JPH0873482A - Glucide reduced in reducing property and its production and use - Google Patents

Glucide reduced in reducing property and its production and use

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Publication number
JPH0873482A
JPH0873482A JP7182216A JP18221695A JPH0873482A JP H0873482 A JPH0873482 A JP H0873482A JP 7182216 A JP7182216 A JP 7182216A JP 18221695 A JP18221695 A JP 18221695A JP H0873482 A JPH0873482 A JP H0873482A
Authority
JP
Japan
Prior art keywords
sugar
reducing
trehalose
starch
enzyme
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
Application number
JP7182216A
Other languages
Japanese (ja)
Other versions
JP4070250B2 (en
Inventor
Takashi Shibuya
孝 渋谷
Toshiyuki Sugimoto
利行 杉本
Toshio Miyake
俊雄 三宅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hayashibara Seibutsu Kagaku Kenkyujo KK
Original Assignee
Hayashibara Biochemical Laboratories Co Ltd
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Filing date
Publication date
Application filed by Hayashibara Biochemical Laboratories Co Ltd filed Critical Hayashibara Biochemical Laboratories Co Ltd
Priority to JP18221695A priority Critical patent/JP4070250B2/en
Publication of JPH0873482A publication Critical patent/JPH0873482A/en
Application granted granted Critical
Publication of JP4070250B2 publication Critical patent/JP4070250B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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  • Cosmetics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Seasonings (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

PURPOSE: To obtain the subject glucide, containing a glucide having the trehalose structure in the molecule and a starch sugaralcohol together with a nonreducing glucide comprising trehalose, reduced in reducing properties and useful as foods and drinks, cosmetics and medicines, etc. CONSTITUTION: This glucide reduced in reducing properties is obtained by reacting a solution prepared by liquefying a starch with a nonreducing glucide- producing enzyme together with a starch debranching enzyme and/or cyclomaltodextrin glucanotransferase or the nonreducing glucide-producing enzyme and a trehalose liberating enzyme together with the starch debranching enzyme and/or cyclomaltodextrin glucanotransferase therewith, hydrogenating a low-reducing glucide containing a reducing starch saccharide such as glucose together with the resultant glucide having the trehalose structure in the molecule and/or a nonreducing glucide comprising the trehalose. The resultant glucide contains a starch sugaralcohol together with the produced glucide having the trehalose structure in the molecule and/or the nonreducing glucide comprising the trehalose.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、還元性を低減させた糖
質とその製造方法並びに用途に関し、更に詳細には、分
子中にトレハロース構造を有する糖質及び／又はトレハ
ロースからなる非還元性糖質とともに澱粉糖アルコール
を含有してなる実質的に還元性を示さない糖質とその製
造方法並びに用途に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sugar having reduced reducibility, a method for producing the same, and a use thereof, and more specifically, a sugar having a trehalose structure in a molecule and / or a non-reducing sugar. TECHNICAL FIELD The present invention relates to a saccharide containing starch sugar alcohol together with a saccharide, which is substantially non-reducing, a method for producing the same, and a use thereof.

【０００２】[0002]

【従来の技術】グルコースを構成糖とする非還元性糖質
として、古くからトレハロース（α，α−トレハロー
ス）が知られており、その存在は、『アドバンシズ・イ
ン・カーボハイドレイト・ケミストリー（Ａｄｖａｎｃ
ｅｓ ｉｎ Ｃａｒｂｏｈｙｄｒａｔｅ Ｃｈｅｍｉｓ
ｔｒｙ）』、第１８巻、第２０１乃至２２５頁（１９６
３年）アカデミック・プレス社（米国）及び『アプライ
ド・アンド・エンビロメンタル・マイクロバイオロジー
（Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａ
ｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ）』、第５６巻、第３２
１３乃至３２１５頁（１９９０年）などにも記載されて
いるように、少量ながら、微生物、きのこ、昆虫など広
範囲に及んでいる。トレハロースのような非還元性糖質
は、アミノ酸や蛋白質等のアミノ基を有する物質とアミ
ノカルボニル反応を起こさず、含アミノ酸物質を損なわ
ないことから、褐変、劣化を懸念することなく利用、加
工できることが期待され、その工業的製造方法の確立が
望まれている。2. Description of the Related Art Trehalose (α, α-trehalose) has long been known as a non-reducing sugar containing glucose as a constituent sugar, and its existence is described in "Advances in Carbohydrate Chemistry (Advanc)".
es in Carbohydrate Chemis
try), Vol. 18, pp. 201-225 (196).
3 years) Academic Press (USA) and “Applied and Environmental Microbiology”
l Microbiology) ", Vol. 56, No. 32
As described in pages 13 to 3215 (1990) and the like, it covers a wide range of microorganisms, mushrooms, insects, etc., even in a small amount. Non-reducing sugars such as trehalose do not cause aminocarbonyl reaction with substances having amino groups such as amino acids and proteins, and do not damage amino acid-containing substances, so they can be used and processed without fear of browning or deterioration. Is expected, and establishment of an industrial manufacturing method thereof is desired.

【０００３】トレハロースの製造方法としては、例え
ば、特開昭５０−１５４４８５公報で報告されている微
生物菌体を用いる方法や、特開昭５８−２１６６９５公
報で提案されているマルトース・ホスホリラーゼとトレ
ハロース・ホスホリラーゼとの組合せでマルトースを変
換する方法などが知られている。しかしながら、微生物
菌体を用いる方法は、該菌体を出発原料とし、これに含
まれるトレハロースの含量が、通常、固形物当り１５ｗ
／ｗ％（以下、本明細書では、特にことわらない限り、
ｗ／ｗ％を単に％と略称する）未満と低く、その上、こ
れを抽出、精製する工程が煩雑で、工業的製造方法とし
ては不適である。また、マルトース・ホスホリラーゼ及
びトレハロース・ホスホリラーゼを用いる方法は、いず
れもグルコース−１リン酸を経由しており、その基質濃
度を高めることが困難であり、また、両酵素の反応系が
可逆反応で目的物の生成率が低く、更には、両酵素の反
応系を安定に維持して反応をスムーズに進行させること
が困難であって、未だ、工業的製造方法として実現する
に至っていない。As a method for producing trehalose, for example, a method using a microbial cell reported in JP-A-50-154485 or maltose phosphorylase and trehalose-proposed in JP-A-58-216695 is proposed. A method of converting maltose in combination with phosphorylase is known. However, the method using a microbial cell uses the microbial cell as a starting material, and the content of trehalose contained in the microbial cell is usually 15 w per solid matter.
/ W% (hereinafter, in the present specification, unless otherwise specified,
w / w% is simply abbreviated as%), and the process of extracting and purifying this is complicated, which is unsuitable as an industrial production method. Further, in the method using maltose phosphorylase and trehalose phosphorylase, it is difficult to increase the substrate concentration of both enzymes via glucose-1-phosphate, and the reaction system of both enzymes is a reversible reaction. The production rate of the product is low, and furthermore, it is difficult to keep the reaction system of both enzymes stable and to proceed the reaction smoothly, and it has not yet been realized as an industrial production method.

【０００４】これに関係して、『月刊フードケミカ
ル』、８月号、第６７乃至７２頁（１９９２年）、「澱
粉利用開発の現状と課題」の「オリゴ糖」の項におい
て、「トレハロースについては著しく広い応用範囲が考
えられるが、本糖の澱粉糖質からの直接糖転移、加水分
解反応を用いた酵素的生産は、現在のところ学術的には
不可能であるといわれている。」と記載されているよう
に、澱粉を原料とし、酵素反応によってトレハロースを
製造することは、従来、学術的にも不可能であると考え
られてきた。In connection with this, in "Monthly Food Chemical", August issue, pp. 67-72 (1992), "Current status and problems of starch utilization development", "Oligosaccharide", "About trehalose" Although it can be considered to have a remarkably wide range of application, it is said that the enzymatic production of this sugar by direct sugar transfer from starch sugar and hydrolysis reaction is not possible at present academically. ” It has been conventionally considered academically impossible to produce trehalose by enzymatic reaction using starch as a raw material.

【０００５】一方、澱粉を原料として製造される澱粉部
分分解物、例えば、澱粉液化物、各種デキストリン、各
種マルトオリゴ糖などは、通常、その分子の末端に還元
基を有し還元性を示すことが知られている。このような
澱粉部分分解物を、本明細書では、還元性澱粉部分分解
物と称する。一般に、還元性澱粉部分分解物は、固形物
当りの還元力の大きさをデキストロース・エクイバレン
ト（ＤｅｘｔｒｏｓｅＥｑｕｉｖａｌｅｎｔ，ＤＥ）と
して表している。この値の大きいものは、通常、分子が
小さく低粘度で、甘味が強いものの、反応性が強く、ア
ミノ酸や蛋白質などのアミノ基を持つ物質とアミノカル
ボニル反応を起こし易く、褐変し、悪臭を発生して、品
質を劣化し易い性質のあることが知られている。On the other hand, starch partially decomposed products produced from starch as a raw material, such as starch liquefaction, various dextrins, various maltooligosaccharides, etc., usually have a reducing group at the end of the molecule and thus show reducibility. Are known. In the present specification, such a partially decomposed product of starch is referred to as a partially decomposed product of reducing starch. In general, the reducing starch partially decomposed product represents the magnitude of the reducing power per solid substance as Dextrose Equivalent (DE). A substance with a large value usually has a small molecule and low viscosity and has a strong sweetness, but it has a strong reactivity and easily causes an aminocarbonyl reaction with a substance having an amino group such as an amino acid or a protein, causing browning and generating a bad smell. Then, it is known that there is a property of easily degrading the quality.

【０００６】このような還元性澱粉部分分解物の種々の
特性は、ＤＥの大小に依存しており、還元性澱粉部分分
解物とＤＥとの関係は極めて重要である。従来、当業界
では、この関係を断ち切ることは不可能とさえ信じられ
てきた。Various characteristics of such a partially decomposed product of reducing starch depend on the size of DE, and the relationship between the partially decomposed product of reducing starch and DE is extremely important. Traditionally, it has been believed in the industry that it is impossible to break this relationship.

【０００７】これを解決するために、本発明者等は、先
に、特願平５−３４９２１６号明細書で、グルコース重
合度３以上から選ばれる１種又は２種以上の還元性澱粉
部分分解物から分子の末端にトレハロース構造を有する
非還元性糖質を生成する新規非還元性糖質生成酵素（本
酵素を、本明細書を通じて、非還元性糖質生成酵素と称
する。）を開示し、本非還元性糖質生成酵素を利用し
て、還元性澱粉部分分解物から分子の末端にトレハロー
ス構造を有するグルコース重合度３以上の非還元性糖質
とこれを含む低還元性糖質並びにこれら糖質からのトレ
ハロースの製造方法を確立した。[0007] In order to solve this, the present inventors previously mentioned that in Japanese Patent Application No. 5-349216, one or two or more types of reducing starch partial decomposition selected from a glucose polymerization degree of 3 or more. Disclosed is a novel non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end of the molecule from an object (this enzyme is referred to as a non-reducing saccharide-forming enzyme throughout the specification). Using the present non-reducing saccharide-forming enzyme, a non-reducing saccharide having a trehalose structure at the terminal of the molecule from a partially decomposed product of reducing starch and a glucose polymerization degree of 3 or more, and a low reducing saccharide containing the same, A method for producing trehalose from these sugars was established.

【０００８】また、本発明者等は、特願平６−７９２９
１号明細書で、分子の末端にトレハロース構造を有する
グルコース重合度３以上の非還元性糖質のトレハロース
部分とそれ以外の部分との間の結合を特異的に加水分解
する新規トレハロース遊離酵素（本酵素を、本明細書を
通じて、トレハロース遊離酵素と称する。）を開示し、
前述の非還元性糖質生成酵素と本トレハロース遊離酵素
とを併用して還元性澱粉部分分解物から比較的高収量の
トレハロースの製造方法を確立した。しかしながら、こ
れら酵素を利用して還元性澱粉部分分解物から分子中に
トレハロース構造を有する糖質や、トレハロースなどの
非還元性糖質を製造する場合には、未反応の還元性澱粉
部分分解物の残存が避けられないばかりか、新たに、グ
ルコース、マルトースなどの還元性澱粉糖を生成するこ
とも判明した。このような非還元性糖質とともに還元性
澱粉糖を含有する低還元性糖質の還元性を更に低減する
ことが強く望まれる。Further, the present inventors have filed Japanese Patent Application No. 6-7929.
1 discloses a novel trehalose-releasing enzyme that specifically hydrolyzes a bond between a trehalose moiety of a non-reducing sugar having a trehalose structure of 3 or more and a non-reducing sugar having a trehalose structure at the end of the molecule and the other moiety ( This enzyme is referred to as trehalose-releasing enzyme throughout the specification.)
Using the above-mentioned non-reducing saccharide-forming enzyme and the present trehalose-releasing enzyme in combination, a method for producing trehalose in relatively high yield from a partially decomposed product of reducing starch was established. However, when a sugar having a trehalose structure in the molecule or a non-reducing sugar such as trehalose is produced from a partially decomposed product of reducing starch using these enzymes, unreacted partially decomposed product of reducing starch is used. It has been found that not only the remaining sucrose is inevitable, but also reducing starch sugars such as glucose and maltose are newly produced. It is strongly desired to further reduce the reducibility of the low reducing sugar containing the reducing starch sugar together with the non-reducing sugar.

【０００９】[0009]

【発明が解決しようとする課題】本発明は、分子中にト
レハロース構造を有する糖質、すなわち、分子の末端に
トレハロース構造を有する非還元性糖質（以下、本物質
を、本明細書では、α−グリコシルトレハロースと称す
る。）、トレハロースの両グルコースに重合度１若しく
は２以上のグルコースを有している非還元性糖質（以
下、本物質を、本明細書では、α−グリコシル α−グ
リコシドと称する。）及びトレハロースなどの非還元性
糖質とともに還元性澱粉糖を含有する低還元性糖質の還
元性を更に低減せしめた糖質、及び、その製造方法を確
立し、併せて、これら糖質の用途を提供するものであ
る。DISCLOSURE OF THE INVENTION The present invention provides a saccharide having a trehalose structure in a molecule, that is, a non-reducing saccharide having a trehalose structure at the end of the molecule (hereinafter, this substance, in the present specification, α-glycosyl trehalose), a non-reducing saccharide having a degree of polymerization of 1 or 2 or higher on both glucose of trehalose (hereinafter, this substance is referred to as α-glycosyl α-glycoside in the present specification). ) And trehalose and other non-reducing saccharides, as well as a sugar that further reduces the reducing property of a low-reducing saccharide containing a reducing starch saccharide, and a method for producing the saccharide. It is intended to provide a use of sugar.

【００１０】[0010]

【課題を解決するための手段】本発明者等は、前記課題
を解決するために、該低還元性糖質の水素添加法に着目
し、種々研究を続けてきた。その結果、分子中にトレハ
ロース構造を有する糖質及び／又はトレハロースからな
る非還元性糖質とともに還元性澱粉糖を含有する低還元
性糖質を水素添加すると該非還元性糖質が悪影響を与え
ること無く、還元性澱粉糖が対応する澱粉糖アルコール
になり、原料の低還元性糖質の還元性を更に低減し、そ
の還元性を実質的に消滅させることも容易であることを
見いだし、本発明を完成した。[Means for Solving the Problems] In order to solve the above problems, the present inventors have focused on the method for hydrogenating low-reducing sugars and have continued various studies. As a result, hydrogenation of a low-reducing sugar containing a reducing starch sugar together with a sugar having a trehalose structure in the molecule and / or a non-reducing sugar consisting of trehalose may adversely affect the non-reducing sugar. However, it was found that the reducing starch sugar becomes the corresponding starch sugar alcohol, further reducing the reducibility of the low-reducing sugar as a raw material, and substantially eliminating the reducibility. Was completed.

【００１１】また、本発明者等は、原料の製造方法につ
いても検討したところ、分子中にトレハロース構造を有
する糖質及び／又はトレハロースからなる非還元性糖質
とともに還元性澱粉糖を含有する低還元性糖質として
は、例えば、グルコース重合度３以上の還元性澱粉部分
分解物に非還元性糖質生成酵素又は非還元性糖質生成酵
素とともにトレハロース遊離酵素を作用させて得られる
ものが有利に利用でき、とりわけ、澱粉を液化した溶液
に、非還元性糖質生成酵素、又は、非還元性糖質生成酵
素及びトレハロース遊離酵素を作用させるに際し、澱粉
枝切酵素及び／又はシクロマルトデキストリン・グルカ
ノトランスフェラーゼを併用して得られる低還元性糖質
が好都合であり、また、本出願人が特願平６−１４４０
９２号明細書で開示した、マルトースにマルトース・ト
レハロース変換酵素を作用させて得られる低還元性糖質
も好都合であることを見いだし、本発明を完成した。The inventors of the present invention also examined the method for producing the raw material, and found that a sugar containing a trehalose structure in the molecule and / or a non-reducing sugar composed of trehalose and a reducing starch sugar were contained. As the reducing sugar, for example, one obtained by causing a trehalose-releasing enzyme to act on a non-reducing sugar-forming enzyme or a non-reducing sugar-forming enzyme on a partially decomposed product of reducing starch having a glucose polymerization degree of 3 or more is advantageous. In particular, when a non-reducing sugar-forming enzyme, or a non-reducing sugar-forming enzyme and trehalose-releasing enzyme are allowed to act on a liquefied starch solution, starch debranching enzyme and / or cyclomaltodextrin A low-reducing sugar obtained by using glucanotransferase in combination is convenient, and the applicant of the present invention filed Japanese Patent Application No. 6-1440.
The inventors have found that the low-reducing sugar obtained by reacting maltose with maltose-trehalose converting enzyme disclosed in the specification No. 92 is also convenient, and completed the present invention.

【００１２】例えば、澱粉を比較的低ＤＥに液化した溶
液、望ましくは、ＤＥ１５未満の溶液に非還元性糖質生
成酵素を作用させて該低還元性糖質を製造するに際し、
澱粉枝切酵素及び／又はシクロマルトデキストリン・グ
ルカノトランスフェラーゼをともに作用させて得られる
非還元性糖質を含む低還元性糖質は、非還元性糖質生成
酵素だけを作用させた場合と比較して、その還元性をほ
とんど増加させることなく、その分子量を著しく低減
し、粘度を低下し、取扱い容易で、本発明の原料糖質と
して好適であることが判明した。ちなみに、このように
して得られた低還元性糖質にグルコアミラーゼを作用さ
せたところ、その構造中に含まれるトレハロース含量が
大幅に増加していることも判明した。また、澱粉を比較
的低ＤＥに液化した溶液、望ましくはＤＥ１５未満の溶
液に非還元性糖質生成酵素及びトレハロース遊離酵素を
作用させてトレハロースを製造するに際し、澱粉枝切酵
素及び／又はシクロマルトデキストリン・グルカノトラ
ンスフェラーゼをともに作用させて得られるトレハロー
スは、非還元性糖質生成酵素及びトレハロース遊離酵素
だけを作用させた場合と比較して、その収量が大幅に増
加し、本発明の原料として好適であることも判明した。
また、マルトース・トレハロース変換酵素の作用によ
り、マルトースから製造されるトレハロースとマルトー
スとの混合糖質も、本発明の原料糖質として好適である
ことが判明した。このようにして得られる分子中にトレ
ハロース構造を有する糖質及び／又はトレハロースから
なる非還元性糖質含量を高めた低還元性糖質を原料とし
て、これに水素添加することは有利に実施できる。この
ようにして得られる本発明の還元性を低減させた糖質
は、実質的に還元性を示さない糖質、望ましくはＤＥ１
未満の糖質で、安定性が高く、取扱い容易で、広範な用
途に利用でき、例えば、飲食物、化粧品、医薬品など各
種組成物に有利に利用できる。For example, when a non-reducing saccharide-forming enzyme is allowed to act on a solution obtained by liquefying starch to a relatively low DE, preferably a solution having a DE of less than 15,
Low-reducing sugars containing non-reducing sugars obtained by acting together with starch debranching enzyme and / or cyclomaltodextrin / glucanotransferase are compared with the case where only non-reducing sugar-forming enzyme is allowed to act. Then, it was found that the molecular weight thereof was remarkably reduced, the viscosity was lowered, the handling was easy, and the raw material sugar of the present invention was suitable, with almost no increase in its reducibility. By the way, when glucoamylase was allowed to act on the thus-obtained low-reducing sugar, it was also found that the trehalose content contained in the structure was significantly increased. Further, when trehalose is produced by reacting a starch liquefied solution with a relatively low DE, preferably a solution having a DE of less than 15 with a non-reducing sugar-forming enzyme and trehalose-releasing enzyme, starch debranching enzyme and / or cyclomalto The trehalose obtained by allowing both dextrin and glucanotransferase to act together has a significantly increased yield as compared with the case where only the non-reducing sugar-forming enzyme and the trehalose-releasing enzyme are allowed to act, and is used as a raw material of the present invention. It has also been found suitable.
It was also found that a mixed sugar of trehalose and maltose produced from maltose by the action of maltose / trehalose converting enzyme is also suitable as the raw material sugar of the present invention. It is possible to advantageously carry out hydrogenation of a sugar having a trehalose structure in the molecule thus obtained and / or a low-reducing sugar containing trehalose having a high content of non-reducing sugar as a raw material. . The thus-obtained reduced-sugar carbohydrate of the present invention is a substantially non-reducible carbohydrate, preferably DE1.
It has a sugar content of less than 1, has high stability, is easy to handle, and can be used in a wide range of applications. For example, it can be advantageously used in various compositions such as food and drink, cosmetics, and pharmaceuticals.

【００１３】まず、本発明で用いる非還元性糖質生成酵
素としては、澱粉を比較的低ＤＥに液化した溶液に含ま
れるグルコース重合度３以上から選ばれる１種又は２種
以上の還元性澱粉部分分解物からα−グリコシルトレハ
ロースを生成する酵素であればよく、例えば、特願平５
−３４９２１６号明細書に開示されるリゾビウム属、ア
ルスロバクター属、ブレビバクテリウム属、フラボバク
テリウム属、ミクロコッカス属、クルトバクテリウム
属、マイコバクテリウム属及びテラバクター属などに属
する微生物由来の酵素が有利に利用できる。また、必要
ならば、耐熱性の非還元性糖質生成酵素を用いることも
随意であり、例えば、本出願人が特願平６−１６６０１
１号明細書で開示したスルフォロブス属由来の耐熱性非
還元性糖質生成酵素を用いることも有利に実施できる。
また、トレハロース遊離酵素としては、澱粉を液化した
溶液に非還元性糖質生成酵素を作用させて生成されるα
−グリコシルトレハロースを、そのトレハロース部分と
それ以外の部分との間の結合を特異的に加水分解する酵
素であればよく、例えば、特願平６−７９２９１号明細
書で開示したリゾビウム属、アルスロバクター属、ブレ
ビバクテリウム属及びミクロコッカス属などに属する微
生物由来の酵素が有利に利用できる。また、必要なら
ば、耐熱性のトレハロース遊離酵素を用いることも随意
であり、例えば、本出願人が特願平６−１６６１２６号
明細書で開示したスルフォロブス属に属するトレハロー
ス遊離酵素を用いることも有利に実施できる。First, as the non-reducing saccharide-forming enzyme used in the present invention, one or more reducing starches selected from the glucose polymerization degree of 3 or more contained in a solution obtained by liquefying starch to a relatively low DE are used. Any enzyme that produces α-glycosyltrehalose from a partially decomposed product may be used.
Enzymes derived from microorganisms belonging to the genus Rhizobium, Arthrobacter, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium, Terrabactor, etc. Can be used to advantage. If necessary, a thermostable non-reducing saccharide-forming enzyme may be optionally used. For example, the applicant of the present invention has a Japanese Patent Application No. 6-16601.
It is also possible to advantageously use the thermostable non-reducing saccharide-forming enzyme derived from the genus Sulfolobus disclosed in the specification 1.
The trehalose-releasing enzyme is α produced by liquefying starch with a non-reducing saccharide-forming enzyme.
Any enzyme may be used as long as it is an enzyme that specifically hydrolyzes the bond between the trehalose portion and the other portion of glycosyltrehalose, and examples thereof include Rhizobium and Arthulosium disclosed in Japanese Patent Application No. 6-79291. Enzymes derived from microorganisms belonging to the genus Bacter, the genus Brevibacterium and the genus Micrococcus can be advantageously used. If necessary, it is also possible to optionally use a thermostable trehalose-releasing enzyme. For example, it is advantageous to use the trehalose-releasing enzyme belonging to the genus Sulfolobus disclosed by the present applicant in Japanese Patent Application No. 6-166126. Can be carried out.

【００１４】更に、本発明で用いるマルトース・トレハ
ロース変換酵素としては、マルトースからトレハロース
を生成する酵素であればよく、例えば、本出願人が、特
願平６−１４４０９２号明細書で開示されるピメロバク
ター属、シュードモナス属およびサーマス属などに属す
る微生物由来の酵素が有利に利用できる。微生物から、
例えば、非還元性糖質生成酵素及び／又はトレハロース
遊離酵素を調製する方法は、更には、マルトース・トレ
ハロース変換酵素を調製する方法は、これら酵素の産生
能を有する微生物を培養して調製すればよい。Further, the maltose / trehalose converting enzyme used in the present invention may be any enzyme that produces trehalose from maltose. For example, the applicant of the present invention discloses pimelobacter disclosed in Japanese Patent Application No. 6-144092. Enzymes derived from microorganisms belonging to the genera Pseudomonas and Thermus can be advantageously used. From microorganisms,
For example, a method for preparing a non-reducing sugar-forming enzyme and / or a trehalose-releasing enzyme, and further a method for preparing a maltose / trehalose-converting enzyme can be prepared by culturing a microorganism capable of producing these enzymes. Good.

【００１５】微生物の培養に用いる培地は、微生物が生
育でき、該酵素を産生しうる栄養培地であればよく、合
成培地及び天然培地のいずれでもよい。炭素源として
は、微生物が資化しうる物であればよく、例えば、グル
コース、フラクトース、ラクトース、スクロース、マン
ニトール、ソルビトール、糖蜜、還元性澱粉部分分解物
などの糖質、また、クエン酸、コハク酸などの有機酸又
はその塩も使用することができる。培地におけるこれら
の炭素源の濃度は炭素源の種類により適宜選択される。
例えば、還元性澱粉部分分解物の場合には、通常、２０
％以下が望ましく、菌の生育及び増殖からは５％以下が
好ましい。窒素源としては、例えば、アンモニウム塩、
硝酸塩などの無機窒素化合物及び、例えば、尿素、コー
ン・スティープ・リカー、カゼイン、ペプトン、酵母エ
キス、肉エキスなどの有機窒素含有物が用いられる。ま
た、無機成分としては、例えば、カルシウム塩、マグネ
シウム塩、カリウム塩、ナトリウム塩、リン酸塩、マン
ガン塩、亜鉛塩、鉄塩、銅塩、モリブデン塩、コバルト
塩などが適宜用いられる。更に、必要に応じて、アミノ
酸、ビタミンなども適宜用いられる。The medium used for culturing the microorganism may be a nutrient medium in which the microorganism can grow and produce the enzyme, and may be either a synthetic medium or a natural medium. The carbon source may be any substance that can be assimilated by microorganisms, for example, glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, sugars such as partially decomposed products of reducing starch, citric acid, succinic acid. Organic acids or salts thereof such as can also be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of carbon source.
For example, in the case of a partially degraded reducing starch, it is usually 20
% Or less is preferable, and 5% or less is preferable from the viewpoint of growth and proliferation of bacteria. As the nitrogen source, for example, ammonium salt,
Inorganic nitrogen compounds such as nitrates and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract are used. As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt and the like are appropriately used. Furthermore, amino acids, vitamins, etc. may be appropriately used, if necessary.

【００１６】培養は、通常、温度４乃至４０℃、好まし
くは２０乃至３７℃、ｐＨ４乃至１０、好ましくは５乃
至９から選ばれる条件で好気的に行われる。また、耐熱
性酵素を産生する微生物の場合には、通常、温度４０乃
至９０℃、好ましくは５０乃至８０℃、ｐＨ２乃至１
０、好ましくはｐＨ３乃至９から選ばれる条件で行われ
る。培養時間は微生物が増殖し得る以上の時間であれば
よく、好ましくは１０時間乃至１００時間である。ま
た、培養液の溶存酸素濃度には特に制限はないが、通常
は、０．５乃至２０ｐｐｍが好ましい。そのために、通
気量を調節したり、撹拌したり、通気に酸素を追加した
り、また、ファーメンター内の圧力を高めるなどの手段
が採用される。また、培養方式は、回分培養又は連続培
養のいずれでもよい。Culturing is usually carried out aerobically under conditions selected from a temperature of 4 to 40 ° C., preferably 20 to 37 ° C. and a pH of 4 to 10, preferably 5 to 9. In the case of a microorganism that produces a thermostable enzyme, the temperature is usually 40 to 90 ° C, preferably 50 to 80 ° C, and the pH is 2 to 1.
It is carried out under conditions selected from 0, preferably pH 3 to 9. The culturing time may be any time that allows the growth of microorganisms, and is preferably 10 hours to 100 hours. The dissolved oxygen concentration of the culture solution is not particularly limited, but usually 0.5 to 20 ppm is preferable. For that purpose, means such as adjusting the air flow rate, stirring, adding oxygen to the air flow, and increasing the pressure in the fermenter are adopted. Further, the culture method may be either batch culture or continuous culture.

