JP2004300109A - Method for producing animal protein, production apparatus and animal protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing proteins such as collagen, gelatin and collagen peptide from animal body tissue in high purity and yield at a low cost. <P>SOLUTION: The method for the production of animal proteins is provided with a pretreatment step including the defatting and deodorizing treatment of raw material comprising an animal body tissue containing animal proteins, an extraction step to solubilize and dissolve an acid-soluble protein by immersing the raw material in an acid solution or an acid and acidic protease solution to obtain an extract containing the acid-soluble protein, a purification step to obtain purified acid-soluble protein from the extract, and a concentration step. The acid solution concentration of the acid solution or the acid and acidic protease solution in the extraction step is 0.3-2M and the weight ratio of the acid solution to the raw material for extraction is 1.5-10. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００５７】
実施例１
図２（Ａ）に例示する本発明の動物蛋白質の一例である製造工程を用い、コラーゲンを製造した。
すなわち、参考例１と同様の原料を使用してアルカリ浸漬後、サメ皮の表皮を剥いで、サメ皮の真皮層１．１１ｋｇを得た。得られた真皮層１．１１ｋｇ（湿重量）の水分含有量は８６．３％であり、油脂分は０．１％へ、アンモニアは２．２ｐｐｍまで減少していた（いずれもドライ重量比）。これにより、残留油脂分は目標（０．１％以下）を達成し、アンモニア残留分も目標（２ｐｐｍ以下）にほぼ近づいた。
得られた真皮層をミートチョッパー（日本キャリア工業製＃２２ＧＭ−Ｄ）にて最大２ｍｍ角程度に粉砕した。抽出は、３０Ｌ容積の攪拌式容器を用い、粉砕物１．１１ｋｇ（湿重量）に、０．５Ｍ酢酸水溶液８．８８ｋｇ（真皮層重量に対し８倍量）、ペプシン（ブタ胃粘膜由来、シグマ社製）１．５２ｇ（真皮層固形分乾燥重量に対して１％）を添加し、４℃で１２０回転／分、３日間攪拌して酸可溶性コラーゲンの抽出液９．９８ｋｇを得た。なお、得られた抽出液中の固形分乾燥重量は、抽出操作前の、アルカリ浸漬後の真皮層の乾燥重量を１００％としたとき９８％であり、抽出液固形分に含まれる残留油脂分は０．１％、アンモニアは１．４ｐｐｍであった（いずれもドライ重量比）。
得られた抽出液は粘性が高く不溶性残さの測定には不向きなので、１００ｇを採りだし、この抽出液へ、粘度が高いため透過抵抗を低くするために１ｍＭ酢酸５００ｇを加え、ろ紙〔アドバンテック東洋（株）製Ｎｏ．５Ａ、２枚重ね〕にて不溶性残さを減圧ろ過した。不溶性残さはほとんどトレースのみであった。ｐＨは２．６程度であった。
【００５８】
同じく抽出液１００ｇに１ｍＭ酢酸５００ｇを加えて、希釈した抽出液６００ｇについて、図１に示す精製・濃縮装置を使用して限外ろ過操作を行なった。精製・濃縮装置は、限外ろ過装置に（株）ミリポア製ペリコンＸＬバイオマックス−３０（分画分子量３０，０００）を膜モジュールとして備えており、循環液流速２５ｍＬ／分となるように送液ポンプおよび出口側に設けた絞り４を調節し、透過液速度２〜３ｍＬ／分で運転した。図示していないが、限外ろ過装置には、循環液のｐＨおよび電気伝導度を検知するｐＨメータ（例えば堀場製作所製（株）カスタニーＬＡＢ−Ｆ２０ＩＩ）および電気伝導度計（例えば堀場製作所製（株）カスタニーＬＡＢ−ＤＳ）が備えられている。
限外ろ過装置中のチューブなどに循環液が約１００ｍＬ存在するため、循環液供給タンク中の液量は当初約５００ｍＬになっていた。このため、循環液供給タンク中の液量は、常に４００〜５００ｍＬになるよう弱酸性溶液添加タンクより適宜１ｍＭ酢酸（ｐＨ４．０）を循環液に加えて限外ろ過を行ない、約６時間経過後、循環液のｐＨの上昇および塩濃度を示す電気伝導度（測定単位μＳ／ｃｍ^２）の減少が見られなくなった。その後、約１２時間経過時まで限外ろ過を継続したが、循環液のｐＨおよび電気伝導度の変化が見られず、またコーラゲンの純度も所望のレベルに達したので、限外ろ過を終了した。限外ろ過を終了して、得られた循環液中の固形分乾燥重量は、限外ろ過操作前の抽出液中の固形分ドライ重量を１００％として、９９％であり、アンモニア濃度は１．４ｐｐｍから０．８ｐｐｍ（ドライ重量比）へと減少していた。
得られた循環液を−８０℃にて凍結させ、１０Ｐａ、２０℃の条件で１２時間凍結乾燥し、乾燥コラーゲン１０７ｇ（水分０．１％）を得た。収率は、アルカリ浸漬後の真皮層の乾燥重量基準で７０．０％であった。
【００５９】
本実施例で得られた乾燥コラーゲンを１ｍＭ酢酸にて再溶解して、電気泳動法で分子量分布を調べたところ、γ鎖を多く含んでいることを確認した。使用した機器は、アトー（株）製ラピダス・ミニスラブにより、アクリルアミドゲル濃度が７．５％のゲルで泳動した。
また得られた乾燥コラーゲンの比旋光度を測定した。乾燥コラーゲンを０．００５Ｍ酢酸で希釈した０．１％溶液を、４℃にて１２時間スターラ攪拌溶解し、１８，０００ｒｐｍ（重力加速度３６，０００ｇ）、４℃にて４０分程度遠心分離（日立製作所製高速冷却遠心機、２ＯＰＲ−５２０）にかけ、その上清液１０ｍＬについて、比旋光度を測定した（堀場製作所（株）製ＳＥＰＡ−２００、使用条件２０℃）。比旋光度の値は、コラーゲンの純度目安と考える［α］^２０ _Ｄ−３６０°〜−４２０°の間にあった。
【００６０】
なお、１０分間当たり５℃変化させる時間−温度直線勾配での比旋光度の測定においても、５℃から３０℃にかけては、ほぼ−３７０°前後であったが、３０℃近辺から急激に低下し、３５℃では−１５０°前後の値を示した。４０℃より再び温度を低下させても、２０℃の比旋光度［α］^２０ _Ｄは−３６０°には戻らず、−１５０°のままであり、強制的に６０℃温水浴加熱を与えたゼラチンの場合の測定値とおよそ変わらなくなった。既に熱変性によりゼラチンに変性したものと見られる。
また、得られた乾燥コラーゲンの残留油脂分、塩分、アンモニアの含量は、それぞれ０．１％、０．１％、０．５ｐｐｍであり、トリメチルアミンは検出されず、臭い官能評価の結果も、パネラーの７５％が無臭と判定した。
なお、塩分量の測定は強熱残分として得られた灰分として測定した（上記非特許文献１参照）。トリメチルアミン量の測定は、皮から過塩素酸により抽出して得られた溶液について、ピクラート法により比色定量行った（日本食品工業学会・食品分析法編集委員会編、「食品分析法」、光琳、東京、１９８２年１０月、ｐ．６７３−６８１参照）。
臭い官能評価は、パネラーとして２０人使用して下記評価基準で行なった。
○；無臭
△；わずかに臭気あり
×；有臭
【００６１】
実施例２
実施例１と同様に０．５Ｍ酢酸８倍量の酸溶液抽出条件にて得られた酸可溶性蛋白質の抽出液を用いて、精製、濃縮の方法として、実施例１の限外ろ過に代わり、塩析、透析でも行った。得られた抽出液１００ｇに、塩化ナトリウムおよび酢酸がそれぞれ１モル／Ｌおよび０．５モル／Ｌとなるよう加え、析出した沈殿物を１８，０００ｒｐｍ（重力加速度３６，０００ｇ）、４℃にて遠心分離にかけ、固液分離した。分離した沈殿物を１ｍＭ酢酸中に再溶解し、２０倍量の１ｍＭ酢酸中で透析を行い脱塩した。透析外液の塩化ナトリウム濃度が一定の値を示すまでに３時間を要し、その度に透析外液の交換を行い、透析外液の交換は４回行った。従って１回の透析操作に要した時間は１２時間である。透析を終了したコラーゲン溶液に、上記と同様に、再び塩化ナトリウムを１Ｍになるよう加え、塩析による沈殿、遠心分離による固形分の分離、固形分の再溶解、透析して脱塩する、塩析、透析の操作を５回繰り返して、精製を終了した。従って、塩析、透析の正味の所要時間は１２×５＝６０時間（約２．５日）であった。
本実施例の塩析、透析による方法においては、実施例１の限外ろ過と比べて、精製、濃縮の日数はかかるが、同様に、比較的純度の高いコラーゲン溶液が得られることを確認できた。なお、抽出処理投入前の真皮層の乾燥重量基準で、得られたコラーゲン収率は５０％程度であった。
また、従来公知例の酸溶液２０倍量の抽出条件で抽出液を得て、精製操作を塩析、透析で行った場合を、後述する比較例３に示しているが、これと比べると、本実施例の０．５Ｍ酢酸溶液８倍量の酸抽出条件で得られた抽出液を用いることで、塩析、透析の回数が少なくても、比較的純度の高いコラーゲン溶液が精製できることを示している。
【００６２】
実施例３
実施例１〜２と同様に、サメ生皮をアルカリ浸漬し、表皮を剥いで真皮層１．０５ｋｇを得た。真皮層の水分含有量は８７％であり、油脂分は０．１％、アンモニアは２．５ｐｐｍであった。
得られた真皮層をミートチョッパー（日本キャリア工業製＃２２ＧＭ−Ｄ）にて最大長２ｍｍ程度に粉砕した。容積３０Ｌの攪拌式容器を用いて、粉砕物１．０５ｋｇ（湿重量）に、０．５Ｍ酢酸水溶液２１ｋｇ（真皮層重量に対し２０倍量）、ペプシン（ブタ胃粘膜由来、シグマ社製）１．３７ｇ（真皮層固形分重量に対して１％）を添加し、４℃で１２０回転／分、３日間攪拌して抽出液２２．０ｋｇを得た。得られた抽出液中の固形分乾燥重量は、抽出操作前のアルカリ浸漬後の真皮層の乾燥重量を１００％としたとき９７％であり、油脂分は０．１％、アンモニアは１．６ｐｐｍであった（いずれもドライ重量比）。
実施例１と異なり、１ｍＭ酢酸で希釈しないで、直接、不溶性残さをろ過し、抽出液２２．０Ｋｇを得た。ｐＨは２．５程度であった。不溶性残さはトレースであった。限外ろ過は、実施例１と同じ装置、同じ条件で行なった。循環液のｐＨおよび電気伝導度の変化が見られなくなり、またコラーゲンの純度も所望のレベルに達するまでの時間は４０時間（実施例１の３倍以上）であった。これは、抽出操作において、０．５Ｍ酢酸水溶液を真皮層重量に対し２０倍量と実施例１に比べ多くして行った為に、抽出液中の分子量分布がバラツキ、即ちコラーゲン純度が低下し、従って循環液中のコラーゲン濃度も薄いため、所望のレベルに達するまでの限外ろ過操作による循環液側の精製、濃縮の時間が長くなったものと思われる。限外ろ過を終了して、得られた循環液中の固形分乾燥重量は、限外ろ過操作前の抽出液中の固形分ドライ重量を１００％として、７５％であり、アンモニア濃度は１．６ｐｐｍから０．９ｐｐｍ（ドライ重量比）へと減少していた。
【００６３】
得られた循環液を実施例１と同様に凍結乾燥し、乾燥コラーゲン０．３７２ｇ（水分０．１％）を得た。アルカリ浸漬後の真皮層の乾燥重量基準での収率は６０％であった。
得られた乾燥コラーゲンを１ｍＭ酢酸にて再溶解して、電気泳動法で分子量分布を調べたところ、α鎖、β鎖、γ鎖を確認したが、実施例１で得られたコラーゲンに比べるとα鎖のバンドは強いのに対し、γ鎖のバンドは弱かった。また、比旋光度は［α］^２０ _Ｄ−２８０であり、コラーゲン純度の低いものであった。この純度が低かった理由として、抽出工程においてγ鎖の可溶化のみならず、γ鎖が分解され低分子のα鎖になったためと考えられる。γ鎖の分解が促進された理由としては、抽出に供した酸溶液の重量がサメ真皮層に対し２０倍重量とかなり大きいため、酵素反応や加水分解が進みやすい環境であったためと考えられる。
【００６４】
比較例１
比較のため、図２（Ｂ）に例示する従来プロセスの一例によるサメ皮コラーゲンの製造方法を示す。
まず、サメ皮の表皮を剥離して、膨潤処理を施して、コラーゲンが可溶化し易いように含有水分を高めた真皮層４ｋｇ（水分９５．２６％）を使用した。
この真皮層４ｋｇ（乾燥重量１８９．６ｇ）を上記と同様にミートチョッパーにて粉砕し、脱脂を目的に界面活性剤（Ｂａｙｅｒ社製、ＰＲＯＤＵＣＴ Ａ）の１％水溶液を加え、４℃で１２時間撹拌した。脱脂終了後、８，０００回転（重力加速度１３，０００ｇ）、３０分の遠心分離により真皮層を沈殿させ、上清に含まれる界面活性剤溶液を除いた。さらに回収した真皮層に、約２重量倍量（約８ｋｇ）の水を加え撹拌後に上記と同条件で遠心分離して、水洗を行った。水洗後の真皮層重量は３．９６ｋｇ（水分９３．４６％、乾燥重量２５８．９ｇ）であった。原料とした真皮層に対する回収率は、乾燥重量基準では１３７％となり、見掛け上増える結果となった。これは、脱脂後の水洗が不充分で界面活性剤を完全には除去できなかったためと考えられる。
【００６５】
脱脂水洗操作後の真皮層３．９６ｋｇに、０．５Ｍ酢酸水溶液７．９３ｋｇ（真皮層重量に対し２倍量）、さらにペプシン（ブタ胃粘膜由来、シグマ社製）１．９ｇ（真皮層固形分重量の１％）を添加し、４℃で１２０回転／分、３日間攪拌してコラーゲンの可溶化および抽出を行った。この後、不溶性残さを８，０００回転（重力加速度１３，０００ｇ）、３０分の遠心分離により除き、抽出液１０．２ｋｇを得た。不溶性残さはトレースであった。
【００６６】
