阅读说明：本技术 一种医药级增强免疫的绣球菌多糖的制备方法及其应用 (Preparation method and application of pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity ) 是由 黄芬 邱英婷 田�健 诸辉 于 2020-12-25 设计创作，主要内容包括：本发明公开了一种医药级增强免疫的绣球菌多糖的制备方法,包括以下步骤：(1)原料预处理：取适量干燥的绣球菌子实体,将其粉碎；(2)取绣球菌粗多糖,加离子液体溶液搅拌,加入酶水解,水解液过滤,滤液中加入乙醇,静置,分离沉淀并干燥,得到水溶性绣球菌多糖；本发明采用离子液体/复合酶体系高效特异性酶解提取工艺从绣球菌子实体中除去纤维素、脂肪及蛋白质,从而提高绣球菌多糖的产率。采用绿色环保的离子液体/组合酶体系定向水解技术降低了绣球菌多糖的粘度,增加了水溶性,更有利于人体吸收,使其增强免疫、抗肿瘤、降血糖等功效显著。(The invention discloses a preparation method of a pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity, which comprises the following steps: (1) pretreatment of raw materials: taking a proper amount of dried sparassis crispa sporocarp, and crushing the sparassis crispa sporocarp; (2) adding ionic liquid solution into crude Sparassis crispa polysaccharide, stirring, adding enzyme for hydrolysis, filtering hydrolysate, adding ethanol into filtrate, standing, separating precipitate, and drying to obtain water-soluble Sparassis crispa polysaccharide; the invention adopts the high-efficiency specific enzymolysis extraction process of an ionic liquid/complex enzyme system to remove cellulose, fat and protein from the sparassis crispa sporocarp, thereby improving the yield of sparassis crispa polysaccharide. The viscosity of the sparassis crispa polysaccharide is reduced by adopting the green and environment-friendly ionic liquid/combined enzyme system directional hydrolysis technology, the water solubility is increased, the absorption by a human body is facilitated, and the effects of enhancing immunity, resisting tumors, reducing blood sugar and the like are remarkable.)

1. A preparation method of sparassis crispa polysaccharide for enhancing immunity in pharmaceutical grade is characterized by comprising the following steps:

(1) pretreatment of raw materials: taking a proper amount of dried sparassis crispa sporocarp, and crushing the sparassis crispa sporocarp; adding 10-60 times volume of ionic liquid solution, heating to 80-100 ℃, uniformly stirring to obtain mixed solution, adding 0.1-0.3 mass percent of specific endo-cellulase and lysozyme, and stirring the mixed solution for 1-2 hours at the temperature of 40-50 ℃ under the condition that the pH is =5-7 for enzymolysis; adding lipase and bromelain with the mass percentage concentration of 0.1-0.5%, stirring the mixed solution at the temperature of 20-50 ℃ for 0.5-2 h under the condition of pH =3-7 for enzymolysis, centrifuging the enzymolysis solution to separate solid from liquid, and removing filtrate to obtain residue to obtain crude Sparassis crispa polysaccharide;

(2) taking crude Sparassis crispa polysaccharide, adding ionic liquid solution, stirring, adding enzyme for hydrolysis, filtering hydrolysate, adding ethanol into filtrate, standing, separating precipitate, and drying to obtain water-soluble Sparassis crispa polysaccharide.

2. The method for preparing sparassis crispa polysaccharide for pharmaceutical grade boosting immunity as claimed in claim 1, wherein in step (1), sparassis crispa fruiting body is pulverized to 800-1200 mesh by ultramicro pulverizer.

3. The method for preparing sparassis crispa polysaccharide for pharmaceutical grade boosting immunity according to claim 1, wherein in the step (1), the ionic liquid solution is 1-5% by mass of an aqueous solution of ionic liquid, wherein the ionic liquid is one or two of 1-butyl-1-methylpyrrolidine bistrifluoromethylsulfonyl imide and tetrabutyl phosphine tetrachloroferrite.

4. The method for preparing sparassis crispa polysaccharide for pharmaceutical grade boosting immunity according to claim 1, wherein in the step (1), the mass ratio of the specific endo-cellulase to the lysozyme is (2-5): 1, the mass ratio of the lipase to the bromelain is (2-4): 1.