【００１７】このようにして、微生物を培養した後、酵
素を回収する。酵素活性は、培養物の菌体及び除菌液い
ずれにも認められ、菌体及び除菌液を粗酵素液として採
取することも、また、培養物全体を粗酵素液として用い
ることもできる。培養物から菌体を除去するには公知の
固液分離法が採用される。例えば、培養物そのものをそ
のまま遠心分離する方法、あるいは、プレコートフィル
ターなどを用いて濾過分離する方法、平膜、中空糸膜な
どの膜濾過により分離する方法などが適宜採用される。
除菌液をそのまま酵素液として用いることができるが、
一般的には、濃縮して用いられる。濃縮方法としては、
例えば、硫安塩析法、アセトン及びアルコール沈殿法、
平膜、中空糸膜など膜濃縮法などが採用される。After culturing the microorganism in this manner, the enzyme is recovered. The enzyme activity is found in both the bacterial cells and the sterilized solution of the culture, and the bacterial cells and the sterilized solution can be collected as a crude enzyme solution, or the entire culture can be used as a crude enzyme solution. A known solid-liquid separation method is adopted to remove the bacterial cells from the culture. For example, a method of centrifuging the culture itself as it is, a method of separating by filtration using a precoat filter, a method of separating by membrane filtration of a flat membrane, a hollow fiber membrane, etc. are appropriately adopted.
The sterilized solution can be used as it is as the enzyme solution,
Generally, it is concentrated before use. As a concentration method,
For example, ammonium sulfate salting-out method, acetone and alcohol precipitation method,
Membrane concentration methods such as flat membranes and hollow fiber membranes are used.

【００１８】更に、除菌液及びその濃縮物を公知の方法
により固定化することもできる。例えば、イオン交換体
への結合法、樹脂及び膜などとの共有結合・吸着法、高
分子物質を用いた包括法などが採用される。また、培養
物から分離した菌体もそのまま粗酵素として用いること
ができるが、これを固定化して用いてもよい。一例とし
て、これをアルギン酸ナトリウムと混合して、塩化カル
シウム溶液中に滴下して粒状にゲル化させて固定化す
る。この粒状化物をさらにポリエチレンイミン、グルタ
ールアルデヒドで処理して固定化してもよい。菌体から
酵素を抽出して、その抽出液を粗酵素液として用いるこ
ともできる。例えば、超音波による破砕法、ガラスビー
ズ及びアルミナによる機械的破砕法、フレンチプレスに
よる破砕法などで菌体から酵素を抽出し、遠心分離又は
膜濾過などで清澄な粗酵素液を得ることができる。Further, the sterilized liquid and its concentrate can be immobilized by a known method. For example, a binding method to an ion exchanger, a covalent binding / adsorption method with a resin or a membrane, an encapsulation method using a polymer substance, etc. are adopted. Further, the bacterial cells separated from the culture can be directly used as the crude enzyme, but they may be immobilized and used. As an example, this is mixed with sodium alginate and added dropwise into a calcium chloride solution to gel and immobilize into a granular form. This granule may be further treated with polyethyleneimine or glutaraldehyde to be immobilized. It is also possible to extract the enzyme from the cells and use the extract as a crude enzyme solution. For example, the enzyme can be extracted from the bacterial cells by a disruption method by ultrasonic waves, a mechanical disruption method by glass beads and alumina, a disruption method by a French press, etc., and a clear crude enzyme solution can be obtained by centrifugation or membrane filtration. .

【００１９】本酵素液はそのまま用いることができる
が、公知の方法によって更に精製して利用することもで
きる。一例として、培養液の処理物を硫安塩析して濃縮
した粗酵素標品を透析後、東ソー株式会社製『ＤＥＡＥ
−トヨパール』などを用いた陰イオン交換カラムクロマ
トグラフィー、続いて、同社製『ブチルトヨパール』な
どを用いた疎水カラムクロマトグラフィー、同社製『ト
ヨパール ＨＷ−５５』などを用いたゲル濾過クロマト
グラフィーを用いて精製することにより、電気泳動的に
単一な酵素を得ることができる。The enzyme solution of the present invention can be used as it is, but can be further purified by a known method before use. As an example, a crude enzyme preparation obtained by salting out a treated product of the culture solution with ammonium sulfate and concentrating it is dialyzed and then manufactured by Tosoh Corporation “DEAE”.
-Anion exchange column chromatography using "Toyopearl", etc., followed by hydrophobic column chromatography using "Butyl Toyopearl" manufactured by the same company, and gel filtration chromatography using "Toyopearl HW-55" manufactured by the same company. By purification using it, an electrophoretically single enzyme can be obtained.

【００２０】このようにして得られる非還元性糖質生成
酵素は、一般的には、例えば、下記の理化学的性質を有
する。 （１） 作用 グルコース重合度３以上から選ばれる１種又は２種以上
の還元性澱粉部分分解物からα−グリコシルトレハロー
スを生成する。 （２） 分子量 ＳＤＳ−ゲル電気泳動法により、約７６，０００乃至８
７，０００ダルトン。 （３） 等電点 アンフォライン含有電気泳動法により、ｐＩ約３．６乃
至４．６。 （４） 至適温度 ｐＨ７．０、６０分間反応で、３５乃至４０℃付近。 （５） 至適ｐＨ ４０℃、６０分間反応で、ｐＨ約６．４乃至７．２。 （６） 温度安定性 ｐＨ７．０、６０分間保持で、３５乃至４０℃付近まで
安定。 （７） ｐＨ安定性 ２５℃、１６時間保持で、ｐＨ約５．５乃至１１．０。The non-reducing saccharide-forming enzyme thus obtained generally has, for example, the following physicochemical properties. (1) Action α-Glycosyltrehalose is produced from one or more reducing starch partial degradation products selected from the glucose polymerization degree of 3 or more. (2) Molecular weight By SDS-gel electrophoresis, about 76,000 to 8
7,000 daltons. (3) pI of about 3.6 to 4.6 by the electrophoresis method containing isoelectric point ampholine. (4) Optimum temperature: pH 7.0, reaction for 60 minutes, at about 35 to 40 ° C. (5) Optimum pH 40 ° C., reaction for 60 minutes, pH about 6.4 to 7.2. (6) Temperature stability It is stable up to around 35 to 40 ° C when kept at pH 7.0 for 60 minutes. (7) pH stability A pH of about 5.5 to 11.0 when kept at 25 ° C for 16 hours.

【００２１】非還元性糖質生成酵素の活性測定方法は、
基質としてマルトペンタオース１．２５ｗ／ｖ％（５０
ｍＭリン酸緩衝液、ｐＨ７．０）４ｍｌに酵素液を１ｍ
ｌ加え４０℃で６０分間反応させた後、１００℃で１０
分間加熱して反応を停止させ、その反応液を正確に脱イ
オン水で１０倍に希釈し、その希釈液の還元力をソモギ
ー・ネルソン法にて測定する。対照として、あらかじめ
１００℃で１０分間加熱することにより失活させた酵素
液を用いて同様に測定する。上記の測定方法を用いて、
１分間に１μｍｏｌｅのマルトペンタオースに相当する
還元力を減少させる酵素量を１単位と定義した。The method for measuring the activity of the non-reducing saccharide-forming enzyme is as follows:
Maltopentaose as substrate 1.25 w / v% (50
1m of enzyme solution to 4ml of mM phosphate buffer, pH 7.0
1 and then reacted at 40 ° C for 60 minutes and then at 100 ° C for 10 minutes.
The reaction is stopped by heating for 1 minute, the reaction solution is exactly diluted 10 times with deionized water, and the reducing power of the diluted solution is measured by the Somogy Nelson method. As a control, the same measurement is performed using an enzyme solution inactivated by heating at 100 ° C. for 10 minutes in advance. Using the above measurement method,
The amount of enzyme that reduces the reducing power corresponding to 1 μmole of maltopentaose per minute was defined as 1 unit.

【００２２】また、前述のようにして得られるトレハロ
ース遊離酵素は、一般的には、例えば、下記の理化学的
性質を有する。 （１） 作用 α−グリコシルトレハロースのトレハロース部分とそれ
以外のグリコシル部分との間の結合を特異的に加水分解
する。 （２） 分子量 ＳＤＳ−ゲル電気泳動法により、約５７，０００乃至６
８，０００ダルトン。 （３） 等電点 アンフォライン含有電気泳動法により、ｐＩ約３．３乃
至４．６。 （４） 至適温度 ｐＨ７．０、３０分間反応で、３５乃至４５℃付近。 （５） 至適ｐＨ ４０℃、３０分間反応で、ｐＨ約６．０乃至７．５。 （６） 温度安定性 ｐＨ７．０、６０分間保持で、３０乃至４５℃付近まで
安定。 （７） ｐＨ安定性 ２５℃、１６時間保持で、ｐＨ約５．０乃至１０．０。The trehalose-releasing enzyme obtained as described above generally has, for example, the following physicochemical properties. (1) Action The bond between the trehalose moiety of α-glycosyltrehalose and the other glycosyl moieties is specifically hydrolyzed. (2) Molecular weight By SDS-gel electrophoresis, about 57,000 to 6
8,000 daltons. (3) pI of about 3.3 to 4.6 by an isoelectric focusing ampholine-containing electrophoresis method. (4) Optimum temperature pH of around 7.0 at 35 to 45 ° C. for 30 minutes reaction. (5) Optimum pH 40 ° C., reaction for 30 minutes, pH about 6.0 to 7.5. (6) Temperature stability It is stable up to around 30 to 45 ° C by keeping at pH 7.0 for 60 minutes. (7) pH stability pH is about 5.0 to 10.0 when kept at 25 ° C. for 16 hours.

【００２３】トレハロース遊離酵素の活性は次のように
して測定する。基質としてマルトトリオシルトレハロー
ス（別名、α−マルトテトラオシル α−Ｄ−グルコシ
ド）１．２５ｗ／ｖ％（５０ｍＭリン酸緩衝液、ｐＨ
７．０）４ｍｌに酵素液を１ｍｌ加え４０℃で３０分間
反応させた後、ソモギー銅液を加え反応を停止させ、還
元力をソモギー・ネルソン法にて測定する。対照とし
て、あらかじめ１００℃で１０分間加熱することにより
失活させた酵素液を用いて同様に測定する。上記の測定
方法を用いて、１分間に１μｍｏｌｅのグルコースに相
当する還元力を増加させる酵素量を１単位と定義する。The activity of trehalose-releasing enzyme is measured as follows. Maltotriosyltrehalose (also known as α-maltotetraosyl α-D-glucoside) 1.25 w / v% (50 mM phosphate buffer, pH
7.0) 1 ml of the enzyme solution is added to 4 ml, and the mixture is reacted at 40 ° C. for 30 minutes. Then, the Somogyi copper solution is added to stop the reaction, and the reducing power is measured by the Somogy Nelson method. As a control, the same measurement is performed using an enzyme solution inactivated by heating at 100 ° C. for 10 minutes in advance. Using the above measurement method, the amount of enzyme that increases the reducing power corresponding to 1 μmole of glucose per minute is defined as 1 unit.

【００２４】更に、前述のようにして得られるマルトー
ス・トレハロース変換酵素は、下記の理化学的性質を有
する。 （１） 作用 マルトースをトレハロースに変換し、トレハロースをマ
ルトースに変換する。 （２） 分子量 ＳＤＳ−ゲル電気泳動法で、約５７，０００乃至１２
０，０００ダルトン。 （３） 等電点 アンフォライン含有電気泳動法により、ｐＩ約３．８乃
至５．１。 （４） 活性阻害 １ｍＭＣｕ++、Ｈｇ++又は５０ｍＭトリス塩酸緩衝液で
阻害を受ける。 （５） 起源 微生物により産生された酵素である。Further, the maltose-trehalose converting enzyme obtained as described above has the following physicochemical properties. (1) Action Maltose is converted into trehalose and trehalose is converted into maltose. (2) Molecular weight By SDS-gel electrophoresis, about 57,000 to 12
10,000 daltons. (3) pI of about 3.8 to 5.1 by an isoelectric focusing ampholine-containing electrophoresis method. (4) Activity inhibition 1 mM Cu ++ , Hg ++, or 50 mM Tris-HCl buffer is used for inhibition. (5) Origin It is an enzyme produced by a microorganism.

【００２５】マルトース・トレハロース変換酵素の活性
は、次のようにして測定する。基質としてマルトース２
０ｗ／ｖ％（１０ｍＭリン酸塩緩衝液、ｐＨ７．０）１
ｍｌに酵素液１ｍｌを加え、反応温度を２５℃とし、６
０分間反応させた後、１００℃で１０分間加熱して反応
を停止させる。この反応液を正確に５０ｍＭリン酸塩緩
衝液ｐＨ７．５で１１倍に希釈し、その希釈液０．４ｍ
ｌにトレハラーゼ含有溶液（１単位／ｍｌ）を０．１ｍ
ｌ添加したものを４５℃、１２０分間インキュベートし
た後、この反応液中のグルコース量をグルコースオキシ
ダーゼ法で定量する。対照として、予め１００℃で１０
分間加熱することにより、失活させた酵素液及びトレハ
ラーゼを用いて同様に測定する。上記の測定方法を用い
て、増加するグルコース量からマルトース・トレハロー
ス変換酵素により生成するトレハロース量を求め、１分
間に１μｍｏｌｅのトレハロースを生成する酵素量を１
単位と定義する。The activity of maltose / trehalose converting enzyme is measured as follows. Maltose 2 as a substrate
0 w / v% (10 mM phosphate buffer, pH 7.0) 1
Add 1 ml of enzyme solution to ml to bring the reaction temperature to 25 ° C, and
After reacting for 0 minutes, the reaction is stopped by heating at 100 ° C. for 10 minutes. This reaction solution was diluted exactly 11 times with 50 mM phosphate buffer pH 7.5, and the diluted solution 0.4 m
0.1m of trehalase-containing solution (1 unit / ml)
After incubating the added solution at 45 ° C. for 120 minutes, the amount of glucose in this reaction solution is quantified by the glucose oxidase method. As a control, 10 at 100 ℃ beforehand
By heating for a minute, the enzyme solution inactivated and trehalase are similarly measured. Using the above measurement method, the amount of trehalose produced by maltose / trehalose converting enzyme is determined from the increasing glucose amount, and the enzyme amount producing 1 μmole of trehalose per minute is 1
Defined as a unit.

【００２６】次に、本発明で用いる澱粉枝切酵素は、澱
粉を比較的低ＤＥに液化した溶液、望ましくは、ＤＥ１
５未満の液化溶液に作用し、澱粉の枝分かれ結合を加水
分解する酵素であって、公知のプルラナーゼ、イソアミ
ラーゼなどが有利に利用でき、また、市販の酵素剤を利
用することも有利に実施できる。また、シクロマルトデ
キストリン・グルカノトランスフェラーゼは、澱粉を比
較的低ＤＥに液化した溶液、望ましくは、ＤＥ１５未満
の液化溶液に作用し、澱粉糖を糖転移し、不均化（ｄｉ
ｓｐｒｏｐｏｒｔｉｏｎａｔｉｏｎ）反応する酵素であ
って、公知のバチルス属、クレブシーラ属などに属する
微生物由来の酵素が有利に利用でき、また、市販の酵素
剤を利用することも有利に実施できる。Next, the starch debranching enzyme used in the present invention is a solution obtained by liquefying starch to a relatively low DE, preferably DE1.
An enzyme that acts on a liquefied solution of less than 5 and hydrolyzes a branched bond of starch, and known pullulanase, isoamylase, etc. can be advantageously used, and a commercially available enzyme agent can also be advantageously used. . Further, cyclomaltodextrin glucanotransferase acts on a solution in which starch is liquefied to a relatively low DE, preferably a liquefied solution having a DE of less than 15 to transfer sugar of starch to cause disproportionation (di).
It is possible to advantageously use an enzyme that reacts with a bacterium, which is derived from a microorganism belonging to the genus Bacillus, Klebsiella or the like, or to use a commercially available enzyme agent.

【００２７】また、前述の澱粉枝切酵素及び／又はシク
ロマルトデキストリン・グルカノトランスフェラーゼに
加えて、必要に応じて、他のアミラーゼ、望ましくは、
澱粉を比較的低ＤＥに液化した溶液に作用して、主とし
てグルコース重合度３以上のオリゴ糖を生成するアミラ
ーゼ、例えば、α−アミラーゼ、マルトトリオース生成
アミラーゼ、マルトテトラオース生成アミラーゼ、マル
トペンタオース生成アミラーゼ、マルトヘキサオース生
成アミラーゼ、マルトヘプタオース生成アミラーゼなど
を用いることも有利に実施できる。In addition to the above-mentioned starch debranching enzyme and / or cyclomaltodextrin glucanotransferase, if necessary, other amylase, preferably,
Amylases that act on a solution in which starch is liquefied to a relatively low DE to produce oligosaccharides mainly having a glucose polymerization degree of 3 or more, for example, α-amylase, maltotriose-producing amylase, maltotetraose-forming amylase, and maltopentaose. It is also advantageous to use a product amylase, a maltohexaose product amylase, a maltoheptaose product amylase, or the like.

【００２８】本発明で使用される澱粉は、とうもろこし
澱粉、米澱粉、小麦澱粉などの地上澱粉であっても、馬
鈴薯澱粉、甘藷澱粉、タピオカ澱粉などの地下澱粉であ
ってもよい。澱粉を液化するには、通常、澱粉を水に懸
濁した澱粉乳、望ましくは濃度１０％以上、更に望まし
くは約２０乃至５０％とし、これを加熱して機械的に液
化しても、酸又は酵素で液化してもよい。液化の程度
は、比較的低いものが適しており、望ましくはＤＥ１５
未満、更に望ましくはＤＥ１０未満のものが好適であ
る。酸で液化する場合には、例えば、塩酸、燐酸、蓚酸
などで液化し、その後、炭酸カルシウム、酸化カルシウ
ム、炭酸ナトリウムなどで必要ｐＨに中和して利用すれ
ばよい。酵素で液化する場合には、α−アミラーゼ、と
りわけ、耐熱性の液化型α−アミラーゼの使用が適して
いる。The starch used in the present invention may be ground starch such as corn starch, rice starch and wheat starch, or underground starch such as potato starch, sweet potato starch and tapioca starch. To liquefy starch, starch milk is usually suspended in water, and the concentration of starch milk is preferably 10% or more, more preferably about 20 to 50%. Alternatively, it may be liquefied with an enzyme. A relatively low degree of liquefaction is suitable, and DE15 is desirable.
Less than 10 and more preferably less than DE10 are suitable. In the case of liquefying with an acid, for example, it may be liquefied with hydrochloric acid, phosphoric acid, oxalic acid or the like, and then neutralized to a required pH with calcium carbonate, calcium oxide, sodium carbonate or the like before use. When liquefying with an enzyme, it is suitable to use α-amylase, especially a thermostable liquefied α-amylase.

【００２９】このようにして得られる澱粉を液化した溶
液に、非還元性糖質生成酵素を澱粉枝切酵素及び／又は
シクロマルトデキストリン・グルカノトランスフェラー
ゼとともに作用させるか、又は、非還元性糖質生成酵素
及びトレハロース遊離酵素を澱粉枝切酵素及び／又はシ
クロマルトデキストリン・グルカノトランスフェラーゼ
とともに作用させるには、これら酵素が作用しうるｐ
Ｈ、温度で行えばよく、通常、ｐＨ４乃至１０、好まし
くは、ｐＨ５乃至８、温度約１０乃至８０℃、好ましく
は、約３０乃至７０℃で行われる。また、澱粉を液化し
た溶液にこれら酵素を加える順序は問わず、いずれかの
酵素を先に加え、他の酵素をその後に加えて作用させる
ことも、また、これら酵素を同時に加えて作用させるこ
とも随意である。A non-reducing saccharide-forming enzyme is allowed to act on the liquefied starch solution thus obtained together with a starch debranching enzyme and / or cyclomaltodextrin glucanotransferase, or a non-reducing saccharide is formed. In order to allow the generative enzyme and the trehalose-releasing enzyme to act together with the starch debranching enzyme and / or cyclomaltodextrin / glucanotransferase, these enzymes may act.
It may be carried out at H and temperature, usually at pH 4 to 10, preferably at pH 5 to 8, and at a temperature of about 10 to 80 ° C, preferably about 30 to 70 ° C. The order of adding these enzymes to the liquefied starch solution does not matter, either enzyme may be added first and the other enzyme may be added afterwards, or these enzymes may be added simultaneously to act. Is also optional.

【００３０】酵素の使用量は、作用条件、反応時間によ
って適宜選べばよいが、通常、基質である澱粉を液化し
た溶液に対して、固形物グラム当たり、非還元性糖質生
成酵素及びトレハロース遊離酵素の場合、それぞれ約
０．０１乃至１００単位から選ばれ、また、澱粉枝切酵
素の場合、約１乃至１０，０００単位から選ばれ、シク
ロマルトデキストリン・グルカノトランスフェラーゼの
場合、約０．０５乃至５００単位から選ばれる。このよ
うにして得られる非還元性糖質とともに還元性澱粉糖を
含む低還元性糖質は、澱粉を液化した溶液に、澱粉枝切
酵素及び／又はシクロマルトデキストリン・グルカノト
ランスフェラーゼが非還元性糖質生成酵素又は非還元性
糖質生成酵素及びトレハロース遊離酵素とともに作用す
るため、比較的低分子のα−グリコシルトレハロース及
び／又はα−グリコシル α−グリコシドを多量に含有
するか、又はトレハロースを多量に含有する特長を有し
ており、本発明の原料用低還元性糖質として好適であ
る。なお、α−グリコシル α−グリコシドは、本出願
人が特願平６−５４３７７号明細書で開示したα−Ｄ−
オリゴグリコシル α−Ｄ−オリゴグルコシドを含む呼
称である。The amount of the enzyme to be used may be appropriately selected depending on the operating conditions and the reaction time. Usually, the amount of the non-reducing saccharide-forming enzyme and trehalose released per gram of the solid is relative to the solution in which the substrate starch is liquefied. In the case of the enzyme, each is selected from about 0.01 to 100 units, in the case of starch debranching enzyme, selected from about 1 to 10,000 units, and in the case of cyclomaltodextrin glucanotransferase, about 0.05. To 500 units. The low-reducing sugar containing the reducing starch sugar together with the non-reducing sugar thus obtained is a solution in which starch is liquefied and starch debranching enzyme and / or cyclomaltodextrin glucanotransferase is non-reducing. Since it acts together with a saccharide-forming enzyme or a non-reducing saccharide-forming enzyme and trehalose-releasing enzyme, it contains a large amount of a relatively low-molecular α-glycosyltrehalose and / or α-glycosyl α-glycoside, or a large amount of trehalose. It is suitable as a low-reducing sugar as a raw material of the present invention. The α-glycosyl α-glycoside is the α-D-glycone disclosed by the present applicant in Japanese Patent Application No. 6-54377.
It is a name including oligoglycosyl α-D-oligoglucoside.

【００３１】反応液は、常法により、濾過、遠心分離な
どして不溶物を除去した後、活性炭で脱色、Ｈ型、ＯＨ
型イオン交換樹脂で脱塩して精製し、濃縮し、シラップ
状製品とする。更に、乾燥して粉末状製品にすることも
随意である。必要ならば、更に、精製、例えば、イオン
交換カラムクロマトグラフィー、活性炭カラムクロマト
グラフィー、シリカゲルカラムクロマトグラフィーなど
のカラムクロマトグラフィーによる分画、アルコール及
びアセトンなど有機溶媒による分別、適度な分離性能を
有する膜による分離などの方法を１種又は２種以上組み
合わせて精製することにより、非還元性糖質含量を高め
た、本発明の原料用低還元性糖質を得ることも容易であ
る。The reaction solution is filtered, centrifuged, etc. to remove insoluble materials by a conventional method, and then decolorized with activated carbon, H type, OH
It is desalted with a type ion exchange resin, purified, and concentrated to give a syrup-like product. Further, it is optionally dried to give a powder product. If necessary, further purification, for example, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, silica gel column chromatography, fractionation by organic solvents such as alcohol and acetone, a membrane having appropriate separation performance. It is also easy to obtain the low-reducing sugar as a raw material of the present invention having an increased non-reducing sugar content by purifying by combining one or more methods such as separation by.

【００３２】とりわけ、工業的大量生産方法としては、
イオン交換カラムクロマトグラフィーの採用が好適であ
り、例えば、特開昭５８−２３７９９号公報、特開昭５
８−７２５９８号公報などに開示されている強酸性カチ
オン交換樹脂を用いるカラムクロマトグラフィーにより
夾雑糖類を除去し、非還元性糖質含量を高めた、原料用
低還元性糖質を有利に製造することができる。この際、
固定床方式、移動床方式、擬似移動床方式のいずれの方
式を採用することも随意である。In particular, as an industrial mass production method,
Ion exchange column chromatography is suitable for use, and examples thereof include JP-A-58-23799 and JP-A-SHO-5.
A column-chromatography using a strongly acidic cation exchange resin disclosed in Japanese Unexamined Patent Publication No. 8-72598 removes contaminating sugars, and advantageously produces a low-reducing sugar as a raw material with a high non-reducing sugar content. be able to. On this occasion,
It is optional to use any of the fixed bed method, the moving bed method, and the simulated moving bed method.

【００３３】このようにして得られた分子中にトレハロ
ース構造を有する非還元性糖質を含む低還元性糖質を、
必要により、アミラーゼ、例えば、α−アミラーゼ、β
−アミラーゼ、グルコアミラーゼなどや、又はα−グル
コシダーゼで分解し、甘味性を調整したり、粘性を低下
させたりする更なる加工処理を施して、原料用の低還元
性糖質を製造することも随意である。A low-reducing saccharide containing a non-reducing saccharide having a trehalose structure in the molecule thus obtained is
If necessary, an amylase such as α-amylase, β
-Amylase, glucoamylase, or the like, or α-glucosidase to decompose and adjust the sweetness, or to perform a further processing treatment to reduce the viscosity, it is also possible to produce a low reducing sugar as a raw material It is optional.

【００３４】このようにして得られる還元性を低減した
糖質は、本発明の原料糖質として有利に用いられる。原
料糖質としては、分子中にトレハロース構造を有する糖
質及びトレハロースからなる非還元性糖質含量が高いも
のが望ましく、通常、２０％以上、望ましくは４０％以
上、更に望ましくは６０％以上が好適であり、また、そ
のＤＥは低いものが望ましく、通常、ＤＥ７０未満、望
ましくは５０未満のもの、更に望ましくは３０未満のも
のが好適である。本発明の原料糖質は、糖組成によって
も変動するが、一般的には、多量の非還元性糖質を含有
し、甘味を有する程の比較的低分子、低粘度であるにも
かかわらず、そのＤＥが低く、本発明の水素添加工程、
その後の精製、濃縮などの製造工程を容易にし、しか
も、水素必要量を大幅に低減させる特長を有している。The thus-obtained reduced-sugar saccharide is advantageously used as the starting saccharide of the present invention. As the raw material sugar, a sugar having a trehalose structure in the molecule and a high content of non-reducing sugar composed of trehalose are desirable, and usually 20% or more, preferably 40% or more, more preferably 60% or more. A low DE is preferable, and a DE of less than 70 is preferable, a DE of less than 50 is more preferable, and a DE of less than 30 is more preferable. Although the raw material sugar of the present invention varies depending on the sugar composition, it generally contains a large amount of non-reducing sugar and has a relatively low molecular weight and low viscosity enough to have sweetness, , Its DE is low, the hydrogenation process of the present invention,
It has the features of facilitating subsequent manufacturing processes such as purification and concentration, and significantly reducing the required amount of hydrogen.

【００３５】このようにして得られる分子中にトレハロ
ース構造を有する糖質及び／又はトレハロースからなる
非還元性糖質とともに還元性澱粉糖を含む低還元性糖質
を水素添加するには、該非還元性糖質を分解することな
く、これに含まれる還元性澱粉糖が澱粉糖アルコールに
還元されればよく、例えば、原料の糖質を濃度３０乃至
７０％水溶液にし、オートクレーブに入れ、触媒として
ラネーニッケル約８乃至１０％を添加し、攪拌しながら
温度を９０乃至１５０℃に上げて水素添加を完了、望ま
しくは、ＤＥを０．５未満に低減させるまで水素添加を
行い、ラネーニッケルを除去し、次いで、常法に従っ
て、活性炭による脱色、イオン交換樹脂による脱塩など
の精製工程を経た後、濃縮し、シラップ状製品にする。
必要ならば、更に乾燥、粉末状製品にすることも、ま
た、トレハロースなどを晶出させた結晶性粉末状製品に
することも随意である。このようにして製造される本発
明の還元性を低減させた糖質は、α−グリコシルトレハ
ロース、α−グリコシル α−グリコシドなど分子中に
トレハロース構造を有する非還元性糖質及び／又はトレ
ハロースからなる非還元性糖質に加えて、ソルビトー
ル、マルチトール、マルトトリイトール、マルトテトラ
イトール及びマルトペンタイトールから選ばれる１種又
は２種以上の澱粉糖アルコールを含有している。In order to hydrogenate a sugar having a trehalose structure and / or a non-reducing sugar composed of trehalose in the molecule thus obtained, a low reducing sugar containing a reducing starch sugar is hydrogenated. It suffices that the reducing starch sugar contained therein is reduced to a starch sugar alcohol without decomposing the organic sugar, and for example, the raw sugar is made into an aqueous solution of 30 to 70% concentration, put into an autoclave, and Raney nickel is used as a catalyst. About 8-10% was added and the temperature was raised to 90-150 ° C. with stirring to complete the hydrogenation, preferably until the DE was reduced to less than 0.5 to remove Raney nickel, then According to a conventional method, after undergoing purification steps such as decolorization with activated carbon and desalting with an ion exchange resin, the product is concentrated to give a syrup-shaped product.
If necessary, it can be optionally further dried and made into a powdered product, or a crystalline powdered product obtained by crystallizing trehalose or the like. The thus-reduced reduced-sugar carbohydrate of the present invention comprises a non-reducing sugar and / or trehalose having a trehalose structure in its molecule, such as α-glycosyltrehalose, α-glycosyl α-glycoside. In addition to the non-reducing sugar, it contains one or more starch sugar alcohols selected from sorbitol, maltitol, maltotriitol, maltotetriitol, and maltopentitol.

