JP4677250B2 - Extraction method of phycocyanin from cyanobacteria - Google Patents

Description

  The present invention relates to an extraction method capable of obtaining phycocyanin contained in cyanobacteria with high purity.

  Cyanobacteria, particularly Spirulina, is known to contain blue phycocyanin, and various extraction methods for phycocyanin have been proposed for use as food coloring agents.

  As a method for extracting phycocyanin contained in cyanobacteria, for example, there is a method of mixing cyanobacteria and a phosphate buffer, eluting phycocyanin in cyanobacteria into the phosphate buffer, and then removing the residue. (For example, refer to Patent Document 1). However, in the extraction method described in Patent Document 1, it is difficult to extract phycocyanin having high purity due to mixing of pigments such as carotenoids that cross each other.

Japanese Patent Laid-Open No. 52-134058

  An object of the present invention is to provide an extraction method capable of obtaining phycocyanin contained in cyanobacteria with high purity.

  As a result of intensive studies, the inventors have obtained an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension, and then reacting calcium salt and phosphate in this extract to obtain calcium phosphate. It has been found that high-purity phycocyanin can be easily extracted by adsorbing phycocyanin impurities such as carotenoids to the calcium phosphate and removing cyanobacterial algae residues and adsorbates from this extract. The invention has been completed.

  That is, the present invention provides a first step of obtaining an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension, and reacting a calcium salt and a phosphate in the extract to produce calcium phosphate. A phycocyanin from cyanobacteria comprising: a second step of adsorbing phycocyanin impurities on the calcium phosphate to obtain an adsorbate; and a third step of removing cyanobacterial algae residues and adsorbate from the extract. The extraction method is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the extraction method which can obtain the phycocyanin contained in cyanobacteria with high purity can be provided. Further, this extraction method is easy and useful as an industrial extraction method.

  The first step of the present invention is a step of obtaining an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension. Cyanobacteria that can be used for the preparation of this extract include the genus Spirulina, the genus Aphanizomenon, the genus Fischerella, the genus Anabaena, the genus Nostoc, the genus Cinecostis The genus Synechococcus, the genus Tolypothrix, the genus Aphanothace, the genus Mastigoclaus, the genus Plurocapsa, and the like are produced on an industrial scale. Those belonging to Spirulina are desirable.

  Examples of cyanobacteria used in the present invention include raw cyanobacteria and dried cyanobacteria, but phycocyanin is extracted in a step of obtaining an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension. Dry cyanobacteria are preferred because they are easily treated and the amount of phycocyanin that can be extracted is stable.

  Raw cyanobacteria are harvested by, for example, methods such as centrifugation and filtration of algae cultured in water, and usually contain 70 to 90% by weight of water. Cyanobacteria are usually cultured in water with natural light or artificial light, but it is preferable to harvest cyanobacteria that are irradiated with light and undergoing photosynthesis. Especially for cyanobacteria grown in outdoor culture tanks under natural light, photosynthesis continues and the water temperature rises after 10 am, compared to cyanobacteria harvested at night or immediately after the start of light irradiation. More preferred are cyanobacteria harvested from sunset to sunset.

  Examples of the dried cyanobacteria include those obtained by freeze-drying or spray-drying the raw cyanobacteria cultured by the above method.

In the method for extracting phycocyanin from cyanobacteria of the present invention, an extract is obtained in the first step, and calcium phosphate and phosphate are reacted in the extract in the second step to obtain calcium phosphate. Adsorb phycocyanin impurities to obtain an adsorbate. In order to perform such an operation in the second step, for example, the first step and the second step may be performed as follows.
1. The first step is a step of preparing an aqueous suspension containing cyanobacteria and a calcium salt, and obtaining an extract obtained by extracting phycocyanin in cyanobacteria into the aqueous suspension. A step of adding phosphate to the liquid to obtain calcium phosphate and adsorbing phycocyanin impurities to the calcium phosphate to obtain an adsorbate.
2. The first step is a step of preparing an aqueous suspension containing cyanobacteria and phosphate, and obtaining an extract obtained by extracting phycocyanin in cyanobacteria into the aqueous suspension. A step of adding calcium salt to the extract to obtain calcium phosphate and adsorbing phycocyanin impurities to the calcium phosphate to obtain an adsorbate.
3. In the first step, an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension is obtained, and in the second step, phosphate and calcium salt are added to the extract to obtain calcium phosphate and the calcium phosphate. The process of adsorbing phycocyanin impurities to obtain adsorbate.

