Extraction method for efficiently separating white tea polysaccharide
By using ionic liquids and cellulase assisted extraction of white tea polysaccharides, combined with ultrafiltration and gel filtration chromatography, the problems of low extraction efficiency and high cost in the prior art are solved, and efficient, green and environmentally friendly polysaccharide extraction is achieved, and yield and purity are improved.
Patent Information
- Application Number
- CN202510535349.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-27
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, the extraction method of white tea polysaccharides has problems such as poor high temperature selectivity, low yield and high cost of supercritical extraction, making it difficult to achieve efficient, green and environmentally friendly polysaccharide extraction.
The ionic liquid of salt substances at room temperature or near room temperature was extracted, combined with cellulase-assisted extraction, and the plant cell wall structure was destroyed by low-temperature operation, and then purified by ultrafiltration and gel filtration chromatography to reduce solvent residue.
It significantly improves the extraction rate and purity of white tea polysaccharides, reduces production costs and environmental burdens, reduces the risk of degradation of heat-sensitive components, and simplifies the purification process.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for efficiently separating and extracting white tea polysaccharides. Background Art
[0002] White tea, belonging to one of the six traditional tea categories in China, has the quality characteristics of natural appearance, full of white hairs, fresh and clear aroma of hairs, clear soup color, light and sweet taste due to its unique manufacturing process. Tea polysaccharides, also known as tea polysaccharides or tea polysaccharides, are an acidic glycoprotein combined with a large number of mineral elements. At present, they have been proven to have functions such as lowering blood sugar, lowering blood lipids, regulating immunity, anticoagulation, and antioxidation, and are a natural active substance with great development potential.
[0003] However, it is not easy to efficiently extract and purify these polysaccharide components from white tea. The traditional hot water extraction method is one of the most commonly used methods for extracting polysaccharides from plant materials due to its simplicity, feasibility, and low cost. However, the extraction operation requires a long time and high temperature, and has poor selectivity, which will cause the degradation of thermosensitive components and a very low yield. To improve the yield, in recent years, researchers have chosen to use supercritical extraction to extract thermosensitive and volatile compounds. However, due to the large investment in equipment and high operation and maintenance costs of this method, it is difficult to promote on a large scale. For polysaccharide substances with relatively high polarity, the effect of using carbon dioxide alone is not ideal, and a co-solvent needs to be added, increasing the cost and complexity. Summary of the Invention
[0004] Aiming at the problems of poor selectivity at high temperature and low yield of the hot water extraction method and high cost and high technical difficulty of the supercritical extraction method mentioned in the above background art in the prior art, the present invention provides a method for efficiently separating and extracting white tea polysaccharides, using salt substances that are liquid at room temperature or near room temperature, relying on their characteristics of low volatility, good thermal stability, chemical stability, and adjustable solubility to extract white tea polysaccharides. While improving the yield of white tea polysaccharides, it is green and environmentally friendly, and can also effectively reduce the problem of solvent residues.
[0005] The technical solution adopted by the present invention is as follows:
[0006] A method for efficiently separating and extracting white tea polysaccharides, comprising the following steps:
[0007] 1) Dry the selected white tea dry leaves to constant weight, crush and sieve them, then mix them with an ionic liquid, heat up and react, and after standing and filtering, obtain the primary extraction solution of white tea polysaccharides;
[0008] 2) Mix the crude extraction solution of white tea polysaccharides obtained in step 1) with an ionic liquid, heat up and react, and after standing and filtering, obtain the secondary extraction solution of white tea polysaccharides;
[0009] 3) Add cellulase to the secondary extraction solution of white tea polysaccharide obtained in step 2) for enzymatic hydrolysis, cool and centrifuge, and extract the supernatant to obtain the tertiary extraction solution of white tea polysaccharide;
[0010] 4) Slowly add absolute ethanol to the tertiary extraction solution of white tea polysaccharide, let it stand for 8 - 10 h, centrifuge, wash and then dry to obtain crude white tea polysaccharide;
[0011] 5) Dissolve the crude white tea polysaccharide, ultrafilter, subject it to gel filtration chromatography, then concentrate and dry to obtain refined white tea polysaccharide.
