CN116444612B - Milk active peptide APADPGRPTGY and preparation method and application thereof - Google Patents
Milk active peptide APADPGRPTGY and preparation method and application thereof Download PDFInfo
- Publication number
- CN116444612B CN116444612B CN202211544797.2A CN202211544797A CN116444612B CN 116444612 B CN116444612 B CN 116444612B CN 202211544797 A CN202211544797 A CN 202211544797A CN 116444612 B CN116444612 B CN 116444612B
- Authority
- CN
- China
- Prior art keywords
- peptide
- small peptide
- apadpgrptgy
- milk
- seq
- 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 115
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 235000013336 milk Nutrition 0.000 title abstract description 40
- 239000008267 milk Substances 0.000 title abstract description 40
- 210000004080 milk Anatomy 0.000 title abstract description 40
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 27
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 241000186605 Lactobacillus paracasei Species 0.000 claims description 13
- 230000003078 antioxidant effect Effects 0.000 claims description 10
- 239000003963 antioxidant agent Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 235000020247 cow milk Nutrition 0.000 claims description 6
- 230000002519 immonomodulatory effect Effects 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 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 claims description 5
- 102000014171 Milk Proteins Human genes 0.000 claims description 5
- 108010011756 Milk Proteins Proteins 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- 235000021239 milk protein Nutrition 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 4
- 230000004071 biological effect Effects 0.000 claims description 3
- 230000021736 acetylation Effects 0.000 claims description 2
- 238000006640 acetylation reaction Methods 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000011987 methylation Effects 0.000 claims description 2
- 238000007069 methylation reaction Methods 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 22
- 239000011347 resin Substances 0.000 description 20
- 229920005989 resin Polymers 0.000 description 20
- 229920001184 polypeptide Polymers 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 13
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
- 238000001514 detection method Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000000975 bioactive effect Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 238000004949 mass spectrometry Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 8
- 230000029087 digestion Effects 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000010511 deprotection reaction Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 6
- 210000002540 macrophage Anatomy 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000108 ultra-filtration Methods 0.000 description 6
- 235000010633 broth Nutrition 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 229940100601 interleukin-6 Drugs 0.000 description 5
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 4
- 108010077544 Chromatin Proteins 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 210000003483 chromatin Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000004807 desolvation Methods 0.000 description 3
- 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 3
- 230000004957 immunoregulator effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000001694 spray drying Methods 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000000825 ultraviolet detection Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000722885 Brettanomyces Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 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 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001196 time-of-flight mass spectrum Methods 0.000 description 2
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 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
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010046644 Polymeric Immunoglobulin Receptors Proteins 0.000 description 1
- 102100035187 Polymeric immunoglobulin receptor Human genes 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- CUTSCJHLMGPBEJ-UHFFFAOYSA-N [N].CN(C)C=O Chemical compound [N].CN(C)C=O CUTSCJHLMGPBEJ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- -1 antiinflammatory Substances 0.000 description 1
- 238000002792 antioxidant assay Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000006315 carbonylation Effects 0.000 description 1
- 238000005810 carbonylation reaction Methods 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 239000002173 cutting fluid Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- YYVGYULIMDRZMJ-UHFFFAOYSA-N propan-2-ylsilane Chemical compound CC(C)[SiH3] YYVGYULIMDRZMJ-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010064245 urinary gonadotropin fragment Proteins 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dermatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Birds (AREA)
- General Engineering & Computer Science (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a milk-derived active peptide APADPGRPTGY, a preparation method and application thereof. The invention firstly provides a small peptide or a derivative thereof, wherein the amino acid sequence of the small peptide is shown as SEQ ID NO. 1; SEQ ID NO.1: APADPGRPTGY. The small peptide is milk-source active peptide, and has good antioxidation and immunity regulation effects.
Description
Technical Field
The invention relates to an active peptide, a preparation method and application thereof, in particular to a milk-derived active small peptide APADPGRPTGY, a derivative thereof, a preparation method and application thereof.
Background
Bioactive peptide refers to a peptide having a specific physiological activity to a living body, wherein milk protein is one of important sources of bioactive peptide. In recent years, milk-derived active peptides have become a well-known word. On one hand, the milk-derived active peptide has the characteristics of small fragments and easy absorption; on the other hand, it has many potential biological functions, so that it attracts more and more attention, and is one of the hot spots of scientific research. Many milk-derived active peptides have also been well demonstrated for their beneficial effects, including antibacterial, anticancer, antihypertensive, cholesterol-lowering, antidiabetic, antiinflammatory, antidepressant properties. More than 4000 different milk-derived active peptides have been reported in the currently most authoritative milk-derived active peptide database BIOPEP-UMW. However, novel milk-derived active peptides having antioxidant or immunoregulatory functions different from existing polypeptides remain the direction of current need for further research.
