CN116655738A - 基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 - Google Patents
基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 Download PDFInfo
- Publication number
- CN116655738A CN116655738A CN202310434856.9A CN202310434856A CN116655738A CN 116655738 A CN116655738 A CN 116655738A CN 202310434856 A CN202310434856 A CN 202310434856A CN 116655738 A CN116655738 A CN 116655738A
- Authority
- CN
- China
- Prior art keywords
- eggshell membrane
- peptide
- antioxidant
- enzymolysis
- antioxidant peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940012466 egg shell membrane Drugs 0.000 title claims abstract description 52
- 101800000068 Antioxidant peptide Proteins 0.000 title claims abstract description 32
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 241000894006 Bacteria Species 0.000 title claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 38
- 229940088598 enzyme Drugs 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 24
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 23
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 18
- 238000012216 screening Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 9
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 9
- 229940111202 pepsin Drugs 0.000 claims abstract description 9
- 238000012795 verification Methods 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 6
- 238000010521 absorption reaction Methods 0.000 claims abstract description 5
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 5
- 210000001503 joint Anatomy 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 3
- 230000036541 health Effects 0.000 claims abstract description 3
- 231100000252 nontoxic Toxicity 0.000 claims abstract description 3
- 230000003000 nontoxic effect Effects 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 9
- 230000003647 oxidation Effects 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 108010033276 Peptide Fragments Proteins 0.000 claims description 4
- 102000007079 Peptide Fragments Human genes 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000002137 ultrasound extraction Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 230000009849 deactivation Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 241000576429 Forsythia suspensa Species 0.000 claims description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 2
