Stropharia rugoso-annulata glycopeptide extraction method, obtained product and application

Abstract

The invention discloses a stropharia rugoso-annulata glycopeptide extraction method, an obtained product and application. The stropharia rugoso-annulata glycopeptide extraction method comprises the following steps: uniformly drying stropharia rugoso-annulata, crushing, sieving, extracting by adopting a microwave-assisted eutectic solvent, filtering out liquid, carrying out alcohol precipitation to obtain glycoprotein, removing free protein from the glycoprotein, decolorizing, and carrying out enzymolysis to convert the glycoprotein into a glycopeptide component. The extraction method of the stropharia rugosoannulata glycopeptide is provided for the first time, and the method is simple in process, low in cost, easy to control, higher in glycopeptide yield and suitable for industrial production. The obtained glycopeptide has excellent antioxidant activity and liver protection activity, and has certain medicinal value in the field of medicines.

Description

Technical Field

[0001] The invention relates to a method for extracting Stropharia rugosa glycopeptide, the obtained Stropharia rugosa glycopeptide and application thereof. Background Art

[0002] Stropharia, also known as red pine mushroom, wrinkled globe mushroom, etc., is an important edible and medicinal fungus. It belongs to the phylum Basidiomycetes and the genus Stropharia of the family Strophariaceae.

[0003] Due to its high nutritional value and good taste, Stropharia cap mushrooms are highly favored in the international market, and their prices are much higher than other common edible mushrooms. Studies in Japan and other places have shown that the anti-cancer effect of Stropharia cap mushrooms can reach more than 75% and can effectively enhance human health.

[0004] Stropharia is rich in various nutrients such as protein, vitamins, minerals and polysaccharides. Specifically, it is rich in amino acids, essential amino acids and various trace elements. In addition, Stropharia also has significant antioxidant effects, can improve human immunity, and has various pharmacological effects such as antibacterial, hypoglycemic and anti-cancer, and is deeply loved by consumers and researchers.

[0005] At present, the main ways to obtain glycopeptides from traditional Chinese medicine include direct extraction from traditional Chinese medicine, obtaining by enzymatic hydrolysis of glycoproteins from traditional Chinese medicine, and preparing by fermentation. Glycopeptides have the properties of both sugars and polypeptides. Most glycopeptide components are hydrophilic components that are easily soluble in water. Therefore, most glycopeptides are currently extracted by water extraction and alcohol precipitation. There are also alkali extraction methods and salt solution extraction methods. Sometimes, in order to improve the efficiency of glycopeptide extraction, ultrasound and microwave assisted extraction methods can be used. When optimizing the process, the glycopeptide extraction rate is often used as an evaluation indicator. Glycopeptides, as a class of natural products with important biological activities, have attracted much attention in recent years. This type of substance has multiple pharmacological effects such as anti-tumor, immunomodulation, neuroprotection, and anti-inflammatory effects, but there are few reports on the activity of glycopeptide extracts from Stropharia rugosodium. Summary of the invention

[0006] The invention provides a method for preparing a Stropharia capillaris extract, and attempts to extract glycopeptides from Stropharia capillaris for the first time. Compared with a conventional method for extracting plant glycopeptides, the invention adopts a microwave-assisted low eutectic solvent to extract glycoprotein components, and finally obtains Stropharia capillaris glycopeptides through deproteinization, decolorization and enzymatic hydrolysis. The extraction method has a simple process, is easy to control, and has a high glycopeptide yield. The obtained glycopeptides have excellent antioxidant activity and hepatoprotective activity, and have good application prospects.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A method for extracting Stropharia rugosa glycopeptide, the method comprising the following steps:

[0009] (1) drying the Stropharia officinalis at a constant temperature of 25-28° C. for 3-4 h, then heating the temperature to 51° C. at a heating rate of 2-3° C. / h, keeping the temperature for 7-8 h, and then heating the temperature to 60° C. at a heating rate of 0.5-1° C. / h until the moisture content is 12%-13%;

[0010] (2) crushing the dried Stropharia officinalis, passing through a 10-mesh sieve and a 30-mesh sieve, and obtaining a powder with a particle size of 10-30 mesh;

[0011] (3) mixing the Stropharia officinalis powder with a low eutectic solvent solution, extracting the mixture under the assistance of a microwave, and filtering the mixture after the extraction to obtain a filtrate;

[0012] (4) adding ethanol to the filtrate for precipitation, and collecting the precipitate to obtain crude glycoprotein;

[0013] (5) dissolving the crude glycoprotein in water, adding sodium hydroxide solution to adjust the pH to 9-9.5, then adding sodium chloride and heating to react, adjusting the pH to neutral after the reaction, adding ethanol to precipitate, and collecting the precipitate;

[0014] (6) dissolving the precipitate from step (5) with water, decolorizing with a macroporous adsorption resin under ultrasound, filtering and centrifuging after decolorization, and collecting the supernatant to obtain a stropharia glycoprotein solution;

[0015] (7) adding snail enzyme to the stropharia glycoprotein solution for enzymolysis, inactivating the enzyme after enzymolysis, then adding bromelain for enzymolysis, inactivating the enzyme after enzymolysis, and freeze-drying the supernatant to obtain stropharia glycopeptide powder.

