Preparation method of Sparassis crispa extracellular polysaccharide alcohol solution
阅读说明：本技术 一种绣球菌胞外多糖醇溶液的制备方法 (Preparation method of Sparassis crispa extracellular polysaccharide alcohol solution ) 是由 韩丹 杨盼盼 吕旭阳 陈金龙 蒋丽刚 于 2021-09-13 设计创作，主要内容包括：本发明涉及一种绣球菌胞外多糖醇溶液的制备方法,其特征在于采用以下步骤：A、将绣球菌子实体机械粉碎成绣球菌粉末；B、将10～20%的绣球菌粉末、10～20%的甜菜碱、20～40%的多元醇、余量为水,混合后搅拌均匀,得预混物；C、将预混物置于双极性方波高压脉冲电场中进行处理,得待提取混合液；D、将待提取混合液投入反应釜中加热搅拌,加热温度为60-80℃,保温搅拌1～3h后进行抽滤,得绣球菌多糖粗溶液；E、将绣球菌胞外多糖粗溶液与Sevage试剂按照体积比4：1进行混合,搅拌15min后进行离心,取上清液浓缩,得绣球菌胞外多糖醇溶液。本发明绣球菌胞外多糖醇溶液的制备方法具有多糖提取率高的优点。(The invention relates to a preparation method of an Sparassis crispa extracellular polysaccharide alcoholic solution, which is characterized by comprising the following steps of: A. mechanically pulverizing Sparassis crispa fruiting body into Sparassis crispa powder; B. mixing 10-20% of sparassis crispa powder, 10-20% of betaine, 20-40% of polyol and the balance of water, and uniformly stirring to obtain a premix; C. placing the premix in a bipolar square wave high-voltage pulse electric field for treatment to obtain a mixed solution to be extracted; D. putting the mixed solution to be extracted into a reaction kettle, heating and stirring at the temperature of 60-80 ℃, keeping the temperature, stirring for 1-3 hours, and then performing suction filtration to obtain a crude Sparassis crispa polysaccharide solution; E. mixing the crude sparassis crispa exopolysaccharide solution with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa. The preparation method of the Sparassis crispa extracellular polysaccharide alcoholic solution has the advantage of high polysaccharide extraction rate.)
1. A preparation method of a sparassis crispa extracellular polysaccharide alcohol solution is characterized by comprising the following steps:
A. mechanically pulverizing Sparassis crispa fruiting body, and sieving with 80 mesh sieve to obtain Sparassis crispa powder;
B. weighing 10-20% of sparassis crispa powder, 10-20% of betaine, 20-40% of polyalcohol and the balance of water according to the following mass ratio, mixing and uniformly stirring to obtain a premix; the polyalcohol is one of glycerol, butanediol, propylene glycol and xylitol or a mixture of glycerol, butanediol, propylene glycol and xylitol in any ratio;
C. and C, placing the premix obtained in the step B in a bipolar square wave high-voltage pulse electric field for treatment, wherein the treatment conditions are as follows: the intensity of the pulse electric field is 15-60kv/cm, the frequency is 100-800Hz, the processing time is 20min, and the mixed liquid to be extracted is obtained after the processing is finished;
D. c, putting the mixed solution to be extracted obtained in the step C into a reaction kettle, heating and stirring at the stirring speed of 300-600r/min and the heating temperature of 60-80 ℃, carrying out suction filtration after stirring for 1-3 h at a heat preservation temperature, and collecting the filtered solution, namely the crude Sparassis crispa polysaccharide solution;
E. d, mixing the crude sparassis crispa exopolysaccharide solution obtained in the step D with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging at 4000r/min for 10min, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa.
Technical Field
The invention relates to the technical field of cosmetic preparation, and particularly relates to a preparation method of an extracellular polysaccharide alcoholic solution of sparassis crispa.
