| The collagen and collagen active peptides derived from aquatic by-products have been the research hotspots because of their excellent functional properties,and have great potential for application.Using Oreochromis niloticus skin as raw material,acid soluble collagen(ASC)and gastric soluble collagen(PSC)were prepared by acid extraction and enzyme extraction methods.The amino acid sequences of two kinds of collagen,secondary and tertiary structurewere identified,and the thermal stability,relative solubility,rheology and apparent structure were compared.The collagen release from collagen ACE was studied by using collagen prepared as the parent protein.On this basis,we investigated the release rule of ACE inhibitory peptides in collagen alkaline protease system.The main results are as follows.1)The yield of ASC was 10.93%,the yield of PSC was 18.99%,and SDS-PAGE showed that they were typical ? collagen;both of them weretype ? collagen of Oreochromis niloticus ideitified by LC/MS/MS;the FITR spectrum,CD spectra and X-ray diffraction determined the collagen are natural three helix structure;thermal stability,solubility and rheology experiments showed that PSC,the non-helical end peptide of collagen was cut by pepsin,can be applied to high temperature and high salt conditions,and the honeycomb sponge structure is suiTab.for use as a drug carrier.2)Papain?trypsin?bromelain?collagenase?animal protease,alkaline protease were used to hydrolyzed collagen for 4h,the highest hydrolysis degree of collagenase hydrolysates was 17.08%,and the highest ACE inhibitory activity of alkaline enzyme hydrolysateswas 65.14 ±0.88%.3)Shotgunproteomics method was used to analyze thehydrolysate mixture with higher ACE inhibitory activity:bromelain(A)?alkaline protease(B)?and collagenase(C).?The molecular weight of polypeptide in the three samples was 1000Da?2000Da.Polypeptide hydrophilicity ratiowas B(88.7%)>A(85.5%)>C(75.9%);The proportion of hydrophilic amino acids composed of peptides was A(62.9%)>B(52.44%)>C(48.75%);The AHTpin platform predicts that the number of polypeptides with ACE inhibitory activity in the three samples was B(9)>C(6)>A(3).? Three enzymatic hydrolysis of collagen showed a tendency to attack the N terminal and C end of collagen subunits.The low degree of enzymatic hydrolysis of bromelain only showed aggression in these two regions,and it was possible to start enzymolysis of collagen from the N end;the alkaline protease may be enzymolysis alpha 1 subunit first from N end at first,and the enzymatic hydrolysis of the alpha 2 subunit is not biased.The enzyme hydrolysis of the collagen alpha 2 subunit may be greater than the other two enzymes;collagenase is possible to hydrolyse collagen from the C side,then the N end,and finally the intermediate region,and the core region of the collagen subunit can be hydrolysable in depth compared with the other two enzymes.4)Alkaline protease system is easy to combine with collagen,and the binding site is dispersed,which is more inclined to the N and C ends of the alpha 1 subunit,the alpha 2 subunit is not biased,the medium-term to the alpha 1 subunit is twice enzyme solutions,the alpha 2 subunit continues to be enzymolysis and the rate is quicker than the alpha 1 subunit,and the alpha 1 subunit is still in the later period.The enzyme solution of alpha 2 subunit tends to be sTab.by twice enzymatic hydrolysis,and it appears that there is a trend that alpha 1 subunit was hydrolysised from N end to C end,and alpha 2 subunit from C end to N end,during the whole enzymatic hydrolysis process.5)The transformation order of peptides can be deduced through the change of peak area intensity of polypeptide at different times.The neural network was used to predicit peptide content changes of collagen-alkaline protease system.The 3 layer neural network of 60/50/40,R2=0.9481,could be used to predict the content of peptide under certain ACE inhibitory activity. |
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