Application of coral hericium in preparing medicine for treating cancer

Abstract

The present invention provides the application of coral hericium in the preparation of drugs for treating cancer, and relates to the field of microbial fermentation technology. The coral hericium is coral hericium (Hericium coralloides) RT25, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC NO.: 41304. In the present application, it is found for the first time that coral hericium RT25 contains a variety of antioxidant active substances, such as flavonoids and γ-aminobutyric acid, and can treat liver cancer and neuroma in a targeted manner, and effectively inhibit the proliferation of cancer cells. In the present application, the fermentation liquid, mycelium, primordium, and fruiting body of coral hericium RT25 are also used as samples to enrich flavonoids and γ-aminobutyric acid, and then an extract with antioxidant and anti-cancer effects is obtained, which provides a new active ingredient for drugs for the treatment of liver cancer and neuroma.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to the application of coral hericium in the preparation of drugs for treating cancer. Background Art

[0002] Hericium coralloides, also known as Jade Beard, belongs to the Basidiomycetes, Agaricomycetes, Russulales, Hericiaceae, Hericium, and is a famous rare and precious edible and medicinal fungus in my country. Hericium coralloides is beneficial to the five internal organs, nourishing, and aiding digestion. It is mainly used for diseases such as neurasthenia and gastric ulcer. It is an excellent medicinal fungus with extremely high nutritional value and health value.

[0003] Hepatocellular carcinoma is one of the most common malignant tumors in the world, and its prevalence has increased rapidly in recent years. The current treatment effect of hepatocellular carcinoma is limited and far from satisfactory. At the same time, the recurrence and metastasis rate of hepatocellular carcinoma is high. 60-70% of patients will experience recurrence and metastasis 5 years after hepatocellular carcinoma resection.

[0004] Neuroma, also known as Schwann cell tumor, is a benign tumor formed by the Schwann sheath (nerve sheath) of the peripheral nerve. When it occurs in the vestibular nerve or cochlear nerve, it is also called acoustic neuroma. Most patients are middle-aged people between 30 and 40 years old, with no obvious gender difference. It often grows in the posterior roots of spinal nerves. If the tumor is large, 2-3 nerve roots may adhere to or be buried in the tumor. The nerve roots are thick and may also occur in several spinal nerve roots. A small number of patients may have concomitant neurofibromatosis, which can be seen with coffee-colored spots and multiple small nodular tumors on the patient's skin. The size of spinal nerve sheath tumors is usually 2-3 cm.

[0005] However, there are currently no reports on the use of coral monkey head to treat hepatocellular carcinoma or neuroma. Summary of the invention

[0006] The purpose of the present invention is to provide an application of coral hericium in the preparation of a drug for treating cancer, specifically coral hericium (Hericium coralloides) RT25. In the present application, it is found that the fermentation broth obtained by culturing the coral hericium in a culture medium with Schisandra chinensis as an induction factor can be used to treat cancer, especially liver cancer and neuroma-related diseases, providing a new effective ingredient for anti-cancer drugs.

[0007] On the one hand, the present application provides the use of coral hericium in the preparation of a drug for treating cancer, wherein the coral hericium is coral hericium (Hericium coralloides) RT25, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.: 41304.

[0008] Furthermore, the method for culturing the coral hericium is: inoculating the coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium includes Schisandra chinensis.

[0009] In a preferred embodiment, the fermentation culture comprises the following steps:

[0010] Step 1, activating the coral Hericium erinaceus RT25 strain to obtain seed liquid;

[0011] Step 2, inoculating the seed liquid into a fermentation medium for fermentation culture, wherein the fermentation medium includes: 1.6%-2.4% lactose, 0.1%-0.9% peptone, 10%-50% potato, 0.1%-0.5% magnesium sulfate, 0.1%-0.5% dipotassium hydrogen phosphate, 0.001%-0.01% VB1, 0.1%-0.3% Schisandra chinensis, and the fermentation culture conditions include: inoculation amount of 0.1%-3%, fermentation temperature of 18°C-32°C, liquid volume of 24%-60%, rotation speed of 100-180rpm, pH of 4.0-7.0, and fermentation time of 1-10 days.

[0012] In a preferred embodiment, the fermentation medium comprises: 200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and 1000 mL water.

[0013] Preferably, the potatoes are potato juice, and the preparation method of the potato juice comprises: peeling the potatoes, cutting them into pieces, adding water, boiling them for 30 minutes, filtering them with gauze to make up the water to 100 mL, and sterilizing them.

[0014] In a preferred embodiment, the fermentation culture conditions include: inoculation amount of 1%, fermentation temperature of 25°C, liquid volume of 32%, rotation speed of 140 rpm, pH of 6.0, and fermentation time of 7 days.

[0015] Among them, the common method in the art can be used to activate the coral hericium erinaceus strain to obtain the seed liquid.

[0016] In a preferred embodiment, the activation method of the coral hericium erinaceus strain includes: inoculating coral hericium erinaceus blocks into seed liquid culture medium, shaking and culturing at 25° C. and 140 r / min for 7 days.

[0017] Seed liquid culture medium: 20% potato, 2% glucose, 0.3% peptone, 0.3% dipotassium hydrogen phosphate, 0.15% magnesium sulfate, 0.001% VB1, and the balance is water.

[0018] Furthermore, the cancer includes liver cancer and / or neuroma.

[0019] The present application firstly discovered that Hericium coralloides RT25 contains a variety of antioxidant active substances, such as flavonoids and γ-aminobutyric acid, which can specifically treat liver cancer and neuroma and effectively inhibit the proliferation of cancer cells. Furthermore, the present application provides a method for preparing an extract rich in flavonoids or γ-aminobutyric acid.

[0020] On the other hand, the present application also provides a method for preparing a coral hericium extract for treating cancer, the method comprising the following steps:

[0021] Step 1, inoculating coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium includes Schisandra chinensis; the coral hericium is coral hericium RT25, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.: 41304;

[0022] Step 2: Ultrasonic extraction of the coral hericium to obtain the coral hericium extract.

[0023] Furthermore, the coral hericium includes mycelium, primordium and / or fruiting body.

[0024] Furthermore, the ultrasonic extraction conditions include: a solid-liquid ratio of 1:(10-120), an ultrasonic time of 0.5-5h, an ultrasonic temperature of 30°C-80°C; and / or, the extractant of the ultrasonic extraction is ethanol with a concentration of 0%-100%.

[0025] The unit of material-liquid ratio is g:V, g·mL -1 .

[0026] Preferably, the coral hericium erinaceus fermentation broth contains antioxidant active substances; preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid and / or flavonoids.

[0027] In a preferred embodiment, the antioxidant active substance is γ-aminobutyric acid, and the ultrasonic extraction conditions include: ultrasonic time 1.4h, solid-liquid ratio 1:98 (g:V, g·mL -1 ), ultrasonic temperature was 38°C, and the extractant was water (i.e., the ethanol concentration was 0%).

[0028] In a preferred embodiment, the antioxidant active substance is flavonoids, and the ultrasonic extraction conditions include: ultrasonic time 3.5h, solid-liquid ratio 1:83 (g:V, g·mL -1 ), ultrasonic temperature 60°C, ethanol concentration 80%.

