Extraction method of tremella sporocarp exopolysaccharide and application of tremella sporocarp exopolysaccharide in skin repair

Abstract

The invention discloses an extraction method of tremella sporocarp extracellular polysaccharide and application of the tremella sporocarp extracellular polysaccharide in skin repair, and relates to a Ni < 2 + > modified tannic acid modified mesoporous material and a method for preparing high-purity tremella sporocarp extracellular polysaccharide through physical adsorption of protein, the method comprises the following steps: covering the surface of a mesoporous SBA-15 material with tannic acid, and then adding Ni < 2 + > ions for unsaturated chelating; the Ni < 2 + > modified tannic acid modified mesoporous protein adsorption material is prepared; the method comprises the following steps: fully soaking tremella, fully dissolving tremella sporocarp extracellular polysaccharide in water, concentrating and centrifugally separating the sporocarp extracellular polysaccharide solution to remove particle impurities, and removing protein through a mesoporous protein adsorption material. The method adopts pure physical extraction of tremella sporocarp exopolysaccharide, and has the characteristics of simple process, capability of improving resource utilization, suitability for factory continuous production, no organic solvent residue, high product purity, good skin health effect and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a method for extracting extracellular polysaccharide from tremella fruiting bodies and its application in skin repair. Background Art

[0002] Tremella, also known as white fungus, belongs to the Tremella genus of the Tremella family of fungi. It contains 17 kinds of amino acids such as leucine, isoleucine, and proline. It contains 6.7% to 10% protein, 0.6% to 12.8% fat, and 65% to 71.2% carbohydrates (including 60% to 70% Tremella polysaccharides). It is known as the "king of fungi" because of its effects of nourishing yin and moistening the lungs, tonifying the spleen and appetizing, and replenishing qi and clearing the intestines.

[0003] Polysaccharide is the main component of Tremella fruiting body, and it is also one of the most effective and active important components, including extracellular polysaccharide, intracellular polysaccharide and cell wall polysaccharide. Tremella is fully cleaned before it is sold. In the process of cleaning Tremella, a large amount of polysaccharide outside the fruiting body is dissolved in water and discharged as waste, which causes environmental pollution and waste of resources. The existing commercial Tremella polysaccharide is mainly mixed Tremella polysaccharide, among which the cell wall polysaccharide content is the highest, with the characteristics of large molecular weight, high viscosity, easy film formation, etc., but the large molecular weight of polysaccharide leads to poor skin permeability, which affects the application of Tremella polysaccharide on the skin. The extracellular polysaccharide of the fruiting body plays an important role in organisms, including energy storage, cell recognition, cell signal transduction and other functions. The special structure makes the extracellular polysaccharide of Tremella fruiting body have better activity. In medicine and biology, it has various physiological activities such as anti-tumor, anti-viral and anti-inflammatory.

