Tremella polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses tremella polysaccharide as well as a preparation method and application thereof. The preparation method of the tremella polysaccharide provided by the invention comprises the following steps: mixing tremella powder with water, and carrying out ultrasonic extraction treatment to obtain a crude extract; performing cellulase enzymolysis treatment and pectinase enzymolysis treatment on the crude extract to obtain an enzymolysis product; carrying out pyrolysis treatment on the enzymolysis product to obtain a pyrolysis product; carrying out macroporous resin adsorption treatment and ceramic membrane filtration and concentration treatment on the pyrolysis product to obtain a tremella polysaccharide solution; performing low-temperature roller drying treatment on the tremella polysaccharide solution to obtain the tremella polysaccharide. The tremella polysaccharide with high purity, uniform molecular weight distribution and excellent biological activity can be prepared by adopting the method, and the method has important significance in realizing the application of the tremella polysaccharide in the fields of medicines and functional foods.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a tremella polysaccharide and a preparation method and application thereof. Background Art

[0002] Tremella fuciformis (Tremella fuciformis), also known as snow fungus, white fungus, or Tremella fuciformis, belongs to the genus Tremella, Tremellales, and Tremella family. It is widely distributed in nature and is known as the "king of fungi." Tremella fuciformis is rich in protein, polysaccharides, dietary fiber, minerals, trace elements, and vitamins, making it a highly nutritious food. Tremella fuciformis polysaccharides (TFPS), an active ingredient extracted from Tremella fuciformis, exhibit biological activities such as hypoglycemic and hypolipidemic properties, anti-aging, anti-inflammatory, antioxidant, anti-tumor, and anti-thrombotic properties.

[0003] Currently, the methods for extracting Tremella polysaccharides mainly include hot water extraction, ethanol extraction, acid hydrolysis, and alkaline hydrolysis. Among them, the hot water extraction method uses a high-temperature and long-term extraction method. Although it helps dissolve Tremella polysaccharides, it is easy to cause degradation and reduce the biological activity of the final product. The ethanol extraction method is prone to cause structural changes in some Tremella polysaccharides, even affecting their biological activity. The final extracted product still contains a large number of non-target components, and the purity of Tremella polysaccharides is low. The molecular weight distribution of Tremella polysaccharides obtained by acid hydrolysis and alkaline hydrolysis is relatively broad and unstable, which affects the absorption and utilization of Tremella polysaccharides in the body. The product quality is difficult to stably control, limiting its application in the fields of medicine and functional foods. Existing Tremella polysaccharide extraction processes have significant limitations in terms of extraction purity, molecular weight stability, and activity retention. Therefore, a simple and efficient extraction method for obtaining high-purity, uniform molecular weight, and highly active Tremella polysaccharides is urgently needed. Summary of the Invention

[0004] The first purpose of the present invention is to solve the problems existing in the existing Tremella polysaccharide extraction, such as low purity, uneven molecular weight, and large activity loss of the obtained Tremella polysaccharide, and to provide a preparation method of Tremella polysaccharide.

[0005] The second object of the present invention is to provide a Tremella polysaccharide.

[0006] The third object of the present invention is to provide the use of the above-mentioned Tremella polysaccharide in the preparation of a tissue damage repair agent.

[0007] The fourth object of the present invention is to provide a tissue damage repair agent.

[0008] Specifically, the preparation method of Tremella fuciformis polysaccharide provided by the present invention includes: S1, taking Tremella fuciformis powder and mixing it with water to perform ultrasonic extraction treatment to obtain a crude extract; S2, taking the crude extract and performing cellulase enzymolysis treatment and pectinase enzymolysis treatment to obtain an enzymolysis product; taking the enzymolysis product and performing pyrolysis treatment to obtain a pyrolysis product; S3, taking the pyrolysis product and performing macroporous resin adsorption treatment and ceramic membrane filtration and concentration treatment to obtain a Tremella fuciformis polysaccharide solution; S4, taking the Tremella fuciformis polysaccharide solution and performing low-temperature drum drying treatment to obtain the Tremella fuciformis polysaccharide.

