A kind of extraction method of acidic Tremella polysaccharide

Abstract

The present invention relates to a method for extracting acidic Tremella polysaccharide using bilirubin-modified chitosan, the method comprising the steps of: (1) preparing a bilirubin-modified chitosan material; preparing a Tremella extract; (2) passing the Tremella extract through a bilirubin-modified chitosan material chromatography column, whereby the acidic Tremella polysaccharide binds to amino groups on the surface of the bilirubin and is adsorbed on the surface of the modified chitosan, thereby separating the acidic Tremella polysaccharide from other components; then adding a strong ion salt solution to separate the acidic Tremella polysaccharide from the modified chitosan, and filtering the mixture using a filter membrane to obtain an acidic Tremella polysaccharide solution. The method for extracting acidic Tremella polysaccharide using bilirubin-modified chitosan provided by the present invention has a simple process, low production cost, high product quality, and is suitable for further promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterial preparation, and particularly relates to a method for selectively separating acidic Tremella polysaccharide by using a bilirubin-modified chitosan material. Background Art

[0002] Tremella fuciformis, also known as white fungus, belongs to the Tremellaceae family. It contains 17 amino acids, including leucine, isoleucine, and proline. It also 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" for its benefits in nourishing yin and moistening the lungs, tonifying the spleen and appetizing, and invigorating qi and clearing the intestines.

[0003] Polysaccharides are the main component of Tremella fuciformis (Tremella) and one of its most effective and active ingredients. Tremella fuciformis polysaccharides primarily include four types of polysaccharides: acidic heteropolysaccharides, neutral heteropolysaccharides, cell wall polysaccharides, and extracellular polysaccharides. The main chain of Tremella fuciformis polysaccharides is a mannan composed of α-(1-3)-glycosidic bonds. Side chains consisting of glucose, xylose, fucose, and common uronic acid residues are attached to the 2, 4, and 6 positions of the main chain. The active center is the common α-(1-3)-mannan structure. The molecular weight of Tremella fuciformis polysaccharides can reach up to 1.3 to 1.7 million.

[0004] Existing methods for extracting and separating Tremella fuciformis polysaccharides primarily involve the following steps: dissolution, protein removal, decolorization, and alcohol precipitation. Deproteinization typically involves the use of organic solvents such as trichloroacetic acid, tannic acid, and sevage, or an enzymatic method combined with a sevage reagent. These methods utilize organic solvents to denature and precipitate proteins, requiring the use of large quantities of these solvents, resulting in residual solvent and further complicating the separation of Tremella fuciformis polysaccharides from impurities. Decolorization methods primarily employ hydrogen peroxide, adsorption, and ion exchange, but these methods suffer from high decolorization costs and the potential for degradation of Tremella fuciformis polysaccharides.

[0005] In summary, the extraction of Tremella polysaccharides and the separation and removal of impurities are the key to the extraction of Tremella polysaccharides. Most existing extraction methods utilize the differences in the properties of polysaccharides and other impurities in different solvents to achieve the separation of different components. However, these methods have problems such as the use of a large amount of organic solvents, huge solvent usage, and incomplete separation and impurity removal. Therefore, the development of selective adsorption polysaccharide products is of great significance for the high-purity, high-efficiency, and low-cost extraction of Tremella polysaccharides. Summary of the Invention

[0006] The purpose of the present invention is to overcome at least one disadvantage of the above-mentioned prior art and to provide a method for selectively separating and extracting acidic Tremella polysaccharides with a simple extraction process, low production cost and high product quality.

