CN114181326B - Extraction method of nostoc polysaccharide - Google Patents
Extraction method of nostoc polysaccharide Download PDFInfo
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- CN114181326B CN114181326B CN202111603911.XA CN202111603911A CN114181326B CN 114181326 B CN114181326 B CN 114181326B CN 202111603911 A CN202111603911 A CN 202111603911A CN 114181326 B CN114181326 B CN 114181326B
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- nostoc
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- sphaeroids kutz
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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
Abstract
The invention relates to the technical field of extraction, in particular to an extraction method of nostoc sphaeroids kutz polysaccharide, which comprises the steps of drying nostoc sphaeroids kutz to constant weight, crushing to obtain nostoc sphaeroids kutz powder, adding the nostoc sphaeroids kutz powder into a sodium hydroxide solution with the temperature of 40-45 ℃ for soaking, filtering out steam explosion treatment, adding a compound enzyme preparation into a disodium hydrogen phosphate-citric acid buffer solution, and carrying out enzymolysis to obtain nostoc sphaeroids kutz crude polysaccharide, and purifying the nostoc sphaeroids kutz crude polysaccharide.
Description
Technical Field
The invention relates to the technical field of extraction, in particular to an extraction method of nostoc polysaccharide.
Background
Nostoc sphaeroids kutz is a low-level single-cell blue algae of the family nostoc, and is commonly known as Nostoc sphaeroids kutz. According to the description of the 'outline of the invention', nostoc sphaeroids kutz 'has the effects of relieving fever, clearing diaphragm and benefiting intestines and stomach' and is an unobtainable natural pollution-free green food. The nostoc sphaeroids kutz has rich polysaccharide content and various physiological activities, is highly concerned by medical and food science, and the mokai chrysanthemum discovers that the wild nostoc sphaeroids kutz polysaccharide has weak oxidation resistance, but can reduce thymus index and inhibit the reduction of cellular immunity caused by tumors, and has no toxicity to kidney tissues; peng Changan and the like find that nostoc polysaccharide has better inhibition capability on proliferation of HCT-116 cells through an MTT colorimetric method; the filter paper patch method is often adopted to research that nostoc sphaeroids kutz water-soluble polysaccharide has inhibition effect on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and mould; zhu Yuting and the like find that the nostoc polysaccharide subjected to sulfation has an anticoagulant function, but the current extraction method of the nostoc polysaccharide is time-consuming and labor-consuming, and has lower purity and yield.
Disclosure of Invention
The invention aims to: aiming at the technical problems, the invention provides an extraction method of nostoc polysaccharide.
The technical scheme adopted is as follows:
an extraction method of nostoc polysaccharide comprises the following steps:
s1: drying nostoc sphaeroids kutz to constant weight, and pulverizing to obtain nostoc sphaeroids kutz powder;
s2: soaking nostoc sphaeroids kutz powder in 40-45 ℃ sodium hydroxide solution for 2-4 hours, filtering, transferring to steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 6-8MPa, keeping the pressure for 10-15 minutes, opening a valve to release pressure, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 20-40 minutes, adding a complex enzyme preparation, and heating to 55-65 ℃ for enzymolysis for 8-12 hours;
s3: heating the solution to reflux for 5-10h after enzymolysis, centrifuging, adding 5-10 times volume of ethanol into the supernatant, standing at 3-5deg.C for 10-15h, and filtering to obtain nostoc sphaeroids kutz crude polysaccharide;
s4: dissolving nostoc sphaeroids kutz crude polysaccharide with water, adding chloroform-n-butanol solution, oscillating for 20-40min, centrifuging, separating water phase from supernatant, adding chloroform-n-butanol solution, continuing oscillating, repeating the above operation for 3-5 times, concentrating the water phase under reduced pressure, evaporating low boiling point component to obtain concentrated solution, adding saturated sodium chloride solution and ethanol into the concentrated solution, standing for 10-15h at 3-5 ℃, filtering, and vacuum drying the obtained solid at 40-60 ℃.
Further, the molar concentration of the sodium hydroxide solution in the S2 is 0.2-0.6mol/L, and the feed-liquid ratio is 1:80-120.
Further, the complex enzyme preparation in S2 consists of cellulase, papain and neutral protease.
Further, in the S2, the weight ratio of the cellulase to the papain to the neutral protease is 2:3:1.
further, the dosage of the complex enzyme preparation in the S2 is 0.5-1% of the weight of nostoc sphaeroids kutz powder.
Further, the volume concentration of ethanol in S3 is 55-65%.
