CN1238372C

CN1238372C - Use and method for preparing high absorbance phycoerythrobilin using H. ceylanica

Info

Publication number: CN1238372C
Application number: CN 03102038
Authority: CN
Inventors: 江永棉; 周宏农; 郑俊明; 吕志翼
Original assignee: Renyu Biological Technology Co Ltd
Current assignee: Renyu Biological Technology Co Ltd; Zen U Biotechnology Co Ltd
Priority date: 2003-01-27
Filing date: 2003-01-27
Publication date: 2006-01-25
Anticipated expiration: 2023-01-27
Also published as: CN1521185A

Abstract

The present invention relates to an application and a method for producing high absorbency phycoerythrin by using halymenia ceylanica. By using the three-generation alternate characteristics of sexual reproduction, asexual reproduction and vegetative reproduction and the characteristic of carpospore filamentous form stage containing no gelatin in the life history of the halymenia ceylanica, the filamentous form stage of the long-leaf laver is continued under the control conditions of light, temperature and nutrition, and the long-leaf laver is further used as materials for extracting the high absorbency phycoerythrin. The effects of decreasing unit cost and reducing phycoerythrin purifying steps can be achieved by the high weight ratio of the contained phycoerythrin per weight unit.

Description

Utilize H. ceylanica to produce the purposes and the method thereof of high absorbance phycoerythrobilin

Technical field

The invention relates to H. ceylanica (Halymenia ceylanica) production and have the purposes and the method thereof of high absorbance (0D) ratio phycoerythrobilin.Utilize syngenesis, the monogony that H. ceylanica has in the life history and nourish and generate three digenesis and carpospores filament stage do not contain under the feature of various glue classes, under light, temperature and the nutritional condition of control, continue its filament stage, and be raw material with it further, extraction has the phycoerythrobilin of high absorbance ratio.

Background technology

No matter with regard to phycoerythrobilin, algocyan or other natural protein pigment, usually has practical security, the stability that heat, potential of hydrogen etc. is also had certain degree, can be applicable on food and the makeup, simultaneously also can be used as the fluorochrome that the antibody sign is used on the immunology, be applied to clinical diagnosis and cellular biochemical and learn in the research.

Existing market has been developed and used algocyan (phycocyanin) and phycoerythrobilin (phycoerythrin), wherein, the raw material sources of algocyan production mostly are and can give the blue-green algae that a large amount of breedings are cultivated, as spirulina (Spirulina) and patina Microcystis aeruginosa (Microcystis), and it is cultivated and production method is enjoyed patent more.

Phycoerythrobilin is then because the shortage of raw material, or because raw material is unfavorable for pigment production, and exist output to reach expensive defective less, demand to the edible red pigments of the annual tens thousand of pounds in the whole world, must develop a kind of natural, safe, stable red pigments that special fluorescence disengages that has again, after estimating mass production, price will be descended, and more can enlarge its range of application and the market supply.

Algae haematochrome albumen major part is the large-scale thallus of separating from red algae at present, and as laver (Porphyra) or rosetangle algae (Ceramium) etc., small part then extracts from the Porphyridium cruentum (Porphyridium) that can control cultivation in a large number.Its subject matter is:

Though 1, the laver of wild a large amount of red algae resource and cultivation is arranged as the phycoerythrobilin raw material at present, but because most red algaes contain and enrich colloid (agar glue, carrageeman, Furcellaria gum), cause the collection of pigment to go out difficult, especially dried algae raw material is all the more so, the raw material of adding wild algae kind all has seasonal variation on product and output, by manpower is difficult for grasping.

2, at present Porphyridium cruentum be incubated at collecting cell the time, significant difficulty is also arranged, because the collection of unicellular frond one to be the power consumption and operation consuming time, cause production cost very big, and frustule secreted solubility Polysaccharides when cultivating, except the collection of block cell, also have influence on the extraction of pigment.

In order to solve the above problems, United States Patent (USP) 5,358,858 have proposed a kind of method of producing phycoerythrobilin by flagelliform nostoc (Bangiaatropurpurea) and narrow leaf laver (Porphyraangusta) filament, utilize flagelliform nostoc and the thread sporophyte stage of narrow leaf laver in the life history not to contain under the feature of various glue classes, under light, temperature and the nutritional condition by control, continue its filament stage, and be raw material with it further, the extraction phycoerythrobilin, its step is as follows:

1, the cultivation of filament and breeding, from sophisticated flagelliform nostoc of field acquisition or narrow leaf laver gametophyte, it is clean to clean frond with sterilization seawater and writing brush, the back input of drying in the shade slightly contains in SWM-III (as shown in Table 1) culture dish of improveing, the culture dish inner bottom part is covered with cover glass, after treating that spore discharges and is attached to cover glass, at room temperature, the illumination of 1000-4000lux and every day 10-16 hour irradiation incubator in germinate and grow up, grow at last and be the highly branched thread sporophyte of difference.

