JPH04121188A - Method for sterilizing immobilized enzyme utilizing high pressure - Google Patents
Method for sterilizing immobilized enzyme utilizing high pressureInfo
- Publication number
- JPH04121188A JPH04121188A JP2238236A JP23823690A JPH04121188A JP H04121188 A JPH04121188 A JP H04121188A JP 2238236 A JP2238236 A JP 2238236A JP 23823690 A JP23823690 A JP 23823690A JP H04121188 A JPH04121188 A JP H04121188A
- Authority
- JP
- Japan
- Prior art keywords
- high pressure
- immobilized enzyme
- enzyme
- immobilized
- sterilizing
- 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
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 69
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 21
- 230000000694 effects Effects 0.000 claims abstract description 27
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 244000005700 microbiome Species 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 6
- 239000012153 distilled water Substances 0.000 abstract description 4
- 229920003002 synthetic resin Polymers 0.000 abstract description 3
- 239000000057 synthetic resin Substances 0.000 abstract description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 1
- 230000002070 germicidal effect Effects 0.000 abstract 1
- 229910052760 oxygen Inorganic materials 0.000 abstract 1
- 239000001301 oxygen Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 13
- 230000007423 decrease Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 239000000645 desinfectant Substances 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 108010043535 protease S Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 3
- 229920002554 vinyl polymer Polymers 0.000 description 3
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010059881 Lactase Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 2
- 239000001639 calcium acetate Substances 0.000 description 2
- 229960005147 calcium acetate Drugs 0.000 description 2
- 235000011092 calcium acetate Nutrition 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 229940116108 lactase Drugs 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 229920002284 Cellulose triacetate Polymers 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical class NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 108700040099 Xylose isomerases Proteins 0.000 description 1
- NNLVGZFZQQXQNW-ADJNRHBOSA-N [(2r,3r,4s,5r,6s)-4,5-diacetyloxy-3-[(2s,3r,4s,5r,6r)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6s)-4,5,6-triacetyloxy-2-(acetyloxymethyl)oxan-3-yl]oxyoxan-2-yl]methyl acetate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](OC(C)=O)[C@H]1OC(C)=O)O[C@H]1[C@@H]([C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(C)=O)O1)OC(C)=O)COC(=O)C)[C@@H]1[C@@H](COC(C)=O)O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O NNLVGZFZQQXQNW-ADJNRHBOSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- YRIUSKIDOIARQF-UHFFFAOYSA-N dodecyl benzenesulfonate Chemical compound CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 YRIUSKIDOIARQF-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000010069 protein adhesion Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000003566 sealing material Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus For Disinfection Or Sterilisation (AREA)
Abstract
Description
【発明の詳細な説明】
皮！上皮机且分！
本発明は、固定化酵素に高圧装置内で高圧を適用して固
定化酵素に付着している微生物を殺菌することよりなる
高圧を利用した固定化酵素の殺菌方法に関する。[Detailed Description of the Invention] Skin! Epithelial tissue! The present invention relates to a method for sterilizing an immobilized enzyme using high pressure, which comprises applying high pressure to the immobilized enzyme in a high-pressure device to sterilize microorganisms attached to the immobilized enzyme.
皿米ｑ肢歪
従来、固定化酵素を使用して物質の生産あるいは改質が
行われている。Traditionally, immobilized enzymes have been used to produce or modify substances.
しかし、固定化酵素を長期間使用していると固定化酵素
に付着している細菌等の微生物が増殖し、その結果、分
解物の微生物汚染固定化酵素の活性の低下、あるいは処
理効率の低下等の問題が生じてくる。However, when an immobilized enzyme is used for a long period of time, microorganisms such as bacteria attached to the immobilized enzyme proliferate, resulting in microbial contamination of the decomposed product, a decrease in the activity of the immobilized enzyme, or a decrease in treatment efficiency. Such problems arise.
