CN103073652A - Method for extracting polysaccharide of spirulina platensis - Google Patents

Method for extracting polysaccharide of spirulina platensis Download PDF

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Publication number
CN103073652A
CN103073652A CN2012105823684A CN201210582368A CN103073652A CN 103073652 A CN103073652 A CN 103073652A CN 2012105823684 A CN2012105823684 A CN 2012105823684A CN 201210582368 A CN201210582368 A CN 201210582368A CN 103073652 A CN103073652 A CN 103073652A
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ultrafilter
molecular weight
spirulina
polysaccharide
supernatant liquor
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CN103073652B (en
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梅艳
李俊
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Yunnan Plant Pharmaceutical Industry Co., Ltd.
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KUNMING ZHENHUA PHARMACY CO Ltd
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Abstract

The invention discloses a method for extracting polysaccharide of spirulina platensis. The method comprises the steps that spirulina platensis is subjected to an extraction process to form an extracting solution, the extracting solution is filtered through a microfiltration membrane and is subjected to ultrafilteration, concentration and drying, then more than one type of crude polysaccharide of aqueous extract spirulina platensis is obtained, the content of total sugar is larger than or equal to 10%, and the molecular weight is more than 5,000; or the extracting solution is concentrated and decontaminated, a processed solution is centrifuged under the rotating speed lower than or equal to 15,000 r/min, filtered through the microfiltration membrane and subjected to ultrafilteration by a filter material with the molecular weight in a range from 5,000 to 200,000, when no effluent flows out from an outlet of an ultrafilter, the effluent of the ultrafilter and a concentration solution in the ultrafilter are collected to be dried, then more than one type of polysaccharide of aqueous extract spirulina platensis is fromed, the content of total sugar is larger than or equal to 40%, and the molecular weight is in a range from 5,000 to 200,000. According to the method for extracting polysaccharide of spirulina platensis, reasonable processing measures are selected at different stages of a proces route according to different requirements for product purity, and then high purity olysaccharide with the scheduled molecular weight specification can be formed without detection; and moreover, polysaccharide of spirulina platensis with a pluraity of specifications can be formed simultaneosuly. Accordingly, the method has the advanatags which are impossible to be obtained by other traditional methods.

Description

A kind of extracting method of spirulina polysaccharide
Technical field
The present invention relates to obtain simultaneously the extracting method of the spirulina polysaccharide of different kinds of molecules weight range.The polysaccharide of made different size can be used for medicine, food, healthcare products and makeup etc.
Background technology
Spirulina is unicellular algae ancient in the ocean, contains the nutritive substances such as a large amount of amino acid, cyanophycin, carotenoid, gamma linolenic acid, inositol.Spirulina polysaccharide be from spirulina frond, spirulina medium, extract the class separate and have Promote cell's growth, improve immunizing power, the important natural bioactivity substance of antitumor, radioprotective, the anti-ageing function of waiting for a long time, the simultaneously remarkable dual function of enhancing body immunizing power of control various diseases is arranged.
There is a large amount of protein in the spirulina, the same polymer substance that all belongs to polysaccharide, part water soluble protein and polysaccharide are together by solvent extraction out.Removing protein is the committed step that spirulina polysaccharide is purified, and often utilizes the dual solvent system of chloroform propyl carbinol or trichoroacetic acid(TCA) that protein denaturation is separated out.The solvent that the former uses has certain toxicity and the strong volatility of tool, gives environment and the certain harm of operator; The trichoroacetic acid(TCA) that the latter uses has certain toxicity, and solvability like the salt of producing behind the deproteinated and the polyose is removed difficulty, certain toxicity is also arranged, so the crude product utilization that the method obtains is restricted.The characteristic of often utilizing polysaccharide to be insoluble to ethanol is made with extra care the spirulina polysaccharide crude extract, but the resulting purity of polysaccharide of this method is not high, can't eliminate protein, other impurity such as pigment, colloid that also can mix, and also the gains molecular weight ranges is wider.Gel filtration chromatography that utilizes in addition DEAE cellulose column ion exchange chromatography, dextrane gel and sepharose etc. separates to get purity polysaccharide high, different molecular weight ranges.But because column packing is expensive, producing in enormous quantities needs more pillar, the production cost superelevation, and lock out operation can't automatization, and the production cycle is longer.And column chromatography need expend a large amount of acid-alkali regeneration resins, causes secondary pollution, and energy consumption is large, cost is high.This method not too is fit to large-scale polysaccharide raw material preparation, only is fit to laboratory and medium and small test run and uses.
