Method for preparing tremella polysaccharide by combining eutectic solvent and enzymolysis

Abstract

The invention relates to a method for preparing tremella polysaccharide by combining a deep eutectic solvent and enzymolysis, and belongs to the technical field of polysaccharide preparation. The method comprises the steps of tremella pretreatment, first-time enzymolysis, deep-eutectic solvent extraction, second-time enzymolysis, separation, purification and the like. By utilizing the method disclosed by the invention, the extraction rate of the molecular weight polysaccharide in the tremella fuciformis can be obviously improved, and the obtained tremella fuciformis polysaccharide can obviously improve the immunity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polysaccharide preparation, and relates to a method for preparing tremella polysaccharide by combining a eutectic solvent with enzymolysis. BACKGROUND

[0002] Tremella, also known as white fungus, is a fungus of the genus Tremella in the family Tremellaceae, and is known as the "crown of fungi". Tremella is composed of more than 10 thin and wrinkled flat lamellae, and the fruiting body of tremella is generally white or milk white, and is generally in the shape of a chrysanthemum or a cockscomb; after drying, it shrinks, is keratinous, hard and brittle, and is white or milky yellow; the surface of the lamella of the fruiting layer is nearly spherical or nearly oval. Tremella can only colonize and grow vigorously under suitable humidity conditions.

[0003] Tremella has the effects of strengthening essence, tonifying kidney, moistening intestines, benefiting stomach, tonifying qi, nourishing blood, strengthening heart, strengthening body, nourishing brain, refreshing, beautifying, tendering skin, and prolonging life. Tremella can improve the detoxification ability of the liver and protect liver function. Tremella can not only enhance the body's immune capacity against tumors, but also enhance the tolerance of tumor patients to radiotherapy and chemotherapy. Tremella can nourish and tonify the body, moisten the lungs and nourish the stomach, and tonify the lung and qi. Tremella is used to treat deficiency and cough, blood in sputum, dry mouth, post-illness debilitation, and shortness of breath and fatigue.

[0004] Tremella is also a kind of nourishing medicine, and has the characteristics of being moistening without being greasy and sticky. Tremella has the effects of tonifying the spleen and stomach, benefiting qi and clearing the intestines, inducing sleep and strengthening the stomach, nourishing the brain, nourishing yin and clearing heat, and moistening dryness. Tremella is a good tonic for patients with yin deficiency and excessive heat, who cannot be treated with warm and nourishing medicines such as ginseng and deer. Tremella is rich in natural gum, and long-term use of tremella can moisturize the skin and remove yellowish-brown spots and freckles on the face. Tremella is a kind of weight loss food containing dietary fiber, and the dietary fiber in tremella can help the peristalsis of the stomach and intestines and reduce fat absorption.

[0005] Tremella polysaccharide is a plant heteropolysaccharide extracted from the fruiting body of tremella, and is the main active ingredient of tremella. According to different properties, tremella polysaccharide can be divided into acid heteropolysaccharide, neutral heteropolysaccharide, cell wall polysaccharide, intracellular polysaccharide and acid oligosaccharide. The main chain structure of tremella polysaccharide is composed of α-(1→3)glycosidic bond connected mannose, and the side chain is composed of glucuronic acid and xylose, galactose, glucose and fucose. Tremella polysaccharide has rich biological activity and application value, can enhance the body's immune function, inhibit inflammatory reaction and reduce free radical damage. At the same time, as a natural moisturizing factor, tremella polysaccharide can increase the water content of the stratum corneum of the skin, reduce water loss, and has good moisturizing effect.

[0006] At present, the extraction methods of tremella polysaccharide mainly include tremella fruiting body extraction, tremella mycelium extraction and tremella spore fermentation liquid extraction. The tremella polysaccharide is directly extracted from the tremella fruiting body, and the method is relatively simple, but the cultivation conditions of tremella are harsh, the growth cycle is long, the artificial cost is high, the supply of tremella fruiting body is insufficient, and the development of deep processing products of tremella is slow; and because the structure of tremella polysaccharide in tremella fruiting body is greatly affected by the variety and planting environment, the quality of tremella polysaccharide product is unstable, so it is necessary to change the traditional extraction method to increase the yield of tremella polysaccharide and improve the quality thereof. SUMMARY

[0007] The main purpose of the present application is to provide a method for preparing tremella polysaccharide by combining eutectic solvent with enzymolysis, which can improve the extraction rate of medium molecular weight tremella polysaccharide, and the physiological activity of the obtained tremella polysaccharide is higher, which can obviously improve the immunity.

