JPH08224063A - Gelatinized composition of protein - Google Patents
Gelatinized composition of proteinInfo
- Publication number
- JPH08224063A JPH08224063A JP7340187A JP34018795A JPH08224063A JP H08224063 A JPH08224063 A JP H08224063A JP 7340187 A JP7340187 A JP 7340187A JP 34018795 A JP34018795 A JP 34018795A JP H08224063 A JPH08224063 A JP H08224063A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- btgase
- transglutaminase
- btg
- added
- 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.)
- Pending
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 80
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 80
- 239000000203 mixture Substances 0.000 title claims abstract description 46
- 108060008539 Transglutaminase Proteins 0.000 claims abstract description 31
- 102000003601 transglutaminase Human genes 0.000 claims abstract description 31
- 244000005700 microbiome Species 0.000 claims abstract description 12
- 239000002002 slurry Substances 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 17
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims description 8
- 125000002252 acyl group Chemical group 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 238000006276 transfer reaction Methods 0.000 claims description 5
- 230000000813 microbial effect Effects 0.000 claims description 3
- 239000000758 substrate Substances 0.000 abstract description 22
- 241000187747 Streptomyces Species 0.000 abstract description 12
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- 239000002537 cosmetic Substances 0.000 abstract description 3
- 235000018102 proteins Nutrition 0.000 description 67
- 230000000694 effects Effects 0.000 description 30
- 102000004190 Enzymes Human genes 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 27
- 229940088598 enzyme Drugs 0.000 description 27
- 239000000243 solution Substances 0.000 description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 239000000499 gel Substances 0.000 description 18
- 238000001879 gelation Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 13
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- 238000012360 testing method Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
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- 238000001816 cooling Methods 0.000 description 6
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
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- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 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 3
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
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- OEPOKWHJYJXUGD-UHFFFAOYSA-N 2-(3-phenylmethoxyphenyl)-1,3-thiazole-4-carbaldehyde Chemical compound O=CC1=CSC(C=2C=C(OCC=3C=CC=CC=3)C=CC=2)=N1 OEPOKWHJYJXUGD-UHFFFAOYSA-N 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 101710123874 Protein-glutamine gamma-glutamyltransferase Proteins 0.000 description 2
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
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- 239000000839 emulsion Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- -1 etc. Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 150000004698 iron complex Chemical class 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000004898 kneading Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 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
- 239000002245 particle Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000020030 perry Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229940108461 rennet Drugs 0.000 description 1
- 108010058314 rennet Proteins 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000013547 stew Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 235000019465 surimi Nutrition 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- POSZUTFLHGNLHX-KSBRXOFISA-N tris maleate Chemical compound OCC(N)(CO)CO.OCC(N)(CO)CO.OC(=O)\C=C/C(O)=O POSZUTFLHGNLHX-KSBRXOFISA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000013343 vitamin Nutrition 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
- 235000013618 yogurt Nutrition 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Dairy Products (AREA)
- Confectionery (AREA)
- Beans For Foods Or Fodder (AREA)
- Grain Derivatives (AREA)
- Fish Paste Products (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、微生物由来の新規
なトランスグルタミナーゼをタンパクに作用せしめて得
られるタンパクゲル化組成物に関する。TECHNICAL FIELD The present invention relates to a protein gelling composition obtained by reacting a novel transglutaminase derived from a microorganism with a protein.
【０００２】トランスグルタミナーゼは、ペプチド鎖内
にあるグルタミン残基のγ−カルボキシアミド基のアシ
ル転移反応を触媒する酵素である。[0002] Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain.
【０００３】このトランスグルタミナーゼは、アシル受
容体としてタンパク質中のリジン残基のε−アミノ基が
作用すると、分子内及び分子間にε−（γ−Ｇlu）−Ｌ
ys架橋結合が形成される。また水がアシル受容体として
機能するときは、グルタミン残基が脱アミド化されグル
タミン酸残基になる反応を進行させる酵素である。This transglutaminase is intra- and intermolecular ε- (γ-Glu) -L when the ε-amino group of the lysine residue in the protein acts as an acyl acceptor.
A ys crosslink is formed. When water functions as an acyl acceptor, it is an enzyme that promotes a reaction in which a glutamine residue is deamidated to become a glutamic acid residue.
【０００４】また、この新規トランスグルタミナーゼを
利用して製造される本発明のタンパクゲル化組成物は、
従来のゲル状食品、ゲル状化粧料をはじめとしてヨーグ
ルト、ゼリー、チーズ、ゲル状化粧料などとして用いら
れる。The protein gelling composition of the present invention produced by utilizing this novel transglutaminase is
It is used as yogurt, jelly, cheese, gel cosmetics, etc. as well as conventional gel foods and gel cosmetics.
【０００５】更に、本発明のゲル化組成物は、未加熱で
製造でき、熱に安定なゲルであるため、マイクロカプセ
ルの素材、固定化酵素等の担体などとしても広範囲に用
いることができるものである。Further, since the gelling composition of the present invention can be produced without heating and is a heat-stable gel, it can be widely used as a material for microcapsules, a carrier for immobilized enzymes and the like. Is.
【０００６】[0006]
【従来の技術】トランスグルタミナーゼはこれまで動物
由来のものが知られている。例えばモルモットの肝臓
［Connellan et al., ジャーナル・オブ・バイオロジカ
ル・ケミストリー(Journal of Biological Chemistry)
246 巻４号1093〜1098頁(1971)］及び哺乳動物の臓器、
血液に広く分布し［Folk et al.,アドバンセス・イン・
エンザイモロジー（Advances in Enzymology）38巻 109
〜191 頁(1973)、Folk etal.,アドバンセス・イン・プ
ロテイン・ケミストリー（Advances in Protein Chemis
try)31巻１〜133 頁(1977)］、その酵素の特徴も研究さ
れている。2. Description of the Related Art Transglutaminase is known to be derived from animals. For example, guinea pig liver [Connellan et al., Journal of Biological Chemistry]
246, Vol. 4, pages 1093-1098 (1971)] and organs of mammals,
Widely distributed in blood [Folk et al., Advances in
Volume 38: Advances in Enzymology 109
~ 191 (1973), Folk et al., Advances in Protein Chemis.
try 31: 133-133 (1977)], the characteristics of the enzyme have also been studied.
【０００７】しかし、現時点では微生物由来のトランス
グルタミナーゼについては報告されていない。また、動
物由来のトランスグルタミナーゼを用いるタンパク質の
ゲル化組成物の製造法については本発明者等が既に研究
を行なっている（特開昭58−149645号）。However, at present, there is no report on microbial-derived transglutaminase. In addition, the present inventors have already conducted research on a method for producing a gelled composition of a protein using animal-derived transglutaminase (JP-A-58-149645).
【０００８】しかし、この動物由来のトランスグルタミ
ナーゼの産業への利用、特にタンパク質のゲル化組成物
の製造法には以下に述べるような欠点がある。[0008] However, the use of this animal-derived transglutaminase in industry, particularly the method for producing a gelled composition of protein, has the following drawbacks.
【０００９】動物由来のトランスグルタミナーゼは安価
にまた大量に入手するのが困難である。また、ゲル化さ
せるのには、この高価な酵素が基質タンパク質１ｇあた
り、１ユニット以上必要でかつ、基質タンパク濃度が
2.0重量％以上必要であるという制限があること、更に
は、この動物由来のトランスグルタミナーゼはカルシウ
ム（Ｃａ2+）依存性である為に用途が制限される。Animal-derived transglutaminase is difficult to obtain inexpensively and in large quantities. In addition, for gelation, this expensive enzyme requires at least 1 unit per 1 g of substrate protein, and the concentration of the substrate protein is low.
Transglutaminase derived from this animal has a limitation that it needs to be 2.0% by weight or more, and further, its use is limited because it is dependent on calcium (Ca 2+ ).
【００１０】以上のような欠点を有する為に、動物由来
のトランスグルタミナーゼを用いるタンパクゲル化組成
物の製造についての実用化は困難であるのが現状であ
る。Due to the above-mentioned drawbacks, it is currently difficult to put into practical use the production of a protein gelation composition using animal-derived transglutaminase.
【００１１】[0011]
【発明が解決しようとする課題】従来トランスグルタミ
ナーゼの供給は動物に由来しているため実用性を考慮し
た場合、供給量、供給費用、保存費用、精製の困難さ等
の種々の面から不利であり、このままでは産業上の利用
への可能性はほとんど考えられなかった。Conventionally, since transglutaminase is conventionally supplied from animals, it is disadvantageous in various aspects such as supply amount, supply cost, storage cost, and difficulty of purification in consideration of practicality. There was little possibility of industrial use as it was.
【００１２】従って、本発明の課題は、供給量、コスト
の面、精製の容易さ等のいずれの面からも問題はなく、
しかも反応にＣａ2+を必要としない点等、実用性の高い
微生物由来の新規トランスグルタミナーゼをタンパクに
作用させて得られるタンパクゲル化組成物の提供にあ
る。Therefore, the problem of the present invention is that there is no problem in terms of supply amount, cost, easiness of purification, etc.
Moreover, the present invention provides a protein gel-forming composition obtained by reacting a novel transglutaminase derived from a microorganism, which is highly practical in that it does not require Ca 2+ for the reaction, with a protein.
【００１３】[0013]
【課題を解決するための手段】これまで動物由来の酵素
が検討されてきたが実用性に欠けるため、本発明者等は
給源を微生物に求め広く検索を行った結果、ストレプト
ベルチシリウム属などの微生物にＣａ2+非存在下でもペ
プチド鎖内のグルタミン残基のγ−カルボキシアミド基
のアシル転移反応を触媒する従来にない新規なトランス
グルタミナーゼの産生能があることが分かった。また、
この酵素を用いることにより、タンパク質濃度 1.0重量
％以上のタンパク含有溶液又はスラリーをゲル化させて
タンパクゲル化組成物を製造できることを見い出し、本
発明を完成するに至った。[Means for Solving the Problems] Enzymes of animal origin have been studied so far, but they lack practicality. Therefore, the present inventors extensively searched for microorganisms as a source and found that Streptoverticillium spp. It was found that the microorganism described above has the ability to produce an unprecedented novel transglutaminase which catalyzes the acyl transfer reaction of the γ-carboxamide group of the glutamine residue in the peptide chain even in the absence of Ca 2+ . Also,
By using this enzyme, it was found that a protein-containing solution or slurry having a protein concentration of 1.0% by weight or more can be gelated to produce a protein gel composition, and the present invention has been completed.
【００１４】すなわち、本発明は、微生物由来の、Ｃａ
2+非依存性の、ペプチド鎖内のグルタミン残基のγ−カ
ルボキシアミド基のアシル転移反応を触媒する新規トラ
ンスグルタミナーゼを添加して作用せしめて、タンパク
質濃度１重量％以上のタンパク含有溶液又はスラリーを
ゲル化させて得られたことを特徴とするタンパクゲル化
組成物の製造法に関する。That is, the present invention relates to Ca derived from microorganisms.
