JP5410612B2 - Squid viscera extract and its preparation method, mixture and use as marine fishery feed protein source - Google Patents
Squid viscera extract and its preparation method, mixture and use as marine fishery feed protein source Download PDFInfo
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Description
本発明は新規オリゴペプチドに関し、具体的には、イカ内臓から抽出したオリゴペプチド及びその調製方法、さらに、この物質の混合物及び該混合物の海洋水産養殖における飼料タンパク源としての使用に関する。 The present invention relates to novel oligopeptides, and more particularly to oligopeptides extracted from squid viscera and methods for their preparation, as well as mixtures of these substances and their use as feed protein sources in marine aquaculture.
我が国の遠洋漁業の迅速な発展に伴い、イカの漁獲量は年々増加し、２００９年のイカ漁獲量は３０万トンを超え、我が国の主要な水産加工原料になっている。しかし我が国のイカ加工の技術水準は低く、加工処理で松かさイカ、干物、イカ塩辛、缶詰及び調味料などの製品を得た後、一般に２０％〜３０％の廃棄物（内臓、頭、足、表皮、イカスミなど）が生じるが、これら廃棄物はタンパク質、脂肪、多糖などの栄養物質を豊富に含んでおり、珠海市出入国検験検疫局の呉莉敏の研究（非特許文献１）によると、イカ内臓１００ｇ当たりには、脂肪２１．１５ｇ、タンパク質２１．２４ｇ、カルシウム５１．４６ｍｇ、鉄６０９．０７μｇ、リン９５．８８μｇなどが含まれている。ただしこれらの物質は極めて腐乱変質しやすい上に人体に有害な重金属カドミウムを含んでいる。通常はこれら廃棄物の処理方法は埋めてしまうことであるが、これは漁業資源の大きな浪費であるだけでなく、環境汚染の問題もはらんでいる。従って、イカ廃棄物を如何に効果的に処理しその価値を高め総合的に利用するかが、国民と社会にますます重視されるようになってきている。 With the rapid development of Japan's pelagic fishery, squid catches have increased year by year, and the squid catch in 2009 has exceeded 300,000 tons, making it the main fishery processing material in Japan. However, the technical level of squid processing in Japan is low, and after obtaining processed products such as pine squid, dried fish, squid salty, canned foods and seasonings, generally 20% to 30% of waste (internal organs, heads, feet, However, these wastes are rich in nutrients such as proteins, fats and polysaccharides, according to Zhuhai City Immigration and Quarantine Bureau's research (Non-patent Document 1). Each 100 g of squid viscera contains 21.15 g of fat, 21.24 g of protein, 51.46 mg of calcium, 609.07 μg of iron, 95.88 μg of phosphorus and the like. However, these substances are extremely susceptible to decay and alteration, and contain heavy metal cadmium which is harmful to the human body. Usually, these waste disposal methods are buried, but this is not only a huge waste of fishery resources, but also the problem of environmental pollution. Therefore, how to effectively treat squid waste, increase its value and use it comprehensively has become more important to the people and society.
中国水産科学院黄海水産研究所の王彩理ら（非特許文献２）は、バナメイエビに対するイカ内臓液化タンパクの摂餌誘引性について研究し、イカ内臓スラリーの摂餌誘引効果が最も高く、ベタイン、タウリン、アリシンより強いことを発見した。上海水産品加工技術開発センターの王建中ら（非特許文献３）は、イカ内臓の総合利用研究において、イカ内臓とその他の廃棄物を利用し自己酵素による加水分解を行うことで、イカ油や内臓加水分解液を開発すると共に、加水分解液からカドミウムを除去するという難題も解決した。中国海洋大学水生生物製品安全性実験室の劉春娥ら（非特許文献４）は、イカ内臓タンパク質の酵素分解技術の研究において、アミノ酸収率を主な指標とし、様々なプロテアーゼを用いてイカ内臓タンパク質の加水分解技術研究を行い、中性プロテアーゼ、アルカリプロテアーゼ、トリプシン、パパインには良好な分解効果があり、アミノ酸転換率は５０％以上に達することを示した。浙江工商大学の励建栄らの特許であるアメリカオオアカイカ内臓の一種の総合利用技術（中国特許出願第２００６１００５３３７２．６号）では、イカ内臓を蒸煮、酵素液化、加熱、遠心分離するなどの手順により得られた下層イカポリペプチドから、水産動物飼料タンパク源に用いるイカペーストを作製し、クルマエビへの食餌試験を行うことにより、イカペーストを添加すると摂餌量が明らかに増加し、幼エビの生存率と体重とが高まることが示されている。福建融▲しん▼海生物技術有限公司の蘇祖鳳の特許であるイカ内臓を用いた水産飼料摂餌誘引物質の生産方法（中国特許出願第２００８１００７１０７３．４号）では、イカ内臓を蒸煮、静置、自然酵素分解発酵することにより、油分を分離した後の沈殿に８０〜９０℃と６０〜６５℃との二回、低温真空濃縮を行っている。 Wang Ayari et al. (Non-patent Document 2) of the Institute of Fisheries Science of China (Non-Patent Document 2) studied the feeding attraction of squid visceral liquefied protein to lobster shrimp. I found something stronger. In the comprehensive research on the use of squid internal organs, Wang Jianchu et al. (Non-patent Document 3) of the Kamikasui Products Processing Technology Development Center conducted hydrolysis using squid internal organs and other wastes by self-enzyme, Along with the development of a hydrolyzate, the challenge of removing cadmium from the hydrolyzate was solved. Liu Chun, et al. (Non-patent Document 4) of the Aquatic Product Safety Laboratory at China University of Marine Science and Technology (Non-Patent Document 4) used a variety of proteases to study squid visceral protein using the amino acid yield as the main indicator in the study of enzymatic degradation of squid visceral protein. As a result, the neutral protease, alkaline protease, trypsin, and papain have a good degradation effect, and the amino acid conversion rate reaches 50% or more. In Zhejiang University of Commerce and Industry's patent, Zhujiang Sakae et al., Which is a kind of integrated utilization technology of American giant squid viscera (Chinese patent application No. 200610053372.6), procedures for steaming, liquefaction, liquefaction, heating and centrifugation of squid viscera From the lower squid polypeptide obtained by the above, the squid paste to be used as a fish animal feed protein source was prepared, and the diet test on the prawns was carried out. It has been shown that survival and body weight increase. In the method of producing an aquatic feed feeding attractant using Chinese squid organs (Chinese patent application No. 20080081073.4), which is the patent of Soh Soebo of Fujian Yin Shin Biological Technology Co., Ltd. By performing natural enzymatic decomposition fermentation, low-temperature vacuum concentration is performed twice at 80 to 90 ° C. and 60 to 65 ° C. for the precipitate after separating the oil.