Disclosure of Invention
It is an object of the present invention to provide milk-derived active small peptides and derivatives thereof.
It is another object of the present invention to provide a process for the preparation of said small peptides and derivatives thereof.
It is a further object of the present invention to provide the use of said small peptides and derivatives thereof.
In one aspect, the invention provides a small peptide or a derivative thereof, wherein the amino acid sequence of the small peptide is shown as SEQ ID NO. 1;
SEQ ID NO.1：APADPGRPTGY。
the small peptide is active peptide, and has good antioxidation and immunity regulation effects.
According to a specific embodiment of the invention, the derivative is a derivative peptide obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification and/or glycosylation modification on the basis of the side chain group, amino-terminus or carboxy-terminus of the amino acid sequence shown in SEQ ID NO. 1. Preferably, the derivative peptide has substantially the same biological activity (e.g. antioxidant and/or immunity modulating efficacy) as the small peptide of the amino acid sequence shown in SEQ ID NO. 1.
In another aspect, the present invention provides a method for preparing the small peptide or derivative thereof, comprising:
Preparing the small peptide or the derivative thereof by a microbial fermentation method; or alternatively
Preparing the small peptide or its derivative by genetic engineering (which can be obtained directly from cells by separation and purification); or alternatively
The small peptide or derivative thereof is synthesized by chemical means.
According to a specific embodiment of the present invention, there is provided a method for preparing the small peptide or the derivative thereof, comprising:
The small peptide or the derivative thereof is obtained by taking emulsion containing milk protein as a fermentation substrate, inoculating lactobacillus paracasei for fermentation and separating from a fermentation product.
Lactobacillus paracasei (Lactobacillus paracasei) is a gram positive bacterium, is usually subjected to abnormal fermentation, has the characteristics of facultative anaerobism, no movement and no spore, is in the form of bacillus or longbacterium, and is singly or in pairs, and has the width of 2.0-4.0 mu m and the length of 0.8-1.0 mu m. In the invention, lactobacillus paracasei can be utilized to ferment and hydrolyze milk proteins to produce the small peptide, and the small peptide derivative can be obtained by modification optionally.
According to a specific embodiment of the present invention, in the method for producing a small peptide or a derivative thereof according to the present invention, the fermentation substrate comprises: 3 to 10 percent of cow milk concentrated protein and 1 to 10 percent of lactose.
According to a specific embodiment of the present invention, in the method of the present invention for producing a small peptide or a derivative thereof, fermentation conditions are: the inoculation amount is 1-3% at 30-45 ℃, and the bacterial count in the seed liquid is more than 1.5X10 11 CFU/mL.
According to a specific embodiment of the present invention, in the method for producing a small peptide or a derivative thereof of the present invention, the lactobacillus paracasei includes lactobacillus paracasei K56. Lactobacillus paracasei K56 is a public strain of CN107916236a, and has been shown to have antibacterial, anti-obesity, antioxidant, blood pressure regulating, intestinal flora regulating, immune system regulating, etc. effects.
The lactobacillus paracasei K56 strain is adopted for natural fermentation to produce the milk-source active peptide APADPGRPTGY, so that the concentration of the produced target small peptide can be increased, and the production cost can be reduced. The method comprises the following steps: the method comprises the steps of taking cow milk concentrated protein as a raw material, fermenting by using lactobacillus paracasei, concentrating by adopting a membrane technology, and separating and purifying to obtain target small peptide. In the present invention, the small peptide finished product in the form of powder can be obtained by a vacuum freeze-drying technique or a low-temperature spray-drying technique. The amino acid sequence of the peptide can be checked by UPLC-MS.
In some embodiments of the invention, the method for producing milk-derived active peptide APADPGRPTGY by natural fermentation using lactobacillus paracasei K56 strain comprises:
Dissolving 3-10% of milk concentrated protein and 1-10% of lactose in an aqueous solution with the pH value of 7.0-8.0 and the temperature of 25-90 ℃, uniformly mixing, and cooling to room temperature to obtain a milk concentrated protein solution;
inoculating fermentation strain, fermenting to obtain fermentation liquor;
Centrifuging the fermentation liquor, collecting supernatant, and performing ultrafiltration filtration by adopting a membrane with a molecular weight cutoff of less than 3kDa to obtain filtered fermentation liquor;
further separating and purifying the filtered fermentation liquor to obtain the target small peptide.
