A process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis and its application

Abstract

The invention provides a process for preparing Tremella fuciformis polysaccharide by enzymolysis, and the specific steps of the process include pretreatment of Tremella fuciformis fruiting bodies, enzymolysis reaction of Tremella fuciformis fruiting bodies and the like. In the invention, multiple enzymes are used to carry out enzymolysis reaction together, and key conditions such as material-liquid ratio, enzymolysis time, and enzymolysis temperature in the enzymolysis reaction are controlled, so that the obtained Tremella fuciformis polysaccharide has high yield, high purity, and low protein content, and the total sugar content of Tremella fuciformis polysaccharide can reach more than 90%. The invention uses enzymolysis to prepare Tremella fuciformis polysaccharide, and the reaction conditions are mild, so that the use of a large amount of organic reagents is avoided, and the Tremella fuciformis polysaccharide is safe and environmentally friendly; the preparation process is simple, and the reaction process does not rely on equipment, and the production cost is low. Tremella fuciformis polysaccharide prepared by the enzymolysis method of the invention can be widely used in cosmetic products, and has excellent antioxidant and moisturizing effects.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extraction, and in particular to a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis and application thereof. Background Art

[0002] Tremella aurantialba, also known as yellow fungus, golden fungus, brain fungus, etc., is named for its golden fruiting body. It is distributed in Tibet, Yunnan and other places in my country. It has the effects of relieving cough and reducing phlegm, nourishing yin and moistening lungs, calming liver and benefiting stomach, and nourishing qi and nourishing heart. Studies have found that many edible and medicinal fungi have various physiological activities, such as antioxidant, anti-cancer, antibacterial, etc. Fungal polysaccharides are one of the most important bioactive ingredients produced by edible and medicinal fungi. Du Xiuju, Dong Changjin, Zhang Zhicai, Chen Long, Wang Jinhua, Zhu Xinhua and others have studied the anti-tumor and antioxidant effects of the fruiting body and mycelium polysaccharides of Tremella aurantialba.

[0003] Polysaccharides are the main active ingredients of Tremella fuciformis. Studies have shown that Tremella fuciformis polysaccharides have multiple functions such as moisturizing, anti-oxidation, anti-inflammatory, anti-aging, and whitening. At present, there are few types of Tremella fuciformis processed products on the market, and the Tremella fuciformis industry is developing slowly. The research and development of Tremella fuciformis polysaccharide-related products can increase the added value of Tremella fuciformis and promote the development of Tremella fuciformis industry. In the prior art, the methods for extracting Tremella fuciformis polysaccharides mainly include water extraction and alcohol precipitation, acid-base extraction, enzyme method, microwave method, and supercritical fluid extraction method. Water extraction and alcohol precipitation and acid-base extraction require the use of a large amount of organic reagents, and the extracted polysaccharides have low purity and low biological activity; microwave method and supercritical fluid extraction method have high requirements for equipment and are difficult to quantify; and both have problems such as low yield, low total sugar content, and high protein content. Among them, the enzymatic method has mild conditions and is one of the methods currently under research, but how to further improve the yield of polysaccharides, improve purity, reduce impurities, and maintain good Tremella fuciformis polysaccharide activity is still a major problem to be solved in the extraction of Tremella fuciformis polysaccharides. Summary of the invention

[0004] In view of the above problems, the present invention provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, wherein a composite enzyme enzymatic hydrolysis process is adopted to extract Tremella fuciformis polysaccharide, thereby improving the extraction rate and purity of Tremella fuciformis crude polysaccharide; and the extract is tested for its moisturizing property and antioxidant property, which, on the one hand, lays a foundation for subsequent research on separation, purification and structural analysis of crude polysaccharide, and on the other hand, provides a reference for studying its polysaccharide functional components and the comprehensive utilization of Tremella fuciformis and the development of Tremella fuciformis polysaccharide cosmetics.

