CN112724273A - Method for extracting and preparing high molecular weight tremella polysaccharide - Google Patents
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Abstract
The invention discloses a method for extracting and preparing high molecular weight tremella polysaccharide, belonging to the technical field of extraction of bioactive substances, and being characterized by comprising a wall breaking process and a protein removing process; the protein removing process also comprises an extraction process, a separation process, a concentration process and a purification process; the invention has the beneficial effects that: the wall breaking treatment is carried out by using chitinase, a complete cell membrane with biological activity, namely a protoplast, can be effectively reserved while cell walls are removed, the glycoprotein containing tremella polysaccharide and wound on the tremella polysaccharide is isolated in the protoplast, tannic acid or trichloroacetic acid can smoothly pass through the cell membrane to act on the glycoprotein wound on the tremella polysaccharide, and the glycoprotein is separated from the tremella polysaccharide after being modified; the water-soluble characteristic of the separated tremella polysaccharide can smoothly pass through a cell membrane to the outside of the cell membrane, so that the purpose of separating the denatured glycoprotein from the tremella polysaccharide is achieved, and the high yield and low protein impurity of the tremella polysaccharide are ensured.
Description
The technical field is as follows:
the invention belongs to the technical field of extraction of bioactive substances, and particularly relates to an extraction and preparation method of high molecular weight tremella polysaccharide.
Background art:
tremella (Tremellafuciforfor, nis Berk), also known as Tremella, and Chrysanthemum ear, mainly contains protein, carbohydrate, etc., vitamins and various amino acids, etc., and can be used as medicinal and tonic food. Tremella fuciformis is mainly produced in Sichuan province, Guizhou province, Hubei province, Shanxi province, Fujian province, Zhejiang province and the like, and one of the main active ingredients of fruiting bodies of the tremella fuciformis is a tremella polysaccharide which is gradually a research hotspot in recent years. Modern researches have proved that tremella polysaccharide is mainly heteropolysaccharide with alpha- (1-3) -D mannose as main chain, and has multiple biological activities such as immunoregulation, anti-tumor, anti-oxidation and anti-aging, blood sugar and blood fat reduction, anticoagulation and thrombosis, antiulcer, protein synthesis promotion, antivirus, nerve cell growth promotion, memory improvement and the like.
Tremella polysaccharides (Tremella polysaccharides) are heteropolysaccharides with alpha- (1-3) -D mannose as main chain, which are obtained by separating and purifying submerged fermentation spores of Tremella sporophores. According to the content of the polysaccharides, the polysaccharide mainly comprises four types of polysaccharides, namely acidic heteropolysaccharide, neutral heteropolysaccharide, mural polysaccharide and exopolysaccharide.
In recent 30 years, with the research and development of plant extraction technology, the extraction of tremella polysaccharide has been advanced to some extent, the research of active components of polysaccharide is also deepened gradually, and the traditional methods mainly comprise a water extraction method, an acid-base extraction method and an enzymolysis extraction method.
However, the research on the preparation process of the tremella polysaccharide is not mature at present, and further improvement is needed. Although the method for obtaining the tremella polysaccharide by using the water extraction method is simple in operation, some non-sugar substances are proposed in the extraction process, for example, because the tremella polysaccharide is provided with glycoprotein, the glycoprotein is difficult to separate and purify subsequent crude polysaccharide. Although the tremella polysaccharide obtained by the acid-base extraction method contains polysaccharide which cannot be mentioned by the water method, the tremella polysaccharide is easy to decompose and the molecular weight is obviously reduced when the tremella polysaccharide is extracted by acid and base; the structure of the tremella polysaccharide can be damaged, and the further research of the tremella polysaccharide is influenced.
The invention content is as follows:
in order to solve the problems and overcome the defects of the prior art, the invention provides the extraction and preparation method of the high molecular weight tremella polysaccharide, which can effectively solve the problem that the tremella polysaccharide is easy to decompose and structurally destroy during acid-base extraction
The second technical problem to be solved is that: during the extraction process, some non-sugar substances are proposed, such as white fungus polysaccharide with glycoprotein, and the glycoprotein is difficult to separate and purify subsequent crude polysaccharide.
The specific technical scheme for solving the technical problems comprises the following steps: the extraction and preparation method of the high molecular weight tremella polysaccharide is characterized by comprising a wall breaking process and a protein removing process;
the wall breaking process comprises the steps of adding tremella powder into water, and then adding chitinase to carry out wall breaking treatment to obtain a treatment solution with tremella thallus protoplasts;
the protein removing process is to add tannic acid or trichloroacetic acid into the treatment solution to obtain a suspension mixed solution containing tremella polysaccharide.