5. the method for preparing sparassis crispa polysaccharide for pharmaceutical grade boosting immunity according to claim 1, wherein in the step (2), the ionic liquid solution is 0.5-2% by mass of an aqueous solution of an ionic liquid, wherein the ionic liquid is one or two of tributylmethylammonium bistrifluoromethylsulfonyl imide salt and 1-ethyl-3-methylimidazolium sulfate.

6. The method for preparing sparassis crispa polysaccharide for pharmaceutical grade boosting immunity according to claim 1, wherein in the step (2), the enzyme is a combination of dextranase and beta-glucanase, and the mass ratio is 1 (1-3).

7. The application of sparassis crispa polysaccharide for enhancing immunity is characterized by being applied to the following food formula in parts by weight:

15-55 parts of hydrangea powder, 0-20 parts of whey protein powder, 0-20 parts of casein, 0-10 parts of soybean protein isolate, 10-50 parts of plant fat powder, 0-10 parts of fructo-oligosaccharide, 0-10 parts of inulin, 0-5 parts of acerola cherry powder, 0-5 parts of elderberry powder, 0.05-1 part of vitamin and 0.05-5 parts of mineral substances.

8. The application of sparassis crispa polysaccharide for enhancing immunity as claimed in claim 7, wherein the food formula comprises the following raw material formula in parts by weight: 25-45 parts of hydrangea powder, 5-15 parts of whey protein powder, 5-15 parts of casein, 1-5 parts of soybean protein isolate, 20-40 parts of vegetable fat powder, 1-5 parts of fructo-oligosaccharide, 1-5 parts of inulin, 0.5-1.5 parts of acerola cherry powder, 0.5-2.5 parts of elderberry powder, 0.1-0.3 part of vitamin and 2-4 parts of mineral substances.

9. The application of sparassis crispa polysaccharide for enhancing immunity as claimed in claim 7, wherein the food formula comprises the following raw material formula in parts by weight: 35 parts of hydrangea powder, 10 parts of whey protein powder, 10 parts of casein, 3 parts of soybean protein isolate, 30 parts of vegetable fat powder, 3 parts of fructo-oligosaccharide, 3.3 parts of inulin, 1 part of acerola powder, 1.5 parts of elderberry powder, 0.2 part of vitamin and 3 parts of mineral substances.

Technical Field

The invention relates to the technical field of food, in particular to a preparation method and application of pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity.

Background

Sparassis crispa is of the genus Sparassis of the order Sparassiales, family Sparassidaceae. Sparassis crispa is rich in proteins, amino acids, mineral elements and the like, is delicious in taste and rich in nutrition, is rich in active ingredients beneficial to human bodies, and has the reputation of Wan mushroom king and sunshine mushroom. The content of polysaccharide in the sparassis crispa dry product is up to 40-60%, and the sparassis crispa polysaccharide is an important active ingredient of sparassis crispa, and the sparassis crispa polysaccharide is a bioactive substance, and has multiple functions of immunoregulation, anti-tumor, anti-inflammation, antivirus, antioxidation, antiradiation, blood sugar reduction, blood fat reduction, liver protection and the like. The polysaccharide is extracted from the sparassis crispa and is prepared into various foods, medicines and the like, and the polysaccharide has wide market and economic value.

By virtue of good health-care efficacy and nutritional value of sparassis crispa, the sparassis crispa has gradually received great attention of scholars and consumers at home and abroad in recent years. The sparassis crispa polysaccharide extracted by the conventional method is difficult to industrially produce due to low yield, and is insoluble in water due to high viscosity. The problem of how to improve the yield of the Sparassis crispa polysaccharide and reduce the viscosity of the Sparassis crispa polysaccharide so as to increase the water solubility of the Sparassis crispa polysaccharide and enable the Sparassis crispa polysaccharide to be better applied to health care products and foods with special medical application is solved at present. When the sparassis crispa polysaccharide is used as a raw material to prepare health-care products and foods with special medical application, the effective absorption of the sparassis crispa polysaccharide by a human body is influenced due to the poor water solubility of the sparassis crispa polysaccharide, so that the effects of enhancing immunity, resisting tumors and reducing blood sugar are greatly reduced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method and application of pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity. The viscosity of the sparassis crispa polysaccharide is reduced by adopting the green and environment-friendly ionic liquid/combined enzyme system directional hydrolysis technology, the water solubility is increased, the absorption by a human body is facilitated, and the effects of enhancing immunity, resisting tumors, reducing blood sugar and the like are remarkable.