The composition of table one SWM-III

NaNO ₃8.5g/100ml get 2ml

Na ₂HPO ₄0.595g/100ml get 2ml

Na ₂EDTA 0.5g/100ml gets 2ml

0.01625g/100ml get 2ml

FeCl ₃(or FeCl ₃7H ₂O) 0.02885g/100ml gets 2ml

* ¹PI-metal gets 2ml

* ²S-3Vitamins gets 2ml

Soil extract gets 50ml

Tris (10cc/l) gets 500mg

Livere xtract gets 10mg

Seawater pH=7,51 liters

(when transferring pH: transfer to pH≤7 with dense HCl earlier, the NaOH with proper concn transfers to pH7,5 again, produces white precipitate in the time of so can avoiding sterilizing)

* wherein ¹PI-metal is:

H ₃BO ₃ 12.368g

MnCl ₂ 1.385g

ZnCl ₂ 0.109g

2 liters of distilled water

CoCl ₂·6H ₂O 4.479mg

CuCl ₂·2H ₂O 0.034mg

* wherein ²S-3Vitamins is:

ThiaminHCl 0.5g

Capantothenate 0.1g

Nicotinicacid 0.1g

P-aminobenzoicacid 10mg

Biotin 1mg

2 liters of distilled water

Inositol 5g

Folicacid 2mg

Thymine 3mg

B ₁₂ 1mg

(all Stock solution all is stored in the brown bottle, stored refrigerated).

2, after filament scrapes, move to the triangular flask that contains nutrient solution, under above-mentioned growing environment condition, cultivate, breed the agglomerated masses of filament.Smash the agglomerated masses of filament, the measuring bottle that moves to bigger growth continues to develop into more agglomerated masses, and the therefore volume of enlarged culturing gradually, and (300 milliliters of cleaned airs of per minute) a large amount of breedings of in the irradiation culture tank of comparatively large vol, should inflating are cultivated.Screen cloth by the 100-400 mesh filters, and the nutrient solution of filtering is recyclable to be re-used, and follow-up cultivation be there is no obstruction.

3, after the frond results,, after pulverizing again, fully stir extraction, centrifugal, can get clarifying dark red algae protein solution with water or phosphate solution because filar structure can be dry rapidly through vacuum or warm wind; Concentrating and purifying of phycochrome can be removed the partial impurities albumen precipitation by 20% ammoniumsulphate soln, is settled out chromoprotein through the 60-65% ammoniumsulphate soln again in solution, is OD ₅₆₅/ OD ₂₈₀The thick red pigments of=1.4-1.6 is food, makeup available pigment.

4, sedimentary albumen is further analysed purifying (gelfiltration) by the glue filtering layer, use Sephadex G200 resin to separate, the first road program can get OD than being the phycoerythrobilin product of 3.3-3.7, repeat once more to get final product OD than being the phycoerythrobilin of 5.1-5.2, again through colloid electrophoresis method (SDS) electrophoresis, the result of gained shows that purity is about 99%, can be used as immunity inspection reagent.

Though above-mentioned method can avoid traditional laver, rosetangle algae extraction pigment method to need the complicated formality with heating and separation gel value, or one-celled plants extraction process such as Porphyridium cruentum, because its volume is little, the problem that causes the difficulty of collecting and its excretory Polysaccharides to influence the extraction of pigment, its major defect is:

But the dark red algae protein solution that flagelliform nostoc and narrow leaf conchocelis of porphyra directly form, its OD ₅₆₅/ OD ₂₈₀Have only 1.4-1.6, still need carry out concentrating and purifying of phycochrome, obtain the phycoerythrobilin of high OD value, cause processing procedure complexity and cost higher.Therefore, we need develop the filament of other algae kind, the phycoerythrobilin that makes its first dark red algae protein solution that forms just have high OD value.

In above-mentioned background technology, the dark red algae protein solution that tradition utilizes flagelliform nostoc and narrow leaf conchocelis of porphyra directly to form, the problem that its absorbance ratio is on the low side.The inventor creates and utilizes H. ceylanica production to have the purposes and the method thereof of high absorbance ratio phycoerythrobilin, avoids said circumstances to produce.

