JP4048420B2 - Purification method of phycocyanin dye solution - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、フィコシアニン色素液の精製方法に関する。
【０００２】
【従来の技術】
スピルリナ等の藍藻類には、青色を呈するフィコシアニンが含まれていることが知られており、従来から食品用着色料として利用するために種々のフィコシアニンの抽出方法が提案されてきた。
【０００３】
藍藻類に含まれるフィコシアニンを抽出する方法としては、例えば、（１）生の藍藻類をカルシウム・イオンを含む第１水相と接触せしめる工程、（２）第１水相を藍藻類より分離する工程、（３）藍藻類を塩基性化合物含有の第２水相と接触せしめる工程、（４）前記（３）工程によりフィコシアニンを含有する第２水相を残存藍藻類より分離する工程からなる抽出方法が、特公昭６３−５５９２０号公報等に報告されている。しかしながら、該抽出方法では、クロロフィル等のフィコシアニンに対する夾雑色素が除去できず、フィコシアニン本来の強く、鮮やかな青みを有するフィコシアニンが得られない。
【０００４】
【発明が解決しようとする課題】
本発明の課題は、強く、鮮やかな青みを有するフィコシアニン色素液が得られるフィコシアニン色素液の精製方法を提供することにある。
【０００５】
【課題を解決するための手段】
本発明者らは、上記課題を解決すべく鋭意研究した結果、種々の製造方法により得られた夾雑色素含有フィコシアニン色素液に、該夾雑色素に対する凝集剤を加えることにより該夾雑色素が凝集した凝集物が形成されること、該凝集物は遠心分離法、濾過法等により除去できること、得られるフィコシアニン色素液は、強く、鮮やかな青みを有すること等を見出し、本発明をなすに至った。
【０００６】
すなわち、本発明は、フィコシアニンの夾雑色素を含有する藍藻類由来のフィコシアニン色素液中の該夾雑色素を、リン酸水素カルシウムにより凝集させたのち、該色素液から凝集物を除去するフィコシアニン色素液の精製方法であり、該リン酸水素カルシウムがフィコシアニン色素液中でリン酸塩とカルシウム塩とを反応させて得られたものである事を特徴とするフィコシアニン色素液の精製方法を提供するものである。
【０００７】
【発明の実施の形態】
以下に本発明について、詳細に説明する。
本発明で用いるフィコシアニン色素液は、藍藻類由来で、かつ、フィコシアニンに対する夾雑色素を含有していれば、藍藻類の種類や製造方法等に制限はない。藍藻類としては、例えば、スピルリナ（Ｓｐｉｒｕｌｉｎａ）属、アファニゾメノン（Ａｐｈａｎｉｚｏｍｅｎｏｎ）属、フィッシェレラ（Ｆｉｓｈｅｒｅｌｌａ）属、アナベナ（Ａｎａｂａｅｎａ）属、ネンジュモ（Ｎｏｓｔｏｃ）属、シネコキスチス（Ｓｙｎｅｃｈｏｃｙｓｔｉｓ）属、シネココッカス（Ｓｙｎｅｃｈｏｃｏｃｃｕｓ）属、トリポスリクス（Ｔｏｌｙｐｏｔｈｒｉｘ）属、スイゼンジノリ（Ａｐｈａｎｏｔｈｅｃｅ）属、マスティゴクラディス（Ｍａｓｔｉｇｏｃｌａｕｓ）属、プルロカプサ（Ｐｌｅｕｒｏｃａｐｓａ）属等が挙げられるが、工業的規模で生産され、その安全性が確認されているスピルリナ属に属するものが望ましい。
【０００８】
藍藻類は、生の状態のものや、生の藍藻を乾燥したもの等が入手できるが、生の藍藻類が、青みが強く色調も良好なフィコシアニン色素液が得られることから好ましい。生の藍藻類は、例えば、水中で培養された藻を遠心分離、濾過等の方法により収穫され、通常水分を７０〜９０重量％含有している。藍藻類は、通常水中で自然光、又は人工光により培養されるが、光が照射され光合成を行っている状態の藍藻を収穫するのが好ましい。特に自然光下の屋外培養槽で培養されている藍藻においては、夜間若しくは光照射が始まった直後に収穫された藍藻よりは、光合成が継続して行われ、水温も上昇してくる午前１０時以降から日没までに収穫された藍藻がより好ましい。
【０００９】
乾燥処理した藍藻類としては、例えば、前記の方法で収穫した生の藍藻類を、凍結乾燥処理したものや、スプレー乾燥処理したもの等が挙げられる。
【００１０】
フィコシアニン色素液は、その製造方法としては、例えば、
１．藍藻類が懸濁した懸濁液を、特公昭６３−５５９２０号公報に記載されている様な塩基性化合物等の化学的手段で処理し、フィコシアニン色素液を得る方法、
２．藍藻類が懸濁した懸濁液を、超音波照射処理等の機械的手段で処理し、フィコシアニン色素液を得る方法、
３．藍藻類が懸濁した懸濁液を、化学的手段および機械的手段で処理し、フィコシアニン色素液を得る方法、
等が挙げられる。
【００１１】
フィコシアニン色素液の製造方法は、機械的手段による処理を用いた製造方法が好ましく、なかでも、機械的手段による処理と化学的手段による処理を併用する製造方法がより好ましい。
【００１２】
前記藍藻類が懸濁した懸濁液は、懸濁液中の藍藻類の濃度が乾燥固形分として０．１〜２０．０重量％となる範囲で懸濁したものが好ましく、２．０〜８．０重量％となる範囲がより好ましい。
【００１３】
前記藍藻類の懸濁液には、後述する凝集剤の形成成分として使用することができること、藍藻類の細胞壁に作用し、フィコシアニンが抽出され易くなること、食品に使用できることから、カルシウム塩を添加しておくのが好ましく、または、凝集剤の形成成分として使用できることに加え、懸濁液のｐＨを安定させる緩衝効果があること、食品に使用できることから、リン酸塩を添加するのが好ましい。
【００１４】
カルシウム塩としては、例えば、塩化カルシウム、硝酸カルシウム、亜硝酸カルシウム等の水溶性のカルシウム塩が挙げられるが、中でも、塩化カルシウムが好ましい。
【００１５】
カルシウム塩の懸濁液中のカルシウム塩の濃度は、０．０１〜２．０モル／リットルが好ましく、０．１〜０．５モル／リットルがより好ましい。
【００１６】
リン酸塩としては、例えば、リン酸ナトリウム、リン酸二水素ナトリウム、リン酸水素二ナトリウム等のリン酸ナトリウム；リン酸カリウム、リン酸二水素カリウム、リン酸水素二カリウム等のリン酸カリウム；リン酸マグネシウム；リン酸二水素アンモニウム等の水溶性無機塩が挙げられるが、中でも、リン酸ナトリウム、リン酸カリウムが好ましく、リン酸ナトリウムが特に好ましい。更に、リン酸塩は、リン酸二水素ナトリウムとリン酸水素二ナトリウムを併用することが、後述する範囲に懸濁液のｐＨを調節しやすいので好ましい。
【００１７】
リン酸塩の水溶液中のリン酸塩の濃度は、０．００１〜０．５モル/リットルが好ましく、０．０１〜０．１モル/リットルがより好ましい。また、リン酸塩の水溶液のｐＨは、５．０〜８．０の範囲が好ましく、５．２〜６．６がより好ましい。
【００１８】
懸濁液の調製は、懸濁液の温度が４〜４５℃となる範囲で行うのが好ましく、１５〜３５℃がより好ましい。
【００１９】
懸濁液は、調製後すぐに次の工程に用いることができるが、調製後静置するのが好ましい。静置時間は、５分〜８時間が好ましく、１０分〜１時間がより好ましい。
【００２０】
次に、懸濁液に対して、前記▲２▼の製造方法においては機械的処理を、▲３▼の製造方法においては化学的処理と機械的処理とを行う。
【００２１】
前記３．の製造方法における化学的処理と機械的処理の順序は、機械的処理を行った後に化学的処理を行っても良いし、化学的処理を行った後に機械的処理を行っても良いが、化学的処理を行った後に機械的処理を行うのが好ましい。
【００２２】
化学的処理を行う化合物としては、例えば、水酸化ナトリウム、水酸化カリウム、水酸化リチウム等のアルカリ化合物；炭酸ナトリウム、炭酸水素ナトリウム、炭酸カリウム、炭酸水素カリウム、炭酸リチウム等のアルカリ金属の炭酸塩；炭酸水素ナトリウム、炭酸水素カリウム、炭酸水素リチウム等のアルカリ金属の炭酸水素塩；酢酸ナトリウム、酢酸カリウム、酢酸リチウム等のアルカリ金属の酢酸塩等の塩基性化合物が好ましく使用できる。
【００２３】
