CN104447967A - Method for simultaneously extracting phycoerythrin and sulphated porphyra polysaccharide from inferior nori - Google Patents

Abstract

The invention provides a method for simultaneously extracting phycoerythrin and laver sulfated polysaccharide from inferior laver, first extracting phycoerythrin from fresh inferior laver, and then extracting laver sulfate polysaccharide from the residue from which phycoerythrin has been extracted, Phycoerythrin and laver sulfated polysaccharides are simultaneously extracted in one extraction process. The invention can obtain phycoerythrin and laver sulfated polysaccharide in one extraction, the obtained phycoerythrin has high purity, and the laver sulfated polysaccharide has good uniformity. The invention has the advantages of simple operation, simple process, fast separation speed and low cost, improves the utilization efficiency of inferior laver, and realizes repeated use and multiple utilization of resources.

Description

A method for simultaneously extracting phycoerythrin and laver sulfated polysaccharide from inferior laver

technical field

The invention relates to the extraction and separation of algae functional substances, in particular to a method for simultaneously extracting phycoerythrin and laver sulfated polysaccharide from laver.

Background technique

Porphyra is a class of macroalgae with important economic value, and several species are currently widely cultivated in coastal countries of Southeast Asia. According to statistics, the annual output of laver in 2006 was about 1.8×10 ⁴ tons of dry product, and the annual output value was estimated to be about 1.3 billion US dollars (FAO, 2006). my country's laver industry is developing rapidly. From the point of view of distribution area, altar laver is the main one in the south of the Yangtze River, mainly concentrated in Fujian and Guangdong provinces; zebra laver is the main one in the north of the Yangtze River, mainly concentrated in Jiangsu and Shandong provinces. Among them, the cultivation area of laver in Jiangsu Province is about 330,000 mu, and the number of employees is about 80,000. In Nantong, Yancheng and Lianyungang, the main production areas of laver in my country have been formed, with an annual output of 4 billion standard products and a total industry output value of 2 billion. around yuan. The produced laver is processed into products after primary and secondary processing locally, and sold all over the world, becoming an important part of my country's export earnings.

In the production of Porphyra zebra, inferior laver has high glue content and low protein content, which leads to low quality of primary processed products, and bad taste of secondary processed products, so the price is low, which in turn affects the economic benefits of enterprises. These inferior laver are often treated as garbage together with the discarded laver produced in the processing process, which not only wastes resources, but also increases the burden on the environment. Therefore, the high-value processing of these inferior laver and the extraction of high value-added substances have become the key to the sustainable development of the laver industry.

Phycobiliprotein is a water-soluble pigment protein with unique absorption spectrum and fluorescence emission spectrum. Its existence complements the light absorption range of chlorophyll, broadens the absorption of visible light by algae, and is more conducive to the adaptation to the aquatic environment. Porphyrophycoerythrin is a type I R-phycoerythrin. Because of its unique spectral characteristics, stability, high absorption coefficient and quantum yield, phycobiliprotein can be used as a fluorescent probe for biochemical research. Markers for cells and biomacromolecules can also be used as photosensitizers for tumor therapy, and it has even been reported to have immunomodulatory effects. In addition, phycobiliprotein can also be used as a natural pigment for the addition of food and cosmetics, avoiding the possible toxicity hazards caused by chemically synthesized pigments.

Porphyra polysaccharide is one of the main components of its cells, which has the functions of anticoagulation, lowering blood fat, anti-aging and immune regulation. It has been reported that Porphyra polysaccharides can directly scavenge free radicals in the body by reducing MDA levels in mice to improve their antioxidant capacity. Therefore, laver polysaccharide is an ideal important component that can be developed into anti-aging food and medicine. There are a large number of polar groups such as hydroxyl or carboxyl in polysaccharide molecules, which can form hydrogen bonds with water molecules, showing good moisture absorption and moisturizing properties, so it also has broad application prospects in medicine, cosmetics, agriculture, etc. Polysaccharide components that can activate macrophages and enhance immune function can be isolated from Porphyra zebra.