Most of extracting method are in order to improve the yield of polysaccharide, can take to extract under the meta-alkalescence condition, afterwards again by acid adjustment neutralization, as adopting the highly basic such as sodium hydroxide, potassium hydroxide to extract in the disclosed patent " extracting method of spirulina polysaccharide " in 1994 (Granted publication number be CN1056852C); Studied the relative tumour appreciation rate T/C(% of spirulina water extraction polysaccharide in 2012 disclosed patent " application of spirulina polysaccharide " (application number 201210134354.6) in the pharmacodynamics test of S180 mouse transplantation model) be higher than the spirulina alkali-extracted polysaccharide, that is to say that extraction obtains under the neutrallty condition polysaccharide is better than under the alkaline condition the restraining effect of tumour.Although the extracting method of basic solvent can improve the yield of extraction, destroy the structure of polysaccharide when the pH value is higher, finally can not get the strongest active polysaccharide, advantage that can not complete complete performance spirulina polysaccharide.Disclosed patent in 1994 " is extracted the method for protein and polysaccharide " and is used low temperature to soak in (Granted publication number be CN1036067C) to extract and obtains simultaneously cyanophycin and spirulina polysaccharide from spirulina, although the method is simple, extraction efficiency is low but low temperature soaks extraction, and the spirulina content of gained is lower.The middle refrigerated centrifugation that uses of disclosed patent " a kind of extracting method of water-soluble spirulina polysaccharide " (publication number is CN101575626A) in 2008, the method of acid adjustment and enzymolysis obtains spirulina polysaccharide, the method improves the yield of polysaccharide by enzymatic protein.But enzyme is expensive, and production cost increases.It is water miscible polypeptide or water-soluble amino acids that enzymolysis can cause proteolysis.Because molecular weight approaches, and water-soluble equally with polysaccharide, can't effective separation, cause purity of polysaccharide to reduce.And the condition that enzyme extraction method requires is harsh, and inactivation requires height to experimental installation easily, is not suitable for large-scale industrial production.
Although the method for the spirulina polysaccharide of reporting is a lot, cuts both ways.But do not have at present a kind ofly can to guarantee that product impurity is few, purity is high, high biological activity, cheaply, and can practice in the operational path of large production.For today that food and drug safety sex consciousness benefit increase, quality and cost all are the lifelines of enterprise.A kind ofly can apply in the suitability for industrialized production, activity, the production operation that can effectively guarantee spirulina polysaccharide is simple, free of contamination to environment and product, product yield and the high extracting method of purity, to guarantee that product quality and cost are necessary, also be enterprise in the urgent need to.
Summary of the invention
The object of the present invention is to provide that a kind of economy, save energy, non-environmental-pollution, safety non-toxic, extraction yield are high, purity is high, the high-titer of retains biological activity composition, can carry out the spirulina polysaccharide extracting method of suitability for industrialized production.
Technical scheme of the present invention is:
The extracting method that obtains simultaneously the spirulina polysaccharide of multiple pureness specifications carries out according to the following steps:
(1) preparation of spirulina Crude polysaccharides
(1) gets spirulina, add 95% ethanol that 4-10 doubly measures, refluxing extraction 1 ~ 3 time, each 1 ~ 3 hour, the ethanol that inclines, solids dries, adding entry 4-20 doubly measures, extract 1 ~ 5 time, each 2 ~ 10 hours, extracting temperature was 60 ~ 100 ℃, united extraction liquid, centrifugal under≤15000 r/min rotating speeds, supernatant liquor merges, and is for subsequent use;
(2) get above-mentioned (1) and obtain the supernatant liquor amalgamation liquid, cross≤filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand above molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in ultrafilter effluent liquid and the ultrafilter, dry, obtain the water extraction spirulina Crude polysaccharides more than a kind, total sugar content 〉=10%, molecular weight are more than 0.5 ten thousand;
(2) preparation of high purity spirulina polysaccharide
(1) gets spirulina, add 95% ethanol that 4-10 doubly measures, refluxing extraction 1 ~ 3 time, each 1 ~ 3 hour, the ethanol that inclines, solids dries, adding entry 4-20 doubly measures, extract 1 ~ 5 time, each 2 ~ 10 hours, extracting temperature was 60 ~ 100 ℃, united extraction liquid, centrifugal under≤15000 r/min rotating speeds, supernatant liquor merges, and is for subsequent use;
(2) get above-mentioned (1) and obtain the supernatant liquor amalgamation liquid, concentrated, removal of impurities is processed, solution after the processing is centrifugal under≤15000 r/min rotating speeds, gets supernatant liquor, the filtering with microporous membrane of mistake≤1.5 μ m, filtrate is added in the ultrafilter, select the filter material between 0.5 ten thousand to 200,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in ultrafilter effluent liquid and the ultrafilter, dry, obtain the water extraction spirulina polysaccharide more than a kind, total sugar content 〉=40%, molecular weight are 0.5 ten thousand ~ 200,000.
