JPH02303487A - Novel curd enzyme and its production - Google Patents
Novel curd enzyme and its productionInfo
- Publication number
- JPH02303487A JPH02303487A JP12526889A JP12526889A JPH02303487A JP H02303487 A JPH02303487 A JP H02303487A JP 12526889 A JP12526889 A JP 12526889A JP 12526889 A JP12526889 A JP 12526889A JP H02303487 A JPH02303487 A JP H02303487A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- milk
- activity
- clotting
- temperature
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 54
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 230000000694 effects Effects 0.000 claims abstract description 37
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 22
- 244000068988 Glycine max Species 0.000 claims abstract description 22
- 235000013336 milk Nutrition 0.000 claims abstract description 16
- 239000008267 milk Substances 0.000 claims abstract description 16
- 210000004080 milk Anatomy 0.000 claims abstract description 16
- 241000235395 Mucor Species 0.000 claims abstract description 13
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000001110 calcium chloride Substances 0.000 claims abstract description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 238000002523 gelfiltration Methods 0.000 claims abstract description 3
- 108010005090 rennin-like enzyme (Aspergillus ochraceus) Proteins 0.000 claims description 28
- 235000013322 soy milk Nutrition 0.000 claims description 18
- 229910021645 metal ion Inorganic materials 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000002532 enzyme inhibitor Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 239000010949 copper Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 claims description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910017052 cobalt Inorganic materials 0.000 claims description 2
- 239000010941 cobalt Substances 0.000 claims description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 229910052742 iron Inorganic materials 0.000 claims description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 2
- 229910052753 mercury Inorganic materials 0.000 claims description 2
- 229910001453 nickel ion Inorganic materials 0.000 claims description 2
- 229910052712 strontium Inorganic materials 0.000 claims description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims 1
- 229940122618 Trypsin inhibitor Drugs 0.000 claims 1
- 101710162629 Trypsin inhibitor Proteins 0.000 claims 1
- 239000000654 additive Substances 0.000 claims 1
- 229910001424 calcium ion Inorganic materials 0.000 claims 1
- 229910001431 copper ion Inorganic materials 0.000 claims 1
- MUCZHBLJLSDCSD-UHFFFAOYSA-N diisopropyl fluorophosphate Chemical compound CC(C)OP(F)(=O)OC(C)C MUCZHBLJLSDCSD-UHFFFAOYSA-N 0.000 claims 1
- 229940125532 enzyme inhibitor Drugs 0.000 claims 1
- 229960005051 fluostigmine Drugs 0.000 claims 1
- 239000011777 magnesium Substances 0.000 claims 1
- 229910001425 magnesium ion Inorganic materials 0.000 claims 1
- 230000000704 physical effect Effects 0.000 claims 1
- 229910001427 strontium ion Inorganic materials 0.000 claims 1
- 239000002753 trypsin inhibitor Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 19
- 235000019658 bitter taste Nutrition 0.000 abstract description 11
- 235000019606 astringent taste Nutrition 0.000 abstract description 10
- 230000001804 emulsifying effect Effects 0.000 abstract description 9
- 241000233866 Fungi Species 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000001112 coagulating effect Effects 0.000 abstract 1
- 235000013305 food Nutrition 0.000 description 23
- 235000019645 odor Nutrition 0.000 description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 7
- 235000011130 ammonium sulphate Nutrition 0.000 description 7
- 235000019640 taste Nutrition 0.000 description 7
- 238000010586 diagram Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 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 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- -1 bittern to soy milk Chemical class 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical group CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241001131796 Botaurus stellaris Species 0.000 description 1
- 241000195940 Bryophyta Species 0.000 description 1
- 101100311260 Caenorhabditis elegans sti-1 gene Proteins 0.000 description 1
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 1
- 101150039033 Eci2 gene Proteins 0.000 description 1
- 235000015429 Mirabilis expansa Nutrition 0.000 description 1
- 244000294411 Mirabilis expansa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 101100438245 Solanum tuberosum PCM8 gene Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- YYRQHJQOVKTWER-UHFFFAOYSA-M azanium sodium hydrogen phosphate sulfuric acid Chemical compound [NH4+].[Na+].OP([O-])([O-])=O.OS(O)(=O)=O YYRQHJQOVKTWER-UHFFFAOYSA-M 0.000 description 1
- 235000013527 bean curd Nutrition 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
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 235000021185 dessert Nutrition 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000015071 dressings Nutrition 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000012209 glucono delta-lactone Nutrition 0.000 description 1
- 239000000182 glucono-delta-lactone Substances 0.000 description 1
- 229960003681 gluconolactone Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000015220 hamburgers Nutrition 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 235000015141 kefir Nutrition 0.000 description 1
- 235000013310 margarine Nutrition 0.000 description 1
- 239000003264 margarine Substances 0.000 description 1
- 235000010746 mayonnaise Nutrition 0.000 description 1
- 239000008268 mayonnaise Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000013536 miso Nutrition 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 235000011929 mousse Nutrition 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- YFZOUMNUDGGHIW-UHFFFAOYSA-M p-chloromercuribenzoic acid Chemical compound OC(=O)C1=CC=C([Hg]Cl)C=C1 YFZOUMNUDGGHIW-UHFFFAOYSA-M 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 235000019643 salty taste Nutrition 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 235000019614 sour taste Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、豆乳を凝固する新規な凝乳酵素及びその製法
に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel milk-clotting enzyme that coagulates soybean milk and a method for producing the same.
