Study On Simulated Digestion Of Cod Fish Skin Collagen Hydrolyzate In Vitro And Isolation And Purification Of Antioxidant Peptides

In this study, gelatin hydrolysates were obtained after hydrolyzation of codfish collagen by six enzymes and subjected into simulated gastrointestinal digestion, and then the degree of hydrolysis (DH), the antioxidant activity and the composition of free amino acid in each stage of digestion were determined. Besides, the effects of hydrolysates from defferent digestion stages on HSF proliferation and the protective effect of hydrolysates from different digestion stages on HSF proliferation after UVB irradiation were also studied. The transmittance and the characteristics of peptide fractions from the hydrolysates of gastrointestinal digestion which could across Caco-2 cell monolayer were analyzed by HPLC and high resolution mass spectrometry.The antioxidant peptides among the hydrolysates of gastrointestinal digestion were isolated and purified.The major findings were presented as follows:1. Simulated gastrointestinal digestion on codfish collagen hydrolysates were conducted to prepare stable dydrolysates. Then the degree of hydrolysis (DH), the antioxidant activity and the composition of free amino acid in each stage of digestion were determined. Finally the alkaline protease hydrolysis group was chosen for further analysis. The DH of the group of alkaline protease hydrolysis were 26.17% (without electrolyte solution and sodium cholate) and 26.77%(with electrolyte solution and sodium cholate) respectively. The ABTS radical scavenging abilities were 59.17%(without electrolyte solution and sodium cholate) and 80.15%(with electrolyte solution and sodium cholate) respectively (hydrolysates concentration was 0.5 mg/mL). The Ferric reducing antioxidant powers were 209.27 μmol/L (without electrolyte solution and sodium cholate) and 212.85 μmol/L (with electrolyte solution and sodium cholate) respectively. Since that we speculated that electrolyte solution and sodium cholate made no influence on ferric reducing antioxidant power.2. The effects of hydrolysates from different digestion stages on HSF proliferation and the protective effect of hydrolysates from different digestion stages on HSF proliferation after UVB irradiation were studied. The result showed that the hydrolysates were nontoxic to the growth of HSF cells in a certain concentration range. The hydrolysates could promote the proliferation of HSF and effectively restrain UVB induced damage of HSF cells. After hydrolysates were simulated gastrointestinal digestion twice, the cell proliferation rate of alkaline protease hydrolysis group was 118.67%(hydrolysates concentration was 100 μg/mL).The HSF cells survival rate of was alkaline protease hydrolysis group was 63.95% after UVB irradiation, and the HSF cells survival rate of control was 56.72%.3. The Caco-2 cell monolayer was used to research the transmittance and the characteristics of peptide fractions from the hydrolysates of gastrointestinal digestion which could across Caco-2cell monolayer. The transmittance of main peptide peak were about 4.58% according to the analysis by HPLC.170 and 50 peptides were detected out respectively in the hydrolysates before and after the trans-membrane transport of Caco-2cell monolayer by high resolution mass spectrometry combined with de-novo date analysis, and the amino acid sequence of peptides accorded with the basic characteristics of collagen.40 peptide fractions of total hydrolysate were able to across Caco-2cell monolayer, most of them were tetrapeptides accounting for 65%, and the longest peptides were octapeptides. There were 28 peptides whose terminals were hydrophobic. Most peptides were composed of amino acids of GPA, GPS and GPR. The content of AGPR, VGPA and SGPR were more than other peptieds which could across Caco-2cell monolayer. The research datas showed that the simulated gastrointestinal (GI) digestion can prepare the stable protein hydrolysate effectively. The peptide fractions which are highly hydrophobic and have low molecular weights are more easily absorbed by the intestinal cell layer.4. The peptides with hydroxyl radical scavenging activity were isolated and purified from simulated digestion hydrolysate. Peptide A1a3c-p was obtained, and the IC50 value of hydroxyl radical scavenging ability of A1a3C-p was 7.626 μg/mL. The molecular mass and amino acid sequences of A1a3c-p were determined using high resolution mass spectrometry combined with de-novo date analysis.The molecular weights of A1a3c-P peptide was 445.29 Da, and it was composed of four amino acides with the sequence Try-Gly-Cys-Cys (YGCC). Seven kinds of amino acid sequence were determined by high resolution mass spectrometry combined with MaxQuant. The sequences were EDIEHMK. ELIQFFEEIMQRLMR. LLHLHASHVR. PAGNVR、EFSTFDR、NWPICK、NNAEYYK, respectively.