JP5209582B2 - Additives for fish paste products - Google Patents
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- JP5209582B2 JP5209582B2 JP2009198004A JP2009198004A JP5209582B2 JP 5209582 B2 JP5209582 B2 JP 5209582B2 JP 2009198004 A JP2009198004 A JP 2009198004A JP 2009198004 A JP2009198004 A JP 2009198004A JP 5209582 B2 JP5209582 B2 JP 5209582B2
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Description
本発明は、魚肉すり身又は魚肉練製品の品質改善のための添加物およびその使用方法に関する。 The present invention relates to an additive for improving the quality of a fish paste or fish paste product and a method for using the same.
魚肉を原料とする練製品はその弾力が重要な品質の尺度の一つである。この弾力は、「足」、「ゲル形成能」などという尺度でも表現される。この独特の弾力は、塩ずりした肉糊を20〜40℃の適当な温度下に10分〜20時間程度置くことにより得られ、「坐り」と呼ばれる。この坐りによって得られた弾力が、その後の加熱工程で低下することが知られており、これを「戻り」と呼ぶ。品質のよい練製品を製造するために、この坐りと戻りが重要であり、坐りを増強させ、戻りを抑制するための方法が工夫されている。
坐りや戻りの現象は、原料の魚種によって大きく影響され、また同じ魚種でも、鮮度、原料の処理方法、水晒し方法により影響される。例えば、同じスケトウタラでも、高鮮度で丁寧に水晒しされる洋上特級すり身は坐りが強く、戻りが弱いが、比較的低鮮度で、血液や内蔵の混入がみられる陸上すり身では、坐りが弱く戻りが強い傾向にある。水晒しを行ってすり身として多用されているスケトウダラに対して、イトヨリ、キンメダイ、マイワシ、アジ等の魚肉は、ゲル形成能は弱いが、旨味が強いことから、風味改善の点で魅力があり、練製品の原料として使用されている。
A refined product made from fish meat is one of the quality measures whose elasticity is important. This elasticity is also expressed by a scale such as “foot”, “gel-forming ability”, and the like. This unique elasticity is obtained by placing salted meat paste at an appropriate temperature of 20 to 40 ° C. for about 10 minutes to 20 hours, and is called “sitting”. It is known that the elasticity obtained by this sitting decreases in the subsequent heating process, which is called “return”. This sitting and returning is important for producing a quality paste product, and a method for enhancing sitting and suppressing returning is devised.
The phenomenon of sitting and returning is greatly influenced by the fish species of the raw material, and even in the same fish species, it is affected by the freshness, the raw material processing method, and the water exposure method. For example, even in the same walleye cod, a high-quality freshly-exposed offshore surimi is strong and sits weakly, but a relatively low-fresh land surimi with blood and built-in contamination can sit back and return weakly. Tend to be strong. In contrast to walleye pollock, which is often used as a surimi after being exposed to water, fish such as Itoyori, Kinmedai, sardine, and horse mackerel are weak in gel-forming ability, but have strong umami, so they are attractive in terms of flavor improvement, Used as a raw material for paste products.
これら魚種の選択だけでなく、坐りや戻りに影響を及ぼす添加物も使用されている。例えば、特許文献１では、魚肉をカルシウム塩の溶液で処理した後、血漿蛋白、卵白および牛乳の一つ以上を添加している。特許文献２では、魚肉に牛または豚の血漿粉末を添加することにより弾力増強の効果を挙げている。特許文献３では、魚肉にトランスグルタミナーゼ、血清、血漿または卵白を添加して品質を向上している。特許文献４では、魚肉に大豆たん白、卵白および油脂を乳化して添加している。
ゲル形成能が低下する原因の一つに魚肉に含まれるプロテアーゼの影響がある。上記の従来技術のうち、血漿、卵白などはプロテアーゼインヒビター活性を有することが知られている。プロテアーゼにはいくつかの種類があり、それらが含まれている比率は魚種により異なる。スケトウダラ、パシフィックホワイティングなどの魚種ではシステイン系のプロテアーゼが主体であり、アジ、タチウオなどの魚種ではセリン系プロテアーゼが主体であり、トリプシンはこのセリン系プロテアーゼに分類される。また、ホッケ、エソなどでは両方のプロテアーゼが含まれていることが知られている。魚種によって、適したプロテアーゼインヒビターを使用することにより坐りの低下を抑制することができる。
特許文献５には、トリプシンインヒビター活性を有する全脂大豆粉又は大豆ホエーをすり身や練製品に使用することが記載されている。トリプシンインヒビター活性を有する全脂大豆粉又は大豆ホエーを添加することにより魚肉練製品の製造時に戻り防止効果が発揮されると記載されている。各種大豆蛋白質もすり身、練製品の添加物として多用されているが、これら大豆蛋白質は加熱処理が施されているため、トリプシンインヒビター活性を有していない。非加熱の全脂大豆粉など限られた大豆粉のみがトリプシンインヒビター活性を有する。
In addition to the selection of these fish species, additives that affect sitting and returning are also used. For example, in
One of the causes of a decrease in gel forming ability is the effect of proteases contained in fish meat. Among the above conventional techniques, plasma, egg white and the like are known to have protease inhibitor activity. There are several types of proteases, and the proportions in which they are contained vary depending on the fish species. Fish species such as walleye pollock and pacific whiting are mainly cysteine-based proteases, and fish species such as horse mackerel and red-tailed fish are mainly serine-based proteases, and trypsin is classified as this serine-based protease. In addition, it is known that both proteases are included in hockey, esos and the like. Depending on the fish species, sitting down can be suppressed by using a suitable protease inhibitor.
