JP2002027979A - Method for producing alpha-glucosidase inhibitor - Google Patents
Method for producing alpha-glucosidase inhibitorInfo
- Publication number
- JP2002027979A JP2002027979A JP2000214168A JP2000214168A JP2002027979A JP 2002027979 A JP2002027979 A JP 2002027979A JP 2000214168 A JP2000214168 A JP 2000214168A JP 2000214168 A JP2000214168 A JP 2000214168A JP 2002027979 A JP2002027979 A JP 2002027979A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- foods
- water
- glucosidase inhibitor
- inhibitor
- 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.)
- Granted
Links
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- 239000004310 lactic acid Substances 0.000 description 1
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Landscapes
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- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、医薬品、食品、健
康食品などに使用することができる阻害活性が強く、臭
いがなく、摂取し易いα−グルコシダーゼ阻害剤の製造
法に関するものである。TECHNICAL FIELD The present invention relates to a method for producing an α-glucosidase inhibitor which has a strong inhibitory activity, has no odor and is easy to take, which can be used for pharmaceuticals, foods, health foods and the like.
【0002】[0002]
【従来の技術】α−グルコシダーゼ阻害剤は、小腸の微
絨毛に局在するα−グルコシダーゼを阻害し、食後の血
糖値の急上昇及びそれに続くインスリン値の上昇を抑制
することが、Diabate Medicine, 10, 688(1993)に報告
され、人間及び人間以外の動物においても炭水化物(特
に、澱粉由来のオリゴ糖、シュクロース等)の代謝を抑
制するために、例えば血糖上昇抑制作用を示し、過血糖
症状及び過血糖に由来する肥満症、糖尿病などの種々の
疾患の改善に有用である。また、α−グルコシダーゼ阻
害剤を添加して製造した食品は、代謝異常の患者食に適
しており、さらに代謝異常予防食として健康な人にも適
している。BACKGROUND OF THE INVENTION α- glucosidase inhibitors, inhibit α- glucosidase localized in microvilli of the small intestine, to suppress spikes and elevated insulin levels subsequent postprandial blood glucose, Diabate Medicine, 10 , 688 (1993). In humans and non-human animals, it shows an inhibitory effect on carbohydrates (especially, starch-derived oligosaccharides, sucrose, etc.). And it is useful for improvement of various diseases such as obesity and diabetes caused by hyperglycemia. Further, the food produced by adding the α-glucosidase inhibitor is suitable for a patient with a metabolic disorder, and is also suitable for a healthy person as a diet for preventing a metabolic disorder.
【0003】食品に由来するα−グルコシダーゼ阻害剤
としては、例えば特開平9−65836号公報には、動
物性蛋白質又は植物性蛋白質の酵素加水分解物が開示さ
れ、特開平5−17364号公報には茶ポリフェノール
が開示されている。As an α-glucosidase inhibitor derived from food, for example, Japanese Patent Application Laid-Open No. 9-65836 discloses an enzymatic hydrolyzate of an animal protein or a vegetable protein. Discloses tea polyphenols.
【0004】しかしながら、上記特開平9−65836
号公報に記載のα−グルコシダーゼ阻害剤は、活性を示
すためには食品として大量に摂取しなくてはならず、ま
た、特開平5−17364号公報開示技術では、ポリフ
ェノールの精製が煩雑であり、かつ通常摂取する茶では
やはり多量に摂取する必要があった。However, Japanese Patent Application Laid-Open No. 9-65836 describes
The α-glucosidase inhibitor described in Japanese Unexamined Patent Application Publication No. H11-146556 must be ingested in large quantities as a food in order to exhibit activity, and in the technology disclosed in Japanese Patent Application Laid-Open No. HEI 5-17364, purification of polyphenol is complicated. In addition, it was necessary to ingest a large amount of tea which is usually consumed.
【0005】[0005]
【発明が解決しようとする課題】そこで本発明者は食品
からかかる阻害剤を見いだすべく鋭意検討を行った結
果、ササゲ属豆を固定培養した培養物に強い阻害活性が
あることを見い出したが、通常、固定培養物は高塩分下
で培養が実施されるため、食品、特に健康食品として日
常摂取するには不適当であることが判明した。そこで塩
分を抑えてササゲ属豆の固形培養を行ってみた所、細菌
や酵母が繁殖するためか、培養物から臭いが発生してし
まうという問題点が発生した。The inventors of the present invention have conducted intensive studies to find such inhibitors from foods, and as a result, have found that a culture obtained by fixedly cultivating cowpea beans has a strong inhibitory activity. Usually, since the fixed culture is cultured under a high salt content, it has been found that the fixed culture is not suitable for daily consumption as a food, especially as a health food. Thus, when solid culture of cowpea beans was carried out while suppressing the salt content, there was a problem that odor was generated from the culture, probably because bacteria and yeast propagated.
【0006】[0006]
【課題を解決するための手段】かかる問題点について鋭
意研究した結果、蒸煮あるいは煮沸したササゲ属豆を、
培養系の塩分を少量に抑えた5重量%以下という条件で
かびを用いて固形培養して得られる培養物であっても、
それを50℃以上の水で抽出することによって阻害剤の
臭いを消失させることができ、しかも阻害活性を更に向
上させ得ることを見いだし本発明を完成した。As a result of diligent research on such problems, steamed or boiled cowpea beans are
Even if the culture is obtained by solid cultivation using mold under the condition that the salt content of the culture system is reduced to 5% by weight or less,
By extracting it with water at 50 ° C. or higher, it was found that the odor of the inhibitor could be eliminated and the inhibitory activity could be further improved, thus completing the present invention.