得られた抽出液１０．２ｋｇへ塩化ナトリウムおよび酢酸がそれぞれ１モル／Ｌおよび０．５モル／Ｌとなるよう加え、析出した沈殿物を８，０００回転（重力加速度１３，０００ｇ）、３０分の遠心分離にかけて分離した。分離した沈殿物を１ｍＭ酢酸中に再溶解し、２０倍量の１ｍＭ酢酸中で透析を行い脱塩した。透析外液の塩化ナトリウム濃度が一定の値を示すまでに３時間を要し、その度に透析外液の交換を行い、透析外液の交換は４回行った。従って１回の透析操作に要した時間は１２時間である。透析を終了したコラーゲン溶液に、上記と同様に、再び塩化ナトリウムを１Ｍになるよう加え、塩析による沈殿、遠心分離による固形分の分離、固形分の再溶解、透析して脱塩する、塩析、透析の操作を５回繰り返して、精製を終了した。従って、塩析、透析の正味の所要時間は１２×５＝６０時間であった。
その後、実施例１と同様に凍結乾燥し、乾燥コラーゲン１０８．８ｇ（水分１２．１２％）を得た。アルカリ浸漬後の真皮層の乾燥重量基準で収率は５０．４２％であったが、混在している塩分および油分を除去すると収率は４９．１８％であった。残留油脂分、塩分、アンモニアの濃度は、それぞれ０．４７％、２．０％、３ｐｐｍと不純物含量が高く、また０．１％溶液（０．００５Ｍ酢酸中）の比旋光度も［α］^２０ _Ｄ−２５６°と低くコラーゲン純度も低かった。
【００６７】
比較例２
前処理および抽出処理は実施例３２と同様に行ない、得られた抽出液１００ｇを比較例１と同様に塩析、透析を行い脱塩した。透析は、４回の外液交換を行いながら１２時間を要して行った。さらに塩析、透析の操作を５回繰り返して、精製を終了した。
その後、実施例１と同様に凍結乾燥し、乾燥コラーゲン０．２９９ｇ（水分０．１％）を得た。アルカリ浸漬後のサメ皮真皮層の乾燥重量基準で収率は４８．２０％であった。残留油脂分、塩分、アンモニアの濃度は、それぞれ０．１％、２．０％、１．５ｐｐｍであり、１％溶液（０．００５Ｍ酢酸中）の比旋光度も［α］^２０ _Ｄ−２５０°であった。
【００６８】
比較例３
実施例３と同様に処理を行ない、得られた抽出液に対し比較例１と同様に塩析・透析により精製操作を行ったが、比較例１や比較例２に対して２倍となる１０回行なった。
得られた乾燥コラーゲンを１ｍＭ酢酸にて再溶解して、電気泳動法で分子量分布を調べたところ、α鎖、β鎖、γ鎖を確認した。比較例２で得られたコラーゲンよりもさらにα鎖のバンドは弱く、実施例１と同程度にγ鎖のバンドが強かった。
得られた乾燥コラーゲンの比旋光度は［α］^２０ _Ｄ−３６０°となり、純度の高いコラーゲンの所望のレベルに入った。このとき、残留油脂分、塩分、アンモニアの濃度も、それぞれ０．１％、０．１％、０．５ｐｐｍと低下していたが、収率は３０．０％に低下していた。
【００６９】
実施例３で得られたコラーゲンは、比較例１に比べれば残留油脂分が低い点では優れるが、これは前処理工程を界面活性剤による脱脂ではなくアルカリ浸漬で行った効果の寄与が大きい。実施例３２では精製・濃縮工程で塩析、透析に代わり限外ろ過を用いているため、元々塩分が少なく残留塩分を減らすことができたが、比旋光度で示されるコラーゲン純度や、電気泳動で示されるγ鎖の比率は、同じ限外ろ過法で精製を行った実施例１よりも低かった。これは抽出工程における酸溶液の重量比が２０倍と過剰であったためと考えられる。また、比較例２，３で精製を塩析、透析で行った場合には、分子量分布のγ鎖のバンドや、比旋光度を純度の高いコラーゲンの所望のレベルにするためには、１０回以上の塩析、透析を必要とした。
実施例１〜３および比較例１〜３により得られたコラーゲンの諸性質を、表２に示した。
【００７０】
実施例４
サメ生皮を原料に、表皮を剥離して真皮層を得るための温度および時間を検討した。生皮２００ｇ（湿重量、水分含量８３％）を、表３に示す温度に設定した温水約５Ｌに投じ、表３に示す時間で浸漬した。浸漬後は手指および包丁を用いて手作業で表皮を剥離し真皮層を回収し、得られた真皮層を、１１０℃で乾燥させた後の乾燥重量を測定した。
３０℃×１分以下の浸漬では、浸漬前の性状との差異が見られなかった。浸漬の温度および時間の上昇に伴い、真皮層の回収率が向上した。特に、４０℃×５分、または５０℃×３分の浸漬で、手指にて多くの部分を剥離可能であった。６０℃×５分の浸漬を超えると、コラーゲンがゼラチンへ変性し、温水への溶解が起こるため、真皮層の回収率が低下した。
【００７１】
実施例５
実施例４で用いたものと同じサメ生皮を原料に、アルカリ処理工程における浸漬時間および温度の影響を検討した。生皮２００ｇ（湿重量、水分含量８３％）を、１Ｌの石灰の飽和溶液（ｐＨ１１．５）中で、表４に示す温度および日数で浸漬し、表皮を除去した真皮層に含まれる油脂分、アンモニア含量を測定した。
その結果を表４に示した。
５℃で浸漬を行った場合、１４日後に、抽出・精製前の工程で所望とするレベルである油脂分０．２％以下、アンモニア１０ｐｐｍ以下に達し、さらに２１日後に油脂分０．０９％、アンモニア２．９ｐｐｍまで減少した。浸漬の温度を変えた場合、所望のレベルに達するまでに、２０℃では８日、３０℃では２日、４０℃では１日であった。高温であれば短期間で油脂分、アンモニアを除去することができるが、コラーゲンの熱変性を起こさないようにするためには、できるだけ低温で行うことが望ましい。
【００７２】
実施例６
参考例１で得たサメ皮真皮層を原料として、抽出日数の短縮を目的に、高温での加熱抽出を行った。
ビーカ試験レベルではミンチ粉砕の更に細かいペースト状にした原料を用いた場合は、真皮層１ｇ（湿重量、水分含量は８６．３％）に、９ｍＬの０．５モル／Ｌの酢酸水溶液を加え、１００℃で１分間、５分間、１０分間、２０分間加熱し、その後実施例１と同様に抽出、精製し、電気泳動法で分子量分布を調べた。但し少ない材料なので、抽出の浸漬処理には、振盪式のマルチシェーカ（東京理科器械製ＭＭＳ）を用いた。１〜５分の加熱では、実施例１とほぼ同比率でγ鎖が見られたが、１０分間加熱ではγ鎖のバンドが薄くなり、一方２０分間加熱すると電気泳動のバンド測定では、分子量５０，０００以下が多く、γ鎖は全く見られず、分子量１０万のα鎖のバンドもほとんど見られなかった。このことから、１００℃×１〜１０分程度の加熱に抑えれば、γ鎖の解離やゼラチンへの変性が許容できる範囲にあることが判る。
さらに、参考例１で得たサメ皮真皮層１．０ｋｇを原料とした試験では、実施例１に示した方法と同様に、０．５Ｍ酢酸水溶液８．０ｋｇおよびペプシン１．３７ｇを加え、４℃で１２０回転／分の撹拌速度で抽出を行った。ただし、抽出の日数は半分の１．５日に留め、この後、１００℃で５分間の短時間加熱抽出を付加した。その後も実施例１と同様に、残さのろ過分別、限外ろ過による精製、凍結乾燥を行ったところ、乾燥コラーゲン９６ｇ（水分０．１％）を得た。得られたコラーゲンは、実施例１で得られたコラーゲンとほぼ同量のγ鎖を含んでおり、比旋光度、残留油脂分、残留アンモニアともに、純度の高いコラーゲンの所望のレベルに達していた。本短時間加熱工程を付加することで、実施例１で３日要した抽出工程が、半分で済んだ。
【００７３】
【表２】

【００７４】
＊１：膨潤済・表皮なし真皮の乾燥重量基準
＊２：０．１％溶液（５ｍＭ酢酸中）
【００７５】
【表３】

【００７６】
【表４】

【００７７】
【発明の効果】
本発明により、品質、純度が高いコラーゲン、ゼラチン、コラーゲンペプタイドなどの動物蛋白質を、動物の体組織から低コストで収率が高く、かつ多量に分離回収できる製造方法と製造装置を提供することができる。また、本発明により得られる動物蛋白質は、医薬品、化粧液、リンス、トリートメント、工業用途、健康食品、調味料などに好適な材料として使用できる。
【図面の簡単な説明】
【図１】本発明の精製・濃縮装置の一例の構成概略図である。
【図２】（Ａ）は本発明の動物蛋白質の製造方法の一例を示す全体工程図の例示であり、（Ｂ）は従来プロセスの動物蛋白質の製造方法の一例を示す全体工程図の例示である。
【符号の説明】
１：限外ろ過装置
２：循環液供給タンク
３：弱酸性液補充タンク
４：絞り
５：送液ポンプ
６：レベル計
１０：精製・濃縮装置
１１：透過液の流れ
１２：循環液の流れ
１３：弱酸性液の流れ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a production method and a production apparatus for extracting and purifying animal protein from animal body tissues, and an animal protein obtained by the method.In particular, the present invention relates to an extraction method using an acid solution and a purification method using ultrafiltration. It relates to the manufacturing method used.
[0002]
[Prior art]
Gelatin and collagen are extracted from the protein contained in animal bones and skins as raw materials, and are used in many applications such as medicine, food, photography, and industry.
The basic unit of collagen is a γ chain having a molecular weight of about 300,000, which is a triple helical structure of three polypeptide chains (α chains) having a molecular weight of about 100,000.
Further, gelatin is defined as "produced by hydrolyzing collagen obtained from animal bone, skin, ligament or tendon" (see Non-Patent Document 1). The temperature at which gelatin is extracted by hydrolysis is generally said to be 40 ° C. or higher for terrestrial animals. An extract from a collagen hydrolysis reaction solution has a molecular weight of 100,000 when the hydrolysis reaction temperature is relatively low. Gelatin is mainly composed of α-chains before and after, but as the hydrolysis temperature is raised, collagen peptide, which is a low-molecular protein, becomes the main component.
[0003]
In the prior art, as a pretreatment step for extracting proteins from animal body tissues, an alkaline solution, an acid solution, an ethanol solution, an organic solvent, for the purpose of removing water-soluble proteins, oils and fats, odor components and the like in raw materials. They are pickled and mixed with salt. When the body tissue of an animal is made of skin, the dermis layer containing a large amount of collagen may be peeled off from the epidermis, and the target protein may be extracted using the separated dermis layer.
[0004]
Alkaline solution treatment using lime or caustic soda is widely used as a pretreatment for gelatin extraction (see Patent Document 1). In addition, there is a combination example of alkali treatment and salting as a pretreatment for collagen extraction, but it relates to the extraction of so-called alkali-soluble collagen solubilized with an alkaline solution, and the purification method involves salting out the extracted collagen simultaneously. Then, it is desalted (see Patent Document 2).
On the other hand, when the raw material is derived from aquatic animals, removal of odor components is a major problem, and a method of treating with an organic solvent such as ethanol or pretreating with salt is known (Patent Documents 3 and 4). reference).
