WO2011152330A1 - Soybean protein hydrolysate-containing antioxidant and use thereof - Google Patents

Soybean protein hydrolysate-containing antioxidant and use thereof Download PDF

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WO2011152330A1
WO2011152330A1 PCT/JP2011/062321 JP2011062321W WO2011152330A1 WO 2011152330 A1 WO2011152330 A1 WO 2011152330A1 JP 2011062321 W JP2011062321 W JP 2011062321W WO 2011152330 A1 WO2011152330 A1 WO 2011152330A1
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antioxidant
protein hydrolyzate
molecular weight
peptides
soy protein
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貴康 本山
恵子 松林
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不二製油株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/02—Antioxidant

Abstract

Disclosed is an antioxidant which exhibits high antioxidation activity and can be used in food, drink or pharmaceutical products. An antioxidant that exhibits high antioxidation activity can be provided by obtaining a soybean protein hydrolysate wherein the content of peptides having a molecular weight of less than 500 is 50% by weight or more relative to the total of peptides and free amino acids contained therein.

Description

大豆蛋白質加水分解物含有抗酸化剤及びその利用Antioxidant containing soybean protein hydrolyzate and use thereof
　本発明は大豆蛋白質白加水分解物を含有する抗酸化剤、及び大豆蛋白質加水分解物の抗酸化剤としての使用方法に関する。 The present invention relates to an antioxidant containing soybean protein white hydrolyzate, and a method of using soybean protein hydrolyzate as an antioxidant.
　ヒトを含む好気性生物は生存のために呼吸により酸素を取り込むが、このとき体内に取り込まれた酸素の一部はエネルギー代謝の際にスーパーオキシドラジカル、過酸化水素、ヒドロキシラジカル等の活性酸素種に変わる。このような活性酸素種は、元来、細菌やウイルスの感染時におけるマクロファージの病原体除去機構をはじめとする生体防御に関わるなど、健康維持に重要な役割を果たしている。しかし、大気汚染や紫外線などの環境要因や喫煙等の生活習慣、精神的ストレスなどにより生体内でのバランスが崩れ、活性酸素種がひとたび過剰となると生体中の蛋白質や脂質、ＤＮＡなどと反応して、蛋白質の変性や過酸化脂質の生成、遺伝損傷などを起こし、生活習慣病の発症や老化の促進をもたらすと考えられている。以上のことから、生体に備わったメカニズムに加え、食事由来の抗酸化活性成分の摂取が健康維持に重要と考えられるようになり、抗酸化物質は７番目の栄養素として積極的な摂取が奨励されてきている。 Aerobic organisms including humans take oxygen by respiration for survival, but some of the oxygen taken into the body at this time is reactive oxygen species such as superoxide radicals, hydrogen peroxide, and hydroxy radicals during energy metabolism. Changes to. Such reactive oxygen species originally played an important role in maintaining health, such as being involved in biological defense including the pathogen removal mechanism of macrophages during bacterial or viral infection. However, due to environmental factors such as air pollution and ultraviolet rays, lifestyle habits such as smoking, mental stress, etc., the balance in the body is lost, and once the active oxygen species becomes excessive, it reacts with proteins, lipids, DNA, etc. in the body. It is thought to cause protein denaturation, lipid peroxide generation, genetic damage, etc., leading to the onset of lifestyle-related diseases and aging. From the above, in addition to the mechanisms in the body, intake of dietary antioxidant active ingredients is considered important for maintaining health, and antioxidant substances are encouraged to be actively consumed as the seventh nutrient. It is coming.
　大豆は多くの生理機能を有していることが報告されており、抗酸化活性についてもいくつか報告されている。たとえば、大豆イソフラボンは他のフラボノイド類と同様に高い抗酸化活性を有していることが確認されている。また、大豆蛋白質加水分解物の抗酸化活性に関しては、抗酸化活性を示すペプチド配列について同定されている。しかしながら、大豆蛋白質加水分解物から特定のペプチドを大量に単離して抗酸化剤として使用することは製造上困難である。そこで、様々なペプチドの混合物である大豆蛋白質加水分解物そのものを抗酸化剤として用いることができれば有用である。
　その中で、特許文献１には、抗酸化力を増大させた大豆蛋白質の加水分解物が記載され、ペプチドの平均分子量は３ｋＤａ～３０ｋＤａ（３０００～３００００）であることが記載されている。本文献は加水分解しすぎることによる苦味の発現を回避するために、比較的高分子量のペプチドを得ている。 Soybeans are reported to have many physiological functions, and some antioxidant activities have been reported. For example, soybean isoflavone has been confirmed to have high antioxidant activity like other flavonoids. In addition, regarding the antioxidant activity of soybean protein hydrolyzate, peptide sequences exhibiting antioxidant activity have been identified. However, it is difficult in production to isolate a large amount of a specific peptide from soybean protein hydrolyzate and use it as an antioxidant. Therefore, it is useful if soybean protein hydrolyzate itself, which is a mixture of various peptides, can be used as an antioxidant.
Among them, Patent Document 1 describes a hydrolyzate of soybean protein having an increased antioxidant power, and describes that the average molecular weight of the peptide is 3 kDa to 30 kDa (3000 to 30000). This document obtains relatively high molecular weight peptides in order to avoid expression of bitterness due to excessive hydrolysis.
　また特許文献２及び３にも抗酸化力を示す大豆蛋白質の加水分解物が記載されているが、特許文献２に記載されているペプチドの平均分子量は５００～５０００であり、特許文献３のペプチドの平均アミノ酸鎖長は１０～１００個で、この分子量はおおよそ１２００以上に相当する。
　さらに、特許文献４には、抗酸化活性を有する大豆蛋白質加水分解物として、バチルス・ズブチリスやアスペルギルス・オリゼ等由来の種々のプロテーゼによる加水分解物が記載されており、該加水分解物は分子量が２０００以下のものをろ取すること、該加水分解物の遊離アミノ酸の含有割合が２０～３０重量％であることが記載されている。
　これらの大豆蛋白質加水分解物は一定の抗酸化活性を有すると認められるが、該活性は必ずしも抗酸化剤として実用化できるレベルではなく、さらに高い抗酸化活性を示す大豆蛋白質加水分解物が求められている。 Patent Documents 2 and 3 also describe a hydrolyzate of soy protein exhibiting antioxidative activity, but the average molecular weight of the peptide described in Patent Document 2 is 500 to 5000. The average amino acid chain length is 10 to 100, and the molecular weight is approximately 1200 or more.
Furthermore, Patent Document 4 describes hydrolysates by various prostheses derived from Bacillus subtilis, Aspergillus oryzae, etc., as soybean protein hydrolysates having antioxidant activity, and the hydrolysates have a molecular weight. It is described that 2,000 or less are collected by filtration, and the content of free amino acids in the hydrolyzate is 20 to 30% by weight.
Although these soy protein hydrolysates are recognized to have a certain antioxidant activity, the activities are not necessarily at a level that can be put to practical use as an antioxidant, and soy protein hydrolysates that exhibit higher antioxidant activity are required. ing.