[0029] Furthermore, the cancer includes liver cancer and / or neuroma.

[0030] In a preferred embodiment, a method for preparing a coral hericium extract for treating cancer comprises the following steps:

[0031] Step 1, inoculating coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium includes Schisandra chinensis; the coral hericium is coral hericium RT25, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.: 41304; preferably, the step 1 also includes the step of crushing and drying;

[0032] Step 2: ultrasonically extract the fermented coral hericium with ethanol at a concentration of 0%-100% as an extractant, and the ultrasonic extraction conditions include: a solid-liquid ratio of 1: (10-120), an ultrasonic time of 0.5-5h, and an ultrasonic temperature of 30°C-80°C to obtain a coral hericium extract.

[0033] Preferably, the crushing mesh size is 80 meshes. It can be understood by those skilled in the art that the crushing mesh size has no direct impact on the technical effect of the present application, and the crushing mesh size can be selected according to actual conditions during actual application.

[0034] Preferably, the step 2 also includes a centrifugation step.

[0035] Preferably, the step 2 further comprises the steps of concentrating under reduced pressure and precipitating the polysaccharide with alcohol.

[0036] Those skilled in the art can use common methods to further purify the extract of the present application, which is not specifically limited in the present application.

[0037] The coral hericium comprises mycelium, primordium and / or fruiting body, and the mycelium, primordium and / or fruiting body can be obtained by a general culture method.

[0038] In a preferred embodiment, the method for culturing the mycelium, primordium and fruiting body of Hericium erinaceus comprises the following steps:

[0039] Step 1, activating the coral hericium and then fermenting and culturing it to obtain liquid strains (or mycelium);

[0040] Step 2: The mycelium obtained by fermentation is rinsed with sterile water, filtered through gauze, dried at 55° C. to a constant weight, and crushed through an 80-mesh sieve to obtain a mycelium powder sample for later use;

[0041] The liquid strain was inoculated into the bag culture medium and cultured in a four-temperature zone constant temperature humidification incubator. The primordium and fruiting body were collected, dried at 55°C to constant weight, crushed and passed through an 80-mesh sieve to obtain primordium samples and fruiting body samples for later use.

[0042] On the other hand, the present application also provides a coral hericium extract prepared by the method described.

[0043] Furthermore, the coral hericium extract contains antioxidant active substances; preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid and / or flavonoids.

[0044] On the other hand, the present application also provides the use of the method or the coral hericium extract as described in the preparation of a drug for treating cancer; preferably, the cancer is liver cancer and / or neuroma.

[0045] Those skilled in the art can choose to adjust the specific concentration of the coral hericium fermentation liquid according to actual conditions.

[0046] The present invention has the following beneficial effects:

[0047] The present application firstly discovered that Hericium coralloides RT25 contains a variety of antioxidant active substances, such as polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and γ-aminobutyric acid, especially rich in flavonoids and γ-aminobutyric acid, and can specifically treat liver cancer and neuroma, and effectively inhibit the proliferation of cancer cells.

[0048] In this application, the fermentation liquid, mycelium, primordium and fruiting body of Hericium erinaceus RT25 are used as samples to enrich flavonoids and γ-aminobutyric acid, thereby obtaining an extract with antioxidant and anti-cancer effects, providing a new active ingredient for drugs for the treatment of liver cancer and neuroma. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0050] Figure 1 It is the gel electrophoresis diagram of PCR products;

[0051] Figure 2 It is a phylogenetic reverse tree diagram;

[0052] Figure 3 This is a schematic diagram of the fermentation substrate screening experiment results;

[0053] Figure 4 This is a statistical chart of the effects of different fermentation substrates on the quality of bacterial strains;

[0054] Figure 5 This is a comparison chart of bacterial growth between the experimental group and the control group;

[0055] Figure 6 This is a pie chart of the active ingredients of Hericium erinaceus mycelium and fruiting body;

[0056] Figure 7 Response surface diagram and contour diagram of Example 5;

[0057] Figure 8 The response surface diagram and contour diagram of the pairwise interaction of each factor in Example 6;

[0058] Fig. 9 It is the statistical chart of HepG2 inhibition rate;

[0059] Fig.10 It is the statistical graph of PC12 inhibition rate;

[0060] Fig.11 This is the HepG2 IC50 statistical chart;

[0061] Fig.12 It is the PC12 IC50 statistical chart;

[0062] Fig.13 This is a microscopic photo of HepG2 cell staining, where the magnification is 200 times;

[0063] Fig.14 This is a microscopic photograph of PC12 cell staining, where the magnification is 200 times.

[0064] Deposit of biological materials:

[0065] A strain of Hericium coralloides RT25 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 20, 2024, with the deposit number CGMCC NO.: 41304, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. DETAILED DESCRIPTION

[0066] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0067] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0068] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0069] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0070] Test strains:

[0071] Hericium coralloides RT25 was deposited in the General Microbiology Center of China Microorganism Culture Collection on May 20, 2024, with the deposit number CGMCC NO.: 41304. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0072] The control strain Hericium militaris SY26 was purchased from Shenyang Shanhai Nongren Food Trading Co., Ltd.

[0073] The culture medium involved in the following embodiments includes:

[0074] Seed liquid culture medium: 200g potato (boiled juice), 20g glucose, 3g peptone, 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, natural pH, 1000mL distilled water. Sterilize at 121℃ for 30min.

[0075] Method for activation of Hericium erinaceus strain: Use a 9mm hole puncher to punch holes in the plate, and inoculate 3 bacterial blocks in each bottle of seed liquid culture medium. After inoculation, place it in a shaking culture at 25℃ and 140r / min for 7 days to obtain seed liquid.

[0076] Basic fermentation medium (1L): 20g glucose, 3g peptone, 200g potato (boiled juice), 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, and the balance is water.

[0077] Bag cultivation medium: 80% broadleaf sawdust, 20% wheat bran, 1% each of gypsum and brown sugar, seasoned to 65% moisture content, natural pH.

[0078] Bag cultivation method: Use bag cultivation medium to avoid light and let the fungus grow for about 37 days until the fungus bag is full. Move the fungus room and place the mushrooms in a four-temperature zone constant temperature and humidification incubator for fruiting. The bag mouth is open for fruiting. The temperature is controlled at 18℃-20℃. Ventilate twice a day. Harvest when the length of the mushroom spines of the coral hericium fruiting body is more than 0.5cm and the spores have not yet been ejected in large quantities.

[0079] Cultivation methods of coral hericium hyphae, primordium and fruiting bodies:

[0080] Step 1, activating the coral hericium and then fermenting and culturing it to obtain liquid strains (or mycelium);

[0081] Step 2: The mycelium obtained by fermentation is rinsed with sterile water, filtered through gauze, dried at 55° C. to a constant weight, and crushed through an 80-mesh sieve to obtain a mycelium powder sample for later use;

[0082] The liquid strain was inoculated into the bag culture medium and cultured in a four-temperature zone constant temperature humidification incubator. The primordium and fruiting body were collected, dried at 55°C to constant weight, crushed and passed through an 80-mesh sieve to obtain primordium samples and fruiting body samples for later use.