【0007】[0007]
【発明の実施の形態】以下本発明について具体的に説明
する。本発明に用いられるササゲ属豆としては、アズキ
(別名ショウズ)、ササゲ(別名ナガササゲ、ハタササ
ゲ)、リョクトウ(別名ヤエナリ、アオアズキ)、タケ
アズキ(別名ツルアズキ、バカアズキ)等が挙げられ、
かかるササゲ属豆を加工した、粉体、穀粒等いずれも用
いられる。本発明の製造法を実施するに当っては、まず
上記のササゲ属豆を水に浸漬した後、蒸煮あるいは煮沸
するのであるが、かかる浸漬の条件としては、ササゲ属
豆を10〜30℃の水に1〜18時間漬ける。また、蒸
煮あるいは煮沸の条件としては、ササゲ属豆を蒸煮器に
入れて、蓋をして下から水蒸気を送って蒸煮前(浸漬
後)のササゲ属豆に比べて重量が1.02〜2倍となる
まで0.2〜2時間程度蒸煮したり、沸騰水に入れて3
0〜360分間煮沸する。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Examples of cowpea beans used in the present invention include adzuki beans (also known as “shoes”), cowpea (also known as “nagasasa” and “hata-sage”), mungbean (also known as “yenari” and “aoazuki”), and take care (also known as “tsuruazuki” and “bakaazuki”).
Powders, grains, and the like obtained by processing such cowpea beans are used. In carrying out the production method of the present invention, first, the above cowpea beans are immersed in water, and then steamed or boiled. Soak in water for 1-18 hours. The conditions of steaming or boiling are as follows: cowpea beans are put in a steamer, covered, and steam is sent from below to weigh 1.02 to 2 times compared to cowpea beans before steaming (after immersion). Cook for about 0.2 to 2 hours or double in boiling water for 3 times.
Boil for 0-360 minutes.
【0008】ササゲ属豆はもともと自然界で0.1重量
%以下の塩分を含有し、蒸煮あるいは煮沸したササゲ属
豆では0.01重量%以下の塩分を含有するので、その
まま次のかびつけ工程に入ってもよいが、固形培養の安
定性をはかるため、かかる系の塩分が5重量%以下(好
ましくは1重量%以下)になる範囲で塩分を添加しても
よい。かかる塩分が5重量%を越えると後で抽出操作を
行っても塩分が抽出物中に混入し、摂取時に著しく塩辛
くあるいはまずくなるので阻害剤の脱塩処理を施す必要
があり不適当である。Cowpea beans originally contain 0.1% by weight or less of salt in nature, and steamed or boiled cowpea beans contain 0.01% by weight or less of salt. However, in order to ensure the stability of the solid culture, salt may be added in a range where the salt content of such a system is 5% by weight or less (preferably 1% by weight or less). If the salt content exceeds 5% by weight, even if an extraction operation is performed later, the salt content is mixed into the extract and becomes extremely salty or unsatisfactory at the time of ingestion.
【0009】培養系の塩分は、培養物にイオン交換水を
加えて撹拌し、その上澄みを適宜希釈してデジタル塩分
計(積水化学工業社製『SS−31A』)を使用して測
定して求める。添加する塩分としては塩化ナトリウム、
塩化カルシウム、塩化カリウム等が挙げられる。[0009] The salt content of the culture system is measured by adding ion-exchanged water to the culture, stirring the mixture, appropriately diluting the supernatant, and using a digital salt meter (“SS-31A” manufactured by Sekisui Chemical Co., Ltd.). Ask. Sodium chloride,
Examples thereof include calcium chloride and potassium chloride.
【0010】次いで、蒸煮あるいは煮沸の後固形培養を
行うのであるが、まずかび付けから実施する。かかるか
びとしては醤油、もろみ、トウチ、みそ等の製造に用い
られるもので、アスペルギルス(Aspergillu
s)属のかびが好ましく、例えば、アスペルギルス ア
ルバス(Aspergillus albus IFO
4039)、アスペルギルス キャンディダス(Asp
ergillus candidus IFO438
9)、アスペルギルス ニーズランス(Aspergi
llus nidulans ATCC10074)、
アスペルギルスグラウカス(Aspergillus
glaucus ATCC10059)、アスペルギル
ス オリゼー(Aspergillus oryzae
IFO4135)、アスペルギルス フラバス(As
pergillus flavus IFO583
9)、アスペルギルス インディカス(Aspergi
llus indicus ATCC15054)、ア
スペルギルス スルフレウス(Aspergillus
sulphureus ATCC11904)、アス
ペルギルス ニガー(Aspergillus nig
er IFO4343)、アスペルギルス ソジャエ
(Aspergillus sojae IFO420
0)、アスペルギルス タマリ(Aspergillu
s tamariiATCC12669)等が挙げられ
る。具体的には、『ハイ・ソーヤ』、『マイルド・
S』、『スリーダイヤ』、『ダイヤモンドC』、『うす
むらさき』、『宝菌』、『白醤油用菌』、『改良焼酎用
菌』(以上いずれも株式会社樋口松之助商店製)等の市
販品が挙げられる。かび付けする場合のかびの添加量は
特に制限されないが、ササゲ属豆に対して0.0001
〜10重量%程度である。かび付けの方法としては蒸煮
あるいは煮沸したササゲ属豆にかびを振りかけたり、一
度培養した容器や発酵室に残存しているかびと該ササゲ
属豆を接触させる等の方法が挙げられる。[0010] Next, solid culture is performed after steaming or boiling. Such molds are used for the production of soy sauce, moromi, torch, miso and the like, and include Aspergillus (Aspergillus).
