CN112029006A - Tremella polysaccharide and preparation method and application thereof - Google Patents
Tremella polysaccharide and preparation method and application thereof Download PDFInfo
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- CN112029006A CN112029006A CN202010948173.1A CN202010948173A CN112029006A CN 112029006 A CN112029006 A CN 112029006A CN 202010948173 A CN202010948173 A CN 202010948173A CN 112029006 A CN112029006 A CN 112029006A
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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
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- 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
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- 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
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; carrying out solid-liquid separation on a product obtained after enzymolysis, taking a 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 2.5h, 3h or 3.5h, 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 0.11mol/L, 0.12mol/L, 0.13mol/L, 0.14mol/L, 0.15mol/L, 0.16mol/L, or 0.17mol/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: 60g/mL, such as 1:41g/mL, 1:42g/mL, 1:43g/mL, 1:45g/mL, 1:50g/mL, 1:51g/mL, 1:53g/mL, 1:55g/mL, 1:56g/mL, 1:57g/mL, or 1:58g/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: 60g/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 to 0.8 percent of the weight of the tremella powder, such as 0.3 percent, 0.4 percent, 0.5 percent, 0.6 percent or 0.7 percent, but not limited to the recited values, and the values in the numerical range are all 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 2.5h, 3h, 3.5h, 4h, 4.5, 5h or 5.5h, 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 6min or 7min, 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 7100rpm/min, 7200rpm/min, 7500rpm/min, 7600rpm/min or 7900rpm/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 14h, 16h, 18h, 20h, 24h, 28h, 30h or 34h, 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 by volume of 90% to 95%, such as 91%, 92%, 93% or 94%, but not limited to the recited values, and values within the recited ranges apply.
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 7h, 8h, 9h, 10h or 11h, 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 8700rpm/min, 8900rpm/min, 9100rpm/min, 9500rpm/min, 9700rpm/min or 9900rpm/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 7min or 8min, 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: 60g/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 1000Da 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 midsummer 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 100g of tremella powder, adding 0.15mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution into the tremella powder according to a feed-liquid ratio of 1:50g/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 7500rpm/min for 6min, 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 6h, centrifuging at 10000rpm/min for 9min, 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 100g of tremella powder, adding 0.2mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution into the tremella powder according to a feed-liquid ratio of 1:60g/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 8000rpm/min for 5min, 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 12h, centrifuging at 8500rpm/min for 6min, 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 100g of tremella powder, adding 0.1mol/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 7000rpm/min for 8min, standing and precipitating for 12h, 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 9h, centrifuging at 9500rpm/min for 8min, 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 0.15mol/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 0.15mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added at a feed-to-solution ratio of 1:60g/mL in step (1).
Example 8
This example was compared with example 1 except that 0.15mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added in a feed-to-solution ratio of 1:20g/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 0.15mol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was added in a feed-to-solution ratio of 1:80g/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 | ＜1000Da | 1000～5000Da | 5000～10000Da | 10000～50000Da |
| 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 1000Da is 2.93-12.66%.
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 (10)
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 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.
2. The method according to claim 1, wherein the leaching of step (1) is carried out under heating;
preferably, the heating mode is water bath heating;
preferably, the temperature of the water bath heating is 60-80 ℃;
preferably, the water bath heating time is 2-4 h;
preferably, the neutral buffer solution of step (1) comprises 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;
preferably, the material-to-liquid ratio of the tremella powder and the neutral buffer solution in the step (1) is 1: 40-1: 60 g/mL.
3. The preparation method according to claim 1 or 2, characterized in that the addition amount of the pectin-cellulose complex enzyme in the step (2) is 0.2-0.8% of the mass of the tremella powder;
preferably, the pH value of the enzymolysis in the step (2) is 4.2-5.2;
preferably, the temperature of the enzymolysis in the step (2) is 40-60 ℃;
preferably, the enzymolysis time in the step (2) is 2-6 h.
4. The method according to any one of claims 1 to 3, wherein the filtration in the step (3) is performed 3 to 5 times, preferably 3 times;
preferably, in 3 times of filtration, 100-200 meshes of filter cloth is used for the 1 st time, 200-300 meshes of filter cloth is used for the 2 nd time, and 500-700 meshes of filter cloth is used for the 3 rd time;
preferably, the filtration in the step (3) is carried out by using a filter cloth;
preferably, the mesh number of the filter cloth is 100-700 meshes;
preferably, the centrifugation time in the step (3) is 5-8 min;
preferably, the rotation speed of the centrifugation in the step (3) is 7000-8000 rpm/min;
preferably, the standing time in the step (3) is 12-36 h;
preferably, said resting of step (3) is carried out under aseptic conditions;
preferably, the alcohol precipitation in the step (4) uses an ethanol solution;
preferably, the volume concentration of the ethanol solution is 90-95%;
preferably, the volume ratio of the supernatant to the ethanol solution is 1 (2-3);
preferably, the alcohol precipitation time in the step (4) is 6-12 h;
preferably, the rotating speed of the centrifugation in the step (4) is 8500-10000 rpm/min;
preferably, the centrifugation time in the step (4) is 6-9 min.
5. The method according to any one of claims 1 to 4, characterized in that it comprises the following steps:
(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: 60g/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 (1.5-2): 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 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/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.
6. A tremella polysaccharide, characterized in that it is prepared by the method of any one of claims 1-5.
7. The tremella polysaccharide according to claim 6, wherein 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.
8. The tremella polysaccharide according to claim 6 or 7, wherein the percentage of tremella polysaccharide with a molecular weight of 10000-50000 Da is 2.89-4.59%, the percentage of tremella polysaccharide with a molecular weight of 5000-10000 Da is 40.47-76.77%, the percentage of tremella polysaccharide with a molecular weight of 1000-5000 Da is 15.62-42.98%, and the percentage of tremella polysaccharide with a molecular weight of less than 1000Da is 2.93-12.66%;
preferably, the total sugar content in the tremella polysaccharides is greater than 92%.
9. Use of the tremella polysaccharide according to any one of claims 6-8 in the manufacture of a food product, a pharmaceutical product, a nutraceutical product or a cosmetic product.
10. A cosmetic comprising the tremella polysaccharide according to any one of claims 6 to 8.
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