  In the present invention, the first step is to prepare an aqueous suspension containing cyanobacteria, calcium salt and phosphate, and extract the phycocyanin in cyanobacteria into the aqueous suspension. In the obtaining step, the process proceeds to the second step as it is, and calcium phosphate is obtained with the extract, and adsorbate can be obtained by adsorbing phycocyanin impurities to the calcium phosphate. In addition, the 3. In the first step, calcium salt and / or phosphate may be added. In the method for extracting phycocyanin from cyanobacteria of the present invention, As a first step, an aqueous suspension containing cyanobacteria and a calcium salt is prepared as a first step, and an extract obtained by extracting phycocyanin in cyanobacteria into the aqueous suspension is obtained. The extraction time of phycocyanin in cyanobacteria can be shortened, and an extract with less elution of phycocyanin impurities, particularly carotenoids, is preferable. The following description of the first step and the second step is 1. The above method is assumed.

  As a method for obtaining an extract in the first step, for example, an aqueous suspension containing cyanobacteria and a calcium salt is prepared, and this extract is held at 0 to 40 ° C. to extract phycocyanin in cyanobacteria. Methods and the like.

To obtain the aqueous suspension, for example,
First method. Add calcium salt to an aqueous solution containing cyanobacteria.
Second method. Add cyanobacteria to an aqueous solution of calcium salt and suspend.
The second method. This method is preferred. The explanation about the following suspension is the second method. The above method is assumed.

  Examples of the calcium salt used in the preparation of the aqueous solution of the calcium salt used in the present invention include water-soluble calcium salts such as calcium chloride, calcium nitrate, and calcium nitrite, among which calcium chloride is preferable.

  The concentration of the calcium salt in the aqueous suspension is preferably 0.1 to 10% by weight, more preferably 0.1 to 5% by weight, and still more preferably 0.5 to 3% by weight.

  When suspending cyanobacteria in an aqueous solution of calcium salt to obtain an aqueous suspension, the concentration of cyanobacteria is suspended in an aqueous solution of calcium salt within a range of 0.1 to 20% by weight in terms of solid content. Is preferable, and a range of 2 to 8% by weight is more preferable.

  The suspension is preferably prepared in a range where the temperature of the aqueous suspension is 0 to 40 ° C, more preferably 0 to 35 ° C.

  An aqueous suspension containing cyanobacteria and a calcium salt is prepared, and phycocyanin in cyanobacteria is extracted into the aqueous suspension to obtain an extract. Phycocyanin is extracted from cyanobacteria by standing, but may be stirred if necessary. The time required for extraction is preferably 1 to 48 hours, more preferably 1 to 20 hours.

  When an extract is obtained, phycocyanin in cyanobacteria can be efficiently extracted into an aqueous suspension by adding a basic compound to the aqueous suspension or performing ultrasonic irradiation treatment. Both the addition of the basic compound and the ultrasonic irradiation treatment may be performed, or only one of them may be performed. When both are performed, the basic compound may be added after ultrasonic irradiation, or the ultrasonic irradiation treatment may be performed after adding the basic compound, but the ultrasonic wave is added after adding the basic compound. It is preferable to perform irradiation treatment.

  Examples of the basic compound include alkali compounds such as sodium hydroxide, potassium hydroxide and lithium hydroxide; carbonates of alkali metals such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate and lithium carbonate; hydrogen carbonate Examples thereof include alkali metal hydrogen carbonates such as sodium, potassium hydrogen carbonate and lithium hydrogen carbonate; alkali metal acetates such as sodium acetate, potassium acetate and lithium acetate.

