CN105601732A - Method for separating phycobiliprotein from laver processing wastewater - Google Patents

Abstract

The invention discloses a method for separating phycobiliprotein from laver processing wastewater. The method comprises the following steps: 1) filtering the laver processing wastewater to remove impurities to obtain the laver processing wastewater after removing impurities; 2) using ion The exchange packing adsorbs the phycobiliprotein in the laver processing wastewater after removing the impurities, and obtains the ion exchange packing with the phycobiliprotein adsorbed; 3) finally elutes the adsorbed phycobiliprotein with 0.1-0.5M sodium chloride-PBS buffer solution. The ion exchange filler with phycobiliprotein obtains the purified phycobiliprotein. The method of the invention recovers and purifies high-value phycobiliproteins from laver processing wastewater, thereby achieving the purpose of waste utilization, and at the same time reduces environmental pollution caused by direct discharge of laver processing wastewater due to the extraction of phycobiliproteins.

Description

Method for separating phycobiliprotein from laver processing wastewater

technical field

The invention relates to a method for separating phycobiliproteins, in particular to a method for separating phycobiliproteins from laver processing wastewater.

Background technique

Laver has important economic value and is one of the main economic seaweeds in my country. The main varieties of laver cultivated in my country are Porphyra altar and Porphyra variegata, among which Porphyra altar is mainly cultivated in the south of the Yangtze River (Fujian Province and Guangdong Province), and Porphyra variegata is mainly cultivated in the north of the Yangtze River (Jiangsu Province and Shandong Province, mainly in the Nantong, Yancheng and Lianyungang sea areas). 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).

Phycobiliprotein is a water-soluble pigment protein with unique absorption and fluorescence emission spectra. Phycobiliproteins include phycoerythrin, phycocyanin, and allophycocyanin. Phycobiliproteins are covalently bonded together by apoproteins and tetrapyrrole-structured chromophores through thioether bonds. They have a stable structure and can be used as fluorescent probes for biological and medical research and clinical diagnosis and treatment of diseases. Phycobiliproteins can also be used as natural pigments as food and cosmetic additives, avoiding the possible poisoning of chemically synthesized pigments. Studies have shown that phycobiliprotein can significantly improve the activity of human lymphocytes and the immune function of the body, and enhance the body's ability to prevent diseases and fight cancer. Therefore, many foreign companies have successively invested in the development of phycobiliprotein products, and the product prices are very considerable. According to statistics, such products have a market opportunity of more than 10 billion US dollars in the international market. At present, these products have also been used in China, but all rely on imports. The main reason for the high price of phycobiliprotein is the difficulty of separation and purification, so the development of new purification technology is one of the focuses of research.

Porphyra zebra processing includes primary processing and secondary processing. Primary processing includes cleaning, chopping, washing-blending-cake-making-dehydration-drying-peeling, sorting and packaging. After the washing process of chopped Porphyra variegata, due to the rupture of cells at the incision and the release of intracellular substances, a large amount of waste water produced is rich in phycobiliprotein and is red in color, commonly known as "red water". Because the chroma of "red water" exceeds the standard, direct discharge will not only waste resources, but also increase the burden on the environment. If phycobiliproteins can be separated and recovered for discharge, it will not only reduce the chroma of laver processing wastewater, reduce the environmental pollution caused by "red water discharge", but also obtain high-value product phycobiliproteins, which is conducive to the healthy and sustainable development of laver industry. .

Contents of the invention

In order to solve the above-mentioned technical problems, the object of the present invention is to provide a method for separating phycobiliproteins from laver processing wastewater, thereby reclaiming and purifying high-value phycobiliproteins from laver processing wastewater, achieving the purpose of waste utilization, and simultaneously Due to the extraction of phycobiliprotein, the environmental pollution caused by the direct discharge of laver processing wastewater is reduced.

To achieve the above object, the technical scheme of the present invention is as follows:

The invention provides a method for separating phycobiliprotein from seaweed processing wastewater, said method comprising the following steps:

1) first filter laver processing wastewater to remove impurities, and obtain laver processing wastewater after removing impurities;

2) Adsorbing the phycobiliproteins in the laver processing wastewater after the removal of impurities with ion-exchange fillers to obtain ion-exchange fillers adsorbed with phycobiliproteins;

3) Finally, 0.1-0.5M sodium chloride-PBS buffer solution is used to elute the ion-exchange filler adsorbed with phycobiliprotein to obtain purified phycobiliprotein.

Preferably, in step 3), the phycobiliprotein-adsorbed ion-exchange filler is eluted with 0.2-0.3M sodium chloride-PBS buffer, which can effectively adsorb phycobiliprotein and has a high purity.

Preferably, in step 1), the laver processing wastewater is filtered with a filter medium to remove impurities.

More preferably, the filter medium is filter cloth or silk screen.

Preferably, in step 2), the phycobiliprotein in the laver processing wastewater after removing impurities is adsorbed by using 1 to 5% by volume of the ion-exchange filler of the laver processing wastewater, which can more effectively adsorb the phycobiliprotein and save cost.

