Aspergillus schermanii with high cellulase yield and application of aspergillus schermanii in fermentation of Chinese herbal medicines

Abstract

The invention provides aspergillus scherweri for high-yield cellulase and application of the aspergillus scherweri in fermentation of Chinese herbal medicines. The Aspergillus scherweri J-10 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Aspergillus scherweri J-10 is CGMCC No. 41630. The Aspergillus scherweri J-10 disclosed by the invention has the advantages that the Aspergillus scherweri J-10 can be used for preparing the Aspergillus When the aspergillus scherweri provided by the invention is used for fermenting Chinese herbal medicines represented by lucid ganoderma, the release and utilization rate of active ingredients of the Chinese herbal medicines can be remarkably improved. An in-vitro anti-tumor activity result shows that the alcohol extract of the Chinese herbal medicine (represented by ganoderma lucidum sporocarp) fermented by the J-10 strain has a stronger in-vitro inhibition effect on various tumor cells, and a new method and way are provided for deep processing and comprehensive utilization of the Chinese herbal medicine.

Description

Technical Field

[0001] The present invention relates to the technical fields of Chinese herbal medicine processing, microorganisms and their applications, and specifically relates to a strain of Aspergillus chevalieri with high cellulase productivity ( Aspergillus chevalieri ) and its application in the fermentation of Chinese herbal medicine. Background Art

[0002] Chinese herbal medicines have been used as traditional medicines for thousands of years. They are rich in various active substances such as polysaccharides, proteins, saponins, alkaloids, and organic acids, and have immunomodulatory, anti-inflammatory, antidiabetic, and anti-allergic activities, covering various therapeutic fields. However, due to the fact that the active ingredients of traditional Chinese medicines are encapsulated by cell walls, the extraction rate of effective ingredients is relatively low, resulting in waste of resources. Microorganisms can use cellulase, glycosidase to hydrolyze cell walls or macromolecular glycoside complexes to release medicinal ingredients; in addition, microorganisms can also use some active substances of Chinese herbal medicines as metabolic substrates for biotransformation and modification to improve the efficacy and utilization rate of Chinese herbal medicines. After fermentation, some Chinese herbal medicines can also reduce toxic and side effects and even produce new active substances. Compared with general physical or chemical means of processing Chinese herbal medicines, the fermentation of Chinese herbal medicines by microorganisms has its unique advantages. Microbial fermentation can effectively improve the efficacy of some Chinese herbal medicines, reduce toxic and side effects, and may produce completely new effective active ingredients. Most of the active ingredients of Chinese herbal medicines are present inside the plant cell walls, resulting in a low bioavailability of their effective ingredients. During the fermentation process, microorganisms will produce various extracellular enzymes such as cellulase, ligninase, and pectinase, which catalyze the degradation of plant cell walls and promote the release of effective ingredients of Chinese herbal medicines. In addition, the molecular weights of the sexual components in traditional Chinese medicines are usually large and it is not easy to penetrate the blood-brain barrier. The enzymes secreted by microorganisms can also degrade these macromolecular substances, making them more easily absorbed by the body. Fermentation of safflower by Bacillus licheniformis has been proven to hydrolyze the glycosides in safflower into aglycones that are more easily absorbed by the body, and significantly improve its ability to scavenge hydroxyl radicals and inhibit the generation of liver oxides. During the growth and metabolism process of microorganisms, rich primary and secondary metabolites (such as antibiotics, peptides, pigments, enzymes, and growth factors) will be produced. These substances can take the active ingredients or even non-active ingredients of Chinese herbal medicines as precursors, and through various biotransformation effects such as isomerization, methylation, and acetylation, the structure is modified to generate new active ingredients. Ginsenosides are the main physiological active natural products of ginseng. Ginsenoside Rb1, Rb2, Rc, Re, and Rg1 account for more than 80% of the total ginsenosides. Some rare ginsenosides, such as F2 and Rd, have been proven to have high bioavailability and biological activities. However, their contents in natural ginseng are extremely low. Probiotics Bifidobacterium animalissubsp. lactis LT 19-2, which can effectively convert ginsenosides Rb2 and Rb3 in red ginseng into rare ginsenosides Rd, Rh1, F2 and Rg3. In addition, many traditional Chinese medicines have certain toxicity and side effects when used. If they are not used according to syndrome differentiation, they are likely to cause liver and kidney damage. Microbial fermentation can utilize the decomposition and transformation of microorganisms to degrade the toxic substances of some Chinese herbal medicines, or to structurally modify their toxic components. It is a good way to minimize the side effects of traditional Chinese medicines and reduce adverse reactions. Conjugated anthraquinone is the main component that causes severe diarrhea in rhubarb. Kluyveromyces marxianus KM12 fermented rhubarb can convert bound anthraquinone into free anthraquinone, thereby reducing the severe diarrhea side effect caused by rhubarb. At the same time, the active ingredients of Chinese medicinal materials are mostly present in the cytoplasm. When extracting the active ingredients, they will encounter dual resistance from the cytoplasm and cell wall. Therefore, many Chinese medicinal materials can improve the utilization rate of precious medicinal resources and solve the potential problem of environmental pollution of Chinese medicinal materials after fermentation. For example, using Lactobacillus plantarum The herbal residues produced during the production of HM218749 fermented stomachic and digestive tablets were found to have strong anti-Helicobacter pylori activity against mice.

