The Extraction?characteristics And Quality Evaluation Of Collagen From Wood Frog Skin

The study belongs to the national science and technology support project-?special processing technology integration and product?(2013BAD16B09).Frog skin contains a lot of collagen.Extracting collagen from the Rana frog skin can turn waste into treasure,and develop new product value.So this has important economic and environmental benefits.In this paper,the Rana frog skin was used as raw material to improve the extraction rate of collagen by pretreatment and optimization of extraction process.Then the extract was purified,dried,and its quality standard and cytotoxicity were studied.Firstly,the forest frog skin was pretreated,and the optimum conditions of decolorization and degreasing were determined by single factor test.The results of pretreatment showed that the decolorization effect was the best when the forest frog skin was treated with 0.15 M sodium hydroxide solution containing 1.0% hydrogen peroxide at room temperature for 12 h.When 10% butanol solution was used to degrease at 1:75 at room temperature for 5 h,the degreasing effect was the best.Then a single factor test was used to determine the concentration of enzymatic hydrolysate,enzymatic hydrolysis time,enzymatic hydrolysis temperature and the ratio of the hydrolysate to liquid.And collagen protein extraction process was optimized by the response surface method.The multivariate regression equation model was obtained using Box-Behken software:Y=0.92+0.14A+0.14B+0.30C+0.13D+0.030AB+0.084AC+0.089AD+0.088BC+0.11BD+0.079CD-0.17A2-0.20B2-0.30C2-0.20D2.Using the multivariate regression equation model,we obtained the optimum technological parameters were as follows: pepsin addition was 4.94,enzymatic hydrolysis time was 3.42 h,and enzymatic hydrolysis temperature was 54.31 ?.The ratio of enzymatic hydrolysis liquid to material is 44.51: 1.Considering the convenience of practical operation,the optimum technological parameters of extracting collagen are modified as follows: the addition of pepsin is 5,the enzymatic hydrolysis time is 3.5 h,and the enzymatic hydrolysis temperature is 50 ?.The ratio of hydrolysate to feed is 45: 1.Under this condition,the extraction rate is 94.89.Secondly,SDS-polyacrylamide gel electrophoresis,ultraviolet scanning,infrared scanning,differential scanning thermal denaturation were used to compare the extracted collagen and the standard type I collagen.The results showed that the maximum ultraviolet absorption peak of collagen was about 227 nm,and the IR spectrum showed that the collagen contained amide-bond A,amide B,amide I band II,the two curves were very close to each other.In the electrophoretogram,we can see that the collagen of Rana frog skin contains ?,? 1,? 2 chain,which proves that the collagen is type I collagen and has a complete three-stranded helical structure.Finally,the quality evaluation criteria of collagen were determined by sensory evaluation and p H.The cytotoxicity of collagen was determined by MTT method.According to sensory evaluation,the quality evaluation criteria of collagen of Rana forest frog skin should be: no caking,fine velvet texture,high density,white or yellowish yellow,fresh smell of forest frog,but no peculiar smell.There were no impurities or spots.Tthe apparent density was 7.13 × 10-3g/cm3.The p H value was 6.9.MTT test showed that the collagen extracted from the skin of Rana had no cytotoxicity.