Hericium erinaceus strain, liquid submerged fermentation extract triterpenoids thereof and application of liquid submerged fermentation extract triterpenoids

Abstract

The invention discloses a hericium erinaceus strain as well as a liquid submerged fermentation extract triterpenoid and application thereof, and belongs to the technical field of bioengineering. The hericium erinaceus strain 01SC403220714 is preserved in the China General Microbiological Culture Collection Center, and the preservation number of the hericium erinaceus strain 01SC403220714 is CGMCC (China General Microbiological Culture Collection Center) No. 41328. Experimental verification shows that liquid submerged fermentation extract of the strain 01SC403220714 subjected to liquid submerged fermentation contains triterpenoids, total phenolic compounds, total flavonoid compounds, polysaccharides and the like, and the triterpenoids have a good anti-gastric ulcer effect and are remarkable in effect. The invention provides a new material for development and utilization of hericium erinaceus strains and products thereof, and provides a new idea for prevention or treatment of gastric ulcer.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a Hericium erinaceus strain and triterpenoid compounds in its liquid deep fermentation extract and applications thereof. Background Art

[0002] Hericium erinaceus belongs to the class of Basidiomycetes, the order of Russulales, the family of Hericiaceae, and the genus of Hericium. Hericium erinaceus is an important edible and medicinal fungus. It is not only one of the eight "mountain delicacies" in China, but also has been called "mountain delicacies of Hericium erinaceus and sea-flavored bird's nest" since ancient times. It is tender and fragrant, delicious and nutritious. It is also a precious traditional Chinese medicinal material with the effects of nourishing and strengthening the body, aiding digestion, and benefiting the five internal organs. Modern studies have shown that the active ingredients of Hericium erinaceus, such as oligosaccharides and polysaccharides, have significant therapeutic effects on gastric diseases such as gastric ulcers and gastritis. In addition, a variety of drugs for the treatment of gastritis and gastric ulcers have been launched on the market, such as Wei Le Xin Granules, Hericium Polysaccharide Tablets, Hericium erinaceus Jianweiling Tablets, and Compound Hericium Capsules. However, the existing products and research reports all focus on the water extracts, polysaccharides, and oligosaccharides of Hericium erinaceus strains, while the anti-gastric effects of Hericium erinaceus strains and other ingredients need to be explored. Summary of the invention

[0003] The purpose of the present invention is to provide a Hericium erinaceus strain and its liquid deep fermentation extract triterpenoid compounds and applications to solve the problems existing in the above-mentioned prior art. The present invention verifies that the triterpenoid compounds in the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 have good anti-gastric ulcer efficacy and significant effect, providing a new idea for the prevention or treatment of gastric ulcer.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a Hericium erinaceus strain 01SC403220714, the Hericium erinaceus strain 01SC403220714 has a preservation number of CGMCC No.41328, a preservation time of June 5, 2024, a preservation unit of the General Microbiology Center of the China Microbiological Culture Collection Administration, and a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides a method for preparing triterpenoid compounds from liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714, comprising the following steps:

[0007] The Hericium erinaceus strain 01SC403220714 is cultured on a slant, first-level shake flask seed culture, second-level shake flask seed culture, first-level seed fermentation tank culture, second-level seed fermentation tank culture and third-level seed fermentation tank culture to obtain a fermentation liquid and a fermentation mycelium, the mycelium is extracted to obtain a mycelium extract, the mycelium extract is combined with the fermentation liquid, and heated and concentrated to obtain a liquid deep fermentation extract;

[0008] The extract is dried, anhydrous ethanol is added, and ultrasonic extraction is performed to obtain an extract solution, and the extract solution is filtered, concentrated, and dried to obtain a liquid deep fermentation extract triterpenoid compound of Hericium erinaceus strain 01SC403220714;

[0009] The deposit number of the strain 01SC403220714 is CGMCC No.41328.

[0010] Furthermore, the temperature of the slant culture is 25-29°C, and the time is 7-13 days; the conditions of the primary shake flask seed culture and the secondary shake flask seed culture are 180-230rpm, 27±1°C culture for 4-7 days; the conditions of the primary seed fermentation tank culture, the secondary seed fermentation tank culture and the tertiary seed fermentation tank culture are 27±1°C culture for 4-7 days; the fermentation liquid is heated to 100°C and kept warm for 10 minutes.