[0008] The present application adopts the following technical solutions to achieve the above-mentioned purposes: A method for preparing tremella polysaccharide by combining eutectic solvent with enzymolysis, mainly comprising the following steps: Step 1, pretreatment of tremella: dry tremella is added to water at 20℃, and after being fully soaked, impurities are removed, washed clean, torn into small pieces, heated at 80-85℃ for 1-1.5h, then cooled to room temperature, placed at-20℃~-18℃ for 20-24h, taken out and heated at 100℃ for 30-40min, cooled to room temperature, placed at-20℃~-18℃ again for 20-24h, taken out and heated at 100℃ for 30-40min, then cooled to room temperature, to obtain pretreated tremella; Step 2, first enzymolysis: the pretreated tremella is ground into a suspension with water at 60℃, the pH is adjusted to 5.5-6.0, alpha-amylase is added, and enzymolysis is carried out at 60℃ for 1-1.5h; then the temperature is lowered to 50℃, the pH is adjusted to 7.0-7.5, and complex protease is added, and enzymolysis is carried out at 50℃ for 2-2.5h; after the enzymolysis is completed, the temperature is raised to 90℃ for 10min to inactivate the enzyme, then cooled to room temperature, centrifuged, and the precipitate I and supernatant I are reserved for use; the supernatant I is mixed with a mixed solution of chloroform-n-butanol with a volume ratio of 4:1, shaken, and placed at 4-5℃ for 7-8h, the upper liquid is collected, the residual organic solvent is removed, concentrated, dried, mixed with the precipitate I, and a mixture is obtained for use; Step 3, eutectic solvent extraction: the mixture obtained above is mixed with eutectic solvent, placed in a water bath at 60-80℃, and treated with ultrasonic wave for 20-30min; then centrifuged, and the supernatant II is reserved for use; Step 4, second enzymolysis: the pH of supernatant II is adjusted to 5.0 and the temperature is raised to 50℃, then the mannanase is added, and the enzymolysis is carried out at 50℃ for 50-60 min; the pH is maintained at 5.0, the β-glucosidase is added, and the enzymolysis is carried out at 50℃ for 60-70 min; after the enzymolysis is completed, the temperature is raised to 90℃, and the enzyme is inactivated by keeping the temperature for 10 min, and then the temperature is cooled to room temperature, and the second enzymolysis solution is prepared; Step 5, separation and purification: anhydrous ethanol is added to the second enzymolysis solution, and the ethanol content is adjusted to 10%, 30%, 50%, 70% and 90% successively, and each time after the ethanol content is adjusted, the solution is kept for 2 h, and then centrifuged to obtain precipitate II; precipitate II is redissolved in water, and then loaded on a Sephadex G-75 gel column, and eluted with a mixed solution of methanol and water in a volume ratio of 0.1:1, and combined with a detection technology, and the eluate containing polysaccharides with a molecular weight of 25000-60000 is collected, and then concentrated and dried to obtain the target tremella polysaccharide.

[0009] Preferably, the solid-liquid ratio of the pretreated tremella and water in step 2 is 1:2-3 in kg / L.

[0010] Preferably, the addition amount of the α-amylase in step 2 is 3-4% of the weight of the pretreated tremella.

[0011] Preferably, the addition amount of the complex protease in step 2 is 5-8% of the weight of the pretreated tremella.

[0012] Preferably, the volume ratio of the supernatant I and the chloroform-n-butanol mixed solution in step 2 is 1:4.

[0013] Preferably, the solid-liquid ratio of the mixture and the deep eutectic solvent in step 3 is 1:(8-10) in kg / L.

[0014] Preferably, the preparation method of the deep eutectic solvent in step 3 is as follows: choline hydroxide, choline chloride and ethylene glycol are mixed according to a molar ratio of 0.2:1:3, and then stirred at 80℃ in a water bath at 300 rpm for 20-30 min, and then deionized water is added to prepare the deep eutectic solvent.

[0015] Further preferably, the mass fraction of water in the deep eutectic solvent is 50%.

[0016] Preferably, the addition amount of the mannanase in step 4 is 1-1.5 g / L.

[0017] Preferably, the addition amount of the β-glucosidase in step 4 is 2-3 g / L.

[0018] The present application provides a tremella polysaccharide prepared by the above method.

[0019] The application provides application of the tremella polysaccharide prepared by the method in preparation of a product with improved immunity.

