JP4790996B2 - Method for producing functional material from brewing by-products and functional material obtained thereby - Google Patents
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Description
本発明は、醸造副産物からの機能性素材の製造方法およびそれにより得られる機能性素材に関するものであり、詳細には、糠もしくは脱脂糠または玄米をヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素で処理し、次いで、酵母で処理することを特徴とする機能性素材の製造方法ならびにそれにより得られる機能性素材に関する。さらに本発明は、該機能性素材を含有する機能性食品、医薬組成物および化粧組成物にも関する。 The present invention relates to a method for producing a functional material from a brewing by-product and a functional material obtained thereby, and more specifically, koji or defatted koji or brown rice is treated with a hemicellulase enzyme, a liquefaction enzyme, and a saccharification enzyme. Next, the present invention relates to a method for producing a functional material characterized by treating with yeast and a functional material obtained thereby. The present invention further relates to a functional food, a pharmaceutical composition and a cosmetic composition containing the functional material.
ビールや清酒の醸造副産物は、その大部分が産業廃棄物として処理されているのが現状である。例えば、清酒醸造副産物の赤糠は、栄養成分が豊富に含まれているにも拘らず、せいぜい飼料として利用されている程度であり、これまで有効活用の技術開発が行われていなかった。赤糠には特にL−アラビノース、D−キシロース、各種オリゴ糖(例えば、アラビノキシラン)などの有用成分が多く含まれている。これらの成分は、特にダイエットや健康増進のごとき種々の好ましい作用を有するといわれている。従来より、これらの成分を分離、精製あるいは濃縮する方法が多く提案されてきた(特許文献1〜8等参照)。 Most of the brewing by-products of beer and sake are currently treated as industrial waste. For example, red potato, a by-product of sake brewing, has been used as a feed at best, despite the fact that it contains abundant nutritional components, and until now no technology has been developed for effective use. Red cocoons are particularly rich in useful components such as L-arabinose, D-xylose, and various oligosaccharides (for example, arabinoxylan). These components are said to have various favorable effects such as dieting and health promotion. Conventionally, many methods for separating, purifying, or concentrating these components have been proposed (see Patent Documents 1 to 8).
例えば、特許文献1はL−アラビノースの精製方法、特許文献2はL−アラビノースの分離方法に関するもので、いずれもキシロースを資化する微生物を作用させることを特徴とする方法である。特許文献3はL−アラビノースまたはL−アラビノース含有酵素処理物の製造方法およびL−アラビノースの製造方法に関するものであり、特許文献4はL−アラビノースの製造方法ならびにL−アラビノース含有酵素処理物およびその製造方法に関するものであるが、いずれもアラビノ多糖類を酵素処理するL−アラビノースの製造方法でD−キシロースには触れていない。特許文献5は、穀類外皮から水溶性糖類を製造する方法に関するものであり、アラビノ多糖類からL−アラビノース、D−キシロース、そのオリゴ糖を酸処理により製造する方法に関するものである。特許文献6は水溶性アラビノキシランの調製法に関するものであり、特許文献7は免疫力増強物質およびその製造方法に関するものであり、特許文献8はアラビノ多糖類からの水溶性アラビノキシランの製造法およびその機能性に関するものである。
For example, Patent Document 1 relates to a method for purifying L-arabinose, and Patent Document 2 relates to a method for separating L-arabinose, both of which are characterized by the action of microorganisms that assimilate xylose. Patent Document 3 relates to a method for producing L-arabinose or an L-arabinose-containing enzyme-treated product and a method for producing L-arabinose, and Patent Document 4 describes a method for producing L-arabinose, an L-arabinose-containing enzyme-treated product, and a product thereof. Although it relates to the production method, none of them mentions D-xylose in the production method of L-arabinose in which arabinopolysaccharide is treated with an enzyme.
しかしながら、いずれの先行技術も、本発明のような醸造副産物、例えば糠(赤糠、脱脂糠など)、あるいは玄米を原料として、酵素処理および酵母処理の2段階プロセスを用いる、L−アラビノース、D−キシロース、およびアラビノキシラン等のオリゴ糖成分を豊富に含有する機能性素材の効率的な製造技術に関するものではなく、また、これらを示唆するものでもない。
上述のごとく、大量に発生するが用途が無いかあるいはほとんど見出されていない醸造副産物を原料とし、L−アラビノース、D−キシロース、各種オリゴ糖を豊富に含み、健康にとり好ましい機能を有し、かつ安全性が高く、香りも良い機能性素材を得ることが本発明の課題であった。 As described above, brewing by-products that are generated in large quantities but have no use or are rarely found are used as raw materials, are rich in L-arabinose, D-xylose and various oligosaccharides, and have favorable functions for health. It was also an object of the present invention to obtain a functional material having high safety and good fragrance.
本発明者らは、上記事情に鑑みて鋭意研究を重ね、糠もしくは脱脂糠または玄米をヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素を必須として含む酵素で処理し、次いで、酵母で処理することにより、肝機能改善機能、抗アレルギー機能、抗酸化機能、消化吸収抑制機能、血糖値上昇抑制機能などを有する機能性素材が得られることを見出し、本発明を完成するに至った。 The inventors of the present invention have made extensive studies in view of the above circumstances, treating rice bran or defatted rice bran or brown rice with an enzyme containing hemicellulase enzyme and liquefaction enzyme and saccharifying enzyme as essential components, and then treating with yeast. The inventors have found that a functional material having a liver function improving function, an antiallergic function, an antioxidant function, a digestion absorption suppressing function, a blood sugar level increase suppressing function and the like can be obtained, and the present invention has been completed.
本発明によれば、肝機能改善機能、抗アレルギー機能、抗酸化機能、消化吸収抑制機能、血糖値上昇抑制機能などを有し、かつ安全性の高い機能性素材ならびにそれを含有する機能性食品、医薬組成物ならびに化粧組成物が得られる。 According to the present invention, a functional material having a liver function improving function, an antiallergic function, an antioxidant function, a digestion absorption suppressing function, a blood sugar level increasing suppressing function, etc., and a high safety, and a functional food containing the same Pharmaceutical compositions as well as cosmetic compositions are obtained.
すなわち、本発明は、
(1)糠または玄米をヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素を含む酵素系で処理し、次いで、酵母で処理することを特徴とする機能性素材の製造方法;
(2)糠が赤糠である(1)記載の方法;
(3)(1)または(2)記載の方法により得られる機能性素材;
(4)(3)記載の機能性素材を含む機能性食品、医薬組成物または化粧組成物;ならびに
(5)肝機能改善、抗アレルギー、抗酸化、消化吸収抑制、および血糖値上昇抑制からなる群より選択される機能/効能を有するものである(3)記載の機能性素材、(4)記載の機能性食品または医薬組成物
を提供するものである。
That is, the present invention
(1) A method for producing a functional material, comprising treating rice bran or brown rice with a hemicellulase enzyme and an enzyme system containing a liquefaction enzyme and a saccharification enzyme, and then treating with yeast.
(2) The method according to (1), wherein the cocoon is red cocoon;
(3) Functional material obtained by the method according to (1) or (2);
(4) a functional food, pharmaceutical composition or cosmetic composition containing the functional material according to (3); and (5) consisting of liver function improvement, antiallergy, antioxidant, digestion absorption suppression, and blood glucose level increase suppression The functional material according to (3) and the functional food or pharmaceutical composition according to (4), which have a function / efficacy selected from the group.
上述のごとく、本発明は、糠もしくは脱脂糠または玄米をヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素を含む酵素系で処理し、次いで、酵母で処理することを特徴とする機能性素材の製造方法ならびにその方法により得られる機能性素材に関するものである。得られる機能性素材はL−アラビノース、D−キシロース、各種オリゴ糖を豊富に含むものである。したがって、本発明の機能性素材の原料はアラビノキシラン含量が高いものが好ましく、例えば、糠(赤糠もしくは脱脂糠など)または玄米が挙げられる。脱脂糠は赤糠を脱脂したものである。本発明に使用される得に好ましい原料は赤糠または脱脂糠である。 As described above, the present invention provides a method for producing a functional material characterized in that koji or defatted koji or brown rice is treated with an enzyme system containing a hemicellulase enzyme and a liquefying enzyme and a saccharifying enzyme, and then treated with yeast. And a functional material obtained by the method. The functional material obtained is rich in L-arabinose, D-xylose and various oligosaccharides. Therefore, the raw material of the functional material of the present invention preferably has a high arabinoxylan content, and examples thereof include rice bran (red rice bran or defatted rice bran) or brown rice. A defatted lees is obtained by defatting red potatoes. A preferred raw material to be used in the present invention is red cocoon or defatted cocoon.
本発明の方法によれば、原料をヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素を含む酵素系で処理する。本明細書において、この処理工程を「酵素処理」または「酵素処理工程」ともいう。この工程は多糖類の分解ならびにデンプン質からのグルコースの遊離を目的としたものである According to the method of the present invention, the raw material is treated with a hemicellulase enzyme and an enzyme system containing a liquefying enzyme and a saccharifying enzyme. In the present specification, this treatment step is also referred to as “enzyme treatment” or “enzyme treatment step”. This process is aimed at breaking down polysaccharides and releasing glucose from starch.