【００３６】従って、本発明の還元性を低減させた糖質
は、還元性が極めて低く安定であり、他の素材、特にア
ミノ酸、オリゴペプチド、蛋白質などのアミノ酸を有す
る物質と混合、加工しても、褐変することも、異臭を発
生することもなく、混合した他の素材を損なうことも少
ない。また、還元力が低いにもかかわらず低粘度であ
り、平均グルコース重合度が低いものの場合には、良質
で上品な甘味を有している。Therefore, the sugar having reduced reducibility according to the present invention has extremely low reducibility and is stable, and is mixed and processed with other materials, especially substances having amino acids such as amino acids, oligopeptides and proteins. In addition, it does not brown, does not generate an offensive odor, and does not damage other mixed materials. In addition, it has a low viscosity even though it has a low reducing power, and when it has a low average degree of glucose polymerization, it has a good quality and elegant sweetness.

【００３７】また、本発明の還元性を低減させた糖質に
含まれる分子中にトレハロース構造を有する糖質は、ア
ミラーゼ、例えば、すい臓由来α−アミラーゼにより分
解し、低分子非還元性オリゴ糖や低分子マルトオリゴ糖
を生成し、また、これらオリゴ糖も、α−グルコシダー
ゼや小腸酵素でも容易に分解し、グルコース及びトレハ
ロースを生成し、更に、生成したトレハロースはトレハ
ラーゼにより容易にグルコースにまで分解することか
ら、経口摂取により、消化吸収され、カロリー源として
利用される。虫歯誘発菌などによって、醗酵されにく
く、虫歯を起こしにくい甘味料としても利用できる。Further, the carbohydrate having a trehalose structure in the molecule contained in the carbohydrate having reduced reducibility of the present invention is decomposed by amylase, for example, α-amylase derived from pancreas to produce a low molecular weight non-reducing oligosaccharide. And low-molecular maltooligosaccharides are produced, and these oligosaccharides are also easily degraded by α-glucosidase and intestinal enzyme to produce glucose and trehalose, and the produced trehalose is easily degraded to glucose by trehalase. Therefore, by ingestion, it is digested and absorbed and used as a calorie source. It can also be used as a sweetener that is unlikely to be fermented by caries-inducing bacteria and is unlikely to cause caries.

【００３８】また、安定な甘味料であることにより、結
晶高含有製品の場合には、プルラン、ヒドロキシエチル
スターチ、ポリビニルピロリドンなどの結合剤と併用し
て錠剤の糖衣剤として利用することも有利に実施でき
る。また、浸透圧調節性、賦形性、照り付与性、保湿
性、粘性、他の糖の晶出防止性、難醗酵性、糊化澱粉の
老化防止性などの性質を具備している。Further, since it is a stable sweetener, it can be advantageously used as a sugar-coating agent for tablets in combination with a binder such as pullulan, hydroxyethyl starch, or polyvinylpyrrolidone in the case of a product having a high crystal content. Can be implemented. Further, it has properties such as osmotic pressure controllability, shaping property, luster imparting property, moisturizing property, viscosity, crystallization preventing property of other sugars, fermentability, and aging preventing property of gelatinized starch.

【００３９】また、本発明の還元性を低減させた糖質
は、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤
などとして、飲食物、飼料、餌料、化粧品、医薬品など
の各種組成物に有利に利用できる。Further, the reduced reducing sugar of the present invention is used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc., as a food or drink, a feed, a feed, a cosmetic, a pharmaceutical, etc. Can be advantageously used for various compositions.

【００４０】本発明の還元性を低減させた糖質は、その
まま甘味付けのための調味料として使用することができ
る。必要ならば、例えば、粉飴、ブドウ糖、マルトー
ス、蔗糖、異性化糖、蜂蜜、メープルシュガー、イソマ
ルトオリゴ糖、ガラクトオリゴ糖、フラクトオリゴ糖、
ラクトスクロース、ソルビトール、マルチトール、ラク
チトール、ジヒドロカルコン、ステビオシド、α−グリ
コシルステビオシド、レバウディオシド、グリチルリチ
ン、Ｌ−アスパルチル−Ｌ−フェニルアラニンメチルエ
ステル、サッカリン、グリシン、アラニンなどのような
他の甘味料の１種又は２種以上の適量と混合して使用し
てもよく、また必要ならば、デキストリン、澱粉、乳糖
などのような増量剤と混合して使用することもできる。The reduced-sugar sugar of the present invention can be used as it is as a seasoning for sweetening. If necessary, for example, starch syrup, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomalt oligosaccharide, galactooligosaccharide, fructooligosaccharide,
One of the other sweeteners such as lactosucrose, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc. Alternatively, they may be used by mixing with an appropriate amount of two or more kinds, and if necessary, they may be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.

【００４１】また、本発明の還元性を低減させた糖質、
とりわけ、結晶状製品は、そのままで、又は必要に応じ
て、増量剤、賦形剤、結合剤などと混合して、顆粒、球
状、短棒状、板状、立方体、錠剤など各種形状に成型し
て使用することも随意である。Further, a sugar having reduced reducibility according to the present invention,
In particular, the crystalline product, as it is, or if necessary, mixed with a filler, an excipient, a binder, etc., and molded into various shapes such as granules, spheres, rods, plates, cubes, tablets, etc. It is also optional to use.

【００４２】また、本発明の還元性を低減させた糖質の
甘味は、酸味、塩から味、渋味、旨味、苦味などの他の
呈味を有する各種物質とよく調和し、耐酸性、耐熱性も
大きいので、一般の飲食物の甘味付け、呈味改良に、ま
た品質改良などに有利に利用できる。The sugar reducing sweetness of the present invention, which has reduced reducibility, is in good harmony with various substances having other tastes such as sourness, salt to taste, astringency, umami, bitterness, acid resistance, Since it has high heat resistance, it can be advantageously used for sweetening and improving the taste of general foods and drinks, and for improving the quality.

【００４３】例えば、アミノ酸、ペプチド類、醤油、粉
末醤油、味噌、粉末味噌、もろみ、ひしお、ふりかけ、
マヨネーズ、ドレッシング、食酢、三杯酢、粉末すし
酢、中華の素、天つゆ、麺つゆ、ソース、ケチャップ、
焼肉のタレ、カレールウ、シチューの素、スープの素、
ダシの素、核酸系調味料、複合調味料、みりん、新みり
ん、テーブルシュガー、コーヒーシュガーなど各種調味
料として有利に使用できる。For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle,
Mayonnaise, dressing, vinegar, three cups vinegar, powdered sushi vinegar, Chinese sauce, tempura soup, noodle soup, sauce, ketchup,
Sauce of roasted meat, curry roux, stew base, soup base,
It can be advantageously used as various seasonings such as dashi stock, nucleic acid-based seasonings, complex seasonings, mirin, new mirin, table sugar, and coffee sugar.

【００４４】また、例えば、せんべい、あられ、おこ
し、餅類、まんじゅう、ういろう、あん類、羊羮、水羊
羮、錦玉、ゼリー、カステラ、飴玉などの各種和菓子、
パン、ビスケット、クラッカー、クッキー、パイ、プリ
ン、バタークリーム、カスタードクリーム、シュークリ
ーム、ワッフル、スポンジケーキ、ドーナツ、チョコレ
ート、チューインガム、キャラメル、キャンディーなど
の洋菓子、アイスクリーム、シャーベットなどの氷菓、
果実のシロップ漬、氷蜜などのシロップ類、フラワーペ
ースト、ピーナッツペースト、フルーツペースト、スプ
レッドなどのペースト類、ジャム、マーマレード、シロ
ップ漬、糖果などの果実、野菜の加工食品類、福神漬、
べったら漬、千枚漬、らっきょう漬などの漬物類、たく
あん漬の素、白菜漬の素などの漬物の素類、ハム、ソー
セージなどの畜肉製品類、魚肉ハム、魚肉ソーセージ、
かまぼこ、ちくわ、天ぷらなどの魚肉製品、ウニ、イカ
の塩辛、酢こんぶ、さきするめ、ふぐみりん干しなどの
各種珍味類、のり、山菜、するめ、小魚、貝などで製造
されるつくだ煮類、煮豆、ポテトサラダ、こんぶ巻など
の惣菜食品、ヨーグルト、チーズなどの乳製品、魚肉、
畜肉、果実、野菜のビン詰、缶詰類、清酒、合成酒、リ
キュール、洋酒などの酒類、コーヒー、紅茶、ココア、
ジュース、炭酸飲料、乳酸飲料、乳酸菌飲料などの清涼
飲料水、プリンミックス、ホットケーキミックス、即席
しるこ、即席スープなどの即席食品、更には、離乳食、
治療食、ドリンク剤、ペプチド食品、冷凍食品などの各
種飲食物への甘味付けに、呈味改良に、また、品質改良
などに有利に利用できる。Further, for example, various Japanese sweets such as rice crackers, hail, rice cakes, rice cakes, steamed buns, uiro, bean paste, yokan, water yoyo, nishikidama, jelly, castella, hard candy, etc.
Bread, biscuits, crackers, cookies, pies, puddings, butter cream, custard cream, cream puffs, waffles, sponge cakes, donuts, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet and other frozen desserts,
Fruit syrup pickles, syrups such as ice honey, flower paste, peanut paste, fruit paste, pastes such as spreads, jam, marmalade, syrup pickles, fruits such as sugar fruit, processed foods of vegetables, Fukugami pickles,
Pickles such as Betara-zuke, Senmai-zuke, and Rakkyo-zuke, pickle-based pickles, pickled pickles such as Chinese cabbage pickles, livestock products such as ham and sausages, fish ham, fish sausages,
Fish products such as kamaboko, chikuwa, and tempura, sea urchin, salted squid, vinegared konbu, simmered sardines, and other delicacies such as dried sushi, fugu mirin, seaweed, sardines, small fish, and boiled beans made from shellfish. , Side dishes such as potato salad, kelp rolls, dairy products such as yogurt and cheese, fish meat,
Alcoholic beverages such as meat, fruits, vegetables bottled, canned goods, sake, synthetic liquor, liquor, coffee, tea, cocoa,
Soft drinks such as juices, carbonated drinks, lactic acid drinks, lactic acid bacteria drinks, pudding mixes, hot cake mixes, instant foods such as instant shiruko, instant soup, and even baby food,
It can be advantageously used for sweetening various foods and drinks such as therapeutic foods, drinks, peptide foods and frozen foods, for improving taste, and for improving quality.

【００４５】また、家畜、家禽、その他蜜蜂、蚕、魚な
どの飼育動物のために飼料、餌料などの嗜好性を向上さ
せる目的で使用することもできる。その他、タバコ、練
歯磨、口紅、リップクリーム、内服液、錠剤、トロー
チ、肝油ドロップ、口中清涼剤、口中香剤、うがい剤な
ど各種固形物、ペースト状、液状などで嗜好物、化粧
品、医薬品などの各種組成物への甘味剤として、又は呈
味改良剤、矯味剤として、さらには品質改良剤、安定剤
などとして有利に利用できる。It can also be used for the purpose of improving the palatability of feed, feed, etc. for domestic animals, poultry and other domestic animals such as bees, silkworms and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drops, mouthwash, mouthwash, mouthwash, various solid materials such as paste, liquid, etc., preference, cosmetics, pharmaceuticals, etc. Can be advantageously used as a sweetening agent for various compositions, or as a taste improving agent, a corrigent, a quality improving agent, a stabilizer, and the like.

【００４６】品質改良剤、安定剤としては、有効成分、
活性などを失い易い各種生理活性物質又はこれを含む健
康食品、医薬品などに有利に適用できる。例えば、イン
ターフェロン−α、インターフェロン−β、インターフ
ェロン−γ、ツモア・ネクロシス・ファクター−α、ツ
モア・ネクロシス・ファクター−β、マクロファージ遊
走阻止因子、コロニー刺激因子、トランスファーファク
ター、インターロイキンＩＩなどのリンホカイン、イン
シュリン、成長ホルモン、プロラクチン、エリトロポエ
チン、卵細胞刺激ホルモンなどのホルモン、ＢＣＧワク
チン、日本脳炎ワクチン、はしかワクチン、ポリオ生ワ
クチン、痘苗、破傷風トキソイド、ハブ抗毒素、ヒト免
疫グロブリンなどの生物製剤、ペニシリン、エリスロマ
イシン、クロラムフェニコール、テトラサイクリン、ス
プレプトマイシン、硫酸カナマイシンなどの抗生物質、
チアミン、リボフラビン、Ｌ−アスコルビン酸、肝油、
カロチノイド、エルゴステロール、トコフェロールなど
のビタミン、リパーゼ、エラスターゼ、ウロキナーゼ、
プロテアーゼ、β−アミラーゼ、イソアミラーゼ、グル
カナーゼ、ラクターゼなどの酵素、薬用人参エキス、ス
ッポンエキス、クロレラエキス、アロエエキス、プロポ
リスエキスなどのエキス類、ウイルス、乳酸菌、酵母な
どの生菌、ローヤルゼリーなどの各種生理活性物質も、
その有効成分、活性を失うことなく、安定で高品質の液
状、ペースト状又は固状の健康食品や医薬品などに容易
に製造できることとなる。As the quality improver and stabilizer, active ingredients,
It can be advantageously applied to various physiologically active substances which easily lose their activity or the like, health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, Tumor necrosis factor-α, Tumor necrosis factor-β, macrophage migration inhibitory factor, colony-stimulating factor, transfer factor, lymphokines such as interleukin II, and insulin. , Hormones such as growth hormone, prolactin, erythropoietin, egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, biologics such as human immunoglobulin, penicillin, erythromycin, Antibiotics such as chloramphenicol, tetracycline, sprepomycin, kanamycin sulfate,
Thiamine, riboflavin, L-ascorbic acid, liver oil,
Carotenoids, vitamins such as ergosterol, tocopherol, lipase, elastase, urokinase,
Enzymes such as protease, β-amylase, isoamylase, glucanase, lactase, ginseng extract, soft-shelled turtle extract, chlorella extract, aloe extract, propolis extract and other extracts, viruses, lactic acid bacteria, yeasts and other viable bacteria, royal jelly, etc. Physiologically active substances
The stable and high-quality liquid, paste or solid health food or pharmaceutical product can be easily produced without losing the active ingredient and activity.

【００４７】以上述べたような各種組成物に本発明の還
元性を低減させた糖質を含有せしめる方法は、その製品
が完成するまでの工程に含有せしめればよく、例えば、
混和、溶解、融解、浸漬、浸透、散布、塗布、被覆、噴
霧、注入、晶出、固化など公知の方法が適宜選ばれる。
その量は、通常０．１％以上、望ましくは１％以上含有
せしめるのが好適である。The method of incorporating the reduced-reducing sugar of the present invention into various compositions as described above may be incorporated in the steps until the product is completed.
Known methods such as mixing, dissolving, melting, dipping, permeating, scattering, coating, coating, spraying, pouring, crystallization and solidifying are appropriately selected.
The amount is usually 0.1% or more, preferably 1% or more.

【００４８】次に実験により本発明をさらに具体的に説
明する。Next, the present invention will be described more specifically by experiments.

【００４９】まず、新規微生物リゾビウム・スピーシー
ズ Ｍ−１１及びアルスロバクター・スピーシーズ Ｑ
３６からの非還元性糖質生成酵素について説明し、次い
で、公知微生物からの非還元性糖質生成酵素について説
明する。First, the novel microorganism Rhizobium species M-11 and Arthrobacter species Q
The non-reducing saccharide-forming enzyme from 36 will be described, and then the non-reducing saccharide-forming enzyme from a known microorganism will be described.

【００５０】[0050]

【実験１ リゾビウム・スピーシーズ Ｍ−１１からの
非還元性糖質生成酵素の生産】マルトース２．０ｗ／ｖ
％、ペプトン０．５ｗ／ｖ％、酵母エキス０．１ｗ／ｖ
％、リン酸二ナトリウム０．１ｗ／ｖ％、リン酸一カリ
ウム０．１ｗ／ｖ％及び水からなる液体培地をｐＨ７．
０に調整した。５００ｍｌ容三角フラスコにこの培地を
約１００ｍｌずつ入れ、オートクレーブで１２０℃で２
０分間滅菌し、冷却して、リゾビウム・スピーシーズ
Ｍ−１１（ＦＥＲＭ ＢＰ−４１３０）を接種し、２７
℃、１３０ｒｐｍで２４時間培養したものを種培養液と
した。[Experiment 1 Production of non-reducing saccharide-forming enzyme from Rhizobium species M-11] Maltose 2.0 w / v
%, Peptone 0.5 w / v%, yeast extract 0.1 w / v
%, Disodium phosphate 0.1 w / v%, monopotassium phosphate 0.1 w / v%, and water, pH 7.
Adjusted to 0. Approximately 100 ml each of this medium was placed in a 500 ml Erlenmeyer flask and autoclaved at 120 ° C for 2
Sterilize for 0 minutes, cool, and then Rhizobium species
Inoculated with M-11 (FERM BP-4130), 27
What was cultivated at 130 ° C. for 24 hours was used as a seed culture solution.

【００５１】容量３０ｌのファーメンターに種培養の場
合と同組成の培地約２０ｌを入れて滅菌、冷却して温度
３０℃とした後、種培養液１ｗ／ｖ％を接種し、温度３
０℃、ｐＨ６．０乃至８．０に保ちつつ、約２４時間通
気撹拌培養した。培養液の本酵素活性は約１．５単位／
ｍｌであった。培養液の一部を採り遠心分離して菌体と
培養液上清とに分離し、更に菌体を５０ｍＭリン酸緩衝
液（ｐＨ７．０）で元の培養液と同じ液量の懸濁液とし
た後、菌体懸濁液と培養液上清の酵素活性を測定したと
ころ、菌体懸濁液には約０．６単位／ｍｌの酵素活性
が、また、培養液上清には約０．９単位／ｍｌの酵素活
性が認められた。About 20 l of a medium having the same composition as in the case of seed culture was put into a fermenter having a volume of 30 l, sterilized and cooled to a temperature of 30 ° C., and then seed culture 1 w / v% was inoculated and the temperature was adjusted to 3
The culture was carried out with aeration and stirring for about 24 hours while maintaining the temperature at 0 ° C. and pH 6.0 to 8.0. The enzyme activity of the culture solution is about 1.5 units /
It was ml. A part of the culture solution is collected and centrifuged to separate the cells and the culture solution supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0) in the same volume as the original culture solution. Then, the enzyme activity of the bacterial cell suspension and the culture solution supernatant was measured, and it was found that the bacterial cell suspension had an enzyme activity of about 0.6 unit / ml, and the culture solution supernatant had an enzyme activity of about 0.6 unit / ml. An enzyme activity of 0.9 unit / ml was observed.

【００５２】[0052]

【実験２ 酵素の精製】実験１で得られた培養液約１８
ｌを、超高圧菌体破砕装置、大日本製薬株式会社製『ミ
ニラボ』で処理し、含まれる菌体を破砕した。処理液を
遠心分離（１０，０００ｒｐｍ、３０分間）することに
より、約１６ｌの上清を得た。その液に飽和度０．２に
なるように硫安を溶解させ、４℃、１時間放置した後、
遠心分離して上清を回収した。[Experiment 2 Purification of enzyme] About 18 culture solutions obtained in Experiment 1
1 was treated with an ultra-high pressure cell disruption device, "Minilab" manufactured by Dainippon Pharmaceutical Co., Ltd., to disrupt the cells contained therein. The treated solution was centrifuged (10,000 rpm, 30 minutes) to obtain about 16 l of supernatant. Ammonium sulfate was dissolved in the solution to a saturation degree of 0.2, left at 4 ° C. for 1 hour, and then
The supernatant was collected by centrifugation.

【００５３】更に、その液に飽和度０．６になるように
硫安を溶解させ、４℃、２４時間放置した後、遠心分離
して硫安塩析物を回収した。得られた硫安塩析物を１０
ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた後、同じ
緩衝液に対して２４時間透析し、遠心分離して不溶物を
除いた。その透析液（３６０ｍｌ）を２回に分けて、
『ＤＥＡＥ−トヨパール』を用いたイオン交換カラムク
ロマトグラフィー（ゲル量３００ｍｌ）を行った。Further, ammonium sulphate was dissolved in the solution so that the saturation was 0.6, and the mixture was allowed to stand at 4 ° C. for 24 hours and then centrifuged to recover the salted out ammonium sulphate. The obtained ammonium sulfate salted out product was mixed with 10
After being dissolved in mM phosphate buffer (pH 7.0), it was dialyzed against the same buffer for 24 hours and centrifuged to remove insoluble matter. Divide the dialysate (360 ml) into 2 parts,
Ion exchange column chromatography (gel amount 300 ml) using "DEAE-Toyopearl" was performed.

【００５４】本酵素は『ＤＥＡＥ−トヨパール』に吸着
し、食塩を含む同緩衝液でカラムから溶出した。得られ
る酵素活性画分を、２Ｍ硫安を含む同緩衝液に対して透
析し、その透析液を遠心分離して不溶物を除き、得られ
る上清を東ソー株式会社製『ブチルトヨパール ６５
０』を用いた疎水カラムクロマトグラフィー（ゲル量３
００ｍｌ）を行った。吸着した本酵素を硫安２Ｍから０
Ｍのリニアグラジエントによりカラムから溶出させ、酵
素活性画分を回収した。続いて、『トヨパールＨＷ−５
５』を用いたゲル濾過クロマトグラフィー（ゲル量３０
０ｍｌ）を行い、酵素活性画分を回収した。精製の各工
程における酵素活性量、比活性、収率を表１に示す。This enzyme was adsorbed on "DEAE-Toyopearl" and eluted from the column with the same buffer containing sodium chloride. The obtained enzyme-active fraction was dialyzed against the same buffer containing 2M ammonium sulfate, the dialysate was centrifuged to remove insoluble matter, and the resulting supernatant was collected by Tosoh Corporation “Butyl Toyopearl 65”.
0 ”hydrophobic column chromatography (gel amount 3
00 ml). Adsorbed this enzyme from ammonium sulfate 2M to 0
The column was eluted with a linear gradient of M to collect the enzyme active fraction. Then, "Toyopearl HW-5
5 ”gel filtration chromatography (gel amount 30
0 ml) was performed and the enzyme active fraction was collected. Table 1 shows the amount of enzyme activity, specific activity and yield in each purification step.

【００５５】[0055]

【表１】 [Table 1]

【００５６】表１の工程でゲル濾過溶出液として得られ
た精製酵素標品をポリアクリルアミドゲル（ゲル濃度
７．５％）を用いる電気泳動法で純度を検定したとこ
ろ、蛋白バンドは単一であることが示され、得られた酵
素標品は電気泳動的に単一な純度の高い標品であった。The purified enzyme preparation obtained as a gel filtration eluate in the step of Table 1 was subjected to an electrophoresis method using polyacrylamide gel (gel concentration 7.5%) to check its purity. It was shown to be present, and the obtained enzyme preparation was an electrophoretically single, highly pure preparation.

【００５７】[0057]

【実験３ 酵素の性質】実験２で得られた精製酵素標品
をＳＤＳ−ポリアクリルアミドゲル（ゲル濃度１０％）
を用いる電気泳動法に供し、同時に泳動した分子量マー
カー（日本バイオ・ラッド・ラボラトリーズ株式会社
製）と比較して本酵素の分子量を測定したところ、分子
量約７７，０００乃至８７，０００ダルトンであった。[Experiment 3 Enzyme properties] The purified enzyme preparation obtained in Experiment 2 was subjected to SDS-polyacrylamide gel (gel concentration 10%).
The molecular weight of this enzyme was measured by comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that was electrophoresed at the same time, and the molecular weight was about 77,000 to 87,000 daltons. .

【００５８】精製酵素標品を２％アンフォライン含有ポ
リアクリルアミドゲルを用いる等電点電気泳動法に供
し、泳動後、ゲルのｐＨを測定して本酵素の等電点を求
めたところ、等電点は約３．６乃至４．６であった。The purified enzyme preparation was subjected to an isoelectric focusing method using a polyacrylamide gel containing 2% ampholine, and after electrophoresis, the pH of the gel was measured to determine the isoelectric point of the enzyme. The point was about 3.6 to 4.6.

【００５９】本酵素活性に対する温度の影響、ｐＨの影
響は活性測定方法に準じて調べた。結果を図１（温度の
影響）、図２（ｐＨの影響）に示した。酵素の至適温度
は、ｐＨ７．０、６０分間反応で、４０℃付近、至適ｐ
Ｈは、４０℃、６０分間反応で、約７．０であった。本
酵素の温度安定性は、酵素溶液（５０ｍＭリン酸緩衝液
を含む、ｐＨ７．０）を各温度に６０分間保持し、水冷
した後、残存する酵素活性を測定することにより求め
た。また、ｐＨ安定性は、本酵素を各ｐＨの５０ｍＭ緩
衝液中で２５℃、１６時間保持した後、ｐＨを７に調整
し、残存する酵素活性を測定することにより求めた。そ
れぞれの結果を図３（温度安定性）、図４（ｐＨ安定
性）に示した。本酵素の温度安定性は４０℃付近まで安
定であり、ｐＨ安定性は約６乃至９であった。The influence of temperature and pH on the enzyme activity was examined according to the activity measuring method. The results are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme is around 40 ° C at a pH of 7.0 for 60 minutes.
H was about 7.0 after reacting at 40 ° C. for 60 minutes. The temperature stability of the present enzyme was determined by holding the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity. The pH stability was determined by maintaining the enzyme in a 50 mM buffer of each pH at 25 ° C. for 16 hours, adjusting the pH to 7, and measuring the remaining enzyme activity. The respective results are shown in FIG. 3 (temperature stability) and FIG. 4 (pH stability). The temperature stability of this enzyme was stable up to about 40 ° C., and the pH stability was about 6 to 9.

【００６０】[0060]

【実験４ 非還元性糖質の調製】基質として、グルコー
ス、マルトース、マルトトリオース、マルトテトラオー
ス、マルトペンタオース、マルトヘキサオース、又はマ
ルトヘプタオースの２０％水溶液を調製し、それぞれに
実験２で得られた精製酵素を基質固形物グラム当たり２
単位の割合で加え、４０℃、ｐＨ７．０で４８時間作用
させた後、脱塩し、和光純薬工業株式会社製『ワコービ
ーズ ＷＢ−Ｔ−３３０』を用いた高速液体クロマトグ
ラフィーで反応生成物を分析した。高速液体クロマトグ
ラフィーは、室温下で行い、溶離液として水を流速０．
５ｍｌ／分で流し、示差屈折計、東ソー株式会社製『Ｒ
Ｉ−８０１２』で分析した。その結果を表２に示す。[Experiment 4 Preparation of non-reducing sugar] As a substrate, 20% aqueous solution of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, or maltoheptaose was prepared, and Experiment 2 was performed for each. The purified enzyme obtained in step 2 was added per gram of substrate solids.
It is added at a unit ratio, and is allowed to act at 40 ° C. and pH 7.0 for 48 hours, desalted, and then subjected to reaction production by high performance liquid chromatography using “Wako beads WB-T-330” manufactured by Wako Pure Chemical Industries, Ltd. The thing was analyzed. The high performance liquid chromatography is performed at room temperature, and water is used as an eluent at a flow rate of 0.
Flow at 5 ml / min, differential refractometer, "R" manufactured by Tosoh Corporation
I-8012 ". The results are shown in Table 2.

【００６１】[0061]

【表２】 [Table 2]

【００６２】表２の結果から明らかなように、反応物中
には残存するそれぞれの基質と新たに生成したそれぞれ
の糖質ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ、ＰＶからな
り、それ以外の糖質はほとんど検出されない。それぞれ
の生成率はグルコース重合度３のＰＩが比較的低いもの
の、グルコース重合度４以上のＰＩＩ、ＰＩＩＩ、ＰＩ
Ｖ、ＰＶは８５％以上の高い生成率であることが判明し
た。なお、グルコース、マルトースからは、新たな糖質
を生成しないことが判明した。As is clear from the results shown in Table 2, the reaction product is composed of the respective remaining substrates and the newly formed sugars PI, PII, PIII, PIV and PV, and the other sugars are Hardly detected. Each of the production rates was relatively low in PI with glucose polymerization degree of 3, but PII, PIII, PI with glucose polymerization degree of 4 or more.
It was found that V and PV had a high production rate of 85% or more. It was found that glucose and maltose do not produce new sugars.

【００６３】それぞれの反応物から新たに生成した糖質
を精製するため、脱色、脱塩、濃縮後、ナトリウム型強
酸性カチオン交換樹脂、東京有機化学工業株式会社製
『ＸＴ−１０１６』（架橋度４％）を用いたカラム分画
を行った。樹脂を内径２．０ｃｍ、長さ１ｍのジャケッ
ト付ステンレス製カラム３本に充填し、直列につなぎ、
カラム内温度を５５℃に維持しつつ、反応糖液を樹脂に
対して５ｖ／ｖ％加え、これに５５℃の温水をＳＶ０．
１３で流して分画し、新たに生成した糖質含量９７％以
上の高純度画分を採取した。得られた高純度画分を真空
乾燥し、それぞれ高純度糖質標品を調製した。基質原料
に対する収率は、固形物換算で、それぞれＰＩで約９
％、ＰＩＩで約６５％、ＰＩＩＩで約８２％、ＰＩＶで
約８０％、ＰＶで約７７％であった。その純度は、それ
ぞれＰＩで９７．５％、ＰＩＩで９８．６％、ＰＩＩＩ
で９９．５％、ＰＩＶで９８．４％、ＰＶで９８．４％
であった。In order to purify newly produced sugars from each reaction product, after decolorization, desalting and concentration, sodium type strong acid cation exchange resin, "XT-1016" manufactured by Tokyo Organic Chemical Industry Co., Ltd. (degree of crosslinking) Column fractionation using 4%). Resin is packed into three stainless steel columns with a jacket having an inner diameter of 2.0 cm and a length of 1 m, connected in series,
While maintaining the temperature in the column at 55 ° C., 5 v / v% of the reaction sugar solution was added to the resin, and warm water at 55 ° C. was added to the SV0.
Fractionation was performed by pouring at 13, and a newly-produced high-purity fraction having a sugar content of 97% or more was collected. The obtained high-purity fractions were vacuum-dried to prepare high-purity saccharide samples. The yield based on the substrate material is about 9 in terms of solid matter and PI
%, PII was about 65%, PIII was about 82%, PIV was about 80%, and PV was about 77%. The purity is 97.5% for PI, 98.6% for PII, and PIII.
99.5%, PIV 98.4%, PV 98.4%
Met.