[0011] Furthermore, the culture medium formula for the shake flask seed culture is 3-4% glucose, 0.03% KH2PO4, 0.3% peptone and 0.05% NaCl; the culture medium formula for the fermenter culture is 0.3% glucose, 0.03% KH2PO4, 0.3% peptone, 0.05% NaCl, 0.001%-0.003% vitamin B1 and 0.01-0.03% polyether defoamer.

[0012] Furthermore, the solid-liquid ratio of the extract to the anhydrous ethanol is 1 g:30 mL.

[0013] Furthermore, the ultrasonic extraction conditions are as follows: power 500W, extraction time 45min.

[0014] The present invention also provides triterpenoid compounds in a liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 obtained according to the preparation method.

[0015] The present invention also provides the use of the triterpenoid compounds in the liquid deep fermentation extract of the Hericium erinaceus strain 01SC403220714 in the preparation of a medicine for preventing and / or treating gastric ulcer.

[0016] Furthermore, the gastric ulcer includes gastric ulcer induced by ethanol or acetic acid.

[0017] The present invention also provides the use of the triterpenoid compounds of the liquid deep fermentation extract of the Hericium erinaceus strain 01SC403220714 in the preparation of health products or foods with the function of assisting in protecting the gastric mucosa.

[0018] The present invention discloses the following technical effects:

[0019] The present invention provides a Hericium erinaceus strain 01SC403220714. Experimental verification shows that its liquid deep fermentation extract contains triterpenoid compounds, total phenolic compounds, total flavonoid compounds, polysaccharides and other substances, among which the triterpenoid compounds have good anti-gastric ulcer efficacy and significant effect. The present invention provides new materials for the development and utilization of Hericium erinaceus strains and products thereof, and provides new ideas for the prevention or treatment of gastric ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a flow chart for the use of triterpenoid compounds from the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 to fight gastric ulcer. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The Hericium erinaceus strain 01SC403220714 provided by the present invention has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 5, 2024, with a deposit number of CGMCC No.41328, and is classified as Hericium erinaceus. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] Example 1 Identification and preservation of Hericium erinaceus strain 01SC403220714

[0029] 1. Cultivation of strain 01SC403220714

[0030] Preparation of slant culture medium: Wash 200g of potatoes and cut them into 2cm cubes. Add 1000mL of boiled juice and filter it. Then add 30g / L glucose, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate and 24g / L agar.

[0031] Inoculation: Divide the prepared culture medium into test tubes while it is still hot, with the filling volume being one-fifth of the test tubes, plug them with cotton plugs, sterilize them in a pressure cooker at 121℃ for 40 minutes, open the lid and take them out, place them on a slope while they are still hot, and after 10 hours, operate on a sterile operating table, inoculate 3 mm-sized strain 01SC403220714 mother seed blocks into the seed tubes, and culture them in an incubator at 25-29℃ for 7-13 days.

[0032] 2. Sequencing

[0033] After the strain 01SC403220714 was cultured, a certain amount of mycelium was ground into powder by liquid nitrogen, and the total DNA of the strain was extracted according to the instructions of the kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). Then, PCR amplification was performed by pre-denaturation at 94℃ for 4min, annealing at 55.5℃ for 1min, and extension at 72℃ for 4min for 35 cycles; extension at 72℃ for 5min. Finally, the ITS sequence obtained by sequencing was analyzed by online BLAST analysis in the NCBI database and DNA star software. The following genome sequence of the strain was obtained. After comparison, the strain 01SC403220714 was identified as Hericium erinaceus, and the synonym of the same species is Hericium caput-medusae.

[0034] The strain gene sequence is:

[0035] 3'-GAGGTCAGTTGAGATGAaAGTTGTCCAAAGACGGTTAGAAGCAGACCAACGCAA -5'.

[0036] Example 2 Liquid submerged fermentation of Hericium erinaceus strain 01SC403220714

[0037] 1. Liquid deep fermentation

[0038] Process flow: activation of original seed → inoculation of slant strain → primary shake flask seed culture → secondary shake flask seed culture → primary seed fermentation tank culture → secondary seed fermentation tank culture → tertiary seed fermentation tank culture → heating of fermentation liquid → tank placement → combination of Hericium erinaceus liquid deep fermentation liquid and mycelium extract → heating and concentration → liquid deep fermentation extract.