Background
Sparassis Crispa (Sparassis Crispa) is a precious fungus used as both medicine and food, and shares the reputation of "king of Wangu mushroom", and the effective components mainly comprise protein, polysaccharide, ergosterol, glucosylceramide, adenosine and other small molecular compounds; the Sparassis crispa contains a large amount of beta-glucan, antioxidant substances, vitamins and minerals, contains superoxide dismutase and vitamin E, is used for treating prostate diseases, and has various pharmacological activities of improving immunity, resisting tumor, resisting hypertension, resisting diabetes and the like. The main active substance in sparassis crispa is polysaccharide, particularly the sparassis crispa beta-glucan with the highest content. There are mainly 2 forms of native β -glucan present: one is β - (1 → 3) -glucan and the other is β - (1 → 6) -glucan. Wherein beta- (1 → 3) -glucan has a wide range of biological activities, and exists in the form of a linear backbone linked by beta- (1 → 3) glycosidic linkages and a branched structure with beta- (1 → 6) glycosidic linkages. At present, the beta- (1 → 3) -glucan has more biological activity researches and applications, including immunopharmacological activity, hematopoietic function and the like, and is widely applied to the fields of cosmetics, health care products and the like. The content of beta-glucan in the sparassis crispa is very high, the content of beta-glucan in the sparassis crispa water extract can reach 43.6 percent of dry weight according to the detection result of a Japan Tokyo experimental research laboratory, and the content of beta-glucan in the sparassis crispa water extract is 39.3 percent through experiments such as Kim and the like; the content of beta-glucan in the handle part and the flap part of the sparassis crispa is measured to be more than 40 percent by adding the cheap additive and the like.
Chinese patent document CN104448014A discloses an extraction method of sparassis crispa alkali-soluble polysaccharide, which adopts an alkali extraction method to obtain sparassis crispa polysaccharide by selecting sodium hydroxide, ethanol/diethyl ether, sugar solution/trichloroacetic acid and ethanol for multiple extractions. However, the method uses various solvents, has complex steps, low utilization rate and large raw material loss, and still needs to be improved; chinese patent document CN104958314A discloses a preparation method of sparassis crispa SC031 polysaccharide extract with neuroprotective effect, which comprises fermenting sparassis crispa pileus powder, centrifuging, precipitating, freeze-drying to obtain sparassis crispa pileus powder, and extracting recovered sparassis crispa polysaccharide with hot water, wherein the sparassis crispa polysaccharide is prepared by freeze-thaw method, and repeated freezing and thawing process is adopted to break cells and separate out active ingredients, but the method needs repeated freezing, has long production time, has the risk of difficult cell breakage, and has low later-stage hot water extraction efficiency; chinese patent document CN106188333A discloses a method for extracting sparassis crispa polysaccharide. The method adopts an ultrasonic hot water extraction method, and obtains the Sparassis crispa polysaccharide through ultrafiltration membrane separation and purification, and has the problems of difficulty in large-scale industrial production and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of an Sparassis crispa extracellular polysaccharide alcohol solution, which has high extraction rate and has the functions of promoting the proliferation of skin probiotics and improving skin immunity.
In order to solve the technical problems, the invention adopts the following technical scheme: a preparation method of a sparassis crispa extracellular polysaccharide alcohol solution is characterized by comprising the following steps:
A. mechanically pulverizing Sparassis crispa fruiting body, and sieving with 80 mesh sieve to obtain Sparassis crispa powder;
B. weighing 10-20% of sparassis crispa powder, 10-20% of betaine, 20-40% of polyalcohol and the balance of water according to the following mass ratio, mixing and uniformly stirring to obtain a premix; the polyalcohol is one of glycerol, butanediol, propylene glycol and xylitol or a mixture of glycerol, butanediol, propylene glycol and xylitol in any ratio;
C. and C, placing the premix obtained in the step B in a bipolar square wave high-voltage pulse electric field for treatment, wherein the treatment conditions are as follows: the intensity of the pulse electric field is 15-60kv/cm, the frequency is 100-800Hz, the processing time is 20min, and the mixed liquid to be extracted is obtained after the processing is finished;
D. c, putting the mixed solution to be extracted obtained in the step C into a reaction kettle, heating and stirring at the stirring speed of 300-600r/min and the heating temperature of 60-80 ℃, carrying out suction filtration after stirring for 1-3 h at a heat preservation temperature, and collecting the filtered solution, namely the crude Sparassis crispa polysaccharide solution;
E. d, mixing the crude sparassis crispa exopolysaccharide solution obtained in the step D with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging at 4000r/min for 10min, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa.
The Sparassis crispa is purchased from scientific and technological research and development Limited company in Fujian Yijun industry.
