CN113667031A - Preparation method and application of micromolecular dendrobium officinale polysaccharide - Google Patents
Preparation method and application of micromolecular dendrobium officinale polysaccharide Download PDFInfo
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Classifications
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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
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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Abstract
A preparation method of micromolecule Dendrobium officinale polysaccharide comprises taking Dendrobium officinale dry product as initial raw material, and performing eutectic solvent and subcritical extraction to obtain micromolecule Dendrobium officinale polysaccharide. The preparation method of the micromolecule dendrobium candidum polysaccharide can be used for preparing the dendrobium candidum polysaccharide with the molecular weight of less than or equal to 50kD, and the dendrobium candidum polysaccharide with the molecular weight of less than or equal to 50kD also has the advantages of high purity and extraction rate. The dendrobium officinale polysaccharide with the small molecular weight of less than or equal to 50kDa has small molecular weight and high purity, is beneficial to skin absorption, can obviously improve the skin immune barrier effect, and has good anti-inflammatory effect.
Description
Technical Field
The invention relates to the technical field of natural product functional component extraction, and particularly relates to a preparation method and application of micromolecule dendrobium officinale polysaccharide.
Background
Dendrobium officinale is a homologous plant of medicine and food, belonging to the genus of Dendrobium of the family Orchidaceae, and is also one of the dendrobium varieties recorded in 'Chinese pharmacopoeia' 2020 edition, known as the head of the nine major immortals in China, about 1000 dendrobium varieties in the world, 74 dendrobium varieties in China, and the main production places in China are Zhejiang, Yunnan, Anhui and the like, wherein the planting area in Yunnan is the largest.
Traditional Chinese medicine and clinical medicine researches believe that the dendrobium officinale has the effects of tonifying stomach, promoting fluid production, nourishing yin, clearing heat, enhancing immunity, resisting tumors and the like, and is developed into medicines and functional foods with various effects. The dendrobium officinale contains active ingredients such as polysaccharide, alkaloid, amino acid, phenanthrene compounds and the like, in the field of skin surgery medicines and cosmetics, various functional cosmetics have been developed due to the fact that the polysaccharide active ingredients in the dendrobium officinale have the effects of moisturizing, resisting oxidation, delaying senescence and the like, meanwhile, the dendrobium officinale extract is recorded in a 2021 edition cosmetic raw material catalogue, and the application of the dendrobium officinale extract as a cosmetic raw material is clarified.
The traditional extraction method of the dendrobium officinale polysaccharide comprises hot water extraction, enzymatic extraction, ultrasonic extraction, microwave extraction, alkaline extraction and the like. Wherein, the hot water extraction and the enzyme extraction have low cost, low requirement on equipment and wide application. The methods of ultrasonic extraction, microwave extraction and the like have high requirements on equipment, and the alkaline extraction has great damage to the polysaccharide structure. Because the dendrobium officinale has thicker plant cell walls, the conventional extraction method is difficult to directly penetrate the cell walls so as to release more intracellular dendrobium polysaccharides. And because the dendrobium candidum polysaccharide contains more proteins and forms a polysaccharide protein structure with a compact structure, the current methods for removing proteins from the dendrobium candidum polysaccharide comprise a protease method, a Sevage method, a trichloroacetic acid method and an ammonium sulfate precipitation method, wherein the last three methods are easy to cause polysaccharide degradation.
Researches prove that the molecular weight range of the dendrobium officinale polysaccharide is from thousands to millions, the dendrobium officinale polysaccharide is closely related to the extraction, separation and purification processes, and the influence of the molecular weight of the dendrobium officinale polysaccharide on the activity is large. The dendrobium officinale polysaccharide prepared by the process in the prior art cannot meet the requirements of small molecular weight, extraction rate and purity.
Therefore, aiming at the defects of the prior art, the preparation method and the application of the micromolecule dendrobium candidum polysaccharide are necessary to solve the defects of the prior art.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of small-molecular dendrobium officinale polysaccharide, which can prepare high-purity dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50kD, and has high extraction rate.
The above object of the present invention is achieved by the following technical measures:
provides a preparation method of micromolecule dendrobium candidum polysaccharide, which adopts eutectic solvent and subcritical extraction.
According to the preparation method of the micromolecule dendrobium candidum polysaccharide, the eutectic solvent is added into the dendrobium candidum dregs, subcritical extraction is carried out to obtain initial filtrate, and then enzymolysis, elution, concentration and precipitation are carried out to obtain the finished product micromolecule dendrobium candidum polysaccharide.
The hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is glycerol, propylene glycol or butanediol, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: 1-1: 5.
The preparation method of the micromolecule dendrobium officinale polysaccharide specifically comprises the following steps:
step one, sequentially introducing water vapor and high-temperature steam into the dendrobium officinale dry product, then adding absolute ethyl alcohol, and filtering to obtain dendrobium officinale dregs;
step two, adding a eutectic solvent into the dendrobium officinale dregs, performing subcritical extraction, and then filtering and centrifuging to obtain an initial filtrate;
step three, carrying out high-pressure homogenization treatment and enzymolysis treatment on the initial filtrate, heating after the enzymolysis treatment to obtain polysaccharide enzymolysis liquid, then carrying out ultrafiltration on the polysaccharide enzymolysis liquid, and collecting filtrate;
step four, decolorizing the filtrate obtained in the step three by ion exchange resin, and performing deproteinization adsorption treatment to obtain eluent;
and step five, concentrating the eluent obtained in the step four to obtain a concentrated solution, adding ethanol into the concentrated solution until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
Preferably, the first step is specifically: pulverizing dry herba Dendrobii, putting pulverized herba Dendrobii fine powder into an extraction tank, steaming with water vapor, soaking with high temperature steam, cooling to room temperature, adding anhydrous ethanol, stirring, standing, and filtering to obtain residue.
Preferably, the second step is specifically: performing subcritical extraction on the dregs of a decoction obtained in the first step for multiple times, adding a eutectic solvent into each extraction, filtering and collecting an extracting solution after each extraction is completed, finally combining all extracting solutions, and centrifuging and collecting the extracting solution by using a tube centrifuge to obtain an initial filtrate.
Preferably, the third step is specifically: and (3) carrying out high-pressure homogenization treatment on the initial filtrate obtained in the second step for multiple times, adding hydrolase into the initial filtrate after the last high-pressure homogenization treatment is finished for enzymolysis, heating the mixture after the enzymolysis treatment to obtain polysaccharide enzymatic hydrolysate, filtering the polysaccharide enzymatic hydrolysate through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtrate.
Preferably, the fourth step is specifically: and (3) decoloring the filtrate obtained in the step (three) by using D282 type ion exchange resin, performing deproteinization adsorption treatment to obtain decolored polyprotein liquid, adding deionized water to rinse the D282 type ion exchange resin to obtain eluent, and combining the decolored polyprotein liquid and the eluent to obtain eluent.