(3) above-mentioned concentrated, can be to utilize heating to make supernatant liquor amalgamation liquid volume be reduced to below 5 times of spirulina charging capacity.Or with the filtering with microporous membrane of supernatant liquor amalgamation liquid by≤1.5 μ m, filtrate is added in the ultrafilter, select the above filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.
(4) above-mentioned removal of impurities is processed, and can be to add 50 ~ 95% ethanol to alcoholic degree to reach 40-90% in enriched material, stirs evenly, and leaves standstill 12 ~ 72 hours, and centrifugal under≤15000 r/min rotating speeds, collecting precipitation is dissolved in water.Also can in enriched material, add protein precipitant, heat 0.5 ~ 3 hour, in≤5 ℃ of lower refrigerations 12 ~ 72 hours, the centrifugation precipitation adds protein precipitant again in the supernatant liquor under≤15000 r/min rotating speeds, heating, refrigeration, this repeatedly extremely separates out without obvious sediment except miscellaneous operation.The aqueous sodium hydroxide solution of supernatant liquor adding 20% or 10% hydrochloric acid soln adjust pH are to neutral.Described protein precipitant can use wherein one or more of aqueous sodium hydroxide solution of ammoniumsulphate soln or the 0.1-10% of 1-50% trichloroacetic acid solution or 0.1-50%.
Above-mentioned ultrafiltration can be according to the required different molecular weight ranges of product, and the concentrated solution that ultrafiltration is obtained carries out ultrafiltration more repeatedly.When continuous ultrafiltration operated more than 2 times, the ultrafilter effluent liquid was the concentration components between the two kinds of molecular weight in front and back.Concentrated solution in the ultrafilter is the polysaccharide component of selecting molecular weight above.
Step of the present invention (1) is extraction process: utilize alcohol reflux to remove fat-soluble component, improve the extraction yield of polysaccharide.Utilize water directly to extract, do not change the potential of hydrogen of solvent, do not affect the structure of polysaccharide, obtain the strongest active polysaccharide.Step (2) is utilized the solid matter in centrifugal and the filtering with microporous membrane removal solution, the efficient of Effective Raise ultrafiltration.By centrifugal, the millipore filtration of first and the coupling of ultrafiltration, obtain total sugar content 〉=10%, molecular weight is polysaccharide and the water soluble protein mixture more than 0.5 ten thousand, protein in this part also has certain physiologically active, so this mixture can be used as the raw material of the products such as the food that requires composition nutritious, healthcare products, makeup.Compare other traditional thick extracting methods, the method is simple to operate, does not need heating also not need to add reagent, has kept as much as possible the activity of polysaccharide and water soluble protein.Also can obtain in addition low-molecular-weight such as other water-soluble addition products such as water-soluble vitaminss in the leaching process.
If need further to the purity of polysaccharide classification, will contain the solution of crude product, process by removal of impurities, remove protein, improve the content of spirulina polysaccharide.Can generate the salt such as sodium trichloroacetate, sodium-chlor in the process of isolating protein, leave in addition the protein precipitants such as excessive trichoroacetic acid(TCA), ammonium sulfate.Insoluble impurities or the macro-molecular protein colloid of sex change are removed in centrifugal, millipore filtration.Utilize the scope of holding back of ultrafiltration filter material film different molecular weight, from water and other liquid, isolate very little colloid and macromole.Remove whereby the low-molecular-weight water-soluble impurities such as sodium trichloroacetate, sodium-chlor, trichoroacetic acid(TCA), ammonium sulfate, play the effect of desalination.By the enriched material of the macromolecule that stopped, can also use molecular weight larger the film material continue ultrafiltration, play classification and inspissated, finally obtain total sugar content 〉=40%, the high purity polysaccharide of polymolecular weight range.This technique can be produced as the raw material to the demanding healthcare products of material purity and medicine.
The present invention compared with prior art has following advantage:
1, the polysaccharide of traditional separation method acquisition need to detect just and can know general molecular weight ranges, and is the mixture of various molecular weight; And the present invention can select rational treatment measures in the different stage of operational path according to the demand different to product purity, do not need to detect the high purity polysaccharide that can obtain the predetermined molecular weight specification, and can obtain simultaneously plurality of specifications.This is the advantage that other traditional methods can not reach.
2, utilize alcohol reflux to remove the pre-treatment of the fat-soluble component in the spirulina, when having guaranteed the water extraction polysaccharide, impurity reduces, and is more conducive to the extraction of polysaccharide.Utilize water from spirulina, to extract polysaccharide, by the control of temperature, guaranteed that spirulina polysaccharide is not destroyed.The yield in extraction stage and alkaline process extraction yield approach, and far above low-temperature extraction method.The active polysaccharide that obtains is higher than potass extraction.