In another aspect, the invention also provides the use of said small peptides or derivatives thereof for the preparation of a composition having antioxidant and/or immunomodulating efficacy.
In another aspect, the invention also provides a composition comprising a small peptide and/or derivative thereof according to the invention, optionally together with an adjuvant.
The milk-derived active peptide APADPGRPTGY has the beneficial effects that: on the one hand, the anti-oxidation capability is higher; on the other hand, the preparation has better immunoregulatory activity and can improve the quality of life. The small peptide and the derivative thereof have very important significance in developing health care products and medicines with antioxidant function and immunoregulation function.
Drawings
Fig. 1 is a first order mass spectrum of a fragment with a mass to charge ratio of 551.2709 (m/z= 551.2709).
FIG. 2 shows the secondary mass spectrum of a fragment with a mass to charge ratio 551.2709 and the fragmentation of polypeptides az, by.
FIG. 3 is a pie chart of in vitro digestion product analysis of milk-derived active peptide APADPGRPTGY.
Detailed Description
Before the embodiments of the invention are further described, it is to be understood that the invention is not limited in its scope to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art, see in particular Sambrook et al MOLECULAR CLONING：ALABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989and Third edition,2001;Ausubel et al ,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS IN ENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),Academic Press,San Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (P.B. Becker, ed.) Humana Press, totowa,1999, et al.
The invention will now be described in detail with reference to the drawings and specific examples.
EXAMPLE 1 obtaining of active peptide APADPGRPTGY
1. Preparation of milk concentrate protein solution
10% Milk protein concentrate, 10% lactose, 0.5% NACL (all by mass fraction) are dissolved in an aqueous solution having a pH of 7.0-8.0. Sterilizing at 95deg.C for 10min, and cooling to 37deg.C in ice water bath;
2. preparation of fermentation broths
Adding lactobacillus paracasei K56 lyophilized powder to make the addition amount reach 1% of milk concentrated protein concentration (i.e. 1.5X10 11 CFU/L), mixing, placing into an incubator at 37deg.C, standing and fermenting for 4 hr to obtain fermentation broth.
3. Extraction of polypeptides
Heating the fermentation broth in 95 ℃ water bath for 10min for inactivation, cooling by using ice water bath, and centrifuging at low temperature under the following conditions: 8000rcf, 4 ℃, centrifuging for 10min, discarding bottom sediment, and taking supernatant. Taking supernatant, transferring into an inner tube of an ultrafiltration tube for ultrafiltration, and selecting an ultrafiltration membrane with a molecular weight cutoff of 3kDa, wherein the ultrafiltration centrifugation condition is 4800rcf, 4 ℃ and 30min. Collecting the bottom ultrafiltrate, and preserving the obtained polypeptide solution at low temperature under the condition of 4 ℃.
4. Preparation of polypeptide powder
And (3) carrying out spray drying on the polypeptide solution obtained in the step (5) to obtain the cow milk concentrated protein peptide powder, wherein the spray drying conditions are as follows: the air inlet temperature is 180 ℃, the air outlet temperature is 90 ℃, and the flow rate is 25mL/min.
5. Screening of active peptides APADPGRPTGY
1) UPLC analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the analysis method, the chromatographic analysis and the mass spectrometry are carried out on the cow milk concentrated protein peptide by utilizing an ultra-high performance liquid phase, electrospray, a quaternary rod and a time-of-flight mass spectrum, so that the amino acid sequence of all polypeptides in the fermentation broth is obtained.
The same amino acid sequence in the parallel control group is selected, and then the amino acid sequence with lower biological activity probability score in PEPTIDE RANKER is removed from the disclosed amino acid sequence, so as to obtain the sequence of the active peptide APADPGRPTGY.
EXAMPLE 2 Synthesis of active peptide APADPGRPTGY
1. Synthesis of bioactive peptides
1. RINK g (substitution degree 0.3 mmol/g) of the resin was weighed into a 150ml reactor and immersed in 50ml of Dichloromethane (DCM).
After 2.2 hours, the resin was washed with 3 times the resin volume of nitrogen-Dimethylformamide (DMF), then drained, and the resin was drained and ready for use.