〔従来の技術］
従来、大豆より食品素材を製造する方法としては、豆乳
ににがりをはじめとした塩類を添加する方法、グルコノ
デルタラクトンをはじめとした酸類を添加する方法及び
プロテアーゼ等の酵素を使用する方法等が知られている
。[Prior art] Conventionally, methods for producing food materials from soybeans include adding salts such as bittern to soy milk, adding acids such as glucono delta-lactone, and adding enzymes such as protease. Methods of using it are known.
しかしながら、塩類や酸１１を添加する方法により得ら
れた食品素材は食感としてざらつきが強く乳化性も劣り
、且つ、塩味や酸味が残る等、今だ満足できる品質Ｑも
のは得られていないのが現状である。However, food materials obtained by the method of adding salts and acids 11 have a strong texture and poor emulsifying properties, and they still have a salty or sour taste, so it is still not possible to obtain a food material with a satisfactory quality. is the current situation.
酵素を使用する方法としては、プロテアーゼによるもの
（特開昭６２−２３２３４０号）プロテアーゼとマグネ
シウム塩の共存化のよるもの（特開昭６３−２６５号）
が知られているが、酵素法では確かに滑らかな食感を有
するペースト状素材が得られるが、酵素法によるものは
収率が極めて低く、苦み、渋味等の雑味を有する等の問
題点があり、又、プロテアーゼとマグネシウム塩の共存
化によるものは、収率は改善されているもののやはり苦
み、渋味等の雑味を有する問題点がある。Methods using enzymes include those using protease (Japanese Unexamined Patent Publication No. 62-232340) and the coexistence of protease and magnesium salt (Japanese Unexamined Patent Publication No. 63-265).
Although it is known that the enzyme method produces a paste-like material with a smooth texture, the yield of the enzyme method is extremely low, and there are problems such as having unpleasant tastes such as bitterness and astringency. Furthermore, although the yield is improved when protease and magnesium salt coexist, there is still the problem of unpleasant tastes such as bitterness and astringency.
一方、このような背景のもと新規な豆乳凝乳酵素を開発
する試みもなされており、豆乳凝乳酵素としては、特開
昭６１−２８２０７４号、特開昭６２−１７９３８６号
、特開昭５１−４８４５５号が知られている。On the other hand, against this background, attempts have been made to develop new soy milk curdling enzymes. No. 51-48455 is known.
なお、特開昭６１−２８２０７４号は、バシラス属の属
する細菌が生産する凝乳酵素であり、以下に示す本発明
の凝乳酵素とはその起源が異なる上に、その酵素学的性
質も、分子量、最適作用温度、アルカリ側でのｐＨ安定
性が大きく異なっている。In addition, JP-A No. 61-282074 is a milk-clotting enzyme produced by bacteria belonging to the genus Bacillus, and its origin is different from the milk-clotting enzyme of the present invention shown below, and its enzymatic properties are also They differ greatly in molecular weight, optimum operating temperature, and pH stability on the alkaline side.
特開昭６２−１７９３８６号は、桿菌に属する細菌が生
産する凝乳酵素であり、その酵素学的性質は粗酵素であ
るため、酵素の特定は出来ていないが、本発明の凝乳酵
素とは熱安定性が大きく異なっている。JP-A No. 62-179386 describes a milk-clotting enzyme produced by bacteria belonging to Bacillus, and its enzymological property is that of a crude enzyme, so the enzyme cannot be identified, but it does not disclose the milk-clotting enzyme of the present invention. have very different thermal stability.
特開昭５１−４８４５５号は、ロドシュードモナス属に
属する細菌が生産する凝乳酵素であるが、その酵素学的
性質は明らかにされていない。JP-A-51-48455 discloses a milk-clotting enzyme produced by bacteria belonging to the genus Rhodopseudomonas, but its enzymatic properties have not been clarified.
そして、これら凝乳酵素を使用した場合においても、苦
みは比較的弱いものの依然として苦み、渋みについての
問題点は解消されているとは言い難い。Even when these milk-clotting enzymes are used, although the bitterness is relatively weak, it is still difficult to say that the problems with bitterness and astringency have been solved.
また、本発明者等は、先にムコール属等の糸状菌の産生
ずる粗酵素を用いた優れた乳化特性及びテクスチャを有
し、苦味、渋味、異臭のない素材及びその製造方法につ
いて出願している（特願昭６３−２０８２８７号）。In addition, the present inventors have previously filed an application for a material that uses a crude enzyme produced by filamentous fungi such as Mucor that has excellent emulsifying properties and texture, and is free of bitterness, astringency, and off-odor, and a method for producing the same. (Patent Application No. 63-208287).
（発明が解決しようとする課題〕
本発明の課題は、優れた乳化特性を有し、滑らかなテク
スチャーを与え、苦み、渋み等の雑味及び大豆臭等の異
臭のない豆乳又は豆乳類原料由来の新規食品素材を得る
ために有用な新規凝乳酵素並びにその製造法を提供しよ
うとするものである。(Problems to be Solved by the Invention) An object of the present invention is to obtain soymilk or soymilk-based raw materials that have excellent emulsifying properties, provide a smooth texture, and are free from unpleasant tastes such as bitterness and astringency and foreign odors such as soybean odor. The purpose of the present invention is to provide a novel milk-clotting enzyme useful for obtaining novel food materials, as well as a method for producing the same.
