Extraction And Biological Evaluation Of Collagen Extracted From Sika Deer (Cervus Nippon) Hamstring

Collagen is a hydrolyzate with a stable triple helix structure and relatively smallmolecular weight which is degraded faster and absorbed easier by the organism. It hasbeen reported recently that collagen is widely used in the domain of food, cosmetics,pharmaceuticals and so on owing to its biochemical properties and pharmacologicalactions. The wild sika deer is the first class national protected animal, deer townshipdevelops large-scale artificial breeding of sika deer and the related industries such assika deer’s muscle, tendon and blood which can be used as a food material. Dried deertendon is widely used medicinal herbs in traditional Chinese medicine. In this study,collagen was extracted from fresh tendon of sika deer.[Objective] To establish collagen extraction process with fresh sika deer tendonand evaluate its biological activity and provide a scientific basis for the developmentof new collagen products.[Methods] Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) wereextracted from sika deer hamstring (cervus nippon). Concentration of acetic acid，extraction time and the ratio of gardenia to liquor was regarded as the main factors.The extraction technology of acid-solubilized collagen from sika deer (DASC) wasoptimized by orthogonal experimental design. The collagens were reclaimed bysalting out method and the surface topography of DASC and pepsin-solubilizedcollagen from sika deer (DPSC) was observed by environmental scanning electronmicroscopy (ESEM). The extracted collagens were identified by ultraviolet spectra,Fourier transform infrared spectrum (FTIR), SDS-PAGE and tannin test. L929cellviability was detected by MTT assay when exposed to leach liquor of the collagenextracted from the sika deer hamstring (cervus nippon). Skin sensitization test wasused to examine the stimulation of DASC and DPSC on the rabbit skin.[Results] The results showed that the optimum extraction conditions asfollows: concentration of acetic acid (0.5mol/L), extraction time (36h), the ratio of gardenia to liquor (1:40). The yields of DASC and DPSC were7.49%and7.9%based on dry weight basis with salting out method. The yields of DASC were7.4%based on dry weight basis with a simpler salting out method. DASC and DPSC tookon a plicated and platelike structure under the ESEM, platelike structure wasconnected with each other and compact porous texture was formed. Much platelikestructure existed in DASC. DPSC contained much platelike structure andcollagenous fiber bundle. Both ASC and PSC from the sika deer hamstring (cervusnippon) have maximum absorptions near223nm, initially regarded the protein ascollagen. FTIR spectra of DASC, DPSC and CASC exhibited the characteristicpeaks of Amide I, II, III as well as amide A and B. Based on FTIR spectra, the aceticacid and pepsin did not disrupt the triple helical structure of collagen. It was foundthat the major constituents of both the ASC and PSC consisted of chains α1, α2andβ. Both DASC and DPSC showed a low degradation by tannin test. The cellproliferation of DASC leach liquor group and DPSC leach liquor group showed nosignificant difference compared with control group. Leach liquor of DASC andDPSC revealed no effect to L929cells proliferation. Based on Skin sensitization test,no skin irritation was observed after treatment with by DASC and DPSC.[Conclusion] Collagen extraction process was established with fresh sika deertendon and the extracted collagen was identified. It showed no cytotoxicity to L929cells and no skin irritation after treatment with by DASC and DPSC.