The technical scheme of the invention is as follows: a preparation method of sparassis crispa polysaccharide for enhancing immunity in pharmaceutical grade comprises the following steps:

(1) pretreatment of raw materials: taking a proper amount of dried sparassis crispa sporocarp, and crushing the sparassis crispa sporocarp; adding 10-60 times volume of ionic liquid solution, heating to 80-100 ℃, uniformly stirring to obtain mixed solution, adding 0.1-0.3 mass percent of specific endo-cellulase and lysozyme, and stirring the mixed solution for 1-2 hours at the temperature of 40-50 ℃ at the pH value of 5-7 for enzymolysis; adding lipase and bromelain with the mass percentage concentration of 0.1-0.5%, stirring the mixed solution at the temperature of 20-50 ℃ for 0.5-2 h under the condition that the pH value is 3-7, carrying out enzymolysis, carrying out centrifugal treatment on the enzymolysis solution to separate solid from liquid, discarding filtrate, and obtaining residue to obtain crude Sparassis crispa polysaccharide;

(2) taking crude Sparassis crispa polysaccharide, adding ionic liquid solution, stirring, adding enzyme for hydrolysis, filtering hydrolysate, adding ethanol into filtrate, standing, separating precipitate, and drying to obtain water-soluble Sparassis crispa polysaccharide.

Further, in the step (1), the sparassis crispa fruiting body is crushed to 800-1200 meshes by an ultrafine crusher.

Further, in the step (1), the ionic liquid solution used is an aqueous solution of 1-5% by mass of ionic liquid, wherein the ionic liquid is one or two of 1-butyl-1-methylpyrrolidine bistrifluoromethanesulfonylimide and tetrabutylphosphine tetrachloroferrite.

Further, in the step (1), the mass ratio of the specific endo-cellulase to the lysozyme is (2-5): 1, decomposing the cell wall of sparassis crispa to better dissolve intracellular glucan; the mass ratio of the lipase to the bromelain is (2-4): 1, to remove fat and protein from the raw material, thereby improving the yield of Sparassis crispa polysaccharide.

Further, in the step (2), the ionic liquid solution used is an aqueous solution of 0.5-2% by mass of an ionic liquid, wherein the ionic liquid is one or two of tributylmethylammonium bistrifluoromethylsulfonyl imide salt and 1-ethyl-3-methylimidazole ethyl sulfate.

Further, in the step (2), the enzyme is a combination enzyme of dextranase and beta-glucosaccharase, and the mass ratio of the combination enzyme is 1 (1-3), so as to degrade glycosidic bonds, and obtain the low-viscosity Sparassis crispa polysaccharide.

The ionic liquid and the enzyme preparation adopted by the invention are not limited to a specific manufacturer and can be used in the market.

The invention also aims to provide the application of the sparassis crispa polysaccharide for enhancing immunity, which is applied to a food formula with reasonable and scientific composition, has the effect of remarkably enhancing the immunity of organisms, does not have toxic or side effect and does not generate dependence.

The food formula consists of the following raw materials in parts by weight:

15-55 parts of hydrangea powder, 0-20 parts of whey protein powder, 0-20 parts of casein, 0-10 parts of soybean protein isolate, 10-50 parts of plant fat powder, 0-10 parts of fructo-oligosaccharide, 0-10 parts of inulin, 0-5 parts of acerola cherry powder, 0-5 parts of elderberry powder, 0.05-1 part of vitamin and 0.05-5 parts of mineral substances.

Further, the food formula comprises the following raw materials in parts by weight: 25-45 parts of hydrangea powder, 5-15 parts of whey protein powder, 5-15 parts of casein, 1-5 parts of soybean protein isolate, 20-40 parts of vegetable fat powder, 1-5 parts of fructo-oligosaccharide, 1-5 parts of inulin, 0.5-1.5 parts of acerola cherry powder, 0.5-2.5 parts of elderberry powder, 0.1-0.3 part of vitamin and 2-4 parts of mineral substances.