The reaction temperature of the wall breaking process is 38-40 ℃, and the addition amount of the tannic acid or the trichloroacetic acid is 0.2-0.3 per mill of the mass of the treatment fluid
The suspension mixed solution containing the tremella polysaccharide is a mixture of tremella polysaccharide solution and tremella thallus protoplast coated with glycoprotein.
The protein removing process also comprises an extraction process, a separation process, a concentration process and a purification process;
in the extraction process, the temperature of the suspension mixture containing the tremella polysaccharide is raised to 55-60 ℃, and the tremella polysaccharide is extracted from the cell subjected to wall breaking;
the separation process comprises the steps of filtering the mixed solution by using a filtering paper board with the thickness of 0.22 mu m, removing tremella thallus protoplasts containing glycoprotein, and obtaining tremella polysaccharide filtrate;
the concentration process comprises the steps of adding the tremella polysaccharide filtrate into concentration equipment for concentration until the tremella polysaccharide filtrate begins to become viscous;
and in the purification process, the concentrated filtrate is added into ethanol for precipitation of feed liquid, and pure tremella polysaccharide is extracted.
Drying the extracted Tremella polysaccharide in a vacuum oven at 55-60 deg.C until water content is below 7.0%.
The concentration viscosity of the concentration process is 1000mm2/s。
The volume ratio of the extracting solution to the ethanol in the purification procedure is as follows: 1:2-4.
The invention has the beneficial effects that:
the invention carries out wall breaking treatment by using chitinase, can effectively keep a complete cell membrane with biological activity, namely the protoplast, isolates the tremella polysaccharide and glycoprotein wound on the tremella polysaccharide in the protoplast while removing cell walls,
adding tannic acid or trichloroacetic acid, wherein the tannic acid or trichloroacetic acid can smoothly pass through a cell membrane to act on glycoprotein wound on the tremella polysaccharide, and the glycoprotein is denatured and then separated from the tremella polysaccharide; the water-soluble characteristic of the separated tremella polysaccharide can smoothly pass through a cell membrane to the outside of the cell membrane, so that the aim of separating the denatured glycoprotein from the tremella polysaccharide is fulfilled;
the high-molecular weight tremella polysaccharide can be obtained, and simultaneously, the high yield and low protein impurity of the tremella polysaccharide are ensured.
The specific implementation mode is as follows:
in the description of the invention, specific details are given only to enable a full understanding of the embodiments of the invention, but it should be understood by those skilled in the art that the invention is not limited to these details for the implementation. In other instances, well-known structures and functions have not been described or shown in detail to avoid obscuring the points of the embodiments of the invention. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The specific implementation mode of the invention is as follows:
z1, adding the tremella powder into a reaction tank filled with water and heated to 38-40 ℃, and adding chitinase to perform wall breaking treatment;
z2, adding tannic acid or trichloroacetic acid into the reaction tank for deproteinization treatment, wherein the addition amount of the tannic acid or the trichloroacetic acid is 0.2-0.3 per mill of the mass of the treatment solution;
z3, raising the temperature of the reaction tank to 55-60 ℃, and extracting the tremella polysaccharide from the cell subjected to wall breaking; this enables the extraction time to be reduced from 4 hours to 2 hours;
z4, filtering with 0.22 μm filter paper board, removing glucoprotein-containing Tremella thallus protoplast to obtain Tremella polysaccharide extract;
z5, adding the tremella polysaccharide filtrate into a concentration device for concentration until the concentration viscosity is 900-2/s；
Z6, adding the concentrated filtrate into absolute ethyl alcohol for precipitation of feed liquid, and extracting pure tremella polysaccharide;
and Z7, drying the extracted tremella polysaccharide in a vacuum oven at 55-60 ℃ until the water content is controlled below 7.0%.
