29 results about "Sepharose" patented technology

The invention relates to a method for separating and purifying polygonatum sibiricum oligosaccharide. According to the method, the polygonatum sibiricum polysaccharide and the filler or / and the filter aid are fully mixed and then separated and purified; wherein the filler is prepared from Sephadex G-15 (crosslinked glucose gel), DEAE-Sepharose CL-4B (agar gel) and Bio-Gel P-2 (polyacrylamide gel), the filter aid is prepared from kieselguhr, perlite, cellulose, activated carbon and red soil, and the method has the advantages that the Mwlt can be obviously improved; the yield of the 2KDa polygonatum sibiricum oligosaccharide is increased, and the effect of combined use of the filler and the filter aid is optimal. The addition of the filter aid can avoid the blockage of filler pores in the middle and later stages of elution, improve the elution performance of the filler on polysaccharide, and accelerate the elution and purification speed of the polygonatum kingianum oligosaccharide. Experiments show that the oligosaccharide can significantly enhance the proliferative activity and phagocytosis of macrophages, promote RAW264.7 cells to secrete TNF-alpha, IL-6, NO and other cell factors, and improve the cellular immunocompetence. The method has the characteristics of convenience, high efficiency and low cost, and has relatively high application value.

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating active components in tobacco based on liquid-phase segmented separation, SPR, affinity chromatography enrichment and mass spectrometry detection, a product and application. According to the method, liquid chromatography separation, SPR detection screening, affinity chromatography enrichment and mass spectrum identification technologies are fused, and active small molecules are screened and enriched from a complex system by virtue of high sensitivity of SPR and accuracy of mass spectrum. The method comprises the following steps: firstly, carrying out liquid chromatography segmented separation on extract components to improve SPR detection precision and affinity enrichment efficiency; by means of specific binding of Ni Sepharose FF (NTA) resin and a His tag, more target proteins are fixed, and the micromolecule enrichment effect is further enhanced; and finally, identifying the molecular structure by using mass spectrum, and locking the bioactive small molecule combined with the target protein. According to the strategy, efficient enrichment of small molecule ligands is achieved, and a new way is provided for early-stage development of small molecules in drug discovery. 6.

The invention relates to a pharmaceutical composition for reducing dentin hypersensitivity and a preparation method thereof, and belongs to the technical field of biological medicine, the pharmaceutical composition comprises chanterelle extract, rabdosia rubescens leaf extract, eggshell membrane protein peptide and bifidobacterium thermophile fermentation extract; the cantharellus cibarius extract is obtained by sequentially performing enzymolysis on cantharellus cibarius through compound enzyme, ginger protease and serratipeptidase and purifying enzymatic hydrolysate through an AB-8 macroporous resin column. The rabdosia rubescens leaf extract is obtained by ultrasonically extracting rabdosia rubescens leaves with an ethanol water solution and purifying an extracting solution with a composite filler adsorption column. The eggshell membrane protein peptide is obtained by carrying out enzymolysis on a shell membrane by virtue of ginger protease and ficin and purifying an enzymatic hydrolysate by virtue of a G-25 sephadex column. The fermentation extract of the bifidobacterium thermophilus is obtained by purifying fermentation liquor of the bifidobacterium thermophilus through an SP agarose gel FF column. All the components are compounded according to a specific ratio and have a synergistic effect, so that the dentin hypersensitivity can be relieved safely in a multi-effect manner.

The invention provides a hirudin separation and purification method and system. The method comprises the following steps: grinding leeches into slurry; adding trypsin into the serous fluid, and then carrying out hydrolysis; performing counter-current extraction on the enzymatic hydrolysate obtained after hydrolysis; carrying out centrifugal separation on extract obtained by counter-current extraction, and collecting supernate; performing ultrafiltration concentration on the supernate by using an ultrafiltration membrane to obtain a concentrated solution; adsorbing hirudin in the concentrated solution by using DEAE-Sepharose FF resin, eluting, and then collecting an eluent; and carrying out vacuum freeze drying on the eluent to obtain freeze-dried powder. The system comprises a purification tank, a grinding device, an enzymolysis tank, a counter-current extraction device, a centrifugal machine, an ultrafiltration concentrator, an ion exchange chromatography instrument and a freeze dryer. In the hirudin production process, conditions are mild, damage to heat-sensitive components is small, high-toxicity organic solvents are not used, green and environment-friendly effects are achieved, and the prepared hirudin is high in activity retention rate and purity.