Patent Document 5 describes the use of full-fat soybean flour or soybean whey having trypsin inhibitor activity in surimi and paste products. It is described that, by adding full-fat soy flour or soy whey having trypsin inhibitor activity, an anti-return effect is exhibited during the production of fish paste products. Various soy proteins are also frequently used as additives in surimi and paste products, but these soy proteins are not heat-treated and therefore do not have trypsin inhibitor activity. Only limited soy flour, such as unheated full fat soy flour, has trypsin inhibitor activity.
本発明は、魚肉すり身、魚肉練製品の品質改良剤を提供することを課題とする。特にセリン系プロテアーゼを含有する魚肉を用いる場合に適した品質改良剤を提供することを課題とする。 This invention makes it a subject to provide the quality improving agent of fish meat surimi and a fish paste product. In particular, it is an object to provide a quality improving agent suitable for use of fish meat containing a serine protease.
発明者らは、全脂大豆粉をすり身や練製品に使用することについて検討してみたところ、その効果が魚肉によって大きく異なることを見出した。この原因を追究する中で、大豆粉にトリプシンインヒビター活性だけでなく、坐りを抑制する活性があることを見出した。この坐り阻害活性を除去するため種々の方策を試みた結果、リポキシゲナーゼが関与していることを発見し、この遺伝子欠損大豆を用いることで坐り阻害活性を低下あるいは除去させることができ、かつ、トリプシンインヒビター活性を維持できることを見出し、本発明を完成させた。 The inventors have examined the use of full-fat soy flour for surimi and paste products, and have found that the effect varies greatly depending on the fish meat. In pursuit of this cause, it was found that soybean flour has not only trypsin inhibitor activity but also activity to suppress sitting. As a result of various measures to remove the sitting inhibition activity, it was discovered that lipoxygenase was involved, and using this gene-deficient soybean, the sitting inhibition activity could be reduced or eliminated, and trypsin The inventors have found that inhibitor activity can be maintained, and have completed the present invention.
本発明は、以下（１）〜（１０）の魚肉練製品、魚肉練製品の製造方法、魚肉用添加物及びそれを用いた魚肉すり身、練製品を要旨とする。
（１）添加物としてリポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を用いたことを特徴とする坐り工程を経て製造される魚肉練製品。
（２）坐り工程を経て製造される魚肉練製品の製造において、添加物の大豆粉としてリポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を用いる方法。
（３）坐り工程を経て製造される魚肉練製品の製造において、トリプシンインヒビターとしてリポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を用いる方法。
（４）リポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を含有する、トリプシンインヒビター活性が保持され、坐り阻害活性は低下あるいは除去された魚肉用添加物。
（５）（４）の魚肉用添加物が添加された魚肉すり身。
（６）魚肉がセリン系プロテアーゼを含有する魚肉である（５）の魚肉すり身。
（７）魚肉に対してリポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を0.1〜5.0重量％添加したものである（５）又は（６）の魚肉すり身。
（８）（４）の魚肉用添加物が添加された魚肉練製品。
（９）セリン系プロテアーゼを含有する魚肉を原料として含有する魚肉練製品である（８）の魚肉練製品。
（１０）魚肉に対してリポキシゲナーゼ遺伝子を欠損させた大豆を原料とする大豆粉を0.1〜5.0重量％添加したものである（８）又は（９）の魚肉練製品。
The gist of the present invention is the following (1) to (10) fish paste product, a method for producing a fish paste product, an additive for fish meat, a fish paste using the same, and a paste product.
(1) A fish paste product produced through a sitting process, characterized by using soybean flour made from soybeans deficient in the lipoxygenase gene as an additive.
(2) A method of using soybean flour made from soybeans deficient in lipoxygenase gene as additive soybean flour in the production of fish paste products produced through a sitting process.
(3) A method of using soybean flour made from soybeans deficient in the lipoxygenase gene as a trypsin inhibitor in the manufacture of fish paste products manufactured through a sitting step.
(4) An additive for fish meat containing soybean flour made from soybeans deficient in the lipoxygenase gene, having trypsin inhibitor activity retained and reduced or eliminated sitting inhibition activity.
(5) Fish surimi to which the fish meat additive of (4) is added.
(6) The fish meat surimi according to (5), wherein the fish meat is a fish meat containing a serine protease.
(7) The fish meat surimi according to (5) or (6), wherein 0.1 to 5.0% by weight of soybean flour made from soybeans with lipoxygenase gene deficient in fish meat is added.
(8) A fish paste product to which the fish meat additive of (4) is added.
(9) The fish paste product according to (8), which is a fish paste product containing fish meat containing a serine protease as a raw material.
(10) The fish paste product according to (8) or (9), wherein 0.1 to 5.0% by weight of soybean flour made from soybeans deficient in the lipoxygenase gene is added to the fish meat.
