CN101235075B - A method for extracting phycocyanin from Matella slender - Google Patents

Abstract

The invention discloses a method for extracting phycocyanin from phormidium tenue, which comprises preparing sodium nitrate, dibasic potassium phosphate, magnesium sulfate, calcium chloride, citrate, ammonium iron citrate, disodium editate, sodium carbonate and micro element solution into phormidium tenue culture medium, arranging phormidium tenue into the culture medium to cultivate and prepare phormidium tenue powder, extracting and purifying phormidium tenue phycocyanin from the phormidium tenue powder. The invention has simple process, easy operation, high reliability, high yield and high quality, and the equipments are simple equipment and suitable for industrial production.

Description

A method for extracting phycocyanin from Matella slender

technical field

The invention relates to a method for extracting phycocyanin from Matella slenderosa, belonging to the field of biotechnology.

Background technique

S. slenderis is a kind of filamentous cyanobacteria widely distributed in desert habitats. It belongs to Cyanophyta, Cyanophyta, Seglophyta, Oscillatoriaceae, and Schizophyta. At present, the utilization of Matella slenderis is generally limited to artificial algae crusts, and the development of other applications of Matsia slenderness is almost blank. The reason why the algae algae are used to form artificial algae crusts for desert control is mainly because it is rich in phycocyanin. Phycobiliproteins are a class of pigment complex proteins present in the phycobilisomes of some algae (mainly red and blue algae). Phycobiliprotein has strong fluorescence and emits orange-red fluorescence, but itself is red, purple or blue, so it is a colored polypeptide. According to the spectral characteristics, phycobiliproteins can be divided into four categories: phycoerythrin, phycocyanin, phycoerythrin and allophycocyanin. Phycocyanin is one of the blue pigment proteins. In addition to being used in plant photosynthesis research, phycocyanin also has high application and development value: it can be used as a pure natural pigment for food, cosmetics and dye industries; reagent grade phycocyanin can be made into fluorescent reagents for application It is used in the research fields of medical clinical diagnosis, immunology and bioengineering; it can also be made into medicines for medical care. At present, the blue pigments used in food and other industries are mainly chemically synthesized pigments, while natural blue pigments are rarely used. Most of the domestic phycocyanin is extracted from spirulina and large seaweed, and their phycocyanin content is mostly higher than The slender mats are lower. In addition, the cost of cultivating large seaweeds is much higher than the cost of cultivating slender mats.

Contents of the invention

The purpose of the present invention is to provide a method for extracting phycocyanin from Matella slender.

In order to achieve the above object, the present invention provides a method for extracting phycocyanin from Matella slenderosa, comprising the following steps:

A. Preparation of culture medium: Add 0.8-2.5g of sodium nitrate, 0.02-0.05g of dipotassium hydrogen phosphate, 0.06-0.09g of magnesium sulfate, 0.02-0.05g of calcium chloride, 0.004-0.008g of citric acid, and citric acid per liter of water. Ammonium ferric acid 0.004～0.008g, EDTA disodium salt 0.001～0.003g, sodium carbonate 0.01～0.04g, trace element solution 0.5～2ml, wherein trace element solution was added boric acid 270～300mg per 100ml of distilled water, 170-200 mg of manganese chloride tetrahydrate, 18-25 mg of zinc sulfate heptahydrate, 15-30 mg of sodium molybdate dihydrate, 6-10 mg of copper sulfate pentahydrate, completely dissolved and stirred to obtain a culture medium;