The invention discloses a separation and purification method of a phthalate degrading enzyme, which comprises the following steps: extracting an intracellular enzyme solution of a phthalate degrading bacterium as a crude enzyme solution, selecting a protein liquid chromatography system, and sequentially carrying out DEAE ion exchange, HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification and Superdex-200 gel filtration to obtain the phthalate degrading enzyme. And separating and purifying the crude enzyme of the phthalate degrading bacteria to obtain the degrading enzyme. The method is high in feasibility and good in separation and purification effect, and the obtained degrading enzyme can efficiently degrade PAEs, so that a foundation is laid for the attempt of constructing more effective microbial degradation engineering strains in the later period.

The invention provides a detection reagent for frozen semen of a sophora japonica boar. The detection reagent is prepared by the following steps: firstly, obtaining original seminal plasma, and performing fractional precipitation on ammonium sulfate; performing DEAE cellulose filtration gel column chromatography; carrying out gel chromatography by using CM Sepharose FastFlow, and carrying out gel chromatography by using a chromatographic column; the NAGAs of the semen of the sophora japonica pig is separated and purified through the steps of SephadexG-100 chromatography and the like, and monoclonal antibodies JN21 and JN37 are prepared by taking the NAGAs as antigens and can specifically recognize the NAGase of the sophora japonica pig. The prepared double-antibody sandwich ELISA kit can rapidly and quantitatively detect the NAGase content in the semen of the sophora japonica pig, and a new idea is provided for breed conservation, purification and rejuvenation of the sophora japonica pig. The kit is convenient to operate, low in use cost, good in repeatability and suitable for wide popularization and application.

The present disclosure provides a Flammulina velutipes proteoglycan (FPG) and a dual-wavelength screening method and use thereof, and relates to the technical field of biomedicine. In the present disclosure, components such as polysaccharides, proteoglycan (PG), and free proteins in the Flammulina velutipes are extracted through water extraction. The free proteins are removed by protein removal, and the polysaccharides and PG are separated by alcohol precipitation. Sepharose FF ion exchange chromatography and Sephadex G-200 chromatography separation are conducted with water as an eluent. Dual-wavelength detection is conducted to collect an eluate with absorption peaks at both 480 nm and 295 nm wavelengths, such that a component—the FPG, which has a protein structure and a polysaccharide structure, is retained.

The invention discloses a method for purifying atosiban. The method sequentially comprises the following steps: dissolving an atosiban crude product with an acetic acid aqueous solution, and adjusting the pH value to 4.0-5.5; carrying out primary purification by adopting SP Sepharose FF cation exchange resin, and removing a large amount of deleted peptide impurities; 0.5-1.0 mM of TCEP is added for reduction treatment, and disulfide bond mismatch is corrected; carrying out fine purification through C18 reversed-phase preparative chromatography, and removing oxidation by-products; and finally, carrying out nanofiltration desalination and freeze drying to obtain the high-purity atosiban. According to the method disclosed by the invention, the purity of the final product is more than or equal to 99.5%, the total yield is more than or equal to 65%, the maximum single impurity is less than or equal to 0.15%, the unit consumption of acetonitrile is less than or equal to 12 L / kg, the amplification verification of 100 mg to 500 g is completed, the process is stable, the cost is low, the industrial applicability is high, and a reliable technical scheme is provided for the large-scale production of the atosiban raw material medicine.