2+ independent protein-containing solution or slurry having a protein concentration of 1% by weight or more by adding a novel transglutaminase which catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain The present invention relates to a method for producing a protein gelation composition, which is obtained by gelling.
【００１５】[0015]
【発明の実施の形態】以下、本発明を詳細に説明する。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
【００１６】本発明のタンパクゲル化組成物の製造に使
用する新規酵素を産生する微生物であるストレプトベル
チシリウム属の菌を具体的に示すと、ストレプトベルチ
シリウム・グリセオカルネウム(Streptoverticillium g
riseocarneum) ＩＦＯ 12776，ストレプトベルチシリウ
ム・シナモネウム・サブ・エスピー・シナモネウム(Str
eptoverticillium cinnamoneum sub sp. cinnamoneum)
ＩＦＯ 12852，ストレプトベルチシリウム・モバラエン
ス（Streptoverticillium mobaraense）ＩＦＯ13819等
があげられる。[0016] A specific example of a bacterium belonging to the genus Streptoverticillium, which is a microorganism that produces a novel enzyme used in the production of the protein gelling composition of the present invention, is Streptoverticillium griseocarneum (Streptoverticillium g).
riseocarneum) IFO 12776, Streptoverticillium, Cinnamonium, Sub-SP, Cinnamonium (Str
eptoverticillium cinnamoneum sub sp. cinnamoneum)
IFO 12852, Streptoverticillium mobaraense IFO 13819 and the like.
【００１７】これら微生物を培養し、本発明に係わる新
規トランスグルタミナーゼ（以後ＢＴＧase と記す）を
取得するための培養法及び精製法等は、次の通りであ
る。The culture method and purification method for culturing these microorganisms to obtain the novel transglutaminase (hereinafter referred to as BTGase) according to the present invention are as follows.
【００１８】すなわち、その培養形態としては液体培
養、固体培養いずれも可能であるが、工業的には深部通
気撹拌培養を行うのが有利である。That is, either liquid culture or solid culture can be used as the culture form, but industrially, it is advantageous to perform deep aeration stirring culture.
【００１９】又、使用する栄養培地の培養源としては一
般に微生物培養に用いられる炭素源、窒素源、無機塩及
びその他の微量栄養源の他、ストレプトベルチシリウム
属に属する微生物の利用出来る栄養源であれば全て使用
出来る。培地の炭素源としてはブドウ糖、ショ糖、可溶
性デンプン「ラスターゲン」（商品名）、グリセリン、
デキストリン、澱粉等の他、脂肪酸、油脂、有機酸など
が単独で又は組合せて用いられる。窒素源としては無機
窒素源、有機窒素源のいずれも使用可能であり、無機栄
養源としては硝酸アンモニウム、硫酸アンモニウム、尿
素、硝酸ソーダ、塩化アンモニウム等が挙げられる。
又、有機窒素源としては大豆、米、トウモロコシ、小麦
などの粉、糠、脱脂粕をはじめコーンスティープリカ
ー、ペプトン、肉エキス、カゼイン、アミノ酸、酵母エ
キス等が挙げられる。無機塩及び微量栄養素としてはリ
ン酸、マグネシウム、カリウム、鉄、カルシウム、亜鉛
等の塩類の他ビタミン、非イオン界面活性剤、消泡剤等
の菌の生育やＢＴＧase の生産を促進するものであれば
必要に応じて使用出来る。As a culture source of the nutrient medium to be used, in addition to carbon sources, nitrogen sources, inorganic salts and other trace nutrients generally used for culturing microorganisms, nutrient sources that can be utilized by microorganisms belonging to the genus Streptoverticillium If so, all can be used. Glucose, sucrose, soluble starch "Rastergen" (trade name), glycerin,
In addition to dextrin, starch and the like, fatty acids, oils and fats, organic acids and the like are used alone or in combination. Either an inorganic nitrogen source or an organic nitrogen source can be used as the nitrogen source, and examples of the inorganic nutrient source include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, ammonium chloride and the like.
Examples of the organic nitrogen source include flour such as soybean, rice, corn, and wheat, bran, defatted meal, corn steep liquor, peptone, meat extract, casein, amino acids, and yeast extract. As inorganic salts and micronutrients, salts such as phosphate, magnesium, potassium, iron, calcium, zinc, etc., as well as vitamins, nonionic surfactants, antifoaming agents, etc. that promote the growth of bacteria and the production of BTGase can be used. If necessary, it can be used.
【００２０】培養は好気的条件で、培養温度は菌が発育
しＢＴＧase が産生する範囲であれば良く、好ましくは
25〜35℃である。培養時間は条件により異なるがＢＴＧ
aseが最も産生される時間まで培養すれば良く、通常２
〜４日程度である。The culture is carried out under aerobic conditions, and the culture temperature may be within the range where the bacteria grow and BTGase is produced, preferably
25-35 ° C. The culture time varies depending on the conditions, but BTG
It is sufficient to culture until the time when ase is produced most, usually 2
About 4 days.
【００２１】ＢＴＧase は液体培養では培養液中に溶解
されており、培養終了後培養液より固形分を除いた培養
ろ液より採取される。培養ろ液よりＢＴＧase を精製す
るには通常酵素精製に用いられるあらゆる方法が使用出
来る。例えば、エタノール、アセトン、イソプロピルア
ルコール等の有機溶媒による処理、硫安、食塩等による
塩析、透析、限外ろ過法、イオン交換クロマトグラフィ
ー、吸着クロマトグラフィー、ゲルろ過、吸着剤、等電
点分画等の方法が使用出来る。又、これらの方法を適当
に組合せる事によりＢＴＧase の精製度が上る場合は適
宜組合せて行う事が出来る。BTGase is dissolved in the culture medium in liquid culture, and is collected from the culture filtrate obtained by removing the solid content from the culture medium after completion of the culture. To purify BTGase from the culture filtrate, any method usually used for enzyme purification can be used. For example, treatment with organic solvents such as ethanol, acetone, isopropyl alcohol, salting out with ammonium sulfate, salt, etc., dialysis, ultrafiltration method, ion exchange chromatography, adsorption chromatography, gel filtration, adsorbent, isoelectric focusing. Etc. can be used. Further, when the purification degree of BTGase can be improved by appropriately combining these methods, they can be appropriately combined.
【００２２】こうしてこれらの方法によって得られた酵
素液に安定化剤として各種の塩類、糖類、蛋白質、脂
質、界面活性剤等を加え或いは加えることなく、限外ろ
過濃縮、逆浸透濃縮、減圧乾燥、凍結乾燥、噴霧乾燥の
方法を施すことにより液状又は固形の精製ＢＴＧase を
得ることが出来る。Ultrafiltration concentration, reverse osmosis concentration, vacuum drying without addition of various salts, sugars, proteins, lipids, surfactants, etc. as stabilizers to the enzyme solution thus obtained by these methods. A liquid or solid purified BTGase can be obtained by subjecting to freeze-drying or spray-drying.
【００２３】ＢＴＧase の活性測定はベンジルオキシカ
ルボニル−Ｌ−グルタミニルグリシンとヒドロキシルア
ミンを基質としてＣａ2+存在下または非存在下で反応を
行い、生成したヒドロキサム酸をトリクロロ酢酸存在下
で鉄錯体を形成させ 525nmの吸収を測定し、ヒドロキサ
ム酸の量を検量線より求め活性を算出する（ヒドロキサ
メート法）。The activity of BTGase was measured by reacting benzyloxycarbonyl-L-glutaminylglycine with hydroxylamine as a substrate in the presence or absence of Ca 2+ , and the produced hydroxamic acid was treated with an iron complex in the presence of trichloroacetic acid. After being formed, the absorption at 525 nm is measured, the amount of hydroxamic acid is determined from the calibration curve, and the activity is calculated (hydroxamate method).
【００２４】ＢＴＧase 活性は特に記載しないかぎり以
下に記載する方法により測定した。The BTGase activity was measured by the method described below unless otherwise specified.
【００２５】[0025]
【表１】 [Table 1]
【００２６】酵素液の0.05mlに試薬Ａ 0.5mlを加えて混
合し37℃で10分間反応後、試薬Ｂ 0.5mlを加えて反応停
止とＦｅ錯体の形成を行った後 525nmの吸光度を測定す
る。対照としてあらかじめ熱失活させた酵素液を用いて
同様に反応させたものの吸光度を測定し、酵素液との吸
光度差を求める。別に酵素液のかわりにＬ−グルタミン
酸γ−モノヒドロキサム酸を用いて検量線を作成し、前
記吸光度差より生成されたヒドロキサム酸の量を求め、
１分間に１μモルのヒドロキサム酸を生成する酵素活性
を１単位とした。0.5 ml of the reagent A was added to 0.5 ml of the enzyme solution and mixed, and after reacting at 37 ° C. for 10 minutes, 0.5 ml of the reagent B was added to stop the reaction and form an Fe complex, and then the absorbance at 525 nm is measured. . As a control, the absorbance of an enzyme solution preliminarily heat-inactivated and similarly reacted is measured to obtain the difference in absorbance with the enzyme solution. Separately, a calibration curve was prepared using L-glutamic acid γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the above-mentioned absorbance difference,
The enzyme activity for producing 1 μmol of hydroxamic acid per minute was defined as 1 unit.
【００２７】このようにして得られる精製ＢＴＧase の
酵素化学的性質を以下に述べる。尚、ストレプトベルチ
シリウム属内の菌株の種類によりＢＴＧase の酵素化学
的性質について若干の相違点がみられるので、それぞれ
の菌株の生産するＢＴＧase、即ちストレプトベルチシ
リウム・モバラエンス（Streptoverticillium mobaraen
se）ＩＦＯ13819 のトランスグルタミナーゼ（ＢＴＧ−
１と命名）、ストレプトベルチシリウム・グリセオカル
ネウム（Streptoverticillium griseocarneum)ＩＦＯ 1
2776のトランスグルタミナーゼ（ＢＴＧ−２と命名）、
ストレプトベルチシリウム・シナモネウム・サブ・エス
ピー・シナモネウム（Streptoverticillium cinnamoneu
m sub sp. cinnamoneum)ＩＦＯ 12852のトランスグルタ
ミナーゼ（ＢＴＧ−３と命名）についての酵素化学的性
質を記載するとともに、それを包含したものをＢＴＧas
e の酵素化学的性質とする。The enzymatic chemical properties of the purified BTGase thus obtained are described below. Since there are some differences in the enzymatic chemical properties of BTGase depending on the type of strain within the genus Streptoverticillium, the BTGase produced by each strain, namely Streptoverticillium mobaraen.
se) IFO13819 transglutaminase (BTG-
1), Streptoverticillium griseocarneum IFO 1
2776 transglutaminase (designated BTG-2),
Streptoverticillium cinnamoneu
The chemical chemistry of transglutaminase (named BTG-3) of m sub sp. cinnamoneum) IFO 12852 is described, and the inclusion thereof is also referred to as BTGas.