特開２００５－８０６６８号公報JP 2005-80668 A 特開平１０－２０３９９４号公報Japanese Patent Laid-Open No. 10-203994 特開２０００－４８９６号公報Japanese Unexamined Patent Publication No. 2000-4896 特開２００３－２１０１３８号公報Japanese Patent Laid-Open No. 2003-210138
　本発明は、飲食品又は医薬品に利用可能な、高い抗酸化活性を示す抗酸化剤を提供することを課題とした。 This invention made it the subject to provide the antioxidant which shows the high antioxidant activity which can be utilized for food-drinks or a pharmaceutical.
　本発明者は本課題について鋭意検討した結果、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上の大豆蛋白質加水分解物を得ることで、従来よりも高い抗酸化活性を示す抗酸化剤とすることができることを見出し、本発明を完成するに至った。 As a result of intensive studies on this problem, the present inventor obtained a soybean protein hydrolyzate in which the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of peptides and free amino acids. The present inventors have found that an antioxidant having higher antioxidant activity than before can be obtained, and have completed the present invention.
　即ち本発明は、以下の（１）～（１３）を提供する。
（１）生体の酸化がもたらす疾病を予防又は治療する方法において有効成分として使用するための抗酸化物質であって、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含むこと特徴とする抗酸化物質、
（２）生体の酸化がもたらす疾病が動脈硬化、心筋梗塞、ガン、糖尿病、アルツハイマー、又は花粉症である前記（１）記載の抗酸化物質、
（３）前記大豆蛋白質加水分解物は加水分解後の不溶物が除去されたものである、前記（１）記載の抗酸化物質、
（４）ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質をさらに含む、前記（１）記載の抗酸化物質、
（５）生体の酸化がもたらす疾病を予防又は治療するために用いられる飲食品又は医薬品の製造において、有効成分である抗酸化物質として蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を添加することを特徴とする、該飲食品又は医薬品の製造法、
（６）ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質を併用する、前記（５）記載の飲食品又は医薬品の製造法、
（７）有効成分として蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含む抗酸化物質を使用することを特徴とする、生体の酸化がもたらす疾病の予防又は治療する方法、
（８）蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含有することを特徴とする抗酸化剤、
（９）前記大豆蛋白質加水分解物は加水分解後の不溶物が除去されている大豆蛋白質加水分解物である、前記（８）記載の抗酸化剤、
（１０）ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質をさらに含む、前記（８）記載の抗酸化剤、
（１１）前記（８）記載の抗酸化剤が添加された飲食品、
（１２）前記（８）記載の抗酸化剤を有効成分とする医薬品、
（１３）抗酸化剤の製造における、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解の使用方法。 That is, the present invention provides the following (1) to (13).
(1) Antioxidant for use as an active ingredient in a method for preventing or treating diseases caused by oxidation in a living body, wherein the content of peptides having a molecular weight of less than 500 in protein hydrolyzate is the sum of peptides and free amino acids An antioxidant comprising 50% by weight or more of a soy protein hydrolyzate,
(2) The antioxidant substance according to (1), wherein the disease caused by oxidation of the living body is arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer, or hay fever.
(3) The antioxidant substance according to (1), wherein the soy protein hydrolyzate is obtained by removing insoluble matters after hydrolysis,
(4) The antioxidant according to (1), further comprising one or more antioxidants selected from vitamins, polyphenols, carotenoids, saponins and allicin,
(5) In the production of foods and drinks or pharmaceuticals used for preventing or treating diseases caused by oxidation in the living body, the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate as antioxidants as active ingredients is peptides and Adding the soy protein hydrolyzate which is 50% by weight or more based on the total amount of free amino acids,
(6) The method for producing a food or drink or pharmaceutical according to (5) above, wherein one or more antioxidants selected from vitamins, polyphenols, carotenoids, saponins and allicin are used in combination,
(7) An antioxidant substance containing soybean protein hydrolyzate in which the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of peptides and free amino acids is used as an active ingredient. A method for preventing or treating diseases caused by oxidation of a living body,
(8) an antioxidant comprising a soy protein hydrolyzate in which the content of a peptide having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of the peptide and free amino acids;
(9) The antioxidant according to (8), wherein the soy protein hydrolyzate is a soy protein hydrolyzate from which insolubles after hydrolysis have been removed,
(10) The antioxidant according to (8), further comprising one or more antioxidants selected from vitamins, polyphenols, carotenoids, saponins, and allicin,
(11) A food or drink to which the antioxidant according to (8) is added,
(12) A pharmaceutical comprising the antioxidant according to (8) as an active ingredient,
(13) A method for using soy protein hydrolysis in which the content of a peptide having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of the peptide and free amino acid in the production of an antioxidant.
　本発明により、大豆蛋白質加水分解物から特定の抗酸化活性を有するペプチドを単離するのではなく、大豆蛋白質加水分解物（ペプチド混合物）そのものを用いた実用的な、高い抗酸化活性を示す抗酸化剤を提供することができ、これによって生体の酸化がもたらす動脈硬化等の疾病の予防又は治療に役立てることができる。 According to the present invention, a peptide having a specific antioxidant activity is not isolated from a soy protein hydrolyzate, but a practical, high antioxidant activity using a soy protein hydrolyzate (peptide mixture) itself. An oxidizing agent can be provided, which can be used for prevention or treatment of diseases such as arteriosclerosis caused by oxidation of a living body.
蛋白質加水分解物中の分子量５００未満のペプチドの含量とORAC値との相関関係を示すグラフである。It is a graph which shows the correlation with the content of the peptide of molecular weight less than 500 in a protein hydrolyzate, and an ORAC value.
　本発明の抗酸化剤、本発明の生体の酸化がもたらす疾病を予防又は治療する方法において有効成分として使用するための抗酸化物質、本発明の生体の酸化がもたらす疾病を予防又は治療するために用いられる飲食品又は医薬品の製造法、あるいは本発明の生体の酸化がもたらす疾病の予防又は治療する方法は、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含有するか、又は有効成分として使用することを特徴とする。以下、本発明について具体的に説明する。 The antioxidant of the present invention, the antioxidant for use as an active ingredient in the method of preventing or treating the disease caused by oxidation of the living body of the present invention, and the prevention or treatment of the disease caused by oxidation of the living body of the present invention In the method for producing a food or drink or pharmaceutical used, or the method for preventing or treating a disease caused by oxidation of a living body of the present invention, the content of a peptide having a molecular weight of less than 500 in the protein hydrolyzate is the total amount of the peptide and free amino acid. It contains 50% by weight or more of soy protein hydrolyzate or is used as an active ingredient. Hereinafter, the present invention will be specifically described.