[0005] The present invention provides a process for preparing Tremella fuciformis polysaccharide by enzymolysis, and the specific process steps of the process for preparing Tremella fuciformis polysaccharide by enzymolysis are as follows:

[0006] Step S1: pretreatment of golden ear fruiting bodies: drying the golden ear fruiting bodies, crushing them and passing them through a 40-80 mesh sieve, placing the crushed golden ear fruiting bodies into a reactor, adding anhydrous ethanol for soaking, and air-drying the precipitate after the soaking treatment to obtain the pretreated golden ear fruiting bodies;

[0007] Step S2: Enzymatic hydrolysis reaction: the pretreated Tremella fuciformis fruiting bodies are placed in a reactor, and after adding water, they are treated at 70-90°C for 0.5-1.5h, and then cooled to 45-55°C and a reaction enzyme is added for enzymatic hydrolysis reaction. After the enzymatic hydrolysis reaction, the supernatant is centrifuged for the first time, and anhydrous ethanol is added to the supernatant until the concentration of anhydrous ethanol is 80-85%. After reacting at 3-5°C for 22-26h, the precipitate is centrifuged for the second time, and the precipitate is freeze-dried to obtain Tremella fuciformis polysaccharide.

[0008] In order to effectively clean the alcohol-soluble impurities on the surface of the material, preferably, the specific steps of adding anhydrous ethanol for soaking treatment in step S1 are: adding anhydrous ethanol with a material-liquid ratio of 1:10-20g / ml to soak for 22-26h, filtering to separate the material and the liquid, and taking a precipitate; adding anhydrous ethanol with a material-liquid ratio of 1:10-20g / ml again to soak for 22-26h, filtering to separate the material and the liquid, and taking a precipitate.

[0009] Preferably, the concentration of anhydrous ethanol added in step S1 is 95%.

[0010] In the pretreatment step, the present invention can effectively remove alcohol-soluble impurities and other components on the surface of the Tremella fuciformis fruiting body by strictly controlling the conditions such as the crushing mesh size of the Tremella fuciformis fruiting body and the soaking treatment in anhydrous ethanol, which is conducive to the subsequent sufficient enzymatic hydrolysis reaction and improves the extraction purity. At the same time, the inventors also found that the mesh size of the Tremella fuciformis fruiting body will also affect the extraction efficiency of polysaccharides in the subsequent enzymatic hydrolysis reaction, which may be because the Tremella fuciformis fruiting body will swell in the subsequent treatment. If the mesh size is too low, some polysaccharides will not be precipitated, thereby affecting the extraction efficiency.

[0011] Preferably, in step S2, the solid-liquid ratio of the Tremella fuciformis fruiting body to water is 1:70-100 g / ml. In production, if the solid-liquid ratio is too large, the subsequent concentration process will be greatly increased in time and energy consumption, and the loss during concentration will also be further increased (in the transfer of the solution, etc.), resulting in a decrease in yield.

[0012] In the enzymatic reaction step, the present invention effectively improves the yield, total sugar content and purity of Tremella fuciformis polysaccharide by controlling the solid-liquid ratio of Tremella fuciformis fruiting bodies to water, water treatment time, enzymatic hydrolysis temperature, reaction enzyme and other conditions. The inventors found that 70-90°C treatment for 0.5-1.5h is the best water treatment condition, and the extraction rate will decrease if the temperature is too high or too low. The same is true for the treatment time. It may be that the polysaccharide cannot be fully dissolved because the time is too short, and the polysaccharide may be decomposed if the time is too long, thereby reducing the content of Tremella fuciformis polysaccharide finally obtained.

[0013] Preferably, in step S2, the reaction enzyme is one or a mixture of cellulase, papain and pectinase.

[0014] Preferably, in step S2, the reaction enzymes are cellulase, papain and pectinase.

[0015] In order to further improve the yield of Tremella fuciformis polysaccharide, preferably, in step S2, the enzyme dosage of the cellulase is 2500-3500 U, the enzyme dosage of the papain is 1500-2500 U, and the enzyme dosage of the pectinase is 2500-3500 U. More preferably, the enzyme dosage of the cellulase is 3000 U, the enzyme dosage of the papain is 2000 U, and the enzyme dosage of the pectinase is 3000 U.

