CN112029006B - Tremella polysaccharide and preparation method and application thereof - Google Patents
Tremella polysaccharide and preparation method and application thereof Download PDFInfo
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- CN112029006B CN112029006B CN202010948173.1A CN202010948173A CN112029006B CN 112029006 B CN112029006 B CN 112029006B CN 202010948173 A CN202010948173 A CN 202010948173A CN 112029006 B CN112029006 B CN 112029006B
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- 241001506047 Tremella Species 0.000 title claims abstract description 121
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 109
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 108
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 150000004676 glycans Chemical class 0.000 title claims abstract 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 33
- 229940088598 enzyme Drugs 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 31
- 229920002678 cellulose Polymers 0.000 claims abstract description 29
- 239000001913 cellulose Substances 0.000 claims abstract description 29
- 238000002386 leaching Methods 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 23
- 108010059892 Cellulase Proteins 0.000 claims abstract description 20
- 108010059820 Polygalacturonase Proteins 0.000 claims abstract description 20
- 229940106157 cellulase Drugs 0.000 claims abstract description 20
- 108010093305 exopolygalacturonase Proteins 0.000 claims abstract description 20
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 239000007853 buffer solution Substances 0.000 claims abstract description 17
- 238000001914 filtration Methods 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000001556 precipitation Methods 0.000 claims abstract description 14
- 238000005119 centrifugation Methods 0.000 claims abstract description 13
- 230000007935 neutral effect Effects 0.000 claims abstract description 13
- 239000000047 product Substances 0.000 claims abstract description 12
- 239000002537 cosmetic Substances 0.000 claims abstract description 11
- 239000000706 filtrate Substances 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims abstract description 4
- 239000004744 fabric Substances 0.000 claims description 30
- 229920001277 pectin Polymers 0.000 claims description 20
- 239000001814 pectin Substances 0.000 claims description 20
- 235000010987 pectin Nutrition 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000010438 heat treatment Methods 0.000 claims description 16
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 13
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 6
- 238000012869 ethanol precipitation Methods 0.000 claims description 4
- 239000002417 nutraceutical Substances 0.000 claims 1
- 235000021436 nutraceutical agent Nutrition 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 4
- 150000004804 polysaccharides Chemical class 0.000 description 83
- 230000000052 comparative effect Effects 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 10
- 239000000287 crude extract Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000001035 drying Methods 0.000 description 6
- 238000001816 cooling Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000007873 sieving Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 206010008570 Chloasma Diseases 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000003351 Melanosis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000037384 skin absorption Effects 0.000 description 1
- 231100000274 skin absorption Toxicity 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- Polymers & Plastics (AREA)
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Abstract
The invention discloses a tremella polysaccharide and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) leaching the tremella powder by using a neutral buffer solution to obtain a leaching material liquid; (2) adding a pectin-cellulose complex enzyme into the leaching solution for enzymolysis, wherein the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is (1.5-2): 1; (3) filtering after enzymolysis, taking filtrate for centrifugation, standing and taking supernatant; (4) and carrying out alcohol precipitation and centrifugation on the supernatant to obtain a precipitate which is the tremella polysaccharide. The preparation method has low cost and high efficiency, the yield of the tremella polysaccharide is higher than 40 percent, and the tremella polysaccharide prepared by the method is suitable for large-scale commercial production, the polysaccharide with the molecular weight of less than 10000Da accounts for more than 95 percent of the total polysaccharide, the total sugar content is more than 92 percent, and the tremella polysaccharide has wide application prospect in the fields of food, medicine, health care products and cosmetic preparation.
Description
Technical Field
The invention belongs to the technical field of tremella polysaccharide preparation, and relates to tremella polysaccharide and a preparation method and application thereof.
