CN105601732A - Method for separating phycobiliprotein from laver processing wastewater - Google Patents
Method for separating phycobiliprotein from laver processing wastewater Download PDFInfo
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- CN105601732A CN105601732A CN201610192773.3A CN201610192773A CN105601732A CN 105601732 A CN105601732 A CN 105601732A CN 201610192773 A CN201610192773 A CN 201610192773A CN 105601732 A CN105601732 A CN 105601732A
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- phycobniliprotein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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Abstract
The invention discloses a method for separating phycobiliprotein from laver processing wastewater. The method comprises the following steps: (1) filtering laver processing wastewater to remove impurities to obtain laver processing wastewater without impurities; (2) adsorbing phycobiliprotein in the laver processing wastewater without impurities with ion exchange packing so as to obtain phycobiliprotein adsorbed ion exchange packing; and (3) eluting the phycobiliprotein adsorbed ion exchange packing with 0.1-0.5M of sodium chloride-PBS buffer solution to obtain purified phycobiliprotein. According to the method, purified high-active-value phycobiliprotein can be reclaimed from laver processing wastewater, so that the aim of waste utilization can be achieved; meanwhile, environmental pollution caused by direct drainage of laver processing wastewater can be reduced due to extraction of the phycobiliprotein.
Description
Technical field
The present invention relates to a kind of method that separates phycobniliprotein, be specifically related to one and process from laverIn waste water, separate the method for phycobniliprotein.
Background technology
Laver has Important Economic and is worth, and is one of China Main Economic marine alga. China mainly plantsThe laver kind of training has porphyra haitanensis and Porphyra yezoensis, and wherein porphyra haitanensis Main Cultivation is on the south the Changjiang river(Fujian Province and Guangdong Province), Porphyra yezoensis Main Cultivation is in North of Yangtze River (Jiangsu Province and ShandongEconomize, be mainly in Nantong, Yancheng and sea area, Lianyun Harbour). According to statistics, laver in 2006 is produced per yearMeasure approximately 1.8 × 104Ton dry product, annual value of production is estimated approximately 1,300,000,000 dollars (FAO, 2006).
Phycobniliprotein is a kind of water colo(u)r albumen, has unique absorption spectrum and fluorescence and sends outPenetrate spectrum. Phycobniliprotein comprises phycoerythrin, phycocyanin and allophycocyanin. PhycobniliproteinBy the color base of apoprotein and tetrapyrrol(e) structure by together with thioether bond covalent bond, structureStable, can be used as fluorescence probe, for the clinical diagnosis of biology, medical research and diseaseWith treatment etc. Phycobniliprotein also can be used as the additive of natural colouring matter as food, cosmetics,The murder by poisoning of having avoided chemical synthesis pigment to bring. Research shows, phycobniliprotein can significantly be carriedHigh human lymphocyte activity and body's immunity, the function of anti-cancer and cancer prevention ability of enhancing body. CauseThis, external many companies invest and develop phycobniliprotein product in succession, and product price is very considerable.According to statistics, this series products market opportunity of nearly more than $ 10 billion in the international market.At present, domestic these products that also used, but whole dependence on import. Phycobniliprotein price is highHigh main cause is because separation and purification difficulty is studied so develop new purification techniqueOne of emphasis.
Porphyra yezoensis processing comprises time processing and secondary operations, and time processing process comprises clearlyWash, shred, clean-blending-cake processed-dewater-dry-peel off, sorting and packaging etc. ChoppingPorphyra yezoensis is after cleaning process, and due to incision cell rupture, intracellular organic matter discharges,In a large amount of waste water that produce, be rich in phycobniliprotein, take on a red color, be commonly called as " red water ". " red water " byExceed standard in colourity, directly discharge not only can be wasted resource, also can increase environmental pressure. If energyTherefrom separate recovery phycobniliprotein and discharge again, not only can reduce laver processing waste water colourity,Alleviate " red water discharge " pollution to environment, also can obtain high value product phycobniliprotein, favourableIn laver industry sustainable health development.
