CN102174119B - Selenized carrageenan and preparation method thereof - Google Patents
Selenized carrageenan and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing selenized carrageenan by an enzymolysis flocculation method. The method comprises the steps of blending, enzymolysis selenizing reaction, inactivation, flocculation reaction, centrifugal separation, ethanol sedimentation, drying, and the like. The method has the advantages of high catalytic efficiency, stable reaction system, mild temperature, moderate pH, simple process, low cost, no corrosion, high safe performance and high organic selenium combination rate. The selenized carrageenan can fully play the roles of mutual coordination and promotion of selenium and carrageenan, is a food additive and can improve the immune performance and the cancer-preventing, anticancer and antivirus functions.
Description
[technical field]
The invention belongs to the polysaccharide compound technical field.More specifically, the present invention relates to a kind of selenocarrageenan, also relate to the preparation method of said selenocarrageenan.
[background technology]
Selenocarrageenan (Seleno-carrageennan) is a kind of polysaccharide compound that contains selenium that inorganic selenium (Sodium Selenite, selenous acid, sodium selenate, selenium powder) and carrageenin (is the polysaccharide of feedstock production with the red algae plant) prepare through chemosynthesis.It is widely used in fields such as biological medicine, protective foods, beauty and make-up, food, fodder additives, the ecological agriculture as a kind of novel organic selenium compounds, because its purposes is constantly developed, market potential is huge, has development prospect.Selenocarrageenan integrates organoselenium and the many physiological functions of polysaccharide; Can increase body resistance of oxidation, improve immune function of human body, remove interior free yl, anti-ageing, anti-cancer, anticancer, control cardiovascular and cerebrovascular diseases etc.; Especially the effect that has anti AIDS virus (HIV); More cause people's great attention (in accounting for foreign Hou Zhe chief editor " trace element and medical diagnosis on disease and treatment " People's Health Publisher, 2001).
China lacks one of serious area of selenium in the world; Disclose according to the People's Republic of China's " localized disease and environmental factors atlas "; Spread southwester to the Yunnan-Guizhou Plateau from three provinces in the northeast of China; Have the scarce selenium of a broadness, low selenium band, northwest and other area make scarce selenium, low selenium area account for 72% of China's area in addition.Some areas of 22 provinces and cities of China have 700,000,000 populations to live in low selenium area, and then cause the generation of multiple disease.It is 60 μ g that the every day ingestion standard is recommended by international selemium nutrition association in 1976, and it is 50 μ g that Chinese Soclety of Nutrition in 1980 recommends day to adopt the standard of taking the photograph.(Xu Huibi writes " biologic trace element-selenium " Huazhong Institute of Technology press, 1984)
Research shows: inorganic selenium toxicity is bigger, and scope is little between toxic dose and the requirement, and usage quantity needs strict control.Developed countries such as Japan, the U.S. have forbidden using as foodstuff additive at present.And organoselenium toxicity, spinoff are low more than inorganic selenium, and animal experiment shows, organic selenocarrageenan LD
50Be 4.92g/Kg, similar Sodium Selenite LD
50Be 7mg/Kg, its toxicity reduces more than 700 times, and toxicity maximum tolerated dose (MIC) is 16g/Kg; Belong to nontoxic level, using dosage is controlled easily simultaneously, can slowly discharge, absorb; Bioavailability is high, and 1992 through the approval of national foodstuff additive technical committee for standardization (TCST), as new food additive; Be expected to substitute Sodium Selenite and aspect food, healthcare products and clinical treatment, use, therefore how inorganic selenium being converted into organoselenium has very strong realistic meaning.
Do not see selenocarrageenan research and patent report abroad; The more domestic patented claim that relevant organoselenium are arranged; For example CN88103347, CN92107669, CN00111262, CN200410067065, CN200910161061, CN200910162003, CN200610045481 etc., wherein CN92107669 relates to selenizing polysaccharide and preparation method thereof.This method comprises yeast rich in selenium → extract → separate → add alkali homogenate → neutralization → concentrate → ethanol sedimentation → refining → steps such as finished product.This method relates to steps such as strain improvement, cultivation, extraction, separation, requires tight, preparation technology's relative complex, selenium content lower to envrionment conditions, is 400 μ g.Se/g.CN88103347 relates to the method for making of selenocarrageenan.This method comprises with concentrated nitric acid dissolving selenium powder, is mixed with selenium liquid; Prepare carrageenan solutions again.Two kinds of solution are mixed, under temperature 50-80 ℃, carry out selenylation reaction 2-4h, then, add ethanol sedimentation, filter at temperature 20-30 ℃ of reaction 12-24h down, washing, drying obtains the selenocarrageenan of selenium content 8-12mgSe/g.The selenocarrageenan of this method preparation uses the reaction of selenium powder and concentrated nitric acid simultaneously except that selenium content is low, the Selenium hydride by product of generation is a deadly poisonous compound, and is bigger to human harm.CN00111262 discloses selenium compound of polysaccharide and preparation method thereof.This method comprise with polysaccharide compound (red seaweed polysaccharide and carrageenin, chitosan, fucose) and selenous acid in acidic solution through Ba
2+, H
+Katalysis, heat 10-100 ℃, isothermal reaction 5-10h reduces to room temperature.Use sulfuric acid precipitation Ba
2+, filtrating adds liquor ferri trichloridi and removes unreacted selenite radical ion, and placement is spent the night, and the spinning throw out is used the Different concentrations of alcohol fractionation precipitation, and throw out vacuum-drying gets raw product.Directly ultrafiltration apparatus is refining through different films for raw product, gets the selenizing polysaccharide products, selenium content 15-125mgSe/g.The selenocarrageenan of this method preparation, though selenium content improves than the former greatly, step is more; Longer 3-4 of cycle days, the loss amount of carrageenin and selenium was bigger in the preparation process, and the recovery of carrageenin is 17.5-29.4%; The recovery of selenium is 1.8-4.6%; Cause a large amount of resource consumptions and environmental pollution, use barium salt to make catalyzer simultaneously, have certain toxicity.
