CN104479009A - Technology for separating phycocyanin in spirulina by using polyvinyl ether carboxylate through two aqueous phase extraction - Google Patents
Technology for separating phycocyanin in spirulina by using polyvinyl ether carboxylate through two aqueous phase extraction Download PDFInfo
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- CN104479009A CN104479009A CN201410682958.3A CN201410682958A CN104479009A CN 104479009 A CN104479009 A CN 104479009A CN 201410682958 A CN201410682958 A CN 201410682958A CN 104479009 A CN104479009 A CN 104479009A
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- phycocyanins
- polyvinyl ether
- ether carboxylate
- carboxylate salt
- spirulina
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
Abstract
The invention provides a technology for separating phycocyanin in spirulina by using polyvinyl ether carboxylate through two aqueous phase extraction, belonging to the technical field of biochemical separation engineering. The technology comprises the following steps: preparing polyvinyl ether carboxylate; carrying out two aqueous phase extraction on phycocyanin; and obtaining phycocyanin powder after concentrating and freeze-drying a phycocyanin aqueous solution, wherein the phycocyanin recovery rate can be 70-90%. The technology has the beneficial effects that the method is simple; the steps of a purification process are simplified by enriching and separating broken wall liquids of the spirulina by directly using polyvinyl ether carboxylate; the selectivity is high; the spectral purity of phycocyanin obtained through separation is A620/A280>2.7; the powder of fresh or dry spirulina can be adopted as the raw material, thus providing a technical method with the effect of solving the problem of high value and large scale utilization of spirulina resources.
Description
Technical field
The invention provides a kind of preparation method of polyvinyl ether carboxylate salt, for extracting Phycocyanins, C-from breaking wall of spirullina princeps clear liquid.Belong to biochemical separation engineering technical field.
Background technology
Spirulina phycocyanin is present in the phycobilisome of spirulina.Phycobilisome is that specific to red algae and blue-green algae, a kind of supramolecule catches photopigment complex body.Phycocyanins, C-color and luster is in bright blueness, bright-colored, is the first-selected pure natural pigment of food, senior eye shadow, lipstick.Phycocyanins, C-can also regulate and the multiple important enzyme required for synthesized human metabolism, has significantly anti-oxidant, anti-inflammatory effect, regulates human immune system, strengthens function of immune system.Highly purified Phycocyanins, C-with very strong fluorescence, the pure natural fluorescent reagent made, for clinical diagnose, the research field such as immunochemistry and biomedical engineering.
In China, annual spirulina whole nation ultimate production has reached 7000 tons.Yield of spirulina 1/3rd for direct export, other spirulina is used as healthcare products mainly with the form of algae powder, micro-algae algae sheet and perfusion capsule and is used.The mass-producing development and utilization of spirulina is in the elementary process segment, does not also have deep processed product.In spirulina, the extraction of Phycocyanins, C-is also in the laboratory study stage, is not suitable for the good processing method of suitability for industrialized production.At present, the highly purified Phycocyanins, C-commodity that market is sold are all from external import, expensive, and application is restricted.
Therefore, develop leaching process that is easy, Spirulina phycocyanin fast, improving Spirulina phycocyanin product purity is key issue urgently to be resolved hurrily in the mass-producing exploitation of current Spirulina phycocyanin.It is high that polymkeric substance double water-phase isolated protein has biocompatibility, and operational condition is gentle, is easy to carry out the advantages such as continuous operation.The patented technology of the Bi-aqueous extraction Phycocyanins, C-of the relevant preparation algae Phycocyanins, C-reported, such as Chinese patent CN103880950A adopts expensive ionic liquid to form double water-phase, increase production cost, CN102993297A, CN101891809A etc. adopt commercially available PEG to form polymkeric substance double water-phase, be separated the poor selectivity of Phycocyanins, C-, need and to saltout etc. that other extracts means conbined usage and just can obtain high purity product, process is loaded down with trivial details.
The invention provides a kind of polyvinyl ether carboxylate salt Phycocyanins, C-to specificity extracting power, by two-phase extraction mode direct separation and purification Phycocyanins, C-from breaking wall of spirullina princeps liquid, without the need to saltout etc. that other extracts means conbined usage, just can obtain high purity phycocyanin.
Summary of the invention
The object of this invention is to provide a kind of preparation method of polyvinyl ether carboxylate salt, for Bi-aqueous extraction Spirulina phycocyanin, obtain highly purified Phycocyanins, C-, be applied to the research and development of natural pigment, protective foods and new drug.
