CN104479008A - Method for separating and purifying phycocyanin in spirulina by using polyacrylic acid/magnesium oxide hybrid microspheres - Google Patents
Method for separating and purifying phycocyanin in spirulina by using polyacrylic acid/magnesium oxide hybrid microspheres Download PDFInfo
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- CN104479008A CN104479008A CN201410681793.8A CN201410681793A CN104479008A CN 104479008 A CN104479008 A CN 104479008A CN 201410681793 A CN201410681793 A CN 201410681793A CN 104479008 A CN104479008 A CN 104479008A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F120/04—Acids; Metal salts or ammonium salts thereof
- C08F120/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
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- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
- C08K3/20—Oxides; Hydroxides
- C08K3/22—Oxides; Hydroxides of metals
- C08K2003/2217—Oxides; Hydroxides of metals of magnesium
- C08K2003/222—Magnesia, i.e. magnesium oxide
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Abstract
The invention provides a method for separating and purifying phycocyanin in spirulina by using polyacrylic acid/magnesium oxide hybrid microspheres, belonging to the technical field of biochemical separation engineering. The method comprises the following steps: preparing a polymerized emulsion; carrying out polymerization reaction to obtain polyacrylic acid/magnesium oxide hybrid microspheres; adsorbing phycocyanin from broken wall clear liquids of spirulina by using the polyacrylic acid/magnesium oxide hybrid microspheres; desorbing phycocyanin from the adsorption microspheres; concentrating and freeze-drying desorption liquids, thus finally obtaining phycocyanin with certain purity, wherein the phycocyanin recovery rate can be 70-80%. The method has the beneficial effects that the method is simple; the steps of a purification process are simplified by enriching and separating the broken wall liquids of the spirulina by directly using the polyacrylic acid/magnesium oxide hybrid microspheres; the selectivity is high; the spectral purity of phycocyanin obtained through separation is A620/A280>2.0; 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 the preparation method of a kind of polyacrylic acid/magnesium oxide heterozygosis microballoon, for concentration and separation Phycocyanins, C-method from breaking wall of spirullina princeps 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.The patented technology of the relevant preparation algae Phycocyanins, C-reported, the such as common trait of Chinese patent CN1106414A, CN1130028A, CN101003565A, CN00117512.2, CN100434528C etc. is that Phycocyanins, C-extracts and all needs through frond cell wall breaking, centrifugal or filter and remove cell debris and other solia particle, to saltout or iso-electric point slightly carries the stage, then the chromatography method such as spent ion exchange resin is purified, the poor specificity of the separation Phycocyanins, C-of carrier, process is loaded down with trivial details, product purity is low, and production cost is high.
The invention provides a kind of polyacrylic acid/magnesium oxide heterozygosis microballoon Phycocyanins, C-to specific adsorption, can adsorb from breaking wall of spirullina princeps liquid, be separated, purifying Phycocyanins, C-.Through finding prior art literature search, have not yet to see about polyacrylic acid/magnesium oxide heterozygosis microballoon is for the method for concentration and separation Spirulina phycocyanin.
Summary of the invention
The object of this invention is to provide the preparation method of a kind of polyacrylic acid/magnesium oxide heterozygosis microballoon, for separating of Spirulina phycocyanin, obtain the Phycocyanins, C-of higher degree, 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 the preparation method of a kind of polyacrylic acid/magnesium oxide heterozygosis microballoon, for fractionation by adsorption 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 the higher rate of recovery, 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: the preparation method of a kind of polyacrylic acid for separating of Spirulina phycocyanin/magnesium oxide heterozygosis microballoon, step is:
(1) preparation of polymerized emulsion; (2) carry out polyreaction and obtain polyacrylic acid/magnesium oxide heterozygosis microballoon; (3) from breaking wall of spirullina princeps clear liquid, Phycocyanins, C-is adsorbed with polyacrylic acid/magnesium oxide heterozygosis microballoon; (4) Phycocyanins, C-is from desorption adsorption microspheres; (5) desorption liquid obtains Phycocyanins, C-powder through concentrated and lyophilize.