Summary of the invention

Main purpose of the present invention provides a kind of purposes and method thereof of utilizing H. ceylanica to produce the high absorbance phycoerythrobilin, contains the high weight ratio of phycoerythrobilin by its unit weight, reaches the purpose that reduces production costs.

Another purpose of the present invention provides a kind of purposes and method thereof of utilizing H. ceylanica to produce the high absorbance phycoerythrobilin, can extract the phycoerythrobilin with high absorbance ratio by it, reaches the purpose that reduces the phycoerythrobilin purification step.

The object of the present invention is achieved like this: a kind of purposes of utilizing H. ceylanica to produce the high absorbance phycoerythrobilin, the syngenesis that have its life history, monogony and three digenesis of nourishing and generating is characterized in that: utilize its spore filament to produce the phycoerythrobilin of high absorbance ratio; This spore filament is carpospores (carpospore) filament.

This algae kind is H. ceylanica (Halymenia ceylanica), the phycoerythrobilin that extracts from its carpospores filament, the chromatography spectrogram of the 565nm that is drawn through high-effect liquid chromatography (LC) instrument.

This H. ceylanica carpospores filament is produced the phycoerythrobilin of high absorbance ratio, comprises the following step: cultivate H. ceylanica with nutrient solution and contain the sporangial gametophyte of mellow fruit, and obtain carpospores; This carpospores is placed under 15-30 ℃, and the illumination of 500-6000lux and light are that 10: 14 above environment are cultivated down, it is grown up to be filament; Collect this filament; Add acid-base buffer, stirred, centrifugal, to obtain dark chromoprotein solution; And remove impurity in this dark chromoprotein solution, and be settled out chromoprotein with salting-out process.

This nutrient solution is the SWM-III nutrient solution.This SWM-III nutrient solution is for removing organic SWM-III nutrient solution.The method of cultivating this carpospores filament is for revolving floating culture method.The environment of cultivating is 20 ℃, and the illumination of 2000lux and light are 12: 12.

The step of collecting this filament more comprises: collect this filament with the screen cloth collecting method; Dry this filament; And grind this filament, make it become powder.The screen cloth collecting method is to use 20-400 mesh screen cloth.The method of dry this filament is a vacuum method.The condition of dry this filament is Wen Fengfa.The centrifugal condition is 4 ℃ of rotating speed 6000rpm, time 10min and temperature.This acid-base buffer is a potassium phosphate solution, to keep the pH value of solution value between 5-10.Salting-out process is removed impurity for adding 20% ammoniumsulphate soln.Salting-out process precipitation chromoprotein is for adding the 60-65% ammoniumsulphate soln.More comprise with ultra-filtration method (ultrafiltration) purifies and separates and go out phycoerythrobilin.More comprise with glue filter chromatography purifies and separates and go out phycoerythrobilin.Glue filter chromatography is for using the glue filter chromatography of SephadexG200.

Describe in detail below in conjunction with preferred embodiment and accompanying drawing.

Description of drawings

Fig. 1 is that the phycoerythrobilin UV that utilizes high-effect liquid chromatography (LC) instrument (HPLC) to draw absorbs and fluorescent emission chromatography spectrogram;

Fig. 2 is the flagelliform nostoc synoptic diagram of the life history;

Fig. 3 is the narrow leaf laver synoptic diagram of the life history;

Fig. 4 is the sea synoptic diagram of the life history;

Fig. 5-Fig. 7 is the 280nm, the 565nm that are drawn through high-effect liquid chromatography (LC) instrument of pure matter phycoerythrobilin that traditional flagelliform nostoc extracts, the chromatography spectrogram of 615nm.

Fig. 8-Figure 10 is the 280nm, the 565nm that are drawn through high-effect liquid chromatography (LC) instrument of pure matter phycoerythrobilin that traditional narrow leaf laver extracts, the chromatography spectrogram of 615nm.

Figure 11-Figure 13 is the 280nm, the 565nm that are drawn through high-effect liquid chromatography (LC) instrument of pure matter phycoerythrobilin that H. ceylanica of the present invention extracts, the chromatography spectrogram of 615nm.

Embodiment

Following preferred embodiment only is explanation the present invention, can also implement at the embodiment of other algae kind widely, and scope of the present invention is not subjected to the qualification of following preferred embodiment.

