CN107964054B - Acid degradation method of hericium erinaceus active polysaccharide - Google Patents
Acid degradation method of hericium erinaceus active polysaccharide Download PDFInfo
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- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses an acid degradation method of hericium erinaceus polysaccharide, which comprises the following steps: dissolving, namely dissolving the hericium erinaceus polysaccharide in water to prepare a solution; hydrolyzing, adding acid into the aqueous solution for hydrolysis; neutralizing, namely immediately cooling the solution obtained after hydrolysis, then neutralizing by using an alkali solution, and adjusting the pH value to be neutral; centrifuging, namely centrifuging the solution obtained in the step 3); dialyzing, namely dialyzing the centrifuged supernatant; freeze drying to obtain Hericium erinaceus polysaccharide acid hydrolysate. The hericium erinaceus polysaccharide fragment obtained by the method has good solubility and high activity, can stimulate macrophages, improve the release amount of NO, enhance the phagocytic capacity of the macrophages and exert the immune activity, and has important application value in the preparation of foods, functional foods and medicines for enhancing the immunity.
Description
Technical Field
The invention relates to the field of edible fungus processing, in particular to an acid degradation method of hericium erinaceus active polysaccharide.
Background
Hericium erinaceus (Hericium erinaceus) is a famous edible and medicinal fungus, has mild nature and sweet taste, benefits five internal organs when used as a medicine, helps digestion, and can be used for treating neurasthenia, gastritis, gastric ulcer and the like. The hericium erinaceus polysaccharide is one of the main active ingredients, and has the effects of improving immunity, resisting oxidation, tumors, aging, reducing blood sugar and the like.
The active polysaccharide is a main bioactive substance in edible fungi, and the hericium erinaceus beta-glucan H6PC20 with the molecular weight of 200 ten thousand Da is extracted from the fruiting body of the hericium erinaceus in a laboratory, but the polysaccharide has high viscosity and low solubility, so that the deep development and utilization of the polysaccharide are limited. The polysaccharide has the advantages of high purity, light color, large molecular weight, good activity, simple preparation, etc. A macromolecule with in vitro immunocompetence is obtained by separating hericium erinaceus, and the polysaccharide has high viscosity and low solubility in water, so that deep development and utilization of the polysaccharide are limited. The polysaccharide derivative with good solubility and higher activity is prepared by adopting a physical or chemical method to carry out proper degradation modification or chemical modification on the polysaccharide derivative, and the development and application of the polysaccharide in foods, health-care products and even medicines can be expanded.
Only a few documents in the prior art disclose that extracted hericium erinaceus polysaccharide is further degraded or hydrolyzed, for example, CN201610935637 discloses a hericium erinaceus active polysaccharide and a preparation method thereof, wherein the hericium erinaceus polysaccharide is subjected to raw material pretreatment, then ultrasonic degradation and finally freeze drying to obtain the required hericium erinaceus active polysaccharide.
However, a method for preparing a polysaccharide derivative having good solubility and higher activity by degrading a polysaccharide of Hericium erinaceum with an acid has not been found.
Disclosure of Invention
In order to solve the technical problems and obtain a hericium erinaceus polysaccharide product with good solubility and higher activity, the invention provides the following technical scheme:
the invention provides an acid degradation method of hericium erinaceus polysaccharide, which comprises the following steps:
1) dissolving, namely dissolving the hericium erinaceus polysaccharide in water to prepare a solution;
2) hydrolyzing, adding acid into the aqueous solution for hydrolysis;
3) neutralizing, namely immediately cooling the solution obtained after hydrolysis, then neutralizing by using an alkali solution, and adjusting the pH value to be neutral;
4) centrifuging, namely centrifuging the solution obtained in the step 3);
5) dialyzing, namely dialyzing the centrifuged supernatant;
6) freeze drying to obtain Hericium erinaceus polysaccharide acid hydrolysate.
According to a preferred embodiment of the invention, the method has one or more of the following: the hericium erinaceus polysaccharide is hericium erinaceus beta-glucan H6PC20, the solubility of the solution in the step 1) is 1-2g/L, the hydrolysis temperature in the step 2) is 90-100 ℃, the alkali solution in the step 3) is NaOH solution, the solution is centrifuged for 15-20min at 8000-12000 Xg in the step 4), and the solution is dialyzed by a 3500Da dialysis bag in the step 5).