Preferably, the food formula consists of the following raw materials in parts by weight: 35 parts of hydrangea powder, 10 parts of whey protein powder, 10 parts of casein, 3 parts of soybean protein isolate, 30 parts of vegetable fat powder, 3 parts of fructo-oligosaccharide, 3.3 parts of inulin, 1 part of acerola powder, 1.5 parts of elderberry powder, 0.2 part of vitamin and 3 parts of mineral substances.

Furthermore, the hydrangea powder is prepared from the low-viscosity hydrangea polysaccharide obtained according to the technical method through a spray freeze drying technology, and is better in instant solubility.

The invention has the beneficial effects that: since most of glucan is on the cell wall of Sparassis crispa, ultramicro pulverization of Sparassis crispa fruiting body to 800-1200 mesh can mechanically destroy part of cell wall, so that glucan can be better dissolved out. The invention adopts an ionic liquid/compound enzyme system, the ionic liquid can effectively dissolve cellulose in the sparassis crispa and has good biocompatibility with enzyme, the high-efficiency enzymolysis of the sparassis crispa cell walls by specific endo-cellulase and lysozyme is promoted, the dissolution rate of intracellular glucan is improved, and the aim of improving the yield of the sparassis crispa polysaccharide is fulfilled. Because an ionic liquid/combined enzyme system is adopted, the dextran enzyme and the beta-glucanase combined enzyme can efficiently directionally control and degrade the macromolecular glycosidic chain segment in the ionic liquid, and the low-viscosity Sparassis crispa polysaccharide is obtained. The Sparassis crispa polysaccharide prepared by the method is prepared by a latest spray freeze drying technology, so that loss of bioactive substances is reduced, the structure of the product is loose and porous, the instant solubility of the product is improved, the absorption rate of a human body is improved, and the immunity of the human body is enhanced. The invention has reasonable and scientific application formula composition by taking low-viscosity Sparassis crispa polysaccharide as a raw material, has the function of obviously enhancing the immunity of the organism, has no toxic or side effect, and does not generate dependence.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The preparation method comprises the following steps of preparing pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity:

1. pretreating the raw materials by an ionic liquid/compound enzyme system:

taking 1kg of dried sparassis crispa fruiting body, and crushing the sparassis crispa fruiting body into 800 meshes by using an ultrafine crusher; adding 10L of 1 wt% 1-butyl-1-methylpyrrolidine bistrifluoromethanesulfonylimide ionic liquid solution, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 45 ℃, adjusting the pH to 6, and adding a complex enzyme with the mass percentage concentration of 0.2%, wherein the complex enzyme comprises specific endo-cellulase in parts by weight: and (2) 1:1, stirring the mixed solution at 45 ℃ for 1.5h for enzymolysis, and then adding 0.3% of complex enzyme in percentage by weight, wherein the complex enzyme comprises the following components in parts by weight: and (3) mixing the mixed solution at 40 ℃ for 1.5h to carry out enzymolysis, carrying out centrifugal treatment on the enzymolysis solution to separate solid from liquid, discarding filtrate to obtain residue, carrying out repeated enzymolysis and extraction on the solid residue for 3 times, and combining the obtained residues to obtain the crude polysaccharide of the Sparassis crispa.

In the steps, the cell wall structure is broken through the ultrafine grinder to promote the polysaccharide content to be separated out, the cell wall structure is further hydrolyzed in an ionic liquid solution through specific endo-cellulase and lysozyme in the complex enzyme, and fat and protein contained in the Sparassis crispa are hydrolyzed through lipase and bromelain, so that the yield and the purity of the Sparassis crispa polysaccharide are improved.