  After adding the basic compound to the suspension, it is preferable to make it homogeneous by stirring or the like. The temperature of the suspension is preferably in the range of 0 to 40 ° C, more preferably in the range of 15 to 35 ° C. After adding the basic compound to the suspension, it is preferable to further stir or leave it because the phycocyanin easily migrates from the cyanobacteria cells into the mixture of the suspension and the basic compound. The time for stirring and standing may be 10 minutes to 8 hours, and preferably 2 to 5 hours.

  Next, by performing ultrasonic irradiation treatment, the gas vesicles, which are the intracellular structures characteristic of cyanobacteria, are destroyed, the discharge of bubbles derived from the gas vesicles to the outside of the cell, and the association of cyanobacteria on the thylakoid membrane. Phycocyanin existing as a combination (phycobilisome) is preferentially solubilized in the aqueous phase. By this ultrasonic treatment, the transfer of phycocyanin from the cell to the aqueous phase can be promoted, and poor separation due to floating algae in the separation step can be prevented.

  The irradiation method when performing the ultrasonic irradiation treatment is not limited as long as it can destroy cyanobacterial cells and promote the migration of phycocyanin into suspension, and examples thereof include a batch type and a continuous type. Especially, the continuous type which irradiates an ultrasonic wave continuously is preferable. As a continuous ultrasonic irradiation treatment apparatus, for example, a multiple ultrasonic dispersion apparatus for production manufactured by Nippon Seiki Seisakusho Co., Ltd. may be used.

  The ultrasonic irradiation treatment may be performed under conditions that can promote the transition from cyanobacteria to the suspension of phycocyanin, but the amount of work given to the suspension by ultrasonic irradiation is about 1 liter of suspension. On the other hand, 1 to 300 kJ is preferable, 5 to 200 kJ is more preferable, and 10 to 100 kJ is particularly preferable.

  Examples of the conditions for the ultrasonic irradiation treatment include output, frequency, irradiation time, and the like. The ultrasonic frequency is preferably 10 to 100 kHz, more preferably 10 to 50 kHz, and particularly preferably 15 to 30 kHz. The output of ultrasonic irradiation is preferably 50 to 600 W, more preferably 100 to 400 W, and particularly preferably 200 to 400 W. In order to perform the ultrasonic irradiation processing for giving the work amount, the output and the irradiation time may be adjusted as appropriate. When ultrasonically processing one liter of suspension, for example, when the output is 50 W at a frequency of 20 kHz. The irradiation time is preferably 20 seconds to 100 minutes, more preferably 2 minutes to 70 minutes, and particularly preferably 3 minutes 30 seconds to 35 minutes. When the output is 300 W, the irradiation time is preferably 3 seconds to 20 minutes, more preferably 20 seconds to 12 minutes, and particularly preferably 30 seconds to 7 minutes. When the output is 500 W, the irradiation time is preferably 2 seconds to 10 minutes, more preferably 10 seconds to 7 minutes, and particularly preferably 20 seconds to 4 minutes.

  Phosphate is added to the aqueous extract of phycocyanin obtained in the first step in the second step. The phosphate may be added as a solid or in the form of an aqueous solution. Examples of the phosphate include sodium phosphate such as sodium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate; potassium phosphate such as potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; Examples include magnesium phosphate; water-soluble inorganic salts such as ammonium dihydrogen phosphate, among which sodium phosphate and potassium phosphate are preferable, sodium phosphate is particularly preferable, and disodium hydrogen phosphate is most preferable.

  The phosphate is preferably added to the extract so that the phosphate concentration is 1 to 5% by weight in the extract, and is added to the extract so that the concentration is 2 to 3% by weight. Is more preferable.

  Phosphate is added to the extract. When phosphate is added to the extract, the phosphate reacts with the calcium salt in the water extract to cause precipitation of calcium phosphate and adsorbs and adsorbs chlorophyll and other contaminants contaminated with phycocyanin pigment on the calcium phosphate. Form things. Thereby, the purity of the phycocyanin pigment can be increased. After adding the phosphate, it may be allowed to stand or may be stirred if necessary. The time required for the reaction (adsorption) of calcium ions and phosphate ions is preferably 2 to 10 hours, and more preferably 3 to 5 hours.

