CN1099839C - 一种获得蛋白质水解物的方法 - Google Patents
一种获得蛋白质水解物的方法 Download PDFInfo
- Publication number
- CN1099839C CN1099839C CN97194838A CN97194838A CN1099839C CN 1099839 C CN1099839 C CN 1099839C CN 97194838 A CN97194838 A CN 97194838A CN 97194838 A CN97194838 A CN 97194838A CN 1099839 C CN1099839 C CN 1099839C
- Authority
- CN
- China
- Prior art keywords
- substrate
- peptide
- bacterial strain
- enzyme
- polyglutamic
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 96
- 239000003531 protein hydrolysate Substances 0.000 title claims abstract description 11
- 108010009736 Protein Hydrolysates Proteins 0.000 title abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 62
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 56
- 102000035195 Peptidases Human genes 0.000 claims abstract description 34
- 108091005804 Peptidases Proteins 0.000 claims abstract description 34
- 230000008569 process Effects 0.000 claims abstract description 34
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 31
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 29
- 239000004220 glutamic acid Substances 0.000 claims abstract description 29
- 230000009471 action Effects 0.000 claims abstract description 19
- 239000000413 hydrolysate Substances 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims description 45
- 108090000790 Enzymes Proteins 0.000 claims description 45
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical group OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 43
- 102000004169 proteins and genes Human genes 0.000 claims description 43
- 108090000623 proteins and genes Proteins 0.000 claims description 43
- 235000018102 proteins Nutrition 0.000 claims description 42
- 230000001580 bacterial effect Effects 0.000 claims description 36
- 230000000694 effects Effects 0.000 claims description 36
- 108700023418 Amidases Proteins 0.000 claims description 34
- 102000005922 amidase Human genes 0.000 claims description 34
- 150000001412 amines Chemical class 0.000 claims description 34
- 229960002989 glutamic acid Drugs 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 15
- 102000018389 Exopeptidases Human genes 0.000 claims description 13
- 108010091443 Exopeptidases Proteins 0.000 claims description 13
- 108010059378 Endopeptidases Proteins 0.000 claims description 11
- 102000005593 Endopeptidases Human genes 0.000 claims description 11
- 101710123874 Protein-glutamine gamma-glutamyltransferase Proteins 0.000 claims description 11
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 10
- 235000013305 food Nutrition 0.000 claims description 8
- 241000187747 Streptomyces Species 0.000 claims description 6
- 125000000266 alpha-aminoacyl group Chemical group 0.000 claims description 5
- 241000228212 Aspergillus Species 0.000 claims description 4
- 241000186046 Actinomyces Species 0.000 claims description 3
- 241001337994 Cryptococcus <scale insect> Species 0.000 claims description 3
- 241000235035 Debaryomyces Species 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 241000233654 Oomycetes Species 0.000 claims description 3
- 235000013311 vegetables Nutrition 0.000 claims description 3
- 241000191940 Staphylococcus Species 0.000 claims description 2
- 239000003674 animal food additive Substances 0.000 claims description 2
- 239000000796 flavoring agent Substances 0.000 abstract description 5
- 230000006240 deamidation Effects 0.000 abstract 1
- 235000013355 food flavoring agent Nutrition 0.000 abstract 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 40
- 238000006460 hydrolysis reaction Methods 0.000 description 23
- 230000007062 hydrolysis Effects 0.000 description 20
- 238000002360 preparation method Methods 0.000 description 18
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 108010068370 Glutens Proteins 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000004365 Protease Substances 0.000 description 10
- 235000021312 gluten Nutrition 0.000 description 10
- 235000019419 proteases Nutrition 0.000 description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 9
- 241000209140 Triticum Species 0.000 description 9
- 235000021307 Triticum Nutrition 0.000 description 9
- 230000009849 deactivation Effects 0.000 description 9
- 241000193752 Bacillus circulans Species 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 230000002779 inactivation Effects 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 230000002797 proteolythic effect Effects 0.000 description 7
- 241000194108 Bacillus licheniformis Species 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 6