[0083] Among them, coix seed, Fritillaria thunbergii, Poria cocos, licorice, wheat bran, black bean, mulberry leaf, astragalus, Schisandra chinensis, Schisandra chinensis, and Panax notoginseng were all purchased from Chaoyang People's Kangtai Pharmacy (Northern Store); magnesium sulfate, potassium dihydrogen phosphate, anhydrous sodium sulfite, PDA potato agar medium, etc. were all purchased from Beijing Aoboxing Biotechnology Co., Ltd.; phenol, salicin, folin-phenol, and ethanol (analytical grade) were all purchased from Tianjin Komio Chemical Reagent Co., Ltd.; acid buffered saline solution (PBS, pH 7.8) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; hydroxyl radical scavenging ability kit, DPPH free radical scavenging rate kit, and superoxide anion scavenging ability kit were all purchased from Suzhou Greis Biotechnology Co., Ltd.

[0084] Tumor cells: PC12 (neuroma cells) and hepG2 (liver cancer cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0085] Tumor cell culture method: HepG2 cells were placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL streptomycin, and 100 U / mL penicillin at 37°C and 5% CO. 2 The PC12 cells were cultured in a constant temperature incubator at 37°C and 5% CO. 2Culture in a constant temperature incubator.

[0086] Cell viability test (MTT method): In the mitochondria of living cells, exogenous MTT can be reduced by intracellular succinate dehydrogenase to blue-purple crystalline formazan that is insoluble in water and deposited in the cells. This function does not exist in dead cells. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its absorbance value is measured at 570nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals produced is proportional to the number of cells. Specific detection methods include:

[0087] (1) Collect cells in the logarithmic phase. When the cells grow to a density of about 85%, digest them and collect them by centrifugation. Keep the precipitate and add 2 mL of fresh culture medium to disperse the precipitate.

[0088] (2) Adjust the cell suspension concentration (cell concentration is 5×10 5 / mL), add 100μL to each well;

[0089] (3) 5% CO 2 , incubate at 37°C for about 24 hours to allow the bottom of the well to be covered with a cell monolayer (96-well flat-bottom plate);

[0090] (4) Add concentration gradient drugs (before adding samples, the samples must be sterilized by filtering through a 0.22 nm microporous filter membrane). Each concentration of the drug concentration gradient is set up with three replicates, 5% CO 2 , incubate at 37°C for about 24 hours and observe under an inverted microscope;

[0091] (5) Add 10 μL MTT solution (5 mg / mL, i.e., 0.5% MTT) to the wells to which the drug has been added and incubate for another 4 h;

[0092] (6) Stop the culture and remove the culture medium from the wells;

[0093] (7) Add 100 μL of dissolving solution to each well and continue incubation for 3 h to allow the crystals to fully dissolve;

[0094] (8) Measure the absorbance value D(λ) of each well at OD570 nm using an enzyme-linked immunosorbent assay.

[0095] Inhibition rate (%) = 1-(D(λ) treatment-D(λ) blank)) / (D(λ) control-D(λ) blank)

[0096] The following embodiments involve the following instruments:

[0097] BHC-1300IIA / B2 biological safety cabinet (Shanghai Lishen Scientific Instrument Co., Ltd.); vertical constant temperature shaker (Shanghai Shiping Experimental Equipment Co., Ltd.); KQ-50DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 723N visible spectrophotometer (Shanghai Yidian Analytical Instrument Co., Ltd.).

[0098] The detection method involved in the following embodiments includes:

[0099] The determination of strain quality includes the determination of ball diameter, mycelial biomass and ball density.

[0100] Determination of the diameter of bacterial balls: According to the ratio of the number of bacterial balls in each culture medium, 10 bacterial balls are randomly selected and arranged in a long row, and the total length is measured. Repeat 3 times and calculate the average value.

[0101] Determination of mycelial biomass: Determination by differential method. Filter the cultured liquid strain, collect all the bacterial balls in the 250mL shake flask (dry to constant weight before weighing the filter paper), rinse with clean water 3 times, place in a constant temperature drying oven at 60℃ to dry to constant weight, and calculate the average value.

[0102] Determination of bacterial ball density: shake the liquid culture medium from each repeated shaking bottle and place it in a culture dish, count the number of bacterial balls, repeat 3 times and calculate the average value.

[0103] Determination of active ingredient content:

[0104] The polysaccharide content was determined by the phenol-sulfuric acid method, and the polysaccharide standard curve equation was: y = 6.1373x + 0.0284, R 2 =0.9947. 0.01 mL of fermentation sample solution was added with 1.99 mL of pure water. The determination method was carried out according to the polysaccharide determination method optimized by Xie Cunyi et al. The polysaccharide content in the fermentation sample solution was calculated according to the standard curve equation and gradient dilution (Reference: Xie Cunyi, Li Jianmei, Guo Lingling, et al. Comparison and optimization of determination methods for extracellular polysaccharide content in mulberry linterus fermentation solution [J]. Edible Fungi, 2022, 44(6): 69-73.).

[0105] The flavonoid content was determined by aluminum nitrate-sodium nitrite colorimetry, and the flavonoid standard curve equation was: y = 2.6125x + 0.0466, R 2=0.9995. 3 mL of fermentation sample solution was placed in a 25 mL graduated tube, and 2.0 mL of 70% ethanol was added and shaken; 1.0 mL of 5% sodium nitrite solution was added and shaken, and allowed to stand for 6 min; 1.0 mL of 10% aluminum nitrate solution was added and shaken, and allowed to stand for 6 min; 10 mL of 4% sodium hydroxide solution was added, and allowed to stand for 10 min, and then diluted to 25 mL with 70% ethanol, and the absorbance value was measured at 510 nm, and the flavonoid content in the fermentation sample solution was calculated according to the standard curve equation and gradient dilution.

[0106] The polyphenol content was determined by a modified method based on the reference literature, using spectrophotometry, with gallic acid concentration as the horizontal axis and absorbance D as the horizontal axis. 750 As the ordinate, the standard curve equation is: y = 4.379x-0.0059, R 2 =0.9962. 80 μL of fermentation sample solution was mixed with 120 μL of water, 1 mL of Folin phenol was added, and then 0.8 mL of 7.5% Na 2 CO 3 , then add 3 mL of pure water and let stand at 25 ° C for 30 minutes, measure the absorbance of the fermentation sample liquid at 765 nm, and calculate the polyphenol content in the fermentation sample liquid according to the standard curve equation and gradient dilution (Reference: Li Jianmei, Zhu Wanqin, Chai Linshan, et al. Optimization of Cordyceps militaris polyphenol extraction process and its antioxidant activity [J]. Food Research and Development, 2023, 44(8): 111-117.).