Summary of the invention
For solving the problems of the technologies described above, the object of the present invention is to provide a kind of useless from laver processingIn water, separate the method for phycobniliprotein, thereby from laver processing waste water, reclaim the active high value of purifyingPhycobniliprotein, reached the object of twice laid, due to the extraction of phycobniliprotein, fall simultaneouslyLow laver processing waste water directly discharges the pollution to environment.
For achieving the above object, technical scheme of the present invention is as follows:
The invention provides a kind of method that separates phycobniliprotein from laver processing waste water, described sideMethod comprises the following steps:
1) first laver processing waste water is removed by filter to impurity, the laver being removed after impurity addsWork waste water;
2) described in ion-exchange packing absorption, remove in the laver processing waste water after impurity againPhycobniliprotein, obtaining absorption has the ion-exchange packing of phycobniliprotein;
3) finally there is algae courage egg with absorption described in sodium chloride-PBS buffer solution elution of 0.1～0.5MWhite ion-exchange packing, obtains the phycobniliprotein after purifying.
Preferably, in step 3) in, by sodium chloride-PBS buffer solution elution of 0.2～0.3MDescribed absorption has the ion-exchange packing of phycobniliprotein, can effectively adsorb phycobniliprotein, andAnd purity is higher.
Preferably, in step 1) in, laver processing waste water is removed to impurity elimination by filter mediaMatter.
More preferably, described filter medium is filter cloth or bolting silk.
Preferably, in step 2) in, use the ion of 1～5 volume % of laver processing waste water to hand overChange described in filling adsorption and remove the phycobniliprotein in the laver processing waste water after impurity, can more haveEffect ground absorption phycobniliprotein, and cost-saving.
Preferably, described ion-exchange packing is anion exchange filler.
Preferably, described anion exchange filler is Q-Ago-Gel (Q-sepharose).
Preferably, in step 2) in, described in adsorbing, removes after impurity described ion-exchange packingLaver processing waste water in phycobniliprotein before, also comprise described ion-exchange packing carried out clearlyThe step of washing.
Preferably, described ion-exchange packing cleans by the method comprising the following steps:Described ion-exchange packing is washed with water three times, leave standstill, and then suck ion-exchange packingUpper unnecessary moisture.
Preferably, in step 2) in, obtaining absorption has after the ion-exchange packing of phycobniliprotein,Also comprise the step that has the ion-exchange packing water of phycobniliprotein to clean described absorption.
The present invention reclaims the active high value phycobniliprotein of purifying from laver processing waste water, has reached uselessThe object that thing utilizes due to the extraction of phycobniliprotein, has reduced laver processing waste water direct simultaneouslyThe pollution of discharge to environment; Q-Ago-Gel filler can reuse, and cost is low; Operation letterJust, technique is simple, and separating rate is fast, takes full advantage of laver processing waste water, obtains high value algaeWhen biliprotein, alleviate the pollution of laver processing waste water to environment, realized the repetition of resourceUtilize.
Tool of the present invention has the following advantages:
1. operating process is simple, and it is residual that laver processing waste water only needs to remove laver by filter mediaThe graininess such as slag, mud impurity, phycobniliprotein does not need to carry out pretreatment before separating.
2. the present invention can with Q-Ago-Gel (Q-selpharose) filler after protein extractionRecycling after washing, cost is low.
3. phycobniliprotein separating rate is fast, and a complete separation process only needs approximately 1 hour,Required 10 minutes of the balance that comprises post, loading 30 minutes, cleans wash-out 10 10 minutesMinute.
4. the phycobniliprotein productive rate after purifying of the present invention is more than 0.98mg/L, by thisThe bright utilization rate that can greatly improve laver processing waste water.