Background
The tremella polysaccharide is a main active component of tremella, comprises acidic polysaccharide, neutral heteropolysaccharide, acidic oligosaccharide, mural polysaccharide, exopolysaccharide and the like, has the effects of strengthening heart, delaying senescence, resisting thrombus, resisting tumors, improving immunity, removing chloasma and the like, and is widely applied to the fields of food, health care products and cosmetics. The tremella polysaccharide with large molecular weight has good moisture-keeping capacity and excellent film-forming property, and has the effects of oxidation resistance, allergy resistance and inflammation diminishing. However, the cosmetic has the defects of low solubility, difficult skin absorption and sticky feeling brought by application in a formula, and has certain defects in application in cosmetics. The tremella polysaccharide with lower molecular weight has high solubility, is easy to be absorbed by skin, has good moisturizing capability and provides deep moisturizing effect for the skin, so the tremella polysaccharide with lower molecular weight is more suitable for being applied to the field of cosmetics.
The preparation of the low molecular weight tremella polysaccharide generally comprises four steps of extraction, degradation, purification and drying. After the low molecular weight tremella polysaccharide is extracted, the tremella polysaccharide is generally degraded by a chemical degradation method and/or an enzymolysis method, purified by graded alcohol precipitation, centrifugal drying or molecular membrane filtration, and finally dried to obtain the low molecular weight tremella polysaccharide.
CN106117387A discloses a preparation method of low molecular weight tremella polysaccharide, which comprises mixing tremella powder with water, adjusting pH to alkalinity, and extracting to obtain a crude extract; adjusting the temperature and pH value of the crude extract, and adding enzyme for enzymolysis; after enzymolysisCarrying out solid-liquid separation on the obtained product, taking the filtrate, and concentrating to obtain a paste; adding ethanol and H into the paste2O2Polysaccharide degradation, standing for precipitation, centrifuging, washing, and drying to obtain low molecular weight tremella polysaccharide with molecular weight less than 10000 Dalton and total polysaccharide content more than 95%, but with low sugar yield.
CN102161710A discloses a preparation method of low molecular weight tremella polysaccharide, which comprises the steps of cleaning tremella, homogenizing, adding an acid water solution, extracting at a certain temperature and under a certain pressure for 1-3 hours, neutralizing the acid water extracting solution with alkaline water to be neutral, centrifuging, concentrating a supernatant to a proper volume, and finally obtaining the low molecular weight tremella polysaccharide by an ultrafiltration membrane process, wherein the polysaccharide loss is large by the ultrafiltration membrane process, and the process efficiency is low.
In conclusion, how to provide a low-molecular weight tremella polysaccharide preparation method with low cost and high efficiency, which can obtain high-yield and high-purity low-molecular weight tremella polysaccharide, has become one of the problems to be solved in the field of tremella polysaccharide preparation.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides tremella polysaccharide and a preparation method and application thereof, wherein the tremella polysaccharide is low-molecular-weight tremella polysaccharide, and the polysaccharide with the molecular weight of less than 10000Da accounts for more than 95% of the total polysaccharide.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a preparation method of tremella polysaccharide, which comprises the following steps:
(1) leaching the tremella powder by using a neutral buffer solution to obtain a leaching material liquid;
(2) adding a pectin-cellulose complex enzyme into the leaching solution for enzymolysis, wherein the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is (1.5-2): 1;
(3) filtering after enzymolysis, taking filtrate for centrifugation, standing and taking supernatant;
(4) and carrying out alcohol precipitation and centrifugation on the supernatant to obtain a precipitate which is the tremella polysaccharide.
According to the preparation method of the tremella polysaccharide, neutral buffer solution leaching and enzymolysis are combined, the mass ratio of pectinase to cellulase in the pectin-cellulose compound enzyme adopted by enzymolysis is (1.5-2): 1, and therefore the tremella polysaccharide with high quality and low molecular weight can be obtained, and the yield of the tremella polysaccharide can be improved.
In the invention, the mass ratio of pectinase to cellulase in the pectin cellulose compound enzyme is (1.5-2): 1, wherein the typical but non-limiting mass ratio is as follows: 1.6:1, 1.7:1, 1.8:1 or 1.9: 1.
Preferably, the leaching of step (1) is carried out under heated conditions.
Preferably, the heating mode is water bath heating.
Preferably, the temperature of the water bath heating is 60-80 ℃, such as 61 ℃, 62 ℃, 63 ℃, 64 ℃, 68 ℃, 70 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃ or 79 ℃, but not limited to the recited values, and the values in the range are all applicable.
