Method for improving tremella polysaccharide extraction yield and application

Abstract

The invention discloses a method for increasing the extraction yield of tremella polysaccharide and application, dry tremella is used as a raw material, tremella polysaccharide is effectively extracted by utilizing an ultrasonic-cellulase wall breaking coupling green solvent method, the extraction yield of tremella polysaccharide is effectively increased, the method is green and environment-friendly and is easy to control, and the obtained tremella polysaccharide has excellent biological activity and can be used for preparing the tremella polysaccharide. The water loss rate of water can be reduced, and the moisturizing and anti-wrinkle capabilities are good; the caenorhabditis elegans can effectively remove hydroxyl free radicals, DPPH free radicals and ABTS free radicals, shows strong oxidation resistance, can significantly prolong the survival rate of caenorhabditis elegans under hydrogen peroxide stress, and shows that the caenorhabditis elegans has good oxidative stress resistance.

Description

(1) Technical Field

[0001] The present invention relates to a method for improving the extraction yield of tremella polysaccharide and its application. (2) Background Art

[0002] Tremella fuciformis is a fungal fruiting body, belonging to the genus Tremella of the family Tremellaceae. Also known as snow ear and white fungus, it is rich in nutritional value and has the functions of nourishing yin and moistening the lungs, and invigorating the spleen and appetizing. According to relevant data, China is the main producer of Tremella in the world. As a natural health food, Tremella has very rich available resources. Research has found that Tremella contains proteins, polysaccharides, dietary fiber, minerals, various amino acids, flavonoids, etc., and is also rich in natural plant colloids, making it a good skin-moistening food that can be taken for a long time. If the nutrients in Tremella are separated and extracted to make various products, it can not only provide more diversified health choices for people, but also improve the comprehensive development and utilization value of Tremella and maximize its value.

[0003] Among them, tremella polysaccharide, as one of the main active components of Tremella, exists in the cell wall and has many pharmacological effects, such as immunomodulation, hypoglycemic and hypolipidemic effects, antioxidant, anti-aging and anti-radiation properties. However, due to the complex composition and structure of its cell wall, the extraction of polysaccharides is very difficult. Moreover, relevant research shows that the biological activity of polysaccharides is closely related to their physicochemical structure and functional characteristics. Different cell wall-breaking extraction methods will have a significant impact on the yield, physicochemical properties, functional properties and biological activity of polysaccharides. Therefore, how to more effectively extract and separate these polysaccharides from the cell wall with a high proportion of polysaccharides without destroying the polysaccharide activity and explore its functions is the main problem to be solved in current research.

[0004] The cell wall of Tremella, as a basidiomycete, has been proven to be mainly composed of chitin, glucan, mannan and glycoprotein, etc., and chitin, glucan and glycoprotein are covalently cross-linked together. The inner wall of Tremella cells is composed of a rigid hydrophobic complex of α-1,3-glucan and chitin, which makes the cell wall of Tremella have high mechanical strength and stability. It is mainly distributed in the soft hydrated matrix of β-(1,3)-glucan. β-(1,3)-glucan and chitin are connected by covalent bonds to form intra-chain hydrogen bonds and can be assembled into fibrous microfibrils, forming a basket-like scaffold around the cells. This exoskeleton represents the load-bearing structural components of the wall, which resists the huge internal hydrostatic pressure exerted by the cytoplasm and membrane on the wall and is rigid and can withstand heat and alkali treatment. This structural feature of the Tremella cell wall makes it difficult to break and polysaccharides are difficult to release.

[0005] At present, there are various methods for extracting tremella polysaccharides at home and abroad, mainly including physical methods, chemical methods, biological enzyme methods, and composite methods for cell wall breaking. Although these methods have different degrees of cell wall breaking ability, they generally have problems such as low extraction efficiency, low purity, and high energy consumption. In addition, some cell wall breaking means are likely to cause changes in the structural characteristics of polysaccharides, thereby reducing their biological activities. (III) Summary of the Invention

[0006] The purpose of the present invention is to provide a method and application for improving the extraction yield of tremella polysaccharides. Using dried tremella as the raw material, the tremella polysaccharides are effectively extracted by ultrasonic-cellulase coupled green solvent method. Based on breaking through the traditional extraction methods, this method comprehensively applies physical, biological, and chemical methods. The ultrasonic cavitation effect is used to destroy the cell wall, significantly improving the destruction efficiency of the cell wall structure. Combining the use of cellulase, the destruction efficiency of the cell wall structure is significantly improved, thereby releasing more polysaccharides. At the same time, the mechanical effect of ultrasound can accelerate the penetration of the green solvent and the diffusion of substances, and can improve the ability of the hydrogen bond donor (glycerol or 1,4-butanediol) in the green solvent to break the hydrogen bond between the polysaccharide and other compounds, making the polysaccharide more easily released, further improving the efficiency of forming a soluble complex between the polysaccharide and the hydrogen bond acceptor (arginine), thereby realizing efficient polysaccharide release; and the amphoteric solution formed by the combination of the hydrogen bond acceptor (arginine) and the hydrogen bond donor (glycerol or 1,4-butanediol) is convenient for polysaccharide separation. The method of the present invention has its unique extraction mechanism and significant advantages, can obtain tremella polysaccharides with high extraction yield and high biological activity, and provides a new way for the exploration of the functions of tremella polysaccharides.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a method for improving the extraction yield of tremella polysaccharides, and the method comprises the following steps:

[0009] (1) Take dried tremella powder, add a green solvent and cellulase, enzymatically hydrolyze in a water bath shaker at 35 - 95 °C and 100 - 150 rpm for 30 - 150 min, then extract under the condition of ultrasonic power of 50 - 200 W for 3 - 15 min, centrifuge (preferably centrifuge at 8000 rpm for 15 min), and collect the supernatant; the green solvent is prepared by using a hydrogen bond donor and a hydrogen bond acceptor as solutes and deionized water as a solvent, wherein the hydrogen bond acceptor is arginine and the hydrogen bond donor is glycerol or 1,4-butanediol;

[0010] (2) Add 95% ethanol (an aqueous solution of ethanol with a volume concentration of 95%) to all the supernatant collected in step (1), let it stand for alcohol precipitation at 4°C for 6 hours, then centrifuge (preferably centrifuge at 4000 rpm for 10 minutes), dissolve the precipitate in deionized water, remove ethanol by rotary evaporation and concentrate its volume to 1 / 3 - 1 / 2 of that before concentration to obtain a concentrated solution; add Sevag reagent to the concentrated solution, mix well, shake to remove protein for 20 - 30 minutes, centrifuge (preferably centrifuge at 8000 rpm for 5 minutes), remove the precipitate, retain the supernatant, and repeat the alcohol precipitation and protein removal 1 - 3 times (preferably 2 times) according to the above method, collect the supernatant, remove the organic solvent by rotary evaporation, concentrate to 1 / 5 of the original volume under rotary evaporation at 65 - 80°C, and freeze-dry (initial temperature is -30°C, vacuum degree is 80 Pa) until the mass water content is 8% to obtain tremella polysaccharide.

[0011] Further, the solute in the green solvent in step (1) is one of the following: a composition of arginine and glycerol with a molar ratio of 1:2 or 1:4, or a composition of arginine and 1,4-butanediol with a molar ratio of 1:2 or 1:4.

[0012] Further, the green solvent in step (1) is prepared as follows: Shake the solute in a water bath shaker at 60 - 80°C at a rotation speed of 100 - 150 rpm (preferably 125 rpm) for 1 - 2 hours until the solution is clear, and then add deionized water to prepare it.

[0013] Further, the volume water content of the green solvent in step (1) is 60 - 90%, preferably 80%.