[0003] The commonly used microorganisms for Chinese herbal medicine fermentation are mainly lactic acid bacteria, Bacillus, Bifidobacterium, yeast and various medicinal fungi. In traditional fermentation processes, microorganisms mainly come from the environment, raw materials and production equipment, and multiple microorganisms play a synergistic role in the fermentation process. Although natural fermentation is inefficient and lacks specificity, it is still the most commonly used fermentation method. In the modern Chinese medicine fermentation process, researchers combine microorganisms according to the characteristics of the drug, using a single strain or a mixed strain for direct fermentation. Since there are rich and diverse probiotics and probiotic combinations in nature, screening new probiotic strains or constructing synthetic probiotic flora has broad prospects in improving the efficacy of Chinese herbal medicines. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a high-yield cellulase Aspergillus chevalerii ( Aspergillus chevalieri )J-10) and its application in the fermentation of Chinese herbal medicines, and the application of this strain in enhancing the anti-tumor activity of Chinese herbal medicines.

[0005] The present invention adopts the following technical solution: The invention discloses a strain of Aspergillus serrata ( Aspergillus chevalieri ) J-10, the deposit number of the Aspergillus serrata is CGMCC No. 41630, and the classification name is Aspergillus chevalieri , deposited in the China General Microbiological Culture Collection Center, and the preservation date is November 18, 2024.

[0006] The Aspergillus chevalieri J-10 can produce β-glucosidase, cellulase, hemicellulase and lignin peroxidase.

[0007] The present invention also discloses a fermentation inoculum, which comprises the above-mentioned Aspergillus chevalieri J-10.

[0008] The preparation method of the above-mentioned fermentation inoculum comprises the following steps: inoculating Aspergillus chevalieri J-10 on a PDA solid medium, activating it at 28 °C for 5-7 days, streaking the activated strain on the PDA solid medium for 3-5 days, adding sterile water, scraping the spores on the solid medium, and filtering through a gauze to obtain a spore suspension, which is the fermentation inoculum.

[0009] The application of the above-mentioned Aspergillus chevalieri J-10 or the above-mentioned fermentation inoculum in fermenting Chinese herbal medicines.

[0010] Furthermore, the Chinese herbal medicines include Ganoderma lucidum, Panax ginseng, and Lycium barbarum.

