Eurotium cristatum of high-yield cellulase system and application of eurotium cristatum to improvement of drug effect and flavor of fermented Chinese herbal medicine of eurotium cristatum

Abstract

The invention provides eurotium cristatum capable of realizing high yield of a cellulase system and application of the eurotium cristatum in fermentation of Chinese herbal medicines. The preservation number of the eurotium cristatum is CGMCC (China General Microbiological Culture Collection Center) NO.17186 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The eurotium cristatum provided by the invention is used for carrying out fermentation treatment on Chinese herbal medicines represented by radix pseudostellariae, volatile odor substances of the radix pseudostellariae are converted from grass-flavor and earthy-flavor substances into fruit-flavor and flower-flavor substances, and meanwhile, the contents of medicinal components (such as radix pseudostellariae cyclic peptide, Neoechinulin A, echinoling, alkaloid E-7, aspergillus glaucus yellow pigment and the like) are increased. Meanwhile, after fermentation treatment of the eurotium cristatum, the in-vitro antioxidant activity of the radix pseudostellariae alcohol extract and the in-vitro inhibition effect of the radix pseudostellariae alcohol extract on various tumor cells are remarkably enhanced. The invention provides a new thought and approach for deep processing and high-valued comprehensive utilization of the Chinese herbal medicines, and has important significance for improving the additional value and efficacy of the Chinese herbal medicines such as the radix pseudostellariae and the like.

Description

Technical Field

[0001] The present invention relates to the technical fields of Chinese herbal medicine processing, microorganisms and their applications, and in particular to a technology for fermenting Pseudostellaria heterophylla with a specific microbial strain Eurotium cristatum FZ-4. Through the fermentation of Eurotium cristatum FZ-4, the processing and biotransformation of Chinese herbal medicine are carried out, aiming to improve the utilization rate of the medicinal components of Chinese herbal medicine, improve its flavor characteristics, and possibly produce new medicinal molecules. This technology is of great significance for the intensive processing and high-value comprehensive utilization of Chinese herbal medicine, especially Pseudostellaria heterophylla, and provides new ideas and methods for the field of Chinese herbal medicine processing and biotransformation. Background Art

[0002] Fermentation and processing of Chinese herbal medicine is an important technology in the field of Chinese medicine processing. It uses the biotransformation of microorganisms to improve the utilization rate of drug components, reduce the content of substances with toxic and side effects, and may produce new medicinal molecules and new effects. This process mainly includes the release of medicinal components by microorganisms through extracellular cellulase, glycosidase and other enzymes to hydrolyze the cell wall or macromolecular glycoside complexes, the biotransformation and modification of some drug molecules as metabolic substrates, and the synergistic effect between microbial metabolites and the functional components of Chinese herbal medicine. Using microorganisms to ferment Chinese herbal medicine can use the biotransformation method to fully release the active substances of Chinese herbal medicine, reduce the content of substances with toxic and side effects or produce new active metabolites. The main effects of Chinese herbal medicine fermentation include: First, improve the efficacy of Chinese herbal medicine. Most of the active ingredients of Chinese herbal medicine are present inside the plant cell wall, resulting in a low bioavailability of its active ingredients. During the fermentation process, microorganisms produce various extracellular enzymes such as cellulase, ligninase and pectinase, which catalyze the degradation of the plant cell wall, promote the release of the active ingredients of Chinese herbal medicine, and thus improve the bioavailability of the drug. [1] Secondly, most of the active ingredients of Chinese herbal medicine are in macromolecular structures and are difficult to penetrate the intestinal mucosa and be effectively absorbed and utilized by the human body. Enzymes such as glycosidase and protease produced by microorganisms during fermentation can degrade macromolecules into small molecule forms such as oligosaccharides and amino acids, which are then more easily absorbed, transported and utilized. The lignocellulase in probiotics can promote the release of bioactive natural products in Chinese herbal medicine by degrading the plant cell wall and produce oligosaccharide prebiotics. Taking ginseng as an example, studies have found that after fermentation with yeast, the contents of the main active ingredients of ginseng, such as 20(S)-Rg3, Rh2 and F2, have increased significantly, reaching 269.87%, 198.46% and 153.98% of unfermented ginseng respectively, indicating that fermentation can effectively increase the content of the medicinal components of ginseng.

[0003] Second, new active substances are produced. During the metabolic process of microorganisms, a series of primary and secondary metabolites can be produced. These metabolites can react chemically with the active or inactive components in Chinese herbal medicine materials, and then generate new precursor compounds. Taking Panax notoginseng as an example, after fermenting it with Bacillus subtilis, ginsenoside RH4 can be newly generated, thereby enhancing its original medicinal efficacy or endowing it with new pharmacological activities. In addition, studies have found that Lactobacillus plantarum can convert ginsenosides Rb2 and Rb3 into ginsenoside Rd with higher biological activities during the fermentation of red ginseng.