s) Fungi of the genus genus are preferred, for example Aspergillus albus IFO
4039), Aspergillus candidas (Asp
ergillus candidus IFO438
9), Aspergillus needslance (Aspergi)
llus nidulans ATCC 10074),
Aspergillus glaucus
glaucus ATCC10059), Aspergillus oryzae
IFO4135), Aspergillus flavus (As
pergillus flavus IFO583
9), Aspergillus indica
lrus indicus ATCC 15054), Aspergillus sulphleus (Aspergillus).
sulphurus ATCC 11904), Aspergillus niger (Aspergillus nig).
er IFO4343), Aspergillus sojae IFO420
0), Aspergillus tamari (Aspergillus
stamariATCC12669) and the like. Specifically, "High Sawyer", "Mild
S ”,“ Three Diamonds ”,“ Diamond C ”,“ Usumurasaki ”,“ Treasure Bacteria ”,“ Bacteria for White Soy Sauce ”,“ Bacteria for Improved Shochu ”(all of which are manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) Goods. The amount of mold added when molding is not particularly limited.
About 10% by weight. Examples of the method of mold application include sprinkling mold on steamed or boiled cowpea beans, and bringing the cowpea beans into contact with molds remaining in a container or fermentation chamber once cultured.
【0011】かび付け後固形培養を開始する。固形培養
温度は10〜50℃程度(好ましくは25〜45℃)、
湿度80〜100%RH(好ましくは90〜100%R
H)で、12〜120時間(好ましくは48〜96時
間)実施される。更に必要に応じて得られた培養物を5
〜40℃で、2〜120日程度放置して熟成工程を行っ
てもよい。発酵終了後、必要に応じてササゲ属豆表面に
付着しているかびを落す為に水洗する。After molding, solid culture is started. Solid culture temperature is about 10 to 50 ° C (preferably 25 to 45 ° C),
Humidity 80-100% RH (preferably 90-100% RH
H) for 12 to 120 hours (preferably 48 to 96 hours). Further, the obtained culture is optionally
The aging step may be performed by leaving the mixture at 4040 ° C. for about 2 to 120 days. After the fermentation, if necessary, washing is performed with water to remove mold attached to the cowpea.
【0012】次にかかる培養物を50℃以上の水で抽出
するのであるが、好ましくは60℃以上の水で抽出す
る。水の温度が50℃未満では、阻害活性の向上が見ら
れず、更に培養物中に発生する臭い成分の除去ができず
不適当である。Next, the culture is extracted with water at 50 ° C. or more, preferably with water at 60 ° C. or more. If the temperature of the water is lower than 50 ° C., no improvement in the inhibitory activity is observed, and furthermore, the odor components generated in the culture cannot be removed, which is inappropriate.
【0013】上記の50℃以上の水で抽出するには、培
養物に1〜30倍重量(好ましくは2〜15倍重量)の
水を加え、昇温すればよい。抽出時間は90℃までの抽
出温度では5〜20時間、90℃を越えると1〜10時
間程度でよい。また、必要であればオートクレーブ等を
利用して100℃以上で0.2〜5時間抽出することが
できる。抽出法としては、特に制限はないが、通常撹拌
抽出あるいは浸漬抽出が用いられる。得られた抽出液は
清澄濾過、遠心分離、膜分離等により固形分を取除いた
後、必要に応じて活性炭や白土で脱色してから、濃縮乾
固、フリーズドライ、スプレードライ等の方法で粉末化
するのが好ましい。In order to extract with water at 50 ° C. or higher, 1 to 30 times (preferably 2 to 15 times) weight of water is added to the culture, and the temperature is raised. The extraction time may be about 5 to 20 hours at an extraction temperature up to 90 ° C, and about 1 to 10 hours when it exceeds 90 ° C. If necessary, extraction can be performed at 100 ° C. or higher for 0.2 to 5 hours using an autoclave or the like. Although there is no particular limitation on the extraction method, usually stirring extraction or immersion extraction is used. The obtained extract is subjected to clarification filtration, centrifugation, membrane separation, and the like to remove solids, and then, if necessary, decolorized with activated carbon or terra alba, and then concentrated to dryness, freeze-dried, spray-dried, or the like. Pulverization is preferred.
【0014】かくして得られたα−グルコシダーゼ阻害
剤は、血糖上昇抑制作用を有しているので、水、エタノ
ール、エチレングリコール、ポリエチレングリコールな
どの液状担体や、でんぷん、セルロースなどの固形担体
などの無毒性担体で希釈して、アンプル剤、顆粒剤、錠
剤、丸剤、カプセル剤、シロップ剤などの医薬品、健康
食品として代謝異常の患者食又は予防薬、糖尿病の予防
薬、あるいは抗肥満食、ダイエット食として用いること
ができる。さらに、本発明のα−グルコシダーゼ阻害剤
を含有する上記製剤を、食前、食中、食後、食間などに
服用することにより、喫食による血糖濃度の増加を抑制
することができる。Since the thus obtained α-glucosidase inhibitor has an action of suppressing a rise in blood glucose, it is nontoxic to liquid carriers such as water, ethanol, ethylene glycol and polyethylene glycol and solid carriers such as starch and cellulose. Diluted with a pharmaceutically acceptable carrier, and used as pharmaceuticals such as ampoules, granules, tablets, pills, capsules and syrups, as a health food, as a diet or prophylactic for metabolic disorders, as a preventive for diabetes, or as an anti-obesity diet, diet Can be used as food. Furthermore, by taking the above-mentioned preparation containing the α-glucosidase inhibitor of the present invention before, during, after or between meals, an increase in blood glucose concentration due to eating can be suppressed.