It is the enzymatic chemistry of e.
【００２８】a) 至適pH：６〜７ 基質としてベンジルオキシカルボニル−Ｌ−グルタミニ
ルグリシンとヒドロキシルアミンを使用し、37℃、10分
反応で作用至適pH範囲を求めた。尚、ＢＴＧ−１の至適
pHは６〜７にあり、ＢＴＧ−２の至適pHは６〜７付近に
あり、ＢＴＧ−３の至適pHは６〜７付近にある（図１、
図５及び図９参照）。A) Optimum pH: 6 to 7 Benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine were used as substrates, and the optimum pH range for action was determined by reaction at 37 ° C for 10 minutes. In addition, the optimum of BTG-1
The pH is 6-7, the optimum pH of BTG-2 is around 6-7, and the optimum pH of BTG-3 is around 6-7 (Fig. 1,
5 and 9).
【００２９】b) 至適温度：45〜55℃ 基質としてベンジルオキシカルボニル−Ｌ−グルタミニ
ルグリシンとヒドロキシルアミンを使用し、pH６、10分
反応での作用至適温度範囲を求めた。尚、ＢＴＧ−１の
至適温度は55℃付近であり、ＢＴＧ−２の至適温度は45
℃付近であり、ＢＴＧ−３の至適温度は45℃付近にある
（図２、図６及び図10参照）。B) Optimum temperature: 45 to 55 ° C. Benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine were used as substrates, and the optimum temperature range for action at pH 6 and 10 minutes was determined. Incidentally, the optimal temperature of BTG-1 is around 55 ° C., and the optimal temperature of BTG-2 is 45 ° C.
The temperature is around 0 ° C, and the optimum temperature of BTG-3 is around 45 ° C (see Figs. 2, 6, and 10).
【００３０】c) pH安定性：pH５〜９ 37℃、10分間処理でのpH安定性を求めた。尚、ＢＴＧ−
１はpH５〜９で安定であり、ＢＴＧ−２はpH５〜９で安
定であり、ＢＴＧ−３はpH６〜９で安定である（図３、
図７及び図11参照）。C) pH stability: pH stability was determined by treating at pH 5-937 ° C. for 10 minutes. In addition, BTG-
1 is stable at pH 5-9, BTG-2 is stable at pH 5-9, and BTG-3 is stable at pH 6-9 (Fig. 3,
(See FIGS. 7 and 11).
【００３１】d) 温度安定性 pH７で10分間処理での温度安定範囲を求めた。40℃では
80％以上、50℃では50〜80％の活性がそれぞれ残存し
た。尚、ＢＴＧ−１は40℃では88％活性が残存し、50℃
では74％活性が残存し、ＢＴＧ−２は40℃では86％活性
が残存し、50℃では56％活性が残存し、ＢＴＧ−３は40
℃で80％活性が残存し、50℃では53％活性が残存する
（図４、図８及び図12参照）。D) Temperature stability The temperature stability range was determined after 10 minutes of treatment at pH 7. At 40 ° C
More than 80% and 50-80% of the activity remained at 50 ℃. In addition, BTG-1 retains 88% activity at 40 ° C and 50 ° C.
BTG-2 retains 86% activity at 40 ° C, 56% activity remains at 50 ° C, and BTG-3 retains 40% activity at 40 ° C.
80% activity remains at 50 ° C and 53% activity remains at 50 ° C (see FIGS. 4, 8 and 12).
【００３２】e) 基質特異性 ＢＴＧase の各種合成基質とヒドロキシルアミンとの反
応を調べた。E) Substrate specificity The reaction of various synthetic substrates for BTGase with hydroxylamine was investigated.
【００３３】合成基質がベンジルオキシカルボニルアス
パラギニルグリシン、ベンジルオキシカルボニルグルタ
ミン、グリシルグルタミニルグリシンの場合反応しな
い。No reaction occurs when the synthetic substrate is benzyloxycarbonylasparaginylglycine, benzyloxycarbonylglutamine, or glycylglutaminylglycine.
【００３４】しかし、合成基質がベンジルオキシカルボ
ニルグルタミニルグリシンの場合の反応性は最も高い。
この時の各種合成基質濃度は５mMとした。However, the reactivity is highest when the synthetic substrate is benzyloxycarbonylglutaminylglycine.
At this time, the concentration of each synthetic substrate was 5 mM.
【００３５】結果は表−１に示される。なお、表−１中
のＣＢＺはベンジルオキシカルボニル基の略であり、Ｇ
lnはグルタミル基の略であり、Ｇlyはグリシル基の略で
あり、Ａsnはアスパラギニル基の略である。The results are shown in Table 1. CBZ in Table 1 is an abbreviation for benzyloxycarbonyl group, and G
ln is an abbreviation for glutamyl group, Gly is an abbreviation for glycyl group, and Asn is an abbreviation for asparaginyl group.
【００３６】[0036]
【表２】 [Table 2]
【００３７】f) 金属イオンの影響 活性測定系に１mM濃度になるように各種金属イオンを加
えて影響を調べた。なお、ここでいう活性測定系は、前
述のハイドロキサメート法において、カルシウム非存在
下の系を用いた（下記表−２におけるＮｏｎｅ）。F) Effect of metal ions Various metal ions were added to the activity measuring system so as to have a concentration of 1 mM, and the effect was investigated. The activity measuring system used herein was a system in the absence of calcium in the above hydroxamate method (None in Table 2 below).
【００３８】結果は表−２に示される。ＢＴＧase はＣ
ｕ2+及びＺｎ2+により活性が阻害される。The results are shown in Table-2. BTGase is C
The activity is inhibited by u 2+ and Zn 2+ .
【００３９】[0039]
【表３】 [Table 3]
【００４０】g) 阻害剤の影響 各阻害剤を１mMになるように加え、25℃、30分放置後、
活性を測定した。G) Effect of inhibitors Each inhibitor was added to 1 mM and allowed to stand at 25 ° C. for 30 minutes.
The activity was measured.
【００４１】結果は表−３に示される。ＢＴＧase はパ
ラクロロマーキュリー安息香酸（ＰＣＭＢと略する）、
Ｎ−エチルマレイミド（ＮＥＭと略する）及びモノヨー
ド酢酸により活性が阻害される。The results are shown in Table-3. BTGase is parachloromercury benzoic acid (abbreviated as PCMB),
The activity is inhibited by N-ethylmaleimide (abbreviated as NEM) and monoiodoacetic acid.
【００４２】[0042]
【表４】 [Table 4]
【００４３】表−３中ＰＭＳＦはフェニルメチルスルホ
ニルフルオライドの略である。In Table 3, PMSF is an abbreviation for phenylmethylsulfonyl fluoride.
【００４４】h) 等電点： 8.9〜9.9 アンホライン等電点電気泳動により求めた。尚、ＢＴＧ
−１の等電点（pI）は９付近であり、ＢＴＧ−２の等電
点（pI）は 9.7付近であり、ＢＴＧ−３の等電点（pI）
は 9.8付近である。H) Isoelectric point: 8.9-9.9 Determined by ampholine isoelectric focusing. In addition, BTG
The isoelectric point (pI) of -1 is around 9, the isoelectric point (pI) of BTG-2 is around 9.7, and the isoelectric point (pI) of BTG-3.
Is around 9.8.
【００４５】i) 分子量：約38,000〜約41,000 ＳＤＳディスク電気泳動法より求めた。尚、ＢＴＧ−１
の分子量は約38,000であり、ＢＴＧ−２の分子量は約4
1,000であり、ＢＴＧ−３の分子量は約41,000である。I) Molecular weight: about 38,000 to about 41,000 Determined by SDS disk electrophoresis. In addition, BTG-1
Has a molecular weight of about 38,000, and BTG-2 has a molecular weight of about 4
1,000 and the molecular weight of BTG-3 is about 41,000.
【００４６】次に、ＢＴＧase とモルモット肝由来のト
ランスグルタミナーゼ（以後ＭＴＧase と記す）との性
質を比較する。尚、ＭＴＧase は特開昭58−149645号に
記載された方法で調製した。Next, the properties of BTGase and guinea pig liver-derived transglutaminase (hereinafter referred to as MTGase) are compared. MTGase was prepared by the method described in JP-A-58-149645.
【００４７】表−４には各酵素化学的性質の比較を、表
−５にはＣａ2+の活性に及ぼす影響を示す。表−４及び
表−５より明らかのように従来主として研究されている
ＭＴＧase とストレプトベルチシリウム属由来のＢＴＧ
ase とでは酵素化学的性質において種々の差が見られ、
特に温度安定性、分子量、等電点、基質特異性に差が見
られる。また、Ｃａ2+の存在下及び非存在下のいずれに
おいても本発明のＢＴＧase は作用する点等でも明らか
な差が見られる。従って、本発明のＢＴＧaseはＭＴＧa
se とはその性質を明らかに異にするものであり、新規
トランスグルタミナーゼである。因みに、本発明に係わ
る新規トランスグルタミナーゼは、Ｃａ2+の存在下及び
非存在下のいずれにおいてもヨードアセトアミドにより
活性阻害を受ける。Table 4 shows a comparison of the chemical properties of each enzyme, and Table 5 shows the effect on Ca 2+ activity. As is clear from Table 4 and Table 5, MTGase and BTG derived from the genus Streptoverticillium have been mainly studied in the past.
There are various differences in enzymatic chemical properties from ase,
In particular, there are differences in temperature stability, molecular weight, isoelectric point, and substrate specificity. Further, there is a clear difference in that the BTGase of the present invention acts in both the presence and absence of Ca 2+ . Therefore, the BTGase of the present invention is MTGa
se is a novel transglutaminase, which has distinctly different properties. Incidentally, the novel transglutaminase according to the present invention is activity-inhibited by iodoacetamide both in the presence and absence of Ca 2+ .
【００４８】[0048]
【表５】 [Table 5]
【００４９】[0049]
【表６】 [Table 6]
【００５０】次にＢＴＧase を用いるタンパクゲル化組
成物の製法について述べる。Next, a method for producing a protein gel composition using BTGase will be described.
【００５１】ＢＴＧase は、適宜なタンパクゲル化剤の
形態で用いることができ、その調製法には、有効成分と
して先に説明した新規トランスグルタミナーゼを使用す
ることを除いては特別の制限はなく、従って、既に知ら
れている同種の酵素製剤の調製手段を適宜採用してタン
パクゲル化剤を調製することができる。例えば、ＢＴＧ
ａｓｅにデキストリン、炭酸カルシウム、乳糖、ソルビ
トール、マルチトールなどの賦形剤、希釈剤等を適宜添
加配合して粉体、顆粒などの固体状に、また液体状に製
剤化できる。BTGase can be used in the form of an appropriate protein gelling agent, and its preparation method is not particularly limited except that the novel transglutaminase described above is used as an active ingredient. Therefore, the protein gelling agent can be prepared by appropriately adopting already known means for preparing the same kind of enzyme preparation. For example, BTG
An excipient such as dextrin, calcium carbonate, lactose, sorbitol, maltitol, a diluent and the like may be appropriately added to and mixed with the asase to prepare a solid form such as powder or granules, or a liquid form.