[0007] In order to solve the above technical problems, the present invention provides a method for extracting acidic Tremella polysaccharide using bilirubin-modified chitosan, the main feature of which is that the method comprises the steps of:

[0008] (1) preparing a bilirubin-modified chitosan material; preparing a Tremella fuciformis extract;

[0009] (2) The Tremella extract is passed through a bilirubin-modified chitosan material chromatography column, and the acidic Tremella polysaccharide combines with the amino group on the surface of bilirubin and is adsorbed on the surface of the modified chitosan. The Tremella extract can be repeatedly washed with water to separate the acidic Tremella polysaccharide from other components. A strong ion salt solution is then added to destroy the interaction between the acidic Tremella polysaccharide and the modified chitosan, thereby separating the acidic Tremella polysaccharide from the modified chitosan. The solution is filtered using a filter membrane to remove the strong ion salt and other small molecular substances to obtain an acidic Tremella polysaccharide solution, which can be freeze-dried to obtain Tremella polysaccharide powder.

[0010] Preferably, the preparation of the bilirubin-modified chitosan material in step (1) is specifically as follows:

[0011] After the chitosan is swollen with acid, an alkaline solution is added to precipitate the chitosan, which is then washed with deionized water and methanol to obtain swollen chitosan;

[0012] After bilirubin is dissolved in dimethyl sulfoxide, swelled chitosan is added, and concentrated sulfuric acid is added and reacted at 35-45°C for 12-36 hours;

[0013] After the reaction is completed, an alkaline solution is added until it becomes neutral, and a precipitate is precipitated. The precipitate is washed with deionized water and dried to obtain a bilirubin-modified chitosan material;

[0014] The preparation of the Tremella extract in step (1) is specifically as follows:

[0015] After the dried tremella is soaked, washed and chopped, it is boiled in a 0.01-0.1 mol / L alkaline solution at a ratio of wet tremella to alkaline solution of 1:10-100 at 80-100° C. for 2-5 hours, and the supernatant is extracted by centrifugation to obtain a tremella extract.

[0016] Preferably, the chitosan is α-crystal, and the degree of deacetylation is ≥70%.

[0017] Preferably, the alkaline solution is one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0018] Preferably, the filter membrane is a PVDF spiral membrane or a ceramic membrane with a molecular weight cutoff greater than 100 KDa.

[0019] Preferably, the strong ion salt solution is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and zinc chloride solution.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention uses chitosan, a widely available and inexpensive carrier, as a carrier. A carboxylic acid porphyrin compound, bilirubin, is modified on the carrier surface. The bilirubin is fixed to the chitosan surface through an esterification reaction between the carboxylic acid and the chitosan hydroxyl group. The amino groups of the porphyrin are simultaneously exposed, selectively adsorbing the glucuronic acid groups of tremella polysaccharides, thereby separating the acidic tremella polysaccharides from impurities such as pigments and proteins in the tremella polysaccharide extract. Under the action of a strong ionic salt solution, the hydrogen bonds of the adsorbed acidic tremella polysaccharides are broken, and the tremella polysaccharides are separated from the modified chitosan surface, thereby obtaining relatively pure acidic tremella polysaccharides. The modified chitosan structure is not destroyed, thereby enabling the reuse of the modified chitosan. The strong ion salts in Tremella polysaccharide can be separated from the polysaccharide by simple membrane filtration due to the large difference in molecular weight between the two. The process of extracting Tremella polysaccharide is simple, the production cost is low, and the product purity is high. The structure of Tremella polysaccharide will not be destroyed during the production process, and the unique structure and efficacy of Tremella polysaccharide are maintained. At the same time, since chitosan itself has certain antibacterial properties, it can inhibit the proliferation of microorganisms in the polysaccharide extraction process to a certain extent, reducing the cleanliness requirements of the production process and also reducing the production cost to a certain extent. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments.