[0107] The content of triterpene compounds was detected by the vanillin-glacial acetic acid-perchloric acid method: sample treatment, fermentation broth was centrifuged at 8000×g for 5 min, the supernatant was discarded, an appropriate amount of isopropanol was added to the mycelium precipitate, heated to reflux extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum distillation, and then dried to constant weight at 60°C; about 0.5 g of the fruiting body sample was weighed and put into a mortar, 2.5 mL of pH 7.0 phosphate buffer was added and ground into a homogenate, and isopropanol was refluxed for extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum evaporation, and then dried to constant weight at 60°C. 10.4 mg of the standard betulin was weighed, dissolved in chloroform, and diluted to a 25 mL volumetric flask. 0, 0.25 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 4.0 mL of the mother solution were respectively taken in a 10 mL volumetric flask, diluted to a constant volume with chloroform, and 0.2 mL was taken in a test tube. After evaporation, 0.3 mL, 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid were added. After shaking, the mixture was placed in a 60°C water bath for 45 min, then cooled to room temperature in an ice-water bath, and 5 mL of anhydrous acetic acid was added. After fully shaking, the absorbance was measured at 548 nm to obtain the standard curve equation y=0.0022x+0.0687, R 2 =0.9991. 0.2mL of the extracted sample solution was placed in a test tube. The determination method was the same as the standard curve. The triterpene content was calculated based on the absorbance value and the standard curve equation.

[0108] Determination of γ-aminobutyric acid in samples: add 0.5mL borate buffer (0.2mol / L, pH10.0), 1mL 6% phenol, 1mL NaClO solution (active chlorine is 8%-12%) to 0.5mL of sample extract supernatant, mix well and put in boiling water bath for 10min, take out and immediately put in ice bath for 20min, after the solution turns blue-green, add 2.0mL of 60% ethanol solution and it can be used for determination (using the reagent solution without GABA standard solution as reference). Use 1cm colorimetric dish, scan at wavelength 645nm, obtain absorbance value, and calculate γ-aminobutyric acid concentration using standard curve equation.

[0109] Determination of γ-aminobutyric acid: Reference: Shi Xiaofeng, Luan Guangzhong, Cao Wanxin, Colorimetric determination of γ-aminobutyric acid in traditional fermented bean products, Chinese Condiments, 2008 (3), 76-81. Standard curve determination: Accurately weigh 2500.000 mg of γ-aminobutyric acid in a 500 mL volumetric flask and dissolve it in ultrapure water to make a 5.00 mg / mL standard solution. When using, accurately prepare 0, 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00 mg·mL -1 The GABA standard solution series was obtained to obtain the standard curve equation y = 0.9901x + 0.1337, R 2 =0.9996, where x is the concentration of γ-aminobutyric acid (mg / mL) and y is the absorbance value.

[0110] Determination of soluble protein content: weigh about 0.5g of sample and put it into a mortar, add 2.5mL pH 7.0 phosphate buffer and grind it into a homogenate, then transfer it to a centrifuge tube, centrifuge it at 8000r / min for 5min, discard the precipitate and obtain the extract to be tested. Preparation of standard curve: take 6 clean test tubes numbered 1, 2, 3, 4, 5, 6, with a mother liquor concentration of 0.5mg / mL, 0, 0.02mL, 0.04mL, 0.06mL, 0.08mL, 0.1mL, then add 5.0mL Coomassie Brilliant Blue G-250 reagent, shake and mix, place it for 2min and then compare the color at 600nm wave, record the optical density value OD measured in each tube, and use the standard protein content (mg / mL) as the horizontal coordinate and the absorbance as the vertical coordinate to draw a standard curve (y=5.8086x+0.0322, R 2 =0.9905). Then, 0.1 mL of the sample extract was added to the test tubes respectively, 0.1 mL of pH 7.0 phosphate buffer was added to the blank control tube, 5.0 mL of Coomassie Brilliant Blue G-250 reagent was added to each tube, and 0.9 mL of distilled water was shaken well. After 2 minutes, the optical density value OD was colorimetrically determined at a wavelength of 600 nm, and the content of soluble protein was calculated by regression equation.

[0111] Determination of free radical scavenging rate:

[0112] The antioxidant activity of the fermentation broth was expressed by the free radical scavenging rate. The DPPH free radical scavenging rate, superoxide anion scavenging rate, and hydroxyl free radical scavenging rate were determined by referring to the method in the article by Xu Bin et al. In the free radical scavenging rate determination of this experiment, the sample solutions were all original solutions and no dilution was required (References: Xu Bin, Zhou Yongkang, Li Huixing, et al. Analysis of the dose-effect relationship between the antioxidant activity of pupa drug fungus and the active ingredients [J]. Food Industry Science and Technology, 2021, 42(2): 250-255.).

[0113] The DPPH free radical scavenging rate of the test sample solution was tested according to the kit instructions. The DPPH free radical scavenging rate of the test sample solution was calculated according to formula (1).

[0114] DPPH free radical scavenging rate = [1-(D 测定 -D 对照 )÷D 空白 ]×100%(1)

[0115] In the formula, D 测定 D is the absorbance value of the mixed solution of sample and DPPH; 对照 D is the absorbance value of the mixed solution of sample and anhydrous ethanol; 空白 It is the absorbance value of the mixture of DPPH and anhydrous ethanol.

[0116] The superoxide anion clearance rate of the test sample was determined according to the kit instructions, and the superoxide anion clearance rate of the test sample was calculated according to formula (2):

[0117] Superoxide anion clearance rate = [1-(D 测定 -D 对照 )÷D 空白 ]×100%(2)

[0118] In the formula, D 测定 is the absorbance value of the measuring tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube

[0119] The hydroxyl radical scavenging rate of the test sample was determined according to the kit instructions, and the hydroxyl radical scavenging rate of the test sample was calculated according to formula (3):

[0120] Hydroxyl radical scavenging rate = [D 空白－ (D 测定 -D 对照 )]÷D 空白 ×100%(3)

[0121] In the formula, D 测定is the absorbance value of the measuring tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube.

[0122] The total antioxidant capacity was determined using the T-AOC assay kit and the total antioxidant capacity of the test sample was calculated according to formula (4):

[0123] Total antioxidant capacity / (μmol Trolox / g)=0.3×(△A+0.029)÷W×D(4)

[0124] In the formula: △A = A blank - (A determination - A control), where A determination is the absorbance of the sample and ABTS working solution, A control is the absorbance of the mixed solution of the sample and PBS, and A blank is the absorbance of the mixed solution of ABTS working solution and PBS; W is the sample mass in g; D is the dilution factor of the sample.

[0125] All results were expressed as mean ± standard deviation. Excel software was used for data statistics and analysis, with P < 0.05 indicating a significant difference and P < 0.01 indicating an extremely significant difference. Design-Expert 11.0 software was used for model regression analysis and response surface interaction analysis.

[0126] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0127] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.

[0128] Example 1 Bacterial species identification and strain preservation

[0129] In this implementation, a new strain of Hericium erinaceus was obtained through screening and named RT25. Molecular identification and phylogenetic study of the Hericium erinaceus strain RT25 were performed, including genomic DNA extraction, PCR product sequencing and phylogenetic analysis of the Hericium erinaceus strain RT25. After purification, unidirectional sequencing was performed, and the rDNA ITS sequence of the test strain was measured using the GenBank nucleic acid sequence database to establish a phylogenetic reverse tree. Among them, the electrophoresis diagram of the PCR product is shown in the figure below. Figure 1 The reverse phylogenetic tree is shown in Figure 2 shown.

