JP2018046775A - Method for recovering water insoluble substances - Google Patents
Method for recovering water insoluble substances Download PDFInfo
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- JP2018046775A JP2018046775A JP2016184581A JP2016184581A JP2018046775A JP 2018046775 A JP2018046775 A JP 2018046775A JP 2016184581 A JP2016184581 A JP 2016184581A JP 2016184581 A JP2016184581 A JP 2016184581A JP 2018046775 A JP2018046775 A JP 2018046775A
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Landscapes
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Abstract
Description
本発明は、水溶液中の非水溶性物を回収する方法に関し、より詳しくは水溶液中の微生物、特に微細藻類を回収する方法に関するものである。 The present invention relates to a method for recovering water-insoluble substances in an aqueous solution, and more particularly to a method for recovering microorganisms, particularly microalgae, in an aqueous solution.
バイオマスを用いた液体燃料、健康食品、医薬品、香粧品または動物性飼料等の製造は、持続的な経済発展に不可欠な技術である。例えば、光合成により炭化水素を生産する微生物の一種である微細藻類は潜在的生産能力の高さから期待が大きく、微細藻類を培養することで、炭化水素化合物などのバイオマスを産生させる様々な研究が既に行われている。 Production of liquid fuel, health food, pharmaceuticals, cosmetics, animal feed, etc. using biomass is an indispensable technology for sustainable economic development. For example, microalgae, a kind of microorganisms that produce hydrocarbons by photosynthesis, are highly expected due to their high potential production capacity, and various researches have been conducted to produce biomass such as hydrocarbon compounds by culturing microalgae. Already done.
微細藻類を用いてのバイオマスの生産には、効率的な微細藻類の培養方法、微細藻類の回収方法、更には産生するオイル等のバイオマスの抽出方法が開発されておらず、コストが高いという問題点がある。その最大の原因の一つが、微細藻類の効率的な回収方法がないことである。 For the production of biomass using microalgae, efficient microalgae cultivation methods, microalgae recovery methods, and extraction methods for biomass such as oil to be produced have not been developed, resulting in high costs There is a point. One of the biggest causes is the lack of an efficient method for collecting microalgae.
具体的には、微細藻類は通常、液中に浮遊しながら生育するため、微細藻類をバイオマスとして利用するためには、非常に希薄な濃度の微細藻類を大量の液中から回収しなければならない。加えて、微細藻類の生育のためには光エネルギーが必要であるため、十分な光の照射を確保するためには液中に存在する微細藻類の濃度を過度に高くすることが出来ない。 Specifically, since microalgae usually grow while floating in the liquid, in order to use the microalgae as biomass, a very dilute concentration of microalgae must be recovered from a large amount of liquid. . In addition, since light energy is required for the growth of microalgae, the concentration of microalgae present in the liquid cannot be excessively increased in order to ensure sufficient light irradiation.
結果として、液中に浮遊する微細藻類を回収するには、多量の水をろ過する必要があった。また、微細藻類のサイズは一般的に小さく、ろ過も容易ではなかった。このような問題を解決するための回収方法の検討として、沈殿剤を用いる方法、遠心分離機を用いる方法、微細藻類をより大型の生物の餌とした後に、該大型の生物を回収する方法などが試みられたものの、いずれの方法も根本的な解決には至っていない。 As a result, in order to collect the microalgae floating in the liquid, it was necessary to filter a large amount of water. Also, the size of microalgae is generally small and filtration is not easy. As a study of a recovery method for solving such problems, a method using a precipitant, a method using a centrifuge, a method of recovering the large organism after feeding microalgae to a larger organism, etc. However, none of these methods has led to a fundamental solution.
また、生活排水等による水域内への栄養塩の供給と蓄積が要因として起こる富栄養化により、湖沼等の閉鎖性水域では、アオコ等の藻類の異常増殖が発生する。このような藻類の異常増殖により、水道の取水、水産、農業または観光の場としての水環境の利用への障害が生じる。このため、異常増殖した微細藻類を効率的に回収する浄化技術が必要とされている。 In addition, due to eutrophication that occurs due to the supply and accumulation of nutrient salts in the water area due to domestic wastewater and the like, abnormal growth of algae such as sea lions occurs in closed water areas such as lakes. Such abnormal growth of algae creates obstacles to the use of the water environment as a place for water intake, fisheries, agriculture or tourism. Therefore, there is a need for a purification technique that efficiently recovers abnormally grown microalgae.
これまでに、水中の微細藻類等の非磁性物質に対し磁性を付し、磁性フロックを形成させ、その後に、磁気分離装置で水中から微細藻類を分離回収する磁気分離方法が開発されている（非特許文献１）。この磁気分離方法は、高磁場を利用することで高速分離処理が可能で、装置もコンパクトになること、物理的に処理するために化学薬剤を要しないこと等の利点がある。 So far, a magnetic separation method has been developed in which magnetism is applied to nonmagnetic substances such as microalgae in water to form a magnetic floc, and then microalgae are separated and recovered from water with a magnetic separator ( Non-patent document 1). This magnetic separation method is advantageous in that high-speed separation processing is possible by using a high magnetic field, the apparatus is compact, and no chemical agent is required for physical processing.
非特許文献１の技術では、非磁性物質に対し磁性を付し、磁性フロックを形成させるための工程において時間および手間を要し、さらにはマグネタイトと磁性フロックの形成しやすさが藻類の形態によって異なり、微細藻類の種類によって回収率が変化するため、実用化しにくいという問題点がある。 In the technique of Non-Patent Document 1, it takes time and labor to attach magnetism to a non-magnetic substance and form a magnetic floc, and the ease of forming magnetite and magnetic floc depends on the form of algae. Unlikely, since the recovery rate varies depending on the type of microalgae, there is a problem that it is difficult to put into practical use.
本発明は、微細藻類等の非水溶性物を水溶液中から効率的、簡便かつ低コストで回収する方法を提供することを目的とする。 An object of this invention is to provide the method of collect | recovering water-insoluble matter, such as microalgae, from aqueous solution efficiently, simply and at low cost.
本発明者は上記の目的を達成するため、鋭意検討した結果、水溶液中の非水溶性物を効率的に回収する方法を見出した。すなわち、本発明は下記の構成により達成されるものである。 As a result of intensive studies to achieve the above object, the present inventor has found a method for efficiently recovering a water-insoluble material in an aqueous solution. That is, the present invention is achieved by the following configuration.
［１］以下の工程（１）〜（３）を含む、水溶液中に存在する非水溶性物を回収する方法。
（１）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性磁性微粒子を含有させ、刺激を与えて刺激応答性磁性微粒子を疎水性として非水溶性物と結合させ、非水溶性物と刺激応答性磁性微粒子との凝集物を生成させる工程
（２）工程（１）で生成させた凝集物を磁力により水溶液中から分離する工程
（３）工程（２）で分離した凝集物に工程（１）と反対の刺激を与えて刺激応答性磁性微粒子を親水性とし、非水溶性物を刺激応答性磁性微粒子からリリースする工程
［２］以下の工程（１’）〜（３’）を含む、水溶液中に存在する非水溶性物を回収する方法。
（１’）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性ポリマーと磁性微粒子を含有させ、刺激を与えて刺激応答性ポリマーを疎水性として非水溶性物と結合させ、非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集物を生成させる工程
（２’）工程（１’）で生成させた凝集物を磁力により水溶液中から分離する工程
（３’）工程（２’）で分離した凝集物に工程（１’）と反対の刺激を与えて刺激応答性ポリマーを親水性とし、非水溶性物を刺激応答性ポリマーと磁性微粒子からリリースする工程
［３］前記刺激応答性磁性微粒子が、温度変化、ｐＨ変化、光変化およびイオン強度変化から選ばれる少なくとも１つに応答して、親水性と疎水性との相転移を生じる高分子が表面修飾された磁性微粒子である、［１］に記載の方法。
［４］前記刺激応答性ポリマーが、温度変化、ｐＨ変化、光変化およびイオン強度変化から選ばれる少なくとも１つに応答して、親水性と疎水性との相転移を生じる高分子である［２］に記載の方法。
［５］前記親水性と疎水性との相転移を生じる高分子が下限臨界共溶温度を有する温度応答性ポリマーまたは上限臨界共溶温度を有する温度応答性ポリマーである［３］または［４］に記載の方法。
［６］前記下限臨界共溶温度を有する温度応答性ポリマーがＮ−エチル（メタ）アクリルアミド、Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ，Ｎ−ジエチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−テトラヒドロフルフリル（メタ）アクリルアミド、Ｎ−エトキシプロピル（メタ）アクリルアミド、Ｎ−エトキシエチル（メタ）アクリルアミド、Ｎ−１−メチル−２−メトキシエチル（メタ）アクリルアミド、Ｎ−モルホリノプロピル（メタ）アクリルアミド、Ｎ−メトキシプロピル（メタ）アクリルアミド、Ｎ−イソプロポキシプロピル（メタ）アクリルアミド、Ｎ−イソプロポキシエチル（メタ）アクリルアミド、Ｎ−シクロプロピル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−エチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ−（メタ）アクリロイルピペリジン、Ｎ−（メタ）アクリロイルピロリジン、Ｎ−（メタ）アクリロイルモルホリン、Ｎ−（２，２−ジメトキシエチル）−Ｎ−メチル（メタ）アクリルアミド、Ｎ−１−メトキシメチルプロピル（メタ）アクリルアミド、Ｎ−ジ（２−メトキシエチル）（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−エチル（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ−メトキシエトキシプロピル（メタ）アクリルアミド、Ｎ−（１，３−ジオキソラン−２−イル）メチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−（１，３−ジオキソラン−２−イル）メチル（メタ）アクリルアミド、Ｎ−ピロリジノメチル（メタ）アクリルアミド、Ｎ−ピペリジノメチル（メタ）アクリルアミド、Ｎ，Ｎ−ジメチルアミノエチル（メタ）アクリレート（（メタ）アクリル酸２−（ジメチルアミノ）エチル）、Ｎ−メチル−Ｎ−エチルアミノエチル（メタ）アクリレート、Ｎ，Ｎ−ジエチルアミノエチル（メタ）アクリレート、Ｎ，Ｎ−ジメチルアミノプロピル（メタ）アクリレート、Ｎ−メチル−Ｎ−エチルアミノプロピル（メタ）アクリレート、Ｎ，Ｎ−ジエチルアミノプロピル（メタ）アクリレート、Ｎ−２−モルホリノエチル（メタ）アクリレート、Ｎ−２−モルホリノエトキシエチル（メタ）アクリレート、N−ｔ−ブチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミドおよび８−（メタ）アクリロイル−１，４−ジオキサ−８−アザスピロ［４，５］デカンからなる群から選ばれる少なくとも１種類の単重合体又は共重合体である、［５］に記載の方法。
［７］前記上限臨界共溶温度を有する温度応答性ポリマーがアクリロイルグリシンアミド、メタクリロイルグリシンアミド、アクリロイルアスパラギンアミド、メタクリロイルアスパラギンアミド、アクリロイルグルタミンアミドおよびメタクリロイルグルタミンアミドからなる群から選ばれる少なくとも１種類の単重合体又は共重合体である、［５］に記載の方法。
［８］前記非水溶性物が微生物である［１］〜［７］のいずれか１に記載の方法。
［９］前記微生物が、高度不飽和脂肪酸、天然色素、ビタミン、アミノ酸、ミネラル、アルコール類、水素、糖質、脂質、タンパク質、脂肪酸、有機酸および炭化水素から選ばれる少なくとも１の物質を生産する微細藻類である、［８］に記載の方法。
［１０］前記微生物が炭化水素を生産する微細藻類であって、該微細藻類がオーランチオキトリウム、ボトリオコッカス・ブラウニー、シュードコリシスチス・エリプソイディアおよびシキゾリウムから選ばれる少なくとも１の藻類またはこれら藻類の変異株もしくは遺伝子組み換え株である、［９］に記載の方法。
［１１］前記工程（３）または前記工程（３’）において、さらに超音波処理を行う、［１］〜［１０］のいずれか１に記載の方法。
［１２］刺激応答性磁性微粒子を含む、水溶液中に存在する非水溶性物を回収するためのキット。
［１３］刺激応答性ポリマーと磁性微粒子を含む、水溶液中に存在する非水溶性物を回収するためのキット。
[1] A method for recovering a water-insoluble material present in an aqueous solution, comprising the following steps (1) to (3).