In order to more intuitively show the process advantages of the wall breaking process and the protein removing process, the wall breaking process and the protein removing process are compared,
comparative example one:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, no chitinase is added for wall breaking;
comparative example two:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, no chitinase is added for wall breaking; and tannic acid or trichloroacetic acid is not added into the treatment liquid for deproteinization;
comparative example three:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, no chitinase is added for wall breaking; replacing tannic acid or trichloroacetic acid in the protein removing process with strong alkali, and removing protein denaturation with strong alkali;
comparative example four:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, a wall breaking machine is adopted for crushing treatment to carry out wall breaking treatment;
comparative example five:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, a wall breaking machine is adopted for crushing treatment to carry out wall breaking treatment; and tannic acid or trichloroacetic acid is not added into the treatment liquid for deproteinization;
comparative example six:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, a wall breaking machine is adopted for crushing treatment to carry out wall breaking treatment; replacing tannic acid or trichloroacetic acid in the protein removing process with strong alkali, and removing protein denaturation with strong alkali;
comparative example seven:
the preparation method is the same as the embodiment except that: in the preparation process of the comparative example, the wall breaking treatment is carried out by using common strong base;
according to standard QB/T4416-2012, namely sodium hyaluronate for cosmetic raw material, the yield, molecular weight and protein impurity of tremella polysaccharide are respectively detected, and the test data are as follows:
table 1: influence of different processes on extraction of tremella polysaccharide
| Group of | Breaking cell wall | Deproteinization | Yield of Tremella polysaccharide | Molecular weight of Tremella polysaccharide | Protein impurities |
| The invention | Chitinase | Tannic or trichloroacetic acid | 40％-45％ | 150-160WDa | ≤0.005％ |
| Comparative example 1 | Is free of | Tannic or trichloroacetic acid | ≤0.1％ | - | ≤0.05％ |
| Comparative example No. two | Is free of | Is free of | ≤0.1％ | - | ≤0.05％ |
| Comparative example No. three | Is free of | Strong base | 25％-30％ | 80-110WDa | 0.1％-0.5％ |
| Comparative example No. four | Wall breaking machine | Tannic or trichloroacetic acid | 25％-30％ | 110-130WDa | 0.1％-0.5％ |
| Comparative example five | Wall breaking machine | Is free of | 25％-30％ | 110-130WDa | 0.8％-1％ |
| Comparative example six | Wall breaking machine | Strong base | 25％-30％ | 80-110WDa | 0.1％-0.5％ |
| Comparative example seven | Strong base | Strong base | 25％-30％ | 80-100WDa | 0.1％-0.5％ |
From the above table data analysis, it can be seen that:
1. from the data analysis of comparative example one and comparative example two, it can be seen that: because the wall breaking treatment is not carried out in the first and second comparative examples, the tremella polysaccharide in the cells cannot be dissolved out, the yield of the tremella polysaccharide is low, and glycoprotein wound on the tremella polysaccharide cannot be dissolved out; the content of glycoprotein wound on the tremella polysaccharide is low and is lower than 0.05 percent;
2. from the data analysis of comparative example three, it can be seen that: although wall breaking treatment is not carried out, strong alkali for protein removal can still damage cell walls, so that the structures of cell walls and cell membranes are damaged, at the moment, tremella polysaccharide in cells and glycoprotein wound on the tremella polysaccharide are dissolved out, so that a large amount of denatured glycoprotein exists in the tremella polysaccharide, the glycoprotein is difficult to separate and purify subsequent crude polysaccharide, and the strong alkali causes the reduction of the molecular weight of the tremella polysaccharide;
3. from the data analysis of comparative example four, it can be seen that: the wall breaking machine is used for wall breaking treatment, so that the structures of cell walls and cell membranes are damaged, the tremella polysaccharides in the cells and the glycoprotein wound on the tremella polysaccharides are dissolved out, and the tannic acid or trichloroacetic acid is added in the subsequent process to denature the glycoprotein wound on the tremella polysaccharides, so that the glycoprotein wound on the tremella polysaccharides is reduced to a certain extent and is maintained at 0.1-0.5%, but the denatured protein is still difficult to separate and remove;
4. from the data analysis of comparative example five, it can be seen that: the wall breaking machine is used for wall breaking treatment, so that the structures of cell walls and cell membranes are damaged, the tremella polysaccharide in the cells and the glycoprotein wound on the tremella polysaccharide are dissolved out, and a protein removing preparation is not added at the later stage, so that a large amount of glycoprotein is wound on the tremella polysaccharide, and the protein content in the product is higher by 0.8% -1%;
5. from the data analysis of comparative example six and comparative example seven, it can be seen that: as long as strong base is added, the structures of cell walls and cell membranes are damaged whether wall breaking treatment is carried out or not, the tremella polysaccharide in cells and glycoprotein wound on the tremella polysaccharide are dissolved out, so that a large amount of denatured glycoprotein exists in the tremella polysaccharide, the glycoprotein is difficult to separate and purify subsequent crude polysaccharide, and the strong base causes the reduction of the molecular weight of the tremella polysaccharide;
6. the invention can be compared with other comparative examples to know that:
the invention carries out wall breaking treatment by using chitinase, can effectively keep complete cell membranes with biological activity, namely protoplasts, can isolate the tremella polysaccharide and glycoprotein wound on the tremella polysaccharide in the protoplasts while removing cell walls,
at the moment, tannic acid or trichloroacetic acid is added, and the tannic acid or trichloroacetic acid can smoothly pass through a cell membrane to act on glycoprotein wound on the tremella polysaccharide, so that the glycoprotein is denatured and then is separated from the tremella polysaccharide; the separated tremella polysaccharide is a water-soluble substance and can smoothly pass through a cell membrane to the outside of the cell membrane, so that the purpose of separating the denatured glycoprotein from the tremella polysaccharide is achieved, the problem that the denatured protein is difficult to separate and purify subsequent crude polysaccharide is solved, and only the protoplast containing the denatured protein and the liquid containing the tremella polysaccharide are filtered and separated;
the method can obtain the high molecular weight tremella polysaccharide, and simultaneously ensures the high yield and low protein impurity of the tremella polysaccharide.