A method is also known in which a purification and concentration step is directly performed by ultrafiltration using a marine organism as a raw material, but the pretreatment step is a combination of an organic solvent treatment and a salt solution treatment (see Patent Document 5). However, additional safety equipment such as explosion proof is required to prevent the danger of the organic solvent igniting and exploding, which requires a large investment in equipment.In the case of collagen used for food, no matter how small the amount of organic matter remaining, there is no health problem. From the point of view, the adoption of organic solvents is difficult.
[0005]
A method is also conceivable in which the fats and oils and odorous components are not brought to a desired residual level in the pretreatment step including the degreasing / deodorizing treatment or the extraction step, but finally to the desired residual level in the purification / concentration step. However, in such a method, the yield is reduced and the cost is increased. In addition, if acid solution immersion is adopted in the pre-treatment degreasing / deodorizing process, the odor component removal function is equivalent to that of the alkali solution immersion, but the oil / fat component removal performance is inferior. Since proteins are generally weakly acidic, there is a drawback that excessive degradation of the protein occurs and the yield is reduced.
There is also a method of removing fats and oils with a surfactant or an organic solvent without performing alkali immersion, acid immersion, or salting for a short period of time. However, in the degreasing method using a surfactant, the surfactant is adsorbed on the protein, and a large amount of cost is required for washing and removing the surfactant as a post-treatment after protein extraction. Further, the organic solvent treatment is not suitable for the installation cost of the explosion-proof device and the demand of the present age where environmentally friendly materials are required.
[0006]
As described above, in the conventional known technology, in the case of performing ultrafiltration as purification of the extracted acid-soluble collagen, the case where the removal of fats and oils and odor components of the raw material is performed in a treatment step including alkali immersion is this case. The optimum immersion conditions for alkaline immersion have not yet been elucidated.
[0007]
As a method of solubilizing collagen, there are a method of immersing and dissolving in an acid solution and a method of adding a protease to the acid solution to cut the telopeptide portion at the end of collagen to promote solubilization. Furthermore, since the properties of the solubilized collagen partially differ depending on the pH state of the solution to be solubilized, alkali-soluble collagen obtained by solubilization and extraction under an alkaline solution or solubilization under a neutral salt solution is used. There is also a neutral salt-soluble collagen obtained by extraction after extraction, but it is said that these do not become a homogeneous solution because the collagen is partially denatured and gelatinized (see Non-Patent Document 2).
[0008]
As an example of extraction of acid-soluble collagen using an acidic protease, water-soluble proteins and oils and fats in animal body tissues as a raw material are removed by pretreatment with a salt or an alkali, and further extracted under acidic conditions. (See Patent Documents 6 and 7). In recent years, proteases have been industrially used, but the basics of the collagen extraction method remain unchanged.
As a method for recovering and purifying collagen from the above-mentioned extract, collagen is precipitated by a chemical method such as salt addition or pH control, and solid-liquid separation is performed by centrifugation. A method of dissolving and performing dialysis or ultrafiltration to obtain a desired collagen is employed (see Patent Documents 8 and 9). There is also disclosed a method of directly collecting collagen by a physical ultrafiltration method without performing salting out or pH manipulation of the collagen extract (see Patent Document 5).
In the case where alkali-soluble collagen is extracted, the above-mentioned salting-out technique is used for its recovery and purification as in the case of acid-soluble collagen (see Patent Document 2).
[0009]
By the way, as a basic condition for solubilizing collagen from animal body tissue and extracting acid-soluble collagen, the weight concentration ratio between the animal body tissue and the acid used for extraction and the water to be dissolved is adjusted to an optimal range. It is necessary to set and extract efficiently. As a prior art, the weight ratio of the acid solution to 1 part by weight of the animal body tissue is 20 times and the concentration of the acid solution is 0.5 M (see Patent Document 8), and the weight ratio of the acid solution is 10 to 100 times. And a concentration of 0.1 to 5 M (see Patent Document 9).
[0010]
Under the above conditions, the acid solution weight ratio is increased in order to efficiently perform the extraction time in a short time. However, disposal of the used acid solution is difficult, and this is a major problem for mass production. In addition, by contacting with a large amount of acid, the target γ-chain with a molecular weight of about 300,000 (a triple helical structure composed of three α-chains, which are polypeptide chains with a molecular weight of about 100,000) is solubilized. (A state in which they are loosely bonded by hydrogen bonds) and dissociates into α-chains or collagen peptides of lower molecular weight, resulting in a variation in the molecular weight distribution, and hence a disadvantage that the collagen solution is non-uniform and the purity is reduced. Was.
[0011]
In the purification step of the prior art, a high molecular weight fraction containing the desired γ chain is precipitated by salting out or the like, and the precipitate is subjected to dialysis or ultrafiltration for desalting of a solid obtained by centrifugation. Although a method of increasing purity is used, the purification step after extraction is complicated, and high-purity collagen is obtained, but the yield is low.
Further, in the extraction of acid-soluble collagen, if the extraction time is lengthened to reduce the amount of insoluble solids that do not dissolve in the acid solution, the dissociation of γ chains into α chains is promoted, and collagen purity is reduced. There is also a method of re-extracting this insoluble solid by re-introducing it into an acid solution (see Patent Document 5), but the obtained collagen also has a low purity, and it may not be said that the collagen is suitable for the purpose of re-extraction. is there.
As described above, the conventional extraction conditions have a problem in terms of mass production when considering the necessary purification / concentration steps, and conditions for stable and low-cost extraction and purification of high-purity collagen and There is a need for improved methods.
[0012]
Looking at the market for animal proteins isolated from animal body tissues, the market for collagen containing γ chains is expensive but the demand is not large. The market for peptides is relatively cheap, but demand is high.
In the method for producing gelatin, usually, a body tissue of an animal is immersed in a warm water bath to decompose a γ chain to obtain gelatin as an α chain. In addition, the method for producing a collagen peptide involves adding a protease to an extract of the reaction solution decomposing the γ chain, further extracting a low-molecular-weight protein, and using the extract by microfiltration or ultrafiltration. To obtain a collagen peptide having a desired molecular weight. Therefore, the method for producing gelatin and collagen peptide is different from that for producing collagen in not only the extraction step but also the purification / concentration step.
From these points, in addition to collagen containing γ chains, gelatin containing α chains as a main component or a low molecular weight collagen peptide can be produced using the same raw materials and the same equipment, so that equipment costs can be reduced and the purification process can be reduced. There is also a demand for the development of a production method capable of changing the purification conditions and the like corresponding to the target animal protein.
[0013]
[Non-patent document 1]
JISK6503-1996 "Glue and gelatin"
[Non-patent document 2]
Japan glue and gelatin industry association, "glue and gelatin", Maruzen Publishing, January 1987 (p.133-p141)
[Patent Document 1]
JP-A-7-247465 (page 3)
[Patent Document 2]
JP-B-46-15033 (page 3)
[Patent Document 3]
JP-A-2000-50811 (page 2)
[Patent Document 4]
JP-A-2000-256398 (page 6)
[Patent Document 5]
JP 2001-200000 A (pages 5-7)
[Patent Document 6]
Japanese Patent Publication No. 44-11037 (page 1-2)
[Patent Document 7]
Japanese Patent Publication No. 44-1175 (page 1-2)
[Patent Document 8]
JP-A-5-93000 (pages 2-3)
[Patent Document 9]
JP-A-11-246598 (page 3)
[0014]
[Problems to be solved by the invention]
The present invention has been made in view of such problems of the related art, and in producing proteins such as collagen, gelatin, and collagen peptides from animal body tissues, the yield and purity are high and the production can be performed at low cost. The purpose is to provide a manufacturing method.
[0015]
[Means for Solving the Problems]
The present invention provides a pretreatment step in which a tissue containing an animal protein and / or an animal body tissue made of animal skin is used as a raw material, and a pretreatment step is performed for extracting a protein contained in the raw material; The obtained raw material is immersed in an acid solution or an acidic protease solution to solubilize and dissolve the acid-soluble protein, and an extraction step of obtaining an extract containing the acid-soluble protein as a main component, and concentrating the extract, and A method for producing an animal protein comprising a purification / concentration step of purifying an acid-soluble protein, wherein the acid solution or the acid and acid protease solution in the extraction step is adjusted to an acid solution concentration of 0.3 M to 2 M, and is subjected to extraction. And an acid solution weight ratio of 1.5 to 10 times.
Next, the present invention relates to a pretreatment device that performs pretreatment for using a tissue containing an animal protein and / or an animal body tissue consisting of animal skin as a raw material and subjecting the raw material-containing protein to extraction, Extraction apparatus in which the raw material subjected to the above is immersed in an acid solution or an acidic protease solution having a weight ratio of 1.5 to 10 times and a concentration of 0.3 M to 2 M to obtain an extract containing an acid-soluble protein as a main component. And a purification / concentration device for introducing the extract into ultrafiltration and concentrating the circulating fluid of the ultrafiltration to purify the acid-soluble protein.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Examples of the animal body tissue of the raw material used in the present invention include bones and skins of terrestrial animals such as cows and pigs and aquatic animals such as fish, sharks, salmon and whales, and terrestrial animals include ligaments or tendons. But are not limited to these.
The raw material to be used for extracting collagen, gelatin or collagen peptide is usually ground or minced in advance. In the case of animal skin, it may be minced or homogenized, or cut into pieces of about 5 mm square to 50 mm square.
[0017]
In the case of extracting, purifying and concentrating animal protein using animal skin as a raw material, the skin itself may be used as a raw material for extraction, but the dermis layer and epidermis constituting the skin are separated to contain a large amount of collagen. Generally, the dermis layer is subjected to an extraction step.
Methods for peeling the dermis layer from animal skin include mechanical and physical peeling methods.In the present invention, the animal skin is immersed in warm water for a short period of time, and the tissue between the epidermis and dermis layer is removed. The binding force is weakened, and the dermis layer can be efficiently separated.
[0018]
When a terrestrial animal-derived skin is used, it is immersed in warm water of preferably 40 to 70 ° C, more preferably 50 to 60 ° C for 1 minute to 10 minutes. In the case of a skin derived from an aquatic animal, the dermis layer is easily formed by immersion in warm water of preferably 30 to 60 ° C, more preferably 40 to 50 ° C, preferably 1 to 10 minutes, more preferably 3 to 5 minutes. It is easy to separate from the epidermis. The above temperature range is a temperature high enough to weaken the bond between tissues and a temperature range in which thermal denaturation of the protein is acceptable. The immersion time is preferably such that heat denaturation does not occur in the central part of the dermis layer.
In order to separate the dermis layer, it is possible to peel off the epidermis manually after immersion in the above-mentioned warm water. However, after immersion, washing with water and freezing, for example, a slicer (AC-300 manufactured by Tazaki Seisakusho) can be used. it can.
In addition, if the dermis layer is separated after performing the following alkaline immersion at a high temperature using the animal skin itself without separating the dermis layer before the degreasing / deodorizing step, the epidermis and the dermis layer can be easily separated.
[0019]
The pretreatment step of the present invention includes a degreasing / deodorizing step of degreasing / deodorizing the raw material.For example, as a degreasing / deodorizing step, immersion in methanol to remove fats and oils and odorous components of the raw material, as well as water-soluble proteins. It may include several steps such as ethanol immersion, alkali immersion, acid solution immersion, salting, and furthermore, treatment with a surfactant and an organic solvent, but may also include an alkali treatment step.
Note that methanol immersion and ethanol immersion may be included in the organic solvent treatment.
[0020]
Examples of the alkaline agent used for alkali immersion include lime, quicklime, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, and the like.
The alkali immersion is carried out by immersing the raw material in an aqueous alkali solution having a weight of twice or more, preferably 2 to 20 times the weight of the raw material, and keeping the pH at 10 or more, preferably at pH 10 to 13. The concentration of the aqueous alkali solution varies depending on the type of the alkaline agent used, but is usually about 0.001 to 5% by weight, preferably about 0.01 to 1% by weight. In addition, when it is performed in a saturated precipitation state using lime, pH control becomes easy.
Although it is possible to perform the immersion treatment in a closed state, it is possible to efficiently remove the oil and fat and the odor component efficiently by exchanging the liquid, explosion, or stirring as appropriate.
The immersion temperature is preferably 0 to 40 ° C., more preferably 5 to 20 ° C. in the case of raw materials derived from terrestrial animals, and preferably 0 to 30 ° C. in the case of raw materials derived from aquatic animals having a low heat denaturation temperature. Preferably it is 0-10 degreeC. The immersion period is preferably 1 to 3 days, more preferably 1 to 2 days at 40 ° C, and it is preferable that the immersion period be doubled or more every time the holding temperature falls from 40 ° C to 10 ° C.
It is preferable that the apparatus for immersion treatment for degreasing and deodorization is provided with additional equipment capable of controlling the temperature within the above temperature range, or it is preferable to control the temperature of the entire room including the raw materials, the immersion liquid and the apparatus.
The alkali-immersed material is subjected to solid-liquid separation by decanting, filtering, centrifuging or the like of the alkali solution, and then, if necessary, washing and appropriately washing with water, followed by the next step. Further, it is desirable that the pretreated material is further pulverized or shredded as necessary to enhance the effect of the next step of extraction.
