JPH01108986A - Immobilized enzyme and production of polymeric branched dextrin composition using same - Google Patents
Immobilized enzyme and production of polymeric branched dextrin composition using sameInfo
- Publication number
- JPH01108986A JPH01108986A JP26733587A JP26733587A JPH01108986A JP H01108986 A JPH01108986 A JP H01108986A JP 26733587 A JP26733587 A JP 26733587A JP 26733587 A JP26733587 A JP 26733587A JP H01108986 A JPH01108986 A JP H01108986A
- Authority
- JP
- Japan
- Prior art keywords
- cyclodextrin
- enzyme
- immobilized enzyme
- branched dextrin
- material sheet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229920001353 Dextrin Polymers 0.000 title claims abstract description 44
- 239000004375 Dextrin Substances 0.000 title claims abstract description 44
- 235000019425 dextrin Nutrition 0.000 title claims abstract description 44
- 239000000203 mixture Substances 0.000 title claims abstract description 20
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 47
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims abstract description 44
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 12
- 238000005342 ion exchange Methods 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract 2
- 239000008103 glucose Substances 0.000 claims abstract 2
- 229920000642 polymer Polymers 0.000 claims description 22
- 229920002472 Starch Polymers 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 23
- 102000004190 Enzymes Human genes 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 13
- 239000011248 coating agent Substances 0.000 abstract description 4
- 239000003995 emulsifying agent Substances 0.000 abstract description 4
- 238000005349 anion exchange Methods 0.000 abstract description 3
- 238000000576 coating method Methods 0.000 abstract description 3
- 239000012530 fluid Substances 0.000 abstract 2
- 240000008042 Zea mays Species 0.000 abstract 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 abstract 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 abstract 1
- 235000005822 corn Nutrition 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 22
- 239000000243 solution Substances 0.000 description 15
- 239000000758 substrate Substances 0.000 description 15
- 239000000839 emulsion Substances 0.000 description 13
- 230000002195 synergetic effect Effects 0.000 description 10
- 230000014759 maintenance of location Effects 0.000 description 8
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 239000003205 fragrance Substances 0.000 description 6
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 6
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 6
- 235000012141 vanillin Nutrition 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 230000001804 emulsifying effect Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229940097362 cyclodextrins Drugs 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 229920001206 natural gum Polymers 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 229920000881 Modified starch Polymers 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000019426 modified starch Nutrition 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 239000004368 Modified starch Substances 0.000 description 1
- 241000178960 Paenibacillus macerans Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 235000009499 Vanilla fragrans Nutrition 0.000 description 1
- 244000263375 Vanilla tahitensis Species 0.000 description 1
- 235000012036 Vanilla tahitensis Nutrition 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- -1 sucrose fatty acid ester Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Compositions Of Macromolecular Compounds (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、イオン交換素材シートに吸着させた固定化サ
イクロデキストリングルカノトランスフェラーゼとこれ
を用いた乳化安定性及び被覆性に優れた高分子分岐デキ
ストリン組成物を製造する方法に関するものであり、こ
の製品は乳化剤や被覆剤として食品、薬品、化粧品等の
分野で有用である。Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an immobilized cyclodextrin glucanotransferase adsorbed on an ion exchange material sheet and a branched polymer with excellent emulsion stability and coating properties using the immobilized cyclodextrin glucanotransferase. The present invention relates to a method for producing a dextrin composition, and this product is useful as an emulsifier or coating agent in the fields of food, medicine, cosmetics, etc.
（従来の技術）
ワキシースターチにα−アミラーゼを作用させ軽度に分
解した低り、Ｅ、デキストリン（高分子分岐デキストリ
ン）はワキシースターチ以外の澱粉を原料としたデキス
トリンに比べて乾燥時の保香性あるいは乳化安定性に優
れていることがよく知られている。しかし、高分子分岐
デキストリンの乳化安定性については、天然ガム質や化
工澱粉の分解物と比べると同等かそれ以下である。(Prior art) Low-E dextrin (polymer branched dextrin), which is made by applying α-amylase to waxy starch and slightly decomposing it, has better flavor retention when dried than dextrins made from starches other than waxy starch. It is also well known that it has excellent emulsion stability. However, the emulsion stability of polymeric branched dextrins is equal to or lower than that of natural gums and decomposed products of modified starches.