3, with after the coupling processing of spirulina aqueous extract by centrifugal, micro-filtration, ultrafiltration, obtain containing the more polysaccharide crude extract of protein: compare traditional thick extraction, in the operating process without high temperature, without chemical transformation, guarantee that as far as possible polysaccharide and protein active do not have destroyed in the leaching process, keep active constant, and composition is highly enriched.Also can obtain simultaneously the addition product of other low macromolecule water-solubility compositions.
4, after the spirulina aqueous extract concentrates, utilize ethanol, trichoroacetic acid(TCA), sodium hydroxide, ammonium sulphite to make protein denaturation, by the coupling of centrifugal, micro-filtration, ultrafiltration, insoluble impurities or the macro-molecular protein colloid of sex change are removed in first centrifugal, millipore filtration.Utilize the scope of holding back of ultrafiltration filter material film different molecular weight, remove the isolating protein process and cross the salt of the small molecular weight of generation, such as salt such as sodium trichloroacetate, sodium-chlor, even the unnecessary protein precipitant that molecular weight is lower such as the also in the lump removal such as trichoroacetic acid(TCA), ammonium sulphite.Impurity-eliminating effect is thorough, process safety, pollution-free, noresidue.Do not increase new impurity yet, do not have the reagent residue problem of conventional organic solvents method yet.The purity of polysaccharide that obtains high, specification is many, the biological activity of not destroying polysaccharide.This inventive method has effectively remedied conventional chemical isolating protein method and has left the defective that residue is difficult for eliminating.
5, traditional separation method is because the pillar filler is expensive, and the column chromatography operation is discontinuous, is difficult to suitability for industrialized production.The present invention is by the coupling of centrifugal, micro-filtration, ultrafiltration, owing to being subjected to the inhibition of osmotic pressure little, so under quite low pressure difference, still have high flow rate.Therefore, produce the continuous and automatic operation, it is low, with short production cycle to consume energy.Ultra-filtration membrane material itself is nontoxic, it is narrow to have the pore size distribution rate, and separation accuracy is high, and anti-microbe ability is strong, chemical stability is good, physical strength is large, and film is easily regenerated, long service life, the advantage such as easy to clean, therefore reduced production cost, it is convenient to bring to production operation, also can remedy the shortcomings of additive method.
6, the present invention can select the ultrafiltration filter material of different molecular weight shut off value, realize the further grading purification of polysaccharide, has the separation efficiency height, have no side effect, equipment is simple, the advantage such as easy and simple to handle, classification can be carried out continuously: as carrying out first the ultrafiltration of 10,000 molecular weight, ultrafiltration and concentration liquid passes through the ultrafiltration of 20,000 molecular weight again, again with the ultrafiltration of ultrafiltration and concentration liquid by 50,000 molecular weight, again with the ultrafiltration of ultrafiltration and concentration liquid by 80,000 molecular weight, with the ultrafiltration of ultrafiltration and concentration liquid by 100,000 molecular weight, finally can obtain 1-2 ten thousand again, 2-5 ten thousand, 5-8 ten thousand, the polyoses extract of 8-10 ten thousand these four kinds of molecular weight ranges.The present invention can obtain the highly purified spirulina polysaccharide of different kinds of molecules specification simultaneously, and this is that extraction and separation method was not available in the past.
7, chemistry removes albumen and centrifugal-millipore filtration-ultra-filtration technique coupling among the present invention, so that separating obtained polysaccharide yield is high, purity is high, the high-titer of retains biological activity composition.Compare gel filtration chromatography and resinbed and the process for purification commonly used such as analyse, obviously shortening of production cycle, cost and energy consumption is lower, equipment is simple, pollution-free, noresidue.Production operation is simple, but continuous automatic production is more suitable for the needs of suitability for industrialized production.
8, spirulina is swum to float in the water body and is produced, and has absorbed the microorganism in the more water, although also can make the part bacteria inactivation rate in the leaching process.But lingering section can make extracting solution apt to deteriorate, even residually in product the very fast microorganism of product is exceeded standard.The polysaccharide of traditional technology system need to reduce microorganism by means such as high temperature, ultraviolet ray, radiation.And the present invention does not need separately product to be sterilized, millipore filtration and ultra-filtration process itself can effectively be removed microorganism in the product, can suppress virus, bacterium, thermal source, endotoxic generation in the environment, reduce the chance of the finished product microbial contamination, the product stability that strengthens prolongs the storage life.This is that other extraction and separation method are not available.
In a word, require and can obtain multi-level, high-quality product two aspects from the production environment of economy, cleaning, environmental protection, method of the present invention is fit to the large production of spirulina polysaccharide crude extract or extract and uses very much.
Embodiment
Following examples are used for explanation the present invention, but the invention is not restricted to embodiment.
Embodiment 1
Get spirulina 100kg, add 95% ethanol of 4 times of amounts, refluxing extraction 1 time, each 1 hour, the ethanol that inclines, solids dries, and adds 4 times of amounts of entry, extract 1 time, extracted 2 hours, extracting temperature is 60 ℃, and extracting solution is centrifugal under 15000 r/min rotating speeds, supernatant liquor, cross the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, drying obtains water extraction spirulina Crude polysaccharides 17kg, total sugar content 12%, molecular weight are more than 0.5 ten thousand.
