CN112759662A - Separation and purification method of tremella polysaccharide - Google Patents
Separation and purification method of tremella polysaccharide Download PDFInfo
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- CN112759662A CN112759662A CN202110060015.7A CN202110060015A CN112759662A CN 112759662 A CN112759662 A CN 112759662A CN 202110060015 A CN202110060015 A CN 202110060015A CN 112759662 A CN112759662 A CN 112759662A
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- quaternary ammonium
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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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
- B01D15/426—Specific type of solvent
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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 belongs to the field of biological material preparation, and particularly relates to a method for rapidly separating tremella polysaccharide by using a nano particle surface quaternary ammonium salt modified material, which comprises the following steps: adding the nano oxide into an alcohol solution under stirring, adding quaternary ammonium salt, heating for reaction, evaporating to remove the solvent after the reaction is finished, and drying to obtain the nano oxide modified by the quaternary ammonium salt on the surface; the tremella polysaccharide is separated from the nano particles by repeatedly washing the tremella polysaccharide precipitate and other components in tremella through the quaternary ammonium salt modified nano oxide, adding a strong ionic salt to separate the tremella polysaccharide from the nano particles, removing the strong ionic salt and other small molecular substances by membrane filtration to obtain a tremella polysaccharide solution, and drying to obtain tremella polysaccharide powder; the process for extracting the tremella polysaccharide is simple, the production cost is low, the product quality is high, and the tremella polysaccharide is suitable for further popularization and application.
Description
Technical Field
The invention belongs to the field of biological material preparation, and particularly relates to a method for quickly separating tremella polysaccharide by using a nano particle surface quaternary ammonium salt modified material.
Background
Tremella is also called Tremella fuciformis (berk.) berk) and belongs to Tremella of Tremellaceae. Contains 17 amino acids such as leucine, isoleucine, proline, etc. 6.7 to 10 percent of protein, 0.6 to 12.8 percent of fat and 65 to 71.2 percent of carbohydrate (wherein, the tremella polysaccharide is 60 to 70 percent). It is called the crown of bacteria because it has the functions of nourishing yin, moistening lung, invigorating spleen, stimulating appetite, benefiting qi and clearing intestine.
The polysaccharide is the main component of tremella and is one of the most effective and most active important components. The tremella polysaccharide mainly comprises four polysaccharides such as acidic heteropolysaccharide, neutral heteropolysaccharide, cell wall polysaccharide, exopolysaccharide and the like. The main chain of the tremella polysaccharide is mannan composed of alpha- (1-3) -glycosidic bonds, 2, 4 and 6 positions of the main chain are connected with side chains composed of residues such as glucose, xylose, fucose, common uronic acid and the like, and the active center of the tremella polysaccharide is a common structural part of the alpha- (1-3) -mannan. The molecular weight of the tremella polysaccharide reaches 130-170 ten thousand.
The existing extraction and separation method of tremella polysaccharide mainly comprises the following steps: the method comprises the steps of dissolving out, removing protein, decoloring, precipitating with ethanol and the like of the tremella polysaccharide, wherein the protein removal is usually carried out by adopting organic solvents such as trichloroacetic acid, tannic acid and sevage or by adopting a sevage reagent method combined with an enzyme method, the methods are all characterized in that the protein is denatured and precipitated by utilizing the organic solvents, a large amount of the organic solvents are required to be used, the solvent residues are caused, and the difficulty in separating the tremella polysaccharide from impurities is further aggravated. The decolorizing method mainly adopts a hydrogen peroxide method, an adsorption method and an ion exchange method, and the methods have the problems of high decolorizing cost, easy degradation of tremella polysaccharide and the like. In conclusion, the extraction of the tremella polysaccharide and the separation and removal of impurities are the key points of the extraction of the tremella polysaccharide. Zhang Yan[1]Purifying the capsular polysaccharide by adopting a quaternary ammonium salt precipitation method, so that the subsequent purification steps are greatly reduced; yangqi forest[2]The optimal technological conditions for extracting the tea polysaccharide by the CTAB precipitation method are determined through orthogonal experiments, the centrifugation time is 12min, the CTAB addition amount is 25mL, the precipitation time is 6h, and the polysaccharide yield under the conditions is 1.34%. However, in all the methods, quaternary ammonium salts such as CTAB and the like are required to be separated from polysaccharide molecules at the later stage, and the quaternary ammonium salts such as CTAB and the like have surfactant performance and are easy to bubble in the separation process, so that the polysaccharide is extremely difficult to extract in the engineering process, and CTAB has strong irritation to skin and intestinal tracts, and even a very small amount of residues can irreparably damage product quality and functional products, so that the development of the quaternary ammonium salt precipitated polysaccharide extraction method without quaternary ammonium salt residues has important significance for reducing the cost and improving the efficiency of the polysaccharide extraction industry.