一方、サイクロデキストリングルカノトランスフェラー
ゼ（以下ＣＧＴ ａ ｓ ｅと称する）が澱粉に作用し
てサイクロデキストリンを生成することも古くから知ら
れており実用化されている。また、ＣＧＴ ａ Ｓ ｅ
を作用させ製造したサイクロデキストリンは環状構造を
持っているために包接能力があり保香性や乳化性に優れ
ている。しかし、サイクロデキストリン単独では天然ガ
ム質や化工澱粉に比べて乳化安定性は十分ではない。ま
た、サイクロデキストリンは高価なため高分子分岐デキ
ストリンへのサイクロデキストリンの添加は経済的では
ない。On the other hand, it has been known for a long time that cyclodextrin glucanotransferase (hereinafter referred to as CGT ase) acts on starch to produce cyclodextrin, and has been put into practical use. Also, CGT a S e
Cyclodextrin produced by the action of cyclodextrin has a cyclic structure, has inclusion ability, and has excellent fragrance retention and emulsifying properties. However, cyclodextrin alone does not have sufficient emulsion stability compared to natural gum or modified starch. Furthermore, since cyclodextrin is expensive, it is not economical to add cyclodextrin to polymeric branched dextrin.
従来、低純度サイクロデキストリンがあるが、これはり
、巳２０以上のオリゴ糖や低分子デキストリンを含む物
であり、サイクロデキストリン以外のオリゴ糖や低分子
デキストリン単独では保香性や乳化性をもっていない。Conventionally, there is low-purity cyclodextrin, but this contains oligosaccharides and low-molecular-weight dextrins with a diameter of 20 mm or more, and oligosaccharides other than cyclodextrins and low-molecular-weight dextrins alone do not have flavor retention or emulsifying properties.
そのため低純度品の保香性や乳化安定性は主にサイクロ
デキストリンに起因するのみである。Therefore, the fragrance retention and emulsion stability of low-purity products are mainly due to cyclodextrin.
高分子分岐デキストリンにＣＧＴａＳｅをバッチ反応で
作用させサイクロデキストリンを生成ざぜる方法では、
長い反応時間を必要としさらに高濃度基質では高＠度の
酵素添加が必要である。このような問題を解決する手段
として、酵素の固定化という技術がある。中でも、イオ
ン交換樹脂は固定化の簡便さ酵素へのダメージの少なさ
から固定化酵素の担体としてよく用いられている。しか
し、ワキシースターチにα−アミラーゼ等を作用させ軽
度に分解した低り、Ｅ、デキストリンのように高分子量
で粘度が高いものを基質溶液とした場合、樹脂構造のた
めに高流速を得ることが困難で反応効率も低い。そのた
め、ＣＧＴ ａ ｓ ｅを樹脂に固定化した場合酵素の
利用効率は上昇しないという問題点がある。In the method of reacting CGTaSe with polymeric branched dextrin in a batch reaction to generate cyclodextrin,
Long reaction times are required, and high concentrations of substrates require high levels of enzyme addition. As a means to solve these problems, there is a technique called enzyme immobilization. Among these, ion exchange resins are often used as carriers for immobilized enzymes because of their ease of immobilization and little damage to enzymes. However, if the substrate solution is a substance with a high molecular weight and high viscosity such as dextrin, which has been slightly decomposed by the action of α-amylase on waxy starch, it is difficult to obtain a high flow rate due to the resin structure. It is difficult and the reaction efficiency is low. Therefore, there is a problem in that when CGTase is immobilized on a resin, the enzyme utilization efficiency does not increase.
（発明の目的）
従って、本発明の目的は従来の酵素の固定化法の改良を
行うことにあり、あわせて高分子分岐デキストリンの乳
化力をざらに高めた、乳化・被覆安定性に優れた高分子
分岐デキストリン組成物を提供することにある。(Objective of the Invention) Therefore, the object of the present invention is to improve the conventional enzyme immobilization method, and also to improve the emulsifying power of polymer branched dextrin, which has excellent emulsification and coating stability. An object of the present invention is to provide a polymeric branched dextrin composition.
（問題点を解決するための手段）
本発明者らは鋭意研究の結果、イオン交換素材シートを
ＣＧＴ ａ ｓ ｅの固定化担体として用いることによ
り高粘度の高分子分岐デキストリン溶液を高流速で通液
でき、ざらに高流速下においても固定化ＣＧＴ ａ ｓ
ｅの働きにより生成したサイクロデキストリンと高分
子分岐デキストリンとの相乗効果により乳化特性が高い
サイクロデキストリン共存高分子分岐デキストリン組成
物が得られることを見出し本発明を完成した。(Means for Solving the Problems) As a result of intensive research, the present inventors have found that by using an ion exchange material sheet as an immobilization carrier for CGTase, a highly viscous polymer branched dextrin solution can be passed through at a high flow rate. It is possible to immobilize CGT a s even under high flow rates.
