CN2643649Y - Device for producing high absorbency phycoerythrin by longleaf laver - Google Patents

Abstract

The utility model relates to a high-absorbency phycoerythrin production device with long-leaf layer, which comprises a first cultivating trough, a second cultivating trough, a collector, a grinder, a blender and a precipitator; the utility model utilizes the characteristics of the long-leaf layer that has no various glues in the three alternations of generations of sexual reproduction, asexual reproduction and vegetative propagation, and the stage of carpospore protonema in the life cycle; and under the conditions of the controlled light, temperature and nutrition, the protonema stage can be extended, and a high absorbency extraction ratio phycoerythrin is extracted with the raw material of long-leaf layer further. The utility model has the advantages that: the unit cost can be lowered and the purification steps for phycoerythrin can be reduced through the high weight ratio of the phycoerythrin in unit weight.

Description

The utilization laver that comes into leaves produces the device of high absorbance rhodophyll

Technical field

The utility model relates to the device that has high absorbance (OD) ratio rhodophyll with laver (Porphyra dentata) production of coming into leaves.Come into leaves sexual reproduction, asexual reproduction that laver has in the history of life and nourish and generate three alternations of generations and carpospore filamentous stage of utilization do not contain the feature of various glue classes, under light, temperature and the nutritional condition of control, continue its filamentous stage, and be material with it further, extraction high absorbance ratio rhodophyll.

Background technology

No matter with regard to rhodophyll, algocyan or other native protein pigment, usually has practical safety, the stability that heat, acid-base value etc. is also had certain degree, can be applicable on food and the cosmetics, simultaneously also can be used as the fluorchrome 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 rhodophyll (phycoerythrin), wherein, the raw material sources of algocyan production mostly are and can give the blue-green alge that a large amount of breedings are cultivated, as spirulina (Spirulina) and patina Microcystis aeruginosa (Microcystis), and it is cultivated and process units is enjoyed patent more.

Rhodophyll is then because of the shortage of raw material, or be unfavorable for pigment production because of former material, 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 a large amount of productions, 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 rhodophyll raw material at present, but, most red algaes enrich colloid (agar glue, antler glue, furcellaran) because of containing, 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 quality and yield, 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 is to being power consumption and operation consuming time, it is very big to influence production cost, 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 rhodophyll by flagelliform nostoc (Bangiaatropurpurea) and narrow leaf laver (Porphyraangusta) filamentous, utilize flagelliform nostoc and the thread sporophyte stage of narrow leaf laver in the history of life not to contain under the feature of various glue classes, utilize under light, temperature and the nutritional condition of control, continue its filamentous stage, and be raw material with it further, the extraction rhodophyll, its step is as follows:

1, the cultivation of filamentous and breeding, flagelliform nostoc or narrow leaf laver gametophyte from the field acquisition maturation, 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 the thread sporophyte that is the difference racemosus at last.

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=7.5 gets 1 liter

(when transferring pH: transfer to pH≤7 with dense HCl earlier, the NaOH with debita spissitudo 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 redistilled waters

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 redistilled waters

Inositol 5g

Folic acid 2mg

Thymine 3mg

B ₁₂ 1mg

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

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

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

4, the albumen of precipitation 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 rhodophyll product of 3.3-3.7, repeat once more to get final product OD than being the rhodophyll of 5.1-5.2, show that through the result of colloid electrophoresis method (SDS) electrophoresis gained purity is about 99% again, can be used as immunity inspection reagent.

Though above-mentioned method can be avoided the method for traditional laver, rosetangle algae extraction rhodophyll, need complicated formality with heating and separation gel pledge, or from the method for unicellular plants such as Porphyridium cruentum extractions rhodophyll, because its volume is little, cause the Polysaccharides of the difficulty of collecting and its secretion to influence the problem of the extraction of pigment, its major defect is:

From 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 rhodophyll of high OD value, cause processing procedure complexity and cost higher.Therefore we need develop the filamentous of other algae kind, the rhodophyll 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 the laver production of coming into leaves to have the device of high absorbance ratio rhodophyll, avoids said circumstances to produce.