- 244000063299 Bacillus subtilis Species 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000003301 hydrolyzing effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 4
- 240000006439 Aspergillus oryzae Species 0.000 description 4
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 4
- 108010006303 Carboxypeptidases Proteins 0.000 description 4
- 102000005367 Carboxypeptidases Human genes 0.000 description 4
- 102000001554 Hemoglobins Human genes 0.000 description 4
- 108010054147 Hemoglobins Proteins 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 235000021245 dietary protein Nutrition 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 108010007119 flavourzyme Proteins 0.000 description 4
- 229940049906 glutamate Drugs 0.000 description 4
- 229930195712 glutamate Natural products 0.000 description 4
- 230000012447 hatching Effects 0.000 description 4
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 4
- 230000000630 rising effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 3
- 241000590020 Achromobacter Species 0.000 description 3
- 241001480052 Aspergillus japonicus Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000223218 Fusarium Species 0.000 description 3
- 108010073324 Glutaminase Proteins 0.000 description 3
- 102000009127 Glutaminase Human genes 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 108030006244 Protein-glutamine glutaminases Proteins 0.000 description 3
- 241000589516 Pseudomonas Species 0.000 description 3
- 241000233639 Pythium Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 241000187392 Streptomyces griseus Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000012139 lysis buffer Substances 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 2
- 102000004400 Aminopeptidases Human genes 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 2
- 241000193422 Bacillus lentus Species 0.000 description 2
- 240000006432 Carica papaya Species 0.000 description 2
- 235000009467 Carica papaya Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000589565 Flavobacterium Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 2
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 2
- 108030007165 Leucyl endopeptidases Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108010053229 Lysyl endopeptidase Proteins 0.000 description 2
- 102000002568 Multienzyme Complexes Human genes 0.000 description 2
- 108010093369 Multienzyme Complexes Proteins 0.000 description 2
- 241001443590 Naganishia albida Species 0.000 description 2
- 241000233614 Phytophthora Species 0.000 description 2
- 241001149949 Phytophthora cactorum Species 0.000 description 2
- 102000056251 Prolyl Oligopeptidases Human genes 0.000 description 2
- 101710178372 Prolyl endopeptidase Proteins 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 241001655322 Streptomycetales Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 101710151579 Zinc metalloproteinase Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- -1 acyl aspartic acid Zinc Chemical compound 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000007073 chemical hydrolysis Effects 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 108010092515 glycyl endopeptidase Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 108010086105 peptidyl-glutaminase Proteins 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000001810 trypsinlike Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 241000590035 Achromobacter lyticus Species 0.000 description 1
- 241001147825 Actinomyces sp. Species 0.000 description 1
- 108010075409 Alanine carboxypeptidase Proteins 0.000 description 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000131386 Aspergillus sojae Species 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 235000007558 Avena sp Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 108090000087 Carboxypeptidase B Proteins 0.000 description 1
- 102000003670 Carboxypeptidase B Human genes 0.000 description 1
- 102100032407 Carboxypeptidase D Human genes 0.000 description 1
- 108090000018 Carboxypeptidase D Proteins 0.000 description 1
- 108010080937 Carboxypeptidases A Proteins 0.000 description 1
- 102000000496 Carboxypeptidases A Human genes 0.000 description 1
- 108010059081 Cathepsin A Proteins 0.000 description 1
- 102000005572 Cathepsin A Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108030006828 D-glutaminases Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 102000004860 Dipeptidases Human genes 0.000 description 1
- 108090001081 Dipeptidases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010048963 Glutamate carboxypeptidase Proteins 0.000 description 1