〔課題を解決するための手段］
大豆を原料とする食品素材をめぐるこのような技術的背
景を踏え、本発明者らは、滑らかな食感を有し、乳化性
に優れ、苦み、渋み等の雑味及び大豆臭等の異臭のない
大豆由来の新しい食品素材その効率的、な製造方法及び
当該食品素材を使用した新しい食品を開発することを目
標として鋭意研究を重ねた結果、ムコール属に属する糸
状菌が産生ずる特定の凝乳酵素、即ち、ムコール属に属
する糸状菌を固体培養して固体麹の酵素抽出物又は液体
培養して得た酵素含有培養液上清を豆乳又は豆乳類に添
加することにより前記問題点を回避し得ることを見い出
して本発明を完成するに至った。[Means for Solving the Problems] Based on this technical background surrounding food materials made from soybeans, the present inventors have developed a food material that has a smooth texture, excellent emulsifying properties, and has no bitterness or astringency. As a result of intensive research aimed at developing new food materials derived from soybeans, which are free from unpleasant tastes such as soybeans and foreign odors such as soybean odor, and efficient production methods, and new foods using these food materials, we have discovered that Mucor genus A specific milk-clotting enzyme produced by a filamentous fungus belonging to the genus Mucor, that is, an enzyme extract of solid koji obtained by solid culture of a filamentous fungus belonging to the genus Mucor, or an enzyme-containing culture supernatant obtained by liquid culture is used to produce soy milk or soy milk. The present inventors have discovered that the above-mentioned problems can be avoided by adding the above-mentioned compounds to the above, and have completed the present invention.
本発明は、優れた乳化特性を有し、滑らかなテクスチャ
ーを与え、苦み、渋み等の雑味及び大豆臭等の異臭のな
い豆乳又は豆乳類原料由来の新規食品素材を提供する新
規凝乳酵素並びにその製造法を提供することを目的とす
るものである。The present invention is a novel milk-clotting enzyme that provides a novel food material derived from soy milk or soy milk raw materials, which has excellent emulsifying properties, gives a smooth texture, and is free from bitterness, astringency, and other unpleasant tastes, and soybean odor and other off-flavors. The object of the present invention is to provide a method for producing the same.
本発明者らは、目的とする酵素を生産する能力を有する
微性物の検索を自然界から広く行った結果、ムコール属
に属する１菌株が該酵素を効率よく菌体外に大量生産す
ることを見出し、本発明を完成するに到った。The present inventors conducted a wide search in nature for microorganisms that have the ability to produce the desired enzyme, and as a result, we found that a strain belonging to the genus Mucor can efficiently produce the enzyme in large quantities outside the bacterial cell. This finding led to the completion of the present invention.
（１）凝乳酵素の諸性質 本発明の凝乳酵素の理化学的性質は以下の通りである。(1) Properties of milk-clotting enzymes The physicochemical properties of the milk-clotting enzyme of the present invention are as follows.
（］）作用 豆乳に作用し、豆乳を凝固する。(]) Effect Acts on soy milk and coagulates it.
（２）基質特異性 主として豆乳を凝固する。(2) Substrate specificity Mainly used to coagulate soy milk.
（３）至適ｐＨ及び安定ｐＨ範囲
豆乳をｐ Ｈ５，９〜７．０に調整したものについて、
５０℃、での活性を測定した。豆乳は１．２５ｍＭ塩化
カルシウム溶液を添加したものと無添加のものについて
測定をおこなった。その結果至適ｐＨは６、ｌ付近であ
った。ｐ Ｈ６，０以下では、プレインキュヘートの段
階で凝固したため、測定出来なかった。（第１図参照）
また、安定ｐＨ範囲は、酵素溶液をｐＨ３，０〜１１．
０の範囲で３０℃１１時間処理した後ｐＨ６、１，５０
’ｃにて活性を測定した結果ｐ １１３．０〜７．０の
範囲内のいずれのｐＨ値においても６０％以上の残存活
性が認められた。（第２図参照）（４）作用温度範囲及
び最適作用温度豆
乳をｐ Ｈ６，１に調整後、反応温度４０〜７５℃の範
囲で活性を測定した。豆乳は１．２５＋＋＋Ｍ塩化カル
シウムン容液を添加したものと無添加のものについて測
定をおこなった。その結果最適作用温度は塩化カルシウ
ム添加で６０℃１無添加で５７℃であった。作用温度は
、塩化カルシウム添加で５０〜７０℃付近の範囲内の各
温度において５０％以上の相対活性を示し、無添加で４
６〜６４℃付近の範囲内の各温度において５０％以上の
相対活性を示した。(3) Optimal pH and stable pH range For soy milk adjusted to pH 5.9 to 7.0,
Activity was measured at 50°C. Measurements were performed on soymilk with and without 1.25mM calcium chloride solution added. As a result, the optimum pH was around 6.1. At a pH of 6.0 or lower, the sample coagulated at the pre-incubate stage, making measurement impossible. (See Figure 1) The stable pH range is pH 3.0 to pH 11.0.