【００６４】またこれらの新たに生成した高純度糖質標
品の還元力をソモギー・ネルソン法で測定し、ＤＥで表
した。結果は表３にまとめた。The reducing power of these newly-produced high-purity carbohydrate preparations was measured by the Somogy Nelson method and expressed as DE. The results are summarized in Table 3.

【００６５】[0065]

【表３】 [Table 3]

【００６６】表３の結果から明らかなように、いずれの
標品にも僅かな還元力しか認めらなかった。その僅かな
還元力は、その標品中に微量に混入、残存している基質
由来の還元性マルトオリゴ糖に起因するものと推定さ
れ、新たに生成した糖質はいずれも実質的に非還元性で
あると判断される。As is clear from the results shown in Table 3, only a small reducing power was observed in all the preparations. It is presumed that the slight reducing power is due to the reducing maltooligosaccharide derived from the substrate that remains in the trace amount in the preparation, and that the newly produced sugar is substantially non-reducing. Is determined.

【００６７】[0067]

【実験５ メイラード反応】実験４において調製した糖
質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ、又はＰＶの
１０％とグリシン１％と、５０ｍＭリン酸緩衝液（ｐＨ
７．０）とを含む溶液を１００℃で９０分間保ち、冷却
後、この溶液の４８０ｎｍ、１ｃｍセルにおける吸光度
を測定した。対照として、それぞれの原料であるマルト
トリオース、マルトテトラオース、マルトペンタオー
ス、マルトヘキサオース、又はマルトヘプタオースを用
いて、同様に、処理し、４８０ｎｍにおける吸光度を測
定した。それらの結果を表４に示す。[Experiment 5 Maillard reaction] 10% of the carbohydrate preparation, PI, PII, PIII, PIV, or PV prepared in Experiment 4, 1% of glycine, and 50 mM phosphate buffer (pH
The solution containing 7.0) was kept at 100 ° C. for 90 minutes, and after cooling, the absorbance of this solution in 480 nm, 1 cm cell was measured. As a control, each raw material, maltotriose, maltotetraose, maltopentaose, maltohexaose, or maltoheptaose was similarly treated, and the absorbance at 480 nm was measured. The results are shown in Table 4.

【００６８】[0068]

【表４】 [Table 4]

【００６９】表４の結果から明らかなように、新たに生
成した非還元性糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、Ｐ
ＩＶ、ＰＶのいずれもメイラード反応による着色度は極
めて低く、それぞれ原料の基質であるマルトオリゴ糖の
着色度の僅かに３乃至６％程度であり、本発明の新規酵
素によって生成する非還元性糖質はメイラード反応をほ
とんど示さない糖質であることが判明した。As is clear from the results in Table 4, the newly produced non-reducing saccharide standard, PI, PII, PIII, P
Both IV and PV have a very low degree of coloring by the Maillard reaction, which is only about 3 to 6% of the degree of coloring of maltooligosaccharide, which is a substrate of the raw material, and is a non-reducing sugar produced by the novel enzyme of the present invention. Was found to be a carbohydrate that shows almost no Maillard reaction.

【００７０】[0070]

【実験６ グルコアミラーゼによる酵素分解】実験４に
おいて調製した非還元性糖質標品、ＰＩ、ＰＩＩ、ＰＩ
ＩＩ、ＰＩＶ又は、ＰＶのそれぞれ５０ｍｇを、５０ｍ
Ｍ酢酸緩衝液（ｐＨ４．５）１ｍｌに溶解し、１単位の
グルコアミラーゼ（生化学工業株式会社製）を加え、４
０℃で６時間保ち、酵素分解した後、高速液体クロマト
グラフィーで分解物を分析したところ、いずれの標品か
らも分解物としてグルコースとトレハロースのみが検出
された。検出されたグルコース含量、トレハロース含
量、その組成モル比の結果を表５に示す。[Experiment 6 Enzymatic Degradation by Glucoamylase] Non-reducing sugar preparations prepared in Experiment 4, PI, PII, PI
50 mg each of II, PIV or PV, 50 m
Dissolve in 1 ml of M acetate buffer (pH 4.5), add 1 unit of glucoamylase (Seikagaku Corporation), and add 4
After keeping at 0 ° C. for 6 hours and enzymatically decomposing, the decomposition products were analyzed by high performance liquid chromatography. As a result, only glucose and trehalose were detected as decomposition products from all the standards. Table 5 shows the results of the detected glucose content, trehalose content, and their composition molar ratio.

【００７１】[0071]

【表５】 [Table 5]

【００７２】表５の結果から明らかなように、グルコア
ミラーゼにより、非還元性糖質ＰＩはグルコース１分子
とトレハロース１分子に分解され、非還元性糖質ＰＩＩ
はグルコース２分子とトレハロース１分子に分解され、
非還元性糖質ＰＩＩＩはグルコース３分子とトレハロー
ス１分子に分解され、非還元性糖質ＰＩＶはグルコース
４分子とトレハロース１分子に分解され、非還元性糖質
ＰＶはグルコース５分子とトレハロース１分子に分解さ
れることが判明した。As is clear from the results shown in Table 5, the non-reducing sugar PI was decomposed into 1 molecule of glucose and 1 molecule of trehalose by glucoamylase, and the non-reducing sugar PII was decomposed.
Is decomposed into 2 molecules of glucose and 1 molecule of trehalose,
The non-reducing sugar PIII is decomposed into 3 glucose molecules and 1 trehalose molecule, the non-reducing sugar PIV is decomposed into 4 glucose molecules and 1 trehalose molecule, and the non-reducing sugar PV is 5 glucose molecules and 1 trehalose molecule. It turned out to be decomposed into.

【００７３】また、グルコアミラーゼの反応特性を考慮
すると、これら非還元性糖質の構造はトレハロース分子
にグルコース分子がα−１，４−結合、もしくはα−
１，６−結合で結合した糖質で、それぞれ、ＰＩはトレ
ハロース１分子にグルコース１分子が結合したグルコー
ス重合度３の非還元性糖質で、ＰＩＩはトレハロース１
分子にグルコース２分子が結合したグルコース重合度４
の非還元性糖質で、ＰＩＩＩはトレハロース１分子にグ
ルコース３分子が結合したグルコース重合度５の非還元
性糖質で、ＰＩＶはトレハロース１分子にグルコース４
分子が結合したグルコース重合度６の非還元性糖質で、
ＰＶはトレハロース１分子にグルコース５分子が結合し
たグルコース重合度７の非還元性糖質であると判断され
る。なお、同様に、非還元性糖質標品、ＰＩ、ＰＩＩ、
ＰＩＩＩ、ＰＩＶ、又はＰＶにβ−アミラーゼを作用さ
せたところ、非還元性糖質ＰＩ、ＰＩＩは分解されず、
ＰＩＩＩはマルトースの１分子とＰＩの１分子に分解さ
れ、ＰＩＶはマルトースの１分子とＰＩＩの１分子に分
解され、ＰＶはマルトースの２分子とＰＩの１分子に分
解されることが判明した。In consideration of the reaction characteristics of glucoamylase, the structures of these non-reducing sugars are such that the trehalose molecule has a glucose molecule α-1,4-bonded, or α-.
PI is a trehalose 1 which is a non-reducing saccharide having a glucose polymerization degree of 3 in which one molecule of glucose is bound to one molecule of trehalose, and PI is a saccharide bound by 1,6-bond.
Glucose degree of polymerization in which two glucose molecules are bound to a molecule 4
PIII is a non-reducing sugar of which the degree of glucose polymerization is 5 in which 3 molecules of glucose are bound to 1 molecule of trehalose and PIV is 4 molecules of glucose in 1 molecule of trehalose.
A non-reducing saccharide with a glucose degree of polymerization of 6 bound to a molecule,
PV is judged to be a non-reducing sugar having a glucose polymerization degree of 7 in which 5 molecules of glucose are bound to 1 molecule of trehalose. In addition, similarly, non-reducing sugar preparations, PI, PII,
When β-amylase was allowed to act on PIII, PIV, or PV, the non-reducing sugars PI, PII were not decomposed,
It was revealed that PIII was decomposed into one molecule of maltose and one molecule of PI, PIV was decomposed into one molecule of maltose and one molecule of PII, and PV was decomposed into two molecules of maltose and one molecule of PI.

【００７４】以上の結果から、本発明の非還元性糖質生
成酵素による反応は、基質の低分子化及び高分子化を伴
わない、換言すれば、グルコース重合度の変化を伴わな
い、分子内変換反応と判断され、また、この非還元性糖
質生成酵素によって生成した非還元性糖質、ＰＩ、ＰＩ
Ｉ、ＰＩＩＩ、ＰＩＶ及びＰＶは、それぞれ、α−グル
コシルトレハロース、α−マルトシルトレハロース、α
−マルトトリオシルトレハロース、α−マルトテトラオ
シルトレハロース及びα−マルトペンタオシルトレハロ
ースで示されるα−グリコシルトレハロース（Ｇn−
Ｔ：但し、 Ｇはグルコース残基を意味し、ｎは１以上
の整数を意味し、Ｔはα，α−トレハロースを意味す
る。）であると判断される。From the above results, the reaction by the non-reducing saccharide-forming enzyme of the present invention does not involve lowering of the molecular weight of the substrate and higher molecular weight of the substrate, in other words, does not involve the change of the degree of glucose polymerization, the intramolecular reaction. It is judged to be a conversion reaction, and non-reducing sugars, PI, PI produced by this non-reducing sugar-forming enzyme
I, PIII, PIV and PV are α-glucosyltrehalose, α-maltosyltrehalose and α, respectively.
-Α-glycosyltrehalose represented by maltotriosyltrehalose, α-maltotetraosyltrehalose and α-maltopentaosyltrehalose (G n −
T: However, G means a glucose residue, n means an integer of 1 or more, and T means α, α-trehalose. ) Is determined.

【００７５】[0075]

【実験７ 各種の酵素による分解】実験４において調製
した非還元性糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩ
Ｖ、又はＰＶのそれぞれを基質として、ブタすい臓由来
α−アミラーゼ（シグマ社販売）、コメ由来α−グルコ
シダーゼ（同社販売）、又はラット小腸アセトン粉末酵
素（同社販売）のそれぞれに作用させた後、分解物の糖
組成を高速液体クロマトグラフィーで分析した。α−ア
ミラーゼの反応は、それぞれの基質１０ｍｇを、５０ｍ
Ｍリン酸緩衝液（ｐＨ６．９）１ｍｌに溶解し、これ
に、酵素活性１単位加え、３７℃で１８時間保って行っ
た。α−グルコシダーゼの反応は、５０ｍＭ酢酸緩衝液
（ｐＨ４．０）を用いた以外、α−アミラーゼの場合と
同様の条件で行った。ラット小腸アセトン粉末酵素の場
合も、５０ｍＭマレイン酸緩衝液（ｐＨ６．０）を用い
た以外、α−アミラーゼの場合と同様の条件で行った。
α−アミラーゼによる分解物の糖組成を以下の表６に、
α−グルコシダーゼ及びラット小腸アセトン粉末酵素に
よる分解物の糖組成を以下の表７、表８に示す。[Experiment 7 Decomposition by various enzymes] Non-reducing sugar preparations prepared in Experiment 4, PI, PII, PIII, PI
After each of V or PV was used as a substrate, each of α-amylase derived from swine pancreas (sold by Sigma), α-glucosidase derived from rice (sold by the same company), or rat small intestinal acetone powder enzyme (sold by the same company) was acted on. The sugar composition of the decomposed product was analyzed by high performance liquid chromatography. The reaction of α-amylase was carried out by adding 10 mg of each substrate to 50 m
It was dissolved in 1 ml of M phosphate buffer (pH 6.9), 1 unit of enzyme activity was added thereto, and the mixture was kept at 37 ° C. for 18 hours. The reaction of α-glucosidase was performed under the same conditions as in the case of α-amylase, except that 50 mM acetate buffer (pH 4.0) was used. Also in the case of rat small intestine acetone powder enzyme, the same conditions as in the case of α-amylase were used except that 50 mM maleic acid buffer solution (pH 6.0) was used.
The sugar composition of the degradation product by α-amylase is shown in Table 6 below.
Tables 7 and 8 below show the sugar composition of the decomposition products of α-glucosidase and rat small intestine acetone powder enzyme.

【００７６】[0076]

【表６】 [Table 6]

【００７７】[0077]

【表７】 [Table 7]

【００７８】[0078]

【表８】 [Table 8]

【００７９】表６の結果から明らかなように、糖質標
品、ＰＩ及びＰＩＩは、α−アミラーゼによりほとんど
分解されないものの、糖質標品、ＰＩＩＩ、ＰＩＶ、及
びＰＶはα−アミラーゼにより低分子のオリゴ糖、Ｐ
Ｉ、ＰＩＩ、マルトトリオース、マルトース及びグルコ
ースにまで分解されることが判明した。As is clear from the results in Table 6, the sugar preparations, PI and PII, were hardly degraded by α-amylase, but the sugar preparations, PIII, PIV and PV were low molecular weight products by α-amylase. Oligosaccharides, P
It was found to be degraded to I, PII, maltotriose, maltose and glucose.

【００８０】また、表７、表８の結果から明らかなよう
に、糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ、ＰＶ
いずれもα−グルコシダーゼ及びラット小腸アセトン粉
末酵素により、実験６のグルコアミラーゼの場合と同様
に、グルコースとトレハロースにまで分解されることが
判明した。Further, as is clear from the results of Tables 7 and 8, the sugar preparation, PI, PII, PIII, PIV, PV
It was found that both of them were decomposed into α-glucosidase and rat intestinal acetone powder enzyme into glucose and trehalose as in the case of glucoamylase in Experiment 6.

【００８１】また、同様にα−グルコシダーゼ及びラッ
ト小腸アセトン粉末酵素によって分解されたそれぞれの
反応物に、更に、１単位のブタ腎臓由来トレハラーゼ
（シグマ社販売）を加え、ｐＨ５．７、３７℃で１８時
間作用させ、高速液体クロマトグラフィー法で糖組成を
分析したところ、糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、
ＰＩＶ、ＰＶいずれの場合も、α−グルコシダーゼ及び
ラット小腸アセトン粉末酵素により生成したトレハロー
スはトレハラーゼによりグルコースにまで分解すること
が判明した。Similarly, 1 unit of porcine kidney-derived trehalase (sold by Sigma) was added to each reaction product similarly decomposed by α-glucosidase and rat small intestine acetone powder enzyme, and the mixture was added at pH 5.7 and 37 ° C. It was allowed to act for 18 hours and the sugar composition was analyzed by a high performance liquid chromatography method. As a result, it was confirmed that the sugar preparation, PI, PII, PIII,
In both cases of PIV and PV, it was found that trehalose produced by α-glucosidase and rat intestinal acetone powder enzyme is decomposed into glucose by trehalase.

【００８２】上述のように、 （１） 非還元性糖質生成酵素は、グルコース重合度３
以上から選ばれる１種又は２種以上の還元性澱粉部分分
解物から、そのグルコース重合度を変化することなく、
α−グリコシルトレハロースを生成している。 （２） 非還元性糖質ＰＶは、α−アミラーゼにより、
主に非還元性糖質ＰＩＩとマルトトリオースを生じ、非
還元性糖質ＰＩＩは、グルコアミラーゼにより、トレハ
ロース１分子とグルコース２分子を生じている。 これらの結果から、本発明の非還元性糖質生成酵素は、
還元性澱粉部分分解物の還元性末端を非還元性のトレハ
ロース構造に分子内変換する全く新しい作用機作の酵素
であると判断される。As described above, (1) the non-reducing saccharide-forming enzyme has a glucose polymerization degree of 3
From one or two or more reducing starch partial decomposition products selected from the above, without changing the glucose polymerization degree,
Producing α-glycosyltrehalose. (2) The non-reducing sugar PV is converted by α-amylase into
The non-reducing sugar PII and maltotriose are mainly produced, and the non-reducing sugar PII produces one molecule of trehalose and two molecules of glucose by glucoamylase. From these results, the non-reducing saccharide-forming enzyme of the present invention is
It is considered to be an enzyme with a completely new mechanism of action that intramolecularly converts the reducing end of the partially decomposed product of reducing starch into a non-reducing trehalose structure.

【００８３】[0083]

【実験８ 急性毒性】７週齢のｄｄ系マウスを使用し
て、実験４において調製した非還元性糖質標品、ＰＩ、
ＰＩＩ、ＰＩＩＩ、ＰＩＶ、又はＰＶを経口投与して急
性毒性試験を行った。その結果、これら非還元性糖質は
いずれも低毒性の物質で、投与可能な最大投与量におい
ても死亡例は認められなかった。従って、正確な値とは
いえないが、それらのＬＤ50値は、いずれも５０ｇ／ｋ
ｇ以上であった。[Experiment 8 Acute toxicity] Using a 7-week-old dd mouse, the non-reducing sugar preparation prepared in Experiment 4, PI,
An acute toxicity test was conducted by oral administration of PII, PIII, PIV, or PV. As a result, these non-reducing sugars were all low-toxic substances, and no deaths were observed even at the maximum administrable dose. Therefore, although not accurate, their LD 50 values are all 50 g / k.
It was more than g.

【００８４】[0084]

【実験９ アルスロバクター・スピーシーズ Ｑ３６か
らの非還元性糖質生成酵素の生産】リゾビウム・スピー
シーズ Ｍ−１１（ＦＥＲＭ ＢＰ−４１３０）に代え
て、アルスロバクター・スピーシーズ Ｑ３６（ＦＥＲ
Ｍ ＢＰ−４３１６）を用いた以外は、実験１と同様に
ファーメンターで約７２時間培養した。培養液の非還元
性糖質生成酵素の酵素活性は、約１．２単位／ｍｌであ
った。実験１と同様にして菌体懸濁液と培養液上清の酵
素活性を測定したところ、それぞれ約０．５単位／ｍｌ
及び約０．７単位／ｍｌであった。[Experiment 9 Production of non-reducing saccharide-forming enzyme from Arthrobacter species Q36] Instead of Rhizobium species M-11 (FERM BP-4130), Arthrobacter species Q36 (FER
The culture was performed in a fermenter for about 72 hours in the same manner as in Experiment 1 except that MBP-4316) was used. The enzyme activity of the non-reducing saccharide-forming enzyme in the culture broth was about 1.2 units / ml. When the enzyme activity of the bacterial cell suspension and the culture supernatant was measured in the same manner as in Experiment 1, it was about 0.5 units / ml, respectively.
And about 0.7 units / ml.

【００８５】[0085]

【実験１０ 酵素の精製】実験９の方法で得られた培養
液約１８ｌを用いて、実験２と同様に精製した。精製の
各工程結果は表９にまとめた。[Experiment 10 Purification of enzyme] About 18 liters of the culture solution obtained by the method of Experiment 9 was used for purification in the same manner as in Experiment 2. The results of each purification step are summarized in Table 9.

【００８６】[0086]

【表９】 [Table 9]

【００８７】表９の工程で、ゲル濾過溶出液として得ら
れた精製酵素標品を、実験２の場合と同様に電気泳動法
で純度を検定したところ、蛋白バンドは単一であること
が示され、得られた酵素標品は電気泳動的に単一な純度
の高い標品であった。The purified enzyme preparation obtained as a gel filtration eluate in the step of Table 9 was subjected to an electrophoresis assay for purity in the same manner as in Experiment 2 to show that the protein band was single. The obtained enzyme preparation was an electrophoretically single, highly pure preparation.

【００８８】[0088]

【実験１１ 酵素の性質】実験１０で得られた精製酵素
標品を、実験３の場合と同様に、ＳＤＳ−ポリアクリル
アミドゲル電気泳動法で分子量を測定したところ、約７
６，０００乃至８６，０００ダルトンであった。また、
本精製酵素標品の等電点を実験３の場合と同様に等電点
電気泳動法で求めたところ、ｐＩ約３．６乃至４．６で
あった。また、本酵素活性に対する温度の影響、ｐＨの
影響、及び本酵素の温度安定性、ｐＨ安定性について、
実験３の場合と同様にして求めた。結果は、温度の影響
を図５に、ｐＨの影響を図６に、温度安定性を図７に、
ｐＨ安定性を図８に示した。[Experiment 11 Properties of enzyme] The purified enzyme preparation obtained in Experiment 10 was subjected to SDS-polyacrylamide gel electrophoresis to measure its molecular weight in the same manner as in Experiment 3.
It was between 6,000 and 86,000 daltons. Also,
The isoelectric point of this purified enzyme preparation was determined by the isoelectric focusing method as in Experiment 3, and the pI was about 3.6 to 4.6. In addition, regarding the influence of temperature, the influence of pH on the enzyme activity, and the temperature stability and pH stability of the enzyme,
It was determined in the same manner as in Experiment 3. The results are shown in FIG. 5, the temperature effect in FIG. 6, the temperature stability in FIG. 7, and the temperature stability in FIG.
The pH stability is shown in FIG.

【００８９】図から明らかなように酵素の至適温度は４
０℃付近、至適ｐＨは約６．５乃至７．０である。温度
安定性は４０℃付近までであり、ｐＨ安定性は約６．０
乃至９．５である。As is clear from the figure, the optimum temperature of the enzyme is 4
Near 0 ° C., the optimum pH is about 6.5 to 7.0. Temperature stability is up to around 40 ° C, pH stability is about 6.0
To 9.5.

【００９０】[0090]

【実験１２ 非還元性糖質の調製】実験１０で得られた
精製酵素標品を用いて、実験４及び実験６の方法に従っ
て、非還元性糖質の調製とその構造確認の実験を行った
ところ、リゾビウム・スピーシーズ Ｍ−１１由来の非
還元性糖質生成酵素の場合と同様に、グルコース重合度
３以上から選ばれる１種又は２種以上の還元性澱粉部分
分解物からα−グリコシルトレハロースを生成すること
が判明した。[Experiment 12: Preparation of non-reducing sugar] Using the purified enzyme preparation obtained in Experiment 10, an experiment for preparing a non-reducing sugar and confirming its structure were performed according to the methods of Experiments 4 and 6. However, as in the case of the non-reducing saccharide-forming enzyme derived from Rhizobium species M-11, α-glycosyl trehalose is obtained from one or more reducing starch partial degradation products selected from the glucose polymerization degree of 3 or more. It turned out to produce.

【００９１】[0091]

【実験１３ 公知微生物からの非還元性糖質生成酵素の
生産とその性質】公知微生物のうち、本発明の非還元性
糖質生成酵素産生能の確認された表１０に示す特定の微
生物を、マイコバクテリウム・スメグマチス（Ｍｙｃｏ
ｂａｃｔｅｒｉｕｍ ｓｍｅｇｍａｔｉｓ）ＡＴＣＣ１
９４２０の場合に３７℃で培養した以外は、実験１の場
合と同様にファーメンターで２７℃で７２時間培養し
た。それぞれの培養液約１８ｌを用いて、実験２の場合
と同様に、培養液を破砕装置にかけ、その上清を硫安塩
析、透析し、更にイオン交換カラムにかけ、部分精製酵
素標品を得、その性質を調べた。結果を表１０にまとめ
た。[Experiment 13 Production of non-reducing saccharide-forming enzyme from known microorganisms and its properties] Among known microorganisms, the specific microorganisms shown in Table 10 whose non-reducing saccharide-forming enzyme production ability of the present invention was confirmed were Mycobacterium smegmatis (Myco
Bacterium smegmatis) ATCC1
In the same manner as in Experiment 1, except that 9420 was cultured at 37 ° C., it was cultured at 27 ° C. for 72 hours in a fermenter. Using about 18 liters of each culture solution, the culture solution was applied to a crushing device in the same manner as in Experiment 2, the supernatant was subjected to ammonium sulfate salting out, dialyzed, and further applied to an ion exchange column to obtain a partially purified enzyme preparation. I investigated its nature. The results are summarized in Table 10.

【００９２】[0092]

【表１０】 [Table 10]

【００９３】また、これら公知菌由来の部分精製酵素を
用いて、実験１２の方法に従って、非還元性糖質の調製
とその構造確認を行ったところ、いずれの酵素もリゾビ
ウム・スピーシーズ Ｍ−１１由来の非還元性糖質生成
酵素の場合と同様に、グルコース重合度３以上から選ば
れる１種又は２種以上の還元性澱粉部分分解物からα−
グリコシルトレハロースを生成することが判明した。Further, non-reducing sugars were prepared and their structures were confirmed by using the partially purified enzymes derived from these known bacteria in accordance with the method of Experiment 12, and all of the enzymes were derived from Rhizobium species M-11. In the same manner as in the case of the non-reducing saccharide-forming enzyme, the α-
It was found to produce glycosyltrehalose.

【００９４】次に、新規微生物リゾビウム・スピーシー
ズ Ｍ−１１及びアルスロバクター・スピーシーズ Ｑ
３６からのトレハロース遊離酵素について説明し、次い
で、公知微生物からのトレハロース遊離酵素について説
明する。Next, the novel microorganism Rhizobium species M-11 and Arthrobacter species Q
The trehalose-releasing enzyme from 36 will be described, and then the trehalose-releasing enzyme from a known microorganism will be described.

【００９５】[0095]

【実験１４ リゾビウム・スピーシーズ Ｍ−１１から
のトレハロース遊離酵素の生産】澱粉部分分解物、松谷
化学工業株式会社製『パインデックス＃』４２．０ｗ／
ｖ％、ペプトン０．５ｗ／ｖ％、酵母エキス０．１ｗ／
ｖ％、リン酸二ナトリウム０．１ｗ／ｖ％、リン酸一カ
リウム０．１ｗ／ｖ％及び水からなる液体培地をｐＨ
７．０に調整した。５００ｍｌ容三角フラスコにこの培
地を約１００ｍｌずつ入れ、オートクレーブで１２０℃
で２０分間滅菌し、冷却して、リゾビウム・スピーシー
ズ Ｍ−１１（ＦＥＲＭ ＢＰ−４１３０）を接種し、
２７℃、１３０ｒｐｍで２４時間培養したものを種培養
液とした。[Experiment 14 Production of trehalose-releasing enzyme from Rhizobium species M-11] Partial degradation product of starch, "Paindex #" 42.0w / manufactured by Matsutani Chemical Industry Co., Ltd.
v%, peptone 0.5 w / v%, yeast extract 0.1 w /
pH of liquid medium consisting of v%, disodium phosphate 0.1 w / v%, monopotassium phosphate 0.1 w / v% and water
Adjusted to 7.0. Approximately 100 ml of this medium was placed in a 500 ml Erlenmeyer flask and autoclaved at 120 ° C.
Sterilize for 20 minutes, cool, inoculate with Rhizobium species M-11 (FERM BP-4130),
What was cultivated at 27 ° C. and 130 rpm for 24 hours was used as a seed culture solution.

【００９６】容量３０ｌのファーメンターに種培養の場
合と同組成の培地約２０ｌを入れて殺菌、冷却して温度
２７℃とした後、種培養液１ｗ／ｖを接種し、温度２７
℃、ｐＨは６．０乃至８．０に保ちつつ、約７２時間通
気撹拌培養した。About 20 liters of a medium having the same composition as in the case of seed culture was put into a fermenter having a volume of 30 liters, sterilized and cooled to a temperature of 27 ° C., and 1 w / v of a seed culture was inoculated at a temperature of 27
The culture was carried out with aeration and stirring for about 72 hours while maintaining the temperature at 60 ° C. and pH at 6.0 to 8.0.

【００９７】培養液の非還元性糖質生成酵素の酵素活性
は約１．５単位／ｍｌで、本発明のトレハロース遊離酵
素の酵素活性は約２単位／ｍｌであった。培養液の一部
を採り遠心分離して菌体と培養液上清とに分離し、更に
菌体を５０ｍＭリン酸緩衝液（ｐＨ７．０）で元の培養
液と同じ液量の懸濁液とした後、菌体懸濁液と培養上清
との酵素活性を測定したところ、菌体懸濁液には、非還
元性糖質生成酵素の酵素活性が約０．６単位／ｍｌ、ト
レハロース遊離酵素の酵素活性が約０．８単位／ｍｌ認
められ、培養上清には、非還元性糖質生成酵素の酵素活
性が約０．９単位／ｍｌ、トレハロース遊離酵素の酵素
活性が約１．２単位／ｍｌ認められた。The enzyme activity of the non-reducing saccharide-forming enzyme in the culture solution was about 1.5 units / ml, and the enzyme activity of the trehalose-releasing enzyme of the present invention was about 2 units / ml. A part of the culture solution is collected and centrifuged to separate the cells and the culture solution supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0) in the same volume as the original culture solution. After that, the enzyme activity of the bacterial cell suspension and the culture supernatant was measured, and it was found that the bacterial cell suspension had an enzyme activity of a non-reducing sugar-forming enzyme of about 0.6 unit / ml and trehalose. The enzyme activity of the free enzyme was found to be about 0.8 unit / ml, the enzyme activity of the non-reducing sugar-forming enzyme was about 0.9 unit / ml, and the enzyme activity of the trehalose free enzyme was about 1 in the culture supernatant. .2 units / ml was recognized.

【００９８】[0098]

【実験１５ 酵素の精製】実験１４の方法で得られた培
養液約１８ｌを超高圧菌体破砕装置『ミニラボ』で処理
し、含まれる菌体を破砕した。処理液を遠心分離（１
０，０００ｒｐｍ、３０分間）することにより、約１６
ｌの遠心上清液を得た。その液に飽和度０．２になるよ
うに硫安を加え溶解させ、４℃、１時間放置した後、遠
心分離（１０，０００ｒｐｍ、３０分間）することによ
り上清を回収した。[Experiment 15 Purification of Enzyme] About 18 liters of the culture solution obtained by the method of Experiment 14 was treated with an ultrahigh-pressure microbial cell disruptor "Minilabo" to disrupt the cells contained therein. Centrifuge the treatment solution (1
About 10,000 rpm for 30 minutes)
l of centrifugation supernatant was obtained. Ammonium sulfate was added to the solution to a saturation degree of 0.2 to dissolve the solution, and the solution was allowed to stand at 4 ° C. for 1 hour and then centrifuged (10,000 rpm, 30 minutes) to recover a supernatant.

