JP2012211084A - Enzyme composition - Google Patents
Enzyme composition Download PDFInfo
- Publication number
- JP2012211084A JP2012211084A JP2009169348A JP2009169348A JP2012211084A JP 2012211084 A JP2012211084 A JP 2012211084A JP 2009169348 A JP2009169348 A JP 2009169348A JP 2009169348 A JP2009169348 A JP 2009169348A JP 2012211084 A JP2012211084 A JP 2012211084A
- Authority
- JP
- Japan
- Prior art keywords
- glucose
- enzyme
- enzyme composition
- aspergillus oryzae
- gdh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 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
- A61P3/04—Anorexiants; Antiobesity agents
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- 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
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- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
Description
本発明は、酵素組成物に関し、詳細には、摂取することにより胃内及び／又は腸内のグルコースを低減しカロリーの摂取を低減する酵素組成物に関する。 The present invention relates to an enzyme composition, and in particular, to an enzyme composition that reduces ingestion of calories by reducing glucose in the stomach and / or intestine by ingestion.
近年、食の欧米化、飽食の時代を迎え、健康上の理由からカロリーのコントロールが重要である。特に、中高年者では、カロリーの摂取過多はメタボリックシンドロームを介して高血圧、心臓病、痛風、脂肪肝、糖尿病等の疾患リスクを増強することが知られている。そのため、多くの低カロリー製品の開発が進められているが、これらの製品は入手が難しいことや、食事本来の機能である食味や香りが損なわれ食事が楽しめないことが多く、実際の使用は進んでいないのが現状である。
一方、食事を楽しみながらカロリーの摂取を低減するための製品開発も進められている。その代表的なものとして、消化酵素阻害製品がある。これらは茶飲料、サプリメント、健康食品として製品化されているが、未消化の栄養成分である炭水化物や脂質により消化器に膨満感が発生することがある。また、食事を楽しみながらカロリーの摂取を低減する酵素を用いる方法として、酵素反応により生体内において食事由来の炭水化物を難消化性で消化・吸収されにくくエネルギー源となりにくいオリゴ糖に変換する酵素組成物がある。該酵素組成物は、食事前後、あるいは食事と同時に摂取することで、食品中に含まれる、あるいは食品中の炭水化物の分解物を生体内でオリゴ糖に変換し、結果として糖質吸収量を低減するもので、サプリメント、健康食品あるいは消化酵素剤として応用できる（特許文献１参照）。
In recent years, the era of westernization and satiety of food has entered, and control of calories is important for health reasons. Particularly in middle-aged and elderly people, it is known that excessive intake of calories increases the risk of diseases such as hypertension, heart disease, gout, fatty liver and diabetes via metabolic syndrome. For this reason, many low-calorie products are being developed, but these products are difficult to obtain, and the taste and aroma that are the original functions of the meal are often impaired, making it impossible to enjoy meals. The current situation is not progressing.
On the other hand, products are being developed to reduce calorie intake while enjoying meals. A typical example is a digestive enzyme inhibitor product. These are commercialized as tea drinks, supplements, and health foods, but the digestive organs may become full due to undigested nutrients such as carbohydrates and lipids. In addition, as a method of using an enzyme that reduces the intake of calories while enjoying a meal, an enzyme composition that converts a carbohydrate derived from a meal into an oligosaccharide that is difficult to digest, absorb, and does not easily become an energy source in vivo by an enzyme reaction There is. The enzyme composition can be taken before or after meals or at the same time as meals to convert carbohydrate degradation products contained in or in food into oligosaccharides in vivo, resulting in reduced carbohydrate absorption. It can be applied as a supplement, health food, or digestive enzyme agent (see Patent Document 1).
しかし、上記の酵素組成物は、グルコースを含む飲食物を摂取した場合、カロリー源のグルコースはオリゴ糖に変換できないために摂取され、カロリーのコントロールが難しいということがある。また、グルコースは、糖尿病などの生活習慣病にも深く関与しているので、酵素反応を利用してグルコースを低減できれば生活習慣病の予防を期待できる。さらに、酵素反応によりグルコースを低減させて生体に有用な生理活性を有する化合物に変換できれば生体に好ましい効果を期待できる。 However, when the above-described enzyme composition is ingested with food or drink containing glucose, it may be difficult to control calorie because glucose as a calorie source cannot be converted into oligosaccharide. Moreover, since glucose is deeply involved in lifestyle-related diseases such as diabetes, prevention of lifestyle-related diseases can be expected if glucose can be reduced using an enzyme reaction. Furthermore, if glucose can be reduced by an enzymatic reaction and converted into a compound having physiological activity useful for the living body, a favorable effect on the living body can be expected.
