High-purity phycocyanin and method for extracting phycocyanin from spirulina
阅读说明：本技术 一种高纯度藻蓝蛋白及其从螺旋藻中提取的方法 (High-purity phycocyanin and method for extracting phycocyanin from spirulina ) 是由 杨南超 于 2021-02-05 设计创作，主要内容包括：本发明涉及一种高纯度藻蓝蛋白及其从螺旋藻中提取的方法,应用于生物化学技术领域,包括以下步骤：(1)螺旋藻破壁：将螺旋藻粉浸泡于氯化钠溶液中,离心,得上清液；(2)藻蓝蛋白粗提纯：向步骤(1)上清液中加入多功能化硅胶材料I,搅拌,过滤,取上清液,得藻蓝蛋白粗提液；(3)藻蓝蛋白精提纯：向步骤(2)上清液中加入多功能化硅胶材料II,搅拌,过滤,洗脱,干燥,得高纯度藻蓝蛋白。本发明是一种具有低成本易操作、高纯度,适合于大规模生产等优点的藻蓝蛋白提取纯化方法,其具有非常重要的意义。(The invention relates to a high-purity phycocyanin and a method for extracting the phycocyanin from spirulina, which are applied to the technical field of biochemistry and comprise the following steps: (1) breaking the walls of spirulina: soaking the spirulina powder in a sodium chloride solution, and centrifuging to obtain a supernatant; (2) crude purification of phycocyanin: adding the multifunctional silica gel material I into the supernatant obtained in the step (1), stirring, filtering, and taking the supernatant to obtain a crude phycocyanin extracting solution; (3) purifying phycocyanin essence: and (3) adding the multifunctional silica gel material II into the supernatant obtained in the step (2), stirring, filtering, eluting and drying to obtain the high-purity phycocyanin. The phycocyanin extracting and purifying method has the advantages of low cost, easy operation, high purity, suitability for large-scale production and the like, and has very important significance.)
1. A method for extracting high-purity phycocyanin from spirulina is characterized in that: the method comprises the following steps:
(1) breaking the walls of spirulina: soaking the spirulina powder in a sodium chloride solution, and centrifuging to obtain a supernatant;
(2) crude purification of phycocyanin: adding the multifunctional silica gel material I into the supernatant obtained in the step (1), stirring, filtering, and taking the supernatant to obtain a crude phycocyanin extracting solution;
(3) purifying phycocyanin essence: and (3) adding the multifunctional silica gel material II into the supernatant obtained in the step (2), stirring, filtering, eluting and drying to obtain the high-purity phycocyanin.
2. The method of claim 1, wherein the phycocyanin is extracted from spirulina by the following steps: in the step (2), the chemical formula of the multifunctional silica gel material I is as follows: [ (O)3/2)Si(CH2)xS(CH2)yNH(CH2CH2NH)zH]a[Si(O4/2)]b[(CH2)uWSi(O3/2)]c；
Wherein x is an integer from 2 to 12; y is an integer from 3 to 12; z is an integer from 0 to 100; u is an integer from 2 to 12; w is SH or S (CH)2)vSH, v are integers from 2 to 6; a, b, c are integers, and a + c: the ratio of b is between 0.000001 and 100, a and b are always present, and when c is greater than zero, the ratio of c to a + b is between 0.000001 and 10.
3. According to claim 1 or2 the method for extracting high-purity phycocyanin from spirulina is characterized in that: in the step (3), the chemical formula of the multifunctional silica gel material II is as follows: [ [ (O)3/2)Si(CH2)xP(＝O)(OM)2]mFen]a[Si(O4/2)]b[(CH2)uWSi(O3/2)]c[VSi(O3/2)]d；
Wherein M is H, an alkali metal, an alkaline earth metal, x is an integer from 2 to 12; u is an integer from 2 to 12; w is SH or S (CH)2)vSH, v are integers from 2 to 6; v is selected from C1-22Alkyl radical, C1-22Alkyl aryl, C2-20Alkyl sulfide radical, C1-12Alkyl radical, C2-20Alkylene thioether alkyl, C2-20Alkyl thioether aryl radical, C2-20-an alkylene thioether aryl group; a, b, c, d, m, n are integers, and a + c + d: the ratio of b is between 0.000001 and 100, a and b are always present, and when c or d or both are greater than zero, the ratio of c + d to a + b is between 0.000001 and 100; the ratio of m to n is 100 to 0.01.