[0004] The existing extraction and separation methods of Tremella polysaccharide mainly include: dissolution of Tremella polysaccharide-protein removal-decolorization-alcohol precipitation and other steps. In particular, the molecular weight of Tremella fruiting body extracellular polysaccharide is relatively small, which is comparable to the protein size in the polysaccharide. It is difficult to separate it from the polysaccharide by simple pore size filtration. Therefore, the removal of protein in Tremella fruiting body extracellular polysaccharide becomes a difficulty in polysaccharide extraction. The existing protein removal in polysaccharide often adopts organic solvents such as trichloroacetic acid, tannic acid, sevage, or enzyme method combined with sevage reagent method. These methods all use organic solvents to denature and precipitate proteins, which requires a large amount of organic solvents, is costly, and easily causes solvent residues. Therefore, the development of selective physical adsorption of proteins in polysaccharides is of great significance for efficiently extracting high-purity Tremella fruiting body extracellular polysaccharides and maintaining the polysaccharide structure. Wang et al. used metal ion chelating resin to deproteinize cattail polysaccharide (Shi et al. 2019); Espana and Zhang et al. used palygorskite to adsorb proteins in polysaccharides (Espana et al. 2019); Wang et al. used tannic acid-iron III complex network adsorbed on Fe3O4 to construct a magnetic material for adsorbing proteins in polysaccharides. However, these methods have problems such as high filtration pressure, low adsorption flux, low selectivity and large polysaccharide loss. Therefore, a composite filtration material with SBA-15 mesoporous material as the substrate, Ni2+ as the efficient protein chelator, tannic acid as the substrate and chelator medium was constructed. The high specific surface area of ​​the mesoporous material can load more Ni. 2+ , thereby increasing the adsorption of protein; at the same time, the rich pore structure of the mesoporous material can increase the passing speed of crude polysaccharides and reduce the pressure of the filtration process, thus realizing a new method of polysaccharide extraction with high efficiency and high adsorption of physical removal of protein impurities. Compared with the chemical protein removal method, the extracellular polysaccharides extracted from Tremella fuciformis fruiting bodies using this new method maintain the original structure of the polysaccharide, and are applied in skin care, showing higher antioxidant and repair activity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention discloses a method for extracting extracellular polysaccharide from Tremella fuciformis fruiting bodies, comprising the following steps:

[0007] Step 1: Ultrasonic dispersion of SBA-15 mesoporous material in deionized water, adding tannic acid aqueous solution under stirring, adding NiCl2 aqueous solution and centrifuging, washing the precipitate with deionized water and vacuum drying to obtain Ni 2+ Modified tannic acid-modified mesoporous materials;

[0008] Step 2: Take a fresh whole Tremella fuciformis and soak it in water, then remove the Tremella fuciformis, and remove the solids by rotary evaporation and centrifugation;

[0009] Step 3: The concentrated extracellular crude polysaccharide of Tremella fuciformis fruiting bodies flows through an adsorption separation column filled with a mesoporous material, the protein in the crude Tremella fuciformis polysaccharide is adsorbed and removed by the adsorption material, and diatomaceous earth is continuously added to the crude polysaccharide to remove the pigment therein;

[0010] Step 4: After filtering and removing the diatomaceous earth, the crude polysaccharide solution is ultrafiltered to remove small molecular compounds to prepare a Tremella fuciformis extracellular polysaccharide solution with a purity of more than 95%. The solution is vacuum dried at low temperature to obtain Tremella fuciformis fruiting body extracellular polysaccharide powder.

[0011] As a preferred technical solution of the present invention, the adsorption material is Ni 2+ The mesoporous SBA-15 material surface-modified with chelated tannic acid has a pore size of 2-20 nm.

[0012] As a preferred technical solution of the present invention, the Ni2+ is a water-soluble nickel salt, including nickel acetate, nickel chloride, nickel nitrate and nickel sulfate.

[0013] As a preferred technical solution of the present invention, the filter membrane for removing small molecule impurities is a 10KDa PVDF rolled membrane.

[0014] As a preferred technical solution of the present invention, the mesoporous material is loaded into a stainless steel column to form an adsorption column. After the Tremella extracellular polysaccharide passes through the adsorption column, the protein is adsorbed, thereby achieving the purpose of removing protein impurities in the polysaccharide.

[0015] The beneficial effects of the present invention are:

[0016] 1. The method for extracting the extracellular polysaccharide from the Tremella fuciformis fruiting body and its application in skin repair, through a physical method of selectively adsorbing proteins, the extraction process does not introduce chemical reagents, the polysaccharide structure is maintained, and the polysaccharide activity is higher;

[0017] 2. The extraction method of the extracellular polysaccharide of Tremella fuciformis fruiting body and its application in skin repair. The extracted extracellular polysaccharide of Tremella fuciformis has good antioxidant and repair effects, and has good application prospects in antioxidant, anti-aging and repair cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 It is a schematic diagram of a method for extracting extracellular polysaccharides from Tremella fuciformis fruiting bodies and the DPPH free radical scavenging capabilities of different Tremella fuciformis polysaccharides used in skin repair;