People then thought of using laver to extract phycobiliprotein and laver polysaccharide at the same time, and a lot of research has been done for this.

Xiao Haifang and others explored the process of simultaneously extracting proteins and polysaccharides from laver with pulsed ultrasonic waves (Research on the process of simultaneously extracting proteins and polysaccharides from laver with pulsed ultrasonic waves, Food Science, 2007, Vol.28, No.06), Zhu Xiaojun et al. Process for Simultaneous Extraction of Laver Polysaccharides and Phycobiliproteins (Optimization of Ultrasonic-assisted Simultaneous Extraction of Porphyra zebra Polysaccharides and Phycobiliproteins, Food Science, 2008, Vol.29, No.05). Chinese patent CN200810018871.0 discloses a method for synchronous ultrasonic-assisted extraction of Porphyra zebra polysaccharides and proteins. However, these extraction processes all use dried laver powder, and the obtained phycobiliprotein has very low spectral activity, and the polysaccharide will inevitably be polluted by other molecules such as protein during the extraction process, and the composition is complex.

Contents of the invention

The object of the present invention is to provide a method for simultaneously extracting phycoerythrin and laver sulfated polysaccharide from inferior laver, the obtained phycoerythrin has high purity, less pollution of polysaccharide, simple extraction process, and improves utilization rate of inferior laver resources, Improve the economic benefits of laver industry.

In order to achieve the above object, the technical solution adopted by the present invention is:

A method for simultaneously separating and extracting phycoerythrin and sulfated polysaccharides from inferior laver, first extracting phycoerythrin from fresh inferior laver, and then extracting laver sulfated polysaccharides from the residue after extracting phycoerythrin, in one extraction process Simultaneously extract phycoerythrin and laver sulfate polysaccharide.

Among them, the specific steps are as follows:

A. Phycoerythrin Extraction

(1) Mince fresh inferior laver, put into 1% phosphate buffer saline to make the cells swell and burst;

(2) Centrifuge, collect the supernatant, obtain the crude phycoerythrin extract, add solid ammonium sulfate to a final concentration of 0.5M;

(3) Pump the phycoerythrin crude extract into the Phenyl-Sepharose column, wash and remove impurity proteins;

(4) Use ammonium sulfate solution and distilled water to elute phycoerythrin from the Phenyl-Sepharose column in turn;

(5) Add the eluate obtained in step (4) to the DEAE-Sepharose ion exchange column respectively, first wash the column with 50mM, 100mM phosphate buffer, 10mM sodium acetate, 4mM acetic acid, and then wash the column with phosphate containing NaCl Buffer elution to obtain purified phycoerythrin;

B. Extraction of laver sulfated polysaccharides from residues

(1) Utilize the remaining residue after extracting phycoerythrin, under 10 times the volume of 1% phosphate buffer, pH 6.0 acidity, control the temperature at 110-121°C, extract for 1 hour, filter with gauze; add 5 Double the volume of 1% phosphate buffer solution, repeat the above operation, extract and filter again; then combine the two filtrates to obtain the crude polysaccharide extract; concentrate the crude polysaccharide extract by rotary evaporation;

(2) adding ethanol to the concentrated polysaccharide crude extract, collecting the precipitate, and freeze-drying to obtain laver sulfated polysaccharide dry powder.

Wherein, the pH of the 1% phosphate buffer solution in step A (1) is 6.0.

Wherein, the centrifugation in the step A(2) is performed at a speed of 9000-11000g and a temperature of 5-10°C for 10 minutes.

Wherein, step A (4) is to use 0.20, 0.10, 0.05M ammonium sulfate solution and distilled water in sequence to elute phycoerythrin from the Phenyl-Sepharose column at a rate of 3-5 ml/min.

Wherein, the elution with phosphate buffer containing NaCl described in step A (5) is sequentially using 50mM phosphate buffer containing 0.15M NaCl, 50mM phosphate buffer containing 0.20M NaCl, and 0.2M NaCl containing phosphate buffer. Elution with NaCl in 100 mM phosphate buffer.

Wherein, step B (2) is to add 4 times the volume of ethanol for precipitation.