懸濁液を塩基性化合物を添加して処理するには、塩基性化合物の水溶液とした上で懸濁液に加えても良いし、固体の塩基性化合物を直接懸濁液に加えても良いが、塩基性化合物の水溶液とした上で懸濁液に加えるのが好ましい。塩基性化合物の水溶液のｐＨは、８．０〜１２．０の範囲が好ましく、９．０〜１１．０がより好ましい。塩基性化合物としては、炭酸ナトリウムと炭酸水素ナトリウムが好ましく、炭酸ナトリウムと炭酸水素ナトリウムを両方用いるのがより好ましい。炭酸ナトリウムと炭酸水素ナトリウムの使用割合（炭酸ナトリウム：炭酸水素ナトリウム）は、１：９０〜１１０：１が好ましく、１：８〜１３：１がより好ましい。
【００２４】
上記懸濁液に塩基性化合物を混合する際は、懸濁液中の塩基性化合物の濃度が０．００１〜０．１モル／リットルとなる様に混合するのが好ましく、０．００５〜０．０５モル／リットルがより好ましい。塩基性化合物を添加した直後の懸濁液のｐＨは、７．５〜１０．０の範囲が好ましく、８．５〜９．５がより好ましい。
【００２５】
懸濁液に塩基性化合物を添加した後は、攪拌等により均質にする。懸濁液の温度は、４〜４５℃の範囲が好ましく、１５〜３５℃の範囲がより好ましい。懸濁液へ塩基性化合物を添加した後は、更に攪拌または静置するのが、フィコシアニンが藍藻細胞から懸濁液と塩基性化合物の混合液中に移行しやすくなるので好ましい。攪拌、静置の時間は、１０分間〜８時間で良く、２〜５時間が好ましい。
【００２６】
懸濁液の機械的処理は、藍藻の細胞を破砕し、細胞中のフィコシアニンが懸濁液に移行できる方法であれば制限はないが、細胞内に含まれるフィコシアニン以外の夾雑成分の懸濁液への移行が少なく、且つフィコシアニンの懸濁液への移行が多い方法が好ましい。細胞破砕法としては、例えば、超音波照射処理法、プロペラ型高速ホモゲナイザー法、湿式ボールミル法、高圧ホモゲナイザー法等が挙げられる。細胞破砕は、藍藻に特徴的な細胞内構造であるガス胞を破壊し、ガス胞に由来する気泡を細胞外へ排出することにより、分離工程での浮上性藻による分離不良を防止することができること、藍藻のチラコイド膜上の会合体（フィコビリゾーム）として存在しているフィコシアニンを懸濁液へ優先的に可溶化させ、細胞からのフィコシアニンの懸濁液への移行を促進することから超音波照射処理により行うことが好ましい。
【００２７】
超音波照射処理を行う際の照射方法は、藍藻類の細胞を破壊し、フィコシアニンを懸濁液中に抽出させることができれば制限はなく、バッチ式や連続式等が挙げられるが、なかでも、連続的に超音波を照射する連続式が好ましい。連続式の超音波照射処理装置としては、例えば（株）日本精機製作所の生産用多連式超音波分散装置等が挙げられる。
【００２８】
超音波照射処理は、懸濁液中の藍藻類からフィコシアニンを抽出させることができるような条件で行えば良いが、前記▲２▼の機械的処理のみを行う時の超音波照射により懸濁液に与えられる仕事量としては、懸濁液１リットルに対して、１００〜３０００ｋＪが好ましく、２００〜２０００ｋＪがより好ましく、３００〜１０００ｋＪが特に好ましい。
【００２９】
超音波照射処理の条件としては出力、周波数、照射時間等が挙げられる。超音波の周波数は１０〜１００ｋＨｚの領域で行うことが好ましく、１５〜５０ｋＨｚの領域がより好ましく、１５〜３０ｋＨｚの領域が特に好ましい。超音波照射装置の出力は、１００〜３０００Ｗが好ましく、２００〜２０００Ｗがより好ましく、３００〜１０００Ｗが特に好ましい。
【００３０】
前記仕事量を与える超音波照射処理を行うには、出力と照射時間とを適宜調整すればよいが、懸濁液１リットルを超音波処理する時には、例えば、周波数２０ｋＨｚにおいて、出力が１００Ｗの時は、１７〜５００分の照射時間が好ましく、３５〜３４０分がより好ましく、５０〜１７０分が特に好ましい。出力が６００Ｗの時は、３〜８５分の照射時間が好ましく、５分３０秒〜６０分がより好ましく、８分３０秒〜３０分が特に好ましい。出力が１０００Ｗの時は、２〜５０分の照射時間が好ましく、３分３０秒〜３４分がより好ましく、５分〜１７分が特に好ましい。具体的には、例えば、後述する実施例２に示すように、周波数が１９．５ｋＨｚ、超音波照射部の容積が２０ｍＬ、出力が６００Ｗの連続式超音波破砕装置を用いて破砕処理するには、懸濁液を３ｍＬ／秒の速度で超音波照射部に送り破砕処理を行う工程を２回行えば良い。
【００３１】
前記▲３▼の機械的処理および化学的処理を行う時の超音波照射により懸濁液に与えられる仕事量としては、懸濁液１リットルに対して、１〜３００ｋＪが好ましく、５〜２００ｋＪがより好ましく、１０〜１００ｋＪが特に好ましい。
【００３２】
超音波照射処理の条件としては出力、周波数、照射時間等が挙げられる。超音波の周波数としては、１０〜１００ｋＨｚが好ましく、１０〜５０ｋＨｚがより好ましく、１５〜３０ｋＨｚが特に好ましい。超音波照射の出力は、５０〜６００Ｗが好ましく、１００〜４００Ｗがより好ましく、２００〜４００Ｗが特に好ましくい。
【００３３】
前記仕事量を与える超音波照射処理を行うには、出力と照射時間とを適宜調整すればよいが、懸濁液１リットルを超音波処理する時には、例えば、周波数２０ｋＨｚにおいて、出力が５０Ｗの時は、２０秒〜１００分の照射時間が好ましく、２分〜７０分がより好ましく、３分３０秒〜３５分が特に好ましい。出力が３００Ｗの時は、３秒〜２０分の照射時間が好ましく、２０秒〜１２分がより好ましく、３０秒〜７分が特に好ましい。出力が５００Ｗの時は、２秒〜１０分の照射時間が好ましく、１０秒〜７分がより好ましく、２０秒〜４分が特に好ましい。具体的には、例えば、後述する実施例１に示すように、周波数が１９．５ｋＨｚ、超音波照射部の容積が１０ｍＬ、出力が３００Ｗの連続式超音波破砕装置を用いて破砕処理するには、懸濁液を５ｍＬ／秒の速度で超音波照射部に送り破砕処理すればよい。
【００３４】
前記のようにして得られたフィコシアニン色素液は、そのままでも用いることができるが、色素液中の藍藻類の残渣を分離除去して使用しても良い。残渣を分離除去する手段としては、固液分離できる手段であれば、例えば、ろ紙やろ布等のろ材を用いたろ過方法や、上澄を回収するデカンテーション法、フィルタープレス法、遠心分離方法等が挙げられる。なかでも、工業的に大量処理の可能な遠心分離法、フィルタープレス法等の上澄を回収する方法が好ましく、特に遠心分離法が好ましい。
【００３５】
遠心分離は、懸濁液から残渣を除去できる条件であれば良いが、重力加速度が１，０００〜３０，０００Ｇで１０秒〜２時間の条件が好ましく、重力加速度が３，０００〜１５，０００Ｇで１〜３０分間の条件がより好ましい。遠心分離機としては、ディスラッジ型遠心分離機、アルファ型遠心分離機、シャープレス型遠心分離機があるが、作業性が向上することから、ディスラッジ型遠心分離機とアルファ型遠心分離機の組み合わせによる連続遠心分離が好ましい。
【００３６】
次に、凝集剤について説明する。
本発明の精製方法で用いる凝集剤としては、フィコシアニン色素の夾雑色素を凝集させることができるものであれば特に制限無く使用することができる。フィコシアニン色素液への凝集剤の添加方法としては、例えば、フィコシアニン色素液に添加する時点で夾雑色素に対して凝集作用を有する凝集剤を添加しても良いし、フィコシアニン色素液に凝集剤の原料となる化合物を添加し、該色素液中で凝集剤としても良い。なかでも、フィコシアニン色素液に凝集剤の原料となる化合物を添加し、該色素液中で凝集剤とするのが、凝集剤が形成される際に夾雑色素の巻き込みが起こり、夾雑色素の凝集効果が増加するので好ましい。
【００３７】
凝集剤としては、例えば、２価若しくは３価の電荷を有する金属イオンのリン酸塩及びリン酸水素塩があり、特にアルカリ土類のリン酸塩やアルカリ土類のリン酸水素塩が好ましく使用できる。アルカリ土類のリン酸塩としては、リン酸マグネシウム、リン酸カルシウム、リン酸バリウム等が挙げられ、アルカリ土類のリン酸水素塩としては、例えば、リン酸水素マグネシウム、リン酸二水素マグネシウム等のリン酸水素マグネシウム；リン酸水素カルシウム、リン酸二水素カルシウム等のリン酸水素カルシウム；リン酸水素バリウム、リン酸二水素バリウム等のリン酸水素バリウム等のアルカリ土類のリン酸水素塩が挙げられる。凝集剤としては、なかでも、リン酸水素カルシウム、リン酸二水素カルシウムが好ましい。
