JPH07286112A - Preparation of phycoerythrin from bangia fusco-purpurea and porphyra angusta - Google Patents

Abstract

PURPOSE: To economically, rapidly and effectively obtain phycoerthrin by applying treatment such as extraction treatment or the like to filaments obtained by culturing spores of Bangia or Porphyra angusta.

CONSTITUTION: Matured red algae thalli comprising gemetophytes of Bangia or Porphyra angusta are cultured in an SWM-III medium to recover spores which are, in turn, irradiated with light with a luminous intensity of 1,000-4,000 lx at 15-25°C for 10-16 hr to germinate paraphyses. Subsequently, the paraphyses are destructed to form fine segments and, further, these fine segments are cultured in a larger tank under the above mentioned condition until the cultured thalli become a desired amt. and these paraphyses are dried and ground to obtain a powder. Water and/or a phosphate soln. is added to this powder to obtain a transparent red pigment protein soln. containing phycoerthrin and a gel like phycoerthrin concentrate is salted-out from the soln.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for preparing phycoerythrin from beet paste and koseki glue.

[0002]

Natural pigment proteins obtained from plants can be safely used in foods and beverages. The pigment is stable under mild heating and stable in acidic or basic solutions. Therefore, they can be used in foods and cosmetics as colorants. Furthermore, the pure form of the pigments can be used for fluorescent labeling of antibodies used as diagnostic agents in immunological, clinical, cell biology and biochemical research.

Phycocyanin and phycoerythrin are two types of pigment proteins currently in use and are used in many fields. The main raw material of phycocyanin is easily growing cyanobacteria such as Spirulina and Microcystis, and many methods for culturing algae and methods for preparing phycocyanin have been developed, and phycocyanin can be supplied without problems. Has been. However, the amount of phycoerythrin is still low, the raw materials available are low, and the processing is difficult for commercial production of phycoerythrin, which makes it expensive.

Most of phycoerythrin is extracted from red algal fronds, for example, the genus Amanori and the genus Squid, and the amount of phycoerythrin extracted from the genus Ginomori obtained by tank culture is small.

Although the amount of wild red algae and cultivated Amanori used as raw materials for phycoerythrin is increasing, most of them contain pasty substances with a high gel content and therefore Extracting phycoerythrin is extremely difficult, especially in the case of dried algae. Furthermore, the quantity and quality of wild algae is influenced by season and temperature. These factors make it more difficult to produce phycoerythrin from wild and cultivated Clams.

The recovery of single cells is usually labor-intensive and time-consuming, and soluble polysaccharides secreted during the culture of algae hinder the recovery of cells and affect the extraction of phycoerythrin. Extraction of phycoerythrin is also difficult.

[0007]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an economical, rapid and effective method for preparing phycoerythrin.

[0008]

It has been found that the filamentous plants do not contain a gel in the generational alternations of shiitake and kosugi and therefore can be maintained under some controlled conditions. The present invention takes advantage of this finding to provide a method for preparing phycoerythrin from spinach and husk glue of the filamentous stage.

That is, the method of the present invention comprises the following steps: a)
Preparing mature autumn leaves of Ushikake Nori and Kosuji Nori; b) Culturing the above leaves in SWM-III medium to obtain spores; c) Temperature, illuminance and lighting time of 15-
25 ° C, 1000 Lux (1x) to 4000 Lux (1x) and 10:14 to 1
Culturing spores under conditions of 6: 8 (lighting: extinguished) time to obtain filaments; d) cutting filaments into fragments, and culturing the filaments in the desired amount Culturing in larger vessels under the above conditions until
e) collecting, drying and crushing the culture filaments into a powder; f) adding the powder to a liquid selected from the group consisting of water and a phosphate solution to obtain a solution containing phycoerythrin. And g) salting out phycoerythrin from the solution.

[0010]

The present invention will be described in detail below with reference to preferred embodiments.

According to the method of the present invention, the mature leaflets of Nokogushi or Nokogure nori are collected from the sea and washed with sterile seawater. After air-drying for a short time, add to the medium (SWM-III medium). After a few hours, the spores are released from the fronds of the pickled or kosoji glue. Separated free spores from the first medium, temperature 25 ℃, illuminance 500 ~ 1001x, lighting / off time 1
Place in a growth chamber from 0:14 to 16: 8. After the spores germinate into branched filaments, the filaments are transferred to a flask containing SWM-III medium and cultured under the above conditions until colonies are formed. The filamentous colonies are then cut into small pieces using a sterile grinder and transferred to a wider space, eg a tank, for further growth. After moving to a wider space, more filaments are generated. The filamentous colonies are cut again and grown further until the desired amount is obtained. In this case, when culturing filamentous colonies in a larger tank, fresh air (300 ml / min) must be supplied to the tank. The filaments are then collected and filtered through a 100-400 mesh screen. The medium can be collected and reused.