[0021]
The pretreatment step of the present invention includes a degreasing / deodorizing step for removing water-soluble proteins, oils and fats, and odor components, and / or a peeling step for removing a dermis layer from the skin. Is preferably provided in the preceding stage, but may be provided between the extraction step and the purification / concentration step.
In some cases, the pretreatment step, the extraction step, and the purification / concentration step are performed using a plurality of factories having different steps due to process restrictions and circumstances. In this case, additional steps such as freeze-solidification and thawing are added from the viewpoint of preventing heat denaturation of the material and reducing transportation costs between the respective steps, but the present invention can be used in such a case as well. it can. That is, the present invention may be a continuous production process or a distributed production process.
[0022]
Normally, the allowable limit value of residual oil and fat components and odor components (for example, residual ammonia concentration) of animal protein varies depending on the product application used, but excluding seasoning applications, experiments, medical care, cosmetic liquids, rinses, treatments and industrial In use, it is desired that the residual oil and fat content of the protein contained after purification is 0.1% or less (dry weight ratio) and the residual ammonia concentration is 2 ppm or less (dry weight ratio).
In the present invention, the above pretreatment step can reduce the residual fat and oil to 0.1% or less (dry weight ratio) and the residual ammonia concentration to 1 ppm or less (dry weight ratio), depending on the nature of the raw materials. Will be possible.
[0023]
In the extraction method of the present invention, acid-soluble collagen and acidic protease-soluble collagen are mainly used. Here, the acidic protease-soluble collagen includes gelatin and collagen peptides obtained by adding a protease of a proteolytic enzyme such as pepsin in an acid solution to solubilize the telopeptide moiety at the molecular end of the γ chain. In the present invention, unless otherwise required, they are collectively referred to as acid-soluble collagen.
[0024]
Examples of the acid solution include organic acids such as acetic acid, citric acid, and lactic acid, and inorganic acids such as hydrochloric acid and sulfuric acid.
Examples of the acidic protease include pepsin derived from animal gastric mucosa and commercially available acidic protease [Newase, trade name, manufactured by Amano Enzyme Co., Ltd .; Morcin F, trade name, manufactured by Kikkoman Corp .; Trade name protease YP-SS, etc.].
[0025]
In the extraction step of the present invention, the weight concentration ratio of the body tissue of the animal, the acid used for extraction and the water used as the solvent among the three, that is, the weight ratio of the acid solution or the acid and acid protease solution to the material and the acid concentration are determined. It is preferable to set the optimum range and perform extraction efficiently.
In the present invention, the acid solution concentration is preferably 0.3 M to 2 M, more preferably 0.5 to 10 times, more preferably 2 to 8 times the weight ratio of the material. ２2M. Within this range, acid-soluble collagen having a high γ-chain content and high purity can be obtained, and thus, in the purification / concentration process, the conventional extraction method having low collagen purity requires salting out, precipitation, centrifugation, and salt removal. There is also an advantage that only complicated steps such as microfiltration or ultrafiltration are required without using complicated processes such as dialysis or ultrafiltration steps.
There is also an advantage in the case where after the extraction of the present invention is performed, purification and concentration are performed by conventional processes such as salting out and dialysis. Under the extraction conditions of the present invention, collagen having a high γ-chain content can be relatively easily solubilized and extracted, and the salting-out and dialysis are repeated many times to provide a conventional process for purifying and concentrating collagen to a desired purity. In this case, the number of repetitions can be reduced, so that the production cost in the conventional collagen purification process is low, and high purity and high quality are obtained.
If the weight ratio is less than 1.5 times, the raw materials are not sufficiently dispersed in the acid solution, and the extraction of the acid-soluble collagen does not proceed well. On the other hand, when the weight ratio exceeds 10 times, the γ chain is decomposed and easily dissociated into the α chain, and the collagen purity is reduced. If the acid solution concentration is less than 0.3 M, the acid concentration is too low to extract sufficiently. On the other hand, if it exceeds 2M, not only the solubilization of the γ chain, but also the decomposition of the γ chain and the dissociation into the α chain proceeds, and the pH of the acid solution tends to be 2.0 or less. It is difficult to adjust the pH to be closer to neutral in the step to be performed.
[0026]
The amount of the acidic protease to be added is preferably 0.1 to 10 g, more preferably 0.8 to 1.2 g, based on 100 g of the dry weight of the raw material substrate to be extracted. When the amount is less than 0.1 g, the acidic protease does not sufficiently act. On the other hand, when the amount exceeds 10 g, not only the cleavage reaction of the telopeptide but also the decomposition reaction of the collagen itself proceeds in parallel, and the collagen purity is reduced.
The extraction conditions when an acid and an acidic protease solution are used as the extract are the same as the extraction conditions using the acid solution.
The present invention can be similarly applied to neutral salt-soluble collagen and alkali-soluble collagen. Specifically, in the case of neutral salt-soluble collagen, the acid may be replaced with a neutral salt, and in the case of alkali-soluble collagen, the acid may be replaced with an alkali.
[0027]
In the extraction of the present invention, for terrestrial animal-derived materials, the extraction temperature is preferably maintained at 0 to 40 ° C, more preferably 0 to 35 ° C, and for aquatic animal-derived materials, the extraction temperature is preferably 0 to 30 ° C. More preferably, the temperature is maintained at 0 to 25 ° C. Extraction does not proceed below the above temperature range. On the other hand, when the temperature exceeds the above temperature range, thermal denaturation of collagen is likely to occur.
The extraction time is preferably 1 to 3 days, more preferably 2 to 3 days. If it is less than 1 day, the extraction is insufficient, while if it is more than 3 days, the dissociation of the γ chain into the α chain proceeds, resulting in a decrease in purity.
It is desirable that the extraction device including the extraction container and the auxiliary devices such as the acid solution tank are provided with additional equipment capable of controlling the temperature within the above-mentioned temperature range, or the temperature of the entire room including the raw materials, the solution and the device is controlled. Is preferred.
In addition, since the solubilized and extracted collagen is a loose bond in which the molecular chains of the three polypeptide chains are connected by hydrogen bonds, especially when it is desired to extract high-purity collagen, the extraction temperature is set in the pretreatment step. It is desirable to lower the holding temperature by 5 ° C. or more so as to minimize dissociation (gelatinization) of acid-soluble collagen into α chains due to thermal denaturation during extraction. It is also possible to finely crush or shred the material to be compatible with the purpose of enhancing the extraction effect, and to reduce the possibility of heat denaturation and the possibility of expanding the variation in molecular weight distribution, which is a concern as a side effect of miniaturization and pulverization. , Reduction and suppression.
[0028]
For the purpose of shortening the number of extraction days, high-temperature heating extraction can be added to the extraction conditions in the extraction step under the above conditions. The heating temperature is preferably 50C to 150C, more preferably 80C to 100C, and the heating time is preferably 1 minute to 10 minutes, more preferably 1 to 2 minutes. Because of the time of about several minutes, the acid-soluble collagen can be extracted without causing significant thermal denaturation and little decrease in purity. When the time is less than 1 minute, solubilization and dissolution are not performed, and the remaining extraction residue increases. On the other hand, when the time exceeds 10 minutes, thermal denaturation is promoted and dissociation of γ chain into α chain, or Furthermore, the decomposition into low molecular weight proteins is promoted, and it is no longer suitable for extracting the target collagen.
For example, assuming a case where a few kg of raw material derived from aquatic animals is used for immersion in an acid solution or an acidic protease solution at a holding temperature of 0 to 40 ° C. for 3 days to perform an extraction treatment, The immersion is performed at 0 to 40 ° C. for 1 to 2 days. During this immersion period, the structure in the pulverized raw material becomes loosely bonded, further miniaturization and pulverization proceeds, and an acid solution per unit weight of the raw material is used. Since the surface area in contact with is increased, the remaining immersion temperature and time are, for example, flash-like heating at 100 ° C. for about 5 minutes to promote solubilization and dissolution of protein collagen and denaturation. Can be done less.
When protein collagen is extracted from a test raw material using an acidic protease solution, protease, which is a protease, is generally alive at 60 ° C or higher, and when the immersion temperature is increased to 60 ° C or higher, the extraction effect of the protease decreases. However, this method gives a short-time flash heating, and is hardly affected by the addition in the latter half of the extraction step.
In addition, although it is a beaker test level, when using a raw material of about several g and preliminarily refining the raw material in the form of a paste to 0.1 mm or less in order to perform an extraction treatment, it is immersed in heat at 100 ° C. for several minutes. It has been confirmed that the protein containing the γ-chain can be solubilized, dissolved and extracted even when the reaction is directly performed. This is because the action similar to the action and effect in the first half of the extraction described above is previously given by pulverizing into a paste form, and since the weight and volume of the raw material are small, heating is applied to an acid solution and an acid solution. It can be understood that the dispersion is carried out evenly with the fine raw materials dispersed therein.
[0029]
Further, in the extraction step of the present invention, insoluble solids that remain without being solubilized, so-called extraction residues, may be generated in a considerable amount. The material to be extracted is preferably ground in advance with a meat chopper or the like so that such insoluble solids are reduced and solubilization is performed uniformly. The maximum length of the pulverized material is preferably 1 cm or less, more preferably 2 mm or less. It is desirable that no processing heat is generated or that the processing heat is hardly transmitted to the test raw material during the processing of the pulverization and miniaturization of the raw material, and it is preferable to perform the processing while cooling. As long as heat is not generated, the paste may be pulverized and refined to 0.1 mm or less. In addition, although the production cost is increased, the raw material can be freeze-dried in advance, finely pulverized, and subjected to an extraction treatment.
Within the range of the combination of the acid solution weight ratio and the concentration according to the present invention, by performing the above-mentioned pulverization treatment, it is possible to obtain an acid-soluble collagen with a low extraction residue, a high yield and a high purity.
Further, in the extraction operation, it is preferable to use an extraction vessel with stirring or shaking (shinto) by immersing the raw material in an acid solution or an acid and an acidic protease solution, and while stirring or shaking the mixture of the raw material and the acid solution, Disperse and disperse the raw material and solution uniformly, and extract so that the acid-soluble protein from the raw material can be solubilized and dissolved evenly in the extraction container with little unevenness and dispersion. It is preferable to operate. Here, the stirring includes air stirring in addition to mechanical stirring. When the extraction processing capacity is relatively small, a method of shaking and shaking the entire extraction container can be employed.
[0030]
In the above preferable extraction conditions, the concentration of the acid-soluble collagen in the extract is high and the viscosity of the extract is high, so that it is slightly easier to handle in the filtration and purification / concentration steps of the extraction residue that is an insoluble solid from the extract. Although inferior, when the acid solution used for the extraction is increased in advance and the solution viscosity is lowered as in the conventional example, the purity of the obtained collagen is lowered. In order to improve the handleability of an extract having a high solution viscosity, a weakly acidic solution close to neutral may be added as needed in the stage of filtration of the extraction residue from the extract and in the purification / concentration step. Even if a weakly acidic solution is added in the purification / concentration step, the purification / concentration step is shorter than the extraction step, and the weak acid solution to be added is neutralized because the purpose is to optimize the viscosity. Since it may be close, dissociation of the collagen from the γ chain to the α chain or collagen peptide is not significantly promoted. Further, considering the later use of the product, it is desirable that the acid-soluble collagen is as weakly acidic as possible as close to neutrality as possible. Therefore, the above-mentioned production method of the present invention is advantageous in terms of the use of the product.
Extraction residue, which is an insoluble solid in the extraction, is preferably removed beforehand from the obtained extract before being subjected to purification and concentration in the next step, and is usually used as a solid-liquid separation method. Filtration, vacuum filtration, centrifugation and the like can be mentioned. In the present invention, since purification and concentration using an ultrafiltration method are performed, also in order to prevent clogging of the ultrafiltration membrane, a pretreatment step that hardly generates an extraction residue, along with setting conditions in the extraction step, It is desirable to pay close attention to the filtration and removal of the generated extraction residue.
As described above, according to the present invention, acid-soluble collagen can be easily extracted and purified with high purity and high quality and in high yield, and high productivity can be obtained.
[0031]
In the present invention, an ultrafiltration treatment is performed in order to purify collagen from the acid-soluble collagen solution obtained by the above extraction method.
Since the extracted acid-soluble collagen solution contains not only gelatin but also collagen peptide in addition to the acid solution and the collagen solubilized and dissolved therein, the ultrafiltration has a molecular weight cut-off of 30,000 to 100,000. When the ultrafiltration membrane is used, collagen containing a large amount of γ-chain having a molecular weight of 300,000 can be concentrated in the circulating fluid side without being permeated, and a mixture of gelatin having a molecular weight of 100,000 and collagen peptide having a molecular weight of 100,000 or less than the permeated solution can be obtained. Can be obtained. In addition, when ultrafiltration is performed using an ultrafiltration membrane having a molecular weight cutoff of 5,000 to 30,000, collagen and gelatin can be concentrated in the circulating fluid side without being permeated, and a collagen peptide can be obtained from the permeated liquid. it can.