Further, the preparation method of the micromolecule dendrobium officinale polysaccharide specifically comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 40-100-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 1-10 min by using water vapor, continuously introducing high-temperature steam of 2-30 m/s for 5-40 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 1-15 times of that of the dendrobium officinale powder, stirring, standing for 1-10 h, and filtering to obtain medicine residues;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2-5 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, eutectic solvent is added according to the ratio of material to liquid of 1: 10-1: 40g/ml each time, the volume of the water content of the eutectic solvent is 30-90%, the extraction parameters are that the temperature is 130-200 ℃, the pressure is 2-4 Mpa, and the time is 10-120 min;
step three, carrying out high-pressure homogenization treatment for 2-5 times on the initial filtrate obtained in the step two, wherein the pressure of each high-pressure homogenization treatment is 40-80 MPa, the temperature is 25-50 ℃, the high-pressure homogenization treatment is carried out for 5-20 min each time, after the last high-pressure homogenization treatment is finished, adding hydrolase, the weight ratio of the hydrolase to the initial filtrate is 0.2-5.0%, carrying out enzymolysis treatment for 0.5-2 h at the temperature of 45-65 ℃, heating to 90-105 ℃ after the enzymolysis treatment, continuing the treatment for 0.5-2 h to obtain polysaccharide enzymolysis liquid, then filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight of 50kDa, and collecting the filtered liquid;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of a sample loading diameter of the resin is 1: 10-1: 30 and the flow rate is 0.3 BV/h-3.0 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.5-3 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 55-75 ℃ and the vacuum degree of-0.2 MP-0.7 MPa until the relative density is 1.20-1.50 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 80-95%, stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
Furthermore, the preparation method of the micromolecule dendrobium officinale polysaccharide specifically comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 50-80-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 3-8 min by using water vapor, continuously introducing high-temperature steam of 5-20 m/s for 10-30 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 2-10 times of that of the dendrobium officinale powder, stirring, standing for 2-8 h, and filtering to obtain recovered ethyl alcohol and dregs;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, the eutectic solvent is added according to the ratio of material to liquid of 1: 12-1: 30g/ml each time, the water content volume of the eutectic solvent is 40-85%, the extraction parameters are that the temperature is 150-190 ℃, the pressure is 2.5-3.5 Mpa, and the time is 20-100 min;
step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 50-70 MPa, the temperature is 30-40 ℃, each time of high-pressure homogenization treatment is 8-12 min, adding hydrolase after the last time of high-pressure homogenization treatment is finished, the weight ratio of the hydrolase to the initial filtrate is 0.3-2.5%, performing enzymolysis treatment at the temperature of 50-60 ℃ for 0.8-1.2 h, heating to 95-102 ℃ after the enzymolysis treatment, continuing the treatment for 0.8-1.2 h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight of 50kDa, and collecting filtrate;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 12-1: 25 and the flow rate is 0.4 BV/h-2.5 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.8-2.5 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 60-70 ℃ and the vacuum degree of-0.25-0.6 MPa until the relative density is 1.25-1.40 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 85-92%, then stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
Preferably, the above hydrolases consist of trypsin, alpha-amylase and beta-glucan hydrolase.
In the first step, absolute ethyl alcohol is added, the mixture is stirred, then is kept stand and is filtered, and recovered ethyl alcohol and herb residue are respectively obtained.
In the fifth step, the ethanol added into the concentrated solution is the recovered ethanol in the first step.
Preferably, the drying is at least one of freeze drying, vacuum drying at 35 ℃ and forced air drying at 35 ℃.
Preferably, the purity of the small-molecular dendrobium officinale polysaccharide is greater than or equal to 96% of the dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50 kD.
The preparation method of the micromolecule dendrobium officinale polysaccharide provided by the invention takes a dendrobium officinale dry product as an initial raw material, and the micromolecule dendrobium officinale polysaccharide is finally obtained through eutectic solvent and subcritical extraction. The preparation method of the micromolecule dendrobium candidum polysaccharide can be used for preparing the dendrobium candidum polysaccharide with the molecular weight of less than or equal to 50kD, and the prepared dendrobium candidum polysaccharide with the molecular weight of less than or equal to 50kD has high purity and high extraction rate.
The second purpose of the invention is to avoid the defects of the prior art and provide the application of the small-molecular dendrobium officinale polysaccharide in preparing the product of the dendrobium officinale polysaccharide as the effective component for improving the skin immune barrier. The product can significantly improve skin immunity barrier.
The above object of the present invention is achieved by the following technical measures:
the application of the small-molecule dendrobium officinale polysaccharide in preparing a product with the dendrobium officinale polysaccharide as an effective component for improving the skin immune barrier is provided, and the product is daily cosmetics or skin surgery medicines.
The third purpose of the invention is to avoid the defects of the prior art and provide the application of the small-molecular dendrobium officinale polysaccharide in preparing the product taking the dendrobium officinale polysaccharide as an anti-inflammatory effective component, wherein the product is daily cosmetics or skin surgery medicines.
The above object of the present invention is achieved by the following technical measures:
provides the application of the micromolecule dendrobium candidum polysaccharide in preparing the product of the dendrobium candidum polysaccharide as an anti-inflammatory effective component, wherein the product is a daily cosmetic or a skin surgery medicine.
The dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50kDa has small molecular weight and high purity, is beneficial to skin absorption, can obviously improve the skin immune barrier effect and has good anti-inflammatory effect.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples, which are not intended to limit the present invention in any way. The starting reagents used in the examples of the present invention are all those conventionally purchased unless otherwise specified.
Example 1.
A method for preparing micromolecular Dendrobium officinale polysaccharide comprises taking Dendrobium officinale dry product as initial raw material, and extracting with eutectic solvent and subcritical manner. Adding a eutectic solvent into the dendrobium officinale dregs, performing subcritical extraction to obtain an initial filtrate, and performing enzymolysis, elution, concentration and precipitation to obtain the finished product of the small-molecular dendrobium officinale polysaccharide.
The hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is glycerol, propylene glycol or butanediol, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: 1-1: 5.
The preparation method of the micromolecule dendrobium officinale polysaccharide provided by the embodiment specifically comprises the following steps:
step one, sequentially introducing water vapor and high-temperature steam into the dendrobium officinale dry product, then adding absolute ethyl alcohol, and filtering to obtain dendrobium officinale dregs;
step two, adding a eutectic solvent into the dendrobium officinale dregs, performing subcritical extraction, and then filtering and centrifuging to obtain an initial filtrate;
step three, carrying out high-pressure homogenization treatment and enzymolysis treatment on the initial filtrate obtained in the step two, heating after the enzymolysis treatment to obtain polysaccharide enzymolysis liquid, then carrying out ultrafiltration on the polysaccharide enzymolysis liquid, and collecting filtrate;
step four, decolorizing the filtrate obtained in the step three by ion exchange resin, and performing deproteinization adsorption treatment to obtain eluent;
and step five, concentrating the eluent obtained in the step four to obtain a concentrated solution, adding ethanol into the concentrated solution until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
Compared with the single subcritical extraction or eutectic solvent extraction or complex enzyme assisted eutectic solvent extraction process in the prior art, the extraction rate of the obtained dendrobium officinale polysaccharide is higher through subcritical and eutectic solvent combined extraction, and the viscosity of the obtained dendrobium officinale polysaccharide solution is reduced because the molecular weight of the obtained dendrobium officinale is smaller.
The steam explosion technology in the prior art can generate toxic substances in the process of breaking the cell wall of the dendrobium officinale, and the invention utilizes high-temperature steam to break the cell wall of the dendrobium officinale, promotes the dissolution of dendrobium officinale polysaccharide in cells and the dispersion of fat-soluble ingredients, and is beneficial to improving the extraction rate of polysaccharide and removing the fat-soluble ingredients. In addition, the preparation conditions are mild, and the equipment is simple, so that the process is more environment-friendly.
Compared with the traditional enzyme extraction and strong acid hydrolysis processes in the prior art, the method can reduce the viscosity of the polysaccharide under high-pressure homogenization treatment, and simultaneously combines enzyme hydrolysis to enable the high-molecular-weight dendrobium officinale polysaccharide to be hydrolyzed more completely, so that the obtained dendrobium officinale polysaccharide has smaller molecular weight, and the removal rate of protein and pigment components in the dendrobium officinale polysaccharide is higher.
According to the preparation method disclosed by the invention, in the first step, the dendrobium officinale cells are subjected to wall breaking by using high-temperature steam, so that the dissolution of polysaccharide in the cells is promoted, the fat-soluble components are dispersed, and the improvement of the extraction rate of the polysaccharide and the removal of the fat-soluble components are facilitated. In the second step, the dendrobium officinale polysaccharide is extracted through the combined action of the subcritical solvent and the eutectic solvent, so that the extraction rate can be greatly improved. According to the invention, high-pressure homogenization is used in the third step to reduce the viscosity of the polysaccharide and simultaneously combine enzyme hydrolysis, so that the high-molecular-weight dendrobium officinale polysaccharide is promoted to be more completely hydrolyzed, the viscosity of the dendrobium officinale polysaccharide solution is reduced, and the dendrobium officinale polysaccharide with smaller molecular weight is obtained, so that the removal rate of protein and pigment components in the dendrobium officinale polysaccharide in the fourth step is higher.