[0020] Specifically, the product includes but is not limited to food, medicine or feed for aquaculture.

[0021] The application has the following beneficial effects: In the preparation process of the tremella polysaccharide, the tremella is first pretreated by heating and freezing, so that the properties of the polysaccharide are changed to some extent, which is more conducive to the later extraction and enzymolysis; after the impurities are removed through enzymolysis, the tremella polysaccharide with different molecular weights is obtained through extraction by using a eutectic solvent, secondary enzymolysis, column separation and impurity removal and separation, and the target tremella polysaccharide is obtained. The tremella polysaccharide prepared by the application has high physiological activity and can significantly improve immunity, and the activity of the tremella polysaccharide obtained by the traditional preparation method (enzymolysis or eutectic solvent extraction) is higher. DETAILED DESCRIPTION

[0022] The application will be further illustrated by specific examples, and it should be understood that the examples are only used to illustrate the application and not used to limit the scope of the application. After reading the application, those skilled in the art can modify various equivalent forms of the application, which all fall within the scope of the claims of the application. The raw materials used in the following embodiments are all commercially available products, wherein the enzyme activity of alpha-amylase is 4000U / g, the CAS number is 9000-90-2; the enzyme activity of complex protease is 120u / mg, the product number is S10155; the enzyme activity of mannanase is 1000U / g, the CAS number is 37288-54-3; and the enzyme activity of beta-glucosidase is 100U / g, the CAS number is 9001-22-3, which are all purchased from a biotechnology company in Shanghai.

[0023] Example 1 Step 1, pretreatment of tremella: dry tremella is added into water at 20℃, and after being soaked, impurities are removed, washed clean, torn into small pieces, heated at 85℃ for 1h, then cooled to room temperature, placed at-18℃ for 24h, taken out and heated at 100℃ for 30min, cooled to room temperature, placed at-18℃ for 24h again, taken out and heated at 100℃ for 30min, then cooled to room temperature, and the pretreated tremella is obtained; Step 2, first enzymolysis: 1 kg of pretreated tremella was ground into a suspension with 3 L of water at 60 ℃, the pH was adjusted to 6.0, 40 g of α-amylase (4000 U / g, CAS No. 9000-90-2) was added, and the mixture was enzymolyzed at 60 ℃ for 1 h; then the temperature was lowered to 50 ℃, the pH was adjusted to 7.5, 80 g of protease (120 u / mg, product No. S10155) was added, and the mixture was enzymolyzed at 50 ℃ for 2 h; after the enzymolysis was completed, the temperature was raised to 90 ℃ for 10 min to inactivate the enzymes, then the mixture was cooled to room temperature, centrifuged, and the precipitate I and the supernatant I were reserved; the supernatant I was mixed with a chloroform-n-butanol mixed solution (volume ratio 4:1) at a volume ratio of 1:4, and then placed in a separatory funnel; after being shaken vigorously, the mixture was allowed to stand at 4 ℃ for 7 h, the upper liquid was collected, the residual organic solvent was removed, the mixture was concentrated and dried, and then mixed with the precipitate I to obtain a mixture for use; Step 3, deep eutectic solvent extraction: 0.2 moles of choline hydroxide, 1 mole of choline chloride and 3 moles of ethylene glycol were mixed according to the molar ratio, and then stirred at 300 rpm in a water bath at 80 ℃ for 30 min; deionized water was added to prepare a deep eutectic solvent with a water content of 50% (mass fraction); 1 kg of the mixture was added to 8 L of the deep eutectic solvent, and then placed in a water bath at 60 ℃, and treated with ultrasonic waves (power 200 W) for 30 min; then the mixture was centrifuged, and the supernatant II was reserved; Step 4, second enzymolysis: the pH of the supernatant II was adjusted to 5.0 and the temperature was raised to 50 ℃, 1.5 g / L of mannanase (1000 U / g, CAS No. 37288-54-3) was added, and the mixture was enzymolyzed at 50 ℃ for 50-60 min; the pH was maintained at 5.0, 3 g / L of β-glucosidase (100 U / g, CAS No. 9001-22-3) was added, and the mixture was enzymolyzed at 50 ℃ for 60 min; after the enzymolysis was completed, the temperature was raised to 90 ℃ for 10 min to inactivate the enzymes, and then the mixture was cooled to room temperature, and the second enzymolysis solution was reserved; Step 5, separation and purification: anhydrous ethanol was added to the second enzymolysis solution, and the ethanol content was adjusted to 10%, 30%, 50%, 70% and 90% successively, and the mixture was allowed to stand for 2 h after each adjustment of the ethanol content, and then centrifuged to obtain a precipitate II; the precipitate II was redissolved in water, and then loaded onto a Sephadex G-75 gel column, eluted with methanol-water (volume ratio 0.1:1), and the eluate containing polysaccharides with a molecular weight of 25,000-60,000 was collected by combining detection techniques, concentrated, and dried to obtain the target tremella polysaccharide.