Summary of the invention

Main purpose of the present utility model provides a kind of usefulness laver that comes into leaves and produces the device of high absorbance rhodophyll, contains the high weight ratio of rhodophyll by its Unit Weight, reaches the purpose that reduces unit cost.

Another purpose of the present utility model provides a kind of usefulness laver that comes into leaves and produces the device of high absorbance rhodophyll, can extract by it to have high absorbance ratio rhodophyll, reaches the purpose that reduces the rhodophyll purification step.

The purpose of this utility model is achieved in that a kind of utilization laver that comes into leaves produces the device of high absorbance rhodophyll, and it is characterized in that: it includes first culture tank, second culture tank, gatherer, mill, agitator and settling vessel; Have in this first culture tank and cultivate the laver that comes into leaves and contain the sporangial gametophytic culture fluid of mellow fruit; This second culture tank that the cultivation carpospore grows into filamentous closes on this first culture tank, and illumination, light that this second culture tank has 500-6000lux are 10: 14 above environment that reach 15-30 ℃; The gatherer of collecting filamentous places this second culture tank; The mill that grinds filamentous closes on this gatherer; This agitator closes on this mill; This settling vessel that is settled out rhodophyll from dark chromoprotein solution closes on this agitator.

This device also includes the liquid chromatography (LC) instrument.This culture fluid is the SWM-III culture fluid.This SWM-III culture fluid is for removing organic SWM-III culture fluid.Second culture tank of cultivating this carpospore filamentous is for revolving floating culture apparatus.The culture environment of this second culture tank is 20 ℃, and the illumination of 2000lux and light are 12: 12.The gatherer of collecting this filamentous is a screen cloth.This screen cloth is the screen cloth of 20-400 mesh.This mill more includes drier.This drier is vacuum desiccator or warm wind drier.The settling vessel that precipitates this rhodophyll is the salt analysis apparatus.Be provided with the ammonium sulfate of 60-65% in this salt analysis apparatus.It also includes purifier, and this purifier is a glue filtering layer analysis apparatus.This glue filtering layer analysis apparatus is for using the glue filtering layer analysis apparatus of Sephadex G200.It also includes purifier, and this purifier is a ultrafilter.

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

Description of drawings

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

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

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

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

Fig. 5-Fig. 7 is the 280nm, the 565nm that are drawn through high-effect liquid chromatography (LC) instrument of pure matter rhodophyll 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 rhodophyll 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 rhodophyll that the laver that comes into leaves of the present utility model extracts, the chromatography spectrogram of 615nm.

Figure 14-Figure 18 is that the utility model utilization laver that comes into leaves produces the device and the using method schematic diagram thereof of high absorbance rhodophyll.

Embodiment

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

As everyone knows, phycobniliprotein is from a kind of water soluble fluorescence proteinochrome in the alginite.Because of it has special photoluminescent property, mainly applied to widely on the fluorometric reagent of flow cell instrument at present.In the various phycobniliprotein, rhodophyll is wherein a kind of, also is the highest a kind of of fluorescence intensity in the natural goods, therefore has many fluorescence detection devices to adopt this type of pigment.

Fig. 1 UV that high-effect liquid chromatography (LC) instrument (HPLC) draws for rhodophyll 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：50％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 precision high pressure pump housing, separator tube, detecting instrument and logger.

Consult shown in Figure 2, viewing head is delivered vegetables the history of life, sexual reproduction is fertilized after 4 with the sperm 3 that anthreid 2 (being male gametophyte 1) discharges for producing archicarp 6 (being female gametophyte 5), can develop into carpospore 8 at carpospore cyst 7, after carpospore 8 maturations of the inside, promptly can shed, become germination carpospore 9, under side form shape plastid b effect, grow into thread sporophyte 10 and conchospore 11, it emits conchospore 12, form position 15 at single spore and further grow into the young 13, single-row frond 14, under starlike plastid a effect, form single spore 16, single spore 16 further forms upright body (gametophyte) 18, multiple row frond 17 is grown into male gametophyte 1.

Asexual reproduction then is to become to sprout into the young 13, single-row frond 14 and small-sized frond by single spore 16 direct developments, by the time can produce single spore 16 again in the time of certain, single spore 16 is sprouted into single-row frond 14 and small-sized frond again, so circulation is until under the suitable environmental condition, single-row frond 14 and the single spore 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 sexual reproduction more from generation to generation.