The invention discloses a preparation method of high-purity sugarcane leaf polysaccharide, which comprises the following steps: separating sugarcane leaf crude polysaccharide by using an alkaline H2O2 method, and then separating and purifying by using a DEAE-Sepharose Fast Flow anion exchange column to obtain the high-purity sugarcane leaf polysaccharide SLP-2. The monosaccharide composition of the high-purity sugarcane leaf polysaccharide SLP-2 is mainly Xyl, Ara and Gal, and the main chain structure of the high-purity sugarcane leaf polysaccharide SLP-2 is composed of glycosidic bonds such as Araf-(1-), Xylp-(1-),-> 3, 4)-Galp-(1-),-> 5)-Araf-(1-),-> 4)-Xylp-(1-),-> 4)-Glcp-(1-),-> 3, 4)-Xylp-(1-) and the like. The research on the efficacy of a diabetic cardiomyopathy rat model induced by feeding with streptozotocin combined with high-sugar and high-fat feed finds that the high-purity sugarcane leaf polysaccharide SLP-2 has the effects of reducing the blood sugar of the diabetic cardiomyopathy rat and improving the heart function of the diabetic cardiomyopathy rat. In conclusion, the preparation process is simple, the polysaccharide composition and structure are clear, and the SLP-2 has good effects of reducing blood sugar and improving diabetic cardiomyopathy and heart injury, has a certain application prospect in the field of medicines, and is worthy of further research.

Anchor molecules for peptide synthesis in aqueous solutions are disclosed. Wherein the anchor molecules are independently present and may be covalently coupled to a variety of hydrophilic solid supports, such as CM agarose and carboxymethyl cellulose, by amido bonds under aqueous phase conditions using a coupling agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) prior to the start of peptide synthesis. The anchor molecule has a general structure of Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n-NH2, wherein Fmoc-AA represents an amino acid protected by Fmoc, and the general structure of the anchor molecule is shown as Fmoc-AA-NH-(CH2) n-NH2. The anchoring strategy includes an acid labile ester bond located between the carboxyl terminal of Fmoc-AA and the benzyl alcohol group of the solid support, which ester bond remains stable during peptide extension and is cleavable under strongly acidic conditions, achieving final peptide release. The anchor molecule may be functionalized to interact with the ion exchange resin. The present invention enables efficient solid phase peptide synthesis (SPPS) in an aqueous phase system, enhances compatibility with hydrophilic resins, and provides improved environmental sustainability by reducing dependence on organic solvents.

The application discloses a kind of white snail glycosaminoglycan-growth factor composite active substance directional extraction purification process, belong to biological active substance extraction technical field.Core breakthrough is in at: through-10 ℃ acetone circulation degreasing and 20kHz ultrasonic coordination papain-neutral protease compound system (mass ratio 1:0.8), realize glycosaminoglycan and growth factor synchronous efficient release, extraction rate reaches 97.2%;Using DEAE-Sepharose Fast Flow and Q Sepharose Fast Flow double column chromatography gradient elution, combined with 1000Da ultrafiltration membrane refining, composite active substance purity is ≥99.6%;Through-Vc mild depolymerization technology regulates glycosaminoglycan molecular weight to 3.8~23.8kDa, retains its heparin-like structure activity;Using porous medium enhanced vacuum freeze-drying process, so that composite active substance activity retention rate is ≥98.5%.Product glycosaminoglycan content is ≥35mg / g, growth factor (EGF class) activity is ≥1200IU / mg, DPPH free radical clearance rate is ≥65%, to skin fibroblast proliferation promotion rate is ≥72%, no organic solvent residue and heavy metal pollution.

The invention provides a preparation method and application of a scylla paramamosain allergen Scy p1 hypoallergenic derivative specific I gG antibody, a New Zealand white rabbit is subjected to immune treatment by using a Scy p1 hypoallergenic derivative, and antiserum is purified by using a Protei n A Sepharose affinity chromatography column, so that the purity of the antibody is greatly improved, and the obtained I gG antibody is ensured to have high specificity; the purified antibody is subjected to molecular weight detection through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), and an immunoglobulin heavy chain and an immunoglobulin light chain containing 50kDa and 25kDa are defined, so that the quality of the IgG antibody is verified, and a reliable basis is provided for subsequent application; the obtained specific I gG antibody effectively blocks combination of Scy p 1 and I gE in whole blood of a patient in vitro, effectively improves allergic symptoms of mice in vivo, ensures that the prepared I gG antibody is safer and is expected to be used for immunotherapy, and side effects possibly occurring in the treatment process are reduced.