既存の飼料タンパク源の製造技術は主に、酵素分解後に高温蒸煮、濃縮する方法が取られているが、こうした工程では、タンパク質が変性しやすく、コラーゲンが生成され、栄養成分が低下するばかりか、水産動物に対する製品の摂餌誘引性が大きく損なわれてしまう。蘇祖鳳らの方法を採用し、ＳＪＮ２−２０００複効蒸発器を利用して酵素分解液に二次濃縮を行い、蒸発温度を９０℃以下に制御し、濃縮温度を大きく下げたとしても、やはりこの難題を根本から解決することはできない。 Existing feed protein source production techniques mainly involve high-temperature steaming and concentration after enzymatic degradation, but in these processes, protein is easily denatured, collagen is produced, and nutritional components are reduced. In addition, the attractiveness of the product to aquatic animals is greatly impaired. Even if the method of Sozoen et al. Is used, the SJN2-2000 double-effect evaporator is used to perform secondary concentration on the enzyme decomposition solution, the evaporation temperature is controlled to 90 ° C or lower, and the concentration temperature is greatly reduced. The difficult problem cannot be solved fundamentally.
本発明の目的は、高温蒸煮方法を放棄し、限外ろ過膜（ＵＦ膜）を利用してイカ内臓から新規オリゴペプチドを抽出することであり、もう一つの目的は、このオリゴペプチドの調製方法を開発すること、さらにこのオリゴペプチドを含む混合物とその使用法を開発することである。 The object of the present invention is to abandon the high-temperature steaming method and extract a novel oligopeptide from the squid viscera using an ultrafiltration membrane (UF membrane), and another object is a method for preparing this oligopeptide And to develop a mixture containing this oligopeptide and its use.
我々は、スルメイカ（Ｔｏｄａｒｏｄｅｓ ｐａｃｉｆｉｃｕｓ）内臓を有機溶剤で脱脂した後、アルカラーゼ（登録商標）とトリプシンで酵素分解し、その後、限外ろ過膜を用いて脱脂済み酵素分解液をろ過し、さらにゲルクロマトグラフィーで精製し、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含むオリゴペプチドを得た後、これに、ソルビン酸カリウムを加えて均一に撹拌し、乾燥させた。本発明のオリゴペプチド、大豆粕粉末、コーンミール、でん粉、魚粉などを配合比率に従い造粒して得られた混合物は海洋水産養殖飼料のタンパク源として使用でき、魚・エビ・貝類などに大変良い摂餌誘引作用がある。これにより本発明の目的を実現した。 We degreased viscera (Todarodes pacificus) viscera with an organic solvent, followed by enzymatic degradation with Alcalase (registered trademark) and trypsin, and then filtered the degreased enzymatic digestion solution using an ultrafiltration membrane, followed by gel chromatography. papermaking rectification in chromatography, after obtaining an oligopeptide comprising the amino acid structure fragments -ILGGSDPKHYTG-, this was uniformly stirred with potassium sorbate, dried. The mixture obtained by granulating the oligopeptide of the present invention, soybean meal powder, corn meal, starch, fish meal, etc. according to the blending ratio can be used as a protein source for marine aquaculture feed and is very good for fish, shrimp, shellfish, etc. There is a feeding attraction effect. This achieves the object of the present invention.
本発明のイカ内臓から抽出したオリゴペプチドは、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含み、相対分子量範囲は１４００〜５８００であり、下記に述べる方法により調製して得られることを特徴とする。（１）スルメイカ内臓を破砕し、水を加えて均一に混ぜ、石油エーテルかｎ−ヘキサン、もしくは石油エーテルとｎ−ヘキサンの混合溶剤で脱脂し、有機溶剤層を除去し、水層を室温下に１〜３時間放置し、５０〜６０℃で１〜３時間加熱し、温度５０〜６０℃の水を加え、ｐＨを８．０〜８．５に調節し、その後５０〜６０℃の温度保持状態において、原料１ｇ当たり２０００〜３０００ユニットの比率によりアルカラーゼ（登録商標）で１〜３時間酵素分解させた後、原料１ｇ当たり２０００〜３０００ユニットの比率によりトリプシンで１〜３時間酵素分解させ、不溶性物質を除去し、酵素分解液を得る。（２）手順（１）で得られた酵素分解液を分画分子量１８０００Ｄａの限外ろ過膜でろ過し、次にゲルクロマトグラフィーで分離・精製して、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含むオリゴペプチドを得る。 The oligopeptide extracted from the squid viscera of the present invention contains the amino acid structural fragment -ILGGSDPKHYTG-, has a relative molecular weight range of 1400 to 5800, and is characterized by being prepared by the method described below. (1) Crush the squid viscera, add water and mix uniformly, degrease with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, remove the organic solvent layer, and keep the aqueous layer at room temperature 1 to 3 hours, heat at 50 to 60 ° C. for 1 to 3 hours, add water at a temperature of 50 to 60 ° C., adjust pH to 8.0 to 8.5, and then at a temperature of 50 to 60 ° C. In the holding state, after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, enzymatic degradation with trypsin at a rate of 2000 to 3000 units per gram of raw material for 1 to 3 hours, Insoluble substances are removed to obtain an enzyme decomposition solution. (2) Oligopeptide containing amino acid structural fragment -ILGGSDPKHYTG- by filtering the enzymatic degradation solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, followed by separation and purification by gel chromatography Get.
本発明のイカ内臓から抽出したオリゴペプチドは、ソルビン酸カリウムなどの防腐剤を加えることもでき、手順（２）で得られたオリゴペプチド１０００ｇ当たりソルビン酸カリウム０．０２５〜０．０５０ｇを加え、均一に混ぜて乾燥させることが好ましい。試験調製においては、オリゴペプチドの純度が５１％〜６３％の時が、コスト制御に有利である。 The oligopeptide extracted from the squid viscera of the present invention can be added with a preservative such as potassium sorbate, and 0.025 to 0.050 g of potassium sorbate is added per 1000 g of the oligopeptide obtained in the procedure (2). It is preferable to uniformly mix and dry. In the test preparation, it is advantageous for cost control when the purity of the oligopeptide is 51% to 63%.
