| Collagen is a macromolecular structural protein widely existed in living organisms,which has a unique triple helix structure.Because of excellent biocompatibility,low immunity and easy degradation,collagen has been widely used in functional foods and biomedical materials.Fiber recombination is the most important molecular behavior of collagen.Under the conditions of appropriate temperature,p H value and ionic strength,the collagen molecules gathers spontaneously to form fiber structure because of hydrogen bond and hydrophobicity.Compared with single molecule of collagen,collagen fiber has better thermal stability and mechanical properties.In order to further improve the inherent performance of collagen materials and achieve functional diversification,the graft modification of collagen side chain is often used to regulate the structure and performance of collagen materials by other functional small molecules or polymers,in order to better adapt to production and life.However,the modification of side chaining branch often results in the loss of the inherent fiber recombination performance of collagen.Therefore,it is of great scientific significance to improve the performance of collagen materials by modifying the side chains while maintaining the composite properties of collagen fibers.In this study,collagen was grafted with n-acryloxysuccinimide,and the research was focused on the in vitro fiber recombination of acrylic acid grafted collagen.On the one hand,the grafted collagen was adjusted by different concentrations of acrylic acid to realize the in vitro fiber recombination.On the other hand,the grafted collagen modified with high graft density was mixed with natural collagen,and the formation of co-assembled fiber products was verified by mercapto nanogold labeling combined with electron microscope observation.The main research contents was as follows:(1)Firstly,Whether the modified collagen at different graft density can realize fiber recombination in vitro is the focus of this chapter.Grass skin acid soluble collagen(ASC)was used as raw material,under alkaline conditions(p H 8.2).Natural collagen was grafted respectively according to N-succinimide acryloyl oxygen radicals(NHS-AA)and collagen epsilon-amino mole ratio of 2,10,20,40.The acrylic acid grafted(AA-g-Col)could be obtained at the low temperature(4 ℃)reaction after8 h dialysis purification,TNBS method is used to determine the grafting rate is respectively: 19%,31%,54%,68%.The results of CD showed that the grafted collagen with different graft rates had similar spectral structure to the natural collagen,indicating that the graft density would not affect the integrity of the triple helix structure of collagen.According to the differential scanning calorimetry(DSC)data,the separation temperature of the grafted collagen trihelix pyrolysis slightly decreased with the increase of grafting density,which was caused by the increase of interchain steric hindrance and the propylene acylation of the amino group of the collagen side chain.Compared with natural collagen self-assembly dynamic showed that the nucleation period and growth period of AA-g-cols was much longer.With the increase of grafting rate,the nucleation period and the end time of AA-g-cols moved backward significantly,and AA-g-col(68)did not have self-assembly performance.Chloramine T colorimetry showed that the degree of fiber recombination of AA-g-col(19),AA-g-col(31)and AA-g-col(54)were 75±4%,63±2% and 17±4%,respectively,which were all lower than that of natural collagen(87±5 %).AA-g-col(68)had no fiber recombination products.The study of rheological frequency scanning mode shows that the elastic modulus(G’)of natural collagen,AA-g-col(19),AA-g-col(31)and AA-g-col(54)are all higher than their viscosity modulus,indicating that these systems are all in gel state.The G’ in AA-g-col(68)system indicates the solution state.Transmission electron microscopy observed that there was no significant difference in d-cycle length between natural collagen and grafted collagen fibers in terms of fiber morphology,while the grafted collagen fibers were observed that the particle size of the grafted collagen fibers decreased by scanning electron microscopy,which may be caused by the decreased fiber recombination capacity because of the increase of graft density.(2)In vitro fiber recombination of collagen grafted by acrylic acid induced by natural collagen.AA-g-col(AA-g-Col)with high graft density was prepared by acid soluble collagen(ASC)from grass fish skin.The results of circular dichroism showed that the graft modification of acrylic acid does not affect the integrity of collagen triple helix structure.After blending AA-g-col with natural collagen,the co-assembly was started under appropriate conditions.Self-assembly dynamic showed the curve was observed to be segment-shaped.It was speculated that the co-assembly of natural collagen and AA-g-col formed hybrid fibers in this process,and the participation degree of AA-g-col was higher than that of natural collagen in this stage.Natural collagen continues to be assembled to form pure collagen fibers when AA-g-col is consumed.In the DSC image,it was also observed that there were two absorption peaks when AA-g-col molecules were simply blended with natural collagen fibers,while AA-g-col/natural collagen co-assembled fiber products had only a single peakwhich indicated the formation of hybrid fibers.Acryl functional groups also exist on the surface of co-assembled fibers since AA-g-col has acryl functional groups while natural collagen molecules do not.Therefore,Michael addition reaction between mercapto gold nanoparticles and acryl functional groups was used to realize the labeling,thus providing visual evidence of hybrid fiber formation.Firstly,the gold nanoparticles with uniform size were prepared by citrate reduction method.After the gold nanoparticles were modified with hexanedimercaptan,the gold surface was sealed with ethyl mercaptan to avoid non-covalent bond adsorption,thus to obtain the mercapto gold nanoparticles.Then natural collagen system and AA-g-Col/natural collagen blend system were isolated from fibrosis after assembly,and respectively with sulphur nano gold reaction,the centrifugal purification using TEM observation,the results showed that the fiber of the two systems have obvious D periodic structures,but AA-g-Col/natural collagen hybrid fiber surface has obvious nanometer gold particles,and no natural collagen fiber surface,thereby directly confirmed AA-g-Col/natural collagen formation of hybrid fiber. |