[0004] Third, the toxicity of Chinese herbal medicine is reduced. Chinese herbal medicine often contains macromolecular toxic substances, which will produce certain toxic and side effects after being taken. Through microbial fermentation, the structure of toxic components can be transformed to reduce toxicity. For example, although Aristolochia debilis is traditionally used for lowering blood pressure and relieving pain, the aristolochic acid it contains can cause kidney diseases. After being fermented with medicinal fungi, the content of aristolochic acid is significantly reduced, and the safety is improved. Fermenting the mother drug of Huafengdan with Bacillus, Pediococcus and Enterobacter can significantly reduce the content of highly toxic alkaloids, and then reduce the potential toxic and side effects of the mother drug of Huafengdan.

[0005] Fourth, resources are saved and the environment is protected. Chinese herbal medicine residues are rich in nutrients such as protein, nitrogen source, carbon source and carbohydrates, which can provide suitable conditions for the growth and reproduction of microorganisms, thereby reducing production costs and saving precious medicinal resources. For example, using the herbal residues generated during the production of Jianweixiaoshi tablets fermented by Lactobacillus plantarum HM218749, it was found that the supernatant after fermentation showed strong anti-Helicobacter pylori activity against mice.

[0006] Chinese herbal medicine resources are abundant, but the traditional processing methods cannot fully utilize the medicinal components in them. Especially for low-grade Chinese herbal medicine materials, they are often wasted. Therefore, it is particularly important to find a processing method that can effectively improve the medicinal efficacy of Chinese herbal medicine and improve its flavor. Summary of the Invention

[0007] The present invention relates to Eurotium cristatum FZ-4 with a high-yield cellulase system and its application in fermenting Chinese herbal medicine to improve the medicinal efficacy and flavor. Through its efficient enzyme systems such as cellulase, hemicellulase, and glucosidase, this strain performs solid-state fermentation on Chinese herbal medicine, significantly improving the dissolution rate of the medicinal components of Chinese herbal medicine, improving its flavor characteristics, and generating new medicinal molecules.

[0008] The present invention first uses a PDA medium modified with esculin as a screening culture condition to preliminarily screen out strains with the ability to produce β-glucosidase. On the PDA medium modified with esculin, strains that can secrete β-glucosidase will form black transparent circles around their colonies. According to the size of the black circle, it can be effectively determined whether the strain has the ability to produce β-glucosidase. Through screening, 5 strains of Eurotium guanidum with strong ability to produce β-glucosidase were obtained. On the basis of the 5 strains of Eurotium guanidum that produced β-glucosidase that were screened, the activities of cellulase, hemicellulase and lignin peroxidase produced by different strains were further compared. Through comparative analysis, the strain Eurotium guanidum FZ-4 with high comprehensive enzyme production activity was selected.

[0009] On the other hand, the volatile odor substances of Chinese herbal medicines (represented by Pseudostellaria heterophylla) before and after fermentation with Eurotium cristatum FZ-4 were analyzed by GC×GC / MS technology. The results showed that the unpleasant odor substances such as 2-butylcyclohexanone and geosmin in Pseudostellaria heterophylla disappeared or decreased after fermentation, and a series of aroma components with rose and wood fragrance were generated, which changed the smell of fermented Pseudostellaria heterophylla from grassy and earthy to fruity and floral.

[0010] On the other hand, under suitable fermentation conditions, the various extracellular enzymes produced by Eurotium guanidinum FZ-4 can catalyze the degradation of the cell wall of Pseudostellaria heterophylla and promote the release of active ingredients. At the same time, Eurotium guanidinum FZ-4 may produce a series of primary and secondary metabolites during the metabolic process, which react chemically with the active or inactive ingredients in Pseudostellaria heterophylla to generate new precursor compounds, thereby enhancing its original efficacy or giving it new pharmacological activity.

[0011] On the other hand, the fingerprint of fermented Chinese herbal medicine alcohol extract represented by Radix Pseudostellariae was constructed based on HPLC analysis. By comparing and analyzing the absorption peaks of different substances in the alcohol extract of Radix Pseudostellariae before and after fermentation, it was found that the composition and content of substances in the alcohol extract of Radix Pseudostellariae showed significant changes during the fermentation process. After LC / MS detection and spectral analysis, a variety of pharmacologically active compounds were successfully identified, such as Radix Pseudostellariae cyclopeptides, alkaloids, etc.

[0012] In a specific embodiment, the antioxidant activity and anticancer activity of the alcohol extract of Chinese herbal medicine represented by Radix Pseudostellariae were evaluated by in vitro experiments. The results showed that the alcohol extract of Radix Pseudostellariae had no significant effect on DPPH free radicals, hydroxyl free radicals, ABTS free radicals, and hydroxyl radicals. + The free radical scavenging ability and total antioxidant capacity showed strong antioxidant activity. At the same time, the fermented Pseudostellariae ethanol extract also significantly enhanced the growth inhibition ability of Hela cells, Caco-2 cells and PC-9 cells.