[0023] The method for separating and purifying Tremella polysaccharide provided by the present invention mainly comprises the following steps:

[0024] Preparation of bilirubin-modified chitosan stationary phase

[0025] After the chitosan is swollen with acid, a strong base solution is added to precipitate the chitosan, and the chitosan is washed with deionized water and methanol; wherein the acid can be acetic acid;

[0026] After bilirubin is dissolved in dimethyl sulfoxide, the swollen chitosan is added, and concentrated sulfuric acid is slowly added, and the reaction is carried out at 35-45°C for 12-36 hours;

[0027] After the reaction is completed, a strong alkaline solution is added until neutral, and a precipitate is precipitated. The precipitate is washed with deionized water and dried to obtain bilirubin-modified chitosan;

[0028] Add bilirubin-modified chitosan to water, stir slowly and continuously with a glass rod to drive away the bubbles in the stationary phase, drain with a glass rod, pour it into the chromatography column along the glass rod, tap the column lightly to drive away the bubbles in the chromatography column, open the piston, let the water flow out, let the bilirubin-modified chitosan settle naturally, and close the piston when the water in the column is almost dry.

[0029] Stationary phase adsorption of acidic Tremella polysaccharide

[0030] After soaking, washing, and chopping the dried tremella, the dried tremella is boiled in a 0.01-0.1 mol / L strong alkaline solution at a ratio of wet tremella to alkaline solution of 1:10-100 (W:W) at 80-100° C. for 2-5 hours, centrifuged at a speed of 10,000 r / min, and the supernatant is extracted to obtain a tremella extract;

[0031] The supernatant was pumped into a chromatography column filled with bilirubin-modified chitosan via a peristaltic pump. The piston of the chromatography column was opened to allow the tremella extract to flow through the chromatography column. After the pumping was completed, the tremella extract was replaced with deionized water as the mobile phase, and the pumping was continued until the mobile phase effluent was clear and transparent.

[0032] Shedding, purification and drying of acidic Tremella polysaccharide

[0033] Switch the mobile phase to a strong ion salt solution until the Tremella polysaccharide is completely eluted, and introduce the eluate into a membrane filtration system. Use a PVDF spiral membrane or ceramic membrane with a molecular weight cutoff of 100 KDa to separate the strong ion salt from the acidic Tremella polysaccharide.

[0034] The obtained Tremella polysaccharide is concentrated by rotary evaporation and then freeze-dried to finally obtain relatively pure acidic Tremella polysaccharide.

[0035] Example 1

[0036] Chitosan was swollen in a 2% acetic acid solution for 2 hours. After the chitosan gradually became a transparent solution, 0.25 mol / L sodium hydroxide solution was added and the pH was adjusted to 7. The chitosan precipitated and washed with deionized water and methanol three times each before use. 0.5 g of bilirubin was dissolved in 50 mL of dimethyl sulfoxide, the swollen chitosan was added, and 0.2 mL of concentrated sulfuric acid was slowly added. The reaction was carried out at 40° C. for 24 hours. After the reaction, 2 mol / L sodium hydroxide solution was added until neutral. A precipitate was formed, which was washed with deionized water and freeze-dried in a vacuum freeze-drying process to obtain bilirubin-modified chitosan. The bilirubin-modified chitosan was added to water until it was completely covered. The precipitate was stirred slowly and continuously with a glass rod to dispel bubbles in the stationary phase. The solution was then drained with a glass rod and poured into a chromatography column along the glass rod. The column was gently tapped to dispel bubbles in the column. The stopcock was opened to allow the water to flow out. The bilirubin-modified chitosan was allowed to settle naturally. The stopcock was closed when the water in the column was almost dry.

[0037] After the dried tremella is soaked, washed, and chopped, it is boiled at 100°C for 2 hours in a 0.1 mol / L sodium hydroxide solution at a ratio of wet tremella to alkaline solution of 1:10 (W:W), and then centrifuged at a speed of 10,000 r / min to remove solid matter, and the supernatant is extracted to obtain a tremella extract; the supernatant is pumped into a chromatography column filled with bilirubin-modified chitosan through a peristaltic pump, the chromatography column piston is opened to allow the tremella extract to flow through the chromatography column, and after the pumping is completed, the tremella extract is replaced with deionized water as the mobile phase, and the pumping is continued until the mobile phase effluent is clear and transparent.