In summary, the following steps: the invention carries out wall breaking treatment by using chitinase, can effectively keep complete cell membranes with biological activity, namely protoplasts, can isolate the tremella polysaccharide and glycoprotein wound on the tremella polysaccharide in the protoplasts while removing cell walls,
adding tannic acid or trichloroacetic acid, wherein the tannic acid or trichloroacetic acid can smoothly pass through a cell membrane to act on glycoprotein wound on the tremella polysaccharide, and the glycoprotein is denatured and then separated from the tremella polysaccharide; the water-soluble characteristic of the separated tremella polysaccharide can smoothly pass through a cell membrane to the outside of the cell membrane, so that the aim of separating the denatured glycoprotein from the tremella polysaccharide is fulfilled;
the high-molecular weight tremella polysaccharide can be obtained, and simultaneously, the high yield and low protein impurity of the tremella polysaccharide are ensured.
Claims (6)
1. A method for extracting and preparing high molecular weight tremella polysaccharide is characterized by comprising a wall breaking process and a protein removing process;
the wall breaking process comprises the steps of adding tremella powder into water, and then adding chitinase to carry out wall breaking treatment to obtain a treatment solution with tremella thallus protoplasts;
the protein removing process is to add tannic acid or trichloroacetic acid into the treatment solution to obtain a suspension mixed solution containing tremella polysaccharide.
2. The method for extracting and preparing high molecular weight tremella polysaccharide as claimed in claim 1, wherein the reaction temperature of the wall breaking process is 38-40 ℃, and the addition amount of tannic acid or trichloroacetic acid is 0.2-0.3 ‰ of the mass of the treatment solution;
the suspension mixed solution containing the tremella polysaccharide is a mixture of tremella polysaccharide solution and tremella thallus protoplast coated with glycoprotein.
3. The method for extracting and preparing high molecular weight tremella polysaccharide as claimed in claim 1, further comprising an extraction process, a separation process, a concentration process and a purification process after the protein removing process;
in the extraction process, the temperature of the suspension mixture containing the tremella polysaccharide is raised to 55-60 ℃, and the tremella polysaccharide is extracted from the cell subjected to wall breaking;
the separation process comprises the steps of filtering the mixed solution by using a filtering paper board with the thickness of 0.22 mu m, removing tremella thallus protoplasts containing glycoprotein, and obtaining tremella polysaccharide filtrate;
the concentration process comprises the steps of adding the tremella polysaccharide filtrate into concentration equipment for concentration until the tremella polysaccharide filtrate begins to become viscous;
and in the purification process, the concentrated filtrate is added into ethanol for precipitation of feed liquid, and pure tremella polysaccharide is extracted.
4. The method for extracting and preparing high molecular weight Tremella polysaccharide according to claim 3, wherein the extracted Tremella polysaccharide is dried in a vacuum oven at 55-60 deg.C until water content is controlled below 7.0%.
5. The method for extracting and preparing high molecular weight Tremella polysaccharides as claimed in claim 3, wherein the concentration viscosity of the concentration process is 900-2/s。
6. The method for extracting and preparing high molecular weight tremella polysaccharide as claimed in claim 3, wherein the volume ratio of the extracting solution to the ethanol in the purification process is: 1:2-4.
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Cited By (1)
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| CN116649577A (en) * | 2023-06-28 | 2023-08-29 | 临沂欣宇辉生物科技有限公司 | Method for preparing anti-aging nutrition by extracting natural tremella aurantialba polysaccharide |
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| US20180186899A1 (en) * | 2005-06-14 | 2018-07-05 | Genis Hf. | Compositions of partially deacetylated chitin derivatives |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116649577A (en) * | 2023-06-28 | 2023-08-29 | 临沂欣宇辉生物科技有限公司 | Method for preparing anti-aging nutrition by extracting natural tremella aurantialba polysaccharide |
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