After treatment at 30°C for 11 hours at pH 6, 1,50
As a result of measuring the activity at p 113.0 to 7.0, residual activity of 60% or more was observed at any pH value within the range of 113.0 to 7.0. (See Figure 2) (4) Action temperature range and optimum action temperature After adjusting the pH of soymilk to 6.1, the activity was measured at a reaction temperature in the range of 40 to 75°C. Measurements were carried out on soymilk with and without addition of 1.25+++M calcium chloride solution. As a result, the optimum operating temperature was 60°C with the addition of calcium chloride and 57°C without the addition of calcium chloride. The action temperature shows a relative activity of 50% or more at each temperature in the range of around 50 to 70°C with the addition of calcium chloride, and a relative activity of 40% without the addition of calcium chloride.
It showed a relative activity of 50% or more at each temperature within the range of around 6 to 64°C.
示した。Indicated.
（５）分子量
精製凝乳酵素の分子量はセファデックスＧ−１００ゲル
ろ過により約３５．０００である。(5) Molecular Weight Purification The molecular weight of the milk-clotting enzyme is approximately 35,000 as determined by Sephadex G-100 gel filtration.
（６）熱安定性
酵素溶液をｐＨ６，ｉ３０分間各温温度加熱処理後、ｐ
Ｈ６，１，５０℃にてそれぞれの残存活性を測定した。(6) After heating the thermostable enzyme solution to pH 6, i for 30 minutes at each temperature, p
The residual activity of each was measured at H6, 1, and 50°C.
その結果３５℃までは１００％、４０℃で８０％、５０
℃、で１０％の残存活性を示し、６０℃でほぼ失活した
。（第４図参照）
（力 凝乳酵素に対する金属イオンの影響（イ） ｐ
Ｈ６，１，５０℃、において各金属イオン１ｍＭ添加し
た場合の本酵素の凝乳活性を以下の第１表に示す。As a result, 100% up to 35℃, 80% at 40℃, 50%
It showed 10% residual activity at 60°C, and almost lost its activity at 60°C. (See Figure 4) (Force Effect of metal ions on milk-clotting enzyme (a) p
Table 1 below shows the milk curdling activity of this enzyme when 1mM of each metal ion was added at 50°C and 1°C.
いずれの金属イオンでも凝乳活性の増大が認められた。An increase in milk curd activity was observed for all metal ions.
第１表 凝乳・活性に対する金属イオンの影響Ｎｏｎｅ
１．００
にＣ１１０３
ＦｅＣ１ｔ １３４
ＣａＣＩｚ ２４５
ＳｎＣ１ｚ ２５０
ＭｎＣ１ｚ １２５
ＭｇＣｈ ２３４
ＨｇＣ１ｚ １４３
ＺＩＩＣＩ２ １８８
ＢａＣｌｚ １８８
ＣｕＣＩｚ ２３４
ＣＯＣ１２１１８
ＡＩＣ１３３１３
（ロ）１ｍＭの金属塩を添加した酵素液を３０℃１３０
分間放置した後、ｐＨ６゜１．５０℃にて活性を測定し
た結果を第２表に示す。Table 1 Effect of metal ions on milk curd/activityNone
1.00 to C1103 FeC1t 134 CaCIz 245 SnC1z 250 MnC1z 125 MgCh 234 HgC1z 143 ZIICI2 188 BaClz 188 CuCIz 234 COC12 118 AIC13313 (b) Heat the enzyme solution containing 1mM metal salt at 30℃130
After standing for a minute, the activity was measured at pH 6° and 1.50° C. The results are shown in Table 2.
第２表 金属イオンによる酵素の活性化、阻害Ｎｏｎｅ
１００
ＭＣｌ １０３
ＰｅＣＩｓ ６５
ＣａＣ１ｚ １３０
ＳｎＣＩｘ ６７
ＭｎＣ１ｚ ８５
門ｇｃ１． １０３
Ｈ１０３Ｈ５８
ＺｎＣ１＝ ６３
ＢａＣ１ｚ ８９
ＣｕＣ１ｔ １１９
ＣｏＣ１ｚ １２９
ＡＩＣ１ｚ １２５
第２表の結果から明らかなようにこの場合においては本
酵素の凝乳活性はカルシウム、銅。Table 2 Enzyme activation and inhibition by metal ionsNone
100 MCl 103 PeCIs 65 CaC1z 130 SnCIx 67 MnC1z 85 phylum gc1. 103 H103H58 ZnC1= 63 BaC1z 89 CuC1t 119 CoC1z 129 AIC1z 125 As is clear from the results in Table 2, in this case, the milk curdling activity of this enzyme is based on calcium and copper.
コバルト、アルミニウム、ニッケルイオンによ゛ り活
性化され、鉄、ストロンチウム、水銀、亜鉛イオンによ
り阻害される。It is activated by cobalt, aluminum, and nickel ions, and inhibited by iron, strontium, mercury, and zinc ions.
（８）酵素阻害剤による酵素の阻害
ｐＨ６，ｌ 、３０℃において各種酵素阻害剤により３
０分間処理した後、ｐＨ６，１，５０℃で凝乳活性を測
定した結果を以下の第３表に示す。(8) Inhibition of enzymes by enzyme inhibitors At pH 6.1 and 30°C, various enzyme inhibitors
After treatment for 0 minutes, the milk curd activity was measured at pH 6, 1, and 50° C. The results are shown in Table 3 below.
何れの阻害剤に対しても著しい阻害は認められなかった
。No significant inhibition was observed with any of the inhibitors.