The invention discloses a preparation method of blackberry oligosaccharide, which comprises the following steps: dehydrating blackberry, grinding into powder, dissolving in water, mixing with ethanol, carrying out rotary evaporation and freeze-drying, sequentially carrying out protein removal and pigment removal, respectively carrying out elution by using DEAE-Sepharose FF ion layer coefficient and G100 chromatographic column, collecting the eluent, and carrying out freeze-drying to obtain the blackberry oligosaccharide. The blackberry oligosaccharide prepared by the preparation method is complete in component preservation, clear in structure and controllable in quality, and meanwhile, the prepared blackberry oligosaccharide also has the functions of promoting the growth of probiotics and improving the activity of the probiotics. Therefore, the blackberry oligosaccharide with a brand new effect is extracted from the blackberries, and more basis is provided for application of the blackberries in the food field.

The present invention provides a preparation method and use of golden squid ink peptidoglycan, which belongs to the field of marine biomedicine. The technical solution takes squid ink and successively homogenizes and defats it, ultrasonically treats the defatted squid ink, soaks and centrifuges it at low temperature to take the supernatant, ultrafilters it to remove impurities with an ultrafiltration membrane, precipitates it with anhydrous ethanol, removes protein with the sevag method, and freezes and dries it to obtain a crude extract of golden squid ink polysaccharide; the crude extract is separated and purified by a DEAE-Sepharose Fast Flow anion exchange chromatography column, the protein-sugar peak eluate is collected, the dialysis method is used for desalting and concentrating, and freeze-drying is performed to obtain golden squid ink peptidoglycan. The preparation method is not only simple and efficient, but also the obtained product has a significant ovarian repair effect. In addition, the experiment found that the golden squid ink peptidoglycan prepared by the present invention can improve intestinal peristalsis, repair liver oxidative damage, and also has the effect of inhibiting pancreatic cancer proliferation, migration and invasion. Based on the above beneficial findings, the scope of use of golden squid ink peptidoglycan is further expanded.

The present invention relates to a chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof, a preparation method thereof, and a method for purifying tetanus immunoglobulin or a fragment thereof. The chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof comprises: an affinity capture ligand selected from TeNT-Hc-C869A subunit antigen, and a chromatographic filler selected from any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads 4FF, or epoxy-activated Seplife FF for protein binding / coupling. The chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof provided by the present invention can improve the purity and titer of the purified tetanus immunoglobulin or a fragment thereof.

The present invention discloses a purification method for simultaneously obtaining two high-purity cobra analgesic peptides, and belongs to the field of biomedical technology. The purification method comprises dissolving cobra venom lyophilized powder in Tris-HCl buffer, and purifying it successively through CM-Sepharose cation exchange column and size exclusion chromatography column, wherein the CM-Sepharose cation exchange column is balanced with Tris-HCl buffer before loading, and the pH value of Tris-HCl buffer is 7.3-7.7. The present invention is purified only by two-step column chromatography steps of CM-Sepharose cation exchange column and size exclusion chromatography column, and no organic solvent is used in the purification process, and no high concentration salt is used, and all operations are performed at room temperature or below, which can maximize the biological activity of cobra analgesic peptide.

The invention discloses a method for separating human fibrinogen from cryoprecipitate, which comprises the following steps: step a, cryoprecipitate dissolving solution pretreatment: mixing the cryoprecipitate with a Tris-HCl buffer solution containing 20-40 mM of sodium citrate according to a mass ratio of 1: 3, and centrifuging at 4 DEG C to remove insoluble substances; step b, cation exchange chromatography: loading the supernatant to a pre-balanced SP Sepharose FF column, and eluting the target protein by using a gradient buffer solution containing 0.3-0.5 M of NaCl; c, heparin affinity chromatography: adjusting the pH value of the eluent obtained in the step b to 6.8, loading the eluent to a Heparin HP column, carrying out gradient elution by adopting 0.15-1.0 M linear NaCl, and collecting a main peak; and step d, ultrafiltration and concentration: concentrating to 50-80 mg / mL by using a 100 kDa ultrafiltration membrane, and freeze-drying and storing. The fibrinogen separation method provided by the invention has the advantages of high efficiency, high recovery rate, high purity and high activity, is suitable for industrial production, and can meet the clinical requirements on high-quality fibrinogen preparations.