【0015】摂取量としては、乾燥粉末として、0.0
01〜10g/日が好ましく、特に0.01〜3g/日
が好ましい。[0015] The intake amount is 0.0
It is preferably from 01 to 10 g / day, particularly preferably from 0.01 to 3 g / day.
【0016】本発明の製造法で得られたα−グルコシタ
ーゼ阻害剤は、塩分が少なく、臭いがないので、例え
ば、以下のような食品に添加可能である。 (1)農水産加工品 はるさめ、こしあん、こんにゃく、パン、麺類(即席め
ん、パスタ、生めん、乾めん)、餅、シリアル食品、大
豆加工品(豆腐、豆乳、納豆、凍豆腐)、水産加工品
〔練り製品、(かに風味)蒲鉾、(魚肉)ハム、(魚
肉)ソーセージ、(魚肉)ウィンナー、ふりかけ、お茶
づけのり〕、卵含有食品(スープ、丼等)、缶詰(シー
チキン、オイルサーディン、焼鳥)、レトルト食品(カ
レー、シチュー、スパゲティー)、みそ汁、スープ (2)乳製品 牛乳、加工乳、乳酸菌飲料、バター、チーズ、練乳、粉
乳 (3)調味料 味噌、醤油、うま味(風味)調味料、(粉末)天然調味
料、ソース、ドレッシング、焼き肉のたれ、みりん、カ
レー、シチュー、香辛料、スパイス、ヨーグルトThe α-glucosidase inhibitor obtained by the production method of the present invention has a low salt content and no odor, and thus can be added to, for example, the following foods. (1) Agriculture and fishery products Harusame, Koshian, konjac, bread, noodles (instant noodles, pasta, raw noodles, dried noodles), mochi, cereal foods, soybean products (tofu, soymilk, natto, frozen tofu), fishery products [kneaded products , (Crab flavor) kamaboko, (fish meat) ham, (fish meat) sausage, (fish meat) wiener, sprinkle, tea sauce), egg-containing food (soup, bowl, etc.), canned (sea chicken, oil sardine, yakitori), Retort food (curry, stew, spaghetti), miso soup, soup (2) Dairy products Milk, processed milk, lactic acid beverage, butter, cheese, condensed milk, powdered milk (3) Seasoning miso, soy sauce, umami (flavor) seasoning, ( Powder) Natural seasonings, sauces, dressings, grilled meat sauce, mirin, curry, stew, spices, spices, yogurt
【0017】(4)健康食品(栄養補助食品) サポニン含有食品(オタネニンジン根含有食品、エゾ
ウコギ含有食品) 糖含有食品〔オリゴ糖(フラクトオリゴ糖含有食品、
イソマルトオリゴ糖含有食品、ガラクトオリゴ糖含有食
品)、多糖類(シイタケ含有食品、ムコ多糖、蛋白含有
食品、コンドロイチン硫酸含有食品、マンネンタケ(霊
芝)含有食品、キチン、キトサン含有食品)〕 ミネラル含有食品(カルシウム含有食品、アルファル
ファ含有食品、プルーンエキス食品、β−カロチン含有
食品) 油脂含有食品 ビタミンE含有油脂〔麦(小麦、鳩麦)胚芽油、大豆胚
芽油、米胚芽油〕、エイコサペンタエン酸含有食品、大
豆レシチン含有食品、γ−リノレン酸含有食品(月見草
油、ボラージ油)、ドコサヘキサエン酸含有食品 蛋白質含有食品 大豆蛋白含有食品、カゼイン、ホエー蛋白、鯉加工食品 タウリン かき加工食品、シジミ加工食品、緑イ貝加工食品(4) Healthy foods (dietary supplements) Saponin-containing foods (pancreatic ginseng root-containing foods, eleuthero-containing foods) Sugar-containing foods [oligosaccharides (fructooligosaccharide-containing foods,
Isomaltooligosaccharide-containing foods, galactooligosaccharide-containing foods), polysaccharides (shiitake-containing foods, mucopolysaccharides, protein-containing foods, chondroitin sulfate-containing foods, Mannentake (reishi) -containing foods, chitin, chitosan-containing foods)] Mineral-containing foods ( Foods containing calcium, foods containing alfalfa, foods containing prune extract, foods containing β-carotene) Foods containing fats and oils Foods containing fats and oils containing vitamin E [wheat (wheat, barley) germ oil, soybean germ oil, rice germ oil], foods containing eicosapentaenoic acid, Soy lecithin-containing food, γ-linolenic acid-containing food (evening primrose oil, borage oil), docosahexaenoic acid-containing food Protein-containing food Soy protein-containing food, casein, whey protein, carp processed food Taurine, persimmon processed food, processed seafood, green ai Shellfish processed food
【0018】(5)その他 スッポン加工食品、アミノ酸代謝異常用食品、流動食
(病食) また、下記のような、糖を多量に含有する食品にも添加
可能であるが、本発明の効果が明確に発現しない場合も
あり、下記のような食品に添加する場合は、食品の製造
時に糖含有量をできるだけ低くしたり、人工甘味量を用
いて低糖分としたものに添加するのが好ましい。 (6)菓子 ケーキ、ムース、(粉末)デザート、アイスクリーム、
飴、チョコレート、グミ、キャンディー、クッキー、ウ
エハース、ゼリー (7)飲料 清涼飲料(炭酸飲料、果実飲料、スポーツドリンク、栄
養飲料)、嗜好飲料(コーヒー、ココア、麦汁)(5) Others Processed foods for turkey, foods for abnormal amino acid metabolism, liquid foods (disease foods) In addition, they can be added to foods containing a large amount of sugar as described below, but the effects of the present invention are not In some cases, it is not clearly expressed, and when it is added to the following food products, it is preferable to add the sugar content as low as possible during the production of the food product or to reduce the sugar content using artificial sweetness. (6) Confectionery cake, mousse, (powder) dessert, ice cream,
Candy, chocolate, gummy, candy, cookies, wafers, jelly (7) Beverages Soft drinks (carbonated drinks, fruit drinks, sports drinks, nutritional drinks), favorite drinks (coffee, cocoa, wort)
【0019】上記(1)〜(7)における添加量として
は、上記食品に対して、乾燥粉末として、0.01〜8
0重量%が好ましく、特に1〜70重量%が好ましい。
更に本発明の効果を阻害しない範囲で、甘味剤、保存
剤、分散剤、着色剤、酸化防止剤等も併用することがで
きる。更に、その他の公知のα−グルコシダーゼ阻害剤
であるバリエナミンやアミノシクリトールなどを併用し
てもよい。The amount of addition in the above (1) to (7) is 0.01 to 8 as a dry powder with respect to the food.