Technical problem to be solved by this invention is to provide a kind of preparation method of polyvinyl ether carboxylate salt, for extracting and separating Phycocyanins, C-from breaking wall of spirullina princeps liquid.Its method is simple, and fast, cost is low, and be separated Phycocyanins, C-and have higher purity, raw material both can adopt fresh spirulina also can adopt spirulina powder, thus provides a kind of method solving Spirulina phycocyanin resource extentization and utilize.
Technical scheme of the present invention: a kind of isolation technique of polyvinyl ether carboxylate salt aqueous two-phase extraction Spirulina phycocyanin, step is:
(1) preparation of polyvinyl ether carboxylate salt; (2) Bi-aqueous extraction Phycocyanins, C-; (3) the Phycocyanins, C-aqueous solution obtains Phycocyanins, C-powder through concentrated and lyophilize.
Described scheme, in step 1, 100g polyoxyethylene glycol and sodium block is put in round-bottomed flask, the weight ratio of sodium block and polyoxyethylene glycol is 0.02 ~ 0.05:1, reaction 5 ~ 8 hours under 25 DEG C ~ 45 DEG C and magnetic agitation condition, slowly instill dehydrated alcohol in the reactive mixture, until the sodium block in flask is eliminated, then the N of 200 ~ 500ml is added in the reactive mixture, dinethylformamide and 5.8 ~ 24g sodium chloroacetate, weight ratio between sodium chloroacetate and polyoxyethylene glycol is 0.06 ~ 0.24:1, react 15 ~ 24 hours at 90 ~ 120 DEG C, then vacuum distilling 1 hour at 80 ~ 100 DEG C, add 500 ~ 1000ml dehydrated alcohol, filter and obtain polyvinyl ether carboxylate salt.
Described scheme, in step 2, the preparation of breaking wall of spirullina princeps clear liquid: adopt water or phosphoric acid buffer to make extraction agent, the concentration of phosphoric acid buffer is 25 ~ 100mmol/L, the solid-to-liquid ratio of algae powder or fresh algae and extracting solution is 1:50 ~ 1:150, after stirring, at being placed in-10 DEG C ~-20 DEG C after freezing certain hour, 37 DEG C of dissolvings, multigelation like this 4-6 time, the broken wall clear liquid of centrifugal acquisition after melting, in broken wall clear liquid, protein concn is 1.0 ~ 5.6mg/ml, and Phycocyanins, C-purity is 0.5 ~ 0.72.
Described scheme, in step 2, adds polyvinyl ether carboxylate salt and inorganic salt in breaking wall of spirullina princeps clear liquid, polyvinyl ether carboxylate salt: inorganic salt: the weight ratio of broken wall clear liquid is 0.05 ~ 0.25:0.2 ~ 0.35:1, concussion mixing 2 minutes, leave standstill phase-splitting, Phycocyanins, C-is enriched in phase.To be 30000 daltonian ultrafiltration membrance filters through molecular weight cut-off containing Phycocyanins, C-upper, remove polyvinyl ether carboxylate salt and inorganic salt, obtain the Phycocyanins, C-aqueous solution, purity be 2.8 ~ 3.7, and protein concentration is 3.15 ~ 5.6mg/ml.
Described scheme, in step 3, to original volume 1/3rd, obtains Phycocyanins, C-powder by Phycocyanins, C-aqueous solution vacuum concentration at 60 DEG C after lyophilize.
Beneficial effect of the present invention: the present invention compared with prior art, mainly has the following advantages: the 1. extraction adsorption selectivity of polyvinyl ether carboxylate salt to Phycocyanins, C-is high, is applicable to the industrialization production requirements in different scales and place; 2. polyvinyl ether carboxylate salt directly can extract and obtain highly purified Phycocyanins, C-from breaking wall of spirullina princeps clear liquid, and extract means without the need to other and coordinate, production cost is low; 3. the raw material preparing Phycocyanins, C-can be the fresh algae of spirulina or dry algae powder, effectively solves the utilization of spirulina data and high-valued problem.
Embodiment
Embodiment 1
(1) preparation of breaking wall of spirullina princeps clear liquid
Get and originate in the spirulina plalensis dry powder that century-old biotechnology company limited is grant in Dongtai city, extraction agent is made with deionized water, the solid-to-liquid ratio of algae powder and deionized water is 1:50, at being placed in-20 DEG C after freezing 4 hours, 37 DEG C melt, multigelation like this 4 times, blue green algae disrupted solution whizzer after thawing under 6000 turns/min centrifugal 10 minutes, getting supernatant liquor is broken wall clear liquid, and in clear liquid, protein concn is 1.0mg/ml, Phycocyanins, C-purity A
620/ A
280for 0.72mg/ml.