本発明は、かかる事情に鑑みなされたもので、酵素反応を利用してグルコースを低減してカロリーの摂取を低減し、またグルコースを整腸効果があると言われるグルコン酸に変換する酵素組成物を提供することを課題とする。なお、本願の明細書、特許請求の範囲、要約書及び図面において記載されるグルコースは、D-グルコースを意味する。 The present invention has been made in view of such circumstances, and an enzyme composition that uses an enzyme reaction to reduce glucose to reduce caloric intake and convert glucose to gluconic acid, which is said to have an intestinal effect. It is an issue to provide. In addition, glucose described in the specification, claims, abstract, and drawings of the present application means D-glucose.
本発明者らは、食事を摂取しながらカロリーの摂取を低減できる酵素反応を種々検討した結果、所定の酸化還元酵素は胃内条件下又は腸内条件下においてもグルコースを基質とする酸化還元反応を触媒できることを見出し本発明を完成するに至った。すなわち、本発明は、酸化還元酵素のフラビンアデニンジヌクレオチド結合型グルコース脱水素酵素、グルコースオキシダーゼ、NAD(P)依存型グルコース脱水素酵素及びピロロキノリンキノン依存型グルコース脱水素酵素から選ばれる１種以上を含み、摂取により胃内及び／又は腸内のグルコースを低減させることを特徴とする酵素組成物を要旨とする。 As a result of various studies on enzyme reactions that can reduce caloric intake while ingesting meals, the present inventors have found that a given oxidoreductase is a redox reaction using glucose as a substrate even under gastric or intestinal conditions. As a result, the present invention has been completed. That is, the present invention provides at least one oxidoreductase selected from flavin adenine dinucleotide-binding glucose dehydrogenase, glucose oxidase, NAD (P) -dependent glucose dehydrogenase and pyrroloquinoline quinone-dependent glucose dehydrogenase. And an enzyme composition characterized by reducing glucose in the stomach and / or intestine by ingestion.
上記の発明において、フラビンアデニンジヌクレオチド結合型グルコース脱水素酵素は、Aspergillus oryzaeが生産するものとしてもよい。また、Aspergillus oryzae BB-56（NITE BP-236）が生産するものとしてもよい。 In the above invention, the flavin adenine dinucleotide-binding glucose dehydrogenase may be produced by Aspergillus oryzae. Alternatively, Aspergillus oryzae BB-56 (NITE BP-236) may be produced.
上記の酵素組成物は、腸溶剤としてもよい。また、上記の酵素組成物を含ませ消化酵素剤、サプリメントあるいは健康食品の原料としてもよい。 The enzyme composition may be an enteric solvent. Further, the enzyme composition described above may be included and used as a raw material for digestive enzyme agents, supplements or health foods.
上記の酵素組成物を用いることを特徴とする生体内でグルコースをグルコン酸に変換する方法を要旨とする。 The gist is a method for converting glucose into gluconic acid in vivo, characterized by using the above enzyme composition.
本発明の酵素組成物は、摂取することにより胃内及び／又は腸内のグルコースを低減させるので、カロリーの摂取を低減でき、肥満の防止や血糖値上昇の防止を図ることができ、ひいてはカロリーの摂取過多が一因となる心筋梗塞等の心疾患、高血圧、脳血管疾患、高脂血症、糖尿病、痛風等の生活習慣病の予防を期待できる。また、本発明の酵素組成物は、酸化還元酵素の作用でグルコースがグルコン酸に変換されるので、グルコン酸又はD-グルコノ-δ-ラクトンによる腸内細菌叢の改善等の整腸効果を期待できる。 Since the enzyme composition of the present invention reduces the glucose in the stomach and / or the intestine when ingested, it can reduce the intake of calories, can prevent obesity and prevent an increase in blood sugar level, and thus calories. Prevention of heart disease such as myocardial infarction, hypertension, cerebrovascular disease, hyperlipidemia, diabetes, gout and other lifestyle-related diseases that are caused by excessive intake of. In addition, since the enzyme composition of the present invention converts glucose into gluconic acid by the action of oxidoreductase, it expects an intestinal regulating effect such as improvement of intestinal flora by gluconic acid or D-glucono-δ-lactone. it can.