  The pH when adsorbing phosphate and cyanobacteria to obtain an adsorbate in the extract is preferably 4-8, more preferably 5-6, because the amount of phycocyanin obtained is increased. The pH can be adjusted, for example, by adding a basic compound or an acidic compound to the extract. Examples of the basic compound include alkali compounds such as sodium hydroxide, potassium hydroxide and lithium hydroxide; carbonates of alkali metals such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate and lithium carbonate; hydrogen carbonate Examples thereof include alkali metal hydrogen carbonates such as sodium, potassium hydrogen carbonate and lithium hydrogen carbonate; alkali metal acetates such as sodium acetate, potassium acetate and lithium acetate. Examples of the acidic compound include citric acid, hydrochloric acid, lactic acid, acetic acid, and the like. The pH of the extract can also be adjusted by adding a basic compound or acidic compound to the aqueous suspension in advance. If the pH of the aqueous suspension at this time is adjusted to 7 to 6, the pH of the extract is preferably 5 to 6.

  In the third step, the cyanobacterial residue and the adsorbate are removed from the extract after completion of the second step, but if the extract contains a chelating agent prior to the third step, the amount of phycocyanin recovered will be increased. It is particularly preferable because it can be used. This is because phycocyanin includes phycocyanin C and allophycocyanin. Allophycocyanin is adsorbed on calcium phosphate, and allophycocyanin adsorbed on this calcium phosphate is removed together with calcium phosphate in the next third step, but before the third step. The inventor believes that when the chelating agent is contained in the extract, the chelating agent is adsorbed on the calcium phosphate, so that allophycocyanin is separated from the calcium phosphate and remains in the extract.

  The chelating agent may be added before the third step. For example, the chelating agent may be added to the water suspension of cyanobacteria in the first step, or may be added to the prepared extract. It may be added to the extract before obtaining the adsorbate in two steps, or may be added after obtaining the adsorbate. In the present invention, it is preferably added at the time of suspension adjustment.

  Examples of the chelating agent include organic carboxylates such as sodium citrate, sodium oxalate, sodium tartrate, sodium gluconate; nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA) Amino carbonates such as dihydroxyethyl glycine (DFG), triethanolamine (TEA), N- (2-hydroxyethyl) iminodiacetic acid (HEIDA), hydroxyethylenediaminetetraacetic acid (HEDTA), etc .; Examples thereof include ether carboxylates such as sodium carboxymethyltaltronate (CMT) and sodium carboxymethyloxysuccinate (CMOS). Of these, sodium citrate and sodium ethylenediaminetetraacetate are more preferable.

  The addition amount of the chelating agent is preferably 5 to 100% by weight, more preferably 10 to 40% by weight based on the amount of calcium chloride used.

  In the third step, cyanobacteria residues and adsorbates are removed from the extract. Examples of means for removing these include various methods, such as a filtration method using a filter medium such as filter paper or filter cloth, a decantation method performed by collecting the supernatant from the precipitate, a centrifugation method, and the like. It is done. Of these, separation by centrifugation is preferable.

  Centrifugation may be performed under conditions that can remove cyanobacterial algae residues and adsorbates from the extract, but the centrifugal acceleration is preferably 10 to 2 hours at a gravitational acceleration of 1,000 to 30,000 G, and the gravitational acceleration is Centrifugation conditions of 3,000 to 10,000 G for 1 to 30 minutes are more preferable. There are disperse type centrifuges, alpha type centrifuges, and shear press type centrifuges as centrifuges. However, since the workability is improved, the disperse type centrifuges and alpha centrifuges are improved. Combination continuous centrifugation is preferred.

  The phycocyanin thus obtained can be used in this state, but may be further concentrated. As a concentration method, concentration by ultrafiltration is preferable because the low molecular weight dye, organic impurities, and inorganic ion content contaminated in the solution can be reduced and the degree of purification can be improved. The ultrafiltration membrane used for ultrafiltration preferably has a fractional molecular weight of 10,000 to 30,000, more preferably 5,000 to 20,000.