0% by weight is preferable, and 1 to 70% by weight is particularly preferable.
Further, a sweetener, a preservative, a dispersant, a coloring agent, an antioxidant and the like can be used in combination as long as the effects of the present invention are not impaired. Further, other known α-glucosidase inhibitors such as varienamine and aminocyclitol may be used in combination.
【0020】[0020]
【実施例】以下本発明について例を挙げて具体的に説明
する。尚、以下の記述で「%」とあるのは、特に断りの
ない限り重量%である。 実施例1 アズキ1000gを25℃の水10リットルに3時間浸
漬して重量を1100gにした後、蒸煮器に入れて10
0℃で1時間蒸煮して2000gの蒸煮したアズキ〔塩
分含有量は検出限界(0.01%)以下〕を得た。かか
る蒸煮したアズキを発酵室の竹かごの上に並べて市販の
醤油製造用のかび(株式会社樋口松之助商店製『マイル
ド・S』)10gを均一にふりかけて添加して、35
℃、湿度100%RHで96時間固形培養した。培養終
了後培養物表面のかびを水洗し、80℃で24時間風乾
して培養物800gを得た。該培養物を4リットルの水
に1時間浸漬後、該浸漬液をオートクレーブに入れて、
内温125℃で1時間抽出した。得られた抽出液からざ
るを用いて大きな残渣を除去した。得られた濾液と、該
残渣に再度4リットルの水を加えてざるで濾過して得ら
れた濾液とを合わせて、遠心分離(条件1000×g)
した。得られた上澄み液に活性炭100gを加えて撹拌
して脱色し、濾過して抽出液4リットルを得た。その後
ロータリーエバポレータで濃縮乾固して阻害剤240g
を得た。かかる阻害剤の阻害活性、臭い、摂取の容易さ
を以下のように評価した。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to examples. In the following description, “%” means “% by weight” unless otherwise specified. Example 1 1000 g of adzuki bean was immersed in 10 liters of water at 25 ° C. for 3 hours to make the weight 1100 g, and then put in a steamer for 10 hours.
Steamed at 0 ° C. for 1 hour to obtain 2,000 g of steamed adzuki beans (salt content is below the detection limit (0.01%)). The steamed red beans are arranged on a bamboo basket in a fermentation room, and 10 g of a commercially available mold for soy sauce production (“Mild S” manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) is evenly sprinkled and added.
Solid cultivation was carried out for 96 hours at 100 ° C. and 100% humidity. After completion of the culture, the mold on the surface of the culture was washed with water and air-dried at 80 ° C. for 24 hours to obtain 800 g of the culture. After immersing the culture in 4 liters of water for 1 hour, the immersion liquid was put into an autoclave,
Extraction was performed at an internal temperature of 125 ° C. for 1 hour. A large residue was removed from the resulting extract using a sieve. The obtained filtrate was combined with the filtrate obtained by adding 4 liters of water to the residue again, and then centrifuged (condition: 1000 × g).
did. 100 g of activated carbon was added to the obtained supernatant, stirred, decolorized, and filtered to obtain 4 liter of extract. After that, it was concentrated to dryness with a rotary evaporator and the inhibitor was 240 g.
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated as follows.