本発明のリポキシゲナーゼ遺伝子欠損大豆粉からなる魚肉用添加物は、トリプシンインヒビター活性が保持され、かつ、坐り阻害活性が低下あるいは除去されているので、魚肉すり身又は魚肉練製品に添加することにより、それらの物性を向上させることができる。特に、坐りが強い魚肉とセリン系プロテアーゼを含む魚肉を混合して練製品を製造する場合に、坐りに影響を及ぼすことなくセリン系プロテアーゼ活性を抑制することができるため、複数の魚肉を混合して用いる場合に非常に有用である。 The additive for fish meat comprising the lipoxygenase gene-deficient soybean powder of the present invention retains trypsin inhibitor activity and has reduced or eliminated sitting inhibition activity. Therefore, by adding to fish surimi or fish paste products, The physical properties of can be improved. In particular, when producing a paste product by mixing fish meat with strong sitting and fish meat containing serine protease, serine protease activity can be suppressed without affecting sitting, so multiple fish meat can be mixed. It is very useful when used.
本発明において、魚肉練製品とは、カマボコ、ちくわ、さつま揚げ、魚肉ソーセージ等の、魚肉を主成分とする通常の水産練製品を指す。魚肉練製品は魚肉に副原料、例えば澱粉、グルテン、食塩、糖類、糖アルコール、調味料、香辛料、着色料等を添加して製造される。練製品の原料となるすり身は、原料魚から採肉、水晒し、脱水、砕肉等の工程により製造される。水晒ししない落し身も練製品の原料として使用される。練製品はすり身又は落し身などに副原料を添加し、擂潰、調味、成形、加熱、冷却等の工程を経て製造される。坐り工程は、通常成型後、15〜50℃、好ましくは20〜40℃の温度下に10分〜20時間、置く工程をいい、この工程により魚肉の弾力が高まる。 In the present invention, the fish paste product refers to a normal fish paste product mainly composed of fish meat, such as sea cucumber, chikuwa, fried fish cake, and fish sausage. Fish paste products are produced by adding auxiliary ingredients such as starch, gluten, salt, sugar, sugar alcohol, seasonings, spices, coloring agents and the like to fish meat. Surimi, which is a raw material for paste products, is produced from raw fish by processes such as meat extraction, water exposure, dehydration, and crushed meat. Lost meat that is not exposed to water is also used as a raw material for paste products. A paste product is produced by adding auxiliary materials to surimi or sacrificial meat, etc., and undergoing processes such as crushing, seasoning, molding, heating, and cooling. The sitting step is usually a step of placing at a temperature of 15 to 50 ° C., preferably 20 to 40 ° C. for 10 minutes to 20 hours after molding, and this step increases the elasticity of fish meat.
本発明においてリポキシゲナーゼ遺伝子欠損大豆粉とは、リポキシゲナーゼ遺伝子欠損大豆の乾燥生丸大豆を破砕し粒径を整えた大豆粉末であり、全脂大豆粉に分類される。リポキシゲナーゼ遺伝子欠損大豆粉として市販されている未加熱の大豆粉も同様に利用することが出来る。大豆を原料とする素材として脱脂大豆、濃縮大豆蛋白、分離大豆蛋白などがあるが、これらは通常製造段階で加熱処理を施しているため、トリプシンインヒビター活性を有していない。したがって、リポキシゲナーゼ遺伝子欠損大豆を原料としていても、トリプシンインヒビター活性が失活するような処理を施した大豆粉は本発明の大豆粉として使用することはできない。トリプシンインヒビター活性を有するリポキシゲナーゼ遺伝子欠損大豆粉としては、LKソイ・ファインS（フードブリッジ・ジャパン製）などが販売されている。
一方、大豆由来の植物蛋白質製品は、大豆に含まれる各種の蛋白質を用途により振り分けて製造された大豆蛋白質の粉状または繊維状の製品であり、全脂大豆粉のような大豆そのものを粉にしたものと製法が基本的に異なる。これらについても高温での加熱など、トリプシンインヒビター活性を失活させる条件で加工された大豆粉は、リポキシゲナーゼ遺伝子欠損大豆を原料としていても、本発明の大豆粉としては不適当である。
In the present invention, the lipoxygenase gene-deficient soybean powder is a soybean powder obtained by crushing dry raw whole soybeans of lipoxygenase gene-deficient soybean and adjusting the particle size, and is classified as full-fat soybean powder. Unheated soybean flour commercially available as lipoxygenase gene-deficient soybean flour can also be used in the same manner. There are defatted soybean, concentrated soybean protein, isolated soybean protein, and the like as raw materials from soybean, but these do not have trypsin inhibitor activity because they are usually heat-treated at the production stage. Therefore, even if lipoxygenase gene-deficient soybean is used as a raw material, soybean powder that has been treated to deactivate trypsin inhibitor activity cannot be used as the soybean powder of the present invention. As a lipoxygenase gene-deficient soybean flour having trypsin inhibitor activity, LK Soy Fine S (manufactured by Food Bridge Japan) and the like are sold.
On the other hand, vegetable protein products derived from soybeans are soy protein powders or fibers produced by sorting various proteins contained in soybeans according to their use. Soy beans such as full fat soybean flour are used as flour. The manufacturing method is basically different. In these cases, soy flour processed under conditions that inactivate trypsin inhibitor activity, such as heating at a high temperature, is unsuitable as the soy flour of the present invention even if lipoxygenase gene-deficient soybean is used as a raw material.