According to a preferred embodiment of the present invention, the acid in step 2) is hydrochloric acid, sulfuric acid, trifluoroacetic acid.
According to a preferred embodiment of the invention, the acid is trifluoroacetic acid.
According to a preferred embodiment of the invention, the final concentration of trifluoroacetic acid is 0.1-1.0 mol/L.
According to a preferred embodiment of the invention, the final concentration of trifluoroacetic acid is 0.1-0.5 mol/L.
According to a preferred embodiment of the invention, the hydrolysis time is 30-90 min.
According to a preferred embodiment of the invention, the hydrolysis time is 30-50 min.
In another aspect, the invention provides an acid hydrolysis product of hericium erinaceus polysaccharide, which is prepared by the method.
Preferably, the molecular weight of the Hericium erinaceum polysaccharide acid hydrolysate is 1.97 × 106-6.66×105Da or 1.65X 106-8.80×105Da。
The invention also provides application of the hericium erinaceus polysaccharide acid hydrolysis product in preparing foods, health-care products or medicines for improving immunity.
Advantageous effects
1. The method of the invention carries out appropriate chemical degradation modification on the macromolecular hericium erinaceus beta-glucan H6PC20, and the prepared hericium erinaceus polysaccharide fragment has good solubility and high activity.
2. By adopting the degradation method of the macromolecular hericium erinaceus polysaccharide obtained by the invention, the obtained hericium erinaceus polysaccharide fragment can stimulate macrophages, improve the release amount of NO (nitric oxide), enhance the phagocytic capacity of the macrophages, exert the immunocompetence and expand the development and application of the polysaccharide in foods, health-care products and even medicines.
Drawings
The above and other objects, advantages and features of the present invention will become more apparent to those skilled in the art from the following detailed description of specific embodiments thereof, taken in conjunction with the accompanying drawings.
FIG. 1 is a liquid chromatogram of degraded H6PC20, wherein,
FIG. 1a is a liquid chromatogram of a product after citric acid degradation of H6PC 20;
FIG. 1b is a liquid chromatogram of the product of trifluoroacetic acid degradation of H6PC 20;
FIG. 1c is a liquid chromatogram of the product of hydrochloric acid degradation of H6PC 20;
FIG. 1d is a liquid chromatogram of the product of sulfuric acid degradation of H6PC 20.
Figure 2 shows the trifluoroacetic acid (TFA) degradation reaction time as a function of the relative molecular mass of the H6PC20 degradation product.
FIG. 3 shows the relationship between trifluoroacetic acid degradation reaction time and macrophage NO release after stimulation with the degradation product of H6PC 20.
Figure 4 shows trifluoroacetic acid concentration as a function of H6PC20 degradation product molecular weight.
FIG. 5 shows the relationship between trifluoroacetic acid concentration and the amount of NO released from macrophages stimulated by the degradation product of H6PC 20.
Detailed Description
The invention is further illustrated by the following specific examples in which the materials and equipment are well known to those skilled in the art and are either commercially available or readily available or available.
Example 1: acid species screening
Weighing 4 parts of 100mg macromolecular hericium erinaceus beta-glucan H6PC20, respectively placing the weighed parts in test tubes with plugs, and adding water to dissolve the components to prepare a solution of 2 g/L; then adding citric acid, hydrochloric acid, sulfuric acid and trifluoroacetic acid respectively, wherein the final concentration of the acid is 0.5 mol/L. Placing the test tube with the plug in a water bath at 100 ℃, hydrolyzing for 2h, immediately placing the acidic polysaccharide solution in an ice bath for cooling after complete hydrolysis, then neutralizing with 1M NaOH solution, and adjusting pH to be neutral. Centrifuging at 12000 Xg for 15min to remove impurities. The centrifuged supernatant was dialyzed against 3500Da dialysis bag to remove the acid. Freeze drying to obtain acid hydrolysis product, and measuring the molecular weight distribution diagram of macromolecular Hericium erinaceus polysaccharide H6PC20 after acid hydrolysis by high performance liquid chromatography.
The effect diagram of the hericium erinaceus beta-glucan H6PC20 degraded by citric acid, trifluoroacetic acid, hydrochloric acid and sulfuric acid is shown in figure 1 (wherein, the diagram a is a liquid chromatogram of a product obtained after the H6PC20 is degraded by citric acid, the diagram b is a liquid chromatogram of a product obtained after the H6PC20 is degraded by trifluoroacetic acid, the diagram c is a liquid chromatogram of a product obtained after the H6PC20 is degraded by hydrochloric acid, and the diagram d is a liquid chromatogram of a product obtained after the H6PC20 is degraded by sulfuric acid).