[0016] In the present invention, by using three enzymes to perform enzymolysis reaction on the Tremella fuciformis fruiting body at the same time, the yield of Tremella fuciformis polysaccharide is greatly improved, and the polysaccharide has high purity and low protein content. Cellulase and pectinase can effectively remove the Tremella fuciformis cell wall, so that the intracellular polysaccharide can be fully dissolved, and papain can remove the protein substances contained in the first two enzymes and the Tremella fuciformis itself, so that the obtained product does not contain protein, so as to eliminate the risk of allergies caused by protein. However, excessive use of enzymes will increase the cost of the enzymes and the enzymes cannot fully remove allergens, and too little use will reduce the yield of polysaccharides.

[0017] In order to further improve the yield of polysaccharides, preferably, in step S2, the reaction time of the enzymatic hydrolysis reaction is 45-50 minutes. When the reaction time is too long, the content of the prepared polysaccharide is reduced. The inventors analyzed that it may be due to the long reaction time that the enzyme will further decompose the polysaccharide in combination with the temperature, resulting in a decrease in the polysaccharide content.

[0018] Preferably, in step S2, the centrifugal rate of the first centrifugation is 7000-9000 r / min, and the centrifugal time is 25-35 min; the centrifugal rate of the second centrifugation is 7000-9000 r / min, and the centrifugal time is 25-35 min.

[0019] In order to avoid high temperature from destroying the structure of Tremella fuciformis polysaccharide and affecting its activity, preferably, the freeze-drying is vacuum freeze-drying. More preferably, the specific conditions of the vacuum freeze-drying are: vacuum degree <10Pa, temperature -50°C, and drying time of 12-24h.

[0020] The second aspect of the present invention provides an application of Tremella fuciformis polysaccharide prepared by the above enzymatic hydrolysis method in cosmetics. Tremella fuciformis polysaccharide is used in cosmetics to have moisturizing and anti-oxidation effects, providing a reference for studying its polysaccharide functional components and the comprehensive utilization of Tremella fuciformis and the development of Tremella fuciformis polysaccharide cosmetics.

[0021] The raw materials and equipment used in this application, unless otherwise specified, are all commonly used raw materials and equipment in this field, and are all commercially available products. The methods used in this application, unless otherwise specified, are all conventional methods in this field.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses an enzymatic hydrolysis method to prepare Tremella fuciformis polysaccharide, the reaction conditions are mild, the use of a large amount of organic reagents is avoided, and it is safe and environmentally friendly; the preparation process is simple, and the reaction process does not rely on equipment, and the production cost is low.

[0024] (2) In the present invention, multiple enzymes are used to carry out enzymolysis reaction together, and key conditions such as the material-liquid ratio, reaction enzyme, enzymolysis time, and enzymolysis temperature in the enzymolysis reaction are controlled. The resulting Tremella fuciformis polysaccharide has high yield, high purity, and low protein content. The total sugar content of Tremella fuciformis polysaccharide can reach more than 90%.

[0025] (3) The Tremella fuciformis polysaccharide prepared by the enzymatic hydrolysis method of the present invention has high adaptability and can be widely used in cosmetic products, and has excellent antioxidant and moisturizing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a product picture of Tremella fuciformis polysaccharide according to Example 1 of the present invention;

[0027] Figure 2 This is a glucose standard curve diagram when the polysaccharide content is tested by the sulfuric acid-phenol method of the present invention;

[0028] Figure 3 This is a protein standard curve diagram when the protein content is measured by the Coomassie Brilliant Blue method in the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The present invention is further explained below in conjunction with specific implementation methods.

[0030] Example

[0031] Example 1

[0032] This embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are as follows:

[0033] Step S1: Pretreatment of Tremella fuciformis fruiting bodies: drying 10 g Tremella fuciformis fruiting bodies to constant weight, crushing and then passing through a 60-mesh sieve, placing the sieved Tremella fuciformis fruiting bodies into a reactor, adding 95% anhydrous ethanol with a solid-liquid ratio of 1:15 g / ml to soak for 24 hours, filtering to separate the solid-liquid, and taking a precipitate; adding 95% anhydrous ethanol with a solid-liquid ratio of 1:15 g / ml again to soak for 24 hours, filtering to separate the solid-liquid, and taking a precipitate; air-drying the precipitate after soaking to constant weight to obtain the pretreated Tremella fuciformis fruiting bodies;