[0039] 1.1 Activation of stock and inoculation of slant strains

[0040] Wash 200g of potatoes and cut them into 2cm cubes. Add 1000mL of water, boil and filter the juice, add 30g / L glucose, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate and 24g / L agar, boil and stir constantly, dispense into test tubes while hot, plug the bottles and place them in an autoclave for sterilization, steam pressure 1.1 atmospheres, 121℃, time 30min, take them out and naturally cool to 40-45℃, spread them into a slope, blank culture for 1 day, and use them only when there are no bacteria. Operate on a sterile operating table, inoculate the 3mm-sized strain 01SC403220714 mother seed block into the seed tube, and place it in an incubator at 25-29℃ for 7-13 days.

[0041] 1.2 Primary shake flask seed culture

[0042] Weigh 3-4% glucose, 0.03% KH2PO4, 0.3% peptone, and 0.05% NaCl by mass ratio, heat and dissolve the above reagents with distilled water, put 100mL in a 500mL triangular flask, plug with cotton, put into the autoclave and sterilize at 1 atmosphere and 121℃ for 30min, cool to room temperature and place for one day for use. Quickly remove the bevel and the first-level shaking bottle stopper under the clean bench in the sterile room, pick the bevel bacteria block into the shaking bottle with an inoculation needle and plug it with cotton; place the first-level shaking bottle in a shaking bottle machine at 180-230rpm and 27±1℃ for 4-7 days and take it out for use.

[0043] 1.3 Secondary shake flask seed culture

[0044] Weigh 3-4% glucose, 0.03% KH2PO4, 0.3% peptone, and 0.05% NaCl by mass ratio, heat and dissolve the above reagents with distilled water, put 500mL in a 2000mL triangular flask, plug it with cotton, put it in a sterilizer and sterilize it at 1 atmosphere and 121℃ for 30min, cool it to room temperature and place it for one day for use. Quickly remove the first-level shake flask stopper and the second-level shake flask stopper under the clean bench in the sterile room, pick the first-level shake flask bacteria block into the second-level shake flask with an inoculation needle and then plug it; place the second-level shake flask in a shaker at 180-230rpm and 27±1℃ for 4-7 days and take it out for use.

[0045] 4. Cultivation of first, second and third level seed fermentation tanks

[0046] The culture medium formula of the first, second and third fermentation tanks is the same. Glucose 0.3%, KH2PO4 0.03%, peptone 0.3%, NaCl 0.05%, trace vitamin B1 0.001%-0.003% and polyether defoamer 0.01-0.03% are weighed according to the mass ratio. The fermentation tank is disinfected and sterilized, and the culture medium formula liquid is passed through the material pipe by means of pressure difference, and is pressed into the tank to be sterilized through the filter, and sufficient water is added according to the calculated material volume, and the tank lid is covered and tightened, and sterilized at 120-130℃ for 30-40min, cooled, and when the tank pressure is lower than the air pressure, the secondary shake flask bacteria are aseptically inoculated, and after culturing at 27-29℃ for 5-7 days, the primary fermentation tank is transferred to the sterilized secondary fermentation tank, and after culturing at 27±1℃ for 4-7 days, the secondary fermentation tank is transferred to the sterilized tertiary fermentation tank, and cultured at 27±1℃ for 4-7 days, the fermentation tank is heated to 100℃, kept warm for 10min, cooled, and the tank is released after the valve is opened to increase the pressure, the fermentation liquid and the mycelium water extract are combined, and heated and concentrated to obtain the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714.

[0047] 2. Detection of chemical components in liquid deep fermentation extract

[0048] 2.1 Determination of triterpenoid content

[0049] 2.1.1 Drawing a standard curve

[0050] Accurately weigh the oleanolic acid reference substance, add methanol to make a solution containing 0.2 mg per 1 mL, which is the reference substance solution. Accurately measure 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the reference substance solution, respectively, and place them in 15 mL stoppered test tubes, evaporate to dryness, cool, accurately add 0.2 mL of the newly prepared vanillin glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70°C water bath for 15 minutes, immediately cool in an ice bath for 5 minutes, take out, accurately add 4 mL of ethyl acetate, shake well, use the corresponding reagent as a blank, and measure the absorbance at a wavelength of 546 nm according to the UV-visible spectrophotometry method, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0051] 2.1.2 Determination of triterpenoid content

