| The research on the denaturation and refolding behaviors of natural collagen is an important branch of the analysis of the triple helix formation mechanism of natural collagen,and is also of great value for the applications of collagen and its denatured products in the fields of biological materials,medicine,and food.Many scholars have conducted a large number of studies on the cooling renaturation behavior of heat-denatured collagen and proposed various mechanisms for the formation of the triple helix conformational fold of collagen during cooling renaturation.However,for another type of collagen denaturation process,that is,the denaturation and renaturation under the action of denaturants such as urea and guanidinium salts strong polar compounds are rarely involved.Therefore,in this study,urea was used as a denaturant to prepare urea-induced denatured collagen,which was removed by dialysis to eliminate the inhibitory effect,so that the denatured collagen was refolded,and the effects of p H,temperature and polar solvents on the overlapping behavior were explored.On this basis,the structure and properties of the refolded products were characterized,and the potential applications of the refolded products were explored.The main research contents and results are as follows:(1)Effect of environmental factors on refolding behavior of urea-denatured collagen.Urea-denatured collagen was obtained from bovine Achilles tendon collagen and PBS solution containing 8mol/L urea as denaturant.To study the influence of environmental factors on collagen refolding behavior,urea was removed by dialysis.The results showed that in 8mol/L urea system,natural collagen showed obvious dissociation and denaturation behavior of peptide chain.After urea removal,collagen peptide chain began to show refolding and renaturation characteristics,and the refolded products had different degrees of triple helix structure.In the refolding process,the environmental factors of dialysis system significantly affected the refolding behavior of renaturated products.Compared with p H value,the temperature of dialysis system has a significant effect on refolding behavior,and reducing dialysis temperature is beneficial to improve the ratio of triple helix reconstruction The introduction of organic solvents with different polarities also has a significant effect on refolding behavior.Reducing the polarity of dialysis system can significantly increase the ratio of triple helix reconstruction in refolding products.(2)Structural analysis of refolded collagen products denatured by urea.The molecular structure of refolded collagen in different environmental systems was systematically analyzed by circular dichroism analysis,free amino and carboxyl analysis and Sirius Red colorimetric analysis.The results showed that the primary structure of urea-denatured collagen remained intact,and the secondary structure of its product showed a trend of transformation to natural collagen structure after refolding.However,there are still obvious differences between the structure of specific gravity folded collagen and natural collagen,which are as follows: the proportion of ?-helix in refolded products is lower than that of natural collagen,while?-folding is opposite;In addition,the content of free groups(amino and carboxyl)of the refolded products increased obviously,while the apparent viscosity decreased.After in-depth investigation of environmental factors,it is found that reducing environmental temperature and polarity is not only beneficial to the transformation of secondary structure of refolded products from ?-folding to ?-helix,but also conducive to the improvement of triple helix reconstruction ratio and free group content;On the contrary,reducing the ambient temperature and polarity reduces the apparent viscosity of refolded products to some extent.(3)Performance analysis of collagen refolding products denatured by urea.Based on the structure exploration of the refolded products,the performance analysis was further carried out.The results showed that the properties of refolded products were obviously different from those of natural collagen based on structural changes.From the aspects of physical and chemical properties,molecular behavior,biology and mechanical properties,they are summarized as follows: First,the solubility of refolded products is obviously increased;Second,although the refolded product has molecular self-assembly behavior,the self-assembly ability,assembly degree and gel viscoelasticity of the product are obviously reduced;Third,the ability of refolding products to promote cell proliferation is enhanced and the function of promoting cell adhesion and migration is weakened Fourthly,the compression resistance of refolded products is improved.In addition,reducing the environmental temperature is helpful to further improve the water solubility,self-assembly degree,cell proliferation promoting ability and compression resistance of the product,but reducing the environmental polarity will weaken the compression performance of the product. |
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