[0024] Example 2 Step 1, pre-treatment of Tremella fuciformis: dry Tremella fuciformis was added into water at 20℃, after soaking and removing impurities, the clean Tremella fuciformis was torn into small pieces, heated at 85℃ for 1h, then cooled to room temperature, placed at -20℃ for 20h, heated at 100℃ for 40min, cooled to room temperature, placed at -20℃ for 20h again, heated at 100℃ for 40min, then cooled to room temperature, the pre-treated Tremella fuciformis was obtained; Step 2, first enzymatic hydrolysis: 1kg of pre-treated Tremella fuciformis was ground into suspension with 2-3L of water at 60℃, the pH was adjusted to 5.5, 30g of α-amylase (4000U / g, CAS No. 9000-90-2) was added, and the mixture was hydrolyzed at 60℃ for 1.5h; then the temperature was lowered to 50℃, the pH was adjusted to 7.0, 50g of protease (120u / mg, product No. S10155) was added, and the mixture was hydrolyzed at 50℃ for 2.5h; after the enzymatic hydrolysis, the temperature was raised to 90℃ for 10min to inactivate the enzymes, then cooled to room temperature, centrifuged, and the precipitate I and supernatant I were reserved; the supernatant I was mixed with chloroform-n-butanol mixed solution (volume ratio 4:1) according to the volume ratio 1:4, and placed in a separatory funnel, after shaking vigorously, the mixture was placed at 5℃ for 8h, the upper liquid was collected, after removing the residual organic solvent, the mixture was concentrated, dried, mixed with the precipitate I, and the mixture was reserved; Step 3, deep eutectic solvent extraction: choline hydroxide, choline chloride and ethylene glycol were mixed according to the molar ratio of 0.2:1:3, and the mixture was prepared into a deep eutectic solvent with water content of 50% (mass fraction) by stirring at 300rpm in a water bath at 80℃ for 20min; 1kg of the above mixture was added into 10L of the deep eutectic solvent, and the mixture was placed in a water bath at 80℃, and treated with ultrasonic wave (power 300W) for 20min; then centrifuged, and the supernatant II was reserved; Step 4, second enzymatic hydrolysis: the pH of the supernatant II was adjusted to 5.0 and the temperature was raised to 50℃, 1g / L of mannanase (1000U / g, CAS No. 37288-54-3) was added, and the mixture was hydrolyzed at 50℃ for 50-60min; the pH was maintained at 5.0, 2g / L of β-glucosidase (100U / g, CAS No. 9001-22-3) was added, and the mixture was hydrolyzed at 50℃ for 70min; after the enzymatic hydrolysis, the temperature was raised to 90℃ for 10min to inactivate the enzymes, then cooled to room temperature, and the second enzymatic hydrolysate was reserved; Step 5, isolation and purification: add anhydrous ethanol to the secondary enzymatic hydrolysate, adjust the alcohol content to 10%, 30%, 50%, 70%, and 90% successively, and stand for 2 h after each adjustment of the alcohol content, centrifuge to obtain precipitate II; resuspend precipitate II in water, load on a Sephadex G-75 gel column, elute with methanol-water (volume ratio 0.1:1), combine the eluate containing polysaccharides with a molecular weight of 25,000-60,000 by detection technology, concentrate, dry, and obtain the target tremella polysaccharide.