[0020] Figure 2It is a method for extracting extracellular polysaccharides from Tremella fuciformis fruiting bodies of the present invention and a representative diagram of the tail fin growth area of ​​different Tremella fuciformis polysaccharides used in skin repair;

[0021] Figure 3 The invention discloses a method for extracting extracellular polysaccharides from Tremella fuciformis fruiting bodies and a representative diagram of the tail fin growth area of ​​different Tremella fuciformis polysaccharides used in skin repair. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] The method for separating and purifying the extracellular polysaccharide of Tremella fuciformis fruiting body provided by the present invention mainly comprises the following steps:

[0025] 1. Ni 2+ Preparation of mesoporous materials modified by modified tannic acid

[0026] 0.1-1g of SBA-15 mesoporous material was ultrasonically dispersed in 250mL of deionized water, and 750mL of tannic acid aqueous solution (2mg / mL) was added under stirring, and stirred for 10-30min. Then, 150mL of NiCl2 (1mg / mL) aqueous solution was added, and stirring was continued for 10-30min. The precipitate was washed with deionized water for 5 times and then vacuum dried to obtain Ni 2+ Modified tannic acid-modified mesoporous materials.

[0027] 2. Polysaccharide extraction method for selective protein adsorption by mesoporous materials

[0028] Take 1 kg of fresh whole Tremella fuciformis and soak it in water to dissolve as much extracellular polysaccharide as possible in the water, then remove the Tremella fuciformis, concentrate the extracellular polysaccharide aqueous solution of Tremella fuciformis fruiting body to 1 / 10-1 / 50 of the original by rotary evaporation, remove solids by centrifugation, and then pass the concentrated extracellular polysaccharide of Tremella fuciformis through a flask filled with Ni 2+The adsorption separation column of a mesoporous material modified with modified tannic acid is repeatedly passed through 2-4 times, the protein in the crude tremella polysaccharide is adsorbed and removed by the adsorption material, diatomaceous earth is continuously added to the crude polysaccharide to remove the pigment therein, after filtering and removing the diatomaceous earth, the crude polysaccharide solution is passed through a 5KDa ultrafiltration fiber column to remove small molecular compounds, and a tremella extracellular polysaccharide solution with a purity of more than 95% is prepared, and the solution is vacuum dried at low temperature to obtain tremella fruiting body extracellular polysaccharide powder.

[0029] Example 1

[0030] 1. Ni 2+ Preparation of mesoporous materials modified by modified tannic acid

[0031] 1 g of SBA-15 mesoporous material was ultrasonically dispersed in 250 mL of deionized water, and 750 mL of tannic acid aqueous solution (2 mg / mL) was added under stirring, and stirred for 10 min. Then, 150 mL of NiCl2 (1 mg / mL) aqueous solution was added, and stirring was continued for 30 min. The precipitate was washed with deionized water for 5 times and then vacuum dried to obtain Ni 2+ Modified tannic acid-modified mesoporous materials.

[0032] 2. Polysaccharide extraction method for selective protein adsorption by mesoporous materials

[0033] Take 1kg of fresh whole Tremella fuciformis and soak it in 5L of water. Turn on the water pump to make the soaking water flow to increase the dissolution of the extracellular polysaccharide of Tremella fuciformis fruiting body. Soak for 3h. Take out the Tremella fuciformis, concentrate the extracellular polysaccharide aqueous solution of Tremella fuciformis fruiting body to 1 / 10 of the original by rotary evaporation, remove the solids by centrifugation, and then flow the concentrated extracellular crude Tremella fuciformis polysaccharide through a Ni-filled 2+ The adsorption separation column of the mesoporous material modified by modified tannic acid was repeatedly passed through for 3 times, and the protein in the crude polysaccharide was adsorbed and removed by the adsorption material. Diatomaceous earth with a concentration of 2% of the crude polysaccharide was added to remove the pigment therein. After filtering and removing the diatomaceous earth, the crude polysaccharide solution was filtered through a 5KDa ultrafiltration fiber column to remove small molecular compounds, and the solution was dried at low temperature and vacuum to obtain the extracellular polysaccharide powder of Tremella fuciformis fruiting body. The extracellular polysaccharide yield of Tremella fuciformis fruiting body was 1.1%, the polysaccharide content was 96.3%, the transmittance of the 0.5% polysaccharide solution was 92.5%, and the viscosity was 93mPa·S.