Next, the collected and filtered filamentous material of the pickled seaweed or the kosoji seaweed is rapidly dried in a vacuum or hot air, and crushed into a powder. Add powder to phosphate solution or water and mix well. The debris precipitate is removed by centrifugation to give a clear red pigment solution. Next, ammonium sulfate is added to remove unnecessary proteins as a 20% to 30% saturated solution, and then precipitation is performed with a 60% to 65% ammonium sulfate saturated solution to obtain crude phycoerythrin. The crude phycoerythrin obtained is 1.4-
It has an absorbance of 565 nm value (OD565) / absorbance of 280 nm value (OD280) of 1.6, is food grade, and is a pigment that can be used for cosmetics.

The crude precipitated phycoerythrin can be further purified by gel filtration chromatography. For example, after purification by Sephadex G200 chromatography once, the phycoerythrin formed has an OD565 / OD280 ratio of 3.3-
Reach 3.7. By repeating the filtration process, the OD565 / OD280 ratio of phycoerythrin reaches 5.1-5.2. Phycoerythrin purity is about 99% when tested by SDS electrophoresis
Is. This shows that the phycoerythrin produced by the method of the present invention can be used as a reagent for immunoassay.

Example 1 Mature leaflets of pickled scallions were collected from the sea and washed with sterilized seawater using a brush. The washed fronds were briefly air dried and then placed in Petri dishes containing SWM-III medium with several coverslips on the bottom. After the spores are released from the leaves of the pickled nose and fallen onto the cover glass, the cover glass carrying the released spores is transferred to a new Petri dish containing the same medium, and the temperature, illuminance, and illumination / light-off time are set, respectively. Placed in a growth chamber at 20 ° C, 20001x and 12:12. After several days, germination occurred and branched filaments were formed. The branched filaments were removed from the cover glass, placed in a flask containing SWM-III medium, and cultured under the above conditions until filament colonies were formed. The colonies were placed in a sterile blender and transferred to a larger tank for further growth. This large tank was supplied with fresh air (300 ml / min). After 40 days, the pickled paste filaments were collected and filtered through a 200 mesh screen. The weight of the dried pickled seaweed was 31.4 times that of the inoculated filaments.

(Example 2) The growth conditions of pickled ginseng were set to temperature.
The same method as in Example 1 was repeated except that the temperature was 20 ° C., the illuminance was 20001 ×, and the illumination / extinguishing time was 14:10. After culturing for 40 days, the weight of the dried laver was 30.8 times the weight of the inoculated filaments.

(Example 3) The growth conditions of pickled seaweed were set to temperature.
The same method as in Example 1 was repeated except that the temperature was 20 ° C., the illuminance was 40001 ×, and the lighting / extinguishing time was 12:12. After culturing for 40 days, the weight of the dried laver was 30.5 times the weight of the inoculated filaments.

(Embodiment 4) The growth conditions of pickled seaweed are set to temperature.
The same method as in Example 1 was repeated except that the temperature was 20 ° C., the illuminance was 40001 ×, and the illumination / extinguishing time was 14:10. After culturing for 40 days, the weight of the dried laver was 30.1 times the weight of the inoculated filaments.

(Example 5) The growth conditions of pickled seaweed were set to temperature.
The same method as in Example 1 was repeated except that the temperature was 15 ° C., the illuminance was 20001 ×, and the illumination / extinguishing time was 12:12. After culturing for 40 days, the weight of the dried laver was 21.6 times the weight of the inoculated filaments.

(Example 6) The growth conditions of pickled seaweed were set to temperature.
The same method as in Example 1 was repeated except that the temperature was 25 ° C., the illuminance was 20001 ×, and the illumination / extinguishing time was 12:12. After culturing for 40 days, the weight of the dried laver was 18.1 times the weight of the inoculated filaments.

(Embodiment 7) The growth conditions of pickled seaweed are set to temperature.
The same method as in Example 1 was repeated except that the temperature was 25 ° C., the illuminance was 40001 ×, and the illumination / extinguishing time was 14:10. After culturing for 40 days, the weight of the dried laver was 15.6 times the weight of the inoculated filaments.