In summary, the method for preparing the small-molecule dendrobium officinale polysaccharide can prepare the small-molecule dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50kD, the obtained small-molecule dendrobium officinale polysaccharide has high purity, and the extraction rate of the method is high.
Example 2.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
crushing a dry dendrobium officinale product, putting crushed dendrobium officinale fine powder into an extraction tank, steaming the dendrobium officinale fine powder by using water vapor, continuously introducing high-temperature steam for soaking, cooling to normal temperature, adding absolute ethyl alcohol, stirring, standing, and filtering to obtain medicine residues;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for multiple times, adding a eutectic solvent into each extraction, filtering and collecting an extracting solution after each extraction is completed, finally combining all extracting solutions, and centrifuging and collecting the extracting solution by using a tube centrifuge to obtain an initial filtrate;
step three, carrying out multiple times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, adding hydrolase into the initial filtrate after the last high-pressure homogenization treatment is finished for enzymolysis, heating the mixture after the enzymolysis treatment to obtain polysaccharide enzymatic hydrolysate, filtering the polysaccharide enzymatic hydrolysate through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtrate;
and step four, carrying out decoloration and deproteinization adsorption treatment on the filtrate obtained in the step three through D282 type ion exchange resin to obtain decolored polyprotein liquid, then adding deionized water to carry out leaching on the D282 type ion exchange resin to obtain leacheate, and combining the decolored polyprotein liquid and the leacheate to obtain eluent.
And step five, concentrating the eluent obtained in the step four to obtain a concentrated solution, adding ethanol into the concentrated solution until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
Wherein, the hydrogen bond acceptor of the eutectic solvent is betaine, and the hydrogen bond donor is glycerol, propylene glycol or butanediol. The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is preferably 1: 1-1: 5.
The hydrolase of the invention consists of trypsin, alpha-amylase and beta-glucan hydrolase. The hydrolase consists of 1 part of trypsin, 1 to 5 parts of alpha-amylase and 1 to 5 parts of beta-glucan hydrolase in parts by weight.
The drying may be at least one of freeze drying, vacuum drying at 35 ℃ or forced air drying at 35 ℃.
The preparation method of the small-molecule dendrobium officinale polysaccharide can prepare the small-molecule dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50 kD. The purity of the obtained micromolecule dendrobium officinale polysaccharide is more than or equal to 96%, and the extraction rate of the method is high.
Example 3.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 40-100-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 1-10 min by using water vapor, continuously introducing high-temperature steam of 2-30 m/s for 5-40 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 1-15 times of that of the dendrobium officinale powder, stirring, standing for 1-10 h, and filtering to obtain medicine residues;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2-5 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, the eutectic solvent is added according to the ratio of material to liquid of 1: 10-1: 40 each time, the water content volume of the eutectic solvent is 30-90%, the extraction parameters are that the temperature is 130-200 ℃, the pressure is 2-4 Mpa, the time is 10-120 min, and the unit of the material to liquid ratio is g/ml;
step three, carrying out high-pressure homogenization treatment for 2-5 times on the initial filtrate obtained in the step two, wherein the pressure of each high-pressure homogenization treatment is 40-80 MPa, the temperature is 25-50 ℃, the high-pressure homogenization treatment is carried out for 5-20 min each time, after the last high-pressure homogenization treatment is finished, adding hydrolase, the weight ratio of the hydrolase to the initial filtrate is 0.2-5.0%, carrying out enzymolysis treatment for 0.5-2 h at the temperature of 45-65 ℃, heating to 90-105 ℃ after the enzymolysis treatment, continuing the treatment for 0.5-2 h to obtain polysaccharide enzymolysis liquid, then filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight of 50kDa, and collecting the filtered liquid;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 10-1: 30 and the flow rate is 0.3 BV/h-3.0 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.5-3 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 55-75 ℃ and the vacuum degree of-0.2 MP-0.7 MPa until the relative density is 1.20-1.50 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 80-95%, stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
Wherein in the step one, absolute ethyl alcohol is added, stirred, kept stand and filtered to respectively obtain recovered ethyl alcohol and dregs of a decoction. And in the fifth step, adding the ethanol into the concentrated solution to obtain the recovered ethanol in the first step.
In the embodiment, the recovered ethanol recovered in the first step is used as the ethanol in the fifth step, so that the production cost can be reduced. Meanwhile, the micromolecule dendrobium officinale polysaccharide obtained by the embodiment has low molecular weight, higher purity and high extraction rate.
Example 4.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 50-80-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 3-8 min by using water vapor, continuously introducing high-temperature steam of 5-20 m/s for 10-30 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 2-10 times of that of the dendrobium officinale powder, stirring, standing for 2-8 h, and filtering to obtain recovered ethyl alcohol and dregs;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, the eutectic solvent is added according to the ratio of material to liquid of 1: 12-1: 30g/ml each time, the water content volume of the eutectic solvent is 40-85%, the extraction parameters are that the temperature is 150-190 ℃, the pressure is 2.5-3.5 Mpa, and the time is 20-100 min;
step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 50-70 MPa, the temperature is 30-40 ℃, each time of high-pressure homogenization treatment is 8-12 min, the weight ratio of the initial filtrate is 0.3-2.5% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 50-60 ℃ for 0.8-1.2 h, heating to 95-102 ℃ after the enzymolysis treatment, continuing the treatment for 0.8-1.2 h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtrate;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 12-1: 25 and the flow rate is 0.4 BV/h-2.5 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.8-2.5 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 60-70 ℃ and the vacuum degree of-0.25-0.6 MPa until the relative density is 1.25-1.40 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 85-92%, then stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
In the sample of the micromolecular dendrobium officinale polysaccharide product prepared by the method in the embodiment 1 or 2, the micromolecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa prepared by the method in the embodiment has good purity and extraction rate results.
Example 5.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
step one, crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 60-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 5min by using water vapor, continuously introducing high-temperature steam of 6-18 m/s for treatment for 15-20 min, cooling to normal temperature, adding absolute ethyl alcohol which is 3-8 times of the mass ratio of the absolute ethyl alcohol to the dendrobium officinale powder, stirring, standing for 3-6 h, and filtering to obtain recovered ethyl alcohol and dregs of a decoction respectively.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 5 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; the hydrogen bond acceptor of the eutectic solvent in this example is betaine, the hydrogen bond donor is glycerol, and the molar ratio of the two is 1: 1. Wherein, eutectic solvent is added according to the proportion of 1: 15-1: 25g/ml, the volume of the water content of the eutectic solvent is 50-80%, the extraction parameters are that the temperature is 160-180 ℃, the pressure is 3.0Mpa, and the time is 30-60 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 60Mpa, the temperature is 33 ℃, each time of high-pressure homogenization treatment is 10min, the weight ratio of the initial filtrate is 0.5-2.0% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 55 ℃ for 1h, heating to 100 ℃ after the enzymolysis treatment, continuing the treatment for 1h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid. The hydrolase of this example consists of 1 part trypsin, 1 part alpha-amylase and 1 part beta-glucan hydrolase, in parts by weight.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 15-1: 20 and the flow rate is 0.5 BV/h-2.0 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 1-2 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.3 MP to-0.5 MPa until the relative density is 1.30 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 90%, stirring to separate out polysaccharide floc, filtering and freeze-drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
In the sample of the small-molecule dendrobium officinale polysaccharide prepared by the method in example 1 or 2, the purity and extraction rate of the small-molecule dendrobium officinale polysaccharide with the molecular weight of less than 50kDa prepared by the method in this embodiment are both better.
Example 6.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 40-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 1min by using water vapor, continuously introducing high-temperature steam of 30m/s for 5min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 3 times of that of the dendrobium officinale powder, stirring, standing for 1h, and filtering to respectively obtain recovered ethyl alcohol and dregs.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 5 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, the eutectic solvent is added according to the ratio of 1:10g/ml, the volume ratio of the water content of the eutectic solvent is 90%, the extraction parameters are that the temperature is 130 ℃, the pressure is 2Mpa, and the time is 10 min.