[0016] Furthermore, in step (1), the water content of the stropharia is relatively high and is not suitable for processing by sun drying, so it is recommended to use a drying machine or an electric heating blast dryer for mechanical drying. In addition, different drying methods have a significant effect on the quality and volatile flavor components of the stropharia, and the drying method used in the present invention can increase the content of the glycopeptide of the stropharia.

[0017] Furthermore, in step (2), the use of a screen to filter the mushroom during the crushing process is a common process step, the purpose of which is to remove larger particles or impurities and ensure the quality of the final product. In the crushing and screening process of the mushrooms, screens with different apertures are usually used to achieve this goal. After multiple experimental verifications, the particle size range of 10-30 mesh has the highest extraction rate and is more suitable in process production.

[0018] Furthermore, in step (3), the deep eutectic solvent (DES) is a green solvent that is non-toxic and biodegradable, and is therefore environmentally friendly and safe for the human body. Microwave heating can significantly improve the efficiency of DES extraction and obtain a higher extraction rate in a shorter time.

[0019] Furthermore, in step (3), the low eutectic solvent system used is betaine and butanediol, and the molar ratio of betaine to butanediol is 1-3: 1. The low eutectic solvent solution is obtained by uniformly mixing the low eutectic solvent with water, and the content of the low eutectic solvent in the low eutectic solvent solution is 55-65wt%.

[0020] Furthermore, in step (3), the ratio of Stropharia rugosa powder to the low eutectic solvent solution is 1 g: 20-40 mL.

[0021] Furthermore, in step (3), the Stropharia rugosa is extracted twice with the assistance of microwaves, wherein the microwave power of the first microwave extraction is 50-150 W, the microwave temperature is 90-110° C., and the extraction time is 10-20 min; and the microwave power of the second microwave extraction is 160-250 W, the microwave temperature is 130-150° C., and the extraction time is 10-20 min.

[0022] Furthermore, in step (4), the alcohol precipitation method is a commonly used refining process in the extraction process of traditional Chinese medicine, and its basic principle is to use the characteristics that the active ingredients can be dissolved in ethanol while the impurities are insoluble in ethanol. This method can effectively remove non-alcohol-soluble impurities in the solution and improve the purity and quality of the active ingredients of the medicine. In the present invention, ethanol is added to an ethanol volume concentration of 80-85%, and a high concentration of system alcohol concentration is used, which can not only improve the extraction rate of glycoproteins but also remove the low eutectic solvent remaining in the solution.

[0023] Furthermore, in step (5), free protein in the crude glycoprotein solution is removed by salting out with NaOH and NaCl. The concentration of the NaOH solution is 1-10wt%. 3-5g of NaCl is added to every 100ml of the solution. After adding NaCl, the reaction is carried out under boiling for 30-50min. The present invention makes full use of the property that protein is easily precipitated under alkaline conditions, thereby improving the removal effect of free protein. After the removal is completed, the pH is adjusted to 7 with HCl to further ensure the neutral environment of the solution for subsequent ethanol precipitation operations. After adding anhydrous ethanol, the low solubility of ethanol is utilized to dissolve polysaccharides in ethanol while proteins are insoluble, thereby achieving the separation of glycoproteins.

[0024] Furthermore, in step (5), ethanol is added to a volume concentration of 80-85% to precipitate the glycoprotein, and the filtered precipitate is washed with anhydrous ethanol and ether respectively to better remove free protein therein.

[0025] Furthermore, in step (6), an activated macroporous adsorption resin is added to the solution at a volume ratio of 1:0.5-2 for decolorization. The macroporous adsorption resin is preferably macroporous adsorption resin X-5. The process of activating the macroporous adsorption resin is as follows: first, add the dry macroporous adsorption resin to a certain volume of anhydrous ethanol and soak it for 24 hours to fully swell it, slowly stir it, remove impurities and broken resin particles, rinse it with water until there is no alcohol taste, then soak it in a dilute HCl solution for 5 hours, rinse it with water until it is neutral, soak it in a dilute NaOH solution for 5 hours, and wash it with water again until it is neutral. Before use, use filter paper to absorb the moisture on the surface of the resin and keep it dry.

[0026] Furthermore, in step (6), ultrasound-assisted decolorization of the macroporous adsorption resin can significantly improve its foaming and emulsifying properties and improve its adsorption capacity for pigments. The ultrasonic power is 100-150W, the decolorization temperature is 40-60°C, and the decolorization time is 20-30min.

[0027] Furthermore, in step (6), after decolorization, the liquid is first filtered out with a filter cloth, and then the liquid is centrifuged at 5800-6200 rpm for 10-12 min to obtain the supernatant.

[0028] Furthermore, in step (7), the cell wall breaking rate of the snail enzyme is 80-90%, and the enzyme activity of the bromelain is 280-320u / mg.

[0029] Furthermore, in step (7), the optimal conditions for the two enzymes to hydrolyze proteins are different: the hydrolysis temperature of snail enzyme is 43-46° C., and the hydrolysis time is 8-10 h; the hydrolysis temperature of bromelain is 58-62° C., and the hydrolysis time is 5-7 h.

[0030] Furthermore, in step (7), the amount of snail enzyme used is 3-6% of the mass of the Stropharia capillaris glycoprotein solution; the amount of bromelain used is 3-6% of the mass of the Stropharia capillaris glycoprotein solution.