[1] Zhang Yan, Lizhou, Doujinxiang, chiffon, Chenjin Ying, Klebsiella pneumoniae membrana clamping polysaccharide extraction and purification and influence on cell immune activity, biological engineering report, 2005, 21 (3): 461-464.
[2] The tea polysaccharide is extracted from Yangqi forest, Liuzhou dong, ren Jian and Boqinghua by adopting a CTAB precipitation method, and the food and fermentation process is 2004 and 30 (10): 139-142.
Disclosure of Invention
Aiming at the technical problems, the invention provides the method for separating and purifying the tremella polysaccharide, which has the advantages of simple extraction process, low production cost and high product quality.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for rapidly separating tremella polysaccharide by using a nano particle surface quaternary ammonium salt modified material comprises the following steps:
adding the nano oxide into an alcohol solution under stirring, adding quaternary ammonium salt according to the mass ratio of the nano oxide to the quaternary ammonium salt of 1-50:1, heating for reaction, evaporating to remove the solvent after the reaction is finished, and drying to obtain the nano oxide modified by the quaternary ammonium salt on the surface;
the tremella polysaccharide extract is subjected to repeated washing to separate the tremella polysaccharide precipitate from other components such as protein, pigment and fat in tremella, then strong ionic salt is added to separate the tremella polysaccharide from nano particles, the strong ionic salt and other small molecular substances are removed by membrane filtration to obtain a tremella polysaccharide solution, and the tremella polysaccharide powder is obtained by drying.
Preferably, the nano inorganic oxide is nano TiO2、SiO2、Al2O3C, MgO, kaolin, montmorillonite and bentonite, and comprises mixture or compound of multiple compounds.
As further preferred, the quaternary ammonium salts include 3- (trimethoxysilyl) propyl-octadecyl dimethyl ammonium chloride, hexadecyltrimethyl ammonium bromide, dodecyl dimethyl tertiary amine, 2, 3-epoxypropyl trimethyl ammonium chloride.
Further preferably, the alcohol solution comprises methanol, ethanol, propanol, butanol, propylene glycol, and glycerol solution.
Further preferably, the strong ionic salt includes sodium chloride, potassium chloride, calcium chloride, magnesium chloride and zinc chloride.
Preferably, the filter membrane is a PVDF spiral membrane or a ceramic membrane with the molecular weight cutoff larger than 1000 KDa.
The preparation method of the tremella extract comprises the following steps:
after the tremella is fully soaked, the tremella: adding water at a ratio of 1:1-50 into a wall breaking machine, filtering with 80 mesh filter press cloth after wall breaking, and diluting the filtrate to obtain Tremella extractive solution.