The present invention was completed by discovering that a cyclodextrin-coexisting polymer branched dextrin composition with high emulsifying properties can be obtained through the synergistic effect of the cyclodextrin produced by the action of e and the polymer branched dextrin.
以下本発明について具体的に説明する。The present invention will be specifically explained below.
本発明で使用するＣＧＴ ａ ｓ ｅは、既知のものを
任意に用いることができる。例えば、バチルス・マセラ
ンスの生産する酵素が代表的なものとして知られている
。Any known CGT ase can be used in the present invention. For example, the enzyme produced by Bacillus macerans is known as a typical example.
固定化担体に用いるイオン交換素材シートは既知のもの
、例えばＬＫＢ社ＩＥＡＥゼータプレツブ、ＱＡＥゼー
タプレツブ、ＳＰゼータプレツブあるいはこれらと同等
のものを用いることができる。しかし酵素の等電点が反
応１）Ｈより低い場合はアニオン交換基、逆の場合はカ
チオン交換基のものを使用しなければならない。例えば
、マセランスＣＧＴａｓｅでは、弱塩基性のＤＥＡＥゼ
ータプレツブを用いることができる。As the ion exchange material sheet used as the immobilization carrier, known ones such as LKB's IEAE Zeta Pret, QAE Zeta Pret, SP Zeta Pret, or equivalent ones can be used. However, if the isoelectric point of the enzyme is lower than reaction 1) H, an anion exchange group must be used, and in the opposite case, a cation exchange group must be used. For example, in macerans CGTase, a weakly basic DEAE zeta pretub can be used.
固定化は酵素溶液をイオン交換素材シートカートリッジ
へ通液することによって行う。基本的には通常のイオン
交換基を持ったものと同様に行う。Immobilization is performed by passing the enzyme solution through an ion exchange material sheet cartridge. Basically, it is carried out in the same way as for those with ordinary ion exchange groups.
すなわち交換基を酸またはアルカリ溶液で遊離型にした
俊洗浄し前処理を行う。このように調整した当該イオン
交換素材シートカートリッジにＣＧＴａＳｅ溶液（活性
０．（）５〜１７Ｕ／ｍｌ好ましくは０．５〜２Ｕ／ｍ
ｌ＞を流速５ｖ＝１〜１００ 好ましくは５Ｖ＝５〜１
５で通液する。活性が漏れる県会、流出液を再循環させ
固定化してもよい。なお、本発明において、酵素活性は
、可溶性澱粉５％濃度を基質としてｐＨ６，０，５゛Ｏ
℃の反応条件で１分間に１ｍｇのサイクロデキストリン
を生成する酵素量を１単位（Ｕ）として表わすことにす
る。サイクロデキストリンの定量は高速液体クロマトグ
ラフィーで７ミノカラム（信和化工製つルトロンＮＨ２
＞を用い、溶媒はアセトニトリル／水＝６５／３５で、
検出は示差屈折計で行う。That is, pretreatment is performed by washing the exchange group in a free form with an acid or alkaline solution. A CGTaSe solution (activity 0.() 5 to 17 U/ml, preferably 0.5 to 2 U/ml) is added to the ion exchange material sheet cartridge prepared in this way.
l> at a flow rate of 5V=1 to 100, preferably 5V=5 to 1
Pass the liquid through step 5. If the activity leaks, the effluent may be recirculated and immobilized. In addition, in the present invention, the enzyme activity is measured at pH 6.0.5゛O using 5% soluble starch as a substrate.
The amount of enzyme that produces 1 mg of cyclodextrin per minute under the reaction conditions of °C is expressed as 1 unit (U). Cyclodextrin was quantitatively determined by high-performance liquid chromatography using a 7-mino column (Shinwa Kako Tsurutron NH2).
>, the solvent was acetonitrile/water = 65/35,
Detection is performed using a differential refractometer.