【００５２】次に、ＢＴＧase を用いて行なうタンパク
ゲル化方法について説明する。Next, a protein gelation method using BTGase will be described.
【００５３】まず、基質となるタンパク質は、リジン残
基及びグルタミン残基を有し、上述の酵素の触媒をうけ
るものであれば、その起源、性状に制約されるものでは
なく、植物性タンパク質、動物性タンパク質、微生物タ
ンパク質、藻類タンパク質などいかなるものでも使用で
きる。植物性タンパク質としては特にその種類は限定し
ないが、例えば油糧種子の脱脂物及びそれらより分離し
たタンパク質などを挙げることができる。また、動物性
タンパク質としては、特にその種類は限定しないが、た
とえば乳タンパク、ゼラチン、コラーゲン、血清アルブ
ミン等を例示することができる。First, if the protein serving as a substrate has a lysine residue and a glutamine residue and is catalyzed by the above-mentioned enzyme, it is not restricted by its origin and properties, and it is a plant protein, Any of animal protein, microbial protein, algal protein, etc. can be used. The type of vegetable protein is not particularly limited, and examples thereof include defatted oil seeds and proteins separated from them. The animal protein is not particularly limited in its kind, and examples thereof include milk protein, gelatin, collagen, serum albumin and the like.
【００５４】また、本発明に用いる蛋白質としては前記
以外にもプロテアーゼなどで部分的に切断したタンパク
質、合成ペプチドおよび各種の化学修飾したタンパク質
でも、グルタミン残基、リジン残基を有する条件が満た
されれば、この酵素の基質とすることができる。In addition to the above-mentioned proteins, the protein used in the present invention can be partially cleaved with a protease, synthetic peptides and various chemically modified proteins, but the condition of having a glutamine residue or a lysine residue is satisfied. For example, it can be a substrate for this enzyme.
【００５５】これらのタンパク質の１重量％以上、好ま
しくは３重量％以上の溶液又はスラリーであれば、ＢＴ
Ｇase の添加により高粘性物、あるいはゲル状物が形成
され、１重量％以下であれば、溶液状又は沈殿状の架橋
高分子化物が得られる。このように、本発明に従ってタ
ンパク質の溶液又はスラリーにＢＴＧase を作用させる
と、高粘性物またはゲル状物が形成される。そこで、本
明細書においてまた本発明に関して、文脈上別異に解さ
れない限り、タンパクゲル化組成物は広義に定義され、
いわゆる典型的なゲル化物のみならず、それ程にゲル化
していない高粘性化物をも包含するものと定義する。同
様に文脈上別異に解されない限り、ゲル化は狭義のいわ
ゆるゲル化および高粘性化を包含するものと定義する。If the solution or slurry of 1% by weight or more, preferably 3% by weight or more of these proteins is BT,
A high-viscosity substance or a gel-like substance is formed by the addition of Gase, and if it is 1% by weight or less, a crosslinked polymerized substance in a solution state or a precipitate state can be obtained. Thus, when BTGase is allowed to act on the protein solution or slurry according to the present invention, a highly viscous substance or gel-like substance is formed. Thus, herein and also with respect to the present invention, unless the context requires otherwise, protein gelling compositions are broadly defined,
It is defined to include not only so-called typical gelled substances but also highly viscous substances which are not so gelled. Similarly, unless the context otherwise requires, gelling is defined to include so-called gelling and thickening in a narrow sense.
【００５６】ＢＴＧase はタンパク１ｇに対して0.01〜
2000ユニット添加、好ましくは 0.1〜200 ユニット添
加、反応溶液のpHは４〜10、好ましくは５〜８に調整
し、５〜80℃、好ましくは40〜60℃で10秒〜24時間、好
ましくは10分〜２時間インキュベートすると架橋高分子
化物ないしはゲル状物を得ることができる。このよう
に、本発明に係わるＢＴＧase は低い酵素濃度でゲル化
できる（基質タンパク質１ｇあたり0.01ユニット以上あ
ればよい）、及び低い基質濃度で使用できる（基質タン
パク質濃度１重量％以上であればよい）等の特徴を有す
る新規な酵素である。BTGase is 0.01 to 1 g of protein.
2000 unit addition, preferably 0.1-200 unit addition, pH of reaction solution adjusted to 4-10, preferably 5-8, 5-80 ° C, preferably 40-60 ° C for 10 seconds-24 hours, preferably By incubating for 10 minutes to 2 hours, a crosslinked polymer or gel can be obtained. Thus, the BTGase according to the present invention can be gelled at a low enzyme concentration (0.01 unit or more per 1 g of substrate protein), and can be used at a low substrate concentration (substrate protein concentration 1% by weight or more). It is a novel enzyme having features such as.
【００５７】このＢＴＧase 処理により十分なゲル化組
成物が得られるが、更に必要により反応終了後のゲル化
組成物を60〜200 ℃で１分間〜24時間加熱処理すること
により更に強固なゲル化組成物が得られる。前記タンパ
ク含有溶液又はスラリーは、単にタンパクと水との混合
物に限らず、タンパク、水および油脂を混合した水中油
型又は油中水型エマルジョンであってもよく、各種塩
類、澱粉、少糖類、多糖類、香料、保湿剤、着色料など
もＢＴＧase による架橋高分子化及びゲル化を阻害しな
い範囲で適宜選択して添加することができる。Sufficient gelling composition can be obtained by this BTGase treatment. If necessary, the gelling composition after completion of the reaction can be heat treated at 60 to 200 ° C. for 1 minute to 24 hours to further strengthen gelation. A composition is obtained. The protein-containing solution or slurry is not limited to a mixture of protein and water, and may be an oil-in-water type or a water-in-oil type emulsion in which protein, water and fats and oils are mixed, and various salts, starch, oligosaccharides, Polysaccharides, fragrances, humectants, colorants and the like can be appropriately selected and added within a range that does not inhibit the cross-linking polymerization and gelation by BTGase.
【００５８】またタンパク質の種類と量を調整すること
によって架橋高分子化物の架橋度を変えることができ、
これにより、生成するゲル化組成物の物性及び含水量を
目的と用途に応じて変えることができる。The degree of crosslinking of the crosslinked polymer can be changed by adjusting the type and amount of protein.
This makes it possible to change the physical properties and water content of the resulting gelled composition depending on the purpose and application.
【００５９】[0059]
【実施例】以下に本発明の実施例について述べる。Embodiments of the present invention will be described below.
【００６０】実施例１（ＢＴＧase の作成（その１）） ストレプトベルチシリウム・モバラエンス（Streptover
ticillium mobaraense）ＩＦＯ13819 を培地組成ポリペ
プトン 0.2％、グルコース 0.5％、リン酸二カリウム
0.2％、硫酸マグネシウム 0.1％からなる水性培地（pH
７） 200mlに接種し30℃、48時間培養し、得られた種培
養液をポリペプトン 2.0％、「ラスターゲン」 2.0％、
リン酸二カリウム 0.2％、硫酸マグネシウム 0.1％、酵
母エキス 0.2％、消泡剤としてポリオキシアルキレング
リコールの「アデカノール」（商品名、旭電化社製品）
0.05％からなる培地20Ｌ（pH７）に加え30℃で３日間培
養後ろ過し、培養液18.5Ｌ得た。このものの活性は0.35
ユニット／mlであった。Example 1 (Preparation of BTGase (1)) Streptoverticillium mobaraens (Streptover)
ticillium mobaraense) IFO13819 medium composition polypeptone 0.2%, glucose 0.5%, dipotassium phosphate
Aqueous medium consisting of 0.2% and 0.1% magnesium sulfate (pH
7) Inoculate 200 ml and incubate at 30 ℃ for 48 hours, and use the resulting seed culture solution for polypeptone 2.0%, "Rastergen" 2.0%,
Dipotassium phosphate 0.2%, magnesium sulfate 0.1%, yeast extract 0.2%, polyoxyalkylene glycol "Adecanol" as a defoaming agent (trade name, product of Asahi Denka Co., Ltd.)
The solution was added to 20 L of a 0.05% medium (pH 7), cultured at 30 ° C. for 3 days, and then filtered to obtain 18.5 L of a culture solution. Its activity is 0.35
Units / ml.
【００６１】培養液を塩酸でpH6.5 に調製し、予め0.05
Ｍリン酸緩衝液（pH6.5)で平衡化しておいたメタアクリ
ル酸系ポーラス型陽イオン交換樹脂の「アンバーライト
ＣＧ−５０」（商品名、ローム・アンド・ハース社製
品）のカラムに通した。この操作でトランスグルタミナ
ーゼは吸着された。さらに同緩衝液で不純蛋白質を洗い
流した後、さらに0.05〜 0.5Ｍの同緩衝液の濃度勾配を
つくり、通液して溶出液を分画回収し、比活性の高い分
画を集めた。The culture solution was adjusted to pH 6.5 with hydrochloric acid and adjusted to 0.05
Passed through a column of “Amberlite CG-50” (trade name, product of Rohm and Haas Co.), a methacrylic acid-based porous cation exchange resin equilibrated with M phosphate buffer (pH 6.5) did. By this operation, transglutaminase was adsorbed. Further, after washing away the impure protein with the same buffer, a concentration gradient of 0.05 to 0.5 M of the same buffer was further prepared, and the eluate was fractionated and collected by passing the fractions with high specific activity.
【００６２】電導度を10ms以下になるように希釈後「ブ
ルーセファロースＣＬ−６Ｂ」（商品名、ファルマシア
・ファインケミカル社製）のカラムに通した。この操作
でトランスグルタミナーゼは吸着された。更に0.05Ｍリ
ン酸緩衝液（pH７）で不純蛋白質を洗い流した後、０〜
１Ｍの食塩濃度勾配をつくり通液して溶出液を回収し比
活性の高い画分を集めた。After diluting so that the electric conductivity was 10 ms or less, it was passed through a column of "Blue Sepharose CL-6B" (trade name, manufactured by Pharmacia Fine Chemical Co.). By this operation, transglutaminase was adsorbed. After washing away the impure proteins with 0.05M phosphate buffer (pH 7),
An eluate was collected by passing through a 1M salt concentration gradient and a fraction having high specific activity was collected.
【００６３】限外濾過膜の「ＡＩＬ1010」（商品名、旭
化成工業(株)製）を使い濃縮し、 0.5Ｍの食塩を含む0.