Preferably, the quaternary ammonium salt modified nano oxide is added into a chromatographic column, and the tremella extract passes through the chromatographic column, so that the quaternary ammonium salt modified nano oxide is combined with tremella polysaccharide to generate a precipitate.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the inorganic metal oxide is used as a carrier, quaternary ammonium salt is modified on the surface of the carrier, the quaternary ammonium salt modified oxide is used as a stationary phase, and the tremella polysaccharide is separated from impurities such as pigment and protein of tremella after being precipitated by utilizing the characteristic that the quaternary ammonium salt adsorbs and precipitates the polysaccharide. The precipitated tremella polysaccharide is separated from the stationary phase under the action of the strong ionic salt solution, and the quaternary ammonium salt in the stationary phase cannot fall off, so that the separation of the quaternary ammonium salt and the polysaccharide is realized, and the stationary phase can be reused. The strong ion salt in the tremella polysaccharide can be separated from the polysaccharide through simple membrane filtration due to small molecular weight. The process for extracting the tremella polysaccharide is simple, the production cost is low, and the product quality is high. The structure of the tremella polysaccharide is not damaged in the production process, and the specific structure and effect of the tremella polysaccharide are maintained; meanwhile, after the quaternary ammonium salt of the nano particle is modified, the quaternary ammonium salt has antibacterial performance, so that the microbial biomass in the polysaccharide extraction process can be effectively reduced, and the influence of microorganisms on the product quality in the production process is avoided.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
The invention provides a method for separating and purifying tremella polysaccharide, which mainly comprises the following steps:
1. preparation of quaternary ammonium salt modified oxide nanoparticle stationary phase
Adding a certain amount of nano oxide into a round-bottom flask, adding 95% ethanol/propylene glycol/isopropanol, stirring vigorously, adding quaternary ammonium salt according to the mass ratio of the nano oxide to the quaternary ammonium salt of 1-50:1, heating for reaction, removing the solvent by rotary evaporation, drying at 60-120 ℃ to obtain quaternary ammonium salt modified nano oxide particles, adding a certain amount of water into the quaternary ammonium salt modified nano oxide, continuously stirring by using a glass rod to remove bubbles in the nano particles, draining by using the glass rod, pouring into a chromatographic column along the glass rod, knocking the column by using a glass tube to remove bubbles in the chromatographic column, opening a piston, flowing out water, naturally settling the nano particles, and closing the piston when the column is quick-dried.
2. Stationary phase adsorption and precipitation of tremella polysaccharide
After the tremella is fully soaked, the tremella: adding water into a wall breaking machine in a ratio of 1:1-50, filtering with 80-mesh filter press cloth after wall breaking, diluting the filtrate, pumping into a chromatographic column by a peristaltic pump, opening a piston of the chromatographic column to allow the tremella solution to flow through the chromatographic column, after pumping, replacing the tremella solution with deionized water as a mobile phase, and continuing pumping until the effluent of the mobile phase is clear and transparent.
3. Exfoliation, purification and drying of Tremella polysaccharides
Switching the mobile phase to be strong ion salt solution of 10% sodium chloride or potassium chloride and the like until the tremella polysaccharide is completely eluted, introducing the eluent into a membrane filtration system, selecting a PVDF roll-type membrane or a ceramic membrane with the molecular weight cutoff of 300KDa, separating the sodium chloride from the tremella polysaccharide, removing the low molecular weight tremella polysaccharide, and retaining the middle and high molecular weight tremella polysaccharide. And drying the obtained tremella polysaccharide after rotary evaporation to finally obtain the tremella polysaccharide with the molecular weight of more than 1000 KDa.
Example 1
50g of nano TiO are taken2Adding into a round-bottom flask, adding 50mL of 95% ethanol/propylene glycol, stirring vigorously, adding 5g of 3- (trimethoxysilyl) propyl-octadecyl dimethyl ammonium chloride (TSA), heating to 80 deg.C, refluxing for 3h, rotary evaporating to remove solvent, and drying at 90 deg.C for 2h to obtain quaternary ammonium salt modified nanometer TiO2TSA, 50g of nano TiO2-TSA is dispersed in 150g of water, stirred continuously with a glass rod, drained with a glass rod, poured along the glass rod into a chromatography column, knocked against the column with a glass tube, to dislodge the bubbles in the column, the piston is opened, water is run off, the nanoparticles settle naturally, and the piston is closed until the column is quick-drying. After the tremella is fully soaked, the tremella: adding water into a wall breaking machine at a ratio of 1:2, filtering with 80-mesh filter press cloth after wall breaking, and filtering the filtrate with a filter press at a ratio of 1: 5 diluting, pumping into a chromatographic column by a peristaltic pump, opening a piston of the chromatographic column to enable the tremella solution to flow through the chromatographic column, switching the tremella solution into deionized water as a mobile phase after pumping is finished, and continuing pumping until effluent of the mobile phase is clear and transparent. Continuously switching the mobile phase to be a 10% sodium chloride strong ion salt solution until the tremella polysaccharide is completely eluted, introducing the eluent into a membrane filtration system, selecting a PVDF ceramic membrane with the molecular weight cutoff of 300KDa, separating small molecular substances such as sodium chloride, pigments and the like from the tremella polysaccharide, removing the low molecular weight tremella polysaccharide, and retaining the middle and high molecular weight tremella polysaccharide. And carrying out rotary evaporation on the obtained tremella polysaccharide, and then carrying out vacuum freeze drying until the water content is 3-10%, thus finally obtaining the tremella polysaccharide with the molecular weight of more than 1000 KDa. The yield of the tremella polysaccharide is 12.3%, the transmittance is 93.5%, and the viscosity is 1.3 Pa.S.