本発明の酵素組成物は、酸化還元酵素のフラビンアデニンジヌクレオチド結合型グルコース脱水素酵素（以下、「FAD-GDH」と略すことがある）、グルコースオキシダーゼ（以下、「GO」と略すことがある）、NAD(P)依存型グルコース脱水素酵素（以下、「NAD(P)-GDH」と略すことがある）及びピロロキノリンキノン依存型グルコース脱水素酵素（以下、「PQQ-GDH」と略すことがある）から選ばれる１種以上を含む。これらの酸化還元酵素の給源は、特に限定されず、微生物、動物、植物等の何れをも用いることができるが、たとえば微生物が給源である場合、以下の微生物が酵素の給源として例示できる。
FAD-GDH(EC.1.1.99.10)は、Aspergillus oryzae、Aspergillus terreus、Penicillium italicum、Penicillium lilacinoechinulatumを例示できる。GO (EC.1.1.3.4)は、Aspergillus niger、Penicillium amagasakiense、Penicillium notatum、Penicillium chrysogenum、Penicillium purpurogenum、Penicillium vitale、Phanerochaete chrysosporiumを例示できる。NAD(P)-GDH(EC.1.1.1.47)は、Bacillus licheniformis、Bacillus megaterium、Bacillus subtilis、Burkholderia cepacia、Haloferax mediterranei、Sulfolobus solfataricus、Thermoplasma acidophilumを例示できる。PQQ-GDH (EC.1.1.5.2) は、Acetobacter aceti、Acinetobacter baumannii、Acinetobacter calcoaceticus、Gluconobacter oxydans subsp. suboxydans、Pseudomonas aeruginosa、Pseudomonas fluorescensを例示できる。
The enzyme composition of the present invention is an oxidoreductase flavin adenine dinucleotide-binding glucose dehydrogenase (hereinafter sometimes abbreviated as “FAD-GDH”), glucose oxidase (hereinafter abbreviated as “GO”). ), NAD (P) -dependent glucose dehydrogenase (hereinafter abbreviated as “NAD (P) -GDH”) and pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter abbreviated as “PQQ-GDH”) 1) or more selected from. The source of these oxidoreductases is not particularly limited, and any of microorganisms, animals, plants, and the like can be used. For example, when microorganisms are sources, the following microorganisms can be exemplified as enzyme sources.
Examples of FAD-GDH (EC.1.1.99.10) include Aspergillus oryzae, Aspergillus terreus, Penicillium italicum, and Penicillium lilacinoechinulatum. GO (EC.1.1.3.4) can be exemplified by Aspergillus niger, Penicillium amagasakiense, Penicillium notatum, Penicillium chrysogenum, Penicillium purpurogenum, Penicillium vitale, Phanerochaete chrysosporium. NAD (P) -GDH (EC.1.1.1.47) can be exemplified by Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis, Burkholderia cepacia, Haloferax mediterranei, Sulfolobus solfataricus, Thermoplasma acidophilum. Examples of PQQ-GDH (EC.1.1.5.2) include Acetobacter aceti, Acinetobacter baumannii, Acinetobacter calcoaceticus, Gluconobacter oxydans subsp. Suboxydans, Pseudomonas aeruginosa, and Pseudomonas fluorescens.
一般的に、消化管内は嫌気性条件にあることが知られている。したがって、上記の酸化還元酵素のうち、GOは嫌気条件化において、メディエーターを介して酸化反応することが知られているが、通常、反応には酸素が必要であり、消化管内のような嫌気性条件での作用が十分ではないことがある。また、NAD(P)-GDHやPQQ-GDHは、反応にNAD(P)、PQQの補酵素の添加が必要となる。NAD(P)やPQQの補酵素は非常に高価である点、更にNAD(P)-GDHとPQQ-GDHは菌体内酵素であるため、純度の高い酵素を取得するためには高度な精製技術が必要となる点から、実用面での懸念がある。一方、FAD-GDHは、反応に酸素を必要とせず、嫌気条件化におかれている消化管内でも十分な作用が期待される点、補酵素であるFADは酵素に結合しているため、反応に補酵素の添加が必要でない点、菌体外酵素であるため、経口投与に必要な純度の高い酵素の調製に有利である点から、FAD-GDHがより好ましい。 In general, it is known that the digestive tract is in an anaerobic condition. Therefore, among the above oxidoreductases, GO is known to undergo an oxidation reaction via a mediator under anaerobic conditions, but usually requires oxygen for the reaction and is anaerobic as in the digestive tract. The effect on conditions may not be sufficient. In addition, NAD (P) -GDH and PQQ-GDH require the addition of NAD (P) and PQQ coenzymes in the reaction. NAD (P) and PQQ coenzymes are very expensive, and NAD (P) -GDH and PQQ-GDH are intracellular enzymes, so advanced purification techniques are required to obtain highly pure enzymes. There is a practical concern because of the need for On the other hand, FAD-GDH does not require oxygen for the reaction, and is expected to work well in the digestive tract under anaerobic conditions. FAD, which is a coenzyme, is bound to the enzyme. FAD-GDH is more preferred because it is an extracellular enzyme and is advantageous for preparing a highly pure enzyme required for oral administration.