[0034] Example 2

[0035] 1. Ni 2+ Preparation of mesoporous materials modified by modified tannic acid

[0036] 0.1 g of SBA-15 mesoporous material was ultrasonically dispersed in 250 mL of deionized water, and 750 mL of tannic acid aqueous solution (0.5 mg / mL) was added under stirring, and stirred for 20 min. Then, 150 mL of NiCl2 (0.2 mg / mL) aqueous solution was added, and stirring was continued for 20 min. The precipitate was washed with deionized water for 5 times and then vacuum dried to obtain Ni 2+ Modified tannic acid-modified mesoporous materials.

[0037] 2. Polysaccharide extraction method for selective protein adsorption by mesoporous materials

[0038] Take 1kg of fresh whole Tremella fuciformis and soak it in 3L of water. Turn on the water pump to make the soaking water flow to increase the dissolution of the extracellular polysaccharide of Tremella fuciformis fruiting body. Soak for 5h. Take out the Tremella fuciformis, concentrate the extracellular polysaccharide aqueous solution of Tremella fuciformis fruiting body to 1 / 20 of the original by rotary evaporation, remove the solids by centrifugation, and then flow the concentrated extracellular crude polysaccharide of Tremella fuciformis fruiting body through a flask filled with Ni 2+ The adsorption separation column of the mesoporous material modified by modified tannic acid was repeatedly passed through twice, and the protein in the crude polysaccharide was adsorbed and removed by the adsorption material. Diatomaceous earth with a concentration of 2% of the crude polysaccharide was added to remove the pigment therein. After filtering and removing the diatomaceous earth, the crude polysaccharide solution was filtered through a 5KDa ultrafiltration fiber column to remove small molecular compounds, and the solution was dried at low temperature and vacuum to obtain the extracellular polysaccharide powder of Tremella fuciformis fruiting body. The yield of Tremella fuciformis extracellular polysaccharide was 2.4%, the polysaccharide content was 95.2%, the transmittance of 0.5% polysaccharide solution was 90.3%, and the viscosity was 106mPa·S.

[0039] Example 3

[0040] 1. Ni 2+ Preparation of mesoporous materials modified by modified tannic acid

[0041] 0.5 g of SBA-15 mesoporous material was ultrasonically dispersed in 250 mL of deionized water, and 750 mL of tannic acid aqueous solution (2 mg / mL) was added under stirring, and stirred for 30 min. Then, 150 mL of NiCl2 (2 mg / mL) aqueous solution was added, and stirring was continued for 30 min. The precipitate was washed with deionized water for 5 times and then vacuum dried to obtain Ni 2+ Modified tannic acid-modified mesoporous materials.

[0042] 2. Polysaccharide extraction method for selective protein adsorption by mesoporous materials

[0043] Take 1kg of fresh whole Tremella fuciformis and soak it in 10L of water. Turn on the water pump to make the soaking water flow to increase the dissolution of the extracellular polysaccharide of Tremella fuciformis fruiting body. Soak for 10h. Take out the Tremella fuciformis, concentrate the extracellular polysaccharide aqueous solution of Tremella fuciformis fruiting body to 1 / 50 of the original by rotary evaporation, remove the solids by centrifugation, and then pass the concentrated extracellular crude polysaccharide of Tremella fuciformis fruiting body through a Ni-filled2+ The adsorption separation column of the mesoporous material modified by modified tannic acid was repeatedly passed through 4 times, and the protein in the crude Tremella polysaccharide was adsorbed and removed by the adsorption material. Diatomaceous earth with a concentration of 2% of the crude polysaccharide was added to remove the pigment therein. After filtering and removing the diatomaceous earth, the crude polysaccharide solution was passed through a 5KDa ultrafiltration fiber column to remove small molecular compounds, and the solution was dried at low temperature and vacuum to obtain the Tremella fruiting body extracellular polysaccharide powder. The Tremella fruiting body extracellular polysaccharide yield was 1.8%, the polysaccharide content was 97.5%, the transmittance of the 0.5% polysaccharide solution was 93.1%, and the viscosity was 126mPa·S.