4. The method of claim 1, wherein the phycocyanin is extracted from spirulina by the following steps: the step (1) comprises the following steps of: 10-50 g/ml, adding the spirulina powder into 0.01-0.1M sodium chloride solution, soaking for 2-8h, and centrifuging at the rotating speed of 6000rpm for 30 min.
5. The method of claim 3, wherein the phycocyanin is extracted from spirulina by the following steps: in the step (2) and the step (3), the particle diameters of the multifunctional silica gel material I and the multifunctional silica gel material II are both 200-500 um, the stirring time is 2-8h, the stirring temperature is 10-40 ℃, and the mesh number of filter cloth used for filtering is 200 meshes.
6. The method of claim 1, wherein the phycocyanin is extracted from spirulina by the following steps: in the step (2) and the step (3), the solid-to-liquid ratios of the multifunctional silica gel material I and the multifunctional silica gel material II to the phycocyanin-containing liquid are as follows: 10-25 g/L.
7. The method of claim 1 or 3, wherein the phycocyanin is extracted from spirulina by the following steps: the elution in the step (3) comprises:
a. adding 0.005-0.01M phosphate buffer solution into the filtered solid, stirring for 0.5-2 h, wherein the solid-to-liquid ratio is 1: 50-1000 g/ml, filtering by using 200-mesh filter cloth, and keeping the solid;
b. adding 0.05-0.5M phosphate buffer solution into the solid obtained in the step a, stirring for 2-4 hours, wherein the solid-to-liquid ratio is 1: 1-10 g/ml, filtering with 40-mesh filter cloth, and keeping the liquid;
c. and (c) repeating the step (b) twice on the solid obtained in the step (a), and reserving the liquid.
8. The method of claim 7, wherein the phycocyanin is extracted from spirulina by the following steps: and d, drying the liquid obtained in the step b and the step c in a freeze dryer to obtain the high-purity phycocyanin.
9. The method of claim 7, wherein the phycocyanin is extracted from spirulina by the following steps: in the step a and the step b, the pH value of the phosphate buffer solution is 6-7.
10. A high purity phycocyanin extracted according to any one of claims 1 to 9.
Technical Field
The invention relates to the technical field of biochemistry, in particular to high-purity phycocyanin and a method for extracting the phycocyanin from spirulina.
Background
The spirulina is a filamentous microalgae rich in protein, vitamins, essential amino acids, mineral substances and essential fatty acids, the abundant phycocyanin is one of the very rare proteins in the natural world, the phycocyanin is not only bright in color, but also is a protein rich in nutrition, the amino acid composition is complete, the essential amino acid content is high and accounts for 37.42% of the total amount of the amino acid, the phycocyanin can help to regulate and synthesize various important enzymes required by human metabolism, and has important effects on inhibiting the growth of cancer cells and promoting the regeneration of human cells, and meanwhile, the phycocyanin can also regulate the immune system of a human body, enhance the functions of the immune system and improve the resistance of the human body to diseases, so the phycocyanin is visually called as 'food diamond' by food experts.
Phycocyanin is the only natural blue pigment approved by FDA in the United states, can be used as an additive of food and cosmetics, high-purity phycocyanin has strong fluorescence characteristic, the fluorescence intensity is 30 times to 100 times stronger than that of common fluorescein, and the phycocyanin can be prepared into a fluorescence probe for scientific research and detection; with the fact that phycocyanin is used as a functional component and is continuously proved by scientific research institutions in the aspects of resisting cancer, diminishing inflammation, enriching blood, protecting liver, maintaining ovary, resisting oxidation and increasing leucocytes, the separation and purification technology of phycocyanin becomes a hot point of research of people at present.