Further, the volume ratio of chloroform to n-butanol in the chloroform-n-butanol solution in S4 is 4:1.
further, the addition amount of the chloroform-n-butanol solution in the S4 is 25-30% of the volume of the nostoc sphaeroids kutz crude polysaccharide solution.
Further, the volume concentration of ethanol in S4 is 75-100%.
Further, the addition amount of the saturated sodium chloride solution and the ethanol in the S4 is respectively 0.05-0.1 times and 3-5 times of the volume of the concentrated solution.
The invention has the beneficial effects that:
the invention provides an extraction method of nostoc sphaeroids kutz polysaccharide, wherein the cell wall of nostoc sphaeroids kutz is provided with an inner layer and an outer layer, the inner layer is mainly composed of cellulose, the outer layer is a colloid sheath, a layered structure is easy to form, the structure of the cell wall of nostoc sphaeroids kutz is relatively firm, high-temperature and high-pressure steam instantaneously releases pressure during steam explosion to damage the structure of the cell wall of nostoc sphaeroids kutz, a composite enzyme preparation composed of cellulase, papain and neutral protease can decompose the cell wall on one hand, promote the release of nostoc sphaeroids kutz polysaccharide, and decompose impurity proteins in the nostoc sphaeroids kutz polysaccharide on the other hand, so that the purity of nostoc sphaeroids kutz polysaccharide is improved, and the enzyme method is combined with chloroform-n-butanol solution, so that the loss rate of nostoc sphaeroids kutz polysaccharide is low, the consumption of chloroform-n-butanol solution is reduced to a certain extent, and the purity of nostoc sphaeroids kutz polysaccharide prepared by the method reaches more than 97%, and the yield is relatively high, and has relatively wide application prospect.
Detailed Description
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
a method for extracting nostoc polysaccharide comprises the following steps:
100g of nostoc sphaeroids kutz is baked to constant weight and then crushed to obtain nostoc sphaeroids kutz powder, the nostoc sphaeroids kutz powder is soaked in sodium hydroxide solution with the temperature of 45 ℃ and the mol/L of 0.5 to be filtered out after 4 hours of soaking, and the feed liquid ratio is 1:120, transferring the mixture into steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 8MPa, maintaining the pressure for 15min, opening a valve for pressure relief, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 30min, and adding cellulose, papain and neutral protease according to the weight ratio of 2:3:1, heating the solution to reflux for 10 hours after the enzymolysis is finished, centrifuging, adding 10 times of 65% ethanol in volume, standing for 15 hours at 4 ℃, filtering to obtain nostoc sphaeroids kutz crude polysaccharide, dissolving nostoc sphaeroids kutz crude polysaccharide with water, adding 30% of chloroform-n-butanol solution (the volume ratio of chloroform to n-butanol is 4:1), vibrating for 40 minutes, centrifuging, separating out water phase from the supernatant, adding chloroform-n-butanol solution, continuing vibrating, repeating the operation for 5 times, evaporating low-boiling components from the water phase under reduced pressure to obtain concentrated solution, adding 0.1 times of saturated sodium chloride solution and 5 times of 75% ethanol into the concentrated solution, standing for 15 hours at 4 ℃, filtering, and performing vacuum drying on the obtained solid at 50 ℃.
Example 2:
a method for extracting nostoc polysaccharide comprises the following steps:
100g of nostoc sphaeroids kutz is baked to constant weight and then crushed to obtain nostoc sphaeroids kutz powder, the nostoc sphaeroids kutz powder is soaked in sodium hydroxide solution with the temperature of 45 ℃ and the mol/L of 0.6 for 4 hours and then filtered, and the feed liquid ratio is 1:120, transferring the mixture into steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 8MPa, maintaining the pressure for 15min, opening a valve for pressure relief, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 40min, and adding cellulose, papain and neutral protease according to the weight ratio of 2:3:1, heating the solution to reflux for 10 hours after the enzymolysis is finished, centrifuging, adding 10 times of 65% ethanol into the supernatant, standing for 15 hours at 5 ℃, filtering to obtain nostoc sphaeroids kutz crude polysaccharide, dissolving the nostoc sphaeroids kutz crude polysaccharide with water, adding 30% of chloroform-n-butanol solution (the volume ratio of chloroform to n-butanol is 4:1), vibrating for 40 minutes, centrifuging, separating out the water phase from the supernatant, adding chloroform-n-butanol solution, continuing vibrating, repeating the operation for 5 times, evaporating the low-boiling component from the water phase under reduced pressure to obtain concentrated solution, adding 0.1 times of saturated sodium chloride solution and 5 times of 75% ethanol into the concentrated solution, standing for 15 hours at 5 ℃, filtering, and vacuum drying the obtained solid at 60 ℃.