(1) An aqueous solution containing a water-insoluble substance contains stimuli-responsive magnetic fine particles that undergo a phase transition between hydrophilic and hydrophobic in response to a stimulus, and the stimulus-responsive magnetic fine particles are rendered hydrophobic by applying a stimulus. Step (2) of generating an aggregate of water-insoluble material and stimulus-responsive magnetic fine particles by binding with water-insoluble material (2) Step of separating the aggregate generated in step (1) from an aqueous solution by magnetic force (3 ) Step [2] and subsequent steps in which the aggregate separated in step (2) is stimulated opposite to step (1) to make the stimulus-responsive magnetic fine particles hydrophilic and the water-insoluble matter is released from the stimulus-responsive magnetic fine particles The method of collect | recovering the water-insoluble matter which exists in aqueous solution including process (1 ')-(3') of these.
(1 ') An aqueous solution containing a water-insoluble material contains a stimulus-responsive polymer that undergoes a hydrophilic and hydrophobic phase transition in response to a stimulus and magnetic fine particles, and the stimulus-responsive polymer is made hydrophobic by applying a stimulus. In step (2 ′), the agglomerates formed in step (1 ′) are magnetically bonded to an aqueous solution by binding to a water-insoluble material and forming an aggregate of the water-insoluble material, stimulus-responsive polymer, and magnetic fine particles. Step (3 ′) for separating from the solution The stimulus separated in the step (2 ′) is subjected to a stimulus opposite to that in the step (1 ′) to make the stimulus-responsive polymer hydrophilic, and the water-insoluble material is used as the stimulus-responsive polymer. Step 3 of releasing from magnetic fine particles [3] The stimulus-responsive magnetic fine particles undergo a phase transition between hydrophilicity and hydrophobicity in response to at least one selected from temperature change, pH change, light change and ionic strength change. Magnetic particles with surface-modified polymer In a method according to [1].
[4] The stimulus-responsive polymer is a polymer that causes a phase transition between hydrophilicity and hydrophobicity in response to at least one selected from temperature change, pH change, light change, and ionic strength change [2] ] Method.
[5] The polymer that causes a phase transition between hydrophilicity and hydrophobicity is a temperature-responsive polymer having a lower critical solution temperature or a temperature-responsive polymer having an upper critical solution temperature [3] or [4] The method described in 1.
[6] The temperature-responsive polymer having the lower critical eutectic temperature is N-ethyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N, N-diethyl (meth). Acrylamide, N-methyl-Nn-propyl (meth) acrylamide, N-tetrahydrofurfuryl (meth) acrylamide, N-ethoxypropyl (meth) acrylamide, N-ethoxyethyl (meth) acrylamide, N-1-methyl- 2-methoxyethyl (meth) acrylamide, N-morpholinopropyl (meth) acrylamide, N-methoxypropyl (meth) acrylamide, N-isopropoxypropyl (meth) acrylamide, N-isopropoxyethyl (meth) acrylamide, N-cyclo Propyl (meta Acrylamide, N-methyl-N-ethyl (meth) acrylamide, N-methyl-N-isopropyl (meth) acrylamide, N- (meth) acryloylpiperidine, N- (meth) acryloylpyrrolidine, N- (meth) acryloylmorpholine, N- (2,2-dimethoxyethyl) -N-methyl (meth) acrylamide, N-1-methoxymethylpropyl (meth) acrylamide, N-di (2-methoxyethyl) (meth) acrylamide, N-2-methoxy Ethyl-Nn-propyl (meth) acrylamide, N-2-methoxyethyl-N-ethyl (meth) acrylamide, N-2-methoxyethyl-N-isopropyl (meth) acrylamide, N-methoxyethoxypropyl (meth) Acrylamide, N- (1,3-dioxolane-2 Yl) methyl (meth) acrylamide, N-methyl-N- (1,3-dioxolan-2-yl) methyl (meth) acrylamide, N-pyrrolidinomethyl (meth) acrylamide, N-piperidinomethyl (meth) acrylamide, N , N-dimethylaminoethyl (meth) acrylate (2- (dimethylamino) ethyl (meth) acrylate), N-methyl-N-ethylaminoethyl (meth) acrylate, N, N-diethylaminoethyl (meth) acrylate, N, N-dimethylaminopropyl (meth) acrylate, N-methyl-N-ethylaminopropyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N-2-morpholinoethyl (meth) acrylate, N- 2-morpholinoethoxyethyl (meth) acrylate At least one simple substance selected from the group consisting of N-tert-butylacrylamide, N, N-dimethylacrylamide and 8- (meth) acryloyl-1,4-dioxa-8-azaspiro [4,5] decane. The method according to [5], which is a polymer or a copolymer.
[7] The temperature-responsive polymer having the upper critical eutectic temperature is at least one single member selected from the group consisting of acryloyl glycinamide, methacryloyl glycinamide, acryloyl asparagine amide, methacryloyl asparagine amide, acryloyl glutamine amide, and methacryloyl glutamine amide. The method according to [5], which is a polymer or a copolymer.
[8] The method according to any one of [1] to [7], wherein the water-insoluble material is a microorganism.
[9] The microorganism produces at least one substance selected from polyunsaturated fatty acids, natural pigments, vitamins, amino acids, minerals, alcohols, hydrogen, carbohydrates, lipids, proteins, fatty acids, organic acids and hydrocarbons. The method according to [8], which is a microalgae.
[10] The microalgae in which the microorganisms produce hydrocarbons, and the microalgae are at least one algae selected from aurantiochitotrium, botryococcus brownie, pseudocollistis ellipsoidia, and cikizolium, or these [9] The method according to [9], which is a mutant or genetically modified strain of algae.
[11] The method according to any one of [1] to [10], wherein sonication is further performed in the step (3) or the step (3 ′).
[12] A kit for recovering a non-water-soluble material present in an aqueous solution, including stimulation-responsive magnetic fine particles.
[13] A kit for recovering a water-insoluble material present in an aqueous solution, comprising a stimulus-responsive polymer and magnetic fine particles.
本発明の方法では、非水溶性物を含有する水溶液中に、刺激に応答して親水性と疎水性の相転移をする刺激応答性磁性微粒子を含有させるか、または刺激応答性ポリマーと磁性微粒子を含有させて、刺激応答性磁性微粒子に刺激を与えるか、または刺激応答性ポリマーと磁性微粒子に刺激を与えることにより、迅速且つ効率的に非水溶性物と刺激応答性磁性微粒子との凝集物、または非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集物を調製することができる。 In the method of the present invention, an aqueous solution containing a water-insoluble substance contains stimulus-responsive magnetic fine particles that undergo a phase transition between hydrophilic and hydrophobic in response to a stimulus, or a stimulus-responsive polymer and magnetic fine particles. And agglomerates of water-insoluble matter and stimulus-responsive magnetic fine particles quickly and efficiently by stimulating the stimulus-responsive magnetic fine particles or stimulating the stimulus-responsive polymer and the magnetic fine particles Alternatively, an aggregate of a water-insoluble substance, a stimulus-responsive polymer, and magnetic fine particles can be prepared.
このようにして得られた凝集物を磁力により水溶液中から分離した後、前記刺激とは反対の刺激を該凝集物に与えて刺激応答性磁性微粒子または刺激応答性ポリマーを親水性とし、非水溶性物を刺激応答性磁性微粒子からリリースするか、または非水溶性物を刺激応答性ポリマーと磁性微粒子からリリースすることで、簡便且つ効率的に水溶液中から非水溶性物を分離、回収することができる。 After the aggregate obtained in this way is separated from the aqueous solution by magnetic force, a stimulus opposite to the stimulus is applied to the aggregate to make the stimulus-responsive magnetic fine particles or stimulus-responsive polymer hydrophilic, and the water-insoluble Release and release non-water-soluble substances from aqueous solutions easily and efficiently by releasing the active substances from the stimulus-responsive magnetic fine particles or by releasing the non-water-soluble substances from the stimulus-responsive polymer and the magnetic fine particles. Can do.