[0034] Step S2: enzymolysis reaction: the pretreated Tremella fuciformis fruiting body is placed in a reactor, water is added at a solid-liquid ratio of 1:80 g / ml, the mixture is treated at 80°C for 45 minutes, the temperature is lowered to 50°C, and then a reaction enzyme is added for enzymolysis reaction, the enzyme dosage of cellulase is 3000U, the enzyme dosage of papain is 2000U, the enzyme dosage of pectinase is 3000U, and the enzymolysis reaction time is 45 minutes; after the enzymolysis reaction, the supernatant is obtained by first centrifugation, the centrifugation rate is 8000r / min, and the centrifugation time is 30 minutes; anhydrous ethanol is added to the supernatant until the concentration of anhydrous ethanol is 80%, and the mixture is reacted at 4°C for 24 hours, and then a precipitate is obtained by second centrifugation, the centrifugation rate is 8000r / min, and the centrifugation time is 30 minutes; the precipitate is vacuum freeze-dried to obtain Tremella fuciformis polysaccharide, and the specific conditions of vacuum freeze-drying are: vacuum degree <10Pa, temperature is -50°C, and drying time is 20 hours.

[0035] In step S2, the reaction enzymes are cellulase, papain and pectinase, the total mass of the three enzymes is 7.07% of the mass of the pretreated Tremella fuciformis fruiting body; the mass ratio of cellulase, papain and pectinase is 4:4:3. Cellulase was purchased from Zhenghong Biotechnology Co., Ltd.; papain was purchased from Tianchen Chemical Products Co., Ltd.; and pectinase was purchased from Zhenghong Biotechnology Co., Ltd.

[0036] Example 2

[0037] This embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Embodiment 1. The difference from Embodiment 1 is that in the enzymatic hydrolysis reaction of step S2, the solid-liquid ratio of Tremella fuciformis fruiting body to water is 1:70.

[0038] Example 3

[0039] This embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Embodiment 1. The difference from Embodiment 1 is that in the enzymatic hydrolysis reaction of step S2, the solid-liquid ratio of Tremella fuciformis fruiting body to water is 1:100.

[0040] Example 4

[0041] This embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Embodiment 1. The difference from Embodiment 1 is that in the enzymatic hydrolysis reaction of step S2, the enzymatic hydrolysis reaction time is 50 min.

[0042] Example 5

[0043] The present embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those of Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the enzyme dosage of cellulase is 2500U, the enzyme dosage of papain is 1500U, and the enzyme dosage of pectinase is 2500U; that is, the total mass of the three enzymes is 5.73% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase, papain and pectinase is 3.7:2.5:3.7.

[0044] Example 6

[0045] The present embodiment provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the enzyme dosage of cellulase is 3500U, the enzyme dosage of papain is 2500U, and the enzyme dosage of pectinase is 3500U; that is, the total mass of the three enzymes is 8.4% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase, papain and pectinase is 5:4:5.

[0046] Comparative Example 1

[0047] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those in Example 1. The difference from Example 1 is that the specific steps of the soaking treatment in step S1 are: putting the sieved Tremella fuciformis fruiting bodies into a reactor, soaking them in 95% anhydrous ethanol with a feed-liquid ratio of 1:15 g / ml for 24 hours, filtering to separate the feed and liquid, and taking the precipitate; and air-drying the treated precipitate to constant weight to obtain the pretreated Tremella fuciformis fruiting bodies.

[0048] Comparative Example 2

[0049] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those of Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the solid-liquid ratio of Tremella fuciformis fruiting body to water is 1:120.

[0050] Comparative Example 3

[0051] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those of Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the solid-liquid ratio of Tremella fuciformis fruiting body to water is 1:60.

[0052] Comparative Example 4

[0053] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the enzymatic hydrolysis reaction time is 60 minutes.

[0054] Comparative Example 5

[0055] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzyme is added after the temperature is lowered to 65°C for enzymatic hydrolysis reaction.