- 239000002537 cosmetic Substances 0.000 claims description 2
- 235000013373 food additive Nutrition 0.000 claims description 2
- 239000002778 food additive Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 4
- 241000555712 Forsythia Species 0.000 abstract description 3
- 230000003796 beauty Effects 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 230000002195 synergetic effect Effects 0.000 abstract description 3
- 238000001356 surgical procedure Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- 235000006708 antioxidants Nutrition 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 12
- 235000011130 ammonium sulphate Nutrition 0.000 description 12
- 238000000502 dialysis Methods 0.000 description 12
- 229920001184 polypeptide Polymers 0.000 description 11
- 239000012528 membrane Substances 0.000 description 10
- 210000003278 egg shell Anatomy 0.000 description 8
- 229910052739 hydrogen Inorganic materials 0.000 description 8
- 239000001257 hydrogen Substances 0.000 description 8
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 102000002322 Egg Proteins Human genes 0.000 description 6
- 108010000912 Egg Proteins Proteins 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000011033 desalting Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000012153 distilled water Substances 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
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000413 hydrolysate Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000003032 molecular docking Methods 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 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 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000004034 Kelch-Like ECH-Associated Protein 1 Human genes 0.000 description 2
- 108090000484 Kelch-Like ECH-Associated Protein 1 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011208 chromatographic data Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 238000002878 ADMET assay Methods 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 101150116862 KEAP1 gene Proteins 0.000 description 1
- 241001071917 Lithospermum Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000002792 antioxidant assay Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 239000010791 domestic waste Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010846 tandem mass spectrometry analysis Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing 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