「ヘミセルラーゼ系酵素」とは、ヘミセルロース分解活性を有する酵素であればいずれの酵素であってもよく、精製されていても、あるいは精製されていなくてもよい。またその起源はいずれのものであってもよい。「ヘミセルラーゼ系酵素」として、例えば、ヘミセルラーゼ「アマノ」90(天野エンザイム株式会社製)、ヘミセルラーゼM(エイチビィアイ株式会社製)等の市販酵素剤を利用することもできる。 The “hemicellulase enzyme” may be any enzyme as long as it has hemicellulose-degrading activity, and may or may not be purified. The origin may be any. As the “hemicellulase-based enzyme”, for example, commercially available enzyme agents such as hemicellulase “Amano” 90 (manufactured by Amano Enzyme Co., Ltd.), hemicellulase M (manufactured by HBI Co., Ltd.) and the like can be used.
「液化酵素」とはデンプン質を液化させる活性を有する酵素であればいずれの酵素であってもよく、精製されていても、あるいは精製されていなくてもよい。またその起源はいずれのものであってもよい。「液化酵素」として、例えば、コクゲンL(大和化成株式会社製)、オリエンターゼAO40(エイチビィアイ株式会社製)等の市販酵素剤を利用することもできる。 The “liquefaction enzyme” may be any enzyme as long as it has an activity to liquefy starch, and may be purified or not purified. The origin may be any. As the “liquefying enzyme”, for example, commercially available enzyme agents such as Kokugen L (manufactured by Daiwa Kasei Co., Ltd.) and orientase AO40 (manufactured by HBI Corporation) can be used.
「糖化酵素」とはデンプン質を糖化させる活性を有する酵素であればいずれの酵素であってもよく、精製されていても、あるいは精製されていなくてもよい。またその起源はいずれのものであってもよい。「糖化酵素」として、例えば、グルク100S(天野エンザイム株式会社製)、グルターゼAD、グルターゼS(いずれもエイチビィアイ株式会社製)等の市販酵素剤を利用することもできる。 The “saccharifying enzyme” may be any enzyme as long as it has an activity of saccharifying starch, and may or may not be purified. The origin may be any. As the “saccharifying enzyme”, for example, commercially available enzyme agents such as Gluc 100S (manufactured by Amano Enzyme Co., Ltd.), Glutase AD, and Glutase S (all manufactured by HTV Corporation) can be used.
「ヘミセルラーゼ系酵素ならびに液化酵素および糖化酵素を含む酵素系」とは、上記の「ヘミセルラーゼ系酵素」、「液化酵素」および「糖化酵素」を必須とし、その他の酵素を含んでいてもよい酵素混合物をいう。その他の酵素としては、原料の分解を促進、あるいは原料中の脂質や蛋白を分解する作用を有するものであってもよい。また、原料からのグルコース遊離を促進するもの、製造された機能性素材に好ましい風味を与えるものであってもよい。その他の酵素としては、例えば、ペクチナーゼ、マンナナーゼ、セルラーゼ、α−ガラクトシダーゼ、フィターゼ、アラバナーゼ、リパーゼ、プロテア−ゼ等が挙げられる。目的に合わせてこれらの酵素を単独あるいは複数種混合して用いることができる。これらの酵素は精製されていても、あるいは精製されていなくてもよい。またそれらの起源はいずれのものであってもよい。これらの酵素として、例えば、ペクチナーゼXP−534(ナガセケムテックス株式会社製)、ペクチナーゼG「アマノ」(天野エンザイム株式会社製)、セルロシンGM5(マンナナーゼ)(エイチビィアイ株式会社製)、リパーゼA−10FG(ナガセケムテックス株式会社製)、リパーゼAY「アマノ」30G(天野エンザイム株式会社製)、スミチームPHY(フィターゼ)(新日本化学株式会社製)、セルロシンTP25(キシラナーゼ)(エイチビィアイ株式会社製)等の市販酵素剤を利用してもよい。 The “hemicellulase-based enzyme and the enzyme system containing a liquefying enzyme and a saccharifying enzyme” are the above-mentioned “hemicellulase-based enzyme”, “liquefying enzyme” and “saccharifying enzyme”, and may contain other enzymes. An enzyme mixture. Other enzymes may have an action of promoting the decomposition of raw materials or decomposing lipids and proteins in the raw materials. Moreover, what promotes the release | release of glucose from a raw material and gives a favorable flavor to the manufactured functional raw material may be sufficient. Examples of other enzymes include pectinase, mannanase, cellulase, α-galactosidase, phytase, arabanase, lipase, and protease. Depending on the purpose, these enzymes can be used singly or in combination. These enzymes may or may not be purified. Further, their origin may be any. As these enzymes, for example, pectinase XP-534 (manufactured by Nagase ChemteX Corporation), pectinase G “Amano” (manufactured by Amano Enzyme Co., Ltd.), cellulosin GM5 (mannanase) (manufactured by HI Corporation), lipase A-10FG ( Nagase ChemteX Corporation), Lipase AY “Amano” 30G (Amano Enzyme Co., Ltd.), Sumiteam PHY (Phitase) (Shin Nihon Chemical Co., Ltd.), Cellulosin TP25 (Xylanase) (manufactured by IB Corporation), etc. An enzyme agent may be used.
酵素処理工程における処理条件(例えば、原料に対する酵素量、反応温度、pH、反応時間等)は目的に応じて適宜選択することができる。原料に対する酵素の割合(重量比)の例は、ヘミセルラーゼ酵素1/20ないし1/50、液化酵素1/50、糖化酵素1/100、リパーゼ、フィターゼ1/500である。酵素量が少なすぎると反応が不十分であり、あるいは反応に長時間を要することとなり、酵素量が多すぎると経済的に好ましくない。反応温度は使用酵素の至適温度を考慮して決定すべきであり、例えば、15〜60℃が一般的であろう。反応pHも使用酵素の至適pHを考慮して決定すべきであり、一般的には3ないし10の範囲であろう。実施例に示すごとく、pH調整用バッファーを使用しなくてもよい。反応時間も適宜決定されうるが、あまり短いと反応が不十分であり、あまり長いと雑菌の混入等の問題が生じるので好ましくない。 The treatment conditions in the enzyme treatment step (for example, the amount of enzyme relative to the raw material, reaction temperature, pH, reaction time, etc.) can be appropriately selected according to the purpose. Examples of the ratio (weight ratio) of the enzyme to the raw material are hemicellulase enzyme 1/20 to 1/50, liquefaction enzyme 1/50, saccharification enzyme 1/100, lipase, and phytase 1/500. If the amount of the enzyme is too small, the reaction is insufficient, or the reaction takes a long time. If the amount of the enzyme is too large, it is not economically preferable. The reaction temperature should be determined in consideration of the optimum temperature of the enzyme used, for example, 15 to 60 ° C. will generally be used. The reaction pH should also be determined in view of the optimum pH of the enzyme used and will generally be in the range of 3-10. As shown in the examples, a pH adjusting buffer may not be used. The reaction time can be determined as appropriate, but if it is too short, the reaction is insufficient, and if it is too long, problems such as contamination with germs occur.
酵素処理工程により得られた酵素処理物を酵母で処理する。本明細書において、この処理工程を「発酵」、「酵母処理」、「発酵工程」または「酵母処理工程」ともいう。この酵母工程の特徴は、ペントース(L−アラビノース、D−キシロースなど)非発酵(資化)性でグルコース、フルクトース発酵(資化)性である清酒酵母でペントースを濃縮し、製造するアルコールで微生物汚染を防止することと、酵母を増殖させて酵母菌体を多く得ることである。酵母菌体については、特にその細胞壁成分由来の食物繊維が豊富な機能を有することが見出されており、また酵母菌体自体にも種々の栄養素が含まれ、胃腸にも良いとされている。さらに酵母により発酵させることで好ましい発酵香も生じる。 The enzyme-treated product obtained by the enzyme treatment step is treated with yeast. In the present specification, this treatment step is also referred to as “fermentation”, “yeast treatment”, “fermentation step” or “yeast treatment step”. The characteristic of this yeast process is that pentose (L-arabinose, D-xylose, etc.) is a non-fermentative (utilizing) glucose, fructose fermenting (utilizing) sake yeast that concentrates pentose and produces microbial microorganisms It is to prevent contamination and to proliferate yeast to obtain many yeast cells. Regarding yeast cells, it has been found that dietary fibers derived from cell wall components have abundant functions, and the yeast cells themselves contain various nutrients and are considered good for the gastrointestinal tract. . Furthermore, preferable fermented aroma is produced by fermenting with yeast.