[0130] The test strain Hericium coralloides RT25 was submitted to Shanghai Sangon Biotechnology Co., Ltd. for strain identification, and the strain was finally identified as Hericium (Hericium erinaceus), Hericium coralloides. Currently, the strain Hericium coralloides RT25 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 20, 2024, with the deposit number CGMCC NO.: 41304.

[0131] Example 2 Fermentation substrate screening experiment

[0132] Different fermentation substrates have different effects on the strain quality of Hericium erinaceus RT25. In this embodiment, the fermentation substrate of Hericium erinaceus RT25 was screened, and the specific method includes:

[0133] The fermentation substrates Coix chinensis Tod., Fritillaria thunbergii, Wolfiporia cocos, Glycyrrhiza uralensis, Wheat bran, Glycinemax, Folium Mori, Astragalus mongholicus Bunge, Schisandra chinensis, and Panax notoginseng were crushed by a grinder, sieved through a 60-mesh sample sieve, and set aside. 0.2 g·100 mL -1 Add them into the basic fermentation medium respectively, set the pH to natural, sterilize at 121℃ for 30min to obtain the substrate medium. The blank control medium is the basic fermentation medium.

[0134] The above substrate culture medium and basic fermentation culture medium were used to ferment Hericium erinaceus RT25, and the culture process was as follows: step 1, activating Hericium erinaceus RT25 to obtain seed liquid; step 2, inoculating the seed liquid with an inoculation amount of 1% into the above substrate culture medium containing different fermentation substrates for fermentation culture, and liquid culture was carried out at 25°C, 140r / min, and natural pH. After 7 days, the quality of the strains of each formula (mycelial biomass, ball diameter, and ball density) was measured. The results are as follows Figure 3 , as shown in Table 1, the content of Table 1 is used to make Figure 4 .

[0135] Table 1 Effects of different fermentation substrates on strain quality

[0136]

[0137]

[0138] like Figure 3 , Table 1 and Figure 4 As shown, Schisandra chinensis can significantly promote the growth of strains, while Fritillaria thunbergii, Coix seeds and Panax notoginseng have significant inhibitory effects. Among them, when Schisandra chinensis is used as a fermentation substrate, the mycelial biomass is the highest, significantly higher than other substrates, the ball diameter is not large (the ball diameter does not block the muzzle during inoculation, and can ensure sufficient mycelial volume), the ball density is significantly higher than other substrates, the ball density is moderate, and it is suitable for production as a liquid strain. Therefore, Schisandra chinensis is selected as a fermentation substrate for subsequent fermentation.

[0139] Example 3 Fermentation formula verification

[0140] In this example, the coral Hericium erinaceus fermentation group without adding Schisandra chinensis was used as the control group, and the fermentation medium with Schisandra chinensis was used as the test group. The strain RT25 was used as an example to test the quality of the strain, and the mycelium was collected and cultivated in bags to obtain primordia and fruiting bodies. The fermentation mycelium state of the strain, the growth rate of fruiting mycelium, the number of days for fruiting mycelium to grow fully, the number of days for primordia to form, the fresh weight of fruiting bodies, and the dry weight of fruiting bodies were recorded. The results are shown in Table 2. Figure 5 shown.

[0141] Control group: The strain RT25 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation amount was 1%, the fermentation time was 7 days, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25°C.

[0142] Experimental group: The strain RT25 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), with an inoculation size of 1%, a fermentation time of 7 days, a shaking table speed of 140 r / min, a pH of 6.0 (natural), a shaking bottle liquid volume of 80 mL / 250 mL, and a culture temperature of 25°C.

[0143] From Table 2, Figure 5 It can be seen that the fermentation liquid of the experimental group has more uniform bacterial balls and a larger density than that of the control group. The mycelium growth rate, fruiting body size and fruiting body weight of the experimental group are significantly improved compared with those of the control group, indicating that Schisandra chinensis has a promoting effect on the growth of coral hericium. The fermentation formula is highly feasible and has broad application prospects.

[0144] Table 2 Comparison between the control group and the experimental group

[0145]

[0146] Example 4: Mining of antioxidant active ingredients

[0147] In this example, the effects of Schisandra chinensis and strain RT25 on the antioxidant active components in mycelium and fruiting bodies and their changes were further explored, and strain SY26 was used as a control.

[0148] Strain RT25 experimental group: The strain RT25 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), with an inoculation size of 1%, a fermentation time of 7 days, a shaking table speed of 140 r / min, a pH of 6.0 (natural), a shaking bottle liquid volume of 80 mL / 250 mL, and a culture temperature of 25°C.

[0149] Strain RT25 control group: The strain RT25 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation size was 1%, the fermentation time was 7 days, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25°C.

[0150] Strain SY26 experimental group: The strain SY26 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation size was 1%, the fermentation time was 7 d, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25 ° C.

[0151] Strain SY26 control group: The strain SY26 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation amount was 1%, the fermentation time was 7 d, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25 ° C.

[0152] After obtaining fermented mycelium using the above-mentioned fermentation method, the fermented mycelium of the RT25 control group and the experimental group of the above-mentioned strain was inoculated into the bag cultivation medium to obtain the RT25 strain fruiting body control group and the experimental group; the mycelium of the RT26 control group and the experimental group was inoculated into the bag cultivation medium to obtain the RT26 strain fruiting body control group and the experimental group, and then the content of antioxidant active ingredients therein was measured respectively.

[0153] The comparison results of active ingredients and free radical scavenging rates in the mycelium and fruiting bodies of the test strain RT25 and the control strain SY26 are shown in Table 3. This example proves that after adding Schisandra chinensis as a fermentation factor, the active ingredients and free radical scavenging rates in the sample liquid of the test group are improved, indicating that adding Schisandra chinensis as an inducing factor has a certain synergistic effect on the content of active substances and antioxidant activity in the mycelium and fruiting bodies of Hericium erinaceus.

[0154] Pearson correlation analysis was used to determine the correlation between antioxidant active substances and antioxidant activity (reference: Ge Xinhui, Meng Jikun, et al., Analysis of phenolic composition and antioxidant activity of wild edible fungi in Wutai Mountain, Chinese Edible Fungi. 2022, 41(04)) (Table 4), and the percentage of each active substance in the total antioxidant active substance content was calculated ( Figure 6 ), the correlation analysis in Table 4 shows that the antioxidant capacity of coral hericium mycelium and fruiting body is the result of the synergistic effect of multiple active ingredients, among which γ-aminobutyric acid, polysaccharides, flavonoids, polyphenols and other active ingredients have different correlations with antioxidant capacity: γ-aminobutyric acid is extremely significantly positively correlated with DPPH free radical scavenging rate (P=0.888), and is extremely significantly positively correlated with hydroxyl free radical scavenging rate (P=0.945); polysaccharides are extremely significantly positively correlated with hydroxyl free radical scavenging rate. There was a significant positive correlation between flavonoids and hydroxyl radical scavenging rate (P=0.825), and a significant positive correlation with DPPH free radical scavenging rate (P=0.68); polyphenols were significantly positively correlated with DPPH free radical scavenging rate and hydroxyl free radical scavenging rate (P=0.737, P=0.812), and positively correlated with superoxide anion (P=0.705).