And step three, performing 2 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 40Mpa, the temperature is 25 ℃, each time of high-pressure homogenization treatment is 5min, the weight ratio of the initial filtrate is 0.2 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 45 ℃ for 0.5h, heating to 90 ℃ after the enzymolysis treatment, continuing the treatment for 0.5h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:10 and the flow rate is 0.3BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water with the volume ratio of 0.5 times of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.2 MPa until the relative density is 1.50 to obtain a concentrated solution, adding recovered ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 80%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is glycerol, and the molar ratio of the two is 1: 3. The hydrolase of this example consists of 1 part trypsin, 5 parts alpha-amylase and 5 parts beta-glucan hydrolase in parts by weight. The drying in this example was specifically vacuum drying at 35 ℃.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, glycerin was obtained from New Ouichi Material (Nanjing) Co., Ltd, ion exchange resin type D282 Cangzhou Baoyen adsorbent science Co., Ltd, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any Eden Biotechnology Co., Ltd, of Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa is 95.72%, and the extraction rate of the dendrobium officinale polysaccharide is 51.96%.
Example 7.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 100-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 10min by using water vapor, continuously introducing high-temperature steam of 2m/s for 40min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 15 times of that of the dendrobium officinale powder, stirring, standing for 10h, and filtering to respectively obtain recovered ethyl alcohol and dregs;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 3 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, eutectic solvent is added according to the proportion of 1:30g/ml, the volume of the water content of the eutectic solvent is 40-30%, the extraction parameters are that the temperature is 200 ℃, the pressure is 4Mpa, and the time is 120 min;
step three, performing 5 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 80Mpa, the temperature is 50 ℃, the time of each time of high-pressure homogenization treatment is 20min, the weight ratio of the initial filtrate is 5.0 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 65 ℃ for 2h, heating to 105 ℃ after the enzymolysis treatment, continuing the treatment for 2h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid;
step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:30 and the flow rate is 3.0BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water of which the volume ratio to the filtrate is 3 times that of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 75 ℃ and the vacuum degree of-0.7 MPa until the relative density is 1.20 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 95%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In this embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is propylene glycol, and the molar ratio of the two is 1: 5. The hydrolase of this example consists of 1 part trypsin, 3 parts alpha-amylase and 2 parts beta-glucan hydrolase, in parts by weight. The drying in this example was specifically 35 ℃ forced air drying.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, propylene glycol was obtained from Dow chemical, ion exchange resin type D282, Canon sorbent material technology Inc., betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of Conni Erlen biotechnology Inc. of Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the embodiment is 97.29%, and the extraction rate of the dendrobium officinale polysaccharide is 54.87%.
Example 8.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing a dried dendrobium officinale product, sieving the crushed dendrobium officinale product with a 50-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 3min with water vapor, continuously introducing 5m/s high-temperature steam for 10min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 1 time to the dendrobium officinale powder, stirring, standing for 2h, and filtering to respectively obtain recovered ethyl alcohol and dregs of a decoction.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging and collecting the extract by using a tube centrifuge to obtain initial filtrate; wherein, eutectic solvent is added according to the ratio of 1:12g/ml, the water content volume of the eutectic solvent is 40%, the addition amount of the eutectic solvent is 1:12 according to the ratio of material to liquid, the water content volume of the eutectic solvent is 40%, the extraction parameters are 150 ℃, the pressure is 2.5Mpa and the time is 20 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 50Mpa, the temperature is 30 ℃, each time of high-pressure homogenization treatment is 8min, the weight ratio of the initial filtrate is 0.3 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 50 ℃ for 0.8h, heating to the temperature of 95 ℃ after the enzymolysis treatment, continuing the treatment for 0.8h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:12 and the flow rate is 0.4BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water with the volume ratio of 0.8 times of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 60 ℃ and the vacuum degree of-0.25 MP until the relative density is 1.25 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 85%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 1. The hydrolase of this example consists of 1 part trypsin, 3 parts alpha-amylase and 4 parts beta-glucan hydrolase, in parts by weight. The drying in this example was specifically 35 ℃ forced air drying.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular-weight dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa obtained in the embodiment is 98.02%, and the extraction rate of the dendrobium officinale polysaccharide is 53.65%.
Example 9.
A preparation method of micromolecule dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale products, sieving the crushed dendrobium officinale products with a 80-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 8min with water vapor, continuously introducing high-temperature steam of 20m/s for 30min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 10 times of that of the dendrobium officinale powder, stirring, standing for 8h, and filtering to respectively obtain recovered ethyl alcohol and dregs of a decoction.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, eutectic solvent is added according to the ratio of material to liquid of 1:40g/ml, the water content volume of the eutectic solvent is 85%, the extraction parameters are 190 ℃, 3.5Mpa and 100 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 70Mpa, the temperature is 40 ℃, each time of high-pressure homogenization treatment is 12min, the weight ratio of the initial filtrate is 2.5 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 60 ℃ for 1.2h, heating to 102 ℃ after the enzymolysis treatment, continuing the treatment for 1.2h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:25 and the flow rate is 2.5BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water of which the volume ratio to the filtrate is 2.5 times that of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 55 ℃ and the vacuum degree of-0.6 MPa until the relative density is 1.40 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 92%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 2.
The hydrolase of this example consists of 1 part trypsin, 3 parts alpha-amylase and 4 parts beta-glucan hydrolase, in parts by weight. The drying in this example was carried out by air-blast drying at 35 ℃ and then freeze-drying.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa of the embodiment is 96.63%, and the extraction rate of the dendrobium officinale polysaccharide is 52.69%.
Example 10.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale products, sieving the crushed dendrobium officinale products through a 90-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 9min by using water vapor, continuously introducing 24m/s high-temperature steam for treating for 28min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 4 times of that of the dendrobium officinale powder, stirring, standing for 6h, and filtering to respectively obtain recovered ethyl alcohol and dregs.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, eutectic solvent is added according to the ratio of material to liquid of 1:11g/ml, the volume ratio of the water content of the eutectic solvent is 67%, the extraction parameters are 187 ℃, the pressure is 3.3Mpa and the time is 52 min.
And step three, performing 4 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 70Mpa, the temperature is 41 ℃, each time of high-pressure homogenization treatment is 13min, the weight ratio of the initial filtrate is 1.8% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 58 ℃ for 1.6h, heating to 99 ℃ after the enzymolysis treatment, continuing the treatment for 1.8h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:21 and the flow rate is 2.0BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water with the volume ratio of 1.3 times of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.5 MPa until the relative density is 1.40 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 84%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 2. The hydrolase of this example consists of 1 part trypsin, 3 parts alpha-amylase and 4 parts beta-glucan hydrolase, in parts by weight. The drying in this example is specifically carried out by vacuum drying at 35 ℃ and then freeze drying.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa of the embodiment is 97.11%, and the extraction rate of the dendrobium officinale polysaccharide is 53.22%.
Example 11.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale products, sieving the crushed dendrobium officinale products with a 60-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 5.5min by using water vapor, continuously introducing 17m/s high-temperature steam for treating the dendrobium officinale fine powder for 27min, cooling the dendrobium officinale fine powder to the normal temperature, adding absolute ethyl alcohol with the mass ratio of 7 times of that of the dendrobium officinale powder, stirring the mixture, standing the mixture for 6h, and filtering the mixture to obtain recovered ethyl alcohol and dregs of a decoction respectively.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, eutectic solvent is added according to the ratio of material to liquid of 1:21g/ml, the volume ratio of the water content of the eutectic solvent is 75%, the extraction parameters are 182 ℃, 3.1Mpa and 50 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 61Mpa, the temperature is 37 ℃, the high-pressure homogenization treatment is performed for 9min each time, the weight ratio of the initial filtrate is 1.1% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 53 ℃ for 1h, heating to 98 ℃ after the enzymolysis treatment, continuing the treatment for 1h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:19 and the flow rate is 0.9BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water with the volume ratio of 1.2 times of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 68 ℃ under the vacuum degree of-0.4 MPa until the relative density is 1.33 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 87%, stirring until polysaccharide floccules are separated out, filtering the polysaccharide floccules, and drying in vacuum at 35 ℃ to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 2.
The hydrolase of this example consists of 1 part trypsin, 3 parts alpha-amylase and 4 parts beta-glucan hydrolase, in parts by weight.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa obtained by the embodiment is 96.45%, and the extraction rate of the dendrobium officinale polysaccharide is 52.31%.