[0052] Accurately weigh an appropriate amount of the sample to be tested and place it in a stoppered conical flask, add 30 mL of ethanol, ultrasonically treat for 45 minutes, filter, place the filtrate in a 50 mL volumetric flask, use an appropriate amount of ethanol to wash the filter and filter residue in batches, add the washing liquid into the same volumetric flask, add ethanol to the scale, shake well, and the test solution is obtained. Accurately measure 0.2 mL of the test solution, place it in a 15 mL stoppered test tube, evaporate, and cool. Subsequent operations are the same as "2.2.1 Draw a standard curve", measure the absorbance, read the content of oleanolic acid in the test solution from the standard curve, and calculate the total triterpene content.

[0053] The total triterpenoid content was calculated according to the formula:

[0054] Where: W: target content in the sample, unit is g / 100g; C: target concentration in the sample solution, unit is mg / L; C0: target concentration in the blank control, unit is mg / L; V: fixed volume, unit is mL; N: dilution factor; m: sample volume, unit is g.

[0055] 2.2 Determination of total phenolic compound content

[0056] 2.2.1 Drawing the standard curve

[0057] Accurately pipette 1mL, 2mL, 3mL, 4mL, and 5mL of 100mg / L gallic acid standard stock solution into a 100mL volumetric flask, dilute to the mark with water, and shake well. The concentrations are 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, and 5μg / mL, respectively. Use a pipette to pipette 1.0mL of gallic acid working solution, water, and test solution into 10mL graduated test tubes, add 1.25mL of Folin phenol (10%) to each test tube and shake well. React for 3-8min, add 4mL of 10% sodium carbonate solution, dilute to the mark with water, and shake well. Place in a 30℃ water bath for 60min, take out, cool and dilute to the mark, and use a 10mm cuvette to measure the absorbance with a spectrophotometer at a wavelength of 760nm. Make a standard curve based on the absorbance of the gallic acid working solution and the concentration of each working solution.

[0058] 2.2.2 Determination of total phenolic compound content

[0059] Accurately weigh an appropriate amount of the sample to be tested into a centrifuge tube, add 5 mL of 60% ethanol aqueous solution preheated at 70°C, stir thoroughly with a glass rod, immediately transfer to a 70°C water bath, extract for 10 minutes (stir every 5 minutes), take out and cool to room temperature, centrifuge at 4000r / min for 10 minutes, and transfer the supernatant to a 50mL volumetric flask. Re-extract the residue with 5 mL of 60% ethanol aqueous solution, combine the extracts to the scale, shake well, and filter through a 0.45μm filter. According to the method under the standard curve preparation, from "add 1.25 mL of folin phenol (10%) and shake well", operate in the same way, measure the absorbance, read the gallic acid content in the test solution from the standard curve, and calculate the total phenol content.

[0060] The total polyphenol content was calculated according to the formula:

[0061] Where: X: total polyphenol content in the sample, unit is g / 100g; A: absorbance of sample extract L; A0: absorbance of reagent blank solution; k: slope of gallic acid standard curve; m: sample weight, unit is g; V: volume of sample extract, unit is mL; d: dilution factor.

[0062] 2.3 Determination of total flavonoid content

[0063] 2.3.1 Drawing a standard curve

[0064] Accurately pipette 0, 0.50, 1.00, 2.00, 3.00, 4.00 mL of 150ug / mL rutin standard solution, transfer to a 10mL graduated colorimetric tube, add 30% ethanol solution to 5 mL, add 0.3 mL of 5% sodium nitrite solution, shake and place for 5 minutes, add 0.3 mL of 10% aluminum nitrate solution, shake and place for 6 minutes, add 2 mL of 1.0 mol / L sodium hydroxide solution, dilute to the scale with 30% ethanol, mix, place for 15 minutes, and measure the absorbance at 510 nm. Draw a standard curve with rutin content (ug) as the horizontal axis and absorbance as the vertical axis.