Preferably, the time for heating in the water bath is 2-4 h, such as 2.5 h, 3 h or 3.5 h, but not limited to the values listed, and the values in the range apply.
Preferably, the neutral buffer solution of step (1) comprises a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.
Preferably, the concentration of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 0.1-0.18 mol/L, such as 0.11 mol/L, 0.12 mol/L, 0.13 mol/L, 0.14 mol/L, 0.15 mol/L, 0.16 mol/L, or 0.17 mol/L, but not limited to the enumerated values, and the numerical values in the range are all applicable.
Preferably, the material-to-liquid ratio of the tremella powder and the neutral buffer solution in step (1) is 1: 40-1: 60 g/mL, such as 1:41 g/mL, 1:42 g/mL, 1:43 g/mL, 1:45 g/mL, 1:50 g/mL, 1:51 g/mL, 1:53 g/mL, 1:55 g/mL, 1:56 g/mL, 1:57 g/mL, or 1:58 g/mL, but is not limited to the recited values, and the numerical values in the numerical range are all applicable.
According to the invention, when the material-liquid ratio of the tremella powder to the neutral buffer solution is lower than 1:40g/mL, the leaching effect is poor, the leaching effect can be improved by increasing the material-liquid ratio, but after a certain value is reached, the leaching effect is not obviously changed any more, and the higher material-liquid ratio can enlarge the volume of an enzymolysis system and influence the enzymolysis effect, so that the tremella polysaccharide yield is high by optimizing the material-liquid ratio and controlling the material-liquid ratio to be 1: 40-1: 60 g/mL, and waste caused by meaningless excessive use of the neutral buffer solution can be avoided.
Preferably, the addition amount of the pectin-cellulose complex enzyme in the step (2) is 0.2-0.8% of the tremella powder by mass, such as 0.3%, 0.4%, 0.5%, 0.6% or 0.7%, but not limited to the recited values, and the values in the value range are applicable.
Preferably, the pH of the enzymatic hydrolysis in step (2) is 4.2-5.2, such as 4.3, 4.4, 4.5, 4.6, 4.9, 5.0, or 5.1, but not limited to the recited values, and values within the range are applicable.
Preferably, the temperature of the enzymolysis in step (2) is 40-60 ℃, such as 42 ℃, 44 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃ or 58 ℃, but not limited to the enumerated values, and the numerical values in the numerical range are all applicable.
Preferably, the enzymolysis time in step (2) is 2-6 h, such as 2.5 h, 3 h, 3.5 h, 4 h, 4.5, 5 h or 5.5 h, but not limited to the recited values, and the values in the numerical range are all applicable.
Preferably, the filtration in the step (3) is performed by using a filter cloth.
Preferably, the filtration in the step (3) is performed 3 to 5 times, preferably 3 times.
Preferably, the mesh number of the filter cloth is 100 to 700 meshes, such as 200 meshes, 300 meshes, 400 meshes, 500 meshes or 600 meshes, but not limited to the listed values, and the values in the numerical range are all applicable.
Preferably, in 3 times of filtration, 100-200 mesh filter cloth is used for the 1 st time, 200-300 mesh filter cloth is used for the 2 nd time, and 500-700 mesh filter cloth is used for the 3 rd time.
Preferably, the centrifugation in step (3) is carried out for 5-8 min, such as 6 min or 7 min, but not limited to the values listed, and the values in the range apply.
Preferably, the centrifugation in step (3) is performed at 7000-8000 rpm/min, such as 7100 rpm/min, 7200 rpm/min, 7500 rpm/min, 7600 rpm/min or 7900 rpm/min, but not limited to the recited values, and any value within the range of values is applicable.
Preferably, the standing time in the step (3) is 12-36 h, such as 14 h, 16 h, 18 h, 20 h, 24 h, 28 h, 30 h or 34 h, but not limited to the enumerated values, and the values in the numerical range are all applicable.
Preferably, said resting of step (3) is carried out under aseptic conditions.
Preferably, the alcohol precipitation in step (4) uses an ethanol solution.
Preferably, the ethanol solution has a concentration of 90% to 95% by volume, such as 91%, 92%, 93% or 94%, but not limited to the recited values, and values within the recited range are applicable.