The traditional phycocyanin separating and purifying technology generally adopts the method of ultrasonic treatment, repeated freeze thawing, enzymolysis, high-pressure homogenization and the like to crack cells to obtain crude phycocyanin extract, and then (NH) is added4)2SO4The precipitation method is combined with various chromatographic methods for use, and the method has the defects of complicated steps, large protein loss amount, difficulty in large-scale popularization, higher than 50% of production cost in the purification process and the like.
The new technology developed in recent years, such as aqueous two-phase extraction technology, reverse micelle extraction technology, expanded bed adsorption, chitosan affinity precipitation-activated carbon adsorption-DEAE Sephadex column chromatography and the combined use of different technologies (such as salting-out combined aqueous two-phase extraction technology and isoelectric point combined aqueous two-phase extraction), provides a new method for the separation and purification of phycocyanin, but has the corresponding disadvantages: compared with the salt-added back extraction method, the difficulty of the reverse micelle extraction method is reduced, but the method is still not mature, part of phycocyanin is lost, and the extraction rate is low; the double-aqueous phase extraction method has the advantages of easy amplification and operation, time saving, energy consumption and cost reduction, mild separation process conditions and the like, but phycocyanin is not easy to be completely separated from the polymer; the expanded bed adsorption method has the advantages of integration, high recovery rate, simplified operation, reduced cost and shortened operation time, but the technology is immature and the operation has uncertainty; the salting-out and the aqueous two-phase extraction method can greatly improve the purity of the phycocyanin, but the operation steps are complicated and the time consumption is long; the algae toxin and heavy metal pollution can be effectively removed by combining isoelectric point with aqueous two-phase extraction, but the molecular structure of the protein is easy to change; the chitosan affinity precipitation-activated carbon adsorption-DEAE Sephadex column chromatography method has short time consumption and simple and convenient operation, but has very high requirements on the operation of instruments.
Therefore, the phycocyanin extracting and purifying method which has the advantages of low cost, easy operation, high purity, suitability for large-scale production and the like is developed, and has very important significance.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a method for extracting high-purity phycocyanin from spirulina, which utilizes a low-concentration sodium chloride solution to carry out swelling wall breaking on spirulina powder so as to release phycocyanin and other substances, and then utilizes two known multifunctional silica gel materials to purify the phycocyanin, and comprises the following steps:
(1) breaking the walls of spirulina: soaking the spirulina powder in a sodium chloride solution, and centrifuging to obtain a supernatant;
(2) crude purification of phycocyanin: adding the multifunctional silica gel material I into the supernatant obtained in the step (1), stirring, filtering, and taking the supernatant to obtain a crude phycocyanin extracting solution;
(3) purifying phycocyanin essence: and (3) adding the multifunctional silica gel material II into the supernatant obtained in the step (2), stirring, filtering, eluting and drying to obtain the high-purity phycocyanin.
In certain embodiments of the present invention, the chemical formula of the multifunctional silica gel material I in the step (2) is: [ (O)3/2)Si(CH2)xS(CH2)yNH(CH2CH2NH)zH]a[Si(O4/2)]b[(CH2)uWSi(O3/2)]c；
Wherein x is an integer from 2 to 12; y is an integer from 3 to 12; z is an integer from 0 to 100; u is an integer from 2 to 12; w is SH or S (CH)2)vSH, v are integers from 2 to 6; a, b, c are integers, and a + c: b is in the range of 0.000001 to 100, a and b are always present, and when c is greater than zero, the ratio of c to a + b is in the range of 0.000001 to 10010。
In certain embodiments of the present invention, the chemical formula of the multifunctional silica gel material II in the step (3) is: [ [ (O)3/2)Si(CH2)xP(＝O)(OM)2]mFen]a[Si(O4/2)]b[(CH2)uWSi(O3/2)]c[VSi(O3/2)]d；
Wherein M is H, an alkali metal, an alkaline earth metal, x is an integer from 2 to 12; u is an integer from 2 to 12; w is SH or S (CH)2)vSH, v are integers from 2 to 6; v is selected from C1-22Alkyl radical, C1-22Alkyl aryl, C2-20Alkyl sulfide radical, C1-12Alkyl radical, C2-20Alkylene thioether alkyl, C2-20Alkyl thioether aryl radical, C2-20-an alkylene thioether aryl group; a, b, c, d, m, n are integers, and a + c + d: the ratio of b is between 0.000001 and 100, a and b are always present, and when c or d or both are greater than zero, the ratio of c + d to a + b is between 0.000001 and 100; the ratio of m to n is 100 to 0.01.