(Embodiment 8) The growth conditions of pickled seaweed are set to temperature.
The same method as in Example 1 was repeated except that the temperature was 30 ° C., the illuminance was 10001 ×, and the illumination / extinguishing time was 10:14. After culturing for 40 days, the weight of the dried laver was 19.3 times the weight of the inoculated filaments.

(Embodiment 9) The growth conditions of pickled seaweed are set to temperature.
The same method as in Example 1 was repeated except that the temperature was 30 ° C., the illuminance was 10001 ×, and the illumination / extinguishing time was 16: 8. After culturing for 40 days, the weight of the dried laver was 14.3 times the weight of the inoculated filaments.

(Example 10) 5 g of a filamentous material of a pickled seaweed was sampled, dried rapidly with warm air, and then pulverized with an automatic pulverizer to give 2.4 g of a powder. Powder and 10 mM potassium phosphate solution 70
ml was mixed well. The mixture is then 6000rp for 10 minutes at 4 ° C.
Separated by centrifugation at m. This gave a clear, red pigment solution. Solid ammonium sulphate
17 g was added to the solution to form a 20% saturated solution of ammonium sulphate and unnecessary proteins were removed to obtain a more pure pigment protein solution. 113 g of solid ammonium sulfate was added to the pigment solution to form a 65% solution of ammonium sulfate, which was then centrifuged under the same conditions to obtain a phycoerythrin precipitate. The precipitated phycoerythrin was then dialyzed against 10 mM potassium phosphate, which gave a crude phycoerythrin solution with OD565 / DO280 1: 5.

Next, the crude phycoerythrin was added to the column length.
Purified by 120 cm Sephadex G200 gel filtration chromatography. A total of 100 tubes of 6 ml of eluate were obtained. From the absorbance at 565 nm, it was found that the eluate in the 35th to 37th tubes contained phycoerythrin. The phycoerythrin solution thus obtained was measured for the absorbance ratio (OD
Ratio) 3.5. By repeating the purification, the OD
The final phycoerythrin solution with a ratio of 5.1 was obtained. The purity of phycoerythrin when analyzed by SDS electrophoresis was 99%, indicating that this phycoerythrin can be used for immunoassay.

(Example 11) Except that Kosugi Nori was used in place of Ushikake Nori and the conditions for growing Kosuji Nori were set to a temperature of 20 ° C, an illuminance of 40001x, and an illumination / light-off time of 12:12. The above method was repeated. After culturing for 40 days, the weight of the dried Kosuji glue was 32.4 times the weight of the inoculated filaments.

(Example 12) The method of Example 11 was repeated, except that the growth conditions for Kosuji seaweed were set to a temperature of 20 ° C, an illuminance of 40001x and an illumination / extinguishing time of 14:10. After culturing for 40 days,
The weight of the dried Kosuji glue was 30.1 times the weight of the inoculated filaments.

(Example 13) The method of Example 11 was repeated, except that the conditions for growing Kosuji seaweed were 20 ° C in temperature, 20001x in illuminance and 12:12 in lighting / extinguishing time. After culturing for 40 days,
The weight of the dried kosuji glue was 18.3 times the weight of the inoculated filaments.

(Example 14) The method of Example 11 was repeated, except that the growth conditions of Kosuji seaweed were set to a temperature of 20 ° C, an illuminance of 20001x and an illumination / extinguishing time of 14:10. After culturing for 40 days,
The weight of the dried kosuji glue was 28.8 times the weight of the inoculated filaments.

(Example 15) The method of Example 11 was repeated, except that the growth conditions for Kosuji seaweed were set to a temperature of 15 ° C, an illuminance of 20001x and an illumination / extinguishing time of 12:12. After culturing for 40 days,
The weight of the dried Kosuji glue was 20.1 times the weight of the inoculated filaments.

(Example 16) The method of Example 11 was repeated, except that the growth conditions of Kosuji seaweed were set to a temperature of 25 ° C, an illuminance of 20001x and an illumination / extinguishing time of 12:12. After culturing for 40 days,
The weight of the dried kosuji glue was 17.1 times the weight of the inoculated filaments.

(Example 17) The method of Example 11 was repeated, except that the growth conditions for Kosuji seaweed were 25 ° C, an illuminance of 40001x, and an illumination / extinguishing time of 14:10. After culturing for 40 days,
The weight of dried Kosuji glue was 20.3 times the weight of the inoculated filaments.