In order to solve existing technological disadvantage, the inventor is through test of many times, and method of the present invention has been accomplished in research.
[summary of the invention]
[technical problem that will solve]
The purpose of this invention is to provide a kind of selenocarrageenan.
Another object of the present invention provides a kind of preparation method of selenocarrageenan.
[technical scheme]
The present invention realizes through following technical proposals.
The present invention relates to a kind of preparation method of selenocarrageenan.
The step of said method is following:
(1) batching
With 1000-5000 weight parts water dissolving 100-500 weight part carrageenin, add 0.1-0.8 weight part AMS or saccharifying enzyme then, stir, add 20-80 weight part selenium compound or selenium powder again, stir;
(2) enzymolysis selenylation reaction
The pH regulator of the reaction mixture that step (1) is obtained with 0.8-1.2M alkali aqueous solution and 0.8-1.2M aqueous acid is to 3-7, then temperature 40-70 ℃ with stirring condition under carry out enzymolysis selenylation reaction 0.1-30h;
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 80-95 ℃, and stirring reaction 0.5-1h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Use again 8-12% (weight/volume) sodium acetate aqueous solution with the pH regulator of step (3) terminated enzymolysis selenylation reaction liquid to 5-9; 0.1-2% (weight/volume) flocculation agent that adds the 4-100 weight part then; Temperature 30-50 ℃ with stirring condition under react 2-10h; Place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add ethanolic soln in the centrifuged supernatant that obtains toward step (5); The concentration of ethanol in whole solution is controlled at 60-80% (volume) to be precipitated; Decompress filter then; The filter cake that obtains adopts spraying or boulton process to carry out drying, obtains the selenocarrageenan of selenium content 20-100mg.Se/g.
According to another kind of preferred implementation of the present invention, described selenium compound is Sodium Selenite, selenous acid or sodium selenate.
According to another kind of preferred implementation of the present invention, the enzyme work of described AMS is 4000-20000IU/g, and the enzyme work of described saccharifying enzyme is 50000-100000IU/g.
According to another kind of preferred implementation of the present invention, described flocculation agent is selected from Poly aluminum Chloride (PAC), polyaluminium sulfate, poly-ferric chloride or bodied ferric sulfate.
According to another kind of preferred implementation of the present invention, when step (6) ethanol sedimentation, different alcohol concn obtains the selenocarrageenan of different molecular-weight average in whole precipitation solution:
(1) alcohol concn is 70-80% (volume), and the molecular-weight average of selenocarrageenan is 100-3500Da;
(2) alcohol concn is 65-70% (volume), and the molecular-weight average of selenocarrageenan is 3500-8000Da;
(3) alcohol concn is 60-65% (volume), and the molecular-weight average of selenocarrageenan is more than the 8000Da.
According to another kind of preferred implementation of the present invention, described alkali is selected from sodium hydroxide, Pottasium Hydroxide, yellow soda ash or sodium hydrogencarbonate; Described acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid.
According to another kind of preferred implementation of the present invention; Described spraying drying is that said filter cake water is adjusted to solid content 10-20% (weight/volume); Through spray-drier, controlling its inlet temperature is 170-200 ℃ then, and temperature out is 160-190 ℃.
According to another kind of preferred implementation of the present invention, described vacuum-drying is directly to put into vacuum drying oven to described filter cake, under the condition of vacuum tightness greater than 0.09Pa and temperature 50-70 ℃, carries out drying.
A preferred embodiment of the invention, described filter cake separates with the laggard capable membrane ultrafiltration of water dissolution, obtains refining selenocarrageenan, and the selenium content of the refining selenocarrageenan of its different molecular weight is following:
(1) selenium content of the selenocarrageenan of molecular weight 100-3500Da is 60-150mg.Se/g;
(2) selenium content of the selenocarrageenan of molecular weight 3500-8000Da is 50-100mg.Se/g;
(3) selenium content of the selenocarrageenan more than the molecular weight 8000Da is 30-60mg.Se/g.
The invention still further relates to the selenocarrageenan that adopts preparation method of the present invention to obtain.
Described selenocarrageenan has following characteristics:
(1) molecular-weight average: 100-20000Da;
(2) selenium content: 20-150mgSe/g;
(3) specific rotation :+30-+90 degree;
(4) spectral response curve:
1. UV spectrum: charateristic avsorption band is arranged at wavelength 291nm place;
2. ir spectra: at wavelength 1383.90cm
-1And 607.83cm
-1There is charateristic avsorption band at the place;
3. proton nmr spectra: charateristic avsorption band is arranged at chemical shift 4.8734ppm and 1.9200ppm place;
4. carbon-13 nmr spectra: charateristic avsorption band is arranged at chemical shift 76.744ppm and 76.576ppm place.
Below the present invention will be described in more detail.
The present invention relates to a kind of preparation method of selenocarrageenan.