【００３８】
凝集剤の添加量は、夾雑色素を凝集させるに十分な量を適宜添加すればよいが、フィコシアニン色素液１００重量部に対して、０．１〜１０重量部が好ましく、１．〜５重量部がより好ましい。フィコシアニン色素液への凝集剤の添加時期としては、例えば、得られたフィコシアニン色素液に凝集剤を加えても良いし、フィコシアニン色素液の製造中に凝集剤を加えても良い。凝集剤は固体の状態で加えても良いし、凝集剤を溶液とした上で加えても良い。
【００３９】
フィコシアニン色素液に凝集剤の原料となる化合物を添加し、該色素液中で凝集剤とするには、例えば、アルカリ土類の金属塩とリン酸塩等の原料を該色素液に添加すれば良い。
【００４０】
リン酸塩としては、例えば、リン酸ナトリウム、リン酸二水素ナトリウム、リン酸水素二ナトリウム等のリン酸ナトリウム；リン酸カリウム、リン酸二水素カリウム、リン酸水素二カリウム等のリン酸カリウム；リン酸マグネシウム；リン酸二水素アンモニウム等の水溶性無機塩が挙げられる。なかでも、リン酸ナトリウム、リン酸カリウムが好ましく、リン酸ナトリウムが特に好ましい。リン酸塩は、固体の状態で用いても良いし、水溶液とした状態で用いても良い。
【００４１】
リン酸塩は、リン酸塩の濃度が色素液中で０．００１〜０．５モル/リットルの濃度になるよう添加するのが好ましく、０．０１〜０．１モル/リットルがより好ましい。また、リン酸塩を加えた後の色素液のｐＨが、５．０〜８．０の範囲に入るようリン酸塩を加えるのが好ましく、５．３〜７．０の範囲がより好ましい。
【００４２】
アルカリ土類金属塩としては、例えば、塩化カルシウム、硝酸カルシウム、亜硝酸カルシウム等のカルシウム塩、塩化マグネシウム、硝酸マグネシウム等のマグネシウム塩、塩化バリウム、硝酸バリウム等のバリウム塩等が挙げられる。アルカリ土類金属塩は、カルシウム塩が好ましく、なかでも、塩化カルシウムが好ましい。また、アルカリ土類金属塩は固体の状態で用いても良いし、水溶液とした状態で用いても良い。
【００４３】
アルカリ土類金属塩は、色素液中で濃度が、０．０１〜２．０モル／リットルになる様に使用するのが好ましく、０．１〜０．５モル／リットルがより好ましい。
【００４４】
フィコシアニン色素液に凝集剤の原料となる化合物、例えば、リン酸塩とアルカリ金属塩好ましくはカルシウム塩とを添加し、該色素液中で凝集剤とするには、例えば、（１）フィコシアニン色素液を製造中にリン酸塩とカルシウム塩を添加しても良いし、（２）フィコシアニン色素液を製造後にリン酸塩とカルシウム塩を添加しても良いし、（３）フィコシアニン色素液を製造中にカルシウム塩を添加しておき、製造後リン酸を添加しても良いし、（４）フィコシアニン色素液を製造中にリン酸塩を添加しておき、製造後カルシウム塩を添加しても良いが、（３）の方法が、夾雑色素の凝集効果が高いこと、カルシウム塩が藍藻類の細胞壁に作用し、フィコシアニンが抽出されやすくなることから好ましく、（４）の方法が、夾雑色素の凝集効果が高いこと、リン酸塩が、製造中の液のｐＨを安定させる緩衝作用を有することから好ましい。
【００４５】
（３）または（４）の方法において、フィコシアニン色素液を調製後にリン酸塩またはアルカリ金属塩、好ましくはカルシウム塩を添加した後は、すぐ次の工程に進めるが、攪拌または攪拌後静置するのが好ましい。静置時間は、５分〜２４時間が好ましく、３０分〜２０時間がより好ましい。また、色素液の温度は、４〜４５℃が好ましく、４〜２５℃がより好ましい
【００４６】
（３）または（４）の方法において、フィコシアニン色素液を調製後にリン酸塩またはアルカリ金属塩を添加した後のｐＨは、ｐＨは５．０〜７．０が好ましい。リン酸塩とカルシウム塩を含有した後は、色素液中でリン酸カルシウム、リン酸水素カルシウム及び水素イオンを生じ、その結果、抽出液のｐＨが５．０未満に低下することがある。ｐＨが５．０未満になると、フィコシアニンの回収率が低下することがあることから、この段階でさらにリン酸塩を加えて抽出液のｐＨを５．０〜７．０に調整するのが好ましい。
【００４７】
次に、色素液中の凝集物の分離処理を行い、フィコシアニン色素液中に含まれる夾雑色素の凝集物や、藍藻類の残渣を分離除去する。分離処理を行う手段としては、種々の方法が挙げられ、例えば、ろ紙やろ布等のろ材を用いたろ過方法や、沈殿から上澄を回収することにより行うデカンテーション法、遠心分離方法等が挙げられる。なかでも、遠心分離による分離が好ましい。
【００４８】
遠心分離は、抽出液から残渣を除去できる条件であれば良いが、重力加速度が１，０００〜３０，０００Ｇで１０秒〜２時間の遠心分離条件が好ましく、重力加速度が３，０００〜１０，０００Ｇで１〜３０分間の遠心分離条件が、より好ましい。遠心分離機としては、ディスラッジ型遠心分離機、アルファ型遠心分離機、シャープレス型遠心分離機があるが、作業性が向上することから、ディスラッジ型遠心分離機とアルファ型遠心分離機の組み合わせによる連続遠心分離が好ましい。
【００４９】
分離処理により得られたフィコシアニン抽出液は、この状態で使用に供することも可能であるが、更に、濃縮しても良い。濃縮方法としては、溶液に夾雑している低分子性色素、有機不純物、及び無機イオン含量を低下させ、精製度を向上させるため、限外濾過による濃縮が好ましい。限外濾過に用いる限外濾過膜は、分画分子量が１，０００〜３０，０００のものが好ましく、５，０００〜２０，０００のものがより好ましい。
【００５０】
本発明の抽出方法で得られるフィコシアニン抽出液は、糖類、塩類等、例えばグリセロール、クエン酸ナトリウム等を加えて安定化させ溶液状の色素液として提供可能である。さらにこのフィコシアニン濃縮液は、乾燥工程を経ることにより、乾燥粉末にすることもできる。乾燥方法は、フィコシアニンが変性劣化しない条件であれば何れでも良いが、熱風噴霧乾燥、凍結乾燥が特に好ましく用いられる。
【００５１】
本発明の抽出方法は、工業的にフィコシアニンを大量生産できる方法であり、得られるフィコシアニンは、従来にない鮮やかな青色を有する色素である。例えば色差計を使用した色調測定においても、従来の工業的抽出方法で得られた色素と明確に区別することが出来る。
【００５２】
【実施例】
次に実施例、比較例により本発明を具体的に説明する。例中において、「部」「％」は特に断りのない限り、重量基準である。
【００５３】
実施例１
人工光を使用し、７日間連続光照射下の培養槽でスピルリナを培養生産し、収穫した。得られた生の藻体１ｋｇ（固形分量１８．０％）を１％塩化カルシウム溶液６Ｌ（０．０９モル／リットル）に加え、攪拌機により２０℃で４５分間攪拌を行いスピルリナ懸濁液を得た。炭酸ナトリウム３ｇと炭酸水素ナトリウム３ｇを５０ｍｌの水に溶解し、スピルリナ懸濁液に添加（懸濁液中の塩基性化合物の濃度は、０．０１モル／リットル）した。この懸濁液を２分間攪拌後、２０℃で５時間、静置した。この懸濁液を連続式超音波破砕装置〔（株）日本精機製作所ＲＵＳ−３００ＴＣＶＰ型、周波数１９．５ｋＨｚ、超音波照射部の容積１０ｍＬ、出力３００Ｗ〕に導き、懸濁液を５ｍＬ／秒の速度で超音波照射部に送り超音波処理を行い、抽出液を得た。得られた抽出液にリン酸二水素ナトリウムが６．８ｇ／リットル、リン酸水素二ナトリウムが２．２ｇ／リットルの濃度になる様に加え、攪拌後、遠心分離機に導き、重力加速度が１０，０００Ｇで、１５分間の遠心分離を行った。得られたフィコシアニンの抽出液は、分画分子量１５，０００の分離膜を使用した限外濾過により低分子成分及び塩類を除去した後、凍結乾燥を行い、フィコシアニン色素乾燥物を４５ｇを得た。これを、フィコシアニン色素１とする。
【００５４】
フィコシアニン色素１の０．１ｇを１００ｍｌの水に溶解したフィコシアニン色素液を調製し、下記に示す測定方法に従い吸光度と色調を測定した。吸光度と色調の測定結果を、藻類乾燥重量１００ｇあたり得られた色素乾燥物の重量と共に、第１表に示す。
【００５５】
（吸光度の測定）