Example 3:
a method for extracting nostoc polysaccharide comprises the following steps:
100g of nostoc sphaeroids kutz is baked to constant weight and then crushed to obtain nostoc sphaeroids kutz powder, and the nostoc sphaeroids kutz powder is soaked in a sodium hydroxide solution with the temperature of 40 ℃ and the mol/L of 0.2 for 2 hours and then filtered, wherein the feed liquid ratio is 1:80, transferring the mixture into steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 6MPa, keeping the pressure for 10min, opening a valve to release pressure, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 20min, and adding cellulose, papain and neutral protease according to the weight ratio of 2:3:1, the dosage of the compound enzyme preparation is 0.5 percent of the weight of nostoc powder, heating to 55 ℃ for enzymolysis for 8 hours, heating the solution to reflux for 5 hours after the enzymolysis is finished, centrifuging, adding 5 times of 55 percent of ethanol into the supernatant, standing for 10 hours at 3 ℃, filtering to obtain nostoc crude polysaccharide, dissolving the nostoc crude polysaccharide with water, adding 25 percent of chloroform-n-butanol solution (the volume ratio of chloroform to n-butanol is 4:1), oscillating for 20 minutes, centrifuging, separating out the water phase from the supernatant, adding chloroform-n-butanol solution, continuing oscillating, repeating the operation for 3 times, concentrating the water phase under reduced pressure, evaporating low-boiling components to obtain concentrated solution, adding 0.05 times of saturated sodium chloride solution and 3 times of 75 percent of ethanol into the concentrated solution, standing for 10 hours at 3 ℃, filtering, and vacuum drying the obtained solid at 40 ℃.
Example 4:
a method for extracting nostoc polysaccharide comprises the following steps:
100g of nostoc sphaeroids kutz is baked to constant weight and then crushed to obtain nostoc sphaeroids kutz powder, the nostoc sphaeroids kutz powder is soaked in sodium hydroxide solution with the temperature of 45 ℃ and the mol/L of 0.2 for 4 hours and then filtered, and the feed liquid ratio is 1:80, transferring the mixture into steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 8MPa, keeping the pressure for 10min, opening a valve to release pressure, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 40min, and adding cellulose, papain and neutral protease according to the weight ratio of 2:3:1, the dosage of the compound enzyme preparation is 0.5 percent of the weight of nostoc powder, heating to 65 ℃ for enzymolysis for 8 hours, heating the solution to reflux for 10 hours after the enzymolysis is finished, centrifuging, adding 5 times of 65 percent ethanol into the supernatant, standing for 15 hours at 3 ℃, filtering to obtain nostoc crude polysaccharide, dissolving the nostoc crude polysaccharide with water, adding 25 percent of chloroform-n-butanol solution (the volume ratio of chloroform to n-butanol is 4:1), oscillating for 40 minutes, centrifuging, separating out the water phase from the supernatant, adding chloroform-n-butanol solution, continuing oscillating, repeating the operation for 3 times, concentrating the water phase under reduced pressure, evaporating low-boiling components to obtain concentrated solution, adding 0.1 times of saturated sodium chloride solution and 3 times of 75 percent ethanol into the concentrated solution, standing for 10 hours at 5 ℃, filtering, and vacuum drying the obtained solid at 60 ℃.
Example 5:
a method for extracting nostoc polysaccharide comprises the following steps:
100g of nostoc sphaeroids kutz is baked to constant weight and then crushed to obtain nostoc sphaeroids kutz powder, and the nostoc sphaeroids kutz powder is soaked in a sodium hydroxide solution with the temperature of 40 ℃ and the mol/L of 0.6 for 2 hours and then filtered, wherein the feed liquid ratio is 1:120, transferring the mixture into steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 6MPa, maintaining the pressure for 15min, opening a valve for pressure relief, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 20min, and adding cellulose, papain and neutral protease according to the weight ratio of 2:3:1, heating the solution to reflux for 5 hours after the enzymolysis is finished, centrifuging, adding 10 times of 55-volume percent ethanol into supernatant, standing for 10 hours at 5 ℃, filtering to obtain nostoc sphaeroids kutz crude polysaccharide, dissolving nostoc sphaeroids kutz crude polysaccharide with water, adding 30 percent of chloroform-n-butanol solution (the volume ratio of chloroform to n-butanol is 4:1) into the nostoc sphaeroids kutz crude polysaccharide, vibrating for 20 minutes, centrifuging, separating out water phase from supernatant, adding chloroform-n-butanol solution, continuously vibrating, repeatedly performing the operation for 5 times, concentrating the water phase under reduced pressure to obtain a concentrated solution, adding 0.05 times of saturated sodium chloride solution and 5 times of 75-volume percent ethanol into the concentrated solution, standing for 15 hours at 3 ℃, filtering, and vacuum drying the obtained solid at 40 ℃.