[0012] In the present invention, by selecting ionic liquid to extract white tea leaves and using cellulase for assisted extraction, the extraction efficiency and yield are greatly improved, the co - extraction of impurities is reduced, and the subsequent purification process is simplified; it can be operated at a lower temperature to reduce the degradation risk of thermosensitive components. After removing small - molecule impurities by ultrafiltration, gel filtration chromatography is used for fine purification to ensure the final purity of the product.
[0013] Preferably, the present invention selects old - leaf white tea with a low tea grade.
[0014] Preferably, in step 1), it passes through a 40 - mesh sieve.
[0015] Furthermore, in step 1), the ionic liquid is one or more of 1 - ethyl - 3 - methylimidazolium chloride, tetrabutylammonium hexafluorophosphate, and choline acetate.
[0016] Furthermore, in step 1), the concentration of the ionic solution is 5 - 10 vol%.
[0017] Furthermore, in step 1), the addition ratio of white tea powder to ionic liquid is 1:(25 - 40) (w / v).
[0018] Furthermore, in step 1), the reaction temperature is 70 - 80 °C and the reaction time is 2 - 3 h.
[0019] Furthermore, in step 3), the addition amount of cellulase is 0.5 - 5% of the weight of the white tea powder.
[0020] Furthermore, in step 3), the temperature of the enzymatic hydrolysis reaction is 45 - 50 °C, the pH value is 5.0 - 5.2, and the enzymatic hydrolysis reaction time is 3 - 5 h.
[0021] Furthermore, in step 4), the addition amount of absolute ethanol is 4 - 5 times the volume of the tertiary extraction solution of white tea polysaccharide.
[0022] Furthermore, in step 4), after centrifugation, it is washed successively with absolute ethanol and acetone, and the number of washing times is 3 - 5 times.
[0023] Preferably, in step 4), the centrifugation speed is 4000-4500 rpm and the centrifugation time is 20-25 min.
[0024] Furthermore, in step 5), the molecular weight cut-off of the ultrafiltration membrane is 10-15 kDa.
[0025] Preferably, in step 5), Sephadex G-200 is selected for gel filtration chromatography.
[0026] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:
[0027] 1) The present invention uses ionic liquids for extraction, effectively destroying the plant cell wall structure under relatively mild conditions, significantly improving the extraction rate of white tea polysaccharides; introducing specific enzymes for assisted extraction further enhances the destruction effect on the cell wall structure, promotes the release of polysaccharides, further improves the extraction efficiency, reduces the co-extraction of impurities, and simplifies the subsequent purification process.
[0028] 2) The present invention operates at a relatively low temperature, reducing the degradation risk of thermosensitive components.
[0029] 3) The present invention uses ultrafiltration to remove small molecule impurities, and then uses gel filtration chromatography for fine purification to ensure a high concentration of the finally produced white tea polysaccharide product.
[0030] 4) The ionic liquids used in the present invention can be recycled, reducing production costs and environmental burdens. Specific Embodiments
[0031] All features disclosed in this specification, or all steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any manner.
[0032] Example 1
[0033] An extraction method for efficient separation of white tea polysaccharides
[0034] 1) Ionic liquid extraction
[0035] Dry the old white tea leaves until constant weight, pulverize and pass through a 40-mesh sieve. Then, weigh 10 g of white tea powder and add it to 300 ml of an aqueous solution containing 5% 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl). Stir and extract at 70 °C for 2 h. After standing for 3 h, filter to obtain the primary extract of white tea polysaccharides; then, continue to add 300 ml of 5% [EMIM]Cl solution to the primary extract of white tea, raise the temperature to 70 °C, stir and react for 2 h, stand for 3 h, and filter to obtain the secondary extract of white tea polysaccharides.