Embodiment 2
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, and adds 20 times of amounts of entry, extract 5 times, each 10 hours, extracting temperature was 100 ℃, united extraction liquid, centrifugal under 9000 r/min rotating speeds, supernatant liquor merges, and crosses the filtering with microporous membrane of 1.2 μ m, and filtrate is added in the ultrafilter, select the filter material of 10,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Dry respectively, obtain the water extraction spirulina Crude polysaccharides 8kg of molecular weight more than 50,000, total sugar content 15%; The water extraction spirulina Crude polysaccharides 10kg that molecular weight 1-5 is ten thousand, total sugar content 25%.
Embodiment 3
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, add 20 times of amounts of entry, extract 5 times, each 10 hours, extracting temperature was 100 ℃, united extraction liquid, centrifugal under 15000 r/min rotating speeds, supernatant liquor merges, and heating makes its volume be reduced to 5 times of spirulina charging capacity.Ethanol to the alcoholic degree of adding 50% reaches 40% in concentrated solution, stirs evenly, and leaves standstill 12 hours, siphons away supernatant liquor, and collecting precipitation is dissolved in water.Centrifugal under the 15000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, dry, obtain water extraction spirulina polysaccharide 1.7kg, total sugar content 〉=55%, molecular weight are more than 0.5 ten thousand.
Embodiment 4
Get spirulina 100kg, add 95% ethanol of 4 times of amounts, refluxing extraction 1 time, each 1 hour, the ethanol that inclines, solids dries, and adds 4 times of amounts of entry, extract 1 time, extracted 2 hours, extracting temperature is 60 ℃, and extracting solution is centrifugal under 3000 r/min rotating speeds, supernatant liquor merges, and heating makes its volume be reduced to 1 times of spirulina charging capacity.Ethanol to the alcoholic degree of adding 95% reaches 90% in concentrated solution, stirs evenly, and leaves standstill 72 hours, siphons away supernatant liquor, and collecting precipitation is dissolved in water.Centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.2 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Dry respectively, obtain total sugar content 78%, molecular weight is 0.5 ten thousand-50,000 water extraction spirulina polysaccharide 1kg; Total sugar content 72%, molecular weight are the water extraction spirulina polysaccharide 0.6kg more than 50,000.
Embodiment 5
Get spirulina 100kg, add 95% ethanol of 4 times of amounts, refluxing extraction 1 time, each 1 hour, the ethanol that inclines, solids dries, add 4 times of amounts of entry, extract 4 times, each 2 hours, extracting temperature was 60 ℃, united extraction liquid, centrifugal under 3000 r/min rotating speeds, supernatant liquor merges, and heating makes its volume be reduced to 3 times of spirulina charging capacity.The trichloroacetic acid solution of adding 50% in enriched material, heated 0.5 hour, in 1 ℃ of lower refrigeration 72 hours, centrifugal under the 9000r/min rotating speed, the trichloroacetic acid solution heated and stirred of supernatant liquor adding 10% 1 hour, in 1 ℃ of lower refrigeration 72 hours, centrifugal under the 9000r/min rotating speed, without Precipitation, supernatant liquor adds 10% aqueous sodium hydroxide solution adjust pH to neutral.Solution is centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 0.05 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 100,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.8kg of total sugar content 78%, 0.5 ten thousand-50,000 molecular weight; Total sugar content 85%, the water extraction spirulina polysaccharide 0.3kg of 5-10 ten thousand molecular weight; Total sugar content 75%, molecular weight are the water extraction spirulina polysaccharide 0.56kg more than 100,000.
Embodiment 6
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, add 20 times of amounts of entry, extract 5 times, each 10 hours, extracting temperature was 100 ℃, united extraction liquid, centrifugal under 9000 r/min rotating speeds, supernatant liquor merges, and heating makes its volume be reduced to 2 times of spirulina charging capacity.The ammoniumsulphate soln of adding 50% in enriched material, heated 3 hours, in 5 ℃ of lower refrigerations 12 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 30% ammoniumsulphate soln, heats 2 hours, in 5 ℃ of lower refrigerations 12 hours, centrifugal under the 9000r/min rotating speed, without Precipitation, supernatant liquor adds 10% aqueous sodium hydroxide solution adjust pH to neutral.Solution is centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 0.05 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 100,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.5kg of total sugar content 78%, 0.5 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.5kg of 5-10 ten thousand molecular weight; Total sugar content 65%, molecular weight are the water extraction spirulina polysaccharide 0.6kg more than 100,000.