[0155] Table 3 Active ingredients and antioxidant activity of Hericium erinaceus

[0156]

[0157]

[0158] Table 4 Correlation analysis

[0159]

[0160] Note: ***, **, * represent 1%, 5%, 10% significance levels respectively

[0161] Depend on Figure 6 Analysis of the increase in γ-aminobutyric acid showed that after the addition of Schisandra chinensis to induce fermentation, the γ-aminobutyric acid content in the active ingredients of RT25 mycelium increased by 21%, while the γ-aminobutyric acid content in the active ingredients of the control strain SY26 mycelium increased by 7%; the percentage of γ-aminobutyric acid in the active ingredients of RT25 fruiting bodies increased by 7%, which was two percentage points higher than that of the control strain SY26 fruiting bodies. Therefore, the experimental strain RT25 can better utilize the Schisandra chinensis fermentation medium than the control strain SY26, and promote the biosynthesis of its antioxidant active substance γ-aminobutyric acid, that is, the Schisandra chinensis induction factor helps the synthesis of the antioxidant active substance γ-aminobutyric acid in coral hericium, and the effect on the strain RT25 is particularly significant.

[0162] Example 5 Single factor test of the process for preparing γ-aminobutyric acid-rich extract from the fruiting body of strain RT25

[0163] This example provides a process for preparing an extract rich in γ-aminobutyric acid from a fruiting body, and further optimizes the process.

[0164] The cultivation process of coral hericium fruiting body includes:

[0165] Step 1: A 250 mL triangular flask is used for seed culture. The seed liquid culture medium is filled with 100 mL. The coral Hericium erinaceus RT25 is inoculated and cultured at 25 ° C and 140 rpm for 7 days. The culture medium is filled with mycelial balls the size of fish eggs. When the culture liquid is clear, the coral Hericium erinaceus seed liquid is obtained.

[0166] Step 2: Inoculate the coral hericium erinaceus seed liquid into the culture medium (200g potato (boiled juice), 19g lactose, 3g peptone, 2.4g Schisandra chinensis, 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, 1000mL water), inoculation amount 1%, fermentation time 7d, shaking table speed 140r / min, pH 6.0 (natural), shaking bottle liquid volume 80mL / 250mL, temperature 25°C, until the culture medium is full of rice-sized mycelial balls, until the culture solution is clear, and the coral hericium erinaceus liquid strain is obtained.

[0167] Step 3: Inoculate the liquid bacteria into the bagged culture medium. The culture medium formula is: 80% broadleaf sawdust, 20% wheat bran, 1% gypsum, 1% brown sugar, 65% water content, natural pH, and culture in a four-temperature zone constant temperature humidification incubator. Collect the fruiting bodies for later use.

[0168] The process for preparing an extract rich in γ-aminobutyric acid comprises the following steps:

[0169] Sample preparation and extraction: Rinse the coral Hericium fruiting body with water three times, drain, dry at a constant temperature of 55°C to constant weight, crush and pass through an 80-mesh sieve, weigh 0.5 g of sample powder, use 70% ethanol by volume as the extractant, and the solid-liquid ratio is 1:30 (g:V, g / mL), and perform ultrasonic treatment. The ultrasonic time is 3 h and the ultrasonic temperature is 40°C. Centrifuge at 8000 rpm and 4°C to obtain the supernatant, and set aside at 4°C.

[0170] In this embodiment, based on the above initial extraction conditions, the single factor experiment was set to ethanol volume fraction 0%, 10%, 25%, 40%, 55%, 70%, 80%, 90%, 100%, ultrasonic time 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5h, solid-liquid ratio 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70 (g:V, g / mL), ultrasonic temperature 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and the effect of different single factors on the content of γ-aminobutyric acid was determined.

[0171] In this example, a single factor experiment was conducted on the above process to investigate the effects of different extraction material-liquid ratios, ultrasonic time, ethanol volume fraction, and ultrasonic temperature on the content of γ-aminobutyric acid in the Hericium erinaceus RT25 fruiting body extract (Table 5), with 3 replicates for each level.

[0172] Table 5 Single factor test of γ-aminobutyric acid extraction from coral Hericium erinaceus fruiting body

[0173]

[0174] As can be seen in Table 5, with the increase of the material-liquid ratio, the γ-aminobutyric acid content first increases and then decreases. When the material-liquid ratio increases to 1:100 (g:V, g / mL), the γ-aminobutyric acid content is the largest. Considering the solvent dosage, processing difficulty, and production cost, it is more appropriate to select a material-liquid ratio of 1:100 (g:V, g / mL).

[0175] As the ultrasonic time increased, the γ-aminobutyric acid content first increased rapidly and then gradually decreased, reaching a peak value when the ultrasonic time was 1.5 hours. It was determined that the appropriate ultrasonic time was 1.5 hours.

[0176] With the increase of ethanol volume fraction, the content of γ-aminobutyric acid first decreases and then gradually increases, that is, γ-aminobutyric acid is easily soluble in water and slightly soluble in hot ethanol, and distilled water is selected as the extraction solvent.

[0177] When the temperature increases from 30℃ to 40℃, the content of γ-aminobutyric acid increases. Increasing the temperature can promote the molecular movement of the substance and accelerate the precipitation of γ-aminobutyric acid. The content reaches a peak at 40℃. When the temperature continues to rise, the content of γ-aminobutyric acid decreases. Therefore, the appropriate ultrasonic temperature is 40℃.

[0178] In summary, according to the results of the single factor test, the response surface factor level design table 6 was used to conduct the experiment, and the three-factor three-level response surface optimization was carried out. According to the Box-Benhnken design principle, A (ultrasonic time), B (solid-liquid ratio), and C (ultrasonic temperature) were used as independent variables. 2 is the response value. The response surface test design results are shown in Table 7, and the analysis results are shown in Table 8.

[0179] Table 6 Response surface factor levels

[0180]

[0181] The results are shown in Table 7, and the multivariate regression equation fitting model is:

[0182] Y 2 =58.3-3.22A-2.7B-5.92C+8.34AB-1.65AC+6.82BC-16.29A 2 -13.38B 2 -17.24C 2 (5)

[0183] The coefficient of determination R of the model 2 =0.967 8, Corrected determination coefficient R 2 adj = 0.926 5, indicating that 92.65% of the changes in the content of γ-aminobutyric acid in the fruiting bodies of Hericium erinaceus can be predicted by this model.

[0184] Table 7 Response surface experimental design results

[0185]

[0186] From Table 8, we can see that the model F = 23.4, P < 0.001, indicating that the model is extremely significant, the lack of fit item F = 1.79, P = 0.287 7 > 0.05, indicating that the lack of fit test is not significant, the equation fit is good, and the difference between the model and the test value is small. According to the F values ​​of each item in Table 8, we can see that: C, AB, A 2 , B 2 , C 2 The influence on the content of γ-aminobutyric acid is extremely significant, A and BC have a more significant influence, and the other items have no significant influence. From the size of the F value, it can also be judged that the order of influencing the content of γ-aminobutyric acid is: ultrasonic temperature>ultrasonic time>solid-liquid ratio.