本発明の固定化酵素では、固定化酵素量と基質濃度と流
速を選択組み合わせることにより目標とするサイクロデ
キストリン含量の高分子分岐デキストリン組成物を製造
することができる。すなわち０通液する基質溶液は、ワ
キシーコーンスターチをり、Ｅ、＝１０以下となるよう
に液化したものを用いる。液化の手段は酸処理でも酵素
処理でもよい。Ｄ、Ｅ、１０以上では高分子分岐デキス
トリンとしての特徴がなくなる。基質濃度は１〜５０％
好ましくは５〜３０％濃度を用いる。第１図に示したよ
うに基質濃度が高いとサイクロデキストリンを生成量は
低くなる。ｐＨ及び反応温度は使用した酵素がサイクロ
デキストリンを生成する条件で行えばよい。例えば、マ
セランスＣＧＴａｓｅの場合、ｐＨ６、反応温度５０℃
で行う。■流速は５Ｖ＝１００以下で行う。第１図に示
したように流速が速いほどサイクロデキストリン含量は
低くなる。■固定化酵素量が多いほど（イオン交換素材
シートカートリッジｄ当り最大２２００Ｕ ）サイクロ
デキストリン生成量は高くなる。With the immobilized enzyme of the present invention, a polymer branched dextrin composition having a target cyclodextrin content can be produced by selectively combining the amount of immobilized enzyme, substrate concentration, and flow rate. In other words, the substrate solution to be passed through is one obtained by liquefying waxy cornstarch so that E=10 or less. The means of liquefaction may be acid treatment or enzyme treatment. When D and E are 10 or more, the characteristics as a polymeric branched dextrin are lost. Substrate concentration is 1-50%
Preferably a concentration of 5-30% is used. As shown in FIG. 1, when the substrate concentration is high, the amount of cyclodextrin produced is low. The pH and reaction temperature may be such that the enzyme used produces cyclodextrin. For example, in the case of Macerans CGTase, pH 6, reaction temperature 50°C
Do it with (2) The flow rate is 5V=100 or less. As shown in FIG. 1, the faster the flow rate, the lower the cyclodextrin content. ■The larger the amount of immobilized enzyme (maximum 2200 U per ion exchange material sheet cartridge d), the higher the amount of cyclodextrin produced.
酵素固定化シートカートリッジは１週間連続運転中、更
にその後４℃で１力月間冷蔵しても活性は安定である。The activity of the enzyme-immobilized sheet cartridge is stable even if it is continuously operated for one week and then refrigerated at 4° C. for one month.
このようにして調製した高分子分岐デキストリン組成物
の乳化安定性はサイクロデキストリン含量が２％以上で
相乗効果が現れる。しかし、３０％以上あるいはり、Ｅ
、１０以上でデキストリンが低分子化しているため相乗
効果がなくなる。また、サイクロデキストリン濃度が低
いところでは、単にサイクロデキストリンと高分子分岐
デキストリンを混合した場合よりも相乗効果は大きくな
る。The emulsion stability of the polymer branched dextrin composition thus prepared exhibits a synergistic effect when the cyclodextrin content is 2% or more. However, if the E
, 10 or more, the synergistic effect disappears because the dextrin has a low molecular weight. Further, where the cyclodextrin concentration is low, the synergistic effect is greater than when simply mixing cyclodextrin and polymer branched dextrin.
更に、本発明により調製した組成物を乳化剤に使用して
製造したエマルジョンは耐酸・耐塩性に優れている。Furthermore, emulsions produced by using the composition prepared according to the present invention as an emulsifier have excellent acid and salt resistance.
次ぎに本発明の実施例を示すが、本発明はこれにより制
限される。ものではない。Next, examples of the present invention will be shown, but the present invention is limited thereby. It's not a thing.
実施例１．固定化酵素の調製
弱塩基性アニオン交換素材シートカートリッジ（ＬＫＢ
社製ＤＥＡＥゼータプレツブ２５０）にマセランスＣＧ
Ｔａｓｅ酵素液１３４００ｄ （１）Ｈ＆、 ２、活性
０，９１Ｕ／ｄ＞を流速５Ｖ＝８で通液し酵素を吸着固
定化し、本発明の固定化酵素を得た。固定化活性■はカ
ートリッジｍｌ当り４８．４Ｕであった。Example 1. Preparation of immobilized enzyme Weak basic anion exchange material sheet cartridge (LKB
DEAE Zeta Pretub 250) and Macerance CG
Tase enzyme solution 13400d (1) H&, 2, activity 0.91 U/d> was passed through the solution at a flow rate of 5V=8 to adsorb and immobilize the enzyme to obtain the immobilized enzyme of the present invention. The immobilization activity (■) was 48.4 U per ml of cartridge.
実施例２．固定化酵素を使用する高分子分岐デキストリ
ン組成物の製造法
実施例１で得た固定化酵素にり、Ｅ、５のワキシーコー
ンスターチ液化液（豊年製油■製ＦＺ−１００＞５％、
２０％濃度の各溶液（ｐｌ−１６，０＞を５０℃で流速
を変えて通液した（ＳＶ＝１〜ｉｏｏ ）。生成したサ
イクロデキストリン量をＨＰＬＣで定Ｕしてサイクロデ
キストリン含ｄを求めた。Example 2. Method for producing a polymeric branched dextrin composition using an immobilized enzyme Using the immobilized enzyme obtained in Example 1, liquefied waxy cornstarch in E.