（大豆蛋白質原料）
　本発明に係る大豆蛋白質加水分解物は、大豆蛋白質原料をプロテアーゼにより加水分解したものである。ここで大豆蛋白質原料の一例として、丸大豆や脱脂大豆等から蛋白質成分を水で抽出し、オカラ成分を除去した全脂豆乳や脱脂豆乳が挙げられる。大豆蛋白質原料の別の一例として、これら豆乳から、限外ろ過膜による処理や酸を用いた等電点沈殿等により蛋白質を濃縮した分離大豆蛋白質が挙げられる。大豆蛋白質原料のさらに別の例として、大豆から酸洗浄やエタノール洗浄によりホエー成分を除去して蛋白質を濃縮した濃縮大豆蛋白質や、大豆を粉砕した大豆粉が挙げられる。これらの大豆蛋白質原料は殺菌・乾燥した物でもよい。尚、最終的に得られる大豆蛋白質原料は、蛋白質を乾燥重量で８０重量％以上含むことが好ましく、例えば分離大豆蛋白質等が好ましい。
　分離大豆蛋白質は一般には以下の様に調製されるものである。すなわち、脱脂大豆に水を加え、中性付近にて抽出を行い、オカラを分離して豆乳を得る。次に豆乳をｐＨ４．５付近に調整し、等電点沈殿物を回収する。沈殿物に水及びアルカリ剤を加え、固形分濃度５～１５重量％、ｐＨ５．７～８．０好ましくはｐＨ６．８～７．５付近の水溶液を得る。この様にして得られた分離大豆蛋白質は、溶液をそのまま以下の工程に用いても良いし、未殺菌の物又は殺菌した物を乾燥後に改めて溶解して用いても良い。還元糖を予め水溶液とし、これに大豆蛋白質原料を溶解させることも可能である。ただし、分離大豆蛋白質は上記製法のみに限定されるものではなく、該製法を種々改変したものであってもよいことは無論である。 (Soy protein raw material)
The soy protein hydrolyzate according to the present invention is a soy protein raw material hydrolyzed with a protease. Here, as an example of the soy protein raw material, there are full-fat soy milk and defatted soy milk from which protein components are extracted with water from whole soybeans, defatted soybeans, etc., and the okara components are removed. As another example of the soy protein raw material, isolated soy protein obtained by concentrating the protein from these soymilks by treatment with an ultrafiltration membrane or isoelectric point precipitation using an acid can be mentioned. As another example of the soy protein raw material, concentrated soy protein obtained by removing whey components from soybean by acid washing or ethanol washing to concentrate the protein, and soybean powder obtained by pulverizing soybean. These soy protein raw materials may be sterilized and dried. In addition, it is preferable that the soybean protein raw material finally obtained contains 80 weight% or more of protein by dry weight, for example, isolation | separation soybean protein etc. are preferable.
Isolated soy protein is generally prepared as follows. That is, water is added to defatted soybean, extraction is performed near neutrality, soy milk is separated to obtain soy milk. Next, the soy milk is adjusted to around pH 4.5, and the isoelectric point precipitate is collected. Water and an alkaline agent are added to the precipitate to obtain an aqueous solution having a solid content concentration of 5 to 15% by weight, pH 5.7 to 8.0, preferably pH 6.8 to 7.5. The isolated soybean protein thus obtained may be used as it is in the following steps, or may be used after dissolving an unsterilized product or a sterilized product after drying. It is also possible to make reducing sugar an aqueous solution in advance and dissolve the soy protein raw material in this solution. However, the separated soybean protein is not limited to the above-described production method, and it goes without saying that the production method may be variously modified.
（大豆蛋白質加水分解物）
　本発明に係る大豆蛋白質加水分解物は、上記大豆蛋白質原料をプロテアーゼ処理することによって得られるペプチド混合物である。該蛋白質加水分解物は分解度がより高いことが好ましく、特に加水分解物中におけるペプチド及び遊離アミノ酸の合計量に占める分子量５００未満のペプチドの割合が高いことが好ましい。具体的には、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上であることが重要であり、６０重量％以上であることがさらに好ましい。
　この分子量５００未満のペプチドは、アミノ酸が２～３分子結合したジペプチドおよびトリペプチドから実質的に構成されるものである。
　分子量５００未満のペプチドの含量は、ペプチド用ゲルろ過クロマトグラフィーにより蛋白質加水分解物における分子量５００未満のペプチド及び遊離アミノ酸画分の割合を測定した後、アミノ酸分析により算出した蛋白質加水分解物中の遊離アミノ酸含量を差し引くことにより算出するものとする。
　上記のように特定される大豆蛋白質加水分解物は分子量５００未満のペプチド以外のペプチド及び遊離アミノ酸の割合をできるだけ低減したものが好ましい。すなわち、蛋白質加水分解物中の遊離アミノ酸含量はペプチド及び遊離アミノ酸の合計量に対して１２重量％以下であることが好ましく、５重量％以下であることがより好ましく、３重量％以下がさらに好ましい。さらに、該蛋白質加水分解物中のペプチド体はより低分子であることが望ましいことから、蛋白質加水分解物中の分子量５００以上の画分割合がペプチド及び遊離アミノ酸の合計量に対して４０重量％以下であることが好ましく、３８重量％以下であることがより好ましく、３５重量％以下がさらに好ましい。 (Soy protein hydrolyzate)
The soybean protein hydrolyzate according to the present invention is a peptide mixture obtained by subjecting the soybean protein raw material to protease treatment. The protein hydrolyzate preferably has a higher degree of degradation, and in particular, the ratio of peptides having a molecular weight of less than 500 to the total amount of peptides and free amino acids in the hydrolyzate is preferably high. Specifically, it is important that the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more with respect to the total amount of peptides and free amino acids, and more preferably 60% by weight or more. preferable.
The peptide having a molecular weight of less than 500 is substantially composed of a dipeptide and a tripeptide in which 2 to 3 amino acids are bonded.
The content of the peptide having a molecular weight of less than 500 is determined by measuring the ratio of the peptide having a molecular weight of less than 500 and a free amino acid fraction in the protein hydrolyzate by gel filtration chromatography for peptides, and then calculating the release in the protein hydrolyzate calculated by amino acid analysis. It shall be calculated by subtracting the amino acid content.
The soy protein hydrolyzate identified as described above preferably has a reduced ratio of peptides and free amino acids other than peptides having a molecular weight of less than 500 as much as possible. That is, the free amino acid content in the protein hydrolyzate is preferably 12% by weight or less, more preferably 5% by weight or less, and still more preferably 3% by weight or less based on the total amount of the peptide and free amino acid. . Furthermore, since it is desirable that the peptide in the protein hydrolyzate has a lower molecular weight, the fraction ratio of the molecular weight of 500 or more in the protein hydrolyzate is 40% by weight with respect to the total amount of peptide and free amino acid. It is preferably at most 38% by weight, more preferably at most 35% by weight.
（プロテアーゼ）
　本発明に係る大豆蛋白質加水分解物を得るために使用するプロテアーゼは、動物起源、植物起源又は微生物起源を問わず、プロテアーゼの分類において「金属プロテアーゼ」、「酸性プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼ、好ましくは「金属プロテアーゼ」、「チオールプロテアーゼ」、「セリンプロテアーゼ」に分類されるプロテアーゼの中から適宜選択することができる。特に２種類以上、あるいは３種類以上の異なった分類に属する酵素を、順次若しくは同時に作用させる分解方法が分子量５００未満のペプチドの割合を増加させることができ、好ましい。 (Protease)
The protease used to obtain the soy protein hydrolyzate according to the present invention, regardless of animal origin, plant origin or microbial origin, is classified into "metal protease", "acidic protease", "thiol protease", " It can be appropriately selected from proteases classified as “serine protease”, preferably proteases classified as “metal protease”, “thiol protease” and “serine protease”. Particularly, a degradation method in which enzymes belonging to two or more, or three or more different classifications are acted on sequentially or simultaneously can increase the proportion of peptides having a molecular weight of less than 500.