[0011] The present invention also discloses a method for improving the release and utilization of active ingredients after fermenting Chinese herbal medicines, which comprises the following steps: (1) adding 5% by mass of glucose to the Chinese herbal medicine powder, and then adding distilled water at a material-liquid ratio of 1:4 and stirring evenly; (2) inoculating the above-mentioned Aspergillus chevalieri J-10 or the above-mentioned fermentation inoculum into the solution obtained in step (1) for fermentation.

[0012] Furthermore, the inoculation amount in step (1) is 3%.

[0013] Furthermore, the fermentation temperature in step (2) is 28 °C and the fermentation time is 10 days.

[0014] The application of a Chinese herbal medicine product fermented by the above method in preparing an anti-tumor drug. Description of the Drawings

[0015] Figure 1 (A) shows the colony morphology of the "Golden Flower" strain on a PDA culture medium plate; Figure 1 (B) shows the colony morphology of the "Golden Flower" strain screening for high-yield β-glucosidase on a esculin culture medium plate.

[0016] Figure 2 shows the activities of the cellulase systems (A: β-glucosidase; B: hemicellulase; C: cellulase; D: lignin peroxidase) of the 10 selected "Golden Flower" strains.

[0017] Figure 3 is a phylogenetic tree constructed based on the 18S rDNA sequence of strain J-10.

[0018] Figure 4 HPLC chromatograms of Ganoderma lucidum ethanol extracts at different fermentation times.

[0019] Figure 5 Total ion chromatograms of Ganoderma lucidum before and after fermentation obtained by HPLC-QTOF-MS / MS analysis technology in the negative ion mode (where A represents Ganoderma lucidum LZ0 in the non-fermented group; B represents Ganoderma lucidum LZ10 fermented for 10 days).

[0020] Figure 6 In vitro effects of fermented Ganoderma lucidum ethanol extracts on the viability of different cancer cells (A: RAW 264.7; B: Hela; C: PC-9; D: Caco-2). Among them, 0d represents 0 days of fermentation, and 10d represents 10 days of fermentation. Embodiment Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and are not used as limitations of the present invention.

[0022] Example 1 Screening of "Jinhua" bacteria with high β-glucosidase production Take 10 g of Anhua dark tea, resuspend it with sterile normal saline (0.9%), and place it in a constant temperature shaker at 28 °C and incubate it with shaking at a speed of 180 rpm for 30 min. Use a pipette to aspirate 100 μL of the dark tea suspension, evenly coat it on the PDA solid medium plate, and incubate it upside down in a constant temperature and humidity incubator at 28 °C for 3 d. Observe the colony growth on the plate.

[0023] Dilute the above dark tea suspension with normal saline (0.9%) to 10 -1 、10 -2 、10 -3 , use a pipette to aspirate 100 μL of the diluted dark tea suspension and coat it on the aesculin plate (peptone 5.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, ferric citrate 0.5 g / L, aesculin 3.0 g / L, agar 1.5 g / L, pH (25 °C): 7.3 ± 0.1), screen 10 strains of "Jinhua" bacteria with the largest enzyme-producing color-changing circle, pick single colonies and store them on the slant for later use.

[0024] As can be seen from Figure 1 , among the 10 samples of Anhua dark tea collected, strains that can produce β-glucosidase were initially screened using the aesculin solid medium plate as the screening culture condition. β-Glucosidase can promote the decomposition of aesculin to produce esculetin, and the reaction of esculetin with Fe 2+ in the medium will generate a black compound, which can be used to screen strains producing β-glucosidase. Dilute the bacterial suspension concentration to 10-1 After that, 10 kinds of "golden flower" bacteria with larger enzyme-producing color-changing circles were screened out on the esculin solid medium plate, numbered J01, J02, J03, J04, J05, J06, J10, J07, J08, J09, and J10 respectively.