[0014] Further, the volume addition amount of the green solvent in step (1) is 60 - 140 mL / g based on the mass of dry tremella powder (preferably 60 mL / g); the mass addition amount of the cellulase is 0.4 - 2.4% based on the mass of dry tremella powder (preferably 0.4%). The activity of the cellulase is preferably 10000 U / g.

[0015] Further, the dry tremella powder in step (1) is obtained by placing the dried tremella in a grinding machine for pulverization and passing through a 100-mesh sieve to obtain tremella powder.

[0016] Further, in step (1), enzymatic hydrolysis is carried out at 95°C and 125 rpm for 30 minutes; the ultrasonic power is 200 W and the extraction is carried out for 9 minutes.

[0017] Further, in step (2), the volume ratio of the added 95% ethanol to the volume of the supernatant is 4:1. The Sevag reagent is chloroform-n-butanol with a volume ratio of 4:1, and the addition amount of the Sevag reagent to the volume of the concentrated solution is 1:4.

[0018] In the second aspect, the present invention provides a tremella polysaccharide prepared by the above method.

[0019] In a third aspect, the present invention provides an application of the tremella polysaccharide in the preparation of a moisturizing product.

[0020] In a fourth aspect, the present invention provides an application of the tremella polysaccharide in the preparation of an anti-wrinkle product.

[0021] In a fifth aspect, the present invention provides an application of the tremella polysaccharide in the preparation of an antioxidant, and the antioxidant can scavenge hydroxyl radicals, DPPH radicals or ABTS radicals.

[0022] In a sixth aspect, the present invention provides an application of the tremella polysaccharide in the preparation of an antioxidant stress preparation. The antioxidant stress preparation includes a preparation that can significantly prolong the survival rate of Caenorhabditis elegans under hydrogen peroxide stress.

[0023] The technical key point of extracting tremella polysaccharide by ultrasonic-cellulase wall-breaking coupling with a green solvent using dry tremella powder as the raw material is that under mild conditions, the cavitation effect of ultrasound generates a large number of bubbles, and when the bubbles burst, local high temperature, high pressure and strong shear force are caused, which destroy the cell wall and cell membrane, accelerate the dissolution of polysaccharides, and improve the degradation activity of cellulase on cellulose; at the same time, mechanical vibration accelerates the penetration of the solvent and the diffusion of substances, reduces the dosage of the solvent and the enzyme, and further improves the extraction rate of polysaccharides. The green solvent is clean, environmentally friendly, pollution-free, low in price, makes it easy to separate tremella polysaccharide, reduces energy consumption and environmental pollution.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] 1. The method of the present invention effectively improves the extraction rate of tremella polysaccharide.

[0026] Different extraction processes have a great influence on the extraction rate of tremella polysaccharide. The present invention uses ultrasonic-cellulase wall-breaking coupling with a green solvent method to extract tremella polysaccharide. The process is green and environmentally friendly, simple and easy to operate, and the extraction rate of polysaccharide is as high as over 47%. It is 5.153 times higher than that of tremella polysaccharide extracted by traditional hot water extraction, 3.107 times and 3.688 times higher than that by enzymatic hydrolysis or ultrasonic-assisted extraction method respectively, and 1.937 times higher than that by ultrasonic-assisted enzymatic method.

[0027] 2. The method of the present invention is green and environmentally friendly and easy to control.

[0028] Regarding the characteristics that conventional crushing methods often have poor effects and low efficiency on fungal cell walls, the present invention utilizes an ultrasonic-cellulase wall-breaking coupled green solvent method. Through the local high temperature and strong shear force generated by the bubble rupture during ultrasonic cavitation effect and the mechanical effect of ultrasonic waves, the cell walls and cell membranes of Tremella fuciformis are damaged, reducing energy consumption. At the same time, ultrasonic waves accelerate mass transfer and reaction rate, significantly shortening the extraction time. Combining with cellulase, it can specifically degrade the glycosidic bonds of cell walls and break cell walls, further improving the dissolution of polysaccharides. The coupled green solvent replaces toxic and harmful chemical reagents. By stabilizing the polysaccharide structure through hydrogen bonds in the green solvent, it can also inhibit protein denaturation, reduce its binding to polysaccharides, and obtain polysaccharides with higher purity. Compared with traditional extraction methods, it avoids the use of strong acids, strong bases and toxic solvents, reduces environmental pollution and solvent residues, and meets various standards. At the same time, compared with traditional high-temperature and high-pressure extraction, this method avoids the breakage of polysaccharide chains and protects the activity of polysaccharides. At the same time, the process parameters (ultrasonic power, enzyme concentration, solvent ratio) of this method are easy to optimize and control, suitable for industrial scale-up production, and have strong process scalability.

[0029] 3. The Tremella fuciformis polysaccharide obtained by the present invention has excellent biological activities.

[0030] The Tremella fuciformis polysaccharide prepared by the method of the present invention can reduce the water loss rate and effectively inhibit the contraction of the tail tissue of zebrafish under water shortage and ultraviolet irradiation, having good moisturizing and anti-wrinkle abilities;

[0031] The Tremella fuciformis polysaccharide prepared by the method of the present invention can effectively scavenge hydroxyl radicals, DPPH radicals and ABTS radicals, showing strong antioxidant ability. At the same time, the scavenging rates of DPPH radicals, hydroxyl radicals and ABTS radicals are 1.932 times, 2.108 times and 0.691 times that of traditional hot water extraction respectively.

[0032] The Tremella fuciformis polysaccharide prepared by the method of the present invention can significantly prolong the survival rate of Caenorhabditis elegans under hydrogen peroxide stress, indicating its good antioxidant stress ability. (IV) Description of the Drawings

[0033] Figure 1 Glucose standard curve.

[0034] Figure 2 Column chart of the influence of the ratio and dosage of green solvent on the extraction yield (%) of Tremella fuciformis polysaccharide.

[0035] Figure 3 Column chart of the influence of different single factors (water bath time, solid-liquid ratio, temperature, cellulase addition amount, water content of green solvent, wall-breaking time and wall-breaking power) on the extraction yield (%) of Tremella fuciformis polysaccharide.

[0036] Figure 4, Column chart of water loss rate (%) of DTFP and WTFP at different concentrations.

[0037] Figure 5 , Curve chart of the effect of DTFP and WTFP on the scavenging rate (%) of DPPH free radicals.

[0038] Figure 6 , Curve chart of the effect of DTFP and WTFP on the scavenging rate (%) of hydroxyl free radicals.

[0039] Figure 7 , Curve chart of the effect of DTFP and WTFP on the scavenging rate (%) of ABTS free radicals.

[0040] Figure 8 , Survival curve of the effect of DTFP and WTFP on the lifespan of C.elegans under oxidative stress.

[0041] Figure 9 , Column chart of the effect of DTFP and WTFP on the average lifespan of C.elegans under oxidative stress.

[0042] Figure 10 , Tail photos of zebrafish under water shortage; a, b, c, d, e represent normal control, model control, positive control, WTFP and DTFP respectively.

[0043] Figure 11 , Column chart of the inhibition rate (%) of DTFP and WTFP on the caudal fin shrinkage of zebrafish under water shortage.

[0044] Figure 12 , Tail photos of zebrafish under ultraviolet irradiation; a, b, c, d, e represent normal control, model control, positive control, WTFP and DTFP respectively.

[0045] Figure 13 , Column chart of the inhibition rate of DTFP and WTFP on the caudal fin shrinkage of zebrafish under ultraviolet irradiation. (V) Specific implementation manners

[0046] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0047] Example 1, Screening of extraction conditions for tremella polysaccharide

[0048] 1 Materials and reagents

[0049] Small tremella fuciformis on basswood from Gutian, Fujian (Shenzhen Shengbaoge Food Co., Ltd.), without pests, diseases and rot. The dried small tremella fuciformis on basswood was crushed with a pulverizer and passed through a 100-mesh sieve to obtain dry tremella powder, which was sealed and stored in a dry and low-temperature environment.