本発明の水溶液中の微細藻類の回収方法は、以下の工程（１）〜（３）を含む、水溶液中に存在する非水溶性物を回収する方法である。
（１）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性磁性微粒子を含有させ、刺激を与えて刺激応答性磁性微粒子を疎水性として非水溶性物と結合させ、非水溶性物と磁性微粒子との凝集物を生成させる工程
（２）工程（１）で生成させた凝集物を磁力により水溶液中から分離する工程
（３）工程（２）で分離した凝集物に工程（１）と反対の刺激を与えて刺激応答性磁性微粒子を親水性とし、非水溶性物を刺激応答性磁性微粒子からリリースする工程
The method for recovering microalgae in an aqueous solution of the present invention is a method for recovering a water-insoluble material present in an aqueous solution, including the following steps (1) to (3).
(1) An aqueous solution containing a water-insoluble substance contains stimuli-responsive magnetic fine particles that undergo a phase transition between hydrophilic and hydrophobic in response to a stimulus, and the stimulus-responsive magnetic fine particles are rendered hydrophobic by applying a stimulus. Step (3) (Step (3)) in which the aggregate formed in the step (1) is separated from the aqueous solution by a magnetic force. The step of applying the opposite stimulus to the step (1) to the aggregate separated in 2) to make the stimulus-responsive magnetic fine particles hydrophilic, and releasing the water-insoluble matter from the stimulus-responsive magnetic fine particles
また、本発明の水溶液中の微細藻類の回収方法は、以下の工程（１’）〜（３’）を含む、水溶液中に存在する非水溶性物を回収する方法である。
（１’）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性ポリマーと磁性微粒子を含有させ、刺激を与えて刺激応答性ポリマーを疎水性として非水溶性物と結合させ、非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集物を生成させる工程
（２’）工程（１’）で生成させた凝集物を磁力により水溶液中から分離する工程
（３’）工程（２’）で分離した凝集物に工程（１’）と反対の刺激を与えて刺激応答性ポリマーを親水性とし、非水溶性物を刺激応答性ポリマーと磁性微粒子からリリースする工程
In addition, the method for recovering microalgae in an aqueous solution of the present invention is a method for recovering a water-insoluble material present in an aqueous solution, including the following steps (1 ′) to (3 ′).
(1 ') An aqueous solution containing a water-insoluble material contains a stimulus-responsive polymer that undergoes a hydrophilic and hydrophobic phase transition in response to a stimulus and magnetic fine particles, and the stimulus-responsive polymer is made hydrophobic by applying a stimulus. In step (2 ′), the agglomerates formed in step (1 ′) are magnetically bonded to an aqueous solution by binding to a water-insoluble material and forming an aggregate of the water-insoluble material, stimulus-responsive polymer, and magnetic fine particles. Step (3 ′) for separating from the solution The stimulus separated in the step (2 ′) is subjected to a stimulus opposite to that in the step (1 ′) to make the stimulus-responsive polymer hydrophilic, and the water-insoluble material is used as the stimulus-responsive polymer. Release from magnetic fine particles
以下、各工程について説明する。 Hereinafter, each step will be described.
工程（１）および工程（１’）
工程（１）は、非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性磁性微粒子を含有させ、刺激を与えて該刺激応答性磁性微粒子を疎水性とすることにより、非水溶性物と刺激応答性磁性微粒子とを疎水性相互作用により結合させて、非水溶性物と刺激応答性磁性微粒子との凝集物を生成させる工程である。
Step (1) and Step (1 ′)
In the step (1), an aqueous solution containing a water-insoluble substance contains stimulation-responsive magnetic fine particles that undergo a phase transition between hydrophilic and hydrophobic in response to stimulation, and the stimulation-responsive magnetic fine particles are given with stimulation. Is a step of forming agglomerates of the water-insoluble substance and the stimulus-responsive magnetic fine particles by combining the water-insoluble substance and the stimulus-responsive magnetic fine particles by hydrophobic interaction.
また、工程（１’）は、非水溶性物を含有する水溶液に、刺激応答性ポリマーと磁性微粒子を含有させ、刺激を与えて刺激応答性ポリマーと磁性微粒子を疎水性とすることにより、非水溶性物と刺激応答性ポリマーと磁性微粒子とを疎水性相互作用により結合させて、非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集物を生成させる工程である。 Further, the step (1 ′) includes a stimulus-responsive polymer and magnetic fine particles in an aqueous solution containing a water-insoluble substance, and the stimulus-responsive polymer and the magnetic fine particles are made hydrophobic by applying a stimulus to make the non-aqueous substance non-aqueous. In this step, the water-soluble material, the stimulus-responsive polymer, and the magnetic fine particles are combined by hydrophobic interaction to form an aggregate of the water-insoluble material, the stimulus-responsive polymer, and the magnetic fine particles.
非水溶性物としては、微生物および動物細胞が好ましい。微生物としては、高度不飽和脂肪酸、天然色素、ビタミン、アミノ酸、ミネラル、アルコール類、水素、糖質、脂質、タンパク質、脂肪酸、有機酸および炭化水素から選ばれる１つ以上の物質を生産する微細藻類、細菌または菌類が挙げられ、該物質を生産する微細藻類が好ましい。また、動物細胞としては、幹細胞が挙げられ、人工多能性幹細胞（ｉＰＳ細胞）または胚性幹細胞（ＥＳ細胞）が好ましい。 As the water-insoluble material, microorganisms and animal cells are preferable. Microorganisms include microalgae that produce one or more substances selected from highly unsaturated fatty acids, natural pigments, vitamins, amino acids, minerals, alcohols, hydrogen, carbohydrates, lipids, proteins, fatty acids, organic acids and hydrocarbons. And bacteria or fungi, and microalgae that produce the substance are preferred. Examples of animal cells include stem cells, and artificial pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) are preferable.
具体的には、天然物の色素の一種であるアスタキサンチン生産能を有する微細藻類［例えば、ヘマトコッカス藻（Ｈａｅｍａｔｏｃｏｃｕｓ ｐｌｕｖｉａｌｉｓ）、クロレラ・ゾフィンギエンシス（Ｃｈｌｏｒｅｌｌａ ｚｏｆｉｎｇｉｅｎｓｉｓ）、クロロコックム属（Ｃｈｌｏｒｏｃｏｃｃｕｍ ｓｐ．）、ファフィア・ロドチーマ（Ｐｈａｆｆｉａ ｒｈｏｄｏｚｙｍａ）、モノラフィディウム属（Ｍｏｎｏｒａｐｈｉｄｉｕｍ ｓｐ．）など］、高度不飽和脂肪酸の一種であるＤＨＡ（ドコサヘキサエン酸）またはＥＰＡ（エイコサペンタエン酸）の生産能を有する微細藻類［例えば、ピンギオクリシス属（Ｐｉｎｇｕｉｏｃｈｒｙｓｉｓ ｓｐ．）、パブロバ属（Ｐａｖｌｏｖａ ｓｐ．）、フェオダクチラム・トリコヌタム（Ｐｈａｅｏｄａｃｔｙｌｕｍ ｔｒｉｃｏｒｎｕｔｕｍ）、ナンノクロロプシス・オーシャニカ（Ｎａｎｎｏｃｈｌｏｒｏｐｓｉｓ ｏｃｅａｎｉｃａ）など］が挙げられ、特に、炭化水素の生産能を有する微細藻類［例えば、オーランチオキトリウム属（Ａｕｒａｎｔｉｏｃｈｙｔｒｉｕｍ ｓｐ．）、ボツリオコッカス・ブラウニー（Ｂｏｔｒｙｏｃｏｃｃｕｓ ｂｒａｕｎｉｉ）、クロレラ属（Ｃｈｌｏｒｅｌｌａ ｓｐ．）、シリンドロテカ属（Ｃｙｌｉｎｄｒｏｔｈｅｃａ ｓｐ．）、ドナリエラ・プリモレクタ（Ｄｕｎａｌｉｅｌｌａ ｐｒｉｍｏｌｅｃｔａ）、イソクリシス属（Ｉｓｏｃｈｒｙｓｉｓ ｓｐ．）、ナンノクロリス属（Ｎａｎｎｏｃｈｌｏｒｉｓ ｓｐ．）、ナンノクロロプシス属（Ｎａｎｎｏｃｈｌｏｒｏｐｓｉｓ ｓｐ．）、ネオクロリス・オレオアブンダンス（Ｎｅｏｃｈｌｏｒｉｓ ｏｌｅｏａｂｕｎｄａｎｓ）、ニッチア属（Ｎｉｔｚｓｃｈｉａ ｓｐ．）、フェオダクチラム・トリコルヌタム（Ｐｈａｅｏｄａｃｔｙｌｕｍ ｔｒｉｃｏｒｎｕｔｕｍ）、シゾキトリウム属（Ｓｃｈｉｚｏｃｈｙｔｒｉｕｍ ｓｐ．）、テトラセルミス・スエシカ（Ｔｅｔｒａｓｅｌｍｉｓ ｓｕｅｃｉｃａ）、シュードコリシスチス・エリプソイディア（Ｐｓｅｕｄｏｃｈｏｒｉｃｙｓｔｉｓ ｅｌｌｉｐｓｏｉｄｅａ）およびマイクロシスティス属（Ｍｉｃｒｏｃｙｓｔｉｓ ｓｐ．）など］が好ましく、より好ましくは該微細藻類がオーランチオキトリウム、ボトリオコッカス・ブラウニー、シュードコリシスティス・エリプソイディアおよびシゾキトリウムから選ばれる微細藻類またはこれら微細藻類の変異株もしくは遺伝子組み換え株である。 Specifically, microalgae having the ability to produce astaxanthin, which is a kind of natural product pigment (for example, Haematococus pluvialis, Chlorella zofingiensis, Chlorococcum sp.) -Rhodocima (Phaffia rhodozyma), Monoraphidium genus (Monoraphidium sp.), Etc.], microalgae having the ability to produce DHA (docosahexaenoic acid) or EPA (eicosapentaenoic acid), which is a kind of highly unsaturated fatty acid [for example, Pinuiochrysis sp., Pavlova sp., Pheodactylum triconatum (Ph) and the like, and in particular, microalgae capable of producing hydrocarbons [e.g., Aurantiochytrium sp., Botriococcus sp., Botriococcus sp. Botryococcus braunii), Chlorella sp., Cylindrotheca sp., Donaliella primolectina, Isochrysno chlorosis sp. Nannochloropsi sp ), Neochloris oleoabundans, Nitzschia sp., Phaeodactylum tricornutum, Schizomistrum schimitrum Ellipsoidia (Pseudochoristis elispoidea) and Microcystis sp., Etc.] are preferred, more preferably the microalgae is selected from Aurantiochytrium, Botryococcus brownie, Pseudocollistis ellipsoidia and Schizochytrium Or a mutant or genetically modified strain of these microalgae.