本発明のイカ内臓から抽出したオリゴペプチドの調製方法は、以下の手順を含むことを特徴とする。
（１）スルメイカの内臓を破砕し、水を加えて均一に混ぜ、石油エーテルかｎ−ヘキサン、もしくは石油エーテルとｎ−ヘキサンの混合溶剤で脱脂し、有機溶剤層を除去し、水層を室温下に１〜３時間放置し、５０〜６０℃で１〜３時間加熱し、温度５０〜６０℃の水を加え、ｐＨを８．０〜８．５に調節し、その後５０〜６０℃の温度保持状態において、原料１ｇ当たり２０００〜３０００ユニットの比率によりアルカラーゼ（登録商標）で１〜３時間酵素分解させた後、原料１ｇ当たり２０００〜３０００ユニットの比率によりトリプシンで１〜３時間酵素分解させ、不溶性物質を除去し、酵素分解液を得る。
（２）手順（１）で得られた酵素分解液を分画分子量１８０００Ｄａの限外ろ過膜でろ過し、次にゲルクロマトグラフィーで分離・精製して、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含むオリゴペプチドを得る。
The method for preparing an oligopeptide extracted from the squid viscera of the present invention is characterized by including the following procedure.
(1) Crush the internal organs of squid, add water and mix uniformly, degrease with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, remove the organic solvent layer, and leave the aqueous layer at room temperature Let stand for 1 to 3 hours below, heat at 50 to 60 ° C. for 1 to 3 hours, add water at a temperature of 50 to 60 ° C., adjust pH to 8.0 to 8.5, then adjust to 50 to 60 ° C. In the temperature holding state, after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, enzymatic degradation with trypsin at a rate of 2000 to 3000 units per gram of raw material for 1 to 3 hours Insoluble substances are removed to obtain an enzyme decomposition solution.
(2) Oligopeptide containing amino acid structural fragment -ILGGSDPKHYTG- by filtering the enzymatic degradation solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, followed by separation and purification by gel chromatography Get.
本発明のイカ内臓から抽出したオリゴペプチドの調製方法は、手順（２）で得られたオリゴペプチドに、ソルビン酸カリウムなどの防腐剤を加えることもでき、オリゴペプチド１０００ｇ当たりソルビン酸カリウム０．０２５〜０．０５０ｇを加え、均一に混ぜて乾燥させることが好ましい。 In the preparation method of the oligopeptide extracted from the squid viscera of the present invention, a preservative such as potassium sorbate can be added to the oligopeptide obtained in the procedure (2), and 0.025 potassium sorbate per 1000 g of the oligopeptide. It is preferable to add ~ 0.050g, and to mix uniformly and to dry.
イカ内臓から抽出したオリゴペプチドの分子質量計算方法：
ｌｏｇＭｗ＝−ｂＫａｖ＋ｃ
ここで、実効分配係数Ｋａｖは排除された程度を示し、Ｍｗは物質の分子質量を示し、ｂ、ｃは定数でゲルカラム自身の性質に関係し、標準タンパク質試料によりｂ、ｃを計算することができる。
Molecular mass calculation method for oligopeptides extracted from squid viscera:
logMw = −bKav + c
Here, the effective partition coefficient Kav indicates the degree of exclusion, Mw indicates the molecular mass of the substance, b and c are constants and are related to the properties of the gel column itself, and b and c can be calculated from a standard protein sample. it can.
ゲルクロマトグラフィーの分離・精製及び分子質量測定の操作は以下の通りである。
（１）中・大型のゲルクロマトグラフ・カラムを用いて分離・精製を行う。スルメイカの内臓を上記の本発明方法に従い脱脂処理した後、酵素分解を行い、分画分子量１８０００Ｄａの限外ろ過膜で、イカ内臓の酵素分解液をろ過する。中・大型のゲルクロマトグラフを用いて分離し、このとき、ゲルクロマトグラフ型番号：Ｓｅｐｈａｄｅｘ Ｇ−２５、カラム型番号：２００×１０ｃｍ、緩衝液は０．０５ｍｏｌ／Ｌリン酸塩緩衝液（０．１６ｍｏｌ／Ｌ ＮａＣｌを含む）、ｐＨ＝７．０、溶媒流速は１０ｍＬ／ｍｉｎ、ベッドボリュームは約１２０００ｍＬであり、試料を濃度５０〜１００ｍｇ／ｍＬの溶液に調合し、０．４５μｍの精密ろ過膜でろ過し、５００〜１０００ｍＬを取って試料注入し分離・精製し、下記に述べる手順の方法で分析測定する。
（２）小型ゲルクロマトグラフを用いて分子質量の測定を行う。分析条件選択：ゲルクロマトグラフ型番号: Ｓｅｐｈａｄｅｘ Ｇ−２５、カラム型番号：１００×１ｃｍ、緩衝液は０．０５ｍｏｌ／Ｌリン酸塩緩衝液(０．１６ ｍｏｌ／ＬＮａＣｌを含む)、ｐＨ＝７．０、溶媒流速０．２０ｍＬ／ｍｉｎ、測定波長２８０ｎｍ、ベッドボリューム約６０ｍＬ。
（３）標準タンパク質試料を濃度５ｍｇ／ｍＬの溶液に調合し、０．３０μｍの精密ろ過膜でろ過し、０．５ｍＬを取って試料注入し、（Ｋａｖ、ｌｏｇＭｗ）の対応点3つ（２８．５， ４．１６）、（５３．７，３．１３）、（７４．９，２．２６）を得、計算により公式：ｌｏｇＭｗ＝−０．０４１Ｋａｖ ＋５．３３（Ｒ２＝０．９９９８）を得た。
（４）手順（１）で分離・精製したスルメイカ内臓の酵素加水分解液を、０．３０μｍの精密ろ過膜でろ過した後、濃度を１０ｍｇ／ｍＬに調整する。小型ゲルクロマトグラフを用いて、０．５ｍＬを取り試料注入し、イカ内臓から抽出したオリゴペプチドの直線状の（Ｋａｖ、ｌｏｇＭｗ）点、それぞれ（５３．３，３．１５）、（３８．２，３．７６）を得、対応する分子質量を計算すると１４００及び５８００であった。
The operations of gel chromatography separation / purification and molecular mass measurement are as follows.
(1) Separation and purification are performed using medium and large gel chromatograph columns. After degreasing the viscera of cuttlefish in accordance with the above-mentioned method of the present invention, enzymatic decomposition is performed, and the enzymatic decomposition solution of squid viscera is filtered through an ultrafiltration membrane having a molecular weight cut off of 18000 Da. Separation using medium and large gel chromatograph. At this time, gel chromatograph type number: Sephadex G-25, column type number: 200 × 10 cm, buffer solution is 0.05 mol / L phosphate buffer solution (0.16 mol) / L NaCl included), pH = 7.0, solvent flow rate is 10 mL / min, bed volume is about 12000 mL, sample is prepared in a solution of 50-100 mg / mL concentration, and 0.45 μm microfiltration membrane is used. Filter, take 500-1000 mL, inject sample, separate and purify, and analyze and measure by the method described below.