3. An amount of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and shaken on a decolorizing shaker for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the resin was washed four times with 3 volumes of DMF and then drained.
4. A small amount of resin is taken and detected by ninhydrin (ninhydrin nine-well) method (two drops of each of detection A and detection B are reacted for 1min at 100 ℃), and the resin is colored, which indicates that the deprotection is successful.
5. The method comprises the steps of weighing an appropriate amount of amino acid Asn and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Asn and the 1-hydroxy-benzotriazole (HOBT), adding 3ml of N, N-Diisopropylcarbodiimide (DIC), shaking uniformly for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ to react.
After 6.2 hours, a certain amount of acetic anhydride head (acetic anhydride: DIEA: dcm=1:1:2, v: v) was used for half an hour, then washed four times with 3 times the resin volume of DMF, and dried for use.
7. An amount of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and shaken on a decolorizing shaker for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the mixture was washed four times with DMF and then dried.
8. A small amount of resin is taken and detected by ninhydrin (ninhydrin nine-well) method (two drops of each of detection A and detection B are reacted for 1min at 100 ℃), and the resin is colored, which indicates that the deprotection is successful.
9. Weighing the right amount of the second amino acid and the right amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the second amino acid and the right amount of HOBT, adding 2.5ml of DIC, shaking uniformly for 1min, adding the solution into a reactor after the solution is clarified, and placing the reactor into a shaking table at 30 ℃ for reaction.
After 10.1 hours, a small amount of resin is taken for detection, ninhydrin method is used for detection (two drops of detection A and detection B are respectively reacted for 1min at 100 ℃), and if the resin is colorless, the reaction is complete; if the resin is colored, this indicates incomplete condensation and the reaction is continued.
11. After completion of the reaction, the resin was washed four times with DMF and then drained, a quantity of 20% piperidine (piperidine/dmf=1:4, v: v) was added to the reactor and placed on a decolorizing shaker and shaken for 20min to remove the Fmoc protecting groups from the resin. After deprotection, the sample was washed four times with DMF and then drained to examine whether the protection had been removed.
12. Amino acids Tyr, gly, thr, pro, arg, gly, pro, asp, ala, prog and Ala were sequentially attached according to steps 9-11.
13. After the last amino acid has been taken up, the deprotection is achieved, the resin is washed four times with DMF and then pumped off with methanol. Then with 95 cutting fluid (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropyl silane: the bioactive peptide was cleaved from the resin (10 ml cleavage solution per gram of resin) and spun down four times with glacial ethyl ether (cleavage solution: ethyl ether=1:9, v: v).
Thus, bioactive peptide APADPGRPTGY was synthesized artificially.
2. Confirmation of bioactive peptides
1) UPLC analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the above analysis methods, the bioactive peptide APADPGRPTGY is subjected to chromatographic analysis and mass spectrometry using ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide APADPGRPTGY is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by breaking conditions are shown in figure 2, and the bioactive peptide of the peak can be obtained with a mass-to-charge ratio of 551.2709 and a retention time of 10.07min.
3) Results
As can be seen from FIG. 2, according to the breaking condition of az and by, the fragment sequence with mass-to-charge ratio 551.2709 is Ala-Pro-Ala-Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr (APADPGRPTGY) and is recorded as SEQ ID NO.1. The fragment corresponds to the 397 th to 407 th residue sequence of Polymeric immunoglobulin receptor protein, and the sequence is shown as SEQ ID NO.2.