　このプロテアーゼの分類は、酵素科学の分野において通常行なわれている活性中心のアミノ酸の種類による分類方法である。各々の代表として「金属プロテアーゼ」にはBacillus由来中性プロテアーゼ、Streptomyces由来中性プロテアーゼ、Aspergillus由来中性プロテアーゼ、『サモアーゼ』等が挙げられ、「酸性プロテアーゼ」にはペプシン、Aspergillus由来酸性プロテアーゼ、『スミチームＦＰ』等が挙げられ、「チオールプロテアーゼ」にはブロメライン、パパイン等が挙げられ、「セリンプロテアーゼ」にはトリプシン、キモトリプシン、ズブチリシン、Streptomyces由来アルカリプロテアーゼ、『アルカラーゼ』、『ビオプラーゼ』等が挙げられる。これら以外の酵素でも作用ｐＨや阻害剤との反応性により、その分類を確認することができる。活性中心が異なる酵素間では、基質への作用部位が大きく異なるため、「切れ残り」を減らし、効率よく酵素分解物を得ることができる。また、異なった起源の（起源生物）の酵素を併用することで、更に効率よく酵素分解物を製造することができる。同分類でも起源が異なれば、基質である蛋白質への作用部位も異なり、結果として分子量５００未満のペプチドの割合を増やすことができる。これらプロテアーゼはエキソプロテアーゼ活性が少ないことが好ましい。 This classification of protease is a classification method based on the type of amino acid at the active center, which is usually performed in the field of enzyme science. As representatives of each, “metal protease” includes Bacillus-derived neutral protease, Streptomyces-derived neutral protease, Aspergillus-derived neutral protease, “Samoase”, etc., and “acidic protease” includes pepsin, Aspergillus-derived acidic protease, “ “Sumiteam FP” and the like, “thiol protease” includes bromelain, papain and the like, and “serine protease” includes trypsin, chymotrypsin, subtilisin, Streptomyces-derived alkaline protease, “alkalase”, “bioprese” and the like . Even with enzymes other than these, the classification can be confirmed by the action pH and the reactivity with inhibitors. Since the site of action on the substrate is greatly different between enzymes having different active centers, it is possible to reduce “uncut residue” and efficiently obtain an enzyme degradation product. In addition, enzymatic degradation products can be produced more efficiently by using enzymes of different origins (origin organisms) together. Even in the same classification, if the origins are different, the site of action on the protein as a substrate is also different, and as a result, the proportion of peptides having a molecular weight of less than 500 can be increased. These proteases preferably have low exoprotease activity.
　プロテアーゼ処理の反応ｐＨや反応温度は、用いるプロテアーゼの特性に合わせて設定すれば良く、通常、反応ｐＨは至適ｐＨ付近で行ない、反応温度は至適温度付近で行なえば良い。概ね反応温度は２０～８０℃、好ましくは４０～６０℃である。反応後は酵素を失活させるのに十分な温度（６０～１７０℃程度）まで加熱し、残存酵素活性を失活させる。 The reaction pH and reaction temperature of the protease treatment may be set in accordance with the characteristics of the protease to be used. Usually, the reaction pH is carried out near the optimum pH, and the reaction temperature may be carried out around the optimum temperature. In general, the reaction temperature is 20 to 80 ° C., preferably 40 to 60 ° C. After the reaction, the enzyme is heated to a temperature sufficient to inactivate the enzyme (about 60 to 170 ° C.) to inactivate the residual enzyme activity.
　プロテアーゼ処理後の反応液は、そのまま又は濃縮して用いることもできるが、通常、殺菌し、噴霧乾燥、凍結乾燥等して乾燥粉末の状態で利用する。殺菌は、加熱殺菌が好ましく、加熱温度は１１０～１７０℃が好ましく、１３０～１７０℃が更に好ましい。加熱時間は３～２０秒間が好ましい。また、反応液を任意のｐＨに調整してもよい。プロテアーゼ処理時やｐＨ調整時に発生する不溶物（沈殿物や懸濁物）を遠心分離やろ過等により除去してもよい。不溶物の除去は、大豆蛋白質加水分解物の抗酸化活性を向上させることができるため、好ましい。更に活性炭や吸着樹脂により精製してもよい。 The reaction solution after the protease treatment can be used as it is or after being concentrated, but is usually sterilized, spray-dried, freeze-dried, etc. and used in the form of a dry powder. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170 ° C, more preferably 130 to 170 ° C. The heating time is preferably 3 to 20 seconds. Moreover, you may adjust a reaction liquid to arbitrary pH. Insoluble matters (precipitates and suspensions) generated during protease treatment or pH adjustment may be removed by centrifugation or filtration. Removal of insolubles is preferable because it can improve the antioxidant activity of the soy protein hydrolyzate. Furthermore, you may refine | purify with activated carbon or adsorption resin.
（抗酸化作用）
　本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物は、高い抗酸化活性を示し、生体の酸化がもたらす疾病を予防又は治療するための有効成分として用いることができる。該加水分解物が持つ抗酸化活性については、非特許文献１に記載の方法にて測定される「ORAC」（Oxygen Radical Absorbance Capacity：活性酸素吸収能力）の値（単位：μmol TE/g）を指標とすることができる。なお、単位の「TE」は“Trolox equivalent”の略であり、「Trolox」は標準物質である「6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid」の登録商標である。ORAC値が高いほど活性酸素吸収能力すなわち抗酸化活性が高いことを示し、本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドが高い大豆蛋白質加水分解物のORAC値は３５０μmol TE/g以上であることが好ましく、４５０μmol TE/g以上がより好ましく、５００μmol TE/g以上がさらに好ましい。 (antioxidant effect)
Soy protein hydrolyzate in which the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate according to the present invention is 50% by weight or more based on the total amount of peptides and free amino acids exhibits high antioxidant activity, It can be used as an active ingredient for preventing or treating diseases caused by oxidation. About the antioxidant activity which this hydrolyzate has, the value (unit: micromol TE / g) of "ORAC" (Oxygen Radical Absorbance Capacity: active oxygen absorption capacity) measured by the method of a nonpatent literature 1 is used. It can be an indicator. The unit “TE” is an abbreviation of “Trolox equivalent”, and “Trolox” is a registered trademark of “6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid” which is a standard substance. . The higher the ORAC value, the higher the active oxygen absorption capacity, that is, the higher the antioxidant activity, and the higher the peptide with a molecular weight of less than 500 in the protein hydrolyzate according to the present invention, the higher the ORAC value of the soy protein hydrolyzate is 350 μmol TE / g or more It is preferably 450 μmol TE / g or more, more preferably 500 μmol TE / g or more.