As can be seen from the liquid chromatogram in FIG. a, citric acid is very weak in acidity and can only degrade H6PC20 to 1.227X 106Da, the degradation effect on polysaccharide is not obvious, the subsequent treatment is more complicated, and the dialysis time is long; as can be seen from the liquid chromatogram charts in fig. c and d, the degradation components are more when the sulfuric acid and the hydrochloric acid are degraded, and the components are less separated from each other and difficult to separate; from the liquid chromatogram in FIG. b, it can be seen that trifluoroacetic acid can degrade H6PC20 to 7.016X 105Da, single component, so trifluoroacetic acid degradation is preferred in terms of degradation effect, but this does not indicate that other acids are not suitable for the process of the present invention.
Example 2: trifluoroacetic acid degradation time determination
Weighing 5 parts of 100mg hericium erinaceus beta-glucan H6PC20, respectively putting into test tubes with plugs, and adding water to dissolve the components to prepare a solution of 1 g/L; adding trifluoroacetic acid solution into each tube to make the final concentration of trifluoroacetic acid be 0.5mol/L, sealing the tube, placing in a water bath kettle at 90 ℃, and reacting for 30min, 50min, 70min, 90min and 110min respectively. After the reaction is finished, cooling, centrifuging at 8000 Xg for 20min to remove precipitate, taking the centrifuged supernatant, dialyzing with a 3500Da dialysis bag, removing trifluoroacetic acid, and freeze-drying in vacuum to obtain the degraded hericium erinaceus polysaccharide fragment. And (3) measuring the molecular weight of different polysaccharide fragments by using a high performance liquid chromatography, and measuring the activity of the hericium erinaceus polysaccharides with different molecular weights for stimulating macrophage RAW264.7 to release NO in vitro.
The relative molecular mass of the polysaccharide molecular fragments obtained after reaction at a trifluoroacetic acid concentration of 0.5mol/L and a temperature of 90 ℃ for different times is shown in FIG. 2. As can be seen from the figure, the relative molecular mass of the polysaccharide is reduced along with the extension of the reaction time, the molecular mass is rapidly reduced within 50-90 min, then the molecular mass changes little along with the extension of the reaction time, the reduction rate of the relative molecular mass is slowed down within 90-110 min, and the degradation has a certain effect within 90min of the reaction.
The effect of the degraded polysaccharide molecular fragments obtained at different degradation times on stimulating NO release from RAW264.7 macrophages under the conditions of acid final concentration of 0.5mol/L and reaction temperature of 90 ℃ is shown in FIG. 3. The results show that the NO release amount of RAW264.7 macrophage stimulated by the molecular fragments corresponding to trifluoroacetic acid degradation reaction for 30min and 50min is obviously increased compared with that of a control group, and the results show that the trifluoroacetic acid can degrade macromolecular hericium erinaceus polysaccharide into molecular fragments with higher activity within 50min of degradation, so that the phagocytic capacity of the macrophage is enhanced; and when the reaction time is prolonged to more than 70min, the activity of the degraded molecular fragments is reduced. The molecular weight distribution result shows that the peak area occupied by the macromolecular polysaccharide is large and the relative content is high in the initial degradation stage; along with the prolonging of the degradation time, the proportion of the polysaccharide with large molecular weight of polysaccharide fragments degraded in the later period is lower, and the result further shows that the hericium erinaceus polysaccharide mainly plays a role in the development of the immunocompetence of the macromolecular polysaccharide component.
Example 3: determination of trifluoroacetic acid concentration
Weighing 5 parts of 100mg hericium erinaceus beta-glucan H6PC20, respectively putting into test tubes with stoppers, and adding water to dissolve to prepare a solution of 2 g/L. Respectively adding trifluoroacetic acid solution with final concentration of 0.1, 0.3, 0.5, 0.7, 1.0mol/L, sealing tube, and placing in 90 deg.C water bath for reaction for 50 min. After the reaction is finished, cooling, centrifuging at 12000 Xg for 20min to remove precipitates, taking supernate, dialyzing with a 3500Da dialysis bag, removing trifluoroacetic acid, and carrying out vacuum freeze drying to obtain the polysaccharide fragment of the degraded hericium erinaceus. And (3) measuring the molecular weight of different polysaccharide fragments by using a high performance liquid chromatography, and measuring the activity of the hericium erinaceus polysaccharides with different molecular weights for stimulating macrophage RAW264.7 to release NO in vitro.