The betaine is purchased from DANISCO company, INCI is betaine, and the serial number of the betaine in Chinese catalogue of names of used cosmetic raw materials (2015 edition) is 06665; the glycerol is purchased from Jiali oil & fat company Limited, INCI is glycerol, and the number in Chinese catalogue of names of used cosmetic raw materials (2015 edition) is 02421; the butanediol is purchased from Japan Co-Ltd, INCI name 1, 2-butanediol, and the serial number in Chinese catalogue of names of used cosmetic raw materials (2015 edition) is 00003; the propylene glycol is purchased from Shell company Limited in China, the INCI name is propylene glycol, and the serial number in the directory of names of used cosmetic raw materials (2015 edition) in China is 00006; xylitol is purchased from Zhejiang Huakang pharmaceutical industry Co., Ltd, INCI is named as xylitol, and in Chinese catalogue of names of used cosmetic raw materials (2015 edition), the number is 04786.
The bipolar square wave high-voltage pulse electric field adopts an OSU-4L type high-voltage pulse electric field of the State university of Ohio, the electric field is a continuous fluid processing type device and consists of a pulse power supply, a digital oscilloscope and a sample processing chamber 3.
The invention adopts the high-voltage pulse electric field to combine with the natural eutectic solvent to prepare the Sparassis crispa extracellular polysaccharide alcoholic solution, and the high-voltage pulse electric field can effectively destroy Sparassis crispa cells, is beneficial to the release of Sparassis crispa polysaccharide, is dissolved in the solvent and improves the extraction efficiency of the polysaccharide. Meanwhile, a high-voltage pulse electric field is adopted to cause part of high molecular weight polysaccharide components to be damaged, so that the relative molecular mass of the polysaccharide components is reduced, and the biological activity of the polysaccharide components is enhanced. The invention adopts a proper natural eutectic solvent to improve the dissolution rate of the polysaccharide by providing or accepting external electrons or protons to form hydrogen bonds, thereby improving the extraction efficiency of the polysaccharide in the solvent.
The extraction rate of the Sparassis crispa extracellular polysaccharide in the traditional method (adopting hot water, alkaline solution extraction and the like) is generally 10.5-22.3%. The extraction rate of polysaccharide in the Sparassis crispa extracellular polysaccharide alcoholic solution prepared by combining a high-voltage pulse electric field with a specific natural eutectic solvent is not lower than 26.0%, and the extraction rate of Sparassis crispa extracellular polysaccharide is remarkably improved.
According to the invention, specific betaine and polyhydric alcohol are selected, but the natural eutectic solvent is not prepared in advance, but is synchronously prepared in the extraction process, and in the heating and stirring process of the step D, the sparassis crispa polysaccharide is extracted while the natural eutectic solvent is formed by the betaine and the polyhydric alcohol, so that the steps are effectively simplified, and the energy loss is reduced.
The betaine and the polyhydric alcohol adopted by the invention have natural raw material sources, the betaine is selected as a natural eutectic solvent hydrogen bond acceptor, and any one of glycerol, butanediol, propylene glycol and xylitol is prepared as a natural eutectic solvent hydrogen bond donor. The method has the advantages that the extraction efficiency of the Sparassis crispa extracellular polysaccharide can be improved, the subsequent purification process is reduced, and the obtained Sparassis crispa extracellular polysaccharide alcohol solution can be directly added into cosmetics for use.
The Sparassis crispa extracellular polysaccharide alcoholic solution prepared by the method has the effects of moisturizing and regulating skin immunity. The polysaccharide solution is liquid, can be directly added into skin care products, and has good safety and high stability.
In order to verify that the alcohol solution of extracellular polysaccharide of Sparassis crispa prepared by the present invention obtained in example 1 has polysaccharide extraction rate and skin immunity regulating effect, the present invention was subjected to the following tests.
1. Measurement of Sparassis crispa polysaccharide in Sparassis crispa extracellular polysaccharide alcoholic solution prepared in example 1 and calculation of extraction rate.
The method adopts a phenol-sulfuric acid method to measure the extraction rate of the sparassis crispa extracellular polysaccharide. The phenol-sulfuric acid method is characterized in that polysaccharide or oligosaccharide is hydrolyzed into monosaccharide by concentrated sulfuric acid, the monosaccharide is rapidly dehydrated into a furfural derivative, the furfural derivative can perform a color reaction with phenol under the condition of strong acid, and the generated orange yellow substance has the maximum absorption value at 490 nm. Because the light absorption value and the polysaccharide concentration form a linear relation in a certain concentration range, the polysaccharide content can be measured by a colorimetric method.