05Ｍリン酸緩衝液（pH７）を用いて平衡化させた。An ultrafiltration membrane "AIL1010" (trade name, manufactured by Asahi Kasei Kogyo Co., Ltd.) was used to concentrate the solution, and 0.5 M sodium chloride was added.
Equilibration was performed using 05M phosphate buffer (pH 7).
【００６４】得られた濃縮液を同緩衝液で予め平衡化し
ておいた「セファデックスＧ−75」（商品名、ファルマ
シア・ファインケミカル社製）を含むカラムに通し、同
緩衝液を流して溶出液を分画した。The obtained concentrated solution was passed through a column containing "Sephadex G-75" (trade name, manufactured by Pharmacia Fine Chemicals Co., Ltd.) which had been equilibrated with the same buffer solution in advance, and the same buffer solution was flowed to the eluate. Was fractionated.
【００６５】この結果活性画分は単一のピークとして溶
出された。このものの比活性は培養ろ液に対し 625倍で
あり、回収率は47％であった。As a result, the active fraction was eluted as a single peak. Its specific activity was 625 times that of the culture filtrate, and the recovery was 47%.
【００６６】実施例２（ＢＴＧase の作成（その２）） 実施例１と同様にしてストレプトベルチシリウム・グリ
セオカルネウム（Streptoverticillium griseocarneum)
ＩＦＯ 12776を30℃で３日間培養後ろ過し培養液19Ｌを
得た。このものの活性は0.28ｕ／mlであった実施例１と
同様な方法で酵素を生成してＳＤＳディスク電気泳動で
単一の酵素を得た。Example 2 (Preparation of BTGase (2)) Streptoverticillium griseocarneum was prepared in the same manner as in Example 1.
IFO 12776 was cultured at 30 ° C. for 3 days and then filtered to obtain 19 L of culture medium. The activity of this product was 0.28 u / ml. An enzyme was produced in the same manner as in Example 1 and a single enzyme was obtained by SDS disk electrophoresis.
【００６７】実施例３（ＢＴＧase の作成（その３）） 実施例１と同様にしてストレプトベルチシリウム・シナ
モネウム・サブ・エスピー・シナモネウム(Streptovert
icillium cinnamoneum sub sp. cinnamoneum)ＩＦＯ 12
852を30℃で３日培養後ろ過し、培養液18.5Ｌを得た。
このものの酵素活性は 0.5ｕ／mlであった。Example 3 (Preparation of BTGase (3)) In the same manner as in Example 1, Streptoverticillium cinnamonium sub-SP cinnamonium (Streptovert)
icillium cinnamoneum sub sp. cinnamoneum) IFO 12
852 was cultured at 30 ° C. for 3 days and then filtered to obtain 18.5 L of culture solution.
Its enzyme activity was 0.5 u / ml.
【００６８】実施例１と同様な方法で酵素を精製してＳ
ＤＳディスク電気泳動で単一の酵素を得た。The enzyme was purified in the same manner as in Example 1 to obtain S.
Single enzyme was obtained by DS disk electrophoresis.
【００６９】 実施例４（タンパクゲル化組成物（その１）） （１）特開昭58-149645 号の実施例１に記載された方法
により調製または購入した食品タンパク類、すなわち
(i) αS1−カゼイン、(ii)Ｎａ−カゼイネート、(iii)
大豆11Ｓグロブリン、(iv)大豆７Ｓグロブリン、(v) 分
離状大豆タンパク「アジプロンＳ−２」（商品名、味の
素(株)製）、(vi)水抽出大豆タンパク、(vii) 酸沈澱大
豆タンパク、(viii)大豆タンパク粒子、(ix)大豆タンパ
クミセル、(x) ゼラチンの各５及び10重量％の水溶液ま
たは懸濁液５mlに、実施例１で調製したＢＴＧase （凍
結乾燥品、比活性2.50ｕ／mg protein) をタンパク１mg
当り0.02ｕ加え、55℃、１時間振盪インキュベートし
た。Example 4 (Protein Gel Composition (Part 1)) (1) Food proteins prepared or purchased by the method described in Example 1 of JP-A-58-149645, that is,
(i) α S1 -casein, (ii) Na-caseinate, (iii)
Soybean 11S globulin, (iv) Soybean 7S globulin, (v) Separated soybean protein "Adipron S-2" (trade name, manufactured by Ajinomoto Co., Inc.), (vi) Water-extracted soybean protein, (vii) Acid-precipitated soybean protein , (Viii) soy protein particles, (ix) soy protein micelles, and (x) gelatin in 5 ml each of 5 and 10 wt% aqueous solutions or suspensions, the BTGase prepared in Example 1 (lyophilized product, specific activity 2.50) was added. u / mg protein) is 1 mg of protein
0.02 u was added per well, and the mixture was incubated at 55 ° C. for 1 hour with shaking.
【００７０】室温放置後、サンプルの入った試験管を倒
置し、流れ落ちるかどうかでゲル化を判定した。結果は
表−６に示した。After being left at room temperature, the test tube containing the sample was inverted and gelation was judged by whether or not it flowed down. The results are shown in Table-6.
【００７１】（２）ＢＴＧase の基質とするためウサギ
ミオシンを次のように調製した。(2) Rabbit myosin was prepared as a substrate for BTGase as follows.
【００７２】Perry の方法（Perry, S.V. (1975), “Me
thods in Enzymology ” vol.2,pp.582-588, AcademicP
ress, New York）に従い、ウサギの骨格筋25ｇより３倍
量の0.45Ｍ ＫＣｌ、５mM ＡＴＰ−ＭｇＣｌ2 、50mM
リン酸緩衝液（pH 6.4）中で０℃、30分間ミオシンを抽
出し、以下希釈沈殿によって集め 0.5Ｍ ＫＣｌ、20mM
Tris-maleate(pH7.5)溶液に透析し、105 ×ｇで60分間
遠心分離した上清を精製ミオシンとして使用した。Perry's method (Perry, SV (1975), “Me
thods in Enzymology ”vol.2, pp.582-588, AcademicP
ress, New York), 0.45M KCl, 5mM ATP-MgCl 2 , 50mM 3 times as much as 25g of rabbit skeletal muscle.
Myosin was extracted in phosphate buffer (pH 6.4) at 0 ° C for 30 minutes and collected by dilution precipitation below 0.5M KCl, 20mM
The supernatant was dialyzed against a Tris-maleate (pH 7.5) solution and centrifuged at 10 5 × g for 60 minutes, and the supernatant was used as purified myosin.
【００７３】タンパク濃度は 1.5％であった。これに上
記(1）と同様の条件でＢＴＧase を添加しゲル化能を調
べた。結果は表−６に示した。The protein concentration was 1.5%. BTGase was added to this under the same conditions as in (1) above, and the gelling ability was examined. The results are shown in Table-6.
【００７４】（３）ＢＴＧase の基質とするためエビミ
オシンを次のように調製した。(3) Shrimp myosin was prepared as follows as a substrate for BTGase.
【００７５】新鮮（生）甘えび（体長約５cm）の皮をむ
きエビ屈曲筋肉をとり出し、ミンチ後、氷水洗浄し、更
に冷却下0.1mM ＤＴＴ、0.1mM ＰＭＳＦ存在下でホモジ
ナイズし、遠心分離でアクトミオシンを抽出分離した。
更に105 ×ｇで60分間超遠心操作によりアクチンを除き
ミオシンに富んだ画分を得た。更に希釈沈殿／超遠心操
作を繰り返し、エビ精製ミオシンを得た。この精製ミオ
シンはＣａ−ＡＴＰase 活性がなくアクチンとの結合能
も消失していることから、変性ミオシンであることがわ
かった。Peel fresh (raw) sweet shrimp (body length about 5 cm), take out the shrimp flexion muscle, mince, wash with ice water, homogenize in the presence of 0.1 mM DTT and 0.1 mM PMSF under cooling, and centrifuge. Then, actomyosin was extracted and separated.
To obtain a fraction rich in myosin except actin by further 10 5 × g for 60 minutes ultracentrifugation. The dilution precipitation / ultracentrifugation operation was further repeated to obtain shrimp purified myosin. This purified myosin had no Ca-ATPase activity and had lost the ability to bind to actin, indicating that it was denatured myosin.
【００７６】このタンパク濃度 3.6％の変性エビミオシ
ン溶液５ml（緩衝液、 0.5Ｍ ＫＣｌ、５mM ＣａＣｌ
2 、25mM Tris-ＨＣｌ（pH7.5 ）５mM ＤＴＴ）に対し
3.6ｕのＢＴＧase （実施例１と同様の方法で調製）を
添加し、35℃の水浴中に浸漬することによって反応を開
始し、最大35分間反応させた。5 ml of a denatured shrimp myosin solution having a protein concentration of 3.6% (buffer, 0.5 M KCl, 5 mM CaCl
2 , for 25 mM Tris-HCl (pH 7.5) 5 mM DTT)
3.6 u of BTGase (prepared in the same manner as in Example 1) was added, and the reaction was started by immersion in a 35 ° C water bath, and allowed to react for a maximum of 35 minutes.
【００７７】以上のゲル化能の実験結果をまとめると表
−６のようになった。Table 6 shows a summary of the above gelling ability experimental results.
【００７８】尚、比較例として、ＭＴＧase によるゲル
化能試験結果も示した。尚、ＭＴＧase の添加量は基質
たんぱく質１mg当り 0.1ｕとした。As a comparative example, the results of gelation ability test with MTGase are also shown. The amount of MTGase added was 0.1 u per 1 mg of substrate protein.
【００７９】[0079]
【表７】 [Table 7]
【００８０】 実施例５（タンパクゲル化組成物（その２）） ゼラチン（新田ゼラチン製）に５及び10重量％溶液とな
るように 0.1Ｍトリス−ＨＣｌbuffer（pH7.6)を加え、
60℃、３分で完全にゼラチンを溶解し、実施例４（１）
におけると同じＢＴＧase を0.02ｕ／mgタンパク質加え
よく撹拌後37℃、１時間反応させた後、沸とう水浴中に
10分間加熱した直後の状態を観察した。Example 5 (Protein Gelation Composition (Part 2)) 0.1 M Tris-HCl buffer (pH 7.6) was added to gelatin (manufactured by Nitta Gelatin Co., Ltd.) to prepare a 5 and 10 wt% solution.
Gelatin was completely dissolved at 60 ° C for 3 minutes, and Example 4 (1) was used.
Add 0.02u / mg protein to the same BTGase as in 1), stir well, react at 37 ℃ for 1 hour, and then in a boiling water bath.
The state immediately after heating for 10 minutes was observed.
【００８１】尚、ＢＴＧase を添加しない以外は全く同
一の処理をしたものを対照とした。結果は表−７に示し
た。The same treatment as that described above except that BTGase was not added was used as a control. The results are shown in Table-7.
