CN115746160A - Method for reducing viscosity of polysaccharide - Google Patents
Method for reducing viscosity of polysaccharide Download PDFInfo
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- CN115746160A CN115746160A CN202211525324.8A CN202211525324A CN115746160A CN 115746160 A CN115746160 A CN 115746160A CN 202211525324 A CN202211525324 A CN 202211525324A CN 115746160 A CN115746160 A CN 115746160A
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for reducing viscosity of polysaccharide, belonging to the technical field of preparation of natural products. The method of the invention comprises the following steps: adding the gracilaria verrucosa powder into the aqueous solution, stirring and extracting to obtain a gracilaria verrucosa polysaccharide extracting solution; concentrating, adding ethanol into the concentrated solution, stirring, centrifuging, and filtering to obtain precipitate; drying and crushing the obtained precipitate to obtain gracilaria fragile polysaccharide; putting into a reaction kettle, adding water, stirring, reacting, and cooling to obtain low-viscosity gracilaria polysaccharide solution; centrifuging and filtering the low-viscosity gracilaria polysaccharide solution, purifying, drying, grinding and packaging. The prepared low-viscosity crispy gracilaria verrucosa has obviously reduced apparent viscosity and obviously increased solubility, and does not have the characteristic of high-concentration gelatinization any more. The method is simple, efficient, economical and environment-friendly, improves the fluidity of the high-viscosity polysaccharide solution, and has wide application range.
Description
Technical Field
The invention relates to a method for reducing viscosity of polysaccharide, belonging to the technical field of preparation of natural products.
Background
Polysaccharides are natural polymeric carbohydrates polymerized from more than 20 monosaccharides, and can be classified into primary, secondary, tertiary, and quaternary structures. The polysaccharide molecules exist in solution in the form of random coils, the compactness of which is related to the composition and the connection form of the monosaccharides; when such molecules rotate in a solution, they occupy a large space, and the probability of collision between the molecules increases, and the friction between the molecules increases, thereby having a high viscosity. Part of the polysaccharide has a high viscosity even at very low concentrations. The viscosity research of the polysaccharide aqueous solution shows that the polysaccharide aqueous solution is closely related to the primary structure of the polysaccharide, such as molecular weight, the connection mode of glycosidic bonds, the existence of branches and the like, and is also closely related to the higher structure, such as the conformational change of the polysaccharide and the change of polysaccharide-water interaction.
The high viscosity of the polysaccharide gives the polysaccharide good gelling property and the efficacy of relaxing bowel and the like, but the high viscosity also increases inconvenience for the production and application of the polysaccharide, and particularly the solubility of the polysaccharide after drying is poor. Meanwhile, the high viscosity property also limits the application range and concentration of the product and increases energy consumption, and the progress of solving the problem is slow. Therefore, certain technologies and methods are needed to effectively reduce the viscosity of the polysaccharide, increase the solubility, enhance the fluidity of the polysaccharide solution, and simultaneously ensure low cost, greenness and no pollution, so that the application range of the polysaccharide is enlarged, and the benefit is increased.
Disclosure of Invention
In order to solve the problems of high viscosity and poor solubility of the macromolecular polysaccharide, the invention provides a method for reducing the viscosity of the polysaccharide, which comprises the steps of carrying out heat treatment on the gracilaria bursa-pastoris and then carrying out polysaccharide extraction.
In one embodiment, the thermal treatment includes, but is not limited to, hydrothermal treatment or enzyme-assisted hydrothermal treatment.
The invention also provides the application of the method in the preparation of low-viscosity polysaccharide.