- 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
- A61Q19/08—Anti-ageing preparations
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Birds (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用,属于生物技术领域。通过LC‑MS/MS鉴定连翘内生菌LQ1协同胃蛋白酶水解的蛋壳膜所产生的活性肽,通过ToxinPred、admetSAR和BIOPEP‑UWM程序分析对活性肽进行初步筛选,分子对接技术再筛选,得到无毒、水溶性高、肠道吸收好最具抗氧化活性潜力的肽序列后,合成进行体外抗氧化验证。该抗氧化肽的氨基酸序列为IRDGWPH、STDVPRDPWVWG或EKIWHHTF,均有较高的抗氧化能力,可应用于食品、美容用品及保健品领域,为后续充分利用蛋壳膜资源筛选天然抗氧化肽提供了一定的理论基础。
Description
技术领域
本发明属于生物技术领域,涉及蛋壳膜抗氧化肽,具体涉及基于菌酶联合制备的蛋壳膜抗氧化肽及其筛选方法和应用。
背景技术
鸡蛋壳是生活及工业废品,是目前给环境治理带来困难的污染源之一。鸡蛋壳膜是禽蛋壳膜副产物,又称凤凰衣,其组成成分主要为各种蛋白质、脂质。糖和钠等,通过分析其主要成分,发现蛋膜是一种潜在的多肽和氨基酸资源,具有很高的利用价值。目前国内外对蛋壳膜的水解多为酸法或碱法。而蛋白酶具有微量高效安全无害等特点,是用来水解蛋壳膜的最佳选择。目前国际上也有研究人员利用酸-酶结合法、碱-酶结合法或者酶法来对蛋壳膜进行水解,而后对其水解产物进行抑菌或抗氧化活性进行研究。但是对于蛋壳膜利用菌酶联合水解,对水解产物的抑菌及抗氧化双活性的研究却鲜有报道。目前,食品及医疗美容行业所使用的抑菌防腐剂多为化学试剂,抗氧化也是医疗美容行业的一个经久不衰的话题。而蛋壳膜作为废弃资源,若能将其利用,得到具有抑菌及抗氧化双活性的产物,将会对环境、食品、医疗、美容等行业都大有裨益。
发明内容
针对现有技术中的上述不足,本发明提供了一种基于菌酶联合制备的蛋壳膜抗氧化肽及其筛选方法。该发明可有效解决现有的蛋壳膜抗氧化肽制备和筛选效率差的问题。
为了实现上述目的,本发明采用的具体方案为:
一种蛋壳膜抗氧化肽,其为如下任意一种氨基酸序列所示的抗氧化肽:IRDGWPH;
STDVPRDPWVWG;EKIWHHTF。
上述蛋壳膜抗氧化肽的制备方法,是以蛋壳膜为原料,采用菌酶联合制备,所述菌采用连翘内生菌LQ1,所述酶为胃蛋白酶;所述连翘内生菌LQ1的保藏编号为CGMCCNO.8728。
作为对上述技术方案的进一步优化,所述制备方法具体包括以下步骤:
(1)将蛋壳膜60℃ 烘干粉碎后过筛,得蛋壳膜粉末;
(2)将蛋壳膜粉末与水以1:10~50的比例混合,在70〜100 ℃下溶解10〜60min后冷却至室温;调节溶液的pH为6.0,加入连翘内生菌LQ1菌液,37℃下发酵30-36h后去除菌体,加入胃蛋白酶,在40℃下酶解2〜8h,超声处理;然后对溶液进行灭酶处理后离心,调节溶液PH至中性;得蛋壳膜水解液,将溶液过滤、浓缩;
(3)对浓缩液进行LC-MS/MS鉴定,获得酶解肽段序列,然后利用计算机对肽段模拟筛选,通过ToxinPred程序、admetSAR程序和 BIOPEP-UWM程序分析对抗氧化肽进行初步筛选,再经分子对接技术再筛选,得到无毒、水溶性高、肠道吸收好且最具抗氧化活性潜力的肽序列后,合成进行体外抗氧化验证。
作为对上述制备方法的进一步优化, 步骤(1)中,蛋壳膜粉粉末的粒径为120-140目。
作为对上述制备方法的进一步优化,步骤(2)中,所述胃蛋白酶酶解条件为:酶添加量为40u/mg、酶解时间为4h、酶解温度为40℃、酶解pH值为4。
作为对上述制备方法的进一步优化,步骤(2)中,连翘内生菌LQ1的菌液加量为所述混合液体积的5~6.1%,所述菌液浓度为1.5×108cfu/mL。
作为对上述制备方法的进一步优化,步骤(2)中,所述超声波处理的温度为30~60℃,超声提取30~90min;超声提取功率为60-90W,超声频率为30-50KHz。
作为对上述制备方法的进一步优化,步骤(2)中,所述离心温度为3~4℃,离心转速为10000~12000r,离心时间为10~15min。
一种抗氧化剂,包括上述蛋壳膜抗氧化肽。
上述蛋壳膜抗氧化肽或抗氧化剂在食品添加剂、化妆品、保健品中的应用。
本发明所产生的有益效果为:
本申请中的蛋壳膜抗氧化肽,IRDGWPH、STDVPRDPWVWG、EKIWHHTF均有较高的DPPH自由基清除活性;羟自由基清除率和较高总抗氧化能力,通过对本申请中的组方进行蛋壳膜抗氧化肽提取与筛选获得的抗氧化肽还具有一定抑菌性,菌酶协同技术可以综合微生物发酵和酶解有两大优势。连翘内生菌LQ1发酵可分泌纤维素酶和蛋白酶,调节动物肠道健康。蛋白酶酶解,则主要通过酶促反应将蛋白质中的二硫键打开,以获得可溶性多肽及生物活性肽。因此在自然界植物中筛选可降解蛋壳膜的菌种,协同优势降解蛋壳膜的胃蛋白蛋白酶,增加蛋壳膜生物活性肽的制备率具有极高研究意义。大量生物活性肽的活性验证过程费时、费力。相比之下,本发明通过计算机辅助多肽活性的生物信息学筛选能够克服传统方法中的缺陷,减少验证数量精准筛选所需生物活性肽进行构建与生物验证。