【００９９】更に、その液に硫安を飽和度０．６になる
ように溶解させ、４℃、２４時間放置した後、遠心分離
して硫安塩析物を回収した。得られた硫安塩析物を１０
ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた後、同じ
緩衝液に対して２４時間透析し、遠心分離し、不溶物を
除いた。その透析液（３６０ｍｌ）を２回に分けて、
『ＤＥＡＥ−トヨパール』を用いたイオン交換カラムク
ロマトグラフィー（ゲル量３００ｍｌ）を行った。Further, ammonium sulphate was dissolved in the solution to a saturation degree of 0.6, allowed to stand at 4 ° C. for 24 hours, and then centrifuged to recover an ammonium sulphate salt-out product. The obtained ammonium sulfate salted out product was mixed with 10
After dissolved in mM phosphate buffer (pH 7.0), it was dialyzed against the same buffer for 24 hours and centrifuged to remove insoluble matter. Divide the dialysate (360 ml) into 2 parts,
Ion exchange column chromatography (gel amount 300 ml) using "DEAE-Toyopearl" was performed.

【０１００】本発明のトレハロース遊離酵素、非還元性
糖質生成酵素とも『ＤＥＡＥ−トヨパール』に吸着し、
食塩を含む同緩衝液でカラムから異なる食塩濃度におい
てそれぞれ溶出した。『ＤＥＡＥ−トヨパール』からの
溶出パターンを図９に示す。非還元性糖質生成酵素は食
塩濃度約０．２Ｍで、トレハロース遊離酵素は食塩濃度
約０．３Ｍで溶出し、それぞれの酵素活性画分を回収
し、以下、両酵素を別々に精製した。Both the trehalose-releasing enzyme and the non-reducing sugar-forming enzyme of the present invention were adsorbed on "DEAE-Toyopearl",
The same buffer containing salt was eluted from the column at different salt concentrations. The elution pattern from "DEAE-Toyopearl" is shown in FIG. The non-reducing saccharide-forming enzyme was eluted at a salt concentration of about 0.2 M, and the trehalose-releasing enzyme was eluted at a salt concentration of about 0.3 M. The respective enzyme active fractions were collected, and then both enzymes were separately purified.

【０１０１】非還元性糖質生成酵素活性画分を２Ｍ硫安
を含む同緩衝液に対して透析し、その透析液を遠心分離
し不溶物を除き、得られる上清を『ブチルトヨパール
６５０』を用いた疎水カラムクロマトグラフィー（ゲル
量３００ｍｌ）を行った。吸着した本酵素を２Ｍから０
Ｍ硫安のリニアグラジエントでカラムより溶出させ、酵
素活性画分を回収した。続いて、『トヨパール ＨＷ−
５５』を用いたゲル濾過クロマトグラフィー（ゲル量３
００ｍｌ）を行い、非還元性糖質生成酵素活性画分を回
収した。The non-reducing saccharide-forming enzyme active fraction was dialyzed against the same buffer containing 2M ammonium sulfate, the dialysate was centrifuged to remove insoluble matter, and the resulting supernatant was collected as "Butyl Toyopearl".
Hydrophobic column chromatography using "650" (gel amount 300 ml) was performed. Adsorbed this enzyme from 2M to 0
Elution was performed from the column with a linear gradient of M ammonium sulfate, and the enzyme active fraction was collected. Then, "Toyopearl HW-
55 ”gel filtration chromatography (gel amount 3
00 ml) was performed and the non-reducing saccharide-forming enzyme active fraction was collected.

【０１０２】トレハロース遊離酵素の精製は、『ＤＥＡ
Ｅ−トヨパール』から溶出したトレハロース遊離酵素活
性画分を用いて、上記の非還元性糖質生成酵素の精製方
法と同様に、２Ｍ硫安を含む緩衝液に対して透析し、次
いで疎水カラムクロマトグラフィー、ゲル瀘過クロマト
グラフィーを行った。Purification of trehalose-releasing enzyme was performed according to "DEA
The trehalose-releasing enzyme active fraction eluted from "E-Toyopearl" was dialyzed against a buffer containing 2M ammonium sulfate in the same manner as in the above-mentioned method for purifying a non-reducing sugar-forming enzyme, and then subjected to hydrophobic column chromatography. , Gel filtration chromatography was performed.

【０１０３】精製の各工程における酵素活性量、比活
性、収率を、非還元性糖質生成酵素の場合は表１１に、
本発明のトレハロース遊離酵素の場合は表１２に示す。The amount of enzyme activity, specific activity and yield in each purification step are shown in Table 11 in the case of non-reducing saccharide-forming enzyme.
Table 12 shows the case of the trehalose-releasing enzyme of the present invention.

【０１０４】[0104]

【表１１】 [Table 11]

【０１０５】[0105]

【表１２】 [Table 12]

【０１０６】表１１及び表１２の工程でそれぞれゲル瀘
過溶出液として得られた、精製非還元性糖質生成酵素標
品及び精製トレハロース遊離酵素標品をポリアクリルア
ミドゲル（ゲル濃度７．５％）を用いる電気泳動法で純
度を検定したところ、蛋白バンドは単一であることが示
され、得られた酵素標品は電気泳動的に単一な純度の高
い標品であった。Purified non-reducing saccharide-forming enzyme preparations and purified trehalose-releasing enzyme preparations obtained as gel filtration eluates in the steps of Table 11 and Table 12 were subjected to polyacrylamide gel (gel concentration 7.5%). ) Was used to analyze the purity, the protein band was found to be single, and the obtained enzyme preparation was electrophoretically single and highly pure.

【０１０７】[0107]

【実験１６ トレハロース遊離酵素の性質】実験１５の
方法で得られた精製トレハロース遊離酵素標品をＳＤＳ
−ポリアクリルアミドゲル（ゲル濃度１０％）を用いる
電気泳動法に供し、同時に泳動した分子量マーカー（日
本バイオ・ラッド・ラボラトリーズ株式会社製）と比較
して本酵素の分子量を測定したところ、分子量約５８，
０００乃至６８，０００ダルトンであった。[Experiment 16 Properties of trehalose-releasing enzyme] The purified trehalose-releasing enzyme preparation obtained by the method of Experiment 15 was subjected to SDS.
-It was subjected to an electrophoresis method using a polyacrylamide gel (gel concentration 10%), and the molecular weight of this enzyme was measured by comparison with a molecular weight marker (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.) that was electrophoresed at the same time. ，
It was between 000 and 68,000 daltons.

【０１０８】精製酵素標品をポリアクリルアミドゲルを
用いる等電点電気泳動法に供し、泳動後、ゲルのｐＨを
測定して本酵素の等電点を求めたところ、等電点は約
３．３乃至４．３であった。The purified enzyme preparation was subjected to an isoelectric focusing method using a polyacrylamide gel, and after the electrophoresis, the pH of the gel was measured to determine the isoelectric point of the present enzyme. The isoelectric point was about 3. It was 3 to 4.3.

【０１０９】本酵素活性に対する温度の影響、ｐＨの影
響を活性測定方法に準じて調べた。結果を図１０（温度
の影響）、図１１（ｐＨの影響）に示した。酵素の至適
温度は、ｐＨ７．０、３０分間反応で、４５℃付近、至
適ｐＨは、４０℃、３０分間反応で、約６．０乃至７．
５であった。本酵素の温度安定性は、酵素溶液（５０ｍ
Ｍリン酸緩衝液を含む、ｐＨ７．０）を各温度に６０分
間保持し、水冷した後、残存する酵素活性を測定するこ
とにより求めた。また、ｐＨ安定性は、本酵素を各ｐＨ
の５０ｍＭ緩衝液中で２５℃、１６時間保持した後、ｐ
Ｈを７に調整し、残存する酵素活性を測定することによ
り求めた。それぞれの結果を図１２（温度安定性）、図
１３（ｐＨ安定性）に示した。本酵素の熱安定性は約４
０℃付近までであり、ｐＨ安定性は約５乃至１０であっ
た。The effects of temperature and pH on the enzyme activity were examined according to the activity measuring method. The results are shown in FIG. 10 (effect of temperature) and FIG. 11 (effect of pH). The optimum temperature of the enzyme is about 45 ° C. at a reaction of pH 7.0 for 30 minutes, and the optimum pH is about 6.0 to 7.
It was 5. The temperature stability of this enzyme is the enzyme solution (50m
The pH was determined by measuring the remaining enzyme activity after maintaining M phosphate buffer (pH 7.0) at each temperature for 60 minutes and cooling with water. In addition, the pH stability of this enzyme is
After maintaining in 50 mM buffer solution at 25 ° C for 16 hours, p
It was determined by adjusting H to 7 and measuring the remaining enzyme activity. The respective results are shown in FIG. 12 (temperature stability) and FIG. 13 (pH stability). The thermostability of this enzyme is about 4
Up to around 0 ° C., pH stability was about 5-10.

【０１１０】[0110]

【実験１７ α−グリコシルトレハロースからのトレハ
ロースの調製】基質として用いるα−グリコシルトレハ
ロースは、実験４の方法に従って調製した。即ち、マル
トトリオース、マルトテトラオース、マルトペンタオー
ス、マルトヘキサオース及びマルトヘプタオースから選
ばれる還元性澱粉部分分解物の２０％水溶液に実験１５
の方法で得られた精製非還元性糖質生成酵素標品を基質
固形物グラム当りそれぞれ２単位の割合で加え、４０
℃、ｐＨ７．０で４８時間作用させた後、常法に従っ
て、加熱失活、瀘過、脱色、脱塩、濃縮し、ナトリウム
型強酸性カチオン交換樹脂『ＸＴ−１０１６』を用いた
イオン交換カラムクロマトグラフィーを行った。樹脂を
内径２．０ｃｍ、長さ１ｍのジャケット付ステンレス製
カラム３本に充填し、直列につなぎ、カラム内温度を５
５℃に維持しつつ、反応糖液を樹脂に対して５ｖ／ｖ％
加え、これに５５℃の温水をＳＶ０．１３で流して分画
し、末端にトレハロース構造を有するグルコース重合度
が３以上の非還元性糖質の高純度標品を調製した。得ら
れた高純度標品のうち、グルコシルトレハロース標品の
純度は９７．６％で、マルトシルトレハロース標品の純
度は９８．６％で、マルトトリオシルトレハロース標品
の純度は９９．６％で、マルトテトラオシルトレハロー
ス標品の純度は９８．３％で、マルトペンタオシルトレ
ハロース標品の純度は９８．１％であった。Experiment 17 Preparation of Trehalose from α-Glycosyltrehalose α-Glycosyltrehalose used as a substrate was prepared according to the method of Experiment 4. That is, an experiment was conducted using a 20% aqueous solution of a partially decomposed reducing starch selected from maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose.
The purified non-reducing saccharide-forming enzyme preparation obtained by the above method was added at a rate of 2 units per gram of substrate solids, and
After being allowed to act at ℃, pH 7.0 for 48 hours, it was deactivated by heating, filtered, decolorized, desalted and concentrated according to a conventional method, and an ion exchange column using sodium-type strongly acidic cation exchange resin "XT-1016" was used. Chromatography was performed. The resin was packed into three stainless steel columns with an inner diameter of 2.0 cm and a length of 1 m, which were connected to each other in series and the column internal temperature was adjusted to 5
While maintaining the temperature at 5 ° C, react sugar solution to resin at 5v / v%
In addition, hot water at 55 ° C. was flown with SV0.13 to fractionate this to prepare a high-purity preparation of a non-reducing sugar having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more. Among the obtained high-purity preparations, the purity of glucosyltrehalose preparation was 97.6%, the purity of maltosyltrehalose preparation was 98.6%, and the purity of maltotriosyltrehalose preparation was 99.6%. The purity of the maltotetraosyltrehalose preparation was 98.3%, and the purity of the maltopentaosyltrehalose preparation was 98.1%.

【０１１１】上記５種の非還元性糖質（α−グリコシル
トレハロース）の２０％水溶液を調製し、それぞれに実
験１５で得られた精製トレハロース遊離酵素を基質固形
物グラム当り２単位の割合で加え、４０℃、ｐＨ７．０
で４８時間作用させた後、脱塩し、『ワコービーズ Ｗ
Ｂ−Ｔ−３３０』を用いた高速液体クロマトグラフィー
で反応生成物を分析した。対照として、マルトトリオー
ス、マルトテトラオース、マルトペンタオース、マルト
ヘキサオース、マルトヘプタオースに精製トレハロース
遊離酵素を同様に作用させ、高速液体クロマトグラフィ
ーで分析した。それらの結果を表１３に示す。A 20% aqueous solution of the above-mentioned five non-reducing sugars (α-glycosyltrehalose) was prepared, and the purified trehalose-releasing enzyme obtained in Experiment 15 was added to each in a ratio of 2 units per gram of the substrate solid. , 40 ° C, pH 7.0
After 48 hours of working at room temperature, desalting is performed and "Wako beads W
The reaction product was analyzed by high performance liquid chromatography using "BT-330". As a control, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were similarly treated with purified trehalose-releasing enzyme and analyzed by high performance liquid chromatography. The results are shown in Table 13.

【０１１２】[0112]

【表１３】 [Table 13]

【０１１３】表１３の結果から明らかなように、 （１） トレハロース遊離酵素は、α−グリコシルトレ
ハロースのトレハロース部分とグリコシル部分との間の
結合を特異的に加水分解し、トレハロースとグルコース
重合度が１以上の還元性糖質とを生成する。 （２） マルトオリゴ糖は、トレハロース遊離酵素によ
って全く作用を受けない。 これらの結果から、本発明のトレハロース遊離酵素は、
α−グリコシルトレハロースのトレハロース部分とその
他のグリコシル部分との間の結合を極めて特異的に加水
分解し、トレハロースを遊離する全く新しい作用機構の
酵素であると判断される。As is clear from the results in Table 13, (1) trehalose-releasing enzyme specifically hydrolyzes the bond between the trehalose portion and the glycosyl portion of α-glycosyltrehalose, and the degree of trehalose-glucose polymerization is increased. It produces one or more reducing sugars. (2) Maltooligosaccharides are not affected by the trehalose-releasing enzyme at all. From these results, the trehalose-releasing enzyme of the present invention is
It is considered to be an enzyme having a completely new mechanism of action, which hydrolyzes the bond between the trehalose portion of α-glycosyltrehalose and the other glycosyl portion in a very specific manner to release trehalose.

【０１１４】次いで、それぞれの反応物からトレハロー
スを精製するため、脱色、脱塩、濃縮し、ナトリウム型
強酸性カチオン交換樹脂『ＸＴ−１０１６』を用いたカ
ラム分画を行い、トレハロース含量９７％以上の高純度
画分を採取した。得られた高純度画分を濃縮して濃度約
６５％にし、２５℃で２日間放置して含水トレハロース
結晶を晶出させ、分蜜し、真空乾燥して、トレハロース
含量９９％以上の高純度標品を調製した。原料基質に対
するそれぞれの収率は、固形物換算で、グルコシルトレ
ハロースから９．５％、マルトシルトレハロースから１
４．９％、マルトトリオシルトレハロースから１６．０
％、マルトテトラオシルトレハロースから１８．５％、
マルトペンタオシルトレハロースから１７．７％であっ
た。得られたそれぞれの高純度トレハロース標品用い
て、市販の試薬トレハロース（和光純薬工業株式会社販
売）を標準品として、融点、融解熱、比旋光度、赤外線
吸収スペクトル、粉末Ｘ線回折パターン及びブタ腎臓由
来トレハラーゼでの分解性について比較したところ、調
製したすべての高純度トレハロース標品は、融点９７．
０±０．５℃、融解熱５７．８±１．２ＫＪ／ｍｏｌ
ｅ、比旋光度＋１８２±１．１°で、試薬トレハロース
の実測値とよく一致し、また、赤外線吸収スペクトル及
び粉末Ｘ線回折パターンについても、試薬トレハロース
のスペクトル又はパターンとよく一致した。更に、ブタ
腎臓由来トレハラーゼによって、高純度トレハロース標
品は試薬トレハロースと同様にグルコースに分解され
た。以上の結果から明らかなように、α−グリコシルト
レハロースに本発明のトレハロース遊離酵素を作用させ
生成した糖質はトレハロースであると確認された。Next, in order to purify trehalose from each reaction product, decolorization, desalting and concentration were performed, and column fractionation was performed using a sodium-type strongly acidic cation exchange resin "XT-1016", and the trehalose content was 97% or more. A high-purity fraction of was collected. The obtained high-purity fraction was concentrated to a concentration of about 65%, and allowed to stand at 25 ° C. for 2 days to crystallize hydrous trehalose crystals, smashed and vacuum-dried to obtain a high-purity trehalose content of 99% or more. A preparation was prepared. The respective yields based on the raw material substrate were 9.5% from glucosyltrehalose and 1 from maltosyltrehalose in terms of solid matter.
4.9%, 16.0 from maltotriosyltrehalose
%, 18.5% from maltotetraosyltrehalose,
It was 17.7% from maltopentaosyl trehalose. Using each of the obtained high-purity trehalose preparations, using a commercially available reagent trehalose (sold by Wako Pure Chemical Industries, Ltd.) as a standard product, melting point, heat of fusion, specific rotation, infrared absorption spectrum, powder X-ray diffraction pattern and When the degradability with trehalase derived from pig kidney was compared, all the prepared high-purity trehalose preparations had a melting point of 97.
0 ± 0.5 ° C., heat of fusion 57.8 ± 1.2 KJ / mol
e, the specific rotation was + 182 ± 1.1 °, which was in good agreement with the measured value of the reagent trehalose, and the infrared absorption spectrum and the powder X-ray diffraction pattern were also in good agreement with the spectrum or pattern of the reagent trehalose. Furthermore, the trehalase derived from pig kidney decomposed the high-purity trehalose preparation into glucose similarly to the reagent trehalose. As is clear from the above results, it was confirmed that the saccharide produced by reacting α-glycosyltrehalose with the trehalose-releasing enzyme of the present invention was trehalose.

【０１１５】[0115]

【実験１８ 還元性澱粉部分分解物からのトレハロース
の調製】５％ワキシーコーンスターチ懸濁液を加熱糊化
させた後、ｐＨ４．５、温度５０℃に調整し、これにイ
ソアミラーゼ（株式会社林原生物化学研究所製）を澱粉
グラム当り４，０００単位の割合になるように加え、２
０時間反応させた。その反応液をオートクレーブ（１２
０℃、１０分間）し、次いで６０℃に冷却し、これを東
ソー株式会社製『トヨパール ＨＷ−５０Ｓ』を用いた
ゲル瀘過クロマトグラフィー（ゲル量７５０ｍｌ）でグ
ルコース重合度３５乃至１０の還元性澱粉部分分解物を
調製した。[Experiment 18 Preparation of Trehalose from Partially Degraded Reducing Starch] After gelatinizing a suspension of 5% waxy cornstarch by heating, the pH was adjusted to 4.5 and the temperature was adjusted to 50 ° C. Chemical Laboratory) at a rate of 4,000 units per gram starch and add 2
The reaction was allowed for 0 hours. The reaction solution was autoclaved (12
(0 ° C., 10 minutes), then cooled to 60 ° C., and subjected to gel filtration chromatography (gel amount 750 ml) using “Toyopearl HW-50S” manufactured by Tosoh Corporation to reduce glucose polymerization degree 35 to 10 A partially degraded starch was prepared.

【０１１６】得られた還元性澱粉部分分解物、又はグル
コース重合度３のマルトトリオースを、１０ｍＭリン酸
緩衝液（ｐＨ７．０）で１％濃度に調整し、これに実験
１５の方法で調製した精製非還元性糖質生成酵素標品及
び精製トレハロース遊離酵素標品をそれぞれ基質固形物
当り４単位の割合で加え、４０℃で２４時間作用させた
後、一部を採り、脱塩し、高速液体クロマトグラフィー
で反応生成物を分析した。The obtained partially decomposed product of reducing starch or maltotriose having a glucose polymerization degree of 3 was adjusted to a concentration of 1% with 10 mM phosphate buffer (pH 7.0) and prepared by the method of Experiment 15. The purified non-reducing saccharide-forming enzyme preparation and the purified trehalose-releasing enzyme preparation were added at a ratio of 4 units per substrate solid, and allowed to act at 40 ° C. for 24 hours, and then aliquoted and desalted, The reaction products were analyzed by high performance liquid chromatography.

【０１１７】残りの反応液は、更に、５０℃、ｐＨ４．
５に調整した後、グルコアミラーゼ（生化学工業株式会
社製）を基質固形物当り５０単位の割合で加え、２４時
間作用させ、同様に脱塩し、高速液体クロマトグラフィ
ーで反応生成物を分析した。それらの結果を表１４に示
す。The remaining reaction solution was further heated at 50 ° C. and pH 4.
After adjusting to 5, glucoamylase (manufactured by Seikagaku Corporation) was added at a ratio of 50 units per substrate solid, allowed to act for 24 hours, similarly desalted, and the reaction product was analyzed by high performance liquid chromatography. . The results are shown in Table 14.

【０１１８】[0118]

【表１４】 [Table 14]

【０１１９】表１４に示すように、非還元性糖質生成酵
素及びトレハロース遊離酵素を作用させた後のトレハロ
ース生成率は、グルコース重合度３のマルトトリオース
では４．２％と低い値であったが、グルコース重合度１
０乃至３４．１の澱粉部分分解物では６６．１乃至８
０．８％の高い値が得られた。また、原料の還元性澱粉
部分分解物のグルコース重合度が高い程、得られるトレ
ハロース純度が高いことも判明した。更に、該両酵素を
作用させた反応液にグルコアミラーゼを作用させ、残存
する末端にトレハロース構造を有するグルコース重合度
が３以上の非還元性糖質をトレハロースとグルコースと
に分解することにより、生成するトレハロース純度がよ
り高まることも判明した。As shown in Table 14, the trehalose production rate after the action of the non-reducing saccharide-forming enzyme and the trehalose-releasing enzyme was 4.2% in maltotriose having a glucose polymerization degree of 3, which was a low value. But glucose degree of polymerization 1
66.1 to 8 for partially degraded starch from 0 to 34.1
A high value of 0.8% was obtained. It was also found that the higher the degree of glucose polymerization of the reduced starch partially decomposed material, the higher the trehalose purity obtained. Further, glucoamylase is allowed to act on the reaction solution which has been acted on by both enzymes, and a non-reducing sugar having a trehalose structure at the remaining terminal and having a glucose polymerization degree of 3 or more is decomposed into trehalose and glucose. It was also found that the purity of trehalose is higher.

【０１２０】[0120]

【実験１９ メイラード反応】実験１７の方法で得られ
た高純度トレハロース標品（純度９９．５％）の１０％
とグリシン１％と、５０ｍＭリン酸緩衝液（ｐＨ７．
０）とを含む溶液を１００℃で９０分間保ち、冷却後、
この溶液の４８０ｎｍ、１ｃｍセルにおける吸光度を測
定した。対照として、グルコース、マルトースを用い
て、同様に処理し、４８０ｎｍにおける吸光度を測定し
た。結果を表１５に示す。[Experiment 19 Maillard reaction] 10% of the high-purity trehalose preparation (purity 99.5%) obtained by the method of Experiment 17
And glycine 1% and 50 mM phosphate buffer (pH 7.
0) and containing the solution at 100 ℃ for 90 minutes, after cooling,
The absorbance of this solution in 480 nm and 1 cm cell was measured. As a control, glucose and maltose were treated in the same manner, and the absorbance at 480 nm was measured. The results are shown in Table 15.

【０１２１】[0121]

【表１５】 [Table 15]

【０１２２】表１５の結果から明らかなように、トレハ
ロース標品は、メイラード反応による着色度は僅かであ
り、グルコースやマルトースの着色度の僅か０．４乃至
０．６％程度であり、本発明のトレハロース標品はメイ
ラード反応をほとんど示さない糖質であることが判明し
た。従って、本糖質は、アミノ酸と混合しても、アミノ
酸を損なうことが少ない糖質である。As is clear from the results shown in Table 15, the trehalose preparation had a slight degree of coloring due to the Maillard reaction, which was only 0.4 to 0.6% of the degree of coloring of glucose or maltose. It was found that the trehalose preparation of No. 1 was a sugar that showed almost no Maillard reaction. Therefore, the present carbohydrate is a carbohydrate that is less likely to damage the amino acid when mixed with the amino acid.

【０１２３】[0123]

【実験２０ 生体内での利用試験】厚治等が、『臨床栄
養』、第４１巻、第２号、第２００乃至２０８頁（１９
７２年）で報告している方法に準じて、実験１７の方法
で得られた高純度トレハロース標品（純度９９．５％）
３０ｇを２０ｗ／ｖ％水溶液とし、これをボランティア
６名（健康な２６才、２７才、２８才、２９才、３０
才、３１才の男性）にそれぞれ経口投与し、経時的に採
血して、血糖値及びインスリン値を測定した。対照とし
ては、グルコースを用いた。その結果、トレハロース
は、グルコースの場合と同様の挙動を示し、血糖値、イ
ンスリン値ともに、投与後、約０．５乃至１時間で最大
値を示した。トレハロースは、容易に消化吸収、代謝利
用されて、エネルギー源になることが判明した。[Experiment 20 In-vivo utilization test] Kouji et al., "Clinical Nutrition", Vol. 41, No. 2, pp. 200-208 (19
1972), a high-purity trehalose preparation obtained by the method of Experiment 17 (purity 99.5%) according to the method reported in (1972).
30 g of 20 w / v% aqueous solution was prepared by 6 volunteers (healthy 26 years old, 27 years old, 28 years old, 29 years old, 30 years old).
Aged 31 and 31 years old) and blood samples were taken over time to measure blood glucose level and insulin level. Glucose was used as a control. As a result, trehalose behaved similarly to the case of glucose, and both the blood glucose level and the insulin level showed maximum values about 0.5 to 1 hour after administration. It was found that trehalose can be easily digested, absorbed, and metabolized and used as an energy source.

【０１２４】[0124]

【実験２１ 急性毒性試験】マウスを使用して、実施例
Ａ−５、Ａ−７およびＡ−８の方法で得られた還元性を
低減させた糖質粉末を経口投与して急性毒性試験を行っ
た。その結果、いずれの標品も低毒性の物質で、投与可
能な最大投与量においても死亡例は認められなかった。
従って、正確な値とはいえないが、それらのＬＤ５０値
は、５０ｇ／ｋｇ以上であった。[Experiment 21 Acute toxicity test] An acute toxicity test was carried out by orally administering the sugar powder with reduced reductivity obtained by the method of Examples A-5, A-7 and A-8 using a mouse. went. As a result, all the preparations had low toxicity, and no deaths were observed even at the maximum dose that could be administered.
Therefore, their LD50 values were 50 g / kg or more, though not accurate.

【０１２５】[0125]

【実験２２ アルスロバクター・スピーシーズ Ｑ３６
からのトレハロース遊離酵素の生産】リゾビウム・スピ
ーシーズ Ｍ−１１（ＦＥＲＭ ＢＰ−４１３０）に代
えて、アルスロバクター・スピーシーズ Ｑ３６（ＦＥ
ＲＭ ＢＰ−４３１６）を用いた以外は、実験１４と同
様に、ファーメンターで約７２時間培養した。培養液の
非還元性糖質生成酵素の酵素活性は約１．３単位／ｍｌ
で、本発明のトレハロース遊離酵素の酵素活性は約１．
８単位／ｍｌであった。実験１４と同様にして菌体懸濁
液と培養上清との酵素活性を測定したところ、菌体懸濁
液には、非還元性糖質生成酵素の酵素活性が約０．５単
位／ｍｌ、トレハロース遊離酵素の酵素活性が約０．５
単位／ｍｌ認められ、培養上清には、非還元性糖質生成
酵素の酵素活性が約０．８単位／ｍｌ、トレハロース遊
離酵素の酵素活性が約１．３単位／ｍｌ認められた。[Experiment 22 Arthrobacter Species Q36
Production of trehalose-releasing enzyme from Rhizobium species M-11 (FERM BP-4130) instead of Arthrobacter species Q36 (FE
In the same manner as in Experiment 14 except that RM BP-4316) was used, the culture was performed for 72 hours in a fermenter. The enzyme activity of the non-reducing saccharide-forming enzyme in the culture solution is about 1.3 units / ml.
The enzyme activity of the trehalose-releasing enzyme of the present invention is about 1.
It was 8 units / ml. When the enzyme activity of the bacterial cell suspension and the culture supernatant was measured in the same manner as in Experiment 14, the enzymatic activity of the non-reducing sugar-forming enzyme was found to be about 0.5 unit / ml in the bacterial cell suspension. , The enzyme activity of trehalose-releasing enzyme is about 0.5
The enzyme activity of the non-reducing saccharide-forming enzyme was about 0.8 unit / ml, and the enzyme activity of the trehalose-releasing enzyme was about 1.3 unit / ml.

【０１２６】[0126]

【実験２３ 酵素の精製】実験２２の方法で得られた培
養液約１８ｌを用いて、実験１５と同様の方法で精製し
た。精製の各工程結果は非還元性糖質生成酵素の場合は
表１６に、トレハロース遊離酵素の場合は表１７にまと
めた。[Experiment 23 Purification of enzyme] About 18 liters of the culture solution obtained by the method of Experiment 22 was used for purification in the same manner as in Experiment 15. The results of each purification step are summarized in Table 16 for the non-reducing saccharide-forming enzyme and Table 17 for the trehalose-releasing enzyme.