(2) The molecular mass is measured using a small gel chromatograph. Analysis condition selection: gel chromatograph type number: Sephadex G-25, column type number: 100 × 1 cm, buffer solution is 0.05 mol / L phosphate buffer solution (containing 0.16 mol / L NaCl), pH = 7. 0, solvent flow rate 0.20 mL / min, measurement wavelength 280 nm, bed volume about 60 mL.
(3) A standard protein sample is prepared in a 5 mg / mL solution, filtered through a 0.30 μm microfiltration membrane, 0.5 mL is taken, and the sample is injected, and three corresponding points of (Kav, logMw) (28 .5, 4.16), (53.7, 3.13), (74.9, 2.26) and obtained by calculation: log Mw = −0.041 Kav + 5.33 (R 2 = 0.9998) )
(4) After the enzyme hydrolyzate of squid viscera separated and purified in step (1) is filtered through a 0.30 μm microfiltration membrane, the concentration is adjusted to 10 mg / mL. Using a small gel chromatograph, 0.5 mL was sampled and injected, and the linear (Kav, log Mw) points of the oligopeptide extracted from the squid viscera were respectively (53.3, 3.15), (38.2, 3.76) was obtained and the corresponding molecular masses were calculated to be 1400 and 5800.
本発明のイカ内臓から抽出したオリゴペプチドを含む混合物は、全質量分率１００％で計算すると、イカ内臓から抽出したオリゴペプチド６％〜８％、大豆粕粉末５０％〜６０％、魚粉３％〜６％、コーンミール２７％〜３３％、及び造粒用でん粉２％〜５％からなることを特徴とする。使用する大豆粕粉末、魚粉、コーンミール及び造粒用でん粉は水産飼料用であり、市販されている。 The mixture containing the oligopeptide extracted from the squid viscera of the present invention is calculated with a total mass fraction of 100%. The oligopeptide extracted from the squid viscera is 6% to 8%, soybean meal powder 50% to 60%, and fish meal 3%. It consists of ~ 6%, cornmeal 27% ~ 33%, and granulating starch 2% ~ 5%. The soybean meal powder, fish meal, corn meal and starch for granulation used are for marine feed and are commercially available.
本発明の混合物の調製方法では、上記の比率により、イカ内臓から抽出したオリゴペプチド、大豆粕粉末、魚粉、コーンミールを均一に混ぜ、配合材料：水＝１：４（質量比）の比率で適量の水を加え、均一に混ぜ、噴霧乾燥し、乾燥エキス粉末を得、配合比率により乾燥エキス粉末に造粒用でん粉を加えて造粒し、乾かして混合物を得る。 In the preparation method of the mixture of the present invention, the oligopeptide extracted from the squid viscera, soybean meal powder, fish meal, and corn meal are uniformly mixed according to the above ratio, and the ratio of the blending material: water = 1: 4 (mass ratio). An appropriate amount of water is added, mixed uniformly, and spray-dried to obtain a dry extract powder, and granulated by adding starch for granulation to the dry extract powder according to the blending ratio, and dried to obtain a mixture.
本発明の混合物は、海洋水産養殖（海洋魚類、エビ、貝類などを含む）飼料のタンパク源として使用でき、特に水産海洋魚類の飼料タンパク源に適している。 The mixture of the present invention can be used as a protein source for marine aquaculture (including marine fish, shrimp, shellfish, etc.) feed, and is particularly suitable as a feed protein source for marine marine fish.
本発明のイカ内臓から抽出したオリゴペプチドの調製過程における温度は６０℃を超えず、乾燥時には噴霧乾燥装置を用いて乾燥し、短時間で効果がよいため、この物質が加水分解又は変性しないことを保証している。本発明の混合物は魚類に強い食餌誘引性があり、その主な成分であるイカ内臓から抽出したオリゴペプチドには、強力な魚臭があり、水産養殖飼料のタンパク源に最適である。本発明が提供する混合物を利用して海洋魚類を養殖すると、魚類の摂餌量を大幅に高め、魚類個体の単位重量を増やすことができる。 The temperature in the preparation process of the oligopeptide extracted from the squid viscera of the present invention does not exceed 60 ° C., and when dried, it is dried using a spray drying device and is effective in a short time, so that this substance does not hydrolyze or denature. Guarantee. The mixture of the present invention has a strong diet-attracting property for fish, and the oligopeptide extracted from the squid viscera, which is the main component, has a strong fishy odor and is optimal as a protein source for aquaculture feed. When a marine fish is cultivated using the mixture provided by the present invention, the amount of fish consumed can be greatly increased, and the unit weight of the individual fish can be increased.
具体的な実施方法：
以下の実施例は本発明についてのさらなる説明であり、本発明に対する制限ではない。実施例中の主な原材料、主な計器設備、主な生物学的及び化学的試薬などは以下の通りである。
Specific implementation method:
The following examples are further illustrations of the present invention and are not a limitation on the present invention. Main raw materials, main instrumentation, main biological and chemical reagents and the like in the examples are as follows.
主な原材料：
広州市黄沙海産品市場からスルメイカ（Ｔｏｄａｒｏｄｅｓ ｐａｃｉｆｉｃｕｓ）内臓を収集し、−１５℃で保存し、使用の１２時間前に室温で解凍した。「大江牌」通常魚用配合飼料は上海大江（集団）股▲ふん▼有限公司より購入した。使用した大豆粕粉末、コーンミールは済寧双華工貿有限公司より購入、使用した魚粉は栄成新希望魚粉有限公司より購入した。
Main raw materials:
The squid (Todarodes pacificus) internal organs were collected from the Guangzhou Huangsha Seafood Market, stored at -15 ° C, and thawed at room temperature 12 hours before use. “Oe 牌” regular fish feed was purchased from Shanghai Oe (Group) Co., Ltd. The used soybean meal powder and corn meal were purchased from Jining Shuanghua Industrial Trade Co., Ltd., and the used fish meal was purchased from Rongcheng New Hope Fish Powder Co., Ltd.