The application provides a detection reagent for frozen semen of Huai pig, and first acquires the raw semen, and then carries out the following steps: ammonium sulfate fractionation precipitation, DEAE cellulose filter gel column chromatography, CM Sepharose FastFlow gel chromatography and Sephadex G-100 chromatography to separate and purify the NAGase of the semen of the Huai pig, and monoclonal antibodies JN21 and JN37 are prepared by taking the NAGase as an antigen, and the monoclonal antibodies can specifically recognize the NAGase of the Huai pig. The prepared double-antibody sandwich ELISA kit can rapidly and quantitatively detect the NAGase content in the semen of the Huai pig, and provides a new thought for Huai pig breeding and purification and rejuvenation. The kit is convenient to operate, low in use cost, good in repeatability and suitable for being widely applied.

The invention relates to a preparation method of recombinant human bone morphogenetic protein-2. The preparation method comprises the following steps: culturing host cells expressing recombinant human bone morphogenetic protein-2, inducing the host cells to express the recombinant human bone morphogenetic protein-2, breaking the cells and collecting precipitates to obtain crude protein, and purifying the crude protein to recover the recombinant human bone morphogenetic protein-2. The purification comprises affinity chromatography and ion exchange chromatography in sequence, a filler for the affinity chromatography comprises heparin sepharose gel, and a filler for the ion exchange chromatography purification comprises any one or a combination of at least two of diethylamino sepharose gel, Q sepharose gel or QAE sepharose gel. The brand-new purification process is designed, the purification efficiency is improved, the endotoxin content is reduced, the purity of the target protein can reach 95% or above, and the endotoxin content is lower than 10 EU / mg.

The invention relates to a chromatographic filler for purifying tetanus immune globulin or a fragment thereof, a preparation method of the chromatographic filler and a method for purifying the tetanus immune globulin or the fragment thereof. The invention relates to a chromatographic filler for purifying tetanus immune globulin or a fragment thereof, which comprises an affinity capture ligand selected from a TeNT-Hc-C869A subunit antigen, and any one of a chromatographic filler which is selected from a group consisting of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads 4FF or epoxy-activated Seplife FF and is used for binding / coupling a protein. The invention further discloses a preparation method of the chromatographic filler for purifying the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin. The chromatographic filler for purifying the tetanus immune globulin or the fragment thereof, provided by the invention, can improve the purity and the titer of the purified tetanus immune globulin or the fragment thereof.

The invention belongs to the technical field of protein extraction, and particularly relates to a method for extracting Gp96 from an HCC-LM3 cell strain. The method comprises the following steps: cracking HCC-LM3 cells, sequentially carrying out ultrasonic treatment and salting-out on a lysate, collecting a precipitate, redissolving, and dialyzing; and loading the dialyzed solution on a Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column, washing until the flow-through liquid does not contain protein, eluting by adopting a glycine-hydrochloric acid buffer solution, collecting the eluent, loading the eluent on a HiTrap-Q Sepharose ion exchange chromatography column, and carrying out second elution. According to the present invention, the reagent type of each link is limited, about 100 [mu] g of the heat shock protein Gp96 carrying the antigen information of the liver cancer can be extracted from 1 * 10 < 10 > HCC-LM3 cells, the purity is 90%, and after the heat shock protein Gp96 is combined with DC cells, the proliferation and the activation of DCT can be promoted, and the liver cancer cells can be specifically killed.

