CN101220080B - Method for extracting phycocyanin from Microcoleus vaginatus - Google Patents

Abstract

The invention discloses a method for extracting a phycocyanin from a microcoleus vaginatus, which comprises the following steps: preparing the microcoleus vaginatus culture medium with a sodium nitrate, a potassium phosphate dibasic, a magnesium sulfate, a calcium chloride, a citric acid, a ferric ammonium citrate, an EDTA, a sodium carbonate and a trace element solution according to a certain proportion, culturing the microcoleus vaginatus in the culture medium and making the microcoleus vaginatus powder, extracting a phycocyanin from the microcoleus vaginatus powder and purifying the phycocyanin. The method for extracting a phycocyanin from a microcoleus vaginatus has the advantages of simplicity, convenient operation, safety and reliability, high yield, good product quality and simple operated equipment, which is suitable for industrial production.

Description

A kind of method for extracting phycocyanin from Microtheca sheath

technical field

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

Background technique

Microsheathing algae belongs to Cyanophyta, Cyanophyta, Seglophytes, Oscillatoriaceae, Microcoleum genus. It is the dominant population in biological crusts in arid and semi-arid regions. At present, the utilization of Microcoleans generally only stays in artificial algae crusts. The reason why Microcoleans is used to form artificial algal 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 phycocyanins are extracted from spirulina and large seaweed, and their phycocyanin content is mostly higher than that of sheaths. Microcoleum is lower. In addition, the cost of cultivating macroalgae is far higher than the cost of cultivating Microcoleum.

Contents of the invention

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

In order to achieve the above object, the present invention provides a method for extracting phycocyanin from Microtheca sheaths 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 Microcoletheca: The first is the first-level culture, inserting the species of Microcoletheca into the culture medium for ventilation and culturing, adjusting the airflow at 1.5~3.5L.min ^-1 , and controlling the light intensity at 65~ 85μE.m ^-2 .s ^-1 , temperature controlled at 22-35°C, cultured for 5-10 days, then transferred to secondary culture; followed by secondary culture, the culture obtained from the primary culture was inserted into a new culture Culture in liquid with aeration, adjust the airflow at 2.5-5L.min ^-1 , aseptically treat, control the light intensity at 75-95μE.m ^-2 .s ^-1 , control the temperature at 22-35°C, and transfer after 5-10 days of cultivation. Enter the tertiary culture; the third is the tertiary culture, insert the culture obtained from the second-level culture into the new culture medium for ventilation and culture, adjust the airflow at 3~6L.min ^-1 , aseptic treatment, light intensity control At 70-100μE.m ^-2 .s ^-1 , the temperature is controlled at 22-35°C, and after 5-10 days of cultivation, it will be used as the inoculation source for continued cultivation; The algal species obtained after the first-grade culture are connected to a small circulation culture pool, 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 Microtheca sheaths after cultivating in the small circulation culture tank for 4 to 7 days to the large circulation culture tank, and the volume ratio of the culture solution in the small circulation culture tank to the large circulation culture tank is 1: 20-25, control the temperature in the large circulation culture pool at 22-35°C, use natural light for illumination, and control the light intensity at 120-180μE.m ^-2 .s ^-1 to filter Microcoletheca sheaths after 10-20 days of cultivation Harvesting the algae pulp of Microcoletheca sheath;

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

C. Preparation of Microcoletheca sheath powder: the alga pulp is centrifuged to obtain Microcoletheca sheath algae mud, which is concentrated, dehydrated, dried and pulverized to obtain Microcoletheca sheatha powder;

D. Extraction of Microcoletheca phycocyanin: Add 0.05 to 0.1 mol.L ^-1 of 7 to 10 times the weight of Microcolethecus powder, and a phosphate buffer solution with a pH value of 6.0 to 8.0, fully Stir well, then freeze at -10~-20°C, take it out after freezing, put it at 20~30°C to melt, wait until it is completely melted, then freeze it, repeat freezing and thawing 3~5 times , and then centrifuged at 3000-6000rpm for 10-15min, and the supernatant was collected, which was 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 present invention has the following advantages and outstanding effects: the method is easy to implement, economical and quick, convenient to operate, high in output, good in product quality, safe and reliable, and the equipment used is simple and suitable for industrial scale production.

Detailed ways

The specific implementation steps of extracting phycocyanin from Microcoletheca sheaths 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, wherein trace element solution is added boric acid 286mg, manganese chloride tetrahydrate 181mg, zinc sulfate heptahydrate 22.2mg, sodium molybdate dihydrate 25.2mg, copper sulfate pentahydrate 7.9 mg per 100ml of distilled water. mg, completely dissolved and then stirred to obtain a culture medium.