Preferably, the volume ratio of the supernatant to the ethanol solution is 1 (2-3), such as 1:2.2, 1:2.4, 1:2.6, or 1:2.8, but not limited to the values listed, and the values in the numerical range apply.
Preferably, the alcohol precipitation time in step (4) is 6-12 h, such as 7 h, 8 h, 9 h, 10 h or 11 h, but not limited to the recited values, and the values in the range are all applicable.
Preferably, the centrifugation in step (4) is carried out at 8500-10000 rpm/min, such as 8700 rpm/min, 8900 rpm/min, 9100 rpm/min, 9500 rpm/min, 9700 rpm/min or 9900 rpm/min, but not limited to the values listed, and values within the range of values are applicable.
Preferably, the centrifugation in step (4) is carried out for 6-9 min, such as 7 min or 8 min, but not limited to the values listed, and the values in the range apply.
In the present invention, as a preferred preparation method, the method comprises the steps of:
(1) leaching the tremella powder by using a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, wherein the concentration of the buffer solution is 0.1-0.18 mol/L, the ratio of leaching materials to leaching solutions is 1: 40-1: 60 g/mL, the leaching is carried out under the water bath heating condition, the water bath temperature is 60-80 ℃, and the water bath time is 2-4 hours;
(2) adjusting the temperature of the leaching solution to 40-60 ℃ and the pH value to 4.2-5.2, adding a pectin cellulose complex enzyme for enzymolysis, wherein the mass ratio of pectinase to cellulase in the pectin cellulose complex enzyme is (1.5-2): 1, the addition amount of the pectin cellulose complex enzyme is 0.2-0.8% of the mass of the tremella powder, and the enzymolysis time is 2-6 hours;
(3) filtering for 3 times by using filter cloth after enzymolysis, using 100-200 meshes of filter cloth for the 1 st time, using 200-300 meshes of filter cloth for the 2 nd time, using 500-700 meshes of filter cloth for the 3 rd time, taking filtrate, centrifuging at 7000-8000 rpm/min for 5-8 min, and standing and precipitating for 12-36 h under an aseptic condition;
(4) and taking the supernatant, adding an ethanol solution with the volume concentration of 90-95% for ethanol precipitation, wherein the volume ratio of the ethanol solution to the supernatant is (2-3): 1, performing ethanol precipitation for 6-12 h, centrifuging at 8500-10000 rpm/min for 6-9 min, and taking the precipitate to obtain the tremella polysaccharide.
In a second aspect, the invention provides a tremella polysaccharide, which is prepared by the preparation method of the first aspect.
Preferably, the molecular weight of the tremella polysaccharide is not higher than 50000 Da.
Preferably, the tremella polysaccharide contains more than 95% of total polysaccharide, wherein the polysaccharide has a molecular weight of less than 10000 Da.
Preferably, in the tremella polysaccharides, the percentage of tremella polysaccharides with molecular weight of 10000-50000 Da is 2.89% -4.59%, the percentage of tremella polysaccharides with molecular weight of 5000-10000 Da is 40.47% -76.77%, the percentage of tremella polysaccharides with molecular weight of 1000-5000 Da is 15.62% -42.98%, and the percentage of tremella polysaccharides with molecular weight less than 1000 Da is 2.93% -12.66%.
Preferably, the total sugar content in the tremella polysaccharides is greater than 92%.
In a third aspect, the invention provides an application of the tremella polysaccharide of the second aspect in preparing food, medicine, health care products or cosmetics.
In a fourth aspect, the present invention provides a cosmetic comprising the tremella polysaccharide of the second aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the preparation method of the tremella polysaccharide, neutral buffer solution leaching and enzymolysis are combined, a pectin cellulose compound enzyme is adopted for enzymolysis, the mass ratio of pectinase to cellulase in the pectin cellulose compound enzyme is set to be (1.5-2): 1, so that the yield of the tremella polysaccharide is higher than 40% and is 45.2% at most, and the preparation method is low in cost and high in efficiency, and is suitable for large-scale commercial production;
(2) the tremella polysaccharide of the invention has the advantages that the polysaccharide with the molecular weight below 10000Da accounts for more than 95 percent of the total polysaccharide, the total sugar content is more than 92 percent and is up to 94.3 percent, and the tremella polysaccharide has wide application prospect in the fields of food, medicine, health care products and cosmetic preparation.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
In the invention, the pectinase and cellulase used are pectinase (SPE-010) and cellulase (SPE-017) respectively, and are purchased from the Xiansheng group.