フィコシアニン色素液を水で１０倍希釈し、６１８ｎｍ（フィコシアニンの吸収極大波長）と２８０ｎｍ（タンパク質の吸収極大波長）における吸光度を測定し、更に、波長６１８ｎｍの吸光度（Ａａｔ６１８ｎｍ）の１００倍値から、色素乾燥物１％溶液の色価（Ｅ１％）を求めた。波長６１８ｎｍの吸光度及びこの色価が、波長６１８ｎｍの吸光度（Ａａｔ６１８ｎｍ）の測定値が高いほど色素乾燥物中のフィコシアニン含有量が高いことを示す。また、Ａａｔ２８０ｎｍとＡａｔ６１８ｎｍとの比（Ａａｔ２８０ｎｍ／Ａａｔ６１８ｎｍ）は、色素乾燥物中のフィコシアニン単位量当たりのタンパク質含有量を示し、この値が小さい程、夾雑タンパク質が除かれ純度の高いフィコシアニンが得られていることを示す。尚、吸光度の測定は、（株）島津製作所製のＵＶ２２００型を用いて行った。
【００５６】
（色調の測定）
色調の測定には、波長６１８ｎｍの吸光度が０．６になる様にフィコシアニン色素液を水で希釈したものを用いた。色調は、ハンター（Ｈｕｎｔｅｒ）の表色法に従い、ａ値とｂ値とを測定し、これらの値から（ａ２＋ｂ２）１／２値を算出した。ｂ値は青色の強さを表し、−ｂ値が大きいほど、青みが強いことを示す。（ａ２＋ｂ２）１／２は彩度を表し、この値が大きいほど、色合いが鮮やかであることを示す。尚、色調の測定は、日本電色工業（株）製ＳＺ−Σ９０型を用いて行った。
実施例２
人工光を使用し、７日間連続光照射下の培養槽でスピルリナを培養生産し、収穫した。得られた藻体１ｋｇ（固形分量１８．０％）をｐＨ６．５の０．０１モル／Ｌのリン酸ナトリウム緩衝液６Ｌに加え、攪拌機により２０℃で３０分間攪拌を行い、スピルリナ懸濁液を得た。この懸濁液を連続式超音波破砕装置（（株）日本精機製作所ＲＵＳ−６００ＴＣＶＰ型、周波数１９．５ｋＨｚ、超音波照射部の容積２０ｍＬ、出力６００Ｗ）に導き、懸濁液を３ｍＬ／秒の速度で超音波照射部に送り超音波処理を行った。この超音波処理をさらにもう１回行った。超音波処理後、得られた抽出液に塩化カルシウムを、０．０９モル／Ｌになる様に加え、攪拌後、１５℃で１２時間静置した。静置後、遠心分離機に導き、重力加速度が１０，０００Ｇで、２０分間の遠心分離を行った。遠心分離後、上澄みを回収し、抽出液を得た。得られたフィコシアニンの抽出液は、分画分子量１５，０００の分離膜を用いた膜分離装置を使用して低分子成分及び塩類を除去した後、凍結乾燥を行い、フィコシアニン色素乾燥物４７ｇを得た。これを、フィコシアニン色素２とする。実施例１と同様にして、フィコシアニン色素液を調製し、吸光度と色調を測定した。結果を第１表に示す。
【００５７】
実施例３
実施例１で用いた生のスピルリナ藻体１ｋｇを１％塩化カルシウム水溶液６Ｌ（０．０９モル／リットル）に加え、攪拌機により２０℃で４５分間攪拌を行いスピルリナ懸濁液を得た。スピルリナ懸濁液からスピルリナを濾布により濾過分離し、該スピルリナを炭酸ナトリウムと炭酸水素ナトリウムをそれぞれ０．５ｇ／Ｌ濃度で含有する水溶液６Ｌ（水溶液中の塩基性化合物の濃度は０．０１モル／リットル）に懸濁した。懸濁後、リン酸二水素ナトリウムが６．８ｇ／リットル、リン酸水素二ナトリウムが２．２ｇ／リットルの濃度になる様に加え、２０℃で２時間攪拌後、次いで１時間放置した。この懸濁液を傾斜して上清を回収し回収液を得ると共に、残存藻類に、該残存藻類の乾燥重量に対し５重量％の割合で濾過助剤として硫酸アルミナを添加した後、減圧濾過を行い回収液を得た。これらの回収液を合わせた液を、遠心分離機に導き、重力加速度が１０，０００Ｇで、１５分間の遠心分離を行った。得られたフィコシアニンの抽出液は、限外濾過処理及び凍結乾燥を実施例１と同様に行い、フィコシアニン色素乾燥物３８ｇを得た。これをフィコシアニン３とする。実施例１と同様にして、フィコシアニン色素液を調製し、吸光度と色調を測定した。結果を第１表に示す。
【００５８】
比較例１
リン酸二水素ナトリウムと、リン酸水素二ナトリウムとを添加しなかった以外は実施例１と同様にして、フィコシアニン色素乾燥物３５ｇを得た。これを、比較対照用フィコシアニン色素１´とする。実施例１と同様にして、比較対照用フィコシアニン色素液を調製し、吸光度と色調を測定した。結果を第２表に示す。
【００５９】
比較例２
塩化カルシウムを添加しながった以外は実施例２と同様にして、フィコシアニン色素乾燥物４８ｇを得た。これを、比較対照用フィコシアニン色素２´とする。実施例１と同様にして、比較対照用フィコシアニン色素液を調製し、吸光度と色調を測定した。結果を第２表に示す。
【００６０】
比較例３
リン酸二水素ナトリウムと、リン酸水素二ナトリウムとを添加しなかった以外は実施例３と同様にして、フィコシアニン色素乾燥物５９ｇを得た。これを、比較対照用フィコシアニン色素３´とする。実施例１と同様にして、比較対照用フィコシアニン色素液を調製し、吸光度と色調を測定した。結果を第２表に示す。
【００６１】
【表１】
【表２】
実施例１〜３で得られたフィコシアニン色素は２８０ｎｍと６１８ｎｍの吸光度比（２８０ｎｍの吸光度／６１８ｎｍの吸光度）が、比較例１〜３で得られた色素と比較して小さな値を示し、夾雑タンパク質が除かれフィコシアニンの純度が高いことが示された。
【００６４】
また、実施例１〜３で得られたフィコシアニン色素は、鮮やかな青色を呈した。色差計による色調の測定値では、実施例１〜３で得られたフィコシアニン色素は、比較例１〜３で得られたフィコシアニン色素と比較して、青色の強さを示す−ｂ値がより大きな値を示し、色の鮮やかさを示す（ａ２＋ｂ２）１／２も大きな値を示した。
【００６５】
【発明の効果】
本発明は、藍藻類、特にスピルリナから青みが深く、色調の鮮やかなフィコシアニンを高純度で、かつ、大量安価に生産できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying a phycocyanin dye solution.
[0002]
[Prior art]
Cyanobacteria such as Spirulina are known to contain phycocyanin exhibiting a blue color, and various extraction methods for phycocyanin have been proposed for use as food coloring agents.
[0003]
Examples of a method for extracting phycocyanin contained in cyanobacteria include, for example, (1) a step of bringing raw cyanobacteria into contact with a first aqueous phase containing calcium ions, and (2) separating the first aqueous phase from cyanobacteria. (3) an extraction step comprising contacting the cyanobacteria with a second aqueous phase containing a basic compound; (4) an extraction step comprising separating the second aqueous phase containing phycocyanin from the remaining cyanobacteria by the step (3). The method is reported in Japanese Patent Publication No. 63-55920. However, this extraction method cannot remove phycocyanin such as chlorophyll, and phycocyanin having a strong and vivid blueness inherent to phycocyanin cannot be obtained.