[0009] Furthermore, in step S1, the mixing ratio of the tremella powder and water is 1 g: (40-80) mL.

[0010] Furthermore, in step S1, the temperature of the ultrasonic extraction treatment is 10°C to 40°C, the ultrasonic power is 100W to 400W, and the ultrasonic time is 20min to 50min.

[0011] Furthermore, in step S2, the amount of cellulase added in the cellulase enzymatic hydrolysis treatment is 0.5 wt% to 1.5 wt% based on the mass of the crude extract.

[0012] Furthermore, in step S2, the temperature of the cellulase enzymatic hydrolysis treatment is 65° C. to 75° C., the pH value is 4.5 to 6.5, and the enzymatic hydrolysis time is 1 hour to 2 hours.

[0013] Furthermore, in step S2, the amount of pectinase added in the pectinase enzymatic hydrolysis treatment is 0.5 wt% to 1.5 wt% based on the mass of the crude extract.

[0014] Furthermore, in step S2, the temperature of the pectinase enzymatic hydrolysis treatment is 50°C to 60°C, the pH value is 3.3 to 4, and the enzymatic hydrolysis time is 1 hour to 2 hours.

[0015] Furthermore, in step S2, the temperature of the pyrolysis treatment is 95° C. to 100° C., and the time is 15 min to 50 min.

[0016] Furthermore, in step S3, the macroporous resin in the macroporous resin adsorption treatment is selected from one or more of macroporous resin AB-8, macroporous resin HP-20, macroporous resin XAD-4 and macroporous resin XAD-7.

[0017] Furthermore, in step S3, in the macroporous resin adsorption treatment, the addition ratio of the macroporous resin to the pyrolysis product is 1 g: (20-40) mL.

[0018] Furthermore, in step S3, the temperature of the macroporous resin adsorption treatment is 50° C. to 70° C., the stirring speed is 150 r / min to 200 r / min, and the time is 1.5 h to 3 h.

[0019] Furthermore, in step S3, the pore size of the ceramic membrane in the ceramic membrane filtration and concentration treatment is 80 nm to 120 nm.

[0020] Furthermore, in step S4, the temperature of the low-temperature drum drying treatment is not greater than 70°C, the rotation speed is 10r / min to 15r / min, the feed rate is 0.3L / min to 0.7L / min, and the residence time is 15s to 30s.

[0021] The present invention provides tremella polysaccharide prepared by the tremella polysaccharide preparation method.

[0022] Furthermore, the weight average molecular weight of the Tremella polysaccharide is 1000kDa to 3162kDa.

[0023] The present invention provides the use of the Tremella polysaccharide in preparing a tissue damage repair agent.

[0024] The tissue damage repair agent provided by the present invention comprises the above-mentioned tremella polysaccharide and agar oligosaccharide, and the polymerization degree of the agar oligosaccharide is 2-10.

[0025] Furthermore, the added mass ratio of the Tremella polysaccharide and agar oligosaccharide is (1-5):1.

[0026] Beneficial effects:

[0027] In the preparation method provided by the present invention, ultrasound is used to provide energy for crushing instead of the traditional hot water leaching method, so that the biological activity of tremella polysaccharide is maintained while the tremella polysaccharide is fully released and freed; then, cellulase and pectinase are used to perform enzymatic hydrolysis on the obtained crude extract to further release the tremella polysaccharide, so that the tremella polysaccharide in the enzymatic hydrolysis product can be quickly and evenly cracked under the conditions of high temperature and short time, and the molecular weight of the tremella polysaccharide can be accurately regulated; then, a process combining macroporous resin adsorption treatment and ceramic membrane filtration and concentration treatment is used to purify the obtained pyrolysis product, and the tremella polysaccharide within a specific molecular weight range is enriched; and the tremella polysaccharide in the obtained tremella polysaccharide solution is structurally stable during low-temperature drum drying treatment, with only a small amount of or no degradation occurring, so that tremella polysaccharide with high purity, relatively uniform molecular weight distribution and excellent biological activity can be prepared, which is of great significance for realizing the application of tremella polysaccharide in the fields of medicine and functional foods.