Weighing 100mg of glucose, adding distilled water to dissolve, transferring to a 1000mL volumetric flask to fix the volume to the scale, and preparing into a 100 mu g/mL glucose standard solution. Respectively taking 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL and 1.4mL of glucose standard solution, supplementing to 2.0mL with distilled water, sequentially adding 1.0mL of 5% phenol solution, rapidly adding 5.0mL of concentrated sulfuric acid solution, standing for 10min, shaking up, cooling, measuring the light absorption value at 490nm, and using distilled water as blank control. And drawing a standard curve which is formed by taking the concentration as an abscissa and the light absorption value as an ordinate, namely the glucose standard curve. By linear regression, the linear equation for the polysaccharide is: y =9.96X + 0.58.
Adding 4 times of 100% ethanol into the Sparassis crispa extracellular polysaccharide alcoholic solution, standing, centrifuging, collecting precipitate, washing with acetone and ethanol, and using to obtain Sparassis crispa polysaccharide. Redissolving Sparassis crispa polysaccharide with distilled water, adding water to a constant volume of 250mL, properly diluting, measuring by using a phenol-sulfuric acid method, substituting the obtained absorbance value into a glucose standard curve, converting into glucose mass, and calculating the polysaccharide extraction rate by using glucose.
Polysaccharide extraction rate = glucose mass converted/mass of sparassis crispa dry powder 100%
Sample 1 is a Sparassis crispa extracellular polysaccharide alcoholic solution prepared by the method of example 1, sample 2 is a Sparassis crispa extracellular polysaccharide alcoholic solution prepared by a natural eutectic solvent method without using a high-voltage pulse electric field, and sample 3 is a Sparassis crispa polysaccharide extracted by distilled water without using a natural eutectic solvent with using a high-voltage pulse electric field.
The polysaccharide extraction rate of sample 1, the polysaccharide extraction rate of sample 2 and the polysaccharide extraction rate of sample 3 were respectively determined and calculated to be 26.6%, 20.2% and 14.2%. The method proves that the extraction rate of the extracellular polysaccharide of the Sparassis crispa polysaccharide alcohol solution prepared by the high-voltage pulse electric field assisted natural eutectic solvent method is high.
2. Effect of Sparassis crispa extracellular polysaccharide alcohol solution on growth and proliferation of beneficial bacteria in skin obtained in example 1
The Sparassis crispa extracellular polysaccharide alcohol solution prepared in the invention has a promoting effect on the growth and proliferation of Bifidobacterium bifidum, Bifidobacterium adolescentis, Lactobacillus acidophilus and Bifidobacterium infantis, and the product prepared in example 1 is used as an example, and the experiment is as follows:
0%, 1.0%, 2.0%, 3.0% and 4.0% mass fraction of Sparassis crispa extracellular polysaccharide alcohol solution is added into MRS culture medium respectively, then 1.0% of activated 4 strains (the concentration is 98 CFU/mL) are inoculated in turn, each group of 3 parallel experiments are cultured for 24h at 38 ℃, and an ultraviolet visible spectrophotometer is used for measuring the absorbance value (OD 600 nm) of each culture solution at the wavelength of 600 nm. The number of bifidobacterium bifidum strains CICC 8061, the number of bifidobacterium adolescentis strains CICC 8060, the number of lactobacillus acidophilus strains CICC8063 and the number of bifidobacterium infantis strains CICC 8066 are purchased from the China center for industrial microorganism strain preservation and management; the MRS culture medium is purchased from Qingdao Haibo biotechnology limited; the UV-visible spectrophotometer used was purchased from Youngco (Shanghai) instruments Inc., model UV-2100 PC.
The OD600nm values of the four bacteria in alcoholic solution culture of extracellular polysaccharide of Sparassis crispa at different concentrations are shown in Table 1.
Table 1: OD600nm values of four bacteria in alcoholic solution culture solution of extracellular polysaccharide of Sparassis crispa with different concentrations
Mass fraction of sparassis crispa polysaccharide alcohol solution in culture solution
0%
1.0%
2.0%
3.0%
4.0%
Bifidobacterium bifidum
0.02
0.26
0.47
0.72
0.98
Bifidobacterium adolescentis
0.03
0.24
0.42
0.80
1.05
Lactobacillus acidophilus
0.05
0.18
0.57
0.65
0.72
Bifidobacterium infantis
0.04
0.42
0.55
0.62
0.68
As can be seen from table 1, the typical skin probiotics bifidobacterium bifidum, bifidobacterium adolescentis, lactobacillus acidophilus and bifidobacterium infantis all showed a growing trend with the increase of the addition amount of the Sparassis crispa extracellular polysaccharide alcohol solution. The experimental result can show that the polysaccharide plays a good role in promoting the growth and proliferation of probiotics.