[0038] The mobile phase is switched to a 10% sodium chloride strong ion salt solution until the tremella polysaccharide is completely eluted, and the eluate is introduced into a membrane filtration system. A PVDF ceramic membrane with a molecular weight cutoff of 100 KDa is selected to separate the sodium chloride small molecule substance from the acidic tremella polysaccharide; the obtained acidic tremella polysaccharide is subjected to rotary evaporation and then freeze-dried to finally obtain the acidic tremella polysaccharide, with a yield of 6.3% of the acidic tremella polysaccharide, a transmittance of 87.5% of a 0.5% solution, and a viscosity of 0.85 Pa·s.

[0039] Example 2

[0040] Chitosan was swollen in a 2% acetic acid solution for 2 hours. After the chitosan gradually became a transparent solution, 0.25 mol / L sodium hydroxide solution was added and the pH was adjusted to 7. The chitosan precipitated and washed with deionized water and methanol three times before use. 0.5 g of bilirubin was dissolved in 50 mL of dimethyl sulfoxide, the swollen chitosan was added, and 0.2 mL of concentrated sulfuric acid was slowly added. The reaction was carried out at 35° C. for 36 hours. After the reaction was completed, 2 mol / L sodium hydroxide solution was added until neutral. A precipitate was precipitated, washed with deionized water, and freeze-dried in a vacuum to obtain bilirubin-modified chitosan. The bilirubin-modified chitosan was added to water until it was completely covered. The precipitate was stirred slowly and continuously with a glass rod to dispel bubbles in the stationary phase. The solution was drained with a glass rod and poured into a chromatography column along the glass rod. The column was gently tapped to dispel bubbles in the column. The stopcock was opened to allow water to flow out. The bilirubin-modified chitosan was allowed to settle naturally. The stopcock was closed when the water in the column was almost dry.

[0041] After being soaked, washed, and chopped, the dried tremella is boiled in a 0.01 mol / L sodium hydroxide solution at a ratio of wet tremella to alkaline solution of 1:100 (W:W) at 80°C for 5 hours, and then centrifuged at a speed of 10,000 r / min to remove solid matter, and the supernatant is extracted to obtain a tremella extract; the supernatant is pumped into a chromatography column filled with bilirubin-modified chitosan through a peristaltic pump, the chromatography column piston is opened to allow the tremella extract to flow through the chromatography column, and after the pumping is completed, the tremella extract is replaced with deionized water as the mobile phase, and the pumping is continued until the mobile phase effluent is clear and transparent.

[0042] The mobile phase was switched to an 8% sodium chloride strong ion salt solution until the tremella polysaccharide was completely eluted, and the eluate was introduced into a membrane filtration system. A PVDF ceramic membrane with a molecular weight cutoff of 100 KDa was selected to separate the sodium chloride small molecule substance from the acidic tremella polysaccharide. The obtained acidic tremella polysaccharide was subjected to rotary evaporation and then freeze-dried to finally obtain the acidic tremella polysaccharide. The yield of the acidic tremella polysaccharide was 4.7%, the transmittance of a 0.5% solution was 89.5%, and the viscosity was 0.78 Pa·s.

[0043] Example 3

[0044] Chitosan was swollen in a 2% acetic acid solution for 2 hours. After the chitosan gradually became a transparent solution, 0.25 mol / L sodium hydroxide solution was added and the pH was adjusted to 7. The chitosan precipitated and washed with deionized water and methanol three times before use. 0.5 g of bilirubin was dissolved in 50 mL of dimethyl sulfoxide, the swollen chitosan was added, and 0.3 mL of concentrated sulfuric acid was slowly added. The reaction was carried out at 45° C. for 16 hours. After the reaction, 2 mol / L sodium hydroxide solution was added until neutral. A precipitate was formed, which was washed with deionized water and freeze-dried in a vacuum freeze-drying process to obtain bilirubin-modified chitosan. The bilirubin-modified chitosan was added to water until it was completely covered. The solution was stirred slowly and continuously with a glass rod to dispel bubbles in the stationary phase. The solution was then drained with a glass rod and poured into a chromatography column along the glass rod. The column was gently tapped to dispel bubbles. The stopcock was opened to allow the water to flow out. The bilirubin-modified chitosan was allowed to settle naturally. The stopcock was closed when the water in the column was almost dry.