非水溶性物を含有する水溶液としては、例えば、河川水、湖沼水、井戸水、水道原水、地下水、下水、廃水および公園の水等の環境中に存在する水試料、並びに動物細胞、微細藻類、細菌または菌類の培養液等が挙げられる。なお、水溶性物を含有する水溶液は必要に応じて、油水分離、濾過またはｐＨ調整等の前処理を行ってもよい。 Examples of aqueous solutions containing water-insoluble materials include water samples present in the environment such as river water, lake water, well water, raw tap water, groundwater, sewage, wastewater, and park water, as well as animal cells, microalgae, Examples include a culture solution of bacteria or fungi. The aqueous solution containing the water-soluble material may be subjected to pretreatment such as oil / water separation, filtration or pH adjustment, if necessary.
後述する実施例においては、非水溶性物として微細藻類であるオーランチオキトリウムを用いた結果を示したが、本発明の方法はｉＰＳ細胞またはＥＳ細胞等の動物細胞の回収にも好適である。 In the examples described later, the results of using auranthiochytrium, which is a microalgae, as a water-insoluble material have been shown. However, the method of the present invention is also suitable for recovering animal cells such as iPS cells or ES cells. .
刺激応答性磁性微粒子とは、刺激応答性の機能を有する磁性微粒子をいう。刺激応答性磁性微粒子は、例えば、温度変化、ｐＨ変化、光変化およびイオン強度変化から選ばれる少なくとも１つに応答して、親水性と疎水性との相転移を生じる高分子が表面修飾された磁性微粒子であることが好ましい。 The stimulus-responsive magnetic fine particles refer to magnetic fine particles having a stimulus-responsive function. The stimulus-responsive magnetic fine particle has a surface-modified polymer that causes a phase transition between hydrophilicity and hydrophobicity in response to at least one selected from, for example, temperature change, pH change, light change, and ionic strength change. Magnetic fine particles are preferred.
刺激応答性ポリマーとは、刺激応答性の機能を有するポリマーをいう。刺激応答性ポリマーは、例えば、例えば、温度変化、ｐＨ変化、光変化およびイオン強度変化から選ばれる少なくとも１つに応答して、親水性と疎水性との相転移を生じる高分子であることが好ましい。 The stimulus-responsive polymer refers to a polymer having a stimulus-responsive function. The stimulus-responsive polymer is, for example, a polymer that causes a phase transition between hydrophilicity and hydrophobicity in response to at least one selected from, for example, temperature change, pH change, light change, and ionic strength change. preferable.
磁性微粒子は、酸化鉄、またはフェライトからなる粒子でもよく、例えば多価アルコールとマグネタイトから製造した粒子のように、酸化鉄、フェライト、またはマグネタイトとその他の無機物、有機物とからなる粒子でもよい。 The magnetic fine particles may be particles made of iron oxide or ferrite, and may be particles made of iron oxide, ferrite, or magnetite and other inorganic substances or organic substances such as particles produced from polyhydric alcohol and magnetite.
磁性微粒子は、例えば、特表２００２−５１７０８５号公報等に開示された方法によって製造することができる。すなわち、鉄（ＩＩ）塩、または鉄（ＩＩ）塩及び金属（ＩＩ）塩を含有する水溶液を、磁性酸化物の形成のために必要な酸化状態下に置き、水溶液のｐＨを７以上に維持して、酸化鉄、またはフェライト磁性体粒子を形成する方法である。また、金属（ＩＩ）塩を含有する水溶液と鉄（ＩＩＩ）塩を含有する水溶液をアルカリ性条件下で混合することによっても製造することができる。 The magnetic fine particles can be produced by, for example, a method disclosed in JP-T-2002-517085. That is, an aqueous solution containing an iron (II) salt or an iron (II) salt and a metal (II) salt is placed under an oxidation state necessary for forming a magnetic oxide, and the pH of the aqueous solution is maintained at 7 or more. Thus, iron oxide or ferrite magnetic particles are formed. It can also be produced by mixing an aqueous solution containing a metal (II) salt and an aqueous solution containing an iron (III) salt under alkaline conditions.
あるいは、磁性微粒子は、多価アルコールおよびマグネタイトから製造することもできる。この多価アルコールは、構成単位に水酸基を少なくとも２個有し、鉄イオンと結合可能なアルコール構造体であれば、特に制限なく使用することができる。例えば、デキストラン、ポリビニルアルコール、マンニトール、ソルビトールまたはシクロデキストリンなどが挙げられる。例えば、特開２００５−０８２５３８号公報に、デキストランを用いた磁性微粒子の製造方法が開示されており、この方法によって製造することもできる。また、グリシジルメタクリレート重合体のように、エポキシ基を有し、開環後多価アルコール構造体を形成する化合物も使用できる。 Alternatively, the magnetic fine particles can be produced from a polyhydric alcohol and magnetite. The polyhydric alcohol can be used without particular limitation as long as it is an alcohol structure having at least two hydroxyl groups in the structural unit and capable of binding to iron ions. For example, dextran, polyvinyl alcohol, mannitol, sorbitol, cyclodextrin and the like can be mentioned. For example, Japanese Patent Application Laid-Open No. 2005-082538 discloses a method for producing magnetic fine particles using dextran, and can be produced by this method. Moreover, the compound which has an epoxy group and forms a polyhydric alcohol structure after ring-opening like a glycidyl methacrylate polymer can also be used.
磁性微粒子の磁気分離が可能であれば特に限定されないが、回収効率の観点から、磁性微粒子の粒径は１〜１０μｍに調製することが好ましく、３〜７μｍであることがより好ましい。 Although it will not specifically limit if the magnetic separation of a magnetic fine particle is possible, From a viewpoint of collection | recovery efficiency, it is preferable to adjust the particle size of a magnetic fine particle to 1-10 micrometers, and it is more preferable that it is 3-7 micrometers.
磁性微粒子の粒子径測定を行える機器としては、粒子径・粒度分布測定装置（Ｚｅｔａｓｉｚｅｒ Ｎａｎｏ ＺＳ、Ｍａｌｖｅｒｎ社製）、および透過型電子顕微鏡（日立透過電子顕微鏡 ＨＴ７７００、日立ハイテクノロジーズ社製）が挙げられる。 Examples of devices that can measure the particle size of magnetic fine particles include a particle size / particle size distribution measuring device (Zetasizer Nano ZS, manufactured by Malvern), and a transmission electron microscope (Hitachi Transmission Electron Microscope HT7700, manufactured by Hitachi High-Technologies). .
本発明において使用できる磁性微粒子の市販品としては、例えば、Ｄｙｎａｂｅａｄｓ（登録商標）、ｎａｎｏｍａｇ（登録商標）、ｄｅＳｔａｒｓおよびＭＡＣＳ（登録商標）などが挙げられる。 Examples of commercially available magnetic fine particles that can be used in the present invention include Dynabeads (registered trademark), nanomag (registered trademark), deStars, and MACS (registered trademark).
親水性と疎水性との相転移を生じる高分子としては、溶液中で可逆的に凝集状態と溶解状態とに変化する温度応答性ポリマーが挙げられる。温度応答性ポリマーとしては、ポリマーの重合度により溶解・析出する温度（下限臨界共溶温度または上限臨界共溶温度）を制御することができる、下限臨界共溶温度を有する温度応答性ポリマーまたは上限臨界共溶温度を有する温度応答性ポリマーであることが好ましく、下限臨界共溶温度を有する温度応答性ポリマーであることがより好ましい。 Examples of the polymer that causes a phase transition between hydrophilicity and hydrophobicity include a temperature-responsive polymer that reversibly changes in an aggregated state and a dissolved state in a solution. As the temperature-responsive polymer, the temperature-responsive polymer having the lower critical eutectic temperature or the upper limit that can control the temperature at which the polymer dissolves and precipitates (lower critical eutectic temperature or upper critical eutectic temperature) depending on the degree of polymerization of the polymer. A temperature-responsive polymer having a critical solution temperature is preferable, and a temperature-responsive polymer having a lower critical solution temperature is more preferable.
下限臨界共溶温度を有する温度応答性ポリマーは、下限臨界共溶温度が好ましくは１０〜５０℃であるポリマーであれば、特に制限はなく、公知の温度応答性ポリマーを用いることができる。下限臨界共溶温度を有する温度応答性ポリマーとしては、アクリルアミド系ポリマーおよびコポリマー、窒素含有アクリレート系ポリマーおよびコポリマー、並びに、オキシアルキレン鎖を有するビニルエーテル系ポリマー及びコポリマーが好ましく挙げられ、アクリルアミド系ポリマー及びコポリマー、並びに、窒素含有アクリレート系モノマー及びコポリマーがより好ましく挙げられる。 The temperature-responsive polymer having the lower critical solution temperature is not particularly limited as long as the lower critical solution temperature is preferably 10 to 50 ° C., and a known temperature-responsive polymer can be used. Preferred examples of the temperature-responsive polymer having a lower critical eutectic temperature include acrylamide polymers and copolymers, nitrogen-containing acrylate polymers and copolymers, and vinyl ether polymers and copolymers having an oxyalkylene chain. Acrylamide polymers and copolymers And nitrogen-containing acrylate monomers and copolymers are more preferable.
