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Preparation And Characterization Of Freshwater Fish Collagens And Their Protein Films

Posted on:2016-01-17Degree:MasterType:Thesis
Country:ChinaCandidate:L L TangFull Text:PDF
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Extracting collagen and gelatin from fish processing by-products to prepare the protein films has became a new way for efficient utilization of freshwater fish resources.Therefore,in this study,acid souble collagens?ASCs?were isolated from tilapia,grass carp and silver carp skins and the based protein films were prepared,then their physicochemical properties were investigated.The correlation between film-forming properties of and primary structure of collagen was further explored.In addition,dialdehyde starch?DAS?was added to improve the mechanical properties and water resistance of tilapia skin gelatin films,on this basis,the effect of collagen hydrolysate?CH?on the properties of gelatin films and growth of human skin keratinocytes?HaCat?cell were also investigated.Firstly,ASCs were extracted from tilapia,grass carp and silver carp skins,the physicochemical properties of ASCs were examined.Similar SDS-PAGE patterns,amino acid composition and UV spectrum were observed in the obtained skin ASCs,but differences were found in the peptide maps.The reduced viscosity value at 25 oC and the thermal transition temperature?Tmax?of tilapia skin ASC were higher than that of grass carp and silver carp skin ASCs.On the other hand,the nucleation time of fibril formation of skin collagen from tilapia,grass carp and silver carp was about 2 min,6 min and 3 min,respectively.It is concluded that difference in the physicochemical properties of ASCs from freshwater fish skins could be correlated with the primary structures.Nextly,the film-forming ability of three ASCs was investigated.As a result,the tensile strength?TS?of tilapia skin collagen films could reach up to 51.24 MPa,which was higher than that of silver carp and grass carp skins collagen films.And the color values of tilapia skin collagen films were closer to that of white standard plate.Moreover,compact,smooth and homogeneous distribution was observed on the upper surface of collagen films based ASCs from tilapia and silver carp skins,but significant coarser with different sizes of bump structure was found on the upper surface of grass carp skin collagen films.On the other hand,the silver carp COL1A1 and COL1A2 gene sequences were cloned using RT-PCR and RACE technology.The full-length cDNA of COL1A1 and COL1A2 were 4923 bp and 4616 bp,which encoded 1448and 1352 amino acids,respectively.Phylogenetic tree shows that the?genes of silver carp is close to grass carp,but far from tilapia.Amino acid sequence analysis indicates that there are more Pro in the C-peptide and G-X-Y sequences of triple-helical domain of tilapia COL1A1.It could be speculated that the?1 chain play an important role in the film-forming process of collagen.On the other hand,the effect of pH and DAS on the mechanical properties and water resistance of gelatin films prepared from tilapia skins was investigated.With increasing pH value of film-forming solution,the TS of films firstly increased,but a decrease of TS was observed after pH 7,where the maximum was 23.78 MPa.Moreover,the matter solubility?MS?and protein solubility?PS?of gelatin films decreased gradually.The TS of gelatin films increased when incorporating DAS into film-forming solution,while their MS and PS declined significantly.On the other hand,the mechanical properties and water resistance of films were effectively improved by adding 1.5%DAS at pH 7.Based on the SDS-PAGE analysis,the crosslinks between gelatin proteins and DAS occurred during the film-forming process.At last,the effect of CH on the properties of tilapia skin gelatin films and growth of HaCat cell was examined.The mechanical properties and water resistance of gelatin films crosslinked with DAS degreased with increasing CH.According to the results of SDS-PAGE and FTIR,Excessive CH?>6%,w/w?destroyed the secondary structure of gelatin film at some extent,reducing the content of triple helix structure and increasing random coil and?sheet in the gelatin film.However,moderate amount of CH?2%-6%,w/w?could promote the proliferation of HaCat cells cultured on tilapia skin gelatin films.
Keywords/Search Tags:acid souble collagen, collagen films, cloning, primary structure, gelatin films, crosslinking, cell proliferation
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