[0031] Furthermore, in step (7), snail enzyme can be completely inactivated by bathing in water at 70°C for 10 minutes; bromelain can be completely inactivated by bathing in water at 80°C for 10 minutes.

[0032] The present invention uses microwave-assisted deep eutectic solvent extraction to extract glycopeptide components from Pleurotus eryngii for the first time, which not only improves the extraction efficiency and purity of glycopeptides, but also reduces environmental pollution and production costs. The obtained glycopeptide has excellent antioxidant activity and hepatoprotective activity, so the Pleurotus eryngii glycopeptide obtained according to the method of the present invention and the use of the Pleurotus eryngii glycopeptide in the preparation of hepatoprotective products and / or antioxidant products are also within the scope of protection of the present invention.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. The present invention helps to evenly remove moisture from the mushroom body through precise temperature control, reasonable drying time arrangement and gradual heating method during the drying process, avoids quality degradation or loss of nutrients due to excessively high temperature, and ensures a high extraction rate of glycopeptides.

[0035] 2. The present invention uses a low eutectic solvent as solvent for extraction. The low eutectic solvent has the advantages of high efficiency, environmental protection, mild conditions, and maintaining the physicochemical properties and biological activity of polysaccharides in the extraction of plant polysaccharides. It is a very promising green extraction technology, but it is rarely used in the extraction process of glycopeptides.

[0036] 3. The present invention adopts a microwave-assisted low eutectic solvent to extract glycopeptides. While the low eutectic solvent itself has excellent extraction efficiency and good biological activity, microwave radiation can also penetrate the cell wall, increase the internal pressure of the cell, cause the cell wall to rupture, promote the dissolution of glycopeptides, and further improve the extraction rate of glycopeptides.

[0037] 4. The present invention uses NaOH and NaCl salting out to remove free protein in the crude glycoprotein solution, which is simple to operate and has significant effects, thereby improving the purity of the glycoprotein.

[0038] 5. The present invention uses ultrasound-assisted macroporous adsorption resin decolorization. X-5 resin has excellent decolorization ability and can effectively remove pigments in the solution. The decolorization process can be further optimized by ultrasound-assisted technology. For example, the use of ultrasound-assisted methods can significantly improve the mass transfer efficiency of the resin, shorten the adsorption equilibrium time, and does not affect the recycling of the resin.

[0039] 6. The present invention adopts the method of enzymolysis of glycoprotein by snail enzyme and bromelain in batches to obtain glycopeptides, which can obtain higher hydrolysis efficiency, more comprehensive glycoprotein decomposition, higher yield and solubility, and more biologically active ingredients, and is suitable for industrial production.

[0040] 7. The present invention is the first to extract glycopeptide components from Pleurotus ostreatus, which has significant antioxidant and liver protection activities and has certain medicinal value in the medical field. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] In the following examples and comparative examples, the snail enzyme used was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a cell wall breaking rate of 90%; the bromelain used was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity of 300u / mg.

[0043] Example 1

[0044] S1. Select 3000g of fresh stropharia, scrape off the scales and mud on the mushroom feet with bamboo strips, spread them in a dryer, keep them at 26°C for 4 hours, then increase the temperature to 51°C at a rate of 2-3°C / h, keep them at 51°C for 8 hours, and then increase the temperature to 60°C at a rate of 0.5-1°C / h, and dry them until the moisture content of the stropharia is 12-13%;

[0045] S2. The dried stropharia were put into a grinder in batches and powdered, first through a 10-mesh sieve, then through a 30-mesh sieve, and the stropharia powder was taken between 10-30 meshes;

[0046] S3. Prepare a low eutectic solvent solution, weigh 234.3 g of betaine, 90.1 g of butanediol and 216.3 g of deionized water, mix them together, heat and stir at 60 ° C for 30 min until it becomes transparent and particle-free, and cool to room temperature to obtain a low eutectic solvent solution with a concentration of 60 wt%;

[0047] S4. Stropharia officinalis powder and low eutectic solvent solution were weighed at a solid-liquid ratio of 1:30 (g / mL), mixed and put into a microwave synthesizer, first extracted at a microwave power of 100 W and 100°C for 15 min, then filtered, and the filtrate was added with a low eutectic solvent solution at a solid-liquid ratio of 1:30 (g / mL), extracted at a microwave power of 200 W and 140°C for 15 min, filtered, and the two filtrates were combined, centrifuged at 6000 rpm for 6 min, and the supernatant was collected;

[0048] S5. The supernatant was concentrated, anhydrous ethanol was slowly added to a volume concentration of 82% ethanol, the mixture was stirred for 1 h, and the mixture was allowed to stand at 4°C for 24 h. After the standing time was complete, the mixture was centrifuged, the supernatant was discarded, the precipitate was collected, water was added to redissolve it, the mixture was centrifuged again, and the supernatant was collected to obtain a crude glycoprotein solution with a concentration of 5 mg / mL;

[0049] S6. The crude sugar protein solution was heated to boiling in a water bath, and a 2 wt % NaOH solution was added while stirring to adjust the solution to pH = 9, and then NaCl solid was added to a NaCl concentration of 5% (g / ml), mixed and boiled for 30 min, cooled to room temperature, adjusted to pH = 7 with HCl, and anhydrous ethanol was added to an ethanol volume concentration of 82%, and allowed to stand at 4°C overnight, filtered, and the precipitate was washed with anhydrous ethanol and ether, respectively, and the process was repeated 3 times, centrifuged, and the precipitate was collected;