（他の抗酸化物質との併用）
　本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物をその他の抗酸化活性を示す物質又はこれらを含むエキスを１種以上併用することにより、抗酸化活性を相乗的に高めることができる。他の抗酸化物質を併用する形態は特に限定されず、予め本発明に係る大豆蛋白質加水分解物と混合し、混合製剤化したものを使用することができるし、また、本発明において大豆蛋白質加水分解物を使用する際に他の抗酸化物質を別途添加して使用することも可能である。
　他の抗酸化物質としては、例えばビタミン類、ポリフェノール類、カロチノイド類、サポニン類、アリシン等が挙げられる。ビタミン類としては、ビタミンＣ、ビタミンＡ、ビタミンＥ等が挙げられる。ポリフェノール類としては、イソフラボン、ケルセチン、ミリセチン、ケンフェロール、ヘスペリジン、ナリンジン、アントシアニン、カテキン、クリシン、アピゲニン、ルテオリン等のフラボノイド類や、セサミン、セサミノール、セサミノール、エピセサミン、セサモール、セサモリン等のリグナン類のほか、ピクノジェノール、クルクミン、クロロゲン酸、没食子酸、ロズマリン酸、エラグ酸、クマリン、フェルラ酸、ショウガオール、カカオマスポリフェノール等が挙げられる。カロチノイド類としては、カロテン（α－，β－，γ－，δ－）やリコピン等のカロテン類、あるいは、ルテイン、アスタキサンチン、ゼアキサンチン、カンタキサンチン、フコキサンチン、アンテラキサンチン、ビオラキサンチン、カプサイシン等のキサントフィル類等が挙げられる。サポニン類としては、キラヤサポニン等の生薬由来のサポニンや、大豆サポニン、茶サポニン等が挙げられる。これらの抗酸化物質を用いる場合、これらが含まれる植物エキスを代わりに用いることができ、例えばアントシアニンを用いる場合はブルーベリーエキス、カシスエキス、アサイーエキス、エルダーベリーエキスなどを用いることができる。上記抗酸化活性を示す物質又はエキスのうち、特にビタミンＣ、ピクノジェノール、アサイーエキス、クルクミン又はセサミンを併用するとより相乗効果が強く好ましい。 (Combination with other antioxidants)
A soy protein hydrolyzate in which the content of a peptide having a molecular weight of less than 500 in the protein hydrolyzate according to the present invention is 50% by weight or more with respect to the total amount of the peptide and free amino acids or other substances exhibiting antioxidant activity Antioxidant activity can be synergistically enhanced by using one or more extracts containing sucrose. The form in which other antioxidants are used in combination is not particularly limited, and can be used by previously mixing with the soy protein hydrolyzate according to the present invention to prepare a mixed preparation. In the present invention, the soy protein hydrolyzate can be used. When using the decomposition product, it is also possible to add other antioxidants separately.
Examples of other antioxidants include vitamins, polyphenols, carotenoids, saponins, and allicin. Vitamins include vitamin C, vitamin A, vitamin E, and the like. Polyphenols include flavonoids such as isoflavone, quercetin, myricetin, kaempferol, hesperidin, naringin, anthocyanin, catechin, chrysin, apigenin, luteolin, and lignans such as sesamin, sesaminol, sesaminol, episesamin, sesamol, sesamoline. In addition, pycnogenol, curcumin, chlorogenic acid, gallic acid, rosmarinic acid, ellagic acid, coumarin, ferulic acid, gingerol, cacao mass polyphenol and the like can be mentioned. Examples of carotenoids include carotenes (α-, β-, γ-, δ-) and lycopene, or xanthophylls such as lutein, astaxanthin, zeaxanthin, canthaxanthin, fucoxanthin, anthaxanthin, violaxanthin, capsaicin and the like. And the like. Examples of saponins include saponins derived from herbal medicines such as Kiraya saponin, soybean saponins, tea saponins and the like. When these antioxidants are used, plant extracts containing them can be used instead. For example, when anthocyanins are used, blueberry extract, cassis extract, acai extract, elderberry extract and the like can be used. Of the substances or extracts exhibiting the antioxidant activity, it is particularly preferable to use vitamin C, pycnogenol, acai extract, curcumin or sesamin in combination with a strong synergistic effect.
（飲食品における利用）
　本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上の大豆蛋白質加水分解物は、これを有効成分とする抗酸化剤として、様々な飲食品に利用することができる。例えば、飲食品自体の酸化防止を目的として飲食品に添加することができる。また、本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上の大豆蛋白質加水分解物を添加することにより摂取した者に生理機能としての抗酸化力が発揮され、生体の酸化がもたらす下記の疾病を予防しうる飲料、タブレット、フードバー、サラダ用ドレッシング、肉製品、スナック菓子、デザート、菓子類及び栄養補助剤などを得ることができる。 (Use in food and drink)
Soy protein hydrolyzate having a content of peptides with a molecular weight of less than 500 in the protein hydrolyzate according to the present invention of 50% by weight or more based on the total amount of peptides and free amino acids is used as an antioxidant. It can be used for various foods and drinks. For example, it can add to food-drinks for the purpose of antioxidant of food-drinks itself. In addition, the protein hydrolyzate according to the present invention has a physiological content for a person who has taken it by adding soy protein hydrolyzate having a content of a peptide having a molecular weight of less than 500 to 50% by weight or more based on the total amount of peptides and free amino acids. To obtain beverages, tablets, food bars, salad dressings, meat products, snack confectionery, desserts, confectionery, nutritional supplements, etc. that can exert the antioxidant power as a function and prevent the following diseases caused by oxidation of the living body Can do.
（医薬品における利用）
　本発明に係る蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上の大豆蛋白質加水分解物は、医薬品としても利用することができる。生体の酸化がもたらす疾病として動脈硬化、心筋梗塞、ガン、糖尿病、アルツハイマー、花粉症などが挙げられるが、これらの疾病を予防及び治療するための医薬品として利用することができる。医薬品の形態として供する場合は、液体、散薬、錠剤、カプセル等の種々の形態で使用することができる。 (Use in medicine)
The soy protein hydrolyzate in which the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate according to the present invention is 50% by weight or more based on the total amount of peptides and free amino acids can also be used as a pharmaceutical product. Diseases caused by oxidation of the living body include arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer, hay fever and the like, and can be used as pharmaceuticals for preventing and treating these diseases. When used as a pharmaceutical form, it can be used in various forms such as liquid, powder, tablet, capsule and the like.
　以下、実施例により本発明をより具体的に説明する。 Hereinafter, the present invention will be described more specifically with reference to examples.
（測定方法）
　まず実施例で用いた各種測定方法について以下にまとめる。 (Measuring method)
First, various measurement methods used in the examples are summarized below.
＜蛋白質含量＞
　１０５℃、１２時間乾燥した大豆蛋白質画分の重量に対して、ケルダール法により測定した蛋白質量の重量を、乾燥物中の蛋白質含量として「％」で表した。なお、窒素換算係数は６．２５とした。 <Protein content>
The weight of the protein mass measured by the Kjeldahl method with respect to the weight of the soybean protein fraction dried at 105 ° C. for 12 hours was expressed as “%” as the protein content in the dried product. The nitrogen conversion factor was 6.25.
