, animal and livestock manure and the like to realize large-scale artificial cultivation, is shown as explosive fruiting, and fungus residues after fruiting can be used for fertilizing soil and has ecological significance. Secondly, the Lepista sordida Mongolica L1 has potential application prospect in food processing, medicine processing or health-care product processing, can be used for preparing glycocholic acid micromolecular non-sugar extracts, can be widely applied to preparing anti-tumor, liver-protecting, antioxidant and other medicaments, and has wide application prospect.

2. The invention provides a preparation method of a small-molecule non-saccharide extract in lepista sordida, which is designed aiming at the property of the small-molecule non-saccharide extract; unlike the complicated steps of the existing preparation method of the Lepista sordida extract, the method is simple and easy to operate, adopts less extraction solvent types, and has short time consumption and good extraction effect.

The main component of the non-saccharide extract obtained by the method is glycocholic acid, which is that cholic acid compounds are firstly extracted from lepista sordida, and the extract has good anti-tumor and liver-protecting effects; the glycocholic acid compound is mainly obtained by adopting a chemical synthesis mode, and the glycocholic acid compound is extracted from fungi for the first time, so that the glycocholic acid compound has better application prospect, the application value of the lepidoptera comita is fully developed, and the utilization rate of the medicinal fungi is improved.

Drawings

FIG. 1 shows the sporophore sporulation and basidiophore electron microscopy morphology of Mongolian L1;

FIG. 2 shows the growth state of the strain Mongolian L1 mycelium and the morphology of domesticated and cultivated fruiting bodies;

FIG. 3 shows a laboratory pilot and greenhouse pilot fruiting experiment of the strain Mongolian L1;

FIG. 4 is a HPLC of the combined liquid chromatography-anion of Lepista sordida extract;

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

FIG. 6 is a HPLC chromatogram of the combined liquid chromatography-cation of Lepista sordida extract;

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

FIG. 8 is the result of the area integration ratio of the chromatographic peaks of example 4.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example 1 domestication and identification of Lepista sordida (Lepista) strain Mongolian L1

1. Separation and domestication cultivation of Mongolian L1 strain

(1) The experimental method comprises the following steps: the inventor performs tissue separation and purification on wild fungus fruiting bodies collected from inner Mongolian grasslands in a sterile environment, optimizes culture conditions and cultures fruiting bodies. Then, the fruiting body after the mushroom screening is re-organized and purified, strains are produced and cultivated to fruiting, and after laboratory pilot-plant test and greenhouse pilot-plant test, the cultivated strain suitable for large-area popularization is screened out, and the cultivated strain is named Mongolian L1.

(2) Experimental results and analysis: the edges of the L1 colony of the Mongolian flowers are neat, the hyphae grow in a divergent shape, are compact and thick, and are light purple in the later growth period, and the creeping hyphae are mainly used, and a small amount of aerial hyphae exist (see figure 2).

2. Authentication

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

(2) Experimental results and analysis:

A. morphological identification:

the fungus fruiting body is medium and large, the fungus cover is flat and hemispherical to flat, sometimes the middle part is slightly concave, the cover is thin, the fungus cover is immersed in water when wet, the fungus cover is stained white to light purple, and the old rear edge is always wavy or flap-shaped. The fungus flesh is light purple, and the fungus folds are light purple, thin, directly growing, sometimes slightly prolonged, and not equal in length. The stipe is the same as the cap color, is often bent near the base and is internally solid. The spore print is light earthy yellow, the spore is elliptical under the 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 was identified as Lepista sordida by comparing the records of Lepista sordida (2009) in China fungus and the like.

B. And (3) identifying strain molecules:

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

Thus, the fungus was identified as a new strain of Lepista sordida (Lepista) in combination with the morphological and molecular identification described above. Subsequently, the Lepista sordida (Lepistasordida) strain Mongolian L1 is preserved for 2022, 4 months and 28 days, and the preservation place is China center for type culture Collection (university of Wuhan, china) with a 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) Cultivation and fermentation of Lepista sordida

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

Inoculating mycelium of Lepista sordida into 100ml of liquid culture medium, inoculating mycelium of Lepista sordida into the liquid culture medium, shake-culturing at 20-22 ℃ for 60h to obtain seed liquid, inoculating the seed liquid into 200ml of liquid culture medium according to 10% of inoculation amount, shake-culturing for 72h, reducing the culture temperature to 10 ℃ for shake-culturing for 48h, recovering the culture temperature to 20 ℃ for 72h, and continuing culturing at 150rpm/min to obtain fermentation liquor.

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

After culturing for 72 hours, the culture temperature is reduced to 10 ℃ for shake culture for 48 hours, then the culture temperature returns to 20 ℃ for continuous culture for 72 hours, and the culture solution is stored in a 4 ℃ environment for low-temperature treatment for 60 hours, so that the step is helpful for inducing the lepista sordida to express target products in the adverse environment.

