Biological Properties Of Collagen Extracted From Black Carp And Study Of Thermal Denaturation

Collagen is a class of structural proteins which exists in organisms widely. In recentyears, collagen-based biological material is used widely in medical tissue engineering. Fishcollagen is a new natural collagen resources have potential applications in biomedicalmaterials. However, compared to mammalian collagen, fish collagen thermal stabilitygreatly limits its scope of application. In this study, the black fish skin as raw materials, wegot acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC), systematic study of itsstructural features, the basic physical and chemical properties, thermal stability, and cellbiological properties of the system and mammalian sources pigskin enzyme-solublecollagen (PPSC). On this basis, the further development of collagen from black carp skinthermal denaturizing behavior and properties of collagen by the degree of degeneration, inorder to provide a theoretical basis for the actual production, application and storage ofcollagen from freshwater fish.Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted withthe yield of15.5and26.5%on the basis of dry weight from the skin of black carp, at lowtemperature. By analysis of UV spectroscopy, Fourier transform infrared spectroscopy andSDS-PAGE analysis showed that black carp collagen samples are typical type I collagenand have complete triple helix structure. By amino acid composition analysis shows, ASCand PSC were similar in amino compositions and subunit compositions, but they wereslightly different in the secondary structure. By DSC detection shows, both of ASC andPSC, either rehydrated in distilled water or in acetic acid, had a lower denaturationtemperature than pig skin collagen (PPSC). The intrinsic viscosity of different collagensdecreased in the order of PPSC>PSC>ASC. Under the action of collagenase, both of ASCand PSC showed only partial degradation, but they revealed more sensitive compared withPPSC. Black carp skin collagens did not induce a significant cytotoxic effect according tothe results of in vitro cytotoxicity test.Analysis PSC thermal denaturation behavior though DSC research under differentconditions. The results show that collagen moisture content, swelling time, the swellingsystem acidity strength, the type of acid, ionic strength, and other factors would result in achange of the thermal denaturation temperature of the collagen. Low moisture content ofsolid thermal stability of the collagen is best (139.14℃), but after solvent swelling, thedenaturation temperatures dropped significantly. Acid swelling in different solventswelling system, the system can significantly reduce collagen thermal stability and the thermal denaturation temperature gradually reduce with the increase of acidity. Swelling inwater system, the ionic strength is (50mM) can effectively improve the thermal stability ofcollagen (43.26℃), however, as the ionic strength gradually increases, its thermal stabilitya declining trend.N-PSC as raw materials, respectively in different under the condition of moderateheat treatment (30,180min under33℃and40℃under30min) and get samples ofdifferent degree of degeneration of collagen. By DSC, the GPC-LLS, FT-IR, SDS-PAGEanalysis are described obtained low-partially denatured collagen (LD-PSC), ofhigh-partially denatured collagen (HD-PSC) and full denatured collagen (FD-PSC)(p<0.05). The turbidity experiments show that the in vitro self-assembly capacity graduallyweakened with the increase in the degree of degeneration of collagen, full denaturedcollagen samples complete loss of self-assembly capabilities. Different degree ofdenaturation of collagen was self-assembled products fiber diameter size as follows:N-PSC（107.8±6.1nm）＞LD-PSC（93.8±5.4nm）＞HD-PSC（80.7±5.9nm）（p＜0.05）;D-period sizes respectively:64.6nm±3.5nm，60.9±5.5nm，52.2816±5.2nm（PPSC is67.5345±4.1nm）. Enzyme degradation experiment in vitro showed that trypsin and pepsinto not modified collagen hydrolysis almost no effect, but with the increase of degree ofdegeneration and collagen protein of collagen protease, trypsin and stomach eggs, thestudy showed that collagen in vitro enzyme degradation of performance, the self-assemblyproperties and in vitro product structure heat denaturation and degree of degeneration andthe influence of the change.N-PSC, LD-PSC, HD-PSC and FD-PSC as raw material, the molded collagen spongematerial was prepared under the same conditions. Respectively, by the SEM observation,the mechanical strength, swelling ratio, enzymatic degradation and cell culture in vitro tocharacterize the material properties. The results show that with the increase of degree ofdegeneration, the sponge material to the porous structure of the original features and waterswelling stability decline; Mechanical tensile properties and resistance to enzymaticdegradation in performance; But its promoting cell proliferation ability has increased.