[0028] In some specific embodiments, in the ceramic membrane filtration and concentration treatment in step S3, the pore size of the ceramic membrane used is preferably 80 nm to 120 nm; at this time, the weight-average molecular weight of the obtained Tremella polysaccharide is 1000 kDa to 3162 kDa, and its synergistic combination with agar oligosaccharides with a degree of polymerization of 2 to 10 can achieve excellent antioxidant, moisturizing and cell migration promotion effects, showing great application potential in tissue damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing the experimental results of liquid chromatography detection of Tremella polysaccharide provided in Example 1 of the present invention;

[0030] Figure 2 The GPC spectrum of Tremella polysaccharide provided in Example 1 of the present invention;

[0031] Figure 3 This is a graph showing the experimental results of promoting tissue repair provided in Example 2 of the present invention (magnification is 10 times);

[0032] Figure 4 Figure 2 (12h) shows the experimental results of promoting tissue repair provided by Example 2 of the present invention;

[0033] Figure 5 Figure 3 (12h) shows the experimental results of promoting tissue repair provided by Example 2 of the present invention. DETAILED DESCRIPTION

[0034] In the first aspect, the preparation method of Tremella fuciformis polysaccharide provided by the present invention specifically includes: S1, taking Tremella fuciformis powder and mixing it with water to perform ultrasonic extraction treatment to obtain a crude extract; S2, taking the crude extract and performing cellulase enzymolysis treatment and pectinase enzymolysis treatment to obtain an enzymolysis product; taking the enzymolysis product and performing pyrolysis treatment to obtain a pyrolysis product; S3, taking the pyrolysis product and performing macroporous resin adsorption treatment and ceramic membrane filtration and concentration treatment to obtain a Tremella fuciformis polysaccharide solution; S4, taking the Tremella fuciformis polysaccharide solution and performing low-temperature drum drying treatment to obtain the Tremella fuciformis polysaccharide.

[0035] In the present invention, in step S1, the mixing ratio of the tremella powder and water is preferably 1g:(40-80)mL, specifically 1g:40mL, 1g:45mL, 1g:50mL, 1g:60mL, 1g:70mL, 1g:80mL or any value therebetween. At this time, it is possible to ensure that the crude extract contains a certain concentration of tremella polysaccharide while achieving full release and freeing of tremella polysaccharide, which is conducive to the subsequent processing process.

[0036] In the present invention, in step S1, the ultrasonic extraction treatment refers to the process of extracting Tremella polysaccharide by utilizing the physical effect of ultrasound, and the conditions specifically include a temperature of preferably 10°C to 40°C, specifically 10°C, 15°C, 20°C, 23°C, 25°C, 30°C, 38°C, 40°C or any value therebetween; an ultrasonic power of preferably 100W to 400W, specifically 100W, 110W, 125W, 150W, 200W, 250W, 300W, 400W or any value therebetween; and an ultrasonic time of preferably 20min to 50min, specifically 20min, 23min, 28min, 30min, 35min, 40min, 50min or any value therebetween.

[0037] In the present invention, in step S2, the amount of cellulase added in the cellulase enzymatic hydrolysis treatment is preferably 0.5 wt% to 1.5 wt%, specifically 0.5 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.3 wt%, 1.5 wt%, or any value therebetween, based on the mass of the crude extract. In this case, the cellulase can achieve a more ideal enzymatic hydrolysis effect.

[0038] In the present invention, in step S2, the cellulase enzymatic hydrolysis treatment refers to the process of using cellulase to decompose cellulose in the crude extract to promote the release of Tremella polysaccharide, and the conditions specifically include a temperature of preferably 65°C to 75°C, specifically 65°C, 68°C, 69°C, 70°C, 73°C, 75°C or any value therebetween; a pH value of preferably 4.5 to 6.5, specifically 4.5, 4.8, 5, 5.2, 5.6, 6, 6.5 or any value therebetween; and an enzymatic hydrolysis time of preferably 1h to 2h, specifically 1h, 1.1h, 1.3h, 1.5h, 1.8h, 2h.