[0025] Example 2 Determination of the enzyme-producing ability of "golden flower" bacteria The 10 strains of "golden flower" bacteria with high β-glucosidase production were cultured in liquid PDA medium at 28 °C and 180 rpm for 72 h. A large number of mycelial pellets could be seen in the medium. The culture solution was inoculated into fresh PDA liquid medium at a ratio of 1%, and secondary culture was carried out under the same medium conditions. The second culture solution was centrifuged (8000 rpm, 4 °C, 10 min), and the supernatant was taken to measure the enzyme activities of its β-glucosidase, cellulase, hemicellulase, and lignin peroxidase.

[0026] Determination of β-glucosidase enzyme activity: Precisely pipette 25 μL of the supernatant, 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 Na2CO3 (1 mol / L) to terminate the reaction, observe the color change, and measure the absorbance at 405 nm with an enzyme-labeling instrument. 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): Formula (1) Y: Absorbance at 405 nm; K: Slope of the p-nitrophenol standard curve; b: Intercept of the p-nitrophenol standard curve; V1: Total volume of the reaction system (mL); t: Reaction time (min); V2: Volume of the enzyme in the reaction system (mL); M: Molecular mass of p-nitrophenol 139.109.

[0027] Determination of cellulase activity: Weigh 40 g of anhydrous sodium hydroxide, dissolve it in deionized water, and then make up the volume to 500 mL. Accurately weigh 6.5 g of 3,5-dinitrosalicylic acid, transfer it into a 1 L volumetric flask, add 325 mL of sodium hydroxide solution, then add 45 g of glycerol, and make up the volume to 1 L to obtain DNS solution. Under the condition of 50 °C water bath, add 30 μL of crude enzyme solution to 60 μL of 10 mg / mL carboxymethyl cellulose sodium solution, mix evenly, and then keep it in a 50 °C water bath for 30 min. Subsequently, add 150 μL of DNS solution, mix evenly, and then keep it in a 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 evenly, 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 a 100 °C water bath as the blank control; use glucose as the standard product to draw a standard curve, and calculate the cellulase activity according to formula (2): Formula (2) Where U: cellulase activity; A: glucose content (μg); V: enzyme solution volume (μL); t: reaction time (min).

[0028] Determination of hemicellulase activity: Under the condition of 50 °C water bath, add 30 μL of crude enzyme solution to 60 μL of xylan (5 mg / mL) solution, mix evenly, and then keep it in a 50 °C water bath for 30 min. Subsequently, add 150 μL of DNS solution, mix evenly, and then keep it in a 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 evenly, 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 a 100 °C water bath as the blank control; use xylose as the standard product to draw a standard curve, and calculate the hemicellulase activity according to formula (3): Formula (3) In the formula, A1: absorbance of the enzyme reaction solution; A2: absorbance of the blank sample; K: slope of the standard curve; C: intercept of the standard curve; t: enzymatic hydrolysis time (min).

[0029] Determination of lignin peroxidase activity: Under the condition of 30 °C water bath, 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), add 20 μL of H2O2 solution (20 mmol / L) and react for 3 min, and then measure the absorbance at 310 nm. Use distilled water instead of the H2O2 solution as the sample blank. Calculate the lignin peroxidase activity according to formula (4): Formula (4) Wherein, A1: sample absorbance; A0: sample blank absorbance; ε: molar absorption coefficient: ε 310 = 9300 (mol / L / min); t: reaction time (min); V1: total volume of reaction solution (mL); V2: total volume of fermentation solution (mL); n: dilution multiple.

[0030] Depend on Figure 2 It can be seen that the activities of β-glucosidase, hemicellulase, cellulase and ligninase of 10 strains of high-yielding β-glucosidase Jinhua fungus were determined. Figure 2 It can be seen that the three strains of "Jinhua" bacteria with the highest β-glucosidase activity are: J-10, J-02, and J-08, and their β-glucosidase activities are: 0.00258, 0.0172, and 0.0169 U / mL, respectively. The three strains of "Jinhua" bacteria with the highest hemicellulase activity are: J-07, J-10, and J-05, and their hemicellulase activities are: 49.3, 47.5, and 39.0 U / mL, respectively. The three strains of "Jinhua" bacteria with the highest cellulase activity are: J-01, J-8, and J-10, and their cellulase activities are: 33.5, 28.9, and 28.8 U / mL, respectively. The three strains of "Jinhua" bacteria with the highest lignin peroxidase activity are: J-10, J-01, and J-07, and their enzyme activities are: 151.4, 122.5, and 99.1 U / mL, respectively. The activities of four enzymes of 10 strains of J. japonica were comprehensively evaluated, and J-10 was selected as the "Jinhua" strain with the best enzyme production capacity for subsequent fermentation experiments.