Embodiment 7
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, add 20 times of amounts of entry, extract 5 times, each 10 hours, extracting temperature was 100 ℃, united extraction liquid, centrifugal under 9000 r/min rotating speeds, supernatant liquor merges, and heating makes its volume be reduced to 4 times of spirulina charging capacity.The aqueous sodium hydroxide solution of adding 20% heated 0.5 hour in enriched material, in 1 ℃ of lower refrigeration 72 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 10% aqueous sodium hydroxide solution, heats 1 hour, in 1 ℃ of lower refrigeration 48 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 0.1% aqueous sodium hydroxide solution, heats 3 hours, in 1 ℃ of lower refrigeration 24 hours, centrifugal under the 9000r/min rotating speed, without Precipitation, supernatant liquor adds 10% hydrochloric acid soln adjust pH to neutral.Solution is centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 80,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.Re-use the above filter material of 100,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.5kg of total sugar content 78%, 0.5 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.5kg of 5-8 ten thousand molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.3kg of 8-10 ten thousand molecular weight, total sugar content 65%, molecular weight are water extraction spirulina polysaccharide 0.2 kg more than 100,000.
Embodiment 8
Get spirulina 100kg, add 95% ethanol of 4 times of amounts, refluxing extraction 1 time, each 1 hour, the ethanol that inclines, solids dries, add 4 times of amounts of entry, extract each 2 hours 3 times, extracting temperature is 60 ℃, and united extraction liquid is centrifugal under 3000 r/min rotating speeds, supernatant liquor merges, and by the filtering with microporous membrane of 0.05 μ m, filtrate is added in the ultrafilter, select the filter membrane of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.The trichloroacetic acid solution of adding 1% in enriched material, heated 0.5 hour, in 1 ℃ of lower refrigeration 12 hours, centrifugal under the 15000r/min rotating speed, the trichloroacetic acid solution heating of supernatant liquor adding 1% 0.5 hour, in 1 ℃ of lower refrigeration 12 hours, centrifugal under the 15000r/min rotating speed, without Precipitation, supernatant liquor adds 10% aqueous sodium hydroxide solution adjust pH to neutral.Solution is centrifugal under the 15000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 100,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.6kg of total sugar content 78%, 0.5 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.4kg of 5-10 ten thousand molecular weight; Total sugar content 65%, molecular weight are the water extraction spirulina polysaccharide 0.6kg more than 100,000.
Embodiment 9
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, add 20 times of amounts of entry, extract each 10 hours 5 times, extracting temperature is 100 ℃, and united extraction liquid is centrifugal under 9000 r/min rotating speeds, supernatant liquor merges, and by the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter membrane of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.The ammoniumsulphate soln of adding 0.1% in enriched material, heated 3 hours, in 5 ℃ of lower refrigerations 72 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 0.1% ammoniumsulphate soln, heats 3 hours, in 5 ℃ of lower refrigerations 72 hours, centrifugal under the 9000r/min rotating speed, without Precipitation, supernatant liquor adds 10% aqueous sodium hydroxide solution adjust pH to neutral.Solution is centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.2 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 100,000 molecular weight concentrated solution is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.5kg of total sugar content 78%, 0.5 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.5kg of 5-10 ten thousand molecular weight; Total sugar content 65%, molecular weight are the water extraction spirulina polysaccharide 0.6kg more than 100,000.
Embodiment 10
Get spirulina 100kg, add 95% ethanol of 10 times of amounts, refluxing extraction 3 times, each 3 hours, the ethanol that inclines, solids dries, add 20 times of amounts of entry, extract each 10 hours 5 times, extracting temperature is 100 ℃, and united extraction liquid is centrifugal under 9000 r/min rotating speeds, supernatant liquor merges, and by the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter membrane of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.The aqueous sodium hydroxide solution of adding 20% heated 1 hour in enriched material, in 5 ℃ of lower refrigerations 72 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 10% aqueous sodium hydroxide solution, heats 2 hours, in 5 ℃ of lower refrigerations 72 hours, centrifugal under the 9000r/min rotating speed, supernatant liquor adds 0.1% aqueous sodium hydroxide solution, heats 3 hours, in 5 ℃ of lower refrigerations 72 hours, centrifugal under the 9000r/min rotating speed, without Precipitation, supernatant liquor adds 10% hydrochloric acid soln adjust pH to neutral.Solution is centrifugal under the 9000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 10,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 80,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.Re-use the above filter material of 100,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect concentrated solution and effluent liquid in the ultrafilter.Difference is dry, obtains the water extraction spirulina polysaccharide 0.5kg of total sugar content 78%, 1 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.5kg of 5-8 ten thousand molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.3kg of 8-10 ten thousand molecular weight; Total sugar content 65%, molecular weight are water extraction spirulina polysaccharide 0.2 kg more than 100,000.