（本頁以下余白）
第３表 酵素阻害剤による酵素の阻害
Ｎ ｏ ｎ ｅ −１００
ＰＣＭ８ ２ ９１
εＤＴＡ ２ ７５
ＤＦＰ ３ ８２
ＳＴＩ １ ７５
ＰＣＭＢ（ｐ−ｃｈｌｏｒｏｍｅｒｃｕｒｉｂｅｎｚｏ
ｉｃ ａｃｉｄ）。(Left space on this page) Table 3 Inhibition of enzymes by enzyme inhibitors None -100 PCM8 2 91 εDTA 2 75 DFP 3 82 STI 1 75 PCMB (p-chloromercuribenzo
ic acid).
ＥＤＴＡ （エチレンジアミンテトラ酢酸）。EDTA (ethylenediaminetetraacetic acid).
ＤＦＰ（ｄｉｉｓｏｐｒｏｐｙｌ ｆｌｕｏｒｏｐｈｏ
ｓｐｈａｔｅ）。DFP (diisopropyl fluorophore)
(spate).
５ＴＩ（Ｓｏｙ−ｂｅａｎ ｔｒｙｐｓｉｎ １ｎｈｉ
ｂｉｔｏｒ）。5TI(Soy-bean trypsin 1nhi)
bitor).
ＰＩ（Ｐｏｔａｔｏ １ｎｈｉｂｉｔｏｒ） ０本発明
の新規凝乳酵素は、上記性質を有する、ものであればそ
の由来を問わないものであるが本酵素の具体例としては
、例えばムコール属に属する微生物由来のものが挙げら
れる
（ＩＩ）酵素活性の測定法
本明細書における酵素活性は、豆乳５滅に酵素液０．５
ｍｌを加え、すばやく混和したのち、一定温度ｔこ保ち
、基質が凝乳するまでの時間を測定した。PI (Potato 1nhibitor) 0 The novel milk-clotting enzyme of the present invention is not limited to its origin as long as it has the above-mentioned properties, but specific examples of the present enzyme include those derived from microorganisms belonging to the genus Mucor. (II) Enzyme activity measurement method In this specification, the enzyme activity is measured by measuring soybean milk with 0.5 ml of enzyme solution.
ml was added and mixed quickly, the temperature was maintained at a constant temperature, and the time until the substrate curdled was measured.
１分間に基質５戚を凝乳させる酵素量をもって１凝乳ユ
ニツトとしたものであり、以下の式により算出される。One curdling unit is defined as the amount of enzyme that curds the substrate 5 in 1 minute, and is calculated by the following formula.
（ＩＩＩ）凝乳酵素の製造 本発明の凝乳酵素の製造方法について以下に述べる。(III) Production of milk-clotting enzyme The method for producing the milk-clotting enzyme of the present invention will be described below.
１、使用微生物
本発明の凝乳酵素を生産するために使用する菌株として
はムコールエスピーストレインＭｕｃｏｒｓｐ、５ｔｒ
ａｉｎ） ５１２１ （ＦＥＲＭ Ｐ−１０２２１）を
挙げることができる。1. Microorganisms used The strain used for producing the milk-clotting enzyme of the present invention is Mucorsp strain Mucorsp, 5tr.
ain) 5121 (FERM P-10221).
この菌株は、次の菌学的性質を有する。This strain has the following mycological properties.
サブロー培地、ポテトデキストロース培地、ツエペック
ドックス培地、麦芽エキス培地、いずれの培地でも生育
は速く、表面は、羊毛状を呈する。Growth is fast on any medium, including Sabouraud medium, potato dextrose medium, Zwepek Dox medium, and malt extract medium, and the surface exhibits a woolly appearance.
集落は、白色〜黄褐色。菌子は、隔壁が無く、気化菌子
で、はふく菌糸を形成、高さ０．５〜３．５μで滑面。The colony is white to yellowish brown. Mycelia have no septa, are vaporized, form fluffy hyphae, and have a smooth surface with a height of 0.5 to 3.5 μm.
接合胞子は球状で、有軸、黒褐色でφ７０〜８０μｍ、
胞子のう柄は、車軸房状に分枝している。Zygospores are spherical, axial, blackish brown, φ70-80μm,
The sporangium is branched into an axle-tufted shape.
胞子のうば、球状でφ２０〜７０μｍ０２、培養及び粗
酵素液の採取
固体培養にて生産する場合は、原料としてフスマ、穀類
の糠、大豆、脱脂大豆、米、小麦等の穀類を単独あるい
は組み合わせて使用することが出来る。これら原料に対
し６０〜１５０χ（Ｗ／Ｗ） 、望ましくは８０〜１２
０χ（Ｗ／Ｗ）散水し、殺菌後、菌を摂取し２０〜３５
″Ｃ５望ましくは２５〜３０℃にて１〜４日間、望まし
くは２〜３日好気的な条件で培養を行う。The spores are spherical and have a diameter of 20 to 70 μm.Culture and crude enzyme solution collectionWhen producing by solid culture, use grains such as bran, cereal bran, soybeans, defatted soybeans, rice, wheat, etc. alone or in combination as raw materials. It can be used. 60-150χ (W/W) for these raw materials, preferably 80-12
Sprinkle water at 0χ (W/W), ingest bacteria after sterilization, and reach 20 to 35
``C5'' Culture is carried out under aerobic conditions, preferably at 25-30°C for 1-4 days, preferably 2-3 days.