[0031] Example 3 Identification of strains of “Golden Flower Fungus”

[0032] The J-10 strain was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing results were subjected to sequence homology alignment using Nucleotide Blast. The sequence of strain J-10 had 100% similarity with Aspergillus chevalieri ( Aspergillus chevalieri ) (sequence ID: CN645257.1) in the database. From Figure 3 it can be seen that the "golden flower fungus" J-10 strain isolated and purified from Anhua dark tea and Aspergillus chevalieri ( Aspergillus chevalieri ) clustered together with a support rate of 85%, indicating a relatively high similarity between the J-10 strain and it. It was identified as Aspergillus chevalieri ( Aspergillus chevalieri ), and this strain was included in the list of functional strains for traditional Chinese fermented foods in China and was suitable for the fermentation of Ganoderma lucidum fruit bodies. It was named Aspergillus chevalieri( Aspergillus chevalieri ) J-10.

[0033] Example 4 Fermentation treatment of Chinese herbal medicines The J-10 strain preserved on the slant was inoculated onto a PDA solid medium and incubated in an inverted position in an incubator at 28 °C for 5 - 7 d. The activated strain was streaked on a PDA plate and also incubated in an inverted position in an incubator at 28 °C for 3 - 5 d. In a laminar flow hood, 5 mL of sterile water was added to the plate, and the spores on the plate were gently scraped off with a sterile inoculation loop. After filtration through four layers of gauze, a spore suspension was obtained. The spores in the spore suspension were counted using a hemocytometer under an optical microscope, and the concentration of the spore suspension was diluted to 10 6 / mL and then used for the fermentation of Ganoderma lucidum. The Ganoderma lucidum slices were placed in an oven at 40 °C and dried to a constant weight, and then pulverized into Ganoderma lucidum fruit body powder using a pulverizer. 30 g of Ganoderma lucidum fruit body powder was weighed into a glass bottle, 5% (w / w) glucose based on the Ganoderma lucidum fruit body powder was added, and distilled water was added at a solid-liquid ratio of 1∶4 and stirred evenly. It was sterilized at 121 °C for 20 min. After cooling to room temperature, the spore suspension of Aspergillus chevalieri J-10 was inoculated at an inoculation amount of 3% in a laminar flow hood. After stirring evenly, it was sealed with eight layers of sterile gauze and placed in an incubator at 28 °C with constant temperature and humidity for 10 d. Samples were taken once every 2 d under sterile conditions and numbered as LZ0, LZ2, LZ4, LZ6, LZ8, LZ10, and the samples were stored in a -20 °C refrigerator. The control group was not inoculated with Aspergillus chevalieri the J-10 spore suspension, and other conditions were the same as those in the fermentation group. It was placed in an incubator at 28 °C with constant temperature and humidity for 10 d. After the fermentation was completed, the Ganoderma lucidum samples at different fermentation times were put into a freeze dryer for freeze-drying. The freeze-dried samples were sealed and stored for later use.