下限臨界共溶温度を有する温度応答性ポリマーとしては、具体的には、例えば、Ｎ−エチル（メタ）アクリルアミド、Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ，Ｎ−ジエチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−テトラヒドロフルフリル（メタ）アクリルアミド、Ｎ−エトキシプロピル（メタ）アクリルアミド、Ｎ−エトキシエチル（メタ）アクリルアミド、Ｎ−１−メチル−２−メトキシエチル（メタ）アクリルアミド、Ｎ−モルホリノプロピル（メタ）アクリルアミド、Ｎ−メトキシプロピル（メタ）アクリルアミド、Ｎ−イソプロポキシプロピル（メタ）アクリルアミド、Ｎ−イソプロポキシエチル（メタ）アクリルアミド、Ｎ−シクロプロピル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−エチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ−（メタ）アクリロイルピペリジン、Ｎ−（メタ）アクリロイルピロリジン、Ｎ−（メタ）アクリロイルモルホリン、Ｎ−（２，２−ジメトキシエチル）−Ｎ−メチル（メタ）アクリルアミド、Ｎ−１−メトキシメチルプロピル（メタ）アクリルアミド、Ｎ−ジ（２−メトキシエチル）（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−ｎ−プロピル（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−エチル（メタ）アクリルアミド、Ｎ−２−メトキシエチル−Ｎ−イソプロピル（メタ）アクリルアミド、Ｎ−メトキシエトキシプロピル（メタ）アクリルアミド、Ｎ−（１，３−ジオキソラン−２−イル）メチル（メタ）アクリルアミド、Ｎ−メチル−Ｎ−（１，３−ジオキソラン−２−イル）メチル（メタ）アクリルアミド、Ｎ−ピロリジノメチル（メタ）アクリルアミド、Ｎ−ピペリジノメチル（メタ）アクリルアミド、Ｎ，Ｎ−ジメチルアミノエチル（メタ）アクリレート（（メタ）アクリル酸２−（ジメチルアミノ）エチル）、Ｎ−メチル−Ｎ−エチルアミノエチル（メタ）アクリレート、Ｎ，Ｎ−ジエチルアミノエチル（メタ）アクリレート、Ｎ，Ｎ−ジメチルアミノプロピル（メタ）アクリレート、Ｎ−メチル−Ｎ−エチルアミノプロピル（メタ）アクリレート、Ｎ，Ｎ−ジエチルアミノプロピル（メタ）アクリレート、Ｎ−２−モルホリノエチル（メタ）アクリレート、Ｎ−２−モルホリノエトキシエチル（メタ）アクリレート、N−ｔ−ブチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミドおよび８−（メタ）アクリロイル−１，４−ジオキサ−８−アザスピロ［４，５］デカンよりなる群から選ばれる単量体を用いた単重合体並びに共重合体（コポリマー）が挙げられる。 Specific examples of the temperature-responsive polymer having a lower critical eutectic temperature include N-ethyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, and N, N. -Diethyl (meth) acrylamide, N-methyl-Nn-propyl (meth) acrylamide, N-tetrahydrofurfuryl (meth) acrylamide, N-ethoxypropyl (meth) acrylamide, N-ethoxyethyl (meth) acrylamide, N -1-methyl-2-methoxyethyl (meth) acrylamide, N-morpholinopropyl (meth) acrylamide, N-methoxypropyl (meth) acrylamide, N-isopropoxypropyl (meth) acrylamide, N-isopropoxyethyl (meth) Acrylamide, N-shi Ropropyl (meth) acrylamide, N-methyl-N-ethyl (meth) acrylamide, N-methyl-N-isopropyl (meth) acrylamide, N- (meth) acryloylpiperidine, N- (meth) acryloylpyrrolidine, N- (meta ) Acryloylmorpholine, N- (2,2-dimethoxyethyl) -N-methyl (meth) acrylamide, N-1-methoxymethylpropyl (meth) acrylamide, N-di (2-methoxyethyl) (meth) acrylamide, N 2-methoxyethyl-Nn-propyl (meth) acrylamide, N-2-methoxyethyl-N-ethyl (meth) acrylamide, N-2-methoxyethyl-N-isopropyl (meth) acrylamide, N-methoxyethoxy Propyl (meth) acrylamide, N- (1,3 Dioxolan-2-yl) methyl (meth) acrylamide, N-methyl-N- (1,3-dioxolan-2-yl) methyl (meth) acrylamide, N-pyrrolidinomethyl (meth) acrylamide, N-piperidinomethyl (meta) ) Acrylamide, N, N-dimethylaminoethyl (meth) acrylate (2- (dimethylamino) ethyl (meth) acrylate), N-methyl-N-ethylaminoethyl (meth) acrylate, N, N-diethylaminoethyl ( (Meth) acrylate, N, N-dimethylaminopropyl (meth) acrylate, N-methyl-N-ethylaminopropyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N-2-morpholinoethyl (meth) Acrylate, N-2-morpholinoethoxyethyl Monomer selected from the group consisting of ru (meth) acrylate, Nt-butylacrylamide, N, N-dimethylacrylamide and 8- (meth) acryloyl-1,4-dioxa-8-azaspiro [4,5] decane Examples thereof include a homopolymer using a polymer and a copolymer (copolymer).
下限臨界共溶温度を有する温度応答性ポリマーが表面修飾された磁性微粒子としては、具体的には、例えば、ＪＮＣ社製「Ｔｈｅｒｍａ−Ｍａｘ（登録商標）」（以下、「サーママックス」という場合がある。）［例えば、ＪＮＣ社製「Ｔｈｅｒｍａ−Ｍａｘ（登録商標）ＬＣ Ｃａｒｂｏｘｙｌｉｃ ａｃｉｄ」（以下、「ＴＭ−ＬＣ」という場合がある。）］等が挙げられる。 Specific examples of magnetic fine particles whose surface is modified with a temperature-responsive polymer having a lower critical eutectic temperature include, for example, “Therma-Max (registered trademark)” manufactured by JNC (hereinafter referred to as “thermamax”). (For example, “Therma-Max (registered trademark) LC Carboxylic acid” (hereinafter sometimes referred to as “TM-LC”) manufactured by JNC).
ＪＮＣ社製「Ｔｈｅｒｍａ−Ｍａｘ（登録商標）」は、酸化鉄磁性微粒子表面にＮ−イソプロピルアクリルアミド（以下、「ＮＩＰＡＭ」という場合がある。）のポリマーが修飾されている。本明細書において、単に「サーママックス」という場合は、温度応答性磁性微粒子を示しており、「サーママックス溶液」、「サーママックス原液」という場合は、温度応答性磁性微粒子にバッファー等を含んでいることを示している。 In “Therma-Max (registered trademark)” manufactured by JNC, a polymer of N-isopropylacrylamide (hereinafter sometimes referred to as “NIPAM”) is modified on the surface of iron oxide magnetic fine particles. In this specification, “thermamax” simply indicates temperature-responsive magnetic fine particles, and “thermamax solution” and “thermamax undiluted solution” indicate that the temperature-responsive magnetic fine particles include a buffer or the like. It shows that.
ＮＩＰＡＭのポリマー水溶液はポリマーの重合度により下限臨界共溶温度を制御することができ、３２℃前後に下限臨界共溶温度を有し、この温度未満では水に分散するが、これ以上の温度では凝集し容易に磁気で回収できるようになる。 The NIPAM polymer aqueous solution can control the lower critical eutectic temperature depending on the degree of polymerization of the polymer, has a lower critical eutectic temperature around 32 ° C., and disperses in water below this temperature, but at higher temperatures Aggregates and can be easily recovered magnetically.
したがって、サーママックスは、その粒子表面を被覆しているＮＩＰＡＭにより、溶液温度３２℃を境として凝集と分散の両状態の間で可逆的に変化する。そのため、粒子が均一に溶液中に分散している３２℃未満の分散状態では、磁気分離は容易ではないが、溶液温度を３２℃以上にすると磁性微粒子は凝集塊を形成し始め、磁気分離が容易に可能となる。 Therefore, the thermamax reversibly changes between both agglomerated and dispersed states at a solution temperature of 32 ° C. due to NIPAM coating the particle surface. For this reason, magnetic separation is not easy in a dispersion state of less than 32 ° C. in which the particles are uniformly dispersed in the solution. However, when the solution temperature is raised to 32 ° C. or more, the magnetic fine particles start to form aggregates, and magnetic separation does not occur. Easy to do.
上限臨界共溶温度を有する温度応答性ポリマーは、分散状態では回収困難な磁性微粒子に前記温度応答性ポリマーを表面修飾することで、上限臨界共溶温度未満に温度を低下させることにより、回収可能な大きさの塊に刺激応答性磁性微粒子を凝集させる能力を有していることが必要である。逆に、上限臨界共溶温度以上に温度を上昇させることにより、凝集した刺激応答性磁性微粒子を分散させることができる。本発明の方法に好適に用いることができる温度応答性ポリマーの重合度は、通常５０〜１００００である。 A temperature-responsive polymer having an upper critical eutectic temperature can be recovered by lowering the temperature below the upper critical eutectic temperature by surface-modifying the temperature-responsive polymer to magnetic fine particles that are difficult to recover in a dispersed state. It is necessary to have the ability to agglomerate stimulus-responsive magnetic fine particles in a large-sized lump. On the contrary, the aggregated stimulus-responsive magnetic fine particles can be dispersed by raising the temperature to the upper critical solution temperature or higher. The degree of polymerization of the temperature-responsive polymer that can be suitably used in the method of the present invention is usually 50 to 10,000.
上限臨界共溶温度を有する温度応答性ポリマーとしては、具体的には、例えば、アクリロイルグリシンアミド、メタクリロイルグリシンアミド、アクリロイルアスパラギンアミド、メタクリロイルアスパラギンアミド、アクリロイルグルタミンアミドおよびメタクリロイルグルタミンアミドからなる群から選ばれる少なくとも１種類の単重合体又は共重合体（コポリマー）が挙げられる。 The temperature-responsive polymer having the upper critical eutectic temperature is specifically selected from the group consisting of, for example, acryloyl glycinamide, methacryloyl glycinamide, acryloyl asparagine amide, methacryloyl asparagine amide, acryloyl glutamine amide, and methacryloyl glutamine amide. There may be mentioned at least one homopolymer or copolymer (copolymer).