[0013] In summary, the present invention provides a technology for fermenting Chinese herbal medicines using specific microbial strains. This technology screens strains with specific enzyme activities and performs solid-state fermentation on Chinese herbal medicines, not only improving the utilization rate of the medicinal components and flavor characteristics of Chinese herbal medicines, but also potentially generating new medicinal molecules. This technology provides new ideas and methods for the intensive processing and high-value comprehensive utilization of Chinese herbal medicines.

[0014] Application effects of the present invention: (1) Improve the dissolution rate of active ingredients in Chinese herbal medicines, thereby increasing the medicinal value; (2) Improve the flavor characteristics of Chinese herbal medicines, eliminate unpleasant odors, and endow them with a more pleasant flavor; (3) Significantly enhance the antioxidant and anticancer activities of Chinese herbal medicines, and improve their health care functions or medicinal effects. Description of the drawings

[0015] Figure 1 : Morphological diagrams of the colonies of 5 strains of Eurotium cristatum and 5 strains of Monascus purpureus on aesculin-modified PDA medium.

[0016] Figure 2 : Detection results of the activities of four cellulases produced by 5 strains of Eurotium cristatum during the fermentation process. A: β-glucosidase; B: Cellulase; C: Hemicellulase; D: Lignin peroxidase.

[0017] Figure 3 : Heat map of the differential analysis of volatile odor substances of Pseudostellaria heterophylla after fermentation treatment with Eurotium cristatum FZ-4 for different times.

[0018] Figure 4 : Comparison chromatogram of the liquid phase of the ethanol extract of Pseudostellaria heterophylla before and after fermentation treatment with Eurotium cristatum FZ-4. The red line represents the group fermented for 15 days, TZS15; the black line represents the group fermented for 0 days, TZS0.

[0019] Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 : Secondary mass spectrometry diagram of the drug components with increased content in the positive ion mode of the ethanol extract of Pseudostellaria heterophylla before and after fermentation treatment with Eurotium cristatum FZ-4 analyzed by HPLC-QTOF-MS / MS.

[0020] Figure 11 : In vitro antioxidant activity analysis diagram of the ethanol extract of Pseudostellaria heterophylla before and after fermentation treatment with Eurotium cristatum FZ-4. A: DPPH; B: Hydroxyl radical; C: ABTS + ; D: Total antioxidant capacity.

[0021] Figure 12 : In vitro inhibitory effects of the ethanol extracts of Pseudostellaria heterophylla before and after fermentation with Eurotium cristatum FZ-4 on cancer cell proliferation. A: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group on the proliferation of HeLa cells; B: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group on the proliferation of HeLa cells; C: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group on the proliferation of Caco-2 cells; D: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group on the proliferation of Caco-2 cells; E: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group on the proliferation of PC-9 cells; F: Inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group on the proliferation of PC-9 cells. Detailed implementation mode

[0022] The following is a detailed description of the implementation mode of the present invention to specifically illustrate how to use Eurotium cristatum FZ-4 with high cellulase productivity to improve the flavor of Chinese herbal medicines, increase the yield of active pharmaceutical ingredients, generate new bioactive ingredients and enhance the antioxidant and anti-tumor activities of Chinese herbal medicines. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0023] Example 1: Screening of high-yield β-glucosidase strains Five Monascus strains and five Eurotium cristatum strains were taken out from the slant medium, inoculated onto the solid PDA medium, and then inverted and placed in a constant temperature and humidity incubator for 3-5 days at 28°C; a small amount of mycelium was selected and transferred to a fresh PDA medium for secondary activation, and continued to be cultured for 3-5 days under the same conditions; spores were picked from the solid PDA medium, diluted with 0.9% sterile physiological saline, and accurately counted using a hemocytometer until the concentration of the spore suspension reached 10 6 cells / mL for subsequent experiments. Esculin-modified PDA medium: 1 L of solid PDA medium, 1 g of esculin, and 0.5 g of ferric ammonium citrate. The spore suspension was spotted on the esculin-modified PDA medium at 100 μL per drop, with 3 drops spotted on each medium, and the color change of the medium was observed after culturing at 28°C for 5 days. If the color of the medium changed to brownish-black, it could be determined that the strain had the ability to produce β-glucosidase.

[0024] As Figure 1As shown in the figure, on the aesculin-modified PDA medium, strains capable of secreting β-glucosidase will form a black transparent circle around their colonies. According to the size of the black circle, it is possible to effectively determine whether the strain has the ability to produce β-glucosidase. Through screening, 5 strains of Eurotium cristatum with strong β-glucosidase-producing ability were obtained.

[0025] Example 2: Determination of the cellulase-producing ability of Eurotium cristatum The 5 strains of Eurotium cristatum producing β-glucosidase were each activated twice in liquid PDA medium. The culture solution was centrifuged (8000 rpm, 4 °C, 10 min), and the resulting supernatant was the crude enzyme solution. The enzyme activities of its β-glucosidase, cellulase, hemicellulase, and lignin peroxidase were measured.