[0056] Comparative Example 6

[0057] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the enzyme dosage of cellulase is 4000U, the enzyme dosage of papain is 3000U, and the enzyme dosage of pectinase is 4000U; that is, the total mass of the three enzymes is 9.75% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase, papain and pectinase is 5:4:5.

[0058] Comparative Example 7

[0059] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the enzyme dosage of cellulase is 2000U, the enzyme dosage of papain is 1000U, and the enzyme dosage of pectinase is 2000U; that is, the total mass of the three enzymes is 4.58% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase, papain and pectinase is 5:3:5.

[0060] Comparative Example 8

[0061] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzymes are cellulase and pectinase, the enzyme addition amount of cellulase is 3000U, and the enzyme addition amount of pectinase is 3000U; that is, the total mass of the two enzymes is 5.14% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase to pectinase is 1:1.

[0062] Comparative Example 9

[0063] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, and the specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzymes are cellulase and papain, the enzyme addition amount of cellulase is 3000U, and the enzyme addition amount of papain is 2000U; that is, the total mass of the two enzymes is 4.50% of the mass of the Tremella fuciformis fruiting body after pretreatment; the mass ratio of cellulase and papain is 4:3.

[0064] Comparative Example 10

[0065] This comparative example provides a process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis. The specific steps are the same as those in Example 1. The difference from Example 1 is that in the enzymatic hydrolysis reaction of step S2, the reaction enzyme is cellulase, and the enzyme dosage of cellulase is 7000U, that is, its mass is 6.07% of the mass of the Tremella fuciformis fruiting body after pretreatment.

[0066] Performance Testing

[0067] 1. Determination of the yield of Tremella fuciformis polysaccharide

[0068] The calculation formula of Tremella fuciformis polysaccharide yield (Y%) is:

[0069]

[0070] (M1: crude polysaccharide mass; M0: pre-treated material mass)

[0071] 2. Determination of total sugar content

[0072] The total sugar content was determined by the modified sulfuric acid-phenol method.

[0073] The determination method is as follows:

[0074] Take 1.00mL of the sample to be tested in a test tube, add 5.00mL of the color developing solution, and shake evenly. Place it in a boiling water bath and keep it warm for 30-35 minutes, take it out, place it in a cold water bath, and cool it to room temperature. Measure its absorbance value at 490nm.

[0075] Determination of glucose standard curve (curve diagram see Figure 2): Take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mL of 0.1 mg / mL glucose solution respectively into stoppered test tubes, make up to 1 mL with distilled water, then add 5.00 mL of color developing solution respectively, shake evenly. Place in a boiling water bath to keep warm for 30-35 minutes, take out, place in a cold water bath, and cool to room temperature. Measure its absorbance at 490 nm.

[0076] 3. Protein content determination

[0077] The Coomassie brilliant blue method for determining protein content uses the principle of protein-dye combination. Coomassie brilliant blue G-250 dye binds to protein in an acidic solution, causing the position of the dye's maximum absorption peak to change from 465nm to 595nm, and the color of the solution to change from brown-black to blue. The amount of protein bound to it can be determined by measuring the increase in light absorption at 595nm.

[0078] Determination of protein standard curve (curve diagram see Figure 3 ): Accurately pipette 0, 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 mL of standard protein solution into a 1 mL stoppered test tube, add 0.15 mol / L NaCl solution to 0.1 mL, then add 5 mL of Coomassie Brilliant Blue G-250 solution respectively, shake well, let stand at room temperature for 10 min, and measure the absorbance at a wavelength of 595 nm.

[0079] Determination of sample protein content: Take 0.5 mL of sample solution and add 0.15 mol / L NaCl solution to make up to 1 mL, operate according to the standard curve preparation steps, measure the absorbance value and calculate the protein content.

[0080] The Tremella fuciformis polysaccharides of Examples 1-6 and Comparative Examples 1-10 were tested for total sugar content and protein content. The test results are shown in Table 1 below.