[0044] Example 4 Effect of Tremella fuciformis fruiting body extracellular polysaccharide on skin care

[0045] In vitro antioxidant activity of exopolysaccharides from Tremella fuciformis fruiting bodies

[0046] Based on the above-mentioned embodiments 1 to 3, further description is given.

[0047] The antioxidant activity of the extracellular polysaccharide of Tremella fuciformis fruiting body was detected, and the specific steps were as follows:

[0048] When scavenging DPPH free radicals, firstly, different concentrations of Tremella fuciformis fruiting body extracellular polysaccharide solutions were prepared, and the concentration ranges included 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, and 2.0 mg / mL;

[0049] Then, 0.1 mM DPPH methanol solution was selected, 50 μL of polysaccharide solution was added to 150 μL of DPPH solution, and the mixture was allowed to stand in a dark environment at 25°C for 30 minutes. The absorbance of the reaction system was measured at 517 nm using an enzyme marker, and the scavenging of DPPH free radicals was reflected by the absorbance.

[0050] After measuring the absorbance, using ascorbic acid (Vc) as a positive control, the calculation expression for the scavenging rate of DPPH free radicals is as follows:

[0051]

[0052] Among them, Q(DPPH) is the scavenging rate of DPPH free radicals of the extracellular polysaccharide solution of Tremella fuciformis fruiting body, A1 is the absorbance of the extracellular polysaccharide solution of Tremella fuciformis fruiting body in DPPH free radical scavenging, A2 is the absorbance of methanol instead of DPPH, and A3 is the absorbance of the control solution (methanol instead of extracellular polysaccharides of Tremella fuciformis fruiting body).

[0053] In order to better reflect the advantages of this method in maintaining the activity of extracellular polysaccharides from Tremella fuciformis fruiting bodies, high molecular weight Tremella fuciformis polysaccharides on the market and extracellular polysaccharides from Tremella fuciformis fruiting bodies extracted by sevage organic solvent method were selected to compare their antioxidant activities in scavenging DPPH free radicals.

[0054] The test results of DPPH free radical scavenging ability are as follows Figure 1 (A: extracellular polysaccharide of Tremella fuciformis fruiting body from which protein is removed by physical adsorption of mesoporous materials; B: high molecular weight Tremella fuciformis polysaccharide; C: extracellular polysaccharide of Tremella fuciformis fruiting body from which protein is removed by chemical method);

[0055] The DPPH free radical scavenging test is widely recognized as a method for evaluating the in vitro antioxidant activity of natural substances. Figure 1 It can be seen that the DPPH free radical scavenging rate increases with the increase of polysaccharide concentration, and the Tremella extracellular polysaccharide from which protein is removed by the mesoporous material physical adsorption method of this patent has a better ability to scavenge DPPH free radicals than large molecular weight Tremella polysaccharides and Tremella fruiting body extracellular polysaccharides from which protein is removed by chemical method.