The step of said method is following:
(1) batching
With 1000-5000 weight parts water dissolving 100-500 weight part carrageenin, add 0.1-0.8 weight part AMS or saccharifying enzyme then, stir, add 20-80 weight part selenium compound or selenium powder again, stir.
Carrageenin is called carrageeman, X 5189 again.Carrageenin is the lyophilic colloid that from some red algae sea grass, extract, calcium, potassium, sodium, the ammonium salt of the polyose sulfuric ester that its chemical structure is made up of semi-lactosi and Anhydrogalactose.Because wherein the difference of sulfuric ester combining form can be divided into κ type (kappa), ι type (lota), λ type (lambda).It is insoluble to cold water, but the solvable blob of viscose shape that is expanded into is insoluble to organic solvent, is soluble in hot water and becomes translucent colloidal solution; In the presence of potassium ion, can generate heat reversible gel; Concentration forms low viscous colloidal sol when low, and near Newtonian fuid, concentration raises and forms HV colloidal sol, then is non-Newtonian fluid.
The enzyme of the O-glucose key in AMS (EC3.2.1.1.) ability hydrolyzed starch, glycogen and the relevant polysaccharide, for example the grand mcroorganism Engineering Co., Ltd in Shandong, Henan Yangshao Biochemical Engineering Co., Ltd., Lianyun Harbour unit rise the AMS that Industry Co.,Ltd produces.
Saccharifying enzyme Gluco-Amylase claims glucoamylase (EC3.2.1.3) again, is the high-performance bio catalyzer that makes through fermentation with the black mold dissociant.Saccharifying enzyme can be under normal temperature condition cuts the α-1.4 of starch molecule and α-1.6 glycosidic link, and makes starch be converted into glucose.Every is the production process that raw material needs saccharification again with starch, all can use saccharifying enzyme to improve the mashing yield with it.Its series product have two types of solid and liquid; Be applicable to Dian Fentang, alcohol, brewage, industry such as monosodium glutamate, glucose, organic acid and microbiotic, the for example saccharifying enzyme produced of Sichuan Province's hill bio tech ltd, the magnificent prosperous and powerful Bioisystech Co., Ltd in east, Beijing, Xingtai Wanda's biotechnology ltd.
Enzyme activity is defined as 1 gram enzyme powder or 1ml enzyme liquid under 40 ℃ of conditions with pH4.6, and it is 1 enzyme work unit that 1 hour decomposing soluble starch produces the enzyme amount of 1mg glucose.
In the present invention, the enzyme work of the AMS of use is 4000-20000IU/g.Preferably, the enzyme work of the AMS of use is 8000-15000IU/g.The enzyme work of the saccharifying enzyme that uses is 50000-100000IU/g.Preferably, the enzyme work of the saccharifying enzyme of use is 60000-80000IU/g.
According to the present invention, 100-500 weight part carrageenin uses 0.1-0.8 weight part AMS or saccharifying enzyme, and promptly every gram calorie draws the collagen material to throw in the 5-20IU AMS; Every gram calorie draws the collagen material to throw in the 50-300IU saccharifying enzyme.If AMS or saccharifying enzyme that 100-500 weight part carrageenin uses are lower than 0.1 weight part, then can make enzymolysis selenylation reaction time lengthening; If AMS or saccharifying enzyme that 100-500 weight part carrageenin uses are higher than 0.8 weight part, then can effectively hydrolyzing, the selenocarrageenan polysaccharide recovery is reduced.
In the present invention, described selenium compound is selected from Sodium Selenite, selenous acid or sodium selenate.Preferably, described selenium compound is Sodium Selenite or sodium selenate.The granularity of said selenium powder is the 80-200 order normally, preferably the 100-150 order.
According to the present invention, 100-500 weight part carrageenin uses 20-80 weight part selenium compound or selenium powder.If selenium compound or selenium powder that 100-500 weight part carrageenin uses are lower than 20 weight parts, then can make the organoselenium combination rate on the low side; If selenium compound or selenium powder that 100-500 weight part carrageenin uses are higher than 80 weight parts, then can make the selenium wastage of material, cause environmental pollution.
Preferably, 100-500 weight part carrageenin uses 30-60 weight part selenium compound or selenium powder.More preferably, 100-500 weight part carrageenin uses 40-50 weight part selenium compound or selenium powder.
(2) enzymolysis selenylation reaction
The pH regulator of the reaction mixture that step (1) is obtained with 0.8-1.2M alkali aqueous solution and 0.8-1.2M aqueous acid is to 3-7, then temperature 40-70 ℃ with stirring condition under carry out enzymolysis selenylation reaction 0.1-30h.
Described alkali is selected from sodium hydroxide, Pottasium Hydroxide, yellow soda ash or sodium hydrogencarbonate.Preferably, described alkali is selected from sodium hydroxide, Pottasium Hydroxide or yellow soda ash.
Described acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid.Preferably, described acid is hydrochloric acid or nitric acid.
The concentration of said alkali aqueous solution and aqueous acid is not crucial, only need be convenient to the pH of conditioned reaction mixture.
Preferably, described enzymolysis selenylation reaction carries out 2-15h under the condition of pH4-6 and temperature 45-60 ℃.More preferably, described enzymolysis selenylation reaction carries out 1-10h under the condition of pH4.5-5.5 and temperature 50-55 ℃.In this reaction, the purpose of stirring is can make in the reaction process reactant to remain abundant contact, carries out according to our expection thereby guarantee to react, and guarantees the reaction acceleration.