[0050] Arginine, 1,4 - butanediol, glycerol, ABTS (2,2 - azino - bis(3 - ethylbenzothiazoline - 6 - sulfonic acid) diammonium salt), DPPH (2,2 - diphenyl - 1 - picrylhydrazyl), potassium persulfate, ferrous chloride, salicylic acid, H2O2, 95% ethanol, absolute ethanol, etc. are all of analytical grade; cellulase (activity 10000U / g) and tricaine (3 - ethoxycarbonylaniline methanesulfonate, ≥98%) are purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; hyaluronic acid (H823435 hyaluronic acid) is purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; wild AB - type zebrafish eggs fertilized for two days are purchased from the experimental zebrafish scientific research service platform of Jingke Instruments Business Department in Jiang'an District, Wuhan City.

[0051] 2 Experimental Instruments

[0052] Small - scale high - speed crusher (model WK - 400A, Beijing Guoweihejiao Medical Equipment Co., Ltd.), AL04 type electronic balance, SHA - B water bath shaker, DHG - 9146A electrothermal constant temperature forced - air drying oven, CT14R0 high - speed centrifuge VS - 840K - U, clean bench, intelligent biochemical incubator, stirrer, microplate reader, UV1800PC ultraviolet - visible spectrophotometer, DS - 5510DTH ultrasonic cleaner.

[0053] 3 Experimental Methods

[0054] 3.1 Establishment of Quantitative Analysis Method for Tremella Polysaccharide

[0055] The total sugar content is determined by the phenol - sulfuric acid method (refer to national standard GB / T 15672 - 2009), and the extraction yield of its polysaccharide is calculated. The specific steps are as follows:

[0056] (1) Drawing of glucose standard curve: Respectively pipette 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL of glucose standard aqueous solution (100mg / L) into 5mL EP tubes, and make up each tube to 0.5mL with deionized water. At the same time, use 0.5mL of deionized water as the blank control, with two controls in each group. Then add 0.5mL of 5% phenol aqueous solution by volume and 2.5mL of 98% sulfuric acid by mass concentration. After vortex - mixing evenly, place it in a 37°C water bath for 30min. Take an appropriate amount of the reaction solution and measure the absorbance value at 490nm. The curve equation is obtained by calculating the regression equation.

[0057] (2) Calculation method for the extraction yield of Tremella polysaccharide

[0058] The calculation formula for the extraction yield of Tremella polysaccharide is as follows:

[0059] Extraction yield of Tremella polysaccharide (mg / 100g) = C × V × F / M × 10

[0060] C is the concentration of tremella polysaccharide in the extract calculated according to the glucose standard curve (μg / mL); V represents the volume of the extract (mL); F represents the dilution factor; M represents the mass of dry tremella powder (g).

[0061] 3.2 Selection of green solvent in the method of extracting tremella polysaccharide by ultrasonic - cellulase wall - breaking coupling with green solvent

[0062] The green solvent is prepared with a hydrogen - bond donor and a hydrogen - bond acceptor as solutes and deionized water as the solvent to form a solvent with a volume water content of 80%. Among them, arginine is selected as the hydrogen - bond acceptor, and glycerol or 1,4 - butanediol is selected as the hydrogen - bond donor. The solutes in the green solvent are combinations of one of the following molar ratios: arginine: glycerol = 1:2, arginine: 1,4 - butanediol = 1:2, arginine: glycerol = 1:4, arginine: 1,4 - butanediol = 1:4.

[0063] Preparation of the green solvent: Shake the solutes in a water - bath shaker at 60 - 80°C at a speed of 125 rpm for 1 - 2 h until the solution is clear, and then add deionized water to adjust to a green solvent with a volume water content of 80%.

[0064] Weigh 1 g of dry tremella powder, initially add the above - mentioned green solvent and 0.02 g of cellulase according to the solid - liquid ratio (1:60, 1:100, 1:140, g / mL), shake in a water - bath shaker at 35°C and 125 rpm for 120 min, and then centrifuge at 8000 rpm for 15 min. Take the supernatant (i.e., the extract) to measure its volume, calculate the polysaccharide extraction rate by the method in step 3.1, and select the best green solvent.

[0065] 3.3 Process optimization for extracting tremella polysaccharide by ultrasonic - cellulase wall - breaking coupling with green solvent

[0066] Carry out the following optimization experiments with the green solvent prepared with arginine: glycerol with a molar ratio of 1:4 selected in step 3.2 as the solute.

[0067] A Water content of the green solvent

[0068] Weigh 1 g of dry tremella powder into a conical flask, add 60 mL of green solvent with different water contents (60%, 70%, 80%, 90%, 100%), add 0.02 g of cellulase, enzymatically hydrolyze in a water - bath shaker at 35°C and 125 rpm for 120 min, then break the wall in an ultrasonic wall - breaker for 10 min, the ultrasonic power is 100 W, the mode is selected as the pulse mode, centrifuge at 8000 rpm for 15 min, take the supernatant (i.e., the extract), and calculate the extraction rate of tremella polysaccharide as the screening index.

[0069] B Solid - liquid ratio

[0070] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the volume of the green solvent was changed to 40 mL, 60 mL, 80 mL, 100 mL, 120 mL, and 140 mL, with other operations being the same.

[0071] C Cellulase content

[0072] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the amount of cellulase added was changed to 0.004, 0.008, 0.012, 0.016, 0.02, and 0.024 g, with other operations being the same.

[0073] D Water bath temperature

[0074] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the water bath temperature was changed to 35 °C, 50 °C, 65 °C, 80 °C, and 95 °C, with other operations being the same.

[0075] E Water bath time

[0076] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the water bath time was changed to 30 min, 60 min, 90 min, 120 min, and 150 min, with other operations being the same.

[0077] F Cell wall breaking time

[0078] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the cell wall breaking time was changed to 3 min, 6 min, 9 min, 12 min, and 15 min, with other operations being the same.

[0079] G Ultrasonic power

[0080] On the basis of A, the volume water content of the green solvent was selected to be 80%, and the ultrasonic power was changed to 50 W, 75 W, 100 W, 125 W, 150 W, 175 W, and 200 W, with other operations being the same.

[0081] 3.4 Purification of tremella polysaccharide

[0082] 3.4.1 Extraction of tremella polysaccharide by ultrasonic-cellulase coupled with green solvent for cell wall breaking

[0083] Weigh 1 g of dried tremella powder into a conical flask, add 60 mL of green solvent with a volume water content of 80% (the solute is arginine: glycerol with a molar ratio of 1:4), add 0.004 g of cellulase, enzymatically hydrolyze in a water bath shaker at 95 °C and 125 rpm for 120 min, then break the cell wall in an ultrasonic cell disruptor for 9 min, with an ultrasonic power of 200 W, the mode selected as pulse mode, centrifuge at 8000 rpm for 15 min, and collect the supernatant.

[0084] All the collected supernatants were added with 95% ethanol at a volume ratio of 1:4, and after standing for 6 hours at 4°C for alcohol precipitation, the precipitate was dissolved in deionized water, and the ethanol was removed by rotary evaporation and the volume was concentrated to 1 / 3 of the original volume to obtain a concentrated solution. 1 / 4 volume of Sevag reagent (chloroform-n-butanol, 4:1, V / V) was added to the concentrated solution, and after sufficient mixing, the protein was removed by oscillation for 20-30 minutes, centrifuged at 8000rpm for 5 minutes, the precipitate was removed, the supernatant was retained, and the alcohol precipitation and protein removal were repeated 2 times according to the above method. The supernatant was collected, the organic solvent was removed by rotary evaporation, and the volume was concentrated to 1 / 5 of the original volume by rotary evaporation, which was recorded as green extracted Tremella polysaccharide (DTFP for short), and the polysaccharide content was 12.28 mg / mL.