In certain embodiments of the invention, step (1) is a step of mixing the components in a solid-to-liquid ratio of 1: 10-50 g/ml, adding the spirulina powder into 0.01-0.1M sodium chloride solution, soaking for 2-8h, and centrifuging at the rotating speed of 6000rpm for 30 min.
In some embodiments of the invention, in the step (2) and the step (3), the particle sizes of the multifunctional silica gel material I and the multifunctional silica gel material II are both 200-500 um, the stirring time is 2-8h, the stirring temperature is 10-40 ℃, and the mesh number of filter cloth used for filtering is 200 meshes.
In certain embodiments of the present invention, the solid-to-liquid ratios of the multifunctional silica gel material I and the multifunctional silica gel material II to the phycocyanin-containing liquid in steps (2) and (3) are both: 10-25 g/L.
In certain embodiments of the invention, the eluting in step (3) comprises:
a. adding 0.005-0.01M phosphate buffer solution into the filtered solid, stirring for 0.5-2 h, wherein the solid-to-liquid ratio is 1: 50-1000 g/ml, filtering by using 200-mesh filter cloth, and keeping the solid;
b. adding 0.05-0.5M phosphate buffer solution into the solid obtained in the step a, stirring for 2-4 hours, wherein the solid-to-liquid ratio is 1: 1-10 g/ml, filtering with 40-mesh filter cloth, and keeping the liquid;
c. and (c) repeating the step (b) twice on the solid obtained in the step (a), and reserving the liquid.
In some embodiments of the present invention, the liquid obtained in step b and step c is dried in a freeze-dryer to obtain high-purity phycocyanin.
In certain embodiments of the present invention, the phosphate buffer of step a and step b has a pH of 6 to 7.
The invention also provides the high-purity phycocyanin extracted by the method.
Compared with the prior art, the invention has the following advantages:
1. the method adopts the low-concentration sodium chloride solution to carry out swelling wall breaking on the spirulina powder, and has the advantages of short time, simple operation, no equipment requirement, low energy consumption and environmental protection.
2. The multifunctional silica gel material has customizable particle size, simple and fast filtration, low cost of the used filter cloth compared with a filter membrane, and large-scale application of the separation and purification process in a large-scale stirring tank.
3. The invention utilizes the sequence of the adsorption of the multifunctional silica gel material on impurities and phycocyanin, controls the material dosage ratio, is used for realizing directional adsorption and elution, and the obtained phycocyanin has high purity, the crude purification A620/A280 is more than 2 and is higher than the food grade, and the fine purification A620/A280 is more than 5 and is higher than the reagent grade.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1:
the embodiment provides a method for extracting high-purity phycocyanin from spirulina, which comprises the following steps:
synthesis of multifunctional silica gel material I:
0.4mol of diethylenetriamine and 0.4mol of 3- (3-chloropropyl) thiopropyl trimethoxy silane are added into a 500mL three-neck flask provided with a glass plug and a condensation reflux device, the mixture is refluxed for 2.5h at 130 ℃, cooled to 70 ℃, added with 40mL of methanol and refluxed for 1 h, cooled, added with 125mL of xylene and 90g of amorphous silica gel (200 plus 500 mu m,makall) at 125 deg.c for 2h, adding 0.2mol of 3-mercaptopropyltrimethoxysilane, heating for 5h, cooling, filtering, washing with methanol for 5 times, and drying to obtain the compound of formula I, wherein x is 3, y is 3, z is 2, u is 3, and W is SH.