In one embodiment, the application comprises the steps of:
(1) Cleaning and drying the gracilaria verrucosa, pulverizing and sieving to obtain gracilaria verrucosa powder;
(2) Adding the fragile gracilaria powder prepared in the step (1) into an aqueous solution, and heating and extracting to obtain a fragile gracilaria polysaccharide extracting solution;
(3) Filtering the gracilaria verrucosa polysaccharide extracting solution obtained in the step (2) to obtain filtrate A; concentrating the filtrate A to obtain a concentrated solution A; adding ethanol into the concentrated solution A, and stirring to obtain a suspension A; standing the suspension, centrifuging, and filtering to obtain precipitate;
(4) Drying and crushing the precipitate obtained in the step (3) to obtain coarse gracilaria crispa polysaccharide;
(5) Putting the Gracilaria verrucosa crude polysaccharide obtained in the step (4) and a free radical inhibitor into a reaction kettle, adding water, stirring, and cooling after the reaction is finished to obtain a Gracilaria verrucosa crude polysaccharide mixed solution;
(6) Centrifuging and filtering the mixture of the coarse gracilaria verrucosa polysaccharide obtained in the step (5) to obtain filtrate B; purifying the filtrate B to obtain purified polysaccharide;
(7) Drying, grinding and packaging the purified polysaccharide.
In one embodiment, the ratio of the gracilaria verrucosa powder to the aqueous solution in step (2) is 1: (20-120) g/ml.
In one embodiment, the temperature of the aqueous solution during the extraction in step (2) is 50-100 deg.C, and the extraction time is 0.5-6h.
In one embodiment, the extraction method in step (2) comprises one of water extraction, acid extraction, alkali extraction and enzyme extraction, ultrasonic-assisted extraction, pulsed electric field-assisted extraction and microwave-assisted extraction.
In one embodiment, the aqueous solution used in the water extraction process is purified water; the aqueous solution used in the acid extraction process is an acid solution.
In one embodiment, the acid is one or more of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, perchloric acid, phosphoric acid, or formic acid.
In one embodiment, the aqueous solution used in the caustic extraction is an alkaline solution, and the alkali includes, but is not limited to, one or more of sodium hydroxide, potassium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate, and ammonium hydroxide.
In one embodiment, the aqueous solution used in the enzyme extraction is an enzyme solution; the enzyme includes but is not limited to one or more of amylase, pectinase and cellulase.
In one embodiment, the acid solution has a pH of 1 to 3; the concentration of the alkali solution is (0.01-2) mol/L, and the pH value of the filtrate A is 6-8.
In one embodiment, the mass fraction of ethanol in suspension a in step (3) is 50-90%.
In one embodiment, the mass volume ratio of the Gracilaria verrucosa crude polysaccharide to water in step (5) is 1 (1-100) g/ml; the reaction temperature is 120-180 ℃, and the reaction time is 5-60min after the central temperature of the raw materials of the reaction kettle reaches the reaction temperature. The final concentration of the free radical inhibitor is 0-500 Mm.
In one embodiment, the free radical inhibitor in step (5) includes, but is not limited to, one or more of Butylated Hydroxyanisole (BHA), 2, 6-di-tert-butyl-p-cresol (BHT), propyl Gallate (PG), tert-butylhydroquinone (TBHQ), 2, 6-tetramethylpiperidine oxide (TEMPO), 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), disodium Ethylenediaminetetraacetate (EDTA), dimethylsulfoxide (DMSO).
In one embodiment, the purification method in step (6) is one of ultrafiltration concentration, dialysis, ethanol precipitation separation, column chromatography, and the like; the aperture of the ultrafiltration membrane is 300-10000Da; the aperture of the dialysis bag is 300-10000Da; the mass fraction of the ethanol is 30-95%; the filler for column chromatography includes, but is not limited to, one or more of agarose gel and its different ligand derivatives, dextran gel and its different ligand derivatives, and macroporous resin.