[0026] Precisely pipette 25 μL of the crude enzyme solution, add 100 μL of 4-nitrophenyl-β-D-glucopyranoside (5 mmol / L) preheated for 10 min, and mix well by pipetting. Incubate in a 50 °C water bath for 30 min. Add 125 μL of Na 2 CO 3 (1 mol / L) to terminate the reaction, observe the color change, and measure the absorbance at 405 nm with an enzyme-linked immunosorbent assay reader. Use the crude enzyme solution inactivated in a 100 °C water bath for 10 min as the blank control; use p-nitrophenol as the standard control, draw the standard curve, and calculate the β-glucosidase enzyme activity according to formula (1): Y: Absorbance at 405 nm; K: Slope of the p-nitrophenol standard curve; b: Intercept of the p-nitrophenol standard curve; V 1 : Total volume of the reaction system (mL); t: Reaction time (min); V 2 : Volume of the enzyme in the reaction system (mL); M: Molecular mass of p-nitrophenol 139.109.

[0027] Under the condition of a 50 °C water bath, add 30 μL of the crude enzyme solution to 60 μL of a 10 mg / mL carboxymethyl cellulose sodium solution, mix well, and incubate in a 50 °C water bath for 30 min. Then add 150 μL of DNS solution, mix well, and incubate in a 100 °C water bath for 10 min. Immediately after the reaction ends, cool it in an ice bath to room temperature, and add 1260 μL of distilled water. After mixing, take 200 μL of the reaction solution into a 96-well plate and measure its absorbance at 540 nm. Use the crude enzyme solution inactivated in a 100 °C water bath for 10 min as the blank control; use glucose as the standard, draw the standard curve, and calculate the cellulase enzyme activity according to formula (2-2): U: Cellulase enzyme activity; A: Glucose content (μg); V: Enzyme solution volume (μL); t: Reaction time (min).

[0028] The method for determining the activity of hemicellulase is as follows: Add 30 μL of crude enzyme solution to 60 μL of xylan (5 mg / mL) solution under the condition of 50 °C water bath. After uniform mixing, keep it in the 50 °C water bath for 30 min. Then add 150 μL of DNS solution, and after uniform mixing, keep it in the 100 °C water bath for 10 min. Immediately after the reaction, cool it in an ice bath to room temperature, and add 1260 μL of distilled water. After mixing, take 200 μL of the reaction solution into a 96-well plate and measure its absorbance at 540 nm. Use the crude enzyme solution inactivated after 10 min in the 100 °C water bath as the blank control; Use xylose as the standard product to draw the standard curve, and calculate the hemicellulase activity according to formula (3): In the formula, A 1 : Absorbance of the enzyme reaction solution; A 2 : Absorbance of the blank sample; K: Slope of the standard curve; C: Intercept of the standard curve; t: Enzymolysis time (min).

[0029] Add 30 μL of crude enzyme solution to 200 μL of veratryl alcohol solution (10 mmol / L) and 400 μL of tartaric acid-sodium tartrate buffer solution (250 mmol / L, pH 3.0) under the condition of 30 °C water bath. Add 20 μL of H 2 O 2 solution (20 mmol / L) and react for 3 min, then measure the absorbance at 310 nm. Use distilled water instead of H 2 O 2 solution as the sample blank. Calculate the lignin peroxidase activity according to formula (4): In the formula, A 1 : Absorbance of the sample; A 0 : Absorbance of the sample blank; ε: Molar extinction coefficient: ε310 = 9300 (mol / L / min); t: Reaction time (min); V 1 : Total volume of the reaction solution (mL); V 2 : Total volume of the fermentation broth (mL); n: Dilution factor.

[0030] As Figure 2 shown, on the basis of 5 strains of Eurotium cristatum that can produce β-glucosidase, further compare the enzyme activities of the crude enzyme solutions of different strains. Through comparative analysis, select the strain Eurotium cristatum FZ-4 with high enzyme production activity among them.

[0031] Eurotium cristatum FZ-4 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 25, 2019. It was taxonomically named Eurotium cristatum, with the deposit number CGMCC No. 17186, and the deposit address is No. 3, Courtyard 1, Beizhan West Road, Chaoyang District, Beijing.