Brief description of the drawings
Fig. 1 be in the present invention for separating of the crude protein liquid of laver processing waste water existThe abosrption spectrogram of 400-800nm;
Fig. 2 separates phycobniliprotein that wash-out the obtains absorption light at 400-800nm in the present inventionSpectrogram.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention. Should be understood that these embodimentOnly be not used in and limit the scope of the invention for the present invention is described. In addition should be understood that and readingAfter content of the present invention, those skilled in the art can do various changes or repair the present inventionChange, these equivalent form of values fall within limited range of the present invention equally.
Unless specialized, reagent used in following examples all can be purchased and obtain from regular channel.
Embodiment 1
1. measure 200ml laver processing waste water, with impurity such as bolting silk elimination laver residue, mud,Obtain crude protein liquid, be placed in the blue lid bottle of 250ml, be placed in 4 DEG C of refrigerators for subsequent use.
2. according to formula albumen (Pro)=OD562/ 0.0157, calculate albumen in crude protein liquidContent is 4.39mg/L, and under room temperature, adopts ultraviolet specrophotometer to measure the crude protein extractingThe absorption spectrum of liquid, Fig. 1 is the absorption spectrum of crude protein liquid at 400-800nm, in figureThere are a lot of absworption peaks at 400-450nm place, illustrates that foreign protein is a lot.
3. get 2ml and be stored in Q-Ago-Gel (Q-selpharose) filler in alcohol to 50mlCentrifuge tube, adds the distilled water of 10 times of volumes, rocks and makes Q-Ago-Gel (Q-selpharose)Filler scatters. Leave standstill, Q-Ago-Gel (Q-selpharose) filler is sunk. By upperClear sucking-off. In triplicate, fully to remove alcohol.
4. in 20 DEG C, Q-Ago-Gel (Q-selpharose) filler that washes away alcohol is addedIn crude protein liquid, rock fast, make phycobniliprotein be adsorbed to Q-Ago-Gel(Q-selpharose) on filler.
5. leave standstill and make filling settlement, abandon supernatant. By solidifying the Q-agarose that has adsorbed phycobniliproteinGlue (Q-selpharose) filler is transferred to 10ml centrifuge tube, with the washing of 10ml distilled water.6. respectively with 0.1M, the 0.2M of 10ml phosphate buffer preparation, the sodium chloride solution of 0.3MWash-out phycobniliprotein. And under room temperature, adopt ultraviolet specrophotometer to measure the phycobniliprotein extractingAbsorption spectrum, Fig. 2 is the absorption spectrum of the phycobniliprotein that obtains of wash-out at 400-800nm,According to phycoerythrin, phycocyanin and allophycocyanin absorbing wavelength scope (referring to NiuJF, WangGC，LinXZ，etal.Large-scalerecoveryofC-phycocyaninfromSpirulinaplatensisusingexpandedbedadsorptionchromatography[J].JournalofChromatographyBAnalyticalTechnologiesintheBiomedical&LifeSciences, 2007,850 (1-2): 267-276.), in known figure, peak 1 is allophycocyanin,Peak 2 is phycocyanin, and peak 3 and peak 4 are phycoerythrin, and the algae courage egg after separation and purification is describedBai Chundu is higher.
7. measure crude protein liquid, Q-Ago-Gel with BAC protein determination kit(Q-selpharose) filling adsorption waste liquid after phycobniliprotein and wash-out gained phycobniliproteinProtein concentration, in table 1. Crude protein liquid total protein content is 878 μ g, purifying gained algae courage eggWhite total protein concentration is 197 μ g, and productive rate is 0.98mg/L.
Table 1 laver processing waste water absorption front and back and effluent volume and protein concentration
Embodiment 2
1. measure 1L laver processing waste water, with impurity such as filter cloth elimination laver residue, mud,Obtain crude protein liquid, be placed in the 1L round bottle of bottom with blow vent, be placed in 4 DEG C of refrigeratorsFor subsequent use.
2. according to formula protein concentration (mg/L)=OD562/ 0.0157, calculate crude protein liquidMiddle protein content is 4.39mg/L.