SEQ ID NO.2：MSRLFLACLLAIFPVVSMKSPIFGPEEVTSVEGRSVSIKC YYPPTSVNRHTRKYWCRQGAQGRCTTLISSEGYVSDDYVGRANLTNFPESGTFVVDISRLTHKDSGRYKCGLGISSRGLNFDVSLEVSQETAIPASPSVVKGVRGGSVTVSCPYNPKDANSAKYWCHWEEAQNGRCPRLVESRGLMKEQYEGRLVLLTEPGNGTYTVILNQLTDQDAGFYWCVTDGDTRWISTVELKVVQGEPSLKVPKNVTAWLGEPLKLSCHFPCKFYSFEKYWCKWSNRGCSTLPTQNDGPSQAFVSCDQNSQVVSLNLDTVTKEDEGWYWCGVKEGPRYGETAAVYVAVESRVKGSQGAKQVKAAPAGAAIQSRAGEIQNKALLDPSFFAKESVKDAAGGPGAPADPGRPTGYSGSSKALVSTLVPLALVLVAGVVAIGVVRARHRKNVDRISIRSYRTDISMSDFENSRDFEGRDNMGASPEAQETSLGGKDEFATTTEDTVESKEPKKAKRSSKEEADEAFTTFLLQAKNLASAATQNGPTEA
EXAMPLE 3 antioxidant Activity assay of milk-derived active peptides
1. Determination of DPPH radical scavenger of milk-derived active peptide APADPGRPTGY
1. Experimental reagent and instrument
Cow milk concentrated protein (MPC) powder, a constant natural commercial company (Shanghai); lactobacillus paracasei (K56), biosciences inc; neutral protease (enzyme activity 1.1X106 u/g), xia Cheng (Shanghai) Biotechnology Co., ltd; lactose, national pharmaceutical group chemical company, inc; sodium chloride, shanghai Lingfeng chemical reagent Co., ltd; gastropeptone Peptone, BBI life sciences ltd; DPPH detection kit, shanghai Biyun Tian Biotech company.
Electronic balance, sartorius germany; constant temperature water bath box, shanghai-a constant technology company; 3kda ultrafiltration tube, millipore company; DR-200Bc enzyme labeling instrument, dekkera instruments, inc.; JB-VS-1300U ultra-clean bench, shanghai Yingming clean equipment limited company; LRH-250F biochemical incubator, shanghai-Heng science instruments Co., ltd; GI36T autoclave, xiamen micro instruments inc; froma 700 fridge, sameiser's technology (china) limited; 96-well cell culture plate, millipore company, usa; RO15 pure water system, likang biomedical science and technology control Co., ltd; GL-22M high-speed refrigerated centrifuge, shanghai Lu Xiangyi centrifuge instruments Inc.
2. Experimental method
9.858Mg of DPPH is dissolved in absolute ethyl alcohol and the solution is fixed to a 25mL brown volumetric flask to prepare a 1mmol/L solution, the solution is refrigerated and placed, and the solution is diluted 10 times to 0.1mmol/L by absolute ethyl alcohol.
50. Mu.L of each peptide solution of different concentration was pipetted into a 96-well plate and 5 replicates were set. Adding 100 mu L of DPPH ethanol solution (0.1 mmol/L) into each hole, oscillating uniformly, reacting for 30min at room temperature in a dark place, and measuring absorbance A1 at 517 nm; adding anhydrous 100 mu L ethanol solution into each hole, oscillating uniformly, reacting at room temperature in a dark place for 30min, and measuring absorbance A2 at 517 nm; 1.0mL of deionized water is used as a blank control instead of the sample, 2.0mL of absolute ethyl alcohol is added to measure the absorbance A0, and the absorbance A0 is calculated according to a formula.
Clearance (%) = [1- (a 1-A2)/A0 ] ×100%
3. Experimental results and analysis
The measurement results are shown in Table 1.
TABLE 1 determination of DPPH radical scavenging Rate by milk-derived active peptide APADPGRPTGY
| Experimental grouping | Experimental results (%) |
| APADPGRPTGY 0mg/ml | 0.0884±0.0311 |
| APADPGRPTGY 1mg/ml | 0.2196±0.0235** |
| APADPGRPTGY 0.5mg/ml | 0.1759±0.0160** |
| APADPGRPTGY 0.1mg/ml | 0.0931±0.0127 |
Note that: * There was a very significant difference (P < 0.01) compared to the negative control group.
The experimental results are shown in table 1, and the total antioxidant activity of the polypeptide APADPGRPTGY in vitro is measured by measuring the DPPH free radical scavenging rate, so that the experimental group added with the milk-derived active peptide APADPGRPTGY has a certain degree of reduction of the light absorption value compared with the blank group, and has better capability of reducing the oxidized substances. As can be seen from Table 1, it was found that the total antioxidant capacity of milk-derived active peptide APADPGRPTGY increased with increasing polypeptide concentration, and that the total antioxidant level of milk-derived active peptide APADPGRPTGY was optimal at a concentration of 1 mg/mL. Thus, the milk-derived active polypeptide APADPGRPTGY of the invention is considered to have significant antioxidant capacity.