[0065] 2.3.2 Determination of total flavonoid content

[0066] Weigh an appropriate amount of the mixed sample into a 50mL Erlenmeyer flask with a stopper, add 30mL of ethanol solution (60% by volume), extract at 40°C for 50min by ultrasonic, filter into a 50mL volumetric flask, add 10mL of ethanol solution (60% by volume) to the Erlenmeyer flask, extract at 40°C for 20min by ultrasonic, filter, combine the filtrate, wash the residue with 10mL of hot ethanol solution (60% by volume), put the filtrate in a volumetric flask, cool to room temperature, dilute to the mark with ethanol solution (60% by volume), shake well, and set aside. Pipette 5.0mL of sample solution into a 25mL stoppered colorimetric tube, add ethanol solution (60% by volume) to 5.0mL, add 0.3mL of 5% sodium nitrite solution, shake and let stand for 5min, add 0.3mL of 10% aluminum nitrate solution, shake well and let stand for 6min, add 2mL of 1.0mol / L sodium hydroxide solution, make up to the scale with 30% ethanol, mix well, let stand for 15min, measure the absorbance at 510nm, and calculate the total flavonoid content of the sample solution according to the standard curve.

[0067] The total flavonoid content was calculated according to the formula:

[0068] Where: X: total flavonoid content in the sample, g / 100g; M: mass of the sample, in g; m1: total flavonoid mass of the sample solution calculated on the calibration curve, in mg; V1: volume of the sample absorbed during measurement, in mL; V: extraction volume of the sample, in mL.

[0069] 2.4 Determination of crude polysaccharide content

[0070] 2.4.1 Drawing a standard curve

[0071] Pipette 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of 100mg / L standard glucose working solution into a 20mL stoppered test tube, and fill to 1.0mL with distilled water. Add 1.0mL of phenol solution to the test solution, then quickly add 5.0mL of sulfuric acid and let stand for 10min. Use a vortex oscillator to mix the reaction solution thoroughly, then place the test tube in a 30℃ water bath to react for 20min, and measure the absorbance at 490nm. Use the glucose mass concentration as the horizontal axis and the absorbance value as the vertical axis to develop a standard curve.

[0072] 2.4.2 Determination of crude polysaccharide content

[0073] Weigh an appropriate amount of sample and place it in a 50mL stoppered centrifuge tube. Soak the sample with 5mL of water, slowly add 20mL of anhydrous ethanol, and shake it with a vortex oscillator to mix it evenly. Place it in an ultrasonic extractor for ultrasonic extraction for 30 minutes. After the extraction is completed, centrifuge it at 4000r / min for 10 minutes and discard the supernatant. Wash the insoluble matter with 10mL of 80% ethanol solution and centrifuge it. Transfer the above insoluble matter into a round-bottom flask with water, add 25mL of water, and extract it ultrasonically at 120W for 90 minutes. Cool to room temperature. Pipette 0.1mL of sample determination solution into a 20mL stoppered test tube, add water to 1mL, add 1mL of phenol solution, 5mL of sulfuric acid, let it stand for 10min, mix it evenly, bath it in 30℃ water for 20min, and measure the absorbance at 490nm. Calculate the crude polysaccharide content of the sample solution according to the standard curve.

[0074] The crude polysaccharide content was calculated according to the formula:

[0075] In the formula: m1: sugar content in the sample solution obtained from the standard curve, in μg; V1: sample fixed volume, in mL; V2: volume of the sample solution transferred during colorimetric determination, in mL; m2: sample mass, in g; 0.9: correction factor for converting glucose to dextran.

[0076] The contents of various chemical components in the liquid deep fermentation extract of the Hericium erinaceus strain 01SC403220714 of the present invention are measured and are shown in Table 1.

[0077] Table 1 Chemical composition content of liquid deep fermentation extract of strain 01SC403220714

[0078]

[0079] 3. Extraction and detection of triterpenoids

[0080] Take the liquid deep fermentation extract of strain 01SC403220714, dry it, add anhydrous ethanol at a solid-liquid ratio of 1:30 (g:mL), place it in an ultrasonic cleaning machine, set the ultrasonic power to 500W, ultrasonic extraction for 45 minutes, filter the extract, concentrate and dry it, dissolve the obtained dry material in chloroform, and add concentrated sulfuric acid. It was found that the chloroform layer appeared red, while the sulfuric acid layer had green fluorescence, which proved that the obtained dry material was a triterpenoid compound.

