Preparation method of high-purity phycocyanin

Abstract

The invention discloses a preparation method of high-purity phycocyanin, and is characterized in that the preparation method comprises the following steps: (1) taking a fresh spirulina powder, fully mixing with a phosphate buffer solution evenly, repeatedly freezing and thawing for 7-10 times to break and remove cell walls, centrifuging to remove spirulina mud, and thus obtaining a supernatant; (2) adopting a two-step precipitation method with a 20%-30% ammonium sulfate and a 40%-60% ammonium sulfate to obtain a phycocyanin crude extract; (3) after dialyzing the crude extract, loading the sample onto a weak anion exchange column DEAE Sepharose FF, carrying out gradient elution after ion exchange, collecting an outflow component with A620 / A280 of more than 3; and (4) dialyzing the collected sample, then loading the sample onto a strong anion exchange column SOURCE30Q, carrying out gradient elution after ion exchange, collecting an outflow component with A620 / A280 of more than 4, again carrying out one-time ammonium sulfate precipitation concentration, and thus obtaining the high-purity phycocyanin having the purity of more than 4.5. The extraction purification method is simplified in process, wide in source of the raw material spirulina, simple in required equipment, and high in purity of the product, and has quite high application value.

Description

technical field

[0001] The invention belongs to the field of protein engineering or biochemical separation and purification, and specifically relates to a preparation method of high-purity phycocyanin. Background technique

[0002] Phycocyanin (phycocyanin) is a photosynthetic pigment present in cyanobacteria cells, composed of chromophore-containing α and β subunits, each of which has a molecular weight of about 20 kDa. In phycocyanin, these two subunits are usually combined into monomers (αβ), trimers (αβ) 3 and hexamers (αβ) 6 , so the molecular weight ranges from 44 to 260 kDa. Phycocyanin is a natural blue colorant, which is widely used in health drinks, candy colorants and cosmetics, and some are used as biochemical tracers because of their fluorescent immunity. In addition, phycocyanin has been shown to have important pharmacological effects, including hepatoprotective, antioxidant, anti-inflammatory, anticancer, and antidiabetic activities. Due to its huge commer...

Examples

Embodiment 1

[0029] A preparation method of high-purity phycocyanin, the steps are as follows:

[0030] (1) Take freshly dried spirulina powder, mix it with 0.05mol / L phosphate buffer at a ratio of 1:15 (mass / volume), stir magnetically at room temperature for 2 hours, place it at -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 8 times in this way, centrifuged at 4°C and 8000rpm for 30min, and the supernatant was taken.

[0031] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 30%, let stand at 4°C for 8 hours, centrifuge at 4°C and 10,000rpm for 30min, take the supernatant, then add solid ammonium sulfate to 45% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 4°C and 10,000rpm for 30min, and take the precipitate.

[0032] (3) Dissolve the precipitate in step (2) with a sma...

Embodiment 2

[0037] A preparation method of high-purity phycocyanin, the steps are as follows:

[0038] (1) Take fresh and dried spirulina powder, mix it with 0.05mol / L phosphate buffer at a ratio of 2:25 (mass / volume), stir magnetically at room temperature for 2 hours, place it in -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 10 times in this way, centrifuged at 4°C, 10,000 rpm for 30 min, and the supernatant was taken.

[0039] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 25%, let stand at 4°C for 8 hours, centrifuge at 4°C and 10000rpm for 30min, take the supernatant, then add solid ammonium sulfate to 50% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 8000rpm for 30min at 4°C to collect the precipitate.

[0040] (3) Dissolve the precipitate in step (2) with a sm...

Embodiment 3

[0045] A preparation method of high-purity phycocyanin, the steps are as follows:

[0046] (1) Take fresh and dried spirulina powder, mix it with 0.05mol / L phosphate buffer saline in a ratio of 1:10 (mass / volume), stir magnetically at room temperature for 2 hours, place it at -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 8 times in this way, centrifuged at 4°C and 12000rpm for 30min, and the supernatant was taken.

[0047] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 30%, let stand at 4°C for 8 hours, centrifuge at 12000rpm for 30min at 4°C, take the supernatant, then add solid ammonium sulfate to 55% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 12000rpm for 30min at 4°C to collect the precipitate.

[0048] (3) Dissolve the precipitate in step (2) wit...

technical field

[0001] The invention belongs to the field of protein engineering or biochemical separation and purification, and specifically relates to a preparation method of high-purity phycocyanin. Background technique

[0002] Phycocyanin (phycocyanin) is a photosynthetic pigment present in cyanobacteria cells, composed of chromophore-containing α and β subunits, each of which has a molecular weight of about 20 kDa. In phycocyanin, these two subunits are usually combined into monomers (αβ), trimers (αβ) 3 and hexamers (αβ) 6 , so the molecular weight ranges from 44 to 260 kDa. Phycocyanin is a natural blue colorant, which is widely used in health drinks, candy colorants and cosmetics, and some are used as biochemical tracers because of their fluorescent immunity. In addition, phycocyanin has been shown to have important pharmacological effects, including hepatoprotective, antioxidant, anti-inflammatory, anticancer, and antidiabetic activities. Due to its huge commer...

Examples

Embodiment 1

[0029] A preparation method of high-purity phycocyanin, the steps are as follows:

[0030] (1) Take freshly dried spirulina powder, mix it with 0.05mol / L phosphate buffer at a ratio of 1:15 (mass / volume), stir magnetically at room temperature for 2 hours, place it at -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 8 times in this way, centrifuged at 4°C and 8000rpm for 30min, and the supernatant was taken.

[0031] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 30%, let stand at 4°C for 8 hours, centrifuge at 4°C and 10,000rpm for 30min, take the supernatant, then add solid ammonium sulfate to 45% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 4°C and 10,000rpm for 30min, and take the precipitate.

[0032] (3) Dissolve the precipitate in step (2) with a sma...

Embodiment 2

[0037] A preparation method of high-purity phycocyanin, the steps are as follows:

[0038] (1) Take fresh and dried spirulina powder, mix it with 0.05mol / L phosphate buffer at a ratio of 2:25 (mass / volume), stir magnetically at room temperature for 2 hours, place it in -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 10 times in this way, centrifuged at 4°C, 10,000 rpm for 30 min, and the supernatant was taken.

[0039] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 25%, let stand at 4°C for 8 hours, centrifuge at 4°C and 10000rpm for 30min, take the supernatant, then add solid ammonium sulfate to 50% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 8000rpm for 30min at 4°C to collect the precipitate.

[0040] (3) Dissolve the precipitate in step (2) with a sm...

Embodiment 3

[0045] A preparation method of high-purity phycocyanin, the steps are as follows:

[0046] (1) Take fresh and dried spirulina powder, mix it with 0.05mol / L phosphate buffer saline in a ratio of 1:10 (mass / volume), stir magnetically at room temperature for 2 hours, place it at -20°C to freeze completely, take out Melt in a 37°C water bath, stir gently with a glass rod to avoid air bubbles, and freeze at -20°C after completely melting. Freezing and thawing was repeated 8 times in this way, centrifuged at 4°C and 12000rpm for 30min, and the supernatant was taken.

[0047] (2) Add solid ammonium sulfate to the supernatant of step (1) to a saturated concentration of 30%, let stand at 4°C for 8 hours, centrifuge at 12000rpm for 30min at 4°C, take the supernatant, then add solid ammonium sulfate to 55% saturation Concentration, after standing at 4°C for 8 hours, centrifuge at 12000rpm for 30min at 4°C to collect the precipitate.

[0048] (3) Dissolve the precipitate in step (2) wit...

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