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 80-95 ℃, and stirring reaction 0.5-1h loses activity enzyme, and termination reaction is placed and reduced to room temperature.Enzyme is an active protein, protein denaturation at high temperature, and enzyme loses the hydrolyzation catalysis ability.
(4) flocculation reaction
Use again 8-12% (weight/volume) sodium acetate aqueous solution with the pH regulator of step (3) terminated enzymolysis selenylation reaction liquid to 5-9; 0.1-2% (weight/volume) flocculation agent that adds the 4-100 weight part then; Temperature 30-50 ℃ with stirring condition under react 2-10h; Place then and spend the night, obtain the flocculence product.
According to the present invention, described flocculation agent should be appreciated that it is more such inorganic macromolecule compounds, and they generate throw out through unconjugated free selenium in absorption, bridge formation, crosslinked action and the reaction mixture, thereby can effectively remove these free selenium.
Described flocculation agent is selected from Poly aluminum Chloride (PAC), polyaluminium sulfate, poly-ferric chloride or bodied ferric sulfate.Preferably, described flocculation agent is selected from Poly aluminum Chloride (PAC), polyaluminium sulfate or poly-ferric chloride.
Above-mentioned inorganic polymer flocculant is the one type of novel tap water treatment agent that grows up in the later stage eighties 20th century; Suitability for industrialized production has been carried out in U.S.A, day, West Europe and domestic Qingdao at present; Compare with traditional aluminum chloride, Tai-Ace S 150, iron(ic)chloride; A large amount of complex ions can be provided, and flocculation performance improves many times.
In the present invention, the concentration of said flocculation agent is 0.1-2% (weight/volume).If the concentration of said flocculation agent is lower than 0.1% (weight/volume), then free selenium clearance rate reduces.If the concentration of said flocculation agent is higher than 2% (weight/volume), then waste flocculation agent.
Described flocculation reaction is reacted 2-10h under the condition of pH 5-9 and temperature 30-50 ℃; Preferably, described flocculation reaction is reacted 4-8h under the condition of pH 5.5-8.0 and temperature 35-45 ℃, and more preferably, described flocculation reaction is reacted 5-6h under the condition of pH 6.0-7.0 and temperature 38-42 ℃.
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted.
In the present invention, use whizzer that reaction solution is separated with the flocculence product.The whizzer that the present invention uses is the whizzer that the people that sell in the market generally use.
(6) ethanol sedimentation
Add ethanolic soln in the centrifuged supernatant that obtains toward step (5); The concentration of ethanol in whole solution is controlled at 60-80% (volume) to be precipitated; Decompress filter then; The filter cake that obtains adopts spraying or boulton process to carry out drying, obtains the selenocarrageenan of selenium content 20-100mg.Se/g.
With the ethanol sedimentation selenocarrageenan time, control precipitation solution different ethanol concentration 60-80% (volume) can obtain the selenocarrageenan of different molecular-weight average.
(1) alcohol concn is 70-80% (volume), and the molecular-weight average of selenocarrageenan is 100-3500Da;
(2) alcohol concn is 65-70% (volume), and the molecular-weight average of selenocarrageenan is 3500-8000Da;
(3) alcohol concn is 60-65% (volume), and the molecular-weight average of selenocarrageenan is more than the 8000Da.
In the present invention, described decompress filter normally leans on the tap aspirator draw air, makes decompression in the filter flask, forms pressure difference in the bottle with on the B liquid level, thereby can accelerate filtering rate.Therefore, described decompress filter is the decompress filter method that the technician in present technique field knows.The decompress filter equipment that the present invention uses comprises B, filter flask, safety flack and tap aspirator, also is the normally used decompress filter equipment of technician in present technique field.
Described spraying drying is that said filter cake water is adjusted to solid content 10-20% (weight/volume), and through spray-drier, controlling its inlet temperature is 170-200 ℃ then, and temperature out is 160-190 ℃.Described spray-drier for example is that Shanghai generation biological plant far away Engineering Co., Ltd, Changzhou are unified drying plant ltd, Shanghai reaches the spray-drier that journey experimental installation ltd produces.
Described vacuum-drying is directly to put into vacuum drying oven to described filter cake, under the condition of vacuum tightness greater than 0.09Pa and temperature 50-70 ℃, carries out drying.Described vacuum drying oven for example is that Beijing Li Kangdasheng development in science and technology ltd, Changzhou are unified the vacuum drying oven that new Medical Instruments ltd produces in drying plant ltd, Jiaxing.
A preferred embodiment of the invention, described filter cake separates with the laggard capable membrane ultrafiltration of water dissolution, obtains the purified selenocarrageenan, and the selenium content of the refining selenocarrageenan of its different molecular weight is following:
Adopt the ultrafiltration membrance filter method to filter in the present invention.Ultrafiltration membrance filter is that a kind of ultra-filtration membrane that adopts is the membrane filtering method of impellent with the pressure difference.Ultra-filtration membrane is generally processed by Mierocrystalline cellulose and verivate thereof, polycarbonate, SE, pvdf, polysulfones, polyacrylonitrile, polymeric amide, polysulfonamides etc., and for example the Zhaoyuan gold converges the ultra-filtration membrane that film Science and Technology Ltd., Beijing Hydranautics company produce.
The selenium content of the refining selenocarrageenan of different molecular weight is following:
(1) selenium content of the selenocarrageenan of molecular weight 100-3500Da is 60-150mg.Se/g;
(2) selenium content of the selenocarrageenan of molecular weight 3500-8000Da is 50-100mg.Se/g;
(3) selenium content of the selenocarrageenan more than the molecular weight 8000Da is 30-60mg.Se/g.