[0085] 3.4.2 Tremella polysaccharides extracted by hot water

[0086] The green solvent in the above 3.4.1 was replaced with deionized water, and the remaining steps were the same to obtain Tremella polysaccharide, which was recorded as water-extracted Tremella polysaccharide (abbreviated as WTFP), and the polysaccharide content was 4.84 mg / mL.

[0087] 3.5 In vitro moisturizing ability test of Tremella polysaccharide

[0088] The Tremella polysaccharides extracted by different methods in step 3.4 were used to prepare polysaccharide solutions of different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL) with deionized water.

[0089] Take 1mL of polysaccharide solution and add it to 1.5mL EP tube. Expose the EP tube to a desiccator (water-absorbing silica gel) with an ambient humidity of 43%, dehydrate it at 25℃ for 12h, and measure its mass change within 12h. Glycerol and water with a volume ratio of 1:3 were used as positive controls, and deionized water was used as blank controls to determine the water loss rate of the samples.

[0090] Water loss rate calculation formula: (M2 / M1)×100%

[0091] Where M1 represents the mass of the sample before placement (g), and M2 represents the mass of the lost water (g).

[0092] 3.6 Detection of DPPH free radical scavenging rate of Tremella polysaccharide

[0093] Sample solution: The Tremella polysaccharides extracted by different methods in step 3.4 were prepared with deionized water to prepare polysaccharide solutions of different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL).

[0094] DPPH solution: Prepare 0.1 mM DPPH solution in anhydrous ethanol.

[0095] Vc solution: Prepare Vc solutions with different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) using deionized water, and store them in the dark.

[0096] Add reagents according to the following grouping conditions and mix well. Place them in the dark at room temperature for 30 min, and measure the absorbance at 517 nm. Set 3 parallels for each group. Specifically as follows:

[0097] Sample group: 2 mL of sample solution + 2 mL of DPPH solution, and record the absorbance value as A1;

[0098] Positive group: 2 mL of Vc solution + 2 mL of DPPH solution, and record the absorbance value as A2;

[0099] Control group: 2 mL of sample solution + 2 mL of absolute ethanol, and record the absorbance value as A3;

[0100] Blank group: 2 mL of H2O + 2 mL of DPPH solution, and record the absorbance value as A4.

[0101] The DPPH scavenging rate is calculated according to the formula:

[0102] Scavenging rate of the sample group (%) = [1 - (A1 - A3) / A4] × 100%

[0103] Scavenging rate of the positive group (%) = [1 - (A2 - A3) / A4] × 100%

[0104] 3.7 Detection of hydroxyl radical scavenging rate of tremella polysaccharide

[0105] Sample solution: Prepare polysaccharide solutions with different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) of tremella polysaccharide obtained by different extraction methods in step 3.4 using deionized water.

[0106] Salicylic acid solution: Prepare a 1.8 mmol / L salicylic acid solution using absolute ethanol.

[0107] FeSO4 solution: Prepare a 1.8 mmol / L ferrous sulfate solution using deionized water.

[0108] Vc solution: Prepare Vc solutions with different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) using deionized water, and store them in the dark.

[0109] Add reagents according to the following grouping conditions and mix well. Place them in a water bath at 37°C in the dark for 30 min, and measure the absorbance at 510 nm. Set 3 parallels for each group. Specifically as follows:

[0110] Sample group: 1 mL of sample solution + 2 mL of FeSO4 solution + 1.5 mL of salicylic acid solution + 0.1 mL of 0.03% H2O2, and the absorbance value is denoted as A1;

[0111] Positive group: 1 mL of Vc solution + 2 mL of FeSO4 solution + 1.5 mL of salicylic acid solution + 0.1 mL of 0.03% H2O2, and the absorbance value is denoted as A2;

[0112] Control group: 1 mL of sample solution + 2 mL of FeSO4 solution + 1.5 mL of salicylic acid solution + 0.1 mL of H2O, and the absorbance value is denoted as A3;

[0113] Blank group: 1 mL of H2O + 2 mL of FeSO4 solution + 1.5 mL of salicylic acid solution + 0.1 mL of 0.03% H2O2, and the absorbance value is denoted as A4.

[0114] The hydroxyl radical scavenging rate is calculated according to the formula:

[0115] Scavenging rate of sample group (%) = [1 - (A1 - A3) / A4] × 100%

[0116] Scavenging rate of positive group (%) = [1 - (A2 - A3) / A4] × 100%

[0117] 3.8 Detection of ABTS radical scavenging ability of tremella polysaccharide

[0118] Sample solution: Prepare polysaccharide solutions with different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) of tremella polysaccharide obtained by different extraction methods in step 3.4 with deionized water.

[0119] Potassium persulfate solution: Prepare a 2.6 mmol / L potassium persulfate solution with deionized water.

[0120] ABTS working solution: Prepare a 7.4 mmol / L ABTS solution with deionized water, mix it with pH 7.4 phosphate buffer solution at a volume ratio of 1:1, and store it at room temperature in the dark for 12 hours to obtain the ABTS working solution.

[0121] Vc solution: Prepare Vc solutions with different concentrations (4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) of Vc with deionized water and store them in the dark.

[0122] Add reagents according to the following grouping conditions and mix well. Let it stand for 6 min under dark conditions, and measure the absorbance at 734 nm. Set 3 parallels for each group. Specifically as follows:

[0123] Sample group: 0.2 mL of sample solution + 0.8 mL of ABTS working solution, and the absorbance value is recorded as A1;

[0124] Positive group: 0.2 mL of Vc solution + 0.8 mL of ABTS working solution, and the absorbance value is recorded as A2;

[0125] Blank group: 0.2 mL of deionized water + 0.8 mL of ABTS working solution, and the absorbance value is recorded as A3.

[0126] The ABTS free radical scavenging rate is calculated according to the formula:

[0127] Scavenging rate of the sample group (%) = [(A3 - A1) / A3] × 100%

[0128] Scavenging rate of the positive group (%) = [(A2 - A1) / A3] × 100%

[0129] 3.9 Detection of the effect of Tremella polysaccharide on the lifespan of C. elegans under oxidative stress

[0130] M9 buffer: Accurately weigh 15.0 g of Na2HPO4·12H2O, 3.0 g of KH2PO4, 5.0 g of NaCl, and 0.12 g of MgSO4, dissolve with 800 mL of water and then make up to 1000 mL, mix well, sterilize at 121 °C under high-pressure steam for 30 min to obtain.

[0131] Polysaccharide solution: Prepare solutions with different concentrations (2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) of Tremella polysaccharide obtained by different extraction methods in step 3.4 with M9 buffer, filter and sterilize through a 0.22 μm filter membrane, and store at -20 °C.

[0132] Vc solution: Prepare Vc with a concentration of 0.5 mg / mL with deionized water, filter and sterilize through a 0.22 μm filter membrane, and store at -20 °C;

[0133] Composition of NGM medium: 2.5 g of peptone, 3 g of NaCl, 17 g of agar, 25 mL of 1 mol / L PBS buffer (pH 6.0), 975 mL of deionized water, add 1 mL of 5 mg / mL cholesterol solution (solvent is ethanol) sterilized through a 0.22 μm filter membrane, 1 mL of 1 mol / L MgSO4 aqueous solution, and 1 mL of 1 mol / L CaCl2 aqueous solution after sterilization.

[0134] LB liquid medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, adjust to neutral with 5 M NaOH aqueous solution, and the solvent is water.

[0135] Lysis solution: 0.5 mL deionized water, 0.3 mL NaClO, 0.2 mL 10 mol / L NaOH aqueous solution, to be prepared and used immediately.