本発明におけるリポキシゲナーゼ遺伝子欠損大豆とは、大豆あるいは大豆加工製品青臭みを発生させ、また、大豆油脂を酸敗させる主要因のリポキシゲナーゼ酵素を、育種によって欠失させた大豆をさす。大豆に含まれるリポキシゲナーゼ酵素にはL-1、L-2、L-3のアイソザイムが存在しており、その一部あるいはすべてを欠損させた大豆が、リポキシゲナーゼ遺伝子欠損大豆となる。この大豆にはリポキシゲナーゼ活性以外の機能は、普通の大豆と変わることなく備わっており、普通の大豆として食品等に使用可能である。 The lipoxygenase gene-deficient soybean in the present invention refers to soybeans that have been bred with the lipoxygenase enzyme, which is the main factor that generates soybeans or a blue odor of processed soybean products, and that also oxidizes soybean oil. The lipoxygenase enzyme contained in soybean has L-1, L-2, and L-3 isozymes, and soybean lacking part or all thereof is lipoxygenase gene-deficient soybean. This soybean has functions other than the lipoxygenase activity without changing from ordinary soybean, and can be used as food in foods and the like as ordinary soybean.
大豆粉を練製品に増量のために使用する場合もあるが、生大豆粉を使用する主な目的は大豆粉が有するトリプリンインヒビター活性を利用するためである。魚肉には種々のプロテアーゼが含まれているが、スケトウダラ、パシフィックホワイティングなどの魚種ではシステイン系のプロテアーゼが主体であり、アジ、タチウオ、エソ、ホッケなどの魚種ではセリン系プロテアーゼが主体であり、トリプシンはこのセリン系プロテアーゼに分類される。また、ホッケ、エソなどでは両方のプロテアーゼが含まれている。したがって、セリン系プロテアーゼを含有する魚肉を練製品原料として用いる場合に、大豆粉を添加物として使用すると、プロテアーゼによる魚肉の弾力の低下を抑制することができる。 In some cases, soy flour is used to increase the weight of paste products, but the main purpose of using raw soy flour is to utilize the triprin inhibitor activity of soy flour. Fish meat contains various proteases, but fish species such as walleye pollock and pacific whiting are mainly cysteine proteases, and fish species such as horse mackerel, prickly fish, esos and hockey are mainly serine proteases. Yes, trypsin is classified into this serine protease. Moreover, both proteases are included in hockey and esos. Therefore, when using fish meat containing serine-based protease as a raw material for paste products, the use of soy flour as an additive can suppress a decrease in fish meat elasticity caused by protease.
ところが、実施例に示すように、通常の生大豆を原料とする大豆粉を魚肉に添加して練製品を製造しようとすると、坐りを抑制してしまう。もともと坐りの弱い魚種に添加した場合には目立たないが、品質の良いスケトウダラすり身のような本来坐りが強い魚肉に添加すると、この坐り抑制作用が強く現れる。本発明は、リポキシゲナーゼ遺伝子欠損大豆を用いることで、この坐り抑制作用を低下、除去させる技術である。本来トリプシンインヒビター活性によって、練製品の坐りを向上させるために添加される大豆粉のリポキシゲナーゼが逆の効果を示すことを見出し、リポキシゲナーゼ遺伝子欠損大豆を用いることで、その効果を除去することに成功したものである。 However, as shown in the Examples, when trying to produce a paste product by adding soybean powder made from normal raw soybeans to fish meat, sitting is suppressed. Although it is not conspicuous when it is added to fish species that are originally poorly seated, when it is added to fish meat that has a strong sitting, such as high-quality ground pollock surimi, this anti-sitting effect appears strongly. The present invention is a technique for reducing and eliminating this sitting-inhibiting action by using soybean deficient in lipoxygenase gene. Originally found that lipoxygenase in soybean flour added to improve the sitting of paste products by trypsin inhibitor activity showed the opposite effect, and succeeded in removing the effect by using lipoxygenase gene-deficient soybean Is.
本発明のリポキシゲナーゼ遺伝子欠損大豆を原料とする大豆粉からなる魚肉用添加物は魚肉すり身への添加物又は練製品製造時の添加物として使用することができる。魚肉すり身に添加する場合、使用する魚肉の魚種によって添加量を調節する。セリン系プロテアーゼ活性が高い魚肉の場合、多く使用し、活性が低い魚肉の場合、少量の使用でよい。具体的には、魚肉重量当たり0.1〜5.0重量％、好ましくは0.1〜3重量％用いる。糖類、リン酸塩、調味料等の公知の成分と併用してもよい。また、血漿、卵白などの他のタイプのプロテアーゼインヒビターと併用してもよい。練製品添加物として製品にする場合、単独で添加しても他の副原料などとの混合物として添加してもよい。添加量は、原料の魚種、その品質、最終製品に要求される物性などにより変動するため、一概に特定できないが、通常、魚肉に対して、0.1〜5.0重量％である。添加量が0.1重量％未満であると、効果は脆弱となり、5.0％を超えて添加すると魚肉特有のテクスチャーが失われる恐れがある。
本発明は複数の種類の魚肉を混合して用いる場合に特に有用であり、混合する魚肉の種類、量比によって、大豆粉の添加量を調節する。
本発明の大豆粉を添加する時期は、すり身製造時、練製品製造時にいずれでもよく、最終製品に要求される弾力に応じて添加量を調節する。
The additive for fish meat made of soybean powder made from the lipoxygenase gene-deficient soybean of the present invention can be used as an additive to fish meat surimi or as an additive for producing paste products. When adding to surimi fish meat, adjust the amount depending on the type of fish used. In the case of fish meat having a high serine protease activity, a large amount is used, and in the case of fish meat having a low activity, a small amount may be used. Specifically, it is used in an amount of 0.1 to 5.0% by weight, preferably 0.1 to 3% by weight, based on the weight of fish meat. You may use together with well-known components, such as saccharides, a phosphate, and a seasoning. Moreover, you may use together with other types of protease inhibitors, such as plasma and egg white. When making into a product as a paste product additive, it may be added alone or as a mixture with other auxiliary materials. The amount added varies depending on the fish species of the raw material, its quality, physical properties required for the final product, etc., and thus cannot be specified generally, but is usually 0.1 to 5.0% by weight with respect to the fish meat. If the amount added is less than 0.1% by weight, the effect becomes brittle, and if added over 5.0%, the texture unique to fish may be lost.