The molecular-weight average of above-mentioned selenocarrageenan is measured with performance liquid gel chromatography (HPGPC) method, polysaccharide molecular weight M and partition ratio K
α v=k
1-k
2Ln M is relevant, k
1, k
2Be constant, M is a molecular weight, and concrete operations are with reference to (Miu Jin come etc., the research of selenocarrageenan physico-chemical property and molecular structure, the research and development 2002 of marine bioactivity material).
The invention still further relates to the selenocarrageenan that adopts preparation method of the present invention to obtain, it is characterized in that it has following characteristics:
(1) molecular-weight average: 100-20000Da;
(2) selenium content: 20-150mgSe/g;
(3) specific rotation :+30-+90 degree;
(4) spectral response curve:
1. UV spectrum: charateristic avsorption band is arranged at wavelength 291nm place;
2. ir spectra: at wavelength 1383.90cm
-1And 607.83cm
-1There is charateristic avsorption band at the place;
3. proton nmr spectra: charateristic avsorption band is arranged at chemical shift 4.8734ppm and 1.9200ppm place;
4. carbon-13 nmr spectra: charateristic avsorption band is arranged at chemical shift 76.744ppm and 76.576ppm place.
The chemical structure of described carrageenin and selenium compound reaction and carrageenin and selenocarrageenan is following:
This reaction is to constitute on unit pyranohexose or the galactose molecule structure at carrageenin to carry out, wherein the unitary C of pyranohexose
4Substitution reaction on the position, or 3, the C on 6-inner ether-D-galactose units position
3Or C
6Addition reaction on the position.
In the present invention, adopt chemical dialysis method to measure the content of organoselenium in the selenocarrageenan.For example use the dialysis tubing of the 500Da that goes up sea base star bio tech ltd or the sale of the sharp Science and Technology Ltd. of the prosperous think of in Wuhan, under normal temperature condition, dialyse.Earlier the selenocarrageenan of preparation is packed in the previously prepd dialysis tubing, in zero(ppm) water, dialyses, during constantly change water, all appear outside the bag until the unconjugated inorganic selenium of inspection, measure the organic selenium content in the bag again.Concrete dialysis and measuring method are referring to document (Hao Sue etc., the research of organic selenium content measuring method in the selenium yeast, physical and chemical inspection-chemical fascicle 1999).The present invention prepares organic selenium content >=90% in the selenocarrageenan, organic selenium content >=95% of refining selenocarrageenan.
In the present invention, adopt conventional ultraviolet, ir spectra and nmr analysis method to identify selenocarrageenan of the present invention.The result of ultraviolet, ir spectra and nmr analysis selenocarrageenan of the present invention sees accompanying drawing 1-4.
Accompanying drawing 1 is the UV spectrum of selenocarrageenan (a) and carrageenin (b).
As can beappreciated from fig. 1, selenocarrageenan of the present invention is in the UV spectrum of 200-500nm wave band and comparing of carrageenin, and said selenocarrageenan has tangible absorption peak at the 291nm place, and carrageenin does not have this absorption peak.This shows that the 291nm absorption peak confirms to have introduced selenium in the carrageenan molecule.
Accompanying drawing 2 is ir spectras of selenocarrageenan (a) and carrageenin (b).
As can beappreciated from fig. 2, selenocarrageenan ir spectra of the present invention is compared with carrageenin, and also there is 1383.90cm in said selenocarrageenan ir spectra
-1With 607.83cm
-1Two other absorption peak.Analysis confirmation, 1383.90cm
-1Absorption peak belongs to the stretching vibration of carrageenin Se=0,607.83cm
-1Absorption peak belongs to pyranohexose unit C in the carrageenin polysaccharide molecule
4Sulphur on the position is replaced by selenium, and 3,6-inner ether-D-galactose units is at C
3Position or C
6The selenium that introduce the position.
Accompanying drawing 3 is proton nmr spectras of selenocarrageenan (a) and carrageenin (b).
As can beappreciated from fig. 3, the hydrogen of selenocarrageenan of the present invention and carrageenin spectrum is identical basically, and the basic configuration that still keeps polysaccharide sulfate is described.But said selenocarrageenan hydrogen spectrum is compared with carrageenin hydrogen spectrum, and said selenocarrageenan hydrogen spectrum also exists 4.8734ppm and two other absorption peak of 1.9200ppm.Analysis confirmation, 4.8734ppm absorption peak belong to the unitary C of pyranohexose
4Sulphur is absorbed by the substituted selenium hydrogen of selenium on the position; Or belonging to carrageenin polysaccharide 3, the interior ehter bond of 6-inner ether-D-galactose units is opened by selenium and is connected in this unit C
3Selenium hydrogen absorbs on the position.1.9200ppm absorption peak belongs to carrageenin polysaccharide 3, the interior ehter bond of 6-inner ether-D-galactose units is opened by selenium and is connected in this unit C
6Selenium hydrogen absorbs on the position.
Accompanying drawing 4 is carbon-13 nmr spectras of selenocarrageenan (a) and carrageenin (b).
As can beappreciated from fig. 4, selenocarrageenan carbon spectrum of the present invention is compared with carrageenin, and said selenocarrageenan carbon spectrum also exists 76.744ppm and two other peak of 76.576ppm.Analysis confirmation, in the selenylation reaction process, the unitary C of pyranohexose in the described carrageenin polysaccharide molecule
4Sulphur on the position is by selenium replacement and 3, the C of 6-inner ether-D-galactose units
3Or C6 introducing selenium causes the C chemical shift to change.