[0039] In the present invention, in step S2, the amount of pectinase added in the pectinase enzymatic hydrolysis treatment is preferably 0.5wt% to 1.5wt%, specifically 0.5wt%, 0.6wt%, 0.8wt%, 1wt%, 1.2wt%, 1.4wt%, 1.5wt% or any value therebetween, based on the mass of the crude extract. In this case, the pectinase can achieve a more ideal enzymatic hydrolysis effect.

[0040] In the present invention, in step S2, the pectinase enzymatic hydrolysis treatment refers to the process of using pectinase to decompose pectin in the crude extract to promote the release of Tremella polysaccharide, and the conditions specifically include a temperature of preferably 50°C to 60°C, specifically 50°C, 52°C, 53°C, 56°C, 58°C, 60°C or any value therebetween; a pH value of preferably 3.3 to 4, specifically 3.3, 3.4, 3.6, 3.8, 4 or any value therebetween; and an enzymatic hydrolysis time of preferably 1h to 2h, specifically 1h, 1.1h, 1.3h, 1.5h, 1.7h, 1.9h, 2h or any value therebetween.

[0041] In the present invention, in step S2, the pyrolysis treatment refers to the process of using high temperature conditions to achieve rapid and uniform cracking of Tremella polysaccharide in the enzymatic hydrolysis product, and the conditions specifically include a temperature of preferably 95°C to 100°C, specifically 95°C, 96°C, 97°C, 98°C, 99°C, 100°C or any value therebetween; a time of preferably 15min to 50min, specifically 15min, 18min, 20min, 23min, 25min, 30min, 35min, 40min, 50min or any value therebetween.

[0042] In the present invention, in step S3, specific examples of the macroporous resin in the macroporous resin adsorption treatment include but are not limited to: one or more of macroporous resin AB-8, macroporous resin HP-20, macroporous resin XAD-4 and macroporous resin XAD-7.

[0043] In the present invention, in step S3, during the macroporous resin adsorption treatment, the addition ratio of the macroporous resin to the pyrolysis product is preferably 1 g:(20-40) mL, specifically 1 g:20 mL, 1 g:23 mL, 1 g:28 mL, 1 g:30 mL, 1 g:35 mL, 1 g:40 mL, or any value therebetween. In this case, the macroporous resin can fully adsorb impurity molecules in the pyrolysis product, thereby achieving a more excellent purification effect.

[0044] In the present invention, in step S3, the macroporous resin adsorption treatment refers to the process of using macroporous resin to adsorb impurity molecules in the pyrolysis product to achieve the purification of Tremella polysaccharide, and the conditions specifically include a temperature of preferably 50°C to 70°C, specifically 50°C, 53°C, 55°C, 58°C, 60°C, 65°C, 70°C or any value therebetween; a stirring speed of preferably 150r / min to 200r / min, specifically 150r / min, 153r / min, 155r / min, 158r / min, 160r / min, 180r / min, 190r / min, 200r / min or any value therebetween; and a time of preferably 1.5h to 3h, specifically 1.5h, 1.7h, 2h, 2.3h, 2.7h, 2.9h, 3h or any value therebetween.

[0045] In the present invention, in step S3, the pore size of the ceramic membrane used in the ceramic membrane filtration and concentration treatment is preferably 80 nm to 120 nm, and specifically can be 80 nm, 83 nm, 85 nm, 90 nm, 95 nm, 100 nm, 110 nm, 120 nm, or any value therebetween. In this case, the Tremella polysaccharide within the molecular weight range of 1000 kDa to 3162 kDa can be better enriched, thereby improving the molecular weight uniformity of the obtained Tremella polysaccharide.