酵母処理工程における反応条件(反応温度、pH、時間、ならびに使用酵母、通気条件等)も適宜決定されうるが、使用酵母のグルコース消費ならびに増殖などの特性に適した条件とすべきである。使用酵母としては、サッカロミセス属、シゾサッカロミセス属、カンジダ属、クルイベロミセス属などが挙げられるが、機能性食品や医薬組成物への用途を考慮すると、サッカロミセス属酵母が好ましく、特に清酒酵母、ビール酵母が好ましい。酵母接種量も適宜選択できるが、一般的には107ないし108個/mlのオーダーであろう。通気は必要に応じて行うことができる。 Although the reaction conditions (reaction temperature, pH, time, used yeast, aeration conditions, etc.) in the yeast treatment step can be appropriately determined, the conditions should be suitable for the characteristics of the yeast used such as glucose consumption and growth. Examples of yeast used include Saccharomyces genus, Schizosaccharomyces genus, Candida genus, Kluyveromyces genus, etc., considering the use for functional foods and pharmaceutical compositions, Saccharomyces genus yeast is preferred, especially sake yeast, Beer yeast is preferred. The amount of yeast inoculation can also be selected as appropriate, but will generally be on the order of 10 7 to 10 8 cells / ml. Ventilation can be performed as needed.
上で説明した本発明の製造方法により得られる機能性素材は、L−アラビノースおよびD−キシロースならびにアラビノキシランなどのオリゴ糖を多く含み、酵母菌体も多く含むものであり、以下に詳述するような豊富な機能を有するものである。本発明の機能性素材は上記方法で得られた酵母処理物をそのまま使用してもよく、あるいはこれをさらに処理加工して用いてもよい。例えば、さらに濃縮または希釈してもよく、凍結乾燥、加熱乾燥等の乾燥処理に付して使用してもよく、あるいはさらに固形分を水などで抽出して用いてもよい。その形態は特に限定されず、例えば、溶液、懸濁液、半固体(例えば、ペースト状)、固体(例えば、粉末、顆粒)等であってもよい。実施例で説明するように、本発明の機能性素材は、健康によいとされる諸活性/諸機能を示し、典型的には、肝機能改善機能、抗アレルギー機能、抗酸化機能、消化吸収抑制機能および血糖値上昇抑制機能などの種々の生理活性/機能および薬理学的活性/機能を示す。また本発明の機能性素材の安全性は高く、食品や医薬品への用途が考えられる。ここで、「機能性食品(素材)」とは、「食品(素材)が本来持っている栄養機能(第一次機能)、味・香りなどの感覚機能(第二次機能)に加えて、生体防御や疾病の防止・回復、体調リズムの調整、老化抑制などの生体調節機能(第三次機能)があることに注目し、これらの生体調節を科学的に解明し、機能を発揮できるように設計・加工された食品(素材)」を意味する。 The functional material obtained by the production method of the present invention described above contains a large amount of oligosaccharides such as L-arabinose and D-xylose and arabinoxylan, and also contains a large amount of yeast cells, which will be described in detail below. It has abundant functions. As the functional material of the present invention, the processed yeast product obtained by the above method may be used as it is, or it may be used after further processing. For example, it may be further concentrated or diluted, may be used after being subjected to a drying treatment such as freeze-drying or heat-drying, or may further be used after extracting a solid content with water or the like. The form is not particularly limited, and may be, for example, a solution, a suspension, a semi-solid (for example, a paste), a solid (for example, a powder or a granule), and the like. As will be described in Examples, the functional material of the present invention exhibits various activities / functions that are considered to be good for health, and typically has a liver function improving function, an antiallergic function, an antioxidant function, and digestive absorption. Various physiological activities / functions and pharmacological activities / functions such as an inhibitory function and a blood glucose level elevation inhibiting function are shown. In addition, the functional material of the present invention is highly safe and can be used for foods and pharmaceuticals. Here, “functional food (material)” means “nutrient function (primary function) inherent to food (material), sensory function (secondary function) such as taste and aroma, Pay attention to biological regulation functions (tertiary functions) such as biological defense, prevention and recovery of diseases, adjustment of physical rhythm, suppression of aging, etc., so that these biological regulation can be elucidated scientifically and functions can be demonstrated Means food (material) that has been designed and processed.
本発明により得られる機能性素材を種々の用途に用いることができる。また本発明の機能性素材は香りが良いものである。1の用途としては本発明の機能性素材を食品中に用いて種々の機能性食品あるいは健康食品を得ることができる。例えば、そのまま、あるいはペレット、粉末、顆粒などの形態として使用してもよく、食品添加物、調味料、ふりかけとして使用してもよい。また、本発明の機能性素材を食材中に含有せしめて使用してもよい。 The functional material obtained by the present invention can be used for various applications. The functional material of the present invention has a good scent. As one use, various functional foods or health foods can be obtained by using the functional material of the present invention in foods. For example, it may be used as it is or in the form of pellets, powders, granules or the like, or may be used as a food additive, seasoning or sprinkle. Moreover, you may use the functional raw material of this invention, making it contain in a foodstuff.
さらなる用途としては本発明の機能性素材を医薬組成物中に用いることができる。一般的には、かかる医薬組成物は本発明の機能性素材のほかに医薬用担体または賦形剤を含むであろう。その形態も特に限定されず、例えば、固体(錠剤、粉末、顆粒等)、半固体(パスタ等)、液体(溶液、懸濁液、エマルジョン等)、あるいはカプセルに封入されたものであってもよい。これらの剤形のための担体または賦形剤も当業者によく知られており、適宜選択されうる。 As a further use, the functional material of the present invention can be used in a pharmaceutical composition. In general, such pharmaceutical compositions will include a pharmaceutical carrier or excipient in addition to the functional material of the present invention. The form is not particularly limited, and for example, it may be solid (tablet, powder, granule, etc.), semi-solid (pasta etc.), liquid (solution, suspension, emulsion, etc.), or encapsulated in a capsule. Good. Carriers or excipients for these dosage forms are also well known to those skilled in the art and can be selected as appropriate.
もう1つの用途としては本発明の機能性素材を化粧組成物中に用いることができる。本発明の機能性素材はフェルラ酸を豊富に含み、グルコースをほとんど含有しないか含有しないので、グルコースによる皮膚へのべたつきがなく、フェルラ酸による活性酸素消去効果および皮膚細胞の成長促進効果があり、化粧品素材としての用途がある。したがって、本発明は、本発明の機能性素材を含む化粧組成物に関するものである。本発明の化粧組成物は、本発明の機能性素材のほかに化粧品に常用される担体、例えば、水、グリセリン、アルコール類、油脂などを含有するものであり、さらに香料、着色料、防腐剤などを適宜添加してもよい。本発明の機能性素材を含む化粧組成物の形状は特に限定されず、例えばローション、乳液、クリーム、軟膏などの形状とすることができる。 As another application, the functional material of the present invention can be used in a cosmetic composition. Since the functional material of the present invention is rich in ferulic acid and contains little or no glucose, there is no stickiness to the skin by glucose, and there is an active oxygen scavenging effect and a skin cell growth promoting effect by ferulic acid, There are uses as cosmetic materials. Therefore, the present invention relates to a cosmetic composition containing the functional material of the present invention. The cosmetic composition of the present invention contains a carrier commonly used in cosmetics in addition to the functional material of the present invention, such as water, glycerin, alcohols, oils and fats, and further includes a fragrance, a colorant and a preservative. Etc. may be added as appropriate. The shape of the cosmetic composition containing the functional material of the present invention is not particularly limited, and for example, it can be a lotion, emulsion, cream, ointment or the like.
これらの機能性食品や医薬組成物は、本発明の機能性素材が有する機能/効能を有するものであるが、それらの典型的な機能/効能としては、例えば、肝機能改善、抗アレルギー、抗酸化、消化吸収抑制および血糖値上昇抑制が挙げられる。 These functional foods and pharmaceutical compositions have the functions / effects of the functional material of the present invention, and typical functions / effects thereof include, for example, liver function improvement, anti-allergy, anti-allergy, Oxidation, digestion absorption inhibition, and blood glucose level elevation inhibition are mentioned.
以下に実施例を示して本発明をより詳細かつ具体的に説明するが、これらの実施例は本発明を限定するものと解してはならない。 The present invention will be described in more detail and specifically with reference to the following examples, but these examples should not be construed as limiting the present invention.