[0034] Example 5 Fingerprint and mass spectrometry analysis of Ganoderma lucidum Precisely weigh 0.2 g of the freeze-dried sample in Example 4 and place it in a 50 mL centrifuge tube. Add 10 mL of absolute ethanol, sonicate for 1 h, centrifuge at 4000 rpm for 15 min, take the supernatant and filter it through a 0.22 μm organic filter membrane. The filtrate is the Ganoderma lucidum alcohol extract, which is used for HPLC and HPLC-QTOF-MS / MS analysis. Chromatographic conditions: Use acetonitrile (A) - 1% acetic acid aqueous solution (B) as the mobile phase for gradient elution. Select an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm) chromatographic column. Column temperature: 30 °C; Use an ultraviolet detector, 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: Scan in the negative ion mode, the scanning range is 50 - 600 m / z, the collision energy is 10.00, 20.00, 40.00 eV, the drying gas flow rate is 10 L / min, the gas used in the experiment is nitrogen (purity > 99.9%), the collision gas is high-purity nitrogen (purity > 99.999%). The drying gas temperature is 350 °C, the spray pressure is 40 psig, the capillary voltage is 3.5 kV, and the acquisition frequency is 1 spectra / sec.

[0035] Table 1 Gradient elution program of the mobile phase As Figure 4 shown, HPLC fingerprint can be used to quickly distinguish the differences in fermentation products. Use a high-performance liquid chromatograph to detect the ethanol extract of Ganoderma lucidum samples at a wavelength of 245 nm, and superimpose the obtained chromatograms to draw a fingerprint ( Figure 4 ). The results show that in the first 4 days of fermentation, the components of the Ganoderma lucidum alcohol extract did not change significantly. After 6 days of fermentation, it can be observed that the intensities of 3 absorption peaks (peak 2, peak 3, peak 4) increased significantly, and 6 new absorption peaks (peak 1, peak 6, peak 7, peak 8, peak 9, peak 10) were generated. Moreover, as the fermentation time extended, the peak areas of these peaks increased significantly. The above results indicate that fermenting Ganoderma lucidum with Aspergillus chevalieri J-10 not only greatly increased the content of existing components in the Ganoderma lucidum alcohol extract but also enriched the components of the Ganoderma lucidum alcohol extract. Since these changes mainly started after 6 days of fermentation, the 6th day of fermentation was selected as the time node, and the fermentation process was divided into two stages: the early fermentation stage (0 - 6 d) and the late fermentation stage (6 - 10 d).

[0036] Use HPLC-QTOF-MS / MS to further analyze the active components in Ganoderma lucidum before and after fermentation in the negative ion mode. Ganoderic acids usually have one or more hydroxyl and carboxyl groups. In the negative ion mode, the fragmentation pathway of ganoderic acids has high sensitivity and characteristic.Figure 5 This is the total ion chromatogram (TIC) of Ganoderma lucidum before and after fermentation in the negative ion mode. By comparing the retention time, precursor ions, and product ions with the database and literature reports, a total of 14 compounds were identified. As shown in Table 2, 7 of them were identified as ganoderic acids (Compound 1 Ganoderenic acid C, Compound 3 Ganoderenic acid G, Compound 4 Ganoderic acid V1, Compound 5 Ganoderic acid GS-1, Compound 6 Ganoderic acid J, Compound 7 Ganoderic acid A, Compound 9 Ganoderic acid F), and their contents all increased after fermentation. In addition to ganoderic acids, 6 newly generated compounds were identified in the ethanol extract of Ganoderma lucidum after fermentation, namely: Baicalein (Compound 10), 5,3-Dihydroxy-2-methoxy-6,7-methylenedioxyisoflavone (Compound 12), Emodin (Compound 13), 5,7,3',5'-Tetrahydroxy-3,6,8,4'-tetramet-hoxyflavone 3'-glucoside (Compound 14), Luteolin (Compound 15), Echinulin (Compound 16), and they may be Aspergillus chevalieri produced by the metabolism of J-10. Luteolin is a natural flavonoid compound with various pharmacological activities such as anti-inflammatory, anti-allergic, anti-tumor, antibacterial, and antiviral. Among them, Echinulin and emodin are the main secondary metabolites of Eurotium cristatum. Echinulin is a cyclic dipeptide with a triphenylmethylated indole moiety. Previous studies have found that Echinulin can exert its immune function through the NF-κB pathway and has the potential to be a new type of immunotherapeutic agent. Emodin has a significant inhibitory effect on the activity of α-glucosidase and has hypoglycemic activity.