(Example 18) The method of Example 11 was repeated, except that the growth conditions of Kosuji seaweed were set to a temperature of 30 ° C, an illuminance of 10001x and an illumination / extinguishing time of 12:12. After culturing for 40 days, the weight of the dried Kosuji glue was 14.0 times the weight of the inoculated filaments.

(Example 19) As a culturing substance, Kosuzu seaweed was used in place of Noshi seaweed, and the conditions for growing Kosugi seaweed were as follows.
The method of Example 11 was repeated except that After culturing for 40 days, the weight of dried Kosuji glue was 14.1 times the weight of the original branched sporophyte filaments.

(Example 20) The method of Example 11 was repeated, except that the conditions for growing Kosuji seaweed were a temperature of 30 ° C, an illuminance of 10001x, and an illumination / extinguishing time of 16: 8. After culturing for 40 days, the weight of dried kosoji glue was 13.3 times the weight of the inoculated filaments.

(Example 21) The method of Example 10 was repeated except that Kozue Nori was used as the raw material in place of Nokushi Nori. The phycoerythrin solution in tubes 45-59 was collected. The phycoerythrin obtained had an OD565 / OD280 of 5.2 and a purity of 99% as analyzed by SDS-PAGE (SDS-polyacrylamide gel electrophoresis).

[0036]

The method of the present invention obviously has the following advantages. 1. Unlike the previously developed methods of extracting phycoerythrin from the leaves of the genus Amaranthus or Squid, the method of the present invention does not require heating or gel removal operations.
It only requires drying, crushing, dissolving, and then gel filtration separation. 2. In the present invention, the recovery of the cultured filaments can be easily carried out by using a net, but unlike the genus recovery method of unicellular algae, the genus recovery method is difficult in recovery and purification because gel polysaccharide is secreted. Occurs. 3. The culture medium after collecting the pickled or kosoji glue can be recovered and used for the subsequent inoculation, and the waste generated in another algae cultivation method can be reduced. 4. The gel-like phycoerythrin obtained from the salting-out process is in a stabilized form and therefore can be stored directly or lyophilized after dialysis. 5. Cultivated Nori and Nori seaweed as raw materials for phycoerythrin production can provide a more uniform and reliable stable product due to the consistency of culture conditions. 6. A mixture of phycocyanin and allophycocyanin can be obtained via gel filtration for purification of phycoerythrin. The mixture can then be separated into pure phycocyanin and allophycocyanin by DEAE-Sephacel ion exchange chromatography and sodium chloride gradient elution.

Claims (9)

[Claims] 1. A method for preparing a crude gel-type phycoerythrin concentrate from red algae, which comprises the following steps: a) Mature red algae selected from the group consisting of squash and kosujiri gametes. Preparing fronds; b) culturing the red algae in SWM-III medium to recover spores; c) temperature, illuminance and daily illumination time 15-25 respectively
Under conditions that are ℃, 10001x ~ 40001x and 10 ~ 16 hours,
Culturing the spores to germinate the filaments; d) destroying the filaments into fine fragments, and culturing the filaments under the above conditions until the desired amount of cultured filaments is obtained. E) collecting the culture filaments, drying and crushing into powder; f) adding the powder to a liquid selected from the group consisting of water and a phosphate solution. To obtain a transparent red pigment protein solution containing phycoerythrin; and g) salting out the gel type phycoerythrin concentrate from the transparent red pigment protein solution described above. 2. The method according to claim 1, further comprising the steps of dialyzing the gel-type phycoerythrin concentrate and purifying phycoerythrin by gel filtration. 3. The method according to claim 1, wherein the SWM-III medium is an inorganic SWM-III medium. 4. The culture condition of step c) is a temperature of 20 ° C. and an illuminance of 20.
The lighting time is 001x and the lighting time is 12 hours per day.
The method described. 5. The method according to claim 1, wherein in step e) the drying of the spinach filaments is carried out under vacuum. 6. The method according to claim 1, wherein, in step e), the filaments of pickled weeds are dried using warm air. 7. The method according to claim 1, wherein in step e), the cultured red algae filaments are collected using a 200 mesh net. 8. The method according to claim 1, wherein in step g), phycoerythrin is salted out using 20% to 30% ammonium sulfate and 60% to 65% ammonium sulfate. 9. The method of claim 2, wherein the gel filtration is Sephadex G200 gel filtration.