(2) preparation of polyvinyl ether carboxylate salt
100g polyoxyethylene glycol PEG-1000 (purchased from Hai'an petroleum chemical plant) and 5g sodium block (purchased from Nanjing Chemistry Reagent Co., Ltd.) is put in the round-bottomed flask of 1000ml, reaction 5 hours under 25 DEG C and magnetic agitation condition, slowly instill dehydrated alcohol (purchased from Nanjing Chemistry Reagent Co., Ltd.) in the reactive mixture, until the sodium block in flask is eliminated, then the N of 200ml is added in the reactive mixture, dinethylformamide (purchased from Zhengzhou Chao An Chemical Co., Ltd.) and 5.8g sodium chloroacetate (purchased from Yancheng prize chemical industry company limited), react 15 hours at 90 DEG C, then vacuum distilling 1 hour at 80 DEG C, add 500ml dehydrated alcohol, filter and obtain polyvinyl ether carboxylate salt.
(3) two-phase extraction
0.5g polyvinyl ether carboxylate salt and 0.2g anhydrous magnesium sulfate (purchased from Nanjing Datang chemical industry limited liability company) is added in 10g breaking wall of spirullina princeps clear liquid, concussion mixing 2 minutes, leave standstill phase-splitting, Phycocyanins, C-is enriched in phase, upper is that 30000 daltonian ultra-filtration membranes (quite that strainer company limited) filter through molecular weight cut-off, removes polyvinyl ether carboxylate salt and magnesium sulfate, obtains the Phycocyanins, C-aqueous solution, purity is 2.8, and protein concentration is 3.15mg/ml.
(4) lyophilize obtains Phycocyanins, C-powder
The Phycocyanins, C-aqueous solution vacuum concentration at 60 DEG C obtained in abovementioned steps (3), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 2.9, protein recovery is 75%.
Embodiment 2
(1) preparation of breaking wall of spirullina princeps liquid
Get and originate in the fresh spirulina that Jiangsu great Feng grants century-old bio tech ltd, make extraction agent with 50mmol/L phosphoric acid buffer, the solid-to-liquid ratio of spirulina and deionized water is 1:100, and at being placed in-10 DEG C after freezing 4 hours, 37 DEG C melt, multigelation like this 5 times.Blue green algae disrupted solution whizzer after thawing under 6000 turns/min centrifugal 10 minutes, getting supernatant liquor is broken wall clear liquid, and in clear liquid, protein concn is 3.1mg/ml, Phycocyanins, C-purity A
620/ A
280be 0.5.
(2) preparation of polyvinyl ether carboxylate salt
100g polyoxyethylene glycol PEG-3000 (purchased from Hai'an petroleum chemical plant) and 3.5g sodium block (purchased from Nanjing Chemistry Reagent Co., Ltd.) is put in the round-bottomed flask of 1000ml, reaction 7 hours under 35 DEG C and magnetic agitation condition, slowly instill dehydrated alcohol (purchased from Nanjing Chemistry Reagent Co., Ltd.) in the reactive mixture, until the sodium block in flask is eliminated, then the N of 400ml is added in the reactive mixture, dinethylformamide (purchased from Zhengzhou Chao An Chemical Co., Ltd.) and 18g sodium chloroacetate (purchased from Yancheng prize chemical industry company limited), react 20 hours at 105 DEG C, then vacuum distilling 1 hour at 90 DEG C, add 200ml dehydrated alcohol, filter and obtain polyvinyl ether carboxylate salt.
(3) two-phase extraction
1.7g polyvinyl ether carboxylate salt and 3g anhydrous sodium sulphate (purchased from Nanjing Datang chemical industry limited liability company) is added in 10g breaking wall of spirullina princeps clear liquid, concussion mixing 2 minutes, leave standstill phase-splitting, Phycocyanins, C-is enriched in phase, by upper be that 30000 daltonian ultra-filtration membranes (quite you strainer company limited) filter through molecular weight cut-off, remove polyvinyl ether carboxylate salt and sodium sulfate, obtain the Phycocyanins, C-aqueous solution, purity is 3.3, and protein concentration is 4.6mg/ml.