Described scheme, in step 1, the diameter of magnesium oxide nanoparticle is 5 ~ 50nm, and nonionogenic tenside is sorbitan fatty(acid)ester Span-80, peregal AEO
9, in polyoxyethylene nonylphenol ether OP-10 any one, weight ratio between magnesium oxide nanoparticle and nonionogenic tenside is 1:0 ~ 2.5, vegetables oil comprises the one in soybean oil, Oleum Gossypii semen, tea-seed oil, rapeseed oil, sweet oil, sunflower seed oil, the weight ratio of nano particle and vegetables oil is 1:10 ~ 20, in acrylic acid aqueous solution, acrylic acid bulking value concentration is 20 ~ 70%, the volume ratio of acrylic acid aqueous solution and vegetables oil is 1:1 ~ 2, with Fluko FA25 type Homogenizing pump dispersion and emulsion, obtain w/o type milk sap.
Described scheme, in step 2, in above-mentioned milk sap, add the Sodium Persulfate (volumeter with acrylic acid aqueous solution) of 0.2 ~ 0.5%, stir 2 hours under 100 ~ 200r/min mixing speed at 30 DEG C ~ 40 DEG C, filter the polyacrylic acid/magnesium oxide heterozygosis microballoon obtaining 50 ~ 500nm size distribution.
Described scheme, in step 3, for the cell wall breaking of spirulina, water or phosphoric acid buffer is adopted to make extraction agent, the concentration of phosphoric acid buffer is 25 ~ 100mmol/L, and 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 3, in 500ml Erlenmeyer flask, add polyacrylic acid/magnesium oxide heterozygosis microballoon and breaking wall of spirullina princeps clear liquid, the weight ratio of microballoon and shell-broken liquid is 1:10 ~ 25, at ambient temperature, in rotating speed be 150r/min shaking table on shake absorption 40 ~ 90 minutes, the microballoon of collecting by filtration saturated adsorption Phycocyanins, C-.
Described scheme, in step 4, the pH value microballoon of saturated adsorption Phycocyanins, C-being placed in 100ml is 7.0, concentration is the phosphoric acid buffer of 500 ~ 1000mmol/L, in rotating speed be 150r/min shaking table on concussion 15 minutes, be product by collected by centrifugation supernatant liquor.
Described scheme, in step 5, contains Phycocyanins, C-elutriant vacuum concentration and lyophilize by what collect, namely obtains purity A
620/ A
280the Phycocyanins, C-powder of >2.0.
Beneficial effect of the present invention: the present invention compared with prior art, mainly have the following advantages: 1. by flow process: the operating method using polyacrylic acid/magnesium oxide heterozygosis microballoon to extract Phycocyanins, C-from breaking wall of spirullina princeps clear liquid is batch operation mode, be applicable to the industrialization production requirements in different scales and place; 2. polyacrylic acid/the adsorption selectivity of magnesium oxide heterozygosis microballoon to Phycocyanins, C-is high, and production cost is low, renewable, is applicable to heavy industrialization application; 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 1.0mg/ml.
(2) preparation of polyacrylic acid/magnesium oxide heterozygosis microballoon
In the round-bottomed flask of 1000ml, putting into the nano oxidized magnesium granules of 20g (can Lehner rice novel material company limited purchased from Boluo County, median size 50nm), the soybean oil (purchased from Zibo Kun Xing grain and oil company limited) of 200ml vinylformic acid (purchased from the Shanghai Shi Jian Industrial Co., Ltd.) aqueous solution and 200ml, in acrylic acid aqueous solution, acrylic acid bulking value concentration is 20%, with Fluko FA25 type Homogenizing pump dispersion and emulsion, add the Sodium Persulfate (purchased from Linyi City sky science, industry and trade company limited) of 0.4g, stir 2 hours under the mixing speed of 200r/min at 30 DEG C, filter the polyacrylic acid/magnesium oxide heterozygosis microballoon obtaining 50 ~ 500nm size distribution.
(3) microballoon adsorbs Phycocyanins, C-from breaking wall of spirullina princeps liquid
In 500ml Erlenmeyer flask, add 4g polyacrylic acid/magnesium oxide heterozygosis microballoon and 100ml breaking wall of spirullina princeps clear liquid, at ambient temperature, in rotating speed be 150r/min shaking table on shake absorption 40 minutes, the microballoon of collecting by filtration saturated adsorption Phycocyanins, C-.