このような点を改善するために、固定化酵素を殺菌剤あ
るいは洗浄剤で洗浄する方法が行われていた０例えば、
特開昭５６−９２７９１号公報には、固定化酵素をジオ
クチルエチレントリアミン等の置換ジエチレントリアミ
ンの希薄水溶液と接触させて殺菌する方法が、また特開
昭５９−１７５８７９号公報には、細菌で汚染された酵
素を多価アルコールに浸漬して殺菌する方法が、さらに
特開昭５９−９８６８９号公報には固定化酵素をラウリ
ルベンゼンスルホン酸塩とエタノールとを含む混液で処
理して固定化酵素を洗浄殺菌する方法が開示されている
。しかし、これらの固定化酵素を殺菌剤や洗浄剤で洗浄
殺菌する方法は、その洗浄殺菌に長時間を要したり、あ
るいは洗浄殺菌効率が必ずしも充分ではなく、また固定
化酵素の活性を低下させたり、洗浄殺菌後固定化酵素に
残存付着している洗浄剤あるいは殺菌剤を完全に除去す
る操作を必要とするなどの種々の解決しなければならな
い問題点があった。In order to improve this problem, methods have been used to wash the immobilized enzyme with disinfectants or detergents.
JP-A-56-92791 discloses a method of sterilizing an immobilized enzyme by contacting it with a dilute aqueous solution of substituted diethylenetriamine such as dioctylethylenetriamine; There is a method of sterilizing the immobilized enzyme by soaking it in polyhydric alcohol, and Japanese Patent Application Laid-Open No. 59-98689 discloses a method of washing the immobilized enzyme by treating the immobilized enzyme with a mixed solution containing laurylbenzenesulfonate and ethanol. A method of sterilizing is disclosed. However, methods of washing and sterilizing these immobilized enzymes with disinfectants or detergents require a long time to wash and sterilize, or the washing and sterilization efficiency is not always sufficient, and the activity of the immobilized enzyme may be reduced. There are various problems that need to be solved, such as the need for an operation to completely remove the detergent or disinfectant remaining on the immobilized enzyme after washing and sterilization.
また、特開平１−２２８４５４号公報では、固定化酵素
に紫外線を照射して付着する微生物を殺菌する方法が開
示されている。しかし、紫外線照射で固定化酵素に付着
している微生物を完全に殺菌することは困難であった。Further, Japanese Patent Application Laid-open No. 1-228454 discloses a method of sterilizing attached microorganisms by irradiating an immobilized enzyme with ultraviolet rays. However, it has been difficult to completely sterilize microorganisms attached to immobilized enzymes by UV irradiation.
゛ しよ゛と る
本発明は、このような従来行われている固定化酵素の殺
菌方法の問題点を解決することを目的としてなされたも
のである。The present invention has been made with the aim of solving these problems of conventional methods of sterilizing immobilized enzymes.
すなわち、本発明の目的は、効率よく、しかも固定化酵
素の活性を損わずに固定化酵素に付着する微生物を殺菌
する新規な方法を提供することにある。That is, an object of the present invention is to provide a novel method for efficiently sterilizing microorganisms adhering to immobilized enzymes without impairing the activity of the immobilized enzymes.
ｆ ″ るための
本発明者らは、上記課題を解決するために固定化酵素に
適用できる種々の殺菌方法について検討をしたところ、
固定化酵素を高圧装置に入れ、高圧を通用すると、固定
化酵素の酵素活性を損なうことなく固定化酵素に付着し
ている微生物だけを殺菌することができることを見出し
、本発明をなすに至った。In order to solve the above problem, the present inventors investigated various sterilization methods that can be applied to immobilized enzymes, and found that
The present inventors have discovered that by placing an immobilized enzyme in a high-pressure device and applying high pressure to it, only the microorganisms attached to the immobilized enzyme can be sterilized without impairing the enzyme activity of the immobilized enzyme, which led to the present invention. .
すなわち、本発明は、固定化酵素に高圧装置内で高圧を
適用して酵素活性を低下させることな（付着する微生物
を殺菌する方法に関する。That is, the present invention relates to a method for sterilizing attached microorganisms without reducing enzyme activity by applying high pressure to an immobilized enzyme in a high-pressure device.