(4) lyophilize obtains Phycocyanins, C-powder
The Phycocyanins, C-aqueous solution vacuum concentration at 60 DEG C obtained in abovementioned steps (3), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 3.6, protein recovery 55%.
Embodiment 3
(1) preparation of breaking wall of spirullina princeps liquid
Get the spirulina powder originating in spirulina base, Lijiang, yunnan Cheng Hai source, make extraction agent with 150mmol/L phosphoric acid buffer, the solid-to-liquid ratio of spirulina and deionized water is 1:150, and at being placed in-20 DEG C after freezing 4 hours, 37 DEG C melt, multigelation like this 6 times.Blue green algae disrupted solution whizzer after thawing under 6000 turns/min centrifugal 10 minutes, getting supernatant liquor is broken wall clear liquid, the Phycocyanins, C-purity A in clear liquid
620/ A
280be 0.77, shell-broken liquid protein concn is 4.6mg/ml.
(2) preparation of polyvinyl ether carboxylate salt
In the round-bottomed flask of 1000ml, put into 100g polyoxyethylene glycol PEG-4000 (purchased from Hai'an petroleum chemical plant) and 5g sodium block (purchased from Nanjing Chemistry Reagent Co., Ltd.), reaction 8 hours under 45 DEG C and magnetic agitation condition, slowly instill dehydrated alcohol (purchased from Nanjing Chemistry Reagent Co., Ltd.) in the reactive mixture, until the sodium block in flask is eliminated, then the N of 300ml is added in the reactive mixture, dinethylformamide (purchased from Zhengzhou Chao An Chemical Co., Ltd.) and 24g sodium chloroacetate (purchased from Yancheng prize chemical industry company limited), react 24 hours at 120 DEG C, then vacuum distilling 1 hour at 100 DEG C, add 500ml dehydrated alcohol, filter and obtain polyvinyl ether carboxylate salt.
(3) two-phase extraction
2.2g polyvinyl ether carboxylate salt and 3.2g anhydrous phosphoric acid potassium (purchased from Nanjing Datang chemical industry limited liability company) is added in 10g breaking wall of spirullina princeps clear liquid, concussion mixing 2 minutes, leave standstill phase-splitting, Phycocyanins, C-is enriched in phase, by upper be that 30000 daltonian ultra-filtration membranes (quite you strainer company limited) filter through molecular weight cut-off, remove polyvinyl ether carboxylate salt and potassiumphosphate, obtain the Phycocyanins, C-aqueous solution, purity is 3.7, and protein concentration is 5.6mg/ml.
(4) lyophilize obtains Phycocyanins, C-powder
The Phycocyanins, C-aqueous solution vacuum concentration at 60 DEG C obtained in abovementioned steps (3), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 3.7, protein recovery 75%.
Claims (3)
1. an isolation technique for polyvinyl ether carboxylate salt aqueous two-phase extraction Spirulina phycocyanin, is characterized in that step is: the preparation of polyvinyl ether carboxylate salt; Aqueous two-phase extraction Phycocyanins, C-; The Phycocyanins, C-aqueous solution is through concentrating and obtaining Phycocyanins, C-powder after lyophilize.
(1) preparation of polyvinyl ether carboxylate salt: put into polyoxyethylene glycol and sodium block in round-bottomed flask, the weight ratio of sodium block and polyoxyethylene glycol is 0.02 ~ 0.05:1, reaction 5 ~ 8 hours under 25 DEG C ~ 45 DEG C and magnetic agitation condition, slowly instill dehydrated alcohol in the reactive mixture, until the sodium block in flask is eliminated, then the N of 200 ~ 500ml is added in the reactive mixture, dinethylformamide and 5.8 ~ 24g sodium chloroacetate, react 15 ~ 24 hours at 90 ~ 120 DEG C, then vacuum distilling 1 hour at 80 ~ 100 DEG C, add 200 ~ 500ml dehydrated alcohol, filter and obtain polyvinyl ether carboxylate salt.
(2) two-phase extraction: add polyvinyl ether carboxylate salt and inorganic salt in breaking wall of spirullina princeps clear liquid, polyvinyl ether carboxylate salt: inorganic salt: the weight ratio of broken wall clear liquid is 0.05 ~ 0.25:0.2 ~ 0.35:1, concussion mixing 2 minutes, leave standstill phase-splitting, Phycocyanins, C-is enriched in phase.