In the present invention, the porphyra algae are crushed, and the water-soluble substances are extracted with a Phenyl-Sepharose expansion column under the condition of a certain concentration of salt ions to obtain a phycoerythrin product, and then the DEAE-Sepharose ion exchange column is used to purify the phycoerythrin. The pure product of phycoerythrin is obtained, and then the remaining residue of extracting phycoerythrin is used for the extraction of laver sulfated polysaccharide. The spectral purity of the purified phycoerythrin is greater than 3.2, and the yield is 0.66 mg/g; the yield of the obtained sulfated polysaccharide is 32.9 mg/g.

The invention firstly purifies active phycoerythrin and then extracts sulfated polysaccharides, thereby achieving the purpose of comprehensive utilization of biomass, and at the same time, due to the extraction of phycobiliproteins, the pollution of protein and starch and other sugars during the extraction of sulfated polysaccharides is reduced. Therefore, the method can not only obtain reagent-grade phycoerythrin with a spectral purity higher than 3.2, but also simultaneously obtain sulfated polysaccharides with better uniformity.

The present invention has the following advantages:

1. The operation process is simple. The extracted raw materials only need to be chopped, freeze-thawed, and filtered to obtain the crude extract. Phycobiliproteins do not need to be pretreated before separation.

2. The present invention uses the residue after protein extraction to extract polysaccharides. On the one hand, the purpose of fully utilizing resources is achieved. On the other hand, after protein extraction, protein pollution during polysaccharide extraction is reduced, killing two birds with one stone.

3. The separation speed of phycobiliprotein is fast. A complete separation process only takes 3.5 hours, including 30 minutes for column equilibration, 60 minutes for sample loading, 50 minutes for washing, and 60 minutes for elution.

4. The present invention proposes a simple and efficient method for comprehensive utilization of laver, the purified phycoerythrin spectral purity is greater than 3.2, the yield is 0.66mg/g, the yield of sulfated polysaccharide is 32.9mg/g, and the polysaccharide has good uniformity , the utilization rate of laver can be greatly improved by the present invention.

The invention utilizes inferior laver to simultaneously extract phycobiliprotein and laver sulfated polysaccharide, and has the advantages of high spectral purity of obtained phycoerythrin and little pollution of laver sulfated polysaccharide. The operation is simple, the process is simple, the separation speed is fast, and the cost is low, the utilization efficiency of inferior laver is improved, and the repeated use and multiple utilization of resources are realized.

Description of drawings

Figure 1 is the absorption spectrum at 250-800nm of the crude extract of Porphyra zebra.

Figure 2A is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column with 0.2M ammonium sulfate solution.

Figure 2B is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column by 0.1M ammonium sulfate solution.

Figure 2C is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column with 0.05M ammonium sulfate solution.

Figure 2D is the absorption spectrum at 250-800nm of phycoerythrin eluted from Pheny-Sepharose expansion column by distilled water.

Fig. 3A is the absorption spectrum at 250-800nm of phycoerythrin purified by ion exchange column.

Fig. 3B is the fluorescence emission spectrum of phycoerythrin after ion exchange resin purification.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present invention.

Embodiment 1, the extraction and purification method of phycoerythrin

1. Weigh 100g wet weight of fresh inferior laver and chop it up, add 1% phosphate buffer saline 10 times the weight of the algal body, overnight at 4°C to swell the cells.

2. Centrifuge for 10 minutes at a rotational speed of 9000g and a temperature of 5-10°C, collect all the supernatant, and obtain 340ml of crude phycoerythrin extract. All residues were collected and stored in a -20°C refrigerator for extraction of sulfated polysaccharides.

The OD value of the crude extract of phycoerythrin was measured, and then according to the formula R-PE=155.8OD498.5-40.0OD614-10.5OD651, the crude extract contained 53.4mg of phycoerythrin, and the yield was 5.34mg/g.

Fig. 1 is the absorption spectrum at 250-800nm of the crude extract of Porphyra variegata phycoerythrin.