上記の酸化還元酵素がFAD-GDHの場合、Aspergillus oryzaeが産生するものを用いることができる。Aspergillus oryzaeは、Aspergillus oryzae BB-56、Aspergillus oryzae IAM2603、Aspergillus oryzae IAM2628、Aspergillus oryzae IAM2683、Aspergillus oryzae IAM2736、Aspergillus oryzae IAM2706及びAspergillus oryzae NBRC30113を例示できる。特に、Aspergillus oryzae BB-56は、FAD-GDHの生産性に優れる好ましい生産菌である。また、Aspergillus oryzae BB-56の生産するFAD-GDHは、基質特異性が高く好ましい。Aspergillus oryzae BB-56は、国際公開番号 WO 2007/139013 A1に記載があり、寄託番号がNITE BP-236として下記の国際寄託機関に寄託されているので入手が容易である。
寄託機関：NITEバイオテクノロジー本部 特許微生物寄託センター(〒292-0818 日本国千葉県木更津市かずさ鎌足2-5-8)
寄託日(受領日)：2006年5月17目
受託番号：NITE BP-236
When the oxidoreductase is FAD-GDH, those produced by Aspergillus oryzae can be used. Examples of Aspergillus oryzae include Aspergillus oryzae BB-56, Aspergillus oryzae IAM2603, Aspergillus oryzae IAM2628, Aspergillus oryzae IAM2683, Aspergillus oryzae IAM2736, Aspergillus oryzae IAM2706, and Aspergillus oryzae NBRC30113. In particular, Aspergillus oryzae BB-56 is a preferable producing bacterium excellent in productivity of FAD-GDH. Further, FAD-GDH produced by Aspergillus oryzae BB-56 is preferable because of high substrate specificity. Aspergillus oryzae BB-56 is described in International Publication No. WO 2007/139013 A1, and the deposit number is NITE BP-236, which is easily deposited because it is deposited with the following international depository.
Depositary: NITE Biotechnology Headquarters, Patent Microorganism Deposit Center (2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan)
Deposit date (receipt date): May 17, 2006 Deposit number: NITE BP-236
Aspergillus oryzae BB-56のCYA培地での形態は、表１に示す通りである。 The form of Aspergillus oryzae BB-56 in CYA medium is as shown in Table 1.
Aspergillus oryzae BB-56が生産するFAD-GDHは、以下の特性を有している。
(1)作用：電子受容体存在下でグルコースの水酸基を酸化してD-グルコノ-δ-ラクトンを生成する反応を触媒する、
(2)分子量：SDS-ポリアクリルアミド電気泳動による分子量が約100KDa、ゲルろ過クロマトグラフィーによる分子量が約400KDa、
(3)基質特異性：マルトース、D-フルクトース、D-マンノース及びD-ガラクトースに対する反応性が低い。
(4)至適pH：7付近、
(5)至適温度：60℃付近、
(6)ｐＨ安定性：pH3.0〜7.0の範囲で安定、
(7)温度安定性：40℃以下で安定。
FAD-GDH produced by Aspergillus oryzae BB-56 has the following characteristics.
(1) Action: catalyses the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce D-glucono-δ-lactone.
(2) Molecular weight: about 100 KDa molecular weight by SDS-polyacrylamide electrophoresis, about 400 KDa molecular weight by gel filtration chromatography,
(3) Substrate specificity: low reactivity with maltose, D-fructose, D-mannose and D-galactose.
(4) Optimal pH: around 7,
(5) Optimal temperature: around 60 ℃
(6) pH stability: stable in the range of pH 3.0-7.0,
(7) Temperature stability: stable at 40 ° C or lower.
上記のAspergillus oryzae IAM2603、Aspergillus oryzae IAM2628、Aspergillus oryzae IAM2683、Aspergillus oryzae IAM2736及びAspergillus oryzae IAM2706は、IAMカルチャーコレクション（独立行政法人 理化学研究所バイオリソースセンター微生物材料開発室（JCM））で保管される菌株で入手が容易である。また、Aspergillus oryzae NBRC30113は、NBRC（独立行政法人 製品評価技術基盤機構バイオテクノロジー本部生物遺伝資源部門）で保管される菌株で入手が容易である。 The above Aspergillus oryzae IAM2603, Aspergillus oryzae IAM2628, Aspergillus oryzae IAM2683, Aspergillus oryzae IAM2736 and Aspergillus oryzae IAM2706 are obtained as strains stored in the IAM Culture Collection (RIKEN BioResource Center Microbial Materials Development Office (JCM)) Is easy. In addition, Aspergillus oryzae NBRC30113 is a strain that is stored at NBRC (Biotechnology Division, Biotechnology Headquarters, National Institute of Technology and Evaluation).