The present invention is particularly useful when a plurality of types of fish meat are mixed and used, and the amount of soybean powder added is adjusted according to the type and amount ratio of the fish meat to be mixed.
The soybean flour of the present invention may be added at any time during the production of surimi or during the manufacture of the paste product, and the amount added is adjusted according to the elasticity required for the final product.
本発明においては、加熱等によりセリンプロテアーゼインヒビター活性が失活せず、維持された状態で粉体化されていることが重要である。非加熱の全脂大豆粉とリポキシゲナーゼ遺伝子欠損大豆粉のセリンプロテアーゼインヒビター活性を、同じセリンプロテアーゼであるトリプシンの阻害活性の指標として、50％阻害活性(50% Inhibitory concentration;IC50)を用い解析した。
すなわち、トリプシン活性を、合成基質としてBoc-Gln-Ala-Arg-MACを使用して測定した。トリプシンインヒビター活性は、トリプシンを20mM Tris pH7.5/0.1M NaClにて20μg/mlに希釈したもの50μlに、被検物質（研究用試薬大豆トリプシンインヒビター(STIH)、非加熱の全脂大豆粉、又は、リポキシゲナーゼ遺伝子欠損大豆粉を各濃度(10μg〜0.01μg/ml)に希釈したもの750μlを加え、37℃で3分間プレインキュベートした。その後、合成基質6.24mg/mlを200μl加え反応液とし、37℃で6分間インキュベートした。その後1mM pAPMSFを1.5ml加え反応を停止し、Ex380nm,Em460nmで蛍光強度を測定した。このときの反応液中に含まれる重量あたりのIC50を比較することで、各種大豆粉の活性を評価した。
IC50は大豆粉の各濃度の阻害率からA:50%阻害をはさむ高濃度側濃度、B:50%阻害をはさむ低濃度側濃度、C:Bでの阻害率、D:Aでの阻害率を用い、計算式
IC50=10^(Log(A/B)*(50-C)/(D-C)+Log(B))
に沿って算出し
リポキシゲナーゼ遺伝子欠損大豆粉および乾燥生丸大豆粉のIC50は概ね0.6〜0.4μg/mlという値であった。トリプシンインヒビター活性が多少弱くても添加量を多くすればいいともいえるが、実用性を考えれば、添加量は少ないほどよく、トリプシンインヒビター活性をID50が0.8μg/ml以下程度に保持することが好ましい。
In the present invention, it is important that the serine protease inhibitor activity is not inactivated by heating or the like and is powdered in a maintained state. Serine protease inhibitor activity of unheated full fat soybean flour and lipoxygenase gene-deficient soybean flour was analyzed using 50% inhibitory activity (IC50) as an index of the inhibitory activity of trypsin, the same serine protease.
That is, trypsin activity was measured using Boc-Gln-Ala-Arg-MAC as a synthetic substrate. Trypsin inhibitor activity was determined by adding 50 μl of trypsin diluted to 20 μg / ml with 20 mM Tris pH 7.5 / 0.1 M NaCl, the test substance (research reagent soybean trypsin inhibitor (STIH), non-heated whole fat soybean flour, Alternatively, 750 μl of lipoxygenase gene-deficient soybean flour diluted to each concentration (10 μg to 0.01 μg / ml) was added and preincubated for 3 minutes at 37 ° C. Then, 200 μl of synthetic substrate 6.24 mg / ml was added to form a reaction solution, The mixture was incubated for 6 minutes at 37 ° C. After that, 1.5 ml of 1 mM pAPMSF was added to stop the reaction, and the fluorescence intensity was measured at Ex380 nm and Em460 nm, by comparing the IC50 per weight contained in the reaction solution. The activity of soy flour was evaluated.
IC50 is from the inhibition rate of each concentration of soybean flour, A: High concentration side concentration that sandwiches 50% inhibition, B: Low concentration side concentration that sandwiches 50% inhibition, C: Inhibition rate at B, D: Inhibition rate at A To calculate
IC50 = 10 ^ (Log (A / B) * (50-C) / (DC) + Log (B))
The IC50 of the lipoxygenase gene-deficient soybean flour and dried raw whole soybean flour was approximately 0.6 to 0.4 μg / ml. Although it can be said that the addition amount can be increased even if the trypsin inhibitor activity is somewhat weak, considering the practicality, the addition amount is preferably as small as possible, and it is preferable to keep the trypsin inhibitor activity at an ID50 of about 0.8 μg / ml or less.
以下に本発明の実施例を記載するが、本発明はこれらに何ら限定されるものではない。 Examples of the present invention will be described below, but the present invention is not limited thereto.