Embodiment 11
Get spirulina 100kg, add 95% ethanol of 8 times of amounts, refluxing extraction 2 times, each 2 hours, the ethanol that inclines, solids dries, add 12 times of amounts of entry, extract each 6 hours 3 times, extracting temperature is 80 ℃, and united extraction liquid is centrifugal under 6000 r/min rotating speeds, supernatant liquor merges, and by the filtering with microporous membrane of 0.45 μ m, filtrate is added in the ultrafilter, select the filter membrane of 10,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.With half of concentrated solution, drying, obtain the above water extraction spirulina Crude polysaccharides 9.0kg of total sugar content 20%, 1 ten thousand molecular weight;
In other half concentrated solution, add 10% aqueous sodium hydroxide solution, heated 1 hour, in 2 ℃ of lower refrigerations 24 hours, centrifugal under the 6000r/min rotating speed, supernatant liquor adds 10% aqueous sodium hydroxide solution, heats 1 hour, in 2 ℃ of lower refrigerations 24 hours, centrifugal under the 6000r/min rotating speed, without Precipitation, supernatant liquor adds 10% hydrochloric acid soln adjust pH to neutral.Solution is centrifugal under the 6000r/min rotating speed, get supernatant liquor, merge, cross the filtering with microporous membrane of 0.8 μ m, filtrate is added in the ultrafilter, select the filter material of 10,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 80,000 molecular weight partial concentration liquid is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter, re-use the above filter material of 50,000 molecular weight the effluent liquid of ultrafilter is carried out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.Distinguish dry each several part concentrated solution, obtain the water extraction spirulina polysaccharide 0.3kg of total sugar content 78%, 1 ten thousand-50,000 molecular weight; Total sugar content 80%, the water extraction spirulina polysaccharide 0.3kg of 5-8 ten thousand molecular weight; Total sugar content 65%, molecular weight water extraction spirulina polysaccharide 0.2 kg more than 80,000.

Claims (7)

1. the extracting method of a spirulina polysaccharide is characterized in that carrying out according to the following steps:
(1) gets spirulina, add 95% ethanol that 4-10 doubly measures, refluxing extraction 1 ~ 3 time, each 1 ~ 3 hour, the ethanol that inclines, solids dries, adding entry 4-20 doubly measures, extract 1 ~ 5 time, each 2 ~ 10 hours, extracting temperature was 60 ~ 100 ℃, united extraction liquid, centrifugal under≤15000 r/min rotating speeds, supernatant liquor merges, and is for subsequent use;
(2) get above-mentioned (1) and obtain the supernatant liquor amalgamation liquid, cross≤filtering with microporous membrane of 1.5 μ m, filtrate is added in the ultrafilter, select the filter material of 0.5 ten thousand above molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in ultrafilter effluent liquid and the ultrafilter, dry, obtain the water extraction spirulina Crude polysaccharides more than a kind, total sugar content 〉=10%, molecular weight are more than 0.5 ten thousand;
A kind of extracting method of spirulina polysaccharide is characterized in that carrying out according to the following steps:
(1) gets spirulina, add 95% ethanol that 4-10 doubly measures, refluxing extraction 1 ~ 3 time, each 1 ~ 3 hour, the ethanol that inclines, solids dries, adding entry 4-20 doubly measures, extract 1 ~ 5 time, each 2 ~ 10 hours, extracting temperature was 60 ~ 100 ℃, united extraction liquid, centrifugal under≤15000 r/min rotating speeds, supernatant liquor merges, and is for subsequent use;
(2) get above-mentioned (1) and obtain the supernatant liquor amalgamation liquid, concentrated, removal of impurities is processed, solution after the processing is centrifugal under≤15000 r/min rotating speeds, gets supernatant liquor, the filtering with microporous membrane of mistake≤1.5 μ m, filtrate is added in the ultrafilter, select the filter material between 0.5 ten thousand to 200,000 molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in ultrafilter effluent liquid and the ultrafilter, dry, obtain the water extraction spirulina polysaccharide more than a kind, total sugar content 〉=40%, molecular weight are 0.5 ten thousand ~ 200,000.
2. the extracting method of spirulina polysaccharide according to claim 2 is characterized in that described concentrating is that heating makes supernatant liquor amalgamation liquid volume be reduced to below 5 times of spirulina charging capacity.
3. the extracting method of spirulina polysaccharide according to claim 2, it is characterized in that described concentrating is with the filtering with microporous membrane of supernatant liquor amalgamation liquid by≤1.5 μ m, filtrate is added in the ultrafilter, select the above filter material of 0.5 ten thousand molecular weight to carry out ultrafiltration, when going out to ultrafilter outlet aneroid, collect the concentrated solution in the ultrafilter.