[0032]
In the ultrafiltration step of the present invention, in the purification / concentration step, the extract is subjected to an ultrafiltration device, and the residual liquid containing the acid-soluble protein that does not pass through the ultrafiltration membrane is concentrated as necessary to form a circulating liquid. Circulation and ultrafiltration may be performed again. By circulating the residual liquid in this manner, the effect of being able to continuously concentrate is obtained.
[0033]
When circulating the circulating fluid, it is desirable to previously adjust the concentration of the acid-soluble protein in the circulating fluid (viscosity of the circulating fluid) to an appropriate upper limit or less. The appropriate upper limit is determined in consideration of the range of permeation resistance in which the ultrafiltration membrane to be used can operate, the likelihood of clogging of the filtration membrane, and the performance of the circulating fluid circulation pump (also called a liquid feed pump) to be used. Can be selected.
For the adjustment, a means for mixing with the extract and a means for adding a weakly acidic solution for controlling the total amount of the circulating liquid described below can also be used. The detection and / or adjustment of the concentration of the acid-soluble protein (viscosity of the circulating fluid) in the circulating fluid may be performed intermittently or continuously.
[0034]
In the purification / concentration step employing the ultrafiltration method of the present invention, since the acid solution in the extract passes through the ultrafiltration membrane and is discharged as a permeate, the animal protein is concentrated on the circulating fluid side to reduce the purity. When the raising operation is continued, the amount of the acid solution on the circulating liquid side decreases. However, in the operation of purification and concentration, it is desirable that the circulating liquid maintain a constant total amount, and the pH of the circulating liquid is desirably as weak as possible to be as neutral as possible in view of the application of the product to be used later.
For this reason, in the present invention, the pH is closer to neutral than the acid solution used in the step performed immediately before the purification / concentration step, independently of the extract, and the total amount detection step of detecting the total amount of the circulating liquid. It is preferable that the method further includes a step of adding a weakly acidic solution to the circulating liquid, and that the total amount of the circulating liquid be maintained and controlled within a predetermined range. The detection of the total amount and / or the addition of the weakly acidic solution may be performed continuously or intermittently. By having these steps, the pH of the circulating fluid after concentration can be made weakly acidic simultaneously with the concentration of the animal protein in the circulating fluid.
As a means for detecting the total liquid amount, a level meter is specifically mentioned. The weakly acidic solution adding means includes a weakly acidic solution replenishing tank.
[0035]
In the purification / concentration step of the present invention, at least one selected from the group consisting of the concentration of the acid-soluble protein contained in the circulating fluid, the purity of the acid-soluble protein, the viscosity of the circulating fluid, and the density of the circulating fluid is used. It is preferable to control the circulation of the circulating liquid and terminate the purification operation when the target purity is reached.
For the detection of the concentration and purity of the acid-soluble protein, the concentration and purity of the acid-soluble protein contained in the circulating fluid may be directly detected. The purity of the resulting acid-soluble protein can also be estimated in terms of conversion. The control of the circulation of the circulating fluid can be performed by a mixing operation with the extract, adjusting the pressure of the circulating fluid (difference between the circulating fluid outlet and the inlet). The above element detection and circulation control may be performed continuously or intermittently. It is natural that the control accuracy and stability of the purification / concentration process are improved while referring to the data for each production lot of the product.
Examples of the means for detecting the concentration of the acid-soluble protein include an ultraviolet absorption measuring instrument, a differential refractive index detector, and a light scattering detector. Examples of means for detecting the purity of the acid-soluble protein include a polarimeter (for example, SEPA-200 manufactured by Horiba, Ltd.) and a circular dichroism measuring device. As a means for detecting the viscosity of the circulating liquid, a rotary viscometer, a falling ball viscometer, and the like can be given. As a means for detecting the density of the circulating liquid, a vibration type densitometer, a floating type specific gravity meter and the like can be mentioned.
[0036]
As the acid solution in the circulating fluid is replaced with a weakly acidic solution, the acid-soluble protein in the circulating fluid is also purified and concentrated to a desired purity level, and the acidity of the solution and the purity have a high correlation with each other. Show. Therefore, by using a pH meter or an electric conductivity meter to detect that the pH or electric conductivity of the circulating fluid changes and becomes constant, it is possible to convert and estimate the purity of the animal protein. The above conversion and estimation can be performed by referring to the purity data of the product obtained according to the time change from the start of ultrafiltration, particularly when the production amount of the animal protein is the same for each lot.
It is desirable that these detecting means be built in an animal protein purification / concentration device to continuously detect and control the state of the circulating fluid. Even if it can be substituted for a desired purpose by detection, it may be used.
[0037]
In purification using ultrafiltration, clogging of the ultrafiltration membrane is a major problem in mass production. To prevent this, filtration of insoluble residues and separation and removal from the extract by microfiltration are generally performed. However, in the present invention, as a method of avoiding the stoppage of the operation of purification / concentration due to clogging, a plurality of ultrafiltration devices may be provided, and a configuration may be adopted in which the circulating liquid of each ultrafiltration is communicated. it can. With this configuration, a compact, inexpensive, and hardly clogging process can be achieved.
The molecular weight cut-off of each ultrafiltration membrane can be selected depending on the target animal protein, and may be the same or different.
When multiple ultrafiltration devices are used, the frequency of clogging of all devices at the same time is low. In addition, when one device is clogged, the circulating fluid is appropriately short-circuited, the remaining device is operated, and during that time, clogging can be removed and the device can be returned, so that there is little need to stop operation. This is a suitable process for continuous mass production.
The detection of the clogging is preferably performed while detecting the pressure difference between the circulating liquid inlet and the circulating liquid outlet of the ultrafiltration device.
[0038]
Further, ultrafiltration in which the fractionation molecular weights of the ultrafiltration membranes are different from each other is constituted in two stages, the first stage comprises an ultrafiltration membrane having a fractionation molecular weight of 30,000 to 100,000, and the second stage comprises: It is equipped with an ultrafiltration membrane having a cut-off molecular weight of 5,000 to 30,000, and the permeate obtained by the first-stage ultrafiltration is introduced into the circulating fluid of the second-stage ultrafiltration, whereby the first-stage ultrafiltration is performed. Collagen containing γ-chain from the circulating fluid of the first stage permeate, that is, gelatin containing α-chain as the main component from the circulating fluid of the second stage, and a lower molecular weight than the α-chain than the permeate of the second stage. Collagen peptides can be produced. Here, the introduction of the extract into the first stage ultrafiltration and / or the introduction of the first stage permeate into the second stage ultrafiltration may be performed intermittently or continuously. Is also good.
In the case where a large amount of collagen peptide is produced, a method of adding a protease to a first-stage permeate and / or a second-stage circulating solution containing a large amount of gelatin and hydrolyzing the same may be used.
[0039]
The holding temperature in the purification and concentration steps is basically the same as the holding temperature in the extraction step. In the case of raw materials derived from terrestrial animals, it is preferably kept at 0 to 40 ° C, more preferably at 0 to 35 ° C, and Is preferably maintained at 0 to 30 ° C, more preferably 0 to 25 ° C. Further, it is preferable to lower the holding temperature in the pretreatment step by 5 ° C. or more. The extract or purification / concentration device to be used for purification is desirably equipped with additional equipment capable of controlling the temperature within the above temperature range. Alternatively, the extract, the device and a storage tank for the purified acid-soluble protein solution, and furthermore, the purification In a subsequent post-process, it is preferable to control the temperature of a sterilizing device and a drying device that are appropriately used as necessary, a storage of product materials, and the entire room including these.
[0040]
In some cases, an insoluble solid that does not dissolve in the acid solution or the acidic protease solution is generated. In the present invention, hydrolyzable gelatin and collagen peptide can be extracted from the insoluble solid by immersing in warm water.
In addition, from the viewpoint of the animal protein product market, the demand for collagen is low, but the demand for gelatin and collagen peptides is high, so that insoluble solids remain in the extraction of acid-soluble proteins. In some cases, it may be more convenient to extract and purify a large amount of gelatin or collagen peptide by immersing the remaining insoluble solid material in hot water to extract and purify a large amount of gelatin or collagen peptide.
[0041]
Alternatively, the raw material that has been subjected to pretreatment including degreasing and deodorization is divided into two groups in advance, and one group is subjected to extraction and purification of an acid-soluble protein obtained by dipping in an acid solution or an acid and an acidic protease solution, Another group, along with the insoluble residue of the above-mentioned acid extraction, is immersed in warm water for extraction and purification, and production and distribution are performed so as to obtain hydrolyzable gelatin and collagen peptides. The apparatus and equipment used for the concentration can also be used for a production process that is shared and shared by both.
[0042]
In the present invention, gelatin and collagen peptides extracted by hydrolysis by immersion in a warm water bath are distinguished from collagen, gelatin, and collagen peptides of acid-soluble proteins obtained by immersion in an acid solution or an acid and acid protease solution. Therefore, an animal protein obtained by immersion in warm water is called a warm water soluble protein.
For example, the insoluble solids isolated from the acid-soluble protein extract by decanting, filtration, centrifugation, etc., and / or raw materials that have been subjected to pretreatment including degreasing / deodorizing treatment may be appropriately washed as necessary. Immersed in warm water in warm water to solubilize, dissolve and extract the warm water soluble protein. The temperature of hot water extraction is preferably 50 ° C to 100 ° C, more preferably 80 to 90 ° C for raw materials derived from terrestrial animals, and preferably 40 ° C to 100 ° C, more preferably 80 to 90 ° C for raw materials derived from aquatic animals. The hot water extraction time is preferably 1 to 20 hours, more preferably 2 to 5 hours.
Gelatin that does not pass through the ultrafiltration membrane is introduced into the ultrafiltration apparatus equipped with an ultrafiltration membrane having a molecular weight cut-off of 5,000 to 30,000 by introducing the extract of the hot water-soluble protein obtained by immersion in hot water. And a collagen peptide that permeates the filtration membrane can be purified. In the same manner as described above, the residual liquid containing the warm water-soluble protein that does not pass through the ultrafiltration membrane is concentrated, circulated as a circulating liquid, and ultrafiltrated again to obtain gelatin from the circulating liquid and collagen peptide from the permeated liquid. You can also.
[0043]
It is also possible to add a protease during hot water extraction and hydrolyze to positively obtain a collagen peptide. As the protease, in addition to the acidic protease described above, a neutral protease and an alkaline protease can also be used. For example, protease Amano (trade name, manufactured by Amano Enzyme Co., Ltd.), GOLD-BNP (trade name, manufactured by Godo Shusei Co., Ltd.), and Shin Nippon Chemical Co., Ltd. Sumiteam (trade name), Protin, Deskin, Samoase (trade name) manufactured by Daiwa Kasei Co., Ltd., Orientase (trade name) manufactured by HBI Co., Ltd. PNT, Novozyme, bioseparase manufactured by Nagase ChemteX Corporation, and the like. The amount of protease added is preferably 0.1 to 10 g, more preferably 0.8 to 1.2 g, based on 100 g of the dry weight of the substrate. Regarding the hydrolysis conditions, the substrate concentration is preferably 0.1 to 50% by weight, more preferably 1 to 30% by weight in the aqueous solution. For the purpose of not increasing the activity of the protease, and since the enzyme is generally alive at 60 ° C. or higher, it is desirable to control the temperature to be lower than the temperature at the time of simple hot water extraction without adding the above protease, and to improve the purity and the like. The temperature is preferably 10 to 60 ° C, more preferably 25 to 50 ° C, and the time is preferably 1 to 50 hours, more preferably 2 to 10 hours, for the purpose of improving quality such as variation in molecular weight distribution.
[0044]
As described above, the extraction of hot-water-soluble protein is different from the extraction of acid-soluble protein in that the raw materials are immersed, solubilized and dissolved using hot water instead of the acid solution used for extraction. Or an extraction container and apparatus for acid-soluble proteins. In the case of dual use or common use, the storage tank for the acid solution supplied to the extraction vessel may be switched to a hot water supply tank, but it is preferable to separately provide a hot water supply tank or a hot water supply source in addition to the acid solution storage tank. It is desirable to keep.
[0045]
The ultrafiltration device used for purification and concentration of the extract obtained by immersing in hot water is, in terms of device specifications, an ultrafiltration membrane and a device used for purification and concentration of the above-mentioned acid-soluble protein gelatin and collagen peptide. Therefore, the equipment for purifying and concentrating the hot water soluble protein may be newly provided, or may be shared with or used as an ultrafiltration apparatus for purifying the acid soluble protein. These are determined on account of production and production costs.
[0046]
In this way, for example, the pretreated raw materials are divided into two groups, and one group of raw materials is first subjected to the extraction and purification of acid-soluble proteins, and the apparatus used is washed and washed. Then, using the insoluble residue separated and recovered from the acid extract and / or one other group of raw materials, a process for extracting and purifying hot water-soluble proteins can be performed. Above is convenient. Since the insoluble residue obtained by solid-liquid separation from the acid-soluble protein extract is relatively small, it is collected and stored at a low temperature, and when the insoluble residue for each production lot accumulates, it is immersed in hot water at once. It is of course conceivable to perform extraction, purification and concentration of the hot water soluble protein.