酵素処理に使用する酵素のスクリーニングその1
小麦由来のアラビノキシラン(Megazyme社製)2gを50mMリン酸緩衝液(pH5.0)10mlに溶かし、食品用市販酵素剤であるヘミセルラーゼ剤、ペクチナーゼ剤、キシラナーゼ剤、セルラーゼ剤、フィターゼ、リパーゼ、α−ガラクトシダーゼ、マンナナーゼ、アラバナーゼ、α−アミラーゼ、グルコアミラーゼなどの市販酵素剤をそれぞれ100mgを加え、50℃で18時間反応後のL-アラビノース(以下「アラビノース」ともいう)、D-キシロース(以下「キシロース」ともいう)、アラビノキシランのオリゴ糖が遊離してくる酵素剤をそれぞれスクリーニングした。アラビノース、キシロース、アラビノキシランのオリゴ糖の定性分析は薄層クロマトグラフィー(TLC プラスティックシート Sirica gel 60 F254 (メルク社製)、展開溶媒 酢酸エチル:ピリジン:水=12:5:4で展開し、濃硫酸で発色した)、定量分析は液体クロマトグラフィー(HPLCシステム(ギルソン社製)、カラム Asahipak NH2P-50 4E(昭和電工社製)、SEDEX-55 蒸発光散乱検出計(SEDERE社製)で行った。
Screening of enzymes used for enzyme treatment (1)
Dissolve 2 g of wheat-derived arabinoxylan (Megazyme) in 10 ml of 50 mM phosphate buffer (pH 5.0), hemicellulase agent, pectinase agent, xylanase agent, cellulase agent, phytase, lipase, α -L-arabinose (hereinafter also referred to as “arabinose”), D-xylose (hereinafter referred to as “the arabinose”) after adding 100 mg each of commercially available enzyme agents such as galactosidase, mannanase, arabanase, α-amylase, glucoamylase and reacting at 50 ° C. for 18 hours. The enzyme agent from which arabinoxylan oligosaccharides are released was also screened. Qualitative analysis of oligosaccharides of arabinose, xylose, and arabinoxylan was developed with thin layer chromatography (TLC plastic sheet Sirica gel 60 F254 (Merck), developing solvent ethyl acetate: pyridine: water = 12: 5: 4, concentrated sulfuric acid. Quantitative analysis was performed with liquid chromatography (HPLC system (Gilson), column Asahipak NH2P-50 4E (Showa Denko), SEDEX-55 evaporative light scattering detector (SEDERE)).
その結果、アラビノースが多く遊離する酵素剤はスミチームARS、スミチームPX(新日本化学工業株式会社製)、ヘミセルラーゼ「アマノ」90、セルラーゼA「アマノ」3(天野エンザイム株式会社製)、セルロシンHC100(エイチビィアイ株式会社製)などであり、キシロースが多く遊離する酵素剤はペクチナーゼG「アマノ」、ヘミセルラーゼ「アマノ」90(天野エンザイム株式会社製)、セルロシンTP25、セルロシンGM5、セルロシンT2(エイチビィアイ株式会社製)、スミチームACH(新日本化学工業株式会社製)、アラビノキシランのオリゴ糖が多く遊離する酵素剤はペクチナーゼXP-534(ナガセケムテックス株式会社製)、VERON191(レーム・エンザイム社製)などであることが分かった。
次に赤糠、脱脂糠(築野食品工業株式会社製)を用いて同じ反応条件で各種酵素剤を組合せてアラビノース、キシロースの単糖が多い条件、アラビノキシランのオリゴ糖が多い条件を検討した結果、赤糠ではペクチナーゼXP-534とセルロシンGM5を組合わせると単糖が、脱脂糠ではスミチームARSとセルロシンGM5を組合わせると単糖とオリゴ糖が効率よく遊離してくることが分かった。
As a result, the enzyme agents that release a large amount of arabinose are Sumiteam ARS, Sumiteam PX (Shin Nippon Chemical Industry Co., Ltd.), Hemicellulase “Amano” 90, Cellulase A “Amano” 3 (Amano Enzyme Co., Ltd.), Cellulosin HC100 ( Enzyme agents that release a large amount of xylose are pectinase G “Amano”, hemicellulase “Amano” 90 (manufactured by Amano Enzyme), cellulosin TP25, cellulosin GM5, cellulosin T2 (manufactured by HBI Corporation) ), Sumiteam ACH (manufactured by Shin Nippon Chemical Industry Co., Ltd.), enzyme agents that release a large amount of arabinoxylan oligosaccharides are pectinase XP-534 (manufactured by Nagase ChemteX Corporation), VERON191 (manufactured by Rame Enzyme), etc. I understood.
Next, as a result of examining conditions under which the amount of monosaccharides of arabinose and xylose is high and conditions of a large amount of oligosaccharides of arabinoxylan by combining various enzyme agents under the same reaction conditions using red koji and defatted lees In red rice bran, it was found that monosaccharides were released efficiently when pectinase XP-534 and cellulosin GM5 were combined, and in defatted silkworms, Sumiteam ARS and cellulosin GM5 were combined.
また赤糠酵素反応時にリパーゼ剤、フィターゼ剤を同時に酵素処理し、苦味が出なく風味が良くなる酵素剤として、リリパーゼA-10FG(ナガセケムテックス株式会社製)、スミチームPHY(新日本化学工業株式会社製)を選択した。更に赤糠、脱脂糠のデンプン質をグルコースまで効率よく分解する酵素剤として、液化型アミラーゼ剤のコクゲンL(大和化成株式会社製)と糖化型アミラーゼ剤のグルク100S(天野エンザイム株式会社製)を組合わせると良いことが分かった。 In addition, lipase A-10FG (manufactured by Nagase ChemteX Corporation), Sumiteam PHY (Nippon Chemical Industry Co., Ltd.) are enzyme enzymes that simultaneously treat lipase and phytase during the red yeast enzyme reaction to improve the taste without bitterness. Selected). Furthermore, as an enzyme agent that efficiently degrades starch from red koji and defatted koji to glucose, liquefied amylase agent Kokugen L (manufactured by Yamato Kasei Co., Ltd.) and saccharified amylase agent gluc 100S (manufactured by Amano Enzyme Co., Ltd.) It turned out to be a good combination.
機能性素材の調製その1
上記予備試験の酵素処理条件の結果を受けて、以下の条件で赤糠と脱脂糠の酵素処理を行った。なお、本実施例により赤糠から得られた機能性素材の粉末乾燥品を「赤糠発酵物1」といい、脱脂糠から得られた機能性素材の粉末乾燥品を「脱脂糠発酵物1」という。
Preparation of functional materials 1
In response to the results of the enzyme treatment conditions in the preliminary test, red candy and defatted candy were subjected to enzyme treatment under the following conditions. In addition, the powder dried product of the functional material obtained from the red koji according to this example is referred to as “red koji fermented product 1”, and the powder dried product of the functional material obtained from the degreased koji is referred to as “fat koji fermented product 1 "
赤糠10kgと水50LとペクチナーゼXP-534を500g、リリパーゼA-10FGを20g加え、pH7、50℃で18時間、更にセルロシンGM5を500g、スミチームPHYを20g、コクゲンLを200g、グルク100Sを100g加え、pH4.5、55℃で18時間酵素処理を行った。脱脂糠10kgと水50LとスミチームARSを500g加え、pH3.5、55℃で18時間、更にセルロシンGM5を500g、コクゲンLを200g、グルク100Sを100g加え、pH4.5、55℃で18時間酵素処理を行った。なお各pH調整は食品添加用の乳酸、乳酸ナトリウムで行った。酵素処理後のグルコース、フルクトース含量を測定した(表1中発酵前の欄)。 Add 10kg of red potato, 50L of water, 500g of pectinase XP-534, 20g of lipase A-10FG, pH 7, 18 hours at 50 ° C, 500g of cellulosin GM5, 20g of Sumiteam PHY, 200g of Kokugen L, 100g of Gluc 100S In addition, the enzyme treatment was performed at pH 4.5 and 55 ° C. for 18 hours. Add 10kg of defatted rice cake, 50L of water and 500g of Sumiteam ARS, pH 3.5, 18 hours at 55 ° C, add 500g of cellulosin GM5, 200g of Kokugen L, 100g of Gluc 100S, 18 hours at pH 4.5, 55 ° C Processed. Each pH adjustment was performed with lactic acid for food addition and sodium lactate. The glucose and fructose content after the enzyme treatment was measured (in the column before fermentation in Table 1).
赤糠及び脱脂糠酵素処理液に清酒酵母である泡無し酵母B29を1×108個植菌し、発酵後の酵母数を更に増加させ(清酒酵母の各種機能性増強)、生成するアルコールで雑菌の繁殖を抑えるためにグルコースを各5kg加えて、25℃で5日間撹拌培養を行った。発酵後の酵母数(cells/ml)、アルコール濃度(Alc;%)、グルコース量(Glu;%)、フルクトース量(Fru;%)と滴定酸度(TA;ml)、滴定アミノ酸度(AA;ml)を測定し、発酵前後での分析値の比較を表1に示す。 Inoculate 1 × 10 8 froth-free yeast B29, a sake yeast, into the red and defatted enzyme treatment solution, further increase the number of yeast after fermentation (enhanced various functions of sake yeast), In order to suppress the propagation of various bacteria, 5 kg of glucose was added, and stirring culture was performed at 25 ° C. for 5 days. Yeast count after fermentation (cells / ml), alcohol concentration (Alc;%), glucose level (Glu;%), fructose level (Fru;%) and titrated acidity (TA; ml), titrated amino acid level (AA; ml) Table 1 shows a comparison of analytical values before and after fermentation.