＜遊離アミノ酸及びペプチドの含量＞
　大豆蛋白質加水分解物の分子量分布を、以下のゲルろ過カラムを用いたＨＰＬＣ法により測定した。
　ペプチド用ゲルろ過カラムを用いたHPLCシステムを組み、分子量マーカーとなる既知のペプチドをチャージし、分子量と保持時間の関係において検量線を求めた。なお、分子量マーカーは、オクタペプチドとして[β-Asp]-Angiotensin IIのβ-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe（分子量１０４６）、ヘキサペプチドとしてAngiotensin IVのVal-Tyr-Ile-His-Pro-Phe（分子量７７５）、ペンタペプチドとしてLeu-EnkephalinのTyr-Gly-Gly-Phe-Leu（分子量５５５）、トリペプチドとしてGlu-Glu-Glu（分子量４０５）、遊離アミノ酸としてPro（分子量１１５）を用いた。蛋白質加水分解物（１％）を１０，０００rpm、１０分で遠心分離した上清を、ゲルろ過用溶媒で２倍に希釈し、その５μlをＨＰＬＣにアプライした。
　蛋白質加水分解物中の遊離アミノ酸及び分子量５００未満のペプチド画分の割合（％）は、全体の吸光度のチャート面積に対する、分子量５００未満の範囲（時間範囲）の面積の割合によって求めた（使用カラム：Superdex Peptide 7.5/300GL（ＧＥヘルスケア・ジャパン株式会社製）、溶媒：１％SDS/10mMリン酸緩衝液、ｐＨ８．０、カラム温度２５℃、流速０．２５ml/min、検出波長：２２０nm）。
　また蛋白質加水分解物中の分子量５００以上のペプチド画分の割合（％）は、上記と同様に、全体の吸光度のチャート面積に対する、分子量５００以上の範囲の面積の割合によって求めた。
　次に、アミノ酸分析により蛋白質加水分解物中の遊離アミノ酸含量の測定を行った。蛋白質加水分解物（４mg/ml）を等量の３％スルホサリチル酸に加え、室温で１５分間振とうした。１０，０００rpmで１０分間遠心分離し、得られた上澄みを０．４５μmフィルターでろ過し、アミノ酸分析器「JLC500V」（日本電子（株）製）にて、遊離アミノ酸を測定した。蛋白質加水分解物中の遊離アミノ酸含量はケルダール法にて得られた蛋白質含量に対する割合として算出した。
　以上より得られた、「遊離アミノ酸及び分子量５００未満のペプチド画分の割合」から「遊離アミノ酸含量」を差し引いた値を、蛋白質分解物中の「分子量５００未満のペプチドの含量」とした。 <Contents of free amino acids and peptides>
The molecular weight distribution of the soy protein hydrolyzate was measured by the HPLC method using the following gel filtration column.
An HPLC system using a gel filtration column for peptides was assembled, a known peptide serving as a molecular weight marker was charged, and a calibration curve was obtained in relation to the molecular weight and the retention time. The molecular weight markers are [β-Asp] -Angiotensin II β-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (molecular weight 1046) as octapeptide and Angiotensin IV Val-Tyr- as hexapeptide. Ile-His-Pro-Phe (molecular weight 775), Leu-Enkephalin Tyr-Gly-Gly-Phe-Leu (molecular weight 555) as pentapeptide, Glu-Glu-Glu (molecular weight 405) as tripeptide, Pro as free amino acid (Molecular weight 115) was used. A supernatant obtained by centrifuging a protein hydrolyzate (1%) at 10,000 rpm for 10 minutes was diluted 2-fold with a gel filtration solvent, and 5 μl thereof was applied to HPLC.
The ratio (%) of the free amino acid in the protein hydrolyzate and the peptide fraction having a molecular weight of less than 500 was determined by the ratio of the area of the molecular weight less than 500 (time range) to the total absorbance chart area (column used). : Superdex Peptide 7.5 / 300GL (manufactured by GE Healthcare Japan Ltd.), solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, column temperature 25 ° C., flow rate 0.25 ml / min, detection wavelength: 220 nm) .
Further, the ratio (%) of the peptide fraction having a molecular weight of 500 or more in the protein hydrolyzate was determined by the ratio of the area having a molecular weight of 500 or more to the total absorbance chart area as described above.
Next, the free amino acid content in the protein hydrolyzate was measured by amino acid analysis. Protein hydrolyzate (4 mg / ml) was added to an equal volume of 3% sulfosalicylic acid and shaken at room temperature for 15 minutes. Centrifugation was performed at 10,000 rpm for 10 minutes, and the obtained supernatant was filtered with a 0.45 μm filter, and free amino acids were measured with an amino acid analyzer “JLC500V” (manufactured by JEOL Ltd.). The free amino acid content in the protein hydrolyzate was calculated as a ratio to the protein content obtained by the Kjeldahl method.
The value obtained by subtracting the “free amino acid content” from the “ratio of free amino acids and peptide fractions having a molecular weight of less than 500” obtained above was defined as “content of peptides having a molecular weight of less than 500” in the proteolysate.
＜ORAC値の測定＞
　得られたサンプルについてそれぞれ、１００mg秤量し５０％エタノールに溶解させ１００mlに定容し、非特許文献１記載の方法に準じて測定を行った。定量は標準物質として「Trolox」（登録商標、キシダ化学（株）より購入）を用い、各測定値を用いて算出した。 <Measurement of ORAC value>
Each of the obtained samples was weighed 100 mg, dissolved in 50% ethanol and fixed to 100 ml, and measured according to the method described in Non-Patent Document 1. The quantification was calculated using each measured value using “Trolox” (registered trademark, purchased from Kishida Chemical Co., Ltd.) as a standard substance.
（製造例１）　分離大豆蛋白質の調製
　低変性脱脂大豆から以下のように分離大豆蛋白質を調製した。
（１）低変性脱脂大豆の温水抽出スラリーを遠心分離機にてオカラ画分を除き脱脂豆乳とした。
（２）得られた脱脂豆乳のｐＨを４．５に調整して等電点沈殿し、遠心分離機にて酸沈殿カードを得て中和した。
（３）得られた各画分を中和して、１２０℃で１０秒間加熱殺菌を行った。 (Manufacture example 1) Preparation of isolation | separation soybean protein The isolation | separation soybean protein was prepared from the low modified | denatured defatted soybean as follows.
(1) Warm water extraction slurry of low-denatured defatted soybeans was defatted soymilk by removing the okara fraction with a centrifuge.
(2) The pH of the obtained defatted soymilk was adjusted to 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge.
(3) The obtained fractions were neutralized and sterilized by heating at 120 ° C. for 10 seconds.