大豆粉のカマボコへの添加効果（坐り単品試験）
市販の全脂大豆粉（ソーヤフラワーNSA：日清オイリオ製）（図中では生大豆と表記）およびリポキシゲナーゼ遺伝子欠損大豆粉（LKソイ・ファインS：フードブリッジ・ジャパン製）（図中では欠損大豆と表記）について、常法により調整されたスケトウダラのすり身に対する添加試験を行った。すなわち、冷凍すり身(市販の無塩冷凍すり身、FAグレード)を解凍し、フードカッターを用いてこれを粗擂り、次いで食塩をすり身に対して1重量％添加して塩擂り、さらに2種類の大豆粉（非加熱の全脂大豆粉、リポキシゲナーゼ遺伝子欠損大豆粉）のいずれかをすり身に対して0.3重量％添加し、すり身に対して40% の加水を行い、本擂りを行った（コントロールは、いずれの大豆粉も添加しないで調製した）。その練り肉を、ポリ塩化ビニリデンフィルムに充填し、30℃で60分加熱した後90℃で40分間加熱してカマボコを調製した。本条件は坐りがよく促進される条件である。
また、上記の大豆粉に替えて、２種の大豆粉を単独あるいは、比率を変えて混合して0.3重量％添加したもの、および、試薬のリポキシゲナーゼである15-Lipoxygenase(sigma社製)を添加したものも調製した。
得られたカマボコのジェリー強度を測定した。ジェリー強度は上記の各カマボコを厚さ2.5cmの輪切りにし、5mm径球状のプランジャーを用いて測定した破断強度(w値、g)と、破断までの距離(L値、cm)を掛け合わせたJ.S.(g・cm)で表した。
Effect of soybean powder added to the sea urchin (sitting single item test)
Commercially available full-fat soy flour (Soya Flour NSA: Nisshin Oilio) (shown as raw soy in the diagram) and lipoxygenase gene-deficient soy flour (LK Soy Fine S: Food Bridge Japan) (deficient soy in the diagram) In addition, an addition test was conducted on a surimi of walleye pollack prepared by a conventional method. In other words, frozen surimi (commercial unsalted frozen surimi, FA grade) is thawed and coarsely crushed using a food cutter, then 1% by weight of salt is added to the surimi and salted, and two more soybeans One of the flour (non-heated whole fat soybean powder, lipoxygenase gene-deficient soybean powder) was added to the surimi at 0.3% by weight, and the surimi was hydrated at 40% (control was It was prepared without adding any soy flour). The kneaded meat was filled into a polyvinylidene chloride film, heated at 30 ° C. for 60 minutes, and then heated at 90 ° C. for 40 minutes to prepare a sea urchin. This is a condition that facilitates sitting well.
Also, instead of the above-mentioned soybean flour, two types of soybean flour are added alone or mixed at different ratios and added at 0.3% by weight, and the reagent lipoxygenase 15-Lipoxygenase (manufactured by sigma) is added A prepared product was also prepared.
The jelly strength of the resulting sea urchin was measured. Jelly strength is obtained by multiplying each of the above-mentioned clams into a 2.5 cm thick slice and multiplying the breaking strength (w value, g) measured using a 5 mm diameter spherical plunger and the distance to the breaking (L value, cm). JS (g · cm).
結果を図１〜３に示した。図１に示したように全脂大豆粉を添加したものでは、坐りが著しく低下するのに対して、リポキシゲナーゼ遺伝子欠損大豆粉では、コントロールと比較して遜色無いジェリー強度が認められた。図２に示したように、リポキシゲナーゼ遺伝子欠損大豆粉の割合が増加するに従い、坐りの低下がなくなることが確認された。図３に示すように、15-Lipoxygenaseの添加により、濃度依存的に坐りが低下することが認められた。また、加熱により酵素活性を失活すると坐りを阻害する活性も失活した。
これらの結果より、通常の生の大豆を粉体化してすり身に添加した場合、著しくカマボコのゲル強度は低下する。このゲル強度の低下は、リポキシゲナーゼ遺伝子欠損大豆の粉を添加した場合には認められず、試薬のリポキシゲナーゼでも同じ作用が確認されたことから、リポキシゲナーゼが関与することが確認された。
The results are shown in FIGS. As shown in FIG. 1, in the case of adding full-fat soy flour, sitting was significantly reduced, whereas in lipoxygenase gene-deficient soy flour, a comparable jelly strength was observed compared to the control. As shown in FIG. 2, it was confirmed that as the ratio of the lipoxygenase gene-deficient soybean flour increases, the decrease in sitting is eliminated. As shown in FIG. 3, it was recognized that the addition of 15-Lipoxygenase lowered the sitting in a concentration-dependent manner. In addition, when the enzyme activity was deactivated by heating, the activity of inhibiting sitting was also deactivated.
From these results, when normal raw soybeans are pulverized and added to surimi, the gel strength of sea urchins is significantly reduced. This reduction in gel strength was not observed when lipoxygenase gene-deficient soybean flour was added, and the same action was confirmed with the reagent lipoxygenase, confirming that lipoxygenase was involved.