表1より赤糠発酵液、脱脂糠発酵液共に酵母数が増加し、グルコース、フルクトースが清酒酵母によりほとんど資化されて無くなり、アルコールに変換され、更に分析値には出してないが、アラビノース、キシロースは酵母発酵前後により全く変化が無く、糖質のデンプン質が減少し、アラビノース、キシロースが濃縮され、酵母数が増加し、生成したアルコールにより微生物汚染が防止できる目的が達成された素材が開発できた。滴定酸度は赤糠発酵液よりも脱脂糠発酵液の方が高くなりやや酸っぱい素材となったが、両者共に香りは発酵後の良いものであった。 From Table 1, the number of yeast increases in both red koji fermented liquor and defatted koji fermented liquid, and glucose and fructose are almost assimilated by sake yeast and converted to alcohol. There is no change in xylose before and after yeast fermentation, the starch content of saccharides is reduced, arabinose and xylose are concentrated, the number of yeasts is increased, and the material that achieves the purpose of preventing microbial contamination with the generated alcohol has been developed did it. The titrated acidity of the defatted koji fermented liquid was higher than that of the red koji fermented liquid, and both became a slightly sour material, but both had good aroma after fermentation.
粉末乾燥は各発酵液を2倍に加熱濃縮(100℃、5時間)し、スプレードライヤーSD-1000(東京理化器械株式会社製)で行い、赤糠発酵物1を7.48kgと脱脂糠発酵物1を8.25kg得た。乾燥物の一般成分値を表2に示す。 Powder drying is performed by heating and concentrating each fermented liquid twice (100 ° C, 5 hours) and using a spray dryer SD-1000 (manufactured by Tokyo Rika Kikai Co., Ltd.). 1.25 kg of 1 was obtained. Table 2 shows the general component values of the dried product.
表2より食品成分表の米糠の成分と比較すると発酵物はいずれも酵母数が多くなり、酵母細胞壁成分と酵母細胞質成分由来の食物繊維とタンパク質の含量が多くなった。また両者共にデンプン質が分解されていることにより糖質の含量が少なくなった。赤糠発酵物1ではリパーゼ剤の処理により脂質の分解は一部となった。 Compared with the rice bran component of the food composition table from Table 2, all fermented products had a higher number of yeasts, and the content of dietary fiber and protein derived from the yeast cell wall component and yeast cytoplasm component increased. In both cases, the starch content was reduced due to the degradation of starch. In red koji fermented product 1, the degradation of lipid became part due to the treatment with the lipase agent.
また各発酵物の遊離のアラビノース、キシロース含量と不溶性残渣中に残存するアラビノース、キシロースの多糖類の含量は、72%硫酸、室温で1時間撹拌、その後4%硫酸、121℃オートクレーブで1時間加水分解を行った後、アラビノース、キシロース含量を測定した結果をそれぞれ表3に示す。 The free arabinose and xylose content of each fermented product and the content of arabinose and xylose polysaccharide remaining in the insoluble residue were 72% sulfuric acid, stirred at room temperature for 1 hour, then 4% sulfuric acid, 121 ° C autoclaved for 1 hour. Table 3 shows the results of measurement of the arabinose and xylose contents after the decomposition.
表3より赤糠発酵物1の可溶性(遊離/遊離+残渣)アラビノース、キシロースの割合はそれぞれ12%、4%となり、脱脂糠発酵物1の可溶性アラビノース、キシロースの割合はそれぞれ13%、3%となった。赤糠発酵物1、脱脂糠発酵物1共に可溶性アラビノキシランのオリゴ糖も薄層クロマトグラム、液体クロマトグラムで酵素処理後、酵母発酵で資化されないオリゴ糖が存在していることがわかったが定量することはできなかった(標準物質が無いため)。 From Table 3, the percentages of soluble (free / free + residue) arabinose and xylose in red koji fermented product 1 are 12% and 4%, respectively, and the proportions of soluble arabinose and xylose in defatted koji fermented product 1 are 13% and 3%, respectively. It became. Both the red koji fermented product 1 and the defatted koji fermented product 1 were found to contain soluble arabinoxylan oligosaccharides that were not assimilated by yeast fermentation after enzyme treatment in thin-layer chromatograms and liquid chromatograms. Could not (because there is no reference material).
酵素処理に使用する酵素のスクリーニングその2
平成15年産日本晴の玄米2gと50mMリン酸緩衝液10mlに、食品用市販酵素剤であるヘミセルラーゼ剤、ペクチナーゼ剤、キシラナーゼ剤、セルラーゼ剤、フィターゼ、リパーゼ、α−ガラクトシダーゼ、マンナナーゼ、アラバナーゼなどの市販酵素剤をそれぞれ100mgを加え、各種酵素剤の至適pHと至適温度で18時間反応後、反応前後の濁度(HACH社製、2100N Turbidimeter)、グルコース量、直接還元糖量を測定した。その結果、スミチームARS、スミチームPX、スミチームACH(新日本化学工業株式会社製)、ペクチナーゼG「アマノ」、ヘミセルラーゼ「アマノ」90(天野エンザイム株式会社製)、ペクチナーゼXP-534(ナガセケムテックス株式会社製)、セルロシンTP-25、セルロシンGM5(エイチビィアイ株式会社製)、VERON191(レーム・エンザイム社製)などの酵素剤が濁度、グルコース量、直接還元糖の上昇率が高いことが分かった。中でもセルロシンTP-25の濁度の上昇率が高く、玄米の最外部に作用する働きが強い酵素剤である結果となった。
Screening of enzymes used for enzyme treatment 2
Commercially available hemicellulase agent, pectinase agent, xylanase agent, cellulase agent, phytase, lipase, α-galactosidase, mannanase, arabanase, etc., which are commercially available food enzymes, in 2 g of Nihonbare rice produced in 2003 and 10 ml of 50 mM phosphate buffer 100 mg of each enzyme agent was added, and after reacting for 18 hours at the optimum pH and temperature of each enzyme agent, the turbidity before and after the reaction (HACH, 2100N Turbidimeter), glucose amount, and direct reducing sugar amount were measured. As a result, Sumiteam ARS, Sumiteam PX, Sumiteam ACH (manufactured by Nippon Chemical Industry Co., Ltd.), pectinase G “Amano”, hemicellulase “Amano” 90 (manufactured by Amano Enzyme Co., Ltd.), pectinase XP-534 (Nagase ChemteX Corporation) It was found that enzyme agents such as Cellulosin TP-25, Cellulosin GM5 (manufactured by HIBI Co., Ltd.) and VERON191 (manufactured by Rame Enzyme) have high turbidity, glucose amount and direct reducing sugar increase rate. Above all, the rate of increase in turbidity of cellulosin TP-25 was high, resulting in a strong enzyme that acts on the outermost part of brown rice.
次に赤糠、脱脂糠各10gと50mMリン酸緩衝液50mlに、スミチームARS、スミチームPX、スミチームACH、ペクチナーゼG「アマノ」、ヘミセルラーゼ「アマノ」90、ペクチナーゼXP-534、セルロシンTP-25、セルロシンGM5、VERON191の各種酵素剤500mgを加え、各種酵素剤の至適pHと至適温度で18時間反応後、清酒酵母B29を5×107個植菌し、グルコースを5g加えて25℃で4日間発酵を行い、遊離するアラビノース、キシロース含量と滴定酸度を測定した。その結果、ペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90が単独で多くのアラビノース、キシロースを遊離し、発酵液の滴定酸度を低くできることが分かった。 Next, red cocoon, defatted cocoon 10 g each and 50 mM phosphate buffer 50 ml, Sumiteam ARS, Sumiteam PX, Sumiteam ACH, pectinase G `` Amano '', hemicellulase `` Amano '' 90, pectinase XP-534, cellulosin TP-25, Add 500 mg of cellulosin GM5 and VERON191 enzyme agents, react for 18 hours at optimum pH and temperature of each enzyme agent, inoculate 5 × 10 7 sake yeast B29, add 5 g of glucose at 25 ° C Fermentation was carried out for 4 days, and free arabinose, xylose content and titratable acidity were measured. As a result, it was found that pectinase G “Amano” and hemicellulase “Amano” 90 independently release a large amount of arabinose and xylose, and the titrated acidity of the fermentation broth can be lowered.
更に赤糠、脱脂糠各10gと50mMリン酸緩衝液50mlに、セルロシンTP-25、ペクチナーゼG「アマノ」、ヘミセルラーゼ「アマノ」90の3種類の酵素剤500mgを組合せの順番を変化させて加え、各酵素剤の至適pH、至適温度で各18時間反応後、清酒酵母B29を5×107個植菌し、グルコースを5g加えて25℃で4日間発酵を行い、遊離するアラビノース、キシロース含量と滴定酸度を測定した。その結果、赤糠、脱脂糠発酵液共に先にセルロシンTP-25で反応し、次にペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90を同時に反応させることにより、多くのアラビノース、キシロースを遊離し、発酵液の滴定酸度を低くできることが分かった。 Furthermore, 500 mg of 3 types of enzyme agents, Cellulosin TP-25, Pectinase G “Amano”, and Hemicellulase “Amano” 90, were added to 10 g each of red koji and defatted koji and 50 mM phosphate buffer, changing the order of combination. After each reaction for 18 hours at the optimum pH and temperature of each enzyme agent, inoculate 5 × 10 7 sake yeast B29, add 5 g of glucose, ferment at 25 ° C. for 4 days, and release arabinose, The xylose content and titratable acidity were measured. As a result, both red koji and defatted koji fermented liquor were reacted with cellulosin TP-25 first, and then pectinase G “Amano” and hemicellulase “Amano” 90 were reacted simultaneously to release a large amount of arabinose and xylose. It was found that the titratable acidity of the fermentation broth can be lowered.