[0004]
[Problems to be solved by the invention]
The subject of this invention is providing the purification method of the phycocyanin dye liquid from which the phycocyanin dye liquid which has strong and vivid blueness is obtained.
[0005]
[Means for Solving the Problems]
As a result of diligent research to solve the above-mentioned problems, the present inventors have aggregated the contaminating dyes by adding an aggregating agent for the contaminating dyes to the contaminating dye-containing phycocyanin dye solutions obtained by various production methods. The present inventors have found that a product is formed, the aggregate can be removed by a centrifugal separation method, a filtration method, and the like, and that the obtained phycocyanin dye solution has a strong and vivid bluish color.
[0006]
That is, the present invention removes aggregates from the dye solution after aggregating the contaminated pigment in the phycocyanin pigment solution derived from cyanobacteria containing the phycocyanin contaminant pigment with calcium hydrogen phosphate. A method for purifying a phycocyanin dye solution, wherein the calcium hydrogen phosphate is obtained by reacting a phosphate with a calcium salt in a phycocyanin dye solution. Is to provide.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
As long as the phycocyanin pigment solution used in the present invention is derived from cyanobacteria and contains a contaminating pigment for phycocyanin, there is no limitation on the type of cyanobacteria and the production method. Examples of cyanobacteria include, for example, the genus Spirulina, the genus Aphanizomenon, the genus Fischerella, the genus Anabena, the genus Nestococcus, and the genus Synechocystis (Tolypothrix) genus, Suphanzinori genus, Mastigo cladis genus, Plurocapsa genus, etc., but those belonging to the genus Spirulina produced on an industrial scale and whose safety has been confirmed. Is desirable.
[0008]
The cyanobacteria can be obtained in a raw state or dried from the raw cyanobacteria, but the raw cyanobacteria are preferable because a phycocyanin pigment solution having a strong bluish color and a good color tone can be obtained. 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 cultured in an outdoor culture tank 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.
[0009]
Examples of dried cyanobacteria include those obtained by freeze-drying or spray-drying raw cyanobacteria harvested by the above method.
[0010]
How to make phycocyanin dye solution Law and For example,
1. A method of obtaining a phycocyanin dye solution by treating a suspension of cyanobacteria with a chemical means such as a basic compound described in JP-B-63-55920,
2. A method of obtaining a phycocyanin dye solution by treating a suspension of cyanobacteria with mechanical means such as ultrasonic irradiation treatment,
3. A method for treating a suspension of cyanobacteria with chemical means and mechanical means to obtain a phycocyanin pigment solution;
Etc.
[0011]
The production method of the phycocyanin dye solution is preferably a production method using treatment by mechanical means, and more preferably a production method using both treatment by mechanical means and treatment by chemical means.
[0012]
The suspension in which the cyanobacteria are suspended is preferably a suspension in which the concentration of cyanobacteria in the suspension is 0.1 to 20.0% by weight as a dry solid content. A range of 8.0% by weight is more preferable.