Example 1
The embodiment provides a preparation method of tremella polysaccharide, which comprises the following specific processes:
(1) drying commercially available tremella, taking dried tremella, removing impurities, crushing, sieving with a 80-mesh sieve, taking 100 g of tremella powder, adding 0.15 mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution into the tremella powder according to a feed-liquid ratio of 1:50 g/mL, heating in a water bath at the temperature of 70 ℃ for 3 hours to obtain a crude extract;
(2) cooling the crude extract to 50 ℃, adjusting the pH to 4.7, adding pectin cellulose complex enzyme (the mass ratio of pectinase to cellulase is 1.75: 1), adding the pectin cellulose complex enzyme into the mixture in an amount of 0.5 percent of the mass of the tremella powder, and carrying out enzymolysis reaction for 4 hours;
(3) filtering with filter cloth for 3 times after enzymolysis, using 200 mesh filter cloth for 1 time, 200 mesh filter cloth for 2 times, and 700 mesh filter cloth for the third time, centrifuging the filtrate at 7500 rpm/min for 6 min, standing and precipitating for 24 h;
(4) and taking the supernatant, adding an ethanol solution for alcohol precipitation, wherein the volume concentration of the ethanol solution is 95%, the volume ratio of the ethanol solution to the supernatant is 1:2.5, carrying out alcohol precipitation for 6 h, centrifuging at 10000 rpm/min for 9 min, and taking the precipitate to obtain the tremella polysaccharide.
Example 2
The embodiment provides a preparation method of tremella polysaccharide, which comprises the following specific processes:
(1) drying commercially available tremella, taking dried tremella, removing impurities, crushing, sieving with a 80-mesh sieve, taking 100 g of tremella powder, adding 0.2 mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution into the tremella powder according to a feed-liquid ratio of 1:60 g/mL, heating in a water bath at the temperature of 80 ℃ for 2 hours to obtain a crude extract;
(2) cooling the crude extract to 40 ℃, adjusting the pH to 5.2, adding pectin cellulose complex enzyme (the mass ratio of pectinase to cellulase is 1.5: 1), adding the pectin cellulose complex enzyme into the mixture in an amount of 0.2 percent of the mass of the tremella powder, and carrying out enzymolysis reaction for 6 hours;
(3) filtering with filter cloth for 3 times after enzymolysis, using 100 mesh filter cloth for 1 time, 300 mesh filter cloth for 2 times, 500 mesh filter cloth for the third time, centrifuging the filtrate at 8000 rpm/min for 5 min, standing and precipitating for 36 h;
(4) and taking the supernatant, adding an ethanol solution for alcohol precipitation, wherein the volume concentration of the ethanol solution is 90%, the volume ratio of the ethanol solution to the supernatant is 1:2, carrying out alcohol precipitation for 12 h, centrifuging at 8500 rpm/min for 6 min, and taking the precipitate to obtain the tremella polysaccharide.
Example 3
The embodiment provides a preparation method of tremella polysaccharide, which comprises the following specific processes:
(1) drying commercially available tremella, taking dried tremella powder, removing impurities, crushing, sieving with a 80-mesh sieve, taking 100 g of tremella powder, adding 0.1 mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution into the tremella powder according to a feed-liquid ratio of 1:40g/mL, heating in a water bath at the temperature of 60 ℃ for 4 hours to obtain a crude extract;
(2) cooling the crude extract to 60 ℃, adjusting the pH to 4.2, adding pectin cellulose complex enzyme (the mass ratio of pectinase to cellulase is 2: 1), adding 0.8% of tremella powder by mass, and performing enzymolysis for 2 h;
(3) filtering with filter cloth for 3 times after enzymolysis, using 150 mesh filter cloth for 1 time, using 250 mesh filter cloth for 2 times, using 600 mesh filter cloth for the third time, centrifuging the filtrate at 7000 rpm/min for 8 min, standing and precipitating for 12 h, collecting supernatant, and freeze drying to obtain Tremella polysaccharide;
(4) and taking the supernatant, adding an ethanol solution for alcohol precipitation, wherein the volume concentration of the ethanol solution is 92%, the volume ratio of the ethanol solution to the supernatant is 1:3, carrying out alcohol precipitation for 9 h, centrifuging at 9500 rpm/min for 8 min, and taking the precipitate to obtain the tremella polysaccharide.