[0032] Example 3: Solid-state fermentation of Pseudostellaria heterophylla Eurotium cristatum FZ-4 was used for the solid-state fermentation of Pseudostellaria heterophylla. The specific method was as follows: Pseudostellaria heterophylla was dried in a forced-air drying oven at 65 °C for 4 h, pulverized, and sieved through a 40-mesh sieve to obtain Pseudostellaria heterophylla powder. Exactly 40.0 g of the Pseudostellaria heterophylla powder was placed into a 250-mL food-grade canning bottle, and deionized water was added at a mass ratio of Pseudostellaria heterophylla: deionized water of 2:1. It was sterilized at high temperature (121 °C, 20 min), and after cooling, 4 mL of the Eurotium cristatum FZ-4 spore suspension (1×10 5 cells / mL) was inoculated, stirred evenly, sealed, and placed in a constant temperature and humidity incubator at 28 °C for static fermentation for 15 days. Sampling was carried out in a laminar flow hood on the 0th, 3rd, 6th, 9th, 12th, and 15th days of fermentation, packaged and sealed with a sterile sampling bag, freeze-dried, and ground into powder to obtain freeze-dried powder of fermented Pseudostellaria heterophylla for subsequent index detection.

[0033] Example 4: Detection of volatile flavor substances 0.5 g of the freeze-dried powder sample of fermented Pseudostellaria heterophylla, 2 g of NaCl solution, 6 mL of ultrapure water, and 10 μL of 2-octanol (10 mg / L) were placed in a headspace vial. The vial was placed in an automatic constant-temperature magnetic water bath, equilibrated at 70 °C for 15 min, and then the extraction head was inserted for extraction for 45 min under the same conditions, with shaking maintained during the extraction process. After the extraction was completed, the extraction head was inserted into the gas chromatography injection port and desorbed at 250 °C for 3 min. After extracting one sample, the extraction head needed to be aged for 3 min. A comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry was used, which has advantages such as high chromatographic resolution, high resolution, and high sensitivity. Chromatographic column conditions: An HP-INNOWAX capillary chromatographic column (specification 0.25 μm × 30 m × 0.25 mm) was used. The temperature programming: The initial temperature of the column oven was 40 °C and held for 3 min; it was raised to 150 °C at a rate of 3 °C / min and held for 3 min; then it was raised to 250 °C at a rate of 5 °C / min and held for 2 min. Interface temperature: 250 °C; carrier gas: helium (He); flow rate: 1 mL / min, splitless injection. Mass spectrometry conditions: Quadrupole temperature: 150 °C; EI ion source was selected; electron energy: 70 eV; ion source temperature was 200 °C; transfer line temperature 250 °C; interface temperature 240 °C; SCAN mode was selected; mass scanning range 30 - 450 amu. The content of volatile odor substances was calculated according to formula (5).

[0034] As Figure 3 shown, the fermentation treatment of Eurotium cristatum FZ-4 significantly changed the composition of volatile odor substances in Pseudostellaria heterophylla. Newly generated polyaromatic substances such as phenylethyl alcohol, hinokitiol, and β-jasmol added fresh odors such as rose, wood, and citrus to the fermented Pseudostellaria heterophylla. In addition, the contents of original substances such as hexanal and phenylacetaldehyde were increased after fermentation, further enriching the fresh and sweet odors. At the same time, the fermentation treatment of Eurotium cristatum FZ-4 effectively removed the unpleasant odor components in Pseudostellaria heterophylla, such as the rancid and earthy odors caused by 2-butylcyclohexanone and geosmin, as well as the fishy, grassy, and greasy odors brought by nonanal, decanal, and n-octanal.

[0035] Example 5: Preparation of ethanol extract of fermented Pseudostellaria heterophylla Take 0.1 g of freeze-dried powder of fermented Pseudostellaria heterophylla at different fermentation times, add 10 mL of methanol, extract by ultrasonic wave (power 300 W, temperature 25-45 °C) for 1 h, centrifuge (3900 g, 10 min), and collect the supernatant, which is the ethanol extract of fermented Pseudostellaria heterophylla.

[0036] Example 6: Fingerprint and mass spectrometry analysis of ethanol extract of fermented Pseudostellaria heterophylla The ethanol extract of fermented Pseudostellaria heterophylla was centrifuged at 10000 g for 10 min. After the supernatant was filtered through a 0.22 μm organic filter membrane, the filtrate was used for HPLC and HPLC-QTOF-MS / MS analysis. Chromatographic conditions: Gradient elution was carried out with acetonitrile (A)-1% acetic acid aqueous solution (B) as the mobile phase. The chromatographic column was an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm) chromatographic column, column temperature: 30 °C; an ultraviolet detector was used, detection wavelength: 254 nm; injection volume: 10 μL, mobile phase flow rate: 1.0 mL / min. The gradient elution program is shown in Table 1. Mass spectrometry conditions: Scanning was carried out in the negative ion mode, the scanning range was 50-600 m / z, the collision energy was 10.00, 20.00, 40.00 eV, the dry gas flow rate was 10 L / min, the gas used in the experiment was nitrogen (purity > 99.9%), the collision gas was high-purity nitrogen (purity > 99.999%), the dry gas temperature was 350 °C, the spray pressure was 40 psig, the capillary voltage was 3.5 kV, and the acquisition frequency was 1 spectra / sec.