[0047]
FIG. 1 shows an example of an apparatus for purifying and concentrating an acid-soluble collagen solution of the present invention. In FIG. 1, a purification / concentration device 10 is provided with an ultrafiltration membrane in the ultrafiltration device 1, and the extract put in the circulating liquid supply tank 2 is sent to the ultrafiltration device 1 by a liquid sending pump 5. The permeated liquid 11 that has flowed in and is subjected to ultra-filtration by the application of the liquid sending resistance by the throttle 4 is recovered, and the collagen-containing solution that is not permeated passes through the ultrafiltration device 1 and is appropriately concentrated to become a circulating liquid 12, which becomes a circulating liquid 12. Circulated in the supply tank 2. The total amount of the circulating liquid in the circulating liquid supply tank 2 is detected by the level meter 6, and the weakly acidic solution 13 is supplied to the circulating liquid supply tank 2 from the weakly acidic solution addition tank 3 as necessary.
[0048]
As a standard of collagen purity, in the measurement of molecular weight distribution using an electrophoresis method, at least a band at a position corresponding to 300,000 molecular weight of γ chain is generally used as a criterion for determining collagen.
In terms of physical properties, the rotation angle (so-called specific rotation, scientific chronology, physical chemistry department, object 102, 1980) of the polarization plane when the NaD line passes through the optical rotatory substance is negative in the case of a collagen solution. Utilizing the fact that the angle in the negative direction becomes larger than that of the gelatin solution, the purity of the collagen is added to the molecular weight distribution, and the specific rotation may be evaluated. In the present invention, the specific rotation [α]²⁰ _DIs larger than -360 ° in the negative direction, as a guideline for collagen having high purity.
[0049]
The above purified, concentrated acid-soluble protein solution and hot water-soluble protein solution may be accompanied by sterilization and sterilization steps, depending on the product application and the necessity of storage, and also include liquid storage and dry storage. The form is taken as appropriate. Further, in the biochemical biotechnology field such as cell culture and the use of pharmaceutical capsules, allergen removal of purified animal proteins may be performed at the same time. In the case of food and medical use, it may be performed in a sterile room from the viewpoint of hygiene management.
As a disinfection and sterilization, filtration with a filter of about 0.2 μm is generally used, and there is also a method of irradiation with ultraviolet rays or γ rays for sterilization.
When stored in a liquid form, it is appropriately diluted with a dilute organic acid or dilute inorganic acid, and in the case of a collagen solution, it is usually stored at a concentration of about 0.005% to 1%. In cosmetics, glycerin may be added at a collagen concentration of about 0.1% to prevent association between γ chains and improve moisture retention.
Gelatin is generally stored dry, but when collagen is also required to be stored for a long period of time, a drying process may be performed with less risk of quality deterioration and bacterial growth. Drying methods include freeze drying and spray drying. In the spray-drying method, a method of pulverizing a solution having a solution concentration of about 10% is also known, and the production efficiency is better than freeze-drying if some measures are taken to avoid thermal denaturation, such as under reduced pressure steam. Further, a method of extruding the solution into a multi-row pellet shape or noodle noodle shape by using an extrusion method, increasing the surface area, and effectively drying the solution can be adopted.
[0050]
The final product can be used in many applications, such as pharmaceuticals, medical products, cosmetics, rinses, treatments, health foods, seasonings, and industrial applications, utilizing some of the properties of animal proteins. For example, it is known that it has a protective effect on gastrointestinal mucosa, a promoting effect on bone formation, a suppressing effect on blood pressure, a growth effect, an improving effect on immunity, etc. for pharmaceuticals and medical use, preventing gastric ulcer hemorrhage, preventing osteoporosis and hyperlipidemia. It can be used for medicines and health foods for such purposes. For cosmetics, the purpose is to promote the beauty of the skin by using the moisturizing effect. For some rinsing and treatment applications of cosmetics, foaming properties that use the high viscosity together with the water and moisturizing effects of the scalp and hair. Can be used for effects.
[0051]
【Example】
Hereinafter, the effects of the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
[0052]
Reference Example 1
As a raw material, 6.0 kg of shark rawhide (wet weight, water content 83%, fat / oil content 0.4%, ammonia 88 ppm (oil / fat content, ammonia dry weight ratio)) was kept warm at 5 ° C. in 12 kg of lime liquid having a pH of 11.5. For 3 weeks. The pH of the lime solution was measured daily using a pH meter (Castany LAB-F20 II manufactured by Horiba Seisakusho Co., Ltd.), and the pH was maintained at 11.5 by appropriately adding lime or water, and the solution was changed daily during the immersion period. After immersion, the shark skin was washed with water, frozen, and the skin was peeled off with a slicer (AC-300, manufactured by Tasaki Seisakusho Co., Ltd.) to remove 1.11 kg (water content 86.3%) of the shark skin dermis layer. Obtained.
[0053]
Reference Example 2
Using the same raw material and lime solution as in Reference Example 1, immersion was performed at the same temperature and for the same period to obtain 1.05 kg of a shark skin dermis layer (water content: 85.7%). During the immersion period, only the pH was adjusted, and unlike Reference Example 1, the solution was sealed and no liquid exchange was performed.
[0054]
Reference Example 3
The same raw material as in Reference Example 1 was used, and immersion was performed at the same temperature and for the same period using an acetic acid solution having a pH of 4.0 to obtain 2.12 kg of a dermis layer of shark skin. The pH was measured with a pH meter (manufactured by Horiba, Ltd., Castany LAB-F20 II), and the pH was maintained in the range of 3.9 to 4.1 by appropriately adding water or acetic acid. The solution was sealed and liquid exchange was not performed.
[0055]
The residual oil and fat and residual ammonia contained in the dermis layer obtained in Reference Examples 1 to 3 were measured, and the results are shown in Table 1.
The measurement of the amount of residual fats and oils was performed by measuring the weight of the hexane extract after the hot hydrochloric acid treatment (see Non-Patent Document 1). The amount of residual ammonia was measured by colorimetric determination of the solution obtained by extracting perchloric acid from the skin with perchloric acid by the method of Russel (Sugawara and Soejima, "Quantitative Determination of Proteins", Gakkai Shuppan Center, Tokyo, Japan). September 1971, pp. 51-52).
The oil and fat content of the alkali immersion in Reference Examples 1 and 2 was greater than the reduction effect of the acid immersion of Reference Example 3. In particular, when alkali immersion was performed while exchanging the liquid in Reference Example 1, the content decreased to 0.09% on the 21st day. Ammonia also decreased from 20.5 ppm to 2.9 ppm in Reference Example 1. A similar effect was observed in the acid immersion of Reference Example 3 (from 20.5 ppm to 3.1 ppm), indicating that long-term immersion also had a deodorizing effect, but was less effective in degreasing. The dry weight of the shark skin after the immersion treatment was 98% for the alkali immersion and 65% for the acid immersion, indicating that the shark skin was already dissolved in the immersion liquid.
It should be noted that it is sufficiently possible to add explosive gas by immersion in an alkali, and in this case, it was confirmed that the explosion could be performed in a number of days close to the lower limit of the scope of the claims.
[0056]
[Table 1]

[0057]
Example 1
Collagen was produced using a production process which is an example of the animal protein of the present invention illustrated in FIG. 2 (A).
That is, after the same raw material as in Reference Example 1 was immersed in an alkali, the skin of the shark skin was peeled off to obtain 1.11 kg of a dermal layer of the shark skin. The moisture content of the obtained dermis layer (1.11 kg (wet weight)) was 86.3%, the fat and oil content was reduced to 0.1%, and the ammonia was reduced to 2.2 ppm (all in dry weight ratio). . As a result, the residual fat and oil achieved the target (0.1% or less), and the ammonia residual almost approached the target (2 ppm or less).
The obtained dermis layer was pulverized with a meat chopper (# 22GM-D manufactured by Nippon Carrier Industry Co., Ltd.) into a maximum of about 2 mm square. Extraction was carried out using a 30 L stirred container, and 1.11 kg (wet weight) of pulverized material, 8.88 kg of 0.5 M acetic acid aqueous solution (8 times the weight of the dermis layer), pepsin (porcine gastric mucosa, Sigma) 1.52 g (1% based on the dry weight of the solid content of the dermis layer) was added and stirred at 4 ° C. at 120 rpm for 3 days to obtain 9.98 kg of an acid-soluble collagen extract. In addition, the dry weight of the solid content in the obtained extract is 98% when the dry weight of the dermis layer after the alkali immersion is 100% before the extraction operation, and the residual oil and fat contained in the solid content of the extract is 98%. Was 0.1% and ammonia was 1.4 ppm (all in dry weight ratio).
The obtained extract is so viscous that it is not suitable for measurement of insoluble residue, so 100 g is taken. To this extract, 500 g of 1 mM acetic acid is added to reduce the permeation resistance due to the high viscosity, and filter paper [Advantech Toyo ( No. 5A, two sheets overlapped], and the insoluble residue was filtered under reduced pressure. The insoluble residue was almost trace only. The pH was around 2.6.
[0058]
Similarly, 500 g of 1 mM acetic acid was added to 100 g of the extract, and 600 g of the diluted extract was subjected to ultrafiltration using the purification / concentration apparatus shown in FIG. The purification / concentration apparatus is equipped with an ultrafiltration apparatus equipped with Pellicon XL Biomax-30 (fraction molecular weight 30,000) manufactured by Millipore Co., Ltd. as a membrane module, and sends the liquid at a circulating liquid flow rate of 25 mL / min. The pump and the throttle 4 provided on the outlet side were adjusted, and the pump was operated at a permeate speed of 2 to 3 mL / min. Although not shown, the ultrafiltration apparatus includes a pH meter (for example, Castani LAB-F20II, manufactured by Horiba, Ltd.) and an electric conductivity meter (for example, manufactured by Horiba, Ltd.) (Castany LAB-DS).
Since about 100 mL of the circulating liquid is present in a tube or the like in the ultrafiltration device, the amount of the liquid in the circulating liquid supply tank was initially about 500 mL. For this reason, 1 mM acetic acid (pH 4.0) is appropriately added to the circulating liquid from the weakly acidic solution adding tank so that the amount of liquid in the circulating liquid supply tank is always 400 to 500 mL, and ultrafiltration is performed. Thereafter, the electric conductivity (measurement unit μS / cm) indicating the rise of the pH of the circulating fluid and the salt concentration²) No longer decreased. Thereafter, the ultrafiltration was continued until about 12 hours had elapsed. However, no change in the pH and electric conductivity of the circulating fluid was observed, and the purity of the collagen also reached the desired level. . After the ultrafiltration was completed, the dry weight of the solid content in the obtained circulating liquid was 99%, where the dry weight of the solid content in the extract before the ultrafiltration operation was 100%, and the ammonia concentration was 1. It decreased from 4 ppm to 0.8 ppm (dry weight ratio).
The obtained circulating liquid was frozen at −80 ° C. and freeze-dried at 10 Pa and 20 ° C. for 12 hours to obtain 107 g of dried collagen (water content: 0.1%). The yield was 70.0% based on the dry weight of the dermis layer after alkali immersion.
[0059]
The dried collagen obtained in this example was redissolved in 1 mM acetic acid, and the molecular weight distribution was examined by electrophoresis. As a result, it was confirmed that the collagen contained a large amount of γ chains. The instrument used was electrophoresed on a gel having an acrylamide gel concentration of 7.5% using Rapidos Minislab manufactured by ATTO Corporation.
The specific rotation of the obtained dried collagen was measured. A 0.1% solution of dried collagen diluted with 0.005 M acetic acid was dissolved by stirring with a stirrer at 4 ° C. for 12 hours, and centrifuged at 18,000 rpm (gravity acceleration 36,000 g) at 4 ° C. for about 40 minutes (Hitachi, Japan). The supernatant was subjected to a high-speed cooling centrifuge (manufactured by Seisakusho Co., Ltd., 2 OPR-520), and the specific rotation was measured for 10 mL of the supernatant (SEPA-200 manufactured by Horiba, Ltd., use conditions: 20 ° C.). The value of specific rotation is considered to be a measure of collagen purity [α]²⁰ _DIt was between -360 ° and -420 °.
[0060]
In addition, also in the measurement of the specific rotation with a time-temperature linear gradient of changing 5 ° C. per 10 minutes, from 5 ° C. to 30 ° C., it was approximately −370 °, but it dropped sharply from around 30 ° C. , At 35 ° C., the value was around −150 °. Even if the temperature is lowered again below 40 ° C., the specific rotation [α] at 20 ° C.²⁰ _DDid not return to -360 °, remained at -150 °, and remained approximately the same as the measured value for gelatin which was forcibly heated to 60 ° C in a hot water bath. It is considered that gelatin was already denatured by heat denaturation.
The residual collagen, fat, and ammonia contents of the obtained dried collagen were 0.1%, 0.1%, and 0.5 ppm, respectively. Trimethylamine was not detected. Of the samples was determined to be odorless.