The immune function of mice test that carry out the Jiangsu Inst of Atomic Medical Sciences shows; Selenocarrageenan of the present invention can improve peripheral white blood cell order, half serum haemolysis numerical value and antibody-producting cell numerical value; It is active to strengthen il-1 (IL-1), interleukin-2 (IL-2), NK cell, TNFa, and the competence for added value of thymus gland T lymphocyte, spleen B lymphocyte, thoracic cavity macrophage phagocytic function, carbon clearance value, tardy parasexuality reaction etc.The mouse tumor inhibition test that carry out the Jiangsu Inst of Atomic Medical Sciences shows that selenocarrageenan of the present invention all has very strong immunity and anti-tumor function to cancer of the stomach SGC7901, lung cancer A549, intestinal cancer LS-174T, thyroid carcinoma K1 and FTC-133, leukemia cell SHI-1, liver cancer HepA, mammary cancer MCF-7, prostate cancer DU-145, sarcoma S-180, neuroblastoma SH-SY5Y.
[beneficial effect]
The invention has the beneficial effects as follows:
The method that the present invention prepares selenocarrageenan can reach carrageenin recovery 70-85%, selenium recovery 70-80%, the selenium content 150mg.Se/g of said selenocarrageenan; Production cycle 1-2 days, and prior art can only reach carrageenin recovery 17.5-29.4%, selenium recovery 1.8-4.6%; Selenium content 125mg.Se/g; Production cycle 3-4 days, therefore, the cost of the inventive method reduced 50-70%.This shows that beneficial effect of the present invention compared with prior art is very tangible.
[description of drawings]
Fig. 1 selenocarrageenan (a) and carrageenin (b) ultraviolet spectrogram;
Fig. 2 selenocarrageenan (a) and carrageenin (b) infrared spectrogram
Fig. 3 selenocarrageenan (a) and carrageenin (b) nucleus magnetic resonance H spectrogram
Fig. 4 selenocarrageenan (a) and carrageenin (b) nucleus magnetic resonance C spectrogram
[embodiment]
Embodiment 1: the present invention prepares selenocarrageenan
Said preparing method's step is following:
(1) batching
With 1000mL water dissolution 100g carrageenin, add the AMS of 0.2g enzyme 4000IU/g alive then, promptly enzyme is lived and is drawn the collagen material for the every gram calorie of 8IU/; Stir; Add 33g weight part selenous acid again, being equivalent to add the selenium amount is that 0.2g selenium/every gram calorie draws the collagen material, stirs;
(2) enzymolysis selenylation reaction
The pH regulator to 6.5 of the reaction mixture that step (1) is obtained with 1M aqueous sodium hydroxide solution and 1M aqueous hydrochloric acid carries out enzymolysis selenylation reaction 15h then under 40 ℃ of temperature and stirring condition;
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 80 ℃, and stirring reaction 1h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Use the pH regulator to 5.5 of 10% (weight/volume) sodium acetate aqueous solution again with the enzymolysis selenylation reaction liquid of step (3); Add the 4g Poly aluminum Chloride (PAC) then; Be equivalent to Poly aluminum Chloride (PAC) concentration 0.4% (weight/volume); Under 30 ℃ of temperature and stirring condition, react 5h, place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add 3 times of volumes 95% (v/v) ethanolic soln in the centrifuged supernatant that obtains toward step (5), the concentration of ethanol in whole solution is controlled at 72% (volume) precipitates, then decompress filter; The collecting precipitation thing; It is 10% (g/v) that water is regulated soluble solid, carries out spraying drying, and the inlet temperature of control spray-drier is 170 ℃; Temperature out is 160 ℃, obtains the selenocarrageenan bullion of selenium content 80mg.Se/g.
Embodiment 2: the present invention prepares selenocarrageenan
Said preparing method's step is following:
(1) batching
With 5000mL water dissolution 100g carrageenin, add the saccharifying enzyme of 0.5g enzyme 6000IU/g alive then, promptly enzyme is lived and is drawn the collagen material for the every gram calorie of 300IU/; Stir; Add 20g weight part Sodium Selenite again, being equivalent to add the selenium amount is that 0.1g selenium/every gram calorie draws the collagen material, stirs;
(2) enzymolysis selenylation reaction
The pH regulator to 3.0 of the reaction mixture that step (1) is obtained with 1M aqueous sodium hydroxide solution and 1M aqueous hydrochloric acid carries out enzymolysis selenylation reaction 20h then under 50 ℃ of temperature and stirring condition;
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 85 ℃, and stirring reaction 0.5h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Use the pH regulator to 7.0 of 10% (weight/volume) sodium acetate aqueous solution again with the enzymolysis selenylation reaction liquid of step (3); Add the 5g polyaluminium sulfate then; Be equivalent to polyaluminum sulfate al concn 0.1% (weight/volume); Under 50 ℃ of temperature and stirring condition, react 10h, place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add 4 times of volumes 95% (v/v) ethanolic soln in the centrifuged supernatant that obtains toward step (5); The concentration of ethanol in whole solution is controlled at 76% (volume) to be precipitated; Decompress filter then, the collecting precipitation thing is directly put into vacuum drying oven with throw out; Under the condition of vacuum tightness, carry out vacuum-drying, obtain the selenocarrageenan bullion of selenium content 90mg.Se/g greater than 50 ℃ of 0.09Pa and temperature.