  The phycocyanin obtained by the extraction method of the present invention can be stabilized by adding saccharides, salts, etc., for example, glycerol, sodium citrate and the like, and can be provided as a solution-like dye solution, and further dried through a drying step. It can also be a powder. Any drying method may be used as long as phycocyanin is not denatured and deteriorated, but hot air spray drying and freeze drying are particularly preferably used.

  The extraction method of the present invention is a method capable of mass-producing industrially high-purity phycocyanin, and the obtained phycocyanin is an unprecedented pigment having a bright blue color.

  Next, the present invention will be specifically described with reference to Examples and Comparative Examples. In the examples, “parts” and “%” are based on weight unless otherwise specified.

Example 1
After adding 500 g of spirulina dried alga body (spray-dried product) produced in an outdoor culture tank to 10 L of 1% calcium chloride (anhydrous) solution, a uniform suspension is obtained by stirring for 15 minutes and then left at 20 ° C. for 15 hours. Under the conditions, phycocyanin in cyanobacteria was extracted into a solution to obtain an extract. After adding 250 g of sodium dihydrogen phosphate to this extract and stirring with a stirrer for 0.5 hours, the mixture was allowed to react at 20 ° C. for 2.5 hours to form calcium phosphate and phycocyanin impurities were added to the calcium phosphate. Adsorbed material was obtained by adsorption. Thereafter, the extract was guided to a centrifuge and centrifuged at a gravitational acceleration of 10,000 G for 15 minutes to remove cyanobacterial algae residues and adsorbates from the extract. The obtained phycocyanin extract was subjected to ultrafiltration using a separation membrane having a fractional molecular weight of 10,000 to remove low molecular components and salts, and then freeze-dried to obtain 46.6 g of a dried phycocyanin pigment. . This is designated as phycocyanin dye 1.

  A phycocyanin dye solution in which 0.1 g of phycocyanin dye 1 was dissolved in 100 ml of water was prepared, and the absorbance and color tone were measured according to the measurement method described below. The measurement results of absorbance and color tone are shown in Table 1 together with the weight of the dried pigment obtained per 100 g of algae dry weight.

(Measurement of absorbance)
Phycocyanin dye solution diluted 10 times with water, 618 nm (maximum absorption wavelength of phycocyanin C type), 650 nm (maximum absorption wavelength of allophycocyanin), 280 nm (maximum absorption wavelength of protein) and 446 nm (maximum absorption wavelength of β-carotene) The absorbance at was measured. It shows that the phycocyanin content in a pigment | dye dried material is so high that the light absorbency (Aat618nm) of wavelength 618nm and the total value of wavelength 650nm are high. It shows that the protein content in a pigment | dye dried material is so low that the measured value of the light absorbency (Aat280nm) of wavelength 280nm is low, and the highly purified phycocyanin is obtained. The lower the measured value of the absorbance at a wavelength of 446 nm (Aat 446 nm), the lower the content of β-carotene in the dried pigment product, indicating that phycocyanin with high purity is obtained.

  The ratio of Aat 650 nm to Aat 618 nm (Aat 280 nm / Aat 618 nm) indicates the allophycocyanin content per unit amount of phycocyanin C in the dried pigment, and the smaller this value, the less the allophycocyanin content and the higher the purity of phycocyanin C. It shows that it is obtained. The ratio of Aat 446 nm to Aat 618 nm (Aat 446 nm / Aat 618 nm) indicates the β carotene content per unit amount of phycocyanin C in the dried pigment, and the smaller this value, the lower the β carotene content and the higher the purity of phycocyanin C. It shows that it is obtained. The ratio of Aat 280 nm to Aat 618 nm (Aat 280 nm / Aat 618 nm) indicates the protein content per unit amount of phycocyanin C in the dried pigment, and the smaller this value, the less the protein content and the higher the purity of phycocyanin C. Indicates that The absorbance was measured using a UV2200 model manufactured by Shimadzu Corporation.

