CN106831947B - 一种牡蛎来源的新型功能肽及其用途 - Google Patents
一种牡蛎来源的新型功能肽及其用途 Download PDFInfo
- Publication number
- CN106831947B CN106831947B CN201710007455.XA CN201710007455A CN106831947B CN 106831947 B CN106831947 B CN 106831947B CN 201710007455 A CN201710007455 A CN 201710007455A CN 106831947 B CN106831947 B CN 106831947B
- Authority
- CN
- China
- Prior art keywords
- functional
- functional peptide
- oyster
- peptide
- aho
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 241000237502 Ostreidae Species 0.000 title abstract description 44
- 235000020636 oyster Nutrition 0.000 title abstract description 43
- 235000013376 functional food Nutrition 0.000 claims abstract description 6
- 239000002516 radical scavenger Substances 0.000 claims abstract description 6
- 239000004475 Arginine Substances 0.000 claims abstract description 5
- 229940123457 Free radical scavenger Drugs 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 238000002360 preparation method Methods 0.000 claims description 12
- 230000002292 Radical scavenging effect Effects 0.000 claims description 5
- 239000013543 active substance Substances 0.000 claims description 4
- 150000003254 radicals Chemical class 0.000 abstract description 7
- 235000018102 proteins Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 230000000694 effects Effects 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 235000013372 meat Nutrition 0.000 description 10
- 239000004365 Protease Substances 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 7
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108090000145 Bacillolysin Proteins 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 108091005507 Neutral proteases Proteins 0.000 description 4
- 102000035092 Neutral proteases Human genes 0.000 description 4
- 108090000526 Papain Proteins 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000007760 free radical scavenging Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 235000019834 papain Nutrition 0.000 description 4
- 229940055729 papain Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108091005658 Basic proteases Proteins 0.000 description 3
- 108010004032 Bromelains Proteins 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000019835 bromelain Nutrition 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000548230 Crassostrea angulata Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000237852 Mollusca Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000020247 cow milk Nutrition 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 2
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 description 1
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 1