EXAMPLE 4 immunomodulatory Activity assay of milk-derived active peptides
1. Determination of cytokine release from macrophages after LPS stimulation by milk-derived active peptide APADPGRPTGY
1. Experimental reagent and instrument
PBS, nanj Kaiki Biotech Co., ltd; DMEM incompletely high sugar culture broth; nanj Kaiki Biotech Co., ltd; fetal bovine serum, GIBCO limited; macrophage RAW264.7, cell site of Shanghai national academy of sciences; lipopolysaccharide (LPS, E.Coli O111: B4), sigma Co., ltd; mouse interleukin 6 (IL-6) ELISA kit, division of biological engineering (Shanghai).
Centrifuge 5414D mini high-speed Centrifuge, eppendorf limited; GL-22M high-speed refrigerated centrifuge, shanghai Lu Xiangyi centrifuge instruments Inc.; electronic balances Sartorius, germany; constant temperature water bath box, shanghai-a constant technology company; DR-200Bc enzyme labeling instrument, dekkera instruments, inc.; JB-VS-1300U ultra-clean bench, shanghai YingMing clean equipments Limited company.
2. Experimental method
DMEM incomplete medium (containing penicillin 80U/mL; streptomycin 0.08 g/L) containing 10% fetal bovine serum was prepared, RAW264.7 cells were prepared to a concentration of 2X 105/m L using this medium, and the cell suspension was inoculated in 25cm 2 disposable flasks or 96-well plates. Culturing in a saturated vapor carbon dioxide incubator with the temperature of 37 ℃ and the concentration of C02 of 5%. After 24h the medium was changed. Passaging was performed when the flask was observed with an inverted microscope to be substantially full of cells in the flask bottom. At the time of passage, the old culture medium was carefully aspirated, 1.5ml of trypsin digest was added, the mixture was placed in an incubator at 37℃for accurate digestion for 2min, and 2ml of medium was added to terminate the reaction. Repeatedly and gently blowing the cells to make the cells growing on the bottom wall fall off and scatter. The cells at the bottom of the tube were collected by centrifugation at 800g for 2min, and the cells were resuspended to a concentration of 2X 105 by adding the medium, and cultured.
Early stage of experiment: RAW264.7 cells were cultured in DMEM medium containing milk-derived active peptide APADPGRPTGY at a concentration of 0g/L, 0.1g/L, 0.5g/L, and 1.0g/L for 24 hours, and LPS solution was added to each flask at a final concentration of 100. Mu.g/L at 24 hours, and after further culturing for 2 hours, the cytokine IL-6 concentration in the supernatant of the medium was measured by ELISA kit.
3. Experimental results and analysis
The measurement results are shown in Table 2.
TABLE 2 determination of IL-6 release by macrophages after LPS stimulation by milk-derived active peptide APADPGRPTGY
| Experimental grouping | Experimental results (pg/mL) |
| APADPGRPTGY 0mg/ml | 12.93±0.03 |
| APADPGRPTGY 1mg/ml | 4.11±0.02** |
| APADPGRPTGY 0.5mg/ml | 6.70±0.03* |
| APADPGRPTGY 0.1mg/ml | 9.88±0.12 |
Note that: * Compared with the negative control group, there was a very significant difference (P < 0.01); * Compared with the negative control group, the negative control group has significant difference (P < 0.05).
When the organism is stimulated by external LPS, macrophages can reach local tissues where pathogens are located through chemotaxis, rapidly phagocytize foreign bodies and generate a large amount of inflammatory factors such as IL-6. The experimental results are shown in Table 1, and the in vitro immunoregulatory activity of polypeptide APADPGRPTGY is measured by measuring the IL-6 release of macrophages after LPS stimulation, so that the experimental group added with milk-derived active peptide APADPGRPTGY has a certain degree of reduction in the concentration of IL-6 in cell culture supernatant compared with the blank group, which indicates that the release level of macrophage pro-inflammatory factor is reduced, and the damage of inflammation to the organism is reduced. As is clear from Table 1, the immunomodulatory ability of milk-derived active peptide APADPGRPTGY increased with increasing polypeptide concentration, and polypeptide APADPGRPTGY had the best immunomodulatory ability at a concentration of 1 mg/mL. Thus, the milk-derived active polypeptide APADPGRPTGY of the invention can be considered to have significant immunomodulatory capacity.
EXAMPLE 5 stability of milk-derived active peptide and core fragment verification experiment
1. In vitro simulated gastrointestinal digestion experiment of milk-derived active peptide APADPGRPTGY
1. In vitro digestion of lyophilized powder of milk-derived active peptide APADPGRPTGY
1.5Mg of the sample powder was dissolved in 1.5mL of ddH 2 O, pepsin (enzyme: substrate=1:50, w/w) was added to the sample (concentration: 1 mg/mL), and then the pH was adjusted to 2.0, and the mixture was subjected to a water bath at 37℃for 90 minutes. Subsequently, the pH of the hydrolysate was adjusted to 7.5, pancreatin (enzyme: substrate=1:25, w/w) was added, and the mixture was water-bath at 37℃for 150min. Finally, the reaction was stopped by inactivating the enzyme with a hot water bath at 95℃for 5 min. In the experiment, a control group was set up and the same pH and temperature treatments were performed at the same time, except that pepsin and pancreatin were not added. The control and sample groups were lyophilized in vacuo to a powder and stored at-80 ℃ for subsequent analysis.
UPLC-MS analysis
The UPLC conditions are as follows:
Instrument: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quaternary rod, time-of-flight mass spectrometer
Chromatographic column specification: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 DEG C
Ultraviolet detection wavelength: 210nm of
Sample injection amount: 2 mu L
Gradient conditions: and (3) solution A: water containing 0.1% formic acid (v/v), solution B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometry analysis
The mass spectrometry conditions were as follows:
Ion mode: ES+
Mass range (m/z): 100. 1000 (1000)
Capillary voltage (CAPILLARY) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (deg.c): 115
Desolvation temperature (deg.c): 350
Desolventizing gas flow (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Internal scan time (sec): 0.02.
According to the analysis method, chromatographic analysis and mass spectrometry are carried out on the in-vitro digestion products of the milk active peptide APADPGRPTGY by utilizing an ultra-high performance liquid phase, electrospray, a quaternary rod and a time-of-flight mass spectrum. Comparing the polypeptide sequences of the polypeptides in the control group with those in the experimental group to detect the stability of the milk-derived active peptide.
3. Experimental results and analysis
FIG. 3 is a pie chart of in vitro digestion product analysis of milk-derived active peptide APADPGRPTGY. The legend is arranged according to the content.
As shown in FIG. 3, the proportion of the original sequence of the milk-derived active peptide APADPGRPTGY in the experimental group is more than 99%, which indicates that pepsin and pancreatin do not hydrolyze. The control group had many more short peptides than the experimental group, these short peptides were degraded in the experimental group, whereas the whole peptide was not degraded. Experiments prove that the milk-derived active peptide APADPGRPTGY has stability in the gastrointestinal tract digestion process.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (7)
1. A small peptide, the amino acid sequence of which is shown as SEQ ID NO. 1;
SEQ ID NO.1：APADPGRPTGY。
2. A derivative of a small peptide, wherein the derivative is a derivative peptide which is obtained by methylation and/or acetylation modification on the basis of the amino terminal or the carboxyl terminal of the amino acid sequence shown in SEQ ID NO.1 and has the same biological activity as the small peptide of the amino acid sequence shown in SEQ ID NO. 1.
3. A method of preparing the small peptide of claim 1, the method comprising:
preparing the small peptide by a genetic engineering method; or alternatively
The small peptides are synthesized by chemical means.
4. A method of preparing the small peptide of claim 1, the method comprising:
inoculating Lactobacillus paracasei with deposit number DSM27447 to ferment by using emulsion containing milk protein as ferment substrate, and separating from ferment product to obtain the small peptide of claim 1;
wherein the fermentation substrate comprises: 3% -10% of cow milk concentrated protein and 1% -10% of lactose.
5. The method of claim 4, wherein the fermentation conditions are: the inoculation amount is 1% -3% at 30-45 ℃, and the bacterial count in the seed liquid is more than 1.5X10 11 CFU/mL.
6. Use of the small peptide of claim 1 for the preparation of a composition having antioxidant and/or immunomodulatory effects.