培養終了後、５〜２０倍、望ましくは１０倍量程度の水
を加え攪拌、酵素抽出することにより粗酵素液が得られ
る。After completion of the culture, a crude enzyme solution is obtained by adding 5 to 20 times, preferably about 10 times the amount of water, stirring, and enzymatic extraction.
液体培養にて生産する場合は、窒素源としては、有機窒
素含有物として、各種アミノ酸、マルトエキス、ペプト
ン、肉エキス等がまた無機窒素化合物として、塩化アン
モニウム、硫酸アンモニウム、硝酸アンモニウム等が単
独又は組み合せて使用される。炭素源としては、グルコ
ース、シュークロース、糖蜜等、通常用いるものは全て
使用出来る。When producing by liquid culture, nitrogen sources include organic nitrogen-containing substances such as various amino acids, malt extract, peptone, meat extract, etc., and inorganic nitrogen compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, etc. alone or in combination. used. As the carbon source, all commonly used carbon sources such as glucose, sucrose, and molasses can be used.
その他、ミネラル、ビタミン類等を適宜添加使用するこ
とが出来る。培養温度は２５〜３５℃１望ましくは２７
〜３２℃が好適である。培養ｐｔｉは、６，０〜７、０
、望ましくは６．５〜６．８が好適である。In addition, minerals, vitamins, etc. can be added as appropriate. The culture temperature is 25-35℃, preferably 27℃.
~32°C is preferred. Culture pti is 6,0-7,0
, preferably 6.5 to 6.8.
培養は、２〜７日間、好気的条件下で行う。培養終了後
、培養液中の菌体等を遠心分離にて除去し、培養上滑を
粗酵素液として使用する。Cultivation is carried out under aerobic conditions for 2-7 days. After the culture is completed, bacterial cells and the like in the culture solution are removed by centrifugation, and the culture supernatant is used as a crude enzyme solution.
３、酵素の精製
通常の酵素精製法を通用できるが、例えば硫安塩析によ
り得られた沈澱蛋白を硫酸アンモニウム−リン酸ナトリ
ウムバッファーで溶解し、不溶物を遠心分離で除去した
後、ブチル、トヨバール６５０５カラムに添加し、同じ
バッファーで未吸着区分を溶出させた後、硫安濃度１．
０Ｍ〜ＯＭ直線濃度勾配をかけて酵素を溶出して、およ
そ０．７Ｍ硫安で溶出される単一の活性画分を集め、濃
縮、透析することにより精製凝乳酵素を得ることができ
る。3. Enzyme purification Ordinary enzyme purification methods can be used. For example, precipitated proteins obtained by salting out ammonium sulfate are dissolved in ammonium sulfate-sodium phosphate buffer, insoluble matter is removed by centrifugation, and then butyl, Toyovar 6505 After adding it to the column and eluting the unadsorbed fraction with the same buffer, the ammonium sulfate concentration was 1.
Purified milk-clotting enzyme can be obtained by eluting the enzyme by applying a 0M to OM linear concentration gradient, collecting a single active fraction eluted with approximately 0.7M ammonium sulfate, concentrating and dialysis.
本発明により、滑らかな食感を有し、乳化性に優れ、苦
み、渋み等の雑味及び大豆臭等の異臭のない大豆由来の
全く新しい食品素材を効率的に製造することを可能にす
る新規かつ有用な凝乳酵素及びその製造方法を提供する
ことができた。そして、本発明の凝乳酵素を使用して得
られた大豆由来の食品素材は、特に優れた食品の乳化特
性を有し、各種食品の新素材として利用できる利点があ
る。また、当該食品素材を使用することにより、品質の
優れた多種食品を製造することが可能となり、例えば、
納豆、味噌、豆腐等の大豆を原料とする食品、チーズ、
ヨーグルト、アイスクリーム、プリン、ケフィール等の
乳製品、マヨネーズ、ドレッシング、マーガリン等に使
用する乳化剤、油脂類の代替品として使用することが出
来る。更には、スープ、コロッケ、ハンバーグ等の惣菜
、ムース、ゼリー等デザート食品、カマボコ、ソーセー
ジ等練り製品、菓子、麺類等に使用し、品質の優れた食
品を製造できる利点を有するものである。The present invention makes it possible to efficiently produce a completely new food material derived from soybeans that has a smooth texture, excellent emulsifying properties, and is free from unpleasant tastes such as bitterness and astringency, and foreign odors such as soybean odor. A new and useful milk-clotting enzyme and a method for producing the same could be provided. The soybean-derived food material obtained using the milk-clotting enzyme of the present invention has particularly excellent food emulsifying properties, and has the advantage of being usable as a new material for various foods. In addition, by using the food material, it is possible to produce a wide variety of foods with excellent quality, such as:
Foods made from soybeans such as natto, miso, and tofu, cheese,
It can be used as an emulsifier and a substitute for oils and fats in dairy products such as yogurt, ice cream, pudding, and kefir, mayonnaise, dressing, and margarine. Furthermore, it has the advantage that it can be used for prepared foods such as soups, croquettes, and hamburgers, dessert foods such as mousse and jelly, paste products such as kamaboko and sausages, confectionery, noodles, etc., and can produce foods of excellent quality.
次に、実施例により、本発明を具体的に説明する。 Next, the present invention will be specifically explained with reference to Examples.
