Extraction Of Rana Skin Collagen By Enzyme And The Study Of Its Activity

In this study,we used the Rana skin which is take from the remaining frogs after the removal of Rana oil for extraction.Collagen is the main protein of the body,mainly stored in connective tissue,with the functions of support organs,protect the body and so on,because of its excellent biocompatibility,biodegradability and low antigenicity,they widely applicated in food,health care products,cosmetics and so on.In this paper,the technology of extracting collagen from Rana skin was optimized,and its physical and chemical properties and physiological activities were studied.It provided a theoretical and technical support for the scientific use of Rana skin,promoting deep transformation and improving product added value.1.The results showed that the protein content of the skin was about 71%,the fat content was about 4.56%,the ash content was about 5.6%,and the water content was about 6.6%.Hydroxyproline was the unique content of collagen,the determination of collagen in the skin of the rana is about 35.75%,accounting for 50% of the total protein content.2.Frog skin contains a lot of pigment,fat and non-collagen protein,they have a great impact on the extraction of collagen,in order to ensure the quality of the collagen and extraction rate,the study perform a series of pretreatment of decolorization,dehybridization,and degreasing.Lipase was used to remove lipid of the Rana skin,and the pretreatment scheme was optimized by single factor orthogonal test.The effects of different extraction techniques on the extraction of collagen were compared.The effects of 0.02 mol / L Na OH(1% H2O2)solution as the decolorizing liquid were studied at room temperature for 18 h.Under the condition of this,the decolorization effect and the collagen loss rate was 1.9%.Under the condition,the removal rate of the protein was 92.59% and the collagen loss rate was 0.3%.After the treatment was carried out at room temperature for 18 h at 7.5% Na Cl solution.The decoction of decolorizing protein was washed,dried and weighed,and degreased by lipase.The final degreasing program was determined as follows: the ratio of material to liquid was 1:30,the amount of enzyme was 5%(dry weight ratio),p H 9.0,treated at 37 ° C for 70 minutes,and the conditional detachment rate reached 81.01%.3.The enzymatic extraction of collagen from the skin of the Rana.First compare the extraction of several different proteases to determine the best enzyme source.The optimum extraction conditions were as follows: the ratio of material to liquid was1:64,and the amount of pepsin was determined by single factor test and response surface methodology.The optimum extraction conditions were as follows: The amount of pepsin 0.34%(dry weight ratio),p H 2.0,37 ? extract for 35 h.The enzyme solution for ultrafiltration,salting out,dialysis,concentration,purification before obtain collagen.The extraction rate of collagen was 80.16%.Collagen purity was 94%.4.The results of collagen analysis showed that SDS-PAGE was consistent with the structure of type ? collagen.The results showed that there were two ?-chains,two?-chains and one ?-Chain,the molecular weight is at least 80 KDa,and the electrophoretic band is similar to Bovine Achille tendon collagen.The full-band scanning results of UV spectroscopy show that the characteristic absorption peak of collagen is about 235 nm.The results of Fourier transform infrared spectroscopy The results showed that the collagen protein of Rana skin had a complete supercoiled structure in the five functional groups of amide A,amide B,amide ?,amide ? and amide ?.The amino acids compension result showed that the amino acid composition contained 17 amino acids in the collagen of the Rana skin,which had the highest glycine content,about one-third of the total amino acid content,and contained the amino acid proline content was 12.70,higher than most proteins,wherein the amino acid composition of collagen compliance,compared with the feed composition bovine Achilles tendon collagen amino acids,the amino acid composition indicated that the Rana skin collagen similar to collagen ?;Rana skin moisture content of the collagen sample was 6%,hygroscopicity and good moisture retention.5.Study on Rana skin collagen DPPH ·,OH ·,O2-· scavenging action and reducing power to determine its antioxidant capacity in vitro.The results showed that skin collagen Rana excellent reduction ability,each free radical scavenging rate was increased with the collagen solution concentration rise,it showed a significant dose-dependent manner.