本発明では、固定化酵素として従来知られている種々の
固定化酵素が用いられる。このような固定化酵素として
は、ラクターゼ、グルコースイソメラーゼ、プロテアー
ゼ等を水に不溶性の担体、例えばキトサン、キチンある
いは樹脂等に化学結合させるかあるいはセルローストリ
アセテート、ポリアクリルアミドゲル等に包括させて固
定化させたものを例示することができる。これらは、バ
ルクの状態であってもカラム等に充填された状態であっ
ても撹拌槽に収納された状態であってもあるいは流動層
、膜型等となった状態であってもさらにこれらの装置か
ら固定化酵素を取り出した状態であっても使用すること
ができる。In the present invention, various conventionally known immobilized enzymes are used as immobilized enzymes. Such immobilized enzymes include lactase, glucose isomerase, protease, etc., which are chemically bonded to a water-insoluble carrier such as chitosan, chitin, or resin, or are immobilized by being wrapped in cellulose triacetate, polyacrylamide gel, etc. Examples can be given. These substances may be in a bulk state, packed in a column, etc., stored in a stirring tank, or in a fluidized bed, membrane type, etc. The immobilized enzyme can be used even after being removed from the device.
高圧装置は、従来食品等の高圧装置として知られている
装置を使用することができる。このような装置としては
、例えば、三菱重工■製高圧試験装置がある。この装置
によると、圧力媒体として水を使用し最高圧力１０，０
００ｋｇ／ｃｄ、使用温度常温〜６０°Ｃで高圧処理を
行うことができる。また、■丸善発行、大杉治部他著「
高圧実験技術とその応用」第２６１頁（１９８０年）、
食品資材研究会発行宮用金二部著「食品の物性」第６集
第９９頁（１９８０年）等に記載されている高圧発生装
置を用いることもできる。As the high-pressure device, a device conventionally known as a high-pressure device for food products and the like can be used. An example of such a device is a high-pressure test device manufactured by Mitsubishi Heavy Industries, Ltd. According to this device, water is used as the pressure medium and the maximum pressure is 10.0
High-pressure treatment can be performed at 00 kg/cd and a working temperature of room temperature to 60°C. In addition, published by Maruzen, written by Osugi Osugi et al.
"High Pressure Experimental Technology and Its Applications", p. 261 (1980),
It is also possible to use a high-pressure generator described in "Physical Properties of Foods" Vol. 6, page 99 (1980), written by Miyayokin, published by the Food Materials Research Group, Nippon.
本発明を完成させるに当り、次の高圧発生装置を用い実
験を行った。In completing the present invention, experiments were conducted using the following high pressure generator.
１、 高圧発生装置 三菱重工業株式会社製高圧試験装置を使用した。1. High pressure generator A high pressure test device manufactured by Mitsubishi Heavy Industries, Ltd. was used.
（１） この装置の主な仕様は次の通りである。(1) The main specifications of this device are as follows.
ａ）最高仕様圧力１０．０００ｋｇ／ｃｉｉｂ）使用温
度 常温〜６０°Ｃ
Ｃ）処理室寸法 ５４閣φＸ２００ ｘｍｄ）圧力媒体
水
（２）次の試験条件で試験を行った。a) Maximum specification pressure: 10.000 kg/ciib) Operating temperature: Room temperature to 60°C C) Processing chamber dimensions: 54 mm φX200 x md) Pressure medium: Water (2) Tests were conducted under the following test conditions.
ａ）圧力 ０〜４，０００ ｋｇ／ｃｈ１ｂ）温度 ２
０〜６０゛Ｃ
Ｃ）時間 ０〜３０分
（３）試料
（１）酵素溶液（対照）。酵素溶液としてプロテアーゼ
Ｓ １００Ｕ／＋ｄをｐＨ７，０のリン酸緩衝液に溶解
し、■０Ｉ１１ビニール製シールバック材に封入したも
のを用いた。a) Pressure 0 to 4,000 kg/ch1b) Temperature 2
0-60°C C) Time 0-30 minutes (3) Sample (1) Enzyme solution (control). The enzyme solution used was one in which 100 U/+d of protease S was dissolved in a phosphate buffer solution of pH 7.0 and sealed in a 0I11 vinyl seal back material.