3. The experimental verification of the Sparassis crispa extracellular polysaccharide alcoholic solution obtained in example 1 on the aspect of improving skin immunity.
The effect of the Sparassis crispa extracellular polysaccharide alcohol solution on the cell density of the female mouse NZBHF 1/J epidermal Langerhans cell closest to the animal model of human SLE is also used to characterize the effect of the Sparassis crispa extracellular polysaccharide alcohol solution on the improvement of skin immunity, taking the product prepared in example 1 as an example, the experiment is as follows:
dividing 12 NZBHF 1/J female mice with the age of 2 months into 6 dexamethasone groups, and injecting dexamethasone 1.0mg/kg intraperitoneally daily; the sparassis crispa exopolysaccharide is 6, the sparassis crispa exopolysaccharide alcoholic solution is injected in an abdominal cavity every day at 3.0mg/kg, and the sparassis crispa exopolysaccharide alcoholic solution is continuously taken for 6 months from 2 months of age of a mouse to 8 months of age of the mouse; SPF grade C57BL/6 inbred female mice, 6, 8 months old, served as a placebo.
Shearing the ear of the same mouse under the condition of full-anesthesia of mouse ether, separating the skin on both sides of the ear by using a dissecting microscope, scraping the cartilage on both sides, putting into a biological EDTA separating solution with pH7.2, soaking for 2h at the constant temperature of 38 ℃, separating the true epidermis under the microscope, fixing the epidermis in acetone at 4 ℃ for 20min, and washing for 3 times with PBS phosphate buffer solution for 3min each time. Adding anti-mouse Ia antigen OX3 with the concentration of 1:50, anti-mouse Ia antigen OX4 with the concentration of 1:200 and mouse epidermis into eppendorf microtubes respectively, overnight at 4 ℃, washing with PBS, adding biotinylated goat anti-mouse IgG serum, incubating at 38 ℃ for 2h, washing with PBS, adding ABC (ovalbumin/biotin/enzyme) complex, incubating for 1h, and washing with PBS. Under a dissecting microscope, the epidermis is laid on a glass slide, the substrate 3-amino-9-ethyl carbazol (AEC) is added for color development and drying at room temperature, and then the glycerol gelatin is sealed.
The cells were observed under a microscope, and the cells were positive if they appeared in orange color. Epidermal langerhans cell density (LC/mm 2) = average number of LCs per field of view/epidermal area, as determined by ocular grid calibrated with micrometer. The epidermal LC density data for each experimental group of mice is presented in table 2. The spontaneous SLE model female mouse NZBWF1/J used was purchased from Nanjing university-Nanjing biomedical research institute; the SPF grade C57BL/6 pure line mouse is female, 8 months old, and purchased from the center of Guangdong province medical experimental animals; the dissecting microscope was purchased from Shenzhen Bo Shida optical instruments, Inc., model number BD-60T.
TABLE 2 average value of epidermal LC density (LC/mm) of mice in each experimental group2）
Antigens
OX3
OX4
Dexamethasone group
208±57
254±33
Sparassis crispa extracellular polysaccharide group
572±38
482±42
C57BL/6 pure line (blank control)
340±52
302±35
As can be seen from Table 2, compared with the mouse NZWF 1/J in the dexamethasone group, the mouse NZWF 1/J in the Sparassis crispa extracellular polysaccharide group has obviously increased epidermal LC density, probably because the Sparassis crispa extracellular polysaccharide promotes the generation of GM-CSF, simultaneously stimulates immature granulocytes and macrophages to differentiate and mature, improves the immune function of an organism, and dexamethasone serving as an immunosuppressant is widely applied to treatment and research of autoimmune diseases of human beings and animals; compared with C57BL/6 pure line mice, the extracellular polysaccharide mice NZBHF 1/J also have significant increase of epidermal LC density.
Experiments prove that the sparassis crispa extracellular polysaccharide alcohol solution can promote the increase of skin Langerhans cell density, so that the skin immunity can be improved, and the skin physique can be improved.
In conclusion, the preparation method of the Sparassis crispa extracellular polysaccharide alcoholic solution has the advantage of high polysaccharide extraction rate, and the prepared Sparassis crispa extracellular polysaccharide alcoholic solution has the effects of promoting skin probiotic proliferation and improving skin immunity.