- 102100025961 Glutaminase liver isoform, mitochondrial Human genes 0.000 description 1
- 101710138819 Glutaminase liver isoform, mitochondrial Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 102000004407 Lactalbumin Human genes 0.000 description 1
- 108090000942 Lactalbumin Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000863031 Lysobacter Species 0.000 description 1
- 241000863030 Lysobacter enzymogenes Species 0.000 description 1
- 102100033320 Lysosomal Pro-X carboxypeptidase Human genes 0.000 description 1
- 102100033468 Lysozyme C Human genes 0.000 description 1
- 108010070551 Meat Proteins Proteins 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108010084695 Pea Proteins Proteins 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 241000589538 Pseudomonas fragi Species 0.000 description 1
- 241001622909 Pythium intermedium Species 0.000 description 1
- 241001505297 Pythium irregulare Species 0.000 description 1
- 241001622892 Pythium periilum Species 0.000 description 1
- 241001622893 Pythium periplocum Species 0.000 description 1
- 241001385948 Pythium sp. Species 0.000 description 1
- 241000918584 Pythium ultimum Species 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 244000000231 Sesamum indicum Species 0.000 description 1
- 235000003434 Sesamum indicum Nutrition 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000520730 Streptomyces cinnamoneus Species 0.000 description 1
- 241000499056 Streptomyces griseocarneus Species 0.000 description 1
- 241000218483 Streptomyces lydicus Species 0.000 description 1
- 241001495137 Streptomyces mobaraensis Species 0.000 description 1
- 241000187177 Streptomyces thermovulgaris Species 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 108010070926 Tripeptide aminopeptidase Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 240000006677 Vicia faba Species 0.000 description 1
- 235000010749 Vicia faba Nutrition 0.000 description 1
- 235000002098 Vicia faba var. major Nutrition 0.000 description 1
- 229920002494 Zein Polymers 0.000 description 1
- 108010055615 Zein Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 125000000254 aspartoyl group Chemical group 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000011118 depth filtration Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- AJFXNBUVIBKWBT-UHFFFAOYSA-N disodium;boric acid;hydrogen borate Chemical class [Na+].[Na+].OB(O)O.OB(O)O.OB(O)O.OB([O-])[O-] AJFXNBUVIBKWBT-UHFFFAOYSA-N 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005428 food component Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 108010057284 lysosomal Pro-X carboxypeptidase Proteins 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000019702 pea protein Nutrition 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
- C12Y304/11001—Leucyl aminopeptidase (3.4.11.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
- A23L27/22—Synthetic spices, flavouring agents or condiments containing amino acids containing glutamic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
- C12Y304/11004—Tripeptide aminopeptidase (3.4.11.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/15—Peptidyl-dipeptidases (3.4.15)
- C12Y304/15001—Peptidyl-dipeptidase A (3.4.15.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/15—Peptidyl-dipeptidases (3.4.15)
- C12Y304/15005—Peptidyl-dipeptidase Dcp (3.4.15.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/16—Serine-type carboxypeptidases (3.4.16)
- C12Y304/16002—Lysosomal Pro-Xaa carboxypeptidase (3.4.16.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/16—Serine-type carboxypeptidases (3.4.16)
- C12Y304/16005—Carboxypeptidase C (3.4.16.5), i.e. carboxypeptidase Y
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/16—Serine-type carboxypeptidases (3.4.16)
- C12Y304/16006—Carboxypeptidase D (3.4.16.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17001—Carboxypeptidase A (3.4.17.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17002—Carboxypeptidase B (3.4.17.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17003—Lysine carboxypeptidase (3.4.17.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17004—Gly-Xaa carboxypeptidase (3.4.17.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17006—Alanine carboxypeptidase (3.4.17.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17011—Glutamate carboxypeptidase (3.4.17.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21004—Trypsin (3.4.21.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21019—Glutamyl endopeptidase (3.4.21.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21026—Prolyl oligopeptidase (3.4.21.26), i.e. proline-specific endopeptidase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/2105—Lysyl endopeptidase (3.4.21.50)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21057—Leucyl endopeptidase (3.4.21.57)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22025—Glycyl endopeptidase (3.4.22.25)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24033—Peptidyl-Asp metalloendopeptidase (3.4.24.33)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01043—Peptidyl-glutaminase (3.5.1.43)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