リポキシゲナーゼ活性確認試験
大豆粉に含まれるリポキシゲナーゼ活性を、メチレンブルーを用いた比色試験により評価した。リポキシゲナーゼ遺伝子欠損大豆粉(LKソイ・ファインS：フードブリッジ・ジャパン製）および全脂大豆粉（ソーヤフラワーNSA：日清オイリオ製）のトリプシンインヒビター活性を測定した。すなわち、0.5％ホウ酸 pH9.0、0.3％リノール酸、0.05％メチレンブルーをそれぞれ5：1：1の割合で混合し、その混合液に市販の全脂大豆粉（ソーヤフラワーNSA：日清オイリオ製）およびリポキシゲナーゼ遺伝子欠損大豆粉（LKソイ・ファインS：フードブリッジ・ジャパン製）の100,50,25,12.5,5,2,5 mg/mlの溶液上清を0.5の割合で添加し25℃で20分間反応させた。その後、Final 10％となるようにオルトフェナントロリンを加え反応を停止し、比色により両者の比較を行った。
図４に示したように、全脂大豆粉（図中、生大豆と表記）には明らかにリポキシゲナーゼ活性が認められたが、リポキシゲナーゼ遺伝子欠損大豆粉（図中、欠損大豆と表記）には活性は認められなかった。
Lipoxygenase activity confirmation test Lipoxygenase activity contained in soybean flour was evaluated by a colorimetric test using methylene blue. The trypsin inhibitor activity of lipoxygenase gene-deficient soybean flour (LK Soy Fine S: manufactured by Food Bridge Japan) and full fat soybean flour (Soya Flower NSA: manufactured by Nisshin Oilio) was measured. In other words, 0.5% boric acid pH 9.0, 0.3% linoleic acid, 0.05% methylene blue were mixed at a ratio of 5: 1: 1, respectively, and commercially available full-fat soy flour (Soya Flour NSA: made by Nisshin Oilio) ) And 100,50,25,12.5,5,2,5 mg / ml solution supernatant of lipoxygenase gene-deficient soybean flour (LK Soy Fine S: Food Bridge Japan) at a rate of 0.5 For 20 minutes. Thereafter, orthophenanthroline was added so that the final concentration would be 10%, the reaction was stopped, and both were compared by colorimetry.
As shown in FIG. 4, lipoxygenase activity was clearly observed in full-fat soy flour (shown as raw soybean in the figure), but active in lipoxygenase gene-deficient soybean flour (shown as defective soybean in the figure). Was not recognized.
トリプシンインヒビター（TI）活性確認試験
大豆粉に含まれるトリプシンインヒビターの活性を合成基質を用いて評価した。リポキシゲナーゼ遺伝子欠損大豆粉(LKソイ・ファインS：フードブリッジ・ジャパン製）および全脂大豆粉（ソーヤフラワーNSA：日清オイリオ製）のトリプシンインヒビター活性を測定した。コントロールとしてトリプシンインヒビター試薬(Trypsin Inhibitor：nacalai tesque)を用いた。
トリプシンの阻害活性をIC50で評価した。プロテアーゼとしてトリプシン(30USPunit/mg 和光純薬)を、基質としてBoc-Gln-Ala-Arg-MAC(ペプチド研究所)を用い、分解に伴う蛍光の変化を求め、阻害活性を求めた。IC50は全脂大豆粉の各濃度の阻害率からA:50%阻害をはさむ高濃度側濃度、B:50%阻害をはさむ低濃度側濃度、C:Bでの阻害率、D:Aでの阻害率を用い、
IC50=10^(Log(A/B)*(50-C)/(D-C)+Log(B))
に沿って算出した。
結果を図５および表１に示した。全脂大豆粉、リポキシゲナーゼ遺伝子欠損大豆粉のいずれもトリプシンインヒビター活性が認められた。
Trypsin inhibitor (TI) activity confirmation test The activity of trypsin inhibitor contained in soybean flour was evaluated using a synthetic substrate. The trypsin inhibitor activity of lipoxygenase gene-deficient soybean flour (LK Soy Fine S: manufactured by Food Bridge Japan) and full fat soybean flour (Soya Flower NSA: manufactured by Nisshin Oilio) was measured. A trypsin inhibitor reagent (Trypsin Inhibitor: nacalai tesque) was used as a control.
The inhibitory activity of trypsin was evaluated by IC50. Using trypsin (30 USPunit / mg Wako Pure Chemicals) as a protease and Boc-Gln-Ala-Arg-MAC (Peptide Institute) as a substrate, the change in fluorescence accompanying degradation was determined, and the inhibitory activity was determined. IC50 is based on the inhibition rate of each concentration of whole fat soy flour, A: High concentration side concentration holding 50% inhibition, B: Low concentration side concentration holding 50% inhibition, C: Inhibition rate at B, D: A Using the inhibition rate,
IC50 = 10 ^ (Log (A / B) * (50-C) / (DC) + Log (B))
Calculated along with
The results are shown in FIG. All fat soy flour and lipoxygenase gene-deficient soy flour showed trypsin inhibitor activity.
２種類の魚肉を用いたカマボコへの添加試験
本発明のリポキシゲナーゼ遺伝子欠損大豆を原料とする大豆粉の作用を確認するため、セリン系プロテアーゼが多く含まれるタチウオすり身と上級スケトウダラすり身を混合した単品試験を行った。タチウオすり身はセリン系のプロテアーゼが混入しており、セリン系プロテアーゼインヒビターを添加しない状態では単品のジェリー強度の測定が困難なすり身である。一方、上級スケトウダラすり身は良好なジェリー強度が得られ、プロテアーゼの混入が少ないすり身である。この両者をタチウオ４：スケトウダラ６の比率で混合し試験に供した。
常法により調整されたすり身に対する添加試験を行った。すなわち、冷凍すり身(市販の無塩冷凍すり身)を解凍し、フードカッターを用いてこれを粗擂り、次いで食塩をすり身に対して1重量％添加して塩擂り、さらに２種の大豆粉（リポキシゲナーゼ遺伝子欠損大豆粉および全脂大豆粉）のいずれかをすり身に対して0.3重量％添加し、本擂りを行った。その後ポリ塩化ビニリデンフィルムに充填し、90℃で40分間加熱し、カマボコを調整した。コントロールは大豆粉を添加しないで調製した。得られたこれら３種類のカマボコのジェリー強度を実施例１と同様の方法で測定した。
Addition test to sea cucumber using two kinds of fish meat In order to confirm the action of soybean powder using the lipoxygenase gene-deficient soybean of the present invention as a raw material, a single item test in which Tachio surimi containing a large amount of serine-based protease and advanced pollock surimi were mixed Went. The sea urchin surimi is mixed with serine-based protease, and it is difficult to measure the jelly strength of a single product without adding a serine-based protease inhibitor. On the other hand, the high-grade walleye surimi is a surimi that has good jelly strength and is less contaminated with proteases. Both of these were mixed at a ratio of prickly fish 4: walleye pollack 6 and used for the test.