【００８２】[0082]
【表８】 [Table 8]
【００８３】 実施例６（タンパクゲル化組成物（その３）） ＢＴＧase の基質とするため、絹蛋白質水溶液を以下の
方法で調製した。脱脂ずみの絹糸2.33ｇを 9.3Ｍ臭化リ
チウム（ＬｉＢｒ）溶液 100mlに加え、40℃で一晩撹拌
すると絹糸は可溶化した。この溶液に対し吸引濾過、対
水透析を行い粗絹蛋白質水溶液（約２重量％）を得た。Example 6 (Protein Gelling Composition (3)) A silk protein aqueous solution was prepared by the following method in order to use it as a substrate for BTGase. 2.33 g of the defatted silk thread was added to 100 ml of a 9.3 M lithium bromide (LiBr) solution, and stirred at 40 ° C. overnight, solubilized the silk thread. The solution was subjected to suction filtration and dialysis against water to obtain a crude silk protein aqueous solution (about 2% by weight).
【００８４】予め試験管内に最終濃度が0.01ｕ、0.02
ｕ、0.04ｕ／mgタンパク質となるように実施例４（１）
におけると同じＢＴＧase を入れておき、シェアリング
によるゲル化をさけるため静かに絹蛋白質水溶液を加え
た。対照としてＢＴＧase 未添加のものも用意した。The final concentration was 0.01u, 0.02 in advance in a test tube.
u, 0.04 u / mg protein Example 4 (1)
The same BTGase as in 1 was put in advance, and a silk protein aqueous solution was gently added to avoid gelation due to sharing. A control without BTGase was also prepared as a control.
【００８５】各々の試験管を室温で一晩放置後試験管内
の試料の状態を観察し表−８の結果を得た。After leaving each test tube at room temperature overnight, the state of the sample in the test tube was observed and the results shown in Table 8 were obtained.
【００８６】[0086]
【表９】 [Table 9]
【００８７】 実施例７（タンパクゲル化組成物（その４）） 市販牛乳（粗タンパク 2.9％）を約５倍（粗タンパク1
4.5％）に減圧濃縮して得た濃縮牛乳１Ｌ（リットル）
に対して、実施例４(1) に示したと同じＢＴＧase を２
ｕ加えて撹拌し、55℃、30分インキュベートした。生じ
たゲル状物を80〜95℃、20分加温し残存酵素を失活させ
た後、冷却するとプリン状のゲル食品を得た。Example 7 (Protein Gelling Composition (Part 4)) Commercial milk (crude protein 2.9%) was added about 5 times (crude protein 1).
1 L (liter) of concentrated milk obtained by vacuum concentration to 4.5%)
On the other hand, the same BTGase as shown in Example 4 (1)
u was added, stirred, and incubated at 55 ° C. for 30 minutes. The resulting gel-like product was heated at 80 to 95 ° C for 20 minutes to inactivate the residual enzyme, and then cooled to obtain a purine-like gel food product.
【００８８】要すれば、10％程度まで砂糖を添加しても
同様のゲル状物を得ることができた。If necessary, a similar gel-like product could be obtained even if sugar was added up to about 10%.
【００８９】 実施例８（タンパクゲル化組成物（その５）） 市販牛乳（粗タンパク 2.9％、油脂 3.2％、水分89％）
を約５倍に減圧濃縮し、濃縮牛乳（約10Ｌ）とし、これ
に30％のグルコノデルタラクトン溶液 100mlを加え、速
やかに混合した後、pH6.0 以上であることを確認してか
ら、実施例４（１）に示したと同じＢＴＧase を 100ｕ
加えて、撹拌し、45℃、45分間インキュベーター中に静
置してゲル化させた。かかる後にゲルを壊わさないよう
にゲルを80〜95℃迄加熱し、ＢＴＧase の失活とグルコ
ノデルタラクトンのグルコン酸への分解を行ない、ゲル
のpHを４〜５に調整した。そして冷却後、カード状のゲ
ルを約８cm角にカッティングし、酸塩法で２％程度の塩
濃度にしてPen. caseicolum （ペニシリウム・カゼイコ
ラム）のスターターを接種し、15℃、３週間、RH85％で
熟成させ、チーズを得た。Example 8 (Protein gelling composition (5)) Commercial milk (crude protein 2.9%, fats and oils 3.2%, water content 89%)
Concentrated milk about 5 times under reduced pressure to make concentrated milk (about 10 L), add 100 ml of 30% glucono delta lactone solution to it, mix quickly, and after confirming that the pH is 6.0 or more, The same BTGase as shown in Example 4 (1) was added to 100 u.
In addition, the mixture was stirred and allowed to stand in an incubator at 45 ° C. for 45 minutes for gelation. After that, the gel was heated to 80 to 95 ° C. to prevent the gel from being broken, the BTGase was inactivated and gluconodeltalactone was decomposed into gluconic acid, and the pH of the gel was adjusted to 4 to 5. Then, after cooling, cut the curd-like gel into about 8 cm square, inoculate with a starter of Pen. Caseicolum (Penicillium casei column) to a salt concentration of about 2% by the acid salt method, and leave at RH85 for 3 weeks at 15 ℃. Aged at%, cheese was obtained.
【００９０】尚、グルコノデルタラクトンを用いない場
合は、乳酸菌（Lactobacillus acidphillus,ラクトバチ
ルス・アシドフィラス）を添加し、ＢＴＧase でゲル化
後、40℃で２〜５時間発酵させても同じようなチーズが
得られた。When glucono delta lactone is not used, lactic acid bacteria (Lactobacillus acidphillus, Lactobacillus acidophilus) is added, gelled with BTGase, and then fermented at 40 ° C. for 2 to 5 hours to obtain the same cheese. was gotten.
【００９１】本法で得られるチーズは、高価な子牛のレ
ンネットを使用せずに製造することができ、またその物
性は、かなりしなやかな弾性をもつ品質の良いものであ
った。The cheese obtained by this method can be produced without using expensive veal rennet, and the physical properties thereof are of good quality with fairly supple elasticity.
【００９２】 実施例９（タンパクゲル化組成物（その６）） 実施例８の濃縮乳（１Ｌ）を５℃前後に冷却して、Stre
ptococuss thermophillus （ストレプトコッカス・サー
モフィラス）からなるスターター（５％程度）をすばや
く添加混合し、更に実施例４（１）におけると同様のＢ
ＴＧase を１ｕ（約0.01ｕ／ｇタンパク質に相当）加え
て撹拌し、35℃、１時間インキューベーターの中で静置
ゲル化させた。次にゲル温度を50℃とし、この温度に40
分間保持し、S.thermophillus によって酸を生成せしめ
かつフレーバーを増加せしめた後、更に75〜85℃に加温
せしめＢＴＧase を失活させた。Example 9 (Protein Gelling Composition (Part 6)) The concentrated milk (1 L) of Example 8 was cooled to about 5 ° C. and then Stre
A starter (about 5%) consisting of ptococuss thermophillus was rapidly added and mixed, and the same B as in Example 4 (1) was used.
TGase (1 u (corresponding to about 0.01 u / g protein)) was added, and the mixture was stirred and allowed to stand in an incubator at 35 ° C for 1 hour for gelation. Next, set the gel temperature to 50 ° C,
After being kept for a minute, acid was produced by S. thermophillus and the flavor was increased, and further heated to 75 to 85 ° C. to inactivate BTGase.
【００９３】冷却すると軽い酸味を持つ品質の優れたヨ
ーグルト様食品が得られた。Upon cooling, a good quality yogurt-like food product with a light sourness was obtained.
【００９４】 実施例10（タンパクゲル化組成物（その７）） 市販豆乳（明治乳業 (株) 製「サングロー豆乳」、粗タ
ンパク 3.1％）を約 2.5倍に減圧濃縮し、更に20℃以下
に冷却して得られた濃縮豆乳（粗タンパク７．７５％）
１Ｌに対し、実施例４（１）に示したと同様のＢＴＧａ
ｓｅ を４ｕ（0.05ｕ／ｇタンパク質に相当）加えてプ
ラスチック容器に充填し、フタをしシールした後、55℃
の湯浴中で30分加温し、酵素反応させゲル化した。しか
る後に高周波誘電加熱装置（電子レンジ、2450メガヘル
ツ、波長12cm）を用いて加熱した。Example 10 (Protein Gelling Composition (Part 7)) Commercially available soymilk (“Sunglow soymilk” manufactured by Meiji Dairy Co., Ltd., crude protein 3.1%) was concentrated under reduced pressure about 2.5 times and further to 20 ° C. or lower. Concentrated soymilk obtained by cooling (crude protein 7.75%)
For 1 L, BTGa similar to that shown in Example 4 (1)
Add 4 u of se (corresponding to 0.05 u / g protein) into a plastic container, cover with a lid and seal, then 55 ℃
The mixture was heated in a hot water bath for 30 minutes to cause an enzyme reaction and gelation. Then, it was heated using a high frequency dielectric heating device (microwave oven, 2450 MHz, wavelength 12 cm).
【００９５】通常の絹ごし豆腐、木綿豆腐と比較すると
しなやかで、型くずれしない品質の良い豆腐様ゲルがで
きた。A tofu-like gel of good quality which was supple and did not lose its shape as compared with ordinary silken tofu and cotton tofu was obtained.
【００９６】 実施例11（タンパクゲル化組成物（その８）） 丸大豆 6.5kgを20kg位の水に浸漬し、常温で１晩充分吸
水膨潤させたものを、水を加えながら磨砕機ですりつぶ
し「ご」を得た。これに更に水を25kg加え、ごを薄め少
量の消泡剤を添加し煮釜に移し、スチームを吹き込んで
加熱した。加熱条件は５分かけて 100℃まで上げ、３〜
５分保つ方法がよい。煮込み後おから絞り機でおからを
除き濃厚豆乳（粗タンパク 7.0％、油分 8.1％、水分75
％）30kg得た。Example 11 (Protein Gelling Composition (Part 8)) 6.5 kg of whole soybeans were immersed in about 20 kg of water and allowed to absorb and swell overnight at room temperature, and then ground with a grinder while adding water. Got "go". To this, 25 kg of water was further added, and the rice was diluted, a small amount of antifoaming agent was added, and the mixture was transferred to a boiling pot, and steam was blown into it for heating. The heating condition is raised to 100 ℃ over 5 minutes,
It is better to keep it for 5 minutes. After the stew, remove the okara from the okara squeezing machine to remove the thick soymilk (crude protein 7.0%, oil 8.1%, moisture 75
%) 30 kg was obtained.