(3) Preparation of crude extract

The fermentation broth after the low temperature treatment was filtered, and mycelium pellet was removed, leaving only the liquid portion of the fermentation broth (i.e., the supernatant as described above). And then carrying out alcohol precipitation on the liquid part, removing the precipitate and leaving supernatant.

Dialyzing the supernatant, collecting 200-500Da supernatant, and rotary evaporating to remove ethanol and water to obtain crude extract.

(4) Extraction of active ingredients

Ethanol is used as a solvent to extract the crude extract, soaking is carried out for 12 hours, filter paper is used for filtering, and impurities are removed to obtain a primary extract.

(5) Dialysis treatment of primary extract

Taking a 2-section dialysis bag, intercepting primary extracting solution with the molecular weight of 200-500Da, sealing, suspending the dialysis bag in a beaker filled with distilled water, and stirring with a magnetic stirrer to promote solution exchange until dialysis is balanced. And removing macromolecular substances in the primary extract after the dialysis treatment to obtain a secondary extract.

(6) Purification treatment

The secondary extract was subjected to high performance liquid chromatography using a Hypersil C18 column (specification 2.1X100 mm,5 μm; thermo Fisher Scientific, waltham, USA) at 30℃and a two-dimensional nano-liquid phase system at a flow rate of 0.2ml/min.

In the elution process, water is adopted as the eluent A, a methanol elution system is adopted as the eluent B, and the volume ratio of the two eluents is as follows: 1 (4-10), and collecting purified product, namely Lepista sordida ethanol extract. And (3) identifying the structure of the purified small molecule non-saccharide extract by adopting liquid chromatography-mass spectrometry.

(7) Mass spectrometry analysis

Carrying out mass spectrometry on the ethanol refined extract, and carrying out full scanning on positive ions and negative ions in a nuclear mass ratio range of 50-1500m/z by electrospray mass spectrometry, wherein nitrogen is used as atomizing gas, and the flow rate is 6L/min; the temperature is 180 ℃; pressure was 1.0Bar and data acquisition and processing used LC/MS data analysis software supplied with the instrument (version 4.1). The nuclear mass ratio data corresponding to a particular elemental composition is calculated using formula prediction software provided with the instrument. It is required that the error between the measured nuclear mass ratio and the standard nuclear mass ratio of the substance is not more than 5ppm.

2. Experimental results and analysis

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

(3) In the control group, after the steps of adding 0.1% fulvic acid and changing temperature are not carried out, the refined extract of the lepista sordida extracted from the control group is detected, and the following steps are found: the glycocholic acid compound was not found in the control extract, which indicates that: lepista sordida L1, which is not subjected to temperature change treatment and 0.1% fulvic acid addition treatment, cannot express enough extractable glycocholic acid substances.

Example 3 in vitro tumor inhibition experiments with Lepista sordida small molecule non-saccharide extracts

1. Culturing tumor cells:

human liver cancer cell line SMMC (purchased from the medical college of Weifang) was cultured with RPMI1640 medium containing 10% fetal bovine serum, 100U/ml penicillin, 100 μg/ml streptomycin at 37℃and 5% CO ₂ In the incubator under the conditions of (2)Culturing, and obtaining the human liver tumor cells after the cells grow in logarithmic phase.

2. Sample set arrangement:

the experimental and control groups were each set with at least 3 replicates according to the following procedure:

(1) experiment group 1: preparing a Lepista sordida ethanol extract according to the complete experimental steps, and taking the Lepista sordida ethanol extract as a sample; (2) experiment group 2: basically the same as the method of the experiment group 1, only the steps of temperature changing treatment and 0.1% humic acid addition are omitted, and the extract is collected as a sample; (3) positive control group: chemically synthesized glycocholic acid was used as a sample.

Samples of each experimental group were dissolved in sterile distilled water, respectively, and prepared solutions were prepared for use.

3. Determination of tumor inhibiting Activity

(1) Detection of apoptosis: after the samples of the experimental group 1, the experimental group 2 and the positive control group are acted on liver cancer SMMC cells for 72 hours by using a dosage of 350 mu mol/L, the liver cancer SMMC cells are detected by a flow cytometer after being stained.

(2) The calculation method for inhibiting tumor comprises the following steps: analysis line t-test analysis was performed with SPSS software, with P <0.05 statistically significant.

(3) Apoptosis was detected using an annexin V-FITC index.

4. Experimental results and analysis

(1) As can be seen from the results in table 1, the apoptosis rate of the experimental group 1 was equivalent to that of the positive control group, and the apoptosis rate of the experimental group 1 was higher than that of the positive control group; the result shows that the main component of the non-sugar extract prepared by the method is the biosynthesized glycocholic acid, and the biosynthesized glycocholic acid has higher activity of inhibiting tumor than the chemically synthesized glycocholic acid, because the molecular structure of the biosynthesized glycocholic acid is higher than the biological activity of the chemically synthesized glycocholic acid, the variable-temperature fermentation production of the glycocholic acid by using the lepidoptera comita has good application prospect, and brings a new idea for preventing and treating liver cancer.