[0050] S7. First, add the dry macroporous adsorption resin X-5 to a certain volume of anhydrous ethanol and soak it for 24 hours to fully swell it, slowly stir it to remove impurities and broken resin particles, repeatedly rinse it with deionized water until there is no alcohol smell, then soak it in 4wt% HCl for 5 hours, rinse it with deionized water until it is neutral, soak it in 4wt% NaOH for 5 hours, rinse it again with deionized water until it is neutral, and finally use filter paper to absorb the moisture on the surface of the resin for use;

[0051] S8. The precipitate in S6 was redissolved in water to obtain a solution with a concentration of 3 mg / mL, and then added to the newly activated macroporous adsorption resin X-5 at a volume ratio of 1:1 between the solution and the resin, and ultrasonically treated at 50°C for 25 min with an ultrasonic power of 125 W;

[0052] S9. After the ultrasonic treatment is completed, the macroporous adsorption resin X-5 is first filtered out with a filter cloth, and then the filtrate is centrifuged at 6000 rpm for 10 min, and the supernatant is collected to obtain a stropharia glycoprotein solution;

[0053] S10. Add snail enzyme to the above-obtained Stropharia capensis glycoprotein solution, the amount of snail enzyme is 5% of the mass of the Stropharia capensis glycoprotein solution, react in a constant temperature box at 45°C for 8 hours, then inactivate the enzyme in a water bath at 70°C for 10 minutes, then add bromelain thereto, the amount of bromelain is 5% of the mass of the Stropharia capensis glycoprotein solution, react in a constant temperature box at 60°C for 6 hours, then inactivate the enzyme in a water bath at 80°C for 10 minutes, centrifuge at 6000 rpm, take the supernatant, measure the sugar content and protein content, and lyophilize to obtain Stropharia capensis glycopeptide powder.

[0054] Example 2

[0055] The method of Example 1 was used to extract Stropharia officinalis glycopeptide, except that in step S3, 351.4 g of betaine, 90.1 g of butanediol and 294.4 g of deionized water were weighed and mixed together, heated and stirred at 60° C. for 30 min until a transparent state without particles was obtained, and cooled to room temperature to obtain a low eutectic solvent solution with a concentration of 60 wt %.

[0056] Example 3

[0057] The method of Example 1 was used to extract Stropharia officinalis glycopeptide, except that in step S3, 117.2 g of betaine, 90.1 g of butanediol and 138.2 g of deionized water were weighed and mixed together, heated and stirred at 60° C. for 30 min until a transparent state without particles was obtained, and cooled to room temperature to obtain a low eutectic solvent solution with a concentration of 60 wt %.

[0058] Example 4

[0059] The Stropharia capillaris glycopeptide was extracted according to the method of Example 1, except that: in step S4, Stropharia capillaris powder and a low eutectic solvent solution were weighed at a solid-liquid ratio of 1:20 (g / mL), mixed and put into a microwave synthesizer, first extracted at a microwave power of 50 W and 90° C. for 10 min, then filtered, and the filter residue was added with a low eutectic solvent solution at a solid-liquid ratio of 1:20 (g / mL), extracted at a microwave power of 160 W and 130° C. for 20 min, filtered, and the two filtrates were combined, centrifuged at 6000 rpm for 6 min, and the supernatant was collected.

[0060] Example 5

[0061] The Stropharia capillaris glycopeptide was extracted according to the method of Example 1, except that: in step S4, Stropharia capillaris powder and a low eutectic solvent solution were weighed at a solid-liquid ratio of 1:40 (g / mL), mixed and put into a microwave synthesizer, first extracted at a microwave power of 150 W and 110° C. for 20 min, then filtered, and the filter residue was added with a low eutectic solvent solution at a solid-liquid ratio of 1:40 (g / mL), extracted at a microwave power of 250 W and 150° C. for 10 min, filtered, and the two filtrates were combined, centrifuged at 6000 rpm for 6 min, and the supernatant was collected.

[0062] Example 6

[0063] The glycopeptide of Pseudomonas aeruginosa was extracted according to the method of Example 1, except that: in step S8, the precipitate in S6 was redissolved in water to obtain a solution with a concentration of 3 mg / mL, and then added to the newly activated macroporous adsorption resin X-5 at a volume ratio of 1:2 of the solution to the resin, and ultrasonically treated at 60°C for 30 min with an ultrasonic power of 100 W.

[0064] Example 7

[0065] The Stropharia officinalis glycopeptide was extracted according to the method of Example 1, except that: in step S8, the precipitate in step S6 was redissolved in water to obtain a solution with a concentration of 3 mg / mL, which was added to the newly activated macroporous adsorption resin X-5 at a volume ratio of 2:1, and ultrasonic treatment was performed at 40°C for 20 min using an ultrasonic power of 150 W.

[0066] Comparative Example 1

[0067] The glycopeptide of Stropharia rugosa was extracted according to the method of Example 1, except that: in step S1, 3000 g of fresh Stropharia rugosa was selected, the mushroom scales and the mud at the mushroom feet were scraped off with bamboo sticks, and the mushrooms were spread in a dryer and dried at 60° C. until the water content was 12-13 wt %.