The molecular weight of the polysaccharide molecular fragments obtained by degradation at different acid concentrations at a reaction temperature of 90 ℃ for 50min is shown in FIG. 4. When the acid concentration was increased to 1mol/L, the molecular mass was degraded to 6.66X 105Da can reduce the macromolecular polysaccharide by one order of magnitude when the acid concentration is 0.7-1.0M, and the degradation effect is good; after 1mol/L trifluoroacetic acid is degraded, the peak area of the main polysaccharide fragment is detected to be very low, and other degraded molecular fragments can not be detected by the set HPLC analysis condition due to too low molecular weight, so that the acid concentration is not suitable to be increased in consideration of the yield of the degraded polysaccharide.
The release amount of NO from macrophage stimulated by polysaccharide molecular fragment degraded by different acid concentration under the reaction conditions of 90 deg.C and 50min is shown in FIG. 5. Compared with the control group, the polysaccharide molecular fragments degraded by trifluoroacetic acid at 0.1M, 0.3M and 0.5M obviously enhance the phagocytic capacity of macrophages, but the NO release amount is obviously reduced relative to the reduction of molecular mass along with the increase of the concentration of the trifluoroacetic acid.
The results show that when the concentration of trifluoroacetic acid is 0.1M, 0.3M and 0.5M, the corresponding molecular fragments stimulate the NO release amount of RAW264.7 macrophage to be obviously increased compared with the contrast group, which indicates that the degradation is within the range of 0.1M-0.5M of acid concentration, the trifluoroacetic acid can degrade macromolecular hericium erinaceus polysaccharide into molecular fragments with higher activity, and the phagocytic capacity of the macrophage is enhanced; and as the acid concentration increases, the activity of the degraded molecular fragments decreases. The molecular weight distribution result shows that when the acid concentration is low, the peak area occupied by the macromolecular polysaccharide is large, and the relative content is high; when the concentration of trifluoroacetic acid is increased, the proportion of the degraded polysaccharide fragment high molecular weight polysaccharide is lower, further showing that the hericium erinaceus polysaccharide mainly plays a role in the high molecular weight polysaccharide component in the immunocompetence.
In a word, the hericium erinaceus polysaccharide fragment obtained by the acid degradation method has important application value in foods, functional foods and medicines for enhancing immunity.
The foregoing is a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as falling within the scope of the present invention.
Claims (3)
1. An acid degradation method of hericium erinaceus polysaccharide comprises the following steps:
1) dissolving, namely dissolving the hericium erinaceus polysaccharide in water to prepare a solution;
2) hydrolyzing, adding acid into the aqueous solution for hydrolysis;
3) neutralizing, namely immediately cooling the solution obtained after hydrolysis, then neutralizing by using an alkali solution, and adjusting the pH value to be neutral;
4) centrifuging, namely centrifuging the solution obtained in the step 3);
5) dialyzing, namely dialyzing the centrifuged supernatant;
6) freeze drying to obtain Hericium erinaceus polysaccharide acid hydrolysate;
the method simultaneously comprises the following conditions: the hericium erinaceus polysaccharide is hericium erinaceus beta-glucan, the concentration of the solution in the step 1) is 1-2g/L, the hydrolysis temperature in the step 2) is 90-100 ℃, the alkali solution in the step 3) is a NaOH solution, the solution is centrifuged for 15-20min at 8000-12000 Xg in the step 4), and the solution is dialyzed by a 3500Da dialysis bag in the step 5);
the acid is trifluoroacetic acid;
the final concentration of the trifluoroacetic acid is 0.1-0.5 mol/L;
the hydrolysis time is 30-50 min.
2. An acid hydrolysate of a polysaccharide of Hericium erinaceum produced by the method according to claim 1.
3. Use of the hericium erinaceus polysaccharide acid hydrolysate of claim 2 in the preparation of food, health care products or medicines for improving immunity.
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| "Immunomodulatory effects of Hericium erinaceus derived polysaccharides are mediated by intestinal immunology";Xiaotong Sheng等;《Food & Function》;20170224;第1-31页 * |
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