Embodiment 3: the present invention prepares selenocarrageenan
Said preparing method's step is following:
(1) batching
With 5000mL water dissolution 500g carrageenin, add the AMS of 0.5g enzyme 20000IU/g alive then, promptly enzyme is lived and is drawn the collagen material for the every gram calorie of 20IU/; Stir; Add 40g weight part selenous acid again, being equivalent to add the selenium amount is that 0.05g selenium/every gram calorie draws the collagen material, stirs;
(2) enzymolysis selenylation reaction
The pH regulator to 7.0 of the reaction mixture that step (1) is obtained with 1M potassium hydroxide aqueous solution and 1M aqueous sulfuric acid carries out enzymolysis selenylation reaction 30h then under 70 ℃ of temperature and stirring condition;
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 85 ℃, and stirring reaction 0.5h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Use the pH regulator to 9.0 of 10% (weight/volume) sodium acetate aqueous solution again with the enzymolysis selenylation reaction liquid of step (3); Add the 100g poly-ferric chloride then; Be equivalent to poly-ferric chloride concentration 2.0% (weight/volume); Under 50 ℃ of temperature and stirring condition, react 10h, place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add 2 times of volumes 95% (v/v) ethanolic soln in the centrifuged supernatant that obtains toward step (5), the concentration of ethanol in whole solution is controlled at 63% (volume) precipitates, then decompress filter; The collecting precipitation thing; It is 20% (g/v) that water is regulated soluble solid, carries out spraying drying, and the inlet temperature of control spray-drier is 200 ℃; Temperature out is 190 ℃, obtains the selenocarrageenan bullion of selenium content 70mg.Se/g.
Embodiment 4: the present invention prepares selenocarrageenan
Said preparing method's step is following:
(1) batching
With 4000mL water dissolution 200g carrageenin, add the saccharifying enzyme of 0.2g enzyme 50000IU/g alive then, promptly enzyme is lived and is drawn the collagen material for the every gram calorie of 50IU/; Stir; Add 80g weight part Sodium Selenite again, being equivalent to add the selenium amount is that 0.2g selenium/every gram calorie draws the collagen material, stirs;
(2) enzymolysis selenylation reaction
The pH regulator to 4.0 of the reaction mixture that step (1) is obtained with 1.2M aqueous sodium hydroxide solution and 1.2M aqueous hydrochloric acid carries out enzymolysis selenylation reaction 15h then under 60 ℃ of temperature and stirring condition;
(3) deactivation
Step (2) enzymolysis selenylation reaction liquid is heated to 95 ℃, and stirring reaction 0.5h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Use the pH regulator to 7.5 of 10% (weight/volume) sodium acetate aqueous solution again with the enzymolysis selenylation reaction liquid of step (3); Add the 40g bodied ferric sulfate then; Be equivalent to polyaluminum sulfate al concn 1% (weight/volume); Under 40 ℃ of temperature and stirring condition, react 2h, place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add 5 times of volumes 95% (v/v) ethanolic soln in the centrifuged supernatant that obtains toward step (5); The concentration of ethanol in whole solution is controlled at 80% (volume) to be precipitated; Decompress filter then, the collecting precipitation thing is directly put into vacuum drying oven with throw out; Under the condition of vacuum tightness, carry out vacuum-drying, obtain the selenocarrageenan bullion of selenium content 100mg.Se/g greater than 70 ℃ of 0.09Pa and temperature.
Embodiment 5: said selenocarrageenan refining
The selenocarrageenan bullion that takes by weighing 5g embodiment 1-4 preparation is used the 1000mL water dissolution; Fully after the dissolving; Ultra-filtration membrane and support equipment thereof that the solution that obtains uses Beijing Hydranautics company or Wuxi City Ultrafilter Equipment Factory to produce carry out membrane ultrafiltration and separate under normal temperature condition.According to the size of raw product molecular weight select the to be complementary ultra-filtration membrane in aperture, obtain the purified selenocarrageenan.The selenium content that adopts the method for describing in this specification sheets to measure them is respectively 98mg.Se/g, 100mg.Se/g, 80mg.Se/g and 150mg.Se/g.
Claims (10)
1. the preparation method of a selenocarrageenan is characterized in that the step of said method is following:
(1) batching
With 1000-5000 weight parts water dissolving 100-500 weight part carrageenin, add 0.1-0.8 weight part AMS or saccharifying enzyme then, stir, add 20-80 weight part selenium compound or selenium powder again, stir;
(2) enzymolysis selenylation reaction
The pH regulator of the reaction mixture that step (1) is obtained with 0.8-1.2M alkali aqueous solution and 0.8-1.2M aqueous acid is to 3-7, then temperature 40-70 ℃ with stirring condition under carry out enzymolysis selenylation reaction 0.1-30h;
(3) deactivation
The enzymolysis selenylation reaction liquid of step (2) is heated to 80-95 ℃, and stirring reaction 0.5-1h loses activity enzyme, and termination reaction is placed and reduced to room temperature;
(4) flocculation reaction
Again with sodium-acetate in g; Sodium acetate soln in ml 8-12% sodium acetate aqueous solution with the pH regulator of step (3) terminated enzymolysis selenylation reaction liquid to 5-9; The flocculation agent that adds the 4-100 weight part then is in gram, and flocculant solution is in ml 0.1-2% flocculant solution, temperature 30-50 ℃ with stirring condition under react 2-10h; Place then and spend the night, obtain the flocculence product;
(5) spinning
The flocculence product that step (4) obtains carries out centrifugal, removes the free selenium bonded throw out of flocculation agent and unreacted;
(6) ethanol sedimentation
Add ethanolic soln in the centrifuged supernatant that obtains toward step (5); The volumetric concentration of ethanol in whole solution is controlled at 60-80% to be precipitated; Decompress filter then, the filter cake that obtains adopt spraying or boulton process to carry out drying, obtain the selenocarrageenan of selenium content 20-100mg.Se/g.