【００９７】これに実施例４(1) に示したと同じＢＴＧ
ase を 200ｕ(0.1ｕ／ｇタンパク質に相当) 加えて直ち
にケーシングチューブ（塩化ビニリデンチューブ）に充
填し、37℃、30分湯浴中で加熱した。次に90℃以上の湯
浴中に移し、加熱（30〜60分）し、流水中で豆腐様ゲル
を得た。The same BTG as shown in Example 4 (1)
200 u (corresponding to 0.1 u / g protein) of ase was added and immediately filled in a casing tube (vinylidene chloride tube) and heated in a hot water bath at 37 ° C for 30 minutes. Next, the mixture was transferred into a hot water bath at 90 ° C. or higher and heated (30 to 60 minutes) to obtain a tofu-like gel in running water.
【００９８】 実施例12（タンパクゲル化組成物（その９）） 表−９のレシピーでカマボコを試作し、レオメーター
（不動工業 (株) 製）による物性測定と官能評価（ｎ＝
10）を実施した。なお、ＢＴＧase は実施例４（１）に
おけると同様のものを使用したが、その添加量はすり身
乾物１ｇに対して20ｕであり、酵素反応はＢＴＧase 無
添加のコントロールのすわり工程と同様に34℃、２時間
とし、反応終了後85℃、30分間加熱して製品とした。Example 12 (Protein Gelling Composition (Part 9)) A Kamaboko was prototyped according to the recipe of Table-9, and physical properties were measured by a rheometer (Fudo Kogyo Co., Ltd.) and sensory evaluation (n =
10) was carried out. The same BTGase as in Example 4 (1) was used, but the addition amount was 20 u per 1 g of the dried surimi, and the enzyme reaction was at 34 ° C as in the control sitting step without addition of BTGase. After 2 hours, the product was heated at 85 ° C. for 30 minutes after completion of the reaction.
【００９９】[0099]
【表１０】 [Table 10]
【０１００】物性測定および官能評価の結果を表−10及
び11に示す。The results of physical property measurement and sensory evaluation are shown in Tables 10 and 11.
【０１０１】[0101]
【表１１】 [Table 11]
【０１０２】[0102]
【表１２】 [Table 12]
【０１０３】以上のようにＢＴＧase を添加して試作し
たカマボコは筋原線維蛋白質の間にε−（γ−Ｇlu）Ｌ
ys架橋が生成するためコントロールに比べて破断強度が
増し、好ましい食感となることがわかった。[0103] As described above, Kamaboko prepared by adding BTGase produced ε- (γ-Glu) L between myofibrillar proteins.
It was found that since ys crosslinks were generated, the breaking strength was increased as compared with the control, and a favorable texture was obtained.
【０１０４】 実施例13（タンパクゲル化組成物（その10）） 表−12のレシピーでソーセージを試作し、レオナー（
(株) 山電製）による物性測定と官能評価（ｎ＝10）を
実施した。なお、ＢＴＧase は実施例４（１）における
と同様のものを使用したが、その添加量は豚肉乾物１ｇ
に対して１ｕであり、酵素反応は55℃、２時間とし、反
応終了後、80℃、30分間加熱して製品とした。尚、ＢＴ
Ｇase を添加しないものをコントロールとした。Example 13 (Protein Gelation Composition (Part 10)) A sausage was trial-produced according to the recipe shown in Table-12, and leonar (
Physical property measurement and sensory evaluation (n = 10) were performed by Yamaden Co., Ltd. The same BTGase as in Example 4 (1) was used, but the addition amount was 1 g of pork dry matter.
1 u, the enzymatic reaction was carried out at 55 ° C. for 2 hours, and after the reaction was completed, the product was heated at 80 ° C. for 30 minutes to obtain a product. In addition, BT
The one to which Gase was not added was used as a control.
【０１０５】[0105]
【表１３】 [Table 13]
【０１０６】物性測定および官能評価の結果を表−13及
び14に示す。The results of physical property measurement and sensory evaluation are shown in Tables 13 and 14.
【０１０７】[0107]
【表１４】 [Table 14]
【０１０８】[0108]
【表１５】 [Table 15]
【０１０９】以上のようにＢＴＧase を添加して試作し
たソーセージは、ＢＴＧase のゲル形成能によりコント
ロールに比べて粘弾性に富んだ歯ごたえの好ましい食感
となることがわかった。As described above, it was found that the sausage prepared by adding BTGase as a trial product has a viscoelasticity and a preferable texture with chewy texture as compared with the control due to the gel forming ability of BTGase.
【０１１０】実施例14（高粘性化物（その１）） 表−15のレシピーでホイッピング・クリームを試作し、
絞り出し特性を評価した。なお、ＢＴＧase は実施例４
（１）におけると同様のものを使用したが、その添加量
はカゼイン・ナトリウム乾物１ｇに対して１ｕであり、
ホイップ操作は万能混合撹拌機（ (株) 三栄製作所製）
を用い、７〜９℃で実施した。尚、ＢＴＧase 無添加の
ものをコントロールとした。Example 14 (Highly viscous product (No. 1)) A whipping cream was made as a trial according to the recipe shown in Table-15,
The squeezing characteristics were evaluated. In addition, BTGase is Example 4
The same as in (1) was used, but the addition amount was 1 u per 1 g of casein sodium dry matter,
Whip operation is a universal mixer (manufactured by Sanei Seisakusho Co., Ltd.)
Was carried out at 7-9 ° C. The control without BTGase was used as a control.
【０１１１】[0111]
【表１６】 [Table 16]
【０１１２】それぞれのホイッピング・クリームを用い
てガラス板上に花柄を描いて状態を観察したところ、Ｂ
ＴＧase を添加したホイッピング・クリームでは画線の
鋭い造花が可能となった。When the flower pattern was drawn on the glass plate using each whipping cream and the state was observed, B
With whipping cream to which TGase was added, artificial flowers with sharp streaks were possible.
【０１１３】 実施例１５（タンパクゲル化組成物（その11）） 表−16のレシピーでアイスクリームを試作し室温に置い
た時の形態変化を観察し、メルトダウン耐性を評価し
た。Example 15 (Protein Gelling Composition (Part 11)) Ice cream was prototyped according to the recipe in Table 16 and morphological changes were observed when the ice cream was placed at room temperature to evaluate meltdown resistance.
【０１１４】なお、ＢＴＧase は実施例４（１）におけ
ると同様のものを使用したが、その添加量は脱脂粉乳乾
物１ｇに対して25ｕであり、酵素反応はアイスクリーム
ミックスの殺菌工程（68℃、30分間）で実施した。殺菌
後、５℃で一晩エージングさせたアイスクリームミック
スをアイスクフリーザー（三菱重工 (株) 製）を用い、
品温−２〜４℃でオーバーラン90％までフリージングを
行い、コーンに充填後、−40℃で硬化させ製品とした。The BTGase used was the same as in Example 4 (1), but the addition amount was 25 u per 1 g of the nonfat dry milk powder, and the enzyme reaction was the sterilization step (68 ° C) of the ice cream mix. , 30 minutes). After sterilization, the ice cream mix that had been aged overnight at 5 ° C was used with an ice freezer (manufactured by Mitsubishi Heavy Industries, Ltd.)
Freezing was performed at a product temperature of −2 to 4 ° C. to an overrun of 90%, filled in a cone, and cured at −40 ° C. to obtain a product.
【０１１５】尚、ＢＴＧase を添加しない以外は全く同
様の操作を行って試作したアイスクリームをコントロー
ルとした。[0115] The ice cream produced by the same procedure as described above was used as a control except that BTGase was not added.
【０１１６】[0116]
【表１７】 [Table 17]
【０１１７】コントロールは室温静置後15分で形崩れし
てしまったが、ＢＴＧase を添加したアイスクリームは
30分以上も形崩れを起こさず、しかもコントロールと同
様、滑らかな口ざわりをしていた。The control lost its shape after 15 minutes of standing at room temperature, but the ice cream with BTGase added
He did not lose shape for more than 30 minutes, and had a smooth mouth as well as the controls.
【０１１８】 実施例16（タンパクゲル化組成物（その12）） 試験管内に所要量の牛皮由来アテロコラーゲン粉末（高
研 (株) 製）をとり、0.1Ｍ Tris-ＨＣｌバッファー（p
H7.5)２mlを加え、55℃の水浴中に15分間保持した後撹
拌することにより３〜10％アテロコラーゲン溶液を調製
した。高濃度溶液が冷却によるゲル化をおこさないうち
に実施例４（１）におけると同様のＢＴＧase を0.05ｕ
／mgタンパク質となるよう添加し、55℃で60分間インキ
ュベートした。全体のコントロールとしてＢＴＧase を
添加しない10％アテロコラーゲン溶液についても同様に
インキュベートした。インキュベート終了直後、室温で
60分放置後、更にその後 100℃の水浴中に15分保持後に
試験管内の様子を観察した。 その結果を表−17に示し
た。Example 16 (Protein Gelation Composition (12)) A required amount of cowhide-derived atelocollagen powder (manufactured by Koken Co., Ltd.) was placed in a test tube, and 0.1M Tris-HCl buffer (p
2 ml of H7.5) was added, and the mixture was kept in a water bath at 55 ° C for 15 minutes and then stirred to prepare a 3-10% atelocollagen solution. Before the high-concentration solution did not gel by cooling, 0.05 u of the same BTGase as in Example 4 (1) was used.
/ Mg protein and incubated at 55 ° C for 60 minutes. As a whole control, a 10% atelocollagen solution without addition of BTGase was similarly incubated. Immediately after incubation, at room temperature
After being left for 60 minutes, and then kept in a water bath at 100 ° C. for 15 minutes, the inside of the test tube was observed. The results are shown in Table-17.
【０１１９】[0119]
【表１８】 [Table 18]
【０１２０】 実施例17（タンパクゲル化組成物（その13）） 生オキアミ凍結肉（大洋漁業 (株) 製）１kgをフローズ
ンカッターにより細砕し、これに食塩30ｇ、ソルビトー
ル（味の素 (株) 製） 100ｇ、新ねり味（味の素 (株)
製）50ｇ、みりん40ｇ、黒レイショ澱粉50ｇを加えさら
に2000ｕのＢＴＧase （実施例４（１）におけると同様
のもの）を 300mlの冷水に可溶化後加えて、ステファン
社製カッターにて約６分混練した。混練直後の温度は５
〜６℃に制御した。Example 17 (Protein gelling composition (13)) 1 kg of raw krill frozen meat (manufactured by Taiyo Fisheries Co., Ltd.) was crushed with a frozen cutter, and 30 g of salt and sorbitol (manufactured by Ajinomoto Co., Inc.) ) 100g, New Neri taste (Ajinomoto Co., Inc.)
50 g, mirin 40 g, and black potato starch 50 g, and 2000 u of BTGase (the same as in Example 4 (1)) was solubilized in 300 ml of cold water, and then added with a Stefan cutter for about 6 minutes. Kneaded The temperature immediately after kneading is 5
Controlled to ~ 6 ° C.