[0136] C. elegans culture: Inoculate 100 μL of the glycerol stock solution of E. coli OP50 into 100 mL of LB liquid medium, and culture it in a shaker at 37 °C for 12 h. When the medium becomes turbid, take it out and store it in a refrigerator at 4 °C for later use. Pipette 50 μL of the E. coli OP50 bacterial solution onto the NGM medium plate. After it dries, inoculate the wild-type N2 strain of Caenorhabditis elegans (C. elegans) onto the plate in pieces. After culturing at a constant temperature of 20 °C for 72 h, a large number of adult worms and larvae can be seen on the medium.

[0137] C. elegans synchronization: Select a plate with well-growing C. elegans, pipette 1.5 mL of M9 buffer onto the plate, and wash the plate several times until most of the worms are washed off. Pipette 1 mL of the C. elegans suspension into a 1.5 mL EP tube, centrifuge at 3000 rpm for 1 min, discard the supernatant, and resuspend the precipitate with 0.25 mL of deionized water. Then add 0.15 mL of the lysis solution, vortex for 5 min, centrifuge at 4000 rpm for 1 min, discard the supernatant, add 1 mL of M9 buffer to resuspend, vortex and wash, centrifuge at 4000 rpm for 1 min, and repeat the washing until there is no smell of sodium hypochlorite. Place the lysed eggs on an NGM medium plate not coated with E. coli OP50, and culture them in an incubator at 20 °C for 12 - 18 h to obtain L1-stage C. elegans. Wash the worms with M9 buffer, place them on an NGM medium plate coated with E. coli OP50, and culture them in an incubator at 20 °C for 48 h to obtain synchronized L4-stage C. elegans for subsequent experiments.

[0138] C. elegans lifespan experiment: Spread 150 μL of the E. coli OP50 bacterial solution (OD600 = 0.8) on the NGM medium and let it dry for later use. Divide it into an experimental group, a positive control group, and a blank control group, and spread 200 μL of the above-mentioned polysaccharide solution, Vc solution, and M9 buffer respectively. Inoculate about 150 L4-stage C. elegans synchronized and cultured above on each plate and culture at 20 °C for 48 h. Then prepare a 96-well plate, add 100 μL of M9 buffer and 200 μL of 1 mmol / L hydrogen peroxide aqueous solution to each well. Use an inoculation loop to pick up the C. elegans from different groups and transfer them to the 96-well plate. Set 3 replicates for each group and culture at 20 °C. Observe the number of surviving C. elegans in each group with a magnifying glass every 40 min.

[0139] 3.10 Detection of the moisturizing function evaluation of Tremella polysaccharide on zebrafish under water-deficient conditions

[0140] Wild AB strain zebrafish that were developmentally normal and 2 days post-fertilization were used as experimental animals. Normal zebrafish embryos were selected under a stereomicroscope. When the fertilized eggs developed to 2 days, they were randomly divided into a blank control group, an NaCl model control group, a positive drug hyaluronic acid (HA) control group, and a sample group, with 10 zebrafish in each group. The zebrafish embryos were transferred to a 96-well cell culture plate, 1 fish per well. And three replicate groups were established. Except for the blank control group which was added with zebrafish culture water, the remaining groups were added with NaCl at a final concentration of 1.2 g / L to construct a skin water deficiency model. The HA group was added with 450 μg / mL of HA, and the sample group was added with two polysaccharides obtained by different extraction methods in step 3.4 at a concentration of 50 μg / mL. After culturing the zebrafish in each group in a constant temperature incubator at 28 °C for 24 h, they were observed and photographed under a microscope. The tail fin area of the zebrafish was calculated using Image-Pro Plus (NIH, Bethesda, MA, USA).

[0141] Anti-shrinkage rate of tail area (%) = (sample group - model group) / (blank control group - model group) × 100%

[0142] 3.11 Detection of the anti-wrinkle function evaluation of Tremella polysaccharide on zebrafish

[0143] Wild AB strain zebrafish that were developmentally normal and 2 days post-fertilization were used as experimental animals. Normal zebrafish embryos were selected under a stereomicroscope. When the fertilized eggs developed to 2 days, they were randomly divided into a blank control group, a model control group, a positive drug vitamin E (V E ) control group, and a sample group, with 8 zebrafish in each group added to a 6-well plate. The solutions were prepared according to Table 1, 3 mL of sample was added to each well. After drug administration, the 6-well plate was placed in an incubator at 28 °C for dark incubation for 2 h. The experimental groups were irradiated 3 times under ultraviolet light, with each irradiation time of 15 min and an interval of 30 min, (Minglang Lighting JT5-8F power: 8 W, power factor ≥ 0.65). Then, they were incubated in the incubator in the dark for 22 h. After 22 h, the 6-well plate was taken out, the fish were anesthetized with tricaine, then placed on a slide loaded with 2% methyl cellulose by body position, and then the tail fin photos of each fish were taken with a stereomicroscope, and the tail fin area of each fish was measured using software Image.

[0144] Table 1 Anti-wrinkle experiment of zebrafish in each group

[0145]

[0146]

[0147] Formula for the inhibition rate of zebrafish tail fin shrinkage:

[0148] Q = [(A1 - A2) / (A3 - A2)] × 100%

[0149] Wherein, Q is the inhibition rate of zebrafish caudal fin shrinkage of the sample group compared with the model control group, and A1, A2, and A3 are the average values of the zebrafish caudal fin areas of the sample group, the model control group, and the blank group, respectively.

[0150] 4 Experimental Results

[0151] 4.1 Quantitative Analysis Method of Tremella Polysaccharide

[0152] The glucose standard curve plotted with glucose concentration as the abscissa and absorbance value as the ordinate is shown in Figure 1 as follows. Its linear regression equation is y = 10.085x - 0.0293, and R 2 = 0.9743.

[0153] 4.2 Effects of Different Green Solvents on the Extraction Yield of Tremella Polysaccharide

[0154] Arginine was selected as the hydrogen bond acceptor and mixed with different hydrogen bond donors (glycerol or 1,4-butanediol) at specified molar ratios (arginine:glycerol = 1:2, arginine:1,4-butanediol = 1:2, arginine:glycerol = 1:4, arginine:1,4-butanediol = 1:4) to prepare different green solvents, and Tremella polysaccharide was extracted respectively. The effects of different extractants on the extraction yield of Tremella polysaccharide at different liquid-to-solid ratios are shown in Figure 2 .

[0155] It can be seen from Figure 2 that there is no obvious difference among different extractants when the liquid-to-solid ratio is 1:60 and 1:140. When selecting arginine and glycerol with a molar ratio of 1:4 as solutes to prepare a green solvent with a volume water content of 80% and a liquid-to-solid ratio of 1:100, there is a difference, and the extraction rate of Tremella polysaccharide is the highest. Therefore, the following ultrasonic-cellulase wall-breaking coupled green solvent extraction experiment of Tremella polysaccharide selects this green solvent for extraction process optimization.

[0156] 4.3 Single-Factor Experiments on the Extraction of Tremella Polysaccharide by Ultrasonic-Cellulase Wall-Breaking Coupled Green Solvent Method

[0157] The effects of water bath enzymolysis time, cellulase addition amount, water content of green solvent, liquid-to-solid ratio, extraction temperature, wall-breaking time and ultrasonic power on the extraction yield of Tremella polysaccharide are shown in Figure 3 .