Each solution (pl-16,0) with a concentration of 20% was passed through the solution at 50°C while changing the flow rate (SV = 1 to ioo).The amount of cyclodextrin produced was determined by HPLC to determine the cyclodextrin content. Ta.
比較例として弱塩基性アニオン交換樹脂アンバーライト
ＩＲＡ−９３（オルガノ製）を用いてＣＧＨａｓｅの固
定化を行った。樹脂９（Ｍ！を直径２ｃｔｎ長ざ２８
ｃｍのカラムに詰め、流速５Ｖ＝３゜７以外は上記シー
トの場合と同様に固定化を行った。固定化活性■は湿樹
脂”Ｉｄ当り９８ｔＪであつた。上記と同様に基質溶液
を流し、生成したサイクロデキストリンを定量した。As a comparative example, CGHase was immobilized using a weakly basic anion exchange resin Amberlite IRA-93 (manufactured by Organo). Resin 9 (M! diameter 2ctn length 28
It was packed in a 5 cm column, and immobilization was carried out in the same manner as in the case of the sheet, except that the flow rate was 5 V = 3°7. The immobilization activity (2) was 98 tJ per wet resin Id. The substrate solution was poured in the same manner as above, and the produced cyclodextrin was quantified.
以上の結果を第１図に示した。シート固定化、樹脂固定
化いずれの場合も流速が遅くなるとサイクロデキストリ
ン生成ｍが増加した。また、基質濃度が低くなるとサイ
クロデキストリン生成量が増加した。しかし、同じ基質
濃度、流速での比較では樹脂固定化よりもシート固定化
の方がサイクロデキストリンを多く生成した。すなわち
、シート固定化では樹脂固定化に比べ単位当りの酵素活
性は半分であるにもかかわらず、生成したサイクロデキ
ストリン含量は２倍であった。The above results are shown in Figure 1. In both cases of sheet immobilization and resin immobilization, as the flow rate decreased, cyclodextrin production m increased. Furthermore, as the substrate concentration decreased, the amount of cyclodextrin produced increased. However, when compared at the same substrate concentration and flow rate, sheet immobilization produced more cyclodextrin than resin immobilization. That is, although the enzyme activity per unit was half in sheet immobilization compared to resin immobilization, the produced cyclodextrin content was twice as high.
実施例３．固定化酵素の安定性
実施例１で得たＣＧＴ ａ ｓ ｅを固定化したシート
カートリッジ（本発明の固定化酵素）にり、Ｅ。Example 3. Stability of immobilized enzyme: CGTase obtained in Example 1 was immobilized on a sheet cartridge (immobilized enzyme of the present invention).
５の高分子分岐デキストリン（豊年製油■ＩｕＦＺ−１
００）を２０％Ｉ！度、ｐＨ６、＠度５０℃、流速５Ｖ
＝２．５で１週間通液した。これを冷蔵 庫で１力
月保管後、再び同一条件で通液した。サイクロデキスト
リンの生成量］（対固形分％）を第２図に示した。いず
れも１０％前１変で安定していた。No. 5 polymer branched dextrin (Honen Oil ■ IuFZ-1
00) to 20%I! degree, pH 6, @ degree 50℃, flow rate 5V
= 2.5 for one week. After storing this in the refrigerator for one month, the solution was passed again under the same conditions. The amount of cyclodextrin produced (% of solid content) is shown in FIG. Both were stable at 1 change before 10%.
比較例として、上述の基質溶液５０ｄに酵素量１３Ｕを
添加しバッチ反応を行った。反応開始１卦２４時間でサ
イクロデキストリン生成含量は９゜８％でプラトーに達
した。As a comparative example, a batch reaction was carried out by adding 13 U of enzyme to 50 d of the above-mentioned substrate solution. One 24 hours after the start of the reaction, the content of cyclodextrin produced reached a plateau at 9.8%.
酵素活性当りの高分子分岐デキストリン組成物生産量は
バッチ反応（０，７７ｏ／Ｕ）に比ベシート固定化（８
，７１；ｌ／Ｕ以上）したものは１１倍以上高くなった
。The amount of polymer branched dextrin composition produced per enzyme activity was higher in the batch reaction (0.77o/U) than in the batch reaction (0.77o/U).
, 71; l/U) was more than 11 times higher.