The amount of salt was measured as ash obtained as a residue on ignition (see Non-Patent Document 1). The amount of trimethylamine was measured colorimetrically by the picrate method on the solution obtained by extracting perchloric acid from the skin (edited by the Japan Society of Food Industry, Food Analysis Method Editing Committee, “Food Analysis Method”, Kourin) , Tokyo, October 1982, pp. 673-681).
The odor sensory evaluation was performed according to the following evaluation criteria using 20 panelists.
○: Odorless
△: Slight odor
×: Odor
[0061]
Example 2
Using the extract of the acid-soluble protein obtained under the same conditions as in Example 1 but under an acid solution extraction condition of 0.5 M acetic acid 8 times, as a purification and concentration method, in place of the ultrafiltration of Example 1, Salting out and dialysis were also performed. Sodium chloride and acetic acid were added to 100 g of the obtained extract at a concentration of 1 mol / L and 0.5 mol / L, respectively, and the precipitate was deposited at 18,000 rpm (gravity acceleration 36,000 g) at 4 ° C. The mixture was centrifuged and separated into solid and liquid. The separated precipitate was redissolved in 1 mM acetic acid, dialyzed against 20 volumes of 1 mM acetic acid, and desalted. It took 3 hours for the sodium chloride concentration of the dialysate to show a constant value, and each time the dialysate was replaced, and the dialysate was changed four times. Therefore, the time required for one dialysis operation is 12 hours. To the collagen solution after completion of the dialysis, sodium chloride is again added to 1 M in the same manner as described above, precipitation by salting out, separation of solids by centrifugation, re-dissolution of solids, dialysis and desalting, The operation of analysis and dialysis was repeated five times to complete the purification. Therefore, the net required time for salting out and dialysis was 12 × 5 = 60 hours (about 2.5 days).
In the method by salting out and dialysis of this example, it takes more days for purification and concentration than the ultrafiltration of Example 1, but it can be confirmed that a collagen solution having relatively high purity can be obtained. Was. In addition, the collagen yield obtained was about 50% based on the dry weight of the dermis layer before the introduction of the extraction treatment.
In addition, a case where an extract was obtained under an extraction condition of a 20-fold amount of an acid solution of a conventionally known example, and a purification operation was performed by salting out and dialysis is shown in Comparative Example 3 described later. The use of the extract obtained under the acid extraction conditions of 8 times the amount of 0.5 M acetic acid solution of this example shows that a relatively high-purity collagen solution can be purified even if the number of times of salting out and dialysis is small. ing.
[0062]
Example 3
In the same manner as in Examples 1 and 2, shark rawhide was immersed in alkali and the epidermis was peeled off to obtain 1.05 kg of a dermis layer. The moisture content of the dermis layer was 87%, the fat content was 0.1%, and the ammonia was 2.5 ppm.
The obtained dermis layer was ground to a maximum length of about 2 mm with a meat chopper (# 22GM-D manufactured by Nippon Carrier Industry). Using a 30 L stirred container, 21 kg of a 0.5 M acetic acid aqueous solution (20 times the weight of the dermis layer), pepsin (derived from porcine gastric mucosa, manufactured by Sigma) were added to 1.05 kg (wet weight) of the pulverized material. .37 g (1% based on the weight of the solid content of the dermis layer) was added, and the mixture was stirred at 4 ° C. at 120 rpm for 3 days to obtain 22.0 kg of an extract. The dry weight of the solid content in the obtained extract is 97% when the dry weight of the dermis layer after alkali immersion before the extraction operation is 100%, the fat and oil content is 0.1%, and the ammonia is 1.6 ppm. (All in dry weight ratio).
Unlike Example 1, the insoluble residue was directly filtered without diluting with 1 mM acetic acid to obtain 22.0 kg of an extract. The pH was around 2.5. The insoluble residue was a trace. Ultrafiltration was performed under the same apparatus and under the same conditions as in Example 1. Changes in the pH and electrical conductivity of the circulating fluid were no longer observed, and the time required for the collagen purity to reach the desired level was 40 hours (three times or more that of Example 1). This is because the 0.5 M acetic acid aqueous solution was increased to 20 times the weight of the dermis layer in comparison with Example 1 in the extraction operation, so that the molecular weight distribution in the extract was uneven, that is, the collagen purity was reduced. Therefore, since the collagen concentration in the circulating fluid is low, it is considered that the time required for purification and concentration on the circulating fluid side by an ultrafiltration operation until the desired level is reached is prolonged. After the ultrafiltration, the dry weight of the solid content in the obtained circulating liquid is 75%, where the dry weight of the solid content in the extract before the ultrafiltration operation is 100%, and the ammonia concentration is 1. It decreased from 6 ppm to 0.9 ppm (dry weight ratio).
[0063]
The obtained circulating liquid was freeze-dried in the same manner as in Example 1 to obtain 0.372 g of dried collagen (water content: 0.1%). The yield based on the dry weight of the dermis layer after alkali immersion was 60%.
When the obtained dried collagen was redissolved in 1 mM acetic acid and the molecular weight distribution was examined by electrophoresis, α-chain, β-chain, and γ-chain were confirmed, but compared with the collagen obtained in Example 1, The band of the α chain was strong, while the band of the γ chain was weak. The specific rotation is [α]²⁰ _D-280, indicating a low collagen purity. It is considered that the reason why the purity was low was not only the solubilization of the γ chain but also the decomposition of the γ chain into a low-molecular α chain in the extraction step. It is considered that the reason why the decomposition of the γ chain was promoted was that the acid solution subjected to the extraction was 20 times the weight of the shark dermis layer, which was much larger than that of the shark dermis layer.
[0064]
Comparative Example 1
For comparison, a method for producing shark skin collagen according to an example of a conventional process illustrated in FIG. 2B is shown.
First, 4 kg (95.26% water) of the dermis layer in which the skin of shark skin was peeled off and subjected to swelling treatment to increase the water content so that collagen was easily solubilized was used.
4 kg (dry weight: 189.6 g) of this dermis layer was pulverized by a meat chopper in the same manner as described above, and a 1% aqueous solution of a surfactant (PRODUCT A, manufactured by Bayer) was added for the purpose of degreasing, followed by 12 hours at 4 ° C. Stirred. After the completion of defatting, the dermis layer was precipitated by centrifugation at 8,000 rotations (gravity acceleration: 13,000 g) for 30 minutes, and the surfactant solution contained in the supernatant was removed. Further, about 2 times by weight (about 8 kg) of water was added to the collected dermis layer, stirred, centrifuged under the same conditions as above, and washed with water. The weight of the dermis layer after washing with water was 3.96 kg (water content 93.46%, dry weight 258.9 g). The recovery rate for the dermis layer used as the raw material was 137% on a dry weight basis, which resulted in an apparent increase. This is probably because the water washing after degreasing was insufficient and the surfactant could not be completely removed.
[0065]
To 3.96 kg of the dermis layer after the degreased water washing operation, 7.93 kg of 0.5 M acetic acid aqueous solution (2 times the weight of the dermis layer), and 1.9 g of pepsin (derived from pig gastric mucosa, manufactured by Sigma) (dermis layer solid) (1% of the weight per minute) and stirred at 4 ° C. for 120 rotations / minute for 3 days to perform solubilization and extraction of collagen. Thereafter, the insoluble residue was removed by centrifugation at 8,000 rotations (gravity acceleration 13,000 g) for 30 minutes to obtain 10.2 kg of an extract. The insoluble residue was a trace.
[0066]
Sodium chloride and acetic acid were added to 10.2 kg of the obtained extract at 1 mol / L and 0.5 mol / L, respectively, and the deposited precipitate was rotated at 8,000 rpm (gravity acceleration 13,000 g) for 30 minutes. For centrifugation. The separated precipitate was redissolved in 1 mM acetic acid, dialyzed against 20 volumes of 1 mM acetic acid, and desalted. It took 3 hours for the sodium chloride concentration of the dialysate to show a constant value, and each time the dialysate was replaced, and the dialysate was changed four times. Therefore, the time required for one dialysis operation is 12 hours. To the collagen solution after completion of the dialysis, sodium chloride is again added to 1 M in the same manner as described above, and precipitation by salting out, separation of solids by centrifugation, re-dissolution of solids, dialysis and desalting, The operation of analysis and dialysis was repeated five times to complete the purification. Therefore, the net required time for salting out and dialysis was 12 × 5 = 60 hours.
After that, it was freeze-dried in the same manner as in Example 1 to obtain 108.8 g of dried collagen (water content: 12.12%). The yield was 50.42% based on the dry weight of the dermis layer after alkali immersion, but the yield was 49.18% when the mixed salt and oil were removed. Concentrations of residual fat, salt, and ammonia are as high as 0.47%, 2.0%, and 3 ppm, respectively, and the specific optical rotation of a 0.1% solution (in 0.005 M acetic acid) is [α].²⁰ _DThe collagen purity was as low as -256 °.
[0067]
Comparative Example 2
Pretreatment and extraction were performed in the same manner as in Example 32, and 100 g of the obtained extract was subjected to salting out and dialysis in the same manner as in Comparative Example 1 to desalinate. The dialysis was performed for 12 hours while exchanging the external solution four times. Further, the operations of salting out and dialysis were repeated five times to complete the purification.
Then, it freeze-dried similarly to Example 1, and obtained 0.299 g (0.1% of water) of dry collagen. The yield was 48.20% based on the dry weight of the shark skin dermis layer after alkali immersion. The concentrations of the residual fat, salt, and ammonia are 0.1%, 2.0%, and 1.5 ppm, respectively, and the specific rotation of a 1% solution (in 0.005 M acetic acid) is also [α].²⁰ _D-250 °.
[0068]
Comparative Example 3
The same treatment as in Example 3 was carried out, and the obtained extract was subjected to purification by salting out and dialysis in the same manner as in Comparative Example 1, but was twice as large as Comparative Examples 1 and 2. Performed twice.
The obtained dried collagen was redissolved in 1 mM acetic acid, and the molecular weight distribution was examined by electrophoresis. As a result, α chain, β chain, and γ chain were confirmed. The α chain band was weaker than that of the collagen obtained in Comparative Example 2, and the γ chain band was as intense as in Example 1.
The specific rotation of the obtained dried collagen is [α]²⁰ _D-360 °, the desired level of high purity collagen. At this time, the concentrations of the residual fats, oils, salts, and ammonia also decreased to 0.1%, 0.1%, and 0.5 ppm, respectively, but the yield decreased to 30.0%.
[0069]
The collagen obtained in Example 3 is superior to Comparative Example 1 in that the residual fat and oil content is low, but this is largely due to the effect of performing the pretreatment step by alkali immersion instead of degreasing with a surfactant. In Example 32, ultrafiltration was used in place of salting out and dialysis in the purification / concentration step, so that the original salt was low in salt content and the residual salt content could be reduced. However, collagen purity indicated by specific rotation, electrophoresis, etc. Is lower than that of Example 1 in which purification was performed by the same ultrafiltration method. This is considered to be because the weight ratio of the acid solution in the extraction step was excessive, that is, 20 times. In addition, when purification was performed by salting out and dialysis in Comparative Examples 2 and 3, in order to bring the γ-chain band of the molecular weight distribution and the specific rotation to a desired level of high-purity collagen, 10 times. The above salting out and dialysis were required.
Table 2 shows various properties of the collagens obtained in Examples 1 to 3 and Comparative Examples 1 to 3.
[0070]
Example 4
Using shark rawhide as a raw material, the temperature and time for peeling the epidermis and obtaining a dermis layer were examined. 200 g of rawhide (wet weight, water content 83%) was poured into about 5 L of warm water set to the temperature shown in Table 3 and immersed for the time shown in Table 3. After immersion, the epidermis was manually peeled off using a finger and a kitchen knife, and the dermis layer was collected. The obtained dermis layer was dried at 110 ° C., and the dry weight was measured.
The immersion at 30 ° C. × 1 minute or less did not show any difference from the properties before immersion. As the immersion temperature and time increased, the recovery of the dermis layer improved. In particular, by immersion at 40 ° C. × 5 minutes or 50 ° C. × 3 minutes, many parts could be peeled off with fingers. When the immersion was performed at 60 ° C. for 5 minutes, the collagen was denatured into gelatin and dissolved in warm water, so that the recovery rate of the dermis layer was reduced.
[0071]
Example 5
Using the same shark rawhide as the raw material used in Example 4, the effects of immersion time and temperature in the alkali treatment step were examined. 200 g of rawhide (wet weight, water content 83%) was immersed in 1 L of a saturated solution of lime (pH 11.5) at the temperature and days shown in Table 4 to remove oil and fat contained in the dermis layer from which the epidermis was removed. The ammonia content was measured.
Table 4 shows the results.
When immersion is performed at 5 ° C., after 14 days, the fat and oil content reaches 0.2% or less and ammonia 10 ppm or less, which are the levels desired in the process before extraction and purification, and the oil and fat content 0.09% after 21 days. , Ammonia to 2.9 ppm. When the immersion temperature was changed, it took 8 days at 20 ° C., 2 days at 30 ° C. and 1 day at 40 ° C. to reach the desired level. If the temperature is high, fats and oils and ammonia can be removed in a short period of time, but in order to prevent the thermal denaturation of collagen, it is desirable to perform the process at as low a temperature as possible.