Example 2
Taking 50g of mesoporous SiO2Adding into a round-bottom flask, adding 95% ethanol50mL of propylene glycol, adding 3g of dodecyl dimethyl tertiary amine (12DMA) after stirring vigorously, heating to 80 ℃, reacting and refluxing for 3h, centrifugally separating, washing for 3 times by using methanol, and drying for 2h at 70 ℃ to obtain quaternary ammonium salt modified mesoporous SiO2-12DMA, 30g nano SiO2-12DMA was dispersed in 150g of water, stirred constantly with a glass rod, drained with a glass rod, poured along the glass rod into the column, tapped with a glass tube to dislodge air bubbles from the column, the piston was opened, water was run off to allow the nanoparticles to settle naturally, and the piston was closed until the column was dry. After the tremella is fully soaked, the tremella: adding water into a wall breaking machine in a ratio of 1:10, filtering by using 80-mesh filter press cloth after wall breaking is finished, and filtering the filtrate by using a filter press in a ratio of 1:1, diluting, pumping into a chromatographic column by a peristaltic pump, opening a piston of the chromatographic column to enable a tremella solution to flow through the chromatographic column, switching the tremella solution into deionized water as a mobile phase after pumping is finished, and continuing pumping until effluent of the mobile phase is clear and transparent. Continuously switching the mobile phase to be a 10% sodium chloride strong ion salt solution until the tremella polysaccharide is completely eluted, introducing the eluent into a membrane filtration system, selecting a PVDF ceramic membrane with the molecular weight cutoff of 300KDa, separating small molecular substances such as sodium chloride, pigments and the like from the tremella polysaccharide, removing the low molecular weight tremella polysaccharide, and retaining the middle and high molecular weight tremella polysaccharide. And carrying out rotary evaporation on the obtained tremella polysaccharide, and drying the tremella polysaccharide with hot air at 50-80 ℃ until the water content is 3-10%, thus obtaining the tremella polysaccharide with the molecular weight of more than 1000 KDa. The yield of the tremella polysaccharide is 22.5%, the transmittance is 96.2%, and the viscosity is 1.4 Pa.S.
Example 3
Taking 200g of mesoporous TiO2Adding 10g of 2, 3-epoxypropyltrimethylammonium chloride (GTA) into 200mL of 10% sodium hydroxide 95% ethanol/propylene glycol solution, stirring and refluxing for 6h in a water bath at 80 ℃, after centrifugal separation, washing for 3 times by using ethanol, washing for 3 times by using 0.1M HCl solution, and drying for 6h at 70 ℃ to obtain the quaternary ammonium salt modified mesoporous TiO2GTA, 40g mesoporous TiO2-GTA is dispersed in 150g of water, continuously stirred with a glass rod, drained with a glass rod, poured along the glass rod into a chromatographic column, knocked against the column with a glass tube, bubbles are removed from the chromatographic column, a piston is opened, water is drained off, and the nano particles are allowed to settleThe particles naturally settle and the piston is closed until the column is quick-dried. After the tremella is fully soaked, the tremella: adding water into a wall breaking machine at a ratio of 1:20, filtering with 80-mesh filter press cloth after wall breaking, and filtering the filtrate with a filter press at a ratio of 1:1, diluting, pumping into a chromatographic column by a peristaltic pump, opening a piston of the chromatographic column to enable a tremella solution to flow through the chromatographic column, switching the tremella solution into deionized water as a mobile phase after pumping is finished, and continuing pumping until effluent of the mobile phase is clear and transparent. Continuously switching the mobile phase to be a 10% sodium chloride strong ion salt solution until the tremella polysaccharide is completely eluted, introducing the eluent into a membrane filtration system, selecting a PVDF ceramic membrane with the molecular weight cutoff of 300KDa, separating small molecular substances such as sodium chloride, pigments and the like from the tremella polysaccharide, removing the low molecular weight tremella polysaccharide, and retaining the middle and high molecular weight tremella polysaccharide. And carrying out rotary evaporation on the obtained tremella polysaccharide, and then carrying out vacuum freeze drying until the water content is 3-10%, thus finally obtaining the tremella polysaccharide with the molecular weight of more than 1000 KDa. The yield of the tremella polysaccharide is 16.3%, the transmittance is 94.6%, and the viscosity is 1.6 Pa.S.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for rapidly separating tremella polysaccharide by using a nano particle surface quaternary ammonium salt modified material is characterized by comprising the following steps:
adding the nano oxide into an alcohol solution under stirring, adding quaternary ammonium salt according to the mass ratio of the nano oxide to the quaternary ammonium salt of 1-50:1, heating for reaction, evaporating to remove the solvent after the reaction is finished, and drying to obtain the nano oxide modified by the quaternary ammonium salt on the surface;
the tremella polysaccharide is separated from the nano particles by repeatedly washing the tremella polysaccharide precipitate and other components in tremella through the quaternary ammonium salt modified nano oxide, adding the strong ionic salt to separate the tremella polysaccharide from the nano particles, removing the strong ionic salt and other small molecular substances by membrane filtration to obtain a tremella polysaccharide solution, and drying to obtain the tremella polysaccharide powder.