Example 12.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale products, sieving the crushed dendrobium officinale products with a 60-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 5min by using water vapor, continuously introducing high-temperature steam of 12m/s for 18min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 5 times of that of the dendrobium officinale powder, stirring, standing for 4h, and filtering to respectively obtain recovered ethyl alcohol and dregs.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, eutectic solvent is added according to the ratio of material to liquid of 1:20g/ml each time, the water content volume of the eutectic solvent is 60%, the extraction parameters are that the temperature is 180 ℃, the pressure is 3.0Mpa, and the time is 45 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 60Mpa, the temperature is 35 ℃, each time of high-pressure homogenization treatment is 10min, the weight ratio of the initial filtrate is 1.0 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 55 ℃ for 1h, heating to 100 ℃ after the enzymolysis treatment, continuing the treatment for 1h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:15 and the flow rate is 1.0BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water of which the volume ratio to the filtrate is 2 times that of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.5 MPa until the relative density is 1.30 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 90%, then stirring until polysaccharide floccules are separated out, filtering the polysaccharide floccules, and drying in vacuum at 35 ℃ to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is propylene glycol, and the molar ratio of the two is 1: 4. The hydrolase of this example consists of 1 part trypsin, 2 parts alpha-amylase and 1 part beta-glucan hydrolase, in parts by weight.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, propylene glycol was obtained from Dow chemical, ion exchange resin type D282, Canon sorbent material technology Inc., betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of Conni Erlen biotechnology Inc. of Fushan.
The method of the embodiment is adopted to prepare the small-molecular-weight dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa obtained by the embodiment is 98.21%, and the extraction rate of the dendrobium officinale polysaccharide is 54.26%. Compared with other examples, the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa is the highest in the embodiment.
Example 13.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 60-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 5min by using water vapor, continuously introducing high-temperature steam of 10m/s for 20min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 8 times of that of the dendrobium officinale powder, stirring, standing for 3h, and filtering to respectively obtain recovered ethyl alcohol and dregs.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, the eutectic solvent is added according to the ratio of material to liquid of 1:18 each time, the water content volume of the eutectic solvent is 40-65%, the extraction parameters are 160 ℃, the pressure is 3.0Mpa, the time is 60min, and the unit of the material to liquid ratio is g/ml.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 60Mpa, the temperature is 35 ℃, each time of high-pressure homogenization treatment is 10min, the weight ratio of the initial filtrate is 1.5% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 55 ℃ for 1h, heating to 100 ℃ after the enzymolysis treatment, continuing the treatment for 1h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:15 and the flow rate is 1.0BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water of which the volume ratio to the filtrate is 2 times that of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.5 MPa until the relative density is 1.30 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 90%, then stirring until polysaccharide floccules are separated out, filtering the polysaccharide floccules, and freeze-drying to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 5. The hydrolase of this example consists of 1 part trypsin, 1 part alpha-amylase and 5 parts beta-glucan hydrolase, in parts by weight.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa of the embodiment is 98.00%, and the extraction rate of the dendrobium officinale polysaccharide is 52.17%.
Example 14.
A preparation method of micromolecular dendrobium officinale polysaccharide comprises the following steps:
step one, crushing the dry dendrobium officinale products, sieving the crushed dendrobium officinale products with a 60-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 5min by using water vapor, continuously introducing high-temperature steam of 18m/s for 15min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 8 times of that of the dendrobium officinale powder, stirring, standing for 3h, and filtering to respectively obtain recovered ethyl alcohol and dregs.
Step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2 times, adding a eutectic solvent into each extraction, extracting according to subcritical extraction parameters, filtering and collecting extract after each extraction is finished, finally combining all the extract, and centrifuging the extract by using a tube centrifuge to collect initial filtrate; wherein, the eutectic solvent is added according to the ratio of 1:25g/ml, the water content volume of the eutectic solvent is 40-80%, the extraction parameters are 160 ℃, 3.0Mpa and 30 min.
And step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 60Mpa, the temperature is 35 ℃, each time of high-pressure homogenization treatment is 10min, the weight ratio of the initial filtrate is 2.0 percent after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 55 ℃ for 1h, heating to 100 ℃ after the enzymolysis treatment, continuing the treatment for 1h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting the filtered liquid.
And step four, passing the filtrate obtained in the step three through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1:20 and the flow rate is 2.0BV/h to obtain decolorized polyprotein liquid, then eluting the D282 type ion exchange resin by using deionized water of which the volume ratio to the filtrate is 2 times that of the filtrate to obtain eluent, and combining the decolorized polyprotein liquid and the eluent to obtain eluent.
And step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 65 ℃ and the vacuum degree of-0.5 MPa until the relative density is 1.30 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 90%, then stirring until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
In the present embodiment, the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is butanediol, and the molar ratio of the two is 1: 2. The hydrolase of this example consists of 1 part trypsin, 2 parts alpha-amylase and 2 parts beta-glucan hydrolase, in parts by weight. The drying in this example is specifically freeze drying.
The dry dendrobium officinale product of this example was obtained from Kunming plant research institute, butanediol was obtained from Dow chemical, D282 type ion exchange resin Cangzhou Baoyen sorbent material science and technology Limited, and betaine, trypsin, alpha-amylase and beta-glucan hydrolase were obtained from any of the Conni Erlen biotechnology Limited in Fushan.
The method of the embodiment is adopted to prepare the small-molecular dendrobium officinale polysaccharide with the molecular weight of less than 50kDa, wherein the purity of the dendrobium officinale polysaccharide with the molecular weight of less than 50kDa of the embodiment is 96.57%, and the extraction rate of the dendrobium officinale polysaccharide is 51.68%.
Comparative example 1
Step one, taking the dendrobium officinale fine powder, directly adding 6 times of absolute ethyl alcohol, fully stirring, standing for 4 hours, adding the absolute ethyl alcohol, stirring, standing, and filtering to obtain medicine residues;
steps two to five are the same as those of example 12.
Comparative example 2
Step one, step three, step four and step five are the same as the example 12, wherein the step two is to put the dendrobium officinale herb residue of the step one into a subcritical extraction tank, add water according to the material-liquid ratio of 1:20g/ml, extract the temperature of 180 ℃, the pressure of 3Mpa, the time is 4min, filter, extract 1 time repeatedly, discard the filter residue, filter the extract with a tubular centrifuge, collect the filtrate.
Comparative example 3
Step one, step three, step four and step five are the same as the embodiment 12, step two is that the dendrobium officinale dregs in the step one are put into an extraction tank, eutectic solvent (composed of betaine and propylene glycol according to the molar ratio of 1:20 g/ml) with the water content volume of 60% is added, the extraction temperature is 100 ℃, the extraction is carried out for 60min, the filtration is carried out, the extraction is carried out for 1 time repeatedly, the filter residue is discarded, the extracting solution is filtered by a tubular centrifuge, and the filtrate is collected.
Comparative example 4
Step one, step two, step four and step five are the same as example 12, step three is to take the filtrate of step two, homogenize under 60Mpa, 35 deg.C for 10min, circulate 3 times, use the ultrafiltration membrane of 50kDa of relative molecular weight cut-off to filter polysaccharide solution, collect the filtrate, spare.
Comparative example 5
The first step, the second step, the fourth step and the fifth step are the same as the embodiment 12, the third step is to take the filtrate in the second step, add 1.0 percent of hydrolase (trypsin, alpha-amylase and beta-glucan hydrolase according to the weight ratio of 1:2:1) at the temperature of 55 ℃, increase the temperature to 100 ℃ after 1 hour of enzymolysis treatment for 1 hour, process the polysaccharide enzymolysis liquid for 1 hour, filter the polysaccharide enzymolysis liquid through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collect the filtrate.
The dendrobium candidum polysaccharides obtained in examples 6 to 14 of the invention and the dendrobium candidum polysaccharides obtained in comparative examples 1 to 5 are subjected to related preparation processes or product decolorization rate, viscosity, deproteinization rate, polysaccharide retention rate, polysaccharide extraction rate and molecular purity tests.
