Purification And Identification Of Collagen And Keratin From Grass Carp Scales

The grass carp scales contains abundant collagen and keratin with important economic worthy.The main components of of fresh grass carp scales were analyzed, and the extract technique of collagen and keratin was optimized by single factor and orthogonal test design, and the purified collagen and keratin from grass carp scales were identified. The results are as following:(1) Fresh grass fish scales contains 59.280% water,25.360% total protein,0.240% fat, and 15.120% total ash respectively. There are 17 kinds of amino acids were detected, accounts for a total percentage of 28.531/100% of dry weight. The optimal process for the pretreatment was using 10% sodium chloride solution soak 12h under 4℃ to removing the impurity proteins. Again used 0.1 mol/L sodium hydroxide solution soak 12h to removing the fat. Then used 1% hydrogen peroxide solution soak 12h to removing the pigment. Finally, used 4% citric acid solution soak 6h by 1:14 material and liquid ratio to removing the ash content.(2) The separation and purification process route of grass carp fish scale collagen and keratin was obtained as follows:first, using citric acid to extracting the acid soluble collagen under 4℃ condition, and the residue using pepsin to extracting the enzyme soluble collagen protein under 4℃ condition, acid soluble collagen and enzyme soluble collagen extract solution using the method of dialysis after salting-out precipitation for purification. At last the residue after extracting enzyme soluble collagen used for keratin extraction by alkaline, keratin extract solution using the method of dialysis after isoelectric point precipitation for purification.(3) Grass carp fish scales were soaked in 0.2 mol/L citric acid solution for 24h to extract acid soluble collagen after pretreatment. The solid-liquid ratio is 1:17.5. The yield of acid soluble collagen is up to 6.410% under this condition. The residue after acid soluble collagen extraction was soaked in 1% citric acid solution, and 2.5% of substrate heavier pepsin was added to extract enzyme soluble collagen, the extracting time is 28h and the solid-liquid ratio is 1:10. Under this condition the yield of enzyme soluble collagen is 10.210%. The residue after enzyme soluble collagen extraction soaked in 9% sodium hydroxide solution to extract keratin, the solid-liquid ratio is 1:15 and the extraction temperature is 60℃.(4) The UV scanning spectrum of grass carp fish scales acid soluble collagen and enzyme soluble collagen uv scanning spectrum has no impurity peak, respectively emerge a strong absorption peak at 227.0 nm and 225.2 nm, accord with the characteristic of type I collagen. The third spiral structure is intact, the molecular weight of single peptides is above 100kD, and the relative molecular mass of collagen molecules composed by three peptides is more than 300kD; There amino acid composition were consistent to type I collagen amino acid composition, no cystine detected and content less of tyrosine. The content of amino acids was slightly lower than type I collagen, among them the content of proline was about 20% lower than the type I collagen. The ultraviolet absorption peak position grass carp fish scales keratin appeared at 218.2nm and 280.4nm, its molecular weight is about 48kD, secondary structure belongs to the alpha helix structure, peptide bond is not destroyed. The amino acid composition were consistent to wool keratin, contains many cystine.