[0036] 2) Enzymatic reaction-assisted extraction
[0037] Add 200 mg of cellulase to the secondary extraction solution of white tea polysaccharide obtained in step 1), carry out enzymatic hydrolysis treatment at 45 °C and pH = 5.0 for 3 h. After cooling to room temperature, centrifuge at 4000 rpm for 20 min, and remove the supernatant to obtain the tertiary extraction solution of white tea polysaccharide.
[0038] 3) Impurity removal
[0039] Add 4 times the volume of absolute ethanol to the tertiary extraction solution of white tea polysaccharide obtained in step 2), let it stand for 8 h, centrifuge at 4000 rpm for 20 min, and collect the precipitate.
[0040] Wash the precipitate with absolute ethanol and acetone three times in sequence to obtain crude white tea polysaccharide.
[0041] 4) Refinement
[0042] Filter the crude white tea polysaccharide prepared in step 3) through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, then perform fine purification through a Sephadex G-200 gel filtration chromatography column, and carry out rotary evaporation and concentration at 50 °C in a water bath and 60 r / min until it is concentrated to 10% of the original volume, and then freeze-dry to obtain white tea polysaccharide.
[0043] 5) Result detection
[0044] Detect the total sugar content (phenol-sulfuric acid method), protein content (Bradford method), ultraviolet absorption spectra (at 260 nm and 280 nm), and molecular weight distribution (gel filtration chromatography) of the white tea polysaccharide prepared in step 4).
[0045] Comparative example 1
[0046] The difference between this comparative example and Example 1 is only that in this comparative example, in step 1), ionic liquid is not used for extraction, and the extraction reagent is only water, and the rest of the process is the same as that in Example 1. Among them, the specific operation of step 1) in this comparative example is as follows:
[0047] Dry the dry white tea old leaves to constant weight, crush and pass through a 40-mesh sieve, then weigh 10 g of white tea powder, add it to 300 ml of aqueous solution, stir and extract at 70 °C for 2 h, let it stand for 3 h and then filter to obtain the primary extraction solution of white tea polysaccharide; then, continue to add 300 ml of aqueous solution to the primary extraction solution of white tea, raise the temperature to 70 °C, stir and react for 2 h, let it stand for 3 h, and filter to obtain the secondary extraction solution of white tea polysaccharide.
[0048] Table 1 Influence of ionic solution extraction on product performance
[0049]
[0050] From the data in Table 1, it can be concluded by comparison that using ionic liquid as the extractant can significantly improve the release efficiency of polysaccharides. At the same time, due to the certain selectivity of ionic liquid, it can reduce the co-extraction of non-target components and protect the original structure of polysaccharides, avoiding excessive degradation.
[0051] Comparative Example 2
[0052] The difference between this comparative example and Example 1 is only that cellulase was not used for assisted extraction in this comparative example, and the rest of the process was the same as that in Example 1.
[0053] Table 2 Influence of enzymatic hydrolysis reaction-assisted extraction on the performance of white tea polysaccharide products
[0054]
[0055] From the data in Table 2, it can be concluded by comparison that assisted extraction with cellulase can improve the total sugar content and yield of the finally produced white tea polysaccharides to a certain extent. At the same time, the impurity content decreases and the molecular weight distribution band becomes narrower. It is speculated that the reason is that cellulase can further destroy the cell wall structure, promote the release of polysaccharides, and significantly improve the extraction efficiency and yield. At the same time, the enzyme has selectivity and can also reduce the co-extraction of non-target components to a certain extent and increase the product concentration.
[0056] Comparative Example 3
[0057] The difference between this comparative example and Example 1 is only that in this comparative example, the concentration of the aqueous solution of 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl) in step 1) was 2%, and the rest of the process was the same as that in Example 1.
[0058] Comparative Example 4
[0059] The difference between this comparative example and Example 1 is only that in this comparative example, the concentration of the aqueous solution of 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl) in step 1) was 15%, and the rest of the process was the same as that in Example 1.