2. Preparing low-viscosity Sparassis crispa polysaccharide liquid by enzymolysis of an ionic liquid/combined enzyme system:

adding 15 times volume of 0.8 wt% of tributyl methyl ammonium bis (trifluoromethyl) sulfonyl imide salt ionic liquid solution into the residue Sparassis crispa crude polysaccharide obtained in the step 1, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 50 ℃, adjusting the pH to 5, adding a combined enzyme with the mass percent concentration of 0.07%, wherein the combined enzyme comprises dextranase and beta-glucanase 1:1 in parts by weight, uniformly stirring to obtain a mixed solution, stirring the mixed solution at 50 ℃ for enzymolysis for 3 hours, centrifuging an enzymolysis solution in a centrifuge (the rotation speed is 4000r/min) for 15min, separating solid from liquid, discarding residues, and obtaining a filtrate to obtain a low-viscosity Sparassis crispa polysaccharide liquid.

3. Concentrating and precipitating with ethanol:

concentrating the low-viscosity Sparassis crispa polysaccharide liquid obtained in the step (2), inactivating at 90 ℃ for 10min, and cooling to room temperature; adding 95% ethanol solution with 3 times volume, standing, and filtering to obtain precipitate.

4. Freezing and spray drying:

and (3) drying the precipitate obtained in the step (3) in a freezing spray dryer, and crushing to 60 meshes to obtain the final Sparassis crispa polysaccharide powder product 1.

Example 2

The preparation method comprises the following steps of preparing pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity:

1. pretreating the raw materials by an ionic liquid/compound enzyme system:

taking 1kg of dried sparassis crispa fruiting body, and crushing the sparassis crispa fruiting body into 1000 meshes by using an ultrafine crusher; adding 15L of 1 wt% tetrabutyl phosphine tetrachloroferrite ionic liquid solution, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 45 ℃, regulating the pH to 6, and adding a complex enzyme with the mass percent concentration of 0.3%, wherein the complex enzyme comprises the following specific endo-cellulase in parts by weight: and (2) mixing the lysozyme with the mixed solution at the temperature of 45 ℃ for 1.5h for enzymolysis, and then adding a complex enzyme with the mass percentage concentration of 0.3%, wherein the complex enzyme comprises the following components in parts by weight: and (3) stirring the mixed solution at the temperature of 40 ℃ for 1.5h to carry out enzymolysis, centrifuging the enzymolysis solution to separate solid from liquid, discarding filtrate to obtain residue, repeatedly carrying out enzymolysis and leaching on the solid residue for 3 times, and combining the obtained residues to obtain the crude polysaccharide of the Sparassis crispa.

In the steps, the cell wall structure is broken through the ultrafine grinder to promote the polysaccharide content to be separated out, the cell wall structure is further hydrolyzed in an ionic liquid solution through specific endo-cellulase and lysozyme in the complex enzyme, and fat and protein contained in the Sparassis crispa are hydrolyzed through lipase and bromelain, so that the yield and the purity of the Sparassis crispa polysaccharide are improved.

2. Preparing low-viscosity Sparassis crispa polysaccharide liquid by enzymolysis of an ionic liquid/combined enzyme system:

adding 15 times volume of 0.6 wt% 1-ethyl-3-methylimidazolium ethyl sulfate ionic liquid solution into the residue Sparassis crispa crude polysaccharide obtained in the step 1, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 50 ℃, adjusting the pH to 5, adding a combined enzyme with the mass percent concentration of 0.07%, wherein the combined enzyme comprises dextranase and beta-glucanase which are 1:2 in parts by weight, uniformly stirring to obtain a mixed solution, stirring the mixed solution at 50 ℃ for enzymolysis for 3 hours, centrifuging the enzymolysis solution in a centrifuge (the rotation speed is 4000r/min) for 15min, separating solid from liquid, discarding residues, and obtaining a filtrate to obtain a low-viscosity Sparassis crispa polysaccharide liquid.

3. Concentrating and precipitating with ethanol:

concentrating the low-viscosity Sparassis crispa polysaccharide liquid obtained in the step (2), inactivating at 90 ℃ for 10min, and cooling to room temperature; adding 95% ethanol solution with 3 times volume, standing, and filtering to obtain precipitate.

4. Freezing and spray drying:

and (3) drying the precipitate obtained in the step (3) in a freezing spray dryer, and crushing to 60 meshes to obtain a final Sparassis crispa polysaccharide powder product 2.