This invention belongs to the field of biotechnology. Specifically, it discloses a method for preparing recombinant protein using rice seeds. The method involves dehulling rice seeds to obtain rice flour, which is then enzymatically hydrolyzed using cellulase and neutral protease in a buffer solution. This process efficiently hydrolyzes the cellulose in the rice cell wall and some impurities. The buffer solution reduces protein salting out and increases the dissolution rate of recombinant human lactoferrin. Activated carbon is used to initially adsorb and remove impurities such as natural pigments and phenolic substances from the rice seeds. The mixture is then subjected to chromatography using CM Sepharose FF, SP Sepharose XL, and CM Sepharose FF columns, effectively improving the purity and recovery rate of the recombinant human lactoferrin. Simultaneously, the enzymatic hydrolysis and fractionation chromatography effectively reduce the iron saturation in the recombinant human lactoferrin.

According to the hucho taimen roe micro-injection device and method, an injection pipe and a discharge pipe are arranged side by side and integrally manufactured, needle tips are arranged at the lower ends of the injection pipe and the discharge pipe, and a liquid storage tank is communicated with the discharge pipe through a connecting pipe. The method comprises the following steps: 1, semen taking; 2, healthy roes are taken, and a container containing the roes is placed on the ice-water mixture; 3, mixing 1000 + / -10 eggs with the seminal fluid, gently and uniformly stirring, and slowly adding water until the eggs are submerged; 4, standing for 2 minutes, washing the eggs with water at 10 DEG C, and removing redundant sperms; 5, injecting clean water again; 6, pouring 1.2% sepharose gel into a culture dish, and after the sepharose gel is coagulated, poking out a semicircular groove with the diameter of 4-5mm; seventhly, the fertilized eggs absorb water to expand, within 0-15 minutes after meeting water, the fertilized eggs are fixed in the semicircular grooves, and injection is conducted through a micro-injection device; 8, timely picking out the eggs which are damaged, severely leaked, not fertilized or have water mildew infection symptoms; and 9, incubating the fertilized eggs after injection. The microinjection device is used for microinjection of hucho taimen fertilized eggs.

The invention relates to a method for separating plasma small extracellular vesicles (sEV) subsets and application of the method in treatment of sepsis cardiomyopathy, and belongs to the technical field of extraction of plasma small extracellular vesicles. According to the method disclosed by the invention, propylene sephadex S-400 and S-500 are combined to be filled into a chromatographic column, and different sEV subgroups in plasma are separated through a dual-size exclusion chromatography (d-SEC), so that the sEV with high purity, complete structure and heterogeneity is obtained. According to the method, more different high-purity plasma subgroups sEV can be conveniently, economically and practically obtained, and good technical support and method models are provided for clinical and basic research. Furthermore, it is found that the healthy human plasma B subgroup sEV has a remarkable cardiac function protection effect in an LPS-induced sepsis cardiomyopathy model, inflammatory factor expression can be reduced, cardiac function indexes can be improved, and a new treatment means is provided for treatment of sepsis cardiomyopathy.

The application belongs to the technical field of food deep processing, and particularly relates to a red date degraded polysaccharide, a degraded polysaccharide component, and a preparation method and application thereof. The application provides a preparation method of the red date degraded polysaccharide, in which the red date polysaccharide is degraded by using an ultrasonic wave assisted method combined with H2O2-Vc, and the red date degraded polysaccharide is separated and purified by using a DEAE-Sepharose Fast Flow gel column and a Sephacryl S-100 chromatographic column. The red date degraded polysaccharide and the polysaccharide component have the following characteristics: after the degradation treatment, the fragmentation degree of the polysaccharide surface is deepened, the degradation does not change the basic structure of the polysaccharide, and the apparent morphological characteristics change to a certain extent; the degradation treatment can improve the antioxidant capacity of the polysaccharide and is inversely proportional to the polysaccharide molecular weight.

