The Extraction And Purification And Properties Study Of Collagen From The Chinese Forest Frog (Rana Chensinensis)

Collagen is a three spiral-shaped fibrous protein which is composed of three polypeptide chains;it is rich in animal bone,connective tissue and skin,the polypeptide chains is known as collagen protein.Rana chensinensis is an important special economic animal,after Ovum Oil extraction,the Rana skin is left as waste while which is rich in collagen protein.Research shows that collagen has an unparalleled biocompatibility and biodegradability and low antigenicity than synthetic polymer.Collagen has widespread applications,mainly for biomedical materials,food,nutrition,health care products,cosmetics,etc.In order to make full use of this resource,the Rana skin collagen proteins were extracted,purified and the properties were studied.After measure and calculation,it was found that the Rana skin contains of not less than 26%collagen protein,and is a good collagen extraction material.To make accuracy,and to establish formula for determination Rana skin collagen protein contents,the digestion method has been amended.And the formula was been added a correction factorλ=1.23.Ultimately,the suitable formula for the Rana chensinensis skin collagen protein content is:M₀=(C×dilution multiple×total volume/conversion factor)×λ.In order to achieve the purpose of extraction,the test use pepsin undermines part of peptide bond to make the collagen into acetic acid soluble collagen peptides.And the enzyme acid extraction conditions were optimized,the results as follows:m(pepsin):m(raw materials) =1:50,extraction temperature 37℃,soaking time 72 h,extraction buffer 50 ml 0.5mol/L ice acetic acid,extracted twice,the final extraction rate could reach 81.66%,orthogonal test analysis of variance showed that enzymatic hydrolysis temperature is an important factor in the rate of extraction,the impact on the results of the most notable.The collagen protein extracting solutions were purified.After ultrafiltration concentration, salting-out precipitation,ion-exchange column chromatography and gel filtration chromatography purification steps,collagen concentration reached 34.5 g/L,purification fold was 2.326,the purity reached 96.1%,Collagen total mass is 104 g,and recovery was 39.8%.Through Vacuum freeze-drying,Rana chensinensis collagen products were got.It is a loose,white slightly yellow poly-film,and it had a very low density about 0.002 g/cm³, furthermore,it is viscous and highly absorbent of water.Through serials test,some parameters of Rana chensinensis collagen protein were obtained.The moisture content was 16.44 percent,isoelectric point is 5.8,and it has an obvious ultraviolet absorption peak at 273.5 nm.the SDS-PAGE electrophoresis indicated that there are two obvious bands and their molecular weight were 107 KD and 89 KD respectively,and these facts proved that the experiment have obtained twoαprotein components of collagen molecules.It is the first time in China to use the Rana chensinensis skin as raw materials for collagen protein purification and property determination,and it is a meaningful work in the exploration of Rana.