B. Cultivation of slender algae: the first is the primary cultivation. When the slender algae species are inserted into the culture medium for aerated culture, the airflow is adjusted at 1.5~3.5L.min ^-1 , and the light intensity is controlled at 65~85μE.m ^{- 2} .s ^-1 , the temperature is controlled at 22-35°C, and after 5-10 days of cultivation, transfer to the secondary culture; followed by the secondary culture, insert the culture obtained from the primary culture into the new culture medium Aeration culture, adjust the airflow at 2.5~5L.min ^-1 , aseptic treatment, control the light intensity at 75~95μE.m ^-2 .s ^-1 , control the temperature at 22~35℃, transfer to Tertiary cultivation; the third is tertiary cultivation, in which the culture obtained from the secondary cultivation is inserted into a new culture medium for ventilation and cultivation, the airflow is adjusted at 3-6L.min ^-1 , aseptically treated, and the light intensity is controlled at 70 ～100μE.m ^-2 .s ^-1 , the temperature is controlled at 22～35℃, and after 5～10 days of cultivation, it will be used as the inoculation source for continuous cultivation; The obtained algae species are connected to the small circulation culture tank, the temperature is controlled at 22-35°C, the natural light is used for illumination, the light intensity is controlled at 120-180μE.m ^-2 .s ^-1 , and the culture medium is stirred; followed by the large circulation culture Pond culture, the slender algae after cultivating in the small circulation culture pond for 4 to 7 days are transferred to the large circulation culture pond, the volume ratio of the culture solution in the small circulation culture pond and the large circulation culture pond is 1: 20～25; control The temperature is 22-35°C, the natural light is used for illumination, and the light intensity is controlled at 120-180 μE.m ^-2 .s ^-1 . After culturing for 10-20 days, the algae are filtered to obtain the algae pulp;

When inoculating the above-mentioned Matella slenderosa, it is inserted at a ratio of 0.1 to 0.5 g of wet weight per liter of culture solution.

C. Preparation of slender mat algae powder: centrifuge the algae pulp to obtain slender mat algae mud, then concentrate, dehydrate, dry and pulverize the fine mat algae powder to obtain slender mat algae powder;

D. Extraction of phycocyanin from Algae spp.: add 7-10 times the weight of 0.05-0.1mol.L ^-1 phosphate buffer solution with a pH value of 6.0-8.0 to the powder of S. Freeze at 10～-20°C, take it out after freezing, put it at 20～30°C to melt, wait until it is completely melted, and then freeze it, repeat the freezing and thawing process for 3 to 5 times, and then set it at 3000～6000rpm Centrifuge for 10-15 minutes, collect the supernatant, which is the crude phycocyanin extract;

E. Purification: Add ammonium sulfate with a saturation of 50-70% to the crude extract of phycocyanin in an ice bath for 20-40 minutes in the dark, and then centrifuge at 3000-6000 rpm for 15 minutes at 3-5°C. ~25min, discard the supernatant, dissolve the precipitate with one volume of 0.05~0.1mol.L ^-1 phosphate buffer solution with a pH value of 6.0~8.0, dialyze in the dark for 20~30h, centrifuge, and take the supernatant Equilibrate DEAE-52 ion exchange column with 0.05～0.1mol.L ^-1 phosphate buffer solution with pH value of 6.0～8.0, and use 0.05～0.1mol.L ^-1 phosphate buffer solution with pH value of 6.0～8.0 And 0.1 ~ 0.3mol.L ^-1 NaCl solution elution, use to collect the blue fraction, freeze-dried to obtain purified phycocyanin.

The invention has the following advantages and outstanding effects: easy method, convenient operation, fast economy, safety and reliability, high output, good product quality, and the equipment used is simple and suitable for industrial scale production.

Detailed ways

The specific implementation steps of extracting the phycocyanin of slender mats are as follows:

A. Preparation of culture medium

Add 1.5g of sodium nitrate, 0.04g of dipotassium hydrogen phosphate, 0.075g of magnesium sulfate, 0.036g of calcium chloride, 0.006g of citric acid, 0.006g of ferric ammonium citrate, 0.001g of disodium edetate, Sodium carbonate 0.02g, trace element solution 1ml. Add 286 mg of boric acid, 181 mg of manganese chloride tetrahydrate, 22.2 mg of zinc sulfate heptahydrate, 25.2 mg of sodium molybdate dihydrate, and 7.9 mg of copper sulfate pentahydrate into the trace element solution per 100 ml of distilled water, and stir well after completely dissolving.