（１）粗酵素液の調整
ムコール エスピー ストレイン５１２１（ＦＥＲＭ
Ｐ−１０２２１）を１００（Ｗ／Ｗ）％散水したふすま
培地６０ｇに接種し、３０℃５３日間培養を行った。培
養終了後１０倍量の水を加え攪拌、抽出し、粗酵素液６
００ｄをえた。この酵素液は２ユニント／ｍｌの凝乳活
性を含有していた。(1) Preparation of crude enzyme solution Mucor SP Strain 5121 (FERM
P-10221) was inoculated into 60 g of bran medium sprinkled with 100 (W/W)% water, and cultured at 30°C for 53 days. After culturing, add 10 times the volume of water, stir, and extract to obtain crude enzyme solution 6.
I got 00d. This enzyme solution contained 2 units/ml of milk curdling activity.
（２）粗酵素液の調整
上記（１）で得られた粗酵素液を硫安８０％飽和状態に
して２〜３時間４℃で放置後、沈澱蛋白を遠心分離で回
収した。この沈澱蛋白中の蛋白含量は９１．５ｍｇであ
った。この沈澱蛋白には１３．１ユニツト／■の凝乳活
性を含有していた。(2) Preparation of Crude Enzyme Solution The crude enzyme solution obtained in the above (1) was saturated with ammonium sulfate to 80% and left at 4° C. for 2 to 3 hours, and then the precipitated protein was collected by centrifugation. The protein content in this precipitated protein was 91.5 mg. This precipitated protein contained 13.1 units/■ of milk curdling activity.
（３）酵素の精製
上記（２）で得ら屁た沈澱蛋白をρ■イ６．１の１．０
Ｍ硫酸アンモニウム−２０１１ＩＭリン酸ナトリウムバ
ファーで溶解し、不純物を遠心分離で除去した後、ブチ
ル−トヨバール６５０５カラム（容量１３Ｊｌｊりに添
加した。同じバファーで未吸着区分を溶出させた後、硫
安濃Ｊｔ １．０ ＭからＯＭ直線濃度勾配をかけて酵
素を溶出させた。およそ０．７Ｍ硫安で溶出される単一
の活性画分を集め、濃縮、透析し、精製凝乳酵素２０．
０ｍｇを得た。この精製酵素は３７．７ユニツト／ｍｇ
の凝乳活性を含有していた。また、本精製酵素は前記の
（１）の諸性質を有していた。(3) Purification of enzyme The precipitated protein obtained in (2) above was
After dissolving with M ammonium sulfate-2011 IM sodium phosphate buffer and removing impurities by centrifugation, it was added to a butyl-Toyovar 6505 column (volume 13 Jlj). After eluting the unadsorbed fraction with the same buffer, ammonium sulfate concentrated Jt 1 The enzyme was eluted using a linear gradient from .0 M to OM. A single active fraction eluted at approximately 0.7 M ammonium sulfate was collected, concentrated, dialyzed, and purified milk-clotting enzyme 20.
0 mg was obtained. This purified enzyme contains 37.7 units/mg.
It contained milk curdling activity. In addition, this purified enzyme had the above-mentioned properties (1).
〔参考例〕 食品素材の製造
上記実施例で得た精製酵素０．２ ｍｇをｐ ［（６，
１に調整した豆乳５００ｄに添加し、５５℃で１０分間
反応させた。反応終了後、１５００ｘｇで１５分間遠心
分離し、水分８１％のペースト状の食品素材を２００ｇ
得た。本食品素材は、優れた乳化特性を有し、滑らかな
テクスチャーを与え、苦み、渋み等の雑味及び大豆臭等
の異臭のない食品素材であった。[Reference example] Production of food material 0.2 mg of the purified enzyme obtained in the above example was added to p[(6,
It was added to 500 d of soymilk adjusted to 1 and reacted at 55° C. for 10 minutes. After the reaction is complete, centrifuge at 1500xg for 15 minutes to collect 200g of pasty food material with 81% water content.
Obtained. This food material had excellent emulsifying properties, gave a smooth texture, and was free of unpleasant tastes such as bitterness and astringency and foreign odors such as soybean odor.
第１図は凝乳酵素のｐＨと活性の関係を示す図であり、
第２図は当該酵素のｐ Ｉ（安定性を示す図である。又
、第３図は当該酵素の温度と活性の関係を示す図であり
、第４図は当該酵素の温度安定性を示す図である。Figure 1 is a diagram showing the relationship between pH and activity of milk-clotting enzyme.
Figure 2 is a diagram showing the p I (stability) of the enzyme. Figure 3 is a diagram showing the relationship between temperature and activity of the enzyme, and Figure 4 is a diagram showing the temperature stability of the enzyme. It is a diagram.