[0158] It can be seen from Figure 3It can be seen that in the single-factor experiment, among the three factors of water bath time, solid-liquid ratio, and enzyme addition amount, within the selected 5-level range, there were no statistical differences in the extraction yields of Tremella polysaccharides at each level of each factor. For the three factors of extraction temperature, cell wall breaking time, and ultrasonic power, as each level increased to the maximum, the extraction yield of Tremella polysaccharides increased significantly without an inflection point. Among the 7 factors, only the water content of the green solvent had an inflection point on the extraction yield of Tremella polysaccharides. According to the principle of central composite experiment, response surface optimization experiments could not be carried out. Therefore, the optimized technological parameters for extracting Tremella polysaccharides by ultrasonic-cellulase cell wall breaking coupling with green solvent method were as follows: the water content of the green solvent was 80%, the solid-liquid ratio was 1 g:60 mL, the cellulase accounted for 0.4% of the mass of dry Tremella powder, the water bath temperature was 95 °C, the water bath time was 30 min, the cell wall breaking time was 9 min, and the ultrasonic power was 200 W. The following experiments were carried out by extracting Tremella polysaccharides with these optimized parameters.

[0159] 4.4 In vitro moisturizing ability of Tremella polysaccharides

[0160] The water losses of the Tremella polysaccharides DTFP and WTFP solutions extracted by the two different methods in 3.4 at different concentrations (0.25 mg / mL - 4 mg / mL) are shown in Table 2, and the water loss rates are shown in Figure 4 . It was found that except for 1 mg / mL WTFP, within the first 4 h, at different concentrations, the water loss rates of the two Tremella polysaccharides were less than those of the deionized water of the blank control and the glycerol water of the positive control, and there were significant differences, showing good in vitro moisturizing ability and no dependence on concentration (such as Figure 4 ). The moisturizing ability of 1 mg / mL WTFP was poor, and 0.25 mg / mL DTFP had the best long-lasting moisturizing ability and could maintain moisture for 6 h.

[0161] Table 2 Water losses (g) of two Tremella polysaccharide solutions at different concentrations

[0162]

[0163] 4.5 Scavenging rate of DPPH free radicals by Tremella polysaccharides

[0164] The full name of DPPH free radical is 2,2-diphenyl-1-picrylhydrazyl radical (2,2-Diphenyl-1-picrylhydrazyl Radical). Due to its stable free radical characteristics, characteristic absorption peak, and specific reaction with antioxidant substances, the DPPH free radical experiment can effectively evaluate the antioxidant ability of samples and is widely used as a standard free radical model to measure the antioxidant activities of natural products, foods, drugs, etc. The results of detecting the scavenging rates of DPPH free radicals by two Tremella polysaccharides at different concentrations in 3.6 are shown in Figure 5 .

[0165] It can be seen from Figure 5 that when the concentration of Tremella polysaccharide (DTFP) extracted by green extraction is 0.25 mg / mL, the DPPH free radical scavenging rate is 0.54%. With the continuous increase of the concentration, the DPPH free radical scavenging rate also continuously increases. When the concentration is 4 mg / mL, the scavenging rate reaches 91.43%, approaching the scavenging rate of Vc in the positive group. The polysaccharide of Tremella fuciformis Berk. extracted by water extraction (WTFP) also has scavenging ability. With the increase of the concentration, the scavenging rate shows an overall upward trend, but it is not as good as DTFP. It shows that the DPPH free radical scavenging rate of Tremella polysaccharide has a concentration-dependent relationship, and the antioxidant activity of DTFP is better than that of WTFP.

[0166] 4.6 Scavenging rate of Tremella polysaccharide on hydroxyl radicals

[0167] In living organisms, hydroxyl radicals are one of the main sources of oxidative damage. By scavenging ·OH, the damage of oxidative stress to cells can be effectively reduced. Through the Fenton reaction (Fe 2+ + H2O2 → Fe 3+ + ·OH + OH - ), ·OH can be generated stably and controllably, providing a reliable reaction system for the experiment. By the reaction of salicylic acid with ·OH to generate colored substances, the concentration change of ·OH can be quantitatively determined, and thus the scavenging rate of the sample can be calculated. The ·OH generated by the Fenton reaction is similar to the free radicals produced in living organisms, so the experimental results can indirectly reflect the antioxidant potential of the sample in living organisms. The results of detecting the scavenging rate of hydroxyl radicals of two kinds of Tremella polysaccharides with different concentrations by 3.7 are as Figure 6 .

[0168] Figure 6 It can be seen that when the concentration of DTFP is 0.25 mg / mL, its hydroxyl radical scavenging rate is 71.15%. When the concentration gradually increases, its hydroxyl radical scavenging rate also gradually increases, reaching 100% complete scavenging. The hydroxyl radical scavenging rate of WTFP shows a state of first decreasing and then increasing. When the concentration is 1 mg / mL, the lowest scavenging rate is 18.22%. When the concentration is 4 mg / mL, its free radical scavenging rate is the highest, which is 73.54%. The overall scavenging rate is weaker than that of DTFP. It shows that the hydroxyl radical scavenging rate of DTFP has a concentration-dependent relationship, while WTFP does not show a concentration-dependent relationship, and the antioxidant activity of DTFP is better than that of WTFP.

[0169] 4.7 Scavenging rate of Tremella polysaccharide on ABTS free radicals

[0170] ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) generates stable blue-green ABTS radicals under the action of an oxidant (such as potassium persulfate). The ABTS radical scavenging rate experiment is a simple, rapid, and reliable method for evaluating antioxidant capacity, which is widely used in the fields of food, medicine, cosmetics, etc. By measuring the scavenging ability of the sample for ABTS + the antioxidant activity of the sample can be evaluated, providing a scientific basis. The results of detecting the scavenging rates of ABTS radicals by two tremella polysaccharides with different concentrations using 3.8 are as follows Figure 7 .

[0171] Figure 7 It can be seen that when the concentration of DTFP is 0.25 mg / mL, the ABTS radical scavenging rate is 10.63%. When the concentration gradually increases, the ABTS radical scavenging rate also gradually increases, reaching a maximum of 36.03%. When the concentration of WTFP is 0.25 mg / mL, the ABTS radical scavenging rate is 2.30%. When the concentration gradually increases, the ABTS radical scavenging rate also gradually increases, reaching a maximum of 19.84%. This shows that the ABTS radical scavenging rate of tremella polysaccharides has a concentration-dependent relationship, and the antioxidant activity of DTFP is better than that of WTFP.

[0172] 4.8 Effect of tremella polysaccharides on the lifespan of C. elegans under oxidative stress

[0173] From Figure 8 , Figure 9 it can be known that the maximum survival time (T max ) of nematodes in the hydrogen peroxide stress state blank control (M9) group is only 480 min. The positive control (Vc) group can extend the T max of nematodes to more than 760 min. Except for 0.5 mg / mL of WTFP (tremella polysaccharide extracted with deionized water), all concentrations of tremella polysaccharides (0.25 - 2.0 mg / mL) extracted with two different solvents can shift the survival curve of nematodes to the right under stress conditions. The average survival time of nematodes in the blank control group is 282.45 min ± 11.52 min, and the average survival time of nematodes in the positive control group is 562.67 min ± 4.96 min. The average survival times of nematodes in the two tremella polysaccharide groups with different concentrations are significantly higher than those in the blank control group. The T maxThey were 328.00 min ± 42.33 min, 308.36 min ± 18.93 min, 393.33 min ± 78.62 min, and 422.30 min ± 42.14 min respectively. The T of DTFP solutions with different concentrations (2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL) max They were 307.74 min ± 66.55 min, 346.98 min ± 24.57 min, 432.98 min ± 23.36 min, and 577.82 min ± 13.11 min respectively. In summary, Tremella polysaccharides of all selected concentrations could extend the lifespan of C. elegans under stress conditions. Meanwhile, the effect of DTFP was better than that of WTFP, and the best effect was achieved when the concentration of DTFP was 2 mg / mL, which could extend the lifespan of C. elegans by 2.04 times. This indicates that Tremella polysaccharides have a significant antioxidant stress effect, and the activity of Tremella polysaccharides extracted by green solvents is better than that of Tremella polysaccharides extracted by deionized water.