Preferably, the ion exchange packing is an anion exchange packing.

Preferably, the anion exchange filler is Q-sepharose.

Preferably, in step 2), before the ion-exchange filler adsorbs the phycobiliprotein in the laver processing wastewater after removing impurities, a step of washing the ion-exchange filler is also included.

Preferably, the ion-exchange packing is cleaned by a method comprising the following steps: washing the ion-exchange packing three times with water, standing still, and then absorbing excess water on the ion-exchange packing.

Preferably, in step 2), after obtaining the ion-exchange filler adsorbed with phycobiliprotein, the step of washing the ion-exchange filler adsorbed with phycobiliprotein with water is also included.

The invention reclaims and purifies active high-value phycobiliproteins from laver processing wastewater, achieves the purpose of waste utilization, and at the same time, due to the extraction of phycobiliproteins, reduces the environmental pollution caused by the direct discharge of laver processing wastewater; Q-agarose gel filler Reusable, low cost; simple operation, simple process, fast separation speed, full use of laver processing wastewater, obtaining high-value phycobiliproteins, reducing environmental pollution of laver processing wastewater, and realizing resource reuse.

The present invention has the following advantages:

1. The operation process is simple. The laver processing wastewater only needs to be filtered with a filter medium to remove particulate impurities such as laver residue and sludge, and no pretreatment is required before the separation of phycobiliproteins.

2. The Q-selpharose filler after protein extraction used in the present invention can be reused after washing, and the cost is low.

3. The separation speed of phycobiliprotein is fast. A complete separation process only takes about 1 hour, including 10 minutes for column equilibration, 30 minutes for sample loading, 10 minutes for washing, and 10 minutes for elution.

4. The yield of the purified phycobiliprotein of the present invention is above 0.98 mg/L, and the utilization rate of laver processing wastewater can be greatly improved by the present invention.

Description of drawings

Fig. 1 is the absorption spectrogram at 400-800nm of the crude protein liquid of the laver processing waste water that is used for separation in the present invention;

Fig. 2 is the absorption spectrum at 400-800nm of the phycobiliprotein obtained by separation and elution in the present invention.

detailed description

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.

Unless otherwise specified, the reagents used in the following examples are commercially available from regular channels.

Example 1

1. Measure 200ml of laver processing wastewater, filter out laver residue, sludge and other impurities with a sieve to obtain a crude protein solution, put it in a 250ml blue cap bottle, and put it in a 4°C refrigerator for later use.

2. According to the formula protein (Pro)=OD ₅₆₂ /0.0157, the protein content in the crude protein solution is calculated to be 4.39mg/L, and the absorption spectrum of the crude protein solution extracted by ultraviolet spectrophotometer is measured at room temperature, as shown in Figure 1 The absorption spectrum of the crude protein solution at 400-800nm, there are many absorption peaks at 400-450nm in the figure, indicating that there are many impurities.

3. Take 2ml of Q-Sepharose (Q-selpharose) stored in alcohol and fill it into a 50ml centrifuge tube, add 10 times the volume of distilled water, and shake to disperse the Q-Sepharose (Q-selpharose) filler. Let stand to make the Q-sepharose filler sink. Aspirate the supernatant. Repeat three times to fully remove the alcohol.

4. Add the alcohol-washed Q-Sepharose (Q-selpharose) filler to the crude protein solution at 20°C, and shake quickly to make the phycobiliprotein adsorb to the Q-Sepharose (Q-selpharose) filler superior.

5. Let the filler settle and discard the supernatant. The Q-selpharose filler adsorbed with phycobiliprotein was transferred to a 10ml centrifuge tube and washed with 10ml of distilled water. 6. The phycobiliproteins were eluted with 0.1M, 0.2M, 0.3M sodium chloride solutions prepared with 10ml of phosphate buffer respectively. And at room temperature adopt the absorption spectrum of the phycobiliprotein that extracts by ultraviolet spectrophotometer measurement, Fig. 2 is the absorption spectrum of the phycobiliprotein that eluting obtains at 400-800nm, according to phycoerythrin, phycocyanin and allophycocyanin Absorption wavelength range (see: NiuJF, WangGC, LinXZ, et al. Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bedad absorption chromatography [J]. Journal of ChromatographyBAnalyticalTechnologiesintheBiomedical&LifeSciences, 2007, 850 (1-2) can be known as blue peak 1, 267 in the figure of allocyanin , peak 2 is phycocyanin, and peaks 3 and 4 are phycoerythrin, indicating that the purified phycobiliprotein has a higher purity.

7. Determination of crude protein liquid, Q-Sepharose (Q-selpharose) packing waste liquid after phycobiliprotein was adsorbed by BAC protein assay kit, and protein concentration of phycobiliprotein obtained by elution, see Table 1. The total protein content of the crude protein solution was 878 μg, the total protein content of the purified phycobiliprotein was 197 μg, and the yield was 0.98 mg/L.