【０１２７】[0127]

【表１６】 [Table 16]

【０１２８】[0128]

【表１７】 [Table 17]

【０１２９】表１６及び表１７の工程で、それぞれゲル
瀘過溶出液として得られた精製非還元性糖質生成酵素及
び精製トレハロース遊離酵素を、実験１５の場合と同様
に電気泳動法で純度を検定したところ、蛋白バンドは単
一であることが示され、得られた両精製酵素は電気泳動
的に単一な純度の高い標品であった。Purified non-reducing saccharide-forming enzyme and purified trehalose-releasing enzyme obtained as gel filtration eluates in the steps of Table 16 and Table 17, respectively, were purified by electrophoresis in the same manner as in Experiment 15. As a result of the assay, the protein band was shown to be single, and both of the obtained purified enzymes were electrophoretically single and highly pure samples.

【０１３０】[0130]

【実験２４ 酵素の性質】実験２３の方法で得られた精
製トレハロース遊離酵素を、実験１６の場合と同様にＳ
ＤＳ−ポリアクリルアミドゲル電気泳動法で分子量を測
定したところ、約５７，０００乃至６７，０００ダルト
ンであった。また、本酵素の等電点を実験３の場合と同
様に等電点電気泳動法で求めたところ、等電点は３．６
乃至４．６であった。また、本酵素活性に対する温度の
影響、ｐＨの影響、及び本酵素の温度安定性、ｐＨ安定
性について、実験１６の場合と同様にして求めた。結果
は、温度の影響を図１４に、ｐＨの影響を図１５に、温
度安定性を図１６に、ｐＨ安定性を図１７に示した。[Experiment 24 Properties of enzyme] The purified trehalose-releasing enzyme obtained by the method of Experiment 23 was treated with S in the same manner as in Experiment 16.
When the molecular weight was measured by DS-polyacrylamide gel electrophoresis, it was about 57,000 to 67,000 daltons. Further, the isoelectric point of the present enzyme was determined by the isoelectric focusing method as in Experiment 3, and the isoelectric point was 3.6.
To 4.6. Further, the influence of temperature, the influence of pH on the activity of the present enzyme, and the temperature stability and pH stability of the present enzyme were determined in the same manner as in Experiment 16. As a result, the effect of temperature is shown in FIG. 14, the effect of pH is shown in FIG. 15, the temperature stability is shown in FIG. 16, and the pH stability is shown in FIG.

【０１３１】図から明らかなように酵素の至適温度は４
５℃付近、至適ｐＨは約６．０乃至７．５である。温度
安定性は４５℃付近までであり、ｐＨ安定性は約５．０
乃至１０．０である。As is clear from the figure, the optimum temperature of the enzyme is 4
Around 5 ° C., the optimum pH is about 6.0 to 7.5. Temperature stability is up to around 45 ° C, pH stability is about 5.0
To 10.0.

【０１３２】[0132]

【実験２５ α−グリコシルトレハロースからのトレハ
ロースの調製】実験２３の方法で得られた精製酵素を用
いて、実験１７の方法に従って、末端にトレハロース構
造を有するグルコース重合度が３以上の非還元性糖質か
らのトレハロースの調製の実験を行ったところ、リゾビ
ウム・スピーシーズ Ｍ−１１由来のトレハロース遊離
酵素の場合と同様に、α−グリコシルトレハロースから
トレハロースを遊離することが判明した。[Experiment 25 Preparation of Trehalose from α-Glycosyl Trehalose] The purified enzyme obtained by the method of Experiment 23 was used according to the method of Experiment 17, and a non-reducing sugar having a trehalose structure at the terminal and a degree of glucose polymerization of 3 or more. Experiments on the preparation of trehalose from qualities revealed that trehalose was released from α-glycosyltrehalose, similar to the case of trehalose-releasing enzyme from Rhizobium species M-11.

【０１３３】[0133]

【実験２６ 公知微生物からのトレハロース遊離酵素の
生産とその性質】公知微生物のうち、本発明のトレハロ
ース遊離酵素産生能の確認されたブレビバクテリウム・
ヘロボルムＡＴＣＣ１１８２２及びミクロコッカス・ロ
ゼウスＡＴＣＣ１８６を、実験１４の場合と同様にファ
ーメンターで２７℃で７２時間培養した。それぞれの培
養液約１８ｌを用いて、実験１５の場合と同様に、培養
液を破砕装置で処理し、その遠心上清を回収し、続い
て、硫安塩析、透析、イオン交換カラムクロマトグラフ
ィーし、得られた部分精製酵素標品の性質を調べた。こ
れらの結果を、前述のリゾビウム・スピーシーズ Ｍ−
１１及びアルスロバクター・スピーシーズ Ｑ３６の場
合とともに表１８にまとめた。[Experiment 26: Production of trehalose-releasing enzyme from known microorganisms and its properties] Among known microorganisms, Brevibacterium
Heroborum ATCC 11822 and Micrococcus roseus ATCC 186 were cultured in a fermenter at 27 ° C. for 72 hours as in Experiment 14. About 18 liters of each culture broth was used to treat the culture broth with a crushing device in the same manner as in Experiment 15, and the centrifuged supernatant was recovered, followed by ammonium sulfate salting out, dialysis, and ion exchange column chromatography. The properties of the partially purified enzyme preparation obtained were examined. Based on these results, the above-mentioned Rhizobium species M-
11 and the cases of Arthrobacter species Q36 are summarized in Table 18.

【０１３４】[0134]

【表１８】 [Table 18]

【０１３５】また、これらの公知微生物由来の部分精製
酵素を用いて、実験２５の方法に従って、末端にトレハ
ロース構造を有するグルコース重合度が３以上の非還元
性糖質からのトレハロースの調製の実験を行ったとこ
ろ、リゾビウム・スピーシーズＭ−１１由来のトレハロ
ース遊離酵素の場合と同様に、α−グリコシルトレハロ
ースからトレハロースを遊離することが判明した。Using these partially purified enzymes derived from known microorganisms, according to the method of Experiment 25, an experiment for the preparation of trehalose from a non-reducing sugar having a trehalose structure at the terminal and a degree of glucose polymerization of 3 or more was carried out. As a result, it was found that trehalose was released from α-glycosyltrehalose as in the case of trehalose-releasing enzyme derived from Rhizobium species M-11.

【０１３６】[0136]

【実験２７ トレハロース高含有糖質を製造するための
澱粉の液化程度と使用酵素の影響】澱粉からトレハロー
ス高含有糖質を製造するために、澱粉の液化程度と使用
する酵素の組合わせの影響を調べた。濃度２０％のとう
もろこし澱粉乳に炭酸カルシウムを０．１％加えてｐＨ
６．５に調整した後、これにα−アミラーゼ、ノボ社販
売『ターマミール』を澱粉当たり０．１乃至２．０％を
加え、９５℃で１５分間反応させ、１２０℃にオートク
レーブして１０分間保って、ＤＥ２．５乃至２０．５の
液化溶液とし、これを急冷したものに実験２の方法で調
製した精製非還元性糖質生成酵素を澱粉グラム当たり５
単位及び実験１５の方法で調製した精製トレハロース遊
離酵素を澱粉グラム当たり１０単位加え、更に澱粉枝切
酵素イソアミラーゼ（株式会社林原生物化学研究所販
売）を澱粉グラム当たり５００単位及び／又はシクロマ
ルトデキストリン・グルカノトランスフェラーゼ（株式
会社林原生物化学研究所販売）を澱粉グラム当たり５単
位加えてｐＨ６．０、４５℃で２４時間反応させた。本
反応液を９５℃、１０分間加熱した後、冷却し、次い
で、グルコアミラーゼを澱粉グラム当たり１０単位加え
てｐＨ５．０で１０時間反応させた。本反応液を高速液
体クロマトグラフィーで分析し、糖質中のトレハロース
含量（％）を求めた。対照として、澱粉液化溶液に非還
元性糖質生成酵素及びトレハロース遊離酵素だけを同様
に作用させ、同様に、グルコアミラーゼを作用させて、
高速液体クロマトグラフィーで分析した。結果は、表１
９に示す。[Experiment 27 Degree of liquefaction of starch for producing trehalose-rich saccharide and effect of enzyme used] Effect of combination of degree of liquefaction of starch and enzyme used for producing trehalose-rich saccharide from starch Examined. Add 20% corn starch milk with 0.1% calcium carbonate to pH
After adjusting to 6.5, 0.1-2.0% of α-amylase, “Tarmamir” sold by Novo Co., per starch is added to the mixture, reacted at 95 ° C. for 15 minutes, autoclaved at 120 ° C. for 10 minutes. The solution was kept in a liquefied solution of DE 2.5 to 20.5, and the solution was rapidly cooled, and the purified non-reducing saccharide-forming enzyme prepared by the method of Experiment 2 was added to the liquefied solution at 5% per gram of starch.
Units and 10 units of purified trehalose-releasing enzyme prepared by the method of Experiment 15 were added per gram of starch, and further 500 units of starch debranching enzyme isoamylase (available from Hayashibara Biochemical Research Institute) and / or cyclomaltodextrin. -Glucanotransferase (available from Hayashibara Biochemical Laboratories, Inc.) was added at 5 units per gram of starch, and the reaction was carried out at pH 6.0 and 45 ° C for 24 hours. This reaction solution was heated at 95 ° C. for 10 minutes and then cooled, and then 10 units of glucoamylase was added per gram of starch and reacted at pH 5.0 for 10 hours. This reaction solution was analyzed by high performance liquid chromatography to determine the trehalose content (%) in the sugar. As a control, only the non-reducing sugar-forming enzyme and trehalose-releasing enzyme were allowed to act on the starch liquefaction solution in the same manner, and similarly, glucoamylase was allowed to act,
Analyzed by high performance liquid chromatography. The results are shown in Table 1.
9 shows.

【０１３７】[0137]

【表１９】 [Table 19]

【０１３８】表１９の結果から明らかなように、澱粉か
らトレハロース高含有糖質を製造するには、その液化の
程度は比較的低いものが適しており、望ましくは、ＤＥ
１５未満、更に望ましくはＤＥ１０未満が好適であるこ
とが判明した。また、それに使用する酵素は、非還元性
糖質生成酵素及びトレハロース遊離酵素だけを作用させ
るよりも、非還元性糖質生成酵素及びトレハロース遊離
酵素を澱粉枝切酵素及び／又はシクロマルトデキストリ
ン・グルカノトランスフェラーゼとともに作用させた方
が、澱粉からのトレハロース収量を約２乃至４倍にも向
上することとなり、澱粉からトレハロースを工業的に大
量生産する上で、極めて有利であることが判明した。As is clear from the results shown in Table 19, a relatively low degree of liquefaction is suitable for producing a trehalose-rich sugar from starch, and DE is preferably
It has been found that less than 15, more preferably less than DE 10, is suitable. In addition, the enzyme used for the non-reducing saccharide-forming enzyme and trehalose-releasing enzyme acts on the starch debranching enzyme and / or cyclomaltodextrin / gluca rather than the action of only the non-reducing saccharide-forming enzyme and trehalose-releasing enzyme. It was found that the action with notransferase increases the yield of trehalose from starch by about 2 to 4 times, which is extremely advantageous for industrially mass-producing trehalose from starch.

【０１３９】以下、本発明の還元性を低減させた糖質の
製造方法を実施例Ａで、該糖質を含有せしめた組成物を
実施例Ｂで示す。Hereinafter, the method for producing a sugar having reduced reducibility according to the present invention will be shown in Example A, and the composition containing the sugar will be shown in Example B.

【０１４０】[0140]

【実施例Ａ−１】馬鈴薯澱粉を濃度約２０％の澱粉乳と
し、これに蓚酸を０．３％加えてオートクレーブし、冷
却し、炭酸カルシウムでｐＨ６．５に中和して、ＤＥ約
１２の液化溶液を得た。本液化溶液に実験２の方法で得
た精製非還元性糖質生成酵素を澱粉グラム当たり２単位
及びイソアミラーゼを澱粉グラム当たり３００単位加
え、温度４５℃で２４時間反応させた、本反応液を９５
℃に加熱して酵素を失活させた後、冷却し、濾過して得
られる濾液を、常法に従って、活性炭で脱色し、Ｈ型及
びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃
縮して濃度約５０％のシラップを固形物当たり約９０％
の収率で得た。本品は、α−グリコシルトレハロースと
ともに還元性澱粉糖を含むＤＥ約８の低還元性糖質であ
る。本低還元性糖質シラップをオートクレーブに入れ、
ラネーニッケル１０％を添加し、攪拌しながら温度を９
０乃至１２０℃に上げ、水素圧を２０乃至１２０ｋｇ／
ｃｍ2に上げて水素添加を完了させた後、ラネーニッケ
ルを除去し、次いで、常法に従って、脱色、脱塩して精
製し、濃縮して、濃度７０％のシラップを固形物当たり
約８０％の収率で得た。本品は、分子中にトレハロース
構造を有する非還元性糖質とともに澱粉糖アルコールを
含有する還元性を低減させたＤＥ１未満の糖質で、温和
で上品な甘味、比較的低粘度、適度な保湿性を有し、甘
味料、呈味改良剤、品質改良剤、安定剤、賦形剤などと
して、各種飲食物、化粧品、医薬品など各種組成物に有
利に利用できる。Example A-1 Potato starch was made into starch milk having a concentration of about 20%, 0.3% of oxalic acid was added thereto, and the mixture was autoclaved, cooled, and neutralized to pH 6.5 with calcium carbonate to give a DE of about 12%. A liquefied solution of was obtained. 2 units of the purified non-reducing saccharide-forming enzyme obtained by the method of Experiment 2 and 300 units of isoamylase were added to the liquefied solution per 300 g of the starch, and the reaction was carried out at a temperature of 45 ° C. for 24 hours. 95
After heating to ℃ to inactivate the enzyme, it was cooled and filtered, the filtrate obtained was decolorized with activated carbon according to a conventional method, desalted with H-type and OH-type ion exchange resins, and purified. Concentrate about 50% syrup to about 90% solids
It was obtained in a yield of. This product is a low reducing sugar having a DE of about 8 containing a reducing starch sugar together with α-glycosyltrehalose. Put this low reducing sugar syrup in an autoclave,
Raney nickel 10% was added and the temperature was raised to 9 while stirring.
Raise the hydrogen pressure to 0 to 120 ℃, 20 to 120 kg /
After Raising to cm 2 to complete the hydrogenation, the Raney nickel was removed and then decolorized, desalted and purified according to the usual method and concentrated to obtain 70% concentration of syrup of about 80% per solid. Obtained in yield. This product is a non-reducing saccharide with a trehalose structure in the molecule and a starch sugar alcohol with a reduced reducibility that is less than DE1 and has a mild and elegant sweetness, relatively low viscosity, and moderate moisturizing effect. As a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

【０１４１】[0141]

【実施例Ａ−２】タピオカ澱粉を濃度約２５％澱粉乳と
し、これにα−アミラーゼ、ナガセ生化学工業株式会社
製『ネオスピターゼ』を澱粉グラム当たり０．２％加
え、８５乃至９０℃で約２０分間反応させ、次いで１２
０℃にオートクレーブし、急冷してＤＥ約４の液化溶液
を得、これに実験９の方法で得た精製非還元性糖質生成
酵素を澱粉グラム当たり５単位、プルラナーゼ（株式会
社林原生物化学研究所販売）を澱粉グラム当たり１００
単位及びマルトテトラオース生成アミーゼ（株式会社林
原生物化学研究所製）を澱粉グラム当たり２０単位加
え、ｐＨ６．５、温度４０℃で３６時間反応させた。本
反応液を、実施例Ａ−１と同様に、加熱して酵素を失活
させた後、精製し、濃度約６０％に濃縮した。本濃縮液
を原糖液とし、非還元性糖質の含量を高めるため、カル
シウム型強酸性カチオン交換樹脂、東京有機化学工業株
式会社製『ＸＴ−１０１６』を用いたカラムクロマトグ
ラフィーを行った。樹脂を内径５．４ｃｍのジャケット
付きステンレス製カラム４本に充填し、直列につなぎ樹
脂層長全長２０ｍとした。カラム内温度を５５℃に維持
しつつ、糖液を樹脂に対して５ｖ／ｖ％加え、これに５
５℃の温水をＳＶ０．２で流して分画し、非還元性糖質
高含有のグルコース重合度４乃至６の画分を採取し、精
製、濃縮し、濃度約５０％のシラップを得た。本品は、
α−グリコシルトレハロースとともに還元性澱粉糖を含
むＤＥ５．４の低還元性糖質である。本低還元性シラッ
プを、実施例Ａ−１の方法に準じて、水素添加し、精製
し、濃縮して、濃度７０％のシラップを固形物当たり約
５０％の収率で得た。本品は、分子中にトレハロース構
造を有する非還元性糖質とともに澱粉糖アルコールを含
有する還元性を低減させたＤＥ１未満の糖質で、温和で
上品な甘味、比較的低粘度、適度な保湿性を有し、甘味
料、呈味改良剤、品質改良剤、安定剤、賦形剤などとし
て、各種飲食物、化粧品、医薬品など各種組成物に有利
に利用できる。[Example A-2] Tapioca starch was used as a starch milk having a concentration of about 25%, and 0.2% of α-amylase, "Neoseptase" manufactured by Nagase Seikagaku Co., Ltd. was added to the starch milk at 85 to 90 ° C. Allow to react for 20 minutes, then 12
It was autoclaved at 0 ° C and rapidly cooled to obtain a liquefied solution of DE about 4, and 5 units of the purified non-reducing saccharide-forming enzyme obtained by the method of Experiment 9 was added per gram of starch, pullulanase (Hayashibara Biochemical Research Co., Ltd. 100) per gram starch
A unit and maltotetraose-forming amize (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) were added in an amount of 20 units per gram of starch and reacted at pH 6.5 and a temperature of 40 ° C. for 36 hours. This reaction solution was heated to deactivate the enzyme and then purified and concentrated to a concentration of about 60% in the same manner as in Example A-1. Using this concentrated solution as a raw sugar solution, column chromatography was performed using calcium-type strongly acidic cation exchange resin "XT-1016" manufactured by Tokyo Organic Chemical Industry Co., Ltd. in order to increase the content of non-reducing sugars. The resin was packed in four jacketed stainless steel columns having an inner diameter of 5.4 cm and connected in series to give a resin layer length of 20 m. While maintaining the temperature in the column at 55 ° C, 5% v / v% of sugar solution was added to the resin.
Fractionation was carried out by flowing warm water at 5 ° C. with SV0.2, and fractions with a high degree of non-reducing sugar and a degree of glucose polymerization of 4 to 6 were collected, purified and concentrated to obtain syrup having a concentration of about 50%. . This product is
It is a low reducing sugar of DE 5.4 containing reducing starch sugar together with α-glycosyltrehalose. This low-reducing syrup was hydrogenated, purified, and concentrated according to the method of Example A-1, to obtain a syrup having a concentration of 70% in a yield of about 50% based on solids. This product is a non-reducing saccharide with a trehalose structure in the molecule and a starch sugar alcohol with a reduced reducibility that is less than DE1 and has a mild and elegant sweetness, relatively low viscosity, and moderate moisturizing effect. As a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

【０１４２】[0142]

【実施例Ａ−３】とうもろこし澱粉を濃度約３０％の澱
粉乳とし、これに炭酸カルシウム０．１％加え、ｐＨ
６．５に調整し、α−アミラーゼ、ノボ社製『ターマミ
ール６０Ｌ』を澱粉グラム当たり０．３％加え、９５℃
で１５分間反応させ、次いで１２０℃にオートクレイブ
し、急冷してＤＥ約４の液化溶液を得、これに実験２の
方法で得た精製非還元性糖質生成酵素を澱粉グラム当た
り４単位、イソアミラーゼを澱粉グラム当たり３００単
位及びシクロマルトデキストリン・グルカノトランスフ
ェラーゼ（株式会社林原生物化学研究所販売）を澱粉グ
ラム当たり５単位加え、ｐＨ６．３、温度４５℃で４８
時間反応させた。本反応液を９５℃で１０分間保った
後、冷却し、これにβ−アミラーゼを澱粉グラム当たり
１０単位加えてｐＨ５．５、温度５５℃で１６時間反応
させた。本反応液を、加熱して酵素を失活させた後、常
法に従って、脱色、脱塩して精製し、濃縮して濃度約５
０％のシラップを得た。本品は、末端にトレハロース構
造を有する非還元性糖質及びα−グリコシル α−グル
コシドなどの非還元性糖質とともに還元性澱粉糖を含む
低還元性糖質である。本低還元性シラップを、実施例Ａ
−１の方法に準じて、水素添加し、精製し濃縮して、濃
度７０％のシラップを固形物当たり約８０％の収率で得
た。本品は、分子中にトレハロース構造を有する非還元
性糖質とともに澱粉糖アルコールを含有する還元性を低
減させたＤＥ１未満の糖質で、温和で上品な甘味、比較
的低粘度、適度な保湿性を有し、甘味料、呈味改良剤、
品質改良剤、安定剤、賦形剤などとして、各種飲食物、
化粧品、医薬品など各種組成物に有利に利用できる。[Example A-3] Corn starch was made into starch milk having a concentration of about 30%, to which 0.1% of calcium carbonate was added to adjust the pH.
Adjusted to 6.5, added 0.3% of α-amylase, "Tarmamir 60L" manufactured by Novo Co., per gram of starch, and heated at 95 ° C.
Reaction for 15 minutes, then autoclave to 120 ° C. and quench to obtain a liquefied solution of DE about 4, to which 4 units of purified non-reducing saccharide-forming enzyme obtained by the method of Experiment 2 were added per gram of starch, Add 300 units of isoamylase per gram of starch and 5 units of cyclomaltodextrin glucanotransferase (sold by Hayashibara Biochemical Laboratory Co., Ltd.) per gram of starch, and add 48 at pH 6.3 and a temperature of 45 ° C.
Allowed to react for hours. The reaction solution was kept at 95 ° C. for 10 minutes and then cooled, and 10 units of β-amylase was added thereto per gram of starch, and the reaction was carried out at pH 5.5 and a temperature of 55 ° C. for 16 hours. This reaction solution is heated to deactivate the enzyme, and then decolorized, desalted and purified according to a conventional method, and concentrated to a concentration of about 5
A 0% syrup was obtained. This product is a low-reducing sugar containing a reducing starch sugar together with a non-reducing sugar having a trehalose structure at the terminal and a non-reducing sugar such as α-glycosyl α-glucoside. This low reducing syrup was tested in Example A.
Hydrogenated, purified and concentrated according to method -1 to give syrup with a concentration of 70% in a yield of about 80% based on solids. This product is a non-reducing saccharide with a trehalose structure in the molecule and a starch sugar alcohol with a reduced reducibility that is less than DE1 and has a mild and elegant sweetness, relatively low viscosity, and moderate moisturizing effect. Having sweetness, sweetener, taste improver,
Various foods and drinks as quality improvers, stabilizers, excipients, etc.
It can be advantageously used for various compositions such as cosmetics and pharmaceuticals.

【０１４３】[0143]

【実施例Ａ−４】実施例Ａ−３の方法で得たシラップを
濃度約５５％にして原糖液とし、非還元性糖質の含量を
高めるため、実施例Ａ−２の方法に準じて塩型強酸性カ
チオン交換樹脂を用いるカラムクロマトグラフィーを行
って、非還元性糖質高含有のグルコース重合度３乃至６
の画分を採取し、精製、濃縮し、濃度約５０％のシラッ
プを得た。本品は、末端にトレハロース構造を有する非
還元性糖質及びα−グリコシル α−グルコシドなどの
非還元性糖質を多量含むとともに還元性澱粉糖を含むＤ
Ｅ８の低還元性糖質である。本低還元性シラップを実施
例Ａ−１の方法に準じて、水素添加し、精製し、濃縮し
て濃度７０％のシラップを固形物当たり約３０％の収率
で得た。本品は、分子中にトレハロース構造を有する非
還元性糖質とともに澱粉糖アルコールを含有する還元性
を低減させたＤＥ１未満の糖質で、温和で上品な甘味、
比較的低粘度、適度な保湿性を有し、甘味料、呈味改良
剤、品質改良剤、安定剤、賦形剤などとして、各種飲食
物、化粧品、医薬品など各種組成物に有利に利用でき
る。Example A-4 A syrup obtained by the method of Example A-3 was made into a raw sugar solution with a concentration of about 55%, and the content of non-reducing sugar was increased. Column chromatography using a salt-type strongly acidic cation exchange resin to give a glucose content of 3 to 6 with a high content of non-reducing sugars.
Was collected, purified and concentrated to obtain syrup having a concentration of about 50%. This product contains a large amount of non-reducing saccharides having a trehalose structure at the terminal and non-reducing saccharides such as α-glycosyl α-glucoside, and D containing a reducing starch saccharide.
It is a low reducing sugar of E8. This low-reducing syrup was hydrogenated, purified, and concentrated according to the method of Example A-1 to obtain a syrup having a concentration of 70% in a yield of about 30% based on solids. This product is a sugar with a reducing property of less than DE1 that contains a starch sugar alcohol together with a non-reducing sugar having a trehalose structure in its molecule and has a mild and elegant sweetness,
It has a relatively low viscosity and moderate moisturizing properties, and can be advantageously used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. in various compositions such as food and drink, cosmetics, and pharmaceuticals. .

【０１４４】[0144]

【実施例Ａ−５】とうもろこし澱粉を濃度約３０％の澱
粉乳とし、これに実施例Ａ−３の方法に準じて、α−ア
ミラーゼを作用させてＤＥ４の液化溶液を得、次いで、
実験２の方法で得た精製非還元性糖質生成酵素を澱粉グ
ラム当たり５単位、実験１５記載の精製トレハロース遊
離酵素を澱粉グラム当たり１０単位及びイソアミラーゼ
を澱粉グラム当たり５００単位加え、ｐＨ６．０、温度
４０℃で４８時間反応させた。本反応液には、トレハロ
ースを７６．３％含有していた。本反応液を加熱して酵
素を失活させた後、常法に従って、脱色、脱塩して精製
し、濃縮して約４５％のシラップを得た。本トレハロー
ス高含有低還元性糖質シラップを、実施例Ａ−１の方法
に準じて、水素添加し、精製し、濃縮して濃度約８５％
にして助晶機にとり、撹拌しつつ徐冷して助晶し、これ
をプラスチック製バットに取り出し、室温で２日間放置
し、晶出熟成させてブロックを調製した。次いで、本ブ
ロックを切削機にて粉砕してトレハロース含水結晶とと
もに澱粉糖アルコールを含有する還元性を低減させたＤ
Ｅ１未満の糖質粉末を、原料澱粉に対して固形物当たり
８０％の収率で得た。本品は、取扱いが容易であり、甘
味料、呈味改良剤、品質改良剤、安定剤、賦形剤などと
して、各種飲食物、化粧品、医薬品など各種組成物に有
利に利用できる。[Example A-5] Corn starch was used as starch milk having a concentration of about 30%, and α-amylase was allowed to act on it to obtain a liquefied solution of DE4 according to the method of Example A-3.
The purified non-reducing saccharide-forming enzyme obtained by the method of Experiment 2 was added at 5 units per gram of starch, the purified trehalose-releasing enzyme described in Experiment 15 was added at 10 units per gram of starch, and the isoamylase was added at 500 units per gram of starch, and the pH was 6.0. The reaction was carried out at a temperature of 40 ° C. for 48 hours. The reaction solution contained 76.3% trehalose. The reaction solution was heated to deactivate the enzyme, and then decolorized, desalted, and purified according to a conventional method to concentrate and obtain about 45% syrup. The trehalose-rich low-reducing sugar syrup was hydrogenated, purified, and concentrated to a concentration of about 85% according to the method of Example A-1.
Then, the block was prepared by accelerating the crystallization by taking it into an auxiliary crystallizer, slowly cooling it with stirring to promote an auxiliary crystal, and taking it out into a plastic vat and allowing it to stand at room temperature for 2 days for crystallization. Next, this block was crushed with a cutting machine to reduce the reducibility of trehalose containing water-containing crystals and starch sugar alcohol.
Sugar powder less than E1 was obtained in a yield of 80% based on solids based on the raw starch. The product is easy to handle and can be advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc. in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

【０１４５】[0145]

【実施例Ａ−６】タピオカ澱粉を濃度約３０％の澱粉乳
とし、これに実施例Ａ−２の方法に準じて、α−アミラ
ーゼを作用させてＤＥ５の液化溶液を得、次いで、実験
１０の方法で得た精製非還元性糖質生成酵素を澱粉グラ
ム当たり３単位、実験２３の方法で得た精製トレハロー
ス遊離酵素を澱粉グラム当たり５単位及びプルラナーゼ
を２００単位及びシクロマルトデキストリン・グルカノ
トランスフェラーゼを澱粉グラム当たり３単位加え、ｐ
Ｈ６．０、温度４５℃で４８時間反応させた。本反応液
には、固形物当たりトレハロースを８４．７％含有して
いた。本反応液を加熱失活し、常法に従って、脱色、脱
塩して精製し、濃縮しながら連続晶析させ、得られるマ
スキットをバスケット型遠心分離機で分蜜し、結晶を少
量の水でスプレーし洗浄してトレハロース含水結晶を固
形物当たり約５５％の収率で得た。この方法で得られた
母液には多量のトレハロース及びトレハロース構造を有
する非還元性糖質とともに還元性澱粉糖を含有してお
り、これを濃縮して濃度５０％のシラップを得た。本低
還元性糖質シラップを、実施例Ａ−１の方法に準じて、
水素添加し、精製し、濃縮して、濃度７０％のシラップ
を固形物当たり約３０％得た。本品は、トレハロース及
びトレハロース構造を有する非還元性糖質とともに澱粉
糖アルコールを含有する還元性を低減させたＤＥ１未満
の糖質で、温和で上品な甘味、比較的低粘度、適当な保
湿性を有し、甘味料、呈味改良剤、品質改良剤、安定
剤、賦形剤などとして、各種組成物に有利に利用でき
る。[Example A-6] Tapioca starch was used as starch milk having a concentration of about 30%, and α-amylase was allowed to act on it to obtain a liquefied solution of DE5 according to the method of Example A-2. 3 units per gram starch of the purified non-reducing saccharide-forming enzyme obtained by the above method, 5 units per gram of the purified trehalose-releasing enzyme obtained by the method of Experiment 23, 200 units of pullulanase and cyclomaltodextrin-glucanotransferase. 3 units per gram of starch, p
The reaction was carried out at H 6.0 and a temperature of 45 ° C. for 48 hours. This reaction liquid contained 84.7% trehalose per solid matter. This reaction solution is inactivated by heating, decolorized, desalted and purified according to a conventional method, continuously crystallized while concentrating, and the resulting mass kit is mashed with a basket type centrifuge and the crystals are washed with a small amount of water. After spraying and washing, trehalose hydrous crystals were obtained in a yield of about 55% based on solids. The mother liquor obtained by this method contained a large amount of trehalose and a non-reducing saccharide having a trehalose structure as well as a reducing starch sugar, which was concentrated to obtain syrup having a concentration of 50%. This low-reducing sugar syrup was prepared according to the method of Example A-1.
Hydrogenated, purified and concentrated to give about 30% syrup at 70% concentration per solids. This product is a trehalose and a non-reducing saccharide having a trehalose structure and a starch sugar alcohol, and a sugar with a reducing property of less than DE1, which has a mild and elegant sweetness, a relatively low viscosity, and an appropriate moisturizing property. It can be advantageously used in various compositions as a sweetener, taste improver, quality improver, stabilizer, excipient, etc.