主な計器設備：
発酵槽：ＬＡＢＦＯＲＳ実験室卓上式小型発酵槽、生産地；スイス
均質装置：予華Ａ８Ｇ型、生産地；河南省鞏義市
据置型遠心分離機：Ｂｅｃｈｍａｎ Ｊ２−Ｈｓ高速冷凍遠心分離機、生産地；米国
中・大型ゲルクロマトグラフ型番号：Ｓｅｐｈａｄｅｘ Ｇ−２５、カラム型番号：２００×１０ ｃｍ、緩衝液は０．０５ｍｏｌ／Ｌリン酸塩緩衝液（０．１６ ｍｏｌ／Ｌ ＮａＣｌを含む）、ｐＨ＝７．０、溶媒流速５０ｍＬ／ｍｉｎ、ベッドボリューム約１２０００ｍＬ
８３５−５０アミノ酸自動分析計（日本日立社）
クロマトグラフＷａｔｅｒｓ ２６９０、質量分析装置Ｗａｔｅｒｓ Ｐｌａｔｆｏｒｍ ＺＭＤ ４０００
Main instrument equipment:
Fermenter: LABFORS laboratory tabletop small fermenter, production area; Swiss homogenous equipment: Preliminary A8G type, production area; Shenyang city stationary type centrifuge: Bechman J2-Hs high-speed freezing centrifuge, production area; US medium / large gel chromatograph model number: Sephadex G-25, column model number: 200 × 10 cm, buffer is 0.05 mol / L phosphate buffer (containing 0.16 mol / L NaCl), pH = 7.0, solvent flow rate 50mL / min, bed volume about 12000mL
835-50 amino acid automatic analyzer (Hitachi, Japan)
Chromatograph Waters 2690, Mass Spectrometer Waters Platform ZMD 4000
主な生物学的及び化学的試薬：
トリプシン：１００万ユニット（酵素活性）／ｇ、大連保税区聯合博泰生物技術有限公司、製品番号：ＲＭ００１０２
アルカラーゼ（登録商標）（Ａｌｃａｌａｓｅ）：５０万ユニット（酵素活性）／ｇ、Ｎｏｖｏｚｙｍｅｓ社
有機試薬：有機試薬は広州試剤公司から購入（分析用試薬）
無機試薬：無機試薬は広州試剤公司から購入（分析用試薬）
純水：実施例で用いた水は全て純水であり、ＭｉｎｉＱ純水調製システムで調整した。
Main biological and chemical reagents:
Trypsin: 1 million units (enzyme activity) / g, Dalian Bonded Zone Hirogoe Biotechnology Co., Ltd., product number: RM00102
Alcalase (registered trademark) (Alcalase): 500,000 units (enzyme activity) / g, Novozymes organic reagent: Organic reagent purchased from Guangzhou Reagents Co., Ltd. (analytical reagent)
Inorganic reagents: Inorganic reagents purchased from Guangzhou Reagents (analytical reagents)
Pure water: All of the water used in the examples was pure water, and was adjusted with a MiniQ pure water preparation system.
スルメイカ内臓２０ｋｇを収集し、予華Ａ８Ｇ型均質装置で撹拌・破砕し、水２Ｌを加え、さらに沸点範囲６０〜９０℃の石油エーテル（２０Ｌ）を加えて撹拌・脱脂し、石油エーテル層を除去し、水層を室温に３時間放置し、５０℃のオーブンで３時間加熱した。温度５０℃の水８Ｌを加え、均一に混ぜ、飽和ＮａＯＨ溶液でｐＨを８．５近くに調節し、さらに５ｍｏｌ／Ｌ濃度のＮａＯＨ溶液でｐＨを８．５に調節し、５０℃の温度保持状態において、イカ内臓を発酵槽に入れ、原料３０００ユニット／ｇで６×１０７ユニットのアルカラーゼ（登録商標）を加えて３時間酵素分解させた後、さらに原料３０００ユニット／ｇＫで６×１０７ユニットのトリプシンを加えて３時間酵素分解させた。Ｂｅｃｈｍａｎ Ｊ２−Ｈｓ高速冷凍遠心分離機を用いて５０００回転／分で遠心分離し、不溶性物質を除去した。分画分子量１８０００Ｄａの限外ろ過膜で、脱脂した酵素分解液をろ過すると、オリゴペプチドを豊富に含む成分が得られ、次に中・大型Ｓｅｐｈａｄｅｘ Ｇ−２５ゲルクロマトグラフで分離・精製すると、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含むオリゴペプチドが得られた。オリゴペプチド１０００ｇ当たりソルビン酸カリウム０．０２５ｇの割合で、ソルビン酸カリウム０．００５９ｇを加え、乾燥後、イカ内臓から抽出したオリゴペプチドを得た。 Collect 20 kg of Japanese squid viscera, stir and crush it with a precious A8G homogenizer, add 2 L of water, add petroleum ether (20 L) with a boiling range of 60-90 ° C, stir and degrease, and remove the petroleum ether layer The aqueous layer was left at room temperature for 3 hours and heated in an oven at 50 ° C. for 3 hours. Add 8 L of water at a temperature of 50 ° C., mix evenly, adjust the pH to 8.5 with a saturated NaOH solution, adjust the pH to 8.5 with a 5 mol / L NaOH solution, and maintain the temperature at 50 ° C. In this state, the squid viscera was placed in a fermenter, 6 × 10 7 units of Alcalase (registered trademark) was added at 3000 units / g of raw material, and enzymatically decomposed for 3 hours, and then 6 × 10 7 at 3000 units / gK of raw material. Unit trypsin was added and enzymatically degraded for 3 hours. The insoluble material was removed by centrifugation at 5000 rpm using a Bechman J2-Hs high-speed refrigerated centrifuge. When the defatted enzyme digestion solution is filtered through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, a component rich in oligopeptides is obtained, and then separated and purified by a medium / large Sephadex G-25 gel chromatograph, the amino acid structure An oligopeptide containing the fragment -ILGGSDPKHYTG- was obtained. 0.0059 g of potassium sorbate was added at a rate of 0.025 g of potassium sorbate per 1000 g of oligopeptide, and after drying, an oligopeptide extracted from the squid viscera was obtained.
イカ内臓から抽出したオリゴペプチド８０ｇ、大豆粕粉末６００ｇ、魚粉３０ｇ、コーンミール２７０ｇを量って取り、水４Ｌを加え、均一に混ぜた後、噴霧乾燥し、乾燥エキス粉末を得、造粒用でん粉２０ｇを量って取り、造粒し、乾かして混合物を得た。 Take 80 g of oligopeptide extracted from squid viscera, 600 g of soybean meal powder, 30 g of fish meal, 270 g of corn meal, add 4 L of water, mix uniformly, and then spray dry to obtain dry extract powder for granulation Weighed 20 g of starch, granulated and dried to obtain a mixture.