The decoloring rate testing and calculating method specifically comprises the following steps:
and measuring the absorbance of the dendrobium officinale polysaccharide solution before and after decolorization by taking the dendrobium officinale polysaccharide solution as a detection wavelength with the wavelength of 323 nm. And (3) calculating the decolorization ratio: the decolorization ratio (%) (absorbance before decolorization-absorbance after decolorization)/absorbance before decolorization × 100%.
The viscosity testing method of the invention specifically comprises the following steps:
and (3) directly testing the dendrobium candidum polysaccharide by adopting an Ubbelohde viscometer, wherein the testing operation scheme is according to the Ubbelohde viscosity operation method, and the molecular weight is larger when the viscosity value is larger.
The deproteinization rate testing and calculating method of the invention comprises the following steps:
the protein test of the invention adopts a Coomassie brilliant blue G-250 method to determine the protein content, and bovine serum albumin is taken as a standard substance to obtain a standard curve: y is2＝0.0007x2+0.0057，R20.9996. In the formula: x is the number of2Is the protein content, y2Is the absorbance. The linear range is 0mg to 1.0 mg. The deproteinization rate was calculated as: deproteinization rate (%) (pre-deproteinization protein content-deproteinized protein content)/pre-deproteinization protein content × 100%.
The method for measuring the content of the dendrobium officinale polysaccharide, the extraction rate of the dendrobium officinale polysaccharide and the retention degree of the dendrobium officinale polysaccharide comprises the following steps:
preparing a glucose solution: weighing a proper amount of glucose standard substance, placing the glucose standard substance in a 105 ℃ oven to dry to constant weight, weighing 100mg of glucose standard substance, using distilled water to fix the volume to a scale in a 100mL volumetric flask, and placing the volumetric flask in a 4 ℃ refrigerator for later use.
Drawing a standard curve: precisely transferring glucose standard stock solution 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, placing in a 10mL volumetric flask, adding distilled water to constant volume, and preparing into glucose standard solutions containing glucose 0mg/mL, 0.02mg/mL, 0.04mg/mL, 0.06mg/mL, 0.08mg/mL and 0.1 mg/mL. Transferring 1mL of each glucose standard solution, placing the glucose standard solutions in a 1mL glass test tube with a plug, sequentially adding 0.5mL of 5 wt% phenol solution and 2.5mL of concentrated sulfuric acid, fully swirling, cooling to room temperature, and taking 1mL of distilled water as a blank control according to the operation. Measuring absorbance values of the solutions at 490nm wavelength respectively; obtaining regression equation y as 10.25X-0.013, R2When the concentration of the glucose standard substance is 0.9997, the concentration of the glucose standard substance in the range of 0.00mg/m to 0.10mg/mL shows a good linear relationship with the absorbance value.
The dendrobium candidum polysaccharide of each example and comparative example is dissolved in distilled water, diluted to a certain mass concentration, 1mL of the solution is absorbed into a test tube with a plug, the operation is carried out according to the standard curve making step, the absorbance is measured, the mass fraction of the crude polysaccharide is calculated according to the standard curve, and the polysaccharide extraction rate (%) is (CV multiplied by dilution) multiplied by 0.9/M multiplied by 100 according to the following calculation formula. Wherein C is the concentration corresponding to the measured absorbance, V is the volume of the extracting solution, 0.9 is the orthogonal coefficient of the dendrobium officinale polysaccharide converted by glucose, and M is the mass of the raw material.
The content of the dendrobium officinale polysaccharide takes glucose as a standard substance, and is tested according to a phenol-sulfuric acid method, and the polysaccharide retention rate (%) is multiplied by 100 as the absorbance value of polysaccharide after removal/before removal.
The method for measuring the molecular weight of the polysaccharide comprises the following steps:
and (3) performing molecular weight determination on the dendrobium officinale polysaccharide by adopting high performance liquid chromatography combined with multi-angle laser scattering. The chromatographic conditions were as follows, using dextran (dextran) as standard: TSK-GELG5000PWXL chromatography column (7.8 mm. times.300 mm); the mobile phase is ultrapure water; the sample injection amount is 20 mu L; the flow rate is 0.6 mL/min; the column temperature was 30 ℃. Detecting by using a 2414 refractive index detector and an 8-angle laser scattering instrument to detect the purity of the dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50 kDa.
The method for testing the repairing effect of dendrobium officinale polysaccharide on SDS-induced cell damage specifically comprises the following steps:
the cells used are in particular HaCaT cells. HaCaT cells are cultured in complete medium and prepared to have a concentration of 1 × 106Cell suspension per mL, plated in 96-well plates at 100 μ L per well. Placing in an incubator at 37 deg.C and 5% CO2After incubation for 24h under conditions, 50. mu.g/mL of the solution was added to each well-1The culture was continued for 24 hours. The SDS solution was aspirated by a pipette gun, and after washing the residue with the complete medium, the complete medium (blank control) and the sample solution having a concentration of 1000.0. mu.g.mL were added at 200. mu.L/well-1、250.0μg.mL-1、62.5μg.mL-1The dendrobium officinale polysaccharide solution. After further culturing for 24 hours, the cells were washed with the complete medium, 100. mu.L of MTT solution was added to each well after washing, and the cells were cultured in an incubator for 4 hours. After the MTT solution was aspirated by a pipette, 150. mu.L of the DMSO solution was added to each well, and the 96-well plate was placed on a shaker and shaken at medium speed for 5 min. Measuring the absorbance at 490nm by using an enzyme-linked immunosorbent assay (ELISA), wherein the cell survival rate (X,%) is calculated as X ═ S-CK)/(YCK-CK). times.100%, wherein S is the absorbance of the test group; CK is blank control group absorbance; YCK is the absorbance of the negative control group.
The method for testing the effect of the dendrobium officinale polysaccharide on LPS (Lipopolysaccharide) induced RAW264.7 cell inflammation comprises the following steps:
the NO test is carried out by adopting a RAW264.7 cell LPS model, and the samples are samples of each example and a control sample, and the specific method is as follows:
(1) cell culture: complete medium culture, add 2mL complete medium, scrape cells down with cell scraper. And uniformly collecting the mixture to a centrifuge tube by blowing and beating, and centrifuging for 3min at the speed of 1200 r/min.
(2) Plate paving: the cells were digested and resuspended according to the procedure described above, the density of the cell suspension being approximately 5x 104one/mL. 2mL of cell suspension is added to each well of the 6-well plate, i.e., the plating density of the cells is 105Per well.
(3) Adding a sample: approximately 24 hours after cell plating, 5. mu.g/mL LPS solution was added. The medium was aspirated from each well of the 6-well plate. Leave 1 well as a negative control and add 2mL of the prepared LPS solution per well. The 6-well plate was placed in an incubator and incubation was continued for 24 hours.
(4) Administration: the medium was aspirated from each well of the 6-well plate. Except for the negative control group, 1 well was left as a model group, and the other wells were grouped by the amount of the drug and the administration concentration, each 2mL of the complete medium containing the drug was added. The 6-well plate was placed in an incubator and incubation was continued for 24 hours. Pipette 50. mu.L of supernatant into 96 plates and add Grice reagent to detect NO content.
(5) Test absorbance: and measuring the light absorption value (OD) by a microplate reader at 540 nm.
(6) Data processing and calculation: and (3) processing the light absorption value measured by the experiment by using Excel software, calculating the OD value of the cell after the samples to be measured with different concentrations are processed, and calculating the NO content by taking the concentration as the ordinate and the absorbance as the abscissa.
The technical effects of the dendrobium officinale polysaccharides obtained in examples 6 to 14 of the invention and the dendrobium officinale polysaccharides obtained in comparative examples 1 to 5 are shown in the first table:
comparison of technical effects of first and second polysaccharides
As can be seen from table one, the preparation method of the present invention, as in examples 6 to 14, has significant advantages in the decolouring rate of the residue in step one, the extraction rate of the dendrobium officinale polysaccharide, the decolouring rate in step four, the decolouring and deproteinization, the retention rate of the dendrobium officinale polysaccharide in step four, and the molecular weight, compared with comparative examples 1 to 5.