（２）固定化酵素、前記プロテアーゼＳをキトサンビー
ズ（キトバール、商品名）にグルタルアルデヒドで架橋
した固定化酵素を用い、これをカゼイン液液中に浸漬し
て汚染させたものを使用した。この汚染固定化酵素を蒸
留水で水洗して前処理し、この５ｇ−ｗｅｔを５ｔａＭ
酢酸カルシウム水溶液１０Ｍ１とともにビニール製シー
ルバック材に封入したものを用いた。(2) The immobilized enzyme used was an immobilized enzyme in which the protease S was cross-linked with chitosan beads (Chitovar, trade name) with glutaraldehyde, which were immersed in casein liquid and contaminated. This contaminated immobilized enzyme was pretreated by washing with distilled water, and this 5g-wet was washed with 5taM
A sample sealed in a vinyl seal back material with a 10 M1 aqueous calcium acetate solution was used.
これらの試料は、それぞれ６パツクずつ使用した。Six packs of each of these samples were used.
（３）試験方法
上記試料を高圧発生装置に入れ、（１）高圧を適用した
ときの圧力の変化による酵素の残存活性及び生菌数、（
２）高圧を適用したときの温度の変化による酵素の残存
活性及び生菌数、（３）さらに、時間の経過による酵素
の残存活性及び生菌数について試験した。(3) Test method The above sample was placed in a high pressure generator.
2) The residual activity of the enzyme and the number of viable bacteria due to changes in temperature when high pressure was applied, and (3) The residual activity of the enzyme and the number of viable bacteria over time were further tested.
（４）試験結果 前記実験を行った結果を第１〜６図に示す。(4) Test results The results of the above experiments are shown in FIGS. 1 to 6.
第１図によると、酵素液（対照）をＯ〜４０００ｋｇ／
ｃｆ１１の高圧を適用するとその活性はいちじるしく低
下するのに対し、固定化酵素に高圧を適用してもその活
性はほとんど低下しないことが分る。また、このさいの
生菌数は、第２図にみられるように高圧発生装置の圧力
が３０００ｋｇ／ｃ−ｆｉ１以上になるといちしろしく
低下し、殺菌効果が生じている。このことから固定化酵
素に高圧を通用するとその活性を低下させることなく殺
菌を行うことができ、この殺菌効果は３０００ｋｇ／ｃ
ｍ２以上でいちじるしく高まることが分る。According to Figure 1, the enzyme solution (control) was
It can be seen that when high pressure is applied to cf11, its activity decreases markedly, whereas when high pressure is applied to the immobilized enzyme, its activity hardly decreases. Moreover, as shown in FIG. 2, the number of viable bacteria at this time decreases significantly when the pressure of the high pressure generator exceeds 3000 kg/c-fi1, and a sterilizing effect is produced. Therefore, when high pressure is applied to the immobilized enzyme, it can be sterilized without reducing its activity, and this sterilization effect is 3000 kg/c.
It can be seen that it increases significantly above m2.
次に、第３図によると、酵素液を高圧３０００ｋｇ／ｃ
ｄ に１０分間保持し、その間高圧装置の保持温度を
２０〜６０°Ｃに保持すると、酵素液の場合は酵素活性
が最初の２０°Ｃのときからいちじるしく低下し、さら
に温度が上昇するにつれてその活性は少しずつ低下して
いるのに対し、固定化酵素は温度が上昇しても活性はほ
とんど低下せず、最初の活性を維持できることが分る。Next, according to Figure 3, the enzyme solution was heated to a high pressure of 3000 kg/c.
d for 10 minutes, during which time the temperature of the high-pressure device was maintained at 20 to 60°C. In the case of an enzyme solution, the enzyme activity significantly decreased from the initial temperature of 20°C, and as the temperature rose further, the enzyme activity decreased significantly. It can be seen that the activity of the immobilized enzyme decreases little by little, whereas the activity of the immobilized enzyme hardly decreases even when the temperature rises, and the initial activity can be maintained.