上記の酸化還元酵素は、メディエーターを含む系で作用が顕著になるので、メディエーターを加えることができる。メディエーターは、電子受容体又は電子供与体として機能し、摂取される生体に害がないもので、チトクローム、ユビキノン、クロロフィル、ポリフェノール類、4-ヒドロキシ安息香酸メチル(HBAMe)等やこれらの誘導体、ビタミンK誘導体を例示できる。 Since the action of the above oxidoreductase becomes remarkable in a system including a mediator, a mediator can be added. Mediators function as electron acceptors or electron donors and are not harmful to the ingested living body, such as cytochrome, ubiquinone, chlorophyll, polyphenols, methyl 4-hydroxybenzoate (HBAMe) and their derivatives, vitamins Examples include K derivatives.
本発明の酵素組成物は、例えば消化酵素剤、サプリメント、健康食品の原料として供することができる。消化酵素剤は、配合禁止とならない他の酵素、例えば、特開２０００−３２５０４５号公報に記載の酵素を配合することが可能である。また、サプリメントは、配合禁止とならない他の成分と組み合わせることが可能である。本発明の酵素組成物の製品形態は、経口で摂取できれば特に限定がなく、粉末剤、細粒剤、顆粒剤、丸剤、錠剤、カプセル剤、マイクロカプセル化したものを例示できる。また、本発明の酵素組成物は、胃内条件下又は腸内条件下のいずれにおいてもグルコースを基質として酸化還元反応を触媒できるが、飲食物は膵液の消化酵素及び腸液の消化酵素によってグルコースに分解されるので、腸溶剤が好ましい。腸溶剤は、腸溶皮膜を固形剤に噴霧・被覆し、あるいはカプセルそのものを腸溶性にして得ることができる。腸溶皮膜には、特に限定はないが、セルロースヒドロキシプロピルメチルフタレート、酢酸フタル酸セルロース、ヒドロキシプロピルメチルセルロースアセテートサクシネート等を例示できる。また、本発明の酵素組成物は、製剤化のための助剤、例えば賦形剤、結合剤、滑沢剤、界面活性剤、流動性促進剤等を適宜含むことができる。 The enzyme composition of the present invention can be used, for example, as a raw material for digestive enzyme agents, supplements, and health foods. The digestive enzyme agent can be blended with other enzymes that are not prohibited to be blended, for example, enzymes described in JP-A-2000-325045. In addition, the supplement can be combined with other components that are not prohibited to be blended. The product form of the enzyme composition of the present invention is not particularly limited as long as it can be taken orally, and examples thereof include powders, fine granules, granules, pills, tablets, capsules, and microcapsules. The enzyme composition of the present invention can catalyze the oxidation-reduction reaction using glucose as a substrate under both gastric conditions and intestinal conditions. However, food and drink are converted into glucose by pancreatic digestive enzymes and intestinal digestive enzymes. Intestinal solvents are preferred because they decompose. The enteric solvent can be obtained by spraying and coating an enteric coating on a solid agent or making the capsule itself enteric. The enteric film is not particularly limited, and examples thereof include cellulose hydroxypropyl methyl phthalate, cellulose acetate phthalate, hydroxypropyl methyl cellulose acetate succinate, and the like. Moreover, the enzyme composition of the present invention can appropriately contain auxiliary agents for formulation, for example, excipients, binders, lubricants, surfactants, fluidity promoters and the like.
本発明の酵素組成物の投与量は、摂取するヒトの年齢、性別、体重、肥満度、血糖値等を考慮して適宜増減でき、1,000〜400,000U（単位）/回、好ましくは2000〜40,000U/回、より好ましくは10,000〜20,000U/回である。本発明の酵素組成物は、飲食物に含まれるグルコース及び生体内で消化酵素により分解されたグルコースに作用するため、食前、飲食物と同時又は食後に摂取することが好ましい。 The dosage of the enzyme composition of the present invention can be appropriately increased or decreased taking into account the age, sex, body weight, obesity, blood glucose level, etc. of the human being ingested, and is 1,000 to 400,000 U (unit) / time, preferably 2000 to 40,000. U / times, more preferably 10,000 to 20,000 U / times. Since the enzyme composition of the present invention acts on glucose contained in food and drink and glucose decomposed by digestive enzymes in vivo, it is preferably taken before or at the same time as or after meal.