[0068] Comparative Example 2

[0069] The method of Example 1 is used to extract the Stropharia officinalis glycopeptide, except that in step S200, the dried Stropharia officinalis is put into a grinder in batches for powder treatment, and the powder is passed through a 10-mesh sieve to obtain the sieved powder.

[0070] Comparative Example 3

[0071] The method of Example 1 was used to extract Stropharia officinalis glycopeptide, except that in step S3, 117.2 g of betaine, 180.2 g of butanediol and 693.9 g of deionized water were weighed and mixed together, heated and stirred at 60° C. for 30 min until a transparent state without particles was obtained, and cooled to room temperature to obtain a low eutectic solvent solution with a concentration of 30 wt %.

[0072] Comparative Example 4

[0073] The method of Example 1 was used to extract Stropharia officinalis glycopeptide, except that in step S3, 117.2 g of betaine, 38 g of propylene glycol and 103.5 g of deionized water were weighed and mixed together, heated and stirred at 60° C. for 30 min until a transparent state without particles was obtained, and cooled to room temperature to obtain a low eutectic solvent solution with a concentration of 60 wt %.

[0074] Comparative Example 5

[0075] The Stropharia capillaris glycopeptide was extracted according to the method of Example 1, except that: in step S4, Stropharia capillaris powder and low eutectic solvent solution were weighed according to a solid-liquid ratio of 1:30 (g / mL), mixed and put into a microwave synthesizer, extracted at a microwave power of 100 W and 100° C. for 40 min, filtered, and the filtrate was centrifuged at 6000 rpm for 6 min, and the supernatant was collected.

[0076] Comparative Example 6

[0077] The method of Example 1 was used to extract the Stropharia capensis glycopeptide, except that in step S10, snail enzyme was added to the Stropharia capensis glycoprotein solution obtained above, and the amount of snail enzyme was 5% of the mass of the Stropharia capensis glycoprotein solution. The mixture was reacted in a 45° C. incubator for 14 hours, and then the enzyme was inactivated in a 70° C. water bath for 10 minutes. The mixture was centrifuged, the supernatant was taken, the sugar content and protein content were measured, and the mixture was freeze-dried to obtain the Stropharia capensis glycopeptide powder.

[0078] Comparative Example 7

[0079] The method of Example 1 was used to extract the stropharia glycopeptide, except that in step S10, bromelain was added to the stropharia glycoprotein solution obtained above, and the amount of bromelain was 5% of the mass of the stropharia glycoprotein solution. The mixture was reacted in a 60° C. incubator for 14 hours, and then the enzyme was inactivated in a water bath at 80° C. for 10 minutes. The mixture was centrifuged, the supernatant was taken, the sugar content and protein content were measured, and the mixture was freeze-dried to obtain the stropharia glycopeptide powder.

[0080] The Stropharia officinalis glycopeptides obtained in the above examples and comparative examples were evaluated in the following manner:

[0081] 1. Determination of glycopeptide content in Stropharia rugosa

[0082] 1.1 Determination of polysaccharide content in Stropharia officinalis glycopeptide

[0083] ① Standard curve drawing

[0084] The sugar content was determined by the phenol-sulfuric acid method, and a standard curve was drawn. Accurately pipette 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of a glucose standard solution with a mass concentration of 0.1 mg / mL into a centrifuge tube, add ultrapure water to make up to 100 μL, mix well, and obtain standard solutions with mass concentrations of 0.00 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, and 0.10 mg / mL, respectively. Add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, place in a 100℃ water bath for 5 minutes, wait until the temperature in the water bath drops to room temperature, and take it out. Accurately pipette 150 μL into a 96-well plate, use ultrapure water as a blank, measure the absorbance at 490 nm, draw a standard curve of concentration and absorbance, and obtain the regression equation.

[0085] y=0.0041x+0.161,R 2 =0.996. This indicates that glucose has a good linear relationship with absorbance in the concentration range of 0-100 μg / mL.

[0086] ② Preparation of sample solution to be tested

[0087] 5 mg of the lyophilized powder of Stropharia rugosa glycopeptide prepared in each example and comparative example was put into a 10 mL volumetric flask, and water was added to make up the volume. After fully dissolving, a sample solution was obtained.

[0088] ③Determination of sample solution

[0089] Take 100 μL of sample solution in a centrifuge tube, add 100 μL of 6% phenol solution, slowly add 500 μL of concentrated sulfuric acid, mix well, place in a 100℃ water bath for 5 minutes, wait until the temperature in the water bath drops to room temperature, then take out. Accurately pipette 150 μL into a 96-well plate and measure the absorbance at 490 nm.

[0090] Substitute the absorbance into the above regression equation to calculate the solution concentration, and then obtain the polysaccharide extraction rate (W) according to the following formula:

[0091]

[0092] Where:

[0093] W—polysaccharide extraction rate, %;

[0094] C—polysaccharide mass concentration in sample solution, μg / mL;

[0095] V—total volume of sample solution,

[0096] M—mass of freeze-dried powder of Stropharia rugosa glycopeptide.