またこのさいの生菌数は、第４図にみられるように５０
°Ｃ以上でいちじるしく低下している。このことから、
高圧装置の保持温度は５０°Ｃ以上が好適である。Also, the number of viable bacteria at this stage was 50, as shown in Figure 4.
It decreases significantly above °C. From this,
The holding temperature of the high pressure device is preferably 50°C or higher.
さらに、高圧装置の保持時間についてみると、第５図に
示すように固定化酵素を圧力３０００ｋｇ／ｄ、温度６
０″Ｃに保持したとき保持時間５〜３０分の間ではほと
んど最初の活性が保持され、また第６図に示すように生
菌数は５分間の保持でほとんどなくなることが分る。Furthermore, regarding the holding time of the high-pressure device, as shown in Figure 5, the immobilized enzyme was held at a pressure of 3000 kg/d and a temperature of 6.
When held at 0''C, most of the initial activity is retained during the holding time of 5 to 30 minutes, and as shown in Figure 6, the number of viable bacteria almost disappears after holding for 5 minutes.
これらの実験から、固定化酵素を高圧装置に保持し、高
圧を適用すると、固定化酵素特有の性質としてその活性
を損なわずに固定化酵素に付着する微生物を殺菌するこ
とができ、この殺菌効果は圧力３０００ｋｇ／ｃｄ以上
、保持温度５０℃以上のときに顕著であることが分る。From these experiments, we found that by holding the immobilized enzyme in a high-pressure device and applying high pressure, microorganisms attached to the immobilized enzyme can be sterilized without impairing its activity, which is a unique property of the immobilized enzyme, and this sterilizing effect It can be seen that this is noticeable when the pressure is 3000 kg/cd or higher and the holding temperature is 50°C or higher.
固定化酵素は、塩化ビニール、ポリエチレン等柔軟な合
成樹脂製の袋に蒸留水、緩衝液等と共に充填して高圧発
生装置に収納し、高圧を適用するとよい。適用条件とし
ては前記実験の結果から圧力３０００ｋｇ／ｃｆｆｌ、
保持温度５０″Ｃ以上とすると酵素活性を損なわず殺菌
を行うことができる。なお、高圧発生装置内で高圧の適
用によって酵素活性力ぐ低下しないのは、前記実験の結
果から酵素液では認められず固定化酵素特有の作用のよ
うに思われる。The immobilized enzyme is preferably filled in a bag made of a flexible synthetic resin such as vinyl chloride or polyethylene together with distilled water, a buffer solution, etc., stored in a high pressure generator, and then high pressure is applied. Based on the results of the above experiment, the application conditions are a pressure of 3000 kg/cffl,
If the holding temperature is 50"C or higher, sterilization can be carried out without impairing the enzyme activity. Furthermore, the result of the above experiment shows that the enzyme activity does not decrease significantly when high pressure is applied in the high pressure generator. This seems to be an action unique to immobilized enzymes.
このようにして高圧を適用した固定化酵素は、合成樹脂
製の袋等から取り出し、蒸留水、緩衝液等で洗浄し、再
び使用することができる。The immobilized enzyme subjected to high pressure in this manner can be taken out from a synthetic resin bag, washed with distilled water, a buffer solution, etc., and used again.