本発明に係る酸化還元酵素は、以下に示すようにグルコースを基質として酸化還元反応を触媒し、グルコースを酸化してD-グルコノ-δ-ラクトン（グルコン酸無水物）を生成する。
グルコース＋受容体→D-グルコノ-δ-ラクトン＋還元型受容体
D-グルコノ-δ-ラクトンは、その後平衡反応によりグルコン酸に変換される。この結果、酸化還元酵素により飲食物中のグルコースは、グルコン酸に変換される。グルコン酸は、蜂蜜に多く含まれ、その他果物やワイン、味噌、醤油等の発酵食品等にも含まれ、食品添加物として酸味料やpH調整剤に利用されている。D-グルコノ-δ-ラクトンは、摂取により腸内ビフィズス菌が増加したとの報告（ビフィズス 1994; 8:29-35)やグルコン酸は摂取により便秘傾向の健康成人女性の摂取期間中の排便回数、排便日数の増加が見られたとの報告(腸内細菌学雑誌 1997; 11:1-9)がある。したがって、本発明の酵素組成物の摂取により、グルコースが低減し、カロリーの摂取量を低減する他、腸内細菌叢の改善等の整腸効果を期待できる。特に、グルコン酸は、酸味が強く経口で摂取することが好まれないので、生体内でグルコン酸に変換でき有用である。
As shown below, the oxidoreductase according to the present invention catalyzes a redox reaction using glucose as a substrate, and oxidizes glucose to produce D-glucono-δ-lactone (gluconic anhydride).
Glucose + receptor → D-glucono-δ-lactone + reduced receptor
D-glucono-δ-lactone is then converted to gluconic acid by an equilibrium reaction. As a result, glucose in food and drink is converted into gluconic acid by the oxidoreductase. Gluconic acid is abundant in honey, and also in fermented foods such as fruits, wine, miso, and soy sauce, and is used as a food additive in acidulants and pH adjusters. Reports of intestinal bifidobacteria increase with ingestion of D-glucono-δ-lactone (Bifidos 1994; 8: 29-35) and the number of stools during ingestion of healthy adult women who tend to have constipation due to ingestion There is a report that the number of days of defecation was increased (Intestinal Bacteriology 1997; 11: 1-9). Therefore, ingestion of the enzyme composition of the present invention can reduce glucose, reduce calorie intake, and expect an intestinal effect such as improvement of intestinal flora. In particular, gluconic acid has a strong sour taste and is not preferred to be taken orally, so it can be converted into gluconic acid in vivo and is useful.
本発明の酵素組成物が適用できるのはヒトに限らず、イヌ、ネコ等の他の哺乳動物にも適用できる。 The enzyme composition of the present invention can be applied not only to humans but also to other mammals such as dogs and cats.
次いで、本発明を実施例を挙げて説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is given and this invention is demonstrated, this invention is not limited to a following example.
（FAD-GDHの酵素活性）
FAD-GDH活性の検出は、下記の反応系で行った。
(1)D-グルコース+PMS→D-グルコノ-δ-ラクトン+還元型PMS
(2)2還元型PMS+NTB→2PMS+Diformazan
尚、式中のPMSは、Phenazine methosu1fateを表し、NTBはNitrotetrazorium blueを表す。
反応(1)において、グルコースの酸化に伴って還元型PMSが生成し、更に反応(2)において還元型PMSによるNTBの還元により生成したDifomazanを570mの波長で測定する。
酵素活性(ユニット)は以下の計算式によって算出される。
(Enzyme activity of FAD-GDH)
Detection of FAD-GDH activity was performed in the following reaction system.
(1) D-glucose + PMS → D-glucono-δ-lactone + reduced PMS
(2) 2-reduced PMS + NTB → 2PMS + Diformazan
In the formula, PMS represents Phenazine methosu1fate, and NTB represents Nitrotetrazorium blue.
In reaction (1), reduced PMS is produced with the oxidation of glucose, and further, Difomazan produced by reduction of NTB by reduced PMS in reaction (2) is measured at a wavelength of 570 m.
The enzyme activity (unit) is calculated by the following formula.
FAD-GDHの酵素活性は、以下の方法で上記の計算式を用いて行った。
0.22%(W/V)トリトンX-100を含む50mM PIPES-NaOH緩衝液pH6.5 2.55mL、1M グルコース溶液0.09mL、3mM PMS溶液0.2mL及び6.6mM NTB溶液0.1mLを混合し、37℃で5分間保温後、検体0.1mLを添加し、反応を開始した。酵素反応の進行と共に570nmに吸収を持つDifomazanが生成される。1分間あたりの570nmにおける吸光度の増加を測定することによりFAD-GDH活性を測定した。
The enzyme activity of FAD-GDH was performed using the above formula using the following method.