スルメイカ内臓２０ｋｇを収集し、予華Ａ８Ｇ型均質装置で撹拌・破砕し、水１２ Ｌを加え、さらに石油エーテルとｎ−ヘキサン混合溶剤（１：１）２０Ｌを加えて撹拌・脱脂し、石油エーテル層を除去し、水層を室温に１時間放置し、６０℃のオーブンで１時間加熱した。温度６０℃の水８Ｌを加え、均一に混ぜ、飽和ＮａＯＨ溶液でｐＨを８．０近くに調節し、さらに希薄濃度のＮａＯＨ溶液でｐＨを８．０に調節した。６０℃の温度保持状態において、イカ内臓を発酵槽に入れ、４×１０７ユニットのアルカラーゼ（登録商標）を加えて１時間酵素分解させた後、さらに４×１０７ユニットのトリプシンを加えて１時間酵素分解させた。Ｂｅｃｈｍａｎ Ｊ２−Ｈｓ高速冷凍遠心分離機を用いて５０００回転／分で遠心分離し、不溶性物質を除去した。分画分子量１８０００Ｄａの限外ろ過膜で、脱脂した酵素分解液をろ過すると、オリゴペプチドを豊富に含む成分が得られ、次に中・大型Ｓｅｐｈａｄｅｘ Ｇ−２５ゲルクロマトグラフで分離・精製すると、アミノ酸構造フラグメント−ＩＬＧＧＳＤＰＫＨＹＴＧ−を含むオリゴペプチドが得られた。オリゴペプチド１０００ｇ当たりソルビン酸カリウム０．０５０ｇの割合で、ソルビン酸カリウム０．００７８ｇを加え、乾燥後、イカ内臓から抽出したオリゴペプチドを得た。 Collect 20 kg of cuttlefish viscera and stir and crush it with a precious A8G type homogenizer, add 12 L of water, add 20 L of petroleum ether and n-hexane mixed solvent (1: 1), stir and degrease, petroleum ether The layer was removed and the aqueous layer was left at room temperature for 1 hour and heated in an oven at 60 ° C. for 1 hour. 8 L of water at a temperature of 60 ° C. was added, mixed uniformly, the pH was adjusted to about 8.0 with a saturated NaOH solution, and the pH was further adjusted to 8.0 with a dilute NaOH solution. With the temperature maintained at 60 ° C., the squid viscera was placed in the fermenter, 4 × 10 7 units of Alcalase (registered trademark) was added and enzymatically decomposed for 1 hour, and then 4 × 10 7 units of trypsin was added. Enzymatic degradation was allowed for hours. The insoluble material was removed by centrifugation at 5000 rpm using a Bechman J2-Hs high-speed refrigerated centrifuge. When the defatted enzyme digestion solution is filtered through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, a component rich in oligopeptides is obtained, and then separated and purified by a medium / large Sephadex G-25 gel chromatograph, the amino acid structure An oligopeptide containing the fragment -ILGGSDPKHYTG- was obtained. 0.0078 g of potassium sorbate was added at a rate of 0.050 g of potassium sorbate per 1000 g of oligopeptide, and after drying, an oligopeptide extracted from the squid viscera was obtained.
イカ内臓から抽出したオリゴペプチド６０ｇ、大豆粕粉末５００ｇ、魚粉６０ｇ、コーンミール３３０ｇを量って取り、水４Ｌを加え、均一に混ぜた後、噴霧乾燥し、乾燥エキス粉末を得、造粒用でん粉５０ｇを量って取り、造粒し、乾かして混合物を得た。 Weigh 60 g of oligopeptide extracted from squid viscera, 500 g of soybean meal powder, 60 g of fish meal and 330 g of cornmeal, add 4 L of water, mix uniformly, and then spray dry to obtain a dry extract powder for granulation. 50 g of starch was weighed out, granulated and dried to obtain a mixture.
実施例１のイカ内臓から抽出したオリゴペプチド６５ｇ、大豆粕粉末５３５ｇ、魚粉３０ｇ、コーンミール３３０ｇを量って取り、水４Ｌを加え、均一に混ぜた後、噴霧乾燥し、乾燥エキス粉末を得、造粒用でん粉４０ｇを量って取り、造粒し、乾かして混合物を得た。 65 g of the oligopeptide extracted from the squid viscera of Example 1, 535 g of soybean meal powder, 30 g of fish meal, and 330 g of cornmeal were added and mixed with 4 L of water, mixed uniformly, and then spray-dried to obtain a dry extract powder. Then, 40 g of starch for granulation was weighed, granulated and dried to obtain a mixture.
実施例２のイカ内臓から抽出したオリゴペプチド７５ｇ、大豆粕粉末５７５ｇ、魚粉５０ｇ、コーンミール２８０ｇを量って取り、水４Ｌを加え、均一に混ぜた後、噴霧乾燥し、乾燥エキス粉末を得、造粒用でん粉を２０ｇ量って取り、造粒し、乾かして混合物を得た。 75 g of the oligopeptide extracted from the squid viscera of Example 2, 575 g of soybean meal powder, 50 g of fish meal, and 280 g of cornmeal were added and mixed with 4 L of water, mixed uniformly, and then spray dried to obtain a dry extract powder. Then, 20 g of starch for granulation was weighed, granulated and dried to obtain a mixture.
アミノ酸組成分析：
実施例１と２で得られた、イカ内臓から抽出したオリゴペプチドをそれぞれ加水分解管に入れ、６ｍｏｌ／Ｌの塩酸溶液を加え、真空密封した。１１０℃で２４ｈ加水分解させ、冷ましてから定容・ろ過・蒸気乾燥し、０．０２ ｍｏｌ／Ｌの塩酸溶液を加えて空気中に３０ ｍｉｎ置き、８３５−５０アミノ酸自動分析計（日本日立社）を用いて、トリプトファン以外のアミノ酸含有量を測定し、次の結果を得た。
Ｉ：Ｌ：Ｇ：Ｓ：Ｄ：Ｐ：Ｋ：Ｈ：Ｙ：Ｔ＝１．０５：１．０２：３．０１：０．９６：０．９８：１．００：０．９７：０．９８：０．９５：１．００
Amino acid composition analysis:
The oligopeptides extracted from the squid viscera obtained in Examples 1 and 2 were each put into a hydrolysis tube, a 6 mol / L hydrochloric acid solution was added, and the mixture was vacuum-sealed. Hydrolyzed at 110 ° C. for 24 hours, cooled, then fixed volume, filtered, steam dried, added 0.02 mol / L hydrochloric acid solution and placed in air for 30 min, 835-50 amino acid automatic analyzer (Nippon Hitachi, Ltd.) ) Was used to measure the content of amino acids other than tryptophan and the following results were obtained.