[0013]
In the suspension of cyanobacteria, calcium salt is added because it can be used as a component for forming a flocculant described later, acts on the cell wall of cyanobacteria, facilitates extraction of phycocyanin, and can be used in foods. In addition to being able to be used as a component for forming a flocculant, it is preferable to add phosphate because it has a buffering effect to stabilize the pH of the suspension and can be used in foods.
[0014]
Examples of the calcium salt include water-soluble calcium salts such as calcium chloride, calcium nitrate, and calcium nitrite, among which calcium chloride is preferable.
[0015]
The concentration of the calcium salt in the calcium salt suspension is preferably 0.01 to 2.0 mol / liter, and more preferably 0.1 to 0.5 mol / liter.
[0016]
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 them, sodium phosphate and potassium phosphate are preferable, and sodium phosphate is particularly preferable. In addition, it is preferable to use sodium dihydrogen phosphate and disodium hydrogen phosphate in combination as the phosphate because the pH of the suspension is easily adjusted within the range described below.
[0017]
The concentration of the phosphate in the aqueous phosphate solution is preferably 0.001 to 0.5 mol / liter, more preferably 0.01 to 0.1 mol / liter. The pH of the aqueous phosphate solution is preferably in the range of 5.0 to 8.0, more preferably 5.2 to 6.6.
[0018]
The suspension is preferably prepared in the range where the temperature of the suspension is 4 to 45 ° C, more preferably 15 to 35 ° C.
[0019]
The suspension can be used in the next step immediately after preparation, but it is preferable to stand still after preparation. The standing time is preferably 5 minutes to 8 hours, and more preferably 10 minutes to 1 hour.
[0020]
Next, the suspension is subjected to mechanical treatment in the production method (2), and chemical treatment and mechanical treatment in the production method (3).
[0021]
Above 3. Of chemical treatment and mechanical treatment in the manufacturing method Is The chemical treatment may be performed after the mechanical treatment, or the mechanical treatment may be performed after the chemical treatment, but the mechanical treatment is preferably performed after the chemical treatment.
[0022]
Examples of the chemical treatment 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 A basic compound such as an alkali metal hydrogen carbonate such as sodium hydrogen carbonate, potassium hydrogen carbonate or lithium hydrogen carbonate; an alkali metal acetate such as sodium acetate, potassium acetate or lithium acetate can be preferably used.
[0023]
In order to treat the suspension by adding a basic compound, the aqueous solution of the basic compound may be added to the suspension, or the solid basic compound may be added directly to the suspension. However, it is preferable to add an aqueous solution of a basic compound to the suspension. The pH of the aqueous solution of the basic compound is preferably in the range of 8.0 to 12.0, and more preferably 9.0 to 11.0. As the basic compound, sodium carbonate and sodium hydrogen carbonate are preferable, and it is more preferable to use both sodium carbonate and sodium hydrogen carbonate. The ratio of sodium carbonate and sodium bicarbonate used (sodium carbonate: sodium bicarbonate) is preferably 1:90 to 110: 1, and more preferably 1: 8 to 13: 1.
[0024]
When mixing the basic compound with the suspension, it is preferable to mix so that the concentration of the basic compound in the suspension is 0.001 to 0.1 mol / liter. .05 mol / liter is more preferred. The pH of the suspension immediately after adding the basic compound is preferably in the range of 7.5 to 10.0, and more preferably 8.5 to 9.5.
[0025]
After adding the basic compound to the suspension, it is homogenized by stirring or the like. The temperature of the suspension is preferably in the range of 4 to 45 ° 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.
[0026]
The mechanical treatment of the suspension is not limited as long as it disrupts cyanobacterial cells and the phycocyanin in the cells can be transferred to the suspension, but the suspension of contaminant components other than phycocyanin contained in the cells is not limited. A method in which there is little transfer to, and there is much transfer to a suspension of phycocyanin is preferred. Examples of the cell disruption method include an ultrasonic irradiation treatment method, a propeller type high-speed homogenizer method, a wet ball mill method, and a high-pressure homogenizer method. Cell disruption can prevent poor separation due to floating algae in the separation process by destroying gas vesicles, which are the intracellular structures characteristic of cyanobacteria, and discharging bubbles derived from gas vesicles to the outside of the cells. Ultrasonic irradiation because phycocyanin existing as an aggregate (phycobilisome) on the thylakoid membrane of cyanobacteria is preferentially solubilized in the suspension and promotes the transfer of phycocyanin from the cell to the suspension. It is preferable to carry out by treatment.
[0027]
The irradiation method when performing the ultrasonic irradiation treatment is not limited as long as the cells of cyanobacteria can be destroyed and phycocyanin can be extracted into the suspension, and examples include a batch type and a continuous type. A continuous type in which ultrasonic waves are continuously applied 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.
[0028]
The ultrasonic irradiation treatment may be performed under conditions that allow phycocyanin to be extracted from the cyanobacteria in the suspension. However, the suspension is obtained by ultrasonic irradiation when only the mechanical treatment of (2) is performed. The work applied to the suspension is preferably 100 to 3000 kJ, more preferably 200 to 2000 kJ, and particularly preferably 300 to 1000 kJ with respect to 1 liter of the suspension.
[0029]
Examples of the conditions for the ultrasonic irradiation treatment include output, frequency, irradiation time, and the like. The ultrasonic frequency is preferably in the range of 10 to 100 kHz, more preferably in the range of 15 to 50 kHz, and particularly preferably in the range of 15 to 30 kHz. The output of the ultrasonic irradiation device is preferably 100 to 3000 W, more preferably 200 to 2000 W, and particularly preferably 300 to 1000 W.
[0030]
In order to perform the ultrasonic irradiation processing that gives 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 100 W at a frequency of 20 kHz. The irradiation time is preferably 17 to 500 minutes, more preferably 35 to 340 minutes, and particularly preferably 50 to 170 minutes. When the output is 600 W, the irradiation time is preferably 3 to 85 minutes, more preferably 5 minutes 30 seconds to 60 minutes, and particularly preferably 8 minutes 30 seconds to 30 minutes. When the output is 1000 W, the irradiation time is preferably 2 to 50 minutes, more preferably 3 minutes 30 seconds to 34 minutes, and particularly preferably 5 minutes to 17 minutes. Specifically, for example, as shown in Example 2 described later, the crushing process is performed using a continuous ultrasonic crushing apparatus having a frequency of 19.5 kHz, an ultrasonic irradiation unit volume of 20 mL, and an output of 600 W. The process of sending the suspension to the ultrasonic irradiation unit at a rate of 3 mL / second and performing the crushing process may be performed twice.
[0031]
The amount of work given to the suspension by ultrasonic irradiation when performing the mechanical treatment and chemical treatment of (3) is preferably 1 to 300 kJ, more preferably 5 to 200 kJ with respect to 1 liter of the suspension. More preferably, 10 to 100 kJ is particularly preferable.
[0032]
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.
[0033]
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. Specifically, for example, as shown in Example 1 to be described later, to crush using a continuous ultrasonic crusher having a frequency of 19.5 kHz, an ultrasonic irradiation unit volume of 10 mL, and an output of 300 W The suspension may be sent to the ultrasonic irradiation unit at a rate of 5 mL / second and crushed.
[0034]
The phycocyanin dye solution obtained as described above can be used as it is, but it may be used after separating and removing cyanobacteria algae residues in the dye solution. As a means for separating and removing the residue, any means capable of solid-liquid separation can be used. If For example, a filtration method using a filter medium such as filter paper or filter cloth, a decantation method for collecting the supernatant, a filter press method, a centrifugation method, and the like can be given. Among them, a method of collecting the supernatant such as a centrifugal separation method and a filter press method capable of industrially processing in large quantities is preferable, and a centrifugal separation method is particularly preferable.