[0072]
Example 6
Using the shark skin dermis layer obtained in Reference Example 1 as a raw material, heat extraction was performed at a high temperature for the purpose of shortening the extraction days.
At the beaker test level, when using a finer raw material obtained by minced pulverization, add 9 mL of 0.5 mol / L acetic acid aqueous solution to 1 g of dermis layer (wet weight, water content: 86.3%). After heating at 100 ° C. for 1 minute, 5 minutes, 10 minutes and 20 minutes, extraction and purification were performed in the same manner as in Example 1, and the molecular weight distribution was examined by electrophoresis. However, since it is a small material, a shaking multi-shaker (MMS manufactured by Tokyo Rika Instruments) was used for the immersion treatment for extraction. When heated for 1 to 5 minutes, γ chains were observed at substantially the same ratio as in Example 1. However, when heated for 10 minutes, the band of γ chains became thinner, and when heated for 20 minutes, the band weight of electrophoresis showed a molecular weight of 50. In most cases, no γ chain was observed, and almost no α chain band having a molecular weight of 100,000 was observed. From this, it can be seen that if the heating is suppressed to 100 ° C. for about 1 to 10 minutes, the dissociation of the γ chain and the denaturation to gelatin are within an acceptable range.
Further, in a test using 1.0 kg of the shark skin dermis layer obtained in Reference Example 1 as a raw material, 8.0 kg of a 0.5 M acetic acid aqueous solution and 1.37 g of pepsin were added in the same manner as in the method shown in Example 1. Extraction was performed at 120 ° C. at a stirring speed of 120 revolutions / minute. However, the number of days for extraction was reduced to half of 1.5 days, and thereafter, a short-time heat extraction at 100 ° C. for 5 minutes was added. Thereafter, the residue was filtered and separated, purified by ultrafiltration, and freeze-dried in the same manner as in Example 1 to obtain 96 g of dry collagen (water content: 0.1%). The obtained collagen contained approximately the same amount of γ chain as the collagen obtained in Example 1, and both the specific rotation, the residual oil and fat, and the residual ammonia reached the desired level of high-purity collagen. . By adding this short-time heating step, the extraction step that required three days in Example 1 was reduced to half.
[0073]
[Table 2]

[0074]
* 1: Based on the dry weight of the swelled, non-skinned dermis
* 2: 0.1% solution (in 5 mM acetic acid)
[0075]
[Table 3]

[0076]
[Table 4]

[0077]
【The invention's effect】
According to the present invention, it is possible to provide a production method and a production apparatus capable of separating and recovering animal proteins such as collagen, gelatin, and collagen peptides having high quality and purity from animal body tissues at a low cost with a high yield and in a large amount. it can. Further, the animal protein obtained by the present invention can be used as a material suitable for pharmaceuticals, cosmetic liquids, rinses, treatments, industrial uses, health foods, seasonings, and the like.
[Brief description of the drawings]
FIG. 1 is a schematic view of the configuration of an example of the purification / concentration device of the present invention.
FIG. 2 (A) is an example of an overall process diagram showing an example of a method for producing an animal protein of the present invention, and FIG. 2 (B) is an example of an overall process diagram showing an example of a method for producing an animal protein in a conventional process. is there.
[Explanation of symbols]
1: Ultrafiltration device
2: Circulating fluid supply tank
3: Weak acid solution replenishment tank
4: Aperture
5: Liquid pump
6: Level meter
10: Purification / concentration equipment
11: Flow of permeate
12: Flow of circulating fluid
13: Flow of weakly acidic liquid

Claims (25)

Using a tissue containing animal protein and / or an animal body tissue consisting of animal skin as a raw material, a pretreatment step including degreasing and deodorizing treatment of the raw material, and immersing the pretreated raw material in an acid solution or an acid and acid protease solution An animal protein comprising an extraction step of solubilizing and dissolving the acid-soluble protein to obtain an extract containing the acid-soluble protein, and a purification / concentration step of purifying and concentrating the acid-soluble protein from the extract. In the production method, the relationship between the raw material and the solution to be subjected to the extraction step is such that the weight ratio of the acid solution or the acid and the acidic protease solution to the raw material is 1.5 to 10 times, and the concentration of the acid is 0.3 M to 2 M. A method for producing an animal protein. The temperature of the pretreatment step, the extraction step and the purification / concentration step is maintained at 0 to 40 ° C. for the terrestrial animal and 0 to 30 ° C. for the aquatic animal, and the extraction step and / or purification / concentration. The method for producing an animal protein according to claim 1, wherein the temperature of the step is kept at least 5 ° C lower than the temperature of the pretreatment step. The purification / concentration step comprises a purification / concentration method using an ultrafiltration membrane, and the extract is put into an ultrafiltration apparatus, and a residual liquid containing an acid-soluble protein that does not pass through the ultrafiltration membrane is removed. The method for producing an animal protein according to claim 1 or 2, wherein the animal protein is concentrated, circulated as a circulating solution, and then subjected to ultrafiltration again. In the purification / concentration step, there is provided a total amount detecting means for detecting a total amount of the circulating liquid, and a weakly acidic solution adding means for adding a weakly acidic solution having a pH closer to neutral than the solution used in the extracting step to the circulating liquid. The method for producing an animal protein according to claim 3, wherein the total amount of the circulating fluid is maintained and controlled intermittently or continuously within a predetermined range. In the purification / concentration step, a group of the concentration of the acid-soluble protein contained in the circulating fluid, the purity of the acid-soluble protein, the viscosity of the circulating fluid, the density of the circulating fluid, the acidity of the circulating fluid, and the electric conductivity of the circulating fluid 5. The method according to claim 3, further comprising a means for continuously or intermittently detecting at least one selected from the group consisting of: and a means for controlling circulation of a circulating fluid, and controlling purification and concentration of the acid-soluble protein. A method for producing the animal protein according to the above. In the purification / concentration step, the ultrafiltration membrane has a plurality of ultrafiltration devices having the same or different molecular weight cutoffs, and the circulating liquids of the ultrafiltration can communicate with each other. Item 6. The method for producing an animal protein according to any one of items 5. In the purification / concentration step, an ultrafiltration membrane having a molecular weight cut off of 30,000 to 100,000 is used for ultrafiltration, and collagen and / or permeate of the ultrafiltration are removed from the circulating liquid of the ultrafiltration. The method for producing an animal protein according to any one of claims 3 to 6, wherein a gelatin or collagen peptide is obtained more. In the above purification / concentration step, an ultrafiltration membrane having a molecular weight cutoff of 5,000 to 30,000 is used for ultrafiltration, and collagen or gelatin and / or The method for producing an animal protein according to any one of claims 3 to 6, wherein a collagen peptide is obtained from the permeate. In the above-mentioned purification / concentration step, ultrafiltration having different molecular weight cutoffs of the ultrafiltration membrane is constituted in two stages, and the first stage is provided with an ultrafiltration membrane having a molecular weight cutoff of 30,000 to 100,000, The second stage is equipped with an ultrafiltration membrane having a molecular weight cut-off of 5,000 to 30,000, and the permeate obtained in the first stage ultrafiltration is intermittently or circulated into the circulating fluid of the second stage ultrafiltration. 7. The method according to any one of claims 3 to 6, wherein the collagen is continuously fed to obtain collagen from the first-stage circulating fluid, gelatin from the second-stage circulating fluid, and / or collagen peptide from the second-stage permeate. 2. The method for producing an animal protein according to claim 1. The method for producing an animal protein according to any one of claims 1 to 9, wherein in the extraction step, the temperature for immersion in an acid solution or an acid and an acid protease solution is 50 to 150 ° C and heating is performed for 1 to 10 minutes. In the extraction step, the insoluble residue remaining without being solubilized and dissolved in the extract and / or the raw material subjected to the pretreatment including the degreasing / deodorizing treatment is immersed in warm water to remove the warm water soluble protein. An extract is obtained by solubilization and dissolution, and the extract is introduced into an ultrafiltration apparatus comprising an ultrafiltration membrane having a molecular weight cut-off of 5,000 to 30,000, and contains warm water soluble protein which does not pass through the ultrafiltration membrane. The residual liquid was concentrated and circulated as a circulating liquid, and again subjected to ultrafiltration to obtain gelatin from the circulating liquid, and / or a collagen peptide from the permeated liquid. A method for producing an animal protein according to any one of claims 1 to 10. The method for producing an animal protein according to claim 11, wherein the temperature of the hot water immersion is 50 to 100C for raw materials derived from terrestrial animals, and 40 to 100C for raw materials derived from aquatic animals. In the pretreatment step, when the animal body tissue is an animal skin, the animal skin is immersed in warm water at a temperature range of 40 to 70 ° C for terrestrial origin and 30 to 60 ° C for aquatic origin for 1 to 10 minutes. The method for producing an animal protein according to any one of claims 1 to 12, further comprising a peeling step of peeling the epidermis to obtain a dermis layer. In the pretreatment step, the body tissue of the animal or the dermis layer is used as a raw material, and the material is immersed in an alkaline solution. The method for producing an animal protein according to any one of claims 1 to 13, wherein the minimum immersion time is twice or more each time the temperature is lowered by 10 ° C based on the minimum immersion time of 1 day at 40 ° C. A pretreatment device for performing pretreatment including degreasing and deodorizing treatment of a raw material using a tissue containing animal protein and / or an animal body tissue composed of animal skin, and converting the raw material into an acid solution or an acid and acid protease solution An animal protein comprising an extraction device for solubilizing and dissolving the acid-soluble protein by immersion to obtain an extract containing the acid-soluble protein, and a purification / concentration device for purifying and concentrating the acid-soluble protein from the extract. In the manufacturing apparatus, the relationship between the raw material and the solution to be subjected to the extraction step is adjusted such that the weight ratio of the acid solution or the acid and the acidic protease solution to the raw material is 1.5 to 10 times, and the acid concentration is in the range of 0.3 M to 2 M. An apparatus for producing an animal protein, comprising: The purification / concentration device includes a method for purification and concentration using an ultrafiltration membrane, and the extract is charged into an ultrafiltration device, and contains an acid-soluble protein that does not pass through the ultrafiltration membrane. The apparatus for producing an animal protein according to claim 15, wherein the residual liquid is concentrated, circulated as a circulating liquid, and subjected to ultrafiltration again. In the above-mentioned purification / concentration apparatus, a total amount detecting means for detecting the total amount of the circulating liquid of the ultrafiltration, and a weakly acidic solution in which a weakly acidic solution whose pH is closer to neutral than the acid solution used in the extraction step is added to the circulating liquid. 17. The animal protein production apparatus according to claim 16, further comprising a solution adding means, wherein the total amount of the circulating fluid is maintained and controlled intermittently or continuously within a predetermined range. In the above-mentioned purification / concentration apparatus, a group of the concentration of the acid-soluble protein contained in the circulating fluid, the purity of the acid-soluble protein, the viscosity of the circulating fluid, the density of the circulating fluid, the acidity of the circulating fluid, and the electric conductivity of the circulating fluid. 18. The animal protein production device according to claim 16, further comprising means for detecting at least one selected from the group consisting of: and a circulating fluid circulation controlling means for controlling the concentration of the acid-soluble protein in the circulating fluid. The purification / concentration device, wherein the ultrafiltration membrane has a plurality of ultrafiltration devices having the same or different molecular weight cutoffs, and the plurality of ultrafiltration circulating liquids can communicate with each other. Item 18. An apparatus for producing an animal protein according to any one of Items 18. In the extraction device, the insoluble residue remaining without being dissolved and dissolved in the extract and / or the raw material subjected to the pretreatment including the degreasing / deodorizing treatment is immersed in warm water to remove the warm water soluble protein. A hot water extraction device for solubilizing and dissolving to obtain an extract, and the extract is put into an ultrafiltration device comprising an ultrafiltration membrane having a molecular weight cutoff of 5,000 to 30,000, and hot water not passing through the ultrafiltration membrane. A purification / concentration apparatus comprising a purification / concentration device for concentrating the residual liquid containing the soluble protein, circulating it as a circulating liquid, ultrafiltrating again, and obtaining gelatin from the circulating liquid and / or collagen peptide from the permeated liquid. Claims: A protein production device is additionally provided, and / or an extraction device and an ultrafiltration device used for production of the acid-soluble protein are used and commonly used for production of hot water-soluble protein. Animal protein production apparatus according to any one of 16 claims 19. The animal protein production device according to any one of claims 15 to 20, further comprising a skin peeling device that peels an animal epidermis to separate a dermis layer. 15. At least one animal protein selected from the group consisting of collagen, gelatin, and collagen peptide obtained by the method for producing an animal protein according to any one of claims 1 to 14. A cosmetic comprising at least one animal protein selected from the group consisting of collagen, gelatin and collagen peptide according to claim 22. A medicament comprising at least one animal protein selected from the group consisting of collagen, gelatin and collagen peptide according to claim 22. A health food comprising at least one animal protein selected from the group consisting of collagen, gelatin and collagen peptide according to claim 22.

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