Mix 50 mM PIPES-NaOH buffer pH 6.5 2.55 mL containing 0.22% (W / V) Triton X-100, 0.09 mL of 1 M glucose solution, 0.2 mL of 3 mM PMS solution and 0.1 mL of 6.6 mM NTB solution at 37 ° C. After incubating for 5 minutes, 0.1 mL of the sample was added to start the reaction. As the enzymatic reaction proceeds, Difomazan having an absorption at 570 nm is generated. FAD-GDH activity was measured by measuring the increase in absorbance at 570 nm per minute.
〔実施例１〕（胃内模型を用いたFAD-GDHの作用）
生体内での反応を1/30サイズとした1/30模型サイズの胃内模型（塩酸添加無し）を用い、胃内条件下でFAD-GDHがグルコースに作用し、グルコースをグルコン酸に変換できるかを検討した。グルコースの量は、市販の清涼飲料１本（350mL）相当のグルコース含量を参考に設定した。
胃内模型は、以下のように作製した。
20%グルコース（1.1mol/L）3.3mL、3%胃粘膜ムチン1.6mL、13.2%NaCl+0.22%CaCl2・2H2O3.3mL、0.1mol/L酢酸緩衝液（pH4.0）40mLからなる溶液を37℃、20分間予備加温し、次いで0.2%ペプシン（Sigma-Aldrich社製）1.6mLを加え、胃内反応を模擬的に再現した。次いで、前記溶液に1.2gフェリシン化カリウム（MW:329.3、和光純薬工業社製）をメディエーターとして加え、酵素溶液添加前に予め2mLのサンプルを採取した後、FAD-GDH酵素溶液（207単位/mL、Aspergillus oryzae BB-56の生産するFAD-GDH）3.3mLを加え反応を開始し、反応時間の0分、5分、30分、60分、90分、120分毎に1mLづつサンプルを採取した。各サンプル1mLに20%TCA0.2mLを加えて反応を停止させた。反応停止後、各サンプルは、グルコースの定量及びグルコン酸の定量に供した。図１に胃内模型を用いグルコースにFAD-GDHを作用させる方法を示した。
[Example 1] (Action of FAD-GDH using intragastric model)
Using a 1/30 model stomach model (without addition of hydrochloric acid) with a 1/30 size reaction in vivo, FAD-GDH can act on glucose under gastric conditions and convert glucose to gluconic acid We examined whether. The amount of glucose was set with reference to the glucose content equivalent to one commercially available soft drink (350 mL).
The intragastric model was produced as follows.
It consists of 3.3 mL of 20% glucose (1.1 mol / L), 1.6 mL of 3% gastric mucosa mucin, 13.2% NaCl + 0.22% CaCl 2 · 2H 2 O 3.3 mL, and 40 mL of 0.1 mol / L acetate buffer (pH 4.0). The solution was pre-warmed at 37 ° C. for 20 minutes, and then 1.6 mL of 0.2% pepsin (Sigma-Aldrich) was added to simulate the intragastric reaction. Next, 1.2 g potassium ferricinized (MW: 329.3, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the solution as a mediator, and a 2 mL sample was collected in advance before adding the enzyme solution, and then FAD-GDH enzyme solution (207 units / mL, FAD-GDH produced by Aspergillus oryzae BB-56) 3.3 mL was added to start the reaction, and 1 mL sample was taken every 0 min, 5 min, 30 min, 60 min, 90 min, and 120 min of the reaction time did. The reaction was stopped by adding 0.2 mL of 20% TCA to 1 mL of each sample. After stopping the reaction, each sample was subjected to determination of glucose and gluconic acid. FIG. 1 shows a method of causing FAD-GDH to act on glucose using an intragastric model.
〔実施例２〕（腸内模型を用いたFAD-GDHの作用）
生体内での反応を1/30サイズとした1/30模型サイズ腸内模型を用い、腸内条件下でFAD-GDHがグルコースに作用し、グルコースをグルコン酸に変換できるかを検討した。グルコースの量は、市販の清涼飲料１本（350mL）相当のグルコース含量を参考に設定した。
腸内模型は、以下のように作製した。
20%グルコース（1.1mol/L）3.3mL、精製水36mL、3%胃粘膜ムチン1.6mL、13.2%NaCl+0.22%CaCl2・2H2O3.3mL、0.1mol/L酢酸緩衝液（pH4.0）6.6mLからなる溶液を37℃、20分間予備加温し、次いで0.2%ペプシン（Sigma-Aldrich社製）1.6mL、0.1mol/L塩酸4mLを加え、さらに1.0g炭酸水素ナトリウム、0.1gタウロデオキシコール酸ナトリウム（Sigma-Aldrich社製）、50mgパンクレアチン（天野エンザイム社製）を加え、腸内反応を模擬的に再現した。次いで、前記溶液に0.35gDCIP（2,6-ジクロロインドフェノール、MW:290.1、同仁化学社製）をメディエーターとして加え、酵素溶液添加前に予め2mLのサンプルを採取した後、FAD-GDH酵素溶液（207単位/mL、Aspergillus oryzae BB-56株の生産するFAD-GDH）3.3mLを加え反応を開始し、反応時間の0分、5分、30分、60分、90分、120分毎に1mLづつサンプルを採取した。各サンプル1mLに20%TCA0.2mLを加えて反応を停止させた。反応停止後、各サンプルは、グルコースの定量及びグルコン酸の定量に供した。図２に腸内模型を用いグルコースにFAD-GDHを作用させる方法を示した。
[Example 2] (Function of FAD-GDH using intestinal model)
We investigated whether FAD-GDH can act on glucose under intestinal conditions and convert glucose to gluconic acid using a 1/30 model intestinal model with a 1/30 size reaction in vivo. The amount of glucose was set with reference to the glucose content equivalent to one commercially available soft drink (350 mL).