Synthesis of multifunctional silica gel material II:
20kg of silica gel (300-,) And 42L of water, stirring and mixing at 100 ℃, adding 16mol of vinyl trimethoxy silane, heating and stirring the reaction mixture for 5 hours, cooling, filtering the solid, fully washing with water, and drying to generate vinyl silica gel solid; putting phosphorous acid (3280g,40mol) and RO (10L) water into a 50L reaction kettle, starting stirring, putting vinyl silica gel (1.4-2.0mmol/g, 4.0kg) and adding 40ml of tert-butyl hydroperoxide, keeping the room temperature and stirring for 40min, starting heating, setting the temperature of an oil bath kettle at 130 ℃, starting adding 8ml of tert-butyl hydroperoxide every 15 min when the temperature of the oil bath kettle reaches and the liquid refluxes, removing the liquid after cooling, adding 30L of water into the solid, stirring for 30min, filtering, and adding water into the solidAdding more water (30L) to the solid, stirring the mixture for 30min, filtering, repeating the process for 3 times, drying, adding 5g of the product into 100ml of 100ppm ferric sulfate solution, stirring at 60 ℃ for 6H, filtering, washing and drying to obtain a component of formula II, wherein M is H; x is 3; the integer c is 0 and the integer d is 0.
Breaking the walls of spirulina: adding 50g Spirulina powder and 1L sodium chloride solution with concentration of 0.05M into 2L beaker, soaking for 6 hr, centrifuging the solution at 6000rpm for 30min, and collecting supernatant.
Crude purification of phycocyanin: adding 10g of multifunctional silica gel material I into the supernatant, stirring at 10 ℃ for 6h, and filtering with 200-mesh filter cloth to obtain crude phycocyanin extract, wherein A620/A280 is 2.9.
Purifying phycocyanin essence:
step one, adding 10g of multifunctional silica gel material II into crude phycocyanin extracting solution, stirring for 4 hours at 40 ℃, and filtering with 200-mesh filter cloth to leave solid;
secondly, adding 1L of 0.01M phosphate buffer (pH 6-7) into the solid filtered in the first step, stirring for 0.5h, and filtering with 200-mesh filter cloth to leave the solid;
step three, adding 20ml of 0.5M phosphate buffer (pH 6-7) into the solid obtained in the step two, stirring for 2 hours, filtering with 40-mesh filter cloth, and reserving liquid;
fourthly, repeating the third step twice on the solid obtained in the second step, and reserving liquid;
fifthly, drying the liquid obtained in the third and fourth steps under a freeze dryer to obtain high-purity phycocyanin, wherein A620/A280＝5.8。
Example 2:
the embodiment provides a method for extracting high-purity phycocyanin from spirulina, which comprises the following steps:
synthesis of multifunctional silica gel material I:
0.4mol of polyethylene polyamine and 0.4mol of 3- (3-chloropropyl) thiopropyltrimethoxysilane are added into a 500mL three-neck flask provided with a glass plug and a condensation reflux device,refluxing at 130 deg.C for 2.5h, cooling to 70 deg.C, adding 40mL of methanol, refluxing for 1 h, cooling, adding 125mL of xylene and 90g of spherical silica gel (300-,makall) at 125 ℃ for 2h, adding 0.2mol of 3-mercaptopropyltrimethoxysilane, continuously heating for 5h, cooling, filtering, washing with methanol for 5 times, and drying to obtain the component of the chemical formula I, wherein x is 3, y is 3, z is 9, u is 3, and W is SH.