実施例４．酵素の固定化量
実施例１と同様にＤＥＡＥ−ゼータプレツブ１５に酵素
活性として１３０Ｕ、１３００Ｕ、６５００Ｕを固定化
した。基質としてり、６．２の高分子分岐デキストリン
（豊年製油ｎ製ＡＰＤＥＸ５０）を３０％濃度、ｐＨ６
，５０℃、５Ｖ−４゜３で通液した。生成した高分子分
岐デキストリン組成物のサイクロデキストリン含量は各
々２．０％、５．３％、１０％であった。固定化酵素量
が多くなるとサイクロデキストリン含量も高くなつた。Example 4. Amount of enzyme immobilized In the same manner as in Example 1, 130 U, 1300 U, and 6500 U of enzyme activity were immobilized on DEAE-zeta pretub 15. As a substrate, 6.2 polymer branched dextrin (APDEX50 manufactured by Hounen Oil Co., Ltd.) was used at a concentration of 30%, pH 6.
, 50°C, 5V-4°3. The cyclodextrin contents of the polymer branched dextrin compositions produced were 2.0%, 5.3%, and 10%, respectively. As the amount of immobilized enzyme increased, the cyclodextrin content also increased.
試験例１．保香性
実施例４で製造したサイクロデキストリン１０％含有す
る高分子分岐デキストリン組成物（ａ）の３０％溶液３
ｇにバニラエツセンスの主成分であるバニリンを３Ｎ添
加した。この溶液を凍結真空乾燥し粉末０．９ｇ得た。Test example 1. 30% solution 3 of polymer branched dextrin composition (a) containing 10% cyclodextrin produced in Fragrance Retention Example 4
3N of vanillin, which is the main component of vanilla essence, was added to g. This solution was freeze-dried in vacuum to obtain 0.9 g of powder.
比較例としてり、Ｅ、２の高分子分岐デキストリン（ｂ
）とり、Ｅ、１２のデキストリン（ｃ）を上述の（ａ＞
の代わりに用いて同様にして凍結乾燥粉末を得た。As a comparative example, E, 2 polymer branched dextrin (b
) and E, 12 dextrin (c) as above (a>
A lyophilized powder was obtained in the same manner using instead of .
保香性の評価法として、残存バニリン量を調べた。すな
わち、粉末を水２．１ｇで溶解しアミラーゼ処理しデキ
ストリンを分解復、クロロフォルムでバニリンを抽出し
ガスクロマトグラフィー（ＧＣ）で定量し残存バニリン
量を求めた。なお、屹燥前の水溶液（ａ＞ （ｂ）
（ｃ＞をアミラーゼ処理した（多のクロロフォルムに
よるバニリンの回収率は１００％であった。As a method for evaluating fragrance retention, the amount of residual vanillin was investigated. That is, the powder was dissolved in 2.1 g of water and treated with amylase to decompose and recover dextrin. Vanillin was extracted with chloroform and quantified by gas chromatography (GC) to determine the amount of remaining vanillin. In addition, the aqueous solution before drying (a> (b)
(c) was treated with amylase (the recovery rate of vanillin by chloroform was 100%).
これらの結果、バニリン残存率は（ａ）、（ｂ）、（Ｃ
）各々８０％、６７％、４３％であった。As a result, the vanillin residual rate is (a), (b), (C
) were 80%, 67%, and 43%, respectively.
なお、ＧＣの分析は、カラム；直径３＃ＩＸ長ざ１Ｔｒ
Ｌ１充填剤： ＦＦＡＰ １％Ｕｎｉｐｏｒｔ ＨＰ
６０／８０．温度：インジエクタ−２００℃ カラム１
５０℃、検出ＦＩＤで行った。In addition, for GC analysis, column; diameter 3#IX length 1Tr
L1 filler: FFAP 1% Uniport HP
60/80. Temperature: Injector - 200℃ Column 1
The test was carried out at 50°C using a detection FID.
試験例２．乳化性における相乗効果
大豆白絞油１５ｄと第１表に示した各種水溶液１５ｄを
５０ｄ容量の遠心管に取りヤマト科学製ウルトラディス
バイザーＬＫ２１を用いて回転数１０１００００ｒｐ分
間で乳化を行った。この遠心管を回転数５０００ｒｐｍ
で１０分間遠心し残った乳化層の割合で乳化安定性を評
価した（乳化層の割合が高いほど安定性に優れている）
。Test example 2. Synergistic effect on emulsifying property 15 d of soybean white squeezed oil and 15 d of various aqueous solutions shown in Table 1 were placed in a 50 d centrifugal tube and emulsified at a rotational speed of 1,010,000 rpm using Yamato Scientific's Ultra Disvisor LK21. Rotate this centrifuge tube at 5000 rpm.