（製造例２）　大豆蛋白質加水分解物の調製　（大豆蛋白質加水分解物Ａ、Ｂ）
　製造例１で得られた分離大豆蛋白質の１０％溶液を作成し、『アルカラーゼ』（ノボザイムズジャパン（株）製）を０．２％、若しくは０．５％添加し、５５℃、１５分間酵素反応を行った。酵素反応後に９０℃、２０分間の加熱処理により反応を停止させた後、凍結乾燥を行い試料とした（大豆蛋白質加水分解物Ａ、Ｂ）。 (Production Example 2) Preparation of soy protein hydrolyzate (soy protein hydrolyzate A, B)
A 10% solution of the isolated soybean protein obtained in Production Example 1 is prepared, and 0.2% or 0.5% of “Alcalase” (manufactured by Novozymes Japan) is added, and the temperature is 55 ° C. for 15 minutes. Enzymatic reaction was performed. After the enzyme reaction, the reaction was stopped by a heat treatment at 90 ° C. for 20 minutes, and then lyophilized to obtain samples (soy protein hydrolysates A and B).
（製造例３）　大豆蛋白質加水分解物の調製　（大豆蛋白質加水分解物Ｃ、Ｄ）
　製造例１で得られた分離大豆蛋白質の３％溶液を作成し、金属プロテアーゼ『サモアーゼ』（起源；Bacillus thermoproteolyticus Rokko、大和化成（株））を蛋白質当たり１％又は２％加え、ｐＨ９．０、５８℃で６０分間酵素反応を行った。
　次にセリンプロテアーゼ『ビオプラーゼ』（起源；Bacillus sp．、ナガセケムテックス（株））を蛋白質当たり０．５％又は１％加え、ｐＨ７．５、５８℃で６０分間酵素反応を行った。
　次に金属プロテアーゼ『スミチームＦＰ』（起源；Aspergillus oryzae、新日本化学工業（株））を蛋白質当たり０．５％又は１％加え、ｐＨ７．５、５８℃で６０分間酵素反応を行った。
　以上の酵素反応後、酵素反応液を９０℃、２０分間の加熱処理により反応を停止させた後、凍結乾燥を行い試料とした（大豆蛋白質加水分解物Ｃ、Ｄ）。 (Production Example 3) Preparation of soy protein hydrolyzate (soy protein hydrolyzate C, D)
A 3% solution of the isolated soybean protein obtained in Production Example 1 was prepared, and 1% or 2% of the metal protease “Samoase” (origin; Bacillus thermoproteolyticus Rokko, Daiwa Kasei Co., Ltd.) was added per protein, pH 9.0, The enzyme reaction was performed at 58 ° C. for 60 minutes.
Next, 0.5% or 1% of a serine protease “bioprelase” (origin: Bacillus sp., Nagase ChemteX Co., Ltd.) was added per protein, and an enzyme reaction was performed at pH 7.5 and 58 ° C. for 60 minutes.
Next, metal protease “Sumiteam FP” (origin: Aspergillus oryzae, Shin Nippon Chemical Industry Co., Ltd.) was added at 0.5% or 1% per protein, and the enzyme reaction was performed at pH 7.5, 58 ° C. for 60 minutes.
After the above enzyme reaction, the reaction was stopped by heating the enzyme reaction solution at 90 ° C. for 20 minutes, and then freeze-dried to obtain a sample (soy protein hydrolyzate C and D).
（製造例４）　大豆蛋白質加水分解物の調製　（大豆蛋白質加水分解物Ｅ、Ｆ、Ｇ）
　製造例１で得られた分離大豆蛋白質の３％溶液を作成し、製造例３と同じ『サモアーゼ』を蛋白質当たり１％、１．５％又は２％加え、ｐＨ９．０、５８℃で６０分間酵素反応を行った。
　次に製造例３と同じ『ビオプラーゼ』を蛋白質当たり０．５％、０．７５％又は１％加え、ｐＨ７．５、５８℃で６０分間酵素反応を行った。
　次に製造例３と同じ『スミチームＦＰ』を蛋白質当たり０．５％、０．７５％又は１％加え、ｐＨ７．５、５８℃で６０分間酵素反応を行った。
　以上の酵素反応後、酵素反応液を９０℃、２０分間の加熱処理により反応を停止させ、さらに酵素反応により生じた不溶物を遠心分離及び孔径１．０μｍのメンブレンフィルターによるろ過で除去し、凍結乾燥を行い試料とした（大豆蛋白質加水分解物Ｅ、Ｆ、Ｇ）。 (Production Example 4) Preparation of soy protein hydrolyzate (soy protein hydrolyzate E, F, G)
A 3% solution of the separated soybean protein obtained in Production Example 1 was prepared, and 1%, 1.5% or 2% of the same “samoyase” as in Production Example 3 was added per protein, and pH 9.0, 58 ° C. for 60 minutes. Enzymatic reaction was performed.
Next, 0.5%, 0.75%, or 1% of the same “bioplase” as in Production Example 3 was added, and the enzyme reaction was performed at pH 7.5, 58 ° C. for 60 minutes.
Next, the same “Sumiteam FP” as in Production Example 3 was added at 0.5%, 0.75%, or 1% per protein, and the enzyme reaction was performed at pH 7.5, 58 ° C. for 60 minutes.
After the above enzyme reaction, the reaction was stopped by heating the enzyme reaction solution at 90 ° C. for 20 minutes, and the insoluble matter generated by the enzyme reaction was removed by centrifugation and filtration with a membrane filter having a pore size of 1.0 μm, and frozen. It dried and it was set as the sample (soy protein hydrolyzate E, F, G).
（試験例１）
　製造例２～４にて得られた大豆蛋白質加水分解物Ａ～Ｇについて蛋白質含量（％）、ペプチド及び遊離アミノ酸の合計量に占める分子量５００未満のペプチド含量（％）、分子量５００以上のペプチド含量及び遊離アミノ酸含量、並びに、ORAC値（μmol TE/g）を測定した（比較例１～４、実施例１～３）。各蛋白質加水分解物の調製条件及び分析結果を表１及び図１にまとめた。 (Test Example 1)
About soybean protein hydrolyzate A to G obtained in Production Examples 2 to 4, protein content (%), peptide content (%) with a molecular weight of less than 500 in the total amount of peptides and free amino acids, peptide content with a molecular weight of 500 or more The free amino acid content and the ORAC value (μmol TE / g) were measured (Comparative Examples 1 to 4, Examples 1 to 3). The preparation conditions and analysis results for each protein hydrolyzate are summarized in Table 1 and FIG.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　図１に示すとおり、分子量５００未満のペプチド含量とORAC値には高い相関が見られ、相関係数は０．９２であった。比較例３と比較例４を比較すると、分子量５００未満のペプチド含量については比較例４が８％程度高いにもかかわらず、ORAC値に差はほとんど見られなかった。一方で比較例３と酵素反応条件は同一であるが、酵素反応後に不溶物を除去した実施例１では、ORAC値が大きく向上した。 As shown in FIG. 1, a high correlation was found between the content of peptides having a molecular weight of less than 500 and the ORAC value, and the correlation coefficient was 0.92. When comparing Comparative Example 3 and Comparative Example 4, there was almost no difference in ORAC value for the peptide content of molecular weight less than 500, although Comparative Example 4 was about 8% higher. On the other hand, although the enzyme reaction conditions were the same as in Comparative Example 3, the ORAC value was greatly improved in Example 1 in which insolubles were removed after the enzyme reaction.