[0046] In the present invention, in step S4, the low-temperature drum drying refers to the process of evaporating water in the Tremella polysaccharide solution by rotating the drum and transferring heat on its surface, and the conditions specifically include: the temperature is preferably not more than 70°C, more preferably 50°C to 60°C, specifically 50°C, 52°C, 54°C, 55°C, 58°C, 60°C or any value therebetween; the rotation speed is preferably 10r / min to 15r / min, specifically 10r / min, 11r / min, 12r / min, 13r / min, 14r / min, 15r / min or any value therebetween; the feed rate is preferably 0.3L / min to 0.7L / min, specifically 0.3L / min, 0.4L / min, 0.5L / min, 0.6L / min, 0.7L / min or any value therebetween; the residence time is preferably 15s to 30s, specifically 15s, 20s, 25s, 30s or any value therebetween.

[0047] In a second aspect, the Tremella polysaccharide provided by the present invention is prepared by the above-mentioned Tremella polysaccharide preparation method.

[0048] In the present invention, the weight average molecular weight of the Tremella polysaccharide is preferably 1000kDa to 3162kDa, and specifically can be 1000kDa, 1150kDa, 1500kDa, 2000kDa, 2150kDa, 2500kDa, 3000kDa, 3162kDa, or any value therebetween. In this case, the Tremella polysaccharide can better cooperate with agar oligosaccharide to exert more excellent biological activities of anti-oxidation, moisturizing, and promoting cell migration.

[0049] In a third aspect, the present invention provides the use of the above-mentioned Tremella polysaccharide in the preparation of a tissue damage repair agent.

[0050] In a fourth aspect, the tissue damage repair agent provided by the present invention specifically includes the above-mentioned Tremella polysaccharide and agar oligosaccharide, and the polymerization degree of the agar oligosaccharide is 2 to 10, specifically 2, 4, 6, 8, 10 or any integer value therebetween.

[0051] In the present invention, the added mass ratio of the Tremella polysaccharide and the agar oligosaccharide is preferably (1-5):1, specifically 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1 or any value therebetween. In this case, the Tremella polysaccharide and the agar oligosaccharide have a better synergistic effect, thereby giving the tissue damage repair agent a better repair effect.

[0052] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0053] Example 1

[0054] This example is used to illustrate a method for preparing Tremella polysaccharide, which specifically includes:

[0055] (1) After cleaning and removing impurities from the Tremella fuciformis, the Tremella fuciformis was placed in an oven for drying, and pulverized by a pulverizer, and passed through an 80-mesh sieve to obtain Tremella fuciformis powder.

[0056] (2) Tremella powder and ultrapure water were mixed at a material-liquid ratio of 1:60 (g:mL), and ultrasonic extraction was performed at room temperature and 200 W for 30 min to obtain a crude extract.

[0057] (3) Cellulase (Xiasheng (Beijing) Biotechnology Development Co., Ltd., product number FDG-2225) was added to the crude extract at an addition amount of 0.5 wt%, and the cellulase was enzymatically hydrolyzed for 1.5 h at a pH of 5.0 and a temperature of 70°C to obtain a primary enzymatic hydrolysis product; pectinase (Xiasheng (Beijing) Biotechnology Development Co., Ltd., product number FDY-2212) was added to the primary enzymatic hydrolysis product at an addition amount of 0.5 wt%, and the pectinase was enzymatically hydrolyzed for 1.5 h at a pH of 3.5 and a temperature of 55°C to obtain a secondary enzymatic hydrolysis product.

[0058] (4) The secondary enzymatic hydrolysis product was subjected to pyrolysis treatment at 90°C and 100 rpm for 20 min. After cooling to room temperature, the product was centrifuged at 8000 rpm for 20 min, and the supernatant was collected to obtain the pyrolysis product.

[0059] (5) Macroporous resin AB-8 (McLean, product number A875381) was added to the pyrolysis product at a ratio of 1:30 (g:mL). The adsorption treatment was carried out at a temperature of 60°C and a stirring speed of 180 r / min for 2 h. The macroporous resin in the polysaccharide solution was removed by filtration to obtain a crude polysaccharide solution.

[0060] (6) The crude polysaccharide solution was filtered and concentrated using a ceramic membrane with a pore size of 100 nm. During the treatment process, 5 times the volume of pure water was slowly and uniformly added to obtain a Tremella polysaccharide solution.