【０１２１】このオキアミ肉ペーストを塩化ビニリデン
製のケーシングチューブ（クレハ化学 (株) 製）に充填
し、50℃にて、１時間インキュベート後、沸とう湯浴中
で25時間加熱した。加熱後流水中で冷却した後、物性測
定をした。即ちサンプルを厚さ３cmに切断し、直径７mm
の球形プランジャーを使用して、不動工業社製レオメー
ターにて測定を行ない、破断強度を求めた。尚、コント
ロールは、ＢＴＧaseを予め、高温加熱変性して失活せ
しめたものを用い、同様の方法で調製した。This krill meat paste was filled in a vinylidene chloride casing tube (manufactured by Kureha Chemical Co., Ltd.), incubated at 50 ° C. for 1 hour, and then heated in a boiling water bath for 25 hours. After cooling in running water after heating, physical properties were measured. That is, the sample is cut to a thickness of 3 cm and the diameter is 7 mm.
Was measured with a rheometer manufactured by Fudo Kogyo Co., Ltd. using a spherical plunger of No. 1, and the breaking strength was determined. The control was prepared by a similar method using BTGase which had been inactivated by heat denaturation at a high temperature in advance.
【０１２２】その結果を表−18に示した。The results are shown in Table-18.
【０１２３】[0123]
【表１９】 [Table 19]
【０１２４】すなわち、ＢＴＧase を加えたオキシアミ
肉のかまぼこ試作品はＢＴＧase を予め失活したコント
ロール区よりも格段に高い破断強度を示すことが認めら
れた。That is, it was confirmed that the kamaboko prototype of oxyami meat added with BTGase showed a markedly higher breaking strength than the control group in which BTGase was inactivated in advance.
【０１２５】[0125]
【発明の効果】本発明に係わる微生物由来のＢＴＧase
は安価に供給され、かつ精製も容易であるので実用性が
大である。そして、このＢＴＧase を用いることによ
り、カルシウム非存在下で又カルシウム存在下でも酵素
（ＢＴＧase)濃度及び基質濃度が非常に低いところで品
質の優れたゲル化組成物を製造できる。EFFECTS OF THE INVENTION BTGase derived from the microorganism of the present invention
Is inexpensive and is easy to purify, so it is highly practical. By using this BTGase, a gelled composition with excellent quality can be produced in the absence of calcium or in the presence of calcium, where the enzyme (BTGase) concentration and substrate concentration are very low.
【図１】本発明のＢＴＧ−１の至適pH曲線を示すもので
ある。FIG. 1 shows an optimum pH curve of BTG-1 of the present invention.
【図２】本発明のＢＴＧ−１の至適温度曲線を示すもの
である。FIG. 2 shows an optimum temperature curve of BTG-1 of the present invention.
【図３】本発明のＢＴＧ−１のpH安定曲線を示すもので
ある。FIG. 3 shows a pH stability curve of BTG-1 of the present invention.
【図４】本発明のＢＴＧ−１の温度安定曲線を示すもの
である。FIG. 4 shows a temperature stability curve of BTG-1 of the present invention.
【図５】本発明のＢＴＧ−２の至適pH曲線を示すもので
ある。FIG. 5 shows an optimum pH curve of BTG-2 of the present invention.
【図６】本発明のＢＴＧ−２の至適温度曲線を示すもの
である。FIG. 6 shows an optimum temperature curve of BTG-2 of the present invention.
【図７】本発明のＢＴＧ−２のpH安定曲線を示すもので
ある。FIG. 7 shows a pH stability curve of BTG-2 of the present invention.
【図８】本発明のＢＴＧ−２の温度安定曲線を示すもの
である。FIG. 8 shows a temperature stability curve of BTG-2 of the present invention.
【図９】本発明のＢＴＧ−３の至適pH曲線を示すもので
ある。FIG. 9 shows an optimum pH curve of BTG-3 of the present invention.
【図１０】本発明のＢＴＧ−３の至適温度曲線を示すも
のである。FIG. 10 shows an optimum temperature curve of BTG-3 of the present invention.
【図１１】本発明のＢＴＧ−３のpH安定曲線を示すもの
である。FIG. 11 shows a pH stability curve of BTG-3 of the present invention.
【図１２】本発明のＢＴＧ−３の温度安定曲線を示すも
のである。FIG. 12 shows a temperature stability curve of BTG-3 of the present invention.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 // Ａ２３Ｃ 9/12 Ａ２３Ｃ 9/12 9/154 9/154 11/10 11/10 Ａ２３Ｇ 9/02 Ａ２３Ｇ 9/02 Ａ２３Ｌ 1/19 Ａ２３Ｌ 1/19 1/317 1/317 Ａ 1/325 １０１ 1/325 １０１Ｄ １０１Ｂ １０２ １０２Ｚ (72)発明者 田中 晴生 神奈川県川崎市川崎区鈴木町１−１ 味の 素株式会社中央研究所内 (72)発明者 内尾 良輔 東京都中央区京橋１−５−８ 味の素株式 会社内 (72)発明者 松浦 明 愛知県春日井市松本町539−２ (72)発明者 安藤 裕康 愛知県江南市古知野町千丸221 (72)発明者 梅田 幸一 岐阜県羽島郡笠松町北及字北山1984−25Continuation of front page (51) Int.Cl. 6 Identification number Office reference number FI technical display location // A23C 9/12 A23C 9/12 9/154 9/154 11/10 11/10 A23G 9/02 A23G 9 / 02 A23L 1/19 A23L 1/19 1/317 1/317 A 1/325 101 1/325 101D 101B 102 102Z (72) Inventor Haruo Tanaka 1-1 Suzuki-cho, Kawasaki-ku, Kanagawa Prefecture Ajinomoto Co., Inc. Company Central Research Institute (72) Inventor Ryosuke Uchio 1-5-8 Kyobashi, Chuo-ku, Tokyo Ajinomoto Co., Inc. (72) Inventor Akira Matsuura 539-2 Matsumotocho, Kasugai City, Aichi Prefecture (72) Inventor Hiroyasu Ando Aichi Prefecture 221 Chimaru, Kochino-cho, Konan-shi (72) Inventor Koichi Umeda Kitayama, Kitayama, Kitayama, Kasamatsu-cho, Hashima-gun, Gifu Prefecture 1984-25
Claims (3)
チド鎖内のグルタミン残基のγ−カルボキシアミド基の
アシル転移反応を触媒する新規トランスグルタミナーゼ
を添加して作用せしめて、タンパク質濃度１重量％以上
のタンパク含有溶液又はスラリーをゲル化させて得られ
たことを特徴とするタンパクゲル化組成物。A novel transglutaminase which catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain, which is derived from a microorganism and is Ca 2 + -independent, is added to act on the protein concentration. A protein gelling composition obtained by gelling a solution or slurry containing 1% by weight or more of protein.
含有溶液又はスラリー中のタンパク質１ｇに対して０．
０１〜２０００ユニット添加使用されたことを特徴とす
る特許請求の範囲第１項記載のタンパクゲル化組成物。2. The novel transglutaminase is added to the protein-containing solution or slurry in an amount of 0.
The protein gelling composition according to claim 1, which is used by adding 01 to 2000 units.
来の、ヒドロキサメート法により測定したときにＣａ2+
非依存性の、ペプチド鎖内のグルタミン残基のγ−カル
ボキシアミド基のアシル転移反応を触媒するものである
ことを特徴とする請求項１または２記載のタンパクゲル
化組成物。3. A novel transglutaminase of microbial origin, Ca 2+ when measured by the hydroxamate method.
The protein gelling composition according to claim 1 or 2, which catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine residue in a peptide chain, which is independent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7340187A JPH08224063A (en) | 1987-03-04 | 1995-12-27 | Gelatinized composition of protein |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4915787 | 1987-03-04 | ||
| JP62-49157 | 1987-03-04 | ||
| JP7340187A JPH08224063A (en) | 1987-03-04 | 1995-12-27 | Gelatinized composition of protein |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62165067A Division JPH0665280B2 (en) | 1987-03-04 | 1987-07-01 | Protein gelling agent and protein gelling method using the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08224063A true JPH08224063A (en) | 1996-09-03 |
Family
ID=26389518
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7340187A Pending JPH08224063A (en) | 1987-03-04 | 1995-12-27 | Gelatinized composition of protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08224063A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997049302A1 (en) * | 1996-06-26 | 1997-12-31 | Swift-Eckrich, Inc. | Low-fat, ground meat food products and methods for making same |
| JP2000004786A (en) * | 1998-06-24 | 2000-01-11 | Ajinomoto Co Inc | Cheese whey protein having improved palatability, its production and utilization thereof |
| JP2012065637A (en) * | 2010-09-22 | 2012-04-05 | Ideamar Kk | Gel-like food, method for producing the same and material for gel-like food |
| CN102781246A (en) * | 2010-03-04 | 2012-11-14 | 味之素株式会社 | Coffee whitener, process for producing same, and process for producing beverage |
| JP5696661B2 (en) * | 2009-05-13 | 2015-04-08 | 不二製油株式会社 | Manufacturing method of paste products |
| JP2016500266A (en) * | 2012-12-14 | 2016-01-12 | ヒルズ・ペット・ニュートリシャン・インコーポレーテッド | Method for preparing food composition |
| JP2018038330A (en) * | 2016-09-08 | 2018-03-15 | 味の素株式会社 | Whip cream |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5959151A (en) * | 1982-09-29 | 1984-04-04 | Ajinomoto Co Inc | Preparation of novel gelatinous food |
-
1995
- 1995-12-27 JP JP7340187A patent/JPH08224063A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5959151A (en) * | 1982-09-29 | 1984-04-04 | Ajinomoto Co Inc | Preparation of novel gelatinous food |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997049302A1 (en) * | 1996-06-26 | 1997-12-31 | Swift-Eckrich, Inc. | Low-fat, ground meat food products and methods for making same |
| JP2000004786A (en) * | 1998-06-24 | 2000-01-11 | Ajinomoto Co Inc | Cheese whey protein having improved palatability, its production and utilization thereof |
| JP5696661B2 (en) * | 2009-05-13 | 2015-04-08 | 不二製油株式会社 | Manufacturing method of paste products |
| CN102781246A (en) * | 2010-03-04 | 2012-11-14 | 味之素株式会社 | Coffee whitener, process for producing same, and process for producing beverage |
| US8617632B2 (en) | 2010-03-04 | 2013-12-31 | Ajinomoto Co., Inc. | Coffee whitener, process for producing same, and process for producing beverage |
| JP2012065637A (en) * | 2010-09-22 | 2012-04-05 | Ideamar Kk | Gel-like food, method for producing the same and material for gel-like food |
| JP2016500266A (en) * | 2012-12-14 | 2016-01-12 | ヒルズ・ペット・ニュートリシャン・インコーポレーテッド | Method for preparing food composition |
| JP2018038330A (en) * | 2016-09-08 | 2018-03-15 | 味の素株式会社 | Whip cream |
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