(Measurement of color tone)
The color tone was measured by diluting a phycocyanin dye solution with water so that the absorbance at a wavelength of 618 nm was 0.6. As for the color tone, the a value and the b value were measured according to Hunter's color specification method, and (a2 + b2) 1/2 value was calculated from these values. The b value represents the intensity of blue, and the greater the −b value, the stronger the blueness. (A2 + b2) 1/2 represents saturation, and the larger this value, the brighter the hue. The color tone was measured using an SZ-Σ90 type manufactured by Nippon Denshoku Industries Co., Ltd.

Example 2
Instead of 10 L of 1% calcium chloride (anhydrous) solution, 5 L of a solution of 50 g trisodium citrate crystals (food additive, Wako Pure Chemical Industries, equivalent to 0.5%) in 10 L of 1% calcium chloride (anhydrous) solution 62.5 g of a dried phycocyanin dye was obtained in the same manner as in Example 1 except that was used. This is designated as phycocyanin dye 2. The absorbance and color tone were measured in the same manner as in Example 1, and the results are shown in Table 1.

Comparative Example 1
500 g of spirulina dried alga (spray-dried product) is added to 10 L of a 0.01 molar phosphate buffer solution, pH 6.0 (Agricultural Chemistry Experiments, edited by Kyoto University), and a uniform suspension is obtained by stirring for 15 minutes. Thereafter, phycocyanin in cyanobacteria was extracted into the solution under static conditions at 20 ° C. for 15 hours to obtain an extract. To this extract, 150 g of sodium dihydrogen phosphate was added, stirred with a stirrer for 0.5 hour, and then allowed to react at 20 ° C. for 2.5 hours while standing. This was then guided to a centrifuge and centrifuged at a gravitational acceleration of 10,000 G for 15 minutes. The obtained phycocyanin extract was subjected to ultrafiltration using a separation membrane having a fractional molecular weight of 10,000 to remove low molecular components and salts, and then freeze-dried to obtain 43.1 g of a dried phycocyanin pigment. . This is designated as Phycocyanin Dye 3. The absorbance and color tone were measured in the same manner as in Example 1, and the results are shown in Table 1.

Claims (9)

A first step of obtaining an extract obtained by extracting phycocyanin in cyanobacteria into an aqueous suspension, and reacting a calcium salt and a phosphate in the extract to produce calcium phosphate, and forming phycocyanin into the calcium phosphate A second step of adsorbing impurities to obtain an adsorbate, a third step of removing cyanobacterial algae residues and adsorbate from the extract, and a step of adding a chelating agent to the extract prior to the third step; A method for extracting phycocyanin from cyanobacteria, comprising: The first step is a step of preparing an aqueous suspension containing cyanobacteria and a calcium salt, and obtaining an extract obtained by extracting phycocyanin in cyanobacteria into the aqueous suspension. The method for extracting phycocyanin from cyanobacteria according to claim 1, which is a step of adding calcium phosphate to the solution to obtain calcium phosphate and adsorbing phycocyanin impurities to the calcium phosphate to obtain an adsorbate. The method for extracting phycocyanin from cyanobacteria according to claim 1, wherein the cyanobacteria are spirulina subjected to a drying treatment. The method for extracting phycocyanin from cyanobacteria according to claim 1, wherein the calcium salt is calcium chloride. The method for extracting phycocyanin from cyanobacteria according to claim 2, wherein the concentration of cyanobacteria in the aqueous suspension is 2 to 8 wt% in terms of solid content and the concentration of calcium salt is 0.1 to 10 wt%. . The method for extracting phycocyanin from cyanobacteria according to claim 1, wherein the phosphate is disodium hydrogen phosphate. The method for extracting phycocyanin from cyanobacteria according to claim 2, wherein the concentration of phosphate in the extract is 1 to 5% by weight. Method of extracting phycocyanin from blue-green algae of claim 1 wherein the chelating agent is an organic carboxylic acid salt. The method for extracting phycocyanin from cyanobacteria according to claim 8, wherein the organic carboxylate is sodium citrate and / or sodium ethylenediaminetetraacetate.