Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof

A technology of allophycocyanin and allophycocyanin, which is applied in the field of simultaneous separation of high-purity phycocyanin and allophycocyanin, can solve the problem of not being able to separate phycocyanin and allophycocyanin at one time, and cannot reach the field of biomedicine Use requirements, product purity is only 2.1, etc., to achieve the effect of facilitating large-scale industrial production, broad prospects for industrialization, and easy promotion

Inactive Publication Date: 2012-01-25
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, ion exchange chromatography and size exclusion chromatography used alone or in series are commonly used and preferred purification methods for the separation of phycocyanin in Spirulina, but the required fillers are expensive, and they cannot be separated from phycocyanin and allophycocyanin at one time. blue protein
Recently, some researchers have obtained purified phycocyanin by multiple extractions using two-phase extraction (Pail G and Raghavarao, Biochem Eng J, 2007, 34: 156-164), but this technology is still not mature enough and needs to be further optimized
The national invention patent with the patent number 200610171008.X and titled "Method for Simultaneous Preparation of Phycocyanin and Allophycocyanin" provides a method for simultaneous preparation of phycocyanin and allophycocyanin by hydroxyapatite column chromatography method, but the purity of the resulting product is only 2.1, which cannot meet the requirements for use in the field of biomedicine

Method used

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  • Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof
  • Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof
  • Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Separation and Purification of Phycocyanin and Allophycocyanin

[0039] (1) Preparation of hydroxyapatite:

[0040] Add 500mL of 0.4M calcium chloride and 0.4M dipotassium hydrogen phosphate dropwise at a rate of 5ml / min to a beaker containing 200ml of 0.4M calcium chloride solution, and keep stirring. After finishing, let the sediment stand, pour off the supernatant, and rinse with enough tap water. Add 2M potassium hydroxide to the precipitate while stirring to ensure that the pH of the precipitate is > 8. After standing for 12 hours, wash it with a large amount of tap water, discard the fine particles, and put it into a piston chromatography column with a size of 1.5cm×20cm. Then balance with 0.001M phosphate buffer solution (pH=6.8) for 3-5 times, wherein the volume ratio of hydroxyapatite precipitate and 1mM phosphate buffer solution (pH=6.8) is about 1:1.

[0041] (2) Separation of phycocyanin and allophycocyanin by hydroxyapatite column chromatography...

Embodiment 2

[0047] Example 2 Separation and purification of selenium-containing phycocyanin and selenium-containing allophycocyanin

[0048] The preparation steps of hydroxyapatite are the same as above. Selenium-containing phycocyanin and selenium-containing allophycocyanin are separated and prepared by the following steps:

[0049] (1) Preparation of phycobiliprotein crude extract: take commercial sources or self-cultured selenium-enriched Spirulina. The self-cultured selenium-enriched Spirulina culture method is as follows: add Zarrouk culture solution in the Erlenmeyer flask, inoculate Spirulina and adjust the initial A of algae 560 =0.20 (Cultivated in a light incubator, temperature (30±1)°C, pH 8-9, light intensity 72 μmol m -2 ·s -1 ; Add sodium selenite at 7-9 days respectively, and the measured selenium concentration is 100, 150, 200mg·L -1 , starting from the 7th day, the cumulative amount of selenium added every 24 hours was 450mg·L -1 Cultivate until 11d and harvest, filt...

Embodiment 3

[0054] Example 3 Separation and Purification of Tellurium-Containing Phycocyanin and Allophycocyanin

[0055] The preparation steps of hydroxyapatite are the same as above. Separation and preparation of tellurium-containing phycocyanin and allophycocyanin through the following steps:

[0056] (1) Preparation of phycobiliprotein crude extract: take commercially sourced spirulina powder or self-cultured spirulina. The culture method of tellurium-rich Spirulina is as follows: add Zarrouk culture solution to the Erlenmeyer flask, inoculate S. platensis and adjust the initial A 560 =0.20 (Cultivated in a light incubator, temperature (30±2)°C, pH=8~9, light intensity 72μmol·m -2 ·s -1 ; Add sodium tellurite at 7-9 days respectively, and measure the tellurium concentration as 200, 250, 250mg·L -1 , added every 24 hours from the 7th day, the cumulative amount of tellurium added was 700mg·L -1 ; Cultivate until harvested on 11d, filter with 400 mesh gauze. ) in 1mM phosphate buff...

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Abstract

The invention discloses a method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof. The method comprises the following steps of: repeatedly freezing and thawing spirulina dry powder, ultrasonically crushing, and centrifuging, wherein supernate is a crude phycobiliprotein extracting solution; then adding ammonia sulfate, and taking out the supernate when the saturation degree is 25-35%; then, adding ammonia sulfate, taking out precipitates when the saturation degree is 60-70%, dialyzing for precipitation to obtain a primarily purified phycobiliprotein extracting solution, and loading the primarily purified phycobiliprotein extracting solution to a hydroxyapatite column; and collecting 0.02-0.03M phosphate buffered solution which contains 0.1-0.2M sodium chloride and has the pH value of 6.5-7.2, and 0.1-0.12M phosphate buffered solution which contains 0.1-0.2M sodium chloride and has the pH value of 6.5-7.2, wherein the 0.02-0.03M phosphate buffered solution is phycocyanin and the 0.1-0.12M phosphate buffered solution is allophycocyanin. The method disclosed by the invention can be used for simultaneously preparing phycocyanin with a purification factor reaching 4.5 and allophycocyanin with a purification factor reaching 3 as well as analogues containing selenium and tellurium, and the purification scale of the method is amplified so as to acquire high-purity protein of a gram level through primary chromatography, so that the purification cost is greatly reduced, and the method can be used for producing the phycocyanin and the allophycocyanin.

Description

technical field [0001] The invention belongs to the fields of health products, functional food and biomedicine, and specifically relates to a method and application for simultaneously separating high-purity phycocyanin and allophycocyanin. Background technique [0002] In the 21st century, there has been an upsurge in the development of marine lake and marsh resources worldwide, and the development of marine drugs has great potential for development. Among them, phycobiliproteins, which are widely and abundantly present in algae phycobilisomes, have attracted the attention of many scholars because of their safety, non-toxicity, multi-faceted development and application value, and high biological activity. Phycobiliproteins are a kind of pigment complex protein, which can be divided into Phycocyanins (PC), Allophycocyanins (APC), Phycoerythrin (Phycoerythrin, PE) and Phycoerythrin according to their absorption spectrum properties. (Phycoerythrocyanin, PEC). Phycobiliprotein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/405C07K1/36C07K1/34C07K1/30C07K1/16
Inventor 陈填烽郑文杰蒋洁杨芳黄家兴
Owner JINAN UNIVERSITY
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