[0061] (7) The tremella polysaccharide solution was dried using a drum dryer at a temperature of 60°C, a feed rate of 0.4 L / min, and a rotation speed of 10 r / min, and the residence time of the liquid was 20 s to obtain tremella polysaccharide powder.

[0062] (8) Tremella polysaccharide powder was added to a 0.1 mol / L sodium nitrate solution (containing 0.01 wt% sodium azide) at a final concentration of 2 mg / mL to obtain a test solution. The test solution was filtered through a 0.22 μm microporous filter membrane and then subjected to liquid chromatography. The molecular weights Mp, Mn, Mw, and Mz of the Tremella polysaccharide powder were calculated based on the measured viscosity and peak time using GPC software. The results are as follows: Figure 1 and 2 shown.

[0063] Depend on Figure 1 and 2 The results shown show that the Tremella polysaccharide prepared by the above method has almost no impurity peaks and high purity. The number average molecular weight of Tremella polysaccharide is 2958 kDa, and 95.88% of the polysaccharide molecules have a molecular weight between 1000 kDa and 3162 kDa.

[0064] Example 2

[0065] This example is used to illustrate the biological activity of the Tremella polysaccharide powder provided in Example 1. The test specifically includes:

[0066] 1. Free radical scavenging effect

[0067] (1) According to the following groups, take 10 μL of the test solution and mix it with 190 μL of 0.1 mM DPPH ethanol solution:

[0068] a. Blank control group: Ultrapure water containing 5% (v / v) glycerol, 3% (v / v) 1,3-propylene glycol, and 0.5% (v / v) phenoxyethanol is the test solution:

[0069] b. Single Tremella polysaccharide group: The Tremella polysaccharide powder provided in Example 1 was added to ultrapure water containing 5% (v / v) glycerol, 3% (v / v) 1,3-propylene glycol, and 0.5% (v / v) phenoxyethanol to prepare test solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL;

[0070] c. Single agar oligosaccharide group: Agar oligosaccharide powder was added to ultrapure water containing 5% (v / v) glycerol, 3% (v / v) 1,3-propylene glycol, and 0.5% (v / v) phenoxyethanol to prepare test solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL;

[0071] d. Composite formula group: The Tremella polysaccharide powder and agar oligosaccharide powder provided in Example 1 (Blue Brain Technology (Xiamen) Co., Ltd., product number AN46, the same below) were mixed in a mass ratio of 3:1 and dissolved in ultrapure water containing 5% (v / v) glycerol, 3% (v / v) 1,3-propylene glycol and 0.5% (v / v) phenoxyethanol to prepare test solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL and 0.2 mg / mL (based on the total mass of Tremella polysaccharide powder and agar oligosaccharide powder).

[0072] (2) The mixed solution was reacted in the dark for 30 minutes, and then the absorbance was measured at 517 nm. The clearance rate (%) was calculated according to the following formula. The results are shown in Table 1.

[0073] Clearance rate = (1-A x / A0)×100%

[0074] Among them, A x is the absorbance measured for the single Tremella polysaccharide group, single agar oligosaccharide group or the compound formula group, and A0 is the absorbance measured for the blank control group.

[0075] Table 1.

[0076]

[0077] From the results shown in Table 1, it can be seen that the Tremella polysaccharide powder prepared by the method provided in Example 1 of the present invention has a certain free radical scavenging effect, and the Tremella polysaccharide cooperates with agar oligosaccharides with a molecular weight of less than 5 kDa to achieve a better free radical scavenging effect.

[0078] 2. Moisturizing effect

[0079] (1) Prepare the test solutions according to the following groups:

[0080] a. Blank control group: ultrapure water is the test solution;

[0081] b single Tremella polysaccharide group: Take the Tremella polysaccharide powder provided in Example 1 and mixed with ultrapure water to prepare a test solution having a concentration of 2 mg / mL;

[0082] c. Single agar oligosaccharide group: Mix agar oligosaccharide with ultrapure water to prepare a test solution with a concentration of 2 mg / mL;

[0083] d. Composite formula group: After mixing Tremella polysaccharide powder and agar oligosaccharide in a mass ratio of 3:1, the mixture was dissolved in ultrapure water to prepare a test solution having a concentration of 2 mg / mL (based on the total mass of Tremella polysaccharide powder and agar oligosaccharide powder);

[0084] e. Positive control group: Hyaluronic acid, glycerol and ultrapure water were mixed separately to prepare a test solution with a concentration of 2 mg / mL.