Example 4
Compared with the example 1, the difference of the embodiment is only that in the pectin cellulose compound enzyme added in the step (2), the mass ratio of the pectinase to the cellulase is 1.5:1, and the rest is the same as the example 1.
Example 5
Compared with the example 1, the difference of the embodiment is only that in the pectin cellulose compound enzyme added in the step (2), the mass ratio of the pectinase to the cellulase is 2:1, and the rest is the same as the example 1.
Example 6
This example was compared with example 1 except that 0.15 mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added in a feed-to-solution ratio of 1:40g/mL in step (1), and the procedure was otherwise the same as in example 1.
Example 7
This example is similar to example 1 except that 0.15 mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added at a feed-to-solution ratio of 1:60 g/mL in step (1).
Example 8
This example was compared with example 1 except that 0.15 mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added in a feed-to-solution ratio of 1:20 g/mL in step (1), and the procedure was otherwise the same as in example 1.
Example 9
This example was compared with example 1 except that 0.15 mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added in a feed-to-solution ratio of 1:80 g/mL in step (1), and the procedure was otherwise the same as in example 1.
Comparative example 1
Compared with the example 1, the comparative example is only different from the example 1 in that the mass ratio of the pectinase to the cellulase in the pectin cellulose compound enzyme added in the step (2) is 1:1, and the rest is the same as the example 1.
Comparative example 2
Compared with the example 1, the comparative example is only different from the example 1 in that the mass ratio of the pectinase to the cellulase in the pectin cellulose compound enzyme added in the step (2) is 3:1, and the rest is the same as the example 1.
Comparative example 3
Compared with the example 1, the comparative example only differs from the example 1 in that the enzyme added in the step (2) is pectinase, namely the addition of cellulase is omitted, the addition amount of the pectinase is the same as that of the pectin cellulose compound enzyme in the example 1, and the rest is the same as that of the example 1.
Comparative example 4
Compared with the example 1, the comparative example only differs from the example 1 in that the enzyme added in the step (2) is cellulase, namely, the addition of pectinase is omitted, the addition amount of the cellulase is the same as that of the pectin cellulose compound enzyme in the example 1, and the rest is the same as that of the example 1.
Test example 1 Total sugar content and yield
The tremella polysaccharides prepared in examples 1-9 and comparative examples 1-4 were prepared into 0.5% (w/w) aqueous solutions, their total sugar content was determined with reference to national standard GB/T15672-2009, "determination of total sugar content in edible fungi", and the tremella polysaccharide yield was calculated according to formula (1), with the results shown in table 1.
TABLE 1
| Numbering | Total sugar content (%) | Yield of Tremella polysaccharide (%) |
| Example 1 | 94.3 | 45.2 |
| Example 2 | 93.6 | 44.4 |
| Example 3 | 93.9 | 43.6 |
| Example 4 | 94.0 | 44.1 |
| Example 5 | 93.1 | 43.8 |
| Example 6 | 93.5 | 44.2 |
| Example 7 | 93.2 | 43.7 |
| Example 8 | 92.3 | 41.1 |
| Example 9 | 92.1 | 41.8 |
| Comparative example 1 | 90.1 | 30.3 |
| Comparative example 2 | 89.5 | 29.8 |
| Comparative example 3 | 89.3 | 29.5 |
| Comparative example 4 | 88.9 | 31.2 |
As shown in table 1, the total sugar content and yield of the tremella polysaccharides prepared in examples 1 to 9 are higher than those of the tremella polysaccharides prepared in comparative examples 1 to 4, and the types and the amounts of the enzymes are important for enzymolysis of the tremella polysaccharides.