[0081] 4. Extraction and detection of polysaccharides

[0082] Take the liquid deep fermentation extract of strain 01SC403220714, dry it, add anhydrous ethanol at a solid-liquid ratio of 1:10 (g:mL), shake it with a vortex oscillator to mix it evenly, and put it in an ultrasonic extractor for ultrasonic extraction for 30 minutes. After the extraction, centrifuge it at 4000r / min for 10 minutes, and discard the supernatant. The insoluble matter is washed with 10mL 80% ethanol solution and centrifuged. The insoluble matter is transferred to a beaker, distilled water is added at a solid-liquid ratio of 1:20 (g:mL), and extracted at 70℃ for 3h. Cool to room temperature, centrifuge at 7000r / min for 10min, concentrate the extract to 1 / 4 of the original volume, and add anhydrous ethanol to a final ethanol concentration of 70%. Overnight at 4℃, centrifuge at 4000r / min for 10min to collect the precipitate and dry it. The obtained dry matter is dissolved in distilled water, 6% phenol is added, and concentrated sulfuric acid is added. It is observed that the solution is orange-yellow, proving that the obtained dry matter is crude polysaccharide.

[0083] Example 3: Triterpenoid compounds in the liquid submerged fermentation extract of strain 01SC403220714 have anti-gastric ulcer activity

[0084] 40 Kunming mice, half male and half female, were randomly divided into 4 groups, 10 mice in each group, namely the model group, the low, medium and high dose groups of triterpenoid compounds fermented by liquid submerged fermentation of strain 01SC403220714 (0.1, 0.2, 0.4 g / kg). The model group was gavaged with distilled water, and the other groups were gavaged with corresponding doses for 5 consecutive days. On the 6th day, the mice were allowed to drink water freely and fast for 24 hours. After 30 minutes of normal administration in each group, each mouse was gavaged with 0.2 mL of anhydrous ethanol. One hour later, the mice were killed by cervical dislocation, the abdominal cavity was opened and the cardia was ligated to free the stomach, the stomach was cut open along the greater curvature of the stomach, and the stomach viscera were carefully washed with saline, soaked in 10% neutral formalin solution, fixed for 10 minutes, placed on filter paper to absorb the liquid, and then the gastric mucosa was observed under an optical microscope. The ulcer area and ulcer inhibition rate were calculated.

[0085] Ulcer area: S = π × d1 × d2 × 1 / 4, where π is the pi, d1 and d2 are the maximum horizontal and vertical diameters passing through the center of the ulcer, respectively.

[0086] Ulcer inhibition rate (%) = (average ulcer area of ​​model group - average ulcer area of ​​drug administration group) / average ulcer area of ​​model group × 100.

[0087] Table 2 Effects of triterpenoids from submerged fermentation of strain 01SC403220714 on gastric ulcer in mice

[0088]

[0089] Note: Compared with the model group, * Indicates p < 0.05, the difference is significant. **Indicates p < 0.01, the difference is extremely significant.

[0090] From the experimental results in Table 2, it can be seen that compared with the model group, the triterpenoids produced by submerged liquid fermentation of strain 01SC403220714 can significantly reduce the area of ​​gastric ulcer in a dose-dependent manner, indicating that the triterpenoids produced by submerged liquid fermentation of strain 01SC403220714 have a good protective effect on gastric ulcer in mice caused by ethanol.

[0091] Example 4

[0092] The effects of triterpenoid compounds and polysaccharides from the liquid submerged fermentation extract of strain 01SC403220714 on rats with acetic acid-induced gastric ulcer were compared and observed.

[0093] Wistra male rats were fasted but not watered for 48 h, anesthetized with ether, fixed in supine position, the abdomen was depilated, disinfected with iodine and alcohol, and a 2.5 cm incision was made in the middle of the xiphoid process. The duodenum was gently hooked out under the right liver with curved hemostatic forceps, the stomach was gently pulled out of the abdomen, and a 0.25 mL syringe was used to pierce 0.4-0.5 mm below the rat's gastric serosa at the junction of the gastric body and the pyloric antrum on the ventral side of the stomach. 0.05 mL of 10% acetic acid solution was injected, the stomach was gently returned, the abdominal wall muscle layer and skin were sutured, and the rat was fed normally after disinfection.