（試験例２）大豆蛋白質加水分解物と抗酸化成分との併用による相乗効果
　次に、製造例４で得られた大豆蛋白質加水分解物Ｇと、抗酸化活性が高いことが既に分かっている抗酸化物質又はこれを含むエキスとの相乗効果について、ORAC値を測定することにより検証した。
　サンプルはビタミンＣと、各種ポリフェノールの代表例として大豆イソフラボン、カテキン、ブルーベリーエキス、カシスエキス、ピクノジェノール、アサイーエキス、クルクミン、セサミンを用いた。方法は、それぞれのサンプル１００mgを１００mlに定容した溶液と大豆蛋白質加水分解物Ｇ１００mgを１００mlに定容した溶液を１：１で混合し、規定の希釈倍率にて測定を行った。 (Test Example 2) Synergistic effect of combined use of soybean protein hydrolyzate and antioxidant component Next, soybean protein hydrolyzate G obtained in Production Example 4 and anti-oxidant that has already been known to have high antioxidant activity. The synergistic effect with the oxidizing substance or the extract containing the same was verified by measuring the ORAC value.
As samples, vitamin C and soybean isoflavone, catechin, blueberry extract, cassis extract, pycnogenol, acai extract, curcumin, and sesamin were used as representative examples of various polyphenols. In the method, a solution in which 100 mg of each sample was made up to 100 ml and a solution in which 100 mg of soybean protein hydrolyzate G was made up to 100 ml were mixed at a ratio of 1: 1, and the measurement was performed at a specified dilution ratio.
　ORAC値の測定の結果を下記表２に示した。表１によると大豆蛋白質加水分解物Ｇは単独でのORAC値は５５１μmol TE/gであるが、各種の抗酸化成分と併用することにより、いずれも各成分が単独で持つ抗酸化性（無添加（－）データ参照）を相乗的に増強していることが確認された（添加（＋）データ参照）。さらに、抗酸化性の相乗効果は大豆蛋白加水分解物Ｇと食品成分を１：１で混合した物の値であるため、食品成分の添加量を半分に減らしても抗酸化効果は食品成分単独よりも高い値を示していることになる。特に、ビタミンＣ、ピクノジェノール、アサイーエキス、クルクミン、セサミンと併用した場合には、これら単独の抗酸化活性の２倍以上の増強効果を示し、高い相乗効果を示すことが確認された。 The results of the ORAC value measurement are shown in Table 2 below. According to Table 1, the soy protein hydrolyzate G alone has an ORAC value of 551 μmol TE / g, but when used in combination with various antioxidant components, each component has its own antioxidant properties (no addition) (See (-) data) was confirmed to be synergistically enhanced (see added (+) data). Furthermore, since the synergistic effect of antioxidant properties is the value of a mixture of soybean protein hydrolyzate G and food ingredients in a ratio of 1: 1, even if the amount of food ingredients added is reduced by half, the antioxidant effect is the food ingredient alone It shows a higher value. In particular, when used in combination with vitamin C, pycnogenol, acai extract, curcumin, and sesamin, it was confirmed that the single antioxidant activity was enhanced more than twice, and a high synergistic effect was exhibited.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

Claims (13)

  1. 生体の酸化がもたらす疾病を予防又は治療する方法において有効成分として使用するための抗酸化物質であって、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含むこと特徴とする抗酸化物質。 An antioxidant for use as an active ingredient in a method for preventing or treating a disease caused by oxidation of a living body, wherein the content of a peptide having a molecular weight of less than 500 in a protein hydrolyzate is based on the total amount of the peptide and free amino acids An antioxidant substance comprising a soy protein hydrolyzate that is 50% by weight or more.
  2. 生体の酸化がもたらす疾病が動脈硬化、心筋梗塞、ガン、糖尿病、アルツハイマー、又は花粉症である請求項１記載の抗酸化物質。 The antioxidant substance according to claim 1, wherein the disease caused by oxidation of the living body is arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer's disease, or hay fever.
  3. 前記大豆蛋白質加水分解物は加水分解後の不溶物が除去されたものである、請求項１記載の抗酸化物質。 The antioxidant substance according to claim 1, wherein the soy protein hydrolyzate is obtained by removing insoluble matter after hydrolysis.
  4. ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質をさらに含む、請求項１記載の抗酸化物質。 The antioxidant of claim 1, further comprising one or more antioxidants selected from vitamins, polyphenols, carotenoids, saponins and allicin.
  5. 生体の酸化がもたらす疾病を予防又は治療するために用いられる飲食品又は医薬品の製造において、有効成分である抗酸化物質として蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を添加することを特徴とする、該飲食品又は医薬品の製造法。 In the production of foods and drinks or pharmaceuticals used for preventing or treating diseases caused by oxidation of living organisms, the content of peptides having a molecular weight of less than 500 in protein hydrolysates as antioxidants which are active ingredients is peptides and free amino acids. A method for producing a food or beverage or pharmaceutical product comprising adding soy protein hydrolyzate in an amount of 50% by weight or more based on the total amount.
  6. ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質を併用する、請求項５記載の飲食品又は医薬品の製造法。 The method for producing a food or drink or medicine according to claim 5, wherein one or more antioxidants selected from vitamins, polyphenols, carotenoids, saponins and allicin are used in combination.
  7. 有効成分として蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含む抗酸化物質を使用することを特徴とする、生体の酸化がもたらす疾病の予防又は治療する方法。 It is characterized by using an antioxidant substance containing soybean protein hydrolyzate in which the content of peptides having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of peptides and free amino acids as the active ingredient A method for preventing or treating a disease caused by oxidation of a living body.
  8. 蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物を含有することを特徴とする抗酸化剤。 An antioxidant comprising a soy protein hydrolyzate in which the content of a peptide having a molecular weight of less than 500 in a protein hydrolyzate is 50% by weight or more based on the total amount of peptides and free amino acids.
  9. 　前記大豆蛋白質加水分解物は加水分解後の不溶物が除去されている大豆蛋白質加水分解物である、請求項８記載の抗酸化剤。 The antioxidant according to claim 8, wherein the soy protein hydrolyzate is a soy protein hydrolyzate from which insolubles after hydrolysis have been removed.
  10. 　ビタミン類、ポリフェノール類、カロチノイド類、サポニン類及びアリシンから選択される１種以上の抗酸化物質をさらに含む、請求項８記載の抗酸化剤。 The antioxidant according to claim 8, further comprising at least one antioxidant selected from vitamins, polyphenols, carotenoids, saponins and allicin.
  11. 　請求項８に記載の抗酸化剤が添加された飲食品。 Food or drink to which the antioxidant according to claim 8 is added.
  12. 　請求項８に記載の抗酸化剤を有効成分とする医薬品。 A pharmaceutical comprising the antioxidant according to claim 8 as an active ingredient.
  13. 　抗酸化剤の製造における、蛋白質加水分解物中の分子量５００未満のペプチドの含量がペプチド及び遊離アミノ酸の合計量に対して５０重量％以上である大豆蛋白質加水分解物の使用方法。 A method for using a soy protein hydrolyzate in which the content of a peptide having a molecular weight of less than 500 in the protein hydrolyzate is 50% by weight or more based on the total amount of the peptide and free amino acids in the production of the antioxidant.
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