下限臨界共溶温度を有する温度応答性ポリマーおよび上限臨界共溶温度を有する温度応答性ポリマーは、例えば、上記モノマーを有機溶媒又は水に溶解し、不活性ガスで系中を置換した後、重合温度まで昇温し、有機溶媒中であればアゾビスイソブチロニトリル等のアゾ系開始剤、過酸化ベンゾイル等の過酸化物、水系であれば過硫酸アンモニウム、過硫酸カリウム、２，２’−アゾビス（２−アミジノプロパン）二塩酸塩、４，４’−アゾビス（４−シアノ吉草酸）等の重合開始剤を添加し、攪拌下加熱を続けることにより得ることができる。その後、貧溶媒中で再沈殿を行い、析出したポリマーをろ取することで、製造したポリマーを精製することができる。 The temperature-responsive polymer having the lower critical eutectic temperature and the temperature-responsive polymer having the upper critical eutectic temperature are polymerized after, for example, dissolving the monomer in an organic solvent or water and replacing the system with an inert gas. The temperature is raised to an temperature, and in an organic solvent, an azo initiator such as azobisisobutyronitrile, a peroxide such as benzoyl peroxide, and an aqueous solvent such as ammonium persulfate, potassium persulfate, 2,2′- It can be obtained by adding a polymerization initiator such as azobis (2-amidinopropane) dihydrochloride or 4,4′-azobis (4-cyanovaleric acid) and continuing heating with stirring. Thereafter, reprecipitation is performed in a poor solvent, and the produced polymer can be purified by filtering the precipitated polymer.
刺激に応答して親水性と疎水性との相転移を生じる高分子を磁性微粒子の表面に固定する方法としては、例えば、反応性の官能基を介して該高分子を磁性微粒子に結合させる方法等の当技術分野で周知の方法が挙げられる（ＡＤＶ．Ｐｏｌｙｍ．Ｓｃｉ．，Ｖｏｌ．４、ｐ１１１、１９６５や、Ｊ．Ｐｏｌｙｍｅｒ Ｓｃｉ．，Ｐａｒｔ−Ａ，３，ｐ１０３１，１９６５に記載されている）。 Examples of a method for fixing a polymer that causes a phase transition between hydrophilicity and hydrophobicity in response to a stimulus to the surface of the magnetic fine particle include, for example, a method of binding the polymer to the magnetic fine particle via a reactive functional group (It is described in ADV.Polym.Sci., Vol.4, p111,1965 and J.Polymer Sci., Part-A, 3, p1031,1965). .
非水溶性物を含有する水溶液中への刺激応答性磁性微粒子の添加量は、水溶液中の非水溶性物と等量以上を添加することが好ましい。短時間で非水溶性物を吸着するためには、非水溶性物の濃度が低い場合でも高い回収率とするように刺激応答性磁性微粒子の添加量を大きくすることが好ましい。回収対象の非水溶性物を含有する水溶液の非水溶性物の濃度の範囲と、目標とする回収率に対応する刺激応答性磁性微粒子の必要量は予備試験により確認した後、添加量を設定することが好ましい。 It is preferable that the amount of the stimulus-responsive magnetic fine particles added to the aqueous solution containing the water-insoluble material is equal to or greater than that of the water-insoluble material in the aqueous solution. In order to adsorb the water-insoluble material in a short time, it is preferable to increase the amount of the stimulus-responsive magnetic fine particles so as to obtain a high recovery rate even when the concentration of the water-insoluble material is low. After confirming the range of the concentration of the water-insoluble material in the aqueous solution containing the water-insoluble material to be collected and the required amount of stimuli-responsive magnetic fine particles corresponding to the target recovery rate, set the amount to be added. It is preferable to do.
前記刺激応答性磁性微粒子に付与する刺激としては、例えば、温度変化、ｐＨ変化、光変化およびイオン強度変化等が挙げられ、各刺激の条件は適宜調整すればよい。非水溶性物と磁性微粒子との凝集物が生成したことは、目視、吸光度の測定、透過率の測定または磁石による磁気回収等の手段により確認することができる。 Examples of the stimulus applied to the stimulus-responsive magnetic fine particles include a temperature change, a pH change, a light change, and an ionic strength change, and the conditions of each stimulus may be appropriately adjusted. The formation of aggregates of water-insoluble materials and magnetic fine particles can be confirmed by means such as visual observation, measurement of absorbance, measurement of transmittance, or magnetic recovery using a magnet.
工程（１）または（１’）においては、非水溶性物と刺激応答性磁性微粒子との凝集を促進、または非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集を促進する凝集促進剤を含有する水溶液（以下、凝集促進剤溶液ともいう）を、非水溶性物と刺激応答性磁性微粒子、または非水溶性物と刺激応答性ポリマーと磁性微粒子を含有する水溶液に添加してもよい。凝集促進剤としては、例えば、Ｎａ２ＳＯ４が挙げられる。 In the step (1) or (1 ′), an aggregation promoter that promotes aggregation of the water-insoluble material and the stimulus-responsive magnetic fine particles, or promotes aggregation of the water-insoluble material, the stimulus-responsive polymer, and the magnetic fine particles. May be added to an aqueous solution containing a water-insoluble substance and stimulus-responsive magnetic fine particles, or a water-insoluble substance, a stimulus-responsive polymer, and magnetic fine particles. . Examples of the aggregation accelerator include Na 2 SO 4 .
凝集剤促進剤溶液の添加量としては特に限定されないが、非水溶性物と刺激応答性磁性微粒子、または非水溶性物と刺激応答性ポリマーと磁性微粒子が所望の温度で容易に凝集する程度まで、非水溶性物と刺激応答性磁性微粒子、または非水溶性物と刺激応答性ポリマーと磁性微粒子を含有する水溶液中に凝集剤促進剤溶液を添加すればよい。 The addition amount of the flocculant accelerator solution is not particularly limited, but is not limited to the degree that the water-insoluble substance and the stimulus-responsive magnetic fine particles, or the water-insoluble substance, the stimulus-responsive polymer, and the magnetic fine particles easily aggregate at a desired temperature. The flocculant accelerator solution may be added to an aqueous solution containing the water-insoluble material and the stimulus-responsive magnetic fine particles, or the water-insoluble material, the stimulus-responsive polymer, and the magnetic fine particles.
工程（２）および工程（２’）
工程（２）は、工程（１）で生成させた凝集物を磁力により磁気分離する工程である。また、工程（２’）は、工程（１’）で生成させた凝集物を磁力により磁気分離する工程である。
Step (2) and Step (2 ′)
Step (2) is a step of magnetically separating the aggregates generated in step (1) by magnetic force. Step (2 ′) is a step of magnetically separating the aggregates produced in step (1 ′) by magnetic force.
磁気分離は、永久磁石、電磁石、超電導磁石または磁気カラムなどの従来公知の方法を用いることができる。凝集物の分離に用いる磁石等の磁力は、用いる磁性微粒子の有する磁力の大きさによって異なる。磁力は、目的の刺激応答性磁性微粒子を磁集可能な程度の磁力を適宜使用できる。磁石の素材としては、例えば、上述した磁性微粒子の素材で構成されたものを使用することができる。例えば、マグネ社製ネオジム磁石等が利用できる。 For magnetic separation, a conventionally known method such as a permanent magnet, an electromagnet, a superconducting magnet, or a magnetic column can be used. The magnetic force of a magnet or the like used for separating the aggregates varies depending on the magnitude of the magnetic force of the magnetic fine particles used. As the magnetic force, a magnetic force capable of collecting the target stimulus-responsive magnetic fine particles can be appropriately used. As a material of the magnet, for example, a material composed of the above-described magnetic fine particle material can be used. For example, a neodymium magnet manufactured by Magne can be used.
凝集物を磁気分離するには、凝集物の入った反応容器の外側から磁石を接触することによって、凝集物が磁石に吸着する。この状態で、上澄み液を排出することにより凝集物を分離することができる。なお、回収効率の観点から、非水溶性物に適した水等の溶媒を反応容器に添加し、工程（３）または工程（３’）に供することが好ましい。 In order to magnetically separate the agglomerates, the agglomerates are adsorbed to the magnets by contacting the magnet from the outside of the reaction vessel containing the agglomerates. In this state, the aggregate can be separated by discharging the supernatant. From the viewpoint of recovery efficiency, it is preferable to add a solvent such as water suitable for a water-insoluble material to the reaction vessel and use it in the step (3) or the step (3 ').
工程（３）および工程（３’）
工程（３）は、工程（２）で分離した凝集物に工程（１）と反対の刺激を与えて刺激応答性磁性微粒子を親水性とすることによる刺激応答性磁性微粒子の表面における力学的な変化を利用して非水溶性物を刺激応答性磁性微粒子からリリースする工程である。
Step (3) and Step (3 ′)
In the step (3), the aggregate separated in the step (2) is subjected to a stimulus opposite to that in the step (1) to make the stimulus-responsive magnetic fine particles hydrophilic. This is a process of releasing a water-insoluble material from stimulus-responsive magnetic fine particles using change.
また、工程（３’）は、工程（２’）で分離した凝集物に工程（１’）と反対の刺激を与えて刺激応答性ポリマーを親水性とすることによる刺激応答性ポリマーの力学的な変化を利用して、非水溶性物を刺激応答性ポリマーと磁性微粒子からリリースする工程である。 Further, in the step (3 ′), the stimuli-responsive polymer mechanically formed by applying the stimulus opposite to that in the step (1 ′) to the aggregate separated in the step (2 ′) to make the stimulus-responsive polymer hydrophilic. This is a process of releasing a water-insoluble substance from a stimulus-responsive polymer and magnetic fine particles by utilizing such changes.
ここで、特開２０００−１４０６３２号公報には、刺激応答性高分子と標的物質に対して特異的に相互作用する物質を含み、刺激応答材料高分子が物理的刺激により構造変化を起こすことで、標的物質に対して特異的に相互作用する物質の相互作用が化学的もしくは物理的環境の変化を受け、標的物質とその相互作用力が温度等の物理的刺激により可逆的に変化する複合化材料を有する充填剤を用いる、標的物質の分離方法が記載されている。 Here, Japanese Patent Laid-Open No. 2000-140632 includes a substance that specifically interacts with a stimulus-responsive polymer and a target substance, and the stimulus-responsive material polymer causes a structural change due to physical stimulation. A complex in which the interaction of a substance that specifically interacts with the target substance is affected by changes in the chemical or physical environment, and the target substance and its interaction force are reversibly changed by a physical stimulus such as temperature. A method for separating a target substance using a filler with material is described.