[0037] Table 1 Gradient elution program of mobile phase As Figure 4As shown, there are more substance absorption peaks in the group fermented for 15 days compared to the group fermented for 0 days; according to the differential analysis of the spectra, at the same injection concentration, after fermentation with Eurotium cristatum FZ-4, the peak areas of some original components of Pseudostellaria heterophylla significantly increase, and at the same time, new metabolites appear.

[0038] Meanwhile, 12 differential substances were identified from the group fermented for 15 days and the group fermented for 0 days by LC / MS, including 8 Pseudostellaria heterophylla cyclopeptides, 3 alkaloids, and aspergillus flavipes pigment ( Figures 5 to 10 ). Among them, after fermentation with Eurotium cristatum FZ-4, the content of Pseudostellaria G decreases, while the contents of Pseudostellaria A, Pseudostellaria B, Pseudostellaria C, Pseudostellaria E, Heterophyllin A, Heterophyllin B, and Heterophyllin D all increase; at the same time, Neoechinulin A, echinochrome, alkaloid E-7, and aspergillus flavipes pigment are newly added to the fermentation products.

[0039] Example 7: Determination of antioxidant indexes of ethanol extract of fermented Pseudostellaria heterophylla (1) DPPH radical scavenging rate Reagent preparation: Preparation of 0.2 mM DPPH solution: 0.0197 g of DPPH is made up to 250 ml with absolute ethanol (it is best to prepare it freshly and use it immediately). Determination method: Take 1 mL of the test solution, add 1 mL of 0.2 mM / L DPPH reagent to a test tube and mix well. React in the dark for 30 min, and measure the absorbance value A at 517 nm using an ultraviolet spectrophotometer. 1 Add 1 mL of absolute ethanol to 1 mL of the test solution, and measure the absorbance value at 517 nm as A 空白 ; Add 1 mL of absolute ethanol to 1 mL of DPPH solution and measure the absorbance value at 517 nm as A 对照 . Calculate the DPPH radical scavenging rate according to formula (6).

[0040] (2) Hydroxyl radical scavenging rate Prepare 9 mmol / L ethanol-salicylic acid solution, 9 mmol / L FeSO 4 solution, and 8.8 mmol / L H 2 O 2 solution. Determination method: Pipette 0.2 mL of FeSO 4 solution and 0.2 mL of H 2 O 2The solution is placed in a 2 mL centrifuge tube. After mixing, 0.2 mL of ethanol-salicylic acid solution, the test solution, and deionized water are added respectively. After mixing, the reaction is carried out in the dark at 37 °C for 15 min, and then the absorbance A is measured at 510 nm. x In the blank group, 1 mL of deionized water is used to replace the H 2 O 2 solution, and the absorbance is denoted as A 空白 ; In the control group, 1 mL of methanol is used to replace the test solution, and the absorbance is denoted as A 对照 . The hydroxyl radical scavenging rate is calculated according to formula (7).

[0041] (3) ABTS + radical scavenging rate ABTS working solution: Take 5 mL of 7 mmol / L ABTS solution and 5 mL of 2.45 mmol / L potassium persulfate solution, mix them evenly, and react in the dark for 12 h. Then dilute its absorbance value to 0.20 ± 0.05 (734 nm). The working solution is prepared and used immediately. Measurement method: Add 0.2 mL of the test solution to a 10 mL centrifuge tube, then add 3.8 mL of ABTS working solution, let it stand in the dark for 15 min, and measure its absorbance at 734 nm, denoted as A i . Mix 0.2 mL of methanol with 3.8 mL of ABTS working solution, and measure its absorbance at 734 nm, denoted as A 对照 . Mix 0.2 mL of the test solution with 3.8 mL of methanol solution, and measure its absorbance at 734 nm, denoted as A 空白 . Calculate the ABTS+ radical scavenging rate according to formula (8).

[0042] (4) Determination of total antioxidant capacity - FRAP method Reagent preparation: Prepare 0.3 mol / L acetate solution, 0.01 mol / L TPTZ solution, 0.02 mol / L FeCl 3 solution and 1 mmol / L FeSO 4 solution, and dilute the FeSO 4 solution to different concentrations (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mmol / L). FRAP working solution: Mix the above three solutions in a volume ratio of 10:1:1. Measurement method: Take 0.4 mL of FeSO 4 solution, add 3.6 mL of FRAP working solution, mix well and incubate in a water bath at 37 °C for 10 min, and measure the absorbance at 593 nm. Take the FeSO 4 solution concentration as the abscissa and the absorbance as the ordinate to establish the FeSO 4Equivalent standard curve. Take 0.4 mL of the sample solution, add 3.6 mL of the FRAP working solution, mix well, incubate in a water bath at 37 °C for 10 min, and measure the absorbance at 593 nm. Substitute the absorbance value into the standard curve to calculate the FeSO 4 equivalent.

[0043] As Figure 11 shown in A, the DPPH radical scavenging rate of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group was 63.6%. With the extension of the fermentation time, its DPPH radical scavenging rate showed a significant increasing trend, and the DPPH radical scavenging rate of the ethanol extract of Pseudostellaria heterophylla in the 6-day fermentation group reached 92.7%.

