Isolation And Characterization Of Collagen From The Skin Of Bighead Crap And Study The Behavior Of Self-assembled In Vitro

In this study, the compositions and some properties of acid-soluble collagen (ASC)and pepsin-solubilised collagen (PSC) from bighead carp (Aristichthys nobilis) wereinvestigated. PSC was used for the study of the self-assembly dynamics and productstructure analysis in vitro under different conditions. On this basis, further performance ofthe product self-assembly were determined and compared for exploring the mechanisms ofcollagen self-assembly behavior in vitro and the relevance of collagen materialperformance from fish resources. This was used for providing theoretical guidance toimprove fish source collagen performance and expanding its scope of application.At low temperatures, ASC and PSC were extracted with the yield of20.6%and30.0%on the basis of dry weight, from bighead carp skin respectively. The pigskinenzyme-soluble collagen (PPSC) was extracted using the same method, which yield is38.7%. Both collagens comprised two different α chains (α1and α2) which were classifiedas type collagen. ASC and PSC were similar in amino compositions, protein patterns,ultraviolet and FTIR spectra, but they were different from those of pig skin collagen(PPSC). Both of ASC and PSC had a lower denaturation temperature than PPSC, but theywere higher than that of other freshwater fish. ASC and PSC showed a maximum solubilityat pH5. No changes in solubility were observed in the presence of NaCl up to3%(w/v).However, a sharp decrease in solubility was found with NaCl above3%(w/v). ASC, PSCand PPSC revealed only partial degradation in collagenase solution and ASC showed moreresistive to collagenase, compared to PSC and PPSC. Both of ASC and PSC did not inducea significant cytotoxic effect according to the results of in vitro cytotoxicity test.Using turbidity experiments and the degree of self-assembly to study the PSC，sdynamic behavior of self-assembly under different conditions. in vitro. It suggested that thePSC，s self-assembly behavior was affected by the system pH, ionic strength, temperatureand concentration of collagen and other factors. In low concentration (1mg/ml), with theincrease of pH, the speed of collagen assembly improved significantly, but in highconcentration (≥3mg/ml), the effect of pH factor in the collagen self-assembly speed andassembly extent weakened; below the collagen denaturation temperature, the speed anddegree of collagen self-assembly was positively correlated with the temperature of thesystem, the higher the temperature, the faster the speed assembly and the higher the degreeof self-assembly; Adjusting the ionic strength of the system by regulating the NaCl content,the system ionic weaken the kinetic behavior of the collagen self-assembly in vitro. The results show that the behavior of PSC self-assembly in vitro affected by the ionic strengthof significantly. The lower the ionic strength of the system the higher the speed and degreeof collagen assembly. In high ionic strength conditions, the self-assembly behavior ofcollagen was significantly inhibited.SEM and TEM were used to observe the PSC self-assembly product structure analysisunder different conditions and comparison of the size of self-assembled fiber. The resultsshowed that PSC assembled product showed a typical D cycle which staggered the columnstripe fibrous structure, but its D cycle value was below the D cycle value of pigskincollagen, suggesting that fish collagen self-assembled is in a regular and orderlyarrangement acts which was different from mammals. The fiber diameter was measuredand statistical analysis results showed that the PSC assembled fiber diameter affected bythe assembled conditions. Under neutral pH conditions, the average particle diameter of thefibers of the assembled product was the maximum (183.4±63.5nm),the particle sizedistribution is relatively uniform; when collagen concentration was1mg/mL, assembledfiber diameter (153.8±37.2nm), its particle size distribution is relatively concentrated, butafter the collagen concentration was up to3mg and5mg/ml, collagen increase in theaverage particle diameter, respectively (183.4±63.5nm,175.0±66.0nm) and its particlesize distribution the uniformity decreased significantly. As the collagen prolongation of theassembly time, assembly of the product fiber diameter gradually increased, but alsoincreases the particle size distribution unevenness. The ionic strength of the system did notsignificantly affect the uniformity of the particle size distribution of the fibers of collagenassembly, but impact of the average particle diameter value. As the NaCl content was0mmol/l, the fiber particle diameter of the highest value. Assembling the temperature ofthe system increases, the average particle diameter of the assembled product is graduallyreduced, but the particle size distribution homogeneity little change.Assembled product in different assembly conditions to further develop the thermaldenaturation temperature, in vitro enzymatic degradation of performance analysis theprobe assembly conditions on the properties of the product. The experimental resultsshowed that the self-assembled by in vitro treatment, the resistance to enzymaticdegradation of the collagen product performance and thermal stability than the originalsample with varying degrees of improvement, indicating that the biological stability of thecollagen sample is improved by the self-assembly process. However, the assemblyconditions affect this change of collagen performance At apparent differences of thebiological properties of the assembled product, in essence, by the uniformity of theassembled product fiber particle size and particle size distribution. Assembled product fiberlarger the particle size distribution is more uniform, the biology better performanceperformance.