Consult shown in Figure 3ly, observe narrow leaf laver in the history of life, because its flagelliform nostoc cardinal principle history of life with Fig. 2 is identical, so do not describe in detail.We can learn that generally be sexual reproduction and two alternations of generations of asexual reproduction the history of life 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 filamentous stage, and be raw material with it further, the extraction rhodophyll.

Consult shown in Figure 4, consult sea (Nemalion) history of life, the history of life of algae is except being with sexual reproduction and two alternations of generations of asexual reproduction, 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 is that thallus archicarp 40 carries out the series division with red orpiment capsule 41 after fertilizations, form carposporophyte (carposporephyte) 42, carpospore (carpospore) 43, tetrasporophyte (tetrasporuphyte) 44, tetrasporangium (tetrasporangium) 45, be tetraspore (tetraspore) 46 at last, wherein carpospore 43 and tetraspore 46 all can be sprouted into filamentous 47, but the characteristic difference of these two kinds of filamentouss, because its flagelliform nostoc with Fig. 2 is identical substantially the history of life, so do not describe in detail.

The utility model is exactly to utilize to have the laver that comes into leaves from generation to generation that nourishes and generates, its carpospore filamentous stage does not contain the feature of various glue classes, under light, temperature and the nutritional condition of control, continues its filamentous stage, and be raw material with it further, extract and have high OD value rhodophyll.

Consult Figure 14-shown in Figure 180, device of the present utility model and using method thereof are as follows:

1, the cultivation of filamentous and breeding, the laver that comes into leaves of maturation 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 first incubator 100 of organic SWM-III culture fluid 102, first incubator, 100 inner bottom parts are covered with sheet glass 103, after treating that carpospore 101 (carpospore) discharges and is attached to sheet glass 103, under 15-30 ℃, the illumination of 500-6000lux, light are more than 10: 14, and every days 10 above irradiation first incubator 100 in the growth of germinateing, grow the filamentous that is the difference racemosus at last, as shown in figure 14.

2, after filamentous 201 scrapes, move to second culture tank 200 that contains culture fluid, under above-mentioned isometric growth environmental condition, cultivate, can breed the loose little agglomerated masses of filamentous, and the therefore volume of enlarged culture gradually, in the irradiation culture tank of larger volume, the device 202 of should inflating is inflated, and this little agglomerated masses is suspended in (is called and revolves floating the cultivation) a large amount of breeding cultivations in the culture fluid 204.Screen cloth 203 by the 20-400 mesh filters, and the culture fluid of filtering is recyclable to be re-used, and follow-up cultivation be there is no obstruction, as shown in figure 15.

3, after frond 303 results, because of filar structure can be dry rapidly through vacuum or warm wind device 301, again after automatic grinding mill 302 is pulverized, get the powder of 2.4 grammes per square metres, add 70 milliliters, potassium phosphate solution or other acid-base buffer 304 of 10mm, fully stir with agitator 303, to keep the pH value of solution value between 5-10, fully stir, again with use centrifuge 305 under 6000rpm, 10min, 4 ℃ the state, the dark red algae protein solution 306 that can clarify.Concentrating and purifying of phycochrome can be removed partial impurities albumen precipitation 308 by 20% ammonium sulfate 307, is settled out chromoprotein through 60%-65% ammonium sulfate 309 again in solution, and it is OD ₅₆₅/ OD ₂₈₀=2.66 thick red pigments 310, it is the pigment that food-grade or cosmetics can be used.The dark red algae protein solution of above-mentioned clarification, also can grind with the wet frond of direct results, add the phosphoric acid potassium solution after, get with the centrifuge centrifuging and taking again, and concentrating with purifying of phycochrome can be undertaken by ultrafiltration method (ultrafiltration), as Figure 16-shown in Figure 180.

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

Consult Fig. 5-shown in Figure 7,280nm, the 565nm that the pure matter rhodophyll 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 rhodophyll 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 rhodophyll 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 rhodophyll 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 rhodophyll that the utility model takes out from the laver that comes into leaves, the chromatography spectrogram of 615nm.