[0025] Example 3 Step 1, pretreatment of tremella: add dry tremella to water at 20°C, soak thoroughly, remove impurities, wash clean, tear into small pieces, heat at 85°C for 1 h, cool to room temperature, place at -18°C for 24 h, take out and heat at 100°C for 30 min, cool to room temperature, place at -20°C for 20 h again, take out and heat at 100°C for 40 min, and cool to room temperature to obtain pretreated tremella; Step 2, first enzymatic hydrolysis: grind 1 kg of pretreated tremella into a suspension with 2-3 L of water at 60°C, adjust the pH to 6.0, add 40 g of α-amylase (4000 U / g, CAS No. 9000-90-2), and enzymatically hydrolyze at 60°C for 1 h; then cool to 50°C, adjust the pH to 7.0, add 60 g of complex protease (120 u / mg, product No. S10155), and enzymatically hydrolyze at 50°C for 2.5 h; after the enzymatic hydrolysis is completed, heat to 90°C to inactivate the enzymes for 10 min, cool to room temperature, centrifuge, and reserve the precipitate I and supernatant I; mix the supernatant I with a chloroform-n-butanol mixed solution (volume ratio 4:1) at a volume ratio of 1:4, place in a separatory funnel, shake vigorously, stand at 5°C for 7 h, collect the upper liquid, remove the residual organic solvent, concentrate, dry, and mix with the precipitate I to obtain a mixture for use; Step 3, deep eutectic solvent extraction: mix choline hydroxide, choline chloride, and ethylene glycol at a molar ratio of 0.2:1:3, continuously stir at 300 rpm in a water bath at 80°C for 25 min to prepare a deep eutectic solvent with a water content of 50% (mass fraction); mix 1 kg of the mixture with 9 L of the deep eutectic solvent, place in a water bath at 70°C, and assist with ultrasonic wave treatment (power 300 W) for 25 min; then centrifuge and reserve the supernatant II; Step 4, second enzymolysis: adjust the pH of supernatant II to 5.0 and warm to 50℃, add 1.3 g / L of mannanase (1000 U / g, CAS No. 37288-54-3) and keep the temperature at 50℃ for 50 min; maintain the pH at 5.0, add 2.5 g / L of β-glucosidase (100 U / g, CAS No. 9001-22-3) and keep the temperature at 50℃ for 60 min; after the enzymolysis, warm to 90℃, keep the temperature for 10 min to inactivate the enzyme, cool to room temperature, and the second enzymolysis solution is ready for use; Step 5, separation and purification: add anhydrous ethanol to the second enzymolysis solution, adjust the ethanol content to 10%, 30%, 50%, 70% and 90% respectively, and stand for 2 h after each adjustment, then centrifuge to obtain precipitate II; dissolve precipitate II in water, and then load it on a Sephadex G-75 gel column, elute with methanol-water (volume ratio 0.1:1), and combine the eluate containing polysaccharides with molecular weight of 25000-60000 by detection technology, concentrate and dry to obtain the target tremella polysaccharide.

[0026] Comparative Example 1 Grind dry tremella to obtain tremella powder for use; add 10 L of eutectic solvent (mix choline chloride and ethylene glycol according to a molar ratio of 1:3, heat and stir at 80℃, add deionized water to prepare a eutectic solvent with a water content of 30% (mass fraction), place it in a 70℃ water bath, and treat it with ultrasonic waves (power 300 W) for 30 min, then centrifuge to obtain supernatant; add anhydrous ethanol to the supernatant, adjust the ethanol content to 10%, 30%, 50% and 70% respectively, and stand for 2 h after each adjustment, then centrifuge to obtain the precipitate; dissolve the precipitate in water according to a solid-liquid ratio of 1:20 (g / mL), mix it with a chloroform-n-butanol mixed solution (volume ratio 4:1) according to a volume ratio of 1:4, shake vigorously, stand for 7-8 h, collect the upper solution, concentrate and dry to obtain tremella polysaccharide.

[0027] Comparative Example 2 Step 1, eutectic solvent extraction: grind dry tremella to obtain tremella powder for use; mix choline hydroxide, choline chloride and ethylene glycol according to a molar ratio of 0.2:1:3, heat and stir at 80℃, add deionized water to prepare a eutectic solvent with a water content of 50% (mass fraction); mix 1 kg of the above obtained tremella powder with 10 L of the eutectic solvent, place it in a 70℃ water bath, and treat it with ultrasonic waves (power 300 W) for 25 min; then centrifuge to obtain supernatant II for use; Step 2, enzymatic hydrolysis: adjust the pH of supernatant II to 5.0 and warm to 50℃, add 1.3 g / L of mannanase (1000 U / g, CAS No. 37288-54-3), and hydrolyze at 50℃ for 50 min; maintain pH 5.0, add 2.5 g / L of β-glucosidase (100 U / g, CAS No. 9001-22-3), and hydrolyze at 50℃ for 60 min; after the enzymatic hydrolysis is completed, warm to 90℃, and incubate for 10 min to inactivate the enzyme, then cool to room temperature, and the secondary enzymatic hydrolysis solution is prepared; Step 3, separation and purification: add anhydrous ethanol to the secondary enzymatic hydrolysis solution, and adjust the ethanol content to 10%, 30%, 50%, 70%, and 90%, respectively, and stand for 2 h after each adjustment of the ethanol content, then centrifuge to obtain precipitate II; resuspend precipitate II in water, and then load onto a Sephadex G-75 gel column, elute with methanol-water (volume ratio 0.1:1), combine the eluate containing polysaccharides with a molecular weight of 25,000-60,000, and concentrate, dry, and the target tremella polysaccharide is obtained.