(4) Phycocyanins, C-is from desorption microballoon
The pH value microballoon of saturated adsorption Phycocyanins, C-being placed in 100ml is 7.0, concentration is the phosphoric acid buffer of 500mmol/L, in rotating speed be 150r/min shaking table on concussion 15 minutes, with whizzer under 4000 turns/min centrifugal 10 minutes, get supernatant liquor.
(5) lyophilize obtains Phycocyanins, C-powder
The supernatant liquor vacuum concentration at 60 DEG C obtained in abovementioned steps (4), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 2.3, protein recovery is 80%.
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 0.45.
(2) preparation of polyacrylic acid/magnesium oxide heterozygosis microballoon
In the round-bottomed flask of 1000ml, putting into the nano oxidized magnesium granules of 20g (can Lehner rice novel material company limited purchased from Boluo County, median size 50nm) and 20g sorbitan fatty(acid)ester Span-80 (purchased from Jiangsu Hai'an Petrochemical Plant), vinylformic acid (purchased from the Shanghai Shi Jian Industrial Co., Ltd.) aqueous solution of 200ml and the rapeseed oil (purchased from Ya Mei bio tech ltd, Hunan) of 400ml, in acrylic acid aqueous solution, acrylic acid bulking value concentration is 50%, with Fluko FA25 type Homogenizing pump dispersion and emulsion, add the Sodium Persulfate (purchased from Linyi City sky science, industry and trade company limited) of 1.0g, stir 2 hours under the mixing speed of 100r/min at 30 DEG C, filter the polyacrylic acid/magnesium oxide heterozygosis microballoon obtaining 50 ~ 500nm size distribution.
(3) microballoon adsorbs Phycocyanins, C-from breaking wall of spirullina princeps liquid
In 500ml Erlenmeyer flask, add 4g polyacrylic acid/magnesium oxide heterozygosis microballoon and 40ml breaking wall of spirullina princeps clear liquid, at ambient temperature, in rotating speed be 150r/min shaking table on shake absorption 70 minutes, the microballoon of collecting by filtration saturated adsorption Phycocyanins, C-.
(4) Phycocyanins, C-is from desorption microballoon
The pH value microballoon of saturated adsorption Phycocyanins, C-being placed in 100ml is 7.0, concentration is the phosphoric acid buffer of 800mmol/L, in rotating speed be 150r/min shaking table on concussion 15 minutes, with whizzer under 4000 turns/min centrifugal 10 minutes, get supernatant liquor.
(5) lyophilize obtains Phycocyanins, C-powder
The supernatant liquor vacuum concentration at 60 DEG C obtained in abovementioned steps (4), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 3.5, protein recovery 85%.
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 100mmol/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 0.72, shell-broken liquid protein concn is 5.6mg/ml mg/ml.
(2) preparation of polyacrylic acid/magnesium oxide heterozygosis microballoon
In the round-bottomed flask of 1000ml, putting into the nano oxidized magnesium granules of 20g (can Lehner rice novel material company limited purchased from Boluo County, median size 50nm) and 25g polyoxyethylene nonylphenol ether OP-10 (purchased from Jiangsu Hai'an Petrochemical Plant), vinylformic acid (purchased from the Shanghai Shi Jian Industrial Co., Ltd.) aqueous solution of 200ml and the tea-seed oil (purchased from Ya Mei bio tech ltd, Hunan) of 300ml, in acrylic acid aqueous solution, acrylic acid bulking value concentration is 70%, with Fluko FA25 type Homogenizing pump dispersion and emulsion, add the Sodium Persulfate of 0.8g, stir 2 hours under the mixing speed of 150r/min at 40 DEG C, filter the JDN-8 microballoon obtaining 50 ~ 500nm size distribution.
(3) microballoon adsorbs Phycocyanins, C-from breaking wall of spirullina princeps liquid
In 500ml Erlenmeyer flask, add 4g polyacrylic acid/magnesium oxide heterozygosis microballoon and 60ml breaking wall of spirullina princeps clear liquid, at ambient temperature, in rotating speed be 150r/min shaking table on shake absorption 90 minutes, the microballoon of collecting by filtration saturated adsorption Phycocyanins, C-.