3.Pheny-Sepharose expanded column extraction. First equilibrate the Pheny-Sepharose expanded column with 0.5M ammonium sulfate solution. At room temperature, add solid ammonium sulfate to the crude phycoerythrin extract to a final concentration of 0.5M, and pump it into the expansion column from bottom to top at a speed of 15ml/min, and the phycobiliprotein is captured by the adsorbent. Then wash the column with 0.5M ammonium sulfate solution to wash away impurities and cell debris until the effluent has no color.

4. Sequentially use 0.20, 0.10, 0.05M ammonium sulfate solution and distilled water to elute phycoerythrin from top to bottom at a speed of 15ml/min. Collect all the eluate, measure its volume after dialysis, and measure the absorption spectrum at 250-800nm.

Figure 2A is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column with 0.2M ammonium sulfate solution.

Figure 2B is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column by 0.1M ammonium sulfate solution.

Figure 2C is the absorption spectrum at 250-800nm of phycoerythrin eluted from a Pheny-Sepharose expanded column with 0.05M ammonium sulfate solution.

Figure 2D is the absorption spectrum at 250-800nm of phycoerythrin eluted from Pheny-Sepharose expansion column by distilled water.

0.20, 0.10, and 0.05M ammonium sulfate solution are shown in Table 1 for the purity and content of samples eluted from the expansion column. The total protein content was 91.2 mg, and the yield was 0.912 mg/g.

Table 1 Yield and purity of phycoerythrin eluted with different ammonium sulfate concentrations

5. Purification by DEAE-Sepharose ion exchange column. Pump the eluate obtained in the previous step into the DEAE-Sepharose ion exchange column, first wash the column with 50mM, 100mM phosphate buffer (PH 6.8), then wash the column with 10mM sodium acetate, wash the column with 4mM acetic acid, and then wash the column with 50mM Add 0.15M NaCl to phosphate buffer, 0.20M NaCl to 50mM phosphate buffer, and 0.20M NaCl to 100mM phosphate buffer for elution. Collect the red eluent, measure the volume, and determine the absorption spectrum at 250-800nm.

Fig. 3A is the absorption spectrum at 250-800nm of phycoerythrin purified by ion exchange column.

Fig. 3B is the fluorescence emission spectrum of phycoerythrin after ion exchange resin purification.

The purity and content of the samples eluted from the ion-exchange column with different concentrations of phosphate buffer and sodium chloride are shown in Table 2. The spectral purity of the purified phycoerythrin is greater than 3.2, the total protein amount is 66 mg, and the yield is 0.66 mg/g.

Table 2 Yield and purity of phycoerythrin purified under different salt ion conditions

Embodiment 2, the extraction of sulfated polysaccharide

Put the residue obtained after extracting the crude phycoerythrin extract into a conical flask, add 10 times the volume of 1% phosphate buffer with a pH of 6.0, control the temperature at 110-121°C for 1 hour, and then 8 layers of gauze Filtration; add 5 times the volume of 1% phosphate buffer solution with pH 6.0 to the filter residue again, boil for another 1 hour at the same temperature; filter and combine the two filtrates to obtain the crude polysaccharide extract; rotate the crude polysaccharide extract Evaporate to a quarter of the original volume; add 4 times the volume of alcohol, collect the precipitate, and freeze-dry. The results are shown in Table 3.

Table 3 Yield and sulfate group content of Porphyra polysaccharides under different extraction conditions

It can be seen that 3.29 grams of sulfated polysaccharides can be obtained with a yield of 3.29% by using 100 g of fresh algal body wet weight to extract the remaining residue after extracting phycoerythrin.

During the extraction process, after the crude polysaccharide extract was concentrated, 3% trichloroacetic acid was added, left standing overnight at 4°C, and centrifuged, but no precipitation was obtained, indicating that there was little protein contamination in the crude polysaccharide extract.

Add 2 times, 4 times and 8 times the volume of ethanol in sequence to the crude polysaccharide extract, let stand at 4°C overnight, centrifuge, and collect the polysaccharide precipitate step by step. The polysaccharide precipitation obtained by 2 times the volume accounts for the vast majority, the amount of polysaccharides obtained by 4 times the volume is relatively low, and no polysaccharide precipitation can be obtained when the alcohol treatment is 8 times the volume. It shows that 4 times the volume of ethanol can basically precipitate the polysaccharide.