2. preparation method according to claim 1 is characterized in that described selenium compound is Sodium Selenite, selenous acid or sodium selenate.
3. preparation method according to claim 1 is characterized in that the enzyme work of described AMS is 4000-20000IU/g, and the enzyme work of described saccharifying enzyme is 50000-100000IU/g.
4. preparation method according to claim 1 is characterized in that described flocculation agent is selected from Poly aluminum Chloride (PAC), polyaluminium sulfate, poly-ferric chloride or bodied ferric sulfate.
5. preparation method according to claim 1 is characterized in that different alcohol concn obtains the selenocarrageenan of different molecular-weight average in whole precipitation solution when step (6) ethanol sedimentation:
When (1) the ethanol volumetric concentration was 70-80%, the molecular-weight average of selenocarrageenan was 100-3500Da;
When (2) the ethanol volumetric concentration was 65-70%, the molecular-weight average of selenocarrageenan was 3500-8000Da;
When (3) the ethanol volumetric concentration was 60-65%, the molecular-weight average of selenocarrageenan was more than the 8000Da.
6. preparation method according to claim 1 is characterized in that described alkali is selected from sodium hydroxide, Pottasium Hydroxide, yellow soda ash or sodium hydrogencarbonate; Described acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid.
7. preparation method according to claim 1; It is characterized in that described spraying drying is that said filter cake water is adjusted to solid content is that solid substance is in g; Solid substance solution is in ml 10-20%; Through spray-drier, controlling its inlet temperature is 170-200 ℃ then, and temperature out is 160-190 ℃.
8. preparation method according to claim 1 is characterized in that described vacuum-drying is directly to put into vacuum drying oven to described filter cake, under the condition of vacuum tightness greater than 0.09Pa and temperature 50-70 ℃, carries out drying.
9. preparation method according to claim 1 is characterized in that described filter cake separates with the laggard capable membrane ultrafiltration of water dissolution, obtains refining selenocarrageenan, and the selenium content of the refining selenocarrageenan of its different molecular weight is following:
(1) selenium content of the selenocarrageenan of molecular weight 100-3500Da is 60-150mg.Se/g;
(2) selenium content of the selenocarrageenan of molecular weight 3500-8000Da is 50-100mg.Se/g;
(3) selenium content of the selenocarrageenan more than the molecular weight 8000Da is 30-60mg.Se/g.
10. the selenocarrageenan that obtains according to the said preparation method of each claim among the claim 1-9 is characterized in that it has following characteristics:
(1) molecular-weight average: 100-20000Da;
(2) selenium content: 20-150mgSe/g;
(3) specific rotation :+30-+90 degree;
(4) spectral response curve:
1. UV spectrum: charateristic avsorption band is arranged at wavelength 291nm place;
2. ir spectra: charateristic avsorption band is arranged at wavelength 1383.90cm-1 and 607.83cm-1 place;
3. proton nmr spectra: charateristic avsorption band is arranged at chemical shift 4.8734ppm and 1.9200ppm place;
4. carbon-13 nmr spectra: charateristic avsorption band is arranged at chemical shift 76.744ppm and 76.576ppm place.
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| CN102911278B (en) * | 2012-10-18 | 2014-05-14 | 成都连接流体分离科技有限公司 | Membrane concentration process used for carrageenan production |
| WO2014202686A1 (en) * | 2013-06-19 | 2014-12-24 | Pancosma S.A. | Elemental selenium nanoparticles and production method |
| CN104286532B (en) * | 2014-10-11 | 2016-08-17 | 恩施土家族苗族自治州农业科学院 | A kind of selenium-rich cold water fish forage |
| CN105853458A (en) * | 2016-04-25 | 2016-08-17 | 湖南中硒生物科技有限公司 | Vitamin E selenium and preparation method thereof |
| CN106676145A (en) * | 2016-12-15 | 2017-05-17 | 钦州市钦南区科学技术情报研究所 | Carrageenan and preparation method thereof |
| CN108276603B (en) * | 2018-01-26 | 2018-11-16 | 青岛鑫康达生物科技有限公司 | A kind of preparation method of high Se content small molecule selenide of carragheen |
| CN111087484B (en) * | 2019-07-15 | 2020-08-25 | 郑州市御合源生物科技有限公司 | Bonded selenium polysaccharide and preparation method and application thereof |
| CN110317100A (en) * | 2019-07-22 | 2019-10-11 | 唐山津国农业技术开发有限公司 | The production technology of organic selenium Liquid Fertilizer |
| CN110839899A (en) * | 2019-11-15 | 2020-02-28 | 扬州大学 | Method for synthesizing selenose by photocatalytic selenium transfer |
| CN112225915B (en) * | 2020-10-28 | 2022-11-18 | 青岛鹏洋生物工程有限公司 | Industrialized powder preparation process of seleno-carrageenan |
| CN112220046B (en) * | 2020-10-28 | 2022-09-09 | 青岛鹏洋生物工程有限公司 | Compound nutrition enhancer containing seleno-carrageenan and preparation method thereof |
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