The cell wall of the dendrobium officinale can be broken through the treatment of the first step, so that water-soluble dendrobium officinale polysaccharide and fat-soluble pigment in cells are dissolved out, and the fat-soluble pigment is dissolved in ethanol, therefore, when the decolorization rate of the medicine residue in the first step is higher, the higher the cell wall breakage is, the higher the extraction rate of the water-soluble dendrobium officinale polysaccharide and the fat-soluble pigment is, the higher the decolorization rate of the medicine residue is, the higher the extraction rate of the dendrobium officinale polysaccharide is reflected indirectly. The table one shows that the decolorization ratio of the process is higher than that of comparative examples 1 to 5, and compared with the untreated dendrobium officinale powder (the process of comparative example 1), the decolorization ratio of the process can be different by 1.39 times at most.
The extraction rate of the dendrobium officinale polysaccharide obtained by adopting the combined subcritical and eutectic solvent extraction process and parameters thereof is obviously higher than that of a single subcritical (namely the process of comparative example 2) or eutectic solvent process (namely the process of comparative example 3) and is also higher than that of an extraction process (namely the process of comparative example 5) adopting an eutectic solvent assisted enzyme method. In table one, the viscosity of the dendrobium officinale polysaccharide in the third step of the invention is below 3 η w, while the viscosity of the dendrobium officinale polysaccharide in the comparative examples is greater than 3 η w, the viscosity of the dendrobium officinale polysaccharide is related to the molecular weight, and when the viscosity of the dendrobium officinale polysaccharide is higher, the molecular weight is higher, so that the molecular weight of the dendrobium officinale polysaccharide can be obviously reduced under the combination of high-pressure homogenization treatment and enzyme hydrolysis in the third step. Because the dendrobium officinale polysaccharide obtained in the third step of the invention has smaller molecular weight and is more favorable for being eluted from the ion exchange resin, the decolorization, deproteinization and polysaccharide retention effects of the fourth step of the first embodiment 6 to 14 are obviously higher than those of the first embodiment 1 to 5.
In addition, in the first table, the dendrobium officinale polysaccharides with molecular weights of 50kDa or less obtained in examples 6 to 14 of the present invention are all over 96%, wherein the highest content is 98.21%, and the highest content in the comparative example is only 93.24%, so the preparation method of the small molecular dendrobium officinale polysaccharide of the present invention has a significantly better effect.
The dendrobium candidum polysaccharides obtained in the embodiments 6 to 14 and the dendrobium candidum polysaccharides obtained in the comparative examples 1 to 5 are subjected to performance tests on SDS-induced cell damage and LPS-induced NO content.
The results of comparing the dendrobium officinale polysaccharides obtained in examples 6 to 14 of the present invention with those obtained in comparative examples 1 to 5 on SDS-induced cell damage and LPS-induced NO content are shown in Table II.
Second, the results of comparing the content of NO induced by SDS and LPS on the cell damage induced by the dendrobium officinale polysaccharide
| Experimental group | SDS damaged cell survival (%) | NO content (μmol/L) |
| Blank group | 101.00±0.12 | 15.32±1.01 |
| Model set | 78.29±1.12### | 48.45±1.06### |
| Example 6 | 93.86±1.15** | 28.12±0.39** |
| Example 7 | 98.24±0.96** | 23.96±1.04*** |
| Example 8 | 95.47±0.86** | 27.37±1.11*** |
| Example 9 | 94.52±1.02** | 26.58±0.74*** |
| Example 10 | 97.01±1.33** | 25.62±0.42*** |
| Example 11 | 96.38±0.62** | 25.33±0.55*** |
| Example 12 | 96.44±0.56** | 25.76±1.00*** |
| Example 13 | 97.17±1.08** | 27.33±0.82*** |
| Example 14 | 95.68±0.87** | 26.57±0.98*** |
| Comparative example 1 | 90.82±1.42* | 30.36±1.12* |
| Comparative example 2 | 90.45±1.06* | 34.98±1.22 |
| Comparative example 3 | 88.63±1.06 | 32.71±0.94* |
| Comparative example 4 | 90.12±0.88* | 32.39±0.61* |
| Comparative example 5 | 87.92±1.61 | 34.52±1.38 |
Note: compared with the blank group, the model group # is p < 0.05, the # is p < 0.01, and the # is p < 0.001; compared with the model group, the sample group was p < 0.05, p < 0.01, and p < 0.001.
From the second table, the small-molecular-weight dendrobium officinale polysaccharide obtained by the preparation method disclosed by the invention has the advantages that the survival rate of SDS-induced cells can be improved more remarkably (p is less than 0.01 or p is less than 0.001) compared with the dendrobium officinale polysaccharide obtained by the comparative examples 1 to 5, the NO secretion generated by LPS-induced cells is inhibited, the damage degree of the SDS-induced cells is further effectively repaired, and the inflammatory reaction is further relieved, so that the dendrobium officinale polysaccharide prepared by the invention is small in molecular weight, and can more easily enter the cells, so that the cell factor is regulated and controlled, and the biological activity effect is exerted.
Example 15.
Use of the small-molecule dendrobium officinale polysaccharide prepared by the methods of examples 6 to 14 in preparation of a product of dendrobium officinale polysaccharide as an effective component for improving skin immune barrier, wherein the product is a daily cosmetic or a skin surgery medicine.
The second table shows that the micromolecular dendrobium officinale polysaccharide can obviously improve the survival rate of SDS-induced cells, so that the damage degree of the SDS-induced cells can be effectively repaired, and the micromolecular dendrobium officinale polysaccharide can be used as an effective component of daily cosmetics or skin surgical medicines for improving skin immune barriers.
Example 16.
Use of the small molecule Dendrobium officinale polysaccharide prepared by examples 6 to 14 in the preparation of a product containing Dendrobium officinale polysaccharide as an anti-inflammatory active ingredient, wherein the product is a daily cosmetic or a skin surgery medication.
The second table shows that the product of the dendrobium officinale polysaccharide prepared by the molecular dendrobium officinale polysaccharide disclosed by the invention as an anti-inflammatory effective component can inhibit LPS (lipopolysaccharide) to induce cells to generate NO secretion, so that the inflammatory reaction is relieved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of micromolecule dendrobium officinale polysaccharide is characterized in that eutectic solvent is adopted to be combined with subcritical extraction.
2. The method for preparing small-molecule dendrobium officinale polysaccharide according to claim 1, which is characterized in that: adding a eutectic solvent into the dendrobium officinale dregs, performing subcritical extraction to obtain an initial filtrate, and performing enzymolysis, elution, concentration and precipitation to obtain the finished product of the small-molecular dendrobium officinale polysaccharide.
3. The method for preparing small-molecule dendrobium officinale polysaccharide according to claim 2, which is characterized in that: the hydrogen bond acceptor of the eutectic solvent is betaine, the hydrogen bond donor is glycerol, propylene glycol or butanediol, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: 1-1: 5.
4. The preparation method of the small-molecule dendrobium officinale polysaccharide according to claim 1, 2 or 3, which is characterized by comprising the following steps:
step one, sequentially introducing water vapor and high-temperature steam into the dendrobium officinale dry product, then adding absolute ethyl alcohol, and filtering to obtain dendrobium officinale dregs;
step two, adding a eutectic solvent into the dendrobium officinale dregs, performing subcritical extraction, and then filtering and centrifuging to obtain an initial filtrate;
step three, carrying out high-pressure homogenization treatment and enzymolysis treatment on the initial filtrate, heating after the enzymolysis treatment to obtain polysaccharide enzymolysis liquid, then carrying out ultrafiltration on the polysaccharide enzymolysis liquid, and collecting filtrate;
step four, decolorizing the filtrate obtained in the step three by ion exchange resin, and performing deproteinization adsorption treatment to obtain eluent;
and step five, concentrating the eluent obtained in the step four to obtain a concentrated solution, adding ethanol into the concentrated solution until polysaccharide floccules are separated out, filtering and drying the polysaccharide floccules to obtain the micromolecule dendrobium officinale polysaccharide.