(3) by above-mentioned be 30000 daltonian ultrafiltration membrance filters containing Phycocyanins, C-upper through molecular weight cut-off, remove polyvinyl ether carboxylate salt and inorganic salt, obtain the Phycocyanins, C-aqueous solution, purity is 2.8 ~ 3.7, and protein concentration is 3.15 ~ 5.6mg/ml.
(4) Phycocyanins, C-aqueous solution vacuum concentration at 60 DEG C, to original volume 1/3rd, is obtained Phycocyanins, C-powder after lyophilize.
2. the preparation method of polyvinyl ether carboxylate salt according to claim 1, is characterized in that the molecular-weight average of the polyoxyethylene glycol adopted can be 500,1000,2000,3000,4000.
3. two-phase extraction method according to claim 1, is characterized in that, can for anhydrous sodium sulphate, without the acid of water sulphur Magnesium, anhydrous slufuric acid ammonium, anhydrous chlorides of rase sodium, anhydrous phosphoric acid potassium for the inorganic salt extracted.
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Cited By (10)
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| CN105820237A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for two aqueous phase extraction separation of phycocyanin through dextran and sodium potassium tartrate |
| CN105820236A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through two-aqueous phase extraction of polyethylene glycol oleate/inorganic salt |
| CN105820238A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate |
| CN106366183A (en) * | 2016-11-30 | 2017-02-01 | 江南大学 | Separation technology for aqueous two-phase extraction of spirulina phycocyanin from polyvinylether phenylacetamide |
| CN106380513A (en) * | 2016-11-30 | 2017-02-08 | 江南大学 | Separation technique of Spirulina phycocyanin by polyvinylether palmic acid double-water-phase extraction |
| CN106432481A (en) * | 2016-11-30 | 2017-02-22 | 江南大学 | Separation technology for performing two aqueous phase extraction on spiral seaweed phycocyanin by octadecyl polyvinylether |
| CN106749630A (en) * | 2016-11-30 | 2017-05-31 | 江南大学 | A kind of isolation technics of polyvinylether benzoic ether aqueous two-phase extraction spirulina phycocyanin |
| CN107298710A (en) * | 2017-07-06 | 2017-10-27 | 浦江县欧立生物技术有限公司 | A kind of extracting method of spirulina phycocyanin |
| CN107312085A (en) * | 2017-07-06 | 2017-11-03 | 浦江县欧立生物技术有限公司 | A kind of phycocyanin blood sugar lowing polypeptide |
| CN107549553A (en) * | 2017-08-15 | 2018-01-09 | 浦江县美泽生物科技有限公司 | The preparation method of cyanophycin polypeptide compound |
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Cited By (10)
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| CN105820237A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for two aqueous phase extraction separation of phycocyanin through dextran and sodium potassium tartrate |
| CN105820236A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through two-aqueous phase extraction of polyethylene glycol oleate/inorganic salt |
| CN105820238A (en) * | 2016-04-18 | 2016-08-03 | 东台市赐百年生物工程有限公司 | Method for separating phycocyanin through aqueous two-phase extraction of methoxyl group polyethylene glycol/potassium sodium tartrate |
| CN106366183A (en) * | 2016-11-30 | 2017-02-01 | 江南大学 | Separation technology for aqueous two-phase extraction of spirulina phycocyanin from polyvinylether phenylacetamide |
| CN106380513A (en) * | 2016-11-30 | 2017-02-08 | 江南大学 | Separation technique of Spirulina phycocyanin by polyvinylether palmic acid double-water-phase extraction |
| CN106432481A (en) * | 2016-11-30 | 2017-02-22 | 江南大学 | Separation technology for performing two aqueous phase extraction on spiral seaweed phycocyanin by octadecyl polyvinylether |
| CN106749630A (en) * | 2016-11-30 | 2017-05-31 | 江南大学 | A kind of isolation technics of polyvinylether benzoic ether aqueous two-phase extraction spirulina phycocyanin |
| CN107298710A (en) * | 2017-07-06 | 2017-10-27 | 浦江县欧立生物技术有限公司 | A kind of extracting method of spirulina phycocyanin |
| CN107312085A (en) * | 2017-07-06 | 2017-11-03 | 浦江县欧立生物技术有限公司 | A kind of phycocyanin blood sugar lowing polypeptide |
| CN107549553A (en) * | 2017-08-15 | 2018-01-09 | 浦江县美泽生物科技有限公司 | The preparation method of cyanophycin polypeptide compound |
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