[0037] Table 2 Active ingredients of Ganoderma lucidum after fermentation in the negative ion mode by HPLC-QTOF-MS / MS Example 6 Determination of the in vitro anti-tumor activity of the ethanol extract of Ganoderma lucidum before and after fermentation Take out the cryopreservation tubes of RAW 264.7, Hela, PC-9, and Caco-2 cells from the liquid nitrogen tank, place them in a 37 °C constant temperature water bath and shake quickly to thaw. In the laminar flow hood, aspirate the thawed cell suspension into a centrifuge tube, centrifuge (4 min at 1000 r), discard the upper cryopreservation medium, add 1 mL of pre-warmed DMEM medium containing 10% fetal bovine serum, gently and slowly disperse the cells with a pipette, transfer them 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 them in a 5% carbon dioxide, 37 °C constant temperature incubator and culture for 24 h, then observe the cell state and replace with fresh medium.

[0038] When the cell density grows to 70%-80%, passage the cells. Use a pipette to aspirate and discard the culture medium, aspirate 4 mL of PBS buffer (0.01 M, pH 7.2) and wash twice, add 1 mL of DMEM medium containing 10% fetal bovine serum, 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, then add 1 mL of DMEM medium containing 10% fetal bovine serum to resuspend the cells, aliquot the cell suspension into 2 10 cm culture dishes, add 10 mL of DMEM medium containing 10% fetal bovine serum to each culture dish, and blow the cells evenly with a pipette. After observing under the microscope, place them in a 5% carbon dioxide, 37 °C constant temperature incubator for culture. After the cells are passaged 2-3 times and the cell viability recovers, perform the CCK-8 assay.

[0039] Under the microscope, observe that RAW 264.7, 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 medium, wash twice with PBS buffer (0.01 M, pH 7.2), gently scrape the cells with a cell scraper, then add an appropriate amount of DMEM medium containing 10% fetal bovine serum to dilute, count the cells, and inoculate 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 culture for 24 h.

[0040] Accurately weigh the ethanol extract of Ganoderma lucidum, prepare a stock solution with a concentration of 1 mg / L using DMEM medium containing 10% fetal bovine serum, and then dilute it with DMEM medium containing 10% fetal bovine serum to 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, 0.5 mg / L, and 1 mg / L respectively. Take out the 96-well plate after the culture ends, 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 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, 0.5 mg / L, and 1 mg / L to the experimental groups. 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, 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 ends, transfer it to a microplate reader for detection. Set the wavelength at 450 nm, and calculate the cell viability according to formula (6).