3. getting Q-Ago-Gel (Q-selpharose) filler that 20ml is stored in alcohol arrivesIn the sampling bottle of 500ml, add the distilled water of 10 times of volumes, rock and make Q-Ago-Gel(Q-selpharose) filler scatters. Leave standstill, filler is sunk. By supernatant sucking-off. Repeat threeInferior, fully to remove alcohol.
4. in 25 DEG C, Q-Ago-Gel (Q-selpharose) filler that washes away alcohol is addedEnter in crude protein liquid, bottom ventilation blows Q-Ago-Gel (Q-selpharose) fillerRise, phycobniliprotein is fully contacted, just with Q-Ago-Gel (Q-selpharose) fillerBe adsorbed on Q-Ago-Gel (Q-selpharose) filler in phycobniliprotein.
5. leave standstill and make Q-Ago-Gel (Q-selpharose) filling settlement, abandon supernatant. WillQ-Ago-Gel (Q-selpharose) filler that has adsorbed phycobniliprotein is transferred to 500mlSampling bottle, with the washing of 200ml distilled water.
Respectively with 0.1M, the 0.2M of 50ml phosphate buffer preparation, 0.3M, 0.4M andThe sodium chloride solution wash-out phycobniliprotein of 0.5M.
7. measure crude protein liquid, Q-Ago-Gel with BAC protein determination kit(Q-selpharose) filling adsorption waste liquid after phycobniliprotein and wash-out gained phycobniliproteinProtein concentration. Crude protein liquid total protein content is 4.39mg, the total egg of purifying gained phycobniliproteinWhite amount is 1.05mg, and productive rate is 1.05mg/L.
The present invention can separate and obtain high value phycobniliprotein from laver processing waste water; Q-agarose is solidifyingGlue (Q-sepharose) filler can reuse, and cost is low. The present invention is easy and simple to handle, techniqueSimply, separating rate is fast, takes full advantage of laver processing waste water, the high value of acquisition phycobniliproteinAlleviate the pollution of laver processing waste water to environment simultaneously, realized the recycling of resource.
Above-described is only the preferred embodiment of the present invention, it should be pointed out that for this areaThose of ordinary skill, without departing from the concept of the premise of the invention, can also doGo out some distortion and improvement, these all belong to protection scope of the present 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.
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Cited By (5)
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| CN107686199A (en) * | 2017-09-18 | 2018-02-13 | 连云港紫金海藻产业研究发展中心 | The circulation comprehensive of seaweed processing waste water utilizes system and technical method |
| CN107867774A (en) * | 2017-10-24 | 2018-04-03 | 连云港紫金海藻产业研究发展中心 | The circulation comprehensive of seaweed processing waste water utilizes system and technical method |
| CN107935266A (en) * | 2017-11-30 | 2018-04-20 | 连云港紫金海藻产业研究发展中心 | The circulation comprehensive of seaweed processing waste water utilizes system and technical method |
| CN108101277A (en) * | 2017-11-30 | 2018-06-01 | 连云港紫金海藻产业研究发展中心 | The circulation comprehensive of seaweed processing waste water utilizes system and technical method |
| CN114702561A (en) * | 2021-12-20 | 2022-07-05 | 中国科学院海洋研究所 | Method for comprehensively extracting phycobiliprotein and carrageenan from delicate solieria |
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| CN107686199A (en) * | 2017-09-18 | 2018-02-13 | 连云港紫金海藻产业研究发展中心 | The circulation comprehensive of seaweed processing waste water utilizes system and technical method |
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| CN114702561A (en) * | 2021-12-20 | 2022-07-05 | 中国科学院海洋研究所 | Method for comprehensively extracting phycobiliprotein and carrageenan from delicate solieria |
| CN114702561B (en) * | 2021-12-20 | 2023-05-26 | 中国科学院海洋研究所 | Method for comprehensively extracting phycobiliprotein and carrageenan from weak and weak plumeria |
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