本发明涉及一种获得用作调味剂的蛋白质水解物的方法。更特别的,本发明提供了从蛋白质底物获得富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的一种方法,该方法包括将该底物进行脱酰胺处理且将该底物经过一种特异性作用蛋白水解酶的作用的步骤。
Description
本发明涉及一种获得用作调味剂的蛋白质水解物的方法。特别是,本发明提供一种从蛋白质底物获得富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的方法,该方法包括将该底物经过一种脱酰胺过程,且将该底物经过一种特异性作用蛋白质水解酶的作用的步骤。
不同的食物产品,如汤、调味汁和调味品,包含通过蛋白质物质水解获得的调味剂。传统地,这种水解是利用强盐酸后再用氢氧化钠中和的方法进行。然而,这种化学水解法导致在水解过程中获得的氨基酸的严重降解,且在此化学反应过程中形成一些危险的副产物。因此,对通过化学水解所获得调味剂的使用的日益担心导致了酶法水解工艺的发展。
酶法水解工艺以获得高水解度(DH)为目的,且通常使用非特异性作用蛋白水解酶复合物(即:非特异性作用内肽酶和外肽酶)来实现。在这方面,如WO94/25580描述了使用一种获自米曲霉(Aspergillusoryzae)的非特异性作用酶制剂水解蛋白质的一种方法。特异性作用蛋白水解酶尚未被用于此目的,因为这种酶仅导致一种不充分的水解。
目前仅报道了几种特异性作用蛋白水解酶。例如,已报道了优选地在谷氨酰肽键(谷氨酸特异性蛋白酶)处切割肽的蛋白水解酶获自金黄色葡萄球菌(Staphylococcus aureus)[Drapeau G.R;Boily Y.和Houmard J;生物化学杂志(J.Biol.Chem.)1972,247(20)6720-6726],获自放线菌属(Actinomyces sp.)[Mosolova O.V.,Rudenskaya G.N.,Stepanov V.M.,Khodova O.M.和Tsaplina I.A.;Biokhimiya 1987,52(3)414-422],获自灰色链霉菌(Streptomyces griseus)[Yoshida N.,Tsuruyama S.,Nagata K,Hirayama K,Noda K.和Makisumi S.;生物化学杂志(J.Biochem.)1988,104,451-456],获自热普通链霉菌(Streptomyces thermovulgaris)[Khaldarova N.Y.,Rudenskaya G.N.,Revina L.P.,Stephanov V.M.和Egorov N.S.;Biochimiia Moscow Eng.1989,54(1)32-38],获自枯草芽孢杆菌(Bacillus subtilis)[Niidome T.,Yoshida N.,Ogata F.,lto A.和Noda K.;生物化学杂志(J.Biochem.),1990,108,965-970]以及获自地衣芽孢杆菌(Bacillus lichenforms)[WO91/13554 A1]。
谷氨酸特异性蛋白酶最初应用于蛋白质结构研究。然而,已报道了谷氨酸特异性金黄色葡萄球菌蛋白酶用于修饰酪蛋白的溶解性和结构性质的用途[Chobert J-M.,Sitohy M.Z.和Whitaker J.R.;农业食品化学杂志(J.Agric.Food Chem.)1988,36,220-224],以及WO 91/13554A1描述了地衣芽孢杆菌用于获得蛋白质有限的特异性水解的用途。
虽然谷氨酰胺(Gln)几乎没有味道,谷氨酸(Glu)无论是游离的还是肽结合的,均对蛋白质水解产物的香味和口味起重要作用。谷氨酸可以通过将游离谷氨酰胺转化为谷氨酸来形成。这种转化(即脱酰胺)在使用强盐酸的传统化学水解过程中自然发生。
此外,谷氨酸可以通过谷氨酰胺酶(L-谷氨酰胺酶,EC3.5.1.22或D-谷氨酰胺酶,EC3.5.1.35)的作用从游离的谷氨酰胺形成。然而,由于其自发地转变成焦谷氨酰胺,大量谷氨酰胺会损失掉。
与谷氨酰胺酶相反,肽聚谷氨酰胺酶(peptidoglutaminase)(PG酶)作用于肽结合的谷氨酰胺。已知两种类型的肽聚谷氨酰胺酶。肽酰谷氨酰胺酶(EC3.5.1.43;肽聚谷氨酰胺酶I)特异于在α-氨基上有取代的谷氨酰胺,以及蛋白质-谷氨酰胺谷氨酰胺酶(EC 3.5.1.44;肽聚谷氨酰胺酶II)特异于在羧基位置或在α-氨基和羧基位置上均有取代的谷氨酰胺。获自日本曲霉(Aspergillus japonicus),环状芽孢杆菌(Bacilluscirculans),Cryptococcus albidus和克洛德巴利酵母(Debaryomyceskloecheri)的肽聚谷氨酰胺酶已被报道。[Kikuchi M.和Sakaguchi K.;农业生物化学(Agr.Biol.Chem.)1973,37(4),719-724]。
已知肽聚谷氨酰胺酶用于改造食品蛋白质。蛋白质结构的修饰改善其功能性质且扩展了作为食品组分的用途。因此,US5,082,672描述了用肽聚谷氨酰胺酶脱酰胺处理食品蛋白质的一种方法,通过此方法大豆蛋白质的溶解性、乳化作用、泡沫以及其它功能性质得以改善。然而,US5,082,672也报道了由于其大的分子尺寸及/或独特构象使得肽聚谷氨酰胺酶对完整大豆蛋白的脱酰胺能力受到限制。因此,US5,082,672指出底物的初始降解使用一种获自地衣芽孢杆菌的非特异性作用内/外肽酶复合物AlcalaseTM水解,且/或加热处理,并发现水解大豆蛋白的前热处理和后热处理产生最高的脱酰胺程度。
US3,857,967描述了用获自环状芽孢杆菌的肽聚谷氨酰胺酶制备食品和饮料的方法。同样,为了获得最高的脱酰胺程度,US3,857,967指出利用非特异性作用内/外肽酶的蛋白质底物的初始降解。
Mimouni等人[Minouni B.,Raymond J.Merle-Desnoyers A.M,AzanzaJ.L.和Ducastaing A.;谷物科学杂志(J.Cereal Science)1994,21,153-165]描述了用于改善小麦面筋的功能性质的联合酸脱酰胺和酶水解方法。更特别地,Mimouni等人描述了与使用非特异性作用内肽酶联合的酸脱酰胺方法。
已发现通过将蛋白质材料经过联合的脱酰胺作用处理及特异性作用的蛋白质水解酶作用后可获得具有极佳口味和功能的蛋白质水解物。
相应地,本发明提供了从蛋白质底物获得富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的一种方法,其包括以下步骤:底物经过脱酰胺处理;且将底物经过一种特异性作用蛋白水解酶作用。
在一优选的实施方案中,本发明提供了用于从蛋白质底物获得富含谷氨酸的水解物的一种方法,其包括以下步骤:将底物经过一种优选地切割谷氨酰-肽键的肽聚谷氨酰胺酶的作用且将底物经过一种蛋白水解酶作用,随后再经过一种或多种非特异性作用内和/或外肽酶作用。
本发明涉及一种获得富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的方法,其包括以下步骤:
(i)将该底物经过一种脱酰胺过程;且
(ii)将该底物经过一种特异性作用蛋白水解酶的作用。
此两步骤,(i)和(ii),可以同时完成,或步骤(ii)也可在步骤(i)后进行。
通过本发明的方法可以获得口味和功能极佳的蛋白水解物。特别的,可获得极佳口味的蛋白水解物是由于谷氨酸(Glu),无论是游离的还是肽结合的,均对蛋白水解物的口味和风味起重要的作用。然而,通过本发明的方法,也可以获得功能改善了的蛋白质水解物,特别是在改善的溶解性、改善的乳化性质、增加的水解程度以及改善的泡沫性质方面。脱酰胺过程
通过放出氨进行的酰胺(谷氨酰胺或天冬酰胺)到荷电的酸(谷氨酸或天冬氨酸)的转化称为脱酰胺。脱酰胺可以是非酶法或酶法脱酰胺过程。
在优选的实施方案中,脱酰胺按一种酶法脱酰胺过程来进行。特别是通过将底物经过一种转谷氨酰胺酶或肽聚谷氨酰胺酶作用的方法进行酶法处理。转谷氨酰胺酶
在优选的实施方案中脱酰胺是用酶法脱酰胺过程来进行的,其中的底物经过一种转谷氨酰胺酶的作用。谷氨酰胺酶可以是任何方便的来源,包括那些来源自哺乳动物的,见如:JP1050382和JP5023182,包括活化的第XIII因子,见如:WO93/15234;那些来源自鱼类的,见如:EP555,649以及那些来源于微生物的,见如:EP379,606,WO96/06931和WO96/22366。
在另一特殊的实施方案中,转谷氨酰胺酶来源于一种卵菌纲(Oomycete),包括疫霉属(Phytophthora)菌株,特别是恶疫霉(Phytophthora cactorum),腐霉属(Pythium)菌株,特别是畸雌腐霉(Pythium irregulare),腐霉属(Pythium sp.),间型腐霉(Pythiumintermedium),终极腐霉(Pythium ultimum)和周雄腐霉(Pythiumperiilum)(或缠器腐霉(P.periplocum))。
在另一特殊的实施方案中,转谷氨酰胺酶是细菌来源的,且优选的源自芽孢杆菌菌株,特别是枯草芽孢杆菌,链轮丝菌属(Streptoverticillium)菌株,特别是茂原链轮丝菌(Streptoverticilliummobaraensis),灰肉色链轮丝菌(Streptoverticillium griseocarneum),肉桂链轮丝菌(Streptoverticillium cinnamoneum)以及链霉菌属菌株,特别是利迪链霉菌。