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- DYBIDOHFRRUMLW-CIUDSAMLSA-N Cys-Leu-Cys Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CS)C(O)=O DYBIDOHFRRUMLW-CIUDSAMLSA-N 0.000 description 1
- -1 DPPH free radical Chemical class 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000470791 Micromonospora maritima Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- 108010001441 Phosphopeptides Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- JVYIGCARISMLMV-HOCLYGCPSA-N Val-Gly-Trp Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N JVYIGCARISMLMV-HOCLYGCPSA-N 0.000 description 1
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- INAPMGSXUVUWAF-GCVPSNMTSA-N [(2r,3s,5r,6r)-2,3,4,5,6-pentahydroxycyclohexyl] dihydrogen phosphate Chemical compound OC1[C@H](O)[C@@H](O)C(OP(O)(O)=O)[C@H](O)[C@@H]1O INAPMGSXUVUWAF-GCVPSNMTSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 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 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 1
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 238000001196 time-of-flight mass spectrum Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000002211 ultraviolet spectrum 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
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种牡蛎来源的新型功能肽及其用途。所述的功能肽的氨基酸序列为半胱氨酸‑亮氨酸‑半胱氨酸‑缬氨酸‑亮氨酸‑天冬酰胺‑谷氨酰胺‑谷氨酰胺‑赖氨酸‑缬氨酸‑甘氨酸‑色氨酸‑丙氨酸‑精氨酸。本发明的功能肽具有良好的清除生物体内自由基的功能，因此本发明的功能肽可以用于制备自由基清除剂、以及具有自由基清除功能的功能食品和功能饲料。
Description
技术领域
本发明属于功能肽领域，具体涉及一种牡蛎来源的新型功能肽及其用途。
背景技术
牡蛎(Oyster)，属软体动物门(Mollusca)，瓣鳃纲(Lanellibranchia)，异柱目(Anisomyaria)牡蛎科(Ostreidae)动物，是一种食用价值和药用价值都极高的海洋双壳贝类。牡蛎的种类繁多，目前全世界已经发现有100多种，而中国沿海地区盛产牡蛎，约有20多种，是我国四大养殖贝类之一。
牡蛎不仅风味鲜美，且营养成分全，可作药用，中医典籍中也有用牡蛎治病的记载。根据已有报道，干牡蛎肉中，蛋白质含量约占45％～52％，氨基酸组成完善。据世界粮农组织评定，牡蛎肉中必需氨基酸完全程度和质量比例优于牛乳和人乳。干牡蛎肉中脂肪含量约为7％～11％，多为具有生理活性的复合磷脂、磷酸肌醇、二十碳五烯酸(EPA)，二十二碳六烯酸(DHA)等，这些成分都可以防止动脉硬化、抗血栓以及抗衰老等作用。牡蛎中总糖含量为19％～38％，据报道，牡蛎中的糖原可直接被机体吸收，从而可以减轻胰腺负担，对糖尿病的防止十分有效。此外，还含有丰富的维生素A、B1、B2、D等，含碘量比牛乳或蛋黄高200倍。此外，牡蛎壳中含有80％～95％的钙盐(碳酸钙、磷酸钙和硫酸钙)，同时也含有镁、铁、硅、铝和锌等金属元素。
牡蛎等海洋生物已经成为功能能肽的重要来源。功能肽又称活性肽，通常包含3到20个氨基酸残基，它们的生理活性依赖于它们的氨基酸组成和序列，由于其特殊的生理功能也越来越受到人们的关注，一些生物活性肽已应用于保健品、药品等领域。有研究表明：人们摄取蛋白质通过消化道的多种酶水解后，不仅仅像以前认为的那样以氨基酸的形式吸收，更多的是以肽的形式被直接吸收，而且二肽和三肽的吸收速度要比同一组成的游离氨基酸更快。功能肽不仅可以提供人体生长发育所需要的营养物质，还具有重要的生理功能，如清除自由基、促进矿物质吸收、防治肝病脑病、抗菌，抑制血管紧张素转化酶活力、提高人体免疫力以及延缓衰老等功能。目前，国内商品化的生物活性肽产品，活性成分明确的，除酪蛋白磷酸肽外还不多见。目前虽有较多文献报道利用牡蛎制备含功能肽的水解蛋白，但其中有效成分并不明确。深入研究牡蛎水解蛋白的有效成分，发现活性良好的新结构活性肽类，明确其分子结构，对于强效新产品的开发、明确活性产生机理、优化生产工艺、强化质量控制，乃至牡蛎资源的高效利用都有重要的意义。
发明内容
本发明的第一个目的是提供一种具有良好的清除生物体内自由基功能的牡蛎来源的功能肽。
本发明的功能肽，含14个氨基酸残基，其氨基酸序列为半胱氨酸-亮氨酸-半胱氨酸-缬氨酸-亮氨酸-天冬酰胺-谷氨酰胺-谷氨酰胺-赖氨酸-缬氨酸-甘氨酸-色氨酸-丙氨酸-精氨酸(CLCVLNQQKVGWAR，其序列如SEQ ID NO.1所示)，是一个新结构肽类。