Synthesis of multifunctional silica gel material II:
20kg of silica gel (300-,) And 42L of water, stirring and mixing at 100 ℃, adding 16mol of vinyl trimethoxy silane, heating and stirring the reaction mixture for 5h, cooling and filtering the solid, fully washing with water, drying to generate a vinyl silica gel solid, adding sodium phosphite (4880g,40mol) and RO (10L) water into a 50L reaction kettle, starting stirring, adding vinyl silica gel (1.4-2.0mmol/g, 4.0kg) and 40ml of tert-butyl hydroperoxide, stirring at room temperature for 40min, starting heating, setting the temperature of an oil bath kettle at 130 ℃, starting to add 8ml of tert-butyl hydroperoxide every 15 min when the temperature of the oil bath kettle reaches and the liquid refluxes, cooling and removing the liquid, adding 30L of water into the solid, stirring for 30min, filtering, adding more water (30L) into the solid, stirring the mixture for 30min, and filtering. Repeating the process for 3 times, drying, adding 5g product into 100ml 100ppm ferric sulfate solution, stirring at 25 deg.C for 4 hr, filtering, washing, and drying to obtain a component of formula II, wherein M is Na; x is 3; the integer c is 0 and the integer d is 0.
Breaking the walls of spirulina: adding 50g Spirulina powder and 1L sodium chloride solution with concentration of 0.1M into 2L beaker, soaking for 4 hr, centrifuging the solution at 6000rpm for 30min, and collecting supernatant.
Crude purification of phycocyanin: adding 10g of multifunctional silica gel material I into the supernatant, stirring at 30 ℃ for 4h, and filtering with 200-mesh filter cloth to obtain crude phycocyanin extract, wherein A620/A280 is 3.5.
Purifying phycocyanin essence:
step one, adding 15g of multifunctional silica gel material II into crude phycocyanin extracting solution, stirring for 6 hours at 20 ℃, and filtering with 200-mesh filter cloth to leave solid;
secondly, adding 1L of 0.01M phosphate buffer (pH 6-7) into the solid filtered in the first step, stirring for 0.5h, and filtering with 200-mesh filter cloth to leave the solid;
step three, adding 20ml of 0.5M phosphate buffer (pH 6-7) into the solid obtained in the step two, stirring for 2 hours, filtering with 40-mesh filter cloth, and reserving liquid;
fourthly, repeating the third step twice on the solid obtained in the second step, and reserving liquid;
fifthly, drying the liquid obtained in the third and fourth steps under a freeze dryer to obtain high-purity phycocyanin, wherein A620/A280＝6.3。
Example 3:
the embodiment provides a method for extracting high-purity phycocyanin from spirulina, which comprises the following steps:
synthesis of multifunctional silica gel material I:
0.4mol of polyethylene polyamine and 0.4mol of 3- (3-chloropropyl) thiopropyl trimethoxy silane are added into a 500mL three-neck flask provided with a glass plug and a condensation reflux device, the mixture is refluxed for 2.5h at 130 ℃, then cooled to 70 ℃,40 mL of methanol is added, the mixture is refluxed for 1 h, 125mL of xylene and 90g of spherical silica gel (300-,500 mu m) are added after cooling,makall) at 125 ℃ for 2h, adding 0.2mol of 3-mercaptopropyltrimethoxysilane, continuously heating for 5h, cooling, filtering, washing with methanol for 5 times, and drying to obtain the component of the chemical formula I, wherein x is 3, y is 3, z is 9, u is 3, and W is SH.
Synthesis of multifunctional silica gel material II:
20kg of silicon was added to a 200L reactorThe glue (300-500 μm,) And 42L of water, stirring and mixing at 100 ℃, adding 16mol of vinyl trimethoxy silane, heating and stirring the reaction mixture for 5h, cooling and filtering the solid, fully washing with water, drying to generate a vinyl silica gel solid, adding sodium phosphite (4880g,40mol) and RO (10L) water into a 50L reaction kettle, starting stirring, adding vinyl silica gel (1.4-2.0mmol/g, 4.0kg) and 40ml of tert-butyl hydroperoxide, stirring at room temperature for 40min, starting heating, setting the temperature of an oil bath kettle at 130 ℃, starting to add 8ml of tert-butyl hydroperoxide every 15 min when the temperature of the oil bath kettle reaches and the liquid refluxes, cooling and removing the liquid, adding 30L of water into the solid, stirring for 30min, filtering, adding more water (30L) into the solid, stirring the mixture for 30min, and filtering. Repeating the process for 3 times, drying, adding 5g product into 100ml 100ppm ferric sulfate solution, stirring at 25 deg.C for 4 hr, filtering, washing, and drying to obtain a component of formula II, wherein M is Na; x is 3; the integer c is 0 and the integer d is 0.