采用本申请中的提取方法,可将蛋壳膜中的抗氧化肽充分提取筛选,以提蛋壳膜抗氧化肽提取的效率。
附图说明
图1是电泳结果图;其中,1为20% 硫酸铵沉淀后的样品,2为80% 硫酸铵沉淀后的样品;
图2是蛋白色谱图;
图3是多肽IRDGWPH与蛋白KEAP1的对接结构示意图;
具体实施方式
一种基于菌酶联合制备的蛋壳膜抗氧化肽及其筛选方法,包括以下将条件:蛋壳膜60℃ 烘干粉碎,得蛋壳膜粉末蛋壳膜粉末与水以1:10~50的比例混合,在70〜100 ℃下溶解10〜60min后冷却至室温,得混合液;调节溶液的pH为6;添加1.5×108cfu/mL连翘内生菌LQ1菌液,添加量为所述混合液体积的5-6.1%,37℃下发酵30-36h,去除菌体。接着加入胃蛋白酶,酶添加量为40u/mg、酶解时间为4h、酶解温度为40℃、酶解pH值为4下进行酶解,结束后灭酶离心,收集上清液,将上清液减压浓缩,得浓缩液,对浓缩液进行通过LC-MS/MS(liquid chromatography coupled with tandem mass spectrometry,LC-MS/MS)法鉴定的酶解肽段序列,然后利用计算机对肽段模拟筛选, 通过ToxinPred程序、admetSAR程序和BIOPEP-UWM程序分析对抗氧化活性肽的吸收、分配、代谢、排泄和毒性等特性进行初步筛选,再经分子对接技术再筛选,得到无毒、水溶性高、肠道吸收好最具抗氧化活性潜力的肽序列后,合成进行体外抗氧化验证。
本申请筛选得到的抗氧化肽序列为:IRDGWPH(SEQ ID NO:1);STDVPRDPWVWG(SEQID NO:2); EKIWHHTF(SEQ ID NO:3)。
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述。
实施例
蛋壳膜抗氧化肽的制备方法,包括以下步骤:
(1)收集新鲜鸡蛋壳,将蛋壳膜里面附着的粘液洗干净后将蛋壳膜剥落,用蒸馏水洗干净后于60℃烘箱烘干。将烘干之后的蛋壳膜用破碎机粉碎,过120目筛之后避光保存备用,制得的蛋壳膜粉。
(2)将20g ESM粉末与1000mL无菌水混合,在85 ℃下溶解40min后冷却至室温;调节溶液的pH为6,取1.5×108CFU/mL的连翘内生菌LQ1菌液50mL,接入混合液中,37℃发酵36h后,调节其发酵液混合物pH值为4后加入胃蛋白酶,添加量为40 u/mg,在40 ℃ 下酶解4 h后高温灭酶,12000 r/min离心20 min取上清,调节溶液pH至中性后用0.45 μm滤膜过滤得ESM粗酶解液。
(3)将(2)中酶解液进行硫酸铵分级沉淀
将1000 mL酶解液平均分装于10个锥形瓶中,在冰浴中缓慢加入事先碾碎的硫酸铵细粉末,分别收集硫酸铵饱和度为20%、40%、60%、80% 的蛋白沉淀。
(4)透析除盐与活性测定
通过透析除去上述沉淀中的硫酸铵。先加入适量PBS缓冲液将上述沉淀复溶,将其加进处理好的透析袋(截留分子量500 D)中,密封完毕后将之置于装满透析液的容器中,放于磁力搅拌器上透析。每3 h更换一次透析液加快透析过程,将透析液更换4-6次后吸取透析袋内少许样液,利用氯化钡与硫酸铵反应可产生白色沉淀的原理检测检测样液中是否还有硫酸铵残留。硫酸铵完全透析除净后透析结束并收集透析袋内样液。全程不可用手直接接触透析袋;使用之前要对透析袋进行检漏;透析袋使用前后都要用50% 乙醇和纯净水分别煮沸20-30 min。透析完成后将样液于冷冻干燥机中冻干后,称取一定量的样品用PBS缓冲液复溶后对其进行SDS-PAGE凝胶电泳分析并分别进行抗氧化活性测定。根据测定结果确定进一步纯化的样品。
分别取0.1 g样品溶于4 mL水中,对其DPPH清除率、羟自由基清除效果、总抗氧化能力进行测定。空白对照液为PBS缓冲液,阳性对照为VC溶液,结果见表1,80% 硫酸铵沉淀样品的总抗氧化能力、DPPH清除率、羟自由基清除效果最好,达到16039.3U/ml、96.3%、84.28%。
表1 不同硫酸铵饱和度下沉淀样品的抗氧化能力对比。
电泳结果见图1,从图中可以看出经20% 硫酸铵沉淀后的样品,分子量较大,在98KD左右,80% 硫酸铵沉淀后的样品,分子量相对较小,主要集中在14-22 KD之间。由此可知菌酶联合蛋壳膜降解液中发挥抗氧化活性的物质主要为小分子肽,接下来将对其进行进一步纯化。
(6)葡聚糖凝胶Sephadex-G25湿法装柱过滤层析
葡聚糖G-25利用分子筛原理可使不同分子量大小的物质先后从层析柱中流出,从而达到分离不同分子量蛋白质的目的。将上述所得80% 硫酸铵处理的ESM酶解液沉淀用0.9% NaCl复溶,进一步进行层析纯化,根据色谱数据工作站中的波动峰可将收集到的属于同一峰值内的样品进行合并。
色谱数据工作站谱线参数:满屏里程(mV)500、起始峰宽水平3、最小峰面积100。其结果如图2所示共出现3个主要峰,表示80% 硫酸铵沉淀样品中主要存在3种不同分子量大小的多肽。将其冻干后收集1峰样品0.98 g,2峰样品1.69g,3峰样品0.87 g。
称取各峰样品0.1 g加入4 mL蒸馏水复溶,此次测定空白液为0.9% NaCl溶液,阳性对照为VC溶液,结果如表2所示,2峰和3峰样液对羟自由基清除效果优于1峰,3峰样品效果最好,3峰样品大部分为小分子肽(5~8 KD)。将3峰酶解液冷冻干燥备测。
表2 各峰的抗氧化能力对比。