本発明の方法と特開２０００−１４０６３２号公報に記載の分離方法との異なる点は、標的物質に対する特異的なリガンドまたは特異的な官能基を利用せず、刺激応答性磁性微粒子の表面、または凝集時の刺激応答性ポリマーの表面と標的物質の表面の疎水性相互作用により結合する点である。また、分離方法もカラムではなく磁気分離を使用する点においても異なる。 The difference between the method of the present invention and the separation method described in JP-A No. 2000-140632 is that a specific ligand or a specific functional group for a target substance is not used, and the surface of stimulation-responsive magnetic fine particles, or This is the point of binding by the hydrophobic interaction between the surface of the stimulus-responsive polymer during aggregation and the surface of the target substance. Also, the separation method is different in that magnetic separation is used instead of a column.
前記工程（１）または工程（１’）と反対の刺激を与えるとは、具体的には、例えば、工程（１）または工程（１’）における刺激が、水溶液の温度を上昇させる刺激であった場合、工程（３）または工程（３’）における刺激は水溶液の温度を低下させる刺激を与えることを意味する。また、例えば、工程（１）または工程（１’）における刺激が、水溶液のｐＨを上昇させる刺激であった場合、工程（３）または工程（３’）における刺激は、水溶液のｐＨを低下させる刺激を与えることを意味する。 Giving a stimulus opposite to that in the step (1) or step (1 ′) is specifically, for example, the stimulus in the step (1) or step (1 ′) is a stimulus that increases the temperature of the aqueous solution. In this case, the stimulus in the step (3) or the step (3 ′) means giving a stimulus for lowering the temperature of the aqueous solution. For example, when the stimulus in the step (1) or the step (1 ′) is a stimulus that increases the pH of the aqueous solution, the stimulus in the step (3) or the step (3 ′) decreases the pH of the aqueous solution. It means giving a stimulus.
水溶液の温度を上昇させる刺激を与える方法としては、例えば、温水を添加する方法および反応容器を加温する方法が挙げられる。また、水溶液の温度を低下させる刺激を与える方法としては、例えば、冷水を添加する方法および反応容器を冷却する方法が挙げられる。 Examples of the method for giving a stimulus for increasing the temperature of the aqueous solution include a method of adding warm water and a method of heating the reaction vessel. Moreover, as a method of giving the stimulus which reduces the temperature of aqueous solution, the method of adding cold water and the method of cooling a reaction container are mentioned, for example.
工程（３）または工程（３’）においては、さらに超音波処理または撹拌等を行うことにより、非水溶性物の回収効率を向上することができる。非水溶性物を磁性微粒子からリリースしたことは、目視、吸光度の測定、透過率の測定または磁石による磁気回収等の手段により確認することができる。 In the step (3) or the step (3 '), the recovery efficiency of the water-insoluble material can be improved by further performing ultrasonic treatment or stirring. The release of the water-insoluble material from the magnetic fine particles can be confirmed by means such as visual observation, measurement of absorbance, measurement of transmittance, or magnetic recovery using a magnet.
本発明の水溶液中に存在する非水溶性物を回収するためのキットは、例えば、以下の試薬から構成される。
試薬Ａ：刺激応答性磁性微粒子の分散液、または刺激応答性ポリマーと磁性微粒子との分散液
試薬Ｂ：凝集促進剤溶液（一例として、１ＭのＮａ２ＳＯ４水溶液が挙げられる）
試薬Ｃ：希釈用バッファー（上記試薬Ａ及びＢの希釈、並びに非水溶性物を含有する水溶液の希釈に使用可能な緩衝液である。一例として、トリス塩酸緩衝液、リン酸緩衝液等が挙げられる。）
試薬Ｄ：回収対象物質（非水溶性物）の標準品（一例として、精製した微細藻類が挙げられる。）。
The kit for recovering the water-insoluble material present in the aqueous solution of the present invention is composed of, for example, the following reagents.
Reagent A: Dispersion of stimulation-responsive magnetic fine particles, or dispersion of stimulation-responsive polymer and magnetic fine particles Reagent B: Aggregation accelerator solution (for example, 1M Na 2 SO 4 aqueous solution may be mentioned)
Reagent C: Buffer for dilution (a buffer that can be used to dilute the above-mentioned reagents A and B and an aqueous solution containing a water-insoluble substance. Examples include Tris-HCl buffer and phosphate buffer. )
Reagent D: Standard product of the substance to be collected (water-insoluble material) (as an example, purified microalgae can be mentioned).
以下、実施例によって本発明を具体的に説明するが、本発明はこれらによって何ら限定されることはない。 EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these.
オーランチオキトリウムの濃度の測定は、株式会社島津製作所社製ＵＶ−１７００ ＰｈａｒｍａＳｐｅｃを用いて、測定波長４０３ｎｍで測定した。また、本実施例で使用した水は、ミリポア社製純水製造装置「Ｅｌｉｘ ＵＶ ３５」によって精製された導電率１４．７ＭΩｃｍの精製水（池田理化社製、品番１−４７０４−０１、型番Ｗ−２０）である。 The concentration of auranthiochytrium was measured at a measurement wavelength of 403 nm using UV-1700 PharmaSpec manufactured by Shimadzu Corporation. The water used in this example was purified water having a conductivity of 14.7 MΩcm purified by Millipore's pure water production apparatus “Elix UV 35” (Ikeda Rika Co., Ltd., part number 1-4704-01, model number W). -20).
［実施例１］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を固定化した磁性微粒子（粒径２．８μｍ、ダイナビーズ：Ｍ−２７０、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。
[Example 1]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and 60 ml sample bottle and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich) 2.0) (10 mg / ml) of magnetic fine particles (particle size: 2.8 μm, Dynabeads: M-270, manufactured by Veritas Corp.) added thereto, and in a water bath with a liquid temperature of 50 ° C. for 2 minutes The mixture was stirred at a rotation speed of 300 rpm.
その後、攪拌を停止し、磁性微粒子と一緒にオーランチオキトリウムを４，０００Ｇのネオジウム磁石にて回収した。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図２に示す。この結果から、磁性微粒子と一緒にオーランチオキトリウムを一部回収できることがわかった。 Thereafter, stirring was stopped, and auranthiochytrium was collected together with the magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. From this result, it was found that a part of auranthiochytrium can be recovered together with the magnetic fine particles.
［実施例２］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を固定化した磁性微粒子（粒径１．０μｍ、ダイナビーズ：ＭｙＯｎｅ、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。
[Example 2]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and 60 ml sample bottle and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich) 2.0 ml (10 mg / ml) of magnetic fine particles (particle size: 1.0 μm, Dynabeads: MyOne, manufactured by Veritas Corp.) immobilized thereon, and rotated for 2 minutes in a water bath having a liquid temperature of 50 ° C. The mixture was stirred at several 300 rpm.
その後、攪拌を停止し、磁性微粒子と一緒にオーランチオキトリウムを４，０００Ｇのネオジウム磁石にて回収した。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図３に示す。この結果から、磁性微粒子と一緒にオーランチオキトリウムを一部回収できることがわかった。 Thereafter, stirring was stopped, and auranthiochytrium was collected together with the magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. From this result, it was found that a part of auranthiochytrium can be recovered together with the magnetic fine particles.
［実施例３］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）の水溶液を０．３ｍｌ（濃度１．０質量％）、磁性微粒子（粒径４．０μｍ、森下弁柄工業社製：ＭＴＢ−３０）を０．０２２ｇ添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。なお、ＰＮＩＰＡＭはポリ（Ｎ−イソプロピルアクリルアミド）の略号である。
[Example 3]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and 60 ml sample bottle and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich) ) 0.3 ml (concentration: 1.0% by mass), 0.022 g of magnetic fine particles (particle size: 4.0 μm, manufactured by Morishita Bengaryo Kogyo Co., Ltd .: MTB-30) and water temperature of 50 ° C. The mixture was stirred for 2 minutes at 300 rpm in the bath. Note that PNIPAM is an abbreviation for poly (N-isopropylacrylamide).
その後、攪拌を停止し、磁性微粒子と一緒にオーランチオキトリウムを４，０００Ｇのネオジウム磁石で回収した。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図４Ａに示す。図４Ａに示すように、磁性微粒子回収後の上清は、透明であった。 Thereafter, stirring was stopped, and auranthiochytrium was collected together with the magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. 4A. As shown in FIG. 4A, the supernatant after collecting the magnetic fine particles was transparent.
磁気分離後の磁性微粒子に３．０ｍｌの蒸留水を添加し、氷浴下、超音波洗浄器で１０分間、回転数３００ｒｐｍにて攪拌した。その後、攪拌を停止し、磁性微粒子だけを４，０００Ｇのネオジウム磁石にて回収した。磁性微粒子回収後の試料を図４Ｂに示す。図４Ｂに示すように、磁性微粒子回収後の上清から、オーランチオキトリウムが溶出したことが確認された。 3.0 ml of distilled water was added to the magnetic fine particles after magnetic separation, and the mixture was stirred in an ultrasonic bath for 10 minutes at 300 rpm in an ice bath. Then, stirring was stopped and only the magnetic fine particles were collected with a 4,000 G neodymium magnet. FIG. 4B shows a sample after collecting the magnetic fine particles. As shown in FIG. 4B, it was confirmed that auranthiochytrium was eluted from the supernatant after collecting the magnetic fine particles.
［実施例４］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を固定化した磁性微粒子（粒径２．８μｍ、ダイナビーズ：Ｍ２７０、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）および、熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を０．１ｍｌ（０．５ｍｇ／ｍｌ）添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。
[Example 4]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and 60 ml sample bottle and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich) ) 2.0 μm (10 mg / ml) of magnetic fine particles (particle size: 2.8 μm, Dynabeads: M270, manufactured by Veritas) and a thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich): 1 ml (0.5 mg / ml) was added, and the mixture was stirred in a water bath with a liquid temperature of 50 ° C. for 2 minutes at 300 rpm.