Has the advantages that:
the invention takes polysaccharide as raw material, and can obviously reduce the viscosity of polysaccharide solution through heat treatment; the method has certain broad spectrum property for reducing the viscosity of the polysaccharide, and has no requirements on the source of the polysaccharide and the extraction mode of the polysaccharide; the effect of adding water for redissolving after the viscosity of the polysaccharide is reduced by heat treatment is better than that of the untreated polysaccharide; the invention mainly reduces the viscosity of the polysaccharide by influencing the high-level structure of the polysaccharide, and has no great influence on the primary structure of the polysaccharide and the composition of monosaccharide.
Drawings
FIG. 1 is the apparent viscosity of Gracilaria gracilaria polysaccharide GCP1 before and after hydrothermal treatment.
FIG. 2 is the apparent viscosity chart of Gracilaria verrucosa polysaccharide GCP1 and Gracilaria verrucosa polysaccharide GCP3 with low viscosity.
FIG. 3 is the apparent viscosity of Gracilaria verrucosa polysaccharide GCP4 before and after hydrothermal treatment.
Detailed Description
Example 1 extraction of Gracilaria verrucosa polysaccharide with Hot Water as raw Material A Low-viscosity Gracilaria verrucosa polysaccharide was prepared by hydrothermal method
100g of gracilaria verrucosa powder is weighed and added into 6L of preheated purified water, and stirring extraction is carried out for 3h at 95 ℃ to obtain gracilaria verrucosa polysaccharide extracting solution. Filtering the gracilaria verrucosa polysaccharide extracting solution by using 8 layers of gauze to obtain a filtrate A, and performing rotary evaporation and concentration to obtain a concentrated solution A, wherein the volume ratio of the filtrate A to the concentrated solution A is 2. Slowly adding absolute ethanol into the concentrated solution A, continuously stirring until the mass fraction of the ethanol is 70% to obtain a suspension A, standing at 4 ℃ for precipitation for 12h, and centrifuging at 5000r/min for 10min to obtain a precipitate; the obtained precipitate is freeze-dried and pulverized to obtain gracilaria polysaccharide GCP1, and the apparent viscosity is 1.41Pa.s at shear rate of 1/s.
1g of Gracilaria verrucosa rough polysaccharide GLP1 is taken and put into a 25mL reaction kettle, 10mL of deionized water is added and stirred evenly, after 40min of reaction at 150 ℃, the mixture is rapidly cooled in ice bath, and the low-viscosity Gracilaria verrucosa polysaccharide solution is obtained. Filtering the crude polysaccharide hydrolysate by using filter paper to obtain filtrate B; and (4) performing ultrafiltration purification on the filtrate B by a 10000Da ultrafiltration membrane to obtain trapped fluid. And (3) carrying out rotary evaporation and concentration on the trapped fluid to obtain a concentrated solution B, wherein the volume ratio of the filtrate B to the concentrated solution 2 is 6. Drying the precipitate, grinding, and packaging to obtain low viscosity gracilaria polysaccharide GCP2 with apparent viscosity of 0.083Pa.s at shear rate of 1/s.
FIG. 1 shows the apparent viscosity of Gracilaria bursa polysaccharides GCP1 before and after hydrothermal treatment, and it can be seen that the apparent viscosity of the polysaccharides after hydrothermal treatment is significantly reduced, which proves that the hydrothermal method is one of the effective methods for reducing the viscosity of the polysaccharides.
Example 2 extraction of Gracilaria verrucosa polysaccharide GCP1 with Hot Water as raw Material and hydrothermal preparation of Low-viscosity Gracilaria verrucosa polysaccharide
Weighing 0.3g of crude gracilaria verrucosa polysaccharide GCP1, putting into a 10mL total nitrogen screw colorimetric tube, adding 20mM TEMPO, adding 6mL purified water, stirring uniformly, carrying out oil bath reaction at 165 ℃ for 10min, and naturally cooling to obtain a low-viscosity gracilaria verrucosa polysaccharide solution. Filtering the low-viscosity gracilaria verrucosa polysaccharide solution by filter paper, collecting filtrate, and performing ultrafiltration purification on the filtrate by a 3000Da ultrafiltration membrane to obtain trapped fluid. And (3) carrying out rotary evaporation and concentration on the trapped solution to obtain a concentrated solution, wherein the volume ratio of the filtrate to the concentrated solution is 5. Adding anhydrous ethanol into the concentrated solution until the final concentration of ethanol is 85%, standing at 4 deg.C for precipitation for 12 hr, and centrifuging at 6500r/min for 8min to obtain precipitate. Drying the precipitate, grinding, and packaging to obtain low viscosity gracilaria polysaccharide GCP3 with apparent viscosity of 0.0062Pa.s at shear rate of 1/s.