【0021】(阻害活性) ・ラット小腸からの二(三)糖加水分解酵素(α−グル
コシダーゼ)の調製 冷凍保存しておいたラット小腸(空腸)を解凍し、粘膜
をピンセットで押出すように採取した。該粘膜に5倍重
量の5mMエチレンジアミン四酢酸を含む0.1Mリン
酸カリウム緩衝液(pH7.0)を加え、冷却しながら
ホモゲナイズした。その後遠心分離(4℃、21000
×g、60分)し、得られた沈殿物に5倍重量になるよ
うに1%トリトンX−100を含む0.1Mリン酸カリ
ウム緩衝液(pH7.0)を加え、可溶化処理(4℃、
60分)を行った。これを超遠心分離(4℃、1100
00×g、90分)し、この上清を0.01Mリン酸カ
リウム緩衝液(pH7.0)で透析(4℃、24時間)
し、酵素液とした。(Inhibitory activity) Preparation of di- (tri) sugar hydrolase (α-glucosidase) from rat small intestine The frozen rat small intestine (jejunum) is thawed, and the mucous membrane is extruded with forceps. Collected. A 0.1 M potassium phosphate buffer (pH 7.0) containing 5 times the weight of 5 mM ethylenediaminetetraacetic acid was added to the mucous membrane, and homogenized while cooling. Thereafter, centrifugation (4 ° C., 21000
× g, 60 minutes), and a 0.1 M potassium phosphate buffer (pH 7.0) containing 1% Triton X-100 was added to the obtained precipitate so as to have a 5-fold weight, and solubilization treatment (4 ℃,
60 minutes). This is ultracentrifuged (4 ° C, 1100
00 × g, 90 minutes), and the supernatant is dialyzed against 0.01 M potassium phosphate buffer (pH 7.0) (4 ° C., 24 hours)
And used as an enzyme solution.
【0022】・酵素(α−グルコシダーゼ)活性の測定 酵素活性は市販のキットを用い、基質としてはシュクロ
ースを用いた。標準反応液組成は、60mM基質溶液
(シュクロースを0.1Mリン酸カリウム緩衝液pH
6.3に溶解したもの)0.7ml、被験物質溶液(そ
れぞれの分画成分の水、有機溶剤を完全に除去した後、
50%ジメチルスルホキシド水溶液に溶解)0.2m
l、上記酵素液0.1ml(計1.0ml)とした。こ
れを37℃、15分間反応させ、2Mトリス塩酸緩衝液
(pH7.0)1.5mlを用いて反応を停止させ試験
液とした。次に96穴マイクロプレートに1穴あたり発
色試薬〔グルコースBテストワコー(和光純薬製)〕2
00μlに試験液50μl(酢酸エチル等は留去したも
の)を加え、37℃で30分間インキュベートした後、
マイクロプレートリーダ(BIO RAD社製、MOD
EL550)で490nmの吸光度を測定した。基質溶
液の代りに0.1Mリン酸カリウム緩衝液(pH6.
3)を加えた時の吸光度をブランク値とし、この値を差
し引いた値をA490sとした。被験液の代りに50重量%
ジメチルスルホキシド水溶液を加えた時の吸光度をコン
トロール値(A 490c)とし、下式によりα−グルコシダ
ーゼ阻害活性を求めた。測定は2回行い、平均値を測定
値とした。 α−グルコシダーゼ阻害活性(%)=[(A490c−
A490s)/A490c]×100Measurement of enzyme (α-glucosidase) activity The enzyme activity was measured using a commercially available kit.
Source was used. Standard reaction solution composition is 60 mM substrate solution
(Sucrose in 0.1 M potassium phosphate buffer pH
0.7 ml dissolved in 6.3) and a test substance solution
After completely removing the water and organic solvent of each fraction component,
Dissolved in 50% dimethyl sulfoxide aqueous solution) 0.2m
1, 0.1 ml of the above enzyme solution (1.0 ml in total). This
This was allowed to react at 37 ° C. for 15 minutes, followed by 2M Tris-HCl buffer.
(PH 7.0) Stop the reaction with 1.5 ml and test
Liquid. Next, one hole is generated in a 96-well microplate.
Color reagent [Glucose B Test Wako (Wako Pure Chemical Industries)] 2
Add 50 μl of test solution to 00 μl (ethyl acetate and the like were distilled off)
) And incubated at 37 ° C. for 30 minutes,
Micro plate reader (BIO RAD, MOD
The absorbance at 490 nm was measured using EL550). Substrate solution
0.1 M potassium phosphate buffer (pH 6.
The absorbance at the time of adding 3) is defined as a blank value.
A minus the value490sAnd 50% by weight instead of test liquid
The absorbance when adding dimethyl sulfoxide aqueous solution
Troll value (A 490c) And α-glucosida by the following formula
The inhibitory activity was determined. Measure twice and measure the average
Value. α-glucosidase inhibitory activity (%) = [(A490c−
A490s) / A490c] × 100
【0023】(臭い) 得られた阻害剤の臭いを以下のように評価した。 ○・・・全く無臭である。 △・・・かすかに臭いがする。 ×・・・強い臭いがする。(Odor) The odor of the obtained inhibitor was evaluated as follows.・ ・ ・: It is completely odorless. Δ: Smell slightly. ×: Smell strong.
【0024】(摂取し易さ) ○・・・塩辛くなく、ササゲ属豆のうまみが出て摂取し
易い。 ×・・・塩辛いあるいはササゲ属豆特有の臭いが強くて
1g以上摂取するのは苦痛である。(Ease of ingestion) ・ ・ ・: Not salty, and the taste of cowpea beans is easy to ingest. ×: Salty or cowpea peculiar odor is strong and it is painful to take in 1 g or more.