また赤糠、脱脂糠各10gと50mMリン酸緩衝液50mlに、セルロシンTP-25、ペクチナーゼG「アマノ」、ヘミセルラーゼ「アマノ」90の3種類の酵素剤をそれぞれ100、200、500mgとコクゲンLを200mgとグルク100Sを100mg加え、赤糠では更にリパーゼAY「アマノ」30G(天野エンザイム株式会社製)またはリリパーゼA-10FGをそれぞれ20mgとスミチームPHYを20mg加え、各酵素剤の至適pH、至適温度で各18時間または42時間反応後、清酒酵母B29を5×107個植菌し、グルコースを5g加えて25℃で4日間発酵を行い、遊離するアラビノース、キシロース含量と滴定酸度を測定した。その結果、赤糠ではセルロシンTP-25を200〜500mgとコクゲンLを200mgとグルク100Sを100mgでpH5、60℃で18時間反応後、ペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90を200〜500mgとスミチームPHYを20mgでpH4.5、50℃で18〜42時間反応後、リパーゼAY「アマノ」30Gを20mgでpH7、50℃で18時間反応し、酵母で発酵後のアラビノース、キシロースが多く遊離する条件であることが分かった。一方、脱脂糠ではセルロシンTP-25を100〜200mgとコクゲンLを200mgとグルク100Sを100mgでpH5、60℃で18時間反応後、ペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90を100〜200mgでpH4.5、50℃で18〜42時間反応し、酵母で発酵後のアラビノース、キシロースが多く遊離する条件であることが分かった。また赤糠発酵液と脱脂糠発酵液の遊離するアラビノース、キシロース、滴定酸度も同程度であった。
In addition, 10 g each of red koji and defatted koji and 50 ml of 50 mM phosphate buffer, 100, 200 and 500 mg of Kelogen L, 3 types of enzyme agents, cellulosin TP-25, pectinase G “Amano” and hemicellulase “Amano” 90, respectively. 200 mg and 100 mg of Gluc 100S, and in Akamine, lipase AY “Amano” 30G (manufactured by Amano Enzyme Co., Ltd.) or lipase A-10FG 20 mg each and
機能性素材の調製その2
上記予備試験の酵素処理、発酵条件の結果を受けて、以下の条件で赤糠と脱脂糠の酵素処理、発酵を行った。なお、本実施例により赤糠から得られた機能性素材の凍結乾燥品を「赤糠発酵物2」という。
Preparation of functional materials 2
In response to the results of the enzyme treatment and fermentation conditions in the preliminary test, the enzyme treatment and fermentation of red koji and defatted koji were performed under the following conditions. In addition, the freeze-dried product of the functional material obtained from red koji according to this example is referred to as “red koji fermented product 2”.
赤糠2kgと水10LとセルロシンTP-25を40gまたは100gとコクゲンLを40gとグルク100Sを20gでpH5、60℃で18時間反応後、ペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90を40gまたは100gとスミチームPHYを4gでpH4.5、50℃で42時間(酵素剤の量が少ない区分)または18時間(酵素剤の量が多い区分)反応後、リパーゼAY「アマノ」30Gを4gでpH7、50℃で18時間反応させた。脱脂糠2kgと水10LとセルロシンTP-25を40gとコクゲンLを40gとグルク100Sを20gでpH5、60℃で18時間反応後、ペクチナーゼG「アマノ」とヘミセルラーゼ「アマノ」90を40gでpH4.5、50℃で42時間反応させた。なおpH調整は食品添加用の塩酸、水酸化ナトリウムで行った。赤糠酵素処理液2区分と脱脂糠酵素処理液1区分に清酒酵母B29を5×107個植菌し、グルコースを1kg加えて25℃で4日間撹拌発酵を行い、遊離するアラビノース、キシロース、グルコース含量と酵母数とアルコール濃度と滴定酸度と滴定アミノ酸度を測定した(表4)。その結果、赤糠発酵液2区分と脱脂糠発酵液1区分の分析値はほぼ同程度であった。そこで赤糠の酵素剤の量が少ない区分の発酵物を加熱処理することなくそのまま真空凍結乾燥機TF10-80TPA(株式会社宝製作所製)で乾燥を行い、赤糠発酵物2 1.66kgを得た。この赤糠発酵物2と素材の上記赤糠発酵物1との各種成分比較を表4に示す。
After reaction with 2 kg of red potato, 10 g of water and 40 g of cellulosin TP-25 or 100 g and 40 g of Kokugen L and 20 g of Gluc 100S at
表4より赤糠発酵物2は、赤糠の可溶性(遊離/遊離+残渣)アラビノース、キシロースの割合はそれぞれ35%、23%となった。また赤糠発酵物1と比べ遊離アラビノースで3倍、遊離キシロースで6倍それぞれが多い素材が開発できた。この赤糠発酵物2の可溶性アラビノキシランのオリゴ糖も液体クロマトグラムで酵素処理後、酵母発酵で資化されないオリゴ糖が存在しているが定量することはできなかった。その他今回の赤糠発酵液(赤糠発酵物2を凍結乾燥する前)の酵母数は前回と同程度であるが、アルコールの生成がやや高くなり、グルコースは全くなくなっていた。滴定酸度は前回の4割程度と低くすることができ、酸っぱさが際立たない、発酵香の良い、嗜好性の優れた素材となった。 According to Table 4, fermented red koji 2 had red koji soluble (free / free + residue) arabinose and xylose ratios of 35% and 23%, respectively. In addition, compared to fermented red koji 1, a material that was 3 times more free arabinose and 6 times more free xylose could be developed. The oligosaccharides of soluble arabinoxylan of this red koji fermented product 2 also existed as oligosaccharides not assimilated by yeast fermentation after enzyme treatment in the liquid chromatogram, but could not be quantified. In addition, the number of yeast in this red koji fermentation broth (before freeze-drying red koji fermented product 2) was about the same as the previous one, but the production of alcohol was slightly higher and glucose was completely lost. The titratable acidity can be lowered to about 40% of the previous time, and it has become a material with excellent fertility and taste that does not stand out sourness.
in vitro試験
(a)二糖類分解酵素抑制試験
得られた赤糠発酵物、脱脂糠発酵物の消化吸収抑制効果を評価するため、次に調製方法を示す小腸粗酵素を用いてマルターゼ活性阻害率、スクラーゼ活性阻害率を測定した。
Rat intestinal acetone powder(シグマ社製)に10倍容のマレイン酸緩衝液(pH6.0)を加え、5000rpm、30分間の遠心分離後、得た上清を小腸粗酵素とした。また、粉末試料に3.33倍容のマレイン酸緩衝液(pH6.0)を加え14000rpm、20分間の遠心分離後、得た上清を70℃湯浴中で残留酵素を失活させたものを評価した。
in vitro test
(A) Disaccharide-degrading enzyme inhibition test In order to evaluate the digestion and absorption inhibition effect of the obtained red koji fermented product and defatted koji fermented product, the maltase activity inhibition rate, sucrase activity using the small intestine crude enzyme which shows the preparation method The inhibition rate was measured.
Rat intestinal acetone powder (manufactured by Sigma) was added with 10 volumes of maleic acid buffer (pH 6.0), centrifuged at 5000 rpm for 30 minutes, and the resulting supernatant was used as a small intestine crude enzyme. In addition, after adding 3.33 volumes of maleic acid buffer (pH 6.0) to the powder sample and centrifuging at 14000 rpm for 20 minutes, the supernatant obtained was evaluated by inactivating the residual enzyme in a 70 ° C hot water bath. did.
マルターゼ活性阻害率は終濃度1%マルターゼ溶液に小腸粗酵素と抽出物を混合し、スクラーゼ活性阻害率は終濃度1%スクロース溶液に小腸粗酵素と抽出物を混合し、それぞれ37℃で1時間反応させ、反応後の溶液中のグルコース生成量をグルコースcIIテストワコー(和光純薬工業株式会社製)により測定した。また、阻害率は抽出物を混合しないBlankの値を基準に以下の計算式より導き出した。その結果を表5に示す。
活性阻害率(%)=100−B/A×100
A:Blankのグルコース生成量、B:抽出物のグルコース生成量
The maltase activity inhibition rate is the mixture of the small intestine crude enzyme and the extract in the final concentration 1% maltase solution. The sucrase activity inhibition rate is the mixture of the small intestine crude enzyme and the extract in the final concentration 1% sucrose solution, each at 37 ° C for 1 hour. The amount of glucose produced in the solution after the reaction was measured with a glucose cII test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Moreover, the inhibition rate was derived from the following calculation formula based on the blank value without mixing the extract. The results are shown in Table 5.