[0035]
Centrifugation may be performed under the condition that the residue can be removed from the suspension, but the gravitational acceleration is preferably 1,000 to 30,000 G and preferably 10 seconds to 2 hours, and the gravitational acceleration is 3,000 to 15,000 G. The condition of 1 to 30 minutes is more preferable. There are disperse type centrifuges, alpha centrifuges, and shear press centrifuges as centrifuges. Combination continuous centrifugation is preferred.
[0036]
Next, the flocculant will be described.
The flocculant used in the purification method of the present invention can be used without particular limitation as long as it can agglomerate the phycocyanin pigment. As a method for adding the flocculant to the phycocyanin dye liquid, for example, a flocculant having an aggregating action on the contaminating dye at the time of addition to the phycocyanin dye liquid may be added. It is also possible to add a compound that becomes a coagulant in the dye solution. In particular, adding a compound that is a raw material for the flocculant to the phycocyanin dye liquid and using it as the flocculant in the dye liquid causes the inclusion of the contaminated dye when the flocculant is formed, and the coagulation effect of the contaminated dye Is preferable.
[0037]
Examples of the aggregating agent include phosphates and hydrogen phosphates of metal ions having a divalent or trivalent charge, and alkaline earth phosphates and alkaline earth hydrogen phosphates are particularly preferably used. it can. Examples of alkaline earth phosphates include magnesium phosphate, calcium phosphate, and barium phosphate. Examples of alkaline earth hydrogen phosphates include phosphorous such as magnesium hydrogen phosphate and magnesium dihydrogen phosphate. Magnesium hydrogen hydrogen; calcium hydrogen phosphate such as calcium hydrogen phosphate and calcium dihydrogen phosphate; alkaline earth hydrogen phosphates such as barium hydrogen phosphate such as barium hydrogen phosphate and barium dihydrogen phosphate . Among them, calcium hydrogen phosphate and calcium dihydrogen phosphate are preferable as the flocculant.
[0038]
The addition amount of the flocculant may be appropriately added in an amount sufficient to aggregate the contaminating dye, but is preferably 0.1 to 10 parts by weight with respect to 100 parts by weight of the phycocyanin dye solution. -5 parts by weight is more preferred. As the addition timing of the flocculant to the phycocyanin dye liquid, for example, the flocculant may be added to the obtained phycocyanin dye liquid, or the flocculant may be added during the production of the phycocyanin dye liquid. The flocculant may be added in a solid state, or may be added after the flocculant is made into a solution.
[0039]
In order to add a compound that becomes a raw material of the flocculant to the phycocyanin dye liquid and to make the flocculant in the dye liquid, for example, by adding raw materials such as an alkaline earth metal salt and phosphate to the dye liquid good.
[0040]
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. Of these, sodium phosphate and potassium phosphate are preferable, and sodium phosphate is particularly preferable. The phosphate may be used in a solid state or an aqueous solution.
[0041]
The phosphate is preferably added so that the concentration of the phosphate is 0.001 to 0.5 mol / liter in the dye solution, and more preferably 0.01 to 0.1 mol / liter. Moreover, it is preferable to add a phosphate so that the pH of the dye solution after adding the phosphate falls within a range of 5.0 to 8.0, and a range of 5.3 to 7.0 is more preferable.
[0042]
Examples of the alkaline earth metal salt include calcium salts such as calcium chloride, calcium nitrate, and calcium nitrite, magnesium salts such as magnesium chloride and magnesium nitrate, and barium salts such as barium chloride and barium nitrate. The alkaline earth metal salt is preferably a calcium salt, and calcium chloride is particularly preferable. The alkaline earth metal salt may be used in a solid state or in an aqueous solution state.
[0043]
The alkaline earth metal salt is preferably used so that the concentration in the dye solution is 0.01 to 2.0 mol / liter, more preferably 0.1 to 0.5 mol / liter.
[0044]
In order to add a compound as a raw material of the flocculant to the phycocyanin dye liquid, for example, a phosphate and an alkali metal salt, preferably a calcium salt, to form the flocculant in the dye liquid, for example, (1) phycocyanin dye liquid Phosphate and calcium salt may be added during production, (2) Phospocyanine pigment solution may be added after production of phycocyanin pigment solution, or (3) Phycocyanin pigment solution is being produced Calcium salt may be added to the product, and phosphoric acid may be added after production. (4) Phosphate may be added during production of the phycocyanin dye solution, and calcium salt may be added after production. However, the method (3) is preferable because the aggregation effect of the contaminating pigment is high, and the calcium salt acts on the cell wall of cyanobacteria and the phycocyanin is easily extracted. The method (4) is preferable. It is highly effective, phosphate is preferable because of having a buffering action to stabilize the pH of the liquid during manufacture.
[0045]
In the method (3) or (4), after the phycocyanin dye solution is prepared, the phosphate or alkali metal salt, preferably calcium salt is added, and then the process proceeds to the next step. Is preferred. The standing time is preferably 5 minutes to 24 hours, more preferably 30 minutes to 20 hours. The temperature of the dye solution is preferably 4 to 45 ° C, more preferably 4 to 25 ° C.
[0046]
In the method (3) or (4), the pH after adding the phosphate or alkali metal salt after preparing the phycocyanin dye solution is preferably 5.0 to 7.0. After containing phosphate and calcium salt, calcium phosphate, calcium hydrogen phosphate and hydrogen ions are generated in the dye solution, and as a result, the pH of the extract may be lowered to less than 5.0. When the pH is less than 5.0, the recovery rate of phycocyanin may decrease. Therefore, it is preferable to further adjust the pH of the extract to 5.0 to 7.0 by adding phosphate at this stage. .
[0047]
Next, a separation process of the aggregates in the dye solution is performed to separate and remove the aggregates of contaminating pigments contained in the phycocyanin dye solution and the residues of cyanobacteria. Examples of the means for performing the separation treatment include various methods, such as a filtration method using a filter medium such as filter paper and 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.
[0048]
Centrifugation is not particularly limited as long as the residue can be removed from the extract. Centrifugation conditions of 10 to 2 hours at a gravitational acceleration of 1,000 to 30,000 G are preferable, and a gravitational acceleration of 3,000 to 10, Centrifugation conditions at 1 000 G for 1 to 30 minutes are more preferred. There are disperse type centrifuges, alpha centrifuges, and shear press centrifuges as centrifuges. Combination continuous centrifugation is preferred.
[0049]
The phycocyanin extract obtained by the separation treatment can be used in this state, but may be further concentrated. As the concentration method, concentration by ultrafiltration is preferred in order to reduce the content of low molecular weight dyes, organic impurities, and inorganic ions contaminated in the solution and improve the degree of purification. The ultrafiltration membrane used for ultrafiltration preferably has a fractional molecular weight of 1,000 to 30,000, more preferably 5,000 to 20,000.
[0050]
The phycocyanin extract obtained by the extraction method of the present invention can be stabilized by adding sugars, salts, etc., for example, glycerol, sodium citrate and the like, and can be provided as a solution-like dye solution. Furthermore, this phycocyanin concentrate can be made into a dry powder through a drying step. Any drying method may be used as long as phycocyanin is not denatured and deteriorated. Hot air spray drying and freeze drying are particularly preferably used.
[0051]
The extraction method of the present invention is a method capable of industrially mass-producing phycocyanin, and the obtained phycocyanin is an unprecedented pigment having a bright blue color. For example, even in color tone measurement using a color difference meter, it can be clearly distinguished from a dye obtained by a conventional industrial extraction method.