The emulsion stability was evaluated by the percentage of the emulsified layer that remained after centrifugation for 10 minutes (the higher the percentage of the emulsified layer, the better the stability).
.
第１表に示したように、Ｄ、６．１０以下の高分子分岐
デキストリンとサイクロデキストリンの相乗効果は認め
られたが、Ｄ、Ｅ、１２ではサイクロデキストリンとの
相乗効果は認められなかった。As shown in Table 1, a synergistic effect was observed between the polymer branched dextrin of D, 6.10 or less, and cyclodextrin, but no synergistic effect with cyclodextrin was observed for D, E, and 12.
第１表
＊ ：サイクロデキストリン
＊＊ ： ５０００ｒｐｍで１０分間遠心侵の残存乳化
層デキストリン水溶液の固形分濃度３０％（Ｗ／Ｗ）試
験例３．混合との違い
試験例１で使用した本発明によりｉ、ｌ１１したサイク
ロデキストリン１０％含有高分子分岐デキストリン（Ａ
）、市販の純品サイクロデキストリン（Ｃ）、Ｄ、Ｅ、
５の高分子分岐デキストリンと（Ｃ）を混合した物（Ｂ
）、を第２表に示したような組成で試験例２と同様に乳
化を行い安定性を評価した。溶液のサイクロデキストリ
ン濃度が２．３％では、サイクロデキストリンと高分子
分岐デキストリンの相乗効果が認められた。サイクロデ
キストリン濃度が１％では、単に混合したもの（Ｂ）で
は相乗効果が認められなかったが、本発明によりｗ４製
したもの（ＡＪでは相乗効果が認められた。Table 1*: Cyclodextrin**: Residual emulsified layer after centrifugation at 5000 rpm for 10 minutes Solid content concentration of dextrin aqueous solution 30% (W/W) Test Example 3. Differences from mixing The polymer branched dextrin (A
), commercially available pure cyclodextrin (C), D, E,
A mixture of polymer branched dextrin in No. 5 and (C) (B
) was emulsified in the same manner as in Test Example 2 with the composition shown in Table 2, and the stability was evaluated. When the cyclodextrin concentration of the solution was 2.3%, a synergistic effect between cyclodextrin and polymer branched dextrin was observed. At a cyclodextrin concentration of 1%, no synergistic effect was observed in the simple mixture (B), but a synergistic effect was observed in the product made from w4 according to the present invention (AJ).
＊ ：サイクロデキストリン
＊＊ ： ５０００ｒｐｍで１０分間遠心後の残存乳化
層試験例４．乳化の耐酸・耐塩性
５％食塩水あるいはｐＨ３，５に調製した酢酸添加水あ
るいは純水に試験例３で用いた本発明（Ａ＞を３０％濃
度、シヨ糖脂肪酸エステル（ＨＬＢ６）を１％濃度、キ
サンタンガム（大日本製薬■製エコーガム）を０．２５
％濃度で各々溶解し、試験例２と同様に乳化を行った。* : Cyclodextrin ** : Residual emulsified layer test example 4 after centrifugation at 5000 rpm for 10 minutes. Acid resistance and salt resistance of emulsification The present invention used in Test Example 3 (30% concentration of A>, 1% sucrose fatty acid ester (HLB6) Concentration, xanthan gum (Echo gum manufactured by Dainippon Pharmaceutical ■) 0.25
% concentration, and emulsification was performed in the same manner as in Test Example 2.
乳化安定性は回転数２００Ｏｒｐｍで１分間遠心し残っ
た乳化層の割合で評価した。本発明品は耐酸・耐塩性が
あった。The emulsion stability was evaluated by the percentage of the emulsion layer remaining after centrifugation at a rotation speed of 200 rpm for 1 minute. The product of the present invention had acid and salt resistance.
第３表
〔発明の効果〕
本発明の固定化酵素を用いることによりバッチ法と比較
して１１倍以上の効率で、また従来の樹脂固定化酵素の
４倍以上の効率で、サイクロデキストリンを含む高分子
分岐デキストリンを製造することができ十分実用に耐え
れるものである。Table 3 [Effects of the Invention] By using the immobilized enzyme of the present invention, the efficiency is 11 times or more compared to the batch method, and the efficiency is 4 times or more than the conventional resin-immobilized enzyme. It is possible to produce a polymeric branched dextrin and is of sufficient practical use.