转谷氨酰胺酶应以有效量加入到蛋白质底物中。转谷氨酰胺酶可以通常用于脱酰胺过程的量加入。目前考虑转谷氨酰胺酶的有效量在从约0.01到约5%(w/w)的相对于底物量的酶制剂的范围内,优选的在从约0.1到约1%(w/w)的相对于底物量的酶制剂的范围内。
酶法处理(孵育)可在任何方便的不使酶制剂失活的温度下进行,优选地在从约20℃到约70℃的范围内。
根据现有的实践,酶制剂可以被适当的失活,这或是通过升高培养混合物的温度到使酶失活的温度,如到高于约70℃,或是类似地通过降低培养混合物的pH到使酶失活的值,如低于约4.0。肽聚谷氨酰胺酶
在另一优选的实施方案中,脱酰胺是用一种酶法脱酰胺过程进行,其中的底物经过一种肽聚谷氨酰胺酶的作用。
在更特别的实施方案中,用于本发明的方法的肽聚谷氨酰胺酶可以是肽聚谷氨酰胺酶I(肽酰谷氨酰胺酶;EC3.5.1.43)或肽聚谷氨酰胺酶II(蛋白质谷氨酰胺谷氨酰胺酶;EC3.5.1.44)或其混合物。肽聚谷氨酰胺酶可以源自曲霉属的菌株,特别是日本曲霉,芽孢杆菌属的菌株,特别是环状芽孢杆菌,隐球酵母属(Cryptococcus)菌株,特别是Cryptococcus albidus,或德巴利酵母属(Debaryomyces)菌株,特别是克洛德巴利酵母菌。
肽聚谷氨酰胺酶应以有效量加入到蛋白质底物中。肽聚谷氨酰胺酶可以通常用于脱酰胺过程的量加入。目前考虑的肽聚谷氨酰胺酶的有效量范围以从约0.01到约100.000PG酶单位(PGase Unit)每100g底物的量,特别是从约0.1到约10.000PG酶单位每100g底物的量加入到底物中。
酶法处理(孵育)可在任何方便的不使酶制剂失活的温度下进行,优选地在从约20℃到约70℃的范围内。
根据现有的实践,酶制剂可以被适当的失活,这或是通过升高培养混合物的温度到使酶失活的温度,如到高于约70℃,或是类似地通过降低培养混合物的pH到使酶失活的值,如低于约4.0。特异性作用蛋白水解酶
本发明的方法包括将底物经过特异性作用蛋白水解酶作用的步骤。更特别的,特异性作用蛋白水解酶可以是一种特异性作用内肽酶或特异性作用外肽酶。特异性作用蛋白水解酶可以是任何可得的来源的。内肽酶
在优选的实施方案中,特异性作用蛋白水解酶是一种内肽酶,如:
(i)一种谷氨酰内肽酶(EC3.4.21.19);
(ii)一种赖氨酰内肽酶(EC3.4.21.50);
(iii)一种亮氨酰内肽酶(EC3.4.21.57);
(iv)一种甘氨酰内肽酶(EC3.4.22.25);
(v)一种脯氨酰内肽酶(EC3.4.21.26);
(vi)胰蛋白酶(EC3.4.21.4)或一种胰蛋白酶样内肽酶;或
(vii)一种肽酰天冬氨酸金属内肽酶(EC3.4.24.33)。
项(i)的谷氨酰内肽酶(EC3.4.21.19)是一种蛋白水解酶,优选地切割谷氨酰肽键,其优选的源自芽孢杆菌属的菌株,特别是地衣芽孢杆菌和枯草芽孢杆菌,葡萄球菌属的菌株,特别是金黄色葡萄球菌,链霉菌属的菌株,特别是热普通链霉菌和灰色链霉菌,或是放线菌属的菌株。
项(ii)的赖氨酰内肽酶(EC3.4.21.50)可以是优选的源自无色杆菌属(Achromobacter)的菌株,特别是水解无色杆菌(Achromobacterlyticus),溶杆菌属(Lysobacter)的菌株,特别是产酶溶杆菌(Lysobacterenzymogenes),或是假单胞菌属(Pseudomonas)的菌株,特别是铜绿假单胞菌(Pseudomonas aeruginosa)。
项(iii)的亮氨酰内肽酶(EC3.4.21.57)可以是植物来源的。
项(iv)的甘氨酰内肽酶(EC3.4.22.25)可优选的源自番木瓜属植物(Carica papaya)。
项(v)的脯氨酰内肽酶(EC3.4.21.26)可优选的源自黄杆菌属(Flavobacterium)的菌株,或可以是植物来源的。
项(vi)的胰蛋白酶样内肽酶可优选的源自镰孢霉属(Fusarium)的菌株,特别是尖镰孢霉(Fusarium oxysporum)如WO89/06270或WO94/25583所述。
项(vii)的肽酰天冬氨酸金属内肽酶(EC3.4.24.33)可优选的源自假单胞菌属的菌株,特别是霉实假单胞菌(Pseudomonas fragi)。外肽酶
在另一优选的实施方案中,特异性作用蛋白水解酶是一种外肽酶,其可以作用于肽的任一末端,即可以是氨肽酶或可以是羧肽酶。
在一优选的实施方案中,特异性作用蛋白水解酶是一种氨肽酶,如:
(i)一种亮氨酰氨肽酶(EC3.4.111);或
(ii)一种三肽氨肽酶(EC3.4.11.4)。
在另一优选的实施方案中,特异性作用蛋白水解酶是一种羧肽酶,如:
(i)一种脯氨酸羧肽酶(EC3.4.16.2);
(ii)一种羧肽酶A(EC3.4.17.1);
(iii)一种羧肽酶B(EC3.4.17.2);
(iv)一种羧肽酶C(EC3.4.16.5);
(v)一种羧肽酶D(EC3.4.1 6.6);
(vi)一种赖氨酸(精氨酸)羧肽酶(EC3.4.17.3);
(vii)一种甘氨酸羧肽酶(EC3.4.17.4);
(viii)一种丙氨酸羧肽酶(EC3.4.17.6);
(ix)一种谷氨酸羧肽酶(EC3.4.17.11);
(x)一种肽酰二肽酶A(EC3.4.15.1);或
(xi)一种肽酰二肽酶(EC3.4.15.5)。
特异性作用的内或外肽酶应当以有效量加入蛋白质底物。蛋白水解酶可以通常用于蛋白质水解过程中的量加入,目前考虑内肽酶的有效量是以范围从约0.05到约15CPU/100g底物的量,特别是以范围从约0.1到约5CPU/100g底物,或以范围从约0.001到约0.5AU/100g底物,特别是范围从约0.01到约0.1AU/100底物的量。
酶法处理(孵育)可在任何方便的不使酶制剂失活的温度下进行,优选地在从约20℃到约70℃的范围内。
根据现有的实践,酶制剂可以被适当的失活,这或是通过升高培养混合物的温度到使酶失活的温度,如到高于约70℃,或是类似地通过降低培养混合物的pH到使酶失活的值,如低于约4.0。非特异性作用蛋白水解酶
在一更特别的实施方案中,本发明的方法包括附加的步骤:
(iii)将底物经过一种或多种非特异性作用内和/或外肽酶作用的处理。
在此步骤中,底物经过传统水解步骤。这一附加步骤可以与步骤(i)和(ii)同时完成,或其在步骤(i)和(ii)之后完成。特别的,底物可以是步骤(i)和(ii)的反应混合物。
在一优选的实施方案中,非特异性作用内和/或外肽酶源自曲霉属菌株,特别是黑曲霉菌株,米曲霉菌株,或是酱油曲霉菌株(Asperglliussoyae),或是芽孢杆菌属菌株,特别是解淀粉芽孢杆菌菌株(Bacillusamyloliquefaciens),迟缓芽孢杆菌(Bacillus lentus)菌株,地衣芽孢杆菌(Bacillus licheniformis)菌株或枯草芽孢杆菌菌株。
非特异性作用内和/或外肽酶以范围从约0.05到约15CPU/100g底物的量,特别是以范围从约0.1到约5CPU/100g底物的量加入到底物中。
酶法处理(孵育)可在任何方便的不使酶制剂失活的温度下进行,优选地在从约20℃到约70℃的范围内。
根据现有的实践,酶制剂可以被适当的失活,这或是通过升高培养混合物的温度到使酶失活的温度,如到高于约70℃,或是类似地通过降低培养混合物的pH到使酶失活的值,如低于约4.0。蛋白质底物
用本发明方法处理的蛋白质底物可含有完整的蛋白质,或其可含有预水解的蛋白质(肽),或其可为上述物质的混合物。同样,蛋白质底物可以是蔬菜或动物来源的。
优选的蛋白质底物为蔬菜来源的,特别是大豆蛋白质,谷物蛋白质,如:小麦面筋,玉米面筋,大麦,黑麦,燕麦,水稻,玉米醇溶蛋白,棉籽蛋白质,油菜籽蛋白质,花生,苜蓿蛋白质,豌豆蛋白质,蚕豆蛋白质,芝麻蛋白质或向日葵。
动物来源的蛋白质底物可特别是乳清蛋白,酪蛋白、肉类蛋白、鱼类蛋白、红血细胞,卵蛋白、明胶或乳白蛋白。工业应用
利用本发明的方法获得的富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物可以用于不同的工业应用,特别是需要功能性蛋白质掺入的地方。
因此,另一方面,本发明提供了一种用本发明的方法获得的富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的食品。
在另一方面,本发明提供了一种用本发明的方法获得的富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的动物食品添加剂。酶活性的鉴定肽聚谷氨酰胺酶活性
肽聚谷氨酰胺酶活性可以根据Cedrangoro等人的方法来测定[Cedrangoro等人,
Enzymologia 1965,29,143],且如US3,857,967中所述。
根据此方法,用1N的NaOH调节0.5ml的此酶样品至pH6.5,装满一小容器中。按Cedrangoro等人的方法,容器中加入1ml pH10.8的硼酸盐缓冲液,释放出的氨由5N硫酸吸收,利用Nessler试剂使混合物形成颜色,在420mγ处测量所形成的颜色的光吸收值。
在这些条件下能产生1γmol/分钟氨的酶的量为一个PG酶单位。
此外,肽聚谷氨酰胺酶活性可以按如实施例2(下面)所述的方法测定蛋白水解活性:酪蛋白蛋白酶单位(CPU)
利用酪蛋白为底物可测定蛋白水解活性。一酪蛋白蛋白酶单位(CPU)定义为在标准条件(即:25℃及pH9.5下温育30分钟)下每分钟放出1mM初级氨基基团(用与丝氨酸标准比较确定)的酶的量。
更详细描述此分析方法的文件AF 228/1只要需要可从NovoNordisk A/S丹麦获得,此处引用该文件作为参考。蛋白水解活性:Anson单位(AU)
也可以用变性血红蛋白为底物测定蛋白水解活性。在用于蛋白水解活性测定的Anson-血红蛋白方法中,消化变性血红蛋白,未消化的血红蛋白用三氯乙酸(TCA)沉淀。TCA溶解产物的量用苯酚试剂测定,其能与酪氨酸或色氨酸产生蓝色。
一个Anson单位(AU)定义为:在标准条件下(即:25℃,pH7.5以及反应时间为10分钟)以初始速度消化血红蛋白以使每分钟释放出的TCA溶解产物的量与苯酚试剂所产生的颜色和千分之一等量的酪氨酸相同。
更详细描述此分析方法的文件AF4/5只要需要就可从Novo NordiskA/S丹麦获得,此处引用作为参考。蛋白水解活性:亮氨酸氨肽酶单位(LAPU)
也可用亮氨酰-对-硝基苯胺为底物测定蛋白水解活性。一旦水解,对-硝基苯胺释放出来使溶液变黄。利用对-硝基苯胺为发色团测定活性,用光度计在405nm测产生的黄色。