Breaking the walls of spirulina: adding 50g Spirulina powder and 1L sodium chloride solution with concentration of 0.1M into 2L beaker, soaking for 4 hr, centrifuging the solution at 6000rpm for 30min, and collecting supernatant.
Crude purification of phycocyanin: adding 10g of multifunctional silica gel material I into the supernatant, stirring at 30 ℃ for 4h, and filtering with 200-mesh filter cloth to obtain crude phycocyanin extract, wherein A620/A280 is 3.9.
Purifying phycocyanin essence:
firstly, adding 25g of multifunctional silica gel material II into crude phycocyanin extracting solution, stirring for 6 hours at 20 ℃, and filtering with 200-mesh filter cloth to leave solid;
secondly, adding 1L of 0.01M phosphate buffer (pH 6-7) into the solid filtered in the first step, stirring for 0.5h, and filtering with 200-mesh filter cloth to leave the solid;
step three, adding 20ml of 0.5M phosphate buffer (pH 6-7) into the solid obtained in the step two, stirring for 2 hours, filtering with 40-mesh filter cloth, and reserving liquid;
fourthly, repeating the third step twice on the solid obtained in the second step, and reserving liquid;
fifthly, drying the liquid obtained in the third and fourth steps under a freeze dryer to obtain high-purity phycocyanin, wherein A620/A280＝6.8。
The synthesis methods of the multifunctional silica gel material I and the multifunctional silica gel material II in embodiments 1 to 3 of the present invention are disclosed in chinese patent CN110368905A and chinese patent CN111939875A, respectively, and the present invention relates to that other chemical formulas of the multifunctional silica gel material I and the multifunctional silica gel material II can be synthesized by referring to the above patents, and the present invention is not described again, and the present invention is mainly directed to the application of the multifunctional silica gel material I and the multifunctional silica gel material II in the phycocyanin extraction process, and by the extraction method of the present invention, high purity phycocyanin can be obtained from spirulina, wherein crude purification a620/a280 is greater than 2, which is higher than a food grade, and refined purification a620/a280 is greater than 5, which is higher than a reagent grade.
In the embodiment of the invention, the spirulina powder contains 50% of protein, 20% of polysaccharide, 10% of phycocyanin, 7% of water, 2% of chlorophyll, 0.5% of carotenoid, 2% of grease, 5% of insoluble cell wall and 1.5% of nucleic acid; the multifunctional silica gel material I is prepared by loading silica with organic functional groups of amino (NH2) and Sulfydryl (SH), wherein a large number of hydroxyl (OH) are arranged on the silica, organic components in phycocyanin can be adsorbed to different degrees according to the polarity of the hydroxyl (OH), the adsorption is sequential according to the structural difference of the organic matters, the addition amount of the multifunctional silica gel material I can determine which organic matter is adsorbed first, researches show that the multifunctional silica gel material I mainly adsorbs impurities of chlorophyll and part of heteroproteins, the purity of the phycocyanin can be controlled within a food-grade range (A620/A280 is more than 2) by controlling the parameter dosage within a certain range (for example, the solid-liquid ratio of the multifunctional silica gel material I to the phycocyanin-containing liquid is controlled within 10-25 g/L), and meanwhile, the multifunctional silica gel material II mainly adsorbs the phycocyanin and a small amount of the heteroproteins, polysaccharide and the like, wherein different substances can be eluted by different concentrations of phosphate buffer solutions, and phycocyanin can be just eluted by the phosphate buffer solution with the pH value of 6-7 in the embodiment of the invention, so that high-purity phycocyanin (A620/A280 is more than 5) can be obtained.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.