本発明の方法は、牛乳をラクターゼを担体に固定化した
固定化酵素を使用し、グルコースとラクトースとに分解
するさいの固定化酵素の殺菌、あるいは同様に乳糖、ホ
エー等に作用させて乳糖分解乳、甘味シロップ製造のさ
いの固定化酵素の殺菌等に用いることができる。さらに
プロテアーゼ、イソメラーゼ等を担体に固定化した固定
化酵素の殺菌等にも用いることができる。The method of the present invention uses an immobilized enzyme in which lactase is immobilized on a carrier, and sterilizes the immobilized enzyme when decomposing milk into glucose and lactose, or similarly acts on lactose, whey, etc. to decompose lactose. It can be used to sterilize immobilized enzymes during the production of milk and sweet syrup. Furthermore, it can also be used to sterilize immobilized enzymes such as protease, isomerase, etc. on carriers.
次に本発明の実施例を示す。Next, examples of the present invention will be shown.
実施例１
カラムＣ６ｃｍ （直径）Ｘ１８Ｃ１（長さ）］に、３
００ ｄの固定化プロテアーゼＳを充填し、これにダウ
ンフローで１０％カゼインを反応温度４０’Ｃ，Ｓシー
３で連続通液を行った。時間経過と共に菌の生育が始ま
り、固定化酵素への乳タンパクの付着と細菌による汚染
蓄積が生した。１２時間通液後、細菌数は１０ｂオーダ
ーとなった。このときカラムから固定化酵素を取り出し
、リン酸緩衝液（ｐＦＩ７．０）で洗浄して乳固形分を
除去した。これを５ｗＭ酢酸カルシウム水溶液とともに
ビニール製シールバック材に封入し、高圧発生装置に充
填し、圧力３０００ｋｇ／ａｌｌ保持温度５０℃で１０
分間高圧を適用した。その後、固定化酵素を袋から取り
出し、無菌のリン酸緩衝液（ｐＨ７，０）で洗浄した。Example 1 Column C6cm (diameter) x 18C1 (length)], 3
00 d of immobilized protease S was filled, and 10% casein was continuously passed through it in down flow at a reaction temperature of 40'C and S Sea 3. Bacteria began to grow over time, resulting in milk protein adhesion to the immobilized enzyme and bacterial contamination accumulation. After passing the solution for 12 hours, the number of bacteria was on the order of 10 b. At this time, the immobilized enzyme was taken out from the column and washed with phosphate buffer (pFI 7.0) to remove milk solids. This was sealed in a vinyl sealing material along with a 5wM calcium acetate aqueous solution, filled into a high pressure generator, and kept at a pressure of 3000kg/all and a holding temperature of 50°C for 10 minutes.
High pressure was applied for minutes. Thereafter, the immobilized enzyme was taken out from the bag and washed with sterile phosphate buffer (pH 7.0).
この酵素活性はほとんど低下せず、付着菌数は０に近い
状態であった。この固定化酵素を再びカラムに充填し、
脱脂乳の通液を開始した。This enzyme activity hardly decreased, and the number of attached bacteria was close to zero. This immobilized enzyme was packed into the column again,
Pour of skim milk was started.
発！９と１果
本発明の方法によると、固定化酵素に用いる担体の形状
や構造が異なっていても固定化酵素に高圧を適用するこ
とにより酵素活性を低下させることなく、固定化酵素に
付着し繁殖している細菌等の微生物を殺菌することがで
きる。この方法は、殺菌剤、洗浄剤等を使用する化学的
方法にくらべて殺菌後の固定化酵素の後処理が簡単で、
しかも酵素活性の低下がほとんどみられず、殺菌効果が
高く工業的に有利な固定化酵素の殺菌方法である。Depart! 9 and 1 According to the method of the present invention, even if the carrier used for the immobilized enzyme has a different shape or structure, by applying high pressure to the immobilized enzyme, the carrier can be attached to the immobilized enzyme without reducing the enzyme activity. It can sterilize microorganisms such as bacteria that are breeding. This method simplifies the post-treatment of the immobilized enzyme after sterilization compared to chemical methods that use disinfectants, detergents, etc.
Furthermore, there is almost no decrease in enzyme activity, and the method has a high bactericidal effect and is an industrially advantageous method for sterilizing immobilized enzymes.