その後、攪拌を停止し、生成した磁性微粒子と一緒にオーランチオキトリウムを４，０００Ｇのネオジウム磁石にて回収した。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図５Ａに示す。図５Ａに示すように、磁性微粒子回収後の上清は、透明であった。 Thereafter, stirring was stopped, and auranthiochytrium was collected together with the produced magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. 5A. As shown in FIG. 5A, the supernatant after collecting the magnetic fine particles was transparent.
磁気分離後の磁性微粒子に３．０ｍｌの蒸留水を添加し、氷浴下で２分間、回転数３００ｒｐｍにて攪拌した。その後、攪拌を停止し、磁性微粒子を４，０００Ｇのネオジウム磁石にて回収した。磁性微粒子回収後の試料を図５Ｂに示す。図５Ｂに示すように、磁性微粒子回収後の上清から、オーランチオキトリウムが溶出したことが確認された。 3.0 ml of distilled water was added to the magnetic fine particles after magnetic separation, and the mixture was stirred for 2 minutes in an ice bath at a rotation speed of 300 rpm. Then, stirring was stopped and magnetic fine particles were collected with a 4,000 G neodymium magnet. FIG. 5B shows a sample after collecting the magnetic fine particles. As shown in FIG. 5B, it was confirmed that auranthiochytrium was eluted from the supernatant after collecting the magnetic fine particles.
［実施例５］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を固定化した磁性微粒子（粒径１．０μｍ、ダイナビーズ：ＭｙＯｎｅ、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）および、熱応答性ポリマー（ＰＮＩＰＡＭ、シグマ−アルドリッチ社製）を０．１ｍｌ（０．５ｍｇ／ｍｌ）添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。
[Example 5]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and 60 ml sample bottle and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich) ) 2.0 μm (10 mg / ml) of magnetic fine particles (particle size: 1.0 μm, Dynabeads: MyOne, manufactured by Veritas) and thermoresponsive polymer (PNIPAM, manufactured by Sigma-Aldrich): 1 ml (0.5 mg / ml) was added, and the mixture was stirred in a water bath with a liquid temperature of 50 ° C. for 2 minutes at 300 rpm.
その後、攪拌を停止し、生成した磁性微粒子と一緒にオーランチオキトリウムを４，０００Ｇのネオジウム磁石にて回収した。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図６Ａに示す。図６Ａに示すように、磁性微粒子回収後の上清は、透明であった。 Thereafter, stirring was stopped, and auranthiochytrium was collected together with the produced magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. 6A. As shown in FIG. 6A, the supernatant after collecting the magnetic fine particles was transparent.
磁気分離後の磁性微粒子に３．０ｍｌの蒸留水を添加し、氷浴下で２分間、回転数３００ｒｐｍにて攪拌した。その後、攪拌を停止し、磁性微粒子だけを４，０００Ｇのネオジウム磁石にて回収した。磁性微粒子回収後の試料を図６Ｂに示す。図６Ｂに示すように、磁性微粒子回収後の上清から、オーランチオキトリウムが溶出したことが確認された。 3.0 ml of distilled water was added to the magnetic fine particles after magnetic separation, and the mixture was stirred for 2 minutes in an ice bath at a rotation speed of 300 rpm. Then, stirring was stopped and only the magnetic fine particles were collected with a 4,000 G neodymium magnet. A sample after the collection of the magnetic fine particles is shown in FIG. 6B. As shown in FIG. 6B, it was confirmed that auranthiochytrium was eluted from the supernatant after collecting the magnetic fine particles.
［比較例１］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと磁性微粒子（粒径２．８μｍ、ダイナビーズ：Ｍ−２７０、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）添加し、室温で２分間、回転数３００ｒｐｍにて攪拌した。
[Comparative Example 1]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and magnetic fine particles (particle size: 2.8 μm, Dynabeads) in a 60 ml sample bottle 2.0 ml (10 mg / ml) of M-270 (manufactured by Veritas) was added and stirred at room temperature for 2 minutes at a rotation speed of 300 rpm.
その後、攪拌を停止し、４，０００Ｇのネオジウム磁石にて、生成した磁性微粒子と一緒にオーランチオキトリウムの回収を試みた。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図７に示す。図７に示すように、４，０００Ｇのネオジウム磁石にて磁性微粒子と一緒にオーランチオキトリウムを回収できないことがわかった。 Thereafter, the stirring was stopped, and an attempt was made to collect auranthiochytrium together with the produced magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. As shown in FIG. 7, it was found that a 4,000 G neodymium magnet cannot collect auranthiochytrium together with magnetic fine particles.
［比較例２］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと磁性微粒子（粒径１．０μｍ、ダイナビーズ：ＭｙＯｎｅ、ベリタス社製）を２．０ｍｌ（１０ｍｇ／ｍｌ）添加し、室温で２分間攪拌した。
[Comparative Example 2]
Aulanthiochytrium algae culture solution (0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and magnetic fine particles (particle size: 1.0 μm, Dynabeads: 60 ml sample bottle) 2.0 ml (10 mg / ml) of MyOne (manufactured by Veritas) was added and stirred at room temperature for 2 minutes.
その後、攪拌を停止し、４，０００Ｇのネオジウム磁石にて磁性微粒子と一緒にオーランチオキトリウムの回収を試みた。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図８に示す。図８に示すように、４，０００Ｇのネオジウム磁石にて磁性微粒子と一緒にオーランチオキトリウムを回収できないことがわかった。 After that, the stirring was stopped, and an attempt was made to collect auranthiochytrium together with the magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. As shown in FIG. 8, it was found that a 4,000 G neodymium magnet cannot recover auranthiochytrium together with magnetic fine particles.
［比較例３］
６０ｍｌのサンプル瓶にオーランチオキトリウム藻類培養液（濃度０．０３質量％、ｐＨ：９．９７、高砂熱学工業社より入手）を３．０ｍｌと磁性微粒子（粒径４．０μｍ、森下弁柄工業社製：ＭＴＢ−３０）を０．０２２ｇ添加し、液温が５０℃のウォーターバス中で、２分間、回転数３００ｒｐｍにて攪拌した。
[Comparative Example 3]
Auranthiochytrium algae culture solution (concentration 0.03% by mass, pH: 9.97, obtained from Takasago Thermal Engineering Co., Ltd.) and magnetic fine particles (particle size 4.0 μm, Morishita valve) in a 60 ml sample bottle 0.022 g (manufactured by Kako Kogyo Co., Ltd .: MTB-30) was added, and the mixture was stirred in a water bath having a liquid temperature of 50 ° C. for 2 minutes at 300 rpm.
その後、攪拌を停止し、４，０００Ｇのネオジウム磁石にて磁性微粒子と一緒にオーランチオキトリウムの回収を試みた。回収前のオーランチオキトリウム藻類培養液を図１に、回収後の試料を図９に示す。図９に示すように、４，０００Ｇのネオジウム磁石にて磁性微粒子と一緒にオーランチオキトリウムを回収できないことがわかった。 After that, the stirring was stopped, and an attempt was made to collect auranthiochytrium together with the magnetic fine particles with a 4,000 G neodymium magnet. The aurantiochytrium algae culture solution before recovery is shown in FIG. 1, and the sample after recovery is shown in FIG. As shown in FIG. 9, it was found that a 4,000 G neodymium magnet cannot recover auranthiochytrium together with magnetic fine particles.
Claims (13)
（１）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性磁性微粒子を含有させ、刺激を与えて刺激応答性磁性微粒子を疎水性として非水溶性物と結合させ、非水溶性物と刺激応答性磁性微粒子との凝集物を生成させる工程
（２）工程（１）で生成させた凝集物を磁力により水溶液中から分離する工程
（３）工程（２）で分離した凝集物に工程（１）と反対の刺激を与えて刺激応答性磁性微粒子を親水性とし、非水溶性物を刺激応答性磁性微粒子からリリースする工程 The method of collect | recovering the water-insoluble matter which exists in aqueous solution including the following processes (1)-(3).
(1) An aqueous solution containing a water-insoluble substance contains stimuli-responsive magnetic fine particles that undergo a phase transition between hydrophilic and hydrophobic in response to a stimulus, and the stimulus-responsive magnetic fine particles are rendered hydrophobic by applying a stimulus. Step (2) of generating an aggregate of water-insoluble material and stimulus-responsive magnetic fine particles by binding with water-insoluble material (2) Step of separating the aggregate generated in step (1) from an aqueous solution by magnetic force (3 ) The step of giving the opposite stimulus to the step (1) to the aggregate separated in the step (2) to make the stimulus-responsive magnetic fine particles hydrophilic, and releasing the water-insoluble matter from the stimulus-responsive magnetic fine particles
（１’）非水溶性物を含有する水溶液に、刺激に応答して親水性と疎水性の相転移をする刺激応答性ポリマーと磁性微粒子を含有させ、刺激を与えて刺激応答性ポリマーを疎水性として非水溶性物と結合させ、非水溶性物と刺激応答性ポリマーと磁性微粒子との凝集物を生成させる工程
（２’）工程（１’）で生成させた凝集物を磁力により水溶液中から分離する工程
（３’）工程（２’）で分離した凝集物に工程（１’）と反対の刺激を与えて刺激応答性ポリマーを親水性とし、非水溶性物を刺激応答性ポリマーと磁性微粒子からリリースする工程 A method for recovering a water-insoluble material present in an aqueous solution, comprising the following steps (1 ′) to (3 ′).
(1 ') An aqueous solution containing a water-insoluble material contains a stimulus-responsive polymer that undergoes a hydrophilic and hydrophobic phase transition in response to a stimulus and magnetic fine particles, and the stimulus-responsive polymer is made hydrophobic by applying a stimulus. In step (2 ′), the agglomerates formed in step (1 ′) are magnetically bonded to an aqueous solution by binding to a water-insoluble material and forming an aggregate of the water-insoluble material, stimulus-responsive polymer, and magnetic fine particles. Step (3 ′) for separating from the solution The stimulus separated in the step (2 ′) is subjected to a stimulus opposite to that in the step (1 ′) to make the stimulus-responsive polymer hydrophilic, and the water-insoluble material is used as the stimulus-responsive polymer. Release from magnetic fine particles
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