Activity inhibition rate (%) = 100−B / A × 100
A: Blank glucose production, B: Extract glucose production
表5より赤糠発酵物(1、2共に)、脱脂糠発酵物1はいずれもスクラーゼ活性抑制、マルターゼ活性抑制効果が認められた。これらの発酵物においてグルコースを低減し、アラビノース、キシロース、アラビノキシランのオリゴ糖を濃縮している結果と考えられる。原料の赤糠、脱脂糠には共存する糖類の影響で両活性を上昇する傾向が認められた。 From Table 5, both the red koji fermented product (both 1 and 2) and the defatted koji fermented product 1 were found to have sucrase activity inhibition and maltase activity inhibition effects. It is considered that glucose is reduced in these fermented products, and oligosaccharides of arabinose, xylose, and arabinoxylan are concentrated. There was a tendency for both activities to be increased due to the influence of the coexisting sugars in the raw red koji and defatted koji.
(b)DPPHラジカル消去能試験
赤糠発酵物、脱脂糠発酵物の抗酸化能を評価するため、粉末試料に20倍容の50%EtOHを加え、室温で3時間抽出し、3000rpm、5分間の遠心分離後、得た上清をDPPHラジカル消去能試験により行った。
(B) DPPH radical scavenging ability test In order to evaluate the antioxidant capacity of fermented red koji and fermented koji koji, 20 times volume of 50% EtOH was added to the powder sample, extracted at room temperature for 3 hours, 3000 rpm, 5 minutes After centrifugation, the obtained supernatant was subjected to DPPH radical scavenging ability test.
2M 酢酸緩衝液(pH5.5)25μL、99.5% EtOH 475μL、抽出物 500μL、1,1-Diphenyl-2-picrylhydrazyl(DPPH)125μLを混合し、DPPH添加30分後のOD517nmを測定した。ラジカル消去能を算出するために検体の代わりに水を混合したものも測定した。
以下のラジカル消去能計算式からラジカル消去能を算出した。
ラジカル消去能(%)=100−(OD517B/OD517A)x100
OD517A:水(対照)、OD517B:検体
また、高いDPPHラジカル消去能をもつことが知られているグルタチオンを標準に用い、発酵物、対照のラジカル消去能をグルタチオン量に換算した。その結果を表6に示す。
25 μL of 2M acetate buffer (pH 5.5), 475 μL of 99.5% EtOH, 500 μL of extract, and 125 μL of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were mixed, and OD517nm was measured 30 minutes after addition of DPPH. In order to calculate radical scavenging ability, water mixed in place of the specimen was also measured.
The radical scavenging ability was calculated from the following formula for radical scavenging ability.
Radical scavenging ability (%) = 100− (OD517 B / OD517 A ) × 100
OD517 A : Water (control), OD517 B : Specimen Glutathione, which is known to have high DPPH radical scavenging ability, was used as a standard, and the radical scavenging ability of the fermented product and the control was converted to the amount of glutathione. The results are shown in Table 6.
表6より赤糠発酵物(1、2共に)、脱脂糠発酵物1はいずれもDPPHラジカル消去能、すなわち抗酸化能が認められた。原料の赤糠、脱脂糠共に抗酸化能があるが、発酵させることによりその活性を高める効果があることが分かった。 From Table 6, both the red koji fermented product (both 1 and 2) and the defatted koji fermented product 1 were found to have DPPH radical scavenging ability, that is, antioxidant ability. Both raw red koji and defatted koji have antioxidant capacity, but it has been found that fermenting has the effect of increasing their activity.
安全性試験
(a)急性経口毒性スクリーニング試験
赤糠発酵物1、脱脂糠発酵物1のラットに対する急性経口毒性を検討するため、雌3匹(6週齢)のSlc:Wistar系(SPF)動物を用いて試験を実施した。用量は2000mg/kgの1用量とした。赤糠発酵物1、脱脂糠発酵物1を2000mg精秤後、注射用蒸留水を加え10mLに定容した懸濁液を投与前16時間絶食した動物に胃ゾンデを用いて単回投与した。投与後の観察期間は7日間とし、動物の生死、一般状態および体重推移について観察するとともに、観察終了時に病理解剖検査を行った(表7)。
Safety test
(A) Acute oral toxicity screening test To examine the acute oral toxicity of fermented red koji fermented product 1 and defatted koji fermented product 1 to rats, three female (6 weeks old) Slc: Wistar (SPF) animals were used. The test was conducted. The dose was 1 dose of 2000 mg / kg. After weighing precisely 2000 mg of fermented red koji fermented product 1 and defatted koji fermented product 1, a suspension of 10 mL of distilled water added for injection was given to the animal fasted for 16 hours before administration using a stomach tube. The observation period after administration was 7 days, and the animals were observed for life and death, general condition, and body weight change, and pathological anatomical examination was performed at the end of the observation (Table 7).
(b)復帰突然変異スクリーニング試験
赤糠発酵物1、脱脂糠発酵物1の遺伝子突然変異誘発性を検討するため、ネズミチフス菌(Salmonella typhimurium)TA100およびTA98株を用いた復帰突然変異スクリーニング試験を行った。
(B) Reverse mutation screening test In order to examine the gene mutagenicity of fermented red koji fermented product 1 and defatted koji fermented product 1, a reverse mutation screening test using Salmonella typhimurium TA100 and TA98 strains was conducted. It was.
被験物質の調製は、適切な溶媒に溶解あるいは懸濁させ調製原液を調製し、必要に応じ、ろ過除菌を行った。また、試験用量は、39.1、78.1、156、313、625、1250、2500および5000μg/プレートの8用量(公比2)を使用した。被験物質の生育阻害作用を確認するため、プレート上の試験菌株の生育状態について実態顕微鏡で観察し、さらに被験物質の析出状態を肉眼で観察した。次いで、復帰突然変異により生じたコロニー数を計測し、復帰突然変異コロニー数が陰性対照の2倍以上に増加し、かつその増加に用量依存性が認められた場合に陽性と判断する事とした(表7)。 The test substance was prepared by dissolving or suspending in a suitable solvent to prepare a preparation stock solution, and sterilizing by filtration if necessary. As test doses, 8 doses (common ratio 2) of 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg / plate were used. In order to confirm the growth inhibitory action of the test substance, the growth state of the test strain on the plate was observed with an actual microscope, and the deposition state of the test substance was further observed with the naked eye. Next, the number of colonies generated by back mutation was counted, and when the number of back mutation colonies increased more than twice that of the negative control and the increase was confirmed to be dose-dependent, it was determined to be positive. (Table 7).
(c)残留農薬多成分一斉分析
常法に従い、赤糠発酵物1の抽出、濃縮、カラム精製等の前処理を行った後、GC-MS測定により77種の残留農薬のスクリーニング分析を行った(表7)。
(C) Residual pesticide multi-component simultaneous analysis According to the conventional method, after pretreatment such as extraction, concentration, column purification, etc. of red koji fermented product 1, screening analysis of 77 pesticide residues was performed by GC-MS measurement. (Table 7).
(d)重金属分析
赤糠発酵物1中の重金属を以下の方法で測定した。
ヒ素、鉛、カドミウムを原子吸光光度法、総水銀を還元気化原子吸光光度法、総クロムをジフェニルカルバジド吸光光度法、シアンをピリジンピラゾロン吸光光度法により測定した(表7)。
(D) Heavy metal analysis The heavy metal in the red yeast rice fermented material 1 was measured with the following method.
Arsenic, lead and cadmium were measured by atomic absorption spectrophotometry, total mercury was measured by reductive vapor atomic absorption spectrophotometry, total chromium was measured by diphenylcarbazide spectrophotometry, and cyan by pyridinepyrazolone spectrophotometry (Table 7).
表7より赤糠発酵物1、脱脂糠発酵物1共に急性経口毒性スクリーニング試験、復帰突然変異スクリーニング試験共に陰性の結果であった。赤糠発酵物は残留農薬77成分一斉分析の結果、77成分は定量限界以下であった。砒素、鉛、水銀、カドミウム、シアン、クロムの重金属分析の結果は問題になる含量ではなかった。すなわち、赤糠発酵物1、脱脂糠発酵物1は共に食品素材としての安全性に問題がないことが分かった。赤糠発酵物2についても同様の結果を得た。従って、本発明の機能性素材の安全性が確認された。 From Table 7, both the red koji fermented product 1 and the defatted koji fermented product 1 were negative in both the acute oral toxicity screening test and the reverse mutation screening test. As a result of simultaneous analysis of 77 pesticide residues, 77 components were below the limit of quantification. The results of heavy metal analysis of arsenic, lead, mercury, cadmium, cyan, and chromium were not problematic. That is, it was found that both the red koji fermented product 1 and the defatted koji fermented product 1 have no problem in safety as food materials. Similar results were obtained for fermented red koji 2. Therefore, the safety of the functional material of the present invention was confirmed.
