Method for separating and purifying phycocyanin by means of combination of two aqueous phase extraction and ultrasonic waves

Abstract

The invention provides a method for separating and purifying phycocyanin by means of combination of two aqueous phase extraction and ultrasonic waves. The method comprises the particular operating steps that spirulina is subjected to wall breaking through a repeated freezing and thawing method, supernatant is collected, and a crude phycocyanin extracting solution is obtained; glucan and inorganic salt two aqueous phase extraction is adopted, the crude phycocyanin extracting solution is put in ultrasonic waves for ultrasonic treatment for 15 min to 30 min, standing is conducted for 10 min to 15 min, the upper phase is taken and subjected to dialysis in a dialysis membrane with the molecular mass cut-off of 5,000 for 12 h to 20 h, freeze drying is conducted, and phycocyanin powder is obtained. The method for purifying the phycocyanin by means of combination of two aqueous phase extraction and the ultrasonic waves has the advantages of being high in selectivity and high in system phase-separation speed, operation is easy and convenient, equipment is conventional, and the method is suitable for industrial production. The phycocyanin purity of the phycocyanin powder prepared through the method reaches up to (A620 / A280) 3.78, and the total yield reaches up to 90.6%.

Description

technical field

[0001] The invention belongs to the technical field of biochemical separation, and relates to a method for separating and purifying phycocyanin, in particular to a method for separating and purifying phycocyanin in combination with two-phase extraction and ultrasonic waves. Background technique

[0002] Phycocyanin (referred to as PC) is one of the rare pigment proteins in nature. It has a bright color (sky blue), good water solubility, and is rich in eight essential amino acids needed by the human body. It has fluorescence itself, and has anti-radiation, Anti-oxidation, anti-tumor and non-toxic side effects and other advantages, so it has been widely concerned and applied in cosmetics, food, dyes, medicine and other fields. Spirulina is a filamentous microalga rich in protein, vitamins, essential amino acids, minerals and essential fatty acids. The cell wall is composed of polysaccharides, which is easy to digest and absorb. PC in Spirulina is mainly in the ...

Examples

Embodiment 1

[0020] (1) Preparation of crude extract

[0021] Add 1g of spirulina powder to 100g of deionized water and soak for 6 hours, freeze at -20°C for 3 hours, then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.78.

[0022] (2) Aqueous two-phase extraction

[0023] Take 33.5g of the crude extract containing phycocyanin, 9g of dextran and 7.5g of ammonium sulfate into the conical flask. , the upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 20 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 12 hours to obtain 13.2 g of phycocyanin powder, wherein the purity of phycocyanin was 3.65, and the recovery rate was 88.9%.

Embodiment 2

[0025] (1) Preparation of crude extract

[0026] Add 1g of spirulina powder to 80g of deionized water and soak for 8 hours, then freeze at -15°C for 2 hours, then thaw at 25°C, repeat the freezing and thawing process 4 times to break the wall, then centrifuge the resulting solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.75.

[0027] (2) Aqueous two-phase extraction

[0028] Take 34g of the crude extract containing phycocyanin, 10g of dextran and 6g of ammonium sulfate and add them to the Erlenmeyer flask. The upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 12 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 15 hours to obtain 14.6 g of phycocyanin powder, wherein the purity of phycocyanin was 3.78, The recovery rate was 90.6%.

Embodiment 3

[0030] (1) Preparation of crude extract

[0031] Add 1g of spirulina powder to 150g of deionized water and soak for 7 hours, freeze at -18°C for 5 hours, then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.73.

[0032] (2) Aqueous two-phase extraction

[0033] Take 29.5g of the crude extract containing phycocyanin, 12.5g of dextran and 8g of magnesium sulfate and add them to the Erlenmeyer flask. , the upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 18 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 15 hours to obtain 13.2 g of phycocyanin powder, wherein the purity of phycocyanin was 3.50, the recovery rate was 89.3%.

technical field

[0001] The invention belongs to the technical field of biochemical separation, and relates to a method for separating and purifying phycocyanin, in particular to a method for separating and purifying phycocyanin in combination with two-phase extraction and ultrasonic waves. Background technique

[0002] Phycocyanin (referred to as PC) is one of the rare pigment proteins in nature. It has a bright color (sky blue), good water solubility, and is rich in eight essential amino acids needed by the human body. It has fluorescence itself, and has anti-radiation, Anti-oxidation, anti-tumor and non-toxic side effects and other advantages, so it has been widely concerned and applied in cosmetics, food, dyes, medicine and other fields. Spirulina is a filamentous microalga rich in protein, vitamins, essential amino acids, minerals and essential fatty acids. The cell wall is composed of polysaccharides, which is easy to digest and absorb. PC in Spirulina is mainly in the ...

Examples

Embodiment 1

[0020] (1) Preparation of crude extract

[0021] Add 1g of spirulina powder to 100g of deionized water and soak for 6 hours, freeze at -20°C for 3 hours, then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.78.

[0022] (2) Aqueous two-phase extraction

[0023] Take 33.5g of the crude extract containing phycocyanin, 9g of dextran and 7.5g of ammonium sulfate into the conical flask. , the upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 20 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 12 hours to obtain 13.2 g of phycocyanin powder, wherein the purity of phycocyanin was 3.65, and the recovery rate was 88.9%.

Embodiment 2

[0025] (1) Preparation of crude extract

[0026] Add 1g of spirulina powder to 80g of deionized water and soak for 8 hours, then freeze at -15°C for 2 hours, then thaw at 25°C, repeat the freezing and thawing process 4 times to break the wall, then centrifuge the resulting solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.75.

[0027] (2) Aqueous two-phase extraction

[0028] Take 34g of the crude extract containing phycocyanin, 10g of dextran and 6g of ammonium sulfate and add them to the Erlenmeyer flask. The upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 12 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 15 hours to obtain 14.6 g of phycocyanin powder, wherein the purity of phycocyanin was 3.78, The recovery rate was 90.6%.

Embodiment 3

[0030] (1) Preparation of crude extract

[0031] Add 1g of spirulina powder to 150g of deionized water and soak for 7 hours, freeze at -18°C for 5 hours, then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant The liquid is the crude extract containing phycocyanin, and the measured phycocyanin purity (A620 / A280) is 0.73.

[0032] (2) Aqueous two-phase extraction

[0033] Take 29.5g of the crude extract containing phycocyanin, 12.5g of dextran and 8g of magnesium sulfate and add them to the Erlenmeyer flask. , the upper phase was placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 18 hours to obtain a phycocyanin aqueous solution, which was then freeze-dried at -15°C for 15 hours to obtain 13.2 g of phycocyanin powder, wherein the purity of phycocyanin was 3.50, the recovery rate was 89.3%.