Detailed Description
Example 1: a preparation method of a sparassis crispa extracellular polysaccharide alcohol solution is characterized by comprising the following steps:
A. mechanically pulverizing Sparassis crispa fruiting body, and sieving with 80 mesh sieve to obtain Sparassis crispa powder;
B. weighing 20% of sparassis crispa powder, 20% of betaine, 40% of polyol and the balance of water according to the following mass ratio, and uniformly stirring after mixing to obtain a premix; the polyalcohol is one of glycerol, butanediol, propylene glycol and xylitol or a mixture of glycerol, butanediol, propylene glycol and xylitol in any ratio;
C. and C, placing the premix obtained in the step B in a bipolar square wave high-voltage pulse electric field for treatment, wherein the treatment conditions are as follows: the intensity of the pulse electric field is 60kv/cm, the frequency is 800Hz, the processing time is 20min, and the extracting solution is obtained after the processing;
D. c, putting the extracting solution obtained in the step C into a reaction kettle, heating and stirring at the stirring speed of 600r/min and the temperature of 80 ℃, keeping the temperature and stirring for 3 hours, then performing suction filtration, and collecting the filtered filtrate to obtain a crude Sparassis crispa extracellular polysaccharide solution;
E. d, mixing the crude sparassis crispa exopolysaccharide solution obtained in the step D with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging at 4000r/min for 10min, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa. Sevage reagent is 5: 1 chloroform: a mixture of n-butanol. The Sparassis crispa adopted in the embodiment is purchased from technical research and development Limited company in Fujian Yijun industry.
Example 2: a preparation method of sparassis crispa exopolysaccharide is characterized by comprising the following steps:
A. mechanically pulverizing Sparassis crispa fruiting body, and sieving with 80 mesh sieve to obtain Sparassis crispa powder;
B. weighing 10% of sparassis crispa powder, 10% of betaine, 20% of polyol and the balance of water according to the following mass ratio, and uniformly stirring after mixing to obtain a premix; the polyalcohol is one of glycerol, butanediol, propylene glycol and xylitol or a mixture of glycerol, butanediol, propylene glycol and xylitol in any ratio;
C. and C, placing the premix obtained in the step B in a bipolar square wave high-voltage pulse electric field for treatment, wherein the treatment conditions are as follows: the intensity of the pulse electric field is 15kv/cm, the frequency is 100Hz, the processing time is 20min, and the extracting solution is obtained after the processing;
D. c, putting the extracting solution obtained in the step C into a reaction kettle, heating and stirring at the stirring speed of 300r/min and the temperature of 60 ℃, keeping the temperature and stirring for 1h, then performing suction filtration, and collecting the filtered filtrate to obtain a crude Sparassis crispa extracellular polysaccharide solution;
E. d, mixing the crude sparassis crispa exopolysaccharide solution obtained in the step D with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging at 4000r/min for 10min, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa. Sevage reagent is 5: 1 chloroform: a mixture of n-butanol. The Sparassis crispa adopted in the embodiment is purchased from technical research and development Limited company in Fujian Yijun industry.
Example 3: a preparation method of sparassis crispa exopolysaccharide is characterized by comprising the following steps:
A. mechanically pulverizing Sparassis crispa fruiting body, and sieving with 80 mesh sieve to obtain Sparassis crispa powder;
B. weighing 15% of sparassis crispa powder, 15% of betaine, 30% of polyol and the balance of water according to the following mass ratio, and uniformly stirring after mixing to obtain a premix; the polyalcohol is one of glycerol, butanediol, propylene glycol and xylitol or a mixture of glycerol, butanediol, propylene glycol and xylitol in any ratio;
C. and C, placing the premix obtained in the step B in a bipolar square wave high-voltage pulse electric field for treatment, wherein the treatment conditions are as follows: the pulse electric field intensity is 40kv/cm, the frequency is 400Hz, the processing time is 20min, and the extracting solution is obtained after the processing;
D. c, putting the extracting solution obtained in the step C into a reaction kettle, heating and stirring at the stirring speed of 450r/min and the temperature of 70 ℃, keeping the temperature and stirring for 1-3 hours, then performing suction filtration, and collecting the filtered filtrate to obtain a crude Sparassis crispa extracellular polysaccharide solution;
E. d, mixing the crude sparassis crispa exopolysaccharide solution obtained in the step D with a Sevage reagent according to a volume ratio of 4: 1, stirring for 15min, centrifuging at 4000r/min for 10min, and concentrating the supernatant to obtain the extracellular polysaccharide alcoholic solution of Sparassis crispa. Sevage reagent is 5: 1 chloroform: a mixture of n-butanol. The Sparassis crispa adopted in the embodiment is purchased from technical research and development Limited company in Fujian Yijun industry.