As everyone knows, phycobiliprotein is from the intravital a kind of water soluble fluorescence proteinochrome of algae.Because of it has special photoluminescent property, mainly applied to widely on the fluorescent reagent of wandering cells instrument at present.In the various phycobiliprotein, phycoerythrobilin is wherein a kind of, also is the highest a kind of of fluorescence intensity in the natural goods, therefore has many fluorescence detection methods to adopt this type of pigment.

Fig. 1 UV that high-effect liquid chromatography (LC) instrument (HPLC) draws for phycoerythrobilin utilizes absorbs and fluorescent emission chromatography spectrogram, and its chromatography condition is as described below:

HPLC column：HYDROCELL DEAE NP10

Column size：50*4.6mm

Buffer A：10mMK-PBS pH6.0

Buffer B：10mMk-PBS，0.5M NaCl pH6.0

Gradient：0％Buffer B→12min→50％Buffer B

Detection：565nm

Flow rate：1ml/min

And high-effect liquid chromatography (LC) instrument mainly is made up of the high degree of accuracy high pressure pump housing, separator tube, detecting instrument and register.

Consult shown in Figure 2, viewing head is delivered vegetables the life history, syngenesis is fertilized after 4 with the sperm 3 that antheridium 2 (being male mating partner 1) discharges for producing archicarp 6 (being female mating partner 5), can develop into carpospores 8 at carpospore cyst 7, after carpospores 8 maturations of the inside, promptly can shed, become thread sporophyte 9, under side waist shape chromatoplast b effect, grow into sporozoite body 10 and shell sporozoite capsule 11, its shell sporozoite 12 of emitting forms position 15 at unit cell and further grows into the young 13, single-row frond 14 is under starlike chromatoplast a effect, form unit cell 16, unit cell 16 further forms upright body (mating partner) 18, multiple row frond 17 is grown into male mating partner 1.

Monogony then is to sprout into the young 13, single-row frond 14 and small-sized frond by monospore 16 direct developments, by the time can produce monospore 16 again in the time of certain, monospore 16 is sprouted into single-row frond 14 and small-sized frond again, so circulation is until under the suitable envrionment conditions, single-row frond 14 and the monospore 16 that small-sized frond produced just can be sprouted into large-scale frond 17.Simultaneously, single-row frond 14 and small-sized frond also can be transformed into large-scale frond 17, enter syngenesis more from generation to generation.

Consult shown in Figure 3ly, observe narrow leaf laver in the life history, because its flagelliform nostoc cardinal principle life history with Fig. 2 is identical, so do not describe in detail.We can learn that generally be syngenesis and two digenesis of monogony the life history of above-mentioned two kinds of algae, conventional art is exactly to utilize the thread sporophyte stage not contain the feature of various glue classes, under light, temperature and the nutritional condition of control, continue its filament stage, and be raw material with it further, the extraction phycoerythrobilin.

Consult shown in Figure 4ly, observe sea (Nemalion) life history, because its flagelliform nostoc cardinal principle life history with Fig. 2 is identical, so do not describe in detail.The life history of algae is except being with syngenesis and two digenesis of monogony, the part algae also has more one and nourishes and generates from generation to generation in addition, promptly produce tetrasporic sporophyte (being tetrasporophyte), its process forms carposporophyte (carposporephyte) for the archicarp after fertilization, carpospores (carpospore), tetrasporophyte (tetrasporophyte), tetrasporangium (tetrasporangium), be tetraspore (tetraspore) at last, wherein carpospores all can sprout into filament with tetraspore, but the characteristic difference of these two kinds of filaments.

The present invention utilizes to have the H. ceylanica from generation to generation of nourishing and generating, its carpospores filament stage does not contain the feature of various glue classes, under light, temperature and the nutritional condition of control, continue its filament stage, and be raw material further with it, extract and have high OD value phycoerythrobilin, its step is as follows:

1, the cultivation of filament and breeding, sophisticated H. ceylanica is contained the sporangial gametophyte of mellow fruit, it is clean to clean frond with sterilization seawater and writing brush, after drying in the shade slightly, drop into and do not contain in the incubator of organic SWM-III nutrient solution, the incubator inner bottom part is covered with sheet glass, after treating that carpospores (carpospore) discharges and is attached to sheet glass, under temperature 15-30 ℃, the illumination of 500-6000lux, light is to germinate in the incubator of 10: the 14 above and every days of irradiation more than 10 hours to grow up, and grows at last to be the highly branched filament of difference.