[0028] Comparative Example 3 Step 1, pretreatment of tremella: add dry tremella to water at 20℃, soak thoroughly, remove impurities, wash clean, tear into small pieces, and heat at 85℃ for 1 h, then cool to room temperature to obtain pretreated tremella; Step 2, first enzymatic hydrolysis: grind 1 kg of pretreated tremella into a suspension with 2-3 L of water at 60℃, adjust the pH to 6.0, add 40 g of α-amylase (4000 U / g, CAS No. 9000-90-2), and hydrolyze at 60℃ for 1 h; then cool to 50℃, adjust the pH to 7.0, add 60 g of protease (120 u / mg, product No. S10155), and hydrolyze at 50℃ for 2.5 h; after the enzymatic hydrolysis is completed, warm to 90℃ to inactivate the enzyme for 10 min, then cool to room temperature, centrifuge, and the precipitate I is prepared; Step 3, deep eutectic solvent extraction: mix choline hydroxide, choline chloride, and ethylene glycol according to a molar ratio of 0.2:1:3, add deionized water, and prepare a deep eutectic solvent with a water content of 50% (mass fraction) by continuously stirring at 300 rpm in a 80℃ water bath for 25 min; mix 1 kg of the mixture with 10 L of the deep eutectic solvent, and treat in a 70℃ water bath with the aid of ultrasonic waves (power 300 W) for 25 min; then centrifuge, and the supernatant II is prepared; Step 4, second enzymolysis: adjust the pH of supernatant II to 5.0 and warm to 50℃, add 1.3 g / L of mannanase (1000 U / g, CAS No. 37288-54-3) and keep the temperature at 50℃ for 50 min; maintain the pH at 5.0, add 2.5 g / L of β-glucosidase (100 U / g, CAS No. 9001-22-3) and keep the temperature at 50℃ for 60 min; after the enzymolysis is completed, warm to 90℃, keep the temperature for 10 min to inactivate the enzyme, cool to room temperature, and the second enzymolysis solution is ready for use; Step 5, separation and purification: add anhydrous ethanol to the second enzymolysis solution, and adjust the ethanol content to 10%, 30%, 50%, 70% and 90% successively, each time after adjusting the ethanol content, stand for 2 h, centrifuge, and obtain precipitate II; resuspend precipitate II with water, load on a Sephadex G-75 gel column, elute with methanol-water (volume ratio 0.1:1), combine the eluate containing polysaccharides with molecular weight of 25000-60000, concentrate, dry, and the target tremella polysaccharide is obtained.

[0029] Comparative Example 4 Step 1, pretreatment of tremella: add dry tremella to water at 20℃, soak thoroughly, remove impurities, wash clean, tear into small pieces, heat at 85℃ for 1 h, cool to room temperature, place at -18℃ for 24 h, take out and heat at 100℃ for 30 min, cool to room temperature, place at -20℃ for 20 h again, take out and heat at 100℃ for 40 min, cool to room temperature, and then pulverize to obtain pretreated tremella; Step 3, deep eutectic solvent extraction: mix choline chloride and ethylene glycol according to a molar ratio of 1:3, continuously stir at 300 rpm in a water bath at 80℃ for 25 min to prepare a deep eutectic solvent with a water content of 50% (mass fraction); mix 1 kg of pretreated tremella with 10 L of the deep eutectic solvent, place in a water bath at 70℃, and assist with ultrasonic wave (power 300 W) treatment for 25 min; then centrifuge, and take the supernatant II for standby; Step 4, second enzymolysis: adjust the pH of supernatant II to 5.0 and warm to 50℃, add 1.3 g / L of mannanase (1000 U / g, CAS No. 37288-54-3) and keep the temperature at 50℃ for 50 min; maintain the pH at 5.0, add 2.5 g / L of β-glucosidase (100 U / g, CAS No. 9001-22-3) and keep the temperature at 50℃ for 60 min; after the enzymolysis is completed, warm to 90℃, keep the temperature for 10 min to inactivate the enzyme, cool to room temperature, and the second enzymolysis solution is ready for use; Step 5, isolation and purification: add anhydrous ethanol to the secondary enzymatic hydrolysate, and adjust the alcohol content to 10%, 30%, 50%, 70%, and 90%, respectively, and stand for 2 h after each adjustment. Centrifuge to obtain a precipitate. Resuspend the precipitate in water, and then load it on a Sephadex G-75 gel column. Elute with methanol-water (volume ratio 0.1:1), and combine the eluate containing polysaccharides with a molecular weight of 25,000-60,000. Concentrate and dry the eluate to obtain the target tremella polysaccharide.