[0044] As Figure 11 shown in B, the hydroxyl radical scavenging rate of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group was 45%. With the extension of the fermentation time, its hydroxyl radical scavenging rate showed a significant increasing trend, reaching the maximum value at 15 days of fermentation. Compared with the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group, the hydroxyl radical scavenging rate increased by 14.7%.

[0045] As Figure 11 shown in C, the ABTS + radical scavenging ability of the ethanol extract of Pseudostellaria heterophylla decreased first and then gradually increased during the fermentation process. The ABTS + radical scavenging ability of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group was 87%, while the ABTS + radical scavenging ability reached the highest value of 98% at the end of fermentation.

[0046] As Figure 11 shown in D, the total antioxidant capacity of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group was 0.60 mmol / L. After fermentation with Eurotium cristatum FZ-4, its total antioxidant capacity gradually increased. The total antioxidant capacity of the ethanol extract of Pseudostellaria heterophylla reached the highest value of 0.99 mmol / L at 15 days of fermentation. Compared with the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group, the total antioxidant capacity of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group increased by 0.65 times.

[0047] Based on the above analysis, it can be concluded that the fermentation treatment with Eurotium cristatum FZ-4 significantly enhanced the antioxidant activity of the ethanol extract of Pseudostellaria heterophylla.

[0048] Example 8: Determination of the in vitro anti-tumor activity of the ethanol extract of Pseudostellaria heterophylla before and after fermentation Take out the cryopreservation tubes of Hela, PC-9, and Caco-2 cells from the liquid nitrogen tank, place them in a 37°C constant temperature water bath and shake rapidly to thaw. In the laminar flow hood, aspirate the melted cell suspension into a centrifuge tube, centrifuge (4 min, 1000 r), discard the upper cryopreservation solution, add 1 mL of pre-warmed DMEM medium containing 10% fetal bovine serum, gently and slowly disperse the cells with a pipette, transfer to a culture dish, and then aspirate 4 mL of medium and add it to the culture dish. Shake crosswise to evenly disperse the cells in the dish. After observing under an inverted microscope, place it in a 5% carbon dioxide, 37°C constant temperature incubator for 24 h, observe the cell status, and replace with fresh medium. When the cell density reaches 70% - 80%, passage the cells. Use a pipette to aspirate and discard the culture medium, aspirate 4 mL of PBS buffer and wash twice, add 1 mL of medium, gently scrape the cells with a cell scraper, transfer the cell suspension to a centrifuge and centrifuge (1000 r, 2 min), discard the upper layer of medium, add 1 mL of medium to resuspend the cells, aliquot the cell suspension into 2 10-cm culture dishes, add 10 mL of medium to each culture dish, and blow the cells evenly with a pipette. After observing under the microscope, place it in a 5% carbon dioxide, 37°C constant temperature incubator for culture. After the cells have been passaged 2 - 3 times and the cell viability has recovered, perform the CCK-8 assay. Observe under the microscope that the Hela, PC-9, and Caco-2 cells basically cover more than 80% of the bottom of the culture dish and are in good condition. Aspirate and discard the old medium, wash twice with PBS buffer, gently scrape the cells with a cell scraper, then add an appropriate amount of medium to dilute, and perform cell counting. Seed the cells at a density of 1×10 5 cells / mL into a 96-well plate, and add 100 μL of cell suspension to each well. Add an appropriate amount of PBS buffer (0.01 M, pH 7.2) to the periphery of the 96-well plate, and place it in a 5% carbon dioxide, 37°C constant temperature incubator for 24 h.

[0049] After freeze-drying the ethanol extract of fermented Pseudostellaria heterophylla, a stock solution with a concentration of 1 mg / L was prepared using DMEM medium containing 10% fetal bovine serum, and then diluted with DMEM medium containing 10% fetal bovine serum into sample working solutions with concentrations of 0.0078 mg / L, 0.0156 mg / L, 0.0313 mg / L, 0.0625 mg / L, 0.125 mg / L, 0.25 mg / L, 0.25 mg / L, 0.5 mg / L, and 1 mg / L respectively. Take out the 96-well plate at the end of the culture, aspirate and discard the old medium. After washing each well with PBS, add 100 μL of DMEM medium containing 10% fetal bovine serum to the blank group, and add 100 μL of the sample working solution to the experimental group. Set 6 parallels for each group, place them in an incubator with 5% carbon dioxide at 37 °C for 24 h. Aspirate and discard the old medium, wash with PBS, then add 100 μL of 10% CCK-8 indicator to each well, and then place it in an incubator with 5% carbon dioxide at 37 °C for incubation for 1 h. After the incubation is completed, transfer it to a microplate reader for detection. Set the wavelength at 450 nm, and calculate the cell viability according to formula (9).