(4) Phycocyanins, C-is from desorption microballoon
The pH value microballoon of saturated adsorption Phycocyanins, C-being placed in 100ml is 7.0, concentration is the phosphoric acid buffer of 1000mmol/L, in rotating speed be 150r/min shaking table on concussion 15 minutes, with whizzer under 4000 turns/min centrifugal 10 minutes, get supernatant liquor.
(5) lyophilize obtains Phycocyanins, C-powder
The supernatant liquor vacuum concentration at 60 DEG C obtained in abovementioned steps (4), to original volume 1/3rd, is obtained Phycocyanins, C-powder product after lyophilize.Phycocyanins, C-purity A in product
620/ A
280be 3.0, protein recovery 75.5%.
Claims (3)
1., by a method for polyacrylic acid/magnesium oxide heterozygosis microballoon separation and purification Spirulina phycocyanin, it is characterized in that step is: the preparation of polymerized emulsion; Carry out polyreaction and obtain polyacrylic acid/magnesium oxide heterozygosis microballoon; From breaking wall of spirullina princeps clear liquid, Phycocyanins, C-is adsorbed with polyacrylic acid/magnesium oxide heterozygosis microballoon; Phycocyanins, C-is from desorption adsorption microspheres; Desorption liquid obtains Phycocyanins, C-powder through concentrated and lyophilize.
(1) preparation of polymerized emulsion: in round-bottomed flask, put into nano oxidized magnesium granules and nonionogenic tenside, acrylic acid aqueous solution and vegetables oil, weight ratio between nano oxidized magnesium granules and nonionogenic tenside is 1:0 ~ 2.5, the weight ratio of nano particle and vegetables oil is 1:10 ~ 20, in acrylic acid aqueous solution, acrylic acid bulking value concentration is 20 ~ 70%, the volume ratio of acrylic acid aqueous solution and vegetables oil is 1:1 ~ 2, with Fluko FA25 type Homogenizing pump dispersion and emulsion, obtain w/o type milk sap;
(2) in above-mentioned milk sap, add the Sodium Persulfate (volumeter with acrylic acid aqueous solution) of 0.2 ~ 0.5%, stir 2 hours under 100 ~ 200r/min mixing speed at 30 DEG C ~ 40 DEG C, filter the polyacrylic acid/magnesium oxide heterozygosis microballoon obtaining 50 ~ 500nm size distribution;
(3) from breaking wall of spirullina princeps clear liquid, adsorb Phycocyanins, C-with polyacrylic acid/magnesium oxide heterozygosis microballoon: in 500ml Erlenmeyer flask, add polypropylene/magnesium oxide heterozygosis microballoon and breaking wall of spirullina princeps clear liquid, the weight ratio of microballoon and shell-broken liquid is 1:10 ~ 25.At ambient temperature, in rotating speed be 150r/min shaking table on shake absorption 40 ~ 90 minutes, the microballoon of collecting by filtration saturated adsorption Phycocyanins, C-;
(5) pH value microballoon of saturated adsorption Phycocyanins, C-being placed in 100ml is 7.0, concentration is the phosphoric acid buffer of 500 ~ 1000mmol/L, in rotating speed be 150r/min shaking table on concussion 15 minutes, be product by collected by centrifugation supernatant liquor.
(6) lyophilize: by collect containing Phycocyanins, C-liquid carry out 60 DEG C at vacuum concentration to original volume 1/3rd, obtain purity A after lyophilize
620/ A
280the Phycocyanins, C-powder of >2.0.
2. the preparation method of polyacrylic acid according to claim 1/magnesium oxide heterozygosis microballoon, is characterized in that adopting nano oxidized magnesium granules to make emulsifying agent, can add sorbitan fatty(acid)ester Span-80, peregal AEO
9, the nonionogenic tenside such as polyoxyethylene nonylphenol ether OP-10 is as co-emulsifier.
3. the preparation method of breaking wall of spirullina princeps clear liquid according to claim 1, is characterized in that, the vegetables oil of employing comprises the one in soybean oil, Oleum Gossypii semen, tea-seed oil, rapeseed oil, sweet oil, sunflower seed oil.
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| CN112812176A (en) * | 2021-01-13 | 2021-05-18 | 江南大学 | Method for extracting phycocyanin from spirulina by low-salt flocculation method |
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| CN101560254A (en) * | 2009-05-19 | 2009-10-21 | 江南大学 | Enrichment and separation method of blue green algae phycocyanin |
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