Comparative example 1:
substantially the same as in example 1, except that the steam explosion treatment was not performed at the time of extraction.
Comparative example 2:
substantially the same as in example 1, except that no cellulase was added during the extraction.
Comparative example 3:
substantially the same as in example 1, except that papain was not added at the time of extraction.
Comparative example 4:
substantially the same as in example 1, except that no neutral protease was added at the time of extraction.
Performance test:
the data relating to the nostoc polysaccharide samples obtained by the methods of examples 1-5 and comparative examples 1-4 are shown in table 1 below:
and (3) determining the nostoc polysaccharide sample by adopting a phenol-sulfuric acid method.
Table 1:
as can be seen from the table 1, the purity of nostoc polysaccharide obtained by the method reaches more than 97%, the yield is higher, and the steam explosion treatment improves the yield of nostoc polysaccharide by comparison with comparative example 1, the addition of cellulase improves the yield of nostoc polysaccharide by comparison with comparative example 2, and the addition of papain and neutral protease improves the purity of nostoc polysaccharide by comparison with comparative examples 3-4.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. The extraction method of nostoc polysaccharide is characterized by comprising the following steps:
s1: drying nostoc sphaeroids kutz to constant weight, and pulverizing to obtain nostoc sphaeroids kutz powder;
s2: soaking nostoc sphaeroids kutz powder in 40-45 ℃ sodium hydroxide solution for 2-4 hours, filtering, transferring to steam explosion equipment, sealing, introducing saturated steam until the pressure in the steam explosion equipment reaches 6-8MPa, keeping the pressure for 10-15 minutes, opening a valve to release pressure, collecting the obtained solid, adding the obtained solid into disodium hydrogen phosphate-citric acid buffer solution, stirring for 20-40 minutes, adding a complex enzyme preparation, and heating to 55-65 ℃ for enzymolysis for 8-12 hours;
s3: heating the solution to reflux for 5-10h after enzymolysis, centrifuging, adding 5-10 times volume of ethanol into the supernatant, standing at 3-5deg.C for 10-15h, and filtering to obtain nostoc sphaeroids kutz crude polysaccharide;
s4: dissolving nostoc sphaeroids kutz crude polysaccharide with water, adding chloroform-n-butanol solution, oscillating for 20-40min, centrifuging, separating water phase from supernatant, adding chloroform-n-butanol solution, continuing oscillating, repeating the above operation for 3-5 times, concentrating the water phase under reduced pressure, evaporating low boiling point component to obtain concentrated solution, adding saturated sodium chloride solution and ethanol into the concentrated solution, standing at 3-5deg.C for 10-15 hr, filtering, and vacuum drying at 40-60deg.C to obtain solid;
the complex enzyme preparation in S2 consists of cellulase, papain and neutral protease;
the weight ratio of the cellulase to the papain to the neutral protease in the S2 is 2:3:1.
2. the extraction method of nostoc polysaccharide according to claim 1, wherein the molar concentration of the sodium hydroxide solution in S2 is 0.2-0.6mol/L, and the feed-liquid ratio is 1:80-120.
3. The extraction method of nostoc polysaccharide according to claim 1, wherein the amount of the complex enzyme preparation in S2 is 0.5-1% by weight of nostoc powder.
4. The extraction method of nostoc polysaccharide according to claim 1, wherein the volume concentration of ethanol in S3 is 55-65%.
5. The extraction method of nostoc polysaccharide according to claim 1, wherein the volume ratio of chloroform to n-butanol in the chloroform-n-butanol solution in S4 is 4:1.
6. the extraction method of nostoc polysaccharide according to claim 1, wherein the chloroform-n-butanol solution in S4 is added in an amount of 25-30% of the volume of the nostoc crude polysaccharide solution.
7. The extraction method of nostoc polysaccharide according to claim 1, wherein the volume concentration of ethanol in S4 is 75-100%.
8. The extraction method of nostoc polysaccharide according to claim 1, wherein the addition amount of saturated sodium chloride solution and ethanol in S4 is 0.05-0.1 times and 3-5 times of the volume of the concentrated solution, respectively.
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