一亮氨酸氨肽酶单位(LAPU)是在下列条件下每分钟分解1μ(微)M底物的酶的量:26mM亮氨酰-对-硝基苯胺为底物,0.1MTris缓冲液(pH8.0),40℃,反应时间10分钟。
实施例
本发明用下列实施例进一步阐明,其不是意在以任何方式限制本发明如权利要求所述的范围。实施例1
蛋白质溶解性提高和通过脱酰胺释放谷氨酸水解
小麦面筋(WG)获自Cargill(JOB 5141),脱酰胺小麦面筋(DWG)获自StaPro Consultancy B.V.,Lemdijk 32,9422 TH Smilde,NL。通过用89g水与11g面筋混合制成8%蛋白质悬浮液。用NaOH调节pH至6.5。谷氨酸/天冬氨酰特异性蛋白酶(SP446)(可如WO91/13554中所述获得),或赖氨酸/精氨酸特异性蛋白酶(SP387)(可如WO89/06270中所述获得)加到悬浮液中。对SP446剂量为0.01AU/克蛋白质,对SP387为0.006AU/克蛋白质。以20LAPU/克蛋白质的量向一些水解物中加入FLavourzymeTM(一种可获自Novo Nordisk A/S,丹麦的非特异性作用蛋白酶制剂,含有内和外肽酶活性,且通过米曲霉发酵获得)。在无需进一步调整pH50℃条件下进行水解18小时。通过85℃加热15分钟使酶失活。调pH到5,离心水解物。测定在上清中的蛋白质以及游离谷氨酸的含量。蛋白质脱酰胺
用Kjeldahl分析法测定蛋白质,使用Kjeldahl因子为6.25。谷氨酸的测定
通过使用适于谷氨酸测定的Boehringer Mannheim试剂盒(Cat.No.139092)测定游离谷氨酸的含量。将此方法改造成适于微滴定板上的应用。
结果
水解物 | 蛋白质溶解度%WG DWG | 谷氨酸含量mg/lWG DWG |
SP446 | 18 54 | 0 0 |
SP387 | 35 44 | 0 0 |
SPU446+FlavourzymeTM | 34 87 | 1000 1000 |
当比较小麦面筋(WG)和脱酰胺小麦面筋(DWG)时,结果表明脱酰胺作用增加了面筋对特异性蛋白酶的敏感性,从而使更多的蛋白质变得可溶。通过加入FlavourzymeTM到特异性蛋白酶上,由于脱酰胺作用谷氨酸的释放增加一倍。
实施例2酶法脱酰胺作用和谷氨酸释放环状芽孢杆菌的发酵
ATCC21590菌株的环状芽孢杆菌培养物在容量为400ml内含200ml培养基的摇瓶中生长,培养基含有1%聚胨;0.5%乳糖;0.025%MgSO4·7H2O;0.005%FeSO4·7H2O;0.025%KH2PO4和17%Na2HPO4·12H2O,pH调至7.2。发酵在30℃下以搅拌速度270转/分进行20小时。以4000转/分离心收集细胞于1升摇瓶中。冷冻细胞。从环状芽孢杆菌中纯化肽聚谷氨酰胺酶
所有步骤均在室温下操作。
冷冻的环状芽孢杆菌细胞融化后悬浮于裂解缓冲液(50mMTris/HCl;25%(w/v)蔗糖;1mM EDTA,pH8.0)直到获得均质悬浮液,每升裂解缓冲液100g湿细胞。LysozymeTM(Fluka 62971,10mg/ml)和DNAse I(Sigma DN-25,10mg/ml)溶于裂解缓冲液。加入100mlLysozymeTM溶液,10ml1.0M MgCl2和1ml DNAseI溶液到每升细胞悬浮液中。使酶作用1小时。
用Seitz深层过滤板过滤悬浮液,滤液移至10mM KH2PO4/NaOH,pH8.0(缓冲液A)的Sephadex G25柱(Pharmacia)上。酶溶液加到用缓冲液A平衡过的SOURCE Q柱(Pharmacia)上并以缓冲液A中的线性NaCl梯度(0→500mM)洗脱。如下述分析柱流分的肽聚谷氨酰胺酶II活性,且合并有活性的流分。所合并的流分的280nm吸光值为1.78,因此估计其蛋白质含量为1.8mg/ml。
从SDS-PAGE凝胶推断肽聚谷氨酰胺酶II合并液中蛋白质的纯度约为25%。因此制剂中含有约0.5mg/ml的纯肽聚谷氨酰胺酶II。肽聚谷氨酰胺酶鉴定方法
通过使用适于氨测定的Boehringer-Mannheim试剂盒(Cat.No.1112732)测量在N-叔丁氧羰基(N-t-BOC-Gln-Pro;SIGMA No.B-4406)γ-羧酰胺的水解过程中形成的氨确定酶活性。在此试剂盒中,氨是通过测定由谷氨酸脱氢酶消耗的NADH来确定的,不加入N-t-BOC-Gln-Pro的空白也进行测量以抵消其它消耗NADH的酶的影响。小麦面筋水解
200mg小麦面筋蛋白质加入到9ml沸水中,冷却后,调节pH到7.0。再加入如上述的250μl肽聚谷氨酰胺酶II制剂(PEP)。以0.04AU/g蛋白质的量加入来自实施例1的谷氨酸/天冬氨酸特异性蛋白酶(SP446),且以20LAPU/克蛋白质的量加入来自实施例1的FlavourzymeTM。
在50℃不调节pH条件下水解进行18小时。不加入肽聚谷氨酰胺酶的空白也同样处理。离心水解物且如实施例1所述测定谷氨酸。
用如下述方式测定小麦面筋蛋白质的水解(DH)程度。水解程度(DH)的测定
通过上清液与(OPA)(邻苯二甲醛,Sigma)的反应测定蛋白质的水解程度,如Adler-Nissen[Adler-Nissen J.;食物蛋白质的酶水解(Enzymic Hydrolysis of Food Proteins);Elsevier应用科学出版社,1986]所述。对OPA试剂,160mg OPA溶于4ml乙醇,并移入容积200ml含有7.62g十水合四硼酸二钠和200mg十二烷基硫酸钠以及176mg二硫苏糖醇的烧瓶中,用水加至烧瓶的标记。试剂是光敏感的。
25μl适当稀释的上清液与200μl OPA试剂在微滴定板的孔中混合,且在25℃下反应恰好2分钟。在微孔板读数仪上测定340nm吸光值,且与95mM L-丝氨酸标准溶液在减去空白值(与OPA试剂反应的水)后的吸光值比较。为找出真实的水解程度,在上清液中测得的丝氨酸当量用由Adler-Nissen用于三硝基苯磺酸方法的推荐因子修正[Adler-Nissen J.;农业和食品化学(Agricultural and Food Chemistry),1979,27(6),1256],其与所述的OPA方法有相同的反应。水解程度在水解混合物中总蛋白质的量的基础上计算出(不以溶解的蛋白质为基础)。结果
水解 | DH% | 谷氨酸mg/l |
不加PEP | 40 | 131 |
加PEP | 43 | 171 |
如表所示,使用肽聚谷氨酰胺酶制剂的水解增加水解的程度以及谷氨酸的释放。
Claims (25)
1.一种从蛋白质底物获得一种富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的方法,包括以下步骤:
(i)将该底物经过一种脱酰胺过程;且
(ii)将该底物经过一种优先切割谷氨酰肽键的蛋白水解酶的作用。
2.如权利要求1的方法,其中的脱酰胺过程按一种非酶法脱酰胺进行。
3.如权利要求1的方法,其中的脱酰胺过程按一种酶法脱酰胺进行。
4.如权利要求3的方法,其中的酶法脱酰胺过程通过将底物经过一种转谷氨酰胺酶的作用来进行。
5.如权利要求4的方法,其中的转谷氨酰胺酶源自卵菌纲。
6.如权利要求4的方法,其中的转谷氨酰胺酶是细菌来源的。
7.如权利要求3的方法,其中的酶法脱酰胺过程通过将底物经过一种肽聚谷氨酰胺酶的作用来进行。
8.如权利要求7的方法,其中的肽聚谷氨酰胺酶是肽聚谷氨酰胺酶I或肽聚谷氨酰胺酶II或其混合物。
9.如权利要求7的方法,其中的肽聚谷氨酰胺酶源自曲霉属的菌株,芽孢杆菌属的菌株,隐球酵母属的菌株,或德巴利酵母属的菌株。
10.如权利要求7的方法,其中的肽聚谷氨酰胺酶以范围从0.01到100.000的PG酶单位每100g底物的量加入到底物中。
11.如权利要求1的方法,其中优先切割谷氨酰肽键的蛋白水解酶源自芽孢杆菌属的菌株,葡萄球菌属的菌株,链霉菌属的菌株,或是放线菌属的菌株。
12.如权利要求1的方法,其中优先切割谷氨酰肽键的蛋白水解酶以范围从0.05到15CPU/100g底物的量加入到底物中。
13.如权利要求1的方法,其中优先切割谷氨酰肽键的蛋白水解酶是谷氨酰内肽酶。
14.如权利要求1的方法,其中权利要求1的步骤(i)和步骤(ii)是同时完成的。
15.如权利要求1的方法,其中权利要求1的步骤(ii)是在权利要求1的步骤(i)之后完成的。
16.如权利要求1的方法,其方法包括附加步骤:
(iii)将底物经过一种或多种非特异性作用内和/或外肽酶的作用。
17.如权利要求16的方法,其中步骤(i),(ii)和(iii)是同时完成的。
18.如权利要求16的方法,其中步骤(iii)是在步骤(i)-(ii)之后完成的。
19.如权利要求16的方法,其中步骤(iii)的非特异性作用内和/或外肽酶源自曲霉属菌株,或是芽孢杆菌属的菌株。
20.如权利要求16的方法,其中的非特异性作用内和/或外肽酶以范围从0.05到15CPU/100g底物的量加入到底物中。
21.如权利要求1的方法,其中的蛋白质底物为蔬菜来源的。
22.如权利要求1的方法,其中的蛋白质底物为动物来源的。
23.一种由权利要求1的方法获得的蛋白水解物。
24.一种含有按权利要求1的方法获得的富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的食品。
25.一种含有按权利要求1的方法获得的富含游离谷氨酸和/或肽结合的谷氨酸残基的水解物的动物食品添加剂。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK0585/1996 | 1996-05-20 | ||
DK58596 | 1996-05-20 | ||
DK0585/96 | 1996-05-20 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN01119457A Division CN1326687A (zh) | 1996-05-20 | 2001-05-31 | 一种获得蛋白质水解物的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1219106A CN1219106A (zh) | 1999-06-09 |
CN1099839C true CN1099839C (zh) | 2003-01-29 |
Family
ID=8095235
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN97194838A Expired - Fee Related CN1099839C (zh) | 1996-05-20 | 1997-05-20 | 一种获得蛋白质水解物的方法 |