[0081] Table 1

[0082] Example Polysaccharide yield (%) Total sugar content (%) Example 1 32.16 92.5 Example 2 30.2 89.5 Example 3 31.5 90.3 Example 4 30.51 90.0 Example 5 29.8 90.5 Example 6 30.12 87.5 Comparative Example 1 30.5 85.4 Comparative Example 2 22.4 87.2 Comparative Example 3 29.4 86.5 Comparative Example 4 27.6 88.5 Comparative Example 5 20.5 85.4 Comparative Example 6 30.56 88.5 Comparative Example 7 25.31 87.4 Comparative Example 8 27.85 86.7 Comparative Example 9 27.45 90.1 Comparative Example 10 25.96 85.9

[0083] The actual picture of the polysaccharide prepared in Example 1 is as follows Figure 1As shown, it is a light yellow powder. According to the data of polysaccharide yield and total sugar content in Table 1 above, the Tremella polysaccharide prepared in Example 1 has the characteristics of high polysaccharide yield and high total sugar content, low impurity content and high purity. The solid-liquid ratio was adjusted in Example 2, Example 3 and Comparative Example 2 and Comparative Example 3. According to the data of Examples 2 and 3, the appropriate solid-liquid ratio adjustment does not have a very large effect on the yield, but when the solid-liquid ratio is too large or too small (see Comparative Example 2 and Comparative Example 3), the exchange loss becomes larger or the dissolution amount is not enough, and the polysaccharide yield is greatly reduced. The enzymolysis time was adjusted in Example 4 and Comparative Example 4. When the enzymolysis time changed less, the polysaccharide yield decreased slightly, but there was still a polysaccharide yield of 30.51% (see Example 4), but when the enzymolysis time was too long (see Comparative Example 4), the polysaccharide content showed a more obvious decrease, which may be due to the excessively long reaction time, the enzyme will further decompose the polysaccharide in conjunction with the temperature, resulting in a decrease in the polysaccharide content. The amount of enzyme used was adjusted in Example 5, Example 6 and Comparative Examples 6-10. According to the data in Table 1 (see Comparative Example 6 and Comparative Example 7), when the amount of enzyme used is insufficient, the polysaccharide dissolution will not be complete, but if the amount used is too much, not only the polysaccharide content will decrease, but also the production cost will increase; at the same time, only when the three enzymes are used in combination can the best effect be achieved (see Comparative Examples 8-10 in Table 1). In Comparative Example 5, the temperature of enzymatic hydrolysis was adjusted, at which time the enzyme would be inactivated, and only the polysaccharide dissolved in water would result in a significant reduction in the polysaccharide content.

[0084] 4. Determination of moisturizing properties

[0085] A 0.5% aqueous solution of Tremella fuciformis polysaccharide and sodium hyaluronate was placed in a constant temperature and low humidity (dry silica gel) desiccator, and the water loss was measured after 75 hours.

[0086] The Tremella fuciformis polysaccharide of Example 1 was subjected to the above moisture retention performance test. The measured results are shown in Table 2.

[0087] Table 2

[0088] Sodium Hyaluronate Tremella polysaccharide Initial Value 30 30 After 75 hours 23.56 23.06 Water loss rate 21.47％ 23.13％

[0089] 5. Determination of hygroscopicity

[0090] Tremella polysaccharide, sodium hyaluronate powder and 0.5 g of glycerol that were freeze-dried to constant weight were placed in a constant temperature and humidity desiccator (25° C., 43% relative humidity). The remaining powder was placed at room temperature and humidity, and the mass of water absorbed after 72 hours was measured.

[0091] The Tremella fuciformis polysaccharide of Example 1 was subjected to the above hygroscopic performance test. The results are shown in Table 3.

[0092] Table 3

[0093] Sodium Hyaluronate Tremella polysaccharide glycerin Initial Value 0.5 0.5 0.5 After 75 hours 0.55 0.58 0.65 Water absorption 10％ 11％ 22％

[0094] 6. Determination of antioxidant properties

[0095] (1) Preparation of sample solution

[0096] Weigh 0.5 g of freeze-dried and spray-dried Tremella fuciformis polysaccharides respectively, dissolve them in distilled water, filter the precipitate, and make up to volume in a 100 mL volumetric flask.