【0025】実施例2 実施例1の終了後、実施例1と同じ発酵室において、か
び(株式会社樋口松之助商店製『マイルド・S』)の替
りに、実施例1で用いた竹かごに付着していた培養物中
のかびで、同例と同じ蒸煮したアズキを35℃、湿度1
00%RHで72時間かびを固形培養し、培養終了後培
養物表面のかびを水洗し、80℃で24時間風乾して培
養物780gを得た。該培養物を4リットルの水に1時
間浸漬後、該浸漬液をオートクレーブに入れて、125
℃で1時間抽出した。その後実施例1と同様に処理して
抽出液4リットルを得、ロータリーエバポレータで濃縮
乾固して阻害剤234gを得た。かかる阻害剤の阻害活
性、臭い、摂取の容易さを実施例1と同様に評価した。Example 2 After the completion of Example 1, in the same fermentation chamber as in Example 1, the bamboo basket used in Example 1 was attached instead of the mold ("Mild S" manufactured by Higuchi Matsunosuke Shoten Co., Ltd.). The same steamed red bean as in the same example was grown at 35 ° C and a humidity of 1
The mold was solid-cultured at 00% RH for 72 hours. After completion of the culture, the mold on the surface of the culture was washed with water and air-dried at 80 ° C. for 24 hours to obtain 780 g of the culture. After the culture was immersed in 4 liters of water for 1 hour, the immersion liquid was placed in an autoclave,
Extracted at C for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 4 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain 234 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.
【0026】実施例3 実施例1において固形培養の温度を37℃にした以外は
同様に培養し培養物745gを得た。かかる培養物を陶
器の器に入れて、蓋をして35℃で40日間後発酵させ
て培養物740gを得た。該培養物を4リットルの水に
1時間浸漬後、該浸漬液をオートクレーブに入れて、1
25℃で1時間抽出した。その後実施例1と同様に処理
して抽出液3.8リットルを得、ロータリーエバポレー
タで濃縮乾固して阻害剤220gを得た。かかる阻害剤
の阻害活性、臭い、摂取の容易さを実施例1と同様に評
価した。Example 3 Culture was carried out in the same manner as in Example 1 except that the temperature of the solid culture was changed to 37 ° C., to obtain 745 g of a culture. The culture was placed in a pottery vessel, covered, and post-fermented at 35 ° C. for 40 days to obtain 740 g of the culture. After immersing the culture in 4 liters of water for 1 hour, the immersion liquid was placed in an autoclave and
Extracted at 25 ° C. for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 3.8 liters of the extract, and concentrated to dryness using a rotary evaporator to obtain 220 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.
【0027】実施例4 実施例1におけるオートクレーブに代えて、鍋を用いて
抽出温度を60℃にして抽出した。その後実施例1と同
様に処理して抽出液3リットルを得、ロータリーエバポ
レータで濃縮乾固して阻害剤153gを得た。かかる阻
害剤の阻害活性、臭い、摂取の容易さを実施例1と同様
に評価した。Example 4 Extraction was carried out at 60 ° C. using a pot instead of the autoclave in Example 1. Thereafter, the same treatment as in Example 1 was carried out to obtain 3 liters of the extract, which was then concentrated to dryness with a rotary evaporator to obtain 153 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.
【0028】実施例5 実施例1においてアズキに替えてタケアズキを用いた以
外は同様に固形培養して培養物730gを得た。該培養
物を3リットルの水に1時間浸漬後、該浸漬液をオート
クレーブに入れて、125℃で1時間抽出した。その後
実施例1と同様に処理して抽出液3.5リットルを得、
ロータリーエバポレータで濃縮乾固して阻害剤220g
を得た。かかる阻害剤の阻害活性、臭い、摂取の容易さ
を実施例1と同様に評価した。Example 5 Solid culture was carried out in the same manner as in Example 1 except that Takeazuki was used instead of adzuki to obtain 730 g of a culture. After the culture was immersed in 3 liters of water for 1 hour, the immersion liquid was placed in an autoclave and extracted at 125 ° C. for 1 hour. Thereafter, the mixture was treated in the same manner as in Example 1 to obtain 3.5 liters of the extract.
Concentrate to dryness with a rotary evaporator and make inhibitor 220g
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.
【0029】実施例6 実施例1においてアズキに替えてリョクトウを用いた以
外は同様に固形培養して培養物715gを得た。該培養
物を3リットルの水に1時間浸漬後、該浸漬液をオート
クレーブに入れて、125℃で1時間抽出した。その後
実施例1と同様に処理して抽出液3.2リットルを得、
ロータリーエバポレータで濃縮乾固して阻害剤193g
を得た。かかる阻害剤の阻害活性、臭い、摂取の容易さ
を実施例1と同様に評価した。Example 6 Solid culture was carried out in the same manner as in Example 1 except that mung bean was used instead of adzuki to obtain 715 g of a culture. After the culture was immersed in 3 liters of water for 1 hour, the immersion liquid was placed in an autoclave and extracted at 125 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was performed to obtain 3.2 liters of the extract.
Concentrate to dryness with a rotary evaporator and make 193 g of inhibitor
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.
【0030】比較例1 アズキ1000gを水10リットルに3時間浸漬した
後、蒸煮器に入れて100℃で1時間蒸煮して2000
gの蒸煮したアズキ(塩分0.01%以下)を得た。か
かる蒸し米に塩を200g添加(塩分10%)してから
実施例1と同様に発酵させて培養物900gを得た。該
培養物を実施例1と同様に抽出し、同様に処理して抽出
液4.1リットルを得、ロータリーエバポレータで濃縮
乾固して固形物320gを得た。かかる固形物の阻害活
性、臭い、摂取の容易さを実施例1と同様に評価した。Comparative Example 1 1000 g of adzuki bean was immersed in 10 liters of water for 3 hours, then put in a steamer and steamed at 100 ° C. for 1 hour to obtain 2,000
g of steamed red bean (0.01% or less in salt content) was obtained. 200 g of salt was added to the steamed rice (salt content: 10%) and fermented in the same manner as in Example 1 to obtain 900 g of a culture. The culture was extracted in the same manner as in Example 1 and treated in the same manner to obtain 4.1 L of an extract, which was then concentrated to dryness with a rotary evaporator to obtain 320 g of a solid. The inhibitory activity, odor, and ease of ingestion of the solids were evaluated in the same manner as in Example 1.