(7)ESM抗氧化肽提取及分析
ESM酶解液冷冻状态下取出全部至离心管中;加入适量0.1% TFA充分溶解;使用Oasis HLB脱盐柱脱盐;使用冷冻离心浓缩干燥器干燥多肽;加入适量50% ACN+0.5% TFA充分溶解;使用Oasis MCX脱盐柱脱盐;使用冷冻离心浓缩干燥器干燥多肽;取1/10样品用50μL蒸馏水充分溶解多肽;使用Pierce Quantitative Colorimetric Peptide Assay定量试剂盒进行浓度测定并进行液相串联质谱分析。
表3 EASY-nLC液相梯度。
色谱分离时间:60 min;A相:2% 乙腈0.1% 甲酸;B相:80% 乙腈0.1% 甲酸;流速:300 nL/min;
MS 扫描范围(m/z):300-1500,采集模式:DDA;Top 20(选择母离子中信号最强的20个进行二级碎裂);一级质谱分辨率:60000,AGC target:3e6,最大注入时间:20 ms,碎裂方式HCD;二级分辨率:15000,AGC target:1e5,最大注入时间:50 ms,fixed first mass:100 m/z;minimum AGC target:8e3,Intensitythreshold:1.6e5,动态排除时间:18 s。
通过液相串联质谱分析发现酶解后的ESM小分子肽中,肽链上氨基酸数目在2~10个以内的寡肽(oligopeptide)肽段占95%,该法得到的寡肽所占比例较高,此类肽更易被消化吸收,且具有大分子蛋白质所没有的一些理化特性。分子量较小的肽段结构更易于改造修饰,人工合成成本较低,这些特点为ESM活性多肽药物的开发利用提供了广阔的前景。
表4 搜库结果列表。
(8)ESM酶解液抗氧化活性肽的筛选及理化性质评价
通过ToxinPred程序(http://crdd.osdd.net/raghava//toxinpred/)来预测所得肽序列的潜在毒性。通过admetSAR程序(http://lmmd.ecust.edu.cn/admetsar2/)来预测所得肽序列的吸收、分配、代谢、排泄和毒性等特性,通过BIOPEP-UWM程序(http://www.uwm.edu.pl/bioch emia/index.php/pl/biopep)来预测所得肽序列的潜在的生物活性。
表5所筛选的具有抗氧化性肽的ADMET分析和活性预测。
(9)分子对接筛选
采用ChemBioDraw Ultra 14.0绘制多肽结构,将结构导入ChemBio3D Ultra 14.0进行能量最小化,将Minimum RMS Gradient设置为:0.001,将小分子保存为mol2格式。从PDB数据库下载Keap1(PDB ID:1ZGK)的蛋白结构,使用Pymol2.3.0去除蛋白结晶水、原始配体等。将多肽和蛋白结构上传至HPEPDOCK进行对接,采用Pymol 2.3.0进行作图。
如图3所示,IRDGWPH与KEAP1的结合能为-226.255kcal/mol,证明具有较好的结合作用。IRDGWPH的H7与蛋白的R380、形成氢键,氢键的长度为3.5Å、2.4Å;IRDGWPH的P6与蛋白的R380、N382、R415形成氢键,氢键的长度分别为2.1Å、3.5Å、3.4Å;IRDGWPH的R2与蛋白的R415、Y525、G527形成氢键,氢键的长度分别为3.3Å、3.5Å、3.5Å;IRDGWPH的I1与蛋白的Y572形成氢键,氢键的长度为3.3Å;以上位点可能为多肽与作用的关键位点。
(10)合成蛋壳膜抗氧化肽进行体外抗氧化验证。
合成STDVPRDPWVWG、IRDGWPH、EKIWHHTF,以同浓度维生素C为阳性对照,PBS缓冲液为阴性对照。
表6 体外抗氧化验证。
对比例
一种常见抗氧化剂维生素C
本发明实施例与对比例制备得到0.1mg/ml溶液。检测结果表明,同等浓度下两者抗氧化能力相当。
需要说明的是,以上所述的实施方案应理解为说明性的,而非限制本发明的保护范围,本发明的保护范围以权利要求书为准。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对本发明作出的一些非本质的改进和调整仍属于本发明的保护范围。
Claims (10)
1.一种蛋壳膜抗氧化肽,其为如下任意一种氨基酸序列所示的抗氧化肽:IRDGWPH;
STDVPRDPWVWG;EKIWHHTF。
2.根据权利要求1所述的蛋壳膜抗氧化肽的制备方法,其特征在于:是以蛋壳膜为原料,采用菌酶联合制备,所述菌采用保藏编号为CGMCC NO.8728的连翘内生菌LQ1,所述酶为胃蛋白酶。
3.根据权利要求2所述的制备方法,其特征在于:具体包括以下步骤:
(1)将蛋壳膜60℃ 烘干粉碎后过筛,得蛋壳膜粉末;
(2)将蛋壳膜粉末与水以1: 10~50的比例混合,在70〜100 ℃下溶解10〜60min后冷却至室温,得混合液;调节溶液的pH为6.0,加入连翘内生菌LQ1菌液,37℃下发酵30-36h后去除菌体,加入胃蛋白酶,在40℃下酶解2〜8h,超声处理;然后对溶液进行灭酶处理后离心,调节溶液pH至中性,得蛋壳膜水解液,过滤减压浓缩;
(3)对浓缩液进行LC-MS/MS鉴定,获得酶解肽段序列,然后利用计算机对肽段模拟筛选,通过ToxinPred程序、admetSAR程序和 BIOPEP-UWM程序分析对抗氧化肽进行初步筛选,再经分子对接技术再筛选,得到无毒、水溶性高、肠道吸收好且最具抗氧化活性潜力的肽序列后,合成进行体外抗氧化验证。