[0085] (2) According to 2mg / cm 2 The amount of each test solution was evenly applied in a desiccator, and placed under constant temperature and humidity conditions of 25°C and 45% RH for 48 hours. The test solution was weighed at 0, 1, 6, 24, and 48 hours, and the moisturizing rate (%) was calculated according to the following formula. The results are shown in Table 2.

[0086] Moisturizing rate = m x / m0×100%

[0087] Among them, m x is the residual water mass of the sample, and m0 is the water mass of the sample.

[0088] Table 2.

[0089]

[0090] From the results shown in Table 2, it can be seen that the Tremella polysaccharide provided in Example 1 is compounded with agar oligosaccharide, and the two cooperate synergistically to achieve an excellent moisturizing effect, and the moisturizing effect is better than that of hyaluronic acid and glycerol.

[0091] 3. Promote cell migration

[0092] (1) Prepare the test solutions according to the following groups:

[0093] a. Blank control group: DMEM medium (Gibco, catalog number 11965092, hereinafter) containing 1% fetal bovine serum (Gibco, catalog number 10091148, hereinafter the same) is the test solution;

[0094] b single Tremella polysaccharide group: Take the Tremella polysaccharide powder provided in Example 1 and mixed with DMEM medium containing 1% fetal bovine serum to prepare a test solution having a concentration of 0.2 mg / mL;

[0095] c. Single agar oligosaccharide group: agar oligosaccharide was mixed with DMEM medium containing 1% fetal bovine serum to prepare a test solution with a concentration of 0.2 mg / mL;

[0096] d. Composite formula group: Tremella polysaccharide powder and agar oligosaccharide were mixed uniformly in a mass ratio of 3:1, dissolved in DMEM medium containing 1% fetal bovine serum, and a test solution with a concentration of 0.2 mg / mL was prepared (based on the total mass of Tremella polysaccharide powder and agar oligosaccharide powder).

[0097] Among them, agar oligosaccharide is prepared by the method provided in "1. Free radical scavenging effect".

[0098] (2) Healthy Hacat cells in the logarithmic growth phase were taken, the original culture medium was removed, and the cells were washed with sterile PBS. Then, 1 mL of trypsin digestion solution (Gibco, catalog number 25300120) was added to digest the cells. After the cells were observed to be completely wrinkled and rounded under a microscope, DMEM medium containing 1% fetal bovine serum was added to terminate the digestion and obtain a cell suspension.

[0099] (3) The cell suspension was added to a 15 mL centrifuge tube and centrifuged at 800 rpm / min at room temperature for 5 min. After counting the blood cells, the supernatant was discarded and an appropriate amount of DMEM medium containing 10% fetal bovine serum was added to resuspend the cells. The cells were then plated at 2 × 10 5The cells were inoculated into a 12-well culture plate at an inoculum volume of 1 mL / well and cultured at 37°C and 5% CO2 for 24 hours. A 200 μL pipette tip was used to vertically stroke the cell surface from one end of the well to the other, remove the old culture medium, and wash the cell surface three times with sterile PBS. The scratched cells were removed and each test solution was added at a volume of 1 mL / well. The cells were cultured at 37°C and 5% CO2 for 24 hours. Microscope photos were taken at 12 and 24 hours to calculate the cell migration rate. The results are shown in Figure 2. Figures 3-5 shown.

[0100] Depend on Figures 3-5 The results shown show that compared with the administration of Tremella polysaccharide or agar oligosaccharide alone, the combined administration of Tremella polysaccharide and agar oligosaccharide in the composite formula group can synergistically cooperate to significantly enhance the cell migration rate (p<0.01).