[0052]
【Example】
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.
[0053]
Example 1
Using artificial light, Spirulina was cultured and harvested in a culture tank under continuous light irradiation for 7 days and harvested. 1 kg of the obtained raw algal bodies (solid content: 18.0%) is added to 6 L (0.09 mol / liter) of 1% calcium chloride solution and stirred at 20 ° C. for 45 minutes to obtain a spirulina suspension. It was. 3 g of sodium carbonate and 3 g of sodium hydrogen carbonate were dissolved in 50 ml of water and added to the Spirulina suspension (the concentration of the basic compound in the suspension was 0.01 mol / liter). The suspension was stirred for 2 minutes and then allowed to stand at 20 ° C. for 5 hours. This suspension was introduced into a continuous ultrasonic crusher (Nippon Seiki Seisakusho RUS-300TCVP type, frequency 19.5 kHz, volume of ultrasonic irradiation unit 10 mL, output 300 W), and the suspension was 5 mL / second. Ultrasonic treatment was performed at a speed to the ultrasonic irradiation unit to obtain an extract. To the obtained extract, sodium dihydrogen phosphate was added to a concentration of 6.8 g / liter and disodium hydrogen phosphate to a concentration of 2.2 g / liter. After stirring, the mixture was introduced into a centrifuge and the gravitational acceleration was 10 Centrifugation was performed at 1,000 G for 15 minutes. The obtained phycocyanin extract was lyophilized after removing low molecular components and salts by ultrafiltration using a separation membrane having a molecular weight cut off of 15,000 to obtain 45 g of a dried phycocyanin pigment. This is designated as Phycocyanin Dye 1.
[0054]
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.
[0055]
(Measurement of absorbance)
The phycocyanin dye solution is diluted 10-fold with water, and the absorbance at 618 nm (maximum absorption wavelength of phycocyanin) and 280 nm (maximum absorption wavelength of protein) is measured. The color value (E1%) of a 1% dry product solution was determined. The absorbance at a wavelength of 618 nm and this color value indicate that the higher the measured value of the absorbance at a wavelength of 618 nm (Aat 618 nm), the higher the phycocyanin content in the dried pigment. 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 in the dried pigment product. Indicates that The absorbance was measured using a UV2200 model manufactured by Shimadzu Corporation.
[0056]
(Measurement of color tone)
For the measurement of the color tone, a phycocyanin dye solution diluted with water was used so that the absorbance at a wavelength of 618 nm was 0.6. The color tone is determined by measuring the a value and the b value according to Hunter's color specification method. 2 + B 2 ) 1/2 The value was calculated. The b value represents the intensity of blue, and the greater the −b value, the stronger the blueness. (A 2 + B 2 ) 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
Using artificial light, Spirulina was cultured and harvested in a culture tank under continuous light irradiation for 7 days and harvested. 1 kg of the obtained alga body (solid content: 18.0%) was added to 6 L of 0.01 mol / L sodium phosphate buffer having a pH of 6.5, and the mixture was stirred with a stirrer at 20 ° C. for 30 minutes to give a spirulina suspension. Got. This suspension was guided to a continuous ultrasonic crusher (Nippon Seiki Seisakusho RUS-600TCVP type, frequency 19.5 kHz, ultrasonic irradiation unit volume 20 mL, output 600 W), and the suspension was 3 mL / second. The ultrasonic treatment was performed at a speed to the ultrasonic irradiation unit. This sonication was performed once more. After sonication, calcium chloride was added to the obtained extract so as to be 0.09 mol / L, and the mixture was stirred and allowed to stand at 15 ° C. for 12 hours. After standing still, it was guided to a centrifuge and centrifuged at a gravitational acceleration of 10,000 G for 20 minutes. After centrifugation, the supernatant was collected to obtain an extract. The obtained phycocyanin extract was lyophilized after removing low molecular components and salts using a membrane separator using a separation membrane with a molecular weight cut off of 15,000 to obtain 47 g of dried phycocyanin dye. It was. This is designated as phycocyanin dye 2. In the same manner as in Example 1, a phycocyanin dye solution was prepared, and absorbance and color tone were measured. The results are shown in Table 1.
[0057]
Example 3
1 kg of the raw spirulina alga used in Example 1 was added to 6 L of 1% calcium chloride aqueous solution (0.09 mol / liter), and stirred at 20 ° C. for 45 minutes to obtain a spirulina suspension. Spirulina was separated from the Spirulina suspension with a filter cloth, and 6 L of an aqueous solution containing the Spirulina at a concentration of 0.5 g / L each of sodium carbonate and sodium bicarbonate (the concentration of the basic compound in the aqueous solution was 0.01 mol). / Liter). After suspension, sodium dihydrogen phosphate was added to a concentration of 6.8 g / liter and disodium hydrogen phosphate to a concentration of 2.2 g / liter, stirred at 20 ° C. for 2 hours, and then allowed to stand for 1 hour. The suspension is decanted and the supernatant is recovered to obtain a recovered liquid. At the same time, alumina sulfate is added to the remaining algae as a filter aid at a ratio of 5% by weight with respect to the dry weight of the remaining algae, followed by vacuum filtration. The recovered liquid was obtained. The liquid obtained by combining these recovered liquids was 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 treatment and lyophilization in the same manner as in Example 1 to obtain 38 g of a dried phycocyanin pigment. This is designated as Phycocyanin 3. In the same manner as in Example 1, a phycocyanin dye solution was prepared, and absorbance and color tone were measured. The results are shown in Table 1.
[0058]
Comparative Example 1
35 g of dried phycocyanin dye was obtained in the same manner as in Example 1 except that sodium dihydrogen phosphate and disodium hydrogen phosphate were not added. This is designated as a comparative phycocyanin dye 1 ′. In the same manner as in Example 1, a phycocyanin dye solution for comparison and control was prepared, and the absorbance and color tone were measured. The results are shown in Table 2.
[0059]
Comparative Example 2
48 g of dried phycocyanin dye was obtained in the same manner as in Example 2 except that calcium chloride was not added. This is designated as a comparative phycocyanin dye 2 ′. In the same manner as in Example 1, a phycocyanin dye solution for comparison and control was prepared, and the absorbance and color tone were measured. The results are shown in Table 2.
[0060]
Comparative Example 3
59 g of a dried phycocyanin dye was obtained in the same manner as in Example 3 except that sodium dihydrogen phosphate and disodium hydrogen phosphate were not added. This is designated as comparative phycocyanin dye 3 ′. In the same manner as in Example 1, a phycocyanin dye solution for comparison and control was prepared, and the absorbance and color tone were measured. The results are shown in Table 2.
[0061]
[Table 1]
[Table 2]
The phycocyanin dyes obtained in Examples 1 to 3 have a small absorbance value between 280 nm and 618 nm (absorbance at 280 nm / absorbance at 618 nm) as compared with the dyes obtained in Comparative Examples 1 to 3, and are contaminating proteins. Was removed, indicating that the purity of phycocyanin was high.
[0064]
Moreover, the phycocyanin pigment | dye obtained in Examples 1-3 exhibited bright blue. In the measured value of the color tone by the color difference meter, the phycocyanin dyes obtained in Examples 1 to 3 have a greater -b value indicating the intensity of blue than the phycocyanin dyes obtained in Comparative Examples 1 to 3. Value and color vividness (a 2 + B 2 ) 1/2 Also showed a large value.
[0065]
【The invention's effect】
INDUSTRIAL APPLICABILITY The present invention can produce phycocyanin with deep blueness and vivid color from cyanobacteria, particularly spirulina, with high purity and in large quantities at low cost.
Claims (6)
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