[0060] Table 3 Influence of the concentration of ionic liquid on the performance of white tea polysaccharide products
[0061]
[0062]
[0063] From the data in Table 3, it can be concluded by comparison that either too high or too low concentration of ionic liquid will lead to a decrease in extraction efficiency and an increase in impurity content. The possible reasons are as follows: when the ionic concentration is too low, due to insufficient dissolution ability and permeability, the cell wall structure cannot be effectively destroyed, resulting in insufficient release of polysaccharides, more residual impurities, and a decrease in yield; when the ionic liquid concentration is too high, it will lead to the co-extraction of non-target components, diluting the proportion of white tea polysaccharides and increasing the impurity content.
[0064] Unless otherwise specified, the raw materials and equipment used in the present invention are all common raw materials and equipment in the art; unless otherwise specified, the methods used in the present invention are all conventional methods in the art.
[0065] The above embodiments only represent the specific implementation manners of the present application, and the description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application.
Claims
1. An extraction method for efficient separation of white tea polysaccharides, characterized in that, It includes the following steps: 1) Dry the white tea dry tea leaves to constant weight, crush and sieve them, then mix with ionic liquid, heat up and react, let it stand and filter to obtain the primary extract of white tea polysaccharide; 2) Mix the crude white tea polysaccharide solution obtained in step 1) with ionic liquid, heat up and react, let it stand and filter to obtain the secondary extract of white tea polysaccharide; 3) Add cellulase to the secondary extract of white tea polysaccharide obtained in step 2) for enzymatic hydrolysis, cool and centrifuge, extract the supernatant to obtain the tertiary extract of white tea polysaccharide; 4) Slowly add absolute ethanol to the tertiary extract of white tea polysaccharide, let it stand for 8 - 10 h, centrifuge, wash and dry to obtain crude white tea polysaccharide; 5) Dissolve, ultrafilter, gel filtration chromatography, concentrate and dry the crude white tea polysaccharide in sequence to obtain refined white tea polysaccharide.
2. The extraction method for efficient separation of white tea polysaccharides according to claim 1, characterized in that, In step 1), the ionic liquid is one or more of 1 - ethyl - 3 - methylimidazolium chloride, tetrabutylammonium hexafluorophosphate, and choline acetate.
3. The extraction method for efficient separation of white tea polysaccharide according to claim 1 or 2, characterized in that, In step 1), the concentration of the ionic solution is 5 - 10 vol%.
4. The extraction method for efficient separation of white tea polysaccharide according to claim 3, characterized in that, In step 1), the addition ratio of white tea powder to ionic liquid is 1:(25 - 40) (w / v).
5. The extraction method for efficient separation of white tea polysaccharides according to claim 1, characterized in that, In step 1), the reaction temperature is 70 - 80 °C and the reaction time is 2 - 3 h.
6. The extraction method for efficient separation of white tea polysaccharide according to claim 1, characterized in that, In step 3), the addition amount of cellulase is 0.5 - 5% of the weight of white tea powder.
7. The extraction method for efficient separation of white tea polysaccharides according to claim 1 or 6, characterized in that, In step 3), the temperature of the enzymatic hydrolysis reaction is 45 - 50 °C, the pH value is 5.0 - 5.2, and the enzymatic hydrolysis reaction time is 3 - 5 h.
8. The extraction method for efficient separation of white tea polysaccharides according to claim 1, characterized in that, In step 4), the addition amount of absolute ethanol is 4 - 5 times the volume of the tertiary extract of white tea polysaccharide.
9. The extraction method for efficient separation of white tea polysaccharides according to claim 8, characterized in that, After centrifugation, wash with absolute ethanol and acetone in sequence, and the number of washing times is 3 - 5 times.
10. The extraction method for efficient separation of white tea polysaccharide according to claim 1, characterized in that, In step 5), the cut - off molecular weight of the ultrafiltration membrane is 10 - 15 kDa.