4. the extracting method of the spirulina polysaccharide described in according to claim 4, it is characterized in that according to the required different molecular weight ranges of product, the concentrated solution that ultrafiltration is obtained carries out ultrafiltration more repeatedly, when continuous ultrafiltration operates more than 2 times, the ultrafilter effluent liquid is the concentration components between the two kinds of molecular weight in front and back, and the concentrated solution in the ultrafilter is the polysaccharide component of selecting molecular weight above.
5. the extracting method of spirulina polysaccharide according to claim 2 is characterized in that it is to add 50 ~ 95% ethanol to alcoholic degree to reach 40-90% in enriched material that removal of impurities is processed, and stirs evenly, left standstill 12 ~ 72 hours, centrifugal under≤15000 r/min rotating speeds, collecting precipitation is dissolved in water.
6. the extracting method of spirulina polysaccharide according to claim 2, it is characterized in that it is to add protein precipitant in enriched material that removal of impurities is processed, heated 0.5 ~ 3 hour, in≤5 ℃ of lower refrigerations 12 ~ 72 hours, the centrifugation precipitation added protein precipitant again in the supernatant liquor under≤15000 r/min rotating speeds, heating, refrigeration, this repeatedly extremely separates out without obvious sediment except miscellaneous operation, and the aqueous sodium hydroxide solution of supernatant liquor adding 20% or 10% hydrochloric acid soln adjust pH are to neutral.
7. the extracting method of spirulina polysaccharide according to claim 7 is characterized in that described protein precipitant is wherein one or more of aqueous sodium hydroxide solution of ammoniumsulphate soln or the 0.1-20% of 1-50% trichloroacetic acid solution or 0.1-50%.
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CN103694370A (en) * 2013-12-31 2014-04-02 福建省神六保健食品有限公司 Method for preparing spirulina polysaccharides
CN103819577A (en) * 2014-03-24 2014-05-28 福州大学 Method for preparing spirulina platensis polysaccharide
CN103951762A (en) * 2014-05-23 2014-07-30 吉林农业大学 Extraction and separation method of extracellular scleroglucan
CN104311685A (en) * 2014-10-21 2015-01-28 汕头大学 Spirulina phatensis polysaccharide and extraction method thereof
CN105294870A (en) * 2015-06-09 2016-02-03 深圳海王药业有限公司 Spriulina polysacchride and preparation method thereof
CN108084291A (en) * 2018-01-19 2018-05-29 贵州独秀峰药业有限公司 The method that polysaccharide is extracted in dendrobium candidum
CN109593128A (en) * 2018-12-30 2019-04-09 张德智 Use the method for fresh spirulina industrialization coproduction phycocyanin, spirulina polysaccharide and protein feed
CN111904975A (en) * 2020-09-04 2020-11-10 佛山蓝强生物科技有限公司 Algal polysaccharide composition and preparation method and application thereof
CN114195907A (en) * 2021-10-20 2022-03-18 中国科学院南海海洋研究所 Low molecular weight spirulina polysaccharide and preparation method and application thereof

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CN103694370A (en) * 2013-12-31 2014-04-02 福建省神六保健食品有限公司 Method for preparing spirulina polysaccharides
CN103694370B (en) * 2013-12-31 2015-10-28 福建省神六保健食品有限公司 A kind of preparation method of spirulina polysaccharide
CN103819577B (en) * 2014-03-24 2016-05-04 福州大学 A kind of preparation method of spirulina polysaccharide
CN103819577A (en) * 2014-03-24 2014-05-28 福州大学 Method for preparing spirulina platensis polysaccharide
CN103951762A (en) * 2014-05-23 2014-07-30 吉林农业大学 Extraction and separation method of extracellular scleroglucan
CN104311685A (en) * 2014-10-21 2015-01-28 汕头大学 Spirulina phatensis polysaccharide and extraction method thereof
CN105294870A (en) * 2015-06-09 2016-02-03 深圳海王药业有限公司 Spriulina polysacchride and preparation method thereof
CN105294870B (en) * 2015-06-09 2017-07-25 深圳海王药业有限公司 A kind of spirulina polysaccharide and preparation method thereof
CN108084291A (en) * 2018-01-19 2018-05-29 贵州独秀峰药业有限公司 The method that polysaccharide is extracted in dendrobium candidum
CN109593128A (en) * 2018-12-30 2019-04-09 张德智 Use the method for fresh spirulina industrialization coproduction phycocyanin, spirulina polysaccharide and protein feed
CN109593128B (en) * 2018-12-30 2020-04-03 张德智 Method for industrial co-production of phycocyanin, spirulina polysaccharide and protein feed by using fresh spirulina
CN111904975A (en) * 2020-09-04 2020-11-10 佛山蓝强生物科技有限公司 Algal polysaccharide composition and preparation method and application thereof
CN114195907A (en) * 2021-10-20 2022-03-18 中国科学院南海海洋研究所 Low molecular weight spirulina polysaccharide and preparation method and application thereof

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