[0050] As Figure 12 shown in , when the concentration of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group exceeded 0.5 mg / mL, it could significantly inhibit the proliferation of HeLa cells (P < 0.001), and showed a good dose-dependence; but when the concentration was lower than 0.125 mg / mL, the viability of HeLa cells increased slightly compared with the control group.

[0051] As Figure 12 shown in , when the concentration of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group was lower than 0.125 mg / mL, it had no significant inhibitory effect on HeLa cells. When the concentration exceeded 0.5 mg / mL, it had an obvious inhibitory effect on HeLa cells (P < 0.05), and when the concentration was 2 mg / mL, the inhibitory effect on HeLa cells was significantly stronger than that of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group.

[0052] As Figure 12 shown in , when the concentration of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group was 0.0625 mg / mL, the survival rate of Caco-2 cells had no significant difference from that of the control group; but when the concentration exceeded 0.125 mg / mL, the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group could significantly inhibit the proliferation of HeLa cells (P < 0.001), and showed a good dose-dependence.

[0053] As Figure 12As shown in Figure D, after the Pseudostellaria heterophylla was fermented by Eurotium cristatum FZ-4, its ethanol extract showed a stronger inhibitory effect on the proliferation of Caco-2 cells, and could still significantly reduce the survival rate of Caco-2 cells at the lowest concentration of 0.0625 mg / mL (P<0.001); the inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group on the viability of Caco-2 cells was stronger than that in the 0-day fermentation group, indicating that the fermentation treatment with Eurotium cristatum FZ-4 could significantly enhance the inhibitory effect of the ethanol extract of Pseudostellaria heterophylla on Caco-2 cells.

[0054] As Figure 12 shown in Figure E, when the concentration of the ethanol extract of Pseudostellaria heterophylla in the 0-day fermentation group exceeded 0.125 mg / mL, it could significantly inhibit the proliferation of PC-9 cells (P<0.001), and showed a good dose-dependence; when the concentration of the ethanol extract was 2 mg / mL, the cell survival rate was only 34.15%.

[0055] As Figure 12 shown in Figure F, when the concentration of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group was 2 mg / mL, the survival rate of PC-9 cells was 17.00%, and when the concentration was reduced to 0.0625 mg / mL, the survival rate of PC-9 cells was 89.04%; at each concentration gradient, the inhibitory effect of the ethanol extract of Pseudostellaria heterophylla in the 15-day fermentation group on the viability of PC-9 cells was stronger than that in the 0-day fermentation group.

Claims

1. A strain of Eurotium cristatum FZ-4 that produces high cellulase and can be used to ferment Chinese herbal medicines, characterized by: The above-mentioned Eurotium cristatum FZ-4 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on February 25, 2019, and was classified and named Eurotium cristatum ( Eurotium cristatum ), the deposit number is CGMCC NO.17186, and the deposit address is No. 3, Yard 1, Beizhan West Road, Chaoyang District, Beijing.

2. The use of Eurotium cristatum FZ-4 as claimed in claim 1, characterized in that: Any one or more of the following: 1) Application in cellulase production system; 2) Application in the preparation of enzyme preparations with cellulase activity; 3) Application in improving the efficacy of fermented Chinese herbal medicines; 4) Application in improving flavor of fermented Chinese herbal medicines.

3. The use according to claim 2, characterized in that: The cellulase system includes beta-glucosidase, cellulase, hemicellulase and lignin peroxidase.

4. The use according to claim 2, characterized in that: The Chinese herbal medicines include but are not limited to Pseudostellaria heterophylla, Ginseng, Lycium barbarum, Angelica sinensis, and Astragalus membranaceus.

5. The use according to claim 2, characterized in that: The drug effects include antioxidant activity and anti-tumor activity.

6. The use according to claim 2, characterized in that: The flavor improvement refers to changing the composition and content of volatile odor substances.

7. A method for fermenting Chinese herbal medicine using the Eurotium cristatum FZ-4 of claim 1, characterized in that: The Chinese herbal medicine is dried, crushed and then sieved to obtain Chinese herbal medicine powder; deionized water is added to the Chinese herbal medicine powder, sterilized at high temperature, inoculated with Aspergillus niger after cooling, stirred evenly, and allowed to ferment to obtain a fermentation product; methanol is added to the fermentation product, ultrasonic extraction is performed, and centrifugation is performed, and the obtained supernatant is the fermented Chinese herbal medicine alcohol extract.

8. The method according to claim 7, characterized in that: The Chinese herbal medicines include but are not limited to Pseudostellaria heterophylla, Ginseng, Lycium barbarum, Angelica sinensis, and Astragalus membranaceus.

9. A fermented Chinese herbal medicine product, characterized in that: Prepared by the method described in any one of claims 7 to 8.

10. The use of the fermented Chinese herbal medicine product according to claim 9, characterized in that: Any one or more of the following: 1) Application in the preparation of antioxidant drugs; 2) Application in the preparation of anti-tumor drugs.

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