[0174] 4.9 Evaluation of the moisturizing function of Tremella polysaccharides on zebrafish under water-deficient conditions

[0175] The effects of two Tremella polysaccharides (DTFP and WTFP) on the anti-shrinkage rate of the tail area of zebrafish under water-deficient conditions are shown in Figure 10 After statistical analysis, the results are shown in Figure 11 . From Figure 11 , it can be seen that the anti-shrinkage rate of the tail area of zebrafish under water-deficient conditions by DTFP was 192.09%, higher than 120.27% of hyaluronic acid (positive control), although there was no difference between the two (p > 0.05). The anti-shrinkage rate of the tail area of zebrafish under water-deficient conditions by WTFP was only 21.49%, much lower than 120.27% of hyaluronic acid (positive control). At the same concentration, the anti-shrinkage rate of the tail area of zebrafish under water-deficient conditions by DTFP was much higher than that of WTFP, and the difference between the two was significant (p < 0.05), indicating that DTFP could effectively inhibit the dehydration and contraction of zebrafish tail tissues, and its moisturizing and anti-wrinkle abilities for zebrafish under water-deficient conditions were better than those of WTFP. It is speculated that the reason may be that DTFP extracted by ultrasonic-cellulase wall-breaking coupling green solvent method may delay water loss and maintain the water content of tail tissues by enhancing the epidermal barrier function or forming a hydrophilic film layer, thereby reducing the shrinkage rate. It is also possible to stabilize the cell membrane structure through the osmotic regulation mechanism, reduce cell shrinkage caused by water deficiency, and maintain the integrity of the tail morphology. This suggests its application potential in the field of biological water retention.

[0176] 4.10 Evaluation of the anti-wrinkle function of Tremella polysaccharides on zebrafish under ultraviolet irradiation

[0177] The effects of two Tremella polysaccharides (DTFP and WTFP) on the inhibition rate of caudal fin shrinkage of zebrafish under ultraviolet irradiation are shown inFigure 12 , after counting the results are shown in Figure 13 . From Figure 13 , it can be seen that the inhibition rate of DTFP on the caudal fin shrinkage of zebrafish under ultraviolet irradiation is 117.42%, much higher than 77.77% of vitamin E (positive control), and there is no significant difference between the two (p>0.05). The inhibition rate of WTFP on the caudal fin shrinkage of zebrafish under ultraviolet irradiation is only 112.51%, which is also higher than that of vitamin E, but there is no significant difference (p>0.05). At the same concentration, the inhibition rate of DTFP on the caudal fin shrinkage of zebrafish under ultraviolet irradiation is higher than that of WTFP, but there is no difference between the two (p>0.05), indicating that both DTFP and WTFP can effectively inhibit the contraction of zebrafish tail tissue under ultraviolet irradiation, suggesting their application potential in the field of biological anti-wrinkle.

[0178] Example 2. Extraction of Tremella polysaccharide by ultrasonic-cellulase wall-breaking coupling green solvent method

[0179] Based on the optimal process parameters optimized by the ultrasonic-cellulase wall-breaking coupling green solvent method in Example 1, the dried Tremella was placed in a grinding machine for pulverization, passed through a 100-mesh sieve to obtain Tremella powder. The optimal extraction process parameters of the ultrasonic-cellulase wall-breaking method coupling green solution method were used to extract Tremella polysaccharide, and finally the in vitro moisturizing ability and free radical scavenging ability of the extracted Tremella polysaccharide were measured. Specifically as follows:

[0180] (1) Place 1 g of dry Tremella powder (the same as in Example 1) in a conical flask, add 60 mL of a green solvent with a volume water content of 80% (the solute is arginine:glycerol with a molar ratio of 1:4 = 1:4) at a solid-liquid ratio of 1 g:60 mL, then add 0.004 g of cellulase, and then incubate and enzymolyze at 95 °C and a rotation speed of 125 rpm in a water bath shaker for 30 min, and then extract in an ultrasonic wall-breaking instrument under the condition of an ultrasonic power of 200 W for 9 min. Centrifuge (8000 rpm, 15 min), and take the supernatant as the extract, and the polysaccharide extraction rate is 47.72%.

[0181] (2) Add 95% ethanol to all the supernatant in step (1) at a volume ratio of 1:4, let it stand for alcohol precipitation at 4°C for 12 h, and then centrifuge (4000 rpm, 10 min). Take the precipitate after centrifugation, dissolve it in deionized water, and concentrate its volume to 1 / 2 of the original; obtain the concentrated solution. Add 1 / 4 times the volume of Sevag reagent (chloroform - n-butanol, 4:1, V / V) to the concentrated solution, mix well, shake to remove proteins for 20 min, centrifuge (8000 rpm, 5 min), remove the precipitate, retain the supernatant, and repeat the alcohol precipitation and protein removal 2 times according to the above method. Collect the supernatant, remove the organic solvent by rotary evaporation, and concentrate by rotary evaporation to 1 / 5 of the original volume. Obtain Tremella polysaccharide solution, and freeze-dry it (initial temperature is -30°C, vacuum degree is 80 Pa) until the mass water content is 8%, and obtain 0.317 g of Tremella polysaccharide.

[0182] Detected by the method of Example 1, the Tremella polysaccharide has a long-lasting moisturizing ability of 6 h; the scavenging rate of DPPH free radicals is 91.43%, the scavenging rate of hydroxyl free radicals is 100%, and the scavenging rate of ABTS free radicals is 36.03%; it can shift the growth curve of nematodes to the right under stress conditions, and T max is extended to more than 577 min.

[0183] Comparative Example 1, Hot water extraction

[0184] Take 2 g of the dried Tremella powder in Example 1 in a conical flask, add deionized water according to the liquid-to-solid ratio of 60:1 (mL / g), place it in a water bath at 95°C for extraction for 30 min, centrifuge at 8000 rpm for 10 min to obtain the supernatant. Take 1.0 mL of the supernatant, prepare Tremella polysaccharide by the method of step (2) in Example 1, and detect the polysaccharide extraction rate and free radical scavenging rate. The results are shown in Table 3.

[0185] Table 3 Polysaccharide extraction rate and free radical scavenging ability of different extraction processes

[0186]

[0187] As can be seen from Table 3, different extraction processes have a great influence on the extraction rate of Tremella polysaccharide. The ultrasonic - cellulase coupling green solvent extraction process can significantly improve the extraction rate of Tremella polysaccharide, which is 5.153 times higher than that of traditional hot water extraction of Tremella polysaccharide, and has a stronger free radical scavenging ability.

[0188] Comparative Example 2, Enzymatic extraction

[0189] Precisely weigh 2.0 g of the dried Tremella powder in Example 1, add 20 mL of phosphate buffer solution with pH 4.5, 0.03 g of cellulase, 0.01 g of pectinase, and 0.04 g of neutral protease, then place it in a water bath at 50°C for 60 min, and calculate the extraction rate of Tremella polysaccharide by the method of Example 1. The results are shown in Table 4.

[0190] Table 4 Yield of Tremella polysaccharide by different extraction processes

[0191]

[0192] As can be seen from Table 4, the extraction process of ultrasonic - cellulase coupling green solvent method can significantly improve the yield of Tremella polysaccharide, which is 3.107 times higher than that of the enzymolysis method for Tremella polysaccharide.

[0193] Comparative Example 3: Ultrasonic - assisted hot water extraction

[0194] Weigh 1.0 g of the dried Tremella powder in Example 1, add deionized water according to the solid - liquid ratio of 1 g:90 mL, water - bath at 100 °C for 4 h, then ultrasonicate for 9 min at an ultrasonic power of 200 W. Centrifuge at 5000 rpm for 10 min to obtain the supernatant. Take 1.0 mL of the supernatant and detect the extraction rate of polysaccharide by the method in Example 1. The results are shown in Table 5.