[0187] Table 8 Analysis of variance of regression model

[0188]

[0189] Note: * indicates that the correlation reaches a significant level (P < 0.05); ** indicates that the correlation reaches an extremely significant level (P < 0.01)

[0190] The 3D response surface illustrates the interaction between the dependent variable and the two test variables when the other factors are fixed at zero level. The interaction surface plot of equation (5) was made using Design-Expert 11.0 software, and the result was Figure 7 , after fixing the value of one of the factors A, B, and C in the regression model, the interactions between the two factors formed a convex surface opening downward; within the value range of A, B, and C, γ-aminobutyric acid Y showed a trend of first increasing and then decreasing, and there was a maximum point; from the contour map, it can be seen that the slope of the response surface of the interaction between ultrasonic time and liquid ratio (AB), and liquid ratio and ultrasonic temperature (BC) is steep, and the effect on γ-aminobutyric acid Y is significant; the AC interaction has no significant effect on Y, and the interaction effect is AB>BC>AC, which is consistent with the results of variance analysis (Table 8).

[0191] The first-order partial derivative of equation (5) was calculated, and the optimal extraction process of γ-aminobutyric acid from coral Hericium erinaceus fruiting body was obtained as follows: ultrasonic time 1.451h, solid-liquid ratio 1:97.982 (g:V, g·mL-1), and ultrasonic temperature 38.283℃. Considering the actual operation factors, the optimized process was adjusted to ultrasonic time 1.4h, solid-liquid ratio 1:98 (g:V, g·mL-1), and ultrasonic temperature 38.28℃. The optimized process was used and the experiment was repeated three times. The average value of γ-aminobutyric acid content was 63.826mg / g, which was 116.48% of 54.795 before optimization and 107% of the predicted value (59.277mg / g). This shows that the model is feasible and can provide a reference for coral Hericium erinaceus fermentation process.

[0192] Example 6 Optimization of the process for preparing an extract rich in total flavonoids from the fruiting body of strain RT25

[0193] In this example, the process for preparing an extract rich in total flavonoids from the fruiting body of the strain was optimized, and the preparation method of the fruiting body was the same as that in Example 5.

[0194] The process for preparing an extract rich in total flavonoids comprises the following steps:

[0195] Sample preparation and extraction: Rinse the coral Hericium fruiting body with water for 3 times, drain, dry at 55℃ to constant weight, crush through 80 mesh sieve, weigh 0.5g sample powder, use 70% ethanol by volume as extraction agent, solid-liquid ratio of 1:50 (g:V, g / mL), perform ultrasonic treatment, ultrasonic time for 3h, ultrasonic temperature of 60℃, centrifuge at 8000rpm, 4℃ to obtain supernatant, and set aside at 4℃.

[0196] In this embodiment, based on the above initial extraction conditions, the single factor experiment was set to ethanol volume fraction 0%, 10%, 25%, 40%, 55%, 70%, 80%, 90%, 100%, ultrasonic time 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5h, solid-liquid ratio 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:80, 1:100, 1:120 (g:V, g / mL), ultrasonic temperature 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and the effect of different single factors on the total flavonoids content was determined.

[0197] In this example, a single factor experiment was conducted on the above process to investigate the effects of different extraction material-liquid ratios, ultrasonic time, ethanol volume fraction, and ultrasonic temperature on the total flavonoid content in the Hericium erinaceus RT25 fruiting body extract (Table 9), with 3 replicates for each level.

[0198] Table 9 Single factor experiment on total flavonoids extraction from coral Hericium erinaceus fruiting body

[0199]

[0200] As can be seen in Table 9, with the increase of the solid-liquid ratio, the total flavonoids content first increased and then decreased. When the solid-liquid ratio increased to 1:50 (g:V, g / mL), the total flavonoids content was the largest. Considering the solvent dosage, processing difficulty and production cost, it is more appropriate to select a solid-liquid ratio of 1:50 (g:V, g / mL).

[0201] With the extension of ultrasound time, the total flavonoids content first increased and then decreased, reaching a peak value when the ultrasound time was 3.5 hours. It was determined that 3.5 hours was the appropriate ultrasound time.

[0202] With the increase of ethanol volume fraction, the total flavonoids content first increased and then reached saturation, and the appropriate ethanol volume fraction was 80%.

[0203] When the temperature increases from 30℃ to 60℃, the total flavonoid content increases, reaches a peak value at 60℃, and decreases as the temperature continues to rise. Therefore, the appropriate ultrasonic temperature is 60℃.

[0204] In summary, according to the results of the single factor test, the response surface factor level design table 10 was used to conduct the experiment, and the response surface optimization of four factors and three levels was carried out. According to the Box-Benhnken design principle, A (ethanol volume fraction), B (ultrasonic time), C (solid-liquid ratio), and D (ultrasonic temperature) were used as independent variables, and the total flavonoids Y 2 is the response value. The response surface test design results are shown in Table 11, and the analysis results are shown in Table 12.

[0205] Table 10 Response surface factor levels

[0206]

[0207] The results are shown in Table 11, and the multivariate regression equation fitting model is:

[0208] Y 2 =7.44+0.1608A+0.0337B+0.5209C+0.0244D-0.0475AB-0.3388AC+0.0889AD-0.8495BC-0.7

[0209] 081BD-0.6811CD-2.24A 2 -1.11B 2 -1.63C 2 -0.6687D 2 (6)

[0210] The coefficient of determination R of the model 2 =0.954 3, Corrected determination coefficient R 2 adj = 0.908 6, indicating that 90.86% of the changes in the total flavonoids content in the fruiting bodies of Hericium erinaceus can be predicted by this model.

[0211] Table 11 Response surface experimental design results

[0212]

[0213] From Table 12, we can see that the model F = 20.88, P < 0.0001, indicating that the model is extremely significant, the lack of fit item F = 3.74, P = 0.1077 > 0.05, indicating that the lack of fit test is not significant, the equation fit is good, and the difference between the model and the test value is small. According to the F values ​​of each item in Table 12, we can see that: C, BC, BD, CD, A 2 , B 2 , C 2 The influence on the total flavonoid content is extremely significant, and the influence on other items is not significant. According to the size of the F value, it can also be judged that the order of influencing the total flavonoid content is: solid-liquid ratio>ethanol volume fraction>ultrasonic time>ultrasonic temperature.

[0214] Table 12 Analysis of variance of regression model

[0215]

[0216] Note: * indicates that the correlation reaches a significant level (P < 0.05); ** indicates that the correlation reaches an extremely significant level (P < 0.01)

[0217] The 3D response surface illustrates the interaction between the dependent variable and the two test variables when the other factors are fixed at zero level. The interaction surface plot of equation (6) was made using Design-Expert 11.0 software, and the result was Figure 8 After fixing the value of one of the factors A, B, C, and D in the regression model, the interaction between the two factors formed a convex surface with an opening downward; within the value range of A, B, C, and D, the total flavonoids Y 2 The results showed that the response surface of the interaction between ultrasonic time and liquid-to-solid ratio (BC), ultrasonic time and ultrasonic temperature (BD), and liquid-to-solid ratio and ultrasonic temperature (CD) had a steep slope, which had an obvious influence on the total flavonoids Y 2 The interaction of AB, AC and AD has a significant effect on Y 2 The effect was not significant, and the interaction effect was BC>BD>CD>AC>AD>AB, which was consistent with the results of analysis of variance.