The invention can obtain phycoerythrin and laver sulfated polysaccharide in one extraction, and the obtained phycoerythrin has high spectral purity, and the laver sulfated polysaccharide has no protein pollution and good uniformity. The invention has the advantages of simple operation, simple process, fast separation speed and low cost, improves the utilization efficiency of inferior laver and realizes the comprehensive utilization of resources.

The above is the description of the embodiments of the present invention, through the above description of the disclosed embodiments, those skilled in the art can realize or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (6)

1. one kind is extracted the method for phycoerythrin and laver sulfated polysaccharide from inferior laver simultaneously, it is characterized in that, first phycoerythrin is extracted from fresh inferior laver, from the residue being extracted phycoerythrin, extract laver sulfated polysaccharide again, in a leaching process, extract phycoerythrin and laver sulfated polysaccharide simultaneously.

2., by the method extracting phycoerythrin and laver sulfated polysaccharide from inferior laver according to claim 1 simultaneously, it is characterized in that, concrete steps are as follows:

A. phycoerythrin extracts

(1) by fresh inferior laver chopping, put into 1% phosphate buffered saline buffer and make that its cell is swelling to break;

(2) centrifugal, collect supernatant liquor, obtain phycoerythrin crude extract, adding solid ammonium sulfate to final concentration is 0.5M;

(3) phycoerythrin crude extract is pumped into Phenyl-Sepharose post, foreign protein is removed in cleaning;

(4) use ammoniumsulphate soln, distilled water from Phenyl-Sepharose post wash-out phycoerythrin successively;

(5) elutriant of gained in step (4) is added DEAE-Sepharose ion exchange column respectively, first wash post with 50mM, 100mM phosphate buffered saline buffer, 10mM sodium-acetate, 4mM acetic acid respectively, then with the phosphate buffered saline buffer wash-out containing NaCl, the phycoerythrin of purifying is obtained;

B. from residue, laver sulfated polysaccharide is extracted

(1) remaining residue after utilization extraction phycoerythrin, under 10 times of volume 1% phosphate buffered saline buffers, pH6.0 acidity, control temperature, at 110 ~ 121 DEG C, extracts 1 hour, filtered through gauze; In filter residue, add 1% phosphate buffered saline buffer of 5 times of volumes, repeat aforesaid operations, again carry out extracting, filtering; Then merge twice filtrate, obtain liquid of extracting polysaccharide; Liquid of extracting polysaccharide rotary evaporation is concentrated;

(2) in the liquid of extracting polysaccharide after concentrated, add ethanol, collecting precipitation, lyophilize, obtain laver sulfated polysaccharide dry powder.

3., by a kind of method extracting phycoerythrin and laver sulfated polysaccharide from laver according to claim 2 simultaneously, it is characterized in that, centrifugal in steps A (2) be rotating speed be 9000 ~ 11000g, temperature be 5 ~ 10 DEG C of conditions under centrifugal 10 minutes.

4. by a kind of method extracting phycoerythrin and laver sulfated polysaccharide from laver according to claim 2 simultaneously, it is characterized in that, steps A (4) be use 0.20 successively, 0.10,0.05M ammoniumsulphate soln, distilled water with the speed of 3 ~ 5ml/min from Phenyl-Sepharose post wash-out phycoerythrin.

5. by the method extracting phycoerythrin and laver sulfated polysaccharide from laver according to claim 2 simultaneously, it is characterized in that, use described in steps A (5), containing the phosphate buffered saline buffer wash-out of NaCl, is successively with 50mM phosphate buffered saline buffer, the 50mM phosphate buffered saline buffer containing 0.20MNaCl, the 100mM phosphate buffered saline buffer wash-out containing 0.2M NaCl containing 0.15M NaCl.

6. by a kind of method extracting phycoerythrin and laver sulfated polysaccharide from laver according to claim 2 simultaneously, it is characterized in that, step B (2) is that the ethanol adding 4 times of volumes precipitates.