[0097] 1.2 Determination of protein content in Stropharia officinalis glycopeptide

[0098] ① Preparation of Coomassie Brilliant Blue G250 solution

[0099] Weigh 50 mg of Coomassie Brilliant Blue G250, measure 25 mL of anhydrous ethanol, and 50 mL of 85% (W / V) phosphoric acid, dissolve the Coomassie Brilliant Blue powder in anhydrous ethanol, mix and stir with phosphoric acid, and add water to make up to 500 mL.

[0100] ②Drawing of standard curve

[0101] The protein content was determined by the Coomassie Brilliant Blue method, and a standard curve was drawn. Accurately pipette 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of a BSA standard solution with a mass concentration of 1 mg / mL into a test tube, add ultrapure water to 1 ml, mix well, and obtain standard solutions with mass concentrations of 0.00 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, and 0.10 mg / mL. Use a pipette to accurately pipette 1 mL of each standard sample into a test tube, then pipette 5 mL of Coomassie Brilliant Blue G250 solution into the test tube, mix thoroughly, let stand for 5 min, measure the absorbance at 595 nm, draw a standard curve of concentration and absorbance, and obtain the regression equation.

[0102] Y=0.0071X+0.0552,R 2 =0.9997. This indicates that the protein has a good linear relationship with absorbance in the concentration range of 0-100 μg / mL.

[0103] ③ Preparation of sample solution

[0104] 5 mg of the freeze-dried powder of Stropharia rugosa prepared in each example and comparative example was put into a 10 mL volumetric flask, and water was added to make up the volume. After fully dissolved, a sample solution was obtained.

[0105] ④Determination of sample solution

[0106] Take 1 mL of the sample solution in a test tube, add 5 mL of the prepared Coomassie Brilliant Blue G250 solution, shake evenly, let stand for 5 minutes, and measure the absorbance at 595 nm.

[0107] Substitute the absorbance into the above regression equation to calculate the solution concentration, and then obtain the protein extraction rate (D) according to the following formula:

[0108]

[0109] Where:

[0110] D is the protein extraction rate, %;

[0111] C—protein concentration in sample solution, μg / mL;

[0112] V—total volume of sample solution, mL;

[0113] M—mass of freeze-dried powder of Stropharia rugosa glycopeptide.

[0114] 1.3 Determination of the extraction rate of glycopeptides from Stropharia rugosa

[0115] The glycopeptide extraction rate in Stropharia capillaris glycopeptide = the polysaccharide extraction rate in Stropharia capillaris glycopeptide + the protein extraction rate in Stropharia capillaris glycopeptide.

[0116] 1.4 Experimental Results

[0117] The results of the determination of the extraction rate of Stropharia officinalis glycopeptide are shown in Table 1 below.

[0118] Table 1 Determination results of glycopeptide content in Stropharia rugosa

[0119] Stropharia Polysaccharide extraction rate / % Protein extraction rate / % Glycopeptide extraction rate / % Example 1 14.14 4.82 18.96 Example 2 12.01 4.26 16.27 Example 3 12.71 3.33 16.04 Example 4 12.09 2.58 14.67 Example 5 12.16 2.68 14.84 Example 6 12.24 3.09 15.33 Example 7 14.02 4.65 18.67 Comparative Example 1 10.23 2.58 12.81 Comparative Example 2 11.08 2.99 14.07 Comparative Example 3 10.97 2.96 13.93 Comparative Example 4 10.14 3.07 13.21 Comparative Example 5 10.12 2.48 12.60 Comparative Example 6 11.10 2.71 13.81 Comparative Example 7 9.88 2.11 11.99

[0120] From Table 1, it can be seen that the glycopeptide content of Pseudomonas aeruginosa in each embodiment is significantly higher than that in each comparative example, and the glycopeptide content of the optimally treated embodiment 1 is the highest. It can be seen that the extraction rate of Pseudomonas aeruginosa glycopeptide can be significantly improved by the method of the present invention.

[0121] 2. In vitro antioxidant test

[0122] 2.1. DPPH in vitro antioxidant activity test

[0123] (1) Sample solution preparation

[0124] Take 50 mg of the freeze-dried Stropharia officinalis glycopeptide powder of Example 1 and Comparative Example 1, Comparative Example 3, Comparative Example 6, and Comparative Example 7, add 10 mL of water, use a vortex to mix evenly to ensure full dissolution, centrifuge 13 portions of Stropharia officinalis glycopeptide solution at 8000 rpm for 6 min, and take the supernatant for determination.

[0125] (2) Detection

[0126] ① Preheat the ELISA instrument for 15 minutes and adjust the wavelength to 517nm.

[0127] ②First select 6 samples for testing. If the absorbance of multiple groups of controls is similar, it means that the concentration is too high. The samples need to be diluted to a certain multiple with 80% methanol extract before testing.

[0128] ③ Add reagents to the EP tube as shown in Table 2.

[0129] Table 2 Dosage of reagents added to EP tube in DPPH antioxidant activity detection experiment

[0130] Reagent name Measurement group (μL) Control group (μL) Blank group (μL) Sample solution 200 200 80% Methanol 200 200 Working fluid 200 200

[0131] ④ Mix well, react at 25℃ in the dark for 30min; centrifuge at 6000rpm at room temperature for 6min, take 150μl into a 96-well plate, and read the absorbance A at 517nm.