The invention belongs to the technical field of heparin, and particularly relates to a process method for preparing heparin based on an enzyme method. The process method comprises the following steps: (1) pretreating heparin; (2) carrying out crosslinking on the Phe-Leu-Asn-Gln-Asp and the sepharose, and carrying out cross-linking on the obtained product and the sepharose; adding the cross-linked gel into a sodium phosphate buffer solution with the pH value of 7.2-7.5, swelling, and absorbing the supernatant; (3) dissolving the pretreated heparin in a buffer solution with the volume 1-1.2 times that of the pretreated heparin, and absorbing the supernatant; (4) after the active center site of the heparin is adsorbed on the pentapeptide of the gel, centrifuging and taking the precipitate; (5) collecting trapped fluid; (6) carrying out alcohol precipitation and impurity removal on the trapped fluid, and drying and weighing the obtained precipitate; and (7) adding 10-12 times of 1 mol / L NaCl solution into the precipitate for dissolving, collecting filtrate, and freeze-drying the filtrate to obtain the heparin prepared based on the enzyme method. The heparin product prepared by the method is good in clarity, free of opalescence and colorless.

The invention discloses a method for extracting polysaccharide and protein from microalgae. The method comprises the following steps: carrying out ultrasonic-assisted compound enzyme enzymolysis to carry out microalgae wall breaking, separating and concentrating enzymatic hydrolysate, separating polysaccharide and protein, carrying out polysaccharide purification by using a DEAE-52 cellulose ion exchange column, and carrying out protein purification by using a DEAE-Sepharose ion exchange column. According to the method, the polysaccharide and the protein in the microalgae are cooperatively extracted through an ultrasonic-enzyme method, the extraction rate of the polysaccharide and the protein in the microalgae is remarkably increased, the method is easy to operate, cost is saved, universality is achieved, the method is suitable for extraction of all microalgae in the market, and the purity of the polysaccharide reaches 90% or above. According to the extraction method, vitamin C is added in the ultrasonic process to protect the physiological activity of the polysaccharide, and the extracted polysaccharide has good antioxidant activity.

The application discloses a method for separating and purifying low-molecular-weight polysaccharides from Polygonatum sibiricum. The method comprises the following steps: mixing the polysaccharides with fillers and / or filter aids, and then separating and purifying the polysaccharides. The fillers comprise Sephadex G-15 (cross-linked glucose gel), DEAE-Sepharose CL-4B (agarose gel) and Bio-Gel P-2 (polyacrylamide gel), and the filter aids comprise diatomite, perlite, cellulose, activated carbon and red clay. The method can significantly improve the yield of the low-molecular-weight polysaccharides with a molecular weight of less than 2 KDa, and the effect of the combination of the fillers and the filter aids is the best. The addition of the filter aids can avoid the blockage of the pores of the fillers in the middle and late stages of elution, improve the elution performance of the fillers on the polysaccharides and accelerate the elution and purification speed of the low-molecular-weight polysaccharides. The experiments show that the low-molecular-weight polysaccharides can significantly enhance the proliferation activity and phagocytosis of macrophages, promote the secretion of cytokines such as TNF-alpha, IL-6 and NO by RAW264.7 cells and improve the immune activity of the cells. The method has the characteristics of convenience, high efficiency and low cost and has high application value.

The invention belongs to the technical field of extraction and preparation of natural products, and discloses camellia seed cake polysaccharide with immunological enhancement and antioxidant activity as well as a preparation method and application of the camellia seed cake polysaccharide. The method comprises the following steps: extracting camellia seed cake polysaccharide by adopting an ultrasonic-assisted method and a water extraction and alcohol precipitation method, determining the content of crude polysaccharide by adopting a phenol-sulfuric acid method, desalting, removing impurities, purifying and grading the camellia seed cake crude polysaccharide by adopting a DEAE-Sepharose FF ion exchange column and a Chromdex 75PG gel filter column to obtain camellia seed cake pure polysaccharide, and determining the molecular weight of the polysaccharide by adopting an exclusion chromatography method. And finally, determining the structure of the compound through chemical methods such as high performance liquid chromatography, full methylation combined gas chromatography-mass spectrometry, infrared spectroscopy, nuclear magnetic resonance and the like, a chromatographic method and a spectrum method. In-vitro experiments and in-vivo experiments prove that the camellia seed cake polysaccharide has immunological enhancement and antioxidant activity at the same time, and can be widely applied to the fields of medicines, daily chemical products, feeds and the like.