【0031】比較例2 実施例1で得られた培養物800gを4リットルの水に
1時間浸漬後、40℃で1時間抽出した。その後実施例
1と同様に処理して抽出液3.7リットルを得、ロータ
リーエバポレータで濃縮乾固して固形物80gを得た。
かかる固形物の阻害活性、臭い、摂取の容易さを同じく
評価した。Comparative Example 2 800 g of the culture obtained in Example 1 was immersed in 4 liters of water for 1 hour and then extracted at 40 ° C. for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 3.7 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain 80 g of a solid.
The inhibitory activity, odor and ease of ingestion of such solids were also evaluated.
【0032】 〔表1〕 α−グルコシダーゼ 臭い 摂取のし易さ 阻害活性(%)* 実施例1 90 ○ ○ 実施例2 91 ○ ○ 実施例3 91 ○ ○ 実施例4 90 ○ ○ 実施例5 92 ○ ○実施例6 92 ○ ○ 比較例1 88 × ×比較例2 10 △ × *被験物の反応系での濃度10mg/ml[Table 1] α-Glucosidase Odor Ease of Ingestion Inhibition Activity (%) * Example 1 90 ○ ○ Example 2 91 ○ ○ Example 3 91 ○ ○ Example 4 90 ○ ○ Example 5 92 ○ ○ Example 6 92 ○ ○ Comparative Example 1 88 × × Comparative Example 2 10 △ × * Concentration of the test substance in the reaction system 10 mg / ml
【0033】[0033]
【発明の効果】本発明では、蒸煮あるいは煮沸したササ
ゲ属豆を、培養系の塩分が5重量%以下となる条件でか
びで固形培養して得られた培養物から50℃以上の水で
α−グルコシダーゼ阻害剤を抽出することにより、摂取
し易く、臭いもなく、強い阻害活性を示すα−グルコシ
ダーゼ阻害剤が製造できる。According to the present invention, a steamed or boiled cowpea bean is solid-cultured in a mold under the condition that the salt content of the culture system is 5% by weight or less. -By extracting the glucosidase inhibitor, an α-glucosidase inhibitor which is easy to ingest, has no odor, and exhibits strong inhibitory activity can be produced.
フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) A61P 3/04 A61P 3/04 3/10 3/10 43/00 111 43/00 111 //(C12N 9/99 (C12N 9/99 C12R 1:66) C12R 1:66) Fターム(参考) 4B018 LB01 LB02 LB04 LB05 LB06 LB07 LB08 LB09 MD57 ME03 MF13 4C087 AA01 AA02 BC06 CA10 CA11 MA01 NA14 ZA70 ZC20 ZC35 4C088 AB59 AC04 AD16 BA09 CA25 MA07 NA14 ZA70 ZC20 ZC35Continuation of the front page (51) Int.Cl. 7 Identification symbol FI Theme coat II (reference) A61P 3/04 A61P 3/04 3/10 3/10 43/00 111 43/00 111 // (C12N 9/99 ( C12N 9/99 C12R 1:66) C12R 1:66) F term (reference) 4B018 LB01 LB02 LB04 LB05 LB06 LB07 LB08 LB09 MD57 ME03 MF13 4C087 AA01 AA02 BC06 CA10 CA11 MA01 NA14 ZA70 ZC20 ZC35 4C088 AB59 AC04 ZA70 ZC20 ZC35
Claims (2)
養系の塩分を5重量%以下の条件下で、かびを用いて固
形培養し、得られた培養物から50℃以上の水で抽出す
ることを特徴とするα−グルコシダーゼ阻害剤の製造
法。1. A steamed or boiled cowpea bean is solid-cultured using a mold under conditions in which the salt content of the culture system is 5% by weight or less, and the obtained culture is extracted with water at 50 ° C. or higher. A method for producing an α-glucosidase inhibitor, characterized in that:
illus)属であることを特徴とする請求項1記載の
α−グルコシダーゼ阻害剤の製造法。2. The method of claim 2, wherein the mold is Aspergillus.
2. The method for producing an α-glucosidase inhibitor according to claim 1, wherein the agent belongs to the genus (illus).
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JP2005348650A (en) * | 2004-06-10 | 2005-12-22 | Nagase Chemtex Corp | Diet food containing mung bean protein decomposition product |
WO2008090999A1 (en) * | 2007-01-22 | 2008-07-31 | Up Well Co. Ltd. | Glucosidase inhibitor |
JP2008239522A (en) * | 2007-03-26 | 2008-10-09 | Kaneka Corp | Antiallergic agent |
WO2011016220A1 (en) * | 2009-08-03 | 2011-02-10 | 株式会社カネカ | Dipeptidyl peptidase-4 inhibitor |
JP2022030599A (en) * | 2020-08-07 | 2022-02-18 | 井村屋グループ株式会社 | Glucose metabolism enzyme inhibitor derived from vigna angularis, and method for manufacturing the same |
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JP2022030599A (en) * | 2020-08-07 | 2022-02-18 | 井村屋グループ株式会社 | Glucose metabolism enzyme inhibitor derived from vigna angularis, and method for manufacturing the same |
JP7429169B2 (en) | 2020-08-07 | 2024-02-07 | 井村屋グループ株式会社 | Method for producing adzuki bean-derived glycolytic enzyme inhibitor |
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