[0132] ⑤ The in vitro antioxidant activity of each embodiment and comparative example of the Stropharia officinalis glycopeptide was characterized by the DPPH free radical scavenging rate (K). The results are shown in Table 3. The calculation formula of the DPPH free radical scavenging rate is:

[0133]

[0134] Among them, A1 is the absorbance value of the measurement group;

[0135] A2 is the absorbance value of the control group;

[0136] A0 is the absorbance value of the blank group.

[0137] Table 3 DPPH free radical scavenging rate of Pleurotus eryngii glycopeptide

[0138] Stropharia officinalis glycopeptide DPPH free radical scavenging rate (%) Example 1 74.30 Comparative Example 1 63.26 Comparative Example 3 68.73 Comparative Example 6 66.61 Comparative Example 7 62.06

[0139] It can be seen from the above table that the DPPH antioxidant activity of the Stropharia officinalis glycopeptide of Example 1 is significantly better than that of the comparative examples.

[0140] 2.2. ABTS in vitro antioxidant activity test

[0141] (1) Sample solution preparation

[0142] Take 50 mg of the freeze-dried Stropharia officinalis glycopeptide powder of Example 1 and Comparative Example 1, Comparative Example 3, Comparative Example 6, and Comparative Example 7, add 10 mL of water, use a vortex to mix evenly to ensure full dissolution, centrifuge at 8000 rpm for 6 min, and take the supernatant for determination.

[0143] (2) Preparation of ABTS solution

[0144] Weigh 0.0384g ABTS and dissolve it in water. Make the volume in a volumetric flask to 10ml. Weigh 0.0134g potassium persulfate and dissolve it in water. Make the volume in a volumetric flask to 10ml. Mix the two in equal volumes and react for 12-16 hours in the dark. Dilute it with PBS to an absorbance of 0.7±0.02 before use.

[0145] (3) Detection

[0146] ① Preheat the ELISA instrument for 15 minutes and adjust the wavelength to 731nm.

[0147] ② Add the sample solution and ABTS working solution directly into the 96-well plate and add reagents as shown in Table 4.

[0148] Table 4 Reagent dosage of EP tube in ABTS antioxidant activity test experiment

[0149] Reagent name Blank group (μL) Blank control group (μL) Measurement group (μL) Determination of control group (μL) water 20 20 sample 20 20 Anhydrous ethanol 100 100 Working fluid 100 100

[0150] ③After each group is fully mixed, react in the dark at 25°C for 6 min and read the absorbance A at 731 nm.

[0151] ④ The in vitro antioxidant activity of each embodiment and comparative example of the Stropharia officinalis glycopeptide was characterized by the ABTS free radical scavenging rate (I), and the results are shown in Table 5. The calculation formula of the ABTS free radical scavenging rate is:

[0152]

[0153] Among them, A1 is the absorbance value of the blank group;

[0154] A2 is the absorbance value of the blank control group;

[0155] A3 is the absorbance value of the determination group;

[0156] A4 is the absorbance value of the control group.

[0157] Table 5 Free radical scavenging rate of Pleurotus eryngii glycopeptide ABTS

[0158] Stropharia officinalis glycopeptide ABTS free radical scavenging rate (%) Example 1 72.08 Comparative Example 1 61.06 Comparative Example 3 65.87 Comparative Example 6 64.96 Comparative Example 7 60.26

[0159] It can be seen from Table 5 that the ABTS antioxidant activity of the Stropharia officinalis glycopeptide of Example 1 is significantly better than that of the comparative examples.

[0160] 3. Hepatoprotective activity test

[0161] The freeze-dried Stropharia officinalis glycopeptide powders of Example 1 and Comparative Examples 1, 3, 6 and 7 were prepared with water to prepare a solution with a concentration of 6.4 mg / mL.

[0162] HSC cells: HSC cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured in high-glucose DMEM containing 10% fetal bovine serum to prepare 3×10 4 HSC cells were cultured at 2×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL per well, and 3 replicate wells were set up. 10 μL of the 6.4 mg / mL stropharia glycopeptide solution was added to make the final mass concentration 640 μg / mL. The wells without the stropharia glycopeptide solution were used as the control group, and the same volume of simple cell culture medium was used as the blank group. They were pre-cultured in a 37°C, 5% CO2 incubator for 24 hours. After the cells adhered to the wall, the supernatant was discarded. 10 μL of MTT solution (5 mg / mL) was added to each well, and the culture was continued at 37°C for 4 hours, and the supernatant was discarded. 150 μL of DMSO was added to each well, and it was gently shaken with an oscillator at room temperature for 5 minutes. The absorbance OD value was measured at a wavelength of 450 nm using an enzyme marker to calculate the cell inhibition rate.

[0163] Hep-G2 cells: Hep-G2 cells were cultured at 2×10 4 The density of cells / well was inoculated in a 96-well plate, 90 μL per well, and 3 replicate wells were set up. 10 μL of the stropharia glycopeptide solution with a mass concentration of 6.4 mg / mL was added to make the final mass concentration 640 μg / mL. The wells without the stropharia glycopeptide solution were used as the control group, and the same volume of simple cell culture medium was used as the blank group. After culturing for 24h and 48h, 10 μL of CCK-8 was added to each well and placed in a 37°C, 5% CO2 incubator for 4h. The absorbance OD value at 450nm was detected with an enzyme marker to calculate the cell inhibition rate.