本发明所述的功能肽是以牡蛎(Crassostrea gigas)为原料，用酶制剂，包括SCSIO 01819酶制剂、中性蛋白酶、碱性蛋白酶、胰蛋白酶、木瓜蛋白酶和菠萝蛋白酶等，作为催化剂，在催化牡蛎蛋白的水解反应，制备所得新型清除自由基活性的水解蛋白中的有效成分。经超滤、色谱方法将其从水解蛋白中分离纯化后，使用基质辅助的飞行时间质谱方法(MALDI-TOF-TOF)分析，鉴定其分子量为1618.83Da，氨基酸序列为半胱氨酸-亮氨酸-半胱氨酸-缬氨酸-亮氨酸-天冬酰胺-谷氨酰胺-谷氨酰胺-赖氨酸-缬氨酸-甘氨酸-色氨酸-丙氨酸-精氨酸(CLCVLNQQKVGWAR)。经NCBI中的SWISS-PROT数据库的检索，未发现明显同源的序列，该结果可以表明这种纯化的肽是从牡蛎产生的新结构肽。
本发明的第二个目的是提供上述功能肽在制备自由基清除剂中的应用。
一种自由基清除剂，其特征在于，包括上述功能肽作为活性物质。
本发明的第三个目的是提供上述功能肽在制备具有自由基清除能力的功能食品或功能饲料中的应用。
一种具有自由基清除能力的功能食品或功能饲料，其特征在于，包括上述功能肽作为活性物质。
本发明的功能肽具有良好的清除生物体内自由基的功能，因此本发明的功能肽可以用于制备自由基清除剂、以及具有自由基清除功能的功能食品和功能饲料。
附图说明：
图1是AHO–II-5-2ODS HPLC色谱图；
图2是目标功能肽(CLCVLNQQKVGWAR)的MALDI-TOF-TOF质谱图。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：牡蛎酶解蛋白的制备
1、原料和试剂
实施例中的牡蛎肉购自广州市黄沙水产市场市场，酶解前粉碎分袋包装并密封，于-20℃保存。
SCSIO 01819酶制剂为自制，参照专利《一种海洋来源小单孢菌SCSIO 01819、酶制剂溶液及其制备方法和应用》(申请号：CN201510366099.1，公开(公告)号：CN104894034A)中的方法制备，即为该专利中的SCSIO 01819酶制剂溶液-2。中性蛋白酶(酶活力为40万U/g)，碱性蛋白酶(酶活力为40万U/g)，木瓜蛋白酶(酶活力为40万U/g)，菠萝蛋白酶(酶活力为40万U/g)，均购自广西南宁庞博生物工程有限公司；胰蛋白酶(酶活力为250U/mg),购自美国sigma公司；DEAE-52纤维素阳离子交换树脂购自whatman公司，Sephadex G-15凝胶树脂购自GE公司。乙醇、甲醛、异丙醇、氢氧化钠、磷酸氢二钠、磷酸二氢钠等其他试剂均为国产分析纯。试验用水均为超纯水。
2、牡蛎的脱脂处理
将粉粹的牡蛎肉与异丙醇按照1g：4ml(w/v)的比例混合，在35℃下反应3小时，过滤，在4500×g下离心20min，取下层脱脂的牡蛎肉，晾干，-20℃下保存。
3、牡蛎肉酶解蛋白的制备
步骤2脱脂后的牡蛎肉在表1所示的蛋白酶的最适酶解条件下分别进行酶解，水解时间4小时，酶底比4000U/g。酶解结束后，在4℃，4500×g下离心，除去不溶性杂质，再在15000r/min下低温离心，所得上清液进行过滤，使其透过0.22um的滤膜。将所得样品溶液(滤液)冻干即得牡蛎水解蛋白，-20℃下保存备用。
表1.六种酶催化牡蛎蛋白水解的温度和pH值
4.DPPH自由基清除作用的测定。
本实验采用用DPPH实验评价样品的自由基清除能力。根据文献(Hsu,K.C.；Lu,G.H.；Jao,C.L.,Antioxidative properties of peptides prepared from tuna cookingjuice hydrolysates with orientase(Bacillus subtilis).Food ResearchInternational 2009,42,(5-6),647-652.)的方法，测定牡蛎肉酶解物的清除DPPH自由基活性的能力。将步骤3的牡蛎水解蛋白溶解于水中得样品溶液，取100μl样品溶液加入到96孔板第一行，使其终浓度为5mg/ml，用超纯水进行浓度梯度稀释,再分别加入100μl浓度为2x10-4mol/L的DPPH乙醇溶液，每个浓度做三个平行实验，空白试验是由无水乙醇代替样品溶液。将混合物放置37℃在黑暗处反应30min。在517nm处测定吸光值。DPPH自由基的清除率按照以下公式进行计算：
式中：S为样品加DPPH的反应后的吸光度；SB为样品加的无水乙醇吸光度；C为无水乙醇加DPPH的吸光度；CB为空白对照的吸光度
根据结果绘制量效曲线，并计算DPPH清除活性的EC50值(半数清除浓度)。用SCSIO01618酶制剂、木瓜蛋白酶(papain)、中性蛋白酶(neutral protease)、胰蛋白酶(trypsin)、碱性蛋白酶(alcalase)、菠萝蛋白酶(bromelain)制备的牡蛎水解蛋白，EC50值分别为：0.62mg/ml，0.88mg/ml，0.71mg/ml，1.41mg/ml，1.92mg/ml，0.89mg/ml。
实施例2从酶解蛋白中分离制备新型功能肽
1、取实施例1中用SCSIO 01819酶制剂酶解得到的牡蛎水解蛋白5g，溶解于10mL水制成样品溶液，用截留分子量为3kDa的超滤膜过滤，截留部分和滤液分别冻干，得两个样品：AHO-I(分子量>3kDa),AHO-II(分子量<3kDa)。
2、用离子交换柱对AHO-II(200mg)进行分离，产生了5个色谱片段(AHO–II-1～5)，分别冻干后，检测其DPPH自由基清除活性的EC50值，分别为3.53mg/ml，2.33mg/ml，2.88mg/ml，1.62mg/ml，0.461mg/ml(AHO–II-5)。
所述的用离子交换柱进行分离，其分离条件为将AHO-II溶液加入预平衡的DEAE-52纤维素柱(1.6×50cm)，并依次用150mL的0.1，0.5和1.0M NaCl溶液洗脱，流速为1.0mL/min。按5mL/份收集洗脱流分(5mL)，根据UV谱图(检测波长280nm)，合并为五个片段(AHO–II-1至AHO–II-5)，分别使用D101大孔树脂对每个片段脱盐，然后后冻干，测试DPPH自由基清除活性。
3、取上一步骤中活性最强的AHO–II-5(52mg)，用G15凝胶柱进行分离，获得4个组分片段AHO–II-5-1～4，分别冻干后，检测其DPPH自由基清除活性的EC50值，分别为1.17mg/ml，0.53mg/ml(AHO–II-5-2)，4.31mg/ml，0.93mg/ml，2.36mg/ml.