[0101] Based on the above experimental results, it can be seen that the method provided in Example 1 of the present invention can be used to prepare high-purity Tremella polysaccharide with a molecular weight uniformly distributed between 1000 kDa and 3162 kDa, and the combination of Tremella polysaccharide and agar oligosaccharide exhibits excellent biological activity in anti-oxidation, moisturizing, and promoting cell migration, showing great application potential in tissue damage repair.

[0102] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for preparing Tremella polysaccharide, characterized in that: The preparation method comprises: S1. Mix Tremella powder with water and perform ultrasonic extraction to obtain a crude extract; S2. Perform cellulase enzymolysis and pectinase enzymolysis on the crude extract to obtain an enzymatic hydrolysis product; perform pyrolysis on the enzymatic hydrolysis product to obtain a pyrolysis product; S3. Perform macroporous resin adsorption and ceramic membrane filtration and concentration on the pyrolysis product to obtain a Tremella polysaccharide solution; S4. Perform low-temperature drum drying on the Tremella polysaccharide solution to obtain the Tremella polysaccharide.

2. The method for preparing Tremella polysaccharide according to claim 1, wherein In step S1, the mixing ratio of the Tremella powder and water is 1 g: (40-80) mL; Optionally, the temperature of the ultrasonic extraction treatment is 10° C. to 40° C., the ultrasonic power is 100W to 400W, and the ultrasonic time is 20min to 50min.

3. The method for preparing Tremella polysaccharide according to claim 1, wherein In step S2, the amount of cellulase added in the cellulase enzymatic hydrolysis treatment is 0.5 wt% to 1.5 wt% based on the mass of the crude extract; Optionally, the temperature of the cellulase enzymatic hydrolysis treatment is 65° C. to 75° C., the pH value is 4.5 to 6.5, and the enzymatic hydrolysis time is 1 h to 2 h; Optionally, the amount of pectinase added in the pectinase enzymatic hydrolysis treatment is 0.5 wt% to 1.5 wt% based on the mass of the crude extract; Optionally, the temperature of the pectinase enzymatic hydrolysis treatment is 50° C. to 60° C., the pH value is 3.3 to 4, and the enzymatic hydrolysis time is 1 h to 2 h; Optionally, the temperature of the pyrolysis treatment is 95° C. to 100° C., and the time is 15 min to 50 min.

4. The method for preparing Tremella polysaccharide according to claim 1, wherein In step S3, the macroporous resin in the macroporous resin adsorption treatment is selected from one or more of macroporous resin AB-8, macroporous resin HP-20, macroporous resin XAD-4 and macroporous resin XAD-7; Optionally, in the macroporous resin adsorption treatment, the addition ratio of the macroporous resin to the pyrolysis product is 1 g: (20-40) mL; Optionally, the temperature of the macroporous resin adsorption treatment is 50° C. to 70° C., the stirring speed is 150 r / min to 200 r / min, and the time is 1.5 h to 3 h; Optionally, the pore size of the ceramic membrane in the ceramic membrane filtration and concentration treatment is 80 nm to 120 nm.

5. The method for preparing Tremella polysaccharide according to claim 1, wherein In step S4, the temperature of the low-temperature drum drying process is not greater than 70°C, the rotation speed is 10 rpm to 15 rpm, the feed rate is 0.3 L / min to 0.7 L / min, and the residence time is 15 s to 30 s.

6. A Tremella polysaccharide, characterized in that The tremella polysaccharide is prepared by the preparation method of tremella polysaccharide according to any one of claims 1 to 5.

7. The Tremella polysaccharide according to claim 6, characterized in that The weight average molecular weight of the Tremella polysaccharide is 1000kDa to 3162kDa.

8. Use of the Tremella polysaccharide according to claim 6 or 7 in the preparation of a tissue damage repair agent.

9. A tissue damage repair agent, characterized in that: The tissue damage repair agent comprises the Tremella polysaccharide and agar oligosaccharide according to claim 6 or 7, and the polymerization degree of the agar oligosaccharide is 2 to 10.

10. The tissue damage repair agent according to claim 9, characterized in that The added mass ratio of the tremella polysaccharide and agar oligosaccharide is (1-5):1.

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