CN01119457A Pending CN1326687A (zh) | 1996-05-20 | 2001-05-31 | 一种获得蛋白质水解物的方法 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN01119457A Pending CN1326687A (zh) | 1996-05-20 | 2001-05-31 | 一种获得蛋白质水解物的方法 |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0969736B1 (zh) |
JP (1) | JP2000515003A (zh) |
CN (2) | CN1099839C (zh) |
BR (1) | BR9709006A (zh) |
DE (1) | DE69736247T2 (zh) |
WO (1) | WO1997043910A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105661553A (zh) * | 2009-04-02 | 2016-06-15 | 诺维信公司 | 用于制备基于乳的蛋白质水解产物的方法 |
Families Citing this family (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1227363C (zh) * | 1997-05-16 | 2005-11-16 | 诺沃奇梅兹生物技术有限公司 | 具有脯氨酰二肽氨肽酶活性的多肽和编码该多肽的核酸 |
JP4128632B2 (ja) | 1997-05-16 | 2008-07-30 | ノボザイムス,インコーポレイティド | アミノペプチダーゼ活性を有するポリペプチドおよびそれをコードする核酸 |
US6756221B1 (en) | 1999-06-03 | 2004-06-29 | Amano Enzyme Inc. | Protein-deamidating enzyme, microorganism producing the same, gene encoding the same, production process therefor, and use thereof |
US6251651B1 (en) * | 1998-06-04 | 2001-06-26 | Amano Pharmaceutical Co., Ltd. | Protein-deamidating enzyme, gene encoding the same, production process therefor, and use thereof |
EP1163853B1 (en) * | 1999-11-29 | 2007-02-21 | Kyowa Hakko Food Specialties Co., Ltd. | Method of strengthenig the taste of sodium chloride, agent for strengthening the taste of sodium chloride, sodium chloride-taste seasoning and food having strengthened taste of sodium chloride |
DK1106696T3 (da) * | 1999-12-03 | 2005-08-01 | Amano Enzyme Inc | Proteindeamiderende enzym, mikroorganisme producerende dette, gen kodende derfor, fremgangsmåde til fremstilling af enzymet og anvendelse deraf |
EP1312268A1 (en) * | 2001-11-19 | 2003-05-21 | Société des Produits Nestlé S.A. | Flavouring compositions |
DK175501B1 (da) | 2002-12-02 | 2004-11-15 | Green Earth | Anlæg og fremgangsmåde til kontinuerlig hydrolyse af et proteinholdigt animalsk eller vegetabilsk råmateriale |
DK1836907T3 (en) * | 2005-01-13 | 2015-08-24 | Ajinomoto Kk | PROCESSED MEAT PRODUCTS OR FISH PAST PRODUCTS AND PROCEDURES FOR PRODUCING THEREOF |
US7485323B2 (en) | 2005-05-31 | 2009-02-03 | Gelita Ag | Process for making a low molecular weight gelatine hydrolysate and gelatine hydrolysate compositions |
US8222372B2 (en) * | 2007-04-16 | 2012-07-17 | Novozymes A/S | Whey protein hydrolysate |
JP5263156B2 (ja) | 2007-07-13 | 2013-08-14 | 不二製油株式会社 | グルテン用分散性改良剤及びグルテンの分散液 |
CN101513218B (zh) * | 2009-03-10 | 2011-09-07 | 周靖波 | 从小麦胚芽中提取抗氧化活性小肽营养粉的方法 |
CA2993053A1 (en) * | 2009-04-27 | 2010-11-04 | Novartis Ag | Antagonistic activin receptor iib (actriib) antibodies for increasing muscle growth |
CN102379359B (zh) * | 2010-08-30 | 2013-09-25 | 内蒙古伊利实业集团股份有限公司 | 酶法水解酪蛋白的方法及水解产物 |
CN103354718B (zh) * | 2010-12-06 | 2016-03-23 | 卡吉尔公司 | 用于液化谷物蛋白的方法 |
AU2014209477B2 (en) | 2013-01-22 | 2017-03-30 | Mars, Incorporated | Flavor composition and edible compositions containing same |
AU2014215729B2 (en) | 2013-02-05 | 2015-11-26 | Oatly Ab | Liquid oat base |
CN103549113A (zh) * | 2013-10-30 | 2014-02-05 | 深圳市伟崇科技发展有限公司 | 一种改性植物蛋白的制备方法及其用途 |
RU2611378C2 (ru) * | 2015-05-08 | 2017-02-21 | Федеральное государственное автономное образовательное учреждение высшего профессионального образования "Казанский (Приволжский) федеральный университет" (ФГАОУВПО КФУ) | Применение рекомбинантного штамма bacillus subtilis тв-06 в качестве продуцента глутамилэндопептидазы, вещества с тромболитическими и антикоагулянтными свойствами |
CN104982891A (zh) * | 2015-06-11 | 2015-10-21 | 广东厨邦食品有限公司 | 一种酱油原油的制备方法 |
CN105248835B (zh) * | 2015-09-02 | 2019-09-20 | 广东美味鲜调味食品有限公司 | 玉米蛋白水解物及其制备方法与在纯粮酿造食醋中的应用 |
CN116284392A (zh) | 2016-03-10 | 2023-06-23 | 艾科赛扬制药股份有限公司 | 活化素2型受体结合蛋白及其用途 |
CN106434799A (zh) * | 2016-09-28 | 2017-02-22 | 华南理工大学 | 一种蛋白质纳米颗粒及其制备方法 |
FR3057180B1 (fr) * | 2016-12-12 | 2018-10-12 | Constellium Issoire | Procede d'amelioration du mouillage d'une surface d'un substrat solide par un metal liquide |
WO2021254943A1 (en) * | 2020-06-17 | 2021-12-23 | Dsm Ip Assets B.V. | Umami flavour composition |
CN113801908B (zh) * | 2021-08-23 | 2024-04-09 | 杭州娃哈哈科技有限公司 | 一种乳清蛋白源缓解体力疲劳功能肽及其制备方法 |
CN115381083A (zh) * | 2022-08-09 | 2022-11-25 | 武汉新华扬生物股份有限公司 | 一种复合酶制剂及其在提高荔枝汁蛋白含量中的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5082672A (en) * | 1989-06-21 | 1992-01-21 | The United States Of American As Represented By The Secretary Of Agriculture | Enzymatic deamidation of food proteins for improved food use |
EP0480104A1 (en) * | 1989-09-01 | 1992-04-15 | Protein Technologies International, Inc. | Enzyme modified protein and process for its production |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3857967A (en) * | 1971-05-10 | 1974-12-31 | Kikkoman Shoyu Co Ltd | Preparation of food and beverages with peptidoglutaminase |
ATE126970T1 (de) * | 1990-03-09 | 1995-09-15 | Novo Nordisk As | Proteinhydrolysate. |
-
1997
- 1997-05-20 BR BR9709006A patent/BR9709006A/pt active Search and Examination
- 1997-05-20 JP JP09541414A patent/JP2000515003A/ja active Pending
- 1997-05-20 WO PCT/DK1997/000230 patent/WO1997043910A1/en active IP Right Grant
- 1997-05-20 CN CN97194838A patent/CN1099839C/zh not_active Expired - Fee Related
- 1997-05-20 EP EP97921649A patent/EP0969736B1/en not_active Expired - Lifetime
- 1997-05-20 DE DE69736247T patent/DE69736247T2/de not_active Expired - Fee Related
-
2001
- 2001-05-31 CN CN01119457A patent/CN1326687A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5082672A (en) * | 1989-06-21 | 1992-01-21 | The United States Of American As Represented By The Secretary Of Agriculture | Enzymatic deamidation of food proteins for improved food use |
EP0480104A1 (en) * | 1989-09-01 | 1992-04-15 | Protein Technologies International, Inc. | Enzyme modified protein and process for its production |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105661553A (zh) * | 2009-04-02 | 2016-06-15 | 诺维信公司 | 用于制备基于乳的蛋白质水解产物的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN1326687A (zh) | 2001-12-19 |
DE69736247D1 (de) | 2006-08-10 |
EP0969736B1 (en) | 2006-06-28 |
AU2765097A (en) | 1997-12-09 |
CN1219106A (zh) | 1999-06-09 |
AU722915B2 (en) | 2000-08-17 |
DE69736247T2 (de) | 2007-05-24 |
BR9709006A (pt) | 1999-08-03 |
WO1997043910A1 (en) | 1997-11-27 |
JP2000515003A (ja) | 2000-11-14 |
EP0969736A1 (en) | 2000-01-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1099839C (zh) | 一种获得蛋白质水解物的方法 | |
US6036983A (en) | Method of obtaining protein hydrolysates | |
Hrckova et al. | Enzymatic hydrolysis of defatted soy flour by three different proteases and their effect on the functional properties of resulting protein hydrolysates | |
Wouters et al. | Relevance of the functional properties of enzymatic plant protein hydrolysates in food systems | |
Kong et al. | Enzymatic preparation and functional properties of wheat gluten hydrolysates | |
Clemente et al. | Protein quality of chickpea (Cicer arietinum L.) protein hydrolysates | |
Lee et al. | Characterization of hydrolysates produced by mild-acid treatment and enzymatic hydrolysis of defatted soybean flour | |
Cole et al. | Purification and partial characterisation of a novel trypsin-like cysteine protease from Metarhizium anisopliae | |
CN102046782A (zh) | 产生酪蛋白水解物的方法 | |
US20090297661A1 (en) | Methods For Flavor Enhancement | |
Puspitojati et al. | Changes in amino acid composition during fermentation and its effects on the inhibitory activity of angiotensin-I-converting enzyme of jack bean tempe following in vitro gastrointestinal digestion. | |
Rezvankhah et al. | The effects of combined enzymatic and physical modifications of lentil protein applying Alcalase, Flavourzyme, microbial transglutaminase, and ultrasound: Antioxidant, antihypertension, and antidiabetic activities | |
WO2002068671A1 (fr) | Désamidation et dénaturation d'une protéine du lait | |
Gilmartin et al. | Production of cod (Gadus morhua) muscle hydrolysates. Influence of combinations of commercial enzyme preparations on hydrolysate peptide size range | |
KR0158207B1 (ko) | 가수분해된 식물성 단백질의 생산 방법 및 이로부터의 생성물 | |
JP2958801B2 (ja) | 脱苦味された機能性ペプチドの製造法 | |
Haard | Enzymic modification of proteins in food systems | |
JPH0360480B2 (zh) | ||
AU722915C (en) | A method of obtaining protein hydrolysates | |
Nielsen et al. | Enzymic modification of food protein | |
Márquez et al. | Effect of dry heat on the in vitro digestibility and trypsin inhibitor activity of chickpea flour | |
Kitabatake et al. | Limited proteolysis of ovalbumin by pepsin | |
Cigić et al. | Preparation and characterization of chicken egg white hydrolysate | |
Gill et al. | An assessment of the potential of peptidoglutaminases I and II in modifying the charge characteristics of casein and whey proteins | |
JP2502531B2 (ja) | 改質蛋白の製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C53 | Correction of patent for invention or patent application | ||
COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: NOVO NORDISK A/S TO: NUOWOQIMEIZI CO.,LTD. |
|
CP03 | Change of name, title or address |
Address after: Denmark bagsvaerd Applicant after: Novo Jymes A/S Address before: Denmark Applicant before: Novo Nordisk A/S |
|
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20030129 |