Table 1 The volume of eluent and protein concentration before and after adsorption of laver processing wastewater

Example 2

1. Measure 1L of seaweed processing wastewater, filter out seaweed residue, sludge and other impurities with a filter cloth to obtain a crude protein solution, put it in a 1L round bottle with a vent at the bottom, and put it in a 4°C refrigerator for later use.

2. According to the formula protein concentration (mg/L) = OD ₅₆₂ /0.0157, the protein content in the crude protein liquid is calculated to be 4.39 mg/L.

3. Take 20ml of Q-Sepharose (Q-selpharose) stored in alcohol and stuff it into a 500ml sampling bottle, add 10 times the volume of distilled water, shake to make the Q-Sepharose (Q-selpharose) filler disperse open. Let stand to allow the filling to sink. Aspirate the supernatant. Repeat three times to fully remove the alcohol.

4. At 25°C, add the Q-selpharose filler that has been washed off the alcohol into the crude protein solution, and blow the Q-selpharose filler with air at the bottom to make the algae The biliprotein is in full contact with the Q-sepharose filler, so that the phycobiliprotein is adsorbed onto the Q-selpharose filler.

5. Allow the Q-Sepharose filler to settle and discard the supernatant. Transfer the Q-selpharose filler adsorbed with phycobiliprotein to a 500ml sampling bottle, and wash with 200ml distilled water.

6. The phycobiliproteins were eluted with 0.1M, 0.2M, 0.3M, 0.4M and 0.5M sodium chloride solutions prepared with 50ml of phosphate buffer respectively.

7. Determine the protein concentration of the crude protein solution, the waste liquid after the phycobiliprotein is adsorbed by the Q-sepharose filler, and the phycobiliprotein obtained by elution with the BAC protein assay kit. The total protein content of the crude protein solution was 4.39 mg, the total protein content of the purified phycobiliprotein was 1.05 mg, and the yield was 1.05 mg/L.

The invention can separate and obtain high-value phycobiliprotein from laver processing wastewater; the Q-sepharose filler can be reused and the cost is low. The invention has the advantages of simple operation, simple process and fast separation speed, fully utilizes laver processing wastewater, obtains high-value phycobiliproteins, reduces environmental pollution of laver processing wastewater, and realizes resource reuse.

The above is only the preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the inventive concept of the present invention, some modifications and improvements can also be made, and these all belong to the present invention. protection scope of the invention.

Claims (10)

1. a method that separates phycobniliprotein from laver processing waste water, described method comprises followingStep:

1) first laver processing waste water is removed by filter to impurity, the laver processing being removed after impurity is uselessWater;

2) described in ion-exchange packing absorption, remove the algae courage in the laver processing waste water after impurity againAlbumen, obtaining absorption has the ion-exchange packing of phycobniliprotein;

3) finally there is phycobniliprotein with absorption described in sodium chloride-PBS buffer solution elution of 0.1～0.5MIon-exchange packing, obtain the phycobniliprotein after purifying.

2. the method that separates phycobniliprotein from laver processing waste water according to claim 1,It is characterized in that, in step 3) in, described in sodium chloride-PBS buffer solution elution of 0.2～0.3MAbsorption has the ion-exchange packing of phycobniliprotein.

3. the method that separates phycobniliprotein from laver processing waste water according to claim 2,It is characterized in that, in step 1) in, laver processing waste water is removed to impurity by filter media.

4. the method that separates phycobniliprotein from laver processing waste water according to claim 3,It is characterized in that, described filter medium is filter cloth or bolting silk.

5. according to separating algae courage described in any one in claim 1 to 4 from laver processing waste waterThe method of albumen, is characterized in that, in step 2) in, with 1～5 volume % of laver processing waste waterIon-exchange packing absorption described in remove the phycobniliprotein in the laver processing waste water after impurity.

6. the method that separates phycobniliprotein from laver processing waste water according to claim 5,It is characterized in that, described ion-exchange packing is anion exchange filler.

7. the method that separates phycobniliprotein from laver processing waste water according to claim 6,It is characterized in that, described anion exchange filler is Q-Ago-Gel.

8. according to separating algae courage described in any one in claim 1 to 4 from laver processing waste waterThe method of albumen, is characterized in that, in step 2) in, described in adsorbing, removes described ion-exchange packingBefore phycobniliprotein in laver processing waste water after decontamination, also comprise described ion-exchange packingThe step of cleaning.

9. the method that separates phycobniliprotein from laver processing waste water according to claim 8,It is characterized in that, described ion-exchange packing cleans by the method comprising the following steps: willDescribed ion-exchange packing washes with water three times, leaves standstill, and then sucks on ion-exchange packing manyRemaining moisture.

10. according to separating algae courage described in any one in claim 1 to 4 from laver processing waste waterThe method of albumen, is characterized in that, in step 2) in, obtaining absorption has the ion of phycobniliprotein to hand overChange after filler, also comprise and have the ion-exchange packing water of phycobniliprotein to clean described absorptionStep.