[0041] Formula (6) The results are as Figure 6 shown in Aspergillus chevalieri As shown in A, the ethanol extract of Ganoderma lucidum after J-10 fermentation shows a promoting effect at low concentrations and an inhibitory effect at high concentrations on the activity of RAW 264.7 cells. It can promote the growth of RAW 264.7 cells within the concentration range of 0 - 0.0156 mg / L. However, when the concentration exceeds 0.125 mg / L, the viability of RAW 264.7 cells significantly decreases, and the half-maximal inhibitory concentration is 0.28 mg / L. As Figure 6 shown in B, with the increase in the concentration of the ethanol extract of Ganoderma lucidum after fermentation, the cell viability of Hela cells decreases, and it shows a concentration-dependent manner ( P <0.05). The half-maximal inhibitory concentration of the ethanol extract of Ganoderma lucidum on Hela cells before fermentation is 0.43 mg / L, while after fermentation it is 0.17 mg / L. And at each concentration gradient, the inhibitory effect of the ethanol extract of Ganoderma lucidum after fermentation on the cell viability of Hela cells is stronger than that of the non-fermented ethanol extract of Ganoderma lucidum. This indicates that Aspergillus chevalieri J-10 fermentation can significantly enhance the inhibitory effect of the ethanol extract of Ganoderma lucidum on Hela cervical cancer cells. With the increase in the concentration of the ethanol extract of Ganoderma lucidum after fermentation, the cell viability of Hela significantly decreases, and it shows a concentration-dependent manner ( P<0.05). At each concentration gradient, the toxicity of the ethanol extract of fermented Ganoderma lucidum to PC-9 cells was stronger than that of the unfermented ethanol extract of Ganoderma lucidum. Moreover, the half inhibitory concentration of the ethanol extract of Ganoderma lucidum before fermentation on PC-9 cells > 1 mg / L, while that after fermentation was 0.14 mg / L. This indicates that Aspergillus chevalieri Fermentation by J-10 can significantly enhance the inhibitory effect of the ethanol extract of Ganoderma lucidum on PC-9 human lung cancer cells ( Figure 6 C). In addition, with the increase in the concentration of the ethanol extract of Ganoderma lucidum before and after fermentation, there was no significant change in the cell viability of Caco-2, which indicates that Aspergillus chevalieri The ethanol extract of Ganoderma lucidum before and after fermentation by J-10 has no inhibitory effect on the proliferation of Caco-2 cells ( Figure 6 D).

[0042] Through the above embodiments, the present invention uses a strain of Aspergillus chevalieri ( Aspergillus chevalieri J-10) to ferment Chinese herbal medicines, significantly improving the release and utilization rate of the active ingredients of Chinese herbal medicines, and further enhancing the anti-tumor activity of Chinese herbal medicines. This method provides a new method and idea for the deep processing and comprehensive utilization of Chinese herbal medicines, and has broad application prospects.

[0043] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A strain of Aspergillus chewartii that produces high cellulase Aspergillus chevalieri ) J-10, characterized by: The deposit number of the Aspergillus serrata is CGMCC No. 41630, and the classification name is Aspergillus chevalieri , deposited in the China General Microbiological Culture Collection Center, and the preservation date is November 18, 2024.

2. The Aspergillus shewanella J-10 according to claim 1, characterized in that: The Aspergillus shewanella J-10 can produce beta-glucosidase, cellulase, hemicellulase and lignin peroxidase.

3. A fermentation agent, characterized in that: The fermentation bacterial agent includes the Aspergillus shewanella J-10 described in claim 1.

4. The method for preparing the fermentation agent according to claim 3, characterized in that: The Aspergillus shewanella J-10 was inoculated into a PDA solid culture medium and activated at 28°C for 5-7 days. After streaking and culturing the activated strain on the PDA solid culture medium for 3-5 days, sterile water was added, and spores on the solid culture medium were scraped off. The spore suspension obtained after filtering through gauze was the fermentation agent.

5. Use of the Aspergillus shewanella J-10 according to claim 1 or the fermentation agent according to claim 3 in fermenting Chinese herbal medicine.

6. The use according to claim 5, characterized in that: The Chinese herbal medicines include ganoderma lucidum, ginseng and wolfberry.

7. A method for improving the release and utilization of active ingredients in Chinese herbal medicine after fermentation, characterized in that: The steps include: (1) Add 5% by weight of glucose to the Chinese herbal medicine powder, and then add distilled water at a solid-liquid ratio of 1:4 and stir evenly; (2) Inoculating the Aspergillus shewanella J-10 described in claim 1 or the fermentation agent described in claim 3 into the solution obtained in step (1) for fermentation.

8. The method according to claim 7, characterized in that: The inoculation amount in step (1) is 3%.

9. The method according to claim 7, characterized in that: The fermentation temperature in step (2) is 28° C. and the fermentation time is 10 days.

10. Use of a Chinese herbal medicine product obtained by fermentation according to the method of claim 7 in the preparation of anti-tumor drugs.

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