血糖値上昇抑制効果確認試験
4週齢Sprague-Dawley系雄性ラット(Jc1;SD、日本クレア株式会社製)を用い、室温23±1℃、湿度55±7%、明暗周期12時間(明期8〜20時)、MF固形飼料(オリエンタル酵母工業株式会社製)および水は自由に与え、4週間予備飼育後(8週齢、264.6±5.1g)実験に供した。胃および門脈にカテーテルを留置した無麻酔・無拘束下のラットを5匹ずつ2群に分け、更に24時間飼育、次に16時間絶食後、15%スクロース溶液を11.25ml/kgラット/時間の速度で2時間投与し門脈血中グルコース濃度が一定になることを確認した。赤糠発酵物2と対照の赤糠をそれぞれ3倍量の水で30分間抽出し、14000rpm、20分間の遠心分離後、250メッシュでろ過し、更にNo.5Aのろ紙でろ過したろ液を濃縮後の試料をそれぞれ胃内に投与し(赤糠発酵物抽出液;0.36g/kgアラビノース+キシロース相当量、赤糠;同様な操作の対照)、その後10分おきに3時間まで門脈カテーテルより門脈血0.05mlを採取して、血漿中のグルコース濃度を測定した(図1;赤糠発酵物抽出液投与ラット、図2;対照赤糠投与ラット)。
Blood glucose level increase inhibitory effect confirmation test
4-week-old Sprague-Dawley male rats (Jc1; SD, manufactured by Clea Japan Co., Ltd.), room temperature 23 ± 1 ° C, humidity 55 ± 7%, light / dark cycle 12 hours (light period 8-20 hours), solid MF Feed (Oriental Yeast Co., Ltd.) and water were freely given and subjected to the experiment after preliminary breeding for 4 weeks (8 weeks old, 264.6 ± 5.1 g). Unanesthetized and unrestrained rats with catheters placed in the stomach and portal vein were divided into 2 groups of 5 each and then reared for 24 hours, then fasted for 16 hours, then 15% sucrose solution 11.25 ml / kg rat / hour It was confirmed that the glucose concentration in the portal vein blood became constant after administration for 2 hours at a rate of 5%. Extract the red koji fermented product 2 and the control red koji for 30 minutes each with 3 times the amount of water, centrifuge at 14000 rpm for 20 minutes, filter through 250 mesh, and filter with the No. 5A filter paper. Each concentrated sample was administered into the stomach (red bean ferment extract; 0.36 g / kg arabinose + xylose equivalent, red bean; similar operation control), then portal catheter up to 3 hours every 10 minutes Further, 0.05 ml of portal vein blood was collected, and the glucose concentration in the plasma was measured (FIG. 1; rat administered with red yeast ferment extract extract, FIG. 2; rat administered with control red coral).
図1、2より、赤糠発酵物抽出液では門脈血のグルコース濃度は、投与前に比べ投与後20分で有意な低下が認められた(図1中アステリスクで示す)。対照の赤糠では門脈血のグルコース濃度は有意な低下が認められず、投与後10分、100分で有意な上昇が認められた。アラビノースとキシロースの合計濃度で0.36g/kgラットではラット小腸のα−グルコシダーゼ阻害作用があることが分かった。従って本発明の赤糠発酵物は糖尿病食後過血糖を抑制することができる素材であることが分かった。 1 and 2, the glucose concentration in the portal vein blood was significantly decreased 20 minutes after administration in the red yeast ferment extract (indicated by an asterisk in FIG. 1). In the control red rabbit, the glucose concentration in the portal vein blood was not significantly decreased, but was significantly increased at 10 minutes and 100 minutes after administration. In the total concentration of arabinose and xylose, 0.36 g / kg rat was found to have an inhibitory effect on α-glucosidase in the rat small intestine. Therefore, it was found that the fermented red koji product of the present invention is a material capable of suppressing hyperglycemia after diabetic diet.
肝機能改善効果確認試験
6週齢SD系雄性ラット(日本クレア株式会社製)を1週間予備飼育後、表8の配合の飼料で、飼料と水は自由摂取させ、室温23±2℃、湿度60±10%、12時間の明暗サイクルの環境下で20日間の飼育実験を行った。
Liver function improvement effect confirmation test
6 weeks old SD male rats (CLEA Japan, Inc.) were preliminarily raised for 1 week, then feed and water were freely consumed with the diet shown in Table 8, room temperature 23 ± 2 ° C, humidity 60 ± 10%, 12 Breeding experiments were conducted for 20 days in an environment with a light-dark cycle.
飼育21日目に肝臓と盲腸重量、尾静脈血清については総コレステロールと中性脂肪(TG)を、腹部大動脈血漿についてはGOT(グルタミックオキザロアセティクトランスアミナーゼ活性)、GPT(グルタミックピルビックトランスアミナーゼ活性)、グルコース、HDL−コレステロール(HDLcho)を測定した(表9)。 On day 21 of breeding, liver and cecal weights, total cholesterol and triglycerides (TG) for tail vein serum, GOT (glutamic oxaloacetic transaminase activity) and GPT (glutamic pyrvic transaminase activity) for abdominal aorta plasma ), Glucose and HDL-cholesterol (HDLcho) were measured (Table 9).
表9より、赤糠発酵物1(C群)と脱脂糠発酵物1(D群)はセルロース(A群)と比較して血漿中のGOT、GPTの酵素活性が有意に低下しており、共に肝機能改善効果が認められた。赤糠発酵物1はHDL−コレステロールをやや増加させる傾向があり、体重あたりの盲腸重量も有意に増加しており、何らかの食物繊維的効果があると考えられる。 From Table 9, the fermented red koji fermented product 1 (Group C) and defatted koji fermented product 1 (Group D) have significantly reduced enzyme activities of GOT and GPT in plasma compared to cellulose (Group A). Both showed improvement of liver function. The red koji fermented product 1 has a tendency to slightly increase HDL-cholesterol, the cecal weight per body weight is also significantly increased, and it is considered that there is some dietary fiber effect.
アレルギー低減効果確認試験
4週齢のアレルギー発症Brown Norway ssn slcラット(日本エルエスシー株式会社製)の8匹を1群として4群を用い、MF固形飼料と水は自由摂取させ、毎日各飼料(水、赤糠、赤糠発酵物、脱脂糠発酵物の4群)を60mgとなるように胃ゾンデで投与し、室温23±2℃、湿度60±10%、12時間の明暗サイクルの環境下で6週間(4週齢〜10週齢)の飼育実験を行った。1週間毎の血漿IgA、IgEと糞中のsIgAと10週齢時の脾臓、肝臓重量を測定した。その結果、体重は各投与群とも順調に増加した。また脾臓、肝臓重量、糞中sIgAにも各投与群に差は認められなかった。各投与群の血漿中のIgAの変化を図3、図4に、IgEの変化を図5に示す。
Allergy reduction effect confirmation test
Alleviate Brown ssn slc rats at 4 weeks of age (4 from 1 group), using 4 groups as one group, MF solid feed and water ad libitum, and each feed (water, red potato, 4 groups of fermented red koji and fermented koji koji were administered with a gastric sonde to a dose of 60 mg, and the temperature was 23 ± 2 ° C, humidity was 60 ± 10%, and the environment was 12 hours light-dark cycle for 6 weeks (4 Breeding experiments were conducted between 10 weeks of age and 10 weeks of age. Plasma IgA, IgE, sIgA in feces and spleen and liver weights at 10 weeks of age were measured every week. As a result, body weight increased steadily in each administration group. In addition, there was no difference in spleen, liver weight, and fecal sIgA among the administration groups. 3 and 4 show changes in plasma IgA in each administration group, and FIG. 5 shows changes in IgE.
図3より、赤糠発酵物1投与群は3週間目以降血漿IgA値が上昇し、特に5週間目では顕著に上昇したことがわかる。その他の投与群では差は認められなかった。また図4より、赤糠発酵物1投与群では投与3週間目以降有意に血漿IgA値が増加し、体液性免疫機構が活性化されたことがわかる。さらに図5より、赤糠発酵物2で10週齢で血漿中のIgEの低下が有意に認められ、免疫機構が活性化された。従って赤糠発酵物2には免疫賦活機能があることがわかった。従って本発明の赤糠発酵物にはアレルギー発症ラットにおいてアレルギー低減効果があることが確認できた。 From FIG. 3, it can be seen that the plasma IgA value increased in the group administered with red yeast fermented product 1 after the 3rd week, and particularly increased in the 5th week. There was no difference in the other groups. Further, FIG. 4 shows that in the group administered with fermented red koji, the plasma IgA level significantly increased after the third week of administration, and the humoral immune mechanism was activated. Furthermore, from FIG. 5, in the red koji fermented product 2, a decrease in plasma IgE was significantly observed at 10 weeks of age, and the immune mechanism was activated. Therefore, it was found that fermented red koji 2 has an immunostimulatory function. Therefore, it was confirmed that the red koji fermented product of the present invention has an allergy reducing effect in allergic rats.
本発明は、健康に好ましい種々の機能を有し、安全性の高い機能性素材を提供するものであり、食品や医薬、あるいは化粧品の分野において利用可能である。 The present invention provides functional materials having various functions preferable for health and high safety, and can be used in the fields of foods, medicines, and cosmetics.
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