[0045] After the dried tremella is soaked, washed, and chopped, it is boiled at 100°C for 3 hours in a 0.1 mol / L sodium hydroxide solution at a ratio of wet tremella to alkaline solution of 1:100 (W:W), and then centrifuged at a speed of 10,000 r / min to remove solid matter, and the supernatant is extracted to obtain a tremella extract; the supernatant is pumped into a chromatography column filled with bilirubin-modified chitosan through a peristaltic pump, the chromatography column piston is opened to allow the tremella extract to flow through the chromatography column, and after the pumping is completed, the tremella solution is replaced with deionized water as the mobile phase, and the pumping is continued until the mobile phase effluent is clear and transparent.

[0046] The mobile phase was switched to a 5% potassium chloride strong ion salt solution until the tremella polysaccharide was completely eluted, and the eluate was introduced into a membrane filtration system. A PVDF ceramic membrane with a molecular weight cutoff of 100 KDa was selected to separate the sodium chloride small molecule substance from the acidic tremella polysaccharide. The obtained acidic tremella polysaccharide was subjected to rotary evaporation and then freeze-dried to finally obtain the acidic tremella polysaccharide. The yield of the acidic tremella polysaccharide was 7.2%, the transmittance of a 0.5% solution was 90.2%, and the viscosity was 0.82 Pa·s.

[0047] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description is to be regarded as illustrative rather than restrictive.

Claims

1. A method for extracting acidic Tremella polysaccharide using bilirubin-modified chitosan, characterized in that: The method comprises the steps of: (1) Preparing bilirubin-modified chitosan material; preparing Tremella fuciformis extract; (2) The Tremella extract passes through a bilirubin-modified chitosan material chromatography column, and the acidic Tremella polysaccharide combines with the amino groups on the surface of bilirubin and adsorbs on the modified chitosan surface, thereby separating the acidic Tremella polysaccharide from other components; Then, a strong ion salt solution is added to separate the acidic Tremella polysaccharide from the modified chitosan, and the mixture is filtered through a filter membrane to obtain an acidic Tremella polysaccharide solution; The preparation of the bilirubin-modified chitosan material in step (1) is specifically as follows: After the chitosan is swollen with acid, an alkaline solution is added to precipitate the chitosan, which is then washed with deionized water and methanol to obtain swollen chitosan; After bilirubin is dissolved in dimethyl sulfoxide, swelled chitosan is added, and concentrated sulfuric acid is added and reacted at 35-45°C for 12-36 hours; After the reaction is completed, an alkaline solution is added until it becomes neutral, and a precipitate is precipitated. The precipitate is washed with deionized water and dried to obtain a bilirubin-modified chitosan material; The preparation of the Tremella extract in step (1) is specifically as follows: After the dry tremella is soaked, washed and chopped, it is boiled in a 0.01-0.1 mol / L alkaline solution at a ratio of wet tremella: alkaline solution = 1:10-100 at 80-100° C. for 2-5 hours, and the supernatant is extracted by centrifugation to obtain a tremella extract.

2. The method for extracting acidic Tremella polysaccharide from bilirubin-modified chitosan according to claim 1, wherein The chitosan is of α-crystal form and has a deacetylation degree of ≥70%.

3. The method for extracting acidic Tremella polysaccharide by bilirubin-modified chitosan according to claim 1, wherein The alkaline solution is one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide.

4. The method for extracting acidic Tremella polysaccharide by bilirubin-modified chitosan according to claim 1, wherein The filter membrane is a PVDF spiral membrane or a ceramic membrane with a molecular weight cutoff greater than 100KDa.

5. The method for extracting acidic Tremella polysaccharide by using bilirubin-modified chitosan according to claim 1, wherein The strong ion salt solution is one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride and zinc chloride.