2, after filament scrapes, move to the large-scale culture tank that contains nutrient solution, under above-mentioned growing environment condition, cultivate, can breed the loose little agglomerated masses of filament, and the therefore volume of enlarged culturing gradually, in the irradiation culture tank of comparatively large vol, should inflate and make this little agglomerated masses be suspended in the nutrient solution (be called and revolve floating the cultivation) a large amount of breedings to cultivate.Screen cloth by the 20-400 mesh filters, and the nutrient solution of filtering is recyclable to be re-used, and follow-up cultivation be there is no obstruction.

3, after the frond results, because filar structure can be dry rapidly through vacuum or warm wind, after pulverizing through automatic grinding mill again, get the powder of 2.4 g of weights, adding 70 milliliters, potassium phosphate solution or other acid-base buffer of 10mM fully stirs, with keep the pH value of solution value fully stir between the 5-10, centrifugal under the state with 6000rpm, 10min and 4 ℃ again, can get clarifying dark red algae protein solution.Concentrating and purifying of phycochrome can be removed the partial impurities albumen precipitation by 20% ammoniumsulphate soln, is settled out chromoprotein through the 60-65% ammoniumsulphate soln again in solution, and it is OD ₅₆₅/ OD ₂₈₀=2.66 thick red pigments is food, makeup available pigment.

Above-mentioned clarifying dark red algae protein solution also can grind with the wet frond of direct results and add behind the phosphoric acid potassium solution again that centrifuging and taking gets, and concentrating with purifying of phycochrome can be undertaken by ultra-filtration method (ultrafiltration).

4, sedimentary albumen is further analysed purifying (gelfiltration) by the glue filtering layer, use 120 centimeters long Sephadex G200 resin to separate, the first road program can get OD than being phycoerythrobilin product more than 4.5, repeat to get final product to such an extent that the OD ratio is 5.3 above phycoerythrobilin once more, show that through the result of colloid electrophoresis method (SDS) electrophoresis gained purity is about 99% again, can be used as immunity inspection reagent.

Consult Fig. 5-shown in Figure 7,280nm, the 565nm that the pure matter phycoerythrobilin that extracts from flagelliform nostoc for tradition is drawn through high-effect liquid chromatography (LC) instrument (HPLC), the chromatography spectrogram of 615nm,

Consult Fig. 8-shown in Figure 10,280nm, the 565nm that the pure matter phycoerythrobilin that extracts from narrow leaf laver for tradition is drawn through high-effect liquid chromatography (LC) instrument, the chromatography spectrogram of 615nm.In fact, the phycoerythrobilin that from different algae kinds, is extracted, it draws also difference to some extent of chromatography spectrogram through high-effect liquid chromatography (LC) instrument, just as the mankind's finger print.Therefore, we can tell phycoerythrobilin by this characteristic and by which kind of algae kind are produced.

Consulting Figure 11-shown in Figure 13, is the 280nm, the 565nm that are drawn through high-effect liquid chromatography (LC) instrument of pure matter phycoerythrobilin that the present invention takes out from H. ceylanica, the chromatography spectrogram of 615nm.

Table two is the comparison sheet of traditional flagelliform nostoc, narrow leaf laver and H. ceylanica of the present invention extraction phycoerythrobilin.

The ratio of phycoerythrobilin weight (milligram) is contained in first unit's of classifying as algae weight (gram), the phycoerythrobilin that representative can output under identical algae weight, and we can know and learn: H. ceylanica of the present invention, its ratio that contains phycoerythrobilin is for the highest.

Second and third classifies the absorbance ratio (OD that extracts phycoerythrobilin from the algae kind as ₅₆₅/ OD ₂₈₀) and (OD ₆₁₅/ OD ₅₆₅), the former represents the ratio of phycoerythrobilin and other lipidated protein, the latter represents the purity ratio of algocyan and phycoerythrobilin, (OD ₅₆₅/ OD ₂₈₀) the high more expression phycoerythrobilin of ratio purity is high more, its value added is high more, has food or cosmetic colors utility value more, and also favourable more follow-up system is purifying, and the immunity inspection that can be applicable to medical treatment is with on the reagent; And can absorb the fluorescence that phycoerythrobilin sends because of algocyan, cause conduct fluorescence color when food or cosmetic colors purposes not good, therefore (OD ₆₁₅/ OD ₅₆₅) low more expression algocyan content is few more, and is better on the fluorescence color effect.Similarly, we can obviously find out: H. ceylanica of the present invention, it extracts phycoerythrobilin and has the highest absorbance ratio.Therefore, utilize H. ceylanica filament of the present invention to extract phycoerythrobilin, it has the advantage of high phycoerythrobilin content and high absorbance ratio than the filament of conventional hair dish and narrow leaf laver.