所述的G15凝胶柱分离，其分离条件为AHO–II-5样品溶解于5mL去离子水中，并通过预先用纯水平衡的Sephadex G-15凝胶过滤柱(2.6×100cm)纯化。用去离子水以1mL/min的流速洗脱柱，按每份2.5mL收集洗脱液，根据UV(280nm)检测的色谱图，将洗脱液合并为4个片段(AHO–II-5-1、AHO–II-5-2、AHO–II-5-3、AHO–II-5-4)，分别冻干冻干保存。
4、取上一步骤制备的样品片段中活性最强的片段AHO–II-5-2(12mg)，用HPLC(ODS柱)分离，收集22分钟处色谱峰流分(图2)，得目标功能肽溶液，冻干后得到目标功能肽，测试目标功能肽的DPPH活性，其EC50值为0.22mg/mL。
所述的HPLC分离，其分离条件为AHO–II-5-2样品溶于1mL0.1％三氟乙酸(TFA)水溶液中。色谱仪为(RP-HPLC)Agilent1200 HPLC型高效液相色谱仪，色谱柱为Zorbax，SB C-18柱(柱尺寸：4.6mm×250mm，5μm粒径，Agilent)，检测波长280nm。溶剂系统：A，0.1％三氟乙酸水溶液，B，乙腈(默克公司，色谱纯)。流速：1.0mL/min；洗脱程序：0-5分钟，5％B；5-50min，5％B-95％B每次分离进样量50mL，收集保留时间22min处色谱峰片段(目标功能肽)。共分离45次，合并峰片段后冻干。
5、结构鉴定
使用基质辅助-飞行时间质谱方法(MALDI-TOF-TOF)，鉴定目标功能肽的分子量为1618.83Da，分析目标功能肽的质谱数据(图1)，确认目标功能肽的氨基酸序列为：半胱氨酸-亮氨酸-半胱氨酸-缬氨酸-亮氨酸-天冬酰胺-谷氨酰胺-谷氨酰胺-赖氨酸-缬氨酸-甘氨酸-色氨酸-丙氨酸-精氨酸(CLCVLNQQKVGWAR)。经NCBI中的SWISS-PROT数据库的检索，未发现明显同源的序列，该结果可以表明这种纯化的肽是从牡蛎产生的新结构肽。
序列表
<110> 中国科学院南海洋洋研究所
<120> 一种牡蛎来源的新型功能肽及其用途
<160> 1
<210> 1
<211> 14
<212> PRT
<213> 牡蛎（ostrea gigas thunberg）
<400> 1
Cys Leu Cys Val Leu Asn Gln Gln Lys Val Gly Trp Ala Arg
1 5 10 14
Claims (5)
1.一种功能肽，其特征在于，其氨基酸序列为半胱氨酸-亮氨酸-半胱氨酸-缬氨酸-亮氨酸-天冬酰胺-谷氨酰胺-谷氨酰胺-赖氨酸-缬氨酸-甘氨酸-色氨酸-丙氨酸-精氨酸。
2.权利要求1所述的功能肽在制备自由基清除剂中的应用。
3.一种自由基清除剂，其特征在于，包括权利要求1所述的功能肽作为活性物质。
4.权利要求1所述的功能肽在制备具有自由基清除能力的功能食品或功能饲料中的应用。
5.一种具有自由基清除能力的功能食品或功能饲料，其特征在于，包括权利要求1所述的功能肽作为活性物质。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710007455.XA CN106831947B (zh) | 2017-01-05 | 2017-01-05 | 一种牡蛎来源的新型功能肽及其用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710007455.XA CN106831947B (zh) | 2017-01-05 | 2017-01-05 | 一种牡蛎来源的新型功能肽及其用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106831947A CN106831947A (zh) | 2017-06-13 |
| CN106831947B true CN106831947B (zh) | 2020-06-12 |
Family
ID=59117061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710007455.XA Active CN106831947B (zh) | 2017-01-05 | 2017-01-05 | 一种牡蛎来源的新型功能肽及其用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106831947B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396048A (zh) * | 2018-02-08 | 2018-08-14 | 舟山海研食品科技有限公司 | 牡蛎活性多肽的制备方法 |
| CN109608520B (zh) * | 2018-11-15 | 2020-09-08 | 中国科学院南海海洋研究所 | 一种珍珠来源的功能肽及其用途 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110132498A (ko) * | 2010-06-02 | 2011-12-08 | 건국대학교 산학협력단 | 패류 유래 기능성 펩티드의 분리 정제 방법 및 기능성 펩티드의 용도 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101533308B1 (ko) * | 2012-08-24 | 2015-07-03 | 경희대학교 산학협력단 | 안지오텐신-i 전환 효소 저해능을 나타내는 펩타이드를 유효성분으로 포함하는 심혈관계 질환 예방 또는 치료용 약학적 조성물 |
-
2017
- 2017-01-05 CN CN201710007455.XA patent/CN106831947B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110132498A (ko) * | 2010-06-02 | 2011-12-08 | 건국대학교 산학협력단 | 패류 유래 기능성 펩티드의 분리 정제 방법 및 기능성 펩티드의 용도 |
Non-Patent Citations (2)
| Title |
|---|
| Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage;Zhong-Ji Qian等;《Bioresource Technology》;20070927;第99卷(第9期);第3365-3370页 * |