7. A composition comprising the small peptide of claim 1, optionally together with an adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211544797.2A CN116444612B (en) | 2022-11-30 | 2022-11-30 | Milk active peptide APADPGRPTGY and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211544797.2A CN116444612B (en) | 2022-11-30 | 2022-11-30 | Milk active peptide APADPGRPTGY and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116444612A CN116444612A (en) | 2023-07-18 |
| CN116444612B true CN116444612B (en) | 2024-07-16 |
Family
ID=87130858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211544797.2A Active CN116444612B (en) | 2022-11-30 | 2022-11-30 | Milk active peptide APADPGRPTGY and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116444612B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107141346A (en) * | 2017-06-22 | 2017-09-08 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide ATLEDSPEVI and its preparation method and application |
| CN107916236A (en) * | 2016-10-05 | 2018-04-17 | 益福生医股份有限公司 | Lactobacillus paracasei subspecies paracasei K56 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL127238A (en) * | 1996-06-04 | 2001-07-24 | Univ California | Cellular internalization of pigr stalk and associated ligands |
| FR2841473B1 (en) * | 2002-06-27 | 2004-09-17 | Ingredia | USE OF AT LEAST ONE PEPTIDE OF CASE S2 WITH INHIBITORY ACTIVITY OF THE ENGIOTENSIN I CONVERSION ENZYME FOR THE PREPARATION OF DRUGS, FOODS AND FOOD SUPPLEMENTS |
| CN102964427B (en) * | 2012-12-12 | 2014-01-15 | 上海交通大学 | Bioactive polypeptide QEPVL, and preparation and application thereof |
| CN103232528B (en) * | 2012-12-12 | 2014-12-24 | 上海交通大学 | Bioactive polypeptide DELQ and preparation method and application thereof |
| CN116114758A (en) * | 2020-11-26 | 2023-05-16 | 内蒙古伊利实业集团股份有限公司 | Lactobacillus paracasei K56 for regulating blood pressure and application thereof |
-
2022
- 2022-11-30 CN CN202211544797.2A patent/CN116444612B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916236A (en) * | 2016-10-05 | 2018-04-17 | 益福生医股份有限公司 | Lactobacillus paracasei subspecies paracasei K56 |
| CN107141346A (en) * | 2017-06-22 | 2017-09-08 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide ATLEDSPEVI and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116444612A (en) | 2023-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110938129A (en) | Bioactive polypeptide SKLVPVGYGIRKL, and preparation method and application thereof | |
| CN112759636A (en) | Bioactive peptide with amino acid structure ESLKGVDPKFLR, and preparation method and application thereof | |
| CN112812168A (en) | Bioactive peptide GLNMCRQCF, and preparation method and application thereof | |
| CN112745380A (en) | Bioactive peptide with amino acid structure RAGLQFPVGRVH, and preparation method and application thereof | |
| CN116444612B (en) | Milk active peptide APADPGRPTGY and preparation method and application thereof | |
| CN112501140A (en) | Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof | |
| CN116444608B (en) | Milk active peptide DPSFFAKE and preparation method and application thereof | |
| CN116444609B (en) | Milk active peptide LPPPLPSRWPL and preparation method and application thereof | |
| CN116444619B (en) | Milk active peptide YPFPGPIHNSLP and preparation method and application thereof | |
| CN116444611B (en) | Milk active peptide TDPLFKG and preparation method and application thereof | |
| CN116444610B (en) | Milk active peptide DASAQFIR and preparation method and application thereof | |
| CN116444649B (en) | Milk active peptide GITDPLFKGM, GITDPLFKG and its obtaining method and use | |
| CN112661830B (en) | Bioactive peptide with amino acid structure AIRNDEELNKLLGR, and preparation method and application thereof | |
| CN112724237B (en) | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof | |
| CN112500468B (en) | Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof | |
| CN112480232A (en) | Bioactive peptide VSLADLQNDEVAFR, and preparation method and application thereof | |
| CN112480233A (en) | Bioactive peptide IAHPKLGKRIR, and preparation method and application thereof | |
| CN112625112A (en) | Bioactive polypeptide PAAPAQLPKKI, and preparation method and application thereof | |
| CN112592396A (en) | Bioactive peptide VDPFSKKDW, and preparation method and application thereof | |
| CN112661829B (en) | Bioactive peptide GGYGSGGGSGGYGSRRF, and preparation method and application thereof | |
| CN112480231B (en) | Bioactive peptide KQSLPPGLAVKDLK, and preparation method and application thereof | |
| CN112812171B (en) | Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof | |
| CN112812170B (en) | Bioactive polypeptide with amino acid structure LLPKKTE as well as preparation method and application thereof | |
| CN112778410B (en) | Bioactive peptide SAPRHGSLGFLPRK, and preparation method and application thereof | |
| CN112745381B (en) | Bioactive peptide with amino acid structure NIRNIGKTLVTR, and preparation method and application thereof |
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 |