4.根据权利要求3所述的制备方法,其特征在于:步骤(1)中,蛋壳膜粉粉末的粒径为120〜140目。
5.根据权利要求3所述的制备方法,其特征在于:步骤(2)中,所述胃蛋白酶酶解条件为:酶添加量为40u/mg、酶解时间为4h、酶解温度为40℃、酶解pH值为4。
6.根据权利要求3所述的制备方法,其特征在于:步骤(2)中,连翘内生菌LQ1的菌液加量为所述混合液体积的5-6.1%,所述菌液浓度为1.5×108cfu/mL。
7.根据权利要求3所述的制备方法,其特征在于:步骤(2)中,所述超声波处理的温度为30~60℃,超声提取30~90min;超声提取功率为60~90W,超声频率为30~50KHz。
8.根据权利要求3所述的制备方法,其特征在于:步骤(2)中,所述离心温度为3~4℃,离心转速为10000~12000r,离心时间为10~15min。
9.一种抗氧化剂,包括权利要求1所述的蛋壳膜抗氧化肽。
10.权利要求1所述的蛋壳膜抗氧化肽或权利要求9所述的抗氧化剂在食品添加剂、化妆品、保健品中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310434856.9A CN116655738A (zh) | 2023-04-21 | 2023-04-21 | 基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310434856.9A CN116655738A (zh) | 2023-04-21 | 2023-04-21 | 基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116655738A true CN116655738A (zh) | 2023-08-29 |
Family
ID=87723152
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310434856.9A Pending CN116655738A (zh) | 2023-04-21 | 2023-04-21 | 基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116655738A (zh) |
-
2023
- 2023-04-21 CN CN202310434856.9A patent/CN116655738A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101589761B (zh) | 一种工业用大麻籽抗氧化肽的制备方法及应用 | |
CN103882083B (zh) | 一种抗氧化胶原肽的制备方法 | |
CN108047313B (zh) | 鸡血球抗氧化肽及其发酵制备方法 | |
CN112877391B (zh) | 一种具有促益生菌生长和抗氧化作用的牛骨蛋白糖肽及其制备方法与应用 | |
Chen et al. | Preparation, characterization and the in vitro bile salts binding capacity of celery seed protein hydrolysates via the fermentation using B. subtilis | |
CN111635920A (zh) | 一种二级仿生酶解技术制备驴骨胶原蛋白肽粉的方法 | |
CN111269290A (zh) | 一种鲟鱼抗炎肽的制备方法 | |
CN107574214A (zh) | 一种抗衰老的乳清蛋白肽的制备方法 | |
CN106701877A (zh) | 源于牡蛎的ace抑制肽的制备方法 | |
CN116731108B (zh) | 草菇抗氧化肽及其应用 | |
CN116715724B (zh) | 来源于草菇子实体的抗氧化肽及其应用 | |
CN111087447B (zh) | 一种鳄鱼抗氧化肽复合物及其制备方法和应用 | |
CN116655738A (zh) | 基于菌酶联合制备的蛋壳膜抗氧化肽及其制备方法和应用 | |
CN115057916B (zh) | 一种马氏珠母贝肉抗氧化多肽及其制备方法与应用 | |
CN113481270B (zh) | 一种从扇贝裙边中提取糖肽的方法 | |
CN114163494A (zh) | 一种拮抗氧化应激损伤的章鱼抗氧化肽的制备方法和应用 | |
CN107173815A (zh) | 一种北太平洋鱿鱼缠卵腺抗氧化酶解寡肽的用途 | |
CN112741199B (zh) | 一种具有提高性功能功效的牡蛎肽及其制备方法 | |
CN109021072B (zh) | 一种红毛藻抗氧化肽及其制备方法 | |
CN108851093B (zh) | 从罗汉果废液中分离水溶性膳食纤维和蛋白的方法 | |
CN111073944A (zh) | 一种基于Caco-2细胞模型的活性肽定向制备方法 | |
CN116751248B (zh) | 抗氧化肽及其在制备清除自由基的药物中的应用 | |
CN114350732B (zh) | 一种具备抗氧抗炎的蛋清蛋白肽 | |
CN107760746A (zh) | 一种工业化生产杏仁活性肽及制备方法 | |
CN112048534B (zh) | 一种具有提高白藜芦醇功效活性的马鲛鱼蛋白水解物及其制备方法 |
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 |