The plain analysis of components of each algae pink colour of table two

The algae kind	Ba	Pa	Hc
The algae kind	Ba	Pa	Hc	RPE mg/g algae powder	53.5	38.89	46.59
Extinction ratio A ₅₆₅/A ₂₈₀	1.40	1.54	1.44	RPE mg/g algae powder	53.5	38.89	46.59
Extinction ratio A ₅₆₅/A ₂₈₀	1.40	1.54	1.44	Extinction ratio A ₆₁₅/A ₅₆₅	0.19	0.53	0.10

Wherein

RPE: a kind of phycoerythrin, possess hydrophilic property and water with form the stable aqueous solution, its maximum UV absorbing wavelength and fluorescent emission wavelength respectively are 566nm and 575nm.

Ba: conventional hair dish (Bangia atropurpurea)

Pa: traditional narrow leaf laver (Porphyra angusta)

Hc: H. ceylanica of the present invention (Halymenia ceylanica)

The present invention describes by enumerating a preferred embodiment; but the present invention is not limited to the embodiment that enumerated, and apparent, other does not break away under the disclosed spirit; the equivalence of being finished changes or modifies, and all should be included within protection scope of the present invention.

In sum, embodiments of the invention can reach desired effect, and its disclosed concrete structure not only be not seen in like product again, also be not disclosed in application before, have novelty, creativeness and practicality.

Claims

1, a kind of purposes of utilizing H. ceylanica to produce the high absorbance phycoerythrobilin, have syngenesis, monogony the life history of H. ceylanica algae kind and three digenesis of nourishing and generating, it is characterized in that: utilize its spore filament to produce the phycoerythrobilin of high absorbance ratio; This spore filament is the carpospores filament.

2, a kind of method of utilizing H. ceylanica to produce the high absorbance phycoerythrobilin, it is characterized in that: it comprises the following step:

(1) cultivates the gametophyte of the synthetic ripe carpospore cyst of H. ceylanica with nutrient solution, and obtain carpospores;

(2) this carpospores is placed under temperature 15-30 ℃, illumination is that illumination and the light of 500-6000lux is that 10: 14 above environment are cultivated down, it is grown up to be filament;

(3) collect this filament;

(4) add acid-base buffer, stirred, to obtain dark chromoprotein solution with centrifugal;

(5) remove impurity in this dark chromoprotein solution with salting-out process, and be settled out phycoerythrin.

3, method according to claim 2 is characterized in that: the nutrient solution of this step (1) is the SWM-III nutrient solution.

4, method according to claim 3 is characterized in that: this SWM-III nutrient solution is for removing organic SWM-III nutrient solution.

5, method according to claim 2 is characterized in that: the method for this carpospores filament of cultivation of this step (2) is for revolving floating culture method.

6, method according to claim 2 is characterized in that: the culture environment of this step (2) is 20 ℃, and the illumination of 2000lux and light are 12: 12.

7, method according to claim 2 is characterized in that: this filament of collection of this step (3) comprises following step more successively: collect this filament, dry this filament and grind this filament with the screen cloth collecting method, make it become powder.

8, method according to claim 7 is characterized in that: this screen cloth collecting method is to use the screen cloth of 20-400 mesh.

9, method according to claim 7 is characterized in that: the method for dry this filament is a vacuum method.

10, method according to claim 7 is characterized in that: the condition of dry this filament is warm wind.

11, method according to claim 2 is characterized in that: the centrifugal condition of this step (4) is 4 ℃ of rotating speed 6000rpm, time 10min and temperature.

12, method according to claim 2 is characterized in that: the acid-base buffer of this step (4) is a potassium phosphate solution, to keep the pH value of solution value between 5-10.

13, method according to claim 2 is characterized in that: the salting-out process of this step (5) is removed impurity for adding 20% ammoniumsulphate soln.

14, method according to claim 2 is characterized in that: the salting-out process precipitation chromoprotein of this step (5) is for adding the 60-65% ammoniumsulphate soln.

15, method according to claim 2 is characterized in that: it also comprises with ultra-filtration method purifies and separates and goes out phycoerythrobilin.

16, method according to claim 2 is characterized in that: it also comprises with glue filter chromatography purifies and separates and goes out phycoerythrobilin.

17, method according to claim 16 is characterized in that: this glue filter chromatography is for using the glue filter chromatography of SephadexG200.