[0030] Performance test I. Effects of different tremella polysaccharides on the immune performance of mice 1. Test design The following tests were conducted according to existing literature. SPF Balb / c male mice weighing 20±2 g were used, license number: SYXK(Lu)2025 0026, and they were adaptively fed for 7 days before the test. The mice were intraperitoneally injected with cyclophosphamide (80 mg / kg) for 5 consecutive days, and the mice were considered to be successfully modeled when they showed symptoms such as sparse hair, no significant increase in body weight, and listlessness. The successfully modeled mice were randomly divided into a model group and test groups 1-7, with 10 mice in each group. Another 10 mice (intraperitoneally injected with normal saline) were used as a blank group.

[0031] The test groups 1-7 were orally administered with tremella polysaccharides, and the blank group and the model group were orally administered with an equal amount of normal saline. The mice were administered once a day for 20 consecutive days.

[0032] After the last administration for 2 h, the mice in each group were sacrificed and weighed, and the complete thymus and spleen were isolated, washed with normal saline, dried, and weighed to calculate the immune organ index.

[0033] In the formula, M1 is the mass of the immune organ (thymus or spleen), g; and M2 is the mass of the mouse, g.

[0034] The mice were taken blood from the orbit, and the serum was separated to determine the contents of serum immunoglobulin G (IgG), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum.

[0035] 2. Results and analysis As can be seen from the results in Table 1 and Table 2, the tremella polysaccharide prepared in Examples 1-3 can obviously improve the immune organ index of mice, and can improve the levels of IgG, IL-6, TNF-α and IFN-γ cytokines in the serum of immunosuppressed mice, indicating that the tremella polysaccharide prepared in the application can enhance the cellular immunity and humoral immunity levels of mice. In Comparative Example 1, the tremella polysaccharide extracted by the traditional eutectic solvent, in Comparative Example 2, the tremella was not pretreated and primary enzymolysis, in Comparative Example 3, the tremella was not pretreated by freezing and did not contain the primary enzymolysis enzyme solution, in Comparative Example 4, the tremella was not subjected to primary enzymolysis, and the hydrolytic enzyme was different in secondary enzymolysis. Compared with the above, the activity of the tremella polysaccharide obtained in Comparative Example 3 is stronger, and the effect is better than that of other comparative examples, but compared with the application, the effect is still poor.

[0036] Table 1 Effect of different tremella polysaccharides on immune organ index of mice Note: * indicates P < 0.05 compared with the model group.

[0037] Table 2 Effect of different tremella polysaccharides on immune performance of mice Note: * indicates P < 0.05 compared with the model group.

[0038] II. Effect of different preparation methods on extraction rate of tremella polysaccharide Based on the above pharmacological experimental results, the tremella polysaccharide prepared in the application has higher physiological activity, and the effect of each factor in the preparation method of the application on the extraction rate of tremella polysaccharide is investigated.

[0039] 1. Effect of different tremella sources on extraction rate The commercially available tremella from different manufacturers is prepared into tremella polysaccharide according to the method described in Example 1, and the extraction rate of the tremella polysaccharide is investigated. The results are shown in Table 3.

[0040] As can be seen from the results in Table 1, the extraction rates of the target tremella polysaccharide prepared by the method described in the application from different sources of tremella are not much different, and are all above 15%.

[0041] Table 3 Comparison of extraction rates of different tremella polysaccharides 2. Effect of water content in eutectic solvent on extraction rate of tremella polysaccharide The same source of tremella is taken to investigate the effect of water content in the eutectic solvent on the extraction rate according to the preparation method described in Example 1. The results are shown in Table 4.