Example 3

The preparation method comprises the following steps of preparing pharmaceutical-grade sparassis crispa polysaccharide for enhancing immunity:

1. pretreating the raw materials by an ionic liquid/compound enzyme system:

taking 1kg of dried sparassis crispa fruiting body, and crushing the sparassis crispa fruiting body into 1200 meshes by using an ultrafine crusher; adding 10L of 1.5 wt% 1-butyl-1-methylpyrrolidine bistrifluoromethanesulfonylimide ionic liquid solution, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 45 ℃, adjusting the pH to 6, and adding a complex enzyme with the mass percentage concentration of 0.2%, wherein the complex enzyme comprises specific endo-cellulase in parts by weight: and (2) mixing lysozyme with the ratio of 4:1, stirring the mixed solution at the temperature of 45 ℃ for 1.5h for enzymolysis, and then adding a complex enzyme with the mass percentage concentration of 0.3%, wherein the complex enzyme comprises the following lipase in parts by weight: and (3) stirring the mixed solution at the temperature of 40 ℃ for 1.5h to carry out enzymolysis, centrifuging the enzymolysis solution to separate solid from liquid, discarding filtrate to obtain residue, repeatedly carrying out enzymolysis and leaching on the solid residue for 3 times, and combining the obtained residues to obtain the crude polysaccharide of the Sparassis crispa.

In the steps, the cell wall structure is broken through the ultrafine grinder to promote the polysaccharide content to be separated out, the cell wall structure is further hydrolyzed in an ionic liquid solution through specific endo-cellulase and lysozyme in the complex enzyme, and fat and protein contained in the Sparassis crispa are hydrolyzed through lipase and bromelain, so that the yield and the purity of the Sparassis crispa polysaccharide are improved.

2. Preparing low-viscosity Sparassis crispa polysaccharide liquid by enzymolysis of an ionic liquid/combined enzyme system:

adding 15 times volume of 0.7 wt% of tributyl methyl ammonium bistrifluoromethylsulfonyl imide ionic liquid solution into the residue Sparassis crispa crude polysaccharide obtained in the step 1, heating to 85 ℃, uniformly stirring to obtain a mixed solution, cooling to 50 ℃, adjusting the pH to 5, adding a combined enzyme with the mass percentage concentration of 0.07%, wherein the combined enzyme comprises beta-dextranase and beta-glucosaccharase in parts by weight, uniformly stirring to obtain a mixed solution, stirring the mixed solution at 50 ℃ for enzymolysis for 3h, centrifuging the enzymolysis solution in a centrifuge (the rotation speed is 4000r/min) for 15min, separating solid from liquid, discarding residues, and obtaining a filtrate to obtain a low-viscosity Sparassis crispa polysaccharide liquid.

3. Concentrating and precipitating with ethanol:

concentrating the low-viscosity Sparassis crispa polysaccharide liquid obtained in the step (2), inactivating at 90 ℃ for 10min, and cooling to room temperature; adding 95% ethanol solution with 3 times volume, standing, and filtering to obtain precipitate.

4. Freezing and spray drying:

and (3) drying the precipitate obtained in the step (3) in a freezing spray dryer, and crushing to 60 meshes to obtain a final Sparassis crispa polysaccharide powder product 3.

Comparative example 1

The difference from example 1 is that the fruit body of Sparassis crispa was pulverized to 200 mesh, and the rest of the procedure was not changed.

Comparative example 2

In contrast to example 1, 10L of a 1% wt 1-butyl-1-methylpyrrolidine bistrifluoromethanesulfonylimide ionic liquid solution was replaced in step 1 with 10L of distilled water, and the rest of the procedure was unchanged.

Comparative example 3

Unlike example 1, the enzyme added in step 1 was a single specific endo-cellulase, and the other processes were not changed.

Comparative example 4

The difference from example 1 is that the enzyme added in step 1 is a single lysozyme and the other processes are not changed.

Comparative example 5

Different from the example 1, 15 times of volume of the ionic liquid solution of 0.8 percent weight of tributyl methyl ammonium bis (trifluoromethyl) sulfonyl imide salt is replaced by 15 times of volume of distilled water in the step 2, and other processes are not changed,

comparative example 6

Different from the example 2, the enzyme added in the step 3 is single dextranase, other processes are not changed, and the final sparassis crispa polysaccharide powder product 4 is obtained.