【０１４６】[0146]

【実施例Ａ−７】実施例Ａ−６の方法で得た加熱失活し
た反応液に、グルコアミラーゼを基質グラム当たり１０
単位加え、ｐＨ５．０、温度５０℃で１０時間反応させ
た。本反応液を加熱失活し、常法に従って、脱色、脱塩
して精製し、濃縮して濃度４５％のシラップを得た。本
トレハロース高含有低還元性糖質シラップを、実施例Ａ
−１の方法に準じて、水素添加し、精製し、次いで、濃
度約７０％に濃縮した後、助晶機にとり撹拌しつつ徐冷
して助晶し、晶出率約４０％のマスキットを得た。本マ
スキットを乾燥塔上のノズルより１５０ｋｇ／ｃｍ2の
高圧にて噴霧し、同時に乾燥塔の上部より８５℃の熱風
を送風し、底部に設けた移送金網コンベア上に結晶粉末
を補集した。コンベアの下より４５℃の温風を送りつ
つ、該粉末を乾燥塔外に徐々に移動させて、取り出し
た。この結晶粉末を熟成塔に充填して温風を送りつつ１
０時間熟成させ、結晶化と乾燥を完了し、トレハロース
含水結晶及びソルビトールを含有する還元性を低減させ
たＤＥ１未満の糖質粉末を、原料の澱粉に対して、固形
物当たり約７５％の収率で得た。本品は、取扱いが容易
であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦
形剤などとして、各種飲食物、化粧品、医薬品など各種
組成物に有利に利用できる。Example A-7 The heat-inactivated reaction solution obtained by the method of Example A-6 was supplemented with 10% of glucoamylase per gram of substrate.
A unit was added, and the mixture was reacted at pH 5.0 and temperature of 50 ° C. for 10 hours. The reaction solution was inactivated by heating, decolorized, desalted and purified by a conventional method, and concentrated to obtain syrup having a concentration of 45%. This trehalose-rich low-reducing sugar syrup was prepared according to Example A.
According to the method of -1, hydrogenate, purify, then concentrate to a concentration of about 70%, then slowly cool while stirring in an auxiliary crystallizer to promote the crystallization, and a mass kit with a crystallization rate of about 40% is prepared. Obtained. This mass kit was sprayed from a nozzle on the drying tower at a high pressure of 150 kg / cm 2 , and at the same time, hot air of 85 ° C. was blown from the upper part of the drying tower, and the crystal powder was collected on a transfer wire mesh conveyor provided at the bottom. The powder was gradually moved to the outside of the drying tower while taking out hot air at 45 ° C. from below the conveyor, and was taken out. This crystal powder is packed in an aging tower and hot air is blown 1
After aging for 0 hours, crystallization and drying were completed, and a sugar powder of less than DE1 containing trehalose hydrous crystals and sorbitol and having a reduced reducibility was collected at a concentration of about 75% based on the solid content of starch. Got at a rate. The product is easy to handle and can be advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc. in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

【０１４７】[0147]

【実施例Ａ−８】リゾビウム・スピーシーズ Ｍ−１１
（ＦＥＲＭ ＢＰ−４１３０）の変異株を実験１の方法
に準じて、ファーメンターで約７０時間培養した。培養
後、ＳＦ膜を用いて除菌瀘過し、約１００ｌの培養瀘液
を回収し、更に、その瀘液をＵＦ膜濃縮し、非還元性糖
質生成酵素（約４１０単位／ｍｌ）とトレハロース遊離
酵素（約４９０単位／ｍｌ）とを含む濃縮酵素液約５ｌ
を回収した。とうもろこし澱粉を濃度約３３％の澱粉乳
とし、これに実施例Ａ−３の方法に準じて、α−アミラ
ーゼを作用させてＤＥ約４の液化溶液を得、次いで、前
記方法で調製した非還元性糖質精製酵素とトレハロース
遊離酵素とを含む濃縮液を澱粉グラム当たり０．０２ｍ
ｌ、イソアミラーゼを澱粉グラム当たり５００単位及び
シクロマルトデキストリン・グルカノトランスフェラー
ゼを澱粉グラム当たり５単位加え、ｐＨ６．２、温度４
０℃で４８時間反応させた。本反応液を加熱失活し、次
いで、グルコアミラーゼを基質グラム当たり１０単位加
え、ｐＨ５．０、温度５０℃で１０時間反応させた。本
反応液には、固形物当たりトレハロースを８５．６％含
有していた。本反応液を加熱失活し、常法に従って、脱
色、脱塩して精製し、濃縮して濃度４５％のシラップを
得た。本トレハロース高含有低還元性糖質シラップを、
実施例Ａ−１の方法に準じて、水素添加し、精製し、次
いで、実施例Ａ−５の方法に準じて、濃縮、晶出させて
ブロックを調製し、切削機にて粉砕して、トレハロース
含水結晶とともにソルビトールを含有する還元性を低減
させたＤＥ１未満の糖質粉末を原料澱粉に対して固形物
当たり約８０％の収率で得た。本品は、取扱いが容易で
あり、各種飲食物、化粧品、医薬品など各種組成物など
に有利に利用できる。[Example A-8] Rhizobium species M-11
The mutant strain of (FERM BP-4130) was cultured in a fermenter for about 70 hours according to the method of Experiment 1. After culturing, sterilization filtration was performed using an SF membrane, about 100 l of the culture filtrate was recovered, and the filtrate was concentrated on a UF membrane to obtain a non-reducing saccharide-forming enzyme (about 410 units / ml). About 5 liters of concentrated enzyme solution containing trehalose-releasing enzyme (about 490 units / ml)
Was recovered. Corn starch was made into starch milk having a concentration of about 33%, and α-amylase was allowed to act on it to obtain a liquefied solution of DE about 4 according to the method of Example A-3, and then the non-reducing method prepared by the above method was used. Concentrated liquid containing saccharide-purifying enzyme and trehalose-releasing enzyme was 0.02 m per gram of starch.
l, 500 units of isoamylase per gram of starch and 5 units of cyclomaltodextrin glucanotransferase per gram of starch were added, pH 6.2, temperature 4
The reaction was carried out at 0 ° C for 48 hours. The reaction solution was inactivated by heating, and then 10 units of glucoamylase was added per gram of substrate, and the reaction was carried out at pH 5.0 and a temperature of 50 ° C. for 10 hours. This reaction liquid contained 85.6% trehalose per solid matter. The reaction solution was inactivated by heating, decolorized, desalted and purified by a conventional method, and concentrated to obtain syrup having a concentration of 45%. This trehalose high content low reducing sugar syrup,
According to the method of Example A-1, hydrogenated, purified, then, according to the method of Example A-5, concentrated, crystallized to prepare a block, crushed with a cutting machine, A sugar powder containing less than DE1 and containing sorbitol together with trehalose hydrous crystals and having a reduced reducibility was obtained in a yield of about 80% based on the solid material based on the starting starch. The product is easy to handle and can be advantageously used for various compositions such as various foods and drinks, cosmetics and pharmaceuticals.

【０１４８】[0148]

【実施例Ａ−９】グルコース２ｗ／ｖ％、ポリペプトン
０．５ｗ／ｖ％、酵母エキス０．１ｗ／ｖ％、リン酸二
カリウム０．１ｗ／ｖ％、リン酸一ナトリウム０．０６
ｗ／ｖ％、硫酸マグネシウム０．０５ｗ／ｖ％、炭酸カ
ルシウム０．５ｗ／ｖ％及び水からなる液体培地をファ
ーメンターにとり、加熱滅菌、冷却し、これにピメロバ
クター・スピーシーズ Ｒ４８（ＦＥＲＭ ＢＰ−４３
１５）を植菌し、温度２７℃で約４０時間、通気攪拌培
養した。この培養液のマルトース・トレハロース変換酵
素の活性は、培養液ｍｌ当たり０．５５単位であった。
この培養液１８ｌから回収した湿重量０．１８ｋｇの菌
体を１０ｍＭリン酸緩衝液（ｐＨ７．０）に懸濁した。
この菌体懸濁液約１．５ｌを、超音波破砕装置で処理
し、菌体を破砕した。この菌体破砕懸濁液を遠心分離機
にかけ、その上清を回収し、更にその液をＵＦ膜濃縮
し、マルトース・トレハロース変換酵素をｍｌ当たり約
１８単位有する濃縮酵素液約５００ｍｌを回収した。１
５％とうもろこし澱粉乳（ｐＨ５．５）に、α−アミラ
ーゼ、ナガセ生化学工業株式会社製『スピターゼＨＳ』
を澱粉グラム当たり２単位加えて攪拌し、加熱糊化、液
化させ、直ちにオートクレーブ（１２０℃）を２０分間
行った後、温度５５℃、ｐＨ５．０に調整した。これに
イソアミラーゼを澱粉グラム当たり３００単位及びβ−
アミラーゼ（ナガセ生化学工業株式会社製）を澱粉グラ
ム当たり２０単位の割合になるように加え、２４時間反
応させ、マルトース含量約９２％の糖液を得た。その反
応液を１００℃で２０分間加熱した後、温度２０℃、ｐ
Ｈ７．０に調整し、これに前記方法で調製したマルトー
ス・トレハロース変換酵素を固形物グラム当たり１．５
単位の割合になるよう加え、７２時間反応させた。その
反応液を９５℃で１０分間保った後、冷却し、常法に従
って活性炭で脱色、濾過し、Ｈ型、ＯＨ型イオン交換樹
脂により脱塩して精製し、更に濃縮して濃度約５０％の
シラップを得た。Example A-9 Glucose 2 w / v%, polypeptone 0.5 w / v%, yeast extract 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06
A liquid medium consisting of w / v%, magnesium sulfate 0.05 w / v%, calcium carbonate 0.5 w / v% and water was placed in a fermenter, sterilized by heating and cooled, and Pimellobacterium species R48 (FERM BP-43 was used.
15) was inoculated and cultivated with aeration and stirring at a temperature of 27 ° C. for about 40 hours. The activity of maltose / trehalose converting enzyme in this culture was 0.55 units per ml of culture.
The wet cells of 0.18 kg recovered from 18 l of this culture solution were suspended in 10 mM phosphate buffer (pH 7.0).
About 1.5 liters of this microbial cell suspension was treated with an ultrasonic crusher to crush the microbial cells. The disrupted cell suspension was subjected to a centrifuge, the supernatant was recovered, and the solution was subjected to UF membrane concentration to recover about 500 ml of a concentrated enzyme solution having about 18 units of maltose / trehalose converting enzyme per ml. 1
5% corn starch milk (pH 5.5), α-amylase, Nagase Seikagaku Co., Ltd. "Spitase HS"
2 units per gram of starch were added and stirred to gelatinize by heating and liquefy, and immediately after autoclaving (120 ° C) for 20 minutes, the temperature was adjusted to 55 ° C and pH 5.0. To this is added 300 units of isoamylase per gram of starch and β-
Amylase (manufactured by Nagase Seikagaku Corporation) was added at a ratio of 20 units per gram of starch and reacted for 24 hours to obtain a sugar solution having a maltose content of about 92%. After heating the reaction solution at 100 ° C. for 20 minutes, the temperature was 20 ° C., p
It was adjusted to H7.0, and maltose / trehalose converting enzyme prepared by the above method was added thereto at 1.5
It was added so that the ratio of the units would be sufficient, and the reaction was carried out for 72 hours. The reaction solution was kept at 95 ° C for 10 minutes, cooled, decolorized with activated carbon according to a conventional method, filtered, purified by desalting with an H-type or OH-type ion exchange resin, and further concentrated to a concentration of about 50%. Got a syrup.

【０１４９】本品は、固形物当たり、トレハロースを約
６４％含有していて還元力がＤＥ１８．０と低い。本シ
ラップを実施例Ａ−１の方法に準じて、水素添加し、精
製し、濃縮して、濃度約７０％のシラップを固形物当た
り約８０％の収率で得た。本品は、トレハロースととも
にマルチトール及び少量のソルビトールを含有する還元
性を低減させたＤＥ１未満の糖質シラップで、温和な甘
味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、
安定剤、賦形剤などとして、各種飲食物、化粧品、医薬
品、など各種組成物に有利に利用できる。This product contains about 64% of trehalose per solid matter and has a low reducing power of DE18.0. This syrup was hydrogenated, purified and concentrated according to the method of Example A-1 to obtain a syrup having a concentration of about 70% in a yield of about 80% based on solids. This product is a sugar syrup of less than DE1 that contains maltitol and a small amount of sorbitol together with trehalose and has reduced reducibility. It has mild sweetness, moderate viscosity and moisturizing property, and is a sweetener and a taste improver. ,
As a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics, pharmaceuticals and the like.

【０１５０】[0150]

【実施例Ｂ−１ 甘味料】実施例Ａ−７の方法で得た還
元性を低減させた糖質粉末１重量部に、α−グリコシル
ステビオシド、東洋精糖株式会社販売『αＧスイート』
０．０１重量部及びＬ−アスパルチル−Ｌ−フェニルア
ラニンメチルエステル、味の素株式会社販売『アスパル
テーム』０．０１重量部を均一に混合し、顆粒成型機に
かけて、顆粒状甘味料を得た。本品は、甘味の質が優
れ、蔗糖の約２倍の甘味度を有し、甘味度当たりカロリ
ーは、蔗糖の約１／２に低下している。本甘味料は、そ
れに配合した高甘味度甘味物の分解もなく、安定性に優
れており、低カロリー甘味料として、カロリー摂取を制
限している肥満者、糖尿病者などのための低カロリー飲
食物などに対する甘味付けに好適である。また、本甘味
料は、虫歯誘発菌による酸の生成が少なく、不溶性グル
カンの生成も少ないことより、虫歯を抑制する飲食物な
どに対する甘味付けにも好適である。Example B-1 Sweetener 1 part by weight of the sugar powder having the reduced reducibility obtained by the method of Example A-7 was mixed with α-glycosyl stevioside, “αG sweet” sold by Toyo Seika Co., Ltd.
0.01 parts by weight of L-aspartyl-L-phenylalanine methyl ester and 0.01 parts by weight of "Aspartame" sold by Ajinomoto Co., Inc. were uniformly mixed and subjected to a granulating machine to obtain a granular sweetener. This product has an excellent sweetness quality and has a sweetness that is about twice that of sucrose, and the calorie per sweetness is reduced to about 1/2 of that of sucrose. This sweetener has excellent stability without decomposition of the high-intensity sweeteners mixed in it, and as a low-calorie sweetener, it is a low-calorie food and drink for obese people, diabetics, etc. whose calorie intake is restricted. It is suitable for sweetening things. Further, the present sweetener is suitable for sweetening foods and beverages that suppress tooth decay because it produces less acid due to cavities-inducing bacteria and less insoluble glucan.

【０１５１】[0151]

【実施例Ｂ−２ ハードキャンディー】濃度５５％蔗糖
溶液１００重量部に実施例Ａ−１の方法で得た還元性を
低減させた糖質含有シラップ３０重量部を加熱混合し、
次いで減圧下で水分２％未満になるまで加熱濃縮し、こ
れにクエン酸１重量部及び適量のレモン香料と着色料と
を混和し、常法に従って成型し、製品を得た。本品は、
歯切れ、呈味良好で、蔗糖の晶出、変形も起こらない高
品質のハードキャンディーである。[Example B-2 Hard candy] To 100 parts by weight of a 55% sucrose solution, 30 parts by weight of a sugar-containing syrup having a reduced reducibility obtained by the method of Example A-1 was heated and mixed,
Then, the mixture was heated and concentrated under reduced pressure until the water content was less than 2%, 1 part by weight of citric acid and an appropriate amount of lemon flavor and color were mixed and molded according to a conventional method to obtain a product. This product is
It is a high-quality hard candy that is crisp, has a good taste, and does not crystallize or deform sucrose.

【０１５２】[0152]

【実施例Ｂ−３ チョコレート】カカオペースト４０重
量部、カカオバター１０重量部、蔗糖３０重量部、実施
例Ａ−８の方法で得た還元性を低減させた糖質粉末２０
重量部を混合してレファイナーに通して粒度を下げた
後、コンチェに入れて５０℃で２昼夜練り上げる。この
間に、レシチン０．５重量部を加え充分に混和分散させ
た。次いで、温度調節器で３１℃に調節し、バターの固
まる直前に型に流し込み、振動機でアワ抜きを行い、１
０℃の冷却トンネルを２０分間くぐらせて固化させた。
これを型抜きして包装し製品を得た。本品は、吸湿性が
なく、色、光沢共によく、内部組織も良好で、口中でな
めらかに溶け、上品な甘味とまろやかな風味を有する。[Example B-3 Chocolate] 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, 30 parts by weight of sucrose, and sugar powder 20 obtained by the method of Example A-8 and having reduced reducibility.
After mixing parts by weight and passing through a refiner to reduce the particle size, the mixture is put in a conche and kneaded at 50 ° C. for 2 days and nights. During this period, 0.5 part by weight of lecithin was added and thoroughly mixed and dispersed. Then, adjust the temperature to 31 ° C with a temperature controller, pour into the mold immediately before the butter hardens, and remove the foam with a vibrator.
It was solidified by passing through a 0 ° C. cooling tunnel for 20 minutes.
This was die-cut and packaged to obtain a product. This product has no hygroscopicity, has good color and gloss, has a good internal structure, melts smoothly in the mouth, and has an elegant sweetness and mellow flavor.

【０１５３】[0153]

【実施例Ｂ−４ チューインガム】ガムベース３重量部
を柔らかくなる程度に加熱溶融し、これに蔗糖４重量部
及び実施例Ａ−５の方法で得た還元性を低減させた糖質
粉末３重量部とを加え、更に適量の香料と着色料とを混
合し、常法に従って、ロールにより練り合わせ、成形、
包装して製品を得た。本品は、テクスチャー、風味とも
良好なチューインガムである。[Example B-4 Chewing gum] 3 parts by weight of a gum base was heated and melted to a softness, and 4 parts by weight of sucrose and 3 parts by weight of a sugar powder obtained by the method of Example A-5 and having a reduced reducibility were obtained. Is added, and further an appropriate amount of flavor and color is mixed, followed by kneading with a roll according to a conventional method, molding,
The product was packaged. This product is a chewing gum with good texture and flavor.

【０１５４】[0154]

【実施例Ｂ−５ 加糖練乳】原乳１００重量部に実施例
Ａ−３の方法で得た還元性を低減させた糖質含有シラッ
プ３重量部及び蔗糖１重量部を溶解し、プレートヒータ
ーで加熱殺菌し、次いで濃度７０％に濃縮し、無菌状態
で缶詰して製品を得た。本品は、温和な甘味で、風味も
よく、乳幼児食品、フルーツ、コーヒー、ココア、紅茶
などの調味用に有利に利用できる。Example B-5 Sweetened condensed milk 3 parts by weight of the sugar-containing syrup having a reduced reducibility obtained by the method of Example A-3 and 1 part by weight of sucrose were dissolved in 100 parts by weight of raw milk, and the mixture was mixed with a plate heater. The product was heat-sterilized, then concentrated to a concentration of 70%, and aseptically canned to obtain a product. The product has a mild sweetness and a good flavor, and can be advantageously used for seasoning baby food, fruit, coffee, cocoa, tea and the like.

【０１５５】[0155]

【実施例Ｂ−６ 乳酸菌飲料】脱脂粉乳１７５重量部、
実施例Ａ−５の方法で得た還元性を低減させた糖質高含
有粉末８０重量部及び特開平４−２８１７９５号公報で
開示されているラクトスクロース高含有粉末５０重量部
を水１，２００重量部に溶解し、６５℃で３０分間殺菌
し、４０℃に冷却後、これに、常法に従って、乳酸菌の
スターターを３０重量部植菌し、３７℃で８時間培養し
て乳酸菌飲料を得た。本品は、風味良好な乳酸菌飲料で
ある。また、本品は、オリゴ糖を含有し、乳酸菌を安定
に保持するだけでなく、ビフィズス菌増殖促進作用をも
有する。[Example B-6 Lactic acid bacterium beverage] 175 parts by weight of skim milk powder,
80 parts by weight of a sugar-rich powder having a reduced reducibility obtained by the method of Example A-5 and 50 parts by weight of a lactosucrose-rich powder disclosed in JP-A-4-281795 are added to 1,200 parts of water. Dissolve in 1 part by weight, sterilize at 65 ° C. for 30 minutes, cool to 40 ° C., inoculate with 30 parts by weight of a lactic acid bacterium starter in accordance with a conventional method, and culture at 37 ° C. for 8 hours to obtain a lactic acid bacterium drink It was This product is a lactic acid bacterium beverage with a good flavor. In addition, this product contains oligosaccharides and not only stably holds lactic acid bacteria, but also has a bifidobacteria growth promoting action.

【０１５６】[0156]

【実施例Ｂ−７ 粉末ジュース】噴霧乾燥により製造し
たオレンジ果汁粉末３３重量部に対して、実施例Ａ−５
の方法で得た還元性を低減させた糖質粉末５０重量部、
蔗糖１０重量部、無水クエン酸０．６５重量部、リンゴ
酸０．１重量部、Ｌ−アスコルビン酸０．１重量部、ク
エン酸ソーダ０．１重量部、プルラン０．５重量部、粉
末香料適量をよく混合撹拌し、粉砕し微粉末にしてこれ
を流動層造粒機に仕込み、排風温度４０℃とし、これ
に、実施例Ａ−６の方法で得たトレハロース高含有シラ
ップをバインダーとしてスプレーし、３０分間造粒し、
計量、包装して製品を得た。本品は、果汁含有率約３０
％の粉末ジュースである。また、本品は異味、異臭がな
く、長期に安定であった。[Example B-7 Powdered juice] 33 parts by weight of orange juice powder produced by spray drying was used, and Example A-5 was used.
50 parts by weight of a reduced sugar powder obtained by the method of
Sucrose 10 parts by weight, anhydrous citric acid 0.65 parts by weight, malic acid 0.1 parts by weight, L-ascorbic acid 0.1 parts by weight, sodium citrate 0.1 parts by weight, pullulan 0.5 parts by weight, powdered flavor An appropriate amount was thoroughly mixed and stirred, pulverized into a fine powder, charged into a fluidized bed granulator, and the exhaust air temperature was set to 40 ° C., and the trehalose-rich syrup obtained by the method of Example A-6 was used as a binder. Spray, granulate for 30 minutes,
The product was obtained by weighing and packaging. This product has a fruit juice content of about 30.
% Powdered juice. In addition, this product had no offensive taste or odor and was stable for a long period of time.

【０１５７】[0157]

【実施例Ｂ−８ カスタードクリーム】コーンスターチ
１００重量部、実施例Ａ−６の方法で得た還元性を低減
させた糖質含有シラップ１００重量部、マルトース８０
重量部、蔗糖２０重量部及び食塩１重量部を充分に混合
し、鶏卵２８０重量部を加えて撹拌し、これに沸騰した
牛乳１，０００重量部を徐々に加え、更に、これを火に
かけて撹拌を続け、コーンスターチが完全に糊化して全
体が半透明になった時に火を止め、これを冷却して適量
のバニラ香料を加え、計量、充填、包装して製品を得
た。本品は、なめらかな光沢を有し、温和な甘味で美味
である。Example B-8 Custard Cream 100 parts by weight of corn starch, 100 parts by weight of syrup containing a reduced sugar obtained by the method of Example A-6 and maltose 80
Parts by weight, 20 parts by weight of sucrose and 1 part by weight of salt are thoroughly mixed, 280 parts by weight of chicken egg are added and stirred, 1,000 parts by weight of boiled milk are gradually added to this, and the mixture is further heated to stir. Then, when the cornstarch was completely gelatinized and the whole became translucent, the heat was turned off, this was cooled, an appropriate amount of vanilla flavor was added, and the product was weighed, filled and packaged. This product has a smooth luster and is mildly sweet and delicious.

【０１５８】[0158]

【実施例Ｂ−９ ういろうの素】米粉９０重量部に、コ
ーンスターチ２０重量部、蔗糖４０重量部、実施例Ａ−
５の方法で得た還元性を低減させた糖質粉末８０重量部
及びプルラン４重量部を均一に混合してういろうの素を
製造した。ういろうの素と適量の抹茶と水とを混練し、
これを容器に入れて６０分間蒸し上げて抹茶ういろうを
製造した。本品は、照り、口当りも良好で、風味も良
い。また、澱粉の老化も抑制され、日持ちも良い。[Example B-9 Uironomoto] 90 parts by weight of rice flour, 20 parts by weight of corn starch, 40 parts by weight of sucrose, Example A-
80 parts by weight of the sugar powder having reduced reducibility obtained by the method of 5 and 4 parts by weight of pullulan were uniformly mixed to produce iuirojin. Knead Uironomoto, an appropriate amount of matcha and water,
This was put in a container and steamed for 60 minutes to produce matcha uiro. This product has good shine, mouthfeel and good flavor. In addition, the aging of starch is suppressed and the shelf life is good.

【０１５９】[0159]

【実施例Ｂ−１０ あん】原料あずき１０重量部に、常
法に従って、水を加えて煮沸し、渋切り、あく抜きし
て、水溶性夾雑物を除去して、あずきつぶあん約２１重
量部を得た。この生あんに、蔗糖１４重量部、実施例Ａ
−９の方法で得た還元性を低減させた糖質含有シラップ
５重量部及び水４重量部を加えて煮沸し、これに少量の
サラダオイルを加えてつぶあんをこわさないように練り
上げ、製品のあんを約３５重量部得た。本品は、色焼け
もなく、舌ざわりもよく、風味良好で、あんパン、まん
じゅう、だんご、もなか、氷菓などのあん材料として好
適である。[Example B-10 Apricot] 10 parts by weight of raw azuki bean was added with water according to a conventional method, and boiled, astringently cut, and drained to remove water-soluble impurities, and about 21 parts by weight of azuki bean paste. Obtained. This raw bean paste, 14 parts by weight of sucrose, Example A
-9. Add 5 parts by weight of the sugar-containing syrup having reduced reducibility and 4 parts by weight of water obtained by the method of No. 9 and boil, add a small amount of salad oil to this, and knead so as not to break the mash. About 35 parts by weight of bean paste was obtained. This product has no color burn, has a good texture, and has a good flavor, and is suitable as a material for bean paste such as bean paste, manju, dango, monaka, and frozen dessert.

【０１６０】[0160]

【実施例Ｂ−１１ パン】小麦粉１００重量部、イース
ト２重量部、砂糖５重量部、実施例Ａ−７の方法で得た
還元性を低減させた糖質含有粉末１重量部及び無機フー
ド０．１重量部を、常法に従って、水でこね、中種を２
６℃で２時間発酵させ、その後３０分間熟成し、焼き上
げた。本品は、色相、すだちともに良好で適度な弾力、
温和な甘味を有する高品質のパンである。Example B-11 Bread 100 parts by weight of wheat flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of sugar-containing powder having reduced reducibility obtained by the method of Example A-7 and 0 of inorganic food. 1 part by weight is kneaded with water according to the usual method, and 2 seeds are added.
It was fermented at 6 ° C. for 2 hours, then aged for 30 minutes and baked. This product has good hue and sueda, and has moderate elasticity.
High quality bread with mild sweetness.

【０１６１】[0161]

【実施例Ｂ−１２ ハム】豚もも肉１，０００重量部に
食塩１５重量部及び硝酸カリウム３重量部を均一にすり
込んで、冷室に１昼夜堆積する。これを水５００重量
部、食塩１００重量部、硝酸カリウム３重量部、実施例
Ａ−８の方法で得た還元性を低減させた糖質含有粉末４
０重量部及び香辛料からなる塩漬液に冷室で７日間漬け
込み、次いで、常法に従い、冷水で洗浄し、ひもで巻き
締め、燻煙し、クッキングし、冷却包装して製品を得
た。本品は、色合いもよく、風味良好な高品質のハムで
ある。[Example B-12 Ham] 1,000 parts by weight of pork thigh meat are uniformly rubbed with 15 parts by weight of salt and 3 parts by weight of potassium nitrate, and are deposited in a cold room for one day. 500 parts by weight of water, 100 parts by weight of salt, 3 parts by weight of potassium nitrate, and sugar-containing powder 4 obtained by the method of Example A-8 and having reduced reducibility
The product was dipped in a salted solution consisting of 0 part by weight and a spice in a cold room for 7 days, then washed with cold water, wrapped with a string, smoked, cooked, and cold-packed according to a conventional method to obtain a product. This product is a high quality ham with good color and flavor.