I: L: G: S: D: P: K: H: Y: T = 1.05: 1.02: 3.01: 0.96: 0.98: 1.00: 0.97: 0. 98: 0.95: 1.00
液体クロマトグラフィー／質量分析計（ＬＣ―ＭＳ）を用いた分子質量の測定：
液体クロマトグラフフィー条件：クロマトグラフＷａｔｅｒｓ ２６９０、検出器：Ｗａｔｅｒｓ ９９６、分析カラムＬｉｃｈｒｏｓｐｈｅｒ Ｃ−１８ （２．６×２５０ｍｍ）、移動相：メタノール−水−０．５％酢酸勾配溶出、測定波長２２０ｎｍ、カラム温度３０℃、試料注入量、１０μＬ、流速０．３ｍＬ／ｍｉｎ。質量分析条件：質量分析装置Ｗａｔｅｒｓ Ｐｌａｔｆｏｒｍ ＺＭＤ ４０００、イオン化法ＥＳＩ＋、キャピラリー電圧４．８０ｋＶ、コーン電圧３２ Ｖ、Ｇａｓ Ｆｌｏｗ：４．２Ｌ／ｈ、イオン源温度１２０℃、溶媒脱気温度２５０℃、質量範囲：１０００〜８０００ｍ／ｚ、光電子増倍管（ＰＭＴ）電圧６７０Ｖ、Ａｎａｌｙｓｅｒ Ｖａｃｕｕｍ２．６ｅ〜５ｍＢａｒ。
Measurement of molecular mass using liquid chromatography / mass spectrometer (LC-MS):
Liquid chromatographic conditions: chromatograph Waters 2690, detector: Waters 996, analytical column Lichlorosphere C-18 (2.6 × 250 mm), mobile phase: methanol-water-0.5% acetic acid gradient elution, measurement wavelength 220 nm, Column temperature 30 ° C., sample injection volume 10 μL, flow rate 0.3 mL / min. Mass spectrometry conditions: Mass spectrometer Waters Platform ZMD 4000, ionization method ESI + , capillary voltage 4.80 kV, cone voltage 32 V, Gas Flow: 4.2 L / h, ion source temperature 120 ° C., solvent degassing temperature 250 ° C. Mass range: 1000-8000 m / z, photomultiplier tube (PMT) voltage 670V, Analyzer Vacuum 2.6e-5mBar.
液体クロマトグラフィー／質量分析計（ＬＣ―ＭＳ）を用いてオリゴペプチドの分子質量を測定する際、分子イオンピークは現れず、一部の基を除去した後のフラグメントピークのみ得られた。 When the molecular mass of the oligopeptide was measured using a liquid chromatography / mass spectrometer (LC-MS), no molecular ion peak appeared, and only a fragment peak after removing some groups was obtained.
アミノ酸配列測定：エドマン（Ｅｄｍａｎ）分解法でイカオリゴペプチドのアミノ酸配列を測定した。配列解析によりオリゴペプチドは−ＩＬＧＧＳＤＰＫＨＹＴＧ−の順序の連結構造を持つことが示された。 Amino acid sequence measurement: The amino acid sequence of the squid oligopeptide was measured by the Edman degradation method. Sequence analysis showed that the oligopeptide had a linking structure in the order of -ILGGSDPKHYTG-.
イカ内臓から抽出したオリゴペプチドのアミノ酸について、アミノ酸組成分析、ゲルクロマトグラフィー分析、オリゴペプチド分子質量測定及びアミノ酸配列測定を行ったことにより、その中に−ＩＬＧＧＳＤＰＫＨＹＴＧ−オリゴペプチド構造フラグメントが含まれ、相対分子質量は１４００から５８００の間であるという結果が得られた。 By performing amino acid composition analysis, gel chromatography analysis, oligopeptide molecular mass measurement and amino acid sequence measurement for amino acids of oligopeptide extracted from squid viscera, it contains -ILGGSDDPKHYTG-oligopeptide structural fragment, The result was that the molecular mass was between 1400 and 5800.
ボラの食餌実験
２００９年４月、１齢のボラ５０尾を、本発明の飼料タンパク源を添加しない対照群（１０尾）、実施例１〜３の混合物を１０％添加した実験群（各１０尾）、及び新鮮なイカ内臓液を１０％添加した実験群（１０尾）に分け、かつ同一の一般飼料（「大江牌」通常魚用配合飼料）を給餌した。１．５立方メートル程度の池５つに魚を入れ、最初の一ヶ月は毎日水を１／５換え、後の半月は毎日水を１／３換え、７日、１５日、３０日及び４５日後の総体重と相応の時間間隔内の飼料の総消費量を記録し、これらの重量の変化状況と飼料の消費状況を分析した。このうち１７日目に対照群のボラが１尾死亡したため、データは１０尾に換算した後のデータである。結果は表１の通りである。新鮮なイカ内臓液は、イカ内臓を３時間自己酵素により分解させて得られた、未加工の物質である。表１から明確に見て取れるように、本発明の飼料タンパク源を添加しない対照群及び一般の新鮮なイカ内臓酵素分解液を添加した実験群と比べ、イカ内臓から抽出したオリゴペプチドを含む本発明の混合物は、飼料タンパク源としてボラの摂餌量を大幅に高め、ボラ個体の単位重量を増やすことができた。
Bora Feeding Experiment April 2009, 50 groups of 1 year old mullet, 10 control groups to which the feed protein source of the present invention was not added, 10% of the mixture of Examples 1 to 3 (each 10 groups) Tail) and an experimental group (10 fish) to which 10% of fresh squid visceral fluid was added, and the same general feed (“Oe Kashiwa” normal fish formula feed) was fed. Put fish in 5 ponds of about 1.5 cubic meters, change the water by 1/5 every day for the first month, and change the water by 1/3 every day for the next half month, after 7, 15, 30, and 45 days The total body weight and the total consumption of feed during the corresponding time interval were recorded, and the change in weight and the consumption of feed were analyzed. Of these, the control group was killed on the 17th day, so the data is the data after conversion to 10 fish. The results are shown in Table 1. Fresh squid visceral fluid is a raw material obtained by degrading squid viscera with autoenzymes for 3 hours. As can be clearly seen from Table 1, compared to the control group to which the feed protein source of the present invention was not added and the experimental group to which a general fresh squid visceral enzyme digestion solution was added, the oligopeptide extracted from the squid viscera was included. The mixture was able to greatly increase the food intake of mullet as a feed protein source and increase the unit weight of mullet individuals.