Claims (1)
にて３０℃、１時間処理した後の残存活性を測定した後
の安定ｐＨ範囲はｐＨ３．０〜７．０の範囲内のいずれ
のｐＨ値においても６０％以上であった （４）作用温度範囲及び最適作用温度 最適作用温度は塩化カルシウム添加で６０℃、無添加で
５７℃でり、作用温度は、塩化カルシウム添加で５０〜
７０℃付近の各温度において５０％以上の相対活性を示
し、無添加で４６〜６４℃付近の各温度において５０％
以上の相対活性を示した。 （５）分子量 分子量は約３５，０００（ゲルろ過法による）ある。 （６）熱安定性 酵素溶液をｐＨ６．１、３０分間各温度で加熱処理後ｐ
Ｈ６．１、５０℃にて残存活性を測定した。その結果３
５℃までは１００％、４０℃で８０％、５０℃で１０％
の残存活性を示し、６０℃でほぼ失活する。 （７）凝乳活性に対する金属イオンの影響 （イ）ｐＨ６．１、５０℃で金属イオン１ｍＭ添加する
場合において特にアルミニウム、銅、マグネシウム、ス
トロンチウム及びカルシウムイオンにより凝乳活性が増
大する。 （ロ）１ｍＭの金属塩を添加した酵素溶液を３０℃、３
０分間放置した後ｐＨ６．１、５０℃にて活性を測定し
た結果カルシウム、銅、コバルト、アルミニウム及びニ
ッケルイオンにより活性化され、鉄、ストロンチウム、
水銀及び亜鉛イオンにより阻害される。 （８）酵素阻害剤による酵素の阻害 ｐＨ６．１、３０℃、３０分間処理したｐＨ６．１、５
０℃で活性を測定した結果Ｐ−クロロメルクリ安息香酸
、エチレンジアミンテトラ酢酸、ジイソプロピルフルオ
ロリン酸、大豆トリプシンインヒビター及びポテトイン
ヒビターにより著しい阻害を受けない。 ２、凝乳酵素がムコール属に属する微生物由来のもので
ある請求項１記載の凝乳酵素。 ３、ムコール属に属する凝乳酵素生産菌を培地に培養し
、培養物から請求項１又は２記載の凝乳酵素を採取する
ことを特徴とする請求項１又は２記載の凝乳酵素の製造
法。 ４、ムコール属に属する微生物がムコールエスピースト
レイン５１２１（ＦＥＲＭＰ−１０２２１）である請求
項３記載の凝乳酵素の製造方法。[Scope of Claims] 1. Milk-clotting enzyme having the following physical properties (1) Action This enzyme acts on soymilk and coagulates the soymilk. (2) Substrate specificity Mainly coagulates soy milk. (3) Optimal pH and stable pH range The optimal pH is around 6.1, and the enzyme solution is
The stable pH range after measuring the residual activity after treatment at 30°C for 1 hour was 60% or more at any pH value within the range of pH 3.0 to 7.0 (4) Action temperature Range and optimum operating temperature The optimum operating temperature is 60°C with addition of calcium chloride and 57°C without addition, and the operating temperature is 50°C with addition of calcium chloride.
Shows relative activity of 50% or more at each temperature around 70°C, and 50% at each temperature around 46 to 64°C without additives
The above relative activity was shown. (5) Molecular weight The molecular weight is approximately 35,000 (according to gel filtration method). (6) After heating the thermostable enzyme solution at pH 6.1 for 30 minutes at each temperature, p
Residual activity was measured at H6.1 and 50°C. Result 3
100% up to 5℃, 80% at 40℃, 10% at 50℃
It shows residual activity of , and almost loses its activity at 60°C. (7) Influence of metal ions on milk curdling activity (a) When 1 mM of metal ions are added at pH 6.1 and 50° C., milk curdling activity is increased especially by aluminum, copper, magnesium, strontium and calcium ions. (b) The enzyme solution containing 1mM metal salt was heated at 30°C for 30 minutes.
After standing for 0 minutes, the activity was measured at pH 6.1 and 50°C. As a result, it was activated by calcium, copper, cobalt, aluminum and nickel ions, and activated by iron, strontium,
Inhibited by mercury and zinc ions. (8) Inhibition of enzyme by enzyme inhibitor pH 6.1, pH 6.1, 5 treated at 30°C for 30 minutes
The activity was measured at 0°C and was not significantly inhibited by P-chloromercribenzoic acid, ethylenediaminetetraacetic acid, diisopropylfluorophosphate, soybean trypsin inhibitor, and potato inhibitor. 2. The milk-clotting enzyme according to claim 1, wherein the milk-clotting enzyme is derived from a microorganism belonging to the genus Mucor. 3. Production of the milk-clotting enzyme according to claim 1 or 2, characterized in that a milk-clotting enzyme-producing bacterium belonging to the genus Mucor is cultured in a medium, and the milk-clotting enzyme according to claim 1 or 2 is collected from the culture. Law. 4. The method for producing a milk-clotting enzyme according to claim 3, wherein the microorganism belonging to the genus Mucor is Mucor sp strain 5121 (FERMP-10221).
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12526889A JP2791098B2 (en) | 1989-05-18 | 1989-05-18 | Novel curdling enzyme and its production method |
| US07/396,662 US4996064A (en) | 1988-08-24 | 1989-08-22 | Novel foodstuff from soymilk and method for production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12526889A JP2791098B2 (en) | 1989-05-18 | 1989-05-18 | Novel curdling enzyme and its production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02303487A true JPH02303487A (en) | 1990-12-17 |
| JP2791098B2 JP2791098B2 (en) | 1998-08-27 |
Family
ID=14905878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12526889A Expired - Fee Related JP2791098B2 (en) | 1988-08-24 | 1989-05-18 | Novel curdling enzyme and its production method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2791098B2 (en) |
-
1989
- 1989-05-18 JP JP12526889A patent/JP2791098B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2791098B2 (en) | 1998-08-27 |
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