| Sequence Determination of an Antioxidant Peptide Obtained by Enzymatic Hydrolysis of Oyster Crassostrea madrasensis (Preston);K. K. Asha等;《INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS》;20160314;第22卷(第3期);第421-433页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106831947A (zh) | 2017-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Characterization, preparation, and purification of marine bioactive peptides | |
| Chi et al. | Isolation and characterization of three antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) heads | |
| US8450100B2 (en) | Collagen peptide having immune-enhancing activity from Cyanea nozakii and preparation method and uses thereof | |
| Pan et al. | Purification and characterisation of a novel angiotensin-I converting enzyme (ACE)-inhibitory peptide derived from the enzymatic hydrolysate of Enteromorpha clathrata protein | |
| Samarakoon et al. | Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate | |
| Paiva et al. | Isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides from Ulva rigida C. Agardh protein hydrolysate | |
| Chen et al. | Isolation, purification and the anti-hypertensive effect of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from Ruditapes philippinarum fermented with Bacillus natto | |
| Alemán et al. | Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate | |
| Toopcham et al. | Characterization and identification of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from tilapia using Virgibacillus halodenitrificans SK1-3-7 proteinases | |
| CN109400678A (zh) | 一种刺参来源的抗氧化和dpp-iv抑制活性肽 | |
| CN101589761B (zh) | 一种工业用大麻籽抗氧化肽的制备方法及应用 | |
| Ko et al. | Effect of angiotensin I-converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of Styela plicata | |
| Oh et al. | Antihypertensive effect of surimi prepared from olive flounder (Paralichthys olivaceus) by angiotensin-I converting enzyme (ACE) inhibitory activity and characterization of ACE inhibitory peptides | |
| Zhi et al. | Novel antioxidant peptides from protein hydrolysates of scallop (Argopecten irradians) mantle using enzymatic and microbial methods: Preparation, purification, identification and characterization | |
| JP5154964B2 (ja) | ローヤルゼリー分解酵素含有物 | |
| WO2012027926A1 (zh) | 鱿鱼内脏提取的肽及其制备方法、组合物和用途 | |
| CN106831947B (zh) | 一种牡蛎来源的新型功能肽及其用途 | |
| Tian et al. | Isolation and purification of antioxidant and ACE‐inhibitory peptides from yak (Bos grunniens) skin | |
| Taniguchi et al. | Identification and characterization of multifunctional cationic peptides from enzymatic hydrolysates of soybean proteins | |
| CN109206483A (zh) | 一种贻贝来源的ace抑制及抗肿瘤活性肽 | |
| CN107082807B (zh) | 具有ace抑制功能的牦牛骨蛋白肽及制备方法和应用 | |
| Tian et al. | Production and identification of peptides with activity promoting osteoblast proliferation from meat dregs of Pinctada martensii | |
| CN109694409B (zh) | 一种血管紧张素转化酶抑制活性功能肽及其应用 | |
| Chen et al. | In vitro antioxidant effects of Porphyra haitanensis peptides on H 2 O 2-induced damage in HepG2 cells | |
| CA2195053C (en) | Agents for inhibiting accumulation of visceral fat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |