Isolation,Purification And Functional Study Of Collagen Polypeptide From Rana And Preparation Of Microcapsules

This thesis is from the Science and Technology Department of Jilin Provin ce?20160204022NY?:"Study on the key technology of structural modification and stability of collagen frog residues in Rana chensinensis".In this study,the bioactive Rana collagen polypeptide?molecular weight less than 3500Da?obtain ed by enzymatic hydrolysis was used as raw material for separation,purificatio n and functionalization.Firstly,the gel chromatography separation conditions w ere optimized to obtain three components I,II and III.The amino acid sequen ce was identified byhigh resolution liquid chromatography-mass spectrometry?L C-MS?,and the dominant sequences of each component were screened.Secondl y,the antioxidant andACE inhibitory activities of components I,II and III wer e studied,and the structure of the antioxidants and ACE inhibitory activities w ere investigated by combining the obtained amino acid sequences.Finally,the R ana sinensis peptide/microporous starch-alginate microcapsules were prepared by using the Rana chensin collagen peptide as the core material,and the sustaine d release effect in the simulated gastrointestinal fluid in vitro was investigated.The research content and corresponding results of this paper are as follows:1.Separation and purification of collagen protein peptides by using dextra n gel chromatography separation technology,optimizing the three main conditio ns of chromatographic medium specification,loading amount and elution flow r ate to obtain components I,II and III,and then The morphology of each com ponent was analyzed by scanning electron microscopy.Finally,the three compo nents were subjected to LC-MS to obtain the dominant amino acid sequence of each component.The results were as follows:?1?The chromatographic separati on conditions were as follows:the chromatographic medium Sephadex G25,the loading amount of 1500?g,and the elution flow rate of 60 mL/h,the separati on effect was the best,and three components were obtained.?2?Scanning elect ron microscopy analysis of mixed peptides and I,II,III showed that the surfac e morphology of the original mixed peptide was spherical,and the surface mor phology of I,II and III components was similar,all of which were flocculent,f laky and rod-shaped.Mixed,the difference between them is not obvious.?3?F rom the amino acid sequence detected by LC-MS,the dominant amino acid se quences in the I component were selected as NMDMPPNK,LDAQVSALEGAR,LEVLEIIMAIFK,respectively named I₁,I₂,I₃;the dominant amino acid sequenc e in the II component was NALSPLK,MLDEVPK and MNAQNVGK are nam ed II₁,II₂,and II₃,respectively;the dominant amino acid sequences in the III component are GGLVGIK,EISSLK,MAAISPK,and MANSQLK,which are na med III₁,III₂,III₃,and III₄,respectively.2.The antioxidant activity of the isolated peptide components I,II and III was studied,and the DPPH scavenging ability,hydroxyl radical scavenging abil ity,superoxide anion scavenging ability and reducing power were investigated,and further exploration was carried out.Antioxidant structure-activity relationshi p.The results showed that:?1?DPPH clearance rates of components I,II and III were 74.3%,50.8%,and 59.0%,respectively;the reducing powers were 0.68,0.32,and 0.33,respectively;the scavenging rates of hydroxyl radicals were5.6%and 39.0,respectively.%,47.1%;the scavenging rates for superoxide ani ons were 33.4%,12.4%,and 11.5%,respectively.In general,I has the highest antioxidant activity.?2?LC-MS results showed that the three component amino acid sequences contained Leu,Pro,Lys,Arg,which may be the main reason f or the antioxidant activity of all three components.?3?The N-terminus of the dominant sequence in I is the amide residue asparagine Asn and Leu of the ac idic amino acid,while the N-terminus of the dominant sequence in II and III i s mostly the neutral amino acid Met,which may be the reason that antioxidant activity of the I component is a little higher than the II and III components.3.Determination of ACE inhibitory activity of peptides I,II and III by hi gh performance liquid phase,and explore its ACE inhibition structure-activity r elationship.The results showed that when the concentration of inhibitor was be tween 0 and 10 mg/mL,the inhibitory rate of ACE of I,II and III increased gradually with the increase of inhibitor concentration,and the ACE inhibitory a ctivity of III was always greater than I.Component II.In the dominant amino acid sequence of I,II and III detected by LC-MS,the dominant sequence of each component has a C-terminus of Lys or Arg,and each sequence has a C-t erminal tripeptide position containing at least one hydrophobic amino acid.This may be the main reason why all three components have ACE inhibitory activi ty.The III component has the highest ACE inhibitory activity,probably becaus e of its small molecular weight,high activity of active sites,and rapid molecul ar movement.4.In order to improve the bioavailability of the collagen polypeptide of R ana chensin in gastrointestinal fluid and enhance its sustained release effect,so dium alginate was used as the wall material,and microporous starch was used as the carrier of adsorption core material,which was prepared by the method of sharp hole coagulation bath.The results of microcapsules and simulated gast rointestinal fluids in vitro showed that the cumulative release of Rana chensin/microporous starch-alginate microcapsules and Rana sylvestris-alginate microcaps ules in simulated gastric fluid for 4 h was At 32%and 41%in simulated inte stinal fluid,the cumulative release was 88%and 83.6%,respectively.The micr ocapsules with carrier release in the gastric juice is less,and the release amou nt in the intestinal juice is slow,indicating that the carrier microcapsule can pl ay a certain sustained release effect.