Figure 2 shows the apparent viscosities of gracilaria verrucosa polysaccharide GCP1 and low viscosity gracilaria polysaccharide GCP3, and it can be seen from figure 2 that the apparent viscosity of polysaccharide is significantly reduced after hydrothermal treatment, which proves that the hydrothermal method is one of the effective methods for reducing polysaccharide viscosity.
Example 3 enzyme-assisted preparation of Low-viscosity Gracilaria polysaccharide from Gracilaria crude polysaccharide by hydrothermal method
Weighing 50g of gracilaria verrucosa powder, adding into 3L of purified water, adding 25U/mL amylase and 25U/mL cellulase according to a final concentration meter, treating at 60 ℃ for 1h, extracting at 90 ℃ for 2h, cooling to room temperature, centrifuging at 8000r/min for 10min, collecting supernatant, and concentrating the collected supernatant at 60 ℃ under reduced pressure to obtain a concentrated solution A, wherein the volume ratio of the supernatant to the concentrated solution A is 3.
And slowly adding absolute ethyl alcohol into the concentrated solution A, continuously stirring until the mass fraction of the ethyl alcohol is 85% to obtain a suspension A, and placing the suspension A in a refrigerator at the temperature of 4 ℃ for overnight. Centrifuging the suspension 1 at 5500r/min for 12min, filtering, collecting the filter residue, re-dissolving the filter residue with a small amount of water, concentrating, vacuum freeze drying, pulverizing to obtain Gracilaria bursa polysaccharide GCP4, and measuring the apparent viscosity at a shear rate of 1/s to be 1.30Pa.s.
Weighing 1g of crude gracilaria verrucosa polysaccharide GCP4, putting into a 25mL reaction kettle, adding 6mL of deionized water, stirring uniformly, reacting at 180 ℃ for 30min, and rapidly cooling in an ice bath to obtain a low-viscosity gracilaria verrucosa polysaccharide solution. Filtering the low-viscosity gracilaria verrucosa polysaccharide solution by using filter paper, collecting filtrate B, and purifying by using a sephadex column G25 column chromatography to obtain eluent. And (3) carrying out rotary evaporation and concentration on the eluent liquid to obtain a concentrated liquid B, wherein the volume ratio of the eluent to the concentrated liquid B is 4. Adding absolute ethanol into the concentrated solution B until the final concentration of ethanol is 95% to obtain suspension B, standing at 4 deg.C for precipitation for 12h, and centrifuging at 8000r/min for 10min to obtain precipitate. Drying the precipitate, grinding, packaging and packaging to obtain the low viscosity gracilaria polysaccharide GCP5, and measuring its apparent viscosity at shear rate of 1/s to be 0.0073Pa.s.
FIG. 2 shows the apparent viscosity of low-viscosity gracilaria polysaccharide GCP4 before and after hydrothermal treatment, and it can be seen from FIG. 3 that the apparent viscosity of polysaccharide is significantly reduced after hydrothermal treatment, which proves that the hydrothermal method is one of the effective methods for reducing the viscosity of polysaccharide.