Comparative example 7

Different from the example 2, the enzyme added in the step 3 is single beta-glucosaccharase, other processes are not changed, and the final Sparassis crispa polysaccharide powder product 5 is obtained.

The crude sparassis crispa polysaccharides obtained in step 1 of the above examples 1, 2 and 3 and comparative examples 1, 2, 3 and 4 were respectively weighed, and the respective yields were calculated, and the data obtained are shown in table 1 below:

TABLE 1

As can be seen from the data in Table 1, the comparison between example 1 and comparative example 1 shows that the finer the mesh number of the crushed Sparassis crispa fruiting body is, the higher the yield of Sparassis crispa polysaccharide product is, which means that the ultra-micro mechanical crushing damages the cell wall of Sparassis crispa fruiting body more, so that the polysaccharide is dissolved out more; comparing example 1 with comparative example 2, it can be seen that the yield of the polysaccharide product of sparassis crispa is much higher in the ionic liquid/complex enzyme system than in the pure water/complex enzyme system; the comparison between example 1 and comparative examples 3 and 4 shows that the complex enzyme has a remarkable effect of damaging the cell walls of the sparassis crispa fruiting body compared with a single enzyme.

The final sparassis crispa polysaccharide powder products obtained in the above examples 1, 2 and 3 and comparative examples 5, 6 and 7 were prepared into aqueous solutions with mass percentage concentrations of 0.1%, 0.2%, 0.5% and 1.0%, respectively, and the viscosity was measured by a rotational viscometer, and the data obtained are shown in the following table 2:

TABLE 2

As can be seen from the data in Table 2, when the mass percentage concentrations of the aqueous solutions are the same, the comparison between the example 1 and the comparative example 5 shows that the viscosity of the Sparassis crispa polysaccharide product prepared by the ionic liquid/combined enzyme system is greatly reduced compared with that prepared by pure water/combined enzyme system, and the liquid/combined enzyme system is more beneficial to the enzymolysis reaction of Sparassis crispa polysaccharide; the viscosity of the Sparassis crispa polysaccharide products prepared in examples 1, 2 and 3 is much lower than that of the Sparassis crispa polysaccharide products prepared in comparative examples 6 and 7, which shows that the hydrolysis effect of the combined enzyme of dextranase and beta-glucanase is better than that of a single enzyme, the product viscosity is low, and the molecular weight is lower, so that the product is easier to absorb by human bodies.

The final Sparassis crispa polysaccharide powder products 1, 2, 3 and 4, 5 obtained in the above examples 1, 2, 3 and comparative examples 6, 7 were each prepared as an aqueous solution having a mass percentage concentration of 0.2%, and the state of the aqueous solution was observed.

The aqueous solutions of the Sparassis crispa polysaccharide products 1, 2 and 3 obtained in examples 1, 2 and 3 were found to be highly transparent, clear and transparent, whereas the aqueous solution of the Sparassis crispa polysaccharide product 4 obtained in comparative example 6 had a small amount of suspended flocs, and the aqueous solution of the Sparassis crispa polysaccharide product 5 obtained in comparative example 7 had not very good transparency, but was not clear and transparent, further showing that the Sparassis crispa polysaccharide product obtained by hydrolysis with a combination of dextranase and beta-glucanase had good water solubility, clear and transparent appearance, and was more easily favored by the market.

Example 8

An application formula of sparassis crispa polysaccharide for enhancing immunity comprises the following raw materials in parts by weight:

35 parts of hydrangea powder, 10 parts of whey protein powder, 10 parts of casein, 3 parts of soybean protein isolate, 30 parts of vegetable fat powder, 3 parts of fructo-oligosaccharide, 3.3 parts of inulin, 1 part of acerola powder, 1.5 parts of elderberry powder, 0.2 part of vitamin and 3 parts of mineral substances.

The raw material Sparassis crispa powder is Sparassis crispa polysaccharide powder product 2 prepared in example 2, and other raw materials are all sold in the conventional market. The formula is comprehensive and balanced in nutrition, and the components synergize to achieve the effect of improving the immunity of the organism.