【０１６２】[0162]

【実施例Ｂ−１３ 粉末ペプチド】濃度４０％食品用大
豆ペプチド溶液、不二製油株式会社製『ハイニュート
Ｓ』１重量部に、実施例Ａ−８の方法で得た還元性を低
減させた糖質粉末２重量部を混合し、プラスチック製バ
ットに入れ、５０℃で減圧乾燥し、粉砕して粉末ペプチ
ドを得た。本品は、風味良好で、プレミックス、冷菓な
どの製菓用材料としてのみならず、経口流動食、経管流
動食などの離乳食、治療用栄養剤などとしても有利に利
用できる。[Example B-13 Powdered Peptide] The reducibility obtained by the method of Example A-8 was reduced to 1 part by weight of a 40% concentration soybean peptide solution for foods, "High New S" manufactured by Fuji Oil Co., Ltd. 2 parts by weight of sugar powder were mixed, put in a plastic vat, dried under reduced pressure at 50 ° C., and pulverized to obtain a powdered peptide. The product has a good flavor and can be advantageously used not only as a material for confectionery such as premixes and frozen desserts, but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient.

【０１６３】[0163]

【実施例Ｂ−１４ 化粧用クリーム】モノステアリン酸
ポリオキシエチレングリコール２重量部、自己乳化型モ
ノステアリン酸グリセリン５重量部、実施例Ａ−５の方
法で得た還元性を低減させた糖質粉末２重量部、α−グ
リコシル ルチン１重量部、流動パラフィン１重量部、
トリオクタン酸グリセリル１０重量部及び防腐剤の適量
を、常法に従って加熱溶解し、これにＬ−乳酸２重量
部、１，３−ブチレングリコール５重量部及び精製水６
６重量部を加え、ホモゲナイザーにかけ乳化し、更に香
料の適量を加えて撹拌混合しクリームを製造した。本品
は、抗酸化性を有し、安定性が高く、高品質の日焼け止
め、美肌剤、色白剤などとして有利に利用できる。[Example B-14 Cosmetic cream] Polyoxyethylene glycol monostearate 2 parts by weight, self-emulsifying type glyceryl monostearate 5 parts by weight, reduced reducing sugar obtained by the method of Example A-5 Powder 2 parts by weight, α-glycosyl rutin 1 part by weight, liquid paraffin 1 part by weight,
10 parts by weight of glyceryl trioctanoate and an appropriate amount of a preservative are dissolved by heating according to a conventional method, and 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 6 parts of purified water are dissolved therein.
6 parts by weight were added, emulsified by applying a homogenizer, an appropriate amount of flavor was further added, and the mixture was stirred and mixed to produce a cream. The product has antioxidant properties and high stability, and can be advantageously used as a high-quality sunscreen, skin-refining agent, skin-whitening agent and the like.

【０１６４】[0164]

【実施例Ｂ−１５ 固体製剤】ヒト天然型インターフェ
ロン−α標品（株式会社林原生物化学研究所製）を、常
法に従って、固定化抗ヒトインターフェロン−α抗体カ
ラムにかけ、該標品に含まれるヒト天然型インターフェ
ロン−αを吸着させ、安定剤であるウシ血清アルブミン
を素通りさせて除去し、次いで、ｐＨを変化させて、ヒ
ト天然型インターフェロン−αを実施例Ａ−７の方法で
得た還元性を低減させた糖質粉末を５％含有する生理食
塩水を用いて溶出した。本液を精密濾過し、約２０倍量
の株式会社林原商事販売無水結晶マルトース粉末『ファ
イントース』に加えて脱水、粉末化し、これを打錠機に
て打錠し、１錠（約２００ｍｇ）当たりヒト天然型イン
ターフェロン−αを約１５０単位含有する錠剤を得た。
本品は、舌下錠などとして、一日当たり、大人１乃至１
０錠程度が経口的に投与され、ウイルス性疾患、アレル
ギー性疾患、リューマチ、糖尿病、悪性腫瘍などの治療
に有利に利用できる。とりわけ、近年、患者数の急増し
ているエイズ、肝炎などの治療剤として有利に利用でき
る。本品は、本発明の非還元性糖質と無水結晶マルトー
スが共に安定剤として作用し、室温で放置してもその活
性を長期間よく維持する。[Example B-15 Solid formulation] A natural human interferon-α preparation (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was applied to an immobilized anti-human interferon-α antibody column according to a conventional method, and contained in the preparation. Human natural interferon-α was adsorbed and the stabilizer, bovine serum albumin, was passed through to be removed, and then the pH was changed to obtain human natural interferon-α obtained by the method of Example A-7. Elution was performed using a physiological saline solution containing 5% of sugar powder having reduced sex. This solution is subjected to microfiltration and added to about 20 times the amount of anhydrous crystalline maltose powder "Finetose" sold by Hayashibara Shoji Co., Ltd., dehydrated and powdered, and then tableted with a tablet machine to give 1 tablet (about 200 mg). A tablet containing about 150 units of human natural interferon-α was obtained.
This product is used as a sublingual tablet for adults 1 to 1 per day.
About 0 tablets are orally administered and can be advantageously used for treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., for which the number of patients is increasing rapidly in recent years. In this product, the non-reducing sugar of the present invention and anhydrous crystalline maltose both act as stabilizers, and their activity is maintained well for a long period of time even when left at room temperature.

【０１６５】[0165]

【実施例Ｂ−１６ 糖衣錠】重量１５０ｍｇの素錠を芯
剤とし、これに実施例Ａ−８の方法で得た還元性を低減
させた糖質粉末４０重量部、プルラン（平均分子量２０
万）２重量部、水３０重量部、タルク２５重量部及び酸
化チタン３重量部からなる下掛け液を用いて錠剤重量が
約２３０ｍｇになるまで糖衣し、次いで、同じ還元性を
低減させた糖質粉末６５重量部、プルラン１重量部及び
水３４重量部からなる上掛け液を用いて、糖衣し、更
に、ロウ液で艶出しして光沢の在る外観の優れた糖衣錠
を得た。本品は、耐衝撃性にも優れており、高品質を長
期間維持する。[Example B-16 Sugar-coated tablet] A plain tablet having a weight of 150 mg was used as a core, and 40 parts by weight of sugar powder having reduced reducibility obtained by the method of Example A-8 and pullulan (average molecular weight 20
10,000) 2 parts by weight, 30 parts by weight of water, 25 parts by weight of talc, and 3 parts by weight of titanium oxide were used for sugar coating until the tablet weight became approximately 230 mg, and then the same reducing sugar was reduced. Sugar coating was carried out using an overcoating liquid consisting of 65 parts by weight of fine powder, 1 part by weight of pullulan and 34 parts by weight of water, and was further polished with a wax solution to obtain a sugar-coated tablet having an excellent glossy appearance. This product has excellent impact resistance and maintains high quality for a long time.

【０１６６】[0166]

【実施例Ｂ−１７ 練歯磨】 配合 第２リン酸カルシウム ４５．０重量部 プルラン ２．９５重量部 ラウリル硫酸ナトリウム １．５重量部 グリセリン ２０．０重量部 ポリオキシエチレンソルビタンラウレート ０．５重量部 防腐剤 ０．０５重量部 実施例Ａ−５の方法で得た還元性を低減させた糖質粉末 １２．０重量部 マルチトール ５．０重量部 水 １３．０重量部 上記の材料を常法に従って混合し、練歯磨を得た。本品
は、適度の甘味を有しており、特に子供用練歯磨として
好適である。[Example B-17 Toothpaste] Blended dicalcium phosphate 45.0 parts by weight Pullulan 2.95 parts by weight Sodium lauryl sulfate 1.5 parts by weight Glycerin 20.0 parts by weight Polyoxyethylene sorbitan laurate 0.5 parts by weight Antiseptic Agent 0.05 parts by weight Sugar powder with reduced reducibility obtained by the method of Example A-5 12.0 parts by weight Maltitol 5.0 parts by weight Water 13.0 parts by weight The above materials were prepared according to a conventional method. Mix to obtain a toothpaste. The product has a moderate sweetness and is particularly suitable as a toothpaste for children.

【０１６７】[0167]

【実施例Ｂ−１８ 流動食用固体製剤】実施例Ａ−７の
方法で製造した還元性を低減させた糖質粉末５００重量
部、粉末卵黄２７０重量部、脱脂粉乳２０９重量部、塩
化ナトリウム４．４重量部、塩化カリウム１．８重量
部、硫酸マグネシウム４重量部、チアミン０．０１重量
部、アスコルビン酸ナトリウム０．１重量部、ビタミン
Ｅアセテート０．６重量部及びニコチン酸アミド０．０
４重量部からなる配合物を調製し、この配合物２５グラ
ムずつ防湿性ラミネート小袋に充填し、ヒートシールし
て製品を得た。本品は、１袋分を約１５０乃至３００ｍ
ｌの水に溶解して流動食とし、経口的、又は鼻腔、胃、
腸などへ経管的使用方法により利用され、生体へのエネ
ルギー補給用に有利に利用できる。[Example B-18 Solid formulation for liquid edibles] 500 parts by weight of sugar powder having reduced reducibility produced by the method of Example A-7, 270 parts by weight of powdered egg yolk, 209 parts by weight of skim milk powder, sodium chloride 4. 4 parts by weight, potassium chloride 1.8 parts by weight, magnesium sulfate 4 parts by weight, thiamine 0.01 parts by weight, sodium ascorbate 0.1 part by weight, vitamin E acetate 0.6 parts by weight and nicotinic acid amide 0.0.
A compound consisting of 4 parts by weight was prepared, and 25 g of this compound was filled in a moisture-proof laminated pouch and heat-sealed to obtain a product. This product is about 150 to 300m per bag
Orally, or nasal cavity, stomach,
It is used in the intestine and the like by a tube-based method, and can be advantageously used for supplying energy to the living body.

【０１６８】[0168]

【実施例Ｂ−１９ 外傷治療用膏薬】実施例Ａ−５の方
法で製造した還元性を低減させた糖質粉末２００重量部
及びマルトース３００重量部に、ヨウ素３重量部を溶解
したメタノール５０重量部を加え混合し、更に１０ｗ／
ｖ％プルラン水溶液２００重量部を加えて混合し、適度
の延び、付着性を示す外傷治療用膏薬を得た。本品は、
ヨウ素による殺菌作用のみならず、トレハロースによる
細胞へのエネルギー補給剤としても作用することから、
治癒期間が短縮され、創面もきれいに治る。Example B-19 Trauma plaster 50 parts by weight of methanol prepared by dissolving 3 parts by weight of iodine in 200 parts by weight of reduced sugar powder and 300 parts by weight of maltose produced by the method of Example A-5 Add 10 parts / mixing
200 parts by weight of a v% pullulan aqueous solution was added and mixed to obtain a plaster for treating wounds, which has a proper extension and shows adhesiveness. This product is
Not only the bactericidal action of iodine, but also the action of trehalose as an energy supplement to cells,
The healing period is shortened and the wound is healed neatly.

【０１６９】[0169]

【発明の効果】上記から明らかなように、本発明の分子
中にトレハロース構造を有する糖質及び／又はトレハロ
ースからなる非還元性糖質とともに澱粉糖アルコールを
含有してなる還元性を低減させた糖質は、安定性に優
れ、良質で上品な甘味を有している。また、経口摂取に
より消化吸収され、カロリー源となる。とりわけ、これ
に含まれるトレハロースは、よく代謝利用される。従っ
て、本発明の還元性を低減させた糖質は、甘味料、呈味
改良剤、品質改良剤、安定剤、賦形剤などとして、各種
飲食物、化粧品、医薬品など各種組成物に有利に利用で
きる。また、本発明の還元性を低減させた糖質の原料用
低還元性糖質としては、澱粉を液化した溶液に、非還元
性糖質生成酵素を澱粉枝切酵素及び／又はシクロマルト
デキストリン・グルカノトランスフェラーゼとともに作
用させることにより、澱粉からの分子中にトレハロース
構造を有する糖質やトレハロースなどの非還元性糖質の
収量を高め、比較的低分子、低粘度で取扱い容易な低還
元性糖質が得られ、また、マルトースにマルトース・ト
レハロース変換酵素を作用させることにより、トレハロ
ースとマルトースとの混合糖質が得られ、これらは、い
ずれも本発明の原料糖質として好適であり、本発明の工
業実施を極めて容易にする。EFFECTS OF THE INVENTION As is clear from the above, the reducing property of the starch of the present invention containing a starch sugar alcohol together with a sugar having a trehalose structure and / or a non-reducing sugar consisting of trehalose is reduced. Carbohydrates have excellent stability, good quality and elegant sweetness. Also, it is digested and absorbed by ingestion and becomes a calorie source. In particular, the trehalose contained in it is often metabolically utilized. Therefore, the sugar having reduced reducibility according to the present invention is advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc., in various compositions such as various foods, drinks, cosmetics and pharmaceuticals. Available. Further, as the low-reducing sugar for use as a raw material for sugar with reduced reducibility according to the present invention, a non-reducing sugar-forming enzyme is added to a starch debranching enzyme and / or cyclomaltodextrin in a liquefied starch solution. By acting together with glucanotransferase, the yield of non-reducing sugars such as trehalose having trehalose structure in the molecule from starch is increased, and relatively low molecular weight, low viscosity and easy-to-handle low reducing sugar Quality is obtained, and by treating maltose with maltose / trehalose converting enzyme, a mixed sugar of trehalose and maltose can be obtained, which are all suitable as raw material sugars of the present invention. Greatly facilitates industrial implementation of.

【０１７０】本発明の確立は、安価で無限の資源である
澱粉から、従来望むべくして容易に得られなかった非還
元性糖質、還元性を低減させた糖質を工業的に大量かつ
安価に提供できる全く新しい道を拓くこととなり、それ
が与える影響は、食品、化粧品、医薬品業界は言うに及
ばず、農水畜産業、化学工業にも及びこれら産業界に与
える工業的意義は計り知れないものがある。The present invention was established by industrially producing a large amount of non-reducing sugars and sugars with reduced reducibility which were not easily obtained as desired from starch, which is an inexpensive and unlimited resource. It will open up a completely new way to provide at low cost, and its impact will not only be on the food, cosmetics and pharmaceutical industries, but also on the agricultural, aquatic and livestock industries, and the chemical industry, and its industrial significance to these industries is immeasurable. There are some that are not.

【図面の簡単な説明】[Brief description of drawings]

【図１】リゾビウム・スピーシーズ Ｍ−１１由来の非
還元性糖質生成酵素の活性に及ぼす温度の影響を示す図
である。FIG. 1 is a diagram showing the effect of temperature on the activity of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.

【図２】リゾビウム・スピーシーズ Ｍ−１１由来の非
還元性糖質生成酵素の活性に及ぼすｐＨの影響を示す図
である。FIG. 2 is a graph showing the effect of pH on the activity of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.

【図３】リゾビウム・スピーシーズ Ｍ−１１由来の非
還元性糖質生成酵素の温度安定性を示す図である。FIG. 3 is a view showing the temperature stability of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.

【図４】リゾビウム・スピーシーズ Ｍ−１１由来の非
還元性糖質生成酵素のｐＨ安定性を示す図である。FIG. 4 is a diagram showing pH stability of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.

【図５】アルスロバクター・スピーシーズ Ｑ３６由来
の非還元性糖質生成酵素の活性に及ぼす温度の影響を示
す図である。FIG. 5 is a graph showing the effect of temperature on the activity of a non-reducing saccharide-forming enzyme derived from Arthrobacter species Q36.

【図６】アルスロバクター・スピーシーズ Ｑ３６由来
の非還元性糖質生成酵素の活性に及ぼすｐＨの影響を示
す図である。FIG. 6 is a graph showing the effect of pH on the activity of a non-reducing saccharide-forming enzyme derived from Arthrobacter species Q36.

【図７】アルスロバクター・スピーシーズ Ｑ３６由来
の非還元性糖質生成酵素の温度安定性を示す図である。FIG. 7 is a view showing temperature stability of a non-reducing saccharide-forming enzyme derived from Arthrobacter species Q36.

【図８】アルスロバクター・スピーシーズ Ｑ３６由来
の非還元性糖質生成酵素のｐＨ安定性を示す図である。FIG. 8 is a diagram showing pH stability of a non-reducing saccharide-forming enzyme derived from Arthrobacter species Q36.

【図９】ＤＥＡＥ−トヨパールからの本発明のトレハロ
ース遊離酵素と非還元性糖質生成酵素の溶出パターンを
示す図である。FIG. 9 is a diagram showing elution patterns of trehalose-releasing enzyme and non-reducing sugar-forming enzyme of the present invention from DEAE-Toyopearl.

【図１０】本発明のリゾビウム・スピーシーズ Ｍ−１
１由来のトレハロース遊離酵素の酵素活性に及ぼす温度
の影響を示す図である。FIG. 10: Rhizobium species M-1 of the present invention
It is a figure which shows the influence of the temperature on the enzyme activity of the trehalose free enzyme derived from 1.

【図１１】本発明のリゾビウム・スピーシーズ Ｍ−１
１由来のトレハロース遊離酵素の酵素活性に及ぼすｐＨ
の影響を示す図である。FIG. 11: Rhizobium species M-1 of the present invention
On the enzymatic activity of trehalose-releasing enzyme derived from 1
It is a figure which shows the influence of.

【図１２】本発明のリゾビウム・スピーシーズ Ｍ−１
１由来のトレハロース遊離酵素の安定性に及ぼす温度の
影響を示す図である。FIG. 12: Rhizobium species M-1 of the present invention
It is a figure which shows the influence of the temperature on the stability of the trehalose free enzyme derived from 1.

【図１３】本発明のリゾビウム・スピーシーズ Ｍ−１
１由来のトレハロース遊離酵素の安定性に及ぼすｐＨの
影響を示す図である。FIG. 13: Rhizobium species M-1 of the present invention
It is a figure which shows the influence of pH on the stability of the trehalose free enzyme derived from 1.

【図１４】本発明のアルスロバクター・スピーシーズ
Ｑ３６由来のトレハロース遊離酵素の酵素活性に及ぼす
温度の影響を示す図である。FIG. 14: Arthrobacter species of the present invention
It is a figure which shows the influence of the temperature on the enzyme activity of the trehalose free enzyme derived from Q36.

【図１５】本発明のアルスロバクター・スピーシーズ
Ｑ３６由来のトレハロース遊離酵素の酵素活性に及ぼす
ｐＨの影響を示す図である。FIG. 15: Arthrobacter species of the present invention
It is a figure which shows the influence of pH on the enzyme activity of the trehalose free enzyme derived from Q36.

【図１６】本発明のアルスロバクター・スピーシーズ
Ｑ３６由来のトレハロース遊離酵素の安定性に及ぼす温
度の影響を示す図である。FIG. 16: Arthrobacter species of the present invention
It is a figure which shows the influence of the temperature on the stability of the trehalose releasing enzyme derived from Q36.

【図１７】本発明のアルスロバクター・スピーシーズ
Ｑ３６由来のトレハロース遊離酵素の安定性に及ぼすｐ
Ｈの影響を示す図である。FIG. 17: Arthrobacter species of the present invention
P on the stability of trehalose-releasing enzyme derived from Q36
It is a figure which shows the influence of H.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ａ６１Ｋ 31/70 ＡＤＤ ＡＤＳ 31/715 ＡＤＡ 47/26 Ｄ Ｂ 47/36 Ｄ Ｂ Ｃ０８Ｂ 37/00 Ｇ 7433−4Ｃ Ｃ１２Ｐ 19/14 Ｚ 7432−4Ｂ 19/18 7432−4Ｂ ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location A61K 31/70 ADD ADS 31/715 ADA 47/26 D B 47/36 D B C08B 37/00 G 7433-4C C12P 19/14 Z 7432-4B 19/18 7432-4B

Claims (14)

【特許請求の範囲】[Claims] 【請求項１】 分子中にトレハロース構造を有する糖質
及び／又はトレハロースからなる非還元性糖質とともに
澱粉糖アルコールを含有してなる還元性を低減させた糖
質。1. A sugar having a reduced reducibility, which comprises a starch sugar alcohol together with a sugar having a trehalose structure in the molecule and / or a non-reducing sugar consisting of trehalose. 【請求項２】 分子中にトレハロース構造を有する糖質
が、分子の末端にトレハロース構造を有する非還元性糖
質であるか、又は、分子の内部にトレハロース構造を有
する非還元性糖質であることを特徴とする請求項１記載
の還元性を低減させた糖質。2. The saccharide having a trehalose structure in the molecule is a non-reducing saccharide having a trehalose structure at the end of the molecule, or a non-reducing saccharide having a trehalose structure inside the molecule. The sugar having reduced reducibility according to claim 1, characterized in that 【請求項３】 澱粉糖アルコールが、ソルビトール、マ
ルチトール、マルトトリイトール、マルトテトライトー
ル及びマルトペンタイトールから選ばれる１種又は２種
以上の糖アルコールである請求項１又は２記載の還元性
を低減させた糖質。3. The reduction according to claim 1, wherein the starch sugar alcohol is one or more sugar alcohols selected from sorbitol, maltitol, maltotriitol, maltotetriitol and maltopentitol. Carbohydrate with reduced sex. 【請求項４】 分子中にトレハロース構造を有する糖質
及び／又はトレハロースからなる非還元性糖質とともに
還元性澱粉糖を含有する低還元性糖質を水素添加し、生
成する分子中にトレハロース構造を有する糖質及び／又
はトレハロースからなる非還元性糖質とともに澱粉糖ア
ルコールを含有する糖質を採取することを特徴とする還
元性を低減させた糖質の製造方法。4. A trehalose structure in a molecule produced by hydrogenating a sugar having a trehalose structure and / or a non-reducing sugar composed of trehalose in a molecule together with a low reducing sugar containing a reducing starch sugar. A method for producing a sugar having reduced reducibility, which comprises collecting a sugar containing a starch sugar alcohol together with a non-reducing sugar consisting of a saccharide having a salt and / or trehalose. 【請求項５】 分子中にトレハロース構造を有する糖質
及び／又はトレハロースからなる非還元性糖質とともに
還元性澱粉糖を含有する低還元性糖質が、還元性澱粉部
分分解物に非還元性糖質生成酵素又は非還元性糖質生成
酵素とともにトレハロース遊離酵素を作用させて得られ
るものであることを特徴とする請求項４記載の還元性を
低減させた糖質の製造方法。5. A low-reducing sugar containing a reducing starch sugar together with a sugar having a trehalose structure in the molecule and / or a non-reducing sugar consisting of trehalose is non-reducing to a partially decomposed product of reducing starch. The method for producing a saccharide having reduced reducibility according to claim 4, which is obtained by allowing a trehalose-releasing enzyme to act together with a saccharide-forming enzyme or a non-reducing saccharide-forming enzyme. 【請求項６】 還元性澱粉糖が、グルコース、マルトー
ス、マルトトリオース、マルトテトラオース及びマルト
ペンタオースから選ばれる１種又は２種以上の還元性澱
粉糖であることを特徴とする請求項４記載の還元性を低
減させた糖質の製造方法。6. The reducing starch sugar is one or more reducing starch sugars selected from glucose, maltose, maltotriose, maltotetraose and maltopentaose. A method for producing a sugar having reduced reducibility as described above. 【請求項７】 トレハロースからなる非還元性糖質とと
もに還元性澱粉部分分解物を含有する低還元性糖質が、
マルトースを含有する還元性澱粉部分分解物にマルトー
ス・トレハロース変換酵素を作用させて得られるもので
あることを特徴とする請求項４記載の還元性を低減させ
た糖質の製造方法。7. A low-reducing sugar containing a partially degraded reducing starch together with a non-reducing sugar consisting of trehalose,
The method for producing a sugar having reduced reducibility according to claim 4, which is obtained by allowing maltose-trehalose converting enzyme to act on a partially degraded product of reducing starch containing maltose. 【請求項８】 澱粉を液化した溶液に、非還元性糖質生
成酵素を澱粉枝切酵素及び／又はシクロマルトデキスト
リン・グルカノトランスフェラーゼとともに作用させる
か、又は非還元性糖質生成酵素及びトレハロース遊離酵
素を澱粉枝切酵素及び／又はシクロマルトデキストリン
・グルカノトランスフェラーゼとともに作用させ、得ら
れる分子中にトレハロース構造を有する糖質及び／又は
トレハロースからなる非還元性糖質とともに還元性澱粉
糖を含有してなる低還元性糖質を水素添加し、生成する
分子中にトレハロース構造を有する糖質及び／又はトレ
ハロースからなる非還元性糖質とともに澱粉糖アルコー
ルを含有する糖質を採取することを特徴とする還元性を
低減させた糖質の製造方法。8. A non-reducing saccharide-forming enzyme is allowed to act on a liquefied starch solution together with a starch debranching enzyme and / or cyclomaltodextrin glucanotransferase, or a non-reducing saccharide-forming enzyme and trehalose release. The enzyme is allowed to act together with starch debranching enzyme and / or cyclomaltodextrin glucanotransferase, and the resulting molecule contains a reducing starch sugar together with a sugar having a trehalose structure and / or a non-reducing sugar consisting of trehalose. And a sugar containing a starch sugar alcohol together with a sugar having a trehalose structure in the produced molecule and / or a non-reducing sugar composed of trehalose are collected. The method for producing a sugar having reduced reducibility. 【請求項９】 澱粉を液化した溶液が、濃度１０ｗ／ｗ
％以上の澱粉をＤＥ１５未満に液化した溶液である請求
項８記載の還元性を低減させた糖質の製造方法。9. A solution obtained by liquefying starch has a concentration of 10 w / w.
9. The method for producing a sugar having reduced reducibility according to claim 8, which is a solution in which a starch content of not less than 15% is liquefied to less than DE15. 【請求項１０】 分子中にトレハロース構造を有する糖
質が、分子の末端にトレハロース構造を有する非還元性
糖質であるか、又は、分子の内部にトレハロース構造を
有する非還元性糖質であることを特徴とする請求項８又
は９記載の還元性を低減させた糖質の製造方法。10. The saccharide having a trehalose structure in the molecule is a non-reducing saccharide having a trehalose structure at the end of the molecule, or a non-reducing saccharide having a trehalose structure inside the molecule. 10. The method for producing a sugar with reduced reducibility according to claim 8 or 9. 【請求項１１】 分子中にトレハロース構造を有する糖
質及び／又はトレハロースからなる非還元性糖質ととも
に還元性澱粉糖を含有する低還元性糖質を水素添加し、
分子中にトレハロース構造を有する糖質及び／又はトレ
ハロースからなる非還元性糖質とともに澱粉糖アルコー
ルを含有する糖質を生成せしめることを特徴とする該低
還元性糖質の還元性を低減させる方法。11. A low reducing sugar containing a reducing starch sugar is hydrogenated together with a sugar having a trehalose structure in the molecule and / or a non-reducing sugar consisting of trehalose,
A method for reducing reducibility of a low-reducing sugar, which comprises producing a sugar containing a starch sugar alcohol together with a sugar having a trehalose structure in the molecule and / or a non-reducing sugar consisting of trehalose . 【請求項１２】 分子中にトレハロース構造を有する糖
質及び／又はトレハロースからなる非還元性糖質ととも
に澱粉糖アルコールを含有してなる還元性を低減させた
糖質を含有せしめた組成物。12. A composition comprising a sugar having a trehalose structure in a molecule and / or a non-reducing sugar composed of trehalose and a sugar having reduced reducibility and containing a starch sugar alcohol. 【請求項１３】 還元性を低減させた糖質が、分子中に
トレハロース構造を有する糖質及び／又はトレハロース
からなる非還元性糖質とともに還元性澱粉糖を含有する
低還元性糖質を水素添加し、得られた糖質である請求項
１２記載の組成物。13. A saccharide having reduced reducibility is a saccharide having a trehalose structure in a molecule and / or a non-reducing saccharide consisting of trehalose and a low reducing saccharide containing a reducing starch saccharide. The composition according to claim 12, which is the sugar obtained by adding. 【請求項１４】 組成物が、飲食物、化粧品又は医薬品
である請求項１２又は１３記載の組成物。14. The composition according to claim 12, which is a food or drink, a cosmetic or a pharmaceutical product.
JP18221695A 1994-06-27 1995-06-27 Carbohydrate with reduced reducibility, its production method and use Expired - Lifetime JP4070250B2 (en)

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* Cited by examiner, † Cited by third party
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US5916371A (en) * 1996-12-10 1999-06-29 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Crystalline powdery saccsharide, its preparation and uses
US6200783B1 (en) 1997-10-16 2001-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for producing trehalose and sugar alcohols
US6268353B1 (en) 1998-09-03 2001-07-31 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method for inhibiting the formation of volatile aldehydes including their related compounds and/or the decomposition of fatty acids including their related compounds, and uses thereof
US6497862B2 (en) 2000-03-02 2002-12-24 Kabushiki Kaisha Hayashibara Composition for inhibiting body odor and uses thereof
US6576281B1 (en) 1997-12-26 2003-06-10 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method for inhibiting trimethylamine formation
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US7186535B1 (en) 1998-09-11 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
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US6126962A (en) * 1996-12-10 2000-10-03 Kabushiki Kaisha Hayashibara Seibutus Kagaku Kenkyujo Crystalline powdery saccharide, its preparation and uses
US5916371A (en) * 1996-12-10 1999-06-29 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Crystalline powdery saccsharide, its preparation and uses
US6200783B1 (en) 1997-10-16 2001-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for producing trehalose and sugar alcohols
US6576281B1 (en) 1997-12-26 2003-06-10 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method for inhibiting trimethylamine formation
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US6268353B1 (en) 1998-09-03 2001-07-31 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method for inhibiting the formation of volatile aldehydes including their related compounds and/or the decomposition of fatty acids including their related compounds, and uses thereof
US7186535B1 (en) 1998-09-11 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US7582463B2 (en) 1998-09-11 2009-09-01 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US7575900B2 (en) 1998-09-11 2009-08-18 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US6497862B2 (en) 2000-03-02 2002-12-24 Kabushiki Kaisha Hayashibara Composition for inhibiting body odor and uses thereof
JP2003183304A (en) * 2001-10-30 2003-07-03 Roquette Freres Soluble hydrogenated starch derivative containing indigestible food cellulose
WO2004071472A1 (en) * 2003-02-13 2004-08-26 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo SKIN PREPARATION FOR EXTERNAL USE CHARACTERIZED BY CONTAINING SUGAR DERIVATIVE OF α,α-TREHALOSE
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