B. Cultivation of slender algae

(1) Primary culture: Put the slender algae species into a triangular flask (150-200ml) filled with culture medium, culture statically or with aeration. During aeration culture, adjust the airflow to 2.5L.min ^-1 Sterile treatment was carried out by cotton balls, the light intensity was controlled at 70μE.m ^-2 .s ^-1 , and the temperature was controlled at 30°C. After 7-10 days of cultivation, transfer to secondary culture.

(2) Secondary culture: put the culture obtained from the primary culture into a culture bottle (500-1000ml) filled with culture medium, culture with ventilation, adjust the airflow to 3L.min ^-1 , aseptically treat, light Strong control at 80μE.m ^-2 .s ^-1 , temperature at 30°C. After culturing for 7-10 days, transfer to the tertiary culture.

(3) Tertiary cultivation: transfer the culture obtained from the secondary cultivation into a culture bottle (5,000-20,000ml) filled with culture medium, culture with ventilation, adjust the airflow to 5L.min ^-1 , aseptically treat, light Strong control at 90μE.m ^-2 .s ^-1 , temperature at 30°C. After 7 to 10 days of cultivation, it can be used as an inoculation source for continued cultivation.

(4) Continue to train

①Cultivation in a small circulation culture pool (1-2m ³ ): use the algal species obtained after the tertiary culture, and insert them into the small circulation culture pool. The glass greenhouse is used to control the temperature at 30°C, the natural light is used for illumination, and the shading net is used to control the light intensity at 120-180 μE.m ^-2 .s ^-1 , and the culture medium is stirred by an impeller or a submersible pump.

②Large circulation culture tank (40-60m ³ ) cultivation: transfer the 4-7 days of slender algae cultured in the small circulation culture tank to the large circulation culture tank, and the difference between the culture fluid in the small circulation culture tank and the large circulation culture tank The volume ratio is 1:20-25, and other culture conditions are the same as those of the small circulation culture tank.

C, the preparation of slender mat algae powder

Pump the fine mat algae cultured in the large circulation culture tank for 15 to 20 days to the filter device—the vibrating inclined sieve. Slender mat algae mud. Thin mat algae powder is obtained by concentrating, dehydrating, drying and pulverizing the slim mat algae mud.

D. Extraction of slender mat phycocyanin

Add 0.05-0.1 mol.L ^-1 phosphate buffer solution with a pH value of 6.0-8.0, 7-10 times the weight of the algae powder, and stir well. Then put it in the freezer at -10~-20°C, take it out after freezing and put it in the thaw at 20~30°C, and freeze it after it is completely melted. After repeated freezing and thawing for 3 to 5 times, most of the phycocyanin was extracted from the cells of Pseudocladon algae, and then centrifuged at 3000 to 6000 rpm for 10 to 15 minutes. Collect the supernatant, which is the crude phycocyanin extract.

E. Purification

Under ice-bath conditions, add ammonium sulfate with a saturation of 50-70% to the crude extract of phycocyanin and salt out in the dark for 20-40 minutes, then centrifuge at 3000-6000 rpm for 15-25 minutes at 3-5°C, discard Remove the supernatant, dissolve the precipitate with one volume of 0.05-0.1mol.L ^-1 phosphate buffer solution with a pH value of 6.0-8.0, dialyze in the dark for 20-30 hours and then centrifuge, take the supernatant and wash it with 0.05-0.1 mol.L ^-1 , pH 6.0-8.0 phosphate buffer equilibrated DEAE-52 ion exchange column, and 0.05-0.1 mol.L ^-1 phosphate buffer pH 6.0-8.0 and 0.1-0.3 mol.L ^-1 NaCl solution elution, using an automatic fraction collector to collect the blue fraction, freeze-dried to obtain purified phycocyanin.