[0195] Table 5 Yield of Tremella polysaccharide by different extraction processes

[0196]

[0197] As can be seen from Table 5, the extraction process of ultrasonic - cellulase coupling green solvent method can significantly improve the yield of Tremella polysaccharide, which is 3.688 times higher than that of the ultrasonic - assisted extraction method for Tremella polysaccharide.

[0198] Comparative Example 4: Ultrasonic - assisted enzyme extraction

[0199] Prepare a pH 5.0 citric acid - sodium citrate buffer solution: Weigh 2.10 g of solid citric acid, dissolve it in 100 mL of deionized water, and make up the volume to 1 L to obtain a 0.1 mol / L solution A. Weigh 29.41 g of solid sodium citrate, dissolve it in 100 mL of deionized water, and make up the volume to 1 L to obtain a 0.1 mol / L solution B. Take 82 mL of solution A and 118 mL of solution B, mix them and add deionized water to a total volume of 1 L.

[0200] Precisely weigh 1.0 g of the dried Tremella powder in Example 1, add 0.004 g of cellulase, mix it according to the ratio of 1 g:90 mL with the pH 5.0 citric acid - sodium citrate buffer solution, ultrasonicate at an ultrasonic power of 200 W for 9 min, and then inactivate the enzyme in a boiling water bath for 10 min. Centrifuge at 5000 rpm for 10 min to obtain the supernatant. Take 1.0 mL of the supernatant and detect the extraction rate of polysaccharide by the method in Example 1. The results are shown in Table 6.

[0201] Table 6 Yield of Tremella polysaccharide by different extraction processes

[0202]

[0203] As can be seen from Table 6, the extraction process of the ultrasonic-cellulase coupling green solvent method can significantly improve the extraction yield of tremella polysaccharide, which is 1.937 times higher than that of tremella polysaccharide by the ultrasonic-assisted enzyme method.

[0204] Example 3

[0205] Take 1 g of the dried tremella powder in Example 1, add 40 mL of green solvent with a volume water content of 90% (the solute is arginine: glycerol with a molar ratio of 1:4), then add 0.012 g of cellulase, mix well, and incubate and enzymolyze at 35 °C and 125 rpm on a water bath shaker for 120 min to obtain an enzymolyzed mixture;

[0206] Put the enzymolyzed solution into an ultrasonic cell breaker for cell breaking, with a cell breaking power of 50 W and a cell breaking time of 3 min. Then centrifuge the broken solution (8000 rpm, 15 min), take the supernatant, and detect the polysaccharide extraction yield by the method in Example 1, which is 40.12%.

[0207] Example 4

[0208] Take 1 g of the dried tremella powder in Example 1, add 60 mL of green solvent with a volume water content of 70% (the solute is arginine: glycerol with a molar ratio of 1:4), then add 0.016 g of cellulase, mix well, and incubate and enzymolyze at 50 °C and 125 rpm on a water bath shaker for 90 min to obtain an enzymolyzed mixture;

[0209] Put the enzymolyzed mixture into an ultrasonic cell breaker, with a cell breaking power of 100 W and a cell breaking time of 6 min. Then centrifuge the broken solution (8000 rpm, 15 min), take the supernatant, and detect the polysaccharide extraction yield by the method in Example 1, which is 41.74%.

[0210] Example 5

[0211] Take 1 g of the dried tremella powder in Example 1, add 140 mL of green solvent with a volume water content of 60% (the solute is arginine: glycerol with a molar ratio of 1:4), then add 0.024 g of cellulase, mix well, and incubate and enzymolyze at 65 °C and 125 rpm on a water bath shaker for 150 min to obtain an enzymolyzed mixture;

[0212] Put the enzymolyzed mixture into an ultrasonic cell breaker for cell breaking, with a cell breaking power of 175 W and a time of 15 min. Then centrifuge the broken solution at 8000 rpm for 15 min, take the supernatant, and detect the polysaccharide extraction yield by the method in Example 1, which is 40.89%.

[0213] Example 6

[0214] Take 1 g of the dried tremella powder of Example 1, add 100 mL of a green solvent with a water content of 80% by volume (the solute is arginine: glycerol with a molar ratio of 1:4), then add 0.02 g of cellulase, mix well, and incubate and enzymatically hydrolyze at 65 °C and 125 rpm on a water bath shaker for 120 min to obtain an enzymatically hydrolyzed mixture;

[0215] Put the enzymatically hydrolyzed mixture into an ultrasonic cell disruptor for cell disruption, with a cell disruption power of 200 W and a cell disruption time of 9 min. Then centrifuge the disrupted solution (8000 rpm, 15 min), take the supernatant, and detect the polysaccharide extraction rate using the method of Example 1, which is 41.96%.

Claims

1. A method for improving the extraction yield of tremella polysaccharide, characterized in that, The method includes the following steps: (1) Take dried tremella powder, add a green solvent and cellulase, enzymatically hydrolyze in a water bath shaker at 35 - 95 °C and 100 - 150 rpm for 30 - 150 min, then extract for 3 - 15 min under the condition of an ultrasonic power of 50 - 200 W, centrifuge, and collect the supernatant; the green solvent is prepared with a hydrogen bond donor and a hydrogen bond acceptor as solutes and deionized water as the solvent, where the hydrogen bond acceptor is arginine and the hydrogen bond donor is glycerol or 1,4 - butanediol; (2) Add 95% ethanol to all the supernatant collected in step (1), let it stand at 4 °C for alcohol precipitation for 6 h, then centrifuge, dissolve the precipitate in deionized water, remove ethanol by rotary evaporation and concentrate its volume to 1 / 3 of the volume before concentration to obtain a concentrated solution; add Sevag reagent to the concentrated solution, mix well, shake to remove protein for 20 - 30 min, centrifuge, remove the precipitate, retain the supernatant, continue to repeat alcohol precipitation and protein removal 1 - 3 times according to the above method, collect the supernatant, remove the organic solvent by rotary evaporation, concentrate to 1 / 5 of the original volume under rotary evaporation at 65 - 80 °C, and freeze - dry until the mass water content is 8% to obtain tremella polysaccharide.

2. The method according to claim 1, characterized in that, In step (1), the solute in the green solvent is one of the following: a composition of arginine and glycerol with a molar ratio of 1:2 or 1:4, a composition of arginine and 1,4 - butanediol with a molar ratio of 1:2 or 1:

4.

3. The method according to claim 1, wherein The green solvent in step (1) is prepared as follows: Shake the solute in a water bath shaker at 60 - 80 °C at a rotation speed of 100 - 150 rpm for 1 - 2 h until the solution is clear, and then add deionized water to prepare it.

4. The method according to claim 1, characterized in that In step (1), the volume water content of the green solvent is 60 - 90%; the volume addition amount of the green solvent is 60 - 140 mL / g based on the mass of the dried tremella powder; the mass addition amount of the cellulase is 0.4 - 2.4% based on the mass of the dried tremella powder.

5. The method according to claim 1, characterized in that, In step (2), the volume ratio of the added 95% ethanol to the volume of the supernatant is 4:1; the Sevag reagent is a chloroform - n - butanol with a volume ratio of 4:1, and the addition amount of the Sevag reagent to the volume of the concentrated solution is 1:

4.

6. A tremella polysaccharide prepared by the method according to claim 1.

7. Use of the tremella polysaccharide according to claim 6 in the preparation of a moisturizing product.

8. Use of the tremella polysaccharide according to claim 6 in the preparation of an anti - wrinkle product.

9. Use of the tremella polysaccharide according to claim 6 in the preparation of an antioxidant.

10. Use of the tremella polysaccharide according to claim 6 in the preparation of an antioxidant stress preparation.

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