(2) The apoptosis rate of experimental group 1 was significantly increased compared to experimental group 2, and the results were significantly different, which suggests that: the refined extract for inhibiting the tumor active ingredient prepared by the application exists under the conditions of temperature-changing culture and humic acid addition, so that the temperature-changing culture and the humic acid addition are important experimental steps.

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

Note that: n=3, ratio P <0.05 to control group

Example 4 optimization experiment of temperature Change treatment conditions of preparation method

1. The experimental method comprises the following steps:

(1) 12 experimental groups were set, and the temperature change treatments were performed at 5℃and 10℃and 15℃for 24 hours, 48 hours, 72 hours and 96 hours, respectively, and the other steps were the same as those in example 2. The yield of glycocholic acid is expressed by a chromatographic peak area integration method.

2. Experimental results and analysis

From the experimental results of FIG. 8, it was revealed that the condition of the temperature change treatment for 48 hours at 10℃was optimal, and the yield of glycocholic acid in the extract prepared under this condition was the highest, so that the condition of the temperature change treatment of example 2 was selected to be 10℃and treated for 48 hours.

Example 5 optimization of Medium

1. Experiment setting:

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

TABLE 2 Lepista sordida liquid fermentation orthogonality factor level Table L9 (3 ⁴ )

2. Experimental results and analysis

As can be seen from the results of Table 3, the electrodeThe optimal formula of the liquid culture medium obtained by differential analysis is A3B3C1D3, so that the proper formula of mycelium fermentation is as follows: glucose 3%, yeast powder 0.3%, fulvic acid 0.1%, wheat straw juice 15% and MgSO ₄ 0.05％，KH ₂ PO ₄ 0.1% of vitamin B1 and 0.01%.

TABLE 3 Effect of Medium composition on Mongolian L1 liquid fermentation

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Sequence listing

<110> Qingdao university of agriculture

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

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> ITS sequence of Lepista sordida (ITS of Lepistasordida)

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gcttttgata cttggttggg ttgtgctggc tttttggagc atgtgcacgc ctagcgccat 60

ttttaccacc tgtgcacctt ttgtagattt gaaacaactc tcgaggaaac tcggtttgag 120

gaatgctgtg tgcaaacatg gctttccttg tgtttcaagt ctatgttttt attatacccc 180

ataagaatgt aatagaatgt tattaatggg ctttatgcct ttaaattaat acaactttca 240

acaacggatc tcttggctct cgcatcgatg aagaacgcag cgaaatgcga taagtaatgt 300

gaattgcaga attcagtgaa tcatcgaatc tttgaacgca ccttgcgctc cttggtattc 360

cgaggagcat gcctgtttga gtgtcattaa attctcaacc ttttcagctt ttgcaagttg 420

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. Lepista sordida (Lepista sordida) LLepista sordida) The flower is named as Mongolian flower L1, and the preservation number is CCTCC M2022511.

2. The use of lepidocrocea according to claim 1 for the preparation of glycocholic acid-based extracts.

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

s1, inoculating the lepista sordida thallus according to claim 1 into a liquid culture medium, culturing at 20-22 ℃ to obtain seed liquid, inoculating the seed liquid into the liquid culture medium, and shake culturing for 2-4 d;

s2, performing cooling treatment: reducing the culture temperature to 5-15 ℃ for shake culture for 24-72 h; and then carrying out rewarming treatment: returning the culture temperature to 20-22deg.C, and continuously culturing for 60-80 hr at shaking table rotation speed of 150rpm/min to obtain primary fermentation broth;

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

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

s5, performing preliminary purification treatment on the supernatant to obtain a crude extract; and then carrying out preparation HPLC purification on the crude extract solution, and separating to obtain a refined extract, namely the glycocholic acid extract.

4. The method for producing a non-sugar extract of lepista sordida according to claim 3, wherein in step S2, the conditions for the cooling treatment are: culturing at 10 ℃ for 48 hours.

5. The method for producing a non-sugar extract of lepista sordida according to claim 3, wherein in step S3, the conditions of the low-temperature refrigeration treatment are: and (5) carrying out low-temperature treatment for 60h at the temperature of 4 ℃.

6. A method for preparing a non-saccharide extract of lepista sordida according to claim 3, characterized in that the liquid medium comprises the following components in mass percent: 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 0.05-0.1% of MgSO ₄ And 0.01-0.05% of vitamin B1, wherein the solvent is water.

7. The method for preparing a non-sugar extract of lepista sordida according to claim 3, wherein in step S5, the preliminary purification treatment method is as follows: alcohol precipitation is carried out on the supernatant, and the supernatant is reserved after precipitation is removed; dialyzing and collecting 200-500Da supernatant, removing ethanol and water by rotary evaporation to obtain crude extract, and extracting with ethanol.

8. A process for the preparation of a non-sugar extract of lepista sordida according to claim 3, characterized in that HPLC purification uses a methanol-water elution system with a volume ratio of water to methanol of 1: (4-10), the flow rate of the extract was 0.2ml/min.

9. The use of lepista sordida according to claim 1 for the preparation of an anti-tumor and liver-protecting medicament.