第１図は固定化酵素に高圧を適用したときの圧力と残存
酵素活性との関係を、また第２図は、圧力と残存生菌数
との関係を示す。第３図は、固定化酵素に高圧を適用し
たときの保持温度と残存酵素活性との関係を、第４図は
保持温度と残存生菌数との関係を示す。第５図は同様に
保持時間と残存酵素活性との関係を、第６図は保持時間
と残存生菌数との関係を示す。
図中、○−○は、高圧を固定化酵素に適用した場合を、
・・・・・・・・は酵素溶液（対照）に適用した場合を
それぞれ示す。FIG. 1 shows the relationship between pressure and residual enzyme activity when high pressure is applied to the immobilized enzyme, and FIG. 2 shows the relationship between pressure and the number of remaining viable bacteria. FIG. 3 shows the relationship between the holding temperature and residual enzyme activity when high pressure is applied to the immobilized enzyme, and FIG. 4 shows the relationship between the holding temperature and the number of remaining viable bacteria. Similarly, FIG. 5 shows the relationship between the retention time and the remaining enzyme activity, and FIG. 6 shows the relationship between the retention time and the number of remaining viable bacteria. In the figure, ○−○ indicates the case where high pressure is applied to the immobilized enzyme.
. . . shows the case where it was applied to an enzyme solution (control).
Claims (3)
性を低下させず付着する微生物を殺菌することを特徴と
する高圧を利用した固定化酵素の殺菌方法(1) A method for sterilizing an immobilized enzyme using high pressure, which is characterized by applying high pressure to the immobilized enzyme in a high-pressure device to sterilize attached microorganisms without reducing enzyme activity.
である請求項（１）に記載の高圧を利用した固定化酵素
の殺菌方法(2) The method for sterilizing immobilized enzymes using high pressure according to claim (1), wherein the high pressure of the high pressure device is 3,000 kg/cm^2 or more.
１）または（２）に記載の高圧を利用した固定化酵素の
殺菌方法(3) Claim in which the holding temperature of the high pressure device is 50°C or higher (
Method for sterilizing immobilized enzymes using high pressure as described in 1) or (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2238236A JP2813837B2 (en) | 1990-09-07 | 1990-09-07 | Sterilization method of immobilized enzyme using high pressure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2238236A JP2813837B2 (en) | 1990-09-07 | 1990-09-07 | Sterilization method of immobilized enzyme using high pressure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04121188A true JPH04121188A (en) | 1992-04-22 |
| JP2813837B2 JP2813837B2 (en) | 1998-10-22 |
Family
ID=17027169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2238236A Expired - Lifetime JP2813837B2 (en) | 1990-09-07 | 1990-09-07 | Sterilization method of immobilized enzyme using high pressure |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2813837B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5618689A (en) * | 1995-05-25 | 1997-04-08 | Nestec S.A. | Enhanced procedures for preparing food hydrolysates |
| EP1421185A2 (en) * | 2001-08-07 | 2004-05-26 | Boston Biomedica, Inc. | Rapid cryobaric sterilization and vaccine preparation |
| US9926383B2 (en) | 2006-05-05 | 2018-03-27 | Leukocare Ag | Biocompatible three dimensional matrix for the immobilization of biological substances |
-
1990
- 1990-09-07 JP JP2238236A patent/JP2813837B2/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5618689A (en) * | 1995-05-25 | 1997-04-08 | Nestec S.A. | Enhanced procedures for preparing food hydrolysates |
| EP1421185A2 (en) * | 2001-08-07 | 2004-05-26 | Boston Biomedica, Inc. | Rapid cryobaric sterilization and vaccine preparation |
| EP1421185A4 (en) * | 2001-08-07 | 2005-12-21 | Boston Biomedica Inc | Rapid cryobaric sterilization and vaccine preparation |
| US9926383B2 (en) | 2006-05-05 | 2018-03-27 | Leukocare Ag | Biocompatible three dimensional matrix for the immobilization of biological substances |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2813837B2 (en) | 1998-10-22 |
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