Claims (2)

1. a method for extracting phycocyanin from Matella slenderis, is characterized in that comprising the steps: A. Preparation of culture medium: Add 0.8-2.5g of sodium nitrate, 0.02-0.05g of dipotassium hydrogen phosphate, 0.06-0.09g of magnesium sulfate, 0.02-0.05g of calcium chloride, 0.004-0.008g of citric acid, and citric acid per liter of water. Ammonium ferric acid 0.004～0.008g, EDTA disodium salt 0.001～0.003g, sodium carbonate 0.01～0.04g, trace element solution 0.5～2ml, wherein trace element solution was added boric acid 270～300mg per 100ml of distilled water, 170-200 mg of manganese chloride tetrahydrate, 18-25 mg of zinc sulfate heptahydrate, 15-30 mg of sodium molybdate dihydrate, 6-10 mg of copper sulfate pentahydrate, completely dissolved and stirred to obtain a culture medium; B. Cultivation of slender algae: the first is the primary culture, when the slender algae species are inserted into the culture medium for static culture or aerated culture, the airflow is adjusted at 1.5~3.5L.min ^-1 , and the light intensity is controlled at 65~85μE .m ^-2 .s ^-1 , the temperature is controlled at 22-35°C, after 5-10 days of cultivation, transfer to the secondary culture; followed by the secondary culture, the culture obtained from the primary culture is inserted into a new Aerated culture in the culture medium, adjust the airflow at 2.5~5L.min ^-1 , aseptic treatment, control the light intensity at 75~95μE.m ^-2 .s ^-1 , control the temperature at 22~35℃, after 5~10 days of cultivation Transfer to the third-level culture; the third is the third-level culture, the culture obtained from the second-level culture is inserted into the new culture medium for aerated culture, the airflow is adjusted at 3-6L.min ^-1 , aseptic treatment, light intensity Controlled at 70-100μE.m ^-2 .s ^-1 , temperature at 22-35°C, cultured for 5-10 days as the source of inoculation for continued cultivation; the fourth is continued cultivation, the first is cultivation in a small circulation culture pool, and the The algal species obtained after the tertiary cultivation are connected to a small circulation culture tank, the temperature is controlled at 22-35°C, the natural light is used for illumination, the light intensity is controlled at 120-180μE.m ^-2 .s ^-1 , and the culture medium is stirred; followed by Cultivate in a large circulation culture tank, transfer the slender algae after 4 to 7 days of cultivation in the small circulation culture tank to the large circulation culture tank, and filter the slender mats after 10 to 20 days of cultivation in the large circulation culture tank to obtain the slender algae pulp; C. Preparation of slender mat algae powder: centrifuge the algae pulp to obtain slender mat algae mud, concentrate, dehydrate, dry and pulverize the fine mat algae powder to obtain slender mat algae powder; D. Extraction of phycocyanin from Algae spp.: add 7-10 times the weight of 0.05-0.1mol.L ^-1 phosphate buffer solution with a pH value of 6.0-8.0 to the powder of S. Freeze at 10～-20°C, take it out after freezing, put it at 20～30°C to melt, wait until it is completely melted, and then freeze it, repeat the freezing and thawing process for 3 to 5 times, and then set it at 3000～6000rpm Centrifuge for 10-15 minutes, collect the supernatant, which is the crude phycocyanin extract; E. Purification: Add ammonium sulfate with a saturation of 50-70% to the crude extract of phycocyanin in an ice bath for 20-40 minutes in the dark, and then centrifuge at 3000-6000 rpm for 15 minutes at 3-5°C. ~25min, discard the supernatant, dissolve the precipitate with one volume of 0.05~0.1mol.L ^-1 phosphate buffer solution with a pH value of 6.0~8.0, dialyze in the dark for 20~30h, centrifuge, and take the supernatant Equilibrate DEAE-52 ion exchange column with 0.05～0.1mol.L ^-1 phosphate buffer solution with pH value of 6.0～8.0, and use 0.05～0.1mol.L ^-1 phosphate buffer solution with pH value of 6.0～8.0 And 0.1 ~ 0.3mol.L ^-1 NaCl solution elution, use to collect the blue fraction, freeze-dried to obtain purified phycocyanin. 2. The method for extracting phycocyanin from Matella slenderosa according to claim 1, characterized in that: the volume ratio of the culture solution in the small circulation culture tank to the large circulation culture tank is 1:20-25.