ざらに、本発明によって製造した高分子分岐デキストリ
ン組成物は従来の分岐デキストリンやサイクロデキスト
リンと比較して保香性・乳化安定性に優れたものである
のみならず、単に両者を混合した以上の効果を示す。ま
た、天然ガム質や従来の乳化剤よりも安定な乳化をつく
り耐酸・耐塩性に優れている。In general, the polymeric branched dextrin composition produced according to the present invention not only has superior fragrance retention and emulsion stability compared to conventional branched dextrins and cyclodextrins, but also has superior fragrance retention and emulsion stability compared to the conventional branched dextrins and cyclodextrins. Show effectiveness. It also creates a more stable emulsion than natural gums or conventional emulsifiers, and has excellent acid and salt resistance.
第１図は流速と基質濃度のサイクロデキストリン生成に
及ぼす影響を示したものである。
図中の○は本発明品、Δは従来の樹脂固定化酵素を使用
したものである。また、実線は基質ｍ度５％、破線は基
質濃度２０％での結果を示している。
第２図は本発明品イオン交換素材シート固定化酵素の安
定性を調べたもので、高分子分岐デキストリン組成物中
のサイクロデキストリン含量の変化を示している。
第１図
ライク０テ゛各ストリン’１ｌ−ｌ
（奸固形ケ２）Figure 1 shows the effects of flow rate and substrate concentration on cyclodextrin production. In the figure, ○ indicates the product of the present invention, and Δ indicates the use of a conventional resin-immobilized enzyme. Further, the solid line shows the results at a substrate concentration of 5%, and the broken line shows the results at a substrate concentration of 20%. FIG. 2 shows the stability of the enzyme immobilized on the ion exchange material sheet of the present invention, and shows changes in the cyclodextrin content in the polymer branched dextrin composition. Figure 1 Like 0 items, each string'1l-l (puppet ke 2)
Claims (3)
ゼをイオン交換素材シートに吸着させることを特徴とし
た固定化酵素。(1) An immobilized enzyme characterized by adsorbing cyclodextrin glucanotransferase to an ion exchange material sheet.
ゼをイオン交換素材シートに吸着させた固定化酵素に、
ぶどう糖当量（Ｄ．Ｅ．）１０以下のワキシースターチ
液化液を接触させることを特徴とする高分子分岐デキス
トリン組成物の製造法。(2) Immobilized enzyme with cyclodextrin glucanotransferase adsorbed on an ion exchange material sheet,
A method for producing a polymer branched dextrin composition, which comprises contacting a liquefied waxy starch solution having a glucose equivalent (D.E.) of 10 or less.
と９８〜７０重量％の分岐デキストリンを含有する特許
請求範囲第（２）項記載の高分子デキストリン組成物の
製造法。(3) A method for producing a polymer dextrin composition according to claim (2), wherein the composition contains 2 to 30% by weight of cyclodextrin and 98 to 70% by weight of branched dextrin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62267335A JP2610452B2 (en) | 1987-10-23 | 1987-10-23 | Method for producing polymer branched dextrin composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62267335A JP2610452B2 (en) | 1987-10-23 | 1987-10-23 | Method for producing polymer branched dextrin composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01108986A true JPH01108986A (en) | 1989-04-26 |
| JP2610452B2 JP2610452B2 (en) | 1997-05-14 |
Family
ID=17443390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62267335A Expired - Fee Related JP2610452B2 (en) | 1987-10-23 | 1987-10-23 | Method for producing polymer branched dextrin composition |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2610452B2 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5225043A (en) * | 1975-08-20 | 1977-02-24 | Japan Maize Prod | Process for preparing cyclodextrine |
| JPS61185196A (en) * | 1985-02-14 | 1986-08-18 | Shokuhin Sangyo Baioriakutaa Syst Gijutsu Kenkyu Kumiai | Production of cyclodextrine |
| JPS61188617A (en) * | 1985-02-15 | 1986-08-22 | Ascii Corp | General purpose input/output interface |
-
1987
- 1987-10-23 JP JP62267335A patent/JP2610452B2/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5225043A (en) * | 1975-08-20 | 1977-02-24 | Japan Maize Prod | Process for preparing cyclodextrine |
| JPS61185196A (en) * | 1985-02-14 | 1986-08-18 | Shokuhin Sangyo Baioriakutaa Syst Gijutsu Kenkyu Kumiai | Production of cyclodextrine |
| JPS61188617A (en) * | 1985-02-15 | 1986-08-22 | Ascii Corp | General purpose input/output interface |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2610452B2 (en) | 1997-05-14 |
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