[0056] The repair effect of Tremella fuciformis fruiting body exopolysaccharide based on zebrafish Figure 2 (Sample No. 1: extracellular polysaccharide of Tremella fuciformis fruiting body with protein removed by chemical method; Sample No. 2: high molecular weight Tremella fuciformis polysaccharide; Sample No. 3: extracellular polysaccharide of Tremella fuciformis fruiting body with protein removed by physical adsorption method)

[0057] Representative diagram of the tail fin growth area of ​​different Tremella polysaccharides Figure 3 (Sample No. 1: extracellular polysaccharide of Tremella fuciformis fruiting body with protein removed by chemical method; Sample No. 2: high molecular weight Tremella fuciformis polysaccharide; Sample No. 3: extracellular polysaccharide of Tremella fuciformis fruiting body with protein removed by physical adsorption method);

[0058] The zebrafish genome is 87% similar to that of humans. Its skin structure and function are highly similar to those of humans. It also has advantages such as small individual differences, short development cycle, transparent and easy-to-observe embryos, and lower testing costs. It has become a model animal for the efficacy evaluation of cosmetic raw materials. Figure 2 It can be seen that Tremella polysaccharide has a good repair effect on the tail fin of zebrafish, and as the concentration of Tremella polysaccharide increases, the repair effect continues to increase. According to the area of ​​zebrafish tail fin growth, the statistics are as follows Figure 3 As shown, similar to the trend of removing DPPH, the extracellular polysaccharides extracted from Tremella fuciformis fruiting bodies by the patented method of removing proteins by physical adsorption of mesoporous materials have better repair effect, indicating that the extracellular polysaccharides of Tremella fuciformis extracted by the patented method have good antioxidant and repair effects, and have good application prospects in antioxidant, anti-aging and repair cosmetics.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting extracellular polysaccharides from Tremella fuciformis fruiting bodies, characterized in that: It includes the following steps: Step 1: Ultrasonic dispersion of SBA-15 mesoporous material in deionized water, adding tannic acid aqueous solution under stirring, adding NiCl2 aqueous solution and centrifuging, washing the precipitate with deionized water and vacuum drying to obtain Ni 2+ Modified tannic acid-modified mesoporous materials; Step 2: Take a fresh whole Tremella fuciformis and soak it in water, then remove the Tremella fuciformis, and remove the solids by rotary evaporation and centrifugation; Step 3: The concentrated extracellular crude polysaccharide of Tremella fuciformis fruiting bodies flows through an adsorption separation column filled with a mesoporous material, the protein in the crude Tremella fuciformis polysaccharide is adsorbed and removed by the adsorption material, and diatomaceous earth is continuously added to the crude polysaccharide to remove the pigment therein; Step 4: After filtering and removing the diatomaceous earth, the crude polysaccharide solution is ultrafiltered to remove small molecular compounds to prepare a Tremella fuciformis extracellular polysaccharide solution with a purity of more than 95%. The solution is vacuum dried at low temperature to obtain Tremella fuciformis fruiting body extracellular polysaccharide powder.

2. A method for extracting exopolysaccharide from Tremella fuciformis fruiting bodies according to claim 1, characterized in that, The adsorption material is Ni 2+ The mesoporous SBA-15 material surface-modified with chelated tannic acid has a pore size of 2-20 nm.

3. A method for extracting exopolysaccharide from Tremella fuciformis fruiting bodies according to claim 1, characterized in that, The Ni 2+ Water-soluble nickel salts include nickel acetate, nickel chloride, nickel nitrate and nickel sulfate.

4. A method for extracting exopolysaccharide from Tremella fuciformis fruiting bodies according to claim 1, characterized in that, The filter membrane for removing small molecule impurities is a 10KDa PVDF spiral membrane.

5. A method for extracting exopolysaccharide from Tremella fuciformis fruiting bodies according to claim 1, characterized in that: The mesoporous material is loaded into a stainless steel column to form an adsorption column. After the Tremella extracellular polysaccharide passes through the adsorption column, the protein is adsorbed, thereby achieving the purpose of removing protein impurities in the polysaccharide.

6. An exopolysaccharide powder of Tremella fuciformis fruiting bodies prepared according to the method for extracting exopolysaccharides from Tremella fuciformis fruiting bodies according to any one of claims 1 to 5.

7. An application of the Tremella fuciformis fruiting body extracellular polysaccharide powder according to claim 6 in skin care.

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