B. Cultivation of Microsheathing algae

(1) Primary culture: put Microtheca sp. into a conical flask (150-200ml) filled with culture medium, culture statically or with aeration. During aeration culture, adjust the airflow to 2.5L.min ^-1 , the airflow passes through cotton balls for sterile treatment, the light intensity is controlled at 70μE.m ^-2 .s ^-1 , and the temperature is controlled at 30°C. After 7-10 days of cultivation, transfer to secondary cultivation.

(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 7 to 10 days of cultivation, transfer to the tertiary cultivation.

(3) Tertiary cultivation: transfer the culture obtained from the secondary cultivation into a culture bottle (5000-20000ml) filled with culture medium, culture with aeration, adjust the air flow to 5L.min ^-1 , aseptically treat, light intensity The temperature is controlled at 90μE.m ^-2 .s ^-1 and 30°C. After 7-10 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 Microtheca sheaths cultured in the small circulation culture tank for 4 to 7 days to the large circulation culture tank, and cultivate in the small circulation culture tank and the large circulation culture tank. The volume ratio of the solution is 1:20-25, and the other culture conditions are the same as those of the small circulation culture tank.

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

C. Preparation of Microcoletheca powder

After 15 to 20 days of culture in the large circulation culture tank, Microcoletheca sheaths are pumped to the filter device—the vibrating slope sieve. After being centrifuged by a centrifuge, the algae mud of Microcoletheca sheaths is obtained. The microsheathing algae mud is concentrated, dehydrated, dried and pulverized to obtain the microsheathing algae powder.

D. Extraction of Microcoletheca phycocyanin

Add 0.05-0.1 mol.L ^-1 phosphate buffer solution with a pH value of 6.0-8.0 in 7 to 10 times the weight of the microsheathing algae powder, and stir well. Then freeze it at -10~-20°C, take it out after freezing, put it at 20~30°C to melt, and freeze it after it is completely melted. So repeated freezing and thawing 3 to 5 times. Most of the phycocyanin is extracted from the cells of Pseudocladon algae, then centrifuged at 3000-6000rpm for 10-15min, and the supernatant is collected, 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 Microcoleena sheathing, 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 Microcoletheca: firstly, primary culture, insert Microcoletheca species into the culture medium for static culture or aeration culture, adjust the airflow at 1.5-3.5L.min ^-1 , and control the light intensity At 65-85μE.m ^-2 .s ^-1 , the temperature is controlled at 22-35°C, and after 5-10 days of cultivation, it is transferred to the secondary culture; the second is the secondary culture, and the culture obtained from the primary culture is inserted into the Culture in the new culture medium with aeration, adjust the air flow at 2.5~5L.min ^-1 , aseptically treat, control the light intensity at 75~95μE.m ^-2 .s ^-1 , control the temperature at 22~35℃, and cultivate for 5~ After 10 days, 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, The light intensity is controlled at 70-100μE.m ^-2 .s ^-1 , and the temperature is controlled at 22-35°C. After 5-10 days of cultivation, it is used as the inoculation source for continued cultivation; the fourth is continuous cultivation, first of all, small circulation culture tank cultivation , put the algal species obtained after the tertiary cultivation into a small circulation culture tank, control the temperature at 22-35°C, use natural light for illumination, control the light intensity at 120-180μE.m ^-2 .s ^-1 , and stir the culture medium Next is the cultivation of the large circulation culture tank, the Microcoletheca sheaths after cultivating in the small circulation culture tank for 4 to 7 days is transferred to the large circulation culture tank, and the temperature is controlled at 22～35°C in the large circulation culture tank, using Illuminate with natural light, control the light intensity at 120-180 μE.m ^-2 .s ^-1 and culture Microcoletheca for 10-20 days to collect Microcoletheca algae pulp by filtering; C. Preparation of Microcoletheca sheath powder: the alga pulp is centrifuged to obtain Microcoletheca sheath algae mud, which is concentrated, dehydrated, dried and pulverized to obtain Microcoletheca sheatha powder; D. Extraction of Microcoletheca phycocyanin: Add 0.05 to 0.1 mol.L ^-1 of 7 to 10 times the weight of Microcolethecus powder, and a phosphate buffer solution with a pH value of 6.0 to 8.0, fully Stir well, then freeze at -10~-20°C, take it out after freezing, put it at 20~30°C to melt, wait until it is completely melted, then freeze it, repeat freezing and thawing 3~5 times , and then centrifuged at 3000-6000rpm for 10-15min, and the supernatant was collected, which was 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. A kind of method for extracting phycocyanin from Microcoletheca sheaths according to claim 1, is characterized in that: the volume ratio of culture solution in the small circulation culture tank and the large circulation culture tank is 1: 20～25 .