Claims (10)
1. A method for reducing the viscosity of gracilaria verrucosa polysaccharide is characterized by comprising the following steps:
(1) Cleaning and drying the Gracilaria verrucosa, crushing and sieving for later use to obtain Gracilaria verrucosa powder;
(2) Adding the fragile gracilaria powder prepared in the step (1) into an aqueous solution, and heating and extracting to obtain a fragile gracilaria polysaccharide extracting solution;
(3) Filtering the gracilaria verrucosa polysaccharide extracting solution obtained in the step (2) to obtain filtrate A; concentrating the filtrate A to obtain a concentrated solution A; adding ethanol into the concentrated solution A, and stirring to obtain a suspension A; standing the suspension, centrifuging, and filtering to obtain precipitate;
(4) Drying and crushing the precipitate obtained in the step (3) to obtain coarse gracilaria crispa polysaccharide;
(5) Putting the gracilaria verrucosa crude polysaccharide obtained in the step (4) and a free radical inhibitor into a reaction kettle, adding water, stirring, and cooling after the reaction is finished to obtain a gracilaria verrucosa crude polysaccharide mixed solution;
(6) Centrifuging and filtering the mixture of the coarse gracilaria verrucosa polysaccharide obtained in the step (5) to obtain filtrate B; purifying the filtrate B to obtain purified polysaccharide;
(7) Drying, grinding and packaging the purified polysaccharide.
2. The method of claim 1, wherein the ratio of the gracilaria friable powder to the aqueous solution in step (2) is 1: (20-120) g/ml; the extraction temperature is 50-100 deg.C, and the extraction time is 0.5-6h.
3. The method of claim 1, wherein the extraction in step (2) is a combination of one or more of water extraction, acid extraction, alkali extraction, enzyme extraction, ultrasound-assisted extraction, pulsed electric field-assisted extraction, or microwave-assisted extraction.
4. The method according to claim 3, wherein the aqueous solution used in the water extraction method is distilled water; the aqueous solution used in the acid extraction method is an acid solution; the aqueous solution used in the alkali extraction method is an alkali solution; the aqueous solution used in the enzyme extraction method is an enzyme solution.
5. The method of claim 4, wherein the acid solution is one or more of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid, perchloric acid, phosphoric acid, or formic acid; the alkali solution is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and ammonium hydroxide; the enzyme is one or more of amylase, pectinase and cellulase.
6. The method of claim 3 or 4, wherein the acid solution has a pH of 1-3; the concentration of the alkali solution is (0.01-2) mol/L, and the pH value of the filtrate A is 6-8.
7. The method for reducing the viscosity of polysaccharide according to claim 1, wherein the mass fraction of ethanol in suspension A in step (3) is 50-90%.
8. The method for reducing the viscosity of polysaccharides in accordance with claim 1, wherein the mass/volume ratio of the Gracilaria bursa crude polysaccharide to water in step (5) is 1 (1-100) g/ml, the final concentration of the free radical inhibitor is ≤ 500mM; the free radical inhibitor in the step (5) is one or more of butyl hydroxy anisol, 2, 6-di-tert-butyl-p-cresol, propyl gallate, tert-butyl hydroquinone, 2, 6-tetramethyl piperidine oxide, 3-methyl-1-phenyl-2-pyrazoline-5-ketone, ethylene diamine tetraacetic acid disodium and dimethyl sulfoxide.
9. The method for reducing the viscosity of polysaccharide according to claim 1, wherein the purification method in step (6) is ultrafiltration concentration, dialysis, ethanol precipitation separation or column chromatography.
10. Use of a process according to any one of claims 1 to 9 for the preparation of a low viscosity polysaccharide or product thereof.
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| CN101475651A (en) * | 2009-01-16 | 2009-07-08 | 伍曾利 | Preparation of Gracilaria gigas Harvey polysaccharides |
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| CN101613419A (en) * | 2008-06-23 | 2009-12-30 | 广东海洋大学 | The method of ultrasonic acid extraction of sulfated polysaccharide from gracilaria tenuistipitata |
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