[0164] LX2 cells: LX2 cells were cultured at 1×10 5The cells were inoculated at a density of 100 μL / well in a 96-well plate. Three replicate wells were set up. After 24 hours, the cells were observed to be uniformly attached to the wall, the supernatant was discarded, and 10 μL of the stropharia glycopeptide solution with a mass concentration of 6.4 mg / mL was added to make the final mass concentration 640 μg / mL. The wells without the stropharia glycopeptide solution were used as the control group, and the same volume of simple cell culture medium was used as the blank group. After 24 hours of culture, the cell supernatant was discarded, and the cells were rinsed 2-3 times with PBS buffer, and then 100 μL MTT solution (0.5 mg / mL) was added to each well, and incubated at 37°C for 4 hours. The culture medium and MTT were carefully removed, and 100 μL DMSO solution was added to each well. The crystals were oscillated for 10 minutes to dissolve. The absorbance OD value was measured at 490 nm using an enzyme reader to calculate the cell inhibition rate.

[0165] The cell inhibition rate was calculated as follows: cell inhibition rate (%) = (OD control group - OD drug group) / (OD control group - OD blank group) * 100%.

[0166] The experimental results are shown in Table 6 below:

[0167] Table 6 Determination of hepatoprotective activity of Stropharia rugosa glycopeptide

[0168] sample HSC cell inhibition rate Hep-G2 cell inhibition rate LX2 cell inhibition rate Example 1 64.53 56.22 59.02 Comparative Example 1 52.35 50.18 50.38 Comparative Example 3 58.44 53.35 55.22 Comparative Example 6 54.13 52.06 54.18 Comparative Example 7 51.03 49.33 48.95

Claims

1. A method for extracting stropharia glycopeptide, characterized in that The following steps are involved: (1) drying the Stropharia officinalis at a constant temperature of 25-28° C. for 3-4 h, then heating the temperature to 51° C. at a heating rate of 2-3° C. / h, keeping the temperature for 7-8 h, and then heating the temperature to 60° C. at a heating rate of 0.5-1° C. / h until the moisture content is 12%-13%; (2) crushing the dried Stropharia officinalis, passing through a 10-mesh sieve and a 30-mesh sieve, and obtaining a powder with a particle size of 10-30 mesh; (3) mixing the Stropharia officinalis powder with a low eutectic solvent solution, extracting the mixture under the assistance of a microwave, and filtering the mixture after the extraction to obtain a filtrate; (4) adding ethanol to the filtrate for precipitation, and collecting the precipitate to obtain crude glycoprotein; (5) dissolving the crude glycoprotein in water, adding sodium hydroxide solution to adjust the pH to 9-9.5, then adding sodium chloride and heating to react, adjusting the pH to neutral after the reaction, adding ethanol to precipitate, and collecting the precipitate; (6) dissolving the precipitate from step (5) with water, decolorizing with a macroporous adsorption resin under ultrasound, filtering and centrifuging after decolorization, and collecting the supernatant to obtain a stropharia glycoprotein solution; (7) adding snail enzyme to the stropharia glycoprotein solution for enzymolysis, inactivating the enzyme after enzymolysis, then adding bromelain for enzymolysis, inactivating the enzyme after enzymolysis, and freeze-drying the supernatant to obtain stropharia glycopeptide powder.

2. The extraction method according to claim 1, characterized in that: In step (3), the low eutectic solvent is betaine and butanediol in a molar ratio of 1-3:1, and the concentration of the low eutectic solvent solution is 55-65wt%.

3. The extraction method according to claim 1, characterized in that: In step (3), the ratio of Stropharia officinalis powder to the low eutectic solvent solution is 1 g: 20-40 mL.

4. The extraction method according to claim 3, characterized in that: In step (3), microwave extraction is performed twice, wherein the microwave power of the first microwave extraction is 50-150 W, the microwave temperature is 90-110° C., and the extraction time is 10-20 min; and the microwave power of the second microwave extraction is 160-250 W, the microwave temperature is 130-150° C., and the extraction time is 10-20 min.

5. The extraction method according to claim 1, characterized in that: In steps (4) and (5), ethanol is added until the ethanol volume concentration is 80-85%.

6. The extraction method according to claim 1 or 5, characterized in that: In step (5), 3-5 g of NaCl is added to every 100 ml of the solution; after adding NaCl, the mixture is reacted under boiling for 30-50 min.

7. The extraction method according to claim 1, characterized in that: In step (6), an activated macroporous adsorption resin is added to the solution at a volume ratio of 1:0.5-2 for decolorization; preferably, the ultrasonic power is 100-150 W, the decolorization temperature is 40-60° C., and the decolorization time is 20-30 min.

8. The extraction method according to claim 1, characterized in that: In step (7), the amount of snail enzyme is 3-6% of the mass of the Stropharia cap glycoprotein solution, the hydrolysis temperature of snail enzyme is 43-46° C., and the hydrolysis time is 8-10 h; the amount of bromelain is 3-6% of the mass of the Stropharia cap glycoprotein solution, the hydrolysis temperature of bromelain is 58-62° C., and the hydrolysis time is 5-7 h.

9. The Stropharia capillaris glycopeptide obtained by the method for extracting Stropharia capillaris glycopeptide according to any one of claim 1.

10. Use of the American ginseng glycoprotein according to claim 9 in the preparation of liver protection products and / or antioxidant products.

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