The intestinal model was produced as follows.
20% glucose (1.1 mol / L) 3.3 mL, purified
〔実施例３〕グルコースの定量及びグルコン酸の定量
実施例１及び実施例２で採取したサンプルについて、グルコースの定量及びグルコン酸の定量をHPLCにより行った。分析のためのHPLCの条件は以下に示した。
（グルコース定量）
カラム（Column） ：SCR-101N （ID7.9mm×300mm, 10μm）
移動相（Mobile phase） ：distilled water
流量（Flow rate） ：0.6 mL/min
分析時間（analysis time） ：20 min
測定（Detection） ：示差屈折率検出法
注入量（Injection amount）：5μL
（グルコン酸定量）
カラム（Column） : Aminex HPX-87H (300×7.8mm) Bio-Rad製
移動相（Mobile phase） : 8 mmol/L H2SO4
流量（Flow Rate） : 0.7 mL/min
分析時間（Analysis time） : 20 min
測定（Detection） :紫外吸収法（210 nm）
注入量（Injection amount）: 10 μL
[Example 3] Quantification of glucose and gluconic acid The samples collected in Example 1 and Example 2 were subjected to HPLC and glucose determination by HPLC. The HPLC conditions for analysis are shown below.
(Glucose determination)
Column: SCR-101N (ID7.9mm × 300mm, 10μm)
Mobile phase: distilled water
Flow rate: 0.6 mL / min
Analysis time: 20 min
Measurement (Detection): Differential refractive index detection method Injection amount: 5μL
(Quantification of gluconic acid)
Column: Aminex HPX-87H (300 × 7.8mm) Bio-Rad Mobile phase: 8 mmol / L H2SO4
Flow rate: 0.7 mL / min
Analysis time: 20 min
Measurement: UV absorption method (210 nm)
Injection amount: 10 μL
HPLCで定量したデータに基づき算定したグルコース残存率及びグルコン酸変換率を図３及び図４に示した。グルコース残存率は、反応前に採取したサンプルのグルコース量を100%とした相対値を示す。また、グルコン酸変換率は、反応前に採取したサンプル中のグルコース量から100%変換した際のグルコン酸量を100%とした相対値で示した。その結果、胃内模型及び腸内模型のいずれにおいてもグルコースが低減し、グルコン酸の生成が確認できた。すなわち、FAD-GDHは、胃内条件下及び腸内条件下のいずれにおいてもグルコースに作用してグルコン酸に変換し、特に腸内条件下での作用が顕著なことが明らかとなった。 The residual glucose rate and gluconic acid conversion rate calculated based on the data determined by HPLC are shown in FIGS. The residual glucose rate is a relative value with the amount of glucose of the sample collected before the reaction as 100%. Further, the gluconic acid conversion rate was shown as a relative value with the gluconic acid amount being 100% converted from the glucose amount in the sample collected before the reaction. As a result, glucose was reduced in both the gastric model and the intestinal model, and the production of gluconic acid was confirmed. That is, it was revealed that FAD-GDH acts on glucose and converts it to gluconic acid under both gastric and intestinal conditions, and the action under intestinal conditions is particularly remarkable.
本発明の酵素組成物は、グルコースを低減させることによりカロリーの摂取を低減でき、肥満の防止や血糖値上昇の防止を図ることができるので、食品分野や医薬分野で有用である。 Since the enzyme composition of the present invention can reduce intake of calories by reducing glucose, and can prevent obesity and blood sugar level, it is useful in the food and pharmaceutical fields.
微生物名：Aspergillus oryzae BB-56、寄託番号：NITE BP-236 Microorganism name: Aspergillus oryzae BB-56, Deposit number: NITE BP-236
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