2. The method for rapidly separating tremella polysaccharide from nanoparticle surface quaternary ammonium salt modified material according to claim 1, wherein the nano inorganic oxide is nano TiO2、SiO2、Al2O3C, MgO, kaolin, montmorillonite and bentonite, and comprises mixture or compound of multiple compounds.
3. The method for rapidly separating tremella polysaccharide from the nanoparticle surface quaternary ammonium salt modified material according to claim 1, wherein the quaternary ammonium salt comprises 3- (trimethoxysilyl) propyl-octadecyl dimethyl ammonium chloride, hexadecyltrimethyl ammonium bromide, dodecyl dimethyl tertiary amine, and 2, 3-epoxypropyl trimethyl ammonium chloride.
4. The method for rapidly separating tremella polysaccharide from the nanoparticle surface quaternary ammonium salt modified material according to claim 1, wherein the alcohol solution comprises methanol, ethanol, propanol, butanol, propylene glycol, and glycerol solution.
5. The method for rapidly separating tremella polysaccharide from the nanoparticle surface quaternary ammonium salt modified material according to claim 1, wherein the strong ionic salt comprises sodium chloride, potassium chloride, calcium chloride, magnesium chloride and zinc chloride.
6. The method for rapidly separating tremella polysaccharide with the nano particle surface quaternary ammonium salt modified material as claimed in claim 1, wherein the filtering membrane is a PVDF roll-type membrane or a ceramic membrane with a molecular weight cutoff of more than 1000 KDa.
7. The method for rapidly separating tremella polysaccharide from the nanoparticle surface quaternary ammonium salt modified material according to claim 1, wherein the preparation method of the tremella extract comprises the following steps:
after the tremella is fully soaked, the tremella: adding water at a ratio of 1:1-50 into a wall breaking machine, filtering with 80 mesh filter press cloth after wall breaking, and diluting the filtrate to obtain Tremella extractive solution.
8. The method for rapidly separating tremella polysaccharide from the quaternary ammonium salt modified material on the surface of the nanoparticle according to claim 1, wherein the quaternary ammonium salt modified nano oxide is added into a chromatographic column, and the tremella extract passes through the chromatographic column, so that the quaternary ammonium salt modified nano oxide and the tremella polysaccharide are combined to generate a precipitate.
9. The method for rapidly separating tremella polysaccharide from the nano particle surface quaternary ammonium salt modified material according to claim 1, wherein a heating reflux manner is adopted in the preparation process of the surface quaternary ammonium salt modified nano oxide, the reaction temperature is 60-100 ℃, and the reaction time is 1-24 hours.
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Effective date of registration: 20220530 Address after: 350000 floor 3, plot 25, Qianshan edible fungus processing base industrial park, Chengdong street, Gutian County, Fuzhou City, Fujian Province Patentee after: Gutian Zhuangxin fungi Co.,Ltd. Address before: No.15, shangxiadian Road, Cangshan District, Fuzhou City, Fujian Province Patentee before: FUJIAN AGRICULTURE AND FORESTRY University |