Test example 2 detection of molecular weight distribution
The molecular weight distribution of the tremella polysaccharides prepared in examples 1-9 of the present invention was determined by high performance gel permeation chromatography, with reference to the study of liu bang et al (liu bang, ningglong, buka. the tremella polysaccharide quality control study based on high performance gel permeation chromatography [ J ]. chinese herbal medicine, 2011, 42(009): 1732-1735.), and the results are shown in table 2.
TABLE 2
| Numbering | ＜1000 Da | 1000~5000 Da | 5000~10000 Da | 10000~50000 Da |
| Example 1 | 3.17% | 32.71% | 61.23% | 2.89% |
| Example 2 | 2.93% | 33.56% | 59.87% | 3.64% |
| Example 3 | 12.66% | 42.98% | 40.47% | 3.89% |
| Example 4 | 3.21% | 31.95% | 60.79% | 4.05% |
| Example 5 | 3.11% | 32.31% | 59.99% | 4.59% |
| Example 6 | 5.69% | 33.36% | 56.98% | 3.97% |
| Example 7 | 8.59% | 26.12% | 61.27% | 4.02% |
| Example 8 | 3.67% | 15.62% | 76.77% | 3.94% |
| Example 9 | 4.17% | 34.26% | 58.36% | 3.21% |
As shown in Table 2, the molecular weight distribution of the tremella polysaccharides prepared in examples 1-9 is: the percentage of tremella polysaccharide with molecular weight of 10000-50000 Da is 2.89% -4.59%, the percentage of tremella polysaccharide with molecular weight of 5000-10000 Da is 40.47% -76.77%, the percentage of tremella polysaccharide with molecular weight of 1000-5000 Da is 15.62% -42.98%, and the percentage of tremella polysaccharide with molecular weight of less than 1000 Da is 2.93% -12.66%, and it can be seen that polysaccharides with molecular weight of less than 10000Da in the tremella polysaccharide prepared by the method account for more than 95% and the highest 97.11% of total polysaccharides, and the preparation method can be used for preparing low-molecular weight tremella polysaccharide.
In conclusion, the preparation method of the tremella polysaccharide has low cost and high efficiency, and the yield of the tremella polysaccharide is higher than 40 percent and is up to 45.2 percent, so that the tremella polysaccharide is suitable for large-scale commercial production. The tremella polysaccharide of the invention has the advantages that the polysaccharide with the molecular weight below 10000Da accounts for more than 95 percent of the total polysaccharide, the total sugar content is more than 92 percent and is up to 94.3 percent, and the tremella polysaccharide has wide application prospect in the fields of food, medicine, health care products and cosmetic preparation.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (30)
1. The preparation method of the tremella polysaccharide is characterized by comprising the following steps:
(1) leaching the tremella powder by using a neutral buffer solution to obtain a leaching material liquid;
(2) adding a pectin-cellulose complex enzyme into the leaching solution, carrying out enzymolysis for 2-6 h at 40-60 ℃ and pH of 4.2-5.2, wherein the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is (1.5-1.75): 1, and the addition amount of the pectin-cellulose complex enzyme is 0.2-0.8% of the mass of the tremella powder;
(3) filtering after enzymolysis, taking filtrate for centrifugation, standing and taking supernatant;
(4) precipitating the supernatant with ethanol, and centrifuging to obtain precipitate as Tremella polysaccharide;
the molecular weight of the tremella polysaccharide is not higher than 50000 Da.
2. The method according to claim 1, wherein the leaching of step (1) is carried out under heating.
3. The method of claim 2, wherein the heating is performed by water bath heating.
4. The preparation method according to claim 3, wherein the temperature of the water bath heating is 60-80 ℃.
5. The preparation method of claim 3, wherein the water bath heating time is 2-4 h.
6. The method according to claim 1, wherein the neutral buffer solution of step (1) comprises a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.
7. The method according to claim 6, wherein the concentration of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 0.1 to 0.18 mol/L.
8. The preparation method according to claim 1, wherein the feed-to-liquid ratio of the tremella powder and the neutral buffer solution in the step (1) is 1: 40-1: 60 g/mL.