[0218] The first-order partial derivative of equation (2) was calculated, and the optimal extraction process of total flavonoids from coral Hericium erinaceus fruiting body was obtained as follows: ethanol volume fraction 80.36%, ultrasonic time 3.51 h, solid-liquid ratio 1:83.20 (g:V, g·mL -1 ), ultrasonic temperature 60.18℃. Considering the actual operation factors, the optimized process was adjusted to 80% ethanol volume fraction, 3.5h ultrasonic time, and 1:83 solid-liquid ratio (g:V, g·mL -1 ), ultrasonic temperature 60℃, optimized process, repeated test three times, the average value of total flavonoids content was 7.045mg / g, which was 108.77% of the predicted value (6.477mg / g), and the measured value was 8.024, which was 108.68% of 7.383 before optimization, indicating that the model is feasible and can provide a reference for the process of preparing total flavonoids-rich extract from coral Hericium erinaceus fruiting body.

[0219] Example 7 Application of coral hericium in treating cancer

[0220] In this embodiment, the fermented mycelium, primordium, and fruiting body of the strain RT25 experimental group, the strain RT25 control group, the strain SY26 experimental group, and the strain SY26 control group (the culture method is the same as in Example 4) are dried to constant weight, crushed by a grinder through an 80-mesh sieve, and the mycelium, primordium, and fruiting body powders are weighed to obtain an extract rich in γ-aminobutyric acid and an extract rich in total flavonoids according to the optimized process of Example 5 and Example 6. The extract was centrifuged at 8000r / min for 10min, the supernatant was collected, and the rotary evaporator was concentrated under reduced pressure in a 60°C water bath, and polysaccharides were removed by alcohol precipitation with 60% ethanol, centrifuged, and the supernatant was collected, and then distilled under reduced pressure to obtain GABA and total flavonoids crude extract mother liquors, respectively. The crude extract was diluted (diluted 0, 3, 5, 10, 15, 20, 30, 40 times) into extract samples with different GABA and total flavonoid concentrations. The MTT test was used to obtain the proliferation of tumor cells (HpeG2, PC12) at 24, 48, and 72 hours to observe the anti-tumor effect of coral monkey head on HepG2 and PC12. The anti-tumor absorbance and survival rate of the extract samples are shown in Table 13. The graph of Table 13 shows Fig. 9 and Fig.10 .

[0221] Table 13 Antitumor effect of coral hericium extract

[0222]

[0223]

[0224] From Table 13, Fig. 9 , Fig.10 It can be seen that the extract with Schisandra chinensis added has a faster and better tumor inhibition effect than the control extract without Schisandra chinensis added, and the inhibition rate curve of tumor cells presents an "S" curve or an "L" curve, both of which are typical inhibition rate curves. And through IC50 and the highest inhibition rate, it can be seen that the extract has a more significant inhibitory effect on the growth of HepG2. The HepG2 proliferation inhibition rate is positively correlated with the total flavonoid concentration and the γ-aminobutyric acid concentration. The mycelium extract inhibition rate can reach more than 95%, the fruiting body more than 97%, and the primordium up to 99%. The MTT test results showed that compared with the 0μg / mL group, the inhibition rate of HepG2 and PC12 cells in the flavonoid and γ-aminobutyric acid (0.5-33μg / mL) treatment group was significantly increased, the difference was statistically significant (P<0.05), and it was concentration-time dependent.

[0225] In this example, the half-inhibitory concentration (IC50) of each treatment group of Hericium erinaceus strain RT25 and SY26 on HepG2 and PC12 at 24, 48, and 72 hours was calculated, as shown in Table 14. Fig.11 and Fig.12, the smaller the IC50, the stronger the inhibitory effect. As shown in Table 14, the inhibitory effect of the RT25 mycelium experimental group (treated with Schisandra chinensis) on HepG2 was obvious at 24h and 48h, and the IC50 at 72h was slightly lower than that of the control, but the maximum inhibition rate was slightly higher; the half-inhibitory concentration IC50 of the total flavonoids extract of the control strain SY26 on HepG2, the experimental group treated with Schisandra chinensis had a significant effect on 24h compared with the control group, and the IC50 at 48 and 72h was slightly higher, but the maximum inhibition rate was higher than that of the control; after adding Schisandra chinensis induction factor to RT25 and SY26 strains, the IC50 was smaller than that of the control, indicating that Schisandra chinensis helps to inhibit the proliferation and growth of liver cancer cells HepG2 and neuroma cells PC12; RT25 primordium and fruiting body have a certain inhibitory effect on the growth of HepG2 and PC12.

[0226] In this embodiment, HepG2 and PC12 cells were stained. The cell staining photos of HepG2 and PC12 are shown in FIG. Fig.13 and Fig.14 As shown in the results, it was found that with the increase of the concentration of flavonoids and γ-aminobutyric acid, the number of colony formation of HepG2 and PC12 cells decreased successively. Compared with the 0 μg / mL group, the difference was statistically significant (P<0.05). It can be further and more intuitively seen that flavonoids and γ-aminobutyric acid have a significant inhibitory effect on the growth of HepG2 and PC12 cells.

[0227] Table 14 IC50 of coral hericium extract

[0228]

[0229] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing a coral hericium extract for treating cancer, characterized in that: The method comprises the following steps: Step 1, inoculating coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium comprises: 1.6%-2.4% lactose, 0.1%-0.9% peptone, 10%-50% potato, 0.1%-0.5% magnesium sulfate, 0.1%-0.5% dipotassium hydrogen phosphate, 0.001%-0.01% VB1, and 0.1%-0.3% Schisandra chinensis; the coral hericium is coral hericium RT25, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.: 41304; Step 2: Ultrasonic extraction of the fermented coral hericium to obtain coral hericium extract; the ultrasonic extraction conditions include: ultrasonic time 1.4 h, solid-liquid ratio 1:98 (g:V, g•mL -1 ), ultrasonic temperature 38℃, extractant water, or, ultrasonic time 3.5h, solid-liquid ratio 1:83 (g:V, g•mL -1 ), ultrasonic temperature 60℃, ethanol concentration 80%; The cancer is liver cancer and / or neuroma.

2. The method according to claim 1, characterized in that The coral hericium includes mycelium, primordium and / or fruiting body.

3. The coral hericium erinaceus extract prepared according to the method of claim 1 or 2.

4. The extract according to claim 3, characterized in that The coral hericium extract contains antioxidant active substances, and the antioxidant active substances are polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or gamma-aminobutyric acid.

5. The extract according to claim 4, characterized in that The antioxidant active substance is gamma-aminobutyric acid and / or flavonoids.

6. Use of the method according to claim 1 or 2 or the coral hericium extract according to any one of claims 3 to 5 in the preparation of a drug for treating cancer, wherein the cancer is liver cancer and / or neuroma.

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