The preparation method comprises the following steps: the preparation method comprises the steps of premixing vitamins and minerals uniformly, adding acerola cherry powder and elderberry powder, mixing uniformly, adding soybean protein isolate, fructo-oligosaccharide and inulin, mixing uniformly, adding whey protein powder and casein, mixing uniformly, adding hydrangea powder and vegetable fat powder, and mixing uniformly to obtain the application formula product M of the immunity-enhancing hydrangea polysaccharide.

< evaluation test of Immunity-enhancing Effect >

Female mice of 6 weeks of age were selected for the experiment and divided into 4 groups by body weight. The human recommended dose of the sparassis crispa polysaccharide application formula product M is 0.5g/kg/d, and the equivalent dose of a mouse is 10 times of the human recommended dose. The human body recommended dose is 5 times, 10 times and 30 times respectively as low, medium and high dose groups. The gavage method is adopted, the gavage is performed once a day, and the control group is filled with distilled water. After each group of mice continuously takes the product M for 30 days, the mice freely eat and drink water, the proper temperature (25 +/-1.0 ℃) and humidity and silence of the feeding environment are ensured, and all indexes are measured.

On day 30 of administration, body weight was weighed. The mice are sacrificed, the spleen and the thymus are taken and weighed, and the organ coefficient is calculated, wherein the ratio of the spleen (thymus) to the spleen (thymus) is weight/weight; the toe swelling degree was also measured, and the results are shown in Table 3.

TABLE 3 Effect of product M on mouse thymus, spleen quality and toe swelling

As can be seen from Table 3, after the product M of the present invention is orally administered for 30 days, the thymus ratio and the spleen/body weight ratio of each dose group are not significantly different (p >0.05) compared with the blank control group after the mice are administered with different doses of the test substance for 30 days, which indicates that the spleen and the thymus can normally exert their physiological functions without causing adverse stimulation to the spleen and the thymus when the product M of the present invention is administered. Meanwhile, after the product M is taken, the toe swelling difference values of the low, medium and high dose groups are obviously different from those of a blank control group (p is less than 0.05), which shows that the product M can obviously improve the cellular immunity of the organism.

Further, on day 30 of administration, 1ml of 20% (V/V) suspension of chicken red blood cells was intraperitoneally injected into each mouse, the abdominal cavity was dropped into a tablet, incubated at 37 ℃ for 30min, fixed, stained, and then macrophages were counted under a microscope, and the phagocytosis index and phagocytosis rate were calculated. Phagocytosis index is the total number of chicken red blood cells phagocytosed per number of phagocytic cells counted; the phagocytosis rate is the number of phagocytes that phagocytose chicken erythrocytes/the number of phagocytes counted × 100%, and the results are shown in table 4.

TABLE 4 Effect of product M on phagocytic function of mice

The phagocytic function of the macrophages can be judged by the total number of phagocytic cells of the abdominal cavity injection chicken erythrocytes of the mice. As can be seen from table 4, the phagocytic index and rate were significantly higher in the low, medium and high dose groups than in the blank control group (p <0.05) after the oral administration of the product M for 30 days.

Further, on day 30 of the administration, the mouse NK cell activity was examined, and the results are shown in Table 5.

TABLE 5 Effect of product M on NK cell Activity in mice

As can be seen from Table 5, the NK cell activities of the low, medium and high dose groups were significantly different (p <0.05) from those of the blank control group after administration of the product M of the present invention, indicating that the product M has a promoting effect on NK cell activity.

In conclusion, the application formula of the sparassis crispa polysaccharide for enhancing immunity provided by the invention can remarkably enhance the immunity of organisms; the proportion of the raw materials is reasonable, and the maximum synergistic effect can be exerted under the proportion; meanwhile, the composition has a simple formula, so the composition has no toxic or side effect, and patients are not easy to generate dependence. The preparation method of the composition is simple, and various parameters are controllable, so that the composition can be produced in a large scale.

The above are merely characteristic embodiments of the present invention, and do not limit the scope of the present invention in any way. All technical solutions formed by equivalent exchanges or equivalent substitutions fall within the protection scope of the present invention.