バスの食餌実験
２００９年８月、生後４５日のオオクチバス５０尾を、本発明の飼料タンパク源を添加しない対照群（１０尾）、実施例１〜３の混合物を１０％添加した実験群（各１０尾）、及び新鮮なイカ内臓液１０％を添加した実験群（１０尾）に分け、かつ同一の一般飼料（「大江牌」通常魚用配合飼料）を給餌した。１．５立方メートル程度の池５つに魚を入れ、最初の一ヶ月は毎日水を１５％換え、後の一ヶ月は毎日水を２０％換え、７日、１５日、３０日及び６０日後の総体重と相応の時間間隔内の飼料の総消費量を記録し、これらの重量の変化状況と飼料の消費状況を分析した。このうち５日目に対照群のバスが１尾死亡したため、データは１０尾に換算した後のデータである。結果は表２の通りである。新鮮なイカ内臓液は、イカ内臓を３時間自己酵素により分解させて得られた、未加工の物質である。表２から明確に見て取れるように、本発明の飼料タンパク源を添加しない対照群及び一般の新鮮なイカ内臓酵素分解液を添加した実験群と比べ、イカ内臓から抽出したオリゴペプチドを含む本発明の混合物は、飼料タンパク源としてバスの摂餌量を大幅に高め、バス個体の単位重量を増やすことができた。
Feeding experiment of bass In August 2009, 50 largemouth basses on the 45th day of birth were added to the control group (10 fishes) to which the feed protein source of the present invention was not added, and to the experimental group (each of which was 10% of the mixture of Examples 1 to 3). 10 fish) and an experimental group (10 fish) to which 10% of fresh squid visceral fluid was added, and the same general feed ("Oe tsumugi" normal fish mixed feed) was fed. Put fish in 5 ponds of about 1.5 cubic meters, change 15% of water every day for the first month, change 20% of water every day for the following month, 7 days, 15 days, 30 days and 60 days later The total body weight and the total consumption of the feed within the corresponding time interval were recorded, and the change of the weight and the consumption of the feed were analyzed. Of these, one bus in the control group died on the 5th day, so the data is data after conversion to 10 fish. The results are shown in Table 2. Fresh squid visceral fluid is a raw material obtained by degrading squid viscera with autoenzymes for 3 hours. As can be clearly seen from Table 2, compared to the control group to which the feed protein source of the present invention was not added and the experimental group to which a general fresh squid visceral enzyme digestion solution was added, the oligopeptide extracted from the squid viscera was used. The mixture significantly increased the amount of food consumed by the bath as a feed protein source and increased the unit weight of the individual bath.
Claims (6)
（１）スルメイカ（Ｔｏｄａｒｏｄｅｓ ｐａｃｉｆｉｃｕｓ）の内臓を撹拌し、破砕し、水を加えて均一に撹拌し、石油エーテルかｎ−ヘキサン、もしくは石油エーテルとｎ−ヘキサンの混合溶剤で脱脂し、有機溶剤層を除去し、水層を室温下に１〜３時間放置し、５０〜６０℃で１〜３時間加熱し、温度５０〜６０℃の水を加え、ｐＨ８．０〜８．５に調節し、その後５０〜６０℃に保温した状態において、原料１ｇ当たり２０００〜３０００ユニットの比率によりアルカラーゼ（登録商標）で１〜３時間酵素分解させた後、原料１ｇ当たり２０００〜３０００ユニットの比率によりトリプシンで１〜３時間酵素分解させ、不溶性物質を除去して酵素分解液を得る工程、および
（２）手順（１）で得られた酵素分解液を、分画分子量１８０００Ｄａ限外ろ過膜でろ過し、次にＳｅｐｈａｄｅｘ Ｇ−２５ゲルクロマトグラフィーで精製して、オリゴペプチドを得る工程により調製して得られることを特徴とするイカ内臓抽出物。 Include oligopeptides comprising an amino acid structure fragments -ILGGSDPKHYTG-, relative molecular weight range Ru der 1400-5800, a squid viscera extract,
(1) The viscera of squid (Todarodes pacificus) is stirred , crushed, added with water, stirred uniformly, degreased with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, and an organic solvent layer The aqueous layer was allowed to stand at room temperature for 1 to 3 hours, heated at 50 to 60 ° C. for 1 to 3 hours, added with water at a temperature of 50 to 60 ° C., adjusted to pH 8.0 to 8.5, Thereafter, in a state where the temperature is kept at 50 to 60 ° C., enzymatic degradation is performed with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per 1 g of the raw material, and then 1 trypsin at a rate of 2000 to 3000 units per 1 g of the raw material. A step of enzymatic decomposition for ˜3 hours to remove insoluble substances to obtain an enzymatic decomposition solution, and (2) the enzymatic decomposition solution obtained in step (1) is subjected to a molecular weight cutoff of 180 Filtered through a 00Da ultrafiltration membrane, then Sephadex G-25 was manufactured by Seiko gel chromatography, squid in 臓抽 out thereof, characterized in that it is obtained by prepared by obtaining the o Rigopepuchi de.
（１）スルメイカ（Ｔｏｄａｒｏｄｅｓ ｐａｃｉｆｉｃｕｓ）の内臓を撹拌し、破砕し、水を加えて均一に撹拌し、石油エーテルかｎ−ヘキサン、もしくは石油エーテルとｎ−ヘキサンの混合溶剤で脱脂し、有機溶剤層を除去し、水層を室温下に１〜３時間放置し、５０〜６０℃で１〜３時間加熱し、温度５０〜６０℃の水を加え、ｐＨ８．０〜８．５に調節した後、５０〜６０℃に保温した状態において、原料１ｇ当たり２０００〜３０００ユニットの比率によりアルカラーゼ（登録商標）で１〜３時間酵素分解させた後、原料１ｇ当たり２０００〜３０００ユニットの比率により、トリプシンで１〜３時酵素分解し、不溶性物質を除去して酵素分解液を得る工程、および
（２）手順（１）で得られた酵素分解液を、分画分子量１８０００Ｄａ限外ろ過膜でろ過し、次にＳｅｐｈａｄｅｘ Ｇ−２５ゲルクロマトグラフィーで精製して、オリゴペプチドを得る工程を含むことを特徴とするイカ内臓抽出物の調製方法。 Include oligopeptides comprising an amino acid structure fragments -ILGGSDPKHYTG-, relative molecular weight range is 1400 to 5800, a process for preparing a squid in 臓抽 out thereof,
(1) The viscera of squid (Todarodes pacificus) is stirred , crushed, added with water, stirred uniformly, degreased with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, and an organic solvent layer After leaving the aqueous layer at room temperature for 1 to 3 hours, heating at 50 to 60 ° C. for 1 to 3 hours, adding water at a temperature of 50 to 60 ° C., and adjusting the pH to 8.0 to 8.5. In the state kept at 50 to 60 ° C., after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, trypsin at a rate of 2000 to 3000 units per gram of raw material. 1 to 3 o'clock enzymatic decomposition to remove insoluble substances to obtain an enzymatic degradation solution, and (2) the molecular weight of the enzyme degradation solution obtained in step (1) is 18000 Da filtered through a ultrafiltration membrane, and then refining with Sephadex G-25 gel chromatography, process for the preparation of squid viscera extract characterized in that it comprises a step of obtaining O Rigopepuchi de.
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