5. The method for preparing small-molecule dendrobium officinale polysaccharide according to claim 4, wherein the method comprises the following steps:
the first step is specifically as follows: crushing the dry dendrobium officinale products, putting the crushed dendrobium officinale fine powder into an extraction tank, steaming the dendrobium officinale fine powder by using water vapor, continuously introducing high-temperature steam for soaking, cooling to normal temperature, adding absolute ethyl alcohol, stirring, standing, and filtering to obtain medicine residues;
the second step is specifically: performing subcritical extraction on the dregs of a decoction obtained in the step one for multiple times, adding a eutectic solvent into each extraction, filtering and collecting an extracting solution after each extraction is finished, finally combining all extracting solutions, and centrifuging and collecting the extracting solution by using a tube centrifuge to obtain an initial filtrate;
the third step is specifically as follows: performing high-pressure homogenization treatment on the initial filtrate obtained in the second step for multiple times, adding hydrolase into the initial filtrate after the last high-pressure homogenization treatment is finished for enzymolysis, heating the mixture after the enzymolysis treatment to obtain polysaccharide enzymatic hydrolysate, filtering the polysaccharide enzymatic hydrolysate through an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtrate;
the fourth step is specifically: and (3) decoloring the filtrate obtained in the step (three) by using D282 type ion exchange resin, performing deproteinization adsorption treatment to obtain decolored polyprotein liquid, adding deionized water to rinse the D282 type ion exchange resin to obtain eluent, and combining the decolored polyprotein liquid and the eluent to obtain eluent.
6. The preparation method of the small-molecule dendrobium officinale polysaccharide according to claim 5, which comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 40-100-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 1-10 min by using water vapor, continuously introducing high-temperature steam of 2-30 m/s for 5-40 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 1-15 times of that of the dendrobium officinale powder, stirring, standing for 1-10 h, and filtering to obtain medicine residues;
step two, performing subcritical extraction on the dregs of a decoction obtained in the step one for 2-5 times, adding a eutectic solvent according to the ratio of material to liquid of 1: 10-1: 40g/ml each time, wherein the eutectic solvent has a water content of 30-90% by volume, and the extraction parameters comprise that the temperature is 130-200 ℃, the pressure is 2-4 MPa, and the time is 10-120 min;
step three, carrying out high-pressure homogenization treatment for 2-5 times on the initial filtrate obtained in the step two, wherein the pressure of each high-pressure homogenization treatment is 40-80 MPa, the temperature is 25-50 ℃, the high-pressure homogenization treatment is carried out for 5-20 min each time, after the last high-pressure homogenization treatment is finished, adding hydrolase, the weight ratio of the hydrolase to the initial filtrate is 0.2-5.0%, carrying out enzymolysis treatment for 0.5-2 h at the temperature of 45-65 ℃, heating to 90-105 ℃ after the enzymolysis treatment, continuing the treatment for 0.5-2 h to obtain polysaccharide enzymolysis liquid, then filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight of 50kDa, and collecting the filtered liquid;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 10-1: 30 and the flow rate is 0.3 BV/h-3.0 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.5-3 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 55-75 ℃ and the vacuum degree of-0.2 MP-0.7 MPa until the relative density is 1.20-1.50 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 80-95%, stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
7. The preparation method of the small-molecule dendrobium officinale polysaccharide according to claim 6, which comprises the following steps:
crushing a dry dendrobium officinale product, sieving the crushed dendrobium officinale product with a 50-80-mesh sieve to obtain dendrobium officinale fine powder, putting the dendrobium officinale fine powder into an extraction tank, steaming and soaking the dendrobium officinale fine powder for 3-8 min by using water vapor, continuously introducing high-temperature steam of 5-20 m/s for 10-30 min, cooling to normal temperature, adding absolute ethyl alcohol with the mass ratio of 2-10 times of that of the dendrobium officinale powder, stirring, standing for 2-8 h, and filtering to obtain recovered ethyl alcohol and dregs;
step two, performing subcritical extraction on the medicine residues obtained in the step one for 2 times, adding a eutectic solvent according to the material-liquid ratio of 1: 12-1: 30g/ml each time, wherein the volume ratio of the water content of the eutectic solvent is 40-85%, and the extraction parameters comprise the temperature of 150-190 ℃, the pressure of 2.5-3.5 MPa and the time of 20-100 min;
step three, performing 3 times of high-pressure homogenization treatment on the initial filtrate obtained in the step two, wherein the pressure of each time of high-pressure homogenization treatment is 50-70 MPa, the temperature is 30-40 ℃, each time of high-pressure homogenization treatment is 8-12 min, the weight ratio of the initial filtrate is 0.3-2.5% after the last time of high-pressure homogenization treatment is finished, performing enzymolysis treatment at the temperature of 50-60 ℃ for 0.8-1.2 h, heating to 95-102 ℃ after the enzymolysis treatment, continuing the treatment for 0.8-1.2 h to obtain polysaccharide enzymolysis liquid, filtering the polysaccharide enzymolysis liquid by using an ultrafiltration membrane with the relative molecular weight cutoff of 50kDa, and collecting filtrate;
step four, enabling the filtrate obtained in the step three to pass through D282 type ion exchange resin, carrying out decolorization and deproteinization adsorption treatment under the conditions that the height ratio of the sample loading diameter of the resin is 1: 12-1: 25 and the flow rate is 0.4 BV/h-2.5 BV/h to obtain decolorized polyprotein liquid, then leaching the D282 type ion exchange resin by using deionized water with the volume ratio of 0.8-2.5 times of that of the filtrate to obtain leacheate, and combining the decolorized polyprotein liquid and the leacheate to obtain eluent;
and step five, concentrating the eluent obtained in the step four, concentrating the eluent at the concentration temperature of 60-70 ℃ and the vacuum degree of-0.25-0.6 MPa until the relative density is 1.25-1.40 to obtain a concentrated solution, adding ethanol into the concentrated solution, adjusting the ethanol concentration in the concentrated solution to 85-92%, then stirring until polysaccharide floc is separated out, filtering and drying the polysaccharide floc to obtain the micromolecule dendrobium officinale polysaccharide.
8. The method for preparing small-molecule dendrobium officinale polysaccharide according to claim 4, wherein the method comprises the following steps: the hydrolase consists of trypsin, alpha-amylase and beta-glucan hydrolase;
in the first step, adding absolute ethyl alcohol, stirring, standing, and filtering to obtain recovered ethyl alcohol and dregs respectively;
in the fifth step, the ethanol added into the concentrated solution is the recovered ethanol in the first step;
the drying is at least one of freeze drying, vacuum drying at 35 ℃ or forced air drying at 35 ℃;
the micromolecule dendrobium officinale polysaccharide is dendrobium officinale polysaccharide with the molecular weight of less than or equal to 50kD, and the purity of more than or equal to 96 percent.
9. Use of the small molecule dendrobium officinale polysaccharide obtained according to any one of claims 1 to 8 for the preparation of a product with dendrobium officinale polysaccharide as an effective component for improving skin immune barrier, wherein the product is a daily cosmetic or a skin surgery medicine.
10. Use of the small-molecule dendrobium officinale polysaccharide obtained according to any one of claims 1 to 8 in the preparation of a product with dendrobium officinale polysaccharide as an anti-inflammatory active ingredient, wherein the product is a daily cosmetic or a skin surgery medicine.
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| CN115819639B (en) * | 2023-01-10 | 2024-03-22 | 仲恺农业工程学院 | Preparation method and application of dendrobium candidum polysaccharide with small molecular weight |
| CN115819639A (en) * | 2023-01-10 | 2023-03-21 | 仲恺农业工程学院 | Preparation method and application of small-molecular-weight dendrobium officinale polysaccharide |
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| CN116656758B (en) * | 2023-06-26 | 2024-03-22 | 上海植纳生物科技有限公司 | Dendrobium oligosaccharide and fresh dendrobium juice as well as preparation methods and applications thereof |
| CN116656758A (en) * | 2023-06-26 | 2023-08-29 | 上海植纳生物科技有限公司 | Dendrobium oligosaccharide and fresh dendrobium juice as well as preparation methods and applications thereof |
| CN117695198A (en) * | 2024-02-05 | 2024-03-15 | 宝萃生物科技有限公司 | Method for preparing dendrobium candidum extract by coupling eutectic solvent with ultrasound and heating |
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