[0042] The prior art usually uses pure eutectic solvent or eutectic solvent containing 20% or 30% of water to extract polysaccharides, and the extraction rate is relatively high, and the increase of water content will affect the extraction of polysaccharides; but for the present application, the extraction rate is not significant, and when the water content in the conventional eutectic solvent is increased to 50%, the extraction rate is the highest, and when the water content is increased to 70%, the extraction rate decreases.

[0043] Table 4 Comparison of extraction rates of tremella polysaccharides

Claims

1. A method for preparing Tremella fuciformis polysaccharide using a combination of eutectic solvent and enzymatic hydrolysis, characterized in that, Includes the following steps: Step 1, Pre-treatment of white fungus: Soak dried white fungus in water at 20℃ until it expands, then heat it at 80-85℃ for 1-1.5 hours, cool it, place it at -20℃~-18℃ for 20-24 hours, then heat it at 100℃ for 30-40 minutes, cool it, place it at -20℃~-18℃ again for 20-24 hours, then heat it at 100℃ for 30-40 minutes, cool it, and you will get the pre-treated white fungus; Step 2, First enzymatic hydrolysis: Grind the pretreated tremella with water at 60℃ into a suspension, adjust the pH to 5.5-6.0, add α-amylase, and hydrolyze at 60℃ for 1-1.5h; then cool to 50℃, adjust the pH to 7.0-7.5, add complex protease, and hydrolyze at 50℃ for 2-2.5h; after the enzymatic hydrolysis is completed, heat to 90℃ to inactivate the enzyme for 10min, cool, centrifuge, and set aside precipitate I and supernatant I; mix supernatant I with a chloroform-n-butanol mixed solution at a volume ratio of 4:1, shake, and let stand at 4-5℃ for 7-8h, collect the upper liquid, remove residual organic solvent, concentrate, dry, and mix with precipitate I to obtain a mixture for later use; Step 3, eutectic solvent extraction: Add the mixture obtained in step 2 to the eutectic solvent and mix. Place in a water bath at 60-80℃, sonicate for 20-30 minutes, centrifuge, and take the supernatant II for later use. Step 4, Second enzymatic hydrolysis: Adjust the pH of the supernatant II obtained in Step 3 to 5.0 and heat to 50℃. Add mannanase and incubate at 50℃ for 50-60 min. Maintain pH 5.0, add β-glucosidase, and incubate at 50℃ for 60-70 min. After enzymatic hydrolysis, heat to 90℃ and incubate for 10 min to inactivate the enzyme. Cool and use the second enzymatic hydrolysate for later use. Step 5, Separation and Purification: Add ethanol to the secondary enzymatic hydrolysate, and adjust the ethanol content to 10%, 30%, 50%, 70%, and 90% sequentially. After each adjustment, allow the mixture to stand for 2 hours, centrifuge, and obtain precipitate II. Redissolve precipitate II in an appropriate amount of water, load it onto a Sephadex G-75 gel column, and elute with a methanol-water mixture with a volume ratio of 0.1:

1. Using detection technology, retain the eluent containing polysaccharides with a molecular weight of 25,000-60,000, concentrate, and dry to obtain the target Tremella polysaccharide.

2. The method as described in claim 1, characterized in that, In step 2, the ratio of pretreated white fungus to water is 1:2-3 (kg / L).

3. The method as described in claim 1, characterized in that, In step 2, the amount of α-amylase added is 3-4% of the weight of the pretreated white fungus.

4. The method as described in claim 1, characterized in that, In step 2, the amount of complex protease added is 5-8% of the weight of the pretreated tremella.

5. The method as described in claim 1, characterized in that, In step 2, the volume ratio of supernatant I to the chloroform-n-butanol mixed solution is 1:

4.

6. The method as described in claim 1, characterized in that, In step 3, the ratio of the mixture to the eutectic solvent is 1:(8-10) in kg / L.

7. The method as described in claim 1, characterized in that, The method for preparing the eutectic solvent in step 3 is as follows: choline hydroxide, choline chloride, and ethylene glycol are mixed in a molar ratio of 0.2:1:3, and then stirred continuously at 300 rpm for 20-30 minutes in an 80°C water bath. Deionized water is then added to prepare the eutectic solvent.

8. The method as described in claim 1, characterized in that, The mass fraction of water in the eutectic solvent is 50%.

9. The method as described in claim 1, characterized in that, In step 4, the amount of mannanase added is 1-1.5 g / L.

10. The method as described in claim 1, characterized in that, In step 4, the amount of β-glucosidase added is 2-3 g / L.

Images