[0094] From the day after surgery, the rats were randomly divided into 5 groups, 10 in each group, namely the model group, the low-dose and high-dose groups of triterpenoid compounds fermented by liquid submerged fermentation of strain 01SC403220714 (0.1 g / kg, 0.4 g / kg), and the low-dose and high-dose groups of polysaccharides fermented by liquid submerged fermentation of strain 01SC403220714 (0.1 g / kg, 0.4 g / kg). The model group was gavaged with distilled water, and the other groups were gavaged with corresponding doses for 14 consecutive days. Two hours after the last administration, the rats were decapitated, the pylorus and cardia were ligated, the stomach was immersed in 1% formaldehyde solution, fixed for 10 minutes, cut open along the greater curvature of the stomach, everted and washed, and the ulcer formation was observed on the mucosal surface of the corresponding part of the acetic acid solution injection. The average of the longest and shortest diameters of the ulcer was taken as the ulcer diameter, and the ulcer inhibition rate was calculated.

[0095] Ulcer inhibition rate (%) = (average ulcer diameter of model group - average ulcer diameter of drug administration group) / average ulcer diameter of model group × 100.

[0096] From the experimental results in Table 3, it can be seen that compared with the model group, the triterpenoids and polysaccharides produced by submerged liquid fermentation of strain 01SC403220714 can significantly reduce the diameter of gastric ulcers in rats and improve the inhibition rate of gastric ulcers. In addition, the triterpenoids produced by submerged liquid fermentation of strain 01SC403220714 have a significantly better inhibitory effect on gastric ulcers in mice caused by ethanol than polysaccharides.

[0097] Table 3 Effects of triterpenoids and polysaccharides from submerged fermentation of strain 01SC403220714 on gastric ulcer in mice

[0098]

[0099] Note: Compared with the model group, * Indicates p<0.05, the difference is significant. ** Indicates p < 0.01, the difference is extremely significant.

[0100] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Hericium erinaceus strain 01SC403220714, characterized in that: The preservation number of the Hericium erinaceus strain 01SC403220714 is CGMCC No.41328.

2. A method for preparing triterpenoid compounds from liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714, characterized in that: The following steps are involved: The Hericium erinaceus strain 01SC403220714 is cultured on a slant, first-level shake flask seed culture, second-level shake flask seed culture, first-level seed fermentation tank culture, second-level seed fermentation tank culture and third-level seed fermentation tank culture to obtain a fermentation liquid and a fermentation mycelium, the mycelium is extracted to obtain a mycelium extract, the mycelium extract is combined with the fermentation liquid, and heated and concentrated to obtain a liquid deep fermentation extract; The extract is dried, anhydrous ethanol is added, and ultrasonic extraction is performed to obtain an extract solution, and the extract solution is filtered, concentrated, and dried to obtain triterpenoid compounds of liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714; The preservation number of the Hericium erinaceus strain 01SC403220714 is CGMCC No.41328.

3. The preparation method according to claim 2, characterized in that: The temperature of the slant culture is 25-29°C, and the time is 7-13 days; the conditions of the primary shake flask seed culture and the secondary shake flask seed culture are 180-230rpm and 27±1°C for 4-7 days; the conditions of the primary seed fermentation tank culture, the secondary seed fermentation tank culture and the tertiary seed fermentation tank culture are 27±1°C for 4-7 days; the fermentation liquid is heated to 100°C and kept warm for 10 minutes.

4. The preparation method according to claim 2, characterized in that: The culture medium formula of the shake flask seed culture is 3-4% glucose, 0.03% KH2PO4, 0.3% peptone and 0.05% NaCl; the culture medium formula of the fermenter culture is 0.3% glucose, 0.03% KH2PO4, 0.3% peptone, 0.05% NaCl, 0.001%-0.003% vitamin B1 and 0.01-0.03% polyether defoamer.

5. The preparation method according to claim 2, characterized in that: The solid-liquid ratio of the extract to the anhydrous ethanol is 1 g:30 mL.

6. The preparation method according to claim 2, characterized in that: The conditions of the ultrasonic extraction are: power 500W, extraction time 45min.

7. Triterpenoid compounds in the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 obtained by the preparation method according to any one of claims 2 to 6.

8. Use of the triterpenoid compounds of the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 according to claim 7 in the preparation of drugs for preventing and / or treating gastric ulcers.

9. The use according to claim 8, characterized in that: The gastric ulcer includes ethanol or acetic acid induced gastric ulcer.

10. Use of the triterpenoid compounds of the liquid deep fermentation extract of Hericium erinaceus strain 01SC403220714 according to claim 7 in the preparation of health products or foods with auxiliary protection function of gastric mucosa.

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