9. The method according to claim 1, wherein the number of filtration in the step (3) is 3 to 5.
10. The method according to claim 9, wherein the number of filtration in the step (3) is 3.
11. The method according to claim 10, wherein the filter cloth of 100 to 200 mesh is used for the 1 st filtration, the filter cloth of 200 to 300 mesh is used for the 2 nd filtration, and the filter cloth of 500 to 700 mesh is used for the 3 rd filtration.
12. The method according to claim 1, wherein the filtration in step (3) is performed by using a filter cloth.
13. The method as claimed in claim 12, wherein the filter cloth has a mesh size of 100 to 700 mesh.
14. The method according to claim 1, wherein the centrifugation in step (3) is carried out for 5-8 min.
15. The method according to claim 1, wherein the rotation speed of the centrifugation in the step (3) is 7000 to 8000 rpm.
16. The preparation method according to claim 1, wherein the standing time in the step (3) is 12-36 h.
17. The method of claim 1, wherein the standing of step (3) is performed under aseptic conditions.
18. The method according to claim 1, wherein the alcohol precipitation in step (4) is performed using an ethanol solution.
19. The method of claim 18, wherein the ethanol solution has a volume concentration of 90% to 95%.
20. The preparation method according to claim 18, wherein the volume ratio of the supernatant to the ethanol solution is 1 (2-3).
21. The preparation method according to claim 1, wherein the alcohol precipitation time in the step (4) is 6-12 h.
22. The method according to claim 1, wherein the rotation speed of the centrifugation in the step (4) is 8500-10000 rpm.
23. The method according to claim 1, wherein the centrifugation in step (4) is carried out for 6-9 min.
24. The method of any one of claims 1-23, comprising the steps of:
(1) under the water bath heating condition, leaching the tremella powder by using a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, wherein the concentration of the buffer solution is 0.1-0.18 mol/L, the leaching material-liquid ratio is 1: 40-1: 60 g/mL, the water bath heating temperature is 60-80 ℃, and the water bath heating time is 2-4 hours;
(2) adjusting the temperature of the leaching solution to 40-60 ℃ and the pH value to 4.2-5.2, adding a pectin cellulose complex enzyme for enzymolysis, wherein the mass ratio of pectinase to cellulase in the pectin cellulose complex enzyme is (1.5-1.75): 1, the addition amount of the pectin cellulose complex enzyme is 0.2-0.8% of the mass of the tremella powder, and the enzymolysis time is 2-6 h;
(3) filtering for 3 times by using filter cloth after enzymolysis, using 100-200 meshes of filter cloth for the 1 st time, using 200-300 meshes of filter cloth for the 2 nd time, using 500-700 meshes of filter cloth for the 3 rd time, taking filtrate, centrifuging at 7000-8000 rpm for 5-8 min, and standing and precipitating for 12-36 h under an aseptic condition;
(4) and taking the supernatant, adding an ethanol solution with the volume concentration of 90-95% for ethanol precipitation, wherein the volume ratio of the ethanol solution to the supernatant is (2-3): 1, performing ethanol precipitation for 6-12 h, centrifuging at 8500-10000 rpm for 6-9 min, and taking the precipitate to obtain the tremella polysaccharide.
25. A tremella polysaccharide, characterized in that it is prepared by the method of any one of claims 1-24.
26. The tremella polysaccharide according to claim 25, wherein polysaccharides with a molecular weight below 10000Da account for more than 95% of the total polysaccharides.
27. The tremella polysaccharide according to claim 25, wherein the percentage of tremella polysaccharide having a molecular weight of 10000-50000 Da is 2.89-4.59%, the percentage of tremella polysaccharide having a molecular weight of 5000-10000 Da is 40.47-76.77%, the percentage of tremella polysaccharide having a molecular weight of 1000-5000 Da is 15.62-42.98%, and the percentage of tremella polysaccharide having a molecular weight of less than 1000 Da is 2.93-12.66%.
28. The tremella polysaccharide according to claim 25, wherein the total sugar content in the tremella polysaccharide is more than 92%.
29. Use of the tremella polysaccharide according to any one of claims 25-28 in the manufacture of a food product, a pharmaceutical product, a nutraceutical product or a cosmetic product.
30. A cosmetic product, characterized in that it comprises the tremella polysaccharide according to any one of claims 25 to 28.
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