An addition test was conducted on surimi prepared by a conventional method. In other words, frozen surimi (commercial unsalted frozen surimi) is thawed and coarsely crushed using a food cutter, then 1% by weight of salt is added to the surimi and salted, and two soy flours (lipoxygenase) are added. Either gene-deficient soybean flour or whole-fat soybean flour) was added to the surimi and 0.3% by weight was added to the main body. After that, it was filled in a polyvinylidene chloride film and heated at 90 ° C. for 40 minutes to adjust the surface. The control was prepared without adding soy flour. The jelly strength of these three kinds of lobsters obtained was measured in the same manner as in Example 1.
図６に示すように、コントロールではジェリー強度が２００のすり身において、リポキシゲナーゼ遺伝子欠損大豆粉および全脂大豆粉を添加したすり身では、いずれもジェリー強度が向上することが示された。すなわち、トリプシンインヒビター活性に基づくと考えられる大豆粉の効果が両者に確認された。 As shown in FIG. 6, it was shown that in the surimi having a jelly strength of 200 in the control, both the lipoxygenase gene-deficient soybean flour and the whole fat soybean flour were improved in the jelly strength. That is, the effect of soybean flour considered to be based on trypsin inhibitor activity was confirmed by both.
２種類を混合した魚肉を用いたトリプシンインヒビター（TI）活性確認試験
本発明の大豆粉のトリプシンインヒビター活性を確認するため、タチウオ４：スケトウダラ６の比率で混合したすり身の内在のプロテアーゼによる蛋白質の分解の指標として遊離アミノ酸量を測定した。すなわち、冷凍すり身(市販の無塩冷凍すり身)を解凍し、5倍量の緩衝液(20mM Tris pH7.5/100mM NaCl)とともにホモジナイズした。リポキシゲナーゼ遺伝子欠損大豆粉および全脂大豆粉を0.3重量％添加し、無添加のホモジナイズ液とともにカマボコの戻りを引き起こす60℃で30分間加熱した。その後、一部を回収し、等量の15%トリクロロ酢酸を加え、蛋白質画分を除去し、等量の0.5M Na2CO3でｐＨを調整し遊離アミノ酸量をlowry法で求めた。コントロールは大豆粉を添加しないで調製した。
Trypsin inhibitor (TI) activity confirmation test using fish meat mixed with two kinds In order to confirm the trypsin inhibitor activity of the soybean flour of the present invention, protein degradation by the protease of the surimi mixed in the ratio of prickly fish 4: walleye pollack 6 The amount of free amino acid was measured as an index of That is, frozen surimi (commercial unsalted frozen surimi) was thawed and homogenized with 5 times the amount of buffer (20 mM Tris pH 7.5 / 100 mM NaCl). Lipoxygenase gene-deficient soybean flour and full-fat soybean flour were added at 0.3% by weight, and the mixture was heated at 60 ° C. for 30 minutes to cause the reversion of the sea urchin with the additive-free homogenizing solution. Thereafter, a part was recovered, an equal amount of 15% trichloroacetic acid was added, the protein fraction was removed, the pH was adjusted with an equal amount of 0.5M Na 2 CO 3 , and the free amino acid amount was determined by the lowry method. The control was prepared without adding soy flour.
図７に示したように、リポキシゲナーゼ遺伝子欠損大豆粉は、全脂大豆粉と同様に、すり身の内在プロテアーゼによる、蛋白質の分解を抑制した。この試験によっても、リポキシゲナーゼ遺伝子欠損大豆粉にプロテアーゼ阻害活性が保持されていることが示された。 As shown in FIG. 7, the lipoxygenase gene-deficient soybean flour suppressed protein degradation by surimi protease in the same manner as full-fat soybean flour. This test also showed that the lipoxygenase gene-deficient soybean flour retains protease inhibitory activity.
本発明のリポキシゲナーゼ遺伝子欠損大豆を原料とする大豆粉は、リポキシゲナーゼによる坐り抑制活性を防ぎ、かつ、トリプシンインヒビター活性が維持されているので、各種魚肉のすり身や練製品の製造において、品質改良効果を有する添加物として広く使用することができる。特に、複数の魚肉を混合して用いる場合でも使用が制限されることがない。 The soybean flour made from the lipoxygenase gene-deficient soybean of the present invention prevents the sitting inhibition activity by the lipoxygenase and maintains the trypsin inhibitor activity, so that it has a quality improving effect in the production of surimi and paste products of various fish meats. It can be widely used as an additive having. In particular, the use is not limited even when a plurality of fish meats are mixed and used.
Claims (10)
10. The fish paste product according to claim 8 or 9, wherein 0.1 to 5.0% by weight of soybean flour made from soybeans deficient in the lipoxygenase gene is added to the fish meat.
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