[0097] (2) Sample measurement

[0098] Accurately measure 1.0 mL of the sample solution and place it in a 10 mL stoppered graduated test tube, add 3.0 mL of ABTS + free radical working solution, and place it in the dark for 6 minutes. Use PBS buffer as a blank and measure the absorbance at a wavelength of 734 nm.

[0099] (3) Calculation of antioxidant activity of Tremella fuciformis polysaccharide extract

[0100] The antioxidant activity of Tremella fuciformis polysaccharide in the extract was calculated according to formula (2):

[0101]

[0102] Where:

[0103] A0——Absorbance after mixing 1mL PBS solution with 3.0mL ABTS+ free radical working solution

[0104] A——Absorbance after mixing 1mL sample solution with 3.0mL ABTS+ free radical working solution

[0105] The above antioxidant performance test was performed on Tremella fuciformis polysaccharide of Example 1. The results are shown in Table 4.

[0106] Table 4

[0107] Dilute 250 times VC Tremella polysaccharide Blank control Free radical scavenging rate 99.71％ 85.72％ 0.00％

[0108] In the present invention, the polysaccharide obtained in Example 1 was subjected to in vitro moisturizing and antioxidant performance analysis. Tremella polysaccharide has good moisturizing performance. The in vitro antioxidant experiment shows that Tremella polysaccharide has a good free radical scavenging rate. According to the above experimental method, Tremella polysaccharide has a free radical scavenging rate of about 85%.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis, characterized in that: The specific process steps of the process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis are as follows: Step S1: pretreatment of golden ear fruiting bodies: drying the golden ear fruiting bodies, crushing them and passing them through a 40-80 mesh sieve, placing the crushed golden ear fruiting bodies into a reactor, adding anhydrous ethanol for soaking, and air-drying the precipitate after the soaking to obtain the pretreated golden ear fruiting bodies; Step S2: Enzymatic hydrolysis reaction: the pretreated Tremella fuciformis fruiting bodies are placed in a reactor, and after adding water, they are treated at 70-90° C. for 0.5-1.5 h, and then cooled to 45-55° C. and then a reaction enzyme is added to perform an enzymatic hydrolysis reaction, and after the enzymatic hydrolysis reaction, the supernatant is centrifuged for the first time to be taken, anhydrous ethanol is added to the supernatant until the concentration of anhydrous ethanol is 80-85%, and then reacted at 3-5° C. for 22-26 h, and then the precipitate is centrifuged for the second time to be taken, and the precipitate is freeze-dried to obtain Tremella fuciformis polysaccharide; In step S2, the reaction enzymes are cellulase, papain and pectinase.

2. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: The specific steps of adding anhydrous ethanol for soaking treatment in step S1 are: adding anhydrous ethanol with a solid-liquid ratio of 1:10-20 g / ml and soaking for 22-26 hours, filtering to separate the solid-liquid and taking a precipitate; adding anhydrous ethanol with a solid-liquid ratio of 1:10-20 g / ml again and soaking for 22-26 hours, filtering to separate the solid-liquid and taking a precipitate.

3. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the solid-liquid ratio of the Tremella fuciformis fruiting body to water is 1:60-100 g / ml.

4. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the reaction enzyme is one or a mixture of cellulase, papain and pectinase.

5. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the enzyme dosage of the cellulase is 2500-3500U, the enzyme dosage of the papain is 1500-2500U, and the enzyme dosage of the pectinase is 2500-3500U.

6. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the reaction time of the enzymatic hydrolysis reaction is 45-50 min.

7. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the centrifugal speed of the first centrifugation is 7000-9000 r / min, and the centrifugal time is 25-35 min; the centrifugal speed of the second centrifugation is 7000-9000 r / min, and the centrifugal time is 25-35 min.

8. The process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to claim 1, characterized in that: The freeze drying is vacuum freeze drying; the specific conditions of the vacuum freeze drying are: vacuum degree <10Pa, temperature -50°C, and drying time of 12-24 h.

9. Use of Tremella fuciformis polysaccharide prepared by the process for preparing Tremella fuciformis polysaccharide by enzymatic hydrolysis according to any one of claims 1 to 8 in cosmetics.

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