Table two is the comparison sheet of traditional flagelliform nostoc, narrow leaf laver and the laver extraction rhodophyll that comes into leaves of the present utility model.

First unit's of classifying as algae weight (gram) contains the ratio of rhodophyll weight (milligram), the rhodophyll that representative can output under identical algae weight, and we can know and learn: the laver that comes into leaves of the present utility model, its ratio that contains rhodophyll is for the highest.

Second and third classifies the absorbance ratio (OD that extracts rhodophyll from the algae kind as ₅₆₅/ OD ₂₈₀) and (OD ₆₁₅/ OD ₅₆₅), the former represents the ratio of rhodophyll and other lipidated protein, the latter represents the purity ratio of algocyan and rhodophyll, (OD ₅₆₅/ OD ₂₈₀) the high more expression rhodophyll of ratio purity is high more, its surcharge is high more, has food or cosmetic colors value more, the also favourable more follow-up purifying of making, and the immunity inspection that can be applicable to medical treatment is with on the reagent; Because algocyan can absorb the fluorescence that rhodophyll sends, 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: the laver that comes into leaves of the present utility model, it extracts rhodophyll and has the highest absorbance ratio.Therefore, utilize the conchocelis of porphyra that comes into leaves of the present utility model to extract rhodophyll, it has the advantage of high rhodophyll content and high absorbance ratio than the filamentous 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	Pd
				RPE mg/g algae powder	53.5	38.89	44.98
Extinction ratio A 565/A 280	1.40	1.54	1.96
				Extinction ratio A 615/A 565	0.19	0.53	0.21

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)

Pd: the laver that comes into leaves of the present utility model (Porphyra dentata)

The utility model is to describe by enumerating a preferred embodiment; but the utility model is not limited to the embodiment that enumerated; apparently; other does not break away under the spirit that the utility model discloses; the equivalence of being finished changes or modifies, and all should be included within the protection domain of the present utility model.

In sum, embodiment of the present utility model 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 (16)

1, a kind of utilization laver that comes into leaves produces the device of high absorbance rhodophyll, and it is characterized in that: it includes first culture tank, second culture tank, gatherer, mill, agitator and settling vessel; Have in this first culture tank and cultivate the laver that comes into leaves and contain the sporangial gametophytic culture fluid of mellow fruit; This second culture tank that the cultivation carpospore grows into filamentous closes on this first culture tank, and illumination, light that this second culture tank has 500-6000lux are 10: 14 above environment that reach 15-30 ℃; The gatherer of collecting filamentous places this second culture tank; The mill that grinds filamentous closes on this gatherer; This agitator closes on this mill; This settling vessel that is settled out rhodophyll from dark chromoprotein solution closes on this agitator.

2, device according to claim 1 is characterized in that: this device also includes the liquid chromatography (LC) instrument.

3, device according to claim 1 is characterized in that: this culture fluid is the SWM-III culture fluid.

4, device according to claim 3 is characterized in that: this SWM-III culture fluid is for removing organic SWM-III culture fluid.

5, device according to claim 1 is characterized in that: second culture tank of cultivating this carpospore filamentous is for revolving floating culture apparatus.

6, device according to claim 1 is characterized in that: the culture environment of this second culture tank is 20 ℃, and the illumination of 2000lux and light are 12: 12.

7, device according to claim 1 is characterized in that: the gatherer of collecting this filamentous is a screen cloth.

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

9, device according to claim 1 is characterized in that: this mill more includes drier.

10, device according to claim 9 is characterized in that: this drier is a vacuum desiccator.

11, device according to claim 9 is characterized in that: this drier is warm wind drier.

12, device according to claim 1 is characterized in that: the settling vessel that precipitates this rhodophyll is the salt analysis apparatus.

13, device according to claim 12 is characterized in that: the ammonium sulfate that is provided with 60-65% in this salt analysis apparatus.

14, device according to claim 1 is characterized in that: it also includes purifier, and this purifier is a glue filtering layer analysis apparatus.

15, device according to claim 14 is characterized in that: this glue filtering layer analysis apparatus is for using the glue filtering layer analysis apparatus of Sephadex G200.

16, device according to claim 1 is characterized in that: it also includes purifier, and this purifier is a ultrafilter.

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